Advances in Carbohydrate Chemistry and Biochemistry, Volume 42

Advances in Carbohydrate Chemistry and Biochemistry

Volume 42

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Advances in Carbohydrate Chemistry and Biochemistry Editors R. STUART TIPSON

DEREK HORTON

Board of Advisors LAURENS ANDERSON J. ANGYAL STEPHEN E. BALLOU CLINTON GUYG. S. DUTTON ALLAN B. FOSTER

BENGT LINDBERG HANSPAULSEN NATHAN SHARON MAURICESTACEY ROYL. WHISTLER

Volume 42

1984

ACADEMIC PRESS, INC. (Harcourt Brace Jovanovich, Publishers)

Orlando San Diego New York London Toronto Montreal Sydney Tokyo

COPYRIGHT @ 1984, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMIMED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR A N Y INFORMATION STORAGE AND RETRIEVAL SYSTEM, WlTHOUT PERMISSION IN WRITINO FROM THE PUBLISHER.

ACADEMIC PRESS,INC.

Orlando, Florida 32887

United Kingdom Edition published by ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London NWl7DX

LIBRARY OF CONGRESS CATALOG CARDNUMBER: 4 5 - 1 1 3 5 1

I S B N 0-12-007242-4 PRINTED IN THE UNITED STATES OF AMERICA 84 85 86 87

9 (I 7 6 5 4 3 2 1

CONTENTS CONTRIBUTORS ... PREFACE ......

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ix xi

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Dexter French (1918-1981) JOHNH . PAZUR Text . . . . . . . . . . . . . . . . . . . . . . . . . Students and Post-Doctoral Fellows of Dr . Dexter French

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1 11

The Composition of Reducing Sugars in Solution STEPHEN J . ANCYAL

I. Introduction

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

II. Methods for Studying the Composition of Sugars in Solution . . . . . . . . III. Relative Stabilities of the Various Forms . . . . . . . . . . . . . . . . .

IV. Composition in Aqueous Solution: Aldoses . . . . . . . . . . . . . . . V. Composition in Aqueous Solution: Ketoses . . . . . . . . . . . . . . . VI. Composition in Aqueous Solution: Substituted and Derived Sugars . . . . VII . Solutions in Solvents Other than Water . . . . . . . . . . . . . . . . VIII. Tabulated Data . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

.

.

15 17 24 34 37 42 60 62

Synthesis of Branched-chain Sugars JUJI

I. I1. I11. IV .

YOSHIMURA

Introduction . . . . . . . . . . . . . . . . . . . . . . General Syntheses. and Selectivities of Reactions Therein . Synthesis of Naturally Occurring. Branched Sugars . . . . Remarks Not Relating to Synthesis . . . . . . . . . . .

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

69 78 118 131

Sugar Analogs Having Phosphorus in the Hemiacetal Ring HIROSHI YAMAMOTOAND SABURO INOKAWA I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 I1. Monosaccharides Having a Phosphinediyl or Phosphonyl Group in the Pyranose Ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 I11. Monosaccharides Having a Phosphonyl Group in the Furanose Ring . . . . 176 IV . Biological Activities of Monosaccharides Having Phosphorus in the Hemiacetal Ring . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 V . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 VI . Table of Some Properties of Sugar Analogs Having Phosphorus in the Hemiacetal Ring . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

V

CONTENTS

vi

Carbon-13 Nuclear Magnetic Resonance Data for Oligosaccharides KLAUSBOCK.CHRISTIAN PEDERSEN. AND HENRIK PEDERSEN I . Introduction 1I.Tables . . .

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193 195

Ketonucleosides KOSTAS ANTONAKIS

I . Introduction . . . . . . . . . . . . . . . . . . . . Synthesis. . . . . . . . . . . . . . . . . . . . . Stability . . . . . . . . . . . . . . . . . . . . . Structure and Spectroscopic Properties . . . . . . Stereospecific Reduction . . . . . . . . . . . . . . VI. Nucleophilic Additions . . . . . . . . . . . . . . VII . Biological Interest . . . . . . . . . . . . . . . . 11 I11. IV . V.

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

227 231 245 249 252 257 261

Plant Cell-Walls PRAKASH M. DEYAND KEN BRINSON I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. The Primary Cell-Wall . . . . . . . . . . . . . . . . . . . . . . . . I11. The Pectic Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . IV . The Hemicelluloses . . . . . . . . . . . . . . . . . . . . . . . . . . V. Non-Cellulosic D-Clucans . . . . . . . . . . . . . . . . . . . . . . . VI . Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Cell-Wall Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . VIII. Cell-Wall-bound Enzymes . . . . . . . . . . . . . . . . . . . . . . . IX . Interconnections Between the Constituent Polymers in Primary Cell-Walls ofDicots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Discussion on the Albersheim Model for Primary Cell-Wall Structure ofDicots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI . Interconnections Between the Constituent Polymers in Primary Cell-Walls of Monocots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI1. Cell-Wall Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . XI11. Cell-Wall and Fruit Ripening . . . . . . . . . . . . . . . . . . . . . . Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

266 269 277 287 293 294 298 300 302 309 314 315 339 382

L- Arabinosidases

AKIRAKAJI

I. Introduction . I1. Classification .

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383 384

CONTENTS 111. a-L-Arabinofuranosidase . . . IV. Endo-(1+5).a.~.arabinanase .

AUTHOR INDEX SUBJECT INDEX

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386 392

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

STEPHEN J. ANGYAL, School of Chemistry, University of New South Wales,Kensington, N .S. W . 2033, Australia ( 15) KOSTAS ANTONAKIS, lnstitut de Recherches Scienti.ques sur le Cancer du C.N.R.S., B.P. 8, 94800 Villejug France (227) KLAUS BOCK,Department of Organic Chemistry, The Technical Universityof Denmark, DK-2800 Lyngby, Denmark (193) KENBRINSON, Department of Biochemistry,Royal Holloway College (Universityof London), Egham Hill, Egham, Surrey TW20 OEX, England (265) PRAKASH M. DEY,Department of Biochemistry, Royal Holloway College (University of London), Egham Hill, Egham, Surrey TW20 OEX, England (265) SABURO INOKAWA, Department of Chemistry,Faculty of Science, Okayama University, Tsushima, Okayama 700,Japan (135) AKIRAKAJI,'Faculty of Agriculture, Kagawa University, Kagawa 761-07, Japan (383) JOHN H. PAZUR, Paul M. Althouse Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802 (1) CHRISTIAN PEDERSEN, Department of Organic Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark (193) HENRIK PEDERSEN, Department of Organic Chemistry,The Technical Universityof Denmark, DK-2800 Lyngby, Denmark (193) HIROSHI YAMAMOTO, Department of Chemistry, Faculty of Science, Okayama University, Tsushima, Okayama 700,Japan (135) JUJI YOSHIMURA, Laboratory

of Chemistryfor Natural Products, Tokyo Institute of Technology,Midoriku, Yokohama 227, Japan (69)

' Present address: Fujitsuka-cho3-9-32, Takamatsu 760, Japan. ix

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PREFACE In this volume, S. J. Angyal (Kensington, Australia) discusses the use of 'H-n.m.r. spectroscopy in determining the composition of reducing sugars in solution. Applications of the n. m.r. technique have greatly advanced our understanding of the tautomeric behavior of sugars. Angyal has himself contributed much in this field, and his article complements from a modern perspective the information, deduced largely from such classical methods as polarimetry, provided by H. S. Isbell and W. W. Pigman in Vols. 23 and 24. Branched-chain sugars were largely a curiosity when the natural occurrence of those then known was treated by F. Shafizadeh in Vol. 11, but the great variety of these actually now shown to exist has stimulated intense efforts by organic chemists to develop methods for their synthesis. J. Yoshimura (Yokohama, Japan) here treats in depth the application of a wide range of synthetic procedures for the generation of specific branching in sugar structures. Sugar analogs having atoms other than oxygen in the hemiacetal ring have become of high interest from the standpoint of synthetic challenge and for their biochemical implications. H. Yamamoto and S. Inokawa (Okayama, Japan) introduce to this Series recent work directed toward such analogs having phosphorus as the ring atom. K. Bock and C. and H. Pedersen extend the article in Vol. 41 by the first two authors, on the 13C-n.m.r. spectroscopy of monosaccharides, to a compilation of such data for oligosaccharides that should prove of great value as a source ofreference. K. Antonakis (Villejuif, France) presents a discussion of ketonucleosides, compounds of interest in synthesis and in biological roles; they have not hitherto been comprehensively examined in this Series. The nature of the plant cell-wall is still surprisingly little understood; P. M. Dey and K. Brinson (Egham, England) bring into current perspective an article thereon by Shafizadeh and McGinnis in Vol. 26. As part of a continued series of articles on classes of enzymes acting on carbohydrates, A. Kaji (Takamatsu, Japan) here discusses L-arabinosidases, thus adding to the detailed treatment of other such enzymes in earlier volumes (P-L-glucosiduronase, by G. A. Levvy and C. A. Marsh in Vol. 14; aand P-D-galactosidases, by K. Wallenfels and 0. P. Malhotra in Vol. 16; and a-D-mannosidase, by S. M.Snaith and G. A. Levvy in Vol. 28). The life and work of Dexter French, who contributed so much to our knowledge of starch, is sensitively treated by his student J. H. Pazur (University Park, Pennsylvania). The pioneering discovery by French and Rundle, in the carbohydrate field, of a helical biopolymer in complexes of amylose predates the widely celebrated work with proteins and nucleic acids where the concept of a helical conformation revolutionized xi

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PREFACE

our understanding of the structure and function of these natural macromolecules. The Editors note with regret the deaths of an unusually large number of well known carbohydrate chemists, including Konoshin Onodera, Leslie F. Wiggins, and Fred Shafizadeh.

Kensington, Maryland Columbus, Ohio May, 1984

R. STUARTTIPSON DEREK HORTON

Advances in Carbohydrate Chemistry and Biochemistry

Volume 42

1918- 1981

ADVANCES I N CARBOHYDRATE CHEMISTRY A N D

BIOCHEMISTRY, VOL.

42

DEXTER FRENCH 1918-1981 It was with a sense of pride and honor that the writer accepted the invitation of the Editors ofAdvances to record some of the highlights and achievements in the life and career of Professor Dexter French. Professor French did indeed have a distinguished career in biochemistry at Iowa State University in research, in teaching, and in administration. In research, he contributed greatly to the advancement of knowledge in carbohydrate chemistry and enzymology; in teaching, he taught elementary biochemistry and advanced courses equally well and with much enthusiasm; and in administration, he fostered the development of excellence in Biochemistry at the Iowa State University and recruited highly qualified staff members for the Department. This article is written with a feeling of warm affection, much admiration, and great respect for Professor French, his life and his accomplishments. One may be confident that such sentiments prevail in his many students, research associates, and professional colleagues. The author first met Dr. French in 1946 on arriving at Iowa State University (at that time Iowa State College) as a new graduate student in the Department of Chemistry. French, a first-year Assistant Professor, was a young man eager to develop a research program, and highly motivated towards scientific discovery. In stature, he was of medium height and slightly above average weight, and in appearance, he had a round face, medium colored hair, brown eyes, and a fair complexion. He had a boyish appearance and a pleasant smile that he maintained throughout life, and he was often mistaken for an undergraduate student. In short, he had the appearance of the “wonder-boy” scientist which, indeed, he was. It was a requirement of the graduate program in Chemistry at Iowa State that all new students should discuss research projects with several faculty members before selecting a thesis advisor. My conversation with Dr. French quickly convinced me that he would be ideal as a thesis advisor. He was articulate, and thorough in the presentation of his research projects; he was very enthusiastic about his research; and he was most optimistic that many discoveries would be made. The decision to study with Dr. French was a good one, and led to many years of a rewarding professional association.

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JOHN H. PAZUR

Over the years, the scientific contributions of Professor French have been a source of much inspiration to researchers in laboratories around the world. Many new theories and concepts in carbohydrate chemistry and enzymology have been developed and utilized by French and his associates. These theories and concepts have led to new experiments and discoveries, many of which have had practical applications. His thoughts and ideas about research on carbohydrates, and about the scientific method, were often expressed at professional meetings and conferences, and were always informative and most refreshing. Dr. French had that remarkable gift of being able to inspire research workers and graduate students to higher levels of achievement. Many of his students have gone on to make important and significant contributions to knowledge of carbohydrate chemistry and enzymology. Dr. French’s creativity in research was evident early in his career in studies on the X-ray crystallography of starch and starch derivatives. These studies were conducted while he was a graduate student at Iowa State, and resulted in the concept of a helical structure for amylose, long before the concept was seized on by the nucleic acid chemists. Also, the iodine potentiometric-titration method for determining the content of amylose and amylopectin in starch was conceived and developed. The method is widely used for analyzing starches from new plant sources and from new varieties of cereal grains. Later in his career, procedures for preparing new oligosaccharides, such as the Schardinger dextrins (cyclomalto-oligosaccharides), maltoheptaose, maltotriose, and planteose, were devised. Such compounds proved to be very useful for enzymological studies. French’s contributions to the elucidation of the biochemical pathways for the synthesis of oligosaccharides and polysaccharides are important and significant. The demonstration that the D-glucosyltransfer mechanism is operative in the synthesis of oligosaccharides and polysaccharides was first achieved by Professor French and associates. In these studies, radioactive isotopes were utilized, and the results convincingly showed the occurrence of this mechanism. The mode of enzymic degradation of starch and glycogen was investigated by French, and the concept of multiple attack per single encounter of enzyme and substrate was formulated. Several practical modifications in paper chromatography and other analytical methods were introduced, to facilitate the structural characterization of oligosaccharides and polysaccharides. Very recently, evidence for the cluster model to represent the structure of starch was obtained by electron microscopy on the native starch-granule and by kinetic measurements on the hydrolysis of starch and starch derivatives by acids. Dexter French was born on February 23, 1918, in Des Moines, Iowa, and was the second child of Raymond Albert and Minnie Emily

OBITUARY-DEXTER

FRENCH

3

(Omerod) French. At an early age Dexter and the family moved to Dubuque, Iowa, when Dr. Raymond French was appointed to the staff ofthe Biology Department of the University of Dubuque. Dexter received his elementary and secondary education in the Dubuque school system. In 1935, he enrolled at the University of Dubuque, and he graduated in 1938 with a B.S. degree magna cum laude with a double major in chemistry and mathematics. He entered Iowa State University in 1938 for graduate study in chemistry, and he was awarded the Ph.D. degree in 1942. His dissertation on “An Investigation of the Configuration of Starch and Its Crystalline Degradation Products” was begun under the direction of Professor R. S. Bear, and completed under Professor R. E. Rundle. Dexter French was married to Mary Catherine Martin on June 17,1939. Dexter and Mary Catherine were the parents of seven children, Alfred (1943), David (1945), Walter (1948), Barbara (1949, deceased), Jean (1951), Nancy (1956), and Carol (1957). Dexter French devoted much of his professional career to research on starch, and on enzymes of starch synthesis and hydrolysis. As starch is an important substance in foods, in alcohol production, and in textile manufacture and other non-food uses, considerable information on starch existed prior to his studies. Starch is the most abundant chemical substance in cereal grains, and is, accordingly, a major, annually renewable, energy source. Starch is a mixture of two polymers, amylose and amylopectin, both of which are composed of D-glucose units joined together by a-(1-4) linkages in amylose, and by a-(1-4) and a-(1-6) linkages in amylopectin. The relative proportions of the polymers in starch markedly influence the physical properties, and, in turn, the uses of a specific starch. The contributions of French and coworkers to our knowledge of starch included a method for determining the two components of starch, the determination of structure by crystallographic methods, the elucidation of pathways ofbiosynthesis, and the development of methods for the conversion of starch into new products by chemical and enzymic reactions. On the basis of the results of X-ray studies on starch and the starchiodine complex, French and his associates concluded that the amylose and amylopectin components of starch bind different proportions of iodine, and that it should be possible to determine the amylose and amylopectin content of starch by potentiometric titration. Such a method for determining the ratio of amylose to amylopectin in starches was developed. The method has been widely used in plant-breeding programs for the development of new varieties of corn (maize) and other cereal grains. Varieties that produce a starch containing essentially 100% of amylopectin, and others producing a starch having 80% of amylose, have become available. The foregoing starches are respectively

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JOHN H. PAZUR

called waxy-maize and high-amylose starch, and have many special industrial applications. Waxy-maize starch is ideally suited for use in the formulation of puddings, jellies, instant foods, and similar products. The high-amylose starch has been used in the manufacture of edible films for packaging of foods, adhesives for glass fibers, and binders for paper. The corn wet-milling companies produce the new starches on a commercial scale, and have been responsible for developing many of the applications. The corn-producing state of Iowa must certainly have benefited from the discoveries of Professor Dexter French, resulting in the increased industrial uses of starch. After receiving his Ph.D., Dr. French spent two years, 1942 to 1944, as a post-doctorate fellow in the laboratories ofprofessors J. D. Edsall and E. J. Cohn at Harvard Medical School. During this period, French worked in the area of amino acids and proteins, and he became especially interested in relating the structure of amino acids and proteins to chemical reactivity. With Dr. Edsall, he published an excellent review on the reactions of formaldehyde with amino acids and proteins. In this stage of his career, his interest was aroused in proteins that possess enzymic activity. In later years, much of his research was devoted to enzymes and their mode of action, and to the molecular mechanisms and theoretical aspects of enzyme action. Dr. French spent 1945 as a research chemist with Corn Products Co., (at present, CPC International) at Argo, Illinois, working on projects of importance in the manufacture and utilization of starch. After one year with the Corn Products Co., he joined the Faculty of the Chemistry Department at the Iowa State University as Assistant Professor of Chemistry. In 1951, he was promoted to Associate Professor of Chemistry, and, in 1955, to Professor of Chemistry. When the Department of Biochemistry and Biophysics was formed at the Iowa State University in 1960, he became Professor of Biochemistry, and three years later, was appointed Chairman of the Department. He held the latter post until 1971, at which time he returned to full-time teaching and research. Dr. French possessed the special talent of being able to train graduate students, research associates, and post-doctorates in the performance of high-quality research. In his career, he directed the programs of 15 post-doctoral fellows, 20 doctoral students, and 17 master’s students. In 1946, when he joined the faculty of the Department of Chemistry at Iowa State, three students who had already begun their program were assigned to Dr. French, and one new student (the writer) elected to study in his laboratory. Many important discoveries dealing with the Schardinger dextrins and the amylases were made, and were described in scientific journals recognized for scholarly research. Dr. French was establishing a new and independent research program, and the period

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5

was characterized by much enthusiasm, constant activity, great excitement, and many achievements. New experiments were devised daily, and performed promptly. New data were accumulating constantly, and new concepts formulated regularly. It was with satisfaction that the members of the group worked hard and long hours in order to contribute to the program. The research experiments were under the watchful eye of Dexter French, as were the interpretations of the experimental data and the writing of the manuscripts. A notable achievement of the period was the demonstration that the action of Bacillus macerans amylase is reversible. It had been known for a long time that the enzyme converts starch into cyclic compounds. It was found by French that the D-glucosidic bonds of the cyclic compounds could be opened by the enzyme, and the resulting unit transferred to a cosubstrate, to yield a new product. The term “coupling reaction” was proposed for describing the reverse reaction of B. macerans amylase, and this term has subsequently been used in the literature. The enzyme catalyzes redistribution, as well as coupling and cyclizing reactions. The coupling reaction has proved to be extremely useful for synthesizing novel types of oligosaccharides. Thus, it has been used to synthesize linear malto-oligosaccharides terminated at the reducing ends with units having different structures, and labeled with radioactive carbon. Among the oligosaccharides that were prepared were malto-oligosaccharides terminated at the “reducing” end with a unit of sucrose, isomaltose, methyl a-D-glucoside, or methyl P-D-glucoside. The enzyme has been used to produce radioactive malto-oligosaccharides having a D-glucose14Cresidue at the reducing end. These oligosaccharides have proved to be extremely valuable for elucidating the mechanism of hydrolysis of linear chains of starch by amylolytic enzymes. Other achievements included the development of new methods for preparing Schardinger dextrins (cyclomalto-oligosaccharides),the determination of their structure by X-ray crystallographic methods, the preparation of linear malto-oligosaccharides, the use of the latter oligosaccharides as substrates for studying the action of amylases, the application of &nity-chromatography principles to the purification of B. macerans amylase by adsorbing the enzyme on starch and eluting with “Schardinger P-dextrin,” and many modifications in paper chromatography for facilitating the separation of complex mixtures of carbohydrates. Professor French was intensely interested in the mechanism of hydrolysis of starch, glycogen, and other polymers of D-glucoseby various types of enzymes, and by acids. He was also interested in elucidating the biochemical pathways for the synthesis of these polymers, and in methods for characterization of the compounds. A very clever method for studying the mechanism of an enzyme reaction was introduced in

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1953, a method based on the sequential use of paper chromatography and enzyme sprays. The compounds under study were first separated on a paper chromatogram, and then a solution of the enzyme was sprayed directly onto the compounds on the paper. The action of the enzyme on the compounds was observed by detecting the products of enzyme action with an appropriate color reagent, and by comparisons of Rpvalues. Because the reaction was performed directly on the paper chromatogram, losses of substrates, enzymes, or products, due to transfers and other manipulations, were eliminated. Micro amounts of substances can be detected on paper chromatograms and, accordingly, small amounts of substrates can be used in performing the enzymic experiments. Utilizing this procedure, the nature of the oligosaccharides that function as primers for phosphorylase in the synthesis of starch was investigated. It was found that oligosaccharides of D-glucose that are composed of four or more D-glucose units joined by a - ~1-4) - ( linkages function as efficient primers for plant phosphorylase. However, the linear trisaccharide of D-glucose was a poor primer, and maltose and D-glucose did not function as primers. It should be emphasized that French was a pioneer in the use of paper chromatography for separating carbohydrates. Shortly after the discovery of the technique, he utilized the method for elucidating the action of various types of amylases on maltooligosaccharides. Also, many improvements in the chromatographic method were made by French and his associates, and these modifications are widely used at the present time in research on carbohydrates. In 1963, a modification in the paper chromatography and enzyme spray method was introduced, namely, the use of two-dimensional chromatography interspersed with spraying of the chromatogram with an enzyme solution. In this procedure, the compounds under study were first separated in one direction on a paper chromatogram, dried thoroughly, and then sprayed with a solution of the enzyme. Enzyme action was allowed to proceed for a short time, and then the chromatogram was developed in the second direction. Appropriate standards and spray reagents were employed in order to identify the products of enzyme action. The work on the isolation and determination of structure of novel oligosaccharides by French and his coworkers is worthy of comment. Good examples of the oligosaccharides characterized structurally by straightforward but elegant methods are panose [O-a-D-glucopyranosyl(1+6)-O-a-D-glucopyranOSyl-( 1~4)-a-D-glucopyranoSe] and planteose [O-a-D-galactopyranosyl-( 1+6)-a-~-fructofuranosyl P-D-glucopyranoside]. For determining the structure of these oligosaccharides, specific enzymes and standard reactions of carbohydrate chemistry were employed for degrading the oligosaccharides. Paper chromatography was

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7

used for separating and identifying the reaction products. Many other oligosaccharides containing D-galactosyl, maltosyl, or sucrose units were prepared, isolated, and characterized. Structural studies were conducted not only on oligosaccharides but also on polysaccharides. Extensive use was made of such enzymes as beta amylase, salivary amylase, and pullulanase, of known substrate specificity, for determining the fine structure of starch and glycogen. The initial work on the Schardinger dextrins by French and associates was concerned with the isolation and characterization of a-,p-, and y-dextrins. The structures of these compounds were deduced by French and coworkers by crystallographic techniques as being cyclic D-glucose oligomers of 6 , 7 , and 8 residues joined bya-(l-4)- glucosidic linkages, and not 5, 6, and 7 residues as reported by others. The importance of these compounds for studying the enzymology of starch has already been mentioned. Later, French and his associates demonstrated that B. macerans amylase synthesizes other cyclic dextrins from starch. Four new compounds of this type were isolated and characterized. The new cyclic dextrins were called delta, epsilon, zeta, and eta cyclic dextrins and were found to be composed of 9, 10, 11, and 12 D-glucosyl residues, respectively. The discovery of the new types of cyclic dextrins, and the studies on the biosynthesis of the new compounds, have enhanced our understanding of the transferase mechanism visualized for the action of B. macerans amylase. The investigations carried out by Professor French and his students were based on sound experimental approaches and on intuitive theoretical considerations. The latter often resulted in new experiments for testing a hypothesis. On the basis of theoretical considerations, Professor French proposed a model for the structure of the amylopectin molecule, and the distribution of the linear chains in this molecule. This model was tested by utilizing enzymes that selectively cleave the linear chains, and the results substantiated the theoretical deductions. He proposed a theory on the nature and types of reactions occurring in the formation of the enzyme - starch complex during the hydrolysis of starch by amylases. In this theory, the idea of multiple attack per single encounter of enzyme with substrate was advanced. The theory has been supported by results from several types of experiments on the hydrolysis of starch with human salivary and porcine pancreatic amylases. The rates of formation of products, and the nature of the products of the action of amylase on starch, were determined at reaction conditions of unfavorable pH, elevated temperatures, and increased viscosity. The nature of the products was found to be dramatically affected by the conditions utilized for the enzymic hydrolysis, and could be accounted for by the theory of the multiple attack per single encounter of substrate and enzyme.

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A series of thorough and revealing studies was conducted with a variety of amylases, with a view to establishing the size of a combining site of an enzyme. In these studies, deductions were made on the basis of the nature of the products produced from starch by amylases. By use of different types of amylases, it was possit :e to show that a single D-glucose unit of the substrate combines with some amylases, whereas as many as nine D-glucose units of the substrate combine with other types of amylases. A model in which nine structural units of the substrate bind to the enzyme is quite unusual, and should be investigated further. The techniques of paper chromatography were used in these studies, and amylases from Bacillus subtilis, Bacillus polymyxa, human saliva, porcine pancreas, and Aspergillus o y z a e were employed. The many innovations that Dr. French introduced in the important technique of paper chromatography should be re-emphasized. An early example was the introduction of the technique of multiple ascent for separating compounds having low partition coefficients in solvent systems. By use of multiple ascent, it was possible to separate oligosaccharides of relatively high molecular weight for which a solvent could not be found. The development of a formula for calculating the RF values of compounds subjected to multiple-ascent chromatography was also an important advance. With the use of this formula, RF values could be calculated, and used to identify new carbohydrates that were isolated. The innovation of spraying solutions of an enzyme on compounds separated on paper chromatograms, and identifying the products of enzyme action, has already been mentioned. The correlation of RF values of oligosaccharides with molecular weight yielded an important method for determining the molecular weights of oligosaccharides. This procedure was useful for elucidating the structure of new oligosaccharides isolated by French and by other investigators. French and coworkers developed a formula that could be used for selecting solvents for optimal resolution of a mixture of carbohydrates. This formula has proved to be extremely useful. Also, the use of high temperatures for developing paper chromatograms is important. The high temperatures allow for the separation of compounds that could not be separated by other methods. In addition, the length of time required for developing such chromatograms is much lessened. Chromatographic procedures used by French and his coworkers utilized not only paper supports but also supports of charcoal, cellulose, cellulose derivatives, and dextran derivatives. Studies by French and associates on the structure of starch by X-ray crystallography, and, more recently, on the structure of the starch granule by electron microscopy, have resulted in the proposal of new models for the structure of starch and of starch granules. The X-ray studies

OBITUARY -DEXTER FRENCH

9

yielded evidence for a double-helical structure for amylose and the linear chains of amylopectin. Such a structure is consistent with the physical and chemical properties of starch. The electron-microscopy studies revealed the nature of the orientation of the linear chains in waxy-maize starch granules, and showed that the linear chains form segments of highly ordered structure, and the branch points are clustered in confined regions. The term “cluster model” for this type of structure was proposed. Prior to this proposal, it had been generally accepted that the starch molecule was best represented by a randomly branched structure. The evidence for the randomly branched structure came from methylation analyses and from the nature of the fragments produced from starch by enzymes. Although the suggestion of a cluster model for the structure of the starch granule had been made in the earlier literature, definitive evidence for such a model was forthcoming from the experiments of French and coworkers. The earlier experimental data from methylation and enzymic degradation of starch are in harmony with the cluster model. In the opinion of the author, the cluster model for starch is the most revolutionary idea on the structure of starch that has been advanced to date. This model adequately accounts for many of the chemical reactions, enzymic susceptibility, and physical properties of starch, and should prove useful for planning future research on starch. Dr. Dexter French held many offices in biochemical societies, was a Visiting Professor at several institutions, and was honored with many awards. In 1959, he was elected Chairman of the Division of Carbohydrate Chemistry of the American Chemical Society. In 1960, h e was awarded the honorary degree of Doctor of Science by the University of Dubuque for his scholarly researches in carbohydrate chemistry and enzymology. Also in 1960, he was a lecturer on glycogen metabolism at the annual symposium of the Ciba Foundation. In 1962, h e was a National Science Foundation Senior Fellow and Visiting Professor at the Lister Institute in London, England, and at the University of Paris, France. In 1964, he was honored with the Hudson Award of the Division of Carbohydrate Chemistry of the American Chemical Society for his work on the cyclic dextrins, including their structure, their properties, and their enzymic synthesis and interconversions. In 1970, he received the Award of Merit of the Japanese Society of Cereal Science for his significant contributions to starch science. In 1974, he was recognized by the American Association of Cereal Chemists for his work on starch chemistry with the Alsberg-Schoch Award. In 1977, he received the Iowa Award of the Iowa Section of the American Chemical Society for outstanding research in Chemistry performed by a resident of Iowa. In 1978, an issue of Carbohydrate Research was published in honor of Dexter French, on the occasion of his 60th birthday, by the Editors of the

10

JOHN H. PAZUR

journal, with contributions by his colleagues in the field of carbohydrate chemistry. In 1980, he received the Award for Advancements in the Application of Agricultural and Food Chemistry of the Division of Agricultural and Food Chemistry of the American Chemical Society. Dr. French was honored by Iowa State University in his appointment as Charles F. Curtis Distinguished Professor in 1968. His research was supported by grants from the National Institutes of Health, National Science Foundation, the U. S. Department of Agriculture, the Corn Industries Research Foundation, and the Corn Refiners Association. He served as a consultant to government agencies and industrial companies. He was a member of the study section for Physiological Chemistry of the National Institutes of Health, and was for many years a consultant with the National Starch and Chemical Company of New Jersey. Dr. French was a prolific contributor of research articles to professional chemical and biochemical journals. He published many reviews and “methods” articles in Annual Reviews of Biochemistry, Methods in Enzymology, Advances in Carbohydrate Chemistry and Biochemistry, Starch Chemistry and Technology, and The Enzymes. Two articles that were published in this Advances are ‘The Raffinose Family of Oligosaccharides” in Vol. 9 and “The Schardinger Dextrins” in Vol. 12. He was a member of the Editorial Advisory Boards of the Journal of Biological Chemistry and Carbohydrate Research, and of the Board of Advisors for Advances in Carbohydrate Chemistry and Biochemistry. Dexter was a member of several professional societies, including the American Chemical Society, the American Association of Biological Chemists, and the Association of Cereal Chemists. He participated actively in the programs of these societies. He was also an honorary member of the Japanese Society of Starch Science. Although science consumed much of his time, there were other activities that Dexter enjoyed immensely. He loved classical music, and found music a great source of relaxation and inspiration. He was able to play the flute, piano, and organ. In his college days, he had considered majoring in music and making music a professional career. He was very active in the musical activities at Iowa State University and in the Ames community. He was president of the Ames Town and Gown Concert Association in 1969- 1970, and he was instrumental in bringing skilled musicians to the area. Another activity that he greatly enjoyed was gardening. Every year, he had a lush garden that was his pride and joy. He loved to fish, and made frequent trips to the Northern States of the U. S. and to Canada. He liked to travel in his recreation vehicle, and he and Mary Catherine often made trips to places far away from Ames, including Mexico, to visit their children and to enjoy the beauties of the country. On Thanksgiving Day, November 26,1981, after valiant resistance for

OBITUARY-DEXTER

FRENCH

11

many years, Dr. Dexter French succumbed to multiple myeloma in the privacy of his home. He is survived by his wife, Mary Catherine, three sons, three daughters, and five grandchildren. Mary Catherine was an important factor in Dexter’s career. She is a most delightful person, and was a staunch supporter and a constant companion of Dexter. Mary Catherine was always a warm and gracious hostess at the many gatherings at the French home, and at Iowa State “get-togethers” at meetings of professional societies. The hospitality emanating from her and from Dexter earned them a special place in the hearts of many in this country and throughout the world. The training and the occupations of the children are in diverse fields. Alfred is a Ph.D. chemist at the U.S.D.A. Southern Regional Laboratory in New Orleans, and carries on structural research on starch and other polysaccharides; David is managing a duPont chemical laboratory in Parkersburg, West Virginia; Walter is a computer expert with Interactive Data Corporation in San Francisco, California; Jean is a nurse at the Swedish Covenant Hospital in Chicago, Illinois; Nancy is a computerprogram analyst at General Dynamics in Fort Worth, Texas; and Carol is living in Minneapolis, Minnesota. Professor Dexter French will be remembered as a creative scientist, an eminent scholar, a wise teacher, a knowledgeable colleague, and a skilful administrator. He was a most able biochemist, an accomplished physical chemist, a successful crystallographer, and an imaginative structural chemist. His universality of capabilities was a rare andvaluable attribute. His passing brings to a close an illustrious career of distinguished service to science, to Iowa State University, and to mankind. All who knew him share a sense of deep and grievous loss. His spirit lives on in his writings, his discoveries, his family, and the many students h e trained in Biochemistry. JOHNH. PAZUR ACKNOWLEDGMENTS It is a pleasure to acknowledge the assistance of Dr. John F. Robyt in providing some of the information for this article, and the cooperation of Mrs. French in the preparation of this tribute to her husband.

STUDENTS AND POST-DOCTORAL FELLOWS OF DR.DEXTER FRENCHO Harvey Dube, Ph.D. (1947), Deceased Melvin Levine, Ph.D. (1947), Deceased Robert McIntire, M.S. (1948), Phillips Petroleum Co.,Bartlesville, OK With present aililiation or address, where known.

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JOHN H. PAZUR

Hans Bolliger, Post-D. (1948- 1949),Research Chemist, Hofmann-LaRoche, Basel, Switzerland Doris Knapp, M.S. (1949), Deceased Ethelda Norberg, Ph.D. (1949), University of Caifornia, Davis, CA John H. Pazur, Ph.D. (1950), Professor of Biochemistry, Pennsylvania State University, University Park, PA Gene Wild, M.S. (1950), Ph.D. (1953), Research Chemist, Eli Lilly Co., Indianapolis, IN William James, M.S. (1952), Ph.D. (1953),Professor of Chemistry, University of Missouri, Rolla, MO Philip Nordin, Ph.D. (1953),Professor of Biochemistry, Kansas State University, Manhattan, KA Robert Suhadolnik, M.S. (1953), Professor of Biochemistry, Temple University, Philadelphia, PA J. Martyn Bailey, Post-D. (1954- 1955), Professor of Biochemistry, George Washington University, Washington, D. C. James Calamari, M.S (1954), 620 Ashford Drive, Indianapolis, IN Russel Summer, Ph.D. (1955) Regional Environmental Engineer, Boise-Cascade, International Falls, MN Joyce Barton, M.S. (1956), Division of Natural Science, University of Saskatchewan, Regina, Saskatchewan, Canada Stig Erlander, Ph.D. (1956), Nutritional Consultant, Pasadena, CA H. B. Wright, Post-D. (1957-1958), Auckland, New Zeland Carol Dahl, M.S. (1958), 679 Jefferson Street, Bryn Mawr, PA Lenorann Lewis Matson, M.S. (1958), 215 Elmwood, Centerville, OH John A. Thoma, Ph.D. (1958), Professor of Biochemistry, University of Arkansas, Fayetteville, AR U. K. Misra, Post-D. (1959 - 1960), Vallabhbhai Pate1 Chest Institute, University of Delhi, Delhi-110 007, India David Genova, M.S. (1960),Sr. Research Chemist, Eastman Kodak Research Laboratories, Rochester, NY R. William Younquist, M.S. (1960), Ph.D. (1962),Research Chemist, Procter andGamble, Cincinnati, OH John A. Effenberger, Ph.D. (1961), Vice Pres. & Director of Technologies, Chemical Fabrics Corp., North Bennington, VT Arden 0. Pulley, Ph.D. (1962), Dentist, St. Louis, MO John Robyt, Ph.D. (1962), Post-D. (1964- 1967), Professor of Biochemistry, Iowa State University, Ames, IA MarciaTripp, M.S. (1962), 3250 North River Drive, Eden, UT Abdullah Mukhtar, Post-D. (1963 - 1967), Starch Enzymologist, CPC International, Argo IL Joseph L. Mancusi, M.S. (1964), Psychiatrist, V.A. Hospital, Alexandria, VA Mary Catherine Smith, M.S. (1966), 115 Armstrong, Ventura, CA Walter Verhue, Post-D. (1 966- 1968), Research Chemist, Procter and Gamble, The Netherlands Melvin Weintraub, Ph.D. (1967),Research Chemist, Abbott Laboratories, North Chicago,

IL Alfred Aslam Khwaja, M.S. (1967), 1206 Tenth Avenue, North Clinton, IA Rajendra Varma, Post-D. (1967 - 1968), Director of the Biochemistry Department, Warren State Hospital, Warren, PA Keiji Kainuma, Post-D. (1968-1970), Visiting Prof. (1977-1978), Director of Starch Enzymology, National Food Research Institute, Tsukuba, Ibaraki, Japan

OBITUARY-DEXTER FRENCH

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Jiun G. Keng, Post-D. (1968- 1969), Research Chemist, CPC International, Argo, IL James R. Runyon, Ph.D. (1968), Research Chemist, Sandoz Chemical Co., Basel, Switzerland Nagavalli Yada Giri, Post-D. (1968- 1971), 16203 Barcelona, Friendswood, TX James Linden, Ph.D. (1969), Research Associate, Colorado State University, Fort Collins,

co

Gary Brammer, Ph.D. (1970), Neurobiochemist, V.A. Center, Los Angeles, CA George E. Smolka, M.S. (1970), Research Chemist, American Maize, Hammond, IN Toshiyuki Watanabe, Post-D. (1970 - 1971), Associate Professor, Tohoko University, Sendai, Japan Shoichi Kikumoto, Post-D. (1972- 1974), Research Chemist, Pfizer Taito, Ltd., Nagoya, Japan Barbara England, M.S. (1973), Project Director, Clinical Chem., Highland Diagnostics, Round Lake, IL Steven Brown, M.S. (1974), Research Associate, University of Kentucky, Lexington, KY Richard Harrington, M.S. (1974), 112 306 Baxter Court, Chaska, MN Yoshiyuki Sakano, Post-D. (1974 - 1975), Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan Yuk-Charn Chan, Ph.D. (1975), Research Biochemist, American Critical Care, McGrew Park, IL Wendy Brown Linder, M.S. (1976), Research Biochemist, Wellcome Res. Labs., Burroughs Wellcome Co., Research Triangle Park, NC Masatake Ohnishi, Post-D. (1976- 1977), Assistant Professor, Kyoto University, Kyoto, Japan James Bolcsak, Ph.D. (1979),Research Biochemist, Celanese Chemical Co., Louisville, KY

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ADVANCES I N CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY. VOL 42

THE COMPOSITION OF REDUCING SUGARS IN SOLUTION

J . ANGYAL BYSTEPHEN School of Chemistry. University of N e w South Wales. Kensington. N.S. W. 2033. Australia

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Methods for Studying the Composition of Sugars in Solution . . . . . . . . . . . . . 1 . Polarimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Nuclear Magnetic Resonance Spectroscopy ......................... 3 . Determination of Acyclic Forms .................................. 4 . Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Relative Stabilities of the Various Forms ............................. 1 . The Pyranose Form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................. 2 . The Furanose Form . . . . . . . . . . . . 3. The Septanose Form . . . . . . . . . . . ............................. ....................... 4 . The aldehydo and keto Forms . . . . . . ....................... 5 . Hydrated Carbonyl Forms . . . . . . . . 6 . Variation of Composition with Temp ....................... ....................... 7 . The Effect of Inorganic Compounds . IV. Composition in Aqueous Solution: Aldo ....................... 1 . Aldohexoses and Aldopentoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Aldoheptoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Aldotetroses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Composition in Aqueous Solution: Ketoses ............................ 1. Hexuloses and Pentuloses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Heptuloses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Composition in Aqueous Solution: Substituted and Derived Sugars . . . . . . . . 1 . Partially 0-Substituted Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Aminohgars . . . . . . . . . . . ................................... 3. ThioSugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Branched-chain Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Sugars with Fused Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W . Solutions in Solvents Other than Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Tabulated Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 17 17 18 20 22 24 24 27 29 29 30 32 33 34 34 35 36 37 37 40 42 43 46 52 54 58 60 62

I. INTRODUCTION Reducing sugars differ from most other organic compounds in one characteristic property . When a pure organic compound that is not a reducing sugar is dissolved in a solvent. one can be reasonably sure that 15

16

STEPHEN J. ANGYAL

the solution will usually contain only one compound; but when a reducing sugar, above an aldotetrose or a 2-pentulose, is dissolved in water, a solution is obtained that always contains at least six compounds: the two pyranoses, the two furanoses, and the acyclic (open-chain) carbonyl form and its hydrate. There are also minute proportions of the septanoses and of dimers. These various forms, often referred to as “tautomeric forms” of the sugar, will be named simply as “forms” in this article, for the sake of brevity. It is to be emphasized that each of these forms is a distinct compound, differing from the other forms in its chemical, physical, and biological properties. Very few of these many compounds that are present in the equilibrium solutions of sugars have ever been isolated. The only method for separating them from the equilibrium mixture is by crystallization, and that depends on the fortuitous presence of seed crystals. In the crystalline state, each form of a sugar is usually stable, and no interconversion occurs (see, however, a-D-lyxose’). In a few cases (D-glucose, D-mannose, D-lyxose, and D-galactose), both pyranose forms have been obtained crystalline. Usually, only one form has ever crystallized, and there are sugars that have thus far been obtained only as syrups (for example, idose and psicose); for the latter, no form of the sugar is yet known in the pure state. The monocyclic furanoses show very little tendency to crystallize; if a reducing sugar is prevented from assuming a pyranose form (for example, the 5-O-methylaldohexoses), it will usually be obtained only as a syrup. Coriose (~-ah-o-3-heptulose)is the only monosaccharide known that can assume pyranose forms, but that nevertheless crystallizes in a furanose form.2 Although most of these forms of sugars have never been isolated, they can be detected in the n.m.r. spectra of the sugars, and their proportion in the equilibrium mixture can be measured. Before the advent of n.m.r. spectroscopy, only a rudimentary knowledge of the composition of sugars in solution was available, but, since 1961, an extensive collection of data has been built up, mainly by the use of n.m.r. spectroscopy. These data are the subject matter of this article. The composition of sugars in solution was reviewed3 in 1969, but since then, much new information has been accumulated. The popular, but erroneous, concept of an aqueous solution of a reducing sugar is of one containing large, and comparable, proportions of the two pyranoses, and only small proportions of the furanoses, but the composition may, in fact, vary within wide limits. At one extreme (for example, glucose), the furanoses can be barely detected, and, at the (1) H. G. Fletcher, Jr., Methods Curbohydr. Chern., 1 (1962) 77-79. (2) T. Taga, K. Osaki, and T. Okuda, Actu Crystullogr., Sect. B, 26 (1970) 991 -997. (3) S. J. Angyal, Angew. Chern., Int. Ed. Engl., 8 (1969) 157-166.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

17

-

other, an aqueous solution of ido-heptulose contains 80% of the furanoses at equilibrium. Furanose contents of -30% are common. At another extreme are found heptuloses, whose aqueous solutions contain only one of the pyranose forms in substantial proportion: in the spectrum of D-gluco-heptulose no signals other than those of the a-pyranose can be detected. The proportion of the acyclic form is usually very low (
11. METHODS FOR STUDYING THE COMPOSITION OF SUGARS IN SOLUTION 1. Polarimetry Before the first application of n.m.r. spectroscopy to sugars, in 1961, polarimetry was the only method available for determining the composition of sugars in s o l u t i ~ nbut , ~ the method has serious limitations. If both pyranose forms had been obtained in the crystalline state and their optical rotations had been measured, and if the proportions of other forms in equilibrium are negligible, the proportion of the a- and ppyranose forms can be calculated from the value of the rotation at equilibrium (for example, for glucose, mannose, and lyxose). However, if the proportions of furanoses are not negligible, mutarotation is not of first order, but “complex,” consisting of an initial, fast change, followed by a slow one. It is possible to calculate the contribution that the slow mutarotation (due to the interconversion of the pyranose forms) makes to the change in rotation. Provided, again, that both pyranose forms are known in the crystalline state, the extent of the slow mutarotation gives an approximate measure of the a :p pyranose ratio at equilibrium (for example, for galactose). The proportion of the furanose forms cannot be de(4) G. R. Gray, Acc. Chem. Res., 9 (1976) 418-424. (5) F. J. Bates and Associates, Polarimety, Sacchurimety and the Sugars, Natl. Bur. Stand. (U.S . ) Circ., C440 (1942) 439-456.

18

STEPHEN J. ANGYAL

termined in this way, however, because the optical rotations of furanoses are unknown. If only one, or none, of the forms is known in the crystalline state, polarimetry does not yield any useful results. It was not even certain, for example, before the advent of n.m.r. spectroscopy, whether the one known crystalline form of D-ribose is the a-or the P-pyranose; its mutarotational change is small, but c ~ m p l e x . ~ 2. Nuclear Magnetic Resonance Spectroscopy

The first nuclear magnetic resonance (n.m.r.) spectra of sugars were published in 1961 by Lenz and Heeschen,s who also estimated the proportion of the pyranose forms. This work had little impact; perhaps, the journal in which it was published was infrequently consulted by carbohydrate chemists, or n.m.r. spectrometers were still a rarity. A few years later, Rudrum and Shaw' and Lemieux and Stevens* described the 'H-n.m.r. spectra, and estimated the proportions of all of the pentoses and of several hexoses. The spectra, and the composition of aqueous solutions of the remaining aldohexoses and of several deoxy sugars, were subsequently reported by Angyal and P i c k l e ~ . In ~ Jthese ~ spectra, most of the signals heavily overlap at 60 or 100 MHz, but the resonances of the anomeric protons, at low field, are well separated from the others, and their proportion can be measured. Because the ketoses have no anomeric hydrogen atom, their lH-n.m.r. spectra offer little useful information. The advent of 13C-n.m.r. spectroscopy, however, provided the solution to that difficulty. The 13C-n.m.r. spectrum of D-fructose was described in 1971 by Doddrell and Allerhand, who gave the proportions of the various forms of that sugar." This work was followed by publications by Perlin and coworkers,12J3Que and Gray,14and Angyal and c o w ~ r k e r s on ~ ~the J ~ spectra of various ketoses and deoxyketoses. In these spectra, most of the signals of the major forms

(6) R.W. Lenz and J. P. Heeschen,]. Polym. Sci., 51 (1961) 247-261. (7) M. Rudrum and D. F. Shaw, J. Chem. Soc., (1965) 52-57. (8) R.U. Lemieux and J. D. Stevens, Can. J . C h . , 44 (1966) 249-262. (9) S.J. Angyal and V. A. Pickles, Aust. J. Chem., 25 (1972) 1695-1710. (10) S.J.AngyalandV. A.Pickles,Aust.J. Chem., 25 (1972) 1711-1718. (11) D. Doddrell and A. Allerhand,J. Am. C h . Soc., 93 (1971) 2779-2781. (12) A. S. Perlin, P. C. M. Hewe du Penhoat, and H. S. Isbell, Ado. Chem. Ser., 117 (1973) 39-50. (13) P. C. M. Herve du Penhoat and A. S.Perlin, Curbohydr. Res., 36 (1974) 11 1 - 120. (14) L. Que, Jr., and G . R. Gray, Biochemistry, 13 (1974) 146-153. (15) S.J. Angyal and G . S.Bethell, Aust. J. C h . , 29 (1976) 1249-1265. (16) S.J. Angyal, G . S. Bethell, D. E. Cowley, andV. A. Pickles, Aust.J.Chem., 29 (1976) 1239- 1247.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

19

are well separated from each other; the anomeric signals, again, appear as a separate group at lower field. In most instances, the anomeric signals are readily assigned. In the 'H-n.m.r. spectra, the coupling constants reveal the identity of most of the anomeric signals. In both 'H- and W-n.m.r. spectra, the aldofuranose signals appear at lower field than those of the aldopyranoses; the signal of the furanose anomer that has a cis hydroxyl group adjacent to the anomeric hydroxyl group appears at lower field in the 'H-n.m.r. s p e c t r ~ mand , ~ at higher field in the W-n.m.r. ~ p e c t r u m ,than ' ~ that of the other anomer. The signals of one of the pyranose forms can be identified by recording the spectrum of the crystalline sugar (if available) soon after dissolution.8 Selective substitution, to prevent the formation of either the pyranose or the furanose forms, will lead to the identification of the furanose or pyranose signals. If none of these methods work, assignments can be obtained by comparing the spectrum of the sugar with those of its methyl glycosides. Assignments of the 13Cspectrum of D-psicose caused difficulties, because the two pyranose signals were close to each other, and of equal intensity, and only one of the methyl pyranosides was known. Selective substitution (methylation of 0 - 3 ) , to change the a : pratio, provided the answer.15 Once the signals have been assigned, their integration provides the proportions of the components in the mixture. For reliable integration of the W-n.m.r. spectra, special precautions need to be taken"J8 in order to overcome differences in relaxation rates and in the magnitude of the nuclear Overhauser effect. Horton and Walaszek18*have shown that even routine, pulse Fourier-transform, 13C-n.m.r. spectra provide reliable determinations of equilibria for sugars in solution, if all of the 13C signals of each form are averaged. N.m.r. spectroscopy has not formerly been well suited for the detection of minor components, the limit of detection, except in special circurnstan~es,'~J~ being 1%.Forms in greater proportion than 1% are, however, readily seen; in several cases, as many as five forms of a sugar were detected, and measured, in a spectrum.'6.20The use of 13C-enriched sugars has emerged as a powerful method for detecting minor components.20a 31P-N.m.r. spectroscopy has been used for the study of the composi-

-

(17) (18) (18a) (19)

C. Williams and A. Allerhand, Curbohydr. Res., 56 (1977) 173-179. A. Allerhand, Pure Awl. Chern., 41 (1975) 255-259. D. Horton and Z. Walaszek, Curbohydr. Res., 105 (1982) 145-153. D. J. Wilbur, C. Williams, and A. Allerhand,]. Am. Chem. Soc., 99 (1977) 5450-

5452. (20) W. Funcke, C. von Sonntag, and C. Triantaphylides, Curbohydr. Res., 75 (1979) 305- 309. (20a) A. S.Serianni, J. Pierce, S.-G.Huang, andR. Barker,].Arn. Chern. Soc., 104 (1982) 4037-4044.

20

STEPHEN J. ANGYAL

tion of fructose phosphates.21 The hydroxyl resonances, which can be observed in aqueous solution at low temperature (- 9"), have also been used to estimate the proportions of various forms.22

3. Determination of Acyclic Forms Nuclear magnetic resonance spectroscopy can detect the presence of

aldehydo and keto forms of sugars; in those rare instances where they occur to the extent of 1%or more in equilibrium, their proportion has thus been determined.1g~20~23~24 However, the percentage of the acyclic forms present in equilibrium is usually very small, and is much below the limit of detection by n.m.r. spectroscopy; other methods have, therefore, to be used. After studying several methods, Swenson and R. Barker concluded that infrared (i.r.) spectroscopy is the most suitable method for this purpose.25They thus determined the proportion of the carbonyl form in glyceraldehyde and in several phosphorylated sugars. However, the limit of detection of i.r. is about the same as that of n.m.r. spectroscopy; they could not detect the keto form in a solution of D-fructose. The first method to provide a reliable figure for the proportion of the aldehydo form was p o l a r ~ g r a p h y . ~Sugars ~ " are reduced at the dropping mercury electrode, giving low limiting currents. From the magnitude of these currents Cantor and Penistonee calculated the proportion of the carbonyl form of several sugars, on the assumption that the limiting currents are a measure of the concentration of the carbonyl form. This interpretation was disproved by W i e ~ n e r ,who ~ ' showed that the limiting current is determined, not by the rate ofdiffusion ofthe aldehydo form to the mercury drop, but by the rate of the transformation of other forms into the reducible acyclic form at the surface of the mercury drop. Accordingly, the values reported by Cantor and PenistonZeare too high; in particular, the prcportion given for the aldehydo form of D-ribose, 8.5%, would readily be seen in the n.m.r. spectrum, but it cannot be detected therein. (21) G. R. Gray, Biochemistry, 10 (1971) 4705-4711. (22) M. C. R. Syrnons, J. A. Benbow, and J. M. Harvey, Carbohydr. Res., 83 (1980) 9 - 20. (23) S. J. Angyal and R. G. Wheen, Aust. J . Chem., 33 (1980) 1001-1011. (24) P. E. Pfeffer and K. 8. Hicks, Carbohydr. Res., 102 (1982) 11-22. (25) C. A. Swenson andR. Barker, Biochemistry, 10 (1971) 3151-3154. (25a) M. Fedoroiiko, Adu. Carbohydr. Chem. Biochem., 29 (1974) 107-171; see pp. 126-162. (26) S. M. Cantor and Q . P. Peniston, J . Am. Chem. SOC., 62 (1940) 2113-2121. (27) K. Wiesner, Collect. Czech. Chem. Commun., 12 (1947) 64-70.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

21

Wiesner and coworkers2* determined, by calculation from polarographic data, the four rate-constants of interconversion between the CY- and /?-pyranose and the aldehydo form of D-glucose, and therefrom, the proportion of the latter. It was found to be 0.0026 f0.0002%.This value, although very small, was proved correct by later investigations. For D-xylose, the value of 0.04% can be calculated from the mutarotation r a t e - c o n s t a n t ~ .The ~ ~ polarographic measurements are conducted in buffered solution, and it was found that the results depend on the concentration of the buffer. The value for xylose was obtained in aphosphate bufferat pH 6.99; in other buffers, at pH 8.22-8.59, higher values were obtained. Measurements in unbuffered solution gave the proportion of the aldehydo form as 0.3% for xylose and 0.04% for glucose.2e It is not clear whether the proportion of the aldehydo form really varies with the nature and the pH of the buffer, or whether the variations are artifacts of the calculations used (which neglected the presence of the furanose and the aldehydrol forms). In solutions of the aldoses, there is too little of the aldehydo form in equilibrium to be detected by its U.V.absorption, but the ketoses give distinct, U.V.signals at 280 nm. These signals have been usedin order to determine the keto content at equilibrium, but they are affected by more strongly absorbing impurities and by the general absorption due to the solvent.25Hence, the results are often too high; for example, the proportion of the keto form of D-fructose was found30 by this method to be 2%, and that of D-fructose 1,6-bisphosphate, as high as 20%.Other ketoses and phosphorylated sugars have been investigated by this method. Circular dichroism does not suffer from the same disadvantage: it is manifested only in the region of an asymmetrical absorption band, and is unaffected by the influence of the rest of the molecule, or by the solvent (if it is not chiral). By using the circular dichroism band at 280 nm, the carbonyl content of numerous sugars has been estimated.31 Even the aldehydo form of D-glucose can be detected be this method. There is some doubt as to the value of the extinction coefficient of the carbonyl group in sugars, and it is not even certain that it is the same for each sugar. The proportions thus determined are, therefore, only approximate, but they are certainly of the right order of magnitude; the relative values, when comparing different sugars, can be regarded as reliable. This is, so far, the only generally applicable method for estimating the proportion of the carbonyl forms in equilibrium. The hydrated aldehydo or keto form can only be detected by n.m.r.

-

-

(28)J. M. Los,L. B. Simpson,andK. Wiesner,].Am. Cbm. Soc., 78 (1956)1564- 1568. (29)T.IkedaandM. Senda, Bull. Chem. Soc.Jpn.,46 (1973)1650-1656,2107-2111. (30)G. Avigad, S.Englard, and L. Listowsky, Curbohydr. Res., 14 (1970)365-373. (31)L. D. Hayward and S.J. Angyal, Curbohydr. Res., 53 (1977)13-20.

22

STEPHEN J. ANGYAL

spectroscopy. If its proportion is < 1%-as it is, in most cases-it not be determined by any of the methods used at present.

can-

4. Other Methods

The composition of mixtures of sugars has been determined by g.1.c. of their trimethylsilyl ethers. The method was introduced by Sweeley and coworkers,32who also used it for the analysis of equilibrium mixtures of sugars in aqueous solution. Their results are intermediate between the composition in water and that in pyridine, the trimethylsilylation having been carried out in pyridine solution. Bentley and B o t l o ~ described k~~ a modification in which the aqueous solution of a sugar is diluted with N,N-dimethylformamide, a solvent in which mutarotation is slow, and then trimethylsilylated at a very low temperature. They claimed that the composition is not changed during this procedure, pure anomers giving essentially single peaks in g.1.c. The mutarotation of D-fructose has been studied by this method,34and the proportion of the keto form was found35 to be 0.5 -0.6%. The composition of D-glucose, D-mannose, and D-galactose during mutarotation was also monitored in this way,36 and a full analysis of the mutarotation of D-galactose was successfully accomplished3' by its aid. (For further mutarotation studies, see Ref. 38.) For single analyses, the method is laborious, because, in contrast to those in n.m.r. spectra, the peaks in the gas-liquid chromatogram cannot be identified by their position and shape; each derivative has to be isolated, and its structure and configuration determined. Once that is done, however, the method is very satisfactory for routine analysis. It is the best method for monitoring the composition during mutarotation; n.m.r. is less suitable, because, at least in the initial stages of mutarotation, the composition usually alters significantly during the time needed to record the spectra. N.m.r. spectroscopy has been used to study the course of the mutarotation of 2-deoxy-~-erythro-pentose in water39 but (32) C. C. Sweeley, R. Bentley, M. Makita, and W. W. Wells, 1.Am. Chem. Sac., 85 (1963) 2497-2507. (33) R. Bentley and N. Botlock, Anal. Biochem., 20 (1967) 312-320. (34) H.-C. Curtius, J. A. Vdlmin, and M. Miiller, Fresenius'Z.Anal. Chem., 243 (1968) 341-349. (35) W. R. Sherman and S. L. Goodwin,]. Chromatogr. Sci., 7 (1969) 167-171. (36) T. E. Acree, R. S. Shallenberger, C. Y. Lee, and J. W. Einset, Carbohydr. Res., 10 (1969) 355-360; C. Y. Lee, T. E. Acree, andR. S. Shallenberger, ibid., 9 (1969) 356- 360. (37) P. W. Wertz, J. C. Garver, and L. Anderson,J. Am. Chem. Soc., 103 (1981) 39163922. (38) G. G. S. Dutton, Ado. Curbohydr. Chem.Biochm., 28 (1973) 11-160; see pp. 40-41. (39) R. U. Lemieux, L. Anderson, and A. H. Conner, Curbohydr. Res., 20 (1971) 59-72.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

23

had to be restricted to working at 0 " ; in other solvents (for example, dimethyl sulfoxide), however, n.m.r. spectroscopy can be used to monitor the mutarotation at ambient t e m p e r a t ~ r e . ~ ~ Caution is necessary when this method is applied to sugars that mutarotate rapidly. Angyal and Picklesg were unable to obtain a gas-liquid chromatogram having only a single peak from pure, crystalline P-D-ribopyranose, a sugar that mutarotates rapidly. The method is not suitable for analysis of the equilibrium mixture from certain ketoses. The trimethylsilylation of ketoses has been studied by Okuda and coworkers.41 The reaction is slow: 2- 120 hours are needed for its completion. The slow reaction of D-fructose had been commented upon p r e v i ~ u s l yIt. ~is~the anomeric hydroxyl group of the ketoses which reacts slowly,43and, while it is free, anomerization and ring opening may occur. The per(trimethy1silyl) derivative of the acyclic form is the preponderant product of the trimethylsilylation of 3-heptuloses, although, in the aqueous equilibrium solution, the keto form occurs only in minute proportion. The size of the g.1.c. peaks correlates poorly with the equilibrium composition, as determined by n.m.r. s p e c t r o ~ c o p y . ~ ~ The composition of aqueous solutions of ~-arabino-2-hexulose(Dfructose) has also been studied by laser-Raman spectroscopy.45 (The composition of solutions of D-fructose appears to have been determined by more methods, and more often, than that of any other sugar.20) There have been several reports of the separation of a-andp-pyranose forms during liquid chromatography of Initially, this was regarded as a nuisance, but systematic attempts were later made to improve this separation. At O " , with 1 : 4 water-acetonitrile as the eluant, many aldoses were separated into two component^.^^ Galactose and altrose showed small peaks probably representing the furanose forms; (40) T. C. Crawford, G. C. Andrews, H. Faubl, and G. N. Chmurny, 1.Am. Chem. Soc., 102 (1980) 2220-2225. (41) T. Okuda, S. Saito, and M. Hayashi, Carbohydr. Res., 68 (1979) 1-13. (42) G. Semenza, H.-C. Curtius, J. Kolinska, andM. Muller, Biochim. Biophys. Acta, 146 (1967) 196-204; L. E. Vidauretta, L. B. Fournier, and M. L. Burks, Anal. Chim. Acta, 52 (1970) 507-518. (43) T. Okuda and K. Konishi, Chem. Commun., (1969) 796-797, 1117-1118. (44) L. Hyvonen, P. Varo, and P. Koivistoinen, ]. Food Sci., 42 (1977) 654-656. (45) M. Mathlouthi and D. V. Luu, Curbohydr. Res., 78 (1980) 225-233. (46) 0.Ramntis and 0. Samuelson, Acta Chem. Scand., Ser. B, 28 (1974) 955-959. (46a) R. W. Goulding,]. Chromatogr., 103 (1975) 229-239. (47) R. Oshima, N. Takai, and J. Kumanstani,J . Chromatogr., 192 (1980) 452-456. (48) V. Kahle and K. Tesaiik,]. Chromatogr., 191 (1980) 121 -128. (49) N. W. H. CheethamandP. Sirimanne,]. Chromatogr.,196 (1980) 171 -175; N. W. H. Cheetham and W. R. Day, tbid., 207 (1981) 439-444. (49a) L. A. T. Verhaar and B. F. M. Kuster,]. Chromatogr., 210 (1981) 279-290.

24

STEPHEN J. ANGYAL

talose gave a complicated pattern. Only a single peak was obtained from ribose and from ketoses. It is apparent that only sugars that mutarotate slowly show a clear separation of anomers under these conditions, and yet it should be possible to separate all forms of any sugar by operating at lower temperatures and by selecting solvents in which mutarotation is slow. The method opens up the exciting prospectqeof separating the various forms of sugars on even a preparative scale.

111. RELATIVE STABILITIES OF THE VARIOUS FORMS Before discussing the composition of solutions of individual sugars, it will be instructive to look at the relative stabilities of the various forms of the sugars. These considerations will give a general overview, and also provide explanations for some of the results.

1. The Pyranose Form Pyranoses are the most stable forms of most sugars in aqueous solutionaS0The difference between the free energies of pyranoses and furanoses is quite substantial; when neither of them has serious steric interactions, it is about 8 kJ/mol. This is to be expected, as six-membered, saturated rings are more stable than five- (or seven-) membered ones; and the substituents of a six-membered ring are all fully staggered, whereas this is not possible in other ring systems. A possible, additional explanation for the greater stability of pyranoses has been suggested by Perlinsl based on the work of Kabayama and Patterson,s2 who suggested that the pyranoses have a special ability to interact with the “structured” component of liquid water. The spacing of the hydroxyl groups on a pyranose ring corresponds to tetrahedrally co-ordinated oxygen atoms hydrogen-bonded into the dynamic, tridymite arrays suggested for water,s3 and mutual stabilization of this water structure and of pyranoses will result, particularly if their hydroxyl groups are predominantly equatorial. Such stabilization would not occur with furanose forms. This theory is consistent with thermodynamic, dielectric, and n.m.r.-spectral studiess4 of simple sugar solutions, and with (50) J. F. Stoddart, Stereochemistry of Carbohydrates, Wiley-Interscience, New York, 1971, pp. 47-108. (51) A. S . Perlin, Can.J.Chem., 44 (1966) 539-550. (52) M. A. Kabayama and D. Patterson, Can. J. Chem., 36 (1958) 563-573. (53) M. D . Danford and H. A. Levy,J. Am. Chem. Soc., 84 (1962) 3965-3966; J. A. Pople, Proc. R. Soc. London, Ser. A, 205 (1951) 163-178. (54) F. Franks, D. S.Reid, and A. Suggett, J. Solution Chem., 2 (1973) 99- 118.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

25

calculations of conformational energy,55Js but it has never been proved and is not generally accepted, although many facts suggest that it may be valid. Thus, the proportion of pyranoses in aqueous solution decreases when the temperature is increased, a process that destroys the water “structure.” It also decreases on changing from water to another solvent (see Section VII). It decreases on partial methylati~n,~‘ a process that would lessen the extent of hydrogen bonding with water. It appears that the conditions favoring the stability of the pyranose form are precisely those under which the equilibrium is usually observed: aqueous solutions, ambient temperature, free hydroxyl groups. Because the conformations of the pyranose ring are well defined, the steric interactions within each isomer can be considered, and the relative stability of different isomers and conformers estimated. Reevess8 was the first to attempt to do this: h e assigned arbitrary “instability factors” to certain features of pyranose forms. Subsequently, AngyaP introduced a simple, semi-quantitative method whereby he estimated the approximate, free-energy content of each chair form by summation of quantitative, free-energy terms for conformational interaction-energies, and also for the anomeric effect. Interactions between axial groups and between vicinal gauche groups were determined, in aqueous solution and at ambient temperature, from the equilibria of cyclitols with their borate complexesso and from the anomeric equilibria of a l d o p y r a n o ~ e sThe ,~~~~~ method is based on the assumptions that the pyranoid ring has the geometry of an ideal cyclohexane chair, and that the various non-bonded interactions are independent of each other. Strictly speaking, neither of these assumptions is valid, but their acceptance appears to introduce only minor errors. Details of the calculations have been previously described in this Series.s2 The calculated, relative free-energies of the aldo-hexo- and -pento-pyranoses are shown in Table I; these values are relative to an imaginary aldopyranose in which there are no conformational interactions. From these values, the a :p ratio can be calculated; despite the simplicity and (55) D . A. Rees and P. J. C. Smith,J. Chem. SOC.,Perkin Trans. 2, (1975) 830-835. (56) L. G. Dunfield and S. G. Whittington, J , Chem. Soc., Perkin Trans. 2,(1977) 654658. (57) W. Mackie and A. S. Perlin, Can. J. Chem., 44 (1966) 2039-2049. (58) R.E.Reeves,J.Am.Chem. Soc., 71 (1949) 215-217; 72 (1950) 1499-1506;Adu. Carbohydr. Chem.,6 (1951) 107-134. (59) S. J. Angyal, Aust. J. Chem., 21 (1968) 2737-2746. (60) S. J. Angyal and D. J. McHugh, Chem. Ind. (London), (1956) 1147-1148. (61) S. J. Angyal, V. A. Pickles, andR. Ahluwalia, Carbohydr. Res., 1 (1966) 365-370. (62) P.L. DuretteandD.Horton,Adu. Carbohydr. Chem.Biochem.,26(1971) 49-125; seepp. 99-101.

STEPHEN J. ANGYAL

26

TABLE I Calculated Conformational Free Energiesse and the Proportion of the a Anomer in Aqueous Equilibrium Solution of Aldopyranoses Free energy (kJ/mol)

a-Pyranose (%'.)"

Aldose

aForm

BForm

Calc.

Foundb

Allose Altrose Galactose Glucose Gulose Idose Mannose Talose Arabinose Lyxose Ribose Xylose

16.1 14.0 11.9 10.0 16.1 15.3 10.5 14.8 8.2 7.8 13.0 8.0

12.3 14.0 10.5 8.6 12.8 16.7 12.3 16.7 9.2 10.0 9.6 6.7

18 50 36 36 21 64 68 68

15 39 32 36 17 52 66 59 63 71 26 37

60 72 21 37

a Percentage of the two pyranose forms, but not of all forms of the aldose. bCalculatedfrom the data in Table 11.

approximate nature of the method, the values calculated agree well with the experimental data. The agreement is less than satisfactory only in those cases (idose, ribose) where there is an accumulation of axial substituents. Apparently, the assumption of additivity of the interaction energies will break down in these cases. There is also good agreement between the calculated and the experimentally determined a :P-pyranose ratios of several deoxyaldoses.1° It may therefore be assumed that the method would also yield useful predictions of this ratio for those deoxyaldoses whose composition has not yet been determined. The method also correctly predicts the proportion of epimeric aldoses in equilibrium with each For ketoses, this simple method of calculation does not give the correct values for the a :P-pyranose ratio. Apparently, the conformational interactions have different values in this case; it is considered15 that both the syn-axial and the gauche interactions are larger on the anomeric carbon atom, which carries two substituents, than they are on that in the aldoses. Owing to these larger interactions, the pyranose forms of the ketoses are less stable than those of the aldoses, and, consequently, for the ketoses (63) S.J. Angyal, 1.Carbohydr. Nucleos. Nucleot., 6 (1979) 15-30.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

27

there is a higher proportion of furanoses in equilibrium than for the homomorphous aldoses. l5 Theoretical calculations applying molecular mechanics have been used by several groups to derive the free energies of pyranose forms.55*56~64~65 Although these calculations provide important information on deformations caused by substituents and on internal rotations, the values derived from them for the a :/?ratio of pyranoses are not in any better agreement with experimental data than are those from Angyal's simple method. The calculations refer to the molecules in the gas phase or in nonpolar solvents; solvation, particularly by water, causes important changes in the free energy that are, at present, not readily calculated. Dunfield and WhittingtonS6 applied an additional, energy value for each equatorial hydroxyl group, and this purely empirical correction improves the calculated a :/? ratio in most cases. 2. The Furanose Form

Calculations of free-energy values, similar to those carried out for p y r a n o s e ~are , ~ ~not practicable for the furanose form. The chair forms of the pyranose ring represent energy minima, separated by substantial, energy barriers; the conformations are therefore clearly defined. By contrast, in five-membered rings, there is little difference in energy between the various envelope and twist conformations.86 When there are several substituents, as on the furanose rings, the steric requirements of the substituents, rather than those of the ring, will confine the molecule to certain energy minima (which may well be intermediate between envelope and twist forms).87 The conformations of methyl furanosides have been discussed in detai168.6e;with slight variations, these discussions would also be valid for the free furanoses. For such a flexible system, a simple calculation of free energies by the use of a set of interaction energies is not possible; the values of interaction energies are probably different for each compound. Nevertheless, calculations of this type have been carried out for the four hexulose 6 - p h o ~ p h a t e s . ~ Assump~" (64) K. S . Vijayalakshmi and V. S. R. Rao, Carbohydr. Res., 31 (1973) 173-181, and earlier papers. (65) S . Melberg and K. Rasmussen, Carbohydr. Res., 69 (1979) 27-38, and earlier papers. (66) J. B. Hendrickson,]. Am. Chem. Soc., 83 (1961) 4537-4547; 85 (1965) 4059. (67) C. Altona, H. R. Buys, and E. Havinga, Recl. Trao. Chim. Pays-Bas, 85 (1966) 973-982. (68) S . J. AngyaI, Carbohydr. Res., 77 (1979) 37-50. (69) N. Cyr and A. S. Perlin, Can.]. Chem., 57 (1979) 2504-2511. (69a) T. A. W. Koerner, Jr., R. J. Voll, L. W. Cary, andE. S . Younathan, Biochemistry, 19 (1980) 2795-2801.

STEPHEN J. ANGYAL

28

tions (some unjustified) were made as to which conformations should be taken into account; the values of interaction energy applicable to the pyranoses were, in most cases, used for these furanoses; and additional, estimated values of interaction energy were introduced. Although the Q :p ratios thus calculated are in good agreement with the experimental results, these calculations can hardly be justified. Despite these difficulties, it is possible to draw some general conclusions about interactions within the furanoses, and about their relative stabilities.1° In a five-membered ring, vicinal carbon atoms cannot be fully staggered; therefore, interactions between vicinal, cis substituents become important. It is a striking fact that removal of the hydroxyl group from C-3 of glucose, to give 3-deoxy-ribo-hexose, causes the furanose content to increase from -0.1% to over 20% (see Table 11); similarly, removal of OH-3 from mannose causes the negligible furanose content to increaseseb to 27%. Obviously, a major interaction has been removed from the furanose forms. The hydroxyl group on C-3 is cis to the side chain, the bulkiest substituent; this arrangement appears to be unfavorable. Indeed, whenever this arrangement is present in a furanose form, its proportion in equilibrium is low. Removal of the hydroxyl group from C-2 of a hexose also increases the furanose content; for example, allose, 8%; and 2-deoxy-ribo-hexose, 27%. The hydroxyl groups on C-2 and C-3 are cis in allose; there is therefore a substantial interaction between vicinal, cis-hydroxyl groups in furanoses. This interaction has been estimated'O to be 3.6 kJ/mol. Even when the hydroxyl groups on C-2 and C-3 are trans, there is some increase in the furanose content on removal of OH-2; for example, galactose, 6%; 2-deoxy-lyxo-hexose, 16% (Table 11). Because of the interaction between cis-hydroxyl groups on C-1 and C-2, the trans anomer will be more stable than the cis anomer (the idofuranoses being an exception). The Q :p ratio is not constant, however, but depends on the configuration of the furanose. When all substituents on the ring are cis to each other (lyxo, manno), the cis-1,2 anomer is particularly disfavored.'O In the ketoses, there is a side chain attached to the anomeric carbon atom. The more-stable furanose anomer is, therefore, the one in which the side chain and the hydroxyl group on the neighboring carbon atom are trans to each other.ls In this anomer, there is little vicinal interaction, but there is one in the other anomer, as well as in the pyranose forms, and this interaction increases when the bulk of the side chain is increased.

-

(69b) P. M. Scensny, S. G .Hirschhorn, and J. R. Rasmussen, Curbohydr. Res., 112 (1983) 307- 312. (70) A. Wiiniewski, J. Gajdus, J. Sokolowski, and J. Szafranek, Curbohydr. Res., 114 (1983) 11-19.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

29

Hence, the proportion of the a-furanose form i n c r e a s e ~ 'in ~ the series 1-deoxy-D-psicose-D-psicose -~-dtr0-3-heptulose,as the side chain changes from CH, to CHzOH to CHOH-CH,OH, from 21 to 39 to 54%.

3. The Septanose Form Very little is known about the septanose form of reducing sugars. It has never been detected in the solution of an unsubstituted sugar. (The report71 that an aqueous solution of D-idose contains 1.6% of a septanose form appears to be e r r o n e ~ u s .The ~ ~ )first demonstration of a septanose in the solution of a reducing sugar was made by Anet,73 who prepared 2,3,4,5-tetra-O-methyl-~-glucose, a sugar that cannot form any other ring, and found that, in aqueous solution at 30°,about one-third of it was in the /3-D-septanose form. The a-D-septanose, of much higher free-energy, was not encountered. This work has been extended by Grindley and coworkers74 to most of the 2,3,4,5-tetra-0-methylaldohexoses. They found that the ratio of septanose to acyclic forms varies considerably; it is controlled by the relative stability of the septanose, rather than by that of the acyclic form. Thus, 2,3,4,5-tetra-O-methyl-~-galactose at 37" is in the septanose form to the extent of 88%in aqueous solution, and 94% in chloroform; for the aZZo isomer, the respective values are 21 and 15%. In the former sugar, the septanose ring has only one pair of cis substituents; in the latter, all hydroxyl groups are cis to each other. The ratio of septanose to acyclic sugar is not necessarily the same for the nonmethylated and for the methylated sugars. In the latter, the methyl groups cause 1,3-parallel interactions in the acyclic form, but not in the septanose form; there are no similar interactions in the acyclic forms of the nonmethylated sugars. The proportion of septanose may, therefore, be less in the nonmethylated than in the methylated sugars. All that can be said, at present, is that the proportion of the septanose form in the equilibrium mixtures of reducing sugars is of the same order of magnitude as that of the aldehydo form, that is, too small to b e detected by n.m.r. spectroscopy. 4. The aldehydo and keto Forms

All y- and S-hydroxyaldehydes and -hydroxyketones are in equilibrium with their cyclic hemiacetals, the position of equilibrium depending on (71) T. B. GrindleyandV.Culasekharam,]. Chem. Soc., Chern. Cornrnun.,(1978) 10731074. (72) S. J. Angyal, unpublished results. (73) E. F. L. J. Anet, Carbohydr. Res., 8 (1968) 164-174. (74) T. B. Grindley, V. Gulasekharam,and D. B. Tulshian, Abstr. Pap. Joint Conf: 2nd, CIC/ACS, Montreal, (1977) CARB 12.

STEPHEN J. ANGYAL

30

the solvent, and on the nature and number of substituents. At first glance, it is, perhaps, surprising that the reducing sugars have such a low proportion of the acyclic form in equilibrium. A study of 5-hydroxy-2-pentanone and 6-hydroxy-2-hexanone, which, except for their lack of an asymmetric carbon atom, could be respectively regarded as a trideoxypentulose and a tetradeoxyhexulose, showed that, in aqueous solution, they are present completely in the acyclic form, although, in organic solvents, almost half of these compounds is present in cyclic hemiacetal forms.7s It is well known, however, that substituents favor cyclic as against acyclic forms,7e and electron-attracting substituents on the a-carbon atom increase the tendency of carbonyl groups to react with hydroxyl groups (for example, the ratio of aldehydrol to aldehyde in aqueous e ~25”, ~ and 17.5 : 1for glycolaldesolution is 1.2 : 1 for a ~ e t a l d e h y d at at 35”). Hence, whereas 6-hydroxy-2-hexanone is found only in the acyclic form, aqueous solutions of the 1-deoxyhexuloses contain only 5% of this form at equilibrium, and those of the hexuloses contain less than 1% (see Table IV). Hemiacetals are more readily formed from aldehydes than from ket o n e ~4-Hydroxybutanal .~~ and 5-hydroxypentanal exist preponderantly as cyclic hemiacetals, containing only 11.4 and 6.1%, respectively, ofthe free aldehyde.79These often-quoted data were, however, obtained for solutions in 3 : 1 1,4-dioxane- water, and, if experience gained with the h y d r o x y k e t o n e ~is~also ~ valid for the hydroxyaldehydes, the proportion of aldehyde would be much higher in aqueous solution (for which data are not yet available). The aldopentoses and aldohexoses, which are completely hydroxylated 4-hydroxypentanals and 5-hydroxyhexanals, respectively, contain only a very small proportion (<0.05%) of the aldehydo form at equilibrium (see Table 11).The aldehyde content of the 2-deoxyaldoses is only slightly higher.

-

5. Hydrated Carbonyl Forms In aqueous solution the (acyclic) aldehydo form is partially hydrated, to give a gem-diol, often referred to as an “aldehydrol.” The extent of hydration is sensitive to the inductive effect of the substituent on C-2. (75) J. E. Whiting and J. T. Edward, Can. 1.Chem., 49 (1971) 3799-3806. (76) N. L. Allinger and V. Zalkow,]. Org. Chem., 25 (1960) 701-704; J. T. Edward, P. F. Morand, and I. Puskas, Can. J. Chem., 39 (1961) 2069-2085. (77) R. P. Bell, Adu. Phys. Org. Chem., 4 (1966) 1-29. (78) G. C. S. Collins and W. 0. George,]. Chem. Soc., B, (1971) 1352-1355. (79) C. D. Hurd and W. H. Saunders, Jr.,]. Am. Chem. SOC., 74 (1952) 5324-5329.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

31

The 2,3,4,5-tetra-O-acetyI-aldehydo-pentoses are hydrated to the extent of 90% in 7 : 3 oxolane - deuterium oxide.80On the other hand, the 2,3,4,5-tetra-0-methylaldohexoses are hydrated74 to a lesser extent in

-

aqueous solution at 37'. When the carbon chain is in the planar, zigzag form, hydration is favored, and the ratio of aldehydrol to free aldehyde is 2 : 1 (mannose and galactose); but when the chain is in a sickle form, hydration is impeded by 1,3-parallel interactions, and the ratio is 1: 2 (glucose and allose). For sugars lacking blocking groups, the aldehydrol form has been observed only in aqueous solutions of ~ - e r y t h r o s e~, ~- t~h r e o s e , ~the ~*,~~ pentose 5-phosphate~,~O~ and 3,6-anhydro-~-allose~l; the signal of the aldehydrol is readily recognizable in the n.m.r. spectrum, because it is absent when the spectrum is recorded for a solution in dimethyl sulfoxide. The ratio of aldehydrol to aldehyde is 10 : 1 at ambient temperature and it has been suggested31Jj3that it would be the same for all aldoses. This may not be true; from a study of the 13C-n.m.r. spectrum of D-( l-13C)idose,it was observed that this ratio is 4 for idose.82D-glycero~ - i d o -l-13C)Heptose ( was found to contain 0.6% of the aldehydrol and 0.06% of the aldehyde in equilibrium solution.8z It appears that, as the length of the chain of the uldehydo form increases, the extent of hydration decreases. For glyceraldehyde, the aldehydro1 : aldehyde ratio23 in deuterium oxide at 24" is 17.5 : 1; for Derythrose it is 10 : 1. For the tetraacetate of aldehydo-L-arabinose in 7 : 3 oxolane - deuterium oxide, the ratio is 9 : 1 whereas, for the corresponding L-fucose derivative, which differs only in the presence of an additional carbon atom at the other end of the chain, it is 3 : 1.The chemical shifts and the coupling constants indicate that these two compounds, surprisingly, adopt different conformations.80 By contrast, the keto forms of the ketoses seem to be hydrated to only a slight extent, if at all. When the proportion of the keto form is 5% or more, as it is for the 1-deoxyhexuloses, the hydrated form should be readily detectable in the 'H-n.m.r. spectrum, but it has not been found. A model compound, 1-deoxy-3,4,5,6-tetra-O-methyl-~-fructose, shows only the signals of the keto form in aqueous solution.16 Ketones are, on the whole, hydrated to a lesser extent than aldehyde^'^; in the case of ketoses having more than four carbon atoms in the chain, there would also be a 1,3-parallel interaction of one of the geminal hydroxyl groups with another hydroxyl group.

-

-

-

-

(80) D. Horton and J. D. Wander, Carbohydr.Res., 16 (1971) 477-479. (8Oa) A. S. Serianni, J. Pierce, andR. Barker, Biochemistry, 18 (1979) 1192-1199. (81) M. H. Randall and S.J. Angyal, J . Chem. SOC., Perkin Trans. 1, (1972) 346-351. (82) R. Barker and A. S. Serianni, personal communication.

STEPHEN J. ANGYAL

32

These conclusions are borne out by the data on some phosphorylated ketosess3 that cannot form pyranoses or furanoses. 5,6-Dideoxy-~-threohexulose l-phosphate (1)contains 96% (by i.r.) or 91% (by n.m.r.) of the keto form, and D-erythro-pentulose 1,5-bisphosphate (2) contains 84% (by ix.) or 88% (by n.m.r. spectroscopys0*)in aqueous solution; in the hydrates, there would be 1,3-parallel interactions. There are no such interactions in the hydrate of 1,5-dihydroxy-2-pentanone1,5-bisphosphate (3),but here one of the carbon atoms vicinal to the carbonyl group carries no hydroxyl group, and there is 84% of the keto form in equilibrium. 1,3-Dihydroxy-2-propanonephosphate has two neighboring hydroxyl groups and no 1,3-parallel interactions: only 55% is in the keto form and 45% is present as the hydrate.83*s4 ChOPO,H, I

c=o

I HOCH

I

HCOH I

7%

CH,OPO,H,

C H,OPO, H,

c=o

c=o

CH,OPO,H,

CH,OPO,H,

I I HCOH I HCOH I

I

7% I

7%

CH,

1

2

3

6. Variation of Composition with Temperature The equilibria between a-and j3-pyranose forms are little affected by changes in t e m p e r a t ~ r eHowever, .~ the proportion of furanose forms is increased considerably as the temperature is raised, as first noted by Isbell and Pigman.85 For example, the proportion of a-D-fructofuranose at 85" is 11% and of the j3-furanose is 33%, whereas, at 27", it is 4 and 21%, re~pectively'~; there is 9% of a-L-sorbofuranose in the equilibrium mixture at 85', but only 2% at 27'. As a further example, the composition39 of 2-deoxy-~-erythro-pentose is 42 : 4 3 : 5 : 10 at 0", but 30 : 30 : 22 : 18 at 90'. It is evident that the furanoses are the high-enthalpy forms,37increasing in proportion with increasing temperature. This increase was put to practical use by Angyal and Bethell15 for the assignment of W-n.m.r. spectra. On occasion, two forms occur in almost equal proportions in the solution of a sugar; it is then difficult to decide which signal in the spectrum should be assigned to which form. The spectrum was then recorded at a higher temperature, and the signals that increased in intensity were assigned to a furanose form, and those that decreased, to a pyranose form. (83) G. R. Gray and R. Barker, Biochemistry, 9 (1970) 2454-2461. (84) S. J. Reynolds, D. W. Yates, and C. I. Pogson, Biochem. J . , 122 (1971) 285-297. (85) H.S.Isbell and W. W. Pigman,J. Res. Natl. Bur. Stand., 20 (1938) 773-798; 16 (1936) 553-554.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

33

The proportion of the acyclic form also increases with increasing temperature; this is true for aldoses and k e t ~ s e s , ' ~ .as ~ 'well as for simple h y d r o ~ y k e t o n e s .This ~ ~ would be expected from considerations of entropy, as the acyclic form has a greater degree of freedom, but studies on y- and S-hydroxyketones show that change in enthalpy contributes even more to the changing position of the equilibrium with increasing temperature. Evidently, cyclization of hydroxyketones is exothermic, and is favored by lower temperature^.'^ The ratio of aldehydrol to aldehyde decreases sharply with increasing 10 : 1 at 24"and 0.85 : 1 at temperature; for example, for e r y t h r ~ s eit, is ~~ 75".The ratio for 2,3,4,5,6-penta-O-methyl-~-glucose in 0.5 M solution decreasese6 from 2.08: 1 at 3" to 0.24: 1 at 74". 7. The Effect of Inorganic Compounds The presence of inorganic salts can profoundly modify the equilibrium proportions of sugars in solution. When a sequence of an axial, an equatorial, and an axial hydroxyl group is present on a pyranose ring, or a sequence of three cis-hydroxyl groups on a furanose ring, the sugar will form complexes with cations.s7If only one form of areducing sugar gives complexes, or several forms give complexes, but to adifferent extent, the composition will change with increasing concentration of the cation; many examples of such a change are known.e8 The strongest complexes are formed by Ca2+,Sr2+,Ba2+,and the lanthanide cations; Na+, K+,and Mg2+form only weak complexes. Complex-formation increases with increasing concentration of both the sugar and the cation; below 0.01 M it is negligible. Hence, salts present as impurities in solutions of sugars will not materially affect their composition. At high concentration, however, cations will change the composition of even weakly complexing sugars; that is, those that lack the a,e,a or cis& arrangement of hydroxyl groups, but have at least one pair of cis-hydroxyl groups. For example, the composition of a 1 .O M solution of D-fructose is only slightly affected by the presence of 1 .O M calcium chloride, but in a 2.0M solution of that salt at 26"it becomes 79% of /I-pyranose, 4% of a-furanose, and 17% of p-furanose; in a 3.0 M solution, it becomes 88 : 2 : 10,the proportion of apyranose being very Only the /I-pyranose form of D-fructose forms a (weak) complex with calcium ions.g0 (86) T. B. Grindley and R. Ponnampalam, unpublished results. (87) S. J. Angyal, Chem. Soc. Reu., 9 (1980) 415-428; Tetrahedron, 30 (1974) 16951702. (88) S. 1. Angyal, Aust. J . Chern., 75 (1972) 1957-1966. (89) J. J. Watters, Ph. D. Thesis, Griffith University, Brisbane, Australia, 1982, p. 85. (90) S . J. Angyal and J. A. Mills, Aust. J . Chern., 32 (1979) 1993-2001.

34

STEPHEN J. ANGYAL

Dilute acids or bases have no noticeable effect on the composition of sugars in aqueous solution; at high concentration, however, they alter it considerably. What is then observed is, in fact, not the equilibrium of the sugars themselves but that of their cations and anions, respectively, sugars being weak bases and weak acids. Thus, the a! :P-pyranose ratio for D-glucose in 2.5 M DCl was foundson to be 45 : 55;the same ratio was obtained when the solvent was formic acid. By contrast, bases have the opposite effect, that is, they increase the proportion of the /3 anomer. In MNaOD the ratio is 10 : 90,and in diethylamine as solvent, 15:85.Analogous changes were also found in the composition of 2-O-methyl-~-glucose.~~~ The effect of alkali on the composition of D-glucose was studied in detail by de Wit and coworkersQobby W-n.m.r. spectroscopy. Gradual addition of potassium hydroxide to an aqueous solution of the sugar causes a gradual increase in theP-pyranose content which levels out at 78% when 1.5equiv. of the base have been simadded. The P-pyranose content of 2,3,4,6-tetra-O-methyl-~-glucose ilarly increases from 39 to 54%. Curiously, the composition of D-mannose is shifted in the opposite direction, from 60% to 75% of a-pyranose when 1.5equiv. of potassium hydroxide have been added. Gradual addition of potassium carbonate to a solution of lactose causes an increase in the proportion of the P-pyranose, which reaches 85% when 20.5 equiv. of the salt have been addedSQocThe authors attributed this increase to the formation of a (sterically impossible) complex with potassium ions!

-

-

IV. COMPOSITION IN AQUEOUS SOLUTION: ALDOSES 1. Aldohexoses and Aldopentoses

The composition of aldohexoses is shown in Table 11.The proportion of the furanose forms of glucose and mannose is too small for them to be detectable in the conventional 'H- or T - n . m . r . spectra. However, the 1% signals of the mannofuranoses were visible when the spectrum was recorded at a field strength of 67.9MHz for a very concentrated (4M) solution of manno nose.'^ By using a 20-mm, n.m.r. tube in a specially constructed probe, with a highly concentrated solution of D-glucose and with W-enriched D-glucose, Williams and Allerhand" detected even the signals of P-D-glucofuranose, present in a proportion of only 0.14%. Glucose, having all of its substituents equatorial in its pyranose forms, shows the highest proportion of pyranoses (>99%) in its equilibrium (90a) H. Sugiyama and T. Usui, Agric. Biol. Chem., 44 (1980) 3001 -3002. (90b) G. de Wit, A. P. G . Kieboom, and H. van Bekkum, R e d . Trau. Chim. Pays-Bas, 98 (1979) 355-361. (9Oc) R. M. Munavu, B. Nasseri-Noori, and H. H. Szmant, Carbohydr. Res., 125 (1984) 253-263.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

35

solution. As the axial hydroxyl groups accumulate, the proportion drops to 70%. Furanoses in which OH-3 and the side chain are cis constitute only a small proportion of the equilibrium mixture of each; the only exception is idose, in which both pyranose forms have such unfavorable interactions that the furanoses, although also disfavored, become important contributors to the equilibrium. The aldopentoses, also shown in Table 11, have, in aqueous solution, compositions similar to those of the homomorphous aldohexoses. The ratio of a-to j?-pyranose agrees well with the calculated values, but for each sugar, the proportions of the furanose and aldehydo forms are higher than those of the homomorphous aldohexoses. The reason for this behavior is an effect noted only a few years ago: cyclic acetals are formed more readily by secondary than by primary hydroxyl groups. This effect is best seen in the formation of aldose anhydridesg1 in which 0-1 is involved. It has been shown that tertiary hydroxyl groups, which occur in branched-chain sugars, form acetals even more readily.91*In the aldopentoses, the pyranose ring contains 0-5 (of the primary hydroxyl group) and its stability is therefore less than in the homomorphous aldohexoses, where the ring is formed by a secondary hydroxyl group. Table I1 also lists the data available on deoxyaldoses. The effect, on the tautomeric equilibrium, of removing a hydroxyl group can be very small, or quite large, depending on which hydroxyl group had been engaged. Thus, aqueous solutions of the 6-deoxyaldohexoses, as would be expected, do not differ significantly in composition from those of the corresponding hexoses. Solutions of the 2- and 3-deoxyaldoses contain more furanose at equilibrium than those of the corresponding aldoses, because removal of a hydroxyl group lowers the vicinal interactions to a greater extent in the five-membered ring, where the carbon atoms are not staggered (see Section 111,2).For the 2-deoxyaldoses, almost equal amounts of a-and j?-furanoses are found, as there is no longer a cis-vicinal interaction between hydroxyl groups in one of the forms. The a- to P-pyranose ratio can be predicted by an approximate calculation (see Section II1,l).

-

2. Aldoheptoses

The equilibrium compositions of aqueous solutions of some aldoheptoses are listed in Table 111. Because the additional carbon atom in the side chain does not introduce additional steric interactions, the composition of solutions of heptoses is similar to that of the homomorphous hexoses, with only one exception, namely, ~-g~ycero-~-ido-heptose.~~ a-D-Idopyranose in solution is a mixture of the 4C, and 'C4 conforma(91) S.J. Angyal and R. J. Beveridge, Curbohydr. Res., 65 (1978) 229-234. (91a) P. KO11, H.-G. John, and J. Schulz,JustusLiebigs Ann. Chern., (1982) 613-625. (92) S. J. Angyal and T. Q . Tran, Aust. J . Chem., 36 (1983) 937-946.

STEPHEN J. ANGYAL

36

ti on^.^ In the 'C, conformer of the heptose, the extended side chain has a serious interaction with OH-4; hence, this conformer is populated to a lesser extent, and the proportion of the a-pyranose form in equilibrium is lowered, compared to that of idose. It may be predicted that this would also be true of D-glycero-L-ido-heptose,the composition of which has not yet been determined. It has been notedQ2that, in aqueous solutions of the D-glycero-L-heptoses, the a-to p-pyranose ratio is somewhat higher than that for the homomorphous hexoses, whereas, for the D-glycero-D-heptosesthe ratio is the same, or even slightly lower. No explanation is apparent for this observation. The composition of aqueous solutions of two octoses has also been reported by two groups: Bilik and coworkers,Q3from the 'H-n.m.r. spectraat 50°,and Angyal andTranQ2,from the W-n.m.r. spectraat 22".The two sets of values are not in good agreement. For D-erythro-L-tdo-octose, Bilik and coworkersQ3found ratiosQ4of 36 : 18 : 24 : 22; Angyal and TranQ2 found 36 : 24 : 27 : 13. For D-threo-L-talo-octose, the results wereQ347% of (a-pyranose a-furanose), 39% of /I-pyranose, 14% of p-furanose, and the ratios wereQ244 : 34 : 12 : 10.

+

3. Aldotetroses The two aldotetroses, erythrose and threose, differ from the other aldoses in their behavior.23 Ring formation, to give furanoses, can occur only through the primary hydroxyl group, and is therefore less favored than with the higher sugars. Consequently, considerable proportions of the aldehydo and aldehydrol forms are found in solution. Like all a- and p-hydroxyaldehydes, the aldehydo form of the aldotetroses readily forms dimers: in concentrated solutions of the tetroses, the signals of the dimers are readily visible in their n.m.r. spectra. In the syrupy state, the tetroses consist mainly of dimers, rather than of furanoses; they have never been crystallized. The composition of a 0.1 M solution of D-threose in water was carefully measured at seven different temperatures.20aAt 25",it was found to be 51.8%ofa-furanose, 37.6%ofp-furanose, 0.96%of aldehyde, and 9.6% of aldehydrol; at 81", the composition is 50.4, 37.8, 4.7, and 7.1%, respectively. Somewhat higher values were found for the aldehydrol in more concentrated (10%)solution.23 In a 1% solution in pyridine, the composition is 58%of a-and 42% of p-furanose, and 3%of free alde-

-

(93) V. Bilik, L. PetruS, and J. Alfddi, Chern. Zoesti, 30 (1976) 698-702. (94) In this case and in all others where the composition is given by four figures, their order is the same as in the Tables, namely, a-pyranose, P-pyranose, a-furanose, Bfuranose.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

37

h ~ d eThe . ~ compositionQ5a ~ of an aqueous solution of D-erythrose at 36" is 25 :63 :-2 :- 10. Glyceraldehyde, the simplest sugar, consists mainly of the aldehydrol in dilute solution; in the crystalline state (DL),or syrupy state (D), it is dirneri~.~~

V. COMPOSITION IN AQUEOUS SOLUTION:KETOSES 1. Hexuloses and Pentuloses

The composition of aqueous solutions of all of the 2-hexuloses, of several 3-hexuloses, 2-pentuloses, and their deoxy derivatives, and some of the hexulosonic acids is shown in Table IV. The anomers of the aldopyranoses occur predominantly in the same chair form. Hence, the difference in their free energies is due solely to the anomeric hydroxyl group's being either axial or equatorial, and is therefore small. By contrast, the anomers of the ketopyranoses have different chair forms: in each one, the side chain is equatorial and the anomeric hydroxyl group is axial. Depending on the disposition of the other hydroxyl groups, the difference in the free energies varies widely. Thus, in a-D-xyb-hexulopyranose (a-D-sorbopyranose; 4), all hydroxyl

-H o

CH,OH OH 4

groups except the anomeric one are equatorial, but in t h e p anomer they would be axial. Hence, the p-pyranose is a very minor component of the equilibrium mixture. At the other extreme, both anomers (5 and 6) of ~-ribo-2-hexulopyranose(D-psicopyranose) have a syn-axial pair of hydroxyl groups; at equilibrium, they are found in about the same proportions. OH

5

6

(95) J. Thiem and H.-P. Wesse1,Justus Liebigs Ann. Chem., (1981) 2216-2227. (95a) A. S. Serianni, E. L. Clark, and R. Barker, Curbohydr. Res., 72 (1979) 79-91.

38

STEPHEN J. ANGYAL

The proportion of the acyclic form is considerably higher than that of the aldoses, but remains below the limit of detection by routine ‘H-n.m.r. spectroscopy. However, at a higher temperature (80”)and very high concentration (3.7 M ) , the signal of the keto form of fructose (3%) and sorbose (2%) is readily visible in the 13C-n.m.r. spectrum.20 When only a furanose ring can be formed, and the side chain thereon and the neighboring hydroxyl group are cis, the unfavorable conformation of the furanose ring causes the appearance of considerable proportions of the keto form (for example, 6-deoxy-~-sorboseand 6-O-methyl-~tagatose15). If the furanose ring is formed by involving a primary hydroxyl group, the situation becomes analogous to that encountered with erythrose and threose (see Section IV,4): considerable proportions of the keto form are found in the equilibrium mixture (for example, the 3-hexuloses and the pentuloses). The equilibrium mixtures of the l-deoxyhexuloses also contain a large proportion of the acyclic form, owing to the lack of an inductive effect from 0-1 (see Section 111,8):at 8 5 ” , 28% of 1-deoxy-D-fructose is in the keto form. This ketose presented the first instance in which signals for five forms of a sugar are visible in the 13C-n.m.r. spectrum: it contains 30 carbon signals, all of which were assigned.“j 1-Deoxy-D-tagatose was also reported to exist, to some extent, in the acyclic form in aqueous solution.g6l-Deoxy-~-threo-%pentulose, having only a primary hydroxyl group available for ring closure, and lacking a hydroxyl group on C-1, is preponderantly in the keto form.g7In none of these cases was the hydrated keto form detected. ~-threo-2,5-Hexodiulose(“5-keto-~-fructose”)is mainly (>95%) in the P-pyranose form (7) in aqueous solutiongs; furanose forms involving

HO

@

*OH

OH

HO 7

both keto groups appear to be less stable. The keto group not participating in the ring formation is hydrated, to form agem-diol; such hydration is favored in a six-membered ring. In anhydrous dimethyl sulfoxide, hydration not being possible, the sugar exists as a tricyclic dimer, of fused P-pyranose and P-furanose rings. When the two carbonyl groups are in 1,5, rather than in 1,4, relationship, the pyranose form involving both (96) W. L. Dills, Jr., and T. R. Covey, Curbohydr. Res., 89 (1981) 338-341. (97) H. Hoeksema and L. Baczynskyj,]. Antibiot., 29 (1976) 688-691. (98) J. Blanchard, C. F. Brewer, S. Englard, and G. Avigad, Biochemistry, 21 (1982) 75-81.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

39

carbonyl groups is favored. Thus, 6-acetamido-6-deoxy-~-xyZo-hexos-5ulose is preponderantly in the j?-pyranose form (8) which has a favorable configuration.gg At equilibrium, there are also small, approximately equal, proportions of the two furanose anomers (9);in these, the aldehyde, not the keto, group had formed the ring.

OH 8

0

Data for four disaccharides containing D-fructose are also included in Table IV, in order to illustrate the changes occurring on going from mono- to di-saccharides. The furanose content increases; this is in accord with the effect of partial 0-substitution. The increase is particularly great where the substituent for turanose, 3-O-a-~-glucopyranosyl-~-fructose, is on 0 - 3 (compare, 3-O-methyl-~-fructose,Section VI,1).Surprisingly, the proportion of the acyclic form also increases quite substantially. Apparently, the acyclic form is the only one that can accommodate the bulky sugar substituent without unfavorable interactions. Equilibrium data are available for four hexulosonic acids40(see Table IV). They are of two different types. The 2-hexulosonic acids are hexuloses in which the hydroxymethyl group on the anomeric carbon atom has been replaced by a carboxyl group. The latter, having a trigonal carbon atom, is conformationally less bulky than the former; hence, the disfavored pyranose and the disfavored furanose forms both become less disfavored, but there is no great change in the composition of an aqueous solution at equilibrium. The 5-hexulosonic acids are hexuloses in which the terminal hydroxymethyl group has been replaced by a carboxyl group. Pyranose forms are, therefore, not possible. If the carboxyl sidechain and the neighboring hydroxyl group are trans to each other, the two furanoses will account for practically all of the sugar (for example, the D - ~ ~ Xisomer, O 10); if they are cis, the furanoses are less stable, and there is a considerable proportion of the keto form present at equilibrium40 (for example, the xylo isomer).

(99) D. E. Kiely and L. Benzing-Nguyen, J . Org. Chern., 40 (1975) 2630-263.

STEPHEN J. ANGYAL

40

~-threo-2,5-Hexodiulosonic acid, having a keto group at both C-2 and C-5 is, like the corresponding diulose, preponderantly in the /3-pyranose form in solution, with the 5-keto group fully hydrated.loO 2. Heptuloses

The composition ofsolutions of the 2-heptuloses has been determined, and discussed, by Angyal and Tran.e2 These ketoses are different from other reducing sugars inasmuch as there are two hydroxymethyl side chains attached to the pyranose ring. In the a-pyranose form, they are cis to each other and will therefore both be equatorial in the preponderant chair form. In the /3-pyranose, however, one or other of the hydroxymethyl groups has to be axial and, in consequence, the /3 anomers are disfavored: in only one solution (that of the altro isomer) was the /3-pyranose detected in the W-n.m.r. spectrum.e2 If the a-pyranose form has no substantial, steric interactions, and the furanose forms are disfavored, the a-pyranose is the only form detectable at equilibrium. ~-gluco-2-Heptulose(11) and ~-manno-2-heptulose exhibit such behavior12 (see Table V). These sugars show no mutarotationlo’; the “equilibrium mixture” has only one detectable component. Similarly, the %-n.m.r. spectrum of a solution of l-deoxy-~-gluco-2heptulose shows the presence of only one form, the a-pyranose.102

H

O

w

c

H OH * o

H

11

On the other hand, if the a-pyranose form has substantial unfavorable interactions, the /3-pyranose being essentially unstable, the furanoses will become the major components of the equilibrium mixture (see Table V). Thus, over 75% of all molecules of D-altro-heptulose (sedoheptulose) and ~4do-2-heptulosein solution are in a furanose form at equilibrium at 22”; even more is present at higher temperatures. In contrast, the 3-heptuloses, several of which have been studiedlo3 (see Table V), are unexceptional. They are, essentially, 2-hexuloses hav(100) G . C. Andrews, B. E. Bacon, J. Bordner, and G . L. A. Hennessee, Carbohydr. Res., 77 (1979) 25-36; 80 (1980) ~ 2 8 . (101) F. B. LaForge,]. B i d . Chem., 28 (1917) 511-516,517-522; W. C. Austin,]. Am. Chem. SOC., 52 (1930) 2105-2112. (102) E. J. Hehre, C. F. Brewer, T. Uchiyama, P. Schlesselmann, and J. Lehmann, Biochemistry, 19 (1980) 3557-3564. (103) T. Okuda, S. Saito, M. Hayashi, N. Nagakura, andM. Sugiura, Chem.Phann. Bull., 24 (1976) 3226-3229.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

41

ing the side chain extended by one carbon atom. In solution, their equilibrium compositions are similar to those of the homomorphous hexdoses, but the furanose form in which the side chain and the vicinal hydroxyl group are trans is favored for the 3-heptuloses (see Section 111,2).Thus, altro-, gluco-, and ido-3-heptulose have a higher proportion of the a-furanose form in solution at equilibrium than have psicose, fructose, and sorbose, respectively. The high proportion of a-furanose (55%) in the solution of ~-ah-o-3-heptulose(“coriose”) is particularly noteworthy, because this is the only monosaccharide known to crystallize in a furanose form, although it is not prevented from forming pyranoses; the crystalline form is the a-furanose.2 ~-altro-2-Heptuloseand ~-ido-2-heptulose, whose solutions contain an even higher proportion of a furanose form, have never been crystallized. The 3-octuloses are homomorphs of the 2-heptuloses, with the side chain on the anomeric center extended by one carbon atom. It is not surprising, therefore, that ~-gZuco-~-glycero-3-octulose is mainly in the a-pyranose form (12), and ~-aZtro-~-glycero-3-octulose, mainlylo4in the p-furanose form (13). For the latter, the proportion of the jl-furanose is further increased, compared to that for aZtro-2-heptulose, by the increased bulk of the side chain. On acetylation, ~-gZuco-~-gZycero-3-octulose gives mainly the heptaacetate of the a-pyranose form; the altro isomer gives mainly the acetate of the jl-furanose form.lo4

voH Hos& C%OH

CH,OH

HOJH

A-

m,oH

HO OH

HCOH

HO

12

&,OH

13

Two biologically important ketoaldonic acids should be mentioned here. N-Acetylneuraminic acid (5-acetamido-3,5-dideoxy-~-glycero-~galacto-nonulosonic acid) is homomorphous with 3-deoxy-gZuco(and manno)-heptulose, and therefore, in solution, would be expected to be overwhelmingly in the “jl-D”-pyranose form (1 4). Actually, although the QH

C&OH 14

(104) E. Westerlund, Carbohydr. Res., 91 (1981) 21-30,

42

STEPHEN J. ANGYAL

“P-D”-pyranose is preponderant (93%), there is also a small proportion (7%) of the “a-D”-pyranose form at equilibrium in aqueous solution.105J06Apparently, removal of the gauche interaction with OH-3 makes the axial carboxyl group more acceptable. (The homomorph of the a-heptulose is designated P-Dby the IUPAC Tentative Rules, because this is in reference to C-8, which is outside the pyranose ring,lo7but, by the British -American Rules, it may be regarded as D-eythro-a-L-ambino, with the configuration of C-6 dictating the a-anomeric designation.) It is noteworthy, however, that, in Nature, N-acetylneuraminic acid is found only in the “a”-pyranoside form.lo8 (Methyl neuraminate exists almost solely in a five-membered ring-formloQ;see Section VI,2.) 3-Deoxy-~-manno-2-octulosonic acidloQa(“KDO”) is homomorphous with 3-deoxy-~-galacto(andtub)-heptulose; a preponderance of the apyranose form (15 ) , accompanied by substantial proportions of the two furanose forms, would therefore be expected. Actually, there is, again, a small proportion of the j3-pyranose form present. The composition found”O in a 0.72 M solution of the ammonium salt at 28”is 64 : 6 : 20 : 10 and, in a 0.18 M solution, 60: 11 : 20: 9. H HO/‘.CH,OH

HO

\

1s

VI. COMPOSITION IN AQUEOUS SOLUTION: SUBSTITUTED AND DERIVED SUGARS Whereas the composition of solutions of the unsubstituted aldoses and ketoses has been systematically investigated, very few studies have been conducted on substituted and on derived sugars, such as amino sugars, (105) H. Friebolin, P. Kunzelmann, M. Supp, R. Brossmer, G. Keilich, and D . Ziegler, Tetrahedron Lett., (1981) 1383-1386. (106) J. Haverkamp, L. Dorland, J. F. G. Vliegenthart, J. Montreuil, and R. Schauer, Abstr. Pup. Int. Symp. Curbohydr. Chem., Qth,London, (1978) 07. (107) W. Pigman and D. Horton, in idem, The Carbohydrates, Vol. l A , Academic Press, New York, 1972, p. 54. (108) J. Montreuil, Adu. Curbohydr. Chem. Biochern., 37 (1980) 157-223. (109) W. Gielen, 2.Physiol. Chem., 348 (1967) 329-333. (109a) F. M. Unger,Ado. Curbohydr. Chern. Biochem., 38 (1981) 323-388. (110) R. Cherniak, R. G. Jones, andD. S.Gupta, Curbohydr. Res., 75 (1979) 39-49; J. F. G. Vliegenthart, personal communication; P. McNicholas, personal communication.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

43

thio sugars, and branched-chain sugars. Accurate information on the composition of solutions of these sugars is seldom available; it is mostly incidental, authors having noted the ratios and proportions of relevant signals in their n.m.r. spectra. These data are often only approximate; in such instances, instead of giving the percentage composition, the ratios found by the authors (such as 2 : 1 or 1 : 3) will be cited. Instances have been encountered in the literature where authors recorded the chemical shifts of both anomers of the pyranose form, but did not indicate their ratio.

1. Partially 0-Substituted Sugars In 1966, Mackie and Perlin5' observed that, in solution, some 2,3-di0-substituted derivatives of sugars exist as furanoses to a greater extent than do their parent sugars; for example, 2,3-di-O-methyl-~-arabinose consists of 17%, 2,3-di-O-methyl-~-galactoseof lo%, and 2,3-di-0methyl-D-altrose of >45%, of the furanose forms at equilibrium in aqueous solution. In dimethyl sulfoxide, the proportion is even higher. Earlier, Bishop and Cooper''' had shown that partially methylated (at 0 - 2 , 0-3, or both) xylose and arabinose derivatives yield a higher proportion of methyl furanosides than do the parent sugars under equilibrating conditions. In particular, 2,3-di-O-methyl-~-arabinose yields 75%of the two furanosides, whereas L-arabinose yields only 28%.These authors suggested that, by increasing the effective size of the substituents on 0 - 2 and 0-3, methylation promotes relatively stronger interactions in the pyranosides than in the furanosides, in which the two trans-methoxyl groups are farther apart. In particular instances, the increased interactions can be clearly defined. Three examples will be considered. The equilibrium composition of 3-O-methyl-~-fructoseat 16.5" was found112 to be 18 : 37 : 11 : 34, compared to 2 : 70 : 5 : 23 for D-fructose at 30".In thep-pyranose, which (16),the methyl group has a 173-paralassumes the 2 C c , ( ~conformation )

PH HO

&-Me Me 16

(111) C. T. Bishop and F. P. Cooper, Can. J. Chem., 41 (1963) 2743-2758. (1 12) T. A. W. Koerner, Jr.,R. J. Voll, L. W. Cary, and E. S.Younathan, Biochem. Biophys. Res. Commun., 82 (1978) 1273-1278.

44

STEPHEN J. ANGYAL

lel interaction, no matter which rotameric form it assumes, with 0 - 2 , 0 - 4 , or C-1 (all three forms are shown in the formula); the stability of the a-pyranose form is thereby lessened. The a-pyranose form exists, to a considerable extent, in the 5C, form,113in which the methyl group can assume an orientation free from such interaction. In the furanose forms, introduction of the methyl group causes some increase in gauche interactions, more in the P than in the a anomer. The outcome of partial methylation is, therefore: less P-pyranose, slightly less P-furanose, and much more a-pyranose and a-furanose. The composition of a solution of 3-O-methyl-~-psicose'~ at 27" is 31 : 7 : 56 : 6, whereas that of D-psicose is 22 :24 :39 : 15.In this case, the methoxyl group in the P-pyranose form (17) is axial, and 1,3-parallel interaction with one of its equatorial neighbors cannot be avoided. There is a similar interaction in the P-furanose form, too, but not in the a anomers. OH

17

There is also an axial methoxyl group flanked by two equatorial hydroxyl groups in both of the pyranose forms of 3-O-methyl-~-allose.The pyranose forms are thereby destabilized, and, in solution, the proportions of the furanose forms are more than doubled,88to give the composition 1 4 : 6 5 : 7 . 5 : 1 3 . 5 a t 3 1 ° . The examples studied by Mackie and Perlin57 are not so clear-cut as those just discussed. There are no 1,3-parallel interactions in the pyranose forms of 2,3-di-O-methyl-~-arabinose or +galactose, but the gauche interactions in these molecules would have been increased by methylation. In 2,3-di-O-methyl-~-altrose,the two methoxyl groups are axial, and, therefore, their interactions with axial hydrogen atoms would be greater than those in the parent sugar. In these examples, the methoxyl groups are trans in the furanose forms, presenting no additional interactions with each other. In the other examples (2,3-di-O-methyl-~glucose, +-mannose, and -D-xylose),furanoses were not observed,57but the proportions of the furanose forms of the parent sugars are so low that even a five-fold increase of the furanose content would have escaped detection. It is, therefore, not known whether methylation of 0 - 2 and 0-3 would increase the proportions of furanose forms in sugars having (113) S. J. Angyal and Y. Kondo, Carbohydr. Res., 81 (1980)35-48.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

45

cis-hydroxyl groups on C-2 and C-3. A report114 that 3-O-methyl-~-glucose and 3-O-methyl-~-xylose exist to a considerable extent in furanose forms proved to be in error,115due to a wrong interpretation ofthe n.m.r. spectra. Mackie and Perlin5' also noted that the a :/.?pyranose ratio increases with the extent of methylation of hydroxyl groups; for example, the equilibrium aqueous solution of 2-O-methyl-~-mannosecontains 75% of the a-pyranose form (D-mannose, 65.5%), that of 2,3-di-O-methyl-~mannose, 80%, and that of 2,3,4,6-tetra-O-methyl-~-mannose, 86%. This seems to be a general phenomenon, probably caused by an increase of the anomeric effect owing to a decrease of the effective dielectric ~ o n s t a n t Other .~ examples are: 2-O-methyl-~-glucose,~~~ 55%; 3-0rnethyl-~-glucose,"~39%; 3,4,6-tri-O-methyl-~-glucose (determined from the optical rotation115a),71%; 2-O-methyl-~-rhamnose,'~~~ 79%; 4-O-methyl-~~-lyxose,~~~~ 73%; and 3-O-rnethyl-~-xylose,"~38%. A similar increase in the proportion of the a-pyranose form occurs on replacing a hydroxyl group by a fluorine atom: in 2-, 3-, 4-, and 6-deoxy2-, 3-, 4-, and 6-fluoro-~-glucose,it lies between 4 1 and 47% (as determined by 19F-n.m.r. spectroscopy). 115d The solution composition of 3deoxy-3-fluoro-~-mannoseis 68% of a- and 32% of J3-pyrano~e."~" When OH-5in aldopentoses and higher aldoses is blocked by substitution, pyranose forms are not possible; the proportion of the a-andp-furanose forms, and, therefore, their relative stability, can then be observed for such sugars as glucose and mannose, where the proportion of furanose forms is very small in solutions of the unsubstituted sugars. Examples 69% of a-furanose and of such sugars are 5-O-methyl-~-mannose,~~~ 31% of p-furanose; 5-O-rnethyl-~-glucose,~~~ 51 : 49; 5,6-di-O-methyl~ - g l u c o s e , ~45 ~ : 55; 5,6-O-isopropy~idene-~-glucose,~~~ 47 :53; 5-O-methyl-~-xylose,"~57 : 43; 5-O-methyl-~-arabinose,"~60 :40; (114) P. J. Garegg, B. Lindberg, and C. G. Swahn, Acta C h m . Scund., Ser. B, 29 (1975) 631-632. (115) B. Lindberg, personal communication, 15 Sept. 1978. (115a) R. L. Sundberg, C. M. McCloskey, D. E. Rees, and G. H. Coleman, ]. Am. Chem. SOC., 67 (1945) 1080-1084. (115b) A. Liptak, Carbohydr.Res., 107 (1982) 300-302; and personal communication. (1 15c) S. M. Srivastava and R. K. Brown, Can. ]. Chrn., 49 (1971) 1339- 1342. (115d) L. Phillips andV. Wray,]. Chem. SOC., B, (1971) 1618-1624; E. M. Bessel, A. B. Foster, J. H. Westwood, L. D. Hall, andR. N. Johnson, Carbohydr. Res., 19 (1971) 39-48; A. D. Barford, A. B. Foster, J. H. Westwood, L. D. Hall, andR. N. Johnson, ibid., 19 (1971) 49-61. (115e) M. Cerny, J , DoleZalova, J. Macovh, J. Pacak, T. Trnka, and M. BudBSinsky, Collect. Czech. Chem. Commun., 48 (1983) 2693-2700. (116) S . J. Angyal and M. H. Randall, unpublished results. (117) K. Horitsu and P. A. J. Gorin, Agric. Biol. Chem., 41 (1977) 1459-1463.

STEPHEN J. ANGYAL

46

2,3,5-tri-O-methyl-~-arabinose,"~ 72 : 28; and 5-O-methyl-~-ribose,~ -28:72. In the absence of pyranose forms, the acyclic forms have to compete, in solution, only with the (much less stable) furanose forms, and should therefore be present in much higher proportion than in solutions of glucose and mannose. 5-O-Methyl-~-glucoseand -mannose, and 5,6-di0-methyl-D-glucose give a red color with the Schiff reagent, in contrast to the unsubstituted sugars,l16 which do not. (6-O-methyL~The composition of 6-O-methyl-~-~yro-2-hexulose tagatose) in aqueous solution at 35" is 21% of a-furanose, 69% ofp-furanose, and 10% of the keto form.I5 The ratio of a-to p-furanose for 6-0methyl-D-psicose was found to be - 2.4 : 1; that of a-to p pyranose for 5-O-methyl-~-psicose, 1.25 : 1;the proportion of the keto form was not determined.I3 Because of their biological importance, the compositions of several sugar phosphates have been determined,4 and were found to be in accord with expectations. Thus, in solution, D-fructose 6-phosphate at 6" existse2as 16% of a-furanose, 82% of B-furanose, and 2.2% of the keto form; for D-fructose 1,6-bisphosphate, the values are 13 : 86 : 0.9. The a :p furanose ratios of the four hexulose 6-phosphates at 16.5 were found to be: D-fructose, 19 :81; D-psicose, 76 : 24; D-tagatose, 1 7 : 83; and L-sorbose, 82 : 18; the proportion of the keto forms was not determined.6gpThe composition of several bisphosphates was also determined117"and was found to be: for D-altro-heptulose 1,7-bisphosphate: 13%of a-pyranose, 13% of a-furanose, 74% ofp-furanose; for D-glyceroD-altro-octulose 1,8-bisphosphate: 7 : 19 : 74; and for D-glycero-D-idooctulose 1,8-bisphosphate: 1 9 : 1 4 : 67. A 0.04 M aqueous solution of D-erythrose 4-phosphate contains 7% of the aldehydo form and 93% of its hydrate4; at 1.O M concentration, however, substantial proportions of three dimeric forms are also present.'le The composition of the four to be: arabinose, 57% of a-, pentose 5-phosphates at 6" was founde0a*e2 40% of p-furanose, 2.2% of aldehydrol, and 0.2% of aldehyde; ribose, 36,64, 0.5,O.l; xylose, 53, 42, 4.7,0.3; and lyxose, 70, 25, 4.3, 0.4%.

-

O

2. Amino Sugars

Replacement of a hydroxyl by an amino group may cause profound changes in the composition of a solution of a sugar. The extent of the change depends on whether the amino group is free, protonated, or acylated; and, even more, on which hydroxyl group has been thus re(117a) F. P. Franke, M. Kapuscinski, J. K. MacLeod, and J. F. Williams, Carbohydr. Res., 125 (1984) 177-184. (118) C . C. Duke, J. K. MacLeod, and J. F. Williams, Carbohydr. Res., 95 (1981) 1-26.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

47

placed. Data on the composition of amino sugars in solution are scarce, but are sufficient to illustrate the variations possible. a. 2-Amino Sugars. -The composition of 2-amino-2-deoxy-~-glucose in aqueous solution is 36% of a-and 64% 0f/3-pyranose"~; that is, it is similar to that of D-glucose. However, the N-acetyl derivative and the hydrochloride of this sugar exist in solution preponderantly as the a anomerlZ0(see Table VI);it appears that the anomeric effect is increased by N-acetylation or protonation. 2-Amino-2-deoxy-~-ga~actose shows similar behavior, but the N-acetyl derivative and the hydrochloride of 2-amino-2-deoxy-~-mannose contains less of the a-pyranose form than does D-mannose (see Table VI). Horton and coworkerslZ0 concluded from these data that the acetamido or ammonium group on C-2 exerts a stabilizing effect on a cis-related hydroxyl group at C-l.2-Acetamido-2deoxy-D-allose and -gulose, however, show a slight increase only of the a-pyranose, and also an increase of the furanose forms, compared to the parent sugars.120*Thefactors affecting the equilibria of these compounds are not clearly understood. Somewhat puzzling is the report that, in aqueous solution, only 13% of 2-amino-2,4-dideoxy-~-lyxo-hexose ("4-deoxymannosamine") hydrocholoride is in the a-pyranose form. lZob This behavior, differing so much from that of the parent compound, is not caused by the presence of different conformations: the 'H-n.m.r. spectra show that both pyranoses are in the 4 C 1 ( ~conformation. ) Methylation of an amino sugar causes the usual increase (see Section VI,1) in the proportion of the a-pyranose: 90% of 2-deoxy-3,4,6-tri-Omethyl-2-(methylamino)-~-glucose hydrochloride is present at equilibrium as the a-pyranose.120c Two groups of workers121J22have, independently, performed calculations of the free energies of these molecules, using semi-empirical, potential functions. The calculated compositions agreed well with those found experimentally. The change of composition on acetylation, and on protonation, of the amino group appears to be caused by electrostatic interactions.

-

(119) A. Neuberger and A. P. Fletcher,J. Chem. SOC.,B, (1969) 178-181. (120) D. Horton, J. S.Jewell, and K. D. Philips,J. Org. Chem., 31 (1966) 4022-4025. (120a) H. Okumura, I. Azuma, M. Kiso, and A. Hasegawa, Curbohydr. Res., 117 (1983) 298-303. (120b) I. Cerny, T. Trnka, and M. Cerny, Collect. Czech. Chem. Commun., 48 (1983) 2386-2394. (120c) C. R. Hall, T. D. Inch, C. Pottage, N. E. Williams, M. M. Campbell, andP. F. Kerr, J. Chem. Soc., Perkin Trans. I , (1983) 1967-1975. (121) T. Taga and K. Osaki, Bull. Chem. S o c . J p . , 48 (1975) 3250-3254. (122) R. Virudachalam and V. S. R. Rao, Curbohydr. Rex, 51 (1976) 135-139.

48

STEPHEN J. ANGYAL

b. 3-Amino Hexoses. -The only instance of a systematic investigation of the composition of diastereoisomeric amino sugars is presented by the work of Fronza and ~oworkers'~3 on the N-benzoyl derivatives of the 3-amino-2,3,6-trideoxy-~-hexoses, sugars important in the chemistry of antibiotics. The results, shown in Table VI,should be compared with the data for the 2-deoxyhexoses in Table 11. When the benzamido group is equatorial, as it is in the L-urubino (18) and the L - Z ~ X O (19) isomers, the

qcwoH

2w0"

HO

NHBz

HO NHBz

18

19

composition is similar, except for a greater proportion of the a-pyranose form, the data on the amino sugars having been obtained in dimethyl sulfoxide, and not in deuterium oxide (see Section VII). Because the steric interactions of an axial benzamido group are greater than those of an axial hydroxyl group, the L-ribo isomer (20) contains a much larger proportion of furanose forms at equilibrium than that found in solutions of 2-deoxy-ribo-hexose. The L - X ~ Z O isomer (21) also possesses an axial benzamido group, but the furanose forms would have an unfavorable, cis interaction between the side chain and the benzamido group, and are therefore not found in substantial proportions.

HON

O

H

pyoH

€I6 20

21

c. 4-Amino and 5-Amino Sugars.-These compounds can form hemiacetal rings containing a nitrogen atom; their complicated behavior has already been fully discussed in this Series,lz4and will be only briefly summarized here. The amino group is more nucleophilic than the hydroxyl group, and has, therefore, a greater tendency to react with the anomeric center. In solution, 5-amino-5-deoxyhexoses and 6-amino-6-deoxyhexulosesare present completely in six-membered ring-forms, containing the nitrogen atom in the ring; 4-amino-4-deoxyaldoses are, to a considerable extent, (123) G . Fronza, C. Fuganti, and P. Grasselli, J. Chern. SOC., Perkin Trans. 1, (1982) 885 - 891. (124) H. Paulsen and K. Todt, Adv. Carbohydr. Chern., 23 (1968) 115-232.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

49

present in five-membered ring forms. These pyranose and furanose forms are in equilibrium with dehydration products (which are Schiff bases), and dimeric forms. The six-membered rings, particularly those of the aminoaldopentoses, are very reactive and aromatize readily; true equilibrium between the anomeric forms has been observed in only a few (nojirimycin) is comparatively cases. Thus, 5-amino-5-deoxy-~-glucose stable, and forms a 63 : 37 mixture of the a- andp-pyranose forms.125The anomeric effect therefore becomes greater, as expected, when the oxygen atom in the ring is replaced by the (less electronegative) nitrogen atom. Methyl neuraminate (methyl 5-arnin0-3,5-dideoxy-~-glt~cero-~galacto-nonulosonate) exists, in solution, almost solely in the Schiff-base form, with a five-membered ring.11Q4-Amino-4-deoxy-~-glucoseand -D-galactose exist in aqueous solution mainly as dimers of the furanose form. The protonated amino group, however, has no nucleophilic activity, and does not form a hemiacetal; an amino sugar can, therefore, be obtained, as a salt, in an otherwise unfavorable form. For example, 6amino-6-deoxy-~-sorbosecan be isolated as the hydrochloride of a furanose form (22) (presumably a).In alkaline solution, immediate ring-expansion to the pyranose form (23) occurs127;this reaction can be reversed under strongly acidic conditions. 4-Amino-4,6-dideoxy-~-glucosehydrochloride forms a 35 : 65 mixture of the a- and P-pyranose forms in solution.12*

C1- H$-

C

CH,OH

Ho&CH20H

OH

H* HO 22

23

The acylamido group has little nucleophilic character, and it is found that a ring containing an acylimino group is formed only under particularly favorable conditions. Formation and opening of such a ring are very slow, and equilibration of furanose and pyranose forms occurs only on heating, or in the presence of acids.lZ7The anomeric composition of the pyranoses is profoundly altered by the introduction of an acylimino group into the ring. 5-Acetamido-5-deoxy-~-xylose shows no mutarotation, and exists as the a-pyranose form in solution.12Q5-(BenzyloxycarB. M. Pinto and S . Wolfe, Tetrahedron Lett., (1982) 3687-3690. H. Paulsen, K. Steinert, and K. Heyns, Chem. Ber., 103 (1970) 1599-1620. H. Paulsen, I. Sangster, and K. Heyns, Chem. Ber., 100 (1967) 802-815. C. L. Stevens, P. Blumberger, F. A. Daniher, D. H. Otterach, and K. G. Taylor, 1.Org. Chem., 31 (1966) 2822-2829. (129) H. Paulsen and F. Leupold, Carbohydr. Res., 3 (1966) 47-57. (125) (126) (127) (128)

STEPHEN J. ANGYAL

50

bonyl)amino-5-deoxy-~-arabinose shows a strong tendency to assume only the P-pyranose form in solution. In such compounds, the N-acyl group is in a position eclipsed with a neighboring, equatorial substituent, and this thereby d e s t a b i l i ~ e sthat ' ~ ~ pyranose anomer which carries an equatorial substituent on C-1. This phenomenon has also been interpreted as an increase in the anomeric effect. Presumably, the compounds described next also assume only one of the pyranose forms, the one that has an axial, anomeric hydroxyl group. However, 5-(benzyloxycarbonyl)amino-5-deoxy-~-ribose exists in solution as a mixture (- 1 : 2) of the a- and P-pyranoses that can be separated by column chromatography. In this case, both pyranoses seem to have axial anomeric hydroxyl groups; it was shownl3O that the acetate of the P-pyranose assumes the 'C,(D) conformation (24), whereas that of the a anomer is in the 4 C 1 ( ~ ) conformation (25). 6-Acetamido-6-deoxy-~-fructose and -L-sorbose e ~ i s t ~ ~in' .the '~~ furanose forms in solution; in such ketoses, both the CYand thep-pyranose forms would have strong vicinal interactions with the N-acetyl group. PhCH,O I

OCH,Ph

6 v

AcO

OAc

24

I

OAc

Aco*oAc

AcO 25

The position of the pyranose - furanose equilibria in solution has been the proportion determined for the four 5-acetamido-5-deoxypentoses: of the pyranose form (which contains the nitrogen atom in the ring) is 65% for the xylo, 50% for the lyxo, 25% for the arabino, and 10%for the ribo isomer,124and this is the order found for the parent pentoses. The corresponding 5-(benzyloxycarbonyl)amino-5-deoxypentoses, however, exist in solution almost exclusively in the pyranose form,'30 reflecting the diminished extent of deactivation of the amide nitrogen atom; but a solution of 5-(benzyloxycarbonyl)amino-5,6-dideoxy-3-0mesyl-L-idose was found to contain 20%of the furanose forms, because the steric effect of the N-acyl group forces it into the particularly unfavorable 4C1(~) conformation (26) of the p-pyranose form.132 The 3,5-diacetamido-3,5-dideoxypentoses favor the pyranose form somewhat more than their 3-hydroxy analogs: in solution, the proportion of the pyranose form is -60-70% for the xylo, 80-90% for the Zyxo,

-

(130) H. Paulsen and F. Leupold, Chern. Bw., 102 (1969) 2804-2821. (131) J. C. Turner, Can.]. Chem., 40 (1962) 826-828. (132) H.Paulsen and M. Friedmann, Chm. Ber., 105 (1972) 731-734.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

51

OCH,Ph

&o I

OH 24

20 - 30% for the arubino, and 15- 20%for the ribo i ~ 0 r n e r . The I ~ ~ furanose forms would have increased gauche interactions, compared to the 3-hydroxy analogs, between substituents on C-2, C-3, and C-4,particularly in the lyxo isomer, where they are all cis. In contrast to the behavior of the hydroxyl group, a secondary acylamido group has less tendency than a primary one to form a cyclic hemiacetal; this is presumably caused by the gauche interaction of the N-acyl group with the side chain on a ring formed by a secondary acylamido group. Thus, in solution, most 5-acetamido-5-deoxyaldohexoses(secondary NAc) are overwhelmingly in furanose forms,134and the 4-acetamido-4-deoxyaldotetroses(primary NAc) are cyclic, the carbonyl form not appearing in appreciable proportion^.'^^ Nevertheless, 4-acetamido-4,5-dideoxy-~-xylose (secondary NAc) contains 4% of the acyclic form in its equilibrium mixture,13s besides the two furanose forms in about equal proportions.

-

d. 6-Amino Hexoses. -Despite the great nucleophilicity of the amino group, the 6-amino-6-deoxyhexoses, in solution, do not form substantial proportions of septanoses. 6-Amino-6-deoxy-~-mannose,for example, exists in solution as a mixture of almost equal proportions of the a-and P-pyranose forms.I3' However, the nucleophilicity is shown by the fact that, in alkaline solution, 6-amino-6-deoxy-~-idoseis converted spontaneously, and almost completely, into134the (all-equatorial) 1,6-anhydropyranose (27). In contrast to the corresponding reaction of the hexoses, anhydride formation involving the amino group requires no heating and no acid. When two amino groups are present on suitable carbon atoms, the drive to form such bicyclic derivatives is very strong. Even 5,6-diamino-5,6dideoxy-D-glucose in solution is in equilibrium with 20-30% of the 1,6-anhydride (28), which has anitrogen atom in both rings, although the (133) J. S. Brimacombe and A. M. Mofti,]. Chem. SOC.,C, (1971) 1634-1638; Carbohydr. Rex, 16 (1971) 167-176. (134) H. Paulsen and K. Todt, Chem. Ber., 99 (1966) 3450-3460. (135) W. A. Szarek and J. K. N. Jones, Can. J. Chem., 43 (1965) 2345-2356. (136) S. Hanessian, Carbohydr. Res., 1 (1965) 178-180. (137) D. Horton and A. E. Luetzow, Carbohydr. Res., 7 (1968) 101-105.

STEPHEN J. ANGYAL

52

conformation of this anhydride is very unfavorable. 134 The corresponding idose derivative is spontaneously and completely converted into its 1,6-anhydride. Interestingly, 4,6-diamino-4,6-dideoxy-~-galactose, which is mainly in the /?-pyranose form as its hydrochloride, is converted into the 1,6-anhydrofuranose (29) in alkaline solution138; again, both rings contain a nitrogen atom. H

HO

I-

27

OI 20

29

3. Thio Sugars Sugars having a sulfur atom in the ring have attracted considerable interest in the past decade. Their chemistry has been discussed by Paulsen and Todt in this Series,124but subsequent developments justify brief mention here. Because the thiol group is more nucleophilic than the amino group, it is to be expected that the thio sugars will show an even greater tendency to assume those forms which have the hetero-atom in the ring. Thus, 5thioaldoses are found only in the pyranose forms, the proportion of furanoses being negligible.124J395-Thio-~-glucosein solution contains 85%of the LY- and 15% of the /?-pyranose form140;the anomeric effect is, therefore, considerably greater than for D-glucose, a result not unexpected, sulfur being less electronegative than oxygen. (There is also 0.005% of the aldehydo form present, as determined by circular d i c h r o i ~ m .It~ has ~ ) been suggested140athat the internal strain due to the replacement of oxygen by sulfur is greater in the /? anomer, and may therefore contribute to the increased preference for the a anomers of these thio sugars. 5-Thio-~-xylose,similarly, contains -85% of the aand 15% of the /?-pyranose in aqueous solution.140a 2-Acetamido-2deoxy-5-thio-~-glucose and -galactose exist in solution almost completely as the a-pyranose; they show no mutarotation, and the signals of (138) H. Paulsen, G. Landsky, andH. Koebernick, Chem. Ber., 111 (1978) 3699-3704. (139) C. E. Grirnshaw,R. L. Whistler, and W. W. Cleland,]. Am. Chem. Soc., 101 (1979) 1521-1532. (140) J. B. Larnbert and S . M. Wharry,]. Org. Chem., 46 (1981) 3193-3196.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

53

only the a-pyranose form were detected in their W-n.m.r. s p e ~ t r a . ' ~ ~ ~ * ~ The composition of a solution of 6-thio-~-fructose'~~ is 15% of a-pyranose, 85% of /.?-pyranose, 0.11% of a-furanose, 0.6% of /I-furanose, and 0.02% of the acyclic form at 25"; the last three figures were calculated from the rate constants for ring-opening and -closing.'3Q In solution, 5-thio-~-riboseconsists of 39%of the a-and 61% of the/.?-pyranose form at 24", as calculated from the optical r0tati0ns.l~~ The extent to which the 4-thioaldoses are in the furanose forms is not certain. It has been claimed that 4-thio-~-ribose'~3 and 4-thio-~-gluare completely in furanose forms, because they show no thiol absorption at 2550 cm-' in their i.r. spectrum. This evidence is not convincing: in the syrupy state, the composition may be different from that in dilute solution. The W-n.m.r. spectrum of a solution of 5 - t h i o - ~ fructose, however, shows141only the signals of the two furanose forms, in the ratio of 11: 89, at 25". The 'H-n.m.r. spectrum of a solution of 6-deoxy-4-thio-~-idosedisplays the signals of one furanose form only, , ~that ~ ~ of 6-deoxy-4-thio-~-gulose presumable that of the /I a n ~ m e rbut shows14esignals of the a- and 8-pyranose, and of one furanose form (presumably /.?) in the ratios of 1: 2 : 1; furanose forms having the gulo configuration are, of course, rather unstable. The 'H-n.m.r. spectrum of 6-deoxy-4-thio-~-altrosedoes not lend itself to easy interpretation, but it appears that there are at least three forms present.145 Whistler and cow o r k e r calculated ~ ~ ~ ~ the proportion of forms that have a free thiol group from the rates of mutarotation and ring closing, and confirmed their results by comparing them with the extent of the initial reaction obtained with 4,4'-dipyridyl disulfide, a reagent for free thiol groups. They found 2 - 3.5% of free thiol (presumably pyranoses) in the solution of 4-thio-~xylose, but less than 0.5% in those of 4-thio-~-arabinose,4-thio-~-ribose, and 5-thio-~-fructose. (140a) J. B. Lambert and S . M. Wharry, Curbohydr. Res., 115 (1983) 33-40. (140b) E. Tanahashi, M. Kiso, and A. Hasegawa, Carbohydr. Res., 115 (1983) 33-40; and A. Hasegawa, personal communication; A. Hasegawa, E. Tanahashi, Y.Hioki, and M. Kiso, Carbohydr. Res., 122 (1983) 168-173. (140c) E. Tanahashi, M. Kiso, andA. Hasegawa,]. Curbohydr. Chem.,2 (1983) 129-137. (141) M. Chmielewski and R. L. Whistler, Curbohydr. Res., 69 (1979) 259-263. (142) C. J. Clayton and N. H. Hughes, Carbohydr.Res., 4 (1967) 32-41. (143) E. J. Reist, D. E. Gueffroy, andL. Goodman,]. Am. Chem. Soc., 86 (1964) 56585663. (144) L. Vegh and E. Hardegger, Helo. Chim. Actu, 56 (1973) 2020-2025. (145) B. Gross and F.-X.Oriez, Curbohydr. Res., 36 (1974) 385-391. (146) R.-A. Boigegrain and B. Gross, Curbohydr. Res., 41 (1975) 135-142. The authors did not assign the anomeric signals, but the chemical shifts and coupling constants are similar to those of the corresponding forms of ~ - g u l o s e . ~

54

STEPHEN J. ANGYAL

Both the a-and the /?-pyranose forms of 1-thio-D-glucose have been crystallized as sodium ~a1ts.I~' The free thio sugars mutarotate very slowly, and are unstable; the a :/?ratio(23 : 77) obtained from the optical rotation is, therefore, somewhat uncertain, but nevertheless shows that the anomeric effect of the thiol group, as expected, is somewhat less than that of a hydroxyl group. In this compound, of course, the sulfur atom is not in the ring. Theoretical calculations on methyl thioglycosides confirmed148that the anomeric effect is smaller than in the oxygen analogs. 4. Branched-chain Sugars

Until the discovery of antibiotic substances, apiose and hamamelose were the only branched-chain sugars that had been found in Nature. The composition of both sugars in aqueous solution has been determined, and it is typical of the effect of branching on the stability of pyranose and furanose forms. Hamamelose is 2-C-(hydroxymethyl)-~-ribose.The bulky branch forces the pyranose forms to exist mainly in the 'C,(D) conformation (30), ,OH

OH 30

and thereby lessens their stability, compared with that of the ribopyranoses, for which the 4C, form is more favorable. The furanoses therefore preponderate; the a-furanose form is least affected by the introduction of the branch, and, therefore, shows the greatest increase in proportion, compared to ribose. The composition of a solution of hamamelose was reported14Qto be 14.5 : 13.5 : 38 : 34 (+ 3), and, more accurately,14Qa 1 2 : 2 1 : 3 8 : 2 9 (+1) at 23". The branched-chain pentose apiose has the peculiar property that it can form two a-furanoses and two /3-furanoses, as it has two hydroxyl groups in the y-position relative to the aldehyde group. Those forms, which are 3-C-(hydroxymethyl)-~-erythrofuranoses(31), are more stable than the 3-C-(hydroxymethyl)-~-threofuranoses (32), because, in the former, OH-2 and the hydroxymethyl group are trans. The composition of apiose in aqueous solution at 31" was found to be 22% of (Y-D(147) (148) (149) (149a)

W. Schneider and H. Leonhardt, Ber., 62 (1929) 1384-1389. S.Vishveshwara and V. S.R. Rao, Carbohydr. Res., 104 (1982) 21 -32. G. Schilling and A. Keller,Justus Liebigs Ann. Chem., (1977) 1475-1479. W. A. Szarek, B. M. Pinto, andT. B. Grindley, Can.]. Chem., 61 (1983) 461-469.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

31

55

32

erythro-, 54% of P-D-erythro-, 9% of a-L-threo-, and 15% of P-L-threofuranose derivatives. 150 Among the many branched-chain sugars isolated from, or synthesized during the search for, antibiotics, there are others that share the peculiar property of apiose. Dihydrostreptose [3-C-(hydroxymethyl)-5-deoxy-~lyxose] can also form four furanoses, but, in the equilibrium mixture, only those two were found151in which ring closure involves the secondary hydroxyl group at C-5 (33), in the ratio of 74% of (Y to 26% ofp: Ring closure through the primary hydroxyl group of the branch would be less f a v ~ r a b l e . ~For ’ streptose (34), the ratio is 79 : 21.

