Advances in Carbohydrate Chemistry and Biochemistry Volume 46
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Advances in Carbohydrate Chemistry and Biochemistry Volume 46
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Advances in Carbohydrate Chemistry and Biochemistry Editors R. STUART TIPSON
DEREK HORTON
Board of Advisors LAURENSANDERSON STEPHEN J. ANCYAL HANSH. BAER CLINTONE. BALLOU JOHN S. BRIMACOMBE
GUY G. S. DUTTON BENGTLINDBERG HANSPAULSEN NATHANSHARON ROY L. WHISTLER
Volume 46
ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers
San Diego New York Berkeley Boston London Sydney Tokyo Toronto
COPYRIGHT 0
1988
BY
ACADEMIC PRESS,
INC.
ALL RIGHTS RESERVED NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL. INCLUDING PHOTOCOPY. RECORDING. OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER
ACADEMIC PRESS, INC. San Diego, California 92101
United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road, London NW 1 7DX
LIBRARYO F CONGRESS CATALOG
ISBN 0-12-007246-7 (alk.
CARD
paper)
PRINTED IN THE UNITED STATES OF AMERICA 88899091
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NUMBER: 45-11351
CONTENTS PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vii
Konoehin Onodera, 1910-1983 TOHRUKOMANO AND
NAOKI KASHIMURA
Text
1
Venancio Deulofeu, 1902-1984 ROSAM. DE LEDERKREMER AND Text
EDUARDO G. GROS
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11
High-Performance Liquid Chromatography of Carbohydrates KEVINB. HICKS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Instrumentation and Stationary Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Separations and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Special Aspects and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17 18 32 63
N.M.R. Spectroscopy of Fluorinated Monosaccharides RENE CSUKAND BRIGITTEI. GLANZER 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Spectroscopy of Fluorinated Monosaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
73 74 80
Applications of Photosensitive Protecting Groups in Carbohydrate Chemistry
URI ZEHAVI 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Hydroxy Functions, Including Diols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Amino Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Carbonyl Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Protection of Phosphoric Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Applications to Biological Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V
179 180 192 195 202 203
CONTENTS
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Inclueion Complexes of the Cyclomalto-oligoeaccharidee (Cyclodextrina) RONALD J. CLARKE,JOHNH. COATES,AND STEPHENF. LINCOLN
I. Introduction . . . , . , , , . . . . . . . , . . . . . . . . . . , . , , , , . . . . . . . . , . . . . . . . . . . . . . . . 11. Historical Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Formation of Inclusion Complexes . . . . . . . , . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . .
IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
205 211 219 249
Hydrolysis and Other Cleavages of Glycosidic Linkages in Polysaccharides CHRISTOPHER J. BIERMANN
I.
11. Liberation of N- and @Linked Carbohydrate Chains . . . . . . . . . . . . . . . . . . . . . . . . . 111.
IV. ............................... V. Formolysis and Acetolysis . v1. Enzymic Hydrolysis . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Reductive Cleavage . .
251 255 256 259 269 270 271
Aqueous, High-Temperature Transformation of Carbohydrates Relative to Utilization of Biomass OLOFTHEANDER AND DAVIDA. NELSON
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Transformation of Monomeric Saccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Transformation of Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Carbohydrate Transformation in the Presence of Amino Compounds . . . . . . . . . . . . . V. Carbohydrate Transformation in Chemical Processes, Including Humus Formation . . I
273 275 295 307 323
Addendum to Article 3: References Published after 1986 (Added at Proof Stage) KEVIN
Text
B. HICKS
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Addendum to Article 4 RENECSUKAND BRIGITTEI. GLANZER Text
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331
Addendum to Article 6 RONALD J. CLARKE,JOHNH. COATES,A N D STEPHEN F. LINCOLN Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
333
AUTHOR INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECTINDEX ..........................................................
337 354
PREFACE l'kaditional chromatographic methods for the separation and purification of carbohydrates of all kinds, ranging from mono- to oligo-saccharides, have permitted many important developments in the carbohydrate field. A major advance that has achieved great utility is high-performance (or so-called highpressure) liquid chromatography. This technique, treated in comprehensive practical detail in the present volume by Kevin B. Hicks (Philadelphia), affords, within an hour, precise analytical and preparative separations of mixtures hitherto separable only with difficulty. The next chapter, by RenC Csuk and Brigitte I. Glinzer (Zhrich), constitutes an extensive treatise on the nuclear magnetic resonance (n.m.r.) spectroscopy of fluorinated monosaccharides [whose early chemistry was surveyed in Vol. 38 (1981) by Anna A. E. Penglis]; the comprehensive data tabulated herein should be especially of value to those working in the field. It continues the coverage, in Advances, of n.m.r. spectroscopy as the key tool for characterization of carbohydrates. It complements articles on the 'H-n.m.r. spectroscopy of carbohydrates by Laurance D. Hall [Vols. 19 (1964) and 29 (1974)], Bruce Coxon [vol. 27 (1972)], and Johannes F. G. Vliegenthart, Lambertus Dorland, and Herman van Halbeek [Vol. 41 (1983)], and on the T-n.m.r. spectroscopy of monosaccharides by Klaus Bock and Christian Pedersen [Vol. 41 (198311, of oligosaccharides by the same authors and Henrik Pedersen [Vol. 42 (1984)], and of polysaccharides by Philip A.J. Gorin in Vol. 38 (1981). Protecting groups remain central to the methodology for synthesis of evermore-complex carbohydrate targets. Herein, Uri Zehavi (Rehovot) discusses a somewhat under-utilized but potentially elegant and useful aspect, namely, that of photosensitive protecting groups capable of selective introduction with accessible reagents and subsequent removal under mild irradiation. The chapter is a useful adjunct to that by Roger W. Binkley on the photochemical reactions of carbohydrates that were adumbrated in Vol. 38 (1981). The next chapter, by Ronald T. Clarke, John H. Coates, and Stephen F. Lincoln (Adelaide) discusses inclusion complexes of the cyclomaltooligosaccharides (cyclodextrins), a unique group of natural cryptands that has attracted great interest within and outside the carbohydrate field in recent years. The article updates the pioneering contribution by Dexter French in Vol. 12 (1957) on these oligosaccharides, then known as the Schardinger dextrins. N o chapters treat widely divergent aspects of the aqueous degradation of carbohydrates. Christopher J. Biermann (Corvallis) discusses aqueous acidic hydrolysis and other cleavages of glycosidic linkages in oligo- and polysaccharides, with specific emphasis on their relation to procedures for determination of chemical structure. In the final chapter, Olof Theander (Uppsala) and David A. Nelson (Richland) provide an informative treatment of the vii
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PREFACE
aqueous, high-temperaturetransformation of starch, cellulose, and other abundant carbohydrates relative to the utilization of biomass as a source of useful chemical feedstocks. This issue pays tribute to two scientists who pioneered the development of carbohydrate chemistry in their respective countries of Japan and Argentina, Tohru Komano (Kyoto) and Naoki Kashimura (Mie) describe the life and work of Konoshin Onodera, and an obituary article on Venancio Deulofeu is contributed by Rosa M. de Lederkremer and Eduardo G. Gros (Buenos Aires).
Kensington. Maryland Columbus, Ohio July, 1988
R. STUART TIPSON DEREKHORTON
Advances in Carbohydrate Chemistry and Biochemistry Volume 46
1902-1984
ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 46
KONOSHIN ONODERA 1910-1983 In the Fall of 1983, Konoshin Onodera, Emeritus Professor of Kyoto University, who had retired from academic activity and lay in bed at his home in Kyoto fighting against nephrosis, received greetings from two carbohydrate chemists. Professor Juji Yoshimura of the Tokyo Institute of Technology conveyed to him the recent activities of the Steering Committee of the International Symposium on Carbohydrate Chemistry, in which he was the national representative from’Japan, having succeeded Dr. Onodera in this position. Professor Susumu Hirase of the Kyoto Institute of Technology informed Dr. Onodera about the successful closing in Sendai of the 6th National Carbohydrate Symposium sponsored by the Japanese Society of Carbohydrate Research that was founded by Dr. Onodera (and, since then, the office which had been maintained by him). Afterwards, on October 6, 1983, to our deep regret, Dr. K. Onodera died of a heart attack. K. Onodera was born on October 5 , 1910, in Tsu City, Japan, a little north of the pearl island in Mie Prefecture, to Sakuemon, his father, and Chiyo, his mother, as the second child of six. His father was an official in the Tsu City office. His family history goes back more than 300 years, to the 16th century, when his ancestors came to Tsu with a princess of a feudal clan from the Sendai district in northeastern Japan who married into another feudal clan family; their house still stands, in the center of Tsu. The Onodera family is famous for its relationship to a historical event which happened on December 14, 1702, the 15th year in the Genroku era. This event was triggered by a group of Ronin soldiers (47 samurai) who lost their clan master. The master was ordered by the Shogun to take the blame for his deed in the shogunate castle, which meant death by hara-kiri. The 47 Ronin took revolutionary action to protest against this decision and to show their devotion to their master, although such an action was prohibited at that time. Junai Onodera was one of the members of the 47 samurai. He sent a letter to his relative, Onodera’s ancestor, asking for care for his soon-to-be bereaved family, because he would surely commit hara-kiri after the event; this letter still remains in Onodera’s house. The event was made into a Kabuki drama, and it has frequently been performed since that time. 1 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Onodera spent his school days in Tsu. He was an honest and active boy in his childhood. In 1927, when he entered the Dai-san (the Third) High School, his family moved to Kyoto from Tsu. At that time, the State High Schools in Japan were numbered, and Dai-san High School was founded in Kyoto. After World War 11, the educational systems were changed, and the Dai-san High School was reorganized, to become the Department of Liberal Arts and Sciences of Kyoto University. Kyoto had been the capital of Japan for more than 1,000 years, as well as the center of the politics, economy, culture, and religion of Japan, so that, since olden times, many young people have gone to Kyoto to be educated. In March, 1932, Onodera graduated from the Dai-san High School, and he entered Kyoto University in April of the same year. In those days, it usually took 3 years to graduate from High School, but he took 5 , the reason being that he was very interested in mountain climbing. He and his group climbed many mountains throughout Japan, and became the pioneers of modern Japanese mountaineering. He was, indeed, famous as a mountain climber, as well as a carbohydrate chemist, in Japan. Onodera chose the Agricultural Chemistry course in the Faculty of Agriculture when he entered Kyoto University, the reason being that his intimate seniors in the mountaineering club were in the same course! There, he could study biochemistry and applied biochemistry. He chose Protein Chemistry, Carbohydrate Chemistry, Lipid Chemistry, Vitaminology, Nutritional Chemistry, Fermentation and Applied Microbiology, and Agronomical Chemistry (including Soil Sciences and Plant Fertilizers). Agricultural chemistry in Japan is known as the course which provides educational background and research on the nature of the composition of animals, plants, and micro-organisms and of their products, such as, vital substances of foods for human consumption; the functions and transformations of chemical entities in biological and food systems; and the chemical and energetic changes associated with these in the course of the activities of living things during both preharvest and postharvest. During Onodera’s time, one of the main biochemical interests in Japan was the chemistry and biochemistry of polysaccharides as a branch of polymer science. Cellulose, starch, and glycogen were investigated intensively. Onodera chose the Laboratory of Biological Chemistry in which to write his graduation thesis in 1934, the last year of his agricultural chemistry course. Onodera’s professor at that time was Dr. Bunsuke Suzuki, who later became a Professor of the University of Tokyo and was well known as a lipid investigator. Studies on the structure of fibroin, carbohydrate metabolism, separation of triglycerides from animals and plants, an analytic
OBITUARY-KONOSHIN ONODERA
3
method for molecular species of fatty acids, and structures of starch or glycosides were made in his laboratory, as in many other laboratories of biochemistry. Onodera’s baccalaureate graduation thesis was on the saponin components included in the bark of Schima liukiuensis, Nakai. Those who knew him in those days used to say that he worked energetically all day, and even if he got good results, he repeated the same experiment again and again, until he got reproducible data; therefore, his results were truly reliable. Because he was so captivated by the study of biochemistry, his mountaineering activities were now limited to long summer vacations and the winter holidays. In March, 1935, he finished the agricultural chemistry course and, in July of the same year, he was appointed a research assistant in the Laboratory of Biological Chemistry, Department of Agricultural Chemistry, Kyoto University, and took his first step as a research investigator. In 1937, Onodera married Miss Yukari Yamada, who was born to a Shoya (mayor) in Goboh City, Wakayama Prefecture. His first work in the Laboratory of Biological Chemistry was on the purification and properties of potato phosphorylase, but he soon changed his subject to the study of amino-containing sugars. It was not well understood at that time whether there were impurities in the products of separation and purification of proteins. Consequently, Onodera made up his mind to devote his whole life to identifying the chemical nature of aminocontaining sugars. In 1939, Dr. Yoshiyuki Inoue became the Professor of the laboratory when Professor B. Suzuki moved to the University of Tokyo. In March, 1940, Onodera joined Professor Inoue’s group, and continued his work in carbohydrate chemistry. In August, 1949, he received his Ph. D. degree from Kyoto University. The title of his thesis was “Biochemical Studies on Starch, Amino-Containing Sugars, and N-Glycosides” (written in Japanese). Onodera tried to separate amino-containing sugars from proteins, but he was unable to obtain constant results at that time. Then, he started to investigate the chemical reactions of amino-containing sugars, and synthesized some of their derivatives in a chemical process. During World War 11, it had been very difficult for him to continue scientific work on a high level. Even under poor conditions, he accomplished a study on the a,@-transformation of glycosides in cooperation with Drs. I. Karasawa and S. Kitaoka. In May, 1944, Onodera was promoted to Associate Professor. After 1950, the condition of scientific research in Japan gradually improved, and Onodera and his associates succeeded in publishing the following results: deamination of D-glucosamine by barium hypobromite (1952); transgly-
4
TOHRU KOMANO AND NAOKI KASHIMURA
cosylation of N-glycosides (1953); synthesis of N-glycosides with Brigl’s anhydride (1954); and studies on p-toluidine N-D-fructoside (1954). Studying in Dr. M. L. Wolfrom’s laboratory, Department of Chemistry, The Ohio State University, Columbus, Ohio, strengthened Onodera’s commitment to be a carbohydrate chemist. In August, 1953, he crossed the Pacific Ocean by ship, and took the transcontinental railway to Columbus. He later told about this journey as follows. “The voyage on the S . S . Cleveland was very pleasant. I got acquainted with many persons and had a good time. This experience was quite effective in raising my expectations to be able to work with Dr. M. L. Wolfrom. I knew the United States of America was a big country, and I realized how big it was only after taking the transcontinental railway. ” He investigated the synthesis of dithioacetals of D-glucuronic acid and 2-amino-2-deoxy-~-galactose, and reported the results in the J . Am. Chem. Soc., Vol. 79 (1957). He also had valuable experiences other than research, because he met many world-famous carbohydrate chemists and became friends with them. Later, he used to talk about the good days at Columbus, and wrote in a poem: “Hospitable were people at the sugar alley, Beautiful was spring on the gentle stream, the star above buckeye tree sparkles friendly, To the old street wanders evening dream.” We also remember him often saying that Dr. M. L. Wolfrom had told him to read the experimental sections of scientific papers before the summaries and introductions. In November, 1956, Onodera returned to Kyoto University and engaged in research on carbohydrate chemistry again. The articles he then published were concerned with an acyl migration in acetohalogeno-glucosaminides (1957); N-acetylation of 2-amino-2-deoxy-~-glucosewith mixed carboxylic acid anhydrides (1960); and N-acylation of unsubstituted D-glycosylamines (1960). Many young research investigators and graduate students joined his group, because his new ideas from experiences in America attracted them. In August, 1960, Onodera was appointed Professor of the Laboratory of Biological Chemistry, Department of Agricultural Chemistry, Kyoto University. He further developed carbohydrate chemistry after he became Professor. In the early period, Drs. S. Kitaoka, H. Ochiai, S. Hirano, and T. Komano worked with him. The work accomplished at that time included the following: N-debenzyloxycarbonylation of 1,3,4,6-tetraO-acetyl-2-(benzyloxycarbonyl)amino-2-deoxy-~-hexopyranoses in the conversion of a,@-acetoxy to glycosyl bromide (1961); oxidative cleavages of 1,Zdiamino sugars and their significance in the mechanism of the aminocarbonyl reactions (1962); and synthesis of 2-amin0-2-deoxy-p-~glucosides via 3,4,6-tri-0-acetyl-2-benzylsulfonamido-2-deoxy-a-~-g1ucopyranosyl bromide (1962).
OBITUARY-KONOSHIN ONODERA
5
Soon after he became Professor, he also became the President of the Kyoto University Alpine Club (1961). The first plan that he scheduled was to conquer an unclimbed summit in the Himalayas. Whoever an alpinist might be, he wants to try once to climb Mt. Everest, and that was Onodera’s dream from his youth. He went to the Himalayas as a leader with six members of the club, and was successful in conquering Indrasan and Deo Tibba. The results were published in the Himalayan Journal, 24 (1963) 90-95, entitled “The Ascent of Indrasan and Deo Tibba,” by Onodera. “The expedition organized and sent made the first ascent of Indrasan (6,221 meters) on October 13,1962. The party also climbed Deo Tibba (6,000 meters).” Onodera stayed in the base camp, encouraged the members, and advised them to move carefully. This great achievement had an important effect not only on young students, including members of his Institute, but also on all Japanese youth, especially alpinists. Also in 1967, Onodera went on another expedition, to India and Bhutan, but, unfortunately, this time he was not successful, as he could not get permission to ascend a peak from Bhutan. Onodera used to work in the laboratory from early in the morning to late at night and would discuss research projects with young coworkers. He used to walk around the laboratory several times a day and hear from them how their work was progressing. He discussed with humor the historical background of past work and persons who contributed to carbohydrate research in his laboratory, as well as in foreign institutions. When a graduate student was disappointed that his experiment was hopeless because the derivatives of carbohydrates had not readily crystallized, he gave the student advice and encouragement to be careful and patient. During his professorship, from 1960 to 1974, he accomplished many tasks. We may classify them roughly as follows: development of a synthetic procedure for nucleosides; establishment of a new oxidation method for sugars; chemistry of sugar moieties of glycosaminoglycans, and their chemical structures; and conformations of sugar moieties of some nucleotide analogs, “sugar nucleotides,” and acidic polysaccharides. He summarized these results in a paper entitled “Retrospect and Prospect in Carbohydrate Chemistry and Biochemistry” (Memoirs of the College of Agriculture, Kyoto University, No. 102, Chemical Series No. 33). He described his philosophy and attitudes on biological chemistry in the Preface section of the Memoirs, stating that “The aim of biological chemistry is, I believe, the eventual elucidation of the structure and function of organic substances in biological environment. This line of consideration naturally led us to make efforts to correlate the structure of biopolymers with the function of them. Our topics have all been started with the hope in mind that studies on stereochemistry of substances will
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TOHRU KOMANO AND NAOKI KASHIMURA
contribute to the elucidation of their biochemical functions. For this purpose, especially, conformational analysis will become a more and more useful tool in chemistry and biochemistry of carbohydrates.” He continued that “In retrospect, I feel that our research areas were more or less in diversity which resulted in vagueness in a way of standpoint for research objectives. In prospect, I really hope that this issue will be of value for stimulating research in the carbohydrate field in our country.” A brief summary of Onodera’s various fields of research begins with the development of a synthetic procedure for nucleosides. This study was made with Drs. H. Fukumi, F. Masuda, T. Yajima, and other coworkers. He considered that investigation of a synthetic procedure for nucleosides and related compounds would contribute to the chemistry and biochemistry of nucleic acid constituents. At that time, he had already started a series of studies on nucleic acid biochemistry with Dr. T. Komano, who developed the subject further into molecular genetics and gene technology. Onodera first attempted to develop a new procedure in which 1-0(trichloroacety1)ated sugars were used instead of acylated sugar halides, expecting that stereospecific synthesis of an N-glycosylic linkage might be possible. Efforts were also concentrated on the stereospecific synthesis of nucleosides with the use of phosphorus pentaoxide or polyphosphoric acid as a dehydrating agent in solution reactions, or ethyl polyphosphate as a catalyst in fusion methods. The results obtained in improved procedures for fusion methods further developed a new method for the novel synthesis of such 1’,2’-cisnucleosides as a-adenosine, and a novel synthesis of several theophylline nucleoside derivatives having a 2’,3’-unsaturated grouping. A second field concerned oxidation and polymerization of sugars with phosphorus pentaoxide. Drs. S. Hirano, N. Kashimura, N. Miyazaki, and other coworkers cooperated to promote this project. In the course of studying the polymerization reaction of reducing mono- and di-saccharides, they found that dimethyl sulfoxide containing phosphorus pentaoxide rapidly oxidizes the alcoholic groups of sugars at room temperature, to produce aldehydes, ketones, or carboxylic acids. This finding, together with the results of Dr. Albright’s work on dimethyl sulfoxideacetic anhydride as an oxidant, was just on the verge of the coming development of dimethyl sulfoxide-mediated oxidation of alcohols that had been pioneered by Dr. J. G. Moffatt and was followed by a number of new dimethyl sulfoxide oxidants. By using this dimethyl sulfoxide-phosphorus pentaoxide mixture, he succeeded in synthesizing a number of aldosuloses and aldosiduloses in good yields. Among them were 1,2 :5,6di-O-isopropylidene-c~-~-ribo-hexofuranos-3-ulose and methyl 2-acetamido-4,6-O-benzylidene-2-deoxy-c~-~-ribo-hexopyranosid-3-ulose that had not been readily obtained by conventional methods.
