Advances in Carbohydrate Chemistry and Biochemistry, Volume 41

Advances in Carbohydrate Chemistry and Biochemistry

Volume 41

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Advances in Carbohydrate Chemistry and Biochemistry Editors R. STUART TIPSON DEREK HORTON

Board of Advisors BENGTLINDBERG HANSPAULSEN NATHAN SHARON MAURICE STACEY ROY L. WHISTLER

LAURENS ANDERSON J. ANGYAL STEPHEN CLINTON E. ~ i L L o u GUYG . S. DUTTON ALLANB. FOSTER

Volume 41

1983

ACADEMIC PRESS A Subsidiary of Harcourt Brace Iovanovich, Publishers

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San Francisco

COPYRIGHT @ 1983, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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LIBRARY OF CONGRESS CATALOG CARD NUMBER: 45- 1 135 1 ISBN 0-1 2-007241 -6 PRINTED IN THE UNITED STATES OF AMERICA 83 84 85 86

9 8 7 6 5 4 3 2 1

CONTENTS CONTRIBUTORS . . . PREFACE. . . . .

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

vii ix

John Kenyon Netherton Jones (1912-1977) A . SZAREK. MAURICESTACEY. AND GEORGE W. HAY WALTER

Text . . . Appendix

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

1 11

Carbon-13 Nuclear Magnetic Resonance Spectroscopy of Monosaccharides KLAUSBOCKAND CHRISTIAN PEDERSEN I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Sampling Techniques . . . . . . . . . . . . . . . . . . . . . . . . . 111. Assignment Techniques . . . . . . . . . . . . . . . . . . . . . . . . IV. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27 28 34 39 44

Structural Chemistry of Polysaccharides from Fungi and Lichens ELIANA BARRETO-BERGTER AND PHILIP A . J . GORIN I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . a-D-Linked Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . 111. p-o-Linked Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Glucans from Lichens . . . . . . . . . . . . . . . . . . . . . . . . . V. Mannans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Galactans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . 2-Acetamido-2-deoxy-o-glucuronic Acid Polymer . . . . . . . . . . . . VIII . 2-Amino-2-deoxy-~-galactopyranan . . . . . . . . . . . . . . . . . . . IX. Heteropolysaccharides Based on o-Mannan Main-Chains . . . . . . . . X . Heteropolysaccharides Based on Galactan Main-Chains . . . . . . . . . XI . Miscellaneous Polysaccharides . . . . . . . . . . . . . . . . . . . .

68 68

72 75

77 87 88

88 89 100 101

Biosynthesis of Cellulose DEBORAH P. DELMER

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. A Survey of Organisms Useful for the Study of Cellulose Biosynthesis

111. IV. V. VI . VII .

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

Structural Considerations Relevant to Biosynthesis Cytological Investigations of Cellulose Biosynthesis The Mechanism of Polymerization . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . Addendum . . . . . . . . . . . . . . . . . . . . .

V

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

105 107 110 116 125 150 152

vi

CONTENTS Capsular Poiysaccharides as Human Vaccines HAROLD J . JENNINGS

I . Introduction . . . . . . . . . . . . . . . . . . . . I1. Structures of Capsular Polysaccharides . . . . . . . I11. Other Important Structural and Physical Features of Capsular Polysaccharides . . . . . . . . . . . . . . IV. Immune Response to Bacterial Infection . . . . . . V. Polysaccharide Vaccines and Immunity . . . . . . VI . Bacterial Virulence . . . . . . . . . . . . . . . . .

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155

. . . . . . . . . . 158 . . . . . . . . . 174 . . . . . . . . . . 186

. . . . . . . . . . 191 . . . . . . . . . 202

High.Resolution. 'H-Nuclear Magnetic Resonance Spectroscopy as a Tool in the Structural Analysis of Carbohydrates Related to Glycoproteins JOHANNES

F. G . \'LIEGENTHART. LAMBERTUS DORLAND. A N D HERMAN VAN HALBEEK

I . General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 209 I1 . Hiyh.resolution. 'H-N.m.r. Spectroscopy of Carbohydrates Related to Glycoproteins of the N-Glycosylic Type . . . . . . . . . . . . . . . . 218 111. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . 371 IV. Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 AUTHORINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

375 393

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ELIANABARRETO-BERGTER, Departamento de Microbiologia Geal, Universidade Federal do Rio de Janeiro, Brazil (67) KLAUSBOCK,Department of Organic Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark (27)

DEBORAH P. DELMER,* MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 (105) LAMBERTUS DORLAND, Department of Bio-Organic Chemistry, University of Utrecht, Utrecht, The Netherlands (209) PHILIP A. J. GORIN,Prairie Regional Laboratory, National Research Council, Saskatoon, Saskatchewan S7N OW9, Canada (67) GEORGE W. HAY,Carbohydrate Research Znstitute and Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada (1) HAROLD J. JENNINGS, Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario KIA OR6, Canada (155) CHRISTIAN PEDERSEN, Department of Organic Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark (27) MAURICE STACEY, 12 Bryony Road, Weoley Hill, Birmingham B29 4BU, England (1) WALTER A. SZAREK, Carbohydrate Research Znstitute and Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada (1) HERMAN VAN HALBEEK, Department of Bio-Organic Chemistry, University of Utrecht, Utrecht, The Netherlands (209) JOHANNES F. G. VLIEGENTHART, Department of Bio-Organic Chemistry, University of Utrecht, Utrecht, The Netherlands (209)

*Present address: ARC0 Plant Cell Research Institute, 6560 Trinity Ct., Dublin, California 94568. vii

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PREFACE In perhaps no other field of biological chemistry has n.m.r. spectroscopy played such an important role as it has in the structural investigation of the carbohydrates. Its use as an investigative tool has had significant implications across the whole range of carbohydrates, from simple sugar derivatives to complex polysaccharides and glycoconjugates. It is therefore fitting that, in Aduances, this technique should constitute a sustained theme of interconnected articles that are devoted to various important groups of carbohydrates and to the implications of rapid advances in instrumental methodology. As well demonstrated by Gorin’s article in Volume 38 of this Series, carbon-13 n.m.r. spectroscopy has proved to be of profound significance in the structural investigation of polysaccharides; it has taken its place in complementing modern versions of such traditional techniques as methylation analysis and periodate oxidation, and may in large measure replace them as our library of reliable reference data for the simple sugar constituents is consolidated. In the present volume, a most significant step in this direction is taken by Klaus Bock and Christian Pedersen (Lyngby, Denmark) in their extensive and careful compilation of carbon-13 data for a wide range of monosaccharides and their derivatives. The data are conveniently arranged in a selection of representative Tables, and the fact that the authors have themselves conducted extensive verification of the data presented offers the user a measure of convenience and confidence that could never be met by the scattered and often conflicting data in the primary literature. In a similar vein, but with reference to proton-n.m.r. spectroscopy, Vliegenthart and coworkers (Utrecht, The Netherlands) have assembled the fruits of their detailed, comparative studies, by state-of-theart, n.m.r. instrumentation, on a large number of carbohydrates related to glycoproteins. Much of this work was conducted with materials isolated in the laboratories of J. Montreuil (Lille, France), who contributed a landmark article on the structure of glycoproteins to Volume 37 of Aduances. The present, complementary article displays the great power of high-field n.m.r. spectroscopy in applications related to glyco-conjugates of considerable complexity. An article by Barreto-Bergter (Rio de Janeiro, Brazil) and Gorin (Saskatoon, Canada) likewise invokes strong emphasis on n.m.r. methods for structure determination, in this instance by use of carbon13 techniques in delineating the structural chemistry of polysaccharides from fungi and lichens. In comparison with the foregoing complex polysaccharides and conjugates, the structure of the world’s most abundant chemical comix

Y

PHE FrlCE

pound, nanwly cellulose, may seem prosaic indeed, and yet it is quite astonishing that. despite a high level of sophistication in our understanding of the mode of hiosynthesis of many rare, complex carbohydrates, we still have remarkably little definitive knowledge of the way in which Nature builds this ubiquitous, plant polysaccharide. In an article that offers challenges to established dogmas and invites fresh thotight, Deborah P. Delmer (now of Dublin, California) challenges the validity of conclusions that have often been taken for granted, and emphiasizes the need for open-minded, new research on the biosynthesis of the cellulose fiber. The dntmatic success of antibiotics for therapeutic control of many microbial infections has tended to overshadow the value of immunochemical approaches. The article by Jennings (Ottawa, Canada) provides an up-to-date discussion of the structures of a variety of bacterial capsular polysaccharides, and sewes to emphasize the important uses that such compounds have as human vaccines. In a voltimc ha\,ing strong emphasis on polysaccharide topics, it is especially appropriate to recognize the life and work of J. K. N. Jones. The article h e r e contributed by Szarek and Hay (Kingston, Ontario, Canada) and Stacey (Birmingham, England) provides a sensitive account of Jones’s work on both sides of the Atlantic, and includes a useful appendix that lists his scientific publications. The Editors note with regret the recent passing of Louis Malaprade, University of Nancy, discoverer of the stoichiometric oxidation of glycols by periodate, a reaction that has had such profound implications in the structural investigation of carbohydrates; and of Karl Freudenberg, Heidelberg, last surviving student of Emil Fischer’s, pioneer of important stereochemical concepts, and a scientist whose extensive contributions to synthesis included the classic, widely used acetone derivatives (isopropylidene acetals) of the monosaccharides. The Subject Index was compiled by Dr. Leonard T. Capell.

R. STUART TIPSON DEREK HORTON

Advances in Carbohydrate Chemistry and Biochemistry

Volume 41

1912 -1977

JOHN KENYON NETHERTON JONES 1912-1977

Many great men have compelled the admiration of their associates, but few have won the respect and the affection of their colleagues and coworkers to the extent achieved by John Kenyon Netherton Jones. Professor Jones was at all times an educator of the highest rank, and an inspiration to a large number of graduate students, from whom he evoked, as a result of his enthusiasm, sincerity, and gentle character, tremendous fealty and dedication. His life belies the popularly accepted quip “nice guys finish last.” On January 28,1912, J. K. N. Jones was born in Birmingham, England, the eldest son of George Edward Netherton Jones and Florence Jones (n6e Goldchild). His father was a shipping agent for the Elder-Dempster line; during the latter part of his life he was in poor health as a consequence of being badly gassed during World War I, and he died in the early 1920’s from tuberculosis. For the next few years, Jones’s mother strove to secure a pension for herself and her seven children, but because a pension was not granted until 1926, shortly before she died from blood poisoning, hardship characterized the early life of the Jones family. The family, now bereft of both parents, was separated, and the six oldest children were made wards of the Ministry of Pensions, and dispersed among five families of relatives. The youngest, Geoffrey David, who had been born after the end of the war in 1918, was not supported by the Ministry of Pensions, and was sent to an orphanage. Ken Jones had a particularly warm affection for his youngest brother, and experienced enormous grief when Geoffrey, a bomber pilot during World War 11, was killed in action in June, 1944. Ken’s school days were happy ones, and although he lived with several aunts and uncles in Birmingham, they afforded him the security and warm affection so necessary to a growing boy. Hereminisced fiequently of the joyous summer days when he was able to cycle out to the home of a paternal uncle, Jack Jones, who, with his wife Lucy, lived in the country near Ross-on-Wye, Herefordshire. He spent his holidays with them, and these visits engendered in him a life-long interest in gardening and an abiding love of plants and flowers. Between 1917 and 1923, Ken attended the local Bordesley Green Council School. He received a scholarship to the Waverley Grammar 1

Copyright 63 1 W by Academic Press,Inc. All nghts of reproduchon in any form reserved. ISBN 0-124x72414

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W. A. SZAREK, M. STACEY, AND G . W. HAY

School, an institution noted for the excellence of its science and engineering students, many of whom progressed to Birmingham University. At both Bordesley Green and Waverley Schools, Ken immersed himself in reading and studying as a means of forgetting the harsh times of his childhood and enduring the loneliness occasioned by the prolonged separation from his brothers and sisters. Later, these two activities became a habit and, eventually, a pleasure for him. Ken inherited his mother’s athletic talents and developed into a fine athlete. In 1929, he was both the captain of the school’s association football team and an athletics champion. He was a typical ectomorph, above average in height, seemingly impervious to weight gain, and walking with a characteristic gait that made him stand out in any gathering of scientists. In 1930, Ken entered Birmingham University, having won a Polytechnic Bursary and a Kitchener Scholarship. He began his studies with great enthusiasm, finding particular enjoyment in the laboratory work. Ken had been advised to take a degree in metallurgy, and he studied this subject in his first year at Birmingham, together with chemistry and physics. He found physical chemistry difficult, and his whole interest turned to organic chemistry. During the vacations, he assisted Dr. W. J. Hickinbottom with his researches; this experience presumably persuaded him to become an organic chemist. Ken completed his studies towards the B.Sc. degree, with first-class honours, in 1933,and received the Frankland Medal for having attained the highest standing in his year. The financial constraints imposed by the dire economic conditions of the time thwarted his endeavor to secure financial assistance to work on platinum compounds with Dr. William W. Wardlaw. Happily for carbohydrate chemistry, Ken was offered a research scholarship to study for his Ph.D. with Professor W. N. Haworth and Dr. E. L. Hirst. Under the supervision of this illustrious team, Ken became engaged in studies related to L-ascorbic acid, and he was allocated to Maurice Stacey, at that time a Research Fellow. The Chemistry Department was aglow with excitement because the determination of structure and the synthesis of L-ascorbic acid had just been achieved, and Ken was assigned the topic of repeating on a large scale the synthesis of L-ascorbic acid, and elucidating the structures of some of the intermediates. When Maurice Stacey departed for London, in late 1933, Ken began a happy collaboration with the late Fred Smith, under Edmund Hirst’s general direction. The long hours expended in the laboratory by this team resulted in rapid progress towards the development of a process for the production of L-ascorbic acid on a commercial scale. In 1937, the rights to a patent (with W. N. Haworth, E. L. Hirst, and F. Smith) on the nitric acid oxidation of

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3

L-sorbose to L-ascorbic acid were sold for a return of $100 sterling to each co-author. The year 1937 was important to Ken Jones both professionally and personally, for, in June of that year, he married Marjorie Ingles Noon, Fred Smith being the best man. The couple first met as teenagers through a family association-Ken’s uncle, Tom, with whom he had lived for a time, had married Majorie’s aunt, Elsie. Later, Ken and Marjorie met at school dances and nurtured their relationship through enjoyable walks and tandem cycling together. Marjorie understood Ken’s commitment to laboratory work, and devotedly spent long evenings, Sundays, and holidays with him in the laboratory, eventually learning enough chemical language to proof-read his writings. Marjorie was an only child; her father was for many years a maintenance and electrical engineer at the offices of the Birmingham Post and Mail,Birmingham’s leading newspaper. In 1936, Ken received the Ph.D. degree. In that same year, when E. L. Hirst was appointed to the Chair of Organic Chemistry at Bristol University, Ken accepted Hirst’s invitation to go with him as an assistant lecturer and to be part of the nucleus of his carbohydrate-research group. Thus began a close and productive partnership that lasted until Hirst moved to Edinburgh in 1948. Jones readily acknowledged that Hirst was his inspiration to diligence in research, and Ken regarded him as a model and a father figure. Ken Jones’s first topic of research at Bristol was the elucidation of the structure of damson gum. Maurice Stacey had collected several pounds of the raw material in Shropshire and had donated it to Hirst. The unravelling of the structure of this complex material was a truly formidable task, but Ken was not daunted by this challenge. He rapidly established himself as a leader, building up, with such colleagues as G. T. Young, a powerful carbohydrate-research team, and they extended their interests to other plant gums, alginates, and unusual starches. Regrettably, the deepening gravity of the war demanded that the major research effort on carbohydrates be suspended in 1940, at which time the Bristol University Chemistry Department, under the headship of Professor W. E. Gamer, was asked to house Professor Bennett of King’s College, London, and his Chemistry Department, and also to find accommodation for sections of the Woolwich Arsenal staff. The organic chemists accepted an invitation to do “war work” with Professors Gamer and Cecil Bawn on problems concerning explosives, such as the use of low-grade toluene to make TNT, and to assist with work on the then-supersecret RDX (hexahydro-l,3,5-trinitro-l,3,5-triazine, a high explosive) and on plastic explosives.

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W. A. SZAREK, M. STACEY, AND G . W. HAY

During this period, Ken supervised the laboratory work at the University, thereby enabling Edmund Hirst to attend to Governmental activities. Under Ken Jones’s leadership, the team, over a period of six years, made a very significant contribution to the war effort, but, because of the nature of the work, received very little publicity. Despite the personal cost to himself, Ken adhered rigidly to the terms of the Official Secrets Act. In 1945, Ken, who was now Lecturer at Bristol University, was again invited by Hirst to move with him to Manchester University, this time as Senior Lecturer in Organic Chemistry. Once more, it became necessary for Hirst to concentrate his efforts on University and Government committee work. Ken Jones, therefore, took charge of the carbohydrate-research group, and supervised the completion of the explosives work. During this interval, Ken enjoyed the able collaboration of Dr. T. G. Halsall in studies on the structures of starch, cellulose, and glycogen, and on the oxidation of carbohydrates by periodate. The close association of Ken with Professor Hirst, which continued at Manchester University until 1948, was a tremendously fruitful one; over 50 joint publications resulted from their research on complex polysaccharides. In 1948, Edmund Hirst moved to Edinburgh University, and Ken returned to Bristol University as Reader in Organic Chemistry. At Bristol, Ken rapidly developed his own carbohydrate-research group and, with great foresight, impressed upon his colleagues the need to apply biochemical methods to the study of natural products, a point of view fully shared by his brilliant assistant (later Professor) Leslie Hough. The decision, in 1951, to accept an invitation to spend six months at the institute of Paper Chemistry, in Appleton, Wisconsin, changed irrevocably the course of Ken’s professional career. i n 1953, he moved to the Chown Research Chair of Chemistry at Queen’s University, Kingston, Ontario, Canada. Of this move, Ken wrote in his Royal Society Record “I stayed (at Appleton) from April to September. The people were very kind and helpful and the weather was hot and sunny. The scenery was good and I liked the large open area. When I saw the advert. for the Chown Research Chair in 1953 I put in for it. I have never regretted moving here. Facilities at first were poor but J. A. McRae, Dean Ettinger and the National Research Council gave me funds to buy apparatus and with the assistance of devoted graduate students we have never looked back.” The Chown Research Chair required the expenditure of a minimum amount of time for administration and teaching, and afforded Ken the maximum possible time for research. One early difficulty in Canada was the restriction of his re-

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5

search effort occasioned by the lack of available Canadian graduates, but this was soon overcome by Ken’s ability to attract research students and postdoctoral researchers from overseas, particularly from Bristol. His research group grew steadily. In the course of time, he did succeed in attracting Canadian graduates into his research group, amongst them being Walter Szarek. Like E. L. Hirst, Professor Jones throughout his career attracted the close collaboration of a number of University colleagues. Thus, at Bristol University, Dr. (later Professor) L. Hough collaborated with him, and in Canada, at Queen’s University, he found senior collaborators in the persons of Dr. M. B. Perry until 1962, and Dr. (later Professor) W. A. Szarek from 1967 to 1977. Despite Professor Jones’s abiding passion for research in carbohydrate chemistry, his life was much broader than his science alone. It encompassed as well an appreciation of the beauties of Nature in general, and a devotion to family life. Three children, Stephanie Netherton, Stephen Howard, and Jonathan Ingles Netherton were born into their family. These children, from 1953 onwards, rapidly became “real” Canadians, enjoying life to the full. The children chose to follow diverse educational paths. Stephanie graduated as a nurse, Stephen as an engineer from Queen’s University, Kingston, and Jonathan as a biologist from Brock University, St. Catharines, Ontario. The Jones family had a charming home on Treasure Island on the St. Lawrence River near Kingston. From their grounds, they could enjoy swimming, boating, fishing, and partaking of the beautiful scenery of the St. Lawrence River. Ken took much pride and joy in cultivating and displaying his flowers and garden. Together with his wife Marjorie, he had an active interest in the cultural affairs of Kingston, such as the promotion of live theatre and the Symphony Orchestra. He was an experienced and extremely eager traveller. Indeed, his travels took him to countries on five continents. He was on sabbatical leave in Brazil for the period September 1967 to March 1968,in South Africa from March 1968 to June 1968, and again in Brazil from January 1976 to June 1976. Ken’s hobbies, which he could share with the family, were simple -music, photography, foreign stamps, chess, and the collecting of plants. He took a general interest in military affairs. He was quite proud of the active role he played in Bristol, where he was a part-time officer in the Royal Corps of Signals attached to the University Training Corps. At the end of the war, he resigned with the rank of Captain. As Chown Research Professor at Queen’s University, Professor Jones, in addition to the supervision of the research of a large number of graduate students, postdoctoral fellows, and fourth-year undergraduates, taught courses in Natural Products Chemistry and Carbohy-

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W. A. SZAREK, M. STACEY, AND G . W. HAY

drate Chemistry. These were graduate-level courses, but were open to fourth-year undergraduates. Occasionally, Ken gave a series of lectures on Carbohydrate Chemistry to students at the Royal Military College in Kingston. Although he was loath to accept administrative responsibilities beyond those inherent in his research operation, Ken, nevertheless, did fulfil such tasks on occasion, at both the Departmental and Faculty level, but administration was not his forte. During his time at Queen’s University, he served briefly as a Member of the Department of Chemistry Graduate Committee, and one term as Chairman of that Division of the School of Graduate Studies and Research which encompassed the physical sciences (Division IV).The Chairmen of the various Divisions are Members of the Council of the School of Graduate Studies. As well, Ken was Secretary of the Committee on Scientific Research, a committee that considered applications from faculty members for financial support of research. He contributed to the design of some of the laboratory renovations (completed in 1964) in Gordon Hall. Professor Jones’s participation in professional societies and affairs outside the University were as follows: Rapporteur for the Royal Society of Canada (Chemical Section) in 1971, and Convenor in 1972; Member of the Advisory Committee to the Atlantic Regional Laboratories of the National Research Council, Halifax, Nova Scotia; Member of the Board of Governors of the Ontario Research Foundation; Member of the Board of Advisors for the British Commonwealth for Advances in Carbohydrate Chemistry and Biochemistry; Member of the Editorial Advisory Board of Carbohydrate Research; Chairman of the Fourth International Conference on Carbohydrate Chemistry, which was held in Kingston in 1967; and a Corresponding Member of the Nomenclature Committee of the Division of Carbohydrate Chemistry, American Chemical Society. Professor Jones was a member of The Chemical Society, the Biochemical Society, the Royal Institute of Chemistry (Associate),the Chemical Institute of Canada, the American Chemical Society, and the New York Academy of Sciences. Professor Jones’s outstanding achievements in carbohydrate chemistry were recognized by his receipt of numerous awards and honors. In 1957, he was elected a Fellow of the Royal Society of London, and, in 1959, a Fellow of the Royal Society of Canada and a Fellow of the Chemical Institute of Canada. The Division of Carbohydrate Chemistry of the American Chemical Society presented him with the Claude S. Hudson Award in 1969. He was the 1975 recipient of the Anselme Payen Award from the Cellulose, Paper, and Textile Division. In March 1975, he was awarded the third Sir Norman Haworth Memorial Medal of The Chemical Society (London).

OBITUARY- JOHN KENYON NETHERTON JONES

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An International Symposium entitled “Perspectives in Carbohydrate Chemistry” was organized in Kingston to honor Professor Jones on the occasion of his 65th birthday and his retirement from the Chair as the Chown Research Professor at Queen’s University. Alas, a few weeks before the Symposium was to commence, he did not survive a second major operation for cancer of the stomach, and the Symposium was held, in May, 1977, as a memorial to him. Over 200 participants attended the Symposium, which was a fitting expression of appreciation of the life and work of a fine scientist and true gentleman. As has already been intimated, J. K.’s earliest publications (with W. N. Haworth and E. L. Hirst) were concerned with L-ascorbic acid (vitamin C) and its analogs. Four papers and one patent resulted from the L-ascorbic acid work at Birmingham. When E. L. Hirst was appointed to the Chair of Organic Chemistry at Bristol University in 1936, he took J. K. with him from Birmingham. At this time, Fred Smith’s work was mainly concerned with the structures of gum arabic and gum tragacanth, topics suggested by Edmund Hirst, and J. K. and Fred agreed to maintain a general collaboration in order to avoid overlap. The early Bristol work indeed owes much to the generous provision by Fred Smith of reference samples of partially methylated sugars. J. K. undertook the major task of elucidating the structures of damson gum and various pectic substances. The next four years was a period of tremendous activity for him, necessitating spending even longer hours than previously in the laboratory, as few research students were available at Bristol. The techniques used for structural determination of the highly complex, acidic carbohydrate polymers were initially those developed by the Birmingham school for the determination of polysaccharide structures : exhaustive methylation of the carbohydrate polymer, or its acid-degraded derivative, followed by partial and complete hydrolysis and then quantitative separation and identification of the constituent methylated mono-, di-, and oligo-saccharides. J. K. realized that new techniques were urgently needed to lessen the enormous amount of labor required for these difficult investigations. J. K. developed new methods of methylation using thallium compounds and methyl iodide, and new oxidative methods for the determination of end groups in saccharide chains. He was quick to perceive the potential of various chromatographictechniques for the separation of complex mixtures of simple saccharides and their methyl ethers. The results were published in a series of papers concerned with the structures of such diverse substances as damson gum, peanut arabinan, pectic acids, cherry gum, slippery-eIm mucilage, and citrus arabinan. The move to Manchester in 1945 made a welcome change, although J. K. had once more to build up a new research team. The cessation of hostilities had

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W. A. SZAREK,

M. STACEY, A N D G. W. HAY

cleared the way for the resumption of his primary interest, namely, polysaccharide chemistry. Salient among the excellent work performed during this period was the elucidation of further major structural features of the complex macromolecules of damson gum, cherry gum, and peach gum. Damson gum is an exudate gathered in the form of resin-like nodules from the bark of the tree Prunus insitia. It is normally isolated in the form of an ash-free, water-soluble, acidic white powder. Hydrolysis by mineral acid afforded D-xylose, L-arabinose, Dgalactose, D-mannose, and D-glucuronic acid. Damson gum differed from gum arabic in its content of D-mannose and D-xylose, and by its lack of L-rhamnose. As in the case of gum arabic, L-arabinose was liberated by autohydrolysis, and was present in the furanoid form. No fewer than 18 methylated saccharide derivatives were isolated from the hydrolyzed, methylated gum and the methylated, degraded gum. The quantitative separation of these constituents and an examination of their modes of union permitted an assignment of structure to a large part of the highly branched molecule. Similar studies were made on cherry gum, to which periodate-oxidation techniques were applied successfully. In the many gum exudates and mucilages studied, striking similarities, and yet often wide differences, in saccharide constituents and their modes of linkage were disclosed. The work was greatly expedited by J. K.’s development of automated fraction-collectors. Column chromatography and paper partition-chromatography were developed into a fine art at Bristol, owing to J. K.’s skill and that of such of his students as A. E. Flood, F. Brown, W. H. Wadman, and L. Hough. Ken’s geographical transition to the New World was accompanied by a concomitant transition in his research emphasis. Although he maintained an interest in polysaccharide chemistry, the publication record from Queen’s University attests to the universality of his interests in carbohydrate chemistry. J. K. made major contributions to synthetic carbohydrate chemistry, stereochemistry , biosynthetic mechanisms, and metabolism of carbohydrates, and the application of such separational techniques as paper and gas-liquid chromatography in the carbohydrate field. The results of his lifetime of research were documented in over 300 scientific publications. Clearly, it would be impractical to review this number of papers individually, and consequently, only a representative sample will be treated. A list of Professor Jones’s publications is appended to this article. During the 1950’s and early 196O’s, J. K. and his coworkers achieved new syntheses of a number of simple sugars. These included D-tag& tose and mpsicose, 5,&dideoxy-~-xylo-and -h-arubino-hexose, derivatives of Dribitol, 5-S-ethyl-5thio-~threo-2-pentulose, wgZycer0-D-

OBITUARY-JOHN

KENYON NETHERTON JONES

9

rnanno-heptose, l-deoxy-~-arabino-3-hexulose, L-arabinoseS-lC, D apiose, 3-acetamido-3-deoxy-~glucose,L-mycarose, L-cladinose, D glycero-Daltro-, L-glycero-L-galacto-, Dglycero-L-gluco-, and Bglycero-L-galacto-octulose,and 3-hexuloses. The classic problem of disaccharide synthesis also attracted J. K.’s attention. Syntheses of 3O-~-D-galactopyranosy~-D-ga~actose, 3-~-~-D-xylopyranosy~-D-xy~ose,

lactose, 2-0- and 5-O-P-D-glUCOpyranOSyl-D-XylOSe,and 4-O-P-D-galactopyranosyl-Dgalactose were included in his achievements. Two accomplishments of particular significance were the synthesis of sugars in which the ring-oxygen atom had been replaced by nitrogen, and the investigation of the reaction of sulfuryl chloride with sugars and their derivatives. The former development was a consequence of J. K.’s study of the microbiological oxidation of sugar derivatives by Acetobacter suboxyans. A series of papers on the oxidation of terminally substituted, polyhydric alcohols was published. In connection with studies of the oxidation of acetamidodeoxyalditols,5-acetamido-5-deoxy-~-arabinose was prepared and, interestingly, was found to exist in two forms, namely, the normal furanoid form, and a pyranoid form in which the ring heteroatom was nitrogen, not oxygen. A number of examples of this new type of compound were synthesized, including 5-acetamido-5-deoxy-~-xylopyranose, methyl 4-acetamido-4-deoxy-~-erythrofuranoside, and methyl 4-acetamido-4deoxy-D and -L-arabinofuranoside. Concomitant with these developments at Queen’s University, researchers in other countries, particularly the United States and West Germany, were synthesizing a variety of analogous compounds in which the oxygen atom of the ring was replaced by nitrogen, phosphorus, selenium, or sulfur. J. K. and his colleagues extensively studied the reaction of sulfuryl chloride with carbohydrates. This work elucidated the stereochemical principles involved in the various transformations, and made available a convenient and effective procedure for the preparation of chlorodeoxy sugars, derivatives that have been found to be extremely valuable intermediates in the synthesis of a wide variety of rare sugars. The synthesis of chlorodeoxy sugars involves the initial formation of chlorosulfuric esters, followed by bimolecular displacement of certain of the chlorosulfonyloxy groups by chloride ion liberated during the chlorosulfation. It is often possible to predict the reactivity of a chlorosulfonyloxy group by a consideration of the steric and polar factors affecting the formation of the transition state. Thus, it has been found that the presence of a vicinal, axial substituent, or of a P-transaxial substituent, on a pyranoid ring inhibits replacement of a chlorosulfonyloxy group; also, a chlorosulfate group at C-2 has been observed to be deactivated to nucleophilic substitution by chloride ion.

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W. A. SZAREK, M. STACEY, AND G . W. HAY

Some of the rare sugars that have been prepared by way of chlorodeoxy derivatives are 4,6-dideoxy-3- 0-methyl-~-xyZo-hexose(D-chalcose), 3,6-dideoxy-D-ribo-hexose (paratose), 3,6-dideoxy-~urabinohexose (tyvelose), methyl 2,3-dideoxy-~-~-glycero-hex-2-enopyranosid-4-ulose, and certain aminodeoxy sugars. Although the bulk of J. K.’s work at Queen’s University concerned reactions of monosaccharides, he nevertheless did not lose interest in polysaccharides and the chemistry of L-ascorbic acid. The polysaccharides examined during the Canadian period included the hemicelluloses of loblolly pine (Pinus taeda) and aspen (PopuEus trernuloides), linseed mucilage from flax (Linurn usztatissimum), the type VIII Pneurnococcus specific polysaccharide, the hemicelluloses and a-cellulose from a specimen of ancient wood from Cedrus penhallowii, an arabinogalactan from Monterey pine (Pinus radiata ), the “gum asafoetida” polysaccharide, a water-soluble arabinogalactan from mountain larch (Larix lylatli, Parl), the glucomannan of bluebell seed (ScyZla n.onsc7ipta L.), polysaccharides from the seeds of the huacra pona palm (friarteauentricosa), cholla gum (Opuntia fulgida),the capsular polysaccharide of Pneumococcus XII, arabinobioses from Acacia nilotica gum, the type-specific polysaccharide from type XIX Pneumococcus, the mucilage from the bark of Ulmus fulua (slipperyelm mucilage), lemon gum (CitrusZiminia), lipopolysaccharides of Proteus, and the galactan from the albumin glands of the snail (Strophocheilus oblongus). In the course of the preparation of C - and 0benzyl derivatives of L-ascorbic acid, it was observed that the L-ascorbate ion acts as an ambident nucleophile; this was the first example of a carbohydrate structure exhibiting this property. The universality of J. K.’s interests was further manifested by his continuing studies of biosynthetic mechanisms and the metabolism of carbohydrates, and the application of such separational techniques as gas-liquid chromatography in the carbohydrate field. In 1961, the mode of the linkage of sugars to amino acids in glycoproteins and “mucoproteins” was obscure. J. K. and his colleagues attempted to determine the nature of the carbohydrate -peptide bonds occurring in natural glycoproteins. In 1967, Walter Szarek returned to Queen’s University as a faculty member, after a three-year absence in the United States, and was asked to direct J. K.’s entire research group while J. K. was on sabbatical leave in Brazil and, subsequently, South Africa. This marked the beginning of the very close association and collaboration between J. K. and Walter Szarek, a partnership that continued until J. K.’s death. The fruit of this close relationship was recorded in the numerous publications that ensued. These were primarily in the area of synthetic carbohydrate chemistry, and involved such topics as synthesis and

OBITUARY- JOHN KENYON NETHERTON JONES

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chemical modification of carbohydrate antibiotics, design of biologically active nucleosides, development of new routes to those sugars and their derivatives that are of interest to biochemistry and chemotherapy, conformationaland mechanistic studies of carbohydrate reactions, microbiological oxidation of sugars and their derivatives, chemical modification of polysaccharides, photochemistry of carbohydrates, stereochemistry, and heterocyclic conformational analysis. In 1976, the long tradition of excellence in carbohydrate chemistry at Queen’s University led to the formation of the Carbohydrate Research Institute. J. K. was a very strong supporter of such an interface between the University, Government, and Industry and was, with Walter Szarek and G. W. Hay, a Founding Member. Regrettably, his deteriorating health at this period obviated his active involvement in the formation and subsequent development of the Institute, which came into being through the efforts, and under the directorship, of Walter Szarek. Professor Jones died on April 13, 1977, after a 10-month struggle with cancer. His family and friends, his carbohydrate chemistry, and his love and appreciation of the beauties of Nature were, for him, his life. His genuine humility precluded any inclination towards extensive eulogizing or ceremony. He was indeed “one among a thousand” (Job, 33: 23). The character of the man was accurately portrayed by the University Chaplain during the Memorial Service at Queen’s University in the words ‘‘. . . our friend, highly regarded and greatly beloved among us . . . cared for all living and growing things and cherished the beauty and wonder of woods and fields, rocks and water flowing by. His loyalty and thoughtfulness toward others taught us that a faithful friend is the medicine of life.” WALTERA. SZAREK MAURICE STACEY GEORGEW. HAY

APPENDIX

The following is a chronological list of the scientific publications of Professor J. K. N. Jones and his colleagues. “Synthesis of Ascorbic Acid and its Analogues: The Addition of Hydrogen Cyanide to Osones,” W. N. Haworth, E. L. Hirst, J. K. N. Jones, and F. SmithJ. Chem. SOC. (1934) 1192- 1197. “Ascorbic Acid and its Analogs,” W. N. Haworth, E. L. Hirst, J. K. N. Jones, and F. Smith, Br. Pat. 443,901 (1936).

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W. A. SZAREK, M. STACEY, AND G. W. HAY

“Gluco-ascorbic Acid,” W. N. Haworth, E. L. Hirst, and J, K. N. Jones,]. Chem. Soc., (1937)549-556. “Pectic Substances. Part I. The Araban and Pectic Acid of the Peanut,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., (1938)496-505. “Analogues of Ascorbic Acid Containing Six-membered Rings,” W. N. Haworth, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1938) 710-715. “The Constitution of Damson Cum. Part I. Composition of Damson Gum and Structure of an Aldobionic Acid (Clycuronosido-2-mannose)Derived from It,” E. L. Hirst and J. K. N. Jones,-/. Chem. Soc., (1938) 1174-1180. “Methylation of a-Methylglucoside by Thallous Hydroxide and Methyl Iodide,” C. C. Barker, E. L. Hirst, and J. K. N. Jones,J. C h m . Soc., (1938) 1695-1698. “Methyl Ethers of Arab-ascorbic Acid and Their Isomerism,” E. G. E. Hawkins, E. L. Hirst, and J. K. N. Jones,J. Chem. Soc., (1939) 246-248. “Pectic Substances. Part 11. Isolation of an Araban from the Carbohydrate Constituents of the Peanut,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., (1939) 452-453. “Pectic Substances. Part 111. Composition of Apple Pectin and the Molecular Structure of the Araban Component of Apple Pectin,” E. L. Hirst and J. K. N. Jones,J. Chem. SOC.,(1939)454-460. “The Constitution of Cherry Gum. Part I. Composition,” J. K. N. Jones,]. Chem. Soc., (1939) 558-563. “Constitution ofthe Mucilage from the Bark of Ulmusfuloa (Slippery Elm Mucilage). Part I. The Aldobionic Acid Obtained by Hydrolysis of the Mucilage,” R. E. Gill, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., (1939) 1469-1471. “The Constitution of Damson Cum. Part 11. Hydrolysis Products from Methylated Degraded (Arabinose-free) Damson Grim,” E. L. Hirst and J. K. N. JonesJ. Chem. Soc., (1939) 1482- 1490. “Pectic Substances. Part IV. Citrus Araban,” G. H. Beaven, E. L. Hirst, and J. K. N. Jones,]. Chem. S o c . , (1939) 1865-1868. “2:3:4-Trimethyl Mannose,” W. N. Haworth, E. L. Hirst, F. A. Isherwood, and J. K. N. Jones,]. Chem. Soc., (1939) 1878-1880. “The Structure of Alginic Acid. Part I,” E. L. Hirst, J. K. N. Jones, and (Miss) W. 0. Jones,]. Chem. Soc., (1939) 1880-1885. “Structure of Alginic Acid,” E. L. Hirst, J. K. N. Jones, and W. 0.Jones, Nnture, 143 (1939) 857. “Molecular Structure of Pectic Acid,” C. H. Beaven and J. K. N. Jones, Chem. Znd. (London),(1939) 363. “The Constitution of Banana Starch,” E. G. E. Hawkins, J. K. N. Jones, and G. T. Young, J . Chem. Soc., (1940)390-394. “The €-Galactan of Larch Wood,” E . L. Hirst, J. K. N. Jones, and W. G. Campbell, Nature, 147 (1941)25-26. “Separation of Methylated Methylglycosides by Adsorption on Alumina. A New Method for End-group Determinations in Methylated Polysaccharides,” J. K. N. Jones, 1. Chein. Soc., (1944) 333-334. “The Condensation of Glucose and p-Diketones,” J. K. N. Jones,]. Chem. Soc., (1945) 116- 119. “The Quantitative Estimation of Xyiose,” L. J. Breddy and J. K. N. Jones,]. Chem. SOC., (1945)738-739. “Nitrogenous Substances Synthesized by Moulds,” A. H. Campbell, M. E. Foss, E. L. Hirst, and J. K. N. Jones, Nature, 155 (1945) 141. “Application of New Methods of End-group Determination to Structural Problems in the Polysaccharides,” F. Brown, S. Dunstan, T. C. Halsall, E. L. Hirst, and J. K. N. Jones, Nature, 156 (1945) 785-786.

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“The Constitution of Damson Gum. Part 111. Hydrolysis Products from Methylated Damson Gum,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1946) 506-512. “Methylation of B-Methylglucopyranoside and ap-Methylxylopyranosidesby Thallous Hydroxide and Methyl Iodide,” C. C. Barker, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1946) 783-784. “Constitution of the Mucilage from the Bark of Ulmusfulua (Slippery Elm Mucilage). Part 11. The Sugars Formed in the Hydrolysis of the Methylated Mucilage,” R. E. Gill, E. L. Hirst, and J. K. N. Jones, J. Chem. SOC., (1946) 1025-1029. “The Chemistry of Pectic Materials,” E. L. Hirst and J. K. N. Jones,Adu. Carbohydr. Chem., 2 (1946) 235-251. “Structure of Starch and Cellulose,” T. G. Halsall, E. L. Hirst, and J. K. N. Jones, Nature, 159 (1947) 97. “Quantitative Estimation of Mixtures of Sugars by the Paper Chromatogram Method,” A. E. Flood, E. L. Hirst, and J. K. N. Jones, Nature, 160 (1947) 86-87. “The Chemistry of Some Plant Gums and Mucilages,” E. L. Hirst and J. K. N. Jones, J . SOC. Dyers Colour., 63 (1947) 249-254. “The Quantitative Determination of Galactose, Mannose, Arabinose, and Rhamnose,” E. L. Hirst, J. K. N. Jones, and E. A. Woods,]. Chem. SOC., (1947) 1048-1051. “The Constitution of Cherry Gum. Part 11. The Products of Hydrolysis of Methylated Cherry Gum,” J. K. N. Jones, J. Chem. SOC., (1947) 1055-1059. “The Synthesis of 3-Methyl and 3:5-Dimethyl L-Arabinose,” E. L. Hirst, J. K. N. Jones, and (Miss) E. Williams,J. Chem. SOC., (1947) 1062-1064. “The Constitution of Egg-plum Gum. Part I,” E. L. Hint and J. K. N. Jones,]. Chem. SOC., (1947) 1064-1068.

“Pectic Substances. Part V. The Molecular Structure of Strawberry and Apple Pectic Acids,” G. H. Beaven and J. K. N. Jones,]. Chem. SOC., (1947) 1218-1221. “Pectic Substances. Part VI. The Structure of the Araban from Arachis hypogea,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1947) 1221-1225. “Pectic Substances. Part VII. The Constitution of the Galactan from Lupinus albus,” E. L. Hirst, J. K. N. Jones, and (MIS.) W. 0. Walder,]. Chem. SOC., (1947) 1225-1229. “Some Derivatives of DGalacturonic Acid,” J. K. N. Jones and M. Stacey,]. Chem.

SOC., (1947) 1340-1341. “Synthesis of Some Derivatives of D and L-Arabinose,” J. K. N. Jones, P. W. Kent, and M. Stacey,]. Chem. Soc., (1947) 1341-1344. “The Quantitative Separation of Methylated Sugars,” F. Brown and J. K. N. Jones,]. Chem. SOC., (1947) 1344-1347. “The Structure of Glycogen. Ratio of Non-terminal to Terminal Glucose Residues,” T. G. Halsall, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1947) 1399-1400. “Oxidation of Carbohydrates by the Periodate Ion,” T. G. Halsall, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1947) 1427-1432. “The Galactomannan of the Lucerne Seed,” E. L. Hirst, J. K. N. Jones, and (MIS.)W. 0. Walder, J . Chem. SOC., (1947) 1443-1446. “The Structure of Starch. The Ratio of Non-terminal to Terminal Groups,” F. Brown, T. G. Halsall, E. L. Hirst, and J. K. N. Jones,J. Chem. Soc., (1948) 27-32. “The Structure of Egg-plum Gum. Part 11. The Hydrolysis Products Obtained from the Methylated Degraded Gum,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1948) 120- 128.

“The +Galactan of Larch Wood (Larix decidua),” W. G. Campbell, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., (1948) 774-777. “The Galactomannan of Carob-seed Gum (Gum Gatto),” E. L. Hirst and J. K. N. Jones, J . Chem. SOC., (1948) 1278-1282. “The Structure of Almond-tree Gum. Part I. The Constitution of the Aldobionic Acid

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W. A. SZAREK, M. STACEY, AND G. W. HAY

Derived from the Gum,” F. Brown, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1948) 1677-1679. “Quantitative Analysis ofMixtures of Sugars by the Method of Partition Chromatography. Part I. Standardisation of Procedure,” A. E. Flood, E. L. Hirst, and J. K. N.Jones,]. Chem. SOC., (1948)1679-1683. “Structure of Acorn Starch,” E. L. Hirst, J. K. N. Jones, and A. J. Roudier,]. Chem. SOC., (1948)1779-1783. “Pectic Substances. Part VIII. The Araban Component of Sugar-beet Pectin,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1948)2311-2313. “Separation and Identification of Methylated Sugars on the Paper Chromatogram,” F. Brown, E. L. Hirst, L. Hough, J. K. N. Jones, and H. Wadman, Nature, 161 (1948)720. “Application of Paper Partition Chromatography to the Separation of Sugars and their Methylated Derivatives on a Column of Powdered Cellulose,” L. Hough, J. K. N. Jones, and W. H. Wadman, Nature, 162 (1948)448. “The Amylose Content of the Starch Present in the Growing Potato Tuber,” T. G. Halsall, E. L. Hirst, J. K. N. Jones, and F. W. Sansome, Biochem.]., 43 (1948)70-72. “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part 11. The Separation and Determination of Methylated Aldoses,” E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chern. SOC., (1949)928-933. “The Polysaccharides ofthe Florideae. Floridean Starch,” V. C. Barry, T. G. Halsall, E. L. Hirst, and J. K. N. Jones,J. Chem. SOC.,(1949)1468-1470. “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part 111. Determination of the Sugars by Oxidation with Sodium Periodate,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1949)1659-1662. “The Constitution of Egg-plum Gum. Part 111. The Hydrolysis Products Obtained from the Methylated Gum,” F. Brown, E. L. Hirst, and J. K. N. Jones,]. Chen. SOC., (1949)1757-1761. “Cholla Gum,” F. Brown, E. L. Hirst, and J. K. N. Jones,], Chem. SOC., (1949)17611766. “Reactions of Nitroparaffins. Part XI. The Reaction of 2-Nitropropane with Formaldehyde and Ammonin,” J. K. N. Jones and T. Urbariski,J. Chem. SOC., (1949)17661767. “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part IV. The Separation of the Sugars and Their Methylated Derivatives on Columns of Powdered Cellulose,” L. Hough, J. K. N. Jones, and W. H. Wadman,J. Chem. SOC., (1949)2511-2516. “Cherry Gum. Part 111. An Examination of the Products of Hydrolysis of Methylated Degraded Cherry Gum, Using the Method of Paper Partition Chromatography,” J. K. N. Jones,J. Chem. Soc., (1949)3141-3145. “The Action ofp-Amylase on Amylopectin and on Glycogen,” T. G. Halsall, E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chern. SOC., (1949)3200-3207. “Pear Cell-wall Cellulose,” E. L. Hirst, F. A. Isherwood, M. A. Jermyn, and J. K. N. Jones,J. Chem. SOC., (1949)s182-sl84. “Composition of the Gum of Sterculia setigera: Occurrence of D-Tagatose in Nature,” E. L. Hirst, L. Hough, and J. K. N. Jones, Nature, 163 (1949)177. “Chromatographic Analysis. The Application of Partition Chromatography to the Separation of the Sugars and their Derivatives,” E. L. Hirst and J. K. N. Jones, Discuss. Faraday SOC., 7 (1949)268-274. “Plant Gums and Mucilages,” J. K. N. Jones and F. Smith,Ado. Carbohydr. Chem., 4 (1949)243-291. “The Structure of Peach Gum. Part I. The Sugars Produced on Hydrolysis of the Gum,” J. K. N. Jones,J. Chem. SOC., (1950)534-537.

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“On the Structure of Knudsen’s Base and of Related Compounds. Part I,” M. E. Foss, E. L. Hirst, J. K. N. Jones, H. D. Springall, A. T. Thomas, and T. Urbairski,]. Chem. SOC.,(1950) 624-628. “The Synthesis of 2:bDimethyl L-Rhamnose; The Action of Sodium Metaperiodate on 2:3- and 3:4-Dimethyl L-Rhamnoses,” F. Brown, L. Hough, and J. K. N. Jones, J. Chem. Soc., (1950) 1125-1127. “The Constitution of Xylan from Esparto Grass (Stipa tenacissima, L.),” S. K. Chanda, E.L. Hirst, J. K. N.Jones, and E. G. V. Percivd,]. Chem. SOC.,(1950) 12891297. “On the Structure of Knudsen’s Base and of Related Compounds. Part 11,”M. E. Foss, E. L. Hirst, J. K. N. Jones, H. D. Springall, A. T. Thomas, and T. Urbairski,]. Chem. SOC.,(1950) 1691-1695. “Grapefruit and Lemon Gums. Part I. The Ratio of Sugars Present in the Gums and Isolated by the Structure of the AIdobionic Acid (4-~-Glucuronosido-~-galactose) Graded Hydrolysis of the Polysaccharides,” J. J. Connell, (Miss) R. M. Hainsworth, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., (1950) 1696-1700. “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part V. Improved Methods for the Separation and Detection of the Sugars and their Methylated Derivatives on the Paper Chromatogram,”L. Hough, J. K. N. Jones, and W. H. Wadman,]. Chem. Soc., (1950) 1702-1706. “Frog-spawn Mucin,” B. F.Folkes, R. A. Grant, and J. K. N. Jones,]. Chem. Soc., (1950) 2136-2140. “The Structure of the Mannan Present in Porphyra umbiliculis,” J. K. N. Jones, J . Chem. SOC.,(1950) 3292-3295. “Composition of Linseed Mucilage,” D. G. Easterby and J. K. N. Jones, Nature, 165 (1950) 614. “Constitution of the Mucilage from the Bark of Ulmu~fuloa (Slippery Elm Mucilage). Part 111. The Isolation of 3-Monomethyl &Galadose from the Products of Hydrolysis,” E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chem.SOC.,(1951) 323-325. “The Synthesis of Sugars from Simpler Substances. Part I. The in oitro Synthesis of the Pentoses,” L. Hough and J. K. N. Jones,]. Chem. Soc., (1951) 1122-1126. “The Synthesis of Sugars from Simpler Substances. Part 11. The Synthesis of DL-Ribose in oitro from D-Glyceraldehyde and Glycollic Aldehyde,” L. Hough and J. K. N. Jones,]. Chem. SOC.,(1951)3191-3192. “Toluene+-sulphonylhydrazones of the Pentose Sugars, with Particular Reference to the Characterisationand Determination of Ribose,” D. G. Easterby, L. Hough, and J. K. N. Jones,]. Chem. SOC.,(1951) 3416-3418. “The Colorimetric Determination of Methylated Sugars: An Improved Micromethod of End-group Assay,” J. K. Bartlett, L. Hough, and J. K. N.Jones, Chem. Ind. (London), (1951) 76. “The Chemical Composition and Properties of Pectins,” J. K. N. Jones, Chem. Ind. (London),(1951) 430-431. “The Origin of the Sugars,” L. Hough and J. K. N. Jones, Nature, 167 (1951) 180. “Some Observations on the Constitution of Gum Myrrh,” L. Hough, J. K. N. Jones, and W. H. Wadman,]. Chem. SOC.,(1952) 796-800. “Methylene Derivatives of &Galactose and &Glucose,” L. Hough, J. K. N. Jones, and M. S. Magson,]. Chem. SOC.,(1952) 1525-1527. “Mannose-containing Polysaccharides. Part 11. The Gdactomannan of Fenugreek Seed (Trigonellafoenurngraecum),” P. Andrews, L. Hough, and J. K. N. Jones,]. Chem. SOC.,(1952) 2744-2750. “The Hemicelluloses Present in Aspen Wood (Populus tremulotdes).Part I,” J. K. N. Jones and L. E. Wise,J. Chem. Soc., (1952) 2750-2756.

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“The Hemicelluloses Present in Aspen Wood (Populus tremuloides). Part 11,” J. K. N. Jones and L. E. Wise,]. Chem. Soc., (1952)3389-3393. “An Investigation of the Polysaccharide Components of Certain Fresh-water Algae,” L. Hough, J. K. N. Jones, and W. H. Wadman,]. Chem. Soc., (1952)3393-3399. “The Reaction of Amino-compounds with Sugars. Part I. The Action of Ammonia on ~ G l u c o s e , ”L. Hough, J. K. N. Jones, and E. L. Richards,]. Chem. Soc., (1952) 38543857. “The Synthesis of Sugars from Simpler Substances. Part 111. Enzymic Synthesis of a Pentose,” L. Hough and J. K. N. Jones,]. Chem. SOC., (1952)4047-4052. “The Synthesis of Sugars from Simpler Substances. Part IV. Enzymic Synthesis of 6-Deoxy-D-fructose and 6-Deoxy-L-sorbose,” L. Hough and J. K. N. Jones, ]. Chem. SOC., (1952)4052-4055. “A Synthesis of 3:4-Dimethyl D-Xylose and 4-Methyl D-Xylose,” L. Hough and J: K. N. Jones,j. Chern. Soc., (1952)4349-4351. “Methylation of Carbohydrate Using Diazomethane,” L. Hough and J. K. N. Jones, Chem. Znd. (London), (1952) 380. “The Enzymatic Synthesis of Methylpentose,” L. Hough and J. K. N. Jones, Chem. Ind. (London), (1952)715. “The Enzymic Synthesis of Heptose Sugars,” L. Hough and J. K. N. Jones, Chem. lnd. (London),(1952)907. “Arabopyranose Residues in Larch E-Galactan,” J. K. N. Jones, Chem. lnd. (London), (1952)954. “Mannose-containing Polysaccharides. Part I. The Galactomannans of Lucerne and Clover Seeds,” L. Hough and J. K. N. Jones,]. Am. Chem. Soc., 74 (1952) 40294032. “Identification of L-Rhamnose in Aspen Wood,” J. K. N. Jones and J. R. Schoettler, Tappi, 35 (1952) 1mA. “Pentahydric Alcohols and their Oxidation Products,’’ J. K. N. Jones, in E. H. Rodd (Ed.), Chemistry of Carbon Compounds, Val. IB, Chap. XIX, Elsevier, Amsterdam, 1952, pp. 1197-1223. “Hexa- and Poly-hydric Alcohols and their Oxidation Products. Carbohydrates and Related Compounds,” J. K. N. Jones, in E. H. Rodd (Ed.), Chemistry of Carbon Compounds, Vol. IB, Chap. XX,Elsevier, Amsterdam, 1952, pp. 1224-1286. “The Synthesis of Sugars from Simpler Substances. Part V. Enzymic Sypthesis of Sedoheptulose,” L. Hough and J. K. N. Jones, j . C h . Soc., (1953) 342-345. “Mannose-containing Polysaccharides. Part 111. The Polysaccharides in the Seeds of In’s achroleuca and I. sibirca,” P. Andrews, L. Hough, and J. K. N. JonesJ. Chem. Soc., (1953) 1186-1192. “The Synthesis of Sugars from Simpler Substances. Part VI. Enzymic Synthesis of D-Idoheptulose,” P. A. J. Gorin and J. K. N. Jones,]. Chem. Soc., (1953) 1537-1538. “The Reaction of Amino-compounds with Sugars. Part 11. The Action of Ammonia on Glucose, Maltose, and Lactose,” L. Hough, J. K. N. Jones, and E. L. Richards,]. Chem. SOC., (1953)2005-2009. “The Synthesis of Sugars from Simpler Substances. Part VII. Enzymic Synthesis of 5-Deoxy-~-xylulose,”P. A. J. Gorin, L. Hough, and J. K. N. Jones,]. Chem. Soc., (1953) 2140-2142. “The Isolation of Oligosaccharides from Gums and Mucilages. Part I,” P. Andrews, D. H. Ball, and J. K. N. Jones,]. Chem.SOC., (1953) 4090-4095. “Structure of the ‘Triuronide’ from Pectic Acid,” J. K.N. Jones, Chem. Ind. (London), (1953)303. “The Galactan of Strychnos nux-uomica Seeds,” P. Andrews, L. Hough, and J. K. N. Jones, ]. Chem. Soc., (1954) 806-810.

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“The Structure of the Oligosaccharides Produced by the Enzymic Breakdown of Pectic Acid. Part I,” J. K. N. Jones and W. W. Reid,]. Chem. SOC., (1954)1361-1365. “The Isolation of Oligosaccharides from Gums and Mucilages. Part 11,” P. Andrews and J. K. N. Jones,]. Chem. SOC., (1954)1724-1726. “The Hemicelluloses of Scots Pine (Pinus syloestris) and Black Spruce (Picea nigru) Woods,” A. R. N. Gorrod and J. K. N. Jones,]. Chem. SOC., (1954)2522-2525. “The Synthesis of Sugars from Smaller Fragments. Part VIII. The Synthesis of D-Idoheptulosan from D-Xylose,” J. K. N. Jones,]. Chem. SOC., (1954)3643-3644. “The Isolation ofOligosaccharides from Gums and Mucilages. Part 111. Golden Apple Gum,” P. Andrews and J. K. N. Jones,]. Chem. Soc., (1954)4134-4138. “A Synthesis of 4-Deoxy-~-erythrohexulose,” P. A. J. Gorin, L. Hough, and J. K. N. Jones,]. Chem. SOC., (1954)4700-4701. “Some Observations on the Browning Reaction Between Glucose and Ammonia,” L. Hough, J. K. N. Jones, and E. L. Richards, Chem. Ind. (London),(1954)545-546. “Colorimetric Estimation of Sugars with Benzidine,” J. K.N. Jones and J. B. Pridham, Biochem. 1..58 (1954) 288-290. “Hemicellulose of Esparto (Stipa tenucissima L.). Part I,” J. K. N. Jones and G. Guzman, An. R. Soc. Esp. Fts. Qutm., Ser. B , 50 (1954)505-516. “An Improved Synthesis of D-Xylose 5-(Barium Phosphate),” P. A. J. Gorin, L. Hough, and J. K. N. JonesJ. Chem. Soc., (1955)582-583. “The Isolation of Oligosaccharides from Gums and Mucilages. Part IV. The Isolation from Lemon Cum,” P. Andrews and J. K. N. of 3-O-~-~-Arabopyranosyl-~-arabinose Jones,]. Chem. SOC., (1955)583-584. “The Synthesis of L-Glycerotetrulose and Related Compounds,” P. A. J. Gorin, L. Hough, and J. K. N. Jones,J. Chem. SOC., (1955)2699-2705. “The Constitution of Gum Myrrh. Part 11,” J. K. N. Jones and J. R. Nunn,J. Chem. Soc., (1955)3001-3004. “The Epimerization of Sugars,” J. K. N. Jones and W. H. Nicholson,]. Chem. Soc., (1955)3050-3053. “The Synthesis of Sugars from Simpler Substances. Part IX.The Enzymic Synthesis P. A. J. Gorin, L. Hough, and J. K. N. Jones,]. Chem. of 5:6-Dideoxy-~threohexulose,” SOC., (1955)3843-3845. “Methylene Derivatives of L-Rhamnose,” P. Andrews, L. Hough, and J. K. N. Jones, J. Am. Chem. Soc., 77 (1955)125-130. “The Structure of Frankincense Gum,” J. K. N. Jones and J. R. Nunn,]. Am. Chem. SOC., 77 (1955)5745-5764. “Chemistry ofthe Carbohydrates,” J. K. N. Jones,Annu. Reo. Biochem., 24 (1955)113 - 134. “A Synthesis of D-Tagatose from D-Galacturonic Acid,” P. A. J. Gorin, J. K. N. Jones, and W. W . Reid, Can.]. Chem., 33 (1955)1116-1118. “Preparation of L-Sorbose from 5-Keto-D-gluconic Acid (L-Sorburonic Acid),” J. K. N. Jones and W . W. Reid, Can. ]. Chem., 33 (1955)1682-1683. “The Analysis of Plant Gums and Mucilages,” J. K. N. Jones and E. L. Hirst, in K. Peach and M. V. Tracey (Eds.), Modern Methods of Plant Anulysis, Vol. 11, SpringerVerlag, Berlin, 1955,p. 275. “Properties of Dextrans Extracted from Plasma and Urine of Dogs,” R. E. Semple, B. J. Excell, and J. K. N. Jones, Fed. Proc., Fed. Am. Soc. Erp. Biol., 14 (1955)443. “The Structure ofthe Oligosaccharides Produced by the Enzymic Breakdown of P e e tic Acid. Part 11,” J. K. N. Jones and W. W. Reid,]. Chem. Soc., (1955)1890-1891. “The Separation ofan Essential Oil and of Methylated Sugars by Thermal Diffusion,” D. H. Ball, R. M. Butler, W. H. Cook, and J. K. N. Jones, Chem. Ind. (London),(1955) 1740-1741.

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“The Synthesis of Sugars from Smaller Fragments. Part X. Synthesis of LClucoheptulose,” J. K. N. Jones and R. B. Kelly, Can. I . Chem., 34 (1956)95-97. “A Synthesis of 5-0-Methyl =Glucose and of2-0-Methyl D-Glyceronamide,” J. K. N. Jones, Can.]. Chem., 34 (1956)310-312. “Fractionation of Polysaccharides,” A. J. Erskine and J. K. N. Jones, Can.].Chem., 34 (1956)821-826. “The Hemicellulose of the Fossilized Wood of Cedrus penhallawii,” J. K. N. Jones and E. Merler, Can. J. Chem., 34 (1956)840. “4-6-OisoPropylidene-rnethyl-a-~-glucoside,” J. K. N. Jones, Can. J . Chern., 34 (1956) 840-842. “The Action of Alkali Containing Metaborates on Wood Cellulose,” J. K. N. Jones, L. E. Wise, and J. P. Jappe, Tappi, 39 (1956)139-141. “The Synthesis of 3-Hexuloses. Part 1.2O-Methyl-~-rylo-3-hexulose,”J. K. N. Jones, J. Am. Chem. SOC., 78 (1956)2855-2857. “The Structure of the Hemicelluloses of Loblolly Pine,” D. H. Ball, J. K. N. Jones, W. H. Nicholson, and T. J. Painter, Tappi, 39 (1956) 438-443. “Reactions of Aliphatic Nitro Compounds. Formation of a Derivative of 1,5-Diazabicyclo(3.3.3)undecane from 1-Nitropropane, Formaldehyde and Ammonia,” J. K. N. Jones, R.Kosinski, H. Piotrowska, and T. Urbariski, Bull. Acad. Pol. Sci. Cl. 3,4 (1956) 509-510. “Reactions of Aliphatic Nitro Compounds. XXVII. On Formation of a Derivative of 1,5-Diazabicyclo(3.3.3}undecane from 1-Nitropropane, Formaldehyde and Ammonia,” J. K. N. Jones, R. Kolinski, €1. Piotrowska, and T. Urbdski, Ron. Chem.,31 (1957) 101109. “The Synthesis ofo-DeoxywS-ethylpoIyols,”J. K. N. Jones and D. L. Mitchell, Can. I. Chem., 36 (1957) 206-211. “The Hemicelluloses of Loblolly Pine (Pinus taeda) Wood. Part I. The Isolation of Five Oligosaccharide Fragments,” J. K. N. Jones and T. J. Painter,]. Chem. Soc., (1957) 669-6’73. “The Hemicelluloses Present in Aspen Wood (Populus tremuloides). Part 111. The Constitution of Pentosan and Hexosan Fractions,” J. K. N. Jones, E. Merler, and L. E. Wise, Can. J . Chem., 35 (1957) 634-645. “The Fractionation of Polysaccharides by the Method of Ultrafiltration,” K. C. B. Wilkie, J. K. N. Jones, B. J. Excell, and R. E. Sernple, Can. l. Ckm.,35 (1957) 7957%. “A Synthesis of 5,6Dideoxy-~-xylohexose(5-Deoxy-X-methyl-D-xylose),”J. K. N. Jones and J. L. Thompson, Can.J . Chem., 35 (1957) 955-959. “The Structure of the Type VIII Pneumococcus Specific Polysaccharide,” J. K. N. Jones and M. B. Perry,J. Am. Chem. SOC., 79 (1957) 2787-2793. ‘‘The Structure of Linseed Mucilage. Part I,” A. J. Erskine and J. K. N. Jones, Can.J. Chem., 35 (1957) 1174-1182. “The Synthesis of Disaccharides,” D. H. Ball and J. K. N. JonesJ. Chem. Soc., (1957) 4871-4873. “Isolation of Disaccharides from Golden Apple Cum,” J. K. N. Jones and B. 0. Lindgren,Acta Chem. S c a d . , 11 (1957) 1365. “The Acidcatalyzed Reversion of L-Arabinose and of DMannose,” J. K. N. Jones and W. H. Nicholson,J. Chem. Soc., (1958) 27-33. “The Acid-catalyzed Reversion of DXylose,” D. H. Ball and J. K. N. Jones,J. Chem. SOC., (1958)33-36. “A Synthesis of3-0-j3-DCalactopyranosyl-~galactose,” D. H. Ball and J. K. N. Jones, 1. Chem. SOC.,(1958)905-907.

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“Carbohydrate Chemistry at Queen’s University,” J. K. N. Jones, Pulp Pap. Mag. Can. Tech. Sect., 59 (1958) 145-147. “The Preparation of Some Derivatives of DRibono-14-lactone and DRibitol,” L. Hough, J. K. N. Jones, and D. L. Mitchell, Can. J. Chem., 36 (1958) 1720-1728. “The Hemicellulosas of Loblolly Pine (Pintts taedu) Wood. Part 11.The Constitution of Hexosan and Pentosan Components,” J. K. N. Jones and T. J. Painter,]. Chem. SOC., (1959) 573-580. “Structural Studies on Clinical Dextrans. Part I. Methylation and Pcriodate Oxidation Studies,” J. K. N. Jones and K. C. B. Wilkie, CanJ. Biochem. Physiol., 37 (1959) 377390. “The Characterization of TriO-tosyl Sucrose,” P. D. Bragg and J. K. N. Jones, Can. J. Chem., 37 (1959)575-578. “The Oxidation of Some Terminal-substituted Polyhydric Alcohols by Acetobacter suboxydans,” L. Hough, J. K. N. Jones, and D. L. Mitchell, Can. J . Chem., 37 (1959) 725-730. “Structure of Some Water-soluble Polysaccharides from Wood,” D. J. Brasch, J. K. N. Jones, T. J. Painter, and P. E. Reid, Proc. Cellul. Con$, 2nd, Syracuse, 1959, 3-15. “Structure of Some Water-soluble Polysaccharides from Wood,” D. J. Brasch, T. J. Painter, P. E. Reid, and J. K. N. Jones, Pulp Pap. Mag. Can., Tech. Sect., 60 (1959)T342T345. “5,6-Dideoxy-~arabino-hexose (S-Deoxy-5C-methyl-L--arabinose),” D. H. Bali, A. E. Flood, and J. K. N. Jones, Can. J. Chem., 37 (1959) 1018-1021. “The Reaction of Sulphuryl Chloride with Glycosides and Sugar Alcohols. Part I,” P. D. Bragg, J.K. N. Jones, and J. C. Turner, Can. J. Chem., 37 (1959) 1412-1416. “The Structure of an Arabogalactan From Monterey Pine (Pinus radiata),” D. J . Brasch and J. K. N. Jones, Can.J. Chem., 37 (1959) 1538-1545. “The Synthesis of S-Deoxy-5S-ethyl-D-threo-pentulose,” J. K. N. Jones and D. L. Mitchell, Can. J. Chem., 37 (1959) 1561-1566. “Separation of Sugars on Ion Exchange Resins,” J. K. N. Jones, R. A. Wall, and A. 0. Pittet, Chem. Ind. (London),(1959) 1196. “Synthesis of Sugars from Smaller Fragments. Part XI. Synthesis of L-Galactoheptulose,” J. K. N. Jones and N. K. Matheson, Can. J. Chem., 37 (1959) 1754-1756. “Investigation of Some Ancient Woods,” D. J. Brasch and J. K. N. Jones, Tappi, 42 (1959) 913-920. “The Reaction of Sodium Metaperiodate with Some Nitrogen Derivatives of Carbohydrates,” M. J. Abercrombie and J. K. N. Jones, Can. J. Chem., 38 (1960)308-309. “Synthesis of Sugars from Smaller Fragments. Part XII. Synthesis of SGlycero-D altro-, cGEycero-Lgalacto-, DClycero-Ggluco-, and DGlycero-cgalacto-odulose,” J . K. N. Jones and H. H. Sephton, Can. J. Chem., 38 (1960) 753-760. “Some Open-chain Derivatives of Glucose and Mannose,” E. J. C. Curtis and J. K. N. Jones, Can. J. Chem., 38 (1980) 890-895. “The Synthesis of 2O-~-~-Glucopyranosyl-~-xylose,” J. K. N. Jones and P. E. Reid, Can.]. Chem., 38 (1960) 944-949. “The Reaction of Sulphuryl Chloride with Glycosides and Sugar Alcohols. Part 11,” J. K. N. Jones, M. B. Peny, and J. C. Turner, Can.]. Chem., 38 (1960) 1122-1129. “The Synthesis of 3O-~-~-Xylopyranosyl-~-xylose and the Recharacterization of Some Benzylidene Derivatives of ~Xylose,”E. J. C.Curtis and J. K. N. Jones, Can.]. Chem., 38 (1960) 1305-1315. “The Polysaccharides of Cryptococcus laurentii (NRRL Y-1401). Part I,” M. J. Abercrombie, J. K. N. Jones, M. V. Lock, M. B. Perry, and R.J. Stoodley, Can.]. Chem., 38 (1960) 1617-1624.

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W. A. SZAREK, M. STACEY, AND G. W. HAY

“A Chemical Procedure for Determination of the C“ Distribution in Some Labelled Carbohydrates,” M. J. Abercrombie and J. K. N. Jones, Can.J . Chem., 38 (1960)1999-

2006. “The Polysaccharides of Cryptococcus laurentii (Y1401).Part 11. Biosynthesis of the Carbohydrates Found in the Acidic Polysaccharide,” M. J. Abercrombie, J. K. N. Jones, and M. B. Perry, Can. J . Chem., 38 (1960)2007-2014. “The Separations of Sugars on Ion-exchange Resins. Part I,” J. K. N. Jones, R. A. Wall, and (in part) A. 0.Pittet, Can. J . Chem., 38 (1960)2285-2289. “The Separations ofSugars on Ion-exchange Resins. Part 11,” J. K. N. Jones and R. A. Wall, Can. J. Chem., 38 (1960)2290-2294. “The Structure of the ‘Gum Asafoetida’ Polysaccharide,” J. K. N. Jones and G. H. S. Thomas, Can. J . Chem., 39 (1961)192-202. “Analysis of Sugar Mixtures by Gas-Liquid Partition Chromatography,” S. W. Gunner, J. K. N. Jones, and M. B. Perry, Chem. Ind. (London), (1961)255-256. “The Demethylation of Sugars with Hydrogen Peroxide,” B. Fraser-Reid, J. K. N. Jones, and M. B. Perry, Can. J . Chem., 39 (1961)555-563. “The Synthesis ofhcetamido-deoxy Ketoses b y Acetobacter suboxydans. Part I,” J. K. N. Jones, M. B. Perry, and J. C. Turner, Can./. Chem., 39 (1961)965-972. “The Carbohydrate-Protein Linkage in Glycoproteins. Part I. The Syntheses of Some Model Substituted Amides and an L-Seryl-Sglucosaminide,” J, K. N. Jones, M. B. Perry, B. Shelton, and D. J. Walton, Can. J . Chem., 39 (1961)1005-1016. “Constitution of a 4-O-Methylglucuronoxylan From the Wood of Trembling Aspen (Populus tremuloides Michx.),” J. K. N. Jones, C. B. Purves, and T. E. Timell, Can. J. Chem., 39 (1961)1059-1066. “The Gas-Liquid Partition Chromatography of Carbohydrate Derivatives. Part I. The Separation of Glycitol and Glycose Acetates,” S. W. Gunner, J. K. N. Jones, and M. B. Perry, C a n . J .Chem., 39 (1961)1892-1899. “The Synthesis of Acetamido-deoxy Ketoses by Acetobacter suboxyduns. Part 11,” J. K. N. Jones, M. B. Perry, and J. C. Turner, Can./. Chem., 39 (1961)2400-2410. “Biogenesis of Carbohydrates in Wood,” J. K. N. Jones, Pure Appl. Chem., 5 (1962)

21 -35. “The Synthesis of Acetamido-deoxy Ketoses by Acetobacter suboxydans. Part 111,” J. K. N. Jones, M. B. Perry, and J. C. Turner, Can.]. Chem., 40 (1962)503-510. “Biosynthesis of Sugars Found in Bacterial Polysaccharides. Part I. Biosynthesis of L-Rhamnose,” J. K. N. Jones, M. B. Perry, and R. J. Stoodley, C a n . ] . Chem., 40 (1962) 856-863. “The Biological and Chemical Synthesis of Polysaccharides,” J. K. N. Jones, Pure Appl. Chem., 5 (1962)469-482. “The Structure of Linseed Mucilage. Part 11,” K. Hunt and J. K. N. Jones, Can. J .

Chem., 40 (1962)1266-L279. “The Reaction of Sulphuryl Chloride with Reducing Sugars. Part I,” H. J. Jennings and J. K. N . Jones, Can. j . Chem., 40 (1962)1408-1414. “The Gas-Liquid Partition Chromatography of Carbohydrate Derivatives. Part 111. The Separation of Amino Glycose Derivatives and of Carbohydrate Acetal and Ketal Derivatives,” H. G. Jones, J. K. N. Jones, and M. B. Perry, Can.]. Chem.,40 (1962)1559 -1563. “Biosynthesis of Sugars Found in Bacterial Polysaccharides. Part 11. Biosynthesis of D-#lycero-D-manno-Heptose,”J. K. N. Jones, M. B. Perry, and R. J. Stoodley, Can. J . Chem., 40 (1962)1798-1804. “The Carbohydrate-Protein Linkage in Glycoproteins. Part 11. The Synthesis ofN-LSeryt-D-glucosamine andN-L-Threonyl-Sglucosamine,” J. K. N. Jones, J. P. Millington, and M. B. Perry, Can. /. Chem., 40 (1962)2229-2233.

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“5-Acetamido-5-deoxyy-~-arabinose: A Sugar Derivative Containing Nitrogen as the Hetero-atom in the Ring,” J. K. N. Jones and J. C. Turner,./. Chem. SOC., (1962)46994703. “Recent Progress in Polysaccharide Chemistry,” J. K. N. Jones, An. Assoc. Bras. Quim., Numero Expec., 21 (1962) 41-55. “Chromatography on Paper,” L. Hough and J. K. N. Jones, Methods Carbohydr. Chem., 1 (1962) 21-31. “Enzymic Methods for Determination of DGlucose. Quantitative Determination of D-Glucose by Oxidation with D-Glucose Aerodehydrogenase,” L. Hough and J. K. N. Jones, Methods Carbohydr. Chern., 1 (1962)400-404. “Determination of isotopic Carbon Distribution in Aldoses. Chemical Oxidation to Carbon Dioxide,” J. K. N. Jones and R. J. Stoodley, Methods Carbohydr. Chem., 2 (1963)489-493. “Structural Studies on the Water-Soluble Arabinogalactans of Mountain and European Larch,” J. K. N. Jones and P. E. Reid,J. Polym. Sci., Part C , (1963) 63-71. “Synthesis of a Sugar Derivative with Nitrogen in the Ring,” J. K. N. Jones and W. A. Szarek, Can.J . Chem., 41 (1963) 636-640. “The Reaction of Chlorosulphate Esters of Sugars with Pyridine,” H. J. Jennings and J. K. N. Jones, Can.J.Chem., 41 (1963) 1151-1159. “The Synthesis of D-glycero-Dmanno-Heptose,”R. K. Hulyalkar. J. K.N. Jones, and M. B. Perry, Can. J. Chem., 41 (1963) 1490-1492. “The Synthesis of 3-Hexuloses. Part 11. Derivatives of 1-Deoxy-Lurabo-3-hexulose (Syn. 6-Deoxy-~-Zyxo4-hexulose),”J. W. Bird and J. K. N. Jones, Can. J . Chem., 41 (1963) 1877-1881. “Synthesis of ~-Arabinose-5-C’~,” R. K. Hulyalkar and J. K. N. Jones, Can. J. Chem., 41 (1963) 1898-1904. “Carbon-Oxygen Fission: Degradation of Polysaccharides,” J. K.N. Jones and M. B. Perry, in K. W. Bentley (Ed.), Elucidation of Structures b y Physical and Chemical Methods, Part 11, Technique of Organic Chemistry, Vol. X I , Interscience Publishers, New York, 1963, pp. 707-750. “The Synthesis of 5#-~-D-Glucopyranosy1-Dxylose and 3,5-Di.0-P-~-glucopyranosyl-D-xylose,” J. K. N. Jones and P. E. Reid, Can. J . Chem., 41 (1963) 2382-2387. “The Occurrence of Dglycero-D.manno-Heptosein the Extracellular Polysaccharide Produced by Azotobacter indicum,” J. K. N. Jones, M. B. Perry, and W. Sowa, Can. J. Chem., 41 (1963)2712-2715. “The Structure of the Extracellular Polysaccharide of Azotobacter indicum,” V. M. Parikh and J. K. N. Jones, Can. J . Chem., 41 (1963)2826-2835. “Synthesis of Methyl 4-Acetamido4-deoxy-~-erythrofuranoside: A Sugar with Nitrogen in a Five-membered Ring,” W. A. Szarek and J. K. N. Jones, Can. /. Chem., 42 (1964)20-24. “The Chemistry of Apiose. Part I,” D. T. Williams and J. K. N. Jones, Can. J . Chem., 42 (1964)69-72. “The Glucomannan of Bluebell Seed (Scylla nonscripta L.),” J. L. Thompson and J. K. N. Jones, Can.]. Chem., 42 (1964) 1088-1091. “Hindered Internal Rotation in Carbohydrates Containing Nitrogen in the Ring,” W. A. Szarek, S. Wolfe, and J. K. N. Jones, Tetrahedron Lett., (1964)2743-2750. “Polysaccharides From the Seeds of the Huacra Pona Palm (Zriartea uentricosa),” W. Sowa and J. K. N. Jones, Can. J . Chem., 42 (1964) 1751-1754. “The L-Ascorbate Ion as an Ambident Nucleophile,” E. Buncel, K.G. A. Jackson, and J. K. N. Jones, Chem. Ind. (London), (1965)89. “Structure of Cholla Gum (Opuntia fulgida),” V. M. Parikh and J. K. N. Jones, J . Polym. Sci., Part C , (1965) 139-148.

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W. A. SZAREK, M. STACEY, AND G. W. HAY

“The C- and 0-Benzylation of L-Ascorbic Acid,” K. G. A. Jackson and J. K. N. Jones, Can. J . Chem., 43 (1965)450-457. “The Oxidation of Sugar Acetals and Thioacetals by Acetobacter suborydans,” D. T. Williams and J. K. N. Jones, Can.]. Chem., 43 (1965)955-959. “Synthesis of 4-Acetamido4-deoxy-sugars,” A. J. Dick and J. K. N. Jones, Can. J.

Chem., 43 (1965)977-982. “The Reaction of Galactose with Hydrazine at Elevated Temperature,” J. K. N. Jones, P. Reid, and J. R. Turvey, Can.]. Chem., 43 (1965)983-985. “The Chemistry of D-Apiose. Part 11. The Configuration of D-Apiose in Apiin,” R. K. Hulydkar, J. K. N. Jones, and M. B. Perry, Can.]. Chem., 43 (1965)2085-2091. “Carbohydrates Containing Nitrogen in a Five-membered Ring and an Attempted Synthesis of a Carbohydrate with Nitrogen in a Seven-membered Ring,” W. A. Szarek and J. K. N. Jones, Can .J. Chem., 43 (1965)2345-2356. “Reactions of Sugar Chlorosulfates. Part V. The Synthesis of Chlorodeoxy Sugars,” H. J. Jennings and J. K. N. Jones, Can.J . Chem., 43 (1965)2372-2386. “Synthesis of 40-~-D-Galactopyranosyl-D-galactose,” E. J. C. Curtis and J. K. N. Jones, Can. J . Chem., 43 (1965)2508-2511. “Reactions of Sugar Chlorosulfates. Part VI. The Structure of Unsaturated Chlorodeoxy Sugars,” H. J. Jennings and J. K. N. Jones, Can.J. Chem., 43 (1965)3018-3025. “Synthesis of 5-Benzamido-5-deoxy-~-xylopyranose,” M. S. Patel and J. K. N. Jones, Cun. J . Chem., 43 (1965)3105-3108. “Direct Displacement of a Primary Tolyl-p-sulfonyloxy Group by the Methoxide Ion: A More Direct Route to 5-0-Methyl-L-arabinose and 3,5-Di4l-methyl-~-arabinose,” S. C. Williams and J. K. N. Jones, Can. J. Chem., 43 (1965)3440-3442. “Oxidation of Sugars with Ruthenium Dioxide-Sodium Periodate: A Simple Method for the Preparation of Substituted Keto Sugars,” V. M. Parikh and J . K. N. Jones, Can.J . Chern., 43 (1965)3452-3453. “Selective Nucleophilic Substitution and Preferential Epoxide Formation,” A. J. Dick and J. K. N. Jones, Can. J . Chem., 44 (1966)79-87. “Cholla Gum. Part I. Structure of the Degraded Cholla Gum,” V. M. Parikh and J. K. N. Jones, Can. J . Chem., 44 (1966)327-333. “The Separation of Aldopentose and Aldohexose Diethyl Dithioacetal Derivatives by Gas-Liquid Partition Chromatography,” D. T. Williams and J. K. N. Jones, Can. J . Chem., 44 (1966) 412-415. “Chlorosulphate as a Leaving Group: The Synthesis of a Methyl Tetrachloro-tetradeoxy-hexoside,” A. G. Cottrell, E. Buncel, and J. K. N. Jones, Chem. Ind. (London), (1%) 552. “Reactions of Sugar Chlorosulfates. Part VII. Some Confonnational Aspects,” A. G. Cottrell, E. Buncel, and J. K. N. Jones, Can.]. C h m . , 44 (1966)1483-1491. “Cholla Gum. Part 11. Structure of the Undegraded Cholla Gum,” V. M. Parikh and J. K. N. Jones, C a n . J .Chem., 44 (1966) 1531-1539. “The Capsular Polysaccharide of Pneumococcus Type XII, SXII,” J. A. Cifonelli, P. Rebers, M. B. Perry, and J. K. N. Jones, Biochemistry, 5 (1966)3066-3072. “A One-step Conversion of Cyclohexene Oxide into cis-l&Dichlorocyclohexane,” J. R. Campbell, J. K. N. Jones, and S. Wolfe, Can.J . Chem., 44 (1966)2339-2342. “A New Synthesis of 3-Acetamido3-deoxy-~glucose,” D. T. Williams and J. K. N. Jones, Cun. J . Chem., 45 (1967)7-9. “A Synthesis of Dihydroxyacetone Phosphate From Dihydroxyacetone,” R. L. Colbran, J. K. N. Jones, N. K. Matheson, and I. Rozema, Carbohydr. Res., 4 (1967)355-358. “The Synthesis, Separation, and Identification of the Methyl Ethers of Arabinose and ‘Their Derivatives,” S. C. Williams and J. K. N. Jones, Can.J.Chem., 45 (1967)275-290.

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“Further Experiments on the Oxidation of Sugar Acetals and Thioacetals by Acetobacter suborydans,” D. T. Williams and J. K. N. Jones, Can.]. Chem., 45 (1967)741744. “Acetonation of D-Xylose Diethyl Dithioacetal,” D. G. Lance and J. K. N. Jones, Can. .J. Chem., 45 (1967)1533-1538. “Reactions of Sugar Chlorosulfates. Part VIII. @Ribose and Its Derivatives,” S. S. Ali, T. J. Mepham, (Miss) I. M. E. Thiel, E. Buncel, and J. K. N. Jones, Carbohydr. Res., 5 (1967)118-125. “Gas Chromatography of Derivatives of the Methyl Ethers of D-Xylose,” D. G. Lance and J. K. N. Jones, Can. 3. Chem., 45 (1967)1995-1998. “The Synthesis of L-Mycarose and L-Cladinose,” G. B. Howarth and J. K. N. Jones, Can. ]. Chem., 45 (1967)2253-2256. “Selective Benzoylation of Benzyl P-L-Arabinopyranoside and Benzyl a-~-Xylopyranoside,” T. Sivakumaran and J. K. N. Jones, Can. 1.Chem., 45 (1967)2493-2500. “Epoxide Ring Opening of Methyl 2,3-Anhydro4-azido4-deoxy-pentopyranosides,” A. J. Dick and J. K. N. Jones, Can.]. Chem., 45 (1967)2879-2885. “The Synthesis of ~-Arcanose,” G. B. Howarth, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1968)62-63. “Isolation of Two kabinobioses From Acacia nilotica Gum,” R. C . Chalk, J. F. Stoddart, W. A. Szarek, and J. K. N. Jones, Con.]. Chem., 46 (1968)2311-2313. “Synthesis of 6-Deoxy3-C-methyl-2-0-methyl-@a~lose,” G. B. Howarth, W. A. Szarek, and J. K. N. Jones, Can. 3. Chem., 46 (1968)3375-3379. “Synthesis of 6-Chloro-4(6’-deoxy-3’C-methyl-2‘,3‘,4‘-~-0-methyl-~-@allopyranosy1)purine: A Branched-chain Sugar Nucleoside,” G. B. Howarth, W.A. Szarek, and J. K. N. Jones, Can. J. Chem., 46 (1968)3691-3694. “Photolysis of Carbohydrate Nitro-olefins,” G. B. Howarth, D. G. Lance, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1968)1349. “Branched-chain Sugar Nucleosides. Synthesis of a Purine Nucleoside of 4-0-AcetylL-arcanose,” G. B. Howarth, W. A. Szarek, and J. K. N. Jones,]. Org. Chem., 34 (1969) 476-477. “Syntheses Related to the Carbohydrate Moiety in Lincomycin,” G. B. Howarth, D. G . Lance, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47 (1969)75-79. “Photolysis of a Carbohydrate Nibbolefin,” G. B. Howarth, D. G. Lance, W. A. Szarek, and J. K. N. Jones, Can. J. Chem., 47 (1969)81-87. “Some Structural Features of the Mucilage From the Bark of Ulmus fulua (Slippery Elm Mucilage),” R. J. Beveridge, J. F. Stoddart, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 9 (1969)429-439. “An Improved Procedure for Oxidation of Carbohydrate Derivatives with Ruthenium Tetraoxide,” B. T. Lawton, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 10 (1969) 456-458. “Synthesis of 8-Deoxy-D+rythro-D-galacto-octose.Determination of the Configuration of Two Octenoses,” D. G. Lance, W. A. Szarek, J. K. N. Jones, and G. B. Howarth, Can.]. Chem., 47 (1969)2871-2874. “Some 0-Isopropylidene Derivatives of @Ribose Diethyl Dithioacetal,” D. G. Lance, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47 (1969)2889-2891. “Synthesis of DChalcose,” B. T. Lawton, D. J. Ward, W. A. Szarek, and J. K. N. Jones, Can. ]. Chem., 47 (1969)2899-2901. “Large Heterocyclic Rings From Carbohydrate Precursors,” J. F. Stoddart, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47 (1969)3213-3215. “A Simple Synthesis of Azidodeoxy-sugars uia Chlorodeoxy-sugars,” B. T. Lawton, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1969)787-788.

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W. A. SZAREK, M. STACEY, AND G . W. HAY

“Reachon of Methyl 4,6C)-Benzy~idene3C-methyl-2-O-p-tolylsulfonyl-a-~-allopyranoside with Sodium Methoxide in Methyl Sulfoxide: Synthesis of 6-Deoxy-34methyl3-O-methyl-aallose (2-Hydroxy-~-cladinose),”G. B. Howarth, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 11 (1969)257-262. “The Synthesis of N-Acetyl-lincosamine (6-Acetamido-6,8-dideoxy-D+rythro-~-galacto-octose), a Derivative of the Free Carbohydrate Moiety in Lincomycin,” G. B. How&, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1969) 1339-1340. “Synthesis of 3-Hexuloses,” K. G. A. Jackson and J. K. N. Jones, Can.J . Chem., 47 (1969)2498-2501. “Separation and Identification of Methyl Ethers of DGlucose and D-Glucitol by GasLiquid Chromatography,” H. G. Jones and J. K. N. Jones, Can.]. Chem., 47 (1969)3269 -3271. E. H. Wi1“Synthesis of Olivomycose (2,6-Dideoxy3C-methyl-~-arabino-hexose),” liams, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47 (1969) 4467-4471. “Addition of Pseudohalogens to Unsaturated Carbohydrates. Part 111. Synthesis of 3DeoxySE-nitromethyl-&allose, a Branchedchain Nitro Sugar,” W. A. Szarek, J. S. Jewell, 1. Szczerek, and J. K. N. Jones, Can.J. Chem., 47 (1969)4473-4476. “Carbohydrate Fluorosulfates,” E. Buncel, H. J. Jennings, J. K. N. Jones, and I. M. E. Thiel, Carbohydr. Res., 10 (1969)331-332. “Structural Feature of Pneumococcus Type XIX Specific Polysaccharide,” T. Miyazaki and J. K. N. Jones, Chem. Pham. Bull., 17 (1969) 1531-1533. “The Isolation and Properties of the Skin-reactive Substance in Aedes aegypti Oral Secretion,” W. H. Newsome, J. K. N. Jones, F. E. French, and A. S. West, Can.J. Biochem., 47 (1969) 1129-1136. ‘?V-(4,6-O-Benzylidene-l-O-methyl-3-oximino-a-~-ribohexopyranos-2-y1)pyridinium p-Toluenesulfonate. A Novel Versatile Carbohydrate Substrate,” W. A. Szarek, B. T. Lawton, and J. K. N. Jones, Tetrahedron Lett., (1970) 4867-4870. “A Facile Synthesis of 4,6-Dideoxy-~-xylo-hexose,” B. T. Lawton, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 14 (1970) 255-258. “The Synthesis of Lincomycin,” G. B. Howarth, W. A. Szarek, and J. K. N. Jones, 1. C h . SOC., C, (1970) 2218-2224. “Synthesis of Deoxy and Aminodeoxy Sugars by Way of Chlorodeoxy Sugars,” B. T. Lawton, W. A. Szarek, and J. K. N.Jones, Carbohydr. Res., 15 (1970) 397-402. “Polysaccharides of Type XIX Pneumococcus. Part I. Isolation of Type Specific Polysaccharide,” T. Miyazaki,T. Yadomae, and J. K. N. Jones,]. Biochem. (Tokyo), 68 (1970) 755-758. “Synthesis of Paratose (3,6-Dideoxy-D.ribo-hexose)and Tyvelose (3,6-Dideoxy-~arabino-hexose),” E. H. Williams, W. A. Szarek, and J. K. N. Jones, Can. ]. Chem., 49 (1971) 796-799. “Some Structural Studies on the Galactan from the Albumen Glands of‘the Snail, Strophocheilus oblongus,” J. H. Duarte and J. K. N. Jones, Carbohydr. Res., 16 (1971) 327-335. “Reaction of Methyl 4,6-Dichloro4,6-dideoxy-a-D-galactopyranoside2,3-Di(chlorosulfate) with Sodium Azide, and with Sodium Bromide, in N,N-Dimethylformamide,” H. Parolis, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 19 (1975) 97-105. “Isolation of Three Oligosaccharides from the Mucilage from the Bark of Ulmusfulua (Slippery-Elm Mucilage). Synthesis of O-(30-Methyl-~-~-galactopyranosyl)-(14)-~rhaanose,” R. J. Beveridge, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 19 (1971) 107-1 16. “Reactions of‘Carbohydrate a-Keto Toluene*-sulphonates. Reaction of Methyl 4,6O-Benzylidene-20-toluenep-suIphonyl-a-D-ribo-hexop~~nosid~-ulose with Triethyl-

OBlTUARY- JOHN KENYON NETHERTON JONES

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amine-Methanol,” A. Dmytraczenko, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1971) 1220-1222.

“Preparation of Unsaturated Carbohydrates from Methyl 4,W-Benzylidene-3chIoro-3-deoxy-pDallopyranoside,and Their Utility in the Synthesis of Sugars of Biological Importance,” E. H. Williams, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 20 (1971) 49-57. ‘‘Structure of Slippery Elm Mucilage (Ulmusfulua),” R. J. Beveridge, J. K. N. Jones, R. W.Lowe, and W . A. Szarek,]. PoZym. Sci., Part C,36 (1971) 461-466. “Studies on Lipopolysaccharides of Proteus,” B. A. Dmitriev, N. A. Hinton, R. W. Lowe, and J. K. N. Jones, Can.]. MicrobioZ., 17 (1971) 1385-1394. “An Evaluation of Methods for the Preparation of 1,2:3,4-Di-O-isopropylidene-a-~galacto-hexodialdo-1,Spyranose. Oxidation of 1,2:3,4DiO-isopropylidene-a-~galactopyranose with Lead Tetraacetate-Pyridine,” D. J. Ward, W.A. Szarek, and J. K. N. Jones, Carbohydr. Res., 21 (1972) 305-308. “Addition of Pseudohalogens to Unsaturated Carbohydrates. Part V. Addition of Iodine Trifluororoacetate,” R. G. S. Ritchie and W. A. Szarek, Can. J. Chem., 50 (1972)

507-511. “Some Reactions of Unsaturated Carbohydrates in the Presence of Iodine,” I. Szczerek, J. S. Jewell, R. G. S. Ritchie, W.A. Szarek, and J. K. N. Jones, Carbohydr. Res., 22 (1972) 163-172. “Amination of Sugar Derivatives with a Mixture of Phthalimide, Triphenylphosphine, and Diethyl Azodicarboxylate,” A. Zamojski, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 23 (1972) 460-462. “Selective Oxidation of a Diol with Methyl Sulfoxide-Acetic Anhydride,” T. B. Grindley, J. W. Bird, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 24 (1972) 212215. “Synthesis of Carbohydrate Furoxan Derivatives,” C. S. Wu, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1972) 1117-1118. “Ethers of Sugars,” J. K. N. Jones and G. W. Hay, in W. Pigman and D. Horton (Eds.), The Carbohydrates, Vol. IA, Academic Press, New York, 1972, pp. 403-422. “Reaction of Some Die-isopropylidenehexoseswith Cyanuric Chloride,” A. Zamojski, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 26 (1973) 208-214. “Reaction of Methyl 2,3-O-lsopropylidene-6~~-tolylsulfonyl-a-DEyxo-hexofuranosid-5-ulose with Triethylamine-Methanol,” A. Dmytraczenko, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 26 (1973) 297-303. “Reductive Cleavage of Carbohydrate p-Toluenesulfonates with Sodium Naphthalene,” H. C. Jarrell, R. G. S. Ritchie, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 51 (1973) 1767-1770. “Conversion of 2-Hexuloses into 3-Heptuloses: Synthesis of D-manno3-Heptulose,” R. W. Lowe, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 28 (1973) 281-293. “The Total Synthesis of Carbohydrates,” W. A. Szarek and J. K. N. Jones, in J. W. ApSimon (Ed.), The Total Synthesis of Natural Products, Wiley-Interscience, New York, 1973, pp. 1-80. “Lipopolysaccharides of Proteus,” J. K. N. Jones, in Mbthodologie de la Structure et du Mbtabolisme des Glycoconjuguk, Colloques Internationaux du Centre National de la Recherche Scientifique, No. 221, June 20-27, 1973, Villeneuve d’Ascq, Vol. 1, pp. 533-543. “A Reinvestigation of the Reaction of Methyl 8-DGlucopyranoside with Sulfuryl Chloride,” D. M. Dean, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 33 (1974) 383-386. “Reaction of Hexopyranoside a-Keto Toluene-p-sulfonates with Triethylamine-

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W. A. SZAREK, M. STACEY, AND G . W. HAY

Methanol,” W. A. Szarek, A. Dmytraczenko, and J . K. N. Jones, Carbohydr. Res., 35 (1974)203-219. “Reaction o f Methyl Yentofuranosides with Sulfiiryl Chloride,” B. Achmatowicz, W. A. Szarek, J. K. N. Jones, and E. H. Williams, Carbohydr. Res., 36 (1974) c14-c16. “Arthur Charles Neish, 1916-1973,” J. K. N. Jones, Biogr. Mem. Fellows R . Soc., 20 (1974)294-315. “Synthesis of Nucleosides by Direct Replacement of the Anomeric Hydroxy-group,” W. A. Szarek, C. Depew, H.C. Jarrell, and J. K. N. Jones,]. Chem. Soc., Chem. Commum, (1975)648-649. “Syntheses Related to Dendroketose,” H. C. Jarrell, W. A. Szarek, J. K. N. Jones, A. Dmytraczenko, and E. B. Rathbone, Carbohydr. Res., 45 (1975) 151-159. “Decarhonylation of Aldehydo Sugar Derivatives with Chlorotris(methyldipheny1phosphine)rhodium(I),” D. J. Ward, W. A. Szarek, and J. K. N. Jones, Chem. Ind. (London), (1976) 162-163. “Syntheses Towards the Carbohydrate Moiety of Lincomycin,” G. R. Woolard, E. B. Rathbone, W. A. Szarek, and J. K. N. Jones,/. Chem. SOC. Perkin Trans. 1 , (1976)950954.

“Synthesis of Carbohydrate-Saccharin Conjugates,” W. A. Szarek, C. Depew, and J. K. N. Jones,/. Heterocycl. Chem., 13 (1976) 1131-1133. “Selective, Reductive Dechlorination of Chlorodeoxy Sugars. Structural Determination of Chlorodeoxy and Deoxy Sugars by I3C Nuclear Magnetic Resonance Spectroscopy,” W. A. Szarek, A. Zamojski, A. R. Gibson, D. M. Vyas, and J. K. N. Jones, Can./. Clnem., 54 (1976)3783-3793. “Oxidation of a Branched-chain Alditol by Acetobocter suboxydans: a Stereospecific Synthesis of L-Dendroketose,” W. A. Szarek, G. W. Schnarr, H. C. Jarrell, and J. K. N. Jones,Carbohydr. Res., 53 (1977)101-108. “Preparation and Activity of Immobilized Acetobacter suboxydans Cells,” G . W. Schnarr, W. it. Szarek, and J. K. N. Jones,AppL Enoiron. Microbiol., 33 (1977)732-734. “Synthesis of Glymsides: Reactions of the Anomeric Hydroxyl Group with NitrogenPhosphorus Betaines,” W. A. Szarek, H. C. Jarrell, and J. K. N. Jones, Carbohydr. Res., 57 (1977) c13-cI6. “Stereospecific Chemical Synthesis of L-Dendroketose Derivatives,” H. C. Jarrell, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 64 (1978)283-288.

ADVANCES IN C N O H Y D R A T E CHEMISTRY AND BIOCHEMISTRY. VOL. 41

CARBON-13 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY OF MONOSACCHARIDES BY KLAUS BOCKAND CHRISTIANPEDERSEN Department of Organic Chemistry. The Technical University of Denmark. DK-2800 Lyngby. Denmark I . Introduction ................................................. I1. Sampling Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Conditions for Optimal Signal-to-Noise Ratio ...................... 3. Referencing of Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Quantitative Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Resolution Enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Assignment Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Comparison with Model Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Isotopic Substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Correlation with Proton Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . RelaxationRates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Paramagnetic Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Protonation Shifts ........................................... IV . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Identity of Monosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Structure Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Conformational Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Relaxation Rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Complexation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27 28 29 30 31 32 33 34 34 35 36 37 38 39 39 39 40 43 43 43 44

I . INTRODUCTION

The first two reports on carbon-13 nuclear magnetic resonance ( 13Cn.m.r.) spectra of carbohydrates appeared1. in 1968 and 1969; since then. 13C-n.m.r. spectroscopy has become increasingly important as a tool for the characterization and structural elucidation of sugars and their derivatives . Although 13C-n.m.r. is closely related to 'H-n.m.r. spectroscopy, especially when both types of spectra are recorded with

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(1) F. J . Weighert. M Jautelat. and J. D. Roberts. Proc. Natl .Acad . Sci . USA. 60 (1968) 1152-1155 . (2) A . S . Perlin and B . Casu. Tetrahedron Lett., (1969) 2921-2924 .

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Copyright @ 1983 by Academic Press. Inc All nghts of reproduction in any form reserved ISBN 0-12-007261-6

28

KLAUS BOCK AND CHRISTIAN PEDERSEN

Fourier-transform instruments, the two techniques are sufficiently different to be valuable complements to each other. In many cases, in particular when dealing with complex molecules, such as polysaccharides, the amount of information obtainable from 'H-n.m.r. spectra is limited, compared to that revealed3 by I3C-n.m.r. spectra. Monosaccharides may also yield 'H-n.m.r. spectra that are poorly resolved, even at high field, and that contain little information. On the other hand, proton-decoupled, I3C-n.m.r. spectra are well resolved and, even if the signals are not assigned, a spectrum will provide an almost unambiguous identification of a compound. The application of I3C-n.m.r. spectroscopy to carbohydrates has already been reviewed many times,4-" and has been discussed in two monograph~.'~.'~ In each of those reviews, limited numbers of chemical-shift data for carbohydrates were given. As, for identification purposes, it is useful to have convenient access to an extensive list of chemical-shift data, the main purpose of the present article is to provide an almost complete collection of W-n.m.r. chemical-shifts of monosaccharides, their methyl glycosides, and acetates; see Tables IV. In addition, examples of shift data for as many different types of monosaccharide derivative as possible will be given; see Tables VIXXI. Nucleosides and nucleotides are not included, but data on compounds of these types have been reported, for example in Refs. 8, 12, and 14. The literature covered in the present article includes most of that published in 1980, together with a few subsequent papers. 11. SAMPLINGTECHNIQUES The fundamental principles of Fourier-transform, n.m.r. spectroscopy have been described in books and reviews.'"-" (3) P. A. J. Gorin,Adc;. Carbohydr. Chem. Biochem., 38 (1980) 13-104. (4) B. Coxon, Dec;. Food Carbohydr., 2 (1980)351-390. (5) A. S. Perlin, M T P Znt. Reo. Sci., Org. Chem. Ser. One., 7 (1976) 1-34. (6) S. N. Rosenthal and J. H. Fendler, Ada. Phys. Org. Chem., 13 (1976)292-424. (7) .4. S. Shashkov and 0. S. Chizhov, Bioorg. Khim., 2 (1976) 437-497. (8) F. W. Wehrli and T. Nishida, Fortsclrr. Chem. Org. Nuturst., 36 (1979) 1-229. (9) R. Barker and T. E. Walker, Methods Carbohydr. Chem., 8 (19130) 151-165. (10) T. D. Inch, Annu. Rep. N M R Spectrosc., 5A (1972)305-352. (11) G. Kotowycz and R. U. Lemieux, Chem. Rev., 73 (1973)669-698. (12) J. B. Stothers, Carbon-13 NMR Spectroscopy, Academic Press, New York, 1972, pp. 458-468. (13) E. Breitmaier, G. Jung, and W. Voelter,Angew. Chem., 83 (1971) 659-672. (14) M.-T. Chenon, R. J. Pugmire, D. M. Grant, R. P. Panzica, and L. B. Townsend, J . Am. Chem. SOC., 97 (1975) 4627-4636. (15) F. W. Wehrli and T. Wixthlin, lntefpretation of Carbon-13 NMR Spectra, Heyden, London. 1976.

13C-N.M.R.SPECTROSCOPY OF MONOSACCHARIDES

29

1. Sample Preparation The solvents most frequently used for the measurement of 13Cn.m.r. spectra are deuterium oxide (D20) and deuteriochloroform (CDCl,). Deuterated dimethyl sulfoxide (Me2SO-d,) is frequently used, especially for oligo- and poly-saccharides,, and a range of other solvents, including pyridine-d, , have also been employed. The 13Cn.m.r. chemical-shifts of carbohydrates cover a range of -200 p.p.m., and, as solvent-induced shifts are usually less than 1 p.p.m., the choice of solvent does not have a large effect on proton-decoupled, 13C-n.m.r.spectra. Exceptions to this are, however, spectra of basic or acidic carbohydrates (amino sugars, and aldonic and uronic acids), which are strongly pH-dependent. Proton-coupled, 13C-n.m.r. spectra may also be affected by a change in solvents owing to their profound effect on the IH-n.m.r. spectra. The concentration of the sample in a particular solvent has little effect on chemical-shift values and, because of the inherently low sensitivity of I3C-n.m.r. spectroscopy, it is advantageous to use as concentrated solutions as possible when measuring these spectra. However, increased concentration, and consequently increased viscosity, causes line broadening due to decreased, spin-lattice relaxation-times (TI values),'* and thus, poorer resolution. Certain solvents that tend to give viscous solutions (for example, Me2SO-d6) may also give decreased resolution. The temperature of the sample solution has a profound effect on the viscosity and, hence, on the resolution; that is, a higher temperature results in better resolution, because of lower viscosity (larger T, values). The most important aspect of temperature changes in the sample is, however, its effect on chemical-shift values. Thus, a series of I3C-n.m.r. spectra recorded for methyl a-aglucopyranoside in D 2 0 solution showedl9 linear changes in chemical shifts of up to 0.015 p.p.m./degree. Hence, when data have to be compared accurately, I3Cn.m.r. spectra should be recorded at the same temperature, and for samples that have reached temperature equilibrium in the probe. It is obvious that the best resolution is obtained from samples that contain no insoluble impurities, and no paramagnetic materials. The line broadening caused by soluble paramagnetic impurities" may be

(16) E. Breitmaier and W. Voelter, 1 3 4 N M R Spectroscopy, Verlag Chemie, Weinheim, 1974. (17) M. L. Martin, J.-J. Delpuech, and G . J. Martin, Practical NMR Spectroscopy, Heyden, London, 1980. (18) K. Bock, L. D. Hall, and C. Pedersen, Can. J . Chern., 58 (1980) 1916-1922. (19) K. Bock, B. Meyer, and M. R. Vignon,J. Magn. Reson., 38 (1980) 545-551.

30

KLAUS BOCK AND CHRISTIAN PEDERSEN

diminished20 by treatment with an ion-exchange resin or by addition of small amounts of (ethylenedinitri1o)tetraacetate(EDTA).Dissolved oxygen also causes some line broadening; it may be removed sufficiently by boiling the solution in the sample tube for 1 minute.

-

2. Conditions for Optimal Signal-to-Noise Ratio The signal-to-noise ratio (s/n) obtained when a l3C-n.m.r. spectrum is recorded for a given sample solution depends, of course, on the type of instrument used, and it is obvious that a high-field instrument, quadrature detection, and large sample tubes are factors that all result in increased s/n in a given time. Increased concentration of the sample results in a larger s/n, but only to a certain extent, as too high a concentration will lead to line broadening, which will, in turn, have an adverse effect on the s/n. The pulse width is an important factor in the measurement of pulsed spectra. The optimal pulse-width may be estimated21from the equation cos a = exp(- T 1 / T ) ,in which a is the pulse width (in degrees), TI the spin-lattice relaxation-time (in s), and T the pulse-repetition time (in s). For monosaccharides in 20% aqueous solution, TI values of the protonated carbon atoms are22 1 s at 30". Using 8 k of computer memory for the acquisition, and a sweep width of 5-6 kHz, T becomes 0.6-0.8 s, and the equation gives an optimum pulse-width of -60". In Fig. 1 is shown a series of spectra measured at different pulse-widths, all other variables being kept constant. The best s/n is seen to correspond to a 63" pulse. If '%-n.m.r. spectra are recorded for very concentrated solutions, or impure samples, the TI values may become small, and, in such cases, a 90"sample pulse will be optimal. The s/n is, of course, directly proportional to the amount of sample present in the sample tube (more correctly, in the volume defined by the receiver coil); hence, a better s/n is obtained when a large sampletube is used. If, however, a limited amount of compound is available, it may be advantageous to use a smaller probe-insert, because this gives a better coupling between the receiver coil and the nuclei in the sample. The increased s/n resulting from measuring the same amount of compound in a 5-mm sample tube rather than in a 10-mm tube is illustrated in Fig. 2. It may be seen that the s/n ratio in the 5-mm insert is -3 times that in the 10-mm. Consequently, with the 5-mm tube, a

-

(20) M.Cohn and T. R. Hughes, Jr.,]. B i d . Chem., 237 (1962) 176-181. (21) R. R. Emst and W. A. Anderson, Rev. Sci. Instrum., 37 (1966) 93-102. (22) K. Bock and L. D. Hall, Carbohydr. Res., 40 (1975) c3-C5.

13C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

31

FIG. 1.-22.63-MHz, 13C-N.m.r. Spectrum of Methyl 8-D-Xylopyranoside in D,O (0.5 M) at 274 K. [All spectra were obtained under the same experimental conditions, but with different pulse-widths:A, 90";B, 63";C, 45"; and D, 27". Obviously, the optimal signal-to-noise ratio is obtained with a 63" pulse-width.The numerals in A indicate the signals of carbon atoms 1-5.1

sln corresponding to that shown in Fig. 2A could have been achieved with only 20,000/9 = -2000 scans, because the s/n is proportional to the square root of the number of scans.

3. Referencing of Signals Carbon-13 chemical-shifts are defined relative to the carbon signal of internal tetramethylsilane (Me,Si); hence, when measuring spectra in organic solvents, Me4Si should be added to the sample solution as the internal reference-~tandard.2~ However, any homo- or hetero-nuclear signal of the solvent, or of an added reference compound, may be used to calculate the 13C-chemical shifts, provided that its shift rela(23) Pure A p p l . Chem., 45 (1976) 217-219.

32

KLAUS BOCK AND CHRISTIAN PEDERSEN

10mm

3

1

2

4

6

5 mm

B

s/n = 24.4/1

FIG.2.-22.63-MHz, %-N.m.r. Spectrum of Methyl /3-D-Xylopyranoside (10 mg) in 40. [A. Measured in a 10-mm sample-tube in 0.9 mL of DzO. B. Measured in a 5-mm sample-tube i n 0.3 mL of 40. Experimental conditions for the acquisition of the two spectra were exactly identical, and both spectra were obtained with 20,000 scans. In A, the numerals 1-5 indicate the signals of corresponding carbon atoms, and 6 indicates the signal of the 0-methyl group.]

tive to Me,Si is known. In aqueous solution, in which MF4Si is insoluble, it is necessary to use a water-soluble reference-compound unless external Me,Si (in a capillary tube) is employed. In most cases, internal 1,4-dioxane (67.4), acetone (30.5),or methanol (49.6 p.p.m.) are used as references when D,O is the solvent. For routine purposes, when accurate chemical-shifts are not important, it may be convenient to use the deuterium signal of the solvent as a heteronuclear reference, thus avoiding addition of any reference compound. 4. Quantitative Analysis It is often assumed that quantitative data cannot be satisfactorily obtained from integrated, I3C-n.m.r. spectra, because of saturation phenomena and nuclear Overhauser effects. However, if spectra are measured under suitable conditions, and if integrals (or peak heights) of

I3C-N.M.R.SPECTROSCOPY OF MONOSACCHARIDES

33

signals from carbon atoms carrying the same number of hydrogen atoms are compared, it is possible to obtain rather accurate information (f5%; that is, comparable to integrals obtained from 'H-n.m.r. spectra) about the relative amounts of components in a mixture. This has been discussed both from a general point of viewz4and, more specifically, with regard to carbohydrate^.^*'^-^^^^^^ It may be concluded that one of the most important conditions for correct integrals is a good s/n, which is, of course most, readily obtained for concentrated solutions. In such samples, the TI values are small, and hence, saturation is less likely. Furthermore, a sufficient digital resolution (at least 5 points per line) is necessary, in order to define the lines in a spectrum. This may be achieved by narrowing the sweep width, by using a sufficiently large computer memory, or by multiplying the free induction decay (f.i.d.) by a sensitivity-enhancement factor corresponding to a line broadening of 2-3 Hz. Obviously, integration can only be performed on signals that are completely separated; hence, high-field instruments are better suited for this purpose. Integrated, 13C-n.m.r. spectra have been used extensively to study mutarotational equilibria of monosaccharides, especially of ketoses, which do not have well-resolved, 'H-n.m.r. ~ p e c t r a , 2 ~ and - ~ also ~ have been used to determine the composition of crude reaction-mixture~.~~

5. Resolution Enhancement Better separation of poorly resolved signals can obviously be achieved by measuring a spectrum at higher field. However, because increased relaxation-times result in sharper lines,18the resolution can also be improved by using a low concentration, a high temperature, and a nonviscous solvent (for example, acetone). Besides, the use of a (24) S. Gillet and J.-J. Delpuech,]. Magn. Reson., 38 (1980)433-445. (25) J. W. Blunt and M. H. G. Munro, Aust. J . Chem., 29 (1976) 975-986. (2%) D. Horton and Z. Wataszek, Carbohydr. Res., 105 (1982) 145-153. (26) D. Doddrell and A. Allerhand,J. Am. Chem. SOC.,93 (1971)2779-2781. (27) L. Que and G. R. Gray, Biochemistry, 13 (1974) 146-153. (28) D. J. Wilbur, C . Williams, and A. Allerhand,J. Am. Chem. SOC.,99 (1977) 54505452. (29) C. Williams and A. Allerhand, Carbohydr. Res., 56 (1977) 173-179. (30) A. S. Perlin, P. C. M. H. du Penhoat, and H. S. Isbel1,Adu. Chem. Ser., 117 (1973) 39-50. (31) S. J. Angyal and G. S. Bethell, Aust. J . Chem., 29 (1976) 1249-1265. (32) W. Funcke, C. von Sonntag, and C. Triantaphylides,Carbohydr. Res., 75 (1979) 305 -309. (33) P. C. M. H. du Penhoat and A. S. Perlin, Carbohydr. Res., 36 (1974) 111-120. (34) K. Bock, C. Pedersen, and H. Thegersen, Acta Chem. Scand., Ser. B , 35 (1981) 441-449.

34

KLAUS BOCK A N D CHRISTIAN PEDERSEN

5-mm insert and sample tube, instead of the usual 10-mm tubes, will, with most instruments, result in sharper lines, in addition to the increased sensitivity mentioned earlier (see Fig. 2). Alternatively, the resolution of a spectrum may be improved by various mathematical methods, readily performed with a computer and normally described in the instruction manuals for the various n.m.r. instruments. A detailed discussion of data processing in Fourier-transform, n.m.r. spectroscopy was given in Reference 35. It should be mentioned that any mathematical improvement of resolution inevitably leads to a loss of s/n. Resolution enhancement is usually not important in proton-decoupled, W-n.m.r. spectra of monosaccharides. However, in the much more complex, proton-coupled, carbon spectra, this technique is useful if the rather small, two- or three-bond, C-H couplings have to be measured. 111. ASSIGNMENT TECHNIQUES The assignment of signals to specific carbon atoms is a necessary prerequisite to the application of 13C-n.m.r. spectroscopy in structural investigations. As assignment techniques have been described in numerous reviews and book~,3*~~'~*'"-" this area will be treated relatively briefly in the present article.

1. Comparison with Model Compounds In earlier publications, the assignment of signals in 13C-n.m.r. spectra of monosaccharides relied mostly on comparison with those of model compounds3697;this approach led to a number of simple, general rules, summarized as follows. ( a ) The anomeric carbon atoms in pyranoses and furanoses, and in their derivatives, resonate at lowest field (90-110 p.p.m.), except in 1-thioglycosides (see Table VI). (b) Carbon atoms carrying primary hydroxyl groups are found at 60-64 p.p.m. (c) Carbon atoms bearing secondary hydroxyl groups, in pyranoses and furanoses, give signals at 65-85 p.p.m. Signals of alkoxylated carbon atoms, including C-5 in pentopyranoses and C-4 in furanoses, are shifted 5-10 p.p.m. to lower field when compared with the corresponding, hydroxy-substituted carbon atoms. (35) J. C. Lindon and A. G . Femge, Prog. Nucl. Magn. Reson. Spectrosc., 14 (1980)2766. (36)A. S. Perlin, B. Caw, and H. J. Koch, Can. /. Chem., 48 (1970)2596-2606. (37) D.E. Dorman and J. D. Roberts,/.Am. Chem. Sac., 92 (1970)1355-1361.

13C-N.M.R.SPECTROSCOPY OF MONOSACCHARIDES

35

A number of more complicated rules on the influence of axial or equatorial substituents on the chemical shifts of a-,p-, or y-carbon atoms may be safely applied to simple, alicyclic m o l e ~ u l e s . 3 ~In~ ~ - ~ ~ the authors’ opinion, however, such rules are generally of limited value for pyranoses or hranoses, because these contain several, mutually interacting substituents, and use of these rules has, in several instances, led to erroneous assignments. 2. Isotopic Substitution

If a compound in which carbon atoms at known positions are substituted with deuterium or carbon-13 is available, the assignment of its l3C-n.m.r. spectrum is greatly facilitated. Substitution with carbon-13 results in a much stronger signal from the enriched carbon atom, and hence, in its unambiguous assignment. In addition, l3C-I3C couplings may be visible in the spectra of I3C-enriched compounds, and these, together with isotope-induced shifts, may assist in the assignment of carbon atoms in positions a or p to the enriched carbon atom.9940-44 In the I3C-n.m.r. spectra of C-deuterated compounds, the deuterium-carrying carbon atom usually gives no signal, due to coupling to deuterium, longer spin-lattice relaxation-time, and quadrupolar broadening of the signal. Furthermore, the p-carbon atoms may be assigned because of the small, deuterium-induced, upfield shift~.4~-~* A convenient procedure for the preparation of glycosides labelled with deuterium at the hydroxyl-bearing carbon atoms has been deve10ped.4~-~~ Introduction of such magnetic nuclei as I9F or 31Pleads to spin-spin (38)D.K. Dalling and D. M. Grant,]. Am. Chem. Soc., 89 (1967)6612-6622. (39)D. E. Doman and J. D. Roberts,]. Am. Chem. Soc., 93 (1971)4463-4472. (40)T.E. Walker, R. E. London, T. W. Whaley, R. Barker, and N. A. Matwiyoff,]. Am. Chem. SOC., 98 (1976)5807-5813. (41)T. E. Walker, R. E. London, R. Barker, and N. A. Matwiyoff, Carbohydr. Res., 60 (1978)9-18. (42)T.E.Walker and R. Barker, Carbohydr. Res., 64 (1978)266-270. (43)A. S. Serianni, E. L. Clark, and R. Barker, Carbohydr. Res., 72 (1979)79-91. (44)G . Excoffier, D. Y. Gagnaire, and F. R. Taravel, Carbohydr. Res., 56 (1977)229238. (45)P. A. J. Gorin, Can. ]. Chem., 52 (1974)458-461. (46)P. A. J. Gorin and M. Mazurek, Can.]. Chem., 53 (1975)1212-1223. (47)H. J. Koch and A. S. Perlin, Carbohydr. Res., 15 (1970)403-410. (48)E.Breitmaier and U. Hollstein, Org. Magn. Reson., 8 (1976)573-575. (49)H. J. Koch and R. S. Stuart, Carbohydr. Res., 67 (1978)341-348. (50) S.-C. Ho, H. J. Koch, and R. S. Stuart,Carbohydr. Res., 64 (1978)251-256. (51)F.Balza, N.Cyr, G. K. Hamer, A. S. Perlin, H. J. Koch, and R. S. Stuart, Carbohydr. Res., 59 (1977)c7-cll.