HO

OH

33 R = CH,OH 34 R = CHO

“y-Octose,” a hydroxyethyl-branched octose that is found as a component of isoquinocycline A, can form four pyranoses (besides two furanoses), because it has two hydroxyl groups in the bposition relative to the aldehyde group. Methyl glycosides (35 and 36) of two of the pyranose forms have been synthesized by Paulsen and S i n n ~ e 1 l . (These l~~ reactions were conducted in the series enantiomeric with “y-octose,” and are thus shown in the formulas.) Hydrolysis under very mild conditions, with 0.5% aqueous trifluoroacetic acid for 18h a t ambient temperature, gave the same anhydro sugar (37) from both glycosides. Apparently, internal attack by the exocyclic hydroxyl group, to yield the unstrained and unhindered anhydride, is more favorable than attack by water. Presumably, the free sugar is also present in the reaction mixture, (150)S.J. Angyd, C. L. Bodkin, J. A. Mills, and P.M. Pojer, Aust. J . Chern., 30 (1977) 1259- 1268. (151)J. R.Dyer, W. E. McGonigal, and K. C. Rice, J . Am. Chem. SOC., 87 (1965)654655.The authors’ tentative anomeric designations have been reversed on the basis of comparison with the n.m.r. spectra of lyxosee and the methyl dihydrostreptosides.’52 (152)S.Umezawa, H.Sano, andT. Tsuchiya, Bull. Chern. S o c . J p . ,48 (1975)556-559. (153)H.Paulsen and V. Sinnwell, Chern. Ber., 111 (1978)869-878.

STEPHEN J. ANGYAL

56

HCOH

ad 85

37

36

but it was not isolated. The two methyl glycosides isomeric with 35 and 36 at the exocyclic carbon atom were also synthesized; on hydrolysis, they did not yield any anhydrides. In those anhydrides, there would have been a 1¶llel interaction between the secondary hydroxyl group and a methyl group. The proliferation of antibiotic^'^^ has resulted in the isolation and synthesis of a great number and variety of branched-chain sugars. The structures and configurations of these were mostly determined by n.m.r. spectroscopy, but, to avoid the complication arising from multiple signals in the spectra of the free sugars, the spectra were recorded for such derivatives as glycosides and acetates. In most cases, the spectrum of the free sugar was not even recorded; even if it was, only signals of the preponderant form were described. Thus, it was stated that, on the basis of its n.m.r. spectrum, evermicose (2,6-dideoxy-3-C-methyl-~-urubinohexose) exists in solution as the P-pyranose form155(38); this was to be

H,C

38

expected as the a-pyranose would have syn-axial methyl and hydroxyl groups. However, the presence of an axial methyl group considerably increases the free energy of the pyranose forms, and therefore, the presence of substantial proportions of the furanose forms would also be expected. Their presence was indicated by the fact that evermicose shows mutarotation, but the published spectral datalg5gave no indication of the presence of the minor forms. Under these circumstances, only three branched-chain sugars, apart (154)H.Grisebach andR. Schmid, Angew. Chem., Int. Ed. Engl., 11 (1972)159-173; S. Umezawa, Ado. Carbohydr. Chem. Biochem., 30 (1974)111-182. (155)I. Dyong andD. Glittenberg, Chem. Ber., 110 (1977)2721-2728.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

57

from those already discussed, have been found for which the composition in aqueous solution has been determined. For 3-deoxy-3-C-nitromethyl-D-allose at 30 the composition, determined from the 'H-n.m.r. spectrum, was givenlSs as a-pyranose, a-furanose, and P-furanose in the ratios 5 : 16 : 79. In the pyranose forms (39), the axial hydroxyl group on O,

CH,NO,

39

C-3 of D-allose has been replaced by a bulkier group; hence, the proportion ofthe furanose forms has become much greater. Undoubtedly, there would also b e a substantial proportion (>20%)of the P-pyranose form present; its signal is, apparently, hidden under the (fairly large) HDO signal. In 1966, the composition of a solution of 6-deoxy-5-C-methyl-~-xylohexose was founde1 to be 8% of a- and 92% of P-pyranose at 40". A subsequent, 300-MHz, 'H-n.m.r. ~ p e c t r u m ' ~ gave a slightly different ratio and the furanose forms were also detected, the composition being 3 :9 5 : 0.8 : 0.8 at 35". Because branching occurs on C-5, one or other of the methyl groups in the pyranose form must be axial, no matter which chair form is assumed. In the P-pyranose (40), the axial methyl group provides the only unfavorable interaction, but in the a anomer, there is syn-axial interaction between the methyl group and OH-1; hence, the a anomer is much the less stable. The axial methyl group lowers the stability of the pyranoses, compared to those of glucose; but the furanoses (42) do not become the major forms, because their stability is also lessened by the cis interaction between OH-4 and the bulky, branched side chain. The closely related 5-C-methyl-~-idose(41 and 43) has the R

I

HsC-COH 1

HO 40R=CH, 4 1 R = CH,OH

42 R = CH, 43 R = CH,OH

(156)W.A.Szarek, J. S.Jewell, I. Szczerek, and J. K.N. Jones, Can.J. Chem.,47 (1969) 4473-4481.

STEPHEN J. ANGYAL

58

solution c o m p o ~ i t i o n 'of ~ ~82.5 : 9.5 : 4:4 at 40" (the a-forms of this compound are homomorphous with the p-forms of 6-deoxy-5-C-methylD-xyb-hexose). The larger proportion of the furanose forms shows that the pyranoses have been further destabilized by replacement of the axial methyl group by an axial hydroxymethyl group. Noviose (6-deoxy-5-C-methyl-4-O-methyl-~-lyxo-hexose) differs from the preceding compounds only in its configuration at C-2; its solution composition at 40"is 26% of a-and 74% of&pyranose.61 TheP-pyranose is somewhat less stable than that of the xylo isomer, owing to the presence of an axial hydroxyl group on C-2; the a-form, on the other hand, is somewhat more stable, because it is a conformational mixture (- 7 : 3) of the two chair forms (44and 45), which are of almost equal free-energy.

WoH "

O

w

Me0

OH

OH

HO

44

45

5. Sugars with Fused Rings

Fusion of another ring to the pyranose and furanose forms can profoundly alter the composition of a solution of a sugar at equilibrium. The classical example of this is presented by the 3,6-anhydro-aldohe~oses.~~~ 3,6-Anhydro-~-glucose,-L-idose, +mannose, and -L-gulose can form pyranoses and furanoses, but the pyranose forms are strained; the equilibrium mixtures contain only the a- and /I-furanose forms in the proportions of 52 : 48, 50 : 50, 79 : 21, and 26 : 74, r e s p e ~ t i v e 1 y . l ~ ~ ~ (The proportions were also determined in pyridine solution.) 3,6-Anhydro-2,4-di-O-methyl-~-glucose and -mannose cannot form furanoses, and appear to be in the aldehydo form to a considerable extent.lS83,6-Anhydro-~-galactose can form furanoses, but they would be even more strained than the pyranoses, as they would contain two transfused, five-membered rings; this sugar also appears to be mainly in the acyclic form,15Qalthough its composition has apparently never been determined. Finally, 3,6-anhydro-~-allose(46) can form neither furanoses (157) G. E. Driver and J. D . Stevens, unpublished results. (158) W. N. Haworth, J. Jackson, and F. Smith,]. Chem. SOC., (1940) 620-632; W. N. Haworth, L. N. Owen, and F. Smith, ibid., (1941) 88-102. (158a) P. Koll, H. Komander, and B. Meyer, ]u.stu.s Liebigs Ann. Chem., (1983) 13101331. (159) A. B. Foster, W. G . Overend, M. Stacey, and G . Vaughan, J . Chem. SOC., (1954) 0367 - 3377.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

59

CH= 0

c;l

HCOH

0

HA

&I3

46

nor pyranoses without considerable strain, and it occurs in dilute solution as the aldehydo and aldehydrol formss1; concentrated solutions contain dimeric forms also. Fusion of a five-membered to a six-membered ring causes strain in the latter, whereas two cis-fused, five-membered rings provide a strain-free system.leOHence, although a solution of rhamnose contains very little of the furanose forms at equilibrium, 2,3-O-isopropylidene-~-rhamnose is mainly in a furanose formlsl; its composition in aqueous solution at 40" is'@ 25 : 10 : 6 5 : trace. As rhamnose has the manno configuration, it has favored pyranose and disfavored furanose forms; hence, it was predictedIe2that other 2,3-O-isopropylidene sugars would have even less of their pyranoses in their equilibrium mixtures. It was, indeed, later reported that a solution of 6-deoxy-2,3-0-isopropylidene-~-gulose contains - 4 0 % of the pyranoses,ls3 and that only the p-furanose form was found in a chloroform solution of 2,3 : 6,7-di-O-isopropylidene-~-gk~cero-D-gdo-heptose. le4 The a-furanose form greatly preponderates in aqueous solutions of D-mannose 2,3-carbonatelg1 and D-lyxose 2,3-carbonateS1; here, the five-membered ring is flatter and more rigid than the dioxolane ring of the isopropylidene derivatives. Fusion of an oxirane ring to a pyranose ring also deforms it, and thereby lowers its stability. The composition of 2,3-anhydro-~-mannose in aqueous solution,1e5as determined by g.1.c. of the trimethylsilyl derivatives, is 23 : 7 : 6 5 : 5 . This is remarkably similar to the composition of a solution of 2,3-O-isopropylidene-~-rhamnose. For 2,3-anhydro-~-allose, the ratios arelee41 : 12 : 5 : 42 (or 41 : 5 : 1 2 : 42). In this case, although the proportion of furanose forms is substantial, there is no clear preponderance of the p-furanose form, presumably because OH-1 and OH-2 are trans but OH-1 is quasi-equatorial; by contrast, in the (preponderant)

-

(160) J. A. Mills,Ado. Carbohydr. Chern., 10 (1955) 1-53. (161) A. S. Perlin, Can. 1.Chern., 42 (1964) 1365-1372. (162) S.J. Angyal, V. A. Pickles, andR. Ahluwalia, Carbohydr. Res., 3 (1967) 300-307. (163) P. M. Collins and B. R. Whitton, Curbohydr. Res., 33 (1974) 25-33. (164) J. S. Brimacombe and L. C. N. Tucker,]. Chem. Soc., C, (1968) 562-567. (165) J. G. Buchanan andD. M. Clode,]. Chern. Soc., Perkin Trans. 1 , (1974) 388-394. (166) J. G. Buchanan, D. M. Clode, and N. Vethaviyasar, ]. Chem. SOC., Perkin Trans. I, (1976) 1449-1453.

60

STEPHEN J. ANGYAL

a-furanose form of 2,3-anhydro-~-mannose,OH-1 and OH-2 are also trans, but OH-1 is quasi-axial. The ratio of a-top-pyranose for 3,4-anhyd r o - ~ - a l t r o s ein ' ~ solution ~ is 32.5 : 67.5. A somewhat different situation is encountered with 2,4-O-methylene'13' The pyranoses (47) would have unand 2,4-O-benzylidene-~-xylose. favorable interactions, and appear not to be formed at all. Attempts to isolate these compounds yielded only condensation products and dimers of the aldehydo form.

I

HO 47

W. SOLUTIONS I N SOLVENTS OTHERTHAN WATER Water is the only solvent in which the composition of sugars has been systematically explored. Stevens'67a has determined the composition of several aldoses in pyridine-d, by 'H-n.m.r. spectroscopy at 300 MHz. There are scattered data on solutions in organic solvents (mainly pyridine, dimethyl sulfoxide, and N,N-dimethylformamide), but only rarely have four (or more) components of such solutions been quantitatively determined. The data that have been encountered are collected in Table VII; undoubtedly, there are others that have been missed. Kuhn and GrassnerlG8were the first to realize that the solution composition of sugars may vary considerably with a change of solvent. They stated that D-fructose in N,N-dimethylformamide exists in furanose forms to the extent of -80%. (This value is probably too high; compare with Table VII.) The only systematic study published on the influence ofsolvents on the solution equilibria of sugars is contained in two articles by Perlin.5'.57 This work showed that, in other solvents, the a:p-pyranose ratio is higher than in water (if the a-anomeric hydroxyl group is axial), and that there is a greater proportion of the furanose forms. The increase in the a-pyranoses is caused by the increased anomeric effect; the possible reason for the increase in the furanose forms has been discussed in Section II1,l. The anomeric effect becomes particularly important in nonpolar solvents; for example, in a chloroform solution of evernitrose (167) S. J. Angyal and K. James, Carbohydr. Res., 15 (1970) 91-100. (167a) J, D. Stevens, unpublished results. (168) R. Kuhn and H. Grassner, Ann., 610 (1957) 122-131.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

61

(2,3,6-trideoxy-3-C,4-O-dimethyl-3-C-nitro-~-urub~no-hexose), equal amounts of the two pyranose forms are found,lsQ despite the serious syn-axial interaction between methyl and hydroxyl groups in the a-form (48).

I

OH

HSc 48

Mackie and PerlinS7found that, when OH-2 is axial, there is a great increase in the proportion of the a-pyranose form in dimethyl sulfoxide, but there is little when it is equatorial (see Table VII). Typical of that small increase is the gradual change in the proportion of the a-pyranose form of lactose on addition of ethanol to the aqueous s01ution~'~:in water, 37%; in 50% ethanol, 40%; and in 80% ethanol, 42.5%. When compared with those in other Tables, the data in Table VII show that Perlin's conclusions are generally valid. 2,3-Anhydro-~-mannose and 2-C-(hydroxymethyl)-~-ribose(hamamelose) are exceptions: there is actually somewhat less furanose in their solutions in dimethyl sulfoxide and pyridine, respectively, than in water; but these can hardly be regarded as typical sugars. When the effect of an organic solvent is combined with partial methylation (see Section VI,l), the proportion of furanoses becomes most significant: 2,3-di-O-methyl-~-galactosein dimethyl sulfoxide contains 38%, 2,3-di-O-methyl-~-arabinose 65%, and 2,3-di-O-methyl-~-altrose 80% of the furanose forms.57 Mention should be made here of the equilibrium between the four methyl glycosides of reducing sugars. The solvent is methanol, and the reaction is not spontaneous, but requires an acid catalyst and, usually, heat; but it is closely related to the equilibrium of the free sugars in aqueous solution. There are more, quantitative data available on the equilibria between methyl glycosides than on the composition of solutions of free sugars, for the obvious reason that the glycosides can be separated from each other. In instances where the equilibrium proportion of methyl glycosides is known, but not that of the free sugars, arough guess can be made as to the latter. In the glycoside equilibrium, there is more a-pyranose (because the anomeric effect is greater) and more furanose (because the solvent is not water) than in the aqueous equilibrium

-

(169) J. Yoshimura, M. Matsuzawa, and M. Funabashi, Bull. Chem. Soc. Jpn., 51 (1978) 2064 - 2067. (170) F. Mayd and T. A. Nickerson,]. Agric. Food Chem., 26 (1978) 207-210.

62

STEPHEN J. ANGYAL

of the free sugars. To give one example: the proportions of the methyl D-xylosides"' in equilibrium at 35" are 65.1 : 29.8 : 1.9 : 3.2, whereas, in an aqueous solution of D-xylose, there is 36.5% of a-pyranose, 63% of /?-pyranose, and less than 1%of the furanoses. Occasionally, however, the relationship is obscure: there are almost equal amounts of the a- and /?-pyranoseforms in an aqueous solution of D-psicose, but, in the methanolysis mixture, there are only traces of the a-pyranoside.15

VIII. TABULATED DATA Tables II-VII contain the data on most of the sugars for which the composition in solution has been determined. Some others which do not fit into any of the Tables (such as the thio and the branched-chain sugars), and some for which the data are not sufficiently accurate to warrant their inclusion in the Tables, are mentioned in the text. The composition of sugars in solution varies considerably with changes in the temperature (see Section 111,6).It is essential, therefore, to record the temperature at which the proportions have been determined. Ideally, all of the compositions should have been listed at the same temperature, but, unfortunately, different authors have used different temperatures for their measurements. In the Tables, therefore, the temperature at which the data were obtained is recorded; where no such figure appears, it was not possible to ascertain this information from the published texts. In those cases where data at several temperatures were published, those recorded closest to 25" are listed. The composition of sugars in solution appears to vary very little with changes in their concentration. Williams and AllerhandI7 determined the composition of D-glucose in the range of 0.11-4.0 M. Up to 2 M, the variation was within the experimental error. In the 4 Msolution, the ratio ofa- to/?-pyranose was 40.2 : 59.7, insteadof37.3 : 62.6 k 1.0 at greater dilution; however, at that high concentration (72% w/v!) the physical properties of the solution must be very different from those of a more dilute one. Somewhat greater changes in the composition of D-glucose were found by HyvBnen and coworker^'^^; the proportion of the pyranose and furanose forms of D-fructose, however, remained constant when the concentration was increased from 20 to 80%. N.m.r. spectra are usually recorded for 5-40% solutions. The concentration is rarely specified in publications, and is not listed in the Tables. All of the 'H-n.m.r. spectra, and most of the 13C-n.m.r.spectra, were recorded for solutions in deuterium oxide, on the tacit assumption that the composition in that solvent would be the same as that in naturalabundance water. It is by no means certain that this assumption is valid, (171) L. HyvOnen, P. Varo, and P. Koivistoinen, J. Food Sd., 42 (1977) 657-659.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

63

as the dielectric constant of the two solvents, and the strength of the hydrogen bonds therein, are different. A s t ~ d y "of~the anomerization of D-glucose in H,O and in D,O showed that the final compositions were the same ( k 1%)in each solvent; but this study did not involve furanose forms. It can only be hoped that the differences in composition between solutions in these two solvents are always within the experimental errors. Some of the early n.m.r. spectra were recorded at 60 MHz, and the separation of signals was not always complete. Some have been re-recorded at 100 MHz, or the W-n.m.r. spectra were recorded in order to confirm, or improve on, the original results. Undoubtedly, many of the data could be improved by re-recording the spectra with modern, highfield instruments. The latest results available are listed in the Tables, with occasional comparisons between 'H- and W-n.m.r. data. The accuracy given by many authors (if it is given at all) is k 2%; actually, 3%may be more appropriate in most cases, although there are instances where special measures were taken to improve the accuracy. Some authors have given their results in decimals; but it is considered here that the decimals have no significance, and the values have been rounded off to the nearest whole number (except for 0.5). TABLE I1 The Composition (%) of Aldo-hexoses and -pentoses, and Some of Their Deoxy Derivatives, in D,O" ~~

Aldose Allosec Altrosed GalactosedSe 6-deoxyGlucose 6-deoxyGulosed Idoseg Mannose 6-deoxyTalosed 6-deoxy-' 2-Deoxy-arabino-hexosej 2-Deoxy-Zyxo-hexosej 2-Deoxy-ribo-hexose 3-Deoxy-ribo-hexosej 3-Deoxy-rylo-hexose

~

Temp. (degrees)

a-

Pyranose Furanose /I- a/3-

31 22 31 31 31 44 22 31 44 44 22 30 44 31 31 31 31

14 27 30 28 38 36 16 38.5 65.5 60 42 44 47.5 40 15 24.5 < 27

77.5 43 64 67 62 64 81 36 34.5 40 29 28 52.5 44 58 55 53.3

3.5 5 13 17 2.5 3.5 -5 0.14-f

_ -

-

3 11.5 14 0.6h 0.3h

-

-

16 16

13 11

Aldehydeb 0.01 0.04 0.02 0.007 0.002 0.002 0.2 0.005 0.006 0.03

_

_

0.008

8 12 5

8 15 15.5 19.5

0.03

(continued) (172) J. Jacin, J. M. Slanski, andR. J. Moshy,J. Chromatogr., 37 (1968) 103-107.

STEPHEN J. ANGYAL

64

TABLEI1 (continued) ~

Temp. (degrees)

Aldose

Arabinose' Lyxose Ribose" Xylose 2-Deoxy-eythro-pentose" 4-Deoxy -eythro-pento~e~ 3-Deoxy-~~-threo-pentoseP

31 31 31 31 30 30

Pyranose

Furanose

a-

/!?-

a-

60 70 21.5 36.5 40 30 50

35.5 28 58.5 63 35 70 22

8-

2.5 2 1.5 0.5 6.5 13.5 <1 13 12 22

Aldehydeb 0.03 0.03 0.05 0.02

6

From the 'H-n.m.r. spectra, Refs. 9 and 10, unless stated otherwise. These references also list equilibrium proportions at 44". Ref. 31, at 20". Ref. 88. From the W-n.m.r. spectrum.72 Wertz and coworkers (Ref. 37) found, by g.1.c. of the 0-(trimethylsilyl)derivatives,32.5:63.8:1.2:2.5at15",and32.0:62.4: 1.8:3.8at25";see also, Ref. 36. fRef. 17, at 43". gFrom the 13C-n.m.r. spectrum7' at 37": 38.5: 33.5 : 12.5 : 15.5. Ref. 19, at 36". Horton and Walaszek18afound, from the W-n.m.r. spectrum at 30", 70% of a- and 30% ofp-pyranose. Ref. 173b.J Pfeffer and coworkers173found, from the 13C-n.m.r. spectrum at 30", 47 : 53 for 2-deoxy-~-arabino-hexose, 38 : 37 : 13: 12 for 2-deoxy-lyxo-hexose, and 26 : 51 : 6 : 17 for 3-deoxy-~ribo-hexose. kIncluded in the figure given for the a-pyranose. 'Liptitk and cow o r k e r ~ ' ~found, ~ * from the W-n.m.r. spectrum at room temperature, 55 : 32 : 7 : 6. Horton and Walaszek18* found, from the W-n.m.r. spectrum at 30", the ratios 19: 64:6: 11. " From the 13C-n.m.r. spectrum.173Lemieux and coworkers38 found, from the lH-n.m.r. spectrum, 42:43:5: 10 at O", 3 8 : 3 5 : 1 3 : 1 4 at 40", and 28 : 32 : 22 : 17 at 90". Ref. 115c. P Ref. 173c.

'

111 TABLE

The Composition" of Some Aldoheptoses in D,O at 22" Pyranose Heptose D-glyCeI"O-D-Qll0D-glycero-L-altroD-glycero-D-galactoD-glycero-L-galactoD-glycero-L-gluco-

a- fi-

14 29 28 36 43

74 41 67 57 57

Furanose

a-

/!?-

5 17 2 3

13 3

-

Aldehydeb

7 4

-

0.01 0.02 (continued)

(173) P. E. Pfeffer, F. W. Parrish, and J. Unruh, Carbohydr. Res., 84 (1980) 13-23. (173a) A. Lipthk, Z. Szurmai, P. NAnhsi, and A. Neszmblyi, Tetrahedron, 38 (1982) 3489-3497. (173b) J. Defaye, A. Gadelle, and S.J. Angyal, Carbohydr. Res., 126 (1984) 165-169. (173c) J. Buddrus, H. Herzog, and H. Bauer, Justus Liebigs Ann. Chem., (1983) 19501958.

COMPOSITION OF REDUCING SUGARS IN SOLUTION

65

TABLE111 (continued) Heptose

Pyranose 01-

8-

Furanose 0/3-

D-ghjCerO-D-gUl!O-" D-glyCerO-D-idOD-glycero-L-mannoD-glycero-D-taloD-glycero-L-talo-d

15 23 70 43 33

80 52 30 34 30

2? 10 13 22

Aldehydeb

3 15

0.02 0.06"

10 15

From the W-n.m.r. spectra (Ref. 92), unless otherwise stated. Ref. 31, at 20". At 60". From the 'H-n.m.r. spectrum at 50" (Ref. 93). Ref. 82. (I

TABLE IV The Composition (%) of Hexuloses, Pentuloses, and Some Related Compounds in DpOo keto Form

Ketose Fructose

l-deoxyPsicose l-deoxySorbose l-deoxy6-deoxyTagatose arabino-3-Hexulosed xylo-3-Hexulose' threo-Pentulose 1-deoxyLactulose Cellobiulose Maltulose

Temp. (degrees) 0 30 31" 80 37 27 27 27 31" 80 27 35 27 31"

37 37 37

Pyranose /I-

a-

-

-4

-

79 71

16 18

83 1 2.5

11 4 7.5

61.5 61 64

71 17 16

15 66 62

-

-

-

by n.m.r.

4 11 5 23 6.5 25 10 32 6 9 39 15 21 17 2 4 1 8 1

2 2.5 2 4 22 27 98 93 87 92

-

25f 25f 25f

85 70 65 53 75 24 29 2 2

Furanose /3-

a-

-

20 7.5 29 10 29 12 22.5

by c.d.b 0.7

0.8 3 6 6

14 0.3 10 0.2

0.25 2 5

-4

6 0.6 0.3

-

14 17 22 80 1.6

References 174 18a 175 20 16 15 16 15 175 20 16 15 15 175

16

B

16 16 97 24 24

1.5

24

8

(continued)

STEPHEN J. ANGYAL

66

TABLE IV (continued)

Ketose Turanose xylo-2-Hexulosonic acid arubino-h xy lo-5-Hexulosonic acid, sodium salt lyxo-

Temp. (degrees)

a-

Pyranose

&

a-

36

<4

39

20

41

ambient ambient

>97 6

-

<3 11

-

ambient ambient

-

-

79 28

10 72

67

Furanose /?-

-

by n.m.r.

by c.d.b 1

References 11

40 40

16

40 40

11

-

Refs. 15 and 16 also give the composition at higher temperatures, and data in earlier papers. At 20" (Ref. 31). From theW-n.m.r. spectrum ofa 4 Msolution. Okudaandcoworkers (Ref. 41) found 81.6 and 18.4%, not counting the keto form, at 30". 'Okuda and coworkers (Ref. 41) found 16.2 and 83.8%, not counting the keto form, at 30O.fDatawere also given for 58". 8 The signalis obscuredin the spectrum. At 58", there is 2.2% of the keto form. The sodium salt and the methyl ester have approximately the same equilibrium composition.

TABLE V The Composition (%) of Heptuloses in D,O" Heptulose

Temp. (degrees) 22 22 22 22 27 27 22 27 37 30 30 30

a

Pyranose a/?70 17 78 100 77 11" >99 39 15 11 -

86

6

-

-

13"

16 16 48

-

Furanose

a21 13 16

89 64 6

-

-

17 11

6 65

-

-

18 54 55.5 17 14

43 15 17.5 35

From the 13C-n.m.r. spectra,ge unless stated otherwise.

* Okuda and coworkers103did not detect the /I-pyranose; they re-

ported 16 : 0 : 18 : 66 at 30". Uncertain within this pair. d Ref. 16; 3% of the keto form, by c.d. at 20" (Ref. 31). Ref. 103.

(174) E. 0.Farhoudi and W. Mauch, Forschungsber. Inst. Zuckerind. Berlin, 4 (1976) 116 pp.; C b m . Abstr., 89 (1976) 163,8621'. (175) G. J. Wolff andE. Breitmaier, Chem.-Ztg., 103 (1979) 232-233.

TABLE VI The Composition (%) of Some Amino Sugars in Solution Sugar ~

Temp. (degrees)

Pyranose

Furanose

Solvent

a-

j%

a-

HsO DzO D,O D20 D,O D20 D,O D,O D,O (CD,),SO (CD3),S0 (CD,),SO (CD3),S0

36 63 68 47 65 43 57 14 17 85 52 16 64

64 37 32 53 35 57 43 72 74 15 32 10 36

&

References

~~~

10 2-Amino-2-deoxy-~-g~ucose" 40 hydrochloride 40 N-acetyl2-Amino-2-deoxy-~-galactose,hydrochloride 40 N-acetyl40 2-Amino-2-deoxy-~-mannose, hydrochloride 40 N-acetyl40 2-Acetamido-2-deoxy-~-allose 23-25 2-Acetamido-2-deoxy-~-gulose 23-25 3-Benzamido-2,3,6-trideoxy-~-arabino-hexose lyro isomer rib0 isomer rylo isomer

3

5 6

8 34

8 40

9

119 120 120, 120ab 120 120 120 120, 120ab 120ab 120ab 123 123 123 123

a From the optical rotation and pK, values. This reference also lists the composition of a derivative that contains a peptide chain on 0-3.

STEPHEN J. ANGYAL

68

TABLE VII The Composition (%) of Some Sugars in Solvents Other than WateP Sugar

Temp. (degrees)

Solvent

Pyranose

Furanose

a-

a-

B-

8-

References

11 13

167a 167a 57 167a,176 176 176a 32 57 32 57 57 167a 10 167a 57 167a 57 167a 167a 57 167a 32 57 177 178 179 179

50 25

23 27

61 36

5 24

Mannose

116

33 32 38 47 38 78 86 87 68 20 33

48 31 57 50 62 22 14 13 13 36 33

7 14

6-deoxyGlucose

25 80 65 116

82.5 88 17 19 10 45 46 39 5 5

15 12 64 51 51 53 43 61 43 26 27 34.5

15 21 20 21.5

35" 48d 52 39.5

tr 6 tr 14 27

54 36 42 13 12

18 23 22 43 58

28 35 36 14f

15

20

35

30

Allose Altrose

44 Galactose

6-deoxyTalose 3-Deox y-xy lo-hexose Arabinose

25 31 80

Lyxose

25

Ribose

25 80

Xylose

25 116

12 23 5 3

15 6 21

4 39 13 33

33 30 24

Fructose Lactulose Maltulose arabino-2-Hexulosonic aci& altro-3-Heptulose 2,3-Anhydromannose 2-C-(Hydroxymethyl)ribose

4 ambient ambient ambient 30