OBITUARY-KONOSHIN
ONODERA
7
Another field of research concentrated on the chemistry of sugar moieties and chemical structures of glycosaminoglycans. Drs. S. Hirano, H. Hayashi, T. Komano, and other coworkers cooperated in this research. At first, an attempt was made to prepare N-acetylneuraminic acid isomers, in which a product from the reaction of oxaloacetic acid with N-acetyl-D-galactosamine was compared with those from those of oxaloacetic acid with N-acetyl-D-glucosamine and N-acetybmannosamine. At the same time, the distribution of sialic acid in biological materials, mainly of plant origin, was also investigated. 2-Amino-2-deoxy-~hexosides and D-glucosiduronic acids were synthesized, and the behavior of those glycosides on acid hydrolysis was investigated in relation to the isolation of unit hexosaminide disaccharides from glycosaminoglycans. He actually succeeded in isolating a 2-amino-2-deoxy-~-galactosidedisaccharide from chondroitin sulfate C. Infrared spectroscopic analysis was applied to study of the disposition of the sulfuric ester groups, and also to sulfonate groups at positions other than C- 1 on the aldopyranose ring. The methylation of heparin, heparitin sulfate, and oligosaccharides from hyaluronic acid was performed in order to analyze the structures. He also studied the constituents and structures of glycosaminoglycans and glycoproteins in human and cow colostrums. A fourth area of study dealt with the conformations of sugar moieties of some nucleotide analogs, “sugar nucleotides,” and some acidic glycosaminoglycans. This research was conducted with Drs. S. Hirano, N. Kashimura, F. Masuda, and other coworkers. ‘H-Nuclearmagnetic resonance spectroscopy was the main tool for conformational analyses of a number of a-nucleosides and a-glycopyranosidesin various solvents, and led to the finding that some derivatives exist in the so-called alternative chair conformation. He also found that chondroitin has a random conformation. Conformational inversion was shown to occur in the molecule of chondroitin 6-sulfate by additional sulfation on C-4 of the 2-acetamido-2deoxy-D-glucopyranosemoiety, whereas it did not occur in the molecule of chondroitin 4-sulfate by additional sulfation on C-6 of the hexosamine moiety. The linkage position of the sulfate group was considered to be important in establishing the conformation with the aid of hydrogen bonds. The D-hexopyranuronic acid moiety of some acidic glycosaminoglycans adopts the * C ~ ( Dconformation, ) whereas the D-glucopyranuronic acid moiety of UDP-D-glucuronic acid adopts the 4CI(D) conformation. Therefore, he assumed that the conformational inversion of the sugar moiety took place when the glycosyl group was transferred from the glycosyl donor to the acceptor. Onodera mentioned in the last part of the Preface section of the Memoirs as follows: “During compiling of this issue, I have been immersed in a pleasant recollection of past years when many collaborators, to whom I
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TOHRU KOMANO A N D NAOKI KASHIMURA
wish to express many thanks, worked with me in the old,’underequipped laboratory.” In fact, in comparison with the well-equipped laboratory of the Department of Chemistry, Ohio State University, the laboratory he inherited was not in good condition. During World War 11, it was not exceptional for many research investigators to leave their laboratories. Fortunately, Onodera was able to stay in the laboratory and continue his education and research. However, it was almost impossible to obtain sufficient supplies of chemical reagents, experimental materials, and equipment for research. Following the war, it took over ten years for the university to regain full activity. He used to talk reminiscently to younger generations, as he looked back on the days after World War 11: “You have to be patient, even though you can’t get a good experimental result. You should feel happy, because it is a time when no one can prohibit you from research on whatever you wish.” It seemed to us that Onodera was now having difficulties going up and down the stairs when he was over 60. Once he told us that he had slipped on a rock when he was climbing a mountain, and had injured his knee; but he used to come to the laboratory on foot when the weather was fine. As his house was halfway up a small hill on the east side of Kyoto City, there were many historical temples and shrines along the way to it, where beautiful trees and gardens could be seen. It was good for him to walk a distance of about 3 kilometers, looking at the view. In the spring, cherry blossoms were in full bloom and, in autumn, the leaves turned red. He really loved these views in Kyoto, and was also very fond of listening to the music of Mozart and Beethoven in his yard and in his large wooden, typical Japanese-style house. Onodera played an important role on the editorial staffs of both the Agricultural Chemical Society and the Biochemical Society of Japan. In the Agricultural Chemical Society, he acted as Chairman of a branch of the Kansai district (west part of Japan) from 1967 to 1969. He was elected a member of the Science Council of Japan from 1968 to 1971. He was awarded the Suzuki prize by the Agricultural Chemical Society of Japan, the highest award of the Society, for his meritorious deeds in carbohydrate chemistry. But what he felt proudest of, and most honored about, was his appointment to the Editorial Board of Carbohydrate Research for 13 years (from 1966 to 1979). He devoted much energy to the 8th International Symposium on Carbohydrate Chemistry, held in the summer of 1975 in Kyoto. Japanese carbohydrate chemists belonged either to the Agricultural Chemical Society (to which Onodera belonged), the Biochemical Society, the Society of Pharmaceutical Science, or the Chemical Society of Japan. Consequently, the scientific papers, on carbohydrates, from Japan were published in various
OBITUARY-KONOSHIN
ONODERA
9
journals. Onodera thought it was necessary to have an opportunity for Japanese carbohydrate chemists to meet together to discuss the matters that they were concerned with. He was appointed Chairman of the 8th Symposium. He took it on himself to establish the Japanese Society of Carbohydrate Research in Japan. The members who took part in establishment of the Society were Drs. S. Umezawa, N. Iseki, J. Yoshimura, K. Anno, T. Yamakawa, A. Misaki, Z. Yoshizawa, Y. Matsushima, S . Hirase, and many other carbohydrate chemists. The Society now has over 600 members, and held its 8th National Symposium in Kyoto in the summer of 1985. Onodera retired from Kyoto University in April 1974, when he was 63 years old, due to regulations for the retirement of members of the Kyoto University Faculty. After the International Symposium on Carbohydrate Chemistry in Kyoto, his health seemed to be getting worse. But nobody, maybe not even he, recognized that he was suffering from a serious disease. We had all thought him to be in excellent health. Because he was an alpinist, he had confidence in his health, as did we. At the end of 1979, he was moved to a hospital, and he remained there under medical care for about 3 months. Afterwards, he had to visit the hospital twice a week for kidney dialysis. Six months later, fortunately, he recovered markedly enough to be able to talk about the good and happy old days. In the spring of 1983, his condition became worse. On April 29th, 1983, the birthday of the Emperor, Onodera was awarded (and received) a memorial medal by the Japanese Government for his achievements as a Professor of Kyoto University. On this commemorative occasion, more than 120 persons who had been associated with him prepared a present for him in order to express their gratitude; and, of course, he received it while lying in bed. He sleeps on a small hill, in Tsu where he was born; he is survived by Yukari, his wife of 46 years, sons Akifumi and Koji, daughters Mizuyo and Fumi, and numerous friends, including many Japanese carbohydrate chemists.
TOHRUKOMANO NAOKIKASHIMURA
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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 46
VENANCIO DEULOFEU 1902-1984
Venancio Deulofeu, first son of Tomas Deulofeu and Camila Gascons, was born on April lst, 1902, in Buenos Aires to a high middle-class family. He died in that city on October 4th, 1984. While he was studying in elementary school, his family made a trip to Spain, where they remained for one year before returning to Argentina. Between 1914 and 1919, the young Venancio attended an official secondary school. He also enjoyed playing the piano, but his music studies were set aside when he started his university career at the Facultad de Ciencias Exactas, Fkicas y Naturales of the Universidad de Buenos Aires, pursuing a degree in Chemistry, a speciality not very extended in the Argentina of the beginning of the century. In 1923, while studying for his degree, he was invited by Prof. A. Sordelli to collaborate in investigations on the isolation and purification of bovine insulin which were being performed at the Instituto Bacteriologico. Deulofeu’s stay at this institute influenced his temperament, and aroused his taste for scientific research. He obtained, with honours, his degree in Chemistry in 1924, continuing at his position with Sordelli and publishing his first papers on subjects related to the chemistry of insulin. The work of ZemplCn on the degradation of acetylated aldononitriles inspired Deulofeu to start investigations in the field of carbohydrates, which was virgin at that time in Latin America. In 1929, by degradation of the tetraacetate of L-xylononitrile, he prepared L-threose, and, in a similar way, he obtained L-erythrose from tetra-0-acetyl-L-arabinononitrile. These reactions formed part of his Doctoral Thesis on “Degradation in the Group of the Monoses,” with which he earned a Doctors’ degree in Organic Chemistry in 1930. The first step in the reaction yields “aldose diamides”, usually as crystalline solids, which upon acid hydrolysis afford an aldose with one carbon less than the original nitrile. The mechanism of the ammonolysis of the acylated nitriles intrigued Deulofeu. Experiments carried out with Hockett and Deferrari, employing labelled ammonia, gave the first proof as to the intramolecular nature of the reaction. A review on the degradation of acylated aldononitriles, written by Deulofeu, was published’ in Advances in Carbohydrate Chemistry. He (1) V. Deulofeu, “The Acylated Nitriles of Aldonic Acids and Their Degradation,” Adu. Carbohydr. Chem., 4 (1949) 119-151.
11 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
12
ROSA M.
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LEDERKREMER AND EDUARDO G. GROS
also found that the 1,l-bis(acy1amido)-1-deoxyalditolsare formed by the action of aqueous or alcoholic ammonia on acylated aldoses. This reaction was systematically extended through the monosaccharide series, and the mechanism for the 0 + N acyl migration was studied by appropriate labelling of the benzoyl groups in the pentabenzoates of D-glucopyranose and D-galactopyranose. From his results, the individual contribution of the acyl groups at the different positions on the chain was established. The contribution of the 2-0-benzoyl group was the lowest, and that for the 4-0-benzoyl group the highest, to afford the corresponding “aldose diamide.” The reaction is very complex, and, by application of chromatographic techniques, cyclic N-acyl-D-aldosylamines were also isolated in most cases. It is interesting that he was self-taught in carbohydrate chemistry, because, at that time, no person in Argentina cultivated this speciality. In 1928, he was married to Irene Escasany, the daughter of a wealthy jeweler, and his companion throughout his life, who passed away one week after Deulofeu. Shortly after his thesis work was accepted, Deulofeu proceeded to the University of Munich, where he worked under Prof. H. Wieland on the structural elucidation of bile acids, a subject that had earned Prof. Wieland the 1927 Nobel prize for Chemistry. Deulofeu’s stay in Germany was interrupted by a call from Prof. B. Houssay, who offered him a position as Assistant Professor of Biological Chemistry at the Facultad de Medicina of the Universidad of Buenos Aires, a position that he held until 1948 as a part-time professor. Deulofeu’s reputation as a leading bio-organic chemist was already established, when, in 1939, he was appointed Professor of Organic Chemistry of the Facultad de Ciencias Exactas y Naturales of the same University. In 1941, invited by the Committee for Interamerican Cultural Relationships, he spent almost one year in the Biochemistry Laboratory, School of Medicine of the St. Louis University, doing research with Prof. E. A. Doisy. For unjustifiable political reasons, he was separated from his professorship in 1952, and accepted the position of Research Director at the private company of E. R. Squibb & Sons, where he remained until 1962. From E. R. Squibb & Sons, he launched a program of grants and fellowships to support biomedical research and to cover the expenses of several graduate students who carried out research in the laboratories of the company. Some of his students at that time have since been appointed to important academic and industrial positions in Argentina and abroad. In 1956, he returned, as Professor of Organic Chemistry, to the University, where he stayed until his retirement in 1968, to become Emeritus Professor thereafter.
OBITUARY-VENANCIO DEULOFEU
13
In 1937, he published, first in collaboration with A. D. Marenzi, and later with A. D. Marenzi and A. 0. M. Stoppani, the book Quimica Biolbgica, which was maintained, and reedited nine times, during the following thirty years, and also translated into Portuguese on two occasions. This book was destined to become the standard reference on its subject for Medicine and Biochemistry students of Latin America. In the chair of Organic Chemistry, besides his extraordinary ability as an organizer, he continued to be active in research. Outside the field of carbohydrates, Deulofeu undertook important investigations in other areas. A series of thirteen publications, starting in 1932, dealt with reactions of amino acids. He also had a long-standing interest in natural products of animal and plant origin, mainly Argentinian plants. About fifty publications, starting in 1939, described his results in this field. He was particularly interested in alkaloids, and in this respect he isolated new bases from Licopodium sururus (saurine and sauroxine), elucidated the structure of y-fagarine from Fagara coco, olivacine and guatambuine from Aspidosperma australe, tubulosine from Pogonopus tubulosus, and the partial structure of ocotein from Ocotea puberula. Pharmacological evaluations were performed on some of the bases isolated; thus, the supposed antimalarial action of a total alkaloid fraction from Pogonopus tubulosus was ruled out. Deulofeu was also concerned with the synthesis of alkaloids. These included D-laudanine, pseudocorydine, and racemic pseudocidamine, which was resolved into its optical isomers. Some of his work in the alkaloid field has been reviewed in a book edited by Manske and Holmes.2 With his italian colleague, G. Marini-Bettolo, he characterized flavonoid glycosides from the ombu (Phytolacca dioica), the big tree of our pampas. One of them, called ombuin, was later synthesized for confirmation of its structure. The plant Zlex paraguariensis, used for making an infusion called mate, very popular in Argentina, Uruguay, Paraguay, and Brazil, was also investigated by Deulofeu. Chlorogenic acids and related compounds were characterized. Studies on the isomerization and lactonization of the acids were performed as an aid to understanding the transformations occurring in the processing of the fresh leaves of the plant. In the field of the components of the venom from toads, he studied the venom of most South American species of the genus Bufo, determining with several coworkers, and also in collaboration with T. Reichstein (University of Basel), their composition in regard to bufadienolides, biogenetic amines, and alkaloid-like compounds. (2) V. Deulofeu, J. H. Comin, and M. J. Vernengo, The Benzylisoquinoline Alkaloids, in R. H. F. Manske and H. L. Holmes (Eds.), The Alkaloids: Chemistry and Physiology, Vol. 10, Academic Press, New York, 1967, pp. 401-461.
14
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LEDERKREMER AND EDUARDO G . GROS
From his time at the Squibb research laboratories, he was interested in the chemistry of antibiotics, and in this area, he elucidated the partial structure of curamycin, an antibiotic produced by Streptomyces curacoi. From curamycin, he isolated and characterized the new natural sugar derivatives D-curacose (4-O-methyl-~-fucose), curacin [4-O-(dichloroisoeverninyl)-2,6-dideoxy-~-arabino-hexose], and curamycose (2,6-di0-methyl-D-mannose). In 1977, he published his last original paper on the structure of a quercetin triglycoside containing D-apiose, isolated from Solanum glaucophylum a plant toxic to cattle. In spite of this, his extraordinary capacity as a reader allowed him to remain up to date in a great variety of topics, not only in those of direct interest to him but in those that were studied by several graduate students working under different supervisors. Among the numerous distinctions that Deulofeu received from Argentina and foreign countries, it seems relevant to mention: Doctor Honoris causa, University of Paris; Honorary Professor, University of Chile; Honorary Professor, University of San Marcos (Peru); elected Member of: Academy of Medicine (Argentina); Academy of Exact, Physical, and Natural Sciences (Argentina); Academy of Exact, Physical, and Natural Sciences (Madrid, Spain); Academy of Sciences (Rio de Janeiro, Brazil); and Academy of Arts and Sciences (Barcelona, Spain). He was also an Honorary Member of various Chemical Societies including those of Argentina, Peru, Colombia, Brazil, Chile, Uruguay, Venezuela, Mexico, Spain, and Switzerland. He was the recipient of several awards from Argentinian and international institutions, such as the Prize “Dr. B. A. Houssay” from the Organization of American States (Washington, D.C.) and a Medal from The International Academy of Lutece (France). Deulofeu was a member of the Editorial Board of several scientific publications, including Medicina, Enzymologia, Anales Asociacibn Quimica Argentina, Tetrahedron, Tetrahedron Letters, Index Chemicus, and Carbohydrate Research, and a member of the Assessor Committee of the Editorial Board of Pure and Applied Chemistry. He liked to travel, and he undertook numerous lecture and scientific meeting tours to several American and European countries, and also to the USSR, Japan, and Australia. Deulofeu was a very social gentleman who enjoyed giving parties for many friends and colleagues from foreign countries; on those occasions, he was very proud to show his collection of “mat6s” (the containers for making the infusion already mentioned) made of sterling silver. Short in stature, but robust in appearance, he projected a vitality and animated interest that made him the central focus of scientific gatherings. A special issue of the journal Carbohydrate Research with contribu-
OBITUARY-VENANCIO DEULOFEU
15
tions from friends and former students was published in February, 1973, in honour of Prof. Deulofeu on the seventieth anniversary of his birth. We hope that this short review of Deulofeu’s scientific activities transmits the feeling that his name is almost synonymous with Organic Chemistry in Argentina. ROSAM. DE LEDERKREMER AND EDUARDO G. GROS Departamento de Quimica Orgdnica Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires Buenos Aires, Argentina
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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL. 46
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF CARBOHYDRATES BY KEVINB. HICKS Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, Pennsylvania 19118
I. Introduction ............................................ ;. ............. 11. Instrumentation and Stationary Phases. ................................... 1. Chromatographic Equipment. ......................................... 2. Stationary Phases.. .................................................. 111. Separations and Applications. ............................................ 1. Analytical Separations ................................................ 2. Additional, Selected Applications ...................................... 3. Preparative, Liquid Chromatography ................................... IV. Special Aspects and Problems.. .......................................... 1. Detectability and Accuracy. ........................................... 2. Combined L.C. Techniques (L.C.-M.S., and L.C.-N.M.R. Spectroscopy) 3. Separation of Carbohydrate Anomers. .................................. 4. Future Trends ....................................................... Addendum: References Since 1986 .......................................
.
17 18 18 23 32 32 50 58 63 63 69 70 71 327
I. INTRODUCTION
This chapter covers advances in so-called high-pressure or high-performance liquid chromatography (often abbreviated as h.p.1.c.) of carbohydrates. The term 1.c. is used here to designate the rapid (<1 h) separation of carbohydrates in modern, liquid-chromatography systems, which include a high-performance, solvent-delivery system using one or more pumps, an injection valve, a column packed with 3-10-pm, monodisperse, resin- or silica gel-based particles, and a high-performance detection-system. These systems are operated at elevated pressures that, depending on the various stationary and mobile phases, the size of the columns employed, and the flow-rates used, may range from -1 to >30 MPa. In this chapter, 1.c. separations of almost all mono-, di-, and oligosaccharides are discussed from both a theoretical and a practical point of view. In addition, the general principles involved in the care and mainte17
18
KEVIN B. HICKS
nance of the relevant instrumentation and stationary phases are covered. A separate Section on preparative 1.c. methods for carbohydrates has been included, and this is the first article to treat this important and growing subject. Other Sections, which provide solutions to problems of detectability and of peak broadening (resolution of anomeric forms) are included, and the current status of emerging 1.c. techniques (1.c.-m.s., and high-performance affinity chromatography) are considered. High-performance, size-exclusion chromatography of oligo- and poly-saccharides will not be discussed here, but it has been described in two reviews.*J It is assumed that the reader is familiar with such common chromatographic concepts as efficiency, selectivity, capacity factors, and theoretical plates, and how these parameters affect and effect chromatographic resolution. Excellent descriptions of these general chromatographic principles have been p ~ b l i s h e d .Other ~ . ~ reviews on various aspects of carbohydrate separations will be cited in the appropriate Sections. 11. INSTRUMENTATION* AND STATIONARY PHASES 1. Chromatographic Equipment
a. Solvent-delivery Systems.-Almost all modern, commercially available pumps and controllers are useful for this kind of carbohydrate analysis. Some, however, because of aspects specifically related to carbohydrate applications, are more useful than others. A majority of the 1.c. methods described here required refractive-index detectors, and these instruments are sensitive to changes in solvent flow, pressure, and composition. Hence, the most useful pumping systems are those that deliver pulse-free and precise solvent-flow. Although most solvent-delivery systems are capable of blending two or more solvents, to afford isocratic mobile phases, few of these systems can blend these solvents accurately, and when the columns on these systems are monitored by refractive index detectors, extremely unstable baselines are the result. Hence, many chromatographers must tediously pre-mix their solvents in order to obtain (1) S. C. Churms, in E. Heftman (Ed.), J . Chromatogr. Library, Elsevier, New York, 1983, pp. B223-B286. (2) S. C. Churms, in G . Zweig and J. Sherma (Eds.), Handbook ofChromatography, Carbohydrates, Vol. I, CRC Press, Boca Raton, FL, 1982, pp. 69-129 and 175-187. (3) K.-P. Hupe, in A. Henschen, K.-P. Hupe, F . Lottspeich, and W. Voelter (Eds.), High Pe$ormance Liquid Chromatography in Biochemistry, VCH Publishers, Deerfield Beach, FL, 1985, pp. 1-15. (4) L. R. Snyder and J. J. Kirkland, Introduction fo Modern Liquid Chromatography, 2nd edn., Wiley-Interscience, New York, 1979, pp. 15-82. * Reference to a brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.
H.P.L.C. OF CARBOHYDRATES
19
stable baselines. Some pumps are now available that are capable of accurately mixing solvents on line, and the purchase of these instruments is recommended. Another consideration in choosing a solvent-delivery system is the matter of the maximum and minimum flow-rate ranges. For extremely precise solvent-metering in microbore-column applications, pumps capable of operating in the 50-100 pL/min range are useful. For preparative chromatography, “dedicated” preparative instrument^^.^ are available that can provide flow rates in excess of 100 mL/min. For most laboratory applications, however, it is not necessary to buy a dedicated microbore or pre~ * ~are caparative chromatograph, as 1.c. systems are now a ~ a i l a b l ethat pable of-performing both functions with only minor changes in plumbing and hardware. Regardless of the type of solvent-delivery system used, solvents should, for best results, be degassed by vacuum, with helium, or by use of commercial degassing instruments. All of these degassing systems work well, but the last is the most convenient, and allows continuous operation. b. Equipment for Preserving Column-Life.-Equipment in this category includes pre-columns, guard columns, silica saturator9J0 columns, and various cartridges containing bonded-phase packings or ion-exchange resins. These can, and should, be used on- or off-line, to prevent sample contaminants from entering the analytical column. The appropriate use of saturator and pre-columns for each type of column stationary-phase will be given in the next Section. When pre-columns are installed instead of sample loops, they can be used as concentratorsI1 for dilute samples, and can be readily backflushed, after injection, to remove unwanted samplecomponents. l 2 Bonded-phase silica and ion-exchange resins in plastic cartridges and mini-columns are very useful for off-line prepurification of samples, especially those for preparative chromatography, when appropriate pre- or guard columns may not be available for on-line clean-up of a sample. (5) N. W. H. Cheetham and P. Sirimanne, Carbohydr. Res., 96 (1981) 126-128. (6) C. D. Warren, A. S. Schmit, and R. W. Jeanloz, Carbohydr. Res., 116 (1983) 171182. (7) K. B. Hicks, S. M. Sondey, D. Hargrave, G . M. Sapers, and A. Bilyk, LC Mag., 3 (1985) 981-984. (8) K. B. Hicks and S. M. Sondey, J . Chrornarogr., 389 (1987) 183-194. (9) D. L. Hendrix, R. E. Lee, Jr., J. G . Baust, and H. James, J . Chromarogr., 210 (1981) 45-53. (10) B. Porsch, J . Chrornatogr., 253 (1982) 49-54. (11) J. A. Polta, D. C. Johnson, and K . E. Merkel, J . Chrornatogr., 324 (1985) 407-414. (12) K. Mopper and L. Johnson, J . Chrornatogr., 256 (1983) 27-38.
KEVIN B. HICKS
20
These have been effectively used to remove lignin13 and hydrophobic metabolite^'^ from plant-derived samples. Bonded-phase mini-columns are also ideal for the prechromatographic purification of a perbenzoylated sugars,15glycopeptides (and derived oligosaccharides),16and peralkylated oligosaccharides.17,18 c. Switching Valves, Fittings, and Filters.-The creative use of switching valves can save chromatographic-run time and extend column life. They can allow elution of the analytical column while the pre-column is being simultaneously cleaned.'* In chromatograms where they are both very early, and very late peaks, such as those of reducing sugars and their degradation products (for example, furfural~),'~ the use of switching valves can lessen the run time by 50 to 70%. The myriad types of 1.c. fittings produced by a great number of vendors have led to confusion among chromatographers about the proper choice for each application. Fortunately, fittings that are more universal in their applications are now available, and one of the most useful of these has a knurled flange on the nut, and a replaceable, polymeric ferrule. These fittings may be sealed by hand and re-used many times without failure. The use of in-line filters between injectors and column can prevent the accumulation of particulate material on the inlet frit of an analytical column, and can avoid back-pressure problems. A second, and often overlooked, site for filter installation is between the column and the detector. Cartridge-type filtration-units that contain readily changed, 0.2-pm filters are commercially available, and they contribute insignificantly to peak broadening. These filters are essential for the prevention of clogged detectors when laboratory-packed columns are used.
d. Injectors.-The choice of injector depends upon the particular application, namely, analytical or preparative chromatography. For the former, fixed-loop injectors are far more accurate than the partially filled loop (universal injector) design. For occasional analytical and preparative G . Bonn, R. Pecina, E. Burtscher, and 0. Bobleter, J . Chromatogr., 287 (1984) 215221.