36

KLAUS BOCK AND CHRISTIAN PEDERSEN

coupling with neighboring carbon atoms, and their I3C-signals may therefore be readily identified.4"s"-"6 Whereas introduction of I3C or deuterium onto carbon atoms requires more-or-less laborious syntheses, 0-deuteration of hydroxyl groups or N-deuteration of amino groups is readily achieved by exchange of protons by deuterons with D,O. In the deuterated carbohydrates thus obtained, only small isotopic-shifts are observed in the '"C-n.m.r. spectra; however, when measured under appropriate conditions, these shifts are very useful for the assignment of 13C-signa1s.50,S7-6i

3. Correlation with Proton Spectra An assignment technique that requires no chemical modification of

the compound studied involves the use of proton-coupled, or off-resonance-decoupled, 'W-n.m.r. spectra. A proton-coupled spectrum, usually measured by the "gated decoupling" technique,'"'' contains information about the I3C-'H coupling-constants, but, as these are large, the 13Cmultiplets may overlap. In an off-resonance-decoupled ~pectrurn,~"-" the C-H couplings are lessened and, hence, overlap of signals is less likely. Both types of spectra show unambiguously how many protons are attached to each I3C nucleus. In addition to the large, one-bond, I3C-H couplings,2,62-65 fully proton-coupled spectra having good resolution will show two- or three-bond, 13C-H COUplings that may be useful for the assignment of signals to certain car(52) K. Bock and C. Pedersen, Acta Chem. Scand., Ser. B , 29 (1975)682-686. (53) V. Wray, J. Chem. Soc., Perkin Trans. 2, (1976) 1598-1605. (54) G . Adiwadjaja, B. Meyer, H. Paulsen, and J. Thiem, Tetrahedron, 35 (1979)3733%.

(55)J. V. O'Conner, H. A. Nunez, and R. Barker, Biochemistry, 18 (1979) 500-507. (56) T. A. W. Koerner, Jr., R. J. Voll, L. W. Cary, and E. S. Younathan, Biochemistry, 19 (1980) 2795-2801. (57) D. Y. Gagnaire and M. Vincendon,J. Chem. Soc., Chem. Commun., (1977) 509510. (58) D. Y. Gagnaire, D. Mancier, and M. Vincendon,Org. Magn. Reson., 11 (1978)344349. (59) P. E. Pfeffer, K. M. Valentine, and F. W. Parrish,J. Am. Chem. SOC., 101 (1979) 1265- 1274. (60) P. E. Pfeffer, F. W. Panish, and J. Unruh, Carbohydr. Res., 84 (1980) 13-23. (61) K. Bock, D. Y. Gagnaire, and M. R. Vignon, C . R . Acad. Sci., Ses. C , 289 (1979) 345-348. (62) K. Bock and C. Pedersen,]. Chem. Soc., Perkin Trans. 2, (1974)293-297. (63)K. Bock and C. Pedersen, Acta Chem. Scand., Ser. B , 29 (1975) 258-264. (64) J. A. Schwarcz and A. S. Perlin, Can. J . Chem., 50 (1972)3667-3676. (65) H. Paulsen, V. Sinnwell, and W. Greve, Carbohydr. Res., 49 (1976)27-35.

13C-N.M.R.SPECTROSCOPY OF MONOSACCHARIDES

37

bon atoms. Two- and three-bond, l3C-H couplings have been discussed in several a r t i ~ l e s , 4 O . ~and * ~ ~in- ~a ~review.72 The most straightforward way of assigning 13Csignals is through selective, proton decoupling. By this technique, one proton is irradiated at its resonance frequency with a low-power, single frequency, causing the signal of the carbon atom to which it is bound to appear as a singlet in the l3C-n.m.r. spectrum, whereas all of the other carbon atoms are coupled to protons, and hence give off-resonance, decoupled multiplets. This is clearly illustrated in Fig. 3. This technique, however, requires a fully assigned, 'H-n.m.r. spectrum having well-dispersed proton-signals (separated by at least 10 Hz), and is therefore best conducted with high-field instruments and for acylated carbohydrates, which afford better-separated proton-signals. With modem, pulsed Fourier-transform instruments, series of selective proton-decouplings may be performed automatically, provided that the correct, decoupling frequencies have been measured.15 Correlation between proton and carbon chemical-shifts and coupling-constants may also be obtained through heteronuclear, twodimensional, n.m.r. ex~eriments.733~~ 4. Relaxation rate^^,^"^

Carbon-13 relaxation-rates of monosaccharides are dominated by dipolar-relaxation mechanisms,18,22 and primarily give information ahor:t molecular m ~ t i o n , in ~ ~addition , ~ ~ to the somewhat trivial distinction between C, CH, CH, , and CH, groups. However, by measuring spectra with a suitable pulse-sequence, the differences in spin-lattice relaxation-rates can be used for the assignment of signals from overlapping C H and CH, groups.77

(66)R. U. Lemieux, T. L. Nagabhushan, and B. Paul, Can.]. Chem., 50 (1972)773-776. (67) A. S. Perlin, N. Cyr, R. G. S. Ritchie, and A. Parfondry, Carbohydr. Res., 37 (1974) cl-c4. (68)J. A. Schwarcz, N. Cyr, and A. S. Perlin, Can.]. Chem., 53 (1975) 1872-1875. (69) R. G. S. Ritchie, N. Cyr, and A. S. Perlin, Can.J . Chem., 54 (1976)2301-2309. (70) N. Cyr and A. S. Perlin, Can.J . Chem., 57 (1979)2504-2511. (71) R. U. Lemieux, Ann. N . Y. Acad. Sci., (1973) 915-934. (72) P. E. Hansen, Prog. Nucl. Magn. Reson. Spectrosc., 14 (1981) 175-296. (73) R. Freeman and G. A. Morris,]. Chem. SOC., Chem. Commun., (1978) 684-686. (74) L. D. Hall and G. A. Moms, Carbohydr. Res., 82 (1980) 175-184. (75) M. F. Czarniecki and E. R. Thomton,]. Am. Chem. Soc., 99 (1977)8279-8282. (76) J. M. Berry, L. D. Hall, and K. F. Wong, Carbohydr. Res., 56 (1977)C16-~20. Lallemand,]. Chem. SOC., Chem. Commun., (1981) 150-152; (77) C. LeCoco and J.-Y. D. M. DoddreIl and D. T. Pegg,]. Am. Chem. SOC., 102 (1980)6388-6390.

86

KLAUS BOCK AND CHRISTIAN PEDERSEN

38

I;,

H-1

~ - n.4 3 n-2

A

14.ti 65

OMe

90 MHz

i

I

C

D

/I

t.

.

I I I

FIG.3.--90-MWz, 'H-N.m.r. Spectrum in Deuteriochloroform (0.1M ) and 22.63-MHz, *T-N.m.r. Spectra of Methyl TetraO-acetyl-a-D-glucopyranosidein Deuteriochloroform (1 M). [A. The W M H z , 'H-n.m.r. spectrum, with the assignment ofthe signals given above the resonances. 8.The 22.63-MHz, 13C-n.m.r., proton-noise-decoupled, lacn.m.r. spectrum, with the assignment of the signals indicated below the resonances. C, D, E, and F show the results of a series of selective, proton decouplings, applied at the frequencies indicated in A, at positions C to F.]

5. Paramagnetic Reagents It is well known from 'H-n.m.r. spectroscopy that the addition of soluble, paramagnetic reagents (notably europium, gadolinium, and cupric complexes) causes large changes in chemical shifts and line widths. Similarly induced changes are observed in 13C-n.m.r.spectra, and their use for assignment of carbon signals have been discussed in ,'~ shift-reagents have general terms by several a u t h o r ~ . ' ~Paramagnetic

13C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

39

also been applied in the study of I3C-n.rn.r. spectra of carbohydrate~.~~-~~

6. Protonation Shifts The chemical shifts observed in the I3C-n.m.r. spectra of aminodeoxy sugars are strongly dependent on the pH of the sample solution, and the spectra of such compounds should, therefore, be measured with control of the pH. Comparison of I3C-n.m.r. spectra, measured at low or high pH, that is, for compounds having protonated or free amino groups, may be used for the assignment of carbons a and p to the amino groups.11J6,81,82 Similar, but smaller, effects are-observed in the spectra of other ionizable compounds, such as aldonic or uronic acid^.^^,^^

IV . APPLICATIONS

1. Identity of Monosaccharides The most important, practical application of 13C-n.m.r.spectroscopy is probably the simple characterization and identification of organic compounds. Because of the simplicity of proton-decoupled carbon spectra, and the sensitivity of carbon-13 chemical-shifts towards structural changes, carbon spectra are extremely well suited for this purpose (see, for example, Ref. a), and it is for this reason that the emphasis of the present article has been placed on presenting chemical-shift data of monosaccharides and their derivatives. Such data are also important for structural studies of oligo- and poly-saccharides? and for the investigation of such mixtures as those arising from r n u t a r o t a t i ~ n ~(see ~ - ~Section ~ II,4) or from other reactionsa3* (78) B. Caw, G. Gatti, N. Cyr, and A. S. Perlin, Carbohydr. Res., 41 (1975) d - C 8 . (79) S. Hanessian and G . Patil, Tetrahedron Lett., (1978) 1031-1034. (80) P. McArdle, J. 0.Wood, E. E. Lee, and M. J. Conneely, Carbohydr. Res., 69 (1979) 39-46. (81) K. F. Koch, J. A. Rhoades, E. W. Hagaman, and E. Wenkert,J.Am. Chem. SOC., 96 (1974) 3300-3305. (82) R. U. Lemieux, K. Bock, L. T. J. Delbaere, S. Koto, and V. S. Rao, Can.].Chem., 58 (1980) 631-653. (83) K . Bock and C. Pedersen, unpublished results. (84) R. C. Beier, B. P. Mundy, and G. A. Strobel, Can.J . Chem., 58 (1980) 2800-2804. (85) W. Voelter, E. Breitmaier, and G. Jung,Angew. Chem., 83 (1971) 1011-1012. (86) S. J. Angyal, G. S. Bethell, D. E. Cowley, and V. A. Pickles, Aust. J . Chem., 29 (1976) 1239-1247. (87) C. F. Midelfort, R. K. Gupta, and H. P. Meloche,]. Biol. Chem., 252 (1977) 34863492.

KLAUS BOCK AND CHRISTIAN PEDERSEN

40

When studying the course of reactions, %-n.m.r. spectra may be used to monitor the progress of a reaction,% or to detect intermediates. The latter was achieved in a study of the Kiliani-Fischer reaction?* 2. Structure Determination The sensitivity of carbon-13 chemical-shifts towards changes in substitution renders W-n.m.r. spectroscopy very useful for the determination of the structures of unknown compounds. This is clearly seen from the large changes in carbon-13 chemical-shifts encountered when deoxy, aminodeoxy, deoxyhalogeno, thio, or unsaturated h n c tions are introduced into monosaccharides (see Tables X-XII, and XIV) and it reflects the influence of electronegativity and polarizability on the chemical shifts. It may be noted that whereas a chlorine and bromine atom situated on C-1 of aldose derivatives causes upfield shifts of 2 and 5 p.p.m., respectively (see Table VI), a much larger effect is observed when substitution takes place at other carbon atoms of pyranoses or furanoses. Thus, replacement of oxygen by chlorine at C 4 or C-6 of galactopyranose causes upfield shifts of 7 and 19 p.p.m., respectively; the corresponding shifts for bromine are -20 and -28 p.p.m., respectively. Similar, carbon-13 chemical-shifts are found in deoxy sugars; but deoxy and deoxyhalogeno carbon atoms can be readily differentiated through the multiplicities of their protoncoupled, W-n.m .r . spectra. A change of ring size is also accompanied by a change of chemical shifts; thus, furanoses and other five-membered rings have chemical shifts downfield from those of the configurationally related, six-membered (see Tables 1-111). Similar relationships are found for five- and six-membered lactonesSR(see Table XX). Acyclic derivatives show chemical shifts at higher field than those of the corresponding cyclic compounds (see Tables XV and XVI). In five-menibered, isopropylidene derivatives that are monocyclic, or fused to a pyranoid ring, the chemical shifts for the quaternary carbon atoms are 108.5111.4 p.p.m., whereas values of 111.4-115.7 p.p.m. are found when they are fused to a furanose ring. Six- and seven-membered, isopropylidene derivatives show the quaternary carbon atoms at 97.1-99.5 and 101- 102 p.p.m., Similar data have been educed from 13C-n.m.r.spectra of benzylidene derivativesw The chemical shifts of the methyl groups of isopropylidene derivatives may also give information concerning the ring size.89The two carbon atoms engaged in

-

-

(88) R. M. Blazer and T. W. Whaley,J. Am. Chem. Soc., 102 (1980)5082-5085. (89) J. G. Buchanan, M. E. Chac6n-Fuertes, A. R. Edgar, S. J. Moorehouse, D. I. Rawson, and R. H. Wightman, Tetrahedron k t t . , (1980)1793-1796. (90)T. B. Grindley and V. Culasekharam, Carbohydc Res., 74 (1979)7-30.

W-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

41

an epoxide give carbon signals at higher field than those of five- and six-membered rings (see Table XIII). Furthermore, the signals of epoxide carbon atoms may be assigned from their large (180-190 Hz), one-bond, C-H coupling-~onstants.~~ Although many pairs of anomers give quite different signals for the anomeric carbon atoms, it has not been found possible to discover a general relationship between the anomeric configuration and the chemical shifts. However, for those furanoses in which the substituents at C-1 and C-2 are trans-oriented, the signals of the anomeric carbon atoms are always found at lower field than in those of the corresponding cis isomers.92For pyranoses, this relationship does not hold, but the anomeric structure of pyranoses can always be determined2s4. 62,83,64,74 from the one-bond coupling-constants, namely, JC--I,H--l. The corresponding coupling-constants of furanoses cannot be used to determine anomeric structures. Alkylation of oxygen leads to a rather large, downfield shift of the a-carbon atom (see Section II1,l and Table VIII), as discussed in reviews3s5Jand in several papers.36,37,93-97 Similarly, formation of cyclic acetals in downfield shifts of the furanose or pyranose car(91) K. S. Kim, D. M. Vyas, and W. A. Szarek, Carbohydr. Res., 72 (1979) 25-33. (92)R. G. S. Ritchie, N. Cyr, B. Korsch, H. J. Koch, and A. S. Perlin, Can.J . Chem., 53 (1975) 1424-1433. (93)P. A. J. Gorin and M. Mazurek, Carbohydr. Res., 48 (1976)171-186. (94) J. Haverkamp, J. P. C. M. van Dongen, and J. F. G. Vliegenthart, Tetrahedron, 29 (1973)3431-3439. (95) J. Haverkamp, J. P. C. M. van Dongen, and J. F. G. Vliegenthart, Carbohydr. Res., 33 (1974)319-327. (96) J. Haverkamp, M. J. A. De Bie, and J. F. G. Vliegenthart, Carbohydr. Res., 39 (1975)201-211. (97) R. Usui, N. Yamaoka, K. Matsuda, K. Tuzimura, H. Sugiyama, and S. Seto, J . Chem. Soc.,Perkin Trans. 1, (1973) 2425-2432. (98) W. Voelter, E. Breitmaier, E. B. Rathbone, and A. M. Stephen, Tetrahedron, 29 (1973)3845-3848. (99) W. A. Szarek, A. Zamojski, A. R. Gibson, D. M. Vyas, and J. K. N. Jones, Can. J . C h m . , 54 (1976)3783-3793. (100) A. S. Shashkov, A. I. Shienok, M. Islomov, A. F. Sviridov, and 0. S. Chizhov, Bioorg. Khim., 3 (1977)1021-1027. (101) A. Lip&, P. Nhhsi, A. Neszmklyi, and H. Wagner, Carbohydr. Res., 86 (1980) 133-136. (102) E. Conway, R. D. Guthrie, S. D. Gero, G. Lukacs, and A.-M. Sepulchre,J. Chem. Soc., Perkin Trans. 2, (1974) 542-546. (103) A. Lip&, P. Fugedi, P. Nhnhsi, and A. NeszmBlyi, Tetrahedron, 35 (1979)11111119. (104) A. NeszmBlyi. A. LipKk, and P. Nbhsi, Carbohydr. Res., 58 (1977) ~ 7 - m . (105) P. J. Garegg, B. Lindberg, and I. Kvamstrom, Carbohydr. Res., 77 (1979)71-78. (106) P. J. Garegg, P.-E. Jansson, B. Lindberg, F. Lindh, J. Lonngren, I. Kvarnstrom, and W. Nimmich, Carbohydr. Res., 78 (1980) 127-132.

42

KLAUS BOCK AND CHRISTIAN PEDERSEN

bon atoms (see Table IX).Introduction of an acyl group onto oxygen causes a smaller (1.5-4p.p.m.), downfield shift of the a-carbon atom than that of an alkyl group. However, as 0-acylation causes the signal of the @-carbonatom to shift upfield (1-5 p.p.m.), the cumulative effect of several acyl groups may be difficult to predict. Acylation effects on simple alcohols have been d i ~ c u s s e d , ~and ~ *systematic '~~ studies of I3C-n.m.r. spectra of carbohydrates selectively 0-acylated in different positions have been reported by several a ~ t h o r s . ' ~ ~ - ~ ~ ~ Just as introduction of a magnetic nucleus into a known position may help in assigning the signals in a 13C-n.m.r.spectrum (see Section III,Z), the placement of an isotope in an unknown position may be determined from isotope shifts or from, for e ~ a m p l e , ' ~ C - *coupling ~C constants, or both. In most cases, the stereochemistry of the quaternary carbon atom in branched-chain carbohydrates cannot be elucidated from 'H-n.m.r. spectra, but 13C-chemical shifts, or long-range, I3C-lH coupling-constants, may often yield valuable inf~rmation."*-"~Likewise, the stereochemistry of acetal carbon atoms of benzylidene derivatives,103* l w and of acetals derived from pyruvic acid,105*10g may be determined from I3C-chemical shifts. Finally, from the 13C-chemicalshifts of glycopyranosides, it is possible to obtain information about the stereochemistry of chiral aglycons. I?'

(107) Y. Terui, K. Tori, and N. Tsuji, Tetrahedron Lett., (1976) 621-622. (108) M. R. Vignon and P. J. A. Vottero, Tetrahedron Lett., (1976) 2445-2448. (109) M. R. Vignon and P. J. A. Vottero, Carbohydr. Res., 53 (1977) 197-207. (110) K. Yoshimoto, Y. Itatani, and Y. Tsuda, Chem. Pharm. Bull., 28 (1980)2065-2076. (111) K. Yoshimoto, Y. Itatani, K. Shibata, and Y. Tsuda, Chem. Pharm. Bull., 28 (1980) 208-219. (112) H. Komura, A. Matsuno, Y. Ishido, K. Kushida, and K. Aoki, Carbohydr. Res., 65 (1978) 271-277. (113) P. E. Pfeffer, K. M. Valentine, B. G. Moyer, and D. L. Gustine, Carbohydr. Res., 73 (1979) 1-8. (114) P. M. Collins and V. R. N. Munasinghe, Carbohydr. Res., 62 (1978) 19-26. (115) J.-C. Depezay, A. Dukault, and M. Saniere, Carbohydr.Res., 83 (1980)273-286. (116) M. MiljkoviC, M. GligorijeviC, T. Satoh, D. GliSin, and R. G. Pitcher, J . Org. Chern., 39 (1974) 3847-3850. (117) K. Sato, M. Matsuzawa, K. Ajisaka, and J. Yoshimura, Bull. Chem. SOC. Jpn., 53 (1980) 189-191. (118) A.-M. Sepulchre, B. Septe, G. Lukacs, S. D. Gero, W. Voelter, and E. Breitmaier, Tetrabdron, 30 (1974) 905-915. (119) S. Seo, Y. Tomita, K.Ton, and J. Yoshimura,J. Am. Chem. Soc., 100 (1978)33313339.

W-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

43

3. Conformational Analysis Relatively little use has yet been made of I3C-n.m.r. spectroscopy in conformational analysis. The most extensive studies have been conducted on furanoses, the conformational equilibria of which may be studied by consideration both of carbon-13 chemical-shifts and of twoand three-bond, C-H coupling-constants.70The conformation of pentopyranoses has been investigated through one-bond, C-H couplingconstantsF3 Other applications of two- and three-bond, C-H couplings are described in Refs. 120 and 121. An experimental method for the determination of long-range, C -H coupling-constants has been describedIe2;this technique can conveniently be used with modem, n.m.r. instruments having full computer-control of the decoupling channels.

4. Relaxation Rates Carbon-13, spin-lattice relaxation-rates may be readily measured with pulsed, Fourier-transform instruments, and they primarily provide information about the molecular motion in s o l ~ t i o n . ~ , ~ ~ ~ ~ , Carbon-13 relaxation-rates have mostly been used to obtain structural information on polysa~charides.~

5. Complexation Carbon-13 chemical-shifts have been used to study the interaction of monosaccharides with such complexing agents as b o r a t e ~ ' ~ *and J~~ calcium ion^.'^^,'^^ Paramagnetic complexing-agents are mentioned in Section III,5.

(120) D. Y. Gagnaire, R. Nardin, F. R. Taravel, and M. R. Vignon, Nouo. J . Chim., 1 (1977) 423-430. (121) R. U. Lemieux and S. Koto, Tetrahedron, 30 (1974) 1933-1944. (122) K. Bock and C. Pedersen,J. Magn. Reson., 25 (1977) 227-230. (123) A. Neszmelyi, K. Ton, and G . Lukacs,J. Chem. SOC.,Chem. Commun., (1977)613 -614. (124) P. A. J. Gorin and M. Mazurek, Carbohydr. Res., 27 (1973) 325-339; Can. J . Chem., 51 (1973)3277-3286. (125) W. Voelter, C. Biirvenich, and E. Breitmaier,Angew. Chem., 84 (1972) 589-590. (126) M. F. Czamiecki and E. R. Thornton, Biochem. Biophys. Res. Commun., 74 (1977) 553-558. (127) L. W. Jaques, J. B. Macaskill, andW. Weltner, ]I.,]. Chem. Phys., 83 (1979) 14121421.

44

KLAUS BOCK AND CHRISTIAN PEDERSEN

V. TABLES* In the following Tables are presented I3C-n.m.r. chemical-shifts of a variety of monosaccharides and their derivatives. As far as possible, complete sets of shift values are given for all of the pentoses, hexoses, methyl glycosides, alditols, and aldonic acids. In addition, the chemical shifts of a selection of the most common types of derivatives of monosaccharides are given. For many compounds, especially free sugars or methyl glycosides, carbon-13 chemical-shifts have been published several times for the same compound. In such cases, references are not necessarily given to all relevant articles, but primarily to those that give a complete assignment. When more than one reference is given to the same compound, the chemical-shift data have been taken from the reference marked with an asterisk in the Table. The majority of the spectra given in the Tables have been unambiguously assigned. Those which are not assigned (indicated with a superscript a ) are included because each constitutes a valuable identification of the compound. For many carbohydrate derivatives, only a few examples of spectra are given. Those references that contain a considerable number of additional data on similar derivatives are marked, or mentioned in footnotes to the Tables. The chemical shifts given in the Tables are, unless otherwise stated, from spectra recorded for solutions in D,O or in deuteriochloroform. Carbon-13 chemical-shifts published for a particular compound may differ considerably (by 1 to 2 p.p.m.), depending on the concentration, the temperature, and the reference standard used. Apart from changes caused by temperature,IYthe variations are generally the same for all of the carbon atoms in a compound, causing a parallel shift of signals. Because of these variations, the values in the Tables have been rounded off to one figure after the decimal point.

* The authors are grateful to Professor S . J. Angyal for a number of suggestions regarding, and corrections to, the data in the Tables. Data on heptoses, heptuloses, and heptitols will be published by S. J. Angyal and coworkers.

I3C-N.M.R.SPECTROSCOPY OF MONOSACCHARIDES

45

TABLE I 13C-N.m.r.Data for Aldoses Compound

C-1

D-Hexopyranoses a-All 93.7 P943 U-Alt 94.7 P92.6 a-Gal 93.2 P97.3 (Y-GlC 92.9 P96.7 CY-GUl 93.6 P94.6 a-Ido 93.2 P93.9 a-Man 95.0 P94.6 a-Tal 95.5 P95.0 D-Pentopyranoses a-Ara 97.6 P93.4 a-Lyx 94.9 P95.0 a-Rib 94.3 P94.7 a-Xyl 93.1 P97.5 D-Hexofuranoses a-All 96.8 P101.6 a-Alt 102.2 P96.2 ff-Gal 95.8 P101.8 P-ClC 103.8

c-2

c-3

C-4

c-5

C-6

References

67.9 72.2 71.2 71.6 69.4 72.9 72.5 75.1 65.5 69.9 73.6" 71.1" 71.7 72.3 71.7' 72.5"

72.0 66.9 72.0 67.7 71.1 66.0 71.3 65.2 70.2 70.3 73.8 69.7 73.8 70.6 76.7 70.6 71.6 70.2 72.0 70.2 72.7" 70.6" 68.8c 70.6' 71.3 68.0 74.1 67.8 70.6c 66.0 69.6" 69.4

37,83,*128" 67.7 61.6 62.1 36,37,63* 74.4 61.6 129 72 .O 129 75.0 62.5 36,37,59,*98 71.4 62.2 36,37,59,*98 76.0 62.0 72.3 61.6 29,36,37,40.46,59,*85,130" 29,36,37,40,46,59,*1306 76.8 61.7 67.2 61.7 83 83 74.6 61.8 73.6" 59.4 83 75.6" 62.1 83 73.4 62.1 28,36,37,40,46,*130,b131 77.2 62.1 28,36,37,46,*1306 72 .O 62.4 59,*131 59,*131 76.5 62.2

72.9 69.5 71.0 70.9 70.8 71.8 72.5 75.1

73.5 69.5 71.4 73.5 70.1 69.7 73.9 76.8

69.6 69.5 68.4 67.4 68.1 68.2 70.4 70.2

67.2 63.4 63.9 65.0 63.8 63.8 61.9 66.1

72.4 76.1 82.4 77.5 77.1 82.2 81.8"

d

73.3 76.9 76.0 75.1 76.6

84.3 83.0 84.3 82.1 81.6 82.8 82.1'

70.2 71.7 72.5 73.4

d

36,37,46,59,*131,132* 36,37,46,59,*131,132* 36,37,83* 36,37,83* 36,48,83* 36,37,48,63,*131 36,37,46,59,*131,133 36,37,46,59,*131,133 83 83 129 129

71.5

63.1 63.3 63.3 63.3 63.3 63.6

d

d

29

d

83 83

(continued)

(128) W. A. Szarek, D. M. Vyas, S. D. Gero, and G. Lukacs, Can. J . Chem., 52 (1974) 3394 -3400. (129) K. Bock and M. Beck Sommer, Acta Chem. Scand., Ser. B , 34 (1980) 389. (130) R. Kasai, M. Okihara, J. Asakawa, K. Mizutani, and 0. Tanaka, Tetrahedron, 35 (1979) 1427- 1432. (131) W. Voelter and E. Breitmaier, Org. Magn. Reson., 5 (1973) 311-319. (132) K. Mizutani, R. Kasai, and 0. Tanaka, Carbohydr. Res., 87 (1980) 19-26. (133) J.-P. Utille and P. J. A. Vottero, Carbohydr. Res., 85 (1980) 289-297.

KLAUS BOCK A N D CHRISTIAN PEDERSEN

46

TABLEI (continued) Compound ~~

C-2

C-1 ~

C-3

C-4

C-5

C-6

References

~

97.3 101.4 a-ldo 102.5 B96.3 a-Tal 101.8 P97.3 DPentofuranoses a-Ara 101.9 896.0 a-Lyx 101.5 &-Rib 97.1 8101.7 m-Erythrose a-Furanose 96.8 P-Furanose 102.4 Hydrate 90.8 DL-Threose a-Furanose 103.4 P-Furanose 97.9 Hydrate 91.1 DL-Glyceraldehyde Hydrate 91.2 Glycolaldehyde Hydrate 91.2 Formaldehyde Hydrate 83.3 CV-GUI

P-

d

d

78.1 78.6 77 .O 76.1 71.6

d

83 83

75.6‘ 75.9 72.7 72.0

80.4 80.3 82.2 81.6 82.7 83.3

62.6 63.2 70.3‘ 63.4 71.7c 63.4 71.6 63.7 63.8

82.3 77.1 77.8 71.7 76 .O

76.5 75.1 71.9 70.8 71.2

83.8 82.2 80.7 83.8 83.3

62.0 62.0 61.9 62.1 63.3

72.4 77.7 74.9

70.6 71.7 73.0

72.9 72.4 64 .O

43 43 43

82.0 77.5 74.6

76.4 76.2 72.2

74.3 71.8 64.4

43 43 43

75.5

63.4

83 83 59,*131 59.*131 83 83 83 48,83* 48,83*

43 43

66.0

43

In dimethyl sulfoxide-d,. Not resolved.

Assignment may have to be reversed.

In pyridine-d,.

TABLE I1

W-N.m.r. Data for Methyl Aldosides ~~

~

~~

~

~

C-2

C-3

C-4

C-5

D-Hexopyranosides a-All 100.0 68.3 P101.9 72.2 a-Alt 101.1 70.0 P100.4 70.7 a-Gal 100.1 69.2 P104.5 71.7 a-Glc 100.0 72.2

72.1 71.4 70.0 70.2 70.5 73.8 74.1

68.0 68.0 64.8 65.6 70.2 69.7 70.6

67.3 74.8 70.0 75.6 71.6 76.0 72.5

Compound

P-

C-1

104.0 74.1 76.8

70.6 76.8

C-6 @Me

References

83 83 55.4 36 83 57.7 36,59,*131 56.0 36,59,*131 58.1 55.9 36,37,40,46,49,59,*60,99,” 130,”134 61.8 58.1 36,37,40,46,49,59,*60,99,” 130,b134

61.7 62.2 61.3 61.7 62.2 62.0 61.6

56.3

58.0

I3C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

47

TABLEI1 (continued) Compound

C-1

100.4 102.6 a-Ido 101.5 101.9 a-Man p101.3 102.2 a-Tal D-Pentopyranosides 105.1 a-Ara p101.0 (Y-LYX 102.0 100.4 a-Rib 103.1 6100.6 a-Xyl p105.1 D-Hexofuranosides a-All 103.8 p109.0 a-Gal 103.8 p109.9 a-Glc 104.0 p110.0 109.7 a-Man p103.6 D-Pentofuranosides 109.2 a-Ara P103.1 109.2 a-Lyx P103.3 103.1 a-Rib 108.0 p103.0 a-Xyl p109.7 D-Tetrofuranosides a-Ery 103.6 p109.6 a-Thr 109.4 p103.8 a-Gul

p-

C-2

C-3

C-4

C-5

65.5 69.1 70.9 71.2 70.6 70.7

71.4 72.3 71.8 71.8 73.3 66.2

70.4 70.5 70.3 68.0 67.1 70.3

67.3 74.9 70.8 73.7 76.6 72.1

71.8 69.4 70.4 69.2 71.0 72.3 74.0

73.4 69.9 71.6 70.4 68.6 74.3 76.9

69.4 70.0 67.7 67.4 68.6 70.4 70.4

67.3 63.8 63.3 60.8 63.9 62.0 66.3

72.3 75.6 78.2 81.3 77.7 80.6 77.9 73.1

69.9 72.7 76.2 78.4 76.6 75.8 72.5 712d

85.9 83.4 83.1 84.7 78.8 82.3 80.5 80.7

72.7 73.8 74.5 71.7 70.7 70.7 70.6 71.W

81.8 77.4 77.0 73.2 71.1 74.3 77.8 81.0

77.5 75.7 72.2 71.0 69.8 70.9 76.2 76.0

84.9 82.9 81.4 82.1 84.6 83.0 79.3 83.6

62.4 62.4 61.5 62.7 61.9 62.9 61.6 62.2

72.8 76.4 80.5 77.4

69.9 71.4 76.4 75.8

73.6 72.6 73.7 72.0

C-6 0-Me

62.0 62.1 60.2 62.1 61.4 62.3

56.3 58.1 55.8 55.9 56.9 55.6

58.1 56.3 55.9 56.7 57.0 56.0 58.3 63.5 63.9 64.1 63.6 64.2 64.7 64.5 64.4

References

135 136 36 36,46,* 13CP 36,*130" 83 46,59,*131 , 1 3 2 , " ~ 46,59,*1 3 1 , 1 3 2 , " ~ 63 63 63,*131,134 36,46,*131,134 36,46,*131,134

56.6 56.4 57.2 55.6 57.0 56.3 57.2 56.8

92 92 92

56.0 56.3 56.9 56.7 55.5 55.3 56.7 56.4

46,92,*137 46,92,*137 92 92 46,92,*137 46,92,*137 92,*138 92,*138

56.7 56.6 55.5 56.2

92 92 92 92

92 92 92 92 92

~

a In dimethyl sulfoxided,. may have to be reversed.

* In pyridined,.

Contain additional data. Assignment

(134) E. Breitmaier, W. Voelter, G. Jung, and C. Tanzer, Chem. Ber., 104 (1971) 11471154. (135) H. Naganawa, Y. Muraoka, T. Takita, and H. Umezawa, J . Antibiot., Ses. A, 30 (1977) 388-396. (136) S. Jacobsen and 0. Mols, Acta Chem. Scand., Ser. B , 35 (1981)163-168. (137) E.Breitmaier, G . Jung, and W. Voelter, Chimia, 26 (1972) 136-139. (138) P. W. K. Woo and R. D. Westland, Carbohydr. Res., 31 (1973) 27-36.

KLAUS BOCK AND CHRISTIAN PEDERSEN

48

TABLE111 'T-N.m.r. Data for Ketoses and Their Methyl Glycosides

Compound

C-1

DHexopyranoses a-Fni 65.9 B64.7 a-Psi 64 .O

B-

64.8

64.5 64.8 864.4 DHexofuranoses a-Fm 63.8 863.6 a-Psi 64.2 B63.3 a-Sor 64.3 a-Tag P63.5 D-Hexopyranosides B-FN 61.8 a-Psi 61.1 B57.7 a-Sor 61.2 &-Tag 61.0 B61.7 D-Hexofuranosides a-Fni 58.7 B60.0 a-Sor 60.7 B57.7 a-Tag 58.8 P60.3 a-Sor a-Tag

C-2

C-3

G4

C-5

C-6

99.1 98.4 992 98.5 99.0 99.1

70.9 68.4 66.4 71.2 71.4 70.7 64.6

71.3 70.5 72.6 65.9 74.8 71.8 70.7

70.0 66.7 69.8 70.3 672 70.1

64.1 58.8 65.0 62.7 63.1 61.0

105.5 102.6 104.0 106.4 102.5 105.7 103.3

82.9 76.4 71.2 75.5 77.0 77.6 71.7

77.0 75.4 71.2 71.8 76.2 71.9 71.8

82.2 81.6 83.6 83.6 78.6 80.0 80.9

61.9 63.2 62.2 63.7 61.6

101.4 100.7 102.6 100.9 102.4 101.4

69.3 67.3 69.7 72.0 69.6 65.5

70.5 72.1 65.7 74.5 71.7 71.5"

70.0 66.7 69.9 70.1 66.8 70.4a

64.7 58.9 65.4 63.0 63.4 61.1

49.3 49.1 48.7 49.2 48.5 49.3

31 31 31 31 31 31

109.1 104.7 1042 109.9 108.7 105.3

81.0 77.7 80.0 80.3 75.2 73.4

78.2 75.9 76.5 772 71.9 71.7

84.0 82.1 78.8 83.4 80.6 82.0

62.1 63.6 61.6 62.1 60.8 61.9

49.1 49.8 49.9 49.3 49.6 49.8

31 31 31 31 31 31

C-OMe

References 31 26,27,31* 27,30,31,*33 27,30,31,*33 31,*27 31,*27 31,*27 26,27,31,*32 26,27,31,*32 27,30,31,*33 27,30,3 1,*33 31,*27 31,*27 31,*27

61.9

Assignments may have to be reversed. For further data, see p. 66.

TABLEIV W-N.m.r. Data for Glycosides of Aromatic Aglycons C-2

C-3

C4

C-5

C-6

References

Phenyl D-glucopyranosides a 97.9 72.0 B 103.1 75.8 a p-NO, 100.5 74.1 B 102.7 76.0 B m-NO, 103.6 76.1 p &NO* 103.3 76.0

73.3 79.5 76.9 80.1 80.0

70.2 72.4 72.5 72.4 72.6 72.4

73.9 79.3 75.8 79.3 79.2 79.5

61.1 63.6 63.5 63.5 63.6 63.5

83 134 134 134 134 134

Compound

C-1

80.1

49

13C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

TABLEIV (continued)

Compound

C-1

C-2

Phenyl D-gdactopyranosides 104.0 73.6 P 75.5 a p-NOI 100.8 103.4 73.2 P p m-NO, 101.2 73.3 73.2 fi o-NOZ 104.1 Phenyl Dmannopyrawsides 73.4 a p-NO, 101.5

C-4

C-5

C-6

References

76.4 712 76.1 76.1 76.3

71.3 70.5 71.1 71.1 71.1

78.3 72.1 78.6 78.7 78.7

63.7 63.0 63.5 63.5

134 134 134 134

63.5

134

72.5

69.5

78.0

63.8

134

C-3

TABLEV 13C-N.m.r. Data for Peracetylated Pyranoses and Furanoses ~

Compound

C1

D-Hexopyranoses P-All 90.1 a-Alt 90.2 ff-Gal 89.5 P91.8 a-Glc 89.2 P91.8 p-Gul 89.7 a-Ido 90.4 a-Man 90.4 ff-Tal 91.4 D-Pentopyranoses a-Ara 92.2 B90.4 a-Lyx 90.7 88.7 a-Rib P90.7 a-Xyl 88.9 P91.7 D-Pentofuranoses a-Ara 99.4 P93.7 a-Lyx 98.0 P93.2 a-Rib 94.1 P98.1 a-Xyl 92.8 P98.9 a

C-2

C-3

C-4

C-5

C-6

References

68.2 68.2 67.2 67.8 69.3 70.5 67.3" 65.9 68.6 65.2"

68.2 66.4 67.2 70.6 69.9 72.8 67.1" 66.2 68.2 66.3"

65.8 66.4 66.2 66.8 68.O 68.1 67.1" 65.9 65.4 65.3"

71.2 64.4 68.5 71.5 69.9 72.8 71.1 66.2 70.5 68.8"

61.9 62.1 61.0 61.0 61.6 61.7 61.3 61.8 62.0 61.5

63 63 83 63 108 108,109* 83 63 62 63

68.2 67.3 68.2 67.1 67.1 69.2 69.3

69.9 68.7 68.2 65.6 66.0 69.2 70.8

67.3 66.9 66.6 66.5 66.0 68.8 68.1

63.8 62.9 61.9 59.3 62.5 60.5 62.5

80.6 75.4 75.0 70.5 70.0 74.1 75.3 79.4

76.9 74.8 70.6 68.5 69.8 70.5 73.8 74.3

82.4 79.7 77.0 77.7 81.6 79.2 75.4 79.9

63.1 64.5 62.4 62.8 63.3 63.6 61.6 62.3

63 63 63 63 63 63+*133* 63,*133b 139 139 139 139 139 139 139 139

Assignments may have to be reversed. * Contains additional data.

(139) B.L. Kam,J.-L. Barascut, and J.-L. Imbach, Carbohydr..Res., 69 (1979) 135-142.

.50

KLAUS BOCK AND CHRlSTlAN PEDERSEN

TABLEVI '"C-N.rn.r.Data for Tetra-0-acetyl-(benmy1)-Dglycopyranos yl Derivatives" ~~

Compound

C-1

C-2

C-3

86.1 87.3 86.5 89.5 87.1 66.8 103.5 105.7 96.3 101.1 94.3 98.8 80.1

69.7 70.3 70.4b 70.2b 72.4 69.4 69.9 70.6 70.4 70.9 70.5 71.1 65.8 70.4 70.8* 69.6b 71.W 68.76 69.4

69.8 72.2 72.P 70.3b 73.0 73.3 69.1 71.4 69.7 72.5 70.1 71.8 71.0 72.1 70.6 73.7b 70.7b 73.56 70.3

68.9 64.6b 67.6 68.5 69.1 69.3

68.2 66.6b 67.6 70.2 68.8 70.4 69.3 68.2 67.4 66.0 69.1 71.0

C-4

C-5

C-6

Me

-

References

~ g l u c oderivatives a-Azide

Pa-Bromide a-Chloride

P@-Cyanide a-Fluoride

Pa-Methoxy

Pa-Phenoxy

Pa-Phenylamino

P-

84 .O

81.8 83.2 a-Methylthio 83.0 P82.3 a-Methoxy, benzoate 96.8 Methyl mglycopyranosides ,&All 99.3 a-Alt 98.2 a-Gal 96.5 P101.5 a-Man 98.1 a-Ara 101.9 P97.6 a-Lyx 98.4 a-Rib 97.5 a-Ethylthio

P-

Pa-Xyl

P-

68.4

69.3 67.5 99.4 68.3 96.4 70.5 101.0 70.2

70.1 73.6 70.W 68.8b 74.9 77.3 69.6 71.5 66.8 71.4 68.1 72.5 72.1 72.8 67.6b 75.6b 68.96 67.76 68.v 75.5b 67.5 71.8 68.1

67.6 67.W 66.8b 67.2 67.8 67.1 67.0 68.2 68.1 68.4 68.2 68.5 68.7 68.7 68.2b

66.1 64.1b 67.0 66.8 65.8 67.9 67.2 66.6 66.1 66.9 68.8 68.3

70.0 68.96 65.7 70.6 68.0 63.2 60.3 59.4 57.9 61.1 57.7 61.3

61.7 61.4 60.8 60.4 61.2 61.8 61.0 61.3 61.5 61.6 61.7 61.8 61.7 62.0 62.0 61.9 62.1 61.8 62.9 62.1 62.2 61.2 61.0 62.1

114.5

55.6 56.6

12.4 55.4

140 62 62 62 62 141 52,62* 52,62* 62,63,*142 62,63,*142 83 62 62 62 143 62 143 62 62

56.0 144 55.0 62 54.8 62,63,*142 56.6 62,*142 54.9 62,63* 63,*142 56.6 55.4 63,*142 54.9 63 56.2 63 55.7 63 54.7 63,*133,142 55.8 63,*133,142

Additional data for related compounds are given in Refs. 145-148. may have to be reversed.

Assignments

(140) T. Takeda, Y. Sugiura, Y. Ogihara, and S. Shibata, Can.1.Chem., 58 (1980)26002603. (141) B. Coxon, Ann. N . Y. Acad. Sci., (1973) 952-970. (142) A. 1. Kalinovskii and E. V. Evtushenko, Khim. Prir. Soedin., 1(1979) 6-8. (143) B. S. Petersen, Ph.D. Thesis, Danrnarks Tekniske Haiskole, Lynehy, 1978. (1M) K. Bock, S. R. Jensen, B. J. Nielsen, and V. Nom, Phytochemistry, 17 (1978) 753757. (145) H. Pauisen, A. Richter, V. Sinnwell, and W. Stenzel, Carbohydr. Res., 64 (1978) 339-364.

i3C-N.M.R. SPECTROSCOPY O F MONOSACCHARIDES

51

TABLEVII 13C-N.m.r.Data for Anhydropyranose Derivatives" Compound

C-1

C-2

C-3 C-4

C-5

C-6 0 - M e C-7

1,6Anhydro-fl-~-hexopyranoses All 101.5 70.2 63.5 70.1 76.8 65.4 101.9 72.9 69.9 70.3 77.6 66.0 Alt Gal 101.3 71.9 70.8 64.9 74.9 64.1 Glc 102.1 70.9 73.3 71.6 76.9 65.8 Gul 101.7 70.5 70.5 69.9 74.9 63.8 101.9 74.7 74.7 71.4 75.8 65.4 Ido Man 101.9 66.6 70.9 72.2 76.4 65.3 1022 69.1 69.2 67.1 74.8 65.1 Tal Per-0-acetylated 1,6anhydr0-/3-~-hexopyranoses 99.0 68.0 62.4 67.7 74.0 64.8 All Alt 99.2 71.8 67.0 69.2 74.7 65.6 Gal 98.7 70.9 67.3 64.6 71.9 64.3 99.5 70.1 69.9 71.0 74.0 65.5 Glc Gul 98.9 68.9 66.7 68.6 71.8 63.9 98.7 72.3 70.1b 70.W 73.5 65.2 Id0 Man 99.2 67.0 67.6 71.8 73.8 65.2 99.0 68.6 66.4 66.0 72.1 65.6 Tal Methyl 3,6anhydro-~hexopyranosides gal 98.6 69.8 77.7 70.5 81.5 69.5 p103.4 72.7 78.4 70.5 81.2 70.9 a-Glc 99.5 71.8 72.0 70.4 76.4 69.8 p104.1 72.5 72.8 71.8 75.3 70.2 2,7-Anhydro-8-n-heptulopyranoses 60.4 107.9 72.8 70.7 70.7 78.3 Alt Gal 61.1 107.1 71.7 71.7 64.9 76.3 Glc 61.4 107.2 71.W 74.4 70.6b 78.3 60.8 107.9 70.4 70.2b 69.9 76.1 Gul Ido 60.5 108.1 74.8 75.4 71.7 76.6 Man 60.9 107.7 66.5 71.3 72.7 78.6 ~

~

References

65,69* 65,69* 65,69* 65,69,*124,149,150 65,69* 65,69* 65,69* 65.69; 69 69 69 69 69 69 69 69 83,*151 83,*151 124 124

58.0 56.2 58.5 56.5 67.0 65.2 66.7 65.1 66.5 66.5 ~~~~

69 69 69 69 69 69

~

Additional data for related compounds are given in Refs. 152 and 153. Assignments may have to be reversed. a

(146) V. Pozsgay and A. Neszmblyi, Carbohydr. Res., 80 (1980) 196-202. (147) B. L. Kam and N. J. Oppenheimer, Carbohydr. Res., 77 (1979)275-280. (148) C. L a t e , A. M.N. Phuoc Du, F. Winternitz, R. Wylde, and F. Pratviel-Sosa, Carhohydr. Res., 67 (1978) 105-115. (149) Y. Halpern, R. Riffer, and A. Broido,J. Org. Chem., 38 (1973) 204-209. (150) N. Gullyev, A. Ya. Shmyrina, A. F. Sviridov, A. S. Shashkov, and 0. S. Chizhov, Bioorg. Khim., 3 (1977) 50-54. (151) A. S. Shashkov, A. I. Usov, and S. V. Yarotskii, Bioorg. Khim., 3 (1977) 46-49. (152) C. Subero, L. Fillol, and M. Marth-Lomas, Carbohydr. Res., 86 (1980)27-32. (153)T. Trnka, M. cerng, A. Ya. Shmyrina, A. S. Shashkov, A. F. Sviridov, and 0.S. Chizhov, Carbohydr. Res., 76 (1979) 39-44.

KLAUS BOCK AND CHRISTIAN PEDERSEN

52

TABLEVIII

'W-N.m.r. Data'' for 0-Substituted Monosaccharide Derivatives Compound

C-1

C-2

C-3

C-4

C-5

C-6

OMe

References

72.W 76.1b 72.8 77.3 71.7 76.1 71.4 75.8

61.4 61.5 62.3 62.3 62.1 62.1 72.6 72.6

58.4 60.9 61.3 61.3 61.6 61.6 60.3 60.3

62,*97 62,*97 37,97,*108 37,97,*108 97 97 97,*154 97,*154

71.0 75.4

72.4 72.4

94' 94'

72.1 76.3 70.5 74.7

61.5 61.5

155 155 155 155

74.1 77.9 73.1 77.2 75.0 78.2 71.3 75.6 72.6 76.1 70.1

61.9 62.4 62.7 62.9 62.4 62.7 64.8 64.8 63.7 63.7 64.4

55 55 55 55 55 55 83 83 156 156

83.0 81.3 81.4 80.8

62.6 63.3 64.5 65.4

56

0-Methyl-D-glucopyranose

90.1 81.3 72.8* 70.5 84.4 76.6h 70.5 96.5 84.1 70.6 93.4 72.6 a 375.1 86.7 70.4 P 97.2 a 493.2 73.0 73.9 80.5 75.8 76.7 80.5 97.1 P 93.3 73.0 74.3 71.4 671.4 75.8 77.2 P 97.3 Methyl tetra-0-methyl-D-glucopyranoside a 93.2 82.6 84.3 80.6 P 105.0 84.6 87.2 80.5 D-GlUCOpyranOSe sulfate a 392.9 71.1 83.1 68.3 73.8 85.2 68.3 P 96.5 70.2 93.1 72.3 73.6 a 675.0 76.5 70.2 P 96.9 Phosphate D-PyranOSeS a Clc 196.3 72.9 74.3 70.9 75.3 76.9 71.2 P 98.9 a Gal 196.5 69.7 70.7 70.7 73.9 70.3 73.0 99.5 P 68.1 72.1 71.6 a Man 197.3 72.6 74.2 68.2 P 96.7 69.9 93.0 72.2 73.3 a G l t 669.9 74.8 76.3 96.7 P 67.1h 94.8 71.3 70.6 CI Man 666.7" 94.4 71.9 73.3 P P Fni 167.4 99.0 69.0 70.4 D-Furanoses a Fni 183.0 77.0 P 66.0 77.4 75.2 a Fru 663.8 105.3 82.6 76.9 P 63.8 102.4 76.2 75.4 a 2-

P

68.1 68.1

56

56

56 56 (continued)

1154) R. Colson, K. N. Slessor, H. J. Jennings, and I. C. P. Smith, Can. ]. Chem., 53

(1975) 1030-1037.

(155) S. Honda, H. Yuki, and K. Tahiura, Carbohydr. Res., 28 (1973) 150-153. (156)P. A. J. Gorin, Can. ]. Chem., 51 (1973) 2105-2109. (157) A. S. Serianni, J. Pierce, and R. Barker, Biochemistry, 18 (1979) 1192-1199. (158) S. A. Abbas, A. H. Haines, and A. G. Wells,]. Chem. SOC., Perkin Trans. I , (1976) 1351- 1357.

I3C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

53

TABLEVIII (continued) Compound

C-1

C-2

C-3

71.9 71.3 97.5 71.7 76.4 102.4 76.7 102.2 82.2 a Ara 575.1 77.0 P 96.3 n-Glyceraldehyde &phosphate, hydrate 91.3 74.9 66.0 n-Glycolaldehyde phosphate, hydrate 90.7 68.2 a Rib 5-

P

C-4

C-5

83.6 82.5 83.1 81.1

65.8 66.6 65.1 66.2

C-6

OMe

References

157 157 157 157 157 157

a Additional data for related compounds are given in Refs. 93, 95, 96, and 158. Assignments may have to be reversed. Contain additional data.

TABLEM W-N.m.r. Dataa for Isopropylidene and Benzylidene Derivatives* Compound

C-1

C-2

C-3

C-4

C-5

C-6

1,2:5,6-Di-O-isopropylidene-a-~-~ucofuranose 3-substituted derivatives OH 105.4 85.2 75.0 81.3 73.3 67.7 105.0 83.3 76.0 79.6 72.4 67.0 OAc OBz 105.2 83.4 76.7 80.0 72.7 67.3 105.3 82.6 81.7 81.3 72.5 67.4 OBn OMe 105.1 83.6 81.9 81.0 72.3 67.1 105.3 83.8 82.8 79.9 72.2 67.6 OMS 105.1 83.4' 82.1" 79.9 71.8 67.1 OTs Cld 104.3 85.4 62.2 79.7 73.2 66.4 104.8 82.3 93.5 80.4 71.7 66.9 F 105.0 79.8 34.7 77.9 76.2 66.1 Deoxyd 1,2 :5,6-DiO-isopropylidene-a-n-allofuranose 103.9 79.7' 75.6 79.1c 72.4 65.8 2,3 :5,6-Di-O-isopropylidene-a-~-mannofuranose 101.1 80.1' 79.7' 85.6 73.4 66.5 l,2 :3,4-DiO-isopropylidene-cr-~-galactopyranose 96.3 70.8" 70.6' 68.3" 71.5 62.1 Methyl 4,6Q-benzylidene-hexopyranosides a-All 100.2 67.9 68.8 78.1 56.9 68.8 a-Alt 101.6 69.6 68.8 76.0 57.8 68.8 99.4 70.8 68.6 76.5 62.8 68.6 8a-Gal 100.8 69.2' 69.5' 76.5 63.0 69.3 104.2 72.8" 71.2' 76.0 66.8 69.3 P99.9 72.4 70.5 80.8 62.0 68.5 a-Glc 104.2 74.2 72.9 80.3 65.9 68.3 8101.7 70.6 68.0 78.5 62.9 68.4 a-Man

C-7

0-Me

References

83,*100,101 83,*100 83 83 83 83 83,*100,101 99,*100 83

99,*100 83,*100 83 83 101.5 101.8 101.8 101.4 101.5 101.5 101.5 101.7

55.7 55.0 56.4 55.7 57.2 54.9 56.8 54.4

102,*128d 102 102 83 83 102 102 102

Additional data for related compounds are given in Refs. 90 and 103-106. For assignment of dependence of chemical shifts on ring size, see Refs. 89 and 90. Assignments may have to be reversed. In dimethyl sulfoxide-d,.

KLAUS BOCK AND CHRISTIAN PEDERSEN

54

TABLEX

13C-N.m.r.Data for Aminodeoxy-, Deoxyhalo- and Thio-substituted Derivatives ~

Compound

~~

C-1

Aminodeoxy-D-pyraw se" o-Glucose a 2-, HCI 89.9 93.5 P Me a 2-,base 99.7 98.7 Me a 3-, base base 99.0 Me (Y 6, a 1-N-Acetyl 79.1 81.8 B a 2-N-Acetyl 92.1 96.2 P Me a 2-N-acetyl 98.6 102.3 P Mannose u 2-, HCI 91.1 91.8 P a 2-iV-A~etyl 94.3 91.3 P Galactose a 2-N-Acetyl 92.2 96.5 P 99.1 M e a 2-N-acetyl Thio-D-pyranoses' B 1-thio-Clc 85.1 73.9 B Sthio-Glc 64.8 a 6-thio-Fru 66.4 P Deoxyhalo-n-pyranoses (I 6-Cl-Gk 93.4 P 97.1

~

~~

C3

C4

C-5

55.3 57.8 54.9 71.7 71.6 71.9 74.3 55.3

70.5 72.8 74.1 54.1 73.1 75.6 80.0 72.0 58.0 75.2 54.3 71.9 56.1 74.6

70.5 70.5 69.7 69.6 71.2 71.9 71.8 71.4 712 70.4 70.9

72.4 76.9 71.7 71.3 712 75.2 79.0 72.8 77.2 72.2 76.3

61.3 61.3 60.6 60.6 41.4 63.1 63.1 61.9 62.0 61.4 61.5

55.3 67.7 56.4 70.3 54.4 70.1 55.3 73.2

67.1 67.0 68.0 67.8

72.8 76.9 73.2 77.5

61.2 61.2 61.7 61.7

51.4 68.6 54.9 72.3 50.8 68.7

69.7 69.0 69.4

71.6 76.3 71.6

62.4 622 62.1

71.4 76.0 73.3* 71.9"

80.6 43.9 68.W 71.7d

62.3 61.0 27.1 30.4

163 83 164 164

71.3 712

71.4 75.6

45.6 45.1

154 154

C-2

79.6 74.4 84.4 85.3

77.9 74.4 72.7d 70.1

72.5 73.6 752 76.5

C-6 OMelNAc References

23.3 23.5 55.6 57.2

42,62* 42,62* 81 81 81 159 159 159,160* 159,160* 161b 82,161b*

23.2 23.2

42,162; 42,162* 160 160

54.9 54.5 54.8

23.2 23.4

160 160 81

(continued)

(159) S . Shibata and H. Nakanishi, Carbohydr. Res., 86 (1980)316-320. (160) D. R. Bundle, H. J. Jennings, and I. C . P. Smith, Can.]. Chem., 51 (1973) 38123819. (161) A. S. Shashkov, A. Yu. Evstigneev, and V. A. Derevitskaya, Carbohydr. Res., 72 (1979) 215-217. (162) T. Yadomae, N. Ohno, and T. Miyazaki, Carbohydr. Res., 75 (1979) 191-198. (163) P. Friis, P. 0.Larsen, and C. E. Olsen,]. Chem. SOC., Perkin Trans. 1 , (1977)661665. (164) M. Chmielewski and R. L.Whistler, Carbohydr. Res., 69 (1979) 259-263.

55

13C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES TABLEX (continued)

Compound

C-1

a 2,6-Br2-Glc

93.1 96.6 101.5 Me B 2-Cl-Glc' Me a 2,6-Br2-Man 94.1 92.3 P 98.9 Me a 4-Cl-Gal' Me a 4,6-C1,-Gale 99.1 103.5 B"

P

C-2

C-3

C-4

C-5

C-6 OMe/NAc References

53.5 56.3 62.3 56.3 60.8 67.1 66.8 68.9

70.9 74.9 75.8 70.1d 71.8d 67.1 66.8 70.3

73.3 73.O 69.9 68.8 69.7 63.9 63.9 62.7

73.6 76.7 75.8 72.1d 75.7d 68.5 68.4 71.8

34.4 33.6 59.7 34.6 33.8 60.0 43.4 42.7

165 165 128 83 83 128 128 128

55.1 53.6 53.9 55.5

" Additional data for related compounds are given in Refs. 145 and 166. * Contains additional data. Additional data for related compounds are given in Refs. 167 and 168. Assignments may have to be reversed. In dimethyl sulfoxide-do. TABLEXI

13C-N.m.r.Data for Some Deoxy Sugars Compound

C-1

C-2

ZDeoxy-Whro-pentose 92.5 34.5 a-Pyranose 35.9 P94.6 98.9 41.9 a-Furanose" 98.9 41.8 P-" Deoxy-D-hexopyranoses 92.1 38.3 a-2-, arabino 94.1 40.5 P91.8 67.4 a&, ribo 69.7 P98.8 a4-,xylo 93.6 74.1" 76.9" P97.1 69.2 a-6-, Galacto 93.3 72.8 P97.3 a-6-,Gluco 93.1 72.9 75.6 P96.8 a&-, Manno 95.0 71.9 94.6 72.4 PDeoxy-whexofuranoses 97.4 73.9 a 3 - , ribo 102.6 76.5 P96.5 77.0 a-5, rylo '77.0 P102.5 a

C-3

C-4

C-5

65.1" 67.3" 71.7 72.0

68.1" 68.3" 86.1 86.6

66.8 63.6 62.3 62.3

68.8 71.4 34.7 39.3 69.3" 73.2" 70.4 74 .O 73.6 76.6 71.1 73.8

72.0 71.7 653 65.3 35.1 35.1 73.0 72.5 76.4 76.1 73.3 72.9

72.8 76.8 73.1 82.8 67.8" 71.3' 67.4 71.9 68.6 73.0 69.4 73.1

61.6 61.9 61.6 61.9 64.6 64.5 16.7 16.7 18.0 18.0 18.0 18.0

62 62 60 60 83 83 37,40,46* 37,40,46* 46 46 37,46.*130,"148 37,46,*130,"148

31.9 33.7 77.0 76.1

77.6 78.O 81.6 79.8

71.6 73.9 31.9 32.6

63.5 63.8 59.5 59.5

60 60 83

C-6

References

83,*137 83,*137 83,*137 83,*137

Assignments may be reversed. In pyridined,.

(165) K. Bock, I. Lundt, and C. Pedersen, Carbohydr. Res., 90 (1981)7-16. (166) B. Paul and W. Korytnyk, Carbohydr. Res., 67 (1978)457-468. (167) J. E. N. Shin and A. S. Perlin, Carbohydr. Res., 84 (1gSO) 315-327. (168) J. E. N. Shin and A. S. Perlin, Carbohydr. Res., 76 (1979) 165-176.

83

KLAUS BOCK AND CHRISTIAN PEDERSEN

S6

TABLEXI1 %-N.rn.r. Data for Methyl Deoxypyranosides Compound DPentopyranosides a-2-Deoxy+m~thro

P j3-2-Deoxy-threo P-3-Deoxy-erythro a-2,3-Dideoxy-glycero

P a3,4-Dideoxy-glycero

P DHexopyranosides a-2-Deoxyurabino

C-1

C-2

C-3

C-4

101.3 99.6 101.5 104.7 99.8 100.3 102.3 104.9

34.6 33.1 372 67.8 27.9 26.0 70.1 68.8

67.9" 65.0" 70.8 36.4 27.4 26.0 28.5" 29.0"

67.4" 689" 702 65.0

65.3 63.6 64.8 68.0 652 66.2 65.1 66.0 25.7" 62.5 23.3" 64.8

100.8 103.2 98.9 106.1 101.4 108.3 103.1 102.4 104.5 100.3 104.3 101.9

39.1 40.7 67.1 68.5 65.8 68.3 67.4 75.6 75.8 72.6 74.5 71.0

70.8 72.9 35.3 39.1 35.7 39.7 33.3 71.1 71.2 73.9 76.7 71.3

73.6 73.6

C-5

OMe

References

56.8 55.6 57.0 57.0 55.7 55.9 57.8 56.9

169 169 169 169 169 169 169 169

56.9 169 59.1 169 a3-Deoxy-n'bo 65.0 55.6 169 B 652 57.7 169 a3-Deoxy-xylo 68.5 57.5 169 P 68.0 59.3 169 u3-Deoxyf yxo 682 57.3 169 a-4-Deoxy-xy lo 36.6 57.5 169 B 35.1 57.9 169 a-6-Deoxy-gluco 76.2 56.2 46 B 76.2 58.3 46 a-6-Deoxy-manno 73.1 55.8 46,*130,b133, 134,145, 147,169,170 B 102.0 71.2 73.0 73.0 73.6 17.6 57.6 130,b169,* 170 a-6-Deoxy-galacto 100.5 69.0 70.6 72.9 67.5 16.5 56.3 46,*82,171 P 104.8 71.5 74.1 72.4 71.9 16.5 58.3 46,*171 a-6-Deoxy-altm 101.3 70.9" 70.9" 70.7" 66.9 17.2 56.3 83 a-2$-Dideoxyerythro 98.1 29.0 26.9 66.0 74.3 61.8 54.9 169 103.5 30.3 30.3 66.1 80.6 62.2 57.0 169 P a-2,3-Dideoxy-threo 98.9 25.4" 23.7" 64.9 71.9 62.9 55.1 169 a3,4-Dideoxy-erythro 100.0 69.8 26.0 26.3 68.5 64.8 55.6 169 106.5 69.9 30.2 26.7 77.4 64.7 57.5 169 B

P

74.6 78.6 73.2 80.5 73.1 80.9 74.0 69.7 73.3 68.7 73.0 69.4

C-6

63.3 63.6 61.5 61.8 64.0 63.8 64.4 66.2 64.5 17.6 17.8 17.7

(continued)

(169) L. Wiebe, Ph.D. Thesis, Danmarks Tekniske Hq~jskole,Lyngby, 1976. (170) L. V. Backinowsky, N. F. Balan, A. S. Shashkov, and N. K. Kochetkov, Cnrbohydr. Res., 84 (1980) 225-235. (171) J.-H. Tsai and E. J. Behrman, Carbohydr. Res., 64 (1978) 297-301. (172) V. Pozsgay and A. Neszrnelyi, Carbohydr. Res., 85 (1980) 143-150. (173) 6. Monneret, C. Conreur, and Q. Khuong-Huu, Carbohydr. Res., 65 (1978)3545. (174) D. R. Bundle,J. Chem. SOC., Perkin Trans. 1 , (1979) 2751-2755. (175) D. R. Bundle and S. J. Josephson, Can. J . Chem., 56 (1978) 2686-2690.

13C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

57

TABLEXI1 (continued) Compound

C-1

C-2

C-3

C-4

102.9 67.3 105.0 67.7 a-2,6-Dideoxy-arabim 98.9 37.7

26.7 30.1

22.8 23.1 77.4

a3,4-Dideoxy-threo

P P

a3,6-Dideoxy-ribo

P a3,6-Dideoxy-xylo

P a3,6-Dideoxyarabim a4,6-Dideoxy-xylo

P a4,6-Dideoxy-ribo

P

a4,6-Dideoxy-lyxo

103.2 99.1 106.2 99.9 106.7 100.8 102.6 106.1 100.8 102.1 101.9

68.7

79.0 72.7 35.4 70.6 39.1 70.5 33.9 69.2 38.1 69.1 34.6 70.7 69.9 42.6 73.1 42.4 68.1 39.1 68.6 39.5 66.0 36.2

41.0 67.4 68.8 63.9 66.4 68.4 75.7 77.5 68.5 71.7 68.8

a Assignments may be reversed. data.

C-5

C-6 OMe

72.2 67.0 79.2 66.8 68.7 17.8

57.1 58.7 55.3

74.5 69.2 76.6 67.0 74.9 68.0 67.3 71.2 60.7 68.1 64.3

59.1 55.6 57.8 55.8 57.6 55.7 57.7 59.7 56.3 57.7 54.7

19.4 17.3 17.6 16.2 16.5 17.9 22.5 22.4 20.4 20.7 21.1

In dimethyl sulfoxided,.

References 169 169 165,*172, 173' 169 169 99,b169,*174 83,*175 83 172,175* 169 169 169 169 172

Contains additional

TABLEXI11 13C-N.m.r.Dataa for Methyl Anhydro-D-glycosides

Compound D-Pentopyranosides a-2,3-Anhydro-lyxo P-2,3-Anhydro-ribo D-Pentofuranosides a-2,3-Anhydro-ribob

C-1

C-2

C-4

C-5

96.4 51.1 56.8 96.3 52.7 52.4

61.4 62.3

59.9 61.9

56.4 56.6

83 83

56.0 55.4 55.6 55.1

78.5 78.6 76.6 76.4

61.5 61.7 59.6 59.6

55.3 54.8 54.6 55.7

91 91 91 91

52.7 54.7 52.9 54.1

76.8 76.4 73.8 73.7

59.9 60.5 61.5 67.8

67.8 68.0 68.3 68.3

54.9 55.8 55.1 56.3

91 91 91 91

67.1 62.1 67.7 61.9

56.3 56.1

83 83

101.1 55.1 101.7 54.8 a-2,3-Anhydro-lyxob 101.5 53.7 Pb 101.8 54.4 4,6-O-Benzylidene-hexopyranosides a-2,3-Anhydro-alld 94.6 49.9 Pb 97.1 50.6 a-29-Anhydro-mannob 96.0 49.8 Pb 99.1 50.1 D-Hexopy ranosides a3,4-Anhydro-galado 97.0 64.4 a3,4-AnhydK1-t& 100.5 65.4

Bb

OMe References

C-3

54.0" 51.8" 52.6" 52.0"

C-6

Additional data for related compounds are given in Ref. 176. In dimethyl sulfoxided,. Assignments may be reversed. (176) M.Chmielewski, J. Mieczkowski, W. Priebe, A. Zamojski, and H. Adamowich, Tetrahedron, 34 (1978) 3325-3330.

58

KLAUS BOCK AND CHRlSTIAN PEDERSEN TABLEXIV

W-N.m.r. Data” for Unsaturated Carbohydrate Derivatives Compound

C-1 ~~

C-2

C-3

C-4

C-5

C-6

OMe

References

~ _ _ _ _

mGlycals 146.6 101.4 63.7’ 68.4’ 65.8 Xylal GDeoxygfucal 144.6 104.4 6 9 9 74.ab 75.7 17.2 Allal 146.2 101.3 62.5 67.0 74.8 61.3 Calactal 144.8 103.4 64.8 65.5 77.9 62.1 Glucal 144.6 103.8 69.7’ 69.2* 79.1 61.0 Methyl 4,6-O-benzylidene-~-hex-%enopyranoses aerythro 96.3 130.9 126.9 75.5 64.2 69.6 56.0 P 97.8 130.1’ 126.8’ 73.6 69.0 76.6 53.3 Methyl 40-acetyi-mpent-Zenopyranosides a-glycero 95.1 129.P 128.7b 64.9 60.1 55.7 P 94.2 130.9 125.1’ 63.4 61.3 55.6 Methyl 4,6-di-O-acetyl-~-hex-%enopyranosides 95.3 129.1’ 127.8’ 65.3 66.9 63.0 55.6 aerythro 63.9 63.0 54.9 72.4 95.6 129.8 125.9 P 62.6 55.2 94.8 130.4 125.0 66.6 62.6 a-threo Methyl U)-acetyl-3,4-dideoxy-~-pent-3-enopyranosides a-glycero 96.1 66.5 121.8 129.3 602 56.1 P 98.9 66.0 120.4 1312 59.4 55.9 Methyl 2,6di-O-acety13,4dideoxy-~-hex-3-enopyranosides aerythro 96.0 66.5‘ 124.3 127.9 66.8’ 65.3 56.0 56.1 71.6 65.9 B 100.2 67.3 124.7 1292 66.3 65.4@ 56.0 98.9 65.3* 122.4 130.8 a-threo 71.8 65.8’ 56.6 98.0 65.1’ 124.2 130.0 P 6-Deoxy-l,2 :3,4di-O-isopropylidene-~-~-am~no-hex-5-enopyranose 97.3 73.2 70.9 72.V 152.4 100.4 Methyl 5,6dideoxy-2~-isopropylidene-a-~-Z~-hex-5-enofuranoside 1072 81.6b 81.P 85.4 132.4 119.1

83 83 177 83 177 83 83 83 83 83 83 83 178 178 178 178 178 178 179 180

” Additional data for related compounds are given in Refs. 176,181,and 182. * Assignments may be reversed.

(177) A. I. R. Burfitt, R. D. Guthrie, and R. W. Irvine,Aust.J. Chem., 30 (1977) 10371043. (178) M.Chmielewski, A. Banaszek, A. Zamojski, and H. Adamowicz, Carbohydr. Res., 83 (1980)3-7. (179) B. Coxon and R. C. Reynolds, Carbohydr. Res., 78 (1980) 1-16. (180) K. Bock and C. Pedersen, Acta Chem. S c a d . , Ser. B, 31 (1977) 248-250. (181) R. D. Guthrie and R. W. Irvine, Carbohydr. Res., 82 (1980)207-224. (182) R. D. Guthrie and R. W. Irvine, Carbohydr. Res., 82 (1980) 225-236. (183)W. Funcke and C. von Sonntag, Carbohydr. Res., 69 (1979)247-251. (184) G. W. Schnarr, D. M. Vyas, and W. A. Szarek, J . Chem. SOC., Perkin Trans. 1 , (1979)496-503. (185) P. Finch and Z. M. Merchant, Carbohydr. Res., 76 (1979)225-232.

13C-N.M.R. SPECTROSCOPY O F MONOSACCHARIDES

59

TABLEXV '3C-N.m.r. Dataa for Some Acyclic Monosaccharide Derivatives Compound

C-1

C-2

C-4

C-3

C-5

C-6

OMe

References

75.46 74.8 72.6b 73.1b 73.7b 72.9 70.7 67.6

73.6b 64.5 73.0 64.6 72.3b 71.8b 64.2 64.2 71.7b 71Zb 64.8 64.8 73.4b 72.6 64.5 73.2b 72.3b 64.5

61.6 61.9 61.3 61.8 61.8 61.7 61.9 61.9

183 183 183 183 183 183 183 183

70.5 70.8 69.7* 69.8 69.8b

71.4b 72.6 69.4b 72.1 69.4b

63.6 62.8 70.0 71.4 71.4

63.2 63.4 63.8

67.6

72.5

71.3

63.2

~~

D-, 0-Methyloximes syn, Rib 152.2

71.4 67.7 anti, Rib 153.4 151.5 71.4 syn, Glc anti, Glc 153.5 67.5 69.8 syn,Gal 153.0 anti,Gal 155.2 66.0 syn, Fru 56.1 161.9 anti, FN 61.6 162.6 D-,Diethyl dithioacetals 54.5 71.6b Arac 54.4 74.2 xyl" 54.7 71.6 GalC 54.1 75.3 Glc" 55.0 73.8 ManC D-, Dimethyl acetals Glc" 104.1 73.6

184 184 184 184 184 53.1, 54.5

184

Additional data for related compounds are given in Ref. 185. Assignments may be reversed. In dimethyl sulfoxided&.

TABLEXVI W-N.m.r. Data for Alditols and Their Acetates Compounds Hexitols Allitol Altritol Galactitol Glucitol Iditol Mannitol Pentitols Arabinitol Ribitol Xylitol

C-1

C-2

C-3

C-4

C-5

C-6

References

63.7 64.4 64.5 63.8 64.1 64.6

73.5 71.8 71.5 74.3 73.1 72.2

73.7 72.2 70.7 71.0 72.5 70.7

73.7 73.0 70.7 72.6 72.5 70.7

73.5 74.0 71.5 72.5 73.1 72.2

63.7 63.4 64.5 64.2 64.1 64.6

184,"186* 184,"186* 184,a186,*187 154,184,"186,*188 184,"186* 184," 186,*187

64.4 63.8 63.9

71.6 73.5 73.2

71.9 73.6 72.0

72.3 73.5 73.2

64.3 63.8 63.9

184,"186,*187 154,184,a186,*187 184,a186,*187 (continued)

(186) S. J. Angyal and R. Le Fur, Carbohydr. Res., 84 (1980) 201-209. (187) W. Voelter, E. Breitmaier, G. Jung, T.Keller, and D. Hiss, Angew. Chem., 82 (1970) 812-813. (188) A. P. G. Kieboom, A. Sinnema, J. M. van Der Tom, and H. von Bekkum, Red. Trau. Chim. PUYS-BUS,96 (1977) 35-37.

KLAUS BOCK AND CHRISTIAN PEDERSEN

60

TABLEXVI (continued) ~

Compounds

C-1

Tetritols Erythritol 64.0 Threitol 63.9 Other alcohols Glycerol 64.0 Ethylene glycol 63.8 Hexitol acetates Allitol 61.8 Altritol 62.1 Galactitol 62.3 Clucitol 62.0 Iditol 61.8 Mannitol 62.0 Pentitol acetates Arabinitol 62.1 Ribitol 61.8 Xylitol 62.0 Tetritol acetates Erythritol 61.9 Threitol 62.0 Other alcohol acetates G lycero 1 62.4 Ethylene glycol 62.4 'I

~~

C-2

C-3

C-4

C-5

73.3 72.9

73.3 72.9

64.0 63.9

73.5 63.8

64.0

69.7 68.4 67.8 69.6 69.3 68.1

69.4 69.1 67.7 68.7 68.9 67.7

69.4 68.7 67.7 69.0 68.9 67.7

69.7 70.0 67.8 68.9 69.3 68.1

68.3 69.6 69.4

68.6 69.4 69.3

68.3 69.6 69.4

61.9 61.8 62.0

69.4 69.4

69.4 69.4

61.9 62.0

69.4 62.4

62.4

C-6

References

184,"186,*187 184,"186* 184:186,*187 186,*187 61.8 61.7 62.3 61.6 61.8 62.0

186 186 186 186 186 186 186 186 186 186 186 186 186

In dimethyl sulfoxided,. TABLEXVII

13C-N.m.r. Data'' for Anhydroalditols _ _ _ _ ~ ~ Compound C-1 c-2 C-3 C-4 C-5

C-6

References

1,4Anhydrohexitols Allitol 72.9 Altritol 74.1 Galactitol 73.7 Glucitol 74.3 Gulitol 72.2 iditol 72.9 Mannitol 71.9 Talttol 73.7

63.4 64.0 63.9 64.5 63.8 63.3 64.O 64.3

27,189* 189 189 189 27,189* 189 27,189* 189

72.1 78.3 77.9 77.3 72.3 77.2 72.3 72.5

72.9 79.1 79.3 76.8 71.3 76.5 71.2 73.3

82.9 86.5 85.8 80.8 81.3 80.9 81.1 82.1

72.5 72.7 72.3 70.3 71.6 71.3 70.3 72.2

(continued ) (189) H. Thggersen, Ph.D. Thesis, Danmarks Tekniske Hgjskole, Lyngby, 1980. (190) B. Matsuhiro and A. B. Zanlungo, Carbohydr. Res., 63 (1978) 297-300. (191) J. C. Goodwin, J. E. Hodge, and D. Weisleder, Carbohydr. Res., 79 (1980) 133141.

61

I3C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES TABLEXVII (continued) Compound

C-1

174-Anhydropentitols 74.1 Arabinitol Lyxitol 72.1 Ribitol 73.1 Xylitol 73.6 1,CAnhydrotetritols 72.3 Erythritol Threitol 73.8 2,!j-Anhydrohexitols 62.9 Allitol Altritol 61.3 Galactitol 60.5 Glucitol 61.1 Iditol 61.0 Mannitol 61.9 1,SAnhydrohexitols 66.1 Allitol Altritol 67.1 Galactitol 70.1 Glucitol 69.8 Gulitol 65.8 Iditol 68.2 Mannitol 70.8 Talitol 71.6

C-2

C-3

C-4

C-5

C-6

References

77.8 72.1 71.9 77.2

79.0 71.4 72.5 77.6

86.5 81.7 82.7 81.8

62.5 61.3 62.3 60.8

71.8 77.2

71.8 77.2

72.3 73.8

84.4 81.5 79.9 81.9 81.3 83.2

72.4 72.5 71.6 77.9 77.6 77.3

72.4 72.7 71.6 79.0 77.6 77.3

84.4 82.1 79.9 85.7 81.3 83.2

62.9 62.5 60.5 62.4 61.0 61.9

189 189 189 27,189* 27,189* 27,189*

66.8 70.4 67.4 70.4 66.6 70.1 70.0 70.1

70.0 70.4 75.1 78.4 71.0 70.8 74.5 70.6

67.1 65.8 70.1 70.7 71.0 70.6 68.2 69.2

77.0 77.2 80.4 81.3 76.6 77.4 81.5 80.4

62.2 62.3 62.3 61.5 62.4 62.0 62.1 63.4

27,189* 27,189* 27,189* 27,189* 189 189 27,189,*190 27,189*

27,189* 27,189* 27,189* 27,189* 27,189* 27,189*

Additional data for related compounds are given in Ref. 191.

TABLEXVIII I3C-N.m.r. Data for Aminodeoxydditols and Aminoanhydrodeoxydditols18g Compound

c-1

c-2

c-3

1-Amino-1-deoxy-D-hexitol hydrochloride 43.6 67.2 71.2 Galactitol Mannitol 43.1 67.6 71.2 1-Amino-1-deoxy-wpentitol hydrochloride 43.2 66.9 71.7 Arabinitol Lyxitol 43.0 67.7 72.3 Ribitol 41.6 68.2 73.1 Xylitol 42.6 68.3 72.1 5-Amino-l,Panhydro-5-deoxy-~-pentitol hydrochloride 74.6 77.5 80.0 Arabinitol Lyxitol 72.0 71.5 71.1 Ribitol 74.5 72.1 73.7 Xylitol 74.1 76.9 78.1

C-4

c-5

C-6

70.0 69.3

70.7 71.0

64.O 63.5

71.1 70.4 72.1 72.1

63.3 63.4 62.8 62.9

82.4 75.9 78.3 77.9

42.4 39.8 42.7 40.4

KLAUS BOCK AND CHRlSTIAN PEDERSEN

62

TABLEXIX 'T-N.m.r. Data for Uronic Acids or Uronolactones Compound

C-2

C-1

C-4

C-3

C-5

C-6

DGlucopyranumnic acid 72.4 71.4 172.9 93.2 72.0 73.4 75.4 173.8 72.2 13 96.9 74.7 76.3 76.9 176.9 73.0 a (pH 7.8) 92.9 72.2 73.5 72.6 177.6 72.7 96.7 75.0 76.5 P Methyl D-ghcopyranosiduronic acid and methyl ester 100.7 71.9 73.8 72.5 71.9 &-Acid a-Ester 100.8 71.9 73.7 72.4 71.9 @-Acid 104.3 73.8 76.5 72.3 75.6 " P-Ester 104.6 73.7 76.3 72.4 75.7 'I D-Glucofuranurono-6,3-lactone ff 99.1 74.8 85.6 76.7 70.4 177.8 P 103.7 74.8 85.6 78.4 70.1 177.9 D-Gdactopyranuronic acid a 93.2 68.7 69.5 70.9 70.5 172.6 B 97.0 72.1 73.1 70.9 74.8 173.5 Methyl (methyl a-mannopyranosid)uronate a 102.3 70.4 71.1 69.2 72.9 a Methyl 2-hexulosonate a-D-urubino 170.6 97.0 70.6' 71.7b 65.7* 63.5 P 170.6 97.0 69.3 69.5 69.1 64.7 a-L-xy lo 170.5 96.6 72.8 73.7 69.5 62.8

0-Me

59 59 59 59

a (pH 1.8)

_

_

-~ ~

~

References

56.7 56.8,54.2 58.5 58.7,56.2

46 46,*192 46 46 59 59 83,*127 83,*127

56.534.1

46

53.5 53.9 53.9

193 193 193

('Not resolved. * Assignments may b e reversed. (192) A. S. Shashkov, A. F. Sviridov, 0. S. Chizhov, and P. KovaC, Carbohydr. Res., 62 (1978) 11-17. (193) T. C. Crawford, G. C. Andrews, H. Faubl, and G. N. Chmumy,]. Am. Chem. SOC., 102 (1980) 2220-2225. (194) H. S. Isbell and M. A. Salam, Carbohydr. Res., 90 (1981)123-126. (195)W. Kondo, F. Nakazawa, and T. Ito, Carbohydr. Res., 83 (1980) 129-134. (196) M. Chmielewski, Tetrahedron, 36 (1980) 2345-2352. (197) S. Berger, Tetrahedron, 33 (1977) 1587-1589. (198) T. Ogawa, J. Wzawa, and M. Matsui, Carbohydr. Res., 59 (1977) c32-c35. (199) G . Schilling and A. Keller,]ustus Liebigs Ann. Chem., (1977) 1475-1479. (200) D. M. Vyas, H. C. Jarrell, and W. A. Szarek, Can.J. Chem., 53 (1975) 2748-2754. (201) A. K. Bhattacharjee, H. J. Jennings, and C. P. Kenny, Biochemistry, 17 (1978) 645 -651. (202) R. Cherniak, R. G. Jones, and D. S. Gupta, Carbohydr. Res., 75 (1979)39-49. (203) V. Eschenfelder, R. Brossmer, and H. Friebolin, Tetrahedron Lett., (1975)30693072. (204) H. J, Jennings and A. K. Bhattacharjee, Curbohydr. Res., 55 (1977) 105-112. (205) L. W. Jaques, B. F. Riesco, and W. Weltner, Jr., Carbohydr. Res., 83 (1980)21-32. (206) M. F. Czamiecki and E. R. Thomton,]. Am. Chem. SOC., 99 (1977) 8273-8279. (207) J. M. Beau, P. Sinay, J. P. Kamerling, and J. F. G. Vliegenthart, Carbohydr. Res., 67 (1978) 65-77.

63

13C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

TABLEXX 13C-N.m.r. Datan for Salts of Aldonic Acids and for Aldonolactones Compound

C-1

C-2

C-3

C4

D-Aldonic acid salts (pH 14) Allonic 179.5 74.7b 74.6b 73.6b Altronic 180.5 74.W 73.9 72.7b Galactonic 180.6 72.4b 72.4b 71.1b Gluconic 179.8 75.2 72.4 73.8 Gulonic 180.4 75.6b 73.9 73.6b Idonic 179.5 73.8 73.2b 72.5b Mannonic 180.3 75.4b 72.2b 72Jb Talonic 180.7 75.3b 74.0" 72.6b Arabinonic 180.5 73.4b 72.6b 72.3b Lyxonic 180.0 75.1b 72.9 72.3b Ribonic 179.9 759 74.6b 73.W Xylonic 180.4 74.2b 74.V 73.6 Erythronic 179.6 74.4b 74.2b 62.8 Threonic 179.4 73.V 73.1b 63.1 Glyceric 179.2 74.4 64.9 D-Hexono-,. .pentono-, and tetrono-1.4lactones Allono 178.7 86.9 70.9 69.5b 81.3 Altrono 176.8 74.8b 73.3b 80.9 74.5b 73.7b Galactono 176.7 Glucono 177.9 73,4b 73.8b 80.5 71.7b 71.V Gulono 178.8 822 Idono 176.5 74.W 71.9 79.9 Mannono 178.8 79.3 71.5b 70.2b Talono 179.3 71.2b 71.V 86.9 Arabinono 176.9 82.0 74.6b 73.2b 82.4 71.3b 70.3b Lyxono 179.0 Ribono 179.3 70.3b 69.8b 87.5 73.9 72.9 Xylono 177.9 812 Erythrono 179.3 73.7 70.5 69.7 Threono 178.0 74.0 73.1 70.4 D-Glucono-1,S-lactone 174.5 82.3b 73.4b 71.7b n-Gluconic acid (pH-3) 176.5 73.1b 72.3b 71.9

C-5

C-6

References

72.7b 72.6b 70.7b 72.0 71.7 71.9 71.6b 72.0 64.1 64.0 63.9 63.6

63.4 63.3 64.3 63.6 63.6 63.9 64 .O 64.4

83 83 83 83,*194 83 83 83 83 83 83 83 83 83,*43 83,*43 195,*43c

69.W 71.2b 69.8b 71.2b 70.4b 68.8b 68.5b 69.4b 60.1 60.6 61.4 59.9

62.8 62.3 62.9 63.2 62.4 62.9 63.4 62.7

83 83 83 83 83 83 83 83 83 83 83 83 83 83

67.9

60.8

83

71.4b

63.5

83

Additional data for related compounds are given in Ref. 196. Assignments may be reversed. Data for acid at pH -3. For further data, see p. 66. (I

(208) Y. Terui, K. Ton, K. Nagashima, and N. Tsuji, Tetrahedron Lett., (1975) 25832586. (209) J. Boivin, M. Pai's, and C. Monneret, Carbohydr. Res., 64 (1978) 271-278. (210) K.-I. Harada, S. Ito, and M.Suzuki, Carbohydr. Res., 75 (1979) ~17-c20. (211) K. Olsson, 0. Theander, and P. h a n , Carbohydr. Res.. 58 (1977) 1-8. (212) S. Mizsak, G. Slomp, A. NeszmBIyi, S. D. Gem, and G. Lukacs, Tetrahedron Lett., (1977) 721-724.

C

CA

TABLEXXI

0" R

W-N.m.r. Data for Some Biologically Significant Monosaccharides Compound

c-1

c-2

c-3

c-4

c-5

C-6

c-7

C-8

c-9

References

r2

U

L-Ascorbic acid

174.2

118.9

156.6

77.3

60.0

63.4

197,*198

97.8 101.5 94.8 95.3

78.3 81.2 75.3 75.7

70.6 71.6 76.9 66.6

81.6 82.5 68.9 68.6

62.9 63.6 65.6 63.6

61.3 62.9 61.1 63.4

199 199 199 199

g

106.1 84.3 82.2 3-Deoxy-D-manno-octulosonicacid, sodium salt a-Pyranose 177.9 97.6 34.8 Me a-Pyranoside 176.5 102.5 35.2 P 174.8 102.4 35.5 A'-Acetyl-D-neuraminic acid, methyl pyranoside 41.0 174.1 101.6 a Acid 40.8 176.1 101.4 P 39.7 170.7 100.1 a Me ester 40.1 171.2 100.1 P

62.3

73.2

200

m

67.8" 67.4" 68.6"

67.4" 67.1" 66.5"

72.4 72.5 74.6

70.5 70.5 70.3

64.2 64.2 65.2

51.9 52.9

201,*202 201 201

69.0 67.1 69.0 67.2

52.9 53.1 52.8 52.6

73.4" 71.1" 73.8" 71.5"

69.2" 69.5" 69.2" 69.0"

72.6" 71.1" 71.5" 70.8"

63.6 64.5 64.0 64.3

203*-206 202,*205-207 203,*204,206 203,*204,206

D-Hamamelose a-Furanose

B a-Pyranose

B

2

3 U

1,u)-Isopropylidene-a-apiose

z

n

Methyl 2,6-dideoxy-3-C-methyl-3-O-methyl-~-ribo-hexopyranoside~ a 98.8 37.8 74.9 78.0 70.8 18.2 P 97.5 35.2 73.0 78.0 64.5 17.9 Methyl 2-deoxy-3C-methyl-a-D-n'bo-hexopyranoside 98.2 40.5 69.4" 71.2 69.7" 62.9 Methyl 4,~-benzylidene-2-deoxy-2C-methyl-~-methyl-~-mannopyranoside a 104.2 37.6 76.6 79.1 63.8 69.1 P 103.8 38.1 79.5 78.9 67.6 68.9 Methyl 3-amino-2,3,6trideoxy-fl-~qlo-hexopyranoside 99.1 34.6 49.7 72.2 69.3 16.5 Methyl 3-acetamido-2,3,6trideoxyhexopyranoside a-tmrabino 97.6 35.0 48.1 74.2 68.6 17.0 a-dyxo 98.2 30.2 45.5 69.9 65.9 16.8 P-L-ribo 99.1 33.5 46.3 72.2" 71.8" 18.6 Methyl 3,4,6-trideoxy-3-(dimethylamino)-~qb-hexopyranoside a 99.6 68.7 60.3 29.3 64.8 21.2 P 104.9 69.9 65.4 28.8 69.5 21.2 Thioglycosides 82.5 80.9 78.1 73.0 70.1 61.7 Ally1 glucosinolatec Lincomycind 89.2 68.8 71.4 69.5 70.0 54.9 N-acetyl-" 88.2 68.8 71.3 69.4 69.5 53.8

21.1 21.9

208 208

25.6

118

54.7 56.9

11.0 5.7

116 116 209

56.0

6

2

+ v)

54.6 54.8 55.9

209,210* 209 209

55.0 56.5

39.9 40.3

67.4 65.7

17.2 20.6

208 208 14.2 13.3

163 212 212

" Assignments may have to be reversed. Additional data for related compounds are given in Refs. 115 and 117. Additional data for related compounds are given in Ref. 211. The carbon chemical-shifts for the pyrrole ring of lincomycin hydrochloride are given in Ref. 212. Pyrrole ring substituted with an N-acetyl group.

2

3

a8 cc

%

5

n Ec

$ i U E

66

KLAUS BOCK AND CHRISTIAN PEDERSEN ADDENDUM

For 'SC-n.m.r. data (Table 111) on B-Sorp and B-Solf, see Ref. 213. For IS-n.m.r. data on D-xylono- and D-mannono-1,5-lactone, and on the four Daldopentonic acids at pH 1-3 (Table XX), see Refs. 214-217.

(213) G.-J. Wolff and E. Breitmaier, Chem. Ztg., 103 (1979) 232-233. (214) A. S . Serianni, H. A. Nunez, and R. Barker,]. Org. Chem., 45 (1980)3329-3341. (215) D. Horton and Z . Waiaszek, Cnrbohydr. Res., 105 (1982) 95-109; 111-129. (216) Z. Wdaszek and D. Horton, Carbohgdr. Res., 105 (1982) 131-143. (217) 2.Wdaszek, D. Horton, and I. Ekiel, Cnrbohydr. Res., 193-201.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 41

STRUCTURAL CHEMISTRY OF POLYSACCHARIDES FROM FUNGI AND LICHENS

BY ELIANABARRETO-BERGTER Departamento de Microbiologia Geral, Uniuersidade Federal do Rio de Janeiro, Brazil

AND

PHILIPA. J. GORIN

Prairie Regional kboratory, National Research Council, Saskatoon, Saskatchewan S7N OW9, Canada

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. a-&Linked Glucans . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . 1. Amylose . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 2. Glycogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pseudonigeran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Glucans Containing a - ~ - ( 1 + 3 )and cw~(1-A)Linkages . . . . . . . . . . . . 5. Pullulan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. P-D-Linked Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Linear /3-D-Glucans 3. Branched-chain P-DIV. Glucans from Lichens

.. .

.. . .. . . . .

.

. .. .

1. Linear Mannans

68

. 68 68 69 69 70 72 72 72

.... . . . . . . ..... ,. .. . .

VI. Galactans . . . VII. 2-Acetamido-2 VIII. 2-Amino-2-deo 1. Rhamnomannans . . . ....................... 89 2. Glucomannans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 3. Galactomannans . . . . . . . . . . . 92 4. Miscellaneous Heteropolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 X. Heteropolysaccharides Based on Galactan Main-Chains . . . . . . . . . . . . . . . 100 1. Glucogalactans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 100 2. Fuco(manno)galactans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Miscellaneous Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 67

Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12007241-6

68

E. BARHETO-BERGTER AND PHILIP A. J. GORIN

I. INTRODUCTION In this Chapter, the authors discuss the chemical structures of the polysaccharides of fungi and lichens investigated from 1967 to the middle of 1980. Work conducted on fungal polysaccharides before 1967 was covered earlier by Gorin and Spencer.' That article and the present one consider the same classes of fungi, except for lichens. Herein, it is convenient to group the polysaccharides in terms of their chemical structures, according to the nature of the component sugars, the predominant linkage and configuration, and, in the case of heteropolymers, the nature of the main chain. Investigations that provided few structural details are not included, except when an unusual structural feature is concerned. Also not discussed are biosynthesis, industrial utilization, and morphological location when related to the immunology of polysaccharides. The chemistry and biochemistry o f fungal polysaccharides have been periodically reviewed.2 Other reviews have appeared on the cell-wall chemistry, morphogenesis, and taxonomy of fungi,3 fungal cell-wall glycoproteins and peptido-polysaccharides,4 and the chemical composition, organization of polymer, and biosynthesis in the cell wall.5 Summaries have also appeared on the use of proton-n.1n.r. spectra of yeast polysaccharides in identification and classification of yeasts," and on 13C-n.m.r. spectroscopy of fungal polysaccharides, as an aid in monitoring the formation of polysaccharides and in their structural identificati~n.~ 11. CY-D-LINKED GLUCANS

1. Amylose A11 reports on starchlike polymers cannot be covered herein. However, the cell walls of Fusicoccum amygdali are stained blue with iodine and attacked by alpha amylase.x Extracts of Sporothrix schenckii

(1) P. A. J. Gorin and J. F. T. Spencer, Ado. Carbohydr. Chem., 23 (1968)367-417. (2) Carbohydr. Chem., Spec. Period. Rep., 3 (1970)236-243; 5 (1972)266-281; 6 (1973) 261-273; 7 (1975)282-293; 8 (1976)254-261; 11 (1979)299-308. (3) S. Bartnicki-Garcia,Annu. Rev. Microbiol., 22 (1968)87-108. (4) J. E. Gander,Annu. Reo. Microbiol. 28 (1974) 103-119. (5) R. F. Rosenberger, in J. E. Smith and D. R. Deny (Eds.),The Filamentous Fungi, Vol. 2, Wiley, New York, 1976, pp. 328-344. (6) P. A. J. Gorin and J. F. T. Spencer, Ado. Appl. Microbiol., 13 (1970)25-89. (7) P. A. J. Gorin, ACS Symp. Ser., 126 (1979) 159-182. (8) K. W. Buck and M. A. Obaidah, Biochem. I., 125 (1971)461-471.

POLYSACCHARIDES FROM FUNGI AND LICHENS

69

and Ceratocystis stenoceras contain 4-0-substituted D-glucopyranosyl units, and the solutions give a blue color with i ~ d i n e . ~

2. Glycogen Glycogens have been isolated from Candida albicans,tOBlastocladiella emersonii," Neurospora crassa,12Allomycesrnacrogynu~,'~ Rhizophydium sphaerotheca, and Monoblepharella elongata.l4 They have p-amylolysis values of 4 5 4 5 % and average chain-length values of 11-14 D-glucosyl units. A c.l. decrease of 15 to 9 corresponded to the different developmental stages of B . emersonii during the 12-20-h culture age-period of resistant, sporangial plants." Polyp o n s circinatus glycogen from young cells hast5a B-amylolysis value of 34%, which decreases with age to 23% (with a x value of 6).

(x)

3. Pseudonigeran Pseudonigeran, isolated following alkaline extraction of cell walls ofAspergillus niger, is a (1-+3)-linked a-D-glucopyranan, as shown by methylation, periodate, and partial-hydrolysis studies. The hot-waterinsoluble polymer contains only 1% of ( 1 - 4 linkages, which are present in the polymer, or are from contaminating material, such as hotwater-soluble nigeran16;this is much less than the 4 to 10%previously reported."J8 Pseudonigeran is present in the cell walls ofAspergilZus nidulans, where it is a reserve material serving for growth of cleistothecia,lS and in Phytophthora infestans,20Cryptococcus, and Schizosaccharomyces spp.*l Many other fungi contain alkali-soluble, (1+3)-linked a-D-glucopyranans; these are summarized in Table I. In Histoplasma capsulatum, the cell wall contains a glucan, whereas the mycelial form con(9)J. 0.Previato, P. A. J. Gorin, R. H. Haskins, and L. R. Travassos, E x p . Mycol., 3 (1979)92-105. (10)H. Yamaguchi, Y.Kanda, and K. Iwata,J. Bacteriol., 120 (1974)441-449. (11)J. Nomnan, G. Wober, and E. C. Cantino, MoZ. Cell. Biochem., 9 (1975)141-148. (12)G.Takahara and K. Matsuda, Agric. BioZ. Chem., 40 (1976)1699-1703. (13)D.B. Coulter and J. M. Aronson, E x p . Mycol., 1 (1977)183-193. (14)D. B. Coulter and J. M.Aronson,Arch. Microbiol., 115 (1977)317-322. (15)J. D.Fontana and G. T. Zanch,J. Bacteriol., 129 (1977)141-148. (16)M. Horisberger, B. A. Lewis, and F. Smith, Carbohydr. Res., 23 (1972)183-188. (17)I. R. Johnston, Biochem. J., 96 (1965)651-658;659-664. (18)S.Hasegawa, J. H. Nordin, and S. Kirkwood,]. B i d . Chem., 244 (1969)5460-5470. (19)B. J. M. Zonneveld, Biochim. Biophys. Acta, 273 (1972)174-187. (20)T.Miyazaki, M. Yamada, and T. Ohno, Chem. Pharm. Bull., 22 (1974)1666-1669. (21)J. S. D.Bacon, D. Jones, V. C. Farmer, and D. M. Webley, Biochim. Biophys. Acta, 158 (1968)313-315.

70

E, RARRETO-BERGTER AND PHILIP A. J. GORIN

TABLEI Fungi Whose Cell Walls Contain Alkali-Soluble, (1+3)-Linked a-D-GlUCOpy~ananS Fungus

Methods used for characterization

References

Tremelln mesentericu Aspergillus nidulons (mycelia) Schizophyllum commune Histoplusma capsulutum Histoplusmu farcinosum Purucoccidioides brasiliensis Blastomyces dermutiditis

X-ray diffraction [a],,,periodate resistance x-ray diffraction, [a],,methylation methylation, [alD,i.r. methylation, i.r., enzymolysis methylation, [ale [a],,, i.r., enzymolysis

24 27 28-30 22 23 31 24

b i n s none.22 Yeast forms preferentially contain the glucan in Histoplusma f a r ~ i n o s u mBlastomyces ,~~ d e r m a t i d i t i ~and , ~ ~ Paracoccid ioides hrusil iEnsi.~.25

4. Glucans Containing a-D-( 1+3) and a-D-( 1 - 4 Linkages

Few reports exist on nigeran, except that it occupies an inaccessible position in the cell wall ofAspergihs niger, and is resistant to enzymic attack.32 An a-D-glucopyranan from fruit bodies of Lentinus edodes is slightly branched, with (1-3) and ( 1 4 4 ) linkages in the ratio of 5.3 : 1. It is partially degraded by amylolytic enzymes, and it was concluded that the (1-4) linkages are concentrated in regions not far from nonreducing ends:{" An a-D-glucopyranan containing (143) and (1-4) linkages in the ratio of 1:2 was obtained on alkaline extraction of the cell walls of Cladosporium herbarum, but the distribution of the linkages thi-oughout (22) F. Kanetsuna, L. M. Carbonell, F. Gil, and I. Azuma, Mycopathol. Mycol. Appl., 54 (1974) 1-13. (23) G. San-Blas and L. M.Carbonel1,J. Bucteriol., 119 (1974) 602-611. (24) F. Kanetsuna and L. M. Carbonel1,J. Bacteriol., 106 (1971) 946-948. (25) F. Kanetsuna and L. M. Carbonell,]. Bocteriol., 101 (1970) 675-680. (26) I. D. Reid and S. Bartnicki-Garcia,]. Gen. Microbiol., 96 (1976) 35-50. (27) B. J. M. Zonneveld, Biochim. Biophys. Acto, 249 (1971) 506-514. (28) J. G. H. Wessels, D. R. Kreger, R. Marchan5 B. A. Regensburg, and 0. M. H. d e Vries, Biochim. Biophys. Acto, 273 (1972) 346-358. (29) D. Siehr, Con. ]. Biochem., 54 (1976) 130-136. (30)J. H. Sietsrna and J. G. H. Wessels, Biochim. Biophys. Acta, 496 (1977) 225-239. (31) F. Kanetsuna, L. M. Carbonell, I. Azuma, and Y. Yamamura,J. Bucteriol., 110 (1972)208-218. (32)K. K. Tung and J. H. Nordin, Biochem. Biophys. Res. Commun., 28 (1967) 519524. (33) M. Shida, T. Uchida, and IL Matsuda, Carbohydr. Res., 60 (1978) 117-127.

71

POLYSACCHARIDES FROM FUNGI AND LICHENS

the polymer was not determined.34Elsinan, isolated from the culture filtrate of Elsinoe leucospila, is a predominantly h e a r ff--0-ghcopyranan having 4-0-and 3-0-substituted units in the ratio of 2.5: 1. It consists mainly of the alternating, repeating sequence 1, although 3 -cr-D-GlCp-(l-4)-cr-D-GlCp-(l-4)-cu-D-Gl~p

-(1-4)-

1

consecutive, (1+4) linkages were evidenced by formation of a small proportion of maltotetraose on partial h y d r ~ l y s i s . Such ~ ~ , ~a~polysaccharide may be present in the hyphal wall of Coprinus mucrorhizus var. m i c r ~ s p o r u sPart . ~ ~ of the cell wall of Neurosporu crussa consists of a glucan having (1-4) and (1+3) linkages and, perhaps, the LY-D configurati~n.~~

1

1

I10

100

I 90

I 00

I

I

70

60

p. p. m. FIG. l.-W-N.m.r.

Spectrum of Pullulan from Tremella mesenterica, at pD 7.0.

(34) T. Miyazaki and Y. Naoi, Chem. Pharm. Bull., 22 (1974) 2058-2063. (35) Y. Tsumuraya, A. Misaki, S. Takaya, and M. Toni, Carbohydr. Res., 66 (1978) 5365. (36) A. Misaki, Y. Tsumuraya, and S. Takaya,Agric. Biol. Chem., 42 (1978) 491-493. (37) C. B. Bottom and D. J. Siehr, Carbohydr. Res., 77 (1979) 169-181. (38) L. Cardimil and G. Pincheira,J. Baetetiol., 137 (1979) 1067-107'2.

72

E. BARRETO-BERGTER AND PHILIP A. J. GORIN

5. Pullulan Pullulan is present in the culture filtrate of Tremella mesentericu NRRL-6158, as shown by methylation, partial hydrolysis,Rgand Wn.m.r.-spectraleoevidence. Its l3C-n.rn.r. spectrum (see Fig. 1)is identical to that of pullulan. The three-unit repeating-sequence 2 was confirmed, because, although it only gives 14 signals in its W-n.ni.r. spectrum, these may be interpreted in terms of 18 signals, some of which overlap. A gliican from Cyttariu harioti Fischer, having (1-6)and (1+4)-linked a-D-glucopyranosyl units in the ratio of I :2.4, also resembles pullulan.41Although pullulans have the overall repeatingunit 2, that of Aureobasidium (Pullularia)pullulans contains a minor sub-unit having 3 consecutive, (1-+4) linkages.42 - a -D - GlC P - ( 1 - 4 ) -

(I-D-

GlCP - (1-

4)-

(2

-D-Glc p- (1-6)

-

2

111. PD-LINKEDGLUCANS 1. Cellulose both cellulose and chitin occur According to infrared (i.r.) in the cell walls of Cerutocystis oliuacea. Based on cytochemical and X-ray data, cellulose occurs in the cell walls of 31 of 47 species of Cerutocystis, and in 4 species of E ~ r o p h i u mConvincing .~~ i.r. evidence was obtained for the presence of cellulose in the cell walls of Europhium ~ u r e u malready , ~ ~ examined by the X-ray technique.

2. Linear I&D-GIuc~~s &D-Giucans are of interest because of their potential, antitumor act i ~ i t yAs . ~they ~ are present in virtually all fungi, the number of investigations conducted on them is considerable, and too great to be covered here. Thus, only those glucans whose structures have been determined in some detail are considered. (39) C. G. Fraser and H. J. Jennings,Can. J . Chem., 49 (1971) 1804-1807. (40) H. J. Jennings and I. C. P. Smith,J. Am. Chem. SOC., 95 (1973) 606-608. (41) N. Waksman, R. M. de Lederkremer, and A. S. Cerezo, Carbohydr. Res., 59 (1977) 505-515. (42) B. J. Catley and W. J. Whelan,Arch. Biochem. Biophys., 143 (1971) 138-142. (43) A. J. Michell and G. Scurfield, Trans. Br. Mycol. SOC., 55 (1970) 488-491. (44) T. R. Jewell, Mycologiu, 66 (1974) 139-146. (45) A. C. M . Weijman, Antonie uan Leeuwenhoek,J . Microbiol. Serol., 42 (1976) 315324. (46) R. L. Whistler, A. A. Bushway, P. P. Singh, W. Nakahara, and R. Tokuzen, Adv. Carbohydr. Chem. Biochem., 32 (1976) 235-275.

POLYSACCHARIDES FROM FUNGI AND LICHENS

73

Pachyman, the major constituent of the cell wall ofPoria COCOS Wolf consists of (l+3)-linked ED-glucopyranosyl units, with only a few (1+6) links in the side chains and one in the main chain. Its numberaverage degree of polymeri~ation~~ is 690, which is higher than previously determined. A (1+3)-linked p-glucan is a major constituent of the conidial wall of Neurospora c r a s ~ aand , ~ ~is similar to that of the mycelial cell-wall. The fruit bodies of Pleurotus ostreatus (Fr.) QuCl contain a glucan having 3-0-substituted p-D-ghCOpyranOSyl, and minor 4-0-substituted a-D-glucopyranosyl, units.49A similar combination of linkages occurs in the cell-wall glucan of Coprinus macrorhizus var. microsporus.50A soluble glucan consisting of 77%of (1-+3)linked @D-ghcopyranosyl units was isolated from the mycelia of Phytophthora cinnamoni.51 The /+D-ghcopyranan from the mycelial form of Paracoccidioides brasiliensis contains 90% of (1-3) linkages.31Although one of the glucans of Saccharomyces cerevisiae is almost linear, it is, for convenience, dealt with in the following section (3). A linear cell-wall glucan(s) from Sporothrix schenckii contains 3 0 , 6 0 , and 4-0-substituted @D-glucopyranosy1 units, the @D-glycosidic configuration being determined by 13C-n.m.r. spectrosc~py,~ by virtue of the C-1 signals, at relatively low field, of 6, 103.8 and 104.8. 3. Branched-chain p-~-Glucans

Cell walls of Saccharomyces cerevisiae were found to contain a ( 1+6)-linked ED-glucopyranan; this was isolated, and identified by i.r. s p e c t r o s ~ o p yand ~ ~ chemical-analysis techniques." The alkali-insoluble glucan from S. cerevisiae contains this and a (1+3)-linked p-Dglucopyranan in the ratio" of 1:5.7. The former, of mol. wt. 2 x lo5,

has 6-0- and 3-0-substituted units in the ratio of 4.4: 1, and contains 14% of 3,6-di-O-substituted units.55(A similar heterogeneity occurred (47) G. C. H o h a n n , B. W. Simson, and T. E. Timell, Carbohydr. Res., 20 (1971)185188. (48) P. R. Mahadevan and U.R. Mahadkar, lndiun J . Exp. BioE., 8 (1970)207-210. (49) H. Sait6, T. Ohki, Y. Yoshioka, and F. Fukuoka, FEBS Lett., 68 (1976) 15-18. (50) C. B. Bottom and D. J. Siehr, Can.J . Biochem., 58 (1980) 147-153. (51) L. P. T. M. Zevenhuizen and S. Bartnicki-Garcia,]. Gen. Microbiol., 61 (1970) 183188. (52) J. S. D. Bacon, V. C. Farmer, D. Jones, and I. F. Taylor, Biochem. J., 114 (1969) 557-567. (53) D. J. Manners and A. J. Masson, FEBS Lett., 4 (1969) 122-124. (54) D. J. Manners, A. J. Masson, and J. C. Patterson,]. Gen. Microbiol., 80 (1974)411417. (55) D. J. Manners, A. J. Masson, J. C. Patterson, H. Bjorndal, and B. Lindberg, Biochem. j., 135 (1973) 31-36.

74

E. BARRETO-BEHGTER A N D PHlLIP A. J. GORIN

in the polysaccharides in the walls of Kloeckera apiculata, Schizosaechurorriyces pombe, Saccharomyces fragilis, and Saccharomyces fermentati.) The latter polysaccharide, of mol. wt. 2.4 x lo5, is less branched, havings6 (143) (85%)and interchain, (1+6) linkages (3%). Smith degradations of total glucans gave glucosylglycero1,j6~5'laminardbiosyiglycerol, and larninaratriosylglycer01.~~ The alkali-soluble P-D-giucopyranan contains 3-0- (80-85%), 6 0 (8- 12%),and 3,6-di0-substituted (3-4%) units'." 'Chc pu-glucopyranans of Candida albicatis serotype B and Candicfu yarupsilosis are mainly linear, with only 10%of branch points, and contain, principally, ( 1 4 6 ) linkages (67 and 63%, respectively).59

-

TABLEI1 Degree of Side-chain Substitution in Glucans Having the General Structure 3 ~

Source of glucan

Sclerotinia glucanicum, exocellulaP1 Pythircm ncunthicum, hyphal wall" Sclerotinia libertinna, exocellulaP Monolinia fructigena, exocellular (mol. wt. 7 x l(r)&la Schizophyllum communewD Claciceps fusiformus (Loveless), exocelluIaP Aweobusidium (Pulluluria) pullulnns, cell wallm Pin'culuria oryzue, cell wall6'

~

~~

~~

Value of n in structure 3

2 2 2 1 2 3 8 5

(56) D. J. Manners, A . J. Masson, and J. C. Patterson, Biochem. I. 135 ,(1973) 19-30. (57) A. Misaki, J . Johnson, Jr., S. Kirkwood, J. V. Scaletti, and F. Smith, Carbohydr. Has.. 6 (1968) 150-164. (58) G . H. Fleet and D. J. Manners,J. Gen. Microbiol., 94 (1976) 180-192. (59) R. J. Yti, C . T. Bishop, F. P. Cooper, F. Blank, and H. F. Hasenclever, Can. J. Cherii., 45 (1967) 2264-2267. (60)K. Buck, A. LV. Cheti, A. G. Dickerson, and E. B. Chain, J. Gen. Microbiol., 51 (1968)337-352. (61) I . j. Goldstein, G. W. Hay, B. A. Lewis, and F. Smith,Abstr. Pap. Am. Chem. SOC. 3fcrt.. 135 (19591 3D. (62) J. H. Sietsma, J. J. Child, L. R. Nesbitt, and R. H. Haskins,]. Cen. Microbiol., 86 (1975) 29-38. (63) Y. Ueno, Y. Hachisuka, H. Esaki, R. Yamauchi, and K. Kato,Agric. B i d . Chem.,44 (1980) 353-359. (Wa) F. Santamaria, F. Reyes, and R. Lahoz,]. Cen. Microbiol., 109 (1978) 287-293. (644h)S. Kikumoto, T. Miyajima, K. Kimura, S. Okubo, and K. Kumatsu, Nippon Nogei Kupaku Kaishi, 45 (1970) 162-168. (65) S . Komatsu, S. Ohkubo, S.Kikumoto, G. Saito, and S. Sakai, Gunn, 60 (1969) 137144. (66) R. G. Brown and B. Lindherg,Acto Chem. Scand., 21 (1967) 2379-2382. (67) T. Nakajima, K. Tamari, K. Matsuda, H. Tanaka, and N. Ogasawara, Agric. Biol. Chem., 36 (1972) 11- 17.

POLYSACCHARIDES FROM FUNGI AND LICHENS

75

A number of P-D-glUCanS have been reported to contain a (143)linked, ~-D-glUCOpyranOSyl main-chain, partially substituted at 0-6 by (single unit) PD-glUCOpyranOSyl groups, as, for example, in repeatingunit 3. The degree of substitution varies with the fungus (see Table p-~-Glcp

I

6

-[p-D-Glcp -(1-t3)]n-@-D-GlCp -(1-+3)3

11). With Claviceps fusiformis, the exocellular glucan has a degree of branching that increases to a maximum of 1, in every third, main-chain residue, after 14 days, and then slowly declines.60The glucans from Aureobasidium (Pullularia) pullulans and Pirikularia oryxae cellwalls contain little branching, and, in the case of the latter glucan, the main chain has a few (1-6) linkages. Lentinan, from Lentinus edodes, appears to have a main chain consisting largely of (1-+3) [and some (1+6)] linkages, together with (143) and (146) links in the side chains.68 The fruit bodies of Auricularia auricula-judae contain two branched FD-glucopyranans having (143) and (1+6) linkages. The soluble one could have a structure similar to that of 3, but the other, which is insoluble in alkali, contains 6-0-substituted units.69

IV. GLUCANSFROM LJCHENS A partially acetylated, (1-+6)-linked P-D-glucopyranan has been extracted from Gyrophora esculenta Miyoshi. The degree and position(s) of substitution have not yet been determined. A similar glucan has been found in Lasallia papulosa (Ach.) Llano.70The unacetylated glucan gives a 13C-n.m.r. spectrum7l similar to that of pustulan from Umbilicaria p ~ s t u l a t a . ~ ~ Common glucans from lichens are lichenan, a p-D-glucan having (143) and (1-4) linkages in the ratio of 3: 7 (approximate repeatingunit 4), and isolichenan, an a-D-glucan having (1-+3) and ( 1 4 4 ) linkages in the ratio of 11 :9. Such components are present in Cetraria ri(68) T. Sasaki and N. Taksuka, Carbohydr. Res., 47 (1976) 99-104. (69) Y. Sone, K. Kakuta, and A. Misaki, Agric. Biol. Chem., 42 (1978)417-425. (70) S. Shibata, Y. Nishikawa, T. Takeda, M. Tanaka, F. Fukuoka, and M. Nakanishi, Chem. Pham. Bull., 16 (1968) 1639-1641. (71) H. Sait6, T. Ohki, N. Takasuka, and T. Sasaki, Carbohydr. Res., 58 (1977)295-305. (72) D. Bassieux, D. (Y.) Gagnaire, and M. (R.) Vignon, Carbohydr. Res., 56 (1977) 19-33.

76

E. BARRETO-BERGTER AND PHILIP A. J. GORIN

churdsonii Hook,73Alectoria sulcata (Lev.) Nyl., and Alectoria sarmentosa. 74 -S-D-GlCP -(1-4)-p-D-Glcp-(1-4)-~-D-Glcp-(l~3)4

Purnieliu caperuta (L.)Ach. provided a cold-water-insoluble amglucopyranan having (1-3)- and (1-+4)-linked residues in equimolar proportions. As there were no successive, (1-+3) and (1-+4)linkages, the glucan is structurally similar to nigeran (repeating unit 5).75 - 0 - D-

GlC p- (1-

3)- (1-D-Glcp - ( 1 - 4 ) 5

From chemical the glucan from Stereocaulon japonicum apparently had a-(1-3) and a-(1-4) linkages in the ratio of 3 : 1, but This technique the ratio was shown to be 2: 1 by a 'T-n.m.r. was useful in showing that a-Dglucans from Sphaerophorus glohosus and Acroscyphus sphaerophoroides had (1-+3) and (1-4) linkages in the ratio of 2 :3, with <6% of (1-+6) linkages, the latter value not being clear from methylation analysis.74 A cold-water-insoluble polysaccharide, evernian, from Evernia prunastri (L.) Ach. was found to be an a-D-glucan having (1-3) and (1-+4) linkages in the ratio7Rof 4 : 1. In a further study, the cold-watersoluble fraction provided a p-Dglucan, precipitated by cetyltrimethylammonium hydroxide, and an a-wglucan in the supernatant liquor. The p-D-glucan contained (1-+3) and (1-4) linkages in the ratio of 3 : 1,and the cr-D-glucan contained these linkages in the ratio of 3 :2, resembling i s o l i ~ h e n a n .The ~ ~ I3C-n.m.r. spectrum of lichenan, obtained at high magnetic field,79corresponded to a (1++4),(1--+4),(1-3) repeating-sequence, giving 15 signals out of a possible 18. The presence of two consecutive, (1-3) linkages is not likely, as there was no splitting of signals associated with (1-3) linkages [whereas there was with those of (1-+4) linkages].