~~~

~~~

~~~

~

2

0.4

6 12 18 1

13 18 21 1

11

40 40

40 103 165

3

~~~~

149 ~

Four more compounds are to be found in Table M. Determined by g.1.c. of the trimethylsilyl derivatives. Also, 2% of the keto form. Also, 2-3% of the keto form. Data are also given for 20,40, and 50". Similar values were found for the sodium salt and for the methyl ester.fAlso, 15% ofthe keto form. (176) T. E. Acree, R. S. Shallenberger, and L. R. Mattick, Carbohydr. Res., 6 (1968) 498- 502. (176a) D. F. Mowery, Jr., Carbohydr. Res., 43 (1975) 233-238. (177) W. Funcke and A. Klemer, Carbohydr. Res., 50 (1976) 9-13. (178) D. J. Nicole, B. Gillet, E.N. Eppiger, and J.-J. Delpuech, TetrahedronLett., (1982) 1669- 1672. (179) P. E. Pfeffer, K.B. Hicks, and W. L. Earl, Carbohydr. Res., 111 (1983) 181-194; P. E. Pfeffer, personal communication.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 42

SYNTHESIS OF BRANCHED-CHAIN SUGARS BYJUJI YOSHIMURA Laboratory of Chemistry for Natural Products, Tokyo Institute of Technology,Midoriku, Yokohama 227, Japan

.

..

.

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . , . . , . , . . . . . . . . . 11. General Syntheses, and Selectivities of Reactions Therein. . . . . . . . . . . . . . . . . 1. Nucleophilic Addition to Glycosiduloses . . . . . . , . . . . . . . . . . . . . . . . . . . . . 2. Addition to C-Alkylidene Glycosides . , , , . . . . . . . . . . . . ... . ... . 3. Nucleophilic Reactions of Sugar Oxiranes . . . . . . . . . . . . . . . . . . . . . 4. Addition to Unsaturated Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. AldolAddition., . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... ....... . 6. Photochemical Addition . . . . . . . . . . . . . . . . . . . 7. Cyclization of Dialdehydes with Nitroalkanes . . . . , . , . . . . . . . . . . . . 8. Rearrangement Reactions . . . . . . . , . . . . . . , . . . . . . . . . . . . , . . . . . . . . . . . 9. Total Synthesis. . . ......................... . . . . . . . . . . . . . . . . . . 111. Synthesis of Naturally curring, Branched Sugars . . . . . . . . . . . . . . . . . . . . . . 1. Methyl-branched Sugars . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . 2. Two-carbon-branched Sugars . , . . , . , . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Formyl- and Hydroxymethyl-branched Sugars. . . . . . , . . . . . . . . . . . . . . . 4. Branched Sugars and Branched Cyclitols Having No Heteroatom at the Branch Point.. . . . . . . . . . . . . . . . . . . . . . . . . .......... IV. Remarks Not Relating to Synthesis . . . . . . . . . . . . .......... 1. Branched-Sugar Nucleosides. . . . . . . . . . . . . . . . . . . . . . . .

.

.

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

.

.

.

.

69 78 78 91 95 97 104 105 107 109 113 118 118 125 128 131 131

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

I. INTRODUCTION The naturally occurring, branched-chain sugars were classified as rare sugars1v2until the 1960's, but the discovery of numerous new sugars as the glycosidic component of antibiotics during the past two decades has stimulated extensive research on their chemistry and biochemistry. Among the branched-chain sugars shown in Table I, apiose (1) and hamamelose (2), respectively found in parsley and witch hazel early in this century, are now known to have widespread occurrence in the plant (1) C. S. Hudson, Ado. Carbohydr. Chem., 4 (1949) 57-74. (2) F. Shafizadeh, Ado. Carbohydr. Chem., 11 (1956) 263-283.

69

70

Re

Hoq>o

FH"

HOCH,

HO

CH"

OH

R

\ o, 28

29 R = CHOHMe(S) 30 R = COMe

HZC 33 R = COMe 34 R = CHOHMe(S)

0

32

31 OH

4

C;H,OH

I

C02R

I Me,HCH,C -COH I

e

HCOH I C0,R

35

Qm2

HO B"

OH

36

37

n

OH 40

HO 41

OH

38

OH

39

TABLE I Principal Branchedchain Sugars and Cyclitols of Natural Occurrence sugars

Trivial name

Source

References

Hydroxymethyl- or Formyl-branched Sugars

l N

3-C-(Hydroxymethyl)-~-g~ycero-tetrose (1)

Apiose

2-C-(Hydroxymethyl)-~-ribose (2)

Hamamelose

5-Deoxy-3-C-formyl-~-lyxose (3) 3-C-Formyl-~-lyxose(4)

Streptose Hydroxystreptose

5-Deoxy-3-C-(hydroxymethyl)-~-lyxose (5)

Dihydrostreptose

Parsley and various plants Hamamelis oirginiana and various plants Streptomycin Hydroxystreptomycin Bluensomycin

3-5 6-10 11,12 13,14 15,16

Methyl-branched Sugars 2-C-Methyl-~-erythrose(6) and derivatives 2-C-Methyl-~-erythrono-1,4-lactone (7) 2,6-Dideoxy-3-C-methyl-~-ribo-hexose (8) 3-methyl ether (9)

Mycarose

%methyl ether (11) 2,6-Dideoxy-3-C-methyI-~-arabino-hexose (12) 3-acetate (13) 2,6-Dideoxy-3-C-methyl-~-arabino-hexose (14) 6-Deoxy-3-C-methyl-~-mannose (15) 6-Deoxy-3-C-methyl-2-O-methyl-~-talose (16)

Cladinose Axenose Arcanose Olivomycose Chromose B Evermicose Evalose Vinelose

6-Deoxy-3-C-methyl-2,3,4-tri-O-methyl-~-mannose (17) 6-Deoxy-3-C-methyl-~-gulose(18)

Nogalose Virenose

2,6-Dideoxy-3-C-methyl-~-xyZo-hexose (10)

Cotylenins Iberian milk-vetch Carbomycin and others Erythromycin Axenom ycins Lankamycin Olivomycins Chromomycin A3 Everninomicins Everninomicin B Acetobacter oinelundii Nogalamycin Virenomycin

17 18 19-22 23 24 25 26 27

28,29 30 31 32,33 34,35

6-Deoxy-5-C-methyl-4-O-methyl-~-Zyxo-hexose (19) 4-C-Methyl-~-glucuronicacid (20) 3-Deoxy-4-C-methyl-3-(methylamino)-~-arabinose (21) 4,6-Dideoxy-3-C-methyl-4-(methylamino)-~-altrose (22) 3-Amino-2,3,6-trideoxy-3-C-methyl-~-lyro-hexose (23) 3-Amino-2,3,6-trideoxy-3-C-methyl-~-xylo-hexose(24) 2,3,6-Trideoxy-3-C-methyl-4-O-methyl-3-nitro-~-arabi~-hexose (25) 2,3,6-Trideoxy-3-C-methyl-4-O-methyl-3-nitro-~-xy~-hexose (26) 2,3,4,6-Tetradeoxy-4-(methoxycarbonyl)~ino-3-C-methyl-3-ni~o-~-arabino-hexose (27)

Noviose Moenuronic acid Garosamine Sibirosamine Vancosamine Evernitrose Rubranitrose Tetronitrose

Novobiocin Moenomycin Gentamicins Sibiromycin Vancomy cin Antibiotic A35512B Everninomicins Rubradirin Tetrocarcins and others

36,37 38-40 41-43 44,45 46,47 48,49 29,50 51-53 54-56

Two-carbon-branchedSugars

2,3,6-Trideoxy-4-C-glycolyl-~-threo-hexose (28) 2,6-Dideoxy-4-C-[1(S)-hydroxyethyl]-~-x~lo-hexose (29) 4-C-Acetyl-2,6-dideoxy-~-rylo-hexose(30) 4,6-Dideoxy-3-C-[1(S)-hydroxyethyll-~-ribo-hexose 3,l’-carbonate (31) 4-C-[1(S)-methoxyethyl]-2,3-O-methylene-~-arabinono-1,5-lactone (32) 4-C-Acety~-6-deoxy-2,3-O-methy~ene-~-galactono-1,5-~actone (33)

Pillarose y-Octose Trioxacarcinose B Aldgarose

6-Deoxy-4-C-[1(S)-hydroxyethyl]-2,3-O-methylene-~-g~actono-1,5-lactone (34) Higher-branched Sugars

(2R,3S)-Z-Isobutylthrearicacid (35)4-(/3-~-glucopyranosyloxy)benzyl diester (35) 2-C-Butyl-2,5-dideoxy-~-arabinono-1,4-lactone 3-(3-methylbutanoate) (36)

Pillaromycin A Quinocycline A Quinocycline B Aldgamycin E Everninomicins Flambamycin Avilamycin A Avilamycin C

37 58,59 60 61,62 29,63 64,65 66,67 67,68

Loroglossine

69

Blastmycinone

Blastmycin

70

Mytilitol Laminitol Valienamine Validamine Validatol

Algae, Mytilus Algae Validamycins Validamycins Validamycins

Branched Cyclitols

1-C-Methyl-scyllo-inositol(37) 1~-4-C-Methyl-myo-inosito~ (38) 1 ~ -1,3,6/2)-6-Amino-4-(hydroxymethyl)-4-cyclohexene-l,2,3-triol(39) ( 1(S)-( 1,2,4/3,5)-1-Amino-5-Olydroxymethyl)cyclohexane-Z,3,4-triol(40) 1~-(2/1,2,4)-4-(Hydroxymethyl)cyclohexane-l,2,3-triol (41) and derivatives

71-73 74-76 77,78 79 80,81

74

JUJI YOSHIMURA

(3) R. R. Watson and N. S. Orenstein, Ado. Carbohydr. Chem. Blochem., 31 (1975) 135-184. (4) E. Vongerichten, Justus Liebigs Ann. Chem., 318 (1901) 121-136; 321 (1902) 71-83. (5) P. Forgacs, J. F. Desonclois, and J. L. Pousset, TetrahedronLett. (1978) 4783-4784. (6) E. Fischer and K. Freudenberg, Ber., 45 (1912) 2709-2726. (7) 0.T. Schmidt and K. Heinz, Justus Liebigs Ann. Chem., 515 (1934) 77-96. (8) W. Mayer, W. Kanz, and F. Loebich, Justus Liebigs Ann. Chem., 688 (1965) 232-238. (9) E. Beck, H. Stransky, and M. Fiirbringer, FEBS Lett., 13 (1971) 229. (10) H. Glick, A. Thanbichler, J. Sellmair, and E. Beck, Carbohydr. Res., 39 (1975) 160-161. (11) R. U. Lemieux and M. L. Wolfrom, Ado. Carbohydr. Chem., 3 (1948) 337-384. (12) H. Umezawa, Recent Advances in Chemistry and Biochemistry ofAntibiotics, Microbial Chemistry Research Foundation, Tokyo, 1964, pp. 67-84. (13) F. H. Stodola, 0.L. Shotwell, A. M. Borud, R. G. Benedict, and A. C. Riley, JrJ. Am. Chem. Soc., 73 (1951) 2290-2293. (14) E. Lederer, Bull. SOC. Chim. Biol., 42 (1960) 1367-1372. (15) B. Bannister and A. D. Argoudelis,J. Am. Chem. Soc., 85 (1963) 234-235. (16) T. Miyaki, H. Tsukiura, M. Wakae, andH. Kawaguchi,J. Antibiot., Ser. A, 15 (1962) 15-20. (17) T. Sassa, M. Togashi, and T. Kitaguchi, Agric. Biol. Chem., 39 (1975) 1735-1744. (18) J. de P. Teresa, J. C. H. Aubanell, A. S.Feliciano, and J. M. M. del Corral, Tetrahedron Lett., (1980) 1359-1360. (19) F. W. Tanner, A. R. English, T. M. Lees, and J. B. Routien, Antibiot. Chemother., 2 (1952) 441-443. (20) D. Vazquez, “The Macrolide Antibiotics,” in J. W. Corcoran and F. E. Hahn (Eds.), Antibiotics, Vol 111, Springer-Verlag, New York, 1975, pp. 459-479. (21) M. Muroi, M. Izawa, and T. Kishi, Chem. Pham. Bull., 24 (1976) 450-462, 463-478. (22) J. Majer, J. R. Martin, R. S.Egan, and J. W. Corcoran,J. Am. Chem. SOC., 99 (1977) 1620-1622. (23) P. F. Wiley and 0. Weaver, J. Am. Chem. SOC., 77 (1955) 3422-3423. (24) F. Arcamone, W. Barbieri, G. Franceschi, S.Penco, and A. Vigevani, J.Am. Chem. Soc., 95 (1973) 2008-2009. (25) G. Roncari and W. Keller-Schierlein, Helo. Chim. Acta, 45 (1962) 138-152; 47(1964) 78-103; 49 (1966) 705-711. (26) Yu. A. Berlin, S. E. Esipov, M. N. Kolosov, M. M. Shemyakin, andM. G. Brazhnikova, Tetrahedron Lett., ( 1964) 1323- 1328. (27) M. Miyamoto, Y. Kawamatsu, M. Shinohara, K. Nakanishi, Y. Nakadaira, and N. S. Bhacca, Tetrahedron Lett., (1964) 2371-2377. (28) A. K. Ganguly and 0. Z. Sarre, Chem. Commun., (1969) 1149-1150. (29) A. K. Ganguly, “Oligosaccharide Antibiotics,” in P. G. Sammes (Ed.), Topics in Antibiotic chemistry, Vol. 2, Wiley, New York, 1978, pp. 59-98. (30) A. K. Ganguly and A. K. Saksema, Chem. Commun., (1973) 531-532. (31) D. Okuda, N. Suzuki, and S. Suzuki,J. Biol. Chem., 242 (1967) 958-966. (32) P. F. Wiley, F. A. MacKeller, E. L. Caron, andR. B. Kelly, TetrahedronLett., (1968) 663 - 668. (33) P. F. Wiley, R.B. Kelly, E. L. Caron, V. H. Wiley, J. H. Johnson, F. A. MacKeller, and S. A. Mizsak,]. Am. Chem. Soc., 99 (1977) 542-549. (34) M. G. Brazhnikova, M. K. Kudinova, V. V. Kulyaeva, N. P. Potapova, and V. I. Ponomalenko, Antibiotiki, 22 (1977) 967-970.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

75

(35) V. V. Kulyaeva, M. K. Kudinova, N. P. Potapova, L. M. Rubasheva, M. G. Brazhnikova, B. V.Rosynoi, and A. R. Bekker, Bioorg. Khim., 4 (1978) 1087-1092. (36) J. W. Hinman, H. Hoeksema, E. L. Caron, and W. G. Jackson,]. Am. Chem. Soc., 78 (1956) 1072-1074. (37) E. Walton, J. 0. Rodin, C. H.Stammer, F.W. Holly, and K.Folkers, ]. Am. Chem. Soc., 80 (1958) 5168-5173. (38) R. Tschesche, D. Lenoir, and H. L. Weidenwulle, Tetrahedron Lett., (1969) 141144. (39) N. Langenfeld and P. Welzel, Tetrahedron Lett., (1978) 1833-1836. (40) P. Welzel, F.-J. Witteler, D. Muller, and W. Riemer, Angew. Chem., 93 (1981) 130- 131. (41) M. J. Weinstein, G. M. Luedemann, E. M. Oden, G. H. Wagman, J. P. Rosselet, J. A. Marquez, C. T. Coniglio, W. Charney, H. L. Herzog, and J. Black,]. Med. Chem., 6 (1963) 463-464. (42) H. Maehr and C. P. Schdner, J. Am. Chem. Soc., 92 (1970) 1697- 1700. (43) D. J. Cooper, PureAppZ. Chem., 28 (1971) 455-467. (44) C. F. Cause, T. P. Preobrazhenskaya, L. P. Invanitskaya, and M. A. Sveshnikova, Antibiotiki, 1 4 (1969) 963-967. (45) A. S. Mesentsev, V. V. Kulyaeva, and L. M. Rubasheva, J . Antibiot., 27 (1974) 866- 873. (46) M. H. McCormick, W. M. Stark, G. E. Pittenger, R. C. Pittenger, and J. M. McGuire, Antibiot. Annu., (1956) 606-611. (47) A. W. Johnson, R. M. Smith, andR. D. Guthrie,]. Chem. Soc.,Perkin Trans. 1, (1972) 2153-2159. (48) H. R. Perkins and M. Nieto, Ann. N.Y. Acad. Sci., 235 (1974) 348-363. (49) M. Debono andR. M. Molloy,]. Org. Chem., 45 (1980) 4685-4687. (50) A. K. Ganguly, 0.Z. Sarre,A. T.McPhai1, andK. D. Onan, Chem. Commun., (1977) 313-314. (51) B. K. Bhuyan, S. P. Owen, and A. Dietz, Antimicrob. Agents C h o t h e r . , (1964) 91 -96. (52) F. Ruesser, Biochemistry, 12 (1973) 1136-1142. (53) S. A. Mizsak, H. Hoeksema, and L. M. Pschigoda,]. Antibiot., 32 (1979) 771-772. (54) T. Tamaoki, M. Kasai, K. Shirahata, S.Ohkubo, M. Morimoto, K. Mineura, S. Ishii, and F. Tomita, 1.Antibiot., 33 (1980) 946-950. (55) K. Kobinata, M. Uramoto, T. Mizuno, andK. Isono,]. Antibiot., 33 (1980) 772-775. (56) A. K. Mallams, M. S.Puar, R. R. Rossman, and A. T. McPhail,]. Am. Chem. Soc., 103 (1981) 3940-3943. (57) M. Asai, E. Mizuta, K. Mizuno, A. Miyake, and S.Tatsuoka, Chem. Pharm. Bull., 18 (1970) 1720- 1723. (58) A. Tulinsky,]. Am. Chem. Soc., 86 (1964) 5368-5369. (59) U. Matern, H. Grisebach, W. Karl, and H. Ashenbach, Eur. ]. Biochem., 29 (1972) 1-5. (60) U. Matern and H. Grisebach, Eur. 1.Biochem., 29 (1972) 5-11. (61) M. P. Kunstmann, L. A. Mitscher, and N. Bohonos, Tetrahedron Lett., (1966) 839846. (62) G. A. Ellestad, M. P. Kunstmann, J. E. Lancaster, L. A. Mitscher, and G. Morton, Tetrahedron, 23 (1967) 3893-3902. (63) A. K. Ganguly, 0. Z. Sarre, A. T. McPhail, and W. Miller, Chem. Commun., (1979) 22-24. (64) L. Ninet. F. Benazet, Y. Charpentie, M. Dubost, J. Florent, J. Lunel, D. Mancy, and J. Preud’homme, Erperlentiu, 30 (1974) 1270-1272. (65) W. D. Ollis, C. Smith, and D. E. Wright, Tetrahedron, 35 (1979) 105- 127.

76

JUJI YOSHIMURA

kingdom, and the chemistry and biochemistry of 1 were discussed in 1975 by Watson and O r e n ~ t e i n2-C-Methyl-~-erythritol .~ (from Conuolvulaceae),s2 2-C-methy~-~-erythrono-l,4-~actone (7), a 2-isobutyl-~threaric acid derivative (35), mytilitol(37), and laminitol(38) have also been found in the plant kingdom. In contrast, only compound 37 is found in the animal kingdom. The occurrence of 3-C-(hydroxymethyl)-~-riburonic acid (42) in a human, bilirubin conjugate was reported,s3 but a supposed synthetic sample was not identical with the natural Vinelose (16) from Acetobacter vineZandii strain 0 was isolated as a cytidine dinucleotide, and a (hydroxymethy1)-branched nonitol was isolated from membrane lipids of thermoacidophile a r c h a e b a ~ t e r i aThe . ~ ~ remaining sugars have been found in various types of antibiotics produced by micro-organisms, mainly strains of Streptomyces. Particularly, mycarose (8)has been found in over fifteen kinds of antibiotics. 3,4-Anhydro and 4-chloro-4-deoxy derivatives of 2-C-methyl-~-erythrose(6) also appear as a partial structure of cotylenins, leaf-growth substances produced by a fungal strain. l7 Deoxyvalidatol and epivalidamine, degradation products of validamycins, are also known as analogs of validatol (41) and validamine (40), respectively.80J'1 Well known intermediates in the biosynthesis of aro(66) F. Buzzetti, F. Eisenberg, H. N. Grant, W. Keller-Schierlein, W. Voser, and H. Ziihner, Erperientia, 24 (1968) 320-323. (67) W. Keller-Schierlein, W. Heilman, W. D. Ollis, and C. Smith, Helu. Chim. Acta, 62 (1979) 7-20. (68) W. Heilman, E.Kupfer, W. Keller-Schierlein, H. Z h n e r , H. Wolf, and H. H. Peter, Helu. Chim. Acta, 62 (1979) 1-6. (69) D. Behr, J. Dahmen, andK. Leander,AdaChem. Scand., Ser. B, 30 (1976) 309-312. (70) H. Yonehara and S. Takeuchi,]. Antibiot., Ser. A, 11 (1958) 254-263. (71) D. Ackermann, Ber., 54 (1921) 1938-1944. (72) B. Wickberg, Acta Chem. Scand., 11 (1957) 506-511. (73) G. Waber and 0. Hoffmann-Ostenhof, Monatsh. Chem., 100 (1969) 369-375. (74) B. Lindberg and J. McPherson, Ada Chem. Scand., 8 (1954) 1875-1876. (75) R. S. Schweiger, Arch. Biochem. Biophys., 118 (1967) 383-387. (76) G. Waber and 0. Hoffmann-Ostenhof, Mrmatsh. Chem., 100 (1969) 369-375. (77) T. Iwasa, H. Yamamoto, and M. Shibata, J. Antibiot., 23 (1970) 595-602. (78) S. Horii and Y. Kameda, Chem. Commun., (1972) 747-748. (79) S.Horii, T. Iwasa, and Y. Kameda,]. Antibiot., 24 (1971) 57-58, 59-63. (80) S. Horii, Y. Kameda, and K. Kawahara,]. Antibiot., 25 (1972) 48-53. (81) Y. Kameda and S. Horii, Chem. Commun., (1972) 746-747. (82) T. Anthonsen, S.Hagen, M. A. Kazi, S.W. Shah, and S. Tager, Acta Chem.Scand., Ser.B, 30 (1976) 91-93. (83) C. C. Kuenzle, Biochem.I., 119 (1970) 411-435. (84) W. Blackstock, C. C. Kuenzle, and C. H.Eugster, Helu. Chim. Ada, 57 (1974) 1003-1009; compare, J. J. Nieuwenhuis and J. H. Jordaan, Tetrahedron Lett., (1977) 369-370. (85) M. De Rosa, S. De Rosa, A. Gambacorta, and J. D. Bu'Lock, Phytochemistry, 19 (1980) 249-254.

77

SYNTHESIS OF BRANCHED-CHAIN SUGARS

matic amino acids from 3-deoxy-~-arabino-heptulosonic acid, such as quinic acid (43) and shikimic acid (44), may be included among branched cyclitols. A few nucleoside antibiotics of branched sugars, such as mildiomycinee (45) and amip~rimycin~' (46) have been reported. Interestingly the unnatural L-dendroketose (47), engaged in a racemic dimer of 1,3-dihydro~y-2-propanone,~~ is selectively metabolized by a microorganism,8e to afford the D isomer.

0

HO,C """"""""""""""""""""OH HO

HOp J H

OH

HO

CO,H

OH

42

43

44

CH,OH

HOCH I CH,OH H,N

0 46

4s

HOH,C 47

From Grisebach's viewpoint of b i o s y n t h e s i ~ ,branched ~~ sugars are now divided into two groups: one group having a hydroxymethyl or formyl branch, which is formed b y intramolecular rearrangement of nucleotide-bound hexosuloses, with ring contraction and expulsion of one carbon atom, and the other having a methyl or two-carbon branch, which arises by transfer of a C, or C, unit from appropriate donors to nucleotide-bound hexosuloses. The chemical synthesis of these sugars (86) S. Harada, E. Mizuta, and T. Kishi, Tetrahedron, 37 (1981) 1317-1327. (87) T.Goto, T.Toya, T. Ohgi, and T.Kondo, Tetrahedron Lett., (1982) 1271 - 1274. (88) L. M. Utkin, Dokl. Akad. Nauk SSSR, 67 (1949) 301 -304. (89) J. Konigstein, D. Anderle, and F. Janecek, Chem. Zuesti, 28 (1974) 701-709. (90) H. Grisebach, Ado. Carbohydr. Chem. Bfochem., 35 (1978) 81-126.

78

JUJIYOSHIMURA

has also been developed from the nucleophilic addition of various carbon nucleophiles to aldosuloses, and syntheses of 1,2, streptose (3), dihydro, (D-1 l ) , olivomycose (12), streptose (5), 8, D-cladinose ( ~ - 9 )D-arcanose noviose (19), garosamine (21), 37, and DL-38were described in reviews that appeared up to 1972. During the past decade, almost all of the remaining branched sugars were synthesized, mainly by the application of new techniques, and the structure of several sugars was finally determined by their synthesis. In addition, better understanding as to the selectivities of reactions used was attained from the data accumulated, and this is important for stereospecific synthesis. The present article concentrates on these advances. Although most of the reactions for the introduction of carbon branching are also applicable for chain extension, the latter will be excluded here. Some of them were described in an article by Hanessian and Pernet.g4

11. GENERAL SYNTHESES, AND SELECTIVITIES OF REACTIONS THEREIN Most of the branched sugars found in Nature have a polar substituent at the branching carbon-atom (Type A); tertiary alcohols are commonest, but, some of them are in the form of a methyl ether [9,11, and nogalose (17)], acetate [chromose B (13)],or cyclic carbonate [aldgarose (31)], and, in several instances [vancosamine (23), the branched sugar (24) in antibiotic A355 12B, evernitrose (25), rubranitrose (26), tetronitrose (27)], an amino or a nitro group is attached to the tertiary carbon atom. Only blastmycinone (36), valienamine (39), 40, 41, and 44 have no substituent at the branching carbon atom (Type B). A diversity (such as formyl, hydroxymethyl, methyl, 1-hydroxyethyl, acetyl, 2-hydroxyacetyl, 1,2-dihydroxyethyl, higher alkyl, and carboxyl groups) is observed in the branchings, but, some of them are chemically interconvertible, and also can be derived from a common intermediate (see Scheme 1). 1. Nucleophilic Addition to Glycosiduloses

a, General Nucleophiles. -The addition of nucleophiles to suitable glycosiduloses has been extensively used for the synthesis of A-type branched sugars.e5Thus, the Grignard reaction was used for 1,06 ~ - 2 , ~ ’ (91) J. S.Brimacombe, Angew. Chem., Int. Ed. Engl., 8 (1969) 401 -409. (92) J. S.Brimacombe, Angew. Chem., Int. Ed. Engl., 10 (1971) 236-248. (93) H. Grisebach and R. Schmid, Angew. Chem.,Int. Ed. Engl., 11 (1972) 159-173. (94) S. Hanessian and A. G. Pernet, Adu. Carbohydr. Chem. Biochem.,33 (1976) 111 185. (95) W. A. Szarek and D. M. Vyas, “General Carbohydrate Synthesis,”in MTP Int. Rev. Sci., Org. Chem. Ser. Two, 7 (1976) 89-130. (96) J. M. J. Tronchet and J. Tronchet, C. R. Acad. Sci., Ser. C, 267 (1968) 626-629. (97) J. S.Burton, W. G. Overend, and N. R. Williams,J. Chem. SOC., (1965) 3433-3445.

SYNTHESIS O F BRANCHED-CHAIN SUGARS

79

Glycosiduloses

Cyanomesyl deriv.

Spiro-aziridine

Branched amino sugar

Branched sugar (A)

Spiro-epoxide

Alkylidene deriv.

Branched nitro sugar

CH,NO, Nitromethyl deriv.

C=CH

- t __ t HO

CHO

Formyl deriv.

-

Ethynyl deriv.

HO

t CH=CH,

Vinyl deriv.

HO

CH,OH

Hydroxymethyl deriv.

-

HO

Acetyl deriv.

1-Hydroxyethyl deriv.

2 -Hydroxyacetyl

1,a-Dihydroxyethyl Oniranyl deriv. deriv. deriv. Scheme 1.-Synthesis of Branched Sugars by the Addition Reaction to Glycosiduloses and the Conversion of Branchings.

3,es 5,esD-8" and &looD-9," ~ - 1 1 19,1°2 , ~ ~21,1°3 ~ and 37,1°4 in which successive ozonolysis of the vinyl group and reduction were used for formyl- and hydroxymethyl-branched sugars. Diazomethane addition, followed by alkaline ring-opening or reduction of the intermediary, J. R. Dyer, W. E.McGonigal, andK. C. Rice,]. Am. Chem. SOC., 87 (1965) 654-655. B. Flaherty, W. G. Overend, andN. R. Williams,]. Chem. Soc., C, (1966) 398-403. G. B. Howarth and J. K.N.Jones, Can. ]. Cbrn., 45 (1967) 2253-2256. G. B. Howarth, W. A. Szarek, and J. K.N. Jones, Carbohydr. Res., 7 (1968) 284289. (102) B. P. Vaterlaus, J. Kiss, and H. Spiegelberg, Helo. Chim. Acta, 47 (1964) 381 -390. (103) W. Meyer zu Reckendorf andE. Bischof, Tetrahedron Lett.,(1970) 2475-2478. (104) T. Posternak, Helv. Chim. A d a , 27 (1944) 457-468. (98) (99) (100) (101)

80

JUJI YOSHIMURA

spiro-epoxide, was also used for 1'05 and ~ - 1 , 2,1°7 ' ~ ~or ~ ~ - 3 8 , ' ~ ~ respectively. In addition, various branched sugars have been synthesized by the use of organolithiumg7~109~110 and the Ref~rmatsky"'-"~ reactions, n i t r ~ m e t h a n e ~ ' ~and - ' ~cyanohydrin ~ s y n t h e s e ~ ,and ~ ~the ~ -base-cata~~~ lyzed addition of acetonitrile.'25-'2e As shown in Scheme 1,the cyanohydrin synthesis was extended to branched amino sugars'27-'z8 by successive mesylation, formation of a spiro-aziridine by reduction with lithium aluminum hydride, and ring opening by catalytic hydrogenation with Raney nickel, and this was further utilized for oxidation to branched nitro sugars.12gIn addition, conversion of a once-introduced branch into another of a different state of oxidation affords a variety of branchings.

(105) A. D. Ezekiel, W. G. Overend, andN. R. Williams, TetrahedronLett., (1969) 16351638. (106) F. Weygand and R. Schmiechen, Chem. Ber., 92 (1959) 535-540. (107) W. G. Overend and N. R. Williams,]. Chem. Soc., (1965) 3446-3448. (108) T. Posternak and J. G. Falbriard, Helo. Chim. Acta, 44 (1961) 2080-2085. (109) A.A. J. Feast, W. G. Overend,andN.R. Williams,]. Chem. Soc.,C,(1966)303-306. (110) R.D. Rees, K. James, A. R.Tatchell, and R. H. Williams, J. Chem. Soc., C, (1968) 2716-2721. (111) Yu. A. Zhdanov, Yu. E. Alexeev, and Kh. A. Khurdanov, Zh. Obshch. Khim., 43 (1973) 186-189. (112) Yu. A. Zhdanov, Yu. E. Alexeev, and E. G. Guterman, Dokl. Akad. Nauk SSSR,211 (1973) 1345- 1346. (113) J. Yoshimura, K. Kobayashi, K. Sato, and M. Funabashi, Bull. Chem. Soc. Jpn., 45 (1972) 1806-1812. (114) A. Rosenthal and G. Schallnhammer, Can.J.Chem., 50 (1972) 1780-1783. (115) G. J. Lourens, Tetrahedron Lett., (1969) 3733-3736. (116) H. P. Albrecht and J. G. Moffatt, Tetrahedron Lett., (1970) 1063-1066. (117) A. Rosenthal, K . 4 . Ong,and D. A. Baker, Carbohydr. Res., 1 3 (1970) 113-125. (118) S . W. Gunner, R. D. King, W. G. Overend, and N. R.Williams, J. Chem. Soc., C, (1970) 1954-1961. (119) A. Rosenthal and K.-S. Ong, Can. J. Chem., 48 (1970) 3034-3038. (120) J. Yoshimura, K. Sato, K. Kobayashi, and C. Shin, Bull. Chem. SOC.Jpn., 46 (1973) 1515-1519. (121) J. Yoshimura, K. Mikami, K. Sato, and C. Shin, Bull. Chem. Soc. Jpn.. 49 (1976) 1686-1689. (122) A. Ishizu, K. Yoshida, and N. Yamazaki, Curbohydr. Res., 23 (1972) 23-29. (123) J.-M. Bourgeois, Helo. Chim. Acta, 56 (1973) 2879-2880. (124) J.-M. Bourgeois, Helo. Chim. Acta, 58 (1975) 363-372. (125) A. Rosenthal and G. Schallnhammer, Carbohydr. Res., 15 (1970) 421 -423; Can.J. Chem., 52 (1974) 51-54. (126) A. Rosenthal and D. A. Baker,]. Org. Chem., 38 (1973) 193-197. (127) J.-M. Bourgeois, Helo. Chim. Ada, 57 (1974) 2553-2561. (128) J.-M. Bourgeois, Helo. Chim. Ada, 59 (1976) 2114-2124. (129) J. Yoshimura, M.Matsuzawa, and M. Funabashi, Bull. Chem. Soc.]pn., 51 (1978) 2064-2067.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

81

Conversion of ethylidene130J31and groups is commonly used for obtaining two-carbon-branched sugars. Oxidation of a nitromethyl group to a formyl group was useds4 for a synthesis of 42. 2-Lithio-1 ,3-dithiane133(48a),as a stable nucleophile, provided a versatile means of effecting chain-extension and -branching in synthetic, carbohydrate chemistry. 134 Catalytic hydrogenation or mercuric oxide boron trifluoride-mediated desulfurization of the addition product of 48a to glycosiduloses gave methyl- or formyl-branched sugars, respectively. Likewise, acetyl or 2-hydroxyacetyl branching can be directly introduced by the use of 48b or 48c, respectively. Thus, the method was actually applied for the synthesis of 2,135 3,13531,136and 42.13' The analogous 4,5-dihydro-2-lithio-5-methyl-l,3,5-dithiazine (49) was less reactive than 48, and desulfurization of the condensation products at the terminal position was successful, but that with glycosiduloses was found i m p o ~ s i b l e .As ' ~ ~newer nucleophiles, 1-methoxyvinyllithium13g(50) for the introduction of acetyl and 2-hydroxyacetyl groups, and 1,l-dimethoxy-2-lithi0-2-propene~~~ (51) for the 1-formylvinyl group, were examined, and successfully applied for the synthesis of pi1larosel4l(28) and142 2, respectively. A similar examination of lithiotetrabutylstannylmethanal (52) gave a mixture of hydroxymethyl- and butyl-branched sugars in low yield.143

b. Stereoselectivities. -It is known that the reaction of glycosiduloses with organolithium or Grignard reagents proceeds stereoselec(130) W. G . Overend, A. C. White, and N. R. Williams, Carbohydr. Res., 15 (1970) 185-195. (131) D. C. Baker, D. K. Brown, D. Horton, andR. G . Nickol, Carbohydr. Res., 32 (1974) 299 - 31 9. (132) J. Yoshimura, Pure A w l . Chem., 53 (1981) 113-128. (133) E. J. Corey and D . Seebach, Angew. Chem., 77 (1965) 1134-1135; D. Seebach, Synthesis, (1969) 17-36. (134) J, D. Wander and D. Horton,Adv. Curbohydr. Chem. Biochem.,32 (1976) 16- 100. (135) H. Paulsen, V. Sinnwell, andP. Stadler, Chem.Ber., 105 (1972) 19 8-1988;Angew. Chem., 84 (1972) 112-113. (136) H. Paulsen and H. Redlich, Angew. Chem., 84 (1972) 112-113 Chem. Ber., 107 (1974) 2992-3012. (137) H. Paulsen and W. Stenzel, Tetrahedron Lett.,(1974) 25-28. (138) H. Paulsen, M. Stube, and F. R.Heiker, Ann., (1980) 825-837. (139) J. S.Brimacombe and A. M. Mather,]. Chem. SOC.,Perkin Trans. I , 1980) 269-272; Tetrahedron Lett., (1978) 1167-1170. (140) J.-C. Depazay and Y. L. Merrer, Tetrahedron Lett.,(1978) 2865-2868. (141) J. S.Brimacombe, R. Hanna, A. M. Mather, andT. J. R.Weakley,]. Chem. SOC.,Perkin Trans. I , (1980) 372-376. (142) J.-C. Depazay and A. Dureault, Tetrahedron Lett.,(1978) 2869-2872. (143) H. Paulsen, E. Sumfleth, V. Sinnwell, N. Meyer, andD. Seebach, Chem. Ber., 113 (1980) 2055-2061.

JUJI YOSHIMURA

82 Me

n

I

,OMe fN)

s v s

Li+ 480 R = H

H2C -C

H,C=C-CH(OEt), LLi

-

49

50

I

Li

51

BU. 'uB

y y H 2

"Sn'

IuB'

Bu

52

48b R = CH, 4 8 c R = CH,OLi

tively in high yield. Thus, the reaction of 1,2:5,6-di-O-isopropylidenea-~-ribo-hexofuranos-3-ulose (53a)or the corresponding pentofuranosand Grig3-ulose (53b)with such nucleophiles as organ01ithium"~J~~ nard r e a g e n t ~ " O J ~ ~ sodium J ~ ~ , b ~ r o h y d r i d e , ' ~ the ~ J ~ Reformatsky ~ reagent,11348 (Refs. 135 and 149), methyl n i t r o a ~ e t a t e ,and ' ~ ~ lithiometaphosphonic acid ester15* gave exclusively products (54)having the D-allo configuration, indicating that the reagents approach from the sterically favored, exo direction with respect to the trioxabicyclo[3.3.0]octane ring-system. Likewise, similar nucleophiles attack methyl 4,6-0benzylidene-2-deoxy-a-~-erythro(56a)and -a-D-threo-hexopyranosid3-ulose (59)from the equatorial direction to afford, selectively, products having the ~ - r i b (57) o ~and ~ ~ D-xzJo'O' ~ ~ ~(60)configurations, respectively. This tendency was also observed with the homologous pyranosides, the methyl 2-acetamid0-2-deoxy-'~~-'~~ (56c)and 2-0-benzoyl4,6-O-benzylidene-a-~-ribo-hexopyranosid-3-uloses~~~ (56b). The stereoselectivity in the Grignard reaction,Q7complementary to that in the r n e t h y l l i t h i ~ m 'and ~ ~ d i a z ~ m e t h a n e reactions, '~~ of methyl 3,4-O-isopropylidene-a-~-erythro-pentopyranosid-2-ulose (62), and that between the Grignard and diazomethane reactions of the /3 anom e P 7 of 62,methyl 2,3-O-isopropylidene-/3-~-erythro-pentopyranosid(144) A. Gonzalez, M. Qrzaez, andR. Mestres. An. Quim., 72 (1976) 954-956. (145) A. Rosenthal and S. N. Mikhailov,]. Carbohydr. Nucleos. Nucleot., 6 (1979) 237245. (146) R. F.Nutt, M. J. Dickinson, F. W. Holly, and E. Walter, J. Org. Chem., 33 (1968) 1789-1795. (147) P. M. Collins, Tetrahedron, 21 (1965) 1809-1815. (148) K. James, A. R. Tatchell, and P. K. Ray,]. Chem. SOC.,C, (1967) 2681 -2686. (149) A.-M. Sepulchre, G . Vass, and S. D. Gero, C. R. Acad. Sd., Ser. C, 274 (1972) 1077- 1080. (150) A. Rosenthal and B. L. Cliff, Carbohydr. Res., 79 (1980) 63-77. (151) H. Paulsen and W. Bartsch, Chem. Ber., 108 (1975) 1229-1238. (152) F. A. Carey and K. 0. Hodgson, Carbohydr. Res., 12 (1970) 463-465. (153) B. R. Baker and D. H. Buss,]. Org. Chem., 30 (1965) 2304-2308; 2308-2311. (154) B. R. Baker and D. H. Buss,]. Org. Chem., 31 (1966) 217-223. (155) J. H. Jordaan and S. Smedley, Carbohydr. Res., 16 (1971) 177-183. (156) R. J. Ferrier, W. G . Overend, G. A. Rderty, H. M. Wall, and N. R. Williams,]. Chem. Soc., C, (1968) 1091-1095.

2

3”

e:

+

0 X

n n

n

a

$:

V\ d

f

“ 2

$

V d

O

\ \ ‘2

P

I

1 I

I1

11

1 I

e:e:e:e:

+

-!x

X

$

84

JUJI YOSHIMURA

4-ulosel30 (63), 53a,b (Refs. 120 and 157), 56a,lS8 56b,120and methyl 3-0-benzoyl-4,6-0-benzylidene-a-~-urub~no-hexopyranosid-2-ulose~~~ (64a) have been described. P h T *

Q

Me2CIo

O '

' O

OMe O

0

+

62

OMe OMe

3

64a R = Bz 64b R = M e

63

It was reported that the reactions of methyl 2,3-di-O-methyl-(65a) and 3; 0-methyl- 2- 0-(methylsulfony1)-6- 0-trityl- a-D- xylo- hexopyranosid4-ulose (65b) with methylmagnesium iodide or methyllithium in ether at - 78 "respectively gave,159stereoselectively, the product of equatorial (e), or axial (a),attack. The contrasting stereoselectivities were explained as due to equatorial attack of the carbanion on 6 5 in the 4C1conformation, fixed by the coordination of magnesium to the carbonyl and vicinal oxygen atoms, and by the axial approach to the OH,-like transition state (66), a conformation lying between 4C, and B1,4, in which the dipole repulsion between C=O and C-3-0 bonds is avoided. This is rather a-approach

,

0 CH,OTr

CH OTr

Me0 OMe I

6sa R = M~ 65b R =Ms

I

,"

OMe

e-approach 66

67a Rz = R3 = OTs, R1 = R4 = H 67b R' = R4 = OTs, R2 = R3 = H 6 7 ~ R' = R3 = OTs, R2 = R4 = H

TsO

0

(157) J. P. Horwitz, N. Mody, andR. Gasser,]. Org. Chem., 35 (1970) 2335-2339. (158) B. Flaherty, S. Nahar, W. G . Overend, and N.$L Williams, J. Chem. SOC., Perkin Trans. 1, (1973) 632-638. (159) M. Miljkovib, M. Gligorijevib, T. Sato, and D. Miljkovib, J. Org. Chem., 39 (1974) 1379-1384.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

85

similar to the situation for a-halocyclohexanones in a low-dielectric solvent, such as ether, that assume that chair conformation in which the halogen atom is in the axial orientation.160 As shown in the Grignard reaction of cis- and trans-4-(tert-butyl)-l-rnethoxycyclohexan0ne,~~~ the preceding coordination of magnesium occurs even in such arigid bicyclic structure as162 1,6-anhydro-2,4-di-O-p-tolylsulfonyl-~-~-hexopyranosid-3-ulose (67). The reaction of the D-ribo (67a), D-ZYXO (67b), and Darabino (67c) diastereoisomers respectively gave axial- (66-69%) and equatorial-attack products (69-81%), and a 1:1mixture ofboth products (62%). The first result indicates a preceding change of the conformation from lC, to B0.3 (68), and successive approach of a carbanion from the exo direction of the Bo.3 conformation. The stereoselectivities in nucleophilic additions to various hexopyranosid-2-le3, -3-164,and - 4 - ~ l o s e s ~ were ~ ~ - 'extensively ~~ examined (see Tables I1 and 111).The results in the Grignard reactions in Table I11 were commonly explained by the approach of the reagent from the sterically favored direction to the magnesium-coordinated conformations (lefthand side in the equilibration formulas), but the concept for the methyl-

70

69

72

71

73

(160) E. L. Eliel, N. L. Allinger, S. J. Angyal, and A. Morrison, Conformational Analysis, Interscience, New York, 1965, p. 460. (161) D. Cuillern-Dron, M.-L. Capman, and W. Chodiewicz, Tetrahedron Lett., (1972) 37-40. (162) M. eerny, M. Kollmann, J. Packk, and M. BudWnsky, Collect. Czech. Chem. Comnun., 39 (1974) 3509-3519. (163) K. Sato and J. Yoshimura, Carbohydr. Res., 73 (1979) 75-84. (164) K. Sato and J. Yoshimura, Bull. Chem. Soc.Jpn., 51 (1978) 2116-2121. (165) M. Matsuzawa, K. Sato, T. Yasumori, and J. Yoshimura, Bull. Chem. SOC. Jpn., 54 (1981) 3505-3509. (166) K. Sato and J. Yoshimura, Carbohydr. Res., 103 (1982) 221-228. (167) J. Yoshimura andK. Sato, Carbohydr. Res., 123 (1983) 341-346.

JUJI YOSHIMURA

86

TABLE I1 Stereoselectivities in the Nucleophilic Reaction of 4,6-0-Benzylidene-~hexopyranosid-2- and -3-uloses Ratio of axial to equatorial attack' and yields (%) of products Aldosiduloses

R

56b 56d 69 70 64a 64b 71 72

Bz Me

Bz Me

NaBH,

CHgNg

MeMgX

0 : 1(55-90)168 1: O(77) 0 : 1(82,94)168 1:O(73) 0 : l(87) 0: l(41)"

0 : l(74) 0 : l(93) 0: l(95)

1: 1.1(93)"* 1: 13.8(89)1se 1: 0(77)"0 1: O(82)l7l 1:O(88)

1: 1.5(87) 0 : l(94) 1: O(50) 1.1: l(94) 1: 1.6(92) 1: l(92) 1: 4.4(95)

1: 21(86)17'

73

0: l(90) 1: 4.4(94) 0 : l(93) 1: 2.1(95) 0 : l(84) 1: O(94) 1: O(93) 1: 3.0(87)

MeLi 1:2.3(97)b 1: 1.8(93) 1: 18(95)b 1: 12(93)

1:2.6(91)

* Axial and equatorial attack are designated on the basis of the 4Clconformation of the individual aldosidulose. b The reaction was conducted in ether at - 78",and the others at room temperature, A ring-expansion product was obtained in 40% yield.

lithium reaction was questionable, because the comparison of 'H-n.m.r. parameters in ether-d,, at -78" with those in chloroform-d (normal conformation on the right) gave contradictory results.167It is noteworthy that the contrasting stereoselectivity of the reverse mode to 65 and homologous aldosid-4-uloses (74 and 75) was observed in the cases of methyl 6-deoxy-2,3-0-methylene-cu-~-ribo(80) and 6-deoxy-2,3-di-O-

OBn

4c,

Tr = Ph,C

(168) Y.Kondo, Carbohydr. Res., 30 (1973) 386-389. (169) Y.Kondo, Agric. B i d . Chem., 39 (1975) 2251-2252. (170) Y. Kondo, N. Kashimura, and K. Onodera, Agric. Biol. Chem., 38 (1974) 25532558. (171) M. MiljkoviC, M. GligorijeviC, and D. MiljkoviC, J. Org. Chem., 39 (1974) 21182120.

SYNTHESIS OF BRANCHED-CHAIN SUGARS TABLE I11 Stereoselectivities in the Nucleophilic Reactions of c~-~-Hexopyranosid-4-u~oses

Ratio of axial to equatorial attack and yields (%) of products Aldosid-4-uloses 74

65a 75 (R = Bn) 75 (R = Me)

76 77 78 79 80 81

CH,N, 1:0(20)b

MeMgX

0 : l(84)‘ 1: 1(72Pd 1:0(15)b 0:1(84)” 1: 2.2(93) 3 : 1(60)b 1: 3.9(93) 2.4 : 1(72)b 1 : 3.6(96) 0 : l(94)’ 0:1(52)b 0:1(89) 1 : O(42) 0 : l(85) 1 :O(82) 0 : l(96) 0 : l(87) 1 :O(92) 0:1(78) 1:0(95) 1: 1.9(70) 1: O(90)” 4.3 : l(95)

MeLi 1 :0(58)“ 1:0(95)” 1 : O(93)”

0:1(80)” 2 : 3(90)” 0 : l(90)“ 1: O(80)” 0:1(96)” 0 : l(96)’

“Axial and equatorial attack are designated on the basis of the 4C1conformation of the individual aldosidulose. * A ring-expansion product was obtained in -65, -24, and 21% yield for 74, 75, and 76, respectively. The reaction was conducted in ether at -78”, and the others at room temperature. Ether-oxolane was used as the solvent.

OMe

OMe

87

JUJI YOSHIMURA

88

methyl-a-~-arabino-hexopyranosid-4-ulose (81).It was concluded that the stereoselectivity of the diazomethane reaction is mainly controlled

o

w

OMe

-g

q

P

77

OMe

*c,

f "C,

OMe

OMe OMe

B 1,4

OMe

81 O S2

by the attractive, electrostatic force between the diazomethyl cation and the neighboring, axial oxygen atom, or the axial lone-pair electrons of 0 - 5 in the transition state.166The reverse selectivity between the reaction of the hexopyranosid-3-uloses 56b,d and 69 (see Table 11) is explicable by the transition states A and B (see Fig. l),and the formation ofthe ring-expansion product, by C. In addition, it is known that the stereo-

SYNTHESIS OF BRANCHED-CHAIN SUGARS

phy% RO

+

N; *

OMe

B

&:; H OMe F

FIG.1.-Transition

0-

...OMe

A

H

89

E

*:, OMe

G

States in the Diazomethane Reaction.

selectivity in the reduction of ~-hexopyranosid-2-uloses with hydride For the p anoanions is controlled by the anomeric c~nfiguration.'~~-"~ mer, equatorial attack is predominant, due to the electrostatic repulsion of the axial approach bisecting the C-1-0-1 and C-1-0-5 torsional angle, whereas, for the (Y anomer, axial attack is predominant due to the (172) G. J. F. Chittenden, Carbohydr. Res., 15 (1970) 101-109. (173) T. D. Inch, G. J. Lewis, and N. R. Williams, Carbohydr. Res., 19 (1971) 17-27.

90

JUJIYOSHIMURA

torsional strain174and the dipole repulsion that would be caused by the equatorial approach. However, the results in the diazomethane reaction of hexopyranosid-2-uloses (64,71, and 72) are completely the reverse of those for reduction with sodium borohydride, and may be rationalized by the transition states D and E. The result for methyl 4,6-O-benzylidene-3-O-methyl-~-~-ribo-hexopyranosid-2-ulose (73) indicated a stronger effect of the axial, 3-methoxyl oxygen atom in the a-position than of the lone-pairs on 0-5 in the &position. Likewise, the predominance of the axial attack, and the formation of ring-expansion products in the cases of 74 and 75, were rationalized by the transition states F and G . The conformations of hexopyranosid-4-uloses are readily changeable, and other results are commonly explained by the axially or quasi-axially oriented oxygen atom or the lone-pair electrons.1seAlthough the reasons for the aforementioned contrasting stereoselectivities in the Grignard and methyllithium reactions are still ambiguous, data on the Grignard and diazomethane reactions of 62 and 63, and other^,'^^-'^^ can be understood in a similar way. On the other hand, it has been reported that such equilibration reactions as the ~ y a n o h y d r i n ~ and ~ ~ Jthat ~ ~with ~ ~ 7nitr0methane”3-”~J~~ ~ give the epimers in various mixtures whose compositions depend on the reaction conditions used. However, selective synthesis of the kinetically controlled products 54a (R’ = CN),12454a (R’ = CH,N0,),181 57a (R’ = CN),lE2and 57b (R’ = CH2N02)183could be achieved by use of a lower temperature, and of a weaker base as the catalyst. Moreover, it was proved that the epimerization of 54a (R‘ = CH2N02)to the thermodynamically controlled epimer 55a (R’ = CH2N02)proceeds by way of the parent 53a, with an activation energy183 of 75 f 8 kJ/mol. The reaction of acetonitrile in liquid ammonia gave only the thermodynamically controlled products, 55a (R’ = CH2CN)12s and 58a (R’ = CH2CN),125probably due to the use of such a strong base as lithium amide. It is interesting that the one-flask cyanomesylation of 56a in pyridine with hydrogen cyanide and then mesyl chloride gave, selec(174) E. C. Ashby and J. T. Laemmle, Chem. Rev., 75 (1975) 521-546. (175) D. Horton andE. K . Just, Carbohydr. Res., 18 (1971) 81-94. (176) I. Izquierdo Cubero and M. D. Portal Olea, Carbohydr. Res., 89 (1981) 65-72. (177) J. Thiem and J. Elvers, Chem. Ber., 111 (1978) 3514-3515. (178) P. J. Garegg and T. Norberg, Acta Chem. Scand., Ser. B, 29 (1975) 507-512. (179) A. Rosenthal and B. L. Cliff, Can. J . Chem., 54 (1976) 543-547. (180) G . Vass, A.-M. Sepulchre, and S . D. Gero, Tetrahedron, 33 (1977) 321-324. (181) K. Sato, J. Yoshimura, and C. Shin, Bull. Chem. Soc. jpn., 54 (1977) 1191-1194. (182) J. Yoshimura, M. Matsuzawa,K. Sato, andM. Funabashi, Chem. Lett., (1977) 14031406. (183) K. Sato, K. Koga, H. Hashimoto, and J. Yoshimura, Bull. Chem. SOC. Jpn., 53 (1980) 2639- 2641.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

91

tively, the mesylate of 57a (€3’ = CN),181whereas that in dichloromethane with aqueous potassium cyanide and sodium hydrogencarbonate afforded,lE4again selectively, the thermodynamically controlled product, namely, the mesylate of 58a (R’ = CN). Likewise, under the former or the latter conditions, 59 gave,185selectively, the mesylate of 61 (R’ = CN) or 60 (R’ = CN), respectively. Also, methyl 2,6-dideoxy-3-0methyl-~-~-threo-hexopyranosid-3-ulose (82) gavels6 methyl 3-C-cyano2,6-dideoxy-3-0-mesyl-4-O-methyl-~-~-xyl~-hexopyranoside (83) or a 1:5 mixture of 83 and its 3-epimer (84), respectively. More data will have to be acquired in order to disclose the factors governing the thermodynamic stability.

Me0 82

83

84

2. Addition to C-Alkylidene Glycosides Addition to the alkenic function of alkylidene glycosides, obtained from glycosiduloses by the Wittig reaction,ls7 is the second useful method for the synthesis of branched sugars, and some fluorinated, branched sugars have been described by Penglis.ls8 Because reagents approach the homomorphous 3-C-cyanomethylene derivative (85a) of (184) T. T. Thang, F. Winternitz, A. Olesker, A. Lagrange, and G . Lukacs, Chem. Commun., (1979) 153- 154. (185) T. T. Thang, F. Winternitz, A. Lagrange, A. Olesker, and G . Lukacs, Tetrahedron Lett., (1980) 4495-4498. (186) J. Yoshimura, T. Yasumori, T. Kondo, K. Sato, and H. Hashimoto, Curbohydr. Res., 106 (1982) c l - c 3 . (187) Yu. A. Zhdanov, Yu. E. Alexeev, and V. G. Alexeeva, Adu. Curbohydr. Chem. Biochem., 27 (1972) 227-292. (188) A. A. E. Penglis,Adu. Carbohydr. Chem. Biochem., 38 (1981) 195-284.

JUJI YOSHIMURA

92

53 from the exo direction, permanganate oxidation of 85a gave,189J90 exclusively, the epimeric 3-C-formyl(86a) and, therefore, 3-C-hydroxymethyl (86b) derivatives of 55b. Likewise, 86b and 55 (R' = Me) were191J92obtained from 85b by way of peroxy acid oxidation to the corresponding spiro epoxide. On the other hand, reduction of 85a, 85b, or 85c gave, selectively, the B-type branched sugars 87a,b (Ref. 193), 87c,d (Refs. 194 and 195), or 87c (Ref. 194),respectively, depending on the method used. Base-catalyzed condensation of ethyl isocyanoacetate with 53 gave 85d by way of the normal addition product [54; R' = CH (NHCHO) CO,Et]; and osmium tetraoxide oxidation of 85d gavelgs 86c, whereas hydrogenation of 85d gave the B-type branched sugar (87e) having a chiral amino acid branch, depending on the (E)or (2)configuration of the

85

86

R' R' X a -0OH b H OH OH c H COC,Et OH

d H

H e H C0,Me

R' RZ a H CN b H H c H SMe d NHCHO COaEt O H C0,Me f H OBn g NBz C0,Me

NH, NH,

R=MeC 'OCH

'

I

'

87

a b c d e

f

R' R" H CH,CN H CH,CH,NH, H H H OH NH, C0,Et NHBz C0,Me

CH,OBn, CH,OTr, or CH,

(189) J. M. J. Tronchet, J.-M. Bourgeois, J.-M., Chalet, R. Graf, R. Gurny, and M. T. Tronchet, Helv. Chim. Acta, 54 (1971) 687-691; J. M. J. Tronchet and J.-M. Bourgeois, i b d , 55 (1972) 2820-2827. (190) M. Funabashi, H. Wakai, K. Sato, and J. Yoshimura, J. Chem. Soc., Perkin Trans. 1 , (1980) 14-19. (191) M. Funabashi, H. Sato, and J. Yoshimura, C h . Lett., (1974) 803-804; Bull. Chem. SOC. Jpn., 49 (1976) 788-790. (192) J. M. J. Tronchet and M. T. Tronchet, Helu. Chim. Acta, 60 (1977) 1984-1989. (193) A.RosenthalandD.A.Baker,J. Org. Chem.,38 (1973) 198-201; TetrahedronLett., (1969) 397-400. (194) J. M. J. Tronchet and R. Graf, Helv. Chim. Ada, 55 (1972) 1140-1150. (195) A. Rosenthal and M. Sprinzl, Carbohydr. Res., 16 (1971) 337-342. (196) A. Jordaan, M. Malherbe, and G. R. Woolard, J. C h . Res., Synup., (1979) 60.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

93

parent double bond.lg7It was foundlg8that treatment of 85b with mercury(I1) acetate and azide ion, followed by reduction of the adduct with sodium borohydride, and hydrogenolysis of the product gave, regioselectively, the branched amino sugar (86d), which has the same configuration as that obtained from 53 by the Bourgeois method.lZ7From this fact, the intramolecular, S N mechanism ~ of the formation of the spiro aziridine from the cyanomesyl derivative was disclosed. In addition to the similar conversion of alkylidene derivatives from other aldosuloses, such as methyl 2,3-O-isopropylidene-6-O-methyl-cu-~-Zyxo-hexopyranosid-4-ulose (88),lQ9 1,2: 5,6-di-O-isopropylidene-a-~-xyZo-hexofuranos-3-dose (89),eoo*zo1 methyl 3,4-O-isopropylidene-P-~-threo-pentofuranosid-2-ulose (90),eooand 62189~202~203 into both A- and B-type FH,OMe

Me&-

88

HCO,

90

I ,CMe, H,CO 89

branched sugars, conversion of 85e into 86e,204osmium tetraoxide oxidation of 85f into 86a,205and addition of phosphonate,206ethyl cyanoacetate,207azido iodide,z08and nitryl iodidezogto the alkenic function of alkylidene derivatives have been reported. The adduct of azido iodide was converted into the branched amino sugar by way of the spiro aziri(197) A. J. Brink, J. Coetzer, A. Jordaan, G . L. Lourens, TetrahedronLett., (1972);53535356; A. J. Brink and A. Jordaan, Carbohydr. Res., 34 (1974) 1-13. (198) J. S. Brimacombe, J. A. Miller, and U. Zakir, Carbohydr. Res., 49 (1976) 233-242; 44 (1975) c 9 - c l l . (199) J. M. J. Tronchetand J.-M. Chalet, Carbohydr. Res., 24 (1972) 263-282,283- 296. (200) A. Rosenthal and D . A. Baker, Carbohydr. Res., 26 (1973) 163-167. (201) J. M. J. Tronchet and D. Schwarzenbach, Carbohydr. Res., 38 (174) 320-324. (202) K. Bischofberger, A. J. Brink, 0.G . De Villiers, R.H. Hall, and A. Jordaan,]. Chem. Soc., Perkin Trans. 1 , (1977) 1472-1476. (203) A. Rosenthal and M. Sprinzl, Can. ]. Chem., 48 (1970) 3252-3256. (204) A. Rosenthal and M. Ratcliffe, Carbohydr. Res., 60 (1978) 39-49. (205) I. Dyong, J. Weigand, and W. Meyer, Tetrahedron Lett., (1981) 2969-2970. (206) J . M. J . Tronchet, J.-R. Neeser, L. Gonzalez, andE. J. Charollais, Helu. Chim.Actu, 62 (1979) 2022-2024. (207) A. Rosenthal and R. H. Alex,]. Carbohydr. Nucleos. Nucleot., 5 (1978) 545-547. (208) J. S. Brimacombe, M. S. Saeed, andT. J. R.Weakley,J. Chem. Soc., Perkin Trans. 1 , (1980) 2061-2064. (209) J. Yoshimura, T. Iida, H. Wakai, and M. Funabashi, Bull. C h . Soc.]pn.,46 (1973) 3207-3209.

JUJI YOSHIMURA

94

dine derivative.208Condensation of 53a210or 56a211with 2-phenyl-%oxazolin-5-one gave, respectively, a 1: 1or 2 : 1 (E, 2) mixture of the condensation products (91 and 92). Methanolysis of 91 with a catalytic amount of sodium acetate gave 85g, which was then hydrogenolyzed with rhodium-on-alumina into 87f, an L-amino acid derivative. This procedure offers a homologous route2I2 to the direct introduction of the amino acid function into a glycosidulose by way of the spiro hydantoin (93).

'zozG w N y N w0-CMe, Ph (Z)-91

Ph

i (Z)-92

II

0 93

In contrast to the aforementioned, nucleophilic addition to glycopyranosiduloses, that to C-alkylideneglycopyranosidesis used for the introduction of an axially oriented branch. Thus, the oxymercuration demercuration of the 3-C-methylene derivative from 59 was used2I3for the synthesis of 12. It was reported that the stereoselectivity in the oxidation with osmium tetraoxide is very high, due to the steric requirement of complex-formation in the intermediary state,214*215 whereas that in the peroxy acid oxidation is moderate.21s Radical deoxygenation of 0-benzoylated, A-type into B-type, branched sugars with tributyltin hydride was found, and the method was applied2" for preparation of an insect sex-attractant. Reductive alkylation of carbonyl compounds as the principal reaction was also reported.218 (210) A. Rosenthal and K. Dooley,]. Carbohydr. Nucleos. Nucleot., l ( 1 9 7 4 ) 61-65. (211) A. Rosenthal and K. Dooley, Carbohydr. Res., 60 (1978) 193-199. (212) H. Yanagisawa, M. Kinoshita, S. Nakada, and S. Umezawa, Bull. Chem. Soc. Jpn., 43 (1970) 246-252. (213) E. H. Williams, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47 (1969) 44674471. (214) D. L. Walker and B. Fraser-Reid,]. Am. Chem. Soc., 97 (1975) 6251-6253. (215) J. Yoshimura and M. Matsuzawa, Carbohydr. Res., 96 (1981) 7-20. (216) J. Yoshimura, K. Sato, and M. Funabashi, Bull. Chem. Soc. Jpn., 52 (1979) 26302634. Neumann,andH. Paulsen,Chem. Ber.,110(1977)2911-2921;H. (217) H.Redlich, J.H. Redlich and J. Xiang-jun, justus Liebigs Ann. Chem., (1982) 717-722. (218) S. S. Hall and F. J. McEnrose,]. Org. Chem., 40 (1975) 271-275.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

95

The chiral s y n t h e s i ~ , by ~ ~the ~ -use ~ ~of~monosaccharides, of optically active natural products having mainly branches of the B-type has now become common in organic chemistry. The reduction of alkylidene derivatives was actually used for the synthesis of thromboxane B,,222canad e n ~ o l i d eand , ~ ~a~degradation product from b ~ r o m y c i n . ~ ~ * 3. Nucleophilic Reactions of Sugar Oxiranes

The reaction of oxiranes with carbon nucleophiles provides a general method for the synthesis of B-type branched sugars. Thus, the diaxial ring-opening of methyl 2,3-anhydro-4,6-0-benzylidene-a-~-mannopy-

--C;clr

OMe -Ph<@

0

OCH,

OCH,

OMe

OMe 2 h - Q

0

Et 94

0 95

CN 99

96

~H,OH 97

90

ranoside (94) and -a-D-allopyranoside (100) with ethylmagnesium chloride (enriched with diethylmagnesium) gave preponderantly 3-ethyl (95) and 2-ethyl (2-C-ethyl analog of 101) derivatives, respectively.22g Oxidation of 95, followed by epimerization at C-3 (96),methylenation by a Wittig reaction, and hydroboration, gave a 1:l mixture of the corre(219) T. D. Inch, Adv. Carbohydr. Chem. Biochem., 27 (1972) 191 -225; B. Fraser-Reid and R. C. Anderson, Fortschr. Chem. Org. Naturst., 39 (1978) 1-61. (220) S. Hanessian, Acc. Chem. Res., 12 (1979) 159-165. (221) H. Ohrui, Yuki Cosei Kagaku, 39 (1981) 275-287. (222) S.Hanessian and P. LavallBe, Can.J . Chem., 55 (1977) 562-565; 59 (1981) 870877. (223) R.C. Anderson and B. Fraser-Reid, Tetrahedron Lett., (1978) 3233-3236. (224) S. Hanessian, P. C. Tyler, G . Dernailly, and Y. Chapleur, J. Am. Chem. SOC., 103 (1981) 6243-6246. (225) T. D. Inch and G . J . Lewis, Carbohydr. Res., 22 (1972) 91-101; 15 (1970) 1-10,

JUJI YOSHIMURA

96

-

sponding 2-C-(hydroxymethyl) derivatives (97 and 98) containing two branch points. Diethyl sodiomalonate (100 101),226,227 hydrogen

Ph<&

OMe

-

Ph-(@r

HO

0 100

OCH,

101

-

cyanide- triethylaluminum (94 99),22sd i m e t h y l m a g n e ~ i u m lith,~~~ ium dimethyl and 48232have also been used as nucleophiles. Reaction of methyl 2,3-anhydro-5-0-trityl-cr-~-ribofuranoside (102a) with 48,233 and of the /3 anomer (102b) with methylmagnesium ~hloride,~34 gave regioselectively 2-C- (103) and 3-C-alkyl(lO4) deriva-

Tro%c@

c-T r O H d j ! ! :

-

T r O MHe a C q

OMe

OH

HO 103

102a R' = H, Rz = OMe l O 2 b R' = OMe, Ra = H

104

tives, respectively, depending on the anomeric configuration. This type of reaction was applied for the total synthesis of (-)-N-methylmayserine23J and rifamycin.236It was found237that the reaction of 100with ethyl 2-(diethy1phosphono)propanoate [(EtO),P(O)CHMeCO,Et] gave, ex(226) S. Hanessian and P. Dextraze, Can. ]. Chem., 50 (1972) 226-232. (227) S. Hanessian, P. Dextraze, and R. Masse, Carbohydr. Res., 26 (1973) 264-267. (228) B. E. Davison and R. D. Guthrie, ]. Chem. SOC., Perkin Trans. 1 , (1972) 658-662. (229) A. Ya. Shimyrina, A. F. Sviridov, 0. S. Chizhov, A. S. Shashkov, and N. K.Kochetkov, Izu. Akad. Nauk SSSR,(1977) 461 -463. (230) S. Hanessian and C.Rancourt, Can. J. Chem., 55 (1977) 11 11 - 11 13. (231) H. Yamamoto, H. Sasaki, and S. Inokawa, Carbohydr. Res., 100 (1982) c44-c45. (232) A.-M. Sepulchre, G. Lukacs, G. Vass, and S. D. Gero, Angew. Chem., 84 (1971) 111 - 112. (233) A. Yamashita and A. Rosowsky,]. Org. Chem., 41 (1976) 3422-3425. (234) S . R . Jenkins andE. Walton, Carbohydr. Res., 26 (1973) 71-81. (235) E. J. Corey, L. 0.Weigel, A. R. Chamberlin, and B . Lipshutz,]. Am. Chem. SOC., 102 (1980) 1439-1441. (236) M. Nakaya, Y. Ikeyama, H. Takao, and M.Kinoshita, Bull. Chem. Soc.Jpn.,53 (1980) 3252-3258. (237) B. J. Fitzsimmons and B . Fraser-Reid,J. Am. Chem. SOC., 101 (1979) 6123-6125.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

97

clusively, one stereoisomer of the methoxycarbonyl-cyclopropane der i v a t i ~ e ~(105), ~ * * from ~ ~ ~which (+) - and (-)-chrysanthemumdicarboxylic acids were selectively synthesized.

pp

101

4. Addition to Unsaturated Sugars

a. Enopyranosides. -Although the alkenic function of the usual enopyranosides, such as 106, is inactive to addition reactions, the allylic acetate moiety of 106 forms an intermediate n-allylpalladium complex with tetrakis(triphenylphosphine)palladium(O) which reacts with carbanions to give,240regio- and stereo-selectively, and regardless of the anomeric configuration, 4-C-substituted derivativese40(107). The conversion of such a cyclopropane derivative of a sugar as241108, produced by the addition of carbenes to glycenoses, into a heptose derivative was reported.242 CH,OAc

AcOb

E 106

CH,OAc

t

107a R R RT =! R' ! O = C0,Me E t 107b R = CO,Me, R' = S0,Ph

'b

HCO,

I

H,CO'

0-CMe, CMe, 108

The addition reaction to branched glycoenopyranosides sometimes offers a stereospecific pathway to A-type, branched sugars. A stereospecific synthesis of the evalose (15) derivative (110) by cis-hydroxylation of methyl 4-0-benzoyl-2,3,6-trideoxy-3-C-methyl-~-~-e~t~~o-hex-2-enopyranoside (log),derived from 57a (R' = Me), with osmium tetraoxide (238) W. Meyer zu Reckendorf and U. Karnprath-Scholtz,Chern. Ber., 105 (1972) 673685. (239) B. Fraser-Reid and B. J. Carthy, Can. J . Chern., 50 (1972) 2928-2934. (240) H. H. Baer and Z. S. Hanna, Can. J . Chern., 59 (1981) 889-906. (241) J. S . Brirnacornbe, E. M. Evans, E. J. Forbes, A. B. Foster, and J. M. Webber, Carbohydr. Res., 4 (1967) 239-243. (242) P. Duckaussory, P. DiCesare, and B. Gross, Synthesis, (1979) 198-200.

98

JUJIYOSHIMURA

was reported.243The 3-epimer (114) of sibirosamine (22) was obtained from 109 as follows.244Successive O-debenzoylation, introduction of a phenylthio group with inversion [by treatment with N-(pheny1thio)succinimide and t r i b u t y l p h o ~ p h i n e ~and ~ ~ ]peroxy , acid oxidation gave the 4-(phenylsulfinyl) derivative (111). Suprafacial [3,3]-sigmatropic rearrangement of 111 with trimethyl phosphite in methanol gave, exclusively, methyl 3,4,6-trideoxy-3-C-methyl-a-~-threo-hex-3-enopyranoside (112) by transfer of the chirality at C-4 to C-2. After O-acetylation of 112, cis-oxyamination with osmium tetraoxide and chloramine-T trihydrate in tert-butyl alcohol gave,246regio- and stereo-specifically, methyl 2 - 0 - acetyl- 4,6 - dideoxy - 3 - C - methyl - 4 - (p-toluenesulfony1amino)a-D-altroside (113), which was converted into 114. cis-Oxyamination was

also used"' for synthesis of the 4-epimer of 21. Regioselectivity in the cis-oxyamination of the 1,e-disubstituted alkenic function is not so high, but, in the case of trisubstituted alkenes, the secondary amino, tertiary alcohol is mainly produced.248 (243) L. Valente, A. Olesker, L. E. S . Barata, R. Rabanal, G . h k a c s , and T. T. Thang, Carbohydr. Res., 90 (1981) 329-333. (244) I. Dyong and G . Schulte, Chern. Ber., 114 (1981) 1484- 1500; Tetrahedron Lett., (1980) 603-606. (245) K. A. M. Walker, Tetrahedron Lett., (1977) 4475-4478. (246) K. B. Sharpless, A. 0.Chong, andK. Oshima,]. Org. Chern., 41 (1976) 177-179;E. Herranz and K. B. Sharpless, ibid., 43 (1978) 2544-2548. (247) I. Dyong and J. Jersh, Chern. Ber., 112 (1979) 1849-1858. (248) G . Schulte, W. Meyer, A. Starkloff, and I. Dyong, Chern. Ber., 114 (1981) 18091821; H. Friege and I. Dyong, ibid., 114 (1981) 1822-1835.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

99

b. Nitro-alkenic Sugars. -Michael-type addition-reaction to unsaturated sugars proceeds with particular ease, that is, under neutral or even slightly acidic conditions, when the double bond carries a nitro group. Thereby, the steric course of the carbanion addition is determined by the substituent that is vicinal to the place of nucleophilic attack, the nucleophile entering with high preference, if not exclusively, from the side opposite thereto. This is nicely borne out by the addition of various nucleophiles to methyl 4,6-0-benzylidene-2,3-dideoxy-3-C-nitro-a-~eqthro-hex-2-enopyranoside(115) or its p anomer, which give24g-251 the thermodynamically less-stable products having the ~ Y - D - ~ u ~configu~IO ~ ~high ~-~~~ ration (116a-f) or the respective p-D-gluco c o m p o ~ n d s , in yields. Similarly, the p-D-threo analog 117 afforded addition products having the P-D-gulacto configuration (118a-f), not the alternative, p-Dtdo c o m p o ~ n d s . ~ ~ ~ * ~ ~ ~

(249) T. Sakakibara and R. Sudoh, Carbohydr. Res., 50 (1976) 191-196;]. Chem. SOC., Chem. Commun., (1974) 69-70. (250) T. Sakakibara,A. Seta, Y.Tachimori, T. Takamoto, andR. Sudoh, Chem. Lett., (1977) 1365-1366. (251) H. H. Baer and Z. S . Hanna, Carbohydr. Res., 85 (1980) 136-142. (252) H. H. Baer and K. S . Ong, Can. J . C h m . , 46 (1968) 2511-2517. (253) H. Paulsen and W. Grewe, C h m . Ber., 107 (1974) 3013-3019.

100

JUJI YOSHIMURA

Michael additions to nitroalkenes of type 119 may similarly be effected with particular ease. Reaction of 119a with vinylmagnesium bromide,254 with dithiane (48a), or with n i t r ~ m e t h a n e , gave ~ ~ ~the . ~ ~5-C-substi~ tuted nitro sugars 120a-c, which, like their unbranched analogs,e57are intramolecularly cyclized upon liberation of the aldehyde function by deacetonation, to yield a mixture of branched nitrocyclitols, the major isomers having the nitro group, as well as the branching substituent, in the equatorial orientation.258Addition of hydrogen cyanide to 119b may similarly be effected, and yet, owing to the presence of triethylamine, and subsequent elimination of nitrous acid, the respective cyano-alkene 121 is formed.253

1200 R = CH=CH, 120b R = CH,NO, 120c R =

3'3

1190 R = B n ll9b R = A c

121

S

c. Pyranoid Enones.-Addition reactions to enones of type 122 or 125 proceed stereoselectively, and yet either 1,2- or 1,4-addition of the nucleophile, or both, may be elicited. Thus, addition of lithiocopper organic reagents{(PhS),CLiCH,OLi-CuI,LiCu[(PhS),CH],, and LiCu [(PhS),CMe],} to methyl 2,3,6-trideoxy-a-~-gZycero-hex-2-enopyranosid-4-ulose (122a) gave only the l,e-adduct, a 1.2:l mixture of the 1,2- and 1,4-adducts (123a), and only the 1,4-adduct (123b), respect i ~ e l y . Similar , ~ ~ reaction of 2-(ethoxycarbonyl)-2-lithio-1,3-dithiolane,

(254) T. Iida, M. Funabashi, and J. Yoshimura, Bull. Chem. SOC. Jpn., 46 (1973) 32033206. (255) M. Funabashi, K. Kobayashi, and J. Yoshimura, J. Org. Chem., 44 (1979) 16181621. (256) M. Funabashi and J. Yoshimura,]. Chem. Soc.,Perkin. Truns. 1, (1979) 1425-1429. (257) J. M. Grosheintz andH. 0.L. Fischer,J. Am. Chem. SOC., 70 (1948) 1479-1484;F. W. Lichtenthaler, Chem. Ber., 94 (1961) 3071 -3085. (258) M. Iwakawa, J . Yoshimura, and M. Funabashi, BUZZ. Chem. SOC. Jpn., 54 (1981) 496-500. (259) H. Paulsen and W. Koebernick, Curbohydr. Res., 56 (1977) 53-66.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

101

a more stable and less-reactive carbanion than 48, with 122a or its p anomer (122b)gave the 1,4-addition products 123c and 124 in high yields, 260,261 the steric course obviously being largely determined by the disposition of the anomeric substituent. The (Y-D analog (125)readily yields the hexosid-4-ulose 126 for analogous reasons.259Cycloaddition of diazomethane to the alkenic function of 125 has been reported.262

oqe oa - oa OMe

R2

R 123a R = CH(SPh), 123b R = [MeC(SPh),], 123c R

=

122a R' = OMe, R2 = H 122b R' = H, Rz = OMe

124 R = s n s

sn

)cCO,Et

XS C0,Et

$HzOTr

$H20Tr

125

126

For such hex-3-enopyranosid-2-ulosesas 127 or 129, reaction with lithium dimethylcuprate,261or with anions of malonic ester-type, methylene components in the presence of bis(2,4-pentanedionate)-Ni(II)cata l y ~ taffords, , ~ ~ ~in each case, the 1,4-addition products (128 and 130, respectively), in which the branching group is in the axial orientation. The methyl-branched pyranosides 126 and 128,readily accessible in this way, have been of use as chiral precursors for the synthesis of multistriatins.264.265

(260) H. Paulsen and W. Koebernick, Chem. Ber., 110 (1977) 2127-2145. (261) M. B. Yunker, D. E. Plaumann, and B. Fraser-Reid, Can. J . Chem., 55 (1977) 40024009. (262) R. M. Srivastava, B. J. Carthy, andB. Fraser-Reid, Tetrahedron Lett., (1974) 21752178. (263) F. Shafizadeh, D. D. Ward, and D. Pang, Carbohydr. Res., 102 (1982) 217-230. (264) S. Hanessian, P. C. Tyler, and Y. Chapleur, Tetrahedron Lett., (1981) 4583-4586. (265) B. J. Fitzsimmons,D. E. Plaumann, and B. Fraser-Reid, Tetrahedron Lett., (1979) 3925-3928.

JUJIYOSHIMURA

102

HzC-

'0

127a R = Tr 127b R = Ac

128

129

1300 R = CH(CO,Et), 130b R = CH(CN)CO,Et 1 3 0 ~R = CH,NO,

d. Pyranoid Enolones. -Branching of such pyranoid enolone esters as 131 or 137, which are readily accessible in large variety from the respective hydroxyglycal esters,266may be readily accomplished, high regio- and diastereo-selectivities being attainable by choice of the conditions or the type of nucleophile, or both. With lithium dimethylcuprate, exclusive 1,4-addition is observed for 131 and 137, but the primary adducts undergo further reactions, such as a distinctly selective 3-2-0-benzoyl migration (132 -133) which results in the elaboration of the respective 4-methyl-branched h e ~ o s i d - 3 - u l o s e(136), ~ ~ ~ or, in the presence of a leaving group in an anomeric position, in a 4-methylbranched enediolone (138). With methyllithium or methylmagnesium bromide, however, almost exclusive 1,2-addition is observed, to give either the enol ester (134) or, under slightly more basic conditions, the 2-methyl-branched hexosid-3-ulose (135) in remarkably clean react i o n ~The . ~ stereochemical ~~ outcome of the conversions 131-135 and 137-139, which is analogous to that of hydride additions to these enolones,268is clearly a result of stereocontrol by the anomeric substituent, the nucleophile entering exclusively from the opposite side. Such 1,5anhydrohex-1-enitol-3-ulosesas 140 may also be considered to be enolones in which the electronic contribution by the ring-oxygen atom induces the aforementioned addition at the carbonyl function instead of at (266) F. W. Lichtenthaler, Pure Appl. Chem.,50 (1978) 1343-1362. (267) F. W. Lichtenthaler and P. Kahler, unpublished results. (268) F. W. Lichtenthaler, U. Kraska, and S. Ogawa, Tetrahedron Lett., (1978) 13231326.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

103

CH,OBz I

BzO Q e -

0

131

132

133

$H,OBz

I

BZO'

M~

Me

OBz

134

135

136

CH,OBz

$H,OBz

YqOBz

138

137

139

the anomeric center. Accordingly, addition of methyllithium to 140 gives L-olivomycal (141) and L-mycaral (142) in 70 and 30% yields, re-

H

O

P

-

H

Meo

p

f

H

HO

0

140

u1

OH O

V

Me 142

spectively; these were c o n ~ e r t e d into ' ~ ~L-olivomycose ~~~~ (12) and Lmycarose (8).This method was also extendedZ7Oto the D series, and to the synthesis of disaccharides. (269) J. Thiem and J. Elvers, Chem. Ber., 112 (1979) 818-822. (270) J. Thiem and J. Elvers, Chem. Ber., 114 (1981) 1442-1454.

104

JUJI YOSHIMURA

5. Aldol Addition The aldol addition of formaldehyde in the presence of sodium hydroxide for the introduction of a hydroxymethyl branch was first applied to 1,2:3,4-di-O-isopropylidene-aldehydo-~-arabinose, and the product (143), formed by a crossed-Cannizzaro reaction, was converted into 1b y H2C4

I

HCO'

CMe,

I

HCO, OC-CH,OH

I

CH,OH 143

removal of the terminal isopropylidene group, followed by periodate oxidation and d e p r o t e c t i ~ n . ~ ' ~ Application of this method to 1,2-0-isopropylidene-a-~-pentodialdo1,4-furanoses [144a,b (Ref. 272) and 144c (Ref. 273)]gave the corresponding 4-C-(hydroxymethyl) derivatives (145a-c), and various nucleosides of them were also synthesized, OHC

Wp R2

HOH,C --t

H

o

0-CMe,

H

z

R2

144 a R ' = OH, R 2 = H

c

I

~

0-CMe,

145

bR'=RZ=H c R' = H, Rz = OH d R' = NMeAc, Rz = H

A similar reaction of an analogous amino sugar (146) at pH 8 and pH 10.5 gave selectively 145d and the tricyclic derivative (147), respectively. The cleavage of the tetrahydrooxazine ring of 147 with iodine and sodium acetate gave the ring-opening compound (148) from which 21 was ~ b t a i n e d . " ~Moreover, it was found that no Cannizzaro reaction accompanies an aldol addition if a weaker base is used. Thus, reaction of 2,3-O-isopropylidene-~-ribose (149) with formaldehyde and potassium (271) D. T. Williams and J. K. N. Jones, Can. J . Chem., 42 (1964) 69-72. (272) D. L. Leland and M. P. Kotick, Carbohydr.Res., 38 (1974) c 9 - c l l . (273) R. D. Youssefyeh, J. P. H. Verheyden, and J. G . Moffatt,]. Org. Chem., 44 (1979) 1301-1309; Tetrahedron Lett., (1977) 435-438. (274) J. J. Wright and C. L. Luce,]. Org. Chem., 43 (1978) 1968-1972.

SYNTHESIS OF BRANCHED-CHAIN SUGARS 0

- "y:3

+:> -

OHC

105

HO&C

HOH,C

0-CMe,

0-CMe,

0-CMe,

146

147

148

carbonate in aqueous methanol for 2 days at 80"gave the 2-C-(hydroxymethyl) derivative (150) in 84% yield, from which 2 was obtained in

-

HOH,Cv

HOH2Cp

OH HOH,C

0,

/o

0,

CMe,

149

/o

CMe,

150

excellent yield.27sCompounds 1and L-1were also derived from 2,3:5,6di-0-isopropylidene-D-mannoseby a similar reaction.276Selective formation of such branched a l d i t o l ~ ~ as' ~2-deoxy-2-(hydroxymethyl)glycerol(l51) and a 2,3,4-trideoxy-2,4-di-C-(hydroxymethyl)pentitol (152) in the formose reaction278was reported. CH,OH

CH,OH

C(H)CH,OH

C (H)CH,OH

CH,OH

YHz C(H)CH,OH

I I

I

I

I

CH,OH 151

152

6. Photochemical Addition Photochemical reactions of carbohydrates have been discussed.279 Therefore, only examples applicable to the synthesis of branched sugars are briefly described here. Although non-stereoselectivity and low yield are general defects of photoreactions, photoaddition of an alcohol to the enones 125 and 122a proceeded with the same stereo- and regio-selectivities as ionic addition ( see Section 11,4,c),and in rather better yields, (275) P.-T. Ho, Tetrahedron Lett., (1978) 1623-1626. (276) P.-T. Ho., Can. J Chem., 57 (1979) 384-386. (277) Y. Shigemasa, M. Kawahara, C. Sakazawa, R. Nakashima, andT. Matsuura,J. Catal., 62 (1980) 107-116. (278) T. Mizuno and A. H. Weiss, Adu. Carbohydr. Chem. Biochem., 29 (1974) 173 - 227. (279) R. W. Binkley, Adu. Carbohydr. Chem. Biochem., 38 (1981) 105- 193.

JUJI YOSHIMURA

106

to give analogszs0(2-C-branch -CHzOH,-C(OH)Me,, -CH(OH)CH,OH, -CH(OH)CH,CH,OH, -CH(OH)CH,CO,Me, and COMe) of 126 and 123, R = CH(OH)Me.zso A similar tendency was observedza1in the reaction of 2-C-methyl derivatives of 125. However, photoaddi, 'on of methanol to 127a gaveese the 4-C-(hydroxymethyl) analog of 125 and its 4-epimer in the ratio of 1:2.7. Addition to 1,2,4,6-tetra-O-acetyl-3-deoxy-a-~-erythro-hex-2e n o p y r a n ~ s e ~(153) * ~ * and ~ ~ ~methyl 4,6-di-O-acety1-2,3-dideoxy-a-~erythro-hex-2-enopyranoside(157) gave various mixtures of regio- and stereo-isomers, 154-156 and 158-159, depending on the reagents

CH,OAc

OMe R 157

R=

158

159

39%

39%

0 R = -C(OH)Me,

66%

(280) B. Fraser-Reid,N. L. Holder, D. R.Hicks, andD. L. Walker, Can.J. Chem.,55 (1977) 3978-3985, and literature cited therein. (281) B. Fraser-Reid, R. C. Anderson, D. R.Hicks, and D. L. Walker, Can. J. Chem., 55 (1977) 3986-3995. (282) B. Fraser-Reid, N. L. Holder, and M. B. Yunker, Chem. Commun., (1972) 12861287. (283) Y. Araki, K. Nishiyama, K. Senna, K. Matsuura, and Y. Ishido, Carbohydr. Res., 64 (1978) 119-126. (284) A. Rosenthal and M. Ratcliffe, Carbohydr. Res., 39 (1975) 79-86.

SYNTHESIS O F BRANCHED-CHAIN SUGARS

107

161 160

~

~ eA similar d result . ~was obtainedzs7 ~ ~ ~in the~ addition ~ of ~ formamide to the enol acetate of 89. Stereospecific formation of the 1,3-dioxolane adduct of 87 from 85b was explained2s8 by the approach of hydrogen, from the less-hindered direction, to the initial radical (160).Photochemical addition to glycosiduloses has also been reported.2se The cycloaddition product (161), obtained from 1,3-diacetoxy-2-propanoneand 1,3dioxol-2-one, was readily converted into DL-1by alkaline hydrolysis.z90 7. Cyclization of Dialdehydes with NitroalkaneseQ1 Extension to such well known nitromethane homologs as nitroethane,z92-z98n i t r o e t h a n ~ land , ~ ~ethyl ~ n i t r o a ~ e t a t eprovides ~ ~ ~ . ~a~simple ~ (285) K. Matsuura, K. Nishiyama, K.Yamada, Y. Araki, and Y. Ishido, Bull. C h . Soc.Jpn., 46 (1973) 2538-2542. (286) K. Matsuura, Y. Araki, Y. Ishido, and S. Sato, Chem. Lett.,(1972) 849-852. (287) A. Rosenthal and M.Ratcliffe, Curbohydr. Res., 54 (1977) 61-73. (288) J. S. Jewel1 and W. A. Szarek, Tetrahedron Lett., (1969) 43-46. (289) P. M. Collins, V. R. N. Munasinghe, and N. N. Oparaeche, J. C h . Soc., Perkin Trans. 1 , (1977) 2423-2428. (290) Y. Araki, J,-I. Nagasawa, and Y. Ishido, Curbohydr. Res., 58 (1977) c4-c6. (291) F. W. Lichtenthaler, in W. Foerst (Ed.), Newer Methods ofPreparatioe Organic Chemistry, Vol. IV, Verlag Chemie, Weinheim, 1968, pp. 155-195; H. H. Baer, Adu. Carbohydr. Chem. Biochem., 24 (1969) 67-138. (292) S. W. Gunner, W. G. Overend, and N. R. Williams, Chem. Ind. (London), (1964) 1523; J. S. Brimacombe and L. W. Doner, J. Chem. SOC., Perkin Trans. 1, (1974) 62 - 65. (293) H. H. Baer and G. V . Rao, Justus Liebigs Ann. Chem., 686 (1965) 210-220. (294) F. W. Lichtenthaler and H. K. Yahya, Carbohydr. Res., 5 (1967) 485-489. (295) F. W. Lichtenthaler, H. Leinert, and U. Scheidegger, Chem. Ber., 101 (1968) 1819-1836. (296) F. W. Lichtenthaler and H. Zinke,Angew. Chem.,Int. Ed. Engl., 5 (1960) 737-738; idem, in W. W. Zorbach and R. S . Tipson (Eds.), Synthetic Procedures in Nucleic Acid Chemistry, Vol. 1, Wiley-Interscience, New York, 1968, pp. 366-368. (297) F. W. Lichtenthaler and H. Zinke,J. Org. Chem., 37 (1972) 1612-1621. (298) M. M. Abuaan, J. S. Brimacombe, and J. N. Low, J . Chem. Soc., Perkin Trans. 1 , (1980) 995-1002; M. M. Abuaan, H. I. Ahmad, J. S. Brimacombe, and T. J. R. Weakly, Carbohydr. Res., 84 (1980) 336-340. (299) F. W. Lichtenthaler and H. Leinert, Chem. Ber., 101 (1968) 1815-1818. (300) S. Zen, Y. Takeda, A. Yasuda, and S . Umezawa, Bull. Chem. Soc. Jpn., 40 (1967) 431 -438. (301) H. Yanagisawa, M. Kinoshita, and S . Umezawa, Bull. Chem. Soc. Jpn., 42 (1969) 1719-1721.

JUJI YOSHIMURA

108

means for the simultaneous introduction of a nitro group and an alkyl branch into a carbocyclic or pyranoid ring, inasmuch as limitations on the scope of the reaction were early established.30eAccordingly, cyclization of dialdehydes 162-164 with nitroethane in the presence of sodium methoxide gives mixtures of C-methyl-branched nitrohexosides or nitroinositols, from which the respective major isomers, each having the nitro and the vicinal hydroxyl groups in equatorial orientation, may be separated either by fractional recrystallization [40% yield on 165b (Refs. 296 and 297), 14% on 167 (Ref. 294)], after acetylation (19% for 166a diacetatees2),or after hydrogenation and acetylation (34% for the amino sugar peracetate derivedes8 from 166b). Unlike dialdehyde l62b (that was exclusively cyclized to the respective D-hexosyl-nucleosides), 162a, 168, and 163b partially gave rise to products epimeric ates3 C-5 andes8 YH,OH

":LJ 0

Hq OH

0

0

0

l62a R = OMe 162 b R = uracil-1-yl

1

EtNOz

O

-Meg

OMe

L

164

R'CH,NO,

CH,OH

"

163a R = M e 163b R = H

EtNO,

OH

\

/

R

hie 165a R = O M e l65b R = uracil-1-yl

l66a R = R1 = M e l66b R = H, R' = M e 1 6 6 ~R = H,R' = C0,Et

167

C-1, obviously owing to base-catalyzed epimerization at the aldehyde stage. As compared with nitromethane cyclizations (wherein such epimerizations have not been observed), nitroalkanes react less readily, thus providing longer exposure of the dialdehyde to the alkaline conditions required for cyclization. N i t r o e t h a n ~ l ,l-nitropr0pane,~~5 ~~~ and ethyl nitroacetate cyclize normally with dialdehydes; for example, 163b gives a mixture of 3-nitrocarboxylic acids, with 166c as one of the principal p r o d ~ c t s . Sur~~~-~~~ prisingly, when treated with ethyl nitroacetate under the same condi(302) F. W. Lichtenthaler, Fortschr. Chern. Forsch., 14 (1970) 556-577.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

109

-

tions, dialdehyde 168 did not give the cyclization products 169a, but those resulting from a C-3 0 - 6 migration of the ethoxycarbonyl portion, that is, the structural isomer303169b. The limits of this nitroalkane cyclization are reached with such nitromethylene components as phenylnitromethane, that carry bulky substituents which preferentially give such mono-addition products as 170 (from glutaraldehyde), not cyclohexane compounds.304

Hfj :; -

bP

I

OMe

0 168

OH

OMe R

OH

1690 R = COzEt, R' = H 169b R = H , R' = COzEt

170

The cyclizing bis(aminoalky1)ation of n i t r ~ m e t h a n ewhich , ~ ~ ~provides a ready entry into pyranoid and cyclohexane nitrodiamines, and, hence, triamino sugars,3os may also be extended to nitroalkanes. Accordingly, glutaraldehyde reacts with nitroethane in the presence of benzylamine to afford the C-methyl-branched nitrodiamine 171 (56%) which may be obtained from the nitrodioll72 in even better yield (83%)by exposure to the same condition^.^^^^^^^ This procedure carries considerable potential for the preparation of C-branched triamino sugars. RHN

R = PhCH,

171

OH

172

8. Rearrangement Reactions It has long been known308 that alkaline degradation of sugars gives branched lactones through the benzilic acid type of rearrangement of (303) F. W. Lichtenthaler and G . Bambach,J. Org. Chem.,37 (1972) 1621-1624. (304) F. W. Lichtenthaler and D. Fleischer,J. Org. Chem., 37 (1972) 1670-1672. (305) F. W. Lichtenthaler, T. Nakagawa, and A. El-Scherbiney, Angew. Chem.,Int. Ed. Engl., 6 (1967) 568-569. (306) F. W. Lichtenthaler, T. Nakagawa, and A. El-Scherbiney, Chem. Ber., 101 (1968) 1837-1845; F. W. Lichtenthaler andT. Nakagawa, ibid.,101 (1968) 1846-1849. (307) T. Nakagawa, T. Sakakibara, and F. W. Lichtenthaler, Bull. Chem. SOC. Jpn., 43 (1970) 3861-3865. (308) J. C. Sowden, Ado. Carbohydr. Chem.,12 (1957) 35-79.

JUJI YOSHIMURA

110

intermediary a-diketones. a-D-Glucosaccharinic acid (173) and “a-Dglucoisosaccharinic” acid y - l a c t o n e ~(174), ~ ~ ~ respectively obtainable from invert sugar and 4-0-substituted D-glucose in 10-20% yields, are the most useful members. The former was used as a building block for a chiral synthesis310of lasalocid A, and the latter was converted into 3,4dideoxy-3-C-methyl-~-ribo-hexose through use of an Arndt - Eistert reaction.311 Although the reaction generally gives a mixture of many acids,1227 and its threo isomer were identified among the products from ~ - x y l o s e .A~branched l~ nucleoside (1 76) was prepared by application313 of the rearrangement to 7-(2,6-dideoxy-3,4-0-isopropylidene-~-~-Z~xohexopyranosyl-2-u1ose)theophylline (175). It is also known314 that treatment with nitrous acid of 3-amino-3deoxy- and 2-amino-2-deoxy-hexopyranosideshaving an equatorially oriented amino group gives formyl-branched furanosides, with expulsion of one carbon atom in a mode similar to that of their b i o s y n t h e s i ~A. ~ ~ branched nucleoside (1 78) was synthesized by rearrangement of the 3-amino-3-deoxy derivative 177, obtained from uridine through nitromethane c y c l i ~ a t i o nBy . ~use ~ ~ of a sulfonyl group, instead of a diazonium group, as the leaving group, methyl 2-amino-2-deoxy-a-~-glucopyranoside was converted31e into methyl 2-acetamido-2,3-dideoxy-3-C-(hydroxymethy1)-a-D-xylofuranoside(181). Treatment of the 2-N,3-0phenyldisulfonyl) derivative of methyl 2-amino-2-deoxy-a-~-glucopyranoside (179) with sodium methoxide gave the corresponding, branched, dialdehydo derivative (180), which, by deprotection and reduction, was converted into 181 in good yield.

HoH2 HOH,C

OH

173

174

(309) R. L. Whistler and J. N. BeMiller, Methods Carbohydr. Chem., 2 (1963) 477-479, 483-484; W. M. Corbett, ibid., 2 (1963) 480-482. (310) R. E. Ireland, R. C. Anderson, R. Bacloud, B. J. Fitzsimmons, G. J. McGarvey, s. Thaisrivongs, and C. S. Wilcox,]. Am. Chem. SOC.,105 (1983) 1988-2006. (311) D. Clittenberg andI. Dyong, Chem. Ber., 109 (1976) 3115-3121. (312) A. Ishizu, B. Lindberg, and 0.Theander,Acta Chem. Scnnd., 21 (1967) 424-432. (313) T. Halmos, J. Herscovici, and K. Antonakis, C. R. Acnd. Sci., Ser. C, 279 (1974) 855-857. (314) J. M. Williams, Ado. Carbohydr. Chem. Biochem., 31 (1975) 9-79. (315) S. Shuto, T. Iwano, H. Inoue, and T. Ueda, Nucleos. Nucleot., 1 (1982) 263-273. (316) K. Tatusta, S. Miyashita, K. Akimoto, and M. Kinoshita, Bull. Chem. SOC.Jpn., 55 (1982) 3254-3256.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

Me$-0

111

COzH 175

176

B = theophyllin-'l -yl

-HoH'c

HOH,C

Q

HO

OH

OH 178

177

B = uracil-1-yl

-

0

-

HO

181

180

On the other hand, the Claisen rearrangement has become popular, due to the readiness of stereoselective, carbon - carbon bond-formation in chiral syntheses. Rearrangement of ethyl 2,3-dideoxy-3-0-vinyl-a-~threo- (182a) and -erythro-hex-2-enopyranosides (182b) in nitrobenzene at 180 gave, stereoselectively, ethyl 2,3,4-trideoxy-2-C-(formylmethyl)-a-D-threo-(1 83a) and -erythro-hex-3-enopyranoside(183b), re~pectively.~~~ O

FH,OH

182a R' = OCH=CH,, R2 = H 182b R' = H, R2 = OCH=CH,

1830 R' = CH,CHO, R2 = H 183b R' = H , RZ = CH,CHO

(317) R.J.FerrierandN.Vethaviyasar,]. Chern. Soc.,PerkinTruns.1,(1973) 1791-1793.

JUJI YOSHIMURA

112

A similar rearrangement of 2-0-substituted methyl 3,4-dideoxy-a-~erythro-hex-3-enopyranosides (184a,b) into 4-C-substituted a-Derythro-hex-2-enopyranosides(185a,b) was used for the chiral synthesis CH,OH

CH,OH

OMe

R

OR 1840 R = C(NMe,)=CH, 184b R = C(OMe)=CH,

b

O

M

e

l8Sa R = CH,CONMe, 185b R = CH,COzMe

of thromboxanes from ~ - g l u c o s e . ~The ~ ~ rearrangement -3~~ was also used for total synthesis of the Prelog-Djerassi l a c t ~ n e , and ~ ~ ' of 322 prostaglandin F2a. Moreover, this reaction was extended to methods for preparing geminal dialkyl sugars and for synthesis of branched amino sugars. Thus, the reaction of 3-C-alkylidene derivatives containing a vinylo~y3~3 (186a) and a t r i c h l o r o a ~ e t i r n i n o(186b) ~ ~ ~ function at the allylic carbon atom gave a 1.5:1mixture of the geminally di-C-substituted derivatives (187 and 188), and one epimer of the vinyl-branched amino sugar (189),

Ph
187 OSiR

+

,

-

..ii=;l-ph<, 0

HCR'

OMe R'

186a R' = OSiR,, Rz = CH,OCH=CH, 186b R' = H, RZ = CH,OC(=NH)CCl,

Q O {P hM e OHCH,C

0

OMe NHCOCCl, 189

OSiR,

188

(318) E. J. Corey, M. Shibasaki, and J. Knolle, Tetrahedron Lett.,(1977) 1625-1626. (319) T. K. Schaaf, D. L. Bussolotti, M. J. Parry, and E. J. Corey, J . Am. Chem. SOC.,103 (1981) 6502-6505. (320) 0. Hernandez, Tetrahedron Lett., (1978) 219-222. (321) R.E. Ireland and J. P. Daub,]. Org. Chem., 46 (1981) 479-485. (322) G . Stork, T. Takahashi,I. Kawamoto, and T. Suzuki,]. Am. Chem. SOC., 100 (1978) 8272-8273. (323) B. Fraser-Reid, R. Tsang, D. B. Tulshian, and K. M. Sun,]. Org. Chem., 46 (1981) 3764 - 3767. (324) I. Dyong, J. Weigand, and H. Marten, Tetrahedron Lett.,(1981) 2965-2968.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

113

respectively. In the former, similar rearrangements at C-2 and C-4 were also examined. A new 1,2-hydride shift in the reaction of methyl 5 , 6 - 0 cyclohexylidene-3-O-(methylsulfonyl)-a-and -P-D-allofuranoside (190) with methylmagnesium iodide was found to give the corresponding 3deoxy-2-C-methyl derivatives (191 and 192), respectively, in which the shift proceeds stereoselectively in the case of the j? a n ~ m e r . ~ ~ ~

Me0

OH

190 a anomer

p anomer

OH

Me

192 37% 2%

191 29%

84%

9. Total Synthesis a. Branched Sugars. -A comprehensive article on the total synthesis of the common sugars from non-carbohydrate precursors has been pub(Ref. 327), l i ~ h e dSynthesis . ~ ~ ~ of such racemic, branched sugars as DL-1 DL-3 (Ref. 98), and DL-8(Refs. 328-330) from branched, acetylenic or alkenic starting-materials has been reported. A new synthesis of DL-1 was (193) achieved from 2-methoxy-4-(methoxycarbonyl)-2,5-dihydrofuran (325) M. Kawana and S. Emoto, Bull. Chem. Soc.]pn., 5 3 (1980) 222-229; Tetrahedron Lett., (1975) 3395-3398. (326) A. Zamojski, A. Banaszek, and G. Grynkiewicz, Adv. Curbohydr.Chem. Biochem., 40 (1982) 1-129. (327) R. A. Raphael and C. M. Roxburg,]. Chem. SOC.,(1955) 3405-3408. (328) H. Grisebach, W. Hofheinz, and N. Doerr, Chem. Ber., 96 (1963) 1823-1826. (329) F. Korte, U. Claussen, andK. Gbhring, Tetrahedron, 18 (1962) 1257-1264. (330) D. M. Lemal, P. D. Pacht, and R. B. Woodward, Tetrahedron, 18 (1962) 12751293.

JUJI YOSHIMURA

114

by cis-hydroxylation to give 194, and successive protection, hydride reduction to afford 195, followed by d e p r ~ t e c t i o n . ~ ~ ~

iI>-OMe

MeO&

-

woMe G

HO 193

OH 194

O

0,

M

e

/o

CMe, 195

Racemic evermicose (14) was synthesized through a common pathway as follows.332The stereospecific epoxidation of ethyl truns-3-hydroxy-3-Cmethyl-~~-glycero-hex-4-enoate (196) (obtained by the Reformatsky reaction of trans-3-penten-2-one with ethyl bromoacetate) with peroxy acid gave mainly the 4,5-epoxide (197) having the DL-I~ZO configuration. Hydrolytic ring-opening of 197 gave, regioselectively, the 1,4-1actone having the DL-urubino configuration (198), which was reduced with diisobutylaluminum hydride, to give DL-14and asmall proportion 0 f ~ ~ - 1 2 . The free acid of 196 was successfully resolved as the quinine salt.

A similar precursor was also for the synthesis of DL-23.The Claisen rearrangement of the vinyl ether (199) of trans-3-penten-2-01, and protection of the aldehyde function of the product, gave trans-3methyl-4-hexanal ethylene acetal(200).Amination of the allylic position of 200 was accomplished by addition of bis(tosy1imino)selenidewith the abstraction of an allylic proton334 followed by [3,3]-sigmatropic rearrangement to give 201 as the minor product, together with the terminal amino derivative (202). cis-Hydroxylation of 201, after conversion of the tosylamino group into an acetamido group, gave 203 in low yield. The structure of 203 was proved to be that of the N-acetyl-di-0-acetyl derivative (204) of DL-23. (331) T. Kinoshita and T. Miwa, Carbohydr. Res., 28 (1973) 175-179. (332) I. Dyong andD. Glittenberg, Chem. Ber., 110 (1977) 2721-2728. (333) I. Dyong and H. Friege, Chem. Ber., 112 (1979) 3273-3281. (334) K. B. Sharpless,T. Hori, L. K. Truesdale, and C. 0.Dietrich, J. Am. Chem. Soc., 98 (1976) 269-271.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

115

bol

Me

Me 199

200

201

TsHNCH, \

k A c

MA

NHAc

204

202

203

Aromatic compounds bearing an unsaturated, six-carbon side-chain having a terminal aldehyde group (205) were for the preparation of optically active 8 and 12. Concurrent, stereoselective cis-hydroxylation and reduction of 205 by fermentation with baker’s yeast gave 206, which was converted into 207 by epoxidation and reduction. Degradation of the aromatic ring of 207 with ozone gave 8 and 12.

A

m

C Me

H

o

-

OAc R

205

206

J OAc

R

OAc 207

b. Branched Cyclito.;. -Compounds 37 and DL-38were synthesized by various Diels- Alder reaction^.^^^-^^^ In 1966, the expression (335)C.Fuganti and P. Grasselli, Chem. Commun., (1978)299-300. (336)J. Wolinsky, R.Novak, and R. Vasileff,]. Org. Chem., 29 (1964)3596-3598. (337)B.A. Bohm, Chem. Rev., 65 (1965)435-466. (338)R. Grewe and S. Kersten, Chem. Ber., 100 (1967)2546-2553.

116

JUJI YOSHIMURA

“pseudo-sugar’’ was proposed in order to designate any carbocyclic analog of a cyclic monosacchride in which the usual ring-oxygen atom is replaced by a methylene group, and the (hydroxymethy1)-branched cyclohexanetetrol (208), obtained from the Diels- Alder adduct of 2-acetoxyfuran and malic anhydride, was named pseudo-a-m-talopyranose.338 Up to the present, pseudo-a- and -P-DL-talopyranoses,340 pseudo-a-34I and - ~ - ~ ~ - g a l a c t o p y r a n o spe~s e, ~u~d~o - a and -~~~ P - ~ ~ - g l u c o p y r a n o s e s ,pseudo-a~~~ and P-~~-rnannopyranoses,~~O pseudo-a-~~-idopyranose,~~~ pseudo-o!-~~-altropyranose,~~~ pseudo$~ ~ - g u l o sand e , purine ~ ~ ~ nucleosides having a pseudo-P-DL-ribofuranosyl moiety346 have been reported, but detailed descriptions of the chemistry of this class of compounds, including amino derivatives, will not be given here. Pseudo-a-D-galactose; (+)- 1L-( 1,2,3/4,5)- 1-(hydroxymethyl)cyclohexanetetrol(209) was found in a fermentation broth of a Streptorny~es.~~‘ CH,OH I

CH,OH

I

OH 208

209

DL-39,DL-40, and DL-41were synthesized from the Diels-Alder adduct (210) of furan and acrylic acid as follows. Sexo,Gendo-Dihydro2endo-(hydroxymethyl)-7-oxabicyclo[ 2.2. llheptane (212), obtained by the oxidation of 210 with hydrogen peroxide to 21 1, followed by reduction with lithium aluminum hydride, was used as the common intermediate. Opening of the 1,4-epoxy ring of 212 with acetic acid containing conc. sulfuric acid gave348213, which was converted into the N,O-pen(339) G.E. McCasland,S.Furuta,andL. J.Durham,J. Org. Chem.,31(1966)1516-1521. (340) S.Ogawa, M. Ara, T. Kondoh, M. Saito, R. Masuda, T. Toyokuni, and T. Suami, Bull. Chm. Soc.Jpn.,53 (1980) 1121-1126. (341) G .E. McCasland, S.Furuta, andL. J. Durham,]. Org. Chem.,33 (1968) 2841 -2844. (342) T. Suami, S. Ogawa, T. Ishibashi, and I. Kasahara, Bull. Chem. Soc. Jpn., 49 (1976) 1388-1390. (343) T. Suami, S. Ogawa, K. Nakamoto, and I. Kasahara, Curbohydr. Res., 58 (1977) 240-244. (344) S.Ogawa, T. Toyokuni, andT. Suami, Chem. Lett., (1980) 713-716. (345) G .E. McCasland, S. Furuta, andL. J. Durham,]. Org. Chem., 33 (1968) 2835-2840. (346) Y. F. Shealy and J. D. Clayton,]. Am. Chm. Soc., 91 (1969) 3075-3083. (347) T. W. Miller, B. H. Arison, and G . Albers-Schtinberg,Biotechnol. Bioeng., 15 (1973) 1075- 1080. (348) S.Ogawa, K. Nakamoto, M. Takahara, Y. Tanno, N. Chida, andT. Suami, Bull. Chem. Soc.Jpn.,52 (1979) 1174-1176.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

117

taacetate of DL-40by S N replacement ~ of the isolated hydroxyl group of the corresponding di-0-isopropylidene derivative (214) by an amino group. Ring cleavage of the triacetate of 212 with hydrogen bromide in acetic acid gave349the dibromide (215), which was converted into DL-41 by way of reduction of 216, obtained by elimination of the primary bromine atom with silver fluoride. Replacement of the bromine atom of 215 by benzoate, by reaction with silver benzoate, followed by elimination of the elements of hydrogen bromide, gave 217, the benzylidene derivative (2 18) of which was selectively converted into the epoxide (219). Diaxial ring-opening of 219 with sodium azide gave 220, which was converted344into DL-39by way of the elimination product (221) of the sulfonate of 220. Moreover, DL-validoxylamine (222a), a component disaccharide of validamycins, was obtained350by the reaction of 219 and di-0-isopropylidenated DL-40. In a similar way, DL-validoxylamine B (222b), in validamycin B, was also synthesized.351

210

21 1 CH,OH

-

212

-

/OCH2

HO

OL-40

'"or 0

OH

213

214

CH, B r Qr

Ac 0

-

-

DL-41

AcO OAc

21 5

OAc

216

(349) S.Ogawa, T. Toyokuni, M. Omata, N. Chida, andT. Suami, Bull. Chem. Soc.Jpn.,53 (1980) 455-457. (350) S . Ogawa, T. Ogawa, N. Chida, T. Toyokuni, and T. Suami, Chem. Lett., (1982) 749-752. (351) S . Ogawa, T. Toyokuni, Y. Iwasawa, Y. Abe, and T. Suami, Chern. Lett., (1982) 279-282.

JUJIYOSHIMURA

118 CH,OBz

Q

AcO

OCH,

-Phq-J

OAc 217

OCH,

-Ph
OAc 218

219

OAc

I OAc 22 1

I OAc 220

HOCH,

h

OH 222a R = H 222b R = O H

111. SYNTHESIS OF NATURALLY OCCURRING, BRANCHED SUGARS 1. Methyl-branched Sugars a. Having a Hydroxyl Group at the Branch Point.-Virenose (la), having an equatorial C-methyl branch, was simply synthesized352by the 6-deoxygenation of methyl 4,6-0-benzylidene-3-C-methyl-cu-~-gulopyranoside (223), obtained by the Grignard reaction of the corresponding (352)N.Hong, K.Sato, and J. Yoshimura, Bull. C h .Soc.Jpn., 54 (1981)2379-2382; Chem. Lett., (1980)1131-1132.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

119

223

glycosid-3-ulose, by treatment with N - b r o m o s ~ c c i n i m i d e ,followed ~~~

by hydrogenolysis and hydrolysis. Vinelose (16) was first derived from 54a (R' = Me) by 6-deoxygenation, 5-epimerization (conversion from the D into the L series) and selective 2-O-methylation, and the structure was confirmed by ~ y n t h e s i s . ~It~ has ' , ~ also ~ ~ been synthesized from the 2-0-methyl derivative of 57d (R' = Me) through 6-deoxygenation and 5-epimerization, by reduction of the corresponding hex-5-enopyranoside.35s The reaction of methyl 4-0-benzyl-2,3,6-trideoxy-a-~-threohexopyranosid-3-ulose (224a) with methylmagnesium iodide gave a 1:2.4mixture of equatorial- and axial-attack products, due to the coordination of magnesium, and therefrom the minor product axenose (10) was ~btained."~

eoM

H3C

R2

R' 2240 R' = OBn, R2 = H 224b R' = H, R2 = OMe

Evermicose (14), having an axial, C-methyl branch, was synthesized by 6-deoxygenation of 58a (R' = Me).357The intermediate was obtained in the ratio of 1:3.4 by epoxidation of the 3-C-methylene derivative of 56a (353) S. Hanessian, Carbohydr. Res., 2 (1966) 86-88; Methods Carbohydr. Chem., 6 (1972) 183-189. (354) M. Funabashi, S. Yamazaki, and J. Yoshimura, Carbohydr.Res., 44 (1975) 275-283; Tetrahedron Lett., (1974) 4331-4334. (355) J. S. Brimacombe, S. Mahmood, and A. J. Rollins, J. Chem. Soc., Perkin Trans. 1, (1975) 1292-1297. (356) L. E. S. Barata, A. J. Marsacoli, L. Valente, A. Olesker, andG. Lukacs, Carbohydr. Res., 90 (1981) 326-328. (357) M. Funabashi, N. Hong, H. Kodama, and J. Yoshimura, Carbohydr. Res., 67 (1978) 139-145.

120

JUJI YOSHIMURA

for the synthesis of followed by reduction. A similar pathway was everlose (15) and n o g a l o ~ e(17). ~ ~ In ~ the latter synthesis, dibutylstannylation was used for regioselective protection of the axial - equatorial, vicinal-diol g r o ~ p i n g . Another ~ ~ ~ - ~synthesis ~~ of 15, by use of cis-hydroxylation has already been mentioned.243 Compound D-17 was synthesized by suitable introduction of a Cmethyl group (to give the desired configuration) into the D-arubino isomer of 53a by a Grignard reaction, but synthesis of L-17 by a similar pathway proved unsuccessful, owing to the impossibility of converting 6 - 0 - benzoyl - 1,2 - 0 - isopropylidene - 3 - C- methyl - 3 - 0- methyl - 5 - 0(methylsulfony1)-a-D-gulofuranose(225) into the corresponding 5,6-

Id;l. OMe 0-CMe,

HCOMB I

H,COBz 225

epoxide, for inversion3s3 at C-5. It is noteworthy that L-17 was obtained364by introduction of a C-methyl group into 1,2-0-cyclohexylidene-6-deoxy-4-O-methyl-~-~-u~ubino-hexopyr~osid-3-ulose (226) by a Grignard reaction. The formation of a 2.3: 1mixture of the products of axial and equatorial attack indicated that 226 exists in the OS, conformation. Moenuronic acid (20) was readily synthesized by stereoselective

226

(358)J. Yoshimura, N.Hong, and K. Sato, Chem. Lett., (1979)1263-1264. (359)N. Hong, M. Funabashi, and J. Yoshimura, Curbohydr. Res., 96 (1981)21-28; Chem. Lett., (1979)687-688. (360)M.A. Nashed and L. Anderson, Tetrahedron Lett., (1976)3503-3506. (361)M. A. Nashed, Curbohydr. Res., 60 (1978)200-205. (362)T.Ogawa and M. Matsui, Tetrahedron, 37 (1981)2363-2370. (363)J. S. Brimacombe and A. J. Rollins, J. Chem. Soc., Perkin Truns. 1 , (1974)15681572. (364)L. Valente, A. Olesker, R. Rabanal, L. E. S. Barata, andG. Lukacs, TetruhedronLett., (1979)1 153- 1 156.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

121

introduction of an axial C-methyl group into 74 by means of methyllithium, followed by catalytic oxidation of the primary hydroxyl The pure compounds 7 (Ref. 366) and branched lower sugars367were synthesized from 54a (R' = Me) by the descending method. Sibirosamine (22) had long been considered to have the D-altro configuration, and some synthetic approaches were reported.244.368.36e HowO from the results of ever, it was revised370to the D - W I U ~ ~configuration the following experiments. Methyl 6-deoxy-3-C-methyl-2-O-(tert-butyldimethylsily1)-a-D-altropyranoside (227), derived from the 3-Cmethyl analog of 95 by protection of OH-2 and 6 - d e o ~ y g e n a t i o nwas ,~~~ converted into the corresponding hex-3-enopyranoside (228), in 50% overall yield, by 4-O-mesylation, displacement with benzeneselenoxide, and syn-oxidative elimination. The cis-oxyamination of 228 and subsequent N-methylation gave the N-tosyl derivative (229) of the methyl glycoside having the D-altro configuration. Selective removal of the tert-butyldimethysilyl group from 228 to give 230, and oxidation with peroxy acid, gave mainly the epoxide (231). Treatment of 231 with N-methyl-p-toluenesulfonamidegave 232 (hav/Me OSiCMe,

-

"

Ye

3

c

v

OSiCMe, 'Me

T'Nm Me

OMe

Me

HO

228

22 7

/ -

-

H3c/x;L
OMe 230

229

OMe 231

T

S

q

Me

OMe

232

(365) K. Sato, K. Kubo, N. Hong, H. Kodama, and J. Yoshimura, Bull. Chern. Soc.Jpn.,55 (1982) 938-942. (366) J. Yoshimura, K. Hara, and M. Yamaura, Carbohydr. Res., 101 (1982) 343-347. (367) J. Yoshimura, K. Hara, M. Yamaura, K. Mikami, and H. Hashimoto, Bull. Chem. SOC. Jpn., 55 (1982) 933-937. (368) J. Yoshimura, N. Hong, A. Rahman, and K. Sato, Chern. Lett., (1980) 777-778. (369) M. Georges, D. MacKay, and B. Fraser-Reid,]. Am. Chern. SOC.; 104(1982) 1101 1103. (370) K. A. Parker and R. E. Babine, Tetrahedron Lett., (1982) 1763-1766.

JWJI YOSHIMURA

122

ing the D-manno configuration)identical with that obtained from a degradation product of sibiromycin. b. Having a Nitrogen Function at the Branch Point.- In Section 11,l,bythe dependence of the stereoselectivity on the reaction conditions in the cyanomesylation of 56a was described. The mesylate of 58a (R' = CN) was converted into the methyl-branched amino sugar 233, by way of the spiro-epimino d e r i ~ a t i v e , ~from ~ ~ Jwhich ~ ~ vancosamine (23) was ~ y n t h e s i z e d by ' ~ ~epimerization at C-5. Application of the stereoselectivity (in the same reaction in pyridine) to methyl 2,6-dideoxy-4-0methyl-cw-~-erythro-hexopyranosid-3-ulose (224b) gave, stereospecifically, the cyanomesyl derivative having the L-rib0 configuration (234a), which was converted into the methyl-branched, amino sugar 234b having the L-arabino configuration. Oxidation of 234b with m-chloroperoxybenzoic acid gave the corresponding nitro sugar (234c), which was hydrolyzed371to evernitrose (25). Compound 166a was also converted into the N-acetate of 23413 by 2-deoxygenation by way of the corresponding g l y ~ a 1 .The ~ ~3-amino-3-C-methyl ~ , ~ ~ ~ analog (24) from antibiotic A35512B was ~ y n t h e s i z e d ~from ' ~ a 3-C-epimeric derivative of 234a. By the use of L-Selectride (lithium tri-sec-b~tylborohydride)~~~ as a reducing reagent, which gives the axial alcohol from alduloses, compound 23 was obtained376from a derivative of 234b by inversion of the

phT% 0

d

o

M

e

Me0

Me I

H2N 233

H

R2

OMe

234a 234b 234c

R' = OMS, R1 = CN R L = Me, R2 = NH, R1 = Me, R2 = NO2

(371) J. Yoshimura, M. Matsuzawa, K. Sato, and Y. Nagasawa, Curbohydr. Res., 76 (1979) 67-78. (372) J. S. Brimacombe, A. S . Mengech, and M. S . Saeed, Carbohydr. Res., 75 (1979) c5-c7. (373) J. S. Brimacombe and A. S . Mengech,]. Chem. SOC., Perkin Trans. 1, (1980) 20542060. (374) J. S. Brimacombe, R. Hanna, and L. C. N. Tucker, Curbohydr. Res., 105 (1982) c l -c3. (375) H. C. Brown and S . Krishnamurthy, J . Am. Chem. SOC., 94 (1972) 7159-7161; S . Krishnamurthy, Aldrichimica Acta, 7 (1974) 55-60. (376) H. I. Ahmad, J. S. Brimacombe, A. S . Mengech, K. M. M. Rahman, andL. C. N. Tucker, Carbohydr. Res., 93 (1981) 288-293; J. S . Brimacombe, A. S. Mengech, K. M. M. Rahman, and L. C. N. Tucker, ibid., 110 (1982) 207-215.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

123

configuration of (2-4. This process was also applied377for a preparation of 18. In addition, synthesis of branched amino sugars from spiro-oxiranes by way of spiro-aziridines was examined.378 It was pointed that rubranitrose (26), reported to be an L sugar from X-ray analysis and c.d. spectrum,53 must be a member of the D series; this conclusion was reached by comparison of the c.d. spectrum and rotational value with those of kijanose (27, tetronitrose), whose configuration was assigned by Hudson’s Rules of I s o r o t a t i ~ nL-26 . ~ ~ was synthesized stereospecifically from the 4-0-methyl analog of 224a, and the result supported the deduction from the Cotton effect of the synthetic specimen.18s Syntheses of L-26by way of the inversion at C-4 of a derivative of3-epi-25, and of D-26 by a similar pathway, were also reported.379 On the other hand, 27, having an amino group vicinal to a methylbranched nitro function, was synthesized as follows.38otrans-Addition of iodine isocyanate to the 2-deoxy analog of 112 followed by treatment with methanol in the presence of sodium methoxide gave the iodo carbamate(235) as a single isomer; this was converted into the aziridine (236) by treatment with methanolic potassium hydroxide. Nucleophilic ringopening of 236 with sodium azide gave the regioisomers (237a and 23713)

-

MeOzCNH

HNboM

235

[HzNQoMe Me

M e 0 2 C H N o

-

M e 0 2 C H N o

OMe

OMe Bn0,CHN

4 N 239

\

236

237a

I

N O o M e

HZN

238

237 b

(377) J. S . Brimacombe, R. Hanna, and L. C. N. Tucker, Carbohydr. Aes., 112 (1983) 320-323. (378) J. S. Brimacombe and K. M. M. Rahman, Carbohydr. Res., 113 (1983) c6-c9. (379) J. S.Brimacombe and K. M. M. Rahman, Carbohydr. Res., 114 (1983) c l -c2. (380) K. Funaki, K. Takeda and E. Yoshii, Tetrahedron Lett., (1982) 3069-3072; Chem. Phann. Bull., 30 (1982) 4031-4036.

JUJI YOSHIMURA

124

in the ratio of 5 : 21. Successive N-(benzyloxycarbony1)ation of 237b, reduction with sodium borohydride and nickel chloride,381 and N-(methoxycarbony1)ation of the product gave methyl 3-(benzyloxycarbony1)amino-2,3,4,6-tetradeoxy-4-(methoxycarbony1)amino-3- Cmethyl-a-D-xylo-hexopyranoside (238). Hydrogenolysis of 238 with triethylsilane and palladium on carbon, followed by oxidation with mchloroperoxybenzoic acid, gave a-D-tetronitroside (230), which was

S0

O

240

M

e

M e0-N' e

\

HO

\

0:4,

0

243a

HO W Me AO

O BnO M

e

Me@

OMe

0Y 00 243b

' 0 6 Me 247

244b

(381) R. B. Boar, D. W. Hawkins, J. F. McGhie, and D. H.R. Barton,J. Chem. SOC., Perkin Trans. 1, (1973) 654-657.

SYNTHESIS O F BRANCHED-CHAIN SUGARS

125

identical with that derived from 27. Thus, almost all of the members of this class of compounds were rapidly synthesized. 2. Two-carbon-branched Sugars Four diastereoisomers of methyl /?-aldgarosides were ~ y n t h e s i z e d ' ~ ~ from methyl 2-0-benzyl-3,4,6-trideoxy-/?-~-erythro-hexopyranosid3-ulose (240), and the configuration of the glycoside of aldgarose31 from aldgamycin was determined by comparison, as shown in 243b having the (S) configuration in the (1-hydroxyethyl) branch. Introduction of a Cacetyl group into 240, by means of 48b, gave 241 and 242 in the ratio of 1.8 : 1. Successive reduction of 241 and 242 with sodium borohydride, carbonylation, and debenzylation gave 243a,b and 244a,b in the ratio of 1 : 0.7 and 1: 0.54, respectively. Later, a C-vinyl group was stereoselectively introduced382into an analogous a-hexopyranosid-3-ulose (245), and this was followed by acetonation, to give 246. Successive epoxidation of 246, reduction, deacetonation, carbonylation, and anomerization gave 243a and 243b in the ratio of 1: 7. A simpler synthesis of 243b from D-glucose through the 1,2-O-isopropylidene-a-~-glycos-3-u~ose (247) has also been reported.383 The structure of pillarose (28) was also determined by synthesis.384 Photochemical addition of ethylene glycol to the ethyl a-D-glycoside of 125 gave 248, which was converted into 249 by 6-deoxygenation, modi-

o ~

CH,OTr

-Oh

OEt 248

BzOH,C

OEt

249

fication of the branched group, and epimerization. However, 249 was not identical with the benzoylated glycoside derived from pillaromycin A. The regioisomers, 251 and 252, were synthesized from methyl 2,3,4,6-tetradeoxy-a-~-glycero-hexopyranosid-4-ulose (250a) through the 4-C-vinyl derivative, and from the 4-C-(methoxycarbonyl)methylene derivative (250b) through osmium tetraoxide oxidation, respectively; compound 251 proved to be identical with natural pillaroside. (382) J. S. Brimacombe, J. Minshall, andC. W. Smith,]. Chem. SOC., Perkin Trans. 1, (1975) 682 - 686. (383) H . Paulsen, B. Sumfleth, and H. Redlich, Chem. Ber., 109 (1976) 1362-1368. (384) D. L. Walker and B. Fraser-Reid, J . Am. Chem. Soc., 97 (1975) 6251-6253.

JUJIYOSHIMURA

126

Later, pillaroside was also synthesized by reaction of 250a 48c 50.y-Octose (29)was also synthesized by introduction of a and C-vinyl group into the corresponding g l y ~ o s - 4 - u l o s e . ~ ~ ~

xq -

BzOH,C Ho%

BzOHaC*

OMe

OMe

OMe

250a X = 0 250b X = CHC0,Me

252

251

The branched lactones 32-34,found in oligosaccharide antibiotics of the orthosomycin familys5 were characterized as their methyl aldonates65.387*388 (253- 255),and synthesized as follows. The introduction of CRMe

CGMe

I HCO,

p H OCH

I I

HOH,C -COH MeOCH

253

I HCO, 1

h C H a OCH

I

HOC-COMe

I

C02Me

I

HCO, p H 1 OCH HOC-CH(OH)Me(S I )

I

HCOH

HCOH

CH,

CH3

I

254

I

255

an equatorial, two-carbon branch into benzyl 2,3-O-methylene-P-~threo-pentopyranosid-4-ulose(256)with vinylmagnesium bromide, followed by epoxidation and reduction, gave the 4-C-[(S)-l-hydroxyethyl] derivative (257a)and the (R)isomer in the ratio of 4.5: 1. Successive hydrogenation of 257a,oxidation with bromine-water, and esterification gave389253. The configuration of methyl eurekanate (254)was determined by synthesis.390The 4-C-acetyl derivative (258),having the D-galacto configuration, was synthesized from 76 by way of the 4-C-[(R,S)-lhydroxyethyl] derivative (257b), and the D-gluco isomer (261)was (385) H. Paulsen, K. Roden, V. Sinnwell, and W. Koebernik, C h .Bet-., 110 (1977) 2146- 2149. (386) H. Paulsen and V. Sinnwell, Chem.Ber., 111 (1978) 869-878. (387) A. K. Ganguly, 0.Z. Sarre, D. Greeves, and J. Morton,]. Am. C h .Soc., 97 (1975) 1982-1985. (388) E. Kupfer, K. Neupert-Laves, M. Dobler, and W. Keller-Schierlein, Helo. Chim. Acta, 63 (1980) 1141-1148. (389) M. Matsuzawa and J. Yoshimura, Carbohydr.Res., 81 (1980) c5-c9. (390) J. Yoshimura and M. Matsuzawa, Carbohydr.Res., 85 (1980) c l-c 4; 100 (1982) 283-295.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

127

257a R = H 257b R = CH,

256

258

Me

259

260

261

obtained from the 4-C-ethylidene derivative (259) of 76 through conversion into 260 by osmium tetraoxide oxidation. The methyl aldonate 254, derived from 258, was identical with that from flambamycin. The structure of 254 was also determined by X-ray analysis.3ss An unequivocal determination was reported on the chirality of the 1-hydroxyethyl group in the branched sugars by a combination of synthetic pathways, by way of C-vinyl and C-ethylidene derivatives,391 and 255, whose configuration was determined by X-ray analysis,392was synthesized3e3 from the (S) isomer of 25713. It is noteworthy that the two-carbon-branched sugars so far known have in common an equatorially oriented branch, and if a 1-hydroxyethyl group is present, it has the (S) configuration. A higher-branched sugar, 35, was synthesized from 262, obtained by the Grignard reaction of 2,3-O-isopropylidene-~-glyceraldehyde by conversion of the methylene derivative 263 into 264 with osmium CH,CHMe,

CH,CHMe,

C(H)OH

C =CH,

I

I

HCO, I /CM% H,CO 262

CH,OH

I

-

I

__t

HCO, I ,CMe, H,CO 263

I I HCO,

Me,HCH,C-COH

I

H,CO

p e ,

264

(391) J. Yoshimura and M. Matsuzawa, Carbohydr. Res., 94 (1981) 173-181. (392) E. Kupfer, K. Neupert-Laves, M. Dobler, and W. Keller-Schierlein, Helu. Chim. Ada, 65 (1982) 3-12. (393) J. Yoshimura, K. Hara, and M. Matsuzawa, Bull. C h m . S o c . J p . ,56 (1983) 21892190.

128

JUJI YOSHIMURA

tetraoxide, giving a 2 : 3 mixture of epimerssQin which 264 was the minor constituent.

3. Formyl- and Hydroxymethyl-branched Sugars

A few new syntheses of this type of sugar were described in Section

II,5. Only new syntheses of 2 and 47 will be briefly mentioned here. Hamamelose (2) was ~ y n t h e s i z e dfrom ' ~ ~ the epimer 265 (from the condensation products of 2,3-O-isopropylidene-~-glyceraldehyde and 51) through spiro-epoxidation of the corresponding 2-C-methylene glycoside (266) and alkaline ring-opening. CH (OEt )2 I I

?Me

H.C=C

265

266

L-Dendroketose (47) was first s y n t h e ~ i z e dfrom ~~~ 5-O-benzyl-4-C.~~~ (benzyloxymethyl)-3,4-O-isopropylidene-~-e~~~~o-pentitol(267a), derived from 143by benzylation and partial hydrolysis. Tritylation of 267a and oxidation of the product gave the corresponding 2-pentulose derivative (268a), which was converted into 3,4-O-isopropyliden-~-dendroketose (269) by hydrogenolysis. A similar pathway was used for the conversion into 269 of 267b, which was obtained from 150 by 0benzoylation, reduction with sodium borohydride, 0-(dimethoxytrityl)ation, and oxidation.3QeCompound 268 is readily epimerized at C-3 CH,OR'

I

HOCH

I I OC-CH20Rz I CH20RS

,OCH Me&,

CH,OR'

-

267a ~1 = H , RZ = R3 = Bn 267b R' = R2 = Bz, RS = CPh(C,H,OMe-p),

I c= 0

I

,OCH MezC, I OC- CH20R2

I

CH20RS

268a R' = Tr, R' = R3 = En 268 b R' = Rz = Bz, R3 = CPh(C,H,OMe-p),

CH,OH

2 69

(394)E.B.Rathbone and G . R.Woolard, Carbohydr. Res., 46 (1976) 183-187. (395)H. C. Jarrell, W. A. Szarek, and J. K. N. Jones, Carbohydr. Rm., 64 (1978) 283-288. (396)P.-T. Ho, Can.J. Chern., 57 (1979) 384-386.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

129

under alkaline conditions.397Direct oxidation of free alditol from 143 with Acetobacter suboxydans afforded398 47. As another pathway, the 2,3-O-isopropylidene derivative (271)of 47 was obtained395through introduction of a C-vinyl group into l-O-benzoyl-2,3-O-isopropylidene~-glycero-2,4-pentodiulo-2,5-furanose (270).

-

dlOB

de2 CH,OBz

0

2 70

OH

0

CH,OH 271

4. Branched Sugars and Branched Cyclitols Having No Heteroatom at the Branch Point Four isomers of blastmycinolactol (274)were synthesized, and the structure of 36 was determined by comparison of them with the compound obtained by degradation of b l a s t m y ~ i nAn . ~ analog ~~ (272)of 95 was converted into pentoside 273 by epimerization at C-5, 6-deoxygenation, periodate oxidation between C-1 and C-2, and glycosidation. Epimerization at c-3of 273, and oxidation at c - 1 , gave 274, which was converted400into deisovalerylblastmycin (276a)and antimycin A (276b) by way of cyclic lactonization of the threonyloxy derivative 275. On the other hand, optically active 43 and 44 were synthesized as follows.4o1Treatment of 2,3,4-tri-0-benzyl-1,5-di-O-tosyl-~-arabinitol (277)with three equimolar proportions of methylenetriphenylphosphorane gave the cyclic phosphorane (278),which was stereoselectively converted into the cyanohydrin (279)by way of the corresponding Cmethylene and inosose derivatives. Compound 279 was converted into 43 and 44by way of the corresponding carboxylic acid (280),and unsaturated nitrile (281),respectively. Compound 39 was derived from the inosose (282),obtained from quebrachitol, through a 21-step convers i 0 1 - 1 .Introduction ~~~ of a hydroxymethyl group into 282 was accomH. C. Jarrell, W. A. Szarek, J. K. N. Jones, A. Drnytrazenko, andE. B. Rathbone, Carbohydr. Res., 45 (1975) 151 - 159. W. A. Szarek, G . W. Schnarr, H. C. Jarrell, and J. K. N. Jones, Carbohydr. Res., 53 (1977) 101-108. S. Aburaki, N. Kinoshita, andM. Kinoshita, Bull. Chem. SOC.Jpn., 48 (1975) 12541259. S. Aburaki and M. Kinoshita, Chem. Lett., (1976) 701-704. H. J. Bestmannand H. A. Heid,Angew. Chem.,83 (1971) 329-331;Angew. Chem., Int. Ed. Engl., 10 (1971) 336-337. H. Paulsen and F. R. Heiker, Angew. Chem., 92 (1980) 930-931.

130

JUJI YOSHIMURA OCH,

Ph-Q

OMe

-Q-q H3C

Bu

OMe

Bu

HO

2 72

H3C

Bu

273

2 74

275 Z = PhCH,OCO

276a R = H 276 b R = Me,CHCH,CO

CH,OTs BnOdH I

HCOBn I HFOBn CH,OTS 2 77

-

PPh,

2 78

J\ 279

281

280

plished via epoxidation with dimethyl sulfoxoniummethylide,403to give 283, which was converted into dimesylate 284 for introduction of unsaturation. Compound 285 was obtained by conversion of 284 into the epoxide with sodium methoxide, followed by deoxygenation. Introduction of an azido group into 285 by the azobis(dicarboxy1ate)method404 gave 286, which was converted into 39 by hydrogenolysis. (403) E. J. Corey and M. Chaykovsky,]. Am. Chem. SOC., 87 (1965) 1353-1364. (404) H. Loibner and E. Zbiral, Helv. Chim. Ada, 59 (1976) 2100-2113.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

Qy

I

Me&-0

OMe

HO

Qy

0

0

___)

I

Me,C- 0

CMe,

131

B z o H z e : B n

MsO

CMe, 283

282

284

J BZOH,C’ 286

BZOH,C’ 285

IV. REMARKS NOTRELATINGTO SYNTHESIS 1. Branched-Sugar Nucleosides

Findings on the antiviral activity of e’-C-methyl- and 3’-C-methyl-cytidines, and on the presence of poly-1 in plant cell-walls have stimulated the synthesis of branched-sugar nucleosideseoby the usual methods, and some of their physiological activities have been summarized.405 The inhibitory activity of adenine nucleoside (287, R = CH,) from 87c on multiplication of the herpes-1 (HF) virus,40sand enhanced growth-inhibitory activity against CCRF-CEM human lymphoblastic leukemia cells in vitro were observed; as the alkyl branch-chain was lengthened, the lipophilic character of the nucleosides increased. Experiments involving incorporation of labeled uridine, thymidine, and leucine revealed407that, at a concentration of 50%, inhibition of RNA synthesis by cordycepin (287, R = NH,) was half that of 287 (R = C4H, or C5Hll), which was almost complete; this applied to synthesis of DNA and protein as well.407At a similar concentration, 3’Gethyl- and 3’-C-butyl-uridine (from 53b) showed 20% inhibition of cultured L-1210 cells in mice.408 “D-Apio-L-furanosy1”uracil and “D-apio-L-furanosyl”thymine showed immunosuppressive activity for rat T-lymphocytes at the same level as 1-P-D-arabinofuranosylcytosine, and similar nucleosides of halogenoura-

-

(405) J. M. J. Tronchet, Biol. Med. (Paris), 4 (1975) 105-114. (406) J. M. J. Tronchet and J. F. Tronchet, Helu. Chfm.Adu, 64 (1981) 425-429. (407) A. Rosowsky, H. Lazarus, and A. Yamashita,]. Med. Chem., 19 (1976) 1265-1270. (408) A. Rosenthal and S. N. Mikhailov, Curbohydr. Res., 79 (1980) 235-242.

JUJI YOSHIMURA

132

cils significantly suppressed growth of human l y m p h o b l a s t ~ How.~~~ ever, they exhibited no inhibitory activity against Herpes simplex It is interesting that the thioguanine a-and P-nucleosides (288 and 289) containing 2,3-dideoxy-3-C-(hydroxymethyl)-~ erythro-pentoHOHZCQ

H O H Z C p s G

R

OH 287

= HOHZCQSG

HOH,C

CH,OH

CH,OH 288

G

CH,OH 289

Ade = adenin-9-yl SG = 2-thioguanin-9-yl

furanose are equally inhibitory to the growth of W - L 2 human lymphoblastoid cells, are phosphorylated and incorporated into the DNA of Mecca lymphosarcoma in mice to the same degree, and are more effective in these tests than the parent analog, 2’-deo~ythio-a-guanosine.~~~ These results indicate that the oxygen atom of the furanose ring and the 2’-methylene group are correspondingly interchangeable, and that acceptance by the enzymes is improved if primary hydroxyl groups are provided at both C-3’ and C-5’. This deduction extends to the biological activity of “pseudosugars” (see Section 1,9). Adenine nucleosides from 145 (Ref. 287) and 85 (R = CH,OH, R’ = Re = H),405and l-[3-deoxy-3-fluoro-3-C-(hy~oxymethyl)-a,~-~-xylof u r a n o s y l ] ~ r a c i were l , ~ ~ ~also synthesized. The last two showed no activity oerssus herpes-1 (HF) virus or L-1210 cells. The discovery of such naturally occurring branched-sugar nucleosides as 45 and 46 will presumably accelerate research on activity- structure relationships in this field. 2. General

Determination of the configuration at the branch point of branched sugars constitutes a very important problem in this field, especially in cases of Type A, for which n.m.r. analysis of coupling patterns provides no decisive information concerning the stereochemistry. A variety of (409) D. K. Parikh andR. R. Watson,]. Med. Chem., 21 (1978) 707-709. (410) P. W. Rabideau, D. K. Parikh, and R.R. Watson,]. Carbohydr. Nucleos. Nucleot., 5 (1979) 537-544. (411) E. M. Acton, R.N. Coerner, H. S . Uh, K. J. Ryan, D. W. Henry, C. E. Cass, and G .A. LaPage,J. Med. Chem., 22 (1979) 518-525. (412) A. J. Brink, 0.C. DeVilliers, and A. Jordaan,Carbohydr.Res., 54 (1977) 285-291.

SYNTHESIS OF BRANCHED-CHAIN SUGARS

133

evidence found for the usual sugars has been applied for this purpose. The chemical method is considered to be the more reliable; in this, the steric requirement in reactions involving two functional groups is examined. Solvolysis of a vicinal sulfonyloxy group by the participation413 of a trans-acetamido group at the branch point by way of formation of an oxazoline intermediate,2s2,297*298 and intramolecular ring-formation, such as of h e m i a ~ e t a l , aminal,lI3 ~~~ i s ~ p r o p y l i d e n e , ’ ~ ~and J~~ o t h e r ~ , ~ between ~ ~ J ~ ’ two groups in cis orientation on a pyranoid or furanoid ring are included in this category. The well known rotational shift of pyranosides due to formation of a cuprammonium complex with an a-diol grouping,414which was extended to a-amino alcohols by the use of tetraminecopper(I1) (Ref. 41 5), seems to be a p p l i ~ a b l e . ’ ~ ~ J ~ ~ J ~ ~ The empirical, “acetyl resonance rule” for secondary acetoxyl and acetamido groups on a pyranoid or cyclohexane ring302s418may be extended to tertiary analogs184*2s7 when the compound contains no neighboring, aromatic ring having a strong magnetic anisotropy (such as benzoyl, benzyl, or tosyl). A lanthanoid-induced shift method, in which shift gradients of ring protons of a tertiary alcohol and the corresponding secondary alcohol are compared, was proposed417 for determination of the configuration of a tertiary alcoholic center. However, the method418 should be applied carefully.183A 13C-n.m.r.-spectral method, in which the chemical shift of a-carbon atoms in axial or equatorial branches is used,419has been widely applied for 1 , 3 - d i t h i a n - 2 - ~ 1methy1,15g,365,421 ,~~~ substituted methy1,422*423 and vinyl derivatives,165but is not applicable for carbonyl carbon atoms.390This method seems to be the most facile, but the conformation of substrates should always be taken into consideration in spectroscopic methods. (413) L. Goodman, Ado. Carbohydr. Chem., 22 (1967) 109-175. (414) R. E. Reeves, Adu. Carbohydr. Chem., 6 (1951) 108-134. (415) S. Umezawa, K. Tatsuta, and T. Tsuchiya, Bull. Chem. SOC. Jpn.. 39 (1966) 12441248. (416) F. W. Lichtenthaler and P. Emig, Tetrahedron Lett., (1967) 577-582. (417) S. D. Gero, D. Horton, A.-M. Sepulchre, and J. D. Wander,]. Org. Chem.,40 (1975) 1061 - 1066. (418) J. J. Nieuwenhuis and J. H. Jordaan, Carbohydr. Res., 51 (1976) 207-213. (419) D. E. Dorman and J. D. Roberts,J. Am. Chem. SOC.,92 (1970) 1355-1361. (420) A.-M. Sepulchre, B. Septe, G. Lukacs, S. D. Gero, W. Voelter, and E. Breitmaier, Tetrahedron, 30 (1974) 905-915. (421) S. Omura, A. Nakagawa, A. NeszmBlyi, S. D. Gero, A.-M. Sepulchre, F. Piriou, and G. Lukacs, J. Am. Chem. SOC., 97 (1975) 4001-4009. (422) P. M. Collins and V. R. N. Munasinghe, Carbohydr. Res., 62 (1978) 19-26. 423) K . Sato, M. Matsuzawa, K. Ajisaka, and J. Yoshimura, Bull. Chem. S o c . J p . ,53 (1980) 189- 191.

134

JUJI YOSHIMURA

It has been noticed that branched sugars are more likely to assume the furanose than the pyranose form,354and it was, in fact, proved'e4 by 'Hand I3C-n.m.r. spectroscopy that, in aqueous solution, hamamelose (2) affords much more of the furanose (71%) than does ribose (24%). More, systematic research is desirable on this subject. ACKNOWLEDGMENT The author is grateful to Dr. F. W. Lichtenthaler of the Technische Hochschule, Darmstadt, for reading the manuscript and giving valuable advice.

(424) S. Schilling and A. Keller,]ustua Liebigs Ann. Chem., (1977) 1475-1479.

ADVANCES I N CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL.

42

SUGAR ANALOGS HAVING PHOSPHORUS IN THE HEMIACETAL RING BYHIROSHI YAMAMOTOAND SABURO INOKAWA Department of Chemistry, Faculty of Science, Okayama Uniuersity, Okayama 700,Japan

I. Introduction

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

135

11. Monosaccharides Having a Phosphinediyl or Phosphonyl Group in the

PyranoseRing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 5-Deoxy-5-phosphino- and -5-phosphinyl-~-xylopyranoses. ............. 2. 5-Deoxy-5-phosphinyl-~-ribopyranose. ............................. 3. 5-Deoxy-5-phosphino- and -5-phosphinyl-~-idopyranoses. .............. 4. 5-Deoxy-5-phosphinyl-~-glucopyranoses. ........................... 5. Structural Analysis of 5-Deoxy-5-phosphino- and -5-phosphinylaldopyranoses by X-Ray Crystallography, and by 400-MHz, Proton Nuclear Magnetic Resonance Spectroscopy and High-resolution Mass Spectrometry ................................................ III. Monosaccharides Having a Phosphonyl Group in the Furanose Ring . . . . . . . . 1. 2,3,4-Trideoxy-4-phosphinylpentofuranoses ......................... 2. 4,5-Dideoxy-4-phosphinylpentofuranoses........................... 3. 4-Deoxy-4-phosphinylaldopentofuranoses . .......................... 4. Structural Analysis of 4-Deoxy-4-phosphinylpentofuranoses by X-Ray Crystallography, and by 400-MHz, Proton Nuclear Magnetic Resonance Spectroscopy and High-resolution Mass Spectrometry . . . . . . . . . . . . . . . IV. Biological Activities of Monosaccharides Having Phosphorus in the Hemiacetal Ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusion ...................................................... VI. Table of Some Properties of Sugar Analogs Having Phosphorus in the Hemiacetal Ring ................................................

138 138 145 145

155

161 176 176 179 181 183 188 189

190

I. INTRODUCTION There exist many naturally occurring sugars in which a hydroxyl group of a monosaccharide is replaced by an amino or a thiol group. These compounds, commonly called amino or thio sugars, play a wide variety of important biological roles. Representative examples of these classes are 2-acetamido-2-deoxy-c~-~-glucose (1) and 7-(5-S-methyl-5-thio-P-~-ribosy1)adenine (vitamin L, ,2). The former is the product ofhydrolysis of 135

136

HIROSHI YAMAMOTO AND SABURO INOKAWA

chitin, and occurs in various mammalian polysaccharides and in certain proteins,' whereas the latter is known to be a factor necessary for lactation.2 No analogous sugars having phosphorus attached to a ring-carbon atom appear to occur in Nature, although numerous sugar phosphates, and some compounds containing a C-P bond, are known (see Section IV).

1

2

Monosaccharides are well known to exist preponderantly in cyclic, hemiacetal forms in solution, and it is generally supposed that, as illustrated in Scheme 1 for aldoses, the equilibrium between the various species depends upon: (I) the reactivities of the -XH, -YH, and -ZH groups towards the carbonyl group (the general order of nucleophilicity is -SH > -PH, > -NH2 > -OH > -NHCOR), and (2) the stability of the hemiacetal rings formed (pyranoid form > furanoid form >> septanoid form). However, the stability of the ring is greatly affected by steric and electronic factors arising both from the nature of the substituents and the configurations of the ring-carbon atoms to which they are attached. Accordingly, if such substituents as amino, thiol, or phosphino are introduced, in the appropriate position, onto a ring-carbon atom of a monosaccharide, corresponding ring closure is expected to take place, to afford sugar analogs having nitrogen, sulfur, or phosphorus in the hemiacetal ring. Indeed, extensive studies on the preparation of such sugar analogs having nitrogen or sulfur in the ring have been carried out by using this method. These analogs are interesting not only from the viewpoint of their physicochemical properties but also from that of their biological activities. For example, 5-amino-5-deoxy-~-ghcose(3, the antibiotic nojirimycin3a4)exhibits antibacterial activity. Also, 5-thio-~glucose (4) has been shown to be a potent, competitive inhibitor of (1) See, for example, M. J. R. Salton, Annu. Reu. Biochem., 35 (1966) 485-520. (2) W. Nakahara, F. Inukai, and S. Ugami, Sci, Pap. Inst. Phys. Chem. Res. (Jpn.), 40 (1943) 433-437; Chem. Abstr., 41 (1947) 6317. (3) S. Inouye,T. Tsuruoka, andT. Niida,J. Antibiot., Ser.A, 19 (1966) 288-292; Chem. Abstr., 66 (1967) 85,989. (4) S . Inouye, T. Tsuruoka, T. Ito, and T. Niida, Tetrahedron, 24 (1968) 2125-2144.

SUGAR ANALOGS HAVING RING PHOSPHORUS

137

CH,ZH

Furanoid form

CH,ZH

Pyranoid form

Acyclic form

It HX Septanoid form Scheme 1

3 x=NH 4

x=s

cellular ~-glucose-transport,~~~ and it is also selectively toxic to hypoxic, radio-resistant, tumor Although the chemistry of these sugar analogs having nitrogen or sulfur in the hemiacetal ring has been well d o ~ u m e n t e d , ’ ~there - ’ ~ are no reviews available on the chemistry and biochemistry of sugar analogs (5) R. L. Whistler and W. C. Lake, Biochem.J.,130 (1970) 919-925. (6) M. Chen and R.L.Whistler, Arch. Biochem. Biophys., 169 (1975) 392-396, and references cited therein. (7) J. H. Kim, S . H. Kim, andE. W. Hahn, Science, 200 (1978) 206-207. (8) C. W. Song, D. P. Guertin, and S. H.Levitt, 1nt.J.Radiat. Oncol.,Biol. Phys., 5 (1979) 965-970; Chem. Abstr., 92 (1980) 16,911. (9) R. Sridhar, E. C. Stroude, and W. R. Inch, In Vitro, 15 (1979) 685-690; Chem. Abstr., 92 (1980) 34,110. (10) H. Paulsen and K. Todt, Ado. Carbohydr. Chem., 23 (1968) 115-232. (11) H. Paulsen, Angew. Chem., Int. Ed. EngE., 5 (1966) 495-516. (12) D. Horton and D. H. Hutson, Ado. Carbohydr. Chetn., 18 (1963) 123-199. (13) S.Inokawa, Kagaku (Kyoto), 24 (1969) 901-913; Chem. Abstr., 72 (1970) 90,757.

138

HIROSHI YAMAMOTO AND SABURO INOKAWA

having phosphorus in the ring. Therefore, the purpose of the present article is to draw attention to this relatively less-explored field of the chemistry of such “P sugars.” The article is divided into Sections, based on the ring size; first, the most thoroughly investigated substances, those having a pyranoid ring, will be discussed (SectionII), and then those with a furanoidring (Section 111);no P-septanoid-ring compound has yet been reported. Finally, some biological aspects will be briefly mentioned (Section IV). The literature has been surveyed up to August, 1983.

11. MONOSACCHARIDES HAVING A PHOSPHINEDIYL OR PHOSPHONYL GROUP IN THE PYRANOSE RING 1. 5-Deoxy -S-phosphino- and -5-phosphinyl-~-xylopyranoses

In order to synthesize monosaccharides having phosphorus in the hemiacetal ring, it is obvious that two fundamental problems have to be solved: (1) how to introduce a suitable phosphine group, having the desired orientation, into an appropriate position on the carbon skeleton of the precursor, and (2) how to accomplish efficient ring-closure in order to yield the desired monosaccharide possessing a ring-phosphorus atom. As direct replacement of the ring-oxygen atom with phosphorus is not feasible for monosaccharides, such a ring-transformation as that illustrated in Scheme 1 is applicable, utilizing an equilibrium shift. Indeed, this method has been employed in most cases for the preparation of monosaccharides having either a nitrogen or a sulfur atom in the hemiacetal ring. For example, 54hio-~-xylopyranose(7), which has been the most thoroughly studied of the monosaccharides having a sulfur-containing hemiacetal ring, was readily by nucleophilic displacement of the p-tolylsulfonyloxy group of the D-xylofuranose derivativela (5) by an RS- ion, followed by reductive conversion into the 5-thio-a-~-xylofuranosederivative (6). Acid hydrolysis resulted in spontaneous ring-expansion to yield 7. Because of the absence of a chiral center at C-5, such pentopyranoses as 7 were judged to be the most amenable to preparation and study. In (14)T. J. Adley and L. N. Owen, Proc. Chem.Soc., (1961)418. (15)J. C.P.Schwarz and K. C. Yule, Proc. Chem.SOC., (1961)417. (16)D.L.Ingles andR. L. Whistler,]. Org. Chm.,27 (1962)3896-3898. (17)R.L. Whistler, M. S.Feather, and D. L. Ingles, J. Am.Chem.SOC., 84 (1962)122. (18)P.A. Levene and A. L. Raymond,]. Elol. Chem.,102 (1933)317-330. (19)R. L.Whistler and C.-C. Wang, J. Org. Chem.,33 (1968)4455-4458. (20)S.Inokawa, H. Yoshida, C . 4 . Wang, and R.L. Whistler, Bull. Chem.Soc. Jpn., 41 (1968)1472-1474. (21) R.L. Whistler, C.-C. Wang, and S.Inokawa,J . Org. Chm.,33 (1968)2495-2497.

SUGAR ANALOGS HAVING RING PHOSPHORUS TsOCH,

~?-Ho~OH 139

HSCH,

(2) (1) NaBnSNa NH,

Q?

*

H,Ot

HO

0-CMe,

0-CMe,

5

7

6

Bn = PhCH,

fact, a similar scheme was employed by Whistler and coworker^^^-^^ in the first synthesis of the phosphorus analogs. To introduce phosphorus into the sugar molecule, application of the Michaelis- Arbuzov reactionZ2was effective in most cases. For this purpose, the hydroxyl group at C-3 of compound 5 was protected with a methyl group to avoid low yields and complication of the reaction (see later). The p-tolylsulfonyloxy group was then replaced by a more reactive leaving-group, leading to an intermediate, such as the 5-bromo d e r i v a t i ~ e l(8), ~ . ~or~better, the 5-iodo compound239 (because of its ease of preparation and also higher reactivity towards the nucleophile in the subsequent step, compared with 8). The reaction of both 5-halogeno compounds (8 and 9) with triethyl phosphite proceeded satisfactorily at 150",to afford the phosphonate 10 (100%yield The same treatment of the p-toluenesulfonate 5 with triethyl phosphite gavele product 10 in lower yield.

5

(1) MeI-Ag,O (2) Bu,N+Bror NaI 0-bMe, 8 X=Br 9 X=I

0-CMe, 10

0-CMe, 1 1 Y =PH, 12 Y = PH,(=O) 13 Y = PH(=O)OH

(22) See G. M. Kosolapoff, Org. React., 6 (1951) 273-338. (23) H. Yarnarnoto, T. Hanaya, S.Inokawa, K. Seo, M.-A. Arrnour, andT. T. Nakashirna, Carbohydr. Res., 114 (1983) 83-93.

140

HIROSHI YAMAMOTO AND SABURO INOKAWA

Reduction of 10 with lithium aluminum hydride (LAH) in ether furnished19 an intermediate, presumably the phosphine derivative l l , which was treated with acid to effect ring enlargement, giving the 5phosphino-D-xylopyranosederivative 14. This compound was immediately converted by air oxidationlQinto the stable crystalline compounds, 5-deoxy-3-O-methyl-5-C-(phosphinyl)-~-xylopyranose (15) and the 5-C-(hydroxyphosphinyl) derivative 16 in overall yields of 15 and 3.5%, respectively, from 10. Compound 16 was obtained in 90%yield from 15 by oxidation with bromine.lQNo mutarotation was o b ~ e r v e d for ' ~ compounds 15 and 16 in water during 48 h. The extremely air-sensitive phosphine 11 was later foundz3 to be also available by reduction of 10 with sodium dihydrobis(2-methoxyethoxy)aluminate (SDMA) in benzene for 1 h at 5" under a nitrogen atmosphere, a procedure that usually converts phosphinates and phosphonates into phosphine oxides (see later). The phosphine 11 was then oxidized with one equivalent of hydrogen peroxide in 2-propanol at 20", to give the 5-phosphinyl derivative 12, together with a small proportion of the further-oxidized product 13. The ring enlargement of the furanose to a pyranoid compound proceeded more efficiently when 12 was heated with oxygen-free, ethanolic 0.5 M hydrochloric acid under nitrogen for 4 h at 100". Compound 15, thus obtained, was then oxidized with an excess of hydrogen peroxide, to afford 16 (65% overall yield from 10). The a-~-pyranoid-~C, structure 18 was p r o p o ~ e d for ' ~ the crystalline product 15 (separated from the reaction mixture in low yield) on the evidence of the splitting patterns of the H-1 signal in the 'H-nuclear magnetic resonance (n.m.r.) spectrum (60 MHz, in deuterium oxide). The exact structure for the oxidized product 16 was not given, although the presence of a phosphinic acid group was supportedlgby infrared (i.r.) absorption at 2260 cm-' and the approximate pK, value (1.61). An unambiguous, structural assignment was made23by converting the anomeric mixture 16 into the tri-O-acetyl-5-C-(methoxyphosphinyl) derivatives 17 by treatment with diazomethane and then with acetic anhydride - pyridine; after chromatographic separation, structures 19 22 [all in the 4 C 1 ( ~conformation] ) were established for each product by analysis of the 400-MHz, 'H-n.m.r. and high-resolution mass spectra (see Section 11,5).The overall yields ofthese products from 10 were: 19 (6.5), 20 (2.7), 21 (4.9),and 22 (3.3%). Besides the four diastereoisomers 19-22, a small proportion of a byproduct was isolated (2.7% overall yield from lo), to which structure 23, namely, 5-C-[(R)-(l-acetoxyethenyl)phosphinyl]-l,2,4-tri-0-acetyl-5deoxy-3-0-methy~-j3-~-xylopyranose-~C, , was assigned24 from the (24) H. Yarnamoto, T. Hanaya, S. Inokawa, and M.-A. Armour, Carbohydr. Res., 124 (1983) 195-200.

SUGAR ANALOGS HAVING RING PHOSPHORUS H*02

11

141

12

i.e' :;q

JH~O+

0

Q m L ( + HO

II

HO

HO

H202

OH

OH

OH

1s

14

16 (1) CH2N2 (2) Ac20- C,H,N

0

0 I1

I

OH 18

0 It

A

c

Me0

o / W

R OMe ,

""""

""m~, 17

OMe P xi::0

Me0

AcO

R 19 R = OAC, R' = H 20 R = H , R' = OAc

OAc

AcO R

21 R = H, R ' = OAc 22 R = OAC, R' = H

23

'H-n.m.r. and mass spectra (see Section 11,5); the corresponding 5-C-[(S)-phosphino] structure had been tentatively assignedz3 to this product. The chemical shifts and the splitting patterns of the ring-proton signals in the n.m.r. spectrum closely resembled those of 20, thus permitting the assignment of the (R)configuration to the phosphinyl group.

142

HIROSHI YAMAMOTO AND SABURO INOKAWA

This minor product 23 and its diastereoisomers were also isolated,e4in 23% yield, when 15 was treated with acetic anhydride -pyridine. Thus, these products were assumede4to have been formed by direct, "double" acetylation of the 5-C-phosphinyl group in 15, rather than by the previously proposede3pathway involving the disproportionation of 15. A similar acetylation reaction is known25for various primary and secondary phosphine oxides. Besides 23 and its diastereoisomers, a complex product resulting from phosphorus -phosphorus dimerization of 15 was isolated, in 25% yield, during the aforementioned acetylation; this appears to be analogous to the formation of tetraphenyldiphosphine monoxide [Ph2P-P( = O ) Ph,] from diphenylphosphine oxide in the presence of acetic anhydride and pyridine at room temperature.26 These results,23therefore, provided further proof for the formation of 5-deoxy-5-C-(phosphinyl)-and -(hydroxyphosphinyl)-D-xylopyranoses (15 and 16) from the 5-phosphinyl-~-xylofuranose precursor 10. Soon after the appearance of a brief report on compounds 15 and 16 by Whistler and Wang,le Inokawa and his coworkerse7 prepared 3- 0-benzyl- 5-deoxy-5-C-[(RS)-ethylphosphinyll-a$- D-xylopyranoses (29) in 27% yield from 3-0-benzyl-5-deoxy-5-iodo172-O-isopropylidene-a-D-xylofuranose28(24) by way of the ethylphosphine oxide 26, according to essentially the same procedures as those described earliere3 for preparation of 15 from 9. Structure 29 was supported by the 'H-n.m.r. spectrum (~O-MHZ, in Me,SO-d,) and i.r. spectrum. Similarly, the Michaelis - Arbuzov reaction of the 5-bromo-3-0methyl compound 8 with diethyl ethylphosphonite at 130- 150" resultedee in a quantitative yield of 5-deoxy-5-C-[(RS)-(ethoxy)ethylphosphinyl]-l,2-O-isopropylidene-3-O-methyl-a-~-xylofuranose, which, upon reduction with SDMA in oxolane for 1hat room temperature under a nitrogen atmosphere, gave an -100% yield of its 5-C-(ethylphosphinyl) derivative 27 (R' =Et). This ethylphosphine oxide 27 was apparently more stable than the phosphine oxide 12, and it clearly showed a characteristic lJH,p value30of 458 Hz at 6 6.92 in the 'H-n.m.r. spectrum (in CDCl,), and typical, i.r. absorptions due to a P-H group3' at 2320 cm-' and a P=O group3, at 1240 crn-'. The hydrolysis of 27 with 0.3 M sulfuric acid for 2 h at 100" gaveee (25) S.A. Buckler and M. Epstein, Tetrahedron, 18 (1962) 1221-1230. (26) S.Inokawa, Y. Tanaka, H. Yoshida, andT. Ogata, Chem. Lett., (1972) 469-470. (27) S. Inokawa, Y. Tsuchiya, K. Seo, H.Yoshida, andT. Ogata, Bull. Chem. Soc.]pn., 44 (1971) 2279. (28) R. C. Young, P. W. Kent, and R. A. Dwek, Tetrahedron, 26 (1970) 3984-3991. (29) K. Seo and S. Inokawa, Bull. Chem. Soc.]pn., 46 (1973) 3301-3302. (30) H. R. Hays.,J. Org. Chem., 33 (1968) 3690-3694. (31) R. A. Chittenden and L. C. Thomas, Spectrochim.Acta, 20 (1964) 489-502. (32) L. C. Thomas and R. A. Chittenden, Spectrochim.Ada, 20 (1964) 467-487.

SUGAR ANALOGS HAVING RING PHOSPHORUS

143

5-deoxy-5-C-(ethylphosphinyl)-3-O-methyl-~-xylopyranose (30) in a relatively good yield (70%). This product was characterized by derivatization to the triacetate, which was considered to be a mixture of the four diastereoisomers with respect to C-1 and the ring-phosphorus atom. A pure, crystalline compound (m.p. 227 - 229") separated from the mixture upon recrystallization from ethanol, and it became apparent,33 by comparison of its 'H-n.m.r. spectrum with those of the structurally similar compounds (see Section 11,5), that this product was the 5-C-[(R)ethylphosphinyll-B-~-xylopyranose 34. Likewise, the use of diethyl butylphosphonite in the Michaelis Arbuzov reaction of 8 gave2e 5-C-(butylphosphinyl)-~-xylopyranose 31 (50% yield) by way of intermediate 27 (R' = Bu). The product was also characterized as the tri-0-acetyl derivative: a single compound (m.p. 218.5 - 220") was r e c o ~ e r e dand , ~ ~structure 35, namely, 5-[(R)-butylphosphinyl]-P-~-xylopyranose, could be assigned33to this product from its n.m.r. spectrum.

0-CMe, 24 R = Bn 2 5 R = Ac or Bz Bz = PhCO

OH

0-CMe,

26 R = Bn, R' = Et 27 R = Me, R' = Et or Bu 28 R = H, A' = Et or Bu Bu = C,H,

29 30 31 32 33

R R R R R

= Bn, R' = Et = M e , R' = Et

= M e , R ' = Bu = H, R' = Et = H , R' = Bu

An analogous procedure was successfully applied34to 5-deoxy-5-iodo1,2-O-isopropylidene-a-~-xylopyranose (42),in which the hydroxyl group on C-3 had been protected with an acetyl or benzoyl group (to give 25), prior to the Michaelis-Arbuzov reaction with an alkylphosphonite. The intermediates 28 (R' = Et or Bu) were hydrolyzed with dilute sulfuric acid, to afford 5-deoxy-5-(ethylphosphinyl)-(32;95% yield) and -5-(butylphosphiny1)-D-xylopyranose(33;91% yield), respectively, as diastereoisomeric mixtures. Acetylation of 32 gave, again, a mixture of four diastereoisomers, from which two crystalline compounds were isolable. Although no exact configurations for C-1 and the ringphosphorus atom of these two products had been presented,34 they could be assigned33 the structures 1,2,3,4-tetra-O-acetyl-5-deoxy-5-C-[(R)ethylphosphinyll-P-~-xylopyranose (36)and its a anomer 37 on the basis (33) S. Inokawa and H. Yamamoto, unpublished results. (34) K. Seo and S . Inokawa, Bull. Chem.SOC.Jpn., 48 (1975) 1237-1239.

HIROSHI YAMAMOTO AND SABURO INOKAWA

144

0

0

34 R = Me, R' = Et 35 R = Me, R' = Bu 36 R = Ac, R' = Et

37 R = E t 38 R = Bu

of their 'H-n.m.r. spectra, melting points, and [a], values (see Table VIII in Section VI). For the per-0-acetyl derivatives of these p y r a n ~ i d - ~ c , sugar analogs, those compounds that have an equatorial acetoxyl group on C-1 normally show a higher melting point and a lower [a], value than those having an axial AcO-1 group. 33 was obLikewise, 5-C-(butylphosphinyl)-5-deoxy-~-xylopyranose tained34from 25 (R = Ac or Bz) by way of the phosphine oxide intermediate 28 (R' = Bu). An acetyl derivative having structure 38, namely, 1,2,3,4-tetra-O- acetyl- 5-C- [(R)-butylphosphinyll-5- deoxy- a -D-xylop y r a n o ~ ecrystallized ,~~ from the diastereomeric mixture of the per-0acetylated products from 33. 0 II

Bu-P-CH,

uT :rg*

f:

' QT

Me0

0-CMe,

39

o*

Bu-P-CH,

+

,CHZ

LQ

SDMA-28

0-CMe,

0-CMe,

40

41

f

BuP(OEt),

0 - CMe, 42

In the course of these experiments, treatment of the 3-O-acetyl-5-C(butylethoxyphosphinyl) compound 39 with sodium methoxide in methanol was found34 to give, in 94% yield, a 5 : 1 mixture of the 5-C-(butylmethoxyphosphinyl)derivative 40 and crystalline 5-C-[(30,P-anhydro) butylphosphinyl] -5-deoxy - 1,2 - 0- isopropylidene - a - D xylo-furanose (41). The structure 41 was based on the n.m.r. spectrum, which showed neither a P-OMe nor an OH signal, and gave the H-3

SUGAR ANALOGS HAVING RING PHOSPHORUS

145

signal at much lower field (S 5.18)than that of 40 (6 4.00). On reduction with 1.2 equivalents of SDMA, both 40 and 41 gave the phosphine oxide 28 (R’ = Bu) in good yields. Compound 41 was obtained almost quantitatively by heating 42 in diethyl butylphosphonite, which provides, at least partly, the complicated reason for the lower yields from the Michaelis- Arbuzov reaction of 5-halogeno- 1,2-O-isopropy~idene-cr-~-xylofuranose where the hydroxyl group on C-3 was not protected (see earlier). 2. 5-Deoxy-5-phosphinyl-~-ribopyranose

Other than the 5-deoxy-5-phosphinyl-~-xylopyranoses described in the previous Subsection, there has been reported35 only one example of a different type of 5-deoxy-5-phosphinylpentopyranose. The Michaelis - Arbuzov reaction of methyl 5-deoxy-5-iodo-2,3-0isopropylidene-~-~-ribofuranoside~~ (43)with diethyl ethylphosphonite gave,35 in 80% yield, the 5-C-[(ethoxy)ethylphosphinyl]derivative which, on treatment with SDMA and then mineral acid, yielded (30%) 5-deoxy-5-C-[(RS)-ethylphosphinyl]-~-ribopyranose (44)as a mixture of diastereoisomers. These compounds showed no mutarotation in methanol during 24 h. Upon treatment with acetic anhydride- pyridine, the product, 44, was converted (90% yield) into a syrup, presumably consisting of four diastereoisomers of the peracetate 45,separation of which was not attempted. Treatment of 45 with sodium methoxide in methanol regenerated 44 quantitatively. 0

p J I; w m ” -

ICH,

0

Ac@ II

(3) H 3 0 t \

/o

CMe, 43

HO

OH 44

AcO

OAc 45

3. 5-Deoxy-5-phosphino-and -5-phosphinyl-~-idopyranoses

If appropriate 5-deoxy-5-phosphino- or -5-phosphinyl-aldohexofuranoses are available as precursors, the analogous ring enlargement described in the previous Subsections would be expected to provide various 5-deoxy-5-phosphino- or -5-phosphinyl-aldohexopyranoses having (35)S. Inokawa, H.Kitagawa, K. Seo, H. Yoshida, and T. Ogata, Carbohydr. Res., 30 (1973)127-132. (36) P. A. Levene and E. T. Stiller,]. B i d . Chem.,106 (1934)421-429.

HIROSHI YAMAMOTO AND SABURO INOKAWA

146

a ring-phosphorus atom. For example, because of the chirality at C-5, 5-deoxy-5-C-[(RS)-phosphino(or phosphinyl)]-~-xyb-hexofuranoses46 and 47 would yield D-glucopyranoses (48) and L-idopyranoses (49), respectively, provided that no epimerization takes place at C-5. Much information concerning such a ring enlargement has been accumulated. For historical reasons, the preparation of L-idopyranose derivatives will be discussed first.

46 Y = PHR or P(=O)HR

47

HOCH,

H

o

a 48

HO O

I

o

S

HO O

H

49

Addition of dimethyl phosphonate to 3-0-acety1-5,6-dideoxy-l,2-0isopropy~idene-6-nitro-a-~-xy~o-hex-5-enofuranose (50) was known to provide3' an 89 : 11mixture of 6-nitro-5-phosphonylhexofuranoses having the D-gluco (51; 40%isolated yield) and the ~ - i d configuration o (53), whereas addition of methanol, ammonia, or phenylmethanethiol to 50 mainly a D-ghco compound. The assignment of the D-ghco configuration to compound 51 was based on the observation of anegative Cotton-effect in optical circular dichroism (c.d.) at 310 nm, which implies the (5A)D-gluco configuration. This effect is present for similar, model compounds, in accordance with the rule of Satoh and KiyoThese results were extended43 to the addition of methyl ethylphos(37)H.Paulsen and W. Grewe, Chern. Ber., 106 (1973)2114-2123. (38)H. Paulsen, Ann., 665 (1963)166-187. (39)H. H. Baer and W. Rank, Can. J . Chm., 43 (1965)3330-3339. (40)R. L.Whistler and R.E. Pyler, Carbohydr. Res., 12 (1970)201 -210. (41)C.Satoh, A. Kiyomoto, andT. Okuda, Carbohydr. Res., 5 (1967)140-148. (42)C.Satoh and A. Kiyomoto, Carbohydr. Res., 23 (1972)450-455. (43)H. Takayanagi, K. Seo, M. Yamashita, H. Yoshida, T. Ogata, and S. Inokawa, Carbohydr. Res., 63 (1978)105-113.

SUGAR ANALOGS HAVING RING PHOSPHORUS

147

phinate to 50, to give, in 95% yield, a 1: 1mixture of the D-gluco (52) and L-ido (54) compounds, from which crystalline 3-0-acetyl-5,6-dideoxy-5C-[(R or S)-ethylmethoxyphosphinyl]-l,2-O-isopropylidene-6-nitro-aD-glucofuranose (52) separated in 20% yield. The D-gluco configuration was assigned to 52 on the evidence of its 'H-n.m.r. spectrum, the chemical shifts and coupling constants of each proton of which closely resembled those of 51.

0

kH

II

QT

EtP(=O)H(OMe) HP(OMe), or 0-CMe, 50

CH,NO,

:: FaNOa

0,NCH

I:

R -8-CH "

'

O

q

T

0-CMe, 51 R = O M e 52 R = E t

HCPR(0Me) +

Q? 0-CMe, 53 R = OMe 54 R = E t

Reduction of 52 with hydrogen in the presence of Raney nickel was a ~ c o m p a n i e dby~ ~cyclization of the phosphinate group and transfer of the acetyl group, to give, in 60%yield, crystalline 6-acetamido-5-C-[(0,P- anhydro)ethylphosphinyl]-5,6- dideoxy-l,2 - 0-isopropyhdene-cDglucofuranose (55). However, hydrogenation of 52 in methanol in the presence of platinum oxide and hydrochloric acid afforded, in 80% yield, the stable hydrochloride 58, which was readily converted into 55 by means of an anion-exchange resin. Deamination of 58 with nitrous acid gave, in 59% yield, the 5-(ethylmethoxyphosphinyl)-~-glucofuranose derivative 59, which was spontaneously converted into the crystalline, 5-C-[(3-O,P-anhydro)ethylphosphinyl]compound 56. Upon treatment with an excess of SDMA, none of these precursors (52,55,56,58, and 59) gave the desired compound 57, but instead, almost complete decomposition. Thus, all attempts to convert the phosphinate group into a phosphine oxide group have remained unsuccessful; see Section 11,4, however, for a successful example of reduction (with SDMA) of compound 130, which is analogous to 55 and 56. Instead of employing the addition of phosphinates to 50, with subsequent reduction, the use of phenylphosphine for the addition reaction to 50 was found to give43.44a mixture of the crystalline ~ - i d compound o 60 (47% yield), the D-gluco isomer 61 (16% yield), and the 1: 2 adduct 62 (34% yield). Because of its negative Cotton-effect in c.d. at 229 nm, and taking into account the results3' using compound 51 (see earlier), the (44) H. Takayanagi,M. Yamashita,K. Seo, H. Yoshida, T. Ogata, and S. Inokawa, Carbohydr. Res., 38 (1974) c19-c21.

148

HIROSHI YAMAMOTO A N D SABURO INOKAWA 52

/

\

H, /Raney Ni or PtO,

H, /PtOz-HC1 0 CH,R

Et -P

II

I -CH

Q7 0-&Me,

SDMA

0-CMe,

55 R = NHAc 56 R = OAC

58 R = NH,*HC1 59 R = O H

/ 0 CH, II I Et-P-CH

ii

qy 0-CMe, 57

major product 60 had been incorrectly assigned43the D-glucoconfiguration. However, the correct configuration (L-ido)of compound 60 was later determined from the precise structural assignments of the monosaccharides 66 - 68 that were derived from 60 as follows. Acid hydrolysis of 60 and of its oxidized derivative 64 afforded the 5-C-(phenylphosphino)-D-xylo-hexopyranose63 (81% yield) and the 5-C-phenylphos-

- qy CH,NO, I HC-PHPh

50

Ql

CH,NO, I PhHP-CH

PhPH2

+

0-CMe, 60

+

1:2 adduct

62

0-CMe, 61

phinyl derivative 65 (67%yield), respectively. After per-0-acetylation of these products, crystalline compounds 66 (26%yield from 60) and 67 (together with a small proportion of 68; 29% yield from 64) were isolated; these compounds were the first examples of hexopyranoses having a ring-phosphorus atom. X-Ray crystallographic analysis indicated45that (45) P.Luger, M. Yamashita, and S. Inokawa, Carbohydr. Res., 84 (1980) 25-33.

SUGAR ANALOGS HAVING RING PHOSPHORUS

ek P-Ph

60

149 Ph

~

Ac,O- C5H5N

CH,NO,

AcO

HO

AcO

OH 66 X = lone pair 67 X = O

63

\

OAc

CH,NO,

I::

HC-PHPh

0-CMe, 64

0 II

0

'--.p h Ac,O- C,H5N * A

c

o CH,NO, n O

A

c

AcO OH 65

68

compound 67 has the structure of 1,2,3,4-tetra-O-acetyI-5,6-dideoxy6-C-nitro-5-C-[(R)-phenylphosphinyl]-~-~-idopyranose, whereas the structures of compounds 66 and 68 were shown by 400-MHz, 'H-n.m.r.and the spectral analysis to be the 5-C-[(S)-phenylpho~phinoI-P-~~ 5-C-[(S)-phenylphosphinyll-a-L-idopyranose d e r i ~ a t i v erespectively; ,~~ see Section II,5 for a discussion. Treatment of the 1:2 adduct 62 with one equiv. of hydrogen peroxide in methanol gave mainly a pure, crystalline compound (m.p. 209210.5") whose structure was presumed to be bis(3-0-acetyl-5,6dideoxy - 1,2-0 - isopropylidene - 6-C-nitro - ~-~-idofuranose-5-yl)phenylphosphine oxide (69);therefore, the original 1 : 2 adduct 62 was assumed to be a mixture consisting mainly of the corresponding phosphine compound. The acid-catalyzed ring enlargement of 61, which could have given the D-gluco epimer of 63, was not attempted, because of the difficulty in separating pure compound 6 1. An alternative method of introducing a phosphinyl group at C-5 of (46) H. Yamamoto, C. Hosoyamada, H. Kawamoto, S. Inokawa, M. Yamashita, M.-A. Armour, and T. T. Nakashima, Carbohydr. Res., 102 (1982) 159-167.

jPPh

HIROSHI YAMAMOTO AND SABURO INOKAWA

150

a2L(?QT CH,NO, \ R

2

0-CMe, 69

5-deoxy-~-xy~o-hexofuranose has been developed as an extension of the reaction of l-(p-tolylsulfonyl)oxy-2-propanone(70) with dimethyl phosphonate (75) in the presence of one equivalent of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); this procedure readily gave4' methyl 1,2epoxy-1-methylethanephosphonate (71), which is an isomer of the (72). antibiotic fosf~nornycin~~ 0 0

I1 H.&--C--CH,OTs

It

HP(OMe),

/O\

0 / \ )PO~HZ

H3C\

H,C-C-CH,

P

DBU

H4c-c H '

I 0 =P(0Me b

72

71

70

Ts = SO,C,H,Me-p

Thus, treatment of 6-O-p-tolylsu~fonyl-a-~-xy~o-hexofuranos-5u 1 0 s e ~(73) ~ with 75 in methanol in the presence of one equivalent of DBU for 20 h at room temperature gave, in 70% yield, a 3 : 1mixture of (5RS)-5,6-anhydro-5-C-(dimethoxyphosphinyl)-~-xylofuranoses (77). The absolute structure of the major product was shown50by X-ray crysCH,OTs I

o=c

Q

0 HP, II,RfR"

-

0 II R'-P-C

l,O

DBU

AM

0-CMe,

73 R = B n 74 R = M e

F,

75 R' = R" = OMe 76 R' = Ph, R" = OEt

77 78 79 80

R R R R

QT

= Bn, = Bn. = Me, = Me,

0-CMe, R' = R" = OMe Rf = Ph, R" = OEt Rf = R" = OMe Rf = Ph, R" = OMe

S. Inokawa, Y. Kawata, K. Yamamoto, H. Kawamoto, H. Yamamoto, K. Takagi, and M. Yamashita, Carbohydr. Res., 88 (1981) 341-344. E.J. Glamkowski, G.Gal, R. Purick, A. J. Davidson, and M. Sletzinger,]. Org. Chem., 35 (1970) 3510-3512, and references cited therein. S. Inouye, Meijt Seika Kenkyu Nempo, (1970) 52-74; Chem. Abstr., 77 (1972) 48,736. S. Kashino, S. Inokawa, M. Hdsa, N. Yasuoka, and M. Kakudo, Acta Crystulbgr., Sect. B, 37 (1981) 1572-1575.

SUGAR ANALOGS HAVING RING PHOSPHORUS

151

tallography to be the (5R) epimer 82. The addition of 75 to 73 is presumed to take place in either the “Cram” or the “anti-Cram” f a s h i ~ n , ~ ’ to yield two diastereoisomers, 82 and 83, with respect to the configuration of C-5. The most likely orientations along the C-5-C-4 bond are illustrated in formula 8 1, which suggests that the anti-Cram type of addition is sterically the more favorable, leading to formation of 82 as the major product.

PhCH, \

M&O 82

OTs

81

83

Similarly, the (5R)-5-deoxy-5-C-[(RS)-(ethoxy)phenylphosphinyl] compound 78 was prepared4’ (69% yield) from 73 and ethyl phenylphosphinate (76). Also, condensation of the 3-O-methyl compound 74 with 75 and 76 (R” = OMe) respectively gave the (5RS)-5-C-[(RS)-phosphinyl] compounds 79 (75% yield) and 80 (46% yield). Hydrogenation of 78 in ethanol in the presence of Raney nickel (W-4) for 2 days at room temperature did not produce the anticipated 5-deoxy5-C-(phosphinyl)hexofuranose 84 but, instead, a i € ~ r d e d , in ~ ~90% *~~ yield, a 1 : 1 mixture of the (5R)-and (5S)-5,6-dideoxy-5-C-[(Rs)-(ethoxy)-phenylphosphinyl]-D-xylo-hexofuranoses($5), which was separated by thin-layer chromatography (t.1.c.). Similarly, (5RS)-5,6-dideoxy-l,2 - 0 - isopropylidene- 5 -C-[(RS)-(methoxy)phenylphosphinyI](51) D. J. Cram and F. A. A. Elhafez,J. Am. Chen. SOC.,71 (1952) 5851 -5859. (52) S. Inokawa, K.Yamamoto, Y. Kawata, H. Kawamoto, H.Yamamoto, K. Takagi, and M. Yamashita, Curbohydr. Res., 86 (1980) c l l - c l 2 . (53) S. Inokawa, K. Yamamoto, H. Kawamoto, H. Yamamoto, M. Yamashita, and P. Luger, Carbohydr. Rex, 106 (1982) 31-42.

HIROSHI YAMAMOTO AND SABURO INOKAWA

152

3-O-methyl-c~-~-xylo-hexofuranose was prepared in 85%yield from 80. Hydrogenation of each component of 85 in the presence of 10%Pd-C at room temperature gave the corresponding (5R)- and (5s)-hexofuranoses 86. Then, the (5R) and (5s)compounds (86) were individually reduced64 with SDMA, to afford a mixture of the (5s)-and (5B)-5,6dideoxy-5-C-(phenylphosphinyl)-~-xylo-hexofuranoses (88 and 89, respectively), along with a small proportion of byproducts (apparently formed by elimination of the phosphinate group). Acid hydrolysis of 88 and 89 was expected to give a mixture of 5,6-dideoxy-5-C-[(RS)-phenylphosphinyll-a,P-~-idopyranose 90 and the D-glum epimer 91.

-

0 CH,OH II I Ph-P-CH EtA

QT

:: 7%

Ph-P-CH

78-

H,

EtA

%\h.

0-CMe,

0-CMe, 85 R = B n 86 R = H 87 R = T H P

84

THP = tetrahydropyran-2-yl

0 II Ph-P-Ii

86 or 8 7 -

SDMA

0 II

Qy

H,C-&H

+

$HS

=

ph-i-'(T

0-CMe,

0-CMe,

p+

89

88

1H'. 0

OH

OH 90

OH 91

(54) H. Yamamoto, K. Yamamoto, S. Inokawa, andP. Luger, Carbohydr.Aes., 113 (1983) 31-43.

SUGAR ANALOGS HAVING N N G PHOSPHORUS

1 S3

Per-O-acetylation of this mixture with acetic anhydride - pyridine gave53five crystalline compounds, to which the structures 1,2,3,4-tetraO-acetyl-5,6-dideoxy-5-C-((S)-phenylphosphiny~]-ac-~-idopyranose (92; 4.5% yield from 86), its p anomer 93 (4.5%), the 5-C-[(R)-phenylphosphinyll-ac epimer 94 (5.7%),the /3 anomer 95 (8.5%), and 2,3,4-tri-Oacetyl- 1,5-anhydro-5,6 - dideoxy- 5 -C- [(S)-phenylphosphinyl]- ~ - i d i t o l (96; 3.9%)were assigned by X-ray crystallographic a n a l y s i ~(for ~ ~ 92, .~~ ~ ~ 92-96); see 93, and 96) and 400-MHz, ‘H-n.m.r. s p e c t r o ~ c o p y(for Section 11.5.

R = H, R’ = OAC 93 R = OAc, R’ = H 92

94 R = H, R’ = OAc 9 5 R = OAc, R’ = H

96

Although a precise configuration [(5R)or (5S)]could not b e assigned to the two starting-materials 86, both compounds were found to give almost the same proportions of the L-idopyranoses 92 -95, plus the byproduct 96; it is noteworthy that no “P sugar” of the D - ~ ~ U C type O was formed from 86. As expected, use of the 1 : 1 mixture 86 as the starting material also resulted in the formation of 92-96 in the same ratios. When the 3-O-(tetrahydropyran-2-y1)derivative 87 was reduced with a smaller proportion (2 equivalents) of SDMA, the yields of the L-idopyranoses 92- 95 were significantly increased (54% total yield from 87); moreover, this procedure totally suppressed both the formation of the over-reduced product 96 and the elimination reaction of the phosphinate group. The following mechanism was proposed54 for the preponderance of the L-idopyranoses 92 - 96. A thermodynamically controlled, more favorable production of the (5s) epimer 88 takes place after an equilibration caused by the strongly basic SDMA during the reduction. This occurs because there apparently exists less steric congestion between the bulky phenylphosphinyl and the 3-hydroxyl (or protected hydroxyl) group in 88 compared with the ( 5 8 ) epimer 89, as illustrated in Scheme 2. This (5s) epimer 88, in turn, readily affords 92-95 on per-O-acetylation, despite the presence of a slightly less favorable, steric requirement for the intermediate 97 compared with the counterpart 98. On the other hand, the formation of a small proportion of 96 from 86 was explained in terms of the further reduction of 88, by an excess of (55) H. Yamamoto, K. Yamamoto, H. Kawamoto, S . Inokawa, M.-A. Armour, and T. T. Nakashima,]. Org. Chem., 47 (1982) 191-193.

X

ti 0

e,

h

m

v,

9

f

0 X

X

N

SUGAR ANALOGS HAVING RING PHOSPHORUS

155

SDMA, to the phosphine 99,which subsequently led to 96,by way of intermediate 100, by transfer of the oxygen atom from C-1 to the phosphorus atom, as in the following, frequently observed, example.56 RR'C=O

+ PH3

H+

RR'CH-P(=O)H,

+

The 5-C-[(R)-phenylphosphinyl]epimer of 96was not isolated, but was presumed to be present in the large quantity of polar substances that remained uneluted in the t.1.c. separation. The 1 : 1 ratio (ofthe combined yields of the compounds) of the (S) to the (R) isomers ofthe ring-phosphorus atom (92,93,96to 94,95)from 86 and 87 suggested that hemiacetal formation from 97 (and 100)to 92-95 (and 96) proceeds at almost the same rate for both 5-C-[(R)-and ( S ) phenylphosphinyll-L-idopyranoses.

-

4. 5-~eoxy-5-ghosphiny~-~-g~ucopyranoses

When either of the two methods in the previous Subsection is employed in order to introduce a phosphino (or phosphinyl) group at C-5 of 5-deoxy-~-xy~o-hexofuranoses, only 5-deoxy-5-phosphino- (or -5-phosphiny1)- L-idopyranoses are produced; for instance, 50 60 (and 64) 63 (and 65), and 73 -+ 78 -, 85 88 90. Therefore, in order to prepare hexopyranoses of the D-gluco type having phosphorus in the hemiacetal ring, an alternative approach had to be devised. In the meanwhile, a new method for preparing various sec-alkanephosphonates (103)from ketones by way of the hydrazone 101 and then the substituted tolylhydrazines 102, had been d e v e l ~ p e d . ~ By ' using this

- -

-

0

101

102

103

method, many sugar derivatives having a phosphorus-carbon bond have been ~ b t a i n e d . ~For ~ - "example, ~ condensation of the D-xylo-hexofuranos-5-dose I04 (prepared"' from 6-deoxy- 1,2-O-isopropyIidene-a-~(56) S. A. Buckler and M. Epstein, 1.Am. C h .SOC., 82 (1960) 2076-2077. (57) S. Inokawa, Y. Nakatsukasa, M. Horisaki, M. Yamashita, H. Yoshida, and T. Ogata, Synthesis, (1977) 179-180. (58) M. Yamashita, Y. Nakatsukasa, M. Yoshikane, H. Yoshida, T. Ogata, and S. Inokawa, Carbohydr. Res., 59 (1977) c 1 2 - c l 4 . (59) M. Yamashita, Y. Nakatsukasa, S.Inokawa, K. Hirotsu, and J. Clardy, Chem.Lett., (1978) 871-872. (60) M. Yamashita, Y. Nakatsukasa, H. Yoshida, T. Ogata, S. Inokawa, K. Hirotsu, and J. Clardy, Carbohydr. Res., 70 (1979) 247-261. (61) H. Ohle and R. Deplanque, Ber., 66 (1933) 12- 18.

HIROSHl YAMAMOTO AND SABURO INOKAWA

156

xylo-hex-5-enofuranose) with p-tolylsulfonylhydrazine in methanol at room temperature gave hydrazone 106 in 80% yield. Similarly, the 3-0methyl derivatives 105 (obtained in 60% yield from 104) afforded hydrazone 107 (70% yield).

TSNHNH,~~~=~!~

y s

y

QT

o=c

0-CMe,

0 R-PH(0Me) II

0-CMe,

I

0

I1

YRt

0-CMe, 1 0 8 R = M e , R' = OMe 1 0 9 R = M e , R' = Ph 110 R = H , R ' = P h

106 R = H 107 R = M e

104 R = H 105 R = M e

by

H,C TsNHNW-C-

3

Treatment of 107 with 75 in the presence of 0.15-0.45 mol. equivalent ofp-toluenesulfonic acid for 40 - 50 hat room temperature gave60the (5RS)-5-[(RS)-dimethoxyphosphinyl]-5-(p-tolylsulfonylhydrazino)hexofuranoses 108 (70%yield). Likewise, compound 107 and methyl phenylphosphinate gave an 100% yield of the 5-[(methoxy)phenylphosphinyl] compound 109, whereas hydrazone 106 afforded four products, namely, two isomers of the xylofuranoses 110 (15%)and two isomers of the 5-C-[(RS)-(U,P-anhydro)phenylphosphinyl]derivatives 111 (51 and 26%). Compound 111 was considered to be produced from 110 during isolation employing sodium hydrogencarbonate, because 110 readily afforded 111 by treatment with sodium methoxide.

-

0 CH, I! I Ph-P-C-NHNHTs

0-&Me, 111

Reduction of compounds 108 and 109 with an excess of sodium borohydride in oxolane respectively gave (SRS)-S-C-[(RS)-dimethoxyphosphinyl]-~-xyb-hexofuranoses112 (21% yield) and the 5-C-[(RS)-(methoxy)phenylphosphinyl] compound 113 (70% yield). The phosphinate 113 was reduced with an excess of SDMA in oxolane at O", to give the 5-C-[(RS)-phenylphosphinyl] derivative (41% yield after purification by t.l,c.), which, on acid hydrolysis, yielded (75%)a mixture of 5,6-di-

SUGAR ANALOGS HAVING RING PHOSPHORUS

157

deoxy-3-O-methyl-5-C-[(RS)-phenylphosphinyl]-~-gZ~c~and - ~ - i d o pyranoses (1 14).Treatment of 114 with acetic anhydride-pyridine gave the per-O-acetylated products, from which two crystalline compounds, 1 , 2 , 4- tri- 0 -acetyl- 5,6-dideoxy- 3 -0-methyl-5 - C-[(S)-phenylphosphinyl]-P-~-glucopyranose-~C~ (1 15; 16% yield,) and, probably, its a anomer 116 (low yield), were isolated after recrystallization from ethanol. The structure of 115 was established by X-ray c r y s t a l l ~ g r a p h y ~ ~ ~ ~ ~ and 400-MHz, 'H-n.m.r. ~ p e c t r o s c o p y(see ~ ~ Section 11,5). The other isomers, which were assumed to be present in the mother liquor, remained uninvestigated.

R 115 R = H , R' = OAc 116 R = OAC, R' = H

By following the same scheme, the first example of a complete glucose structure having a ring-phosphorus atom was prepared.46Treatment of 5,6-anhydro-3-0-benzyl- 1,2-O-isopropylidene-a-~-g~ucofuranose (obtained'j2 from the 6-O-p-tolylsulfonyl compound 117) with sodium phenylmethoxide for 4 days at room temperature gave 3,6-di-O-benzyl-~glucofuranose 118 (70% yield). Compound 118 was oxidized with pyridinium chlorochromate over molecular s i e v e P in dichloromethane for 4 h at room temperature, to afford ketone 119 (90%yield). By using the method described earlier, 119 was converted (100% yield) into a mixture of E- and Z-hydrazones 120 and 121 in the ratio of 7 : 3. Treatment of the hydrazones with methyl phenylphosphinate in the presence (62) A. S. Meyer andT. Reichstein, Helo. Chim. Acta, 29 (1946) 152-163. (63) J. Herscovici and K. Antonakis,]. Chem. Soc., Chem. Commun., (1980) 561-562.

HIROSHI YAMAMOTO AND SABURO INOKAWA

158

of trifluoromethanesulfonic acid yielded (50%) the adduct 122 as a mixture of four diastereoisomers (with respect to C-S and the P atom). The p-tolylsulfonylhydrazino group of 122 was removed by reduction with sodium borohydride in oxolane, to give the intermediate 123 (34% yield), again as a mixture of four diastereoisomers. Reduction of 123 with CH,OR I HO-CH

CH,OBn I

+ 118

Q7

x=c

Q7

C,H, NCr0,CL;

* 0-CMe,

0-CMe,

117 R = T s 118 R = Bn

119 X = O 120 X =NNHTs ( E ) 121 X = NNHTs (2)

I,

120

0 CH,OBn II I Ph-P-C-R

PhP(=O)H(OMe) -

-

heQ 0-CMe,

122 R=NHNHTs 123 R = H

SDMA in toluene for 20 min at 0" under an argon atmosphere gave the 5-C-(phenylphosphinyl) compound 124, which, on acid hydrolysis, afforded the 5-C-(phosphinyl)hexopyranose125. This was treated with acetic anhydride-pyridine, to give the peracetates (126), from which crystalline 1,2,4-tri-O-acetyl-3,6-di-O-benzyl-5-deoxy-5-C-[ (S)-phenylphosphinyl]-P-~-glucopyranose-~C, (127) was isolated in 2% overall yield from 123; none ofthe other diastereoisomers of 127 were obtained. Structure 127 was established by 400-MHz, 'H-n.m.r. spectroscopy (see Section 11,s). Subsequently, the first example of an unsubstituted D-glucose structure having a ring-phosphorus atom was p r e p a ~ - e din~improved ~ . ~ ~ yield by a modification of the same scheme. Condensation of hydrazones 120 and 12 1 with methyl ethylphosphinate in the presence of trifluoromethanesulfonic acid in benzene for 35 h (64) H. Yamamoto, K. Yamamoto, S. Inokawa, M. Yamashita, M.-A. Armour, and T. T. Nakashima, Carbohydr. Res., 102 (1982) c l - c 3 . (65) H. Yamamoto, K. Yamamoto, S. Inokawa, M. Yamashita, M.-A. Armour, and T. T. Nakashima,J. Org. Chem.,48 (1983) 435-440.

SUGAR ANALOGS HAVING RING PHOSPHORUS

0-CMe,

159

OH 125 R = H

124

OAC 127

126

at 2@",followed by reduction with sodium borohydride in oxolane, gave the (.5RS)-5-[(ethoxy)ethylphosphinyl)]compound 128 (58% overall yield) as a diastereomeric mixture. Debenzylation of 128 was accomplished by repeated hydrogenolysis in ethanol in the presence of 10% Pd-C at 4Q0,to give the tricyclic compound 129. Treatment of this product with chlorotriphenylmethane in pyridine for 84 h at 35 - 40" gave the 6-0-(triphenylmethyl) derivative 130 as a 1: 1 mixture of only two diastereoisomers (17% overall yield from 128). The most likely 0 CH,OBn I1 I Et -PN-C-H

120 121)

0 CH,OR 11 I Et-P--C--H H, / Pd - C

(1) EtPH(=O)(OMe)

7 Q-CMe, 128

0-CMe, 129 R = H 130 R=CPh,

structures, namely, (5R)-5-C-[(l?)-(3-O,P-anhydro)ethylphosphinylj-aD-xylo-hexofuranose (130a) and its (5S)-5-C-[(S)-ethylphosphinyl] diastereoisomer (130h), for these products were derived from 400-MHz, 'H-n.m.r.-spectral analysis and inspection of a CPK model. Reduction of 130 with SDMA gave the 5-(ethylphosphinyl) intermediate 131, which was then converted into the 5-(ethylphosphinyl)hexopyranose 132 by acid hydrolysis. Treatment of 132 with acetic

HIROSHI YAMAMOTO AND SABURO INOKAWA

160

anhydride - pyridine, and chromatographic separation of the crude mixture 133, gave crystalline penta-O-acetyl-5-deoxy-5-C-[(R)-ethylphosH,

0-CPh,

,O-CPh,

I

0 CH,OCPh,

II

I

-P-vC-vH

0-CMe,

O-bMe,

131

130b

130a

H,Ot

131

H

-

O

Ac,O

G

- C,H,N

A

c

Ir

o

u OAc

OAc AcO

HO

OAc

OH

133

132

phinyl]-P-~-glucopyranose(134) in 4% overall yield from 130, and the syrupy a anomer 135 (7%yield), as pure products. The rest of the fractions contained the other diastereoisomers, namely, the 5-[(S)ethy~phospiny~]-~-~-glucopyranose derivative 136 (2%yield) and the a anomer 137 (2%yield), along with tetra-O-acetyl-1,5-anhydro-5-deoxy5-C-[(R)-ethylphosphyinyl]-~-glucitol~~ (138; 1%); structures 134 - 138 were established by 400-MHz, n.m.r. spectroscopy (see Section 11,5). AcOCH,

-

O

0

AcOCH,

W AcO R

,

AcO-~,

Et ;$$0 AcO

Et AcOCH, A

R

R

134 R = H, R’ = OAc 135 R = OAc, R‘ = H

136 R = H, R’ = OAc 137 R = OAc, R’ = H

c

L.,:$O

OAcO

qH H

138

In contrast to the r e s ~ l t ,described ~ ~ . ~ ~ earlier, of the similar ring-enlargement of 86 (or 87) to solely the L-idopyranoses 90, only D-glucopyranoses 132a were isolated when the 1 : 1 mixture of the precursors 130a and 130b was subjected to the usual procedure; no per-0-acetyl derivatives of L-idopyranose 132b were present among the reaction products. As a possible explanation of these results, the following mechanism has

SUGAR ANALOGS HAVING RING PHOSPHORUS

161

been proposed.65 Ring closure of the acyclic phosphinyl intermediate 139a to 132a is likely to be much faster than that of the counterpart 139b (to 132b), because the two precursors, 131a and 131b, would be expected to be almost equally derived from 130a and 130b by reduction with SDMA, as illustrated in Scheme 3.The combined yield of the four diastereomers 134- 137 and the glucitoll38 was- 30%.Thus, instead of giving L-idopyranoses 132b by (the sluggish) intramolecular cyclization, most of the epimer 139b presumably yielded intermolecularly condensed, polar products. The formation of a small proportion of the glucitol 138 is explained in terms of further reduction of the phosphinyl group of 131a to the 5phosphino compound by an excess of SDMA, followed by intramolecular oxygen-transfer similar to that described in Scheme 2 for the formation of 96. The fact that ring enlargement of the 5-(ethylphosphinyl)-~-xylohexofuranose 131 gave only D-glucopyranoses 132a suggests that the relative bulkiness of the substituents on P-5and 0 - 6 greatly affects the direction of the ring closure of the key intermediates, resulting in the formation of the P sugars of either the D-gluco or L-ido type. Accordingly, to prepare 5-deoxy-5-phosphinyl-~-glucopyranoses, a relatively large substituent on 0 - 6 and a smaller one on P-5(and also on 0-3)seem to be required in order to produce a larger proportion of the desired intermediates; see formulas 88 and 131a, and illustrations ofthe steric congestion around 0-3 and P-5of these compounds in Schemes 2 and 3.

5. Structural Analysis of 5-Deoxy-5-phosphino-and -5-phosphinylaldopyranoses by X-Ray Crystallography, and by 400-MHz, Proton Nuclear Magnetic Resonance Spectroscopy and High-resolution Mass Spectrometry a. X-Ray Crystallography. -For sugar analogs having phosphorus in the hemiacetal ring, precise, X-ray crystallographic analyses have been performed on the following compounds: L-idopyranoses 67 (for two independent molecules, Ref. 45), 92 (Ref. 53),and 93 (Ref. 53),L-iditol96 (Ref. 54),and L-lyxofuranose 163a (Ref. 66; for a discussion, see Section 111,3).The precise structures of all of these compounds had not been established until the crystallographic results were available, although their gross structural assignments had been made ~ o r r e c t l y ~(for ~ . ~96 ' and 163a) or i n ~ o r r e c t l (for y ~ ~67) by 'H-n.m.r. spectroscopy. Therefore, it was considered that such an X-ray analysis would be of value not only (66) P. Luger, H. Yamamoto, and S.Inokawa, Carbohydr. Res., 110 (1982) 187-194.

(67) H. Yamamoto, Y . Nakamura, H. Kawamoto, S. Inokawa, M. Yamashita, M.-A. Armour, and T. T. Nakashima, Carbohydr. Res., 102 (1982) 185-196.

134 -137 (30%yield from 130)

H,O+ l3Ob

0 II

0

HO%o

P-Et

___t

HO 0-CMe, l3lb

no 139b

no

HO 132b

(-0% yield) Scheme 3

OH

SUGAR ANALOGS HAVING RING PHOSPHORUS

163

FIG.1. - O R T E P Representations3 of a Molecule of 1,2,3.4-Tetra-O-acetyl-5,6-dideoxy-5-C-[(S)-~henylphosphii1yl]-~-~-idopyranose (93).

from the viewpoint ofX-ray crystallography but also from that of molecular biology. An ORTEP8 representation of the molecular structure of 93 is shown in Fig. 1, as a representative example of the 5-phosphinylaldopyranoses. Seven ofthese compounds are in the T 1 (conformation; ~) the methyl and nitromethyl groups on C-5 are linked axially (thus showing the ~ - i d o form), and the phenyl rings on P-5are linked axially (for 67), or equatorially (for 92, 93, and 96). The acetoxyl groups, all linked equatorially (except for AcO-l of 67 and 93), have the usual orientation: the carbonyl bond is almost syn-parallel to the corresponding C - H bond at the pyranoid ring. The geometry of the pyranoid rings of these compounds is that of a regular chair, as indicated by the Cremer puckering parameters (see Table 1) and the ring-torsion angles. The inclination of the (66) C. K. Johnson, ORTEPReport ORNL-3794 (2nd revision), Oak Ridge National Laboratory, Tennessee, U.S.A., 1970. (69) D. Cremer and J. A. Pople,]. Am. Chern. SOC., 97 (1975)1354-1358. (70)G.A. Jeffreyand J. H. Yates, Carbohydr. Res., 74 (1979)319-322.

164

HIROSHI YAMAMOTO AND SABURO INOKAWA 163

96 0-50

FIG.2. -"Newman" Projection Down the P-C(pheny1) Bond in the Equatorial (Left) and Axial (Right) Case, I l l ~ s t r a t i n gthe ~ ~Phenyl Ring. [The digits mark the phenyl rings for compounds 92 (92); 93 (93); 96 (96); 67, mols. 1 and 2 (67a and 67b), and 163a (163).]

phenyl ring for the analogous derivatives 92,93, and 96, for which the phenyl ring is always equatorial, is illustrated in Fig. 2 (Ref. 54). With respect to the P=O bond, the inclination angle is smallest for 96, medium for 92, and almost 90" for 93; thus, it is apparently affected by the steric situation at C-1. When the phenyl ring is linked axially, as in 67 and 163a, a similar change of inclination of the phenyl ring is observed: it is near 90" for 67, and almost 0" for 163a, although these two compounds have slightly different steric situations at the neighboring ring-atoms. The distribution of the bond lengths and the average bond-angles of the pyranoid ring are tabulated for comparison (see Tables I1 and 111, respectively). The lengths of the phosphorus-carbon bonds of these compounds are longer than those of the carbon-carbon bond of the usual hemiacetal ring by a factor of 1.2, which is consistent with the TABLE I Cremer-Pople Puckering Parameters for the 5-(Phenylphosphinyl)-~-idopyranoses and -L-iditol" Compound

Q(pm)

e(')

4(7

67 mol. 1 mol. 2 92 93 96

61 59 69 67 65.3

3.7 3.8 12.5 6.1 13.7

338.7 329.2 20.0 346.5 347.3

Refs. 45, 53, and 54.

165

SUGAR ANALOGS HAVING RING PHOSPHORUS

TABLE I1 Bond Lengths (pm) of the Pyranoid Ring in the 5-(Phenylphosphinyl)-~idopyranoses and -L-iditol (E.s.d. Values in Parentheses)" ~

~

~

_

_

_

_

_

_

Compound

C-1 -C-2

C-2-C-3

C-3-C-4

C-4-C-5

C-5-P-5

C-1-P-5

67 mol. 1 mol. 2 92 93 96

153.0(6) 150.7(6) 151.9(4) 153.6(5) 151(1)

152.4(6) 152.4(7) 153.4(4) 152.5(5) 152.7(7)

154.1(7) 153.6(7) 152.5(4) 153.0(5)

153.9(6) 153.6(6) 153.9(4) 152.9(5) 150(1)

182.5(4) 183.7(5) 181.5(3) 191.3(4) 181.6(5)

184.1(4) 185.0(5) 183.8(3) 183.2(4) 180.3(7)

~~

a

153.2(8)

Refs. 45, 53, and 5 4

reported ratio (- 1.2 : 1)for such phosphorus-containing, six-membered rings as that of 4,4-dimethyl-l-phenyIpho~phorinane.~~~~~ The P-C bonds are slightly longer for the 5-C-[(R)-P] configurations than for the 5-C-[(S)-P]forms. For exocyclic P=O and P - C bonds, all bond lengths are almost identical. The bond angles of C-2 - C-1 -P-5 and C-4 - C-5 P-5 are slightly larger for the (R) than for the (S) configuration. Although it was not as accurate as the analyses just described, an X-ray analysis p e r f ~ r m e d ~on ~ Jcompound j~ 115 established the D-glucopyran~se-~C structure. ,

b. 400-MHz, 'H-N.m.r. Spectroscopy. -The precise structures of these newly prepared sugar analogs having a ring-phosphorus atom could not be determined by the usual 'H-n.m.r. spectroscopy, because of insufficient resolution of the ring-proton signals, even at 100 MHz. However, with the aid of the results obtained by X-ray crystallographic analysis, 400-MHz, 'H-n.m.r. spectroscopy has allowed the establishment of the precise configurations, as well as the most probable conformations, of these sugar analogs. By this method, assignments of the signals are, in most cases, readily made by employing first-order analysis with the aid of a decoupling technique, the effectiveness of which is exemplified by the various spectra (see Fig. 3) of ~ - g l u c o p y r a n o s e134. ~~ The 'H-n.m.r. parameters for these P sugars are summarized in Table IV. In these detailed, spectral data, there are some interesting features with regard to the chemical shifts (6values) and coupling constants (J values) of the ring protons. The general trends of these values are summarized in Table V. (71) A. T. McPhail, J. J. Breen, J. H. Somers, J. C. H. Steele, Jr., andL. D. Quin, Chem. Commim., ( 1 97 1) 1 020 - 1 02 1. (72) A . T . McPhail, J. J. Breen,andL. D. Quin,].Am. Chem. Soc.,93(1971) 2524-2525.

TABLE 111 Bond Andes of the Pyranoid Ring in the 5-(Phenylphosphinyl)-~-idopyranoses and -L-iditol (E.s.d. Values in Parenthesesy

67 mol. 1 mol. 2 92 93 96 a

111.7(4) 113.9(3) 107.4(2) 107.4(2) 109.5(4)

Refs. 45, 53, and 54.

114.8(3) 114.2(4) 111.8(3) 114.4(2) 115.1(5)

113.3(3) 113.0(3) 113.7(2) 110.5(3) 113.9(5)

113.8(4) 116.5(3) 115.5(2) 115.2(3) 115.9(5)

111.4(2) 109.7(4) 108.0(2) 105.5(2) 106.2(3)

101.6(2) 10 1.6(2) 99.8(1) 103.1(2) 10 1.3(3)

, , , , . ,

I

JI.

I

1 , 1 , , , , , , , , , 1 , , , , , , , /

H -2

H-4

H-1

H-3

ti-60 H-6b

H -5

FIG.3. -400-MHz, 'H-N.m.r. Spectra of 1 , 2 , 3 , 4 , 6 - P e n t r a - 0 - a c e t y ~ - 5 - d e o x y - 5 - C - l ( o s e (134). [(a) Ring-proton signals without decoupling, and (b-e) those with decoupling, on irradiation at H-5 (b), at H,-6 (c), at H-4 (d),and at H-3 (e ).]

TABLE IV 400-MHz, 'H-N.m.r. Parameters for 5-Deoxy-5-C-phosphino-and -phosphinyl-aldopyranosesin CDCI, -~

~

~

~

Chemical shifts (6) Compound Enbylb 20 c.

23

m

m

36h 68 92 96' 115 127

134

H-le H-la

H-2

5.27 5.43 5.3 5.94 6.11 2.66 2.50 5.60 5.57

5.45

3.42

5.24

5.63

3.49

5.40

5.63

5.16

5.3

5.38

H-3

H-4

H-5e H-5a

5.87

5.54

5.88

5.80

5.55

5.76

5.60

5.44

5.72

5.78

3.58

5.52

5.83

3.88

5.70

5.72

5.22

5.58

2.55 1.91 2.61 1.95 2.60 1.9 3.57 2.79 2.77 2.15 2.65

2.37

H-6a H-6b

4.58 4.34 1.10'

1.04" 1.06' 3.86 3.70

4.49 4.45

AcO-1,2,3,4,6" R-P 2.16, 2.10, 3.48", 2.08, 3.77d 2.11, 2.09, 3.51', 2.08, 6.19'. 6.08f, 2.26' 2.10, 1.98, 1.98, 195,2.0, 1.16' 2.04, 1.98, 2.02, 1.93, 7.92, 7.62, 7.71' 2.06, 2.01, 2.06, 1.95, 7.80, 7.54, 7.60' - 2.05, 2.07, 2.05, 7.72, 7.54, 7.60' 2.14, 2.05, 3.49, 1.90, 7.74, 7.54, 7.61' 1.88, 1.87, 4.70", 1.86, 4.19"' 7.27" 4.21" 7.32" 7.27" 7.75, 7.47, 7.56' 2.16, 2.07, 2.01, 2.06, 1.99 2.04, 1.19'

References 23 26 33 46 55 55 46 65

65

Entry 2" 19

5.60

5.41

3.62

5.24

-

37h 38h

93 135

5.67 5.60 6.09 5.84 -

5.4

5.3

5.4

5.4

5.3

5.4

5.75

5.68

5.78

5.55

5.45

5.57

2.43 2.13 2.56 2.0 2.56 2.0 3.05 2.50

-

-

1.36'

4.45 4.41

2.22, 2.12, 3.51', 2.09, 3.73d 2.16, 2.00, 2.00, 1.95, 1.75, 1.20' 2.17, 2.02, 2.02, 1.96, 1.7, 0.88P 2.15, 2.09, 2.08, 1.98, 7.78, 7.54, 7.62' 2.21, 2.09, 2.03, 2.07, 1.98 1.71, 1.22'

23 33

33 55 65

Entry 39 21

-

5.22

3.36

4.98

94

5.44 5.95

5.24

5.49

5.10

136

-

5.25

5.65

5.23

138'

5.70 2.73 2.1

4.87

5.22

5.20

5.04

3.54

4.96

4.90

5.75

5.3

4.91

5.65

5.04

Entry 4' 22 66h 95

5.64 6.25 6.15 -

2.52 1.92 3.11 2.58 2.53 2.48 2.22 4.2 3.20 -

1.50

4.75 4.29 4.68 4.29 5.3 5.1 1.56k

2.14, 2.12,3.51",2.09, 3.94d 2.12, 2.03, 2.07, 2.02, 8.00, 7.62, 7.651 2.14, 2.07, 2.05, 2.00, 2.00 2.1, 1.40' - 2.07, 2.09, 2.08, 2.06 1.84, 1.83, 1.37'

23

2.22, 2.12, 3.50', 2.06, 3.88d 2.25, 2.00, 1.96, 198, 8.0, 7.6, 7.6-1 2.23, 2.02, 2.13, 2.01,7.94, 7.65, 7.65-1

23

55 65 33

33 55

(continued)

TABLE IV (continued) Coupling constants (Hz)'

Entry l b 20

23

10.5

-

10.8 36h

-

5.5 0 1.6 0

11 CI

68

-4

0

92 96'

-

10.5 10.75 4.5 11.7 14.0"

115

-

11.0 127

-

11.2 134

-

11.0 Entry 2" 19

2.8

-

8.7 3.8 9.2 2.6 10 3 9.5 3.5 9.5 2.4 9.5 2.5

2.2 0 2.75 0 17.8 2.0 5.3 0 2.7 0.3 2.8 0.3 3.6 0.2

9.6 2.8 9.5 3.0 10.0 3.0

14.2 2.0

9.8 1.0

8.6 9.5

4.5 12.5 4.4 12.0

0.8 3.2

22.5 11.0 20.0 5.3

4

9.5

-

-

14.8

-

14.4 14 -

3.5 11.0 16.8

-

-

3.2

10.1

6.5 4.6

3.4

21.7

8.5 2.0 7.6

9.7

4.5

-

3.7

20.0

7.5

-

16.9

9.8

-

2.7

3.5

7.0

-

15.0

-

-

9.8

12.0 11.5

2.8

-

-

-

-

9.8 11.5

10.0 14.5 11.5 15.0

-

2.7

7.0 6.0 7.4 5.0 -

14.0

-

9.8

10.0

-

-

-

4.5 12.0

-

4.0

11.5 9.3

11.5

3.5 3.0

22.5 12

14.0

-

-

-

-

-

15'

-

19.3' 7.6' 11.0d -

37*

3

38h

3

93

3.1

135

3.2

Entry 3q

21

-

10 2 10 2 11.5 2.1 11.7 0

-

3.6

-

-

10.8 94

-

10.8 136

-

138'

10.5 4.5 10.0 14.0"

0

10.4 0 11 0 15.0 0

17.5' 7.0' 9.7 0.9 12.5 0.3 9.6 2.1 8.6 4.5 11

9.9 11.8

4.7

-

3.1

3.0

14.5 9.6 2.0 9.1 11

10.0 2

10.0

10.0 0 9.6 2.5 9.4 3.4

9.7

4.5 12.0 4.3

-

2.0 0

7.6

-

16.2

8.3 6.3

-

4.5

14.5

16.2 18.5

23.6 10.0 15.6

7.6

14.8

-

-

14.3

-

-

-

-

4

-

-

-

-

12.0

7.0p

21.6

12

-

18.0

3.2 2.0

25.0 12.0

-

14.3

15.5

7.5

-

11.5

23 9 23.1 8.7

-

15'

15.2'

-

18.3' 7.5' 10.5d

-

18' 7.5' 17.6' 7.6'

0

Entry 4'

22

3.0

-

66

2.5

95

3.0

-

15.0 2.0 8.5 1.2 8.6 1.7

9.6 9.1

4.8 12.0

2.1

4.6 -

6.1

-

-

13.7

-

10.5d

-

-

-

a Acetoxyl assignments are interchangeable. * Pyranoses having Ac0,-1 and P=O,. Me0-3. P-OMe. PC=C-H(2) (3]H,p 10.2, '1H.H 2.8 Hz). fP-C=C-H(E) 29.8, ' J H , ~ 2.8 Hz). KP-C(OAc)=C. Measured at 100-MHz.'P-CH,-CH3.jP-Ph(o,m,p). H,-6.' 1.5-Anhydro-~-iditol."O-CH2Ph(]11.8Hz)." 0-C-C,H,. Pyranoses having Ac0,-1 and P=O,. ' 1,5-Anhydro-~Pyranoses having Ac0,-1 and P=O,. p P-CH,-C,H,. glucitol. Pyranoses having Ac0,-1 and P=O,. ' J Values confirmed by double resonance.

HIROSHI YAMAMOTO AND SABURO INOKAWA

172

TABLE V Characteristic Features in the 6 and J Values for the 54 Phosphinyl)aldopyranoses Pyranose-'C, with P(=O,)

Ring proton

He-1

11.2

11.P

Ii.sr

K-1

11.2 1L.P

H-4

6 6 6

He-5

14.5r

H-2

s 15e.P

Ho-5

14,5n

15o.P

2.8-3.2 10-14 2 10.5-11.2 2.4-3.6 5.4- 5.6" 5.6-5.82b 5.25- 5.46a 5.5-5.88b 4.5-6.5 20-22.5 11.5- 14.5 3.5-5.3

Pyranose-'C, with P(=O,)

2.5-3.0 8.5-9 1.2-2.0 10.5-10.8 10-11 5.0-5.2" 4.87-5.0b 4.98-5.2" 5.05-5.3b 4.5-4.8 15-16 12 10-18

For 5-(alkylphosphinyl)aldohexopyranoses.bFor 5-(pheny1phosphinyl)aldohexopyranoses.

The values of the geminal P-C-H coupling constants andJ,,,) d these phosphinyl compounds apparently depend upon the magnitude of their approximate O=P-C-H dihedral angles, as shown in Table V. Thus, the anti orientation of the O=P-C-H group exhibits a lower magnitude of coupling than the gauche orientation. A similar dependence of the geminal P-C-H coupling constant on the dihedral angle has been reported both for linear and cyclic phosphonyl compounds.73-75 Application of these principles to the structural analysis permitted establishment of the configurations of the ring-carbon atoms and the orientations of the protons thereon, and the stereochemistry of the phosphorus atom in these pento- and hexo-pyranoses. c. High-resolution Mass Spectrometry. -Because of the versatile applicability to structural assignments, an extensive, systematic investigation of the mass spectra of carbohydrate derivatives has been undertaken, and a wide variety of general degradation-pathways has been well (73) A. N. Pudovik, I. V. Konovalova, M. G. Zimin, T. A. Kvoinishnikowa, L. I. Vinogradov, and Yu. Yu. Samitov, Zh. Obshch. Khim., 47 (1977) 1696-1703. (74) Yu. Yu. Samitov, E. A. Suvalova, I. E. Boldeskul, Zh. M. Ivanova, and Yu. G. Gololobov, Zh. Obshch. Khim., 47 (1977) 1022- 1027. (75) For a review of 'H-n.m.r. spectroscopy of cyclic phosphorus compounds, see L. D . Quin, The Heterocyclic Chemistry ofphosphorus, Wiley, New York, 1981, pp. 31 9 359.

SUGAR ANALOGS HAVING RING PHOSPHORUS

173

e s t a b l i ~ h e d . ~Detailed "~~ analysis of the mass spectra of some of those sugar analogs having phosphorus in the hemiacetal ring has revealede1 certain characteristic features that are considered to be of use in structural assignments for these phosphorus sugars. As a representative example of these compounds, the analysis of the high-resolution, electron-impact (e.i.) mass spectrum of the D-glucopyranose derivative 134 is shown in Fig. 4. Although not of high intensity, the molecular-ion peaks of these phosphinyl sugar analogs are usually detectable as the protonated species [(M l)+;probably due to the resonance-stabilizedoxoniumform] in the e.i. mass spectra, whereas the molecular ions of the usual carbohydrate derivatives (having a ring-oxygen atom) are barely observable7eowing to their lability. A highly unusual feature in the mass spectra of the per-0-acetylated derivatives of these P sugars is that the most intense peaks (except for the peaks at m/z 43, due to CH,CO+ ions of much higher intensity) are normally those for the fragments still having the phosphorus-containing ring. This is also in striking contrast to the spectra76of per-0-acetylated monosaccharides having an oxygen-containing ring; in these, the intensities of the fragments retaining the hemiacetal ring are generally much lower than those of the ring-ruptured ions. The most probable fragment-ions of the first series (A, according to the nomenclature used by Kochetkov and C h i ~ h o v of ~ ~134 ) are illustrated in Scheme 4; these ions are produced by loss of the substituent from C-1, with subsequent, stepwise elimination of other substituents. The species A31, formed from the molecular ion by successive loss of one acetic acid and two ketene groups, consists of the base peak, suggesting high stability of the phosphorus-containing ring-system. Further elimination of two molecules of acetic acid from this species leads to the species As2 and As3 having the 1,2-dihydr0-l-methylene-1~-phosphorine ring-system, which still possesses high intensity owing to the resonancestabilized forms (see Scheme 4).The presence of such a set of fragment ions of the A series is, in turn, strongly indicative of the 5-deoxy-5C-(phosphiny1)hexopyranose structure. The mass spectrum of the Q anomer (135) closely resembles that of the

+

(76) K. Biemann, D. C. DeJongh, and H. K. Schnoes, J . Am. Chem. SOC., 85 (1963) 1763-1771. (77) N. K. Kochetkov and 0.S. Chizhov, Ado. Carbohydr. Chem., 21 (1966) 39-93. (78) K. Heyns, H. F. Grutzmacher, H. Scharmann, and D. Mueller, Fortschr. Chem. Forsch.,5 (1966) 448-490. (79) O.S. ChizhovandN. K.Kochetkov,MethodsCarbohydr.Chem., 6(1972) 540-554. ( 8 0 ) J. Lonngren and S. Svensson, Ado. Carbohydr. Chem. Biochem., 29 (1974) 41 - 106. (81) H. Yamamoto and S. Inokawa, Phosphorus Sulfur, 16 (1983) 135-141.

h

0

n

I-.

0. N

sn

4

t

4

0

f

0

n

v1 0

N

0

N 0

Y) 0

v

U

h

3

L

I

d

4

rz

SUGAR ANALOGS HAVING RING PHOSPHORUS

+

t

AcOCH,

- m,co OAC

-

OAC

+ OH Et

OAC (1.4%

- CH,CO

Et OH

OH

m/s 391

m/z 451 [2.69; @ I)'] l+

+ OH

AcOCH,

AcO

AcO

OAc

P 75

OH

m/z 307

m/e 349

4)

(66.4%

4)

AcOCH,

HO

-CH CO 1 ACO

HO OH m / z 241

OH

m/z 205

(83.6%;

OH m/z 306

OH

m/z 289

4')

):A

(76.28;

- CH,CO

-H

HO

m / z 187 (70.09; 4 2 )

HO

- CH,CO

HO

-H

Ac 0

m/z 229 4')

m/z 1% (66.7%; 4 ')

-

m/z 265 (19.8% &*)

(16.6%

OH

A0

t----c etc.

Scheme 4

+

anomer 134, but the corresponding peaks of the A series [(M 1)and A, - A3] are of higher intensities than those of 134; this is in accord with the fact that the A, peak in the mass spectrum of a-D-glucose pentaacetate is more intense than in that of its j3 a n ~ r n e r . ' ~ Simultaneous, or stepwise, rupture of two bonds (C-2- C-3 and C-4 C-5) of the pyranoid ring in species A,' (or in its precursors) gives rise to ring-opened fragments of another series, B, which accounts for the peaks at mass 165 (Be1), followed by degradation producing the peaks at m/z 145, 136, 121, 103, 93, and 77. These peaks, of relatively low intensities, imply the minor importance of these ring-opening fragmentations in the e.i. mass spectrum. Besides the ring rupture (series B), there exists another type of cleav-

176

HIROSHI YAMAMOTO AND SABURO INOKAWA

age that gives fragments of appreciable intensities, at m/z 111 and 95, which are produced by the pathway illustrated in Scheme 5. This type of removal of only the heteroatom from the ring (as a phosphinyl group) is another characteristic feature that is normally absent from the spectrum of the usual sugar derivati~es.'~-~O

W+1)

OH I

- CH,CO

- Et P(=0)H

7

0 Ac 0

OAc

OAc

- 2 AcOH - CH,CO

m/z 95 (33.0%) Scheme 5

m/z 111 (29.0%)

Similarly, the mass spectra of the tri-0-acetyl derivative (127) of 3,6di-O-benzyl-5-(phenylphosphinyl)-~-glucopyranose and of those (19 and 20) of 5-(methoxyphosphinyl)xylopyranosehave been analyzed*'; the main fragmentation is, again, the stepwise removal of the substituents from the ring-carbon atoms and C-6 (series A), leading to the formation of similar 1,2-dihydro-A5-phosphorine derivatives.

111. MONOSACCHARIDES HAVING A PHOSPHONYL GROUP IN THE FURANOSE RING 1. 2,3,4-Trideoxy-4-phosphinylpentofuranoses

Many examples of monosaccharides having nitrogen or sulfur in the furanose ring have been reported,1°-13and some of these compounds, for example, the 4-thio-~-ribofuranosyl derivative 140, possess a variety of

OH

HO 140

SUGAR ANALOGS HAVING RING PHOSPHORUS

177

novel biochemical properties.s2 On the other hand, relatively few examples of monosaccharides having phosphorus in the furanose ring have been reported. The main reason for this seems to be the difficulty in preparing their precursors, compared with those for pyranose compounds described in the previous Section. A first, very preliminary study of the preparation of compounds of this class has been r e p ~ r t e d ,utilizing ~ ~ . ~ ~an equilibrium shift that involves ring contraction of a pyranoid to a furanoid ring (see Scheme 1). For the preparation of the precursor, methyl 2,3-dideoxy-(lS)-~~pent-2-enopyranosid-4-uloses4 (141)was hydrogenated in the presence of Pd-C, to give an almost quantitative yield of the pentopyranosid-4ulose 142.Treatment of 142 with p-tolylsulfonylhydrazine, followed by condensation of the product with 75 and 76,respectively, in the presence of p-toluenesulfonic acid, afforded the methyl 4-(dimethoxyphosphinyl)-4-(p-tolylsulfonylhydrazino)pentopyranoside derivative 143 (42% overall yield from 142),and the 4-[(methoxy)phenylphosphinyl] compound 144 (57% yield from 142),respectively. Reduction of 143 and 144 with an excess of sodium borohydride respectively gave methyl 2,3,4-trideoxy-4-C-(dimethoxyphosphinyl)and -4-C-[(methoxy)phenylphosphinyl]-DL-glycero-pentopyranosides[ 145 (78% yield) and 146 (66% yield), respectively]. Treatment of compound 146 with SDMA, followed by acid hydrolysis, gave 2,3,4-trideoxy-4-C-(phenylphosphinyl)-DL-glycero-pentofuranose(147;36% overall yield from 146). As usual, the product 147 was characterized by derivatization to the per-0-acetyl compounds, 1,5-di-O-acetyl-4-C-(phenylphosphinyl)pentofuranoses 148.Although separation was not attempted, the acetates 148 apparently consist of 1,5-di-O-acetyl-2,3,4-trideoxy-4-C-[ (S)-phenylphosphinyl]-P-~-glycero-pentofuranose (148a),its cy anomer (148b), the 4-C-[(R)-phenylphosphinyll-/?-~-glycero-pentofuranose 1,5-diacetate (148c), and its a anomer (148d)(together with enantiomers of these compounds). The most plausible conformers, 148a-d, are speculative, but are presumed by analogy with those of the structurally similar compounds 160- 163 (see Section 111,2); however, assignment of the exact structures, as well as presentation of the yields of the individual components, are not yet possible (because of the low resolution of the 'H-n.m.r. spectra at 60 MHz). (82) A. K. M. Anisuzzarnan andR. L. Whistler, Carbohydr.Res., 55 (1977) 205-214, and references cited therein. (83) M . Yarnashita, M. Yoshikane, T. Ogata, and S. Inokawa, Tetrahedron, 35 (1979) 741 - 743. (84) 0.Achmatowicz, Jr.. P. Bukowski, B. Szechner, Z. Zwierzchowska, and A. Zamojski, Tetrahedron, 27 (1971) 1973-1996.

HIROSHI YAMAMOTO AND SABURO INOKAWA

178

141

142 ( 1 ) TaNHMI,

0 MeO-P

0

II

MeO--P

II

NaBH,

H ' O

O

M

e

c -

143 R=OMe 1 4 4 R = Ph

1 4 5 R = OMe 1 4 6 R = Ph

0 (1)SDMA 146

(2)HCl

-

OR H 147R=H 1 4 8 R = AC

wb CH,OAc

148m

CH,OAc

$0 148c

'4-0 AcO 148b

ppa CH,OAc

OAc

B

148d

SUGAR ANALOGS HAVING RING PHOSPHORUS

179

2. 4,5-Dideoxy-4-phosphinylpentofuranoses A further example of this class of compound was ~ r e p a r e dfrom ~ ~ .an~ ~ acyclic precursor. 2,3-O-Isopropylidene-~-ribose diethyl dithioacetale5 (149), which may be ~ r e p a r e d ~from ~ f ' D-ribose, ~ was used as the starting material for this synthesis. The hydroxyl groups of 149 were first acetylated with acetic anhydride-pyridine, to give 150 (89%),and diacetate 150 was treated with mercuric chloride-cadmium carbonate, to afford the dimethyl acetal 151 (92% yield). This compound was deacetylated with sodium methoxide, providing a quantitative yield of the dimethyl acetal 152.Compound 152 was then converted into the 5-p-toluenesulfonate 153 (96% yield), and 153 was efficiently oxidized (96% yield) to the pentos-4-ulose derivative 154 by means of dimethyl sulfoxide - oxalyl chloride - triethylamines8 in dichloromethane at - 70". In accordance H EtSCSEt

H MeOCOMe

HCO,

HCO,

I

I

H MeOCOMe

II HCO,

I

,CM%

HCO

I

HCOR

I

149 R = H 1 5 0 R = AC

150

HgCl,-CdCO, MeOH

*

I

HCO'

CMez

II

HCOR I 151 R = R ' = A c

153

I

HCO'

CMez

II

c=o I

164

152 R = R' = H 1 5 3 R = H , R' = Ts

with the ~ c h e m e ~described ~ * ~ * in Section I1,3, compound 154 was treated with 1 equivalent of methyl phenylphosphinate in the presence of 1.2equivalents of DBU at room temperature, to give (4RS)-4,5-anhydro-4-C-[(RS)- (methoxy)phenylphosphinyl]-D-erythro-pentosedimethyl acetals 155a (19% yield) and 155b (43% yield); the structures of these epimers were derived from study of their 'H-n.m.r. spectra. The anti-Cram type of addition seems to afford the major product 155b, as had been observed in the formation of the a-D-xybhexofuranose 82 (see formula 81 for the addition reaction). Although hydrogenation of 155a and 155b in the presence of Raney nickel was conducted separately, both compounds gave, in 60% yield, an almost identical mixture of (4RS)-4,,5-dideoxy-2,3-O-isopropylidene-4(85) K . Blumberg, A. Fuccello, and T. van Es, Curbohydr. Res., 70 (1979) 217-232. (86) G . W. Kenner, H. J. Rodda, and A. R. Todd,J.Chem. Soc., (1949) 1613-1620. (87) M. A. Bukhari, A. B. Foster, J. Lehrnann, and J. M. Webber,J. Chem. SOC., (1963) 2291 - 2295. (88) A. J. Mancuso, S..-L.Huang, and D. Swern,]. Org. Chem.,43 (1978) 2460-2482.

HIROSHI YAMAMOTO AND SABURO INOKAWA

180

C-[(methoxy)phenylphosphinyl]-~-erythro-pentosedimethyl acetals (156a and 156b) in the ratio of 3 : 1 (with respect to the configuration of C-4).This result suggested the formation of acommon intermediate, such as (4RS) - 4,s - dideoxy - 2,3- 0isopropylidene - 4 - C- [(RS)- (methoxy)phenylphosphinyl]-~-erythro-pent-4-enose dimethyl acetal, by deoxygenation, prior to the reduction during hydrogenation. Reduction of the mixture of 156a and 156b with SDMA gave a diastereoisomeric mixture of phosphinyl compounds 157, which was immediately hydrolyzed by acid, to afford 4,5-dideoxy-4-C-[(RS)-phenylphosphinyl]-~-ribo-and -L-lyxo-furanose (158). The structure 158 was determined6' by 400-MHz, 'H-n.m.r. spectroscopy of the per-0-acetyl derivatives 159; these are 1,2,3-tri-O-acety1-4,5-dideoxy-4-C-[(RS)H MeOCOMe

H MeOCOMe

HCO,

HCO,

I

154

PhPH(=O)OMe

I

I

H,-Raney Ni

* HCO

I

,C--P(

=O)Ph(OMe)

0l, CH, 1550 (4s) 155b (4R)

I CMe, I 40 H-C-P-Ph I R' HCO/

CH, 156a, b R = OMe 157 R=H

0

RO

I

OR

158 R = H

159 R = A c

phenylphosphinyll-~-ribo-and -L-lyxo-furanoses 160 - 163; see Scheme 6 for their probable conformations (and yields from 156). The exact configuration and conformation of the crystalline product 163a was determined66by X-ray crystallography (see later). As regards the yields of the eight diastereoisomers, the p anomers (160b and 16lb) preponderated on formation of the D-ribofuranoses, whereas, for the L-lyxofuranoses, more of the a anomers (162a and 163a) was produced; this behavior was explained in terms of the thermodynamic stability of precursor 158. The ratio of the combined yields of D-ribofuranoses (160a,b and 161a,b) to the L-lyxofuranoses (162a,b and 163a,b) was 9 : 10, whereas that of the (S) to ( R )isomers of the ring-phosphorus atom (160a,b and 162a,b to 163a,b) was 11 : 26.

SUGAR ANALOGS HAVING RING PHOSPHORUS

181

I

AcO 162a (ZE; 3.6%) M2b (0%)

163a ( E 3 ; 12.5%) 163b (E9;3.2%)

a R = H , R’ = OAc; b R = OAc, R’ = H (conformations in CDCI,; yields) Scheme 6

Possible factors influencing these results were that an almost equimolar (4R and 4s) equilibration had been caused by the strongly basic SDMA during the reduction of 156,but the hemiacetal formation from 157 to 158 proceeded more readily for the [ (R)-phenylphosphinyllpentofuranoses 158, because there is less steric congestion between the P-phenyl and the 2-and 3-hydroxyl groups in the precursors of 158.

3. 4-Deoxy-4-phosphinylaldopentofuranoses Following a scheme similar to that described in Section III,l, compounds having a complete D-ribofuranose structure and a ring-phosphorus atom were prepared.33g89 (164),preparedso Methyl 2,3-O-isopropylidene-c~-~-lyxopyranoside (89) H.Yamamoto, Y. Nakamura, S. Inokawa, M. Yamashita, M.-A. Armour, and T. T. Nakashima, Carbohydr. Res., 118 (1983) c7-c9. (90) M. Bobek and R. L. Whistler, Methods Carbohydr. Chem., 6 (1972) 292-296.

HIROSHI YAMAMOTO AND SABURO INOKAWA

182

from D-galacturonic acid, was oxidized by the procedure of Mancuso and coworkerss8 to give methyl 2,3-O-isopropylidene-P-~-erythro-pentopyranosid-4-u!ose, which was then converted into the p-tolylsulfonylhydrazone. The addition of methyl ethylphosphinate to this hydrazone, followed by reduction with sodium borohydride in oxolane, afforded the phosphinate 165 (28% overall yield from 164).

0,

o,

0,

CMe, 164

so

o,

CMe,

165

166

Compound 165 was reduced with SDMA and the product was hydrolyzed with acid to effect ring contraction, affording 4-deoxy-4-C-[(R,S)ethylphosphinyl]-a,p-~-ribo-and -L-lyxo-furanoses (166), which were characterized by conversion into the peracetates. After separation by chromatography on silica gel, structures 167 - 170 were established for these peracetates by 400-MHz, 'H-n.m.r. spectroscopy and high-resolution mass spectrometry; the structures of these products, their probable conformations, and the yields from 165 are summarized in Scheme 7 . [(S)-P]-D-Tdbo

[(R)-P]-D-n'bO

H

H HA

AcO

Rf

167a ('E, 12%)

167b CE

= 'E,

CH,OAc

p

;

p

ACO (E214%)

12.7%)

168b ( E , == E , , 12.8%)

aR=H,R'=OAc

bR=OAc,R'=H

-

[ (S) PI - L- zyxo

[(R)-P]-L-lyxO

H

Et

R 169a ('E

-

,

AcO 5.3%)

170a (E,

Scheme 7

*

ES,2.8%)

SUGAR ANALOGS HAVING RING PHOSPHORUS

183

n

FIG.5. -ORTEPs8 Representations4of a Molecule of 1,2,3-Tri-O-acetyI-4,5-dideoxy-4C-[(R)-phenyIphosphinyl]-cu-~-lyxofuranose (163a).

It is noteworthy that, by employing this method, the D-ribofuranose derivatives (167 and 168)were preponderantly produced (41.5%)compared with the L-lyxofuranoses (169 and 170; 8.1%yield from 165). 4. Structural Analysis of 4-Deoxy-4-phosphinylpentofuranoses by X-Ray Crystallography, and by 400-MHz, Proton Nuclear Magnetic Resonance Spectroscopy and High-resolution Mass Spectrometry a. X-Ray Crystallography. -The precise structure of the crystalline compound 163a has been established66by X-ray crystallographic analysis, and the ORTEP69representation of the molecular structure is shown in Fig. 5. The fivemembered ring has the E, conformation, with a tendency ~.'~ towards the ,T2 form; the Cremer -Pople puckering p a r a r n e t e r ~ ~are q = 42.0 pm and 42= 102.98", and the asymmetry parameters after Duax and coworkersQ1are ACs = 8.4"for the E, conformation and AC2 = 12.9" for the ,T2 conformation. The acetoxyl group on C-2 and the methyl group on C-4 are linked quasi-equatorially, whereas the Ac0-3 (91) W. L. Duax, C. M. Weeks, and D. C. Rohrer, Top.S t e r e o c h . , 9 (1976)271-383.

184

HIROSHI YAMAMOTO AND SABURO INOKAWA

group is linked axially; thus, C-3 is the out-of-plane atom in the E3 conformation. The endocyclic lengths P-5-C-1 and P-5-C-4 are 186.1 and 182.6 pm, respectively. The bond angle of C-1 -P-5 -C-4 is 94.3", whereas the rest of the angles for the furanoid ring range between 105.4 and 108.5". The plane of the phenyl ring has an orientation parallel to the P-5 - 0-5 bond, and its inclination is shown in Fig. 2 (see Section 11,5). In this orientation, steric collisions with the adjacent acetoxyl group on C-1 are avoided. The acetoxyl group on C-1 differs considerably from the usual, syn-parallel arrangement of the C=O bond with the C-H bond of the corresponding ring-atom.

b. 400-MHz, 'H-N.m.r. Spectroscopy. -The parameters of the n.m.r. spectra for the 4,5-di- and 4-deoxy-4-C-[(R,S)-phenylphosphinyl]-~ribo- and -L-lyxo-furanoses (160- 163 and 167 - 170) are summarized in Table VI, and some important features of these spectral data for the structural analysis are as follows. When the methyl signal of 160- 163 appears at relatively low field (6 1.3- 1.4), the methyl group apparently lies close to the oxygen atom on the phosphorus, whereas a small S value (-0.95) indicates that the two groups are remote; this may be inferred by analogy with the n.m.r. data for similar, cyclic phosphorus c o r n p ~ u n d s . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Relatively large values (7 - 12 Hz) suggest a trans relationship between H-3 and H-4 (thus indicating the D-ribofuranose form), whereas smallerJ,,, values (4.3 - 5.8 Hz) suggest the L-lyxofuranose structure. On the other hand, the large J4,, values (22-24 Hz) for the H-4 signal suggest a trans relationship of the methyl and P=O groups, whereas the small J4,p values (6 Hz) support a cis (or gauche) relationship; these features are generally observed (see Section 11,5).This result permits establishment of the configuration of P-4. Similarly, the large J1,, value (8- 1 2 Hz) of the H-1 signal indicates a cis (or gauche) relationship of the H-1 and P=O groups, whereas the small value (0.8-5 Hz) suggests a trans relationship, thus establishing the configuration of C-1. The significant difference in the magnitudes of and I,,, is indicative of an unsymmetrical conformation with respect to H-2 and H-3. It has have been proposede7that, when these 4-C-(phosphinyl)pentofuranoses a largeJ,,,value (23- 28 Hz) and a smallJ,,, value (0-5Hz), as in 163a,b, the compounds exist in the E, conformation, wherein the approximate dihedralanglesofp-C-4-C-3-HandP-C-1 -C-2-Hare 150and9Oo, respectively; the E, conformation of 163a was later confirmed by X-ray crystallographic analysis.ee On the other hand, reversed magnitudes of and],,,, as with 161a and 162a, support the E2 conformation for the compounds, whereas when these magnitudes are almost the same, as for 160b, this suggests an averaging between the E , and E3 (or ,E and ,E)

TABLE VI 400-MHz, 'H-N.m.r. Parameters for 4-Deoxy- and 4,5-Dideoxy-4-C-(phosphinyl)aldopentofuranosesin CDCI, Chemical shiftsa (6) Compounds

H-1

H-2

H-3

H-4

H-5

160a 160b 161a 161b 162a 163a 163b 167a 167b 168a 168b 169a 170a

5.49 5.15 5.24 5.29 5.33 5.51 5.26 5.45 4.97 4.97 5.43 4.98 5.92

5.72 5.92 5.62 5.75 5.38 5.40 5.78 5.66 5.75 5.36 5.50 4.80

5.46 5.00 5.33 5.71 5.70 5.59 5.39 5.54 5.00 5.20 5.68 5.8"

2.75 2.67 3.01 2.90 2.75 2.50 2.57 2.58 2.62 3.12 2.95 2.88 2.48

1.30d 1.3gd 0.96d 1.06d 0.95d 1.2Sd 1.36d 4.46 4.53 4.47 4.43 4.45" 4.29

H-5'

A~0-1,2,3,5~

-

-

cc

2.23, 2.14, 2.12, 2.16, 2.23, 2.14, 2.21, 2,21, 2.15, -

-

4.37 4.30 4.34 4.34 4.30 4.20

P-CH-Me, P-CH'-Me, P-C-CH,

1.61, 2.22, 2.08, 2.22, 2.28, 2.14,2.17, 2.13, 2.12, 2.08 2.23, 2.13, 2.12, 2.10 2.22, 2.19.2.11, 2.09 2.19, 2.18,2.14,2.11 2.22, 2.17, 2.13.2.09 2.20, 2.16.2.13, 2.02

7.90, 7,58, 7.62" 7.75, 7,58, 7.62' 7.73, 7.57, 7.61' 7.90, 7.6, 7.6' 7.72, 7.56, 7.62' 7.75, 7.57, 7.61' 2.1' 1.92 2.28 2.01 2.05 2.08 2.00 1.94 2.04c 1.83 1.74 1.69

1.39 1.35 1.30 1.29 1.23 1.21 (continued)

TABLE VI (continued)

~

160a 160b 161a 161b 162a 163a 163b 167a 167b 168a

168b 169a 170a

5 5.7 4.6 3.0 10.5 9.4 3.2 4.5 6.0 4.8 3.6 10.0 8.6

11.5 1.4 0.8 8.0 0.6 12.1 5.8 12.5 2.5 2.6 7.8 0.5 10.2

0.5 0 0.6

0 0 0

0.5 0 0.5 0

c

c

3.8 3.0 3.5 3.0 3.2 2.8 3.0 3.6 3.0 3.8 1.0 3.0

11.7 26.6 16.2 27 0 5.0 26.5 10.5 24.6 15.1 3.5 15.2

7.0 12.3 10.5 4.8 4.3 5.8 11.2 6.0 12.0 10.0 4.0 1.8

13.5 0.5 6.0 27.5 22.8 1.0 13.5 1.0 5.5 25 31

7.0 7.5 7.2 7.4 7.5 7.3 7.2 8.5 7.3 4.8 5.2 9.5 11.5

6.0 24.0 22.5 22 6.0 6.5

5.8 7.5 7.0 6.8 6.0 5.2

8.7 6.5 22.0 21.2 20.5 12.0

11.2 12.0 12.0 11.8 12 11.5

14.8 14.2 16.5 16.5 16.5 15.1 14.6 12.0 9.2 14.8 14.8

17.0 12.2 17.0 16.0

15.0 15.0

9.0 11.5

15.0 15.0

15.0

10.5

15.0

c

c

18.5 17.0 17.0 17.0 17.0 16.5

7.8 7.5 7.5 7.5 7.5 7.5

Chemical shifts (6 values) are in parts per million from Me,% Acetoxyl assignments are interchangeable. Values are approximate, or uncertain. CH, . P-C6H5 (0, p , m).f] values confirmed by double resonance.

SUGAR ANALOGS HAVING RING PHOSPHORUS

187

conformations. It was presumeds7 that these shapes would allow minimization of the nonbonded interaction between the phenyl ring on the ring-phosphorus atom and the substituents on the adjacent atoms. When the energy barrier between these two forms (E, and E3) is relatively low, the rapidly interconverting conformations are expected to exist in solution. There have been reported some examples of similar angular dependence of P- C - C - H vicinal coupling-constants upon the dihedral angles in the case of phosphonate compoundsse and P(V)-heterocyclic systems.Q3,Q4 c. High-resolution Mass Spectrometry. -Analysis of the mass spectrum of tri-O-acetyl-4,5-dideoxy-4-f(R)-phenylphosphinyl~-~-~-lyxofuranose (163a) has been made,81 and the most important of the series of degradation pathways are shown in Scheme 8. Here again, the preponderant fragmentation is the A series, to give rise to ions A,-A, of the As-phosphorole structure, followed by removal of the phenylphosphine oxide from the furanoid ring; the molecular-ion peak of 163a was con-

m / z 368 (0%; M)

m/r 326 (2.5%; A,)

m / z 267 (100%; &)

I

- CH,CO

m / z 84 (69.1%)

m / z 207 (57.2%; A:)

m / z 225 (99.6%; A:)

+ H-P=O I Ph

m / z 125 (66.5%)

Scheme 8

(92) C. Benezra, Tetrahedron Left., (1969) 4471 -4474. (93) T. C. Chan and K. T. Nwe, Tetrahdron, 31, (1975) 2537-2542. Awerbouch and Y. Kashman, Tetrahedron, 3 1 (1975) 33-43. (94) 0.

188

HIROSHI YAMAMOTO AND SABURO INOKAWA

firmed by 23Na, field-desorption, mass spectrometry. Careful comparison of these fragmentation patterns of 163a with those of the pyranoid compounds (see Section II,5,c) would provide a convenient method for characterizing phosphorus-containing, furanoid structures of related compounds. The mass spectra of 167b, 168a, and 168b showedae fragmentation patterns similar to that of 163a.

IV. BIOLOGICAL ACTIVITIESOF MONOSACCHARIDES HAVING PHOSPHORUS IN THE HEMIACETAL RING Since the first isolation of 2-aminoethanephosphonic acid (17 1)from rumen ciliate protozoa,e5 related compounds (for example, phosphinolipides.e7172) in considerable number have been found in Nature, and 0

II H,NCH,CH,-P-OR I

OH

171 R = H 1 7 2 R = CH,CH(OCOR')-CH,OCOR"

the biochemistry of such substances containing a carbon - phosphorus ~ ~ * ~ ~these 2bond appears to be drawing increasing i n t e r e ~ t . Besides aminophosphonic acid derivatives and several antibiotics found in Nature [such as f o s f o n o r n y ~ i n(72) ~ ~ and phosphinothricinloO(173), a wide 0 H0,C -CH- C!H,CH,I

NH,

II P -CH, I

OH 173

(95) M. Horiguchi and M. Kandatsu, Nature, 184 (1959) 901-902; Bull. Agric. Chem. S o c . j p . , 24 (1960) 565-570. (96) G . Rouser, C. Kritchefsky, D. Heller, andE. Lieber,j. Am. Oil Chem. Soc., 40 (1963) 425-454. (97) E. Baer and N. Z. Stanacev,]. B i d . Chem., 239 (1964) 3209-3214. (98) M. Tamari, Kogakuno Ryoiki, 31 (1977) 955-965; Chem. Abstr., 88 (1978) 116,400. (99) T. Hori and M. Horiguchi, Bfochemkity of C - P Compounds, Gakkai Shuppan, Tokyo, 1978, pp. 1- 372. (100) E. Bayer, K. H. Gugel, K. Haegel, H. Hagenmaier, S. Jessipow, W. A. Koenig, and H. Zaehner, Helv. Chirn. Acta, 55 (1972) 224-239.

SUGAR ANALOGS HAVING RING PHOSPHORUS

189

variety of synthetic compounds containing a C-P bond are well known to possess useful biological activities, as exemplified by the antibacterial nucleotide analogs'01J02 (174) and the insecticide d i p t e r e ~ ' ~ (175). ~-'~~ 0

0

0

w

II II It HO-P- 0-P-0-P- CH,-OCH, I I I HO OH OH

HO

0 OH It

I

(MeO),P-CH --CCl, se

175

OH

174

In view of these facts, along with the unique activities of sugar analogs having a ring-nitrogen or -sulfur atom (see earlier), biological testing was conducted33 for a large majority of the phosphorus-containing compounds mentioned herein, and the results are summarized in Table VII. On the whole, it may be stated that, although some mild, biological activities were observed for certain compounds, no remarkable result has thus far been obtained. It therefore seems desirable to prepare a far greater variety of sugar analogs having a ring-phosphorus atom, and to explore their various activities. V. CONCLUSION Although to a far smaller extent than that of nitrogen and sulfur analogs, there has obviously been considerable development in the synthesis of carbohydrates having phosphorus in the ring, and valuable physical data in support of their structures have been accumulated. However, much more work is needed in order to clarify the reaction mechanisms discussed, and, consequently, to improve the yields of many of the steps needed for preparing both the precursors and the final products. It would also be desirable to increase the regio- and stereo-selectivities during the introduction of a phosphorus-containing group into carbohydrates, in order to prepare larger quantities of the desired monosaccha(101) T. C. Myers, K . Nakarnura, and A. B. Darnielzadeh, J . Orig. Chem., 30 (1965) 1517- 1520. (102) A. Holy, personal communication. (103)W. Lorenz, U.S. Pat. 2,701,115 (1955); Chem. Abstr., 49 (1955) 7180. (104) W. F. Barthel, P. A. Gang, andS. A. Hal1.J.Am. C h m . SOC., 76 (1954) 4186-4187. (105) W. Lorenz, A. Henglein, andG. SchraderJ. Am. C h m . SOC., 77 (1955) 2554-2556. (106) S . Inokawa, T. Gornyo, H. Yoshida, and T. Ogata, Synthesis, (1973) 364-365. (1 07) T. Gomyo, H. Yoshida, T. Ogata, H. Inokawa, and S. Inokawa, Nippon Kagaku Kaishi, (1974) 1093 - 1096.

190

HIROSHI YAMAMOTO AND SABURO INOKAWA

TABLE VII Biological Activities of Phosphorus Sugars

Compounds

A‘

Bb

19,20,21,22 30,34 43,44,45 51,52,53,62 67 71,75,76 92,93,94,95 134,135 160b 176,177,178h

>loo

44

C‘

_

-

_ -

5 9 - -

>loo

g

8

Ff

E” 8

8

0 0 0 0 0

4,l.O 0 2 0 0

0 0 0

0 0

8

8

8

g

8

8

0

2,2,1

3.1.1

8

-

DA

Anticarcinogenic activity: ED,, Values (pg/mL) obtained by tn oitro screening, using KB cells (derived from a human, epidermoid carcinoma of the mouth) in Eagle’s MEM- 10% calf-serum culture-medium. Values below 4 pg/mL are regarded as being effective; Cancer Chemotherapeutic Center, Tokyo. b.c Antibacterial and antiviral activity, respectively. The -sign indicates “not effective”; Sankyo Pharmaceutical Co., Ltd. d*e*fInsecticidal,fungicidal, and herbicidal activity, respectively. Testing was performed by using various kinds of plants or insects, and the effectiveness is shown in terms of six grades (5-0), grades 5 and 0 corresponding to 100 and 0%, respectively, of the activities of the reference drugs or chemicals; Asahikasei Co., Ltd., and Nissan Kagaku Kogyo Co., Ltd. 8 Under investigation. hRefs. 106 and 107. Me 0 I II C3H,- CH -P(OEt),

HO 0 I II Me- C-P-Ph I

t

Me Ph 176

177

178

rides having phosphorus in the ring. In the near future, the development of new, efficient procedures and reagents may be expected for the preparation of various other kinds of P sugars, such as nucleoside and nucleotide analogs, which are of considerable interest from the viewpoint of both their physicochemical properties and their biological activities.

VI. TABLE OF SOME PROPERTKES OF SUGAR ANALOGS HAVKNC PHOSPHORUS IN THE MEMIACETAL RING The follGwing abbreviations are used in Table WII: C, chloroform; E, ethanol; W, water.

TABLE VIII

Properties of Sugar Analogs Having Phosphorus in the Hemiacetal Ring

[a],

M.p.

Compound

("C)

(degrees)

Rotation solvent

References

Tri-O-acetyl-l,5-anhydro-5-deoxy-5-C-[(S)phenylphosphinyll-~-iditol 158 Tetra-O-acetyl-5-deoxy-~-xy~opyranose 5-C-[ (R)-butylphosphinyl]-a156.5-158.5 5-C-[ (R)-ethylphosphiny1)-a176-178 5-C-[(R)-ethylphosphinyl]-/l232-234.5 Tetra-O-acetyl-5-deoxy-5-C(ethylphosphiny1)-D-ribopyranose syrup Tri-O-acety~-5-deoxy-3-Q-methyl-~-xylopyranose 5-C-[(S)-( 1-acetoxy)ethenylphosphinyl]-/3189-190 5-C-[ (R)-butylphosphinyl]-p218.5-220 5-C-[ (R)-ethylphosphinyl]-/3227-229 5-C-[ (R)-methoxyphosphinyl]-asyrup 5-C-[ (R)-methoxyphosphinyI]-/3194-195 5-C-[ (S)-rnethoxyphosphinyI]-asyrup 5-C-[ (S)-methoxyphosphiny1]-/3syrup Tetra-O-acetyl-5,6-dideoxy-~-idopyranose 6-C-nitro-5-C-[ (R)-phenylphosphinyl]-j?158-157 6-C-nitro-5-C-[(S)-phenylphosphinyl]-a305 (dec.) 6-C-nitro-5-C-[(S)-phenylphosphino]-/3150-152 5-C-[(R)-phenyIphosphinylj-cY138 5-C-[(R)-phenylphosphinyl]-/3168 5-C-[(S)-phenylphosphinylj-a215 5-C-[(S)-phenylphosphinyIj-P199 Tri-O-acetyl-4,5-dideoxy-4-C-[ (R)phenylphosphinyll-a-~-l yxofuranose 155-156 5-Deoxy-3-O-methy~-~-xy~opyranose 5-C-(hydroxyphosphinyl)-a-(or -p-) 192 208-210 J-C-(phosphinyl)-aD-Ghcopyranose tri-O-acetyl-3,6-di-O-benzyl-5-deoxy5-C-[ (S)-phenylphosphinylj-P210 penta-O-acetyl-5-deoxy-5-C-[(R)ethylphosphinylj-csyrup penta-O-acetyl-5-deoxy-5-C-[(R)ethylphosphinyll-p233 tri-O-acetyl-5,6-dideoxy-3-O-methyl5-C-[ (S)-phenylphosphinylj-a164-165 tri-O-acetyl-5,6-dideoxy-3-O-methyl5-C-[(S)-phenylphosphinylj-/?304- 306 o-Ribofuranose tetra-O-acetyl-4-deoxy-4-C-[ (R)ethylphosphinyl]-/3syrup tetra-O-acetyl-4-deoxy-4-C-[ (S)ethylphosphinyll-a145-146 tetra-O-acetyl-4-deoxy-4-C-[ (S)ethylphosphinyl]-,!?syrup tri-O-acetyl-4,5-dideoxy-4-C-[(S)phenylphosphinyl]-/lsyrup

53,54

+ 28 + 26

C

- 22

C C

34 34 34

- 24

C

36

- 10.0

C C

23 29 29 23 23 23 23

-8.1 0.0 27.0 - 17.4 +6.2 -0.14

+

- 8.7 - 3.2

-9.3 -31.8 - 10.3 -7.1 18.4

+

C C C C

C C C C

E E E E

33,43 33,43 43 53 53 53 53 67

- 25.8

+ 35.0

W W

19 19 34 65

+3.65

+ 37.3 + 23.2 -0.20

C

65

C

60

C

60

C

89 89

-0.38

C

33 67

This Page Intentionally Left Blank

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 42

CARBON-13 NUCLEAR MAGNETIC RESONANCE DATA FOR OLIGOSACCHARIDES BYKLAUSBOCK,CHRISTIAN PEDERSEN, AND HENRIK PEDERSEN Department of Organic Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark

I. Introduction

. . . . . . . . 193 ................................................ 195 ..

Table I. Glucobiose Table 11. Oligomers Table 111. Cyclomalto-hexa- to -octa-oses (Cyclodextrins) . . . . . . . . . . . . . . . . Table IV. Oligosaccharides Containing Aldohexoses. . Table V. Oligosaccharides Containing Sucrose Residue Table VI. Oligosaccharides Containing Fructose Table VII. Oligosaccharides Containing Rhamno Table VIII. Oligomers of Xylose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table IX. Oligosaccharides Containing Amino or Acetamido Groups. . . . . . . Table X. (IHl)-Linked Hexopyranoses Containing One Amino Group . . . . . Table XI. Glycosides of Oligosaccharides Containing Simple Aldohexoses . . Table XI. Methyl Glycosides of Oligo Glucose (in Dimethyl Sulfoxide-d,) . . . . . . . . Table XIII. Methyl Glycosides of Oligomers of Table XIV. Acetates of Methyl Glycosides of 0 Table XV. Peracetates of Glycobioses. Table XVI. Oligosaccharides Related t Table XVII. Reduced Oligosaccharides Related to Blood-group Determinants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table XVIII. Oligosaccharides of Glycoproteins . Table XIX. Oligosaccharides Related to Those of Table XX. Oligosaccharides Related to Those of Salmonella (Glycosides) . . . Table XXI. Oligosaccharides Related to Those of Shigellaflexneri . . . . . . . . . Table XXII. Gangliosides ... ..

199

207 209 210 2 11

218

222 223 224

I. INTRODUCTION The compilation of W-nuclear magnetic resonance (n.m.r.)data for oligosaccharides that is presented in the following Tables constitutes a supplement to the data given in an earlier article on the W - n . m . r .spec193

194

KLAUS BOCK et ul.

troscopy of monosaccharides.' Although several reviews have discussed the W-n.m.r. characteristics of oligo- and poly-sa~charides,~-'it seemed that it would be valuable to have an extensive coIlection of '3C-n.m.r. data on oligosaccharides, similar to those on monosaccharides already published.' The present literature-survey covers most of 1982, and all of the data given had been measured for solutions in D,O unless stated otherwise. The data for peracetates of xylobioses and of glucobioses, given in Tables XIV and XV,were recorded for solutions in CDCl,. All data have been copied from the original articles, and may contain some errors in assignment, particularly for data from the older literature, but these may still be valuable for identification of the molecules, as discussed in Ref. 1. However, the data in Tables I and I1 have been parallel-shifted, in order to bring the chemical shifts into accord with the reference compound currently used (1,4-dioxane, 67.40 p.p.m. relative to external Me&). For mutarotated mixtures of trisaccharides and larger oligosaccharides, the complete set of data is given for the a anomer, but only the chemical shifts for the reducing end of the anomer are given, provided that all other chemical shifts (for the remaining units) are identical. When more than one reference is given for a compound, the data are taken from the reference marked with an asterisk in the Table. The following abbreviations are used for the sugar units: Abe, 3,6-dideoxy-~-rylo-hexopyranose; Col, 3,6-dideoxy-~-xyb-hexopyranose; Fruf, D-fructofuranose; Frup, D-fructopyranose; Fuc, 6-deoxy-~-galactopyranose; Gal, D-galactopyranose; GalNAc, 2-acetamido-2-deoxyD-galactopyranose; GalNAcol, 2-acetamido-2-deoxy-~-galactitol; Glc, D-glucopyranose; GlcN, 2-amino-2-deoxy-~-g~ucopyranose; GlcNAc, 2acetamido-2-deoxy-~-g~ucopyranose; Man, D-mannopyranose; NeuAc, N-acetylneuraminic acid; Par, 3,6-dideoxy-~-ribo-hexopyranose; Rha, 6-deoxy-~-mannopyranose; D-Rha, 6-deoxy-~-mannopyranose; Tyv, 3,6-dideoxy-w-arabino-hesopyranose; Xyl, D-xylopyranose; and Xylol, xylitol. Branched structures are indicated by means of square brackets; for (1)K.Bock and C. Pedersen, Adu. Carbohydr. Chem. Biochem., 41 (1983)27-65. (2) P.A. J . Gorin, Ado. Carbohydr. Chem. Blochan.,38 (1980)13-104. (3)B. Coxon, Dev. FoodCurbohydr., 2 (1980)351-390. (4)K. Bock and H. Thegersen, Annu. Rep. N M R Spectrosc., 13 (1982)1-57. (5)A.S.Perlin and B. Caw, in G . 0.Aspinall (Ed.), The Polysacchrides,Vol. 1, Academic Press, New York, 1982,pp. 133-193. (6)R. Barker, H.A. Nunez, P. Rosevear, and A. S. Serianni, MethodsEnzymol., 83 (1982) 58-59. (7)F.W. Wehrli and T. Nishida, Fortschr. Chem. Org. Nuturst., 36 (1979)1-229.

195

CARBON-13 N.M.R. DATA FOR OLIGOSACCHAFUDES

example, a-Glc-(1-+4)-[a-GIc-(1+6)]-a-Glc indicates an a-mglucopyranose residue substituted with two a-D-glucopyranosylgroups, at 0-4and 0-6, respectively. The sampling and assignment techniques used for the measurement of 13C-n.m.r.spectra of oligosaccharides are identical to those discussed previously for monosaccharides'. Particularly for oligosaccharides, it is important, when data are to be compared accurately, that is, with a precision better than k 0 . 5 p.p.m., to measure the spectra at the same temperature.

11. TABLES TABLE I 13C-N.m.r. Data for Glucobioses Compound

C-1

C-2

C-3

C-4 ~

a-Glc( 1-1)a-Glc a-Glc(l-1)P-Glc p-Glc(1-1)P-Glc Cr-Clc(1-2)a-Glc" a-Glc( 1-2)~-~ic= P-Clc(1-2)cy-GICb

P-Clc(l-2)P-Glcb a-Glc( 1-3)a-Clc a-Glc(1-3)P-Glc p-clc(l-3)a-Glcb P-Clc(1-3)P-Glcb