R. Schwarzenbach, J . Chromatogr., 140 (1977) 304-309. P. W. Tang and J. M. Williams, Anal. Biochem., 142 (1984) 37-42. S. J. Swiedler, J. H. Freed, A. L. Tarentino, T. H. Plummer, Jr., and G . W. Hart, J . Biol. Chem., 260 (1985) 4046-4054. M. W. Spellman, M. McNeil, A. G . Darvill, P. Albersheim, and A. Dell, Carbohydr. Res., 122 (1983) 131-153. J. M. Lau, M. McNeil, A. G . Darvill, and P. Albersheim, Carbohydr. Res.; 137 (1985) 111-125.
D. W. Patrick and W. R. Kracht, J . Chrornatogr., 318 (1985) 269-278.
H.P.L.C. OF CARBOHYDRATES
21
applications, the universal design is useful, because any volume up to a few mL may be injected. For automated, or manual, preparative injection, fixed-loop injectors may be used with large loop sizes (up to 10 or 20 mL) that are currently available. Custom loops are also readily made with 0.5 mm (0.020”) or 0.75 mm (0.030) (i.d.) tubing, cut to the appropriate length. e. Column Design.-Because the heart of any chromatographic system is the column, considerable attention has been devoted to improvements, through new designs, in its efficiency and stability. The 10-pm packings and 25-cm-long columns of the 1970’s have given way to the 5-pm (15 cm) and 3-pm (10 cm) columns available today. These improvements have resulted in faster, higher resolution, and more-sensitive separations and analyses. Although most columns are of the traditional, steel-tube-withend-fittings design, newer columns are available as cartridges with reusable end-fittings. Such cartridges that fit into dynamic axial compression fittingss or radial compression modules20.21 are especially useful, because voids that develop in the stationary phase are removed by the changing configuration of the module. The diameter of commercial, 1.c. columns is also evolving toward wider bores. Initial reports that only long, narrow columns (<5 mm) could produce narrow plate-heights, especially with cation-exchange resin columns,22have been disproved,23and most commercial columns of that type are now 8 mm wide. Columns that are 30 cm in length, and up to 25 mm in width are also commercially available, and these columns provide resolution as good as, or better24than, the narrow (4.0 mm) bore models. These columns are useful for preparative purposes (see preparative section).
f. Column-packing Equipment.-Commercially available equipmentz-28 is available for laboratory-packing of analytical and preparative columns. With minimal practice, columns can be packed as efficiently as J. G. Baust, R. E. Lee, Jr., and H. James, J . Liq. Chromatogr., 5 (1982) 767-779. N. W. H. Cheetham and V. E. Dube, J . Chromatogr., 262 (1983) 426-430. M. R. Ladisch and G . T. Tsao, J . Chromatogr., 166 (1978) 85-100. K. Brunt, J . Chrornatogr., 246 (1982) 145-151. M. Verzele and E. Geeraert, J . Chromatogr. Sci., 18 (1980) 559-570. J. Kumanotani, R. Oshima, Y. Yamauchi, N. Takai, and Y. Kurosu, J . Chromatogr., 176 (1979) 462-464. (26) A. D. Jones, I. W. Burns, S. G . Sellings, and J . A. Cox, J . Chromatogr., 144 (1977)
(20) (21) (22) (23) (24) (25)
169- 180. (27) G . J. Manius and R. J. Tscherne, Am. Lab. (Fait$eld, Conn.), 13 (1981) 138-145. (28) S. A. Matlin and L. Chan, J . High Resolut. Chromatogr. Chromatogr. Commun., 8 (1985) 23-27.
22
KEVIN B. HICKS
those from commercial sources, and this can generally be done at a small fraction of the cost for a new column. For information on packing a particular phase, see Section II,2. All modern packing is done with slurries of the stationary phase in an appropriate solvent. For silica-based phases, actual packing takes place at pressures of -28 MPa (4000 lb. in.-2) and requires only a few seconds. Cation-exchange resin columns can frequently be packed by gravity sedimentation, followed by further compression with flow from an analytical pump. The packing of columns is one of the most overlooked sources of cost economics in 1.c. analyses. g. Column Ovens and Post-Column Reaction-Modules.-Many 1.c. columns require elevated temperature for proper performance. Both water jackets and ovens for this purpose are commercially available, but the latter type is more useful and makes leak-detection more practical. Even if an elevated temperature is not required, the enclosure of column, injector, and fittings in a sealed-column compartment (free from sporadic aircurrents and temperature changes) markedly improves the baseline on refractive index detector systems. In the absence of column ovens, enclosing the column, tubing, and fittings with large-diameter Tygon tubing will also improve detector stability and sensitivity. Several post-column reaction-modules are commercially available, and many can also serve as column heaters. For details of specific modules, see references listed in Section IV,ld.
h. Detectors.-An extensive listing of the characteristics of various 1.c. detection systems may be found in Section IV,1. Several general aspects about detectors should be described here, however. In general, the most sensitive detector available for a given separation should be used. Sensitive detection allows injection of very dilute samples and results in greatly extended column-life. Whereas some detector types may be best suited for only a few types of separations, others are more versatile and should be considered first when purchases are made. Photometric detectors that have exchangeable cells for analytical and preparative applications are very useful. Many analytical detectors, especially those of the refractive-index type, have definite limits on flow rates and back pressures, and should not be used for preparative chromatography (flow rates >10 mL/min). One commercially available8refractive-index monitor can be used at flow rates of up to 100 mL/min, but it is equally useful for analytical work at <1 mL/min.
i. Data Systems.-A number of inexpensive, microprocessor-controlled, recording integrators are now available. These are capable of computing unknown concentrations of carbohydrates by using external or
H.P.L.C. OF CARBOHYDRATES
23
internal standard methodology on a peak-height or peak-area basis. Although, when used properly, these integrators are ideal for analytical determinations, they should not be used to monitor preparative separations, because they do not operate in “real time,” but, rather, with an unspecified, delay time. Traditional strip-chart recorders are best for monitoring proper collection-times for preparative chromatography. j. Automated Fraction-Collectors.-These instruments are exceptionally useful for preparative I.c., especially when they are interfaced, through microprocessors, with the rest of the 1.c. system. Certain collect o r ~contain ~ , ~ so-called “intelligent” modes that permit collection on a peak-sensing mode, as well as on time, drop, or volume modes. These systems are especially useful for automated, preparative chromatography of carbohydrates .8 2. Stationary Phases
a. Amine-modified Silica Gels.-These popular stationary phases are silica gels that have been covalently modified with polar aminopropyl or other amine-containing groups or cyanopropyl-containing groups. The development of these packings in the mid-l970’s, an innovation that allowed the development of modern l . ~ analysis . of sugars, has been thoroughly r e v i e ~ e d .Today, ~~.~~ amine-modified silica gels are available in a variety of shapes (spherical or irregular) and sizes (3,5,or 10 pm) and can be purchased in bulk, or in pre-packed columns, from many commercial supp1ie1-s.~~ Moreover, the aminopropyl phase is readily prepared26from 1.c.-grade silica gel, and can be packed into analytical,26or preparative28sized columns with common packing-equipment. For some applications, there is no need to use an amine-bonded silica gel, because, when various amines are added to the mobile phase, ordinary silica-gel columns can be used for sugar ~ e p a r a t i o n s . The ~ ” ~advantages ~ and disadvantages of these in situ amine-modified columns have been d i s c u s ~ e d . ~ ~ ~ ~ When amine-modified silica-gel columns are eluted with a mobile phase of acetonitrile-water, carbohydrates are separate on the basis of normal, phase p a r t i t i ~ n i n g ~increasing ~ - ~ ~ ; the”water content of the mobile phase (29) L. A. T. Verhaar and B. F. M. Kuster, J . Chromatogr., 220 (1981) 313-328. (30) J. F. Pirisino, in J. F. Lawrence (Ed.), Food Constituents and Food Residues, Their Chromatographic Determination, Dekker, New York, 1984, pp. 159-193. (31) K. Aitzetmiiller, J . Chromatogr., 156 (1978) 354-358. (32) B. B. Wheals and P. C. White, J . Chromatogr., 176 (1979) 421-426. (33) H. Binder, J . Chromatogr., 189 (1980) 414-420. (34) M. D’Amboise, D. Noel, and T. Hanai, Carbohydr. Res., 79 (1980) 1-10. (35) L. A. T. Verhaar and B. F. M. Kuster, J . Chromatogr., 234 (1982) 57-64. (36) Z. L. Nikolov and P. J. Reilly, J . Chromatogr., 325 (1985) 287-293.
24
KEVIN B. HICKS
speeds up the elution of the sugars. Early investigators mistakenly called these “reversed-phase” columns, and that unfortunate misnomer is still occasionally used. Besides being useful for normal-phase separations, amine-modified silica-gel columns are useful for weak anion-exchange applications when appropriate buffers are used. Details of the proper use of these columns for separation of sugars, oligosaccharides, glycopeptides, and acidic carbohydrates are given in Section 111. Serious attention must be given to the maintenance of aminopropylbonded phase columns. The silica gel-based packing will dissolve in water-rich mobile phases, leaving voids in the inlet end. To delay this process, a second column packed with silica gello should be placed between the pump and the injector, to saturate the mobile phase with silica gel. The formation of glycosylamines between reducing sugars and stationaryphase amino groups is another common process that leads to column failure. Certain sugars, such as most pentoses, galactose, mannose, and l a c t o ~ e I ~ .readily ~ ~ . ~ *react with, and deactivate, these stationary-phase amino groups unless the mobile phases are buffered to pH 6 with phosphate buffer.1° Although it has been claimed39that in situ,amine-modified silica-gel columns are more durable than covalently bonded types, these columns also dissolve rapidly in their required mobile phases, retention time are quite variable, and, furthermore, they also are not suitable for the analysis of ribose, galactose, arabinose, mannose, and l a c t o ~ e . ~ When an amino-type column becomes contaminated by physically adsorbed, non-polar materials, it may be cleaned by washing with acetonitrile, hexane, and dichloromethane. If column failure is due to covalent interactions, or to dissolution of the stationary phase, there is very little that can be done to regenerate it. Samples should therefore always be cleaned up, and a pre-column and a saturator column should always be used with the analytical column.
b. Cation-Exchange Resins.-During the first half of the 1980’s, there was a dramatic increase in the application of resin-based, 1.c. columns to the separation of carbohydrates. The mechanisms involved in these separations are not based on newly discovered “ion-moderated partitioning” effects, as many reports have claimed, but are derived from the longknown principles developed by Wheaton and Bauman,40Jones and co(37) S. R. Abbott, J . Chromatogr. Sci., 18 (1980) 540-550. (38) C. Brons and C. Olieman, J . Chromatogr., 259 (1983) 79-86. (39) J. G. Baust, R. E.Lee, Jr., R. R. Rojas, D. L. Hendnx, D. Friday, and H. James, J . Chromatogr., 261 (1983) 65-75. (40) R. M. Wheaton and W. C. Bauman, Ann. N.Y. Acad. Sci., 57 (1953) 159-176.
H.P.L.C. OF CARBOHYDRATES
25
w o r k e r ~ , ~Barker ’ - ~ ~ and associates,4 and others,45for the separation of molecules by “low-pressure” column-chromatographic methods. These techniques were refined over the years (for a review of the chronological development, see Refs. 30 and 46-48), and, today, 1.c. columns packed with sulfonated, polystyrene-divinylbenzene spheres of precisely controlled particle sizes (-10 pm) are routinely used for the separation of every class of carbohydrate listed in Section 111, except for the larger oligosaccharides. Pre-packed columns in a variety of particle sizes, ionic forms, and column dimensions are commercially available, and a list of suppliers has been published.30Bulk resins, usually in the H+ form, are also available from many of these vendors, and they can be readily converted into the commonly used CaZ+(Refs. 49 and 50) and Ag+ (Ref. 51) forms. The expense of these resins is related to their size and monodispersity, with the smallest, most precisely sized resins costing the most. Any resin having a particle size of <400 mesh can be used with some success, but those in the 10-15-pm size give the most efficient analytical and preparative separations. Detailed procedures are available for packing various Monodisperse resins pack easily, with the use of only an extra empty column to serve as a reservoir and a union to join the reservoir and packing column together. The soft, 4%-crosslinked resins must be packed with care, to avoid crushing the spheres. The 8%crosslinked resins are, however, more stable, and can be slurry-packed in water at pressures >14 MPa. Preparative columns8 (2.2 x 30 cm) are packed with preparative-sized reservoirs, and the larger (30-60 pm), 4%and 8%-crosslinked resins should be packed into them at pressures of <7 and 14 MPa, respectively. Small (10 pm), monodisperse resins are easiest to pack into preparative columns and can be allowed to settle by gravitation before pumping at 3-4 times the normal flow-rate (-10 mL/min). Hydrogen-form resins are always eluted with dilute, mineral or organic acids (pH 2), and, as such, they are constantly regenerated during use. Hydrochloric acid should not be used, because of its corrosive properties. (41) J. K. N. Jones, R. A. Wall, and A. 0. Pittet, Chem. Ind. (London), (1959) 1196. (42) J. K. N. Jones, R. A. Wall, and A. 0. Pittet, Can. J . Chem., 38 (1960) 2285-2289. (43) J. K. N. Jones and R. A. Wall, Can. J . Chem., 38 (1960) 2290-2294. (44) S. A. Barker, B. W. Hatt, J. F. Kennedy, and P. J. Somers, Carbohydr. Res., 9 (45) (46) (47) (48) (49) (50) (51)
(1969) 327-334. S. J. Angyal, G . S. Bethell, and R. J. Beveridge, Cnrbohydr. Res. 73 (1979) 9-18. R. Wood, L. Cummings, and T. Jupille, J . Chromatogr. Sci., 18 (1980) 551-558. S. J . Angyal, Chem. SOC. Rev., 9 (1980) 415-428. K. B. Hicks, P. C. Lim, and M. J. Haas, J . Chrornatogr., 319 (1985) 159-171. M. R. Ladisch, A. L. Huebner, and G . T. Tsao, J . Chromatogr., 147 (1978) 185-193. L. E. Fitt, W. Hassler, and D. E. Just, J . Chromntogr., 187 (1980) 381-389. H. D. Scobell and K. M. Brobst, J . Chrornatogr., 212 (1981) 51-64.
26
KEVIN B. HICKS
For Ca2+,Pb2+,and Ag+ columns, pure degassed water is employed as the mobile phase. When used under these conditions, each of these columns has a fairly wide range of applications, as will be discussed in Section 111. In general, however, each column is best suited for the following applications: 8%-crosslinked, H+ form: sugar acids and alcohols; 4%, Ca2+:oligosaccharides (d.p. <6); 8%, Ca2+:monosaccharides and sugar alcohols; 4%, Ag+: oligosaccharides (d.p. <12-14); and 8%, Pb2+: monosaccharides (see Addendum). The various mechanisms whereby carbohydrates are separated on cation-exchange columns have been the subject of intensive study. In general, a combination of mechanisms, including exclusion, ligand exchange, and hydrophobic adsorption, appears to be involved. The H+-form columns,48for instance, separate molecules on the basis of ion- and sizeexclusion and hydrophobic adsorption, resulting in the elution of carbohydrates of high molecular weight before small ones, acidic carbohydrates before neutral species, and polar sugars before nonpolar (deoxy, acetylated, or methylated) sugar derivatives. The Ca2+,Pb2+,and Ag+ columns separate carbohydrates on the same basis as the H+ columns, with the addition of an extra mode, namely, ligand e x ~ h a n g e . ~In * .the ~~ last mode, selectivity (and resolution) is brought about by the variable capacity of the hydroxyl groups of each sugar to form complexes with the fixed cation of the stationary phase. Sugar mixtures that cannot be resolved on one ionic form of the resin can often be resolved on another, which will display a different selectivity. Most resin columns should be run at elevated temperatures, because, at room temperature, the partitioning of solutes between the mobile and stationary phases is a diffusionlimited process.54Increasing the temperature speeds up the partitioning process, and decreases the height equivalent per theoretical plate. Moreover, at room temperature, mutarotation of most reducing sugars is slow, relative to the chromatographic process, and individual anomers of each sugar are partially resolved, leading to broad peaks. At temperatures of -85", mutarotation is faster, and reducing sugars are eluted as one sharp peak. Hence, all Ca2+-,Pb2+-,and Ag+-formcolumns should be run at 7585" in order to obtain the highest column-efficiency and resolution. For the separation of sugar acids on H+-form resins, however, high temperature should usually be avoided, because of partial, or complete, conversion of these acids, in situ, into various lactone forms.48 (52) S. J. Angyal, Tetrahedron, 30 (1974) 1695-1702. (53) R. W. Goulding, J . Chromatogr., 103 (1975) 229-239. (54) R. Pecina, G. Bonn, E. Burtscher, and 0. Bobleter, J . Chromatogr., 287 (1984) 245258.
H.P.L.C. OF CARBOHYDRATES
27
Cation-exchange resin columns are relatively easy to maintain, and this is a major reason for their popularity. Unlike silica-based columns, they are not sensitive to extreme pH values, and they do not dissolve in aqueous, mobile phases. As in the case of all 1.c. columns, the key to long life is effective, sample clean-up. Samples should, whenever possible, be pretreated with small amounts of cation-exchange resin in the appropriate ionic form, and pre-columns containing a similar resin in the same ionic form should be used, and changed regularly. Inexpensive, cation-exchange resins (minus 400-mesh size) can be slurry-packed into precolumns, and their use adds very little to peak broadening. Mixed-bed can be used in pre-columns to de-ash samples. When this is done, the anion-exchange resin must be of a weak-base variety, because strongbase resins absorb sugar^.^^^^^ Convenient, cartridge-type pre-columns are also commercially available. Various mobile-phase additives, such as calcium acetate” and calcium eth~lenediaminetetraacetate~~ have been used in order to allow constant regeneration of the Ca2+-formcolumns. This, of course, adds another step to mobile-phase preparation and is unsuitable for preparative use. With proper sample clean-up, use of such additives should be unnecessary. If, however, a column becomes contaminated, the back-pressure will rise to unacceptable limits. If this is due to nonpolar materials that have adsorbed to the packing, the column should be backflushed with 3 : 7 acetonitrile-water. If column failure is due to ionically bound species, it must be emptied, and the resin must be cleaned by treatment with 3 N HCl, converted into the desired ionic form, and repacked as already described. The regeneration of the phase inside the column is not recommended. c. Alkylated (Reversed-Phase) Silica Gels.-These durable phases are extremely useful for the analysis and preparation of less-polar carbohydrates and such carbohydrate derivatives as those encountered in the synthesis laboratory. Quite often, complex mixtures such as these may be readily fractionated on a 15- to 25-cm-long column packed with 3-pm size (&-bonded silica gel, using only pure water, or dilute methanol, as the isocratic eluant. A good example of this, covered in detail in a later Section, is in the separation of isomeric glycosides formed during the reaction of reducing sugars or sugar acids with methanol in the presence of an acid catalyst. Often, reactions can be more effectively examined for (54a) P. L. Keeling and P. James, J . Liq. Chromatogr., 9 (1986) 983-992. (55) M. Tanaka, Carbohydr. Res., 88(1981) 1-8. (56) M. L. Richmond, D. L. Barfuss, B. R. Harte, J. I. Gray, and C. M. S h e , J . Dairy Sci., 65 (1982) 1394-1400. (57) D. C. Woollard, N. Z . J . Dairy Sci. Techno/., 18 (1983) 209-224.
28
KEVIN B. HICKS
particular products by this method than by t.l.c., as in the analysis of synthetic mixtures containing diastereoisomeric pyrimidine nucleoside and isomeric 2’-deo~y-C-nucleosides.~~~ For more-difficult separations, such as the separation of diastereoisomeric guanosine deriva t i v e and ~ ~ coformycin ~~ analogs,57dsimple binary gradients, composed of water (or phosphate buffer) and methanol, provide good separations. Other so-called “reversed-phase” silica-gel phases may have better selectivities for particular separations than the common C18 variety. A “pheny1”-bonded phase, for instance, was found useful for the analysis of synthetic reactions containing adriamycin analogs.57eAlthough these methods are quite useful, they do not replace t.1.c. for the routine monitoring of reactions. In most cases, the greater expense of lac.,combined with its lower speed make it an impractical method for monitoring reactions, especially as many reactions can be monitored simultaneously on one t.1.c. plate. However, when quantitative results are desired, or when separations of similar compounds cannot be achieved by t.l.c., such 1.c. methods as these are advised. The use of 1.c. as a preparative method will be covered later, but it is relevant to mention here that, for nonpolar carbohydrate derivatives, reversed-phase type silica gels also function quite well in this capacity. Various nucleoside derivative^,^^^?^^^ for instance, have been isolated on preparative-sized (2.2 x 50 cm) columns packed with CI8silica gels. Because of the low capacities of columns of these types (maximum of 100-150 mg per injection), they will not soon replace the more-common types of silica-gel chromatography or their “flash”-type modifications, for the isolation of large quantities of reaction products. The same, reversed-phase, silica-gel columns that are used for 80-90% of all other 1.c. applications find only limited use in analysis of polar carbohydrates. This is primarily due to the poor retention of these polar, water-soluble compounds on the nonpolar, alkylated, stationary phases when even the weakest mobile phase, namely, water, is used. Nevertheless, for several carbohydrate applicati~ns,~~~-g these columns are ideal, and therefore deserve consideration. (57a) J. C. Boehm and W. D. Kingsbury, J . O m . Chem., 51 (1986) 2307-2314. (57b) J. C.-Y. Cheng, U. Hacksell: and G. D. Daves, Jr., J . Org. Chem., 51 (1986) 30933098. (57c) R. C. Moschel, K. Hemminki, and A. Dipple, J . Org. Chem., 51 (1986) 2952-2955. (57d) 0. L. Acevedo, S. H. Krawczyk, and L. B. Townsend, J . Org. Chem., 51 (1986) 1050- 1058. (57e) N. Kumar, R. Seshadri, and M. Israel, Carbohydr. Res., 153 (1986) 171-180. (570 E. Rajakyla, J . Chromatogr., 353 (1986) 1-12. (57g) G . D. McGinnis, S. Prince, and J. Lowrimore, J . Carbohydr. Chem., 5 (1986) 83-97.
H.P.L.C. OF CARBOHYDRATES
29
When water is used as the mobile phase, Cia-bonded silica-gel columns are quite useful for the analysis of the more nonpolar or higher-molecularweight carbohydrates, such as methyl glyco~ides,’~ chitinJ9 or starchmderived oligosaccharides, and such N-acetylated or deoxy sugar-containing oligosaccharides as those in human milk6’ and glycopeptides.62For analysis of very nonpolar derivatives, such as peralkylated oligosaccharides,‘j3or many of the pre-column 1.c. derivatives discussed in Section IV,l, such organic modifiers as acetonitrile or primary alcoholsu must be added to the mobile phase to shorten retention times. Conversely, some success has been achieved in enhancing the retention of normally unretained sugars by addition of salts to the mobile p h a ~ e . ~ f j ~ When such hydrophobic bases as alkylamines are added to the aqueous mobile phase, such acidic carbohydrate derivatives as ascorbic acids,66 uronate~,~’ and sulfated oligosaccharides68are retained and separated by ion-pairing mechanisms. A major disadvantage of reversed-phase columns is that they resolve the anomeric forms of reducing sugars, and this leads to broad, complex peaks. Neither the addition of basic mutarotation catalysts, nor elevation of column temperature can satisfactorily correct this problem without destroying the column, or decreasing the resolution, re~pectively.~~ However, nonreducing sugars and those oligosaccharides having chemically reduced, reducing ends, produce sharp peaks on these columns. Alkylated silica-gel columns have low sample-capacity and are therefore of limited use in preparative applications. Reversed-phases are generally very stable, and do not dissolve in aqueous mobile phases, as do normal-phase silica-gel columns. After injection of many samples that contain highly retained, nonpolar materials, resolu(58) N. W. H. Cheetham and G . Teng, Carbohydr. Res., 144 (1985) 169-175. (59) K. Blumberg, F. Liniere, L. Pustilnik, and C. A. Bush, Anal. Biochern., 119 (1982) 407-41 2 . (60) N. W. H. Cheetham, P. Sirirnanne,and W. R. Day, J . Chrornatogr., 207 (1981) 439-
444. (61) V. K. Dua and C. A. Bush, Anal. Biochern., 133 (1983) 1-8. (62) V. K. Dua and C. A. Bush, Anal. Biochern., 137 (1984) 33-40. Franztn, and P. Albersheim, Methods (63) M. McNeil, A. G. Darvill, P. h a n , L.-E. Enzymol., 83 (1982) 3-45. (64) L. A. T. Verhaar, B. F. M. Kuster, and H. A. Claessens, J . Chrornatogr., 284 (1984) 1-11. (65) N. W. H. Cheetharn and G . Teng, J . Chromatogr., 336 (1984) 161-172. (66) C. S. Tsao and M. Young, J . Chromatogr., 330 (1985) 408-411. (67) A. G. J. Voragen, H. A. Schols, J. A. De Vries, and W. Pilnik, J . Chromatogr., 244 (1982) 327-336. (68) A. Heyraud and C. Rochas, J . Liq. Chrornatogr., 5 (1982) 403-412.