-

(73) Y. Nishikawa, S . Kobayashi, S. Shibata, F. Fukuoka, and S. Maekawa,J . J p n . Biochem. Soc., 41 (1969) 632. (74) T. Takeda, M . Furiatsu, S. Shibata, and F. Fukuoka, Chem. Pharm. Bull., 20 (1972) 2445-2449. (7.5) T. Takeda, Y. Nishikawa, and S. Shittata, Chem. Phurm. Bull., 18 (1970) 10741075. (76) 1. Yokota and S. Shibata, Chem. Pharm. Bull., 26 (1978) 2668-2670. (77) 1. Yokota, S. Shibata, and H. Sait6, Carbohydr. Res., 69 (1979)252-258. (78) V. Stefanovich, Lije Sci., 8 (1969) 1223-1233. (79) 1).(Y.)Cagnaire and M. Vincendon, Bull. Soc. Chim. Fr., 5-6 (1977) 479-482.

POLYSACCHARIDES FROM FUNGI AND LICHENS

77

V. MANNANS The cell walls of certain fungi contain water-soluble polysaccharides, in addition to skeletal chitin and ED-glucan. Many yeasts contain mannose, and, sometimes, other sugars, and these are often bound to protein and phosphate. In Saccharomyces cereuisiae, this is a surface component,80and so are the surface antigens in several fungi (for a summary, see Ref. 81). Fully synthesized mannan was found in cell walls, on the plasmalemma, and within the cytoplasm of S. cereuisiae and Candida utilis, as detected by homologous, anti-mannan antibodies, or with con A, both labelled with gold granules.82

1. Linear Mannans Mannans of Hansenula capsulata Y-1842 and H . holstii Y-2448 differ structurally from the phosphonomannan component^.^^ The mannan of H . capsulata is probably a cell-wall component, and it has the linear, repeating-structure 6. Other Pichia and Hansenula mannans have predominantly linear structures.84The 13C-n.m.r. spectrum of 6 (see Fig. 2) is distinguishable from that of the branched-chain mannans5from Saccharomyces rouxii (repeating unit 7.) -cu-~-Manp-(l-cZ)-cu-D-Manp-(l-Z)-cu-D-lManp-(1-6)6

I

2 - c u - ~ - M a n p- (1-

6)-

7

The cell-wall mannan of Candida sp. P.R.L. 1520 is almost linear, with a block type of structure involving successive (1+3)- and (1-6)linked eDmannopyranosy1 units.86 (80) B. Mundkur, E r p . Cell Res., 20 (1960) 28-42. (81) K. 0. Lloyd, Biochemistry, 9 (1970) 2446-2453. (82) M. Horisberger and M. Vonlanthen, Arch. Microbiol., 115 (1977) 1-7. (83) M. E. Slodki, M. J. Safranski, D. E. Hensley, and G. E. Babcock, Appl. Microbiol., 19 (1970) 1019- 1020. (84) P. A. J. Gorin and J. F. T. Spencer, Can. J . Microbiol., 18 (1972) 1709-1715. (85) P. A. J. Gorin, Can. J . Chem., 51 (1973) 2375-2883. (86) P. A. J. Gorin and J. F. T. Spencer, Can. J . Chem., 48 (1970) 198-200.

78

E. BARRETO-BERCTER A N D PHILIP A. J. GORIN

FIG.2.--”C-h.ni.r.

Spectrum of Mannan from Harisenula cupsulotu in DzO. (Nu-

iiwrical values are chemical shifts, &.)

The mannan Erom Rhodotorula glutinis contains87 alternating and (1-+4)-linked P-D-mannopyranosyl residues (repeating structure 8). The absence of consecutive, (1-3) and (1+4) linkages

( 1-3)-

-3-0-Manp - (1-3)

- p - ~ - M a n p - (1-

4)-

a was confirmed by its W-n.ni.r. spectrum (see Fig. 3), which had 12 signals, corresponding to a two-unit repeating-sequence.HR

2. Branched-chain Mannans

The mannan and glycoprotein from the cell wall of Saccharomyces ceretiisioe have been extensively investigated b y Ballou and coworke r ~ The . ~ ~ niannan contains a ( 1+6)-linked, a-D-mannopyranosyl

I 80

1

100

6, (relative

I

60

to Ma,%)

FIG. 3.--13C-N.m.r. Spectruiii of Mannan from Rhodotomla ghtinis.

J. F. T. Spencer, Can. /. Chern., 43 (1965) 950954. (88) J. F. T. Spencer and P. A. J. Corin, Biotechnol. Bioeng., 15 (1973) 1-12. (89)T. S. Stewart, P. B. Mendershausen, and C. E. Ballou, Biochmistrtt, 7 (1968) 1843 - 18.54.

(87) P. A. J. Gorin, K. Horitsu, and

POLYSACCHARIDES FROM FUNGI AND LICHENS

79

main-chain7 substituted on the 0 - 2 atoms by a-DManp, a-D-Manp(1+2)-a-D-Manp, and a-mManp-( 1+3)-a-~-Manp-(1+2)-a-~-Manp side-chains. Mild acetolysis gave rise to penta-, hexa-, and hepta-saccharides containing the di-, tri-, and tetra-saccharide fragments obtained on stronger acetolysis, and these were connected by (1+6) links. These also showed the sequence of the side-chain units of different lengths distributed along the main chain.89Very little (146)linked tetrasaccharide was obtained on partial acetolysis of mannan from S. cerevisiae 4484-24D7thus favoring structure 10. The pentasaccharide was mainly M2+M,, consistent with structure 9, and possibly 10, but not structure 11. The results showed that the side-chain sequence is non-random, but a single repeating-sequence was not proved." M

i

M 1

1

- M- (1-6)-

M 1

I

2 2 M- (1*6)-M- (1-6)-

2 M 1

1

2 M- (1-

6)-

9 M = CI-D-M~

M 1

M 1 t 2 M 1

I

4

M 1

I

2 2 2 -M-(l-6)-M-(l+6)-M-(l*6)-M-(l-+6)10

M

1

2 M 1

M 1

M

i

2

2

1

-M-(1-6)-

1

M- (1-+6)-M- (1-

f

6)-M- (1-6)-

11

(90)L. Rosenfeld and C. E. Bailou, Biochem. Biophys. Res. Commun., 63 (1975) 571579.

80

E. BARRETO-BERGTER AND PHILIP A. J . GORIN

Studies on a-D-mannopyranan structures were greatly facilitated by the use of an a-D-mannosidase (from an Arthrobacter sp.) that preferentially removes side-chain~,~' giving /3-*mannose. Such p-D-mannopyranosyl-containing structures as fl-D-Manp-(1-+2)-p-~-Manp( 1-+2)-@D-Manp-(1-+2)-D-Manp and a-D-Manp-(l-S)-p-~-Manp(1-+2)-P-~-Manp-( 1-+2)-a-~Manp-(1+2)-~-Man are resistant to enzymic attack.= S. cerevisiae mannan was degraded to a (1-6)linked a - ~ m a n n o p y r a n a n . ~ ~ present in mannan preparaTraces of 2-amino-2-deoxy-~-glucose tions arise from 2-acetamido-2-deoxy-~-glucosylunits in the mannanprotein bridge. These are periodate-resistant, and, therefore, probably 3- or 4-O-sub~tituted,~ the latter possibility being proved by methylation, and analysis of the 2-deoxy-O-methyl-2-(methylamino)-mannose fragments by g.l.c., and by the Spinco amino-acid analy~er.9~ A mannan-protein bridge is indicated, consisting of glycosyl units attached to the hydroxyl groups of serine and threonine, as 2-0- and 60-a-D-mannopyranosyl-Dmannose were formed on alkaline p-elimination.% An oligosaccharide fragment corresponding to the bridge region in S . cerevisiae X2180 mannan was obtained by concomitant action of an endo-a-D-(1-+6)-mannanase and an endo-2-acetamido-2-deoxy-p-~glucosidase on the mutant mannan of S . cerevisiae X2180-1A-5, which has an unbranched-polysaccharide outer-chain. It had 12 units, with a 2-acetamido-2-deoxy-~-glucose reducing-end residue, and gave, on partial acetolysis, Dmannose, a-~-Manp-(1-+2)-D-Man,a-D-Manp(1-+3)-~-Man,D-mannotrioses having (1-2) and (1-3) linkages, a Dmannotetraose, and a-DManp-(1-+3)-a-~-Manp-( l-+2)-a-D-Manp(1+3)-p-~-Manp-(1+4)-~-GlcNAc. These were interlinked with (1-6) bonds. The reducing end should arise from a -P-DGlcpNAc(1-+4)-p-~-GlepNAc-Asn-s t n i ~ t u r e The . ~ ~ aforementioned fragment could be fractionated, to give molecules ranging from Man,,GlcNAc to Man,,GlcNAc. Structure 12 was proposedgxfor the glycoprotein from S. cereoisiae X2180.

-

(91) G . If. Jones and C. E. Ballou,]. Biol. Chem., 244 (1969) 1043-1051. (92) P. A. J. Corin, J. F. T. Spencer, and D. E. Eveleigh, Carbohydr. Res., 11 (1969) 387 -398. (93) G. H . Jones and C. E. Ballou,]. Biol. Chem., 244 (1969) 1052-1059. (94)R. Sentandreu and D. H . Northcote, Biochem. J . , 109 (1968) 419-432. (95) P. A. J. Gorin, Con. J . Chem., 49 (1971) 527-529. (96)R. Sentandreu and D. H . Northcote, Carbohydr. Res., 10 (1969) 584-585. (97) T. Nakajima and C. E. Bdlou,J. Biol. Chem., 249 (1974) 7685-7694. (98) T. Nakajima and C. E. Ballou, Biochem. Biophys. Res. Comrnun., 66 (1975) 870879.

[M-(l-a)-M-(1-6)-M-(1-6)-M-(l~ 2 2 2

tI

1 M

t

i

t i

M

M

M

M 3

t1

M

M

M

M

I

Inner core

Cuter chain

M-(I--z)-M-(~+z)-M--

Base-labile oligosaccharides 12 M = LY-D-MWI~

82

E. BARRETO-BERGTEH AND PHILIP A. J . GORIN

Apart from the presence of chitin, no gross structural differences were detected between the bud scars of S . cereuisiue and the rest of the cell wall.99 Branched-chain mannans from Candida species were analyzed by the inethylation technique, and the presence of 2,6-di-O-substituted mannofuranosyl units was suggested on the basis of supposed 3,Ei-di0-methyl-D-mannose isolated.100However, this compound proved to be identical to synthetic 2,4-di-O-methyl-~-mannose,~~~ and it differed from the authentic 3,S-dia-methyl isomer,102which is consistent with 3,6-di-O-substituted D-mannopyranosyl units. Partial-acetolysis studies conducted on yeast D-mannopyranans containing (1-+6)-linked a-Dmannopyranosyl main-chains revealed a large variety of side-chain structures, as represented by oligosaccharides isolated from the acetolyzate (see Table 111). Following purification b y cellulose-column chromatography, the oligosaccharides were identified by their typical, 13C-n.m.r.spectra.85 A number of yeast mannans containing a-and P-D-mannopyranosyl units exist, and the latter structure was detected by distinctive, H-1 signals in 'H-n.1n.r. spectra at fields higher than 6 5.43 (see Table IV). The mannans from Piclzia pastoris and Citeromyces matritensis respectively contain structures 13 and 14. Main chains that were isolated

0-D- M a p 1

I

2 8-D - M p~ 1

p-~-Manp

1

2 B-D-ManP

1

I2

&~-Manp

T 2 - f ~ - ~ - M a n-(1-+6)p 13

1

i

2

- CY -D-Manp - (1-6)

-

14

were prepared by selective, acid hydrolysis of side-chain, p-D-mannopyranosyl units, followed by treatment with a - ~ m a n n o s i d a s eSide .~~ (99) 11. A. Bush arid .M. Horisberger,]. B i d . Chetn., 248 (1973) 1318-1320. (100) R. J . Yu, C . T.Bishop, F. P. Cooper, H . F. Hasenciever, and F. Blank, Can. J. Chem., 47 (1967) 2205-2211. (1011 S. S . Bhattacharjee and P. A. J. Gorin, Can.J. Chem., 47 (1969) 1207-1215. (I&!) I . H. Siddiqui and V. L. N. Mu*, Corbohydr. Res., 8 (1968)477-481.

POLYSACCHARIDES FROM FUNGI AND LICHENS

83

TABLEI11 Oligosaccharide Fragments Obtained Following Partial Acetolysis of Mannans Having (1+6)-Linked a-D-Mannopyranosyl Main-Chains Parent yeast Torulopsis b o m b i ~ o l a ' ~ ~ Torulopsis magnoliae'" Torulopsis apicola'" Torulopsis gropengiesseri'" Saccharomyces rouxii103 Endomycopsis jibuliger'" Trichosporon aculeatum'm Saccharomyces cerevisiaesS Saccharomyces fragiliss5 Hansenula subpelliculosa'"

Oligosaccharide formed" [M-( 1-+2)-]1-7-M [M-( 1+2)-]1+-M [M-(1+2)-]l-s-M [M-( 1+2)-]l-s-M [M-( 1+2)-]l-z-M M-( 1+2)-M M-( 1+3)-M-( 1+2)-M [M-(l+2)-],-7-M M-(1+3)-[M-(1+2)-],-M M-( 1+2)-M M-( 1+2)-M M-(1+3)-[M-(1-+2)-]3-M M-( 1+3)-M [M-( 1+2)1 i-rM

Various Saccharomyces ~ p p . and '~~ Metschnikowia reukaujii'DB M-( 1+3)-M-( 1+3)-M-( 1+2)-M-( 1+2)-M Debaryomyces hanseniF M-( 1+2)-M-( 1+3)-M-( 1+2)-M-( 1+2)-M Hansenula wingei'@' M-( 1+3)-M-( 1+2)-M-( 1+2)-M, M-(l+3)-M-(l+2)-M, 4 possible trisaccharides having (1-2) and (1-3) linkages, M-( 1+2)-M, M-( 1+3)-M a

M

= a-DManp.

chains were characterized by isolation of oligosaccharides by way of partial a c e t o l y s i ~ . 'The ~ ~ fragment obtained from 13 was a-D-Manp(1+2)-P-~-Manp-( l+2)-P-D-Manp-( l+2)-a-~-Manp-( l+z)-D-Man. The positions of glycosidic linkages were determined by methylation studies, and the sequence of glycosidic configurations was elucidated as follows. Fragments were obtained from the pentasaccharide by a Smith degradation involving oxidation with lead tetraacetate in acetic acid, followed by reduction with sodium borohydride and partial hydrolysis (103) P. A. J. Gorin, J. F. T. Spencer, and R. J. Magus, Can. J. Chem., 4 7 (1969) 36593576. (104) P. H. Yen and C. E. Ballou, Biochemistry, 13 (1974) 2420-2427. (105) C. E. Ballou, P. N. Lipke, and W. C. Raschke,J. Bacteriol., 117 (1974) 461-467. (106) J. F. T. Spencer and P. A. J. Gorin, Antonie van Leeuwenhoek, J. Microbiol. Serol., 37 (1971) 75-80. (107) P. A. J. Gorin, J. F. T. Spencer, and S. S . Bhattachajee, Can. 1.Chern., 47 (1969) 1499- 1505.

a4

E. BARRETO-BERGTER AND PHILIP A. J. GORIN

TABLEIV Yeasts Producing Mannans Giving H-1 Signals at Higher Field than 6 5.43 Brettanomyces anomalus bruxellensis dublinensis lambicus Candida obtusa lusitaniae Citeromyces matritensis De baryomyces castelli hansenii kioeckeri PhafF subglobosus Hanseniaspora osmophila Kloeckera africana magna

Pichia farinosa ohmeri pastoris toletana oanrijii Saccharornyces lodderi microellipsodes pre toriensis rosei tiafer Schwanniomyces alluvius Schwanniomyces castelli Torulopsis colliculosa Trichosporon aculeatum

(pH 2,1000) ofthe resulting alditols. Following use of 3 mol of oxidant per mol of pentasaccharide for 3 h, mannotetraosylmannitol, mannotriosylmannitol, and mannobiosylmannitol were formed, and isolated. With a higher proportion of oxidant, acting over a longer period of tirne, 2-@cr-D-mannopyranosyl-D-mannitol was obtained, and tentatively identified by the chemical shift of H-1, and its mobility on a paper chromatogram. Assignments of configuration were made by comparing the specific rotations of consecutively related pairs of compounds that were formed during the degradation. For example, the reduced trisaccharide must contain a p-linkage, as it has a specific rotation ( - 14") lower than that of 2-0-a-D-mannopyranosyl-D-mannitol (+ 14").By consideration of the consecutive increases, or decreases, in specific rotation on going from reduced trisaccharide to reduced tetrasaccharide to reduced pentasaccharide, the last compound must have the structure indicated as the pentasaccharidic part of 13.

3. Phosphonomannans To the phosphonomannan of Hansenula capsulata Y-1842 was attributed the principal structure 15, as, on partial hydrolysis, it gave a mannobiose 6-phosphate (16) which provided 1 mol of formalde-

POLYSACCHARIDES FROM FUNGI AND LICHENS

85

0

1

CH20H

1

It

HO-P-TH,

qH20H

OH 16

15

hyde/mol on periodate oxidation.losHowever, the 13C-n.m.r.spectrum of the mannobiose 6-phosphate contained two C-5 signals having 3bond, '3C, 31P coupling, corresponding to a reducing end having the a,P-pyranose structure 18, instead of a single, coupled C-5' signal from109 structure 16. The parent structure 17 was favored by the results of W-n.m.r. experiments using EuC1, and PrC1, (which shift the resonances of the carbon signals attached to phosphate and those of adjacent carbon atoms). In the 13C-n.m.r. spectrum of partly degraded phosphonomannan, the signals were too broad for coupling to be detected, although the coupled resonances were sometimes lessened in height. On addition of the shift reagent, a signal corresponding to C-5 of an a-D-mannopyranosyl unit was displaced, thus favoring structure"0 17.

1

0

II

HO-P-OCH, I YH,OH OH

@ *-

I

HO

.OH

18 s

2

OH

17

Hansenuh holstii NCYC 560 produces cell-wall, and exocellular, phosphorylated mannans of mol. wt. lo6.The cell-wall mannan contains two branched polysaccharides having (1-2) and (1-+3)linkages, with p-D,in addition to CZ-D,linkages. Phosphoric diesters are present. The exocellular phosphonomannan is composed of 3 polymers, each (108) M. E. Slodki, Biochim. Biophys. Acta, 69 (1963)96-102. (109) P. A. J. Gorin, Can. /. Chem., 51 (1973) 2105-2109. (110) P. A. J. Gorin and M. Mazurek, Can. J . Chem., 52 (1974) 3070-3076.

86

E. BARRETO-BERGTEH AND PHILIP A. J. GORIN

having a (l+6)-lhked a-Bmannopyranosyl main-chain, and side chains having a-(1-2) and a-(143) linkages and snbstituted with rnonophosphoric esters."' The phosphononiannan of H . Aolstii NRRL Y-2448, which contains mannose and phosphorus in the molar ratio of 5 : 1 a i d phosphoric. diesters bridging 0-6 and 0-1 of mannosyl resiclues, was partially hydrolyzed. A product accounting for 65% of the intact polymer appeared to be a-D-&'Ianp-(l-S)-a-D-Manp-( 1-3)-a-DManp-(1+3ba-~-&'Iarip-( 1+2)-D-h'Ian, substituted with a phosphate groiip at C-6 of the nonreducing end.112 The phosphate to ~iiiiniioseratios varied from 1: 144 to 1:9 in phosplioiiomaiinans isolated from baker's yeast, Saccharomyces cereuisiae 238C, Succlzuromyce.s carlsbergensis ATCC 9080, Canclidu albicans 792, Catiditfa stellatoidea, and Kloekeru brevis (H Phaff 5545). The polymer from K . breuis, having the highest content of phosphorus, contained phosphoric diester groups that survived when the polymer w a s extracted under mild conditions. The action of a-D-mannosidase preferentially removes some side chains, leaving a polymeric residue '~ Kloekeru brevis phosphohaving a high phosphorus ~ 0 n t e n t . I From nomannan, 4 fractions were obtained, having mannose to phosphoric cliester ratios of20: 1 to 6.5: 1.The niannose was linked through 0-1to phosphate in the (Y configuration, as shown b y proton-1i.rn.r. spectroscopy. Smith degradation, partial acid-hydrolysis, and "P-n.rn.r. spectroscopy showed that the phosphate was linked to 0 - 6 of a D-mannosyl residue of a 3-unit side-chain, as in stnicture"" 19. a-D-Mannopyranosyl phosphate units constitute the immunodominant group.'lS

i

2

i

i

2

2

-M-(1-6)-M-(l-S)-M-(l-6)-M_I

1-(1-6)-

19

A siniilar, side-chain structure was found present in the phosphononimnan of S . cerecisiae, except that mannobiose, as well as mannose, \vas fomied on partial acid-hydrolysis, and that the phosphate was linked to 0-6 of 3-unit and 4-unit side-chains.114Such a structure (20) ( 1 1 1 ) C;. Sa~i-Blasa i d W.L. Cunninghain, Biochim. Biophys. Actu, 354 (1974) 233-246. ( 1121 H. I<. Bretthaiier, B. J. Kaczorowski, and bf. J. Wiese, Biochenii.str!y, 12 (1973) 1231-1236. i 113) 'I. S. Stewart and C . E. Ballou, Biochemistry, 7 (1968) 1855-1863. i 114) T. .I. Thierne and C. E. Ballou, Biochemistry, 10 (1971) 4121-4129. ( 1 15! \V. C. Haschke and C. E. Ballou, Biochemistry, 10 (1971) 4130-4135.

POLYSACCHARIDES FROM FUNGI AND LICHENS

87

is present in S . cerevisiae X2180 phosphonomannan having a mannose :phosphate ratio of 38 : 1. Other strains lack this tetrasaccharide unit, and, instead, possess a side chain (21) in which the a-D-mannOSyl phosphate group is immunodominant.116 M

=

a-~-Manp

cu-D-Manp-(l-3)-Cf-D-Manp-(l-P

1 6

cu-D-Manp-(l-3)-Cf-D-Manp-(1~2)-cu-D-ManP-(1-22)20

Cf-D-

&p-

(1-P

i

6

Cf-D-Manp-(l-Z)-cu-D-Manp-(1-2)21

As would be expected from the foregoing observations, in phosphate-rich fractions of cell walls of S . cerevisiae, the phosphate in the side chain shields mannosyl residues from attack by a-D-mannosidas e. The polysaccharide from Hansenula polymorpha 52-251 has a block type of structure of the main chain, with (1-2)- and (1+6)-linked WDmannopyranosyl units. The side chains also contain (1+2) links interspersed with (1+6) links. Immunochemical experiments revealed the presence of a-D-ghCOpyranOSyl phosphate determinants,"* in common with the polysaccharide of Hansenula wingei, a yeast of interest because of its sexual, agglutination factors.'W

VI. GALACTANS The malonogalactan of Penicillium citrinium Thom 1131 has a (1+5)-linked @-Dgalactofuranosyl chain, and its resistance to periodate oxidation seemed to point to esterification at OH-2, or OH-3, or both.11gHowever, the 13C-n.m.r.spectrum120appeared to indicate esterification at OH-6 (22). The spectrum of the malonylgalactan contains signals, at 6,66 and 76, that are not present in that of the galactan. These may be interpreted as arising from an a-shift of +3.5 p.p.m. (from the C-6 resonance at 6,62.5), and a @-shiftof - 1.5 p.p.m. (from the C-5 resonance at 6, 77.5).7Presumably, the lack of periodate oxidation was due to the presence of resistant, trans-OH-2,OH-3 groups on a 5-membered ring. (116) L. Rosenfeld and C. E. Ballou,J. Biol. Chem., 249 (1974) 2319-2321. (117) W. J. Colonna and J. 0. Lampen, Biochemistry, 13 (1974) 2741-2748. (118) P. N. Lipke, W. C. Raschke, and C. E. Ballou, Carbohydr. Res., 37 (1974) 23-35. (119) T. Kohama, M. Fujimoto, A. Kuniaka, and H. Yoshino, Agric. B i d . Chem., 38 (1974) 127- 134. (120) M. Ogura, T. Kohama, M. Fujimoto, A. Kuninaka, H. Yoshino, and H. Sugiyama, Agric. Biol. Chem., 38 (1974) 2563-2564.

88

E. BARRETO-BERGTER AND PHILIP A. J . GORIN

HCO-

HO-C -CH2II

0

I

AH

COCH, II

0

22

The nuclei of the acellular, slime mold Physariurn polycephalum contains a P-Dgalactan (d.p. 560) bearing phosphate (2.5%) and sulfate (9.6%) groups. One unit in every 13 is branched, but the main structural-feature is 4-0-substituted Dgalactopyranosyl units.’*l It resembles the exocellular polysaccharide.lZ2

ACID POLYMER VII. 2-ACETAMIDO-2-DEOXY-D-GLUCURONIC A polysaccharide containing 2-acetamido-2-deoxy-~-glucuronic acid residues was isolated from the black yeast Rhinocladiella eliator Mangenot NRRL YB-4613, obtained from a “slime outbreak” at a pulp and paper mill. The exocellular material was purifed by Cetavlon pre~ cipitation, and its specific rotation (- 75”) indicated p - linkages.12Y Methylation analysis of partially degraded polysaccharide, following carboxyl reduction and acid hydrolysis, provided 2-deoxy-3-0methyl-2-(N-methylacetamido)-~glucose, showing the presence of (1-4) linkageslZ4(23).

NHAc

23

VIII. 2-AMINO-2-DEOXY-D-GALACTOPYRANAN The myxomycete Physarium poEycephalum produces hard-walled sclerotia (spherules) that contain 88%of 2-amino-2-deoxy-~-galactose. The positive, specific rotation, and the g.1.c. characterization of 1,5-di(121) D. R. Farr and M. Horisberger, Biochirn. Biophys. Acta, 539 (1978) 37-40. (122) D. R. Fan, H. Amster, and M. Horisberger, Carbohydr. Res., 24 (1972)207-209. (123) P. R. Watson, P. A. Sandford, K. A. Burton, M. C. Cadmus, and A. Jeanes, Carbohydr. Res., 46 (1976) 259-265. (124) L. Kenne, B. Lindberg, K. Peterson, and P. Unger, Carbohydr. Res., 84 (1980) 184- 186.

POLYSACCHARIDES FROM FUNGI AND LICHENS

89

O-acetyl-2-deoxy-2,3,6-tri-O-methyl-2-(N-methylacetamido)galactitol, following a methylation procedure, indicated a (1+4)-linked 2-amino2-deoxy-a-~-galactopyranan repeating-structure (24). The spherule wall consists of two layers of fibrils, having different staining properties, and it appears that the purified wall is composed of 2-amino-2deoxy-D-galactosyl residues; the outer layer is readily removed during p~rificati0n.l~~ P. polycephulum produces a viscous, extracellular p-Dgalactan,121J22 probably analogous to this outer, slime layer. CH,OH I

IX. HETEROPOLYSACCHARIDES BASEDON D-MANNAN MAIN-CHAINS

1. Rhamnomannans Rhamnomannans are present in the cell walls of several Cerutocystis and Graphium ~ p pCells . ~ of Cerutocystis ulmi, the causative agent of Dutch-elm disease, were extracted with hot alkali, and the resulting rhamnomannan was purified by way of its insoluble copper complex, formed with Fehling solution. It consists mainly of a (1-6)linked, a-D-mannopyranosyl main-chain substituted at 0-3 by (singleunit) a-L-rhamnopyranosyl groups126(25). The rhamnomannan is related to phytotoxic glycopeptides formed in culture media. The toxin contains the foregoing polysaccharide structure linked to amino acid residues (7%)by 0-glycosyl linkages to threonine and to serine resid u e ~ . ~ ~ ~ 0-L-Rhap 1

1

-cy-~-Manp-(l-+G)25

(125) D. R. Farr, A. Schuler-Hovanessian. and M. Horisberger, Carbohydr. Res., 59 (1977) 151-154. (126) P. A. J. Gorin and J. F. T. Spencer, Carbohydr. Res., 13 (1970) 339-349. (127) G. Strobel, N. van Alfen, K. D. Hapner, M. McNeil, and P. Albersheim, Biochirn. Biophys. Acta, 538 (1978) 60-75.

90

E. BARRETO-BERGTER AND PHILIP A. J. GORIN

Yeast form\ of the dimorphic yeast Sporothrix schenckii, grown at 370, produce12xrhainnomannan 25, but, at 25", small proportions of 40- and 2,4,di-O-substituted a-D-mannopyranosyl structures129 are formed, as13oin 26, 27, and 28. Structure 25 was formed by alkaline degradation of a peptir~orhaxiinoinannanpresent in the cells and the ciilture niedium .

- a - DManp - (1-

4)-

26 (r - D - Man p

1

1

n L

-(~-~-Manp-(l--)-(~-D-ManP-(l-4)27 CY

- D- Manp 1

1 2

- {y - D- M a p - (1-

a-~-Manp

1

1

-

2 4) - (Y- D- Manp - (1

4)- (Y - D - Mano - (1-

4)-

28

Conitlia of S . scherickii produce a rhamnoniannan having structiire1'$225, h i t the mycelia form dirhamnosyl side-chains, as in 29, that are the main antigenic Cerutocljstis stenoceras, which coexists with S. schenckii in its natural habitat, produces an acidic rhainnomannan containing structure 30, in addition1.'" to 25. The acidic component is probably a feature of S. d?erickii polysaccharides, and there is a general similarity between the two organisms, except that dirhamnosyl side-chains (29) are formed onl! b y S . scherzckii. 134 The foregoing investigations were aided b y l"C-n.m.r. spectroscopy in structural studies and in monitoring polysaccharide composition in the multicomponent system. The signal shifts are often characteristic of a structural component of a mixture. A review of work conducted on S. sclieiickii and related Cerutoc!/stis 'pp \vas published b y Travassos and Lloyd.135 (1281 L. llt.ntlonc;ii, P. A. J . Gorin, K. 0. Lloyd, and L. R. Travassos, Biochernistrcl, 15 (1976) 2423-2431.

129) I,. R. Travassos, P. A. J . Gorin, and K. 0.Lloyd,Znfect. Ztnmun., 8 (1973) 685-693. ( 130) P. A. J. Gorin, R. H . Haskins, L. R . Travassos, and L. Mendonqa-Previato, Curboh p l r . R r s . . 55 (1977) 21-33. I

(

1311

K. 0. I.loytl and M.A. Ritoon,]. I t n t n u n o l . , 107 (1971) 663-671.

132) I,. H. Travassos and L. ~lendoiiqa-Previato,Ztifect. Zmmun., 19 (1978) 1-4. (1.33) K . 0. Lloyd and L. R. Travassos, Corbohydr. Res., 40 (1975) 89-97. (134) L. R. Travassos, L. Mendonca-Previato, and P. A. J. Gorin, Infect. lmmun., 19 (

(

(1978) 1107- 1109. 1353 L. K. TRIV~SSOS and K. 0. Lloyd, Microbial. Rev., 44 (1980) 683-721.

POLYSACCHARIDES FROM FUNGI AND LICHENS

91

L - Rhap

?

1

4

a-L-Rhap

p-~-GlcpA

1

1

1

I

2

2

a-L - Rhap

a-L-Rhap

30

29

2. Glucomannans The glucomannan of Cerutocystis brunnea contains a (1+6)-linked a-D-mannopyranosyl main-chain, substituted at the 0 - 2 atoms mainly by a-D-glucopyranosyl groups (31).The main chain was characterized by conversion of the side chain into its 6-0-tosyl derivative, followed by alkaline elimination, to give the 3,6-anhydro derivative (32).This residue was preferentially hydrolyzed with acid, givinglZ6a (1-6)linked a-D-mannopyranan (33).Glucomannans are present in a number of Cerutocystis ~ p p . including ,~ phytopathogenic Cerutocystis paradoxa and Cerutocystis j’imbriuta. 136 The synthesis of glucomannans, mannans, and galactomannans by the pathogens was found to depend on their original hosts, and on growth and temperature variation which influenced the incorporation of 2-acetamido-Zdeoxy-~glucosyl units into the mannans of C . paradoxu. No correlation was observed between the synthesis of a particular polysaccharide and the host specificity, or the host specialization, in the Ceratocystis isolates.

P\ H HO

O

W

H6

- 7 o J

31

‘ I

6

w

HO

HO

-

kJ

33

HO

32

(136) C. S. Alviano, P. A. J. Gorin, and L. R. Travassos, Exp. Mycol., 3 (1979) 174-187.

92

E. BARRETO-BERGTER AND PHILIP A. J. GORIN

3. Galactomannans Many galactomannans, obtained by alkaline extraction and Fehling precipitation from yeasts, contain (l+6)-linked a-D-mannopyranosyl main-chains. These were isolated by removal of galactopyranosyl side-chains by partial hydrolysis, followed by enzymolysis of remaining a-D-mannopyranosyl groups with a - D - m a n n o ~ i d a s eMethylation .~~ and partial-acetolysis studies showed a great variety of side-chain octosporus galactomannan, s t r u c t ~ r e s . I~n~ Sclaizosaccharonzyces ~'~~ the main chain is partially substituted at the 0 - 2 atoms b y a-D-galactopyranosyl groups (34). In Candida ZipoZyticalnD and Trichosporon fermentans1D7 galactomannans (the latter, 35),the D-galactose-containing side-chain is1oia-D-Galp-( 1+2)-a-D-Manp. The galactomaiman from Schizosaccharomyces pombe cell-wall has a structure similar to that of S . octosporus. It is prescnt in the outer layer, as shown by electron microscopy with the aid of an a-D-galactopyranosyl-binding lectin from Bandeiraea simplicifoZia.1:i9 (Y

-~-Galp 1

1

2 CY-D-M~~

a - D- G a b

t

1

i

2

-cY-D-Manp-(l-s)-u-D-ManP-(l-6)-

2 -n-n-Manp -(1+6)35

34

0- D - Galp 1

I

6 O-D-Mi3IlP 1

i

2 -cu-~-Manp-(l-6)-

36

The galactomannans from Tomlopsis magnoliae, Tordopsis gropengicsseri, and Tordopsis lactis-condensi contain P-D-galactopyranosyl (nonreducing) end-units, as shown by methylation data, low specific rotations,1n3and I3C-n.m.r. evidence. That of T . Zactis-condensi is the best characterized, having repeating structure 36. Signals of C-1 at 6, (137) P. A. J . Goriii and J . F. T. Spencer, Can. J . Chem., 47 (1968) 2299-2304. (138) D. A. Bush, M. Horisherger, I. Horman, and P. Wursch,]. Gen. Microbiol., 81 (1974) 1W-206. (1391 M.Horisberger and J. Rosset, Arch. Microbiol., 112 (1977) 123- 126.

POLYSACCHARIDES FROM FUNGI AND LICHENS

93

-

105 and of C-5 at 6, 76.8 are characteristic of ~-D-galactopyranosyl units, and distinguish them from those containing140a-D-galactopyranosyl units.13' The C-1 shift of the a-D-gaIactopyranosy1groups in the S . octosporus galactomannan (34)is at 8, 102.8. b-D-Galactopyranosylcontaining disaccharides were not obtained on partial acetolysis, and the galactose-mannose linkage was not determined.lo3The C-1 shifts of a-and /?-D-galactopyranosylunits differ from those of C-1 of /?-D-galactofuranosyl units, which lie from 6, 109.5 to 106.6. The galactomannans of S . schenckii and C . stenoceras are closely related, consisting of a core of 2-0- and 2,6-di-O-substituted a-D-mannopyranosyl units, three or four consecutive, (1-2) linkages occurring between them. Methylation and 13C-n.m.r. studies showed that the pD-galactofuranosyl side-chains are linked (1+6) to each other, and are, at least partly, linked (1+2) to the mannan core.141The C-1 resonance of the &D-galactofuranosyl units depends on the linkage to the adjacent unit, and on the nature of the adjacent unit. Serologically active galactomannans, purified by way of their insoluble Fehling complexes, were obtained from mycelial and yeastlike forms of Paracoccidioides brasiliensis and Blastomyces dermaditidis, and shown by methylation analysis to consist of galactofuranosyl and mannopyranosyl units attached to a mannan The dimorphic, black yeast Cladosporium werneckii, the causative agent of tinea nigra, produces a surface peptidophosphonogalactomannan that was investigated in detail by Lloyd and coworker^.'^^-^^^ The compound from the hot-water extract had structure 37 (mol. wt. 150,000-200,000), consisting of three principal chains; ( i )chain a consisting of galactomannan units linked through phosphoric diesters, giving long chains of mol. wt. 50,000-60,000; ( i i ) chain b , which has short, manno-oligosaccharide portions, isolated by treatment with NaBH,-NaOH, and linked to the peptide backbone via 0-glycosylic linkages to serine and theonine; and ( i i i ) chain c , consisting of long galactomannan chains. 0-Acetyl groups were present mainly on chain a , and were detected by proton- and I3C-n.m.r.spectroscopy. Methylation analysis of the complex, yeast antigen indicated both galactofuranosyl and galactopyranosyl (nonreducing) end-units, and manno(140) P. A. J. Gorin and M. Mazurek, Carbohydr. Res., 105 (1982) 283-287. (141) L. Mendonqa-Previato, P. A. J. Gorin, and L. R. Travassos, Infect. Immun., 29 (1980) 934-939. (142) I. Azuma, F. Kanetsuna, Y. Tanaka, Y. Yamamura, and L. M. Carbonell, Mycopathol. Mycol. AppE., 54 (1974) 111-125. (143) K. 0. Lloyd, FEBS Lett., 11 (1970) 91-94. (144) K. 0. Lloyd, Biochemistry, 9 (1970) 3446-3453. (145) K. 0. Lloyd, Biochemistry, 11 (1972)3884-3890. (146) W.-L. Lee and K. 0. Lloyd,Arch. Biochem. Biophys., 171 (1975) 613-623.

94

I:1

E . BARRETO-BERGTER A N D PHILIP A. J. GOHIN -0AC

Galactoinannan ( c chains)

- -P-

G,M,,-

-peptide-

I

[&Im( b chains)

M,Gl n

Phosphonogalactomannan (achains)

37

pyranosyl units that are terminal and E-O-, 3-0-, 6-0-, and ~,li-di-O-sul~stiti~tecl. The specific rotation ( - 24O) of the cornplcx (Dgalactose, 14; D-nlanllOSe, 75; phosphate, 3; and peptide, 11%)indicates a inisture of a- and pD-maiinopyrariosyl units. The niycelial fbnn of Cladosporium Izerlmrunz produces an exocelhilar galactomnnnan having a mannose :galactose ratio o f 3 :2. Methyliition analysis indicated mannopyranosyl and galactofuranosyl nonreduoing end-units, 4-O-substituted galaotopyranosyl or 5 -0-substituted galactofiiranosyl, or both, and 2-0- and 2,4-di-O-substituted mannopyranosyl units. Partial acid-hydrolysis of the galactomannan with 0.01 3f sulfuric acid for 4 h at 100" removed some galactose, and left a nondialyzable fragment containing 30% of galactose, and it was concluded that galactopyranosyl units were present. 14' A galactomai?nan, contaminated with 12- 14% of nigeran, was extracted with alkali froin hyphal walls ofAspergiZlus niger. Methylation data showed the presence of galactofuranosyl end-units, 4-O-substihited galactopyranosyl or S-0-substituted galactofuranosyl units, or both, and 2-0- and 2,6-di-O-substituted rnannopyranosyl units. Partial acid-hydrolysis (at pH 2) at 100" removed furanosyl (nonreducing) end-units, giving new ones of galactopyranose (methylation data), and 4-O-substituted D-galactopyranosyl units were s u g g e ~ t e d . 'How~~ ever, an A. tiiger extract, purified with Fehling solution,149gives a strong '.'C-n.m.r. signal at 6,. 108.4, corresponding to P-Dgalactofuranose residues which are ( l-+5)-linked,150 and of an average length of -4 units. A minor signal at 6, 109.2 could be consistent with C-1 ofp-D-galacto(147) T. Sliyazaki and Y. Naoi, Cheni. Pharm. Bull., 22 (1974) 1360-1365. (1481 P. (-1. Bardalaye anti J . H. Nordin,J. B i d . C h e n ~ .252 , (1977) 2584-2591. (149) E. hf, Rarreto-Bergter, L. R. Travassos, and P. A. J. Gorin, Carbohydr. Kes., 86 (1980) 273-285. (150) P. A. J . Gorin and M. Mazurek, Carbohydr. Res., 48 (1976) 171-186.

POLYSACCHARIDES FROM FUNGI AND LICHENS

95

furanosyl units (l-t6)-linked to the mannan core. Variation ofC-1 shifts occurs, depending on the position of substitution on the a-D-mannOpyranosyl residues. The ratio of galactose to mannose decreases with culture age.149,151 The galactomannan contains a core of (1-+6)-linked a-D-mannopyranosyl units substituted on 0-2 by side chains of a-DManp-, a-D-Manp-(1+=2)-a-D-Manp , and a-D-Manp-(l+S)-a-D-Manp(1-+2)-a-~-Manp.The polysaccharides from Aspergillus fumigatus, Aspergillus terreus, Aspergillusflauus, and Aspergillus nidulans have related ~ t r u c t u r e s . 'The ~ ~ mycelial form of A. fumigatus produces polysaccharides structurally different from those of the spore f 0 1 - m ~ ~ ~ ~ having long, (1+5)-linked (rather than single-unit) P-D-galactofuranosyl side-chains. On degradation with alkali, the major, exocellular glycopeptide from Penicillium charlesii gives mono-, di-, and tri-saccharides, and a phosphonogalactomannan. This contains 90 a-D-mannopyranosyl residues that are 2-0- and 2,6-di-0-substituted7up to 4 consecutive, (1-2) linkages being present. Some 8- 10 (I+5)-linked P-D-galactofuranosyl chains of variable length are attached directly, and not through the 9 to 10 phosphoric diester linkages.153The peptide portion of the glycopeptide is attached to the polysaccharide by O-glycosylic linkage of the terminal D-mannosyl residue to the hydroxyl group of serine or threonine.'% A mutant ofP. charlesii produces a glycopeptide containing a smaller proportion of galactose residues.155An intracellular galactomannan from Penicillium chrysogenum contains (1+5)-linked ~-D-galactofuranosylresidues and some acid-resistant galactosyl residues.

-

4. Miscellaneous Heteropolysaccharides A highly branched, acidic galactomannan was isolated from the oak lichen Evernia prunastri by extraction with cold alkali, followed by successive purification steps with barium hydroxide and Fehling solutions. The polysaccharide contained nonreducing end-units of galactopyranose, mannopyranose, and glucopyranosyluronic acid, with 20-,2,6-di-0-, 3-6-di-0-, and 2,3,6-tri-O-substituted mannopyranosyl (151) M. Rietschel-Berst and J. E. Gander, Fed. Proc., Fed. Am. SOC.E x p . B i d , 32 (1973) 528. (152) E. M. Barreto-Bergter, P. A. H. Gorin, and L. R. Travassos, Carbohydr. Res., 95 (1981) 205-218. (153) J. E. Gander, N. H. Jentoft, L. R. Drewes, and P. D. Rick, J . B i d . Chem., 249 (1974) 2063-2072. (154) P. D. Rick, L. R. Drewes, and J. E. Gander,/. Biol. Chem., 249 (1974) 2073-2078. (155) L. R. Drewes and J. E. Gander,/. BacteP.ioZ, 121 (1975) 675-681. (156) T. Miyazaki and T. Yadomae, Chem. Phamn. Bull., 17 (1969) 361-365.

E. BARRETO-BERGTER AND PHILIP A. J. GOHIN

96

units. The inannosy1 residues have the a-Dconfiguration, and the galactosyl units, the p - ~according , to infrared and enzymic e ~ i d e n c e . ' ~ ' After fractionation with cetylpyridinium chloride, the acidic heteropolycaccharide of Aureobasidium (Pullularia) pullulans contains Dmannose, D-galactose, D-glucose, and D-ghcuronic acid in the ratios of 43 : 35 : 10: 12. It has a main chain of (1-+6)-linked a-D-mannopyranosyl units substituted as in structure15838. O-D-Galf

1

1

a-D-Glc p A

I

5 0-Galf

1

6 S-D-Galf 1

I

6 P - D - G ~p C

0-calf

1

2

D-Gdf

1

1

-

- (Y - D - Man p - (1

1

1

I 3

6)- a- D- Manp - (1-

6)-0 - D -

3 Manp (1-

p - D- Glcp 1

I

3

6)- a-D-Manp - (1-

6) -

38

The purified peptidoheteroglycan from Piricularia oryzae contains D-mannose, wglucose, and =galactose in the ratios of 6: 2: 1, and has a main chain of (1-+6)-linked a-Dmannopyranosyl units. It contains mannosyl-serine and mannosyl-threonine bridges that are alkalilabile. Side chains having 1to 4 mannosyl units are present, and some of these are terminated by glucosyl and galactofuranosyl units. An a - ~ Glcp-(l-+6)-~-Man structure was shown by antiserum inhibition-studies with oligosaccharides, and it was found that the immunodominant 1-+6)-a-~-Manp-( l-t2)-a-D-Manp( 1+2)side-chain is I S Y , I ~ Ucu-~-Glcp-(

man.

A xylomannan (xylose to mannose ratio, 1: 1.2), obtained from Polyporus tumulosus cell-wall by extraction with alkali followed by Fehling precipitation, consists of a (1+3)-linked a-D-mannopyranosyl main-chain principally substituted by xylobiosyl side-chains. Methylation data showed end units that were principally xylopyranosyl, with M. Hranisavljevie-JakovljeviC, and J. Miljkovic-StojanoviC, Carbohydr. Res., 10 (1969)525-533. (158)H. G. Brown and B. Lindberg,Actu Chem. Scund., 21 (1967) 2383-2389. 1159) T. Nakajima, K. Tamari, and K. Matsuda,J. Biochem. (Tokyo), 82 (1977) 16471655. (160) T. Kakajima, H. Sasaki, M. Sato, K. Tamari, and K. MatsudaJ. Biochern. (Tokyo), 82 (1977) 1657-1662. i 157) V. M. hlicovic,

POLYSACCHARIDES FROM FUNGI AND LICHENS

97

a little mannopyranosyl. According to the formation of 2,3-die-methylxylose, the internal, side-chain units can be 44-substituted xylopyranosyl or 50-substituted xylofuranosyl, or both. Also, 2-6-di0methylmannose was only tentatively characterized, so that the linkage between the side chains and main chain is still uncertain.I6l The acidic, exocellular polysaccharide from Tremella mesenterica, isolated by Cetavlon precipitation, has repeating structure 39 with D-

D-xylp

1

I2

D-xylp

1

1

2

-a-n--p

D-Xylp 1

D-XylP 1

2 D-XYlP 1

2 D-xylp 1

1

I

4

O-D-G~CPA 1

4

1

2 2 2 -(1-3)-cy-~-Manp -(l-J)-cy-~-Manp -(1-3)-a-~-Manp -(l-S)-a-D-ManP

-(1-+3)-

39

xylose, D-mannose, D-glucuronic acid and 0-acetyl in the ratios of 7 :5 :1:0.7. Acetate groups occur on C 3 of the glucuronic acid residues, rendering them periodate-resistant.162J63 Although the chemical structure of the acidic polysaccharide from Tremella fuciformis appears closely related, its immunological behavior is different.164*165 An acidic polysaccharide from Auricularia auricula-judae, having D-XYlose, D-mannose, D-glucose, and Dghcuronic acid in the ratios of 1.0:4.1:1.3:1~3, could be structurally similar, although not all of its structural features were determined. However, the polysaccharide contained (nonreducing) shexopyranosyl groups, in addition to those of xylopyranosyl and D-glucopyranosyhronic acid.lB6 Cryptococcus twoformans, the etiologic agent of cryptococcosis, exists as four serological types, namely, A, B, C, or D.167 At present, serotypes B and C are designated as Cryptococcus bacillosporus. 168 (161) S. J. Angyal, V. J. Bender, and B. J. Ralph, Biochim. Biophys. Acta, 362 (1974) 175-187. (162) C. G. Fraser, H. J. Jennings, and P. Moyna, Can.]. Biochem., 51 (1973)219-224. (163) C. G. Fraser, H. J. Jennings, and P. Moyna, Can.]. Biochem., 51 (1973) 225-230. (164) S. Ukai, K. Hirose, T. Kiho, and C. Hara, Chem. Pharm. Bull., 25 (1977)338-344. (165) M. Kakuta, Y. Sone, T. Umeda, and A. Misaki,Agric. Biol. Chem., 43 (1979)16591668. (166) Y. Sone, M. Kakuta, and A. Misaki, Agric. Biol. Chem., 42 (1978) 417-425. (167) D. E. Wilson, J. E. Bennett, and J. W. Bailey, Proc. SOC. E r p . Biol. Med., 12 (1968) 820-823. (168) A. K. Bhattacharjee, K. J. Kwan-Chung, and C. P. J. Glaudemans, Carbohydr. Res., 82 (1980) 103-111.

E. BARRETO-BEHGTER AND PHILIP A. J . GORIN

98

Serotype B contains capsular polysaccharide, 60% of which consists of a (1-+3)-linkeda-D-mannopyranosyl main-chain, two of every three units being substituted at 0-2 by P-Dxylopyranosyl side-chains, the other being disubstituted, at 04 with 0-D-xylopyranosyl and at 0-2 with p-o-glucopyranosyluronicacid units (40). The polysaccharide contains 3 acetyl groups for every 7 sugar residues.1694s serotype C polysaccharide is similar, except for 2 of every 3 units being disubstit~ted,'~ and " its serotype D polysaccharide contains 1 of every 3 mannosy1 residues unsubstituted (and no disubstituted residues171),the reclassification appears to be confirmed. J-D-xylp

$-D-xylp 1

f

1 2

2

- n - ~ - M a n p-(1-3)-a-~-Manp -(1--3)-

8- D-xyl p 1

i

4 a-D-Mano -(1--3)2

t1

8-0- Glcp A 40

The fucoxylomannan (41) of Polyporus pinicola was reinvestigated b y methylation of the polysaccharide and of derived, partially hydrolyzed material.lT2 a-L-FUCP

(Y-L-FUCP

a-L-FUCP

1

1

2

2

2

t 4

d

1

1

1

1

-,3-~-Manp-(1- J)-~-D-Manp-(l-3)-8-D-Manp

t 4 -(1-3)-p-D-Manp-(1-3)-

41

Candida hogoriensis polysaccharides contain a (1+3)-linked a-Dmannopyranosyl main-chain. Fucopyranosyl, rhamnopyranosyl, and galactofuranosyl nonreducing end-units are present, and side-chain components are represented by partial-hydrolysis products, a-L-Rhap1169?A. K. Bhattacharjee, K J. Kwan-Chung, and C. P. J. Glaudemans, fmnunochemi s t r y , 15 (1978)673-679. i170) A . K.Bhattarharjee, K. J. Kwan-Chung,and C . P. J. Glaudemans, Mol. Imrnunol., 16 (1979)531-532. ( 171) .4.K. Bhattacharjee, K. J. Kwan-Chung, and C. P. J. Claudemans, Carbohydr. Res., 73 (1879) 183- 192. (172) K. Axelsson, H . Bjiirndal, and B. Lindberg, Acta Chern. Scund., 23 (1969) 15971600.

99

POLYSACCHARIDES FROM FUNGI AND LICHENS

(1+3)-~-Rha,a-~-Rhap-( 1+2)-~-Fuc,a-~-Fucp-( 1+3)-~-Fuc,and p-DGlcpA-( ~ ~ ) - L - F uThe c . isolation of 2-0-a-~-rharnnopyranosyl-~erythritol after a Smith degradation indicated an a-L-Rhap-(1+4)-p-~GlcpA-( 1 4 ) - ~ - F u c sequence. p 173 Many yeasts contain (2-acetamido-2-deoxy-g1uco)mannans that are liberated on alkaline extraction, and are purifiable by way of their insoluble copper complexes. The percentage of N-acetylated residues, detected through the NAc signal in the p.m.r. spectra, varies from 1to 17% in 18 yeasts investigated (see Table V). Polysaccharides from Pichia bovis and Saccharomyces phaseolosporus contain 2-acetamido-2deoxy-D-glucopyranosyl units having the a-D configuration (specific rotations), and a methylation experiment provided 0-methylated 2TABLEV The Percentage Content of N-Acetyl Groups in (2-Acetamido-2-deoxy-~-gluco)mannans from Yeasts, Based on Magnitude of NAc Signals in Proton-N.m.r. Spectra Chemical shift of N-acetyl signal(s) Yeast Saccharomyces lactis Pichia bovis Hansenula bimundalis var. Americana Pichia salictoria Pichia sp. 3R-38-68 Saccharomyces dobzhanskii Saccharomyces sociasi Saccharomyces drosophila Hansenula fabianii Hansenula beijerinkii Saccharomyces phaseolosporus Hansenula ciferii Saccharomyces wickerhamii Hansenula saturnus Candida freyschussii Hansenula mrakii Pichia strassburgensis Candida pelliculosa NCYC 471

(6)

2.61 2.62 2.66 2.62 2.61 2.66 2.62 2.66 2.66 2.66 2.62 2.66 2.66 2.67 2.62 2.67

N-Acetylated units in polysaccharide (%)

10 10

5.9

4.0

6 4.9 3.8 6 11 13 5 5 17

1.5 1.3

} 10

]

9

}

3

2.61 2.61 2.61 2.62 2.67

(173) P. A. J. Gorin and J . F. T. Spencer, Can. J . Chem., 46 (1968) 3407-3411.

100

E. BARRETO-BERGTER AND PHILIP A. J. GORIN

deoxy- methy lamino) derivatives that were identified by using a Spinco amino acid analyzer, and principally nonreducing end-units were indi~ated."~ 44-Substituted units, also present, probably occur as a bridge between mannan and protein portions, as in baker's yeast glycopr~tein.~~

x. HETEROPOLYSACCHARIDES BASED ON GALACTAN MAIN-CHAINS

1. Glucogalactans Varianose, an exocellular glucogalactan from Penicillium varians G. Smith, contains a high proportion of repeating unit 42; the investigation was facilitated by use of W-n.m,r. spectroscopy. It appears that the structure could be modified by hydrolytic enzymes during culture. The presence of a- and p-D-galactofuranosyl units is unusual.175Glucogalactans have been reported in Sporobolomyces roseus. 176 o - ~ - G l c p-(1-2)-a-D-Gdf 1

2. Fuco(manno)galactans The fruit bodies of several fungi contain fucogalactans or fucomannogalactans having a (l+6)-linked a-Dgalactopyranosyl main-chain partly substituted on 0-2 atoms b y various side-chains. Aqueous extraction of Polyporus ovinus (Schaeff.) gave a glucan and a fucogalactan, which were resolved, and the latter found to contain 20% of L-fucopyranosyl ~ide-chains.'~~ Alkaline extraction of Polyporus fomenturius and Polyporus igniarius gave glucans, glucuronoglucan, and fucomannogalactan, and the latter, on purification, contained a main chain, of which 30-40% was substituted with L-fucopyranosyl, D-galactopyranosyl, and O-cr-D-mannopyranosy1-(1-+3)-L-fucopyranosyl residues.17* The heteropolymer of Polyporus squamosus (Fr.) contained similar side-chains, and additional ones having (1+2)- and (17.1) P. A. J. Gorin, J . F. T. Spencer, and A. J. Finlayson, Carbohydr. Res., 16 (1971) 161- 166. (175) Y.-E. Jansson and B. Lindberg, Carbohydr. Res., 82 (1980)97-102. (176) S . P. Elinov and G . A. Vitovskoya, Biokhimiyn, 32 (1967) 337-340 (English version, 279-282). (177) K. Axelsson and H. Bjomda1,Acta Chem. Scand., 23 (1969) 1815-1842. (178) H. Bjcmdal and B. Lindberg, Carbohydr. Res., 10 (1969)79-85.

POLYSACCHARIDES FROM FUNGI AND LICHENS

101

(1+3)-linked a-D-galaCtOpyranOSe.17sExtraction of Flamnulina uelutipes (Fr.) with cold water gave glucan, xylomannan, and fucomannogalactan. The last had a number-average molecular weight of 9.5 x lo5, and side chains of a-L-fucopyranose and 0-a-D-mannopyranosyl-(1+3)-~-fucopyranose.'~~~~~~ One possible interpretation of the structural data for the fucomannogalactan is that the side chains are single-unit L-fucopyranose and D-mannopyranose residues.lE2A fucogalactan of Rhodotorula glutinis K-24, separated from mannan, containslE3the sugar components in equimolar proportions, and a galactan core.

XI. MISCELLANEOUS POLYSACCHARIDES The extracellular polysaccharide from Aspergillus nidulans is a linear molecule, and, according to methylation and specific-rotation data, contains 4-0-substituted a-D-galactopyranosyl and 4-0-substituted 2acetamido-2-deoxy-a-~-galactopyranosyl units. Periodate oxidation and proton-n.m.r. data showed their ratio to be 1.8:1.A Smith degradation yielded mainly 2-0-(2-acetamido-2-deoxy-a-~-galactopyranosy1)-D-threitol,two (2-hydroxyethylidene) acetals, and D-threitOl, indicating that the different units are distributed evenly along the chain, as in repeating unit 43, because consecutive, 2-acetamido-2-deoxy-~galactosyl units were not detected.lE4A heteropolysaccharide from Aspergillus niger contains galactosyl and 2-amino-2-deoxygalactosyl units in the ratio of 7 :2. The linkage positions and glycosidic configurations are similar to those just given.lE5 The acid component, isolated from Fomes annosus (Polyporus annosus), which forms fruiting bodies and causes wood destruction, is a glucuronoglucan (30-35% uronic acid)lE6similar to those of Polyporus fomentarius (Fr.) and Polyporus igniarius (Fr.).lS7Chains of 4 to 5 p( 1 4 ) - l i n k e dglucuronic acid residues are joined through 03 to a glucan core, in which the glucopyranosyl units are connected by (1+3) and (1-6) linkages. Mucoran, isolated from cell walls of Mucor rouxii, and purified by way of its insoluble, copper complex, consists principally of

-

(179) H. Bjorndal and B. Wagstrom,Acta Chem. Scand., 23 (1969) 3313-3320. (180) T. Mukumoto and H. Yamaguchi, Carbohydr. Res., 59 (1977) 614-621. (181) T. Mukumoto and H. Yamaguchi, Carbohydr. Res., 79 (1980) 142-146. (182) M. Shida, K. Haryu, and K. Matsuda, Carbohydr. Res., 41 (1975) 211-218. (183) K. Fukagawa, H. Yamaguchi, D. Yonezawa, and S. Murao,Agdc. Biol. Chem., 38 (1974) 29-35. (184) P. A. J. Gorin and D. E. Eveleigh, Biochemistry, 9 (1970) 5023-5027. (185) P. C. Bardalaye and J. H. Nordin,J . Bacterial., 125 (1976) 655-669. (186) K. Axelsson and H. Bjorndal,Acta Chem. Scand., 24 (1970) 713-714. (187) H. Bjomdal and B. Lindberg, Carbohydr. Res., 12 (1970) 29-35.

102

E. BARRETO-BERGTER AND PHILIP A. J. GORIN

4 HO

NHAc 43

D-glucuronic acid, D-mannose, and L-fucose in the ratios of 5:3:2. Methylation data and the isolation of 3-O-cy-D-(glucopyranosyluronic acid)-wmannose show that its structure is mainly linear, with a high proportionlH8 of the structure D-Manp-(1+[4)-a-~-GlcpA-(1 4 ) wManp-( l)ln. The nonpathogenic, black yeast Rhinocludiella mansonii NRRL Y6272 produces an extracellular polysaccharide that, after purification by means of Cetavlon (8%yield), contains residues of 2-acetamido-2deoxy-D-glucose and 2-acetamido-2-deoxy-~-glucuronic acid in the molar ratio 0PH9 2 : 1. Methylation analysis, followed by carboxyl reduction, gave a mixture of 2-deoxy4,6-di-O-methyl-2-(N-methylacetamido)-o-glucose and 2-deoxy4U-methyG2-(N-methylacetamido)-~glucose, identified by g.1.c.-m.s. of the derived alditol acetates. Thus, (1+3)-linkages are present, as in structure 44.

-

CO,H

NHAc

NHAc

NH Ac

44 ( 188) S. Bartnicki-Garciaand B. Lindberg, Carbohydr. Res., 23 (1972) 78-85. (189) A. (R.) Jeanes, K. A. Burton, M.C. Cadmus, C . A. Knutson, G . L. Rowin, and P. A. Sandford, Nature, New BWZ., 233 (1971) 259-265; P. A. Sandford, P. R. Watson,

and A. R. Jeanes,Corbohydr. Res., 29 (1973) 153-164.

POLYSACCHARIDES FROM FUNGI AND LICHENS

103

30-Methylgalactose is a component of the polysaccharide from Armilh-ia metlea lW; it is present in a 60-(30-methyl-D-galactopyranosyl)-D-galactosylside-chain that is attached, along with L-fucopyranosyl and 0-a-D-mannopyranosyl-( l+3)-L-fucopyranosy1 side-chains, to 0-2 atoms of a (l-t6)-linked a-D-galactopyranosy1main-chain. It also occurs in the polysaccharide of Lampteromyces j a p o n i c u ~ (see ~ ~Sec~ tion X,2). Cell walls of arthrospores, mycelia, spherules, and spheruleculture filtrates of Coccidioides immitis have 30-methylmannosecontaining components.1s2~1s3 Many articles have appeared concerning the content and location of chitin, which is virtually universal in the cell walls of fungi. Both chitin (17%)and chitosan, which lacks N-acetyl groups, are present in the cell wall of Choanephora curcurbitarum.ls4 Chitin may be detected in bud scars, cell wall, and cytoplasm, near the plasmalemma of Saccharomyces cerevisiae and Cundida utilis, as detected by wheat-germ agglutinin.82 Cyttaria harioti F i s ~ h e r ' ~produces ~,'~ a polysaccharide containing D-glUCOSe, Darabino-hexulosonic acid, and D-fructose in the molar ratios of 98 :6 :1.

(190) H. 0.Bouveng, R. N. Fraser, and B. Lindberg, Carbohydr. Res., 4 (1967) 10-31. (191) K. Fukuda and A. Hamada, Biochim. Biophys. Acta, 544 (1978)445-447. (192) R. W. Wheat and E. Scheer, Infect. lmmun., 15 (1977) 340-341. (193) R. W. Wheat, C. Tritschler, N. F. Conant, and E. P. Lowe, Infect. Immun., 17 (1977) 91-97. (194) D. R. Letoumeau, J. M. Deven, and M. S. Manocha, Can.j . Microbiot., 22 (1976) 486-494. (195) A. Femandez Cirelli and R. M. de Lederkremer, Chem. Ind. (London),(1971) 1139. (196) A. Femandez Cirelli and R. M. de Lederkremer,Carbohydr. Res., 20 (1972)299308.

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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 41

BIOSYNTHESIS OF CELLULOSE*

BY DEBORAH P. DELMER MSU-DOE Plant Research Iaboratory, Michigan State Uniuersity, East Lansing, Michigan 48824**

................................................. 105 . . . . 107 . . . . . . . . . . . . 110 IV. Cytological Investigations of Cellulose Biosynthesis . . . . . . . . . . . . . . . . . . 116 1. The Site of Cellulose Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 2. Orientation of Microfibrils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123 V. The Mechanism of Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 1. Involvement of Glycosyl Esters of Nucleoside Diphosphates . . . . . . . . , . 125 2. Possible Involvement of Lipid Intermediates . . . . . . . . . . . . . . . . . . . . . . 132 I. Introduction

11. A Survey of Organisms Useful for the Study of Cellulose Biosynthesis 111. Structural Considerations Relevant to Biosynthesis ... ....

.

3. Possible Involvement of High-molecular-weightPrecursors to Cellulose 135 4. Genetic Mutations, and Chemical Inhibitors of Cellulose Biosynthesis . . 143 5. Possible Factors Affecting the Lability of the Polymerizing System . . . . . 145 VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 VII. Addendum .................................................. 152

. .. . .

..

I. INTRODUCTION

The topic of the biosynthesis of cellulose was discussed by Shafizadeh and McGinnis' in this Series about a decade ago, and since then, several reviews have appeared in the literature?-' One theme com* This article was written while the author was on a sabbatical leave at the Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel. She is grateful for support for this effort from Michigan State University, from the U. S.-Israeli Binational Agriculture Research and Development Fund (BARD),and from The Hebrew University. ** Present address: ARC0 Plant Cell Research Institute, 6560 Trinity Ct., Dublin, CA 94568. (1) F. Shafizadeh and G. D. McGinnis, Ado. Carbohydr. Chem. Biochem., 26 (1971) 297-349. (2) J. R. Colvin, CRC Crit. Reu. MacromoZ. Sci., 1 (1972)47-84. (3) D. P. Delmer, Recent Adu. Phytochem., 11 (1977)45-77. (4) D. G . Robinson,Adu. Bot. Res., 5 (1977)89-151. (5) G. A. Maclachlan, Trends Biochem. Sci., 2 (1977) 226-228. (6) A. Darvill, M. McNeil, P. Albersheim, and D. P. Delmer, in N. E. Tolbert (Ed.), The Biochemistry ofPZants, Vol. I, Academic Press, New York, 1980, pp. 91-162. (7) J. R. Colvin, in J. Priess (Ed.), The Biochemistry ofPZants, Vol. 111, Academic Press, New Yorlc, 1980, pp. 543-570. 105

Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-0072414

106

DEBORAH P. DELMER

mon to all of these is the lament that, despite the fact that cellulose is the world’s most abundant organic polymer, so little is known about its mode of synthesis. Those working in this field are well aware that the older literature is confusing and contains errors, misinterpretations, and incomplete data. The key to sorting out all of this confusion must come in the recognition that it has been exceedingly difficult to demonstrate convincingly the synthesis of true, microfibrillar cellulose by using cell-free preparations derived from cells that are capable of abundant synthesis ofcellulose in uiuo. As one engaged in studying this process in higher plants, the present author doubts whether this has ever been achieved with cell-free preparations derived from these organisms; and, even in the case of the bacterium Acetobacter xylintina, where convincing synthesis of (l+4)-p-@glucan has been achieved in uitro, the in uitro rates of synthesis are, in general, less than 1% of those observed in z;iuo; and, as will be discussed in Section V , l , it is not even certain that, in all cases, (1+4)-p-@glucan should be equated with microfibrillar cellulose. Such a situation, although frustrating, is also fascinating. Are we supplying the correct substrates in uitro? If so, what is the nature of the extraordinary lability of the cellulose-synthesizing system? What essential features of the intact system are lost when cells are broken, or even just rendered permeable to substrates? The key to unraveling the complexities of the biosynthesis of cellulose will only come when full answers to such questions have been obtained. The importance of the older, more confusing literature in the field is that it established two incontrovertible facts-that the process is undoubtedly complex and that we must look beyond simple conclusions in order to progress further. The real cause for optimism is that these difficulties are at last being recognized, and that the past few years have witnessed a remarkable maturing of the field and have presented us with some intriguing new findings. Elegant visual observations of the process of cellulose biosynthesis by microscopists have confirmed the complexity that the biochemists predicted; new evidence concerning the nature of possible precursors to cellulose has been presented; and, finally, new insights into the reasons for the lability of the system are being explored. The purpose of this article is, therefore, to attempt, first of all, to analyze the older, fragmented literature and assess its major contributions; secondly, to concentrate extensively on new findings, and attempt to coordinate their interpretations; and thirdly, to indicate what gaps in our knowledge of this complex process still exist.

BIOSYNTHESIS OF CELLULOSE

107

11. A SURVEY OF ORGANISMS USEFUL FOR THE STUDY OF CELLULOSEBIOSYNTHESIS Before discussing the literature on the biosynthesis of cellulose, it may prove useful to describe some characteristics of the various organisms available for study, and to attempt to assess the various advantages and disadvantages presented by each. Among the bacteria, only the genus Acetobacter produces abundant quantities of cellulose. Largely due to the pioneering efforts of Hestrin and coworkers in Israel: Acetobacter xylinum has emerged as a classic organism for the study of cellulose biosynthesis. Unlike the algae and higher plants, the cellulose of A . xylinum is not produced as a cell-wall component, but as an extracellular pellicle of essentially pure cellulose. A . xyh u m is a non-motile, strict aerobe; a teleological argument has been made that the pellicle is produced as a means of allowing the cells to maintain an adequate supply of oxygen by virtue of their continued association with the floating pellicle. This argument is supported by the experimental observation that mutants which lack the ability to synthesize cellulose can be readily isolated from cultures grown under vigorous a e r a t i ~ nThe . ~ advantages of studying A . xylinum are numerous: it is a unicellular organism, and is easily propagated; numerous studies have elucidated the details of its pathways of carbohydrate metabolism (see references cited in Ref. 10); and mutants lacking the capacity for cellulose synthesis can be isolated. As disadvantages, it lacks a well-defined, genetic system, and is far removed on the evolutionary scale from higher plants, the major producers of cellulose. However, insufficient information is as yet available to allow speculation as to the direction and extent to which the synthesizing system has evolved. There are a few reports that indicate that a few other genera of bacteria are capable of limited synthesis of cellulose11J2;some fungi are also capable of synthesis of cellulose,13as well as a few rare members (8) S. Hestrin, in I. C. Gunsalus and R. Y. Stanier (Eds.), The Bacteria, Vol. 111, Academic Press, New York, 1962, pp. 373-388. (9) M. Swissa, Y. Aloni, H. Weinhouse, and M. BenzimanJ. Bacteriol., 143 (1980) 1142 -1150. (10) Y. Aloni and M. Benziman, in R. M. Brown, Jr. (Ed.),Cellulose and Other Natural Polymer Systems: Biogenesis, Structure, and Degradation, Plenum, New York, 1982, pp. 341-361. (11) E. Canale-Parola and R. S. Wolfe, Biochirn. Biophys. Acta, 82 (1964)403-405. (12) M. H. Deinema and L. P. T. M. Zevenhuizen,Arch. Mikrobiol., 78 (1971)42-57. (13) J. M. Aronson, in G. C. Ainsworth and A. S. Sussman (Eds.),The Fungi, Vol. I, Academic Press, New York, 1972, pp. 49-76.

108

DEBORAH P. DELMER

of the animal kingdom, most notably the tunicates.I4 However, in none of these organisms has the synthetic process been studied in detail. Many of the algae synthesize cell walls that contain cellulose. A book by Preston15gives an excellent description of the cell-wall structure of these algae, the most widely studied of which are the genera Valonia, Oocystis, Microsterias, Cladophora, Chaetomorpha, Glaucocystis, and Pleurochrysis. To date, surprisingly few biochemical studies have been conducted with these organisms, perhaps partly because of difficulties in culturing these algae in large quantities, and in breaking the cells. However, some of the unicellular algae have served as excellent models for microscope examinations of the process, which have been facilitated by the unusually large, and highly ordered, cellulosic microfibrils present in their cell walls. Among the nonphotosynthetic eukaryotes, several organisms have emerged as possessing good potential for biosynthetic studies. These organisms are: Prototheca zopfii, a colorless alga having a cellulosic cell-wall; the slime molds Polysphondylium pallidum and Dictyostelium discoidum; and the amoeba Acanthamoeba castellanii. The slime molds and Acanthamoeba produce a cellulosic wall during one specific stage of development (encystment); they may therefore be of use for studies of regulation, and may also have the advantage that relatively undamaged, plasma membranes could be isolated from cells before extensive encystment occurs.16All of these organisms hold special promise, because some in uitro synthesis of (1+4)-P-~-glucan from UDP-glucose has been observed on using cell-free preparations, even though no unique conditions for extraction or assay were emp l ~ y e d . ' ~ The - ' ~ zygote of the brown alga Fucus also produces a cellulosic wall upon fertilization,*Obut in uitro studies with this cell type have not been reported to date. Among the higher plants, much enzyme work has been conducted with elongating, etiolated hypocotyls or epicotyls of such legumes as mung beans (Phaseolus aureus) or peas (Pisum satiuum). These tissues have the advantages of being quite easy to grow, possessing cells (14) A. B. Wardrop, Protoplasma, 70 (1970) 73-86. (15) R. D. Preston, The Physical Biology ofPlant Cell Walls, Chapman and Hall, London, 1974. (16) M. L. Phillippi and R. W. Parish, Planta, 152 (1981) 59-69. (17) H. E. Hopp, P. A. Romero, G. R. Daleo, and R. Pont-Lezica, Eur. 1.Biochem., 84 (1978) 561-571. (18) C. Ward and B. E. Wright, Biochemistry, 4 (1965) 2021-2026. (19) J. L. Potter and R. A. Weisman, Biochim. Biophys. Acta, 237 (1971) 65-74. (20) R. S. Quatrano and P. T. Stevens, Plant Physiol., 58 (1976) 224-231.

BIOSYNTHESIS OF CELLULOSE

109

engaged in active, cell-wall synthesis, and yielding extracts relatively free from pigments and deleterious, phenolic compounds. However, such tissues are composed of more than one cell type, and suffer the disadvantage that a number of other cell-wall polymers are also synthesized at this stage of development. Woody tissues, which are rich in secondary-wall cellulose, but also contain large proportions of lignin, have not been exploited for studies on cellulose synthesis. However, another cell type rich in secondarywall cellulose, namely, the developing cotton-fiber, has emerged as a useful experimental system; the fibers constitute a homogeneous celltype which elongates synchronously with time and, at a precisely regulated time in its development, initiates the rapid synthesis of a cellulosic secondary-wall that is free from lignin. Further advantages are that ( a ) the pattern and composition of the developing cell wall has been studied in detail by Meinert and DelmeP and Huwyler and coworkers,2z (b) the flow of carbon in vivo from D-glucose to end products has been extensively analyzed by Carpita and Delmer,23and ( c ) the fibers can be cultured in uitro, with their associated ovules, by techniques devised by Beasley and Ting24 and Waterkeyn and cow o r k e r ~ ?A~ major disadvantage of this system is that extensive growth-facilities must be maintained in order to ensure a constant supply of flowering cotton-plants. Plant cells proliferating in tissue culture have also been used; here, the cell type is somewhat more homogeneous, but a variety of polysaccharides is produced. A great deal of optimism was held for the use of protoplasts derived from such cells; such protoplasts can often regenerate a cell wall, and it was considered that precursors might be directly fed to such cells, or that membranes isolated by gentle, osmotic lysis might be more active for studies of synthesis in vitro. However, experiments by Klein and DelmeP with soybean protoplasts, and by Haass and coworkersz7with tobacco protoplasts, indicated that such protoplasts, although capable of producing cellulose during wall regeneration in uiuo, are unable to use glycosyl esters of nucleotides directly for cellulose synthesis; and, in general, they have yielded re(21) M. Meinert and D. P. Delmer, Plant Physiol., 59 (1977) 1088-1097. (22) H. R. Huwyler, G . Franz, and H. Meier, Planta, 146 (1979) 635-642. (23) N. C. Carpita and D. P. Delmer,J. Biol. Chem., 256 (1981)308-315. (24) C. A. Beasley and I. P. Ting,Am.J. Bot., 61 (1974) 188-194. (25) L. Waterkeyn, E. de Langhe, and A. A. H. Eid, Cellule, 71 (1975)41-51. (26) A. Klein and D. P. Delmer, PZanta, 152 (1981) 105-114. (27) D. Haass, W. Blaschek, H. Koehler, and G . Franz, in D. Robinson and H. Quader (Eds.), Cell Walls, Wissenschaftliche Verlags, Gmb H, Stuttgart, 1931, pp. 109118.

DEBORAH P. DELMER

110

sults in studies in rjitro with isolated membranes similar to those obtained with other plant cells possessing cell walls. 111. STRUCTURAL CONSIDERATIONS RELEVANT TO BIOSYNTHESIS

No critical discussion of biosynthesis can be attempted without consideration of'the information available on the structure of native cellulose. As for studies on biosynthesis, much controversy has existed in the field of structural analyses of cellulose. There seem to be only two points on which all workers are in agreement, namely, that ( a ) native cellulose is a composite of linearly extended chains of (1-+4)-/3-D-glucan (l),and ( b ) strong intrachain and interchain hydrogen-bonding between D-glucosyl residues occurs in such a way as to create a highly insoluble, and partially crystalline, fibrillar structure.

HO~~o~

o OH

CH,OH

~OH OH

n

CH,OH

Cellulose (1-4)-p-D-Glucan 1

For studies of biosynthesis, even this limited information imposes certain necessary considerations. Any product synthesized in vitro should be chemically characterizable as (l+"i)-P-D-glucan. The presence of small proportions of sugars other than D-glucose has often been detected in samples of "purified' cellulose28*'Y; it is not known whether these constituents are truly a part of the covalent'structure. Even so, the proportions are quite low, and, as such, should constitute only a small fraction of the product. The product synthesized in vitro should be insoluble in alkali, but this criterion alone is not acceptable as proof of cellulose synthesis, as other D-glucans, most notably (1-+3)fi-D-glucans, may also be insoluble under these ~ o n d i t i o n s . 4 *The ~*~~~~* product should be degraded by enzymes specific for the p-~-(1+4)glucosidic linkage, but proof must be given that the enzymes used are pure, and this has rarely been offered. Furthermore, proof should be A. Adams and C . T. Bishop, Tappi, 38 (1955) 672-675. (29) 13. T. Dennis and R. D. Preston, Nature, 191 (1961) 667-668. (30) Y . Raymond, G. B. Fincher, and G. A. Maclachlan, Plant Physiol., 61 (1978) 938(28)

942.

(31) U. Heiniger and D. P. Delmer, Plant Physiol., 5941977) 719-723.

BIOSYNTHESIS OF CELLULOSE

111

given that the series of oligosaccharides released, either by enzymic digestion or by partial hydrolysis with acid are, in fact, cello-oligosaccharides; unfortunately, identifications have usuaIly been based solely on comigration during paper chromatography with cello-oligosaccharide standards, with no indication given as to whether the separation procedure used would allow resolution of a cello-oligosaccharide series from a series of oligosaccharides having a different linkage. Having free, vicinal hydroxyl groups, the product should be attacked by periodate, but the high degree of insolubility creates difficulties in achieving complete reaction. Similarly, methylation analyses should yield 2,3,6-tri-O-methyl derivatives Of D-glucose, but, once again, insolubility often leads to low yields. For cellulose, new solvents that are nondegrading have been employed and have been found to improve the yields The product should theoretically give an X-ray diffraction pattern typical of cellulose I (or cellulose 11, if treated with alkali; see later discussion), but the limited yields to date in synthesis in vitro, as well as the impurity of the preparations, usually preclude this analysis. Thus, even a chemical characterization of the products synthesized in vitro is fraught with difficulties, and this problem is responsible for some of the confusion in the literature on biosynthesis. In Section V,1, the question of whether true, microfibrillar cellulose can be expected as a product of in vitro synthesis will be discussed in more detail. Another problem for biosynthesis concerns the fact that certain other, native plant-polysaccharides, most notably the xyloglucans found in higher-plant cell-walls,6 contain backbone structures of (l-A)-P-D-glucan, and it is necessary to consider whether separate enzymes synthesize these structures, and, if so, whether these enzymes could be confused with enzymes specifically involved in the synthesis of cellulose. Much controversy has existed over the structure of the so-called microfibril. From electron-microscope studies, it is clear that all native celluloses exist in linear aggregates, or fibrils, of discrete size. However, although being well defined and precisely regulated within one cell type, the size varies considerably among different organisms, or even at different stages of development within one cell type. The diameters of the fibrils are various: the smallest fibrils noted have a diameter of 1.5 nm, the so-called “sub-elementary fibril,” which has been seen in wood cambium33and quince slime34;the frequently ob-

-

(32) J.-P. Joseleau, G. Chambat, and B. Chumpitazi, Carbohydr. Res., 90 (1981) 339344. (33) R. B. Hanna and W. A. Cote, Jr., Cytobiologie, 10 (1974) 102-116. (34) W. W. Franke and B. Ermen, Z. Naturforsch., Teil B , Z4 (1969)918-922.

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DEBORAH P. DELMER

served, 3.5-nm “elementary fibril” that is found in Acetobucter xyl i n ~ r nin, ~the ~ primary cell-walls of higher plant^,'^ and in the frayed ends of larger micro fibril^^^; and diameters of 5-10 nm for the secondary walls of wood:’ 10-20 nm for such fiber cells as ramie:* and, finally, up to 50-200 nm, characteristic of the cellulose in cell walls of unicellular algae.l5 The controversy partly concerns the questions of whether ( a )a fundamental and constant unit, such as the 3.5-nm elementary fibril proposed by Muled1aler,3~exists in nearly all native celluIoses, and ( b ) larger microfibrils are constituted of aggregates of these elementary fibrils. The various models of microfibrillar structure proposed have been well reviewed by Colvin2and by Shafizadeh and McGinnis.‘ Part of the controversy arises from the fact that crystallite sizes observed by X-ray diffraction analyses do not always correspond to fibril sizes observed by microscopy. The presence of amorphous regions in crystalline cellulose also creates problems in interpretation, as was clearly discussed by Shafizadeh and McGinnis.’ At present, it would seem that many workers accept the concept that the 3.5-nm elementary fibril is a very common (although, perhaps, not universal), discrete entity. Packing analyses indicate that such a fibril would contain -36 Dglucan chains. The question of the existence ofa discrete and uniform elementary fibril is relevant to considerations of biosynthesis. Does such a discrete fibril arise as the result of synthesis by a multi-subunit, enzyme complex, with each subunit responsible €or the polymerization of a single Bglucan chain? Although P r e ~ t o n ’ ~ argued against elementary fibrils, he nevertheless recognized that cellulose is composed of many chains, and he was responsible for first proposing4O the so-called “ordered-granule hypothesis” which envisages such a multi-subunit complex embedded in the plasma membrane (see Fig. 17 in Ref. 1).If the basic fibril is the 3.5-nm elementary fibril, such a complex should contain 36 subunits. A proposal by Haigler and Benziman4I for A. xylinum envisages closely spaced, synthetic sites, each containing 12-15 subunits that synthesize 1.5-nm subelementary fibrils, which then self-associate to form the 3.5-nm elementary fibril (see Fig. I). However, based on existing data, it is (35)R. M. Brown, Jr., J. H. M. Willison, and C. L. Richardson, Proc. Natl. Acod. Sci. CJSA,73 (1976)4565-4569. (36)J. Blackwell and F. J. Kolpak, Macromolecules, 8 (1975)322-326. (37)A. J. Hodge and A. B. Wardrop, Nature, 165 (1950)272-279. (38)A. Vogel, Makromol. Chem., 11 (1953)111-117. (39)K. Mulethaler, Beih. Z. Schweiz. Forstu., 30 (1960) 55-62. (40)R.D.Preston, in M. H. Zimmerman (Ed.),The Formation of Wood in Forest Trees, Academic Press, New York, 1964, pp. 169-188. (41) C. H. Haigler and M. Benziman, in Hef. 10, pp. 273-297.

BIOSYNTHESIS OF CELLULOSE

(1+ 4)-p-D-Glucan

113

polymerizing enzymes

FIG.1.-Proposed Model of Cellulose Assembly in Acetobacter ~ylinum.~' [DGlucan chain aggregates from organized,multiple-enzyme complexes, and extrusion pores crystallize into microfibrils, which then assemble into bundles and the normal ribbon at the cell surface.]

certainly not necessary to invoke one discrete, elementary-fibril size for all organisms; fieeze-fracture studies on algae suggested that enzyme complexes of various sizes can exist (see Section IVJ). X-Ray crystallography has contributed greatly to our understanding of the structure of cellulose. It has been known for many years that cellulose can exist in at least four different, crystalline forms termed42 celluloses I, 11, 111, and IV. Essentially all native forms of cellulose exist in the cellulose I a form thermodynamically less stable than cellulose 11; when native cellulose I is dissolved and recrystallized, or subjected to swelling in alkali ("mercerization"), it is converted into the more stable, cellulose I1 structure. For purposes of biosynthesis, the most critical question addressed by these studies has concerned the orientation of the chains in cellulose I, that is, whether all of the chains are aligned parallel, or antiparallel. There seems to be uniform agreement that the chains of cellulose I1 are arranged in an antiparallel o r i e n t a t i ~ n The . ~ ~ original structure proposed for cellulose I by Meyer and M i s ~ also h ~ possessed ~ antiparallel chains. However, more-refined methods of analysis led to re-evaluation of the structure of cellulose I, and indicated that the most favored models show the chains to be parallel. The most definitive work was performed, with the native cellulose of the alga Valonia, by Gardner and Bla~kwell"~ and Sarko and M ~ g g l i , 4and, ~ for ramie fibers, by Woodcock and S a r k ~all ; ~concluded ~ that the chains of native cellulose I are parallel. French4' also extensively examined the X-ray data for ramie (42) A. Sarko, Tappi, 61 (1978) 59-61. (43) K. H. Meyer and L. Misch, Helo. Chim. Acta, 20 (1937) 232-244. (44) K. H. Gardner and J. Blackwell, Biopolymers, 13 (1974) 1975-2001. (45) A. Sarko and R. Muggli, Macromolecules, 7 (1974) 486-441. (46) C. Woodcock and A. Sarko, Macromolecules, 13 (1980) 1183-1187. (47) A. D. French, Carbohydr. Res., 61 (1978) 67-80.

DEBORAH P. DELMER

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fibers and, although he stated that a parallel model is possible, he concluded that an antiparallel orientation cannot yet be completely excluded. However, the fact that a derivative of native ramie cellulose, namely, cellulose triacetate, has been shown by Stipanovic and Sarkd8 to have the parallel orientation, further strengthens the case for parallel chains in native ramie cellulose. The one serious problem yet to be resolved concerns the mechanism of conversion of cellulose I into cellulose I1 during mercerization. As the cellulose really is not solubilized during the treatment, it is difficult to envisage a mechanism whereby parallel chains can be converted into an antiparallel orientation. Sarko and O k a n ~ proposed *~ a mechanism whereby this might be possible, but details of this conversion are still poorly understood. Because the X-ray patterns of the native celluloses from all organisms examined are quite similar, it now appears that the weight of the present evidence supports the concept that the chains of all native celluloses are in the parallel orientation. This conclusion has very important implications for the mechanism of polymerization and crystallization of native cellulose. If all the chains are oriented in the same direction, and growth proceeds from one end of the microfibril, only one mechanism of polymerization (either addition at the reducing or nonreducing end) need be invoked. If, on the other hand, the chains are antiparallel, some more-complex kind of mechanism must be envisaged that would allow some chains to be elongated from the reducing end, and others from the nonreducing end. For those who study biosynthesis, it is therefore a relief to learn that the weight of evidence favors the parallel model! Another structural consideration relevant to biosynthesis is the fact that, within a chain, each D-glucosyl residue is rotated approximately 180" with respect to its neighbor, as depicted in 1. Thus, cellobiose may actually be considered to be the true repeating-unit within the chain, and it is necessary to consider whether some activated form of the disaccharide (rather than of D-glucose) might be the donor for chain elongation; this point will be considered again in Section V,2. Finally, the question of the average degree of polymerization (d.p.) of the glucan chains must be considered. Determinations of the d.p.of polysaccharides are usually made by viscosity measurements, but native cellulose, being highly insoluble and often in close association with other cell-wall polysaccharides and lignin, presents special prob(48) .4. J . Stipanovic and A. Sarko, Polymer, 19 (1978)3-8. (49) A. Sarko and T. Okano, Proc. Ekmun-Days Int. Symp. Wood Pulp. Chem., 4 (1981)

91-95.

BIOSYNTHESIS O F CELLULOSE

115

TABLEI Average Degree of Polymerization (d.p.)of Native Celluloses d.p. of cellulose Material

Acetobacter ~ y l i n u m ~ ' * ~ * Gossypum hirsutum (cotton fiber)% Primary cell-wall Secondary cell-wall Acanthamoeba castellanis V~lonia~~ Seed hairsa;" Bast Wood from angiosperms";" Wood from g y r n n o ~ p e r m s " ~ ~ ~ ~~

2,000-3,700;5,700 2,000-6,000 13,000-14,000 2,000-6,000 26,500 10,350 9,550 8,200 8,450

~~

Average of all species examined.

lems, as discussed by Goring and Timell.50Purification of the cellulose by sequential extractions may lead to chain breakage, as may some of the derivatization techniques used for solubilizing the cellulose for viscosity measurements. Thus, the values presented in Table I should be considered to be minimum estimates; however, care has been taken only to cite values obtained by techniques that should lead to minimal degradation. A determination of the 6. of the A. xylinum cellulose by Takai and coworkers51yielded a value of 5,700 residues per chain; values obtained by Man-Figini and Pion52were somewhat lower (2,OOO-3,700). The cellulose of primary cell-walls of higher plants, a classic example being the elongating cotton-fiber,%also has a relatively low and heterogeneous d.p.value ranging from 2,000-6,000, as does the cellulose from A ~ a n t h a m o e b aOn . ~ ~the other hand, the native cellulose of the unicellular alga Vdonia has been to have a d.p.of 26,500, with some molecular-weight species approaching 44,000.The d.p.of the secondary-wall cellulose of higher plants is also much higher than that found in the primary walls. M a ~ x - F i g i n i ~ ~ found a d.p.of 13,OOO-14,000 for the secondary-wall cellulose of the cotton fiber, and the homogeneity of molecular weight was surprisingly high. Goring and Tirnell5O examined other, mature seed-hairs and bast fibers, and also found d.p. values ranging from 7,000 to 15,000.The native cellulose of wood and bark tissues showed values (50)D.A. I. Goring and T. E. Timell, Tappi, 45 (1962)454-460. (51)M.Takai, Y. Tsuta, and S. Watanabe, Polym. ]., 7 (1975)137-146. (52)M. Man-Figini and B. G. Pion, Biochim. Biophys. Acta, 338 (1974)382-393. (53) M. Man-Figini,]. Polym. Sci., Part C, 28 (1969)57-67. (54)W. E. Blanton and C. L. Villemez,]. Protozoal., 25 (1978)264-267. (55)A. Palma, G. Biildt, and S. M. Jovanovic,Makromol. Chem., 177 (1976)1063-1072.

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DEBORAH P. DELMER

from 7,000 to 10,0o0, the softwood celluloses having, on the average, slightly lower values than those of the h a r d ~ o o d s . ~ ' The length of microfibrils observed far exceeds the length of even the longest of the D-glucan chains. Thus, it would appear that chains are terminated and re-initiated many times during the course of synthesis of a microfibril. The presence of such ends within the microfibril has been proposed by MiilethaleP as being one cause of the amorphous regions in the microfibril. Unfortunately, we have absolutely no idea as to what regulates chain initiation or termination during cellulose biosynthesis. The surprisingly monodisperse, molecular-weight distribution observed by M a ~ x - F i g i n ifor ~ ~ cotton-fiber, secondary-wall cellulose, and the fact that this distribution was not affected by changes in the rate of synthesis, led her to propose that some kind of template mechanism may govern the chain length. However, it is difficult to envisage such a mechanism for polysaccharide synthesis, and no experimental evidence exists to support the hypothesis. Maclachlanj proposed that cellulases may continually break chains and create new initiation-sites, but direct experimental evidence for this is also lacking. An alternative possibility is that imperfect alignment of the D-glucan chains during polymerization and subsequent crystallization could eventually lead, at the region of an active site, to a strain that could lead to termination. As the cellulose of higher-plant and algal secondary-walls is more highly ordered, the strain may dethis is, however, velop less frequently, and thus lead to a higher at present pure conjecture.

6.;

Iv. CYTOLOGICAL INVESTIGATIONS OF CELLULOSE BIOSYNTHESIS

1. The Site of Cellulose Biosynthesis In the past few years, some impressive observations have been made by microscopists studying cells actively engaged in cellulose synthesis. It is now generally accepted by most workers that, in the bacterium A . xylinum, in most algae, and in all of the higher plants, cellulose is synthesized at the cell surface by an enzyme system localized in the plasma membrane. The notable exception to this conclusion concerns those algae which synthesize a cell wall composed of cellulosic scales; such scales are synthesized intracellularly by way of the Golgi apparatus (see Ref. 57 and references cited therein). a. Observations with Acetobacter xylinum. -In the bacterium A . xyZinum, microscope observations of single cells by Brown and cowork(56) K. Miilethaler,J. Polym. Sci., Purt C, 28 (1969)305-316. (57) R. M . Brown, Jr., and D. K. Romanovicz,Appl. Polym. Symp., 28 (1976) 537-585.

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117

and ZaaP clearly showed the synthesis of a ribbon of cellulose, composed of microfibrils, that is attached parallel to the longitudinal axis of the cell. The microfibrils of the ribbon are extruded from a linear row of pores along one side of the cell, and associate to form the ribbon (see Fig. 1).A. xylinum, possessing no flagellae, is considered to be a nonmotile organism but, in a fascinating, motion picture photographed by Brown and discussed in Ref. 59, the bacterium is clearly propelled forward by the force created in synthesizing the rigid ribbon. In a model presented by Brown,Gothe enzymes responsible for polymerization of the D-glucan chains are presumed to exist below the extrusion pores in the lipopolysaccharide layer of the bacterium, but, to date, no conclusive evidence exists for their precise location; it seems equally probable that they could be located in the inner plasma-membrane. As techniques exist for separating the inner and outer membranes of Gram-negative bacteria,6l and as synthesis of (1+4)-P-~-glucan from UDP-glucose can be demonstrated in vitro with cell-free preparations ofA. x y Z i n ~ m ,the ~ * localization ~~ of the enzyme(s) could be attempted. Early attempts by Cooper and M a n l e ~ ~ ~ to perform such experiments were inconclusive. Haigler and coworkersMalso made the interesting observation that polymerization and crystallization of cellulose microfibrils can be “uncoupled.” This was achieved by incubating A. xylinum cells engaged in cellulose synthesis in the presence of the fluorescent brightener Calcofluor White ST, a compound capable of forming hydrogen bonds with free hydroxyl groups. When present during polymerization, it presumably binds to the newly polymerized, D-glucose residues, and thus prevents microfibril assembly. Hence, they observed production of ordered, but not crystalline, material attached perpendicular to the cell axis in place of the normal ribbons. This material was characterized as non-crystalline cellulose by Benziman and coworkers,G5who also made the interesting observation that the rate of polymerization of D-glucose is enhanced up to 4-fold in the presence of Calcofluor White, implying that crystallization may be the rate-limiting step in cellulose biosynthesis. As the microfibrils produced are, normally, rigid structures, it seems possible that the rate of elongation (58) K. Zaar,J. Cell Biol., 80 (1979) 773-777.

(59) R. M. Brown, Jr.,Proc. Ekman-Days Int. Symp. Wood Pulp. Chem., 3 (1981)3-15. (60) R. M. Brown, Jr., Proc. Philip Morris Sci. Symp., 3rd., (1979) 52-123. (61) M. J. Osbom and R. Munson, Methods Enzymol., 31 (1974) 642-653. (62) L. Glaser,J. Biol. Chem., 232 (1958)627-636. (63) D. Cooper and R. S. J. Manley, Biochim. Biophys. Actu, 381 (1975)97-108. (64) C. Haigler, R. M. Brown, Jr., and M. Benziman, Science, 210 (1980)903-906. (65) M. Benziman, C. H. Haigler, R. M. Brown, Jr., A. R. White, and K. M. Cooper, Proc. Natl. Acad. Sci. USA, 77 (1980) 6678-6682.

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DEBORAH P. DELMER

of an entire microfibril could be constrained by the slowest, individual, catalytic site; when crystalline association between the chains is prevented, such a restriction would be relieved. The rate of cellulose synthesis in A . xylinum has also been shown by Ben-Hayim and Ohadfi6to be stimulated (but only by -30%) by the presence of soluble 0-(carboxymethy1)cellulose (CMC). Haigler and Benziman4I discussed and interpreted these results, and suggested that the high-molecular-weight CMC interferes with synthesis at a higher level of organization, that is, by preventing association of the 3.5-nm fibrils into larger bundles. At this point, it must be mentioned that R. Colvin, who has produced numerous publications on cellulose synthesis in A . xylinum, strongly disagrees with the model of cellulose synthesis proposed by Brown's group and supported by Zaar. Colvin's arguments were presented in a review.' He apparently believes that enzyme(s) localized within the cell produce a soluble precursor (see also, Section V,3) (to cellulose) that is secreted and somehow converted, outside the cell, into crystalline cellulose without the aid of cell-associated enzymes. He thus does not believe that polymerization and crystallization are coupled events that are directed by an enzyme complex within the sudace membranes. There is no question that some evidence now exists for a possible, soluble, high-molecular-weight precursor to cellulose; this topic will be discussed in more detail in Section V,3. Colvin's groiqF?'j*claimed to have observed such an intermediate by electron-microscope observation; however, Willison and coworkers69 argued that these could be artifacts of radiation damage or improper focusing of specimens during microscopy. There is also some reason to question most of Colvin's criticisms of the cytological studies of Brown's group. The images of multiple sites of attachment' of a growing ribbon of cellulose were clearly demonstrated, not only by Brown's group but also by Zaar.jHColvin's techniques of microscopy might not favor the preservation of such structures in intact cells, and certainly not in the membrane preparations that he used for in vitro studies.'" One of his criticisms is, however, partially valid. In the initial study reported by Brown and coworkers,35no carbon source was supplied to the cells before the visual observations were recorded, (66)G. Ben-Ha!-im and I. Ohad,]. Cell Biol., 25 (1965) 191-207. (67) J. H . Colviri and G. G. Leppard, Cun. J . Microhiol., 23 (1977) 701-709. (68)J. R. Colvin, L. L. Sowden, and G. G. Leppard, Cun. J . Microhiol., 23 (1977) 790797. (69)J . H. M . Willison, R. M. Brown, Jr., and S. C. Mueller,]. Microsc. (Oxford), 118 11980) 177-186. (70) J. H. Colvin, PEontu, 149 (1980) 97-107.

BIOSYNTHESIS OF CELLULOSE

119

and thus, Colvin argued7that no cellulose could have been produced under these conditions. However, the observations made by Zaar,58 and all subsequent ones by Brown and coworkers,59~60~64~65 were conducted in the presence Of D-glUCOSe; it seems probable that sufficient, residual mglucose from the growth medium remained associated with the cells described in Brown’s earlier report.70a b. Observations with Algae and Higher Plants.-Several unicellular algae have served as excellent models for cytological studies of cellulose biosynthesis. These algae deposit a layered, cellulosic cell-wall having a highly ordered pattern of deposition of large microfibrils whose direction of orientation alternates with each successive layer deposited. The older cytological studies with these algae have been well reviewed by R ~ b i n s o nSubsequent .~ research has effectively employed the technique of freeze-fracturing the plasma membrane of these algae, in order to look for structures that could constitute the sites of cellulose synthesis. A brief, but succinct, review of these freeze-fracture studies has been presented by Lloyd.” In one of the first examples of such studies, Robinson and Preston72observed ordered arrays of protein subunits (termed “granule bands”) embedded in the plasma membrane of the alga Oocystis. They suggested that these ordered arrays could be the multi-subunit, cellulose synthetase complexes originally proposed to exist by Preston40in his “orderedgranule hypothesis” of 1964. However, subsequent work by Brown and M o n t e ~ i n o sshowed ~ ~ , ~ ~that the structures observed by Robinson and Preston72were situated on the inner face of the cytoplasmic half (P-face) of the plasma membrane. Brown and Montezinos also observed, in addition to these P-face granule-bands, another type of ordered protein-complex on the inner side of the outer half (E-face) of the plasma membrane. These ordered arrays were nearly always observed to be situated at the ends of microfibrils. They called these ordered arrays of protein subunits “terminal complexes,” and proposed that they constituted the cellulose-synthesizing, enzyme complexes. Because of their precise orientation, it was proposed that the granule bands situated on the opposite face of the plasma membrane play some role in the orientation of the microfibrils. Fig. 2A shows a model of these various complexes as interpreted by Montezinos and Brown.74 (70a) M. Benziman, personal communication. (71) C. Lloyd, Nature, 284 (1980) 596-597. (72) D. G . Robinson and R. D. Preston, Planta, 104 (1972)234-246. (73) R. M. Brown, Jr., and D. Montezinos, Proc. Natl. Acad. Sci. USA, 73 (1976) 143147. (74) D. Montezinos and R. M. Brown, Jr.,J. Suprarnol. Struct., 5 (1976)277-290.

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DEBORAH P. DELMER

A

B Rosette

I

.

@

7

lOnm 15nm 2Onm

FIG.2.-A. A Model Depicting the Molecular Architecture of the Plasma Membrane of Oocystis apiculata During Secondary-wall F~rmation.'~ [MF, Cellulosic microfibrils; TC, terminal complexes; PTC, paired, terminal complexes; CR?, regions of possible, transmembrane control; GB, granule bands;TC1, impressions of terminal, complex particles; IP, intramembranous particles, AL, region of membrane phospholipids af-

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121

Similar, and equally elegant, results have been reported by Giddings and for the alga Microsterias denticulata. This group also observed, by freeze-fracture techniques, the so-called “terminal complexes” associated with the ends of microfibrils. These terminal complexes, however, show some morphological differences from those observed in Oocystis (compare the model of Brown and Montezinos, in Fig. 2A, with that of Giddings and coworkers in Fig. 2B). In Microsterias, the complex consists primarily of a hexagonal array of from 3 to 175 rosettes, consisting of 6 particles each, which fractures with the P-face of the membrane. On the E-face of complementary fractures, particles complementary to the central hole of the P-face rosettes were sometimes evident. No structures similar to the granule bands of Oocystis were apparently observed in Microsterias. The studies on Microsterias may be relevant to an older study by Kiermayer and D ~ b b e r s t e i n who , ~ ~ observed, in thin sections of Microcells, flat vesicles containing regular arrays of 20-nm particles; sterias they suggested that these vesicles are Golgi-derived, and ultimately fuse with the plasma membrane, and thus could constitute precursors to the terminal complexes observed by Giddings and coworker^.^^ Less distinctive, cylindrical, terminal complexes have also been reported in another alga, namely, G l a ~ c o c y s t i s . ~ ~ Terminal complexes have also been claimed to have been observed in freeze-fracture studies of higher-plant cells. However, at least to the inexperienced eye of this biochemist, the images seen are far less clear than those observed with the algae. Thus far, such complexes have been reported in cells of corn root^,^*,^^ radish roots,8O and cotton (75) T. H. Giddings, Jr., D. L. Brown, and L. A. Staehelin,J . Cell Biol., 84 (1980) 327339. (76) 0.Kiermayer and B. Dobberstein, Protoplasma, 77 (1973) 437-451. (77) J. H. M. Willison and R. M.Brown, Jr.,J. Cell Biol., 77 (1978) 103-119. (78) S. C. Mueller, R. M. Brown, Jr., and T. K. Scott,Science, 194 (1976) 949-951. (79) S. C. Mueller and R. M.Brown, Jr.,J. Cell Biol., 84 (1980) 315-326. (80) J. H. M. Willison and B. W. W. Grout, Planta, 140 (1978)57-58.

fected by granule bands; and UAL, regions of membrane phospholipids not affected by granule bands.] B. A Model of Cellulose-Fibril Deposition During Secondary-wall Formation in Mic r o s t e r i ~ s[Each . ~ ~ rosette is believed to form one 5-nm microfibril. A row of rosettes forms a set of 5-nm microfibrils which aggregate laterally to form the larger fibrils of the secondary wall. Above: side view. The stippled area in the center of a rosette represents the presumptive site of microfibril formation, although details of its structure, composition, and enzymic activity remain unclear. Below: surface view, with expanded, crosssectional view of cellulose fibrils.]

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DEBORAH P. DELMER

fibers.x’ In general, the “complexes” in plant cells are smaller, and less distinct, than in the algae, consisting usually of one globule at the end of a microfibril, although Mueller and Brown79also claimed to have observed rosettes similar to those found in Microsterius. It is, perhaps, unfair to criticize too strongly the lack of clarity in these freeze-fracture images. The microfibrils of higher-plant cells are considerably smaller than those found in the algae; thus, the size and complexity of such a plant terminal-complex might be expected to be less than those for the algal cells. It is necessary to mention a report by Grout,H2strongly supported by Colvin,’ which proposed that no such terminal complexes exist in plant cells, but rather that cellulose synthesis proceeds by a relatively slow accumulation of cellulose precursors in the outer regions of the plasma membrane, followed by swift, spontaneous precipitation, and crystallization without the aid of enzymes. Grout’s conclusions were based on deep-etch studies of the outer surface of the plasma membrane of tobacco protoplasts engaged in cell-wall regeneration. He saw no terminal complexes, and only saw microfibrils after a lag of 16 hours after wall removal. However, the complexes, if embedded in the membrane, may only be made visible by a fracturing of the two membrane faces. Furthermore, Klein and D e l m e P observed microfibril deposition in soybean protoplasts within minutes after wall removal, and such microfibrils increase in size progressively with time; thus, there seems no need to invoke a sudden crystallization following a long lag. What is the evidence that the terminal complexes observed are really cellulose-synthesizing complexes? Obviously, cytological procedures do not allow the investigator to “see” the process ofpolymerization; thus, all of the evidence available is circumstantial. Evidence in favor of such a role for the terminal complexes is, in my opinion, strongest for the algae Oocystis and Microsterias. There is no doubt that these complexes are found embedded within the plasma membranes at the ends of microfibrils. In Microsterias, the distance between rows of rosettes is equal to the center-to-center distance between parallel fibrils in the wall. Furthermore, the widest fibrils (those in the center o f a band, see Fig. 2b) are found associated with the longest rows ofrosettes, whereas shorter rows of rosettes appear to give rise to narrower fibrils. In Oocystis, the number of particles per complex has been estimated to approximate the number of D-glucan chains per microfibril. The complexes and associated microfibrils are (81) J . H. IM. Willison and R. M . Brown, Jr., Protoplasma, 92 (1977)21-41. (82) B. W. W. Grout, Plonta, 123 (1975)275-282.

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always associated with the innermost, developing layer of the wall, and are perpendicular to the previous layer. Montezinos and BrownB3 found that treatment of Oocystis cells with the divalent-cation chelator (ethylenedinitri1o)tetraacetic acid (EDTA) results in dissociation of the complexes from the microfibrils, and the effect is, at least partially, reversible by Mg2+.Such concentrations in this chelator have been shown by other worker^'^^^*^^^ to inhibit cellulose synthesis in vivo. The possible arguments against a role for the terminal complexes are: ( a ) the technical limitations, which do not allow a correlation of the cytology with direct biochemical measurements of cellulose synthesis, and (b) the possibility that the complexes are artifacts resulting from an unnatural aggregation of plasma-membrane proteins during preparation of the samples. Cytologists counter the latter argument with the facts that ( a ) the images are precise and quite reproducible, (b)the cells undergo little or no previous fixation prior to freezing, and (c) the existence of microfibril impressions indicates that the cells were turgid before freezing. In the present author’s opinion, the evidence at present tends to favor the conclusion that the terminal complexes observed, at least in the algae, do represent the sites of cellulose synthesis.

2. Orientation of Microfibrils The cellulosic microfibrils in the cell walls of algae and higher plants often have a characteristic pattern of orientation. In the thin, primary-cell walls of dividing, non-differentiated, higher-plant cells, the orientation of microfibrils tends to be random. However, as such cells begin to elongate, the innermost, newly formed microfibrils tend to become parallel in a direction transverse to the growth axis. According to the “multinet-growth hypothesis” of Roelofsen and Houwink,S6 the realignment of the microfibrils during elongation occurs passively by the forces generated during elongation, although studies by Roland and coworkerP indicated that such a hypothesis may be a somewhat oversimplified view of the orientation of cell-wall components in elongating cells. However, the most striking cases of precise orientation of microfibrils occur in the secondary walls of plant cells, and in the algae. In these instances, the walls consist of distinct layers of pre(83) D. Montezinos and R. M. Brown, Jr.,Cytobios, 23 (1979) 119-139. (84) H. Quader and D. G . Robinson, Eur. I. Cell Biol., 20 (1979)51-56. (85) D. Montezinos and D. P. Delmer, Planta, 148 (1980)305-311. (86) P. A. Roelofsen and A. L. Houwink, Acta Bot. Need., 2 (1953)218-225. (87) J. C. Roland, B. Vian, and D. Reis, Protoplasma, 91 (1977) 125-141.

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cisely parallel microfibrils, the directions of which alternate in each succeeding layer. Such cells are not elongating, and some mechanism other than passive forces must be invoked in order to account for the precise orientations observed. Because the bacterium A. xylinum extrudes its newly synthesized cellulose as a ribbon into the growth medium, no mechanism is required for orientation of these microfibrils. However, the observation by Brown, in his motion pictures of living A. xylinum cells, that the cells are propelled forward by the elongation of a relatively rigid, cellulosic ribbon, may be relevant to our understanding of the mechanism of orientation of microfibrils in the maturing walls of algae and higher plants. As the synthesizing complex is embedded in a fluidmosaic membrane, it seems probable that the propelling force created by the act of polymerization could move the complex within the membrane and, therefore, that no external source of energy for such movement need be invoked. (If the complexes do move within the membrane in algal and plant cells, it is worth noting that this constitutes a notable difference from the stationary complexes of A . xylinum. ) The question of interest, therefore, is not how the complex in algae and plants moves, but rather, what determines the precise direction of the motion, such that highly ordered patterns of orientation result. Ever since the discovery, by Ledbetter and Porter,"* of microtubules below the surface of the plasma membrane, suggestions have been made that these structures play some role in microfibril orientation. The suggestion arose because of two observations: that (1) the orientation of microtubules has very frequently, but not always, been observed to be parallel to the orientation of the microfibrils most recently synthesized, and (2) agents, such as colchicine, that disrupt microtubules interfere with the orientation, but not the synthesis, of cellulose microfibrils. The literature pertaining to these studies has been well reviewed by Robinson: Schnepf and cow0rkers,8~Hepler and Palevitz,goand Heath?' In sum, the present evidence seems to favor some role for microtubules in orientation; in some cases, such as the , ~ ~a series of papers studies on guard cells by Palevitz and H e ~ l e rand on Oocystis by Robinson and ~ o ~ o r k e r s , Rthe ~ ~ case ~ " ~ for ~ micro(88) M. C. Ledbetter and K. R. Porter,]. Cell Biol., 19 (1963)239-250. (89) E. Schnepf, G. Roderer, and W. Herth, Planta, 125 (1975)45-62. (90)P. K. Hepler and B. A. Palevitz, Annu. Reti. Plant Physiol., 25 (1974)309-362. (91) I. B. Heath,]. Theor. Biol., 48 (1974)445449. (92)B. A. Palevitz and P. K. Hepler, Planta, 132 (1976)71-93. (93) D. G. Robinson and W. Henog, Cytohiologie, 15 (1977)463-474. (94)H. Quader, 1. Wagenbreth, and D. C. Robinson, CytobioZogie, 18 (1978)39-51. (95) D. G . Robinson and H. Quader, Eur. ]. Cell Biol., 21 (1980)229-230.

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tubule involvement would seem to be unequivocal, but many questions remain unanswered. It has been difficult to visualize any connection between these microtubules and structures in the plasma membrane, although a peculiar, nonstaining region surrounding the microtubules, first noted by Behnke,% implies the existence of some connection. Thus, the question of the mechanism by which microtubules interact and direct the movement of the synthetase complex remains unanswered. Also unanswered is whether the granule bands observed in Oocystis play some role in orientation and, if so, what their relationship to microtubules may be.

v. THE

MECHANISMOF POLYMERIZATION

The cytological investigations on cellulose biosynthesis already described support the concept that the overall process of cellulose biosynthesis, that is, polymerization of (l-A)-p-D-glucan chains, and the accompanying organization, crystallization, and orientation of microfibrils, is a complex operation that most probably requires a high degree of structural organization. The need to maintain such organization may explain the relatively slow progress achieved to date by workers attempting in vitro synthesis of cellulose by using cell-free preparations. Because convincing in vitro synthesis of true, microfibrillar cellulose has been difficult, if not impossible, to achieve in so many systems, extensive controversy has existed, even for the straightforward question of what activated form(s)of &glucose serve(s) as precursor to cellulose. The following three sub-sections of this article, therefore, deal with this important question. Discussion will be focused primarily on work using cell-free preparations, but will also consider the results of pertinent experiments designed to trace the path of carbon into cellulose in vivo.

1. Involvement of Glycosyl Esters of Nucleoside Diphosphates The most common donors of activated glycosyl groups for polysaccharide biosynthesis have been found to be glycosyl esters of nucleoside diphosphates, and, therefore, it is no surprise that workers in the field have assumed that a D-glucosyl ester of a nucleoside diphosphate is a probable precursor to cellulose. The two compounds that emerged from early studies as the most likely candidates are GDP-glucose and UDP-glucose. To the best of the author’s knowledge, no other Bglucosy1 ester of a nucleoside diphosphate has been successfully used for in vitro synthesis of (1+4)-p-&glucan. (96)0. Behnke, Cytobiologie, 11 (1975)366-381.

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DEBORAH P. DELMER

A possible role for GDP-glucose emerged from studies by Hassid's group,gi~"x using membrane preparations derived primarily from etiolated hypocotyls of mung beans; they found that GDP-glucose, and no other nucleoside (D-glucosyl diphosphate), could serve as a precursor to (l-+$)-p-D-glucan.Since then, a number of reports on in uitro synfrom GDP-glucose, using plant extracts, thesis of (1+4)-@-~-g1~can have appeared in the literature; previous review^^,"^ documented these earlier studies, and also discussed some of the problems of interpretation of the results. To summarize these discussions, a number of observations have led to questioning whether this enzyme reaction is really involved at all in cellulose biosynthesis; these are as follows. ( ( 1 ) The level of GDP-glucose in most plants seems to be vanishingly small; for example, in an analysis by Carpita and DelmerZ3of quantities and patterns of labeling of glycosyl esters of nucleoside diphosphates in the developing cotton fiber, no significant level of GDP-glucose was detected by chemical analyses, nor was any radioactivity found in any compound resembling GDP-glucose during la. is in marked contrast to the situation beling experiments in c k ~This for UDP-glucose, where a substantial pool is observed in cotton fibers'L3.YYand other plant tissues.'"0-104In the cotton fiber, the UDPgliicose pool is readily labeled, and shows rapid turnover during pulse-chase experiments.'" These observations were complemented by measurement of'the levels of activity of enzymes responsible for the synthesis of GDP-glucose and UDP-glucose in plants. Only two report^'^^*'^^ exist that document an activity for GDP-glucose pyrophosphorylase (EC 2.7.7.34)in plants (pea seedlings only). In the present author's laboratory, this enzyme has never been demonstrable in any other plant extract examined, and conversations with colleagues in the field, as well as a report by Heiniger and F r a n ~ ,indicated '~~ that similar results have been obtained in a variety of laboratories. In contrast, the activity of UDP-glucose pyrophosphorylase (EC 2.7.7.9) is quite high in all plant tissues examined,"Jo5as are the levels of sucrose synthetasej (EC 2.4.1.13), another enzyme capable of synthesizing (97) C . A. Barber, A. D. Elhein, and W. Z . Hassid,J. B i d . Chem., 239 (1964) 40564061. (93)A. 1).Elbein, C . A. Barber, and W. Z. Hassid,J. Am. Chem. Soc., 86 (1964) 3093 10. (991 G. Franz, Phybochemistry, 8 (1969) 737-741. (100) M. A. Elnaghy sand P. Nordin,Arch. Biochem. Biophys., 113 (1966)72-76. (101) F.A. Isherwood and R. R. Selvendran, Phytochemistry, 9 (1970) 2265-2269. (102) M . M. Smith and B. A. Stone, Biochim. Biophys. Acta, 313 (1973)72-94. (103) G. A. Barber and W. Z. Hassid, Biochim. Biophys. Acta, 86 (1964) 397-399. (104) C. Peaud-Lenoel and M. Axelos, Eur. 1. Biochem., 4 (1968)561-567. (105) U . Heiniger and G . Franz, Plant Sci. Lett., 17 (1980)443-450.

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UDP-glucose. It might be suggested that, although pool levels of GDP-glucose are low, the flux through the pool could be high; however, observation of a high capacity for synthesis of GDP-glucose would be expected were this so. (b) The incorporation of radioactivity from GDP-glucose into D-glUcan is linear with time for only a few minutes, and is often stimulated by the addition of unlabeled G D P - m a n n o ~ e . ~ , 'Under ~~-~~ such ~ circumstances, glucomannan, a linear heteropolymer containing p-( 1 4 ) linkages of both glucose and mannose, is produced. V i l l e m e ~ ' ~ ~ J ~ * therefore suggested that the GDP-glucose:(ld)-p-D-glucan synthetase (EC 2.4.1.29)'09 really functions in vivo in the synthesis of the glucomannan. This is a quite plausible explanation; it is puzzling, however, that glucomannans are not common constituents of the primary cell-walls of plants, and most of the tissues wherein this enzyme was observed contain cells having such primary walls. In this regard, it is also of interest that Delmer and coworkers1l0showed that, in the developing cotton-fiber, the GDP-glucose:(ld)-P-D-glucan synthetase is active only during the period of fiber elongation and primary-wall synthesis, and activity declines abruptly with the onset of massive, secondary-wall synthesis of cellulose. Thus, the case against a role for GDP-glucose as a precursor to cellulose seemed to have been established. However, the idea has been resurrected by the studies of Hopp and coworkers," who studied in vitro synthesis of cellulose using membrane preparations derived from the alga Prototheca zopfii. They observed that a combination of UDP-glucose and GDP-glucose was required for synthesis of alkaliinsoluble (14)-p-D-glUCan. This unusual finding has not been observed in our laboratory using plant extracts,"' nor reported elsewhere in the literature, and it remains to be seen whether the observation in Prototheca has general significance. It should also be noted that the characterization of the cellulosic product by Hopp and cow o r k e r ~was ~ ~ not extensive. A considerably stronger case can be made for a role for UDP-glucose as precursor to cellulose. The first convincing report of in vitro synthesis of alkali-insoluble (1+4)-p-Dglucan from UDP-glucose (106) A. D. Elbein,]. Biol. Chem., 244 (1969) 1608-1616. (107) C. L. Villemez, Biochem. I., 121 (1971) 151-157. (108) C. L. Villemez and J. S. Heller, Nature, 227 (1970)80-81. (109) Previously, this enzyme and the one that utilizes UDP-glucose have been designated as cellulose synthetases (EC 2.4.1.29 and EC 2.4.1.12, respectively). These designations should still be considered questionable.