~~~

C-5 ~

C-6

References

~~

94.0

72.0

73.5

70.6

73.0

61.5

101.9 104.0 100.7

72.4 74.3 74.2

73.8 77.4 77.3

70.4 70.9 71.1

73.6 76.8 77.3

61.6 62.3 62.5

3",8,9

97.1 90.4 98.6 97.1 104.4 92.4 103.2 95.1 99.8 93.1 99.8 97.0 103.2 92.7 103.2 96.5

72.7 76.7 72.7 79.5 74.2 81.4 74.2 82.1 72.8 71.3 72.8 74.1 74.1 71.4 74.1 74.1

74.0 72.7 74.0 75.4 76.5 72.5 76.5 76.5 74.1 80.8 74.1 83.2 76.4 03.5 76.4 86.0

70.7 70.7 70.7 70.7 70.4 70.4 70.4 70.4 71.3 70.6 71.3 70.6 70.5 68.9 70.8 68.9

72.7 72.7 72.7 76.7 76.5 71.8 76.5 76.5 72.8 72.2 72.8 76.6 76.4 71.7 76.4 76.4

61.6 61.6 61.6 61.6 61.7 61.7 61.7 61.7 61.8 61.8 61.8 61.8 61.7 61.7 61.7 61.7

8

3",8,9

8

8

8

8 8

8 8

(continued)

(8) T. Usui, N. Yamaoka, K. Matsuda, K.Tuzimura, H. Sugiyama, and S. Seto, /. Chem. SOC.,Perkin Trans. I, (1973) 2425-2432. (9) P. E.Pfeffer, K. M. Valentine, and F. W. Parrish, J . Am. Chem. SOC., 101 (1979) 1265- 1274.

KLAUS BOCK et ul.

196

TABLE I (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

a-Glc(1-4)a-GlcE a-Glc(1-4)P-Glc' P-Glc(1-4)a-Glcb P-Glc(1+4)P-Glcb a-Glc(l-t6)-

100.7 92.8 100.7 96.8 103.6 92.9 103.6 96.8 98.5 92.9 98.5 96.8 103.0 92.5 103.0 96.4

72.8 72.3 72.8 75.0 74.3 72.3 74.3 75.0 72.4 72.4 72.4 75.0 73.7 72.1 73.7 74.7

73.9 74.1 73.9 77.1 76.6 72.4 76.6 75.4 74.1 74.1 74.1 76.2 76.3 73.7 76.3 76.3

70.4 78.5 70.4 78.2 70.6 79.9 70.6 79.8 70.4 70.4 70.4 70.4 70.3 70.3 70.3 70.3

73.6 71.0 73.6 75.6 77.0 71.2 77.0 75.8 72.9 70.4 72.9 75.0 76.3 71.0 76.3 75.3

61.6 61.6 61.6 61.8 61.7 61.0 61.7 61.2 61.6 66.5 61.6 66.5 61.7 69.4 61.7 69.4

a-~1~4 a-Glc(l-6)P-GlcO P-Glc(1-6)a-Glcb P-Glc(1-6)P-Glcb

References 3,8,10',11 3,8.10',11 3,8,9,10°,l 1,12 3,8,9,10',11.12,13' 8 8

8 8

a The published chemical-shift values are obviously too high, and have been corrected to give the terminal C-6 atom a value of 61.6, in agreement with its normal shift. The published chemical shift values are obviously too high, and have been corrected to give the terminal C-6 atom avalue of 61.7, in agreement with its normal shift. Data for a solution in Me,SO-d,.

I1 TABLE 13C-N.m.r. Data for Oligomers of Glucose" Compound

C-1

C-2

C-3

C-4

C-5

C-6

96.3 97.0 92.9

72.5 76.5 72.7

73.8 72.7 73.7

70.6 70.4 70.4

72.3 73.2 70.8

61.5 61.5 67.1

96.8 100.9 100.6

75.0 72.8 72.6

76.7 74.0 74.3

70.4 70.5 78.3

75.1 73.7 72.3

67.1 61.6 61.6

References

Trisaccharides a-Glc(1-2)a-Glc(1-6)CY-GIC p anomer p-Glc a-Glc(l+Q)a-Gk(1-4)-

14

8,10',15

(10) A. Heyraud, M. Rinaudo, M. (R.) Vignon, and M. Vincendon, Biupolymers, 18 (1979) 167-185. (11) D. E.Dorman and J. D. Roberts,]. Am. Chem. Soc., 93 (1971) 4463-4472. (12) J. C. Gast, R. H. Atdla, and R. D. McKelvey, Curbohydr. Res., 84 (1980) 137- 146. (13) S.L. Patt, F. Sauriol, and A. S.Perlin, Curbohydr. Res., 107 (1982) cl-c4. (14) V.Pozsgay, P. Nhnbi, and A. Neszmklyi, Carbohydr. Res., 75 (1979) 310-313. (15) V. Munksgaard, Ph. D. Thesis, Danmarks Farmaceutiske Hejskole, 1981.

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

197

TABLE 11 (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

a-Glcb (~-Glc(l-4)a-Glc(1-4)P-Glcb P-GIc( 1-4) P-Glc(1-4)O-GIC" /3 anomer P-Glc' a-Glc(1-4)a-Glc(l+G)a-Glcb P anomer P-Glcb a-Glc(1-6)(~-GIc(l+4)a-Glcb /?anomer P-Clcb ~r-Glc(1-6)a-Glc(1-6)a-Glc /3 anomer p-Glc P-Glc(1-6)/?-Clc(1-6)a-Glc /3 anomer P-GIC

92.9 100.9 100.5 96.8 103.6 103.4 92.9

72.3 72.8 72.5 75.1 74.2 74.0 72.3

74.1 74.0 74.3 77.1 76.6 75.1 72.4

78.6 70.5 78.3 78.4 70.5 79.5 79.8

71.1 73.7 72.3 75.6 77.0 75.9 71.2

61.6 61.6 61.6 61.8 61.7 61.0 61.0

96.8 100.4 98.6 93.1

75.0 73.4 72.6 72.6

75.3 74.3 73.9 73.9

79.6 70.3 78.1 70.3

75.9 72.3 70.9 70.6

61.1 61.6 61.6 66.8

97.0 98.5 100.3 92.5

75.1 72.3 73.5 72.3

77.0 73.8 73.8 73.8

70.3 70.4 70.4 77.7

75.1 72.3 70.4 70.8

66.8 61.6 66.6 61.6

96.4 98.4 98.6 92.9

74.6 72.1 72.1 72.1

76.9 73.7 74.0 73.7

77.7 70.1 70.9 70.6

75.0 72.5 72.1 72.5

61.6 61.1 66.1 66.4

96.8 102.8 102.8 92.1

74.7 73.0 73.0 71.4

76.7 75.5d 75.6d 72.7

70.2 69.4 69.6 69.6

74.9 74.gd 74.gd 70.4

66.4 60.7 68.5 68.8

95.9

74.0

75.9

69.6

70.8

68.9

102.8 104.2 102.8 92.2

' 73.9 72.2 71.5

76.3d 75.1h 87.8 75.1h

70.3 80.8 68.6 80.8

77.1 74.gh 76.6d 74.gh

61.0f 60.8 61.2f 60.6f

96.8 103.5d 103.0d 103.9 92.0

73.2 72.1 73.2' 73.8' 71.2

75.1h 76.4f 75.0' 75.01 85.2

80.8 70.2 80.7 80.7 68.8

74.gh 77.0 74.6' 74.6' 76.7f

60.6f 61.0h 60.7h 60.5h 61.8

References 8,10",15 10°,12

8

8

15",16

17

Tetrasaccharides P-Glc(1-4)P-Glc(1-3)P-Glc(1-4)a-Glc P anomer P-Glc P-Glc(1-4)P-Glc(1-4)P-Glc(1-3)a-Glc

188

18g

(continued)

(16) T.Takeda, Y.Sugiura, Y.Ogihara, andS. Shibata, Curbohydr. Res., 105 (1982) 271 275. (17) D. Bassieux, D. Y.Gagnaire, and M. (R.) Vignon, Curbohydr. Res., 56 (1977) 19-33. (18) P. Dais and A. S. Perlin, Curbohydr. Res., 100 (1982) 103-116.

KLAUS BOCK

198

et

al.

TABLE I1 (continued) C-1

C-2

C-3

C-4

C-5

C-6

96.6 103.6 103.4 103.4 92.9

73.5' 74.2 74.0 74.0 72.3

88.2 76.6 75.1 75.1 72.4

68.8 70.5 79.4 79.5 79.8

76.71 77.1 75.9 75.9 71.2

61.8 61.7 61.0 61.0 61.0

96.8

95.9

75.0 73.0 73.0 73.0 71.4 73.0 73.0 73.0 74.1

75.3 75.6d 75.6d 75.6d 72.7 75.ad 75Bd 75.8d 75.8

79.6 69.6 69.6 69.7 69.7 69.6 69.6 69.7 69.7

75.9 74.9 74.9 74.9 70.4 74.9 74.9 74.9 74.8

61.1 60.9 68.8 68.8 68.9 60.9 68.8 68.8 68.9

100.8 100.6 100.6 100.6 92.9 100.8 100.6 100.8 100.5 96.8 103.5 103.3 103.3 103.3 92.9

72.8 72.6 72.6 72.6 72.3 72.8 72.6 72.6 72.6 75.0 74.3 74.1 74.1 74.1 72.4

73.9 74.2 74.2 74.2 74.1 73.9 74.2 74.2 74.2 77.1 76.7 75.2 75.2 75.2 72.4

70.5 78.3 78.4 78.4 78.6 70.5 78.3 78.4 78.4 78.4 70.7 79.6 79.6 79.6 80.1

73.7 72.3 72.3 72.3 71.0 73.7 72.3 72.3 72.3 75.6 77.0 75.9 75.9 75.9 71.4

61.6 61.6 61.6 61.6 61.6 61.6 61.6 61.6 61.6 61.8 61.7 61.2 61.2 61.2 61.2

96.8

75.0

75.4

79.9

75.9

61.4

103.4 103.2 103.2 103.2 103.2

74.4 74.3 74.3 74.3 74.3 72.6

76.9 75.4 75.4 75.4 75.4 72.6

70.5 79.5 79.5 79.5 79.6

76.9 76.1 76.1 76.1 76.1 71.4

61.7 61.0 61.0 61.0 61.0 61.0

96.7

75.4

75.5

79.8

76.1

61.2

Compound

References

p anomer P-Glc j.3-Glc(1-4)p-Clc(l+4)/3-Glc(l+4)ff-Glc= p anonier /3-Glc" /?-Gl~(l+6)p-Glc(l--S)j3-Gk(l+6)a-Glc @-Glc(l+6)p-CIc(146)/?-Glc(1+6)p-Clc

' 102.6 102.7 92.0 102.6 102.7 a

10

17

17

Pentasaccharides a-Glc(l44)(~-Gk(l+4)a-Glc(1-4)a-Glc(1-4)a-Glcb a-Glc(1-4)c~-Clc(l+4)~&lc(l+4)a-Glc(l+4)P-Glcb P-Glc(1 4 4 ) /?-Gk(l-4)p-Clc(1-4)/I-Glc(1-4)a-Glc p anomer P-Glc

10O.12

10",12

10

Hexasaccharide p-G1~(1+4)B-Glc(1-4)/?-Gk( 1-4)p-Glc(1-4)P-Gk(l+4)a-GlcC /3 anomer p-clcc

10

CARBON-13 N.M.R. DATA FOR OLJGOSACCHARIDES

199

TABLE I1 (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

100.7 100.6 100.6 100.6 100.6 100.6 96.8

72.8 72.6 72.6 72.6 72.6 72.6 75.1

74.0 74.2 74.2 74.2 74.2 74.2

70.5 78.4 78.4 78.4 78.4 78.4

73.7 72.3 72.3 72.3 72.3 72.3

61.6 61.6 61.6 61.6 61.6 61.6 61.8

References

Heptasaccharide a-Glc(1-4)a-Glc(l+4)a-@lc(l-r4)a-Glc(l+4)a-GIc(1-4)(~-Glc(l+4)p-clcb

e

10

Data for related compounds are given in Refs 14, 16; 17. and 19. [I The published chemical-shift values are obviously too high, and have been corrected to give the terminal C-6 atom a value of 61.6: in agreement with its normal shift. The published chemical-shift values are obviously too high, and have been corrected to give the terminal C-6 atom a value of 61.7, in agreement with its normal shift. Assignments may Rave to be reversed. Signal not reso1ved.f Assignments may have to be reversed. g In Me,SO-d,. h - j Assignments may have to be reversed.

TABLE III '3C-N.m.r. Data for Cyclomalto-hexa- to -octa-oses (Cyclodextrins) Compound (a-G1~(1-+4))6 (a-GI~(l-4))7 Per-0-methyl(~i-GI~(l--nl)), Per-0-methyl(a-Gk(1+4)]7h (a-Gl~(l+4)),

C-1

C-2

C-3

C-4

C-5

C-6

References

101.5 101.9

72.0 72.4

73.5 73.4

81.4 81.3

72.3 72.1

60.7 60.7

20" 20"

98.3

82.0

79.0

81.2

71.3

71.7

21

99.1 101.7

82.4 72.5

82.1 73.1

80.6 80.7

71.2 72.1

71.8 60.6

21 20"

Data for solutions in Me,SO-d, are also given in Ref. 20. In CDCI,.

(19) T. Ogawa andT. Kaburngi, Carbokyds. Res., 103 (1982) 53-64. (20) M. Vincendor,, Bull. Soc. Chirn. Fr., Pt. 2, (1981) 129-134. (21) J. Szejtli, A. LiptBk, I. JodBl, P. Fugedi, P. Nhnbsi, arid A. Neszmblyi, Stuerke, 32 (1980) 165- 169.

KLAUS BOCK et ul.

200

TABLE IV 13C-N.m.r. Data for Oligosaccharides Containing Aldohexoses" Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

103.0 91.9 103.0 95.8 99.0 93.0 99.0 96.9 96.6 97.7 101.4 97.9 104.2 95.3 104.2 95.3 101.7 93.5 01.7 97.5 02.5 92.9 01.0 94.6 101.0 94.5

71.1 70.2 71.1 73.9 69.3 72.3 79.3 74.9 73.0 71.5 73.6 73.1 74.7 71.9 74.7 71.9 75.6 72.3 75.6 72.3 70.6 79.4 71.4 71.0 71.4 71.4

72.6 71.2 72.6 74.5 70.3 73.8 70.3 76.7 74.1 78.8 74.0 73.1 77.6 70.6 77.6 73.4 74.6 73.0 74.6 76.2 70.3 70.3 73.7 69.8 73.7 72.5

68.6 78.4 68.6 78.4 70.0 70.4 70.0 70.3 70.7 66.3 70.6 78.6 71.2 78.5 71.2 78.5 68.4 80.5 68.4 80.5 67.2 67.3 67.5 77.6 67.5 77.3

75.4 71.5 75.4 74.9 71.8 70.9 71.8 75.2 72.6 76.1 73.1 76.3 77.1 72.6 77.1 76.5 78.1 71.7 78.1 75.9 72.8 73.6 77.2 71.7 77.2 75.6

61.1 60.2 61.1 60.2 61.9 66.8 61.9 66.7 61.7 62.2 61.4 61.4 62.0 62.2 62.0 62.2 62.2 62.4 62.2 62.4 61.3 61.4 61.9 61.3 61.9 61.3

9,22",23,24

Disaccharides P-Gal(1-4)a-Glc @-Gal(1-4)&-Gk a-Gal( 1-6)a-Clc a-Gal(l-6)/?-Glc a-Glc( 1-3)P-Gal a-Glc(1-4)P-Gal P-Glc( 1-4)a-Man P-Glc(1-4)&-Man P-Man(l-4)a-Glc P-Man( 1-4)P-Glc a-Man(l-2)a-Man P-Man(l-4)a-Man P-Man(l-4)/?-Man

9,22",23,24 24,25" 24,25' 26 26 27 27 27 27 28 27,29" 27.29"

(22) W. Voelter, V. Bilik, and E. Breitmaier, CoZZect. Czech. Chem. Conzmun., 38 (1973) 2054-2071. (23) H. A. Nunez and R.Barker, Biochemistry, 19 (1980) 489-494. (24) E. Breitmaier, G . Jung, and W. Voelter, Chimiu, 25 (1971) 362-364. (25) G. A. Morris and L. D. Hall,]. Am.Chem. SOC., 103 (1981) 4703-4711. (26) N. K. Kochetkov, V. I. Torgov, N.N. Malysheva, A. S. Shashkov, a n d E . M. Klimov, Tetrahedron, 36 (1980) 1227- 1230. (27) T. Usui, T. Mizuno, K. Kato, M. Tomoda, and G . Miyajima, Agrtc. Biol. Chem., 43 (1979) 863-865. (28) T. Ogawa and H. Yamamoto, Curbohydr. Res.. 104 (1982) 271 -283. (29) B. V. McCleary, F. R. Taravel, and N. W. H. Cheetham, Curbohydr. Res., 104 (1982) 285-297.

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

201

TABLE IV (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

105.2 103.4 92.7

71.9 71.0 72.0

73.4 82.7 72.2

69.4b 69.3b 79.2

75.9 75.9 70.9

61.8 61.8 60.9

30

96.6 99.2 101.2 94.6

74.7 69.3 71.3 70.9

75.2 70.2 73.7 69.8

79.0 70.1 67.4 78.1

75.6 71.8 75.3 71.6

61.1 61.9 67.1 61.4

94.5 99.7 100.7 94.7 99.6 100.8 94.6 101.6 104.2 95.3

71.3 69.3 71.4 71.0 69.3 71.4 71.4 72.0 74.2 71.6

72.5 70.3 73.7 69.7 70.3 73.7 72.5 74.2 77.0 70.7

77.9 70.1 67.5 77.9 70.1 67.5 77.5 68.1 80.6 78.4

75.5 72.1 77.3 70.3 72.1 77.3 74.1 77.0 76.0 72.4

61.4 62.0 61.8 67.4 62.0 61.8 67.3 62.4b 62.0b 62.0b

95.3 101.5 101.7 93.4

72.0 72.0 71.5 72.0

73.6 74.3 73.0 73.0

78.4 68.4 77.9 80.6

76.0 77.9 76.5 71.5

62.4b 62.4b 62.0b 62.0b

97.3 102.5 100.8 92.7 101.6 101.6 95.2

75.3 70.6 78.8 79.6 71.9 71.4 71.9

76.1 70.2 70.2 70.2 74.3 73.0 70.4

80.6 67.1 67.3 67.3 68.2 77.9 77.9

75.7 72.7 73.5 73.5 77.9 76.5 72.4

62.4b 61.3 61.3 61.3 62.0b 62.0b 62.4b

95.2

71.9

73.0

77.9

76.5

62.4b

105.1 104.9 103.5 92.7

72.1 71.1 71.1 72.0

73.5 82.9 82.7 72.2

69.4 69.5b 69.3b 79.2

75.9 75.9 75.9 71.0

61.9 61.9 61.9 60.9

Trisaccharides /?-Gal(1-3)/?-Gal(1-4)CX-GIC /3 anomer /?-Glc a-Gal(1-6)P-Man(1-4)a-Man /?anorner /?-Man a-Gal(l-6)[/?-Man(1+4)]a-Man a-Gal(1-6)[P-Man(l-4)]/?-Man /?-Man(1+4)/?-Glc(l+4)a-Man /? anorner /?-Man P-Man(l-4)/?-Man(1-4)a-Glc /? anorner /?-Glc a-Man(l-r2)a-Man(l-2)a-Man P-Man(l-4)P-Man(1-4)a-Man /? anomer /?-Man

29

29 29 27

27

28 27

Tetrasaccharides /?-Gal(1-3)/?-Gal(1-3)/?-Gal(1-4)a-Glc

30

(continued)

(30) J. G. Collins, J. H. Bradbury, E. Trifonoff,and M. Messer, Curbohydr. Res., 92 (1981) 136- 140.

202

KLAUS BOCK et al. TABLE IV (continued)

Compound /?anomer P-Glc a-Man(1-2)a-Man(1-2)a-Man(1-2)a-Man

C-1

C-2

C-3

C-4

C-5

C-6

96.7 102.5 155.9 100.9 92.7

74.7 70.6 78.8 79.1 79.7

75.4 70.3 75.3 70.3 70.3

79.1 67.2 67.3 67.3 67.3

75.6 72.7 73.5 73.5 73.5

60.9 61.3 61.3 61.3 61.3

102.5 100.8 100.8 100.8 100.8 92.8

70.6 78.8 79.1 79.1 79.1 79.7

70.2 70.2 70.2 70.2 70.2 70.2

67.2 67.3 67.3 67.3 67.3 67.3

72.8 73.5 73.5 73.5 73.5 73.5

61.3 61.3 61.3 61.3 61.3 61.3

References

28

Hexasaccharide a-Man(1-2)a-Man(1-2)a-Man(1-2)a-Man(1-2)a-Man(1-2)a-Man

28

a Data for related compounds are given in Refs. 30,31, and 32. Assignments may have to be reversed.

TABLE V '3C-N.m.r. Data for OligosaccharidesContaining Sucrose Residues ~~~~

~~

Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

92.9 62.2

71.9 104.5

73.4 77.3

70.0 74.8

73.2 82.2

61.0 63.2

3",9,25,33-39

100.6 92.8 62.3 93.7

72.6 71.7 104.5 72.4

73.8 73.8 77.4 73.8

70.2 77.7 74.9 70.5

73.5 71.9 82.2 73.6

61.4 61.0 63.2 61.4

15

Disaccharide a-Glc(1-2)P-Fruf Trisaccharides a-Glc(l--r4)a-Glc(1-2)8-Fruf a-G1c(1@2)-

38"

(31) T. Takamura, T. Chiba, and S. Tejima, Chem. Phann. Bull., 29 (1981) 1027-1033. (32) K. Bock, unpublished results. (33) A. J. Jones, P. Hanisch, and A. K. McPhail, Aust. J . Chem., 32 (1979) 2763-2766. (34) R. U. Lemieux and K. Bock,Jpn. J . Antibiot., 32 (1979) s163-s177. (35) A. Bax, R. Freeman, T. A. Frenkiel, and M.H. Levitt, J . Magn. Reson., 43 (1981) 478-483. (36) K. Bock and R. U. Lemieux, Curbohydr. Res., 100 (1982) 63-74. (37) D. Doddrell and A. Allerhand, 1.Am. C h .SOC., 93 (1971) 2779-2781. (38) H. C. Jarrell, T. F. Conway, P. Moyna, and I. C. P. Smith, Carbohydr. Res., 76 (1979) 45-57. (39) F. R. Seymour,R. D. Knapp, J. E.Zweig, and S.H. Bishop, Carbohydr. Res., 72 (1979) 57 - 69.

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

203

TABLE V (continued) ~~

Compound

C-1

[P-Fruf@-l)]61.7 P-Fruf 62.2 a-Gl~(l+2)92.5 [a-Glc(l-3]101.0 j?-Fruf 62.8 &-Gal(1-6)99.3 a-GI~(l*2)92.9 P-Fruf 62.2 a-Gk(1-6)99.0 ~ y - G l ~ ( l ~ 2 ) - 92.9 P-Fruf 62.2

~~~

C-2

C-3

C-4

C-5

C-6

104.5 104.9 71.8 72.2 104.5 69.3 71.8 104.6 72.3 71.7 104.6

77.9 77.9 73.6 73.9 84.0 70.3 73.5 77.2 73.8 73.7 77.1

75.7 75.7 70.3 70.4 74.0 70.0 70.3 74.8 70.3 70.1 74.8

82.4 82.4 73.1 73.0 82.0 71.8 72.2 82.2 72.6 72.1 82.1

63.4b 63.5b 61.2 61.4 63.0 61.9 66.7 63.3 61.3 66.4 63.2

69.8' 69.7' 71.4" 103.9 72.4 104.4 104.3 104.9 72.2 72.5 71.6 104.4

68.5d 68.9' 73.0" 77.0 73.8 77.9 78.7 77.9 73.9 73.9 73.7 77.3

69.8' 68.6" 69.5' 81.4 70.4 75.8 75.5 75.1 70.3 70.2 78.0 74.8

71.1 69.5' 71.2' 74.4 76.7 82.3 82.3 82.4 72.6 72.1 71.7 82.1

61.3d 66.6 66.2 62.0b 61.3 63.5 63.5 63.5 61.3 66.7 61.0 63.1

References

15O.39 25",37 15

Tetrasaccharides a-Gal(l-+6)a-Gal( 1-6)a-GIc(l*2)P-Fruf a-Glc(1-2)[j?-Fruf(2-+l)j?-Fruf(Z-l)]P-Fruf a-CIc(1-6)a-Gk(1+4)a-Glc(1-2)j?-Fruf

98.2 98.5 92.2 62.6b 93.7 61.5 62.2 62.1 98.9 100.7 92.7 62.1

37

38"

15

a Data for related compounds are given in Refs. 38 and 39. b-e Assignments may have to be reversed.

TABLE VI '%-N.m.r. Data for Oligosaccharides Containing Fructose Compound

C-1

C-2

C-3

C-4

C-5

C-6

61.0 64.2 61.1 93.0 61.1 96.8 103.9

104.3

77.2 68.8 77.8 73.5 77.9 76.5 73.7

75.0 70.2 75.4 70.6 75.5 70.5 69.7

81.9 69.8 82.0 71.5 82.0 75.8 76.0

62.7 64.5 63.2 61.7 63.3 61.7 62.1

References

Disaccharides P-Fruf(2-1)/3-Frup j?-Fruf(2-6)a-Glc P-Fruf(2-6)P-ClC P-GaI(l+4)-

100.0

104.6 72.3 104.6 74.9 71.7

15 15 15 40 (continued)

(40) P. E. Pfeffer andK. B. Hicks, Carbohydr. Res., 102 (1982) 11-22.

KLAUS BOCK

204

et

al.

TABLE VI (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

a-Fruf P-Gal(l-4)P-FrUf P-Gal(1-4)P-Frup a-ClC(1-1)P-Frup a-Cl~(l+3)a-Fruf a-Glc(1-3)P-Fruf a-Glc(1-3)P-Frup (Y-CIC( 1-4)a-Fruf a-Glc(1-4)P-Fruf a-GI~(l+4)P-FKUP P-Glc(1-4)a-Fruf P-Glc(1-4)P-Fruf P-Glc(1-4)P-Frup a-Gk(1-5)P-Frup a-Gk(1-6)a-Fruf a-Glc(1-6)-

63.9 103.4 65.1 101.5 65.1 99.2 69.9 97.6 61.8 99.2 63.1 101.7 64.8 98.9 63.8 99.4 63.8 101.5 65.1 103.5 63.6 103.1 63.6 101.1 65.0 101.5 65.1 99.7 63.9 99.4 63.9

105.6 71.7 103.1 71.7 98.8 72.2 98.6 72.0 105.0 72.2 102.4 72.8 98.5 72.4 106.3 72.4 103.1 73.0 99.4 74.0 105.9 74.0 103.2 74.0 99.1 73.2 99.2 72.6 105.9 72.6 102.9

81.8 73.7 76.1 73.7 67.2 73.7 68.6 73.7 85.5 73.5 81.2 73.7 77.4 74.0 81.3 73.4 76.5 74.1 68.2 76.7 81.7 76.7 76.7 76.7 67.1 74.2 69.2 74.2 82.9 74.2 76.5

86.0 69.7 84.9 69.7 78.3 70.3 70.3 70.1 73.0 70.1 73.1 70.1 71.0 70.7 83.3 70.7 82.4 70.9 79.2 70.6 86.2 70.9 84.9 70.6 78.4 70.9 71.2 70.8 77.3 70.8 75.8

81.4 76.0 80.8 76.0 67.7 72.6 69.8 75.3 82.3 75.1 81.6 73.5 69.8 73.5 82.2 73.5 81.1 73.4 70.3 76.9 81.7 76.9 80.9 76.9 67.7 73.3 80.2 73.1 81.2 73.1 80.1

63.6 62.1 63.6 62.1 63.9 61.3 64.3 61.1 63.5 61.1 63.7 61.3 64.1 61.7 62.6 61.7 63.8 61.8 64.5 61.8 63.6 61.8 63.6 61.8 63.9 61.9 63.4 61.8 68.0 61.8 69.0

100.5 98.9 63.2 100.4 101.1 64.6

72.5 71.8 102.7 72.5 72.4 99.1

73.7 73.9 76.0 73.7 74.1 67.7

70.1 77.6 82.2 70.1 77.6 78.9

73.5 71.6 80.8 73.5 71.6 69.9

61.3 61.3 63.5 61.3 61.3 64.2

P-Fruf

References

40 40 15 15',37 15',37 15',37 38,40" 38,40" 38,40" 40 40 40 38 38',39 38",39

Trisaccharides

a-Glc(1-4)a-Glc( 1-4)P-Fruf a-Glc(1+4)a-Glc(1-4)-

P-FKUP

15 15

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

205

TABLE VII 13C-N.m.r.Data for Oligosaccharides Containing Rhamnose and Simple AldohexosesO Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

105.9 94.1 105.1 93.9 105.5 95.0 105.5 94.5 100.5 94.3 100.5 94.1 104.9 95.0 104.9 94.6 105.3 94.0 104.6 93.9 105.0 95.0 105.0 94.6 104.4 95.0 104.4 94.6 102.5 94.8 102.5 94.6 101.8

72.2 81.7 72.2 82.4 72.4 71.9 72.4 72.4 69.2 71.8 69.2 72.0 72.9 72.0 72.9 72.5 74.5 82.1 74.5 82.4 74.7 71.8 74.7 72.3b 75.1 72.0 75.1 72.5 71.5 72.2 71.5 72.7 71.8

73.7 71.1 73.7 74.2 73.8 81.0 73.8 83.4 69.6 69.6 69.6 72.4 74.0 71.2 74.0 74.0 77.0 70.9 77.0 74.3 76.9 81.0 76.9 83.5 77.2b 71.2 77.2b 74.0 71.6 70.1 71.6 72.8 74.3

69.7 73.6 69.7 73.3 69.9 72.4 69.9 72.4 69.9 82.1 69.9 81.6 69.8 82.3 69.8 81.9 70.5 73.5 70.5 73.2b 70.8 72.5b 70.8 72.3b 70.8 82.5 70.8 82.0 67.7 82.7 67.7 82.3 68.0

76.2 69.3 76.2 73.6 76.3 69.5 76.3 73.0 70.0 68.1 70.0 72.3 76.4 68.1 76.4 71.8 76.7 69.3 76.7 73.8b 76.9 69.5 76.9 73.0 77.0b 68.0 77.0b 71.6 74.1 69.0 74.1 72.2 77.5

62.2 18.1 62.2 17.9 62.6 18.1 62.6 18.1 61.6 17.9 61.6 17.9 62.1 18.2 62.1 18.2 61.7 17.9 61.7 17.9 61.9 18.1 61.9 18.1 61.9 18.2 61.9 18.2 61.9 18.2 61.9 18.2 62.2

41

Disaccharides P-Gal(l-+2)a-Rha P-Cal(1-2)P-Rha /?-Gal(l-+3)a-Rha P-Gal(l-3)P-Rha a-Gal(l-r4)a-Rha a-Gal(l-4)P-Rha P-Gal(1+4)a-Rha P-Gal(l-4)P-Rha P-Glc(l-2)a-Rha P-Glc(l+2)P-Rha P-Glc(l+3)a-Rha P-Glc(1+3)P-Rha P-Glc(l-+4)a-Rha P-Glc(l+4)P-Rha a-Man(l+4)a-Rha a-Man(l44)P-Rha P-Man(1 4 4 ) -

41 41 41 42 42 41',48 41 41 41 41 41 41 41 44 44 45

(continued) (41) P. Colson and R. R. King, Carbohydr. Res., 47 (1976) 1- 13. (42) P. Fugedi, A. Liptak, P. Nanhi, and A. Neszmelyi, Carbohydr. Res., 80 (1980) 233239. (43) V. Pozsgay, P. NBnasi, and A. Neszmelyi, Chem. Commun., (1979) 828-831. (44) V. I. Torgov, V. N. Shibaev, A. S. Shashkov, and N. K. Kochetkov, Bioorg. Khim., 6 (1980) 1860- 1871. (45) B. A. Dmitriev, A. V. Nikolaev, A. S . Shashkov, andN. K. Kochetkov, Carbohydr. Res., 100 (1982) 195-206.

KLAUS BOCK et al.

206

TABLE VII (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

a-Rha 8-Mm(1-4)P-Rha c~-Rha(l-+3)&-Gal c~-Rha(l+3)P-Gal gRha(1-3)&-Gal P-Rha(l-3)P-GaI a-Rha(1-4)a-Gal a-Rha(l-4)&Gal a-Rha(l-6)a-Gal a-Rha(l-6)P-Gal a-Rha(l-6)a-Glc a-Rha(l-6)-

95.1 101.8 94.5 103.6 93.6 103.6 97.5 98.1 93.3 98.1 97.5 103.7 93.5 103.7 98.0 101.7 93.6 101.7 97.8 101.9 93.4 102.1 97.4 102.8 93.4 103.1 94.8 103.1 94.2 102.1 94.5

72.2 71.8 71.8 71.3 70.4 71.3 72.5 73.2 68.1 73.2 72.3 71.7 70.6 71.7 71.7 71.3 70.2 71.3 73.2 71.4 72.9 71.4 75.5 70.9 79.9 71.0 71.5 71.0 72.1 71.2 71.3

71.2 74.3 74.0 71.3 78.4 71.3 81.8 73.9 77.1 73.9 80.4 71.7 78.5 71.7 81.9 71.5 69.6 71.5 74.1 71.7 74.1 71.7 77.2 70.6 70.9 71.0 78.6 71.0 81.2 71.2 71.5

80.8 68.0 80.4 73.2 69.8 73.2 68.9 73.2 67.5 73.2 66.9 73.6 70.0 73.6 69.3 73.3 70.7 73.3 70.0 73.5 71.2 73.5 71.2 72.8 73.2 72.9 72.5 72.9 72.1b 72.8 80.7

68.2 77.5 72.8 70.4 71.7 70.4 76.3 73.5 71.6 73.5 76.1 70.4 72.9 70.4 76.4 69.9 70.3 69.9 74.7 69.9 71.8 69.9 76.1 69.8 69.1 69.9 69.3 69.9 72.7b 70.0 67.3

18.3 62.2 18.3 17.8 62.3 17.8 62.1 17.9 62.3 17.9 62.2 18.0 62.4 18.0 62.2 17.9 68.7 17.9 68.2 17.9 68.5 17.9 68.3 17.6 17.4 17.4 17.4 17.4 17.6 17.3 18.3

100.5 104.5 93.4

69.3 74.3 81.9

70.1 76.7 69.4

69.9 70.3 81.8

71.4 76.5 68.2

61.5 61.5 17.9

P-Gk a-Rha(1-2)a-Rha cr-Rha(l-3)a-Rha a-Rha(l-3)P-Rha a-Rha(l-4)a-Rha

References

45 44",45 44',45 44 44 46 46 47",48 47",48 47",48 47',48 43",49 43",48-50 43",48,50 49

Trisaccharides a-Cal(1-4)[P-Glc(l--n)]cY-Rha

42

(46) N.K. Kochetkov,B.A.Dmitriev,A. V. Nikolaev, N.I?.Bairamova,andA.S.Shashkov, Bioorg. Khirn., 5 (1979) 64-76. (47) L. V. Backinowsky, N. F. Balan, A. S . Shashkov, and N. K. Kochetkov, Carbohydr. Res., 84 (1980) 225-235. (48) C. Laffite, A. M. Nguyen Phouc Du, F. Winternitz, R. Wylde, and F. Pratviel-Sosa, Carbohydr. Res., 67 (1978) 91-103. (45) A. Liptak, P. NBnAsi, A. Neszmelyi, and H. Wagner, Tetrahedron, 36 (1980) 1261 1268. (50) V. Pozsgay, P. NBnbi, and A. NeszmBlyi, Carbohydr. Res., 90 (1981) 215-231.

CARBON-I3 N.M.R. DATA FOR OLIGOSACCHAFUDES

207

TABLE VII (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

a-Gd(l+4)[P-Gl~(l+2)]P-Rha a-Rha(1-3)a-Rha(l-4)a-Gal P anomer PGal a-Rha(1-3)a-Rha(l-+2)a-Rha a-Rha(1-3)a-Rha(1-3)a-Rha a-Rha(l-4)a-Rha(1-3)P-Rha a-Rha(ld3)P-XyI(l44)a-Rha(1-2)Xylol

100.5 104.4 93.3 103.2 101.2 93.2

69.3 74.1 82.5 71.0 70.6 69.9

70.1 76.9 72.4 71.0 79.0 69.1

69.9 70.2 81.4 72.9 72.2 70.2

71.4 76.4 72.4 69.9 69.6 69.9

61.5 61.4 17.9 17.4 17.4 69.3

42

97.4 102.7 102.4 93.4 102.8 102.5 94.6 102.8 102.5 94.1 102.4 106.4 102.4 64.7

72.7 71.0 70.0 79.6 71.0b 70.ab 72.0 71.0b 70.gb 71.6 71.7b 76.0 72.3* 83.2"

73.6 71.2 78.4 70.8 71.1b 79.0 78.5 71.1b 79.0 81.8 72.5b 83.9 71.7b 71.gb

69.6 73.0 72.2 73.4 73.0 72.2 72.4 73.0 72.2 72.6 73.9 69.8 81.5" 72.5"

74.2 69.7 69.7 69.1 69.9" 69.7" 69.2 69.9' 69.7" 73.0 69.1 67.1 68.0 62.6

67.7 17.8 17.6 17.6 16.7 17.5 17.5 16.7 17.5 17.5 18.2d

48

43

50 50 51

18.€id

"Data for related compounds are given in Refs. 43, 47, 48, and 50. Assignments may have to be reversed.

b-d

VIII TABLE '%-N.rn.r. Data for Oligorners of Xylose"

Compound

C-1

C-2

C-3

C-4

C-5

References

97.8 90.9 99.0 98.2 105.9 93.1 104.9 96.5 100.0 93.6

72.7 77.1 72.7 79.4 74.3 81.9 74.3 82.9 72.8 70.8

74.2 72.5 74.2 75.6 76.7 73.0 76.7 74.5 74.3 80.1

70.7 70.7 70.7 70.0 70.4 70.4 70.4 70.4 71.1 70.6

62.7 62.1 62.7 66.2 66 2 61.7 66.2 66.2 62.7 62.4

52

Disaccharides a-Xyl(l-2)a-Xyl a-Xyl(l-2)P-XYl p-XyI(l-+2)a-Xyl P-Xyl(1-2)a-XY 1 a-Xyl(l+S)a-Xyl

52 52 52

52 (continued)

(51) M. Becchi, M. Bruneteau, H. Pontagnier, and G. Michel, Plantu Med., 42 (1981) 265-267. (52) E. Petrakovhand P. KovaE, Chern. Zoesti, 35 (1981) 551-566.

KLAUS BOCK et al

208

TABLE VIII (continued) Compound

C-1

C-2

C-3

C-4

C-5

References

a-Xyl(1-3)P-XYl /?-Xyl(l+3)a-Xyl /.-Xyl(l+3)-

100.0 97.9 104.7 93.3 104.7 97.6 101.4 93.2 101.4 97.7 102.7 92.8 102.7 97.3

72.8 73.8 74.6 72.1 74.6 74.9 72.9 72.4b 72.9 75.1 73.7 72.3b 73.7 74.9

74.3 82.7 76.8 82.9 76.8 85.3 74.2 72.gb 74.2 76.1 76.5 71.gb 76.5 74.9

71.1 70.6 70.4 68.9 70.4 68.9 70.6 79.3 70.6 79.3 70.1 77.5 70.1 77.3

62.7 66.2 66.3 62.1 66.3 65.5 62.8 61.3 62.8 65.5 66.1 59.8 66.1 63.9

52

102.7 102.5 92.8

73.6 73.6 72.2b

76.5 74.5 71.8b

70.0 77.2 77.2

66.1 63.8 59.7

97.3

74.8

74.8

77.2

63.8

102.7 102.5 102.5 92.8

73.5 73.5 73.5 72.2b

76.4 74.5 74.5 71.8b

70.0 77.2 77.2 77.2

66.1 63.8 63.8 59.7

97.3

74.7

74.7

77.2

63.8

102.5 102.5 102.5 102.5 92.8

73.5 73.5 73.5 73.5 72.2b

76.4 74.5 74.5 74.5 71.8b

70.0 77.2 77.2 77.2 77.2

66.1 63.8 63.8 63.8 59.7

97.3

74.7

74.7

77.2

63.8

P-XYl a-Xyl(1-4)a-Xyl a-Xyl(1-4)P-XYl p-Xyl(1-4)a-Xyl P-Xyl(l-+4)P-XYl

52 52 52 52 12",52,53 12',52,53

Trisaccharides P-Xyl(1-4)p-Xyl(1-4)a-Xyl P anomer P-XYl

12O.53

Tetrasaccharides p-Xyl(1-4)P-Xyl(1-4)P-Xyl(1-4)a-Xyl /3 anomer P-XYl

12",53

Pentasaccharides p-Xyl(1-4)P-Xyl(1-4)P-Xyl(1-4)p-Xyl(1-4)a-Xyl p anomer

P-XYl

~

12O.53

~~~

Data for related compounds are given inRef. 53. Assignments may have to be reversed.

(53) J. Hirsch, P. KovBL., and E. PetrBkovB, Curbohydr. Res., 106 (1982) 203-216.

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

209

IX TABLE %-N.m.r. Data for Oligosaccharides Containing Amino or Acetamido Groups" Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

104.1 91.6 104.1 96.2 98.6 89.7 98.6 93.4 102.9 91.8 102.9 96.2 103.3 92.2 103.3 96.6

72.3 55.1 72.3 57.6 56.8 55.0 56.8 57.5 57.0 55.0 57.0 57.5 53.6 51.5 53.6 55.0

73.9 70.6 73.9 74.9 72.8 68.8 72.8 71.2 74.9 70.7 74.9 73.6 72.2 68.5 72.2 72.2

69.9 80.1 69.9 79.7 70.5 77.4 70.5 77.5 71.1 81.3 71.1 80.9 69.0 69.7 69.0 69.0

76.6 71.5 76.6 76.1 77.2 70.9 77.2 75.4 77.3 71.4 77.3 75.9 76.3 70.3 76.3 75.1

62.3 61.3 62.3 61.3 61.1 60.9 61.1 61.1 62.0 61.5 62.0 61.5 62.2 69.0 62.2 69.0

23

100.1 101.4 91.9 101.4 102.3 92.1

70.0 77.3 54.2 69.8 77.9 55.5

70.4 72.5 72.5 71.2 72.0 70.8

73.3 69.1 69.5 73.3 70.8 77.7

67.3 75.9 75.9 68.5 76.8 70.8

15.9 61.8 61.2 16.8 62.7 61.6

96.5 104.3 102.8 92.0

57.9 72.4 56.6 55.0

75.1 74.0 74.0 70.7

77.5 70.0 79.7 81.2

76.7 76.2 76.8 71.4

61.6 62.3 62.0 61.5

96.3

57.6

73.6

80.8

76.0

61.5

Disaccharides P-Gal(l-+4)a-GlcNAc P-Gal(l-+4)P-GIcNAc P-GlcN(1 4 4 ) a-GlcN P-GlcN(1-4)P-GlcN P-GlcNAc(1+4)a-GlcNAc P-GlcNAc(1 4 4 ) P-GlcNAc P-GalNAc(1-6)a-GalNAc j?-GalNAc(l+6)P-GalNAc

23 54 54 23',41,55 23',41,55 41 41

Trisaccharides a-Fuc(1-2)P-Gal(l-+3)a-GkNAc (Y-Fuc(1-2)P-Gal( 1-4)a-GlcNAc P anomer P-GlcNAc /%Gal(144)P-GIcNAc(1-4)a-GlcNAc P anomer P-GlcNAc

56 57

23

(continued)

(54) S. Tsukadaand Y.Inoue, Curbohydr. Res., 88 (1981) 19-38. (55) H. Sait6, T. Mamizuka, R. Tabeta, and S. Hirano, Chem. Lett.,(1981) 1483-1484. (56) A. S. Shashkov, N. P. Arbatsky, V. A. Derevitskaya, andN. K. Kochetkov, Curbohydr. Res., 72 (1979) 218-221. (57) P. R.Rosevear, H. A. Nunez, and R. Barker, Biochemistry, 21 (1982) 1421-1431.

210

KLAUS BOCK et a[.

TABLE IX (continued) Compound a-Man(1-3)p-Man(1-4)a-GlcNAc P anomer P-GIcNAc

C-1

C-2

C-3

C-4

C-5

C-6

References

101.5 103.8 92.0

71.5 71.8 55.2

82.0 71.9 70.6

67.4 68.4 80.9

77.7 74.9 71.6

62.4 62.6 61.7

58

96.4

57.7

73.8

80.5

76.1

61.8

Data for other derivatives are given in Ref. 23.

TABLE X l3C-N.rn.r. Datasg for Some (1ul)-Linked Hexopyranoses Containing One Amino Group' Compound

C-1

C-2

C-3

C-4

C-5

C-6

a-Gal(1-1)a-GlcN a-CaI(l4)p-GlcN p-Gal(l-1)a-GlcN P-Gal(l-1)P-GIcN a-Glc(l-1)a-GlcN a-Glc(l-1)P-GlcN P-ClC(1-1)a-GlcN /3-Glc(l-l)P-GIcN a-Man(l-1)a-GlcN a-Man(l-1)P-CICN P-Man(l-1)a-GkN P-Man(1-1)P-GlcN

94.1 94.8 101.6 104.8 104.6 102.1 101.1 101.1 94.0 94.8 101.4 105.0 104.0 102.1 100.5 101.4 95.5 96.0 102.6 104.2 101.2 101.6 97.9 100.8

70.3 55.9 70.3 57.9 72.0 56.4 71.4 57.1 72.0 55.9 72.5 57.8 74.2 56.3 73.8 57.1 71.3 55.9 70.8 57.6 71.6 56.1 71.7 57.1

71.0 75.1 70.3 76.6 74.1 75.0 73.9 76.5 73.4 74.9 73.8 76.7 77.3 74.8 77.4 76.8 71.7 75.3 71.3 76.5 74.0 74.9 73.9 76.4

69.0 71.2 69.5 70.8 69.6 70.9 69.7 70.8 70.8 70.8 70.6 70.6 70.8 70.6 70.8 70.8 68.0 71.0 67.7 70.8 67.8 70.8 67.9 70.7

72.6 73.6 72.9 77.5 76.5 73.6 76.5 77.4 73.6 73.6 73.8 77.5 76.6 74.2 76.9 77.3 74.6 74.1 74.7 77.5 77.6 73.8 77.6 77.6

62.4 61.8 62.3 61.9 62.2 61.8 62.2 61.8 61.7 61.i 61.5 61.8 61.8 61.7 61.9 61.9 62.2 61.9 61.9 61.9 62.2 61.7 62.2 61.7