KEVIN B. HICKS
30
tion may decrease, but this can generally be corrected by washing the column with methanol and boric acid solutions.69Methods which detail the procedures for packing these columns have been published.27 d. Anion-Exchange Resins and Silica Gels.-Both silica-gel- and polystyrene-based, anion-exchange, 1.c. stationary-phases are commonly used for the separation of the many classes of acidic carbohydrates listed in Section 111. These packings, which bear quaternary ammonium groups, are available in efficient 5- and 10-pm particle sizes. The development of modem anion-exchange phases, their various physical characteristics, and commercial suppliers have been reviewed.46Resin-type and silicabased anion-exchange particles can be packed into columns by using the methods described earlier for cation-exchangers and aminopropyl silica gels, respectively, and by following manufacturer's directions. These columns are often eluted with phosphate buffers, or salt solutions, for separation of simple and complex acidic carbohydrates. When boric acidcontaining buffers are e m p l ~ y e d , ~neutral ~ . ~ ' as well as acidic sugars can be separated by borate-complexation mechanisms. Unfortunately, these separations are rather lengthy (>lh). Silica-based anion-exchangers tend to have short life-times. This may be extended somewhat by proper sample clean-up and by the use of a silica-gel-saturation column, but this rapid degradation remains the biggest disadvantage of these phases. The resin-based phases are very stable, unless operated above 65". Unfortunately, because of the slow diffusion processes in these resins, they must be operated at higher temperatures in order to achieve good efficiencies. A unique anion-exchange column has been developed7*that has a thin (non-diffusion limited) anion-exchange phase coated onto a 10-pm latex bead. When a mobile phase of 0.15 M NaOH is used, neutral carbohydrates are converted into anions, which are separated on the column. Although the resin has low capacity, and probably causes degradation of the carbohydrates, when it is coupled to a triple-pulsed, amperometric detector, the system provides extremely sensitive, high-resolution separations. (69) P. VrBtny, J. Coupek, S. Vozka, and Z. Hostomskii, J . Chrornafogr.,254 (1983) 143155.
(70) S. Honda, M. Takahashi, K. Kakehi, and S. Ganno, Anal. Biochem., I13 (1981) 130138. (71) S. Honda, S. Suzuki, M. Takahashi, K. Kakehi, and S. Ganno, Anal. Biochern., 134 (1983) 34-39. (72) R. D. Rocklin and C. A. Pohl, J . Liq. Chrornafogr., 6 (1983) 1577-1590.
H.P.L.C. OF CARBOHYDRATES
31
e. Miscellaneous Stationary Phases.-Several less well-known, stationary phases have been used for carbohydrate separations. Boronic acidsubstituted silica ge1,73,74 for example, is useful for chromatography of sugars and nucleosides that contain cis-diols. When copper(I1) silicate gel is used as the stationary p h a ~ e , sugars ~ ~ . ~are ~ separated by combined ligand-exchange and normal phase-partition modes. This phase can be readily synthesized by treating 1.c.-grade silica gel with ammoniated copper sulfate solutions. Another useful silica derivative, diol-modified silica gel,38appears to function like aminopropyl silica gel, but it is much more robust, and can be used for separation of pentoses and hexoses that cannot be analyzed on the amine-modified phases. A similar phase, a socalled “polyol” bonded silica gel,76ahas also been used to separate various mono-, di-, and tri-saccharides. As on the previously mentioned diol phase, however, reducing sugars are eluted from this polyol phase in broad, or double, peaks (resolution of anomeric forms). Such mutarotation catalysts as triethylamine can be added to the mobile phase to produce sharp, symmetrical peaks, but this practice is usually destructive to these silica gel-based packings. Although ion-exchange resins77have often been used for normal-phase, partition chromatography in open, glass columns, this technique has not been easily adapted to high-performance and high-pressure applications. However, new resins have now been developed that are of the correct size (5 pm) and rigidity (>50% cross-linking with divinylbenzene)to withstand high pressures and the high flow-rates of acetonitrile-water mobile phases. The application of these resins for the separation of monosacand sialic acids charides, deoxy sugars, 2-acetamido-2-deoxy-~-hexoses, has been d e m o n ~ t r a t e d . It ~ ~is- ~ anticipated ~~ that the latter stationary phases will be used much more frequently in the future.
(73) M. Glad, S. Ohlson, L. Hansson, M.-0. Mhsson, and K. Mosbach, J . Chrornatogr., 200 (1980) 254-260. (74) E. Hagemeier, K.-S. Boos, E. Schlimme, K. Lechtenborger, and A. Kettrup, J . Chrornarogr., 268 (1983) 291-295. (75) J. L. Leonard, F. Guyon, and P. Fabiani, Chrornatographia, 18 (1984) 600-602. (76) F. Guyon, A. Foucault, M. Caude, and R. Rosset, Carbohydr. Res., 140 (1985) 135138. (76a) M. Verzele and F. Van Damme, J . Chrornarogr., 362 (1986) 23-31. (77) 0. Samuelson, Adu. Chrornarogr., 16 (1978) 113-149. (78) S. Honda, S. Suzuki, and K. Kakehi, J . Chrornatogr., 291 (1984) 317-325. (79) S. Honda, T. Konishi, and S. Suzuki, J . Chrornarogr., 299 (1984) 245-251. (80) S. Honda and S . Suzuki, Anal. Biochern., 142 (1984) 167-174. (80a) A. Sugii, K. Harada, and Y. Tomita, J . Chrornarogr., 366 (1986) 412-416.
32
KEVIN B. HICKS
111. SEPARATIONS AND APPLICATIONS
1. Analytical Separations a. Neutral Mono- and Di-saccharides.-A great number of the published 1.c. methods for carbohydrates deal with the simple mono- and disaccharides, and this fact is verified by the many comprehensive reviews that cover this s ~ b j e ~As tsuch, . ~the~reader ~ ~is referred ~ ~ ~to those ~ ~ ~ ~ ~ reviews, which contain details of specific separations of simple and complex mixtures. As has been pointed out,s1 no general conditions and equipment are currently available for the simultaneous 1.c. separation of all of the known monosaccharides. Fortunately, however, this necessity seldom arises, and, instead, methods are more often needed for the separation of the mono- and di-saccharides present in such samples as foods, pharmaceuticals, and hydrolyzates of polysaccharides, glycoconjugates, and “biomass” materials. Specific procedures for these examples are given in Section 111,2. Additional key references that describe advance in the separation of mono- and di-saccharides are given in Table I. In general, the method selected for a given analysis of sugars should be the one that provides the simplest and fastest separation, without sacrificing accuracy of analysis or column-life. Because cation-exchange resin-based columns (Ca2+or Pb2+form) provide convenient, accurate, rapid, reproducible, and sensitive analyses, and use only water as the mobile phase, they are generally the method of choice, despite some claims to the contrary.39Most mixtures of monosaccharides can be resolved on one or the other form of these columns (see Ref. 30 for a general comparison). However, for certain other applications, such as the simultaneous analysis of several disaccharides, aminemodified silica-gel columns must be u ~ e d ,despite ~ ~ . ~their relative instability and requirement for a highly pure, acetonitrile-containing, mobile phase. Reversed-phase silica-gel columns are of little use for separation of polar mono- and di-saccharides, because of their low capacity factors. More-nonpolar sugar derivatives, such as 2-acetamido-2-deoxy derivatives, are retained on &-bonded silica gel,59but are separated into two, or more, broad (difficult to analyze) anomeric peaks. Such columns are very useful for separation of simple g l y c o s i d e ~ .A ~ .column ~ ~ ~ ~ used ~ for monosaccharide separations0combines the stability of ion-exchange resins with the high resolving power of normal, phase-partitioning using acetonitrile-water as the mobile phase, and further use of this method is expected. (81) S. Honda, Anal. Biochem., 140 (1984) 1-47. (82) A. Heyraud and M. Rinaudo, J . Liq. Chromatogr., 4 (Suppl. 2) (1981) 175-293. (82a) K. Robards and M. Whitelaw, J . Chromatogr., 373 (1986) 81-110.
H.P.L.C. OF CARBOHYDRATES
33
TABLEI Selected L. C. Methods for the Analysis of Mono- and Di-saccharides
Class
Members
Aldoses
d o s e and a l t r o ~ ea, r~a~b i n ~ s e , ' ~galactose , ~ ~ . ~ ~and glucose,38.39,83 ribose,39mannose and xylose10.39.83 Ketoses f r u c t o ~ e ,psicose," ~ ~ ~ ~ ~erythro-2-pentulose .~ and sorb o ~ e , ~t a~g, a) t~o ~ e ~ ~ . ~ ' Alditols arabinitol, galactitol, ribitol, mannitol, and glucito1,38.39.46.86 xylitol19.39,86 ,~~~~~ and 2Deoxy sugars 2 - d e o ~ y - a r a b i n o - h e x o s e6-deoxyglucose deoxy-erythro-pento~e,~~ fucose and r h a ~ n n o s e ,2~~.~~ acetarnid0-2-deoxyhexoses~~~~~~
Disaccharides cellobiose and c e l l o b i ~ l o s e(gentiobiose, , ~ ~ ~ ~ ~ ~ isomaltose, kojibiose, laminarabiose, leucrose, melibiose, nigerose, palatinose, sophorose, trehalose, turanose, and xylobio~e),~~.W lactose and l a c t u l o ~ emaltose ,~~~~~ and m a l t ~ l o s e ~ ~ ~ ~ ~
b. Ionic Mono- and Di-saccharides.-Common members in this category are the uronic, aldonic, keto-aldonic, and aldaric acids, which occur in metabolic pathways, biopolymers, and fermentative processes. For analysis of simple mixtures of uronic acids, methods based on ion-exclusion chr~matography*~.~*.~~ are rapid (<15 min) and accurate. When several uronic acids exist in the same sample, separations are best performed on r e ~ i n - ~ or ' , ~ i,l~i c~a - b a s e d ,strong ~~,~~ anion-exchange columns, which provide greater resolution. The silica-based columns are somewhat unsta(83) R. C. Pettersen, V. H. Schwandt, and M. J. Effland, J. Chromatogr. Sci., 22 (1984) 478-484. (84) L. W. Doner, Carbohydr. Res., 70 (1979) 209-216. (85) K.B. Hicks, D. L. Raupp, and P. W. Smith, J. Agric. Food Chem., 32 (1984) 288292. (86) S. Honda, M. Takahashi, S. Shimada, K. Kakehi, and S . Ganno, Anal. Biochem., 128 (1983) 429-437. (87) D. E. Hughes, J . Chromatogr., 331 (1985) 183-186. (88) W. M. Blanken, M. L. E. Bergh, P. L. Koppen, and D. H. van den Eijnden, Anal. Biochem., 145 (1985) 322-330. (89) K.B. Hicks, E. V. Symanski, and P. E. Pfeffer, Carbohydr. Res., 112 (1983) 37-50. (90)Z. L. Nikolov, M. M. Meagher, and P. J. Reilly, J . Chromatogr., 319 (1985) 51-57. (91) N. W. H. Cheetham and P. Sirimanne, J . Chromatogr., 208 (1981) 100-103. (92) A. Hjerpe, B. Engfeldt, T. Tsegenidis, and C. A. Antonopoulos, J. Chromatogr., 259 (1983) 334-337. (93) R. Oshima, Y. Kurosu, and J. Kumanotani, J. Chromatogr., 179 (1979) 376-380. (94) K. Mopper, Anal. Biochem., 86 (1978) 597-601. (95) M. J. Spiro, Anal. Biochem., 82 (1977) 348-352. (%) P. Gacesa, A. Squire, and P. J. Winterbum, Carbohydr. Res., 118 (1983) 1-8.
34
KEVIN B. HICKS
ble, but provide rapid, simultaneous separations of galacturonic, mannuronic, and glucuronic acids.67For the separation of the D-mannuronic and L-guluronic acids that exist in hydrolyzates of alginates, 1.c. on strong anion-exchange silica-gel columns has been recommended.67*% The polystyrene-based resins are more stable, but are not useful for rapid separations. Often as long as 60 min is required to resolve such complex mixtures as that of iduronic, glucuronic, mannuronic, and galacturonic acids.71 Surprisingly few 1.c. methods are yet available for the aldonic acids. These acids have been separated from non-acidic compounds by chromatography on s t r ~ n g ~and ~ - on ~ weakloo-lolanion-exchangers. Separations of ribonic, D-glycero-D-gulo-heptonic, mannonic, glUCOniC, and galactonic acids have been successful on strong cation-exchange resins.48,101,102 Because of their involvement in several economically important fermentation pathways, several methods have been developed to separate the aldulosonic acids. During the biotechnological conversion of ~ - g l u cose into D-fructose by way of ~-arabino-hexos-2-ulose(D-glucosone),Im the levels of the by-products D-gluconic (1)and ~-arabino-2-hexulosonic (2) acids were rapidly (
H.P.L.C. OF CARBOHYDRATES
35
ge1,Io4and strong anion-exchange silica gelIos is difficult, because these acids are eluted in broad peaks. Addition of magnesium ions to the mobile phase tends to speed up the elution of aldaric acids, and helps to provide somewhat sharper peak-geometries.% Peak broadening is partially due to the coexistence of acid and lactone forms, and this effect must be taken into account whenever sugar acids are chromatographed. In fact, many sugar acids can be baseline-separated from their lactone forms on aminopropyl silica ge1,100J01J04 anion exchange,98 and cati~n-exchange~~ columns, allowing the simultaneous determination of each form of the compound in a single sample. If individual levels of acid and lactone are not of interest, all lactones should be converted into the acid form prior to chr~matography.~~ Methods for the 1.c. analysis of ascorbic acids and related compounds in foods, pharmaceuticals, and physiological tissues have been extensively reviewed e l ~ e w h e r e . ' ~Ascorbic J ~ ~ acid may be determined after separation on cation-exchange 1.c. columns (H+form),losbut care must be taken to prevent on-column d e g r a d a t i ~ nThis . ~ ~ system only marginally separates L-ascorbic acid from its C-5 epimer, isoascorbic acid. Although ascorbic acids have little retention on reversed-phase silica gel, the use of ion-pairing reagents improves their retention and allows the separation of ascorbic and isoascorbic acids.Iw Ascorbic acid and its oxidation product, dehydroascorbic acid, have been separated on reversed-phaseIl0and on hydrophilic gel columns."' Both strongly and weakly basic anion-exchangers have been used for ascorbic acid analysis,Io7and the latter"* system has been used to separate, completely and rapidly ( e l 5 min), ascorbic, isoascorbic, dehydroascorbic, dehydroisoascorbic, and threoand erythro-2,3-hexodiulosonicacids. The 2-sulfate and 2-phosphate of Lascorbic acid are of biomedical importance, and their separations have
(104) E. I. Laakso, R. A. Tokola, and E. L. Hirvisalo, J . Chromatogr., 278 (1983) 406411. (105) D. G. Walters, B. G. Lake, D. Bayley, and R. C. Cottrell, J . Chromatogr., 276 (1983) 163- 168. (106) L. A. Pachla, D. L. Reynolds, and P. T. Kissinger, J . Assoc. Ofl.Anal. Chem., 68 (1985) 1-12. (107) L. W. Doner, in J. Lawrence (Ed.), Trace Analysis, Vol. 3, Academic Press, New York, 1984, pp. 113-138. (108) M. Griin and F. Loewus, Anal. Biochem., 130 (1983) 191-198. (109) C. S. Tsao and S. L. Salimi, J . Chromatogr., 245 (1982) 355-358. (110) J. W. Finley and E. Duang, J . Chrornatogr., 207 (1981) 449-453. (111) T. Seki, Y. Yamaguchi, K. Noguchi, and Y. Yanagihara, J . Chromatogr., 332 (1985) 283-286. (112) L. W. Doner and K. B. Hicks, Anal. Biochem., 115 (1981) 225-230.
36
KEVIN B. HICKS
also been noted.I13 An impressive separation of ascorbic acid, 2-0methylascorbic acid, 3,4-dihydroxy-5-methyl tetrone, ascorbic acid 2phosphate, ascorbic acid 2-sulfateYand 6-bromo-6-deoxy-~-ascorbicacid was achieved in less than 15 min on a reversed-phase-ionlpairing sysRelatively few methods exist for the 1.c. separation of sugar phosphates. Simple mixtures containing a sugar phosphate and a neutral aldose can be resolved by anion-Il4or cation-exchange columns.11sGlucosyl phosphate and glucose 6-phosphate have been separated on an ion-pairing, reversed-phase system,116and some low-resolution separations of pentose, hexose, and heptose mono- and di-phosphates have been achieved on strong anion-exchange silica gel. 117-118 The isomeric sugar phsopates, D-fructose 6- and D-glucose 6-phosphateYhave been separated by ion-pairing, reversed phase 1.c.lIsaA variation1ISbof the same system was used to separate inositol tri-, tetra-, penta-, and hexa-phosphates. Various deoxy-2-octulosonic acids [for example, 3-deoxy-~-manno-2octulosonic acid, (KDO)], neuraminic acids, and their derivatives have been chromatographed on 1.c. columns packed with anion-exchange resins,119-122 cation-exchange resins,123amine-modified silica g e 1 ~ , and ~ ~ ~ J ~ ~ reversed-phase silica gels.'" Although class separations of neutral sugars from these acidic compounds may be obtained on cation-exchangers, very little separation of individual sialic acids occurs. KDO and several derivatives of neuraminic acid have been more successfully separated on (113) D. Mauro, D. Wetzel, C. H. Lee, and P. A. Seib, J. Chrornatogr., 187 (1980) 421428. (114) K. Brunt and H. Hokse, J. Chromatogr., 268 (1983) 131-137. (115) R. M. Stikkelrnan, T. T. Tjioe, J. P. van der Wiel, and F. Van Rantwijk, J. Chromatogr., 322 (1985) 220-222. (116) T. T. Tjioe, J. P. van der Wiel, R. M. Stikkelman, A. J. J. Straathof, and F. Van Rantwijk, J . Chromatogr., 330 (1985) 412-414. (117) C. Giersch, J . Chromatogr., 172 (1979), 153-161. (118) S. K. Henderson and D. E. Henderson, J . Chromatogr. Sci., 23 (1985) 222-226. (118a) S. K. Henderson and D. E. Henderson, J . Chromatogr. Sci., 24 (1986) 198-203. (118b) A.4. Sandberg and R. Ahderinne, J. Food Sci., 51 (1986) 547-550. (119) A. K. Shukla, N. Scholz, E. H. Reimerdes, and R. Schauer, Anal Biochem., 123 (1982) 78-82. (120) A. K. Shukla and R. Schauer, J. Chromafogr.,244 (1982) 81-89. (121) A. K. Shukla, R. Schauer, U. Schade, H. Moll, and E. T. Rietschel, J. Chromutogr., 337 (1985) 231-238. (122) A. K. Shukla, R. Schauer, F. M. Unger, U. Zahringer, E. T. Rietschel, and H. Brade, Curbohydr. Res., 140 (1985) 1-8. (123) H. K. B. Silver, K. A. Karim, M. J. Gray, and F. A. Saiinas, J . Chromatogr., 224 (1981) 381-388. (124) S . Diaz and A. Varki, Anal. Biochem., 150 (1985) 32-46. (125) H. Fiedler and H. Faillard, Chromatographia, 20 (1985) 231-234.
H.P.L.C. OF CARBOHYDRATES
37
polystyrene-based, anion-exchange resins eluted with formate, I2l sulfate120.122 or borate buffer.II9 On these columns, retention is affected by the number of acetyl groups present on the neuraminic acid derivative; consequently, mono-, di-, and tri-0-acetylated acids are readily separated, as are many of the N-acetyl and N-glycolyl derivatives. When borate is included in the mobile phase, separations are also affected by the ability of the sialic acids to form stable borate complexes, and this leads to orders of elution different from those produced with formate or sulfate buffers. The more highly 0-acetylated derivatives are also well separated on reversed-phase columns by using an ion-pairing mode. 124 By application of one or more of these methods, mixtures of sialic acids most commonly encountered can be resolved, except for positional isomers. Sulfated disaccharides, such as those resulting from the breakdown of the chondroitin sulfates, have been separated on aminocyanopropyl silica aminopropyl silica gel,126J27 and cation-exchange columns. 128~129 Each of these columns produces distinctly different orders of elution, allowing the ready identification of chondroitin disaccharides by chromatography of unknowns uersus standards on two or three of the different phases. Oversulfated chondroitin disaccharides, containing 2 or 3 sulfate groups, may also be r e s ~ l v e d . ' ~ * Similarly, J ~ ~ J ~ ~ sulfated disaccharides from enzyme- or nitrous acid-treated heparin have been separated on rever~ed-phase,'~~ polar aminocyanopropyl silica gel,132 and strong anionexchange silica gel columns.133Often, these disaccharides are reduced with sodium borotritide prior to chromatography; this results in better chromatographic resolution and the ability to use sensitive, radiochemical-detection methods. c. Simple, Neutral 0ligosaccharides.-Within the area of oligosaccharide separations, the largest variety of 1.c. methods has been developed for the a-(1 + 4)-linked D-glucose oligosaccharides (malto-oligosaccharides), such as those present in starch hydrolyzates. Effective separations have been achieved on columns packed with aminopropyl(126) G. J.-L. Lee and H. Tieckelmann, Anal. Biochem., 94 (1979) 231-236. (127) A. Hjerpe, C. A. Antonopoulos, and B. Engfeldt, J . Chromatogr., 171 (1979) 339344. (128) K. Murata and Y. Yokoyama, Anal. Biochem., 146 (1985) 327-335. (129) K . Murata and Y. Yokoyama, Anal. Biochem., 149 (1985) 261-268. (130) A. Hjerpe, C. A. Antonopoulos, B. Engfeldt, and M. Nurminen, J . Chromatogr., 242 (1982) 193-195. (131) D. C. Seldin, N. Seno, K. F. Austen, and R. L. Stevens, Anal. Biochem., 141 (1984) 291-300. (132) G. J.-L. Lee and H. Tieckelmann, J . Chromatogr., 195 (1980) 402-406. (133) S. R. Delaney, M. Leger, and H. E. Conrad, Anal. Biochem., 106 (1980) 253-261.