(110) D. P. Delmer, C. A. Beasley, and L. Ordin, Plant Physiol., 53 (1974) 149-154. (111) D. P. Delmer, unpublished results.

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came from Glaser's studieP using membrane preparations derived from Acetobacter xylinum. This observation has been repeated numerous times by others with A . x y l i n ~ m The . ~ , GDP-glucose ~~~~ pyrophosphorylase has not been found in A . xylinum, nor does GDP-glucase serve as a substrate for the synthesis of D-glucan in vitro.1° Careful analyses, by Swissa and coworkers: of the flux of carbon through the UDP-glucose pool in uivo in A. xylinum also fully supported a role for UDP-glucose as precursor. However, some caution is in order, as Sanderman and D e k k e F reported that, under the conditions they employed, UDP-glucose served in uitro as a precursor to a water-soluble ( l + Z ) - ~ - ~ g l u c ainn A. xylinum. This finding has been confi~med,"~ but it was also found that, in addition to the water-soluble (l+Z)-p-D-glucan, alkali-soluble and -insoluble (l+i)-P-D-glUcans were also produced from UDP-glucose in uitro. In the slime and A c a n t h a m e b ~ , 'studies ~ in vitro supported a role for UDP-glucose as precursor to (1+4)-P-~glucan,and the reaction products were reasonably well, although not extensively, characterized in these studies. In higher plants, the situation has been greatly complicated by the presence, in almost all plant extracts, of a highly active UDP-glucose:(1+3)-p-~-glucansynthetase (EC 2.4.1.34) which was first demonstrated by Feingold and coworker^"^ in mung-bean seedlings. This enzyme, probably localized in the plasma memb~-ane,"~*"~ displays a relatively low affinity for UDP-glucose (apparent K,,, in the millimolar range), and sigmoidal V uersus S kinetics are often observed, suggesting activation by the substrate, UDP-glucose. The enzyme has often been found to be activated by such p-linked disaccharides as cellobiose and l a m i n a r a b i o ~ e . ~ JThe ~ ~ JDglucan *~ products display a range of solubilities, from water-soluble to alkali-insoluble. The fascinating aspect about this enzyme is its high activity in uitro in extracts derived from tissues that normally contain little or no (1+3)-fi-D-glucan. [Exceptions to this are lily pollen-tube~"~ and cotton fibersY1l8 which contain active enzyme, and also (1+3)-/3-~glucanas a natural wall-constituent.] In most plants, callose, a (1+3)-p-~-glucan, appears as a wound polymer, and the obvious conclusion is that this enzyme is normally latent in most plant cells and is activated by wounding and also (112) H. Sanderman and R. F. H. Dekker, FEBS Lett., 107 (1979) 237-240. (113) Y. Aloni, M. Benziman, and D. P. Delmer, unpublished results. (114) D. S. Feingold, E. F. Neufeld, and W. Z. Hassid,]. Biol. Chern., 233 (1958)783787. (115) R. L. Anderson and P. M. Ray, Plant Physiol., 61 (1978) 723-730. (116) P. H. Quail, Annu. Rev. Plant Physiol., 30 (1979) 425-484. (117) D. Southworth and D. B. Dickinson, Plant Physiol., 56 (1975)83-87. (118) D. P. Delmer, U. Heiniger, and C. Kulow, Plant Physiol., 59 (1977)713-718.

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by the process of preparation of cell-free extracts. The mechanism of such possible activation is not yet understood, although activation of a similar enzyme in fungi by protease has been r e p ~ r t e d . ~ ~ ~ J ~ ~ Nevertheless, this enzyme, fascinating though it may be, has created havoc in the field of cellulose biosynthesis, as numerous claims of the synthesis of cellulose from UDP-glucose in uitro have been erroneous, because of poor characterization of the product, when, in fact, (1+3)-p-~-glucanwas the compound actually produced in vitro. (Primary references have been discussed by Delmer.3) Despite this situation, there are some reports of synthesis of ( 1 4 ) - p - ~ glucan from UDP-glucose where the linkage of the reaction product was reasonably well established; the classic example of this is a Golgilocalized UDP-g1ucose:glucan synthetase first demonstrated by Ray and coworkerslZ1in membranes derived from pea tissue. This enzyme can be distinguished from the UDP-glucose:(1+3)-p-~-glucansynthetase by virtue of its much lower K, for UDP-glucose, and its specific requirement for Mgz+.The two different glucan synthetases, found in many plant tissues, have evolved as marker enzymes in plants for plasma membrane [UDP-glucose:(1+3)-p-~glucansynthetase, called glucan synthetase 111 and Golgi membranes [UDP-glucose:(1 4 ) - p - ~ glucan synthetase (EC 2.4.1.12), called glucan synthetase I], although caution in their use has been advised by Quail.l16For example, glucan synthetase I activity has also been reported by Van der Woude and coworkerslZ2to occur in the plasma membranes of onion stem-tissue. A crucial question, for the higher-plant systems especially, is whether such a UDP-glucose:(l4)-/3-D-glucan synthetase is really a cellulose synthetase. First, the usual localization in Golgi-derived membranes is suspect, although it might be argued that this enzyme is nascent cellulose synthetase en route to the plasma membrane; if it were, it should also be expected to be found more commonly in plasma membrane as well. Second, in tissues engaged in primary-wall synthesis, several other cell-wall polymers, such as the xyloglucan common to dicotyledonous, primary cell-walls and the mixed-linkage (1+3, 14)-p-D-glucan of grass cell-walls, also contain P-D( 1 4 ) linked Dglucosyl residues.6 Ray1= extensively characterized a pea UDP-xy1ose:xylosyl transferase, and found that its activity is stimu(119) M. Fevre and M. Rougier, Planta, 151 (1981)232-241. (120) M. C. Wang and S. Bartnicki-Garcia,Arch.Biochem. Biophys., 175 (1976) 351354. (121) P. M. Ray, T. L. Shininger, and M. M. Ray, Proc. Natl. Acad. Sci. USA, 64 (1969) 605-612. (122) W. J. Van der Woude, C. A. Lembi, D. M. Moore, J. I. Kindinger, and L. Ordin, Plant Physiol., 54 (1974)333-340. (123) P. M. Ray, Biochim. Biophys. Acta, 629 (1980)431-444.

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TABLEI1 “Effect of Concentration of UDP-xylose on Stimulation by U D P - g l u c o ~ e ~ ~ ~ Incorporation of [‘4Clxylose (pmol) at [12C]-UDP-glucoseconcentration ( p M ) UDP-xylose IPM 1

0

1.5

0.25 1.5 10 50

5.0 17 38 43

8.1 23 51 39

250

8.6 46 239 256

A11 assays contained 1 mM MnC1, and 4.5 nCi of UDP-[’*C]xylose, plus sufficient unlabeled UDP-xylose and UDP-glucose to give the con‘I

centrations indicated.

lated by addition of UDP-glucose (see Table 11). Under these conditions, oligosaccharides having structures resembling fragments of xyloglucan can be prepared from the reaction products. He concluded that the UDP-ghcose:( l-*4)-&D-glUcan synthetase probably plays a role in the synthesis of xyloglucan rather than of cellulose. This enzyme is most probably one of the enzymes studied by Maclachlan’s group,’24who studied the synthesis of cellulose, also by using pea tissue. They originally reported a “cell surface cellulose synthetase” activity in pea epicotyl-slicesfZ4; however, later studies30 revealed that, at high concentrations of UDP-glucose (which were used in the initial studies), (1+3)-P-D-glucan was mostly produced, whereas, at low concentrations of UDP-glucose, an increase in P-D-(1-4) linkages was observed. Similar problems exist in studies with cereal tissues; for example, the careful work of Stone and coworkers’02”25 on ryegrass may be cited; this showed that, in addition to (1+3)-p-D-glucan and (1-4)p-D-glucan, a mixed-linkage Dglucan product can be produced from UDP-glucose, and that the ratio of (1-4) to (1+3) linkages in the total D-giucan products increases as the concentration of UDP-glucose is lowe red. In summary, it seems that convincing in zjitro synthesis of cellulose from UDP-glucose using plant extracts has never been conclusively demonstrated. The reader should note that, even for the case ofA. xylinuni and other lower organisms, the in vitro products have been re(1s)C . Shore, Y. Raymond, and C . A. Maclachlan, Plant Physiol, 56 (1975) 34-40. (125J J . A. Cook, C . B. Fincher, F. Keller, and B. A. Stone, in J. J. Marshall (Ed.),Mechunisms of Sacchoride Polymerimtion and Depolymerizution, Academic Press, New York, 1980, pp. 301-315.

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ferred to herein as (1+4)-p-D-g1ucan7not as cellulose. In contrast to the synthesis of chitin, where the in vitro, microfibrillar product has been characterized (chemically, by electron microscopy, and by X-ray diffraction) as authentic hiti in,'^^^'^^ this has not been done for in vitro studies with cellulose biosynthesis. Interestingly, cellulose synthesized in vivo is resistant to solubilization by a mixture of concentrated acetic and nitric acids (the so-called “Updegraff reagent”; for examples of its effects on native cellulose, see Refs. 21 and 26). However, noncrystalline cellulose synthesized in Oocystis in the presence of Calcofluor White is s01ubilized’~~~; and, from discussions with colleagues in the field, it appears that all of the (1+4)-P-Dglucans synthesized in vitro are also solubilized by this reagent, a finding that suggests that these products are noncrystalline. Robinson and Preston12*searched without success for an X-ray diffraction diagram characteristic of cellulose I in the in vitro products derived from UDPglucose in mung-bean seedlings. More significantly, C01vin~~ also did not observe such a pattern from the dried, untreated products of incubation ofA .xylinum membranes with UDP-glucose under conditions where (1+4)-P-D-glUCan is known to be produced. Such a result suggests that true, organized microfibrillar cellulose I is not the in vitro product, and, until proof is otherwise forthcoming, the products should be viewed simply as poly-D-glucose chains that may or may not be some nascent form of cellulose. It is of interest, however, that an X-ray pattern having some reflections characteristic of cellulose 11, as well as electron micrographs of apparent microfibrillar material, were obtained by Colvin after treatment of the products with alkali.70It is possible, therefore, that such “mercerization” can lead to the subsequent crystallization of such a “nascent” form of cellulose, but, as the mercerized product is cellulose 11, this mechanism of microfibril formation certainly does not mimic the in vivo mechanism of assembly. A comprehensive study by Carpita and Delme1-,2~ designed to trace the flow of carbon in vivo from D-glucose to cellulose in the developing cotton fiber, offered strong support for the concept that UDPglucose is a precursor to secondary-wall cellulose in this higher-plant cell. Using cotton fibers cultured in vitro and supplied with radioactive Dglucose, pool sizes and rates of accumulation of label into D-glucose phosphates, UDP-glucose, and end products known, or sus(126) J. Ruiz-Herrera, V. 0. Sing, W. J. Van der Woude, and S. Bartnicki-Garcia, Proc. Natl. Acad. Sci. USA, 72 (1975) 2706-2710. (127) J. Ruiz-Herrera and S. Bartnicki-Garcia, Science, 186 (1974) 357-359. (127a) D. G. Robinson, personal communication. (128) D. G. Robinson and R. D. Preston, Biochim. Biophys. Acta, 273 (1972) 336-345.

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pected, to be derived horn UDP-glucose were measured. Computer analyses of the data allowed construction of a model of carbon metabolism in the cotton fiber that indicated that the flux of carbon through the UDP-glucose pool was more than sufficient to account for the combined rates of synthesis of sucrose, steryl Dglucosides, (1+3)-p-D-glucan, and cellulose. No other glycosyl ester of a nucleoside diphosphate displayed an appropriate pattern of labeling consistent with precursor properties. Thus, in summary, both in vitro and in vivo studies in A. xylinum, and in vitro studies with other, lower organisms, supported the concept that UDP-glucose serves as a precursor to a (1+4)-/3-D-glucan that is most probably related to cellulose; in higher plants, in vitro evidence for such synthesis, either from GDP-glucose or UDP-glucose, is not yet convincing. However, levels of UDP-glucose, and capacity for synthesis of UDP-glucose, are quite high in plant tissues; and in vivo labeling studies strongly support a role for UDP-glucose as precursor to cellulose in higher plants.

2. Possible Involvement of Lipid Intermediates Major credit should be given to Khan and C ~ l v i n who, ' ~ ~ while studying the synthesis of cellulose in A. xytinum, first evolved the concept of a lipid intermediate in polysaccharide synthesis. Since then, a role for lipid intermediates in cell-wall synthesis in bacteria, and in glycoprotein synthesis in both plants and animals, has been well documented.'80 In these cases, the lipids are phosphorylated polyprenols containing, typically, 11 isoprenoid units (undecaprenol) in the prokaryotes, and 16-20 units (dolichols) in the eukaryotes, including higher plants. In addition, the dolichols are distinguished from undecaprenol by having an a-saturated isoprene unit. Despite the impressive progress made in regard to the part played by such lipid-linked saccharides in the synthesis of complex carbohydrates, a role for such compounds in cellulose synthesis has yet to be firmly established.

a. Acetobacter -hum.-The evidence for such an intermediate in the early work by Colvin's group was indirect, consisting mainly of electron-microscope observation of the appearance of fibrillar material following incubation of relatively crude ethanol extracts from

(129) A. W. Khan and J. R. Colvin, Science, 133 (1961) 2014-2015. (130) A. D. Elbein,Annu. Reu. Plant Physiol., 30 (1979)239-272.

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A. xylinum cells with a crude enzyme derived from the growth medium.129J31 Garcia and coworkers,132using EDTA-treated cells, and Kjosbakken and C ~ l v i n , ' using ~ ~ membrane preparations from A. xylinum, demonstrated synthesis, from UDP-glucose, of glycolipids having properties resembling those of polyprenol Dglucosyl phosphate and containing D-glucosyl, mgalactosyl, or cellobiosyl groups. The possibility of the existence of a cellobiosyl-lipid is of special interest, because, as mentioned previously, cellobiose may be considered to be the true repeating-unit in a (1+4)-p-D-glucan. However, for a number of reasons, the significance of the findings with respect to cellulose biosynthesis remains uncertain. First, the glycolipids have never been purified, and conclusively characterized structurally. Second, no evidence for turnover of these lipids or for subsequent incorporation into (1+4)-p-D-glucan has been presented. Third, Benziman's has been unable to reproduce many of the details of the work of Garcia and coworkers.132Fourth, sander man^^'^^ claimed that the disaccharide attached to the lipid is maltose, not cellobiose, and fifth, a report by Couso and indicated that incubation of A. xylinum membranes with UDP-glucose and GDP-mannose results in the synthesis of a prenyl (Dmannosyl-cellobiosyl diphosphate) that most probably plays no role in cellulose synthesis. Also noteworthy, but not conclusive, is the observation that bacitracin, an inhibitor of lipid-linked reactions involved in prokaryotic, cef !-wall synthesis, does not inhibit cellulose synthesis in viuo in A. xyZinwn.10

b. Prototheca zopfii.-Hopp and coworkers17also reported the synthesis, from UDP-glucose, of glycolipids resembling polyprenol (glycosy1 phosphate) by using membranes derived from Prototheca zopfii. The lipids produced were characterized as lipid-P-glucose, lipid-PPglucose, and lipid-PP-oligosaccharidescontaining (1+4)-p-Dglucosyl residues. Evidence was then given that these glycolipids serve as a precursor to a water-soluble polymer that also contains (1+4)-p-D-glu-

(131)A. W.Khan and J. R. Colvin,]. Polym. Sci., 51 (1961)1-9. (132)R. C. Garcia, E. Recondo, and M. A. Dankert,Eur.]. Biochem., 43 (1974)93-105. (133)J. Kjosbakken and J. R. Colvin, in F. Loewus (Ed.),Biogenesis ofPlant Cell Wall Polysaccharides, Academic Press, New York, 1975,pp. 361-371. (133a)M.Benziman, personal communication. (134)H. Sanderman, FEBS Lett., 81 (1977)294-298. (135)R. 0. Couso, L. Ielpi, R. C. Garcia, and M. A. Dankert,Arch. Biochem. Biophys., 204 (1980)434-443.

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cosy1 residues. As already mentioned, they then observed, on addition of GDP-glucose, production of small proportions of alkali-insoluble Dglucan that was briefly, but not conclusively, characterized as ( 1 4 ) p-D-glucan. Although the lipids were not purified, their characterization was sufficiently extensive to indicate that they probably did contain single D-glucosyl or (1-+4)-~-~-glucosyl residues. However, the lack of conclusive characterization of the cellulosic product still leaves open the question of the role of these lipids in cellulose synthesis. It is of interest, however, that, in a subsequent report, these workers showed that coumarin, an inhibitor of cellulose biosynthesis (see Section V,4) did inhibit the transfer of oligosaccharides from the lipids to the water-soluble, polymeric rnate1ia1.I~~ c. Higher Plants.-Essentially no direct evidence exists to support or refute, conclusively, a role for lipid intermediates in cellulose biosynthesis in higher plants. Forsee and E1beinls7reported the synthesis from UDP-glucose of low amounts of a compound resembling polyprenol (D-glucosyl phosphate) by using extracts derived from cotton fibers, but gave no evidence for its further participation in cellulose biosynthesis. Similarly, Pont-Lezica and coworkersz3*were able to demonstrate the presence of a lipid acceptor in several plant-tissues that would accept radioactivity from UDP-D-[14C]glucoseto afford a compound resembling dolichol-P-glucose. As dolichol-P-glucose is also known to participate in protein glycosylation of glycoproteins of the high-mannose type,'"* the observation of synthesis of such a compound does not necessarily implicate its role in D-glucan synthesis. In the present author's laboratory, substantial activity for in vitro synthesis of polyprenol (D-glucosyl phosphate) from UDP-glucose or GDPglucose, using plant extracts,'" has never been detected, despite the fact that synthesis of a compound proved by extensive structural analyses to be dolichol-P-mannose is readily demonstrable by using GDPmannose as the substrate.13gFurthermore, exhaustive analyses of the pattern of labeling of lipids in uiuo, in developing cotton-fibers actively engaged in cellulose synthesis, revealed no substantial levels of labeled D-gliicolipids that might serve as candidates for precursors to cellulose.14uSimilar, negative results were obtained in the presence of specific inhibitors of cellulose, namely, 2,6-dichlorobenzonitrile and (136) H. E. Hopp, P. A. Romero, and R. Pont-Lezica, FEBS Lett., 86 (1978) 259-262. (137) W. T. Forsee and A. D. Elbein,]. Biol. Chem., 218 (1973) 2858-2867. (138) H.Pont-Lezica, C. T. Brett, P. R. Martinez, and M. A. Dankert, Biochem. Biophys. Res. Commun., 66 (1975)980-987. (139) D. P. Delmer, C. Kulow, and M. C. Ericson, Plant Physiol., 61 (1978)25-29. (140) D. Montezinos and D. P. Delmer, unpublished res-ults.

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coumarin (see Section V,4), which were added in the hope of causing accumulation of such precursors. Thus, for higher plants, it may only be concluded that no evidence exists to support a role for such intermediates; if they do exist, they must be present in very low, steadystate levels, and exhibit very rapid turnover, conditions that may have precluded their detection. Knowledge of the direction of chain growth of a polysaccharide is valuable for assessing the mechanism of polymerization. Thus, it is known that “conventional” synthesis of a polysaccharide, involving direct donation of glycosyl groups from glycosyl esters of nucleoside diphosphates, results in growth of the polysaccharide from the nonreducing end of the chain, whereas addition of sugar residues by way of lipid intermediates has been observed to result in growth of the chain from the reducing end.141The synthesis of dextrans utilizing sucrose as the high-energy donor also results in growth from the reducing end of the chain.141Unfortunately, no information is yet available on the direction of growth of cellulose chains, and the high d.p.of these chains is likely to make such analyses exceedingly difficult. 3. Possible Involvement of High-molecular-weight Precursors to Cellulose

In addition to the suggested, but not proved, role for lipid intermediates, much speculation has appeared on the possibility that there may also exist some sort of high-molecular-weight precursor to cellulose. Here again, suggestive evidence exists for such a polymer, but conclusive proof of its existence is still lacking. a. Acetobacter ryZinum.-Early work with A. x y l i n u m by BenHayim and Ohad66gave evidence for a soluble form of “filtrable cellulose.” However, in these experiments, the possibility that some small fibrils of cellulose might have passed through the filters was not rigorously excluded. For many years, Colvin has been an advocate of the existence of a soluble, intermediate polymer in A. x y Z i n ~ r nKjosbak.~ ken and C01vin’~~ incubated A. xy h u m membrane-preparations with radioactive UDP-gIucose, and observed that the radioactive product sedimented as a peak having a density substantially lower than that of cellulose. Although they interpreted this result as indicating that a transient D-glucan of lower density was produced, it seems more probable that, based on the density of the sedimented material (-1.20; even lower than that of pure proteins), they had simply observed a (141) J. F. Robyt, Trends Biochem. Sci., 4 (1979)47-49. (142) J. Kjosbakken and J. R. Colvin, Cen.1. Microbial., 21 (1975) 111-120.

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continued association of the radioactive products with the membrane vesicles. Such vesicles, prepared by sonic disruption of bacteria, are often inverted; therefore, it is possible that UDP-glucose, when accepted as substrate by the outer (inverted) face of the vesicles, leads to synthesis of polymers that are deposited in the interior of the vesicles. N o evidence for any conversion of the “intermediate” product into ~~ cellulose was given. In another report, by King and C ~ l v i n ,a’ radioactive, borate-soluble, polymeric fraction was isolated following incubation of A. xylinum membrane-preparations with radioactive UDPglucose. Some evidence was given that the product was a glucan of molecular weight >30,000; when the radioactive material isolated was re-incubated with the membranes, a small fraction of the radioactivity was transferred to the cellulosic fraction. However, as values were given as the percentage of the total radioactivity supplied, it is difficult to assess the statistical significance of this result. Although of some potential interest, further characterization of this borate-soluble material has not yet been reported. In later s t ~ d i e s , ’ Colvin ~ ~ J ~ and ~ coworkers isolated a fraction, insoluble in 60% ethanol but soluble in water, from the medium in which A . zylinum cells had been grown. Methylation analysis indicated that the material contained a glucan having p-(1-+4) linkages with single glucosyl groups as branches at 0-2 of every third glucosyl residue, on the average. They suggested that this water-soluble glucan was a precursor to cellulose, but gave no proof of a precursor-product relationship. Furthermore, the significance of this polymer with respect to the synthesis of cellulose has since been questioned by Colvin and cow o r k e r ~ , ’and ~ ~ the possible relationship between this polymer and the borate-soluble polymer previously studied is also not clear. Benziman and c o w ~ r k e r soffered ~ ~ ’ ~ additional evidence of a different sort for high-molecular-weight precursors to cellulose in A. xylinum. These workers monitored the kinetics of labeling of various cellular fiactions during incubation of “resting cells” (lacking an external source of nitrogen) in radioactive glucose. Following extraction of the labeled cells with chloroform-methanol, they were able to extract labeled, non-dialyzable, water- and alkali-soluble polymeric materials. During a subsequent chase with unlabeled glucose, radioactivity declined in these fractions, and a corresponding increase in radioactivity (143) G. C . S. King and J. R. Colvin, A w l . Polym. Symp., 28 (1976)623-636. (144) J. R. Colvin, L. Chene, L. C. Sowden, and M. Takai, Can.]. Biockm., 55 (1977) 1057- 1063. (145) L. C. Sowden and J. R. Colvin, Can. 1. Microbiol., 24 (1978) 772-777. (146) J. R. Colvin, L. C. Sowden, V. Daoust, and M. B. Perry, Can. J . Biochem., 57 (1979) 1284-1288.

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Time ( m i d

FIG.3.-Pulse-chase Experiment with Acetobacter ryZinum.1° {Incorporation of D ['4C]glucose (3,300 c.p.m. per nmol) into the water- and alkali-soluble fractions, and its subsequent transfer from these fractions into cellulose. In the pulse, cells were incuo s0" e in buffer at pH 6.0; at the time of the chase, cells bated in 3 mM ~ [ ~ ~ C l g l u c at were diluted in cold buffer, centrifuged, and re-incubated at 30" in buffer either conor lacking &glucose (--).} taining 40 mM unlabeled Dglucose (-)

in cellulose was observed (see Fig. 3).The kinetics of chase suggested that the two fractions (water-soluble and alkali-soluble) were distinct, and that the alkali-soluble polymer(s)served as precursor to the watersoluble material which, in turn, served as precursor to cellulose. The fractions were rendered dialyzable by digestion with crude cellulase, and partially dialyzable by treatment with pronase. Thus, the authors suggested that these polymers might be glucoproteins that serve as precursors to cellulose. Subsequent methylation analysis of these fractions indicated that the radioactivity is mainly located in (1+4)-glucosyl residues, with lesser, and variable, proportions of (1-*2)-glucosyl residues.147Thus, although these fractions have not yet been purified, or completely characterized structurally, they seem to possess properties consistent with a role as precursors to cellulose. b. Protothecu zopfiL-Studies by Hopp and coworkers,17described earlier in connection with the alga Prototheca xopfii, indicated that a radioactive, water-soluble polymer is produced when membrane fractions are incubated with radioactive UDP-glucose. As -40% of the polymer was "hydrolyzed" by pronase treatment, the authors sug(147) U. Rothschild, M. Benziman, and D. P. Delmer, unpublished results.

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DEBORAH P. DELMER

gested that the polymer is a glucoprotein. The existence of p-D-(1+4)glucosyl residues in the polymer was reasonably well established. However, as in the work with A. xylinnm, rigorous proof that the polymer was a glucoprotein is still lacking; and a role for this polymer as preciirsor to cellulose is still not definitely established. The Prototlzecci \ystem does, however, appear to be a promising one for in vitro studies, and it is to be hoped that future work will clarify the nature of this water-soluble polymer.

c. Higher Plants.- Some circumstantial evidence for a high-moleciilar-weight precursor to cellulose was also educed from studies with higher plants. MortimeP" found that radioactive a-cellulose, formed in barley leaves and sugar beet by labeling with radioactive carbon dioxide, contained a distinct fraction of glucan, of higher specific activity, that could be released by extraction of the a-cellulose with hot, dilute trichloroacetic acid. Following a chase with unlabeled CO, , this radioactivity was transferred to the portion of the a-cellulose fraction that was insoluble in this acid. An intermediate role was suggested for this glucan; an alternative interpretation is, however, that the glucosyl residues most recently incorporated were the most accessible to partial hydrolysis with acid, resulting in the release, by trichloroacetic acid, of glucan fragments having low molecular weights. Satoh and coworkers149observed a somewhat comparable phenomenon during i n cioo labeling of mung-bean hypocotyl-segments in radioactive glucose. However, in this instance, a glucan fraction of high specific activity, that could be hydrolyzed by an impure cellulase to yield glucose and (probably) cellobiose, was found associated with membranes and not with the cell-wall fraction, as was the case in Mortimer's study."* Some turnover of the radioactivity in this fraction could be observed during an in oioo, pulse-chase experiment; furthermore, the synthesis ofthe "cytoplasmic" glucan could be inhibited by coumarin, a relatively specific inhibitor of cellulose synthesis (see Section V,4). These studies appear to be of potential importance; nevertheless, definitive, structural characterization of this fraction as (~+4)-~-D-glucan is required, and, as yet, no further analysis of this fraction has been published. In 1975, FranzI5Oreported that incubation of mung-bean membranepreparations with radioactive UDP-glucose resulted in the production of a variety of polymeric products, one of which was identified as a glucoprotein. The radioactivity associated with this presumed glucoprotein could be solubilized by treatment of the alkali-insoluble prod(148)D. C. Mortimer, C a n . / . Bot., 41 (1963) 995-1004. (149) S. Satoh, K. Matsuda, and K. Tamari, Plant Cell Physiol., 17 (1976) 1213-1254. (150) C . Franz, A p p l . Polym. Symp., 28 (1976) 611-621.

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ucts with pronase, or by hydrazinolysis. The released material contained all of its radioactivity in glucose residues; upon digestion with an impure cellulase, compounds migrating coincident with cellobiose and cellotriose standards, during paper chromatography, were released. Some turnover of the “glucoprotein” fraction was indicated in pulse-chase experiments. These results appeared quite promising, but surprisingly have not been pursued further by Franz. As with the results of Satoh and coworkers,149the characterization of the glucan moiety was not complete; crude cellulases were employed and, in both studies, it is uncertain whether the chromatographic techniques employed really allowed resolution of di-, tri-, and tetra-saccharides having different linkages. Quite a different sort of possible, high-molecular-weight precursor to cellulose, namely, ( 1+3)-p-D-g1ucan7has also been considered for higher plants. This suggestion has arisen primarily from studies with developing cotton-fibers by the groups of Delmer, Meier, and Waterkeyn. Meinert and DelmeP first observed the appearance of substantial quantities of (1+3)-glucosyl residues in cell walls of cotton fibers during the early stages of secondary-wall formation. Subsequently, Maltby and coworkers151and Huwyler and coworker^^^*'^^ definitely characterized this material as (1+3)-/%D-glUCan.Some peculiarities in the pattern of labeling of the (1+3)-p-D-glucan in viuo, using cultured cotton-fibers, led Maltby and coworkers151to consider whether this glucan might exhibit turnover and, perhaps, serve as a precursor to cellulose; thus, they observed that, in short-term, labeling experiments, the rate of incorporation of label into (1+3)-p-D-glucan exceeded that expected on the basis of chemical analyses of levels of accumulation of (1+3)-P-D-glUCan in the cell wall at this stage of development. However, in repeated, pulse-chase experiments, significant turnover of this glucan fraction could not be demonstrated (see Fig. 4A). also observed, during short-term labeling Pillonel and of fibers, using intact plants, that (1+3)-p-D-glucan is synthesized at a rate that appears to exceed the accumulation observed in the walls, and they, also, suggested that turnover must occur. In subsequent studies, Meier and coworker^^^^,^^^ were able to demonstrate a slow turnover of the (1+3)-p-D-glUCan fraction in vivo (see Fig. 4B).Tech(151) D. Maltby, N. C. Carpita, D. Montezinos, and D. P. Delmer, Plant Physiol., 63 (1979) 1158-1164. (152) H. R. Huwyler, G . Franz, and H. Meier, Plant Sci. Lett., 12 (1978) 55-62. (153) C. Pillonel, A. J. Buchala, and H. Meier, Planta, 149 (1980)306-312. (154) H. Meier, L. Buchs, A. J. Buchala, and T. Homewood, Nature, 289 (1981) 821822. (155) H. Meier, in Ref. 27, pp. 75-83.

m-s 3 - A -

a 0

c 0

0

60

rnin

120

Ieo

Days after beginning of 14C02 pulse FIG.4.-A. Pulse-chase Experiment using Cotton Fibers (Gossypium hirsuturn) Cultured in oitro; Kinetics of Labeling of Cellulose and (1+3)-/3-~-Glucan.'~~ {Fibers, with (20 mM; 0.08 pCi per pmol). their associated ovules, were incubated in ~-['~C]glucose At the time of the chase, half of the remaining ovules with fibers were briefly rinsed and then incubated in 100 mM unlabeled r>-glucose. Cellulose and (1+3)-p-~-glucan were pulse; (---) chase; (0)cellulose; (0)water-soluanalyzed as d e ~ c r i b e d . ' ~Key: ' (-) ble (1+3)-p-~-glucan; (A) water-insoluble (1-+3)-/3-~-glucan.) B. Pulse-chase Feeding1Mof W02 to Branches of Intact Plants of Gossypium arhoreirm L. ['*C02 (7.4 MBq; 2.0 FCi) was fed in the morning in bright sunlight to branches, each with three or four leaves and one capsule, at 35-40 days post-anthesis, inside poly(ethy1ene)bags. After 30 minutes, -80% of the original radioactivity in the bags had been taken up by the branches which were, however, left inside the bags for another 90 minutes. The latter were then removed, and the plants were kept under normal day and night conditions, when photosynthesis could occur normally, until the capsules were harvested. The radioactivity was determined in the fractions 80%soluble in methanol ( O ) ,and (1+3)-@-~-glucan(callose) (O),and (1-*4)-p-D-glucan (cellulose) (a) fractions of the fibers. Numbers in parentheses are the percent radioactivity in the callose and cellulose type of Dglucans, respectively.]

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nical difficulties with obtaining a clear chase in vivo using intact plants made it hard to decide from these studies whether a quantitative conversion of radioactivity from (1+3)-p-D-ghcan into cellulose had occurred. In the results of Maltby and coworkers,151the chase was effective, as evidenced by a cessation of incorporation of label into cellulose, and in those experiments no turnover was observed. Other cytochemical studies of developing cotton-fibers by Waterk e ~ n indicated '~~ that (1+3)-P-~-glucanis always localized, independent of the age of the fiber, in the innermost wall-layer. The basis of identification of the (1+3)-j%~-glucan was that it showed a specific fluorescence after staining with Aniline Blue, a procedure that is not always specific for (1+3)-P-D-gl~can.'~~ However, Waterkeyn is experienced with the use of this dye, and, assuming that his identification was correct, such a localization for the (1+3)-P-~-glucancould be interpreted to indicate a precursor function. Alternatively, Waterkeyn also offered the suggestion that the glucan could play a role in providing a matrix wherein microfibrils undergo "maturation" and orientation. By chemical analyses of levels of (1+3)-p-~-glucan, Maltby and coworker^'^' found that the maximum level of accumulation occurred during a time when an overlap takes place between the phases of fiber elongation and secondary-wall synthesis, and they similarly suggested that the role of the (1+3)-j3-~-glucancould be in modulating the extensibility of the wall, rather than as a precursor to cellulose. Nevertheless, there are peculiarities in the pattern of labeling of this glucan in vivo that are not readily explained, except by invoking turnover; but, even if such turnover occurs, a specific conversion into cellulose has by no means been proved as yet. A curious analogy in A. rylinurn to the whole (1+3)-P-~-glucanpuzzle in higher plants may also be worth mentioning. In addition to catalyzing the synthesis of (1+3)-/?-D-glucan from UDP-glucose, A. xyZinum membrane-preparations are also active in catalyzing the synthesis from UDP-glucose of water-soluble ( 1 + 2 ) - ~ - ~ - g l u c a n . " ~ * ~ ~ ~ Such a glucan has not been described as a structural component of this organism, although it has been reported to exist in other Grsm-negative bacteria (see references cited in Ref. 112). It has also been observed that some synthesis of this glucan is found in labeling studies in v i ~ dhowever, ~ ~ ; no data as yet exist to support, or refute, an intermediate role for this polymer in cellulose biosynthesis. In summary, much incomplete evidence has been given that offers

(156) L. Waterkeyn, Protoplasma, 106 (1981) 49-67. (157) M. M. Smith and M. E. McCully, Protoplasma, 95 (1978) 229-254.

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suggestions for a role for high-molecular-weight precursor(s) to cellulose. Taken in sum, the evidence is not yet fully convincing, nor does it preclude such a possibility. It is, perhaps, useful to consider the implications for biosynthesis that the existence of such a polymer would have. Based on current concepts of mechanisms of polysaccharide synthesis, it is difficult to envisage a mechanism of microfibril synthesis involving such polymers. If, as Colvin' believes, such precursors associate without the aid of enzymes, to afford crystalline cellulose, it is exceedingly difficult to explain three things: (1 ) as cellulose I1 has a structure much more stable than that of cellulose I, it would be expected that spontaneous crystallization of a soluble form would yield cellulose 11, and yet we know that the native cellulose is cellulose I; ( 2 )the d.p.ofthe cellulose would be expected to be low, and we know that it is not; Colvin' proposed the possible existence of chain ligases, but no evidence exists for such enzymes as of this writing; and ( 3 ) whatever polar group exists that must confer solubility to such an intermediate glucan must be removed, a process that would surely require some kind of enzyme. If, on the other hand, transfer of glucan chains from a (protein?) carrier is mediated by an enzyme, it is difficult to envisage why a process of elongation involving carrier-mediated transfer of oligosaccharides should require such long oligosaccharides, instead of D-glucose or cellobiose. Perhaps, a more likely possibility is that such polymers are primers that are subsequently elongated by a different mechanism, to afford the final D-glucan chains in cellulose. With respect to the possibility that (1+3)-P-D-glucan might serve as an intermediate in cellulose synthesis in higher plants, transfer of Dgliicosyl groups to cellulose by trans-D-glucosylation could 6e envisaged; the spontaneous crystallization of the cellulose might serve to drive this reaction in the appropriate direction. Exoglucanases having transglucosylase activity are known to exist, but the new linkages generated are often random. Meier's group'j5 demonstrated the existence in cotton fibers of a wall-bound exoglucanase that has a preference for (1--d)-~-D-giucansas substrate, but they have yet to demonstrate any trans-D-gliicosylase activity for this enzyme. One attractive argument for the hypothesis that this glucan is a precursor to cellulose is that it could nicely explain the rapid production of (l+d)-P-D-glucan that occurs on wounding of plant cells; if the final conversion of this glucan into cellulose is catalyzed by a very labile enzyme-system, it might be expected that, on cellular damage, the precursor would rapidly accumulate. However, much more study is needed before such a possibility can be considered to be more than just speculation.

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143

4. Genetic Mutations, and Chemical Inhibitors of

Cellulose Biosynthesis The elucidation of metabolic pathways has often been aided by the use of genetic mutations, or specific, chemical inhibitors that result in the blockage of a specific, metabolic reaction along the pathway; such inhibition can sometimes lead to the accumulation of prior intermediates, and thus facilitate their isolation and identification. Mutants of A. xylinum that are incapable of, or impaired in their, ability to synthesize cellulose have been isolated. One such mutant was partially characterized by Swissa and coworker^.^ Although this mutant lacked the capacity to synthesize cellulose, it possessed capability for the normal metabolism of hexose phosphates and UDP-glucose; of considerable interest was the observation that, during in vivo labeling with radioactive D-glucose, it showed enhanced accumulation of label in chloroform-, water-, and alkali-soluble material (as compared to the wild type). Such a finding supports a possibility proposed by these workers, namely, that the water- and alkali-soluble fractions may constitute precursors to cellulose. However, a chemical characterization of these fractions in the mutant strain has yet to be reported. They also found that the mutant is still capable of catalyzing the synthesis, from UDP-glucose, of D-glucans in vitro; this is in contrast to results with another mutant studied by Cooper and M a n l e ~ , ' ~ ~ who found their mutant strain incapable of D-glucan synthesis from UDP-glucose. Incorporation, in whole cells, of label from labeled D glucose into the lipid fraction was also lessened in this mutant. Garcia and also mentioned that they observed a lower capacity for glucolipid synthesis from UDP-glucose with another cellulose-less mutant. Unfortunately, the lack of a good genetic system for A. xylinum has made genetic analyses of these mutants impossible to date; in none of the foregoing was it known how many mutations were involved, or where the specific blocks occurred in these metabolic pathways. To the best of the present author's knowledge, there are, unfortunately, in the algae or higher plants, no known mutants available that are specifically blocked in cellulose synthesis. However, several, relatively specific, chemical inhibitors of the process have been characterized. One of these, coumarin, has been reported to inhibit cellulose synthesis in A. x y Z i n ~ m , ' ~as - 'well ~ ~ as in higher plant^?^,'^^ Relatively (158) D. Cooper and R. S. J. Manley, Biochim. Biophys. Acta, 381 (1975) 109-119. (159) S. Satoh, M. Takahama, and K. Matsuda,Plant Cell Physiol., 17 (1976) 1077-1080. (160) J. Burgess and P. J. Linstead, Planta, 133 (1977)267-273.

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high concentrations (in the millimolar range) are required in order to obtain a substantial inhibition; this appears to be relatively specific for cellulose synthesis, at least in comparison to its effects on the synthesis of other cell-wall polymers. Coumarin has, however, been reported to have some other side effects in plants, as discussed by Montezinos and Delmer.85Nevertheless, it is of some interest that it was effective in inhibiting the synthesis of proposed intermediates in cellulose synt h e ~ i s , ' ~as~ discussed ,'~~ in Section V,3. Some indication that Mg2+may be an important ion for cellulose synthesis comes from studies by Montezinos and as discussed earlier (in Section IVJ). Also of interest is the observation by Quader and RobinsonBdthat calcium ionophores and the cryptates 211 and 212 were potent inhibitors of cellulose synthesis in Oocystis, although their mode of action is at present not understood. One of the most promising inhibitors studied to date is 2,6-dichlorohenzonitrile (DCB), which has been marketed as a herbicide under the names of Casoron and Dichlobenil. Hogetsu and coworkers'61first provided an indication that the mode of action of DCB as a herbicide could be due to its effect on cellulose biosynthesis in plants. Subsequently, Montezinos and DelmeP showed it to be a specific and reversible inhibitor of cellulose synthesis, effective in low concentrations (1-10 pM)in cotton fibers. Meyer and Herthls2also found DCB to be an effective and reversible inhibitor of cell-wall regeneration in tobacco protoplasts. Aloni and Benziman'O reported that DCB also inhibits cellulose synthesis in A. xylinum. Further studies, with cotton fibers and soybean cells,lm indicated that DCB does not inhibit mglucose uptake, or the synthesis of hexose phosphates or UDP-glucose, nor does it affect ATP levels. However, attempts to demonstrate a DCB-induced accumulation of any intermediates beyond the level of UDP-glucose were not successful. Montezinos and Delmef15pointed out that use of this inhibitor for studies of cellulose synthesis should be confined to short-term experiments, as some indication exists that DCB can be metabolized to a derivative that can affect oxidative phosphorylation.lM The documented, herbicidal activity of DCB offers some promise that inhibition of the process of cellulose synthesis could be further exploited as a safe and effective target of herbicide action. (161) T. Hogetsu, H. Shibaoka, and H. Shimo-Koriyama, Plant Cell Physiol., 15 (1974) 389-393. (162) Y. Meyer and W. He&, Plantn, 142 (1978) 253-262. (163) N. C. Carpita, A. Klein, and D. P. Delmer, unpublished results. (164) D. E. Moreland. G . G . Hussey, and F. S. Fanner, Pestic. Bwchem., Physiol., 4 (1974) 356-364.

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5. Possible Factors Affecting the Lability of the Polymerizing System

From the preceding discussions, it is evident that, in all systems studied, and, in particular, in higher plants, attempts to synthesize cellulose in vitro have met with only limited success; this therefore leads to the conclusion that, for poorly understood reasons, the cellulose synthetase complex is a highly labile system. As a conclusion to this article, it may prove useful for future research to discuss possible reasons for this apparent lability. a. Effect of Proteases.-One obvious possibility is that the complex is highly susceptible to proteolytic attack. Chao and Ma~lachlan'~~ reported that, present in extracts of pea seedlings was an endogenous factor, suggested to be a protease, that caused partial inactivation of UDP-glucose:(l4)-/3-D-glucan synthetase activity. (As discussed earlier, it is not certain whether this enzyme functions in synthesis of cellulose or of xyloglucan.) Nevertheless, attempts by these workers to prevent the inactivation by the addition of protease inhibitors or high concentrations of nonspecific protein were unsuccessful. Through conversations with colleagues in the field, as well as personal experience, it is clear that numerous attempts to inhibit protease activity have not resulted in a substantial enhancement of UDP-glucose:(1+4)-p-~glucansynthetase activity. Likewise, the present author knows of no successful attempts to stimulate activity by limited protease treatment, a procedure used with great success for chitin ~ynthetase'~*'~' (EC 2.4.1.16). b. Effect of Poly(ethy1ene Glycol).-It has observed that inclusion of 0.06 molal poly(ethy1ene glycol)-4O00 (PEG-4000) in the isolation medium results in a considerable enhancement of UDP-g1ucose:glucan synthetase activities in membrane preparations derived from cotton fiber~.l@J~~ Polymerization of both &( 1+3)- and p-( 14)-glucosyl residues is enhanced; whereas, in previous work,3l it was possible to detect synthesis only of (1+3)-p-~-glucan,from UDP-glucose in the (165) H.-Y. Chao and G. A. Maclachlan, Plant Physwl., 61 (1978) 943-948. (166) E. Cabib, R. Ulane, and B. Bowers, in B. L. Horecker and E. R. Stadtman (Eds.), Current Topics in Cellular Regulation, Vol. 8, Academic Press, New York, 1974, pp. 1-32. (167) J. Ruiz-Herrera, E. Lopez-Romero, and S. Bartnicki-Garcia,J . Biol. Chem., 252 (1977) 3338-3343. (168) D. P. Delmer, M. Benziman, A. Klein, A. Bacic, B. Mitchell, H. Weinhouse, Y. Aloni, and T. Callaghan,]. Appl. Polym. Sci., in press. (168)A. Bacic and D. P. Delmer, Planta, 152 (1981)346-351.

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presence of PEG4000, it is now routinely observed that -25% of the glucan products contain p-( 14)-glucosyl residues.169Poly(ethy1ene glycols) of lower molecular weight were less effective. A similar enhancement of activity (- 10-fold) by PEG4000 has also been observed for the A. xylinum UDP-glucose:( 1+4)-p-~-glucansynthetase."jRMaclachlan and coworkers170also observed some enhancement of UDPglucose:( 1+4)-P-~-glucan synthetase from pea tissue on using PEG400. Inclusion of PEG in isolation buffers may, therefore, prove to be of considerable help in stabilizing such enzymes. Poly(ethy1ene glycol)~ of high molecular weight are known to promote protein-protein and this could be the mechanism whereby these substances stabilize the activity of a multi-subunit enzyme-complex. It should also be noted, however, that inclusion of PEG4000 in the isolation buffer leads to the production of abnormally large, membrane vesicles that sediment at low centrifugal forces, presumably due to the known ability of PEG to promote membrane fusions. At present, it is not known whether these vesicles contain components only from the plasma membrane, or represent mixed-membrane fusions. c. Attempts to Assay Solubilized Glucan Synthetases-Some attempts to solubilize and purify a UDP-glucose:( 1+4)-p-D-glucan synthetase activity have been made. Tsai and H a ~ s i dwere ' ~ ~ able to solubilize p-( b 3 ) - and p-( l-A)-glucan synthetase activities from membranes of oat seedlings by use of high concentrations of digitonin; they were also able to resolve these activities by chromatography in a column ofhydroxylapatite; however, the solubilized enzymes were quite unstable, and further purification was not attempted. Larsen and BnLmmond'i4 also succeeded in solubilizing, with digitonin, these activities from membranes of Lupinus albus; however, no purification was attempted. Klein16H-1i5 achieved good solubilization of these activities from membranes derived from cultured soybean cells; her soliibil ization procedure involved treatment of the membranes for 15 minutes at 0" with 30 mM cholate at pH 7.8. The solubilized enzymes were quite labile, but could be both stimulated and stabilized by high concentrations of glycerol. Advances in solubilization and re(170) G. Maclachlan, M. Durr, and Y. Raymond,Methodol. Sum. (B)Biochem., 9 (1979) 147-153. (171) L. A. Halper and P. A. Srere, Arch. Biochem. Biophys., 184 (1977) 529-534. (172) J. C. Lee and L. L. Y. Lee,J. Biol. Cliem., 256 (1981)625-631. (173) C. M. Tsai and W. Z . Hassid, Plont Physiol., 47 (1971) 740-744. (174) G. L. Larsen and D. 0. Brurnrnond, Phytochemistry, 13 (1974)361-365. (175) .4. S. Klein, Ph.D. Thesis, Michigan State University, 1981.

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constitution of membrane-bound proteins into artificial lipid vesicles suggest that it would be profitable to pursue these preliminary studies farther. However, to date, no dramatic increases in activity for UDPglucose:( 1+4)-P-D-glucan synthetase have been reported as a result of working with a solubilized form of the enzyme. d. Requirement for an Intact Cell for Activity; Possible Modulation of Activity by a Transmembrane, Electrical Potential.-Assay of cel-

lulose synthetase activity in intact cells has been attempted in a number of laboratories. In higher plants, incubation of pea-epicotyl slices in radioactive UDP-glucose results in production of P-glucan, but the product is mainly ( 1 + 3 ) - P - D - g l u ~ a n . ~Anderson * ~ ~ , ~ ~ ~ and Ray115concluded that this activity occurred primarily at the cut edges of the tissue. Similarly, Delmer and coworkers1lsobserved that intact cottonfibers do not utilize UDP-glucose, but substantial activity for synthesis of (1-*3)-P-D-glUCan is obtained when the cells are damaged; Brett176found a similar phenomenon with suspension-cultured, soybean cells. Klein and Delmel.26*175 observed that preparations of “intact,” soybean protoplasts could utilize UDP-glucose for synthesis of (1+3)-P-D-glucan, but activity was enhanced at least 10-fold when the protoplasts were lysed; therefore, it was difficult to exclude the possibility that the “intact” protoplast-preparation contained a low percentage of damaged protoplasts. The main conclusions from all of these experiments are that (a) intact plant-cells utilize UDP-glucose poorly, or not at all; and (b) when damaged (or even just rendered permeable by treatment with a detergent or dimethyl sulfoxide, or by cold shock”’), they can utilize the substrate for synthesis of (1+3)-P-D-glucan, but not for synthesis of cellulose. This, in turn, leads to the conclusion that the cellulose synthetase complex can only accept UDPglucose from the inner face of the plasma membrane and, unfortunately, loss of cellular integrity results in inactivation of the complex. Carpita and Delmer177*178 proposed that one feature of an intact cell, that is, a transmembrane, electrical potential (A$) may be an important factor for maintaining an active, cellulose-synthetase complex. This hypothesis evolved from observations of another effect of PEG-4000 on cellulose synthesis in cotton fibers; it was found that cutting of intact fibers (just once) with scissors resulted in an essentially total cessation of the synthesis of radioactive cellulose from radioactive D-ghCOSe supplied. [The synthesis of (1+3)-p-D-glucan, was not, however, substantially lessened, suggesting that energy-generating systems neces(176) C. T. Brett, Plant Physiol., 62 (1978) 377-382. (177) N. C. Carpita and D. P. Delmer, Plant Physiol., 66 (1980)911-916, (178) N. C. Carpita, in Ref. 10, pp. 225-242.

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n-n- L n :ontrol

K - MES

4L

K-l

SIVAL

(+)

BTP/N03 (-)

Frc. 5.-Stimulation of UDP-g1ucose:glucan Synthetase Activities by Conditions that Lead to Induction of a Transmembrane, Electrical P0tentia1.l~~ {The experiment was performed by using membrane vesicles prepared from developing cotton-fibers;

into total P-D-glUCanS was meaincorporation of radioactivity from UDP-~-[~~C]glucose sured. Anion and cation concentrations were 50 mM; valinomycin (VAL) was present at 5 p M ;and UDP-glucose at 0.1 mM; 1 pCi per pmol.}

sary for synthesizing activated Dglucose substrates were not seriously impaired by this procedure.] However, if the fibers were cut in the presence of 0.06 molal PEG-4000, the rate of cellulose synthesis observed was 50% of that of uncut fibers. A variety of observations led to the conclusion that this protective effect of PEG is mainly due to a promotion of resealing of the cut fiber-membranes, which thereby results in restoration of an “intact” cell. Thus, it was reasoned that some feature of an intact cell was essential for maintaining active synthesis of cellulose. It was possible to rule out turgor pressure, and to propose, instead, that a transmembrane, electrical potential could be the critical factor required. Results of later studies by Delmer and coworker^^^^*^^^ provided some support for this hypothesis. Thus, it was shown that re-establishment of a transmembrane, electrical potential (positive-inside) across vesicles isolated from cotton fibers resulted in a 4-12-fold stimulation of p-D-glucan synthesis from UDP-glucose (see Fig. 5). Such a potential was established by the addition of K+ in the presence of an imper-

-

(179) D. P. Delmer, N. C. Carpita, A. Bacic, and D. Montezinos, Proc. Ekman-Days Int. Symp. Wood Pulp. Chern., 3 (1981) 25-27.

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149

meant anion, such as 2-(4-morpholino)ethanesulfonicacid (MES), to membrane vesicles incubated in the presence of valinomycin (VAL). VAL is a K+-specific ionophore that allows the free movement of K+ down its concentration gradient, thereby establishing a net, positive-inside potential. Conditions that should lead to a negative-inside potential, such as the addition of an impermeant cation, for example, 1,3-bis[tris(hydroxymethyl)methylamino]propane (“bistrispropane”; BTP) together with a permeant anion such as NO3-, resulted in no substantial stimulation. Other experiments provided further evidence that the effect was truly due to a creation of A$, and not just to stimulation by K+; it was also shown that creation of a ApH across the vesicles did not lead to stimulation; only creation of A$ (positive-inside) was successful. It is of interest that only a positive-inside potential was effective. As UDP-glucose can presumably only be accepted as substrate from the inner face of the plasma membrane, it is presumed that only inverted vesicles are active; therefore, a positive-inside potential in an inverted vesicle mimics the in vivo situation, which is negative-inside. Analyses of the linkages found in the products of the stimulated reaction revealed that polymerization of both p-( 1+3)- and p-( 14)-glucosyl residues was enhanced. Once again, it is necessary to point out that no proof exists at present that this UDP-glucose:(14)-p-&glucan synthetase activity is related to a true, cellulose-synthesizing reaction. Results of Delmer and coworkers,’80with A. xylinum also indicated that the existence of a A$ across the cell membrane may be a crucial factor for maintaining active synthesis of cellulose. In these studies, it was shown that dissipation of A$, by addition of K+ and VAL to EDTA-treated, A. xylinum cells in the presence of the impermeant anion S042-, resulted in an essentially complete inhibition of cellulose synthesis from supplied D-glucose. When the experiments were performed under conditions where energy metabolism could still be driven18’ by a transmembrane ApH (that is, at pH 4.5;ApH -1.3 or 76 mV), the effect of dissipating A$ was specific for cellulose synthesis; it was also reversible, because, when cells were transferred from a high-K+ medium to a high-Na+ medium, cellulose synthesis resumed. Several possible mechanisms can be envisaged to explain how A$ may modulate the activity of the cellulose-synthetase complex. The effects of membrane fluidity on membrane-bound enzymes is well (180) D. P. Delmer, M. Benziman, and E. Padav, Proc. Natl. Acad. Sci USA, 79 (1982) 5282-5286. (181) E. D. Padan, D. Zilberstein, and H. Rottenberg,Eur. J . Biochern., 63 (1976) 533541.

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DEBORAH P. DELMER

documented.’”2 Lelkes’s:’ has shown that changes in AJ, markedly influence the fluidity (and, almost certainly, also, the orientation of lipids) in phospholipid vesicles. Thus, it is quite conceivable that changes in the lipid environment surrounding a highly organized, enzyme complex could result in conformational changes in the complex. Another possibility is that changes in A+ could influence the movement of substrates (UDP-glucose, lipid intermediates, or proteinlinked carriers) within the membrane. In any case, the loss of A+ upon cellular disruption could well be one factor responsible for the loss of enzyme activity in citru. VI. CONCLUSIONS Many difficulties have been encountered in the study of the biosynthesis of cellulose, chief among them being the apparent lability of the cellulose-synthetase system. Based on evidence accumulated to date, a current model of cellulose synthesis184can be envisaged as indicated in Fig. 6. Polymerization of the mglucan chains occurs by way of a multi-subunit, enzyme complex embedded in the plasma membrane; an almost simultaneous association, by means of hydrogen bonds, of the newly formed chains results in formation of partially crystalline microfibrils. This mechanism of polymerization and crystallization results in the creation of microfibrils whose chains are oriented parallel (cellulose I). In A. xylinurn, the complex is apparently immobile, but, in cells in which cellulose is deposited as a cell-wall constituent, it seems probable that the force generated by polymerization of the relatively rigid microfibrils propels the complex through the fluid-mosaic membrane. The direction of motion may be guided through the influence of microtubules. Much controversy has surrounded the question as to the nature of the active form(s) of D-glucose that serve(s) as precursor to cellulose. Current evidence strongly favors a role for UDP-glucose; much suggestive, but by no means conclusive, evidence indicates that lipid- or protein-linked intermediates, or both, may also be involved. Much of the difficulty in studying cellulose biosynthesis may be at(182) H. K. Kimeiberg, Cell Surf. Rev., 3 (1977)205-293. (183)P. I. Lelkes, Biochem. Biophys. Res. Commun., 90 (1979)656-662. (184)D.P. Delnier, in C. C. Black and A. Mitsui (Eds.), CRC Handbook Series ofBiosolar Resources, Vol. I : Basic Principles, CRC Press, Boca Raton, Florida, 1982, pp. 351-355.

BIOSYNTHESIS OF CELLULOSE

Cell wall

1

u\

Plasma membrane

151

Cytoplasm

Mobile cellulose svnthetase

r

D- gtucose 6 - P

\I

0-

-

glucose I P

Individual D-glucan chain

.UDP

w

fructose Growing microfibril

microtubules?) involved in the orienting movement

sucrose

FIG.6.-Hypothetical Model for the Biosynthesis of [Numbers refer to reactions catalyzed by the following enzymes: 1, invertase (EC 3.2.1.26); 2, sucrose synthetase; 3, hexokinase (EC 2.7.1.1); 4, phosphoglucomutase (EC 2.7.5.1); 5, UDPglucose pyrophosphorylase; and 6, 7, and 8, hypothetical reactions in the pathway to cellulose.]

tributed to the apparent lability of the synthesizing complex. Such compounds as glycerol or PEG4000 have been found to offer some protection of UDP-glucose:( ld)-p-D-ghcan synthetase activity. A hypothesis that the activity may be modulated by the transmembrane, electrical potential may offer another clue to the lability of the complex in vitro. Nevertheless, it is clear that many questions concerning the nature of the complex, and of the process in general, remain unanswered. The writing of such an article as this is a tedious process; but, if even just one imaginative young scientist is stimulated to join the quest as a result of reading it, the effort will have been well worth while. The study of cellulose biosynthesis requires both a combination of careful scientific analyses and imagination, and the field can only profit from the entry of more scientists possessing these qualities. An invitation is extended to you to join in.

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VII. ADDENDUM Since this article was sent to press, several noteworthy findings have been reported in the literature. The first concerns the structural identification of the radioactive compounds which appeared to serve as precursors to cellulose inA. xylinum (see Fig. 3). In a note added in proof to Ref. 168, Delmer and coworkers have concluded that the radioactive compounds present in the water- and alkali-soluble fractions analyzed by Swissa and coworkerss and Aloni and Benziman’O consist of a mixture of sugar phosphates (Dfructose 1,6-bisphosphate7 Dglucose 6-phosphate7and Dfructose &phosphate) and fine fibrils of cellulose. During a chase with unlabeled Dglucose, labeled carbon from the sugar phosphates is rapidly converted into cellulose, concomitant with a much slower, apparent “chase” of the fine fibrils of cellulose into the mat of larger aggregates of cellulose produced upon prolonged incubation in high concentrations of Dglucose. Thus, Delmer and coworkers168concluded that, as a result of a quite extensive analysis of the chloroform-methanol-soluble, water-, and alkali-soluble fractions, no positive evidence exists for intermediates beyond the level of UDP-glucose in A. xylinum. The second finding concerns reports by Aloni and coworkers185and Benziman and coworkersla6 of success in achieving high rates of in vitro synthesis of (1+4)-&~glucanfrom UDP-glucose by using membrane preparations derived from A. xylinum. The key to this success lay in the discovery that the A. xylinum enzyme-system can be activated by GTP. Activation by GTP requires the presence of an additional, protein factor; this factor tends to dissociate from the enzyme, but enzyme-factor association can be promoted by PEG-4000 or by Ca*+.Under optimal conditions, that is, in the presence of GTP, factor, and PEG4000 (or Caz+),initial rates of synthesis of (l-;.4)-P-~-glucan that are 200 times higher than any previously reported can be achieved; such rates exceed 40% of the in viuo rate of synthesis of cellulose in A. xylinum. The enzyme system has also been successfully solubilized by using digitonin, and the solubilized enzyme possesses high activity and displays all of the regulatory properties observed for the membrane-bound enzyrne.l8’ These findings offer new hope that future, in uitro studies can lead to a detailed understanding of the (185) Y. Aloni, D. P. Delmer, and M. Benziman, Proc. Natl. Acad. Sci. USA, (1982) in press. (186) M. Benziman, Y. Aloni, and D. P. Delmer, J . A p p l . Polym. Sci., (1982)in press. (187) Y. Aloni, M. Benziman, and D. P. Delmer,J. BWL. Chem., (1983) in press.

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mechanism and regulation of the synthesis of cellulose in A. xylinum. It will certainly also be of interest to know if the activation by poly(ethy1ene glyco1)s of plant D-ghcan synthetases (see Section V, 5,b) relates to a similar, regulatory mechanism for the synthesis of cellulose in higher plants.

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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY VOL. 41

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES BY HAROLDJ . JENNINGS Division of Biological Sciences. National Research Council of Canada. Ottawa. Ontario KIA OR6. Canada

I . Introduction ............................. . . . . . . . . . . . . . . . . .155 I1. Structures of Capsular Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . .158 158 1. Neisseria meningitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 2 . Haemophilus influenzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 3 . Group B Streptococcus ....................................... 170 4 . Streptococcus pneumoniae .................................... 111. Other Important Structural and Physical Features of Capsular Polysaccharides ...................................... 174 . . . . . . . . . . . . 174 1. Structural Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . .175 2. Determinants and Immunological Specifi 3. Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 4 . Molecular Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 183 5 . Location .................................................. IV . Immune Response to Bacterial Infection ............................ 186 187 1. Phagocytosis ............................................... 2 . Role of Complement ......................................... 187 189 3 . Humoral Antibodies to Polysaccharide Vaccines .................... V. Polysaccharide Vaccines and Immunity ............................. 191 1. Streptococcus pneumoniae .................................... 191 ............................ 193 2 . Neisseria meningitidis . . . . . . . 3 . Haemophilus injluenzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 4 . Group B Streptococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 197 5 . Polysaccharide-Protein Conjugates ............................. 6 . Natural Immunity, and Polysaccharide Serological Cross-reactions . . . . . .200 VI . Bacterial Virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 1. Role of the Capsular Polysaccharide ............................. 202 206 2. Polysaccharide Structure and Pathogenicity .......................

I . INTRODUCTION Vaccination has proved to be one of the most useful scientific developments in the control and eradication of human disease . Early vaccines were based on whole-organism preparations. or on protein toxins isolated from different bacteria and. although these methods 155

Copynght @ 1983 by Academic Press. Inc. All nghts of reproductlon in any form reserved. ISBN 0-12-007241-6

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HAROLD J. JENNINGS

are still effective, the discovery of a “specific soluble substance” secreted by pneumococcal organisms during growth,’ and the immunogenicity of these substances (capsular polysaccharides),2 opened the door to a new and important development in vaccine technology. In 1923, Heidelberger and Avery3 demonstrated that this substance was, in fact, a type-specific, polysaccharide antigen that was able to precipitate: quantitatively, antibodies produced in animals by injection of the homologous, whole organisms. Heidelberger5 has given an interesting account of these significant, early discoveries of the immunogenicity of polysaccharides. In subsequent, pioneering work, he and his associates6 demonstrated that, when used as human vaccines, these purified, pneumococcal polysaccharides provide type-specific protection against pneumococcal infections. However, at that critical stage of development, the phenomenal success of the newly discovered antibiotics in treating bacterial diseases overshadowed the early promise of polysaccharide vaccines. Since then, the prophylaxis of bacterial disease has been the subject of renewed, intensified research,’ due in large part to the expanding incidence of antibiotic-resistant, bacterial strains6 Also, clinical and epidemiological studies have demonstrated that the antibiotic treatment of infectious diseases caused by encapsulated bacteria does not always prevent their morbidity and m ~ r t a l i t y Thus, .~ “cured” H. inJuenzue type b rneningitidis is the leading cause of acquired mental retardation,9 and epidemiological statistics indicate that deaths due to pneumococcal pneumonia occur at the same rate as in the pre-antibiotic era.’oCurrent interest in the capsular polysaccharides has evolved simultaneously with this resurgence of interest in the prophylaxis of human, bacterial disease, because of their potential as good immunogens in providing protection against bacterial infections. The concept of using a purified polysaccharide immunogen devoid of its accom(1) A. R. Dochez and 0. T. Avery, J . E r p . Med., 26 (1917)477-493. (2) T. Francis, Jr., and W. S. Tillet,]. E r p . Med., 52 (1930) 573-585. (3) M. Heidelberger and 0. T. Avery,]. E x p . Med., 38 (1923) 73-79. (4) M. Heidelberger and F. E. Kendall,]. E x p . Med., 61 (1935) 563-591. (5) M.Heidelberger, Annu. Reo. Microhiol., 31 (1977) 1-12. (6) C. M . McLeod, R. G. Hodges, M. Heidelberger, and W. G. Bernhard,J. E x p . Med., 82 (1945) 445-465. (7) J. B. Rohbins, Zmmunochemistry, 15 (1978) 839-854. (8) M. Finland, Rec;. Infect. Dis., 1 (1979) 4-21. (9) H. W. Sell, R. E. Merril, 0. E. Doyne, and E. P. Zimsky, Pediatrics, 49 (1972)206211. (10) R. Austrian, in R. F. Beers, Jr., and E. G. Bassett (Eds.),The Role of Immunological Factors in Infectious, Allergic and Autoimmune Processes, Raven Press, New York, 1976, pp. 79-89.

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panying, complex, bacterial mass is technically elegant. Besides their demonstrated immunogenicity in man, these materials are nontoxic, thus avoiding unpleasant (for example, pyrogenic) and, possibly, other deleterious effects associated with whole-cell vaccines. Another important feature of these purified, polysaccharide immunogens is that they can be chemically and physically defined: criteria that add a greater measure of control over their efficacy as vaccines than can be attained by using whole-cell vaccines. As a measure of the success of these vaccines, up to 1978, 130 million individuals had been immunized with capsular polysaccharides, resulting in a high degree of protection and no fatalities or significant adverse effects.' The purpose of this Chapter is to outline the development of bacterial-polysaccharide vaccines, and also to relate the structures of these capsular polysaccharides to their many roles in the immune response to bacterial infection. Because bacterial disease is host-related, this article will be concerned only with bacterial polysaccharides associated with human disease, particularly those capsular polysaccharides currently being used as human vaccines, or those having some immediate potential as human vaccines. The requirements that mediate this decision include clinical importance, the presence of meaningful epidemiological studies, and the identification of a stable, polysaccharide immunogen. Although Klebsiella pneumoniae" and Staphylococcus aureus'* possess defined, capsular polysaccharides, they have not yet satisfied the first two requirements, and will thus be referred to only briefly. The genus Klebsiella is largely restricted to hospital infections, and, because it has 72 serotypes, more-comprehensive epidemiological studies will be needed before the design of a capsularpolysaccharide vaccine is p ~ s s i b l e . ' ~ In a number of pathogenic bacteria (for example, Salmonella and Shigella), the capsular polysaccharide is replaced by the 0-chain polysaccharide of their lipopolysaccharides. Although these 0-chains have been demonstrated to be the immunological equivalent of the capsular poly~accharides,~~ they will not be covered in this Chapter, because they are unique, in that they can only be isolated from bacteria in their high-molecular-weight form, attached to a highly toxic, and physiologically active, lipid A moiety. This circumstance has generally discouraged the development of lipopolysaccharide vaccines,

-

(11) W. Nimmich, Z. Med. Mikrobiol. Zmmunol., 154 (1968) 117-131. (12) W. W. Karakawa and A. J. Kane,]. Zmmunol., 115 (1975) 564-568. (13) J. Z. Montgomerie, Reo. Infect. Dis., 1 (1979) 736-748. (14) 0.Liideritz, 0.Westphal, A. M. Staub, and H. Nikaido, in G . Weinbaum, S. Kadis, and S. J. Ajl (Eds.),Microbial Torins, Vol. IV, Academic Press, New York, 1971, pp. 145-233.

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HAROLD J. JENNINGS

as the removal of lipid A results in the non-immunogenicity of the resultant O-chain. However, the conjugation of these 0-chains to nontoxic, protein carriers has obvious significance in the future development of 0-chain A previous review of microbial p~lysaccharides'~ has been updated by reviews on the structure'8 and immunological r e ~ p o n s e of ' ~ polysaccharides; a pertinent and comprehensive review of vaccines for the prevention of encapsulated bacterial diseases has also been published.' The term polysaccharide has been used throughout this Chapter, although, strictly speaking, some of the phosphorylated, capsular antigens bear a close, structural resemblance to teichoic acids.

11. STRUCTURESOF CAPSULARPOLYSACCHARIDES I. Neisseria meningitidis Neisserin meningitidis is a Gram-negative organism that has been classified serologically20into groups A, B, C, 29-e, W-135,X, Y, and Z. This grouping system depends on the presence of capsular polysaccharides that, although identified some time ago in the case of groups A (Ref. 21) and C (Ref. 22), were not compositionally defined until later.'""'" In these studies, it was established that the group A polysaccharide is a partially 0-acetylated, (1+6)-linked homopolymer of 2acetamido-2-deoxy-D-mannopyranosyl and that groups B and C pol ysaccharides are homopolymers of sialic

(15)S. R. Svenson and .4. A. Lindberg,]. Immunol., 120 (1978)1750-1757. (16)H. J. Jorbeck,S. B. Svenson, and A. A. Lindberg, Infect. Immun., 32 (1981)497502. (17)K. Jann and 0. Westphal, in M. Sela (Ed.), The Antigens, Vol. 111, Academic Press, New York, 1975,pp. 1-125. (18) L. Kenne and B. Lindberg, in G. 0. Aspinall (Ed.), The Polysaccharides, Vol. 2 , Academic Press, New York, in press. (19)C. T.Bishop and H. J. Jennings, in Ref. 18,Vol. 1, 1982,pp. 291-330. (20) E. C . Gotschlich, T.-Y. Lui, and M. S. Artenstein,J. Exp. Med., 129 (1969)13491365. (21) E. A. Kabat, H. Kaiser, and H. Sikorski,]. E x p . Med., 80 (1944)299-307. (22)R. G. Watson, G. V. Marinetti, and H. W. Scherp,]. Immunol., 81 (1958) 337-344. (23)T.-Y. Lui, E. C. Gotschlich, E. K. Jonssen, and J. R. Wysocki,]. B i d . Chem., 246 (1971)2849-2858. (24) T.-Y. Lui, E. C. Gotschlich, F. T. Dunne, and E. K. Jonssen,]. Biol. Chem., 246 (1971)4703-4712.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

159

3-Deoxy-Dmanno-2-octu~osonicacid (KDO) has also been identified as a component of the group 29-e polysaccharidez5Sz6 and the Escherichia coli K6 capsular poly~accharide.~~ Because of the presence of complex, 3-deoxy-2-glyculosonic acid components and phosphoric diester linkages in these polysaccharides, plus their great fragility, particularly to acid treatment, problems were encountered in the application of more-conventional, chemical techniques to their structural elucidation. This situation prompted a search for new techniques with which to tackle these problems, and one that has been used with great success is l3C-nuclear magnetic resonance (l3C-n.m.r.) spectroscopy. The potential of this technique as applied to polysaccharides had been demonstrated in studies on amyloseZ8and the more-complex polysaccharide heparin.29Subsequent studies on the polysaccharides of N. meningitidis and other bacterial polysaccharides (see later) served to consolidate the method as a powerful technique in structural and conformational investigations of polysaccharides. Since the earlier studies, reports of the application of this technique to other bacterial polysaccharides, and to polysaccharides in general, have been prodigious; these, beyond the scope of this article, have been discussed in a previous Volume of this Series.3oMore-pertinent reviews on the application of 13C-n.m.r. spectroscopy to polysaccharides of human pathogenic bacteria have also been p u b l i ~ h e d . ~ ~ * ~ ~ The structures of the repeating units of these capsular polysaccharides of N. meningitidis are shown in Table I. Although all of the polysaccharides are linear and acidic, and contain acetamido groups, they can be divided into two categories, based on their acidic components: those containing phosphoric diester bonds, and those containing 3deoxy-2-glyculosylonic acid residues. Interestingly, except for the group A polysaccharide, on which some structural information was al-

(25) A. K. Bhattachaqjee, H. J. Jennings,and C. P. Kenny, Biochem. Biophys. Res. Commun., 61 (1974)439-443. (26) A. K. Bhattacharjee, H. J. Jennings,and C. P. Kenny, Biochemistry, 17 (1978)645651. (27) F. M. Unger, Ado. Carbohydl-. Chem. Biochem., 38 (1981)323-388. (28) D. E. Dorman and J. D. Roberts,]. Am. Chem. Soc., 92 (1970) 1355-1361. (29) A. S. Perlin, N. M. K. Ng Ying Kin, S. Bhattacharjee, and L. F. Johnson, Can. J . Chem., 50 (1972)2437-2441. (30) P. A. J. Gorin,Adu. Carbohydr. Chem. Biochem., 38 (1981) 13-104. (31) H. J. Jennings,A. K. BhattacharJee,D. R. Bundle,C. P. Kenny, A. Martin, and I. C. P. Smith,]. Infect. Dis., Suppl., 136 (1977) s78-s83. (32) W. Egan, in J. S. Cohen (Ed.),Magnetic Resonance in Biology, Vol. 1, Wiley, New York, 1980, pp. 197-258.

HAROLD J. JENNINGS

160

TABLEI Structures of the Capsular Polysaccharides of Neisseria meningitidis Group

structure

References

0

1I

A

+

6)-a~-ManpNAc-l-O-P-O-

9

33

I OH

I

OAc + 8)aD-NeupAc(2+ + 9)aD-NeupAc(2+ 718

B C

34 34

I

I

OAc + 3)-a~-CalpNAc(l + 7)p~-KDOp(2 + 4(5

29e

26

I

OAc -+ 6)-a~-Galp( 1+ 4)a~-NeupAc(2+ 0

W-135

35

II

X

+~)~D-G~c~NAc-~-O-P-O-

33

I

Y

+

OH fi)-a~-Glcp(l-+4)a~-NeupAc(2-+ (contains OAc groups) 0

35

II Z

+

3)-a&CalpNAc(l

+

l)glycerol-3-O-P-O-

36

I

OH

ready available,23all of the other structures were deduced entirely by I3C-n.m. r. s p e c t r o s ~ o p y . ~ ~ ~ ~ ~ * ~ ~ - ~ ~ Some of the fundamental principles involved in these structural The lacanalyses are outlined here for the group A poly~accharide.~~ n.m.r. spectrum thereof is shown in Fig. 1; although complex, due to the presence of 0-acetyl substituents, it is considerably simplified, to an eight-resonance spectrum (carbonyl signal at 175.8 p.p.m. not (33) D. R. Bundle, I. C. P. Smith, and H. J. Jennings,]. Biol. Chern., 249 (1974) 22752281.

(34) A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and 1. C. P. Smith, J . B i d . Chern.,250 (1975) 1926-1932. (35) A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and 1. C . P. Smith, Can. 1. Biochem., 54 (1976)1-8. (36) H. J. Jennings, K.G. Rosell, and C. P. Kenny, Can.]. Chern., 57 (1979)2902-2907.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

161

p.p.m.

FIG. 1. -Fourier-transformed, %-N.m.r. Spectrum of the Native Polysaccharide Antigen of Croup A Neisseria meningitidis. [Upper, containing ( a )0-acetylated and ( b ) unacetylated residues, and lower, its fully O-deacetylated form.]

shown), on removal of the 0-acetyl groups. This simplicity indicated that the polysaccharide consists of a linear arrangement of a 2-acetamido-mannopyranosyl phosphate repeating-unit. The individual signals in the spectrum of the 0-deacetylated, group A polysaccharide were assigned by using the generally applicable, empirical methodology of comparing the chemical shifts of the signals of the polysaccharide with the corresponding chemical shifts of the anomers of its monomeric or oligomeric constituents. Experience has indicated that the chemical shifts of the monosaccharides are similar to those of the monosaccharide residues within the polysaccharide, except for substituent These effects, caused by the attachment of any substituent to a sugar moiety, cause an increase in the chemical shift of the carbon atom directly involved in the linkage; this increase is usually accompanied by a deckease of smaller magnitude (or, sometimes, an increase) in the chemical shifts (37) H. J. Jennings and I. C. P. Smith, Methods Enzymol., 5OC (1978)39-50. (38) H. J. Jennings and I. C. P. Smith, Methods Carbohydr. Chem., 8 (1980)97-105.

162

HAROLD J . JENNINGS

of the neighboring P-carbon atoms. Thus, these chemical-shift differences serve to determine the position of linkages; similarities in chemical shifts, especially those involving carbon atoms known to be sensitive to change in anomeric configuration, can be employed to determine the configuration of linkages. By using this approach, the l-+6)-linked.33 ( The linkgroup A polysaccharide was shown to be a - ~ age position was also confirmed by the pattern of the two- and threebondi11P-13Ccoupling manifest in the spectrum of the O-deacetylated polysaccharide. Chemical-shift differences between the signals of the native, and the O-deacetylated, group A polysaccharide (see Fig. 1)indicated that the O-acetyl substituents were linked to 0-3 of the 2-acetarnido-2-deoxy-~-mannopyranosyl residues; a comparison of the intensities of' the characteristic methyl signals of the O-acetyl and N-acetyl groups indicated that 70% of these residues were substituted in this way. A similar analysis indicated that the analogous, gronp X polysaccharide is composed of a repeating unit of a-~-(l+4)-linked2acetarnido-2-deoxy-D-glucopyranosyl phosphate.33 The group Z polysaccharide can be included in the same category as the aforementioned polysaccharides, as it also contains phosphoric diesters.36 The structure was shown to be a repeating unit of 10-(2-acetamido-2deoxy-a-D-ga1actopyranosyl)glyceroljoined through phosphoric diester groups at 03 of glycerol and 03 of the 2-amino-2-deoxy-D-galactose residue. However, in this case, the phosphoric diester is not glycosidically linked to the 2-amino-2-deoxy-~-galactoseresidue, and the structure closely resembles that of a teichoic acid. In the second category of meningococcal polysaccharides, those containing 3-deoxy-2-glyculoses, the group B and C polysaccharides are the simplest in structure34;this is illustrated in the 13C-n.m.r.spectrum of the O-deacetylated, group C polysaccharide, shown in Fig. 2. The simple, eleven-resonance spectrum indicated that the group C polysaccharide is a linear polymer of sialic acid. The group B polysaccharide gives a similar, simple, eleven-resonance spectrum. By using the methyl a- and p-D-ketosides of sialic acid as model compounds, and comparing the chemical-shift differences between some of their carbon atoms with those of the sialic residues in the polysaccharides, it was indicated that the group B polysaccharide is (2-+8)-linked, whereas the group C polysaccharide is (2+9)-linked.34 Similarities in the chemical shifts of the configurationally sensitive signals (C-1, C 4 , and C-6) of the polysaccharides with those of the methyl a-D-ketoside, permitted the a-Dconfiguration to be assigned to both polysaccharides. The carboxylate signal (C-1) proved to be extremely useful in these configurational determinations, as it is readily discernible and undergoes a significant, chemical-shift displacement (- 2 p.p.m.) with

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

r

163

I4m7

5

P.P.m FIG.2. -Fourier-transformed, W-N.m.r. Spectrum of the Native Polysaccharide Antigen of Group C Neisseria meningitidis (upper),and its 0-Deacetylated Form (Iower).

change in anomeric config~ration.~~ This characteristic displacement is dependent on the orientation of the carboxylate group (axial or equatorial), and could prove to be generally applicable to 3-deoxy-2glyculose r e s i d ~ e s . ~ ~ , ~ ~ , ~ ~ The 0-acetyl substituents of the native, group C polysaccharide were located by comparing its 13C-n.m.r. spectrum with that of the 0deacetylated, group C polysaccharide (see Fig. 2). The appearance of characteristic muhiplets in some of the signals of the native polysaccharide showed that the 0-acetyl groups are distributed exclusively between 0-7 and 0-8of its sialic acid residues. The group Y and W-135 capsular polysaccharides also contain sialic acid. However, unlike the groups B and C polysaccharides, they are not homopolymers, as they also contain, respectively, D-glucosyl and D-galactosyl residues.35 Structural studies indicated that the group Y (39) H. J. Jennings and A. K. Bhattacharjee, Carbohydr. Aes., 55 (1977) 105-112. (40) H. J . Jennings, K.-G. Rosell, and K. G. Johnson, Carbohydr. Res., 105 (1982)4556.

164

HAROLD J . JENNINGS

polysaccharide has a -+6)-a-D-Glcp-(14)-a-~-NeupAc-(2+repeating unit, whereas that of group W-135 has a -6)-a-D-Galp-(l+4)-a-DNeupAc-(2+ repeating unit.35Interestingly, these two, serologically distinguishable, polysaccharides differ only in the configuration of one hydroxyl group in their respective, disaccharide repeating-units. The group Y polysaccharide contains 0-acetyl groups (1.3mol per sialic acid residue), but the locations of these have not yet been established. The group 29-e polysaccharide is composed of an alternating sequence of 3-deoxy-p-~-manno -2-octulosylonic acid and S-acetamido2-deoxy-a-~-glucopyranosyl residues; linkage is to 0-7 of the former and to 03 of the latter. 0-Acetyl substituents were also located on both 04 and 0-5 of the KDO residues.26It is of interest that, whereas sialic acid has only been found in bacterial polysaccharides, and elsewhere in Nature, as its a-Danomer, there is strong evidence to suggest that KDO probably exists in bacterial polysaccharides in both of its anomeric f ~ r m ~ . ~ ~ , ~ ~ 2. Haemophilus influenzae The Haemophilus influenzae are Gram-negative organisms that can be serologically classified into six types (a through f) on the basis of their type-specific, capsular polysaccharides. Analytical studies indicated that those of types a, b, c, and f a r e poly(sugar phosphates):’ whereas that of type e contains a “hexosamine-uronic acid” component,’2 since characterized as 2-acetamido-2-deoxy-~-mannuronic acid.43*,44 This component sugar is also a constituent of the type d polys a c ~ h a r i d e . Thus, ~ ~ . ~ ~like the meningococcal polysaccharides, the type-specific polysaccharides of H. influenzae may be divided into two groups on the basis of their acidic components, 2-acetamido-2deoxy-D-mannuronic acid replacing the 2-glyculosonic acids of the former. The structures of the type-specific polysaccharides are shown in Table 11, and, except for one particular of type e, are composed of linear arrangements of disaccharide repeating-units. The clinically (41) E. Rosenberg, G . Leidy, J. Jaff, and S. Zamenoff,]. Biol. Chern., 236 (1961) 28412844. (42) A. R. Williamson and S. Zamenoff, J . Biol. Chem., 238 (1963)2255-2258. (43) P. Branefors-Helander, L. Kenne, B. Lindberg, K. Petersson, and P. Unger, Carbohydr. Res., 88 (1981)77-84. (44) F.-Y.Tsui, R. Schneerson, and W. Egan, Carbohydr. Res., 88 (1981)85-92. (45) P. Branefors-Helander,L. Kenne, B. Lindberg, K. Petersson, and P. Unger, Carbohydr. Res., 97 (1981)285-291. (46) F.-P. Tsui, R. Schneerson, R. A. Boykins, A. B. Karpas, and W. Egan, Carbohydr. Res., 97 (1981)293-306.

165

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES TABLEI1 Structures of the Capsular Polysaccharides of Haemopkilus influenzae

Type

References

Structure

0 a

ll

+ 4)p~-Glcp(l+4)D-ribito&O-P-O-

48

I

OH 0

II

b

-+

3)p~-Ribf(l-+ l)~-ribito1(5-O-P-O-

47,s

I

OH 0

II C

d e

4)pD-GIcpNAc(l+ 3fc~D-Gdp(l-O-P-O3 I t OH OAc + 4)p~-GlcpNAc( 1+ 3)pD-ManpANAc(1-+ -+ 3)p~-GlcpNAc( 1+ 4)p~-ManpANAc( 1+ 3 -+

49,50

45,46 43,44

t

2 p~-Fr~p 0

f

1I

.+ 3)p~-GalpNAc( 1+ 4)a~-GalpNAc( 1-O-P-O-

3

t

51,52

I

OH

OAc

important, type b polysaccharide was the first to have its structure determined4'; I3C-n.m.r. spectroscopy played a prominent role in this early investigation. This technique was also extensively used in subsequent structural determinations on the other H. influenzae, typespecific p o l y ~ a c c h a r i d e s . ~ ~ - ~ ~ ~ ~ ~ - ~ ~ (47) R. M. Crisel, R. S. Baker, and D. E. DormanJ. Biol. Chem., 250 (1975)4926-4930. (48) P. Branefors-Helander,C. Erbing, L. Kenne, and B. Lindberg, Carbohydr. Res., 56 (1977) 117-122. (49) P. Branefors-Helander, B. Classon, L. Kenne, and B. Lindberg, Carbohydr. Res., 76 (1979) 197-202. (50) W. Egan, F.-P. Tsui, P. A. Climenson, and R. Schneerson, Carbohydr. Res., 80 (1980) 305-316. (51) P. Branefors-Helander, L. Kenne, and B. Lindqvist, Carbohydr. Res., 79 (1980) 308-3 12. (52) W. Egan, F.-P. Tsui, and R. Schneerson, Carbohydr. Res., 79 (1980)271-277.

166

HAROLD J. JENNlNGS

Except for the anomeric configuration of the ribofuranosyl residue,53 the repeating unit (1) of the type b polysaccharide was proposed by

Crisel and coworker^.^' They established that the type b polysaccharide is composed of ribose, ribitol, and phosphate in the molar ratios of 1: 1: 1, and, by periodate oxidation studies, that the ribitol is linked at both of its hydroxymethyl groups. The 13C-n.m.r.spectrum ofthe type b polysaccharide exhibited ten individual, carbon signals, indicative of a simple, linear arrangement of the disaccharide repeating-unit. The presence of 13C-31Pscalar couplings in five of these signals was also consistent with the structure proposed. Later studiess3 established the chirality (D) of the ribitol residue, and the anomeric configuration (p-D)of the ribofuranosyl residue. Additional structural studies%*confirmed the structure of the type b polysaccharide. The remaining N. infEuenzae polysaccharides in the phosphoric diester category (a, c, and f) were structurally elucidated by. using similar procedures, and were shown to have other structural similarities; all of them are composed of linear, disaccharide phosphate repeatingunits. The type a polysaccharide, which contains D-glucose, D-ribitol, and phosphate in the molar ratios of 1:1:1was shown to be composed of 4-O-~-D-ghcopyranosyl-D-ribitolresidues linked by phosphoric . ~ independent ~ diesters between 0-5of ribitol and 03 of D - g l u ~ o s eIn studies, two different groups of researchers proposed identical structures for the respective type c (Refs. 49 and 50) and type f (Refs. 51 and 52) polysaccharides. The type c polysaccharide was reported to conand phosphate in the tain D-galactose, 2-amino-2-deoxy-D-glucose, (53) P . Branefors-Helander, C. Erbing, L. Kenne, and B. Lindberg,Acta Chem. Scund., Ser. B, 30 (1976)276-277. (54) B. A. Fraser, F.-P. Tsui, and W. Egan. Carbohydr. Res., 73 (19'79)59-65.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

167

molar ratios of 1:1:1. The structure is based on a 34-(2-acetamido-2deoxy-p-D-glucopyranosyl)-a!-D-galactopyranosyl phosphate repeating-unit, and the phosphate is attached to 0-4of the 2-amino-2-deoxyD-glucose residues in the polysaccharide structure. The type c polysaccharide also contains acetyl substituents situated at 03 of 80% of the 2-amino-2-deoxy-D-glucose residues. The structures of the two remaining 2-acetamido-2-deoxy-~-mannuronic acid-containing, H. injluenzae capsular polysaccharides (d and e) were also elucidated independently by the same two groups of re~earchers.~ Both ~ - ~polysaccharides ~ also contain 2-amino-2-deoxyD-glucose, and the structures of both are based on alternating 2-amino2-deoxy-D-glucose and 2-amino-2-deoxy-D-mannuronic acid residues. The type d p o l y ~ a c c h a r i d eis~ composed ~~~~ of a +4)-/3-D-GlcpNAc(1+3)-/3-~-ManpANAc-( l+ repeating unit, with L-alanine, L-serine, or L-threonine linked to the carboxylate group of 2-acetamido-2deoxy-D-mannuronicacid by an amide bond, whereas the type e polyis composed of a differently linked, -*3)-/3-D-GlcpNAc(1+4)-p-~-ManpANAc-(l+ repeating-unit. Strain-dependent structure-variations have also been demonstrated for the type e polysaccharide.43 One particular strain of the type e organism produced a polysaccharide possessing the foregoing structure but having additional, terminal p-D-fmctofuranosyl groups linked to 03 of all of the 2-acetamido-2-deoxy-D-mannuronic acid residues. 3. Group B Streptococcus

L a n ~ e f i e l d ~characterized ~-~' two polysaccharide antigens obtained from Group B Streptococcus: a group antigen common to all strains, and the type-specific, capsular polysaccharides that distinguish four major serotypes, namely, Ia, Ib, 11, and 111. The type-specific polysaccharides were originally isolated by extraction of the whole, streptococcal organisms with hot hydrochloric acid, and all had identical constituents: galactose, glucose, and 2-acetamido-2-deoxyglucose.57-62 This extraction procedure produces immunologicallyincomplete antigens that form a lower molecular weight core to the complete, native (55) R. C. Lancefield,]. Exp. Med., 57 (1933) 571-582. (56) R. C. Lancefield,]. E r p . Med., 59 (1934)441-458. (57) R. C. Lancefield,]. E r p . Med., 67 (1938)25-40. (58) R. C. Lancefield and E. H. Friemer,]. Hyg., 64 (1966) 191-203. (59) H. Russell and N. L. Norcross,]. lmmutwl., 109 (1972)90-96. (60)J. A. Kane and W. W. Karakawa, Infect. lmmun., 19 (1978)983-991. (61) J. Y. Tai, E. C. Gotschlich, and R. C. Lancefield,]. E r p . Med., 149 (1979) 58-66. (62) D. L. Kasper, C. J. Baker, R. S. Baltimore, J. H. Crabb, G. Sc hihan, and H. J. Jennings,]. E x p . Med., 149 (1979) 327-339.

168

HAROLD J. JENNINGS

The latter antigens, which can be obtained by neutral or buffered (pH 7.0) extraction of whole organisms,61.62,65,67a contain additional, terminal sialic acid residues. The presence of these residues proved to be essential for the immunological expression of the native antigens. The native types Ia (Ref. 68, 69), Ib (Ref. 61, 69), and I11 (Ref. 63) antigens contain D-galactose, D-glucose, 2-acetamido-2-deoxy-~-glucose, and sialic acid in the molar ratios of 2 : 1:1:1, and constitute a group of isomeric polysaccharides, whereas the type I1 (Ref. 70) native antigen has identical component sugars, but in the ratios of 3 :2 : 1:1. The structures of the repeating units of the types Ia, Ib, 11, and I11 polysaccharides are shown in Table 111. These structures were elucidated by first determining the structures of the simpler, core antigens, as describedm for the type I11 polysaccharide. A comparison of the methylation analysis of the core with that of the native polysaccharide permitted the position of linkage of the terminal sialic acid residues in the native antigen to be established. The anomeric configurations of the sugar residues were determined by W-n.m.r. spectroscopy. Structurally, all of the Group B streptococcal antigens constitute an interesting group of polysaccharides, both in their relationships to each other, and to other biologically important molecules (glycoproteins). All of the polysaccharides have in their ~ t r u c t u r e s ~a*com~-~~ mon p-~-GlcpNAc-( l+S)-P-~-Galp-(1+4)-p-~-Glcp trisaccharide which forms the repeating unit of the backbone of the type I11 polysaccharide. This was also presumed, in a previously proposed structure:* to be true of the type Ia polysaccharide; however, the evidence on which this structure was based proved not to be definitive, as it was also compatible with an alternative structure in which the 2-amino-2deoxy-0-glucose residue of the trisaccharide becomes a part of the (63)H. J. Jennings, K.-C. Rosell, and D. L. Kasper, Can. ]. Biochem., 58 (1980) 112120. (64)E. H. Friemer,]. E x p . Med., 125 (1967) 381-392. (65) H. W. Wilkinson, Infect. Zmmun., 11 (1975) 845-852. (66) C. J. Baker and D. L. Kasper, Infect. Immun., 13 (1976) 284-288. (67) J. A. Kane and W. W. KarakawqJ. Immunol., 118 (1977) 2155-2160. (67a) C. J. Baker, D. L. Kasper, and C. E. Davies,]. E r p . Med., 143 (1976) 259-5370. (68) H. J. Jennings, K.-G. Rosell, and D. L. Kasper, Proc. Natl. Acad. Sci. U . S . A., 77 (1980) 2931-2935. (69) H . J . Jennings, E. M. Katzenellenbogen, C. Lugowski, and D. L. Kasper, Biochemistry, in press. (70) H. J. Jennings, K.-G. Rosell, and D. L. Kasper,]. Biol. Chem., in press. (71) H. J. Jennings, E. M. Katzenellenbogen, C. Lugowski, and D. L. Kasper, unpublished results.

TABLEI11 Structures of the Capsular Polysaccharides of Group B Streptococcus Type

Structure

References

Ia

+ 4)-p-D-Gkp-(1 + 4)-p-D-Gdp-(1 +

69

3

t p-~GlcpNAc 4

t

Ib

1 c~-~-NeupNAc-(2 + )-P-DGalp + 4)-p-Dklcp-(l + 4)-p-~-Galp-(l+ 3

69

t

1 p-~ClcpNAc 3

t

I1

1 a-D-NeupNAc-(S+ 3)-P-&alp --* 4)-p-D-GlcpNAc-(l+ 3)-B-D-Galp(l+ 4)-p-D-Glcp-(l -* 3 ) - p - ~ - G l ~ p - ( l 2)-8-~-Galp-( + 1+ 6 3

t

I11

1 p-DGdp +4)-p-~-Gl~p 1 --* ( B)-p-D-GlcpNAc-(1 + 3)+-D-G&-( 1 + 4

t

1 a-~-NeupNAc-(2--* 6)-/3-DGdp

70

f

2 a-D-NeupNAc 62

170

HAROLD J. JENNINGS

branches of the type Ia polysaccharide. This was indicated in subsequent, extensive degradation studies on the types Ia and Ib polysaccharides?” the results of which are consistent only with both having trisaccharide branches as shown in Table 111. All of the incomplete core-structures have branches terminating in P-D-galactopyranosyl residues ,63 ,I%-?1 and the fact that the type 111, core antigen has a structure identical to that of the capsular polysaccharide of type 14 S. pneum ~ n i a e is ? ~of some serological significance.62*63 in the native type Ia (Refs. 68 and 69), Ib (Ref. 69), and I11 (Ref. 63) polysaccharide antigens, the terminal P-D-galactopyranosyi residues of the core antigens are completely masked by sialic acid residues, forming branches that terminate in sialic acid residues. These sialic acid residues are linked to 0-3of the P-D-galactopyranosyl residues of the native type Ia (Refs. 68 and 69) and Ib (Ref. 69) polysaccharides and to 0-6 of the type I11 polysaecharide.63 These branches are of considerable serological importance to the group B streptococcal organism, because of their structiiral homology with some important, human serum-glycoproteins. The terminal 3-0-(N-acetyl-a-o-neuraminy~)-~-D-galactopyranosyl group of the types Ia and Ib antigens is the end group in the human $1 and N blood-group substance^,^^ and the 6-O-(N-acetyl-a-~-neuraminyl)-P-D-galactopyranosylmoiety of the type I11 polysaccharide is also a structural feature of human ~erotransferrin.~~ 4. Streptococcus pneumoniae

Strelitococczis pneumoniae are Gram-positive organisms which, like the group B Streptococcus, have a common, group antigen (Cs ~ b s t a n c e ) ,and ~ ~ .different ~~ type-specific, capsular polysaccharides. Unlike the organisms previously described, S. pneumoniae have been identified in a prolific number of immunologically distinguishable types based on these capsular polysaccharides. To date, there are at and these have been designated least 84 known type-spe~ificities,~.’O types 1-84 in the American system. However, on the basis of serological cross-reactivity among these polysaccharides, the organisms have also been conveniently classified into serologically related groups (see Table IV) in the Danish system. The pneumococcal polysaccharides are particularly important, because early investigations into (72) B. Lindberg, J. Likmgren, and D. A. Powel1,Carbohydr. Res., 58 (1977) 177-186. (73) J . E. Sadler, J. C. Paulson, and R. L. Hill,/. BioE. Chem., 254 (1979)2112-2119. (74) G . Spik, B. Bayard, B. Fournet, G . Streker, S. Bouquelet, and J . Montreuil, F E B S . k t t . , 50 (1975)296-299. (75) D. E. Brundish and J. Baddiley, Biochem.]., 110 (1968) 573-581. (76) H. J. Jennings, C. Lugowski, and N. M. Young, Biochemistry, 19 (1980) 47124719.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

171

their immunological and structural properties resulted in the acquisition of knowledge fundamental to the development of human, polysaccharide vaccines. Structural studies on these polysaccharides also provided technical and conceptual contributions to the general problem of polysaccharide structure, and it is a tribute to earlier investigators that these contributions were made without the benefit of modem instrumental methods. The structures of the pneumococcal polysaccharides have been re~iewed.'~,''Because of their number and structural complexity (up to 7 sugar components in their repeating units), they can be dealt with only briefly in this chapter, and only the structures of those used in the current, pneumococcal vaccine (see Section V,1) are listed in Table IV. Two of the pneumococcal polysaccharides [types 14 (Ref. 72) and 37 (Ref. 78)] are neutral; in fact, that of type 14 is a rare example of a neutral-polysaccharide capsule involved in human bacterial The rest of the pneumococcal polysaccharides are acidic, and can be classified according to their common acidic components. Types 1 (Ref. 79), 2 (Ref. 80),3 (Ref. 81), 5 (Ref. 18), 8 (Ref. 82),9A (Ref. 83), 9N (Ref. 84), and 9V (Ref. 85) have D-glucuronic acid residues, whereas types 6A (Ref. 86), 6B (Ref. 87), 11A (Ref. SS), 13 (Ref. 89), 15F (Ref. W),17F (Ref. 91), 19F (Refs. 92 and 93),19A (Ref. 94),23F (Ref. 95), 27 (Refs. 96 and 97), 29 (Ref. 98),and (77) 0. L a m and B. Lindberg, Ado. Carbohydr. Chem. Biochem., 33 (1976) 295-322. (78) J. C. Knecht, G. Schiffman, and R. Austrian,J . Exp. Med., 132 (1979) 475-487. (79) B. Lindberg, B. Lindqvist, J. Lonngren, and D. A. Powell, Carbohydr. Res., 78 (1980) 111-117. (80) L. Kenne, B. Lindberg, and S. Svensson, Carbohydr. Res., 40 (1975) 69-75. (81) R. E. Reeves and W. F. Goebel,]. Biol. Chem., 139 (1941) 511-519. (82) J. K. N. Jones and M. B. Perry,]. Am. Chem. Soc., 79 (1957) 2787-2793. (83) L. G. Bennet and C. T. Bishop, Can. J . Chem., 58 (1980) 2724-2727. (84) H. J. Jennings, K . G . Rosell, and D. J. Carlo, unpublished results. (85) M. B. Perry, V. Daoust, and D. J. Carlo, Can.]. Biochem., 59 (1981) 524-533. (86) P. A. Rebers and M.Heidelberger,J. Biol. Chem., 139 (1941) 511-519. (87) L. Kenne, B. Lindberg, and J. K. Madden, Carbohydr. Res., 73 (1979) 175-182. (88) D. A. Kennedy, J. G. Buchanan, and J. Baddiley, Biochem.]., 115 (1969) 37-45. (89) M. J. Watson, J. M. Tyler, J. G. Buchanan, and J. Baddiley, Biochem.]., 130 (1972) 45-54. (90) M. B. Perry, D. R. Bundle, V. Daoust, and D. J. Carlo, Mol. Immunol., 19 (1982) 235-246. (91) M. B. Perry, personal communication. (92) H. J. Jennings, K . 4 . Rosell, and D. J. Carlo, Can.J. Chem., 58 (1980) 1069-1074. (93) H. Ohno, T. Y. Yadomae, and T. Miyazaki, Carbohydr. Res., 80 (1980) 297-304. (94) C.-J. Lee and B. A. Fraser,J. B i d . Chem., 255 (1980) 6847-6853. (95) M. B. Perry, V. Daoust, and R. Lowe, unpublished results. (96) L. C. Bennet and C. T. Bishop, Can.]. Chem., 55 (1977) 8-16. (97) L. C. Bennet and C. T. Bishop, lmmunochemistry, 14 (1977) 693-696. (98) E. V. Rao, M. J. Watson, J. G. Buchanan, and J. Baddiley, Biochem.]., 111 (1969) 547-556.

TABLEIV Structures of Some of the Capsular Polysaccharides of Streptococcus pneumoniae Contained in the Current, Pneumococcal Vaccine Structureb

Type"

w

1 2

References

79

-+ B)a-Sugp(1 + 4)aD-GalpA(1 S)aD-GalpA(1 + S)a~-Rhap(l S)a~-Rhap(l+ 3)P~-Rhap(l+ 4)aD-GIcp(l -+ 2 -+

-+

4

&a

80

-+

t

3 4

1 a ~ - G l c p A ( l - +6 ) a ~ G l c p + 4)PD-GlCp(l S)pD-GIcpA(1 -+ -+ 4)P~-ManpNAc(l + S)a~-FucpNAc(l-+ S)aD-GalpNAc(l -+

-+

4 ) a ~ - G a l (-+ l

H,C 5

-+

x

81 100

CO,H 18

Z)PD-GkpA(1 -+ B)aL-FucpNAc(1 -+ 4

t -+

2)aD-Gdp(1 -+

a-Sugp(l 3)crD-Glcp(1

1 0 4)P~Glcp II 3)a~-Rhap( 1 -+ 3)-ribitol-(5-O-P-O-

-+

-+

I

OH

86

12F (12)

+ 4)a~-FucpNAc( 1+ 3)p~-GalpNAc(l+ 4)p~-ManpANAc(l+

3

3

t

t

1

1 aDGalp a ~ - G l c p (+ l 2)aDGkp + 4)pD-Gkp(l+ G)pD-GlcpNAc(l+ 3)p~-Galp(l + 4

14

82 a4 101

72

t

1

0

II

19F (19)

2

+ 4)pD-ManpNAc(1+ 4)aD-Gkp(1--* 2)a~-Rhap( 1-O-P-O-

92,93

I

OH 23F (23)

+ 4)p~-Gfcp(l + 4)pD-Galp(l + 4)a~-Rhap(l+

I

2

P

t

1 aL-Rhap a

US. typing system in parentheses. *Sug = 2-Acetamido-l-arnino-2,4,6-trideoxygalactose.

95

174

HAROLD J . JENNINGS

34 (Ref. 99) contain phosphate. Type 27 also contains p y r u ~ a t e ~as ~,~' an additional acid component, and type 4 contains pyruvate as the sole acid component." Type 1 2 F contains 2-acetamido-2-deoxy-~-mannuronic acid as its only acidic component.101 The structure of the type 3 polysaccharide was the first to be established, by Reeves and Goebel,8' and thus this polysaccharide became a model for many immunological investigations. The concept of a repeating unit was also established in this work, and this was elegantly confirmed in later, chemical-degradation studies by Rebers and Heidelberger,H6in which they isolated the tetrasaccharide repeating-unit of the type 6A polysaccharide in crystalline form in 94% yield. Another early study!' in which partial hydrolysis with acid was employed, established the structure of the type 8 polysaccharide as having a tetrasaccharide repeating-unit containing cellobiouronic acid. Other, extremely valuable, early degradative studies were carried out b y Baddiley and coworkers on the types 11A (Ref. 88), 13 (Ref. 89),29 (Ref. 98), and 34 (Ref. 99)polysaccharides; they were able to establish that each was composed of a pentasaccharide phosphate repeating-unit, and to locate the phosphoric diester linkages in these polysaccharides. Finally, the combined use of gas-liquid chromatography and mass spectrometry by Lindberg and coworker^'^^^'^^ has had a profound impact on the structural analysis of polysaccharides. This was demonstrated in work on the type 2 polysaccharide,8° and in subsequent, structural elucidations of other pneumococcal polysaccharides (see Table IV). 111. OTHER IMPORTANT STRUCTURAL AND PHYSICAL OF CAPSULAR POLYSACCHARIDES

FEATURES

1. Structural Heterogeneity There is now abundant structural, spectroscopic, and biosynthetic evidence to suggest that, except for the possibility of minor structural irregularities, the fundamental structures of bacterial polysaccharides consist of fairly small, regular repeating-sequences of from 1to 7 saccharide units. Thus, as the polysaccharides are multivalent antigens, the effect of minor irregularities in their structures would, in general, (99)G. J. F. Chittenden, W. K. Roberts, J. G . Buchanan, and J. Baddiley, Biochem.J., 109 (1968) 597-602. (100) P.-E. Jansson, B. Lindberg, and U. Lindquist,Carbohydr. Res., 95 (1981) 73-80. (101) K. Leontein, B. Lindberg, and J. L(inngren,Can.]. Chem., 59 (1981) 2081-2085. (102) B. Lindberg, Methods Enzymol.,28B (1972) 178-195. (103) B. Lindberg, Methods Enzymol., 5OC (1980) 3-34

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

175

be unimportant to their immunological specificity. However, minor structural irregularities cannot be ignored in other immunological properties, as it has been demonstrated that the presence of extremely small proportions of attached lipid can have a profound effect on the immunogenicity of polysaccharides (see Section 111,4). A larger degree of structural heterogeneity has been documented for some bacterial, capsular polysaccharides, and this is mostly introduced by the distribution of 0-acetyl substituents on these polysaccharides. Only 70% of the 2-amino-2-deoxy-D-mannose residues of the group A meningococcal polysaccharide have 0-acetyl s~bstituents,3~ and this heterogeneity is also exhibited by the group C meningococcal polysac~ h a r i d e . 3In ~ the latter, an a-D-(2+9)-linked homopolymer of sialic acid, the molar ratio of 0-acetyl to sialic acid is 1.2:l-0. These 0acetyl groups are distributed on the sialic acid residues, in a complex pattern, at 0-7 and 08 (monosubstitution and disubstitution), and some of the sialic acid residues remain unsubstituted (see formulas 25). Interestingly, in the group C polysaccharide, the pattern of O-acetyl substitution is dependent on the conditions used to grow the group C organisms31; this could be important in the production of human polysaccharide vaccines. However, it is unlikely that the extent of this structural variation would be sufficient to impart complete, serological specificity to the variant group C polysaccharide and to preclude its use as an effective, group C meningococcal vaccine, because, even the 0-deacetylated group C polysaccharide can function as an effective, group C meningococcal vaccine in man (see Section V,2).

-

2 R=R'=H 3 R = R' = COCH, 4R=H,R=COCH, 5 R=COCH,,R'=H

The Four Different, N-Acetylneuraminic Acid Residues in the Native Polysaccharide Antigen of Croup C Neisseria meningitidis.

2. Determinants and Immunological Specificity An important step in understanding the immunology of polysaccharides consists in establishing which part of the polysaccharide is re-

176

HAROLD J. JENNINGS

sponsible for its immunological specificity. This part of the polysaccharide is called a determinant, and, from early studies by Goebel,lW it became apparent that determinants constitute only a small part of the large polysaccharide molecule. Antibodies made to the type 3 pneumococcal polysaccharide are strongly inhibited by cellobiouronic acid, the disaccharide repeating-unit of the polysaccharide; conversely, antibodies made to a cellobiouronic acid-protein conjugate cross-react with the type 3 pneumococcal polysaccharide. This procedure of using compounds of low molecular weight that are representative of parts of the polysaccharide structure, in order to inhibit the classical, antibody-antigen (polysaccharide) precipitin reaction of Heidelberger and Kendall? was used extensively by Kabat in ~ t u d i e s ' ~on ~ Jthe ~ linear dextran-antidextran reaction. This model system is probably representative of all linear polysaccharides, with the possible exception of those terminating in nonreducing sialic acid groups (see later); as such, it is pertinent to polysaccharide vaccines, as most of the polysaccharides involved are linear. In these definitive studies, this procedure furnished information on the location and size of determinant groups, and on the heterogeneous nature of antibodies in terms of their specificities. By using a series of oligosaccharides of the isomaltose series with human antidextran sera, it was found that the inhibitory power of the oligosaccharides increases with molecular size until it becomes more or less constant at the hexasaccharide, and this was interpreted as a measure of the optimum size of the combining site of the antibody molecule. From the relative, inhibitory powers of each oligosaccharide, it was possible to calculate the contribution, to the binding energy, of each successive D-glucose residue; it was found that, although the terminal mglucose unit contributed most to this binding energy, each succeeding D-glucose unit also made incrementally smaller contributions. The nonreducing, terminal D-glucosyl groups were called immunodominant, although, in fact, they remain a part of the larger determinant. Another important finding in these studiesIo7was the heterogeneous nature of the antibodies in regard to their serological specificities. The antibodies were adsorbed onto Sephadex, and fractionated by elution with isomalto-oligosaccharidesof' different molecular sizes. Inhibition studies on the different fractions indicated that antibodies having a (104) W. F. Goebel,]. Exp. Med., 68 (1938)469-484. (105)E. A. Kabat,]. Immunoi., 84 (1960) 82-85. (106) E. A. Kabat, Experimental Immunochemistry, 2nd edn., Charles C. Thomas, Springfield, Illinois, 1967, pp. 241-267. (107) J. Gelmer and E. A. Kabat, Immunochemistry, 1 (1964)303-316.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

177

specificity both to small (disaccharide)and large (hexasaccharide) determinants were present in the antidextran serum. Branched polysaccharides differ from linear polysaccharides in having many more terminal, glycosyl residues on each polysaccharide molecule. This fact, together with the more exposed, and, thus, more accessible, position of these residues, tends to make them immunodominant, although not exclusively so, as populations of antibodies having specificities to the backbone of the polysaccharide can also be formed. Although the immunodominance of terminal p-D-glucosyluronic acid groups was recognized in early studies,lWthis phenomenon was more definitively resolved in classical studies on the serological determinants of the lipopolysaccharides of SaZmoneZZa; these have been reviewed." As with the capsular polysaccharides, the 0chains of the lipopolysaccharides consist of a linear arrangement of oligosaccharide repeating-units,the majority of which contain unique, terminal 3,6-dideoxyhexosyl groups in each repeating unit. These terminal saccharides are, to a large degree, responsible for the specificity of antibodies made to the Salmonella organisms; however, antibodies having a specificity for the backbone are also detected in these antisera. In serological studies on branched Dmannans, Ballou and coworkerslOgJ1O determined that the participation of the backbone D mannosyl residues in the immunological response to these branched D-mannans is dependent on the length of the branch structure. When the side chains extend to a tetrasaccharide unit, they are able completely to inhibit antibodies made to the homologous Dmannan. Interesting exceptions to the general rule of the immunodominance of branch saccharides have become apparent as regards the capsular polysaccharides of Group B Streptococcus. These are structure-related, and are important in both the human immunologicalresponse to (see Section 111,3),and the virulence of (see Section VI,Z), the Group B streptococcal organisms."' The type I11 polysaccharide has a 6-0(N-acetyl-cu-Dneuraminyl)-P-wgalactopyranosyl branch,= whereas those of types Ia and Ib have terminal 3-0-(N-acetyl-a-Dneuraminy1)p-Dgalactopyranosyl ~ n i t s .Neither ~ ~ . ~ of ~ these terminal-branch disaccharides is immunodominant, and this can probably be attributed to structural homology between the type 111, and the types Ia and Ib polysaccharides and human serum glycoproteins (serotransferrin and (108) M. Heidelberger, Fortschr. Chem. Org. Natumt., 18 (1960) 503-536. (109) C. E. Ballou,]. BioZ. Chem., 245 (1970) 1197-1203. (110) C. E. Ballou, P. N. Lipke, and N. C. Rashke,J. Bacteriol., 117 (1974)461-467. (111) H. J . Jennings, C. Lugowski, and D. L. Kasper, Biochemistry, 20 (1981) 45114518.

178

HAROLD J. JENNINGS

the M and N blood-group substances, respectively)."' The production of antibodies to these determinants would be highly unfavorable, and, consequently, is probably suppressed by the human immune-system. Following this reasoning, it is highly improbable that terminal sialic acid residues can be a part of any determinant responsible for significant amounts of antibody, although these residues exercise confonnational control over these determinants"' (see Section 111,3). This would also imply that nonreducing, terminal sialic acid residues of any linear polysaccharide (for example, groups B and C meningococcal polysaccharides) would not be immunodominant. Many of the bacterial polysaccharides contain small, noncarbohydrate substituents that could be regarded as branches, and that can also be important in the serological reactions of polysaccharides. These substituents have been r e ~ i e w e d , ' ~and , ' ~ the most important, in terms of the formulation of current, polysaccharide vaccines (see Section V,2), is the 0-acetyl substituent. These substituents can be immunodominant, but are not exclusively so; other populations of antibodies having specificities for other sectors of the polysaccharide are usually formed.

3. Conformation It has been established that the primary structures of the capsular polysaccharides are responsible for their serological specificity. Obviously, conformational factors must also piay a role in this specificity. Rees112showed that polysaccharides, like proteins, can have ordered (helical) structures in which interchain and intrachain associations are both involved, and that these polysaccharides undergo temperatureinduced, order-disorder transitions. Rees'I3 also found that the secondary structures are responsible for the physical and biological properties of these polysaccharides. Of special interest to the present discussion is the fact that the ordered conformation of the capsular polysaocharide from the Gram-negative organism Xanthomonus campestris, a plant pathogen, is necessary, in order that the bacteria may bind to the surface of the plant-host cell^."^ A similar dependence of specific binding to antibody molecules on the ordered (helical) structure of capsular polysaccharides has not been established. However, similar, order-disorder transitions have been detected in a number of (112) D. A. Rees, Biochem. I., 126 (1972) 257-273. (113) D. A. Rees, M T P Int. Reu. Sci., Org. Chem., Ser. One, 7 (1973) 251-283; M T P Int. Ret;. Sci., Biochem., Ser. One, 5 (1975) 1-42. (114) E. R. Morris, D. A. Rees, G. Young, M. D. Walkinshaw, and A. Darke,J. MoZ. Biol., 110 (1977) 1-16.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

179

the capsular polysaccharides from Klebsiella, which would suggest that these polysaccharides have some helical character in solution at lower temperature^."^ Helical structures for some of these polysaccharides have also been reported from study of their X-ray fiber, diffraction pattern^.'^^,"^ Interestingly, the K8 polysaccharide has a two-step, temperature transition, the first step of which is due to the breaking of interactions between its terminal Dglucosyluronic acid groups and its backbone saccharides that results in a change in the backbone conformation of the poiysaccharide.l15 Although helical structure in solution has not been demonstrated for capsular polysaccharides associated with human vaccines, it has been identified in oriented fibers and films of the capsular polysa~charides."~,~~~ In an X-ray fiber diffraction study, the pneumococcal type 3 polysaccharide was found to exist in an extended, left-hand, helical conformation, hydrogen bonding being involved in maintenance of this secondary structure."* This was confirmed by X-ray studies of oriented films of both the types 3 and 8 pneumococcal poly~accharides."~The former exists as a two-fold helix, and the latter as a three-fold helix. Extended conformations in solution have also been assigned to the groups A (Ref. 33), X (Ref. 33), and Z (Ref. 36) polysaccharides of N. meningitidis on the basis of large, three-bond (31P-13C)coupling-constants in their 13C-n.m.r. spectra, but the presence of any helical content in solutions of these polysaccharides has not been established. Laser light-scattering techniques were applied to the group C meningococcal polysaccharide, and these studies indicated that it behaves like a random coil in solution.lZ0This conclusion is consistent with data obtained from 13Cn.m.r.-relaxation studies on the same polysaccharide that indicated32 that a fair degree of flexibility is exhibited by the group C polysaccharide in solution. This situation could prove to be representative of the majority of capsular polysaccharides in solution, although, as in the case of amylosic chain conformations,121the occurrence of regions of helical content in these polysaccharides is also a distinct possibility. (115)C. Wolf, V. Elsasser-Beile, S. Stirm, G. G . S. Dutton, and W. Burchard, Biopolymers, 17 (1978) 731-748. (1161 D. H. Isaac, K. H. Gardner, E. D. T. Atkins, V. Elsasser-Beile, and S. Stirm, Carbohydr. Res., 66 (1978)43-52. (117) D. H. Isaac, E. D. T. Atkins, H. Niemann, and S. Stirm, I n t . ] . Biol. Macromol., 3 (1981) 135-139. (118) R. H. Marchessault, K. Imada, T. L. Bluhm, and P. R. Sundararajan, Carbohydr. Res., 83 (1980)287-302. (119) W. T. Winter and I. Adelsky, Biopolymers, 20 (1981) 2691-2694. (120) T. Tsunashima, K. Mom, B. Chu, and T.-Y. Lui, Biopolymers, 17 (1978)251-265. (121) R. C. Jordan, D. A. Brant, and A. Cesbo, Biopolymers, 17 (1978) 2617-2632.