~

a

~~~~

At pD -8.5. Data for acidic solutions are also given in Ref.

59. (58) H. A. Nunez, F. Matsuura, and C. C. Sweeley, Arch. Biochem. Biophys., 212 (1981) 638-643. (59) S. Koto, S. Inada, and S.Zen, Bull. Chem. SOC.Jpn., 54 (1981) 2728-2734.

CARBON- 13 N.M.R. DATA FOR OLIGOSACCHARIDES

21 1

TABLE XI 13C-N.m.r.Data for Glycosides of Oligosaccharides Containing Simple Aldohexoses" Compound

C-1

C-2

C-3

C-4

C-5

C-6

104.1 103.2 101.4 100.4 103.1b 103.zb 104.5 100.0 99.0 105.0 105.0 100.0 101.1 104.4 103.9 104.5 104.0 104.5 103.0 100.1 102.6 101.0 101.0 101.8 100.3 101.8 104.7 102.1 101.3 100.1 102.9

73.8 79.3 69.3b 69.5b 71.2 73.0 74.1 69.6 73.0 79.0 74.4 81.7 74.3b 74.6 74.6 74.2 74.0 74.0 71.7 79.3 70.3 69.8 70.7 71.4 70.8 70.8 75.2 71.4 71.1 72.8 71.1

73.6 73.6 70.0' 71.9 73.0 74.9' 76.4b 80.4 74.2 75.8 77.1 73.3 74.6 77.8 77.2 75.9 77.2 71.0* 71.7 70.8 70.6 78.5 71.4 70.7 71.sb 71.5b 77.3 71.8 71.1 73.9 70.9

69.5 69.6 69.9' 79.8 68.9 78.9 70.1 67.9 71.3 70.8 71.3 71.3 70.9 78.7 71.2 80.3 71.0b 71.2" 67.8 67.8 67.0b 66.4b 70.7 74.5 67.7 67.4 71.0 82.5 72.8 70.4 72.9

76.1 75.9 71.9 70.1' 75.5 74.7' 76.2" 71.1 73.0 77.1 77.1 72.5 73.4b 76.1 77.5 76.4 77.2 76.1 74.1b 73.4b 73.6" 73.0' 74.0 71.4 73.6 71.6b 77.3 68.3 69.5 71.1 69.7

61.7 61.7 61.5 61.5 61.2 60.5 61.6 61.9 61.9 62.5 62.2 62.2 62.3 62.3 62.4 61.8 62.5 70.0 61.8" 61.9' 61.1 61.1 61.3 61.3 61.8 66.5 62.1 18.1 17.4 68.8 17.8

C-7

References

Disaccharides P-Gal(142)/?-GalOMe @-Gal(1 4 4 ) a-GalOMe /?-Gal(1-4)/?-GlcOMe p-Glc(1+3)a-GalOMe a-Glc( 142)0-GlcOMe P-Glc( 1-2)a-GlcOMe a-Glc( 1-4)/?-GlcOMe p-Glc( 1-4)P-GlcOMe /?-Glc(1+6)/?-GlcOMe a-Man( 142)a-ManOMe a-Man( 1 4 3 ) a-ManOMe a-Man( 1+4)a-ManOMe a-Man( 1-6)a-ManOMe P-Glc(l+4)a-RhaOMe a-Rha(1 4 6 ) a-GlcOMe a-Rha( 1 4 2 ) -

61 57.7 62 56.1 9,11",24,62 57.3 63 8 58.9 8 56.2

n

58.7 8,11,64' 58.9 8 58.6 65d 55.7 65d 55.0 65d 55.0 65d 55.7 46 55.9 48 66',67 (continued)

(60) V. K. Srivastava and C. Schuerch, Carbohydr. Res., 106 (1982) 217-224. (61) R. Eby and C. Schuerch, Carbohydr. Rex, 92 (1981) 149- 153. (62) D . D. Cox, E. K. Metzner, L. W. Cary, and E. J. Reist, Carbohydr. Res., 67 (1978) 23-31. (63) Y.V. Wozney, L. V. Backinowsky, and N. K. Kochetkov, Curbohydr. Res., 73 (1979) 282- 286. (64) F. Balza, N. Cyr, G . K. Hamer, A. S . Perlin, H. J. Koch, andR. S.Stuart, Curbohydr. Res., 59 (1977) c 7 - c l l . (65) T. Ogawa and K. Sasajima, Carbohydr. Res., 97 (1981) 205-227. (66) A . Liptak, A. Neszmblyi, and H. Wagner, Tetrahedron Lett.,(1979) 741 -744. (67) K. Bock, S. Josephson, andD. R.Bundle,]. Chern. Soc.,Perkin Trans. 2, (1982) 59-70.

KLAUS BOCK et al.

212

TAEILE XI (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

C-7

a-RhaOMe P-Rha(1-2)a-RhaOMe a-Rha(1-3)a-RhaOMe a-D-Rha(1-3)a-D-RhaOR" P-Rha(1-3)a-RhaOMe a-Rha(l+4)a-RhaOMe p-Rha(l-4)a-RhaOMe

100.5 99.7 99.7 102.9 101.6 103.4 100.6 98.4 101.8 103.0 102.1 101.6 101.8

79.0 70.8 78.6 71.0 70.8 71.5 71.5 69.3 71.6 71.7 71.9 70.5b 71.7

70.9 73.7 73.7 71.1 78.8 71.5 79.6 73.8b 78.7 71.8 72.4 73.8" 70.Sb

73.1 73.1 72.1 73.0 72.2 73.4 72.7 73.Jb 72.1 73.2 81.1 73.4" 83.7

69.2 73.5 69.7 69.6 69.4 70.3 70.3 73.1b 68.5 70.6 68.2 73.0' 68.0

17.7 17.gb 17.7b 17.8 17.8 17.8 17.8 18.0' 17.9' 18.0 18.7 17.8' 17.7'

1O4.gb 72.5 103.3b 81.0' 103.4 81.1" 101.3 69.5 103.gb 71.8' 104.2b 73.1 103.2 73.5b 103.2 73.gb 99.6 70.8

73.8 73.4 73.4 70.1' 76.3h 75.4h 76.3" 76.3" 82.5

69.3 69.5 69.5 69.9' 78.3 79.7 69.9 69.9 68.1

76.5 75.9 75.9 71.9' 73.8' 75.7' 75.9' 75.9" 71.1

61.9' 61.7' 61.6' 57.9 61.5% 61.2' 61.0g 58.0 61.1 61.1 68.9 55.6

References 68

56.0 66",67 69 68 55.9 17 55.9 47.68' 56.0

Triraccharides P-Gal(l42)P-Gal(l42)P-GalOMe a-GaI(1-4)P-Gal(1-4)P-GlcOMe P-Glc(l43)[P-Glc(l+G)]a-GlcOMe

61 62

19

a Data for related compounds are given in Ref. 60. b*c Assignments may have to be reversed. Data for higher oligomers are given in Ref. 65. R = 1-deoxyglycerol-1-yl. f-' Assignments may have to be reversed.

TAEILE XI1 W-N.m.r. Data'O for Solutions in MepSO-d6of Methyl Glycosides of Ohgosaccharides Containing Galactose and Glucose Residues Compound

C-1

C-2

C-3

C-4

C-5

96.5 96.5 105.6

68.7 76.2 70.7

69.2 72.4 72.2

68.3 69.9 68.0

70.9 71.5 75.0

C-6

C-7

60.7 60.3 60.4

54.3

-~

Disaccharides a-Gal(1-2)a-GlcOMe P-Gal(1-2)-

(68) T. Iversen and D. R. Bundle,]. Org. Chem., 46 (1981) 5389-5393. (69) Yu. A. Knirel, A. S. Shashkov, B. A. Drnitriev, N.K. Kochetkov, N.V.Kasyanchuk,and I. Ya. Zhakhrova, Btoorg. Khim., 6 (1980) 1851-1859. (70) A. Temeriusz, B. Piekarska, J. Radomski, and J. Stepinsky, Carbohydr. Res., 108 (1982) 298-301.

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

213

TABLE XI1 (continued) Compound a-GlcOMe a-Gal(1-3)a-GlcOMe P-Gal(1-3)a- GI c0M e

C-2

C-3

C-4

C-5

C-6

C-7

98.8 99.4" 99.8" 104.4 98.9

81.7 69.4b 70.1 70.8 70.3

72.8 69.5b 82.5 72.3" 84.9

69.6 68.9' 68.7' 68.2b 68.3b

71.7 71.1 72.1 75.2 72.2"

60.5 60.4 60.4 60.4 60.4

54.2

95.7 103.3 95.7 104.4 103.6 98.7 103.8 104.5 98.9

68.8 70.7 72.9 70.8 71.0 78.4 70.9 70.9 70.5

68.8 72.9 80.6 73.3 73.3 82.7 73.2 73.2 84.8

68.4 68.4 68.4 68.2 68.2 68.6 68.0 68.0 68.0

70.9 75.4 72.1 75.1 75.3 72.1 75.4 74.9 70.5

60.2 60.2 60.3 60.4 60.4 60.4 60.2 60.2 68.0

C-1

54.4 54.1

Trisaccharides a-Gal(1-2)[/?-Gal(1+3)]a-GlcOMe P-Gal(l-2)[/?-Gal(1-3)]a-GlcOMe p-GaI(1-3)[/?-Gal(l+6)]a-GlcOMe 4-c

54.3

54.1 54.3

Assignments may have to be reversed. TABLE XI11 '3C-N.m.r. Data for Methyl Glycosides of Oligomers of Xylosd

Compound

C-1

C-2

C-3

C-4

C-5

99.1 105.4 103.7 104.9 100.1 105.3 104.8 104.9 101.5 105.2 103.1 105.1

72.7 78.5 74.7 81.8 72.9 72.7 74.6 73.7 73.0 74.1 74.0 74.0

74.2 75.5 76.8 76.4 74.3 82.9 76.9 85.3 74.4 76.0 76.9 75.0

70.7 70.7 70.4 70.2 71.0 70.6 70.4 69.0 70.7 79.4 70.4 77.7

62.6 66.1 66.3 65.9 62.7 66.2 66.4 66.0 62.9 65.4 66.5 64.1

105.5 101.8

75.1 82.0

76.8 76.5

70.6 70.3

66.5 66.2

C-6

References

Disaccharides a-Xyl(l-2)/?-XylOMe /?-XyI(l-2)/?-XylOMe cu-Xyl(l-3)P-XylOMe P-XyI( 1-3)/?-XylOMe a-Xyl(l-4)/?-XylOMe /?-Xyl(1 4 4 ) /?-XylOMe

52.71"

58.5 52,71"

58.1 52,71' 58.4 52,71" 58.4 52,71" 58.4 52,71',72 50.4

Trisaccharides

/?-Xyl( 1-+2)P-Xyl(14 4 ) -

71 (continued)

(71) P. KovaC, J. Hirsch, A. S. Shashkov, A. I. Usov, andS. V. Yarotsky, Carbohydr. Res., 85 (1980) 177-185. (72) P. Kovaf and J. Hirsch, Curbohydr. Res., 100 (1982) 177-193.

KLAUS BOCK et al.

214

TABLE XI11 (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

P-XylOMe P-Xyl(1-2)[P-XyI(1+4)]P-XylOMe a-Xyl(l+3)P-Xyl(l+4)P-XylOMe P-Xyl(1-3)P-XyI(1-4)P-XylOMe P-Xyl(1-3)[P-Xyl(1+4)]P-XylOMe b-Xyl(1-4)p-XyI(l+4)P-XylOMe

105.2 103.9 103.6 105.3 100.1 103.3 105.1 104.8 103.0 105.1 104.2 102.4 104.5 103.1 103.0 105.1

74.2 74.8 74.4 81.9 72.8 72.6 74.1 74.7 73.8 74.2 74.2 73.5 73.6 74.0 74.1 74.1

75.1 77.2 77.2 75.0 74.3 82.6 75.1 77.0 84.9 75.1 76.6 76.6 80.8 76.9 75.0 75.0

78.0 70.8 70.8 77.9 70.9 70.6 77.8 70.5 69.0 77.7 70.4 70.4 74.2 70.4 77.7 77.7

64.1 66.8 66.8 63.9 62.7 66.1 64.2 66.5 66.2 64.2 66.3 66.3 63.4 66.5 64.3 64.1

58.4

104.0 102.5 102.4 105.1 103.1 103.0 103.0 105.1

74.1 73.6 73.6 74.1 74.1 74.1 74.1 74.1

76.6 76.6 80.6 75.0 76.9 75.0 75.0 75.0

70.4 70.4 74.3 77.5 70.4 77.6 77.6 77.6

66.3 66.3 63.7 64.0 66.5 64.2 64.2 64.2

102.9 102.9 102.9 102.9 105.0

74.0 74.0 74.0 74.0 74.0

76.8 74.9 74.9 74.9 74.9

70.4 77.6 77.6 77.6 77.6

66.5 64.2 64.2 64.2 64.2

103.1 103.1 103.1 103.1 103.1 105.3

74.0 74.0 74.0 74.0 74.0 74.0

76.9 75.0 7.5.0 75.0 75.0 75.0

70.5 77.7 77.7 77.7 77.7 77.7

66.5 64.2 64.2 64.2 64.2 64.2

References 71

58.4 71 58.4 71 58.5 71 58.2 71",72 58.3

Tetrasaccharides P-Xyl(l+B)[P-XyI(l+4)]p-Xyl(1-4)/I-XylOMe P-Xyl(1-4)P-Xyl(l+4)/3-Xyl(1-4)P-XylOMe

71 58.4 72 58.5

Pen tasaccharide P-Xyl(l+4)P-Xyl(l+4)P-Xyl( 1-4)p-Xyl(l+.I)P-XylOMe

72

58.4

Hexasaccharide p-XyI(l+4)P-XyI( 1+4)P-Xyl(l+4)P-Xyl(1-4)P-Xyl(1-4)P-XylOMe a

72

58.5

Data for related compounds are given in Refs. 71 and 72.

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

TABLE XIV I3C-N.m.r.Data7Pin CDCI, of Methyl Glycosides of Peracetates of Oligomers of Xylose" C-1

C-2

C-3

C-4

C-5

96.2 101.7 99.7 102.0

70.8 71.5 70.4 71.3

69.1 73.1 70.4 72.6

69.1 73.9 68.4 75.1

58.7 63.8 61.6 62.9

96.3 100.3 101.9 99.5 100.5 101.9

71.2 70.8 71.2 70.2 71.1 71.1

69.0 72.6b 72.ab 70.2 71.9 72.5

69.0 73.2 75.6 68.2 74.2 75.6

58.8 62.9 63.4 61.6 62.5 62.9

71.1 70.8 70.8 71.1 70.4 71.0 71.0 71.3

69.0 72.7 72.0 72.7 70.4 72.0 72.0 72.7

69.0 73.2 75.0 75.6 68.4 74.3 74.9 75.7

58.8 62.9 62.7 63.4 61.6 62.6 62.6 63.0

71.2 70.8 70.8 70.8 71.2 70.4 71.0 71.0 71.0 71.3

69.0 72.6 71.9 71.9 72.6 70.4 72.1 72.1 72.1 72.7

69.0 73.2 74.9 74.9 75.6 68.4 74.3 74.9 74.9 75.7

58.8 63.0 62.5 62.5 63.5 61.7 62.6 62.6 62.6 62.9

71.2 70.8 70.8 70.8 70.8 71.2

69.0 72.7 72.0 72.0 72.0 72.7

69.0 73.2 75.0 75.0 75.0 75.6

58.8 63.0 62.5 62.5 62.5 63.4

Compound

C-6

Disaccharides Cr-XyI( 1-4)D-XyIOMe p-xyl(l-4)/I-XylOMe

56.9 56.8

Trisaccharides c~-XyI(l+4)p-Xyl( 1 4 4 ) 8-XylOMe p-Xyl(1-4)p-Xyl(l-4)P-XylOMe

56.8

56.9

Tetrasaccharides Ci-XyI(1+4)p-XyI(1-4)p-Xyl( 1-4)P-XylOMe P-Xyl(l+4)p-XyI(l-'4)P-XyI(1+4)/I-XylOMe

96.4 100.3b 100.4b 101.9 99.7 100.6 100.6 102.1

56.9

57.0

Pentasaccharides cr-Xyl(l+4)p-XyI(1-4)P-Xyl(1-4)p-XyI(1-4)P-XylOMe p-Xyl(1-4)/l-Xyl(l-+4)p-XyI(1-4)p-XyI( 1-4)p-XylOMe

96.3 100.3 100.3 100.3 101.9 99.7 100.6 100.6 100.6 102.1

56.8

57.0

Hexasaccharides c~-Xyl(l+4),&XyI(l-+4)p-Xyl(l+4)p-XyI( 1-4)p-XyI(1-4)p-XylOMe

96.3 100.3 100.3 100.3 100.3 101.9

56.8

(continued)

215

KLAUS BOCK et 01.

216

TABLE XIV (continued) ~~

~

Compound

C-1

C-2

C-0

C-4

C-5

C-6

B-XyI(1-4)p-Xyl(1-4)/?-Xyl(l+4)b-XyI(l+4)p-XyI(1-4)P-XylOMe

99.5 100.4 100.4 100.4 100.4 102.1

70.5 71.0 71.0 71.0 71.0 71.3

70.5 71.9 71.9 71.9 71.9 72.7

68.4 74.3 75.0 75.0 75.0 75.7

61.7 62.6 62.6 62.6 62.6 63.1

57.0

a Data for other derivatives are also given in Ref. 72. Assignments may have to be reversed.

TABLE XV W-N.m.r. Data for Solutions of Peracetates of Glycobioses in CDCl, ~

Compound

~~

~~

C-1

C-2

C-3

C-4

C-5

C-6

References

95.4 93.8 100.9 92.8 96.0 91.8 101.0 92.1 96.5 92.0 99.; 92.4

71.1 74.4 70.1 76.6 71.1 69.5 70.3 70.1 70.9 70.2 70.7 70.1

69.1 72.3 70.7 72.8 69.2 73.7 70.8 76.9 69.1 73.2 70.8 72.2

69.0 69.0 68.8 68.7 69.0 69.0 68.6 68.6 69.0 73.0 68.6 74.3

58.7 63.1 61.7 63.0 59.0 61.6 61.8 62.4 58.8 64.3 61.8 63.5

92.2

70.1

70.1

68.7

68.4

61.9

74

96.9

71.3

72.9

68.7

72.2

62.0

75

96.3 91.9 101.0 91.8 95.9 91.4

71.3b 71.2b 71.2 71.8b 70.2 71.1

69.4 77.7 73.0 78.9 69.4 75.1

68.0 68.8 68.1 67.6 68.2 72.9

68.5 72.9 71.gb 72.8 68.7 73.1

61.3 61.6 61.8 61.8 61.7 62.7

74

Pentoses" a-Xyl(l+2)-

P-XYI B-Xyl(1-2)-

P-XYI a-Xyl( 1-3)B-XYI /?-Xyl(l+3)-

B-XYl a-Xyl( 1-4)D-XYl p-Xyl( 1-4)P-XYI

73 73 73 73 73 73

Hexoses a-Glc( 1-1)a-Clc B-Glc(1-1)a-Glc a-Glc( 1-3)P-Glc p-Glc( 1 4 3 ) P-Glc a-Glc(1-4)B-Glc

74 74

(73) J.-P. Utille and P. J. A. Vottero, Carbohydr. Res., 98 (1981) 1-9. (74) D . Y. Gagnaire, F. R. Taravel, and M. R. Vignon, Curbohydr. Res., 51 (1976) 157168.

(75) G. Schilling, W.-D. Henkels, K. Kunstler, and K. Weinges, Ann., (1975) 230-239.

CARBON-1 3 N.M.R. DATA FOR OLIGOSACCHARIDES

217

TABLE XV (continued) Compound

C-1

P-GIc(l+4)a-Glc P-GIc(l-t4)-

101.0 89.0 100.7 p-Glc 91.6 CY-CIC(~-G)- 96.0 P-Glc 91.6 P-Gk(l46)100.7 p-Glc 91.7

C-2

C-3

C-4

C-5

C-6

References

71.7 69.4 71.6 72.4 70.8 70.3 71.1 70.4

73.0 69.4 72.9' 70.5' 70.1 72.9 72.9 73.0

67.9 76.1 67.9 75.9 68.5 68.6 68.6 68.6

72.0 70.8 72.0 73.6 67.6 73.5 72.1 74.0

61.6 61.5 61.7 61.7 61.9 66.2 62.0 67.6

74 74 74 74

'

Data for other derivatives of xylobioses are also given in Ref. 73. Assignments may have to be reversed. TABLE XU l3C-N.rn.r.Data for Glycosides Related to Blood-group Determinants

Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

100.1 102.4 100.4 101.8 96.2 103.4 104.3 101.7 103.8 101.9 94.7 103.4 103.9 104.5

69.2 77.4 68.9 56.9 69.1 70.2 71.6 55.5 71.8 56.0 50.5 70.2 57.5 56.4

70.5 74.7 70.3 73.5 70.1 78.3 73.4 83.4 73.4 73.0 68.5 77.9 77.1 86.7

72.8 69.9 72.7 78.5 70.0 65.7 69.4 69.7 69.4 79.6 69.3 65.7 70.4 69.0

67.6 75.8 67.9 76.1 71.7 75.6 76.1 76.2 76.2 75.6 71.8 75.8 76.4 76.4

16.3 61.8 16.1 61.0 61.8 61.8 61.8 61.7 61.8 61.1 61.9 61.8 61.5 61.6

76

99.4 94.1 102.4 100.3 101.0 102.7 100.3

68.7 69.0 73.2 69.0 77.5 55.7 69.1

70.4 70.7 77.6 70.3 74.4 78.4 70.6

72.9 70.2 64.4 72.7 70.0 69.6 72.6

67.5 72.0 75.3 67.3 75.9 76.3 67.7

16.3 62.2 61.7 16.1 62.0 61.7 16.1

Disaccharides ~~-Fuc(l+2)/3-GalOR" a-Fuc(1-4)P-GlcNAcOR" &Gal( 1-3)P-GalOR" p-Gal( 1-3)/3-CIcNAcORa p-Gal(l-4)P-GIcNAcOR" a-GalNAc(1-+3)P-GaIOR" P-GlcN( 1-3)P-GIcNOMe

76 76 76 76 76 76

Trisaccharides ~ - F u c1-2)( [a-Gal(1+3)]P-GalOR" ~ - F u c1-2)( p-Gal( I +3)P-GlcNAcOR" @-Fuc(l+2)-

76

76

77 (continued)

(76) R. U. Lemieux, K . Bock, L. T. J. Delbaere, S. Koto, and V. S. Rao, Can. 1.Chem.. 58 (1980) 631 -653. (77) 0 . Hindsgaul, T. Norberg, J. Le Pendu, and R. U. Lemieux, Carbohydr. Res., 109 (1982) 109-142.

KLAUS BOCK et al.

218

TABLE XVI (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

p-Gal(1-4)p-GlcNAcOR4 a-Fuc(l-+2)[a-GalNAc(1-+3)] KalOR" p-Cal( 1 4 ) [a-Fuc(144)]/I-GlcNAcOR" /I-GaI(1 4 4 ) Ia-Fuc(l+3)]~-GlcNAcOR"

101.3 101.9 99.2 92.3 102.3 103.7 98.9 101.8 102.9 99.6 102.0

77.4 65.3 68.7 50.3 72.9 71.5 68.8 56.7 71.6 68.8 56.9

74.4 73.1 70.6 68.8 76.9 73.3 70.1 77.1 74.5 70.3 76.4

70.0 77.3 72.9 69.4 64.0 69.3 72.9 73.4 69.4 73.0 73.6

76.0 76.1 67.6 72.0 76.0 75.7 67.7 76.4 76.0 67.7 76.0

61.9 61.1 16.2 62.2 61.8 62.5 16.3 60.8 62.5 16.3 60.9

100.4 101.4 98.6 102.7 100.4 101.1 99.6 101.8

69.1 77.4 68.7 56.4 68.3 77.4 68.8 56.9

70.3 74.5 70.0 75.6 70.7 74.4 70.3 76.5

72.8 69.0 72.8 73.1 72.6 69.6 73.0 74.2

67.0 75.6 67.8 76.2 67.7 75.7 67.7 75.7

16.1 62.4 16.2 60.6 16.4 62.3 16.3 60.8

References

76 76

77

Tetrasaccharides cu-Fuc(l+2)/I-Ga1(1+3)[a-Fuc(1-4)jD-GIcNAcOR" a-Fuc(l+2)p-Gal(1-4)]a-Fuc(143)]/3-GlcNAcOR"

~~

76

77

~

R = (CH,),CO,Me.

TABLE XVII W-N.m.r. Datass for Oligosaccharides, Related to Blood-group Determinants, Reduced to Alditols at the Formerly Reducing Residue Compound

c-1

c-2

C-3

C-4

C-5

C-6

104.4 61.2

71.7 52.0

73.1 77.0

69.1 69.9

75.6 69.8

61.6 63.5

101.9 102.9 98.6 104.9 61.3

70.1 79.9 52.2 54.8 70.4 52.1

70.3 72..5 75.3 71.6 72.7 77.1

72.9 69.3 70.1 71.0 77.6 70.2

69.1 75.6 69.6 72.7 76.1 69.8

16.2 61.6 63.4 60.8 61.1 63.4

100.0 101.0

69.7 77.1

70.3 72.4

73.0 69.0

67.5 75.9

15.9 61.5

Disaccharides /3-Gal(1+3)GalNAcol Trisaccharides ~ - F u c1-2)( p-Cal( 1-3)GalNAcol a-GlcNAc( 1+4)/3-Ga1(1-+3)-

GalNAcol

61.1

Hexasaccharides a-Fuc( 1-2)/3-Gal(l-4)-

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

219

TABLE XVII (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

/?-GlcNAc(l+6)la-Fuc(l42)/?-Gal(I +3)]GalNAcol a-Fuc( 14 2 ) /?-Gal(1 4 4 ) /?-GlcNA~(l+6)[a-GlcNA~(l+4)/?-Gal(1-+3)]GalNAcol a-GlcNAc(l44)/?-Gal(1 4 4 ) /?-GlcNAc(l46)[ ~ - F u 1c4( 2 ) /?-Gal(1 4 3 ) j GalNAcol a-GlcNAc(l+4)/?-Gal(1 4 4 ) /?-Gl~NAc(l-+6)[a-GlcNAc(l+4)/?-Gal(1+3)]GalNAcol

102.2 101.8 102.8 60.9 100.1 101.0 102.0 98.5 105.0 61.1 98.9 104.0 102.2 101.9 103.0 61.1 98.9 104.0 101.9 98.6 105.1 61.0

55.9 70.0 79.9 52.1 69.8 77.0 55.9 54.7 70.4 52.1 54.7 70.3 56.0 70.0 80.1 52.3 54.7 70.4 56.0 54.7 70.4 52.1

74.2 70.3 72.5 75.1 70.4 72.4 74.2 71.5 72.7 77.0 71.4 72.7 73.3 70.3 72.7 75.1 71.5 72.7 73.3 71.5 72.7 77.0

77.1 73.0 69.0 71.6 73.1 68.9 77.0 71.0 77.8 71.5 71.2 77.4 79.6 73.0 69.2 71.6 71.2 77.3 79.5 71.2 77.6 71.1

75.6 69.1 75.6 69.7 67.9 76.0 76.0 72.7 76.0 69.5 72.7 76.4 75.5 69.1 75.6 69.7 72.7 76.4 75.5 72.7 76.1 69.6

60.9 16.1 61.5 68.5 16.0 61.8 60.9 60.9 61.1 68.2 60.8 61.1 60.8 16.1 61.7 68.5 60.9 61.0 60.9 60.9 71.0 68.2

TABLE XVIIl W-N.m.r. Data for Oligosaccharides of Glycoproteins Compound

C-2

C-3

C-4

C-5

C-6

100.3 56.2 98.7 77.0 99.8 56.0 101.0 67.9 101.8 55.9 100.8 71.1 101.9 55.9 101.1 70.8"

74.2 70.8" 74.1 79.0 73.8 69.7 74.1 70.3"

70.4" 68.2 70.2 65.5 70.1 77.8 70.1" 69.4

76.6 73.9 76.3 72.8 76.2 69.7 76.1 71.6

61.5 62.4 61.0 61.2 60.7 61.0 61.1 67.1

104.3 100.9

73.9 73.9

69.9

76.6 76.1

62.3 61.4

C-1

C-7

References

Disaccharides b-GlcNAc(1 4 2 ) a-ManOMe ~-GICNAC( 143)a-ManOMe b-GlcNAc(1-4)a-ManOMe /?-GlcNAc(1 4 6 ) a-ManOMe

78 78 55.1 78 55.1 78 55.0

Trisaccharides /?-Gal(1+4)/?-GlcNAc(1 4 2 ) -

72.3 56.3

80.0

79 (continued)

(78) T. Ogawa and S. Nakabayashi, Agric. Biol. Chem., 45 (1981) 2329-2335. (79) K. Bock, J. Amarp, and J. Lonngren, Eur. J . Biochem., 129 (1982) 171-178.

KLAUS BOCK et al.

220

TABLE XVIII (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

a-Man /.?-Gal(1-4)D-GlcNAc(1-6)a-Man /.?-Gal(1-4)/.?-GkNAc(1+6)/.?-Man /.?-GlcNAc(1-2)[/.?-GkNAc(1+4)]a-ManOMe /.?-GkNAc(1-3)[/.?-GIcNAc( 1+6)]a-ManOMe

92.5 104.3 102.9 95.4 104.3 102.8 95.1 99.9 101.9 98.1 99.6 102.0 101.1

78.7 72.3 56.4 71.7 72.3 56.4 72.5 55.7 55.9 78.4 55.9 55.9 71.5

70.7 73.9 73.7 72.0 73.9 73.7 74.4 73.8 73.7 68.7 74.1 74.1 78.8

68.8 69.9 80.0 70.5 69.9 80.0 70.5 70.3 70.0 76.2 70.3 70.3 65.3

73.4 76.7 76.1 73.6 76.7 76.1 76.4 76.2 76.2 71.4 76.1 76.1 67.7

62.9 62.3 61.5 68.2 62.3 61.5 68.0 61.0 61.0 61.0 61.0 61.0 69.3

104.6 72.5 103.2 56.5 104.6 72.5 101.2 56.5 92.8 78.9 104.8 72.3 102.8 56.6 104.8 72.3 101.1 56.8 92.4 79.4

74.1 73.4 74.1 73.2 71.7 73.9 73.6 73.9 73.4 69.0

70.1 70.1 80.1 70.7 69.9 80.0 69.9 80.1 79.7

76.9 76.3 76.9 76.3 73.9 76.7 76.1 76.7 76.1 72.3

62.4 61.6 62.4 61.5 69.1 62.4 61.4 62.4 61.4 62.9

104.8 101.4 101.4 104.8 101.4 98.7 96.1

74.2 73.7 71.3 74.2 73.7 71.1 79.9

70.2 80.3 69.0 70.2 80.3 69.0 67.4

77.0 76.5 75.1 77.0 76.5 74.6 72.0

62.6 61.7 63.2 62.6 61.7 63.2 67.3

C-7

References 79 79 78 78

55.0

Pen tasaccharides D-Gal(1-4)/.?-GIcNAc(1-6)[/.?-Gal(1-4),BGIcNAc( 1-2))a-Man /.?-Gal(1-4)/.?-GlcNAc(1-4)[/.?-Gal(1-4)/.?-GlcNAc(1-2)]a-Man

80.2

79

79

Heptasaccharide /.?-Gal(l-4)/.?-GlcNAc(1-2)a-Man(l43)[/.?-Gal(1-4)P-GlcNAc(l+2)a-Man(1+6)]a-Man

72.7 56.5 78.3 72.7 56.5 78.1 70.2

79

Assignments may have to be reversed.

TABLE XIX W-N.m.r. Data for Oligosaccharides Related to Those of Salmonella Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

102.5 103.2 93.5

71.6 71.6 70.4

71.6 70.2 78.5

67.8 82.5 68.9

74.3 69.3 71.6

61.9 18.1 61.9

44

Trisaccharides a-Man(1-4)a-Rha(1-3)&-Gal

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

221

TABLE XIX (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

97.5 101.9 103.5 93.6

72.4 71.9 71.5 70.4

81.8 74.4 72.1 78.7

69.7 68.1 80.9 69.8

76.2 77.5 69.2 71.9

61.9 62.3 18.2 62.3

97.6 102.7 98.1 93.3

72.5 71.5 72.4 68.4

82.0 71.5 72.6" 77.2

68.9 67.8 82.5 67.5

76.4 74.4 72.4" 71.5

62.2 62.0 18.1 62.3

97.6 103.6 99.5 93.6

72.2 71.3 72.5 70.4

80.5 71.3 74.3 78.4

66.9 73.2 70.8 69.8

76.1 70.4 73.1 70.8

62.1 17.9 61.8 67.7

97.6

72.5

81.7

68.9

75.3

67.6

101.8 102.9 101.3 93.6

71.9 71.5 73.7 69.9

74.4 71.5 73.8 78.3

68.2 79.4 70.5 76.5

77.5 68.2 73.3 72.8

62.4 18.2 61.5 61.8

97.8

73.3

80.8

76.5

76.1

61.6

102.0 103.5 104.5 101.8 95.2

71.8 71.5 71.5 71.8 72.5

74.5 71.9 81.8 74.5 71.9

68.1 80.8 69.7 68.1 80.8

77.5 69.0 76.3 76.6 68.7

62.2 18.2 62.0 69.7 18.5

4s

104.7 103.2 104.4 103.5 102.1 97.5 101.9 103.5 104.4 101.9 103.5 93.6

75.1 71.4 71.4 75.1 71.4 72.7 71.8 71.5 71.5 71.8 71.5 70.3

77.1 71.4 82.0 77.2 71.6 80.1 74.4 71.9 81.8 74.4 71.9 78.7

70.8 82.2 69.6 70.8 82.8 69.8 68.1 80.8 69.8 68.1 80.8 69.8

77.1 68.9 76.6 76.3 68.9 76.6 77.5 69.1 76.3 76.3 69.1 71.9

62.1 18.2 62.1 69.8 18.3 62.1 62.2 18.2 62.2 70.3 18.2 62.2

46

97.7

72.6

81.8

69.1

76.6

62.2

P anomer /?-Gal P-Man(1-4)a-Rha(1-3)a-Gal P anomer /?-Gal a-Man(1-4)P-Rha(l-3)a-Gal P anomer /?-Gal a-Rha(1-3)[a-Glc(1+6)]@-Gal /? anomer P-Gal

45

44

44

Tetrasaccharide P-Man(l-4)cr-Rha(l43)[a-Glc(1-4)ja-Gal P anomer P-Gal

26

Pentasaccharide P-Man(1-4) a-Rha(1-3)P-GaI(l-+6)P-Man(l44)a-Rha Hexasaccharides P-Glc( 1-+4)a-Rha(1 4 3 ) /?-Gal(1-+6)/I-Gk( 1-+4)a-Rha(1-3)P-Gal /?-Man(l+4)a-Rha(1 4 3 ) /3-GaI(1-6)P-Man(1 4 4 ) a-Rha(1-3)@-Gal /? anomer P-Gal

45

(continued)

KLAUS BOCK et al

222

TABLE XIX (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

101.8 103.5 104.3 101.8 103.5 104.3 101.8 103.5 93.5

71.7 71.4 71.4 71.7 71.4 71.4 71.7 71.4 70.3

74.3 71.7 81.9 74.3 71.7 81.9 74.3 71.7 78.6

68.1 80.8 69.7 68.1 80.8 69.7 68.1 80.8 69.7

77.4 69.1 76.3 76.3 69.1 76.3 76.3 69.1 71.7

62.2 18.3 62.1 70.3 18.3 62.1 70.3 18.3 62.2

45

97.6

72.5

81.9

69.1

76.6

62.2

Nonasaccharide P-Man(1-4)a-Rha(l-3)P-Gal(l-6)P-Man(1-4)a-Rha(1-3)P-Gal(l-6)P-Man(l-t4)a-Rha(1-3)a-Gal /? anomer /?-Gal

Assignments may have to be reversed.

XX TABLE W-N.m.r. Data for Glycosides of Oligosaccharidesof Salmonella Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

101.7 99.5 100.8 99.5 102.4 99.4 101.5 102.0 96.5 101.8 102.4 100.5 103.7 98.8 102.1 99.1

64.8 71.2 68.4 71.1 68.6 70.9 64.7 71.2 64.3 68.0 71.3 71.5 71.5 70.1b 69.7 80.5

34.3 79.5 35.8 79.5 34.6 79.1 34.1 79.6 34.1 77.2 71.5 70.3 71.5 78.6 70.2 71.6

69.6 67.0 71.0 67.0 68.1 67.0 69.6 67.2 69.7 66.4 68.4 82.4 73.4 68.7 70.2 68.0

68.1 74.9 70.4 74.9 71.4 74.9 68.0 73.9 67.8 73.8 74.0 67.5 70.6b 72.3 72.2 73.2

16.6 61.9 17.6 61.8 17.9 61.8 16.5 62.1 16.5 62.2 61.7 17.8 18.0 62.2 62.1 61.7

80

102.5 103.1

71.6 71.6

71.6 70.1

67.7 82.5

74.2 69.3

61.9 18.1

Disaccharides a-Abe(l-3)a-ManOPh a-Par(l-3)a-ManOPh a-Tyv(1-3)a-ManOPh a-Abe(1-3)a-ManOMe a-Col(1-3)a-ManOMe a-Man(1-4)a-RhaOR" a-Rha(l-3)a-GalOPh a-Gal(1-2)a-ManOR"

80 80 80 80 80 80 80

Trisaccharides a-Man(l-4)a-Rha(1 4 3 ) -

80

(80) K. Bock, M. Meldal, D. R. Bundle, T. Iversen, B. M. Pinto, P. J. Garegg, I. Kvanstrom, T. Norberg, A. A. Lindberg, and S . B. Svensson, Carbohydr. Res., 130 (1984)35-53.

CARBON-13 N.M.R. DATA FOR OLIGOSACCHARIDES

223

XX (continued) TAHLE ~~

~

Compound

C-1

C-2

C-3

C-4

C-5

C-6

a-GalOPh a-Cal( 1 4 2 ) la-Par(l-3)Ia-ManOR" a-Gal(1 +2)la-Tvv(1-3)la-ManOR"

98.7 101.7 100.2 99.0 101 .9 101.9 99.0

70.1 69.5 67.9 79.4 69.6 68.2 79.5

78.5 70.2 35.4 78.2 70.1 34.2 78.2

68.5 70.2 70.7 67.6 70.2 67.8 67.1

72.8 72.2 70.0 73.8 72.2 71.1 73.8

61.9 62.1 17.3 61.6 62.1 17.6 61.6

101.6 102.6 103.3 98.8 104.6 103.4 104.4 104.4 100.8 102.5 103.3 98.7 102.5 102.5 103.3 98.7

64.8 71.7 71.7 68.6 75.1 71.3 71.3 74.3 68.3 71.6 71.6 68.6 68.7 71.5 71.5 68.7

34.3 79.6 70.3 78.5 77.0 71.3 81.8 77.2 35.7 79.6 70.3 78.5 34.7 79.3 70.4 78.5

69.3 67.0 82.9 70.3 70.7 82.3 69.6 70.6 70.9 67.0 82.8 70.3 68.2 67.1 82.9 70.3

68.1 74.5 69.3 73.0 77.0 68.9 76.3 76.1 70.3 74.5 69.3 73.0 71.6 74.6 69.3 73.0

16.8 62.0 18.3 62.1 61.8 18.2 62.1 69.6 17.6 61.6 18.2 62.1 18.1 61.9 18.3 62.1

References 80

80

Tetrasaccharides a-Abc(l43)a-Man( 1+4)a-Rha(l43)a-GalOPh B-GIc(1 4 4 ) a-Rha(1-3)P-GaI(1+6)/3-GlcOMe a-Par(1-3ja-Man(l+4)a-Rha(1-3)a-GalOPh ~~-Tyv(l-3)a-Man(1-4)a-Rha(1-3)a-GalOPh a

80

46

80

80

R = (CH,),C02Me. Assignments may have to be reversed.

XXI TABLE W-N.m.r. Data for Oligosaccharides Related to Those of Shigellaflexnerf Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

103.5 99.5 102.2 101.4

56.7 79.8 70.9 56.2

74.5 69.5 71.1 82.4

70.6 73.2 72.8 69.7

76.6 68.7 69.5 76.8

61.5 17.5 17.4 61.8

67

103.5 101.9 99.4

56.7 79.4 79.7

74.6 70.7 69.5

70.7 73.1 73.1

76.6 70.0 68.6

61.6 17.6' 17.7"

Disaccharides /3-GkNAc(l42)a-RhaORb a-Rha(1-3)/3-GkNAcORb

67

Trisaccharides /3-GkNAc(l+2)a-Rha(l-2)a-RhaORb

67

(continued)

224

KLAUS BOCK et a1 TABLE XXI (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

References

a-Rha(143)P-GlcNAc(1 4 2 ) a-RhaORb a-Rha(l-3)a-Rha( 1-3)P-GlcNAcORb a-Rha(1-2)a-Rha(1-3)a-RhaORb

102.1 102.8 99.6 103.1 102.1 101.5 103.1 101.6 100.6

71.1 56.5 79.6 71.0 71.0 56.1 70.9 79.2 69.8

71.1 82.0 69.2 71.0 79.0 82.6 71.0 70.9 78.3

72.6 69.8 73.2 72.9 72.1 69.8 72.9 73.1 72.6

69.5 76.7 68.7 69.5 69.8 76.8 70.0 69.6 68.6

17.4 61.6 17.6 17.6' 17.4" 61.7 17.6 17.5 17.7

67

103.0 101.6 102.1 101.5 105.0 102.6 102.6 93.4 103.5 101.8 101.6 100.4 103.1 102.0 102.9 99.4

71.0 78.8 70.9 56.1 72.0 70.6 70.7 79.8 56.7 79.7 79.1 69.9 71.0 71.0 56.4 79.6

71.2 70.9 78.2 82.5 73.4 80.7 78.6 70.7 74.5 70.7 70.7 78.3 71.4 78.8 82.3 69.2

72.9 73.0 72.5 69.8 69.4 71.9 72.1 73.3 70.8 73.1 73.0" 72.6 72.9 72.1 69.8 73.2

70.0 69.3 70.0 76.8 75.8 69.7 70.0 69.2 76.6 70.0" 69.5 68.8 69.5 69.9 76.7 68.8

17.5 17.5 17.2 61.7 61.8 17.7 17.5 17.4 61.5 17.5 17.5 17.5 17.4 17.3 61.5 17.4

67

67

Tetrasaccharides a-Rha( 1-2)a-Rha(l-3)a-Rha(1-3)P-GlcNAcORb p-Gal( 1-3)a-Rha( 1-3)a-Rha( 1 4 2 ) a-Rha /?-GlcNAc(1-2)a-Rha(1-2)a-Rha(1+3)a-RhaORb a-Rha(1-3)a-Rha( 1-3)P-GlcNAc(1-2)a-RhaORb

Data for related compounds are given in Ref. 43. R = (CH,),CO,Me. ments may have to be reversed.

67

43

67

32

Assign-

TABLE XXII W-N.m.r. Data for Gangliosides" Compound

C-1

C-2

C-3

C-4

C-5

C-6

C-7

C-8

C-9

174.8 103.8

100.6 70.0

40.8 76.6

68.9 68.3

52.9 75.6

73.8 61.8

69.1

72.5

63.6

174.6 103.9 103.6

100.9 70.4 74.0

40.8 76.6 75.4

69.2 68.6 80.0

53.0 76.1 75.8

74.0 62.0 61.3

69.4

72.7

63.8

Disaccharide a-NeuAc(2-3)/?-GalORb Trisaccharide a-NeuAc(2-3)P-Gal(l-4)P-GlcORb

CARBON-I 3 N.M.R. DATA FOR OLIGOSACCHARIDES

225

TABLEXXII (continued) Compound

C-1

C-2

C-3

C-4

C-5

C-6

103.6 174.7 103.6 103.6

53.3 102.6 70.8 74.2

70.8 37.8 75.3 75.3

68.9 69.2 78.3 79.6

75.6 52.8 74.9 75.3

61.4 73.9 62.1 61.1

105.6 103.4 174.5 103.4 103.4

71.7 52.8 102.5 70.7 74.1

73.6 81.5 37.8 75.2 75.2

69.2 68.8 69.2 78.1 79.8

75.7 75.7 52.0 75.0 75.7

61.4 61.4 73.6 61.8 61.0

105.5 103.4 174.2 174.2 103.4 103.4 174.9 105.4 103.5 174.8 103.5 103.5

71.6 53.3 101.4 102.0 70.7 73.5 100.7 71.4 52.9 102.6 70.8 73.8

73.5 81.2 41.4 39.4 75.1 75.1 40.7 76.5 81.8 38.0 75.2 75.2

69.2 68.9 69.2 69.2 78.9 79.4 69.2 69.2 68.5 69.2 78.3 79.9

75.7 75.7 52.8 52.2 75.1 75.7 52.9 75.5 75.5 51.9 75.2 75.5

61.8 61.5 73.5 73.5 61.8 61.0 73.8 61.9 61.5 73.8 61.9 61.0

174.6 105.3 103.5 174.1 174.1 103.5 103.5

100.7 70.4 53.3 101.5 101.5 70.4 73.7

40.7 76.5 81.1 41.6 40.0 75.0 75.0

69.1 69.1 68.5 69.1 69.1 78.8 79.4

52.9 75.5 75.5 52.9 52.2 75.0 75.5

73.7 61.8 61.5 73.7 73.7 61.8 60.8

C-7

C-8

C-9

69.2

72.5

63.9

69.2

72.9

64.0

69.2 69.2

72.6 77.7

63.6 62.4

69.2

72.7

63.6

69.2

73.0

63.6

69.1

72.5

63.5

69.1 69.1

72.5 77.1

63.5 62.5

-

Tetrasaccharide P-GalNAc(1 4 4 ) [a-NeuAc(2-3)]P-GaI(1-4)P-GlcORb Pentasaccharide p-Gal( 143)P-GalNac(1 4 4 ) [a-NeuAc(2+3)I/.?-Gal(1 4 4 ) P-GlcORb Hexasaccharides P-Gal(1-3)P-GaINAc(1 4 4 ) [a-NeuAc(2 4 8 ) a-NeuAc(2-3)j/?-Gal(1 4 4 ) P-GIcORb a-NeuAc(2 4 3 ) P-Gal(1 4 3 ) P-GalNAc(1 4 4 ) [a-NeuAc(2-3)]P-Gal(1 4 4 ) P-GlcORb Heptasaccharide a-NeuAc(243)P-Gal(1 4 3 ) P-GalNAc(1 4 4 ) [a-NeuAc(2+8)a-NeuAc(2-+3)]P-Gal(1-4)P-GlcORb

Data from Ref. 8 1. R = ceramide.

(81) L. 0. Sillerud, R. K. Yu, and D. E. Schafer, Biochemistry, 21 (1982) 1260-1271.

This Page Intentionally Left Blank

ADVANCES I N CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY, VOL. 42

KETONUCLEOSIDES

BY KOSTASANTONAKIS lnstitut de Recherche3 Scientifiques sur le Cancer du C.N.R.S.,B.P. 8,94800 VillejuiJ France I. Introduction. . . . . . . ... 11. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Efficient Oxidative Systems in Nucleoside Chemistry 2. Ketopentose Nucleosides . . . . . . . . 3. Ketohexose Nucleosides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Epoxyketonucleosides 5. Unsaturated Keto 111. Stability . . . . . . . . . . 1. AcidicMedia.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Alkaline Media . IV. Structure and Spectroscopic Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Infrared Spectra . . . . 2. lH-N.m.r. Spectra. . . 3. Ultraviolet Spectra. . V. Stereospecific Reduction. . . . . . . . . . . VI. Nucleophilic Additions . 1. To Ketohexose Nucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 2. To Unsaturated Ketohexose Nucleosides. . . . VII. Biological Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

231

237

245 249

257 258 261

I. INTRODUCTION Ketonucleosides constitute a class of nucleosides containing in the sugar moiety a keto group that results from the oxidation ofan asymmetric carbon atom. This article considers all so-called ketonucleosides deriving from aldosyl derivatives, and excludes those in which the keto group of the sugar moiety is involved in the nucleosidic bond (for example, nucleosides of psicose'), which have occasionally been termed ketonucleosides. Keto derivatives of aldopentofuranosylpyrimidines(3and 6 ) were synthesized2 in 1967 by oxidation of the partially protected uridine deriva( 1 ) W. Schroeder and H. Hoeksema, J Am. Chem. SOC., 81 (1959) 1767- 1768. (2) A. F. Cook and J. G. Moffatt,J. Am. Chem. SOC., 89 (1967) 2697-2705.

227

228

KOSTAS ANTONAKIS

tives 1 and 4 (by way of 2 and 5, respectively), whereas ketopyranosylnucleoside 8 derived from a hexose derivative (7) was obtained3 in

1

2

3

4

5

6

Me

Me

I

I

PhHC

OH 7

8

( 3 ) K. Antonakis and F. Leclercq, C. R . Acad. Sci., Ser. C, 271 (1970) 1197-1200; Bull. SOC. Chirn. Fr., (1971) 2142-2144.

229

KETONUCLEOSIDES

1970 by direct oxidation of the sterically hindered aldohexopyranosylpurine. The synthetic procedure currently used consists in the direct oxidation of an isolated hydroxyl group in the sugar moiety of suitably protected nucleosides. In the meantime, the synthesis of some keto derivatives of aldopentose nucleosides by selective-elimination processes has been rep~rted.~-~

+ J ( o=c-NH

o=c-NH OH

I I

CH,

HOH,C-CH

I

CHNH, I

7%

NH I

o=c

NH

I

II

CH,-N-CNH, I Me

CH,NHMe

Blasticidin S

Gougerotin

9

10

CH

uoU

Me,N

OH

Amicetin 11

(4) T. Sasaki, K. Minamoto, and K.Hattori,]. Org. Chern., 38 (1973) 1283-1286. (5) T. Sasaki. K. Minamoto, K. Hattori, andT. Sugiura,]. Curbohydr.Nucleos. Nucleot., 2 (1975) 47-62. ( 6 ) Y. Mizuno, Y. Watanabe, K. Ikeda, and J. A. McCloskey, Chem. Phann. Bull., 23 (1975) 1411-1416.

230

KOSTAS ANTONAKIS

Although keto derivatives of pentose nucleosides have, in most cases. proved to be unstable, keto derivatives of aldohexose nucleosides have, over the past few years, given rise to a new and interesting chemistry; moreover, they provide an advantageous and often unique route to complex nucleosides of biological interest. In particular, nucleosides of branched-chain sugars and amino sugars have been obtained by direct nucleophilic attack, as well as unsaturated ketoglycosyl nucleosides, which have been synthesized for the first time. The biological interest of such molecules was first suggested3e7by the hypothesis that the lateral chain of certain natural nucleoside antibiotics, such as 9, 10, and 11, might be formed by nucleophilic addition to a carbonyl group present in the sugar moiety of the nucleoside. Since then, Suhadolnik and coworkers8 hypothesized that a keto derivative of an aldopentosyladenine is the key intermediate in the biosynthesis ~-P-Darabinofuranosyladenine (14, see Scheme l), and Seto and coworkerse

Y2

N H 2

I

HO 12

13

14

Scheme 1

postulated a ketonucleoside as the key intermediate in the formation of 9, the antibiotic Blasticidin S (see Scheme 2). Biological results published during the past ten years concerning the in uitr010-13 and in u i ~ o ’ ~activity J~ of ketonucleosides themselves have (7) K. Antonakis and M.-J. Arvor, C. R. Acad. Sci.,Ser. C, 272 (1971) 1982-1984. (8) P. B. Farmer, T. Uematsu, H. P. C. Hogenkamp, and R. J. Suhadolnik,]. Biol. Chem., 248 (1973) 1844-1847. (9) H. Seto, K. Furihata, and H. Yonehara, J. Antibiot., 29 (1976) 595-596. (10) K. Antonakis andI. Chouroulinkov, C. R. Acad. Sci., Ser. D, 273 (1971) 2661-2663. (11) K. Antonakis and I. Chouroulinkov, Biochem. Pharmacol., 23 (1974) 2095-2100. (12) C. Aujard, Y. Mouk, E. Chany-Morel, and K. Antonakis, Biochem. Phamacol., 27 (1978) 1037-1042. (13) P. Allard, T. H. Dinh, C. Gouyette, J. Igolen, J.-C.Cherman, and F. Bard-Sinoussi, J . Med. Chem., 24 (1981) 1291-1297. (14) I. Chouroulinkov and K.Antonakis, C. R. Acad. Sci., Ser. D,285 (1977) 1021 - 1024. (15) K. Ant0nakis.T. Halmos, J. Bach, andI. Chouroulinkov, Eur.].Med. Chem.,25 (1980) 237-240.

231

KETONUCLEOSIDES o-Glucose

+

cytosine

OH 15

OH 16

9

Scheme 2

shown that most of them are biologically active. In particular, numerous unsaturated ketonucleosides exhibit interesting antileukemic activity. The object ofthis article is to discuss all known aspects ofthe chemistry and biochemistry of this new class of nucleosides, emphasizing the advantages of using them as synthetic intermediates, and also as models for biological studies. A brief review on keto derivatives of aldohexosyl nucleosides appeared16 in 1975, and a plenary lecture entitled “Recent Developments in the Chemistry of Ketonucleosides” has been given.”

11. SYNTHESIS 1. Efficient Oxidative Systems in Nucleoside Chemistry Several reviews describing methods for oxidation of carbohydrates leading to the corresponding aldehydo and keto sugars have appeared.18-22Also, a short discussion published in 1970 was devoted to (16) K . Antonakis, Chimia, 29 (1975) 59-62. (17) K . Antonakis, Proc. Znt. Round Table, Nucleos. Nucleot. Biol. Appl., 4th, Antwerp, (1981) 1-23. (18) K . Heyns and H. Paulsen, Ado. Carbohydr. Chem., 17 (1962) 169-221. (19) J. S. Brimacombe, Atigew. Chem, Znt. Ed. Engl., 8 (1969) 401-409. (20) R . F. Butterworth and S. Hanessian, Synthesis, 2 (1971) 70-88. (21) R. L. Angustine and D . J. Trecker (Eds), Oxidation, Vol. 2, Dekker, New York, 1971, pp. 1-64. (22) G. H. Jones and J. G. Moffatt, Methods Carbohydr. Chem., 6 (1972) 315-352.

232

KOSTAS ANTONAKIS

the oxidation of nucleosides to the glycosyluronic acid n u c l e o ~ i d e sIt. ~ ~ is, however, noteworthy that, among the various methods described, only certain reagents could be successfully used in the synthesis of ketonucleosides. Most of the known processes either catalyze further reactions, leading to glycosylic cleavage, or proved to be completely inefficient in the nucleoside field. The dimethyl sulfoxide (Me,SO) - dicyclohexylcarbodiimide (DCC) method described by Pfitzner and M ~ f f a t opened t~~ a route to keto derivatives of aldopent~sylpyrimidines,~~~~ as well as to many keto derivatives of h e ~ o s y l - p u r i n e s and ~ ~ ~- p~ y~ r~i -m~i~d i n e ~ . ~ An ~ . ~alternative ~J~ proposed by Swern and coworkers,34requiring the presence of oxalyl chloride as an activating agent, has been used to prepare 4-keto-Zyxohexose C-nucleo~ides.’~ Me,SO -acetic anhydride35 and Me,SO - phosphorus p e n t a ~ x i d e ~ ~ have also been used to obtain so-called 2’- and 3’-ketouridines2 The use of ruthenium t e t r a ~ x i d in e ~a ~mixture of carbon tetrachloride, aqueous sodium hydrogencarbonate, and 5% aqueous sodium metaperiodate permitted the oxidation of a partially protected xylofuranosyladenine to the corresponding (2-keto-threo-pentofurano~yl)adenine,~~ as well as of that of a pyrimidine nucleoside derived from ~ - r h a m n o s e . ~ ~ Activated manganese dioxide, which is generally used for the selective oxidation of allylic alcohols, permitted the synthesis of an unsaturated (3-keto-urubino-hexopyranosyl)pyridine.~3 New oxidative systems for alcohols, involving molecular sieve-assisted (23) C. A. Dekker and L. Goodman, in W. Pigman and D. Horton (Eds), The Curbohydrutes, Vol. IIA, Academic Press, New York, 1970, pp. 1-68, see pp. 38-39. (24) K. E. Pfitzner and J. G. Moffatt,]. Am. Chem. Soc., 85 (1963) 3027-3028; 87 (1965) 5661 -5670. (25) U. Brodbeck and J. G. Moffatt, ]. Org. Chem., 35 (1970) 3552-3558. (26) K. Antonakis and J. Herscovici, C. R. Acud. S c i . , Ser. C, 274 (1972) 2099-2101. (27) J. Herscovici and K. Antonakis,]. Chem. SOC., Perkin Trans. 1, (1974) 979-981. (28) K. Antonakis, Curbohydr. Res., 24 (1972) 229-234. (29) K. Antonakis and M. Bessodes, Curbohydr. Res., 30 (1973) 192-195. (30) J. Herscovici and K. Antonakis,]. Curbohydr. Nucleos. Nucleot., 4 (1977) 65-76. (31) T. Halmos and K. Antonakis, Curbohydr. Res., 68 (1979) 61 -69. (32) J. Herscovici, A. Ollapally, and K. Antonakis, C. R. Acud. Sci.,Ser. C, 282 (1976) 757-759. (33) M. Bessodes, A. Ollapally, and K. Antonakis, Chem. Commun., (1979) 835-836. (34) K. Omura and D. Swern, Tetrahedron, 34 (1978) 1651 -1660; A. J. Mancuso, D. S. Brownfain, and D. Swern,]. Org. Chem., 44 (1979) 4148-4150. (35) J. D. Albright and L. Goldman,]. Am. Chem. Soc., 87 (1965) 4214-4216. (36) K. Onodera, S. Hirano, and N. Nashimura, Curbohydr. Res., 6 (1968) 276-285. (37) V. M. Parikh and J. K. N. Jones, Can.].Chem., 43 (1965) 3452-3453; B. T. Lawton, W. A. Szarek, and J. K. N. Jones, Curbohydr. Res., 10 (1969) 456-458. (38) A. Rosenthal, M. Sprinzl, and D. A. Baker, Tetrahedron Lett.,(1970) 4233-4235. (39) M. Bessodes, These, Universite d e Paris 6, March 1978.

KETONUCLEOSIDES

233

oxidation, reported by Herscovici and A n t ~ n a k i s ~ O proved , ~ ~ to be of great advantage, especially for complex or sensitive nucleosides; ketonucleosides have been isolated from crude mixtures without further purification. The systems of molecular sieves -pyridinium chlorochromate and molecular sieves -pyridinium dichromate moreover permitted preparation, at room temperature, of the first ketoepoxy- and ketothionucleo~ides.~~ Selective oxidation of nucleosides by photolysis of pyruvic esters has also been r e p ~ r t e d .This ~ ~ .photochemical ~~ p r o ~ e d u r eis~ particu~.~~ larly useful for the oxidation of fragile molecules, and has been used to prepare protected “3’-ketothymidines.” 2. Ketopentose Nucleosides

(2-Keto- and 3-keto-pentosyl)-purines and -pyrimidines have been reported. Most of them exhibit great sensitivity toward alkaline media. 2’,5’-Di-O-trityI-3’-ketouridine (2) was obtained in 46% yield from 2’,5’-di-O-trityluridine (1) by the Pfitzner - Moffatt reagentsaZ4The same procedure applied to 3’,5’-di-O-trityluridine2 (4) gave 3’,5’-di-0trityl-2’-ketouridine (5) in 63% yield. These oxidations have also been performed2 by using dimethyl sulfoxide - acetic anhydride35 and dimethyl sulfoxide - phosphorus p e n t a ~ x i d eFree . ~ ~ 3’-ketouridine (3)and 2’-ketouridine (6)were obtained by the action of hydrogen chloride in a cold chloroform solution of tritylketouridines 2 and 5 . The DCC - Me,S024 and the Me2S0- A c methods ~ ~ have~ also~ been applied for the synthesis of 2’- and 3‘-keto derivatives of cytidine.25 N4-AcetyI-2’,5‘-di-O-tritylcytidine(17a) was oxidized by both systems to give N4-acetyl-l-(2,5-di-O-trityl-P-~-erythro-pentofuranosy1-3u1ose)cytosine (I8a) in 86 and 87% yields, respectively. In asimilar way, (19a) was performed the oxidation of N4-acetyI-3‘,5‘-di-O-tritylcytidine with Me,SO- Ac20, to give N4-acetyl-l-(3,5-di-O-trityl-P-~-erythropentofuranosyl-2-ulose)cytosine (20a) in 8 1% yield. (40) J. Herscovici and K. Antonakis, Chern. Cornrnun., (1980) 561 -562. (41) J. Herscovici, M.-J. Egron, and K. Antonakis, J. Chern. SOC., Perkin Trans. 1 , (1982) 1967-1973. J. Herscovici, J.-M. Argoullon, M.-J. Egron, and K. Antonakis, Carbohydr.Res., 112 (1983) 301-306. R. W. Binkley, D. G. Hehemann, and W. W. Binkley, Carbohydr. Res., 58 (1977) c10-c12. R. W. Binkley, D. G. Hehemann, and W. W. Binkley, J. Org. Chern., 43 (1978) 2574 - 2575. R. W. Binkley, Carbohydr.Res., 48 (1976) c l - c 3 ; J . Org. Chem., 42 (1977) 12161221. R.W. Binkley, Ado. Carbohydr. Chem. Biochern., 38 (1981) 105-193.

KOSTAS ANTONAKIS

234

HNR'

HNR'

I

I

0

170 R' = Ac, R2= Tr 17b R' = H, R2 = T r

18 a R' = Ac, €7' = Tr 18b R' = Ac, R2 = H 18 c R1 = H, R' = T r

HNR'

HNR'

I

I

R20Po---! R20P04 \r/ R20 R20

OH OH

19a R ' = Ac, RZ = Tr 19b R' = H, Rz = T r

R20

20a R1 = Ac, R2 = Tr 20b R' = Ac, R2 = H 20c R' = H , R2 = T r

The isomeric di-O-tritylcytidines 17b and 19b have been successfully oxidized by the Me,SO-DCC method to give, respectively, 1-(2,5-diO-trityl-~-~-erythro-pentofuranosyl-3-ulose)cytosine (18c) and 1-(3,5di-~-trity~-~-~-erythro-pentofuranosy~-2-u~ose)cytos~ne (20c). Treatment of 18a and 20a with anhydrous hydrogen chloride in chloroform at 0" afforded free h'4-acetyl-3'- and -2'-ketocytidines, 18b and 20b, in good yields.2s 9-(3,5-0-Isop ro p y lid en e- ~- ~- ~~~eo -pe nto fur a no s y ~ -2 -u~ o s e )a de ni ne (22) was obtained38from 9-(3,5-0-isopropy~idene-~-~-xy~ofuranosy~)adenine (21) by oxidation with ruthenium tetra~xide.~' The syntheses of both 1-(3-deoxy-~-~-g~ycero-pentofuranosy~-2u1ose)uracil (25c) and 1-(3-deoxy-~-~-g~ycero-pentofuranosy~-2u1ose)cytosine (30b) by selective elimination reactions have been rep ~ r t e d .Thus, ~ . ~ the reaction of sulfonyl derivatives of the cytosine nucleoside 26, and uracil nucleosides 23a, 23c, and 28, with sodium benzoate in N,N-dimethylformamide (DMF) leads to 3'-deoxy-Z'-ketonucleosides by way of such (presumed) unsaturated intermediates as 24a, 24c, 29a, and 29b. However, in one instance, the intermediate

KETONUCLEOSIDES

235

21 OH

22

3'-deoxy-2'-O-tosyl-2'-eno nucleoside (24a) could be isolated and chara c t e r i ~ e dAppropriate .~ detritylation or debenzylation, or both, of %a, 27a, and 30a gave the corresponding 1-(3-deoxy-P-~-glycero-pentofuranosyl-2-u1ose)uraciI (25c) and l-(3-deoxy-~-~-g~ycero-pentofuranosy~2-ul0se)cytosine (30b). 7-(3-Deoxy-5-0-trity~-a-~-g~ycero-pentofuran0

f

RocQ 230 R = Bn, R1 = OMS, R Z = OMS 23b R = Bz, R' = OMS, RZ = OMS 23c R = Bn, R' = OTs, RZ = OMS 23d R = Bz, RL = OTS, RZ = OMS M s = rnesyl TS = tosyl Bn = benzyl Bz = benzoyl

240 24 b 24 c 24d

R R R R

= = = =

ORL Bn, Bz, Bn, Bz,

RocQ 250 R = B n 25b R = Bz 25c R = H

R1 = Ts R' = Ts RL = Ms R' = Ts

N3

TroctiJ 0A

N

OMS

RocQ

26

Tr = Ph,C

0

270 R = Tr 27b R = H

KOSTAS ANTONAKIS

236

BnWQ

RWQ

OMS 29a X = SH 29b X = SCH,

28

0

30a R = Bn 30b R = H

osyl-2-u1ose)hypoxanthine(31) was also synthesizeds by a similar proceTrOCH,

31

dure. An interspatial repulsion between the ionized 2-carbonyl group of the nitrogenous base and the electron-rich, sulfonyl group seems to be the probable reason for the formation of 2’- ketonucleosides by this pro~edure.~ A photochemical process consisting in the esterification, with pyruvoyl chloride,4sof the alcohol to be oxidized, followed by photochemical reaction of the ester obtained, has been applied to some thymine nucleosides.43Derivatives (33a and 33b) of 3’-ketothymidine were prepared43 by this method. 0

.Me

0 0

II

II

CH,C-CCl hv

-

HO 32a R = Ts 32b R = Tr

33a R = TS 33b R = Tr

KETONUCLEOSIDES

237

3. Ketohexose Nucleosides This Subsection deals with the preparation of 2’-and 4’-ketohexosylpurines and -pyrimidines, which have proved to b e versatile synthetic intermediates. A 5’-keto derivative of a hexofuranose nucleoside is also described. The synthesis of epoxy-, halogeno-, unsaturated, epimino-, and thio-ketonucleosides will be developed in subsequent Subsections and Sections.

-

a. 2’-Ketonucleosides. 7 - (4,6- 0 -Benzylidene- 3-0-methyl-/?-Darabino-hexopyranosyl-2-u1ose)theophylline(8) was obtained3 in 1970 Me

7-

phHc’d \ 0 OMea

by oxidation of 7-(4,6-0-benzylidene-3-O-methyl-~-~-arabino-hexopyranosy1)theophylline (7) with Me,SO -DCC,24 whereas the Me,SO Ac,O reagent led exclusively to the 2’-(methylthio)methyl ether.47Protected 7-/?-~-fucopyranosyltheophylline (Ma) afforded the 2’-ketonucleoside2s 35a in 50% yield when treated with Me,SO-DCC. 1.r. and

34a R = R1 34b R = RZ

35a R = R1

36a R = R1

R

36b R = RZ

35b

Me

=

R2

Cl

(47) K . Antonakis and F. Leclercq, Bull. SOC. Chim. Fr., (1971) 4309-4310.

238

KOSTAS ANTONAKIS

n.m.r. spectra were used for determination of the structure. Ready hydration of 35a was observed, leading to the corresponding 2'-gern-diol. Treatment with 0.1 M hydrochloric acid gave 7-(6-deoxy-P-~-Zyxo-hexopyranosyl-2-u1ose)theophylline (36a). 6-Chloropurine ketonucleoside 35b has been obtainedes by a similar procedure from 6-chloro-9-(6deoxy - 3,4 - 0-isopropylidene -p-L - galactopyranosy1)purine (34b). Deacetonation with conc. sulfuric acid in 9 : 1 nitromethane-methanol afforded (2-ketofucosyl)chloropurine(36b). 2'-Ketofucosyl nucleosides (38a and 38b) of 5-fluorouracil and 6azauracil have been obtained39 from the partially protected L-fucosylpyrimidines 37a and 37b by oxidation with Me,SO-DCC and Ru0,CHCI,, respectively.

Me,SO-DCC

0 RuO,- CHC1, Me$-0

*

Me&-0

0 380 R = F, X = CH 38b R = H, X = N

37a R = F, X = CH 37b R = H, X = N

b. 4'-Ketonucleosides. -4'-Ketonucleosides 1 -,7 - ,and 9-linked have been by oxidation of the protected L-rhamnopyranosylpurines 40a and 40b with the Me,SO-DCC reagent. 7-(6-Deoxycu-~-lyxo-hexopyranosyl-4-ulose)theophylline (43) and its 2,3-0isopropylidene and 2,3-di-O-(trimethylsilyl) derivatives (41a and 42) have been r e p ~ r t e d .6-Chloro-9-(6-deoxy-2,3-O-isopropylidene-a~~.~~ ~-lyxo-hexopyranosyl-4-ulose)purine (41b) was isolatede6 (from isopropyl alcohol) after removal of 1,3-dicyclohexylurea.

39 R = R'

400 R = R1 40b R = R2

(48) M. Bessodes and K. Antonakis, unpublished results.

41a R = R1 41b R = R'

KETONUCLEOSIDES

239

I

Me,SiO

OSiMe,

OH

HO

42 R = R'

43 R = R'

Me

c1

I

I

1 - (6 - Deoxy - 2,3 - 0 - isopropylidene - a - L - lyxo - hexopyranosyl - 4 u1ose)thymine (45) was synthesized30 by oxidation of the protected Lrhamnosylthymine 44 with the DCC-MezSO system. The same reagent proved to be efficient in the synthesis of 1-(6-deoxy-2,3-0-isopropylidene-a-~-lyxo-hexopyranosyl-4-ulose)-5-fluororouracil~~ (47a) and of the 6-azauracil derivative 47b, respectively obtained from 46a and 46b. 0

I

I

0 , ' 0 CMe, 44

0,

45

/o

CMe,

460 R' = H,RZ = F, X = CH 46b R' = H, R2 = H, X = N

0,

/o

CMe,

470 R' = H. Ra = F. X = CH 47b R' = H, Ra = H, X = N

KOSTAS ANTONAKIS

240

The Me,SO - oxalyl chloride method34 was successfully usedI3 to obtain 2 - (6 - deoxy 2,3- 0-isopropylidene-a-L-lyxo-hexopyranosyl- 4ulose)-8-nitro-v-triazolo[ 1,S-alpyridine (49a) in 62% yield from the partially protected L-rhamnose nucleoside 48a. Treatment of 49a with formic acid gave the free ketonucleoside 50a. Hydrogenation over 10% Pd-C afforded the amino derivative 49b.

-

480 R = NO, 48bR=NH,

49a R = NO, 49b R = NH,

500 R = NO, 5Ob R = NH,

c. 5'-Ketonucleosides. -An exocyclic ketohexosylpurine has been obtained by direct oxidation of an isomeric mixture of partially protected hexof~ranosylpurines.4~ A mixture of Ns-benzoyl-9-(6-deoxy-2,3-0-isopropylidene-/3-D-allofuranosyl)adenine(51) and its C W - L - ~ U ~isomer O 52 was oxidized by the DCC- Me,SO system to give Ne-benzoyl-9-(6deoxy- 2,3 -O-isopropylidene-~-~-ribo-hexofuranosyl-5 -ulose)adenine (53), which was i ~ o l a t e d 'as ~ its (2,4-dinitrophenyl)hydrazone. HNR

HYR

52

53

(49) R . S. Ranganathan, G . H. Jones, and J. G. Moffatt,J. Org. Chm.,39 (1974) 290-298.

KETONUCLEOSIDES

241

4. Epoxyketonucleosides

~ ~ . ~ ~the reaction Epoxynucleosides 55 and 58 were ~ b t a i n e dthrough of the corresponding sulfonates 54 and 57 with sodium methoxide. Oxidation of 7-(3,4-anhydro-6-deoxy-cr-~-ta2O-hexopyranosyl)theophylline (55) with the DCC -Me2S0 reagent gave 7-(3,4-anhydro-6-deoxy-a-~Zyxo-hexopyranosyl-4-ulose)theophylline (56)as a semicrystalline material, whereas 58 could be oxidized in 90 min at room temperature by the molecular sieve - pyridinium dichromate system40 to give pure 7-(2,3anhydro-6-deoxy-~-~-Zyxo-hexopyranosyl-4-ulose)theophylline~~ (59).

54

55

HO

56

V

57

58

59

Me I

These molecules, which constitute the only keto epoxy nucleosides that have thus far been synthesized, have proved to be important, synthetic intermediates.

5. Unsaturated Ketonucleosides l.~~ ketonucleoside, The synthesis of the first r e p ~ r t e d ~unsaturated 6 la, was accomplished by acetylation of the known 7-(6-deoxy-&~-lyxo(50) J. Herscovici and K. Antonakis,]. Chem. Soc., Perkin Trum. I , (1979) 2682-2686. (51) K. Antonakis and M.-J. Arvor-Egron, Carbohydt. Res., 27 (1973) 468-470. (52) K . Antonakis, C. R. Acud. S c i . , Ser. C, 275 (1972) 1101-1103.

242

KOSTAS ANTONAKIS

hexopyranosyl-2-ulose)theophylline2*(36a), followed by elimination of the 4-acetoxyl group. In a similar manner, acetylation of 36b afforded2e 9-( 3 -O-acetyl-4,6-dideoxy-~-~-glycero-hex-3-enopyranosyl-2-ulose) -6chloropurine (61b) in 80% yield (see Scheme 3).

610 R = R1 61b R = R a

60

Me

Scheme 3

When applied to the synthesis of unsaturated 4'-ketonucleoside 66, this method did not give good results, because of the lability of the glycosylic bond. An alternative approach consisted in the oxidation of a free hydroxyl group in partially acetylated nucleosides, initiating the /3-elimination of an acetoxyl g r o ~ p .Thus, ~ ~ .treatment ~ ~ of 62 and 65 with Me,SO-DCC gave, respectively, 7-(3-0-acetyl-4,6-dideoxy-a-~-glycero-hex-3-enopyranosyl-2-ulose)theophylline (63) and 7-(3,6-di-Oacetyl- 2 -deoxy -P-~-glycerohex - 2 - enopyranosyl- 4 -dose)theophylline (66). The same procedure was used15 to obtain 7-(3-0-benzoyl-2-deoxy-6O-triphenylmethyl-~-~-glyce~o-hex-2-enopyranosyl-4-ulose) theophyl-

- $J-

"OQ

AcO

OH

62

AcO 63

(53) J. Herscovici, M. Bessodes, and K. Antonakis,]. Org. Chen., 41 (1976) 3827-3830.

KETONUCLEOSIDES

Q

-

HoQ

(I-i

-0

243

0

OAc

I AcO

OAc

64

AcO

65

66

Me I

line (68a) in 80% yield from the dibenzoate 67a. Detritylation was performed in aqueous acetic acid, leading to the unsaturated ketonucleoside 68b, isolated as crystalline material. Oxidation of the partially benzoylated 6’-deoxy nucleoside 67b with the MezSO-acetic anhydride reagent35 gave 7-(3-0-benzoyl-2,6-dideoxy-~-~-glycero-hex-2-enopyranosyl-4-u1ose)theophyline (68c) in 55% yield.15

dcqo

Me

Me

<+Ie

N,Me

HO

-

OBz

OBz

BzO

67a R = OCPh, 67b R = H

68a R = OCPh, 68bR=OH 6 8 R ~

=H

Treatment of the unsaturated C-nucleoside 69 with activated manganese dioxide afforded 2-(1,5-anhydro-2,6-dideoxy-~-erythro-hex-l-enitol-1 -yl-3-ulose)-8-nitro-u-triazolo[1,5-u]pyridine (70) in 40% yield.’3

H

O

y

-

p

6N,N

/

“095 N/N

/

HO

0 69

70

/

KOSTAS ANTONAKIS

244

The synthesis of 7-(2,3,6-trideoxy-~-~-glycero-hex-2-enopyranosyl-4u1ose)theophylline (71),which constitutes an important intermediate in the synthesis of branched-chain nucleosides (see Section VI),has been achieved42by r e a c t i ~ nof~ the ~ . ~2',3'-anhydroketonucleoside ~ 59 with sodium iodide and sodium acetate. The unsaturated halogenoketonucleosides 72a and 72b were preMe I

59

-OQ

I

I

hie 71

7 2 0 X = C1 72b X = Br

pareds0 by oxidation of the 3',4'-anhydrohexosylpurine 55, followed by the action of lithium halide on the ketoanhydro intermediate 56. Lithium chloride and lithium bromide have been used to obtain 7-(3-chloro3,4,6trideoxy -cY-~-glycerohex- 3-enopyranosyl- 2 -ulose)theophylline (72a) and 7-(3-bromo-3,4,6-trideoxy-~-~-glycero-hex-3-enopyranosyl2-u1ose)theophylline (72b) in 40 and 75% yield, respectively. The use of these compounds as intermediates in the synthesis of new nucleosides is developed in Sections V and VI. Among the unsaturated ketonucleosides may be classified the disaccharide derivative 74, which is an analog of the biologically active compound 68c. The key intermediate for the synthesis of this unsaturated ketodisaccharide nucleoside was the partially protected, disaccharide nucleoside 73, which was prepared by two separate routes.ss Treatment of 73 with the Me,SO-acetic anhydride reagent for two days at room temperature afforded 7-[2,3-di-O-benzoyl-4-0-(3-O-benzoyl-2,6-dideoxy - p - D - glycero - hex - 2 - enopyranosyl- 4 - ulose) - 6 - deoxy - p - D glycopyranosylltheophylline (74), isolated ~ r y s t a l l i n e . ~ ~

(54) H. Paulsen, K. Eberstein, and W. Koebernick, TetrahedronLett.,(1979) 4377-4380. (55) H. Paulsen and K. Eberstein, Chem. Ber., 109 (1976) 3907-3914. (56) T. Halmos, J. Filippi, J. Bach, andK. Antonakis, Carbohydr. Res., 99 (1982) 180- 188.

KFTONUCLEOSIDES

245

Me

Me

I

0

BzO

OBz

74

73

111. STABILITY Pfitzner and Moffatt noted24 that a severe limitation existed for the synthesis of ketopentose nucleosides owing to their instability in certain media, which was attributed to loss of the nitrogenous base by elimination reactionse2 The role of the reaction medium for the successful synthesis and study of the reactions of ketonucleosides has also been emphasized by Herscovici and Antonakis, who r e p ~ r t e d ' ~catalytic . ~ ~ solutions to this problem. Thus, the use of inorganic catalysts proved to be of great efficiency for the oxidation of fragile molecules, especially in the nucleoside field. Significant stability of ketohexose nucleosides in various media has facilitated their direct transformation, leading to important structures of biological interest, such as branched-chain and rare-sugar nucleosides (for example, amino-, epimino-, thio-, and trideoxy-nucleosides).

1. Acidic Media Removal of trityl groups from the tritylated k e t o n u c l e ~ s i d e 2, s ~5, ~~~ 18a, and 20a with anhydrous hydrogen chloride in chloroform at 0" afforded the free ketonucleosides 3, 6, 18b, and 20b in good yields, whereas attempted detritylation with acetic acid led to complete decomposition of the ketopentosylpyrimidines. Also, 5'-0-trityl-3'ketothymidine 33b proved to be unstable under certain mild conditions, as its chromatography on silica gel resulted43in quantitative formation of 75 and 76. In contrast, 1 -(3-deoxy-5-O-trityl-/?-~-ghjcero-pentofuranosyl-2-u1ose)cytosine (27a) could be treated with 80% acetic acid for 6 h at 50" to give ketonucleoside 27b (in 45% yield after purification5). With ketohexose nucleosides, removal of protecting groups in acidic

246

KOSTAS ANTONAKIS 0

76

76

medium was successful in all cases reported. Isopropylidene, as well as benzylidene, groups are generally removed on treatment with 0.1 M hydrochloric acid at room temperature [for example, 41a (Ref. 26) and 35a (Ref. 28)], or with 50 mM sulfuric acid in nitromethane-methanol (35b, Ref. 29). Under these conditions, no glycosylic cleavage was observed. However, attempts to achieve similar deacetalation of the 4'-ketonucleoside 41b were unsuccessfule6; this ketonucleoside was less stable than its analog 41a under the same conditions. Experiments on the stability of some ketohexosyl-purines and -pyrimidines have been reported. On treatment2e of 6-chloro-9-(6-deoxy-/.?-~lyro-hexopyranosyl-2-u1ose)purine(36b) with 0.1 M hydrochloric acid at loo", free 6-chloropurine was detected after 5 min, and hydrolysis was complete after 2 h. Under similar conditions 7-(6-deoxy-/.?-~-lyxohexopyranosyl-2-u1ose)theophylline(36a) was unaffected2s during 22 h. Unsaturated ketonucleosides have been shown to be remarkably stable under acidic conditions. 7-(3-O-Acetyl-4,6-dideoxy-/3-~-glycerohex-3-enopyranosyl-2-ulose)theophylline(6la) proved to be stable in 0.1 M hydrochloric acid, as no glycosylic cleavage had occurred51 after 20 h. Similarly, no decomposition was observed when 7-(3,6-di-O-acetyl-2-deoxy-/3-~-gZycero-hex-2-enopyranosyl-4-ulose)theophylline (66) was treated with 0.1 M sulfuric acid during 48 h at room temperature, and attempted, ionic hydrogenation with triethylsilane - trifluoroacetic acid failed.31 2. Alkaline Media

The use of ketonucleosides as synthetic intermediates is based on their stability, particularly in alkaline media. Unlike ketopentosylpyrimidines, which are rapidly decomposed, ketohexose nucleosides appear to be convenient intermediates for syntheses of branched-chain, deoxy, and rare sugar nucleosides. The action of alkali on the 3'- and 2'-ketouridines 3 and 6 has been studied,2 and it was shown that almost instantaneous elimination of uracil took place either in 0.01 M sodium hydroxide or in buffer of pH 10.8. A similar alkaline instability of the trityl 2'-ketone 5 was also observed, and free uracil could be readily identified. Under the same conditions, the di-0-trityl 3'-ketone 2 was more stable towards alkali as the rate of

KETONUCLEOSIDES

247

release of uracil in 0.1 Mmethanolic sodium hydroxide could be readily monitored. This relative stability, probably due to the presence of the 2’and 5’-O-trityl groups, may be a consequence of steric distortion of the furanose ring, assuming a conformation that does not favor p-elimination.2 Attempts to alkylate the group of ketouridines under alkaline conditions led to extensive decomposition of the ketonesa2 As for the 2‘- and 3’-ketouridine~,~ both 2‘- and 3’-ketocytidine derivatives have been found to be very unstable under basic condition^.^^ Di-0-trityl-3’-, as well as 2‘-, ketones 18a, 18c, 20a, and 2042, were completely degraded within 10 min at room temperature in 0.01 Mmethanolic sodium hydroxide, whereas the detritylated nucleosides 18b and 20b decomposed instantly under the same conditions. The behavior of 5’-O-trityl-3’-ketothymidine (33b) in pyridine at 25“ has also been reported43;rapid formation of free glycal75 and thymine was detected. Several studies on the action of alkalis on ketohexose nucleosides have shown that ketohexosyl-purines or -pyrimidines react slowly, and often without concomitant glycosylic cleavage. Treatment of (6-deoxy-3,4-0-isopropylidene-~-~-Zyxo-hexopyranosyl-2-u1ose)theophylline (35a) with 10 mM sodium methoxide led to slow decomposition of the compound.28Under the same conditions, the corresponding 6-chloropurine derivative 36b underwent2g complete glycosylic cleavage within 30 h. In 0.1 M methanolic sodium hydroxide, the 6-chloropurine ketonucleoside 41b was cleaved after 30 min, whereas the theophylline derivative 4 l a was u n a f f e ~ t e d . ~The ~ * ~stability ’ of the theophylline ketonucleoside 41a may be explained by the establishment of a hydrogen bond between the 4’-gem-diol system (hydrate form of 41a) and the 2-keto group of theophylline. The assigned 4C, (L) conformation would facilitate this bonding, whereas it could not occur with the 6-chloropurine derivative regardless of its conf~rmation.~’ It has also been observed32that, under similar conditions, 1-(6-deoxy2,3-O-isopropylidene-cy-~-Zy~o-hexopyranosyl-4-ulose)thymine (45) is more stable than 41b, but less stable than 41a. Other pyrimidine keto-nucleosides, such as 47a and 47b, could be treated with acetic anhydride - pyridine without glycosylic cleavage.33 The stability of 47a and 47b in this medium permitted elaboration of a new, mild, regioselective synthesis of enol acetates. The reaction of 4’-ketonucleosides of uracil with acetals of N,N-dimethylformamide led,33after addition of acetic anhydride, to the corresponding enol acetate-nucleosides 77a and 77b. Study of the stability of these ketonucleoside derivatives in alkaline media has shown that they can readily revert to the starting ketone.

KOSTAS ANTONAKIS

0, / o CMe, 77a R = F, X = CH 77b R = H, X = CN

Furthermore, by saponification in vitro, the original activity of the ketonucleoside could be restored. Significant stability has also been observed when ketohexose nucleowere treated with sodium sides,57as well as ketopento~ylpyrimidines,~*~~ borohydride (see Section V). Some examples of the behavior of unsaturated ketonucleosides under alkaline conditions have also been reported. The enol acetate 61a is more stable than the parent ketonucleoside 36a. In 0.1 M methanolic sodium hydroxide, free theophylline was detected only after 4 h, by which time, loss of the acetyl group was complete; a reaction time of more than 18 h was needed for complete cleavage of the glycosylic bond.51In alcoholic solution, the unsaturated 4'-ketonucleoside 66 was very sensitive to nucleophilic attack, and decomposed rapidly, with elimination of the nitrogenous base.31Thus, treatment with sodium borohydride at - 70"led to complete decomposition within 10 min; but, when sodium borohydride was added to a solution of 66 in 1,2-dichloroethane containing acetic acid, fast reduction occurred, and no degradation was observed.31 The action of alkali on 7-(6-deoxy-3,4-O-isopropylidene-~-~-lyxohexopyranosyl-2-u1ose)theophylline (35a) has been studied in some detail, and a mechanism for the formation of the branched-chain pentofuranosylpurines 78a and 78b by alkaline degradation of 35a was propo~ed.~~.~~ This mechanism is illustrated in Scheme 4.The reaction occurred in 0.2 M methanolic sodium hydroxide, and the isosaccharinic acid derivatives 78a and 78b obtained constitute the first saccharinic acid nucleosides reported. The configuration of the isomers at the branch-point was established by spectroscopics8 and chemical5Qmeans. (57)K.Antonakis, M.-J.Arvor-Egron, and F. Leclercq, Carbohydr. Res., 25 (1972)518521. (58)T. Halmos and K. Antonakis, Carbohydr. Res., 43 (1975)208-212. (59)T.Halrnos and K. Antonakis, J Chem. SOC., Perkin Trans. I, (1975)2138-2140.

KETONUCLEOSIDES

249

Me

I

CO,RZ

780Ra=H

Scheme 4

78b Ra = M e

IV. STRUCTURE AND SPECTROSCOPIC PROPERTIES

1. Infrared Spectra The appearance of a carbonyl band assigned to the keto group of the sugar moiety is observed in most cases, even when one or more carbonyl groups are present in the nitrogenous base. The C=O band of such purines or pyrimidines generally lies between 1700 and 1650 em-', whereas that of the sugar carbonyl group is situated above 1720 cm-'. However, in some examples reported, the absence of a keto band could be explained either by the ready hydration of the ketone, or by the presence of ester groups, the bands for which hide the carbonyl band. The infrared spectrum of di-O-trityl-3'-ketouridine 2, in comparison with that2 of the parent 1, exhibited an additional carbonyl band at 1775 cm-'. The presence of a 3'-keto function in 18a was confirmed by a carbonyl band at 1780 em-', in addition to those present at 1720 and 1675 cm-l in the i.r. spectrum of the parent nucleoside 17a. Similarly, a new carbony1 band appeared at 1780 cm-' in the spectrumP5of the 2'-ketocytidine derivative 20a. The 5'-O-trityl 3'-ketothymidine 33b exhibited an i.r. absorption at 1778 cm-', attributed to the keto group i n t r ~ d u c e dAlso, . ~ ~ theophylline 4'-ketonucleoside derivative 41a had2e*27 an i.r. spectrum similar to that of the parent 40a, except for a carbonyl band at 1755 cm-'. Thymine 4'-ketonucleoside derivative 45 exhibited acarbonyl band at 1725 cm-', assigned to the C = O and - C ( = O ) - C = C - groups of the base.30 The

250

KOSTAS ANTONAKIS

spectrum of the theophylline ketonucleoside derivative 8 clearly indicated the appearance of a carbonyl absorption at 1740 cm-’ compared to the parent nucleoside 7. The unsaturated 4‘-ketonucleoside 66 exhibited31 two characteristic bands, at 1780 and 1755 cm-’, attributed respectively to the vinylic ester and to the acetyl groups of the sugar moiety. However the band for the keto group introduced, being overlapped by the bands for the carbony1 groups of the nitrogen heterocycle, could not be observed. Similarly, detection of the sugar 2-carbonyl group in 61a was not possible, because of the acetyl band at 1740 em-’.

2. ‘H-N.m.r. Spectra The introduction of a keto group into the sugar moiety of a nucleoside induces changes in the ‘H-n.m.r spectrum. In addition to the disappearance of the signal for the corresponding proton, a deshielding effect of neighboring protons is observed, These changes result in lessened multiplicities which allow first-order analysis of the spectrum, and thus can serve as diagnostic tools in structure elucidation. In the case of 2’-ketonucleosides, this shifting of the H-1’ resonance is further enhanced by the purine or pyrimidine ring. Some characteristic results are as follows. The n.m.r. spectra2 of 2’-ketouridines 2 and 3 showed the H-1’ signal as sharp singlets at 6 5.79 and 5.42, respectively, and J3’,4, as a doublet in both cases, confirming the absence of a proton at C-2’. Similarly, in the n.m.r. spectrum of the 2’-ketocytidine derivative 20a, the appearance of the H-1’ signal as a singlet, whereas the H-1’ signal of the alcohol 19b was a doublet (J1.,2. 5 Hz), indicated the absencez5 of H-2’ in the ketone 20a. A large shift of the signal for the anomeric proton of 18a from S 6.75, for the alcohol 17a, to 6 4.7 was observed. A singlet at S 5.63 was assigned3 to H-1’ in 8, whereas, for the parent alcohol 7, the H-1’ signal was adoublet (J1.,2. 9 Hz) at 65.96. Similarly, in the spectrum2eof 6-chloropurine 2’-ketonucleoside 35b, the H-1’ signal appeared as a singlet at 6 6.35, whereas, in that of the parent nucleoside 34b, the signal of this proton was a doublet (J1t,2. 9 Hz) at 6 5.80. The n.m.r. spectrum of the 4’-ketonucleoside 41a showed clearly the presence of the 4’-keto group. Whereas, for the alcohol 40a, the H-5’ signal was an octet, at 6 4 (J4,,5, andJ5’,6,),the H-5’ signal of the ketonucleo-sidez6 41a appeared as a quartet at 6 4.44 (J5t,6t 7 Hz), confirming the absence of a proton at C-4’. The n.m.r. spectrum of the bromoketonucleoside 72b contained the H-1’ signal as a singlet at 6 7.6, whereas, in that of the parent epoxynucleoside 56, the H-1’ signal appeareds0 as a doublet (J1,,*,9 Hz) at 6 6.1. The n.m.r. spectrum of the unsaturated ketonucleoside 61a showed the H-1‘ signal as a singlet at 6 6.80; in

KETONUCLEOSIDES

25 1

addition, the absence of a signal for H-3’, and the chemical shift (S4.9) of H-4’, confirmed the structural a ~ s i g n m e n t . ~ ~ The structure of other ketonucleosides was also established by study of their n.m.r. spectra [for example, 66 (Ref. 31), 59 (Ref. 42), and 68b, 68c (Ref. 15)],in comparison with those of the parent nucleosides. A techniqueeobased on n.m.r.-spectral analysis of unsaturated ketonucleosides has been used for the structural determination of partially acylated nucleosides; such a determination b y means of n.m.r. studies is often unreliable, owing to the complexity of the signals observed. This method takes advantage of the ready conversion of partially acylated nucleosides into the corresponding a$-unsaturated ketonucleosides (see Section 11,5),the n.m.r. study of which permits ready and unambiguous determination. In fact, the chemical shift of the vinylic proton, compared with that of the anomeric proton, allows deduction of the position of the keto group, and, thus, the position of the free hydroxyl group in the parent nucleoside (for example, comparison ofthe spectra of 67a and 67b with those of 68a and 68c). Conformational inversions in the field of ketonucleosides were estabthat the introlished by n.m.r.-spectral analysis. It has been rep0rted~O7~~ duction of a carbonyl group at C-4 of the sugar moiety of L-rhamnosylpurines and -pyrimidines led to ketonucleosides possessing an axially attached nitrogenous base. Such unusual positions of the base have been observed in the case of a glycosyltheophylline,el and for some derivatives of a rhamnosyl-5-fluoroura~il.48 The comparative study of the n.m.r. spectra of L-rhamnose nucleosides 40a, 40b, and 44 and their 4‘-keto derivatives 41a, 41b, and 45 revealed that the conformation changes from 4C1to ‘C, on introduction of the carbonyl The value of J1.,2t and J28.3, changed from 8 or 9 Hz to 2 or 3 Hz; the latter values indicated the ‘C,conformation for the ketonucleosides. In addition, the H-3’ signal shifted downfield, because of the presence of the 4’-keto group. The ‘C,conformation was favored by the existence of weak interactions between the carbonyl group at C-4’, and the equatorial substituents on C-3’ and C-5’, whereas these interactions were strong in the ,C1conformation. The examination of the spectra of L-rhamnopyranosyl-purines and -pyrimidines having various substituents on 0-2’ and 0 - 3 ’ led to the conclusion that the structural inversion was not due to the strain introduced by the isopropylidene

(60) M. Bessodes, M.-J. Arvor-Egron,A. Ollapally, and K. Antonakis, C. R. Acad. Sci., Ser. C, 280 (1975) 1273- 1275. (61) K. Onodera, S . Hirano, and F. Masuda, Carbohydr. Res., 7 (1968) 27-37.

KOSTAS ANTONAKIS

252

3. Ultraviolet Spectra In contrast to other spectroscopic techniques, U.V. spectroscopy, largely used to detect ketoses, does not constitute a reliable method in the case of ketonucleosides. The residual absorption of the nitrogenous bases, in the region of 300-350 nm, often prevents the appearance of the carbonyl peak. Although a significant shift of the absorption to longer wavelengths has been observed with ketopent~sylpyrimidines,~~ and a slight displacement of the absorption with some unsaturated ketonucleosides has also been r e p ~ r t e d , ' ~ . this ~ ' . ~shift ~ could not be confirmed in all of the cases examined.

v. STEREOSPECIFIC REDUCTION The reduction of ketopentose-, as well as ketohexose-, nucleosides with metal hydrides has been used to obtain biologically important nucleosides and rare sugar nucleosides, by epimerization of a chiral center. Moreover, reduction of unsaturated ketonucleosides with borohydride provides new and direct routes to unsaturated and deoxy-nucleosides. Reduction of the 2',5'-di-O-trityl-3'-ketopentofuranose nucleoside 2 with sodium borohydride in ethanol readily gave a 59% yield of 1-(2,5di-O-trityl-P-D-xylofuranosy1)uracil(79a),and treatment of 3 (detritylated 3'-ketouridine 2) with the same reagent, under the same conditions, afforded 69% of 1-P-D-xylofuranosyluracil (79b). The 2'ketouridine derivative 5 and free 2'-ketouridine (6) were reduced to give 57 and 90% yields, respectively, of 1-(3,5-di-O-trityl-P-~-arabinofuranosy1)uracil (SOa) and 1-P-D-arabinofuranosyluracil (Sob).These results

H . 4

2or3

__L

Rod 5or6

-

OR

79a R = Tr 3 -79bR=H

2

RO 5 -80a 6 -8Ob

R=Tr R=H

emphasize the predominant, directive influence of the uracil ring in the reduction process, the greater stereoselectivity with 2'-ketouridine (6) being a consequence of the proximity of the pyrimidine and the keto group.2

KETONUCLEOSIDES

253

The borohydride reduction of 2'- and 3'-ketocytidine derivatives having or lacking an N4-acetyl group provided a facile route to cytosine nucleosides labeled with tritium at specific positions of the sugar.25Reduction of either 20a or 20c with sodium borohydride in ethanolbenzene afforded the arubino and the rib0 derivatives in the ratio of 4 : 1. Treatment of the reduction mixture with methanolic ammonium hydroxide gave the deacetylation products, 82b and 19b, isolated in crystalline form by preparative, thin-layer chromatography. Reduction of the 3'-ketocytidine derivatives 18a and 18c gave similar resultse5 and rylo and ribo derivatives could be isolated in the ratio of 3 : 2 . Crystalline 1-(2,5-di-O-trity~-~-~-xy~ofuranosy~)cyto~~ne @la) was obtained after complete deacetylation with ammonium hydroxide. Treatment of 20b and 18b with sodium [3H]borohydride in ethanol, followed by deacetylation of the product with ammonium hydroxide, permitted isolation,e5 after purification by column chromatography, of l-P-~-[2-~H]arabinofuranosylcytosine (82d)and l-P-~-[3-~H]xylofuranosylcytosine (81c).

F2

HM' I

x3

0 18a,18 b -R2wQ

POa,2Ob 2 0 c

-

810 R' = H, RZ = Tr 81b R1 = H , R2 = H 81c R' = T, R2 = H

820 R = R' = Ac, R2 = Tr 82b R = R 1 = H , R 2 = T r a 2 ~R = R' = RZ = H

a d R=T,R'=R2=H

Reduction** of 3'-keto-S'-O-tritylthymidine 33b with sodium borohydride in ethanol was also found to be stereospecific, as 80% of 1-(2deoxy-5-O-trityl-~-~-threo-pentofuranosy~)thymine (83) was obtained. 0

a3

KOSTAS ANTONAKIS

254

Conversion of L-fucosyl- and 3’-O-methyl-~-g~ycosyl-purines into nucleosides of two naturally occurring, rare sugars, 6-deoxy-~-taloseand 3-O-methyl-~-mannose, could be achieved5’ by stereospecific reduction at C-2 of 7 - ( 6 - d e o x y - 3 , 4 - 0 - i s o p r o p y l i d e n e - ~ - ~ - l y x o o s y l - 2 u1ose)theophylline (35a) and 7-(4,6-0-benzylidene-3-O-methyl-P-~arabino-hexopyranosyl-2-ulose) theophylline (8). Treatment of 35a and 8 with sodium borohydride in ethanol afforded the expected 7-(6-deoxy3,4-O-isopropylidene-~-~-talopyranosyl)theophylline (84) and 7-(4,6340

-35a

U

ic)I

P

HO

OH

Me&-0

HO

OH

84

O-benzylidene-3-O-methyl-~-~-mannopyranosyl)t~~eophylline (86) in 92 and 94% yield, respectively. 0-CH,

\

7-8-

ph‘H&

OMe

R =

(2xr I

Me

86

The stereospecificity of the reduction of these hexosulose nucleosides, trans to the aglycon, parallels previous o b s e r ~ a t i o n s ~ ~ with - ~ 4several hexopyranosulose derivatives. Attempted, similar reduction of the unprotected 2’-ketonucleoside 36a gave 7-(6-deoxy-/3-~-talopyranosyl)theophylline (87) in 60% yield. This lesser stereospecificity may be ex-

36a -

I

Me 87

(62) P. M. Collins and W. G . Overend,]. Chem. SOC., (1965) 1912-1918. (63) K. Antonakis, Bull. SOC. Chim. Fr., (1969) 122-126. (64) G . J. F. Chittenden, Curbohydr. Res., 7 (1970) 101-109.

KETONUCLEOSIDES

255

plained by the absence of protecting groups, which would have a directive influence. 1 - (6 - Deoxy - 2,3 - 0 - isopropylidene - a- L - Zyxo - hexopyranosyl - 4 u10se)thymine~~ (45) was treated with sodium borohydride in methanol to give a 40% yield of 1-(6-deoxy-2,3-0-isopropylidene-a-~-talopyranosy1)thymine (88), which, on deacetonation with trifluoroacetic acid, gave 1-(6-deoxy-cu-~-talopyranosyl)thymine~~ (89). This result indicates that,

88

a9

in 4‘-ketonucleosides, the nitrogenous base (because of its more-distant position) offers less steric hindrance than in the case of 2’-ketonucleosides. The reduction of unsaturated-ketohexosyl purines has been thoroughly st~died.~~v53 In particular, in the case of the unsaturated 2’-ketonucleosides 61a, 6lb, and 63, a mechanism of the reduction by metal hydride was e ~ t a b l i s h e dby~ study ~ of the n.m.r. spectra of the different deoxynucleosides obtained by the action of sodium borohydride in deuterated solvents and of sodium borodeuteride in nondeuterated solvents. The reduction of both 7-(3-0-acetyl-4,6-dideoxy-~-glycero-hex-3-enopyranosyl-2-u1ose)theophylline (6la) and its 6-chloropurine relative 61b in methanol led, respectively, to 7-(2-0-acetyl-4,6-dideoxy-P-~xylo-hexopyranosy1)theophylline (90a) and (2-0-acetyl-4,6-dideoxy-fi~-xyZo-hexopyranosyl)-6-chloropurine (90b) having OH-2’ equatorial,

61a,61b

A

OR WR HO

ma R = R‘ 90bR=RZ

(65) J. Herscovici, Thhe, UniversitC de Paris 7, 1979.

KOSTAS ANTONAKIS

256

whereas reduction of the a-Lanomer 63 afforded 7-(3-0-acetyl-4,6-dideoxy-a-L-ribo-hexopyranosyl-theophylline(91), having OH-2’ axial.

9lR=R’

From these results, it was concluded that the configuration of the molecule has a definite effect upon the direction of attack on the carbonyl group. Thus, the hydride attacks the keto group of the a anomer, transto the purine, producing an axial OH-2’ (91), whereas, in the case of the p anomer, this attack occurss2 cis, leading to an equatorial OH-2’ (90a and 90b). Fast reduction was observed when the unsaturated 4’-ketonucleoside 66 was treated with sodium borohydride in solution in 1,2-dichloroethane containing acetic acid.31 An unexpected attack from the most-hindered side gave 7-(4,6-di-0-acetyl-2-deoxy-~-~-arabino-hexopyranosy1)theophylline (92) almost exclusively. The mechanism proposed

& <$ CH,OAc

66-

R’=

AcO

“Me

0

92 R = R’

R2=

c1

( 2 1 r ”

implies the formation of a complex between Ac0-6’ and the hydride ion. In this complex, the hydride is favorably situated for intramolecular transfer to the 4‘-keto group, and this explains the favored attack from the most-hindered side of the molecule. Unsaturated bromoketonucleoside 72b was treated with sodium borohydride in methanol - chloroform solution, to afford 7-(3-bromo-3,4,6trideoxy-a-~-erythro-hex-3-enopyranosyl)theophylline (93). As in the

KETONUCLEOSIDES

257

case of 63, the hydride attacks from the side trans to the nitrogenous base.50

VI. NUCLEOPHILIC ADDITIONS In contrast to nucleophilic additions to ketoses, which very often lead to diastereoisomeric products, additions to ketonucleosides have been found to be highly stereoselective, permitting ready synthesis of amino-, epimino-, triazolo-, and other rare-sugar nucleosides. A variety of nucleophiles could also be added to unsaturated ketonucleosides, to afford (by way of a 1,4-addition mechanism) amino-, azido-, aziridino-, and thio-ketonucleosides. The action of metal hydrides on ketonucleosides has been extensively discussed in Section V.

1. To Ketohexose Nucleosides The direct addition of nitromethane, in a mixture of anhydrous methanol and sodium methoxide, to 9-13,5-O-isopropylidene-P-~-threo-hexofuranosyl-2-u1ose)adenine(22) gave 9-(2-C-nitromethyl-~-D-hpo-hexofuranosy1)adenine (94) in 75% yield.38 Reduction of 94 in 5: 5:1 methanol - water -acetic acid in the presence of 10% palladium-oncharcoal, followed by N-acetylation of the resulting aminomethyl group, afforded, in 62% yield, 9-(2-C-acetamidomethyl-3,5-O-isopropyIideneP-D-lyxo-hexofuranosy1)adenine(95).

+ J 4,

22

Q

Me,C-0

$2

JJrJ

Me,C-0 CH,NO, 94

CH,NHAc 95

Addition of nitromethane to 7-(4,6-0-benzylidene-3-O-methyl-P-~urubino-hexopyranosyl-2-u1ose)theophylline(8)and 6-chloro-9-(3,4-0isopropylidene-P-L-lyxo-hexopyranosyl-2-ulose)purine (35b)afforded, respectively, 7-[ 4,6-0-benzylidene-3-O-methyl-2-C-nitrornethyl-~-~gluco (or manno)pyranosyl]theophylline (96)and 6-chloro-9-(3,4-0-isopropylidene-2-C-nitromethylene-P-L-lyxo-hexopyr~osyl)purine (98), both isolated in high yield.se Treatment of 96 with acid permitted the (66) F. Leclercq, M. Bessodes, J. Jurnelet, and K. Antonakis, Nucleot., 1 (1974) 349-356.

1. Carbohydr. Nucleos.

KOSTAS ANTONAKIS

258

elimination of the benzylidene group without any glycosylic cleavage, to give 97. It seems probable that the nitromethyl derivative of 35b under-

0 CH,OH

8

* -

MeNQ MeONa

H+

OMeHO

HO CHZNO1 Me

%R=R1

CH,NOa

WR=R'

goes spontaneous dehydration in the reaction medium to give the nitromethylene-nucleoside 98, whereas similar dehydration of 96 was not c1

98R=RZ

favored, because of the establishment of a hydrogen bond between OH-2' and a carbonyl group of the purine.

2. To Unsaturated Ketohexose Nucleosides Treatment of unsaturated bromoketonucleoside 72b, prepared by a method described in Section 11, with hydrazoic acid led to 7-(3,4,6-trideoxy-3,4- triazolo-a-~-gZycero-hex-3-enopyranosyl-2-ulose)theophylline (99) by a 1,2-addition mechanism50 (see Scheme 5). A 1,4-addition mechanism was postulated50 for the addition of cyclohexylamine to 72b in the presence of nitromethane. An intramolecular elimination of bromine afforded the (unisolated) epimino-ketonucleoside 100, which underwent further attack by nitromethane, to give 7-(3,4-cyclohexylamino-3,4-dideoxy-2-C-nitromethyl-~-~-allopyranosy1)theophylline (101). The unsaturated trideoxyketonucleoside 71, synthesized according to a procedure described in Section 11,has been used as a key intermediate

KETONUCLEOSIDES 72b

+

259

HN,

I Me

Me I

I

Me

N 0 99

Scheme 5

Me

I

“Me 0

Scheme 6

KOSTAS ANTONAKIS

260

to acetamido- and thio-ketonucleosides, as well as to the first trideoxyn~cleosides.~~ Hydroxylamine reacted with 71 to give the oxime of 7-(2,3,4,6-tetradeoxy-~-~-glycero-hex-2-enopyranosyl-4-ulose)theophylline (102), a new type of unsaturated nucleoside. Treatment of 71 with sodium borohydride reduced the carbonyl group, and afforded 7-(2,3,6-trideoxy-P~-eythro-hex-2-enopyranosyl)theophylline (103). The n.m.r. spectrum of acetylated 103 showed H-4',5' to be trans-diaxial and that axial addition of hydride to the carbonyl group of 71 had occurred.4e Addition of ammonia to 71 was less stereospecific than the additionSo of amine to bromoketonucleoside 72b. Thus, treatment of 71 with ammonia in acetonitrile, and acetylation of the product, gave a mixture of eythro and threo acetamido-nucleosides from which only the 7-(2-acetamido - 2,3,6- trideoxy-P-~-threo-hexopyranosyl4-u1ose)theophylline (104) could be isolated.42 Addition of benzenethiol to 71 in the presence of a catalytic amount of tetrabutylammonium fluorides7 afforded only the trans isomer 7-(3,6dideoxy - 2 - S-phenyl- 2 - thio-P-~-eythrohexopyranosyl- 4 -ulose)theophylline (105). In the absence of catalyst, 71 reacted with benzenethiol to give 7-(3,6-dideoxy-2-S-phenyl-2-thio-~-~-t~reo-hexopyranosyl-~u1ose)theophylline (106). These results suggest that the uncatalyzed reaction gave the kinetic product, 106, whereas the catalyzed reaction led42to the thermodynamic product 105.

71

HON O

R 102

103

,

NHAc 104

(67) I. Kuwahima, T. Murofushi, and E. Kakamura, Synthesis, (1976) 602-603.

261

KETONUCLEOSIDES n0.

71

P

-

h

L

TBAF 105

SPh 106

Me I

VII. BIOLOGICAL INTEREST The substantial progress made in synthesis of the complex carbohydrates occurring in medicinally important molecule^^^-^^ is largely due to the discovery of new oxidative procedures that permit ready preparation of aldosuloses. Branched-chain sugars were obtained by nucleophilic additions to various ketopentoses and ketohexoses; subsequent condensation with purines and pyrimidines then afforded the desired natural, or synthetic, antibiotics (see, for example, Refs. 19 and 73). Unfortunately, this procedure did not permit preparation of the corresponding ketonucleosides postulated as key intermediates in many biosynthetic routes. In fact, j?-elimination reactions precluded the coupling of ketoses with nitrogen heterocycles. These di5culties may explain the scarcity of information concerning their biological properties or functions. Nevertheless, the availability of ketonucleosides due to the methods of synthesis described in Section I1 has, over the past decade, produced some interesting biological studies opening large horizons for extensive (68) S.Hanessian andT. H. Haskell, in W. Pigman andD. Horton, The Carbohydrates, Vol. IIA, Academic Press, New York, 1970, pp. 139-211. (69) R. J. Suhadolnik, Nucleoside Antibiotics, Wiley-Interscience, New York, 1970. (70) R. J. Suhadolnik, Nucleosfdes us Biological Probes, Wiley -Interscience, New York, 1979. (71) H. Griesebach, Ado. Curbohydr. Chem. Biochem., 35 (1978) 81-126. (72) S. Umezawa, Adw. Curbohydr. Chem. Biochem., 30 (1974) 111-181. (73) H . P. Albrecht and J. G. Moffatt, Tetruhedron Lett., (1970) 1063-1066.

262

KOSTAS ANTONAKIS

investigations of the mechanism of action of antibiotics, thus contributing to the development of new concepts in the biosynthesis of naturally occurring nucleosides. On studying the biosynthesis of the antibiotics blastidicin S and H, which inhibit protein synthesis, Seto and coworker^^.^^ suggested that the (P-D-glucosyluronic acid)nucleoside 107 isolated from the culture

Ya

Hd

HO

OH

107

medium of Streptomyces griseochromogenes constitutes a precursor to the ketonucleoside intermediate 16. Consequently, oxidation of OH-4 of the hexosyl moiety leading to the 4’-ketohexosylpyrimidine16 occurs subsequent to the oxidation of the hydroxymethyl group of the hexose to the corresponding carboxyl group70 (see Scheme 2). The biosynthesis of 9-/3-D-arabinosyladenine (14), which inhibits the synthesis of DNA in viruses and various tumors,70has been thoroughly studied by Farmer and coworkers.8 2’-Ketoadenosine 13 was postulated to be an intermediate of the biosynthesis (see Scheme 1).This 2’-ketonucleoside 13, formed by oxidation at C-2 of adenosine, should be epimerized to the 2/,3/-enediol nucleoside, which undergoes reduction7O to give 14. The first reportedlo biological activity in the ketonucleoside field concerned 7-(6-deoxy-~-~-lyxo-hexopyranosyl-2-ulose)theophylline (36a), which was found to inhibit the growth of KB cells in uitro, whereas the parent nucleoside 34a was inactive under the same experimental conditions. These findings were confirmed by a comparative study of 7-linked and 9-linked ketohexosylpurines possessing the a,as well as the P, configuration. The inhibitory activity against KB cancerous cells of the 2-ketonucleosides 36a and 36b, the 4’-ketonucleosides 41a and 41b, and the unsaturated ketonucleosides 61a and 61b was demonstrated,” in contrast to the parent nucleosides, which appeared to be inactive, as were those derived by reduction of the carbonyl group. Based on the overall (74)

H.Set0 and H. Yonehara,]. Antibiot., 30 (1977) 1019-1021.

KETONUCLEOSIDES

263

results, it appeared that the 9-linked ketonucleosides 36b, 41b, and 6 l b were more active than the 7-ketonucleosides 36a, 41a, and 6la, whereas no significant difference could be observed between the activity o f a and p anomers. Unsaturated ketonucleosides had the highest inhibitory activity. A thorough investigation12 of the effects of the unsaturated ketonucleoside 61a on KB cells in culture confirmed the high cytotoxic potency of this compound. Moreover, it appeared that, at low doses, where no cytotoxic effect occurs, the ketonucleoside impaired DNA, RNA, and protein synthesis, and strongly inhibited cell multiplication. of a keto Confirming the importance, for the biological group in the sugar moiety, the antiviral activity of some keto-C-nucleosides has also been reported.'3 From this study, it was clear that the presence of a ketone group in these nucleosides, as in 4'-ketonucleoside 50a, gave a compound able to inhibit the replication of rnurine leukemia virus at nontoxic concentrations. The first, in uiuo study on the antitumor activity of ketonucleosides appeared14 in 1977. The action of 1-(6-deoxy-2,3-O-isopropylidenea-~-Zyxo-hexopyranosyl-4-ulose)thymine (45) and of 7-(3-0-acetyl4,6-dideoxy-~-~-gZycero-hex-3-enopyranosyl-2-ulose)theophylline (Sla) against L1210 leukemia in mice was examined comparatively. From the results obtained, it was clear that the unsaturated ketohexosylpurine 61a was much more active than the ketohexosylpyrimidine 45, whereas the parent nucleosides (44 and 34a, respectively) were inactive under the same experimental conditions. A thorough study15 of the structure-activity relationship of the four unsaturated ketonucleosides 61a, 68b, 68c, and 72b showed that all of the compounds examined exhibited significant activity against L1210 leukemia, and the presence of a methyl group on C-5 of the hexose did not appear to be a necessary prerequisite for significant activity. In the meanwhile, nucleosides 68b and 72b proved to be less toxic, and repeated administration of a dose appeared to decrease the toxic effects without affecting the antitumor activity of the compounds. In order to establish the possible mode of action of the tumor-inhibitory, unsaturated ketonucleosides, the reaction of 61a, 68b, 68c, and 72b with a variety of physiologically occurring nucleophiles was examined.7s Glutathione and cysteine reacted much more rapidly, as did other biological nucleophiles. The high rate of reaction with N-acetyl-L-cysteine was measured, and the reaction products were isolated and characterized (see Scheme 7). (75)

T. Halmos, J.-M. Argoullon, and K. Antonakis, Proc. Znt. Round Table, Nucleos. Nucleot. B i d . Appl., 4 t h Antwerp, (1981)204-208.

KOSTAS ANTONAKIS

264

NHAc

I

0

CH,R

___)

OBz

BzO

S

I

I

68b R = O H 68c R = H

y

HO

2

CHNHAc

F N HAc

JOJi

C02H

Me I

Bcheme 7

Interestingly, as in the additionqe of benzenethiol to the unsaturated ketonucleoside 7 1, the addition of N-acetyl-L-cysteine occurred by a 1,4-mechanism. The ability of unsaturated ketonucleosides to react with protein sulfhydryl groups was demonstrated by measuring their inhibitory action towards beef-heart lactate dehydrogenase.75 These results led to the conclusion that the primary targets for unsaturated ketonucleosides are glutathione and reactive thiol groups of proteins. Also, the interaction of these nucleosides with cellular constituents of intact L12 10 leukemia cells was ~ t u d i e d . 7Rapid ~ reaction with plasma membrane and soluble, intracellular thiols was demonstrated, as well as a good correlation between the extent of plasma membrane alkylation in uitro and the biological activity in uiuo. The inhibition of tumor growth by unsaturated ketonucleosides could, therefore, be due to selective alkylation of the SH groups of key proteins that control cell division. Because the plasma membrane is the first barrier encountered by the drugs, it seems important to consider the interaction of unsaturated ketonucleosides with this cellular structure.76

(76) T. Halmos, A. Cardon, andK. Antonakis,

C h .B i d . Interact., 46 (1983) 11-29.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL.

42

PLANT CELL-WALLS

BY PRAKASH M. DEYAND KEN BRINSON Department of Biochemistry, Royal Holloway College (University of London), Egham, Surrey TW20 OEX, England

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

266 269 269 274 275 277 277 285 IV. The Hemicelluloses . . . . . . . . . . . . ........................... 287 1. Dicotyledonous Plants. . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 2. Monocotyledonous Plants. . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 1 V. Non-Cellulosic D-Glucans , . . . . . . . . . . . , . . . . . . . , . . . . . . . . . , . . . . . . . . . . 293 1. Dicotyledonous Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 293 294 298 298 299 300 30 1 301

11. The Primary Cell-Wall. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . The Problems of Elucidating Primary-Wall Structure. . . . . . . . . . . . . . . . . 2. The Types of Cell-Wall Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Methods Used in the Elucidation of Primary-Wall Structure . 111. The Pectic Polysaccharides . . . . . . . , . . . . , . . . . . . . , . . . . . . . . . . . . . . . . . . . 1. Dicotyledonous Plants. . . ............. 2. Monocotyledonous Plants .............

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

302 304 306

2. Noncovalent Bonding of Hemicelluloses to Cellulose Microfibrils . . . . . . . 3. Interconnections Involving the Hydroxy-L-proline-rich, Cell-Wall Glycoprotein . , . . . . , . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 X. Discussion on the Albersheim Model for Primary Cell-Wall Structure 309 ofDicots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 315 .... ... 315 317 3. Intermediates in the Synthesis of Polysaccharides. . . . . . . . . . . . . . . . . . . . 323

XJI. Cell-Wall Biosy 1 . Introduction

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4. Cytological Location of Polysaccharide Synthesis .................... 5. Alterations of Cell-Wall Polymers Outside the Plasma Membrane. . . . . . . 6. Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII. Cell-Wall and Fruit Ripening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The Physiology of Fruit Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Addendum.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

330 337 338 339 339 340 382

I. INTRODUCTION The cell wall is a more or less rigid envelope that encases the plant cell. The wall must be rigid enough to give the plant strength and form, and yet, if necessary, it must yield freely in order to facilitate growth. The network of cell walls, where adjoining cells have a wall in common, provides in a plant the structural framework analogous to both the skin and the bones of an animal. In some plants, especially those having woody tissues, the strength of this cell-wall network is prodigious. However, despite their apparently tough sheathing, the cells of the growing regions of a plant are able to extend to many times their initial The cell wall lies outside the plasma membrane, which defines the boundaries of the cell itself. The wall is freely permeable to most molecules, but the membrane exhibits selective permeability, tending to concentrate certain dissolved molecules and ions inside the cell. The presence of such charged components as acidic polysaccharides within the wall imparts ion-exchange properties to the wall. Water diffuses through the membrane by osmosis, increasing the intracellular pressure and pressing the membrane against the wall. The restraining strength of the wall prevents the elastic cell from bursting. It is this osmotic pressure that powers the growth of the plant; the rate at which the wall yields to the pressure determines the rate of cell enlargement and, consequently, the overall growth-rate of the Two processes are involved in plant growth, namely, cell division and cell elongation. The former process takes place only in specialized tissue, the meristematic regions, characteristically found at root and stem tips and in the buds that form leaves and flowers. Grasses, on the other hand, have a meristematic region between the leaves and the roots. During (1) P. A. Roelofsen, The Plant Cell Wull, Gebr. Borntraeger, Berlin, 1959. (2) H. J. Rogers and H. A. Perkins, Cell Walk and Membranes, Spon, London, 1968. (3) T. W. Goodwin and E. I. Mercer, Introduction to Plunt Biochemistry, Pergamon, Oxford, 1974. (4) R.D. Preston, The Physical Biology ofPlant Cell Walls, Chapman and Hall, London, 1974. (5) P. Albersheim, Sci. Am., 232 (1975) 81-95.

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division of a meristem cell, the replication of the genetic material and separation of the two nuclei are followed by the formation of a transverse string of disconnected vesicles in the cytoplasm separating the nuclei. These vesicles coalesce to form a partition, the middle lamella, dividing the initial cell in two.l-5 Such cell division produces no direct increase in plant volume; the latter is accomplished by elongation (often as much as one hundred-fold) of the daughter cells. The cells become progressively longer with distance from the meristematic region. The cell walls control not only the rate of elongation but also its direction, thus determining cell shape as well as length. At intervals, the walls are pierced by structures called plasmodesmata through which the cytoplasm of adjacent cells can communi~ate.’-~ As long as the cell continues to grow, the wall surrounding it remains relatively thin; in this stage of development, it is called a primary cellwall. The primary cell-wall consists almost entirely of polysaccharides (90% of the structural material), and the remaining 10%is Ray and his a s s o ~ i a t e s , ~working -~ with meristem regions of oat stem, showed that walls do not get thinner or alter significantly in composition, during elongation, and this finding has been verified by A l b e r ~ h e i m . ~ J ~ Because the growing wall maintains its thickness and its strength, new material must, during elongation, be added to it in such a way that the average chemical composition of the wall is not altered. It has been suggested,s although not generally accepted, that auxin stimulates a shift from apposition (deposition of new wall at the cell membrane only) to intussusception (deposition throughout the wall). Intussuscepted polysaccharides would loosen the wall by forcing apart cellulose microfibrils, or providing a “lubricant” to facilitate such slippage. In pea stem and Auena coleoptile-tissue, wall synthesis is entirely by apposition in the absence of auxin; after auxin treatment, a sizable proportion of deposition of hemicellulose, but not of cellulose, occurs throughout the wall.” After a cell has matured, the wall may become thickened and take on a distinctive shape and specialized properties; it is then called a secondary wall. Secondary thickening of plant cell-walls is particularly marked in (6) P. M. Ray, Am. J . Bot., 49 (1962) 928-939. (7) P. M. Ray and A. Abdul-Baki, in F. Wightman and G . Setterfield (Eds.), Biochemistry and Physiology of Plant Growth Substances, Runge, Ottawa, 1968, pp. 647-658. (8) D. B. Baker and P. M. Ray, Plant Physiol., 40 (1965) 345-352. (9) P. M. Ray and A. W. Ruesink, Deo. Biol., 4 (1962) 377-397. (10) P. Albersheim, in J. Bonner and J. E. Varner (Eds.),Plant Biochemistry, Academic Press, New York, 1976, pp. 225-274. (11) P. M. Ray,J. CellBiol., 35 (1967) 659-674.

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woody and fibrous tissues. Each type of mature plant-cell has a characteristic secondary-wall, presumably adapted to the particular function of the cell. The process of differentiation that transforms primary cells into secondary cells is only partially understood. In such thick-walled cells as tracheids, fibers, and vessels in mature tissues, the greater part of the wall is made up of secondary thickening deposited after expansion of the primary wall has stopped. In cells that show localized growth (for example, tip growth), the secondary wall may well start to form in areas removed from the growing zone, while the latter is still active.2 The primary wall is a highly hydrated structure having a relatively sparse distribution of cellulose microfibrils. The cell walls obtained from tissues containing mainly primary walls, including coleoptiles of Auena and Zea mays, cambium initials of Pinus, and young-cotton hairs, are composed of only 25-40% (dry weight) of cellulose.2 The secondary wall is much denser. Woody tissue, for example, wherein there is developed secondary thickening, is composed of 40-60% (dry weight) of cellulose, and, compared with the primary wall, the relative amounts of pectin and hemicellulose are lesse The secondary wall is much less hydrated than the primary wall; mature wood contains only 25-40% (by fresh weight) of water,2Jecompared with 60% for primary-wall preparations from the mesocotyl of corn seedlings.' Secondary-cellulose deposition occurs after cessation of expansion of the primary wall. Layers of the secondary wall, in contrast to the primary wall, display a very orderly, parallel arrangement of the microfibrils. In such plants as flax and hernp,'s2 bamboo,13 ~ i s a l , ' ~ -cotton '~ hairs,l and pine t r a ~ h e i d s ,three ' ~ main layers can be detected in the secondary wall, each made up of cellulose microfibrils arranged in a helical fashion around the cell. In each of these secondary walls, the middle layer of cellulose is considerably thicker than the cellulose layers on each side of it, with a helical direction opposed to those of the latter. It is probable that each of these three layers is, in fact, complex, and made from a number of lamellae, each with its own helix of cellulose microfibrils.'*e In addition to cellulose, hemicelluloses are also laid down during secondary thickening. In angiosperms, these hemicelluloses are preponder-

(12)A. Allsopp and P. Misra, Biochem. I., 34 (1940)1078-1084. (13)J. P. Thornber and D. H. Northcote, Biochem. ]., 81 (1961)455-464. (14)P. Sonntag, Flora ( J e w ) , 99 (1909)203-209. (15)F.Stern,]. Text. Inst., 48 (1957)21-24. (16)R. D. Preston and M. Middlebrook,]. Text. Inst., 40 (1949)715-718.

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antly x y l a n ~ , ' ~and - ~ ~in gymnosperms, they are mainly glucomannans and galactoglucomannans.z1*22 Lignin, a phenolic polymer, is also incorporated into the wall during the secondary-thickening phase. This component permeates the spaces between the plant cells, thereby strengthening the tissue. Lignification is a result of enzymic dehydrogenation and subsequent polymerization of coumaryl, coniferyl, and sinapyl alcohols, the relative proportions of which differ in the lignins from different plant^.^^-^^

11.

THEPRIMARY CELL-WALL

1. The Problems of Elucidating Primary-Wall Structure The biological role of the cell wall was discussed in the previous Section, and it is therefore obvious that a knowledge ofits structure, biosynthesis, and degradation is of profound interest and importance. The biochemistry of plant cell-walls is still at the stage of identifying and elucidating the covalent structures of the macromolecular components of the primary cell-wall. The secondary, tertiary, and quarternary structures of the polysaccharides therein have received only scant attention.2e-32The ultrastructural distribution of polymers within the wall, the integration of newly synthesized macromolecules into the wall, and

(17) G . 0. Aspinall and D. McCrath, j . Chem. SOC., C, (1966) 2133-2139. (18) P. C. Bardalaye and G . W. Hay, Carbohydr. Res., 37 (1974) 339-350. (19) I. R.Siddiqui and P. J. Wood, Carbohydr. Res., 54 (1977) 231-236. (20) R.Toman, Cellul. Chem. Technol., 7 (1973) 351-357. (21) G. 0. Aspinall, R. Begbie, and J. E. McKay,J. Chem. Soc., (1962) 214-219. (22) T. E. Timell, Ado. Carbohydr. Chem., 19 (1964) 247-302; 20 (1965) 409-483. (23) D. H. Northcote, Annu. Rev. Plant Physiol., 23 (1972) 113-132. (24) K. Freudenberg, in A. Kleinzeller (Ed.), Molecular Biology, Biochemistry and Biophysics, Vol. 2, Springer, Berlin, 1968, pp. 45- 122. (25) H. Hibbert, Annu. Reo. Biochem., 11 (1942) 183-202. (26) I. A. Pearl, The Chemistry oflignin, Dekker, New York, 1967. (27) K. V. Sarkanen, in B. L. Browning (Ed.), The Chemistry ofwood, Interscience, New York, 1963, pp. 249-311. (28) W. J. Schubert, inL. P. Miller (Ed.),Phytochemistry, Vol. 3, Van Nostrand-Reinhold, Princeton, N. J., 1973, pp. 132- 153. (29) I. C. M. Dea, E. R.Morris, D. A. Rees, P. Smith, and D. Thom, FEBSLett., 32 (1973) 195- 198. (30) I. C. M. Dea, E. R. Morris, D. A. Rees, E. J. Welsh, H. A. Barnes, and J. Price, Carbohydr. Res., 57 (1977) 249-272. (31) D. A. Rees, Biochem. I.,126 (1972) 257-273 (32) D. A. Rees and E. J. Welsh, Angew. Chem., Int. Ed. Engl., 16 (1977) 214-234.

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the biochemistry of wall growth are other areas in which little has been clearly established. Many studies have been based upon polymers extracted not from primary walls directly but from differentiated tissues containing secondary walls. A number of workers have contributed to our understanding of the structure of the primary cell-wall. Two who are responsible for important, pioneering work are N o r t h ~ o t e and ~~~ L~ am ~ -p ~ ~ r t . North~~-~~ cotez3 considered the cell wall to be a growing, constantly changing, composite material consisting of a dispersed phase of microfibrils within a complex, continuous matrix, and that it was possible, by studying polymer roles in the composite, to relate the chemical and physical properties of the individual constituents to those of the whole wall. In Northcote’s model, during the growth of the cell, the polymers of the wall interact and change, and the resulting alteration in the properties of the wall can be correlated with a variation in its function. A change in the properties of the wall can also occur in response to a variation in the environment of the growing cell, and can be brought about by interactions between cells, by gaseous or aqueous changes in the surrounding medium, or by an increase or decrease in the stresses and strains applied to the cell wall. Although the properties of the wall then become modified, the fundamental, composite structure, consisting of microfibrils dispersed in a complex matrix, is retained; however, there are alterations in the orientation of the microfibrils, changes in the chemical composition of the matrix, and variations in the interaction of the fibrillar and matrix components. Lamportqo considered the primary cell-wall to be a single, “bagshaped” macromolecule having a coherent, cross-linked structure, with bonds both between the hydroxy-L-proline-rich, wall protein “extensin” and wall polysaccharides, and between individual polysaccharides. These pioneering studies have been further developed by other lead(33) D. H. Northcote, Biol.Reu. Cambridge Philos. Soc., 33 (1953) 53- 102. (34) D. H. Northcote, Znt. Reo. Cytol., 14 (1963) 223-265. (35) D. H. Northcote, Essays Biochem., 5 (1969) 89- 137. (36) D. T. A. Lamport, Fed. Proc., 22 (1963) 647. (37) D. T. A. Lamport, 1.Biol. Chem., 238 (1963) 1438- 1440. (38) D. T. A. Lamport, Erp. Cell Res., 33 (1964) 195-206. (39) D. T. A. Lamport, Nature, 202 (1 964) 293 - 294. (40) D. T. A. Lamport, Ado. Bot. Res., 2 (1965) 151-218. (41) D. T. A. Lamport, Nature, 216 (1967) 1322-1324. (42) D. T. A. Lamport, Biochemistry, 8 (1969) 1155-1163. (43) D. T. A. Lamport, Proc. Int. Bot. C o n t , I l t h , Seattle, 1969, p. 121. (44) D. T. A. Lamport, Abstr. Pap. Am. Chem. Soc. Meet., 158 (1969) CARB 47.

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ing groups, including Selvendran and associate^,^^-^^ working with runner-bean hypocotyls, Monro and coworker^,^^-^^ studying lupin hypocotyls, Ray and his colleague^,^^^^^ working with oat coleoptiles, and Albersheim and coworker^.^^-^^ Albersheim’s g r o ~ p investigated ~ ~ - ~ ~ the primary-wall structure of a number of suspension-cultured tissues, including cells of sycamore, Douglas fir, red kidney-bean, wheat, rice, oat, sugar cane, and brome grass, rye-grass endosperm, and barley aleurone layer. They also performed the cardinal service of drawing together the conclusions based on their own work and those of others in the field into a series of important reviews,5J0~s4~65 and, on this basis, constructed a model for the primary cell-wall structure in higher plants. This model structure is discussed later. Significant technical problems face those who wish to study the structure of primary cell-walls. An important consideration is the purity of the cell-wall preparations. Often, cell-wall studies have been carried out on walls obtained from heterogeneous, differentiated tissues containing

(45) R. R. Selvendran, Phytochemisty, 14 (1975) 1011-1017. (46) R. R. Selvendran, A. M. C. Davies, and E. Tidder, Phytochemisty, 14 (1975) 21692174. (47) R. R. Selvendran, Phytochemisty, 14 (1975) 2175-2180. (48) R. R. Selvendran and S. Selvendran, Phytochemisty, 11 (1972) 3167-3171. (49) J. A. Monro, D. Penny, andR. W. Bailey, Phytochemisty, 15 (1976) 1193-1198. (50) J. A. Monro, R. W. Bailey, and D. Penny, Phytochemisty, 15 (1976) 175-181. (51) J. A. Monro, R. W. Bailey, and D. Penny, Phytochemisty, 13 (1974) 375-382. (52) J. A. Monro, R. W. Bailey, and D. Penny, Phytochemisty, 11 (1972) 1597-1602. (53) S. Wada and P. M. Ray, Phytochemisty, 17 (1978) 923-931. (54) J. M. Labavitch and P. M. Ray, Phytochemisty, 17 (1978) 932-937. (55) K. W. Talmadge, K. Keegstra, W. D. Bauer, and P. Albersheim, Plant Physiol., 51 (1973) 158-173. (56) W. D. Bauer, K. W. Talmadge, K. Keestra, and P. Albersheim, Plant Physiol., 51 (1973) 174-187. (57) K. Keegstra, K. W. Talmadge, W. D. Bauer, and P. Albersheim, Plant Physiol., 51 (1973) 188-196. (58) B. M. Wilder and P. Albersheim, Plant Physiol., 51 (1973) 889-893. (59) B. S . Valent and P. Albersheim, Plant Physiol., 54 (1974) 105- 108. (60) D. Burke, P. Kaufman, M. McNeil, and P. Albersheim, Plant Physiol., 54 (1974) 109-115. (61) M. McNeil, P. Albersheim, L. Taiz, andR. L. Jones, Plant Physiol., 55 (1975) 64-68. (62) A. G . Darvill, M. McNeil, and P. Albersheim, Plant Physiol., 62 (1978) 418-422. (63) L. Weinstein and P. Albersheim, Plant Physiol., 6 3 (1979) 425-432. (64) P. Albersheim, Znt. Reo. Biochem., 16 (1978) 127-150. (65) A. G . Darvill, M. McNeiI, and P. Albersheim, in N.E. Tolbert (Ed.), The Plant Cell, Academic Press, New York, 1980, pp. 91 - 162.

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both primary and secondary wall^.^^-^^ The use of cultured cells as a homogeneous tissue, rich in primary walls, is one method of resolving this problem. This approach has been particularly favored by Lamport38-40.42 and Albersheim and his associate^,^^-^^^^^ and has also been employed by some other worker^.^^-'^^ Another approach to obtaining relatively homogeneous, primary-wall tissue is the use of coleoptiles, and

(66) (67) (68) (69) (70) (71) (72) (73) (74) (75) (76) (77) (78) (79)

R.D. Hartley, E. C. Jones, andT. M. Wood, Phytochemisty, 12 (1973) 763-766.

(80) (81) (82) (83) (84)

A. J. Buchala and K. C. B. Wilkie, Phytochemisty, 12 (1973) 499-505. A. J. Buchala and K. C. B. Wilkie, Phytochemtsty, 13 (1974) 1347-1351. See Ref. 48. J.-P. Joseleau and F. Barnoud, Phytochemisty, 14 (1975) 71-75. D. Maltby, N. C. Carpita, D. Montelinos, C. Kulow, andD. P. Delmer, PlantPhysiol.,

SeeRef. 12.

V.W. Tripp, A. T. Moore, and M. L. Rollins, Tert. Res. J., 24 (1954) 956-959. P. Morrall andD. E. Briggs, Phytochemisty, 17 (1978) 1495-1502. M. C. Meinert and D. P. Delmer, Plant Physiol., 59 (1977) 1088-1097. Y. P. Cho and M. J. Chrispeels, Phytochemisty, 15 (1976) 165-169. R. L. Whistler and J. R.Young, Arch. Bfochem. Biophys., 89 (1960) 1-9. W. H. Matchett and J. F. Nance, Am.]. Bot., 49 (1962) 311-318. A. J. Buchala, Phytochemisty, 12 (1973) 1373-1376. A. J. Buchala, Phytochemisty, 13 (1974) 2185-2188. A. J. Buchalaand G. Franz, Phytockmisty, 13 (1974) 1887-1889. A. J. Buchala, G. Franz, and H. Meier, Phytochemisty, 13 (1974) 163-166. A. J. Buchala and H. Meier, Phytochemisty, 11 (1972) 3275-3278. A. J. Buchala, C. G. Frazer, and K. C. B. Wilkie, Phytochemisty, 12 (1972) 28032814.

63 (1979) 1158-1164. (85) D. J. Nevins, R. Yamamoto, and D. J. Huber, Phytochemisty, 17 (1978) 15031505. (86) A. Hillestad and J. K. Wold, Phytochemisty, 16 (1977) 1947-1951. (87) A. Hillestad and T. Engen, Phytochemfsty, 16 (1977) 1953-1956. (88) J. A. Boundy, J. S.Wall, J. E. Turner, J. H. Woychik, andR. J. Dimler,]. Biol. Chem., 242 (1967) 2410-2415. (89) J.-P. Joseleau and F. Barnoud, Phytochemisty, 13 (1974) 1155-1158. (90) C. W. Ford, Phytochemisty, 11 (1972) 2559-2562. (91) G. G. S. Dutton and M. S. Kabir, Phytochemisty, 11 (1972) 779-785. (92) Y.Ghali, A. Youssef, and E. A. El Mobdy, Phytochemisty, 13 (1974) 605-610. (93) G. R. Woolard, E. B. Rathbone and L. Novellie, Phytochemisty, 16 (1977) 961 963. (94) D. Adomako, Phytochemistry, 11 (1972) 1145-1148. (95) N . J. Barras, Phytochemisty, 12 (1973) 1331-1339. (96) H.Hori and S. Sato, Phytockmisty, 16 (1977) 1485-1487. (97) M. F. Heath and D. H. Northcote, Biochem. I., 125 (1971) 953-961. (98) R.M. Roberts, J. J. Cetorelli, E. G. Kirby, and M. Ericson, Plant Physiol., 50 (1972) 531-535. (99) G. B. Hawes and G. A. Adams, Phytochemisty, 11 (1972) 1461-1465. (100) P. Halmer andT. A. Thorpe, Phytochemtsty, 15 (1976) 1585-1588.

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Ray,53s54J01-103 Kauss,104-10e and Monro4e.52and their coworkers, as well as have used them. A further problem is that many of the methods for plant cell-wall preparation, based upon insolubility in aqueous buffers, salt solutions, and organic solvents, undoubtedly remove, or detach, some of the molecules, present in the intact wall, which may have important, structural functions. Care must be taken, for example, to avoid acidic conditions which may cleave glycosidic linkages, particularly those involving arabinofuranose, fucose, and rhamnose."' Dilute acid also hydrolyzes the ester linkages of sugar acetyl groups and the methyl esters of uronic acids. Alkaline solutions may likewise hydrolyze ester linkages, and will also catalyze the transelimination of uronic a c i d ~ , " ~and J ~thep-elimination ~ of glycosidic bonds to serine or threonine residues in g l y c o p r ~ t e i n s . ~ ~Base ~-"~ also removes hemicellulose polysaccharides from their non-covalent association with cellulose.117 Despite the elaborate washing procedures usually employed, cell-wall preparations may also frequently be contaminated with cytoplasmic constituents that sediment with the walls after tissue homogenization. Starch grains and proteins are particularly difficult to remove in this respect. Both incubation with purified alpha amylase (EC 3.2.1.l)55J01 and extraction with chloral hydratells have been utilized for removal of starch from cell-wall preparations. Pronase5'Jo1 has been used to remove proteins. The lack of specificity of the extraction methods makes it difficult to

(101) J. M. Labavitch and P. M. Ray, Plant Physiol., 53 (1974) 669-673. (102) J. M. Labavitch and P. M. Ray, Plant Physiol., 54 (1974) 499-502. (103) M. Jacobs andP. M. Ray, PlantPhysiol., 56 (1975) 373-376. (104) H. Kauss and C. Glaser, FEBS Lett., 45 (1974) 304-307. (105) H. Kauss and D. J. Bowles, Planta, 130 (1976) 169-174. (106) R. W. Bailey and H. Kauss, Planta, 119 (1974) 233-245. (107) A. L. Karr and P. Albersheim, Plant Physiol., 46 (1970) 69-80. (108) N. K. Matheson and H. S. Saini, Phytochemistry, 16 (1977) 59-66. (109) J. F. Nance, Plant Physiol., 51 (1973) 312-317. (110) W. Loescher and D. J. Nevins, Plant Physiol., 50 (1972) 556-563. (1 11) E. A. Davidson, Carbohydrate Chemistry, Holt, Rinehart, and Winston, New York, 1967. (112) P. Albersheim, Biochem. Biophys. Res. Commun., 1 (1959) 253-256. (113) H. Neukom and H. Deuel, Chem. Znd. (London), (1958) 683-684. (114) J. B. Adams, Biochem.].,94 (1965) 368-377. (115) J. B. Adams, Biochem.J.,97 (1965) 345-352. (116) B. Anderson, P. Hoffman, andK. Meyer,]. Biol. Chem., 240 (1965) 156-167. (117) J. D. Blake and G. N. Richards, Carbohydr. Res., 17 (1971) 253-268. (118) M. Knee, Phytochemisty, 12 (1973) 1543-1549.

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PRAKASH M. DEY AND KEN BRINSON

isolate well-defined fragments from the wall, and to determine how these components are linked to each other or to the residual wall-structure. Highly purified enzyme-preparations currently constitute the most effective, if costly, tool for the extraction of more-or-less defined wallpolymers,42~55-5e~e2*63~107 but even these enzymes frequently contain unpredictable, degradative activities, and fail to remove the expected amount of corresponding polysaccharides from the wall for reasons that are often obscure. Once extracted from the wall, the purification of wall polymers to homogeneity poses further problems. Such purification techniques as combined gel-filtration and ion-exchange ~ h r o m a t o g r a p h y ~ ~ * ~ ~ - ~ ~ * e6~e7~ee~101~102 are being continually improved, but, even so, the heterogeneity and complexity of interconnected glycans constitute an exacting problem. Great advances have been made in evolving sensitive techniques for sequencing glycosyl residues in purified polysaccharides. They include methylation followed by gas - liquid chromatography - mass-spectrometric analysis, partial hydrolysis with acid, acetolysis, formolysis, periodate oxidation, and the related procedure of Smith degradation, diborane reduction, and the use of highly purified endoglycanases. Detailed references for these techniques appear later in this article. All of these techniques have greatly advanced the elucidation of the primary structures of extracted, purified polysaccharides, and the major features of wall structure in higher plants are now recognized. Nonetheless, the overall complexity of these structures, and the technical problems associated with their study, suggest that a complete picture of the cell wall will not be available in the near future.

2. The Types of Cell-Wall Polysaccharides Classically, wall polysaccharides have been separated into three fractions: the pectic polysaccharides, which are extracted by hot water, ammonium oxalate solution, weak acids, or chelating agents; the hemicelluloses, which can be extracted by relatively strong alkali; and the residue remaining, which is composed mainly of cellulose. Although these extraction techniques suffer from incomplete and overlapping extraction of the polymers, they are still widely used. Pectic polysaccharides are classified as those polymers composed mainly of galactosyluronic residues, together with covalently or noncovalently bound, neutral fractions. Hemicelluloses are polymers that are probably covalently linked to pectin, and noncovalently associated with cellulose: it has been proposed that they are capable ofstrong hydrogen-

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bonding to cellulose.5s The hemicelluloses of the cell wall are the xyloglucans, heteroxylans, and xy1ans.55-57,61.119,1zo

3. Methods Used in the Elucidation of Primary-Wall Structure The techniques to be described are those used by Albersheim and his coworkers in studies that have led to a model for primary-wall structure. However, most of these techniques are well defined, and have been utilized by many workers in the field of polysaccharide structural research. Cell cultures, mainly from sycamore, were used by Albersheim’s group as a source of homogeneous tissue rich in primary wa11s.55-59*62-63 The cell-suspension cultures were washed with cold potassium phosphate buffer (pH 7.0) and then passed through a French pressure-cell. The wall preparations were isolated by centrifugation at 2,00Og, and finally washed successively with buffer, distilled water, and organic solvents. This procedure yields cell-wall material equal to 1%ofthe fresh weight of the original cells. The first step in fractionating primary walls that has normally been used by Albersheim’s team is treatment of the walls with a purified endo-a-(1-+4)-galacturonase(EC 3.2.1. 15).55This enzyme hydrolyzes a-(1-+4)-linkedgalactosyluronic linkages, resulting in the solubilization of 18% of the mass of the wall. The cell-wall residue remaining could be extracted with alkali, to yield additional pectic polysaccharides and the hemicelluloses, or be treated with a second enzyme, an endo-p(1+4)-glucanase (EC 3.2.1.4) that specifically fragments xyloglucan, a primary-wall h e m i c e l l ~ l o s eThe . ~ ~cell-wall polysaccharides solubilized by enzymes and by alkali are then separately fractionated by ionexchange chromatography on DEAE-Sephadex A-25, and gel-filtration c h r ~ m a t o g r a p h y on ~ ~Bio-Gel J ~ ~ P-2. Sensitive, colorimetric assays were used for the detection of specific residues in column fractions, namely, anthrone for hexosyl residues,lZ1orcinol for pentosyl residues,lZ1m-hydroxybiphenyl for glycosyluronic residues,lZ2Folin -Lowry reagent for proteins,lZ3and the Kivirikko - Liesmaa reagent for hydroxy-L-prolyl residues. lz4 Five noncellulosic, primary cell-wall polysaccharides from dicots have

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(1 19) A. C. Darvill, Ph.D. Thesis, University of Wales, Aberystwyth, Wales, 1976. ( 1 20) J. Darvill, A. G . Darvill, M. McNeil, and P. Albersheim, unpublished results, cited in Ref. 65. (121) 2.Dische, Methods Carbohydr. Chern., 1 (1962) 478-512. (122) N. Blumenkrantz and C. Asboe-Hansen, Anal. Biochem., 54 (1973) 484-489. ( 1 23) 0. H. Lowry. N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chern., 193 (1951) 265-275. (124) K. I. Kivirikko and M. Liesmaa, Scand.]. Clin. Lab. Inwest., 11 (1959) 128-133.

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been highly purified by this combination of techniques, namely, xyloglurhamnogalactcan,56 glucuronoarabinoxylan,120hom~galacturonan,'~~ uronan I (Ref. 125) and rhamnogalacturonan I1 (Ref. 62). The glycosyl composition of the polymers was established by hydrolysis with 2 M trifluoroacetic acid,55 and the glycosyl residues were converted into alditol acetates126for analysis by gas -liquid chromatography (g.1.c.).Glycosyluronic residues, which are subject to degradation during acid hydrolysis of their glycosidic bonds,es were quantitated by conversion into the corresponding dideuterio-labelled hexosyl residues127 before hydrolysis and acetylation. Labelled hexosyl residues were quantitatively distinguished from unlabelled hexosyl residues by g.1.c. -mass spectrometry (g.1.c. -m.s.). Clycosyl-linkage composition analysis was carried out by methylation. The partially methylated aldoses subsequently obtained by hydrolysis were then reduced to the corresponding alditols, and these were acetylatedlZ6;the methylated alditol acetates were quantitated, and tentatively identified, by flame ionization g . l . ~ . , and ~ ~then J ~ ~their structures were confirmed by g.1.c. - m.s.129Chemical ionization-mass spectrometry'30 proved to be of value in augmenting electron ionization-mass spect r ~ m e t r y in '~~ identifying these derivatives (see also, Refs. 130a-d). Before linkages to the glycosyluronic residues were ascertained, the residues were reduced by the carbodiimide method127to their corresponding deuterium-labelled alditols. Various techniques have been utilized for sequencing the glycosyl residues of polysaccharides and oligosaccharides. These techniques are elaborations of the commonplace procedure of converting polysaccharides, by partial hydrolysis, into structurally analyzable oligosaccharides. This conversion is generally achieved by partial hydrolysis with acid, by a c e t ~ l y s i s ,or ' ~by ~ formolysis.s2These reagents may differ in the (125) A. Darvill, M. McNeil, and P. Albersheim, unpublished results, cited in Ref. 65. (126) P. Albersheim, D. J. Nevins, P. D . English, and A. Karr, Carbohydr. Res., 5 (1967) 340-345. (127) R. L. Taylor and H. E. Conrad, Biochemistry, 11 (1972) 1383-1388. (128) P. A . Sandford and H. E. Conrad, Biochemistry, 5 (1966) 1508- 1513. (129) H. Bjorndal, C. G . Hellerqvist, B. Lindberg, G . Fahraeus, and S. Svensson, Angew. Chem., Int. Ed. Engl., 9 (1970) 610-616. (130) M. McNeil and P. Albersheim, Carbohydr. Res., 56 (1977) 239-248. (130a) H. Rauvala, J. Finne, T. Krusius, J. Kiirkkainen, and J. Jiirnefelt, Adu. Carbohydr. Chem. Biochem., 38 (1981) 389-416. (130b) H. R. Morris, A. Dell, and R. A. McDowell, Mass Spectrom., 8 (1981) 463-473. 257 (1982) (130c) L. S. Forsberg, A. Dell, D. J. Wdton, and C. E. Ballou,]. Biol. Chem., 3355-3363. (130d) A. Dell and H. R. Morris, Carbohydr. Res., 115 (1983) 41-52. (131) I. Danishefsky, R. L. Whistler, andF. A. Bettelheirn, in W. W. PigmanandD. Horton (Eds.), The Carbohydrates, Vol. IIA, Academic Press, New York, 1970, pp. 375410.

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rate at which they cause the hydrolysis of different glycosidic linkages and, therefore, the methods yield different sets of oligosaccharides from the same polysaccharide. A similar goal of converting polysaccharides into manageable oligosaccharides was also achieved by utilization of highly purified endoglycanases which included endo-/?-(1 - + 4 ) - g l u ~ a n a s endo-a-( e , ~ ~ l-+4)-galactu r o n a ~ e , ~endo-/?-( ~ J ~ ~ 1 - 4 ) - g a l a c t a n a ~ e , ' ~and ~ endo-a-(1+5)-arabina~iase.~~J~~ Polysaccharides have also been specifically cleaved into sets of analyzable fragments by periodate oxidation and, in particular, by using the related procedure known as Smith d e g r a d a t i ~ n . ' ~ ~ Analysis . ' ~ ~ of the Smith-degradation products yields information regarding the sequence of glycosyl residues in the original polysaccharides, and their chain lengths.

111. THEPECTICPOLYSACCHARIDES 1. Dicotyledonous Plants

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Pectic polysaccharides make up 35% of the dicot primary-wall, and the main components present in these polysaccharides are galactosyluronic residues. The most characteristic, physical property of these polymers is their ability to form gels.32J35J36The middle lamella, which lies between the primary walls of adjacent cells, is particularly rich in pectic polysa~charides.'~~ Other major monosaccharide components found in pectic polysaccharides are rhamnose, arabinose, and galactose. Rhamnose and galacturonic acid are found in the rhamnogalactosyluronic pectic backbone, arabinose and galactose are associated with arabinan, and galactan side chains are apparently covalently linked to the rhamnogalactosyluronic backbone. Apart from these side chains, some of the arabinose and galactose residues may very likely be present in arabinogalactans, but it is unclear whether, in the cell wall, the latter are covalently In the sycamore primary-wall, attached to the rhamnogalacturonan.5s~64 it appears probable that the pectic polymers also contain free g a l a ~ t a n ~ ~ (132) J. M. Labavitch, L. E. Freeman, and P. Albersheim, J. Biol. Chem., 2.51 (1976) 5904 - 59 10. (133) A . Kaji a n d T . Saheki, Biochim. Biophys. Acta, 410 (1975) 354-360; A. Kaji, This Vol., pp. 383-394. (1 34) N. Sharon, Complex Carbohydrates: Their Chemistry, Biosynthesis and Functions, Addison- Wesley, Reading, Massachussetts, 1976. (13.5) G . T. Grant, E. R.Morris, D. A. Rees, P. Smith, and D. Thom, FEBSLett., 32 (1973) 195- 198. (136) H. G . J. Worth, Chem. Reo., 67 (1967) 465-473. (137) M. A. Hall, in M. A. Hall (Ed.), Plant Structure, Function and Adaptation, Macmillan, London, 1976, pp. 49-90.

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and free arabinanlz5as polymers distinct from the galactan and arabinan side chains of rhamnogalacturonan. a. Rhamnogalacturonan I. -Rhamnogalacturonan is considered to be the backbone of the pectic polymers. Studies by Aspinall and his associate^,^^^-^^^ for example, on rapeseed hull, soybean cotyledon, lucerne leaves and stem, and lemon peel, which are tissue sources containing both primary and secondary walls, indicated that galacturonic acid residues are linearly linked a-(1+4), and that many L-rhamnose residues present in the chain are linked through 0 - 2 to galacturonic acid. Methylation analysis of the intact polysaccharides from rapeseed138 and luerne el^^ demonstrated that -50% of the rhamnosyl residues are 2linked, but the other 50% are 2,4-linked. No aldobiouronic acid having a galactosyluronic residue attached to 0 - 4 of a rhamnosyl residue has been isolated, and it is assumed that 0 - 4 of rhamnose is the point ofattachment of other neutral, glycosyl residues, such as 5-linked arabinosyl and several differently linked galactosyl r e ~ i d u e s . ~ The ~ J way ~ ~ in which the various aldobiouronic acids and oligosaccharides containing galacturonic acid and rhamnose, which have been released from rhamnogalacturonan by partial acid hydrolysis, are arranged in the intact polymer has not yet been e ~ t a b l i s h e d . ~ ~ J ~ ~ A polymer termed rhamnogalacturonan I has been isolated by enzymic from primary cell-walls of cultured hydrolysis, by Albersheim’s sycamore cells; it is composeds5 of rhamnose, galacturonic acid, arabinose, and galactose in the molecular ratios of 1 : 2 : 1.5: 1.5.Gel-filtration chromatography of the polysaccharide suggested a molecular weight of the order of 5 X 1Os with a degree of polymerization of 2000. Chromatography in 0.5 MNaCl containing 5 mMEDTA indicated that this large size is not due to noncovalent aggregation. If the backbone of rhamnogalacturonan I is a single, linear chain, this chain contains about 300 rhamnosy1residues and 600 galactosyluronic residues. This is a major, upward revision of the size of rhamnogalacturonan, compared to that envisaged earlier.55 Linkage analysis of the polymer isolated from suspension-cultured, sycamore cells i n d i ~ a t e dratios ~ ~ . ~of~%linked rhamnosyl to 2,4-linked rhamnosyl to 4-linked galactosyluronic residues of 1 : 1 : 4 (see Fig. 1).

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(138) G. 0. Aspinall and K. S.Jiang, Carbohydr. Res., 38 (1974) 247-255. (139) G. 0. Aspinall and J . A. Molloy,]. Chem. Soc., C, (1968) 2994-2999. (140) G. 0.Aspinall, J. W. Cottrell, S.V. Egan.1. M. Morrison, and J. N. C. Whyte,]. Chem. Soc., C, (1967) 1071-1080. (141) G. 0.Aspinall, J. W. T. Craig, and J. L. Whyte, Carbohydr.Rex, 7 (1968) 442-452. (142) G. 0. Aspinall, B. Gestetner, J. A. Molloy, and M. Uddin,]. Chem. Soc., C, (1968) 2554-2559.

. a, I(“;o) 1

0

I

Structure for Pectic Rhamnogalacturonan (I) of Primary Cell-Walls of Dicots (After A l b e r ~ h e i m ” ~[Linear ~). sequences of galacturonic acid (Gal) are interrupted by rhamnosyl (Rha) residues, half of the latter being covalently attached to arabinogalactans.]

FIG. 1. -Proposed

280

PRAKASH M. DEY AND KEN BRINSON

b. Homogalacturonan. -Also within the acidic, pectic polysaccharides from sycamore are regions of unbranched a-(1-*4)-linked galactosyluronic r e s i d ~ e s . ~ Polymers ~ J ~ ~ J of ~ ~this type have been isolated by degradation procedures from sunflower seeds144and apple pectin.145 The a-(1+4)-linked galacturonan released by endogalacturonanase from sycamore cell-walls accounts for 1- 2% of the starting wall-material, and is stable to further enzymic hydrolysis due to esterification of the carboxyl g r o ~ p s ~of~the , ' glycosyluronic ~~ acids. Gel-filtration chromatography of the sycamore homogalacturonan indicated'25 a degree of polymerization > 25. The carboxyl groups of the galactosyluronic residues of the cell-wall, pectic polysaccharides from many plants are known with various degrees of esterito be methyl-esterified138J3QJ41J42J46J47 fication, depending on the species. The distribution of the methyl esters along the galacturonan backbone has not yet been established, but, for the sycamore polysaccharide, the reaction pattern of endogalacturonanase indicated that there are regions that are highly methyl-esterified, as well as regions relatively free from methyl esters.'25 c. Hhamnogalacturonan II. -This polysaccharide fraction was isolated by Albersheim and coworkerss2 from suspension-cultured, sycamore cell-walls by endogalacturonanase treatment.e2 Hydrolysis of the polymer yields the rarely observed, cell-wall sugars 2-O-methyl-Lfucose. %o-methyl-D-xylose, and D-apiose; the two methylated sugars have long been recognized as trace components of pectic polymers in apple,'45 lucerne, lQ8soybean,148and sisal,14Qand apiose has been found in the pectic polymers of Lemna species (see later).150-153 However, this reporte2was the first recorded instance of all three of these sugars being associated in a single pectic polysaccharide. Rhamnogaiacturonan I1 has been found to contain 25 - 50 glycosyl residues.65 The large number and variety of terminal glycosyl residues in the polymer suggested a highly branched molecule. The structure contains 2-linked glucosyluronic, 3-linked apiosyl, 3-linked rhamnosyl, 2,4(113) P. D. English, A. Maglothin, K. Keegstra, and P. Albersheim, Plant Physiol., 49 (1972) 293-297. (144) V. Zitko and C. T. Bishop, Can. J. Chem., 14 (1966) 1275- 1282. (145) A. J . Barrett and D. H. Northcote, Biochem. J., 94 (1965) 617-627. (146) G . 0. Aspinall and R. S. Fanshawe.]. Chem. Soc., C, (1961) 4215-4221. (147) I. R . Siddiqui and P. J. Wood, Curbohydr. Res., 30 (1976) 97-107. (148) G . 0. Aspinall, K. Hunt, and I. M. Morrison,]. Chem. Soc., C, (1967) 1080-1086. (149) G . 0. Aspinall and A. Canas-Rodriguez,]. Chem. Soc.. C, (1958) 4020-4026. (150) R. B. Duff, Biochetn. J., 94 (1965) 768-772. (151) E. Beck, Z. Pflanzenphysiol., 57 (1967) 444-450. (152) D. A. Hart and P. A. Kindel, Biochem.]., 116 (1970) 569-579. (153) D. A. Hart and P. A. Kindel, Biochemistry, 9 (1970) 2190-2196.

PLANT CELL-WALLS

28 1

linked galactosyl, 3,4-linked rhamnosyl, and 3,4-linked fucosyl residues. Terminal sugars include galacturonic acid, galactose, arabinose, rhamnose, 2-O-methylfucose, and 2-0-methylxylose. Indeed, rhamnogalacturonan I1 appears to be the most structurally complex, plant polysaccharide yet found, but little is known of its detailed structure.62

d. Apiogalacturonan. -This apparently rare polymer has been isolated150-153from cell walls of duckweed (Lemna minor). Apiose and galacturonic acid are its only constituent sugars. Apiose-containing galacturonans that probably contain other glycosyl residues have been isolated from other plant genera, including Zostera and Po~idonia,'~~ but they have not been established with any certainty as components of primary cell-walls. The evidence available suggests that the polymer in Lemna consists of an a-(1+4)-linked galacturonan backbone, with side chains of apiobiose, ~-Apif-(1+3)-~-Apif.The degree of methyl esterification of the galactosyluronic residues is low. The nature of the galactosyluronic - apiosyl linkage has not yet been established. e . Arabinan. -An arabinan essentially free from other polysaccharides has been isolated from primary cell-walls of suspension-cultured, sycamore cells following enzymic hydrolysis. l Z 5Methylation analysis of the primary walls of pea cells155also strongly suggested the presence of a primary-wall arabinan. The limited evidence available suggests that these primary-wall arabinans are similar in structure to arabinans that have been isolated from other dicot tissues, containing secondary walls, which include willow,156Rosa glauca bark,157 aspen bark,158 soybean lemon-peel pectin,15Qand mustard cotyledons.160 The arabinans from all of these plant sources exhibit similar structural features. They appear to be highly branched with the L-arabinose residues largely in the furanose form. Glycosidic linkages are uniformly in the a-L-anomeric configuration; 5-,3,5-, and 2,5-linked arabinosyl residues have been detected. Willow arabinan has156a degree of polymerization of 90, and Rosa glauca bark contains two arabinans, having degrees ~' of polymerization of 34 and 100, r e ~ p e c t i v e l y . ~Smith-degradation (154) J. S. D. Bacon and M. V. Cheshire, Biochem. J . , 124 (1971) 555-562. (155) N. R. Gilkes and M. A. Hall, New Phytol., 78 (1977) 1 - 12. (156) S. Karacsonyi, R. Toman, F. JaneCek,and M. Kubafkova, Curbohydr.Res., 44 (1975) 285-290. (157) J.-P.Joseleau, G. Chambat, M. Vignon,and F. Barnoud, Carbohydr.Rex, 58 (1977) 165-175. (158) K. S. Jiang and T. E. Timell, Cellul. Chetn. Technol., 6 (1972) 499-502. (159) G. 0.Aspinall and I. W. Cottrell, Can.]. Chem.,49 (1971) 1019-1022. (160) D. A. Rees and N. G. Richardson, Biochemistry, 5 (1966) 3099-3107.

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studies on mustard-cotyledon arabinan, conducted by Rees and Richardson,16oprecluded the occurrence of regions of long, unbranched chains of 5-linked arabinosyl residues. Methylation analysis indicated that the primary-wall arabinans of cultured s y c a m ~ r e - c e l l and s ~ ~pea ~ cells155share these general structural features. However, glycosyl-linkage analyses of the pectic polysaccharides of sycamore primary c e l l - ~ a l l sand , ~ ~studies using mild acid hydrolysis for selective cleavage of the furanosyl linkages,5s suggested the presence, within the sycamore polymer, of unbranched, 5-linked homoarabinan regions, in contrast to the mustard-cotyledon160arabinan. Clearly, branched arabinans are important, primary cell-wall components, but considerable further study is needed before it will be possible to draw even a tentative structure for the intact-wall component. Isolation and analysis of the arabinan of sycamore cell-wall is of current interest, a study augmented by the availability of two purified enzymes, endo-a-( 1 + 5 ) - a r a b i n a n a ~ e ~and ~ J ~exo-a-arabino~idase.~~ ~

f. Galactan. -No homogalactan has been isolated directly from primary cell-walls. However, glycosyl-linkage studies with the pectic polymers from suspension-cultured, sycamore cells55revealed galactosyl residues having similar linkages, in similar ratios, to those present in homogalactans from heterogeneous tissue-preparations from dicots, for example, citrus pectin,132white willow,A61and beech,162 Pectic galactans appear to be primarily P-D-(l+4)-linked polymers. The ( 1 4 4 ) linkage has been established by methylation a n a l y ~ i s , ' ~ ~ J ~ ~ and the units have been shown to be in the /?-D configuration by (a) the fact that they are susceptible to hydrolysis by a p-( 1+4)-endogalactanase132and (6)the low-positive, optical rotations of the p ~ l y m e r s . ' ~ ~ T h e / ? configuration of some of the galactosidic linkages in oligosaccharides derived by partial, acid hydrolysis from beech galactan162has been established by chromatographic comparison to known standards. Those galactans that have been studied (see the preceding) have degrees of polymerization ranging from 33 in white willowlel to 50 in sycamore-cell p r i m a r y - ~ a l l sThese . ~ ~ ~ values were obtained by vapor pressure and osmosis studies,A61and by comparing the ratio of terminal to intrachain galactose units by methylation a n a 1 y ~ i s . l ~ ~ It is probable that many of the galactosyl residues of pectic polymers are not part of the homogalactans. In rapeseed hull, the galactosyl residues attached to the uronic acid backbone have been shown to occur as p-(1+4)-linked dimers, not as longer oligosaccharides or polymers.'3s (161) R. Toman, S. Karacsonyi, and V. Kovaeik, Carbohydr. Res., 25 (1972) 371-378. (162) H. Meier,Acta Chem. Scand., 16 (1962) 2275-2283. (163) M. McNeil and P. Albersheim, unpublished results, cited in Ref. 65.

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Pectic polysaccharides in Rosa glauca bark157 and lucerne leaves and stems139have been shown to contain 3- and 6-linked galactosyl residues. The sycamore, primary cell-walls contain appreciable proportions of ~ ~ it J ~is ~ terminal and 3-,6-, 3,6-,and 2,6-linked galactosyl r e s i d u e ~ , and possible that many of these residues are components of an arabinogalactan (see later). The galactans from white willow,161beech,ls2 and sycamore-cell prim a r y - ~ a l l contain s ~ ~ ~ 6-linked, as well as 4-linked, galactosyl residues. The beech galactan contains a high proportion of 6-linked residues, but, in the galactans of white willow and sycamore-cell primary-wall, 6linked galactosyl residues account for only 4% of the total galactose present. The presence of a homogalactan in sycamore-cell primary-walls has not been proved, but was inferred from the detection of large proportions of 4-linked galactosyl residues by methylation analysis of the wall, and of pectic fractions from the wa11.55J63In addition, endo-p(1+4)-galactanase releases relatively large proportions of small, p-( 1+4)-linked, galactose-containing oligosaccharides from the primary walls of sycamore cells.132 As with arabinan, the present information is too limited to permit drawing any conclusion regarding the definitive structure of the galactan of primary cell-walls, still less to state with authority whether primarywall galactan contains glycosyl residues other than p-(1+4)-linked galactose. The oligosaccharides Gal-( 1+2)-Xyl, GlcA-(l-*6)-Gal, and GlcA-(1-*4)-Gal have been isolated from soybean pectin,148 and GalA-(1+4)-Gal has been isolated from white willow-bark pectin.ls4 It is not known whether these oligosaccharides are constituents of the polymers of primary cell-walls. g. Arabinogalactan. -No arabinogalactan has been isolated from primary cell-walls of dicots. However, arabinogalactans have been obtained from a number of dicot tissue-preparations containing secondary walls. These include rapeseed ~ o t y l e d o n , ~Japanese-larch ~~*'~~ and soybean cotyledon. 168-170 There is considerable variation in the glycosyl composition of these arabinogalactans. The rapeseed polymer166 con(164) R.Toman, S.Karacsonyi, and M. KubaEkovB, Carbohydr. Res.,43 (1975) 111-116. (165) I. R. Siddiqui and P. J. Wood, Carbohydr. Res., 24 (1972) 1-9. (166) 0. Larm, 0. Theander, and P. Aoman, Acta Chem. Scand., Ser. B, 30 (1976) 627630. (167) G. 0.Aspina1l.R. M. Fairweather, andT. M.Wood,J. Chem. SOC., C , (1968) 21742179. (168) G . 0.Aspinall, R. Begbie, A. Hamilton, and J. N. C. Whyte,]. Chem. Soc., C , (1967) 1065-1070. (169) M . Morita, Agric. B i d . Chem., 29 (1965) 564-573. (170) M. Morita, Agric. Biol. Chem., 29 (1965) 626-630.

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tains 90% of arabinosyl residues, whereas the larch polymer'67 contains 88%of galactosyl residues. Rapeseed c o t y l e d ~ n ' ~and ~ Jsoybean ~~ cotyl e d ~ n ' ~ * arabinogalactans -'~~ contain no rhamnose, and larch arabinog a l a ~ t a n 'contains ~~ only traces of this sugar. Despite this variation, arabinogalactans appear to be structurally related, possessing galactan backbones composed of (3+6)-linked galactosyl residues, with (terminal) arabinofuranosyl groups attached as side chain^.'^^-'^^ The soybeancotyledon polymer, which has been isolated and characterized by both Aspinall and associates'68 and M ~ r i t a , ' ~has ~ *a 'structure ~~ very different from those of all of the other arabinogalactans so far studied. It possesses a p-( 1+4)-linked D-galactosyl backbone, with arabinosyl dimers, Araf-( 1+5)-Araf, linked to 0-3 of some of the galactosyl residues. p-~-Gal-( 1+3)-~-Gal, p-~-Gal-( 1+6)-~-Gal, and p-~-Arap-( 1+3)-~Araf have been isolated from larch a r a b i n ~ g a l a c t a n l ~ by~ acid hydrolysis. Smith degradation of the larch16' and rapeseed'6sJ66 polysaccharides supported the conclusion that the backbones are galactans with the galactosyl residues /I-glycosidically linked to each other through 0 - 3 or 0 - 6 , or both. Arabinogalactans that may have their origin in the primary cell-wall have been isolated from the extracellular medium of suspension-cultured, sycamore168J71J72and tobacco172cells. The general structural features of these polysaccharides are similar to those of other arabinogalactans so far s t ~ d i e d . ' ~ The ~ - ' disaccharides ~~ p-~-Gal-( 1-'3)-~-Gal and p-~-Gal-( 1+6)-~-Galhave been isolated by hydrolysis from both polymers, and the disaccharide P-~-Arap-( 1+5)-~-Araf has been isolated from the extracellular polymer from cultured tobacco-cells.'72 Smith degradation of this cultured, tobacco-cell polymer172supported the conclusion that these arabinogalactans possess (3+6)-linked galactosyl backbones having arabinofuranosyl terminal groups. The presence of arabinogalactans in primary cell-walls is supported primarily by the results of a single with an endogalacturonanasereleased pectic fraction obtained from the walls of suspension-cultured, sycamore cells. The main arabinosyl- and galactosyl-containing components present in this pectic fraction appear to originate from a separate p-( 1+4)-linked galactan and a highly branched arabinan. However, glycosy1 linkages were also detected that were characteristic of arabinogalactans. The endogalacturonanase-released pectic polysaccharides contained 3-, 6-, and 3,g-linked galactosyl residues and terminal groups. These residues were detected in amounts totalling -5% of the pectic fraction. In addition, 3-, 5-, and 2,5-linked arabinofuranosyl residues and (171) G . 0.Aspinall, J. A. Molloy, and J. W. T. Craig, Can. J . Biochen., 47 (1969) 10631070. (172) K. Kato, F. Watanabe, and S. Eda, Agric. Biol. Chem., 41 (1977) 533-538.

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terminal groups were detected in substantial proportions in the pectic polysaccharides, but these could have originated from a branched arabinan. Partial, acid hydrolysis of the endogalacturonanase-released, cell-wall polysaccharides did not significantly alter the proportions of the branched galactosyl residues that were detected. This study did allow the suggestion that the primary cell-walls of sycamore may contain an arabinogalactan similar to that isolated from larch167; sycamore, primary-wall arabinogalactan may, however, possess a lower percentage of arabinosyl sidechains. It should also be critically noted that the residues detected could have arisen from the presence, in the sycamore primarywall, of both a branched arabinan and a branched galactan lacking arabinosyl sidechains. A highly tentative, proposed structure of a pectic arabinogalactan (arabinan - galactan?) in primary cell-walls of dicots is shown in Fig. 2. 2. Monocotyledonous Plants The pectic polymers of monocots have not been as extensively studied as those of the dicot, primary cell-walls; they appear to contain only minor proportions of these polysaccharides. Wada and Ray,53for example, established that the principal matrix-polysaccharides of oat-coleoptile cell-walls are glucuronoarabinoxylans and hemicellulosic P - D - ~ ~ u cans, rhamnogalacturonan being only a minor component. Ray and R ~ t t e n b e r gestimated '~~ that galactosyluronic residues account for 3%of the cell wall in this tissue; corresponding values of 6 and 1.3%have been given for maize c ~ l e o p t i l e "and ~ maize-root rneri~tern,"~ respectively. Homogalacturonans, rhamnogalacturonans, and arabinogalactans have never been isolated from the cell walls of monocots. However, a polysaccharide rich in galacturonic acid and glucose, but lacking galactose, arabinose, or rhamnose, has been isolated from oat-coleoptile cellbut no evidence was presented as to whether the glucosyl residues were covalently linked to the galactosyluronic residues. A disaccharide of galacturonic acid has also been isolated from the cell wall of and 4-linked galactosyluronic residues have been detected in oat- and maize-coleoptile c e l l - ~ a l l s ' ~by~ methylation J~~ analysis. These findings suggest that monocot cell-walls may contain small proportions of homo galact uronan . (173) P. M. Ray and D. A. Rottenberg, Biochem. J . , 9 0 (1964) 646-655. (174) J. E. Dever, Jr., R. S . Bandurski, and A. Kivilaan, Plant Physiol., 43 (1968) 50-56. (175) A. G. Darvill, C. J. Smith, and M. A. Hall, in E. Marre and 0.Ciferri (Eds.), Regulation of Cell Membrane Actioities in Plants, North Holland, Amsterdam, 1977, pp. 275- 282.

47

-0

FIG.2. -Proposed Structure for Pectic Arabinogalactan of Primary Cell-Walls of Dicots (After Albersheim5*"4). [Arabinans and galactans are believed to be homopolysaccharides that are interconnected, although it is possible that these two polymers are individually attached to the rhamnogalacturonan. Xyloglucan molecules are covalently bonded through their reducing ends to the galactan. It is presumed that the arabinogalactan interconnects the xyloglucans and the rhamnogalacturonans, but the details of the interconnections are unknown. Gal = D-galactose; A = ~-arabinohranose.]

PLANT CELL-WALLS

287

Hydrolysis of oat-coleoptile walls yields173 the disaccharide GalA-(1+2)-Rha, and small proportions of 2-0-methyl-D-xylose and 2-0-methyl-L-fucose, sugars characteristic of the rhamnogalacturonan I1 of dicots, have also been obtained from this source.62However, if rhamnogalacturonan I1 is present in monocot cell-walls, it is present at a concentration of, at most, one-tenth of that in the walls of dicots. Galactosyl residues having 3, 6, and 3,6 links, characteristic of arabinogalactans in dicots, are present in the cell walls of suspension-cultured cells from wheat- and rice-root, and oat and brome-grass embryo.60Arabinogalactan possessing similar linkages to the dicot polysaccharide, and bonded to protein, has also been isolated from many r n o n o c ~ t s ,but ~~~-~~~ this glycoprotein, which has lectin properties, is not considered to be a cell-wall component.176 There is no evidence for the presence of pectic arabinans or galactans in monocot, primary c e l l - w a l l ~ . ~ ~

IV. THEHEMICELLULOSES 1. Dicotyledonous Plants

a. Xyloglucan. -This is probably the most extensively studied hemicellulosic polysaccharide of primary cell-walls. It was first isolated by Aspinall and associate^'^^ from the medium of suspension-cultured, sycamore cell-walls, and later from the primary walls of these cells by Albersheim and coworkers.56Before the importance of xyloglucan as a cellwall component was realized, similar polysaccharides, which were termed amyloids, were known to occur as components of the seeds of some plant species. i80-185 The seed sources of xyloglucan, from which the polysaccharide has nasbeen isolated, and characterized, include Tamarindus indi~a,'~~*'~~ turtium (Tropeoleurn majus) ,182-186 and rape (Brassica campes( 1 76) R . L. Anderson, A. E. Clarke, M. A. Jermyn, R . B. Knox, and B. A. Stone, Aust.]. Plant Physiof, 4 (1977) 143-158. (177) G. B. Fincher andB. A. Stone,Aust.J. B i d Sci., 27 (1974) 117-132. (178) E. Maekawa and K. Kitao, Agric. Biol. Chem., 38 (1974) 227-229. (179) H. Neukom and H. Markwalder, Carbohydr. Res., 39 (1975) 387-389. (180) G. 0. Aspinall, T. N . Krishnamurthy, and K.-G. Rosell, Carbohydr. Res., 55 (1977) 11-19 (181) S . E. B. Could, D. A. Rees, and N. J. Wight, Biochem. J . , 124 (1971) 4 7 - 5 3 . (182) D. S . Hus and R. E. Reeves, Carbohydr. Res., 5 (1967) 202-209. (183) P. Kooiman, R e d . Trau. Chim. Pays-Bas, 80 (1961) 849-865. (184) 1. R. Siddiqui and P. J. Wood. Carbohydr. Res., 17 (1971) 9 7 - 1 0 8 . (18.5) I. R . Siddiqui and P. J. Wood, Carbohydr. Res., 5 3 (1977) 8 5 - 9 4 . (186) J. E. Courtois and P. Le Dizet, An. Quim.,70 (1974) 1067-1072.

288

PRAKASH M. DEY AND KEN BRINSON

tris),160~184~185 Their tissue location is not known. Xyloglucans that proba-

bly originate in the primary cell-wall have been isolated from the media ~ ~ (Rosa of suspension-cultured bean (Phaseolus v u l g ~ r i s ) , rose g l a u ~ a ) , and ' ~ ~ ~ y c a m o r e ~cells, ~ * ' ~and ~ from the walls of suspension)~~ cultured sycamore5s and bean (Phaseolus ~ u l g a r i s cells. All of the aforementioned xyloglucans share common structural features, although the glycosyl composition varies somewhat with the source. They all possess a backbone of p-(1+4)-linked D-glucosyl residues and, in the intact, primary cell-wall, this glucan backbone is believed to be hydrogen-bonded to ~ e l l ~ l o ssingle, e ~ ~xylosyl ~ ~ ~residues ~ ~ ~ ; 1+6), to the glucan backbone. In an elegant are glycosidically linked, a-( study of Tamarindus indica xyloglucan conducted by Kooiman,lE3after enzymic degradation of the polysaccharide, almost all of the xylose residues were recovered in the disaccharide a-~-Xyl-( 1 + 6 ) - ~ - G k . The cu-(1+6) nature of this glycosidic linkage has been confirmed in the case of xyloglucans from rapeseedIE1and from the primary cell-walls of suspension-cultured, sycamore cells56 and bean cells.58 In the primary cell-wall xyloglucans, some of the xylosyl residues are terminal, whereas others are substituted with p-( 1+2)-linked D-galactosyl r e ~ i d u e s . ~ ~ Xyloglucans *~~f'~ of seeds appear to share this structural feature; the disaccharide /3-D-GaI-(1+2)-D-Xyl has been isolated following hydrolysis of the xyloglucans from rape,lsO nasturtium,182 and Tamarindus i n d i c ~ 'seeds. ~ ~ In the primary ceil-wall, terminal fucosyl the nature of this groups are linked to these galactosyl linkage has not yet been established, but the likelihood that it is a-L-fucosy1 linked to 0 - 2 of galactose is suggested by the fact that the linkage is hydrolyzed by an enzyme mixture known to contain an a-(1+2)-fucosidase.166J6gThe seed xyloglucans lack these terminal fucosyl g r o ~ p s . ' ~ As ~ - seed ' ~ ~ tissue is likely to contain secondary walls, it has been postulateds5 that fucosyl groups may be removed from the primary cell-wall xyloglucan during secondary differentiation of the wall. However, primary cell-walls may contain xyloglucans, as yet undetected, lacking fucosyl groups. Arabinopyranose, a minor constituent of primary-wall xyloglucans, is ~~-~~ attached to a few glucosyl residues of the polymer b a c k b ~ n e . " *The glycosidic linkage is believed to be Arap-( 1+2)-Glc. The structure of the xyloglucans from the walls and culture medium of suspension-cultured sycamore-cells has been investigated by Albersheim and associates56by utilizing purified endo$-( 1+4)-glucanase for

(187) F. Barnoud, A. Mollard. and G. G. S. Dutton, Physiol. Veg.,15 (1977) 153-161. (188) 0 . P. Bahl,]. B i d . Chem., 245 (1970) 299-304. (189) B. S. Valent, M. McNeil, and P. Albersheim, unpublished results, cited in Ref. 65.

PLANT CELL-WALLS

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digestion of the polysaccharides. The oligosaccharides produced were fractionated by Bio-Gel P-2 chromatography into four, quantitatively major, components: a void peak and the oligosaccharides, A, B, and C, composed of 7, 9, and 22 glycosyl residues respectively. The proposed structures of the oligosaccharides are shown in Fig. 3. The relative, molar proportions of the xyloglucan oligosaccharides A, B, and C suggest that these occur in the xyloglucan chains in the ratios of 10 : 10 : 1.The smallest possible xyloglucan that can be constructed from 10 repeating units each of oligosaccharides A and B and a single unit of oligosaccharide C would contain 182 glycosyl residues. However, the molecular weight of sycamore-cell primary-wall xyloglucan has been estimated as 7600, representing 50 glycosyl r e s i d ~ e sa,value ~ ~ ~that ~~ compares favorably with that found by K ~ o i m a nfor ' ~ the ~ xyloglucan of Tamarindus indica seed. Because the value of 182 glycosyl residues is much greater than the experimentally determined 50 glycosyl residues (based upon molecular-weight determination), it suggests that at least two different xyloglucan species exist in sycamore-cell primary-wall. For example, there could be one species made up of a dimer of oligosaccharide C and another species made up of 3 molecules each of oligosaccharides A and B . If this were true, the ratio of the two species would be one molecule of the former to 7 molecules of the latter. Clearly, the exact structure of the primary-wall xyloglucan is not known. Indeed, the following additional uncertainties concerning the structure of this polysaccharide remain to be elucidated. (I) The Arap-(l+2)-Glc linkage shown in oligosaccharide Cis based solely (and insubstantially) on the finding that equimolar proportions of terminal arabinopyranosyl and 2,4,6-linked glucosyl residues are present. (2) Some glucosyl residues could be attached to 0 - 6 of other glucosyl residues, with an equivalent number of xylosyl residues attached to 0 - 4 of glucosyl residues. This possibility has not been precluded for primary cell-wall xyloglucans. (3) The anomeric nature of the glycosidic linkages has not been carefully determined for cell-wall xyloglucans. They are assumed to be the same as those present in the seed x y l o g l ~ c a n s , ~ ~ ~ J ~ ~ that is, all of the glucosyl and galactosyl residues are P-linked, and the xylosyl residues are a-linked. The fucosyl residues are also presumed to be a-linked.188.189

-

-

b. Glucuronoarabinoxylan. -The first glucuronoarabinoxylan to be clearly established as a constituent of a dicot primary cell-wall was extracted from suspension-cultured sycamore-cells.120This polymer constituted 5 % of the wall, and contained 4-, 2,4-, and 3,4-linked xylosyl residues and terminal groups (totalling 69 mol%), 2-linked arabinofuranosyl residues and terminal groups (totalling 17 mol%), terminal glucosyluronic residues (10 mol%), and terminal 4-O-methyl-~-glucosy~-

‘“1 u Heptasaccharide A

’

G

r?

I

Olrgosaccharide C

Pentaascchdrlde D

Nonvacchartde B

FIG.3. -Proposed Structure of a Portion of the Hemicellulosic Xyloglucan of the Primary Cell-Wall of Dicots (After Alber~heirn~.”~). [Heptasaccharide “A” and nonasaccharide “B” are derived from oligosaccharide “C” by the action of endo-(1+4)-P-~-glucanaseat the bonds indicated by arrows. Pentasaccharide “ D ” is derived from “B” bv the combined action of a-L-fucosidase. a-D-xvlosidase, and b-D-ducosidase. A = Larabinopyranose; F = L-fucose; G = D-glucose; Gal = D-galactose; X = ~-xylose.] I

L

PLANT CELL-WALLS

29 1

uronic residues (2 niol%). Studies designed to establish the covalent structure of this polymer are essential.

2. Monocotyledonous Plants a. Xyloglucan. -There is some evidence for the presence of xyloglucan in the primary cell-walls of monocots, albeit accounting for only -2% of the cell wall, as opposed to 19% in dicots. This evidence is largely based on the isolation of 4,6-linked glucosyl residues from monoresidues is not cot primary c e l l - ~ a l l s The . ~ ~occurrence ~ ~ ~ ~ ~of~ such ~ unequivocal proof of the presence of xyloglucan, but it is suggestive, in that 4,e-linked glucose appears to be present only in the xyloglucan fraction of dicot c e l l - ~ a l l s . ~ ~ . 5 ~ * ~ ~ In addition, a fraction rich in 4,6-linked glucosyl residues has been isolated from oat-coleoptile walls (a preparation that probably consisted largely of primary walls) that had been subjected to digestion with a crude enzyme-preparation containing an endo-/.?-(1 + 4 ) - ~ - g l u c a n a s e . ~ ~ Among the products were the trisaccharide Xyl-( 1+6)-Glc-( 1+4)-Glc and a pentasaccharide having the following structure.

-

XY 1 1

Xyl 1

i

4

6 6 Glc-(144)-Clc-(1-+4)-Glc

Such products would be expected to arise from similar treatment of xyloglucans from dicots. However, if xyloglucan is present in primary cell-walls of monocots, it is clearly there in much smaller proportions than in dicot primary-walls, and it may not assume the structural significance that this polysaccharide has in the latter.

b. Xylan.-Despite the limited number of studies conducted, it is clear that hemicellulosic xylan is a major component of monocot, primary cell-walls. Methylation analysis of primary walls of suspension-cultured cells of various monocot t i s s ~ e s , ~including ~ . ' ~ ~ wheat root, oat embryo, rice root, brome-grass embryo, and rye-grass endosperm, indicated that xylans are major components. These walls possess a high content of 4and 3,d-linked xylosyl residues and (terminal) arabinofuranosyl groups, as well as some 2,4-linked xylosyl residues. Presumably, 2-linked xylosyl, 4-linked galactosyl, terminal galactosyl, 5-linked arabinofuranosyl, gluresidues, which were also cosyluronic, and 4-O-methy~-~-glucosyhronic observed, arise from xylan side chains. The aforementioned residues are typical of those found in xylans that have been isolated, and characterized, from a wide range of monocot

292

PRAKASH M. DEY AND KEN BRINSON

tissues containing secondary walls by Aspinall and associate^,^^^-^^^ Buchala and coworkers,74-75*81Je4*1e5 and others.8QJQ6-1Qe All of these xylans possess backbones composed of p-( 1+4)-linked xylosyl residues. There is a wide variety in the nature of the side chains attached to this xylan backbone: the commonest side chains encountered are single L-arabinofuranosyl groups attached to 0 - 3 of some of the backbone xylosyl residues, or single D-glucosyluronic or 4-O-methyl-~glucosyluronic groups attached to 0 - 2 of some of the backbone xylose unit^.^^.^^,^^.^^^-^^^^^^^ However, oligomeric side chains containing other glycosyl residues are also f o ~ n d Some . xylans ~ contain ~ ~ ~ both terminal D-glucosyluronic and terminal 4-O-methyl-~-ghcosyhronic side chain~.1e0.lQe,197,1Qe A xylan has been isolated from young internodes of the reed Arztndo donax, a tissue in which most of the cells have primary walls.8QThis polysaccharide possesses a backbone of p-( 1+4)-linked D-xylosyl residues with single 4-O-methy~-~-g~ucosyluronic and arabinofuranosyl groups separately attached to backbone xylose units: they are bonded to 0 - 2 and 0 - 3 , respectively, of separate xylosyl residues. A similar xylan was isolated from older tissues, containing secondary walls, of the same plant .8e

c. Glucuronoarabinoxylan. -Glucuronoarabinoxylans have been isolated by Ray and associate^^^.^^ from an oat-coleoptile, cell-wall preparation that was presumed to be low in secondary-wall content, and from maize coleoptile by Darvill and coworkers.200Partial, acid hydrolysis of the oat-coleoptile polysaccharide released most of the glycosyluronic residues as glucosyluronic acid-xylose and 4-0-methylglucosyluronic a c i d - ~ y l o s e .Initial ~ ~ . ~methylation ~ studies of the maize-coleoptile polysaccharide indicated a xylan backbone having arabinofuranosyl and glucosyluronic side chains.200 (190) G. 0. Aspinall and H. Wilkie, ]. Chem. Soc., C, (1956) 1072- 1079. (191) G. 0.Aspinall and R. J . Sturgeon,]. Chnn. Soc., C, (1957) 4469-4471. (192) G. 0.Aspinall, I. M. Cairncross, and K. M. Ross, ]. Chem. Soc., C, (1963) 1721 1727. (193) G. 0.Aspinall and I. M. Cairncross,]. Chem. Soc., C, (1960) 3877-3881. (194) A. J. Buchala, C. J. Frazer, and K. C. B. Wilkie, Phytochemisty, 1 1 (1972) 2803281 4. (195) A . J. Buchala and H. Meier, Phytochemistry, 1 1 (1972) 3275-3278. (196) G. R. Woolard, E. B. Rathbone, and L. Novellie, Phytochemisty, 16 (1977) 957959. (197) G . R. Woolard, E. B. Rathbone, and L. Novellie, Carbohydr. Res., 51 (1976) 239 247. (198) F. Barnoud, G. G. S . Dutton, and J.-P.Joseleau, Carbohydr. Res., 27 (1973) 215223 (199) K. C. B. Wilkie and S . L. Woo, Carbohydr. Res., 54 (1977) 145-162. (200) A. G. Darvill, C. T. Smith, and M. A. Hall, New Phytol., 80 (1978) 503-516.

~

PLANT CELL-WALLS

293

v. NON-CELLULOSIC D-GLUCANS These polymers are distinguished from cellulose by the presence of both p-( 1+3)- and p-(1+4)-linked D-glucosyl residues, lower molecular weights (some noncellulosic glucans are water-soluble), and susceptibility to hydrolysis by p-D-glucanases that cannot hydrolyze cellulose. Unlike cellulose, whose microfibrillar structure and structural role in the cell wall has been clearly established, the function of these polymers as structural components of the wall is still a subject of controversy: there is some evidence that they are energy-reserve materials.110*201~202

1. Dicotyledonous Plants A /3-D-glucan has been isolated from the cell walls of 3-day-old, rnungbean h y p o ~ o t y l sExtracts .~~ of cell walls of older hypocotyls were deficient in this polysaccharide. The glucan contained %linked and 4-linked glucopyranosyl residues in the molar ratio of 1 . 0 : 1.7. However, the hypocotyl tissue from which the glucan was extracted contained both primary and secondary walls, and therefore, the polymer cannot be definitely characterized as a primary cell-wall component. 2. Monocotyledonous Plants Glucans possessing both p-( 1+3)- and/?-(1+4)-linked D-glucosyl residues (mixed p-D-glucans) are among the most studied polysaccharides of the monocot. primary cell-wall, although their role is open to question. On the one hand, their generalized occurrence and the difficulty of removing them from walls suggest apossible structural role. On the other hand, mixed p-D-glucans of oat coleoptile are catabolized, and disappear when this tissue is dark-grown in the absence of an energy source, suggesting a role as an energy-reserve polymer.110.201~202 Mixed &D-glucans are widely distributed in monocots, and they have been isolated from rye,203at,^^^-^^^ and barley207endosperms, and from maize,208barley,209and wheaP0S2l0stems. All of these preparations probably possessed secondary as well as primary cell-walls. (201) D. J. Nevins and W. H . Loescher. Proc. Znt. Conf: Plant Growth Substances, sth, Hirokawa Pub. Co., Tokyo, 1974, pp. 828-837. (202) D. J. Nevins, D. J. Huber, R . Yamamoto, and W. H. Loescher, Plant Physiol.. 60 (1977) 617-621. (203) M. M. Smith and B. A. Stone, Phytochemisty, 12 (1973) 1361 - 1367. (204) D. L. Morris,]. Bid. Chem., 142 (1942) 881-891. (205) F. W . Parrish, A. S. Perlin, and E. T. Reese, Can. J . Chem., 38 (1960) 2094-2104. (206) S. Peat, W. J. Whelan, and J. G . Roberts,]. Chem. Soc., C, (1957) 3196-3924. (207) P. R. Costello and B. A. Stone, Proc, Aust. Biochem. Soc., (1968) 43. (208) A. J. Buchala and H. Meier, Carbohydr. Res., 26 (1973) 421-425. (209) A. J. Buchala and K. C. B. Wilkie. Nnturwissenschaften. 10 (1970) 496-497. (210) A. Kivilaan, R. S. Bandurski, and A. Schulze, Plant Physiol., 48 (1971) 389-393.

294

PRAKASH M. DEY AND KEN BFUNSON

These polysaccharides have also been isolated from cell-wall preparations of maize210and oat53*54,202 coleoptiles, tissues that, it is claimed, are rich in primary walls. However, in contrast, mixed P-D-glucans could be detected in the primary walls of only one out of six suspension-cultured, monocot tissues, namely, rye-grass endosperm (but not in cultured wheat-root, oat-embryo, rice-root, sugar-cane internode, brome-grass embryo, or rye-grass endosperm60).This may have been because, in this investigation, the purified B. subtilis alpha amylase used to remove starch from the walls was contaminated with P-D-glucanase that solubilized the mixedP-D-ghcans.211However, in another study of the walls of cultured wheat-endosperm, conducted by Mares and in which the walls were not treated with a purified, B. subtilis alpha amylase preparation, again no mixed P-D-glucans were found. A controversy, therefore, remains regarding the existence of mixed P-D-glucans in monocot, primary cell-walls. Returning to the monocot mixed glucans that have been isolated, there are more 4-linked than 3-linked glucosyl residues, the ratio differing with the s p e ~ i e s ~ ~ from s ~ 1~. 7 ~to -4.0. ~ The ~ ~ ratio * ~ has ~ ~also . ~ been ~ ~ shown to change with the development ofthe tissue in barley stem20eand oat stem, leaf, and the ratio of 4-linked to 3-linked residues increases with tissue aging. The 3- and 4-linked glucosyl residues also appear to occur within a single, linear, polymer hai in.^^^*^^^-^^^,^^^ Partial, enzymic hydrolysis of oat “primary cell-wall” P-D-glucans suggested that contiguous, 3-linked glucosyl residues are relatively uncomm0n,54,202but the tetrasaccharide j?-~-Glc-( 1-’4)-P-~-Gk-(1+4)-P-~Glc-(1 + 3 ) - ~ - G k has been isolated from the hydrolysis products,202 demonstrating the presence of contiguous, 4-linked, glucosyl residues. A study by Forrest and Wainright216of P-D-glucans isolated from barley endosperm, a tissue containing secondary walls, showed that these polysaccharides contain from 1 to 3% of a peptide. These authors suggested that the peptides are an integral part of the P-D-glucans.

VI. CELLULOSE Cellulose is the most abundant polysaccharide in Nature, and is probably the most-studied cell-wall p ~ l y m e r . ~The ” majority of structural investigations have, however, been conducted with cellulose from sec(21 1) D. J. Huber and D. J. Nevins, Plont Physiol., 60 (1977) 300-304. (212) D. J. Mares and B. A. Stone, At&. I. Biol. Sci., 26 (1973) 793-812. (213) C . J. Fraser and K. C. B. Wilkie, Phytochemistry, 10 (1971) 199-204. (214) K. C. B. Wilkie andS. L. Woo, Carbohydr. Res., 49 (1976) 399-409. (215) A. J. Buchala and K.C. B. Wilkie, Phytochemisty, 10 (1971) 2287-2291. (216) I. S. Forrest andT. Wainright,]. Znst. Brew. (London), 83 (1977) 279-286. (217) D. P. Delmer, Ado. Corbohydr. Chem. Biochem., 41 (1983) 105-153.

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ondary cell-walls; little is known about this glucan in primary cell-walls, but it is usually assumed that cellulose from primary and secondary walls is fundamentally similar in structure, and that monocot and dicot celluloses are also similar.4*z17ae18 Cellulose is composed of long, linear chains ofp-( 1+4)-linked D-glucosyl residue^.^.^^^" The chains aggregate by hydrogen-bonding along their lengths, to form thin, flattened, rod-like structures that are referred ~ ~single ~ ~ ~microfibril -~~~ is estimated to consist of to as m i ~ r o f i b r i l s . A 60 - 7 0 D-glucan chains, giving cross-section dimensions estimated4 as 4.5 X 8.5 nm (see Fig. 4). Estimation of the degree of polymerization of the D-glucan chains within the cellulose fibrils is complicated by the necessity of first solubilizing the D-glucans, and this process is likely to break the chains. One estimate put the degree of polymerization at 6000 to 7000 for cellulose chains derived from cotton fiberszz1(see also, Ref. 217 and references cited therein). It is possible that the D-glucan chains of cellulose have no natural ends; that is, once a chain is initiated, it never ends, except when a fibril is physically separated from its synthetic enzymes. This idea is supported by the electron-microscope observation that the cellulose fibrils do not ~ ~ ~ possible ~ ~ ~ ~ that appear to have natural t e r m i n a t i o n - p o i n t ~ .It~is- also the fibrils have an unlimited length, but that the individual D-glucan chains within the fibrils have a finite length; the ends of the D-glucan chains may overlap, and thus result in fibrils of indeterminate length. The aggregated D-glucans within a fibril are so ordered that they are, in X-Ray diffraction studies of the highly fact, crystalline.4~z17"zzo~zzz~zz3 crystalline cellulose of the cell wall of the alga Vulonia ventricosu indicated that the reducing ends of all of the D-glucan chains within cellulose microfibrils face in the same direction; this is described as a parallel orientation.z1Q*z2z It seems probable that the D-glucan chains of higherplant, primary cell-wall cellulose have a similar, parallel orientation, but this has not been firmly established. Purified, crystalline cellulose isolated from secondary walls appears to contain minor proportions of D-glycosyl residues other than D-ghcosyl, in hemicellulosic chains of "paracrystalline" regions within the microfibril structure.zz3These hemicelluloses contain xylose and probably lesser proportions of arabinose, mannose, and fucose. It was conceived (217a) (218) (219) (220) (221) (222) (223)

K. H. Gardner and J. Blackwell, Biopolymers, 13 (1974) 1975-2001. F. J. Kolpak and J. Blackwell, Macromolecules,9 (1976) 273-278. K. H. Gardner and J. Blackwell, Biochim. Biophys. Acta, 343 (1974) 232-237. F. J. Kolpak and J. Blackwell, Text. Res.]., 45 (1975) 568-572. M. Marx-Figini and G. Schulz, Biochirn. Biophys. Acta, 112 (1966) 74-85. A . Sarko and R. Muggli, Macromolecules,7 (1974) 486- 494. R. D. Preston and J. Cronshaw, Nature, 181 (1958) 248-251.

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FIG.4. -Proposed Arrangement of D-Glucan Chains Within Cellulose Microfibrils of Primary Cell-Walls of Dicots (After AIbersheim8). [A microfibril is believed to contain 60-80 D-glucan chains in an orderly array (lower right). The chains line up next to one another, to form sheets, and also line up one above the other, staggered at half the length of a single D-glucose unit. Within a D-glucan chain, the units are joined by /I-o-glucosidic bonds and the chains are held together by hydrogen bonds between oxygen atoms and the hydrogen atoms ofhydroxyl groups. (In this diagram the hydrogen atoms are not shown and the hydrogen bonds are indicated as if they extended from one oxygen atom to another. In addition, the lengths and angles of the bonds are distorted, because the D-glucose units have been flattened and the chains separated for clarity.) The large number of hydrogen bonds gives cellulose its mechanical strength and its resistance to chemical degradation.]

that the hemicelluloses are mixed with loosely deposited, cellulose molecules around a highly ordered, crystalline core of parallel-oriented, cellulosic, D-glucan chains.223It was assumed that the core does not run continuously along the whole length of the microfibril. It is not clear whether the hemicellulosic chains constitute an integral part of the D-glucan chains, perhaps acting as covalently bonded, termination points at the ends of D-glucan chains joining the latter to the ends of adjacent D-glucan chains along the length of the microfibril, or are merely hydrogen-bonded to the surface of the D-glucan chains in the way in which xyloglucan is known to “coat” the surface of cellulose microfibrils in the There are several hypotheprimary wall of cultured sycamore-cells.5s~5e

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t

5 nm

1 a

b

C

d

FIG.5. -Diagrammatic Representation of Several Different Models of Cellulose Microfibrillar Structure. [(a)Preston and Cronshaw’s conceptzz3of microfibrillar structure. The solid strokes represent the planes of the o-glucan chains. The broken strokes indicate the position of other sugars or sugar derivatives in noncellulosic chains. The central, crystalline core is surrounded by a paracrystalline sheath. (b) Model postulated by Hess and cow o r k e r ~ . The * ~ ~ microfibrils contain a number of elementary fibrils which are segmented into crystalline and paracrystalline regions; such periodicity is apparent after incorporation of iodine or thallium. (c) Microfibril composed of folded cellulose “units” as suggested by Marx-Figini and S ~ h u l zThe . ~ end ~ ~ loops of successive micellae are interlinked, and this region of linkage between the loops corresponds to the paracrystalline regions. (d) Manley’s proposal226of a D-glucan “ribbon” wound into a helix. (After M~hlenthaler.’~’)]

ses223-227regarding the physical arrangement of D-glucan chains and hemicelluloses in cellulose microfibrils; these are illustrated, and explained, in Fig. 5 . The cellulose fibrils of secondary cell-walls have a considerably greater cross-sectional area than those of primary It is possible that primary microfibrils aggregate to form secondary-wall fibrils. Hemicelluloses trapped between aggregating primary, cellulose microfibrils may constitute the origin of a major proportion of the non-D-glucosy1 residues of cellulose obtained from secondary walls. (224) (225) (226) (227)

K. Hess, H. Mahl, and E. Gutter, Kolloid Z., 155 (1957) 1 - 9 See Ref. 221. R. S. J. Manley, Nature, 204 (1964) 1155-1157. K . Muhlenthaler, Annu. Reo. Plant. Physiol., 18 (1967) 1-24.

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VII. CELL-WALL GLYCOPROTEINS 1. Hydroxy-L-proline-rich Glycoproteins of Dicots Primary cell-walls of dicots contain between 5 and 10%of protein that is rich (20%)in hydroxy-~-proline.~*~~*~~* The wall protein also contains a relatively high content of L-alanine, L-serine, and L-threonine, and this feature is found in such animal structural-proteins as collagen.228This characteristic, amino acid composition, together with the fact that it is difficult to extract much of the protein from cell walls under nondegradative conditions,22esuggests that it has a structural role in the wall.228*230 Fragments of hydroxy-L-proline-rich protein obtained from the primary cell-walls of dicots invariably contain arabinosyl and galactosyl residues and a series of hydroxy-L-proline arabinosides; mono-, di-, tri-, and tetra-arabinosides, glycosidically linked to the hydroxyl group of hydroxy-L-proline have been isolated from wall preparations obtained from suspension-cultured sycamore- and t o m a t o - c e l l ~ ,and ~~~ sepa*~~~ rated chromatographically on Chromobeads B.231 The hydroxy-L-proline tetraarabinoside is the preponderant molecular species obtained from the dicot primary cell-wall protein. Little or no nonglycosylated hydroxy-L-proline appears to be p r e ~ e n t . ~ ~This , ~ ~wall . ~ protein ~' is, therefore, clearly a glycoprotein. Structural analysis indicated that the arabinosyl residues are terminal, 2- and 3-linked, and that the structure of the hydroxy-L-proline tetraarabinoside isolated from suspension-cultured tobacco-cells is as follows.55232.233 /3-~-Araf(1-'3)-/3-~-Araf( 1-*2)-/?-~-Araf-(1+2)-/?-~-Araf(1+4)-hydroxy-~-proline

Single galactosyl residues are glycosidically attached to the serine hydroxyl groups of the glycoprotein of suspension-cultured, tomato cellwalls.228,22e This was shown by removing the arabinosides from the intact cell-walls by acid hydrolysis; the hydroxy-L-proline-rich wall-protein, with the arabinosyl residues removed, is susceptible to proteolysis with trypsin. The resulting, solubilized "tryptides" have been separated by cation-exchange and gel-filtration chromatography, and the composition of the tryptides determined by amino acid analysis.230Some of the tryptides have been sequenced by subtractive, N-terminus identification, (228) D. T. A. Lamport, Annu. Reu. Plant Physiol., 21 (1970) 235-270. (229) D. T. A. Lamport, Colloq. Znt. C.N.R.S.,212 (1973) 27-31. (230) D. T.A. Lamport, L. Katona, and S. Roerig, Biochem.]., 133 (1973) 125-132. (231) D. T.A. Lamport and D. H. Miller, Plant Physiol., 48 (1971) 454-456. (232) A. L. Karr, Plant Physiol., 5 0 (1972) 275-282. (233) Y. Akiyama and K. Kato, Agric. B i d . Chem., 41 (1977) 79-81.

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and further partial hydrolysis with acid.230Each of the hydroxy-L-proline-rich, wall-protein tryptides contains a pentapeptide consisting of L-serine-(hydroxy-L-proline), , and most of the tryptides also contain one or more galactosyl residues.230One tryptide, which was found to contain two residues each of galactose and serine, was subjected to degradation by /.-elimination, using several methods. The elimination procedures converted L-serine into either L-alanine or L-cysteic acid, with concomitant release of free galactose.230These results demonstrated the covalent attachment of a single galactosyl residue to each L-seryl residue in the tryptide. Similar evidence has also been obtained for the existence of galactosyl - serine linkages in the hydroxy-L-proline-rich glycoprotein of carrot c e l l - w a l l ~ . ~ ~ The covalent attachment of arabinose and galactose to the hydroxy-Lproline-rich protein of primary cell-walls is now generally ac~ e p t e d ,but ~ the ~ ~evidence . ~ ~ ~available suggests that the glycoprotein is not covalently attached to any of the other cell-wall polymers. This, of course, does not preclude the possibility of the existence of strong, noncovalent forces binding protein to wall p o l y s a ~ c h a r i d e s . ~ ~ ~ ~ ~ ~ ~ A hydroxy-L-proline-rich glycoprotein in which the carbohydrate component is an arabinogalactan is secreted into the culture medium by suspension-cultured s y ~ a m o r e - c e l l sThis . ~ ~ arabinogalactan is similar in structure to a protein-free arabinogalactan also secreted into the culture medium by the same cells.57The hydroxy-L-proline-rich glycoprotein of the primary cell-wall is structurally dissimilar from the glycoprotein present in the culture medium.234 In the cell-wall glycoprotein, the tetraarabinosides account for 80 mol% of the hydroxy-L-proline arabinosides, whereas, in the culture-medium glycoprotein, the corresponding value is only 4 mol%. Furthermore, arabinogalactan accounts for 50% of the culture-medium glycoprotein, but no arabinogalactan can be detected in the cell-wall glycoprotein. This evidence indicated the presence of two hydroxy-L-proline-rich glycoproteins in the primary cell-wall of suspension-cultured sycamorecells; one, a structural glycoprotein found only in the wall, and the second which, in culture, is present in the wall in only small proportions and is mainly found in the culture These two glycoproteins appear to be unrelated structurally.

2. The Hydroxy-L-proline-rich Glycoproteins of Monocots These polymers have not been studied so extensively as those present in dicots. Cell walls of suspension-cultured, monocot tissues, including (234) D. G . Pope, Plnnt Physiol, 59 (1977) 894-900.

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PRAKASH M. DEY AND KEN BRINSON

wheat-root, oat-embryo, rice-root, sugar-cane internode, brome-grass embryo, and rye-grass endosperm, have been reported by Albersheim and associateseoto contain 0.13- 0.16%of hydroxy-L-proline, compared to 2% in the primary walls of suspension-cultured sycamore-cells.60 Maize coleoptiles were reported by Darvill1IQto contain 2 - 3% of hydroxy-L-proline. Total protein in the walls of suspension-cultured monocots and in maize-coleoptile walls was shown by D a r ~ i l l "to~ be equal to, or greater than, the total protein in the walls of suspension-cultured dicots. In contrast to dicots, 65 - 75% of the hydroxy-L-prolyl residues in the hydroxy-L-proline-rich proteins from the primary wall of four monocot species, including Zea mays (pericarp), Auena sativa (coleoptile), Zris kaempferi (pericarp), and Allium porum (pericarp), are reported to have no arabinoside residues attached.231Of the glycosylated hydroxy-L-prolyl residues, the majority are bonded to triarabinosides, although smaller proportions of tetra-, di-, and mono-arabinosides have also been dete~ted.~~~ There is, therefore, a distinct, structural difference between the hydroxy-L-proline-rich glycoproteins of dicots and monocots. In monocots, the degree of polymerization of arabinosides attached to hydroxy-L-prolyl residues is lower, and far fewer hydroxy-L-prolyl residues are glycosylated than in dicots.

VIII. CELL-WALL-BOUND ENZYMES Undoubtedly, proteins and glycoproteins are cell-wall constituents, and some of these molecules possess enzymic activity. The tight binding of enzymes to cell walls is of probable significance in such processes as the breakdown of the walls, and hormone-mediated, cell-elongation growth. The way in which enzymes are bound to walls is, at present, poorly understood, and, frequently, extraction techniques involving maceration or sonication, or both, and extraction media of high ionic strength are needed, in order to dissociate the enzymes from the wall. The relationships between such extracted activities and the activity of the enzymes in situ is at present difficult to interpret. Pierrot and Van Wielink235pointed out that the presence of enzymes in insoluble residues (walls) of plant tissues might be strongly misleading with respect to their location in the intact tissue, because intracellular enzymes could become bound to wall material by adsorption, or by ionic attraction during extraction. Modification of enzymic activities by liberation of such inhibitors as polyphenols during maceration was another factor (235) H. Pierrot and J. E. Van Wielink, Planta, 137 (1977) 235-242.

PLANT CELL-WALLS

30 1

emphasized by these authors.235Some of the enzymes that are apparently bound to cell walls are listed next. 1. Dicots

Enzymes bound to the cell walls of cultured Conuoluulus arvensis cells include acid phosphatase (EC 3.1.3.2),235 acid invertase (EC 3.2.1.26),235N-acetyl-a-D-glucosamiriidase (EC 3.2.1.50),235N-acetyl(EC P-D-glucosaminidase (EC 3.2.1.30),235 a-D-galactosidase 3.2.1.22),235.23s P-D-galactosidase (EC 3.2.1,23),235s23s a-D-glucosidase p-D-ghcosidase (EC 3.2.1.21),235*236 a-D-mannosi(EC 3.2.1.20),235,23e (EC 3.2.1.25),235.23s andp-~dase (EC 3.2.1.24),235*23e/3-~-mannosidase xylosidase (EC 3.2.1.37).235 P-D-Glucosidase and a- and p-D-galactosidase are associated with the cell walls of suspension-cultured ~ y c a m o r e - c e l l sP-D-glucosidase ~~~; and P-D-galactosidase activities increase as the cells go through a period of growth, and then decrease as growth ceases.236 Acid invertase activity is bound to cell walls in radish seedlings,238and light is reported to induce transfer of the enzyme from the cytosol to the cell wall. a-D-Glucosidase and acid invertase are tightly bound to walls in carrot ~ a l l u s - t i s s u eand , ~ ~P-D-glucosidase ~ is bound to bean-hypocotyl walls.240.241 In pea epicotyls, there is a positive correlation between the tissue growth-rate and the levels of activity of wall-bound p-D-glucosidase, a - and P-D-galactosidase, and acid p h ~ s p h a t a s e . ~ ~ ~ 2. Monocots

Cell-wall-bound enzymes in monocots have been less extensively stud-

ied than in dicots. Moreover, the majority of studies with this class of plants has been conducted with oat coleoptiles. Cell-wall-bound glycosidases present in these coleoptiles include p-Dgalactosidase, P-D-glucosidase, P-D-xylosidase, P-L-fucosidase, and WDmannosidase: the enzymes were reported to be activated by auxin and by hydrogen ions, and, therefore, they may be involved in cell A contrary claim has been made by Evans244that neither cell-wall-bound 1236) F. M. Klis, R . Dalhuizen, and K. Sol. Phytochemistry, 13 (1974) 5 5 - 5 7 . (237) K. Keegstra and P. Albersheim. Plant Physiol., 45 (1970) 675-678. (238) M. Zouaghi and P. Rollin, Phytochemisty, 15 (1976) 897-901. (239) D. R. Parr and J. Edelman, Planta, 127 (1975) 1 1 1 -119. (240) D. J . Nevins, Plant Physiol., 46 (1970) 458-462. (241) T. A. Jaynes, F. .4. Haskins, H. J. Gorz, and A. Kleinhoes, Plant Physiol., 4 9 (1972) 277- 279. (242) A. K. Murray and R. S. Bandurski, Plant Physiol.,56 (1975) 143- 147. (243) K. D. Johnson, D. Daniels, M. J. Dowler, and D. L. Rayle, Plant Physiol.,5 3 (1974) 224- 228. (244) M. L. Evans, Plant Physiol., 5 4 (1974) 213-215.

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P-D-galactosidase nor P-D-glucosidase plays an important role in shortterm growth promoted by auxin or acid in oat coleoptiles. Greve and Ordin245purified, and characterized, the wall-bound a-D-mannosidase of this tissue. In maize-root tips, high specific activities of P-D-galactosidase, a-and P-D-ghcosidase, N-acetyl-P-D-glucosaminidase, acid phosphatase, and phosphodiesterase (EC 3.1.4.1) are found in the ceI1-wall fraction.246 There is thus some evidence for the tight binding of enzymes, especially glycosidases, to cell walls in both dicots and monocots. The nature and localization of these enzymes suggest that they may, perhaps, play a role in wall breakdown and such other processes as elongation growth. The membrane systems of plant cells are known to be involved in the transport, and introduction, of polysaccharides into the cell ~ a 1 1 ~ ~ ~ - ~ enzymes localized in the wall may also play a part in the metabolism of these polymers when they are transferred from the membrane system to the wall. IX. INTERCONNECTIONS BETWEEN THE CONSTITUENT POLYMERS I N PRIMARY OF DICOTS CELL-WALLS

In earlier Sections, the individual polymers that make up the constituent parts of the primary cell-wall have been discussed, and partial structures proposed for some of these polymers are shown in Figs. 1- 4. A model of the primary cell-wall of suspension-cultured sycamore-cells, illustrating proposed interconnections between these constituent polymers within the intact wall, has been constructed by Albersheim and his associate^.^^'^^^^^^^^^^ The model, not designed to be spatially or quantitatively accurate, is depicted in Fig. 6. This model suggests that the cellulose fibrils are linked together by four other polysaccharides. Hemicellulose xyloglucans completely coat the surface of the cellulose fibrils, and are held to the fibrils by hydrogen bonds. Hydrogen bonding is supported by the observation that 8 M urea and dilute base, both of which are able partially to extract xyloglucan noncovalently bound in vitro to commercial cellulose, are also able to extract, partially, the xyloglucan from endogalacturonanase-pretreated s y c a m ~ r e - w a l l sThe . ~ ~ reducing ends of some of the xyloglucan mole(245) L. C . Greve and L. Ordin, Plant Physiol., 60 (1977) 478-481, (246) R. W. Parish, Planta. 123 (1975) 15-31. (247) D. H. Northcote, Endeaoour, 30 (1970) 26-33. (248) D. H. Northcote, in J. B. Pridham (Ed.),Plant Carbohydrate Biochemistry, Academic Press, New York, 1974, pp. 165-181. (249) M. Dauwalder, W. G. Whaley, and J. E. Kephart, Sub-cell. Biochem., 1 (1972) 225-231.

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Ib-

FIG. 6. -Tentative Structure of Sycamore Primary Cell-Wall (After Albersheirn and Coworkerss7). [This model is not intended to b e quantitative, but the wall components are presented in appi oximately proper proportions, although the distance between cellulose microfibrils is expanded, in order to allow room to present the interconnecting structures. The key in the Figure depicts regions representing the various wall-components. (a) cellulose microfibrils, (b) rhamnogalacturonan (pectic backbone), (c) xyloglucan, (d) wall protein with arabinosyl tetrasaccharides (e) attached to hydroxyl-L-proline residues, (f) (3+6)-linked arabinogalactan attached to serine residue (9) of the wall protein, (h) total pectic polysaccharide, showing L-arabinan and 4-linked o-galactan side-chains attached to rhamnogalacturonan backbone, and (i) unsubstituted L-serine residues of wall protein.]

cules may b e covalently bonded to arabinogalactan~,~' although the existence of the latter polymers has not been definitely proved. Each xyloglucan chain in the model can bind to only a single arabinogalactan, which, in turn, binds to a single rhamnogalacturonan. It is proposed that each rhamnogalacturonan molecule is connected to several arabinogalactan chains, each radiating from a different cellulose fibril. Similarly, each cellulose fibril is connected to several rhamnogalacturonans by way of several xyloglucan chains. As a result, the cellulose fibrils are extensively cross-linked. The primary cell-wall may be pictured as consisting of cellulose rods embedded in an amorphous matrix of noncellulosic polysaccharides (see Fig. 7). This generalized model for the cell wall of dicots has, by no means, received general acceptance. In particular, the occurrence of arabinoga-

304

FIG.7 .-Model, Devised by A l b e r ~ h e i m , ~of~the ~ ' Interconnections Between Polysaccharides in the Primary Cell-Wall of Dicots. [Many xyloglucan molecules adhere to the surface of the cellulose microfibrils by means of hydrogen bonding. Each xyloglucan molecule binds to a single arabinogalactan chain, which in turn, binds to a single rhamnogalacturonan molecule. Each rhamnogalacturonan chain can receive several arabinogalactan molecules, radiating from different cellulose microfibrils. Similarly, each cellulose microfibril can be connected to several rhamnogalacturonan chains. As a result of this extensive cross-linking, the microfibrils are immobilized in an apparently rigid, wall matrix.]

lactans as constituent polymers within primary walls is highly tentative,5,10*57,s4.s5 and there are a number of open questions concerning the exact nature of the interconnections between the constituent polymers. These questions have been considered in a comprehensive review b y Albersheim and his associate^.^^ A summary of the conclusions reached by this group, and some discussions on their model, now follow. 1. Interconnections Between the Pectic Polymers

Attachment of the neutral pectic polysaccharides, galactan and arabinan, to rhamnogalacturonan has been established from a number of lines of evidence. Purified Colletotrichum lindemuthianum endopolygalacturonase, free from arabinanase and galactanase activities, solubilizes,

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from suspension-cultured sycamore-cells, a wall fraction containing arabinan, galactan, and rhamnogalacturonan I, which binds as a single acidic polymer to DEAE-Sephadex, indicating strong covalent attachment of the neutral polymers to the acidic r h a m n o g a l a ~ t u r o n a n .These ~ ~ J ~ ~polymers can similarly be isolated as a single fraction by chromatography on agarose .Iz5 Further powerful evidence for covalent attachment of these neutral polymers to rhamnogalacturonan I comes from studies in which p-elimination of the glycosyluronic residues of the rhamnogalacturonan moiety, under a variety of conditions, results in a drastic diminution in the apparent molecular size ofboth the arabinan and the galactan.lZ5It is noteworthy that, in agreement with the proposed covalent bonding between these polysaccharides, no arabinan or galactan has ever been extracted65 from a primary cell-wall free from rhamnogalacturonan I. Homogalacturonans have always been assumed to be attached to, or to be a part of, rhamnogalacturonans. Evidence for this association was obtained by the isolation, from sycamore cell-walls, of oligogalactosiduronic acids containing 10 or more galactosyluronic residues in which the reducing ends of the oligosaccharides were covalently attached to single rhamnosyl residues.55 More evidence for the interconnection of these polymers comes from the fact that both the homogalacturonan and rhamnogalacturonans I and 11, with associated arabinan and galactan, are released from sycamore cell-walls by Colletotrichurn lindernuthianurn e n d o p o l y g a l a c t u r ~ n a s e . ~This ~ J ~ finding ~ so far provides the only evidence that rhamnogalacturonan I1 is covalently attached to the other pectic polymers. Pectic polysaccharides probably interact through noncovalent, as well as covalent, links; noncovalent interactions may contribute significantly to interconnections between the pectic polymers and other polymers of the cell wall. Calcium has long been known to confer rigidity to cell walls.13eRees and his postulated an “egg-box’’ model for inclusion of calcium, wherein the ions can fit between two or more chains of nonesterified galactosyluronic residues in such a fashion that they chelate to the oxygen atoms of four galactosyluronic residues distributed between two galacturonan chains, thus packing the ions like eggs within a box composed of galacturonans. Such chelation results in cross-linking of the galacturonan chains, and in increased rigidity. The cross-linking is sensitive to the degree of methyl-esterification of the galacturonan chains: ester groups would interfere with the uronic carboxylate - Ca2+bond-formation. Efforts have been made to correlate the degree of calcium cross-linking of galacturonans to the rate of cellwall elongation, but no such relationship has been established.250 (250) R. W. Stoddard, A. J. Barrett, and D. H . Northcote, Biochm. J . , 102 (1967) 194204.

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The possibility of other types of noncovalent interactions between the pectic polysaccharides and other cell-wall polymers must be borne in mind,300251but there is at present little evidence to indicate that such interactions do exist in the primary cell-wall (see also, Section IX,3). 2. Noncovalent Bonding of Hemicelluloses to Cellulose Microfibrils

In dicot primary cell-walls, it would appear that xyloglucan is the main hemicellulose, and it has been proposed56that this polysaccharide bonds strongly to cellulose fibrils through multiple hydrogen-bonds. The evidence for this hypothesis is convincing. (1) The xyloglucan is quantitatively sufficient to form a monolayer coating of the cellulose fibril^.^^.^^ (2) Space-filling models of xyloglucan show that it is capable of forming multiple hydrogen-bonds to cellulose.56 (3) Xyloglucan may be extracted from xyloglucan -cellulose complexes in the cell wall by such hydrogen-bond-breaking reagents as dilute base or 8 M urea.56 (4) The binding of xyloglucan to the cell wall and to isolated cellulose is reversible.56 (5) Xyloglucan reacts strongly with, and bonds strongly to, isolated cellulose in the absence of enzymes or chemical c a t a l y ~ t s . (6) ~~J~~ Xyloglucan can be extracted from the cell wall, and separated from cellulose fibrils by using enzymes that degrade xyloglucan into fragments that are not long enough to form stable, hydrogen-bonded complexes with cellulose.56(7) Short, enzymically produced, xyloglucan fragments can be induced to form complexes with cellulose, by lessening the water activity of the solvent, thereby lowering the opportunity for the fragments to hydrogen bond with the solvent.5Q The xyloglucan-cellulose bond is presumed to be one of the major interconnections of the dicot primary cell-wall, and it may function to prevent the cellulose fibrils from forming the large aggregates that are characteristic of secondary walls. Xyloglucan chains may also form bridges between cellulose fibrils, to which they are hydrogen-bonded, and other cell-wall polymers. Albersheim and his associate^^^-^^ provided evidence that xyloglucan is covalently attached to the neutral sidechains of the pectic polymers, and proposed'O that the synthesis and degradation of this covalent linkage, during cell-wall elongation induced by auxin, is a key mechanism that allows relative slippage of cellulose fibers during the elongation process. Labavitch and Raylo' and Loescher and Nevinsllo had earlier reported findings that suggested the latter proposition. M ~ N e i isolated, 1 ~ ~ ~ from the walls of pea coleoptiles, a wall fragment that contains the xyloglucan attached to the pectic galactan. (251) S . E. B. Gould, D. A. Rees, N. G . Richardson, and I. W. Steele, Nature, 208 (1965) 876-878. (252) M. McNeil. unpublished findings, cited in Ref. 10.

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The detailed structure, and the significance, if any, of this wall fragment in elongation growth, are areas of further investigation. Xyloglucan is not the only primary cell-wall hemicellulose. The glucuronoarabinoxylan isolated from walls of suspension-cultured sycamore-cells120 is structurally related to other arabinoxylans and xylans, polymers that are also known to be capable of hydrogen-bonding to c e l l ~ l o s eIn. ~addition, ~ ~ ~ ~work ~ ~ by ~ Albersheim and coworkerss6 suggested that glucuronoarabinoxylan contaminated the xyloglucan extracted by urea from sycamore primary cell-wall. It seems probable, therefore, that glucuronoarabinoxylan and, perhaps, xyloglucan molecules bind not only to cellulose but also to each other, to form aggregates. Plant arabinoxylans form aggregates in s o l ~ t i o nand , ~ evidence ~ ~ ~ ~ ~has been presented that, in this state, arabinoxylans exist as mixtures of random coils and aggregated, linear chains.30Such structures can lead to gel formation and may, by this method, be involved in cross-linking of the primary cell-wall polymers.

3. Interconnections Involving the Hydroxy-L-proline-rich, Cell-Wall Glycoprotein The hydroxy-L-proline-rich protein present in the culture medium of suspension-cultured sycamore-cells was shown to b e covalently linked to an arabinogala~tan,~' and this led to speculation regarding the existence of a similar interconnection within the cell wall. However, it has now been established that the hydroxy-L-proline-rich protein of the primary wall is structurally different from that present in the extracellular (see Section VII,l), and thus the latter cannot be used as a model for the cell-wall glycoprotein. There does not appear to be a covalent linkage between the cell-wall glycoprotein and hemicelluloses, or pectic polymers, in lupin hypocotyl cell-walls,49~s0 or between the wall glycoprotein and pectic polymers in mung-bean hypocotyl cell-walls.106On the other hand, there is a very real, if undemonstrated, possibility that non-covalent interconnections link the wall glycoprotein to wall polysaccharides.4e~65 In this connection, plant biochemists and physiologists have become interested in lectins that are glycoproteins or proteins. Their significance in higher plants has been reviewed by Callow,254K a u s ~and , ~Lis ~ ~and Sharon.256Most of the lectins isolated in substantial amounts for biochemical studies have (253) J. D. Blake and G . N. Richards, Carbohydr. Res., 18 (1971) 11 -20. (254) J. A. Callow, Curr. Adu. Plant Sci., 7 (1975) 181 -193. (255) H. Kauss, Fortschr. Bot., 38 (1976) 58-70. (256) H. Lis and N. Sharon, in P. K. Stumpf and E. E. Conn (Eds.), The Biochemistry of Plants: A Comprehensiue Treatise, Vol. 6, Academic Press, New York, 1981, pp. 371-447.

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PRAKASH M. DEY AND KEN BRINSON

been derived from seeds, and, for a while, it appeared that the occurrence oflectins might be a special feature of legume seeds.z55However, a glycoprotein having lectin properties was found to be extractable with 0.5 M phosphate buffer, or EDTA solution containing Triton, from mung-bean-hypocotyl cell-wall preparations. 104~105-257It was shown that the lectin was associated with the wall, and was not a cytoplasmic contaminant.104J05The lectin-binding sites were specific for carbohydrate groups containing a-D-galactose. It could thus be envisaged (on a purely speculative basis) that the interaction of the lectin-binding site with special groups on wall polysaccharides contributes towards the rigidity of the wall. Galactose-containing polysaccharides that may constitute partners for the galactoside-specific lectins are frequent in mung-bean walls.258 The hydroxy-L-proline of the crude, mung-bean-wall preparation is not associated with the l e c t i r ~this ~ ~ indicates ~; a difference between the mung-bean-wall lectin and the lectin isolated from potato tubers. The latter is rich in hydroxy-L-proline, arabinose, and galactosezs0; in this respect, it shows features similar to those of the hydroxy-L-proline-rich glycoprotein (“extensin”) of the primary cell-wall, first reported by Lamportzsl (see Section VI1,l). It has not been clearly established whether extensin is a lectin, but such a lectin property might allow this glycoprotein to bind noncovalently to polysaccharides within the primary-wall structure. There has been speculationzs5 that the lectin associated with the mung-bean hypocotyl-wall may play a role in the extension growth-process, possibly reversibly binding wall polysaccharides involved in “polymer creep” during cell extension. Alternatively, it might function merely as a “gluing substance” between cell-wall polysaccharides, or between the wall and the plasmalemma, or both. Another suggestion is that it is the material that fills the inner channels of p l a s m a d e ~ r n a t a . ~ ~ ~ Protein fractions having lectin activity have been extracted from mitochondria, plasma, and Golgi membranes, and from the endoplasmic reticulum of mung-bean hypocotyls, as well as from total-membrane fractions from a variety of plant tissues.257The carbohydrate specificity of the lectin fractions differs with the membrane type. It is conceivable that, in addition to cell-wall lectin - membrane interactions, there may also be membrane lectin -cell wall, noncovalent bonds. (257) D. J. Bowles and H. Kauss, Plant Sci. Lett., 4 (1975) 411 -418. (258) H. Kauss, in J. B. Pridham (Ed.),Plant Carbohydrate Biochemistry, Academic Press, London, 1974, pp. 191-205. (259) V. Haas and H. Kauss, unpublished findings, cited in Ref. 255. (260) A. K . Allen and A . Neuberger, Biochem.]., 135 (1973) 307-314. (261) D. T. A. Lamport, in F. A. Loewus (Ed.), Biogenesis ofPlant Cell Wall Polysaccharides, Academic Press, New York, 1973, pp. 149-165.

PLANT CELL-WALLS

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Because of the obvious similarity of the combining sites of lectins to those of glycosidases, Lis and Sharon25s considered the possibility that lectins are enzymes that have lost their catalytic sites. It is also possible that, in situ, lectins possess highly labile enzymic activities and that, during purification, the enzymic activity of the lectins may b e lost, while the binding activity is retained. Further speculation by Hankins and Shannones2suggested that lectins in dry seeds are enzymically inactive proteins that acquire catalytic properties during germination: a highlypurified, a-D-galactose-specific lectin from mung beans possessed WDgalactosidase a c t i ~ i t y . It ~ ~is3conceivable that lectins in the cell wall may function as wall-bound glycosidases.

X. DISCUSSION ON THE ALBERSHEIM MODELFOR PRIMARY CELL-WALL OF DICOTS STRUCTURE

Since the discovery of the arabinosyloxy-L-proline-richglycoprotein, sometimes referred to as extensin, in the primary cell-wall by L a m p ~ r t , ~ ~ and its negative correlation with the cell-wall matrix has been regarded by some as an extensin -polysaccharide complex. Two structures for this complex were proposed: that of Lamport,228in which extensin is linked to cellulose microfibrils by an arabinosyloxy-Lproline-linked galactan, and that of Albersheim and associate^,^^^^^^ in which arabino-oligosaccharide side-chains of the hydroxy-L-proline-rich glycoprotein are free, and the glycoprotein is linked through arabinogalactan to the pectic rhamnogalacturonan. The Albersheim group has itself critically reconsidered the existence of such a glycoprotein polysaccharide covalent linkage in the primary cell-wall of dicotss5 (see also, Ref. 234). Added weight has been given to this self-criticism by objections, arising from the work of Bailey and coworkers with the primary wall of intact 1 ~ p i and nmung-bean106 ~ ~ hypocotyls, ~ ~ ~ raised to the early Albersheim model. The results and conclusions of Bailey and coworkers were drawn together in a review,48 and may be summarized as follows. (1) Extraction of the poly(g1ycosiduronic acid) from lupin and mung-bean hypocotyl wall, under conditions that remove, and degrade, poly(g1ycosiduronic affords little of the wall (262) C. N. Hankins and L. M. Shannon, J Biol. Chem., 253 (1978) 7791 -7797. (263) R.Cleland and A. Karlsnes, Plant Physiol., 42 (1967) 669- 671. (264) P. Albersheim, W. D . Bauer, K. Keegstra, and K. W. Talmadge, in F. A. Loewus (Ed.), Biogenesis of Plant Cell Wall Polysaccharides, Academic Press, New York, 1973,pp. 117-147. (265) J. A. Monro, R. W. Bailey, and D. Penny, Phytochetnisty, 1 1 (1972) 1597-1602. (266) J . A. Monro, R. W. Bailey, and D . Penny, Carbohydr. Res., 41 (1975) 153-161. (267) P. Albersheim, H. Neukom, and H. Deuel, Arch. Biochem. Biuphys., 90 (1960) 46-51.

~

310

PRAKASH M. DEY AND KEN BRINSON

protein,losJs5 which is unexpected from the early Albersheim mode1.s7.2s4(2) 10% KOH at 20 - 24" removes hemicellulose without extracting poly(g1ycosiduronic acid).lo6Extractions of hemicellulose (including xyloglucan) should be accompanied by release of both extensin and poly(g1ycosiduronic acid), according to the Albersheim model. Therefore, poly(g1ycosiduronic acid) appears not to be a component of a polymer bridge between xyloglucan and extensin. (3) 6 M Guanidinium thiocyanate (GTC), which is a powerful chaotropic agent,2ee removes about one-third of the 10%KOH-soluble hemicellulose (including xylan) from depectinized lupin h y p o ~ o t y lIf. ~the ~ only linkage between hemicellulose and cellulose microfibrils is by hydrogen bonding of xyloglucan, such reagents as 6 M GTC should extract most of the hemicellulose and protein. (4) Even after extraction with 6 M GTC for 18 h at 2 0 ° , a further fraction of the wall is extracteds0 by 10%KOH at 0 " .Release of the latter fraction may require rupture of very alkali-labile, covalent bonds. The GTC-insoluble material includes most of the "hemicellulose A," which is, however, a minor fraction.s0 ("Hemicellulose A" is the hemicellulose fraction defined in the classical extraction-procedure of O ' D ~ y e as r ~the ~ ~fraction obtained on neutralization of the KOH extract of cell walls with acetic acid. "Hemicellulose B" is obtained on addition of ethanol to the neutral extract after removal of the hemicellulose A.) The hemicellulose A contains > 70% of xylose and only 6% of glucose, and is, therefore, not a xyloglucan of the type in suspension-cultured sycamore-cells which, it is claimed, contains all of the hemicellulose xylose, and binds the hemicellulose to the cellulose micro fibril^.^^*^^ Some 60% of the wall hemicellulose (including that extracted by GTC) may be removed with 10% KOH at O", without extracting the wall protein.sO (5) Extraction of hemicelluloses with 10% KOH at 0" does not cause any changes in amino acid composition of the walls,sosuch as would occur during /?-elimination of 0-galactosyl-L-serine links. It is noteworthy that there is no loss of L-serine, although this does occur at 20"with 10%KOH. Alkalinep-elimination of 0-galactosyl-L-serine links is, therefore, not necessary for extraction of the bulk of the wall hemicelluloses, which is, hence, not dependent on links to the L-serine for its covalent association with the wall. (6) 10%KOH at 18-22' releases most of the hemicellulose not soluble at O", along with most of the wall protein (as measured by hydroxy-L-proline extraction) The time-course of alkali extraction showss0~2ee that material initially extracted at room temperature differs from that extracted after 4 h. Hemicellulosic galactan is (268)W.B. Dandliker, R. Alonso, V. A. De Saussure, F. Kierszenbaum, S.A. Levison, and H. C. Schapiro, Biochemistry, 6 (1967)1460- 1467. (269)M. H. O'Dwyer, Biochm. J . , 20 (1926)656-664.

PLANT CELL-WALLS

31 1

extracted early in this time period, whereas most of the hydroxy-L-proline and bound arabinose is extracted later. The arabinose-containing fraction is obtained by addition of ethanol to the 4-h alkali-extract after neutralization of the base, and removal of a slight, initial precipitate. This arabinose-containing fraction also contains most of the extracted hydroxy-L-proline, and the bound monosaccharide in the fraction is largely arabinose (86%),the arabinose residues being mainly (1+4)-glycosidically linked.50The results suggested that it is unlikely that the arabinose in question is bound in a serine-linked arabinogalactan but, rather, in a hydroxy-L-proline-rich glycoprotein. Because arabinans having (1-4) linkages have not been reported in plants, it was suggested that the arabinose-containing fraction consists of the cell-wall protein with its associated, hydroxy-L-proline-linked arabino-oligosaccharides. (7) Extraction of the wall with 10% KOH does not completely remove either the nonglucosidic polysaccharides or the protein from the wall. There appears to be a strong association between protein and cellulose microfibrils, and the low level of L-serine in the alkali-resistant protein5" suggested that 0-galactosyl-L-serine linkages are not involved. Certainly, a more direct cellulose -protein association than one involving arabinogalactan, poly(g1ycosiduronic acid) or its side chains, or xyloglucan is strongly suggested. (8) If polymer "creep" is involved in the control of cell elongation, bonds controlling the creep should be at right angles to the direction of elongation. In the Albersheim and in a similar model proposed by Davies,"O the xyloglucan chains creep along the cellulose fibrils. As the fibrils are mainly transverse, these models would predict a relatively larger auxin, or low pH-induced, stimulation of radial rather than longitudinal expansion. Such is not observed, and, in fact, low pH induces a decrease in the cell radius. These data led Bailey and associates4e to propose that the following general features should be part of any model of the primary cell-wall of lupin and mung-bean hypocotyls. ( 1 ) There is no covalent linkage between poly(g1ycosiduronic acid) and extensin. (2) Covalent bonds between hemicelluloses and poly(g1ycosiduronic acid) are not involved in binding either into the wall structure. (3) 0-Galactosyl-L-serine links do not play a significant part in binding hemicelluloses to extensin (the hydroxy-L-proline-rich wall-glycoprotein). (4) A large proportion of the hemicellulose is bound into the wall structure by very alkali-labile, covalent bonds. Weak ester links involving uronic acid residues are suggested as a possibility, but significant involvement of 0-galactosyl-L-serine links in attaching hemicelluloses to other wall polymers is precluded. (5) Some of the hemicellulose is attached to the wall by more-alkali-stable (270) P. J . Davies, Bot. Reu., 39 (1973) 139- 171