38
KEVIN B. HICKS
bonded silica aminocyanopropyl-bonded silica gel, 138 in situ amine-modified silica ge1,139J40 pure silica gel,L41 and amin~propyl-"doped,"~~~ (218-bonded, reversed-phase silica gel, polystyrene-based anion-exchangers used in either a normal phase-partition,Iu or ion-chr~matographic~~ mode, or cation-exchange resins used in Ca2+(Refs. 23 and 50) or Ag+ (Refs. 51 and 145) forms. Aminopropyl and aminocyanopropyl silica gel columns both allow the rapid (d.p. 1-15 in 15 min) separation of these oligosaccharides in ascending order when aqueous acetonitrile is used as the mobile phase. The limited solubility of higher-d.p. oligosaccharides, dextrins, and starch in this mobile phase may, however, cause peak broadening, sample precipitation, and eventual fouling of the packing, unless samples are pretreated to remove these insoluble components. Moreover, glycosylamine formation between reducing oligosaccharides and stationary-phase aminopropyl groups10*37 can also lead to column failure (see Section II,2a). Pure silica gel columns that are physically modified in situ with aliphatic amines appear to be more robust in such applications, as the silica-absorbed amine is constantly regenerated. Use of such a stationary phase140 allowed the separation of malto-oligosaccharides from d.p. 2 to 23 in 40 min. Reversed-phase (c18)packings (with pure water as the mobile phase) are also quite durable in these applications, but, unfortunately, the anomeric forms of reducing oligosaccharides are partially resolved on these columns, leading to broad and difficult-to-analyze peaks. Although no practical solvent-modifier or temperature effect has been found to prevent this peak doubling, a chemical modification (aminopropyl doping)143of CIS columns has been used to eliminate this effect. At present, this column appears to be ideal for efficient, reproducible, and economical separation of a-(1 4 4)-linked gluco-oligosaccharides. Cation-exchange resins, which also may be eluted with pure water as the mobile phase, separate (134) R. Schwarzenbach, J . Chromatogr., 117 (1976) 206-210. (135) V. Kahle and K. Tesanl, J . Chromatogr., 191 (1980) 121-128. (136) K. Kainuma, T. Nakakuki, and T. Ogawa, J . Chromatogr., 212 (1981) 126-131. (137) 2. L. Nikolov, J. B. JakovljeviC, 2. M. BoHkov, and N. Sad, StarchlStaerke, 36 (1984) 97-100. (138) F. M. Rabel, A. G . Caputo, and E. T. Butts, J . Chromatogr., 126 (1976) 731-740. (139) K. Aitzetmiiller, Chromatographia, 13 (1980) 432-436. (140) C. A. White, P. H. Corran, and J. F. Kennedy, Carbohydr. Res., 87 (1980) 165-173. (141) S. Iwata, T. Narui, K. Takahashi, and S . Shibata, Carbohydr. Res., 133 (1984) 157162. (142) A. Heyraud and M. Rinaudo, J . Liq. Chromatogr., 3 (1980) 721-739. (143) B. Porsch, J . Chromatogr., 320 (1985) 408-413. (144) J. Havlicek and 0. Samuelson, Anal. Chem., 47 (1975) 1854-1857. (145) J. J. Warthesen, Cereal Chem., 61 (1984) 194-195.
H.P.L.C. OF CARBOHYDRATES
39
oligosaccharides in the order of descending molecular weight, owing to size-exclusion effect^.^.^^ Resins with crosslinking percentages between 4 and 8%, in the Ca2+ or Ag+ form, may be used as stationary phases according to the following general rules: resins with low percentage of crosslinking are less mechanically stable but allow resolution of saccharides of higher d.p. value. Silver-form resins are more mechanically stable than resins in the Ca2+form, and they also allow resolution of higher-d.p. saccharides. All cation-exchange resin (Ca2+and Ag+ form) columns must be operated at temperatures of 75-85' to produce optimum resolution. For high-conversion corn syrups, containing oligosaccharides smaller than maltotetraose, 8% Ca2+-formresins are appropriate. For analysis of low-conversion syrups, 4% Ag+-formresins are used. If samples are carefully de-ionized prior to injection, columns of this type can operate for years without significant loss of resolution, and are therefore often the choice for industrial, quality-control applications. Much of the previous discussion is directly applicable to the methods that are available for the separation of other neutral oligosaccharides. Hence, those oligosaccharides produced from the partial hydrolysis of cellulose, xylans, and dextrans with acid have been separated on reversed-phase p a ~ k i n g s , ~ @ "aminopropyl-bonded j"~~J~~ silica ge1,34914G149 and cation-exchange resins. 13,49~150,151Because of the low solubility of these oligosaccharides in partly aqueous, mobile phases, those stationary phases that use pure water as the mobile phase (cation exchangers and reversed-phase packings) are of especially great utility. The 4%crosslinked, Ag+- and Ca2+-formresins are particularly well suited to separation of cello-oligosaccharides (d.p. 1-8 in <20 min) and can be regenerated or repacked by the user (see Section 11,2,b). With proper care, aminopropyl silica-gel columns can also produce highly efficient and rapid (<15 min) separations of cello-oligosaccharides of d.p. 1-6, with the usual limitations imposed by low solubility of the cello-oligosaccharides in the mobile phase. Aminopropyl-bonded and physically modified amino silica-gel columns have also been used to separate fructo-oligosacfrom inulin (from d.p. 1 to 30 in <40 min), and oligosac(146) (147) (148) (149) (150) (151) (152) (153)
E. K . Gum,,Jr., and R. D. Brown, Jr., Anal. Biochern., 82 (1977) 372-375. R. Niesner, W. Briiller, and 0. Bobleter, Chrornatographia, 11 (1978) 400-402. D. Noel, T. Hanai, and M. D'Amboise, J . Liq. Chrornatogr., 2 (1979) 1325-1336. K. L. Smiley, M. E. Slodki, J. A. Boundy, and R. D. Plattner, Carbohydr. Res., 108 (1982) 279-283. M. R. Ladisch, A. W. Anderson, and G . T. Tsao, J . Liq. Chrornatogr., 2 (1979) 745760. J. Schmidt, M. John, and C. Wandrey, J . Chromatogr., 213 (1981) 151-155. A. Heyraud, M. Rinaudo, and F. R. Taravel, Carbohydr. Res., 128 (1984) 311-320. W. Praznik, R. H. F. Beck, and E. Nitsch, J . Chrornatogr., 303 (1984) 417-421.
40
KEVIN B. HICKS
charides produced from the hydrolysis of ~ h i t i n . ' ~The ~ Jlatter ~ ~ oligosaccharides have also been separated on reversed-phase columns.59 d. Simple, Ionic 0ligosaccharides.-Several methods have been developed for the 1.c. separation of simple, linear, acidic oligosaccharides that contain one or two types of carbohydrate unit. Oligosaccharides in this category include those produced by the partial depolymerization of pectic or alginic acids, K-carrageenan, and similar polysaccharides. Traditionally, these oligomers have been separated by gel-filtrati~n'~~ or anionexchange column-chromatography (see Ref. 156 and those cited therein). Both methods are lengthy, and, because of the tendency of oligoglycuronans to aggregate,157 the former method may not give pure fractions. L.c. methods for the separation of normal, pectic oligoglycosiduronic acids and those possessing 4,5-unsaturated, terminal residues, employ weak and strong anion-exchange s i l i ~ a - , ~strong ~ , ' ~ ~anion-exchange resin-,159and reversed-phase s i l i ~ a - based ~ ~ J ~stationary phases. Strong anion-exchange silica gels, which provide excellent resolution of oligoglycuronans from d.p. 1 through 8 in <30 min (acetate buffers as the mobile phase) are relatively unstable, and have limited lifetimes. Weak anionexchange silica gel (aminopropyl silica gel) eluted with acetate buffers provides somewhat poorer separation^,^' but is more stable. Strong-base anion-exchange resins (polystyrene-based), which are relatively stable and provide high-resolution separations,159 have not been extensively applied to high-performance 1.c. methods, but show considerable promise for future developments. Reversed-phase (CIE)silica gels are excellent stationary-phases for oligoglycuronan separations when a hydrophobic, ion-pairing reagent is used to suppress ionization of the carboxyl groups. Heyraud and Rocha@ separated pectic oligosaccharides having d.p. 1-1 1 by using the ion-pairing reagent tributyldodecylammonium chloride. Voragen and coworkers67used tetrabutylammonium bromide to separate oligoglycosiduronic acids of d.p. 1-7 in <15 min. Although the CIS columns are very stable under these conditions, it has been noted that the bromide salt of this reagent has a deleterious effect on chromatographic (154) P. van Eikeren and H. McLaughlin, Anal. Eiochem., 77 (1977) 5.13-522. (155) S. J. Mellis and J. U. Baenziger, Anal. Eiochem., 114 (1981) 276-280. (156) J.-F.Thibault, J. Chromatogr., 194 (1980) 315-322. (157) M. A. F. Davis, M. J. Gidley, E. R. Moms, D. A. Powell, and D. A. Rees, Int. J. Biol. Macromol., 2 (1980) 330-332. (158) E. A. Nothnagel, M. McNeil, P.Albersheim, and A. Dell, Plant Physiol., 71 (1983) 916-926. (159) P. VrBtnq, 0. Mikeg, P. Strop, J. Coupek, L. RexovB-BenkovB, and D. ChadimovB, J . Chromatogr., 257 (1983) 23-35. (160) T. Romeo and J. F. Preston 111, Carbohydr. Res., 153 (1986) 181-193.
H.P.L.C. OF CARBOHYDRATES
41
hardware: use of the hydroxide is therefore recommended.I6lOther simple, acidic oligosaccharides derived from alginateImand K-carrageenarP have also been separated by ion-pairing, reversed-phase chromatography. Few reports exist on the 1.c. separation of such cationic carbohydrate oligomers as those resulting from partial hydrolysis of chitosan. The neutral, N-acetylated analogs from chitin hydrolyses are, however, well separated on columns of aminopropyl silica ge1154J55 and reversed-phase silica gel.5g e. Complex (and Cyclic), Neutral 0ligosaccharides.-This Section covers those non-ionic oligosaccharides (2d.p.3) from various sources that are heterogeneous with respect to linkage or composition, or both. For a general overview of the use of various I.c. stationary phases in oligosaccharide separations, Section III,l,c should be referred to. Such plant-derived oligosaccharides as those produced from the hydrolysis of heteropolysaccharides and various cell-wall carbohydrate polymers often contain several different sugars and linkages. For the separation of the simplest members of this class, namely, the trisaccharides, stationary phases of amine-modified silica ge1399162 possess greater selectivity than cation-exchange resins, and are therefore recommended. In this system, retention of various trisaccharides is governed by sugar composition and linkage, and some of the precise effects of these parameters on retention have been determined.88J62For example, isomaltotriose and panose, which differ by only one linkage substitution, are readily separated on aminopropyl silica. Reversed-phase stationary phases (c18type) are useful for complex, plant oligosaccharides that are larger than tri- or tetra-saccharides. Retention on these columns is especially sensitive to saccharide linkage, and linear oligosaccharides are often well separated from those that are isomeric, but branched. Chromatography on reversed-phase packings was used to study the branching patterns of dextrans,'63and to compare the structure of a synthetic uersus a mycelial wall-derived hexa-O-P-D-glucopyranosyl-D-glucitol.'bl The complete separation of eight isomeric hexa-0-P-D-glucopyranosyl-D-ghcitols required a preliminary fractionation on a polar, aminocyanopropyl silica-gel column, followed by a second separation on Cl8-bonded silica (161) A. G. J. Voragen, personal communication. (162) 8. L. Nikolov, M. M. Meagher, and P. J. Reilly, J . Chromatogr., 321 (1985) 393-399. (163) C. Taylor, N. W. H. Cheetham, and G . J. Walker, Curbohydr. Res., 137 (1985) 1-12. (164) J. K. Sharp, P. Albersheim, P. Ossowski, b;. Pilotti, P. Garegg, and B. Lindberg, J . Biol. Chem., 259 (1984) 11,341-11,345.
42
KEVIN B. HICKS
gel.16sCombined approaches of this type are often necessary for the complete separation of such complex mixtures. An alternative approach for the separation of plant and microbial oligosaccharides, pioneered by Albersheim and c o w o r k e r ~ , is ~~ toJprepare ~ the peralkylated derivatives of the oligosaccharides, which are subsequently fractionated on c18 columns. Although extra time is required for the derivatization step, the resulting separated products may be directly analyzed by mass spectrometry, which can lead to determination of complete oligosaccharide sequence. Numerous glycoprotein-derived oligosaccharides have been separated by 1.c. techniques, and these methods may be divided into two general categories: those employing amine-modified silica gel and those with c18 (reversed-phase) stationary phases. With these columns, various “highmannose,” “complex,” “hybrid,” and miscellaneous oligosaccharides from o v a l b ~ m i n , ~IgD,168,169 ~ ~ J ~ ~ IgM,167a-acid g l y c o p r ~ t e i n , ~mu~~J~~ cins,171-174 ovarian-cyst glycoproteins,174 ceruloplasmin,~~5 p-D-glucosid u r ~ n a s e , ’fibr~nectin,”~ ~~ slime-mold gly~oprotein,’~~ tumor cells,178
(165) J. K. Sharp, B. Valent, and P. Albersheim, J. Biol. Chem., 259 (1984) 11,31211,320. (166) B. S. Valent, A. G. Darvill, M. McNeil, B. K. Robertsen, and P. Albersheim, Curbohydr. Res., 79 (1980) 165-192. (167) P. I. Clark, S. Narasimhan, J. M. Williams, and J. R. Clamp, Carbohydr. Res., 118 (1983) 147-155. (168) S. J. Mellis and J. U. Baenziger, J. Biol. Chem., 258 (1983) 11,546-11,556. (169) S. J. Mellis and J. U. Baenziger, J. Biol. Chem., 258 (1983) 11,557-11,563. (170) M. L. E. Bergh, P. L. Koppen, D. H. van den Eijnden, 1. Arnarp, and J. Lonngren, Carbohydr. Res., 117 (1983) 275-278. (171) A. Boersma, G. Lamblin, P. Degand, and P. Roussel, Carbohydr. Res., 94 (1981) c7-c9. (172) H. Van Halbeek, L. Dorland, J. F. G. Vliegenthart, W. E. Hull, G. Lamblin, M. Lhermitte, A. Boersma, and P. Roussel, Eur. J . Biochem., 127 (1982) 7-20. (173) G. Lamblin, A. Boersma, M. Lhermitte, P.Roussel, J. H. G. M. Mutsaers, H. Van Halbeek, and J. F. G. Vliegenthart, Eur. J . Biochem., 143 (1984) 227-236. (174) V. K . Dua, V. E. Dube, and C. A. Bush, Biochim. Biophys. Acta, 802 (1984) 29-40. (175) D. R. Howard, M. Natowicz, and J. U. Baenziger, J . Biol. Chem., 257 (1982) 10,86110,868. (176) P. W. Ledger, S. K. Nishimoto, S. Hayashi, and M. L. Tanzer, J. Biol. Chem. 258 (1983) 547-554. (177) C. A. Gabel, C. E. Costello, V. N. Reinhold, L. Kurz, and S . Kornfeld, J. Biol. Chem., 259 (1984) 13,762-13,769. (178) S . R. Hull, R. A. Laine, T. Kaizu, I. Rodriguez, and K. L. Carraway, J. Biol. Chem., 259 (1984) 4866-4877.
H.P.L.C. OF CARBOHYDRATES
43
ovomucoid ,179,180 blood-group glycoproteins,I E 1 and histocompatability antigensI8*have been separated, and often isolated (see also Section III,3 .). Separations of these oligosaccharides on amine-modified silica gel with acetonitrile-water mobile phases were pioneered in 1981 by three separate groups,155,171,183 and most of the reports of the separation of glycoprotein-derived oligosaccharides since published have been extensions of those methods. Oligosaccharides or oligosaccharide-alditols(produced by chemical cleavage of the oligosaccharide from glycoprotein under reductive conditions) having -3 to 15 carbohydrate residues may usually be separated in <60 min on these columns by normal phasethat govern the repartitioning mechanisms. Several factors88.’55~170~171,183 tention of these oligosaccharides are molecular weight, carbohydrate composition, anomeric linkage, and branching patterns. An important advantage of this system over the reversed-phase or cation-exchangetype is the ability to modify and optimize the mobile-phase composition. A preliminary molecular weight (d.p.) fractionation of glycans, using a weak mobile phase (relatively high percentage of water) can be followed by a second fractionation of each collected peak on the same column, using a strong mobile phase (relatively high percentage of acetonitrile) to resolve isomers in each size-class. As previously stated, aminopropyl-bonded silica-gel columns are somewhat unstable and should always be used with the precautions outlined in Sections II,l,b and II,2,a. TurcoIS3found that silica-gel columns modified in situ with 1,6diaminobutaneare more durable than covalently bonded amino columns for the separation of “highmannose” and related oligosaccharides. There have been relatively few examples of the use of reversed-phase for separations of complex, glycoprotein-derivedglycans. Retention in this system is based upon hydrophobic interactions between carbohydrate and stationary phase, and appears to be sensitive to oligosaccharide linkage, composition, and branching, rather than to molecular weight or number of sugar residues. Because most oligosaccharides are relatively polar, little interaction takes place between them and the sta(179) J. P. Parente, G. Strecker, Y. Leroy, J. Montreuil, and B. Fournet, J . Chrornafogr., 249 (1982) 199-204. (180) J. P. Parente, J.-M. Wieruszeski, G . Strecker, J. Montreuil, B. Fournet, H. van Halbeek, L. Dorland, and J. F. G. Vliegenthart, J . Biol. Chem., 257 (1982) 13,17313,176. (181) A. M. Wu, E. A. Kabat, B. Nilsson, D . A. Zopf, F. G . Gruezo, and J. Liao, J . Biol. Chem., 259 (1984) 7178-7186. (182) S.J. Swiedler, G. W. Hart, A. L. Tarentino, T. H. Plummer, Jr., and J. H. Freed, J . Biol. Chem., 258 (1983) 11,515-11,523. (183) S. J. Turco, Anal. Biochem., 118 (1981) 278-283.
44
KEVIN B. HICKS
tionary phase, even when the weakest mobile-phase (water) is used. Reversed-phase columns are, therefore, most useful for separating those complex oligosaccharides that contain higher proportions of (nonpolar) acetyl and methyl groups, or deoxy sugar moieties, and those that have been derivatized with nonpolar groups to enhance detectability. Other complex, neutral oligosaccharides that have been fractionated by 1.c. techniques include those found in human milk21J84J85 and in urine from patients with lysosomal-storage disorders.6J86 An unusual class of oligosaccharides that contain a homogenous sugar composition are the cyclic oligosaccharides, such as the cyclomalto-oligosaccharides186a (the “cyclodextrins”), having a-(1 + 4)-linked D-glucosyl residues, and the cyclosophoroses, containing p-( 1 + 2)-linked D - ~ ~ U C O syl residues. The most common cyclodextrins, those with 6, 7, or 8 Dglucosyl units, have been separated on amine-modified silica-gel columns calcium-form cation-exchange columns,23J88 and reversedphase silica-gel columns.189The cyclosophoroses, having a d.p. up to 40, have been separated18s191 on amine-modified and reversed-phase silicagel columns. All three stationary phases mentioned here separate the cyclic oligosaccharides by different mechanisms, and they provide a unique retention order. Separations that are sensitive to molecular weight are produced on amine-modified silica-gel phases, with large oligosacchandes being eluted last. With reversed phases, separations based on hydrophobicity occur, with the most hydrophobic, least w a t e r - ~ o l u b l e ~ ~ ~ compounds being eluted last. On cation-exchange resin columns, separations are based on a combination of size-exclusion and hydrophobic interactions. Koizumi and used the unique retention-mechanisms of these phases to obtain structural information about newly discovered cyclic oligosaccharides. For instance, the molecular weight
(184) H. Egge, A. Dell, and H. Von Nicolai, Arch. Biochem. Biophys., 224 (1983) 235-253. (185) V. K. Dua, K. Goso, V. E. Dube, and C. A. Bush, J . Chromatogr., 328 (1985) 259269. (186) N. M. K. Ng Ying Kin and L. S. Wolfe, Anal. Biochem., 102 (1980) 213-219. (186a) See R. J. Clarke, J. H. Coates, and S. F. Lincoln, This Volume, pp. 205-249. (187) H. Bender, Carbohydr. Res., 124 (1983) 225-233. (188) H. Hokse, J . Chromatogr., 189 (1980) 98-100. (189) K. Koizumi, Y. Okada, S. Horiyama, T. Utamura, M. Hisamatsu, and A. Amemura, J . Chromarogr., 265 (1983) 89-96. (190) M. Hisamatsu, A. Amemura, K. Koizumi, T. Utamura, and Y. Okada, Curbohydr. Res., 121 (1983) 31-40. (191) K. Koizumi, Y. Okada, T. Utamura, M. Hisamatsu, and A. Amemura, J . Chromatogr., 299 (1984) 215-224. (191a) K. Koizumi, T. Utamura, M. Sato, and Y. Yagi, Carbohydr. Res., 153 (1986) 55-67.
H.P.L.C. OF CARBOHYDRATES
45
(or d.p.), sugar composition, and type of linkages present in a cyclic oligosaccharide may be determined by comparing the 1.c. analysis of its partial hydrolyzate to that of known, standard oligosaccharides. In addition, the same methods can provide information about branched structures. If branching is present in the cyclic oligosaccharide, 1.c. analysis (amine-modified silica-gel phase) of the partial hydrolyzate will reveal multiple peaks of materials having the same approximate d.p. value. of these peaks can verify the presence of simple D-glucosyl or maltosyl side-chains. To identify more-complex branching patterns, additional information from methylation analysis or n.m.r. spectroscopy is required. f. Complex, IoNc Oligosaccharides and G1ycopeptides.-This Section will be divided into four separate areas: sialylated (or phosphorylated) oligosaccharides,glycopeptides, and acidic glycosaminoglycan and acidic plant cell-wall oligosaccharides. The sialylated oligosaccharides of glycoproteins are of considerable biological interest, and this is reflected in the development of many 1.c. methods for their fractionation. Free sialylated oligosaccharides from such biological sources as human milk,192the urine from patients with lysosomal-storage disease,186J93 or in uitro sialyltransferase assays, are often e n c ~ u n t e r e d . ' ~In~ Jother ~ ~ instances, the N- or 0-linked oligosaccharides of glycoproteins are, before 1.c. analysis, liberated by hydrazinolysis (and subsequent reduction),193or alkaline borohydride treatment,'" respectively. In each case, the product is an acidic oligosaccharide-alditol, and it is this species that is most commonly chromatographed. Three types of 1.c. stationary phase have been used for sialylated oligosaccharide separation: weak anion-exchange silica gel (primary amine groups), strong anion-exchange silica-gel (quaternary amine groups), and strong anion-exchange resin beads. Such strong anion-exchangers as MicroPak-AX-10are eluted with phosphate buffer gradients (pH 4) and they separate sialylated oligosaccharides according to the number of sialic acid (192) J.-M. Wieruszeski, A. Chekkor, S . Bouquelet, J . Montreuil, J. Peter-Katalinic, and H. Egge, Carbohydr. Res., 137 (1985) 127-138. (193) S. J. Mellis and J. U . Baenziger, Anal. Biochem., 134 (1983) 442-449. (194) M. L. E. Bergh, P. L. Koppen, and D. H. van den Eijnden, Biochem. J . , 201 (1982) 41 1-415. (195) M. L. E. Bergh, G. J. M. Hooghwinkel, and D. H. van den Eijnden, J . Biol. Chem., 258 (1983) 7430-7436. (196) D. K. Podolsky, J . Biol. Chem., 260 (1985) 8262-8271.