180

HAROLD J. JENNINGS

Because interactions between antibodies and polysaccharides are restricted to comparatively small regions of the polysaccharides, the orientation of the glycosidic linkages between the individual saccharide units is a hndamental parameter; this is the linkage orientation,122,123 and is defined by the torsion angles (A4 and A+) between these saccharides. Although Se1alz4has shown that antibodies made to peptide sequences did not recognize the same sequences when they formed part of a protein helical structure, this type of conformational specificity is not the general rule for polysaccharides. In fact, regions of conformational similarity are found in polysaccharides of different structures, and this is manifested in the extensive, serological crossreactivity of polysaccharides (see Section V,6). These regions of conformational similarity have been demonstrated in X-ray studies of oriented films of the cross-reacting types 3 and 8 pneumococcal polysaccharide^."^ The common cellobiouronic acid unit adopts the same conformation in both polysaccharides, despite the fact that it is the repeating unit of the former and is separated by a 4-O-a-~glucopyranosyl-cu-D-galactopyranosyl spacer in the structure of the type 8 polysaccharide (see Fig. 3). Rees and S k e r ~ - e t tshowed '~~ that disaccharides can generally be fitted into polysaccharide structures without significant changes in their torsion angles, and this is the basis of the use of

FIG. 3.-Conformations of the Types 3 (Lower) and 8 (Upper) Polysaccharides of Streptococcus pneumoniae, Showing the Common Disaccharide Unit. (122) D. A. Rees,J. Chem. SOC., B , (1969)217-226. (123) D. A. Rees,j. Chem. SOC., B , (1970)877-884. (124) M. Sela, B. Schecter, I. Schecter, and F. Barek, Cold Spring Harbor Symp. Quant. Biol., 32 (1967) 537-545. (125) D. A. Rees and R. J. Skerrett, Carbohydr. Res., 7 (1968) 334-348.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

181

hard-~phere,'~~-'~' and other, more sophisticated, calculations,128in predicting the conformations of homog1ucans,lz6diheteroglycans,126 and even more-complex, saccharide sequence^.^^^^^^^ Not all disaccharides maintain their torsion angles in different structural environments, however, and this results in the type of conformationally controlled, and highly serologically-specific, determinants demonstrated by Jennings and coworkers111J31 in the type 111 capsular polysaccharide of group B Streptococcus. The repeating unit of this capsular polysaccharide is shown in Fig. 4. The conformation of the determinant of this polysaccharide, responsible for the population of antibodies involved in the protection of humans against type I11 group B streptococcal infections, is dependent on its terminal sialic acid groups, even though these are not immunodominant (see Section V,4) I I

I

.'

/O

FIG.4.-Proposed Conformation of the Repeating Unit of the Type I11 Polysaccharide Antigen of Group B Streptococcus. (126) D. A. Rees,]. Chem. SOC., B, (1969) 217-226. (127) D. A. Rees and W. E. Scott,J. Chem. SOC., (1971) 469-479. (128) D. A. Rees and P. J. C. Smith,]. Chem. SOC., Perkin Trans. 2, (1975)836-840. (129) R. U. Lemieux, K. Bock, L. T. Delbaere, S. Koto, and V. S. Rao, Can.]. Chern., 58 (1980) 631-653. (130) K. Bock, S. Josephson, and D. R. Bundle,]. Chem. SOC., Perkin Trans. 2, (1982) 59-70. (131) H. J. Jennings, C. Lugowski, K.-G. Rosell, and D. L. Kasper, in D. A. Brant (Ed.), Solution Properties of Polysaccharides, A.C.S. Symp. Ser., 150, American Chemical Society, Washington, D. C., 1980, pp. 161-172.

182

HAROLD J. JENNINCS

and are, most probably, not even a part of the determinant. By using '"C-n.m.r.-spectroscopicdata on the native, and modified-native, type I11 polysaccharides, it was possible to determine that the terminal sialie acid groups exert conformational control over the torsion angles of the penultimate 2-acetamido-2-deoxy4O-~-D-ga~actopyranosy~-~-Dglucopyranosyl unit of the polysaccharide. It is possible that this control is achieved by interactions (possibly hydrogen bonding) between these terminal sialic acid groups and the backbone of the type I11 polysaccharide. It is also possible, and, indeed, probable, that substituent groups smaller than sialic acid groups, such as 0-acetyl, pyruvate, and phosphate, could also confer immunospecificity in polysaccharide determinants by a similar mechanism. 4. Molecular Size One of the most important physical parameters in the effectiveness of capsular polysaccharides as vaccines is their molecular size. This ' ~ ~ demonstrated that the fact was discovered b y Kabat and B e ~ e r ,who molecular weight of native dextrans that are highly immunogenic in man is of the order of several million. They then proceeded to ascertain the dextran of lowest molecular weight that could still retain its immunogenicity; this was achieved by monitoring the increase in serum-antibody titers following the injection of humans with dextrans having different ranges of molecular weight. They found that, in the molecular weight range of 90,OOO and above, the dextrans remained excellent immunogens, whereas, at values of 50,000and below, they exhibited poor immunogenicity. In subsequent, similar experiments on the pneumococcal, type 3 capsular polysaccharide, Howard and cow o r k e r ~ injected '~~ fractions of different molecular weight of the type 3 polysaccharide into mice; the immunogenicity of each fraction was determined by monitoring the number of plaque-forming cells in the spleens of the mice. It was found that the number of such cells was directly related to the molecular size of the fraction; the native polysaccharide was easily the most effective immunogen. In studies on the capsular polysaccharides of N . meningitidis, Gotschlich and coworkersznwere able to obtain the group A and C polysaccharides in their high-molecular-weight form by precipitation directly from the liquid culture by means of Cetavlon. These highmolecular-weight polysaccharides were highly immunogenic in man. However, the group A polysaccharide isolated from cultures concen(132) E. A. Kabat and A. E. Bezer,Arch. Biochem. Biophys., 78 (1958)306-310. (133) J. G . Howard, H. Zola, G. H. Christie, and B. M. Courtenay,]. Immunol., 21 (1971)535-546.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

183

trated by rotary evaporation had lower molecular weights (less than 50,000), and proved to be non-immunogenic in humans. This depolymerization of the group A polysaccharide was attributed to the action of specific enzymes in the culture medium during evaporation. Lui and coworkerslMthen made the interesting and important observation that estimations of the molecular size of the group A polysaccharide by reducing end-group analysis were considerably lower than those obtained by gel filtration; this phenomenon was also reported earlier for the group X polysaccharide of N . m e n i n g i t i d i ~ . 'On ~ ~ the basis of their results, Lui and coworkers postulated134that some form of aggregation was occurring between individual polysaccharide chains, resulting in a macromolecular structure, and that lipid components could be responsible for this aggregation. In subsequent experiments by Gotschlich and coworkers,136aggregation was found to occur in the groups A, B, and C capsular polysaccharides of N. meningitidis and in the type K92 polysaccharide of E. coli. Experiments were then carried out to determine the nature of this aggregation. They showed,136as previously postulated, that a small proportion of lipoidal material was attached to all of these polysaccharides (8040% of di-0-palmitoylglycerol and 10-20% of di-0stearoylglycerol), and that these diO-acylglycerols were glycosylically attached to the reducing eqd of the polysaccharides by phosphoric diester bonds (see Fig. 5). This small proportion of lipid was sufficient to impart micellar behavior to the individual chains of the polysaccharides. These results couid be significant in our perceptions of capsular polysaccharides, in that an apparently minor component can have such a profound effect on their physical (molecular size) and immunological (immunogenicity) properties. In addition, this minor, lipoidal component could be the entity by which these polysaccharides are actually attached to the outer membrane of the bacterium (see Section 111,5).

5. Location

The importance of capsular polysaccharides in the immune response to bacterial infection is due to their location on the outer surface of the bacteria. They are at the interface of the many host-bacte(134) T.-Y. Lui, E. C. Gotschlich, W. Egan, and J. B. Robbins,]. Inject. Dis., Suppl., 136 (1977) S71-S77. (135) D. R. Bundle, H. J. Jennings, and C. P. Kenny,]. Biol. Chem., 249 (1974)47974801. (136) E. C. Gotschlich, B. A. Fraser, 0.Nashimura, J. B. Robbins, and T.-Y.Lui,J. Biol. Chem., 256 (1981) 8915-8921.

r

1

Ac AC

0

FIG.5.-Proposed Structure of the Lipid Functional Group of the Group C Meningococcal Polysaccharide.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

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ria interactions, and constitute the principal antigens in most of the pathogenic, Gram-negative and Gram-positive organisms. However, the outer membranes of encapsulated bacteria are complex, and other antigens, such as protein^^"^^ and lipopolysaccharide~,~~~ can also play a minor, but important, role in the human immune-response to bacterial infection. Physical experiments have established the surface location of capsular polysaccharides on bacteria; from early experiments, previously reviewed,19using the optical microscope to visibilize the interactions of capsules and specific antibody (quellung reaction) with India ink, to improved techniques using electron microscopy to study the latter intera~ti0ns.l~~ Electron microscopy has also been used to study the capsules of bacteria following the incubation of the bacteria with ferritin-conjugated, specific antibody.140When applied to the group B Streptococcus, the surface location of the type Ia, Ib, Ic, 11, and 111 capsules was experimentally confirmed; types Ib and Ic also have additional, surface-protein antigens.141 The definition of a capsular polysaccharide is arbitrary; it is generally described as an envelope of mucilaginous material surrounding the bacterium. A fundamental question concerning these capsules is whether they are actually attached to the bacteria. Although the detection of free, capsular polysaccharide in culture filtrates is indicative of only weak forces of attraction, this conclusion need not necessarily be correct, as the release of capsular polysaccharide from the bacteria could be due to enzymic activity.142Evidence that covalent bonding could be involved in the attachment of some capsular polysaccharides to bacteria was reported by Tai and coworkers61;they had to use muralytic enzymes in order to isolate the type Ib, group B streptococcal polysaccharide. The identification of end-group diO-acylglycerol phosphate moieties on the group A, B, and C meningococcal and type K92 E. coli polysaccharides has also raised the possibility that these esters could be involved in the anchoring of the capsular polysaccharides to the outer membranes of their respective bacteria.13s Endgroup phosphoric esters have also been detected, by 31P-n.m.r. spectroscopy, in the H. injuenzae capsular polysaccharides; these esters

(137) T. M. Buchanan, in M. Inouye (Ed.), Bacterial Outer Membranes, Wiley, New York, 1979, pp. 475-514. (138) H. J. Jennings,A. K. Bhattacharjee, L. Kenne, C. P. Kenny, and G. Calver, Con.J . Biochem., 58 (1980) 128-136. (139) M. E. Bayer and H. Thurow.]. Bacterial., 130 (1977) 911-936. (140) J. Swanson, K. C. Hsu, and E. C. Gotschlich,]. Exp. Med., 130 (1969) 1063-1075. (141) D. L. Kasper and C. J. Baker,]. Infect. Dis., 139 (1979) 147-151. (142) F. A. Troy, Annu. Reu. Microbial., 33 (1979) 519-560.

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could have been part of an original linkage to the outer membrane of the bacteria.143 Iv. IMMUNE RESPONSE TO BACTERIALINFECTION

In order to understand the function of capsular polysaccharides as human vaccines, it is necessary to describe the human immuneresponse to infections caused by encapsulated bacteria. This response consists of a highly complex interplay of different cells and molecular components, and, of necessity, will only be described in an abbreviated and simplified way. More-extensive information on this subject can be obtained in books and review^.'^^-^^^ Once micro-organisms have penetrated subepithelial tissue and have invaded the human circulatory system, the immune mechanism is the last line of defense against proliferation of the bacteria and the eventual establishment of the disease state in the host. The fact that infections are common indicates that the host defenses do not constitute an impenetrable barrier for micro-organisms. In fact, we are all engaged in a constant struggle with invasive bacteria, a struggle in which various strategies employed by micro-organisms play an important role. As surface components of the bacteria, capsular polysaccharides are implicated in the complex, host uerszis micro-organism interactions, and, in particular, are responsible both for the stimulation of the human immune-system against the invading bacteria and for the virulence of the encapsulated bacteria (see Section V1,l). Two excellent reviews have been written on the intriguing subject of bacterial strategy and the human, immunological-defense ~ y s t e m . ~ " The * ' ~ ~process of eliminating the invading, encapsulated bacteria from the circulatory system is based on three factors; phagocytosis, the activation of complement, and the production of humoral antibodies. Cell-mediated immunity, as opposed to humoral immunity, is less important in the critical, acute and early stages of infections due to encapsulated bacteria, but is probably important in long-term immunity. (143)W.Egan, H. Sclineerson, K. E. Werner, and G. ZonJ. Am. Chem. Soc., 104 (1982) 2898-2910. (144) 31. C. Raff; Nature, 242 (1973) 19-23. (145) E. S. Golub, The Cellular Basis of the Immune Response, Sinauer Assocs., Sunderland, Mass., 1977. 1146) W. E. Paul, in J. B. Robbins, R. E. Horton, and E. M. Krause, New Approaches f o r Inducing Natural Immunity to Pyogenic Organisms, Proceedings of a Symposium, DHEW Publ., No. (NIH) 74553. (147) P. J. Baker and B. Prescott, in J. A. Rudbach and P. J. Baker (Eds.),Development in Immunology, Vol. 2, Elsevier-North Holland, New York, 1979, pp. 67-104. (148) P. Densen and G. L. Mandell, Rev. Infect. Dis., 2 (1980)817-838.

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1. Phagocytosis The engulfment and digestion of micro-organisms, termed phagocytosis, is the function of specialized cells in the human circulatory system.149J50 These cells consist of two major types, the largest and most complex of which are the macrophages. The macrophages constantly monitor the subepithelial tissues and all circulatory fluids, and have an important, immediate function, in that they are able to adhere directly to microbes by some kind of rather primitive, recognition mechanism. They are also able to adhere to microbes by a more specific recognition mechanism involving the prior coating of the bacteria with specific antibody, or the third component (C3) of complement. These molecules function as ligands between the macrophages and the bacteria by adhering to specific, macrophage receptor-sites to the Fc portion of the antibody, or to the C3 component of complement. Macrophages are also important in cell-mediated immunity and in the instigation of the immune response to invading micro-organisms. They achieve this by stimulating antibody-producing cells (see Section IV,3) to produce antibodies having specificities for surface components of micro-organisms to which the macrophages have previousIy adhered. Polymorphs are small, less complex cells that are of extreme importance to the immune system, as they are highly specific and short-lived, and can be rapidly produced by the body and delivered to the tissues by chemotactic response. Like the macrophages, they operate in association with complement and antibody through their respective C3 and Fc receptors. 2. Role of Complement The complement system has been r e v i e ~ e d ' ~ it~ is ~ 'composed ~~; of a series of proteins, Cl-C9, present in normal human serum, that serve as important mediators in the host defense. The terminal components, C3-C9, are involved in the destruction of invading microorganisms, but, in order to achieve this, they have to be activated. This activation process can be divided into two pathways, the alternative pathway and the classical pathway, although both pathways can occur simultaneously in the host defense-mechanism. Surface carbohydrates of micro-organisms are able to activate the al(149)C.A. Mims, The Pathogenesis of Infectious Disease, Academic Press, New York, 1977. (150) M. J. Taussig, Processes in Pathology, Blackwell, Oxford, 1979. (151)H. J. Muller-Eberhard and R. D. Schrieber,Adu. Zmmunol., 29 (1980)1-53. (152)J. A. Winkelstein, Reu. Infect. Dis., 3 (1981)289-298.

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ternative pathway, directly generating C3 convertase activity; those important to human immunity to bacterial infection are the cell-wall mucopeptide of Gram-positive o r g a n i ~ m s , ~in ~ ~which - l ~ ~the teichoic * ' ~ the ~ lipopolysaccharides acid moiety is probably f ~ n c t i o n a l , ' ~and of Gram-negative organisms.'58 The convertase splits C3, to give C3b, which binds to the microbe, and a chemotactic agent that serves to attract polymorphs. The polymorphs then migrate towards the site of infection, and are able to adhere readily to the C3b-coated microbes, because of their surface C3b-receptors. The exact, structural basis of the activation of the alternative pathway is as yet but little understood, but it performs an important, immediate function in the destruction of invading microbes, because of the speed at which this defense mechanism can be deployed as compared to the classical pathway. Although antibody is not required for the activation of the alternative pathway, there is evidence to show that it can participate functionally in this mechanism.15z The classical pathway generally requires the presence of antibody ( 1 s ) having a specificity for a surface component of the bacterium, in order that activation may occur. The antibody adheres to the bacterium, and the resulting, immune complex, through a conformational change in the Fc portion of the antibody molecule, activates the first component of complement (Cl).This, in turn, activates C2 and C4, to yield C3b from C3, in which respect, this pathway now converges with that of the alternative pathway. In fact, the generation of C3b at this stage can lead to the activation of the alternative pathway.152Interestingly, in a few isolated cases, the classical pathway can be activated without the participation of antibody by some molecular structures; this has been reported for the lipid A moiety of lipop~lysaccharides,~~~ for a polysaccharide found in ant venom,'6oand for some synthetic oligosaccharides when linked to an 8-methoxycarbonyloctanol carrier.161 (153) J. A. Winkelstein and A. Tomasz,]. Zmmunol., 118 (1977)451-454. (154) P. H. Quinn, F. J. Crossan, J. A. Winkelstein, and E. R. Moxon, Inject. Zmmun., 16 (1977)400-402. (155) J. W. Tauher, M. J. Polley, and J. B. Zabriskie,J. E r p . Med., 143 (1976) 13521366. (156) J. A. Winkelstein and A. Tomasz,J. Zmmunol., 120 (1978) 174-178. (157) B. A. Fiedel and R. W. Jackson, Infect. Zmmun., 22 (1978)286-287. (158) C. Galanos and 0. Luderitz, Eur. J . Biochem., 65 (1976)403-408. (159) D. C. Morrison and L. F. Kline,]. Zmmunol., 118 (1977)362-368. (160) D. R. Schultz, P. I. Arnold, M . X . Wu, T. M. Lo, J. E. Volkanakis, and M. Loos, Mol. Zmmunol., 16 (1979) 253-264. (161) D. R. SchuIk and P. I. Amold,J. Zmmunol., 126 (1981) 1994-1998.

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Capsular polysaccharides are actively involved in the mediation of complement, in that they are able to suppress the activation of the immediate, alternative-pathway mechanism, thus forcing the immune system to use the classical pathway; this is an important factor in the virulence of bacteria (see Section VIJ).

3. Humoral Antibodies to Polysaccharide Vaccines

An important distinction must be made between the humoral response to a pure, capsular polysaccharide, and to the same polysaccharide when it is an integral part of the bacterium. Thus, the immunity received on recovery from infection by encapsulated bacteria, in terms of the polysaccharide antigen, differs from that generated by purposeful immunization with purified capsular-polysaccharide vaccines. Fortunately, with the exception of infants, the polysaccharide vaccines still stimulate protective-antibody levels in humans, despite these differences. In infants, due to the immature nature of their immune systems, these polysaccharide vaccines are of only marginal benefit.7 Some insights into the nature of these different responses in humans can be found in studies on the cellular basis of the immune response to polysaccharides. However, for the purposes of this Chapter, it would be inappropriate to provide a lengthy description of this incompletely understood mechanism; in-depth reviews of this burgeoning field of research can be referred t ~ . ~ ~ - ~ ~ ~ , ~ ~ For most antigens, the production of antibody (immunoglobulin) is based on the cooperative interaction of two types of lymphocyte, called T-cells (thymus-derived) and B-cells (bone marrow-derived). The T-cells, preprimed with macrophage-presented antigen, stimulate the B-cells to secrete copious quantities of antibody. However, on the basis of animal studies, such polysaccharide antigens as the type 111 pneumococcal polysaccharide have been considered to be T-cellindependent, as they are capable of triggering B-cells to produce antibody (IgM) in T-cell-deficient mice.16' These studies also indicated (162) P. J. Baker, H. C. Morse, S. C. Gross, P. W. Stashak, and B. PrescottJ. Infect. Dis., Suppl., 136 (1977) s20-~24. (163) D . E. Mosier, N. M. Zidivar, E. Goldings, J. Mond, 1. Sher, and W. E. Paul, J . Infect. Dis., Suppl., 136 (1977) s14-s20. (164) H. Braley-Mullen, Immunology, 40 (1980) 521-527. (165) E. C. Gotschlich, I. M . Goldschneider, M. L. Lepow, and R. Gold, in E. Huber and R. M. Krause (Eds.),Antibodies in Human Diagnosis and Therapy, Raven Press, New York, 1977, pp. 391-402. (166) P. J. Baker, D. F. Amsbaugh, P. W. Stashak, G. Caldes, and B. Prescott, Reu. Infect. Dis., 3 (1981) 332-341. (167) J. G. Howard, G. H. Christie, B. M. Courtenay, E. Leuchars, and A. J. S. Davies, Cell. Immunol., 2 (1971)614-626.

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that the same type of antibody (IgM) is produced in normal mice by the same polysaccharide z ~ n t i g e n . ' ~ ~ - ' ~ ~ The majority of antibodies can be broadly classified into three main categories ( I s , IgM, and I d ) on the basis of their different structure and functions in the immune response, and IgG is the most important type of antibody in promoting the classical pathway of the complement system."O Further experiments in mice demonstrated that, although pol ysaccharides are capable of functioning as T-cell-independent antigens in athymic mice, in normal mice, polysaccharides do stimulate T-cell 'activity, although the T-cell response differs markedly (more restricted) from that generated by the injection of mice with whole b a ~ t e r i a . 'T-Cells ~ ~ , ~ ~can ~ modulate the immune response in mice b y either an effector or a suppressor mechanism, and, unlike the situation for the whole bacteria, polysaccharides cause the supAlso, unlike the response of pressor mechanism to be d~rninant.'~'~''~ whole bacteria in mice, polysaccharide antigens fail to induce a memory (amnestic) re~ponse.'~" If the results of the foregoing experiments in mice were projected to the human situation in general, the use of polysaccharides as efficacious, human vaccines would show little promise. However, the immunological response to polysaccharides is species-dependent, and, in humans, with the exception of young infants, a fuller range of antibody types is produced. Thus, polysaccharides stimulate the production of IgG antibodies in humans, in addition both to IgM and IgA ant i b ~ d i e s , 'but, ~ ~ as in the mouse experiments, they fail to exhibit a sizable, amnestic response to subsequent, booster injection^.^ Although the presence of this effect would be a decided advantage in immunoprophylaxis, it is not detrimental, because efficacious, antibody levels in humans are maintained for up to 8 years follbwing the injection of pneumococcal poiysa~charides.'~~ Human infants have immature, immune systems in relation to polysaccharide vaccines, (168) B. Anderson and €1.Blombren, Cell. Zmmunol., 2 (1971) 411-424. (169) C . F. Mitchell, F. C. Grumet, and H. D. McDevittJ. E x p . Med., 135 (1972) 126135. (170) E. C. Gotschlich, I . M. Coldschneider, and M. S. Artenstein,J. E x p . Med., 129 (1969) 1367- 1384. (171) P. J. Baker, N. D . Reed, P. W. Stashak, D. F. Amsbaugh, and B. Prescott,]. E x p . Med., 137 (1973) 1431-1441. (172) P. J . Baker, T r Q n S p h f .Rer;., 26 (1975) 3-20. (173) A. Basten and J. G. Howard, Contemp. T o p . Zmmunobiol., 2 (1973)265-291. (174) W. J . Yount, M. M. Domer, H. J. Kunkel, and E. A. Kabac]. E x p . Med., 127 (1968) 633-646. [175) M. Heidelberger, M. M. Dilapi, M. Siegel, and A. W. Walter,]. lmmunol., 65 (1950) 535-541.

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and do not produce the necessary IgG antibodies (see Sections V,5 and 6). This behavior is similar to that exhibited by mice, and, if it is permissible (not yet proved) to use these experiments in mice as models, this immaturity could be attributed to T-suppressor-cell functi~n.’~~

v. POLYSACCHAFUDE VACCINES AND

IMMUNITY

1. Streptococcus pneumoniae

Pneumonia was, and still remains, among the leading causes of death in the United States. It is responsible for lower-respiratory-tract infections in humans, and is also the most common cause of otitis media (a bacterial infection of the middle ear) in children. The high rate of mortality caused by this disease prompted a search for a preventive approach to its control, an investigation in which the pneumococcal, capsular polysaccharides were the first, purified polysaccharides to be used as human vaccine^.^*'^^*'^^ This followed directly from the discovery by Francis and TilleP that the intradermal injection of type 1 and 2 pneumococcal polysaccharides induced serum antibodies in humans. These results led to the demonstration by Heidelberger and his associates: in a large field-trial under epidemic conditions, that a vaccine composed of types 1,2,5,and 7 polysaccharides is efficacious against disease caused by S. pneumoniae. These studies also confirmed the type-specific protection induced in humans by these capsular polysaccharides. Other successful field-trials were subsequently carried out; in one of these, Heidelberger and demonstrated that a hexavalent, polysaccharide vaccine administered in a single injection induced the corresponding, satisfactory, serotypeantibody levels, which persisted for up to 8 years.175This success rapidly led to the commercial licensing of hexavalent, pneumococcalpolysaccharide vaccines. However, interest in the prophylaxis of pneumococcal pneumonia waned at this time, due to the advent of the “sulfa” drugs, and then the unprecedented success of antibiotic therapy. This development engendered such a complacent attitude towards pneumococcal infections that even the accurate serotyping of disease isolates was discontinued in most medical centers. However, subsequent epidemiological studies of pneumococcal pneumonia by conclusively showed that, despite the success of antibi(176) R. Austrian, Reu. Infect. Dis., Suppl., 3 (1981) sl-sl7. (177) M. Heidelberger, C. M. McLeod, and M. M. DilapiJ. E r p . Med., 88 (1948)369372. (178) R. Austrian, Am. J . Med. Sci., 57 (1959) 133-139.

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otic therapy, the disease occurs with the same frequency, and the same mortality rate, as in the pre-antibiotic era. This fact, together with the emergence of antibiotic-resistant strains: led to consideration of reviving the preventative approach to control of the disease; the eventual development and licensing of a pneumococcal-polysaccharide vaccine179gave fruition to the earlier, interrupted research. Because of the diversity of pneumococcal-capsular types (84 have so far been identified), and a reluctance to use them all in a single, multivalent vaccine, the final composition of the vaccine was the result of a compromise in which the number of serotype polysaccharides was limited to fourteen, presumably with minimum loss of effective coverage. This decision was based on epidemiological s t ~ d i e s , ' ~con~,'~~ ducted in the United States, which indicated that 80-9O% of pneumococcal, bacteremic infections are caused by fourteen serotypes (see Table V). However, because of the restricted nature of these epidemiological studies, this vaccine can only be regarded as a core vaccine to which other serotypes may have to be eventually added. This flexibility will probably be required, in order to allow for geographical variations in pneumococcal serotypes, for time-related changes in the prevalence of serotypes in disease isolates, and, also, for the possibility of the tailoring of pneumococcal vaccines in response to agerelated, epidemiological studies (infant ~accine).'.''~ A further factor that enabled the limitation of the number of serotypes used in the vaccine was the occurrence of some structural homology in the pneumococcal polysaccharides, resulting in extensive cross-reactions and cross-immunity in their serological properties. This is exemplified in the Danish serotyping system, which designates capsular types within groups, based on this cross-reactivity. For example, the two types recognized as 6A (Ref. 86) and 6B (Ref. 87), that is, types 6 and 26 in the U. S. system, differ structurally by only one linkage position in a tetrasaccharide phosphate repeating-unit, the a-L-rhamnopyranosyl residues of type 6B being linked to 04 of the ribitol residues instead of to 03 of the same residues, as in the case of type 6A (see Table IV).This structural similarity, accompanied by a degree of conformational retention in the structure, allows for the production of antibodies common to both types. Because of this crossreactivity (see Section V,6), only type 6A is used in the vaccine. Other significant cross-reactions that occur within the polysaccharides used in the vaccine are types 19F (Refs 92 and 93) and 19A (Ref. 94),of which, type 19F is used in the vaccine, and types 9A (Ref. 83), 9V (Ref. (179) R. E. Weibel, P. P. Vella, A. A. McLean, A. F. Woodhour, W. L. Davidson, and M. R. Hilleman, Proc. SOC. E x p . Biol. Med., 156 (1977) 144-150.

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TABLEV Distribution of Pneumococcal Types Responsible for Bacteremic Infection in the United States During 1968-1973 Pneumococcal type" 1 2 3 4 5 6A (6) 7 8 9N (9) 12 14 18 19F (19) =F (23) All other Total

Number of isolates

Percentage of isolates

293 10 237 320 70 160 213 325 132 182 2.40 159 134 117

9.1 0.3 7.3 9.9 2.2 5.0 6.7 10.1 4.1 5.6 7.4 4.9 4.2 3.6 19.6 100.0

633 3225

American type-designation is given in parentheses.

85), and 9L and 9N (Ref. 84), of which, type 9N is used in the vaccine (see Table V). The choice of these serotypes (6A, 19F, and 9N) was made because, of all the serotypes in each cross-reacting group, these occur more frequently in disease isolates. 2. Neisseria nzeningitidis

Meningococcal disease (purulent meningitis) commonly occurs in children, but is also observed in adults. Without antibiotic treatment, the mortality rate is high (85%), and, even with this treatment, cured patients can suffer serious and permanent neurological deficiencies.ls5 These facts, together with the emergence of antibiotic-resistant strains: prompted the rapid development of a commercial vaccine. This vaccine was developed almost simultaneously with the pneumococcal vaccine. In contrast to the pneumococcal vaccine, however, the composition of the meningococcal vaccine was greatly simplified, due to the fact that fewer polysaccharides were required. Based on their capsular polysaccharides, there are only eight different serogroups of N.meningitidis (A, B, C, 29e, W-135, X,Y,and Z), of which, groups A, B, and

194

HAROLD J. JENNINGS

C account for more than 90% of meningococcal d i s e a ~ e . ' ~The ~-~*~ high-molecular-weight, group A and C polysaccharides raise titers of bactericidal antibody in adults,16j although, in young children, their use has been only marginally (see Section V,5). The group A and C polysaccharide vaccines have also been used in numerous, successful, human field-trials.165-1R' Interestingly, because of the lack of a suitable animal model in which to test the efficacy of these vaccines, the standards for their licensure and release were, for the first time, based purely on physiochemical criteria. However, one major problem in the design of a comprehensive, trivalent pol ysaccharide, meningococcal vaccine inclusive of group B is the poor immunogenicity of the group B polysaccharide in man.lW2 Two major reasons have been proposed to account for this phenomenon. One is that the rr-~-(2-+8)-linkedsialic acid homopolymer is rapidly depolymerized in human tissue, because of the action of neuraminidase; the other is that this structure is recognized as "self" by the human immune-system, and, in consequence, the production of antibody having a specificity for this structure is suppressed. The weight of evidence is in favor of the latter explanation. A neuraminidase-sensitive variant of the group C polysaccharide [an a-D-(2+9)-linked, sialic acid homopolymer] having no 0-acetyl groupsIR3still proved to be highly immunogenic in man.Is4In addition, in experiments using tetanus toxoid conjugates of the group B polysaccharide, it was demonstrated that, when conjugated at the nonreducing sialic acid group (thereby producing a neuraminidase-resistant, group B polymer), its immunogenicity was not enhanced.185Finally, the Escherichia coli K92 capsular polysaccharide contains alternating sequences of CPD( 2 4 ) -and -(2-+9)-linked sialic acid.1*6This polysaccharide proved to be immunogenic, but produced only antibodies that cross-reacted with the group C polysaccharide [a-~-(2+9)-linked]. The immune mechanism avoids the production of antibody having a specificity for the cy-1)-(2+8)linkage.'"*'Rg (180) E. C. Gotschlich, in Ref. 10, pp. 91-101. (181) R. Gold, M. L. Lepow, I. M. Goldschneider. and E. C. Gotschlich,]. Infect. Dis., S U ? I ~ >136 ~ . , (1977) S31-S35. (182) F . A. Wyle, M. S. Artenstein, D. L. Brandt, D. L. Tramont, D. L. Kasper, P. Altieri, S. L. Berman, and J. P. Lowenthal,]. Infect. Dis., 126 (1972)514-522. (183) M . A. Apicellq]. Infect. Dis.,129 (1974) 147-153. (1%) s.1. P. Glode,E. B. Lewin,A. Sutton,C. T. Le,E.C. Gotschlick,and J. B. Robbins, J . In-fect.Dis., 139 (1979)52-59. (185)H. J. Jennings and C. Lugowski,]. lmmunol., 127 (1981) 1011-1018. (186) W. Egan,T.-Y. Lui, D. Dorow, J. S. Cohen, J. D. Robbins, E. C. Gotschlich, and J. B. Robbins, Biochemistry, 16 (1977)3687-3692. .

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195

Attempts to surmount this problem have included the use of ( a ) other surface-components of the group B organism (type-specific prot e i n ~ ) , ' ~(b) ~ . 'group ~ ~ B polysaccharide conjugates (see Section V,5), and (c) cross-reacting polysaccharides as alternative vaccines. In the last category, a proposal has been made to use a cross-reacting, E. coli p o l y s a c ~ h a r i d eThe . ~ ~ E. ~ coli K1 polysaccharide is ~ t r u c t u r a l l y 3 ~ ~ ~ ~ and serologically'" identical to the meningococcal group B polysaccharide (see Section V,6), but a form variant (OAc+)of this organism was isolated that produces a polysaccharide randomly 0-acetylated at 0-7 and 0-9 of its sialic acid residues.lgl This form variant (OAc+)of the E. coli K1 organism is more immunogenic in rabbits than the OAcvariant, and produces antibodies having specificities for both the 0acetylated and the nonacetylated polysa~charides.'~~ Human trials using the 0-acetylated K1 polysaccharide as a vaccine are now needed, in order to determine whether this approach to the problem will show any promise.

3. Haemophilus infiuenzae Although there are six capsular types of H. influenzae, the most serious disease is caused'9z by type b. This organism is the cause of meningitis, which occurs exclusively in infants, and even survivors of this disease can suffer severe, and permanent, neurological defect^.^ In consequence, an extensive amount of work has been dedicated to finding a vaccine for this organism, and the capsular polysaccharide was a prime candidate. Heidelberger and coworkers's3 demonstrated that protective antibodies in hyperimmune rabbit antisera could be removed by absorption with the purified H. influenzae type b polysaccharide. Since then, Anderson and coworkers,194and Parke and cow o r k e r ~ , 'were ~ ~ able to induce, in adults, long-lived, complement(187) C. E. Frasch and E. C. Gotschlich,]. E x p . Med., 140 (1974) 87-104. (188) W. D. Zollinger and R. E. Mandrell, Infect. Immun., 18 (1977)424-433. (189) J. B. Robbins, R. Schneerson, J. C. Parke, T.-Y. Lui, Z. T. Handzel, I. Brskov, and F. Brskov, in Ref. 10, pp. 103-120. (190) D. L. Kasper, J. L. Winkelhake, W. D. Zollinger, B. Brandf and M. S. Artenstein, J . Immunol., 110 (1973) 262-268. (191) F. Brskov, A. Sutton, R. Schneerson, L. Wenlu, W. Egan, G . E. Moff, and J. B. Robbins,J. E r p . Med., 149 (1979)669-685. (192) J. C. Parke, R. Schneerson, J. B. Robbins, and J. J. Schlesselman,J. Infect. Dis., S ~ p p l . 136 , (1977) S25-~30. (193) H. E. Alexander, M. Heideiberger, and G . Leidy, Yale 1. Biol. Med., 16 (1944) 425-438. (194) P. Anderson, G . Peter, R. B. Johnston, L. H. Wetterlow, and D. H. SrnithJ. Clin. Inoest., 51 (1972) 39-44.

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mediated, bactericidal antibodies by using the purified type b polysaccharide as a vaccine. However, the development of this pure, polysaccharide vaccine was retarded when it was discovered that the p l y saccharide induced only short-lived immunity in older infants, and little or no protection in younger infants.lS5Current research to obviate this problem has focussed on the use of ( a ) type b polysaccharide-protein conjugates (see Section V,5) and (b) cross-reacting organisms. The latter approach would involve the deliberate colonization of infants with the non-pathogenic, cross-reacting E. coli (see Section V,6).

4. Group B Stseptococcw Group B Streptococcus is a major cause of bacterial meningitis in new-born infant^.'^^'^^ The organisms can be into four distinct serotypes (Ia, Ib, 11, and HI), of which, type I11 is the most important in human disease.Is7 The original isolation of the type 111 capsular polysaccharide was achieved by using acid-extraction proced u r e ~ ~ ~this . ~ resulted '; in the isolation of an immunologically incomplete, core antigen that originated from a native polysaccharide containing labile, terminal sialic acid residuesw The complete, native, type I11 antigen could be isolated by growing the type I11 organism under pH-controlled conditions and isolating the polysaccharide by mild extraction-procedures.62The core antigen is structurally identical to the capsular polysaccharide of type 14 S . pneumoniae,63and, on the basis of serological experiments in animals using the latter organism, it was suggested that antibodies to the core polysaccharide could be functional in the production of protective antibodies against type 111, group B Streptococcus organisms.1s8However, confirmatory evidence for the essential participation ofthe native, type I11 polysaccharide in the development of human immunity to the disease was obtained when it was demonstrated that, in human sera, only antibody directed to the native antigen correlated most highly with opsonic (bactericidal) a ~ t i v i t y . ~ ~ * ' ~ ' I n a disease that is restricted to the newborn, the use of immunoprophylaxis is impractical, due to the time lag before effective levels of (195) I>. H . Smith, G . Peter, D. L. Ungram., A. L. Harding, and P. Anderson, Pediatrics, 52 (1973) 637-645. (1%) T. C . Eickhoff, J . 0. Klein, A. K. Daly, D. Ingall, and M. Finland, New Engl. /. Med., 271 (1964) 1221-1228. (197) C. J. Baker,Ado. Intern. Med., 25 (1930) 475-499. (198) G. W.Fisher, G . M. Lowell, M. H. Cumrine, and J. W. Bass,]. E x p . Med., 148 (1978) 776-786.

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protective antibodies are produced. Therefore, a different vaccination strategy is envisaged for this disease, one in which the target population for the polysaccharide vaccine would be pregnant mothers deficient in antibodies specific for the native, type 111, group B streptococcal polysaccharide. The type I11 polysaccharide is immunogenic in and the baby could acquire immunity by the placental transfer of antibody ( 1 s ) . It has been demonstrated that babies born of mothers having high levels of type I11 polysaccharide-specific antibody are less liable to infectionse2than others. 5. Polysaccharide-Protein Conjugates

The utilization of the previously described, capsular polysaccharides as human vaccines is only partially successful, due to the fact that they are poor immunogens in young children. This situation is highly undesirable, as this section of the population experiences the highest incidence of disease (particularly meningitis) caused by these pathogenic bacteria.' The immunological basis of this phenomenon, discussed in Section IV,3, is the inability of young children to generate a mature and amnestic response involving the production of IgG antibodies to these purified-polysaccharide antigens. A possible solution would be to enhance the immunogenicity of these pure polysaccharides by converting them into thymus-dependent antigens. One method of achieving this objective would be to conjugate them to a protein carrier. The feasibility of this approach is well established. Fifty years ago, Goebel and AverylWcoupled the type 3 pneumococcal polysaccharide to horse serum-globulin by the diazotization of p aminobenzyl ether substituents on the polysaccharide. They demonstrated that this polysaccharide conjugate,2O0and a similar conjugate made with the oligosaccharide repeating-unit (cellobiouronic acid) of the type 3 pneumococcal polysaccharide,2°0were able to induce polysaccharide-specific antibody in rabbits previously unresponsive to the pure polysaccharide. GoebelzO1*zOz also established that the cellobiouronic acid conjugate was able to confer immunity to pneumococcal infection in mice. Other investigators confirmed these results by using the type 3 pneumococcal polysaccharide covalently linked to proteinzwand to erythrocytes,204and noncovalently linked in ionic as(199) W. F. Goebel and 0. T. Avery, ]. E x p . Med., 54 (1931)431-436. (200) 0. T. Avery and W. F. Goebel,]. E x p . Med., 54 (1931)437-447. (201) W. F. Goebel,]. Em. Med., 72 (1940)33-48. (202) W. F. Goebel,]. E r p . Med., 69 (1939)353-364. (203) W. E. Paul, D. H. Katz, and B. Benacerraf,]. lmmunol., 107 (1971)685-688. (204) H. Braley-Mullen,]. lmmunol., 113 (1974) 1909-1920.

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sociation with methylated bovine serum albumin.205In some of these methods, the coupling techniques employed were far too drastic for the highly sensitive polysaccharides currently used as human vaccines. Also, the carrier proteins and the coupling methods employed in the synthesis of these conjugates resulted in the formation of conjugates having constituents or structural features (for example, aromatic groups) highly undesirable for use in human vaccines. More-comprehensive studies on polysaccharide-protein conjugates directed specifically to their use as human vaccines have now been reported; the development of simple, and efficient, coupling procedureszo6has resulted in the formation of linkages to compounds containing more-innocuous, and more-acceptable, structural features. The H. injuenzue type b polysaccharide was conjugated to a number of proteins by Schneerson and coworkers207by using an adipic dihydrazide spacer between the molecules. These conjugates were relatively nontoxic, and, in contrast to the pure polysaccharide, functioned as thymic-dependent (T-cell-dependent) antigens. They produced polysaccharide-specific, serum antibodies in mice and other animals, and the level of these antibodies could be augmented by reinjection of the conjugate. The group C meningococcal polysaccharide was also successfully converted into a thymic-dependent antigen b y Beauvery and coworkers,2°M who linked it directly through the carboxyl groups of its sialic acid residues to the amino groups of tetanus toxoid (amide linkages), using l-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride. The foregoing conjugation methods employed random activation of the many functional groups of the polysaccharides, and more-specific coupling was obtained in the formation of artificial SaZmoneZZa typhimurium and Pseudomonas aeruginosa vaccines. Svenson i n d Lindberg15*.209 synthesized the former by coupling the smaller molecularsize octa- and dodeca-saccharides, obtained by treatment of the 0chain of the Salmonella typhimurium lipopolysaccharide with phage enzymes, to bovine serum albumin. A carboxyl group, unique to the oligosaccharide, was generated on their reducing (end-group) rham(205) 0. J. Plescia, W. Braun, and N. C. Palczuk, Proc. Natl. Acad. Sci. U . S. A., 52 (1964)279-285. (206) C. P. Stowell and Y. C. Lee, Ado. Carbohydr. Chem. Biochem., 37 (1980) 225281. (207) R. Schneerson, 0.Barrena, A. Sutton, and J. B. Robbins,]. E r p . Med., 152 (1980) 361 -376. (208) E. C. Beauvery, F. Miedema, R. W. Van Delft, and J. Nagel, in J. B. Robbins, J. C. Hill, and J. C. Sadoff (Eds.), Seminars in Infectious Disease. Bacterial Vaccines, Vol. 4, Thieme-Stratton, New York, 1982, pp. 268-274. (209) S. B. Svenson and A. A. Lindberg, J . Immunol. Mkthods, 25 (1979)323-335.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

199

nose residues, through which they were linked to the protein by amide linkages by using 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride. The conjugates elicited good antibody responses in rabbit^,'^,^'^ but not in mice,2l0although it was demonstrated that the rabbit antibody was able passively to protect the mice against infection by live, homologous Salmonella organisms.210In subsequent work, Seid and Sadoff prepared a tetanus toxoid conjugate of the (larger molecular size) whole, base-treated lipopolysaccharide of type 5 Pseudomonas aeruginosa by the formation of amide linkages between the carboxyl groups of its few KDO residues and the amino groups of 1,4-diaminobutane spacers pre-attached to the protein. The conjugate proved considerably less toxic than the original lipopolysaccharide, and preliminary, immunological results indicated that IgG antibodies having a specificity for the 0-chain can be readily induced in mice by using this conjugate.211Except for the potentiaI loss of alkali-labile substituents on lipopolysaccharide 0-chains, this method could prove to be applicable to all bacterial lipopolysaccharides, and it has obvious potential in the synthesis of human vaccines. In an attempt to extend the monofunctional-group approach to the conjugation of the meningococcal polysaccharides, and thus to produce conjugates more chemically defined, Jennings and L ~ g o w s k i ' ~ ~ inserted a unique, terminal, free aldehyde group into the groups A, B, and C polysaccharides. This was achieved by controlled, periodate oxidation of the native, group B and C polysaccharides and of the group A polysaccharide premodified by reduction of its terminal, reducing 2-acetamido-2-deoxy-~-mannose residue (see Fig. 6). These monovalent molecules were then specifically coupled to tetanus toxoid by reductive amination, using sodium cyanoborohydride, without activating the other functional groups in the polysaccharide. When used as vaccines in mice and rabbits, the group A and C polysaccharide-tetanus toxoid conjugates produced high-titer antisera having bactericidal activity against the homologous organisms, indicating the potential of these conjugates as human vaccines. In contrast, the group B polysaccharide-tetanus toxoid conjugate failed to elicit detectable, polysaccharide-specific antibodies in these anim a l ~Inhibition . ~ ~ ~ experiments indicated that the antibody produced was not specific for the polysaccharide, but was highly specific for the linkage between the lysine residues of tetanus toxoid and the nonreducing (end-group) heptulosylonic acid group of the oxidized group B polysaccharide. (210) S. B. Svenson and A. A. Lindberg, Infect. lmmun., 32 (1981) 490-496. (211) R. C. Seid, Jr., and J. C. SadofT,/. B i d . Chem., 256 (1981) 7305-7310.

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6 R=R'=H IR=R'=COCH, 8 R=H,R'=COCH, 9 R = COCH,, R' = H

1

L R = OCCH, or H

FIG.6.-Shucture ofthe Group C (uppermost),B (middle), and Terminally Reduced Group A (lowest) Polysaccharide Antigens OfNeisseria meningitidis, Depicting the Positions of Cleavage on Oxidation by Periodate.

6. Natural Immunity, and Polysaccharide Serological Cross-reactions Adult-animal sera contain antibodies to a variety of polysaccharides, including those of human pathogenic bacteria, indicating that, to confer immunity, disease is not required. Robbins and were able to detect antibodies having a specificity for Vibrio cholerue (212)J. B.Robbins, R. Schneerson,M. P. Glode, W. Vann,M. S. Schiffer,T.-Y. Lui, J. C . Parke, and C. Huntley,]. Cell. Clin. Zmmunol., 56 (1975)141-151.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

201

in animals, as the animals were allowed to mature without the possibility of contact with the homologous organism. In addition, in serological studies on a healthy human population, there is frequently detected the presence of antibodies having a specificity for the capsular polysaccharides of groups A, B, and C N. meningitidis and type b H. influenme that cannot be satisfactorily explained by asymptomatic Similar findings were reported for antibodies to the pneumococcal type 3 polysaccharide in children.213These populations of antibodies result from exposure to cross-reacting antigens among the nonpathogenic bacteria found in the nasopharyngeal or gastrointestinal Serological cross-reactions among polysaccharides are a well documented phenomenon, due in large part to the extensive work conducted in this area by Heidelberger and This phenomenon is due to the ability of polysaccharide antigens to promote the formation of heterogeneous populations of antibodies, and to the special property of polysaccharides to retain domains of structural and conformational similarity despite some structural differences. This is well illustrated by the cross-reactions exhibited between the different pneumococcal polysaccharides (see Sections III,3 and VJ). However, cross-reactions between polysaccharides from different species of other organisms have been used to great advantage in probing polysaccharide structures. Thus, an antiserum of one polysaccharide of known structure becomes a reagent to monitor for similar structural features in other polysaccharides. Heidelberger and coworkers have used this type of analysis extensively, and the results have constituted the subject of several reviews.108*214-219 In addition to its analytical value, this phenomenon is probably the basis of the important mechanism by which humans develop natural imrn~nity.~ In fact, it has also been postulated that exposure to these cross-reacting, T-cell-dependent organisms is the most satisfactory explanation for the eventual maturation of the polysaccharide immuneresponse in infants.165It can be shown, for instance, that there is an age-related increase in natural antibodies to the group A meningococcal polysaccharide in children, even though the group A organism is

(213) M. Finland,]. Infect. Dis., 128 (1973) 76-124. (214) M. Heidelberger, Res. lmmunochem. lmmunobiol., 3 (1973) 1-40. (215) M. Heidelberger, in J. B. G. Kwapinski (Ed.), Research in Zmmunochemistry, Vol. 3, University Park Press, Baltimore, 1973. (216) M. Heidelberger, Annu. Reo. Biochem., 36 (1967) 1-12. (217) J. M. Tyler and M. Heidelberger, Biochemistry, 7 (1968) 1384-1392. (218) M. Heidelberger and W. Nimmich,J. lmmunol., 109 (1972) 1337-1344. (219) M. Heidelberger and W. Nimmich, Immunochemistry, 13 (1976) 67-80.

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rarely isolated in the United States.22oFrequently found in normal human flora are cross-reacting organisms that could be responsible for natural immunity to groups A, B, and C N. meningitidis and type b H. infiuenzae. Robbins and coworker^^*^^^ and Egan and coworkers222 identified some of these important organisms; they are listed in Table VI. Serological studies indicated that the capsular polysaccharides of these organisms are responsible for the cross-reactions, and this is confirmed by the structural similarity of some of these polysaccharides (see Table VI) to those from N. meningitidis (see Table I) and H. influenzcre (see Table 11). Schneerson and rob bin^^^^ clearly demonstrated the feasibility of this mechanism by deliberately feeding nonpathogenic E. coli possessing the K-100 capsule to human-adult volunteers, Colonization readily occurred, and antibodies specific for the H. infiuenzae type b polysaccharide were induced.

VI. BACTERIAL VIRULENCE

1. Role of the Capsular Polysaccharide We are constantly in contact with a wide range of micro-organisms in our external environment, but few of these prove to be virulent. The virulence of bacteria is dependent on their ability to invade the human host and to evade the host's immune system, thus allowing the bacteria to propagate within the host. The varied strategies employed by the bacteria to evade the host's immune system have been re~iewed.':'~.'*~ Although the mechanisms behind these different strategies are incompletely understood, research in this area is encouraging, and suggests that an understanding of the pathogenesis of infectious diseases at the molecular and macromolecular level will eventually be possible. Because of their surface location, capsular polysaccharides are important agents in bacterial pathogenesis, as they interact directly with the host's immune system. The initial event in the pathogenesis of most bacterial infections is the attachment of the bacteria to the mucosal surface. This probably occurs by a receptor mechanism that exhibits a high degree of cellular specificity. Capsular polysaccharides have not been implicated in this (220) I. M. Goldschneider, M. L. Lepow, E. C. Gotschlich, F. T. Mauck, F. Bache, and M. Randolph,]. Infect. Dis., 128 (1973)769-776. (221) J. B Robbins, R. L. Myerowitz, J. K.Whishant, M. Argaman, R. Schneerson, Z.T. Handzel, and E. C. Gotschlich, Infect. Zmmun., 6 (1972)651-656. (222) W. Egan, F.-P. Tsui, and H.Schneerson,]. Biol. Chem., submitted for publication. (223) R. S. Schneerson and J. B. Robbins, New Engl.]. Med., 292 (1975) 1093-1095.

TABLEVI

Polysaccharides of Bacteria, Frequently Found in Human Flora, That Cross-react with the PolysaccharideCapsules of Human Pathogenic Bacteria Pathogen Neisseria meningitidis Group A Croup B Group C

Cross-reacting organism Bacillus pumilis Streptococcus fecalis Escherichia coli K 1 Escherichia coli K92

Structure

References

2-acetamido-2-deoxymannosyl phosphate residues

22 1 7 191 186

+ g)cyD-NeupAc(z+

and its OAc' variant

+ 8)a~-NeupAc(2 + g)aD-NeupAc(2+

Haemophilus influenme

0 Type b

Escherichia coli KlOO

+ 3)p~-Ribf( 1-+

II

2)D-ribitol(5-O-P-

222

I

Staphylococcus aureus Bacillus pumilis Bacillus subtilis Lactobacillus plantarum

OH teichoic acids containing ribitol phosphate

7

204

HAROLD J. JENNINGS

mechanism, which probably involves interactions between the glycose moieties of the surface glycoprotein of human cells and surface ~ ~ , ~ ~ ~polysacchaproteins (pili or fimbriae) of the b a ~ t e r i a . ' Capsular rides, however, are very much involved in the pathogenesis of bacteria following the penetration of these bacteria into body tissue. There is abundant accumulated evidence to demonstrate that capsular polysaccharides are important virulence factors in disease caused by Neisseria meningitidis, Haemophilus influenxae, group B Streptococcus, and Streptococcus pneu~oni~e.7~14E~'s5.225~227 This is also the case for the capsular polysaccharides of other pathogenic bacteria, including those in which the high-molecular-weight 0-chains of their lipopolysaccharides are functionally equivalent to the capsular polysaccharides.17 The importance of capsular polysaccharides in pneumococcal infections was demonstrated fifty years ago, when it was shown that the enzymic depolymerization of the capsular polysaccharide on the surface of type 111 S. pneumoniae organisms considerably decreased the virulence of these organisms in mice.z28The property of the capsular polysaccharide that enhances the virulence of bacteria is its ability to mediate the host's immune system. Except for a few cases of molecular mimicry (see Section VI,2), the major mechanism involved in this mediation is its function as an inhibitor of the fast-acting, alternative pathway of complement-induced phagocytosis, thus forcing the immune system to utilize the slower, classical pathway. The complement system is briefly explained in Section IV,2. Because the classical pathway has a requirement for polysaccharide-specific antibody, and because the process of producing this antibody takes a few days, the host is compromised during the initial, acute stages of bacterial infection, and is liable to die, or to acquire serious morbidity effects. Thus, the rationale behind vaccination with capsular polysaccharides is to maintain a long-lasting, effective level of polysaccharide-specific antibody in the host. The function of the polysaccharide capsule in inhibiting the alternative pathway is most satisfactorily and simply explained by the fact that it masks the underlying, bacterial structures (for example, teichoic acids), which are known to be powerful activators of the alternative p a t h ~ a y . ' ~ " -However, '~ although this mechanism is no doubt

(224)E. H. Beachey,]. Infect. Dis., 143 (1981) 325-345. (225) R. Bortolussi, P. Ferrieri, B. Bjorksten, and P. G. Quie, Infect. Immun., 25 (1979) 293-298. (226) C . M. McLeod and M. R. Krauss,]. E r p . Med., 92 (1950) 1-9. (227) C. J . Howard and A. A. Glynn, Immunology, 20 (1971) 767-777. (228)0.T. Avery and R. Dubos,J. E x p . Med., 54 (1931) 73-89.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

205

operative, the concept of the capsule as a simple, physical barrier is inadequate to explain all of the experimental results and observations. For instance, why are not all encapsulated bacteria pathogenic? To understand this mechanism filly, it will first be necessary to comprehend the molecular events leading to the complement-mediated phagocytosis of bacteria. Although most of these events have been elucidated, the critical mechanism whereby the alternative pathway is first activated still remains o ~ s c u ~ However, ~ . ~ ~ from ~ , ~ the~point ~ , of ~ ~ ~ view of the capsular polysacckaride, evidence has been accumulated that helps to reveal some of its functional aspects in the inhibition of complement-mediated phagocytosis of bacteria. Evidence that the capsule creates a simple, physical barrier to the underlying surface of the bacteria can be found in the fact that only encapsulated bacteria are pathogens, and, for any given pathogenic strain of bacteria, its virulence is directly related to the amount of capsu1e.176,226,231 However, the amount of capsule is not the only criterion on which virulence is based, because although heavily encapsulated type 3 pneumococci are extremely virulent in mice, type 37 pneumococci, having the same degree of encapsulation, are In addition, type 12 pneumococci, having very small capsules, are extremely virulent in humans.176On the basis of these results, plus the known species-specificity of bacterial pathogenesis, other physical, compositional, or structural properties must be postulated to account for the role of the polysaccharide capsules in bacterial pathogenesis. Experimentation leading to the delineation of this role is hampered by the complexity of the bacterial surface and by lack of knowledge as to whether the polysaccharide capsules function alone as mediators of the complement system. However, the use of molecular cloning-techniques has considerably simplified the problem, and has demonstrated that not all capsular polysaccharides have the same function in bacterial pathogenesis. Moxon and Vaughn232demonstrated that the polysaccharide capsule of type b H. injluenzue is necessary, and, indeed, sufficient, to perform this function. Type b and d transformants were made from the same, unencapsulated, H. influenme strain, thus giving the transformants DNA homology, except for the regions that determine serotype specificity. As in the clinical situation, the type b (229)D.T.Fearon and K. F. Austen, Proc. Natl. Acad. Sci. U . S . A.,74 (1977)16831687. (230)R. D. Schrieber, M. K. Pangburn,P. H. Lesavre, and H. J. Muller-Ebenhard,Proc. Natl. Acad. Sci. U . S . A., 75 (1978)3948-3952. (231)A. A. Glynn, W. Brumfitt, and C. J. Howard, Lancet, (1977)514-516. (232)E. R. Moxon and K. A. Vaughn,]. Infect. Dis., 143 (1981)517-524.

206

HAROLD J. JENNINGS

transformant proved to be more invasive and virulent in rats than that of type d. This experimental result is also consistent with the structural basis of bacterial pathogenicity. In similar studies by Silver and coworkers,233the cloned genes responsible for synthesis of the polysaccharide capsule of the highly pathogenic E. coZi K 1 organisms were able to synthesize the identical capsule in the non-pathogenic E. coZi K12 organisms. In this case, however, the capsule alone was not able to induce the same virulence properties in the transformed organism nonnally associated with the E. coli K1 bacteria, indicating that the E . coli K1 capsular polysaccharide probably functions in concert with other surface components of the K1 organisms.

2. Polysaccharide Structure and Pathogenicity

That polysaccharide structure could be involved in bacterial pathogenesis can be deduced from the observation that, of all the encapsulated bacteria, only a few are virulent in man, and interestingly, two of these different species of bacteria share a common capsular polysaccharide. Group B N. meningitidis and K1 E . coZi produce the same aD-(2+8)-linked sialic acid homopolymer (see Table V) as their only common surfacecomponent, and both are a major cause of meningitis in children. Some experimental evidence234is also consistent with the structure of polysaccharide capsules’s being implicated in the virulence of bacteria, which stems from their differing abilities to inhibit the activation of complement by way of the alternative pathway. In measuring the survival time of the six serotypes (a, b, c, d, e, and f ) of H . influenme in antibody-free sera containing complement, only the type b organisms were able to survive complement-mediated phagocytosis for any appreciable length of time. Although it has, to date, not been possible to identify any common structural feature among all the polysaccharide capsules of bacteria associated with the most pathogenic human disease, there is one common feature in many of them. The capsular polysaccharide of type I11 group B Streptococcus has terminal sialic acid residues in its struct~ree,6~ asqdo ~ ~ the groups B and C N. rneningitidis and K 1 E . C O Z ~ . ~ ~ ~ ~ ~ The ability of terminal sialic acid residues to inhibit the activation of complement by way of the alternative pathway has been well docu(233)R. P. Silver, C. W. Finn, W. F. Vann, W. Aaronson, R. Schneerson, P. J. Kretschmer, and C. F. Garon, Nature, 289 (1981)696-698. (234) A. Sutton, R. Schneerson, S. Kendail-Morris, and J. B. Robbins, Infect. Immun., 35 (1982)95-104.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

207

mented for erythrocyte-membrane ~ u r f a c e s . ~ ~Edwards ~ - ~ ~ ' and cow o r k e r ~also ~ ~observed ~ this phenomenon on bacterial surfaces. Normally, type I11 group B streptococcal organisms are potent inhibitors of the alternative pathway, but, when they are grown in neuraminidase, which removes the terminal sialic acid residues, they are converted into alternative-pathway activators. F e a r ~ described n ~ ~ ~ a similar experiment for the conversion, using neuraminidase, of sheep erythrocytes into activators of the alternative pathway. In an attempt to delineate the structure-function relationship of the terminal sialic acid residues of the type I11 group B streptococcal polysaccharide with this inhibition, Edwards and coworkers238chemically modified the polysaccharide on the surface of the bacteria. Reduction of the carboxylate groups of the sialic acid residues to hydroxymethyl groups also changed the surface of the type I11 organisms to become alternative-pathway activators, thus indicating that removal of these terminal residues in order to expose underlying, structural features is not required for activation to occur. F e a r ~ nalso ~ ~demonstrated ~ that removal of only C-8 and C-9 of the glycerol end-chain of terminal sialic acid residues of sheep erythrocytes is sufficient to convert them into activators; in this experiment, the charge on the sialic acid residues was retained, thus indicating, by extrapolation to the type I11 group B streptococcal polysaccharide, that the charge alone is not responsible for its inhibitory properties. All of these experiments involving the chemical modification of terminal sialic acid residues gave results that are consistent with the hypothesis that any change in the integrity of the sialic acid residue could alter its capacity to modulate the complement system, and this is also consistent with the work of Varki and K ~ r n f e l dwho , ~ ~showed ~ that the extent of 0-acetylation of sialic acid residues is directly related to the capacity of murine erythrocytes to activate the alternative-complement pathway. However, this hypothesis may be oversimplistic, and there still remains the possibility that there are involved more-complex, structural features in which the sialic acid residues could provide tertiary conformation to those surface structures. Certainly, such conformationally controlled determinants (235) D. T. Fearon, Proc. Natl. Acad. Sci. U . S. A., 75 (1978) 1971-1975. (236) M. K. Pangbum and H. J. Muller-Eberhard,Proc. Natl. Acad. Sci. U . S . A., 75 (1978) 2416-2420. (237) M. D. Kazatchkine, D. T. Fearon, and K. F. Austen,J. Immunol., 122 (1979) 7581. (238)M. S. Edwards, D. L. Kasper, H. J. Jennings, C. J. Baker, and A. NicholsonWeller,J. Zmmunol., 128 (1982) 1278-1283. (239) A. Varki and S. Komfeld,J. E r p . Med., 152 (1980)532-544.

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have been identified in the type I11 group B streptococcal polysaccharide.76.131 Another mechanism whereby capsular polysaccharide could mediate the human immune-system is by molecular mimicry. If a bacterium were able to coat itself with molecules having a structure similar to that of those found in the host’s tissue, the production of antibodies having a specificity for these structures would be suppressed, as they would be recognized as part of “self” by the host. Some organisms (schistosomes) are able to acquire human blood-group determinants on their surfaces in order to circumvent the deleterious effects of the host’s immunological response.%O However, known examples of this type of molecular mimicry in bacteria are very few. Although the capsular polysaccharides of both types Ia and Ib, and type I11 group B Streptococcus have a high degree of structural homology with the respective M and N human blood-group s ~ b s t a n c eand s~~ human ~ ~ serotran~ferrin,6~ there is no evidence that this structural homology interferes with the production of polysaccharide-specific antibody in humans.62The best example of molecular mimicry is probably that of the a-D-(2+8)-linked sialic acid homopolymer, which serves as the capsule for both groups B N . meningitidis and K1 E . coli (see Table VI). This polysaccharide is poorly immunogenic in humans,ls2 and there is evidence to suggest that this poor immunogenicity could be attributed to its structural homology with human glycoprotein.lS5Already highly pathogenic, were it not for the production of antibodies against other surface components of both organisms, they would, indeed, be superpathogens.

(240) 0.L. Goldring, J. A. Clegg, S . R. Smithers, and R. J. Teny, Clin. Enp. Zmmunol., 26 (1976)181-187.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 41

HIGH-RESOLUTION, 'H-NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AS A TOOL IN THE STRUCTURAL ANALYSIS OF CARBOHYDRATES RELATED TO GLYCOPROTEINS BY JOHANNES F. G. VLIEGENTHART, LAMBERTUS DORLAND, AND HERMANVAN HALBEEK Department of Bio-Organic Chemistry, Unioersity of Utrecht, Utrecht, The Netherlands

I. General Introduction ........................................... 209 1. High-resolution, 'H-N.m.r. Spectroscopy ......................... .211 2. Literature Data on High-resolution, 'H-N.m.r. Spectroscopy of Carbohydrates Derived from Glycoconjugates ....................214 11. High-resolution, 'H-N.m.r. Spectroscopy of Carbohydrates Related .218 to Glycoproteins of the N-Glycosylic Type ....................... 1. Carbohydrate Chains of the N-AcetylladosamineType .............. .218 2. Carbohydrate Chains of the Oligomannoside Type ................. .343 3. Additivity Rules ........................................... .365 111. Concluding Remarks .......................................... .371 IV. Experimental ................................................. 373

I. GENERALINTRODUCTION Glycoproteins are biopolymers consisting of a polypeptide backbone bearing one or more covalently linked carbohydrate chains. During the past decade, interest in the structure and function of glycoproteins has increased greatly, as it was found that the carbohydrate chains of these polymers are involved in several important biochemical processes. In particular, their roles in recognition phenomena, in immunological events, and in determining the life-span of cells and glycoproteins must be mentioned. For understanding of the biological function of the glycan chains, detailed knowledge of their structures is a prerequisite. The carbohydrate chains of glycoproteins may be classified according to the type of linkage to the polypeptide backbone. N-Glycosylic chains are attached to the amide group of asparagine (Asn), whereas the 0-glycosylic chains are linked to the hydroxyl group of such amino acid residues as serine (Ser), threonine (Thr), and hydroxylysine (Hyl). As a whole, the carbohydrate chains show a large variety in pri209 Copyright @ 1883 by Academic Press, Inc.

All rights of repmduaion in any form reserved. ISBN 0-1%0072418

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mary structure, as has been discussed by Montreuil,1*2the Kornfelds,3 and Sharon and Lis.* The unambiguous determination of the primary structure of carbohydrates is much more cumbersome than for other biopolymers. The large number of glycosylic linkages possible, in conjunction with the occurrence of branching, yields a fantastically large number of theoretically possible isomers, even for a relatively simple oligosaccharide. This demands a high degree of sophistication in methods used for analysis of the structure. The first analytical problem is encountered at the level of the glycoprotein as such. Owing to natural or artificially introduced (micro)heterogeneity in the carbohydrate chains, it is virtually impossible to obtain the polymer in the form of a single molecular species. Secondly, analysis ofthe complete primary structure can so far not be conducted on intact glycoproteins, and degradation to glycopeptides, oligosacckarides, or oligosaccharide-alditols is obligatory. Thirdly, the fractionation of more or less complex mixtures of (closely) related, partial structures is difficult. Reliable checks for the purity of the compound isolated are indispensable, because, if a sample is considered to be homogeneous, but, in fact, consists of a mixture of closely related components, incorrect structures may be deduced. This might be one of the reasons why it is not exceptional that, for one and the same carbohydrate side-chain of a certain glycoprotein, more than one structure has been reported. Over the years, several strategies have been developed for determination of the structure of carbohydrate chains; for concise reviews, see Refs. 3 and 4.In particular, the refinements of methylation a n a l y s i ~ , ~ , ~ ~ and c h e m i c a P and enzymic degradation method^^-'^ have permitted (1) J. Montreuil, Adz-. Curbohydr. Chern. Biochem., 37 (1980) 157-223. (2) J. Montreuil, in A. Neuberger (Ed.), Comprehensive Biochemistry, Vol. 19 B, Part 11, Elsevier, Amsterdam, 1982, pp. 1-188. (3) R. Komfeld and S. Komfeld, in W. J. Lennalz (Ed.),The Biochemistry ofGlycoproteins und Proteoglycans, Plenum, New York, 1980, pp. 1-34. (4)N. Sharon and H. Lis, in H. Neurath and R. L. Hill (Eds.), The Proteins, 3rd edn., Vol. V, .4cademic Press, New York, 1982, pp. 1-144. ( 5 ) B. Lindberg and J. Liinngren, Methods Enzymol., 50 (1978) 3-33. (54 H. R a ~ i ~ a l J, a , Finne, T. Krusius, J. Kirkkainen, and J . Jamefelt,Ada. Carbohydr. Chem. Biochem., 38 (1981) 389-416. (6)B. Lindbery, J. Lonngren, and S. Svensson, Ado. Carbohydr. Chem. Biochern., 31 (1975) 185-240. (7) G. 0. Aspinall, Pure A p p l . Chem., 49 (1977) 1105-1134. (8) G. Strecker, A. Pierce-Cretel, B. Foumet, G. Spik, and J. Montreui1,Anal. Biochem., I 1 1 (1981) 17-26. (9) Y.-T.Li and S.-C. Li, in M. I. Horowifz and W. Pigman (Eds.),The Glycocunjugates, Vol. I, Academic Press, N e w York, 1977, pp. 51-67. (9a) H. M. Flowers and N. Sharon, Ado. Enzymol., 48 (1979) 29-95. (10) A. Kobata, A n d . Biochem., 100 (1979) 1-14.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

21 1

the unraveling of new structures. The enzymic methods were greatly improved by rigorous purification of isolated exo- and endo-glycosidases. However, the classical methods have some inherent limitations, and are time- and material-consuming. In the past few years, we have had the opportunity to introduce high-resolution, 'H-n.m.r. spectroscopy for determination of the structure of glycan chains of glycopr~teins.'~-'~ In close collaboration with J. Montreuil and his colleagues (Lille, France), we have shown that this technique, in conjunction with methylation analysis, is extremely suitable for structural studies on N-, as well as on 0-,glycosylic glycans. The present article contains a discussion of the high-resolution, 'H-n.m.r. spectra of compounds, derived from N-glycosylically linked carbohydrate chains, comprising the N-acetyllactosamine and oligomannoside types of structure.

1. High-resolution, 'H-N.m.r. Spectroscopy 'H-N.m.r. spectroscopy has contributed significantly to extensions of our knowledge on the structure and conformation of biomolecules as well as on intra- and inter-molecular, interaction processes. N.m.r. spectroscopy is, however, an inherently insensitive technique, and the richness of information contained in an n.m.r. spectrum often limits the size and complexity of the molecules that can usefully be studied. Advances in instrument design have now greatly improved the sensitivity of the spectrometers, so that resonances can readily be observed in aqueous solutions of, for instance, complex carbohydrates, at concentrations of the order13 of 0.05 mM. Increase in the strength of magnetic fields (up t014 14 Tesla) has enabled the study of molecules of larger molecular weight and complexity by inducing better spectral-resolution. Moreover, the availability of sophisticated computerfor example, programs allows an artificial resolution-enhan~ement,'~ by Lorentzian to Gaussian transformation; for general reviews, see (11)J, Montreuil and J. F. G. Vliegenthart, in J. D. Gregory and R. W. Jeanloz (Eds.), Glycoconjugate Research, Proc. lnt. Symp. Glycoconjugates, 4th, Vol. I, Academic Press, New York, 1979, pp. 35-78. (12)J. F. G.Vliegenthart, H. van Halbeek, and L. Dorland, in A. Varmavuori (Ed.), IUPAC Int. Congr. Pure A p p l . Chem., 27th, Helsinki, 1979, Pergamon, Oxford, 1980, pp. 253-262. (13)J. F.G. Vliegenthart, H. van Halbeek, and L. Dorland, Pure A p p l . Chem., 53 (1981) 45-77. (14)M. Llinis, A. de Marco, and J. T. J. Lecomte, Biochemistry, 19 (1980)1140-1145; D. G. Davis and B. F. Gisin, FEBS Lett., 133 (1981)247-251; M.L. Hayes, A. S. Serianni, and R. Barker, Carbohydr. Res., 100 (1982)87-101. (15)R. R. Emst, Ado. Magn. Reson., 2 (1966)1-135.

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Refs. 16-20. Already, a partial interpretation of an n.m.r. spectrum can provide detailed information about molecular structure, as will be shown. For interpretation of the ‘H-n.m.r. spectrum of a carbohydrate chain in terms of primary structural assignments, the concept of “structuraEThis means that the chemical reporter groups” was shifts of protons resonating at clearly distinguishable positions in the spectrum, together with their coupling constants and the line widths of their signals, bear the information essential to permit assigning of the primary structure. In high-resohtion, ‘H-n.m.r. spectra of relatively large, N-glycosylic carbohydrate structures, resonances of the following structural-reporter groups can be discerned. (a) Anomeric Protons ( H - l Atoms). Their chemical shift and coupling constant provide information on the kind of sugar residue, as well as on the type and configuration of its glycosylic linkage. (b) Mannose H-2 and H-3 Atoms. The pattern of their signals is, as a whole, indicative of the type of substitution of the common, mannotriose branching-core. ( c )Sialic Acid H-3 Atoms. Their chemical shifts are characteristic for the type and configuration of the glycosylic linkage of the sialic acid residue, and, in some cases, for the location of the residue in the chain. ( d )Fucose H - 5 and CH, Atoms. The chemical shifts of these protons, together with that of H-1 of the residue, are indicative of the type and configuration of its glycosylic linkage, and of the structural environment, in particular of the residue to which the fucose is attached. ( e )Galactose H-3 and H-4 Atoms. Their chemical shifts are, in some cases, useful for characterizing the type and configuration of the glycosylic linkage between galactose and its substituent. cf)Amino Sugar

(2-Acetamido-2-deoxyglucoseand Sialic Acid) N-Acetyl-CH, Protons. Their chemical shifts are sensitive to even small structural variations, making this region of the spectrum highly informative. The line widths of the signals of the structural-reporter groups are influenced by the local mobility of the protons. This will be illustrated, in particular, for the anomeric-proton signals and for the N-acetyl signals. (16) R. A. Dwek, Nuclear Magnetic Resonance (N.M.R.)in Biochemistry: Applications to Enzyme Systems, Clarendon Press, Oxford, 1973. (17) P. F. Knowles, D. Marsh, and H. W. E. Rattle, Magnetic Resonance of Biomolecules, Wiley, New York. 1976. (18) K . Wuthrich, NMR in Biological Research: Peptides and Proteins, North-Holland, Amsterdam, 1976. (19) L. J. Berliner and J. Reuben, Biological Magnetic Resonance, Vols. 1 and 2, Plenum, New York, 1978. (20) R. G. Shulman, Biological Applications of Magnetic Resonance, Academic Press, New York, 1979.

.‘H-N.M.R. SPECTRA OF

GLYCOPROTEIN CARBOHYDRATES

213

Besides the aforementioned n.m.r. parameters [chemical shift (a), coupling constant and line width], spectral integration can give valuable information. The relative intensities of the structural-reporter-group signals in the n.m.r. spectrum can be used as markers for the purity of the compound. Often, from the spectrum, it can be deduced whether or not the sample consists of more than one carbohydrate structure, and in which molar ratios the components of the mixture, and the sugar residues in each of these, occur. In the ‘H-n.m.r. spectrum of a carbohydrate chain, a broad signal is observed, between 6 -3.4 and -4.0, that is derived from the bulk of nonanomeric, sugar-skeleton protons, but, so far, it could not be resolved into contributions of individual protons. In the case of glycopeptides, additional signals, derived from protons of the amino acid residues, are found in the spectrum. It should be stressed that a high-resolution, ‘H-n.m.r. spectrum of a compound provides a measure of the structural identity which, even if the spectrum cannot be completely interpreted, renders possible a comparison with the spectra of compounds obtained from other sources, allowing a decision as to whether or not the compounds are identical. After recording of this “identity card,” the unimpaired compound may be submitted to chemical and enzymic degradation-procedures. In this article, the 500-MHz, or, sometimes, 360-MHz, ‘H-n.m.r. spectra of some seventy N-glycosylic carbohydrate chains will be discussed. The spectra were recorded in D 2 0 at ambient temperature and at p D -7, unless indicated otherwise; for experimental details, see Section IV. First, the outstanding features of the spectra of fundamental elements of N-acetyllactosamine-type structures will be treated in detail (compounds 1-20); this part also covers the characteristics of the intersecting, GlcNAc residue in this type of structure. Secondly, extensions of these chains with differently linked, sialic acid residues (compounds 21-41), and thirdly, with fucose residues (compounds 42-54), will be discussed. Besides the structural-reportergroup signals of the newly introduced residues, the effects of extension on the remaining signals in the spectra will be traced. Next, some unusual, N-acetyllactosamine-type, N-glycosylic carbohydrate structures (compounds SS-SO) containing a virtually abnormal core-region, namely, PGal(1+4)pGlcNAc( l-*N)Asn, will be considered. Finally, the n.m.r. characteristics of oligomannoside-type carbohydrate chains (compounds 61-72) will be presented; in particular, the second branching-point, and the characteristics and influences of a-(1-2)linked mannose residues occurring in these structures, are the subjects of discussion.

u),

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2. Literature Data on High-resolution, 'H-N.m.r. Spectroscopy of Carbohydrates Derived from Glycoconjugates

The fundamental work of Lemieux and coworkers21introduced the successful application of 'H-n.m.r. spectroscopy to structural problems in the carbohydrate field. Since then, numerous studies have been devoted to the (partial) determination of primary structures of carbohydrates and derivatives thereof, as well as to the elucidation of their conformation in solution, by means of 'H-n.m.r. spectroscopy. An exhaustive discussion of all these contributions is beyond the scope of this article. For comprehensive reviews, see Refs. 22-28. Regarding the applicability of 'H-n.m.r. spectroscopy for elucidation of the structure of the carbohydrate moieties of glycoconjugates, several reports have been published. In 1973, one of the first examples of employment of high-resolution, 'H-n.m.r. spectroscopy for structural studies on intact g l y c ~ l i p i d safforded ~~ the 220-MHz, 'Hn.m.r.-spectral data for some peracetylated galactocerebrosides, determined in three different solvents. In 1979, Falk and described 270-MHz, 'H-n.m.r. spectroscopy of non-degraded, permethylated and permethylated-reduced derivatives of (blood-group active) glycosphingolipids (spectra were recorded in chloroform solution, at probe temperatures of -25 and -40"), as a suitable approach for the determination of the configuration of the anomeric linkages in the sugar chains. According to Karlsson and c o ~ o r k e r s , 3 3 -these ~~~ data, at most, supplement the structural fingerprinting of lipid-linked (21) R. U . Lemieux, R. K. Kullnig, H. J. Bemstein, and W. G. SchneiderJ. Am. Chem. SOC., 80 (1958) 6098-6105. (22) L. D. Hall, Adc. Carbohydr. Chem., 19 (1964) 51-93. (23) T. D. Inch, Annu. Aec. N M A Spectrosc., 2 (1969) 35-82. (23a) P. L. Durette and D. Horton,Adr;.Carbohydr. Chem. Biochem., 26 (1971)49-125. (24) B. Coxon, Adr. Curbohydr. Chem. Biochem., 27 (1972) 7-83. (25) G. Kotowycz and R. U. Lernieux, Chem. Rec., 73 (1973) 669-698. (26) L. D. Hall, Adc. Carbohydr. Chem. Biochem., 29 (1974) 11-40. (27) D. B. Davies, Nucl. Magn. Reson., 9 (1980) 182-203. (28) L. D. Hall, in W. Pigrnan and D. Horton (Eds.), The Carbohydrates: Chemistry und Biochemistry, 2nd edn., Vol. IB, Academic Press, New York, 1980, pp. 13001.326. (29) M. Martin-Lornas and D. Chapman, Chem. Phys. Lipids, 10 (1973) 152-164. (30) K.-E. Falk, K.-A. Karlsson, and B. E. Samuelsson, Arch. Biochem. Biophys., 192 (1979) 164-176. (31) K.-E. Falk, K.-A. Karlsson, and B. E. Sarnuelsson, Arch. Biochem. Biophys., 192 (1979) 177- 190. (32) K.-E. Falk, K.-A. Karlsson, and B. E. Sarnuelsson, Arch. Biochem. Biophys., 192 (1979) 191-202. (32a) K.-E. Falk, K.-A. Karlsson, H. Leffler, and B. E. Samuelsson, FEBS Lett., 101 (1979) 273-276.

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215

oligosaccharides by mass spectrometry. Since 1980, Dabrowski and coworker^^^-^^^ have published 360MHz, 'H-n.m.r. data for a number of underivatized glycosphingolipids (blood-group active glycosylceramides, as well as more-complex compounds related to the Forssman glycolipid); the spectra were recorded at 65" for solutions in dimethyl sulfoxideds containing a trace of DzO. These authors were able to assign all of the anomeric-proton signals, and a number of nonanomeric-proton resonances. The chemical shifts of these protons were found to be dependent on essential, primary, Most of the chemical shifts of the ring prostructural tons were determined by spin-decoupling, and nuclear Overhauser, difference s p e c t r o s ~ o p y . ~ Concerning ~ , ~ ~ - ~ ~ ~ the potential of J-resolved, two-dimensional, 'H-n.m.r. spectroscopy for this purpose, Yamada and drew attention to the fact that, despite the many useful aspects of this method for carbohydrates (see Refs. 28 and 39), these experiments require a fair amount of time and material. In the field of glycoproteins, the following high-resolution, 'Hn.m.r.-spectral studies for solutions in D,O on underivatized, glycan chains related to, or identical with, those that will be described herein deserve mention. Wolfe and coworkers publishedPO in 1974 and4' 1975, 220-MHz, 'H-n.m.r. spectra of two oligosaccharides related to the N-acetyllactosamine type of N-glycosylic carbohydrate chain. (33) K.-A. Karlsson, in Ref. 12, pp. 171-183. (33a) M. E. Breimer, G . C. Hansson, K.-A. Karlsson, and H. Leffler, Biochim. Biophys. Acta, 617 (1980) 85-96. (33b) G . C. Hansson, K.-A. Karlsson, and J. Thurin, Biochim. Biophys. Acta, 620 (1980) 270-280. (34) K.-E. Falk, K.-A. Karlsson, and B. E. Samuelsson,FEBS Lett., 124 (1981) 173-177. (34a) J. Angstrom, M. E. Breimer, K.-E. Falk, I. Griph, G. C. Hansson, K.-A. Karlsson, and H. LeHer,J. Biochem. (Tokyo), 90 (1981) 909-921. (35) J. Dabrowski, H. Egge, P. Hanfland, and S. Kuhn, in C. C. Sweeley (Ed.), Cell Surface Glycolipids, ACS Symp. Ser. 128, American Chemical Society, Washington D. C., 1980, pp. 55-64. (36) J. Dabrowski, H. Egge, and P. Hanfland, Chem. Phys. Lipids, 26 (1980) 187-196. (37) J. Dabrowski, P. Hanfland, and H. Egge, Biochemistry, 19 (1980) 5652-5658. (37a) J. Dabrowski, P. Hanfland, H. Egge, and U. Dabrowski, Arch. Biochem. Biophys., 210 (1981) 405-411. (37b) P. Hanfland, H. Egge, U. Dabrowski, S. Kuhn, D. Roelcke, and J. Dabrowski, Biochemistry, 20 (1981) 5310-5319. (374 J. Dabrowski, P. Hanfland, and H. Egge, Methods Enzymol., 83 (1982) 69-86. (38) A. Yamada, J. Dabrowski, P. Hanfland, and H. Egge, Biochim. Biophys. Acta, 618 (1980) 473-479. (39) L. D. Hall, G. A. Moms, and S. Sukumar, Carbohydr. Res., 76 (1979) c7-cQ. (40) L. S. Wolfe, R. G. Senior, and N. M. K. Ng Ying KinJ. Biol. Chem., 249 (1974)1828 -1838. (41) N. M. K. Ng Ying Kin and L. S. Wolfe, Biochem. Biophys. Res. Commun., 66 (1975) 123- 130.

J. F. G. VLIEGENTHAHT et uZ.

2 16

These compounds were isolated from the 1ivefO and the urine4' of patients suffering from GM,-gangliosidosis type I. Their structures are identical to those of the di- and tri-antennary, asialo oligosaccharides 7 and 10 ofthis article (see Chart 1). The spectra were recorded at 70°, and the chemical shifts were measured in p.p.m. relative to external tetramethylsilane as the standard. In later s t ~ d i e s , 4 *these , ~ ~ authors arrived at a partial interpretation of the 100-MHz, and 90-MHz, 'Hn.m.r. spectra, recorded at 70 and 60°, respectively, of some oligosaccharides stemming from the liver of a patient who died of GMz-gangliosidosis variant 0 (Sandhoff- Jatzkewitz disease). The structures of the isolated hexa- and hepta-saccharides are the same as those respectively denoted 6 and 14 herein, and as that of the minor constituent present in the heptasaccharide mixture of which compound 14 forms the major part (see legend to Fig. 15).The linear, GMz-gangliosidosis tetrasaccharides described in Ref. 43,namely, /3GlcNAc(1+2)aMan(1+3)BMan( 1+4)GlcNAc and /3GlcNAc(lA)crMan(1+3)/3Man(1+4)GlcNAc, are not incorporated in the series of compounds discussed herein. In addition, the authors described43 the W M H z , 'H-n.m.r. data for a trisaccharide (compound 5) isolated from the urine of mannosidos is patients . In 1977 and 1980, we respectively introduced the application of 360-MHz, and 500-MHz, 'H-n.m.r. spectroscopy for elucidation of the structure of underivatized carbohydrate chains obtained from glycoproteins. Since then, several other research groups have become active in this field. For reasons outlined later, the results of their work will be briefly summarized first. In 1979, Kin and Wolfe" published the 220-MHz, 'H-n.m.r. spectra, recorded at 70°, of a mono-antennary glyco-asparagine possessing the same structure as 46 or 47, and of the corresponding oligosaccharide, h a v i n e GlcNAc-2 as the terminal, reducing-sugar residue. Like 46 and 47, the compounds were isolated from the urine of a hcosidosis patient. published some 360-MHz, 'H-n.m.r. data Schachter and for a series of glycopeptides having N-glycosylically linked carbohydrate chains of the N-acetyllactosamine type, and one having a chain of the oligomannoside type; spectra were recorded at a variety of probe temperatures (20,25,30,70, and 85"). The compounds (42) N. M.K. Ng Ying Kin and L. S. Wolfe, Biochem.Biophys. Res. Commun., 59 (1974) 837-844.

(43)N. M. K. Ng Ying Kin and L. S. Wolfe, Carbohydr. Res., 67 (1978) 522-526. (44) N. M. K. Ng Ying Kin and L. S. Wolfe, Biochem.Biophys. Res. Commun., 88 (1979) 696-705. (44a) For the system of numbering used for the sugar residues in N-glycosylic carbohydrate chains, see the footnote on page 221. (45) S. Narasimhan, N. Harpaz, G. Longmore, J. P. Carver, A. A. Grey, and H. Schachter, I. Biol. Chem., 255 (1980) 4876-4884.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

217

a M a n ( l + 3), 'pMan( 1+ 4)pGlcNAc( 1-+ 4)pGlcNAc(1+ N)Asn, / / aMan(1 + 6) a F u c ( 1 + 6) pGlcNAc( 1+ 2)aMan( 1+ 31, aMan(1-+ 6)/

/3Man(1-+ 4)/3GkNAc( 1 + 4)/3GlcNAc( 1 + N)Asn, / aFuc(1 -+ 6)

and pGlcNAc( 1+ 2)aMan( 1-+ 3) \ pGlcNAc( 1+ 4)8Man( 1-+ 4)pGlcNAc(1+ 4)pGlcNAc( 1+ N)Asn, / aMan(1 -+ 6)/ aFuc(1 + 6)

were prepared from a human myeloma IgG; the other two, namely, pGlcNAc( 1 + 2)aMan( 1-+ 3), pGlcNAc( 1 + 4)pMan( 1-+ 4)PGlcNAc( 1+ 4)bGlcNAc( 1-+ N)Asn, pGlcNAc( 1 + 2)aMan(1+ 6f

and aMan(1 + , 1 3 pMan( 1-+ 4)pGlcNAc( 1 -+ 4)pGlcNAc( 1+ N)Asn,

crMan(l-+ 3)

\

aMan( 1+ 6)/

were obtained from hen-egg albumin. Subsequently, Cohen and BallouM exhaustively discussed the 180-MHz7'H-n.m.r. data (40")for a large number of building units (both as oligosaccharides and as glycopeptides) of the oligomannoside type of N-glycosylic carbohydrate chain possessing 1 through 8 mannose residues. Most of the compounds treated in Ref. 46 will also be subject to discussion herein (corresponding to structures 5,62-65, and 68-70). However, some ofthem, for example, pMan( 1 4 ) G l c N A c and aMan( 1-+ 3 ) , pMan( 1+ 4)GlcNAc, aMan( 1+ 6)'

are not included. It should be noted that the pioneering work of Gorin and coworker^^^-^^ on yeast mannans and galactomannans provided, for resonankes characteristic for a series of oligosaccharides, assignments (46) R. E. Cohen and C. E. Ballou, Biochemistry, 19 (1980) 4345-4358. (47) P. A. J. Gorin and J. F. T. Spencer, Can. J. Chem., 46 (1968) 2299-2304. (48) P. A. J. Gorin, M. Mazurek, and J. F. T. Spencer, Can. J. Chem., 46 (1968) 23052310. (49) P. A. J. Gorin, J. F. T. Spencer, and S. S. Bhattachaiee, Can. I . Chem., 47 (1969) 1499-1505.

2 18

J . F. G. VLIEGENTHART et

a1

that proved to be valuable for high-resolution, 'H-n.m.r.-spectral Finally, in addition to 'H-n.m.r. data for structures identical to 62 and 63, Carver and coworkers50 reported the 360-MHq 'H-n.m.r.spectral dsta for two glycopeptides possessing a hybrid type1 of structure, namely, pGlcNAc( 1

2)aMan(1 -+ 3)\ pClcNAc( 1 + 4)@Man(1 4 4)PGlcNAc(1 + 4)PGlcNAc(1 + N)Asn, uMan(1 -+ 3 ) , aMan(1 -+ 6) uhIlan(1 + 6') -+

/

and

4)

pGal( 1 -+ 4)/.3CkNAc(1 + pGlcNAc(1 + 2)aMan(l + 3)\ pGlcNAc( 1 + 4)BMan(1 -+ 4)PGlcNAc(1 -+ 4)@GlcNAc(1 + N)Asn

a.Cfan[l + 3)\ a M ~ n ( -+ 1 6)/

/

aMan(1 + 6)

In this study, n.0.e. difference-spectroscopy was applied to enable the making of some assignments. The spectral data for the compounds, the detailed structures of which have been presented in Section I, 2, are not compiled in the following Sections, because they were obtained from spectra recorded under more or less different experimental conditions (regarding strength of magnetic field, and reference standard, but, most of all, probe temperature) (compare, our experimental conditions, Section IV). This choice was dictated solely by the impossibility of making these data comparable with ours, and is not intended to imply any criticism of the work performed in other laboratories. 11. HIGH-RESOLUTION, IH-N.M.R.SPECTROSCOPY OF CARBOHYDRATES RELATED TO GLYCOPROTEINS OF T H E N-GLYCOSYLIC TYPE

1. Carbohydrate Chains of the N-Acetyllactosamine Type a. Fundamental Elements of Carbohydrate Chains of the N-Acetyllactosamine Type (Compounds 1-20).- Symbols employed for compounds 1-20 are depicted in Chart 1. (50) J. P. Carver, A. A. Grey, F. M.Winnik, J. Hakimi, C. Ceccarini, and P. H. Atkinson, Biochemistry, 20 (1981) 6600-6606.

CHART 1. Symbols Employed for Compounds 1-20 Key to the symbolic notation: 0 = GlcNAc

+ = Man W = Gal

1

0 = c~-NeuAc-(2-*6)

A = c~-NeuAc-(2+3) 0 = Fuc

e A s n

11 2 3

l-

Asn

rAsn 12

4

RAsn

5 13 6 14 7 15 8

16 9

17 10

18

19

20

>y

Thr

220

J. F. G. VLIEGENTHART et al.

The simplest element of the linkage region between an N-glycosylic carbohydrate chain and the polypeptide backbone to which it is attached is pGlcNAc(l+N)Asn (compound 1).Among various sources, 1 may be isolated from the urine of patients with aspartylglucosaminria.^'-% The 360-MHz, 'W-n.m.r. spectrum of compound 1 is presented in Fig. 1.The resonances in this spectrum may be divided into signals from the following groups of protons. The structural-reporter groups resonate at clearly distinguishable positions and provide essential information on the primary structure. The GlcNAc anomeric proton resonates at a lower field (see Table I) than could be expected [the normal range of S for an axial anomeric proton in a free or 0-glycosylically linked GlcNAc residue in the 'Cc,(D) conformation is 4.40-4.75 p.p.m.1. It is reasonable to assume that this effect is due to the electron-withdrawing amide group attached to C-1 (compare with Ref. 54).TheJl3 value (9.8 Hz) is characteristic for a p-glycosylic linkage. The value is relatively large, due to the N-type of glymsylic linkage. The chemical shift of the singlet of the N-acetyl group is typical for GlcNAc linked to Asn. As will be shown later, its position may vary, depending on the nature of the peptide moiety. The nonanomeric protons resonate in the range of 3.4-4.0 p.p.m. For this simple compound, a complete interpretation was achieved, and this was confirmed, and refined, by computer simulation of the spectrum. The n.m.r. data for compound 1 are summarized in Table I. By means of a modified Karplus equation, the 'Cc,(D)conformation of the GlcNAc group was deduced.55 The amino acid protons of Asn resonate apart from the GlcNAc protons. In glycoproteins, substitution at 0-6 of GlcNAc-1 by an (Y-L-FUC group frequently occurs.* Compound 2 can be isolated from the urine of patients suffering from f u ~ o s i d o s i s . ~The ~ , ~360-MHz, *~ 'H-n.m.r. spectrum of 2 is given in Fig. 2. (51) J. N. Isenberg, in E. F. Walborg, Jr. (Ed.), Glycoproteins and Glycolipids in Diseuse Processes, ACS Symp. Ser. 80,American Chemical Society, Washington D. c., 1978, pp. 129-131, and references cited therein. (52) G. Strecker and J. Montreuil, Biochimie, 61 (1979) 1199-1246. (53) In the description of the n.m.r.-spectralfeatures of each compound discussed in this article, only the actual source(s)of the compound used for the n.m.r. investigations is (are)mentioned. For other possible glycoprotein sources of these carbohydrates, the reader is referred to reviews'-'; see also, Section IV. (54) M. Tanaka and I. Yamashina, Carbohydr. Res., 27 (1973) 175-183. (55) L. Dorland, B. L. Schut, J. F. C. Vliegenthart,C. Strecker, B. Fournet, G. Spik, and J. Montreuil, Eur. I . Biochern., 73 (1977) 93-97. (56) G . Strecker, B. Foumet, J. Montreuil, L. Dorland, J. Haverkamp, J. F. G. Vliegenthart, and D. Dubesset, Biochimie, 60 (1978) 725-734.

221

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

TABLEI 'H Chemical Shifts and Coupling Constants for pGlcNAc(l+N)Asn (Compound 1) and aFuc(l-&)pGlcNAc(l+N)Asn (Compound 2) Coupling constant (Hz)"

Chemical shift (p.p.m.)"

Proton Compound 1 Compound 2

Residue ~

Compound 1 Compound 2

~

GlcNAC-1

H-1 H-2 H-3 H-4 H-5 H-6a H-6b NAc H-1 H-2 H-3 H-4 H-5 CHI H-(Y H-P H-P'

(YFUC( 1+6)

Asn

a

5.072 3.821 3.601 3.472 3.527 3.876 3.739 2.013

9.85 9.85 9.50 9.50 2.10 5.55 -11.40

-

3.75 10.30 3.40 0.60 6.55

-

-

6.30 3.80 - 17.25

3.968 2.866 2.932

Chemical shifts and coupling constants measured at 360 MHz and T = 300 K.

Comparison of the spectral data for 1and 2 shows that the chemical shifts of the GlcNAc structural-reporter groups, that is, H-1 and the N-acetyl-CH,-protons, are slightly, but significantly, influenced, whereas theJ,,2 value is but little affected by the extension of 1with a Fuc group. For the Fuc group, the resonances of its H-1, H-5, and CH3-groupprotons are characteristic. TheJ1,2value (3.75Hz) of Fuc is * Coding of monosaccharides for the carbohydrate chains described in this Chapter: Ny8-7

\ N-6- 5 - 4

\

9-3-2-l- Asn N'- 6 - 5 ' - 4

a-

7'

/

'

for 1-54

b D1-C-4

N -c- b-a

- 1I

F

Asn

for 55-60

&"\ 03- B

'3-2-1/

Asn

for 61-72

J . F. G. VLIEGENTHART et al.

222

0 -GlcNAc - (1--N) -Asn 1

1

NAc

CH3 p o I w

anomeric proton

I

60

-1

50 \

I I

\

\ \

I 1 I GlcNAc

n-i

A

I I I

, \

I

\ \

\

Asn H-*

\

GlcNAc

H-2 H-6a

\

H-5

H-6b H-3

H-4

A m

H - 6 H-P

\

I

I

1

5M 2 SO

FIG.1.- Resolution-enhanced, Overall, 360-MHz, IH-N.m.r. Spectrum of Compound 1 (Upper Trace), Supplied with Assignments in Full Detail, Most of Which are Indicated in the Expanded Regions (Lower Trace).[A small amount of sodium 4,4-dimethyl4-silapentane-I-srilfonate (DSS) had been added to the D,O solution, in order to serve as an internal reference for the chemical shifts (S(CH,),, DSS 0). (In the expanded pattern for the Asn P-CH, protons, the signals marked by asterisks originate from DSS.)]

indicative of an a-L-glycosidic bond between this group and the GlcNAc residue. The signals of the nonanomeric protons form a bulk that could be completely interpreted. The refined n.rn.r. data, obtained after spectral simulation, are given in Table I. For both GlcNAc and Fuc, the normal chair conformations [", (D) and ' C 4 ( ~ )respectively] , were cal-

'H4.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

223

0 -GlcNAc - (1-N)-Asn /

a-Fuc-(l-B)

I

'

2 NAC

C H 3 protons

Fuc -CH3 protons

.<*lor*

anomeriL protons

wQar skeleton protons

-I

1

1 Fuc

do

s'o

-8

/

\zo

10

LD

/

\

/

\

\

, /

\

/

,

\

QlcNAc

/

\ \

/

/

0.0

W \

/

Hy5 H-En

H-Bb

H-2

/

i

\

H-3

H-4

\ \

\

/

\

/

\

I

'

I

Fuc

* H-5

H-3

H-4 H-2'

H-p- H-P

I

M' 1

-1

ra

3*

I6

1b

12

10

FIG.2.-Resolution-enhanced, Overall, 36@MHz,'H-N.m.r. Spectrum of Compound 2 (Upper Trace), Supplied with Assignments in Full Detail, Most of Which are Indicated in the Expanded Region (Lower Trace). [Here, and throughout this article, chemical shifts are measured by reference to internal acetone (ti 2.225, relative to DSS in 4 0 at 300 K).]

224

J. F. G. VLIEGENTHART et ul.

~ u l a t e d The . ~ ~ attachment of Fuc to 0-6 of GlcNAc influences the chemical shifts of the GlcNAc H-4, -5, and -6 atoms, in comparison to those of 1. The change in the geminal J6,sr value (from -12.7 to - 11.4 Hz) can be conceived as a strong indication for a (1+6)-glycosidic linkage. Unambiguous proof for the (1-6) linkage was obtained by 1%-n.m.r. spectroscopy5' of compound 2. (57) The type of linkage between Fuc and GlcNAc was unambiguously determined by '3C-n.m.r. spectroscopy. T h e 'SC-n.m.r.-spectral data for compounds 1and 2, acquired at 25 MHz and 33", are as follows.

13CChemical Shifts of Constituent Monosamharides for /3GlcNAc(l+N)Asn (Compound 1) and aFuc(I+WGlcNAc(l+N)Asn (Compound 2) (in p.p.m. relative to internal DSS) Schematic structure of compound ~

1

2

tAsn

Residue

Carbon atom

GlcNAc

c-1 G2 C3

c4 c-5 C-6

'3c Chemical shift

bAsn

80.85 56.82 76.95 7225 80.26 63.25

80.86 56.76 76.86 72.49 79.48 69.93

175.60"

175Mb

177.57"

177.65b

24.80

24.80

-

101.82 70.93 72 25 74.54 69.44 18.00 53.64 37.74 175.27b

0

I1

-C'-NH-C-CHZ-

0 --@-NH-C-CH,

II

*

0

--@-NH-C-$H, Fuc

c-1

Asn

c-2 C-3 C-4 c-5 C-6 C-U

C-b -CO,H

I

53.68 37.68 175%"

Assignments may have to be interchanged. The increment in chemical shift observed for C-6 of GlcNAc in the step from 1to 2 (A8 +6.7 p.p.m.) indicates attachment of a Fuc group at 0-6of GlcNAc. This shift increment is in accord with literature data [P. Colson and R. R. King, Carbohydr. Res., 47 (1976) 1-13]. For ISC-n.m.r.-spectral data on /?GlcNAc(l+N)Asn, see also, K. Dill and A. Allerhand, FEBS Lett., 107 (1979) 26-29, and S. Shibata and H. Nakanishi, Carbohydr. Res., 86 (1980)316-320.

‘H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

225

Differences in the chemical shifts of the methylene protons of Asn in the spectrum of 2 in comparison to that of 1are due to a small variation in the pD for both solutions. Compound 3 has been isolated from the urine of patients with aspartylglucosaminuria51~5z or with Gaucher’s d i s e a ~ e . The ~ , ~ 500~ (58) H. van Halbeek, L. Dorland, G. A. Veldink, J. F. G. Vliegenthart, J.-C. Michalski, J. Montreuil, G. Strecker, and W. E. Hull, FEBS h t t . , 121 (1980) 65-70. (59) J.G.Michalski, J. Monbeuil, G. Strecker, H. van Halbeek, L. Dorland, J. F. G. Vliegenthart, B. Cartigny, and J.-P. Farriaux, Eur. 1.Biochem., (1983) in press.

0 -Man-(l-4)-~-GlcNAc -(I- 4)- 0 -GlcNAc-(1-N)-Asn /

a-Man-(1-

s

1

1

6)’

41

3 NAc -CH3 protons 2

,’

Hm

suosr skeleton pdaa anomeric protons

Man H-2 atoms

0

1

w

n-m - 6

50

al”

P - q

UI

40

20

NAc CH3 protons 2 I

I

anomeric protons

’

Man H-2 atoms

,

3

4

J Am 110

c

d

50

&a

4.6

42

40

I

I L-10

Overall, 500-MHz. ‘H-N.m.r. Spechum of Compound 3. (b) Expanded, Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, ‘H-N.m.r. Spectrum of Compound 3.[The bold numbers in the spectra refer to the corresponding residues in the structure.The relative-intensity scale of the expanded, N-acetyI-proton region differs from that of the other parts of the spectrum, as indicated.]

FIG.3.-(a)

226

J. F. G. VLIEGENTHART et al.

MHz, 'H-n.m.r. spectrum of 3 obtained from the latter source is given in Fig. 3, and the spectral data are presented in Table 11. The substitution at 0 - 4 of GlcNAc-1 by GlcNAc-2 does not significantly affect the chemical shift of its H-1 signal, as compared to 1.The H-1 atom of GlcNAc-2 resonates at 6 4.618; the J1.2 value (8.0 Hz) points to a P-glycosidic linkage between the two GlcNAc residues. The different configurations of the glycosidic bonds of Man-3 and Man-4' are reflected in the chemical shifts of their anomeric protons: 6 4.767 (Man-3: p ) and S 4.915 (Man-4': a).The signal of H-1 of Man-3 appears in the spectrum as a relatively broad singlet due to the small value of J , v 2 (- 0.7 Hz; H-la-H-2e). In contrast, the resonance signal of the H-1 atom of Man-4' is a well resolved doublet, with J1,2 1.8 Hz (H-le-H-2e). This difference in coupling constants is even more clearly observable in the H-2 signals of the respective Man residues. TABLEI1 IH Chemical Shifts" of Structural-reporter Groups of Constituent Monosaccharides, and of Amino Acid Protons for Glyco-asparagines 3and4 Compound and schematic structure

3 Reporter group

Residue

H-1 of

1 2 3 4' 3 4' 1 2 uFuc(1+6) aFuc(1+6) aFuc( 1-43) Asn Asn Asn

H-2 of NAc of

H-1 of H-5 of CH, of H-a of H-P of H-@' of

4

nAS 5.07 1 4.618 4.767 4.915 4.080 3.968 2.014 2.076

-

3.971 2.851 2.931

5.076 4.690 4.770 4.916 4.083 3.967 2.018 2.095 4.877 4.125 1.209 3.987 2.871 2.934

N.m.r. data listed in this Table and the other Tables were acquired at

500 MHz, unless indicated otherwise. In this heading and in those of the other Tables, the compounds involved are represented by the following, Man; 0, aNeuAc(2-+6); A, symbolic notation: 0 , GlcNAc; H, Gal; aNeuAc(2+3); and 0 , Fuc. See also, Chart 1.

+,

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

227

The H-2 atom of M a n 3 resonates at 6 4.080 [typical for mono-~-(l+6) substitution, see later], and that of Man-4' at 6 3.968 [typical for a terminal a-(1+6)-linked Man residue, see later]. For both residues, the J2,3 value is 3.4 Hz. The singlet of the N-acetyl protons of GlcNAc-1 is found at essentially the same position as in the spectrum of 1(6 2.014). The signal at 6 2.076 stems from the corresponding group of GlcNAc-2. The signals of the remaining, sugar-skeleton protons are found in a very narrow spectral-region (3.55-3.95 p.p.m.), and have not yet been completely assigned. Compound 4 may be isolated from the urine of patients with fucosid o ~ i sThe . ~ ~500-MHz7'H-n.m.r. spectrum of 4 is presented in Fig. 4. The spectral data for 4 are compiled in Table 11, to enable comparison with those of its afuco analog 3. The chemical shift of the anomeric-proton signal of Fuc (6 4.877) deviates considerably from that in the spectrum of 2, reflecting the 4,6disubstitution of GlcNAc-1. However, the H-5 and CH3-group signals of this Fuc group occupy essentially the same positions as for 2 (see Table I). Therefore, this set of chemical-shift values of H-1, H-5, and the CH3-group protons may be considered to be characteristic for a Fuc group a-(1+6)-linked to the /3-GlcNAc-1 residue (see later). In turn, the attachment of Fuc to 0-6 of GlcNAc-1 has an interesting influence on the chemical shifts of the H-1 (A6 0.072 p.p.m.) and the N-acetyl protons (A6 0.019 p.p.m.) of GlcNAc-2 as compared to 3. The spectral region wherein the sugar-skeleton protons are found is unaltered in comparison to that for 3.The presence of Fuc in 4 makes the bulk more complex. The methylene resonances of Asn are found at 6 2.934 and 2.871, whereas H-a of the amino acid is observable at 6 3.987, probably due to a small difference in the pD of the solutions of 3 and 4. Compound 5 can be isolated from the urine of patients with mannos i d o ~ i s . ~The ~ , ~500-MHz, ~ , ~ ~ 'H-n.m.r. spectrum of the reducing trisaccharide is given in Fig. 5, and the spectral parameters are listed in Table 111. The observed spectrum of 5 is a superposition of the spectra of the two anomeric forms of this trisaccharide, occurring, under the n.m.r. measuring-conditions applied, in the ratio of C U : = ~ -2: 1. The effect of anomerization is clearly recognizable from the structural-reporter-group signals of the three constituent monosaccharides. (60) L. Dorland, J. Haverkamp, B. L. Schut, J. F. G . Vliegenthart, G. Spik, G. Strecker, B. Foumet, and J. Montreuil, FEBS Lett., 77 (1977) 15-20. (61) H. van Halbeek, L. Dorland, G . A.Veldink, J. F. G . Vliegenthart, G. Strecker, J.-C. Michalski, J. Montreuil, and W. E. Hull, FEBS Lett., 121 (1980) 71-77.

J. F. G . VLIEGENTHART et (11.

228

p -Man-(l--)-B o-Map-(l-S)

/

=

-GlcNAc-(l-4)-8 2 o-Fuc-(l-6) /

-GIcNAc-(l--N)-Asn l

4

I

4

NAc -CH3 protons

I

ii

Man H-2

anomeric protons I

atoms

m - - .

UI

50

10

LO

-4

u

d

*nbW

" *n D

____ ___ . ~. d

Fuc -CHs protons

-

0

I0

A,"""

6-

-f

20

I1

7 -

I I2

FIG.4.-(a) Overall, W M H z , 'H-N.m.r. Spectrum of Compound 4. (b) Expanded, Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 4. [The bold numbers in the spectra refer to the corresponding residues in the structure. The relative-intensity scale o f t h e expanded, N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance has been omitted from the expanded spectrum; its position is indicated by an arrow.]

The H-1 signals of the a! and /3 anomer of GlcNAc-2 are found at 6 5.209 and 4.718, respectively. The intensity of these signals reflects the anomeric ratio. The same ratio is found for the signals of the N-acetyl group of GlcNAc-2, at 6 2.043 (a!anomer) and 2.041 (/3 anomer), respectively.

229

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES cr-Man-(l- 3) 4

\p

-My-(l--t4)-GFNAc 5 2 NAC

-CH3 protons

-

-~ ~-

7-

4

-

Man H-2 atoms

3

20

3,a 4 P

4

2#

0

I I

L-2

1

NAc -CH3 protons 2p

I

anomeric protons

I-

7-

3

I

Man H-2 atom8

q

4.0

'!,.I 51

-

50

48

16

'

42

I---!

20

'

FIG.5.-(a) Overall, 500-MHz, 'H-N.m.r. Spectrum of Compound 5. (b) Expanded, Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 5. [The bold numbers in the spectra refer to the corresponding residues in the structure. Signals of corresponding protons in the a and /.? anomer of 5, occurring in this anomeric mixture in the ratio of 2: 1,coincide, unless otherwise indicated. The relative-intensity scale of the expanded, N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated.]

The H-1 atom of M a n 3 resonates at 8 4.787 for the a anomer of 5, whereas the chemical shift of this H-1 atom for the p anomer of 5 is 4.783 p.p.m. The latter value deviates from those of the corresponding H-1 atoms of the glyco-asparagines 3 and 4 (see Table XI) (GlcNAc-2 p-linked to GlcNAc-1). This effect is due to the a-(1+3) substitution of

J . F. G. VLIEGENTHART et al.

230

TABLE I11 'H-N.m.r. Data for Structural-reporterGroups of Oligosaccharide 5, aMan( 1+3)JPMan(ld4)GlcNAc 4 3 2

Schematic structure

' . , Reporter group

Residue

Anomer of compound

H-1 of

2

Q

P 3

Q

P H-2 of

4 3

Q

,P

Q

P 4

Q

P NAc of

2

Q

P

Chemical shift (p.p.m.)

Jl,* (H4

5.209 4.718 4.787 4.783 5.111 4.244 4.233 4.075 4.071 2.043 2.041

3.5 8.1 0.9 1.0 1.8 0.9 1.0 1.8 1.7 -

-

If, for a certain proton, IA6,-,l s 0.001 p.p.m., only an average 6 value has been listed in this and other Tables in this article, as such a difference in chemical shift due to anomerization, if any, is not detectable at 500 MHz, unless the lines of the signal are extremely sharp.

Man3 (see later). The doublets for this H-1 for both the a andP form of 5 are well resolved, reflecting the relatively large J1,2value (0.95 Hz) for this compound. For the H-2 atom of Man-3, this largeJIs2value leads to two doublets of doublets, at 6 4.244 (a anomer of 5 ) and at 6 4.233 (jI anomer of 5), respectively, instead of to two doublets having relatively broad lines. Nevertheless, the pattern of the H-2 resonance of an a-linked Man residue is clearly distinguishable from that of a p-linked one. The signal at 6 5.111 is ascribed to H-1 of Man-4. The value of its coupling constant 1.8 Hz) indicates an a-linkage between the two Man residues. The influence of anomerization on the chemical shift of H-2 of the nonreducing-end sugar, Man-4 (a 4.075 and 4.071 for the a and jI anomer, respectively) is apparent. The chemical shifts of H-1' and H-2 of Man-4 in the p anomer of 5 differ from those of H-1 and H-2 of Man-4' in 3 and 4. Obviously, the type of linkage of the terminal Man residues [a-(1-+3)or a-(1-+6), respectively] has a large effect upon the chemical shifts of their H-1 and H-2 atoms. Especially, the chemical shift of H-2 of M a n 3 reflects the position of substitution of 3

5

p -GlcNAc- (l-a)-a-Man-( 5

1-

3)

4

-Man-(1-

\j3

/

p -GlcNAc -(l-2)-a-Man-(l5'

6)

41

'

-CH3

I)-GpNAc

=

6

anomeric protons

*

\

I HOD

5

+

S

2,,>

4

4.

3.0

II

3.p

-.._

I

.I

52

-6

50

II II

I!

40

Man H-2 atoms

46

t

I

R

44

I ,

42

#

FIG. 6.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 6. p h e bold numbers in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the a and p anomer of 6, occurring in this anomeric mixture in the ratio of 2: 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum, as indicated. In addition to 6, the sample contained a small proportion of oligosaccharide 14, as can be inferred from the signals marked by asterisks (compare Fig. 15).]

TABLE1V

'H Chemical Shifts of Structural- reporter Groups of Constituent Monosaccharides for Asialo-agalacto Di-, and Asialo Di-, Tri-, Tri'-, and Tetra-antennary Oligosaccharides (Compounds 6, 7, and 10-12) Compound and schematic structure h,

W N

6

Reporter group

Residue

Anomer of compound

H-1of

2

ff

P 3

ff

P 4

a

P 4'

ff

P 5 5'

ff7P

%P

5.213 4.724 4.779 4.770 5.119 4.919 4.558 4.558

7

5.212 4.721 4.775 4.765 5.123 5.121 4.927 4.930 4.582 4.582

10

5.211 4.723 4.767 4.757 5.118 4.923 4.925 4.569 4.584

11

12

5.206 4.721 4.776 4.770 5.133 5.131 4.874

5.205 4.719 4.768 4.761 5.131 5.129 4.870

4.585 4.592

4.572 4.592

4.468 4.471 4.473

6 6' 7 7' H-2 of

8 8' 3

4

N

W

H-3 Of

4' 4

NAc of

4' 2

W

5 5' 7 7'

4.468 4.471 4.473 4.546

-

-

4.463

4.257 4.247

4.259 4.249

4.223 4.212

4.189

4.193

4.218

4.108

4.114

4.114 4.050 4.048 <4.0 2.060 2.057 2.050 2.048 2.046 2.078

C4.0

~4.0

<4.0 2.059 2.055 2.054

<4.0 2.060 2.057 2.052 2.048 2.046

2.052

4.469 4.469 4.471

-

4.555

-

-

4.481 4.263 4.252 4.201 4.198 4.099 ~4.0 C4.0 2.060 2.057 2.057 2.043 2.046

-

2.039 2.040

4.469 4.469 4.471 4.547 4.554 4.464 4.480 4.214 4.203 4.228 4.224 4.099 4.056 4.053 <4.0 2.059 2.056 2.054 2.041 2.045 2.079 2.038 2.040

234

J . F. G. VLIEGENTHART et al.

in the case of mono-substitution by another Man residue: 6 -4.24 for

5, and 6 -4.08 for 3 and 4. Concerning the remaining skeleton-protons of the three residues of 5, it is very probable that, due to anomerization, doubling of signals from protons other than those from structural-reporter groups occurs; however, this could not be traced in the bulk. Compound 6 can be isolated@from the urine of patients with Sandhoff's disease (GM,-gangliosidosis variant 0). The 500-MHq 'Hn.m.r. spectrum of this reducing oligosaccharide is depicted in Fig. 6; its spectral parameters are compiled in Table IV. Compound 6 contains the 3,6-disubstituted Man-3, characteristic for diantennary struetures. Each branch is terminated with a p-(l-*2)-linked GlcNAc residue. As described for compound 5, subspectra of the two anomeric forms constitute the spectrum observed. Anomerization comes to expression in the structural-reporter-group signals of GlcNAc-2 and Man-3. The H-1 atoms of the two a-linked Man residues resonate at 6 5.119 (Man4)and 6 4.919 (Man-4'), respectively. The H-1 signal of Man-4 shows broader lines than that of Man-4'; this may reflect the effect of anomerization, although other influences cannot be excluded. Comparison with compounds 3 and 5 reveals that the substitution at 0 - 2 of Man-4 and -4'by GlcNAc residues causes only small increments in shift for their anomeric protons. However, these substitutions give rise to significant shift-increments for the H-2 atoms. Furthermore, the 6 values of H-1 and H-2 of Man-3 are significantly influenced by these substitutions (A6 - 0.008 and + 0.014 p.p.m., respectively). As will be shown later, for compound 18, the latter effects are attributable to the attachment of GlcNAc-5' rather than of -5. The signals of the H-1 atoms of GlcNAc-5 and -5' are found at 6 4.558. The complexity of the latter signal(s) is probably due to differences in the chemical shifts of the H-1 atoms in the (Y and p anomer of 6. The @type of the linkages between GlcNAc and Man is evident from the J,,* value (8.6 Hz). The N-acetyl signals of the amino sugars of 6 are well separated; for GlcNAc-2, two singlets are observed, at 6 2.059 and at 2.055, respectively corresponding to the (Y and p anomer of 6. The difference in peak height of the other two N-acetyl signals does not reflect an intensity ratio of 2: 1, due to anomerization. In fact, this difference results from a variation in line width. Therefore, each signal belongs to one (62) G . Strecker, M.-C. Herlant-Peers, B. Foumet, J. Montreuil, L. Dorland, J. Haverkamp, J. F. G . Vliegenthart, and J.-P. Farriaux, Eur. J . Biochem., 81 (1977) 165171.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

235

GlcNAc residue. Their assignment is based on spectra of more-complex compounds (for example, 7,10,25, and 26). Compound 7 is a so-called diantennary oligosaccharide; it can be isolated from the urine of patients suffering from GM,-gangliosi~ o s ~ s ,as~well ~ . as~ from64,65 ~ - ~ ~Morquio syndrome type B. The 500MHz, 'H-n.m.r. spectrum of 7 is presented in Fig. 7, and the chemical shifts of the structural-reporter groups are summarized in Table IV. As compared to the corresponding agalacto compound 6, the presence of Gal residues 6 and 6' introduces a change in the chemical shift of the H-1 atoms of the peripheral GlcNAc residues 5 and 5' from 6 4.558 to 4.582. However, this extension has virtually no effect upon the chemical shifts of the H-1 atoms of Man residues 3,4,and 4'. Interestingly, the H-1 atom of Man-4 and also that of Man-4' each yields two separated doublets which must be ascribed to the two anomeric forms of 7. From the relative intensities of these doublets, it may be concluded that the anomers of this octasaccharide occur in the ratio of a :p = 2 : 1.Apparently, the chain length does not affect the anomericequilibrium constant. The anomeric protons of Gal-6 and -6' resonate at 6 -4.47. Three doublets are observed; J1,2of each of these has a value of 7.9 Hz, indicating p bonds between Gal and GlcNAc. The highest-field doublet, at 6 4.468, representing one proton, is attributed to Gal-6. This assignment is based on spectral data for monosialo diantennary structures (compounds 27 and 28). Consequently, the two lower-field doublets, at 6 4.471 and 4.473, which together represent one proton, belong to H-1 of Gal-6' in the a and p anomer of 7, respectively. Doubling of the signals, as observed for the H-1 atoms of Gal-6' and Man-4 and Man-4', also appears for the H-1 and H-2 atoms of Man-3 and for H-1 of GlcNAc-2. No doubling is detectable for the anomeric signal of Gal-6, nor for the H-1 signals of GlcNAc-5 and -5'. However, a possible doubling of the latter signals cannot be completely excluded due to the relatively broad lines. The total pattern of the H-2 signals of Man-3, -4, and -4' is essentially identical to that for 6. (63) E. G. Brunngraber, in Ref. 51, pp. 135-149. (64) H. van Halbeek, L. Dorland, G. A. Veldink, J. F. G. Vliegenthart, G. Strecker, J.-C. Michalski, J. Montreuil, and W. E. Hull, Eur. J . Biochern., (1983)submitted for publication. (Ma) K. Yamashita, T. Ohkura, S. Okada, H. Yabuuchi, and A. Kobata,J. Biol. Chern., 256 (1981)4789-4798;T.Ohkura, K. Yamashita, and A. Kobata, ibid., 256 (1981)

8485-8490. (65)J.-C. Michalski, G. Strecker, H. van Halbeek, L. Dorland, and J. F. G. Vliegenthart, Carbohydr. Res., 100 (1982)351-363.