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PRAKASH M. DEY AND KEN BRINSON

bonds than those broken by 10% KOH at O D . These bonds are ruptured by 10%KOH at 18 - 22”.This hemicellulose fraction, which constitutes no more than 30%of the total hemicellulose, may provide a covalently bonded bridge between extensin and the cellulose fibrils. (6) At least a part of both the wall glycoprotein and the hemicellulose is strongly bound to cellulose fibrils. It was proposed that extensin may be covalently linked to cellulose fibrils, or, alternatively, to another polysaccharide that is itself covalently linked to the fibrils. This means that the cellulose fibrils may only be detached from the other polymers making up the wall matrix by the breaking of covalent bonds. (7) Cellulose fibrils are oriented within the wall of elongating lupin-hypocotyl cells mainly in the transverse direction, that is, at right angles to the direction of cell elongation. Separation of the cellulose fibrils during elongation is permitted by polymer “creep,” that is, sequential cleavage and reformation of unspecified bonds situated at “junction zones” between matrix polysaccharide chains cross-linking the cellulose fibrils, allowing mutual slippage of these cross-linking chains. In the model proposed, these unspecified bonds at the junction zones have a direction parallel to the cellulose fibrils (see Fig. 8). A partial model of the primary cell-wall of lupin hypocotyl, based on the foregoing points, is shown in Fig. 8. This work with hypocotyls challenges the existence of some of the linkages between wall polymers proposed by Albersheim and coworker^^^.^^^ and Lamport,22ebut does not actually indicate the nature of the bonds that are present. Consequently, the proposed wall-structure, shown in Fig. 8, depicts the types of networks that might occur. It shows covalent, extensinpolysaccharide association (A) with unspecified bonding between extensin and the cellulose microfibrils, and the pectin network (B), not involving extensin, but interconnected at either guanidinium thiocyanate-labile or 0”- 10% KOH-labile junctions. Bailey and associates49 attempted to introduce scale into this model, rendering it more relevant to the in uivo organization of wall polymers, through a consideration of cellulose-fibril size, the distance separating fibrils, and the lengths of other polymer molecules that may cross-link the fibrils. These workers calculated the average distance separating cellulose fibrils within the lupin-hypocotyl wall-matrix as 20.8 nm. Pectin chains are over 50.0 nm in length2T1and would therefore readily stretch between two fibrils. An estimated chain length of 50.0 nm, based on a degree of p o l y m e r i ~ a t i o nof~ ~ 150- 200, can be obtained for xylans. Moreover, pectin^^'^*^^^ and x y l a n are ~ ~ both ~ ~ capable of forming net-

-

(271) S. M. Siegel, The Plant Cell Wall, Pergamon, Oxford, 1962. (272) D . A. Rees, Adu. Carbohydr. Chem. Biochem., 24 (1969) 267-332. (273) D. E. Hanke and D. H. Northcote, Biopolymers, 14 (1975) 1 - 13. (274) I. A. Neiduszynski and R. H. Marchessault, Nature, 232 (1971) 46-47.

313

PLANT CELL-WALLS A

B

FIG.8.-Partial Model of Primary Cell-Wall in Lupin Hypocotyl, Proposed by Monro and coworker^.'^ [The half of the Figure labeled (A) represents the extensinhemicellulose network, and the half labeled (B)represents the separate, pectic network, which is believed not to involve the wall glycoprotein (extensin). Thus, the cellulose microfibrils (M) are separately cross-linked by two networks of polymers, the first (A) being composed of the wall glycoprotein and polysaccharide (probably hemicelluloses), and the second (B) being composed of the pectic polymers. These two networks have been separated in the Figure for clarity. This model is tentative and incomplete, as the nature of the linkages between the polymers in these two networks has not yet been identified. The and junction zone symbols used represent extensin (------), polysaccharide chain (-), (==) between polysaccharide chains.]

works from several polymer chains by joining through noncovalent bonds at regions of association termed “junction zones.” Estimates of the chain length of extensin molecules, based on molecular-weight estimations and the assumption that extensin has a poly-L-proline configuration (as found in collagen), vary48-228~275 between 30.3 and 95.0 nm. Although these calculations of polymer chain-length are not based on data directly derived from lupin-hypocotyl cell-walls, it is probable that polymer chain-lengths in these walls are of the same order of magnitude. It is quite possible, though unproved, that a very extensive extensinpolysaccharide complex can exist in plant primary-walls and a series linkages of xylan, pectin, galactan, and hydroxy-L-proline-rich glycopro(275) M. M. Brysk and M. J. Chrispeels, Biochim. Biophys. Acta, 257 (1972) 421 -432.

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tein would be capable of stretching over several cellulose fibrils at an average spacing within the wall matrix of the order of 20.8 nm. Strong forces encountered in the cell wall put wall polymers under tension, and this must be taken into account in constructing models ofthe wall structure. Bailey and coworkers4eproposed that their partial model met the requirements for the stresses likely to be imposed on cellulose fibrils and cross-linking matrix-polymers, in the walls of elongating cells under turgor pressure, better than the model constructed by Albersheim and a s s o ~ i a t e s . ~Bailey's ' * ~ ~ ~ group further pointed out that Albersheim's model did not appear to accommodate chemical bonds having the labilities encountered in the former group's studies with lupin-hypocotyl c e l I - ~ a l l s ,in~ which ~ - ~ ~the ~ alkali-fractionation findings do suggest that linkages other than glycosidic bonds are involved in the cohesion of wall matrix-polymers, and that certain polymers are not glycosidically interconnected. An accurate, cell-wall model must eventually take into account stresses on the cell wall, orientation of wall components, detailed structures of wall polymers, and the exact nature of chemical bonds between wall components. Present knowledge ofprimary cell-wall structure is too inadequate to allow any of the models currently proposed to be other than working hypotheses.

BETWEEN THE CONSTITUENT POLYMERS IN PRIMARY XI. INTERCONNECTIONS CELL-WALLS OF MONOCOTS There have been few studies of polymer interconnections within the primary wall of monocots. An arabinoxylan from the primary wall of cultured, barley-aleurone cells,61 and a glucuronoarabinoxylan from maize-coleoptile primary-wa11,200have been shown to bind reversibly to cellulose in uitro. Because xylans are, quantitatively, the major component of monocot primary cell-walls, this interconnection is an important finding: it is very likely to occur through multiple hydrogen-bonds, analogous to the interconnection between xyloglucan and cellulose in dicot ~ e l l - w a l l sIt. is ~ also ~~~ possible ~ ~ ~ ~that heteroxylans participate in binding other cell-wall polymers to cellulose. In contrast to these findings, a glucuronoarabinoxylan isolated from oat coleoptiles did not bind to cellulose in uitro under reaction conditions that allowed other heteroxylans to bind.s3 This oat heteroxylan had, however, a high percentage of arabinosyl side chains that would be likely to hinder binding sterically. A similar inability to bind to cellulose is exhibited by an arabinose-rich arabinoxylan isolated from cultured, barley-aleurone cell-walls.61 As with dicots, it is probable that monocot xylans bind to each other, as

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well as to cellulose. Such xylans form aggregates in s 0 1 u t i o n ~ ~that J~~ could lead to gel formation and may, in this way, be involved in crosslinking of monocot, primary-wall polymers. Some monocot, primary cell-wall polysaccharides may be cross-linked by esters of ferulic acid (4-hydroxy-3-methoxycinnamic acid). There is evidence for the existence of such cross-links in monocot tissues containing secondary walls, including Ztalia ryegrass stem276and wheat endosperm.277Ferulic acid is also present in barley c e l l - ~ a l land , ~ ~it~has been reported that treatment with base releases ferulic acid from the cell walls of several G r ~ m i n a esupporting ,~~~ the idea that ferulic acid is bound to the wall as an ester. However, ferulic acid has not, so far, been reported to be present specifically in primary cell-walls in either monocots or dicots. XII. CELL-WALL BIOSYNTHESIS 1. Introduction Up to this point, the subject matter of this article has been concerned with the structure and function, in higher plants, of the intact, primary cell-wall and of its constituent polymers. The degradation of wall polymers during the development of plant tissues, including such senescent processes as fruit ripening, involves catabolic pathways. However, biosynthesis probably continues even into senescence; there occurs, for example, continued incorporation of radioactivity from [‘*C]methionine into methyl groups of poly(methy1 galacturonate) in ripening apples,279 and of radioactivity from 14C02into complex polysaccharides in leaves and fruit mesocarp of fruiting-plum spurs.280Further clarification of the mechanisms of synthesis and insertion into the wall of its constituent polymers may enhance understanding of the roles of these polymers within the wall, and of the possible significance oftheir removal. Little is, at present, known about the involvement of biosynthetic steps during senescence, but some understanding has been gained regarding the synthesis of individual wall-components. Glycosyl esters (“sugar nucleotides”) are the glycosyl donors for the formation of wall polysaccharides. Some glycosyl-nucleotides can, in uiuo, be synthesized directly from the corresponding monosaccharide, ATP, and the appropriate nucleoside triphosphate. In addition, some (276) (277) (278) (279) (280)

R.D. Hartley andE. C. Jones, Phytochemisty, 15 (1976) 1157-1160. H. U. Markwalder and H. Neukom, Phytochemisty, 15 (1976) 836-837 G . B. Fincher,]. Inst. Brew. (London), 81 (1975) 116-122. M. Knee, Photochemistry, 17 (1978) 1261-1264. L. Hough and J. B. Pridham, Nature, 177 (1956) 1039.

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PRAKASH M. DEY AND KEN BFUNSON

UDP-glycoses can be produced from UDP-D-glucose by successive structural alterations of the D-glucosyl group. Such nucleotide esters of D-glucose as UDP-D-glucose and ADP-D-glucose can also be synthesized directly from sucrose by the enzyme sucrose synthetase (EC 2.4.1.13). Finally, myo-inositol is considered by some to be an intermediate in the formation of the “sugar nucleotides” containing glucuronic acid, galacturonic acid, arabinose, xylose, and the branched sugar apiose. These are the major pathways considered to be potentially important for the formation of nucleotide substrates for biosynthesis of wall polysaccharides. Much detail is known about the enzymes concerned, and the subject has been comprehensively reviewed by Karr,281Nikaido and Hassid,ea2and

other^.^*^-^^' Although the biochemistry of the synthesis of glycosyl-nucleotides in higher plants is well advanced, the subsequent polymerization reactions, involving their glycosyl groups, to yield the various classes of cell-wall polysaccharides is not. The enzymes catalyzing these processes have not yet been fully characterized, and the involvement of membrane systems, and the mechanism of assembly of the wall itself, are ill-understood. All of the enzymes responsible for formation of cell-wall polysaccharide appear to be membrane-bound, and are usually recovered in the particulate, cell-wall fraction sedimenting at 20,OOOg:none have been purified significantly. Several names have been given to these complex enzyme-systems, which may possess more than one activity, including polysaccharide synthase and glycosyltransferase. The term polysaccharide synthase is preferred for use in this article. The general equation for single steps in the reaction catalyzed by a polysaccharide synthase is

-

NDP-glycosyl

+ acceptor

+

glycosyl-acceptor

+ NDP,

where N is a nucleoside. Polysaccharide synthases may exhibit a high degree of substrate specificity for both the base of the “sugar nucleotide” and the glycosyl group. On transfer, the glycosyl group is joined to the terminal residue of the acceptor by a glycosidic linkage specific with (281) A. L. Karr, in J. Bonner and J. V. Varner (Eds.) Plant Biochemistry, Academic Press, New York, 1976, pp. 405-426. (282) H. Nikaido and W. Z. Hassid, Ado. Carbohydr. Chem. Biochem., 26 (1971) 351483. (283) G . A. Barber, in J. B. Pridham and T. Swain (Eds.), Biosynthetic Pathways in Higher Plants, Academic Press, New York, 1965, pp. 117-121. (284) V. Ginsburg, Ado. Enzymol., 26 (1964) 35-38. (285) L. Glaser, Physiol. Reo., 43 (1963) 215-235. (286) E. F. Neufeld and W. Z. Hassid, Ado. Carbohydr. Chem., 18 (1963) 309-356. (287) S.Passeron and H. Garminatti, An. Soc. Cient. Argent., (1971) 31 -40.

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respect to its position on the glycon ring and its anomeric configuration.281.288 2. The Biosynthesis of Cell-Wall Polymers

a. Cellulose. -This/&( 1+4)-linked D-glucan is synthesized by a wide variety of organisms, ranging from bacteria to higher plants. It is present in the cell wall in the form ofhighly ordered structures called microfibrils that are embedded in a matrix composed of the other interlinked wall polymers, which include pectins, hemicelluloses, and glycoproteins. The possible nature of chemical bonding between cellulose fibrils and these matrix polysaccharides, as well as interconnections between the matrix polysaccharides themselves, in the intact primary-wall have been discussed in Sections IX, X,and XI. There are some reports in the literature4.ss-zz3that purified cellulose contains minor proportions of glycosyl residues other than D-glucosyl, and it is not clear whether such residues are covalently attached to the cellulose fibrils, or are present in hemicelluloses that are tightly and non-covalently bound to the fibrils. Cellulose is present in both primary and secondary cell-walls. However, differences in both the degree of polymerization and the control of chain length between the celluloses from these two sources suggest that the two types of cellulose are formed by different mechanism^.^^^-^^^ Whereas the mechanisms responsible for formation and orientation of cellulose fibrils are not yet known, some information is available concerning the enzymes capable of catalyzing the formation of celluloselike, p-(l+4)-linked D-glucans (see also, Ref. 217). Early reports of such an enzyme system, tightly bound and occurring in a particulate fraction of mung bean, were made by Hassid and assoc i a t e ~ .The ~ ~glycosyl ~ * ~ ~donor ~ was reported to be GDP-D-glucose and the product was characterized as a p-( 1+4)-linked ~-glucan.~e2.Ze~ Liu and HassidZe4solubilized, and partially purified, this enzyme. Hassid and coworkers2e3reported that this mung-bean enzyme-system catalyzes the incorporation of D-glucose from GDP-D-glucose into a polysaccharide having the characteristics of cellulose and that, in the presence of GDP-D-mannose, this incorporation was stimulated. The same enzyme-system, when provided with GDP-D-mannose as the sole substrate, catalyzed the incorporation of GDP-D-mannose into a glucoman(288) A. F. Clark and C. L. Villemez, Plant Physiol., 50 (1972) 371 -374. (289) M. Marx-Figini, Nature, 210 (1966) 754-755. (290) M. Marx-Figini,]. Polym. Sci., Part C, 28 (1969) 57-67. (291) F. S. Spencer and G . A. MacLachlan, Plant Physiol., 49 (1972) 58-63. (292) A.D.Elbein,G.A.Barber,andW.Z.Hassid,].Am. Chem.Soc.,86(1964) 309-310. (293) C.A. Barber,A.D. Elbein, and W. Z. Hassid,]. B i d . Chem.,239 (1964) 4056-4061. (294) T. Liu and W. Z. Hassid,J. B i d . Chem., 245 (1970) 1922-1925.

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PRAKASH M. DEY AND KEN BRINSON

nan. Hassid and C O W O ~ ~ interpreted ~ ~ S ~ these ~ ~ results - ~ ~ to ~ mean that the particulate system contains more than one enzyme that can utilize GDP-D-glucose, that is, one enzyme that catalyzes the incorporation of D-glucose from GDP-D-glucose into cellulose, and a separate enzyme that utilizes GDP-D-glucose as one of the substrates for the synthesis of glucomannan. Some workers disagree with this interpretation. Villemez and Heller29ee noted that GDP-D-glucose may not be present in plant tissues that are synthesizing cellulose, and that periods of maximal utilization of GDP-D-glucose do not coincide with periods of cellulose synthesis. Furthermore, V i l l e m e considered ~ ~ ~ ~ ~ the ~ ~ kinetic ~ consequences of the existence of two enzymes that use the same substrate, namely, GDP-Dglucose, and argued that, if only one enzyme (the enzyme that catalyzes cellulose synthesis) is operative when GDP-D-glucose is present as the sole substrate, and that both enzymes (separate enzymes catalyzing cellulose and glucomannan synthesis, respectively) are operative when GDP-D-mannose is also present, the argument presented by Hassid and c o w ~ r k e r sthe , ~presence ~ ~ ~ ~ of ~ ~GDP-D-mannose should induce an increase in the initial rate of incorporation of D-glucose from GDP-D-ghcose into polysaccharides. Villemez2Q7*2Q8 did not note this effect: working with the mung-bean, particulate-enzyme system, he found, instead, that whereas GDP-D-mannose enhanced the total incorporation of D-glucose from GDP-D-glucose into polysaccharide, it did not enhance the initial rate of this incorporation. On this basis, he concluded that the particulate system contained only one enzyme capable of utilizing GDP-D-glucose as a substrate for polysaccharide synthesis. Heller and solubilized, but did not separate, the D-mannosyltransferase and D-glucosyltransferase activities from this particulate-enzyme system, and found that, when only GDP-D-mannose is provided as the substrate, a/?-(1+4)-linked D-mannanis synthesized and, when only GDP-D-glucose is present, a /I1-*4)-linked ( D-glucan is produced. They further noted, with this transferase system, that incorporation of D-glucose from GDP-D-glucose into polysaccharide is a shortlived reaction that can be greatly extended if GDP-D-mannose is also included in the reaction mixture. These results may be explained on the basis of assuming that gluco(295) A. D. Elbein and W. Z. Hassid, Bfochem.Btophys. Res. Commun.,23 (1966) 311318. (296) C. L. Villemez and J. S. Heller, Nature, 227 (1970) 80-81. (297) C . L. Villemez, unpublished findings, cited in Ref. 298. (298) C. L. Villemez, Btochem.]., 121 (1971) 151-157. (299) J. S. Heller and C. L. Villemez, Btochem. J., 128 (1972) 243-252. (300) J. S. Heller and C. L. Villemez, Btochm. J., 129 (1972) 645-655.

PLANT CELL-WALLS

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mannan (not cellulose) is the normal product of the reactions catalyzed by this particulate-enzyme system and that continued action of the Dglucosyltransferase is dependent upon the action of the D-mannosyltransferase. Further weight was lent to the proposition that this enzyme system does not normally catalyze the synthesis of cellulose, from GDP-D-glucose as the substrate, by the finding of Heller and V i l l e m e ~ ~ ~ ~ that cellobiose constitutes only a small proportion of the acetolysis products of the polysaccharides synthesized when both GDP-D-glucose and GDP-D-mannose are provided as substrates. ~ ~ the ~ This evidence was supported by a report by V i l l e m e ~that p-( l-+4)-linkedD-glucans synthesized from GDP-D-glucose as the sole substrate were a mixture of low-molecular-weight polymers having a maximum degree of polymerization of 40, that is, much smaller molecules than that of naturally occurring cellulose. The presence of GDP-Dmannose as a second substrate led to the disappearance, from the products of enzyme activity, of these low-molecular-weight polymers, and their replacement by high-molecular-weight polysaccharide. Villeme~ contended ~~~ that all of this evidence is consistent with the hypothesis that the particulate-enzyme system of mung bean in question catalyzes, in uiuo, the synthesis of D-glucomannan, and not of cellulose, and that the low-molecular-weight D-glucans, which are produced in uitro when GDP-D-glucose is present as the sole substrate, are not related to cellulose synthesis, but are merely a series of molecular artifacts that result from premature termination of D-glucosyl transfer in the absence of sufficient D-mannose-containing acceptors. V i l l e m e thus ~ ~ ~proposed ~ that GDP-D-glucose is a precursor, in uiuo, of D-ghcomannan, not of cellulose. However, an enzyme that uses GDP-D-glucose as a substrate in order to synthesize a cellulose-like product has been reported to be present in developing cotton fibers during the period when primary-wall cellulose synthesis normally occurs, but is absent from older fibers undergoing active deposition of secondary-wall cellulose.303 Brummond and Gibbons304 demonstrated the presence, in higher plants, of enzymes that utilize UDP-D-gluCOSe to produce a cellulose-like polymer, and Hassid and c0workers3~~ separately reported in mung bean a similar enzyme system in which 80 - 90% of the product consisted of (301) C. L. Villemez, in J. B. Pridham (Ed.), Plant Curbohydmte Biochemistry, Academic Press, New York, 1974, pp. 183-189. (302) C . L. Villemez, unpublished findings, cited in J. Bonner and J. V. Varner (Eds.),Plant Biochemistry, Academic Press, New York, 1976, pp. 405-442. (303) D. P. Delmer, C. A. Beasly, and L. Ordin, Plant Physiol., 53 (1974) 149-153. (304) D. A. Brurnmond and A. P. Gibbons, Biochem. 1..342 (1965) 308-318. (305) C . L.Villemez, G . Franz, and W. Z. Hassid, Plant Physiol., 42 (1967) 1219- 1223.

320

PRAKASH M. DEY AND KEN BRINSON

p-( 1+4)-linked p-( l+3)-linked.

D-glucan, the rest of the D-glucosyl residues being Clark and V i l l e m e ~described ~~~ conditions under which only /I1+4)-linked -( D-glucan is produced from UDP-D-glucose by the mung-bean enzyme-system, and they characterized the product and showed that it is of high molecular weight; 7.5% of the total product possessed301a molecular weight of > 1.2 X 1Os. However, in a contrary finding, Hassid and coworkers307reported that enzymes from mung bean could, indeed, utilize UDP-D-glucose for polysaccharide synthesis, but that the product did not contain p-( 1+4)-linked D-glucosyl residues. An enzyme system that catalyzes the synthesis of p-( l-+linked D-glucans from UDP-D-glucose has also been reported, by Ordin and associate^,^^^-^^^ to be present in oat seedlings. The product contained only small numbers of p-( 1-+3)-linked D-glucosyl residues. These workers, together with Tsai and H a ~ s i d , ~demonstrated ll that high concentrations of UDP-D-glucose favor the formation of p-( 1+3)-linked Dglucans, whereas low concentrations of the nucleotide favor the formation of p-( 1+4)-linked D-glucans. Tsai and Hassid31econfirmed that the particulate-enzyme system from oat seedlings contains two enzymes that utilize UDP-D-glucose: an enzyme that catalyzes the transfer of the Dglucosyl group to ap-(1+3)-linked D-glucan,and a separate enzyme that catalyzes tranfer to a p-( l-*4)-linked D-glucan. From the conflicting nature of the experimental evidence just cited, it is clear that the glycosyl-nucleotide utilized in uiuo by synthetic enzymes as a source of D-glucosyl groups for incorporation into cellulose remains unidentified. The balance of present evidence appears to favor UDP-Dglucose. However, positive confirmation of the role of this nucleotide ester as the substrate for cellulose synthesis awaits further work. An aspect in especial need of clarification is the apparent effect of varying the concentration of UDP-D-glucose on the nature of the D-ghcosidic linkages within the D-glucan p r o d u ~ t . ~ ~It~ -is~ not l l clear whether p-( 1+4)-linked D-glucans and p-( 1+3)-linked D-glucans are synthesized by the same or by separate enzymes; clarification of this question calls for more-refined techniques for the separation and purification of the enzymes involved. Resolution of these problems, and the elucidation of the control mechanisms presumably involved, promise to yield fascinating insight into the mechanisms of assembly of cell-wall polymers.

(306) See Ref. 288. (307) H. M. Flowers, K.K. Batra, J. Kemp, and W. Z. Hassid, Plant Physiol., 43 (1968) 1703 - 1709. (308) L. Ordin and M. A. Hall, Plant Physiol.,43 (1968) 473-476. (309) L. Ordin and M. A. Hall, Plant Physiol., 42 (1967) 205-212. (310) A. Pinsky and L. Ordin, Plant Cell Physiol., 10 (1969) 771-785. (311) C. M. Tsai and W. Z. Hassid, Plant Physiol., 51 (1973) 998-1001. (312) C. M. Tsai and W. Z. Hassid, Plant Physiol., 47 (1971) 740-744.

PLANT CELL-WALLS

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b. Pectins and Hemicelluloses. -These polymers are the matrix polysaccharides of the plant primary cell-wall, in which the cellulose fibrils are embedded. The middle lamella is particularly rich in pectic polysa~charides.'~'The proposed structures of pectic polysaccharides and hemicelluloses have been discussed in Sections 111 and IV,respectively, and the possible nature of linkages between these polymers and cellulose fibrils has been discussed in Sections IX,X, and XI. For a discussion of the biosynthesis of cellulose, see Ref. 217. A particulate-enzyme system capable of producing poly(ga1acturonic acid) was isolated from mung bean by Hassid and associates.313 The D-galactosyluronic donor in this reaction is UDP-D-galacturonic acid; the D-galacturonic acid derivatives of other glycosyl-nucleotides are not utilized as ~ubstrates.~l4 The polymer produced can be completely hydrolyzed by polygalacturonase, to yield D-galacturonic acid. The latter observation suggested that this mung-bean enzyme-activity represents only partial synthesis of a polysaccharide, as the pectic backbone in primary walls contains Methyl esterification takes place after poly(ga1acturonic acid) is formed, and Kauss and c o ~ o r k e r s ~ showed ~ ~ - ~ ~that ' the mung-bean particulate-fraction that contains the galactosyluronic transferase also contains an enzyme responsible for methylating carboxyl groups of poly(ga1acturonic acid). The methyl donor for this reaction is S-adenosyl-L-methionine. The particulate-cell fraction from plants contains a number of other polysaccharide synthases involved in heteroglycan synthesis. For example, an enzyme system that will catalyze the transfer of a D-glucosyluronic acid group from UDP-D-glucuronic acid to polysaccharides has been isolated from corn. The polymer produced in vitro is similar to the D-glucuronic acid-containing hemicellulose fraction obtained from corn.318J1QThe 4-0-methyl derivative of the polymerized D-glucuronic acid is formed by an enzyme present in the same particulate fraction.31s*31Q The methyl donor in the reaction is, again, S-adenosyl-L-methionine. Other reactions that have been demonstrated with preparations from a variety of higher plants include the formation of xylan from UDP-D-XY(313)C.L. Villemez, A. L. Swanson, and W. Z. Hassid, Arch. Biochem. Biophys., 116 (1966)446-452. (314)W.Z.Hassid, Annu. Reu. Plant Physiol., 18 (1967)253-280. (315)H. Kauss and W. Z. Hassid,]. Biol. Chem., 242 (1967)3449-3453. (316)H. Kauss and A. L. Swanson, Z . Naturforsch., 24 (1969)28-33. (317)H. Kauss, Biochirn. Biuphys. Acta, 148 (1967)572-514. (318)H . Kauss and W. Z. Hassid, J . Bid. Chem., 242 (1967)1680- 1684. (319)H. Kauss, Phytochemistry, 8 (1969)985-988.

322

PRAKASH M. DEY AND KEN BRINSON

lose, and of arabinoxylan from UDP-D-xylose and UDP-~-arabinose,~~O the synthesis of galactan from U D P - ~ - g a l a c t o s eand , ~ ~the ~ ~synthesis ~~~ of a p-( 1+3)-linked D-glucan from U D P - ~ - g l u c o s e . ~ ~ ~ In conclusion, it may be stated that present knowledge of the enzymic mechanisms catalyzing the formation of heteroglycans from glycosylnucleotides as glycosyl donors, and of the relevance of such synthetic mechanisms to the formation of the intact, primary cell-wall, is very limited. In particular, no heteroglycan having properties identical to those of known, native polymers of the primary wall has yet been synthesized in uitro. c. Extensin, The Hydroxy-L-proline-rich Glycoprotein of the Cell Wall. -The proposed structure of this glycoprotein has been discussed in Section VII, and its proposed position and role in the intact, primary cell-wall of dicots were discussed in Sections IX and X. Present knowledge suggests that the glycoprotein is an important structural component of the wall, linked covalently to wall-matrix polysaccharides and, possibly, also strongly bonded to cellulose fibrils. However, little is currently known about its synthesis. The protein component may be assembled on the ribosomes by the normal mechanism of protein synthesis.324L-Proline is known to be the precursor of the hydroxy-L-proline found in the g l y ~ o p r o t e i n ,hydroxylation ~ ~ ~ * ~ ~ ~ of the peptide-bound L-proline being catalyzed, in carrot cells, by cytoplasmic enzymes.324 L a m p ~ r and t ~ Karre32 ~~ showed that the particulate-cell fraction from cultured sycamore-cells contains enzymes responsible for the synthesis of the extensin arabino-oligosaccharides; these enzymes catalyze transfer of L-arabinose from UDP-L-arabinose to the hydroxy-L-prolinerich protein, to produce the glycoprotein. The oligosaccharide synthesized by this in uitro system appears identical to the naturally occurring tetra-L-arabinosyl side-chain of extensin which, on the basis of its structural complexity, probably requires the activity of several enzymes for its synthesis.23zFurthermore, it was proposed that the hydroxy-L-prolinerich protein is glycosylated by the sequential transfer of single L-arabinosyl groups, and not by the transfer of preformed oligosaccharides.232

(320) R. W. Bai1eyandW.Z. Hassid, Proc. Natl.Acad. Sci. U.S.A.,56 (1966) 1586-1593. (321) J. M. McNab, C. L. Villemez, and P. Albersheirn, Biochem.]., 106 (1968) 355-360. (322) N. Panyatatos and C. L. Villemez, Btochem. J., 133 (1973) 263-271. (323) D . S. Feingold, E. F. Neufeld, and W. Z. Hassid, J. B i d . Chem., 233 (1958) 783788. (324) M. J. Chrispeels, Plant Physiol., 45 (1970) 223-227. (325) J. Hollernan, Proc. Natl. Acad. Sci. U.S.A.,57 (1967) 50-54. (326) D . T. A. Lamport and D. H. Miller, Symp. SOC. Dm.Btol., Plant Physiol., 48 (1971) 454- 456.

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3. Intermediates in the Synthesis of Polysaccharides a. Introduction. -Glycosyl-nucleotides are used as substrates by the enzymes responsible for the formation of plant polysaccharides, as has been discussed in Section WI,2. However, it is not clear whether the glycosyl-nucleotides are the direct glycosyl donors in the polysaccharidesynthase-catalyzed reactions (see also, Ref. 21 7). The synthesis of bacterial cell-wall p o l y s a c c h a r i d e ~and ~ ~of ~ a mannan from Micrococcus Z y s o d e i k t i ~ u s 3 ~involves ~ J ~ ~ the formation of a glycolipid intermediate. These intermediates serve as glycosyl donors in the formation of polysaccharides. The involvement of glycolipid and glycoprotein intermediates in the synthesis of polysaccharides from glycosyl-nucleotides in plants is considered to be a likely possibility. Such intermediates could act as specific primers, or acceptor substrates, for the formation of polysaccharides. Furthermore, subunits of complex heteropolysaccharides could be assembled on such intermediates, and later incorporated into polysaccharides, or directly cross-linked into the cell wall. Evidence of the involvement of such intermediates in the synthesis of polysaccharides in a number of organisms is presented in Sections XII,3,b and XII,3,c.

b. Intermediates in Polysaccharide Synthesis in Bacteria and Algae. -The first report of the involvement of a glycolipid intermediate in the synthesis of cell-wall polysaccharides in bacteria came from J. S. Anderson and coworkers327in 1965, Later, Higashi and associates330implicated a glycolipid intermediate, characterized as a polyisoprenyl (glycosyl diphosphate), in bacterial-peptidoglycan biosynthesis. S ~ h e r , ~ " . ~ ~ ~ H i g a ~ h iand , ~ ~L~e n n a r ~ , ~and ~ l Jtheir ~ ~ respective coworkers, demonstrated that crude, cell-free extracts of Micrococcus Zysodeikticus catalyze the incorporation of D-mannose from GDP-D-mannose into a glycolipid that was characterized328as undecaprenyl (D-mannosylphosphate). In this mannolipid, the polyisoprenoid component is 55 carbon atoms long, and contains 11 carbon-carbon double bonds; its structure is as shown. The reversible, enzymic formation of this D-mannolipid involves the transfer of the D-mannosyl group from GDP-D-mannose to undecaprenyl (327) J. S. Anderson, M. Matsuhashi, M. A. Haskin, and J. L. Strominger, Proc. Natl. Acad. Sci. U.S.A.,53 (1965) 881-889. (328) M. Scher, W. J. Lennarz, andC. C. Sweeley, Proc. Natl. Acad. Sci. U.S.A.,59 (1968) 1313 - 1320. (329) M. Scher and W. J. Lennarz,]. Biol. Chem.,244 (1969) 2777-2789. (330) Y. Higashi, J. L. Strominger, and C. C. Sweeley, Proc. Natl. Acad. Sci. U.S.A., 57 (1967) 1878 - 1884. (331) W. J. Lennarz and B. Talamo,]. B i d . Chem., 241 (1966) 2707-2719. (332) M. Scher, K. K. Kramer, and W. J. Lennarz, Abstr. Pap. Am. Chem. SOC.Meet., 154 (1967) D43.

PRAKASH M. DEY AND KEN BRINSON

324

& 1CH,OH

Me

Me Me I I (CHzCH=C-CH2)9- CH,CH=C-Me

I

O-P-O-CHzCH=C-CH2-

HO

phosphate.328In a later study,3e9these workers confirmed that this mannolipid is an intermediate in the biosynthesis of a D-mannan occurring in the plasma membrane of M. lysodeikticus. The D-mannolipid, in uitro, serves to donate D-mannosyl groups to terminal, nonreducing sites of endogenous D-mannan by the reaction shown in Scheme 1. 0

0 II [14C]Mannosyl-OPOR + GDP

II

GDP-(L4C]Man + O--POR

b0 II

[“C]Mannosyl-OPOR

A-

D

(

b-

0

(“C]Mannan

11

i 0--POR

A-

- Mannan

acceptor SCHEME 1. -Reactions Involved in the Biosynthesis of o-Mannan from GDP-Mannose. [The acceptor lipid in the reaction, undecaprenyl phosphate, is designated as follows:]

0

II

0--POR

I

0-

The isoprenoid intermediates involved in biosynthesis of peptidoglycans and 0-antigen of lipopolysaccharide in certain other bacterial syst e m ~ ~contain ~ ~a diphosphate , ~ ~ ~ link . ~between ~ ~ the glycosyl and lipid groups. According to the mechanism proposed for heteropolysaccharide biosynthesis involving these intermediates, the diphosphate group remains linked to undecaprenol when the glycosyl group is transferred to an acceptor, and the undecaprenyl diphosphate must be hydrolyzed to undecaprenyl phosphate in order to regenerate the active-carrier lipid.329*330*333s334 It was proposed by Robbins and coworkers335that the energy liberated when inorganic phosphate is released from undecaprenyl diphosphate supplies a “driving force” to the heteropolysaccharide biosynthesis in these bacterial systems. However, as undecaprenyl (D-mannosyl phosphate) serves as a D-mannosyl donor for D-mannan synthesis in M. l y ~ o d e i k t i c u sand , ~ ~ has ~ a lability to acid comparable to that of the aforementioned, diphosphate-containing glycolipid interme(333) A. Wright, M. Dankert, P. Fennessey, and P. W. Robbins, Proc. Natl. Acad. S d . U.S.A., 57 (1967) 1798-1803. (334) M. A. Cynkin and M. J. Osborn, Fed. Proc., 27 (1968) 293. (335) P. W. Robbins, D. Bray, M. Dankert, and A. Wright, Sdence, 158 (1967) 15361542.

PLANT CELL-WALLS

325

diates, it seems equally possible that the glycosyl group in all of these intermediates is activated because of the presence of a double bond in allylic relationship to the phosphate or diphosphate group that Iinks the glycosyl group to the lipid. Clearly, these findings32s,32e.331.33P do not provide a full understanding of the mechanism of D-mannan biosynthesis in M.lysodeikticus, as synthesis de novo was not observed. Perhaps, under proper experimental conditions, synthesis of the entire molecule can proceed by way of undecaprenyl (D-mannosyl phosphate). Such a process might proceed by way of oligosaccharide - lipid intermediates, as suggested by Behrens and Cabib336 and Stewart and B a l l o ~ . ~On ~ ' the other hand, it is equally possible that synthesis of the backbone of the D-mannan molecule involves participation of a diphosphate-containing polyisoprenoid lipid, with undecaprenyl (D-mannosyl phosphate) serving as a D-mannosyl donor only for the introduction of short branches to complete the D-mannan, as the polymer is branched, containing (1+2)-, (1-+3)-,and (1+6)-~-mannosidiclinkages.32eAlternatively, transfer of D-mannose from undecaprenyl (D-mannosyl phosphate) to nonreducing termini could also serve as a completion step in a process in which the backbone of the D-mannan is synthesized by a reaction involving a D-mannosyl-nucleotide as the D-mannosyl donor. Finally, the possibility of a glycoprotein intermediate between undecaprenyl (D-mannosyl phosphate) and the completed D-mannan must be considered. Pont Lezica and associate^^^^-^^' demonstrated that particulate preparations from the green alga Prototheca zopfii catalyze the incorporation of ~-['~C]glucose from UDP-~-['~C]glucose into D-glucohpids in which the lipid moiety was identified as a dolichyl phosphate, a polyisoprenyl phosphate having a chain length ranging from 90 to 105carbon atoms.33e The D-glucolipids have been characterized as dolichyl (D-glucosyl phosphate), dolichyl (D-glucosyl diphosphate), and dolichyl (D-gluco-oligosaccharidyl d i p h o ~ p h a t e s )The . ~ ~ lipid-linked ~ oligosaccharides were a mixture ranging from a disaccharide to a decasaccharide, the D-glucosyl residues being linked by P-D-(1+4)-glucosidic linkages.33sThe particulate-enzyme system catalyzed transfer of the oligosaccharides from the lipid carrier to a protein acceptor, resulting in the formation of a waterN . H. Behrens and E. Cabib,]. Biol. Chem., 243 (1968) 502-509. T. W. Stewart and C. E. Ballou, Biochemistry, 7 (1968) 1855-1863. H. E. Hopp, P. A. Romero, G.R. Daleo, and R. Pont Lezica, Eur. J . Biochem., 84 (1978) 561-571. H. E. Hopp, G . R. Daleo, P. A. Romero, and R. Pont Lezica, Plant Physwl., 61 (1978) 248 - 251. H. E. Hopp, P. A. Romero, and R. Pont Lezica, FEBS Lett., 86 (1978) 259-262. H. E. Hopp, P. A. Romero, G.R. Daleo, and R. Pont Lezica, in L. A. Appelqvist and C. Liljenberg (Eds.),Adoances in Biochemistry and Physiology of Plant Lipids,Elsevier/North Holland Biomedical Press, Amsterdam, 1979, pp. 313-318.

PRAKASH M. DEY AND KEN BRINSON

326

soluble ~ - g l u c o p r o t e i n ~ ~ 8this . 3 ~oligosaccharide ~; transfer was inhibited by coumarin, a known inhibitor of cellulose synthesis in plants.34oWhen GDP-D-glucose was added to the incubation mixture, the soluble D-glucoprotein appeared to serve as a primer for the acceptance of D-glucosyl groups donated by GDP-D-glucose in the formation of an alkali-insoluble polymer having the properties of c e l l ~ l o s e . ~ ~ ~ - ~ ~ ~ On the basis of these findings, Pont Lezica and coworkers341proposed a scheme, involving glucolipids and a glucoprotein as intermediates, for the biosynthesis of cellulose in Prototheca zopfii; this is shown in Scheme 2. Endoplasmic reticulum Dol-f-P-Gk UDP

I \ \

Dol-P-Glc \

-Glc \

Dol-P-P

Protein

Coumarin

I

1 Protein-Glen GDP-Glc GDP

=i

cellulose

Golgi apparatus

SCHEME2. -Proposed Scheme for the Reactions Involved in the Synthesisofcellulose in the Green Alga Prototheca zopfii (AfterHopp and Coworkers3"). [Abbreviationsused are: Do1 = dolichol, Glc = D-glucose, Dol-P = dolichol phosphate, -P-P = diphosphate, and Pi = inorganic phosphate.]

PLANT CELL-WALLS

327

It is significant that, in all of the mechanisms proposed for the biosynthesis of polysaccharides that involve glycolipids as intermediates, already cited, the lipid acceptor for glycosyl groups appears to be a phosphorylated polyisoprenol compound. The further involvement of a glycoprotein as an intermediate between glycolipid and completed polysaccharide has been strongly suggested by the findings with the algal, cellulose-synthesizing system,338-341but the present findings have not revealed a similar role for a glycoprotein in the bacterial, synthetic mechanisms that have been d i s c ~ s s e d . ~The ~ ' - possible ~ ~ ~ involvement of similar intermediates in the biosynthesis of polysaccharides in higher plants will now be considered. c. Intermediates in Polysaccharide Synthesis in Higher Plants. There is considerable evidence that polyisoprenyl phosphates also act as acceptors for glycosyl groups in higher plants. In early studies, K a ~ s s ~ ~ ~ and Villemez and Clark343reported the presence, in mung bean, of a mannosyl-lipid, in which, they presumed, the lipid moiety was of the polyisoprenol type, and they proposed that this glycolipid acts as an intermediate in the incorporation of D-mannose from GDP-D-mannose into D-glucomannan. They were, however, unable to demonstrate the role of this D-mannolipid as a direct, D-mannosyl donor in glucomannan synthesis. Daleo and Pont L e ~ i c and a ~ Delmer ~ ~ and coworkers345confirmed that the mung-bean mannolipid is dolichyl (D-mannosyl phosphate), but its role as a glycosyl donor in polysaccharide formation has not been established. Forsee and Elbein346reported that a particulate fraction from cotton fibers catalyzed the incorporation of D-mannose from GDP-D-mannose into a D-mannolipid that had the properties of a polyisoprenyl (D-mannosylphosphate). A lipid having the properties of a polyisoprenyl phosphate was extracted from the reaction mixture, but its chain length was not characterized. A polyisoprenyl phosphate has been shown to act as an acceptor for D-mannose and 2-acetamido-2-deoxy-~-glucose in mung b e a d 4 ' and peas.348Similar polyisoprenyl phosphates that accept glycosyl groups have been reported in soy bean, wheat germ, and pea seedlings,34ePhaH. Kauss, FEBS Lett., 5 (1969) 81-84. C. L. Villemez and A. F. Clark, Biochem. Biophys. Res. Commun., 36 (1969) 57-63. G . R. Daleo and R. Pont Lezica, FEBS Lett., 74 (1977) 247-250. D. P. Delmer, C. Kulow, and M. C. Ericson, Plant Physiol., 61 (1978) 25-29. W. T. Forsee and A. D. Elbein, I. Biol. Chem., 248 (1973) 2858-2867. L. Lehle, F. Fartaczek, W. Tanner, and H. Kauss, Arch. Biochem. Biophys., 175 (1976) 419-426. (348) C. T. Brett andL. F. Leloir, Biochem. J., 161 (1977) 93-101. (349) R. Pont Lezica, C. T. Brett, P. Romero Martinez, and M. A. Dankert, Biochem. Biophys. Res. Commun., 66 (1975) 980-987. (342) (343) (344) (345) (346) (347)

328

PRAKASH M. DEY AND KEN BRINSON

seolus vulgaris cotyledons,350and Pisum s a t i v ~ m . ~However, ~' none of these glycolipids have been confirmed as direct glycosyl donors in polysaccharide biosynthesis. Glycoproteins possessing an N-glycosylic linkage between 2-acetamido-2-deoxy-~-glucoseand the amide nitrogen atom of an L-asparagine residue of the protein have been identified in cotton fibers,352 mung-bean s e e d l i n g ~ 3 Phaseoh ' ~ ~ ~ ~ ~ vulgaris cotyledons,3s0Pisum sati~ 2 1 7 1 2 and , ~ ~ ~sycamore Pont L e z i ~ isolated, a ~ ~ ~ from the green alga Prototheca zopfii, a similar glycoprotein, which was characterized as a D-mannoprotein possessing N-glycosylic linkages between 2-acetamido-2-deoxy-~-glucosyl residues (linked to the D-mannan) and protein. He355proposed that, in all of these plant systems, these glycoproteins are synthesized from protein and a dolichyl diphosphate-linked polysaccharide composed primarily of D-mannosyl residues, with termiUMP

UDP-GIcNAc

k

I

9 -"c:

UDP-GIcNAc

DOl-f-f-GlcNAc

GDP-Man

Dol-P-f-(GkNAc), DP

Dol-P-Man Dol-f-P-(GlcNAc),-Man

Dol-f-f

Protein

GDP-Man Dol-f-P-(GlcNAc),-Man,

G DP

Protein-Asn-(GIcNAc),-Man.

SCHEME3. -Tentative Scheme for the Formation of Lipid-linked Oligosaccharides and in the Green Alga ProGlycoproteins of D-Mannose and 2-Acetamido-2-deoxy-o-glucose totheca zopfit and Higher Plants (After Pont L e ~ i c a ~[Abbreviations ~~). used are: Do1 = dolichol, Dol-P = dolichol phosphate, Man = D-mannose, GlcNAc = 2-acetamido-2deoxy-o-glucose, Asn = L-asparagine, and P, = inorganic phosphate.]

(350) M. C. Ericson and D. P. Delmer, Plant Phyaiol., 59 (1977) 341-347. (351) L. Beevers and R. M. Mense, Plant Physiol., 60 (1977) 703-708. (352) W. T. Forsee and A. D. Elbein, 1.Bid. Chem.,250 (1975) 9283-9293. (353) W. T. Forsee, G. Valkovich, and A. D. Elbein, Arch. Biochen. Biophys., 174 (1976) 469- 479. (354) M. M. Smith, M. Axelos, and C. Pkaud-LenotA, Biochimfe, 58 (1976) 1195-1211. (355) R. Pont Lezica, Biochem. SOC.Trans., 7 (1979) 334-337.

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nal 2-acetamido-2-deoxy-~-g~ucosy~ residues attached, by way of a diphosphate link, to the dolichol, as the final step in the biosynthesis, from glycosyl-nucleotides, of these glycoproteins, involving as intermediates dolichol-containing glycolipids. This author355 drew up a proposed scheme for this biosynthetic mechanism, which is shown in Scheme 3. It must be stressed that this scheme is highly tentative. None of the glycoproteins in question have been fully characterized, and the proposed involvement of the glycolipids is, at this stage, merely speculative, as the role of dolichol-containing glycolipids as glycosyl donors in synthetic mechanisms in the plant sources cited has not been established. Few of the enzymes catalyzing individual steps in this proposed scheme have been isolated or characterized. Pont L e ~ i c acompared ~ ~ ~ this scheme to the mechanisms for glycosylation of proteins in animal syst e m and ~ ~yeast,357 ~ ~ but it is based on very incomplete data. Furthermore, even if this scheme is accepted as a valid hypothesis, it would be unwise to draw from it any conclusions as to the mechanisms and intermediates involved in the biosynthesis of plant cell-wall polysaccharides, as glycoproteins of this type have not been shown to be components of the primary wall in higher plants. The occurrence of D-glucose in plant glycoproteins has been reviewed,358and it is of considerable interest, because of the potential role of such glycoproteins in the synthesis of plant polysaccharides, especially cellulose. The apparent involvement of a D-glucoprotein as a primer for the synthesis of cellulose from GDP-D-glucose in Prototheca ~ o p f i ihas ~ been ~ ~ .discussed ~ ~ ~ earlier. In pea seedlings, Pont Lezica and identified intermediates, including dolichyl (D-glucosyl phosphate and diphosphate) and an oligosaccharide-lipid containing seven or eight D-glucosyl residues. The oligosaccharide-lipid appears to be a precursor of a membrane-bound glycoprotein tentatively identified as the /I-subunit of the pea i ~ o l e c t i n s . ~The ~ ’ lectin subunit is a D-glucoprotein, but the types of linkages it contains have not yet been established. No evidence relating this glucoprotein to cellulose biosynthesis has been published. The possible role of lectins as “gluing substances” between polysaccharides within the primary cell-wall or, alternatively, as wall-bound enzymes, has already been discussed. The further possibility that lectins are involved in binding glycans during the processes of P. J. Evans and F. W. Hemming, FEBS Lett., 31 (1973) 335-338. P. Jung and W. Tanner, Eur. J. Biochem., 37 (1973) 1 - 6 . R. G . Brown and W. C. Kimmins, Int. Rm. Bfochem.,13 (1977) 183-209. R. Pont Lezica, P. A. Romero, and M. A. Dankert, Plant Physiol., 58 (1976) 675680. (360) P. A. Romero and R. Pont Lezica, Acta Physiol. Lat. Am., 26 (1976) 364-370. (361) C. L. Villemez, J. M. McNab, and P. Albersheirn, Nature, 218 (1968) 878-880. (356) (357) (358) (359)

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glycan transport to, and location in, the intact primary-wall will be considered in Section XII,4,a. d. Conclusions. -The evidence that has been cited suggests that dolichol-containing glycolipids and, possibly, glycoproteins may be intermediates in the synthesis of cell-wall polysaccharides in higher plants. However, this conclusion must be drawn only tentatively. Certainly, such glycolipids and glycoproteins have been isolated from a number of plant sources in which polysaccharide biosynthesis occurs, but, in no higher-plant preparation has, to date, any glycolipid or glycoprotein been demonstrated to serve as a glycosyl donor to any polysaccharide that has been clearly characterized as a cell-wall component, although polyisoprenol phosphates have been shown to accept glycosyl residues donated by glycosyl-nucleotides in several, higher-plant sources. The clearest evidence of the status of dolichol-containing glucolipids and a glucoprotein as intermediates in the synthesis of cellulose from glucosyl-nucleotides has come from studies of the green alga Prototheca and the intermediate role of these glucolipids may be comzopfii,338-341 pared to the established role of D-mannosyl-lipid as an intermediate in the synthesis of D-mannan from GDP-D-mannose in Micrococcus lysodeikticus.328.320 Pont L e ~ i c speculated a ~ ~ ~ that the role of dolichyl phosphates, a-saturated polyisoprenyl phosphates of chain length between 80 and 105 carbon atoms, in glycosyl transfer during biosynthetic mechanisms is a general feature of eukaryotic cells, whereas, in prokaryotic cells, the lipids fulfilling this function are a-saturated polyisoprenyl phosphates of shorter chain-length, such as undecaprenyl phosphate (which contains 55 carbon atoms). In higher plants, the only direct, experimental evidence of incorporation of glycosyl residues from a glycolipid into a polymer comes from studies with pea seedlings,361and, in this case, the polymer product is a glucoprotein lectin component that has not been shown to be a cell-wall component. Confirmation and elucidation of the role of glycolipids and glycoproteins as intermediates in wall-polysaccharide synthesis from glycosyl-nucleotides in higher plants requires further study, especially the isolation and characterization of the enzymes that catalyze individual glycosylation steps in the biosynthetic processes, and identification of the substrates and products of these enzyme activities (see also, Ref. 217). 4. Cytological Location of Polysaccharide Synthesis

Preceding Sections (X11,3,b)and (XII,3,c) have outlined what is known of the synthetic mechanisms by which cell-wall polymers are formed in

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plants. It now remains to discuss the compartments within the plant cell where these synthetic processes occur, and the proposed mechanisms for the assembly of completed polymers into the intact primary-wall. a. Pectic Polymers and Hemicelluloses. -Polysaccharide synthases are located in a particulate-cell fraction. Albersheim and associates361 showed that enzymes responsible for synthesizing polysaccharides from UDP-D-glucose, UDP-D-galactose, UDP-D-glucuronic acid, UDP-D-galacturonic acid, and GDP-D-glucose are present in a single particle obtained from ruptured cells of mung-bean tissue. Their findings led them to suggest that cell-plasma membranes are the site of polysaccharide synthesis. This hypothesis stands in marked contrast to the findings of other workers, including Whaley and MollenhaueP2 and Harris and Northwho suggested that the sites for such synthesis are located in Golgi dictyosomes. The latter a u t h o r P 3 obtained evidence that both pectic polymers and hemicelluloses (but not cellulose), which became labelled when ~-['*C]glucosewas introduced into pea-seedling roots, are synthesized and transported by the Golgi bodies and their associated vesicles. confirmed these conclusions in their work on Ray and polysaccharide synthesis in pea seedlings. They showed, like Albersheim and coworker^,^^' that polysaccharide synthases utilizing GDP-D-ghcose, UDP-D-glucose, and UDP-D-galactose are present in a single subcellular particle. Furthermore, they reported365the isolation of a pure Golgi-dictyosome fraction that retained polysaccharide synthase activity, and found that the product formed in the fraction had a composition similar to the sum of the pectic and hemicellulose fractions of the cell wall, but contained no true cellulose. Robinson and Ray366reported that synthesis of pectic-wall polymers and hemicelluloses takes place in the Golgi system, but that cellulose synthesis occurs in a different cell-compartment. On the basis of present evidence, it seems probable that pectic polymers and hemicelluloses are synthesized b y the Golgi bodies before transport to the cell wall by Golgi vesicles and insertion intact into the wall after fusion of these vesicles with the plasma membrane.247-249 It is possible that membrane lectins play arole in the binding of glycans during these processes. Protein fractions having lectin activity have been (362) W. G . Whaley and H. H. Mollenhauer,J. Cell Biol.,17 (1963) 216-225. (363) P. J. Harris and D. H. Northcote, Biochim. Biophys. Acta, 237 (1971) 56-64. (364) P. M. Ray, T. L. Shinninger, and M. M. Ray, Proc. Natl. Acad. Sci. U.S.A.,64 (1969) 605 - 6 12. (365) W. Eisinger and P. M. Ray, Plant Physiol., Suppl., 49 (1972) 2. (366) D. G . Robinson and P. M. Ray, Plant Physiol., Suppl., 51 (1973) 59.

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found in endoplasmic reticulum, Golgi and plasma membranes of mungbean hypocotyls, and the total-membrane fractions from a number of other plant tissues.257Such lectins may stabilize, and aid the transfer of, polysaccharides during the fusion of the Golgi vesicles with the plasma membrane, and glycosidase activity of the lectins could break and make glycosidic bonds, facilitating transfer of polysaccharides from Golgimembrane lectins to plasma-membrane lectins. There could be similar interactions between plasma-membrane lectins, if these are involved, and cell-wall lectins in transferring and binding polysaccharides into the wall.

b. Cellulose. -In Section XII,4,a, several

were cited that support the concept that cellulose is not synthesized in the Golgi bodies of plant cells. However, in a study of the cellulose-synthesizing system of the green alga Prototheca zopjii, which has been described earlier (see Section X11,3,b), Pont Lezica and coworkers341reported that the enzymes responsible for the D-glucosylation of dolichol derivatives and protein were found in the endoplasmic reticulum-rich fraction, but that cellulose synthase activity, which completes cellulose synthesis utilizing GDP-D-glucose as substrate and a glucoprotein intermediate as primer,3380341was located in the Golgi dictyosome-rich fraction. Cellulose, in the intact primary-wall, occurs in the form of microfibrils embedded in the matrix formed by other wall polymers (see Sections VI, IX, and X). With regard to the synthesis of microfibrils and their deposition into the wall matrix, it is of great interest that, in certain studies, highly ordered arrays of intramembrane particles have been observed on the protoplasmic leaf (P-fracture face) of freeze-fractured, plasma membranes of various plant-cells during wall growth. The cells studied in, ~fern ~ ~ protocluded those of the green alga Micrasterias d e n t i ~ u l a t aof nemata,3e8 and of germinating rootlets of Zea mays and mung bean.369 These intramembrane particles appear as precise, hexagonal arrays of from 3 to 175 rosettes, each rosette being composed of 6 sub-units. These arrays of rosettes are observed at the ends of impressions of cellulose fibrils, and the results suggested that these ordered complexes first appear when secondary-wall synthesis begins. Each row of rosettes appears to be associated with the formation of a fibril, the parallel rows of rosettes forming a band of parallel fibrils. This conclusion is also supported by the observation that the distance between fibrils is equal to the distance between rows of rosettes, namely, -30 nm. Each rosette appears to be composed of 6 particles packed (367) T. H. Giddings, D . L. Brower, andL. A. Staehelin, 1.Cell Bfol., 84 (1980) 327-339. (368) S. Wada and L. A. Staehelin, unpublished findings, cited in Ref. 367. (369) S. C. Mueller and R. M. Brown, I. Cell Biol., 84 (1980) 315-326.

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tightly in a ring having an outside diameter of 22 nm and a central space of 7 - 8 nm diameter. The individual particles measure 8 nm in diameter. Fibril bands composed of various numbers offibriIs can be accounted for by the presence of rosette arrays of various sizes. In the specimens examined, complexes having as few as 3, and as many as 175, rosettes were observed.367 Significantly, the ordered pattern of fibril deposition in the secondary ~ ’ shown to be derived from the structure of the wall of M i c r ~ s t e r i a s ~was complexes located in the plasma membrane. The results indicated that the widest fibrils, those in the center of a band, are formed by the longest rows of rosettes, those in the center of arosette array. The shorter rows of rosettes within an array give rise to narrower fibrils. This proportionality between the width of a secondary cellulose fibril and the number of rosettes involved in its formation provides strong evidence that the rosette structure plays a significant role in the synthesis of cellulose fibrils. A schematic model illustrating this relationship is shown in Fig. 9. It is

-

FIG.9. -Model of Cellulose-fibril Deposition During Secondary Cell-Wall Formation in Micrusterias (After Giddings and coworker^^^'). [Each “rosette” (cellulose-fibril-synthesizing complex?) 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 ofthe secondary wall. Above: side view. The area in the center of a rosette is the presumed site of microfibril formation, although details of its structure, composition, and enzymic activity remain unclear. The “membrane-associated layer” is based on the results of Kiermayer and Dobb e r ~ t e i n . ~ ” . ~This ’ * layer may serve to hold the rosettes together in the hexagonal array. Below: surface view, with expanded, cross-sectional view of cellulose fibrils.]

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suggested that each rosette accounts for one 5-nm microfibril, and that these 5-nm microfibrils aggregate laterally to form the larger, secondary-wall fibrils. The observation of distinct longitudinal striations (periodicity, 5 nm) in the secondary-wall fibrils is consistent with this model. The model proposes hexagonal packing of the 5-nm microfibrils within the larger fibrils. For example, the widest fibrils are considered to be the product of 16 rosettes, and to show five striations on their surface. Such a fibril could contain 16 microfibrils packed three deep in a hexagonal manner, and would have a predicted width of 30 nm; the maximum fibril-width 0bserved3~7is 28.5 nm. In the secondary wall of Micrasterias, there is a noted shift from flat, wide fibrils in the center of a fibril band to more-rounded, narrower fibrils at the periphery. A 5-nm diameter microfibril could accommodate 50 D-glucan chains, although the extent to which substances other than cellulose contribute to the observed diameter is unknown.387These 5-nm microfibrils that emanate from the individual rosettes could be structural equivalents of the “elementary fibrils” composing secondary cellulose-fibrils which were suggested by F r e y - W y ~ s l i n gand , ~ ~ critically ~ reviewed by Prest o The ~ 6~particles ~ making up each rosette could be sub-units of an enzyme system involved in cellulose synthesis. The exoplasmic surface (E-fracture face) of freeze-fractured, Micrasterias plasma-membranes frequently, but not consistently, reveals particles that are complementary to the central hole of the rosettesSB7;these particles may arise from forming microfibrils. It has been suggested that, in freeze-fractured, plasma membranes from corn-seedling tissue,3ee these particles on the outer surface of the exoplasmic leaflet, also displaying apparent complementarity with rosettes on the P-fracture face, represent terminal enzyme-complexes functioning in association with rosettes to synthesize microfibrils. Similar rosette-structures have been detected on the P-fracture faces of membranes of cytoplasmic vesicles in M i c r a s t e r i a ~The . ~ ~ ~freezefracture images obtained suggested that the rosette complexes are first assembled into cytoplasmic vesicles, and are subsequently incorporated into the plasma membrane by a process of vesicle fusion. This supports the evidence obtained by Kiermayer and D ~ b b e r s t e i n ,in~ Micras~~.~~~ terias, that the flat vesicles that contain the rosette complexes are formed by the Golgi apparatus. The pattern of deposition of the bands of secondary fibrils observed by Giddings and coworkers387suggested that the complexes move in the (370) A. Frey-Wyssling, Fortschr. Chem. Org. Naturst., 27 (1969) 1-30. (371) B. Dobberstein and 0. Kiermayer, Protoplasma, 75 (1972) 185-194. (372) 0. Kiermayer and B. Dobberstein, Protoplasma, 77 (1973) 437-451.

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plane of the membrane during deposition, although the possible mechanisms by which the complexes move, and by which their orientation is controlled, are unknown. Each complex may propel itselfby the addition of D-glucosyl (monomer) groups to the growing tips of the associated microfibrils. The results suggested that the array of rosettes within each complex remains fairly cohesive during this movement, and the studies of Kiermayer and D o b b e r ~ t e i n ~ ~revealed ~ J ’ ~ staining material within the complex that could represent a substance serving to keep the hexagonal array intact as it moves within the lipid bilayer of the plasma niembrane. The general direction taken by the complexes does appear to be random, cell shape having already been determined during primary-wall growth.367 The pattern of microfibril deposition during primary-wall formation also seems to be random. The microfibrils, 6 - 8 nm in diameter, appear to be the products of single rosettes that can be visibilized on the P-fracture face of the plasma membrane of Micrasterias during the stage of primary-wall growth.3s7 These rosettes and accompanying microfibrils are shown diagrammatically in Fig. 10. The observed diameter of 6 8 nm, which could accommodate 60 - 80 D-glucan chains, compares

-

FIG.10.-Model of Microfibril Deposition During Primary Cell-Wall Formation in Micrmterias (After Ciddings and coworker^^^'). [Above: side view. Below: surface view. Single “rosettes” apparently give rise to single, randomly oriented microfibrils.]

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favorably with the earlier cited (see Section VI) estimate of cellulose microfibrils consisting of 60 - 70 D-glucan chains giving cross-section dimensions' of 4.5 X 8.5 nm. Vesicles containing small numbers of separate rosettes have been found in the cytoplasm of Micrasterias during primary growth,3e7 suggesting that microfibril-synthesizing units are assembled in cytoplasmic membranes, and are incorporated into the plasma membrane by similar mechanisms during primary- and secondary-wall formation. All of the available evidence suggests that the rosettes represent morphological equivalents of plasma-membrane-bound complexes of enzymes involved in the synthesis of cellulose fibrils in plant cells. Overall, the report of Pont Lezica and coworkers341that cellulose synthase is located in a Golgi-dictyosome fraction of Prototheca (see Section X11,3,b) stands in contrast to the conclusions drawn by the other authors which have been cited. However, relating the evidence, obtained separately by Giddings and associates3s7 and by Kiermayer and D o b b e r ~ t e i n ,that ~ ~ cellulose-synthesizing ~,~~~ rosettes are found in cytoplasmic vesicles that can be observed fusing with the plasma membrane in Micrasterias and higher plant cells, to the report341 of Pont Lezica and coworkers, it is conceivable that cellulose synthesis is initiated by rosette complexes in Golgi vesicles, but that completion of microfibril formation follows fusion of these vesicles with the plasma membrane. An alternative possibility is that the latter finding34l is an experimental artifact, possibly arising from contamination of their Golgi fraction with plasma-membrane components (see also, Ref. 2 17). c. Extensin, the Cell-Wall Glycoprotein. -Little has been published on the cytological location of hydroxy-L-proline-rich glycoprotein synthesis in plant cells. D a ~ h e reported k ~ ~ ~ that the glycoprotein was transported to the cell wall by a mechanism involving smooth membranes. Particle-bound extensin, which is transferred rapidly to the wall, has been reported from cultured carrot374and sycamore232-3escells. The particulate fractions from sycamore cells have been shown to contain the enzymes that catalyze glycosylation of the hydroxy-L-proline-rich protein.232.320 Gardiner and C h r i ~ p e e l implicated s~~~ the Golgi apparatus in glycosylation and transport to the wall of the glycoprotein. On the basis of this limited evidence, the most feasible conclusion is that hydroxy-L-proline-rich protein, synthesized on ribosomes, is glycosylated in the Golgi bodies, transported to the cell surface in Golgi vesi(373) W. V. Dashek, Plant Physiol., 46 (1970) 831 -838. (374) M. J. Chrispeels, Plant Phystol., 44 (1969) 1187-1193. (375) M. G. Gardiner and M. J. Chrispeels, Plant Physiol., Suppl., 51 (1973) 60.

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cles, and inserted into the wall after fusion of these vesicles with the plasma membrane.

5. Alterations of Cell-Wall Polymers Outside the Plasma Membrane Extensive reference has been made earlier in this article to glycosidases bound to the cell walls of higher plants (see Section VIII). Pectin methylesterase (EC 3.1.1.11) is also associated with the wall.37s Such enzymes may be important for in situ alterations of cell-wall polymers. Certain physiological processes, such as growth or the ripening of fruits, which will be dealt with in detail in Section XIII, are accompanied by the specific removal of polymers from the cell wall. The wall-bound glycosidases are probably involved in the removal of polysaccharides, although, apart from polygalacturonase, there is little direct evidence for the presence of endoglycanases. Keegstra and A l b e r ~ h e i mshowed ~ ~ ~ that glycosidases isolated from sycamore cell-walls catalyze the partial degradation of these walls, and Kivilaan and coworkersz10~377 demonstrated the removal from cell walls, by an autolytic enzyme, of a polymer composed of (1-3)- and ( 1 4 4 ) linked D-glucosyl residues. Although correlation between cell growth and the level of wall-bound glycosidases has been demonstrated in higher plants,z37~242~z43 no overall relationship between the level of catalytic activity and functionally important changes in the wall can be drawn at the present level of knowledge. Nothing is known about how, or where, the component wall-polymers are assembled to produce the final wall-structure, or about the forces responsible for the orientation of cellulose microfibrils in the wall. The possibility that intramembraneous, cellulose-synthesizing, enzyme complexes move within the plasma membrane as microfibrils are laid down in the wall structure has been discussed earlier (see Section XII,4,b), but the mechanisms controlling these processes are unknown. It is possible that wall-bound glycosidases catalyze transglycosylation reactions that result in the covalent cross-linking of cell-wall polymers, but there is no evidence for this at present. The possibility that lectins possessing glycosidase activity may be involved in such reactions has already been mentioned (see Section XII,4,a). Finally, it is possible that the glycoproteins, pectins, and hemicelluloses are formed and cross-linked in blocks, and transported to the cell wall, where they react, possibly by non-enzyme-catalyzed mechanisms, with cellulose microfibrils. Such a membrane would presuppose that the (376) W. H. Bryan and E. H. Newcomb, Physiol. Plant., 7 (1954) 290-297. (377) S. Lee, A. Kivilaan, andR. S. Bandurski, Plant Physiol., 42 (1967) 968-972.

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information for intact cell-wall structure is inherent in the structures of the component polymers.

6. Conclusions Although present knowledge of cell-wall biosynthesis in plants is incomplete and, in some areas, contradictory, nevertheless an overall, hypothetical scheme for wall assembly may be projected. Glycosyl esters of nucleotides are synthesized from D-glucose or sucrose by a combination of pathways, and these nucleotide esters serve as glycosyl donors for the synthesis of polysaccharides, probably by way of glycolipid and glycoprotein intermediates. The polysaccharides other than cellulose are transported to apoint outside the plasma membrane by a mechanism that appears to involve the Golgi system, and are then incorporated into the cell wall. Primary (and later secondary) cellulose microfibrils are laid down within this wall matrix by intramembraneous, cellulose-synthesizing enzyme-complexes, which originate in Golgi vesicles but which become located in the plasma membrane, by vesicle fusion, before microfibril deposition commences. The cellulose-synthesizing complexes appear to move freely within the lipid bilayer of the plasma membrane as fibril deposition proceeds, and the pattern of deposition appears to be random. Covalent and non-covalent bonds between component wallpolymers are established by unknown mechanisms. Much of the difficulty in drawing more-exact conclusions about wall biosynthesis arises from the fact that most of the work thus far has involved the individual, biosynthetic steps. Only the group of workers led by A l b e r ~ h e i m has ~ ~ made - ~ ~ a committed attempt to project a universal model for primary cell-wall structures in higher plants (see Section IX) on the basis of characterization of individual wall-components, and this model is by no means undisputed. Thus, workers in the biosynthetic field have found themselves studying the synthesis of natural products of currently unknown, or at least unconfirmed, structure. There is at present no precise information concerning either the control mechanisms that govern wall-biogenesis or the interactions between wall biogenetic-processes and general cellular metabolism. The number of steps involved in the formation of a polysaccharide from a glycosylnucleotide is not known, and it is not clear how cellular control is extended beyond the plasma membrane, or how the cell wall is formed from the component polymers. Thus, it appears that the major questions posed by the problem of cell-wall biosynthesis have yet to be answered (see also, Ref. 217).

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XIII. CELL-WALL A N D FRUIT RIPENING 1. Introduction Changes in cell-wall polysaccharides and the associated enzyme-activities occur in the soft, mesocarp tissue of fleshy fruits during ripening. Such changes are central to the process of tissue softening which, alongside changes in size, shape, color, and flavor, is one of the main physical manifestations of ripening. Transport of fruits from the countries of cultivation to the countries of marketing often poses a related problem that impinges upon the viable shelf-life of the mature fruit. Some tropical and sub-tropical fruits have proved unsuitable for existing bulk methods of delaying fruit-ripening during storage. Some are susceptible to "chill injury," a marked discoloration, with accompanying texture and flavor changes, that renders the fruits commercially valueless upon removal from low-temperature (05 " )s t ~ r a g e , ~ a' ~method - ~ ~ ~of delaying ripening during bulk storage and transport that is much applied to such temperate-zone fruits as apples382 and pears.383Fruits may also become susceptible to infection by microo r g a n i s m ~Nevertheless, .~~~ fruits and their products constitute a commercially significant food-commodity.385-3e3 (378) C. W. Campbell, Proc. Fla. Mango Forum, (1959) 1 1 . (379) E. K. Akamine, Hawaii Farm Sci., 12 (1973) 6 - 12. (380) C . F. Kinman, Bull. P. R. Agric. Exp. Stn., Fed. Stn. Mayaquez, 24 (1968) 30-35. (381) A. K. Mattoo and V. V. Modi, Proc. Znt. Conf: Trop. Sub-Trop. Fruits, London, (1969) 11 1-115. (382) R. B. H. Wills, K. J. Scott, and M. J. Franklin, Phytochemisty, 15 (1976) 18171818. (383) M. Knee,]. Food Sci.Agric., 24 (1973) 1137-1145. (384) S. Krishnarnurthy and H. Subramanyam, Trop. Sci.,15 (1973) 167-193. (385) R. W. Schery, Plantsfor Man, Prentice-Hall, New York, 1972. (386) D. S. Leigh, Report on an Overseas Study Tour of Some Tropical Fruit Areas of the World, N. S . W. Dept. Agric., Australia, 1972. (387) The Markets for Selected Exotic Fruit Products in the U . K . , The Federal Republic of Germany, Switzerland and the Netherhinds, Int. Trade Centre (UNCTAD/GATT) Rep., Geneva, 1971. (388) J. Candia, Proc. Conf Tropical Sub-Tropical Fruits, London, Tropical Products Institute, London, 1970, pp. 23-27. (389) J. Stother, Trop. Prod. Znst. Rep., 1971. (390) L. B. Singh, The Mango, Leonard Hill, London, 1968. (391) A. Jones, Trop. Prod. Znst. Rep., 1973. (392) G . S. Cheema, S . S. Bhat, and K. C. Naik, Commercial Fruit of India, Macmillan, London, 1954. (393) A. C. Hulme, in A. C. Hulme (Ed.), The Biochemisty ofFruits and Their Products, Academic Press, London, 1971, pp. 233-254.

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Tissue softening results from the breakdown of the cell walls in the edible portion of the fruit. Elucidation of the mechanism of this cell-wall breakdown in fruits is of potential value to food technologists seeking chemical or physical means of delaying or retarding ripening processes. The soft, edible tissues of most fruits are composed of parenchyma, unlignified tissue containing primary cell-walls. Sections I to XI1 of this article have dealt with the general features of the structure and biosynthesis of the plant primary cell-wall. The physiology of fruit development and, especially, the breakdown of primary cell-walls in the edible tissues of ripening fruits will be discussed in the following Sections. 2. The Physiology of Fruit Development a. Introduction. -This Section will outline the development of fruits from a meristem through growth to full size, and will culminate in the ripening process that marks the end of maturation and the onset of senescence in the fruit. A fruit that has grown to full size, but in which ripening has not yet begun, is described as a mature fruit, and many fruits are harvested at this stage. In some fruits, such as the mango, apple, and tomato, the process of maturation is separated from ripening and senescence by a “respiratory climacteric,” a period of intense synthetic and metabolic activity that appears to pave the way for the largely degradative changes that constitute the subsequent ripening. This climacteric appears to occur both in fruit allowed to ripen while still attached to the plant, and in fruit detached after the stage of maturity has been reached. In such detached fruit, treatment with ethylene appears to accelerate the onset ofthe climacteric. This climacteric will be dealt with in more detail later (see Section XIII,2,e). In other fruits, such as strawberry and citrus fruits, development of the fruit is continuous, there being no climacteric phase between maturation and r i ~ e n i n g . ~ O ~ - ~ ~ ~

b. Fruit Enlargement during Maturation. -Differentiation of any higher-plant cell from a meristem almost invariably involves an increase in cell size. The increase in volume associated with fruit growth occurs as a result of cell division, together with cell enlargement; cell division predominates in the early stages of growth, whereas cell expansion predominates during later stages. In addition, in some fruits, such as the apple, expansion of intercellular spaces may also contribute to the growth of the fruit during the later stages.307 (394) M. Knee, J. A. Sargent, and D . J. Osborne, J . E r p . Bot., 28 (1977) 377-393. (395) J. B. Biale, Citrus Leaves, 34 (1 954) 6 - 7. (396) J. B. Biale, in W. Ruhland (Ed.),Handbuch der Pfanzenphysiologie, Vol. 12, Part 11, Springer, Berlin, 1960, pp. 536-592. (397) R. J. Weaver, Plant Growth Substances in Agriculture, W. H. Freeman, San Francisco. 1972.

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There is some varietal variation. For example, in the tomato species Lycopersicon pimpinelli$olium, cell division continues right up to maturity, whereas in the variety Lycopersicon esculentum, cell division ceases at anthesis, and all growth following anthesis is a result of cell expansi0n.~Q8 In apple and peach, cell division ceases 3 - 4 weeks after bloom, whereas, in avocado, it is reported to persist to maturity.3e7There have been conflicting reports3e7J99of the duration of cell division in developing strawberries, but an authoritative study by Knee and associates394 presented evidence that cell division continues for 7 days after petal fall, whereas cell expansion continues for at least 28 days. In this study, the onset of ripening was noted 21 - 28 days after petal fall. Complicated patterns of development occur in some fruits in which cell division ceases at different times in different parts of the fruit.3e7 The period of fruit growth varies from 1 - 2 weeks to several years. However, fruits are usually initiated and mature within several months. Two distinct types of fruit-growth curves are observable when such parameters as volume, fresh weight, dry weight, or fruit diameter are plotted as a function of time after anthesis. Apple, pear, tomato, cucumber, and strawberry display a smooth, sigmoid curve,3e7 whereas fig, currant, grape, blueberry, and many stone-fruits (including mango,384 cherry, olive, apricot, peach, and are characterized by a double-sigmoid growth-curve. In the latter type, two periods of rapid growth are separated by an intermediate period when either less growth, or no increase in volume, occurs. A double-sigmoid curve may be regarded as two successive, sigmoid curves. In this case, there are three clearly defined stages of growth. In stage 1 (cell division), the ovary and its contents, except for the embryo and endosperm, grow rapidly. Stage 2 is characterized by rapid growth of embryo and endosperm, lignification of endocarp, and slight growth of the ovary wall. During stage 3, rapid growth of the mesocarp occurs, causing the final swell of the fruit, resulting in maturity.3e7 Increase in fruit size is due mainly to cell enlargement. It is, therefore, not surprising that because auxins (indole-3-acetic acid and its derivatives) control cell e x t e n s i ~ n , they ~ ~ ~have -~~ also ~ been presumed to play

-

(398) (399) (400) (401)

H. B. Houghtaling, Eull. Torrey Bot. Club, 62 (1935) 243-248. A. L. Havis, Am. J . Bot., 30 (1943) 311 -314. R. Cleland, Annu. Reo. Plant Physiol., 22 (1971) 197-222. R. D. Preston, Physical Biology ofPZunt Cell Walkr, Chapman and Hall, London,

1974. (402) F. B. Salisbury and C. Ross, Plant Physiology, Wadsworth, Belmont, California, 1969. (403) L. N . Vanderhoef and R. R. Dute, Plant Physiol., 67 (1981) 146- 149. (404) H. Sol1 and M. Bottger, Plant Physiol. Suppl., 67 (1981) 127.

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a dominant part in determining the growth patterns of some fruits, such as cucumber405and strawberry.406This hypothesis of the predominance of auxins in mediating fruit growth is strengthened by the fact that applications of synthetic auxins can enlarge many fruits, or change their patterns of growth.3Q7,407 Proteinaceous, auxin-binding sites, which are believed to be physiologically relevant in mediating fruit-growth, have been located in the plasma membranes of parenchyma cells in cucumber40s and strawberry.406The proposed mechanisms by which auxins promote cell extension through wall-loosening will be discussed in detail in Section XI11,2,d. However, the general lack of correlation between endogenous-auxin levels and fruit growth3Q7.40Q-411 suggests that, for some species at least, auxins are not the sole controlling factors. The conclusion follows that other classes of plant-growth regulators must play a part. Both gibber ell in^^^^*^^^-^'^ and cytokinins,3Q7*418-420 when applied to fruits, lead to enlargement. Gibberellin and auxin together exhibit a synergistic effect on the growth of tomatoes,421and Sastry and M ~ i r ~ ~ ~ obtained evidence that, in this fruit, the effect of gibberellin may be to increase the amount of auxin in the ovaries. Gibberellin applied to one side of Japanese pears leads to increases in the number and size of cells on the treated side.415 (405) G . Elassar, J. Rudich, D. Palevith, and N. Kedar, Hort. Sci.,9 (1974) 238-239. (406) K. R. Narayanan and B. W. Poovaiah, Plant Physiol., Suppl., 67 (1981) 3. (407) J. C. Crane, Proc. Am. Soc. Hortic. Sci., 54 (1949) 102-104. (408) K. R. Narayanan, K. W. Mudge, and B. W. Poovaiah, Plant Physiol., 67 (1981) 836-840. (409) J. P. Nitsch, Q.Reu. Biol., 27 (1952) 33-45. (410) E. A. Stahly and A. H. Thompson, Uniu. Md. Agric. Exp. Stn. Bull., (1959) A-104. (411) W. B. Collins, K. H. Irving, and W. G . Barker, Proc. Am. Soc. Hortic. Sci., 89 (1966) 243. (412) A. Christodoulou, R. J. Weaver, and R. M. Pool, Proc. Am. Soc. Hotic. Sci., 92 (1968) 301. (413) E. M. Zuluaga, J. Lumelli, and J. H. Christensen, Phyton (Buenos Aires), 25 (1968) 35-41. (414) H. C. Dass and G . S. Randhawa, Am. J. Enol. Vitic., 19 (1968) 56-61. (415) S. Nakagawa, M. J. Bukovac, N. Hirata, and H. Kurooka,]. ]pn. Soc. Hortic. Sci., 37 (1968) 9-11. (416) M. J. Bukovac and S. Nakagawa, Hortic. Sci., 3 (1968) 172-178. (417) D . I. Jackson, Amt. 1.Biol. Sci., 21 (1968) 209-215. (418) R. J. Weaver and J. Van Overbeek, CaZ$ Agric., 17 (1963) 12-16. (419) M. W. Williams andE. A. Stahly,]. Am. Soc. Hortic. Sci., 94 (1969) 17-22. (420) R. J. Weaver, J. Van Overbeek, and R. M. Pool, Hilgardia, 37 (1966) 181 - 189. (421) L. C. Luckwill, in D . Rudnick (Ed.), Cell, Organism and Milieu, Ronald, New York, 1959, pp. 223-251. (422) K. K. S. Sastry and R. M. Muir, Science, 140 (1963) 494-495.

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Fry4z3suggested that gibberellin promotes expansion of culturedspinach cells by suppressing the secretion, from the protoplast, of the enzyme peroxidase. This author postulated that peroxidase stiffens the wall by catalyzing the oxidation of cell-wall phenolic compounds, to form the more-hydrophobic biphenyls, quinones, or polyphenols, any of which might protect the wall polysaccharides against enzymic attack, or solubilization by external water. Suppression of peroxidase secretion into the wall could maintain the phenols in their reduced state, thus making the environment of wall polymers less hydrophobic, and wall polysaccharides might be lost through attack by wall hydrolases, or hydrogen-bonded polysaccharides might be freed from the wall by solubilization because of the increased access of water. The loss of polysaccharides would lead to wall loosening, and facilitate expansion-growth. Fry showed that, in cultured-spinach cells, gibberellic acid inhibits the secretion of peroxidase from the protoplast, and promotes the release from the cell wall of a pectic polymer (containing galacturonic acid, rhamnose, galactose, and arabinose) during cell expansion. Developing fruits are rich sources of cytokinins, which are found in tissues where rapid cell-divisions are occurring, and are considered to play an important role in the regulation of cell division in fruit.397*424 Letham424concluded that shape of the apple at maturity probably depends upon the balance between gibberellins and cytokinins in the immature fruit, and that apple varieties differ in their response to these compounds. Ethylene is now considered to be one of the main plant-hormones involved in fruit development. Many responses formerly believed to result from the presence of auxins are now ascribed to induced ethylene production.425The biosynthetic pathway for formation of ethylene from methionine, in a wide variety of plant tissues, including shoots of mung bean,426tomato,427and pea427;carrot427and tomato428roots; and the fruits of apple,42e.430 tomato,427and avocado,427has been elucidated, and is as follows. Methionine

(423) (424) (425) (426) (427) (428) (429) (430)

-

-

S-adenosylmethionine 1-aminocyclopropane-1 -carboxylic acid (ACC)

+

ethylene

S. C. Fry, Phytochemistry, 19 (1980) 735-740. D. S. Letham, Annu. Reo. Plant Physiol., 18 (1967) 349-364. S. P. Burg and E. A. Burg, Proc. Natl. Acad. Sci. U.S.A.,55 (1966) 262-269. Y. B. Yu, D. 0. Adams, and S. Y. Wang, Plant Physiol., Suppl., 65 (1980) 40. Y.Fuchs, E. Chalutz, I. Rot, and A. K . Mattoo, Plant Physiol., Suppl., 65 (1980) 43. K. J. Bradford, T. C. Hsiao, and S. Y. Wang, Plant Physiol., Suppl., 65 (1980) 40. M. Lieberman and S. Y. Wang, Plant Physiol., Suppl., 65 (1980) 41. D. 0. Adams, S. Y. Wang, and M. Lieberman, Plant Physiol., Suppl., 65 (1980) 41.

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Yu and Wang43' considered that indole-3-acetic acid exerts its stimulating effect on expansion growth by inducing the synthesis of the enzyme catalyzing the conversion of S-adenosylmethionine into ACC, a conclusion at variance with the suggestion of Vioque and coworkers432that indoleacetic acid oxidase and its substrate (IAA) participate in the last reaction in the ethylene biosynthesis pathway, namely, the formation of ethylene from ACC. Application of ethylene to fruits during the cell-enlargement stage of development leads to rapid growth and maturation.3g7~433-43s Hale and associates436suggested that an auxin -endogenous ethylene relationship determines the rate at which grape berries mature. Eisinger and T a i ~ ~ ~ ' reported that cell-wall acidification is a requirement for ethylene-induced, lateral cell-expansion, as well as auxin-induced cell-elongation, in pea internode-tissue, and they43s established that ethylene alters the orientation of cellulose microfibrils from transverse to longitudinal in the wall of cortical parenchyma in pea epicotyls. They concluded that the most-recently deposited (inner 10- 30%)layers of microfibrils control the directionality of cell expansion. Sisler and Isenhour reported ethylene-bonding sites in mung-bean sprouts43gand tomato The exact roles and interactions of hormones in the control of fruit development remain to be determined. The evidence suggests that all classes of plant-growth regulators probably play a part, and that their influence is perhaps effected by changes in their balance. The reason for the slow-growth period (stage 2) that occurs between two periods of rapid growth in fruits exhibiting a double-sigmoid g r o w t h - c u r ~ is e ~still ~ ~ unexplained. Competition between the embryo and pericarp for nutrients was, for many years, presumed to be the cause, but the theory is invalid, because parthenocarpic fruits show the same growth-patterns as those of pollinated fruits that contain seeds. In some species, the growth occurring during the stage of cell division (stage 1) correlates closely with auxin and gibberellin content.3g7 The movement of organic and inorganic nutrients, from such sourcelocations as mature leaves, into fruits (which act as storage organs) is (431) Y. B. Yu and S. Y. Wang, Plant Physiol.,64 (1979) 1074-1077. (432) A. Vioque, M. A. Albi, and B. Vioque, Phytochemisty, 20 (1981) 1473-1475. (433) E. C. Maxie and J. C. Crane, Proc. Am. Soc. Hortic. Sci., 92 (1968) 255. (434) J.C.Crane, N. Marei,andH. M. Nelson,].Am. Soc. Hortic. Sci.,95(1970)367-372. (435) R. E. Byers, H. C. Dostal, and F. H. Emerson, Bioscience, 19 (1969) 903-908. (436) C. R. Hale, B. G . Coombe, and J. S . Hawker, Plant Physiol., 45 (1970) 620-623. (437) W. Eisinger and L. Taiz, Plant Physiol.,Suppl., 67 (1981) 126. (438) L. Taiz and W. Eisinger, Plant Physiol., Suppl., 67 (1981) 126. (439) E. C. Sisler and E. M. Isenhour, Plant Physiol., Suppl., 67 (1981) 52. (440) E. C. Sisler and E. M. Isenhour, Plant Phydol., Suppl., 67 (1981) 51.

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considered to contribute to fruit The osmotic pressure resulting from this accumulation of nutrients in the fruit tissue leads to movement of water into the fruit, causing cell enlargement and growth. Application of auxin has long been known to increase the extent of translocation of organic material to the treated regions of and g i b b e r e l l i n ~ and ~ ~ l cytokinin~~'~*~43 are presumed to induce a similar mobilization of nutrients into fruit tissue. Coombe,444 working with grape, put forward the hypothesis that the accumulation of sugars in the berry effects the onset of the stage of cell enlargement (stage 3). Maxie and Crane433later proposed that this stage of growth is initiated by ethylene. Crane445suggested that, in figs, fruit growth is directly controlled by hormones emanating from the seeds, and also by hormones (synthesized in the shoots) that enter the fruit tissue surrounding the seeds, as these hormones have the ability to attract metabolites into the fruit tissue from other regions of the plant. The differential effect of different growth-regulators on fruit size and shape446requires further elucidation. Application of different growthregulators to young fruits may result in differential attraction of various amino acids, organic acids, and sugars.447The theory that the hormones found in such high concentrations in seeds397mobilize essential metabolites and nutrients into fruit tissue against the competition for such nutrients afforded by developing shoots is widely held. Supporting evidence for this theory is that fruits having few or no seeds cannot usually survive shoot competition, but their development is enhanced if vegetative growth is suppressed.448 c . Wall Changes during Cell-Expansion Growth of Fruits. -The previous Section (XIII,Z,b) described the growth of fruits from a meristem to mature fruit, full-sized but unripe, and the factors that influence growth during this development stage. Little has been published on changes occurring in the cell walls during this part of fruit development, as most studies of changes in fruit cell-walls have been concerned with the ripening process that follows maturation and that, in climacteric fruits, is separated from maturation by the climacteric rise in respiration.

W. Shindy andR. J. Weaver, Nature, 214 (1967) 1024-1025. K. Mothers, Naturwissenschaften, 47 (1960) 337-339. P. E. Kriedmann, Aust. J . Bid. Sci., 21 (1968) 569-571. B. C . Coombe, Plant Physiol., 35 (1960) 241 -250. J. C. Crane, Plant Physiol., 40 (1965) 606-610. R. J. Weaver andR. M. Sachs, in F. Wightman and G. Setterfield (Eds.),Biochemistry and Physiology ofplant Growth Substances, Runge, Ottawa, 1968, pp. 957-974. (447) R. J. Weaver, W. Shindy. and W. M. Kliewer, Plnnt Physiol., 4 4 (1969) 183-188. (448) D. L. Abbott, Ann. Appl. Biol., 48 (1960) 434-438. (441) (442) (443) (444) (445) (446)

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However, in the nonclimacteric strawberry, in which maturation and ripening are continuous processes, Knee and coworkers3B4studied the development of the wall both during maturation and subsequent ripening. Cell division predominated during the first 7 days after petal fall, but cell expansion continued for up to 28 days after petal fall. Cell-wall polysaccharides increased about 1O-fold during fruit development up to 2 1days, after which they remained constant, or declined. The increase in cell volume during this period was about 1000-fold. During cell expansion, radioactivity from ~-['~C]glucose was incorporated into poly(g1ycosiduronic acids), a proportion of which could be extracted from the wall with neutral, aqueous buffers at 20-30", and into D-glucosyl and D-galactosyl residues in the wall. Radioactivity from ~-['~C]proline was also incorporated into the wall, but only 10% of this activity was found in hydroxy-L-proline residues. Correspondingly, wall protein contained a low proportion of hydroxy-L-proline residues. The proportion of radioactivity from 14C0, fixed by fruitlets remained constant in most sugar residues in the wall during cell expansion, but the proportion of radioactivity in galactose fell, indicating turnover of galactosyl residues. Tubular proliferation of the tonoplast, and hydration of middle lamella and wall matrix material, began 7 - 14 days after petal fall. This hydration of the wall was associated with increasing aqueous extractability of wall poly(g1ycosiduronic acids), which became extreme during ripening that followed cessation of cell expansion. Loss of galactosyl and arabinosyl residues from the wall also became marked after cell expansion had ceased, and incorporation of ~-['~C]glucose into wall polysaccharides ceased, but incorporation of ~-['~C]proline into wall protein continued. From these findings, the general conclusion may be drawn that, during the earlier stages of fruit maturation, biosynthetic processes involving polymer addition to the primary wall are predominant, with little evidence of degradative processes leading either to actual loss of glycosyl residues from the wall or to increased solubility of polymers resulting from endoglycanase activity. However, in the later stages of cell expansion (that is, more than 21 days after petal fall), some of the degradative processes that characterize the subsequent ripening process appear to commence. This is especially noticeable for wall poly(uronic acids). At 21 days after petal fall, 90% of poly(uronic acid) is tightly wall-bound, being extractable only after prolonged incubation with buffers containing EDTA, whereas, at 28 days, only 47% of the poly(uronic acid) is tightly wall-bound, and by 35 days this figure for wall-bound poly(uronic acid) has fallen to 28%. Knee and his associates3s4considered that the increasing solubility of wall poly(uronic acid) may be due to the disruption of wall structure resulting from galacturonanase activity, or from loss of calcium-stabilized gel-structure due to increased methylation of

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galacturonan. After 28 days following petal fall, the incorporation of radioactivity into polysaccharides, which indicates biosynthesis of polysaccharides throughout the stage of cell expansion, ceases, indicating the onset of ripening. In developing peaches, the walls of parenchyma cells of the mesocarp increase in thickness to a maximum during maturation, and are then diminished during subsequent ripening. The increase in cell diameter during expansion growth parallels the increase in wall thickness. During this maturation, there is a close correlation between the degree of methyl esterification of pectin and the wall thickness. Little esterified pectin is present in the meristematic tissues of very young peaches until the cessation of cell division, but a high and relatively constant level of esterified pectin is present during cell enlargement.44eDuring subsequent ripening, the degree of esterification and the molecular weight of wall glycuronan decreases,450and the catalytic activities of both endo- and exo-gal a c t u r o n a n a ~ e sappear ~ ~ ~ to remove both galactosyluronic acid and arabinosyl residues from the wall.452 These results suggested that, in the peach, as in the strawberry,394 biosynthetic addition ofwall polymers is predominant during cell expansion, preceding loss of wall polymers by enzymic action during subsequent ripening. In the developing mango, as in the strawberry, the aqueous extractability of glycuronan from the wall appears to increase in the final stage of expansion growth, just preceding maturity of the fruit. 380 Although it appears probable that there is no overall loss of wall polymers, except, perhaps, in the period just preceding full fruit-maturity, and that wall growth is continuous during cell expansion, there is almost certainly a turnover of wall polysaccharides, and the making and breaking of bonds to facilitate wall expansion during the maturation of fruits. The subject of the possible mechanism by which this wall expansion occurs is discussed in the Section that follows.

d. Cell-Wall Loosening during Fruit Maturation. -From the evidence cited in Section XII1,2,c, it seems likely that, during fruit maturation, the primary walls of soft fruits undergo both biosynthetic and degradative processes, although biosynthesis is predominant. The clearest evidence for degradative processes during this development stage is the apparent loss of galactosyl residues in the strawberry primary-wall during cell expansion.3e4 (449) S.M. Siegel, in M. Florkin and E. H. Stotz (Eds.), Comprehensive Biochemistry, Vol. 26A, Academic Press, New York, 1968, pp. 1-51. (450) R. M. McCready and E. A. McComb, Food Res., 19 (1954) 530-538. (451) R. Pressey and J. K. Avants, Plant Physiol., 52 (1973) 252-256. (452) J. Labavitch and E. R. A. Ahmed, Plant Physiol., Suppl., 61 (1978) 116.

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In cell expansion, the processes involved in changes in wall extensibility are of major importance. As outlined in Section X111,2,b, the maturation of fruit is influenced by growth regulators,3Q7,400-422,424,425~ and comparable hormonal control is known to be exercised over the processes of cell-wall extension and loosening during elongation growth in other plant t i s s ~ e s . ~It~is. probable, ~ ~ ~ - although ~ ~ ~ ~ not ~ ~ ~ firmly established, that the extension of the primary walls of fruit parenchyma-cells during cell expansion-growth involves similar mechanisms. The wall-loosening processes initiated during cell expansion may continue into the ripening stage that follows maturation. An outline account of what is known about the biochemistry of cell-wall extension is given next. From the large volume of work conducted in this field, certain fundamental premises have been established: (1) cell enlargement involves a stretching of the wall already present, as well as synthesis of new wall so as to keep the thickness of the wall constant; (2) the driving force for extension is turgor pressure; (3) cell enlargement is an active process that normally requires respiration; (4) continuous synthesis of RNA and protein is needed for cell enlargement; and (5) the rate of cell enlargement in many higher-plant tissues is regulated by auxin. An open question is whether auxin stimulates wall loosening by acting at the level of gene transcription. Auxin certainly stimulates the rate of RNA synthesis in plant s e ~ t i o n , isolated ~ ~ ~ -nuclei,4s7-45Q ~ ~ ~ and chromatin,457.460 and new protein species appear after treatment of intact tissues r~~~ with the h o r m ~ n e . *However, ~ ~ - ~ ~ the ~ findings of H a ~ c h e m e y e and Evans and Ray46sstrongly indicated that the induction of cell-wall exten433-436n441-448

(453) See Ref. 403. (454) Y. Masuda, E. Tanimoto, and S. Wada, Physiol. Plant., 20 (1967) 713-719. (455) J. L. Key and J. C. Shannon, Plant Physiol., 39 (1964) 360-364. (456) T. H. Hamilton, R.J. Moore, A. F. Rumsey,A. R.Meands, and A. R. Schrank,Nature, 208 (1965) 1180-1183. (457) A. G . Matthyse and C. Phillips, Proc. Natl. Acad. Sci. U.S.A.,63 (1969) 897-903. (458) T. J. O’Brien, B. C. Jarvis, J. H. Cherry, and J. B. Hanson, in F. Wightman and G . Setterfield (Eds.), Biochemistry and Physiology ofplant Growth Substances, Runge, Ottawa, 1968, pp. 747-759. (459) R. Roy-Choudhury, A. Datta, and S. P. Sen, Biochim. Biophys. Acta, 107 (1965) 346- 35 1. (460) R. E. Holm, T. J. O’Brien, J. L. Key, and J. H. Cherry, Plant Physiol., 45 (1970) 41 -45. (461) B. D. Patterson and A. J . Trewavas, Plant Physiol., 42 (1967) 1081 - 1086. (462) I. V. Sarkissian andT. C. Spelsburg, Physiol. Plant., 20 (1967) 991-998. (463) M. A. Venis, Nature, 202 (1964) 900-901. (464) A. E. V. Haschemeyer, Proc. Natl. Acad. Sci. U.S.A.,62 (1969) 128-135. (465) M. L. Evans and P. M. Ray, J. Cen. Physiol., 53 (1969) 1-20,

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sion by auxin can occur so rapidly that the inducing agent could hardly be acting at the gene level. In 1971, the wall-acidification h y p ~ t h e s i was s ~ ~first ~ ~proposed, ~~~ independently, by Hager and coworkersqes and Cleland.400Low pH has long been known to induce rapid cell-elongation in growing plantThe hypothesis, which proposes that auxin regulates wall loosening by causing a pH drop in the area of the cell wall, has survived a series of rigorous challenges, as outlined in Cleland’sqeereview. The mechanism of auxin-induced wall-acidification remains to be elucidated. The response of growing plant-tissues to low pH closely resembles the auxin-induced g r o w t h - r e s p ~ n s e suggesting , ~ ~ ~ ~ ~ a~ ~common, ~ ~ ~ ~ wallloosening mechanism. Massive cell-wall extension also occurs in frozen thawed Avena coleoptile sections subjected to low pH and an applied force in place of the missing turgor pressure472;this extension in uitro displays characteristics similar to those of the extension induced in uiuo both by auxin and low pH. This finding in uitro has been cited as apoint of evidence that synthesis of wall polymers is not directly involved in the wall-loosening process, as such synthesis does not occur in the frozenthawed c ~ l e o p t i l e - c e l l s . ~ ~ ~ It is now generally accepted that auxin and low-pH-induced stimulation of elongation growth results from a temporary weakening or relaxation of the wa11.400~46e~470~473-477 There is some evidence that such hormones as auxin activate, within the cell membrane, ion pumps that lower the pH of the wa11,400~468*477~478-481 and it was suggested that the direct (466) R. Cleland, in F. Skoog (Ed.), Plant Growth Substances, Springer-Verlag, Berlin, 1979, pp. 71-78. (467) D. L. Rayle and R.E. Cleland, Cum. Top. Dew. B i d , 11 (1977) 187-214. (468) A. Hager, H. Menzel, and A. Krauss, Planta, 100 (1971) 47-75. (469) J. Bonner, Protoplasma, 21 (1934) 406-423. (470) A. Harrison, Physiol. Plant., 18 (1965) 321-328. (471) D. L. Rayle and R. Cleland, Plant Physiol.,46 (1970) 250-253. (472) D. L. Rayle, P. M. Haughton, andR. Cleland, Proc. Natl. Acad. Sci. U.S.A.,67 (1970) 1814-1817. (473) P. A. Adams, P. B. Kaufman, and H. Ikuma, Plant Physiol., 51 (1973) 1102- 1108. (474) M. L. Evans, Ph.D. Thesis, Univ. of California, Santa Cruz, 1967. (475) M. J . Montague, H. Ikuma, and P. B. Kaufman, Plant Physiol., 51 (1973) 10261032. (476) J. P. Nitsch and C. Nitsch, Plant Physiol., 31 (1956) 94 - 11 1 . (477) D. L. Rayle, Phnta, 114 (1973) 63-73. (478) R. Cleland, Proc. Natl. Acad. Sci. U.S.A.,70 (1973) 3092-3093. (479) M. L. Fisher and P. Albersheirn, Plant Physiol., 53 (1974) 464-468. (480) E. Marre, B. Lado, R. R. Caldogno, and R. Colombo, Plant Sci. Lett., 1 (1973) 179- 184. (481) E. Marre, B. Lado, R. R. Caldogno, and R. Colombo, Plant Sci. Lett., 1 (1973) 185 - 192.

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action of the hormone is on the membrane, so altering its permeability to protons that the wall reactions permitting elongation growth take place at lowered pH. Two hypotheses on the nature of these auxin-induced reactions within the wall have received major attention. The first conceives of a change in wall synthesis, either in amount or pattern of deposition; the second ascribes loosening to the action, at lowered pH, of polysaccharide hydrolases that are induced by auxin. With respect to the first of these hypotheses, auxin enhances the rate of wall synthesis in virtually every tissue in which growth is also promoted.482*483 It seems certain that this stimulation extends to all components of the wall p o l y s a ~ c h a r i d e and ~ . ~ ~to~ wall g l y c ~ p r o t e i n . ~ ~ ~ . ~ ~ ~ Auxin has been shown to stimulate P-D-glucan synthetase activity in several p l a n t - t i s ~ u e s . ~ . ~ ~ ~ Rays suggested that auxin stimulates a shift from apposition (deposition of new wall-material only at the cell membrane) to intussusception (deposition throughout the wall). Intussuscepted polysaccharides would loosen the wall by forcing apart cellulose microfibrils or providing a “lubricant” to facilitate such slippage. In pea stem and Avena coleoptiletissue, wall synthesis is entirely by apposition in the absence of auxin; after auxin treatment, a sizable proportion of hemicellulose, but not of cellulose, deposition occurs throughout the wall.” However, a number of s t u d i e ~ ~have - ~ *indicated ~ ~ ~ that it is unlikely that wall loosening is due simply to an increase in the rate of wall synthesis, because, during the process, it is necessary for wall polymers to slip or “creep” relative to one another. Although cell-wall extension occurs, in Avena coleoptile sections in vitro, in the absence of wall-polymer synthesis,472it would be unwise to conclude from this single finding that synthesis plays no part in extension growth. Wall synthesis may contribute indirectly to wall extension, possibly by maintaining the normal organization of the wall, so that wallloosening steps continue to occur. The low-pH-induced wall-extension in vitro in Avena is irreversible,472and this militates against the concept of elastic extension fixed by wall synthesis because, according to this concept, any wall loosening that is not fixed by wall synthesis would be entirely elastic, and thus reversible. (482) J. Bonner, Proc. Natl. A c Q ~Sci. . U.S.A.,20 (1934) 393-397. (483) G . S. Christiansen and K. V. Thimann, Arch. Biochen., 26 (1950) 230-239. (484) P. M. Ray and D . B. Baker, Plant Physiol., 40 (1965) 353-360. (485) R. Cleland, Plant Physiol., 43 (1968) 1625-1630. (486) S. Kuraishi, S. Uematsu, and T. Yamaki, Plant Cell Physiol., 8 (1967) 527-528. (487) M. A. Hall and L. Ordin, in F. Wightman and G . Setterfield (Eds.),Biochernkity and Physiology ofplant Growth Substances, Runge, Ottawa, 1968, pp. 659-675. (488) G . M. Barklet and M. L. Evans, Plant Physiol., 45 (1970) 143-147.

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35 1

However, the data obtained are in complete agreement with the concept of a reversible cleavage of some acid-labile cross-links, the re-formation of such ruptured bonds in new configurations depending on tension applied to the wall. Such tension would, in viuo, depend upon turgor pressure. Much attention has been given to the attempt to identify an acid-labile bond within the wall structure as the site of wall loosening. Polysaccharide hydrolases are known to play an essential role in wall extension in bacteria,489fungal h y ~ h a e , ~and ~ Opollen tubes,491and the evidence for their similar role in extension in all higher-plant tissues is considerable. For example, the enzymes ( 1 + 3 ) - ~ - ~ - g l u c a n a s e c, ~e~l ~l *~ ~l ~a ~s e , ~ ~ ~ * ~ (1+ 6 ) - P - ~ - g l u c a n a s e , ~(1+6)-a-~-glucanase,~'~ ~~*~~~ exoga~actana~e,~~~ and nonspecific polysaccharide hydro lase^^^^^^^^ have been located bound to wall preparations from various higher-plant sources, and, in the tomato fruit,49Q-s01(1+3)-P-~-glucanase and polygalacturonase are bound to the primary cell-wall. Auxin enhances the activities of each of and the these enzymes in one or more plant-tissues,237~4Qz~4g4-4Q6~~oz~503 distribution of polysaccharide hydrolases in bean hypocotyls mirrors the distribution of It is claimed that both exogenous cellu]ase505,506 and ( 1 + 3 ) - P - ~ - g l u c a n a s e enhance ~ ~ ~ * ~ ~wall ~ extensibility, and exogenous (1+3)-P-~-glucanase can induce a limited amount of cell elongation.495*507*508 Exoglycosidases have also been located bound to cell walls (see Section VIII), but there is no evidence for their involvement in wall extension. Careful consideration of the role of these enzymes indicates that no (489) V. Schwarz, A. Asmus, and M. Frank,]. Mol. Biol., 41 (1969) 419-423. (490) D . S . Thomas and J. T. Mullins, Physiol. Plant., 22 (1969) 347-353. (491) H. P. Roggan and R. G . Stanley, Planta, 84 (1969) 295-303. (492) A. H. Datko and G. A. MacLachlan, Plant Physiol., 43 (1968) 735-742. (493) A. N. J. Heyn, Arch. Biochem. Biophys., 132 (1969) 442-449. (494) D . F. Fan and G. A. MacLachlan, Can. 1.Bot., 44 (1966) 1025-1034. (495) E. Tanirnoto and Y. Masuda, Physiol. Plant., 21 (1968) 820-826. (496) A. N. J. Heyn, Science, 167 (1969) 874-875. (497) M. Katz and L. Ordin, Biochim. Biophys. Acta, 141 (1967) 126-134. (498) S. Lee, A. Kivilaan, and R. S. Bandurski, Plant Physiol., 42 (1967) 968-972. (499) S. J. Wallner and J. E. Walker, Plant Physiol., 55 (1975) 94-98. (500) S . J. Wallner and H. L. Bloom, Plant Physiol., 60 (1977) 207-210. (501) K. C. Gross and S . J. Wallner, Plant Physiol., 63 (1979) 117-120. (502) E. Davies and G . A. MacLachlan, Arch. Biochem. Biophys., 129 (1969) 581 -587. (503) D. F. Fan and G . A. MacLachlan, Can. J . Bot., 44 (1966) 1837- 1844. (504) D. J. Nevins, Plant Physiol., Suppl., 43 (1968) 16. (505) A. C. Olsen, J. Bonner, and D. J. Morre, Planta, 66 (1965) 126-133. (506) A. W. Ruesink, Planta, 89 (1969) 95-107. (507) Y. Masuda, Planta, 83 (1968) 171-184. (508) Y. Masuda and S . Wada, Bot. Mag., 80 (1967) 100-111.

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PRAKASH M. DEY AND KEN BRINSON

single hydrolase is likely to be the wall-loosening factor. Cellulase, for example, increases wall extensibility, but does not cause cell elongation in either Avena coleoptile-sections506~50g or pea e p i c o t y l ~In. ~the ~ ~latter tissue, swelling of the cells was, at first, considered to be cellulase-mediated, but was later discovered to be ethylene-induced. Furthermore, ethylene exerted no effect on cellulase activity,504nor did the distribution of cellulase in the tissue parallel the distribution of A similar situation exists in the case of (1+3)-jI-~-glucanase. Auxin promotes growth, but not (1+3)-jI-~-glucanase activity in pea epicotyls,4Q2.503 and the enzyme only promotes growth in these epicotyls after treatment for several hours at lessened turgor. If the reported 10% increase in (1+3)-jI-~-glucanase activity in Avena coleoptiles following auxin addition is responsible for the reported 600-800% increase in growth rate induced by auxin,511this would indicate truly remarkable kinetics for the enzyme. Elongation growth in plant tissues depends not only upon protein synthesis but also upon respiration. Growth is stopped within 15minutes by KCN or a n a e r o b o s i ~and , ~ ~within ~ 30 minutes by inhibitors ofprotein synthesis.400This indicates that the wall-loosening factors are highly unstable; known polysaccharide hydrolases, in contrast, are highly stable. The half-life of cellulase in pea stems, for example, isso4almost 24 h. In addition, it seems probable that wall-loosening involves a reversible breakage and re-formation of some c r ~ s s - l i n k sCleland400 ~ ~ ~ ~ ~ pro~~; posed that polysaccharide hydrolases alone are unlikely to mediate such a mechanism, claiming their action to be irreversible. Lamport41,42,229 proposed that the extensibility of the cell wall is governed by the number of hydroxy-L-proline - arabinose links in the hydroxy-L-proline-rich glycoprotein of the wall. a,a-Dipyridyl, which limits the synthesis of new h y d r o x y - ~ - p r o l i n eand, , ~ ~therefore, ~ ~ ~ ~ ~ the synthesis of hydroxy-L-proline - arabinose links, causes some extension in soybean h y p o c o t y l ~ . ~ ' ~ There is much evidence that the hydroxy-L-proline-rich glycoprotein is involved both in the mechanical properties of the wall and in rates of growth. Clelands14-51~ showed that, in oat coleoptiles, removal of wall (509) S . Wada, E. Tanirnoto, and Y. Masuda, Plant Cell Physiol., 9 (1968) 269-276. (510) G . A. MacLachlan, A. H. Datko, J. Rolhtt, and E. Stokes, Phytochemisty, 9 (1970) 1023- 1030. (511) Y. Masuda and R. Yamarnoto, Deu. GrowthDifl, 11 (1970) 287-296. (512) J. Hurych and M. Chvapil, Biochim. Biophys. Acta, 97 (1965) 361 -369. (513) N. M. Barnett, Plant Physiol., 45 (1970) 188-191. (514) R. Cleland, Plant Physiol., 42 (1967) 271 -274. (515) R. Cleland, Plant Physiol., 42 (1967) 1165-1170. (516) R. Cleland, Planta, 74 (1967) 197-209.

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glycoprotein increased extensibility and, less directly, Linskens517found that plant cell-walls having little of this glycoprotein have lower tensile strength than walls rich in this component. Application of auxin to peastem segments strongly inhibits synthesis of hydroxy-L-proline-rich protein, but stimulates elongation,518and, in the same tissue, treatment of the apex with ethylene leads to an increase in the wall glycoprotein and an inhibition of longitudinal In the cells of Jerusalem artichoke tubers, also, an increase in the wall glycoprotein occurs as growth slows.52oThese results suggested an inverse relationship between cellular-elongation growth-rate and the hydroxy-L-proline-rich-glycoprotein content of the cell wall. There are, however, contrary findings that challenge this hypothesis. Cleland and K a r l s n e ~for , ~ example, ~~ reported that the hydroxy-L-proline content of growing mung-bean cell-walls increased over the growth period, and Winter and coworkerss1* found that the greatest elongation in excised, pea-stem segments, after treatment with auxin, and a sugar as an energy source, coincided with the greatest content of wall hydroxy-Lproline. The inverse relationship does not hold, therefore, under these circumstances. In some instances, at least, it appears that an increase in wall hydroxy-L-proline is only one of the factors changing with wall extension. Pea seedlings treated with ethylene show typical diminution of elongation growth and increase in diameter (reflecting radial expansion of the cells), and this radial growth is accompanied by an increase in hydroxy-L-proline-rich glycoprotein in the wall,521Moreover, the walls of the cortical parenchyma double in thickness, and some of the new cellulose microfibrils adopt a new longitudinal orientation in place of the previous transverse orientation.521It seems clear that, in such radial-cell expansion, which may be comparable to cell-expansion growth of fruit parenchyma during maturation, there is a whole complex of factors involved in wall changes, and it would be inappropriate to single out one of them, an increase in hydroxy-L-proline-rich glycoprotein, as being solely responsible for enhanced extensibility of the wall. Perhaps, therefore, the importance of wall-bound, hydroxy-L-prolinerich glycoprotein lies in a property other than the breaking of hydroxy-Lproline - arabinose cross-links between the glycoprotein and wall poly(517) H. P. Linskens, Proc. Int. Symp. Pollen Physiol. Fertilism. North Holland, Amsterdam, 1964, pp. 230-236. (518) H. Winter, L. Meyer, E. Hengeveld, and P. K. Wiersma, Acta Bot. Need., 20 (1971) 489-491. (519) I. Ridge and D. J. Osborne,]. E x p . Bot., 21 (1970) 843-856. (520) N . J. King and S . T . Bayley, ]. E x p . Bot., 16 (1965) 294 -303. (521) D. J. Osborne, I. Ridge, and J. A. Sargent, in D. J. Carr (Ed.), Plant Growth Substances, Springer-Verlag. Berlin, 1972, pp. 534-542.

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saccharides during wall extension. Cleland514-51sconsidered that auxin may regulate the synthesis of a pool of substances, necessary for growth and used up during growth, amongst them a hydroxy-L-proline-rich glycoprotein. This author reported that externally fed hydroxy-L-proline inhibits synthesis of the g l y c o p r ~ t e i n , ~and ~ ~ -that ~ l ~auxin and sugar added together increase hydroxy-L-proline f o r m a t i ~ n , and ~ ~ Rays ~.~~~ reported that auxin and a sugar together promote the synthesis of noncellulosic wall-polysaccharides, in growing coleoptiles of several higher plants. Perhaps, the hydroxy-L-proline-rich glycoprotein is required only for the intussusception of matrix polysaccharides and for their correct orientation in the wall structure, in the mechanism ofwall loosening, proposed by Ray,s that has already been discussed. Da~hek~ also ’ ~ proposed that the glycoprotein plays a role in the incorporation of polysaccharides into the wall, even suggesting that matrix polysaccharides may be partly assembled by the glycosylation of hydroxy-L-proline residues in the wall protein. Prestonqo1speculated that the wall glycoprotein may have enzyme properties, and that this enzymic activity may play a role in breaking and making glycosidic linkages in the orientation of wall-matrix polysaccharides and in wall extension. (The possibility that the hydroxy-L-proline-rich glycoprotein may have lectin properties has been discussed in Section IX,3.) If this supposition is correct, the glycoprotein may be involved in reversibly binding polysaccharides that slip or “creep” relative to each other during wall extension. Thus, the glycoprotein may combine such lectin properties with enzymic activity in regulating wall extensibility. A report523 claimed that cell-wall-bound peroxidase is involved in cross-linking hydroxy-L-proline-rich glycoprotein within the wall matrix in carrot-root tissue, and that inhibition of peroxidase activity inhibits the uptake into the wall of synthesized glycoprotein, which arrives at the wall as soluble glycoprotein and is rendered insoluble by binding into the wall matrix. In this regard, it is interesting that the increase in diameter of pea-seedling, cortical-parenchyma cells induced by ethylene is accompanied by an increase in wall-bound, peroxidase activity and in hydroxyL-proline-rich glycoprotein content of the It is possible that ethylene promotes this expansion growth partially through its effect on the wall glycoprotein and that this ethylene effect is peroxidase-mediated. At this stage, the precise relationship between wall glycoprotein and cell extension remains a subject for speculation. Much more information is needed about the nature of the bonding, within the wall matrix, in which the hydroxy-L-proline-rich glycoprotein is involved, and about its (522) R. Cleland, Science, 160 (1968) 192-194. (523) J. B. Cooper and J. E. Varner, Plant Physiol., Suppl., 67 (1981) 125.

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distribution between the lamellae of the growing wall and over the wall area. Albersheim and associatesJ7 suggested that the hydrogen bonds between xylogIucan and cellulose fibrils in dicot primary-walls (see Section IX,2)may be the bonds that are broken during cell-wall extension induced by auxin and low pH, the breaking of such links facilitating the slipping of cellulose fibrils relative to each other. In a separate report,1° this group also put forward the alternative proposition that the key mechanism allowing this slipping or creep of cellulose fibrils, during wall extension induced by auxin, might be the sequential degradation and resynthesis, during extension, of the covalent linkages between neutral side-chains of the pectic polymers and xyloglucan that is hydrogenbonded to cellulose. Labavitch and Ray101J02.524and Loescher and NevinsllO also suggested that covalent bonds between xyIoglucan and pectic side-chains are broken and re-made during auxin-induced elongation-growth of pea-stem sections, and Labavitch and Ray101*102*s24 demonstrated that auxin induces turnover of primary-wall xyloglucan in this tissue; this polymer appears to be both removed from, and re-inserted into, the wall during elongation growth. Perhaps, when bonds between xyloglucan and pectic side-chains are broken, this allows removal of xyloglucan from the wall, and simultaneous slippage of cellulose fibrils, facilitating wall extension which is “fixed” by re-insertion of newly synthesized xyloglucan into the extended wall. The re-insertion of xyloglucan assumes resynthesis of its covalent links to pectic side-chains, thus stabilizing the extended wall. A criticism of this hypothesis is that, if this is really the mechanism regulating creep of cellulose fibrils during wall extension, turnover of xyloglucan would have to be extremely rapid to cope with the rapid elongation induced by auxin. The possibility must also be borne in mind that the auxin-induced turnover of xyloglucan, reported by Labavitch and Ray101*102.524 may be unrelated to any role that this polymer may have in wall extension. However, it should be noted that these authorslo2demonstrated that the turnover of wall polysaccharides other than xyloglucan was not affected by auxin. Even if the auxin-induced turnover of xyloglucan is unrelated to wall extension, this does not precIude the possibility that the sequential breaking and resynthesis of covalent bonds, between pectic side-chains and xyloglucan molecules that remain attached to cellulose fibrils, is the mechanism that regulates the slippage of the fibrils. A l b e r ~ h e i r nand ~ ~coworkers isolated, from pea-stem cell-wall, a fragment composed of the xyloglucan attached to the pectic galactan. Pro(524) J. M. Labavitch, unpublished findings, cited in Ref. 10.

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PRAKASH M. DEY AND KEN BRINSON

posals have been made to determine the detailed structure of this fragment, and to ascertain whether radioactive label disappears more quickly from the xyloglucan portion than from the galactan during auxin-stimulated elongation growth. Such experiments might provide conclusive evidence that the xyloglucan - galactan linkage is cleaved and resynthesized during the growth process, and thus lead to the isolation of an enzyme (or enzymes) that catalyzes this mechanism. These workers considered that it may be possible to form a complex between such an enzyme and the xyloglucan (or a portion of the xyloglucan), in line with their hypothesiss4 that the elongation enzyme is an endotransglycosylase that transfers a portion of a cellulose fibril-interconnecting polysaccharide to itself. They proposed that, ideally, the enzyme would not only catalyze wall extension by breaking bonds that interconnect the cellulose fibrils within the wall matrix, but would also cross-connect new polysaccharide partners, in order to maintain wall strength during growth, as the walls of cells that have undergone elongation are about as strong as walls of unelongated cells. It has been found that the cell wall maintains about the same thickness, that is, the same mass per unit length, during e l ~ n g a t i o n . ~ ~ It is now established that new wall-polymers are synthesized during elongation g r o ~ t h ~ and * ~that . ~existing ~ ~ - polymers ~ ~ ~ cannot be continually weakened, as otherwise, the extended wall would be weaker per unit length than the unextended wall. If growth were catalyzed by the breaking of bonds without resynthesis, every time a wall doubled in length, half of the wall would consist of aged, relatively degraded polymers, and half of new, relatively undegraded polymerss4 and wall strength would not be maintained. Any satisfactory explanation of wall extension must account for the maintenance of wall strength. Albersheims4proposed that the hypothetical endotransglycosylase, after forming an enzyme -polysaccharide complex between itself and part of a cellulose fibril-interconnecting polysaccharide (thus facilitating fibril slippage), then transfers the detached fragment to a new, interconnecting polysaccharide chain. Fibrilinterconnecting bonds are thus repeatedly broken and re-formed by this reversible enzyme activity. This mechanism is illustrated in Fig. 1 1 . Reference has already been made to the endoglycanases known to be bound to cell walls in higher plants. Both Hehre and coworkers525and Albersheims4 made the point that these enzymes are more correctly termed endotransglycosylases than hydrolases, but that, in some cases, they catalyze hydrolysis by transferring a glycosidic bond (more correctly termed a glycosylic bond525)from a glycosyl residue, within a (525) E. J. Hehre, G . Okada and D. S. Genghof, Adu. Chem. Ser., 117 (1973) 309-333.

357

PLANT CELL-WALLS Cellulose microfibrils Cross- 1i n k i n g polysaccharides

L

A

B

C

FIG.11.-Diagrammatic

Representation of Proposed Mechanism for Mutual “Slippage” or “Creep” of Cellulose Microfibrils During Extension of the Primary Cell-Walls of Higher Plants (After Albersheim5). [Extension of the cell-wall might b e regulated by an enzyme acting on bonds between the cross-linking polysaccharides. The enzyme would separate two molecules (A), and would become attached to one of them. The cellulose microfibrils could then shift in relation to one another (B), until the enzyme was able t o join the polysaccharide molecules to new, partner molecules (C). No enzyme capable of controlling such a process has yet been isolated from a plant cell-wall, and which of the cross-linking polysaccharides it might act on is not known.]

polysaccharide chain, to water. These authors pointed out that such enzymes not only catalyze the breaking of glycosylic bonds, but also catalyze the synthesis of such bonds, although Cleland400disputed the reversible nature of endoglycanase activity. Although the last author400 also pointed out that no endoglycanase is known to be activated by a lowering of pH, A l b e r ~ h e i minterpreting ,~~ the evidence that lowering of the wall pH to S induces loosening of the wall in a manner similar to that ~ - ~ ~that ~ . ~it~ is ~ reasonable . ~ ~ ~ that catalyzed by a ~ x i n , ~ 0 0 . 4 0 1 . 4 ~argued the proposed endotransglycosylase be more active at pH 5 than at pH 7. Extending this hypothesis, A l b e r ~ h e i mput ~ ~forward the possibility that the equilibrium, between the polysaccharide fragment attached to the enzyme and the polysaccharide fragment attached to cellulose, favors

358

PRAKASH M. DEY AND KEN BRINSON

the enzyme -polysaccharide fragment attachment at pH 5 and the cellulose - polysaccharide fragment attachment at pH 7, resulting in a wall weaker at pH 5 than at pH 7 .Auxin-induced secretion of H+ through the plasma membrane into the cell wall has been clearly demonstrated in ~ ~ ~soybean hypopea-stem sections,480 Hetianthus c ~ l e o p t i l e s ,and cotyls.52' Verification or confutation of the role of an endotransglycosylase as the auxin - low pH-activated, wall-loosening factor in cell extension awaits further study. Even if the activity of such an enzyme can be demonstrated, the correlation between such activity and extension would need to be rigorously examined, as it is often difficult to be certain whether a positive correlation means a causal relationship between the two processes, or only that the two are affected in a parallel manner by some other agent. Nevertheless, a search for such an enzyme would probably constitute the potentially most fruitful avenue for research in this field at the present time. Interest in the hydrogen bonds between xyloglucan and cellulose fibrils as potentially the acid-labile bonds within the extending primary wall of some dicots was diminished by Valent and Albersheim's finding59that the binding of xyloglucan to cellulose fibrils, in the primary wall of suspension-cultured sycamore-cells, is not affected by changes in hydrogen-ion concentration. Vanderhoef and Dute403argued that the mediation of auxin-induced elongation-growth through proton secretion into the wall is not incompatible with the gene-expression hypothesis, which proposes that auxin regulates wall-loosening and steady-state elongation by action at gene transcription or translation, leading to enhanced ~ a l l - s y n t h e s i s . ~ ~ ~ ~ ~ Vanderhoef and Stah1530and Kazama and K a t ~ u m idemonstrated ~~l that, in soybean and cucumber hypocotyls, respectively, auxin-induced elongation could be separated into two phases, the early burst of growth (simulated by lowering the pH from 6 to 4),and a later phase associated with long-term, steady-state growth. In subsequent e x p e r i m e n t ~ , ~ 3 ~ - ~ ~ ~ (526) J. Mentze, B. Raymond, J. D. Cohne, and D. L. Rayle, Plant Physiol., 60 (1977) 509-51 2 (527) D . L. Rayle and R.E. Cleland, Plant Physiol., 66 (1980) 433-437. (528) J. L. Key, Annu. Aeo. Plant Physiol., 20 (1969) 449-462. (529) A. J. Trewavas, Prog. Phytochem., 1 (1968) 113-124. (530) L. N . Vanderhoef and C. A. Stahl, Proc. Natl. Acad. Sci. U.S.A.,72 (1975) 18221825. (531) H. Kazama and M. Katsurni, Plant Cell Physiol., 17 (1976) 467-473. (532) L. N. Vanderhoef, in C. J. Leaver (Ed.), Genome Organisation and Expression in Plants, Plenum, New York, 1979, pp. 159- 173. (533) L. N. Vanderhoef, in F. Skoog (Ed.), Plant Growth Substances, Springer-Verlag, Berlin, 1980, pp. 90-96. (534) L. N. Vanderhoef, C. A. Stahl, and T. S. Lu, Plant Physiol., 58 (1976) 402-404. (535) L. N. Vanderhoef, C. A. Stahl, C. A. Williams, K. A. Brinkmann, and J. C. Greenfield, Plant Physiol., 57 (1976) 871-819.

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it was confirmed that elongation during the first phase is biochemically distinct from elongation during the second phase, and that this second phase involves enhanced insertion of newly synthesized polysaccharide into soybean hypocotyl-walls. The first phase had the characteristics of the universally accepted “wall-loosening’’ which is mimicked by wall acidification (see Fig. 12). Vanderhoef and ~ 0 ~ 0 r k e r ~ ~ 0 3 proposed ~ 5 3 ~ ~ that ~ 3 ~the - ~aforemen~ ~ tioned findings meet the objections to the gene-activation hypothesis raised by Evans and Ray4e5and H a s ~ h e m e y e r and , ~ ~that ~ the “fast response” to auxin reported by the latter authors refers to the first elongation phase only. Vanderhoef and Dute403demonstrated that, in soybean hypocotyls in which the walls were kept in a “loose” state by maintaining the pH at 4,exogenous auxin induced only the second response, the first response having already been induced by the low pH. They postulated that all data available in the field can be accommodated if the assumption is made that auxin regulates and coordinates both the wall loosening (possibly mediated by proton secretion) and the supply of wall materials, both of which contribute to elongation growth. A second line of evidence that supports a mediating role of gene activation in auxin-induced elongation comes from studies of auxin-effected protein synthesis in elongating cells. Auxin has been shown to induce the synthesis of specific, elongation-associated proteins in soybean h y p o ~ o t y l s Auxin . ~ ~ ~ activity at gene expression in sustained cell-elongation appears to be a real possibility. In conclusion, it must be stated that there remain many unanswered questions concerning the effects of auxin upon cell walls in developing plant-tissues. With regard to the swelling of fruit parenchyma during maturation, equally important is elucidation of the role of ethylene in promoting radial growth of parenchyma cells, a subject on which there has been much less work published than on auxin in elongation growth characteristic of growing shoots and rootlets, As outlined in Section XIII,2, b, fruit maturation is undoubtedly regulated by interaction of the effects of auxin, ethylene, and other plant hormones. Swelling of fruit parenchyma almost certainly occurs by radial growth, and such growth may, in some respects, not be directly comparable to elongation growth of cells in coleoptiles. Reference has already been made to the ethyleneinduced radial-swelling of parenchyma in pea seedling^,^^^.^^^ and to accompanying wall changes.521Interestingly, this ethylene response, like auxin-induced elongation growth, appears to require wall acidificat i ~ n , and ~ ~ the ’ directionality of the ethylene-promoted growth appears to be regulated by the layer of cellulose micro fibril^^^^ most recently deposited. The mechanisms involved here invite considerable further (536) L.L. Zurfluh and T. J. Guilfoyle, Proc. Notl. Acad. S c i . U.S.A., 77 (1980) 357-361.

PRAKASH M. DEY AND KEN BFUNSON

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-

L”..

. . . +wall growth

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

Auxin

I Time

A

p,,,. Q1

c

E t .-c0

............... Time

m

0

W

C

Time

D

FIG.12.-Proposed Events in Cell-Wall Extension During Elongation Growth of Pea Epicotyls (After Vanderhoef and Dute403).[Auxin is postulated to regulate and coordinate both wall “loosening” and wall synthesis during extension. (A) Elongation in the intact seedling. A continuous supply of auxin keeps the wall “loose” by maintaining a low wallpH, and keeps the cells growing by maintaining the supply of material(s) essential for wall growth. Thus, there is a steady rate of elongation. (B) Growth in an excised, elongating segment. Some 30 to 90 min after excision, the elongation rate decreases to a low value in the absence of endogenous auxin. Wall pH increases, so that the wall is not maintained in a Acid-in“loosened” state, and the synthesis of materials for wall growth is terminated. (C) duced growth in auxin-depleted, excised segments. Acid is added at the arrow, and mimics the “wall-loosening’’ component of auxin-regulated elongation, causing a burst of growth. Thus, acid does not induce a steady-state elongation-rate; rather, the rate rises after addition of acid, and then begins to decline. (D) Auxin-induced growth in auxin-depleted, excised segments. The first observable effect of auxin added at the arrow is the burst of growth caused by wall loosening. The elongation rate rises, and then begins to fall, with kinetics very similar to those for acid-induced growth. However, the auxin-induced inser-

PLANT CELL-WALLS

36 1

study. Ethylene is known to play a crucial part in inducing onset of the climacteric rise in respiration in fruit that exhibit the latter phenomenon (a subject that will be dealt with in the following Section). It is possible, and, indeed, likely, that wall changes induced by ethylene during the maturation stage of fruit development may continue into the climacteric period and the subsequent ripening that marks the final senescence of the fruit.

e. The Respiratory Climacteric. -The general, physiological significance ofthe respiratory climacteric in the life of many fruits was referred to in Section XIII,2,a. In such fruits, the changes associated with the climacteric occur when the fruit is allowed to ripen on the plant,3D6JD7 but, as many climacteric fruits, including the mango,384*3e3.538 are usually harvested commercially when mature but unripe, most studies of the climacteric rise in respiration have been conducted in detached fruit.537-542 In mangoes, the respiratory rise commences immediately after harvesting at maturity, with the maximum value for the rate of respiration occurring 2 - 5 days after The chemical changes that take place in detached fruit are directly, or indirectly, related to the oxidative and fermentative activities collectively referred to as biological oxidations. Once the fruit is harvested, respiration, the process concerned with the oxidation of predominantly organic substances by the cell, assumes the dominant role, and the fruit no longer depends on absorption of water and minerals by the root, on conduction by vascular tissues, and on the photosynthetic activity of the leaves. After harvest, the fruit lives an independent life by utilizing substrates accumulated during maturation.385 After fruit set, and through the stages of cell division and cell expansion leading to maturation, there is a constant decrease in the rate of

-

tion of newly synthesized wall-materials begins 5 0 min after auxin addition, and the rate rises again, eventually reaching a steady-state rate. Thus, the two auxin-regulated phases of elongation growth can be individually observed only when exogenous auxin is added to auxin-depleted segments. Their separation occurs because the lag times for the two phases are different; that is, auxin-regulated, wall acidification occurs with a lag near 15 min, whereas supply of auxin-regulated wall-materials begins with a lag near 50 rnin.] (537) A. C. Hulrne, J. D. Jones, and L. S. C. Wooltorton, Proc. R. SOC.London, Ser. B, 158 (1953) 514-535. (538) A. C. Hulme, H. J. C.Rhodes, and L. S. C. Wooltorton, Phytochemisty, 6 (1967) 1343-13.51. (539) J. D. Jones, A. C. Hulme, andL. S. C. Wooltorton, New Phytol., 64 (1965) 158- 167. (540) C. Lance, G. E. Hobson, P. E. Young, and J. B. Biale, Plant Physiol., 42 (1967) 471-478. (541) K. S.Rowan, H.K. Pratt, andR. N . Robertson,Aust.J. B i d . Sci.,11 (1958) 329-370. (542) J . M. Jager and J. B. Biale, Physiol. Plunt., 10 (1957) 79-85.

PRAKASH M. DEY AND KEN BRINSON

362 250

-

-

C

200

c

\ c, .r

3 %

+ 0

150

m

X \

P

v

U

2

100

0 0

0 N

V 0

50

4

8

12

16

T i m e a f t e r h a r v e s t (days)

FIG.13. -Pattern of Post-harvest Respiration at 20” in Mangoes (After Krishnamurthy and S~bramanyam~~‘). [(a)Preclimacteric period, (b) climacteric rise, (c) climacteric peak, (d) over-ripeness (senescence).Solid line (1) shows the pattern obtained in a single fruit, anddotted line (2) shows the pattern obtained by averaging results from randomly selected fruits.]

respiration of many fruits.396.537-543 In early work, Kidd and West544 demonstrated this in the case of the apple, and coined the phrase “climacteric rise” to describe the marked rise in evolution of C 0 2that occurs at the end of maturation. A typical pattern of respiration exhibited by climacteric fruits consists of a short-lived decline in the rate of oxygen uptake and CO, evolution immediately after harvest, followed by a sharp rise. The peak, termed the “climacteric maximum,” is followed by a period of declining respiration referred to as the “post-climacteric” stage. The climacteric pattern of respiration for the mango is shown in Fig. 13. The opinion ofKidd and West544that a cIimacteric occurs with all fruits was challenged by Biale395-396 on the basis of observations with citrus fruits, where no marked ripening changes and no chemical transformations of the type described for climacteric fruits could be detected. In citrus fruits, senescence apparently follows maturation without any intervening transition stage. The possibility that the climacteric occurs on the tree prior to harvest cannot be discounted, but this seems unlikely. Lemons, for example, display a steadily declining rate of respiration,

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irrespective of the stage of development, ranging from dark green to yellow, at h a r v e ~ t Knee . ~ ~ and ~ ~ coworkers394 ~ ~ ~ concluded that the strawberry follows a similar, nonclimacteric course of development. Some, but not all, fruits that are regarded as nonclimacteric are characterized by low respiratory It should be noted that Trout and coworker^^^^.^^^ expressed doubt of the generally accepted classification of citrus fruits as nonclimacteric. Clarification of whether these and other fruits, such as fig, grape, strawberry, and pineapple, exhibit a truly nonclimacteric pattern of ripening may come from further study. In fruits that display it, the climacteric may be regarded as the dividing line between maturation and senescence. The changes characteristic of ripening are linked to the climacteric, and the flesh texture referred to as “eating ripeness” in avocado, banana, and mango, for example, is closely associated with the climacteric peak. Similarly, such color changes as green to yellow in bananas, and green to red in mangoes, occur during the climacteric rise, or immediately after the peak.384.3g3.547-544e Known factors that influence the onset of the climacteric in fruits are temperature (in general, lowering of the temperature delays the onset of the climacteric), 0,and CO, tension (in general, lowering the 0,tension below that of air, or raising the CO, tension, delays the climacteric), and the presence of ethylene.537-542.550 The role of ethylene in fruit ripening is still subject to question. Exogenous ethylene promotes the ripening of fruits, and endogenous ethylene is produced (presumably by the pathway described in Section X111,2,b), along with other volatiles, by fruit during ripening, but Biale543concluded that ethylene is a product of the ripening process, rather than a causal agent of the climacteric rise, Pratt and G o e ~ c hchallenged l~~~ this conclusion, assigning to ethylene the role of the quintessential planthormone initiating the climacteric. The effect of ethylene in inducing increased activity of polysaccharide hydrolases during the climacteric rise in some ripening fruits will be dealt with in Section XIII,2,f. In cucumbers, the compound (aminoethoxy)vinylglycine (AVG) inhibits synthesis of the intermediate ACC (see Section xIII,2,b) and ethylene (543) J. B. Biale, Ado. Food Res., 10 (1960) 293-320. (544) F. Kidd and C. West, Rep. Br. Dep. Sci. Ind. Res. Food Znoest. Board, (1924) 27. (545) S. A. Trout, G. B. Trindale, and F. E. Huelin, Australia Commonwealth Council Sci. Ind. Res. Pam., Vol. 80, 1938. (546) S. A. Trout, F. E. Huelin, and G. B. Tindale, Australia C. S. 1. R. O.,Diu. Food Presero. Transport, Tech. Pap., 14 (1960) 1 - 16. (547) V. K . Leley, N . Narayana, and J. A. Daji, Indian]. Agric. Sci., 13 (1943) 291-299. (548) P. K. Mukherjee, Hortic. Ado., 3 (1959) 95-101. (549) S. Lakshimarayana, N . V. Subhadra, and H. Subramanyam,]. Hortic. Sci.,45 (1970) 133- 143. (550) L. W. Mapson and J. E. Robinson,]. Food Technol., 1 (1966) 215-221. (551) H. K. Pratt and J. D. Goeschl, Annu. Reo. Plant Physiol., 20 (1969) 541 -563.

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from m e t h i ~ n i n eand, , ~ ~ in ~ pears, the same inhibitor strongly inhibits ethylene production, and delays for 5 days the respiratory climacteric and accompanying changes in skin color and flesh firmness. When treated with ethylene, the inhibited pears exhibit a climacteric rise in respiration, soften, and become yellow.553Treatment of the AVG-infiltrated pears with ethylene for 24 h resulted in no recovery of endogenous-ethylene production, but in a stimulation of protein synthesis measured as a 200%increase in leucine incorporation by excised tissue, and a 74% increase in the percentage of ribosomes present as polysomes.553 This evidence provides powerful support for the hypothesis that ethylene synthesis is central to initiation of the climacteric rise and accompanying ripening changes. There is considerable evidence for the involvement of protein synthesis (de novo enzyme-synthesis) at the climacteric stage.539*540*554-560 Reports of the synthesis of ribosomal RNA just prior to the climacteric peak,539*5sg-561-563 and more-recent reports of changes in the levels of different ~ - R N A ’and s~~ messenger ~ RNA’s565*566 related to tomato ripening, support the concept of de nouo enzyme-synthesis catalyzing the climacteric and the final breakdown of fruit cells during subsequent senescence. H ~ l m demonstrated, e ~ ~ ~ in one or more fruits, increased activity, accompanying the climacteric, of enzymes that included malic enzyme, catalase (EC 1 . 1 1.1.6), peroxidase (EC 1.11.1.7), phosphatase (EC 3.1.3.2), invertase (EC 3.2.1.26), alpha amylase (EC 3.2.1.1), pectin methylesterase (EC 3.1.1.1l),polygalacturonase (EC 3.2.1.15),glucose 6-phosphate dehydrogenase (EC 1 . l .1.49), 6-phosphogluconate dehy(552) C. Y. Wang and D. 0.Adams, Plant Physiol., 66 (1980) 841 -843. (553) P. J. Ness and R. J, Romani, Plant Physiol., 65 (1980) 372-376. (554) J. A. Sacher, Plant Physiol., 41 (1966) 701 -708. (555) J. A. Sacher, Annu. Reo. Plant Physiol., 24 (1973) 197-224. (556) C. Frenkel, I. Klein, and D. R. Dilley, Plant Physiol., 43 (1968) 1146-1153. (557) J. Riov, S. P. Monselise, and R. S. Kahan, Plant Physiol., 44 (1969) 631 -635. (558) C. J. Brady, J. K. Palmer, P. B. H. O’Connell, and R. M. Smillie, Phytochemisty, 9 (1970) 1037- 1047. (559) G . H. De-Swardt, J. H. Swanepoel, and A. J. Dubenage, Z. P’anzenphysiol., 70 (1970) 358-365. (560) A. Richmond and J. B. Biale, Plant Physiol., 41 (1966) 1247-1253. (561) N. E. Looney and M. E. Patterson, Phytochemisty, 6 (1967) 1517-1520. (562) L. L. Ku and R. J. Romani, Plant Physiol., 45 (1970) 401 -407. (563) A. Richmond and J. B. Biale, Biochim. Biophys. Acta, 138 (1967) 625-627. (564) I. J. Mettler and R. J. Romani, Phytochemisty, 15 (1976) 25-28. (565) N . Rattanpanone, D. Grierson, and M. Stein, Phytochemisty, 16 (1977) 629-633. (566) N . Rattanpanone, J. Spiers, and D . Grierson, Phytochemisty, 17 (1978) 14851486. (567) A. C. Hulme,]. Food TechnoE., 7 (1972) 343-351.

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drogenase (EC 1.1.1.43),L-aspartate-oxoglutarate amino transferase (EC 2.6.1.l), L-glutamate-1-decarboxylase(EC 4.1.1.15),citrate lyase (EC 4.1.3.6),transaminases, lipoxygenase (EC 1.13.1.13),succinic dehydrogenase (EC 1.3.99.l), malic dehydrogenase (EC 1.1.1.37), and cytochrome-creductase (EC 1.6.99.3).Changes in the level of activity of a number of polysaccharide hydrolases, some of them exhibiting correlation with ethylene evolution and the onset of the climacteric rise, during ripening of a variety of fruits, will be dealt with in Section X111,2,f. From the foregoing evidence, it is clear that the climacteric period consists of an intense burst of metabolic activity, possibly triggered by ethylene and involving considerable synthesis of enzymes that catalyze the senescence of the fruit. The chemical changes in fruit associated with the climacteric include the early hydrolysis of starch followed by the utilization of D-glucose as a respiratory substrate during the post-climacteric period,543.568-570the probable mobilization of h e m i c e l l u l ~ s e s ~and ~ ~insoluble * ~ ~ ~ - (wall-bound) ~~~ p e ~ t i n s ~ ~ ~ , ~ ~ as reserve carbohydrates contributing to the pool of monosaccharide loss of chlorophyll substrates for respiration, changes in acidity,543.570.573 and synthesis of c a r o t e n o i d ~ , ~ ~increase ~*~~~ in*the ~ ' ~ratio of protein nitrogen to total nitr0gen,541*5~3*~'~ and increase in "energy-rich" phosp h a t e ~ . ~ There ~ ~ is. considerable ~ ~ ~ - ~ ~evidence ~ for enhanced oxidative and phosphorylation activity in fruit mitochondria at the climacteric, as opposed to the preclimacteric and senescent stages. In general, mitochondrial oxidation rates (of pyruvate, succinate, malate, and a-ketoglutarate) and ADP : O2uptake ratios reach a maximum with incipient ripeness, and then decline with the onset of s e n e s ~ e n c e . ~ ~ ~ ~ ~ ~ " - ~ ~ ~ (568) A. C. Hulme, Adu. Food Res., 8 (1958) 277-391. (569) A . C. Hulme, Production and Application ofEnzyrne Preparations in Food Manufacture, Society of Chemical Industry, London, 196 1. (570) C. Rolz, S. Deshpande, L. Paiz, L. Oritz, M. C. Fiores, M. Sanchez, and M. D e Ortega, Turrialba, 22 (1972) 65-72. (571) H. R. Barnell, Ann. Bot. (London), 5 (1943) 217-261. (572) H. W. Van Loesecke, Bananas, Interscience, New York, 1950. (573) J. Wolf, 2. Lebensm. Unters. Forsch., 107 (1958) 124-134. (574) B. Borenstein and R. H. Bunnell, Adu. Food Res., 15 (1966) 195-2.10. (575) A. C. Hulme,]. Exp. Bot., 5 (1954) 159-172. (576) G. E. Hobson, Qual. Plant. Muter. Veg., 19 (1969) 1-3. (577) G. E. Hobson, Biochem.]., 116 (1970) 20 P. (578) G. E. Hobson, Phytochemisty, 9 (1970) 2257-2263. (579) C. Lance, G . E. Hobson, R. E. Young, and J. B. Biale, Plant Physiol., 40 (1965) 1 1 16- 1123. (580) C. Lance, G. E. Hobson, R. E. Young, and J. B. Biale, Biochim. Biophys. Acta. 113 (1966) 605-612. (581) A . G. Drouet and C. J. R. Hartmann, Phytochaistry, 16 (1977) 505-508. (582) 0. Kane and P. Marcellin, Plant Physiol., 61 (1978) 634-638.

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The ways in which the various metabolic processes are interrelated, and the mechanisms of control during the climacteric rise, are at present ill-understood. The theory put forward by Solomos and la tie^,^^^ to explain the respiratory burst, invoked the concept of a marked increase in membrane permeability with accompanying cellular decompartmentalization and metabolic deregulation. However, it is an open question as to whether the membrane-permeability changes occurring during ripening584are causative or consequential. Millerd and coworkers585considered that the climacteric rise in respiration during ripening could be brought about by the uncoupling of phosphorylation from respiration. H o b ~ o demonstrated n~~~ that, even in tomatoes subjected to the action of uncoupling agents, production of enzymes necessary for the ripening process continued. He discussed the possibility that “loose” coupling of phosphorylation (the term applied to alack of normal, feed-back control of mitochondria1 respiration by ADP) during the climacteric rise results in a net increase in the synthesis of “energy-rich” bonds at this stage, leading to the formation of additional enzymes necessary for the furtherance of ripening. Pearson and Robertproposed that the respiratory rate is controlled by the ADP : ATP ratio, whereas Barker and sol or no^^^^ supported the view that cellular D-fructose 1,6-bisphosphate concentration is a major, controlling factor. Laties and a s s o ~ i a t e s provided ~ ~ ~ - ~ evidence ~~ for an alternative, cyanide-resistant path of respiration in avocado mitochondria. Uncouplers were considered to stimulate glycolysis to the point where the glycolytic flux exceeds the oxidative capacity of the cytochrome pathway, with the result that the alternative pathway is engaged. However, these authors concluded that the alternative pathway is not required in order to sustain the elevated rate of respiration that characterizes the climacteric. Clarification of the role, if any, of this alternative pathway in fruit ripening awaits further study. Central to enhancing understanding of the initiation of ripening is elucidation of the connection between plant-hormone secretion and the (583) T. Solomos and G. G. Laties, Nature, 245 (1973) 390-391. (584) S. Ben-Yehoshua, Physiol. Plant., 17 (1964) 71 -80. (585) A. Millerd, J. Bonner, and J. B. Biale, Plant Physiol., 28 (1953) 521-531. (586) G. E. Hobson,]. Exp. Bot., 16 (1965) 411-422. (587) J. A. Pearson and R. N . Robertson, Aust. ]. B i d . Sci., 7 (1954) 1-9. (588) J. Barker and T. Solomos, Nature, 196 (1962) 189. (589) T. Solomos and G . G. Laties, Plant Physiol., 54 (1974) 506-511. (590) T. Solomos and G . G. Laties, Plant Physiol., 55 (1975) 73-78. (591) T. SolomosandG. G. Laties, Biochem. Biophys. Res. Commun., 70 (1976) 663-671. (592) A. Theologis and G . G . Laties, Plant Physiol., 62 (1978) 249-255.

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metabolic changes that accompany the climacteric. HobsonSQ3 suggested that adenosine 3’,5’-monophosphate, a “secondary messenger” known to exert a regulatory role in a number of hormonally mediated, metabolic changes in animal systems,5e4-5Q5 may have a regulatory role in fruit ripening, although he presented no evidence in support of this proposition. The presence of cyclic AMP in the tissues of various species of higher plants has been reported by several and Newton and coworkerseo2 appear to have answered the objections of other authorse03-e0sthat the cyclic nucleotide had not been rigorously identified in the tissues of Phaseolus vulgaris seedlings, by unambiguously identifying cyclic AMP by mass spectrometry. In the light of the afore~ interesting ~~ that, in the fruit of mentioned suggestion by H o b ~ o nit, is the Chinese plant Zizyphus jujuba, which contains high levels of cyclic AMP, the level of this compound increases 5000-fold during maturation and ripening of the fruit.‘j06 This isolated finding does not prove a connection between cyclic-AMP level and the initiation of the climacteric, but it is thought-provoking, and suggests a potentially valuable, future line of enquiry in seeking to elucidate the relationship between planthormone secretion and the ripening process. In conclusion, although the controlling mechanisms are currently unknown, the climacteric may well be regarded as a hormonally induced intensification of metabolic activity succeeding maturation of the fruit, during which enhanced respiratory-activity generates the energy required for synthesis (or activation) of enzymes catalyzing cellular breakdown and death. The reported increase in enzyme activities accompanying ripening may result from de nouo enzyme-synthesis induced by the (593) G. E. Hobson, personal communication. (594) M. Kaliner, J. Clin. Inuest., 60 (1977) 951 -963. (595) G. A. Robison, R. W. Butcher and E. W. Sutherland, Cyclic AMP, Academic Press, New York, 1971. (596) E. G. Brown and R. P. Newton, Phytochemisty, 12 (1973) 263-269. (597) P. Raymond, A. Narayanan, and A. Pradet, Biochem. Biophys. Res. Commun., 53 (1973) 1115-1121. (598) M. Giannattasio and V. Macchia, Plant Sci. Lett., I (1973) 259-264. (599) N. J. Brewin and D. H. Northcote, J. Exp. Bot., 24 (1973) 881-888. (600) K . Ashton and G. M. Polya, Biochem. J,. 165 (1977) 27-32. (601) E. G . Brown, T. Al-Najafi, and R.P. Newton, Phytochemisty, 18 (1979) 9 - 14. (602) R. P. Newton, N. Gibbs, C. D. Moyse, J. L. Wiebers, and E. G. Brown, Phytochemistry, 19 ( 1 980) 1909 - 19 1 1. (603) R. A. B. Keates, Nature, 244 (1973) 355-356. (604) N. Amrhein, Planta, 118 (1974) 241-248. (605) P. P. C. Lin, Ado, Cyclic Nucleotide Res., 4 (1974) 439-458. (606) C. Jyong-Chyul and K. Hanabusa, Phytochemisty, 19 (1980) 2747-2748.

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climacteric rise or, alternatively, they may reflect conversion of inactive zymogens into active enzymes. Such increased activities include enzymes that degrade fruit cell-walls, clearly significant in final breakdown and death of the cells, and these changes form the subject of the Section that follows.

f. Cell-Wall Changes during Ripening. -Most fruit is commercially harvested when mature, but unripe. For this reason, food technologists interested in extending the commercial life of fruits are most concerned with potential means of delaying the processes that accompany the respiratory climacteric and attendant ripening. The breakdown of primary cell-walls within the parenchyma tissue clearly leads to tissue softening, one of the major manifestations of ripening. Elucidation of the mechanism by which this softening occurs possibly holds the key to means of delaying ripening. (i) Changes in the Pectic Polymers.-In Section 111, it was pointed out that, in the primary wall of cultured sycamore-cells, pectic polymers constitute 35% of the cell walLs5 These cell walls have been studied in more detail than those from any other dicotyledonous plant and, although it would not be valid at this stage to conclude definitively that the structures that have been demonstrated in these walls apply universally in the walls of all dicots, nevertheless they provide a valuable and detailed model, and it seems likely that other dicot cell-walls are comparaorigible in structure. The group of workers led by nally reported that sycamore-wall, pectic polymers consist of a rhamnogalacturonan backbone with attached p-( 1+4)-linked D-galactan and branched arabinan side-chains. Subsequent findings by this groups2Bso8 indicated that the sycamore pectic-polymers possess a greater degree of complexity than was earlier believed. Rhamnogalacturonan I (see also, Section III,l,a), a fractionso8that accounts for 23% of the pectic polysaccharides, can be isolated from sycamore walls: it has a molecular weight of 200,000, and a backbone composed of D-galactosyluronic and L-rhamnosyl residues in the ratio of 2 : 1.About half of the L-rhamnosyl residues are 2-linked, and are glycosidically attached to C-4 of D-galactosyluronic residues; the other half are 2,4-linked, with a D-galactosyluronic group glycosidically attached at 0 - 2 and side chains averaging 6 residues in length (shorter than originally envisaged) attached to

-

(607)M. McNeil, A. G . Darvill, and P. Albersheim, in W. H e n , H. Grisebach, and G . W. Kirby (Eds.), Progress in the Chemistry of Organic Natural Products, Vol. 37, Springer-Verlag,Vienna, 1979,pp. 191 -249. (608)M. McNeil, A. G. Darvill, andP. Albersheim, Plant Physiol., 66 (1980)1128- 1134.

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0-4. These side chains appear to be more complex and more varied than was originally p r o p o ~ e d ~there ~ - ~ are ~ ; many different side-chains containing variously linked L-arabinosyl or D-galactosyl residues, or both, as well as terminal D-galactosyluronic groups. Rhamnogalacturonan 11, a separate polysaccharide fraction isolatede2 from sycamore cell-walls, constitutes 13% of the pectic polymers, and possesses an even more complex structure, containing 10 different monosaccharides (see Section III,l,c). However, the concept of the pectic polymers as comprising a rhamnogalacturonan backbone with side chains containing mainly galactosyl and arabinosyl groups attached to rhamnosyl units of the backbone still finds broad acceptance within the field. The evidence (based mainly on enzymic hydrolysis and linkage analysis) for the attachment of D-galactosyl residues in the pectic side-chains to xylogtucan hydrogen-bonded to cellulose fibrils in the sycamore wall is c o n v i n ~ i n g .There ~ ~ ~ has ~ ~been *~~ insufficient, detailed analysis of fruit cell-walls to permit the conclusion that the model proposed for cultured, sycamore cell-walls applies in fruit cells, but Kneeeoe suggested that the model holds for the apple. The pectin-degrading enzymes are pectin methylesterase (PME) and galacturonanase (PG). PME catalyzes the de-esterification of methyl galactosyluronate residues (in which the carboxyl group is methyl-esterified) in the pectic backbone. PME appears to occur almost universally in fruits,e10and, in torn at^,^"-^^^ banana,614and avocado,615the activity of this enzyme increases to a maximum, either in the period immediately preceding the climacteric rise, or during the early stages ofripening, and then falls away continuously as ripening proceeds. In mango,381the level ofPME activity doubles during ripening, but, in contrast to the fruits just mentioned, the level of activity in the mesocarp remains high at an advanced stage in ripening.616Presseyel7 suggested that increased PME activity alone would result in decreased solubility of pectin, due to the increase in free carboxyl groups and greater interaction with Cae+ions in the wall. However, it has generally been shown that galacturonanases in (609) M. Knee, Colloq. Znt. CNRS 283 (1975) 341-345. (610) Z. I. Kertesz, The Pictic Substances, Interscience, New York, 1951. (61 1) R. A. Dennison, C. B. Hall, and V. F. Nettles, Proc. Annu. Meet. Am. SOC. Hortic.Sci., 51 (1954) 17-18. (612) R. T. Besford and G. E. Hobson, Phytochemisty, 11 (1972) 2201-2205. (613) G. E. Hobson, Biochm.]., 86 (1963) 358-365. (614) H. 0. Hultin and A. S.Levine,]. Food Sci., 30 (1965) 917-921. (615) M. Awad and R. E. Young, Plant Physiol., 64 (1979) 306-308. (616) M. G. Medina, Arch. Latinoam. Nutr. 18 (1968) 401-410. (617) R. Pressey, in R. L. Orv and A. I. S. Angelo (Eds.), ACS Symp. Ser., 47 (1977) 172.

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fruits require de-esterified pectate as their s u b ~ t r a t e , ~ and ' ~ - there~~~ fore, the action of PME is considered to be a prerequisite for PG activity. Galacturonanases have been reported from a wide variety of fruits. Endo-galacturonanase randomly cleaves the pectic backbone at internal positions within the molecule, whereas exo-galacturonanase sequentially removes galactosyluronic groups from the (nonreducing) end of galacturonan hai ins.^^^-^^' Pressey and Avants625-628considered that the degradation of de-esterified pectin in fruit may be initiated by endogalacturonanase, the oligogalactosiduronates formed by the randomcleaving enzyme being hydrolyzed to galacturonic acid by the exo-galacturonanase. However, this may be an over-simplified concept of the degradation of polygalacturonate as the same authors showed that, in peache2' and pear,62e exo-galacturonanase exhibits maximal activity when acting on relatively high-molecular-weight galacturonan, and cleaves the lower oligogalacturonates only slowly. Moreover, the presence of other residues within the chain, such as rhamnosyl units, or the possible presence of galactosyluronic branches within the pectic backbone, may mean that there are limit products of exo-galacturonanase activity. There may well be enzymes other than galacturonanases involved in the complete breakdown of the pectic backbone. The involvement of endogenous galacturonanases in solubilization of rhamnogalacturonan, leading to dissolution of the middle lamella and cell separation, is now generally accepted as a major, contributory factor in the tissue softening that accompanies fruit ripening. Fruits in which both endo- and exo-galacturonanase activities have been located include pear,e26 peach,e25 and c u ~ u m b e r , ~and ~ ~ -it ~ has ~ ~ been suggested619~e2s~s32 that tomato fruit similarly contains both activities. Other fruits in which galacturonanase activity, of unspecified type, has been demonstrated include avocado,e20*e33 m e d l a ~ - , ~pineapple,s34 ~* cran(618) B. S. Luh, S. J . Leonard, and H. J. Phaff, Food Rex, 21 (1956) 448-455. (619) D . S . Patel and H. J. Phaff, Food Res., 25 (1960) 47-57. (620) D . Reymond and H. J. Phaff,]. Food Sci., 30 (1965) 266-273. (621) I. M. Bartley, Phytochemistry, 17 (1978) 213-216. (622) C. J. Brady, Aust. ]. Plant Physiol., 3 (1976) 163-174. (623) E. F. Jansen and R. Jang, Food Res., 25 (1960) 64-72. (624) R. M. McReady andE. A. McComb, FoodRes., 19 (1955) 530-535. (625) R. Pressey and J. K. Avants, Plant Physiol., 52 (1973) 252-256. (626) R. Pressey and J. K. Avants, Phytochemisty, 15 (1976) 1349-1351. (627) R. Pressey and J. K. Avants, Phytochemistry, 14 (1975) 957-961. (628) R. Pressey and J. K. Avants, Biochim. Biophys. Acta, 309 (1973) 363-369. (629) R. Pressey and J. K. Avants,]. Food Sci., 40 (1975) 937-941. (630) R. F. McFeeters, T. A. Bell, and H. P. Fleming, J. Food Biochem., 4 (1980) 1-9. (631) M. E. Saltveit andR. F. McFeeters, Plant Physiol., 66 (1980) 1019-1023. (632) B. S. Luh, S. J. Leonard, and H. J. PhaK, Food Res., 21 (1956) 448-455. (633) G . Zauberman and M. Schiffmahn-Nadel, Plant Physiol., 49 (1972) 864-865. (634) G. E. Hobson, Nature, 195 (1962) 804-805.

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berry,e34 grape,635and date.633sH o b ~ o was n ~ ~ unable ~ to demonstrate galacturonanase activity in persimmon, tangerine, melon, cucumber, and grape, but, in other reports,629-631,635 the presence of this enzyme was indicated in the last two cases. Conflicting data for galacturonanase activities in fruits at various stages of ripening may arise from the presence in fruit tissues of inhibitors of the enzyme. Several authors635*637-639 have reported inhibitors of galacturonanase in fruit tissues, and Pansolli and B e l l i - D ~ n i n successfully i~~~ separated the inhibitor present in grape from the enzyme by pH-adjustment and ammonium sulfate precipitation. W e ~ r m a n ~ suggested ~ ~ - ” ~ that polyphenols in fruit tissues may also reported partial inhibit the activity, and Pressey and Avants628.640 inhibition of polygalacturonase in tomatoes by interaction with the substrate, poly(ga1actosiduronic acid). They suggested that, at low ionic strength, the enzyme(s) may form relatively stable complexes with the acidic polysaccharide. Because of these inhibitory effects, reports of the complete absence of galacturonanase activity from fruits at all stages during ripening should be regarded with caution. Galacturonanases in the parenchyma have been shown to be wallbound in peach (both endo- and e x o - e n z y m e ~ )apple , ~ ~ ~ (exo-enzyme tomato ( e n d o - e n ~ y m e )and , ~ ~pear ~ (unspecified activity).641 Increases in the level of polygalacturonase activity during ripening have been demonstrated in tomato,499~642-e44 a v o ~ a d opeach645 , ~ ~ ~date,s36 ~ ~ ~ ~ ~ u c u m b e rand , ~pear,641 ~ ~ ~ ~and, ~ ~in tomato,644avocado,615and cucumber,631this increase is associated with the respiratory climacteric following a transient burst of ethylene production. In the mutant “rin” tomato fruit, which does not ripen, this increase in galacturonanase does not occur.644 The prevailing concept is that, in the softening that accompanies ripening, textural changes occur as insoluble, wall-bound “protopectin” of molecular weight, after partial de-esterification by pectin methyl P. Pansolli and M. L. Belli-Donini, Agrochimica, 17 (1973) 365-372. S.Hasegawa, V. P. Maier, H. P. Kaszycki, and J. K. Crawford,]. Food Sci., 34 (1969) 527 -531. N. P. Ponomareva, Obmen Ugleoodou Plodou Ovoschei Ontog.Akad. Nauk Mold. SSR, Inst. FizioZ. Biokhim. Rust., (1967) 33. C. Weurman, Acta Bot. Neerl., 3 (1954) 108-112. C. Weurman,Acta Bot. Need., 2 (1953) 107-110. R. Pressey and J. K. Avants, J Food Sci., 36 (1971) 486-489. A. E. Ahmed and J. M. Labavitch, Plant Physiol., 65 (1980) 1014- 1016. G. A. Tucker, N. G . Robertson, and D . Grierson, Eur. J. Biochem., 112 (1980) 119-124. G . E. Hobson, Biochem.]., 9 2 (1964) 324-332. B. W. Poovaiah and A. Nukaya, Plant Physiol., 6 4 (1979) 534-537. R. Pressey, D. M. Hinton, and J. K. Avants,]. Food Sci., 36 (1971) 1070-1073. T. A. Bell, Bot. Caz., 113 (1951) 216-221.

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esterase, is converted into more-soluble forms, probably by the action of galacturonanases, but possibly also involving other polysaccharide-degrading enzymes. Such solubilization of galacturonan from the wall during ripening has been demonstrated in mango,384*s47*e48 strawb e r r ~ , ~ date,64Q Q4 pear,452.650 peach,450ss45 avocado,450 and apple.652-655Ultrastructural studies showed that this solubilization of galacturonan is accompanied by dissolution of the middle lamella of parenchyma cells, leading to cell separation in tomato,e5estrawberry,3e4 a p ~ l e , ~and s 7 pear,657and that, in the last two fruits, application of endogalacturonanase in vitro to tissue discs from firm, unripe fruit induces ultrastructural changes similar to those that accompany ripening. More-detailed studies conducted with a limited range of fruits suggested that removal of pectic polymers from the primary wall during ripening may not depend only on the action of polygalacturonase; enzymes that degrade pectic side-chains may also be implicated. Furthermore, certain apparently common features of ripening have emerged from these studies, in particular the loss of galactose or arabinose, or both, from the wall, associated with, or actually preceding, solubilization of high-molecular-weight, wall-bound galacturonan. A study of the cell-wall changes associated with ripening of apples has been conducted. Analysis of the polysaccharides and glycoproteins present in the apple wall, by a combination of extractive and chromatographic technique^,^^^^^^^ together with analysis in vitro of the fragments liberated from the wall by the use of various endoglycanases purified from micro-organisms,6e0established that apple-pectin side-chains contain galactosyl and arabinosyl residues, and strongly suggested (but did not confirm) that these side chains link the rhamnogalacturonan backbone to a hydroxy-L-proline-rich wall-glycoprotein containing tetra-ara(647) C. Rolz, M. C. Flores, M. C. D e Ariola, H. Mayorga, and J. F. Menchu, Rep. Unido Expert Group Meet., Salvador, Bahia, Brazil (ID/WG, 88/15) 1971. (648) R. A. Dennison and E. M. Ahmed,]. Food Sci., 32 (1967) 702-705. (649) I. Rouhani and A. Bassiri,]. Hortic. Sci., 51 (1970) 489-493. (650) A. E. Ahmed and J. M. Labavitch, Plant Physiol., 65 (1980) 1009-1013. (651) G . E. Hobson and J. N. Davies, in A. C. Hulme (Ed.), The Biocherntsty ofFruits and Their Products, Vol. 2 , Academic Press, New York, 1971, pp, 459-469. (652) M. Knee, Phytochemisty, 12 (1973) 1543-1549. (653) M. Knee, Phytochemisty, 17 (1978) 1257-1260. (654) M. Knee, Phytochemisty, 17 (1978) 1261-1264. (655) J. J. Doesburg,]. Food Sci. Agric., 8 (1957) 206-213. (656) W. P. Mohr and M. Stein, Can. J . Plant Sci., 49 (1969) 549-553. (657) R. B. Arie, N. Kislev, and C. Frenkel. Plant Physiol., 64 (1979) 197-202. (658) M. Knee, Phytochemisty, 12 (1973) 637-653. (659) M. Knee, Phytochemisty, 14 (1975) 2181-2188. (660) M. Knee, A. H. Fielding, S. A. Archer, and F. Laborda, Phytochemisty 14 (1975) 2213- 2222.

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binosides attached to the hydroxy-L-proline residues. Fractions that may be obtained from apple-parenchyma walls by means of dilute alkaline extraction at 2 0 ° ,followed by column-chromatographic separation techniques, include a neutral polysaccharide fraction containing mainly glucose and xylose residues, a glycoprotein in which the carbohydrate moiety contains mainly glucose and xylose, and a glycoprotein containing uronic acid, arabinose, and galactose, as well as glucose and ~ y l o s e . ~ ~ ~ The neutral polysaccharide may be a hemicellulosic xyloglucan comparable to the sycamore primary-wall x y l o g l ~ c a nand , ~ ~the two glycoproteins may be a xyloglucan - wall glycoprotein fragment and a pectic polymer - xyloglucan -wall glycoprotein fragment, respectively. The three fractions are probably derived from various degrees of alkaline degradation of linkages within the wall, particularly of the linkages between the hemicellulose and other wall polymers. The attachment of xylose and glucose both to protein and to residues characteristic of the pectic polymers led Kneess8 to suggest that extensive cross-linking, possibly covalent, exists between pectic polymers, hemicelluloses, and glycoprotein in the apple wall, with the pectic sidechains providing the links between rhamnogalacturonan and the other wall polymers. He proposed that this cross-linking renders the cell-wall components insoluble and contributes to the structural properties of the unripe fruit-tissue, both in terms of wall rigidity and inter-cell cohesion. The breaking of these cross-links by enzyme activity may be the key process in dissolution of the wall, through solubilization of its component polymers, leading to cell separation. Kneeeoe concluded that the data obtained from analysis of the apple wall are consistent with the model for dicot primary-wall structure proposed by Albersheim and coworker~,55.5'*~~ but conceded that the apparent linkages between polymers in the apple wall might be artifacts of aggregation between polymers occurring after extraction, rather than linkages actually occurring in vivo. Rharnnogalacturonan in the apple wall contains neutral galactan sidechains, whereas the soluble-pectin fraction, which increases during ripening, is a virtually pure rhamnogalacturonan. Bartleyssl found that hydrolysis of the galactan during ripening precedes solubilization of galacturonan, suggesting that galactan acts to stabilize rhamnogalacturonan within the wall, and that hydrolysis of the galactan side-chains is necessary before solubilization of the pectic backbone can occur. Bartleyss1 also proposed that a P-D-galactosidase present in the apple wall (the activity of which increased in parallel to the loss of galactose from the wall) catalyzes hydrolysis of the galactan after another enzyme, of (661) I. M. Bartley, Phytochemistry, 13 (1974) 2107-2111.

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unknown identity, has released the nonreducing ends of the galactan chains from unspecified linkages to other wall polymers. There may be an enzyme present in apples that detaches pectic galactan chains from xyloglucan, although no such activity has so far been demonstrated. The apple P-D-galactosidase possesses the ability to hydrolyze, in uitro, a galactan with /3-( 1-4) links, obtained from potato pectin.ee1 The loss of galactose from the apple wall was accompanied by a loss of arabinose, but to a lesser degree.6s2-6S2 After the removal of these residues early in the ripening processes, galacturonan in the wall decreased, and water-soluble galacturonan increased.s54 Knees54 postulated that cell separation probably depends upon the removal of low-ester galacturonan from the middle lamella by exo-galacturonanase, which has been shown to occur in apple parenchyma,s21 and which possesses the ability to liberate both galacturonan and galacturonic acid from applecortical, cell-wall preparations.e21 Apple tissue appears to contain no endo-galacturonanase activity.ee3 The continued incorporation of methyl groups from [14C]methionineinto poly(methy1 galacturonate) in the wall during ripeningss4 is probably due to synthesis, and insertion into the wall, of new poly(methy1 galacturonate) alongside the removal of de-esterified galacturonan. This suggests a dynamic turnover of the pectic backbone during ripening, as opposed to a simple, continuous loss of galacturonan. Knee and coworkers3Q4found certain comparable changes in the primary wall of strawberry parenchyma during ripening, although the strawberry wall appeared to differ from the apple wall in some respects. In unripe-strawberry walls, there are lower levels of arabinose, galactose, and xylose than in apple walls at the corresponding developmental stage, which may mean that there are fewer pectic side-chains available for cross-linkage of rhamnogalacturonan to cellulose microfibrils. In addition, galacturonan in strawberry walls at the unripe stage appears to be more readily extractable with aqueous extractants than that in the unripe-apple wall. Thus, in contrast to galacturonan in unripe-apple walls, which can only be released by destructive treatments (extraction in neutral, aqueous media containing EDTA at loo", or extraction with dilute alkali), more than half of the total galacturonan of unripe-strawberry walls is extractable by prolonged exposure at 20" to a neutral, aqueous medium containing EDTA. Thus, at least half of the galacturonan of unripe-strawberry walls is weakly bound, and is probably stabiIized in the wall by the presence ofcalcium ions, which interact with free carboxyl groups of galacturonan.3Q4 (662)I. M.Bartley, Phytochemisty, 15 (1976)625-626. (663)W.Pilnik and A. G . J. Voragen, in A. C. Hulme (Ed.), The Biochemkity ofFruits and Their Products, Vol. 1 , Academic Press, New York, 1970,pp. 53-75.

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Freely water-soluble (in the absence of EDTA) galacturonan in the strawberry wall increased greatly during ripening.394This solubilization may be due to the disruption of wall structure by galacturonanase activity,e64or to loss of the calcium-stabilized gel-structure due to increased methylation of g a l a c t u r ~ n a nor , ~both. ~ ~ It may be that, as in the apple, newly synthesized poly(methy1 galacturonate) replaces de-esterified galacturonan removed from the middle lamella by polygalacturonase activity during ripening, leading to cell separation. Associated with the solubilization of galacturonan (which is accompanied b y hydration and swelling of the wall matrix), arabinosyl, galactosyl, and rhamnosyl residues disappeared from the wall fraction, and increased in soluble fractions. This suggests that, as in the apple, degradation of pectic side-chains probably contributes to the solubilization of the rhamnogalacturonan by “disentangling” the latter from other wall polymers. Ahmed and L a b a v i t ~ h ~showed ~ ~ * ~ that, ~ ~in, ~ the~ ripening ~ pear, alongside increase in the level of galacturonanase activity, there is solubilization from the wall of a high-molecular-weight, branched arabinan [consisting of a backbone of a-( l-+5)-linked L-arabinosyl residues, some of which bear a-linked L-arabinosyl side-groups at 0 - 2 or 0-3, or both] covalently linked to galacturonan in an acid-soluble fraction. The branched arabinan appears to have a structure similar to that present in the pectic polymers of the primary wall of cultured s y c a m ~ r e - c e l l s . ~ ~ ~ The wall arabinan is not hydrolyzed to free arabinose. There is also solubilization of another acidic fraction, of lower molecular weight, containing galacturonan free from arabinosyl residues. Treatment of the unripe wall with purified endo-galacturonanase solubilized, from the wall, an acidic, branched arabinan with characteristics similar to those of the polymer solubilized during ripening.e50 There was also a small decrease in the galactose content of the wall during ripening. Ahmed and Labavitche41attempted to pinpoint the enzyme(s) responsible for removal of arabinose from the wall. They were unable to detect arabinanase activity, but there was a slight increase in the level of a - ~ arabinosidase, which appeared to be wall-bound, during ripening. However, these authors seriously challenged the concept that such exo-glycosidases can have a role in the degradation of wall polysaccharides, pointing out that the cellular role of these enzymes is open to speculation. They challenged Bartley’s conclusionee1 that /?-D-galactosidase present in homogenates of apple fruit is responsible for the large decrease in cell-wall galactose that accompanies apple ripening, citing (664) E. J. Cizis, Ph.D. Thesis, Oregon State University, 1964. (665) C. E. Neal,]. Food Sci. Agric., 16 (1965) 604-618.

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Wallner’s findingeee that apple p-D-galactosidase is not able to digest apple cell-walls. The level of P-D-galactosidase activity in pear tissue almost doubled during the course of ripening.s41 It is not clear how exo-glycosidases might act to disrupt cell walls. Even if a glycosidase acted as an exo-glycanase, it is unlikely that it could cleave beyond cell-wall, constituent branch-points, and thus remove interpolysaccharide linkages. Furthermore, the mixed glycosidase extracts containing a-L-arabinosidase obtained from pear tissue did not generate reducing sugar from a purified arabinanas41The most active glycosidases in the pear were a-D-galactosidase and a-D-mannosidase, and it is difficult to assign a role in cell-wall modification to these enzymes, because analysis of pear-fruit cell-walls gave no indication of a-linked galactans or mannans, and little change in wall mannose or galactose content occurred during pear ripening.e50 It is possible that glycosidase activities determined by incubation with p-nitrophenyl substrates give an inaccurate picture of in viuo enzyme-specificity (compare Pharr and coworkersee7). Ahmed and Labavitche41concluded that, at present, no role in ripening-associated, pear cell-wall modification should be assigned to glycosidases, and that the solubilization of both the rhamnogalacturonan backbone and the covalently attached, branched arabinan results from galacturonanase activity. However, pending further study, this conclusion should be treated with caution. If pectic side-chains, including the branched arabinan, are covalently linked to other wall polymers, such as, for example, a xyloglucan, as apple cell-wall analysis strongly suggests,e58 it is difficult to see how arabinan attached to galacturonan could be removed from the wall by galacturonanase activity alone, without the preceding activity of other enzyme(s) which detach the arabinan from linkage to polymers other than the rhamnogalacturonan. The pear wall contains a xyloglucan,ese similar in structure to the xyloglucan of cultured-sycamore ~ e l l - w a l land , ~ ~ap-(1-*4)-linked ~ - x y l a which n~~~ might be linked to pectic side-chains. Xylanase activity could not be detectedin pear fruit, although P-D-xylosidase activity, which increased during ripening, was present.e41Because the breaking of single glycosidic bonds might be significant in facilitating wall-polysaccharide dissociation and solubilization, the possible role of glycosidases in cell-wall metabolism should not be totally discounted until greater understanding of their catalytic activities is gained. (666) S. J. Wallner,J. Am. Soc. Hortic. Sci., 103 (1978) 364-373. (667) D . M. Pharr, H. N. Sox, and W. B. Nesbitt, J . Am. Soc. Hortic. Sci., 101 (1976) 397- 403. (608) A. E. R. Ahrned, Ph.D. Thesis, University of California, Davis, 1978. (669) S. K. Chanda, E. L. Hirst, and E. G . V. Perciva1,J. Chem. Sac., (1951) 1240- 1246.

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Wallner and associates4g9-501~e70 demonstrated that the sharp increase in tomato-galacturonanase activity during the “turning” stage of ripening is accompanied by an increase in the tomato pericarp-wall of a rhamnogalacturonan fraction that may be extracted from isolated walls by 4-h incubation with water at 30 ’. This water-soluble rhamnogalacturonan, which is not present in the cell walls of hard, unripe fruits, has a molecular weight of >20,000,and is almost free from neutral-sugar residues. These authors suggested that this soluble polymer was a product of galacturonanase activity against wall rhamnogalacturonan detached from neutral pectic side-chains. The increase in soluble rhamnogalacturonan is accompanied by an 18%decrease in the total galacturonic acid content of the wall during ripening. It is proposed that this conversion of high-molecular-weight rhamnogalacturonan into a readily water-soluble polymer of lower molecular weight, possibly by a two-stage mechanism with detachment of pectic side-chains preceding galacturonanase activity against the pectic backbone, makes a major contribution to loosening of the wall matrix, with resultant tissue-softening during tomato ripening. This conclusion is strengthened by these authors’ findings4gg-501~s70 that galacturonanase extracted from ripe tomatoes solubilized, from cell walls isolated from unripe tomatoes, a rhamnogalacturonan virtually identical to the watersoluble polymer produced in vivo during ripening, along with galactosyluronic oligosaccharides of a range of chain lengths. The nature of the products suggests endo-galacturonanase activity. It seems likely that, in the ripening fruit, the water-soluble rhamnogalacturonan fraction, which remains associated with the wall during wall isolation, is an intermediate product of endopolygalacturonase activity between the highmolecular-weight, strongly wall-bound, rhamnogalacturonan of the unripe wall and oligogalacturonates in the cytoplasm oftomato cells at an advanced stage in ripening. Apart from that of galacturonanase, significant levels of (1+3)-p-~glucanase and P-D-galactosidase are present in tomato tissue, and both activities increase during ripening,4ggbut Wallner and concluded that neither of these enzymes plays a role in tissue softening, as they have no activity against isolated, tomato cell-walls in uitro. Furthermore, tomato P-D-galactosidase did not degrade the purified b-(1-4)linked galactan obtained from tomato p e c t i c - p ~ l y m e r sThe . ~ ~ripening~ related, 40-60% decrease in wall galactose, and the more modest decline in wall arabinose, are clearly processes separate from rhamnogalacturonan solubilization as, in the nonsoftening, rin-mutant tomato, the post-harvest loss of these neutral sugars occurred in the total absence of galacturonanase activity and rhamnogalacturonan solubilization.501 (670) G. D. Lackey, K. C. Gross, and S. J. Wallner, Plant Physiol., 66 (1980) 532-533.

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However, in normal fruits, rhamnogalacturonan solubilization without the loss of these neutral sugars has not been d e m o n ~ t r a t e d The . ~ ~ ~enzyme(s) responsible for the removal of galactose and arabinose from the wall has not been identified. In subsequent work, Wallner and cow o r k e r demonstrated ~~~~ a diminished level of synthesis of new wall-galactan in ripening tissue, as compared to green, unripe tissues, in both senescing, normal fruits and detached, rin mutants. They postulated that, if the wall pectic-galactan undergoes metabolic turnover, lower levels of synthesis and re-insertion into the wall would account for the net loss of galactose from the wall. Turnover of wall polysaccharides and lessened incorporation of [14C]-labelledprecursors into wall polymers during ripening has been demonstrated in apple,054strawberry,3e4and grape.671 However, if decreased synthesis combined with metabolic turnover is responsible for galactan loss from the tomato wall during ripening, this still leaves open the question of the enzymes responsible for turnover of the galactan and its detachment from the rhamnogalacturonan. In conclusion, it seems clear that, in a wide variety of fruits, increased galacturonanase activity accompanying ripening is responsible for the removal, by solubilization, from the wall, of de-esterified rhamnogalacturonan, and that the resulting dissolution of the middle lamella makes a major contribution to tissue softening. Moreover, in a number of fruits, this solubilization appears to be preceded by loss of galactose and arabinose from the wall. However, specific galactanases and arabinanases responsible for these processes have not been located in fruits, and the enzymic mechanisms responsible for degradation of pectic galactans and arabinans in fruit cell-walls are at present unknown. (ii) Changes in Hemicelluloses. -A xyloglucan similar to the xyloglucan of cultured-sycamore ~ e l l - w a lhas l ~ ~been located in the wall of pear parenchyma,008and analysis of apple-fruit cell-wall strongly suggested the presence of a similar polymer.058 However, there is little evidence of hemicellulose degradation contributing to tissue softening during fruit ripening. In the pear, the wall content of xylose, noncellulosic glucose, mannose, and fucose remains stable during ripening,050and similar results have been obtained in the ripening tomato.501 Knee052 reported losses of wall hemicellulosic-glucan in apples during ripening, but, in a later publication, BartleyGB2reported no change in ripening apple-wall, noncellulosic glucose or xylose. Rolz and coworkerse47could find little change in the total hemicellulosic content of mango soft-tissue during ripening. However, free xylose has been detected in the flesh of ripening mangoes,072and the possibility that this arises as a degradation product of hemicellulosic xyloglucan or xylan cannot be discounted. (671) K. Saito and Z. Kasai, Plant Physiol., 62 (1978) 215-219. (672) K. P. Sankar, Sci. Cult., 29 (1963) 51-59.

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Leley and associates673suggested that mango cell-wall hemicelluloses may be degraded during the later stages of tissue softening, but, as this work did not incorporate detailed, cell-wall analyses, the suggestion is open to question. A water-soluble glucan containing chains of both a-(1-4)- and a-(1+3)-linked D-glucosyl residues, with branching points provided by a-( 1+6)-linked residues has been isolated from ripe-mango mesocarp, but whether or not this polymer is a cell-wall degradationproduct is 0bscure.~~~.67s It does not seem likely that the polymer could be derived from a xyloglucan hemicellulose; the xyloglucans of pearee8 and suspension-cultured sycamoreSe and beanS8cells appear to possess only a /I-(1-*4)-linked D-glucan backbone (see Section IV). A decline in the total hemicellulose content of grape-berry cell-walls during ripening has been reported,676 and Knee and coworkers394 claimed an increase in xylosyl, mannosyl, and glucosyl residues in soluble fractions of the strawberry wall during ripening, suggesting that hemicellulosic polysaccharides were being either degraded, or released from interpolymer bonds. No hemicellulose-degrading enzymes have been detected in fruit tissues. Both peare41 and tomato4gglack xylanase activity, although both contain j?-D-xylosidase and j?-D-glucosidase a c t i v i t i e ~ , ~ whereas ~~*~*~ peare41contains a-D-mannosidase, The improbability that such glycosidases are involved in the degradation of cell-wall polysaccharides has already been discussed. Tomato contains (1+3)-P-~-glucanase activity,499but the likely natural substrates for this enzyme, namely, mixed /I-D-glucans (see Section V), have not been shown to be present in the cell wall of tomato or any other fruit. The (1+3)-linked D-glucosyl residues present in the water-soluble polysaccharide isolated from ripe-mango mesocarp are considered to possess the a-anomeric configuration, and are thus unlikely to provide a substrate for (1+3)-/I-~-glucanaseactivity. Furthermore, it is not known if this mango polymer is derived from the wall, and neither a-nor /I-(1+3)-~-glucanaseactivity has been detected in the mango. The functions of the various, aforementioned enzyme-activities in ripening fruits thus remain obscure at present. (iii) Changes in Cellulose. -Cellulase activity has been detected in pear,e77 l y ~ h e e a, ~v ~o ~~ a d o , banana,677 ~ ~ ~ - ~ ~pineapple,e77 ~ plum,e77 See Ref. 547. A. Das and C. V. N. Rao, Tappi, 47 (1964) 339-345. A. Das and C. V. N . Rao, Aust. J . Chem., 18 (1965) 845-850. S.V. Baltaga and L. V. Yarotskaya, Izv. Akad. Nauk Mold. SSR,Ser. Biol. Khim. Nauk, 3 (1973) 39. (677) G . E. Hobson, Rep. Glasshouse Crops Res. Inst., (1967) 134- 136. (678) E. Pesis, Y. Fuchs, and G . Zauberman, Plant Physiol., 61 (1978) 416-419. (679) M. Awad and P. E. Young, Plant Physioi., 64 (1978) 306-308.

(673) (674) (675) (676)

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peach,677grape,677 marrow,680and oranges8' fruits. Moreover, the level of activity in the soft tissues increases at the climacteric (and continues to increase into over-ripeness) in ~ v o c ~and~ to-o ~ ~ ~ mat^.^^^ However, whether or not this cellulase activity contributes significantly to tissue softening during ripening is open to question. Although, in the peach, small, but distinct, changes in cellulosic-micelle size and in percentage of crystallinity during ripening were presented as evidence of a limited breakdown of cellulose,682there was little correlation between the level of cellulase activity and the extent of tissue softening in the tomato.683Furthermore, in the nonclimacteric, nonripening, rin-mutant tomato, the same post-harvest increase in cellulase activity occurred as occurred at the climacteric in normally ripening tomatoes. Exposure of the rin mutant to ethylene further increased the cellulase activity, but did not induce galacturonanase activity (which was totally absent) or induce tissue softening.644 The cellulose content of apple-parenchyma walls remained constant during ripening,662and Gross and WallnerSo1reported a slight increase in cellulose in the tomato-parenchyma wall during ripening. Although Arie and coworkersss7 reported degradation of cellulose microfibrils in pear, Ahmed and L a b a v i t ~ hin, ~a ~subsequent ~ report, claimed that pear-parenchyma wall-cellulose is stable throughout the period of ripening. Similar contradictory findings have been obtained in ripening mango, Leley and coworkers673reporting degradation of cellulose, but Rolz and cow o r k e r finding ~ ~ ~ ~no evidence of cellulose breakdown in the soft tissue. The balance of the evidence available suggests that the activity of endogenous cellulase in degrading, primary-wall microfibrils does not contribute significantly to fruit tissue-softening accompanying ripening. (iv) Changes in Cell-Wall Glycoprotein. -Little attention has been given to changes in wall glycoprotein during ripening. The results that have been published suggest that the changes are minimal, and make little contribution to tissue softening. Gross and WallnerS0' reported that wall-protein content is stable during tomato ripening. Kneees2noted that wall-glycoprotein content did not change during apple ripening, and, in a separate report,66e demonstrated that hydroxy-L-proline-rich glycoproteins, some of which were associated with galacturonan, were liberated from isolated apple-walls by protease treatment. The tetra-arabinosides covalently attached to the hydroxy-L-proline residues were only slowly degraded by a purified a-L-arabinofuranosidase. Susceptibility of (680)M.V.Tracey, Biochern. I., 47 (1950)431-433. (681)G.A. Rasmussen, Plant Physiol., 56 (1975)765-767. (682)C.Sterling,]. Food Sct., 26 (1961)95-98. (683)G.E.Hobson,J. Food Sct., 33 (1968)588-592.

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381

the hydroxy-L-proline-rich glycoprotein in the wall to attack by protease and arabinosidase did not change during ripening, but galacturonanase pretreatment of isolated walls led to increased release of hydroxy-L-prolyl residues by protease. These findings suggest that some degradation of the glycoprotein by proteolytic enzymes may be possible following solubilization of galacturonan from the wall by galacturonanase activity, but this could not be demonstrated in uivo. However, the amount of an unidentified hexosamine, probably associated with the glycoprotein, was less in walls prepared from ripe fruit than in those of unripe fruit.65e Knee and coworkers3Q4also reported that, in the strawberry, the synthesis of wall glycoprotein increased during ripening, and that incorporation of ~ - [ ~ ~ C j p r o linto i n e the glycoprotein continued into over-ripeness. It seems likely that, if proteolytic hydrolysis of wall glycoprotein does occur in ripening fruit, such activity comes after the glycoprotein has been detached from other wall polymers (such as galacturonan) by the action of other enzymes (such as galacturonanase) that have already initiated the process of tissue softening. Although the glycoprotein may well cross-link and stabilize polysaccharides in the unripe cell-wall, such cross-linking would not appear to be capable of protecting polysaccharides from degradation by polysaccharide-degradingenzymes. (v) Conclusions.-It has already been noted that, in most fruits that have been studied, it is probable that the major contribution to tissue softening during ripening is made by galacturonanase-catalyzed degradation of the pectic rhamnogalacturonan, with resultant dissolution of the middle lamella, allowing cell separation. If galacturonanase activity is genuinely absent from any fruit, an alternative mechanism must be considered, involving detachment of the rhamnogalacturonan from other polymers, particularly the pectic side-chains, by other enzymes, as yet undetected. Such detachment of rhamnogalacturonan from cross-linking polymers could facilitate its solubilization from the wall into the cell cytoplasm or intercellular fluid. Further elucidation of the mechanisms by which arabinose and galactose are removed from the pectic sidechains is crucial to advancing understanding of the means by which the pectic network is degraded. Certainly, degradation of the pectic polymers appears to be the primary process in tissue softening, with breakdown of the other wall-polymers (which is still largely obscure) probably secondary. Galacturonanase is, to date, the only enzyme that has been assigned a definite role in fruit ripening. However, other enzymes must surely be involved, if only to the extent of detaching the pectic backbone from cross-linking polymers, allowing galacturonanase to initiate rapid dissolution of the wall matrix. To solve these problems, it will probably be necessary first to gain greater knowledge of the structure of the intact-fruit, primary cell-wall,

382

PRAKASH M. DEY AND KEN BRINSON

and the exact nature of the linkages within it. Detailed characterization of the constituent polymers of the wall in a wide range of fruits at various ripening stages is needed, alongside more-exacting studies of the effects of purified, hydrolytic enzymes (extracted from fruits) on these components. In conducting these studies, the possibility that fruits of different species possess cell walls of different structure, and, therefore, utilize different, species-specific mechanisms for wall degradation should not be overlooked. Over-ardent espousal of the concept of a “general model” for cell-wall structure applicable to all fruits could seriously misdirect the course of research into the mechanisms of fruit softening, should such a general model ultimately prove not to apply. ACKNOWLEDGMENTS We thank Professor J. B. Pridham for his continual support and advice; K.B. is grateful to Tropical Products Institute, London, for a Research Training Award.

ADDENDUM There is evidences84*s85~es5a for the attachment of phenolic compo1+4)-linked ( D-galactose nents (ferulic and coumaric acids) both to /Iand a-(1+3)-linked L-arabinose in the primary cell-wall, suggesting feruloylation - coumaroylation of pectic neutral side-chains. Earlier papersees-sss had also suggested the attachment of these phenolic compounds to primary-wall polysaccharides which remained uncharacterized. Frysss has implicated a novel phenolic amino acid, for which the name isoditryrosine has been proposed, in providing inter-polypeptide crosslinks in plant cell-wall glycoproteins, such linkages contributing, perhaps, to glycoprotein insolubility. Neither of these topics is discussed in the present article.

(684) S. C. Fry, Pkznta, 157 (1983) 111-123. (685) S. C. Fry, Biochem. J , , 203 (1982) 493-504. (685a) M. M. Smith and R. D. Hartley. Carbohydr. Res., 118 (1983) 65-80. (686) P. J. Harris and R. D. Hartley, Nature, 259 (1976) 508-510. (687) S. C. Fry, Planta, 146 (1979) 343-351. (688) M. M. Smith and T. P. O’Brien, Aust. J . Plant Physiol., 6 (1979) 201 -219. (689) S. C. Fry, Biochm. J , , 204 (1982) 449-455.

ADVANCES I N CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL.

42

L-ARABINOSIDASES BYAKIRAKAJI" Faculty of Agriculture, Kagawa University, Kagawa 761-07,lapan

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Classification

383

2. Endo-L-arabinanase

IV. Endo-( I+S)-cu-L-arabinanase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Occurrence . . . .

3. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

392 393

I. INTRODUCTION In 1928, Ehrlich and Schubert' pointed out the L-arabinanase activity of Takadiastase. Kaji and coworkers2 isolated an L-arabinanase for the first time by zone electrophoresis in 1961. Since then, there has been much research on L-arabinosidases, although it has been delayed in comparison with that on other glycosidases. Studies on glycosidases have strongly emphasized those degrading starch and cellulose, so common in the plant kingdom, together with P-D-galactosidase, an enzyme of animal origin, which decomposes lactose. The quantities of L-arabinose and L-arabinan present in living tissues are relatively small, but L-arabinose residues are widely distributed in heteropolysaccharides and glycoconjugates, constituting one of the components of the middle lamella and cell wall of higher plants. There' Emeritus Professor. Present address: Fujitsuka-cho 3-9-32, Takamatsu 760, Japan. ( 1 ) F. Ehrlich and F. Schubert, Biochem. Z . , 203 (1928) 343-350. (2) A. Kaji, H . Taki, 0.Yoshihara, and A. Shimasaki, Kagawa Doigaku Nogakubu Cakutyutu Hokoku, 12 (1961) 265-268.

383

384

AKIRA KAJI

fore, research on L-arabinosidases is valuable in understanding the structures of these conjugates. Furthermore, it is of potential use in relation to conjugates of L-arabinose having specific physiological activities. A development that advanced this research occurred when Aspergillus niger was selected as an excellent enzyme-producer by Kaji and coworkers3 in 1963, and taking this opportunity, an L-arabinosidase that splits terminal groups was purified and crystallized. This result confirmed that a-L-arabinofuranosidase exists independent of P-D-galactosidase. The second powerful tool that has been developed during research on a-L-arabinofuranosidase is the synthesis of two model substrates, namely, phenyl a-L-arabinofuranoside and p-nitrophenyl a-L-arabinofuranoside. The third advance occurred when exo-type Larabinanase activity was found in various micro-organisms, and in a small number of plants, by many investigators. The fourth step is that research on L-arabinanases of Bacillus subtilis has advanced, and an endo-L-arabinanase has been purified from its culture filtrate.4 The present article describes the occurrence, assay, purification, and properties of L-arabinosidases, classified into exo- and endo-types. Unless otherwise noted, the arabinosides discussed are in the L-furanoid form. In 1976, Dekker and Richards5 reviewed L-arabinanases, among the hemicellulases, and Kajia introduced basic and applied research on Larabinosidases in 1981. Herein are described all known aspects of L-arabinosidases acquired thus far.

11. CLASSIFICATION Table I shows the L-arabinosidases as described in Enzyme Nomenclat ~ r e . ' *Some ~ of them hydrolyze from the nonreducing terminal of the substrate molecule, and some degrade the substrate at random sites. Glycosidases whose action is specific to glycosides of low molecular weight are usually classified as glycohydrolases or simply as glycosidases, and those specific to polysaccharides belong to the glycanohydrolases (glycanases). According to this division, L-arabinosidases are to be classified as follows. (3)A. Kaji, H.Taki, A. Shimasaki, and T. Shinkai, Kagawa Daigaku Nogakubu Gakuzyutu Hokoku, 15 (1963)34-39. (4)A. Kaji and T. Saheki, Bfochim. Biophys. Actu, 410 (1975)354-360. (5)R. F. H. Dekker and G . N . Richards, Adu. Carbohydr. Chem. Biochem., 32 (1976) 277-352;s e e pp. 279-292. (6)A. Kaji, Nippon Nhgei Kagaku Kaishi, 54 (1980)561-567. (7)Enzyme Nomenclature (1972), Elsevier, Amsterdam, 1973,p. 220. (8)Enzyme Nomenclature, Recommendations (1978), Supplement 2, Eur.J. Biochem., 116 (1981)423-435.

TABLE I Classification of Arabinosidaws

EC Number Reference 3.2.1.55

7

3.2.1.99

8

Name a-L-Arabinofuranosidase

Preferred substrate

Action pattern

a-L-arabinofuranosides, hydrolysis of (terminal) arabinans, arabinoxylans, nonreducing a-L-arabinoand arabinogalactans furanosyl groups Endo-(l-r5)a-~-arabinanase (1-6)-arabinan endohydrolysis of a - ~ (1+5)-arabinofuranosidic linkages

386

AKZRA KAJI

1. a - L -Arabinofuranosidase This L-arabinosidase acts on L-arabinosides of low molecular weight, such as the synthetic substrates already mentioned, and L-arabino-oligosaccharides. a. A. niger Type of cr.~-Arabinofuranosidase.~-~~Purified L-arabinsidase from A, niger not only releases the side-chain L-arabinosyl residues of L-arabinan, L-arabinoxylan, and L-arabinogalactan, but is also active towards simple synthetic substrates, and it decomposes > 90% of beet arabinan. These results showed that the enzyme also hydrolyzes (1-5)L-arabinan, but the initial rate of decomposition is lower, and the value of K , is larger than those for beet arabinan; consequently, (1-+5)-~-arabinan is not its best substrate. Similar results were also reported for the a-L-arabinofuranosidase from Corticium rolj.sii.12 a-L-Arabinofuranosidases from other sources have been reported by many investigators. They may have the same properties as the arabinanases ofA. niger and C. rolfsii, although it is difficult to reach a definite conclusion, as details of their action on (1+5)-~-arabinanhave not yet been reported. b. Streptomyces purpuruscens Type of a-~-Arabinofuranosidase.'~a-L-Arabinofuranosidase from S. purpuruscens IF0 3389 acts on such low-molecular-weight L-arabinosides as p-nitrophenyl a-L-arabinofuranoside and L-arabino-oligosaccharides,but does not act on L-arabinan, L-arabinoxylan, or L-arabinogalactan. From the genera1 classification of glycosidases, it is a typical a-L-arabinofuranosidase. 2. Endo-~-arabinanase~

As shown in Table I, this enzyme causes random hydrolysis of L-arabinan. 111. a-L-ARABINOFURANOSIDASE 1. Occurrence A number of organisms, including fungi, bacteria, actinomycetes, protozoa, and plant^,^ release L-arabinose from L-arabinose-containing polysaccharides or from simple substrates, but it is difficult, strictly speaking, to conclude that a-L-arabinofuranosidase is produced by them. Reports of work in which this enzyme was highly purified and its enzymic proper(9) (10) (11) (12) (13)

A . Kaji, K. Tagawa, and K. Matsubara, Agric. Bfol. Chem., 31 (1967) 1023-1028. A . Kaji and K. Tagawa, Biochim. Eiophys. Acta, 207 (1970) 456-464. K. Tagawa and A. Kaji, Carbohydr. Res., 1 1 (1969) 293-301. A. Kaji and 0. Yoshihara, Biochim. Biophys. Acta, 250 (1971) 367-371. K. Komae, A. Kaji, and M. Sato, Agric. B i d . Chem., 46 (1982) 1899-1905.

L-ARABINOSIDASES

387

ties were investigated in detail are relatively few, except for the following organisms: A. niger, C.rolfsii, Rhodotorula Java, s. purpurascens, Streptomyces massasporeus, B. subtilis, and Scopolia japonica. There have been reports of L-arabinosidase activity in various plants. The presence of a-L-arabinofuranosidase was confirmed in callus cultivation of S. japonica, and in an extract of germinating seeds of Lupinus luteus. Organisms producing exo-type a-L-arabinosidases are shown in Table 11. TABLE I1 p H Optima of Some Plant and Microbial a - L -Arabinofuranosidases Origin Plant Scopolia japonica Lupinus luteus Microbial Aspergillus niger Botytis cinerea Sclerotinia libertiana Gloeosporium kaki Corticium rolfsii Coniophora cerebella Lentinus lepideus Trametes versicolor Poria vaporaria Oxyporus populinus Piptoporus betulinus Flammulina velutipes Lentinus edodes Agaricus campestris Botytis fabae

pH optimum 4.8

{::: 3.8-4.0 3.0 3.0 4.0-6.0 2.5 3.0 4.0 3.75 2.5 3.0 3.25 4.5 2.5 5.0 3.8-4.8

References 14 15 3,9,10,16 17,18 17 17 12.19,20 21 21 21 21 21 21 21 21 21 22 (continued)

(14) M. Tanakaand T. Uchida, Biochim. Biophys. Acta, 522 (1978) 531-540. (15) N. K. Matheson and H.S. Saini, Carbohydr. Res., 57 (1977) 103-116. (16) A. Kaji, K. Tagawa, and T. Ichimi, Biochim. Biophys. Acta, 171 (1969) 186- 188. (17) A. Kaji, K. Tagawa, and K. Motoyama, Nippon Nbgei Kagaku Kaishi, 39 (1965) 352 357. (18) R. J. W. Byrde and A. H. Fielding, Nature, 205 (1965) 390-391. (19) A. Kaji and 0. Yoshihara, Appl. Microbiol., 17 (1969) 910-913. (20) A. Kaji and 0. Yoshihara, Agric. Biol. Chem., 34 (1970) 1249-1253. (21) G. Butschak, W. Forster, and A. Gr&, Z. Allg. Mikrobiol., 16 (1976) 507-519. (22) A. Fuchs, J. A. Jobsen, and W. M. Wouts, Nature, 206 (1965) 714-715.

AKIRA KAJI

388

TABLE I1 (Continued) Origin

pH optimum

Clomerella cingulata Sclerotinia sclerotiorum

4.8 3.6-5.8

References