46
KEVIN B. HICKS
groups bound. 197~198~198a Such class-separated oligosaccharides are, however, generally heterogeneous with respect to carbohydrate composition and further chromotography on primary amine-bonded silica is necessary for obtaining pure, homogeneous f r a ~ t i o n s . ' In ~ ~the J ~latter ~ ~ case, the primary amine-bonded silica gel is usually eluted with acetonitrile mixed with one of the following buffers: ammonium hydrogencarbonate,lw triethylammonium a ~ e t a t e , ' ~sodium ~ , ' ~ ~acetate,'& potassium dihydrogenphosphate containing 0.01% of 1,Cdiaminob~tane,'~~ or potassium phosphate. Under these conditions (pH 5.2-7.0), the acidic oligosaccharides are separated on the basis of carbohydrate content, linkage, and composition, allowing the separation of linkage isomers as well as of oligosaccharides of differing mass. A slight modificationlWaof these procedures has been used to differentiate between complex sialylated and sulfated oligosaccharides. Fractionation of acidic oligosaccharides may also be accomplished on polystyrene-based anion-exchangerseluted with sodium chloride gradients.20"*20' In this case, fractionation is similar to that on primary amine-bonded silica gel. In all of the foregoing instances, because of the extreme heterogeneity of many glycoprotein-derivedoligosaccharides, a combination of two or three of the methods described, in conjunction with classical gel-filtration chromatography, is usually necessary for complete purification. Such coupled procedures have allowed the fractionation of sialylated oligosaccharidesfrom b r o n ~ h i a 1 , submax~~~J~ illary,20',202 and colonic mucins,'%tumor-cell g l y c o p r ~ t e i n sf,e~t~~~i n , ' ~ ~ . ' ~ ~ o v ~ m u c o i d ,o~ ~ ~o s o ~ u c ~ov a~l b~u m, i~n , '~~~e~~r u~l o~p l ~a s ~ i m~, ' ~fi-~ ~ ' ~ ~ b r ~ n e c t i n , IgD ' ~ ~ ( h ~ m a n ) , ' glycocalicin,200 ~~J~~ and thyroglobulin.201In addition, phosphorylated oligosaccharides have been similarly fractionated.193.197 One of the most difficult aspects of the study of glycoprotein structure is the remarkable heterogeneity displayed in their glycan chains. The 1.c. (197) J. U. Baenziger and M. Natowicz, Anal. Biochem., 112 (1981)357-361. (198) J. P. Parente, Y. Leroy, J. Montreuil, and B. Fournet, J . Chromarogr., 288 (1984) 147- 155. (198a) P. Cardon, J. Paz Parente, Y. Leroy, J. Montreuil, and B. Fournet, J . Chromatogr., 356 (1986)135-146. (199) G.Lamblin, A. Klein, A. Boersma, Nasio-ud-Din, and P. Roussel, Carbohydr. Res., 118 (1983)cI-c~. (199a) E. D.Green and J. U. Baenziger, Anal. Eiochem., 158 (1986)42-49. (200) T.Tsuji, S. Tsunehisa, Y. Watanabe, K. Yamamoto, H. Tohyama, and T. Osawa, J . Eiol. Chem., 258 (1983)6335-6339. (201) T. Tsuji, K. Yamamoto, Y. Konami, T. Irimura, and T. Osawa, Carbohydr. Res., 109 (1982)259-269. (202) M. L. E. Bergh, P. Koppen, and D. H.van den Eijnden, Carbohydr. Res., 94 (1981) 225-229. (203) A. P. Sherblom and C. E. Dahlin, J . Eiol. Chem., 260 (1985) 1484-1492.
H.P.L.C. OF CARBOHYDRATES
47
separation and isolation of hydrazine- or alkali-generated oligosaccharidealditols from glycoproteins, as just described, is a useful technique, but it does not answer the question of whether all the oligosaccharides come from one, or from several, glycosylation site(s). Hence, as described next, several methods have been developed for the isolation of intact glycopeptides, released from the parent molecule by gentle, protease treatment. By this procedure, the entire glycosylation site(s), is separated or isolated (see Section 111,3), allowing subsequent structural analysis of both the peptide and the carbohydrate portion. Glycopeptides are usually separated on either reversed-phase or aminopropyl-bonded silica gel. The former stationary phase is usually eluted with an organic modifier, such as acetonitrile, in conjunction with an " , ~ ~or~pH 2aqueous buffer at pH 7-7.5 ( p h ~ s p h a t e ~or" b~ ~~ r~a~t e ~ type) 3 (phosphate208*209, phosphate-chlorate,'6J82or trifluoroacetate type210). Under these conditions, glycopeptides are separated largely on the basis of peptide variation. This is supported by the fact that two, nearly identical, 13-amino acid peptides having greatly differing glycan chains (monosaccharide uersus pentasaccharide chain) are only marginally separated by this whereas large glycopeptides that differ in only a single amino acid residue are separated with relative ease.*IoThese characteristics make these systems ideal for the separation of glycosylation sites, that is, homogeneous-peptide regions that contain one homogeneous or heterogeneous glycan chain. Once the homogeneous-peptide sequences (glycosylation sites) have been separated, or isolated, the heterogeneity of the glycan chains may be studied by classical, size-exclusion chromatography after peptide hydrolysis,2o8or by 1.c. analysis. With the latter method, several procedures may be used, including reversed-phase chromatography under special conditions. Because of the low sensitivity of reversed-phase columns to carbohydrate variation, it is necessary to use combinations of weak solvents and elution times207of up to 8 h. In special cases where all of the peptide except for one amino acid may be enzymically digested away, as in ovalbumin,62the carbohydrate heterogeneity may be moderately resolved by using reversed-phase columns eluted with phosphate buffer alone.
(204) T. Takahashi, P. G . Schmidt, and J. Tang, J . B i d . Chem., 258 (1983) 2819-2830. (205) T. Takahashi, P. G. Schmidt, and J. Tang, J . B i d . Chem., 259 (1984) 6059-6062. (206) H. Iwase, S.-C. Li, and Y.-T. Li, J . Chromatogr., 267 (1983) 238-241. (207) H. Iwase, Y. Kato, and K. Hotta, J . Chromatogr., 320 (1985) 426-429. (208) P. Hsieh, M. R. Rosner, and P. W. Robbins, J . B i d . Chem., 258 (1983) 2548-2554. (209) P. Hsieh, M. R. Rosner, and P. W. Robbins, J . Biol. Chem., 258 (1983) 2555-2561. (210) D. Tetaert, N. Takahashi, and F. W. Putnam, Anal. Biochem., 123 (1982) 430-437.
48
KEVIN B. HICKS
As in most of the other complex separations described herein, a combined separation approach is usually needed in order to ensure homogeneity in glycopeptide fractions. Hence, after separation of glycosylation-site glycopeptides by one of the reversed-phase systems previously described, the oligosaccharides may be released by chemical or enzymic treatment, and then separated by one of the methods presented in Section 111,l ,e. Alternatively, these semipurified glycoprotein fractions may be resolved on primary amine-type silica-gel columns, which separate glycopeptides according to variations in carbohydrate, rather than peptide, s t r u ~ t u r e . The ~ ~ ~methods J ~ ~ just described have been used for the separation of glycopeptides from ovalbumin,62~193~2M~207 ribonuclease B,193 cathepsin-D2@' and -B ,205 virus glycoproteins,208tumor-cell antigens,ls2ovomucoid,211orosomucoid,2Mceruloplasmin,210immunoglobulins,210and urine of patients with pathological states.Is6 Sialylated glycopeptides are separated by these systems, with minor modifications,186!206 as are methylated2I1and N-acylated glycopeptides.193 Oligosaccharides produced by the enzymic or chemical hydrolysis of glycosaminoglycuronans have been separated by 1.c. methods based on s i z e - e x ~ l u s i o n , ~ weak ~ ~ ~ ~ani~n-exchange,~'~ ~~ strong anion-ex~ h a n g e , ~and ~ ~normal v ~ ~phase-partition ~.~~~ chr~matography.~'~ Although both sulfated and non-sulfated oligosaccharides from these biopolymers have been chromatographed, most methods have been developed for the latter type, as, for example, the hyaluronic acid-derived oligosac~ h a r i d e s . ~ *Generally, ~ . ~ ~ ~ * ~these ~ ' methods have been used to separate the odd- and even-numbered, oligosaccharides (composed of 2-acetamido-2-deoxy-~-glucopyranose and D-glucopyranuronic acid residues) produced by the action of various endo- and exo-glycanohydrolases on purified hyaluronic acid. Size-exclusion212methods allow the simultaneous quantification of mono-, di-, tetra-, to hexa-saccharides, and unreacted biopolymer. Odd- and even-numbered hyaluronate oligosaccharides (up to decasaccharide) may be separated with much higher resolution, however, on high-performance anion-exchange columns,213,214 which separate oligosaccharides by a combination of molecular weight and charge effects. In addition, retention times of hyaluronate oligosac(211) J. Conchie, A. J. Hay, and J . A. Lomax, Carbohydr. Res., I12 (1983) 281-295. (212) P. J. Knudsen, P. B. Eriksen, M. Fenger, and K. Florentz, J . Chromatogr., 187 (1980) 373-379. (213) P. Nebinger, J . Chromatogr., 320 (1985) 351-359. (214) P. Nebinger, M. Koel, A. Franz, and E. Werries, J . Chromatogr., 265 (1983) 19-25. (215) S. R. Delaney, H. E. Conrad, and J. H. Glaser, Anal. Biochem., 108 (1980) 25-34. (216) M. J. Bienkowski and H. E. Conrad, J . Biol. Chem., 260 (1985) 356-365. (217) I. Takazono and Y. Tanaka, J . Chromatogr., 288 (1984) 167-176.
H.P.L.C. OF CARBOHYDRATES
49
charides in these chromatographic systems may be predicted by additive contributions of the constitutive groups.2i8A disadvantage of anion-exchange chromatographic separations of hyaluronate oligosaccharides (particularly on Dowex-1 X8 resin) is the extremely low recovery (<60%) of sample from the column effluent. Chromatographic separations of sulfated glycosaminoglycuronan oligosaccharides are more difficult than those of the hyaluronate type, because of the complex variety of sulfation patterns that may exist. In the separation of oligosaccharides produced from nitrous acid-depolymerized heparin216 on strong anion-exchange columns, for example, more than 16 different tetrasaccharides (differing in uronic acid composition and sulfation patterns) were resolved and isolated. The method provides high-resolution separations, but requires long run-times (>2 hr) and pre-chromatographic separation of oligosaccharidesaccording to size (by gel-filtration). Nonetheless, it is a powerful tool for sequence analyses of heparin structure, and has also been applied to the separation of enzyme-generated heparin oligosac~harides.~~~~~~~ Likewise, oligosaccharidesfrom chondroitin sulfate show considerable heterogeneity in sulfation patterns, even in samples of “pure” chondroitin 4- or 6-sulfate. The variety of sulfation patterns in tetra- to deca-saccharides has been demonstrated by the separation of multiple “hybrid” oligosaccharides (of identical mass) by chromatography on strong anion-exchange silica-gel eluted with phosphate buffer.21s Complex, acidic oligosaccharides from plant cell-walls have occasionally been fractionated by I.c. methods. Because of the great structural heterogeneity of these oligosaccharides, 1.c. is generally preceded by one of several additional fractionation steps, namely, gel filtration, ion-exchange, or affinity chromatography. Although there is one report of the fractionation of pectic-type oligosaccharides on strong anion-exchange silica-gel columns,Is8the majority of methods reported describe the fractionation of these molecules as their carboxyl-reduced, peralkylated derivatives on reversed-phase silica gel. These methods have been used for fractionating oligosaccharides from the plant cell-wall polysaccharides’rhamnogalacturonanI (Ref. 18) and I1 (Ref. 17), and have also been useful for the fractionation of various acidic fragments of bacterial, extracellular polysa~charides.~~ i7~189166
(218) E. Shimada and G . Matsumara, J . Chrornatogr., 328 (1985) 73-80. (219) K. G . Rice, Y. S. Kim, A. C. Grant, Z. M. Merchant, and R. J. Linhardt, Anal. Biochern., 150 (1985) 325-331. (220) Z. M. Merchant, Y . S. Kim, K. G . Rice, and R. J. Linhardt, Biochem. J., 229 (1985) 369-311.
50
KEVIN B. HICKS
2. Additional, Selected Applications
a. Analysis of Carbohydrates in Foods.-The determination of simple and complex carbohydrates in foods by 1.c. has been aptly discussed in reviews,29,30.221.222 and will be covered here in only a general manner. In addition, reviews of this topic continue to appear on a biannual basis.223-225 The carbohydrates in foods may be divided into two c a t e g ~ r i e s , ~ ~ the (mostly) soluble, metabolically utilizable sugars and starches, and the insoluble, complex “dietary-fiber” carbohydrate that is resistant to human alimentary enzymes. It is the former class of carbohydrates that have received the most attention, and many 1.c. methods for their determination exist (see Table 11). Most of these applications require 1.c. columns packed with either cation-exchange resins or amine-modified silica gel, which provide two different types of selectivities, often leading to complementary information. Because no single type of column will provide the selectivity and resolution required for every food analysis, some care must be taken in choosing the correct column for each application. In general, when an application can be served with a cation-exchange resin column, it should be the column of choice, because of the durability, ease of regeneration, rapid analyses, and complete recovery of carbohydrate from the column. In addition, these columns require pure water or dilute mineral acid as the mobile phase, which is economical and allows highsensitivity detection on refractive index monitors. With proper, sample pre-treatment and the use of pre-columns (see Sections II,l,b and 2,b) cation exchange columns in the H+, Ag+, Ca2+,or Pb2+form can be operated for several years without noticeable loss in efficiency. Saccharides are separated on these systems by a combination of size-exclusion and ligand-exchange mechanisms (see Section II,2,b) and columns with different metal-ion ligands give different selectivities that can be used to optimize specific separations. As summarized p r e v i o ~ s l yAg+ ,~~ columns are optimized for the separation of oligosaccharides, such as W. A. Davis and C. G. Hartford, in G . Charalambous (Ed.), Liquid Chromatographic Analysis of Food and Beverages, Vol. 2, Academic Press, New York, 1979, pp. 353-362. R. Macrae, in G. Birch (Ed.), Analysis of Food Carbohydrates, Elsevier Appl. Sci., London, 1985, pp. 61-89. K. G . Sloman, A. K. Foltz, and J. A. Yeransian, Anal. Chem., 53 (1981) 2 4 7 ~ - 2 4 8 ~ , 265~-266~. A. K. Foltz, J. A. Yeransian, and K. G. Sloman, Anal. Chem., 55 (1983) 1 6 9 ~ - 1 7 1 ~ , 1873- 1 8 8 ~ . J. A. Yeransian, K. G . Sloman, and A. K. Foltz, Anal. Chem., 57 (1985) 2 8 6 ~ - 2 8 8 ~ , 306~-307~.
H.P.L.C. OF CARBOHYDRATES
51
TABLEI1 Selected L. C. Methods for Soluble Food-Carbohydrates Type of Food
Carbohydrates Analyzed
Beer, simple sugars and oligosaccharides Cereal ( b r e a k f a ~ t ) ~ ~ ~fructose, .~~ glucose, sucrose, maltose Chewing gumzz9 sugar alcohols Confe~tionery~3~.23f simple sugars Corn sy~p50.i45.232 simple sugars, psicose, maltulose, oligosaccharides Fruit j~ice227.233.234 sucrose, glucose, fructose Honey,227invert glucose, fructose s~gar,~3~.~3~ maple syrup,227 m0lasses~~6 Ice cream,227.237 lactose, lactulose, galactose, glucose lactose hydrolyzates ,57.238 &k,38.239 whey,85.238 lactulose syrups240~24i
those in corn syrups. Columns in the H+form are especially useful for separation of simple sugars, sugar acids, and sugar alcohols that are present in fermented dairy products. The so-called “heavy-metal” or Pb2+forms of cation-exchange resins are useful for the separation of those monosaccharides found in dietary-fiber carbohydrates. The Ca2+-form columns are useful for the class separation of mono-, di-, tri-, and tetrasaccharides and sugar alcohols. G. K. Buckee and D. E. Long, J. A m . SOC.Brew. Chem., 40 (1982) 137-140. J. L. Iverson and M. P. Bueno, J. Assoc. Ofl.Anal. Chem., 64 (1981) 139-143. L. C. Zygmunt, J. Assoc. Ofl.Anal. Chem., 65 (1982) 256-264. E. C. Samarco and E. S. Parente, J. Assoc. Off.Anal. Chem., 65 (1982) 76-78. S. I. M. Johncock and P. J. WagstaEe, Analyst, 105 (1980) 581-588. D. J. Timbie and P. G. Keeney, J. Food Sci., 42 (1977) 1598-1599. H. D. Scobell, K. M. Brobst, and E. M. Steele, Cereal Chem., 54 (1977) 905-917. J. K. Palmer and W. B. Brandes, J. Agric. Food Chem., 22 (1974) 709-712. P. E. Shaw and C. W. Wilson, 111, J . Sci. Food Agric., 34 (1983) 109-112. M. Wnukowski, Int. Sugar J . , 86 (1984) 170-174. C. E. Damon and B. C. Pettitt, Jr., J . Assoc. Off.Anal. Chem., 63 (1980) 476-480. J. M. Beebe and R. K. Gilpin, Anal. Chim. Acta, 146 (1983) 255-259. I. J. Jeon, S. J. Galitzer, and K. J. Hennessy, J. Dairy Sci., 67 (1984) 884-887. J. F. Pirisino, J. Food Sci., 48 (1983) 742-744, 754. L. A. T. Verhaar, M. J. M. Van Der Aalst, J. A. W. M. Beenackers, and B. F. M. Kuster, J. Chromatogr., 170 (1979) 363-370. (241) F. W. Panish, K. Hicks, and L. Doner, J . Dairy Sci., 63 (1980) 1809-1814.
(226) (227) (228) (229) (230) (231) (232) (233) (234) (235) (236) (237) (238) (239) (240)
52
KEVIN B. HICKS
On Ca2+-formcolumns, some separation of monosaccharides is possible and, for the separation of galactose and glucose in dairy products, this is the column of choice. The separation of several disaccharides, such as sucrose plus maltose plus lactose, in sweetened dairy products cannot be accomplished on single-resin columns, however, and separation on amine-modified silica gel or on dual-resin is recommended. These columns are capable of separating the five major food sugars, namely, D-glucose, D-fructose, sucrose, maltose, and lactose, but are subject to rapid degradation if proper precautions are not used (see Section 11,2,a). Amine modifiers in the mobile phase have been used in conjunction with silica-gel columns3’to provide more-stable columns. In addition, the use of diol-type silica-gel columns for the separation of food sugars has been reported.38These columns provide separations similar to those afforded by aminopropyl silica gel, but they are much more stable. The use of these diol columns, whenever practical, is therefore recommended. b. Analysis of Carbohydrates in Biomass-Conversion Processes.-In biomass “conversion,” plant-derived polysaccharides are partially or totally converted into fermentable sugars by the aid of chemical or enzymic methods. L.c. methods are useful in monitoring such reactions, because they can provide several different types of information, namely, the extent of polysaccharide hydrolysis, the distribution of the various monosaccharides produced, and the amounts of sugar fermentation- or degradation-products, or both, that are present. For monitoring the extent of polysaccharide hydrolysis, 1.c. methods that separate and analyze the non-fermentable oligosaccharides (d.p. 330) derived from cellulose, hemicellulose, and pectins are useful, and have already been described (see Section III,l,c). For determination of the monosaccharide composition of completely hydrolyzed, plant polysaccharides, 1.c. is especially useful and has been applied to the compositional analysis of hydrolyzed plant fiber,242wood pulps,83+243 plant celland cotton fibers.z45In these representative examples, the major sugars of interest, namely, glucose, xylose, galactose, arabinose, and mannose, have traditionally been difficult to resolve by 1.c. The separa(241a) (242) (243) (244) (245)
J. A. M. van Riel and C. Olieman, J . Chrornarogr., 362 (1986) 235-242. J. L. Slavin and J. A. Marlett, J . Agric. Food Chern., 31 (1983) 467-471. F. E. Wentz, A. D. Marcy, and M. J. Gray, J . Chrornatogr. Sci.,20 (1982) 349-352. W. Blaschek, J . Chrornatogr., 256 (1983) 157-163. W. Schwald, R. Conch, G. Bonn,and 0. Bobleter, Chrornatographia, 20 (1985) 3540.
H.P.L.C. OF CARBOHYDRATES
53
tion of these sugars on high-performance aminopropyl silica-gel columns is dependent on the source and condition of the column. Although Binder33was able to separate these sugars efficiently, Yang and coworke r had~ to couple ~ ~ two ~ such columns together in order to achieve satisfactory resolution. Although separation was complete, the peaks were broad, and the detector sensitivity was poor. Moreover, under these conditions, the pentoses and certain hexoses in those samples are knownI0to form covalent linkages with the amino groups of the stationary phase, which leads to improper quantitation and column failure. Separation of these sugars can be accomplished more efficiently on special cation-exchange resins (Pbz+ The accuracy of these methods has been shown to be the equal of that of official, paper-chromatographic methods, and they are much more ~ o n v e n i e n tWhen . ~ ~ samples contain rhamnose, in addition to the other five sugars already listed, total separation is not possible on Pbz+-form columns,z4zand coupled aminopropyl silica-gel columns246or sequential separationsz4 on aminopropyl silica gel, and Pbz+-formcation-exchange columns are required. Biomass samples that have been hydrolyzed by h y d r ~ t h e r m a l or ~~,~~~ acidI9treatment, and the products then fermentedI3J2contain a complex array of alcohols, acids, aldehydes, ketones, and various sugar degradation-products. For analysis of these mixtures, cation-exchange resin columns are often more useful than reversed-phase or amine-modified silica-gel types.54Various ionic forms of cation- exchange resin columns are used to determine sugar degradation-products, such as 2-furaldehyde and 5-(hydroxymethyl)-2-furaldehyde,~3~~9~zz~54~z45~z46 methylglyo~al,~~~ socalled a n h y d r o g l u c ~ s eand , ~ ~acetic ~ and formic acids.19Using an H+-form column, Patrick and KrachtI9 determined 2-furaldehyde, 5-(hydroxymethyl)-2-furaldehyde, total monosaccharides, and acetic acid in wood hydrolyzates in <20 min. By using the more-selective, Pbz+-formof a similar resin, Schwald and coworkersz45were able to separate the individual sugars and the furfurals in a plant hydrolyzate, but, on this column, the acids could not be determined. Proper column selection, therefore, depends on the information that must be obtained in each specific analysis. Liquid-chromatographic techniques have been useful for studying the mode of action of various polysaccharide-degrading enzymes. Rapid and quantitative, 1.c. end-product analysis has been used to study the action
(246) M. T. Yang, L. P. Milligan, and G . W. Mathison, J . Chromatogr., 209 (1981) 316322. (247) G. Bonn and 0. Bobleter, Chromuzogruphia, 18 (1984) 445-448.