5

-CH3 protons

-Gal-(1-4)-fi 0

-GlcNAc-(l-2)-cr-Man-(l- 3) S

4

p -&,I(1- 4)- fi -GI:NAc - (1 0

-+

2)-n-Man- (1-

5

6)

4'

7

anomeric protons

I

c. I c. I

d

.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

237

The N-acetyl signals of GlcNAc-5 and -5' were assigned by comparison with compounds 27 and 28; only the N-acetyl signal of GlcNAc-5' undergoes doubling. These results demonstrate that there is a large difference in the effect of anomerization on the upper- and the lower-branch signals; the effect is far more pronounced for the lower branch. Compound 8 is a diantennary, asialo glycopeptide that can be isolated from various desialylated glyc~proteins.'-~The 500-MHz, 'Hn.m.r. spectrum of this nonreducing compound, obtained from a,-acid g l y c o p r ~ t e i n , ~ -is' ~given in Fig. 8, and the n.m.r. parameters are listed in Table V. Comparison of the spectrum of 8 with that of 7 demonstrates that, for the glycopeptide, the spectrum is less complex, despite the additional residues. The additional, H-1 signal of GlcNAc-1 is found at 6 5.094. This value is similar to the corresponding values for compounds 1-4. Although the resonance position of the anomeric proton of GlcNAc-l can vary considerably (5.05-5.10 p.p.m.),6' due to differences in the pD of the glycopeptide solution and in the amino acid composition of the peptide moiety, the influence of the presence of Lys is only small. A similar phenomenon is observed for the N-acetyl signal of GlcNAc-1; its chemical shift may varye7from 2.004 to 2.014 p.p.m. Despite these variations, the signals can be used for identification purposes, because they occupy unique spectral-positions. The chemical shift of H-1 of GlcNAc-2 is not sensitive to the aforementioned influences; this proton resonates at S 4.616, which is in perfect agreement with its position in the spectrum of glyco-asparagine 3. The substitution pattern of the Man residues 3,4,and 4' is reflected in the chemical shifts of their H-1 and H-2 atoms (compare, compounds 6 and 7; see Table VI). The H-1 signals of GlcNAc-5 and -5' coincide at S 4.582, whereas the H-1 signals of Gal-6 and -6' are distinct from each other. The assignment of the Gal H-1 doublets is based on the spectrum of a diantennary, monosialo glycopeptide (31), and is consistent with that for oligosaccharide 7. (66)H. van Halbeek, L. Dorland, J. F. G . Vliegenthart, K. Schmid, J. Montreuil, B. Foumet, and W. E. Hull, FEBS Lett., 114 (1980) 11-16. (67) B. Foumet, J. Montreuil, G. Strecker, L. Dorland, J. Haverkamp, J. F. G. Vliegenthart, J. P. Binette, and K. Schmid, Biochemistry, 17 (1978) 5206-5214. (68) L. Dorland, J. Haverkamp, J. F. G. Vliegenthart, B. Fournet, G. Strecker, G. Spik, J. Montreuil, K. Schmid, and J. P. Binette, FEBS Lett., 89 (1978) 149-152. (69) K. Schmid, J. P. Binette, L. Dorland, J. F. G. Vliegenthart, B. Foumet, and J. Montreuil, Biochim. Biophys. Acta, 581 (1979) 356-359. (70) L. Dorland and J. F. G. Vliegenthart, in R. Balian, M. Chabre, and P. F. Devaux (Eds.), Membranes and lntercellular Communication (Les Houches, 1979), NorthHolland, Amsterdam, 1981, pp. 183-192.

C

q?

A

N

C.

$'

?

N

!

I N

1

c I

i

f

u

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

239

TABLEV

'H Chemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for Asialo Di-, Tri-, and Tetra-antennary Glycopeptides of the N-Acetyllaetosamine Type (Compounds 8,9, and 13) Compound and schematic structure

8

Reporter group H-1 of

Residue 1 2 3 4 4' 5 5' 6

6'

H-2 of

7 7' 8 8' 3 4

4' H 3 of NAc of

a

4 4' 1 2 5 5' 7 7'

>;:

s:;: G Thr W

>A?n

5.094 4.616 4.765 5.121 4.928 4.582 4.582 4.467 4.473

4.246 4.190 4.109 <4.0 ~4.0 2.004 2.079 2.050 2.046

-

5.092 4.614 4.755 5.120 4.924 4.570 4.580 4.468 4.473 4.545

-

4.462

-

4.209 4.218 4.108 4.045 <4.0 2.003 2.078 2.048 2.015 2.075

-

5.053 4.614 4.757 5.129 4.868 4.573 4.596 4.4 70 4.472 4.547 4.553 4.465 4.481 4.210 4.224 4.092 4.052 <4.0 2.008 2.078 2.054 2.042" 2.079 2.0110

Assignments may have to be interchanged.

The spectrum of 8 shows four, separated, N-acetyl signals, at 6 2.004 (GlcNAc-1, see earlier), 2.046 (GlcNAc-5'), 2.050 (GlcNAc-5), and 2.079 (GlcNAc-2), respectively. The assignments of the N-acetyl signals of 1and 2 were made with the aid of the spectrum of compound 3, and those of the signals of 5 and 5' on the basis of specific shift-increments, introduced by the presence of sialic acid in the monosialo compound 31 (see also, Ref. 66).An interesting, spectral feature is the line width of the N-acetyl signals and of some anomeric-proton resonances

240

J. F. G. VLIEGENTHART et al.

in relation to the position of the corresponding monosaccharide in the carbohydrate chain. The line widths of the H-1 doublet and the N-acetyl singlet of GlcNAc-1 vary upon changing the pD of the glycopeptide solution; consequently, this parameter is of only limited value for the identification of this residue. The doublet of H-1 and the N-acetyl signal of GlcNAc-2 show relatively broad lines in comparison to those of residues 5 and 5', which is ascribed to the rigidity of the intact, core structure of trimannosyl-N,N'-diacetylchitobiose. The line widths of the Gal H-1 signals are small compared to those of the H-1 doublet of the peripheral GlcNAc residues 5 and 5'. This observation is in accord with the relatively large mobility of terminal residues. The H-1 signals of GlcNAc-5 and -5' in the spectra of compounds 7 and 8 show broader lines than those in the spectrum of compound 6 (see Fig. 6), wherein GlcNAc-5 and -5' occupy terminal positions. The somewhat narrower lines of the H-1 doublet of Man-4' compared to that of 4 may indicate that the conformational freedom of Man-4', occurring in a-(1-+6)linkage, is larger than that of Man-4, which is present in an m(1-3) linkage. This may ultimately lead to differences in mobility for the upper and lower branches. Compound 9 is a triantennary, asialo glycopeptide that can be isolated from various desialylated glyc~proteins.'-~The 500-MHz, 'Hn.m.r. spectrum of 9, derived from a,-acid g l y ~ o p r o t e i n , is ~ -given ~ ~ in Fig. 9, and the n.m.r. parameters are compiled in Table V. The spectral features of this glycopeptide bear a high similarity to those of compound 8 (see Table V). The presence of the third N-acetyllactosamine unit gives rise to additional signals for the anomeric protons of GlcNAc-7 (at 6 4.545) and of Gal-8 (at 6 4.462), and to an extraN-acetyl signal, at 6 2.075, belonging to GlcNAc-7. The presence of the additional branch causes some specific changes in the chemical shifts of the H-2 atoms of Man-3 and -4 in comparison to those of 8. The H-2 signal of Man3 undergoes an upfield shift (A6 -0.037 p.p.m.), whereas that of H-2 of Man-4 shifts downfield (A6 0.028 p.p.m.). These effects are rather large, so that their relative posi-

FIG.9.-Shuctural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 9. [The bold numbers in the spectrum refer to the correspondingresidues in the shucture. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum,as indicated. The HOD resonance, as well as the H-1 signal of Man4 (which, at 300 K, is almost hidden under this line), have been omitted from the spectrum; their position is indicated by an arrow.]

0

ul c

0 :

a~ I

= m

Y

T

09

,...be b

4

i

$:

242.22

J. F. G. VLIEGENTHART et al.

tions are interchanged. The spectral resolution at 360 MHz is not sufficient to permit observation of these H-2 signals ~ e p a r a t e l y Fur.~~ thermore, an upfield shift is observed for H-l of Man-3 (A6 -0.010 p.p.m.) as compared to 8. The set of chemical shifts for the H-1 and H-2 atoms of the Man residues are characteristic for triantennary, asialo glycopeptides (see Table VI). Another interesting feature is the appearance of the H-3 signal of Man-4 at a position that is well resolved from the bulk of the skeleton protons. The H-1 signal of GlcNAc-5 shifts upfield (A6 -0.012 p.p.m.) and undergoes a significant line-broadening upon substitution of Man-4 with N-acetyllactosamine in a p-( 1+4) linkage. The chemical shifts and the line widths of the other GlcNAc anomeric signals (1,2, and 5’) remain unchanged with respect to 8. Attachment ofthe third branch causes only slight differences in the chemical shifts of the N-acetyl signals of GlcNAc residues 1,2,5, and 5‘ in comparison to 8, but the small decrement in shift for GlcNAc-5 (A8 - 0.002 p.p.m.) is typical (see later). The assignment of the N-acetyl signal at 8 2.075 to GlcNAc-7 is in agreement with the line width of this singlet, because a peripheral residue will give rise to a sharper line than an internal residue (GlcNAc-2: 6 2.078). Compound 10 is a triantennary, asialo oligosaccharide that has been isolated from the urine of a patient with G M , - g a n g l i o s i d o ~ i s . ~ ~ ~ ~ ~ * ~ ~ The 500-MHz, ‘H-n.m.r. spectrum of 10 is given in Fig. 10, and the n.m.r. parameters are summarized in Table IV. For spectral interpretation, 10 may be conceived of either as an extension of oligosaccharide 7, with an N-acetyllactosamine unit linked to Man-4, or as compound 9 lacking the pGlcNAc( l-+N)Asn-Lys moiety. The attachment of the third N-acetyllactosamine unit to Man-4 of the diantennary oligosaccharide 7 has the same influence upon the structural-reporter groups of its p anomer as was described for the extension of 8 to 9. A difference between the chemical shifts of H-1 of Man-3 for the a and /3 anomer of 10 is clearly visible. This effect is somewhat less pronounced for Man-4‘ in comparison to the spectrum of 7 (see Fig. 7). The doublets ofthe H-1 atoms of GlcNAc-5, -5’, and -7 are well separated, showing a total pattern similar to that observed for the correFIG.10.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, ‘ H 4 . m . r . Spectrum of Compound 10. [The bold numbers in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the (Y and p anomer of 10, occurring in this anomeric mixture in the ratio of 2: 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region (see insertion)differs from that of the other part of the spectrum, as indicated. The HOD resonance has been omitted from the spectrum; its position is indicated by an arrow.]

9

2

N-

in’

1

244

J. F. G. VLIEGENTHART et al.

sponding signals of 9. Among the anomeric protons of the Gal residues 6,6’, and 8, only H-1 of Gal-6’ gives rise to two doublets (due to anomerization). The substitution of the mannotriose branching core by three N-acetyllactosamine units can be recognized on the basis of the chemical shifts of the H-2 signals of the three Man residues (see Table VI). Owing to anomerization, two signals are observed for H-2 of Man-3. The complexity of the H-2 signals of Man-3 and -4, in comparison to 9, is caused by partial overlap of the H-2 signal of Man-3 of the a anomer with the H-2 signal of Man-4 of both anomers of 10. As discussed for 9, H-3 of M a n 4 can be conceived of as being a structural-reporter group typical for the presence of the 7-8 branch. Its signal is doubled; the intensities reflect the anomeric ratio. The pattern of the N-acetyl proton signals of 2,5, and 5’ is identical to that of oligosaccharide 7.As described for 9, the attachment of the third branch to 0-4of Man-4 induces a shift decrement of the N-acetyl signal o f 5 (AS - 0.002 p.p.m.)- The N-acetyl signal of GlcNAc-7 has a relatively narrow line-width (compare 9, Fig. 9). The chemical shifts of the signals of the structural-reporter groups of the additional N-acetyllactosamine unit are identical for the a and f3 anomer of 10. Compound 11has been isolated from the urine of patients suffering from G M , - g a n g l i o s i d ~ s i s or ~ ~f* i~ *~~ m ~ Morquio ~ , ~ ~syndrome type B. This reducing decasaccharide is isomeric with oligosaccharide 10. It contains three N-acetyllactosamine units, the third unit being attached to 0-6 of Man-4’. This structure is denoted as a tri‘-antennary oligosaccharide. In the 500-MHz, ‘H-n.m.r. spectrum of 11 (see Fig. ll),the presence of the third N-acetyllactosamine unit p-( 1-+6)-linkedto Man-4’ is revealed in the pattern of the H-1 and H-2 signals of the Man residues (see Table IV). In comparison to 7,relatively large shift-decrements of the H-1 (A8 - -0.054 p.p.m.) and H-2 signals (A8 -0.015 p.p.m.) of Man-4’ are observed (see Table IV), whereas the chemical shifts of H-1 and H-2 of Man-4 undergo smaller, but significant shift-increments (AS 0.010 and 0.007 p.p.m., respectively). The positions of the H-1 and H-2 signals of Man-3 remain unchanged. The third N-acetyllactosamine unit gives rise to anomeric-proton signals at 8 4.555 (H-1 of GlcNAc-7’) and at 8 4.481 (H-1 of Gal-8’). The FIG. 1 l.-Structural-reporter-group Regions of the Resolution-enhanced, W - M H z , ‘H-N.m.r. Spectrum of Compound 11. [The bold numbers in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the rr and p anomer of 11, occurring in this anomeric mixture in the ratio of 2: 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum, as indicated.]

246

J. F. G . VLIEGENTHART et al.

attachment of the pGal( 1-4)GlcNAc moiety to 0-6 of Man-4' causes a downfield shift of the H-1 signal of GlcNAc-5' (A8 0.010 p.p.m.) and a considerable line broadening of this doublet, when compared with 7 (compare with the chemical shift and line width of the H-1 signal of 5 in 10). The pattern of the N-acetyl proton signals is rather complex. The signal at 6 2.060is attributed to the a! anomer of GlcNAc-2, and that at 8 2.057 belongs partially to the p anomer of GlcNAc-2. The remaining part of the latter singlet belongs to GlcNAc-5 (compare with 7 and 10, see Table IV). Furthermore, four signals occur in the relatively highfield part of this spectral region, at 6 2.039, 2.040, 2.043,and 2.046, with the relative intensity ratios of 2 :1 :2 :1. Therefore, they can be divided into two pairs, the intensities within each pair reflecting the anomeric ratio. The former pair of signals, separated by 1A6,+ I 0.001 p.p.m., stems from GlcNAc-7' in the a and /3 anomer of compound 11, respectively. The latter pair of singlets, having 1A6,+ I 0.003p.p.m., i s ascribed to GlcNAc-5' in the a! and p form of 11 (see also, Fig. 14b). The assignment is based on the more pronounced influence of the anomeric configuration of GlcNAc-2 on the chemical shifts of the structural-reporter groups of the 5'-6' branch, in comparison to those of the 7'-8' branch. Although the influence of attachment of the third N-acetyllactosamine unit, to 0-6 of Man-4', on the chemical shift of the N-acetyl singlet of GlcNAc-5 (A8 0.005 p.p.m.) might be unexpected, it will later be shown that it is consistent with that for comparable extensions. The overall effects of the 7'-8' branch make it tempting to suggest that this branch is in close proximity to the 4-5-6 branch, whereas the anomerization effects suggest that the 4'-5'-6' branch is in the sphere of influence of GlcNAc-2. Compound 12 is a tetra-antennary oligosaccharide that wa? isolated from the urine of a patient with GM1-gangliosidosis.52."4.64a This structure can be conceived of as an extension of the tri- (compound 10)or tri '-antennary (compound 11) oligosaccharide with an N-acetyllactosamine unit. The 500-MHz, 'H-n.m.r. spectrum of 12 is presented in Fig. 12, and its n.m.r. data are given in Table IV. ~

~~

~

~~

FIG. 12.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, IH-N.m.r. Spectrum ofCompound 12. [The bold numbers in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the Q and p anomer of 12, occurring in this anomeric mixture in the ratio of 2 : 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum, as indicated. The HOD resonance, as well as the H-1 signal of M a n 3 for the a anomer of 12, have been omitted from the spectrum; their position is indicated by an arrow.]

d

w-

i

N

(D

P

2

2!

L

.-0 4

0

F

L

I

'-1

248

J. F. G . VLIEGENTHART et al.

For the spectral interpretation, the spectrum of the triantennary oligosaccharide 10 is used as a reference. The attachment of the N-acetyllactosamine unit in 8-(1+6) linkage to Man-4' does not influence the chemical shifts of the H-1 and H-2 signals of Man-3 in the spectrum of the a and fl anomers of the oligosaccharide. Shift increments are observed for the H-1 and H-2 signals of Man-4 (A6 0.012 and 0.008 p.p.m., respectively), and shift decrements for Man-4' (A6 -0.054 and -0.015 p.p.m., respectively). The complete set of chemical shifts of the H-1 and H-2 signals of the Man residues reflects the substitution pattern of the mannotriose branching core. The H-3 signal of Man-4 is unaffected by this substitution. The signals of the anomeric protons of GlcNAc-5 and -7 resonate at the same positions as for 10. The H-1 signals of GlcNAc-5' and -7' are found at 6 4.592 and 4.554, respectively. The anomeric-proton signals of the four terminal Gal residues are all separated, and could be assigned. The signals of Gal-6, -6', and -8 are found at positions similar to those for 10, and the additional doublet at S 4.480 is ascribed to Gal8'. The pattern in the N-acetyl region of the spectrum of 12 is similar to that of 10 with respect to the signals of GlcNAc-2, -5, and -7. The four remaining singlets, at 6 2.045,2.041,2.040, and 2.038, are ascribed to GlcNAc-5' in the 8 and a anomer of 12, and to GlcNAc-7' in the @ and a anomer of 12, respectively. This assignment is in accordance with that for oligosaccharide 11 (see Table IV and Fig. 14b). Considering 12 as an extension of 11, an analogous reasoning can be applied for the interpretation of its spectrum. From the latter step, the effects of introduction of GlcNAc-7 and Gal-8 (for example, the characteristic shifts for the H-2 signals of Man-3 and -4, and for the N-acetyl signal of GlcNAc-5) can be derived. It turns out that, starting from the diantennary oligosaccharide 7,the influences of attachment of the third and fourth N-acetyllactosamine unit are independent and additive. The doubling of signals due to anomerization, as observed for 7, remains unaltered upon extension to tri-, tri'-, and tetra-antennary structures. The newly introduced N-acetyllactosamine units do not show doubling of signals (except the N acetyl signal of GlcNAc-7'), indicating that they are beyond the sphere of influence of the anomeric center of GlcNAc-2. Compound 13 is one of the tetra-antennary, asialo glycopeptides, isolated from asialo a,-acid glycoprotein,"-68 that differ only in amino acid composition. It may be conceived as oligosaccharide 12 having an extension of pGlcNAc(l+N)Asn-Gly-Thr. The 500-MHz, 'H-n.m.r. spectrum of 13 is given in Fig. 13a, and the n.m.r. parameters are shown in Table V.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

249

The presence of GlcNAc-1, N-glycosylically linked to Asn, is evident from the doublet of its H-l atom at 6 5.053 and the singlet of its N-acetyl protons at 6 2.008. The resonance positions of these protons of GlcNAc-1 are influenced by the structure of the peptide moiety, as already mentioned (compare 8 and 9; see Table V). In order to illustrate this influence once again, the spectrum of an analog of 13, ending with PGlcNAc(l+N)[Thr-Pro-lAsn-Lys, is given for comparison in Fig. 13b [GlcNAc-l,6 5.063(H-1),6 2.011 (NAc)]. The H-1 and N-acetyl signals of GlcNAc-2 are found at the same positions as described for the triantennary glycopeptide 9, indicating that GlcNAc-2 is linked p-(1-*4) to GlcNAc-1 (see Table 11). The resonance positions of all other structural-reportergroups of 13, namely, the H-1 doublets of residues 3 to, and including, 8 and 8', the Man H-2 signals, the H-3 signal of Man-4, and the N-acetyl singlets of the peripheral GlcNAc residues, remain unchanged as compared to those of the /3 anomer of the tetra-antennary oligosaccharide 12.It may TABLEVI Chemical-shill Data for the Mannose H-1and H-2Atoms in Asialo Di-, Tri-, and Tetra-antenuary Glycopeptides, and in Asialo Di-, Tn-, TIT-, and Tetra-antennary Oligosacchauides Chemical shift (6) of

H-1 of Man residue

8 2 of Man residue

3

4

4'

3

4

4'

4.765 4.775 4.765

5.121 5.123 5.121

4.928 4.927 4.930

4.246 4.259 4.249

4.190 4.1w

4.109 4.114

4.755 4.767 4.757

5.120

4.924

4.209

4.218

4.108

5'118" 4*923 4.925

4*223 4.212

4.218

4.114

4.776 4.770

5.133 5.131

4.263 4.252

4.201 4.198

4.099

4'874

4.757 4.768 4.761

5.129 5.131 5.129

4.868 4.870

4.210 4.u)3 4.214

4.224 4.228 4.224

4.092 4.099

Schematic Compound

structure

--->... 8 7

Diantenna

a

p

...

Triantenna

?F

9 10

a

fl

...

Tri'-antenna

11

-7-

a

p

...

...

Tetra-antenna

..~

13 12

a

p

... ... ~

a

See footnote to Table 111.

P

250

0

m-

8-

5' 7'

1

I anomeric

x IIL

6 " "6

orotons

d

I

-

OD

? ?

Man H-2 atoms

B

4 ,

4

1

Am H-a

- 6

50

I

3

4

1

i

20

22

48

LYS H - R

46

LL

I/

42

40

FIG.13b.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 13 (peptide moiety: Thr-Pro-Asn-Lys). [The bold numbers in the spectra refer to the corresrmnding residues in the structures. For both spectra, the relative-intensity scale of the N-acetyl-proton region (see insertions) differs from that of the other part as indicated. The HOD resonance, as well as the H-1 signal of Man-3, have been omitted from spectrum (a); their positions are indicated by arrows.]

Man H-3

Man H-2 atoms

3

4

4‘

4‘

4

4

A 3

3 4 Ih

4

-.

1

3-

b

NAc -CH3 protons

L2

L1

4

-8

ID

101

ZQ

1.a

~rn

ia

FIG.14a.--Characteristic Resonance-patterns of the Mannose H-2 (and H-3) Atoms, and of the 2-Acetamido-2deoxyglucose N-Acetyl Methyl Protons, in the 500-MHz, ‘HN.m.r. spectra for Di-, Tri-, and Tetra-antennary Glycopeptides of the N-Acetyllactosamine type.

FIG.14b.--Characteristic Resonance-patterns of the Mannose H-2 (and H-3) Atoms, and of the 2-Acetamido-2-deoxyglucose N-Awtyl Methyl Protons, in the W - M H z , ‘H-N.m.r. Spectra for Di-, Tri-, Tn’-, and Tetra-antennary Oligosaccharides of the N-Acetyllactosamine Type. Signals for Corresponding Protons in the a and p Anomer of Such an Oligosaccharide, Occurring in the Ratio of 2: 1, Coincide, Unless Otherwise Indicated. [The bold numbers in the Figure refer to the corresponding Man (*) and GlcNAc ( 0 )residues. The carbohydrate chains are represented by the symbolic notation, defined in Chart 1 (see also footnote on page 221).] 252

Man H-2 atoms 43

42

41

I

NAc -CH3 protons

Man "-3 ID

zm

zm

IP(

201

rn

c

4

3,a .. "-3,p 4,a

44,,

2.P

I

C

'-8

41

25 3

zio

z'm

"II

zbs

I Z'OL

zm

zm

J. F. G. VLIEGENTHART et a1

2.54

be mentioned that the chemical-shift values of the Man H-1 and H-2 signals unambiguously point to a tetra-antennary type of structure. The sets of chemical shifts of the Man H-1 and H-2 signals, which are highly characteristic for the type of branching of the N-acetyllactosamine type of carbohydrate chain of glycopeptides and related oligosaccharides, are summarized in Table VI. The typical, resonance patterns of the Man H-2 atoms for di-, tri-, tri’-, and tetra-antennary chains are compared in Fig. 14. An independent criterion for recognition of the asialo di-, tri, tri’-, and tetra-antennary structures is found in the resonance patterns of the N-acetyl-proton signals of the glycopeptides and oligosaccharides. For comparison, these characteristic patterns, which may additionally be used as “fingerprints” for recognition of the type of branching of these complex carbohydrate chains, are also depicted in Fig. 14. Interesting differences in line width for various anomeric-proton resonance5 can be observed in a series of di- (S), tri- (9),and tetra-antennary (13) glycopeptides. In particular, the introduction of the 7-8 branch causes a line broadening for H-1 of GlcNAc-5; the effect of the 7 ’ 4 ‘ branch on H-1 of GlcNAc-5’ is even more pronounced. The linebroadening efrects in a homologous series of oligosaccharides (7, 10, 11, and 12) are more difficult to define, owing to anomerization effects. Compound 14 may be isolated from the urine of patients with Sandhoffs disease (GM,-gangliosidosis variant O).62Fundamentally, it is a diantennary oligosaccharide bearing an additional, so-called intersecting GlcNAc residue (9), p-( 1+4)-linked to Man-3. The 500-MHz, ‘H-n.m.r. spectrum of 14 is given in Fig. 15, and the n.m.r. data are presented in Table VII. Comparison with the spectrum of 6 shows that the additional ~~~

~

FIG.15. -Stnictural-reporter-group Regions of the Resolution-enhanced, 500-MHz. iH-N.m.r. Spectrum of Compound 14. [The hold-face numbers in the spectrum refer to the corresponding residues in the structure. Signals of Corresponding protons in the (Y and p anomer of 14, occurring in this anomeric mixture in the ratio of 2: 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum, as indicated. In addition to 14, the sample contained a small proportion of a positionally isomeric oligosac charide that possesses GlcNAc-7 instead of -9, as follows. This can be inferred from the signals marked by asterisks. p-GlcNAc-(l-4) [3 -GlcNAc-(I-

\

2)-a-Man-(l4

3)

\ p -Man- (1-

4)-GIcNAc 2

p -GlcNAc - (I3

2)-0 -Man- (I4‘

6)

/

3

]

2.56

J. F. G. VLIECENTHART et al.

TABLEVII 'H Chemical Shifts of Structural-reporterGroups of Constituent Monosaccharides for Two Oligosaccharides(14 and 15) and a Glympeptide (16) Containing the Intersecting GlcNAc Residue (9) Compound and schematic structure 14

15

16

Reporter group

Residue

oligosaccharide

H-1 of

1 2

a

B 3

a

B 4

a

B 4'

a

B 5 5'

a$

a

6

B aB

6'

a

7 9

B a

B H-2 of

3

a

P 4 4'

H 3 of NAc of'

f f

B

a

B

4 4' I 2

5 5'

5206 4.722 4.713 4.699 5.063 5.060 4.999 5.003 4.555 455.5 4.549 -

-

4.469 4.190 4.177 4.247 4.148

a,B

<4.0

a,B

t4.0

-

B

2.059 2.056 2.059 2.052 2.050

ffB

2.068

a

P 4 a

7 9

Arg

-

5.204 4.721 4.704 4.686 5.059 5.057 5.008 5.020 4.583 4.579 4.591 4.468 4.473 4.477

-

4.466 4.468 4.190 4.175 4.258 4.148 4.142 <4.0 <4 .O

-

2.064 2.060 2.053 2.044 2.039 2.064

5.056 4.613 4. a 7 5.057 4 .w 4.537 4.545

-

4.520 4.463 4.146 4.282 4.141 4.044 <4.0 2.008 2.077 2.057 2.048 2.082 2.063

‘H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

257

GlcNAc-9 has a profound influence on the resonance positions of several reporter groups. For H-1, as well as H-2, of Man-3, two signals are observed, all four of which are shifted upfield as compared to 6 (compare Table IV). The signals of H-1 of Man4 shift to higher field (A6 - 0.057 p.p.m.), whereas the signal of H-2 of this residue shifts downfield (A6 +0.058 p.p.m.). The latter signal may be recognized very readily as belonging to Man-4, because of the specific pattern of the well resolved doublet of doublets for H-2 of an a-linked Man (see earlier). This eliminates any confusion with H-2 of Man-3, which resonates at 6 -4.25 in normal, diantennary structures (6, 7, and 8). The H-1 doublets, as well as the H-2 signal, of Man-4’ are shifted downfield. The resonance positions of the anomeric protons of GlcNAc-2, -5, and -5’ are essentially unchanged with respect to 6. The relatively sharp, H-1 doublet of GlcNAc-9 is found at 6 4.469; its J1,2 value (8.4 Hz) is indicative of a /l-glycosylic linkage. The presence of GlcNAc-9makes the subspectra of the two anomers of 14 more different than those of 6; this is especially illustrated by the relatively large differences in chemical shift for H-1 of Man-4, as well as H-1 of Man-4’, in both anomers of 14. It seems as if the steric requirements of GlcNAc-9 push the two branches towards the sphere of influence of the anomeric center of GlcNAc-2. GlcNAc-9 itself is apparently remote from this center, as no doubling of its signals is observed. In the N-acetyl region of the spectrum, the singlet at 6 2.068 is ascribed to GlcNAc-9. The a and p anomer of GlcNAc-2 give rise to two signals, at 6 2.059 and 2.056, respectively, as described for other reducing oligosaccharides. Considering the strong influence of anomerization on the chemical shifts of the structural-reporter groups of the lower branch (for example, H-1 of Man-4’ in 14), the set of signals at 6 2.052 and 2.050 is assigned to the N-acetyl-group protons of GlcNAc-5’ in the a and /3 form of 14, respectively. Consequently, the rest of the broad singlet at 6 2.059 is attributed to GlcNAc-5; this means that the presence of GlcNAc-9 causes a downfield shift (A6 0.005 p.p.m.) for this signal. Compound 15 is another diantennary oligosaccharide, having the intersecting GlcNAc-9 linked /l-(1+4) to Man-3. It has been isolated from the urine of a patient ~ i t h Morquio ~ ” ~ syndrome type B. The 500-MHz, ‘H-n.m.r. spectrum of 15 is presented in Fig. 16, and the chemical shifts of the structural-reportergroups are included in Table VII. As compared to the corresponding agalacto compound 14, Gal-6 and -6’ introduce downfield shifb of the H-1 atoms of GlcNAc-5 and -5’

2,r+9

NAc /3-%1-(1--

4)-j3-GlcNAc-(1-2)-a-Man-(l-J)

B-Ga,l-(1-

4)- B -GlcNAc-(1-

5

4

2)-a-Ma?-(146)

5

I

I

8

-CH3 protons

\

1s anomeric protons 7 6

I

non 22

Man H-2 atoms

I--7

52

-

5;P r

d

50

48

L6

1L

1 2

FIG.16.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 15. [The bold numbers in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the a and p anomer of 15, occurring in this anomeric mixture in the ratio of 2 : 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum, as indicated.]

il

‘H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

259

(compare the step from 6 to 7), whereas the chemical shift of the anomeric proton of GlcNAc-9 is essentially unaffected. However, for 15, the latter proton gives rise to two doublets. The doublet of this H-1 atom for the p anomer of compound 15 coincides with that of H-1 of Gal-6, at 6 4.468, whereas, for the a form, it is found at 6 4.466. This assignment is based on the relative-intensity ratio of the aforementioned doublets, namely, 2: 1.The H-1 resonance of Gd-6’ is also doubled, due to anomerization. This feature is in accord with the relatively strong (for example, IA6,,] 0.012 p.p.m. for H-1 of Man-4’, as well as for H-1 of GlcNAcd’), anomerization effects present on lowerbranch residues (compare 7), especially when GlcNAc-9 is present. The set of chemical shifts of the H-1 and H-2 signals of Man-3, -4, and -4’ reflects the substitution pattern of the branching point, as outlined for the agalacto analog 14. In the N-acetyl region of the spectrum, five separate singlets are observed. At 6 2.064, two signals coincide that are attributed to GlcNAc9 and to the a anomer of GlcNAc-2. The resonance at 6 2.060 stems from GlcNAc-2 in the p anomer of 15. The N-acetyl signals of GlcNAc5 and -5’ are shifted slightly upfield, as compared to 14 (compare the step from 6 to 7). The relatively large )At& I value for the N-acetyl signals of GlcNAc-5’ is worth mentioning, as it is typical for the presence of GlcNAc-9 in combination with that of Gal-6’. Starting from the spectrum of 7 as a reference, a detailed interpretation of the spectrum of 13 can also be achieved. Essentially the same characteristic influences of GlcNAc-9 may be traced along this route, as described for the step from 6 to 14. Compound 16 is a triantennary glycopeptide containing GlcNAc-9. It may be obtained by pronase digestion of chicken o v ~ t r a n s f e r r i n . ~ ~ ~ ~ ~ The de-Arg analog of 16 was also available for investigation. The 500MHz, ‘H-n.m.r. spectrum of 16 is presented in Fig. €7, and its n.m.r. data are summarized in Table VII. For the spectral interpretation, 16 is conceived as 14 extended with GlcNAc-7, as well as with pGlcNAc(1-N)Asn-Arg. The chemical shift (8 5.056) of the H-1 signal of GIcNAc-l in 16 is distinguishable from that (6 5.070) for its de-Arg analog, illustrating the effect of the peptide moiety on this residue. A similar feature occurs for the N-acetyl signal of GlcNAc-1. However, the H-1 signal and the relatively broad N-acetyl signal of GlcNAc-2 are found at 6 4.613 and 2.077, respectively, for both compounds. This shows that the influence of Arg is restricted to GlcNAc-1. (71) L.Dorland, J. Haverkamp, J. F. G. Vliegenthart, G. Spik, B. Foumet, and J. Montreuii, Eur. 1. Biochem., 100 (1979) 569-574.

1

i.r -GlcNAc - (17

\ \

-CH3 protons

0 -GlcNAc-( 1-2)-@-Man-( 15

4

3)

\

6- GIcNAc -( 1- 4) -6.

"-(1-

4)-0 -GIcNAc-(l-

4)-3! -GlcNAc-(l-

9

0 -GlcNAc-(1-

2)-0 -Man-(1-

5'

2.,

NAc

4)

8) /

N)-Asn'""'' I

A=gOOp)

3

4'

16 Ix114

anomeric protons I

+I Man H-2 atoms I

1

nI I

0-b

51)

46

46

44

42

,

40

FIG.17.-Structural-reporter-group Regions of the Resolution-enhanced, SO-MHz, 'H-N.m.r. Spectrum of Compound 16. [The hold numbers in the spectrum refer to the corresponding residues in the structure. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum, as indicated. In addition to 16, the sample contained a small proportion of a similar glycopeptide lacking GlcNAc-7 (as compared to 16), that is, a glycopeptide analog of compound 14. This can he inferred, for example, from the signals marked by asterisks.]

‘H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

261

Owing to the presence of GlcNAc-7, the H-1 signal of Man-3 is shifted upfield (A6 -0.012 p.p.m.) with respect to the H-1 signal of M a n 3 in the p anomer of oligosaccharide 14. This effect has the same magnitude as described for this signal in the steps from a di- (8) to a tri-antennary (9) glycopeptide, from a di- (7) to a tri-antennary (10) oligosaccharide, and also from a tri’- (11) to a tetra-antennary (12) oligosaccharide (compare Tables IV and V). In consequence, the presence of Gal-8 is not a requirement for the shift effect observed; it can be fully attributed to the attachment of GlcNAc-7 to Man-4 in p-( 1-4) linkage. Similarly, GlcNAc-7 is responsible for the specific changes in the chemical shifts of the H-2 signals of Man-3 and -4 in comparison to 14. The resulting set of chemical shifts for the H-1 and H-2 signals of the Man residues of 16 is characteristic for this kind of substitution of the mannotriose core. The H-1 signal of GlcNAc-5 shifts upfield (A6 - 0.018 p.p.m.) upon substitution of Man-4 with GlcNAc-7 in a p-( 1+4) linkage. Furthermore, the doublet of this anomeric proton has broader lines than the doublet of the H-1 atoms of the other terminal GlcNAc residues. This might reflect the lessened conformational freedom of GlcNAc-5, due to the presence of GlcNAc-7 (compare, for instance, Fig. 9). GlcNAc-7 also exerts some influence on the chemical shift and the line width of the H-1 signal of GlcNAc-9. In the N-acetyl region, the assignment of the signals of GlcNAc-1, -2, -5‘, and -7 is straightforward (compare 9). The signal at S 2.057 is ascribed to GlcNAc-5. It is shifted upfield (A6 - 0.002 p.p.m.) in comparison to 14, which corresponds with the known effect of attachment of GlcNAc-7 (compare 9). In consequence, the signal at 6 2.063 belongs to GlcNAc-9. Apparently, this singlet undergoes an upfield shift (A6 -0.005 p.p.m.) due to the introduction of GlcNAc-7. Comparison of 14 with 15 shows that, for the H-1 signals of GlcNAc5 and -5‘, the expected “agalacto” upfield shifts (AS - 0.025 p.p.m.) occur.)Assuming that this effect is independent of the location of the N-acetyllactosamine unit in the structure, the chemical shift of the H-1 signal of GlcNAc-7 for 16 may be derived from that for 9, correcting for the absence of Gal-8. Compound 17 is a mono-antennary, upper-branch, asialo oligosaccharide that may be regarded as an extension of trisaccharide 5 with an N-acetyllactosamine unit p-( 1+2)-linked to Man-4. This pentasaccharide has been isolated, from the urine of a patient with GM,-gangl i o s i d ~ s i s , ~in~a, mixture ~ , ~ ~ of oligosaccharides consisting of 17 and 18 in the ratio of 3:2. The 500-MHz, ‘H-n.m.r. spectrum of this mixture is given in Fig. 18a, and the n.m.r. data are compiled in Table VIII.

-

\

3

--N

u)

C

a

9

0 *

i

N

N

263

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES TABLEVIII

H Chemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for Asialo Mono-N-acetyllactosaminyl Oligosaccharides (Compounds 17-20) Compound and schematic structure

17 Reporter group

Residue

H-1 of

2

homerof compound a

P 3

a

P 4

a

P 4' 5 5' 6 6'

a

P .B ff,P ff,P

3

5.207 4.719 4.785 4.782

5.216 4.726 4.769 4.760

5.127

-

5.122

-

4.920 4.923

4.920 4.925 4.579

4.577

-

4.468 a a

P 4 4'

a

P

4.242 4.231 4.194 4.190

a

P NAcof

2 5 5'

a

P a ?P a

B

19

20

\

P H-2 of

18

2.043 2.041 2.053

-

5.213 4.725 4.788 4.779

-

5.213 4.725 4.779 4.768 5.106 5.102 4.925 4.929

-

-

4.467

4.470 4.472 4.089 4.079

4.263 4.253

4.583 4.470 4.472 4.263 4.253

4.193

4.071

4.582

4.104 4.107 2.063 2.060

-

2.048 2.047

<4.0 2.058 2.055 2.052

4.112 2.061 2.058 2.048 2.046

FIG. 18a. -Structural-reporter-group Regions of the Resolution-enhanced, 500MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 17 and 18in the Ratio of 3:2. [The bold numbers in the spectrum refer to the corresponding residues in the structures, and the italic numbers to the compounds in the mixture, each of which occurs as an anomeric mixture in the ratio of a : P = 2: 1. (Anomeric-proton signal designated as 4', 18a means: H-1 of Man-4' in the a anomer of compound 18).Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum, as indicated.]

264

J. F. G . VLIEGENTHART et al.

As with 5, the reducing character of compound 17 gives rise to two doublets for H-1 of GlcNAc-2, at 6 5.207 (J1,23.1 Hz) and 6 4.719 (J1,2 8.1 Hz), for the a and /3 anomer, respectively. For the N-acetyl protons of GlcNAc-2, two singlets are observed, also in the anomeric ratio, at 6 2.043 (a)and 2.041 (p anomer of 17).The set of chemical shifts of the H-1 doublets, in conjunction with that of the N-acetyl singlets of GlcNAc-2, is indicative of a mono-a-(l-3) substitution of Man-3 by another Man residue (compare 5 in Table 111, and 18 in Table VIII). The aforementioned chemical shifts are not influenced by the presence of the N-acetyllactosamine unit. Equally, this extension has hardly any influence on the chemical shifts and coupling constants (JIs2 andJ2,3)of the H-1 and H-2 signals of Man-3 in both anomers of the oligosaccharide. The ASCr+values (0.003 p.p.m.) for H-1 of Man-3 in the a and /3 anomers of 17 and 5 are smaller than those observed for all other related oligosaccharides (A&-,, 0.01 p.p.m., compare Tables IV and VII). The H-1 atom of Man4 resonates, as one doublet with relatively broad lines, at a position that is rather low-field for an asialo upper branch (6 5.127). The H-2 signal of Man4 appears as two doublets of doublets in the anomeric ratio. The shift increment (A6 0.12 p.p.m.) of H-2 of Man-4 in comparison to 5 reflects the substitution at 0-2 of Man-4 by GlcNAc-5, as already described for the step from 5 to 6. The single doublet of H-1 of Gal-6 is found at 6 4.468, which is in accord with the diantennary oligosaccharide 7 (see Table IV). The relatively narrow lines of this signal correspond with the terminal position of Gal-6. The doublet at 6 4.577 has relatively broad lines, and must be ascribed to H-1 of GlcNAc-5. Its resonance position deviates by 0.005 p.p.m. from that observed for this proton in the diantennary oligosaccharide 7,and turns out to be characteristic for the peripheral GlcNAc5 in an asialo, mono-N-acetyllactosamine, upper-branch oligosaccharide (see later; compound 19, Table VIII). As already described, the resonance position of the signal of the N-acetyl group of GlcNAc-5 is not influenced by anomerization of the oligosaccharide. Compound 18 is a mono-antennary, lower-branch, asialo oligosaccharide that has been isolated from the urine of patients suffering from G M , - g a n g l i ~ s i d o s i sor~ fromwp65 ~ ~ ~ ~ ~ Morquio ~ syndrome type B. The 500-MHz, 'H-n.m.r. spectrum of the relatively pure pentasaccharide is presented in Fig. 18b. The signals corresponding to 18 in Fig. 18a can readily be traced by comparison of spectra 18a and 18b. The chemical shifts of its structural-reporter groups are summarized in Table VIII. The resonance positions of the H-1 doublets of GlcNAc-2 for the a and p anomer of 18, in combination with those of the N-acetyl signals

NAc

-CH3 protons

j3 -Man-(l-+4)GlcNAc /

j3 -Gal-(l-

4)-j3 -GlcNAc-(1-

6'

a)-a-Man-(l-

6)

4'

5'

'

a

3

18 ,5;p

-

It

anomeric

f 0

protons

I

'

22

1*

It

/-

20

Man. H-2 atoms

i 4,
II

B3

II

1

5.2

- h

5.0

4.8

4.6

4.4

12

/I

FIG.18b.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 18. [The bold numbers in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the a and p anomer of 18, occurring in this anomeric mixture of the ratio of 2 : 1,coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum, as indicated. In addition to 18, the sample contained a very small proportion of 17, as can be inferred from the signals marked by asterisks.]

266

J. F. G. VLIEGENTHART et al.

of GlcNAc-2, are specific for mono-a-(l+6) substitution of Man-3. Also, the chemical shifts for H-1 and H-2 of M a n 3 can be used to determine whether M a n 3 is mono-a-( 1-4) or -a-(1+6) substituted by another Man residue. The values for H-1 and H-2 of Man-3 in the p anomer of 18 are in accord with those observed for the glyco-asparagines 3 and 4, provided that they are corrected for the attachment of GlcNAc-5’ (for H-1, A6 - 0.009 p.p.m.; for H-2, A6 - - 0.005 p.p.m., as may be deduced from comparison of the spectrum of compound 19 with that of 6). The relatively large,J,,2 value for Man-3 in compound 18 (1.1 Hz) gives rise to two doublets of doublets for H-2 of Man-3, resembling the resonance pattern of the H-2 atom of an a-linked Man residue in a reducing oligosaccharide (see Fig. 18a and b). The effects of anomerization on the chemical shifts of the signals of H-1 and H-2 of Man-3 are comparable to those observed for other oligosaccharides (for example, 6, 7 and 10; see Table IV). The H-1 and H-2 signals of Man-4’, the H-1 doublet of Gal-6‘, and the N-acetyl signal of GlcNAc-5’ are doubled. This is in line with the observation that the influence of anomerization is, in general, more pronounced in the signals of the lower- than of the upper-branch residues. The chemical shifts of the structural-reporter groups of Gal-6’ and GlcNAc-5’ are in full accord with those observed for the corresponding protons in the diantennary oligosaccharide 7 (see Table IV). The chemical shift and the line width of the H-1 doublet of GlcNAc-5’ differ considerably from those observed for GlcNAc-5 in the spectrum of 17 (see Fig. 18a). The chemical shifts of the H-1 and H-2 atoms of Man-4’ reflect the incompleteness of the branching core (compare with 6, 7, 19, and 20; see Tables IV and VIII). Compound 19 is a reducing oligosaccharide consisting of the mannotriose branching core bearing an N-acetyllactosamine unit in the upper branch. It has been isolated from the urine of a patient suffering f r ~ mMorquio ~ ~ . ~syndrome ~ type B, as a constituent of a mixture containing compounds 19 and 20 in the ratio of 1:3. The 500-MHz, ‘Hn.m.r. spectrum of the mixture is shown in Fig. 19. The spectral parameters for compound 19, which belong to the signals having the lower intensities in Fig. 19, are listed in Table VIII. For the spectral interpretation, 19 is considered as being structure 17 extended with Man-4‘, a-(l+6)-linked to Man-3. The attachment of Man-4’ leads to chemical shifts for H-1 of the a anomer of GlcNAc-2 and for the N-acetyl protons of 2 for both anomers of 19, which are identical to those for the diantennary compound 7 (at 300 K, the panomeric, H-1 signal of GlcNAc-2 is partially hidden under the HOD line).

‘H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

267

The major effect on Man-3 due to the introduction of Man-4’ is found in the chemical shift of its H-2 signal (A6 0.02 p.p.m.). Man-4’ causes an upfield shift (A6 - 0.005 p.p.m.) and a line broadening of the H-1 signal of Man-4. However, the structural-reporter-group signals of GlcNAc-5 and Gal-6 are unaffected. The well resolved, H-1 doublets of the terminal Man-4’ are found at 6 4.920 (a)and 4.925 (p anomer of 19). The latter value differs from that observed for H-1 of the terminal Man4’ in glyco-asparagine 3. This difference can be ascribed to the influence of the presence of Man-4. The H-2 signal of Man-4’ is hidden in the bulk of the sugar skeleton protons (8 <4.0), which is characteristic for a terminal a(l+6)-linked Man group (see Table 11). Comparison of the spectrum of 19 with that of the diantennary oligosaccharide 7 shows that the absence of GlcNAc-5‘ and Gal-6’ has no influence on the structural-reporter-group signals of Gal-6, GlcNAc-5, and Man-4, but results in changes for those of Man-3 and -4’. In fact, the absence of GlcNAc-5’ is responsible for the alterations, which is evident from comparison with the data for oligosaccharide 6. Compound 20 is a reducing oligosaccharide consisting of the mannotriose branching core bearing an N-acetyllactosamine unit in the lower branch; 20 was obtained as the main component of a mixture also containing 19. As already described, this mixture has been isolated from the urine of a patient ~ u f f e r i n gfrom ~ , ~ Morquio ~ syndrome type B. The 500-MHz, ‘H-n.m.r. spectrum of the mixture is depicted in Fig. 19; the relevant, spectral parameters for 20 are compiled in Table VIII. For the interpretation of the signals having the higher intensities (see Fig. 19), belonging to 20, this oligosaccharide is conceived as structure 18 extended with Man-4 that is a-(1+3)-linked to Man-3. Comparison of the spectral data with those for 18 demonstrates that the chemical shifts of H-1 and H-2 of Man-3 and -4’ have undergone alterations towards values that are typical for a complete branchingcore (see Table VIII). The (terminal) nonreducing position of Man-4 in 20 is revealed by its H-1 (8 5.106 for the a,and 6 5.102 for the p anomer of the oligosaccharide) and H-2 chemical-shift values (6 4.071), which are closely related to those for the corresponding residue in oligosaccharide 5 (see Table 111).The slight, upfield shift of H-1 of Man-4 compared with 5 (A8 -0.007 p.p.m.) is caused by the presence of Man-4’; the same effect is observed in the step from 17 to 19. Furthermore, the narrow lines of the signals of Man-4 reflect its terminal, relatively mobile position in the carbohydrate chain. Comparison of the spectral data for 20 with those for the dianten-

-

0

In c

0

m I

2; z

T

--

cn (0

'.,

* ln

. '

ob

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

269

nary oligosaccharides 6 and 7 reveals that the essential differences are located in the spectral features of M a n 4 In particular, the effect of anomerization is lessened by the attachment of GlcNAc-5.

b. Extensions of Carbohydrate Chains of the N-Acetyllactosamine carbohydrate chains of the N-acetyllactosamine type, attached to, or liberated from, a glycoprotein peptide backbone. bear one or more sialic acid (NeuAc) groups (in a terminal Symbols employed for compounds 21-41 are depicted in Chart 2. Two well defined ways of terminating chains of the N-acetyllactosamine type by a NeuAc residue will be treated here, namely, (i) the ~ ~ ( 2 - 6and ) (ii) the a-(2+3) attachment of NeuAc to a Gal residue that forms part of an N-acetyllactosamine unit. First, the spectral features of structures containing only a-(2+6)-linked NeuAc (compounds 2134) will be discussed. Then, the characteristics and influences of NeuAc in a-(2+3) linkage to Gal will be considered (compounds 3538). Finally, (iii) some structures (compounds 39-41) possessing aNeuAc(2+6)Ga17 as well as aNeuAc(hS)Gal, will be the subjects of discussion.

Type with Sialic Acid Groups (Compounds 21-41).-Many

(i) NeuAc a-(2+6)-Linked to Gal.-Compound 21 is a mono-antennary, upper-branch, sialo oligosaccharide. It was available as the main component (90%)of a mixture of two oligosaccharides also containing 22 that has been isolated from the urine of a sialidosis ~ a t i e n t .The ~~,~~ 500-MHz, 'H-n.m.r. spectrum of the mixture, recorded at pD 7 (as for all of these sialo compounds, except 60)is shown in Fig. 20, and the n.m.r. parameters for 21 are listed in Table IX. Comparison with the spectrum of 17 demonstrates that the attachment of a NeuAc residue a-(2+6)-linked to Gal gives some significant, additional signals in the spectrum, and causes some changes in chemical shifts of structural-reporter groups of other constituent monosac(72) L. Dorland, J. Haverkamp, J. F. G. Vliegenthart, G. Strecker, J.-C. Michalski, B. Foumet, G. Spik, and J. Montreuil, Eur. J . Biochem., 87 (1978) 323-329.

FIG.19.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 19 and 20 in the Ratio of 1 :3. [The bold numbers in the spectrum refer to the corresponding residues in the s t r u c tures, and the italic numbers to the compounds in the mixture, each of which occurs as an anomeric mixture in the ratio of a : p = 2: 1. (Anomeric-proton signal designated as 4', 1% means: H-1 of Man-4' in the a anomer of compound 19.) Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other part of the spectrum, as indicated.]

CHART2 .

Symbols Employed” for Compounds 21-41

31

21 22

32 23 33 24

34 25

35 26 36 27 37 28

38 29

39

30 40

41 For the key to the symbolic notation, see Chart 1. 210

a-NeuAc - (2-

6)- p -Gal- (1-

4)-p -GlcNAc - (1-

Z)-a-Man- (1-

5

8

3)

4

21

o-NeuAc'-(2-6)-p-Ga,l-(l--

/ 4)-p-GlcNAc-(l-2)-a-Ma,p-(1---6) 5'

6

p -Man( 1 -

4)-GlcNAc

5

2

4

Man H-2 atoms

2.210

-

52

h

50

46

411

42

1'

1

NAc -CH3 protons NS"*<

21

N p L .22

H-3e

H-3a

atoms

atoms

2,210

MUAS

k"*C

8 28

.22

A

,a Neuk

.22

2.27~

26

22

la

111

1

FIG. 20.-Strudural-reporter-group Regions of the Resolution-enhanced, ~CIO-MHZ, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 21 and 22 in the Ratio of 9: 1. [The bold numbers in the spectrum refer to the corresponding residues in the s t r u c tures, and the italic numbers to the compounds in the mixture, each of which occurs as an anomeric mixture in the ratio of a! : p = 2: 1. Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relativeintensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The sample was contaminated with a very small proportion of the asialo analog of 22,that is, compound 18, as can be inferred from the signals marked by asterisks (compare Fig. 18b).] 27 1

TABL 'H Chemical Shifts of Shchual-reporter Groups of Constituent Monosaccharides for Sialic Acid (Corn Compound and 21

22

23

24

5318 -4.72 4.774 4.765

5.215 -4.72 4.792 4.784

5.215 -4.72 4.784 4.775 5.107 5.103

h o m e r of Reporter group

Residue

H-1 of

2 3

4 4' 5 5' 6 6'

H-2 of

7 8 3

4 4'

H 3 of H 3 a of H-3eof NAc of

4 4' "NeuAc "NeuAc' 'NeuAc* NeuAc NeuAc' NeuAc* 2

5 5' 7 NeuAc NeuAc' NeuAc*

saccharide

5208 -4.72 4.788 4.785 5.143

-

5.140

-

4.942

4.921

4.602

-

4.606

-

4.602

4.446

-

-

4.446 4.449

-

4.949

4.606

4.446

-

-

4.446 4.450

-

-

-

-

4.244 4.233 4.198 4.196

4.088 4.078

4268 4.258

4.263 4.253

-

4.198

4.073

-

4.106 4.110

-

-

C4.0

-

<4.0

-

1.716 -

-

2.030

-

-

2.672

2.061 2.058

2.059 2.055 2.070

2.063 2.059

2.064

-

-

2.070 2.067

2.030

-

272

-

2.669

2.030

* NeuAc denotes NeuAc linked to Gal&.

~4.0

2.672

-

-

4.119 c4.0

-

-

2.043 2.041 2.069

<4.0 <4.0 1.719

1.714

-

-

2.670

<4.0

-

1.718

-

-

-

-

2.030 -

NeuAc' denotes NeuAc linked to Gal&'.

E M

Oligosaccharides of the N-Acetyllactosamine Type Containing Only a-(2+6)-Linked, pounds 21-30) schematic structure

5.213 -4.72 4.784 4.776

5.213 -4.72 4.784 4.776

5214 -4.72 4.781 4.771

5214 -4.72 4.781 4.771

5.216 -4.72 4.786 4.777

5215 -4.72 -4.77 -4.76

5.137

5.121

5.138

5.124

5.137

5.134

4.921

4.950

4.949

4.952

4.943

4.607

4.557 4 .w

4.583 4.608 4.468 4.446 4.450

4.608 4.608 4.445 4.445 4.449

4.446

-

-

4.446 4.450

4.929 4.932 4.608 4.586 4.446 4.471 4.473

-

-

-

-

4.264 4.253

4859 4.248

4.260 4.249

4.265 4254

4.266 4.255

4.594 4.605 4.443 4.443 4.447 4.573 4.441 4.228 4.217

4.198

4.190

4.197

4.193

4.199

4.221

4.109

4.115

4.114

4.118

4.121

4.119

<4.0 <4.0 1.721 1.719

4.049 <4.0 1.720 1.717 1.706 2.670 2.672 2.670 2.062 2.059 2.069 2.067 2.065 2.102 2.030 2.030 2.028

4.558

-

<4.0 <4 .O 1.718 -

2.669

-

2.059 2.055 2.070 2.053

-

2.030

-

<4.0 <4.0

-

1.718 -

-

<4.0 <4.0

<4.0 <4.0 1.720

-

-

1.719

-

2.671

-

-

2.672 -

2.063 2.059 2.055 2.070 2.066

2.061 2.a58 2.070 2.050 2.018

2.063 2.060 2.053 2.069 2.066

-

2.030

-

2.669

2.031 -

-

2.031

-

NeuAc* denotes NeuAc linked to Gal& 213

-

-

2.669 2.672 2.063 2.060 2.071 2.069 2.066

-

-

2.031 2.031

-

274

J. F. G . VLIEGENTHART et al.

charides. Typical resonances for sialic acid, which do not coincide with the bulk, are those of H-3a (6 1.716)and H-3e (6 2.670), and of the N-acetyl protons (6 2.030). The signals of the H-3 atoms have characteristic patterns: H-3a gives a triplet (IJ3a.4 I = IJ3a,3e I), and H-3e, a doublet of doublets. The configuration of the linkage between NeuAc and Gal may be derived from the chemical shift of H-3e (in p-linkage SH3e = 2.2-2.4 ~ . p . r n . ) . ? The ~ - ~ ~chemical shifts of H-3a and H-3e are sensitive to changes in the p D of the oligosaccharide solution in D20. At pD 2, 6H-3a +- 1.79 and 6H-3e -2.66 (see Ref. 75). The introduction of a sialic acid group at 0-6 of Gal-6 gives rise to a shifi decrement for H-1 of Gal-6 (A8 -0.022 p.p.m.) and to shift increments for H-1 of GlcNAc-5 (A6 0.025 p.p.m.), the N-acetyl protons of GlcNAc-5 {A6 0.016 p.p.m.), H-1 of Man-4 (A6 0.016 p.p.m.), and H-2 of Man4 (A6 0.005 p.p.m.), whereas the line widths of their signals, and the anomerization effects upon these, are unaffected, as compared to 17. These changes in chemical shifts, in combination with the chemical-shift values of the H-3 signals of NeuAc, are specific for the type and configuration of the linkage of NeuAc to Gal-6. Compound 22 is a mono-antennary, lower-branch, sialo oligosaccharide, isolated in admixture with 21 from the urine of a patient with sialidosis. The W - M H z , ‘H-n.m.r. spectrum ofthe mixture is given in Fig. 20; the set of lower-intensity signals belongs to 22.The n.m.r. parameters for 22 are summarized in Table IX. For the interpretation of the n.m.r. spectrum, comparison with the spectra of 18 and 21 is appropriate. The chemical shifts of the H-3 signals of the NeuAc’ residue, that is, NeuAc cu-(2-+6)-linkedto Gal-6’, in 22 differ slightly from those of NeuAc a-(2+6)-linked to Gal-6 in 21 (6H-3e 2.672 as against 2.670, and 6 H - 3 ~1.714 as against 1.716). The 6 value of the N-acetyl protons of NeuAc’ in 22 exactly matches that for NeuAc in 21. The observed shift increments and decrements, due to the introduction of sialic acid, for structural-reporter groups of neighboring residues are of the same order of magnitude as for the step from 17 to 21, but larger for H-1 of Man4’ than for H-1 of M a d . In this case, also (73) J. Haverkamp, L. Dorland, J. F. G. Vliegenthart, J. Montreuil, and R. Schauer, -4bstr. Int. Symp. Carbohydr. Chem., 9th, London, 1978, 281-282. (73a) J. F. G. Vliegenthart, L. Dorland, H. van Halbeek, and J. Haverkamp, in R. Schauer (Ed.),S i a l i c Acids-Chemistry, Metabolism and Function, Cell B i d . Monogr., Vol. 10, Springer, Wien, 1982, pp. 127-172. (74) j. F. G. Vliegenthart, in L. Svennerholm, H. Dreyfus, and P. F. Urban (Eds.), Structure and Function of Ganglwsides, Adu. E x p . Med. Biol., 125 (1980) 77-91. (75) J. Haverkamp, H. van Halbeek, L. Dorland, J. F. G . Vliegenthart, R. Pfeil, and R. Schauer, Eus. J . Bwchem., 122 (1982)305-311.

‘H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

275

the resonance position of H-1 of Man-3 is slightly, but significantly, influenced by the extension of 18 with NeuAc: A6 0.005 p.p.m. for H-1 of Man-3 in the a and p anomer of 22. The observation that the effects of NeuAc in the lower branch reach farther than in the upper branch supports the proposal that these two branches have different conformations (compare oligosaccharides 7,lO-12, and 17 as against 18, and 19 as against 20). Concerning anomerization effects, it is noteworthy that differences in chemical shifts of corresponding protons in the a and p anomer of 22 remain unchanged in comparison to 18, except for the H-1 signals of Man-3 and Man-4‘. The A6,+ for these protons is (for Man-3) almost, or (for Man-4’) completely, nullified by attachment of the NeuAc group. Compound 23 is the sialo analog of 19, bearing NeuAc in a-(2+6) linkage to Gal-6. Compound 24 is the sialo analog of 20, having NeuAc’ in a-(2+6) linkage to Gal-6’. The oligosaccharides 23 and 24 could be obtained as a mixture from the urine of a patient with s i a l i d ~ s i and s~~ from new-born, human m e ~ o n i u m The . ~ ~ 500-MHz, ‘H-n.m.r. spectrum of such a mixture containing 23 and 24 in the ratio of 3: 2 is presented in Fig. 21. The n.m.r. parameters for both compounds are summarized in Table IX. The attachment of NeuAc to Gal-6 of 19 or to Gal-6’ of 20 gives rise to typical sets of H-3 and N-acetyl signals for NeuAc, and to alterations of chemical shifts of structural-reporter groups of neighboring residues, as described for 21 and 22. In 23, no effect is observed on the chemical shift of H-1 of Man-4’ in comparison to 19. Similarly, the signals of H-1 and H-2 of Man-4 in 24 occupy the same positions as in the spectrum of 20. In contrast to the situation for the asialo analog, the H-1 doublet of Man-4’ of 23 is not doubled due to anomerization; however, its line width is not affected. Compounds 25 and 26 are reducing oligosaccharides having incomplete, diantennary structures. They were isolated from new-born, human m e c o n i ~ min~a~mixture containing 25 and 26 in the ratio of 3: 1.The 500-MHz, IH-n.m.r. spectrum of the mixture is shown in Fig. 22, and the n.m.r. data for both compounds are compiled in Table IX. For the interpretation of their n.m.r. spectra, compounds 25 and 26 are considered to be extensions of 23 and 24 with a GlcNAc residue in p-( 1+2) linkage to Man-4’ or -4, respectively. The H-1 atoms and the N-acetyl-group protons for the terminal GlcNAc residues 5’ (compound 25) and 5 (compound 26) resonate at positions similar to those (76) M.-C. Herlant-Peers, J. Montreuil, G. Strecker, L. Dorland, H. van Halbeek, G. A. Veldink, and J. F. G. Vliegenthart, Eur. J . Biochem., 117 (1981) 291-300.

276

J. F. G . VLIEGENTHART et al.

a-NeuAc -(2-6)-p

-Gal-(&-

4)- fl -GlcNAc- (1-

6

2)-a-Man- (1-

3)

i

5

\p a-Man- (1-

6)

/

-Man- (1-

4)-GlcNAc

s

4’

23 a-Man-(l-

3)

4

\p-Man-(l--4)-G1:NAc

a -NeuAc’-(2-6)-

p -Ga,i-(l-

4)-8 -GlcyAc-(l5

5

2)-a-Man-(1-

/

6)

$

It

24

2.0

12

-

b

50

H-3a

atoms H-3e atoms

, 28

“CUlC

4

26

,23

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

277

for compound 6. The attachment of GlcNAc-5' to 0-2 of Man-4' has hardly any effect on the chemical shift of H-1 of Man-4', but the H-2 signal shifts downfield. The observed chemical shifts are in accord with those of 6. The chemical shifts of H-1 and H-2 of Man-4 in 26 are practically identical to those for 6; they are different from the values for 24, owing to the presence of GlcNAc-5 (compare the positions of H-1 and H-2 of Man-4 in 20 and 6). The presence of GlcNAc-5' is revealed by small shift-decrements of H-1 of Man-3 in both anomers of 25, whereas GlcNAc-5 does not affect the chemical shift of H-1 of Man-3 in either the (Y or the /3 anomer of 26. Compounds 27 and 28 are diantennary, monosialo oligosaccharides that could be obtained in a mixture from the urine of a patient with s i a l i d ~ s i sas , ~ well ~ as from new-born, human me~onium.'~ The 500MHz, lH-n.m.r. spectrum of such a mixture containing 27 and 28 in the ratio of 11:9 is presented in Fig. 23. The n.m.r. data are listed in Table IX. For the spectral interpretation, the oligosaccharides are conceived as extensions of 25 and 26, with a Gal residue /3-(1+4)-linked to GlcNAc-5' or -5, respectively. As already described, this attachment almost exclusively influences the chemical shift of H-1 of the penultimate GlcNAc residue (A8 0.027 p.p.m.). The resonance positions of the H-1 signals of the terminal Gal residues are in complete accord with those of the diantennary, asialo oligosaccharide 7. The H-1 signal of Gal-6' in 27 is clearly doubled, due to anomerization, an effect that is also observable for the residues Man-4' (H-1) and GlcNAc-5' (N-acetyl protons). The location of NeuAc in a diantennary, monosialo structure may be directly inferred from the chemical-shift values of the H-1 signals of Man-4 and -4'. The chemical shifts of the N-acetyl signals of GlcNAc-5 and -5' also reflect whether or not a certain branch is terminated with a NeuAc residue in (~-(2+6)linkage to Gal. In principle, the chemical

~

FIG.21.-Structural-reporter-groupRegions of the Resolution-enhanced,S M H z , 'H-N.m.r. Spectrum of a Mixture Containing Compounds 23 and 24 in the Ratio of 3: 2. [The bold numbers in the spectrum refer to the corresponding residues in the structures, and the italic numbers to the compounds in the mixture, each of which occurs as an anomeric mixture in the ratio of a : p = 2: 1. Signals of correspondingprotons in the various components of this mixture coincide, unless otherwise indicated. The relativeintensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated.]

J. F. G . VLIECENTHART et (11.

278 a-NeuAc - (2-

6)- /3 -Gal-(16

-

4)- p -GlcNAc (1-2)-a-Man5

(1-

4

3)

\

\

6 -Man-(%+ 4)-GlcNAc I

f3 -GlcNAc - (1-

2)-a-Man- (1-

/

6)

s

4’

51

25

p-GlcNAc-(l-- 2)-u-Man-(l-+ 3) 5

cr-NeuAc<- (2-

6)- 6 -Gal-(16‘

4

-

4)- p -GlcNAc (l--2)-a-Man-(15’

6)

4‘

26

NAC -CH3 protons *“A<

.25

*“*S..X

H-3e

atoms

5;r” 5;xD 525

5.26

2:25a

H-3a atoms

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

279

shifts of the H-1 signals of Gal-6 and -6', and those of the H-3 signals of NeuAc itself, can also be utilized for the locating of NeuAc. Compound 29 is a diantennary, sialo oligosaccharide that has been isolated from the urine of a sialidosis patient,72as well as from newborn, human m e c o n i ~ m The . ~ ~ 500-MHz, 'H-n.m.r. spectrum of 29 is depicted in Fig. 24, and the n.m.r. data are compiled in Table IX. The chemical shifts of the structural-reporter groups of the sialylated branches of compounds 27 and 28 are found unchanged in this spectrum. This shows that the shift effects introduced by the extension of a branch with sialic acid in ~ ( 2 4 6linkage ) are limited to the branch to which it is attached. The pattern of the signals of the H-1 and H-2 resonances of Man residues 3,4, and 4' is, as a whole, typical ofa diantennary, sialo structure wherein the chemical shifts of the H-1 signals of Man4 and -4' indicate that each branch is terminated with NeuAc in a-(2+6) linkage to Gal. The NeuAc residues are not completely equivalent, as is evident from the differences in the chemical shifts of their H-3e as well as their H-3a signals. It should be noted that this doubling of signals is not caused by anomerization, as is clear from the 1 : 1intensity ratio of the signals. The difference in chemical shift between the H-1 signals of Gal-6 and -6' is slightly smaller than for the asialo analog 7. a-NeuAc* - (2-

6)-

a-NeuAc - (2-6)-

p -Gal- (1B

4)- p -GlcNAc - (17

p -Gal- (1- 4)- p -GlcNAc -(16

5

4)

\,

2)-u-Man- (1-3) 4

\

\

p -Man - (1- 4) - GlcNAc 2

a-NeuAc'-(2-6)-p

-Gal-(16'

4)-p -GlcNAc -(15'

/

2)-a-Man-(1- 6)

3

4)

30

Compound 30 is a triantennary, sialo oligosaccharide terminated with NeuAc a-(2+6)-linked to Gal in the three branches. It has been FIG.22.- Structural-reporter-groupRegions of the Resolution-enhanced, 500-MHz, lH-N.m.r. Spectrum of a Mixture Containing Compounds 25 and 26 in the Ratio of 3 :1. [The bold numbers in the spectrum refer to the corresponding residues in the s t r u c tures, and the italic numbers to the compounds in the mixture. Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance has been omitted from the spectrum; its position is indicated by an arrow. The sample was contaminated with very small proportions of 27 and 28, and, to a very small extent, with an oligosaccharide similar to 25,26,27, or 28, containing a NeuAc group in a-(2+3), instead of a-(2-+6),linkage to Gal. This can be inferred from the signals marked by asterisks.]

J. F. G. VLIEGENTHART et 01.

280

-

u-NeuAc (2-

6)- p -Gal - (18

4)- p -GIcNAc - (I-

2)-u-Man-(I-t 3)

5

\

i

'6-Man-(&/

p -131-(16'

rl)-p-GlcNAc-(l-

2)-a-Man-(l--

4)-GlcNAc

s

6)'

4'

5'

27

fc -GaI-(l6

o-NeuAc'-(2-6)-@

-Gal-(I6'

4)-8 -GlcNAc-(1-

2)-a-Man-(l-

5

4

4)- p -GlcNAc -(l-

3)

\p -Man - (1/

2)-u-Man-(l+

5'

4'

28

NAcCHs protons

H-3a

H-3e atoms

atoms

'

I

4)- GlcNAc 2

3

6)/

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

281

obtained from the urine of a patient suffering from sialidosis, as a minor constituent of a mixture of two oligosaccharides containing 41 as the major part.13 The SW-MHz, 'H-n.m.r. spectrum of this mixture is presented in Fig. 32. The n.m.r. parameters for 30 are compiled in Table IX. For the interpretation of the signals of lower intensity in the spectrum, 30 is considered to be an extension of 10 with three a-(2+6)linked NeuAc residues. The introduction of NeuAc groups a-(2-+6)linked to Gal-6 and -6' influences the 6 values of the structural-reporter groups of the upper-branch residues 6,5, and 4, and the lowerbranch residues 6', 5', and 4' in a way similar to that already described (see Tables IV, VIII, and IX). The attachment of NeuAc*, that is, NeuAc a-(h6)-linked to Gal-8, is revealed in a shift decrement for H-1 of Gal8 (A6 - 0.022p.p.m.) with respect to 10, and an increment for H-1 of GlcNAc-7 (A8 0.027 p.p.m.) (compare Table XIII).The shift of the N-acetyl singlet of GlcNAc-7 from 6 2.078 for 10to 8 2.102 for 30 (A6 0.024 p.p.m.) is highly characteristic for the elongation of 10 with NeuAc* a-(24)-linked to Gal-8. No additional effects of this NeuAc* upon 6H-1 and 6H-2 of the more remote Man4 and Man-3 are observed. The most striking feature of the 500-MHz7'H-n.m.r. spectrum of compound 30 is the Occurrence of three well-resolved, H-3a triplets of NeuAc at 6 1.720, 1.717, and 1.706. The first is assigned to NeuAc a(2+6)-linked to Gal-6, and the second, to NeuAc' linked to Gal-6', in agreement with 29. Consequently, the third signal, at 6 1.706, must be ascribed to H 3 a of NeuAc* in the additional (third) branch of 30. Based on the relative intensities of the H-3e doublets of doublets, the H 3 e signal of this NeuAc* group apparently coincides with the doublet of doublets at 6 2.670 stemming from NeuAc linked to Gal-6, as H 3 e of the lower-branch NeuAc' resonates at 6 2.672. Finally, it is remarkable that the NeuAc* group linked to Gal-8 possesses N-acetyl FIG.23. -Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 27 and 28 in the Ratio of 11:9. [The bold numbers in the spectrum refer to the corresponding residues in the structures, the italic numbers to the compounds in the mixture, each of which occurs as an anomeric mixture in the ratio of Q :#? = 2 :1. Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance, as well as the H-1 signal of GlcNAc-2 for the #? anomers of 27 and 28, have been omitted from the spectrum; their positions are indicated by arrows. The sample was contaminated with a small proportion of 36 or 37. or both, containing ~-(%3)-linked NeuAc, as can be inferred from the signals marked by asterisks (compare Fig. %).I

J. F. G. VLIEGENTHART et al.

282

a-NeuAc-(2-

a-NeuAc '-(2-

4)-a -GicNAc- (1-

2)-a-Man-(l-

3)

6 ) - p' -Gal- (I-- 4)- B -GlcNAc- (1-

8)-a-Man- (1-

6)

6)- O-Gal-(l-

5

8

6'

4

/

4'

5'

29

5 ,

- - b

5 0

L6

' I

' 2

' I

NAGCH3 prOtOnS

H-3e atoms

5

I

t

?a

26

12

20

8

8

FIG.24.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 29. [The bold numbers in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the a and p anomer of 29, occurring in this anomeric mixture in the ratio of 2: 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance has been omitted from the spectrum; its position is indicated by an arrow. T h e sample was con-

I

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

283

protons in a chemical environment different from that of the corresponding protons in the other two NeuAc groups (6 2.028 as against 2.030). It must be emphasized that the extension of a triantennary structure with a NeuAc group a-(2+6)-linked to Gal-8 can be unambiguously established on the basis of both the unique set of chemical shifts for its H-3a, H-3e, and N-acetyl protons, as well as its specific effects upon the chemical shifts of the structural-reporter groups of GlcNAc-7 and Gal-8 (compare Table XIII). Compounds 31 and 32 are diantennary, monosialo glycopeptides that may be obtained from numerous sialo-glycoproteins, for example, from rabbit serotransferrin." Compounds 31 and 32 can occur in different ratios in glycopeptide mixtures thus prepared. It is unclear whether these monosialo compounds reflect partial degradation, or incomplete biosynthesis; the origin of this microheterogeneity is still under investigation. It should be noted that the preparation in vitro of the Asn-Lys analog of the monosialo glycopeptide 31,using 8 as the substrate, by means of a p-D-galactoside a-(2+6)-sialyltransferase (EC 2.4%. 1) may be efficiently monitored by high-resolution, 'H-n.m.r. spectroscopy.7* The spectral parameters for 31 and 32 are listed in Table X. With regard to the spectral interpretation, it may be mentioned that 31 and 32 are the glycopeptide analogs of oligosaccharides 27 and 28, respectively. The presence of a-(2-+6)-linked NeuAc in one of the branches can be derived from the chemical shifts of the anomeric protons of Man-4 and -4', or, alternatively, from those of the N-acetyl protons of GlcNAc-5 and -5', as already described. The chemical shifts of H-1 and of the N-acetyl protons of GlcNAc-1 deviate from those for 8 (see Table V)and 33 (see Table X),illustrating the effect of the peptide moiety (Asn-Ser as against Asn-Lys) on the chemical shifts of the structural-reporter groups of this residue. Compound 33 is a diantennary, sialo glycopeptide that can be obtained from various glyc~proteins'-~; for example, from human sero(77) D. Leger, V. Tordera, G . Spik, L. Dorland, J. Haverkamp, and J. F. G . Vliegenthart, FEBS Lett., 89 (1978) 149-152. (78) D. H. van den Eijnden, D. H. Joziasse, L. Dorland, H. van Halbeek, J. F. G. Vliegenthart, and K. Schmid, Biochem. Biophys. Res. Commun. 92 (1980) 839-845. taminated with small proportions of the monosialo oligosaccharides 27 and 28 (or even with the asialo compound 71,as well as the corresponding proportion of free NeuAc. This is evident from the signals marked by asterisks. Special attention should be paid to the chemical shifts of the N-acetyl signal (6 2.050)and the H-3a signal (6 1.838),and to the typical doublet of doublets for the latter proton, of free NeuAc (p anomer).]

-/ m

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

285

t r a n ~ f e r r i nand , ~ ~ from azHS g l y c ~ p r o t e i n Structural .~~ investigations employing 360-MHz, 'H-n.m.r. spectroscopy have been conducted on such a glycopeptide isolated from rat-liver, plasma membrane.80The 500-MHz, 'H-n.m.r. spectrum of 33 from serotransferrin is presented in Fig. 25, and its n.m.r. parameters are compiled in Table X. For the interpretation of the spectrum, the compound may be conceived of as a glycopeptide analog of oligosaccharide 29, which allows a straightforward assignment of all signals of structural-reporter groups. The resonances of the H-3a atoms of the two NeuAc groups coincide, whereas those of the H-3e atoms are well separated. The assignment of the latter signals was achieved on the basis of the n.m.r. spectrum of the enzymically diantennary, monosialo glycopeptide (Asn-Lys)having the NeuAc group a-(2+6)-linked to Gal-6 (compare 31). Interestingly, the N-acetyl signals of the two NeuAc groups in 33 are observed separately (6 2.029 and 2.028), in contrast to the corresponding signals of oligosaccharides 29 and 30. Compound 34 is a triantennary, sialo glycopeptide terminated with NeuAc a-(2+6)-linked to Gal in all three branches; it could be obtained by exhaustive, in vitro sialylation of compound 9 with P-Dgalactoside a-(2+6)-sialyltran~ferase.'~*~~ The Gln-Asn analog of compound 34,in admixture with compound 53 and a glycopeptide analog of 41,has been isolated from human-plasma ceruloplasmin (see compound 53).The 500-MHz, 'H-n.m.r. spectrum of 34 is given in Fig. 26, and its pertinent spectral parameters are listed in Table X. Compared to the spectrum of 33,an additional set of NeuAc H-3 signals is present, at S 1.706 (H-3a) and S 2.670 (H-3e), which is specific for NeuAc* in ~ ~ ( 2 - 6 linkage ) to Gal-8. This set has also been observed in the spectrum of the oligosaccharide analog of 34,namely, 30.The chemical shift of the N-acetyl singlet of GlcNAc-7 (6 2.101), in combination with its relatively large line-width, is also characteristic for an 0-6 substitution of Gal-8 by a NeuAc group (compare 30).13All other spectral features of 34 are in agreement with those described for 30. The difference in chemical shift between the H-1 signals of Gal-6 and Gal-8 is significantly lessened, as compared to that for the asialo analog 9 (A8 0.001 as against 0.006p.p.m.). This may be attributable to a small, secondary, downfield-shift effect, induced on SH-1 of Gal-8, by the attachment of NeuAc to Gal-6 in a-(2--*6) linkage. Furthermore, it (79) M. Endo, K. Hoare, K. Schmid, H. van Halbeek, L. Dorland, and J. F. G. Vliegenthart,J. Biol. Chem., 258 (1983) in press; K. Schmid, M. Endo, H. van Halbeek, L. Dorland, and J. F. G. Vliegenthart, Fed. Proc., 40 (1981) 1598. (80) H. Debray, B. Fournet, J. Montreuil, L. Dorland, and J. F. G. Vliegenthart, Eur. J . Biochem., 115 (1981) 559-563.

TABLEX 'HChemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for Glycopeptides of the N-Acetyllactosamine Type Containing Only a-(2+6)-Linked Sialic Acid (Compounds 31-34) Compouhd and schematic structure

N

m

m

31"

32"

33

34

5.073 4.620 4.769 5.138 4.928 4.599 4.577 4.445 4.472

5.073 4.620 4.769 5.120 4.944 4.577 4.599 4.470 4.445

5.088 4.616 4.773 5.133 4.949 4.603 4.603 4.442 4.447

5.060 4.615 -4.76 5.133 4.936 4.594 4.602 4.440 4.448

Reporter

H-1of

1

2 3 4 4' 5 5' 6 6'

H-2 of H 3 of

H3a of H-3e of NAc of

7 8 3 4 4’ 4 4’ NeuAc NeuAc’ NeuAc* NeuAc NeuAc’ NeuAc* 1 2

5 5‘ 7 NeuAc NeuAc’ NeuAc*

4.251 4.192 4.112 <4.0 <4.0 1.716 2.670

-

2.005 2.078 2.069 2.046 2.030

Chemical shifts measured at 360 MHz, T = 298 K.

-

1.716

4.254 4.195 4.116 <4 .O <4.0 1.716 1.716

2.670

2.666 2.672

4.251 4.192 4.112 <4.0 <4.0

-

2.005 2.078 2.050 2.067 -

-

2.030

-

2.002 2.081 2.067 2.063

-

2.029 2.028b

Assignments may have to be interchanged.

4.571 4.439 4.220 4.225 4.116 4.050 <4.0 1.717 1.717 1.706 2.670 2.674 2.670 2.008 2.082 2.069 2.065 2.101 2.030 2.030 2.030

288

J. F. G . VLIEGENTHART et al.

I

anomeric

orotons

Man H-2 atoms 7 7

L*l H

52

-

n

so

La

a% 33

46

NAc -CH3 DrOfOnS m-bNmIc

i 2

' I

I,

-

H-3e atoms "I"AC

*WAC

FIG.25.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 33.frhe bold numbers in the spectrum refer to the The relative-intensity scale ofthe corresponding residues in the structure (seepage 2.84). N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The sample contained small proportions of the monosialo analogs of 33,that is, 31 and 32 (or even of the asialo glycopeptide 8),as can be inferred from the signals marked by asterisks.]

is remarkable that the H-1 signal of Man4' undergoes an upfield shift upon extension of the diantennary, sialo glycopeptide 33 with the third branch. The origin of this effect is not yet clear; the third branch may exert a direct, through-space action on Man-4', or it may, more probably [compare the partly sialylated triantennary glycopeptide, lacking NeuAc' in comparison to 34: 6H-1 of Man-4' = 4.927 (Ref.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

289

anomeric Protons 1

I

g. .

',

,s8 .' _.

Man H-2 atoms

I

I

3

1

4

HOD

3

I

L;s

H-3a atoms

34 H-3e atoms

hAc tk"W

I".

&

28

26

'

,I

1

20

22

*-'.-*

cy

I

I8

FIG.26.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, bold numbers in the spectrum refer to the 'H-N.m.r. Spectrum of Compound 34. corresponding residues in the structure (see page 284).The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance, as well as the H-1 signal of Man-3, have been omitted from the spectrum; their position is indicated by an arrow. From the signals marked by asterisks, especially from the low-intensityN-acetyl-proton singlet of GlcNAc-7 at 6 2.077, it can be inferred that the sample contained a small proportion of the disialo analog of 34 that lacks the NeuAc* residue.]

me

78)], lessen the influence of NeuAc' attached to Gal-6' on this Man residue . (ii) NeuAc a-(2+3)-Linked to Gal.-Compound 35 is a mono-antennary, upper-branch, sialo oligosaccharide. It was available as the main component (75%) of a mixture of at least three isomeric oligosaccharides also containing the lower-branch analog of 35, namely, aNeuAc(2+3)PGal( 1+4)/3GlcNAc(l+2)aMan( l-G)PMan( 1+4)Glc

O N

9 ?

c

w

t

c

v c (

$a\W

t

u)

c

0

a

z h

w

t

.3 v

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

291

NAc (7%) (compound 35*),80a and oligosaccharide 21, differing from 35 in the type of linkage between NeuAc and Gal (12%). Small proportions of the corresponding asialo analogs (compound 17 or 18, or both) were also detected in the sample. This mixture was isolated from the urine of a patient with s i a l i d ~ s i sThe . ~ ~ 360-MHz, 'H-n.m.r. spectrum of the mixture is given in Fig. 27; the n.m.r. parameters for 35 are listed in Table XI. Comparison with the spectrum of 17 demonstrates that the attachment of a NeuAc group in a-(2+3) linkage to Gal affords three NeuAc structural-reporter-group signals. The chemical shifts of H-3a (6 1.799) and H-3e (6 2.758) are both clearly discernible from the corresponding ones for NeuAc in a-(2+6) linkage to Gal (21), making these parameters excellently suited for determination of the type (and configuration) of the NeuAwGal linkage, but the N-acetyl protons of NeuAc in a(2+3) linkage resonate at the same position as those of NeuAc a(2+6)-linked to Gal (compare Tables XI11 and XIV). As compared to the spectrum of the asialo analog 17, the introduction of NeuAc at 0-3 of Gal-6 gives rise to a limited number of effects on the chemical shifts of structural-reporter groups of neighboring sugar residues, namely, a shift increment for H-1 of Gal-6 (A6 0.076 (80a) The occurrence of the lower-branch, sialo oligosaccharide 35* in this complex mixture is based upon the presence of an additional H-1 doublet at 6 5.215 that belongs to the a anomer of GlcNAc-2 in a lower-branch oligosaccharide (compare 17 and 18; see Table VIII) in combination with the H-1 signal at 6 4.925 derived from Man-4'. The N-acetyl signal at 6 2.060, which is ascribed to the a anomer of GlcNAc-2 in lower-branch oligosaccharides (compare la), and the relatively intense signal at 6 -4.11, partly stemming from the nearly coinciding H-2 resonances of Man3 (6 -4.08) and -4' (6 -4.10) in such lower-branch compounds (compare 3,18, and 22;see Tables 11, VIII, and IX), support this conclusion. However, the resolution of the 360-MHz, 'H-n.m.r. spectrum available (see Fig. 27) is rather poor; therefore, it is impossible to deduce the detailed n.m.r. data for this lower-branch oligosaccharide with desirable accuracy. The presence of the Gal H-1 doublet(s) at 6 -4.46, next to the signal at 6 -4.44, suggests the occurrence of compound 17 or 18, or both, in the sample (compare with Table VIII).

FIG. 27.-Resolution-enhanced, Overall, 36@MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 35,35*,and 21 in the Ratios of 75: 7: 12. [The bold numbers in the spectrum refer to the corresponding residues in the structures, and the italic numbers to the compounds in the mixture, each of which occurs as an anomeric mixture in the ratio of a : p = 2: 1. Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The signals marked by (doublet at 6 1.32; quartet at 6 4.11) originate from a frequently occurring nonprotein, noncarbohydrate contaminant of unknown structure. In addition, the sample contained small proportions of the asialo analogs of35and 35*,that is, 17 and 18 (see also, footnote 80a).]

TABLEXI 'HChemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for Oligosaccharides of the N-Acetyllactosamine Type Containing Only a-(2+3)-Linked Sialic Acid (Compounds 35-38)

2 w

Compound and schematic structure

Reporter group

H-1 of

Residue

h o m e r of oligosaccharide

2

ff

P 3

ff

P 4

ff

P 4'

35"

36

37

38

5.206 -4.72 -4.79 -4.78

5.214 -4.72 -4.77 -4.76 5.123 5.119 4.929 4.932 4.579 4.587

5.214 -4.72 -4.77 -4.76 5.123 5.119 4.929 4.932 4.587 4.579

5.213 -4.72 4.775 4.763 5.122 5.120 4.923 4.928 4.578 4.578

5.122

a

P 5

5'

a,P B

ff

4.579

-

H-2 of

H-3 of

H-3a of H-3e of

NAc of

6 6'

4.544

3

4.245 4.234 4.197

4 4' 6 6' NeuAc NeuAc' NeuAc NeuAc' 2

5 5' NeuAc NeuAc' Chemical shifts measured at 360 MHz, T = 298 K.

-

4.115

-

1.799 2.758

-

2.043 2.041 2.049

2.029

-

4.546 4.472 4.474 4.260 4.249 4.195 4.117 4.115 <4.0 1.796

-

2.757

-

2.060 2.057 2.049 2.049 2.047 2.031

-

4.469 4.548 4.550 4.260 4.249 4.195 4.117 <4.0 4.117

-

1.799

-

2.757 2.060 2.057 2.052 2.045 2.042 2.031

4.544 4.546 4.547 4.256 4.244 4.192 4.117 4.113 4.115 1.797 1.799 2.758 2.758 2.061 2.058 2.049 2.046 2.043 2.032 2.032

J. F. G. VLIEGENTHART

294

et

al.

3)- @-Gal-(l--- 4)- ,r3 -GlcNAc -(l- 2)-a-Man-(l-

a-NeuAc- (2-

5

6

3)

\

4

>-My-(l-4)-GlcNAc

6 -Gal- (1-

4)- ,r3 -GlcNAc - (1-

2)-a-Man- (1-

5'

6'

6)

4'

36

Ci -Gal-(l-4)-j3-GlcNAc-(l-2)-a-Man-(l-

3)

\

4

5

6

'p-Man-(l-

u-NeuAc ' - (2-

3)- 6 -Gal- (1-

4)- f3 -GlcNAc - (1-

6'

/

2)- a-Man - (ld 6)/

4)-GlcNAc a

=

a'

5'

37 Man H-2 atoms

ammeric protons

i

iAr

4.0

4p

Z.,

L

4,6

4J3

1 6,366 0;3t7m

-I

~

1

5.p 5 3

A

;;:

-1

H-3

6.37

y

I'

rri

-~ 12

so

-b

68

--

.

16

(4

_ I _ -

(2

II

NAC -CH3 protons M

.38

-:37

I

H-3e a t m s nu* .36

H-3s atoms

2.6

/I

HUM 3

7

4370

537P + + i I -

----.-/ 20

FIG.28.- Sbic.tural-reporter-groupRegions of the Resolution-enhanced, 500-MHz, lH-N.m.r. Spectrum of a Mixture Containing Compounds 36 and 37 in the Ratio of 3: 1. [The bold numbers in the spectrum refer to the corresponding residues in the structures, and the italic numbers to the compounds in the mixture, each of which occurs as an anomeric mixture in the ratio of a : p = 2: 1. Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relativeintensity scale of the N-acetyl-proton region differs from that of the other parts of the

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

295

p.p.m.) and a small shift decrement for the N-acetyl protons of GlcNAc-5 (AS -0.004 p.p.m.). Furthermore, the H-3 signal of Gal-6 undergoes a relatively large, downfield shift (A8 -0.45 p.p.m., compare compound 59,Table XXIII), thereby emerging from the bulk of skeleton protons, and becoming a structural-reporter group for the cuNeuAc(2+3)/3Gal(l+.) sequence. In summary, the type of linkage of NeuAc to Gal, namely, a-(2+3) or a-(2+6), can be distinguished on the basis of (i) the set of chemical shifts for the H-3 signals of NeuAc itself, and (ii) the typical effects of attachment of NeuAc on the chemical shifts of structural-reporter groups of neighboring residues (see Tables XI11 and XIV). It should be noted that the anomerization effect upon the structuralreporter group signals of 35 is only observable for those of GlcNAc-2 and Man-3, probably due to the lower resolving power of the 360MHz with respect to the 500-MHz spectrometer. Apparently, the effect of anomerization on the various structural-reporter groups is similar to that for 17,as will later be considered in more detail (for example, 36 and 37). Compounds 36 and 37 are diantennary, monosialo oligosaccharides, each containing an a-(2+3)-linked NeuAc group. They were obtained in a mixture (together with a small amount of 27)from the urine of a patient with s i a l i d ~ s i s . ~The ~ , ' ~500-MHz, 'H-n.m.r. spectrum of this mixture, containing 36 and 37 in the ratio of 3 : 1, is presented in Fig. 28. The n.m.r. data for both oligosaccharides are given in Table XI. For the spectral interpretation, 36 and 37 are conceived of as extensions of the diantennary, asialo oligosaccharide 7.The spectral parameters of the structural-reporter groups of the core residues, that is, GlcNAc-2, and Man-3, -4, and -4', as well as doubling, due to anomerization, of the structural-reporter-group signals of the constituent monosaccharides (including those of GlcNAc-5' and Gal-6') are not influenced by the attachment of a NeuAc group in (w-(2-3) linkage to one of the Gal residues. The unaltered differences in the influence of anomerization on the chemical shifts of upper- and lower-branch N acetyllactosamine structural-reporter groups turn out to be valuable for spectral assignments. For locating an a-(2+3)-linked NeuAc in one of the branches of a diantennary, monosialo structure, the combination of three effects is spectrum, as indicated. The HOD resonance, as well as the H-1 signals of Man-3for both anomers, and that of GlcNAc-2 for the B , anomer, of 36 and 37, have been omitted from the spectrum; their positions are indicated by arrows. The sample was contaminated with a small proportion of 27, containing a-(2+6)-linked NeuAc, as can be inferred from the signals marked by asterisks.]

296

J. F. G. VLIEGENTHART et d.

specific. (i) As already described, the H-1 signal of Gal undergoes a downfield shift (A6 0.077 p.p.m.) upon substitution by NeuAc in a(2-3) linkage. Therefore, the relatively intense, single doublet at 6 4.546 is ascribed to H-1 of Gal-6 in 36;the set of doublets at 6 4.472 and 4.474, occurring in the anomeric ratio, belongs to H-1 of Gal-6' in the a and p anomer of 36,respectively. The latter values of chemical shift are in accord with those for 7 (see Table IV). For the minor component 37,the H-1 resonance of the terminal Gal-6 is found at 6 4.469 (compare 7),whereas the H-1 signal of Gal-6', still doubled due to anomerization, is shifted towards 6 4.548 (aanomer) and 4.550 (p anomer of 37).This shows that the attachment of NeuAc in a-(2-3) linkage does not affect the influences of anomerization. (ii) From the relatively complex pattern at 4.10 < 6 < 4.13 p.p.m., it may be inferred that, besides the H-2 resonance of Man+€',two separate Gal H-3 signals occur in this spectral region, at 6 4.115 and 4.117, which are assigned to Gal-6 in 36 and to Gal-6' in 37,respectively, on the basis of their relative intensities, namely, 3: 1. (iii) The N-acetyl singlet of GlcNAc-5 in 36 is found at 6 2.049, showing a small upfield shift (A6 -0.003 p.p.m.) as compared to the asialo upper branch of 7.The N acetyl signal of GlcNAc-5' in 36 is doubled due to anomerization; the pair ofhr-acetyl singlets is observed at 6 2.049 (coinciding with the signal of GlcNAc-5, see earlier) and 6 2.047 for the a and p anomer of 36, respectively (compare 7). For compound 37, the N-acetyl signal of GlcNAc-5 in the asialo upper branch is found at 6 2.052, whereas both singlets of GlcNAc-5' are shifted upfield, to 6 2.045 and 2.042, on attachment of NeuAc' in a-(2-3) linkage to Gal-6'. The effects of attachment of NeuAc in ~ ~ ( 2 - 3 linkage ) to Gal are restricted to the structural-reporter groups of the N-acetyllactosamine unit to which Gal belongs; as the chemical shifts of corresponding protons of these units in the asialo upper and lower branch of a diantennary structure differ only slightly (compare compounds 7, 8, and 15;see Tables IV, V, and VII), it is advisable to utilize the combination of the three effects just mentioned, in order to establish unambiguously the upper- or lower-branch position of NeuAc in 4-3) linkage to Gal. Also, the H-1 signal of GlcNAc in the N-acetyllactosamine moiety is influenced by sialylation of Gal at 0-3 (A8 - 0.003 p.p.m., and the line width of the resonance is increased), but the signals of the H-1 atoms of GlcNAc-5 in 36 and GlcNAc-5' in 37 remain indistinguishable from each other (see Table XI). Essentially, the resonance positions of the structural-reporter-group signals of NeuAc in 36 and 37 are identical to those described for 35, However, there is a small difference between the chemical shifts of

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

a-NeuAc-(2-3)-8

-Gal-(l-

4)-p -GlcNAc -(I-

2)-u-Man- (1

-+

5

6

297

3)

4

\p-Man-(l-4)-GlcNAc a-NeuAc'-(Z-J)-

8 -Gal- (1- 4)- 8 -GlcNAc- (1

2)-u-May (1-6)

/

s

+

5

6'

4

38 anomeric protons

7

I

40

q p .'.. :.'

,B Man H - 2 atoms H-3 8 ' 6

4.0

I

NAc -CH3 protons

H-3a atoms H-3e atoms

I2B

26

20

Id

FIG.29. -Structural-reporter-groupRegions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 38. [The bold numbers in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the a and /3 anomer of 38,occurring in this anomeric mixture in the ratio of 2: 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance, as well as the H-1 signal of GlcNAc-2 for the /3 anomer of 38, have been omitted from the spectrum; their positions are indicated by arrows.]

298

J. F. G. VLIECENTHART

et

al.

the H-3a signals of the upper- (6 1.796) and lower-branch NeuAc (6 1.799);the relative intensities of the latter signals corroborate that the molar ratio of the mixture of 36 and 37 is 3 :1. Compound 38 is a diantennary, sialo oligosaccharide that has been isolated from the urine of a patient with sialidosis.72The 500-MHz, ‘H-n.m.r. spectrum of 38 is depicted in Fig. 29, and its relevant n.m.r. parameters are included in Table XI. The chemical shifts of the structural-reporter groups of the core monosaccharides, that is, GlcNAc-2, and Man-3, -4, and -4‘, and of those of the sialylated N-acetyllactosamine branches of compounds 36 (5-6) and 37 (5’-6‘), are found unaltered in the spectrum of 38. The attachment of NeuAc in a-(2+3) linkage to Gal-6 or Gal-6’, or both, does not influence the anomerization effect upon any structural-reporter group of the oligosaccharide, neither with respect to its occurrence nor to its magnitude, as compared to the asialo analog 7 (see Table IV). The NeuAc groups in 38 are not completely equivalent, as is evident from the differences in the chemical shifts of their H-3a signals (see Table XI).

(iii) Structures Possessing a-(2+6)- and a-(2+3)-Linked NeuAc Groups-Compound 39 is a diantennary, sialo oligosaccharide having its two branches terminated with NeuAc, but in a mixed type of linkage; it has been isolated from the urine of a patient suffering from s i a l i d o s i ~ . The ~ ~ , ~W ~- M H z , ‘H-n.m.r. spectrum of 39 is presented in Fig. 30, and its spectral data are compiled in Table XII. The spectral features of the upper-branch residues Man-4, GlcNAc5, Gal-6, and NeuAc are essentially identical to those of other oligosaccharides possessing an a-(2+6)-sialylated, upper branch (namely, 21, 23, 25, 27, and 29; see Table IX). The n.m.r. parameters of the structural-reporter groups of the lower-branch residues Man-4‘, GlcNAc-5’, Gal-6‘, and NeuAc’ very closely resemble those of the corresponding residues in 37 and 38, also having NeuAc’ in a-(2+3) linkage to Gal-6’ (see Table XI). The spectrum of 39 (see Fig. 30) clearly illustrates that the sets of chemical shifts for the NeuAc H-3 signals are characteristic for the a(2+3) or a-(2+6) type of linkage to the Gal residue of an N-acetyllactosamine moiety. These values of chemical shift are independent of elongation of the other branch in a diantennary structure by NeuAc in any type of linkage. Compound 40 is a double-branched oligosaccharide containing a disuhstituted Man-4, but lacking the lower branch up to and including Man-4’, as compared to di- or tri-antennary structures; it is an example

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES a-NeuAc - (2-

6)- p -Gal- (I-+ 4)- p -GlcNAc - (16

2)- a-Man- (1 4

5

299

-\ 3)

' p -Man-(l-cl)-GlcNAc /

a-NeuAc'- (2-

3)- p &&-(I+ 6'

/

4)-p -GlcNAc -(I+

2)-a-Man- (1-

5'

I

2

s

6)

4'

39 Man H-2 atoms

anomeric protons

I

H-3

'

6

NAC -CH3 protons Nc"*r NCUk

H-3a atoms

H-3e atoms 5

FIG.30.-Structural-reporter-group Regions of the Resolution-enhanced, W - M H z , 'H-N.m.r. Spectrum of Compound 39.[The bold numbers in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the a and p anomer of 39, occurring in this anomeric mixture in the ratio of 2 :1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance has been omitted from the spectrum; its position is indicated by an arrow. The sample was contaminated with small proportions of the monosialo analogs of 39 lacking NeuAc or NeuAc', or even the asialo analog (oligosaccharide 7). This can be inferred from the signals marked by asterisks.]

300

J. F. G. VLIEGENTHART et al.

TABLEXI1 IH Chemical Shifts of Structural-reporterGroups of Constituent Monosaccharides for Oligosaccharides of the N-Acetyllactosamine Type Containing Both a-(2+3)and a-(2+6)-Linked Sialic Acid (Compounds 39-41)

Compound and schematic structure

Reporter group

Residue

H-1 of

2 3

4 4' 5 5' 6 6' 7 8 H-2 of

3 4 4'

H-3 of

4

4' 6 6' 8

H-3a of' aNeuAc(2-+6) aNeuAc'(Bj6) aNeuAc(2-3) H-3e of aNeuAc(2-6) aNeuAc'(2-6) aNeuAc(2-3) NAc of 2 5 5' 7 aNeuAc(2-6) aNeuAc'(2j6) aNeuAc(2j3)

39

40

41

5.213 -4.72 4.780 4.767 5.136 4.924 4.929 4.606 4.578 4.444

5.207 -4.72 -4.78 -4.77 5.135

5.215 -4.72 -4.77 -4.76 5.134

h o m e r of oligosaccharide

4.547

-

4.263 4.251 4.197 4.119 €4.0 <4.0 <4.0 4.115

-

1.719 1.800 2.668

-

2.756 2.061 2.057 2.069 2.045 2.044

-

-

4.943

4.590

4.594 4.605 4.443 4.443 4.447 4.551 4.545 4.228 4.217 4.221 4.119

-

4.445

4.552 4.546 4.211 4.201 4.219 4.053 4.049

-

~4.0 4.116 1.719 1.801 2.669

-

2.757 2.042 2.040 2.068

-

2.030

2.073 2.030

2.030

2.030

-

A

4.049 <4.0 <4.0 <4.0 4.116 1.720 1.717 1.802 2.670 2.672 2.757 2.062 2.059 2.067 2.067 2.065 2.074 2.030 2.030 2.030

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES cr-NeuAc* - (2-

3)- p -Gal - (1-

4)- p - GlcNAc - (1-

8

7

4)

\

cr-NeuA~-(Z~6)-p-GaI-(l-)4)-p-GlcNAc-(lZ)-cr-Man-(l& 3) 6

301

4

5

\ B -Man-(l-)4)-GlcNAc 3

2

40 anomeric protons

Man H-2 atoms I

I

3.

c'

H-3

NAcCH3 protons

H-3. aims

H-3e atoms

v

m

'H-N.m.r. the corresponding residues in%e structure. Signals o f m n dp anomer of 40, occurring in this anomeric mixture in the ratio of 2: 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance, as well as the H-1 signals of Man-3for both anomers and that of GlcNAc-2 for the p anomer of 40,have been omitted from the spectrum; their positions are indicated by arrows.]

of an oligosaccharide possessing two NeuAc residues in different types of linkage, and it has been obtained from the urine of a patient with ~ialidosis.'~ The 500-MHz, 'H-n.m.r. spectrum of 40 is given in Fig. 31, and its n.m.r. parameters are summarized in Table XII. Mono-a-(1-4)substitution of Man-3 is evident from the presence of

302

J. F. G. VLIEGENTHART et al.

the Man-4 H-1 doublet at 6 5.135, in combination with the absence of a Man-4' H-1 signal in the spectral region 4.85 < 6 < 4.95 p.p.m. This type of substitution of Man-3 is corroborated by the chemical shifts of H-1 of GlcNAc-2 in the a anomer of 40, and of the N-acetyl singlets of this monosaccharide at the reducing end (compare 5, 17,521, and 35; see Tables 111, VIII, IX, and XI). The chemical shifts of the H-2 signals of Man-3 and -4 indicate disubstitution of Man-4 at 0 - 2 and 0-4, as in tri- and tetra-antennary structures (see Table VI and compounds 9,10, 12, 13, 16, 30, and 34). The a-(2+6)-linked NeuAc group (6H-3a 1.719; 6H-3e 2.669) is attached to Gal-6, as may be inferred from the chemical shifts of H-1 of Man-4 (6 5.135), of the N-acetyl signal of GlcNAc-5 (6 2.068), and of the single H-1 signal of Gal-6 (6 4.445). Furthermore, the anomerization effect upon the chemical shift of H-1 of Man-4 is strongly lessened (IA6=+ I < 0.001 p.p.m.) by the attachment of this NeuAc (compare the step from 10 to 30 or to 41). The N-acetyllactosamine unit 7-8 is extended with an a-(2-3)linked NeuAc group (6H-3a 1.801; 6H-3e 2.757). In accordance with the effects of introduction of NeuAc a-(2+3)-linked to an N-acetyllactosamine unit (see 35-39, and Table XIV), the H-1 doublet of Gal-8 is found at 6 4.546; the H-3 signal of the latter residue is observed at 6 4.116, and the N-acetyl singlet of GlcNAc-7 is shifted slightly upfield, to 6 2.073, compared to the triantennary, asialo oligosaccharide 10 (see Table IV). Finally, H-1 of GlcNAc-7 shows a small downfield shift (A6 0.004 p.p.m.) with respect to the asialo 7-8 branch (for example, in 10). It should be noted that the assignment of the doublets at 6 4.552 and 4.546 to the H-1 atoms of GlcNAc-7 and Gal-8, respectively, is facilitated by the considerable difference in the line width of the signals. Compound 41 is a triantennary, sialo oligosaccharide possessing a(2+6)-linked NeuAc groups in the terminal position of bothP-( 1+2)linked N-acetyllactosamine branches, and an a-(2+3)-linked NeuAc as the terminating group of the p-(1+4)-linked part. Compound 41 was available as the main component (92%)of a mixture of two oligoFIG. 32. -Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 41 and 30 in the Ratio of 23 :2. [The bold numbers in the spectrum refer to the corresponding residues in the structures, and the italic numbers to the compounds in the mixture, each of which occurs as an anomeric mixture in the ratio of a : p = 2: 1. Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relativeintensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance, as well as the H-1 signals of Man-3 for both anomers and that of GlcNAc-2 for the B anomer of 41, and also of 30,have been omitted from the spectrum; their positions are indicated by arrows.]

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

p -Gal-(1-

u-NeuAc* -(2-3)-

4)-@-GlcNAc-(l- 4)

\

7

8

a-NeuAc-(2-6)-0

-Gal-(l+ 4)-p -GlcNAc-(16

\

2)-u-Man-(1--3)

5

4

'b-Man-(lu-NeuAc'-(2-

303

6)-p -Gal-(1-

4)- p -GlcNAc-(l---'2)-a-Man-(l-'6)'

a'

/

4)-GlsNAc

s

4'

5'

41

a-NeuAc* - (2-6)-

p -Gal-(1-

4)-p -GlcNAc-(17

8

a-NeuAc -(2-6)-

p -Gal-(+-

4)- p -GlcNAc-(1-

6

5

4)

\ \

2)-u-Man- (14

3)

\ \

p -Man-(l-4)-GlcNAc

u-NeuAc '-(2-

/

6)- p -Gaf-(l- 4)- p -GlcNAc -(l- 2)-u-Man- (16

5'

6)/

s

a

4)

30 anorneric orolons

Man H - 2 atoms

-./Ti c

H."K WAC'

5,41t5::

21

16

/

I

304

J . F. G. VLIEGENTHAHT et al.

saccharides also containing 30. This mixture was isolated from the urine of a patient suffering from s i a l i d ~ s i s . ' ~The * ~ ~500-MHz, ,~~ 'Hn.m.r. spectrum of this mixture is given in Fig. 32,and the n.m.r. parameters for 41 are listed in Table XII. The triantennary type of branching of the trimannosyl part of the oligosaccharide was established on the basis of the chemical shifis of the H-1 and H-2 signals of Man-3, -4, and A', taking into consideration the shift effects upon these due to extension of both the 5-6 and the 5'-6' branch with NeuAc in a-(Z+6) linkage (compare Tables VI and XIII; see also, compound 30). TABLE XI11 Recognition of u-(2-*6)-Linlred, Terminal NeuAc in the Sequence aNeuAc(Z.*G)BCal(l-*4)BGlcNAc(l+z)aMa( l-+y)BMan(1+)

'H Chemical Shifts of Structural-reporter Croups of a-(2+6)-Linked NeuAc (at pD -7) Reporter group H-3a ( X = 2; y = 3) H-3a ( X = 2; y = 6) H-& ( X = 4; y = 3) H-3e ( X = 2; y = 3) H-3e ( X = 2; y = 6) H-3e ( X = 4; y = 3) NAc ( x = 2; y = 3 or 6) NAc ( x = 4; y = 3)

-

6 +s.d.

1.718 20.002 1.717 20.001 1.706 20.002 2.669 20.001 2.672 +0.001 2.670 20.002 2.030 +0.001 2.029 20.002

Influence of a-(2+6)-Linled NeuAc on the Chemical Shifts of Structural-reporter Groups of Neighboring Residues" A8 (p.p.m.) 2s.d. (p.p.m.)

H-1 ofGal ( x = 2 o r 4 ; y = 3 o r 6 ) H-1 of GlcNAc ( x = 2 or 4; y = 3 or 6) NAc of GlcNAc ( x = 2; y = 3 or 6) NAc of GicNAc ( x = 4; y = 3) H-1 of aMan ( x = 2; y = 3) H-lofaMan(x=2; y = 6 ) H-1 ofaMan ( x = 4; y = 3) H-1 of BMan ( x = 2; y = 6) H-2 of aMan ( x = 2; y = 3) H-2ofaMan(x=2; y = 6 ) H-2ofaMan ( x = 4; y = 3) H-2 of PMan ( x = 2; y = 6)

-0.024 *0.001 +0.024 ,0.001 +0.019 20.001 +o.m20.002 +0.014 20.002 +0.020 r0.003b -0.0 +0.008 20.003 +o.m20.002 +0.005 20.002 -0.0 +0.007 20.002

" Values of A6 are mean values ? standard deviation (s.d.), at pD -7 and T = 300 K, calculated from asialo -P (2+6)-sialo steps (see Tables IV, V, VIII, IX, X, XII, X V , and XVIII). Except for Man-4' in triantennary compounds having NeuAc* in a-(2+6) linkage to Gal-8 (see, for example, compound 34 as compared to 9: A6 0.012 p.p.m.).

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

305

The spectral features of the a-(2+6)-sialylated N-acetyllactosamine branches are identical to those of the corresponding parts of 29 and 30, whereas the spectral characteristics of the third branch, bearing a NeuAc in a-(2+3) linkage to Gal-8, are identical to those of the same branch in 40 (see Table XII). The H-3a signals, and also the H-3e signals, of the a-(2+6)-linked NeuAc groups are well separated; furthermore, they are clearly distinguishable from those of NeuAc* a-(2+3)linked to Gal-8. The spectral characteristics of carbohydrate chains terminating in N-acetyllactosamine residues bearing NeuAc linked to Gal are summarized, for the a-(2+6) and a-(2+3) type of linkage, respectively, in Tables XI11 and XIV. c. Extensions of Carbohydrate Chains of the N-Acetyllactosamine Type with Fucose Groups (Compounds 42-54).--Symbols employed for compounds 42-54 are depicted in Chart 3. In many carbohydrate chains of the N-acetyllactosamine type that

TABLEXIV Recognition ofa-(2+3)-Linked7 Terminal NeuAc in the Sequence aNeuAc(2+3)BGal( 1+4)/3GlcNAc( l-rx)crMan(l-*y)/?Man(1-r)

'H Chemical Shifts of Structural-reporterGroups of u-(2+3)-Linked NeuAc (at pD -7) Repo*r

gmup

H-3a ( Z = 2; y = 3) H-3a ( X = 2; y = 6) H-3a ( Z = 4; y = 3) H-3e ( X = 2; y = 3) H-3e ( X = 2; y = 6) H-3e ( X = 4; y = 3) NAc ( x = 2 or 4; y = 3 or 6)

6 +s.d.

1.7% 20.002 1.800 20.001 1.801 20.001 2.758 20.001 2.757 20.001 2.757 20.001 2.030 20.001

Influence of a-(2+3)-Linked NeuAc on the Chemical Shifts of Structural-reporter Groups of Neighboring Residues" bs (p.p.m.) *s.d. (p.p.m.)

H-1 of Gal ( X = 2; y = 3 016) H-1 of Gal ( x = 4; y = 3) H-3 of Gal ( x = 2 or 4; y = 3 or 6) H-1 of GlcNAc ( x = 2; y = 3 or 6 ) H-1 of GlcNAc ( X = 4; y = 3) NAc of GlcNAc ( x = 2 or 4; y = 3 or 6 )

+0.076 20.001 +0.083 20.002 +0.453 k0.003

-0.004 20.002 +0.005 20.002

-0.003 20.001

Values of AS are mean values 2s.d., at pD -7 and T = 300 K, calculated from asialo + (2+3)-sido steps (see Tables IV,V, VIII,XI,XII,and XV).

J. F. G . VLIEGENTHART et al.

306

CHART3 Symbols Employed" for Compounds 42-54

42

>:[:

1 Xaa

45

ASll

Thr

Xaa I

43

44

49

2

__

Asn

L >AS"

51

9

dT thr y

Asn I

G,~Y Thr

46

47

48

5

53 Xaa

Asn

'' For the key to the symbolic notation, see Chart 1.

are potentially N-glycosylically linked to a peptide backbone, one or more Fuc groups occur as terminal mon~saccharide(s).'-~ (i) When a Fuc group is linked to the trimannosyl-N,N'-diacetylchitobiose core, reliable (n.m.r.) evidence e ~ i s t s ' only ~ * ~for ~ an a-(1-6) linkage to GlcNAc-1. Compounds 2 and 4, containing this structural unit, have already been discussed; several others will be treated in this Section.

c

W

n P

m

b

grn

W

A-

E

3

E

t

c

c

--tA P

P

8

z

c

4: Y

--t

6

5-

308

J. F. G. VLIEGENTHART et al.

(ii) Another, well defined type of Fuc linkage is the a-(1+3) attachment to a peripheral, GlcNAc residue forming part of an asialo N-acetyllactosamine branch. Compounds 46 to, and including, 53 contain the structural unit pGal( 1+4)[aFuc( l-S)lpGlcNAc( l-*). (iii) Finally, Fuc may be present as the terminal group in N-acetyllactosamine-type structures, in a-(1+2) linkage to a Gal residue. An example of a compound containing the aFuc(1+2)pGal( 1+4)PGlcNAc(l4.) moiety is the glyco-asparagine 54. (i)Fuc a-(1+6)-Linked to GlcNAc-1.-Compound 42 is a diantennary, monosialo glycopeptide bearing an additional Fuc in a-(1-6) linkage to GlcNAc-1. Compound 42 was obtained from human lactotransferrin8’ in a mixture of glycopeptides consisting of 42 and 52 in the ratio of 3: 1. The 500-MHz, ‘H-n.m.r. spectrum of this mixture is given in Fig. 37, and the n.m.r. data for 42 are compiled in Table XV. For the spectral interpretation, the carbohydrate chain of 42 is regarded as an extension of that of the diantennary, monosialo glycopeptide 31, with a Fuc residue a-(1+6)-linked to GlcNAc-1. The chemical shifts of the Fuc structural-reporter groups, namely, H - l ( 6 4.877), H-5 (6 4.125), and CH, (6 1.208),are in perfect agreement with those reported for 4 (see Table 11). For the protons of the CH, group, two doublets are observed, at 6 1.206 and 1.210, probably due to the heterogeneity of the peptide moiety. The effects on the chemical shifts of structural-reporter groups of neighboring residues, due to the attachment of Fuc in a-(1-6) linkage to GlcNAc-1, are restricted to H-1 (A6 0.062 p.p.m.) and the N-acetyl protons of GlcNAc-2 (At3 0.016 p.p.m., in comparison to 31). All other n.m.r. parameters of 31 are found, essentially unaltered, in the spectrum of 42. The value of the chemical shift of H-1 of Man-4 shows that 42 contains NeuAc in ~ ~ ( 2 - 6linkage ) to Gal-6 (see Tables VI and XIII). Compound 43 is a diantennary, sialo glycopeptide containing a terminal a-(2+6)-linked NeuAc group in each branch, and a Fuc group a-(1+6)-linked to GlcNAc-1. Compound 43 was derived from human lactotransfenin.81It could also be identified as a minor constituent of a complex mixture of glycopeptides that additionally contained compounds 34 and 53 and a glycopeptide analog of 41. This mixture was derived from human-plasma ceruloplasmin; its SO-MHz, ‘H-n.m.r. spectrum is given in Fig. 38. The relevant n.m.r. data for 43 are included in Table XV. As compared to the spectrum of the afuco analog 33, the spectrum of 43 reveals three additional, structural-reporter-group signals, namely,

-

(81) G. Spik, G. Strecker, B. Foumet, S. Bouquelef J. Montreuil, L. Dorland, H. van Halbeek, and J. F. G. Vliegenthart, Eur. 1. Biochem., 121 (1982) 413-419.