~~~~

22 22 23

Sclerotinia fructigena Myrothecium venvcaria Rhodotomla ~ Q V U Clostridium felsineum Bacillus subtilis Streptom yces massasporeus Streptomyces purpurascens

4.0 2.0 5.6 6.5 5.0 6.5

21 24,25 26 27 28 13

2. Assay

To determine enzymic activity, p-nitrophenyl a-L-arabinofuranoside,eephenyl a-~-arabinofuranoside,~~*~~ and beet L-arabinan are used as substrates. Most of the a-L-arabinofuranosidasesso far reported act on each of these three substrates, but there are some enzymes that act exclusively on either the low-molecular-weight or high-molecularweight substrates. When p-nitrophenyl a-L-arabinofuranoside is used, the amount of pnitrophenol released is assayed by measuring the absorption at 400 nm. When phenyl a-L-arabinofuranoside or beet L-arabinan is used, the amount of L-arabinose produced is measured by the Nelson - Somogyi meth~d.~~,~~ In either case, the amount of enzyme that releases one pmol of L-arabinose in one minute under standard conditions is defined as one unit. (23) F. Laborda, A. H. Fielding, and R.J. W. Byrde, J . Gen. Microbiol., 79 (1973) 321329. (24) E. Uesaka, M. Sato, M. Raiju, and A. Kaji,]. Bacteriol., 133 (1978) 1073-1077. (25) I. Kusakabe, T. Yasui, and T. Kobayashi, Nippon Nhgei Kagaku Kaishi, 49 (1975) 295-305. (26) A. Kaji, Y. Anabuki, H. Taki, Y. Oyama, and T. Okada, Kagawa Daigaku Nogakubu Cakuzyutu Hokoku, 15 (1963) 40-44. (27) L. Weinstein and P. Albersheim, Plant Physiol., 63 (1979) 425-432. (28) A. Kaji, M. Sato, 0.Yoshihara, and A. Adachi, Kagawo Daigaku Nogakubu Gakutyutu Hokoku, 34 (1982) 79-85. (29) A. H. Fielding and L. Hough, Carbohydr. Res., 1 (1965) 327-329. (30) H. Bbrjeson, P. Jerkeman, and B. Lindberg, Acta Chem. Scand., 17 (1963) 17051708. (31) S. Sadeh and U. Lehavi, Carbohydr. Res., 101 (1982) 152-154. (32) N. Nelson,]. Biol. Chem., 153 (1944) 375-380. (33) M. Somogyi,]. Biol. Chem., 160 (1945) 61-68; 195 (1952) 19-23.

L-ARABINOSIDASES

389

3. Purification When micro-organisms are used as the enzyme source, the culture medium must contain L-arabinan or L-arabinose. In A. niger, L-arabinose, L-arabinitol, and L-arabinan are inducers of this enzyme.34 Because a-L-arabinofuranosidase is an extracellular enzyme, a crude preparation may be made simply by fractionation of the culture filtrate with ammonium sulfate. The enzyme can be purified from the crude enzyme-preparation by some suitable combination of ion-exchange chromatography, gel filtration, and similar techniques. Three examples of purification procedures, two from micro-organisms and one from a plant, are given here. a. a-L-Arabinofuranosidase from C.rolfsii.le-This enzyme is readily purified, because it is extremely stable over a wide range ofpH. It may be purified by use of ammonium sulfate, DEAE-Sephadex A-50, SE-Sephadex C-50, Sephadex G-200, and QAE-Sephadex A-50. The enzyme thus purified was demonstrated to be homogeneous by disc electrophoresis, and its specific activity had been increased 67-fold. b. a-L-Arabinofuranosidase from S. purpura~cens.'~- This enzyme was purified from the culture filtrate to a homogeneous protein by salting-out with ammonium sulfate, column chromatography on DEAE-cellulose, QAE-Sephadex A-50, and hydroxylapatite, and gel filtration on Sepharose 6B, giving a purification of 120-fold. In the chromatography on DEAE-cellulose at pH 7.5, the L-arabinosidases are eluted in three peaks. The enzymes of two peaks showed the same substrate specificity as the a-L-arabinofuranosidasesfrom A. niger and C . rolfsii, but the L-arabinosidase in the third peak differed in size specificity. This enzyme was purified, and it proved to act exclusively on substrates of low molecular weight. c. a-L-Arabinofuranosidase from S. japonica."- Calluses were cultured in suspension, the culture medium was concentrated, and a crude, enzyme solution was obtained from the medium by ultrafiltration. The crude, enzyme solution was purified 163-fold by means of ammonium sulfate, Sephadex G-150, DEAE-Sephadex A-50, and isoelectric focusing. 4. Properties

a. Effect of pH on Activity and Stability of a-L-Arabinofuranosidase. -As may be seen in Table 11, many reports show pH optima on the acidic (34) K. Tagawa and G . Terui, J. Ferment. Technol., 46 (1968) 693-700.

390

AKLRA KAJI

side; in particular, there are obtainable, from the fungi belonging to the Basidiomycetes, many enzymes that are active at extremely low pH values. The enzyme of C. rolfsii shows high activity even12 at pH 1.1. The enzyme ofA. niger shows high stability35over a p H range of 1.5 to 9.0,and that of C. rolfsii in a pH range12 of 1.5 to 10.0.The enzyme from R. Juua still retains 82% of its activity after being incubatede4 at p H 1.5 for 24 h at 30".

b. Specificity. -The rates of hydrolysis of various substrates by a-Larabinofuranosidase are shown in Table 111. A remarkable point regarding their glycan specificity is that they are exclusively active on the L-arabinofuranosidic linkages. In 1960, Wallenfels and coworkers had found that thep-D-galactosidase of Escherichia coli ML 309 is active on the 0- and p-nitrophenyl a-L-arabinopyranosides. Because of this, there was a time when a-L-arabinofuranosidase was not considered to be an independent enzyme. However, as aresult of substrate-specificity studies using A. niger K1,Kaji andTagawa'O demonstrated that a-L-arabinofuranosidase is different from p-D-galactosidase. As shown in Table IV, the K , value for the reaction of the purified enzyme on phenyl or p-nitrophenyl a-L-arabinofuranosides is small, and the value for that on beet L-arabinan is much smaller than that on (1+5)L-arabinan. TABLE 111 Hydrolysis of Various Substrates by a-L-Arabinofuranosidase Rate of hydrolysisn

S. mamaSubstrate

A. niger"

Phenyl a-L-arabinofuranoside p-Nitrophenyl a-L-arabinofuranoside p-Nitrophenyl a-D-galactopyranoside p-Nitrophenyl/3-D-galactopyranoside p-Nitrophenyl cr-L-arabinopyranoside L-Arabinan (beet) (1-+5)-~-Arabinan L- Arabinoxylan L- Arabinogalactan Gum arabic

282.0

a

0 36.0

C. r01fsii1e*19 R. $a0ae4

124.0

53.0 16.7

sporeuses

S. purpurascenP

1.81

71.5

49.8

0

0

0

0

10.4 3.6 4.9

-

0.83 0.45 0.19 0.41 0

Rates of hydrolysis are given in pmol of arabinose produced per minute per mg of protein.

(35) 0. Yoshihara and A. Kaji, Abstr. Int. Ferment. Symp., 4th, (Kyoto), (1972) p. 241.

0

L-ARABINOSIDASES

391

TABLE IV Properties of a-L-Arabinofuranosidase Mol. wt.

pZ

e

References

A. niger

53,000

3.6

10,16

C . rolfsii

-

-

-

5.3

4.86 mM (PhAraf) 0.26 g/L (BA) 2.86 mM (PhAraf) 8.47 g/L (BA) 28.6 g/L (1,s-A) 9.1 mM (PhAraf) 1.67 mM (p-NPhAraf) 6.7 mM (p-NPhAraf) 0.082 mM (p-NPhAraf)

Enzyme from

R. Paon B. subtilis S. massasporeus S. juponica S. purpurascens

65,000 54,000 62,000 495,000

-

8.0 3.9

12 24 27 28 14 13

Key: PhAraf. phenyl a-L-arabinofuranoside; BA, beet arabinan; 1,5-A, (1+5)-arabinan; p-NPhAraf. p-nitrophenyl a-L-arabinofuranoside. (I

Many of the enzymes tested had a molecular weight of less than 100,000. That of the S . purpurascens enzyrnel3 was 495,000, and those of the Sclerotinia fructigena enzymesz3were 200,000 and 350,000. c. Enzymic Reactions.-As may be seen from the values shown in Tables I11 and IV, a-L-arabinofuranosidase hydrolyzes (nonreducing) terminal L-arabinosyl groups. When beet L-arabinan is used as the substrate, such side chains are quickly excised by the purified enzyme from A. niger K1, and a hydrolysis of >90% is attained." The side-chain L-arabinosyl groups of wheat-flour L-arabino-D-xylan are almost completely split off by the purified a-L-arabinofuranosidase" from A. niger K1. Similar results were reported36for an enzyme preparation from Pectinol R-1 0. In contrast, the a-L-arabinofuranosidase from Pectinol 59-L hydrolyzes only 18% of the L-arabinosidic linkages of wheat ~ - a r a b i n o - ~ - x y l a n . ~ ~ Terminal L-arabinosidic linkages in L-arabinose conjugates are also hydrolyzed by the enzyme. The enzyme of R. Java releases L-arabinose from the polysaccharide of the water shield (Brasenia schreberi J. F. Gmel)3s and from the cotyledon of Tora bean (Phaseolus v u l g ~ r i s ) . ~ ~ Some 7 0 to 80%ofthe side chains of the arabinoxylan in rice cell-wall are composed of L-arabinose. When the a-L-arabinofuranosidase from R. (36) H. Neukom, L. Providoli, H. Gremli, and P. A. Hui, Cereal Chem., 44 (1967) 238244. (37) K. A. Andrewartha, D. R. Phillips, and B. A. Stone, Carbohydr. Res., 77 (1979) 191-204. (38) M. Kakuta and A. Misaki, Agric. Biol. Chem., 43 (1979) 1269-1276. (39) K. Ohtani and A. Misaki, Agric. Biol. Chem., 44 (1980) 2029-2038.

392

AKIRA KAJI

jlava acted upon this polysaccharide, enzymic action on up to 20% of the

arabinosyl groups of its side chains was observed.40 According to investigations made by Graffi and coworker^,^'*^^^^^ when P-peltatin A ([1,2,3,4-tetrahydro-2-(hydroxymethyl)-6,7-(methylenedioxy)-4-(3,4,5-trimethoxyphenyl)naphthalene-3-carboxylic 3,2llactonel-8-yl) a-L-arabinofuranoside (1) is injected43into a mouse with

M e O v O M e -Peltatin A

(I

Me0 -L-arabinofuranoside 1

ascites sarcoma MV 276A, followed by a-L-arabinofuranosidase from A. niger K1, the tumor tissue releases L-arabinose at pH 6.5-6.8. In this way, the antitumor effect of P-peltatin A is activated. The L-arabinose residue of gum arabic is split by a-L-arabinofuranosidase from A. niger to an extremely limited extent, that is," 5%.

Iv. ENDO-(1-'5)-a-L-ARABINANASE 1. Occurrence

Endo-L-arabinanase activity was found for the first time2e in the culture of Clostridiumfelsineum var. sikokianum in 1963. Then, in 1975, it was found that B. subtilis F-11 produces this enzyme well,4and, in 1982, that B. subtilis IF0 3022 is also a producer.44 2. Purification

From a culture filtrate of 23. subtilis strain F-11, Kaji and Saheki4 puri-

fied endo-L-arabinanase to a homogeneous protein by hydroxylapatite (40) N. Shibuya, personal communication. (41) G. Butschak, G. Sydow, A. Graffi, E. Pehl, andH. Sydow, Arch. Geschwulstforsch..46 (1976) 365-375. (42) B. Tschiersch, K. Schwabe, G. Sydow, and A. Graffi, Cancer Treat. Rep., 61 (1977) 1489-1492. (43) K. Schwabe, A. Graffi. and B. Tschiersch, Carbohydr. Res.. 48 (1976) 277-281. (44) 0. Yoshihara and A. Kaji, Agkc. Btol. Chem., 47 (1983) 1935-1940.

L-ARABINOSIDASES

393

TABLE V Purification of Endo-arabinanase from B. subtilis I F 0 3022 ~

Step

Volume (mL)

Total protein (md

Total activity' (units)

(NH,)*SO, CM-Sephadex C-50 Ultrafiltration (I)b Hydroxylapatite Ultrafiltration (1I)b Sepharose 6B

400 430 38 176 14 40

4935 86 52 4.4 2.5 1.8

236 52 44 30 17 14

Yield

("/.I 100

22.2 18.6 12.7 7.2 5.9

Specific activity (units/mg) 0.048 0.60 0.85 6.82 6.80 7.78

(145)-Arabinan was used as substrate in the enzyme assay. Didlter G-O1T was used.

chromatography and Sepharose 6B filtration; however, the yield was very low. In 1978, Weinstein and A l b e r ~ h e i mpurified ~~ this endo-L-arabinanase in a higher yield from the same strain. The purification of B. subtilis IF0 3022 endo-L-arabinanase is ~ u r n m a r i z e din~Table ~ V. Two liters of culture filtrate, with endo-L-arabinanase and exo-type L-arabinosidases, were used for purification. As shown in Table V, the end0-Larabinanase was purified 162-fold. For enzyme assay, (1+5)-~-arabinanis the best ~ u b s t r a t eReducing .~ groups produced after enzymic action are determined by the NelsonSomogyi method. One unit of the enzyme is the amount that liberates one pmol of reducing groups from (1+5)-~-arabinanper minute at 30".

3. Properties

The properties of endo-L-arabinan from B.subtilis F-1 1 and IF0 3022 are shown in Table VI. When this enzyme acts on (1--*5)-~-arabinan,arabino-oligosaccharides are produced in the initial stage of the reaction. The end products are L-arabinobiose and L-arabinose. When the strain F-11enzyme acts on (l+S)-~-arabinan,the extent of decomposition is4 23.3%, whereas, that of beet L-arabinan is only 3.3%. This enzyme is inactive towards phenyl and p-nitrophenyl a-L-arabinofuranoside, arabinoxylan, arabinogalactan, and gum arabic. These results led to the conclusion that endo-L-arabinanase preferentially cleaves 5-linked arabinosyl residues. The action of endo-L-arabinanase on L-arabinan produces arabino-oligosaccharides, from which L-arabinotriose was isolated.27When acting upon cell walls obtained by sycamore-cell cultivation, this enzyme releases L-arabinan.27

394

AKIRA KAJI TABLE VI Properties of Endo-arabinanase from B. subtilis F-11 and I F 0 3022 Property Optimum pH Mol. wt. PI Substrate specificity (1+5)-arabinan beet arabinan p-nitrophenyl a-L-arabinofuranoside arabinose-conjugated polysaccharides potato disc (macerating activity) sycamore cell-wall ~~

ORefs. 4 and 27. Ref. 44.

~~

F-11"

I F 0 302Zb

6.0 32,000 9.3

6.0 33,000 7.9, 9.7

+ +

+ +

-

+

-

+

AUTHOR INDEX Numbers in parentheses are footnote reference numbers and indicate that an author’s work is referred t o although his name is not cited in t h e text. A Abbott, D. L., 345, 348(448) Abdul-Baki, A,, 267, 350(7), 356(6, 7) Abe, Y., 117 Abuaan, M. M., 107, 108(298), 133(298) Aburaki, S., 129 Achmatowicz, O., Jr., 177, 183(84) Ackermann, D., 73(71), 76 Acree, T. E., 22, 64(36), 68 Acton, E. M., 132 Adachi, A., 388, 391(28) Adams, D. 0.. 343,364 Adams, G. A,, 272 Adams, J. B., 273 Adams, P. A., 349, 357(473) Adley, T. J., 138 Adomako, D., 272 Ahluwalia, R., 25, 57(61), 58(61), 59 Ahmad, H. I., 107, 108(298), 122, 133(298) Ahmed, A. E. R., 321, 347, 372(452), 374, 376(641), 378(650, 668), 379(641, 668), 380 Ahmed, E. M., 372, 376(650) Ajisaka, K., 133 Akamine, E. K., 339 Akimoto, K., 110 Akiyama, Y., 298 Albers-Schonberg, G., 116 Albersheim, P., 266, 267(5), 271(5, lo), 272, 273(55, 57). 274(55, 56, 57, 58, 59, 62, 63, 107), 275(55, 56, 57, 58, 59.61, 62, 63). 276(55, 56, 62,65, 120), 277(55, 56, 63, 64, 125), 278(55, 65, 125). 279, 280(55, 62, 65, 125), 281(62, 125), 282(55, 63, 125, 132). 283(55, 125, 132, 163). 284(55), 286, 287(56, 60, 62, 65), 288(56, 58, 65), 289(56, 65, 120, 189), 290, 291(56, 58, 60, 65). 294(60), 296(56, 59), 298(55), 299(57), 300(60), 301, 302(56), 303(57),304(10, 57, 65). 305(55, 65, 125). 306(10, 55, 56, 57, 59), 307(56, 57, 61, 120). 309(57, 65).

310(56, 57, 59, 264). 311(57), 312(57), 314(56, 57, 59, 61), 317(56), 321(55,64,65),322, 329, 330(361), 331(361), 337, 338(56, 57, 58, 59), 348(64), 349, 351(237), 352(321), 355(10,57), 356(64), 357, 358, 368(55, 56, 57, 62, 65), 369(55, 56, 57.62, 65). 373(55,56, 57, 65), 376(57), 378(57), 379(56, 58), 388, 391(27), 393,394(27) Albi, M. A., 344 Albrecht, H. P., 80, 90(116), 261 Albright, J. D., 232, 233(35), 243(35) Alex, R. H., 93 Alexeev, Yu, E., 80, 91 Alexeeva, V. G., 91 Alfoldi, J., 36, 65(93) Allard, P., 230, 232(13), 240(13), 243(13), 263(13) Allen, A. K., 308 Allerhand, A,, 18, 19, 34(19), 62, 64(17, 19), 66(11), 202, 203(37), 204(37) Allinger, N. L., 30,85 Allsopp, A,, 268, 272(12) Al-Najafi, T., 367 Alonso, R., 310 Altona, C., 27 Amrhein, N., 367 Anabuki, Y., 388, 392(26) Anderle, D., 77 Anderson, B., 273 Anderson, J. S., 323, 327(327) Anderson, L., 22, 32(37), 64(37, 39), 120

Anderson, R. C., 95, 106, 110 Anderson, R. L., 287 Andrewartha, K. A., 391 Andrews, G. C., 23, 39(40), 40, 66(40), 68(40) Anet, E. F. L. J., 29 Angustine, R. L., 231 Angyal, S. J., 16, 18, 19(9, 15, 16), 20(16), 21, 23, 25, 26(10, 15, 59), 27(15), 28(10, 16). 29(15), 31(16, 23, 31, 63). 32, 33(16, 23, 31), 35, 36(9, 23, 92), 38(15, 16), 40, 44(15,

395

396

AUTHOR INDEX

88),45(3, 72). 46(15, 116), 52(31), 55(9, 91), 57(61, 72), 58(61), 59(81), 60, 62(15), 64(31, 72, 88),65(15, 16, 31, 92), 66(15, 16, 31, 92), 68(10), 85 Anisuzzman, A. K. M., 177 Anthonsen, T., 76 Antonakis, K., 110, 157, 228, 230(3), 231, 232(3, 7). 233, 237, 238(26, 27, 29, 30), 239(30, 48), 241(40, 42). 242(15, 28, 29, 31), 243(15), 244(42, 50), 245, 246(26, 28, 29, 31), 247(26, 27, 28, 29, 32, 33), 248(31, 51), 249(26, 27, 30), 250(3, 26, 29, 31, 50), 251(30, 31, 42, 48, 51), 252(15, 31, 56), 254(57), 255(30, 52, 53). 256(31, 52), 257(50), 258(50), 260(42, 50), 262(10, ll),263(10, 11, 12, 14, 15), 264(42, 75) Aoman, P., 283, 284(166) Ara, M., 116 Araki, Y.,106, 107 Arbatsky, N. P., 209, 218(56) Arcamone, F., 72(24), 74 Archer, S.A., 372 Argoudelis, A. D., 72(15), 74 Argoullon, J.-M., 233, 241(42), 244(42), 251(42), 260(42), 263, 264(42, 75) Arie, R. B., 372, 380 Arison, B. H., 116 Armour, M.-A., 139, 140(23), 141(23), 141(23, 24), 149, 153, 157(46), 158, 161(55, 65), 165(65), 168(23, 46, 55, 65), 169(23, 55, 65), 179(67), 180(67), 181, 184(46, 55, 65, 67), 187(67), 188(89), 191(23, 65, 67.89) Arnarp, J., 219, 220(79) Arvor, M.-J., 230, 232(7) Arvor-Egron, M.-J., 241, 248(51), 251(51), 254(57) Asai, M., 73(57), 75 Asboe-Hansen, G., 275 Ashby, E. C., 90 Ashenbach, H., 73(59), 75 Ashton, K., 367 Asmus, A., 351 Aspinall, G. O., 269, 278, 280(138, 139, 141, 142), 281, 283(139, 148). 284(167, 168), 285(167), 287, 288(171, 180), 289(180), 292

Atalla, R. H., 196, 197(12), 197(12), 208( 12) Aubanell, J. C. H., 72(18), 74 Aujard, C., 230, 263(12) Avants, J. K., 347, 370,371(625, 629), 372(645) Avigad, G., 21, 38 Awad, M., 369, 371(615), 379, 380(679) Awerbouch, O., 187 Axelos, M., 328 Azuma, I., 47, 67(120a) B Babine, R.E., 121 Bach, J., 230, 242(15), 243(15), 244, 252(15, 56), 263(15) Backinowsky, L. V., 206, 207(47), 211, 2 12(47) Bacloud, R., 110 Bacon, B. E., 40 Bacon, J. S. D., 281 Baczynskyj, L., 38 Baer, E., 188 Baer, H. H., 97, 99, 107, 108(293), 146 Bahl, 0. P., 288, 289(188) Bailey, R. W., 271, 273(49, 52), 307(49, 50, 106). 309(49, 50, 51, 106), 310(50, 106, 265, 266), 311(49, 50), 312(49), 313(49), 314(49, 50, 266), 322 Bairamova, N . I?.., 206, 211(46), 221(46), 223(46) Baker, D. A., 80,81, 82, 90(117, 126), 92, 93, 232, 251(38) Baker, D. B., 267, 350(8), 356(484) Balan, N. F., 206, 204(47), 212(47) Ballou, C. E., 276, 325 Baltaga, S. V.,379 Balza, F., 21 1 Bambach, G., 109 Banaszek, A., 113 Bandurski, R. S., 285, 293, 294(210), 301,337(210, 242), 351 Bannister, B., 72(15), 74 Barata, L. E. S.,98, 119, 120(243) Barber, G. A., 316, 317, 318(293) Barbieri, W., 72(24), 74 Bardalaye, P.C., 269 Barford, A. D., 45 Barker, J., 366

AUTHOR INDEX Barker, R., 19, 20, 21(25), 26(20a), 31(20a), 32(80a), 36(20a), 37, 46(80a, 82). 65(82), 194, 200, 209(23), 210(23) Barker, W. G., 342, 348(411) Barklet, G. M., 350 Barnell, H. R., 365 Barnes, H. E., 269, 306(30), 307(30), 315(30) Barnett, N. M., 352 Barnoud, F., 272, 281, 283(157), 288, 292(89) Barras, N. J., 272 Barre-Sinoussi, F., 230, 232(13), 240(13). 243(13), 263(13) Barrett, A. J., 280, 305 Barthel, W. F., 189 Bartley, I. M., 370, 371(621), 373, 374(621,661), 375,378,380(662) Barton, D. H. R., 124 Bartsch, W., 82 Bassieux, D., 197, 198(17), 199(17) Bassiri, A,, 372 Bates, F.J., 17, 18, 32(5) Batra, K. K., 320 Bauer, H., 64 Bauer, W. D., 271, 272(55, 56, 57), 273(55, 57), 274(55, 56, 57), 275(55, 56, 57), 276(55,56), 277(55, 56), 278(55), 280(55), 282(55), 283(55), 284(55), 287(56), 288(56), 289(56), 291(56), 296(56), 298(55), 299(57), 302(56, 57), 303(57), 304(57), 305(55), 306(55,56, 57), 307(56, 57). 309(57). 310(56, 57, 264), 311(57, 264), 312(57, 264), 314(56,57, 264), 317(56), 321(55), 338(56, 57). 355(57), 368(55, 56, 57), 369(55, 56, 57), 373(55, 56, 57), 376(57), 378(57), 379(56) Bax, A., 202 Bayer, E., 188 Bayley, S. T., 353 Beady, C. A., 319 Becchi, M., 207 Beck,E., 72(9, l o ) , 74, 280, 281(151) Beevers, L., 328 Begbie, R., 269, 283, 284(168) Behr, D., 73(69), 76, 128(69) Behrens, N. H., 325 Bekker, A. R., 72(35), 75

397

Bell, R. P., 30, 31(77) Bell, T. A., 370, 371(630) Belli-Donini, M. L.,371 BeMiller, J. N., 110 Benazet, F., 73(64), 75 Benbow, J. A., 20 Benedict, R. G., 72(13), 74 Benezra, C., 187 Bentley, R., 22, 68(32) Ben-Yehoshus,S., 366 Benzing-Nguyen,L., 39 Berlin, Yu. A., 72(26), 74 Besford, R. T., 369 Bessel, E. M., 45 Bessodes, M., 232, 238(29, 39), 239(48), 242(29), 246(29), 247(29, 33), 250(29), 251(48), 255(53), 257 Bestmann, H. J., 129 Bethell, G. S., 18. 19(15, 16), 20(16), 26(15), 27(15), 28(16), 29(15), 31(16), 32, 33(16), 38(15, 16). 44(15), 46(15), 62(15), 65(15, 16), 66(15, 16) Bettelheim, F. A., 276, 277(131) Beveridge, R. J., 35, 55(91) Bhacca, N. S., 72(27), 74 Bhal, S. S., 339 Bhuyan, B. K., 73(51), 75 Biale, J. B., 340, 361(396), 362(396, 540, 542, 543), 363(395, 396, 540. 542), 364(540), 365(540,543), 366 Biemann, K., 173, 175(76), 176(76) Bilik, V.,36, 65(93), 200 Binkley, R. W., 105, 233, 236(43, 45), 245(43), 247(43), 253(44) Binkley, W. W., 233, 236(43), 245(43), 247(43), 253(44) Bischof, E., 79 Bischoherger, K., 93 Bishop, C.T., 43, 62(111), 280 Bishop, S. H., 202, 203(39), 204(39) Bjdrndal, H.,276 Black, J., 73(41), 75 Blackstock, W., 76, 81(84) Blackwell, J., 295 Blake, J. D., 273, 307, 315(117) Blanchard, J., 38 Bloom, H. L., 351, 377(500) Blumberg, K., 179 Blumberger, P., 49 Blumenkrantz, N., 275

398

AUTHOR INDEX

Boar, R. B., 124 Bobek, M., 181 Bock,K., 194, 202, 211, 212(67), 217, 218(76), 219, 220(79), 222, 223(67, 80), 224(32, 67) Bodkin, C. L., 55 Bohm, B. A., 115 Bohonos, N., 73(61), 75 Boigegrain, R.-A., 53 Boldeskul, I. E., 172 Bonner, J., 349, 350,356(482), 357(469), 366 Bordner, J., 40 Borenstein, B., 365 Borjeson, H., 388 Borud, A. M., 72(13), 74 Botlock, N., 22 Bottger, M., 341, 348(404) Boundy, J. A,, 272 Bourgeois, J.-M., 80, 90(123, 124), 92, 93(127, 189), 122(127, 128) Bowles, D. J., 273, 308(105), 332(257) Bradbury, J , H., 201, 202(30) Bradford, K. J . , 343 Brady, C. J., 364, 370 Bray, D., 324 Brazhnikova, M. G., 72(26, 34, 35), 74, 75 Breen, J. J., 165 Breitmaier, E., 65(175), 66, 133, 200, 211(24) Brett, C. T., 327 Brewer, C. F., 38, 40 Brewin, N. J., 367 Briggs, D. P., 272 Brimacombe, J. S.,51, 59, 78, 81, 93, 97, 107, 108(292, 298), 119, 120, 122, 123, 125, 126(141), 133(292, 298), 231, 261(19) Brink, A. J., 93, 132 Brinkmann, K. A., 358, 359(535) Brodbeck, U., 232, 233(25), 234(25), 237(25), 245(25), 247(25), 248(25), 249(25), 250(25), 252(25), 253(25) Brossmer, R., 42 Brower, D. L., 332, 333(367), 334(367), 335(367), 336(367) Brown, D. K., 81 Brown, E. G., 367 Brown, H . C., 122 Brown, R. G., 329 Brown, R. K., 45, 64(115c)

Brown, R. M., 332,334(369) Brownfain, D. S . , 232, 240(34) Brummond, D. A., 319 Bruneteau, M., 207 Bryan, W. H., 337 Brysk, M. M., 313 Buchala, A. J., 272, 292(74, 75, 81), 293(76, 80). 294(208, 209) Buchanan, J. G., 59, 60(165), 68(165) Buckler, S.A., 142, 155 Buddrus, J., 64 Budeginsky, M., 45, 85, 108(162) Bukhari, M. A., 179 Bukovac, M. J., 342, 348(415, 416) Bukowski, P., 177, 183 Bu’Lock, J. D., 76 Bundle, D. R., 211, 212(67), 222, 222(67, 80), 224(67) Bunnell, R. H., 365 Burg, E. A., 343, 348(425) Burg, S . P., 343, 348(425) Burke, D., 271, 272(60), 287(60), 291(60), 294(60), 300(60) Burks, M. L., 23 Burton, J. S.,78, 80(97), 82(97) Buss, D. H., 82 Bussolotti, D. L., 112 Butcher, R. W., 367 Butschak, G., 387, 388(21), 392(21) Butterworth, R. F., 231 Buys, H. R., 27 Buzzetti, F., 73(66), 76 Byers, R. E., 344, 348(435) Byrde, R. J. W., 387, 388, 391(23)

C Cabib, E., 325 Cairncross, I. M., 292 Caldogno, R. R., 349, 358(480) Callow, J. A., 307 Campbell, C. W., 339 Campbell, M. M., 47 Canas-Rodriguez, A., 280 Candia, J., 339 Cantor, S. M., 20 Capman, M.-L., 85 Cardon, A., 264 Carey, F. A,, 82 Caron, E. L., 72(32, 33), 73(36), 74, 75 Carpita, N. C., 272

AUTHOR INDEX

Carthy, B. J., 97, 101 Cary, L. W., 27, 43, 46(69a), 211, 212(62) Cass, C. E., 132 Casu, B., 194 Cause, C. F., 73(44), 75 Cerny, I., 47 cerny, M., 45, 47, 85, 108(162) Cetorelli, J. J., 272 Chalet, J.-M., 92, 93(189) Chalutz, E., 343 Chambat, G . , 281, 283(157) Chamberlin, A. R., 96 Chan, T. C., 187 Chanda, S . K.,376 Chany-Morel, E., 230, 263(12) Chapleur, Y.,95, 101 Charney, W., 73(41), 75 Charollais, E. J . , 9 3 Charpentie, Y.,73(64), 75 Chaykovsky, M., 130 Cheema, G. S., 339 Cheetham, N. W. H . , 23, 24(49), 200, 201(29) Chen, M., 137 Cherman, J.-C., 230, 232(13), 240(13), 243(13), 263(13) Cherniak, R., 42 Cherry, J. H., 348 Cheshire, M. V.,281 Chiba, T., 202 Chida, N., 116, 117 Chittenden, G. J. F., 89, 254 Chittenden, R. A., 142 Chizhov, 0. S., 96, 173, 176(77, 79) Chmielewski, M., 53 Chmurny, G. N., 23, 39(40), 66(40), 68(40) Cho, Y. P., 272, 299(71) Chodiewicz, W., 85 Chong, A. O., 98 Chouroulinkov, I., 230, 242( 15), 243( 15), 252(15), 262(10, l l ) , 263(10, 11, 14, 15) Chrispeels, M. J., 272, 299(71), 313, 322, 336 Christensen, J. H . , 342,348(413) Christiansen, G. S . , 350, 356(483) Christodowlou, A , , 342, 348(412) Chvapil, M.,352 Clardy, J., 155, 156(60), 157(59, 60). 165(59, 60), 191(60)

399

Clark, A. F., 317, 320, 327 Clark, E. L., 37 Clarke, A. E., 287 Claussen, U., 113 Clayton, C. J., 53 Clayton, J. D., 116 Cleland, R. E., 309, 341, 348(400), 349, 350(472), 352(400), 353, 354(485), 356(485), 357(471), 358 Cleland, W. W., 52,53(139) Cliff, B. L.,82, 90 Clode, D. M.,59, 60(165), 68(165) Coetzer, J., 93 Cohne, J. D., 358 Coleman, G. H.,45 Collins, G. C. S., 30 Collins, J. G., 201, 202(30) Collins, P. M., 59, 82, 107, 133, 254 Collins, W. B., 342, 348(411) Colombo, R., 349, 358(480) Colson, P., 205, 209(41) Coniglio, C. T., 73(41), 75 Conner, A. H., 22, 64(39) Conrad, H. E., 276 Conway, T. F., 202, 203(38), 204(38) Cook, A. F., 227, 232(2), 233(2), 245(2), 246(2), 247(2), 248(2), 249(2), 250(2), 252(2) Coombe, B. G . , 344, 345, 348(436, 444) Cooper, D. J., 73(43), 75 Cooper, F. P., 43, 62(111) Cooper, J. B., 354 Corbett, W . M., 109 Corcoran, J . W., 72(22), 74 Corey, E. J., 81, 96, 112, 130 Costello, P. R . , 293 Cottrell, J. W., 278, 281 Courtois, J. E., 287 Covey, T. R., 38 Cowley, D. E., 18, 19(16), 20(16), 28(16), 31(16), 33(16), 38(16), 65(16), 66(16) Cox, D. D., 211, 212(62) Coxon, B., 194, 195(3), 196(5), 202(3) Craig, J. W. T., 278, 280(141), 284, 287( 17l), 288( 171) Cram, D. J., 151 Crane, J. C., 342, 344, 345, 348(407, 433, 434,445) Crawford, J. K., 371

400

AUTHOR INDEX

Crawford,T. C., 23,39(40),66(40), 68(40) Cremer, D., 163, 183(69) Cronshaw, J., 295, 296(243), 297, 3 17(223) Curtius, H.-C., 22, 23 Cynkin, M. A , , 324, 327(334) Cyr, N., 27,211

D Dahmen, J., 73(69), 76, 128(69) Dais, P., 197 Daji, J. A., 363, 379(547),380(673) Daleo, G . R., 325, 326(338, 341), 327(338, 339, 341), 329(338, 341), 330(338, 341), 332(338, 341). 336(341) Dalhuizen, R., 301 Damielzaheh, A. B., 189 Dandliker, W. B., 310 Danford, M. D., 24 Daniels, D., 301, 337(243) Daniher, F. A., 49 Dankert, M. A., 324, 327(333), 329 Danishefsky, I., 276, 277(131) Darvill, A. G . , 271, 274(62), 275(62), 276(62, 65, 120). 277(125), 278(65, 125). 280(62, 65, 125), 281(62, 125), 282(125), 283(125), 285(119), 287(62, 65), 288(65), 289(65, 120). 291(65, 175), 292, 300, 302(65). 304(65), 305(65, 125), 307(65, 120), 309(65), 314(200), 321(65), 368(62, 65). 369(62, 65), 373(65) Darvill, J., 275, 276(120), 289(120), 307(120) Das, A., 379 Dashek, W. V., 336, 354 Dass, H. C., 342, 348(414) Datko, A. H.,351, 352(492) Datta, A., 348 Daub, J. P., 112 Dauwalder, M., 302, 331(249) Davidson, A. J., 150, 188(48) Davidson, E. A., 273 Davies, A. M. C., 271, 272(62) Davies, E., 351 Davies, J. N., 372 Davies, P. J., 31 1

Davison, B.E., 96 Day, W. R.,23, 24(49) Dea, I. C. M., 269, 306(30), 307(30), 315(30) De Ariola, M. C., 372,378(647), 380(647) Debono, M., 73(49), 75 Defaye, J., 64 DeJongh, D. C., 173, 175(76), 176(76) Dekker, C. A,, 232 Dekker, R. F. H., 384 Delbaere, L. T. J.. 217, 218(76) del Corral, J. M. M., 72(18), 74 Dell, A., 276 Delmer, D. P., 272, 294, 317(217),319, 321(217), 323(217), 327, 328, 330(217), 336(217), 338(217) Delpuech, J.-J., 68 Demailly, G . , 95 Dennison, R.-A,, 369, 372 DeOrtega, M., 365 Depazay, J.-C., 81, 128(142) Deplangue, R., 155 Derevitskaya, V. A., 209, 218(56) DeRosa, M., 76 DeRosa, S., 76 DeSaussure, V. A., 310 Deshpande, S., 365 Desonclois, J. F., 72(5), 74 De-Swardt, G. H., 364 Deuel, H., 273, 309 Dever, J. E., Jr., 285 DeVilliers, 0. G., 93, 132 de Wit, G . , 34 Dextraze, P., 96 DiCesare, P., 97 Dickinson, M. J., 82 Dietrich, C. O., 114 Dietz, A., 73(51), 75 Dilley, D. R., 364 Dills, W. L., Jr., 38 Dimler, R. J., 272 Dinh, T. H., 230, 232(13), 240(13), 243(13), 263(13) Dische, Z., 275 Dmitriev, B. A., 205, 206(45), 211(46), 212, 221(45, 46). 222(45), 223(46) Dmytrazenko, A., 129 Dobberstein. B., 333, 334. 335. 336 Dobler, M., 126, 127(388)

40 1

AUTHOR INDEX

Doddrell, D., 18, 66(11), 202, 203(37), 204(37) Doerr, N., 113 Doesburg, J. J., 372 Doleialovh, J., 45 Doner, L. W., 107, 108(292), 133(292) Dooley, K.,94 Dorland, L., 42 Dorman, D. E., 133, 196, 211(ll) Dostal, H. C., 344, 348(435) Dowler, M. J., 301, 337(243) Driver, G . E., 58 Drouet, A. G., 365 Duax, W. L., 183 Dubenage, A. J., 364 Dubost, M., 73(64), 75 Duckaussory, P., 97 Duff, R . B., 280, 281(150) Duke, C. C., 46 Dunfield, L. G . , 25, 27(56) Dureault, A., 81, 128(142) Dutette, P. L., 25 Durham, J., 116 Dute, R.R., 341, 348(403), 358, 359(403), 360 Dutton, G. G. S., 22, 272, 288, 292 Dwek, R. A,, 142 Dyer, J. R., 5 5 , 7 9 Dyong, I., 56.93.98, 110, 112, 114, 12l(244) E Earl, W. L., 68 Eberstein, K., 244 Eby, R.,211, 212(61) Eda, S., 284 Edelman, J., 301 Edward, J. T., 30 Egan, R.S . , 72(22), 74 Egan, S . V.,278 Egron, M.-J., 233, 241(42), 244(42), 245(41), 251(42), 260(42), 264(42) Ehrlich, F., 383 Einset, J. W., 22, 64(36) Eisenberg, F., 73(66), 76 Eisinger, W., 331, 332(365), 344, 359(437, 438) Elassar, G., 342, 348(405) Elbein, A. D., 317, 318(293), 327, 328

Elevers, J., 90 Elhafez, F. A. A., 151 Eliel, E. L., 85 Ellestad, G. A., 73(62), 75 El Mobdy, E. A., 272 El-Scherbiney, A., 109 Elvers, J., 103 Emerson, F. H., 344, 348(435) Emig, P., 133 Ernoto, S.,113 Engen, T., 272 Englard, S., 21, 38 English, A. R.,72(19), 74 English, P. D., 276, 280 Eppiger, E. N., 68 Epstein, M., 142, 155 Ericson, M. C., 272, 327, 328 Esipov, S. E., 72(26), 74 Eugster, C. H., 76, 81(84) Evans, E. M., 97 Evans, M. L., 301, 348, 349, 350, 357(474), 359 Evans, P. J., 329 Ezekiel, A. D., 80 F Fahraeus, G., 276 Fairweather, R. M., 283, 284(167), 285(167) Falbriard, J. G., 80 Fan, D. F., 351, 352(503) Fanshawe, R. S., 280 Farhoudi, E. O., 65(174), 66 Farmer, P. B., 230, 262 Farr, A. L., 275 Fartaczek, F., 327, 328(347) Faubl, H . , 23, 39(40), 66(40), 68(40) Feast, A. A. J., 80, 82(109) Feather, M. S., 138 Fedoroiiko, M., 20 Feingold, D. S., 322 Feliciano, A. S . , 72(18), 74 Fennessey, P., 324, 327(333) Ferrier, R.J.. 82, 111 Fielding, A. H., 372, 387, 388, 391(23) Filippi, J., 244, 252(56) Fincher, G . B., 287, 315 Finne, J., 276 Fiores, M. C., 365

402

AUTHOR INDEX

Fischer, E., 72(6), 74 Fischer, H. 0. L., 100 Fisher, M. L., 349 Fitzsimmons, B. J., 96, 101, 110 Flaherty, B., 79, 82(99), 84 Fleischer, D., 109 Fleming, H. P., 370, 371(630) Fletcher, A. P., 47, 49(119), 67(119) Fletcher, H. G., Jr., 16 Florent, J., 73(64), 75 Flores, M. C., 372, 378(647), 380(647) Flowers, H . M., 320 Folkers, K., 73(37), 75 Forbes, E. J., 97 Ford, C. W., 272 Forgacs, P., 72(5), 74 Forrest, I. S., 294 Forsberg, L. S . , 276 Forsee, W. T., 327, 328 Forster, W., 387, 388(21), 392(21) Foster, A. B., 45, 58, 97, 179 Fournier, L. B., 23 Franceschi, G., 72(24), 74 Frank, M., 351 Franke, F. P., 46 Franklin, M. J., 339, 356(482) Franks, F., 24 Franz, G., 272, 293(76), 319 Fraser, C. J., 294 Fraser-Reid, B., 94, 95, 96, 97, 101, 106, 112,121,125 Frazer, C. G., 272 Frazer, C. J., 292 Freeman, L. E., 277, 282(132), 283(132) Freeman, R.,202 Frenkel, C., 364,372, 380(657) Frenkiel, T. A,, 202 Freudenberg, K., 72(6), 74, 269 Frey-Wyssling, A,, 334 Friebolin, H., 42 Friedmann, M., 50 Friege, H . , 98, 114 Fronza, G., 48, 67(123) Fry, S . C., 343, 382 Fuccello, A., 179 Fuchs, A., 387, 388(22) Fuchs, Y., 343,379, 380(678) Fugati, C., 48, 67(123), 115 Fiigedi, P., 199, 205, 206(42), 207(42) Funabashi, M., 61, 80, 82(113), 90(113), 92, 93, 94, 100, 119. 120, 133(113, 191), 134(354)

Funaki, K., 123 Funcke, W., 19, 20(20), 23(20), 38(20), 65(20), 68 Furbringer, M., 72(9), 74 Furihata, K., 230,262(9) Furuta, S., 116

G Gadelle, A,, 64 Gagnaire, D. Y.,197, 198(17), 199(17), 216, 217(74) Gajdus, J., 28 Gal, G . , 150, 188(48) Gambacorta, A., 76 Gang, P. A., 189 Ganguly, A. K., 72(28, 29, 30), 73(29, 50, 63), 74, 75, 126 Gardiner, M. G., 336 Gardner, K. H., 295 Garegg, P. J., 45, 90, 119(178), 222, 223(80) Garminatti, H., 316 Garver, J. C., 22, 32(37), 64(37) Gasser, R., 84 Gast, J. C., 196, 197(12), 198(12), 208(12) Genghof, D. S., 356 George, W. 0.. 30 Georges, M., 121 Gero, S. D., 82, 90, 96, 133 Gestetner, B., 278, 280(142) Ghali, Y., 272 Giannattasio, M., 367 Gibbons, A. P., 319 Gibbs, N., 367 Giddings, T. H., 332, 333(367), 334(367), 335(367) Gielen, W., 42 Gilkes, N. R.,281, 282(lSS) Gillet, B., 68 Ginsburg, V., 316 Cizis, E. J., 375 Glamkowski, E. J., 150, 188(48) Glaser, C., 273, 308(104) Glaser, L., 316 Glick, H., 72(10), 74 Gligorijevib, M., 84,86,89(171), 133(159) Glittenberg, D., 56, 110. 114 Goerner, R. N., 132 Goeschl, J. D., 363 GOhring, K., 113 Goldman, L., 232, 233(35), 243(35)

AUTHOR INDEX Gololobov, Yu. G., 172 Gomyo, T., 189, 190(106, 107) Gonzalez, A., 8 2 Gonzalez, L., 9 3 Goodman, L., 53, 133, 232 Goodwin, S. L., 22 Goodwin, T. W., 266, 267(3) Gorin, P. A. J., 45, 46(117), 194 Gorz, H. J., 301 Goto, T., 77 Could, S. E. B., 287, 288(181), 306 Goulding, R. W., 23 Gouyette, C., 230, 232(13), 240(13), 243(13), 263(13) Graf, R.,92, 93(189) Gr&, A., 387, 388(21), 392(21) Grant, G. T., 277, 305(135) Grant, H. N., 73(66), 76 Grasselli, P., 48, 67(123), 115 Grassner, H., 60 Gray, G. R., 17, 18, 20, 32, 46(4) Greenfield, J. C., 358,359(535) Greeves, D., 126 Gremli, H., 391 Greve, L. C., 302 Grewe, R., 115 Grewe, W., 99, 100(253), 146, 147(37) Grierson, D., 364, 371 Griesebach, H., 261 Grimshaw, C. E., 52,53(139) Grindley, T. B., 29, 31(74), 33(74), 54, 64(71) Grisebach, H., 56, 73(59, 60), 75, 77, 78, 110(90), 113, 131(90) Grosheintz, J. M., 100 Gross, B., 53, 97 Gross, K.C., 351, 372(501), 377(501), 378(501, 670). 380 Grutzmacher, H. F., 173, 176(78) Grynkiewicz, G., 113 Gueffroy, D. E., 53 Guertin, D. P., 137 Gugel, K. H., 188 Guilfoyle, T. J., 359 Guillern-Dron, D., 8 5 Gulasekharam, V.,29, 31(74), 33(74), 64(71) Gunner, S. W., 80, 90(118), 107, 108(292), 133(292) Gupta, D. S., 42 Gurny, R . , 92, 93(189) Guterman, E. G., 80