54
KEVIN B. HICKS
patterns of ~ e l l u l a s e s , 2 x~yJl~a n~a~~~e ~ s ,~ endo-glucanases, ~~' and poly(galacturonic acid) l y a ~ e (see s ~ ~Addendum). ~ In addition to the normally suggested, sample pre-treatment procedures used prior to 1.c. injection to avoid column contamination, CI8bonded silica-gel cartridgesI3are particularly useful for removal of lignin-derived degradation-products that occur in biomass-conversion samples. c. Compositional Analysis of Carbohydrate Polymers.-Determination of the monosaccharide composition of complex heteropolysaccharides, glycan chains, and glycoconjugates is of fundamental importance for studying the biological and physicochemical properties of these polymers. For this purpose, many excellent methods based upon gas-liquid chromatography (g.1.c.) currently exist. High-performance liquid-chromatographic methods should not be regarded as replacements for these methods, but as being complementary to them. The primary ruison d'2tre for 1.c. is to allow rapid, high-resolution separations of molecules without need for pre-derivatization. Accordingly, the most useful 1.c. methods take this into consideration. In the compositional analysis of complex carbohydrate, both g.1.c. and 1.c. methods have at least one thing in common; they must both be preceded by an enzymic or chemical hydrolysis step. In many cases, the limiting factor in the accuracy of compositional analysis is the method used to hydrolyze the specific polymer; this topic is outside the scope of this chapter, however, and only general guidelines will be given. The monosaccharide composition of plant polysaccharides, for instance, is still usually accomplished by g.1.c. analysis, although progress is being made in 1.c. separations. No method currently exists for the simultaneous determination of underivatized acidic and neutral sugars found in plant cell-wall polysaccharides. Sugar acids, such as the uronic acids produced by the hydrolysis of alginates, gums, and pectins, may be directly separated and quantitated by anion-ex~hange~~.~'.%*~~~ and o t h e P 1.c. methods. The simultaneous separation of all neutral sugars present in plant cell-wall polysaccharides is also a particularly difficult challenge, and early attempt^^^^.^^^ led to only moderate separations between the various deoxy and neutral sugars present. A subsequent method,244utilizing both aminopropyl silica-gel and cation-exchange columns, now per(248) S. P. Shoemaker and R. D. Brown, Jr., Biochim. Biophys. Acra, 523 (1978) 133-146. (249) A. R. White, A. G . Darvill, W. S. York, and P. Albersheim, J . Chromatogr., 298 (1984) 525-530. (250) G . Annison, N . W. H. Cheetham, and I. Couperwhite, J . Chrornarogr., 264 (1983) 137- 143. (251) E. C. Conrad and I. K. Palmer, Food Technol. (Chicago), 30 (1976) 84, 86, 88-92.
H.P.L.C. OF CARBOHYDRATES
55
mits the complete separation and quantitation of the major sugars (L-rhamnose, L-arabinose, D-xylose, D-mannose, D-galactose, and D-glucose) that exist in plant cell-wall hydrolyzates. Because of their biomedical importance, several methods have been developed to determine the composition of various glycosaminoglycans, such as heparin, heparin sulfate, the chondroitin sulfates, dermatan sulfate, and hyaluronic acid. These polymers are composed basically of repeating disaccharide units, usually containing a hexuronic acid and a 2amino-2-deoxyhexose, both of which may bear substituent groups (N- or C-sulfates and N-acetates). Fortunately, in most cases, these polymers can be uniformly degraded by specific chondroitinases, heparinases, heparanases, and hyaluronidases, or by specific chemical reagents, such as nitrous acid. Separation and identification of the reaction products (mostly disaccharides), by 1.c. reveals the composition of the original polymer. Such analyses have been useful for determining the ratios of isomeric chondroitin or dermatan s ~ l f a t e s ' ~ ~and J ~ hyaluronic ~.~~~ a ~ i d ~ in ' ~various , ~ ~ tissues ~ - ~ and ~ ~physiological fluids. Moreover, the content of "over-sulfated" residues, a phenomenon related to pathological condition^,'^^ may also be determined by the direct 1.c. separation of the mono-, di-, and tri-sulfated disaccharides from various glycos a m i n o g l y ~ a n s . ' ~ ~The J ~ ~position J ~ ~ of sulfate substitution on disaccharides may be determined by comparing 1.c. retention-times before and after treatment with specific chondroitinsulfatases.i29~131 Because of the heterogeneity found in the carbohydrate composition, and the relatively high resolving power of I.c., as many as 9 different chondroitin disaccharides have been separated and identified. 13' Disaccharides produced from nitrous or enzyme-'32v220!255 degraded heparin and heparan sulfate have been separated by liquid-chromatographicprocedures. Fluorescent derivatives of heparin disaccharides have also been prepared, to enhance their d e t e ~ t a b i l i t y(see ~ ~ ~Addendum). Through the combined approach of 1.c. and the sequential applications of specific glycosaminoglycan-lyases, it is possible to determine the levels of several different glycosaminoglycans in the same tissue-sample. Thus, Gurr and coworkers257demonstrated that hyaluronate, chondroitin sulfate G . J.-L. Lee and H. Tieckelmann, J . Chromatogr., 222 (1981) 23-31. A. Hjerpe, C. A. Antonopoulos, and B. Engfeldt, J . Chromatogr., 245 (1982) 365368. S. R. Delaney and H. E. Conrad, Biochem. J . , 209 (1983) 315-322. G . J.-L. Lee, D.-W. Liu, J. W. Pav, and H. Tieckelmann, J . Chromatogr., 212 (1981) 65-73. M. Kosakai and Z. Yosizawa, J . Biochem. (Tokyo),92 (1982) 295-303. E. Gum,G . Pallasch, S. Tunn, C. Tamm, and A. Delbriick, J . Clin. Chem. Clin. Biochem., 23 (1985) 77-87.
56
KEVIN B. HICKS
isomers, and dermatan sulfate in human cartilage could be determined by sequential application of chondroitinase AC and chondroitinase ABC to the sample, followed each time by 1.c. analysis of the enzyme products. Because of their importance to medicine, biochemistry, and biotechnology, glycoproteins are of considerable interest, and accurate compositional analysis of the carbohydrates in these polymers is frequently needed. As with the other macromolecules previously discussed, there is a need for better methods for ensuring complete and quantitative hydrolysis. Because of the diversity in saccharide composition (neutral, acidic, and amino sugars), it is difficult to find optimal hydrolysis conditions. Some progress has been made in this area, and such reagents as anhydrous hydrogen f l u ~ r i d and e ~ others ~ ~ ~ (see ~ ~ Refs. ~ 81 and 260) may be capable of completely hydrolyzing glycosidic linkages without causing degradative reactions. Another, more frequently used, alternative to acid hydrolysis of glycoproteins is methanolysis.92.261 This procedure may give improved yields of liberated monosaccharides, but it results in the formation of several (2 to 4) anomeric forms of glycoside of each, each of which gives rise to a separate 1.c. peak, and this makes quantitation difficult. Once a suitable hydrolysis procedure has been established, several useful 1.c. procedures are available for saccharide separation, and the final choice in method should be based upon the sensitivity and selectivity required. Sensitivity of detection is a major problem, as many glycoprotein samples are available only in sub-milligram quantities. To improve detector-sensitivity, several precolumn derivatization methods have been developed (see Section IV, le). Saccharides from glycoproteins have been converted into p e r b e n z o a t e ~ , dansylhydrazone~,~~~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ and other derivativesza which may be detected at the nanomole to picomole levels. Unfortunately, these added, bulky, chromophoric groups often overshadow the subtle stereochemical features of the original sugar, and separation (selectivity)of these derivatives is often poor. For this reason, l . ~ . methods that separate the underivatized carbohydrates and use postcolumn, detection-enhancementmethods often give the best separations. One such methoda0claimed to separate, on a normal-phase column, all hexoses, amino sugars, and sialic acids known to be present in glycopro(258) (259) (260) (261) (262) (263) (264)
A. J. Mort, Carbohydr. Res., 122 (1983) 315-321. M. P. Sanger and D. T. A. Lamport, Anal. Biochem., 128 (1983) 66-70. V. N. Reinhold, E. Coles, and S. A. Carr, J . Carbohydr. Chem., 2 (1983) 1-18. N.. Jentoft, Anal. Biochem., 148 (1985) 424-433. R. Oshima and J. Kumanotani, J . Chromatogr., 265 (1983) 335-341. F. M. Eggert and M. Jones, J . Chromatogr., 333 (1985) 123-131. G. Rosenfelder, M. Morgelin, J.-Y. Chang, C.-A. Schonenberger, D. G . Braun, and H. Towbin, Anal. Biochem., 147 (1985) 156-165.
H.P.L.C. OF CARBOHYDRATES
57
teins. Another method262must be used in conjunction with this procedure to differentiate between free and N-acetylated amino sugars. d. Structural and Sequence Analysis of Simple and Complex Carbohydrates.-L.c. methodology has played a key role in research on the structure of simple and complex carbohydrates and glycoconjugates. Besides providing high-resolution separation of these molecules for analytical purposes (see appropriate previous Sections), these nondestructive techniques serve as preparative methods (see Section III,3) for the efficient isolation of molecules that can subsequently be identified by chemical and instrumental means. In addition to these quantitative and preparative aspects, I.c. can provide qualitative information about the structure of various carbohydrates. A representative list of some of these methods is given in Table 111. Most of the methods rely on additional enzymic procedures, or specific detection-systems, such as p ~ l a r i m e t r i cor~mass-spec~~ TABLE111 L. C. Methods for Determining Structure or Sequence or Both in Carbohydrates Determination
Type of Carbohydrate
Enantiomeric form general review ,26J monosaccharides,266 partially methylated mo n o s ac~ h aride s~~~ Anomeric form mono- and di-saccharides enzymically released from polysaccharide~~~~" Molecular weight cyclodextrins,lm~lm linear oligosaccharidesZa Glycosyl sequence general m e t h o d ~ , ~ 3neutral .!~ oligosac~harides,~~*~~~~~~ polysaccharide s i d e- ~ h ai n s , '~glycoprotein-derived 3.~~~ in oligo- and oligosac~harides,~~~~~~~ hyaluronic a ~ i d , 2 ~choadroitin ~ , ~ ~ ~sul. ~ ~ ~ poly-saccharides fate,*I5heparin,216v2s6 complex p l a n t - p o l y s a c ~ h a r i d e s , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ complex b a c t e r i a l - p o l y ~ a c c h a r i d e s ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ (265) M. R. Little, J. Biochem. Biophys. Methods, 11 (1985) 195-202. (266) J. Golik, H.-W. Liu, M. Dinovi, J. Furukawa, and K. Nakanishi, Curbohydr. Res., 118 (1983) 135-146. (267) A. Heyraud and P. Salemis, Carbohydr. Res., 107 (1982) 123-129. (267a) J. 0. Baker and M. E. Himmel, J. Chromutogr., 357 (1986) 161-181. (268) S. Hase, T. Ikenaka, and Y. Matsushima, J. Biochem. (Tokyo), 90 (1981) 1275-1279. (269) S. F. Osman and P. D. Hoagland, Curbohydr. Res., 128 (1984) 361-365. (270) W. T. Wang, N. C. LeDonne, Jr., B. Ackerman, and C. C. Sweeley, Anal. Biochem., 141 (1984) 366-381. (271) M. McNeil, A. G. Darvill, and P. Albersheim, Plant Physiol., 70 (1982) 1586-1591. (272) P. W. Tang and J. M. Williams, Curbohydr. Res. 136 (1985) 259-271. (273) P. Aman, M. McNeil, L.-E. Franztn, A. G. Darvill, and P. Albersheim, Curbohydr. Res., 95 (1981) 263-282. (274) W. Dudman, L.-E. Franztn, M. McNeil, A. G. Darvill, and P. Albersheim, Curbohydr. Res., 117 (1983) 169-183.
58
KEVIN B. HICKS
trometric detectors,‘j3to provide structural information, but most of them would not be possible, or at least practical, without the use of modem 1.c. equipment. Especially noteworthy are the methods for determining the sequence of glycosyl residues in a complex oligo- or poly-saccharide. Methods of this type generate a series of overlapping oligomers that are separated and identified by I.c., in conjunction with either direct or indirect mass spectrometry. Most of these methods are still in the developmental stages, and they often require expensive and not-routinely available equipment (see also, Section IV,2) (see Addendum).
3. Preparative, Liquid Chromatography Much of the research on the 1.c. of carbohydrates has focused on analytical, rather than preparative, aspects. In reality, however, the conditions found in the majority of 1.c. methods, namely, no sample derivatization, high-resolution separations, and nondestructive detection-techniques, are ideal for the preparation of pure molecules. Thus, most of the analytical 1.c. methods previously described can also be used to isolate small quantities of pure compounds. This Section will cover the use of analytical-scale equipment for preparative applications, as well as the use of large-scale and “dedicated” preparative instruments for this purpose. Prior to discussion of these applications, a general overview of the preparative 1.c. of carbohydrates will be given. a. General Aspects of Preparative L.C.-The area of preparative 1.c. has been covered in two review^.^^.^^^ Investigators have developed theories to predict resolution of components at various K ’ values, loading capacities, flow rates, theoretical plates, sample volumes, and several other column parameters. The effects of these parameters on preparative, chromatographic resolution is complex and thus far unpredictable, and, as a result, preparative 1.c. remains a highly empirical process. Optimal preparative 1.c. is achieved when the largest amount of pure sample can be obtained in the shortest time. These optimum conditions are reached only by repeated experimentation and parameter-modification. Because the goal of preparative 1.c. is the preparation of pure compounds, a small, high-resolution system will often be more efficient than a large, lowresolution chromatograph which produces fractions that must be repurified. (275) M. Verzele and C. Dewaele, LC Mug., 3 (1985) 22-28.
H.P.L.C. OF CARBOHYDRATES
59
All of the stationary phases previously mentioned may be used for preparative purposes. Aminopropyl-bonded silica gels have the greatest sample 10adability~~ of any of the phases, but, often, the carbohydrates to be isolated have low solubility in the acetonitrile-water mobile phases used in conjunction with them. When using these columns in the preparative mode, care must be taken to use conditions1° that prevent glycosylamine formation between stationary-phase amino groups and reducing sugars. Reactive sugars, such as galactose and ribose, should not be isolated on these columns. Amine modifiers should not be used in mobile phases for preparative chromatography of reducing carbohydrates. Cation-exchange resin and C18-bonded silica columns have lower sample ~ a p a c i t i e s ~ ~than @ J amine-modified ~~ silica-gel columns, but they are more robust, do not covalently interact with sample components, and can be eluted with pure water at low flow-rates. Water is an ideal mobilephase, because of low cost, solute solubility, and ability to be completely removed from collected samples by evaporation. All mobile phases should have these characteristics-including buffers, which should contain volatile c o m p o n e n t ~ . ~ ~ J ~ The level of sample recovery from columns should always be noted. Low recovery may lead to rapid column-failure. Low recovery-values (50-90%) have often been reported61,I6' for normal- and reversed-phase columns. Preparative samples should always be pretreated by passage through a column (by gravity or suction) of a type of bonded phase similar to that used in the preparative separation. This packing material can generally be prepared, or purchased, at nominal cost. Pre-columns are generally recommended for preparative chromatography, but they are often less effective than the pretreatment just described, and they may be degraded quickly. Occasionally, silica gel and bonded-phase material may be leached from the column and be introduced into the purified ~ a m p l e . This ~ J ~ may ~ be removed by centrifugation, filtration, or extraction of the sample with nonpolar solvents, such as hexane. b. Preparative L.C. in Analytical-Scale Equipment.-The majority of preparative 1.c. is carried out on this level. No changes in hardware (except a larger sample-injector loop), column, or detector are necessary, and, because separations of very high resolution are possible, very pure compounds are isolated after only one chromatographic pass. Selected applications of small-scale preparative 1.c. of carbohydrates are given in Table IV. Although the amounts of carbohydrate isolated are low (several p g to 20 mg), they are often enough for subsequent qualitative methods such as lH-n.m.r. spectroscopy. By computer automa-
TABLEIV Isolation of Carbohydrates on Analytical-Scale Columns, and Instrumentation Used
Class of Carbohydrate
Columns and Solvents
Specific Carbohydrates Isolated (wt. in mg) ~____
Mono- and di-saccharides Glycosides
HPX-87P, H2 0 Radial Pak B, Silica gel, MeCN-H20-TEPA Dextropak CIS,H 2 0
Plant oligosaccharides
Dextropak CIS,H2 0
plant cell-wall sugars (0.5)244 sugars in standard mixture (20.0)39 benzylglycosides (3000) isolated on automated system276methyl glycosides from synthetic reaction (8)91 linear malto-, isomalto-, xylo-, and cello-oligosaccharides (1)@
Partisil PAC, MeCNisomalto-oligosaccharides (2)l49 Hz0 Neutral oligosaccharides of AUtech RP-5, H20; oligosaccharides from ovariananimal origin Micro PAK-AX-5, cyst glycoproteins174 MeCN-phosphate buffer; Primary amine-modified complex or high-mannose oligosilica gel, MeCN-H20 saccharides released from bronchial m u c i n ~ , ovomu~~~J~ coid,179Jw ceruloplasmin and o r o s o r n u ~ o i dIgD,'" , ~ ~ ~ ovalbumin,IJJ9167.1s and IgMl67 (smples for IH-NMR-spectral analysis, me) Dextropak C-18, H20; or human-milk oligosaccharides21.61.185 MicroPAK AX-5, phosphate buffer Ionic oligosaccharides of MicroPAK AX-10, phos- sialic acid-containing oligosacanimal origin phate buffer charides from ceruloplasmin,193.197 ovalbumin,193.197 orosomucoid,lWIgD,I@mutins," phosphorylated oligosaccharides from p-glucosiduronase (mg 1 e v e l ~ ) ~ ~ ~ J ~ primary amine-modified sialic acid-containing oligosacsilica gel, MeCNcharides from ceruloplasmin buffer and o r o ~ o m u c o i d ,IgD,I@ ~~~ human milk,[%ovornucoid,l% mucins,277and sulfated oligosaccharides from glycosaminoglycan~~~~J~l Glycopeptides Alltech WRP, or Suprotease-released glycopeptides pelco LC-18, phosfrom ovalbumin,62cathepphate buffer sin,m~20S and virus glycoproteinsZw Microbial oligosaccharides Partisil PAC, MeCNbranched hexa-0-p-D-glucoHzO; Spherisorb-5pyranosyl-D-glucitols from ODs, MeCN-H20 fungal-wall fragments (mg quantities)16-' C18-bondedsilica gel, cyclic glucans (cyclosophoroses) (mg quantities)l9I Hz0
H.P.L.C. OF CARBOHYDRATES
61
t i ~ nthese , ~ ~ analytical ~ systems can be used on an unattended, 24-h basis, and gram quantities of pure carbohydrates can be obtained. Of the many areas where these methods have been useful, the greatest impact has been in the area of complex plant and animal oligosaccharides, glycopeptides, and other glycoconjugates (see Table IV). The isolation of these pure carbohydrates, by the methods described, has allowed their spectroscopic, chemical, and enzymic analysis, in many cases for the first time (see Addendum). c. Preparative L.C. in Large-Scale Equipment.-This area of preparative chromatography is in a rapid stage of development. Clearly its potential lies in the applications that require multigram (or even multi-kilogram) amounts of pure carbohydrate for research, pharmaceutical, or food applications. Formerly, large-scale, preparative 1.c. required a “dedicated” instrument that used columns with “large” sample capacity (5-10 g), low resolving-capability, and requirements for “high” (200-500 mL/min) flow-rates. These inefficient systems have now been largely superseded by the use of efficient, preparative columns (2 x 30 cm), such as the Whatman Magnum 20, Dupont Zorbax Series, and Rainin Dynamax (see Section II,1 ,e) which allow high-resolution, “large-scale” (0.5 to 2.0 g) separations at moderate (10-25 mL/min) flow-rates. Interestingly, the efficiency of these columns is often superior to,24,275 and the back pressure lower than, that of their analytical counterparts. Many of the 1.c. stationary-phases available may be packed into columns by the user at a fraction of the cost of a commercial column (see Section II,l,f). The most notable of these are the cation-exchange resins8J9 and the aminopropyl-bonded silica gels,28which can be readily packed to provide high-resolution separations. The CIS-bonded silica gel and cation-exchange resin stationary-phases are especially useful, as large columns (2 x 25 cm) can be accommodated on analytical chromatographs, at flow rates of 1 to 5 mL/min, without any modifications of equipment. The use of an aminopropyl-bonded-phase silica-gel column (2 x 25 cm) for the high-resolution isolation of gram quantities of malto-oligosaccharides is shown in Fig. 1. The use of a home-packed, Ag+-form,cationexchange resin, preparative column (2.2 x 30 cm) for malto-oligosaccharide isolation is shown in Fig. 2. Other specific examples of large-scale, preparative 1.c. are listed in Table V. Undoubtedly, preparative-1.c. methods will be used routinely in future food-, industrial-, biological-, and biotechnological-carbohydrate applications. (276) A. F. Hadfield, R. N. Dreyer, and A. C. Sartorelli, J . Chromatogr., 257 (1983) 1-1 1. (277) G . Lamblin, A. Boersma, A. Klein, P. Roussel, H. van Halbeek, and J. F. G . Vliegenthart, J . Biol. Chem., 259 (1984) 9051-9058.
2
FIG.1.-Preparative Separation of Amylose-derived Oligosaccharides on Aminopropyl Silica-gel L.c. Column (2 X 25 cm) Eluted with 11 :9 Acetonitrile-Water at 12 mL/min. [Sample size, 500 mg; pressure, 7 MPa. Refractive index detection at 128x. Each numbered peak was collected at 86-98% purity. Numbers above peaks refer to d.p. values. See also, Ref. 8.1 TABLEV Isolation of Carbohydrateson Large-Scale Columns, and Instrumentation Specific Carbohydrates Isolated Class of Carbohydrate
Columns and Solvents
(WW
Mono- and di-saccharides
columns (diameter 1-10 cm) packed with ionexchange resin, H 2 0
Glycosides and simple sugar derivatives
Waters 500A, with C18 column (5.7 X 30 cm), Hz0 Rainin aminopropyl column (2. x 25 cm), MeCNHZ0 size-fractionation of highChromatospac Prep 10 silica gel, 2-propanolmannose oligosaccharides, ethyl acetate-HzO d.p. 1-9 (gram quantities)6 strong anion-exchange sialic acid-containing oligosacsilica-gel columns (-1 x charides derived from ovomucoidIm;sulfated, uronic 50 cm), phosphate buffers acid-containing oligosaccharides from heparin2I6
Plant-derived oligosaccharides Neutral, animal-derived oligosaccharides Acidic, animal-derived oligosaccharides
neutral mono- and di-saccharides from polysaccharide hydrolyzates (100 ketose disaccharides (lactulose, maltulose, cellobiulose) (200 mg)@;fructose (kg s ~ a l e ) ,uronic ~ ~ ~ acidszs .~~~ methyl maltosides,' O-methylated sucrose derivative9 (gram quantities) malto-otigosaccharides, d.p. 37 (gram quantitie#
H.P.L.C. OF CARBOHYDRATES
63
6
I
I
I
I
I
0
10
20
30
40
50
60
'IIme (min)
FIG.2.-Preparative Separation of Same Sample as Used in Fig. 1, on Laboratorypacked, Preparative Column (2.2 X 30 cm) of HPX-42 (Ag+)Resin, Eluted with H 20 at 1.1 mL/min. [Sample size, 12.5 mg; pressure, 700 kPa. Refractive index detection at 128x. Each numbered oligosaccharide was collected at 75-92% purity. Numbers above peaks refer to d.p. values. See also, Ref. 8.1
IV. SPECIAL ASPECTSAND PROBLEMS 1. Detectability and Accuracy
After two decades of quantitative 1.c. analyses, it has been established that, when proper precautions are taken, these methods can provide accurate and reproducible r e s ~ l t s . Quantitative ~~~?~~~ 1.c. measurements are usually as accurate as, and often more precise than, those obtained by spectroph~tornetric,~~~~~~~ paper-chroma tog rap hi^,^^ and gas-liquid-chroma tog rap hi^^^^ methods. Both external and internal standardization have been used to translate peak height or areas into quantitative, solute-concentration values. Because peak heights are easy to measure, many methods use this parameter, and, when slightly overlapping peaks or unsteady baselines are encountered, it is the method of choice. With introduction of (278) P. E. Barker and S. Thawait, Chem. Znd. (London),(1983) 817-821. (279) P. E. Barker, G. A. Irlam, and E. K. E. Abusabah, Chromatographia, 18 (1984) 567574. (280) W. Moody, G. N. Richards, N. W. H. Cheetham, and P. Sirimanne, Carbohydr. Res., 114 (1983) 306-310.