'H-N.M.R. SPECTRA OF CLYCOPROTEIN CARBOHYDRATES

309

the Fuc H-1, H-5, and CH, resonances, in conjunction with downfield-shift effects upon the H-1 andN-acetyl proton signals of GlcNAc2. The step from 33 to 43 is completely analogous to that from 31 to 42. The set of chemical shifts of these five structural-reporter groups is typical for the presence of a Fuc group a-(1+6)-linked to GlcNAc-1 of the N, N '-diacetylchitobiose core-region of carbohydrate chains that are N-glycosylically linked. Compounds 44 and 45 are diantennary glyco-asparagines termianomeric protons

Man H-2 atoms

I

I

H-3 v,44

1

UFUC

11-111

4.44

I

4;44

z2J-AsnhA uFucI1-61

H-5

52

-6

50

&8

46

i 2

6'

NAc -CH3 protons

Fuc -CH3 protons

H-3a atoms

m

M A C 7'.

M"rs:44

FUC1j-W

n

t4..*.;44

18

+

FIG.33.- Structural-reporter-group Regions of the Resolution-enhanced, W-MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 44 and 45 in the Ratio of 5 : 1. [The bold numbers in the spectrum refer to the corresponding residues in the structures (see p. 307), and the italic numbers to the compounds in the mixture. Signals of corresponding protons in the two components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance, as well as the H-1 signal of Man-3, have been omitted from the spectrum; their position is indicated by an arrow. The sample was contaminated with free NeuAc, of which only the p anomer is detectable in this spectrum (SH3a 1.829, S H 3 e 2.210, and GNAc 2.050).]

TABLEXV 'H Chemical Shifts of Structural-reporterGroups of Constituent Monosaccharides for Glycopeptides of the N-Acetyllactosamine Type, Containing Fuc in a-(1+6) Linkage to GlcNAc-1 (Compounds 42-45) Compound and schematic structure 42

Reporter group

Residue

H-1 of

1 2 3 4 4' 5 5' 6 6' 3 4 4'

H-2 of

43

s = i " -' 5.102" 4.682" 4.771 5.134 4.927 4.605 4.579 4.445 4.472 4.256 4.191 4.112

5.045 4.684 -4.77 5.131 4.946 4.607 4.607 4.444 4.446 4.257 4.200 4.112

44 Xaa

otc

Xaa I

&+*

>.2

5.068 4.682 -4.77 5.136 4.922 4.606 4.575 4.444 4.549 4.253 4.197 -4.11b

-*> 45

- Asn

,"-

AS"

5.068 4.682 -4.77 5.118 4.940 4.575 4.606 4.545 4.447 4.253 4.190 -4.11b

H 3 of

H-1 of H-5 of CH, of

6 6' aNeuAc(2+6) aNeuAc(2+3) aNeuAc(2+6) aNeuAc'(2+6) aNeuAc(2+3) aFuc(l+6) aFuc(1+6) aFuc(l+6)

NAc of

1

H-3a of H3e of

2 5 5' aNeuAc(2+6) aNeuAc(2+3)

<4.0 <4.0 1.720

-

2.667

4.877 4.125 1.206" 1.210" 2.006" 2.016" 2.094 2.069 2.048 2.030

2.760 4.876 -4.12b 1.202

-4.11b ~4.0 1.717 1.BOO 2.671 2.758 4.876 -4.12b 1.202

2.020

2.012

2.012

2.094 2.065 2.065 2.030d

2.094 2.069 2.044 2.030" 2.032'

2.094 2.048 2.066 2.030" 2.032'

<4.0 <4.0 1.717" 2.670 2.673 4.873 -4.12 1.200

<4.0 -4.11b 1.717 1.802 2.669

-

Signals stemming from the main component(s)with respect to the heterogeneity of the peptide moiety. Values could not be determined more accurately (kO.01 ppm), due to partial overlapping of the H-5 signal of Fuc, the H-2 signals of Man-4', and the H 3 signals of Gal-6 in the subspectrum of 45 and -6' in that of 44. Signal of two protons. Signal of two methyl groups. Assignments may have to be interchanged. (I

312

J. F. G. VLIEGENTHART et u1.

nated by differently linked NeuAc groups and bearing Fuc in a-(1+6) linkage to GlcNAc-1. These compounds were obtained in a mixture, containing 44 and 45 in the ratio of 5: 1, from horse-pancreatic ribonuc l e a ~ e . ~ The * ~ *500-MHz, *~~ 'H-n.m.r. spectrum of the mixture is presented in Fig. 33; the n.m.r. parameters for 44 and 45 are summarized in Table XV. For the spectral interpretation, the glyco-asparagines are conceived of as extensions of the corresponding oligosaccharides ending in GlcNAc-2 (for example, 39), with an aFuc( 1+6)PGlcNAc( l-+N)Asn moiety. The presence of GlcNAc-1 N-glycosylically linked to Asn is evident from the H-1 doublet at 6 5.068, and the singlet of its N-acetyl protons at 6 2.012. The H-1 and N-acetyl signals of GlcNAc-2 are found at the same positions as described for compound 4 (see Table 11). In combination with the chemical shifts of the Fuc structural-reporter groups (6H-14.876,aH-5 -4.12, and 6CH3 1.202),the chemical shifts of the GlcNAc-2 reporter groups are characteristic for the a(1+6) type of linkage of Fuc to GlcNAc-1. The total pattern of the H-1 and H-2 signals of Man-3, -4, and -4' is indicative of the diantennary type of structure (compare Table VI and Fig. 14a). The spectral parameters of the upper- and lower-branch residues of 44,from Man4-4' up to, and including, the NeuAc residues, are identical with those for the fl anomer of oligosaccharide 39 (see Table XII). The presence in the mixture of the isomeric, minor compound 45, containing NeuAc in a-(2+3) linkage in the upper branch, and NeuAc' in a-(2+6) linkage in the lower branch, is proved by the occurrence of relatively low Gal H-1 doublets at 6 4.545 (Gal-6) and 6 4.447 (Gal-6'), and of N-acetyl signals of GlcNAc-5 and -5' at 6 2.048 (82) B. L. Schut, L. Dorland, J. Haverkamp, J. F. G. Vliegenthart, and B. Foumet, Biochem. Biophys. Res. Commun., 82 (1978) 1223-1228. ~

~~

~

FIG. 34.- Structural-reporter-group Regions of the Resolution-enhanced, W M H z , IH-N.m.r. Spectrum of a Mixture Containing Compounds 46,47, and 54 in the Ratios of 1:6: 1. [The bold numbers in the spectrum refer to the corresponding residues in the structures, and the italic numbers to the compounds in the mixture. Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance has been omitted from the spectrum; its position is indicated by an arrow. In addition, the sample contained one (or more) structurally related, difuco glyco-asparagine(s). The presence of a mono-antennaxy, lower-branch analog of 47 that contains a repeating N-acetyllactosamine moiety, namely, pGal(1 4 X a F u c (1+3)@GlcNAc( 1+3)~Gal(14)GlcNAc,p(1-+2)-linked to M a n 4 , may be suggested, mainly on the basis of the signals marked by asterisks (see Ref. 13).]

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

313

-Gal-(1-4)-,6?-GlcNAc-(1-2)-a-Man-(l-3)

'

a-Fuc -(1-

/

3)

$

\f3

f3 -GlcNAc-(I-

-Edan-(1-4)-

a-Fuc-(1-

4)-p -GlcNAe-(l-

/

N)-As.

=

6)

46

a-Fuc-(l-3)

\ f3 -Gal-(l-4)-@ -GlcNAc-(15'

6'

f3 -Man- (1-

4)- f3 -GlcNAc- (12

%)-a-Man-(l- 6)

a-Fw-(l-6)

4)- f3 -GlcNAc - (1-

/

N)-A!

'

4'

47 f3 -Man- (1-

a-Fuc-(1-

4)- f3 -GlcNAc - (1-

4)-

2)-~-Gal-(l-4)-f3-Gl~NAc-(1-2)-a-Man-(l---6) 6'

41

5'

f3 -GlcNAc- (1- N)-Am a-Fuc-(l-6) 54 anomeric protons I

3,47+54

1

NAc -CH3 protons

2,47+54

I

Man H-2 atoms

4;47+54

547

I'

2,47+54

TABLEXVI 'H Chemical Shifts of Structurat-reporterGroups of Constituent Monosaccharides for Glycopeptides of the N-Acetyllactosamine Type Containing a-(l+3)-Linked Fucose (Compounds 46-51)

Compound and schematic structure 46

48

47

a+:;; 7 50

49

51

11

* 4%

i. *\As"

Reporter group

H-1of'

F

A

s

n

.-*\

c , ) ; c c c A s n

$--hF

Thr

Residue 1

2 3

4 4' 5 5' 6 6'

5.073 4.677 4.775 5.110

5.073 4.687 4.764

-

4.906

-

4.585

-

-

4.585

4.442

-

-

4.449

5.071 4.616 4.756 5.116 4.925 4.568 4.580 4.464 4.474

5.067 4.611 4.758 5.124 4.866 4.570 4.594 4.462 4.470

5.067 4.611 4.758 5.124 4.866 4.570 -4.61" 4.468 4.462

:c

\-Am

5.067 4.611 4.758 5.124 4.866 4.570 4.594 4.468 4.470

G,' Y Thr

H-2 of H 3 of

7 7' 8 8' 3 4 4' 4 4'

W . i

VI

H-1 of H-5 of CH, of H-1 of H-5 of CH, of NAc of

aFuc(l+3) aFuc(l+3) aFuc(l+3) c~Fuc(l+6) aFuc(l+6) aFuc(l+6) 1 2 5 5' 7 7'

4.228 4.187

-

C4.0 5.126 4.830 1.172 4.876 4.126 1.206 2.016 2.090 2.043

-

-

-

-

4.557

-

4.447

-

4.081 4.087 c4.0 5.127 4.830 1.177 4.876 4.126 1.206 2.016 2.095 2.040

-

-

4.212 4.219 4.112 4.038 ~4.0 5.112 4.835 1.176

-

2.013 2.078 2.049 2.045 2.067 -

4.558 4.550 4.447 4.479 4.209 4.222 4.092 4.052 <4 .O 5.112 4.832 1.176

-

2.006 2.076 2.052 2.040 2.067 2.038

4.545 4.550 4.462 4.479 4.209 4.222 4.092 4.052 <4 .O 5.124 4.832 1.178

-

4.545 4.563" 4.462 4.473 4.209 4.222 4.092 4.052 C4.0 5.112 4.832 1.177 -

-

-

-

2.006 2.076 2.052 2.033 2.077 2.038

2.006 2.076 2.052 2.040 2.077 2.029

a Tentative assignments, because of the low amount of compounds 50 and 51 in the mixture with 49, in combination with the relative broadness of the signals.

316

J. F. G . VLIEGENTHART et. al.

and 2.066, respectively. In comparison to the diantennary, asialo glycopeptide 8, these signals show shift influences that are characteristic for the a-(2-3) type of NeuAc linkage to Gal in the upper branch, and the a-(2+6) linkage type in the lower branch (see Tables XI11 and XIV). In accordance with this interpretation, the H-1 signals of Man-4 and - 4 ' for 45 are observed at 6 5.118 and 4.940, respectively. Their relative intensities correspond with those of the Gal H-1 doublets of 45. It should be mentioned that the chemical shifts of the H-1 signals of G a l 4 and -6' in the spectra of 44 and 45 lend independent support for the assignment of the Gal H-1 doublets in the asialo afuco glycopeptide analog 8 (compare 31; see Ref. 66). The coincidence of the H-3a signals of the a-(2+6)-linked NeuAc groups for both compounds is in line with the observation for 33. The assignments of the well separated H 3 a signals of the cr-(2+3)-linked NeuAc groups, and also those of the H 3 e signals of the a-(2+6)linked NeuAc groups, are based on their relative intensities; they are in accord with those for 38 and 33,respectively. Finally, the H-3e signals of the a-(2+3)-linked NeuAc groups are observed separately, in contrast to the situation for 38.The assignment given in Table XV is based on the relative intensities of the signals. (ii) Fuc a-(l+3)-Linked to a Peripheral, GlcNAc Residue.-Compound 46 is a mono-antennary glyco-asparagine bearing two differently linked Fuc groups. Compound 47 is an isomer of 46, deviating in the type of a-glycosidic linkage between the Man residues. Both compounds occur, together with 54, in a mixture (ratios of 46: 47 : 54 = 1:6 : 1)isolated from the urine of a patient suffering from f ~ c o s i d o s i s .The ~~~ 500-MHzY ~ ~ * ~ 'H-n.m.r. spectrum of this mixture is given in Fig. 34. The n.m.r. data for 46 and 47 are presented in Table XVI . The occurrence of the @Man(1+4)@GlcNAc(1+4)[aFuc( 1+6)]pGlcNAc(l+N)Asn moiety in compounds 46 and 47 may be inferred from comparison of the n.m.r. data for 46 and 47 with those for 4 and 42-45 (see Tables I1 and XV). The presence of Fuc a-(1+6)-linked to GlcNAc-1 is obvious from the chemical-shift values of its H-1, H-5, and CH, signals, and from its J1,2 value (3.6 Hz), as well as from the effects on the chemical shifts of H-1 and the N-acetyl protons of GlcNAc-2, as compared to an afuco structure (compare the steps from 3 to 4, from 31 to 42, and from 33 to 43). The chemical shifts of the structural-reporter groups of Fuc in a-(1+6) linkage to GlcNAc-1, together with the influences of its introduction upon the chemical-shift values of reporter groups of neighboring residues, are summarized in Table XVII. The resonance position of H-2 of M a n 3 (6 4.228) indicates that this residue in 46 is substituted only at 0-3by another Man residue (com-

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

317

TABLEXVII Recognition of a-(1+6)-Linked, Terminal Fuc in the Sequence flGlcNAc(l+l)[aFuc( 1+6)wGlcNAc( l+N)Asn 'H Chemical Shifts of Structural-reporter Groups of a-(l+B)-Linked Fuc" Reporter group H-1 H-5 CH,

6 ?s.d.

4.876 kO.001 4.125 20.003 1.207 *0.003b

Influences of a-(1+6)-Linked Fuc on the Chemical Shifts of Structural-reporter Groups of Neighboring ResiduesC

AS (p.p.m.)

2

s.d. (p.p.m.) H-1 of GlcNAc-1 NAc of GlcNAcl H-I of GlcNAc-2 NAc of GlcNAc-2

+O.Old +0.008d +0.068 20.004 +0.016 20.002

a Mean values ?s.d. at T = 300 K; see Tables I, 11, XV, XVI, and XVIII (the mean value for 6H-1 was derived from Tables 11, XV, XVI, and XVIII). The value of 6CH, depends slightly on the composition of the peptide moiety (see, for example, compounds 42 and 52). Values of A6 are mean values +-s.d.,calculated for afuco + (1+6)fuco steps (see Tables I, 11, V, X, XV, XVI, and XVIII); comparison may be made with the values of A6 for H-1 and NAc of H GlcNAc-1, and for H-1 of Gal-a, for a similar step in Table XXIII. These effects are barely traceable, as they are negligible compared to the influences of changes in the pD of the solution, or in the composition of the peptide part.

pare 17 and 21; see Tables VIII and IX). Small differences between the chemical shifts of the structural-reporter groups of GlcNAc-2, and H-1 of Man-3, of 46 as compared to 4 (and to 47!), also reflect mono-a(143) rather than -a-(1+6) substitution of Man-3 in 46 (see later). Concerning the peripheral part of the molecule, 46 may be regarded as an extension ofthe (B anomer ofthe) mono-antennary, upper-branch, asialo oligosaccharide 17, with Fuc a-(1+3)-linked to GlcNAc-5. This additional Fuc manifests itself by its structural-reporter-group signals at positions clearly distinguishable from those of the a-(1+6)-linked Fuc already mentioned: 6H-1 5.126, 6H-5 4.830, and 6CH3 1.172. TheJl,z value of the (1+3)-linked Fuc (4.1 Hz) indicates an a linkage. The rather low-field, resonance position of H-5 of Fuc is indicative of a location of this residue in vicinal position to Gal, both being s ~ b s t i t u e n t s ~of ' , ~GlcNAc. ~ (83) R. U. Lemieux, K. Bock, L. T. J. Delbaere, S. Koto, andV. S. Ra0,Con.J. Chem., 58 (1980)631-653; R. U. Lemieux, D. R. Bundle, and D. A. BakerJ. Am. Chem. SOC., 97 (1975) 4076-4083.

318

J. F. G. VLIEGENTHART et al.

Furthermore, the introduction of this type of Fuc causes some shift effects on structural-reporter groups of neighboring residues. Most specific is the upfield shift of the N-acetyl protons of GlcNAc-5 (AS -0.01 p.p.m.). Also, the H-1 doublet of Gal-6 is shifted upfield (AS - 0.026 p.p.m.) towards F 4.442. Moreover, the chemical shifts of H-1 of Man-4 (A8 -0.017 p.p.m.) and H-1 of GlcNAc-5 (AS + 0.008 p.p.m.), both compared to 17p, are significantly influenced. The introduction of Fuc a-(1+3)-linked to GlcNAc-5 does not cause strong changes in line widths of structural-reporter-group signals of neighboring residues . In 47, the main component of the mixture, Man-3 is mono-a-( 1-6)substituted by Man-4'. This is obvious from the chemical-shift values of H-1 and H-2 of M a n 3 (compare compounds 3,4,18,22,61, and 62; see Tables 11, VIII, IX, and XXIV). The set of chemical shifts for H-1 and the N-acetyl protons of GlcNAc-2 [despite the alterations caused by Fuc in a-(1+6) linkage to GlcNAc-1] is also indicative of the a(1-6) type of mono-substitution of Man-3 (compare 46), as has already been pointed out for mono-antennary oligosaccharides (for example, 17, 18, 21, 22, and 35). The presence of Fuc in a-(1+3) linkage to GlcNAc-5' is revealed in the resonance positions of H-1, H-5, and its CH, protons (6 5,127, 4.830, and 1.177, respectively), as well as in its J1,2 value (4.1 Hz). It should be noted that the chemical shifts of the CH,-group protons of 46 and 47 differ clearly. On comparing the n.m.r. data for 47 with those for the p anomer of 18 (see Table VIII), some characteristic shift increments and decrements of reporter groups may be ascribed to the introduction of Fuc a-(l-+3)-linked to GlcNAc-5'. Although the chemical shift of H-1 of 5' FIG.35.-Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 48. [The bold numbers in the spectrum refer to the corresponding residues in the structure. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other parts of the spectrum, as indicated. The HOD resonance, as well as the H-1 signal of Man-3, have been omitted from the spectrum; their positions are indicated by arrows. The signal marked by 4 originates from a frequently occurring, nonprotein, noncarbohydrate contaminant of unknown strucwre. The sample was contaminated with a small proportion of a diantennary, asiaio glycopeptide, containing Fuc in cr-(1+6) linkage to GlcNAc-I, that is depicted as follows. This can be inferred from the signals marked by asterisks (compare Ref. 84.1 p -Gal-(&-6

4)-p-GlcNAc-(l5

2)-a-Man-(1-3)

'

o-GaI-(l- 4)-p-GlcNAc-(l-l)-a-Man-(1-6) 6'

5'

I'

I

0 -Man- (1-

IS

4)- p -GlcNAc -(I-

4)- p -GlcNAc - (1-

2

*a-Fuc-(1-

/

6)

I

N)-Asn ]

4 t

c

u)

a

0

C

-P .-0 2 E

e m

* *

*

*

J. F. G. VLIEGENTHART et ~ l .

320

remains essentially unaltered, the N-acetyl signal of this residue shows a highly significant, shift decrement upon attachment of Fuc: A6 - 0.007 p.p.m. Furthermore, H-1 of Gal-6' undergoes a shift decrement from 6 4.472 to 4.449. Finally, the chemical shifts of H-1, as well as of H-2 of Man-4', in 47 are different from those in IS@, which may be due to the presence of Fuc. Most of the shift increments and decrements mentioned are similar for the a-(1+3)-linked Fuc in 46 and 47.Therefore, they are characteristic for attachment of Fuc in a-(1+3) linkage to GlcNAc of a PGal( l+4)pGlcNAc( l+2)aMan( 1-) moiety, and the chemical shift of the CH, group of Fuc might be a helpful parameter for its localization in a certain branch. Compound 48 is a triantennary glycopeptide containing a terminal Fuc group a-(1+3)-linked to GlcNAc-7. This compound was derived from asialo a,-acid g l y c o p r ~ t e i n ~(peptide ~-~ moiety: Asn-Lys) and from asialo ceruloplasmirP (peptide moiety: Asn). The 500-MHz, 'Hn.m.r. spectrum of the glycopeptide obtained from the latter source is shown in Fig. 35; its spectral parameters are listed in Table XVI. The type of branching of the trimannosyl-N,N'-diacetylchitobiose core is evident from the set of chemical shifts of the H-1 and H-2 signals of Man-3, -4, and -4' (compare Table VI and Fig. 14a). For the spectral interpretation, 48 is considered to be an extension of its afuco analog, 9. The presence of a Fuc group a-(1+3)-linked to GlcNAc in the structural unit @Gal(l+ri)[aFuc( 1-+3)@GlcNAc(I+.) may be inferred from the chemical shifts and the J1.2 value (3.6 Hz) of the Fuc structural-reporter-group resonances, which are essentially the same as those for 46 and 47 (see Table XVI). However, the chemical shift of H-1 of Fuc a-(l-+3)-linkedto GlcNAc-7 is significantly different from that for Fuc a-(1-+3)-linked to GlcNAc-5 (46)or -5' (47). Among the shift effects brought about by the introduction of Fuc into 9, the shift decrement of the N-acetyl protons of GlcNAc-7 (A6 - 0.008 p.p.m.) is the most typical; the resonance positions of all other N-acetyl signals remain unchanged in comparison to those of 9. Furthermore, H - l of Gal-8 undergoes a significant, upfield shift, from 6 4.462 for 9 to 6 4.447 for 48.The H-1 doublet of GlcNAc-7 itself is shifted downfield (A6 0.012 p.p.m.), and is somewhat broadened. Also, the chemical shift of H-1 of Gal-6 is affected by the attachment of Fuc in a-(1+3) linkage to GlcNAc-7 (A8 -0.004 p.p.m.). This secondary effect probably originates from close spatial proximity of Fuc linked to GlcNAc-7 and the anomeric proton of Gal-6. For the characterization (84) M. Endo, K. Suzuki, K. Schmid, B. Foumet, Y. Karamanos, J. Montreuil, L.

Dorland, H. van Haibeek, and J. F. G. Vliegenthart,]. B i d . Chern., 257 (1982)

8755-8760.

'H-N.M.R. SPECTRA OF CLYCOPROTEIN CARBOHYDRATES

321

of the location of Fuc in this triantennary structure, the combination of these effects, together with the chemical shift of H-1 of Fuc itself, is decisive. Compounds 49,50, and 51 are isomeric, tetra-antennary, monofuco glycopeptides differing in the type of glycosidic linkage of the peripheral GlcNAc to which Fuc is attached. These glycopeptides were prepared in a mixture, containing 49,50, and 51 in the ratios of 12:2 :3, from asialo a,-acid g l y c o p r ~ t e i n . The '~~~ 500-MHz, ~ ~ ~ ~ 'H-n.m.r. spectrum of this mixture is presented in Fig. 36; the pertinent n.m.r. parameters for the three constituents are summarized in Table XVI. The tetra-antennary type of branching of the trimannosyl part of the pentasaccharide core in the three glycan chains may be deduced from the set of chemical-shift values for the H-1 and H-2 signals of Man-3, -4, and -4' (see Fig. 14a, Table VI, and the h c o analog of the compounds, namely, 13). In the main component (49) of the mixture, the terminal Fuc group is a-(1+3)-linked (6H-15.112,6H-5 4.832, and SCH, 1.176;J,,23.8 Hz) to GlcNAc-7. The location of this Fuc group is primarily evident from the shift decrement of the N-acetyl protons of GlcNAc-7 from 6 2.077 to 2.067, which is in accord with that described for the step from 9 to 48 (see also, Table XIX). The decrease of the intensity of the N-acetyl signal at 6 2.077 due to attachment of Fuc to GlcNAc-7 (compare Fig. 36 with Figs. 13 and 14a) gives independent proof of the assignment of the N-acetyl signals at 6 2.079 and 2.078, given before for the tetra-antennary, afuco compound 13 (see Table V). Owing to the introduction of Fuc a-(&+3)-linked to GlcNAc-7, H-1 of Gal-8 (A6 - 0.015 p.p.m.), H-1 of GlcNAc-7 (A6 0.013 p.p.m.), and H-1 of Gal-6 (A6 - 0.006 p.p.m.) show changes in their chemical shifts, in comparison to 13, that are very similar to those observed for the corresponding protons going from 9 to 48. Again, the H-1 signal of GlcNAc-7 is significantly broadened. Compound 49 was earlieP judged to be homogeneous (by 360MHz, 'H-n.m.r. spectroscopy). However, the presence of two minor components, namely, 50 and 51 in the actual mixture can be clearly seen from the N-acetyl region of the 500-MHz spectrum of this Sample; this conclusion is supported by the Occurrence of three partially overlapping Fuc CHS signals, at 1.17 < 6 < 1.18 p.p.m., and of more than one Fuc H-1 signal at 5.11 < 6 < 5.12 p.p.m. The locations of the Fuc groups in 50 and 51 may be inferred from the N-acetyl region of the spectrum. In this region, nine singlets are (85) H. van Halbeek, L. Dorland, J. F. G . Vliegenthart, J. Montreuil, B. Foumet, and K. Schmid, J. B i d . Chm.,256 (1981) 5588-5590.

\

P -Gal 6 - (l-+

4)-

a-Fuc-(1-

3)

p -GlcNAc 5 - (1- z)-cy-Man4 (1- 3)

p -Man-(1-

\ fi-Ga,l-(l-+4)-p-GlcNAc-(l8

p -Gal- (1-

5

4)- 6-GlcNAc - (1-

8’

‘

\

2)-a-Man-(1-

/

6)

/

6)

4)- p -GlcNAc-(12

4)- p -GlcNAct (I--CN)-Asn(NHz) I GfY

3

Thr(COIH)

*’

7‘

50

p -Fl-(l-

4)- p -GlcNAc -(1+ 4)

p -Gal-(1-

4)-p-GlcNAc-(l-

7

2)-a-Man-(l-

5

6

3)

4

\p-Man-(l--

p -Ga~-(1+4)-~-GlcNAc-(l-2)-a-Map-(l-6) 6

/3 - Q,l-(18

/

3

/

CY-FUC - (1- 3)

4)-p -GlcNAc-(1-N)-Am I (NHz) G~Y mr(C0,H)

5

4) - -GICNAC-( 1-

4)-p -GlcNAc-(1-

/

4

6)

7’

51

'H-N.M.R. SPECTRA O F GLYCOPROTEIN CARBOHYDRATES

323

s+fi

anmeric protons 4~ G 5 1 49 7

n

49+50+51

5ox51

*

W+51

h a d r 6

Man H-2 atoms anorneric protons I

1

NAcCH3 protons 5

Fuc -CH3 protons 0 Fur 11-31

F"C,*

31

A

Fur Fuc

-.5

*-

rm

I A v

d . 5

rm

206

ra

rnr

rm

'

mk ocl

11M

IM

IS

,

FIG.36.-Structural-reporter-group Regions of the Resolution-enhanced, 50()-MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 49,50, and 51 in the Ratios of 12 :2 :3. [The bold numbers in the spectrum refer to the corresponding residues in the structures, and the italic numbers to the compounds in the mixture. Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relative-intensity scale, as well as the chemical-shift scale, of the N-acetyl-proton region and the Fuc CH,-proton region (b), differ from those of the other parts (a) of the spectrum, as indicated. The HOD resonance, as well as the H-1 signal of Man3, have been omitted from the spectrum.]

324

J. F. C. VLIEGENTHART et al.

observed. The signals at 6 2.076,2.052, and 2.006, which are of equal intensity, are assigned to GlcNAc-2, -5, and -1, respectively, in accordance with 13 (see Table V). The remaining six signals may be divided into three pairs. The signals of each pair are separated by A6 -0.01 p.p.m. The signal pair at 6 2.077 and 2.067, having an intensity ratio of -2:3, stems from GlcNAc-7 (see earlier). The former signal corresponds to GlcNAc-7 without Fuc (50 and 51), the latter to GlcNAc-7 with Fuc (49). Similarly, GlcNAc-5’ gives rise to a pair of signals, at 6 2.040 and 2.033, without (49 and 51) and with (50) Fuc, respectively. Also, GlcNAc-7’ gives rise to a pair of singlets, at 6 2.038 and 2.029, without (49 and 50) and with (51) Fuc, respectively. (This may be compared with the assignment of the N-acetyl signals of GlcNAc-5’ and -7’ for oligosaccharides 11 and 12; see Table IV and Fig. 14b.) Therefore, 50 has its Fuc cr-(l+3)-linked to GlcNAc-S’, and 51 to GlcNAc-7’. The interpretation of the N-acetyl region is corroborated by the evaluation of the Gal H-1 region of the spectrum (see Fig. 36 and Table XVI). For instance, the H-1 doublet of Gal-8’ for 51 is found at 6 4.473, showing an upfield shift as compared to 13. It should be noted that the N-acetyl signals at 6 2.041 and 2.042 in the spectrum of 13 are suggested as belonging to GlcNAc-7’ and -5‘, respectively, based on the foregoing interpretation. The structural analysis of this glycopeptide sample (from asialo alacid glycoprotein) by W M H z , ‘H-n.m.r. spectroscopy, revealing a new type of microheterogeneity, gives a nice impression of the potency of this technique in detecting and identifying compounds in mixtures, even if they are present in low proportions. Compound 52 is a diantennary glycopeptide bearing NeuAc in a( 2 4 6 ) linkage to Gal-6, and also two differently linked Fuc groups, namely, a-(l-+6)-linked to GlcNAc-1, and a-(1+3)-linked to GlcNAc5’. Compound 52 has been obtained from human lactotransferrin,*l as well as from86human-milk secretory IgA, as the minor constituent of a 3: 1 mixture of glycopeptides, also containing 42. The W - M H z , ‘Hn.m.r. spectrum of this mixture is presented in Fig. 37; the n.m.r. data for 52 are compiled in Table XVIII. For the spectral interpretation, 52 is conceived of as an extension of 42 with a Fuc group a-(l+3)-linked to GlcNAc-5‘. The spectral parameters of the structural-reporter groups of the fucosylated core pentasaccharide of 42 are found unaltered in 52; the same holds for those of the sialylated, upper branch. It may be mentioned that the apparent heterogeneity of the peptide moiety of 52 (and 42) clearly affects the (86) A. Pierce-Crktel, M. Pamblanco, G . Strecker, J. Montreuil, G. Spik, L. Dorland, H. van Halbeek, and J . F. G . Vliegenthart, Eur. J . Biochem., 125 (1982)383-388.

m W

8

N t

m

v -

s n

?

I

*

t

4.V .

W

A-

W

t

W

t

E

t

I

(1

n

t

I

c

t

2

d

t c

t

I

J. F. C. VLIEGENTHART et al.

326

R

Man H-2 atoms

~d-1152

5

4

4

R FurR-LII

H-5

5

24

c 36

.

I

I 0

I)

t I4

11

I

FIG.37.- Structoral-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 42 and 52 in the Ratio of 3: 1. [The bold numbers in the spectrum refer to the corresponding residues ih the structures, and the italic numbers to the compounds in the mixture. Signals of corresponding protons in the two components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The signal marked by 41originates from a frequently wvumng, nonprotein, noncarbohydrate contaminant of unknown structure. For solvent-peak suppression, a water-eliminating, Fourier-transform (w.e.F.t.) pulse-sequence was used. The sample was contaminated with a very small proportion of an asialo analog of 42 or 52, or both, as can be inferred from the signals marked by asterisks.]

chemical shifts of the structural-reporter groups of GlcNAc-1 and -2, as well as that of the CH, protons of Fuc a-(l+fi)-linked to GlcNAc-1. The chemical shifts of the structural-reporter groups of the a-(1+3)linked Fuc group are in accord with those described for other compounds containing Fuc a-(l+3)-linked to GlcNAc-5' (47 and 50, see

TABLEXVIII 'H Chemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for Glycopeptides Containing Both NeuAc and Fuc as Terminating Groups (Compounds 52 and 53) Compound and schematic shucture -

~~

~~

~

52

Reporter group

Residue

H-1 of

1 2 3

4 4' 5 5' 6 6' 7 H-2 of

8 3

H-3 of

4 4' 4 4'

H-3a of

H-1 of H-5 of CH, of H-1 of H-5 of CH, of

NeuAc NeuAc' NeuAc NeuAc' aFuc(1-3) ruFuc(1-3) aFuc(1+3) aFuc(1-6) aFuc(1-6) aFuc(1-6)

NAc of

1

H-3e of

2

5 5' 7 NeuAc NeuAc'

Asn

Xaa

Xaa

5.102" 4 682" 4.771 5.134 4.910 4.605 4.605 4.445 4.447

-

4.256 4.191 4.112 C4.0 <4.0 1.720

-

2.667

-

5.123 4.829 1.179 4.877 4.125 1.206" 1.210" 2.006" 2.016" 2.094 2.069 2.042

-

2.030

5.036 4.615 -4.76 5.131 4.934 4.59 4.6W 4.444 4.446 4.557 4.446 4.218 4.218 4.112 4.043 <4.0 1.717 1.717 2.670 2.673 5.114 4.82b 1.176

-

2.004 2.079 2.065 2.065 2.068 2.030 2.030

Signals stemming from the main component(s) with respect to the heterogeneity of the peptide moiety. Values could not be determined more accurately (20.01 p.p.m.), due to the complexity of the mixture, of which compound 53 is one of the minor components (see also, legend to Fig. 38). (I

328

J. F. G . VLIEGENTHART et al.

Table XVI). The location of this Fuc group may be established on the basis of the shift effects upon structural-reporter groups of neighboring residues: the N-acetyl protons of GlcNAc-5' undergo a characteristic, upfield shift (from 6 2.048 for 42 to 2.042 for 52); H-1 of Gal-6' also shifts upfield (from 6 4.472 for 42 to 4.447 for 52), and, last but not least, H-1 of Man-4' is shifted significantly upfield from 6 4.927 in the asialo, lower branch of 42 to S 4.910 in the asialo, fucosylated, lower branch of 52 (compare 47). Compound 53 is a triantennary glycopeptide that contains two extending units, namely, NeuAc groups a-(2+6)-linked to Gal-6 and -6', and Fuc a-(l-+3)-linked to GlcNAc-7. This compound has been derived from human-plasma ceruloplasminMas a minor component of a mixture also containing compound 43, and another two triantennary glycopeptides, both terminated with a NeuAc group in all three branches, that is, a glycopeptide analog of oligosaccharide 41, and the Gin-Asn analog of compound 34 (compare Ref. 87). The 500-MHz, 'Hn.m.r. spectrum of this mixture is presented in Fig. 38; the pertinent n.m.r. parameters for 53 are listed in Table XVIII. The triantennary type of structure of 53 may be derived from comparison of the chemical shifts of the H-1 and H-2 signals of Man-3, -4, and -4' with those of 9 and 48 (see Table VI). The structural-reportergroup signals for the upper (4-5-6-NeuAc)and lower (4'-5'-6'-NeuAc') branch residues are in accord with those reported for compound 34 (see Table X). The occurrence of an a-(l-+3)-linked Fuc group attached to a peripheral GlcNAc residue is obvious from the set of chemical shifts of its structural-reporter groups (6H-1 5.114, 6H-5 -4.82, and SCH, 1.176; 6 8 - 5 could not be established more accurately, because of the complexity of the mixture of which 53 forms a part, and of its partial coincidence with the broad, HOD signal). The chemical shifts of the N-acetyl signal of GlcNAc-7 (6 2.068) and the H-1 of Gal-8 (6 4.446) prove the attachment of Fuc a-(1+3) to GlcNAc-7. The shift decrements for the latter reporter-groups, compared to 9, are in full accord with those observed for the asialo analog of 53,namely, 48 (see Table XVI). It should be noted that attachment of Fuc in a-(1+3) linkage to GlcNAc-7 also influences the chemical shift of H-1 of the sialylated Gal-6 (compare 34;see Table X).Therefore, it may be concluded that shift effects on structural-reporter groups of neighboring residues, induced by attachment of Fuc a-(1+3) to a peripheral GlcNAc (sum(87)K. Yamashita,C. J. Liang, S. Funakoshi, and A. KobataJ. Biol. Chern., 256 (1981) 1283-1289.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

329

anomeric protons

r

\

I._.....6 Man H-2 atoms

1

H-5

4.353

h

r

n

753

4.

1

H-3

r

7.53

.___

r i I "

"

i.

2.8

22

10

1.8

'V

FIG.38.-Structural-reporter-group Regions of the Resolutionenhand, 500-MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 53 and 43 in the Ratio of 1: 1. [The bold numbers in the spectrum refer to the corresponding residues in the s t r u c tures, and the italic numbers to the compounds in the mixture. Signals of corresponding protons in these two components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance, as well as the H-1 signal of Man-3, have been omitted from the spectrum; their positions are indicated by arrows. In addition to 53 and 43,the sample contains another two triantennary glycopeptides, both terminated with NeuAc in all three branches, that is, the analogs of 53 containing NeuAc* in a-(2-+3)anda-(2+6) linkage to Gal-8, respectively, instead of the Fuc group. In addition, the &co analog of 43 is present in this mixture to a small extent. This can be inferred from the signals marked by asterisks (compare Ref. a).]

marized in Table XIX),and those caused by extension of an N-acetyllactosamine unit in another branch with NeuAc in ~(2-3)or (~-(2-6) linkage (see Tables XIV and XIII,respectively), are independent of each other, and are additive.

330

J. F. G. VLIEGENTHART et al. TABLEXIX Recognition of a-(l+3)-Linked, Terminal Fuc in the Sequence f3Gal(l+4)[aFuc( 1+3)~GlcNAc(I-*x)aMan( l y ) f 3 M a n (1 4 . ) 'H Chemical Shifts of Structural-reporter Groups of rr-(l-+3)-LinkedFuc Reporter p u p

H-1 ( X = 2; y = 3) H-1 ( X = 2; y = 6) H-1 ( X = 4; y = 3) H-1 ( X = 6; y = 6) H-S(x= 2,4,or6;y=3or6) CH, ( x = 2; y = 3) CH, ( x = 2; y = 6) CH, ( x = 4; y = 3) CH, ( x = 6; y = 6)

6 2s.d.

5.126 k0.002 5.124 20.002 5.113 e0.002 5.112 e0.002 4.835 20.015" 1.172 20.002 1.178 e0.002 1.176 50.002 1.177 *0.002

Influence of a-(1+3)-Linked Fuc on the Chemical Shifts of Structural-reporter Groups of Neighboring Residues* bs (p.p.m.) 2s.d. (p.p.m.)

H-1 of ClcNAc ( x = 2, 4, or 6; y = 3 or 6) NAc of GlcNAc ( x = 2,4, or 6;y = 3 or 6) H-1 of Gal ( x = 2 or 4; y = 3 or 6) €I-1 of Man ( x = 2; y = 3 or 6) H-1 of Man ( x = 4; y = 3)

+0.011 k0.003 -0.009 e0.002 -0.025 20.002 -0.017 20.002 -0.004 rt0.002

" 6H-5 is extremely sensitive to changes in the temperature ofthe sample. * Values of A8 are mean values +s.d. at T = 300 K, calculated from afuco 4 (l+3)-fuco steps (see Tables V, VIII, X, XVI, and XVIII).

So far, we have never observed, by n.m.r. spectroscopy, oligosaccharides or glycopeptides bearing Fuc and NeuAc both linked to the same N-acetyllactosamine branch; this is in agreement with the biosynthetic principle of mutual exclusion existing in transferring (1-+3)linked a-fucosyl and (2+6)-linked a-sialyl groups to the same N-acetyllactosamine branch, as formulated by Hill and coworkers.8R (iii) Fuc a-(1+2)-Linked to #3Gal(1+4)GlcNAc.-Compound 54 is a mono-antennary glyco-asparagine bearing two terminating Fuc groups, namely, a-(1-+6)-1inked to GlcNAc-1, and a-(1+2)-linked to Gal-6'. This compound occurred, together with 46 and 47, in the ratios of 1: 1:6, respectively, in a mixture that could be isolated from the urine of a patient with f u c o s i d ~ s i s .The ~ ~ ,W ~ - M H z , lH-n.m.r. spec(88) J. C. Paulson, J . P. Prieels, L. R. Glasgow, and R. L. Hill,]. B i d . Chern., 253 (1978) 5617-5624; T. A. Beyer, J. E. Sadler, J. I. Rearick, J. C. Paulson, and R. L. Hill, Ado. Enzymol., 52 (1981) 23-175.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

331

trum of this mixture is depicted in Fig. 34.The n.m.r. data for 54 are compiled in Table XX, Mono-a-(1+6) substitution of Man3 in 54 is evident from the relative intensities of several structural-reporter-group signals (in the spectrum of the mixture) that are markers for the type of mono-substitution of Man-3; it may most readily be inferred from the N-acetyl singlets of GlcNAc-2 at 6 2.095 (lower branch) and 2.090 (upper branch), occurring in the ratio of 7: 1. As 47, the main component of the mixture, is a lower-branch structure, whereas the other minor component, 46, is an upper-branch glyco-asparagine (see earlier), it TABLEXX 'H Chemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for a Glyco-asparagine (Compound 54) of the N-Acetyllactosamine Type Containing a-(1+2)-Linked Fucose Compound and schematic structure

54 Reporter group

Residue

H-1 of

1 2 3 4 4' 5

J 5.073 4.687 4.764

-

4.918

5'

H-2 of H-1 of H-5 of CHSof H-1 of H-5 of CH3 of NAc of

-4.58"

-

6 6' 3 4 4' aFuc(1-2) aFuc(1+2) aFuc(1-2) aFuc(1-6) aFw(1+6) aFuc(1+6) 1 2 5

4.541 4.081

-

4.087 5.309 -4.22" 1.234 4.876 4.126 1.206 2.016 2.095 -

5'

2.072 ~

~

~

~

" Values could not be determined more accurately (kO.01p.p.m.1, due to the small amount of compound 54 present in the mixture with 46 and 47; moreover, the H-1 doublet of GlcNAc-S', as well as the H-5 signal of Fuc a-(1+2)-linked to Gal-6', are relatively broad-lined.

J. F. G . VLIEGENTHART et 01.

332

was concluded that 54 contains the lower branch. The chemical shifts of the structural-reporter groups of the @Man(1+4)@GlcNAc(1-4)[aFuc(l+G)]@GlcNAc(1 j N ) A s n moiety in 54 are identical to those for 47 (see Table XVI). Concerning the peripheral part of the structure, 54 is considered to be an extension of (the p anomer of) the mono-antennary, lowerbranch oligosaccharide 18, with Fuc a-(1+2)-linked to Gal-6'. This additional Fuc group is characterized by the following set of structuralreporter-group signals: 6H-1 5.309,6H-5 -4.22, and 6CH3 1.234. The jI2 value (-4 Hz) of the Fuc group is indicative of an a-glycosidic linkage. The introduction of this Fuc group causes some shift effects on structural-reporter groups of neighboring residues. The H-1 doublet of Gal-6' is shifted downfield to 6 4.541 (A6 + 0.069 p.p.m.); also, the N-acetyl signal of GlcNAc-5' undergoes a downfield shift (A6 + 0.025 p.p.m.), but a possible influence on the chemical shift of H-1 of 5' cannot be traced, due to the complexity of the mixture. The chemical shifts of the structural-reporter groups of Fuc in a(1-2) linkage to Gal of an N-acetyllactosamine branch, together with the influences of its introduction upon the chemical-shift values of reporter groups of neighboring residues, are summarized in Table XXI. TABLEXXI

Recognition of a-(l-+2)-Linked,Terminal Fuc in the Sequence aFuc(l+B)@Gal(1-+4)BGlcNAc(I+-)

'H Chemical Shifts of Structural-reporter Groups of a-(l+Z)-Linked Fuc" Reporter group H-1 H-5 CH,

6 2s.d.

5.311 20.002 4.224 20.002 1.237 ?0.003

Influences of a-(l--*2)-LinkedFuc on the Chemical Shifts of Structural-reporter Groups of Neighboring Residuesb

A8 (p.p.m.) ? s.d. (p.p.m.) H-1 of Gal H-1 of GIcNAc NAc of GlcNAc

+0.070 20.002 -0.008 20.004 +0.025 ?0.003c

" Mean values 2s.d. at T = 300 K; see Tables XX and XXIII. Values of A8 are mean values ts.d., calculated for afuco + (1+2)-fuco steps (see Tables VIII, XX,XXII, and XXIII). The value of A8 holds for a peripheral GlcNAc residue; for GlcNAc-1, see compound 57 as against 95.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

333

d. Carbohydrate Chains of the N-Acetyllactosamine Type Possessing the j3Gal(1+4)j3GlcNAc(l+N)Asn Moiety (Compounds 5560).-Symbols employed for compounds 55-60 are depicted in Chart 4. Compounds 55 through 60 are glyco-asparagines of the N-acetyllactosamine type lacking the trimannosyl-N,N'-diacetylchitobiose core. These compounds occur, in addition to large amounts of pGlcNAc(l+N)Asn (compound 1)and small amounts of aMan(1+6)pMan(1+4)pGlcNAc( 1+4)pGlcNAc(l+N)Asn (compound 3), in the urine and other body fluids of patients suffering from aspartylglucosaminuria. It has been s ~ g g e s t e d ~that ~ * pGlcNAc( ~ ~ * ~ ~ l+N)Asn can serve as an acceptor compound for various glycosyltransferases, leading to the synthesis of neutral (55-57) or acidic (58-60) glyco-asparagines having in common the PGal(1+4)PGlcNAc( l+N)Asn moiety. From the structural point of view, this series of relatively simple compounds offers the possibility of verifying and refining the n.m.r. parameters of the various units of extension of carbohydrate chains of the N-acetyllactosamine type, namely, Fuc and NeuAc, both in different types of linkage (compare, Tables XIII, XIV, XVII, and XXI). Moreover the parameters of an additional N-acetyllactosamine unit in p-( 1+3) linkage to Gal of the original unit can be established (see compound 60); such a structural unit is known to occur in various oligosac-

CHART 4

Symbols Employeda for Compounds 55-60

55 56

57

58 59 60 a

8-e-

Asn

TAsn r>--ee Asn

-

Asn

Asn Asn

For the key to the symbolic notation, see Chart 1.

(89) H. van Halbeek, L. Dorland, J. F. G . Vliegenthart, G . Strecker, J.-C. Michalski, J. Montreuil, W. Pollitt, and W. E. Hull, Eur. 1. Biochern., (1983) in press.

334

J. F. G. VLIEGENTHART et al.

charides isolated from the urine of patients with GM,-gangliosidosis,1*84a-w and in gly~oproteins.~-~*~~-~~~ Compound 55 is a linear glyco-asparagine isolated from the urine of a patient with aspartylglucosaminuria,5Z~89 as the main component of a mixture also containing 57. The 500-MHz, 'H-n.m.r. spectrum of this mixture is depicted in Fig. 39. The chemical shifts of the structural-reporter groups of 55 are included in Table XXIII. The attachment of Gal-a in /3-(1-+4) linkage to GlcNAc-1 of compound 1 is revealed in a significant downfield shifi of the H-1 signal of this GlcNAc residue (A8 -0.03 p.p.m.) in comparison to 1 (see Table I), as would be expected from the extension of 6 to 7 (see Table IV). The resonance positions of the structural-reporter groups of GlcNAc1, together with its relatively large],, value (9.8 Hz), are characteristic for the p-N-glycosylic linkage to Asn (compare 1).The chemical shift of the H-1 signal of Gal-a (6 4.481) reflects the (terminal) nonre(90)G. Strecker, J.-C. Michalski, B. Foumet, J. Montreuil, H. van Halbeek, L. Dorland, and J. F. G. Vliegenthart, unpublished results. (91) E. Li, R. Gibson, and S. Kornfeld, Arch. Biochem. Biophys., 199 (1980) 393-399. (92) H. Yoshima, S. Takasaki, and A. Kobata,J . Biol. Chem., 255 (1980) 10,793-10,804. (92a) T. Knisius, J. Finne, and H. Rauvala, Eur. J . Biochern., 92 (1978) 289-300.

TABLEXXII

'H Chemical Shifts and Coupling Constants for @Gal(l-+4)f3GlcNAc( l+N)Asn (Compound s * . - - e A n n ) ~

~

Residue

Proton

Chemical shift (p.p.m.)

GIcNAc- 1

H-1 H-2 H-3 H-4 H-5 H-6a H-6b NAc H- 1 H-2 H-3 H-4 H-5 H-6a H-6b H-u

5.101 3.880 3.778 3.754 3.678 3.941 3.837 2.018 4.481 3.549 3.667 3.927 3.708 3.785 3.742

H-P H-p'

2.875 2.942

Gal-a

Asn

3.985

Coupling constant (Hz) 11.2 12.3 13.4 J4.s J5.m J 5 m JOam

11.2

JZ.3 J3

.,

J*.S

Js.ea

15.ab Jeam

JCIB J d l

JB8,

9.8 10.0 8.75 2.05 2.25 4.6 - 12.3 7.9 9.9 3.4 0.9 8.7 3.5 -11.6 6.7 4.5 - 17.2

‘H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES p -Gal- (1-

4)- 0 -GlcNAc - (1-

a

1

N)-Am

a-Fuc - (1-

335

2)- j3-Gal-(l- 4)- j3-GlcNAc -(l- N) - A m a

55

1

57 anomeric protons

n-i 1.55 ,

5‘

52

-A

H-1 1.57

50

I@

46

11

12

NAc -CH3 protons

sugar skeleton protms.55 Fuc -CH3 protons

ti-5 1

I FY 11-21.57

H-4

n 8

H-8a 1

n-2

xh I

4 A M

P-W,

FIG.39.-Structural-reporter-group Regions, Together with the Sugar-skeleton-proton Region, of the Resolution-enhanced, 500-MHz, ‘H-N.m.r. Spectrum of a Mixture Containing Compounds 55 and 57 in the Ratio of 9: 1. [The bold number and letter in the spectrum refer to the corresponding residues in the structures, and the italic numbers to the compounds in the mixture. The relative-intensity scale of the N-acetylproton region differs from that of the other parts of the spectrum, as indicated. The signal marked by 4 originates from a frequently occurring, nonprotein, noncarbohydrate contaminant of unknown structure. The assignments indicated in the bulk region of the spectrum belong to the main component (55)of the mixture. They were proved by double-resonance techniques, namely, by pseudo-INDOR and by spin-tickling experiments (see Ref. 89). Surprisingly, the sample was contaminated with a small proportion of free NeuAc, as can be inferred, among others, from the N-acetyl signal at 6 2.048, marked by an asterisk.]

if

J . F. G . VLIECENTHART et al.

336

ducing position of this group in an N-acetyllactosamine unit; the J1,2 value (7.9 Hz) indicates a p-linkage. For the relatively simple compound 55, complete interpretation of all resonances in the 500-MHz, 'H-n.m.r. spectrum was achieved.89 The interpretation was confirmed, and refined, by computer simulation of the spectrum. The detailed, n.m.r.-spectral data for compound 55 are summarized in Table XXII. By means of a modified Karplus e q ~ a t i o n , 9the ~ 4C1(D) conformation of the GlcNAc residue, as well as of the Gal group, was d e d u ~ e d . 8 ~ Compound 56 is a branched glyco-asparagine possessing GlcNAc-1 as the disubstituted residue; it has been isolated from the urine of a The 360-MHz, 'Hpatient suffering from aspartylgluc~saminuria.~~~~~ n.m.r. spectrum of 56 is presented in Fig. 40. The chemical shifts of the structural-reporter groups of 56 are compiled in Table XXIII. For the spectral interpretation, compound 56 may be conceived of either as an extension of 55 with a Fuc group cr-(l+6)-linked to GlcNAc-1, or as an extension of 2 with Gal-a in /3-(1-+4) linkage to (93)C. A. G. Haasnoot, F. A. A. M. de Leeuw,and C. Altona, Bull. SOC. Chim. Belg., 89 (1980) 125- 131; Tetmhedron, 36 (1980) 2783-2792. -Gal -(1-

4)- f3 -GlcNAe-(1-

N)-Asn

a

a-Fuc -(1-

/

6)

l

56

Fuc -CH3 protons

I

ar"c,,-gl

anomeric protons I-

7

~

-

A

'0

30

I 0

I0

FIG. 40.-Resolution-enhanced, Overall, 36GMHz, 'H-N.m.r. Spectrum of Compound 56. [The bold number and letter in the spectrum refer to the corresponding residues in the structure. The signal marked by 4 originates from a frequently occurring, nonprotein, noncarbohydrate contaminant of unknown structure.]

TABLEXXIII 'H Chemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for Glyco-asparagines Possessing the flGal(l+4)flGlcNAc(l+N)Asn Moiety (Compounds SS-Sa) Compound and schematic structure

I H-Asn

Reporter p u p

Residue

H-1 of

GlcNAcl Gal-a GlcNAob Gal-c aNeuAc(2+6) aNeuAc(2-3) aNeuAc(2+6) aNeuAc(2+3) Gal-a Gd-c Gal-a Gal-c aFuc(1-6) aFuc(1+6) aFuc(1+6) aFuc(1+2) cxFuc(1+2) aFuc(1 4 2 ) GlcNAcl GlcNAc-b aNeuAc(2-6) aNeuAc(2+3)

Ma

H A of

H& of H 3 of H-4 of H-1 of H-5 of CH, of H-1 of H-5 of CH, of NAc of

5.093 4.552

-

2.677

-

-

<3.9

3.665

-

-

3.929

3.923

-

4.918 4.123 1.212

-

I

5.133 4.454 1.709

-

-

Asn

Asn

60 beeeeAsn

~~

5.117 4.538

-

2.018

59

58

TAsn ~

5.101 4.481

57

5.312 4.224 1.240 2.022

5.096 4.557

-

5.094 4.468 4.699 4 .!%a

-

1.796,

1.799

2.759 4.113

2.756 <4 .O 4.121 4.154 3.959

-

3.959

-

-

I

2.042

2.028 -

2.014 2.032

-

2.030

Measured at 360 MHz; T = 295 K. The value could not be determined more accurately (20.05 p.p.m.), due to the small amount of compound 57 present in the mixture with 95.

338

J . F. G . VLIEGENTHART et al.

GlcNAc-1. The set of chemical shifts of the Fuc H-1 and H-5 and the -CH, protons is indicative of this type of linkage to GlcNAc-1 (compare Table XVII). Besides for H-1 of GlcNAc-1, the presence of Gal-a, instead of GlcNAc-2, p-( 1+4)-linked to GlcNAc-1 causes a significant alteration in the chemical shift of H-1 of Fuc, as compared to the mean value for this proton, observed in structures containing the fucosylated N, N'-diacetylchitobiose moiety (see Table XVII). This is an additional proof of the influence of GlcNAc-2 on the chemical shift of H-1 of Fuc a-(l+6)-linked to GlcNAc-1, as described for compound 4 and extensions thereof (see Tables XV and XVI). As compared to 55, attachment of Fuc in a-(1+6) linkage to GlcNAc-1 hardly affects the chemical shifts of the structural-reporter groups of GlcNAc-1, whereas the H-1 signal of Gal-a undergoes a significant, downfield shift (A6 0.057 p.p.m.). The latter shift is in perfect agreement with that observed for H-1 of GlcNAc-2 in the case of attachment of Fuc in a-(146)linkage to GlcNAc-1 in the normal corestructure (compare Table XVII). Compound 57 is a linear glyco-asparagine containing a terminating Fuc group a-(l+2)-linked to Gal of the N-acetyllactosamine moiety (a1).This compound was isolated from the urine of a patient with aspartylglucosamin~ria,8~ as the minor (10%) component of a mixture containing 55 as the main constituent. The 500-MHz, 'H-n.m.r. spectrum of this mixture is presented in Fig. 39; the relevant, n.m.r.-spectral parameters for 57 are summarized in Table XXIII. Compound 57 may be considered to be an extension of 55 with Fuc a-(l+Z)-linked to Gal-a. The a-(1+2)-1inked Fuc group can be recognized from the following set of structural-reporter-group signals : 8H-1 5.312, SH-5 4.224, and 6CH3 1.240 (compare Table XXI). The shift effects brought about by the introduction of this Fuc group, as compared to 55, are similar to those described for the step from 1Sp to 54 (see Table XXI). The H-1 signal of Gal-a undergoes a considerable downfield shift (A8 0.071 p.p.m.); also, the N-acetyl signal of GlcNAc-1 is shifted downfield (A8 0.004 p.p.m.), whereas the chemical shift of the H-1 signal of GlcNAc-1 changes slightly upfield. Compound 58 is a sialo glyco-asparagine containing NeuAc in a(2-6) linkage to Gal-a; it has been isolated from the urine of a patient suffering from aspartylglucosaminuria.51~52~89 The 500-MHz7'H-n.m.r. spectrum of 58 is given in Fig. 41; its pertinent n.m.r.-spectral parameters are compiled in Table XXIII. The spectrum of 58 shows the characteristic features of the peripheral part of a mono-antennary N-acetyllactosamine type of structure bearing a-(2+6)-linked NeuAc (compare compounds 21-24). The presence of NeuAc in a-(2+6) linkage to Gal is obvious from the set of

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES anomeric protons 1 '

a-NeuAc -(2-6)-

II

p -Gal- (1-

4)- 6 -GlcNAc -(I-

a

339

N) - A m

1

58 Sugar skeleton protons

I1.6

-S

44

40

42

36

38

1

NAc -CH3 protons

I.

ID

I 28

26

t 12

20

II

I

FIG. 41.-Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 58. [The bold number and letter in the spectrum refer to the corresponding residues in the structure. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The signals marked by asterisks stem from impurities of unknown origin.]

chemical shifts of its H-3a (6 1.709),H-3e (8 2.677),andN-acetyl-group protons (6 2,028).These values deviate slightly from those given for NeuAc in a-(2-6) linkage to an 0-glycosylically linked N-acetyllactosamine unit (see Table XIII);this illustrates that these parameters are very sensitive to the complete structure of the branch to which NeuAc is attached.

340

J. F. G . VLIEGENTHART et al.

The introduction of NeuAc at 0-6of Gal-a of 55 gives rise to a shift decrement for the H-1 signal of Gal-a (A6 - 0.027 p.p.m.), and to shift increments for H-1 (A6 +0.032 p.p.m.) and the N-acetyl protons of GlcNAc-1 (A6 + 0.024 p.p.m.). The line widths of these signals are not affected. The changes in chemical shifts due to the introduction of a(.2-+6)-linked NeuAc, observed for neighboring, structural-reporter groups, are essentially identical to those deduced for the attachment of NeuAc in a-(2-6) linkage to asialo oligosaccharides or glycopeptides of the N-acetyllactosamine type (see Table XIII). Compound 59 is a sialo glyco-asparagine, containing NeuAc in a(2+3) linkage to Gal-a. This compound has been isolated from the urine of a patient suffering from aspartylglu~osaminuria.~'~~~*~~ The 500-MHz, 'H-n.m.r. spectrum of 59 is depicted in Fig. 42; its n.m.r.spectral parameters are listed in Table XXIII. The presence of NeuAc in ~ ( 2 4 3linkage ) to Gal is evident from the chemical shifts of the NeuAc structural-reporter groups (SH-3a 1.796, 6 H 3 e 2.759, and 6NAc 2.031; compare Table XIV). The observed, downfield shift of the H-1 signal of Gal-a in comparison to the asialo analog of 59, namely, 55, is A6 +0.076 p.p.m., and this, together with the resonance position of H 3 of Gal-a, far apart from the bulk of the skeleton proton signals (6H-3 4.113), gives additional proof of the substitution of Gal at 0-3 by NeuAc. The resonance positions of the structural-reporter groups of GlcNAc-1 are slightly, but significantly, affected (ASH-1 - 0.005 p.p.m., AGNAc - 0.003 p.p.m., as compared to 55), which is in agreement with the limited effects, due to introduction of a-(2+3)-linked NeuAc, on the chemical shifts of structural-reporter groups of neighboring residues, as summarized in Table XIV. Compound 60 is a sialo glyco-asparagine of the N-acetyllactosamine type containing NeuAc in a-(2-+3) linkage to Gal-c. The latter residue forms part of an additional N-acetyllactosamine unit (b-c) which is p(l-+3)-linked to the fundamental structure (55). Compound 60 has been isolated from the urine of a patient with aspartylglucosaminria^^ as the major component of a mixture also containing small amounts of 58 and of the asialo analog of 60. The W - M H z , 'H-n.m.r. spectrum of this mixture is presented in Fig. 43; the pertinent n.m.r.spectral parameters of 60 are compiled in Table XXIII. The presence of the additional, sialylated N-acetyllactosamine unit in p-(1+3) linkage to Gal-a is revealed by the occurrence of the following structural-reporter-group resonances: ( i ) the H-1 doublet of the p-(1+3)-linked GlcNAc-b at 6 4.699, ( i i ) the additional singlet of GlcNAc-b in the N-acetyl-proton region of the spectrum, at 6 2.032, and (iii) the H-1 doublet of Gal-c at 6 4.554.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

341

anomeric protons

r 1

59

sugar skeleton protons

NAc -CH3 protons 1 NIllC

&

30

---

18

.

I -

I

,

+

20

FIG. 42.-Resolution-enhanced, W - M H z , 'H-N.m.r. Spectrum of Compound 59. [The bold number and letter in the spectrum refer to the corresponding residues in the structure. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated.]

The introduction of a (sialylated)N-acetyllactosamine unit p-( 1+3)linked to Gal-a gives rise to very few alterations in chemical shifts, coupling constants, and line widths of structural-reporter groups of neighboring residues that can be used for the localization of additional N-acetyllactosamine units in more-complex structures (compare Refs. 91-92a). The influences of this unit are almost exclusively re-

0 co

n

*'.

9 0 a J

. P

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

343

stricted to the protons of Gal-a. The considerable, downfield shift of the H-4 signal, from 6 3.927 for 55 to 6 4.154 for 60, is most striking. For the assignment of this signal in the spectrum, its typical shape, namely, a relatively broad-lined, virtual doublet (J4,5 of Gal < 1 Hz) is extremely helpful. The combination of a characteristic pattern and a resonance position clearly resolved from the bulk (6 -4.15) makes the Gal H-4 an excellent, structural-reporter group for the recognition of a p-( 1+3) substitution of this Gal residue by, for example, an N-acetyllactosamine unit (see also Ref. 12). Furthermore, the resonance positions of H-1 (A6 - 0.013 p.p.m.) and of H-3 of Gal-a (A6 0.3 p.p.m.) are significantly influenced by this extension. Finally, it should be noted that the N-acetyl signal of GlcNAc-1 undergoes a small upfield shift (AS - 0.004 p.p.m.) due to the elongation of the glycan chain. In 60, the additional N-acetyllactosamine unit is terminated by NeuAc in a-(2+3) linkage to Gal-c; this can be inferred from comparison of the set of chemical shifts of the NeuAc structural-reporter groups for 60 with those reported to be characteristic for the aNeuAc(2+3)~Gal(l+4)~GlcNAc(l+*)sequence (compare Table XIV). As the influences of sialic acid in a-(2+3) linkage to Gal are well described (see Table XIV), the values of chemical shift of the structuralreporter-group signals for an additional, asialo N-acetyllactosamine unit can be predicted.

-

-

2. Carbohydrate Chains of the Oligomannoside Type

In the second family ofN-glycosylically linked carbohydrate chains, the core pentasaccharide aMan(l+S)[aMan( l+6)]/3Man( 1+4)/3GlcNAc(l+4)pGlcNAc(l+N)Asn is substituted by mannose residues.' The n.m.r.-spectral study of such glycopeptides and oligosaccharides is hampered by the similarity of the constituent units. Only the H-1 and H-2 resonances of each of the Man residues can serve as markers for the primary structure. Obviously, this demands high spectral resolution on increase in the number of Man residues. Crucial features for spectral assignments for this type of carbohy~~~

FIG.43.-Structural-reporter-group Regions of the Resolution-enhanced, W M H z , 'H-N.m.r. Spectrum of a Mixture Containing Compounds 60 and 58 in the Ratio of 9: 1. [In contravention of the usual experimental conditions (see Section IV), the glycoasparagine solution used for recording this spectrum had pD -2. The bold number and letters in the spectrum refer to the corresponding residues in the structures, and the italic numbers refer to the compounds in the mixture. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs &om that of the other parts of the spectrum, as indicated.]

J. F. G . VLIEGENTHART

344

et

d.

drate structure are the parameters of the structural-reporter groups of a-(1+2)-linked Man residues, as well as the manifestation of the second branching-point, which is a Man-4' residue, substituted in the same way as Man-3.The complete assignment of all structural-reporter groups for structures containing up to nine Man residues will be discussed. Symbols employed for compounds 61-72 are depicted in Chart 5. Compound 61 is a glyco-asparagine of the oligomannoside type containing four Man residues that has been i ~ o l a t e from d ~ ~the ~ ~urine of a patient with Gaucher's disease, a glucocerebrosidase deficiency.w (94)E. G . Brunngraber, in Ref. 51, pp. 143-144. CHART 5

Symbols Employed* for Compounds 61-72

61

T"'" 68

62

pAsn 69

X

A

;

1

Ser o 7

Vat

I 64

70

-%-a

71

65

66

+--+--L 72

* For the key to the symbolic notation, see Chart 1.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

345

The 500-MHz, 'H-n.m.r. spectrum of 61 is presented in Fig. 44,and its n.m.r. parameters are listed in Table XXIV. For the spectral interpretation, 61 is conceived of as an extension of compound 3 with Man-A and -B. The latter groups, together with Man-4', constitute the second branching-point, which is characteristic for oligomannoside-type structures.' Compared to the spectrum of 3, the resonance positions of H-1 and H-2 of Man-3 are found essentially unaltered; this indicates that 61 contains a mono-a-(1+6)-substituted Man-3 residue (compare the chemical shifts of H-1 and H-2 of Man-3 in other mono-antennary, lower-branch, glyco-asparagines and oligosaccharides, for example, 4,18,22,47, and 54).The H-1 and N-acetyl proton signals of GlcNAc-2 are shifted somewhat upfield, to 6 4.608 and 2.061, respectively, due to extension of the a-(1+6)-linked Man-4' by Man groups only. In this type of compound, these values are not markers for the type of mono-substitution of Man-3, in contrast to structures of the N-acetyllactosamine type,but now, these values proved to be ~-Man-(l-4)-~-GlcNAc-(l-4)-~-GlcNAc-(l-N)-Asn

a-Man-(ld 3)

a

A

\a-Man-(l-

/

a-Man-(l--c 6)

1

6)

"

B

61 anomeric protons

,

Man H-2 atoms -

A

3

NAG -CH3 protons

I

I

2

I

m

h

-6

U

40

; o

FIG.44.-Structural-reporter-gmup Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 61.[The bold numbers and letters in the spectrum refer to the corresponding residues in the structure. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The signal marked by c$ originates from a frequently occurring, nonprotein, noncarbohydrate contaminant of unknown structure. In addition to 61,the sample contained a small proportion of glyco-asparagine 62, as can be inferred from the signals marked by asterisks.]

346

J. F. G . VLIEGENTHART

et al.

TABLEXXIV 'H Chemical Shifts of Structural-reporterGroups of Constituent Monosaccharides for Small Glyco-asparaginesof the Oligomannoside Type (Compounds 3 and 61-63)

Compound and schematic structure

YF

A

62

61

3

s

n

T

A

s

n

?Am

Reporter group Residue H-1 of NAcof

H-1 of

H-2 of

1 2 1 2 3 4 4' A B C 3 4 4' A B C

5.07 1 4.618 2.014 2.076 4.767

5.069 4.608 2.013 2.061 4.770

4.915

4.870 5.076 4.909 4.076 4.140 4.064 3.988 -

-

-

4.080

-

3.968

-

-

-

5.071 4.606 2.012 2.060 4.781 5.099 4.872 5.093 4.908

4.251 4.077 4.144 4.066 3.985 -

63 (ov)

Y-AW 5.070 4.605 2.011 2.061 4.770 5.342 4.871 5.095 4.908 5.052 4.229 4.114 4.143 4.066 3.990 4.066

characteristic for the GlcNAc-2 core residue in glycopeptides of the oligomannoside type. Concerning the characteristics of the peripheral part of the structure, the well resolved doublet at 6 4.909(J1,2 1.8 Hz) and the narrow doublet of doublets at 6 3.988 are attributed to H-1 and H-2, respectively, of' the terminal, a-(1-+6)-linked Man-B group. These values closely resemble those of H-1 and H-2 of the terminal a-(l+6)-linked Man-4' in 3,4, 19& and 23p. The H-1 doublet at 6 5.076 1.8 Hz), in conjunction with the H-2 resonance at 6 4.064, is characteristic for the terminal, a-(l-+3)-linked Man-A group, as can be derived from comparison with the n.m.r.-spectral data for compounds containing a terminal, a-(1--+3)-linkedMan-4, for example, 5, ZOP, and 24P. The H-1 signal of the disubstituted Man-4' is found at 6 4.870 (J1,2 1.8 Hz). The shift decrement (A6 -0.045 p.p.m.) observed for this proton in comparison to 3 is ascribed to the substitution of Man-4' at 0-6, as the presence of an a-(1+3)-linked Man group hardly influences the resonance position of H-1 of the Man residue to which it is attached

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

347

(compare the steps from 18 to 20,Table VIII; and from 22 to 24, Table IX). A similar shift-decrement is observed for H-1 of Man-4' in the steps from di- to tri'-antennary, and from tri- to tetra-antennary, structures of the N-acetyllactosamine type (see Table VI), involving extension with a j3Gal(1+4)GlcNAc moiety in j3-( 1+6) linkage to Man-4'. The H-2 signal of Man-4' is found at 8 4.140, showing a shift increment (A8 0.172 p.p.m.) as compared to 3.This downfield shift can be attributed almost exclusively to the attachment of the a-(1-+3)-linked Man-A, as an a-(1+6) substitution of one Man residue by another hardly affects the H-2 chemical shift of the former (compare the steps from 17 to 19,Table VIII; and from 21 to 23,Table IX). Compound 62 is a glyco-asparagine of the oligomannoside type containing five Man residues that has been isolated from the urine of a patient with Gaucher's it was also obtained from hen-egg albumin.95 The 500-MHz, 'H-n.m.r. spectrum of 62 is presented in Fig. 45; its n.m.r. parameters are compiled in Table XXIV. a-Man-(14

a-Man-(l-

6) /

\a-Man-(l-

/ a-Man-(I-

\ p -Man-(1-

3)

A

3) 4)-p -GlcNAc-(1-

4)-p -GlcNAc-(I-

=

"

6)'

B

NAC -CH3 protons 2

62

I

Man H - 2 atoms

anomeric protons

B

. - d

N)-Am

54

48

C6

41

'

I

t---

-. 10

FIG.45. -Structural-reporter-group Regions of the Resolution-enhanced, !XO-MHz, 'H-N.m.r. Spectrum of Compound 62. [The bold numbers and letters in the spectrum refer to the corresponding residues in the structure. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated. The HOD resonance has been omitted from the spectrum; its position is indicated by an arrow. In addition to 62, the sample contained small proportions of structurally related, oligomannoside-type glyco-asparagines, for example, compound 3; some characteristic, reporter-group signals of the latter compound are marked by asterisks.]

348

J. F. G. VLIEGENTHART et al.

For the interpretation of the spectrum, 62 is conceived of as an extension of 61 with Man4 in a-(1+3) linkage to Man-3, also completing the first branching-point. The additional Man-4 is characterized by its H-1(6 5.099)and H-2 signal (6 4.077). The chemical shifts ofthese H-1 and H-2 signals are essentially the same as those described for the terminal Man-4 in compounds SP (see Table 111), 20p (see Table VIII), and 24P (see Table IX). Owing to the introduction of Man-4, the characteristically shaped H-2 signal of Man-3 in particular undergoes a downfield shift, from 8 4.076 for 61 to 8 4.251, whereas the chemical shift of H-1 of Man-3 is only slightly affected (A8 -0.01 p.p.m.) (compare the influence ascribed to the introduction of Man-A upon the reporter groups of Man-4', in the step from 3 to 61). The effects of introduction of Man-4 into 61 are identical to those described for the steps from 18 to 20 (see Table VIII) and from 22 to 24 (see Table IX). As compared to 61, the chemical shifts of all other structural-reporter groups (including those of GlcNAc-2) remain essentially unaltered, except for the H-1 signal of Man-A. The change in the chemical shift of the latter proton may reflect a spatial effect. It should be noted that, at lower magnetic field strength, the H-1 signals of Man-4 and -A for 62 are indistinguishable from each other?6*50 Compound 63 (ov) is a glyco-asparagine of the oligomannoside type containing six Man residues that has been obtained from hen-egg alb ~ r n i nThe . ~ ~500-MHz7'H-n.m.r. spectrum of 63 (ov) is given in Fig. 46, and its n.m.r.-spectral parameters are included in Table XXIV. For the spectral interpretation, 63 is conceived of as an extension of 62, with Man-C in a-(1+2) linkage to Man-4. The additional Man-C is characterized by its H-1(6 5.052) and H-2 (6 4.066) signals. This set of chemical shifts for a Man residue has not been observed for the foregoing compounds of the oligomannoside type (61 and 62); it proves to be typical for a terminal, a-(l+2)-linked Man group. Owing to the introduction of Man-C, the structural-reporter-group signals of M a n 4 undergo downfield shifts (A8H-l,O.243; A8H-2,0.037 p.p,m.). The H-1 and H-2 signals of Man-3 are both shifted slightly up(95) T. Tai, K. Yamashita, A.-M. Ogata, N . Koide, T. Muramatsu, s. Iwashita, Y. Inoue, and A. Kobata, /. Biol. Chem., 250 (1975) 8569-8575.

FIG.46.- Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of Compound 63. [The bold numbers and letters in the spectrum refer to the corresponding residues in the structure. The relative-intensity scale of the N-acetyl-proton region (see insertion) differs from that of the other parts of the spectrum, as indicated. For solvent-peak suppression, a w.e.F.t. pulse-sequence was used.]

I

*-=====2

1. F. G. VLIEGENTHART et d.

350

field (A6H-1 - 0.011 and A6H-2 - 0.022 p.p.m.). The chemical shifts of the structural-reporter groups of the other residues remain essentially unaltered, in comparison to 62. Compound 64 is a reducing tetrasaccharide that has been isolated from the urine of a patient suffering from m a n n o ~ i d o s i s . The ~ ~ ~500~' MHz, 'H-n.m.r. spectrum of 64 is presented in Fig. 47, and its n.m.r.spectral parameters are summarized in Table XXV. For the spectral interpretation, 64 can be conceived of as an extension of trisaccharide 5, with Man-C in a-(1+2) linkage to Man-4. The structural-reporter groups, H-1 and H-2, of Man-C give rise to relatively sharp-lined signals, at 6 5.050 and 4.069, respectively. This set of chemical shifts is typical for a terminal, a-(1+2)-linked Man group (compare compound 63,see Tabie XXIV). Upon attachment of Man-C to Man-4, the H-1 and H-2 signals of Man-4 undergo downfield shifts (A8 0.245 and 0.034 p.p.m., respectively) and are slightly broadened. The chemical shifts of the structural-reporter groups of the other residues, Man-3 and GlcNAc-2, are unaffected by the presence of Man-C; together, they reflect the a(1-3) type of mono-substitution of Man-3 (compare 5, Table 111; 17, Table VIII; 21, Table IX; 35, Table XI; 40, Table XII; and 46, Table XVI).The effect of anomerization on the chemical shift of structuralwMan-(l- Z)-a-Man-(l- 3) C

4

\'@-Man-(l3

4)-GlcNAc 2

NAc -CH3 protons

64

2,o

anomeric protona r-------

-

Men H-2 atoms

7

Man

W

n-3

FIG.47.-Structural-reporter-groupRegions of the Resolution-enhanced,500-MHz, 'H-N.m.r.Spectrum ofCompound 64. [The bold numbers and the letter in the spectrum refer to the correspondingresidues in the structure. Signals of correspondingprotons in the a and fi anomer of 64, occumng in this anomeric mixture in the ratio of 2 :1, coincide, unless otherwise indicated.]

351

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES TABLEXXV

'H Chemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for Oligosaccharides of the Oligomannoside Type (Compounds 5, and 64-67) Compound and schematic structure

Reporter group Residue

64

65

66"

67

5.209 4.718

5.206 4.718

5.250 4.716

2.043 2.041 4.787 4.783 5.111

4.776 4.772 5.356

5.207 4.719 2.043 2.041 4.774 4.771 5.343

-

-

-

4.899

-

-

-

5.098 5.126

5.050 -

5.296 5.046

5.056 -

-

-

-

h o m e r of compound

-

-

-

4.244 4.233 4.075 4.071

4.224 4.213 4.108 4.105

4.220 4.209 4.087 4.082

4.238 4.227

5.231 4.714 2.050 2.046 4.776 4.772 5.337 4.872 4.869 5.398 5.407 5.142 5.308 5.048 5.058 5.063 5.040 4.239 4.229

4.109"

4.089

4'

-

-

-

A

-

-

-

4.129 4.127 4.068 4.065

4.069 -

4.105 4.066

4.068 -

4.158 4.155 4.106 4.103 4.025 4.109 4.0693

-

-

-

4.073b

-

-

-

4.066b

H-1 of

2

NAc of

2

H-1 of

3

4 4' A

B C Dl Dz

-

4

H-2 of

5

3 4

-

D3

" Measured at 360 MHz, T

=

2.041

2.044" 4.776" 5.348

-

-

295 K. Assignments may have to be interchanged.

reporter groups decreases, going from the reducing residue to the nonreducing end-group; at 500 MHz, neither H-1 nor H-2 of Man-C shows a difference in chemical shiA between the (Y and p anomer of 64.

J. F. C. VLIECENTHART et al.

352

Compound 65 is a linear, reducing pentasaccharide containing four Man residues that has been isolated from the urine of a patient suffering from mannosid~sis.~**~~ The 500-MHz, 'H-n.m.r. spectrum of 65 is depicted in Fig. 48, and its relevant, n.m.r.-spectral parameters are listed in Table XXV. For the spec& interpretation, 65 can be conceived of as an extension of 64 with another a-(1+2)-linked Man group, denoted as ManD, , at the nonreducing end. The chemical shifts, coupling constants, and line widths of the H-1 and H-2 signals of Man-D, are essentially identical to those of the terminal, a-(1-+2)-linked Man-C in 64. The a-(1 4 2 ) substitution of Man-C by Man-D, leads to shift increments of the H-1 and H-2 signals of Man-C : A8 0.246 and 0.036 p.p.m., respectively. These increments are identical to the effects described for H-1 and H-2 of Man-4 due to elongation of 5 to 64 with Man-C (see earlier). Attachment of Man-D, in ~ ~ ( 1 - 2linkage ) to Man-C also influences the chemical shifts of the H-1 and H-2 signals of Man4 (A6 -0.013 and -0.022 p.p.m., respectively). The line widths of the H-1 doublets of the a-linked Man residues in 65 decrease on going from the internal Man-4 by way of Man-C to the terminal Man-D,. Doubling of signals due to anomerization of 65 is manifested only in the signals of GlcNAc-2 and Man-3. The more remote a residue from the reducing end, the less the effect of the anoa-Man-(++a)-a-Man-(l- 2)-a-Man-(1- 3) D1

C

I

\B- M y-

(1-

4 1- GltNAc NAc -CH3 protons

6!i c-

-

2,"ffl

anomeric pmlons

Man H-2 atoms Man H-3atoms

__________-._11 57

-*

.

10

u

..

c2

m

FiG. 48.-Structural-reporr-~oup Regions of the Resolution-enhanced,S

i

I1

v

MHz,

'H-N.m.r. Spectrum of Compound 65. [The bold numbers and letters in the spectrum refer to the correspondingresidues in the structure. Signals of corresponding protons in the a and fi anomer of 65,occurring in this anomeric mixture in the ratio of 2: 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-protonregion differs from that of the other parts of the spectrum, as indicated.]

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

353

meric configuration of GlcNAc-2 on the chemical shift of its structuralreporter groups. Compound 66 is a branched-chain, reducing oligosaccharide containing five Man residues that has been isolated from the urine of a patient with mannosidosis. The 360-MHz, 'H-n.m.r. spectrum of 66 is depicted in Fig. 49; its n.m.r.-spectral features are listed in Table

xxv.

For the interpretation of the spectrum, 66 is considered to be an extension of 64, with an aMan(b3)Man unit (A-4') in a-(1+6) linkage to Man-3. For H-1 of the terminal, a-(1+3)-linked Man-A group, two well-separated doublets are found, at 6 5.098 and 5.126, with relative intensities in the momeric ratio, a :p = 2 :1.The H-2 signal of Man-A is also doubled, due to anomerization. The set of chemical-shift values for H-1 and H-2 of Man-A is very similar to the sets observed for terminal, a-(1+3)-linked Man groups in compounds 5 (Man-4; see Table III), 20 (Man-4; see Table VIII), 24 (Man-4; see Table IX), 61 (Man-A; see Table XXIV), 62 (Man4 and -A; see Table XXIV), and 63 (Man-A; see Table XXIV). However, a value for the anomerization effect as large as 1ASa+, I for H-1 of Man-A in 66 (0.028 p.p.m.) had not been observed before; it indicates a very close spatial proximity of this H-1 to the anomeric center of GlcNAc-2. This is mutually proved by the shift increment (A6 -0.04 p.p.m.) observed for H-1 of GlcNAc-2 in the (Y anomer of 66 in comparison to that in related structures, for example, 5,64, or 65 (see Table XXV). The H-1 doublet ofMan-4' is observed at 6 4.899; its position is only little affected by the attachment of Man-A in a-(1+3) linkage [compare with compounds 3,4,19,23, and with 61,62,and 63,having in eommon a terminal, a-(1-+6)-linked Man group, namely, 4' and B, respectively; see Tables 111, VIII, IX, and XXIVl. However, the chemical shift of the H-2 signal of Man-4' (6 -4.128) differs considerably from that of H-2 of the terminal a-(1+6)-linked group in the aforementioned series of compounds (6 3.98; signal almost hidden in the bulk resonance of skeleton protons). The shift increment observed must be attributed to the substitution of Man-4' by Man-A; it is essentially identical to that described for H-2 of Man3 upon introduction of Man4 (in the steps from 18 to 20, from 22 to 24, and from 61 to 62;see Tables VIII, IX,and XXIV). Thus, the spectral data for compound 66 offered the possibility of unraveling the influences of the attachment of Man-A on the chemical shifts of the structural-reporter groups of neighboring residues, apart from those of attachment of Man-B (compare 66 as against 61-63),as 66 is the only compound reported herein having an incomplete, second branching-point. As described for the step from 17 to 19,the introduction of the a-

-

t

0

t

r(

c

v

4"

-t

t

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

355

3

Man H-2 atoms

awmeric protons

(b)

I

7

4. 4

4

+ 54

-6

52

50

:

I LL

b2

(a +

FIG. 49.-(a) Resolution-enhanced, Overall, 360-MHz, 'H-N.m.r. Spectrum of Compound 66.(b) Expanded, Structural-reporter-group Regions of Spectrum (a). [The bold numbers and letters in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the a and p anomer of 66, occurring in this anomeric mixture in the ratio of 2 :1, coincide, unless otherwise indicated. The signal marked by originates from a frequently occurring, nonprotein, noncarbohydrate contaminant of unknown structure.]

(1-+6)-linked Man4' mainly influences the chemical shift of H-2 of Man-3 (As 0.014 p.p.m.); the chemical shift of H-1 of Man4 is also slightly affected (see Table XXV) (compare compound 19). The presence of Man-A apparently does not alter the magnitude of these effects. The chemical shifts of the structural-reporter groups of Man-C are unaltered with respect to those of the corresponding protons in 64. As already described (compare compounds 61 and 62), disubstitution of Man-3 does not result in a significant alteration of the chemical shift of the N-acetyl signal of GlcNAc-2. In contrast to the situation with compounds of the N-acetyllactosamine type (see earlier), it is impossible to deduce, for glyco-asparagines and oligosaccharides of the oligomannoside type, the type of mono-substitution from the chemical shift of this particular signal. Compound 67 is a double-branched, reducing oligosaccharide of the oligomannoside type containing nine Man residues; it is the largest oligosaccharide thus far isolated from the urine of patients with m a n n o s i d o s i ~ . ~The ~ , ~ 500-MHz, ,~~ 'H-n.m.r. spectrum of 67 is depicted in Fig. 50, and its pertinent, n.m.r.-spectral features are included in Table XXV. (96) K. Yamashita, Y. Tachibana, K. Mihara, S. Okada, H. Yabuuchi, and A. Kobata, 1.Biol. Chem., 255 (1980)5126-5133. (97) F. Matsuura, H. A. Nunez, G. A. Grabowski, and C. C. Sweeley,Arch. Biochem. B i o p h y s . , 207 (1981)337-352; P. F. Daniel, D. F. De Feudis, and I. T. Lott,Eur. I. Biochem., 114 (1981)235-237; H. Egge, J.-C. Michalski, and G. Strecker,Arch. Biochem. B i o p h y s . , 213 (19f32),318-326.

J. F. G. VLIEGENTHART et al.

356

a-Man- (1- 2)-@-Man-(Ic

DI

a-Man-(I-

2) -0-Man- (1-

\p-Man-(ld4)-GI:Nk

2)-@-Man-(l--3) A

D2

\o-Man-(l--

/

a-Man-@- 2)-u-Man-(I4 6)

4p

B

%

-- -

/ 6)

= Man H-2 atoms 1

D1.25

67

-----

3)

I

NAc C H 3 protons

anorneric protons

---

FIG.50.- Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, ‘H-N.m.r. Spectrum of Compound 67.[The bold numbers and letters in the spectrum refer to the corresponding residues in the structure. Signals of corresponding protons in the (Y and p anomer of 67,occurring in this anomeric mixture in the ratio of 2 : 1, coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region diEers from that of the other parts of the spectrum, as indicated.]

The presence of the first branching-point (4-3-4’) in 67 is evident from the chemical shifts of the H-1 and, especially, of the H-2 signal of Man-3 (compare 66).The changes in the chemical shifts of H-1 and of the N-acetyl protons of GlcNAc-2, as compared to 66, reflect the extension of the second branching-point (A-4’-B) with a-(1+2)-linked Man groups, in particular with Man-D, (see later). Analogous to the data for 65, the H-1 signals at S 5.337, 5.308, and 5.048 are ascribed to the upper-branch Man residues 4, C, and D, , respectively; also, the H-2 resonances of these residues are found at the same positions as described for 65 (see Table XXV). The H-1 and H-2 signals of Man-4‘ are found at 6 -4.87 and - 4.16, respectively; these positions are in accord with those of the corresponding protons in 61-63, also possessing a Man-4’ residue disubstituted by A and B. Thus, for the spectral interpretation, the lowerbranch part of 67 can be conceived of as an extension of 61-63 with two a-(1+2)-linked Man groups, Dz and D,. As is usual for terminal,

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

357

a-(1+2)-linked Man groups, the H-1 signals of Man-D, and -D, are ob-

-

-

served at 6 5.05, whereas their H-2 resonances are found at 6 4.07. Owing to the substitution of Man-B by Man-D, in a-(1+2) linkage, the signals of both H-1 and H-2 of the former residue undergo a downfield shift (for H-1, A6 0.234 p.p.m. and for H-2, A6 0.040 p.p.m.; compare Table XXVII). The H-1 and H-2 signals of Man-A also show downfield shifts, due to the attachment of Man-D,. The shift increment for H-2 (A6 -0.04 p.p.m.) is identical to that just described as being typical for the introduction of an cu-(l+e)-linked Man. However, the shift increment for H-l (A6 -0.31 p.p.m.) deviates, and this may arise from a spatial effect (see later). Doubling of the signals of the structural-reporter groups due to anomerization is clearly observable for Man-A (lA6a-BI for its H-1 signal is again larger than I for H-1 of Man-4'; however, the value is lessened as compared to 66 having a terminal, Man-A group), but anomerization effects upon the H-1 and H-2 signals of Man-B are apparently absent. Therefore, the set of doublets at 6 5.058 and 5.063 (relative intensity 2:1, the anomeric ratio) are attributed to H-1 of Man-D, in the a and /3 anomer of 67, respectively. Consequently, the single H-1 doublet at 6 5.040 belongs to Man-D,. Comparison of the n.m.r.-spectral parameters of the three branches of 67 reveals that the anomeric configuration of GlcNAc-2 exerts its influence far more pronouncedly on the A-D, branch than on the other two branches, suggesting that this branch occurs in the sphere of influence of the anomeric center of GlcNAc-2, in contrast to the other two. On the basis of these n.m.r.-spectral features, it is proposed that the favored conformation of 67 in solution is as depicted in Fig. 51. This conformation might offer an explanation for the unexpectedly large, downfield shift of the H-1 signal of Man-A due to elongation of this branch with Man-D2, and also for the observed shifts of the H-1 signals of GlcNAc-2 in the a anomers of 66 and 67 and for that of the N-acetyl protons of this residue in 67, in comparison to 64 and 65. Compounds 63 (IgM) and 68-70 are examples of the oligomannoside type of glycopeptide that differ in the content of a-(1+2)-linked Man residues. These compounds were isolated in a mixture from glycosylation site Asn-563 of an immunoglobulin M obtained from blood a-Man-(1-

Z)-a-Man-(l2)-a-Man-(1-

C

a-Man-(1-

3)

4

\

4

\a-Man-(1-

/

a-Man-(l-6) B

\

' pp -Man-(1-4)-Man-(1-4)-

3)

/

4'

6)

IT$-l p-GlcNAc-(1p-Gl;NAc-(l2

4)-(l4)- 0 -GlcNAc -Gl:NAc -(l1

N )-Ah N)-Asn

358

J. F. G. VLIEGENTHART et al.

FIG.51,- Space-filling, Molecular Model of the Oligomannoside Type of Carbohydrate Chain that Contains 9 Mannose Residues. [Numbers and letters correspond to the coding used in Figs. SO,%, and 54 (see also, footnote on page 221).The relatively close, spatial proximity of the A-Dz branch and the N,N’-diacetylchitobiose core-region (2-1) is clearly illustrated.] a-Man-(1-

Z)-a-Man-(l-

Z)-a-Man-(l-

<

D,

i

a-Man-(l-

3)

\ \,3

3)

-Man+-

4 ) - 0 -GlcNAc -(12

\

a-Man-(1-

4)- p -GkNAc-(l-

[ Tylo., N)-Ah

1

6)

H

68

o-Man- (1<

a-Man-(1\

Z)-o-Man- (1-

3)

\p

3)

-Man+-

\u-Man-(l-6) $-Man-(l!i)

/

2)-o-Man-(1-6)’ s

*‘ 69

?-Man- (1“1

2)-a-Man - (1n

6)’

70

4)- p-CicNAe -(I-

4 ) - p-GlcNAc-(1-

N)-Am

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

359

plasma of a patient (Du) with Waldenstrom's macroglobulinemia.98~9g The 500-MHz, 'H-n.m.r. spectrum of this mixture is presented in Fig. (98) J. Jouanneau and R. Bovrrillon, Biochem. Biophys. Res. Commun., 91 (1979) 1057-

1061. (99) H. van Halbeek, L. Dorland, J. F. G. Vliegenthart, J. Jouanneau, and R. Bourrillon, Biochem. B w p h y s . Res. Commun., 99 (1981) 886-892.

NAc -CH3 protons I

-I 1

anomeric protons C,63+E9

HOD

j

Man H-2 slorns 7

k

, '2'

'-'

*I*HLC

'b 20 I FIG.52.-(a) Overall, 500-MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 63,68,69, and 70. [The signals marked by I$ originate from a frequently occurring, nonprotein, noncarbohydrate contaminant of unknown structure.] (b) Expanded, Structural-reporter-group Regions of the Resolution-enhanced, 500-MHz, 'H-N.m.r. Spectrum of the Aforementioned Mixture. [The bold numbers and letters in the spectrum refer to the corresponding residues in the structures, and the italic numbers to the compounds in the mixture. Signals of corresponding protons in the various components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated.] -n

52

50

TABLEXXVI 1H Chemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for Glycopeptides of the Oligomannoside Type (Compounds 63 and 68-72)

W QI

0

Compound and schematic structure ~

63

~

68

71

70

69

72

(LTF)

OgM)

Asn

Reporter group Residue

H-1of NAc of

H-1 of W QI

0

(SBA)

Xaa

Ser

Xaa

01

5.092 5.092 5.092 5.02" 5.02" 5.02" 5.02" 1 4.608 4.608 4.610 4.621 4.621 4.621 4.621 2 TABLEXXVI 2.007 2.007 2.015 2.00" 2.00" 2.00" 2.00" 1 2.054 2.054 2.066 2.067 2.066 2.054 2.054 2 1H Chemical Shifts of Structural-reporter Groups of Constituent Monosaccharides for Glycopeptides of the Oligomannoside Type -4.77 -4.77 -4.77 -4.78 -4.78 -4.78 -4.78 3 (Compounds 63 and 68-72) 5.336 5.345 5.333 5.334 5.345* 5.345 5.336 4 4.869 structure 4.868 4.869 4.868 4,869 4.869 4.869 4' Compound and schematic ~

63

68

~

69

71

70

72

(LTF)

OgM)

Asn

Reporter group Residue

H-1of NAc of

1 2 1 2

(SBA)

Xaa

Ser

5.02" 4.621 2.00" 2.054

5.02" 4.621 2.00" 2.054

5.02" 4.621 2.00" 2.054

5.02" 4.621 2.00" 2.054

Xaa

01

5.092 4.608 2.007 2.066

5.092 4.608 2.007 2.066

5.092 4.610 2.015 2.067

A

B C

5.09" 4.908 5.052

D 1

DZ D8

H-2 of

3 4 4' A B C

D1 w

5.09" 4.908 5.304 5.044

-

-

4.232 4.W 4.145 4.07b

4.232 4.w 4.145 4.07b

3.986

3.98b

4.07b

4.W 4.07b

5.09" 5.145 5.052

5.044 4 232 4.W 4.145 4.07b 4.026 4.07b

-

D*

-

4

4.07b

5.09" 5.145 5.304 5.044 5.044 4.232 4 .w 4.145 4.076 4.02b 4.W 4.07b

-

4.07b

5.401 5.141 5.059

-

5.059 5.040 4.228 4.W 4.15" 4.10" 4.02" 4.07"

-

4.07" 4.07"

5.401 5.141 5.308 5.047 5.059 5.040 4.228 4.10" 4.15" 4.10" 4.02c 4.10" 4.07c 4.07" 4.07"

5.404 5.143 5.308 5.049 5.061 5.042 4.228 4 .OM 4.156 4.109 4.023 4.109 4.073d 4.0736 4.066d

a Values could not be determined more accurately (20.01 p.p.m.), due to heterogeneity of the peptide moiety (see also, legend to Fig. 52). Values could not be determined more accurately (20.01 p.p.m.), due to the complexity of the mixture of 63, and 68-70. " Values could not be determined more accurately (kO.01 p.p.m.), due to the complexity of the pattern in the spectral region 4.00 < 6 < 4.15 of the mixture of 71 and 72 (LTF). Assignments may have to be interchanged.

362

J. F. C . VLLEGENTHART et 01.

52, and the relevant, n.m.r.-spectral parameters of the compounds are listed in Table XXVI. From the spectrum of this complex mixture of glycopeptides, it may be inferred that all of the constituents have, in common, Man residues 3, 4, 4’, A, and B, and the N,N’-diacetylchitobiose unit. [The smallest component of this mixture differs from 63 (ov), obtained from ovalbumin, only in the peptide part.] The N-acetyl signals of GlcNAc-1 and -2 are both split into two singlets; this reflects the heterogeneity of the peptide moiety. Nevertheless, the chemical shift of the N-acetyl signal of GlcNAc-2 (6 -2.055) is typical for extension of the core pentasaccharide with Man residues only [compare 61,62,63 (ov), and 67; see Tables XXIV and XXV]. Besides the ,&linked Man-3, clearly characterized by its H-2 signal at 6 4.232, only a-linked Man residues occur in the peripheral part of the glycan chain.This is derived from the chemical shifts of their H-1 signals, in combination with their Jl,z vaIues.66The set of chemical shifts of H-1 (6 4.869) and of H-2 (6 4.145) of Man-4’ is indicative of a disubstitution of this residue at 0-3and 0-6 by Man-A and -B, respectively [compare 61, 62,63 (ov), and 67, Tables XXIV and XXV]. The H-1 atom of Man-A gives rise to two doublets, at 6 5.093 and 5.088, in the ratio of 2: 1, resonating in an area that is characteristic for a terminal, nonreducing position of Man-A [compare 61, 62, 63 (ov), 66, and Table XXVXI]. The doubling of the signal can be explained in terms of heterogeneity of the sample. It is tempting to correlate the intensity ratio of the two signals of the anomeric proton of Man-A with the molar ratio of the N-terminal amino acids (see Fig. 52). This suggestion would fit the proposal for the spatial conformation of carbohydrate chains of the aligomannoside type (compounds 66 and 67; see Fig. 51). The H-1 atom of Man-B also gives rise to two doublets, at 6 4.908 (terminal B) and at 6 5.145 (O-2-substituted B), in the ratio of 2: 1. However, from these values, it may be concluded that 33% of the Man-B residues in the glycopeptide sample bear an a-(1+2)-linked Man-D, (6H-15.044), whereas, in the remaining part, Man-B occupies a terminal position in the chain. For H- 1 of M a n 4 in the upper branch, two doublets are observed, at 6 5.345 and 5.336, in the ratio of 1:1. The approximate value of the chemical shift (5.34 p.p.m.) indicates that, in all components of the mixture, Man-4 bears the a-(1+2)-1inked Man-C [see 63 (ov), and 64671. Also, H-1 of Man-C gives rise to two doublets, at 6 5.304 (O-%substituted C) and 5.052 (terminal C). These features can be explained by the presence of Man-D, in 50% of the structures in the glycopeptide mixture. The presence of Man-D, (6H-1 5.044) gives rise to a downfield shift of the H-1 signal of Man-C (A6 0.252 p.p.m.) and to a slight,

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

363

-

upfield shift for H-1 of Man-4 (A6 - 0.01 p.p.m.), as compared to the 4-C branch without D, (compare the step from 64 or 66 to 65 or 67; see Table XXV). The high-resolution, 'H-n.m.r. spectroscopy conducted on this glycopeptide mixture shows that compounds of the oligomannoside type that differ in the number and the position of a-(1+2)-linked Man residues can be identified, even if heterogeneity is present in the peptide part. Compounds 71 and 72 (LTF) are glycopeptides of the oligomannoside type containing eight and nine Man residues, respectively, that were obtained from bovine 1actotransferrin'"O in a mixture of them in the ratio of 2:3. The 500-MHz, 'H-n.m.r. spectrum of this mixture is depicted in Fig. 53,and the pertinent, n.m.r.-spectral parameters of 71 and 72 (LTF) are included in Table XXVI. For the spectral interpretation, 71 and 72 can be conceived of as extensions of 63 with two, or three, terminal a-(1+2)-linked Man groups, respectively. The spectral features of the core residues, GlcNAc-1 and -2, and Man-3, -4, and -4', in 71 and 72 are essentially identical with those of the corresponding residues in compounds 63 and 68-70 (see Table XXVI). Heterogeneity of the peptide moiety produces multiple resonances for the N-acetyl protons, and also relatively broad-lined, anomeric doublets of GlcNAc-1 and of GlcNAc-2. The anomeric signal of Man-A, -B, and -4' at 6 5.401, 5.141, and 4.868, respectively, each appears as a single doublet in the spectrum of the mixture (relative intensities 1: 1: 1).These values of chemical shift are in good agreement with those of the corresponding residues in the p anomer of oligosaccharide 67, indicating that the lower branches of 71 and 72 are terminated with Man-D, and -D3. However, for H-1 of Man-4, two doublets are observed, at 6 5.333 and 5.345, in the intensity ratio of 3 :2. The sum of these intensities equals that of the H-1 signal of Man-A. This feature indicates the presence of a terminal Man-C, in a-(1+2) linkage to Man-4, in the minor component (71)of the mixture, whereas, in the major component (72), Man-C is substituted at 0-2 by another Man residue (D,). This effect of the attachment of Man-D, has been described in the step going from oligosaccharide 64 to 65 (see Table XXV). In accordance with this interpretation, the H-1 signal of Man-C in 72 at 6 5.308 has an intensity equal to that of Man-4 at 6 5.333. The H-1 signal of the terminal, a-(1+2)-linked Man-C in 71 is found at 6 - 5.05, as would be expected from the data for 63, 64, 66, and 69. Obviously, the presence of Man-D, in one of the two compounds (100) H. van Halbeek, L. Dorland, J. F. G. Vliegenthart, G. Spik, A. Chkron, and J. Montreuil, Biochim. Biophys. Acta, 675 (1981) 293-296.

a-Man-(F- 2)-a-Man-(1-

3)

C

a-Man+-

%)-a-Man-(l-

'

3)

\

.\

D2

-GlcNAc- (1-4)2

\a-Man-(lu-Man-(l-

0 -Man- (1-4)-8

2)-a-Man-(1-

6)

/

X7lo-,

8 -G~cNAc-(~- N ) - A ~ ~ I

6)

I [x=lO-l

"

B

U3

71

a-Man-(l-

-

Z)-a-Man-(l-

a-Man-(1 Da

Z)-a-Man-(l-

3)

C

D,

%)-o-Man-(l-

3)

3

'

\

B-Man-(l-

4)-

0 -GlcNAc-(lz

4)- ,9-GIcNAc-(l1

rx i I 0 - I

N)-Asn

I

IX=I0-, a-Man-(1-

2)-a-Man-(1-

6)

H

71 (LTF)

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

365

present in the mixture is reflected in the intensity ratio of the anomeric signals of terminal a-(1+2)-linked Man residues (6 5.05). Three signals are observed, at 6 5.059,5.047, and 5.040 in the ratios of 7:3:5. Based on its relatively low intensity, the signal at 6 5.047 is ascribed to Man-D, in 72. The signal at 6 5.059 partly belongs to ManD2in both 71 and 72, as may be deduced from comparison with 67 (see Table XXV). The anomeric signal of the terminal Man-C in 71 (compare 64 and 66) also contributes to the intensity of the signal at 6 5.059, thereby making this the most intense signal in this area. Finally, the doublet at 6 5.040 is attributed to H-1 of Man-D,. Compound 72 (SBA) is a glycopeptide of the oligomannoside type containing nine Man residues that was obtained from soybean agglutinin.'01,'02 The 500-MHz, 'H-n.m.r. spectrum of 72 (SBA) is depicted in Fig. 54, and its n.m.r.-spectral parameters are listed in Table XXVI. The chemical shifts, coupling constants, and line widths of all structural-reporter-group signals of 72 (SBA)are identical to those of the corresponding signals of 72 (LTF), except for the N-acetyl signals of GlcNAc-1 and -2. The latter signals show a multiplicity that is due to heterogeneity of the peptide moiety.67The carbohydrate part is homogeneous; its spectral data are in excellent agreement with those for the /3 anomer of oligosaccharide 67 (see Table XXV).

-

3. Additivity Rules In Sections 11, 1and 2, have been described the 'H-n.m.r.-speciiic: parameters, namely, the chemical shifts, coupling constants, and line widths of the structural-reporter groups of the monosaccharide constituents, of a wide diversity of N-glycosylic carbohydrate chains of glycoproteins. The coupling constant of the anomeric proton of a given sugar residue (J1,2)provides essential information on the configuration of its (101) H. Lis and N. Sharon, J . Biol. Chem., 253 (1978) 3468-3476. (102) L. Dorland, H. van Halbeek, J. F. G. Vliegenthart, H. Lis, and N. Sharon,J. Biol. Chem., 256 (1981) 7708-7711.

FIG.%.--(a) Overall, 500-MHz, 'H-N.m.r. Spectrum of a Mixture Containing Compounds 71 and 72 in the Ratio of2 :3, Obtained from Bovine Lactotransferrin. [The signals marked by 4 originate from a frequently occurring, nonprotein, noncarbohydrate contaminant of unknown structure.] (b) Expanded, Structural-reporter-group Regions of the Resolution-enhanced, W M H z , 'H-N.m.r. Spectrum of the Aforementioned Mixture. [The bold numbers and letters in the spectrum refer to the corresponding residues in the structures. Signals of corresponding protons in the two components of this mixture coincide, unless otherwise indicated. The relative-intensity scale of the N-acetylproton region differs from that of the other parts of the spectrum, as indicated.]

J, F. G. VLIEGENTHART et al.

366

sugar skeleton protons

( 0 )

II

nlm

I,

NAC

-CH3 wotons

ri

rn

a-Man- (11h

a-Man-(1Y

2)-a-Man-(1c 2)-a-Man-(13)

2)-a-Man+-

*

\p-Man-(f-

4

\a-Man-(l-

/

a-Man-(1-

3)

6)

/

4)-p -GI?NAc-(l-

’

I Xaalo-, I N)-Asn

I Wale-,

q’

6)’

Z)-a-Man-(l-

7!2(SBA)

X

D3

4)-p -Gl:NAc-(l-

Man H 2 atoms

(b)

O123

NAc C H 3 protons

anomerlc protons 7

--__

-

- --

51

r~

6

_ 52

1 __._ ~

so

-

. -4 t-- ;;--

-Fo+-T

-

FIG.%.--(a) Overall, W M H z , *H-N.m.r.Spectrum of Compound 72, Obtained from Soybean Agglutinin. (b) Expanded, Structural-reporter-groupRegions of the Resolution-enhanced, 500-MHz, ‘H-N.m.r. Spectrum of Compound 72. [The bold numbers and letters refer to the corresponding residues in the structure. The relahve-intensity scale of the N-acetyl-proton region differs from that of the other parts of the spectrum, as indicated.]

glycosylic linkage. With H-2 in axial position, a J1,2 value of 2-4 Hz indicates an a,and that of 7-9 Hz, a p, anomer; this applies to such monosaccharides as Gal, GlcNAc, and L-FUC.For Man, having its H-2 atom in equatorial position, the differenqe is more subtle: 1.6 Hz

‘H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

367

for an a,and 0.8 Hz for a p, linkage. In principle, the complete set of vicinal coupling-constants of sugar-ring protons allows the deduction of the conformation of the residue in question, as was shown for compounds 1,2,and 55. The line widths of the structural-reporter-group signals bear information on the “flexibility” of the protons involved and, by extrapolation, on the mobility of individual sugar residues and of branches in the chain. Furthermore, coupling constants, in combination with line widths, give the structural-reporter-group signals their characteristic shapes, which make them recognizable. These parameters facilitate the assignments of resonances belonging to structural-reporter groups. However, it is the chemical shift that makes a certain proton a structural-reporter group. The large amount of information hidden in a chemical shift is readily accessible for purposes of structure determination if a signal occurs outside the bulk of overlapping resonances and thus can be found by inspection. Among the structural-reporter groups, the chemical shift of an anomeric proton is primarily sensitive to the type of sugar, the anomeric configuration of its linkage, the site of glycosidic linkage, the sequence of the component sugars, and the position of the residue in the carbohydrate chain. However, steric factors are also important determinants of the chemical shift of an anomeric proton. The chemical shifts of other structural-reporter groups also show a dependence on many primary, structural parameters. In theory, it is possible to deduce the complete, primary structure of carbohydrate chains from the n.m.r.-spectral parameters of the structuralreporter groups. The availability of consistent series of compounds is helpful in interpretation of the spectra. This is well illustrated by the series of structurally related N-glycosylic carbohydrate chains of glycoproteins, which have been arranged herein by increasing complexity for those of the N-acetyllactosamine and the oligomannoside type. Based on the sets of data listed in Tables I-V, VII-XII, XV, XVI, XVIII-XX, and XII-XXVI, it is possible to establish empirical rules to describe the influence of structural changes on the chemical shifts of the structural-reporter groups involved. These rules permit reliable prediction of the ‘H-n.m.r. spectrum for every partial structure in the most complex one thus far observed. Less certain may be predictions of the spectra of structures built up from the same units as those occurring in the set of reference compounds, but constituting a novel family of structures. Comparison of the available data shows that additivity rules can readily be deduced for the chemical shifts of structural-reporter groups in those cases in which the mannotriose core is complete in the starting compound, as well as in the elongated chain. For compounds

368

J. F. G . VLIEGENTHART et al.

possessing an incomplete, mannotriose branching-unit, the chemical shifts of structural-reporter groups can less readily be rationalized; the influences, on the chemical shifts of neighboring residues, due to the attachment of Man-4 (for example, 17-19) or Man-4' (1k-20) are partly attributable to the drastic, steric changes accompanying these extensions. However, the type of mono-substitution of Man-3 by (a substituted) Man-4, or by (a substituted) Man-4', is obviously characterized by the presence of either the structural-reporter-group signals of Man-4 (6H-1 -5.12; 6H-2 -4.19 for mono-antennary structures) or of Man-4' (SH-1 -4.92; 6H-2 -4.12). The set of chemical shifts of the Man-3 H-2 signal (6 -4.24 and 4.08, respectively), and of the GlcNAc2 H-1 (for example, for the a anomers of reducing oligosaccharides, 6 5.207 and 5.216, respectively) andN-acetyl protons (6 2.042 and 2.062, respectively) gives independent information in this respect. Recognition of the type of branching in the N-acetyllactosamine type of structures possessing an intact mannotriose core unit substituted by one or more GlcNAc residues is possible on the basis of the set of chemical shifts of the H-1 and H-2 signals of Man-3, -4, and -4', as summarized in Table VI. The chemical shift of the H-1 signal of a terminal, p-linked GlcNAc residue is dependent on the type of its linkage to Man. For the p(1+2)-linked GlcNAc residues 5 and 5' in diantennary structures, 6H-1-4.56 is found. Forthe #1+4)-linked I-( GlcNAc-7,6H-14.52is observed (for example, for 16),whereas, for the p-( l-+6)-linked GlcNAc7', 6H-1 4.53 can be expected (see later). For the chemical shift of the H-1 signal of the intersecting GlcNAc-9 residue, p-(l+4)-linked to Man-3, in N-acetyllactosamine-type structures possessing at least GlcNAc-5 and -5', the value 6 4.47 is observed. The relatively highfield, resonance position of this proton is probably due to the special, steric requirements for the attachment of GlcNAc-9, which also cause a disturbance of the total pattern of the Man H-1 and H-2 signals (compare Table VII with VI), rather than to the P-type of linkage of the substituted Man-3. This is a very illustrative example of changes in chemical shifts that are attributable to conformational differences between starting compound and extended chain. As such conformational changes are hardly predictable, it is evident that the prediction of chemical shifts of structural-reporter groups in such cases will also be difficult. This problem is nicely illustrated by the impossibility of predicting the chemical shifts of some structural-reporter groups in hybrid structures containing GlcNAc-9, starting from data for compounds of the N-acetyllactosamine type.50The N-acetyl-proton resonances of GlcNAc residues in terminal position are observed at 6 2.053 (GlcNAc-5 in diantenna), 2.050 (GlcNAc-5' in diantenna), 2.078

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

369

(GlcNAc'l), and 2.066 (GlcNAc-9), whereas 6 2.041 can be expected for GlcNAc-7' (see later). Attachment of Gal in /3-(1-4) linkage to GlcNAc, completing an N acetyllactosamine unit, causes a downfield shift for the signal of GlcNAc H-1 (A8 0.025 p.p.m.) and a small, upfield shift for the GlcNAc N-acetyl protons (A8 - 0.003 p.p.m.), regardless of the type of linkage of the latter residue. The resonance position of H-1 of such a terminal Gal residue primarily reflects the type of linkage of the N-acetyllactosamine unit, containing Gal, to Man-4 or -4' (8H-1 4.47 for Gal-6 and -6', 6H-14.46 for Gal-8, and 6H-14.48 for Gal-8'). The small difference in chemical shifts (A8 -0.005 p.p.m.) between the H-1 signals of Gal-6 and -6' can be ascribed to the different type of linkage between Man-4 and Man-3, in comparison to that between Man-4' and Man-3. The influences of extension of structures of the N-acetyllactosamine type with terminating NeuAc, in ~ ~ ( 2 - 6or ) a-(2+3) linkage to a Gal residue, on the chemical shifts of structural-reporter groups of neighboring residues are compiled in Tables XI11 and XIV. These Tables also contain the characteristics of the structural-reporter groups (H-3a, H-3e7and N-acetyl protons) of NeuAc itself. Similar data have been listed for the effects of attachment of Fuc, as well as for the structuralreporter groups (H-1, H-5, and -CH,) of Fuc in ~~(1-6) linkage to GlcNAc-1 (in Table XVII), for Fuc in ~ ~ ( 1 - 3linkage ) to a peripheral GlcNAc residue (in Table XIX), and for Fuc in 41-2) linkage to Gal (in Table XXI). Concerning structures of the oligomannoside type, the influences of substitution of the Man-4 and Man-4' residues of the core pentasaccharide by Man-A, -B, and -C, and, in turn, those of substitution of the latter by terminating Man-D residues, have been summarized in Table XXVII. Also, the characteristics of the H-1 and H-2 signals of terminal a-(1+2)-, a-(1-+3)-,or a-(l-+6)-linked Man residues have been given. In the foregoing, it has been emphasized that chemical shifts and chemical-shift differences are the most appropriate parameters for correlating the primary, structural characteristics with the set of n.m.r.spectral data. The fact that the chemical shifts of structural-reporter groups are so sensitive to changes in the sequence of the oligosaccharide chain opens the possibility of discriminating between compounds that are very closely related structurally. Under favorable conditions, all components of mixtures of isomers can even be characterized (see, for example, 49-51). However, the chemical shifts of structural-reporter groups are also sensitive to such conformational effects as through-space interactions, or changes in linkage orienta-

-

J. F. G. VLIEGENTHART et al.

370

TABLEXXVII l+l)-Linked, Terminal or Nonterminal Recognition of a-(1-+2)-, a-(1+3)-, or a-( Man in the Sequence aMan(l-*x)yMan(l-+z)Gly 'H Chemical Shifts of Structural-reporter Groups of a-Linked, Terminal Mana Residue

6 2s.d.

Reporter group H-1 ( X H-1 ( X H-1 ( X H-1 ( X H-1 ( X H-1 ( X H-1 ( X H-2 ( x H-2 ( X H-2 ( X H-2 ( X H-2 ( X

Man-D, Man-C or -4 Man-D, Man4 Man-A Man-4' Man-B Man-C, -D, , -D2,or -D, Man-4 Man-A Man-4' Man-B

2; y = a;z = 2; y = a;z = 2; y = a; z = 3; y = 8; z = 3; y = a;z = 6; y = p; z = 6; y = a;z = 2; y = a;z = 3; y = p; z = 3; y = a;z = 6; y = 8; z = 6; y = a;z =

2) 3) = 6) = 4) = 6) =

=

= 4) = 6) = 2, 3, or 6 ) = 4) = 6) = 4) = 6)

5.047 5.057 5.042 5.105 5.09 4.919

4.908

-c0.002 20.003

-+0.002 20.005 20.02b 20.003 ?0.001 20.005 20.003 r0.003 co.01

4.068 4.073 4.065 3.97 3.99 20.01

Principal Influences of a-linked Man on the Chemical Shifts of Structural-reporter Groups of Neighboring Residuese

Neighboring residue

Site of substitution

yMan( 1 4 z )

x =

2

Gly = aMan(143)

x =

2

yMan( 1-4

~

=

y Man ( 1 4 z )

x =6

Reporter group

M (p.p.m.) *s.d. (p.p.m.)

