Advances in Carbohydrate Chemistry and Biochemistry, Volume 42

Advances in Carbohydrate Chemistry and Biochemistry

Volume 42

This Page Intentionally Left Blank

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 ......

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

ix xi

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

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

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

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 . . .

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

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

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 .

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

383 384

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

AUTHOR INDEX SUBJECT INDEX

vii

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

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

386 392

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

395 423

This Page Intentionally Left Blank

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

This Page Intentionally Left Blank

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

xii

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.

2

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

4

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

OBITUARY-DEXTER FRENCH

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

6

JOHN H.PAZUR

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

OBITUARY-DEXTER

FRENCH

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.

8

JOHN H. PAZUR

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.

12

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

13

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

This Page Intentionally Left Blank

.

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 (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 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 -

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-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 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.

282

PRAKASH M. DEY A N D KEN BRINSON

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.

PLANT CELL-WALLS

283

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.

284

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

285

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

289

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.

PLANT CELL-WALLS

295

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.

296

PRAKASH M. DEY AND KEN BRINSON

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

PLANT CELL-WALLS

297

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.

298

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

299

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.

300

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.

302

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

303

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,

PLANT CELL-WALLS

305

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.

306

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

307

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.

308

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

309

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

312

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.

314

PRAKASH M. DEY AND KEN BRINSON

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

PLANT CELL-WALLS

315

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.

316

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.

PLANT CELL-WALLS

317

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.

318

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

319

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

321

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.

PLANT CELL-WALLS

323

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.

PLANT CELL-WALLS

329

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)

330

PRAKASH M. DEY AND KEN BRINSON

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

PLANT CELL-WALLS

331

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.

332

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

333

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.]

334

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

335

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.]

336

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

337

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.

338

PRAKASH M. DEY AND KEN BRINSON

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).

PLANT CELL-WALLS

339

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.

340

PRAKASH M. DEY AND KEN BRINSON

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.

341

PLANT CELL-WALLS

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.

342

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

343

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.

344

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

345

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)

346

PRAKASH M. DEY AND KEN BRINSON

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

PLANT CELL-WALLS

347

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.

348

PRAKASH M. DEY AND KEN BRINSON

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,

PLANT CELL-WALLS

349

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.

350

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

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.

352

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.

PLANT CELL-WALLS

353

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.

354

PRAKASH M. DEY AND KEN BFUNSON

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.

PLANT CELL-WALLS

355

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.

356

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.

PLANT CELL-WALLS

359

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

360

-

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,

PLANT CELL-WALLS

363

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.

364

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

365

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.

366

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

367

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.

368

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

369

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.

370

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

371

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.

372

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

373

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.

374

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

375

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.

376

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

377

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.

378

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

379

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)

380

PRAKASH M. DEY AND KEN BRINSON

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.

PLANT CELL-WALLS

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