403

Guthrie, R. D., 73(47), 75, 96 Gutter, E., 297

H Haas, V., 308 Haegel, K., 188 Hagen, S., 76 Hagenmaier, H., 188 Hager, A., 349 Hahn, E. W., 137 Haisa, M.,150 Hale, C. R., 344, 348(436) Hall, C. B., 369 Hall, C. R.,47 Hall, L. D., 45, 200, 202(25), 203(25) Hall, M. A., 277, 281, 282(155), 285, 291(175), 292, 314(200), 320, 321(137), 350, 356(487) Hall, R. H., 9 3 Hall, S. A., 189 Hall, S. S., 94 Halrner, P., 272 Halmos, T., i10, 230, 232, 242(15, 31), 243(15), 244, 246(31), 248(31), 250(31), 251(31), 252(15, 31, 56), 256(31), 263(15), 264(75) Hamer, G. K., 211 Hamilton, A., 283, 284(168) Hamilton, T. H., 348 Hanabusa, K., 367 Hanaya, T., 139, 140(23), 141(23), 142(23, 24), 168(23), 169(23), 191(23) Hanessian, S., 51, 78, 95, 96, 101, 119, 121(353), 231, 261 Hanisch, P., 202 Hanke, D. E., 312 Hankins, C. N., 309 Hanna, R., 81, 122, 123, 126(141) Hanna, 2.S., 97, 99 Hanson, J. B., 348 Hara, K., 121, 127 Harada, S . , 77 Hardegger, E., 5 3 Harris, P. J., 331, 332(363), 382 Harrison, A,, 349, 357(470) Hart, D. A., 280, 281(152, 153) Hartley, R. D., 272, 315, 382 Hartmann, C. J. R . , 365 Harvey, J. M., 20

404

AUTHOR INDEX

Hascherneyer, A. E. V.,348, 359 Hasegawa, A., 47, 53, 67(120a) Hasegawa, S . , 371 Hashimoto, H., 90, 91, 121, 123(186), 133(183) Haskell, T.H., 261 Haskin, M. A., 323, 327(327) Haskins, F. A., 301 Hassid, W. Z., 316, 317, 318(293), 319, 320,321,322 Hattori, K., 229, 234(4, 5), 235(4), 236(4), 245(5) Haughton, P. M., 349, 350(472) Haverkamp, J., 42 Havinga, E., 27 Havis, A. L., 341 Hawes, G. B., 272 Hawker, J. S., 344, 348(436) Hawkins, D. W., 124 Haworth, W. N., 58 Hay, G . W., 269 Hayashi, M., 23, 40, 66(41, 103), 68(103) Hays, H. R., 142 Hayward, L. D., 21, 31(31),33(31), 52(31), 64(31), 65(31), 66(31) Heath, M. F., 272, 274(97) Heeschen, J. P., 18 Hehemann, D. G., 233, 236(43), 245(43), 247(43), 253(44) Hehre. E. J., 40, 356 Heid, H. A., 129 Heiker, F. R., 81, 129 Heilrnan, W., 73(67, 68), 76 Heinz, K., 72(7), 74 Heller, D., 188 Heller, J. S., 318, 319 Hellerqvist, C. G., 276 Hemming, F. W., 329 Hendrickson. J. B., 27 Hengeveld, E., 353 Henglein, A., 189 Henkels, W.-D., 216 Hennessee, G. L.A., 40 Henry, D. W., 132 Hernandez, O., 112 Herranz, E., 98 Herscovici, J., 110, 157, 232, 233, 238(26, 27, 30), 239(30), 241(40, 42), 242, 244(42, 50), 245, 246(26), 247(26, 27, 32), 249(26, 27, 30), 250(26, 50), 251(30, 42), 255(30,

53), 257(50), 258(50), 260(42, 50), 264(42) Herve du Penhoat, P. C. M., 18, 40(12), 46(13) Herzog, H. L., 64, 73(41), 75 Hess, K., 297 Heyn, A. N. J., 351 Heyns, K., 49, 50(127), 173, 176(78), 231 Heyraud, A., 196, 197(10), 198(10), 199(10) Hibbert, H., 269 Hicks, D. R., I06 Hicks, K. B., 20, 65(24), 68, 203, 204(40) Higashi, Y., 323, 324(330), 327(330) Hillestad, A., 272 Hindsgual, O., 217, 218(77) Hinman, J. W., 73(36), 75 Hinton, D. M., 371, 372(645) Hioki, Y., 53 Hirano, S., 209, 232, 233(36), 251 Hirata, N., 342, 348(415) Hirotsu, K., 155, 156(60), 157(59, 60), 165(59, 60), 191(60) Hirsch, J., 208, 213, 214(71, 72), 215(72), 216(72) Hirschhorn, S . G., 28 Hirst, E. L., 376 Ho, P.-T., 105, 128 Hobson, G. E., 361, 362(540), 363(540), 364(540), 365(540), 366, 367, 369, 370, 371(634), 372, 379, 380(677) Hodgson, K. O., 82 Hoeksema, H., 38, 73(36, 53), 75, 123(53), 227 Hoffman, P., 273 73(76), 76 Hoffman-Ostenhor, 0.. Hofheinz, W., 113 Hogenkamp, H. P. C., 230, 262(8) Holder, N. L., 106 Holleman, J., 322 Holly, F. W., 73(37), 75, 82 Holm, R.E., 348 Holy, A., 189 Hong, N., 118, 119, 120, 121, 133(365) Hopp, H. E., 325, 326(338, 340, 341), 327(338-341), 329(338, 341). 330(338, 341), 332(338, 341), 336(341) Hori, H., 272, 274(96) Hori, T.,114, 188 Horiguchi, M., 188

AUTHOR INDEX Horii, S.,73(73, 78, 79, 80, 81). 76 Horisaki, M., 155 Horitsu, K.,45, 46(117) Horton, D., 19, 25, 31, 42, 47, 51, 64, 65(18a), 67(120), 81, 90, 133, 137 Horwitz, J. P., 8 4 Hosoyamada, C., 149, 157(46), 168(46), 184(46) Hough, L., 315, 388 Houghtaling, H. B., 341 Howarth, C.B., 78, 82(101) Hsiao, T. C., 343 Huang, S.-C., 19, 26(20a), 31(20a), 36(20a) Huang, S.-L., 179, 182(88) Huber, D. J . , 272, 293, 294(202) Hudson, C. S., 69 Huelin, F. E., 363 Hughes, N. H., 53 Hui, P. A., 391 Hulme, A. C., 339, 361(393), 362(537, 538, 539). 363(393, 537, 538, 539), 364(539), 365 369 Hultin, H. 0.. Hunt, K., 280, 283(148) Hurd, C. D., 30 Hurych, J., 352 Hus, D. S., 287, 288(182) Hutson, D. H., 137 Hyvdnen, L., 23, 62

I Ichimi, T., 387, 391(16) Igolen, J., 230, 232(13), 240(13), 243(13), 263(13) Iida, T., 93, 100 Ikeda, K., 229, 236(6) Ikeda, T., 21 Ikeyama, Y.,96 Ikurna, H., 349, 357(473) Inada, S., 210 Inch, T.D., 47, 89, 95 Inch, W. R., 137 Ingles, D. L., 138 Inokawa, H., 189, 190(107) Inokawa, S., 96, 137, 138, 139(20), 140(.23), 141(23), 142(23, 24), 143(29), 144(33, 34), 145, 146, 147(43), 148(43), 149(33), 150, 151(47), 152, 153(53, 54), 155,

405

156(60), 157(46, 59, 60), 158, 160(53, 54). 161(43, 45, 53, 54, 55, 65), 164(54), 165(45, 53, 54, 59, 60, 65), 166(45, 53, 54), 168(23, 26, 33, 46, 55, 65). 169(23, 33, 55, 65), 173, 177(58), 179(47, 52, 66, 67), 180(66, 67), 181(33), 183(66), 184(46, 55, 65, 66, 67), 187(67, 81). 188(89), 189(33), 190(106, 107). 191(23, 29, 33, 34, 43, 53, 54, 60, 65,67, 89) Inoue, H., 110 Inoue, Y.,209 Inouye, S., 136, 150 Inukal, F., 136 Invanitskaya, L. P., 73(44), 75 Ireland, R. E., 1-10, 112 Irving, K. H., 342, 348(411) Isbell, H. S.,18, 32, 40(12) Isenhour, E. M., 344 Ishibashi, T., 116 Ishido, Y.,106, 107 Ishizu, A., 80, llO(122) Isono, K., 73(55), 75 Ito, T., 136 Ivanova, Zh. M., 172 Iversen, T., 212, 222, 223(80) Iwano, T., 110 Iwasa, T., 73(73, 77, 79), 76 Iwasawa, Y.,117 Izawa, M., 72(21), 74 Izquierdo Cubero, I., 90

J Jacin, J., 63 Jackson, D. I., 342, 348(417) Jackson, J., 58 Jackson, W. C., 73(36), 75 Jacobs, M., 272 Jager, J. M., 361, 362(542), 363(542) James, K., 60, 80, 82(110) JaneEek, F., 77, 281 Jang, R., 370 Jansen, E. F., 370 Jiirnefelt, J.. 276 JarreI1, H. C., 128, 129(395), 202, 203(38), 204(38) Jarvis, B.C., 348 Jaynes, T. A,. 301 Jeffrey, G . A., 163, 183(70)

406

AUTHOR INDEX

Jenkins, S. R., 96 Jerkeman, P., 388 Jermyn, M. A., 287 Jersh, J., 98 Jessipow, S., 188 Jewell, J. S., 47, 57, 67(120), 107 Jiang, K. S., 278, 280(138) Jobsen, J. A., 387, 388(22) JodAl, I., 199 John, H. G., 35 Johnson, A. W., 73(47), 75 Johnson, C. K., 163, 183(68) Johnson, J. H., 72(33), 74 Johnson, K. D., 301, 337(243) Johnson, R. N., 45 Jones, A. J., 202, 339 Jones, E. C., 272, 315 Jones, G. H., 231, 240 Jones, J. D., 361, 362(537, 539), 363(537, 539). 364(539) Jones, J. K. N., 51,57, 79,82(101), 94, 104, 128, 129(395), 232, 234(37) Jones, R. G., 42 Jones, R. L., 271, 275(61), 307(61), 314(61) Jordaan, A., 92, 93, 132 Jordaan, J. H., 76, 81(84), 82, 133 Joseleau, J.-P., 272, 281, 283(157), 292(89) Josephson, S., 21 1, 212(67), 223(67), 224(67) Jumelet, J . , 257 Jung, G., 200, 211(24) Jung, P., 329 Just, E. K., 90 Jyong-Chyul, C., 367

K Kabayama, M. A., 24 Kabir, M. S., 272 Kaburagi, T., 199, 212(19) Kahamura, E., 260 Kahan, R. S., 364 Kahle, V.,23 Kaji,A., 277, 282(133), 383, 384, 386(4), 387(3, 9, lo), 388(13), 389(12, 13).390(12, 13, 17, 19, 24), 391(10, 11, 12, 13, 16, 24, 28), 392(4, 11, 26). 393(4), 394(4, 44)

Kakudo, M., 150 Kakuta, M., 391 Kaliner, M., 367 Kameda, Y.,73(78, 79, 80, 811, 76 Kamprath-Scholtz, U.,97 Kandatsu, M., 188 Kane, O., 365 Kanz, W., 72(8), 74 Kapuscinski, M., 46 KarAcsonyi, S., 281, 282, 283(161) Kirkainen, J., 276 Karl, W., 73(59), 75 Karlsnes, A., 309, 353 Karr, A. L., 273, 274(107), 276, 298, 316, 317(281), 322, 336(232) Kasahara, I., 116 Kasai, M., 73(54), 75 Kasai, Z., 378 Kashimura, N.,86 Kashino, S., 150 Kashman, Y., 187 Kasyanchuk, N. V., 212 Kaszycki, H. P., 371 Kato, K., 200, 201(27), 284, 298 Katona, L., 298, 299(230) Katsumi, M., 358 Katz, M., 351 Kaufman, P. B., 271, 272(60), 287(60), 291(60), 294(60), 300(60), 349, 357(473) Kauss, H., 273, 307(106), 308(104, 105, 255), 309(106), 310(106), 321, 327, 328(347), 332(257) Kawaguchi, H., 72(16), 74 Kawahara, K., 73(80), 76 Kawahara, M., 105 Kawamatsu, Y., 72(27), 74 Kawamoto, H., 149, 150, 151(47), 153(53), 157(46), 161(53, 55). 165(53), 166(53), 168(46, 55), 169(55), 179(47, 52, 67), 180(67), 184(46, 55, 67), 187(67), 191(53, 67) Kawamoto, I., 112 Kawana, M., 113 Kawata, Y.,150, 151(47), 179(47, 52) Kazama, H., 358 Kazi, M. A,, 76 Keates, R. A. B., 367 Kedar, N., 342, 348(405) Keegstra, K., 271, 272(55, 56, 57), 273(55,57), 274(55, 56, 57), 275(55,

AUTHOR INDEX 56, 57). 276(55, 56). 277(55, 56), 278(55), 280(55), 282(55), 283(55), 284(55), 287(56), 288(56), 289(56). 291(56), 295(56), 298(55), 299(57), 301, 302(56, 57), 303(57), 304(57), 305(55), 306(55,56,57), 307(56, 57), 309(57), 310(56, 57, 264). 311(57. 264), 312(57, 264), 314(56, 57, 264), 317(56), 321(55), 337, 338(56, 57), 351(237), 355(57), 368(55,56, 57). 369(55, 56, 57), 373(55, 56, 57). 376(57), 378(57), 379(56) Keilich, G., 42 Keller, A,, 54, 68(149), 134 Keller-Schierlein, W., 72(25), 73(66, 67, 68), 74, 76, 126, 127(388) Kelly, R. B., 72(32, 33), 74 Kemp, J., 320 Kenner, G. W., 179 Kent, P. W., 142 Kephart, J. E., 302, 331(249) Kerr, P. F., 47 Kersten, S., 115 Kertesz, Z. I., 369 Key, J. L., 348, 358 Khurdanov, Kh. A., 80 Kidd, F., 362, 363 Kieboom, A. P. G., 34 Kiely, D. E., 39 333, 334, 335, 336 Kiermayer, 0.. Kierszenbaum, F., 310 Kim, J. H., 137 Kimmins, W. C.,329 Kindel, P. A., 280, 281(152, 153) King, N. J., 353 King, R. D., 80, 90(118) King, R. R., 205, 209(41) Kinman, C. F., 339, 347(380) Kinoshita, M., 94, 96, 107, 110, 129 Kinoshita, N., 129 Kinoshita, T., 114 Kirby, E. G., 272 Kishi, T., 72(21), 74, 77 Kislev, N., 372, 380(657) Kiso, M., 47, 53, 67(120a) Kiss, J.. 79 Kitagawa, H., 145 Kitaguchi, T., 72(17), 74, 76(17) Kitao, K., 287 Kivilaan, A,, 285,293,294(210), 337, 351

407

Kivirikko, K. I., 275 Kiyomoto, A , , 146 Klein, I., 364 Kleinhoes, A., 301 Klemer, A,, 68 Kliewer, W. M., 345, 348(447) Klimov, E. M., 200, 221(26) Klis, F. M., 301 Knapp, R. D., 202, 203(39), 204(39) Knee, M., 273, 315, 339, 340, 341, 346, 347(394), 356(483), 363, 369, 372(394, 658). 374(652, 654), 375(394), 376(658), 378(394, 654, 658). 379, 380(659), 381(659) Knirel, Yu.A,, 212 Knolle, J., 112 Knox, R . B., 287 Kobayashi, K., 80, 82(113, 120), 84(120), 90(113), 100, 133(113, 120) Kobayashi, T., 388 Kobinata, K., 73(55), 75 Koch, H. J., 21 1 Kochetkov, N. K., 96, 173, 176(77, 79). 200, 205, 206(44, 45), 207(47), 209, 21 1(46), 212(47), 218(56), 220(44), 221(26, 44, 45, 46). 222(45), 223(46) Kodama, H., 119, 133(365) Koebernick, H., 52 Koebernick, W., 100, 101(259), 106(260), 126, 244 Koener, T. A. W., Jr., 27, 43, 46(69a) Koenig, W. A , , 188 Koga, K., 90, 133(183) Kahler, P., 102 Koivistoinen, P., 23, 62 Kolinska, J., 23 K d l , P., 35, 58 Kollmann, M., 85, 108(162) Kolosov, M. N., 72(26), 74 Kolpak, F. J., 295 Komae, K., 386, 388(13),389(13), 390(13), 391(13) Komander, H., 58 Kondo, T., 77, 91, 123(186) Kondo, Y.,44, 86 Kondoh, T., 116 Konigstein, J., 77 Konishi, K., 23 Konovalova, I. V., 172 Kooiman, P., 287, 288, 289 Korte, F., 113

408

AUTHOR INDEX

Kosolapoff, G. M., 139 Kotick, M. P., 104 Koto, S., 210, 217, 218(76) Kov3, P., 207, 208(52), 213(52), 214(71, 72). 215(72), 216(72) KovAEik, V.,282, 283(161) Kramer, K. K., 323, 325(332), 327(332) Kraska, U., 102 Krauss, A., 349 Kriedmann, P. E., 345, 348(443) Krishnamurthy, S.,122, 339, 341(384), 356(384), 361(384), 362, 363(384), 372(384) Krishnamurthy, T. N., 287, 288(180), 289(180) Kritchefsky, C., 188 Krusius, T., 276 Ku, L. L., 364 KubaEkovA, M., 281, 283 Kubo, K., 121, 133(365) Kudinova, M. K., 72(34, 35), 74, 75 Kuenzle, C. C., 76, 81(84) Kuhn, R., 60 Kulow, C., 272, 327 Kulyaeva, V.V.,72(34,35),73(45), 74,75 Kumada, Y.,73(73), 76 Kumanstani, J., 23 Kiinstler, K., 216 Kunstmann, M. P., 73(61, 62), 75 Kunzelmann, P., 42 Kupfer, E., 73(68), 76, 126, 127(388) Kuraishi, S., 350, 356(486) Kurooka, H., 342,348(415) Kusakabe, I., 388 Kuster, B. F. M., 23 Kuwahima, I., 260 Kvoinishnikowa, T. A., 172

L Labavitch, J., 347, 375 Labavitch, J. M., 271, 273(54), 274(101, 102), 277, 282(132), 283(132), 291(54), 292(54), 294(54), 306, 355, 371, 372, 376(641, 650), 378(650), 379(641), 380 Laborda, F., 372, 388, 391(23) Lackey, G. D., 377, 378(670) Lado, B., 349, 358(480) Laemmle, J. T., 90

Laffite, C., 205(48), 206,207(48), 211(48) LaForge, F. B., 40 Lagrange, A., 91, 122(184) Lake, W. C., 137 Lakshimarayana, S.,363 Lambert, J. B., 52(140a), 53 Lamport, D. T. A., 270, 272, 274(42), 298(41, 42), 299(228, 230), 300(231), 308,309, 312, 313(228), 322, 323(38), 335(326), 352 Lancaster, J. E., 73(62), 75 Lance, C., 361, 362(540), 363(540), 364(540), 365(540) Landsky, G., 52 Langenfeld, N., 73(39), 75 LaPage, G. A., 132 Larm, O., 283, 284(166) Laties, G . G . , 366 Lavalke, P., 95 Lawton, B. T., 232, 234(37) Lazarus, H., 131 Leander, K., 73(69), 76, 128(69) Leclercq, F., 228, 230(3), 232(3), 237, 248, 250(3), 254(57), 257 Lederer, E., 72(14), 74 LeDizet, P., 287 Lee, C. Y.,22, 64(36) Lee, S., 337, 351 Lees, T. M., 72(19), 74 Lehavi, U.,388 Lehle, L., 327, 328(347) Lehmann, J., 40, 179 Leigh, D. S.,339, 356(386) Leinert, H., 107, 108(295, 299) Leisma, M., 275 Leland, D. L., 104 LeIey, V. K., 363, 379(547), 380 Leloir, L. F., 327 Lemal, D. M., 113 Lemieux, R. U., 18, 19(8), 22, 46(8), 64, 72(11), 74, 202, 217, 218(76, 77) Lennarz, W. J., 323, 324(329), 325(328, 329, 331, 332), 327(328, 329, 331, 332), 330(328, 329) Lenoir, D., 73(38), 75 Lenz, R. W., 18 Leonard, S. J., 370 Leonhardt, H., 54 LePendu, J., 217, 218(77) Letham, D. S.,343, 348(424), 349(424)

AUTHOR INDEX Leupold, F., 49, 50 Levene, P. A., 138, 145, 191(36) Levine, A. S., 369 Levison, S . A., 310 Levitt, M. H., 202 Levitt, S. H., 137 Levy, H. A., 24 Lewis, G. J., 89, 95 Lichtenthaler, F. W., 100, 102, 107, 108(294, 295, 296, 297, 299), 109, 133(297, 302) Lieber, E., 188 Lieberman, M., 343 Lin, P. P. C., 367 Lindberg, A., 222, 223(80) Lindberg, B.,45, 73(74), 76, 110, 276, 388 Linskens, H. P., 353 Lipshutz, B., 96 Liptak, A., 45, 64, 199, 205, 206(42), 207(42), 211, 212(66) Lis, H., 307, 309 Listowsky, L., 21 Liu, T., 317 Loebich, F., 72(8), 74 Loescher, W. H., 273, 293(110), 294(202), 306, 355 Loibner, H., 130 Lonngren, J., 173, 176(80), 219, 220(79) Looney, N. E., 364 Lorenz, W., 189 Los, J. M., 21 Lourens, C . J., 80,90(115) Lourens, G. L., 9 3 Low, J. N., 107, 108(298), 133(298) Lowry, D. H., 275 Lu, T. S., 358, 359(534) Luce, C. L., 104 Luckwill, L. C., 342, 348(421) Luedemann, G. M., 73(41), 75 Luetzow, A. E., 51 Luger, P., 148, 151, 152, 153(53, 54). 160(53, 54). 161(45, 53, 54). 164(54), 165(45, 53, 54). 166(45, 53, 54). 179(66), 180(66), 183(66), 184(66), 191(53, 54) Luh, B. S., 370 Lukacs, G . , 91, 96, 98, 119, 120(243), 122(184), 133 Lumelli, J., 342, 348(413)

409

Lunel, J., 73(64), 75 Luu, D. V., 23

M McCasland, G. E., 116 McCleary, B. V.,200, 201(29) McCloskey, C. M., 45 McCloskey, J. A., 229, 236(6) McComb, E. A., 347, 370, 372(450) McCormick, M. H., 73(46), 75 McCready, R. M., 347, 372(450) McDowell, R. A., 276 McEnrose, F. J., 94 McFeeters, R. F., 370, 371(630, 631) McCarvey, G. J., 110 McChie, J. F., 124 McGonigal, W. E., 55, 79 McCrath, D., 269 McCuire, J. M., 73(46), 75 Macchia, V., 367 McHugh, D. J., 25 MacKay, D., 121 McKay, J. E., 269 MacKeller, F. A , , 72(32, 33), 74 McKelvey, R. D., 196, 197(12), 198(12), 208(12) MacLachlan, G. A., 317, 351, 352(492, 503) MacLeod, J. K., 46 McNab, J. M., 322, 329, 330(361), 331(361), 352(321) McNeil, M., 271, 272(60, 62), 274(62), 275(61, 62), 276(62, 65, 120), 277(125), 278(65, 125), 280(62, 65, 125), 281(62, 125), 282(125), 283(125, 163), 287(60, 62, 65), 288(65), 289(65, 120, 189), 291(60, 65), 294(60), 300(60), 302(65), 304(65), 305(65, 125), 306, 307(61, 65, 120), 309(65), 314(61), 321(65), 368(62, 65). 369(62, 65), 373(65) McNicholas, P., 42 McPhail, A. T., 73(50, 56, 63), 75, 123(56), 165, 202 McPherson, J., 73(74), 76 McReady, R. M., 370 Mackie, W., 25, 43, 44, 45, 61, 68(57) MAcova, J., 45 Madusa, F., 251

410

AUTHOR INDEX

Maehr, H., 73(42), 7 5 Maekawa, E., 287 Maglothin, A., 280 Mahl, H., 297 Mahmood, S.,1 1 9 Maier, V. P., 371 Majer, J., 72(22), 7 4 Makita, M., 22, 68(32) Malherbe, M., 9 2 Mallams, A. K., 73(56), 75, 123(56) Maltby, D., 272 Malysheva, N. N.,200, 221(26) Mamizuka, T., 2 0 9 Mancuso,A. J., 179, 182(88), 232,240(34) Mancy, D., 73(64), 75 Manley, R. S. J., 297 Mapson, L. W., 3 6 3 Marcellin, P., 3 6 5 Marchessault, R. H., 3 1 2 Marei, N., 344, 348(434) Mares, D. J., 294 Markwalder, H . U., 287, 315 Marquez, J. A., 73(41), 75 Marre, E., 349, 358(480) Marsacoli, A. J., 1 1 9 Marten, H., 112 Martin, J. R., 72(22), 74 Man-Figini, M., 295, 297(221), 3 1 7 Masse, R., 9 6 Masuda, R., 116 Masuda, Y.,348, 351, 352 Matchett, W. H., 272 Matern, U., 73(59, 60), 75 Mather, A. M., 81, 126(111) Matheson, N. K., 273, 387 Mathlouthi, M., 2 3 Matsubara, K., 386, 387(9) Matsuda, K., 195, 196(8), 197(8), 21 l(8) Matsuhashi, M., 323, 327(327) Matsui, M., 1 2 0 Matsuura, F., 210 Matsuura, K., 106, 107 Matsuura, T., 105 Matsuzawa, M., 61, 80, 85,90, 94, 122, 126, 127, 133(165, 390) Matthyse, A. G., 3 4 8 Mattick, L. R., 6 8 Mattoo, A. K., 339, 343, 369(381) Mauch, W., 65(174), 6 6 Maxie, E. C., 344, 348(433) Mayd, F., 6 1

Mayer, W., 72(8), 7 4 Mayorga, H., 372, 378(647), 380(647) Meands, A. R.,348 Medina, M. G., 3 6 9 Meier, H., 272, 282, 283(162), 292, 293, 294(208) Meinert, M. C., 272 Melberg, S.,2 7 Meldal, M., 222, 223(80) Menchu, J. F., 372, 378(647), 380(647) Mengech, A. S., 1 2 2 Mense, R. M., 3 2 8 Mentze, J., 358 Menzel, H., 3 4 9 Mercer, E. I., 266, 267(3) Merrer, Y.L., 81 Mesentsev, A. S., 73(45), 7 5 Messer, M., 201, 202(30) Mestres, R.,8 2 Mettler, I. J., 3 6 4 Metzner, E. K., 211, 212(62) Meyer, A. S., 157 Meyer, B., 58 Meyer, K., 2 7 3 Meyer, L., 353 Meyer, N., 81 Meyer, W., 93, 9 8 Meyer zu Reckendorf, W., 79, 9 7 Michel, G., 207 Middlebrook, M., 2 6 8 Mikami, K., 80, 1 2 1 Mikhailov, S. N., 82, 131 Miljkovik, D., 84, 86. 89(171), 133(159) Miljkovii., M., 84, 86, 89(171), 133(159) Miller, D. H., 298, 300(231), 322, 336(326) Miller, J. A., 93 Miller, T. W., 116 Miller, W., 73(63), 7 5 Millerd, A,, 3 6 6 Mills, J. A,, 33, 55, 5 9 Minamoto, K.,229, 234(4, 5), 235(4), 236(4), 245(5) Mineura, K., 73(54), 75 Minshall, J., 1 2 5 Misaki, A,, 3 9 1 Mistra, P., 268, 272(12) Mitscher, L. A., 73(61, 62), 7 5 Miwa,T., 1 1 4 Miyajima, G., 200, 201(27) Miyake, A., 73(57), 75

AUTHOR INDEX Miyaki, T., 72( 16), 74 Miyamoto, M., 72(27), 74 Miyashita, S., 110 Mizsak, S. A., 72(33), 73(53), 74, 75, 123(53) Mizuno, T., 73(55, 57), 75, 105, 200, 201(27) Mizuno, Y.,229, 236(6) Mizuta, E., 73(57, 73), 75, 76, 77 Modi, V. V., 339, 369(381) Mody, N., 84 Moffatt, J. G., 80, 90(116), 104, 227, 231, 232(2), 233(2, 24, 25), 234(25), 237(24, 25). 240, 245(2, 25), 246(2), 247(2, 25), 248(2, 25). 249(2, 25), 250(2, 25), 252(2, 25). 253(25), 261 Mofti, A. M., 51 Mohr, W. P., 372 Mollard, A., 288 Mollenhauer, H. H . , 331 Molloy, J. A., 278, 280(139, 142), 283(139), 284, 287(171), 288(171) Molloy, R. M., 73(49), 75 Monro, J. A., 271, 273, 307(49, 50), 309(49, 50, 51), 310(50, 265, 266), 311(49, 50), 312(49), 313, 314(49, 50, 266) Monselise, J. K.,364 Montague, M. J., 349 Montelinos, D., 272 Montreuil, J., 42 Moore, A. T., 272 Moore, R. J., 348 Morand, P. F., 30 Morimoto, M., 73(54), 75 Morita, M., 283, 284(169, 170) Morrall, P., 272 Morris, D. L., 293 Morris, E.R . , 269, 277, 305(135), 306(30), 307(30), 315(30) Morris, C. A., 200, 202(25), 203(25) Morris, H. R., 276 Morrison, A., 85 Morrison, I. M., 278, 280, 283(148) Morton, G., 73(62), 75 Morton, J., 126 Moshy, R. J., 6 3 Mothers, K., 345, 348(442) Motoyama, K., 387, 390( 17) Mode, Y., 230, 263(12) Mowery, D. F., Jr,, 68

411

Moyna, P., 202, 203(38), 204(38) Moyse, C. D., 367 Mudge, K. W., 342,348(408) Mueller, S. C., 332, 334(369) Muggli, R., 295 Muhlenthaler, K., 297 Muir, R. M., 342, 348(422) Mukherjee, P. K., 363 Miiller, D., 73(40), 75, 173, 176(78) Miiller, M., 22, 2 3 Mullins, J. T., 351 Munasingle, V. R. N., 107, 133 Munavu, R. M., 34 Munksgaard, V., 196, 197(15), 202(15), 203(15), 204(15) Murofushi, T., 260 Muroi, M., 72(21), 74 Murray, A. K., 301, 337(242) Myers, T. C., 189

N Nagakura, N., 40, 66(103), 681103) Nagasawa, J.-L, 107 Nagasawa, Y., 122 Nahar, S.,84 Naik, K. C., 339 Nakabayashi, S.,219, 220(78) Nakada, S., 94 Nakadaira, Y., 72(27), 74 Nakagawa, A., 133 Nakagawa, S.,342, 348(415, 416) Nakagawa, T., 109 Nakahara, W., 136 Nakamoto, K., 116 Nakamura, K., 189 Nakamura, Y.,161, 179(67), 180(67), 181, 184(67), 187(67), 188(89), 191(67, 89) Nakanishi, K., 72(27), 74 Nakashima, R., 105 Nakashima, T. T., 139, 140(23), 141(23), 142(23), 149, 153, 157(46), 158, 161(55, 65). 165(65), 168(23, 46, 55, 65), 169(23, 55, 65). 177(58), 179(67), 180(67), 181, 184(46, 55, 65, 67), 187(67), 188(89),191(23, 65, 67, 89) Nakatsukasa, Y., 155, 156(60), 157(59, 60). 165(59, 60), 191(60)

412

AUTHOR INDEX

Nakaya, M., 96 NBnBsi, P., 64, 196, 199(14), 205, 206(42, 43), 204(42,43, 50), 224(43) Nance, J. F., 272, 273 Narayana, N., 363, 379(547), 380(673) Narayanan, A., 367 Narayanan, K. R., 342, 348(406, 408) Nashed, M. A., 120 Nashimura, N., 232, 233(36) Nasseri-Noon, B., 34 Neal, G. E., 375 Neeser, J.-R., 93 Neiduszynski, I., 312 Nelson, H. M., 344, 348(434) Nelson, N., 388 Nesbitt, W. B., 376 Ness, P. J., 364 Neszmdyi, A., 133, 196, 199(14), 205, 206(42, 43), 207(42, 43, 50), 211, 212(66), 224(43) Neszmknyi, A,, 64 Nettles, V. F., 369 Neuberger, A., 47, 49(119), 67(119), 308 Neufeld, E. F., 316, 322 Neukom, H., 273,287,309,315,391 Neumann, J. H . , 94 Neupert-Laves, K., 126, 127(388) Nevins, D. J., 272, 273, 276, 293(110), 294(202), 301, 306, 351, 352(504), 355, 359(504) Newcomb, E. H., 337 Newton, R. P., 367 Nguyen Phouc Du, A. M., 205(48), 206, 207(48), 211(48) Nickerson, T. A., 61 Nickol, R. G., 81 Nicole, D. J., 68 Nieto. M., 73(48), 75 Nieuwenhuis, J. J., 76, 81(84), 133 Niida, T., 136 Nikaido, H., 316 Nikolaev, A. V., 205, 206(45), 211(46), 221(45, 46), 222(45), 223(46) Ninet, L., 73(64), 75 Nishida, T., 194 Nishiyama, K., 106, 107 Nitch, C., 349 Nitsch, J. P., 342, 348(409), 349 Norberg, T., 90, 119(178), 217, 218(77), 222, 223(80) Northcote, D. H . , 268, 269, 270, 272,

274(97), 280, 302, 305, 307(23), 312(35), 331(247, 248), 332(363), 367 Novak, R., 115 Novellie, L., 272, 292 Nukaya, A., 371, 380(644) Nunez, H . A , , 194, 200, 209(23), 210(23) Nutt, R. F., 82 Nwe, K. T., 187 0

O’Brien, T. J., 348 O’Brien, T. P., 382 O’Connell, P. B. H., 364 Oden, E. M., 73(41), 75 O’Dwyer, M. H., 310 Ogata, T., 142, 145, 146, 147(43), 148(43), 155, 156(60), 157(60), 161(43), 165(60), 168(26), 177(58), 189, 190(106, 107), 191(43,60) Ogawa, S., 102, 116, 117(344) Ogawa,T., 117, 120, 199, 200, 201(28), 202(28), 211, 212(19), 219, 220(78) Ogihara, Y., 197, 199(16) Ohgi, T., 77 Ohkubo, S.,73(54), 75 Ohle, H., 155 Ohrui, H., 95 Ohtani, K., 391 Okada, G., 356 Okada, T., 388, 392(26) Okuda, D., 72(31), 74, 125(31) Okuda,T., 16, 23, 40, 41(2), 66(41), 68(103), 146 Okumura, H . , 47,67(120a) Olesker, A., 91, 98, 119, 120(243), 12 2 (184) Ollapally, A., 232, 247(32, 33). 251 Ollis, W. D., 73(65, 67), 75, 76, 126(65) Olsen, A. C., 351 Omata, M., 117 Omura, K., 232, 240(34) Omura, S., 133 Onan, K. D., 73(50), 75 Ong,K.-S., 80, 90(117, 119). 99 Onodera, K., 86, 232, 233(36), 251 Oparaeche, N.N., 107 Ordin, L., 302, 319, 320, 350, 351, 356(487) Orenstein, N. S., 72(3), 74, 76

AUTHOR INDEX Oriez, F.-X., 53 Oritz, L., 365 Osaki, K., 16, 41(2), 47 Osborn, M. J., 324, 327(334) Osborne, D. J., 340 341(394), 346(394), 347(394), 353, 354(521), 359(521), 363(394), 372(394), 374(394), 378(394), 379(394), 381(394) Oshima, K., 98 Oshima, R.,23 Otterach, D. H., 49 Overend, W. G., 58, 78, 79, 80(97), 81, 82(97, 99, 107, log), 84(130), 90(118), 107, 108(292), 133(292), 254 Owen, L. N., 58, 138 Owen, S.P., 73(51), 75 Oyama, Y.,388, 392(26) P Pacak, j., 45, 85, 108(162) Pacht, P. D., 113 Paiz, L., 365 Palevith, D., 342, 348(405) Palmer, J. K., 364 Pang, D., 101 Pansolli, P., 371 Panyatatos, N., 322 Parikh, D. K., 132 Parikh, V. M., 232, 234(37) Parish, R. W., 302 Parker, K. A., 121 Parr, D. R., 301 Parrish, F. W., 64, 195, 196(9), 200(9), 202(9), 211(9), 293, 294(205) Parry, M. j., 112 Passerson, S., 316 Patel, D. S., 370 Patt, S. L., 196 Patterson, B. D., 348 Patterson, D., 24 Patterson, M. E., 364 Padsen, H., 48, 49, 50(124, 127), 51, 52(134), 55, 81, 82(135), 94, 99, lOO(253). 101(259), 106(260), 125(136), 126, 129, 137, 146, 147(37), 231, 244 Pearl, I. A., 269 Pearson, J. A,, 366 Peat, S., 293

413

Peaud-Lenoel, C., 328 Pederson, C., 194 Pehl, E., 392 Penco, S., 72(24), 74 Penglis, A. A. E., 91 Peniston, Q. P., 20 Penny, D., 271, 273(49, 52). 307(49, 50), 309(49, 50, 51). 310(50, 265, 266), 311(49, SO), 312(49), 313(49), 314(49,50,266) Percival, E. G. V., 376 Perkins, H. R., 73(48), 75, 266, 267(2) Perlin, A. S., 18, 24, 25, 27, 40(12), 43, 44, 45, 46(13), 59(51), 61, 68(57), 194, 196, 197, 211, 293, 294(205) Pernet, A. G., 78 Pesis, E., 379, 380(678) Peter, H. H., 73(68), 76 Petrakova, E., 207, 208(52), 213(52) PetruS, L., 36, 65(93) Pfeffer, P. E., 20, 64, 65(24), 68, 195, 196(9), 200(9), 202(9), 203, 204(40), 211(9) Pfitzner, K. E., 232,233(24), 237(24), 245 Phaff, H. J., 370 Pharr, D. M., 376 Philips, K. D., 47, 67(120) Phillips, C., 348 Phillips, D. R., 391 Phillips, L., 45 Pickles, V. A., 18, 19(9, 16), 20(16), 23, 25, 26(10), 28(10, 16), 31(16), 36(9), 38(16), 55(9), 57(61), 58(61), 59, 65(16), 66(16), 68(10) Piekarska, B., 212 Pierce, J., 19, 26(20a), 31(20a), 32(80a), 36(20a), 46(80a) Pierrot, H . , 300, 301(235) Pigman, W. (W.), 32, 42 Pilnik, W., 374 Pinsky, A,, 320 Pinto, B. M., 49, 54 Piriou, F., 133 Pittenger, G. E., 73(46), 75 Pittenger, R. C., 73(46), 75 Plaumann, D. E., 101 Pogson, C. I., 32 Pojer, P. M., 55 Polya, G . M., 367 Ponnampalam, R.,33 Ponomalenko, V. I., 72(34), 74

414

AUTHOR INDEX

Ponomareva, N. P., 371 Pontagnier, H., 207 Pont Lezics, R., 325, 326(338, 340, 341), 327(338-341). 328, 329(338,341, 355), 330(338, 341), 332(338, 341). 336(341) Pool, R. M., 342, 348(412, 420) Poovaiah, B.W., 342, 348(406, 408), 371, 380(644) Pope, D. G . , 299, 307(234), 309(234) Pople, J. A., 24, 163, 183(69) Portal Olea, M. D., 90 Posternak, T., 79, 80 Potapova, N. P., 72(34,35), 74, 75 Pottage, C., 47 Pousset, J. L., 72(5), 74 Pozsgay, V., 196, 199(14), 205, 206(43), 207(43, 50), 224(43) Pradet. A,, 367 Pratt, H. K., 361, 362(541), 363(541), 365(541) Pratviel-Sosa, F., 205(48), 206, 207(48), 211(48) Preobrazhenskaya, T. P., 73(44), 75 Pressey, R., 347, 369, 370, 371(625, 629), 372(645) Preston, R. D., 266, 267(4), 268, 295(4), 296(223), 297(4), 298(4), 314(4), 317(223), 334, 336(4), 341, 348(401), 349(401), 352(401), 354, 357(401) Preud’homme, J., 73(64), 75 Price, J., 269, 306(30), 307(30), 315(30) Pridham, J. B., 315 Providoli, L., 391 Pschigoda, L. M., 73(53), 75, 123(53) Puar, M. S., 73(56), 75, 123(56) Pudovik, A. N., 172 Purick, R., 150, 188(48) Puskas, I., 30 Pyler, R. E., 146

Q Qrzaez, M., 82 Que, L., Jr., 18 Quin, L. D., 165, 172, 184(75)

R Rabanal, R., 98, 120(243) Rabideau, P. W., 132

Radomski, J., 212 Rafferty, G. A., 82 Rahman, A,, 121 Rahman, K. M. M., 122, 123 Raiju, M., 388, 390(24), 391(24) Ramnas, O., 23 Rancourt, G., 96 Randall, M. H . , 31, 45, 46(116), 59(81) Randall, R. J., 275 Randhawa, G. S., 342, 348(414) Ranganathan, R. S., 240 Rank, W., 146 Rao, C. V. N., 379 Rao, G. V., 107, 1081293) Rao, V. S. R., 27, 47, 54, 217, 218(76) Raphael, R. A., 113 Rasmussen, G. A,, 380 Rasmussen, J. R., 28 Rasmussen, K., 27 Ratcliffe, M., 93, 106, 107 Rathbone, E. B., 128, 129, 272, 292 Rattanpanone, N., 364 Rauvala, H., 276 Ray, M. M., 331 Ray, P. K., 82 Ray, P. M., 267, 271, 273(53, 54), 274(101, 102). 285, 287(173), 291(54), 292(53, 54), 294(53, 54), 306(173), 314(53), 331, 332(365, 366), 348, 350(7, 8, 9), 355, 356(6, 7, 484) Rayle, D. L., 301, 337(243), 349, 350(472), 357(471), 358 Raymond, A. L., 138 Raymond, B., 358 Raymond, P., 367 Redlich, H., 81, 94, 125(136) Rees, D. A., 25, 27(55), 269, 277(32), 281, 282, 287, 288(181), 305(135), 306(30), 307(30), 312, 315(30) Rees, D. E., 45, 80, 82(110) Reese, E. T., 293, 294(205) Reeves, R. E.,25, 133, 287, 288(182) Reichstein, T., 157 Reid, D. S., 24 Reist, E. J., 53, 211, 212(62) Reymond, D., 370 Reynolds, S. J., 32 Rhodes, H. J. C., 361,362(538), 363(538) Rice, K.-C., 55, 79 Richards, G . N., 273, 307, 315(117), 384

AUTHOR INDEX Richardson, N. G., 281,282,305(160),306 Richmond, A., 364 Ridge, I., 353, 354(521), 359(521) Riemer, W., 73(40), 75 Riley, A. C., Jr., 72(13), 74 Rinaudo, M., 196, 197(10), 198(10), 199(10) Riov, 364 Robbins, P. W., 324, 327(333) Roberts, J. D., 133, 196, 211(11) Roberts, J. G., 293 Roberts, R. M., 272 Robertson, N. G., 371 Robertson, R. N., 361, 362(541), 363(541), 365(541), 366 Robinson, D. G., 331, 332(366), 367 Robinson, J. E., 3 6 3 Rodda, H. J., 179 Roden, K., 126 Rodin, J. O., 73(37), 75 Roelofsen, P. A., 266, 267(1), 268(1) Roerig, S . , 298, 299(230) Rogers, H. J., 266, 267(2), 268(2) Roggan, H. P., 351 Rohrer, D. C., 183 Rollin, P., 301 Rollins, A. J., 119, 120 Rollins, M. L., 272 Rollitt, J., 352 Rob, C., 365, 372, 378, 380 Romani, R. J., 364 Romero, P. A., 325. 326(338, 340, 341). 327(338-341), 329(338, 341). 330(338, 341), 332(338, 341), 336(341) Romero Martinez, P., 327 Roncari, G., 72(25), 74 Rosebrough, N. J., 275 Rosell, K.-G., 287, 288(180), 289(180) Rosenthal,A., 80, 82, 90(114, 117, 119, 125, 126), 92, 93, 94, 106, 107, 131, 232, 257(38) Rosevear, P. R., 194, 209 Rosowsky, A., 96, 131 Ross, C., 341, 348(402) Ross, K. M., 292 Rosselet, J. P., 73(41), 75 Rossman, R. R., 73(56), 75, 123(56) Rosynoi, B. V., 72(35), 75 Rot, I., 343 Rottenberg, D. A., 285, 287(173), 306(173) J.%

415

Rouhani, I., 372 Rouser, G., 188 Routien, J. B., 72(19), 74 Rowan, K. S . , 361, 362(541), 363(541), 365 (54 1) Roxburg, C. M., 113 Roy-Choudhury, R., 348 Rubasheva, L. M., 72(35), 73(45), 75 Rudich, J., 342, 348(405) Rudrum, M., 18 Ruesink,A. W., 267,350(9),351,352(506) Ruesser, F., 73(52), 75 Rumsey, A. F., 348 Ryan, K. J., 132

S Sacher, J. A,, 364 Sachs, R. M., 345, 348(446) Sadeh, S., 388 Saeed, M.S.,93, 122 Saheki, T.,277, 282(133), 384, 386(4), 392(4), 393(4), 394(4) Saini, H. S., 273, 387 SaitB, H., 209 Saito, K., 378 Saito, M., 116 Saito, S., 23, 40,66(41, 103). 68(103) Sakakibara, T., 99, 109 Sakazawa, C., 105 Saksema, A. K., 72(30), 74 Salisbury, F. B., 341, 348(402) Salton, M. J. R., 136 Saltveit, M. E., 370, 371(631) Samitov, Yu., 172 Samuelson, 0..23 Sanchez, M., 365 Sanford, P. A,, 276 Sangster, I., 49, 50(127) Sankar, K. P., 378 Sano, H., 55 Sargent, J. A., 340, 341(394), 346(394), 347(394), 353, 354(521), 359(521), 363(394), 372(394), 374(394), 378(394), 379(394), 381(394) Sarkanen, K. V., 269 Sarkissian, I. V., 348 Sarko, A., 295 Sarre, 0. Z., 72(28), 73(50, 63), 74, 75, 126 Sasajima, K., 21 1 Sasaki, H., 96

416

AUTHOR INDEX

Sasaki, T., 229, 234(4, 5), 235(4), 236(4), 245(5) Sassa, T., 72(17), 74, 76(17) Sastry, K. K. S., 342, 348(422) Sato, H., 92, 133(191) Sato, K., 80,82(113, 120), 84(120), 85, 86(168), 88(166), 90(113,166), 91, 92, 94, 108(163, 164), 118, 120, 121, 122, 123(186), 133(113,120, 164, 165, 181, 183, 365) Sato, M., 386, 388(13), 389(13), 390(13, 24), 391(13, 24, 28) Sato, S., 107, 272, 274(96) Sato, T., 84, 133(159) Satoh, C., 146 Saunders, W. H., Jr., 30 Sauriol, F., 196 Scensny, P. M., 28 Schaaf, T. K., 112 Schafer, D. E.,225 Schaffner, C. P., 73(42), 75 Schapiro, H. C., 310 Scharmann, H.,173, 176(78) Schauer, R., 42 Scheidegger, U.,107, 108(295) Scher, M., 323,324(329), 325(328, 329, 332), 327(328, 329, 332), 330(328, 329) Schery, R. W., 339, 356(485), 361(385) Schiffmahn-Nadel, M., 370, 371(633) Schilling, G., 54, 68(149), 216 Schilling, S., 134 Schlesselmann, P.,40 Schmid, R., 56, 78 Schmidt, 0. T., 72(7), 74 Schmiechen, R., 80 Schnarr, G . W., 129 Schneider, W., 54 Schnoes, H. K., 173, 175(76), 176(76) Schdllnhammer, G., 80,90(114, 125) Schrader, G., 189 Schrank, A. R., 348 Schroeder, W., 227 Schubert, F., 383 Schubert, W. J., 269 Schuerch, C., 211, 212(60), 212(61) Schulte, G . , 98, 121(244) Schulz, G., 295, 297(221) Schulz, J., 35 Schulze, A., 293, 294(210), 337(210) Schwabe, K., 392 Schwarz, J. C. P., 138

Schwarz, V., 351 Schwarzenbach, D., 93 Schweiger, R. S., 73(75), 76 Scott, K. J., 339, 356(482) Seebach, D., 81 Sellmair, J., 72(10), 74 Selvendran, R. R., 271, 272(48) Selvendran, S., 271, 272(48) Semenza, G . , 23 Sen, S. P., 348 Senda, M., 21 Senna, K., 106 Seo, K., 139, 140(23), 141(23), 142(23), 143(29), 144(34), 145, 146, 147(43), 148(43), 161(43), 168(23), 169(23), lgl(23.29, 34, 43) Septe, B., 133 Sepulchre, A.-M., 82, 90, 96, 133 Serianni, A. S., 19, 26(20a), 31(20a), 32(80a), 36(20a), 37, 46(80a, 82), 65(82), 194 Seta, A., 99 Seto, H., 230, 262 Seto, S., 195, 196(8), 197(8), 211(8) Seymour, F. R., 202, 203(39), 204(39) Shafizadeh, F., 69, 101 Shah, S. W., 76 Shallenberger, R. S., 22, 64(36), 68 Shannon, J. C., 348 Shannon, L. M., 309 Sharon, N., 277,307,309 Sharpless, K. B., 98, 114 Shashkov, A. S., 96, 200, 205, 206(44, 45), 207(47), 209, 211(46), 212(47), 213, 214(71), 218(56), 220(44), 221(26, 44, 45, 46), 222(45), 223(46) Shaw, D. F., 18 Shealy, Y. F., 116 Shemyakin, M. M., 72(26), 74 Sherman, W. R., 22 Shibaev, V. N., 205, 206(44), 220(44), 221(44) Shibasaki, M., 112 Shibata, M., 73(77), 76 Shibata, S., 197, 199(16) Shibuya, N., 392 Shigemasa, Y., 105 Shimasak, A,, 383, 384, 387(3) Shimyrina, A. Ya., 96 Shin,C., 80, 82(120), 84(120). 90, 133(120, 181) Shindy, W., 345, 348(441, 447)

AUTHOR INDEX Shinkai, T., 384, 387(3) Shinohara, M.,72(27), 74 Shinninger, T. L., 331 Shirahata, K., 73(54), 75 Shotwell, 0. L., 72(13), 74 Shuto, S., 110 Siddiqui, I. R., 269, 280, 283, 284(165), 287, 288(184, 185), 289(185) Siegel, S. M.,312, 347 Sillerud, L. O., 225 Simpson, L. B., 21 Singh, L. B., 339 Sinnwell, V., 55, 81, 82(135), 126 Sirimanne, P., 23, 24(49) Sisler, E. C., 344 Slanski, J. M.,6 3 Sletzinger, M.,150, 188(48) Smedley, S., 82 Smillie, R. M.,364 Smith, C., 73(65, 67), 75, 76, 126(65) Smith, C. J., 285, 291(175) Smith, C. T., 292, 314(200) Smith, C. W., 125 Smith, F., 58 Smith, I. C. P., 202, 203(38), 204(38) Smith, M. M.,293, 294(203), 328, 382 Smith, P. J. C., 25, 27(55), 269, 277, 305(135) Smith, R. M.,73(47), 75 Sokolowski, J.. 28 Sol, K., 301 Soll, H., 341, 348(404) Solomos, T., 366 Somers, J. H., 165 Somogyi, M.,388 Song, C. W., 137 Sonntag, P., 268 Sowden, J. C., 109 Sox, H. N., 376 Spelsburg, T. C., 348 Spencer, F. S., 317 Spiegelberg, H., 79 Spiers, J., 364 Sprinzl, M.,92, 93, 232, 257(38) Sridhar, R., 137 Srivastava, R. M.,101 Srivastava, S. M.,45, 64(115c) Srivastava, V. K., 211, 212(60) Stacey, M.,58 Stadler, P., 81. 82(135) Staehelin, L. A,, 332, 333(367), 334(367), 335(367)

417

Stahl, C. A., 358, 359(530, 534, 535) Stahly, E. A., 342, 348(410, 419) Stammer, C. H., 73(37), 7 5 Stanacev, N. Z., 188 Stanley, R. G., 351 Stark, W. M.,73(46), 75 Starkloff, A., 98 Steele, I. W., 306 Steele, J. C. H., Jr., 165 Stein, M.,364, 372 Steinert, K., 49 Stenzel, W., 81 Stepinsky, J., 212 Sterling, C., 380 Stern, F., 268 Stevens, C. L., 49 Stevens, J. D., 18, 19(8), 46(8), 58, 60, 68(167a) Stewart, T. W., 325 Stiller, E. T., 145, 191(36) Stoddard, R. W., 305 Stoddart, J. F., 24 Stodola, F. H., 72(13), 74 Stokes, E., 352 Stone, B. A., 287, 293, 294(203), 391 Stork, G., 112 Stother, J., 339 Stransky, H., 72(9), 74 Strominger, J. L., 323, 324(330), 327(327, 330) Stroude, E. C:, 137 Stuart, R. S., 211 Stube, M.,81 Sturgeon, R. J., 292 Suami, T., 116, 117(344) Subhadra, N. V., 363 Subramanyam, H., 339, 341(384), 356(384), 361(384), 362, 363(384), 37 2 (384) Sudoh, R.,99 Suggett, A., 24 Sugiura, M., 40, 66(103), 68(103) Sugiura, T., 229, 234(5) Sugiura, Y.,197, 199(16) Sugiyama, H., 34, 45(90a), 195, 196(8), 197(8), 211(8) Suhadolnik, R.J., 230, 261, 262(8, 70) Sumfleth, B., 125 Sumfleth, E., 81 Sun, K. M.,112 Sundberg, R.L., 4 5 Supp, M.,42

AUTHOR INDEX

418

Sutherland, E. W., 367 Suvalova. E. A., 172 Suzuki, N., 72(31), 74, 125(31) Suzuki, S., 72(31), 74, 125(31) Suzuki, T., 112 Svensson, S., 173, 176(80), 222, 223(80), 276 Sveshnikova, M. A., 73(44), 75 Sviridov, A. F., 96 Swahn, C. G., 45 Swanepoel, J. H . , 364 Swanson, A. L., 321 Sweeley, C. C., 22, 68(32), 210, 323, 324(330), 325(328), 327(328, 330), 329(328) Swenson, C. A., 20,21(25) Swern, D., 179, 182(88), 232, 240(34) Sydow, G., 392 Sydow, H., 392 Symons, M. C. R.,20 Szafranek, J., 28 Szarek, W. A., 51,54, 57.78, 79, 82(101), 94, 107, 128, 129(395), 232, 234(.37) Szczerek, I., 57 Szechner, B., 177, 183 Szeytli, J., 199 Szmant, H. H., 34 Szurmai, Z., 64 T Tabeta, R.,209 Tachimori, Y., 99 Taga, T.. 16, 44(2), 47 Tagawa, K., 386, 387(9, 10, 12). 389, 390(17), 391(10, 11, 16), 392(11) Tager, S., 76 Taiz, L., 271, 275(61), 307(61), 314(61), 344, 359(437, 438) Takagi, K., 150, 151(47), 179(47, 52) Takahara, M., 116 Takahashi, T., 112 Takai, N., 23 Takamoto, T., 99 Takamura, T., 202 Takao, H., 96 Takayanagi, H., 146, 147(43), 148(43), 161(43), 191(43) Takeda, K., 123 Takeda, T., 197, 199(16) Takeda, Y., 107

Takeuchi, S., 73(70), 76 Taki, H . , 383, 384, 387(3), 388, 392(26) Talamo, B., 323, 325(331), 327(331) Talmadge, K. W., 271, 272(55, 56, 57), 273(55, 57), 274(55, 56, 57), 275(55,56, 57), 276(55, 56), 277(55, 56), 278(55), 280(55), 282(55), 283 (55), 28 4 (55), 287(56), 288 (56), 289(56), 291(56), 294(56), 298(55), 299(57), 202(56, 57), 303(57), 304(57), 305(55), 306(55, 56, 57), 307(56,57, 65), 309(57), 310(56, 57, 264), 311(57, 264), 312(57, 264). 314(56, 57, 264), 317(56), 321(55), 338(56, 57), 355(57), 368(55, 56, 57), 369(55,56,57), 373(55,56,57), 376(57), 378(57), 379(56) Tamaoki, T., 73(54), 75 Tamari, M., 188 Tanahashi, E., 53 Tanaka, M., 387, 389(14), 391(14) Tanaka, Y., 142, 168(26) Tanimoto, E., 348, 351, 352 Tanner, F. W., 72(19), 74 Tanner, W., 327, 328(347), 329 Tanno, Y.,116 Taravel, F. R.,200, 201(29), 216, 217(74) Tatchell, A. R.,80, 82(110) Tatsuoka, S.,73(57), 75 Tatusta, K., 110, 133 Taylor, K. G., 49 Taylor, R. L., 276 Tejima, S.,202 Temeriusz, A., 212 Teresa, J. de P., 72(18), 74 Terui, G., 389 Tesarik, K., 23 Thaisrivongs, S., 110 Thanbichler, A., 72(10), 74 Thang,T. T., 91, 98, 120(243), 122(184) Theander, O., 110, 283, 284(166) Theologis, A., 366 Thiem, J., 37, 90, 103 Thimann, K. V.,350, 356(483) Thegersen, H., 194 Thom, D., 269, 277, 305(135) Thomas, D. S., 351 Thomas, L. C., 142 Thompson, A. H., 342, 348(410) Thornber, J. P., 268 Thorpe, T. A., 272 Tidder, E., 271

419

AUTHOR INDEX

Timell, T. E., 269, 281 Todd, A. R., 179 Todt, K., 48, 50(124), 52(134), 137 Togashi, M., 72(17), 74, 76(17) Toman, R.,269, 281, 282, 283(161) Tomita, F., 73(54), 75 Tomoda, M., 200,201(27) Torgov, V. I., 200, 205, 206(44), 220(44), 221(26, 44) Toya, T., 77 Toyokuni, T., 116, 117(344) Tracey, M. V., 380 Tran, T. Q., 35, 36(92), 40, 65(92), 66(92) Trecker, D. J., 231 Trewavas, A. J., 348,358 Triantaphylides, C.,19, 20(20), 23(20), 38(20), 65(20) Trifonoff, E., 201, 202(30) Trindale, G. B., 363 Tripp, V. W., 272 Trnka, T., 45, 47 Tronchet, J. (F.), 78, 131 Tronchet, J. M. J., 78, 92, 93(189), 131, 132(405) Tronchet, M. T., 92, 93(189) Trout, S.A., 363 Truesdale, L. K., 114 Tsai, C. M., 320 Tsang, R., 112 Tschesche, R., 73(38), 75 Tschiersch, B., 392 Tsuchiya, T., 53, 133 Tsuchiya, Y.,142 Tsukada, S.,209 Tsukiura, H., 72(16), 74 Tsuruoka, T., 136 Tucker, G. A,, 371 Tucker, L. C. N., 59, 122, 1 2 3 Tulinsky, A., 73(58), 75 Tulshian, D. B., 29, 31(74), 33(74), 112 Turner, J. C., 50 Turner, J. E., 272 Tuzimura, K., 195, 196(8), 197(8), 211(8) Tyler, P. C., 95, 101

U Uchida, T., 387, 389(14), 391(14) Uchiyama, T., 40 Uddin, M.,278, 280(142) Ueda, T., 110

Uematsu, S., 350, 356(486) Uematsu, T., 230, 262(8) Uesaka, E., 388, 390(24), 391(24) Ugami, S.,136 Uh, H. S . , 132 Umezawa, H., 72(12), 74, 94, 107 Umezawa, S.,55, 56, 133, 261 Unger, F. M., 42 Unruh, J., 64 Urarnoto, M., 73(55), 75 Usov, A. I., 213, 214(71) Usui, T., 34, 45(90a), 195, 196(8), 197(8), 200, 201(27), 211(8) Utille, J. P., 216, 216(73) Utkin, L. M., 77 V Valent, B. S.,271, 272(59), 274(59), 275(59), 288, 289(189), 296(59), 306(59), 310(59), 314(59), 338(59), 358 Valente, L., 98, 119, 120(243) Valentine, K. M., 195, 196(9), 200(9), 202(9), 211(9) Valkovich, G., 328 van Bekkum, H., 34 Vanderhoef, L. N., 341, 348(403), 358, 359,360 van Es, T., 179 Van Loesecke, H. W., 365 Van Overbeek, J., 342, 348(418, 420) Van Wielink, J. E., 300, 301(235) Varner, J. E., 354 Varo, P., 23, 62 Vasileff, R., 115 Vass, G., 82, 90, 96 Vaterlaus, B. P., 79 Vaughan, G . , 58 Vazquez, D., 72(20), 74 Vegh, L., 53 Venis, M. A., 348 Verhaar, L. A. T., 2 3 Verheyden, J. P. H., 104 Vethaviyasar, N., 59, 111 Vidauretta, L. E., 2 3 Vigevani, A,, 72(24), 74 Vignon, M. (R.), 196, 197(10), 198(10, 17). 199(10, 17), 216, 217(74), 281, 283( 157) Vijayalakshmi, K. S.,27 Villemez, C. L., 317,318,319,320(301),

420

AUTHOR INDEX

321, 322, 327, 329, 330(361), 331(361), 352(321) Vincendon, M., 196, 197(10), 198(10), 199(10) Vinogradov, L. I., 172 Vioque, A., 344 Vioque, B., 344 Virudachalam, R., 47 Vishveshwara, S., 54 Vliegenthart, J. F. G., 42 Voelter, W., 133, 200, 211(24) Voll, R. J., 27, 43, 46(69a) Vhllmin, J. A., 22 Vongerichten, E., 72(4), 74 vonSonntag, C., 19, 20(20), 23(20), 38(20), 65(20) Voragen, A. G. J., 374 Voser, W., 73(66), 76 Vottero, P. J. A., 216, 217(73) Vyas, D. M.,78 W Wada, S.,271, 273(53), 285, 292(53), 294(53), 314(53), 332, 348, 351, 352 Wagman, G. H., 73(41), 75 Wagner, H., 206, 211, 212(66) Wainright, T., 294 Wakae, M., 72(16), 74 Wakai, H., 9 2 , 9 3 Walaszek, Z., 19, 64, 65(18a) Walker, D. L., 94, 106, 125 Walker, J. E., 351,371(499), 377(499), 379(49 9) Walker, K. A. M., 98 Wall, H. M., 82 Wall, J. S.,272 Wallner, S.J., 351, 371(499), 372(501), 376, 377, 378(501), 379(499), 380 Walter, E., 82 Walton, D. J., 276 Walton, E., 73(37), 75, 96 Wander, J. D., 31, 81, 133 Wang, C.-C., 138, 139(19, no), 140(19), 142, 191(19) Wang, C. Y., 364 Wang, S. Y.,343, 344 Ward, D. D., 101 Watanabe, F., 284 Watanabe, Y.,229, 236(6) Watson, R. R., 72(3), 74, 76, 132

Watters, J. J., 33 Weakley, T. J. R., 81, 93, 107, 108(298), 126(141), 133(298) Weaver, O., 72(23), 74 Weaver, R. J., 340, 341(397), 342(397), 344(397), 345(397), 348(397, 412, 418, 420, 441, 446, 447). 361(397), 362(397) Webber, J. M., 97, 179 Weeks, C. M., 183 Wehrli, F. W., 194 Weidenwiille, H. L., 73(38), 75 Weigand, J.. 93, 112 Weigel, L. O., 96 Weinges, K., 216 Weinstein, L., 271, 274(63), 275(63), 277(63), 282(63), 388, 391(27), 393, 394(27) Weinstein, M. J.. 73(41), 75 Weiss, A. H., 105 Wells, W. W., 22, 68(32) Welsh, E. J., 269, 277(32), 306(30), 307(30), 315(30) Welzel, P., 73(39, 40). 75 Wertz, P. W., 22, 32,64(37) Wessel, H.-P., 37 West, C., 362, 363 Westerlund, E., 41 Westwood, J. H., 45 Weurman, C., 371 Weygand, F., 80 Whaley, W. G., 302, 331(249) Wharry, S. M., 52(140a), 53 Wheen, R. G., 20, 31(23), 33(23), 36(23) Whelan, W. J., 293 Whistler, R. L., 52, 53(139), 110, 137, 138, 139(19, 20), 140(19), 142, 146, 177, 181, 191(19), 272, 276, 277( 131) White, A. C., 81, 84(130) Whiting, J. E., 30 Whittington, S. G., 25, 27(56) Whitton, B. R., 59 Whyte, J. L., 278, 280(141) Whyte, J. N. C., 278,283, 284(168) Wickberg, B., 73(72), 76 Wiebers, J. L., 367 Wiersma, P. K., 353 Wiesner, K., 20, 21 Wight, N.J.. 287, 288(181) Wilbur, D. J., 19, 34(19), 64(19)

AUTHOR INDEX Wilcox, C. S . , 110 Wilder, B. M., 271, 272(58), 274(58), 275(58), 288(58), 291(58), 338(58), 379(58) Wiley, P. F.,72(23, 32, 33), 74 Wiley, V. H., 72(33), 74 Wilkie, H., 292 Wilkie, K.C. B., 272, 292(81), 293(80), 294(209) Williams, C. A,, 19, 34(19), 62, 64(17, 19), 358, 359(535) Williams, D. T., 104 Williams, E. H., 94 Williams, J. F.,46 Williams, J. M., 110 Williams, M. W., 342, 348(419) Williams, N. E., 47 Williams, N . R., 78, 79, 80(97), 81, 82(97, 99, 107, 109). 84(130), 89, 90(118), 107, 108(292), 133(292) Williams, R. H., 80, 82(110) Wills, R. B. H., 339, 356(482) Winter, H., 353 Winternitz, F.,91, 122(184), 205(48), 206, 207(48), 211(48) Wisniewski, A., 28 Witteler, F.-J., 73(40), 75 Wober, G., 73(76), 76 Wold, J. K., 272 Wolf, H., 73(68), 76 Wolf, J., 365 Wolfe, S . , 49 Wolff, G. J., 65(175), 66 WoIfrom, M. L., 72(11), 74 Wolinsky, J., 115 Woo, S . L., 292, 294 Wood, P. J., 269, 280, 283, 284(165), 287,288(184, 185), 289(185) Wood, T.M., 272, 283, 284(167), 285(167) Woodward, R. B., 113 Woolard, C . R., 92, 128, 272, 292 Wooltorton, L. S. C., 361, 362(537, 538, 539), 363(537, 538, 539), 364(539) Worth, H. G. J., 277, 305(136) Wouts, W. M., 387, 388(22) Woychik, J. H., 272 Wozney, Y. V., 211 Wray, V., 45 Wright, A., 324, 327(333) Wright, D. E., 73(65), 75, 126(65)

421

Wright, J. J., 104 Wylde, R., 205(48), 206,207(48), 211(48)

Y Yahya, H. K., 107, 108(294) Yamada, K., 107 Yamaki, T., 350, 356(486) Yamamoto, H., 73(77), 76, 96, 139, 140(23), 141(23), 142(23,24), 143, 144(34), 149(33), 150, 151(47), 152, 153(53,54), 157(46), 158, 160(53, 54), 161(53, 54, 55, 65), 164(54), 165(53, 54, 65), 166(53, 54). 168(23, 33, 46, 55, 65), 169(23, 33, 55, 65), 173, 179(47,52, 66, 67), 180(66, 67), 181(33),183(66), 184(46, 55, 65, 66,67), 187(67, 81), 188(89), 189(33), 191(23, 33, 53, 54,65,67,89), 200,201(28), 202(28) Yamamoto, K., 150, 151(47), 152, 153(53, 54), 158, 160(53, 54), 161(53,54,55,65), 164(54), 165(53, 54, 65), 166(53, 54), 168(55, 65). 169(55, 65), 179(47, 52). 184(55, 65), 191(53, 54, 65) Yamamoto, R., 272, 293, 294(202) Yamaoka, N., 195, 196(8), 197(8), 211(8) Yamashita, A., 96, 131 Yamashita, M., 146, 147(43), 148(43), 149, 150, 151(47), 153(53), 155, 156(60), 157(46, 59, 60), 158, 161(43,45,53,65), 165(45,53, 59, 60, 65), 166(45, 53), 168(46, 65), 169(65), 177(58), 179(47, 52, 67), 180(67), 181, 184(46, 65, 67), 187(67), 188(89), 191(43, 53, 60, 65, 67, 89) Yamaura, M., 121 Yamazaki, N., 80, llO(122) Yamazaki, S., 119, 134(354) Yanagisawa, H., 94, 107 Yarotskaya, L. V., 379 Yarotsky, S . V., 213, 214(71) Yasuda, A , , 107 Yasui, T., 388 Yasumori, T.,85, 91, 123(186), 133(165) Yasuoka, N., 150 Yates, D. W., 32 Yates, J. H., 163, 183(70) Yonehara, H., 73(70), 76, 230, 262(9)

422

AUTHOR INDEX

Yoshida, H., 138, 139(20), 142, 145, 146, 147(43), 148(43), 155, 156(60), 157(60), 161(43), 165(60), 168(26), 177(58), 189, 190(106, 107), 191(43, 60) Yoshida, K., 80, llO(122) Yoshihara, O., 383, 386, 387(12), 388, 389(12), 390(12, 19). 391(12, 28), 392, 394(44) Yoshii, E., 123 Yoshikane, M., 155, 177(58) Yoshimura, J., 61, 80,81,82(113, 121), 84(120), 85, 86(168), 88(166), 90(113, 166), 91, 92, 93, 94, 100, 108(163, 164), 118, 119, 120, 121, 122, 123(186), 126, 127, 133(113, 120, 164, 165, 181, 183, 191, 365, 390), 134(354) Younathan, E. S.,27, 43, 46(69a) Young, J. R.,272 Young, P. E., 361, 362(540), 363(540), 364(540), 365(540), 369, 371(615), 379, 380(679) Young, R. C., 142, Youssef, A., 272 Youssefyeh, R.D., 104 Yu, R. K., 225

Yu, Y. B.,343, 344 Yule, K. C., 138 Yunker, M. B., 101, 106 Z Zaehner, H., 188 Zihner, H., 73(66, 68), 76 Zakir, U., 93 Zalkow, V.,30 Zamojski, A., 113, 177, 183 Zauberman, G., 370,371(633), 379, 380(678) Zbiral, E., 130 Zen, S., 107, 210 Zhakhrova, I. Ya, 212 Zhdanov, Yu. A., 80, 91 Ziegler, D., 42 Zimin, M. G., 172 Zinke, H., 107, 108(296, 2971, 133(297) Zitko, V., 280 Zoughi, M., 301 Zuluaga, E. M., 342, 348(413) Zurfluh, L.L.,359 Zweig, J. E., 202, 203(39), 204(39) Zwierzchowska, Z., 177, 183

SUBJECT INDEX

A Aldoheptoses, composition in aqueous solution, 35-36, 64-65 Acetaldehyde in aqueous solution, 30 Aldohexopyranoses, relative free energies Acetamido groups, oligosaccharides of, 25-26 containing, W-n.m.r. data for, 209Aldohexose(s) 210 in aqueous solutions Acetobncter suboxydans in dendroketose composition, 34-35, 63-64 synthesis, 129 n.m.r. spectroscopy, 18 Acetylation of amino sugars, effect on deoxy-, composition in aqueous behavior in solution, 47 Acid invertase in plant cell-walls, 301 solution, 35 ketonucleosides from, 237 - 240 Acid phosphatase in plant cell-walls, 301, oligosaccharides containing 302 W-n.m.r. data for, 200-202, Acids 205- 207 effect on reducing sugars in solution, 34 ketonucleoside stability in, 245 - 246 glycosides of, %-n.m.r. data for, 211-212 Acyclic carbonyl forms of reducing sugars ,5-acetamido-5-deoxy, composition in in solution, 16, 17, 29-30 determination of, 20-22 aqueous solution, 51 -, 5-O-methy1, in aqueous solution, 16 Adenine keto derivatives of, synthesis, 234 -, 2,3,4,5-tetra-O-methyI, in aqueous solution, 29, 31 -, arabinofuranosyl-, biosynthesis of, 230 -, 9-P-D-arabinosyl-,biosynthesis of, 262 Aldol addition in branched-chain sugar synthesis, 104- 105 -, (2-keto-threo-pentofuranosyl)-, Aldopentofuranoses, 4-deoxy-4-phossynthesis of, 232 phinyl, synthesis and structures of, -, 7-(5-S-methyl-5-thio-P-~-ribosyl)-, 181- 183 biological activity and structure of, 135 Aldopentofuranosylpyrimidines, keto Adenine nucleoside, antiviral activity of, derivatives of, 227-229 131 Aldopentopyranoses, relative free S-Adenosyl-L-methionine,as methyl energies of, 25-26 donor, 321 Aldopentoses Agaricus campestris, a-L-arabinofuranosicomposition in aqueous solution, dase of, 387 Alanine, L-, in cell-wall glycoproteins, 298 34-35,63-64 ketonucleosides of, 229 - 230 Albersheim model for plant primary-wall structure, 275, 303, 304, 338 Aldopentosylpyrimidines, keto derivatives discussion of, 309-314 of, 232 Aldopyranose(s) Aldehydo form of reducing sugars in solution, 29-30, 35 aqueous equilibria of, 25, 26 determination of, 20 - 22 n.m.r. spectroscopy, 19 Aldehydrol, formation in aqueous -, 5-deoxy-5-phosphino- and -5-phossolution, 30 phinyl-, Aldgarose ORTEP representation, 163 natural occurrence of, 7 3 structural analysis of, 161-176 structure of, 7 1, 78 Aldoses synthesis of, 81 anhydrides of, formation in aqueous Aldofuranoses, in aqueous solution, n.m.r. solution, 35 spectroscopy, 19 in aqueous solution

-

423

424

SUBJECT INDEX

composition, 21, 34-37 liquid chromatography, 2 3 - 24 hemiacetal formation in (dtagrum), 137 Aldosuloses, synthesis of, 261 Aldotetroses, 1 6 composition in solution, 36-37 -, 4-acetamido-4-deoxy-, composition in aqueous solution, 5 1 Algae, polysaccharide biosynthesis in, 323-327,332,333 Allium porum, cell-wall studies on, 300 Allose composition in aqueous solution, 26, 28, 31, 6 3 composition in nonaqueous solvent, 6 8 -, 2-acetamido-2-deoxy-~-,composition in aqueous solution, 47, 67 -, 2,3-anhydro-o-, composition in aqueous solution, 59-60 -, 3,6-anhydro-~-,composition in aqueous solution, 3 1 , 5 8 - 5 9 -, 3-deoxy-3-C-nitromethyl-~-,composition in aqueous solution, 5 7 -, 3-O-methyl-~-,composition in aqueous solution, 44 Alpha amylase in fruit climacteric, 364 in plant cell-wall purification, 273. 294 Altrose composition in aqueous solution, 26, 6 3 composition in nonaqueous solvents, 6 8 liquid chromatography of, 23 - 24 -, 3,4-anhydro-~-,composition in aqueous solution, 60 -, 6-deoxy-4-thio-o-, composition in aqueous solution, 53 -, 2,3-di-O-methyI-~composition in aqueous solution, 43, 44 composition in nonaqueous solvent, 61 Amicetin, structure of, 229 Aminal, formation of, 133 (Arninoethoxy)vinylglycine (AVG), effect on fruit ripening, 363-364 Amino groups, oligosaccharides containing, W-n.m.r. data for, 209-210 Amino ketonucleosides, synthesis of, 257 Amino nucleosides, synthesis of, 245, 257 Amino sugars biological activity of, 135-137

composition in aqueous solution, 42, 46-52,67 nucleosides of, 230 Amipurim ycin natural occurrence of, 77 structure of, 77 Arnyloids, xyloglucan and, 287 Angiosperms, plant cell-wall formation in, 268- 269 Anthrone reagent, for plant cell-wall residues, 275 Antibiotic A35512B, branched-sugar in, 7 8 Antibiotics branched-chain sugars from, 54-56, 69-77 from ketonucleosides, 261 nucleoside type, 230 synthesis of, 230-231 Antileukemic activity of ketonucleosides, 23 1 Antimycin A, synthesis of, 129 Antitumor activity of ketonucleosides, 262- 264 Antiviral activity of ketonucleosides, 263 Apiogalacturonan, in plant cell-wall, structure, 281 Apiose chemistry and biochemistry of, 76 composition in aqueous solution, 54-55 natural occurrence of, 69, 76 nucleosides, immunosuppressive activity of, 131- 132 poly-, in plant cell walls, 131 structure of, 70 synthesis of, 78, 80, 104, 107, 113-114 D-, in plant cell wall polymers, 280, 281 L-, synthesis of, 80 Apple cell-wall studies on, 280 during ripening, 315, 369 development physiology of, 340, 341, 343,371-376,378,380 Apricot, development physiology of, 341 Arabinan, 383 enzyme for, 3 8 5 , 3 8 6 L-, as endo-L-arabinanase substrate, 394 as a-L-arabinofuranosidase substrate, 390,391 in plant cell-wall, 375 interconnections, 305

SUBJECT INDEX

-

structure, 281 -282, 286 Arabinanase, endo-(1 5 ) - a - ~ occurrence of, 392 in plant cell-wall fractionation, 277, 282,394 properties of, 393, 394 purification of, 392-393 substrates and activity of, 385 Arabinofuranosidase, a - ~ from Aspergillus niger, 386 assay of, 388 effect on cell-wall glycoprotein, 380-381 occurrence of, 386-387 pH optima of, 387-388 properties of, 389 - 392 purification of, 389 substrates and activity of, 385 Arabinofuranoside p-nitrophenyl a-L-, as enzyme substrate, 384,390 as enzyme substrate, 384, phenyl a-L-, 390 Arabinogalactan L-, as a-L-arabinofuranosidase substrate, 390 in plant cell-walls, 283- 285, 287 interconnections, 303 -304, 307, 309,311 structure, 284-285 Arabinoglycose in xyloglucans, 288 Arabinono-l,5-lactone, 4-C-[ l(S)-methylethyl]-2,3-O-methylene-~natural occurrence of, 73 structure of, 71 synthesis of, 126 Arabinooxylan, L-, as a-L-axabinofuranosidase substrate, 390, 391 Arabinopyranoside, p-nitrophenyl a - ~ -as, a-L-arabinofuranosidase substrate, 390 Arabinose composition in aqueous solution, 26, 43,64 composition in nonaqueous solvents, 68 in pectic polysaccharides, 277, 278 removal from plant cell-wall during ripening, 375-376 L-

in living tissue, 383

425

in plant cell-wall polymers, 281 - 283 -, aldehydo-L-, tetraacetate, aldehydrol formation, 31 -, 5-(benzyloxycarbonyl) amino-5deoxy-L-, composition in solution, 49-50 -, 2,3-di-O-methyl-~composition in aqueous solution, 43, 44 composition in nonaqueous solvent, 61 -, 2,3-di-O-methyl-~-,composition in aqueous solution, 43 -, 5-O-methyl-~-,composition in aqueous solution, 45 -, 4-thio-~-,composition in aqueous solution, 53 -, 2,3,5-tri-O-methyl-~-,composition in aqueous solution, 46 -, UDP-L-,in polysaccharide biosynthesis, 322 Arabinose 5-phosphate, composition in aqueous solutions, 46 L-Arabinosidase(s). 383-394 a-,fruit ripening and, 375,376 classification of, 384 exo-a-, in plant cell wall fractionation, 282 Arabinosyloxy-L-proline-richglycoprotein in plant cell wall, 309 Arabinoxylans in plants aggregate formation by, 307 interconnections of, 314 Arcanose natural occurrence of, 72 structure of, 70, 78 D-, synthesis of, 78, 79 Archaebacteria, thermoacidophilic, branched nonitol from, 76 Arndt-Eistert reaction, 110 Arundo donax, cell-wall studies on, 292 L-Aspartate-oxoglutarateaminotransferase in fruit climacteric, 365 Aspen, cell-wall studies on, 281 Aspergillus niger, a-L-arabinofuranosidase from, 384,386,387,390-392 Auxins in fruit ripening, 341 -345, 348350,351,355 Auena coleoptile, cell-wall studies on, 267, 268,300, 349,352 Avocado, development physiology of,

426

SUBJECT INDEX

341, 343, 363, 369, 371, 372, 379, 380 Axenose natural occurrence of, 72 structure of, 70 synthesis of, 119 Azido ketonucleosides, synthesis of, 257 Aziridino ketonucleosides, synthesis of, 257 B Bacillus subtilis

L-arabinanases from, 384, 391 a-L-arabinofuranasidase from, 387, 388 endo-L-arabinanase in, 392-393 Bacteria cell-wall extension in, 51 polysaccharide biosynthesisin, 323-327 Bamboo, cell-wall studies on, 268 Banana, development physiology of, 363, 369,379 Barium ion, effect on reducing sugars in solution, 33 Barley, cell-wall studies on, 271, 293, 294,314,315 Bases effect on reducing sugars in solution, 34 ketonucleoside stability in, 247-248 Bean cell-wall studies on, 271, 288, 301, 328,351 cyclic AMP in, 367 Beech, cell-wall studies on, 282, 283 4,6-0-Benzylidene-~-hexopyranosid-2 and 3-uloses,nucleophilic reactions of, 86 Blasticidin H,biosynthesis of, 262 Blasticidin S biosynthesis of, 230, 262 structure of, 229 Blastmycinolactol isomers, synthesis of, 129 Blastmycinone natural occurrence of, 73 structure of, 71, 78 synthesis of, 129 Blood-group determinants, glycosides related to, W-n.m.r. data for, 217-219 Blueberry, development physiology of, 341

Borate complexes of cyclitols, aqueous equilibria of, 25 Botrytis cinerea, a-L-arabinofuranosidase from, 387 Botrytis fabae, a-L-arabinofuranosidase of, 387 Branched-chain sugars composition in aqueous solution, 43, 54-58 configuration determination of, 132-134 natural occurrence of, 72 - 73 nucleosides of, 131-132, 230, 244, 245,246 in antibiotic synthesis, 261 synthesis of, 69-134 addition to C-alkylidene glycosides, 91-95 by addition to unsaturated sugars, 97- 103 by aldol addition, 104-105 cyclitols, 129-131 by cyclization of dialdehydes with nitroalkanes, 107- 109 formyl- and hydroxymethyl-branched, 128-129 methyl-branched, 118- 128 by nucleophilic addition to glycosiduloses, 78-91 by nucleophilic reactions of sugar oxiranes, 95 -97 by photochemical addition, 105- 107 by rearrangement reactions, 109- 113 two main groups of, 77 - 78 Brome grass, cell-wall studies on, 271, 287,291,300 Butanal, 4-hydroxy, as hemiacetal in solution, 30 C

Calcium and calcium ion effect on reducing sugars in solution, 33 function in cell walls, 305, 346, 369 Canadensolide, synthesis of, 95 Carbon-13 n.m.r. spectroscopy, 18- 19.62 for branched-chain sugars, 133 for oligosaccharides, 193-225 Carbonyl forms, hydrated, of reducing sugars in solution, 30-32 Carrot cell-wall studies on, 336, 354

SUBJECT INDEX development physiology of, 343 Catalase in fruit climacteric, 364 Cell cultures, plant cell-wall studies using, 272 Cell division in plant growth, 266 Cell elongation in plant growth, 266-267 Cell expansion in fruit ripening, 348-349 Cellobiulose, composition in aqueous solution, 65 Ce1Iuh.w on plant cell-walls, 351,352 Cellulose in algal cell-walls, biosynthesis, 325- 327 in plant cell-walls, 274,294-297 biosynthesis, 317-320,332-337 creep of, 356-357 interconnections, 302-303, 306307,312,314-315,338,355 primary cell-walls, 268 structure, 295-297, 317 Chelation in cell-wall structure, 305,346 Chemical ionization-mass spectrometry of plant cell-wdl components, 276 Cherry, development physiology of, 341 Chill injury of fruits, 339 Chiral synthesis, use in branched-sugar synthesis, 95 Chloral hydrate in plant cell-wall purification, 273 Chloroform, sugar composition in, 60-61 Chromose B natural occurrence of, 72 structure of, 70,78 Chrysanthemumdicarboxylic acids, synthesis of, 97 Circular dichroism of reducing sugars in solution, 21 Citrate lyase in fruit climacteric, 365 Citrus fruits, development physiology in, 363 Cladinose natural occurrence of, 72 structure of, 70,78 D-,synthesis of, 78,79 Clostridium felsineum, a-L-arabinofuranosidase of, 388 Clostridium felsineum var. sikokianum, endo-L-arabinanase in, 392 Coleoptiles, plant cell-wall studies using, 272 -273 Configuration of branched sugars, 132-134

427

Conformational free energies in aqueous solutions of aldopyranoses, 26 Coniferyl alcohol polymer in lignin, 269 Coniophora cerebella, a-L-arabinofuranosidase of, 387 Convoloulus amensis, cell-wall enzymes in, 301 Coriose in aqueous solution, 16,41 Corn, cell-wall studies on, 268,285,292, 293,294,300,314,332 Corticiurn rolfsii, a-L-arabinofuranosidase from, 387,389,390,391 Cotton hairs, cell-wall studies on, 268 Cotylenins, methyl-branched sugars from, 70,76 Coumaric acid, attachment to primarywall polysaccharides, 382 Coumaryl alcohol polymers in lignin, 269 “Cram” addition mechanism, 151 Cranberry, development physiology of, 371 Cremer-Pople puckering parameters for 4-deoxy-4-phosphinylpentofuranoses, 183-184 for 5-phosphonylaldopyranoses,163, 164 Cucumber, development physiology of, 341,342,363,370,371 Currant, development physiology of, 341 Cyclic AMP, in plant tissues, 367 Cyclitols equilibria with borate complexes in aqueous solutions, 25 natural occurrence of, 73,77 synthesis of, 115-118, 129-131 Cyclodextrins, %-n.m.r. data for, 199 Cysteine, N-acetyl-L-, ketonucleoside reaction with, 263-264 Cytidine, keto derivatives of, synthesis, 233 - 234 Cytochrome c reductase, in fruit climacteric, 365 Cytokinins in fruit ripening, 342,343,345 Cytosine nucleosides, preparation of, 253 D Date, development physiology of, 371,372 Dendroketose, Lselective metabolism of, 77 structure of, 77 synthesis of, 128-129

SUBJECT INDEX

428

Deoxy nucleosides, synthesis of, 246 Deoxy sugars in aqueous solution, n.m.r. spectroscopy, 18 Dialdehydes, cyclization with nitroalkanes in branched-sugar synthesis, 107- 109 Diazomethane reaction, transition states in, 89 Dicotyledonous plants Albersheim model of cell-wall of, 309 - 314 cell-wall bound enzymes in, 301 b-D-glucan from, 293 hydroxy-L-proline-rich glycoproteins of, 299 primary cell-wall polysaccharides of, 275 hemicelluloses, 287-292 interconnections of, 303 - 309 pectic polysaccharides, 277-287 Diethylamine, reducing sugar behavior in, 34 N,N-Dimethylformamide in g.1.c. of sugar trimethyl ethers, 22 sugar composition in, 60, 68 Dimethyl sulfoxide amino sugar behavior in, 48 n.m.r. spectroscopy of sugars in, 23 sugar composition in, 60,61, 68 Dimethyl sulfoxide-acetic anhydride method for ketonucleoside synthesis, 232 Dimethyl sulfoxide-dicyclohexylcarbodiimide method for keto derivatives, 232,233,238 Dimethyl sulfoxide-phosphorus pentaoxide method for ketonucleoside synthesis, 232 gem-Diol, formation in aqueous solution, 30,38 Dipterex, biological activity and structure of, 189 Dolichyl phosphates in biosynthesis of cell-wall polysaccharides, 325- 330 Double-sigmoid growth-curve of fruits, 341,344 Douglas fir, cell-wall studies on, 271 Duckweed, cell-wall studies on, 281

E “Egg-box” model of cell-wall for calcium inclusion, 305

Enopyranosides in synthesis of branchedchain sugars, 97-98 Enzymes bound in plant cell-walls, 300- 302 Epiminonucleosides, preparation of, 245, 257 Epoxyketonucleosides, synthesis of, 241 Erythritol, %C-methyl-~-,natural occurrence of, 76 Erythrono-l,4-lactone, 2-C-methyl-~natural occurrence of, 72, 76 structure of, 70 synthesis of, 110 , 121 Erythrose D-,

in aqueous solution, 31, 36-37 temperature effects on, 33 -, 2-C-methyL~natural occurrence of, 72, 76 structure of, 70 Erythrose 4-phosphate, D-, composition in aqueous solution, 46 Ethylene, role in fruit development, 343-344,359,363-365,371 Evalose natural occurrence of, 72 structure of, 70 synthesis of, 97, 120 Evermicose composition in aqueous solution, 56 natural occurrence of, 72 structure of, 70 synthesis of, 114, 119-120 Evernitrose composition in chloroform, 60- 61 natural occurrence of, 73 structure of, 70, 78 synthesis of, 122 -, 3-epf-, synthesis of, 123 Extensin in plant cell-wall, 270, 308,309 biosynthesis, 323,336-337 interconnections, 310

F Fern, cellulose biosynthesis in, 332 Ferulic acid attachment to primary-wall polysaccharides, 382 in plant cell-wall polysaccharide cross-linking, 315

SUBJECT INDEX Fig, development physiology of, 341, 345,363 Flambamycin, 127 Flame ionization gas-liquid chromatography ofplant cell-wall components, 276 Flammulina oelutipes, a-L-arabinofuranosidase of, 387 Flax, cell-wall studies on, 269 Folin-Lowry reagent for plant cell-wall proteins, 275 Fosfonomycin, 188 structure of, 150 French, Dexter, obituary o f , 1- 13 Fructose oligosaccharides containing residues of, W-n.m.r. data for, 203-204 D-,

in aqueous solution composition, 21, 38, 62, 6 5 furanose form stability, 32 inorganic compound effects on, 33 laser-Raman spectroscopy, 2 3 n.m.r. spectroscopy, 18, 62 composition in nonaqueous solvents, 60.68 trimethylsilyl ether, mutarotation of, 22,23 -, 6-acetamido-6-deoxy-~-,composition in aqueous solution, 50 -, 1-deoxy, composition in aqueous solution, 6 5

429

physiology of development of, 340- 382 respiratory climacteric in, 361 -368 ripening cell-wall role in, 315, 339-382 galacturonase and, 381 Fucose nldehydo-L-, tetraacetate aldehydrol formation, 31 -, 2-O-methyl-~-,in cell-wall polymers, 280,281,287 Fungal hyphae, cell-wall extension in, 351 Furanose ring, monosaccharides with phosphorus in, 176-188 Furanoses formation from reducing sugars in solution, 16-68 temperature effects on, 32 - 33 stability in solution, 27 - 29 Fused-ring sugars, composition in aqueous solution, 58 -60

C

Galactan in plant cell-wall polymers biosynthesis, 322 interconnections, 305 structure, 282-283 Galactanase(s) endo-/]-(I 4)-, in plant cell-wall fractionation, 277, 282 on plant cell-walls, 351 -, l-deoxy-3,4,5,6-tetra-O-methyl-~-, Calactoglucomannans in plant cell-walls, 269 keto form in aqueous solution, 31 Galactono- 1,5-lactone -, 3-O-a-~-glucopyranosyl-~-, composi-, 4-C-acetyl-6-deoxy-2.3-O-methylenetion in aqueous solution, 3 9 D-, synthesis of, 126 -, 3-O-methyl-~-,composition in -, 6-deoxy-4-C-[ 1(S)-hydroxyethyll-2,3aqueous solution, 39, 43-44 0-methylene-o-, s-thio-~-,composition in aqueous solution, 53 natural occurrence of, 7 3 structure of, 7 1 -, 6-thio-D-, composition in aqueous synthesis of, 126 solution, 53 Galactopyranosides, p-nitrophenyl, (Y-DD-Fructose 1,6-bisphosphate and /]-D-, 390 in aqueous solution, acyclic forms, 21 Galactose in fruit ripening, 366 liquid chromatography of, 23- 24 Fructose phosphates in aqueous solution methyl glycosides of oligosaccharides composition, 4 6 containing, %-n.m.r. data for, n.m.r. spectroscopy, 2 0 212-213 Fruit (s) in pectic polysaccharides, 277, 278 climacteric of, chemical changes with, 365 D-, in aqueous solution, 16 enlargement during maturation, composition, 31, 26, 28, 63 340-345

-

430

SUBJECTINDEX polarimetry, 17 composition in nonaqueous solvents, 68

trimethylsilyl ethers, mutarotation of, 22 -, 2-acetamido-2-deoxy-5-thio-~-,

composition in aqueous solution, 52 - 53 -, 2-amino-&-deoxy-~-, composition in aqueous solution, 47, 67 -, 4-amino-4-deoxy-~-,composition in aqueous solution, 49 -, 3,6-anhydro-o-, composition in aqueous solution, 58 -, 6-deoxycomposition in aqueous solution, 63 composition in methanol, 68 -, 4,6-diarnino-4,6-dideoxy-o, composition in aqueous solution, 52 -, 2,3-di-O-methyl-~composition in aqueous solution, 43, 44

composition in nonaqueous solution, 61

-, pseudo-a-o-, occurrence of, 116 -, UDP-D-,in polysaccharide biosynthesis, 322, 331 Calactosidase a-D-, in plant cell-walls, 301, 376

p-D,383, 384 in plant cell-walls, 301, 302,

natural occurrence of, 70 structure of, 73 synthesis of, 78, 79, 104 Gas-liquid chromatography of trimethylsilyl ethers of sugars, 22 Gel filtration of plant cell-wall polysaccharides, 274, 275 Gibberellins in fruit ripening, 342-345 Gloeosportum kaki, a-L-arabinofuranosidase from, 387 Glomerella cingulata, a-L-arabinofuranosidase of, 388 o-Glucan(s) chains, in plant cell-wall cellulose, 296 /?-,in plant cell-walls, 235, 293-294 biosynthesis, 323 )-n-Glucan synthetase in plant tissues, auxin effects on, 350 Glucanase (1 (1

--

3)-a-D-, 379 ~)-P-D-,on plant cell-walls, 351, 352,377,379 (1 -,4)-p-D-, endo, in primary plant cell-wall fractimation, 275, 277 , plant cell-wdk, 351 (1 -, 6 ) - a - ~ - on (1 6)-p-n-, on plant cell-walls, 351

-

Clucobiose(s) W-n.m.r. data for, 195-196 peracetates, W-n.m.r. data on, 195 Glucomannan(s) formation in cellulose, biosynthesis, 318-319

373-374,376,377

Calactosyluronic residues in plant cell-wall polysaccharides, 280 Calacturonanase effect on plant cell-walls, 346, 347, 369-372,376-377,381

-.

-, endo-a-(1 4, in primary plant cell-wall fractionation, 275, 270 Galacturonans in plant cell-walls, in ripening, 372, 374 role in structure, 305 Gangliosides, 13C-n.m.r.data for, 224- 225 Galacturonic acid in pectic polysaccharides, 277, 278, 281 -, UDP-, in polysaccharide biosynthesis, 331

-, UDP-D-, in poly(galacturonic acid) biosynthesis, 321 Garosamine 4-epimer of, synthesis, 98

in plant cell-walls, 269 Glucopyranose(s) D-, phosphorus derivatives of, physical properties, 191 -, 5-deoxy-5-phosphonyl-~-, synthesis and structures of, 155-161 Glucosaminidase -, N-acetyl-a-D-, in plant cell-walls, 301 -, N-acetyl-P-D-, in plant cell-walls, 301, 302

Glucose methyl glycosides of, oligosaccharides containing, 13C-n.m.r.data for, 212-213

oligomers of, l3C-n.m.r. data for, 196- 199 D-,

in aqueous solution. 16 composition, 21,26, 31, 34, 35, 63

SUBJECT INDEX inorganic compound effects, 34 polarimetry, 17 polarography, 21 in nonaqueous solvents, 62, 68 nucleotide esters of, 338 in plant glycoproteins, 329- 330 trimethylsilyl ethers of, mutarotation, 22 -, 2-acetamido-2-deoxy-a-~-, biological activity of, 135 structure of, 136 -, 2-acetamido-2-deoxy-5-thio-~-, composition in aqueous solution, 52 - 53 -, ADP-D-,biosynthesis of, 316 -, 2-amino-2-deoxy-~-,composition in aqueous solution, 47, 67 -, 4-amino-4-deoxy-~-,composition in aqueous solution, 49 -, 5-amino-5-deoxy-~as antibiotic, 136, 137 composition in aqueous solution, 49 -, 4-amino-4,6-dideoxy-~-,hydrochloride, composition in aqueous solution, 49 -, 3,6-anhydro-~-,composition in aqueous solution, 58 -, 3,6-anhydro-2,4-di-O-methyl-o, composition in aqueous solution, 58 -, 6-deoxy, composition in aqueous solution, 45, 63 -, 2-deoxy-3,4,6-tri-O-methyl-2(methylamino)-D-, composition in aqueous solution, 47 -, 5,6-diamino-5,6-dideoxy-~-, composition in aqueous solution, 51 -52 -, 5,6-di-O-methyl-~-,composition in aqueous solution, 45, 46 -, 2,3-di-O-methyl-~-,composition in aqueous solution, 44 -, 2-, 3-, 4-, and B-fluoro-~-,composition in aqueous solution, 45 -, GDP-D-,in cellulose biosynthesis, 317-320 in polysaccharide biosynthesis, 329, 330 -, 5,6-O-isopropylidene-~-,composition in aqueous solution, 45 2-O-rnethyl-o-, composition in solution, 34, 45 -, 5-O-methyl-~-, composition in

-.

431

solution, 45, 46 -, 3-O-methyl-~-,composition in aqueous solution, 45 -, 2,3,4,5,6-penta-O-methyl-~-, in aqueous solution, 33 -, 2,3,4,5-tetra-o-methyl-~-, in aqueous solution, 34 septanose form of, 29 -, 1-thio-o-, composition in aqueous solution, 54 -, 4-thio-~-,composition in aqueous solution, 53 -, 5-thi0-11 antitumor activity of, 136-137 composition in aqueous solution, 52 -, 3,4,6-tri-O-methyl-~-,composition in solution, 45 -, UDP-D-, biosynthesis of, 316 in cellulose biosynthesis, 319 in polysaccharide biosynthesis, 322, 325,326,331 Glucose 6-phosphate dehydrogenase in fruit climacteric, 364 Glucosidase (Y-D-, in plant cell walls, 301, 302 P-D-.in plant cell-walls, 301, 302, 379 D-Ghcosykransferase in cellulose biosynthesis, 318 Glucuronoarabinoxylans in plant cellwalls, 285 interconnections, 307, 314 purification, 276 structure, 289, 291, 292 L-Glutamate 1-decarboxylase, in fruit climacteric, 365 Glutathione, ketonucleside reaction with, 263,264 Glycanases, endo, plant cell-wall and, 337,346 Glyceraldehyde, composition in solution, 20, 31, 37 Glycobiose peracetates, W-n.m.r. data for, 216-217 Glycolipid, as intermediate in cell-wall Glycolaldehyde in aqueous solution, 30 polysaccharide biosynthesis, 323324,327,338 Glycoproteins hydroxy-L-proline-rich, in plant cell-walls, 298-300

432

SUBJECT INDEX

oligosaccharides of, W-n.m.r. data for, 219- 220 in plant cell-wall, 337,338 Glycoses, UDP-,biosynthesis of, 316 Glycosidases, 383 in cell walls, 337 classification of, 384 lectins and, 309,337 Glycosides of aldohex ose-containing oligosaccharides, 13C-n.m.r.data for, 211 -212 C-alkylidene, branched-chain sugar synthesis by addition to, 91 -95 related to blood-group determinants, %-n.m.r. data for, 217-219 of Salmonella oligosaccharides, W-n.m.r. data for, 222-223 methyl of oligosaccharides containing galactose and glucose, W-n.m.r. data for, 212-213 of reducing sugars, composition in methanol, 61 of xylose oligomers, %-n.m.r. data for, 213-216 Glycosiduloses, nucleophilic addition to, for branched-sugar synthesis, 78-91 Glycosyl esters in plant cell-wall biosynthesis, 315-323,338 Glycosyl residues in plant cell-wall polymers, 274,276 sequencing of, 276-277 Glycosyltransferase in plant cell-wall biosynthesis, 316 Glycosyluronic nucleosides, synthesis of, 232 Glycosyluronic residues in plant cell-wall polymers, 276 Golgi system, polysaccharide biosynthesis in, 331-332,334,336,338 Gougerotin, structure of, 229 Graminae, ferulic acid in, 3 15 Grape, development physiology of, 341, 344,363,371,378,379,380 Gulose in aqueous solution, composition, 63 pyranose form, 63 stability in solution, 26 -, 2-acetamido-2-deoxy-~-composition in aqueous solution, 47,67 -, 3,6-anhydro-~-composition in aqueous solution, 58

-, 6-deoxy-4-thio-~-composition in

aqueous solution, 53 -, 6-deoxy-2,3-0-isopropylidene-~composition in aqueous solution, 59 Gum arabic, as a-L-arabinofuranosidase substrate, 390,392 Gymnosperms, plant cell-wall formation in, 269

H Halogenoketonucleosides, synthesis of,

244 Hamamelose composition in aqueous solution, 54, 134 composition in nonaqueous solution, 61 natural occurrence of, 69,76 structure of, 70 synthesis of, 78, 80, 128 L-, synthesis, 78, 81 Heavy water, reducing sugar composition in, 63 - 64 HeNanthus coleoptiles, development physiology of, 358 Hemiacetal formation of, 30,133 sugar analogs having phosphorus in ring of, 135-191 biological activity, 188- 190 physical properties of, 191 Hemicelluloses in plant cell-walls, 268-269,274-275,287-292 biosynthesis, 321 -322, 331 -332, 337 bonding to cellulose, 306-307 in fruit ripening, 365,378-379 interconnections, 31 1-312 Hemicelluloses A and B from plant cell-walls, 310 Hemp, cell-wall studies on, 268 Heptose -,D-glycero-D-ido-, composition in aqueous solution, 31,35,36,65 2,3:6,7-di-O-isopropykdene-~-glycero0-gUl0-, composition in aqueous solution, 59 Heptulose(s) composition in solution, 29, 40-42,66 -, D-gluco, in aqueous solution, 17 -, tdo-, in aqueous solution, 17

SUBJECT INDEX -, deoxy, composition in solution, 40 - 42 3-Heptulose(s) trimethylsilyl ethers, mutarotation, 23 -, altro, composition in dimethyl sulfoxide, 6 8 -, D-altro, see Coriose Herpes-I virus, adenine nucleoside activity against, 131 Heteroxylans, in plant cell-walls, 275 e-Hexanone, 6-hydroxy-, acyclic form in aqueous solutions of, 30 2,5-Hexodiulose, D-threo-, composition in aqueous solution, 38 o-threo-2,5-Hexodiu~osonic acid composition in aqueous solution, 40 Hexopyranoses with one amino group, 13C-n.m.r. data for, 210 xylo-Hexopyranoside, methyl 3-C-cyano2,6-dideoxy-3-0-mesyI-O-rnethyl-/3L-, synthesis and structure of, 9 1 a-~-Hexopyranosid-4-uloses nucleophilic reactions of, 87 Hexose(s) n.m.r. spectroscopy, 18 -, 5-amino-deoxy-, composition in aqueous solution, 48-49 -, 6-amino-6-deoxy, composition in aqueous solution, 51 -, 3-amino-2,3,6-trideoxy-~-, composition in aqueous solution, 48 -, 3,6-anhydro, composition in aqueous solution, 58, 59 -, 3-benzamido-2,3,6-trideoxy-~-, (4 isomers), composition in aqueous solution, 61 -, 2-deoxy, composition in aqueous solution, 35, 6 3 -, 2-deoxy-lyxo-, furanose stability in solution, 28 -, 2-deoxy-ribo-, furanose stability in solution, 28 -, 3-deoxy-ribo-, furanose stability in solution, 28 -, 3-deoxy-rylo-, composition in pyridine, 68 -, 6-deoxy-5-C-methyl-o-xylo-, composition in aqueous solution, 57, 58 xylo-Hexose, 3-amino-2,3,6-trideoxy-Cmethyl+ natural occurrence of, 7 3 structure of, 70, 71 synthesis of, 122

433

Hexose C-nucleosides, 4-keto-lyxo-, synthesis of, 232 Hexos-5-ulose, 6-acetamido-6-deoxy-~xylo-, composition in solution, 39 Hexosyl purines, keto derivatives of, 232 Hexosyl pyrimidines, keto derivatives of, 232 Hexosyl residues in plant cell-wall polymers, 276 Hexulose(s) composition in aqueous solution, 30, 37-40,65 nucleosides of, reduction of, 254 -, I-deoxy-, acyclic form in solutions of, 30 -, 6-amino-6-deoxy-, composition in aqueous solution, 48 - 49 -, 1-deoxy, hydrated carbonyl forms of,

-.

31.38

arabino-, composition in aqueous solution, 65 -, xylo-, composition in aqueous solution, 65 Hexulose 1-phosphate, 5,6-dideoxy-~threo-, composition in aqueous solution, 32 Hexulose 6-phosphate furanoses, stability in solution, 27-28 Hexulosonic acids, composition in aqueous solution, 37, 39, 40, 66 2-Hexulosonic acid, orabino-, composition in organic solvents, 68 Homogalacturonan from plant cell-walls, 285 interconnections of, 305 purification of, 276 structure of, 280 Hydroxyaldehydes in aqueous solution, 29-30 hemiacetal formation, 30 m-Hydroxybiphenyl reagent for plant cell-wall residues, 275 Hydroxyketones in aqueous solution, 29 hemiacetal formation, 30 temperature effects on, 33 Hydroxy-L-proline in extensin, 270 Hydroxy-L-proline-rich glycoproteins in plant cell-walls, 298 -300 biosynthesis of, 322, 372-373 cell-wall expansion and, 352-355 interconnections involving, 307- 309 in ripening, 380-381

SUBJECTINDEX

434 I

Iditol, tri-0-acetyl-1,5-anhydro-5-deoxy5-C-[(S)-phenylphosphinylj-t-, physical properties of, 191 Idopyranose(s) phosphorus derivatives of, physical properties, 191 -, 5-deoxy-5-phosphino- and 5-phosphinyl-LCremer-Pople puckering parameters for, 164 bond lengths for pyranoid ring of, 165 ORTEP representation, 163 synthesis and structures of, 145-155 -, 5-(phenylphosphinyl)-~mass spectrometry of, 172- 176 n.m.r. spectroscopy of, 165- 172 X-ray crystallography of, 161- 165 Idose lack of crystalline form of, 16 D-, composition in aqueous solution, 26, 29, 31, 35, 63 -, 6-amino-6-deoxy-~-,composition in aqueous solution, 51 -, 3,6-anhydro-o-, composition in aqueous solution, 58 -, 5-(benzyloxycarbonyl)amino-5,6-dideoxy-3-O-mesyl-~-,composition in aqueous solution, 50 -, 6-deoxy-4-thio-~-,composition in aqueous solution, 53 -, J-C-methyl-~-,composition in aqueous solution, 57 - 58 Indole-%acetic acid (IAA), role in fruit development, 344 Infrared spectroscopy of ketonucleosides, 249 - 250 of reducing sugars in solution, 20 Inorganic compounds, effect on reducing sugars in solution, 33-34 Inosose, as vahenamine precursor, 129 Insect sex-attractant, preparation of, 94 Invertase in fruit climacteric, 364 Ion-exchange chromatography of plant cell-wall polysaccharides, 274, 275 Iris, cell-wall studies on, 300 (2R,3S)-2-Isobutylthrearic acid natural occurrence of, 73, 76 structure of, 71 synthesis of, 127-128 Isodityrosine in cross-linkages of plant cell walls, 382

Isoprenoid intermediates in biosynthesis of bacterial polysaccharides, 324-325 Isopropylidene, formation of, 133 K KDO, see 3-Deoxy-~-manno-2-octu~osonic acid Keta forms of reducing sugars, 29 - 30 determination of, 20-22 Ketoaldonic acids, composition in aqueous solution, 41 Ketoepoxynucleosides, synthesis of, 233 2'-Ketofucosyl nucleosides, synthesis of, 238 Ketoglycosyl nucleosides, unsaturated, 230 Ketohexose nucleosides nucleophilic additions to, 257-258 stability of, 245 synthesis of, 237- 240 unsaturated, nucleophilic additions to, 258-260 (3-Keto-arabino-hexopyranosyl)pyridine, synthesis of, 232 Ketonucleosides, 227 - 264 antitumor activity of, 262-264 biological activity of, 230-231, 261 264 definition of, 227 'H-n.m.r. spectra of, 250-251 infrared spectra of, 249-250 nucleophilic additions to, 257-260 stability of, 245-248 in acidic media, 245-246 in alkaline media, 246-248 stereospecific reduction of, 252- 257 structure and spectroscopic properties of, 249-252 synthesis of, 231-244 epoxyketonucleosides, 240 from ketohexoses, 237-240 from ketopentoses, 233-236 oxidative systems in, 231-233 unsaturated ketonucleosides, 241-244 ultraviolet spectra of, 252 unsaturated, 241 -244, 251, 257, 263, 264 nucleophilic additions to, 258 -260 reaction with protein sulfhydryl groups, 264 stability of, 246 2'-Ketonucleosides, synthesis of, 237 - 238

SUBJECT INDEX 4’-Ketonucleosides, synthesis of, 238 - 240 5’-Ketonucleosides, synthesis of, 240 Ketopentose nucleosides, synthesis of, 233-236 Ketoses in aqueous solution acyclic forms, 21 composition, 26-28, 37-42 n.m.r. spectroscopy, 18- 19 temperature effects, 33 keto hydration of, 31 liquid chromatography of, 24 phosphorylated, in aqueous solution, 32 trimethylsilylation of, 23 -, deoxy, in aqueous solution, 17 n.m.r. spectroscopy, 18- 19 Ketothionucleosides, synthesis of, 233

3'-Ketoth y midines protected, synthesis of, 233 synthesis of, 236 2’-Ketouridines alkali effect on, 247-248 stereospecific reduction of, 252 synthesis of, 232 3’-Ketouridines alkali effect on, 247-248 synthesis of, 232 Kidney bean, cell-wall studies on, 271 Kivirikko-Liesmaa reagent for plant cell-wall residues, 275

L Lactose in aqueous solution, inorganic ion effects, 34 composition in nonaqueous solution, 61 Lactulose composition in aqueous solution, 6 5 composition in dimethyl sulfoxide, 68 Laminitol natural occurrence of, 73, 76 structure of, 71, 78,80, 115 Lamport model for plant primary-wall structure, 309 Larch, cell-wall studies on, 283-285 Laser-Raman spectroscopy of D-frUCtOSe solutions, 23 Lect i n s arabinogalactan properties similar to, 287 binding function in plant cell-walls,

435

309-310,329-330,331-332, 337-338 Lemna spp., cell-wall studies on, 280, 281 Lemon cell-wall studies on, 278, 281 development physiology of, 362-363 Lentinus edodes, a-L-arabinofuranosidase of, 387 Lentinus lepideus, a-L-arabinofuranosidase of, 387 Lignin, in plant cell-walls, 269 Liquid chromatography of sugars, pyranose form separation in, 2 3 - 24 Lucerne leaves, cell-wall studies on, 278, 280 Lupin, cell-wall studies on, 271, 309, 311,313 Lupinus luteus, a-L-arabinofuranosidase from, 387 Lychee, development physiology of, 379 Lymphoblastic leukemia cells, adenine nucleoside activity against, 131 Lymphoblastoid cells, thioguanine nucleoside activity against, 132 Lipoxygenase in fruit climacterics, 365 Lyxofuranose L-, X-ray crystallography of, 161 -, tri-O-acetyl-4,5-dideoxy-4-C-[ (R)phenylphosphiny1)-a-~-,physical properties of, 191 Lyxose composition in nonaqueous solvents, 68 D-, in aqueous solution, 16 composition, 64, 66 polarimetry, 22 -, 4-O-methyl-~~-, composition in aqueous solution, 45 Lyxose 2,3-carbonate, D-, composition in aqueous solution, 59 Lyxose 5-phosphate, composition in aqueous solution, 46 M Magnesium ion, effect on reducing sugars in solution, 3 3 Maize, see Corn Malic dehydrogenase, in fruit climacteric, 365 Malic enzyme, in fruit climacteric, 364 MaItulose composition in aqueous solution, 6 5 composition in dimethyl sulfoxide, 68

436

SUBJECTINDEX

Mango, development physiology of, 340, 341, 347, 361, 363, 369, 372, 378-379 o-Mannan, biosynthesis of, 323-325 o-Mannolipid, as intermediate in polysaccharide biosynthesis, 323 -324 Mannosamine, 4-deoxy, composition in aqueous solution, 47 Mannose composition in nonaqueous solvents, 68 D-

in aqueous solution, 16 composition, 26, 28, 31, 34, 45, 47,63 inorganic ion effects, 34 polarimetry, 1 7 trimethylsilyl ethers, mutarotation of, 22 -, 2-amino-2-deoxy-o-, composition in aqueous solution, 47, 67 -, 6-arnino-6-deoxy-o, composition in aqueous solution, 51 -, 2,3-anhydro-ocomposition in aqueous solution, 59, 60 composition in nonaqueous solvents, 61,68 -, 3,6-anhydro-~-,composition in aqueous solution, 58 -, 3,6-anhydro-2,4-di-O-methyl-~-, composition in aqueous solution, 58 -,6-deoxy composition in aqueous solution, 63 composition in dimethyl sulfoxide, 68 -, 3-deoxy-3-fluoro-~-,composition in aqueous solution, 45 -, 2,3-di-O-rnethyl-o-, composition in aqueous solution, 45 -, CDP-Din cellulose biosynthesis, 317-319 in polysaccharide biosynthesis, 323, 327 -, 2-O-methyl-ocomposition in aqueous solution, 45 nucleoside, preparation, 254 -, 5-0-methyl-D-, composition in aqueous solution, 45, 46 -, 2,3,4,6-tetra-O-methyl-o-, composition in aqueous solution, 45 Mannose 2.3-carbonate. D-, composition in aqueous solution, 59

Mannosidase (Y-D-, in plant cell-walls, 301, 376 p-D-, in plant cell-walls, 301 D-Mannosyltransferase in cellulose biosynthesis, 318-319 Marrow, development physiology of, 380 Mass spectrometry of 5-deoxy-5-phosphino- and 5-phosphinyl-~-idopyranoses, 165- 172 Medlar, development physiology of, 371 Melon, development physiology of, 371 Metal hydrides, ketonucleoside reduction by, 252 Methanol, sugar composition in, 61 Methionine, ethylene biosynthesis from, 343-344 inhibition of, 363-364 Methyl 1,Z-epoxy-1-methylethanephosphonate, synthesis and structure of, 150 Methyl furanosides conformation of, 27 (-)-N-Methylmayserine, synthesis of, 96 Michaelis-Arbuzov reaction in phosphorus sugar synthesis, 139, 142, 143, 145 Micrastedas denticulata, cellulose biosynthesis in, 332-336 Micrococcus lysodeikticus, biosynthesis of cell-wall polysaccharides in, 323325,330 Mildiomycin, 135 structure of, 77 Moenuronic acid natural occurrence of, 73 structure of, 70 synthesis of, 120-121 Molecular sieves in ketonucleoside synthesis, 233 Monocotyledonous plants cell-wall-bound enzymes in, 301 -302 o-glucans from, 293-294 hemicelluloses of, 291 -292 hydroxy-L-proline-rich glycoproteins of, 298-299 pectic polysaccharides of, 285, 287 polymer interconnections in cell walls of, 314-315 Monro model of plant primary cell-wall, 313 Mung bean cell-wall studies on, 308, 309, 311, 320,321,327,331

SUBJECTINDEX development physiology of, 343, 344 Mustard, cell-wall studies on, 281, 282 Mycaral, L-, synthesis of, 103 Mycarose natural occurrence of, 72, 76 structure of, 72 synthesis of, 78, 79, 115 D-, synthesis of, 79 L-, synthesis of, 103 Myrothecium verrucarfu, a-L-arabinofuranosidase of, 388 Mytilitol natural occurrence of, 73, 76 structure of, 73 synthesis of, 78, 79, 115

N Nasturtium, cell-wall studies on, 287 Neuraminic acid N-acetyl, composition in aqueous solution, 41-42 methyl, composition in aqueous solution, 42, 49 Newman projection for phenyl ring, 164 Nitroalkanes in branched-chain sugar synthesis, 107- 109 Nitro-alkenic sugars in synthesis of branched-chain sugars, 99- 100 Nogalose natural occurrence of, 72 structure of, 70, 78 synthesis of, 120 D-, synthesis, 120 Nojirimycin biological activity and structure of, 136, 137 composition in aqueous solution, 49 Nonitol, (hydroxymethy1)-branched, natural occurrence of, 76 Noviose composition in aqueous solution, 58 natural occurrence of, 7 3 structure of, 70 synthesis of, 78, 79 Nuclear magnetic resonance spectroscopy of 5-deoxy-5-phosphino- and 5-phosphinyl-L-idopyranoses, 165- 172 of ketonucleosides, 250-251 of reducing sugars in solution, 16, 18-20.21-23,32,34,62-63

437

Nucleosides of branched sugars, 131- 132 Nucleotide analogs, antibacterial, 189

0 0-antigen of bacterial polysaccharides, biosynthesis of, 324 Oat, cell-wall studies on, 267, 271, 285, 287, 291, 292, 294, 300, 301, 320, 352 -353 Octose

Ycomposition in aqueous solution, 55, 56 natural occurrence of, 73 synthesis of, 126 -, D-erythro-L-tab, composition in solution, 36 -, o-threo-L-tolo, composition in solution, 36 Octulose bisphosphates, composition in aqueous solution, 46 3-Octuloses, composition in aqueous solution, 41 2-Octu~osonicacid, 3-deoxy-~-manno-, composition in aqueous solution, 42 Oligosaccharides with amino or acetamido groups, W-n.m.r. data for, 209-210 W-n.m.r. data for, 193-225 Olive, development physiology of, 341 Olivomycal, L-, synthesis of, 103 Olivomycose natural occurrence of, 72 structure of, 70 synthesis of, 78, 94, 114, 115 L-, synthesis of, 103 Orange, development physiology of, 380 Orcinol reagent for plant cell-wall residues, 275 ORTEP representation of phosphorus sugars, 163, 183 0-substituted sugars, composition in aqueous solution, 43- 46 Overhauser effect, W-n.m.r. spectra and, 19 Oxalyl chloride method for preparation of ketonucleosides, 232, 240 Orporus populinus, a-L-arabinofuranosidase of, 387

438

SUBJECTINDEX

P Pea cell-wall studies on, 267, 282, 300, 327-329,331,350,355-356 development physiology of, 343, 344, 350,352,353,358,359 Peach, development physiology of, 341, 347.370-372,380 Pear, development physiology of, 341, 342,370-372,376,378,379 Pectic polysaccharides in plant cell-walls, 274 biosynthesis, 321 -322, 331 -332, 337 of dicotyledonous plants, 277 -285 fruit ripening and, 343,365, 368-378, 373 gel formation, 277 interconnections between, 304 - 306 of monocotyledonous plants, 285, 287 Pectin, galactan in, 282, 283 Pectin galacturonase, role in cell-wall changes, 369 Pectin methylesterase association with plant cell wall, 337, 369,371-372 in fruit climacteric, 364 P-Pelatin A activation by a-L-arabinofuranosidase, 392 structure of, 392 Pentanal, 5-hydroxy, as hemiacetal in solution, 31 Pentanone, 5-hydroxy-2-, acyclic form in solutions of, 30 %Pentanone 1,5-bisphosphate 1,5-dihydroxyhydrate of, 32 structure of, 32 Pentofuranoses -, 4-deoxy-4-phosphinyl mass spectrometry of, 187- 188 n.m.r. spectroscopy of, 184- 187 X-ray crystallography of, 183- 184 -, 4,5-dideoxy-4 phosphinyl synthesis and structures of, 179-181 ORTEP representation of, 184 -, 2,3,4-trideoxy-4-phosphinyl synthesis and structures of, 176-178 Pentose(s) n.m.r. spectroscopy of, 18 -,5-acetamido-5-deoxy-, composition in aqueous solution, 50

-, 5-(benzyloxycarbonyl)amino-, composition in aqueous solution, 50 -,2-deoxy-~-eythro-,composition in solution, 22-23, 32, 64 -, 3-deoxy-~~-threo-, composition in aqueous solution, 64 -, 4-deoxy-erythro-, composition in aqueous solution, 64 -, 3,5-diacetamido-3,5-dideoxy-, composition in aqueous solution, 50-51 -, 2,3,4,5-tetra-O-acetyyl-aldehydo-, in aqueous solution, 31 Pentose 5-phosphates, aldehydrol forms of in aqueous solution, 31 Pentulose(s) composition in aqueous solution, 37-40,65 -, threo-, composition in aqueous solution, 65 -, 1-deoxy-threo-, composition in aqueous solution, 65 2-Pentulose, 16 1-deoxy-D-threo-, composition in aqueous solution, 38 Pentulose 1,5-bisphosphate, D-eythro-, composition in aqueous solutions, 32 Peptidoglycans of bacterial lipopolysaccharides, biosynthesis, 324 Peroxidase in fruit climacteric, 364 gibberellin suppression of, 343 Persimmon, 371 Phosphatase in fruit climacteric, 364 Phosphinediol group on pyranose ring of monosaccharides, synthesis of, 138-176 Phosphinothricin, biological activity and structure of, 189 Phosphodiesterase in plant cell-walls, 302 Phosphonic acid, 2-aminoethane, biological activity and occurrence of, 188-189 Phosphonyl group on pyranose ring of monosaccharides, synthesis of, 138-176 Phosphorus-31 n.m.r. spectroscopy, 19-20 Phosphorus sugars biological activity of, 188- 190 physical properties of, 191 synthesis and structure of, 135-191

SUBJECT INDEX Phosphorylated sugars in aqueous solution, acyclic forms, 20,21 6-Phosphogluconate dehydrogenase in fruit climacteric, 364-365 Photochemical addition in branched-chain sugar synthesis, 105- 107 Photochemical mt.thod for synthesis of ketonucleosides, 236 Pillarose natural occurrence of, 7 3 structure of, 71, 81, 125 synthesis of, 125- 126 Pineapple, development physiology of, 363,370,379 Pinus (pine), cell-wall studies on, 268 Piptopom betulinus, a-L-arabinofuranosidase of, 387 Plant cell-walls, 265-382 acidification hypothesis for, 349 Albersheim model of, 303, 304, 309314 biosynthesis of, polymers of, 315-338 description of, 266 during cell-expansion of fruits, 345-347 enzymes bound in, 300-302,351 fruit ripening and, 339-382 cell-wall loosening, 347-361 hydroxy-L-proline-rich glycoproteins in, 298-300 interconnections, 307 - 309 interconnections in, 302-315 “loosening” of, 347 - 36 1 diagram, 360 polysaccharides of, see Polysaccharides of plant-cell walls preparation of, 273-274 primary, 267 structure, 269-277, 303 Plasma membrane, description of, 266 Plum cell-wall studies on, 315 development physiology of, 341, 379 Polarimetry of reducing sugars in solution, 17-18 Polarography of aldehydo form of sugars, 20 Pollen tubes, cell-wall extension in, 351 Polygalacturonase in fruit climacteric, 364 on plant cell-wall, 337, 351 -, endo, solubilization of pectic polymers by, 304-305, 372

439

substrate for, 321 Poly(ga1acturonic acid), biosynthesis of, 321 Polyisoprenyl phosphates as possible polysaccharide intermediates, 327-330 Polysaccharide hydrolases ethylene effects on, 363 in fruit ripening, 365 role in cell-wall extension, 351 Polysaccharide synthase localization in cell, 331 in plant cell-wall biosynthesis, 316 Polysaccharides of plant cell-walls acidic, 266 alterations outside plasma membrane, 337-338 biosynthesis of, 315-338 cellulose, 294-297 D-glucans, 293-294 hemicelluloses, 268- 269, 274-275, 287- 292 interconnections among, 303 - 309 pectic polysaccharides, 277- 287 purification of, 274 types of, 274-277 Poria uaporaria, a-L-arabinofuranosidase of, 387 Posidonia, galacturonan of, 281 Potassium ion, effect on reducing sugars in solution, 33, 34 Potato, cell-wall studies on, 308 Prelog-Djerassi lactone, synthesis of, 112 Primary plant cell-wall, see under Plant cell-wall Pronase in plant cell-wall purification, 273 2-Propanone phosphate, 1,3-dihydroxy-2hydrate and keto form of, 32 structure of, 32 Prostaglandin Fpa,synthesis of, 112 Protein synthesis in respiratory climacteric of fruits, 365, 366 Prototheca zopfii cell-wall potysaccharide biosynthesis in, 325-326,330,332,336 diagram, 328 Pseudo-sugar biological activity of, 132 definition of, 116 Psicose lack of crystalline form of, 16 nucleosides of, 227

SUBJECTINDEX

440 D-

in aqueous solution composition, 29, 37, 41, 44, 62, 65 n.m.r. spectroscopy, 19 in methanol, 62 -, l-deoxycomposition in aqueous solution, 65 furanose stability in solution, 29 -3-O-rnethyl-o-, composition in aqueous solution, 44 -, S-O-methyl-~-,composition in aqueous solution, 46 -, 6-O-methyl-~-,composition in aqueous solution, 46 Psicose 6-phosphate, D-, composition in aqueous solution, 46 Pyranoid enolones, in branched-chain sugar synthesis, 102- 103 Pyranoid enones in synthesis of branchedchain sugars, 100-102 Pyranose ring, monosaccharides with phosphorus in, 138- 176 Pyranoses aldo-, anomeric equilibria of, 25, 37 formation from reducing sugars in solution, 16-68 stability, 24-27 temperature effects, 32 - 33 methylated, effect on stability of, 25 polarimetry of, 17 Pyridine, sugar composition in, 22, 60, 61,68

Q Quinic acid structure of, 77 synthesis of, 129

R Radish, cell-wall studies on, 301 Rape, cell-wall studies on, 287 Rapeseed, cell-wall studies on, 278, 282-284 Rare-sugar nucleosides, preparation of, 245,246,257 Rearrangement reactions, branched-chain sugar synthesis by, 109-113 Reducing sugars in aqueous solution, 15- 68

acyclic-form determination, 20- 22 compound separation, 16 composition variation with temperature, 32-33 inorganic compound effects on, 33 n.m.r. spectroscopy, 16, 18-20 polarimetry, 17- 18 stability of various forms in, 24-34 in nonaqueous solvents, 60-62,68 Respiratory climacteric in fruits, 361 -368 Rhamnogalacturan I in plant cell walls changes in, 368,370,373-375,377, 381 interconnections, 305, 309 purification, 276 structure, 278-279 Rhamnogalacturan I1 in plant cell-walls, 287 changes in, 369, 370,377, 381 interconnections, 305, 309 purification, 276 structure, 280-281 Rhamnose oligosaccharides containing, I3C-n.m.r. data for, 205 - 207 in pectic polysaccharides, 277, 278 -, 2,3-O-isopropylidene-~,composition in aqueous solution, 59 -, 2-O-methyl-~-,composition in aqueous solution, 45 L-Rhamnose nucleosides, 'H-n.m.r. spectra of, 251 Rhodotorulajluua, a-L-arabinofuranosidase from, 387,388,390-392 Ribofuranose, D-, phosphorus derivatives of, physical properties, 191 Ribopyranose P-D-, g.1.c. of aqueous solutions of, 23 -, 5-deoxy-5-phosphinyl-~-, synthesis and structure of, 145 -, tetra-O-acetyl-5-deoxy-5-C-(ethylphosphiny1)-D-,physical properties of, 191 Ribose composition in aqueous solution, 26, 64,134 composition in nonaqueous solvents, 68 D-

aldehydo form, detection, 20 crystalline form, 18 liquid chromatography of, 24 -,5-(benzyloxycarbonyl)amino-5-deoxy-

SUBJECT INDEX D-,composition in aqueous solution, 50 -, 2-C-(hydroxymethyl)-~composition in aqueous solution, 54 composition in dimethyl sulfoxide, 68 -, 5-O-methyl-~-,composition in aqueous solution, 46 -, I-thio-~-,composition in aqueous solution, 53 -, 5-thio-~-,composition in aqueous solution, 53 Ribose 5-phosphate, composition in aqueous solution, 46 Riburonic acid, 3-C-(hydroxymethyl)-~natural occurrence of, 76 structure of, 77 synthesis of, 81 Rice, cell-wall studies on, 271, 287, 294, 300 Rifamycin, synthesis of, 96 Ripening of fruit, cell-wall role in, 315, 339-382 Rose cell-wall studies on, 281, 283, 288 Rubranitrose natural occurrence of, 73 structure of, 70,78 synthesis of, 123 D-, synthesis of, 123 L-, synthesis of, 123 Ruthenium tetraoxide method for ketonucleoside synthesis, 232 Rye grass, cell-wall studies on, 271, 291, 293,294,300,315

S

Saccharinic acid nucleosides, preparation of, 248 Salmonella oligosaccharides related to those of I3C-n.m.r. data for, 220-222 glycosides of, W-n.m.r. data for, 222-223 Sclerotinafructigena, a-L-arabinofuranosidase of, 388, 391 Sclerotina libertiana, a-L-arabinofuranosidase from, 387 Sclerotina sclerotiorum, a-L-arabinofuranosidase of, 388

441

ScopoZia japonica, a-L-arabinohranosidase from, 387, 389, 391 Senescence of plants, cell-wall changes in, 315 Septanoses from reducing sugars in solution, 16, 29 L-Serine in cell-wall glycoproteins, 298, 299 ShigellaJexnerf, oligosaccharides related to, 13C-n.m.r. data for, 223-224 Shikimic acid structure of, 77, 78 synthesis of, 129 Sibiromycin, degradation product of, synthesis, 122 Sibirosamine 4-epimer of, synthesis, 98 natural occurrence of, 7 3 structure of, 70, 121 synthesis of, 121 Sinapyl alcohol polymer in lignin, 269 Sisal, cell-wall studies on, 268, 280 Smith degradation of plant cell-wall polysaccharides, 277, 281 -282, 284 Sodium borohydride, effect on ketonucleoside stability, 248 Sodium ion, effect on reducing sugars in solution, 33 Solvents, nonaqueous, reducing sugar composition in, 60 - 62, 68 Sorbose composition in aqueous solution, 32, 37, 38, 41, 65 -, 6-acetamido-6-deoxy-~-,composition in aqueous solution, 50 -, 6-amino-6-deoxy-~-,composition in aqueous solution, 49 -, 1-deoxy-L-, composition in aqueous solution, 65 -, 6-deoxy-, composition in aqueous solution, 38, 65 Sorbose 6-phosphate, composition in aqueous solution, 46 Soybean, cell-wall studies on, 278, 280, 281,283,284,327, 358,359 Spinach, gibberellin effects on cell cultures of, 343 Strawberry, development physiology of, 341,342,346,347,363,373-375, 378,379,381 Streptomyces, branched-chain sugars from antibiotics from, 76

442

SUBJECT INDEX

Streptomyces griseochromogenes ketonucleoside intermediate from, 262 Streptomyces massasporew, a-L-arabinofuranosidase from, 387,388,390,391 Streptomyces purpurascens, a-L-arabinofuranosidase from, 386-391 Streptose natural occurrence of, 72 structure of, 70 synthesis of, 78, 79, 81 DL-, synthesis, 113 -, dihydro-, composition in aqueous solution, 55 natural occurrence of, 72 structure of, 70 synthesis of, 78, 79 -, hydroxy natural occurrence of, 72 structure of, 70 Strontium ion, effect on reducing sugars in solution, 33 Succinic dehydrogenase in fruit climacteric, 365 Sucrose nucleotide esters from, 338 oligosaccharides containing residues of, W-n.m.r. data for, 202-203 Sucrose synthetase, nucleotide ester synthesis by, 316 Sugar cane, cell-wall studies on, 271, 294, 300 Sugar nucleotides, biosynthesis of, 315-316 Sugar oxiranes, use in synthesis of branched-chain sugars, 95- 97 Sugars liquid chromatography of separation, pyranose forms during, 23 polarimetry of, 17- 18 reducing composition in solution, 15-68 methods for studying in solutions, 17-24 substituted and derived, composition in aqueous solution, 42 - 60 Sulfhydryl groups, ketonucleoside reaction with, 264 Sunflower seeds, cell-wall studies on, 280 Sycamore, cell-wall studies on, 271, 275, 277-278,280,262-285,288, 298-305,336,337,358,366,369, 394

T Tagatose composition in aqueous solution, 6 5 -, 1-deoxy-o-, composition in aqueous solution, 38 -, 6-O-methyl-~-,composition in aqueous solution, 38, 46 Tagatose 6-phosphate, D-, composition in aqueous solution, 46 Takadiastase, L-arabinanase activity of, 383 Talose composition in aqueous solution, 63 composition in nonaqueous solvent, 68 -, 6-deoxy-, composition in aqueous solution, 63 -,6-deoxy-~-,nucleoside, preparation of, 254 Tamarindus indica, cell-wall studies on, 287- 289 Tangerine, 371 Tautomeric forms of sugars, 16 Tetronitrose natural occurrence of, 73 structure of, 71, 78 synthesis of, 123, 125 Tetroses, in aqueous solution, 17 Theophylline -, 7-(3-0-acetyI-4,6-dideoxy-P-~-glycero-hex-3-enopyranosyl-2-ulose), antitumor activity of, 263 -, 7-(6-deoxy-~-~-Zyro-hexopyranosyl-2dose), biological activity of, 262-263 Thioguanine nucleosides, biological activity of, 132 Thioketonucleosides, synthesis of, 257 Thionucleosides, preparation of, 245 Thio sugars biological activity of, 135- 137 composition in aqueous solution, 43, 52 - 54 Threonine, L-, in cell-wall glycoproteins, 298 Threose, D-, composition in aqueous solution, 31, 36-37 Thromboxane, synthesis of, 95 Thymine, 1-(6-deoxy-2,3-0-isopropylidene-a-~-lyxo-hexopyranosyl-4dose), antitumor activity of, 263 Tobacco, cell-wall studies on, 284 Tomato cell-wall studies on, 298

443

SUBJECT INDEX development physiology of, 340-343, 369,371,372,377,379,380 Trametes uersicolor, a-L-arabinofuranosidase of, 387 Transaminases in fruit climacterics, 365 Transglycosylase, endo-, in cellulose microfibril “creep,” 357 Triamino sugars, C-branched, synthesis of, 109 Triazolo nucleosides, synthesis of, 257 Trideoxy-nucleosides, preparation of, 245 Trimethylsilyl ethers, of reducing sugars in aqueous solution, 22 Trioxacarcinose B natural occurrence of, 7 3 structure of, 71 Turanose, composition in aqueous solution, 39, 66

U Ultraviolet spectra of ketonucleosides, 252 Undecaprenyl (D-mannosyl phosphate) in cell-wall polysaccharide biosynthesis, 323-324,330 Uracil, keto derivatives of synthesis, 234-235

v Validamine natural occurrence of, 73, 76 structure of, 71, 78 synthesis of, 117 Validatol natural occurrence of, 7 3 , 7 6 structure of, 71, 78 synthesis of, 117 Validoxylamine B, synthesis of, 117 Valienamine natural occurrence of, 73 structure of, 7 1, 78 synthesis of, 129 - 130 Vancosamine natural occurrence of, 73 structure of, 70, 78 synthesis of, 114, 122- 123 Vinelose natural occurrence of, 72, 76 structure of, 70 synthesis of, 119

Virenose natural occurrence of, 72 structure of, 70 synthesis of, 118, 123 Vitamin L2,biological activity and structure of, 135- 136

w Wheat cell-wall studies on, 271, 287, 291,293,294,300,315,327 Willow cell-wall studies on, 281, 282, 283

X Xylan(s) in plant cell-walls, 269, 275 structure, 291-292 Xylanase, 379 Xyloglucans from plant cell-walls, 275,373 biosynthesis, 321 -322 interconnections, 302, 303, 306-307, 310,311,315,355-356 purification, 276, 287 structure, 288-290, 291 P-D-Xylosidase, in plant cell-walls, 301, 376,379 X-Ray crystallography of 4-deoxy-4-phosphinylpentofuranoses, 183-184 of 5-deoxy-5-phosphino- and 5-phosphinyl-L-idopyranoses, 165 - 172 Xylobiose peracetates, n.m.r. data for, 194 Xyloglucan, interconnections of in plant cell-walls, 369 Xylopyranose(s) phosphorus derivatives of, physical properties, 191 -, 5-deoxy-5-phosphino- and -5-phosphinyl-D-, synthesis of, 138- 145 -, 5-thio-D-, synthesis and structure of, 138- 139 Xylose composition in nonaqueous solvents, 62,68 oligomers of W-n.m.r. data for, 207-208 methyl glycosides of, W-n.m.r. data for, 213-214 peracetates of, %-n.m.r. data for, 215-216 Xylose D-, in aqueous solution composition, 26, 62.64 polarography, 21

444

SUBJECT INDEX

-, 4-acetamido-4,5-dideoxy-~-, composition in aqueous solution, 51 -, 5-acetamido-5-deoxy-o-, composition in aqueous solution, 49 -, 2,4-O-benzylidene-t-, composition in aqueous solution, 60 -, 2-0-methyh-, in plant cell-wdl polymer, 280,281,287 -, 3-O-methyl-o-, composition in aqueous solution, 45 -, J-O-methyl-~-,composition in aqueous solution, 45 -, 4-thio-o-, composition in aqueous solution, 53

-, 5-thio-~-,composition in aqueous

solution, 52 UDP-D-,in polysaccharide biosynthesis, 321 -322 Xylose 5-phosphate, composition in aqueous solution, 46

Y Yeast, protein glycosylation in, 329 2

Zizyphw jujuba fruit, cyclic AMP in, 367 Zostera, galacturonan of, 281