H-1 ( y = a; z = 2 , 3 , or 6) H-2 ( y = a;z = 2, 3, or 6) H-1(y = a; z = 2) H-2(y=a;z=2) 3H - l ( y = B ; z = 4 ) H-1 ( y = 0 ; z = 6) H-2 ( y = 8; z = 4) H-2 ( y = a;z = 6) H-1 (y = p; z = 4) H-1 (y = a;X. = 6) H-2 ( y = p; z = 4) H-2(y=a;~=6)

+0.244 t0.007d +0.037 ?0.007 -0.012 ?0.003 -0.02 20.01 +0.016 20.005 -0.017 50.003 +0.175 20.005 +0.160 20.003 -0.0 -0.027 ?0.004' +0.021 20.005 +0.015 c0.004e

Mean values &s.d. at T = 300 K; see Tables 11,111, VIII, IX, XXIV, XXV, and XXVI. structure (see 61,62, and 66). Values of A8 are mean values 2s.d. at T = 300 K, calculated for extensions with differently linked Man residues (see Tables 11, 111, VIII, IX,XXIV, XXV, and XXVI). Except for Man-A; due to steric effects, A8H-1 -0.31 p.p.m. for this residue (see compound 67). Values of A8 could not be established separately; only their combination with the influences of attachment of aMan(l-+3) could be deduced, for example, from comparison of 3 with 61. However, see the description of compound 66. "

* 6 Value for H-1 of Man-A is also influenced by spatial

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

371

tions due to elongation of the carbohydrate chain (see 6H-1 of Man-A in 67). Obviously, these effects are not comprised within the additivity rules. Except for this limitation, the additivity rules, correlating the chemical shifts of structural-reporter groups with primary, structural changes, are very useful for the deduction of 'H-n.m.r. spectra of (partial) structures belonging to the class of compounds already discussed. 111. CONCLUDING REMARKS

This article describes the application of high-resolution, 'H-n.m.r. spectroscopy for the characterization of the primary structure of 72 carbohydrate chains derived from glycoproteins. These structures constitute sets of N-glycosylic compounds of the N-acetyllactosamine type and of the oligomannoside type. Most of the 'H-n.m.r. spectra of these compounds were recorded at 500 MHz. The chemical shifts, coupling constants, and line widths of their structural-reporter-group signals were established as far as possible, and the following remarks are relevant. ( i ) The 'H-n.m.r. spectrum is, as a whole, typical for the compound investigated. Provided that it is recorded at a relatively strong magnetic field, such a spectrum can serve as an identity card, which, even without interpretation, is at least suitable for comparison with spectra of other compounds, allowing a conclusion as to whether the compounds are identical or not. The visual inspection of spectra, besides comparing data listed in tables, is extremely helpful for this purpose, as the foregoing atlas of spectra may illustrate. ( i i ) Integration of structural-reporter-group signals permits estimation of the purity of the sample. In this way, it is often easy to decide whether a sample consists of a single compound or a mixture of several components. This is particularly interesting for the detection of natural and artificial (micr0)heterogeneity. ( i i i ) The n.m.r.-spectral parameters of the structural-reporter groups bear the essential information necessary for the elucidation of the primary structure of the carbohydrate chain. If consistent series of structurally related compounds are available for both types of N-glycosylic, carbohydrate structure, a number of empirical, additivity rules for the chemical shifts of the structural-reporter groups can be deduced, as outlined in Section II,3. (iu)Differences in chemical shifts, and also in line widths, of structural-reporter-group signals allow, in several instances, qualitative deduction of spatial structures, in particular regarding the orientation of different branches with respect to each other (for example, the influences of the configuration of the anomeric center of GlcNAc-2 on the

372

J. F. G. VLIEGENTHART et al.

chemical shifts of structural-reporter groups of reducing oligosaccharides). To obtain more details about the structure of the carbohydrate chains in solution, a theoretical approach, such as that given by Lemieux and cow0rkers,8~J~~ is necessary. There is no doubt that, if, by means of more-advanced, n.m.r.-spectral techniques, more details about resonances located in the bulk of the spectrum become available, these data will be useful in elucidating secondary structures. (0) Performance of the 'H-n.m.r.-spectral measurements at 5OQ MHz has the advantage of enabling a more precise determination to be made of the chemical shifts of the structural-reporter groups, and of affording more details of the splitting patterns of their signals. Also, the sensitivity of an n.m.r. apparatus operating at such a strong magnetic field is relatively high, making possible the analysis of 25 nmoles of glycopeptide or oligosaccharide sample.99 (ui)The high-resolution, 360-MHz and 500-MHz, 'H-n.m.r.-spectral data described herein can be used for the interpretation of spectra obtained with n.m.r. apparatus operating at lower magnetic-field strengths (as has been illustrated in, for example, Refs. 50 and 104110). For the analysis of new types of compound, it is advisable to record spectra at the highest magnetic fields available; this is particularly relevant for 0-glycosylic carbohydrate structures of glycoproteins, where only a beginning has as yet been made in the characterization of corresponding oligosaccharide-alditols by means of high-resolution, 'H-n.m.r. spectr~scopy.'~~'~~~~*'''-~~~

(103) R. U. Lemieux, Chem. SOC. Reo., 7 (1978) 423-452. (101) J. P. Carver and A. A. Grey, Biochemistry, 20 (1981) 6607-6616. (105) J. Hakimi, J. P. Carver, and P. H. Atkinson, Biochemistry, 20 (1981) 7314-7319. (105a) R. R. Townsend, E. Hilliker, Y.-T. Li, R. A. k i n e , W. R. Bell, and Y. C. Lee, I . Bwl. Chem., 257 (1982) 9704-9710. (106) M. Kuriyama, T. Ariga, S. Ando, M. Suzuki, T. Yamada, and T. Miyatake,J. Biol. Chem., 256 (1981) 12,316-12,321. (107) J. Arnarp, M. Haraldsson, and J. bnngren, Carbohydr. Res., 97 (1981) 307-313. (108) T. Ogawa and K. Sasajima, Carbohydr. Res., 97 (1981) 205-227. (109) T. G. Warner and J. S. OBrien, in T. Yamakawa, T. Osawa, and S. Handa (Eds.),

Glycoconjugates, Proc. Int. Symp. Glycoconjugates, 6th, Japan Scientific Societies, Tokyo, 1981, pp. 10-11. (110) Y. Inoue and H. Nomoto, in Ref. 109, pp. 505-506. (111) H. van Halbeek, L. Dorland, J. F. C. Vliegenthart, A.-M. Fiat, and P. Jolles, Biochirn. Biophys. Acta, 623 (1980) 295-300. (112) H. van Halbeek, L. Dorland, J. Haverkamp, G. A. Veldink, J. F. G. Vliegenthart, B. Fournet, G. Ricart, J. Montreuil, W. D. Gathmann, and D. Aminoff, Eur. I . Biochem., 118 (1981) 487-495. (113) H. van Halbeek, L. Dorland, J. F. G. Vliegenthart, A.-M. Fiat, and P. JollBs,FEBS Lett., 133 (1981) 45-50.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

373

IV. EXPERIMENTAL Samples of carbohydrate structures dealt with herein stemmed from a wide variety of glycoproteins, and were isolated by a number of research groups (see Acknowledgments). The detailed sources of the compounds generously supplied for our 'H-n.m.r.-spectral investigations are indicated in the text (see also, footnote 53). The pD of solutions of glycopeptides and oligosaccharides in D20 was adjusted to 7, if necessary. Deuterium-exchanged samples were prepared by five dissolutions in D20, and lyophilizations of the solution, finally using 99.96 atom% deuterated water (Aldrich, Milwaukee, WI,USA). For n.m.r.-spectral analysis, 0.1 to 3.0 mM solutions of the compounds in 0.4 mL of DeO were generally used. The 500-MHz, 'H-n.m.r. spectra were recorded with a Bruker WM-500 spectrometer operating in the pulsed, Fourier-transform mode and equipped with a Bruker Aspect 2000 computer having an 80k memory-capacity. The D resonance of D,O was used as the field-frequency lock-signal. The spectra were obtained by using a 90"pulsewidth, and accumulated into 16k addresses with an acquisition time of 3.28 s and a spectral width of 2.5 kHz. Resolution enhancement was achieved by Lorentzian to Gaussian transformation from quadraturephase detection, followed by employment of a 32k-point, complex, Fourier transformation. In general, a few hundred acquisitions were accumulated for each sample. The indicated probe temperature was 300 K, kept constant within 20.1 K. At this temperature, the HOD resonance is at 6 -4.75; the value depends slightly on the concentration of the sample. The 360-MHz7 'H-n.m.r. spectra were recorded with a Bruker HX-360 spectrometer operating in the pulsed, Fourier-transform mode and equipped with a Bruker B-NC 12 computer having a 16k memory-capacity. The D resonance of D 2 0 was used as the field-frequency lock-signal. The spectra were obtained by using a 90" pulsewidth, and accumulated into 16k addresses with an acquisition time of 3.28 s and a spectral width of 2.5 kHz. Resolution enhancement was achieved by Lorentzian to Gaussian transformation from quadraturephase detection. In general, a few hundred acquisitions were accumulated for each sample. Spectra were recorded at probe temperatures of 295 to 300 K.

-

(114) H. van Halbeek, L. Dorland, J. F. G. Vliegenthart, W. E. Hull, G. Lamblin, M. Lhemitte, A. Boersma, and P. Roussel, Eur. I . Biochem., 127 (1982) 7-20. (115) H. van Halbeek, L. Dorland, J. F. G . Vliegenthart, N. K. Kochetkov, N. P. Arbatsky, and V. A. Derevitskaya, Eur. /. Bwchem., 127 (1982) 21-29.

374

J. F. G. VLIEGENTHART et ul.

The chemical shifts (6) are expressed in p.p.m. downfield from internal sodium 4,4-dimethyl-4-silapentane-l-sulfonate (DSS), but were actually measured by reference to internal acetone (6 2.225) with an accuracy of 0.001 p.p.m. at 500 MHz, or of 0.003 p.p.m. at 360 MHz. ACKNOWLEDGMENTS The authors are much indebted to Profs. J. Montreuil, G. Strecker, B. Foumet, G. Spik (Lille, France), K. Schmid (Boston, MA, U. S. A.), N. Sharon (Rehovoth, Israel), A. Kohata(Kobe, Japan), R. Bourrillon (Paris, France), D. H. van den Eijnden (Amsterdam, The Netherlands), and their coworkers for fruitful collaboration. The authors express their gratitude to Bruker h a l y t i s c h e Messtechnik GmbH, Rheinstetten, FRG, and Netherlands Bruker Spectrospin NV, Wormer, The Netherlands, for making the 500MHz facility in Rheinstetten, FRG, accessible for their studies, and, in particular, to Dr. W. E. Hull for recording most of the 5 0 a M H z spectra depicted herein. The remaining 500-MHz spectra and the W M H z spectra described in this article were recorded at the Dutch SOX-n.m.r. facilities in Nijmegen and Groningen, The Netherlands, respectively. The authors also acknowledge the contribution of Dr. C. A. G. Haasnoot, Prof. R. Kaptein, and their coworkers for keeping the instruments employed in excellent condition. The authors thank Dr. G. A. Veldink for his continuous interest and for his valuable discussions, Miss J. H. van Hout and Miss C. L. E. d e Lint for their expert secretarial assistance, and Mr. J. L. den Boesterd for preparing the photographs. This investigation was supported by the Netherlands Foundation for Chemical Research (SON), with financial aid from the Netherlands organization for the Advancement of Pure Kesearch (ZWO), and by the Netherlands Foundation for Cancer Research (KWF), grant UUKC-OC 7913.

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

Aaronson, W., 206 Abbas, S. A., 52,53(158) Adamowicz, H., 57,58(176) Adams, G. A., 110 Adelsky, I.. 179 Adiwaidjaja. G., 36 Ajisaka, K., 42, 65(117) Albersheim, P., 89, 105, 110(6), 111(6), 126(6), 129(6) Alexander, H. E., 195 Allerhand, A., 33.45(28,29), 48(26), 224 Aloni, Y., 107, 123(10), 128(9, lo), 133(10), 136(9, lo), 137(10), 141(113), 143(9, 10). 144, 145, 146(168). 152(168) Altieri, P., 194,208(182) Altona, C., 336 Alviano, C. S., 91 Aman, P., 63,65(211) Aminoff, D., 372 Amsbaugh, D. F., 189, 190(166) Amster, H., 88,89(122) Anderson, B., 190 Anderson, P., 195, 196 Anderson, R. L.,128, 147(115) Anderson, W. A., 30 Ando, S., 372 Andrews, G. C., 62 Angstrom, J., 215 Angyal, S. J., 33,39.48(31), 59, 60(186), 97 Aoki, K., 42 Apicella, M.A., 194 Arbatsky, N. P., 372(115), 373 Argaman, M.,202,203(221) Ariga, T.,372 Amarp, J., 372 Arnold, P. I., 188 Aronson, J. M.,69, 107 Artenstein, M. S., 158, 182(20), 190, 194, 195,208(182)

Asakawa, J., 45,46(130), 47(130), 55(130), 56(130) Aspinall, G . O., 210 Atkins, E. D. T., 179 Atkinson, P. H., 217(50),218,348(50), 368(50),372(50) Austen, K. F., 205,207 Austrian, R., 156, 170(10),171(10), 191, 192(176, 178), 205(176) Avery, 0. T., 156, 197,204 Axelos, M., 126 Axelsson, K., 98,100, 101 Azuma, I., 70, 73(31), 93

B Babcock, G. E., 77 Bache, F., 202 Bacic, A.. 145, 146(168, 169), 148(169), 152(168) Backinowsky, L. V., 56 Bacon, J. S. D., 69, 73 Baddiley, J., 170, 171, 174 Bailey, J. W., 97 Baker, C. J., 167, 168(62), 169(62), 170(62), 185, 196(62), 197(62), 206(62), 207,208(62) Baker, D. A., 317,372(83) Baker, P. J., 186, 189(147),190(147, 166) Baker, R. S., 165, 166(47) Balan, N. F., 56 Ballou, C. E.,78, 79(89), 80,83(89), 86, 87(104), 177, 217, 218(46),348(46) Baltimore, R. S., 167, 168(62), 169(62), 170(62), 196(62), 197(62),206(62), ZOS(62) Balza, F., 35 Banaszek, A., 58 Barascut, J.-L., 49 Barber, G. A., 126 Bardalaye, P. C., 94, 101 Barek, F., 180

375

376

AUTHOR INDEX

Barker, R., 28, 35(9),36(43), 37(40), 45(40), 46(40,43), 52(sS), 53(157), 54(42), 55(40), 63(43). 211 Barrena, O., 198 Barreto-Bergter, E. M.,94,95(149) Bartnicki-Garcia, S., 68,70,73, 102, 129, 131,145 Bass, J. W., 196 Bassieux, D., 75 Basten, A., 190 Bayard, B., 170 Bayer, M. E., 185 Beachey, E. H., 204 Beasley, C. A., 109,127 Beau, J. M.,62,64(207) Beauvery, E. C., 198 Beck Sommer, M.,45 Behnke, O., 125 Behrman, E. J., 56 Beier, R.C., 39 Bell, W. R., 372 Benacerraf, B., 197 Bender, V. J., 97 Ben-Hayim, G., 118,135 Bennet, L. G., 171, 174(96, W),192(83) Bennett, J. E., 97 Benziman, M.,107, 112, l13(41), 117, 118. 119(64, 65),123(10), 128(9, lo), 133(10), 136, 137(10), 141(113), 143(9, lo), 144, 145, 146(168), 149, 152(168) Berger, S., 62.64(197) Berliner, L. J., 212 Berman, S. L., 194,2tB(182) Bernhard, W.G., 156, 191(6) Bernstein, H. J., 214 Beny,J. M.,37,43(76) Bethell, G. S., 33,39,48(31) Beyer, T. A., 330 Bezer, A. E., 182 Bhattacharjee, A. K., 62,64('201,204). 97, 98,159,160(26,31), 163(26,35), 164(26, .%). 175(31.34), 185, 2Of331,34) Bhattacharjee, S. S., 82,83,92(107), 159, 217 Binette, J. P., 217(67,68,69), 237, 240(67,68,69), 242W). 248(67,68), 317(67), 3eo(67,68, 69),321(67), 365(67)

Bishop, C. T., 74,82, 110, 158, 171, 174(96,97), 185(19), 192(83) Bitoon. M. A., 90 Bjiirndal, H., 7 4 9 8 , 100,101 Bjorksten, B., 204 Blackwell, J., 112, 113 Blank, F., 74,82 Blanton, W.E., 115 Blaschek, W., 109 Blazer, R. M., 40 Blombren, H., 190 Bluhm, T. L., 179 Blunt, J. W.,33 Bock, K., 29,30,33,36,37(18, a), 39(34), 40(34,83), 41(62,63), 43(22, 631, 44(19), 45(63,83), 46(83), 47(63, 831, WW, 49(62,63,83), W52,62, 63,83),5W3), 52(62), SGW, a(62, 82, a), sS(62,83), 56(83),57(83, 16% WW, W831, W83). 181,317, 372(83) Boersma, A.. 372(114), 373 Boivin, J., 63, 65(209) Bortolussi, R., 204 Bottom, C. B., 71,73 Bouquelet, S., 170,308,324(81) Boumllon, R., 359,372(99) Bouveng, H. O., 103 Bowers, B., 145 Boykins, R. A., 164, 165(46), 167(46) Braley-Mullen, H., 189, 197 Brandt, D. L., 194,195,208(182) 166(48,49,51), 167(43,45) Branefors-Helander, P., 164, 165(43,45), Brant, D. A., 179 Braun, W., 198 Breimer, M.E., 215 Breitmaier, E.,28(16),29,34(16), 35, 36(16), 39(16), 41,42(16), 43,45(48, 98), 46(48, 131, 134), 47(131), 48(134), 49(134), 51(98),55(137), 56(134), 59, 60(187), 65(118) Brett, C. T., 134,147 Bretthauer, R. K.,86 Broido, A., 51 Brossmer, R., 62,64(203) Brown, D. L., 121 Brown, R. G., 74,96 Brown, R. M.,Jr., 112, 116, 117(35), 118, 119, 120(74), 121, 122,123, 144

AUTHOR INDEX B m f i t t , W., 205 Bnunmond, D. O., 146 Brundish, D. E., 170 Brunngraber, E. G., 217(63),235,344 Buchala, A. J., 139, 140(154) Buchanan, J. G., 40,53(89), 171, 174(88, 89,QW Buchanan, T. M., 185, 186(137), 202(137), 204(137) Buchs, L., 139, 140(154) Buck, K. W.,68,74,75(60) Biildt, G., 115 Biirvenich, C., 43 57(174, 1751, 159, Bundle, D. R., %,a, 160(31),162(33),171, 175(31,33), 179(33), 181, 183,206(31),317, 372(83) Burchard, W., 179 B d t t , A. I. R., 58 Burgess, J., 143 Burton, K. A., 88,102 Bush, D. A., 82, 92 Bushway, A. A., 72

C Cabib, E., 145 Cadmus, M. C., 88,102 Caldes, G., 189, lW(166) Callaghan, T., 145, 146(168), 152(168) Calver, G., 185 Canale-Parola, E., 107 Cantino, E. C., 69 Carbonell, L. M., 70,73(31), 93 Cardimil, L., 71 Carlo, D. J., 171, 173(84,92),192(92), 193(84,85) Carpita, N. C., 109,126,131, 139, 140(151), 141(151), 144, 147, 148 Carver, J. P., 216,217(50),218,348(50), 368(50),372(50) Cary, L. W., 36,SZ(56) Casu, B., 27,34,36(2),39,41(2,36), 45(36),46(36),47(36) Cadey, B. J., 72 Ceccarini, C., 217(50), 218,348(50), 368(50), 372(50) Cerezo, A. S., 72 &m$, M., 51

377

CesBro, A., 179 Chdn-Fuertes, M. E., 40,53(89) Chain, E. B., 74,75(60) Chambat, G., 111 Chw, H.-Y., 145 Chapman, D., 214 Chen, A. W., 74,75(60) Ch6n6, L., 136 Chenon, M.-T., 28 Cherniak, R., 62,64(202) Chkron, A., 363 Child, J. J., 74 Chittenden, G. J. F., 174 Chizhov, 0. S., 28,41(7), 51,53(100), 62 Chmielewski, M., 54,57,58(176), 62, 63(196) Chmurny, G. N., 62 Christie, G. H., 182,189,190(167) Chu, B., 179 Chumpitazi, B., 111 Clark, E. L., 35,36(43), 46(43), 63(43) Classon, B., 165, 166(49) Clegg, J. A., 208 Climenson, P. A., 165,166(50) Cohen, J. S., 194,203(186) Cohen, R. E.. 217,218(46),348(46) Cohn, M., 30 Collins, P. M., 42 Colonna, W. J., 87 Colson, P., 52, 54(154), 59(154), 224 Colvin, J. R., 105,112, 117(7), 118, 122, 128(7), 131, 132, 133(129), 135(7), 136, 142 Conant, N. F., 103 Conneely, M. J., 39 Conreur, C., 56,57(173) Conway, E., 41,53(102) Cook, J. A., 130 Cooper, D., 117, 143 Cooper, F. P., 74,82 Cooper, K. M., 117,119(65) C&, W. A., Jr., 111 CouIter, D. B., 69 Courtenay, B. M., 182,189,190(167) Couso, R. O., 133 Cowley, D. E., 39 Coxon, B., 28,33(4), 34(4), 37(4),41(4), 43(4),50,58,214

378

AUTHOR INDEX

Crabb, J. H., 167, 168(62), 169(62), 170(62),196(62),197(62),206(62), 208(62) Crawford, T. C., 62 Crisel, R. M., 165, 166 Crossan, F. J., 188, 204(154) Cumrine, M. H., 196 Cunningham, W. L., 86 Cyr, N., 35,37, 39,41,43(70), 47(92), 5 l(69) Czarniecki, M. F., 37,43(75), 62, 64(206)

Dennis, D. T., 110 Densen, P., 186,202(148),204(148) Depezay, J.-C., 42,65(115) Derevitskaya, V. A,, 54,372(115),373 Deven, J. M., 103 de Vries, 0. M. H., 70 Dickerson, A. G., 74, 75(60) Dickinson, D. B., 128 Dilapi, M. M., 190, 191(175) Dill, K., 224 Dobberstein, B., 121 Dwhez, A. R., 156 Doddrell, D., 33,48(26) D Doddrell, D. M., 37 Dabrowski, J., 215 Dorland, L., 211,212(12, 13),217(55,56, Dabrowski, U., 215 58,59,60,61,62,64,65,66,67,68, Daleo, G. R., 108, 127, 133(17),137(17) 69, 70, 71, 72, 73, 73a, 75, 76,77,78, Dalling, D. K., 35 79), 220,224(55), 225,227(56), 234, Daly, A. K., 196 235,237, 239(66),240(66,67,68, 69), Daniel, P. F., 355 242(64,67), 2.14(64,65), 246(64), Dankert, M. A,, 133, 134, 143(132) 248(66, 67,68), 254(62),257(64,65), Daoust, V., 136, 171, 173(95),193(85) 259(70),261(64),264(64,65), 266(64, Darke, A., 178 65), 267(64, 65), 269,274, 275(72), Darvill, A., 105, 110(6), 111(6), 126(6), 277(72, 76), 279(72,76), 281(13), 129(6) 283, 285(13, 72, 78), 289(78), Davidson, W. L., 192 291(72),295(72),293(72),301(72), 304(13, 72), 306(13,55), 308,312(13, Davies, A. J. S.,189, 190(167) 66),316(13,56,66), 317(67),318(84), Davies, C. E., 168 Davies, D. B., 214 320(67,68,69), 321(13,67), 324(81), Davis, D. G., 211 328(84),329(84),330(13, 56), 333, 334(89), 335(89),336(89),338(89), DeBie, M. J. A., 41,53(96) 340(89),343(12),344(58,59), 347(58, Debray, H., 285 Defendis, D. J., 355 59), 350(61),352(61),355(61),359, Deinema, M. H., 107 362(66),363,365(67), 372(12, 13, 99, Dekker, R. F. H., 128, 141(112) 114, 115),373 de Larghe, E., 109 Dorman, D. E., 34, 35(37),41(37),45(37), Delbaere, L. T. J., 39, 54(82), 181,317, 46(37), 52(37),55(37), 159, 165, 372(83) 166(47) Dorner, M. M., 190 de Lederkremier, R. M., 72, 103 Dorow, D., 194,203(186) de Leeuw, F. A. A. M., 336 Delmer, D. P., 105, 109, 110(6), 111(6), Doyne, 0. E., 156 Drewes, L. R., 95 122, 123, 126(3,6), 127(3),128(3), Dubesset, D., 217(56),220,227(56), 129(6), 131(21,26), 134(111),137, 139, 140(151), 141(113, 151), 143(85), 316(56),330(56) 144, 145(31),146(168, 169), 147(3, Dubs, R., 204 I l l ) , 148, 149, 150, 151(184), Durr, M., 146 152(168) Dunne, F. T., 158 Delpuech, J.-J., 28(17),29,33(17), du Penhoat, P. C. M. H., 33,40(30), 34(17),36(17),38(17) 48(30,33) de Marco, A., 211 Dureault, A., 42,65(115)

379

AUTHOR INDEX

Durette, P. L.,214 Dutton, G. G. S., 179 Dwek, R. A., 212

E Edgar, A. R., 40, 53(89) Edwards, M. S., 207 Egan, W., 159, 164, 165(44,46), 166(50, 52), 167(44,46), 179(32),183, 186, 194, 195,202,203(186, 191,222) Egge, H., 215,355 Eickhoff, T. C., 196 Eid, A. A. H., 109 Elbein, A. D., 126, 127, 132, 134(130) Elinov, N. P., 100 Elnaghy, M. A., 126 Elsasser-Beile, V., 179 Endo, M., 217(79), 285,318(84), 320, 328(84), 329(84) Erbing, C., 165, 166(48) Ericson, M. C., 134 Ermen, B., 111 Emst, R. R., 30,211 Esaki, H., 74 Eschenfelder, V., 62,64(203) Eveleigh, D. E., 80,92(92), 101 Evstigneev, A. Yu., 54 vtushenko, E. V., 50 xcoffier, G., 35

z

F

Falk, K.-E., 214,215 Fanner, F. S., 144 Fanner, V. C., 69,73 Farr, D. R., 88,89(121, 122) Farriaux, J.-P., 217(62),234,254(62) Faubl, H., 62 Fearson, D. T.,205,207 Feingold, D. S., 128 Fendler, J. H., 28 Femandez Cirelli, A., 103 Ferrieri, P., 204 Ferrige, A. G., 34 Fevre, M., 129 Fiat, A.-M., 372 Fiedel, B. A., 188,204(157) Fillol, L., 51 Finch, P., 58, 59(185) Fincher, G. B., 110, 130(30),147(30)

Finland, M., 156, 192(8),196,201 Finlayson, A. J., 100 Finn, C. W., 206 Finne, J., 210, 334,341(92a) Fisher, G. W., 196 Fleet, G. H., 74 Flowers, H. M., 210 Fontana, J. D., 69 Forsee, W. T., 134 Foumet, B., 170,210,217(55,56,60,62, 66.67, 68,69, 71, 72), 220,224(55), 227(56), 234,237,239(66), 240(66, 67, 68, 69), 242(67),248(66,67, 68), 254(62), 259, 269,275(72), 277(72), 279(72), 285(72), 291(72), 295(72), 298(72), 301(72),304(72),306(55), 308,312(66), 316(56, 66),317(67), 318(84), 320(67-69), 321(67), 324(81), 328(84), 329(84),330(56), 334,362(66), 365(67),372 Francis, T., Jr., 156, 191 Franke, W. W., 111 Franz, G., 109,126,138,139(22) Frasch, C. E., 195 Fraser, B. A,, 166, 171, 183, 185(136), 192(@) Fraser, C. G., 72, 97 Fraser, R. N., 103 Freeman, R., 37 French, A. D., 113 Friebolin, H., 62, 64(203) Friemer, E. H., 167, 168 Friis, P., 54, 65(163) Fiigedi, P., 41,42(103), 53(103) Fujimoto, M., 87 Fukagawa, K., 101 Fukuda, K., 103 Fukuoka, F., 73, 75, 76 Funakoshi, S., 328 Funatsu, M., 76 Funcke, W., 33,48(32),58,59(183)

c Gagnaire, D. Y.,35,36,43,75,76 Galanos, C., 188, 2@4(158) Gander, J. E., 68,95 Garcia, R. C., 133, 143 Gardner, K. H., 113, 179

AUTHOR INDEX

380

Garegg, P. J., 41,42(105,106),53(105,

106) Garon, C. F., 206 Gathmann, W.D., 372 Gatti, G., 39 Gero, S. D.,41,42,45,53(102, la), 55(128),63,65(118,212) Getzner, J., 176 Gibson, A. R., 41,46(99),53(99), 57(99) Gibson, R., 334,341(91) Giddings, T.H., Jr., 121 Gil, F.,70 Gillet, S., 33 Gisin, B. F., 211 Glaser, L.,117 Glasgow, L.R., 330 Glaudemans, C.P.J., 97,98 Gligorijevic, M.,42,65(116) GliSin, D.,42,65(116) Glode, M. P.,194,200,201(212) Glynn, A. A., 204,205 Goebel, W.F.,171,172(81),174, 176,

197 Gold, R., 189,191(165),194(165),

201(165),204(165) Goldings, E., 189 Goldring, 0.L.,208 Goldschneider, I. M.,189,190, 191(165),

194(165),201(165),202,204(165) Goldstein, I. J., 74 Golub, E. S.,186, 189(145) Goodwin, J. C.,60, 61(191) Gorin, P.A. J., 28,29(3),34(3),35(3), 37(3),41(3),43(3),45(46),46(46), 47(46),51(124),52,53(93),55(46), 56(46),62(46),68,69,72(9),77,78, 80, 82(85),83(85),85,87(7),89(6), 90,91(6,126),92(92,103,107), 93(103,137),94,95(149),99, 100(95),101,102(126),159,217 Goring, D. A. I., 115, 116(50) Gotschlich, E. C., 158, 160(23),167, 168(61),182, 183, 185(61,136), 189, 190,191(165),194(165), 195, 201(165),202,203(186,221), 204(165) Grabowski, G.A., 355 Grant, D. M.,28,35 Gray, G. R.,33,48(27),60(27),61(27) Creve, W., 36

Grey, A. A., 216,217(50),218,348(50),

368(50),372(50) Grindley, T. B., 40,53(90) Griph, I., 215 Gross, S. C.,189 Grout, B. W.W., 121,122 Grumet, F.C.,190 Gulasekharam, V.,40,53(90) Gullyev, N.,51 Gupta, D.S.,62,64(202) Gupta, R. K.,39 Gustine, D.L.,42 Guthrie, R. D.,41,53(102),58

H Haasnoot, C. A. G., 336 Haass, D., 109 Hachisuka, Y., 74 Hagaman, E. W., 39,54(81) Haigler, C.H., 112, 113(41),117,118,

119(64,65) Haines, A. H., 52,53(158) Hakimi, J., 217(50),218,348(50),

368(50),372(50) Hall, L. D., 29,30,37(18,22),41(74),

43(22,76),214,215(28) Halper, L.A., 146 Halpern, Y., 51 Hamada, A., 103 Hamer, G. K.,35 Handa, S.,372 Handzel, Z.T.,195,202,203(221) Hanessian, S., 39 Hanfland, P.,215 Hanna, R. B., 111 Hansen, P.E.,37 Hansson, G. C.,215 Hapner, K. D.,89 Hara, C.,97 Harada, K.-I., 63,65(210) Haraldsson, M.,372 Harding, A. L.,196 Harpaz, N., 216 Haryu, K., 101 Hasegawa, S.,69 Hasenclever, H. F., 74,82 Haskins, R. H., 69,73(9),74,90 Hassid, W.2.. 126,128,146

AUTHOR INDEX Haverkamp, J., 41, 52(94), 52(95,96), 217(56,60,62,67,68,71,72,73, 73a, 75, 77), 220,227(56), 234,237, 240(67,68), 242(67),248(67,68), 254(62), 259,269,274,275(72), 277(72), 279(72),283,285(72), 291(72), 295(72),298(72),301(72), 304(72),312,316(56), 317(67), 320(67, 68),321(67),330(56), 365(67), 372 Hay, G. W., 74 Hayes, M. L., 211 Heath, I. EL, 124 Heidelberger, M., 156,171, 172(86),174, 176, 177, 190, 191(175),192(86), 195, 201 Heiniger, U.,110, 126, 128, 145(31), 147(118) HelIer, J. S., 127 Hensley, D. E., 77 Hepler, P. K., 124 Herlant-Peers, M.-C., 217(62,76), 234, 254(62), 275,277,279(76) Herth, W., 124,144 Herzog, W., 124 Hestrin, S., 107 Hill, R. L., 170,330 Hilleman, M. R., 192 Hilliker, E., 372 Hirose, K., 97 Hiss, D., 59, 60(187) Ho, S.-C., 35,36(50) Hoare, K., 217(79), 285 Hodge, A. J., 112 Hodge, J. E., 60, 61(191) Hodges, R. G., 156, 191(6) H o h a n n , G. C., 73 Hogetsu, T., 144 Hollstein, U., 35,45(48),46(48) Homewood, T., 139, 140(151) Honda, S., 52 Hopp, H. E., 108, 127,133, 134,137, 144(136) Horisberger, M., 69, 77, 82,88,89(121, 122), 92, 103(82) Horitsu, K., 78 Horman, I., 92 Horton, D., 33,214 Houwink, A. L., 123 Howard, C. J., 204,205

381

Howard, J. G., 182, 189, 190(167) Hranisavljevi6-Jakovljevib,M., 96 Hsu, K. C., 185 Hughes, T. R., Jr., 30 Hull, W. E., 217(58,61,64, 66),225,227, 235,237, 239(66),240(66),242(64), 244(64),246(64), 2 W W , 257(64), 261(64), 264(64), 266(64), 267(64), 312(66),316(66), 333,334(89), 335(89),336(89), 338(89),340(89), 344(58),347(58), 350(61),352(61), 355(61), 362(66),372(114),373 Huntley, C., 200,201(212) Hussey, G. G., 144 Huwyler, H. R., 109, 139 I Ielpi, L., 133 Imada, K., 179 Imbach, J.-L., 49 Inch, T. D., 28,214 Ingall, D., 196 Inoue, Y., 347(95),548,372 Irvine, R. W., 58 Isaac, D. H., 179 Isbell, H. S., 33,40(30), 48(30), 62, 63(194) Isenberg, J. N., 217(51), 220, 225(51), 333(51), 338(51), 340(51) Isherwood, F. A., 126 Ishido, Y.,42 Islomov, M., 41, 53(100) Itatani, Y., 42 Ito, S., 63, 65(210) Ito, T., 62, 63(195) Iwashita, S., 347(95), 348 Iwata, K., 69

J Jackson, R. W., 188,204(156) Jacobsen, S., 47 Jag, J., 164 Jann, K., 158, 177(17),178(17),204(17) Jansson, P.-E., 41,42(106), 53(106),100, 170(100), 174 Jaques, L. W., 43,62(127), 64(205) Jamefelt, J., 210 Jarrell, H. C., 62, 64(200) Jautelat, M., 27

382

AUTHOR INDEX

Jeanes, A. H., 88, 102 Jennings, H. J., 52, 54(154), 59(154), 62, 64(201, 204), 72, 97, 3,159, lfio(26, 31). 161, 162(33,34, 36), 163(26,35), 164(26,35,40), 167, 168(62), 169(62,69, 70), 170(62,63, 68,69,70, 71), 171, 172(84,92), 175(31, 33,34), 177(63,68, 69), 178(111), 179(33,36), 181(111),183, 185(19), 1cj2(92),193(84),194, 196(62,63), 197(62), 199,206(31, 34, 62, 63),207,208(62,63, 68, 69, 76, 131, 185) Jensen, S. R., 50 Jentoft, N. H., 95 Jewell, T. R., 72 Johnson, J., Jr., 74 Johnson, K. G., 163, 164(40) Johnson, L. F., 159 Johnston, I. R., 69 Johnston, R. B., 195 Jolks, P., 372 Jones, D., 69,73 Jones, G. H., 80 Jones, J. K. N., 41,46(99), 53(99), 57(99), 171, 173(82), 174(82) Jones, R. G., 62,64(202) Jonssen, E. K., 158, 160(23) Jorbeck, H. J., 158 Jordan, R. C., 179 Joseleau, J.-P., 111 Josephson, S. J., 56,57(175), 181 Jouanneau, J., 359,372(99) Jovanovic, S. M., 115 Joziasse, D. H., 217(78), 283,285(78), 289(78) Jung, G., 28,39, 46(134), 47,48(134), 49(134), W137), 56(134), 59, 60(187)

K

Kabat, E. A., d i , 176, 182, 190 Kaczorowski, B. J., 86 Mrkkiiinen, J., 210 Kaiser, H., 158 Kakuta, M., 75, 97 Kalinovskii, A. I., 50

Kam, B. L., 49, 50(147), 51, 56(147) Kamerling, J. P., 62, 64(207) Kanda, Y.,69 Kane, A. J., 157, 167, 168(60) Kanetsuna, F., 70, 73(31), 93 Karakawa, W. W., 157, 167, 168(60) Karamanos, Y.,318(84), 320,328(84), 329(84) Karlsson, K.-A., 214,215 Karpas, A. B., 164, 165(46), 167(46) Kasai, R., 45,46(130),47(130, 132), 55(130), 56(130) Kasper, D. L., 167, 168(62), 169(62,69, 70), 170(62, 63, 68, 69, 70, 71), 177(63, 68,69), 179(111),181(111), 185, 194, 195, 196(62,63), 197(62), 206(62, 63),207, 208(62, 63, 68,69, 131, 182) Kato, K., 74 Katz, D. H., 197 Katzenellenbogen, E. M., 162, 169(69), 170(69, 71), 208(69) Kazatchkine, M. D., 207 Keller, A., 62, 64(199) Keller, F., 130 Keller, T., 59, 60(187) Kendall, F. E., 156, 176 Kendall-Morris, S., 206 Kenne, L., 88, 158, 164, 165(43,45), 166(48,49, 51), 167(43,45), 171(18), 172(18,80), 174(80), 178(18),185, 192(87) Kennedy, D. A., 171, 174(88) Kenny, C. P., 62,64(201), 159,'160(26, 31), 162(34,36), 163(26,35), 164(26, 35), 175(34), 179(36), 183, 185, 206(31, 34) Khan, A. W., 132, 133(129) Khuong-Huu, Q., 56, 57(173) Kieboom, A. P. G., 59 Kiermayer, O., 121 Kiho, T., 97 Kikumoto, S., 74 Kim, K. S., 41, 57(91) Kimelberg, H. K., 150 Kimura, K., 74 Kindinger, J. I., 129 King, G. G. S., 136 King, R. R., 224

AUTHOR INDEX

Kirkwood, S., 69,74 Kjosbakken, J., 133, 135 Klein, A., 109, 122, 131(26),144, 145, 146(168),147, 152(168) Klein, J. O., 196 Kline, L. F., 188 Knecht, J. C., 171 Knowles, P. F., 212 Knutson, C. A., 102 Kobata, A., 210,217(64a), 235,242(64a), 244(64a),246(64a),261(64a), 264(64a),328, 334(64a),341(92), 347(95), 348,355 Kobayashi, S., 76 Koch, H. J., 34,35,36(50),41(36), 45(36), 46(36), 47(36, 92) Koch, K. F., 39,46(49), 54(81) Kochetkov, N. K., 56,372(115), 373 Koehler, H., 109 Koemer, T. A. W., Jr., 36,52(56) Kohama, T., 87 Koide, N., 347(95), 348 Kolpak, F. J., 112 Komatsu, N., 74 Komura, H., 42 Kondo, W., 62, 63(195) Komfeld, R., 210, 220(3),237(3), 240(3), 269(3), 283(3), 306(3),334(3) Komfeld, S., 207, 210,220(3), 237(3), 240(3), 269(3), 283(3), 306(3),334(3), 341(91) Korsch, B., 41,47(92) Korytnyk, W., 55 Koto, S., 39,43, 54(82), 181,317, 372(83) Kotowycz, G., 28, 29(11), 39(11),214 Kov66, P., 62 Krauss, M. R., 204,205(226) Kreger, D. R., 70 Kretschmer, P. J., 206 Krusius, T., 210, 334,341(92a) Kuhn, S., 215 Kullnig, R. K., 214 Kulow, C., 128, 134, 147(118) Kuninaka, A., 87 Kunkel, H. J., 190 Kuriyama, M., 372 Kushida, K., 42 Kvarnstrom, I., 41,42(105, 106), 53(105, 106) Kwan-chung, K. J., 97, 98

383

L Laffite, C., 50(148), 51,55(148) Lahoz,R., 74 Laine, R. A., 372 Lallemand, J.-Y., 37 Lamblin, G., 372(114),373 Lampen, J. O., 87 Lancefield, R. C., 167, 168(61),185(61), 196(55-57) Larm, O., 171 Larsen, G. L., 146 Larsen, P. O., 54,65(163) Le, C. T., 194 LeCoco, C., 37 Lecomte, J. T. J., 211 Ledbetter, M. C., 124 Lee, E. E.,39 Lee, G.-J., 171, 192(94) Lee, J. C., 146 Lee, L. L. Y.,146 Lee, W.-L., 93 Lee, Y.C., 198,372 Lefller, H., 214,215 LeFur, R., 59, sO(l86) Leger, D., 217(77), 283 Leidy, G., 164, 195 Lelkes, P. I., 150 Lembi, C. A., 129 Lemieux, R. U., 28,29(11), 37,39(11), 43, 54(82), 181,214,317,372(83) Leontein, K., 174 Lepow, M. L., 189, 191(165), 194(165), 201(165), 202,204(165) Leppard, G. G., 118 Lesavre, P. H., 205 Letoumeau, D. R., 103 Leuchars, E., 189, 190(167) Lewin, E.B., 194 Lewis, B. A., 69, 74 Lhermitte, M., 372(114),373 Li, E., 334,341(91) Li, S.-C., 210 Li, Y.-T., 210,372 Liang, C. J., 328 Lindberg, A. A., 158, 198,199(15) Lindberg, B., 41,42(105, 106), 53(105, 106), 74,88,96,98, loo, 101,102, 103, 158, 164, 165(43,45), 166(48, 49), 167(43,45), 170, 171(18, 72),

AUTHOR INDEX

384

172(18,79,80, 100). 173(72), 174(80),178(18),192(87),210 Lindh, F., 41,42(106),53(106) Lindon, J. C.,34 Lindquist, U., 172(100),174 Lindqvist, B.,165,166(51),171,172(79) Linstead, P.J., 143 Lipke, P. N.,83,87,177 Lip&, A., 41,42(103,104),53(101,103, 104) Lis, H., 210,220(4),237(4),240(4), 269(4),283(4),306(4),334(4),365 Llinris, M., 211 Lloyd, C., 119 Lloyd, K. O.,77,90,93 Lo,T.M., 188 Gnngren, J., 41,42(106),%(IN),170, 171(72),172(79),173(72),174,210, 372 London, R. E., 35,37(40),45(40),46(40), ~ 4 0 ) Longmore, G., 216 h, M., 188 Lopez-Romero, E., 145 Lott, I. T., 355 Lowe, E. P., 103 Lowe, H., 171,173(95) Lowell, G.M., 196 Lowenthal, J. P.,194,2@3(182) Liideritz, O.,157,188,204(158) Lugowski, C.,168, 169(69), 170(69,71), 177,178(111),181(111),194, 199, 20?3(69,76,131,185) Lui, T.-Y., 158,160(23),179,182(20), 183,185(136),194,195,200, 201(212),2a3(186) Lukacs, G., 41,42,43,45,53(102, 128). 55(128),63,6!5(118,212) Lundt, I., 55, 57(16!5) M McArdle, P., 39 McCulIy, M. E.,141 McDevitt, H. D., 190 McCinnis, G. D.,105,112 McLean, A. A., 192 McLeod, C. M., 156,191(6),204, 205(=) McNeil, M.,89,105,110(6), 111(6), 126(6),129(6)

Macaskill, J. B., 43,62(127) Maclachlan, G.A., 105,110,130(30), 145,146,147(30) Madden, J. K.,171,192(87) Maekawa, S., 76 Magus, R. J., 83, 92(103),93(103) Mahadevan, P. R.,73 Mahadkar, U.R.,73 Maltby, D.,139,140(151),141 Mancier, D., 36 Mandell, G. L.,186,202(148),204(148) Mandrell, R. E.,195 Manley, R. S. J., 117,143 Manners, D. J.,73,74 Manocha, M. S., 103 Marchant, R., 70 Marchessault, R. H.,179 Marinetti, G.V., 158 Marsh, D., 212 Martin, A., 159,160(31),162(34),163(35), 164(35),175(34),206(31,34) Martin, G. J., 28(17),29,33(17),34(17), 36(17),W17) Martin, M. L., 28(17),29,33(17),34(17), 36(17),W17) Martinez, P.R., 134 Martin-Lomas, M., 51,214 Marx-Figini, M., 115,116 Masson, A. J., 73,74 Matsuda, K., 41,52(97),69,70,74,96, 101,138,139(149),143,144(149) Matsuhiro, B., 60,61(190) Matsui, M., 62,64(198) Matsuno, A., 42 Matsuura, F.,355 Matsuzawa, M., 42,65(117) Matwiyoff, N. A., 35,37(40),45(40), 46(40),5!5(40) Mauck, F. T.,202 Mazurek, M., 35,41,43,45(46), 46(46), 47(46),51(124),53(W,55(46), 56(46),62(46),85,93,94,217 Meier, H.,109, 139(22), 140(154), 142 Meinert, M., 109,131(21),139 Meloche, H.P.,39 Mendershausen, P. B.,78,79(89),83(89) Mendonca, L,90 Mendonca-Previato, L., 90.93 Merchant, Z. M., 58,59(185) Meml, R. E.,156

385

AUTHOR INDEX Meyer, B., 29, 36,44(19) Meyer, K. H., 113 Meyer, Y., 144 Michalski, J.-C., 217(58,59,61, 64,65, 72), 225,227,235,242(64),244(64. 6% 246(64),257(64, 65),261(64), 264(64,65),zsS(64, a),267(64,65), 269,275(72),277(72),279(72), 285(72), 291(72), 295(72), 298(72), 301(72), 304(72),333,334(89), 335(89),336(89),338(89),340(89), 344(58,59), 347(58,59),35WU, 352(61),355(61) M i h v i k , V. M., 96 Midelfort, C. F., 39 Mieczkowski, J., 57,58(176) Miedema, F., 198 Mihara, K., 355 Miljkovik, M., 42, 65(116) MiljkovibStojanovik, J., 96 Mims, G. A., 187 Misaki, A., 71, 74, 75,97 Misch, L., 113 Mitchell, A. J., 72 Mitchell, B., 145,146(168),152(168) Mitchell, G. F., 190 Miyatake, T., 372 Miyazaki, T., 54,69,71,94,95, 171, 173(93), 192(93) Mizsak, S., 63,65(212) Mizutani, K., 45,46(130),47(130, 132), 55(130),56(130) Moff, G. E., 195,203(191) Mols, O., 47 Mond, J.. 189 Monneret, C., 56,57(173),63,65(209) Montezinos, D., 119,120(74),123, 134, 139,140(151),141(151), 143(85), 144, 148 Montgomerie, J. Z., 157 Montxeuil, J., 170,210,211,217(52,55, 56,58,60,61,62,64,66,67,68,69,

71.72,73, 76),Zeo(l,2),224(%), zzY52). 227(52, W, 234,235(52), 237(1,2),239(66),240(1,2,66,67, 68,69), 242(52,64,67),!?.44(52,64), 246(52.64), M(66. 67,681, zsl(sZ), 257(64),259,261(52,64),M(52, 64).266(64),267(64), 269(1,2,52), 274,275(72),277(72,76),279(72,

76), 283(1,2),285(72), 291(72), 295(52,72),298(52,72),301(72), 304(52,72),306(1,2,55), 308, 312(66),316(52,56,66),317(67), 318(84), 320(67,68,69),321(67), 324(81),328(W, 329(W, 330(56), 333(52),334(1,2,52, 89), 335(89), 336(52,89),338(52,89),340(52,89), 344(58), 345U). 347(58),350(52,61), 352(52,61),355(61),362(66), 363, 365(67),372 Moore, D. M., 129 Moorehouse, S. J., 40,53(89) Moreland, D. E., 144 Moro, K., 179 Moms, E. R., 178 Moms, G. A., 37,41(74),215 Morrison, D. C., 188 Morse, H. C., 189 Mortimer, D. C., 138 Mosier, D. E., 189 Moxon, E. R., 188,204(154),205 Moyer, B. G., 42 Moyna, P., 97 Miilethaler, K., 112,116 Mueller, S. C., 118, 121, 122 Muggli, R., 113 Mukumoto, T., 101 Muller-Eberhard, H. J., 187,205(151), 207 Munasinghe, V. R. N., 42 Mundkur, B., 77 Mundy, B. P., 39 Munro, M. H. G., 33 Munson, R., 117 Muramatsu, T., 347(95),348 Murao, S., 101 Muraoka, Y., 47 Murty, V. L. N., 82 Myerowitz, R. L., 202,203(221)

N Nagabhushan, T. L., 37 Naganawa, H., 47 Nagashima, K., 63, 65(208) Nagel, J., 198 Nakahara, W.. 72 Nakajima, T., 74,80,96 Nakanishi, H., 54,224

AUTHOR INDEX

386

Iriakanishi, M., 75 Nakazawa, F., 62, 63(195) Ngnasi, P., 41, 42(103, IM), 53(101, 103, 104) Naoi, Y., 71, 94 Narasimhan, S., 216 Nardin, R., 43 Nashimura, O., 183, 185(136) Nesbitt, L. R., 74 Neszmelyi, A,, 41,42(103, lM), 43, 50(146), 51, 53(101, 103, l a ) , 56, 57(172), 63, 65(212) Neufeld, ti. F., 128 Ng Ying Kin, N. M. K., 159,215,216(40, 41) Nicholson-Weller, h., 207 Nielsen, B. J., 50 Niemann, H., 179 Nikaido, H., 157 Nimmich, W., 41,42(106),53(106), 157, 201 Nishida, T., 28 Nishikawa, Y., 75, 76 Nomoto, H., 372 Xorcross, N. L., 167, 168(59) Nordin, J. H., 69,70, 94, 101 Nordin. P., 126 Nom, V., 50 Norman, J., 69 Northcote, D. H., 80 Nunez, H. A,, 36. 52(55), 355

0 Obaidah, M. A., 68 O’Brien, J. S., 372 O’Conner, J. V., 36, 52(55) Ogasawilra, N., 74 Ogata, A.-M., 347(95),348 Ogawa, T., 62,64(198), 372 Ogihara, Y., 50 Ogura, M., 87 Ohad, I., 118, 135 Ohki, T., 73,75 Ohkubo, S., 74 Ohkura, T., 217(64a), 235,242(64a), 244(64a),246(64a), 261(64a), 264(64a),334164a) Ohno, H., 171, 173(93), 192(93) Ohno, N., 54 Ohno, T., 69

Okada, S., 217(64a), 235,242(64a), 244(64a), 246(64a), 261(64a), 264(64a), 334(64a), 355 Okano, T., 114 Okihara, M., 45,46(130), 47(130), 55(130), 56(130) Olsen, C. E., 54,65(163) Olsson, K.,63,65(211) Oppenheimer, N. J., 50(147), 51,56(147) Ordin, L., 127, 129 Brskov, F., 195,203(191) Brskov, I., 195 Osborn, M. J., 117 Padan, E. D., 149 Padav, E., 149 Pais, M., 63, 65(209) Palczuk, N. C., 198 Palevitz, B. A., 124 Palma, A., 115 Pamblanco, M., 324 Pangburn, M. K., 205,207 Panzica, R. P., 28 Parish, R. W., 108, 128(16) Parke, J. C., 195,200,201(212) Parrish, F. W., 36, 39(60), 45(59),46(59, 60),47(59), 55(60), 62(59) Patil, G., 39 Patterson, J. C., 73, 74 Paul, B., 37, 55 Paul, W. E., 186, 189(146),197 Paulsen, H., 36, 50, 55(145), 56(145) Paulson, J. C., 170,330 Peaud-Lenoel, C., 126 Pedersen, C., 29,33,36, 37(18), 39(34), 40(34,83), 41(62, 63),43(63), 45(63, 83), 46(83), 47(63, 83),48(83), 49(62, 63,831, SO(52, 62,63,83), 51(83), 52(62), 53(83),54(62, 83), 55(62, 83), 56(83), 57(83, 165), 58(83),62(83), W83) Pegg, D. T., 37 Perlin, A. S., 27,28,33,34,35,36(2), 37(64), 39,40(5, 30), 41(2, 5,36, 64), 43(70), 45(36), 46(36), 47(36, 92), 48(30, 33), 51(69),55, 159 Perry, M. B., 136, 171, 173(82, 95), 174(82), 193(85) Peter, G., 195, 196 Peterson, K., 88, 164, 165(43,45), 167(43,45)

AUTHOR INDEX Pfeffer, P. E., 36, 39(60),42,45(59), 46(59, 60),47(59), 55(60),62(59) Pfeil, R., 217(75), 274 Phillippi, M. L., 108, 128(16) Phuoc Du, A. M. N., 50(148),51,55(148) Pickles, V. A., 39 Pierce, J., 52, 53(157) Pierce-Crbtel, A., 210,324 Pillonel, C., 139 Pincheira, G., 71 Pion, B. G., 115 Pitcher, R. G., 42, 65(ll6) Plescia, 0. J., 198 Polley, M. J., 188, 204(155) Pollitt, W., 333,334(89), 335(89),336(89), 338(89), 340(89) Pont-Lezica, R., 108, 127, 133(17),134, 137(17), 144(136) Porter, K. R., 124 Potter, J. L., 108, 128(19) Powell, D. A., 170, 171(72),172(79), 173(72) Pozsgay, V., 50(146), 51,56,57(172) Pratviel-Sosa, F., 50(148), 51, 55(148) Prescott, B., 186, 189(147),190(147, 166) Preston, R. D., 108, 110, 112(15), 119, 131 Previato, J. O., 69,73(9) Priebe, W., 57, 58(176) Prieels, J. P., 330 Pugmire, R. J., 28

Q Quader, H., 123, 124(84),144 Quail, P. H., 128, 129 Quatrano, R. S., 108 Que, L., 33,48(27), 60(27),’61(27) Quie, P. G., 204 Quinn, P. H., 188,204(154)

R Raff, M. C., 186, 189(144) Ralph, B. J., 97 Randolph, M.,202 Rao, E. V., 171, 174(98) Rao, V. S., 39, 54(82), 181,317,372(83) Raschke, W. C., 83,86,87, 177

387

Rathbone, E. B., 41,45(98), 51(98) Rattle, H. W. E., 212 Rauvala, H., 210,334,341(92a) Rawson, D. I., 40, 53(89) Ray, M. M., 129 Ray, P. M., 128, 129, 147(115) Raymond, Y., 110, 130(30),146, 147(30) Rearick, J. I., 330 Rebers, P. A., 171, 172(86),174, 192(86) Recondo, E., 133, 143(132) Reed, N. D., 190 Rees, D. A., 178, 180, 181(125) Reeves, R. E., 171, 172(81), 174 Regensburg, B. A., 70 Reid, I. D., 70 Reis, D., 123 Reuben, J., 212 Reyes, F., 74 Reynolds, R. C., 58 Rhoades, J. A., 39, 54(81) Ricart, G., 372 Richardson, C. L., 112, 117(35),118(35) Richter, A., 50, 55(145),56(145) Rick, P. D., 95 Riesco, B. F., 62, 64(205) Rietschel-Berst, M., 95 Riffer, R., 51 Ritchie, R. G. S., 37,41, 47(92), 51(69) Robbins, J . B., 156, 157(7), 158(7), 170(7), 171(7), 183, 185(7, 136), 190(7), 191(7), 192(7),194, 195(7), 197(7), 198,200,201(7,212),202, 203(7, 186, 191,221), 204(7), 206 Robbins, J. D., 194,203(186) Roberts, J. D., 27,34, 35(37),41(37), 45(37), 46(37), 52(37),55(37), 159 Roberts, W.K., 174 Robinson, D. G., 105, 110(4), 119, 123, 124, 126(4), 131, 144 Robyt, J. F., 135 Roderer, G., 124 Roelcke, D., 215 Roelofsen, P. A., 123 Roland, J. C., 123 Romanovicz, D. K., 116 Romero, P. A., 108, 127, 133(17), 134, 137(17), 144(136) Rosell, K.-G., 160, 162(36),163, 164(40), 168, 169(70), 170(63,68,70), 171, 173(84,92), 177(63,68,69), 179(36),

AUTHOR INDEX

388

181, 192(92), 193(84), 196(63), 206(63), 208(63,68,131) Rosenbery, E., 164 Rosenberger, R. F., 68 Rosenfeld, L., 79,87 Rosenthal, S. N., 28 Rosset, J., 92 Rothschild, U., 137 Rottenberg, H., 149 Rougier, M., 129 Roussel, P., 372(114), 373 Rowin, G. L., 102 Ruiz-Herrera, J., 131, 145 Russell, H., 167, 168(59)

S Sadler, J. E., 170 Sadoff, J. C., 199 Safianski, M. J., 77 Saito, G . , 74 Sait6, H., 73, 75, 76 Sakai, S . , 74 Salam, M. A., 62,63(191) Samuelsson, 8.E., 214, 215 San-Blas, C., 70, 86 Sanderman, H., 128, 133, 141(112) Sandford, P. A., 88, 102 Saniere, M., 42, 65(115) Santamaria, F., 74 Sarko, A., 113, 114 Sasajima, K., 372 Sasaki, H., 96 Sasaki, T., 75 Sato, K., 42, 65(117) Sato, M., 96 Satoh, S.,138, 139, 143, 144(149) Satoh, T., 42, 65(116) Scaletti, J. V., 74 Schachter, H., 216 Schauer, R., 217(73,75), 274 Schecter, B., 180 Schecter, I., 180 Scheer, E., 103 Scherp, H. W., 158 Schiffer, M. S.,200,201(212) Schiffman, G., 167, 168(62), 169(62), 170(62), 171, 196(62), 197(62), 206(62),208(62)

Schilling, G., 62,64(199) Schlesselman, J. J., 195 Schmid, K., 217(66,67,68, 69, 78, 79), 237,239(66), 240(66,67,68,69), 242(67), 248(66,67,68), 283, 285(78), 289(78), 312(66), 316(66), 317(67), 318(84), 320167-69), 321(67), 328(84), 329(84), 362(66), 365(67) Schnarr, G. W., 58,59(184), 60(184) Schneerson, R., 164, 165(44,46), 166(50, 52), 167(44,46), 186, 195, 198,200, 201(212), 202, 203(191, 221, 222), 206 Schneider, W. G., 214 Schnepf, E., 124 Schrieber, R. D., 187,205(151) Schuler-Hovanessian, A., 89 Schultz, D. R., 188 Schut, B. L., 217(55, 60),220, 224(55), 227,306(55),312 Schwarcz, J. A., 36, 37(64), 41(64) Scott, T. K., 121 Scott, W. E., 181 Scurfield, G., 72 Seid, R. C., Jr., 199 Sela, M., 180 Sell, H. W., 156 Selvendran, R. R., 121 Senior, R. G., 215,216(40) Sentandreu, R., 80 Seo, S., 42 Septe, B., 42, 65(118) Sepulchre, A.-M., 41,42, 53(102), 65(118) Serianni, A. S., 35, 36(43), 46(43), 52, 53(157), 63(43), 211 Seto, S., 41, 52(97) Shafizadeh, F., 105, 112 Sharon, N., 210, 220(4), 237(4), 240(4), 269(4), 283(4), 306(4), 365 Shashkov, A. S., 28,41(7), 51, 53(100), 54,56,62 Sher, I., 189 Shibaoka, H., 144 Shibata, K., 42 Shibata, S., 50, 54,75, 76, 224 Shida, M., 70, 101 Shienok, A. I., 41, 53(100) Shimo-Koriyama, H., 144 Shin, J. E. N., 55

AUTKOR INDEX Shininger, T. L., 129 Shmyrina, A. Ya, 51 Shore, G., 130 Shulman, R. G., 212 Siddiqui, I. R., 82 Siegel, M., 190, 191(175) Siehr, D. J., 70, 71, 73 Sietsma, J. H., 70, 74 Sikorski, H., 158 Silver, R. P., 206 Simson, B. W., 73 Sinay, P., 62, 64(207) Sing, V. O., 131 Singh, P. P., 72 Sinnema, A., 59 Sinnwell, V., 36, 50, 55(145), 56(145) Skelbaek Petersen, B., 50 Skerrett, R. J., 180,181(125) Slesson, K.N., 52, %(IS), 59(154) Slodki, M. E., 77,85 Slomp, G., 63, SS(212) Smith, D. H.,195, 196 Smith, F., 69,74 59(154), 72, Smith, I. C. P., 52, %(la), 159, 160(31),161, 162(33,34), 163(35),164(35),175(33,34), 179(33),181, 206(31,34) Smith, M. M., 126, 130(102), 141 Smithers, S. R., 208 Sone, Y., 75, 97 Southworth, D., 128 Sowden, L. C., 118; 136 Spencer, J. F. T., 68, 77, 78, 83, 89(6), 91(6, 126), 92(103, 107),93(103, 137), 99, 100, 102(126),217 Spik, G., 170,210,217(55, 60,68,71, 72, 77), 220,224(55),227,237,240(68), 248(68),259,269,275(72), 277(72), 279(72), 283,285(72),,291(72), 295(72), 298(72), 301(72),304(72), 306(55),308,320(68),324(81), 363 Srere, P. A., 146 Staehelin, L. A., 121 Stashak, P. W., 189, 190(166) Staub, A. M.,157 Stefanovich, V., 76 Stenzel, W., 50,55(145),56(145) Stephen, A. M., 41,45(98), 51(98) Stevens, P. T., 10s

389

Stewart, T. S., 78, 79(89), 83(89), 86 Stipanovic, A. J., 114 Stirm, S., 179 Stone, B. A., 126, 130 Stothers, J. B., 28,34(12) Stowell, C. P., 198 Strecker, G., 170,210,217(52,55,56, 58, 59, 60,61,62,64,65,67,68, 72, 76), 220,224(55),225(52), 227(52, 56), 234, 235(52), 237,240(67,68), 242(52, 64,67), 244(52,64,65), !246(52, 64),%8(67,68), 254(62), 257(64, 65), 261(52,64),264(52,64, 65),266(64,65),267(64,65),269(52), 275(72),277(72,76),279(72, 76), 285(72),291(72), 295(52, 72), 298(52, 72), 301(72),304(52,72),306(55), 308,316(52, 56), 317(67),320(67, 68),321(67),324(81),330(56), 333(52),334(52,89),335(89),336(52, 89), 338(52,89),340(52,89),344(58, 59), 347(58,59), 350(52,61),352(52, 61), 355(61),365(67) Strobel, G. A., 39,89 Stuart, R. S., 35, 36(50), 46(49) Subero, C., 51 Sugiura, Y.,50 Sugiyama, H.,41, 52(97), 87 Sukumar, S., 215 Sundararajan,P. R., 179 Sutton, A., 194, 195, 198,203(191),206 Suzuki, K., 318(84),320,328(84),329 Suzuki, M., 63, 65(210),372 Svenson, S. B., 158,198,199(15) Svensson, S., 171, 172(80), 174(80),210 Sviridov, A. F., 41, 51,53(100),62 Swanson, J., 185 Sweeley, C. C., 355 Swissa, M., 107, l28(9),136(9), 143 Szarek, W. A., 41,45,46(99), 53(99,128), 58, 59(184), 55(128), 57(91, W), 60(184),62,64(200)

T Tachibana, Y., 355 Tahiura, K., 52 Tai, J. Y.,167, 168(61),185 Tai, T., 347(95),348

390

AUTHOR IIVDEX

Takahama, M., 143 Takahara, G., 69 Takai, M., 115, 136 Takasaki, S.,334,341(92) Takaya, S., 71 Takeda, T., 50,75,76 Takita, T., 47 Taksuka, X., 75 Tamari, K., 74,96, 138, 139(149), 144(149) Tanaka, H., 74 Tanaka, M., 75,217(54), 220 Tanaka, O., 45,46(130),47(130, 132), 55(1301, 56(130) Tanaka, Y., 93 TPnzer, C., 46(134), 47,48(134), 49(134), 56/134) Taravel, F. R., 35,43 Tduber, J. w., 188, 201(155) Taussig, M. J., 187 Taylor, I. F., 73 Terry, R. J., 208 Terui, Y., 42, 63,65(208) Theander, 0..63,65(211) Thiem, J., 36 Thieme, T. T., 86 Thggersen, H., 33,39(34),40(34), 60, 61(189) Thomton, E. R., 37,43(75),62,64(206) Thurow, H., 185 Till&, W. S., 156, 191 Timell, T. E., 73, 115, 116(50) Ting, I. P., 109 Tokuzen, R., 72 Tomasz, A., 188,204(153, 156) Tomita. Y., 42 Tordera, V., 217(77),283 Ton, K., 42, 43,63, 65(208) Toni, M., 71 Townsend, L. B., 28 Townsend, R. R., 372 Tramont, D. L., 194,208(182) Travassos, L. R., 69, 73(9), 90,91, 93, 94, 9!5(149) Triantaphylides, C., 33,48(32) Tritschler, C., 103 Tmka, T., 51 Troy, F. A., 185 Tsai, C. M., 146 Tsai, J.-H., 56 Tsuda, Y.,42

Tsui, F.-P., 164, 165(44,46), 166(50,52), 167(44,46), 202,203(222) Tsuji, N., 42, 63, 65(208) Tsumuraya, Y., 71 Tsunashima, T., 179 Tsuta, Y., 115 Tung, K. K., 70 Tuzimura, K., 41, 52(97) Tyler, J. M., 171, 174(89),201

U Uchida, T., 70 Ueno, Y.,74 Ukai, S., 97 Ulane, R., 145 Umeda, T., 97 Umezawa, H., 47 Unger, F. M., 159 Unger, P., 88, 164, 165(43,45), 167(43, 45) Ungram, D. L., 196 Unruh, J.. 36, 39(60), 46(60), 55(60) Usov, A. I., 51 Usui, T., 41, 52(97) Utille, J.-P., 45,49(133), 50(133),561133) Uzawa, J., 62, 64(198) V Valentine, K. M., 36,42,45(59), 46(59), 47(59), 62(59) van Alfen, N., 89 Van Delft, R. W., 198 van den Eijnden, D. H., 217(78),283, 285(78), 289(78) van Der Tom, J. M., 59 Van der Woude, W. J., 129, 131 van Dongen, J. P. C.M., 41,52(94), 53(95) van Halbeek, H., 211,212(12, 13), 217(58, 59, 61, 64,65,66, 73, 75, 76, 78, 79), 225, 227, 235,237,239(66), 240(66),242(64), m ( 6 4 , 6 5 ) , 246(64), 248(66),257(64, 65), 261(64),264(64,65), 266(64, 65). 267(64, 65), 274, 275, 277(76), 279(76),281(13), 283,285(13, 78), 289(78), 304(13), 306(13),308, 312(13,66), 316(13,66), 318(84), 320,321(13), 324(81),328(84), 329(84),330(13),333,334(89),

AUTHOR INDEX

335(89),336(89), 338(89),340(89), 343(12), 344(58, 59), 347(58, 59), 350(61), 352(61),355(61), 359, 362(66),363,365,372(12, 13,99, 114, 115),373 Vann, W., 200,201(212) Vann, W. F., 206 Varki, A., 207 Vaughn, K. A., 205 Veldink, G. A., 217(58,61,64, 76), 225, 227,235,242(64), 244(64),246(64), 257(64), 261(64), 264(64), 266(64), 267(64),275,277(76),279(76), 344(58),347(58),350(61),352(61), 355(61),372 Vella, P. P., 192 Vian, B., 123 Vignon, M. R., 29,36,42,43, 44(19), 49(108, 109), 52(108) 75 Villemez, C. L., 115, 127 Vincendon, M., 36, 76 Vitovskoya, G. A., 100 Vliegenthart, J. F. G., 41, 52(94), 53(95, 96),62, 64(207), 211,212(12, 13), 217(55,56,58,59,60,61,62,64,65, 66, 67,68,69, 70, 71, 72, 73, 73a, 74,

75, 76, 77, 78, 79), 220,224(55),225, 227(56), 234,235,237,239(66), 240(66, 67, 68, 69), 242(64,67), 244(64,65), 246(64), 248(66,67,68), 254(62), 257(64,65),259(70), 261(64),264(64,65),266(64,65), 267(64, 65), 269,274,275(72), 277(72, 76), 279(72,76),281(13), 283,285(13, 72, 78), 289(78), 291(72),295(72),298(72), 301(72), 304(13, 72), 306(13, 55), 308,312(13, 66, 74), 316(13, 56, 66),317(67), 318(84), 320(67-69), 321(13, 67), 324(81),328(84), 329(84), 330(13, 56), 333,334(89),335(89),336(89), 338(89),340(89),343(12),344(58, 59), 347(58, 59), 350(61), 352(61), 355(61), 359,362(66),363,365(67), 372(12, 13, 99, 114, 115), 373 Voelter, W., 28(16),29,34(16), 36(16), 39(16), 41,42(16),43,45(98),46(131, 134), 47(131),48(134), 49(134), 51(98),55(137),56(134),59, 60(187), 65(118)

391

Vogel, A., 112 Volkanakis, J. E., 188 Voll, R. J., 36, 52(56) von Bekkum, H., 59 Vonlanthen, M., 77, 103(82) von Sonntag, C., 33,48(32),58, 59(183) Vottero, P. J. A., 42,45,49(108, 109, 133),50(133),52(108),56(133) Vyas, D. M., 41,45,46(99), 53(99,128), 55(128),57(91,99),58, 59(184), 60(184), 62, 64(200)

W Wagenbreth, I., 114 Wagner, H., 41, 53(101) Wagstrom, B., 101 Waksman, N., 72 Wdaszek, Z., 33 Walker, T. E., 28,35(9), 37(40),45(40), 46(40),53(42),55(40) Walkinshaw, M. D., 178 Walter, A. W., 190, 191(175) Wang, M. C., 129 Ward, C.,108, 128(18) Wardrop, A. B., 108, 112 Warner, T. G., 372 Watanabe, S., 115 Waterkeyn, L., 109, 141 Watson, M. J., 171, 174(89, 98) Watson, P. R., 88, 102 Watson, R. G., 158 Webley, D. M., 69 Wehrli, F. W., 28,34(15),36(15),37(15), 38(15) Weibel, R. E., 192 Weighert, F. J., 27 Weijman, A. C. M., 72 Weinhouse, H., 107, 128(9), 136(9), 143(9),145, 146(168),152(168) Weisleder, D., 60,61(191) Weisman, R. A., 108, 128(19) Wells, A. G., 52,53(158) Weltner, W., Jr., 43, 62(127),64(205) Wenkert, E.,39,54(Sl) Wenlu, L., 195, 203(191) Werner, K. E., 186 Wessels?J. G. H., 70 Westland, R. D., 47 Westphal, O., 157, 158, 177(17), 178(17), 204(17)

392

AUTHOR INDEX

Wetterlow, L. H., 195 Whaley, T.W., 35,37(40), 40,45(40), 46(40), 55(40) Wheat, R. W., 103 Whelan, W. J., 72 Whishant, J. K.,202, 203(221) Whistler, R. L.,54, 72 White, A. R., 117, 119(65) Wiebe, L.,56,57(169) Wiese, M.J., 86 Wightman, R. H.,40,53(89) Wilbur, D.J., 33,45(28) Wilkinson, H.W., 168 Williams, C., 33,45(28,29) Williamson, A. R., 164 Willison, J. H. M., 112, 117(35), 118(35), 121,122 Wilson, D.E.,97 Winkelhake, J. L.,195 Winkelstein, J. A., 187, 188(152), 204(153,154,156) Winnik, F. M.,217(50), 218,348(50), 368(50), 372(50) Winter, W. T., 179 Wintemitz, F.,50(148), 51, 55(148) Wirthlin, T.,28,34(15), 36(15), 37(15), W15) Wober, G.,69 Wolf, C.,179 Wolfe, L.S.,215,216(40,41) Wolfe, R. S., 107 Wong, K. F.,37,43(76) Woo, P.W. K.,47 wood, J. o.,39 Woodcock, C., 113 Woodhour, A. F.,192 Wray, V.,36 Wright, B. E.,108, 128(18) w u , M.-C., 188 Wdthrich, K.,212 Wursch, P.,92 Wyide, R., 50(148), 51,55(148) Wyle, F.A., 194,208(182) Wysocki, J . R., 158, lW(23)

Y Yabuuchi, H., 217(64a), 235,?.42(64a), 244(64a), %46(64a),261(64a), 264(64a), 334(64a), 355

Yadomae, T., 54,95 Yadomae, T.Y., 171, 173(93),192(93) Yamada, A., 215 Yamada, M.,69 Yamada, T.,372 Yamaguchi, H.,69, 101 Yamakawa, T.,372 Ymamura, Y., 70, 73(31), 93 Yamaoka, N.,41,52(97) Yamashina, I., 217(54), 220 Yamashita, K.,217(64a), 235,242(64a), 244(64a), 246(64a), 261(64a), 264(64a),328,334(64a), 347(95), 348,

355

Yamauchi, R., 74 Yarotskii, S. V., 51 Yen, P. H., 83,87(104) Yokota, I., 76 Yonezawa, D.,101 Yoshima, H., 334,341(92) Yoshimoto, K., 42 Yoshimura, J., 42,65(117) Yoshino, H., 87 Yoshioka, Y., 73 Younathan, E. S., 36,52(56) Young, G.,178 Young, N. M.,170,208(76) Yount, W.J., 190 Yu, R. J., 74,82 Yuki, H., 52

2

Zaar, K., 117, 118, 119 Zabriskie, J. B., 188,204(155) Zamenoff, S., 164 Zamojski, A.,41,46(99), 53(99),57(99), 58(176) Zancan, G. T., 69 Zanlungo, A. B., 60,61(190) Zevenhuizen, L.P.T. M.,73, 107 Zidivar, N. M.,189 Zilberstein, D.,149 Zimsky, E. P., 156 Zola, H., 182 Zollinger, W. D.,195 Zon, G., 186 Zonneveld, B. J. M., 69,70

SUBJECT INDEX

Akctoria sulcata, polysaccharide, 76 Algae Acanthamoeba, cellulose biosynthesis, cellulose biosynthesis, cytological 108,128 studies, 119-122 Acetobacter xylinum cellulose-containingcell-wall synthecellulose assembly model, 113 sis, 108 cellulose biosynthesis, 106, 107, 116microfibril orientation, 123-125 119,124,128 Allofuranose, 1,2:5,6-di4-isopropylihigh-molecular-weight precursors, dene-a-D-, carbon-13 nuclear mag135-137,141 netic resonance spect~oscopy,53 lipid intermediates involvement, Allomyces macrogynus, glycogen, 69 132, 133 Amylose, fungal, 68 Acroscyphus sphaerophoroides, polysac- Anhydropyranose derivatives, carbon-13 charide, 76 nuclear magnetic resonance specAgglutinin, see Soybean agglutinin troscopy, 51 Albumin, hen-egg, glycoprotein (comAntibodies, humoral, to polysaccharide 'H-n.m.r. spectroscopy, pound a), vaccines, 189-191 344,346,348-350,357,359-361 Apiose, D-,synthesis, 9 Alcohols, oxidation of polyhydric, 9 -, 1,2,-O-isopropylidenear-, carbon-13 Alditols nuclear magnetic resonance specand acetates, carbon-13 nuclear magtroscopy, 64 netic resonance spectroscopy, 59, Arabinan 60 citrus, 7 aminoanhydrodeoxy-, carbon-13 nupeanut, 7 clear magnetic resonance spectros- Arabinofuranoside, methyl 4-acetamido4deoxy-D-, synthesis, 9 COPY, 61 aminodeoxy-, carbon-13 nuclear mag-, methyl 4-acetamido-4deoxy-~-,synnetic resonance spectroscopy, 61 thesis, 9 anhydro, carbon-13 nuclear magnetic Arabinose, 5-acetamido&leoxy-~-. synresonance spectroscopy, 60,61 thesis, 9 Aldonic acids, carbon-13 nuclear magArabinose-5-'C-, L-, synthesis, 9 netic resonance spectroscopy, 63 Annillaria mellea, polysaccharide, Aldonolactones, carbon-13 nuclear mag103 Arthrobacter, polysaccharide, 80 netic resonance spectroscopy, 63 Ascorbic acid Aldoses L-, carbon-13 nuclear magnetic resocarbon-13 nuclear magnetic resonance nance spectroscopy, 64 chemical shifts, 45 synthesis, history, 2, 7 carbon-13 nuclear magnetic resonance Asialo a,-acid glycoprotein spectroscopy, 45,46 glycoprotein (compound 8), 'H-n.m.r. Aldosides S ~ ~ ~ ~ O S219,237-240 COPY, methyl, carbon-13 nuclear magnetic glycoprotein (compound 9), 'H-n.m.r. resonance chemical shifts, 46,47 S ~ ~ C ~ ~ O S219,239-242 COPY, methyl, carbon-13 nuclear magnetic glycoprotein (compound 13), 'H-n.m.1. resonance spectroscopy, 46,47 S ~ ~ C ~ ~ O S C O219,239,248-251 PY, Alectoria sarmentosa, polysaccharide, 76 A

393

SUBJECT INDEX

304

glycoprotein (compound 48), 'H-n.m.r. spectroscopy, 306,319-321 glycoproteins (compounds 49, 50, and 51). 'H-n.m.r. spectroscopy, 306, 314,315,321-324 Asialo ceruloplasmin, glycoprotein (compound 48). 'H-n.m.r. spectroscopy, 306,319-321 Aspartylglucosaminuria glycoprotein (compound 11, 'H-n.m.r. spectroscopy, 219-224 glycoprotein (compound 3). 'H-n.m.r. spectroscopy, 219, 225-226 glycoprotein (compound 55),'H-n.m.r. spectroscopy, 333-337 glyeoprotein (compound 56), 'H-n.m.r. spectroscopy, 333,336-338 glycoprotein (compound 57), 'H-n.m.r. spectroscopy, 333,335,337,338 glycoprotein (compound 58), 'H-n.m.r. spectroscopy, 333,337-339 glycoprotein (compound 59). 'H-n.m.r. spectroscopy, 333,337,340-342 glycoprotein (compound 60),'H-n.m.r. spectroscopy, 333,337,340-343 Aspergillus Jacus, polysaccharide, 95 Aspergillus fumigatus, polysaccharides,

95 Aspergillus nidulans, polysaccharide, 69, 70,95,101 Aspergillus niyer, galactomannan, 94 heteropolysaccharide, 69, 70, 101 Aspergillus terreus, polysaccharide, 95 Aureobusidium pullulans heteropolysaccharide, 96 polysaccharide, 72, 75 Auricularia auricula-judae, polysaccharide, 75, 97

B Bacterial infection, immune response of capsular polysaccharides, 186- 191 Bacterial virulence capsular polysaccharide activity, 189 role of capsular polysaccharides, 202206 Benzonitrile, 2,6-dichloro-, inhibitor of cellulose biosynthesis, 144

Bibliography, John Kenyon Netherton Jones publications, 11-26 Biosynthesis cellulose, 105-153 structural considerations, 110-116 Blastocladiellu emersonii, glycogen, 69 Btastomyces dermatiditis galactomannan, 93 polysaccharide, 70

C Calcium ionophores, inhibitors of cellolose biosynthesis, 144 Calcofluor White, 117, 131 Callose, effect on cellulose biosynthesis, 128,129 Candida, mannans, 82 Candida albicans glycogens, 69 phosphonomannan, 86 polysaccharide, 74 Candida bogoriensis, polysaccharides,

98 Candida lipolytica, galactomannan, 92 Candida parapsilosis, polysaccharide, 74 Candida stellatoidea, phosphonomannan, 86 Candida utilis chitin, 103 polysaccharide, 77 Carbohydrate chains proton nuclear magnetic resonance spectroscopy of glycoprotein-related, 210-374 structural reporter groups, 212 Carbohydrates, unsaturated, carbon-13 nuclear magnetic resonance spectroscopy, 58 Carbon-13 nuclear magnetic resonance chemical-shifts acyclic monosaccharides, 59 alditols and acetates, 59, 60 aldonic acids, 63 aldonolactones, 63 aldoses, 45,46 aminoanhydrodeoxyalditols,61 aminodeoxyalditols, 61 arninodeoxy pyranoses, 54

SUBJECT INDEX anhydroalditols, 60,61 anhydropyranose derivatives, 51 benzylidene and isopropylidene monosaccharide derivatives, 53 biologically significant monosaccharides, 64,65 deoxyhalo pyranoses, 54 deoxy sugars, 55 glycosides of aromatic aglycons, 48, 49 ketoses and methyl glycosides, 48 methyl aldosides, 46,47 methyl anhydro-D-glycosides, 57 methyl deoxypyranosides, 56,57 monosaccharides, 28,31 complexation, 43 peracetylated pyranoses and furanoses, 49 0-substituted monosaccharide derivatives, 52,53 tetraOacetyl(benzoyl)-D-gIycopyranosyl derivatives, 50

thio-D-pyranoses, 54 unsaturated carbohydrates, 58 uronic acids, 62 uronolactones, 62 Carbon-13 nuclear magnetic resonance signals monosaccharide, comparison with model compounds, 34,35 correlation with proton spectra, 36, 37 isotopic substitution, 35,36 relaxation rates, 37, 38 Carbon-13 nuclear magnetic resonance spectroscopy capsular polysaccharides, 159-164 monosaccharides, 27 -65 conformational analysis, 43 effect of paramagnetic reagents, 38 for identification, 39,40 protonation shifts, 39 relaxation rates, 43 for structure determination, 40-42 quantitative data, 32,33 resolution enhancement, 3 3 , M signal-to-noise ratio, 30 solvents, 29, 30 Casoron, see Benzonitrile, 2,6-dichloroCellobiouronic acid, determinant group

395

in polysaccharide immunology, 176, 180 Cellobiouronic acid conjugate, immunogenicity, 197 Cells, intact, cellulose synthetase activity, and transmembrane electrical potential, 147-150 Cellulose biosynthesis, 105-153 chemical inhibitors, 143, 144 cytological studies, 116-123 genetic mutations, 143, 144 hypothetical model, 151 lipid intermediates involvement, 132-135 organisms, 107-110 site, 116-123 structural considerations, 110-116 degree of polymerization of native, 114- 116 fungal, 72 non-crystalline, 117 Cellulose, 0-(carboxymethy1)-, effect on cellulose biosynthesis, 118 a-Cellulose, 138 Cellulose I, structure, 113 Cellulose 11, structure, 113 Cellulose 111, structure, 113 Cellulose IV, 113 Cellulose precursors biosynthesis, 125-132 high-molecular-weight, 135- 142 radioactive compounds, 152 Cellulose-synthetase complex activity, effect of transmembrane electrical potential, 147 lability, factors affecting, 145 Ceratocystis brunnea, polysaccharide, 91 Ceratocystis jimbriata, polysaccharide, 91 Ceratocystis olioacea, polysaccharides, 72 Ceratocystis paradora polysaccharide, 91 Ceratocystis stenoceras amylose, 69 galactomannan, 93 polysaccharide, 69 rhamnomannan, 90 Ceratocystis ulmi, rhamnomannan, 89

396

SUBJECT INDEX

Ceruloplasmin, human-plasmin, glycoprotein (compound 53),'H-n.m.r. spectroscopy, 306,327-330 Cetraria richurdsonii, polysaccharide, 75,76 Chalcose, D-, synthesis, 10 Cherry gum, structure, 7 , 8 Chitin, fungal, 72, 103 Chitosan, fungal, 103 Choanephora curcurbitarum, chitin and chitosan, 103 Citeromyces matritensis, mannan, 82 Cladinose, L-,synthesis, 9 Clodosporium herbarum galactomannan, 94 polysaccharide, 70 Clodosporium werneckii, galactomannan, 93 Claoiceps fusifoonnis, polysaccharide, 75 Coccidioides immitis, polysaccharide, 103 Conformation, capsular polysaccharides, and serological specificity, 178-182 Coprinus macrorhizus, polysaccharide, 71,73 Cotton-fiber cellulose biosynthesis, 109, 147, 148 precursors, 126, 139, 141 Coumarin, inhibitor of cellulose biosynthesis, 143, 144 Cryptate 211, inhibitor of cellulose biosynthesis, 144 Cryptate 212, inhibitor of cellulose biosynthesis, 144 Cryptococcosis, 97 Cryptococcus, polysaccharide, 69 Cryptococcus bacillosporus, polysaccharide, 97, 98 Cryptococcus neofomans, polysaccharide, 97, 98 Cytturia horioti, polysaccharide, 72, 103

D Damson gum, structure, history, 3, 7, 8 DCB, see Benzonitrile, 2,6-dichloro-, Degree of polymerization, cellulose biosynthesis, 114, 115 Deuterated dimethyl sulfoxide, solvent for carbon-13 nuclear magnetic resonance spectroscopy, 29

Deuteriochloroform, solvent for carbon13 nuclear magnetic resonance spectroscopy, 29 Deuterium oxide, solvent for carbon-13 nuclear magnetic resonance spectroscopy, 29 Dextrans, immunogenicity and molecular size, 182 Dichlobenil, see Benzonitrile, 2,6dichloroDictyostelium discoidurn, cellulosic cellwall, 108 Dutch-elm disease, 89

E Elsinan, fungal, 71 Elsinoe leucospila, polysaccharide, 71 Erythrofuranoside, methyl 4-acetamido.l-deoxy-~-,synthesis, 9 Escherichia coli K1, capsular polysaccharide, immune response, 195, 206 Escherichia coli K92,capsular polysaccharide, immune response, 194 Eukaryotes, cellulose biosynthesis, 108 Europhium, polysaccharide, 72 Evernian, lichen, 76 Evernia prunastri galactomannan, 95 polysaccharide, 76

F Flamnulina oelutipes, fucomannogalactan, 101 Fomes annosus, glucuronoglucan, 101 Fourier transform, 28,34 Fuco(manno)galactans, 100, 101 Fucose groups, glycoproteins containing, 'H-n.m.r. spectroscopy, 305-332 Fucosidosis glycoprotein (compound 2), 'H-n.m.r. S ~ ~ C ~ ~ O S219-224 COPY, glycoprotein (compound 4), 'H-n.m.r. spectroscopy, 219,226-228 glycoprotein (compounds 46 and 47, 'H-n.m.r. spectroscopy, 306,313318 glycoprotein (compound 54),'H-n.m.r. S ~ ~ C ~ ~ O S306,330-332 COPY,

SUBJECT INDEX Fucoxylomannan, 98 Fucus, cellulosic cell-wall, 108

Fungi, polysaccharides, 67-103 Fusicoccum amygduli, amylose, 68

G Galactans, 87,88 Galactomannans, 92-95 Galactopyranan, 2-amino-2-deoxy-D-,88, 89 Galactopyranose, 1,2:3,4-di-O-isopropylidene-a-D-, carbon-13 nuclear magnetic resonance spectroscopy, 53 Galactose, 3-0-/%D-galactopyranosyI-D-, synthesis, 9 -, 4-O-P-D-galactopyranosyl-D-,synthesis, 9 Gangliosidosis GMl, glycoprotein (compound 7), 'Hn.m.r. spectroscopy, 219,2.32,233, 235-237 glycoprotein (compound lo), 'Hn.m.r. specboscopy, 219,232,233, 242-244 glycoprotein (compound II), 'Hn.m.r. spectroscopy, 219,232,233, 244-246 glycoprotein (compound 12), 'Hn.m.r. spectroscopy, 219, 232,233, 246-248 glycoprotein (compound 17), 'Hn.m.r. spectroscopy, 219,261-264 glycoprotein (compound 18), 'Hn.m.r. spectroscopy, 219,262-266 GMI, variant 0, see Sanhoffs disease Gaucher's disease glycoprotein (compound 3), 'H-n.m.r. spectroscopy, 219,225,226 glycoprotein (compound 61), 'H-n.m.r. spectroscopy, 344-347 glycoprotein (compound 62), 'H-n.m.r. S ~ ~ C ~ ~ O S C O344,346-348 PY, Genetic mutations, in cellulose biosynthesis, 143, 144 (1+3)-p-~-Glucan biosynthesis, 147 in higher plants, 139-142 in plant cell-walls, 128 (1+4)-fi-D-Ghcan biosynthesis, 117

397

in uitro, 106, 152 cellulose biosynthesis, 110 polymerization mechanism, 125-132 Glucans branched-chain p-D-, 73 -75. lichen, 75, 76 a-Dlinked, 68-72 p-dinked, linear, 72, 73 side-chain substitution, 74, 75 Glucan synthetase I, 129 Glucan synthetase 11,129 Glucan synthetases, assay of solubilized, 146 Glucofuranose, 1,2:5,6-di-O-isopropylidene-a-D-, and derivatives, carbon13 nuclear magnetic resonance spectroscopy, 53 Glucogalactans, 100 Glucomannans, 91 biosynthesis, 127 yeast, 99 Glucopyranan a+-, fungal, 70 B-D-, fungal, 73 Glucopyranoside, methyl tetra-o-acetylWD-, carbon-13 nuclear magnetic resonance spectrum, 38 Glucose, 3-acetamido-3-deoxy-D-,synthesis, 9 Glucosinolate, allyl, carbon-13 nuclear magnetic resonance spectroscopy, 65 Glucuronic acid, 2-acetamido-2-deoxy-~-, polymer, 88 Glucuronoglucan, 101 Glyceraldehyde, D-, 3-phosphate hydrate, carbon-I3 nuclear magnetic resonance spectroscopy, 53 Glycogen, fungal, 69 Glycolaldehyde, D-, phosphate hydrate, carbon-I3 nuclear magnetic resonance spectroscopy, 53 Glycoproteins N-acetyllactosamine-typecarbohydrate chains, high-resolution proton magnetic resonance spectroscopy, 218-343 compound 1, from aspartylglucosaminuria, 'H-n.m.r. spectroscopy, 219224 compound 2, from fucosidosis, 'Hn.m.r. spectroscopy, 219-224

SUBJECT INDEX

398

compound 3, from aspartylglucosaminuria and Gaucher's disease, 'Hn.m.r. spectroscopy, 219,225-226 compound 4, from fucosidosis, 'Hn.m.r. spectroscopy, 219, 226228 compound 5, from mannosidosis, 'Hn.m.r. spectroscopy, 219,227230 compound 6, from Sandhoff's disease, 'H-n.m.r. spectroscopy, 219 231-

234 compound 7, from CM,-gangliosidosis, and Morquio syndrome type B, IH-n.m.r. spectroscopy, 219,232, 233,235-237 compound 8, from asialo a,-acid glycoprotein, 'H-n.m.r. spectroscopy, 219,237-240 compound 9, from asialo a,-acid glycoprotein, 'H-n.m.r. spectroscopy, 219,239-242 compound 10, from CM,-gangiiosidosis, 'H-n.m.r. spectroscopy, 219, 232,233,242-244 compound 11, from GM,-gangliosidosis and Morquio syndrome type B, 'H-n.m.r. spectroscopy, 219, 232, 233, 244-246 compound 12, from CM,-gangliosidosis, 'H-n.m.r. spectroscopy, 219, 232,233,246-248 compound 13, from asialo a,-acid glycoprotein, 'H-n.m.r. spectroscopy, 219,239,248-251 compound 14, from Sandhoffs disease, 'H-n.m.r. spectroscopy, 219, 254-257 compound 15, from Morquio syndrome type B, 'H-n.m.r. spectroscopy, 219,256-259 compound 16, from chicken ovotransferrin, 'H-n.m.r. spectroscopy, 219, 256,259-261 compound 17, and 18, from GM,-gangliosidosis, 'H-n.m.r. spectroscopy, 219,261-264 compound 18, from CM,-gangliosidosis and Morquio syndrome type B, ,H-n.m.r. spectroscopy, 219, 262-266

compound 19, from Morquio syndrome type B, 'H-n.m.r. spectroscopy, 219,263,266-268 compound 20, from Morquio syndrome type B, 'H-n.m.r. spectroscopy, 219,263,267,268 compound 21, from sialidosis, 'Hn.m.r. spectroscopy, 269-274 compound 22, from sialidosis, 'Hn.m.r. spectroscopy, 270-272,274, 275 compound 23, and 24, from sialidosis and new-born human meconium, 'H-n.m.r. spectroscopy, 270-272, 275,276 compound 25, and 26, from new-born human meconium, 'H-n.m.r. spectroscopy, 270, 273,275,277,278 compound 27, and 28, from sialidosis and new-born human meconium, 'H-n.m.r. spectroscopy, 270,273, 277,280 compound 29, from sialidosis and new-born human meconium, 'Hn.m.r. spectroscopy, 270,273, 279, 282 compound 30, from sialidosis, 'Hn.m.r. spectroscopy, 270,273,279, 281,283 compound 31, and 32, from rabbit serotransferrin, 'H-n.m.r. spectrosCOPY, 270,283-287 compound 33 from human serotransferrin, 'H-n.m.r. spectroscopy, 270, 283-288 compound 34,'H-n.m.r. sp&ctroscopy, 270,2&-289 compound 35, 'H-n.m.r. spectroscopy, 270,289-293 compound 36, and 37, from sialidosis, 'H-n.m.r. spectroscopy, 270,292-

298 compound 38, from sialidosis, 'Hn.m.r. spectroscopy, 270,297, 298 compound 39, from sialidosis, IHn.m.r. spectroscopy, 270,298300 compound 40, from sialidosis, 'Hn.m.r. spectroscopy, 270,298, 300-302

SUBJECT INDEX compound 41, from sialidosis, 'Hn.m.r. spectroscopy, 270,300,302 -305 compound 42, from human lactotransferrin, 'H-n.m.r. spectroscopy, 306 -308,310,311,324-326 compound 43, from human lactotransferrin, 'H-n.m.r. spectroscopy, 306 -311,324-326 compound 44, and 45, from horse-pancreatic ribonuclease, 'H-n.m.r. S ~ ~ C ~ ~ O S C O306,307,309-312, PY, 3 16 compound 46, from fucosidosis, 'Hn.m.r. spectroscopy, 306,313-318 compound 47, from fucosidosis, 'Hn.m.r. spectroscopy, 306,313-318 compound 48, from asialo &,-acid glycoprotein and asialo ceruloplasmin, lH-n.m.r. spectroscopy, 306, 314,315,319-321 compound 49, 50, and 51, from asialo a,-acid glycoprotein, 'H-n.m.r. S ~ ~ C ~ T O S 306,314,315,321COPY, 324 compound 52, from human lactotransferrin and human milk, 'H-n.m.r. spectroscopy, 306,324-328 compound 53,from human-plasma ceruloplasmin, 'H-n.m.r. spectrosCOPY, 306,327-330 compound 54,from fucosidosis, 'Hn.m.r. spectroscopy, 3M, 330-332 compound 55, from aspartylglucosaminuria, 'H-n.m.r. spectroscopy, 333-337 compound 56, from aspartylglucosaminuria, 'H-n.m.r. spectroscopy, 333,336-338 compound 57, from aspartylglucosaminuria, 'H-n.m.r. spectroscopy, 333,335,337,338 compound 58, from aspartylglucosaminuria, 'H-n.m.r. spectroscopy, 333,337-339 compound 59, from aspartylglucosaminuria, 'H-n.m.r. spectroscopy, 333,337,340-342 compound 60,from aspartylglucosaminuria, 'H-n.m.r. spectroscopy, 333,337,340-343

399

compound 61, from Gaucher's disease, 'H-n.m.r. spectroscopy, 344-347 compound 62, from Caucher's disease, 'H-n.m.r. spectroscopy, 344,346348 compound 63, from hen-egg albumin, 'H-n.m.r. spectroscopy, 344, 346, 348-350,357,359-361 compound 64,from mannosidosis, 'Hn.m.r. spectroscopy, 344,350,351 compound 65, from mannosidosis, 'Hn.m.r. spectroscopy, 344,351,352 compound 66, from mannosidosis, 'Hn.m.r. spectroscopy, 344,351, 353 -355 compound 67, from mannosidosis, 'Hn.m.r. spectroscopy, 344,351, 355-357 compound 68,69, and 70, from Waldensbom's macroglobulinemia, 'H-n.m.r. spectroscopy, 344,357363 compound 71, and 72, from bovine lactotransferrin, 'H-n.m.r. spectroscopy, 344,360,361,363-366 compound 72, from soybean agglutinin, 'H-n.m.r. spectroscopy, 363365 oligomannoside-type carbohydrate chains, high-resolution proton magnetic resonance spectroscopy, 343-365 proton nuclear magnetic resonance spectroscopy of carbohydrate chains in structural analysis, 210374 Glycopyranosyl derivatives, tetra-0-acetyl(benzoyl)-D-, carbon-13 nuclear magnetic resonance spectroscopy, 50 Glycosides methyl anhydro-, carbon-13 nuclear magnetic resonance spectroscopy, 57 methyl, of ketoses, carbon-13 nuclear magnetic resonance spectroscopy, 48 nibophenyl, carbon-13 nuclear magnetic resonance spectra, 48 phenyl, carbon-13 nuclear magnetic resonance spectra, 48,49

SUBJECT INDEX

400

thio-, carbon-13 nuclear magnetic resonance spectroscopy, 65 Graphiurn, rhamnomannan, 89 Guanosine diphosphate: (l-A)-B-D-glucan synthetase, cellulose biosynthesis, 127 Guanosine diphosphate-glucose, precursor to cellulose biosynthesis, 125132 Guanosine diphosphate-glucose pyrophosphorylase, cellulose biosynthesis, 126 Gum arabic, structure, 8 Gums, see also Cherry gum; Damson gum; Peach gum structure, history, 3, 7

H Haenwphilus influenzae capsular polysaccharide-protein conjugate vaccine, 198 capsular polysaccharides, end groups, 185 structure, 164- 167 polysaccharide vaccine, 195, 196 Hamamelose, D-, carbon-13 nuclear magnetic resonance spectroscopy, 64 Hansenula capsulata Y-1842 phosphonomannan, 84 polysaccharide, 77,78 Hansenula holstii, phosphonomannan, 85, 86 Hansenula holstii Y-2448,polysaccharide, 77 Hansenula polymorphu, polysaccharide, 87 Hansenula wingei, polysaccharide. 87 Heptose, wglycero-manno-, synthesis, 8, 9 Heptulopyranose, 2,7-anhydro-B-~,carbon-I3 nuclear magnetic resonance spectroscopy, 51 Heteropolysaccharides branched, 95-100 galactan main-chains, 100, 101 D-mannan main-chains, 89-100 Hex-Z-enopyranosid4-ulose,methyl 2,3dideoxy-B-mglycero-, synthesis, 10

Hexopyranose, 1,6-anhydro-B-~-,carbon13 nuclear magnetic resonance spectroscopy, 51 -, per-O-acetyl-1,6-anhydro-@~-,carbon-13 nuclear magnetic resonance spectroscopy, 51 Hexopyranoses aminodeoxy, carbon-13 nuclear magnetic resonance spectroscopy, 54 deoxyhalo, carbon-13 nuclear magnetic resonance spectroscopy, 54,55 D-, peracetylated, carbon-13 nuclear magnetic resonance spectroscopy, 49 thio, carbon-13 nuclear magnetic resonance spectroscopy, 54 Hexopyranoside, methyl 3-acetamido2,3,6-trideoxy-a-~-arabino-, carbon13 nuclear magnetic resonance spectroscopy, 65 -, methyl 3-acetamido-2,3,6-trideoxy-aD-Iyro-, carbon-13 nuclear magnetic resonance spectroscopy, 65 -, methyl 3-acetamido-2,3,6-trideoxy-aL-ribo-, carbon-13 nuclear magnetic resonance spectroscopy, 65 -, methyl 3-amino-2,3,6-trideoxy-B-~rylo-, carbon-13 nuclear magnetic resonance spectroscopy, 65 -, methyl 3,6-anhydro-~-,carbon-13 nuclear magnetic resonance spectrosCOPY, 51 -, methyl 4,6-0-benzylidene-, carbon-13 nuclear magnetic resonance spectroscopy, 53 -, methyl 2-deoxy3-C-methyl-a-~-ribo-, carbon-13 nuclear magnetic resonance spectroscopy, 65 -, methyl 2,6-dideoxy3-C-methy13-0methyl-Llr'bo-, carbon-13 nuclear magnetic resonance spec~oscopy,65 -, methyl 3,4,6-trideoxy3-(dimethylamino)-r>-rylo-,carbon-I3 nuclear magnetic resonance spectroscopy, 65 Hexopyranosides. methyl deoxy, carbon13 nuclear magnetic resonance spectroscopy, 56,57 Hexose, 3,6-dideoxy-~arabino-,see Tyvelose -, 5,6-dideoxy-~urabino-,synthesis, 8

SUBJECT INDEX

-, 4,6-dideoxy3-0-methyl-~-xylo-, see Chalcose -, 3,6-dideoxy-Mibo-, see Paratose -, 5,6-dideoxy-~ylo-,synthesis, 8 3-Hexulose, 1-deoxy-L-arabino-, synthesis, 9 3-Hexuloses, synthesis, 9 Histoplasma capsulatum, polysaccharide, 69,70 Histoplasma farcinosum, polysaccharide, 70 I Immunity natural, 200,201 polysaccharide vaccines, 191-202 Immunoglobulin G, production, 190,197 Immunology capsular polysaccharides, 174-178, 186-191 role of complement, 187-189 Infection, see Bacterial infection Inhibitors, of cellulose biosynthesis, 143, 144 Isolichenan, lichen, 75, 76

J Jones John Kenyon Netherton, bibliography, 11-26 obituary, 1-26

K Ketoses, carbon-13 nuclear magnetic resonance spectroscopy, 48 Klebsiella, capsular polysaccharides, conformation and serological specificity, 179 Kloeckera apiculata, polysaccharide, 74 Kloekera brevis, phosphonomannan, 86

L Lactose, synthesis, 9 Lactotransfemin bovine, glycoproteins (compounds 71 and 72), 'H-n.m.r. spectroscopy, 344,360,361,363-366

401

human, glycoprotein (compound 42), 'H-n.m.r. spectroscopy, 306-308, 310,311,324-326 glycoprotein (compound 43), 'Hn.m.r. spectroscopy, 306-311,324 -326 glycoprotein (compound 52), 'Hn.m.r. spectroscopy, 306,324-328 Lumpteromycesjaponicus, polysaccharide, 103 Lusallia papulosa, polysaccharide, 75 Lentinan, fungal, 75 Lentinus edodes, polysaccharide, 70, 75 Lichenan, lichen, 75 Lichens glucans, 75,76 polysaccharides, 67-103 Lincomycin, carbon-13 nuclear magnetic resonance spectroscopy, 65 -, N-acetyl-, carbon-13 nuclear magnetic resonance spectroscopy, 65 Lipid intermediates, in cellular biosynthesis, 132-135 Lipopolysaccharides, serological determinants, 177

M Macrophages, immune action, 187 Magnesium ion, inhibitor of cellulose biosynthesis, 144 Malonogalactan, 87 Mannofuranose, 2,3:5,6di-O-isopropylidene-u-1>-,carbon-13 nuclear magnetic resonance spectroscopy, 53 Mannopyranan, a-D,structure, 80,81 Mannans fungal, branchedchain, 78-84 carbon-I3 nuclear magnetic resonance spectra, 78 linear, 77, 78 yeast, 82-84 Mannopyranoside, methyl 4,W-benzylidene-2-deoxy-2-C-methyla-0methyl-m, carbon-13 nuclear magnetic resonance spectroscopy, 65 Mannosidosis glycoprotein (compound 5), 'H-n.m.r. S~~C~~OS 219,227-230 COPY,

402

SUBJECT INDEX

glycoprotein (Compound 64),'H-n.m.r. spectroscopy, 344,350,351 glycoprotein (compound a), 'H-n.m.r. spectroscopy, 344,351,352 glycoprotein (compound 66),'H-n.m.r. spectroscopy, 344,351,353-355 glycoprotein (compound 67), 'H-n.m.r. spectroscopy, 344,351,355-357 Manniironic acid, 2-acetamido-2deoxyD-, 164-167 Meconium new-born human, glycoproteins (compounds 23 and 24), 'H-n.m.r. spec~ ~ O S C O P Y270-272,275,276 , glycoproteins (compounds 25 and 26), IH-n.m.r. spectroscopy, 270, 273,275,277,278 glycoproteins (compounds 27 and 28), 'H-n.m.r. spectroscopy, 270, 273,277,280 glycoprotein (compound 29), 'Hn.m.r. spectroscopy, 270,273,279, 282 Meningitis, capsular polysaccharide vaccine, for infants, 195-197 Meningococcal disease, polysaccharide vaccines, 193-195 Microfibrils biosynthesis, 116, 117 orientation, of cellulosic, 123-125 structure, 111-1 16 terminal complexes, 119-121 uncoupling, 117 Milk, human, glycoprotein (compound 52), 'H-n.m.r. spectroscopy, 306,324 -328 Monoblephurella elongatu, glycogen, 69 Monosaccharides acyclic, carbon-13 nuclear magnetic resonance spectroscopy, 59 biologically significant, carbon-13 nuclear magnetic spectroscopy, 64, 65 carbon-13 nuclear magnetic resonance chemical shifts, 28 carbon-13 nuclear magnetic resonance spectroscopy, 27-65 0-substituted, carbon-13 nuclear magnetic resonance spectroscopy, 52, 53

Morquio syndrome type B glycoprotein (compound 7), 'H-n.m.r. spectroscopy, 219,232,233,235237 glycoprotein (compound ll),'H-n.m.r. spectroscopy, 219,232, 233, 244246 glycoprotein (compound 15), 'H-n.m.r. ~ p e c t r o ~ c o p219,256-259 y, glycoprotein (compound 18), 'H-n.m.r. spectroscopy, 219,262-266 glycoprotein (compound 19), 'H-n.m.r. spectroscopy, 219,263,266-268 glycoprotein (compound 20), 'H-n.m.r. spectroscopy, 219,263,267,268 Mucilage, slippery-elm, 7 Mucoran, 101, 102 Mucor rouxii, mucorm, 101, 102 Mung bean cellulose biosynthesis, 108 high-molecular-weight precursors, 138 precursors, 126 Mutants, genetic, in cetlulose biosynthesis, 143, 144 Mycarose, L-, synthesis, 9

N Neisseriu meningitidis capsular polysaccharides, conformations, 179 molecular size and immunological specificity, 182-184 structure, 158-164 polysaccharide vaccines, 193-195 structure of polysaccharide antigens, 200 Neuraminic acid, N-acetyl-D, carbon 13 nuclear magnetic resonance spectroscopy, 64 Neurospora crassu, polysaccharide, 69, 71,73 Nigeran, fungal, 69, 70 Nuclear magnetic resonance spectroscopy, see Carbon-13 nuclear magnetic resonance spectroscopy; Proton nuclear magnetic spectroscopy Nucleoside diphosphates, glycosyl esters, in polymerizations, 125-132

SUBJECT INDEX

0 Obituary, John Kenyon Netherton Jones, 1-26 OCtulOSe, D-glycero-D-altro-, synthesis, 9 -, D-glycero-L-galacto-, synthesis, 9 -, L-glycero-L-galacto-, synthesis, 9 -, D-glycero-D-gluco-, synthesis, 9 Octulosonic acid, 3-deoxy-~manno-, 159, 164 sodium salt, carbon-13 nuclear magnetic resonance spectroscopy, 64 Ovotransferrin, chicken, glycoprotein (compound 16), 'H-n.m.r. spectrosCOPY, 219,256,259-261

P Pachyman, fungal, 73 Paracoccidioides brasillensis galactomannan, 93 polysaccharide, 70,73 Paratose, synthesis, 10 P a m l i a caperata, polysaccharide, 76 Pathogenesis, bacterial, capsular polysaccharides role, 202-206 Pathogenicity, polysaccharide structure role, 206-208 Peach gum, structure, 8 Peas, cellulose biosynthesis, 108 precursors, 126 Pectic acids, structure, 7 Penicillium charlesii, polysaccharides,

95 Penicillium chrysogenum, galactomannan, 95 Penicillium citrinium, malonogalactan, 87 Penicillium uatians, glucogalactan varianose, 100 Pentofuranoses, n,peracetylated, carbon-13 nuclear magnetic resonance spectroscopy, 49 Pentopyranoses, D-, peracetylated, carbon-13 nuclear magnetic resonance spectroscopy, 49 Pentopyranosides, methyl deoxy, carbon13 nuclear magnetic resonance spectroscopy, 56

403

2-Pentulose, 5 s -ethylJ-thio-r>-threo -, synthesis, 8 Peptidoheteroglycan, 96 Phagocytosis, macrophages and polymorphs, 187 Phizophydium sphaerotheteca, glycogen, 69 Phosphonomannans, 84-87 Physarium polycephalum, polysaccharide, 88, 89 Phytophthora cinnamoni, polysaccharide, 73 Phytophthora infestans, polysaccharide, 69 Pichia bouis, polysaccharide, 99 Pichia pastoris, mannan, 82 Piricularia oryzae peptidoheteroglycan, 96 polysaccharide, 75 Plant cell-walls microfibril orientation, 123-125 terminal complexes, 121-123 Plants cellulose biosynthesis in higher, highmolecular-weight precursors, 138142 lipid intermediates involvement, 132, 134, 135 Pleurotus ostreatus, polysaccharide, 73 Pneumococcal vaccine, polysaccharides, structure, 171-174 Pneumonia death rate, 156 polysaccharide vaccines, 191-193 Poly(ethy1ene glycol), effect on cellulose synthetase complex, 145-148, 152 Polymerization, see also Degree of polymerization mechanism, factors affecting, 145-150 glycosyl esters of nucleoside diphosphates, 125-132 polysaccharide chain growth, 135 Polymorphs, immune action, 187 Polyporus annosus, see Fomes annosus Polyporus circinatus, glycogen, 69 Polyporus fomentarius, hcomannogalactan, 100 Polyporus igniarus, fucomannogalactan,

100 Polyporus ouinus, fucogalactan, 100

SUBJECT INDEX

404

Polyporus pinicola, fucoxylomannan, 98 Polyporus squamosus, heteropolysaccharide, 100

Polyporus tumulosis, xylomannan, 96 Polysaccharide-protein conjugates, immunogenrcity, 197-200 Polysaccharides branched, immunodominance, 177, 178 capsular, conformation and serological specificity, 178-182 definition, 185 as human vaccines, 155-208 immune response to bacterial infection, 186-191 and immunity, 191-202 immunological specificity determinants, 175-178 location and immune response, 183186 molecular mimicry, 208 molecular size, 182, 183 role in bacterial virulence, 202-206 role of complement, 187-189 role in pathogenicity, 206-208 structural heterogeneity, 174, 175 structures, 158-174 fungi, structural chemistry, 67- 103 lichen, structural chemistry, 67-103 serological cross-reactions, 201-203 structure, 4, 7, 10 Polysphondylium pallidum, cellulosic cell-wall, 108 Poria COCOS, polysaccharide, 73 Proteases, effect on cellulose synthetase complex, 145 Proton chemical shifts additivity rules, 365-371 coupling constants and line widths, 212,213 and coupling constants for compounds 1 and 2,221 structural-reporter groups, 304, 305, 317,330,332,370 of compounds 3 and 4,226,346 of compound 5,230,351 of compounds 6, 7, and 10-12,232, 233 of compounds 8, 9, and 13,239 of compounds 14, 15, and 16,256 of compounds 17-20,263

of compounds 21-30,272,273

of compounds 31-34,286,287 of compounds 35-38,292,293 of compounds 39-41,300 of compounds 42-45,310,311 of compounds 46-51,314,315 of compounds 52 and 53,327 of compound 54,331 of compound 55,334 of compounds 55-60,337 of compounds 61-63,346 of compounds 63, and 68-72,360, 361 of compounds 64-67,351 fucose groups, 305-332 of mannose residues, 249-305 Proton nuclear magnetic resonance spectrometers, 373 Proton nuclear magnetic resonance spectroscopy carbohydrates, 27, 28 high-resolution, literature data, 214218 structural analysis of carbohydrate chains related to glycoproteins, 210-374 structural-reporter groups, 211-213 Protoplasts, cellulose biosynthesis, 109 Prototheca zopjii cellulose biosynthesis, 127 high-molecular-weight precursors, 137, 138 lipid intermediates involvement, 133, 134 cellulosic cell-wall, 108 Pseudonigeran, fungal, 69, 70 Psicose, D-, synthesis, 8 Pullulan fungal, 72 carbon-13 nuclear magnetic resonance spectrum, 71 Pyranosides, methyl deoxy-, carbon-13 nuclear magnetic resonance spectroscopy, 56,57

R Rhamnomannans, 89-91

Rhinocladiella eliator, polysaccharide, 88

SUBJECT INDEX

Rhinocladiella mansonii, polysaccharide, 102 Rhodotorula glutinis, mannan, 78 Rhodotomla glutinis K-24, fucomannogalactan, 101 Ribitol, D,derivatives, synthesis, 8 Ribonuclease, horse-pancreatic, glycoproteins (compounds 44 and 45), 'Hn.m.r. spectroscopy, 306,307,309312,316

S Saccharomyces carlsbergensis, phosphonomannan, 86 Saccharomyces cerevisial chitin, 103 phosphonomannan, 86,87 polysaccharide, 73,77-80 Saccharomyces fermentati, polysaccharide, 74 Saccharomyces fragilis, polysaccharide, 74 Saccharomyces phaseolosporus, polysaccharide, 99 Saccharomyces rortrii, polysaccharide, 77 Sandhoff's disease glycoprotein (compound 6), 'H-n.m.r. spectroscopy, 219,231-234 glycoprotein (compound 14), 'H-n.m.r. spectroscopy, 219,254-257 Schizophyllum commune, polysaccharide, 70 Schizosaccharomyces, polysaccharide, 69 Schizosaccharomyces octosporus, galactomannan, 92 Schizosaccharomyces pombe galactomannan, 92 polysaccharide, 74 Serotransferrin glycoprotein (compound 33) from human, 'H-n.m.r. spectroscopy, 270,283-288 glycoproteins (compounds 31 and 32) from rabbit, 'H-n.m.r. spectroscopy, 270,283-287 structure, 170 Sialic acid, determinant group in polysaccharide immunology, 175,176, 178, 182

405

Sialic acid groups, glycoprotein carbohydrate chains, 'H-n.m.r. spectroscopy, 269-305 Sialidosis glycoprotein (compound 21), 'H-n.m.r. spectroscopy, 269-274 glycoprotein (compound 22), 'H-n.m.r. spectroscopy, 270-272,274,275 glycoprotein (compounds 23 and 24), 'H-n.m.r. spectroscopy, 270-272, 275,276 glycoprotein (compounds 27 and 28), lH-n.m.r. spectroscopy, 270, 273, 277,280 glycoprotein (compound 29), 'H-n.m.r. spectroscopy, 270,273,279,282 glycoprotein (compound 30), 'H-n.m.r. spectroscopy, 270,273,279,281, 283 glycoprotein (compounds 36 and 37), 'H-n.m.r. spectroscopy, 270,292298 glycoprotein (compound 38),'H-n.m.r. spectroscopy, 270,297,298 glycoprotein (compound 39), 'H-n.m.r. spectroscopy, 270,298-300 glycoprotein (compound 40), 'H-n.m.r. spectroscopy, 270,298,300-302 glycoprotein (compound 41), from siaiidosis, 'H-n.m.r. spectroscopy, 270,300,302-305 Slime mold (1+4)-p-D-glucan biosynthesis, 108, 128 polysaccharide, 88 Solvents, for carbon-13 nuclear magnetic resonance spectroscopy, 29,30 Soybean agglutinin, glycoprotein (compound 72), 'H-n.m.r. spectroscopy, 366 Sphuerophorus globosus, polysaccharide, 76 Sporobolomyces roseus, glucogalactans, 100 Sporothrix schenckii amylose, 68 galactomannan, 93 rhamnomannans, 90 polysaccharide, 73 Stereocaulon japonicum, polysaccharide, 76

SUBJECT INDEX

406 Streptococcus Group

B

capsular polysaccharides, conformations, 181 immunological response, 177 structure, 167-170 capsular polysaccharide vaccine, 196, 197 Streptococcus pneumoniae capsular polysarulharides, conformations, 180 structure, 170-174 polysaccharide vaccines, 191- 193 Sugars chlorodeoxy, synthesis, 9 deoxy, carbon- 13 nuclear magnetic resonance spectroscopy, 55 Sulfuryl chloride, reaction with carbohydrates, 9

T Tagatose, D-, synthesis, 8 Tinea nigra, 93 Torulopsis, galactomannans, 92 Transmembrane electrical potential, intact cell, 147-150 Tremella fuciformis, polysaccharide, 97 Trernella mesenterica, polysaccharide, 70, 71, 72, 97 Trichosporon fennentans, galactomannan, 92 Tyvelose, synthesis, 10

U Umbilicaria pustulata, polysaccharide, 75 Uridine diphosphate-glucose, precursor to cellulose biosynthesis, 125-132 Uridine diphosphate-glucose: (1-+3)-p-~-glucan synthetase, in plant extracts, 128 Uridine diphosphate-glucose: (14)-j3-@glucan synthetase assay of solubilized, 146 cellulose biosynthesis, 128, 129, 148 Uridine diphosphate-xy1ose:xyylosyl transferase, 129, 130

Uronic acids, carbon-I3 nuclear magnetic resonance spectroscopy, 62 Uronolactones, carbon-13 nuclear magnetic resonance spectroscopy, 62 V

Vaccines artificial polysaccharide, 198, 199 capsular polysaccharides as human, 155-208 humoral antibodies, 189-191 and immunity, 191-202 Valinomycin, 148, 149 Varianose, 100 Virulence, see Bacterial virulence W

Waldenstrom’s macroglobulinemia, glycoprotein (compounds 68,69, and 70), ‘H-n.m.r. spectroscopy, 344, 357-363 X

Xanthomonas campestris, capsular polysaccharide, conformation and serological specificity, 178 X-Ray crystallography, cellulose structure, 113 Xylomannan, 96 Xylopyranose, 5-acetamido-5-deoxy-~-, synthesis, 9 Xylopyranoside, methyl B-D-, carbon-13 nuclear magnetic resonance spectrum, signal-to-noise ratio, 31, 32 Xylose, 2-O-p-~-glucopyranosyl-~-, synthesis, 9 -, 5-0-p-~-glucopyranosyl-~-, synthesis, 9 -, 3-0-B-D-xylopyranosyl-D-, synthesis, 9

Y Yeasts mannans, 82-84,W phosphonomannan, 86