64
KEVIN B. HICKS
the use of microprocessor-controlled,recording integrators, more peakarea analyses are being reported, especially for analysis of oligosaccharides, such as the m a l t ~ - ~ ~ ~cello-oligosaccharides,49 ’and where each oligosaccharide gives the same peak area per unit weight (for refractive index detectors). The various detection methods used to obtain these quantitative results are described next. a. Refractive Index Detection.-Refractive index ( r i ) detectors, or differential refractometers, are the most commonly used detectors for 1.c. analysis of non-u.v.-absorbing, simple sugars, alditols, and oligosaccharides. These relatively inexpensive instruments are regarded as “universal detectors” because they detect almost all classes of molecules present in a mixture, and this is especially useful in preparative chromatography, where it is essential to be aware of the presence of all of the compounds in a sample. Despite their common use, these detectors have several limitations, namely, sensitivity to changes in solvent composition, temperature, and pressure. These limitations are most apparent, however, when highly sensitive detection is required. Even then, with the use of low-pulsation, solvent-delivery systems, equipped with pulse dampeners, column temperature-control compartments, and other accessories outlined in Section II,1, r.i. detection can be useful and relatively troublefree. R.i. detectors cannot be used in conjunction with solvent gradients, and this remains one of their major shortcomings. The sensitivity of various r.i. detectors has been studied, and the limit of detection for traditional models depends upon the column and the mobile phase being used. For amine-modified silica-gel columns, mono-, di-, and oligo-saccharides may be detected at levels greater than -5 pg.33,39J86.242 For cation-exchange-type 1.c. columns (Ca2+or Pb2+form), the detection limit is almost an order of magnitude 1 0 w e r , ~and ~ . ~these ~ columns are more useful for high-sensitivity work. In most cases, the level at which sugars can be accurately and reproducibly measured is about five times the minimal detectable levels just described. The linear range of detection differs considerably from one laboratory to another, and therefore must be determined for each instrument. Linear ranges of 5 pg to 2 mg have been reported39g83.233.242 for various carbohydrates. Manufacturers have now introduced highly sensitive, r.i. detectors which are capable of signal-tonoise ratios that are 10 to 100 times better281than those of conventional r.i. detectors, allowing the detection of as little92as 15-25 ng of monosaccharide. With these detectors, precise analysis of sugars may be accomplished in the 200-ng to >20-pg range. A side benefit of these detectors is the dramatic lengthening of column life, due to the small amounts of (281) J. Koops and C. Olieman, Neth. Milk Dairy J., 39 (1985) 89-106.
H.P.L.C. OF CARBOHYDRATES
65
samples (and contaminants) which are injected. At this sensitive range, however, special precautions must be taken to prevent contamination of samples, because even trace components (in samples or diluents) will produce 1.c. peaks that are equivalent in size to the sample peaks. b. U.V. Detectom-U.v. detectors are useful in carbohydrate 1.c. analysis, especially for those compounds having carboxyl or other u.v.absorbing groups. Even simple alditols and sugars have been detected at low wavelengths (188-192 nm), although the sensitivity of this technique is no greater than that of r.i. detection (minimum detectable levels for these compounds are -1-12 pg).33987.234.246 Owing to the low u.v.-absorbances of these carbohydrates, accurate analyses are often difficult because of interfering, chromatographic peaks from strongly u.v.-absorbing, trace contaminants in solvents and samples.246Carbohydrates that have higher extinction coefficients and are readily detected are as follows: (compounds, wavelength, minimal detectable level) hexuronic, aldonic acids, and lactones, 210-220 nm, 0.5-10 pg, Refs. 48 and 96; ascorbic acids, 254-268 nm, 25-50 pg, Refs. 112 and 282; sialic acids, 195-200 nm, 200 pg-6 ng, Refs. 120 and 122; oligosaccharides containing 2-acetamido2-deoxyhexose units, 190-195 nm, 0.1-0.3 nm01~~J~O; sialylated oligosaccharides, 195 nm, 0.6 nmolzo2;and unsaturated oligosaccharides from glycosaminoglycans, 232 nm, 10 ng.253
c. Miscellaneous, Direct-Detection Methods.-Other methods for direct, 1.c. detection are based on electrochemical, mass, polarographic, and flame-ionizationdetectors. Electrochemical detectors are most useful for such electroactive molecules as the ascorbic acids, and their application to the detection of reducing sugars is limited.283 A modification of this technique, triple-pulsed amperometry on platinumzu or gold72electrodes, has been used for the direct and sensitive (nanogram level) detection of reducing sugars, alditols, and oligosaccharides. This detector is, however, only compatible with highly basic (pH >lo) mobile phases. Mass detectors, which measure light-scattering from nebulized solute, after solvent evaporation, constitute a type of universal detector that can be used with solvent gradients. Their sensitivity is about equal to that of conventional refractive index detectors when mobile phases that are low in water content are used. Salt-containing mobile-phases cannot be used. Mass detectors have been used to detect reducing and nonreducing sugars, as (282) P. Wimalasiri and R. B. H. Wills, J . Chromatogr., 256 (1983) 368-371. (283) W. Buchberger, K. Winsauer, and C. Breitwieser, Fresenius Z . Anal. Chem., 311 (1982) 517. (284) S . Hughes and D. C. Johnson, J . Agric. Food Chem., 30 (1982) 712-714.
66
KEVIN B. HICKS
well as oligosaccharides.247~z85-z87 Polarimetric d e t e ~ t o r s ~are ~~ useful , * ~ in ~ I.c., because they provide qualitative information (configuration, and enantiomeric or anomeric form) about eluted peaks. Because some sugars (arabinose, galactose, rhamnose, and mannose) occur in Nature in both the D and the L form, the determination of enantiomeric form is an important and required step in the total characterization of naturally occurring sugars or polysaccharide hydrolyzates. Polarimetric, or optical-activity, 1.c. detectors have been used to determine the enantiomeric form of eluted sugars288and partially methylated sugar derivatives267(from permethylated polysaccharide hydrolyzates). The accuracy of such detectors is good, and as most 1.c. solvents are optically inactive, almost any mobile phase can be used in conjunction with them. The sensitivity for these detectors is, however, quite variable, as it is related to the magnitude of the specific rotation for each sugar. For carbohydrates with [a],>50”, minimum detection limits of 100 pg have been reported.267For D-fructose, as little as 100 ng was detectedzE8in 1.c. effluents by use of a highly sensitive, optical-activity detector. Unfortunately, these detectors are not at present readily available from commercial sources. To determine absolute configuration, it is therefore necessary to use alternative chromatographic procedures wherein either indirect methods (converting the enantiomeric pair into diastereoisomers) or direct ones (chromatography on chiral, stationary phases) are used to distinguish between the D and the L form. Sensitive 1.c. and g.1.c. procedures are available for these purposes and they have been reviewed.z65Otherwise, carbohydrates eluted from 1.c. columns may be collected (see Section III,3) and subsequently examined by polarimetry or circular dichroism methodszMin order to establish the enantiomeric form. The use of flame-ionization detectors has been d e s ~ r i b e d . An ~ ~ .important ~~ feature of these detectors, which are commercially available, is their ability to monitor all types of eluted species (so-called “universal” detection). In addition, unlike refractive-index detectors, they may be used in conjunction with gradient-elution programs. d. Post-column Derivatization Methods.-Often, carbohydrate samples are available in such small amounts that direct 1.c.-detection by one of the previous methods is not possible. To enhance carbohydrate detectability, methods based upon post-column derivatization have been developed. In these methods, carbohydrates are separated by using normal 1.c.-techniques, and only the detection system is altered. Several excel(285) M. Lafosse, M. Dreux, L. Morin-Allory, and J. M. Colin, J . High Resolut. Chromatogr. Chromatogr. Commun., 8 (1985) 39-41. (286) R. Macrae and J. Dick, J . Chromarogr., 210 (1981) 138-145. (287) R. Macrae, L. C. Trugo,and J. Dick, Chromatographia, 15 (1982) 476-478. (288) J. C. Kuo and E. S. Yeung, J . Chromatogr., 223 (1981) 321-329.
H.P.L.C. OF CARBOHYDRATES
67
lent reviews29.81,289 on the subject covered advances in this area up to about 1983, and all post-column methods are now being reviewed on a biennial b a s i ~ . *Table ~ , ~ VI ~ ~lists representative methods that have since TABLEVI Post-Column Reagents for Enhancing the Detectability of Carbohydrates
Carbohydrates
Reagent ~~
~
Detection Limit (method)
~
Tetrazolium Blue34~23*~29z reducing sugars reducing sugars Copper bis(phenanthr01ine)~~~~~ alditols 2-tert-Butyl anthraq~inone~~-’+~% mono-, oligo-saccharides C~prammonium29~ reducing Cyanoacetamide hexosamines,m sialicw and uronic7’acids hexosamines general carbohydrate reagent sugars, reducing and nonreducing p -Aminobenzoic sugars, reducing and hydraidem nonreducing Europium salts”* sugar phosphates
10 ng (P)” 0.2 ng (A)b 80 ng (F)’ 0.4 pg (PI 0.2 pg (PI 5.0 ng (E)d 0.1 nmol (F) 1.0 nmol (P) 1.0 nmol (F) 1.0 pmol (E)
Photometric. Amperometric. Fluorometric. Electrochemical detection principles.
(289) R. W. Frei, H. Jansen, U. A. T. Brinkman, Anal. Chem. 57 (1985) 1529~-1539~. (290) R. E. Majors, H. G. Barth, and C. H. Lochmiiller, Anal. Chem., 54 (1982) 3 4 7 ~ 349R, 3 6 0 ~ - 3 6 2 ~ . (291) R. E. Majors, H. G . Barth and C. H. Lochmiiller, Anal. Chem., 56 (1984) 3 2 9 ~ - 3 3 1 ~ , 346R-347~. (292) R. E. A. Escott and A. F. Taylor, J . High Resolut. Chromatogr. Chrornatogr. Commun., 8 (1985) 290-292. (293) N. Watanabe and M. Inoue, Anal. Chem. 55 (1983) 1016-1019. (294) N. Watanabe, J . Chrornatogr., 330 (1985) 333-338. (295) M. S. Gandelman and J. W. Birks, Anal. Chim. Acfa, 155 (1983) 159-171. (2%) M. S. Gandelman, J. W. Birks, U. A. T. Brinkman, and R. W. Frei, J . Chromarogr., 282 (1983) 193-209. (297) G. K. Grimble, H. M. Barker, and R. H . Taylor, Anal. Biochem., 128 (1983) 422428. (298) T. D. Schlabach and J. Robinson, J . Chromarogr., 282 (1983) 169-177. (299) S. Honda, T. Konishi, S. Suzuki, K. Kakehi, and S. Ganno, J. Chromarogr., 281 (1983) 340-344. (300) P. Nordin, Anal. Biochem., 131 (1983) 492-498. (301) K. Mopper, R. Dawson, G. Liebezeit, and H.-P. Hansen, Anal. Chem., 52 (1980) 2018-2022. (301a) S. Honda, K. Enami, T. Konishi, S. Suzuki,and K. Kakehi, J . Chromatogr., 361 (1986) 321-329. (302) P. VrAtnq, U. A. T. Brinkman, and R. W. Frei, Anal. Chern., 57 (1985) 224-229.
KEVIN B. HICKS
68
been used, or developed. Most of these reagents require the use of a postcolumn reactor, and advantages of the various commercial and laboratory-built devices have been d e s ~ r i b e d . ~ ~A~ comparison , ~ ~ ~ - ~ " of several post-column reagents has been published.302 e. Pre-column Derivatizatlon Methods,-Another technique for enhancing the detectability of carbohydrates is to convert them into readily detectable derivatives prior to chromatography. In many cases, this should be a method of last resort, because 1.c. was originally developed to analyze polar, non-derivatized carbohydrates directly. Moreover, existing methods based on g.1.c. would suffice in many applications (except for certain ones, such as oligosaccharides) where highly sensitive detection is needed. Nonetheless, there are a number of useful, pre-column derivatization procedures, and the developments in this area up to about 1983 have been and methods of this type for the following carbohydrates have been described. Simple sugars: p e r b e n z ~ a t e sdan,~~~ sylh ydrazones , N-(p-rnethoxyphenyl)glycosylamines,307products from reductive amination with aniline,269~2704'-(dimethylamino)-4-aminoazobenzene,2u or 2-amin0pyridine~~~; alditols: perbenzoatesY3*phenylglycosides: perbenzoates,261per-p-bromobenzoates and pernaphthoates2%; sialic acids: (p-nitrophenyl)hydra~ones~~~; hexosamines: products from reductive amination with 2 - a m i n 0 p y r i d i n e ~ ~ ~ ~ ~ ~ ~ or 4'-(dimethylamino)-4-aminoazobenzene2u;uronic acids: products from reductive amination with 4'-(dimethylamino)-4-aminoazobenzene2a ; glycosaminoglycan-derived oligosaccharides: product from reductive amination with a n i l i ~ ~ e ~ and ~ ~simple . ~ ' ~ and ; complex oligosaccharides: p e r b e n z ~ a t e s , ~d~a.n~s' y ~ l h y d r a z ~ n e s products , ~ ~ ~ ~ ~ from reductive ami12,263y306
(303) J. H. M. van den Berg, H. W. M. Horsels, and R. S. Deelder, J . Liq. Chromatogr., 7 (1984) 2351-2365. (304) P. Vrhtnf, R. W. Frei, U. A. T. Brinkman, and M. W. F. Nielen, J . Chromarogr., 295 (1984) 355-366. (305) R . Galensa, Z. Lebensm. Unters. Forsch., 178 (1984) 199-202. (306) S. R. Hull and S. J. Turco, Anal. Biochem., 146 (1985) 143-149. (307) M. Batley, J. W. Redmond, and A. Tseng, J . Chromatogr., 253 (1982) 124-128, (308) H. Takemoto, S. Hase, and T. Ikenaka, Anal. Biochem., 145 (1985) 245-250. (309) R. Galensa, Z . Lebensm. Unters. Forsch., 178 (1984) 475-478. (310) J.-M. Dethy, B. Callaert-Deveen, M. Janssens, and A. Lenaers,Anal. Biochem., 143 (1984) 119-124. (31 1) P. A. McNicholas, M. Batley, and J. W. Redmond, J . Chromarogr., 315 (1984) 451456. (312) C. Kodamti, N. Ototani, M. Isemura, and Z. Yosizawa, J . Biochem. (Tokyo), 96 (1984) 1283- 1287. (313) P. F. Daniel, D. F. De Feudis, I. T. Lott, and R. H. McCluer, Curbohydr. Res., 97 (1981) 161-180.
H.P.L.C. OF CARBOHYDRATES
69
nation with 2 - a m i n 0 p y r i d i n e , ~ 4’-(dimethylamino)-4-aminoazoben~~*~~~ zene,2642-aminopyridine, and 7-amino-l-naphth01.~~~ 2. Combined L.C. Techniques (L.C.-M.S. and L.C.-N.m.r. Spectroscopy)
The separative power of 1.c. can be coupled to various types of instruments in order to obtain additional qualitative information about eluted compounds. Two such instruments that may be coupled to 1.c. include n.m.r. and mass spectrometers. Although there has been one report on the use of 1.c.-n.m.r. spectroscopy315in carbohydrate research, it is the 1.c.-mass spectrometry (m.s.) system that has received the bulk of the attention. Mass spectrometers are capable of generating a great deal of information about molecular weights, empirical formulas, isotope ratios, and molecular structure, but the actual on-line coupling of m.s. to 1.c. has been a challenge, and the full potential of this method has yet to be realized. To begin with, 1.c. requires high-pressure, liquid-phase operation, whereas m.s. requires gas-phase and vacuum operation. Despite these diametrically opposed principles, several interfaces have been developed to allow on-line m.s. detection of 1.c. effluents, and these have been The direct, liquid introduction of 1.c. effluents into m.s. ion-sources (one type of interface) creates a huge burden on the vacuum system, and hence, only 1-3% of the total flow from a conventional 1.c. column may be directly injected. Although the sensitivity of such split-stream interfaces is low, they have been used to analyze eluted sialic acids,l2Iand various peralkylated oligosaccharides.166*273 When microbore columns are used, the flow rates are low (
70
KEVIN B. HICKS
ization mass spectrometer^.^^^ These methods are useful for analyzing such small, polar molecules as peptides and sugars. An alternative to direct liquid introduction is the moving belt, or moving-wire, transport Because all 1.c. solvents are evaporated before the sample is transported into the ion source, fewer restrictions are placed on solvent type, flow rates, or buffer composition. This system has been used for analysis of mixtures of pentoses, hexoses, and disaccharides.319 Most of the direct and indirect (transport) interfaces described here use chemical ionization (c.i.) ion-sources, which are not well suited to such polar, non-volatile compounds as tri- and higher oligosaccharides. The thermospray interface, which can operate on an ion-evaporative mode ,317 is capable of producing intact molecular ions from such nonvolatile, polar molecules and should be useful in oligosaccharide analysis. Molecules of this type, however, can also be easily analyzed by fast-atom-bombardment ionization, and use of this technique,321acoupled to direct liquid introduction322and m ~ v i n g - b e l interfaces, t~~~ has been reported. The latter system has been applied to complex oligosaccharide analysis.324
3. Separation of Carbohydrate h o m e r s Although reducing sugars exist in solution as a mixture of two or more tautomeric forms, they often give only one peak when analyzed by I.c., and this is fortunate for analytical purposes. There are, however, instances where the separation of sugar anomers is encountered. Partial or complete separation of anomers of reducing sugars and oligosaccharides has been achieved by ligand-exchange chromatography on cation-exchange resins (metal form),53,267a by normal phase-partition chromatography on either polystyrene-based anion-exchangers (sulfate form)325or aminopropyl-bonded silica gel (in the sulfate135or acetate326form), by combined partition-ligand exchange chromatography on Na+- and Ca2+form cation-exchangeresins78,and by reversed-phase chromatography on alkyl-bonded s i l i ~ a . In ~ *all~these , ~ ~ cases, anomers of individual reducing sugars are separated because their mutarotation rate is low, relative to the speed of the chromatography. Pyranose anomers of some sugars may (321a) A. Dell, Adu. Carbohydr. Chem. Biochem., 45 (1987) 19-72. (322) Y. Ito, T. Takeuchi, D. Ishii, and M. Goto, J . Chromatogr., 346 (1985) 161-166. (323) J. G . Stroh, J. C. Cook, R. M. Milberg, L. Brayton, T. Kihara, Z. Huang, K. L. Rinehart, Jr., and I. A. S. Lewis, Anal. Chem., 57 (1985) 985-991. (324) V. Reinhold, unpublished results. (325) R. Oshima, N. Takai, and J. Kumanotani, J . Chromatogr., 192 (1980) 452-456. (326) M. Moriyasu, A. Kato, M. Okada, and Y.Hashimoto, Anal. Lett., 17 (1984) 689699.
H.P.L.C. OF CARBOHYDRATES
71
be separated at room temperature on one system,s3but, by using a lower temperature (0-4"), nearly all of the a- and P-pyranose anomers of the common aldohexoses and aldopentoses can be separated.78Furanose anomers interconvert at higher rates, and temperatures of -25 to -45" and special solvents were necessary in order to separate the a-and p-furanose anomers of L-fucose and ~ - g a l a c t o s e . ~Because ~ ~ . ~ * ~of the rapidity of these assays, and their accuracy, as verified by n.m.r. spectro~copy~~ and p ~ l a r i m e t r y ,they ~ ~ ~have , ~ ~ ~been used to determine the mutarotation rates of the common s ~ g a r s . ~ ~ , ~ * ~ When resolution of anomers is not desired, it can usually be prevented by the following procedures. Cation-exchange columns (metal forms) should always be run at temperatures 285". Aminopropyl-bonded silicagel columns should not be used in the acetate or sulfate form. On alkylmodified silica, the prevention of anomer separation is not easy, and increasing the temperature, or adding catalysts to increase the mutarotation rate, causes loss of r e t e n t i ~ n When . ~ ~ sugars and oligosaccharides must be analyzed on such columns, they should be reduced by sodium borohydride prior to chromatography, in order to obtain sharp and symmetrical, chromatographic peaks. 4. Future Trends
New advances in the 1.c. of carbohydrates are likely to come from three general areas. The first is in the development of more-durable and stable, stationary phases. At present, a major limitation on the use of commercial columns, especially those of the aminopropyl-bonded silica-gel variety, is their short life-time and ease of fouling. More-durable, resin-based columns that operate with the same solvent system and selectivity as aminopropyl silica-gel columns are currently available,80and will see further use and development. The development of improved phases for supercritical, fluid-type 1.c. will allow this method to be of use for analysis of various carbohydrates.327a A second field of rapid development in the 1.c. of carbohydrates is in practical, preparative chromatography. Early preparative systems used large, expensive columns with low resolving power, and hence, were not extensively applied in carbohydrate research. New research8 is showing that various carbohydrates can be separated on the gram scale, using normal 1.c. equipment and large columns home-packed with relatively (327) M. Moriyasu, A. Kato, M. Okada, and Y. Hashimoto, Anal. Lett., 17 (1984) 15331538.
(327a) T. L. Chester and D. P. Innis, J . High Resolut. Chromatogr. Chromatogr. Commun., 9 (1986) 209-212.
12
KEVIN B. HICKS
inexpensive resins and bonded silica gels. Rapid advances are to be expected in this area (see Addendum). A third area of development in carbohydrate 1.c. analyses is in the combined techniques (see Section IV,3) and other methods that provide qualitative, as well as quantitative, information about sample constituents, such as high-performanceliquid affinity chromatography. The use of specific lectin- and monoclonal antibody-based, stationary phases for analytical and preparative applications is now being considered. The basic concepts of these techniques have been r e v i e ~ e dand ~ ~their ~ . ~applica~~ tions to have been discussed. ACKNOWLEDGMENTS The author thanks Rebecca Haines, Robert Miller, Scott M. Sondey, and Pauline Barnett for assistance in obtaining references and in proofreading the manuscript.
(328) S. Ohlson, M. Glad, and P.-0. Larsson, in I. M. Chaiken, M. Wilchek, and I. Parikh (Eds.), &Enity Chromatography and Biological Recognition, Academic Press, New York, 1983, pp. 241-250. (329) P.-0. Larsson, M. Glad, L. Hansson, M.-0. Mhsson, S. Ohlson,and K. Mosbach, in J. C. Giddings, E. Grushka, J. Cazes, and P. Brown, Adu. Chromatogr., 21 (1983) 41-85. (330) A. J. Muller and P. W. Carr, J . Chromarogr., 284 (1984) 33-51.
ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 46
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES* BY RENB CSUK**AND BRIGITTEI. GLANZER** Instirute of Organic Chemistry, Technical University of Graz, Graz, Austria I. Introduction ............................................................ 11. Spectroscopy of Fluorinated Monosaccharides ............................. 1. IH-N.m.r. Spectroscopy .............................................. 2. W-N.m.r. Spectroscopy .............................................. 3. I9F-N.m.r. Spectroscopy .............................................. 4. Other Nuclei.. ....................................................... 111. Tables ................................................................. 1. Guide to Tables.. .................................................... 2. Tables .............................................................. Addendum .............................................................
73 74 75 77 78 80 80 80 82 33 1
I. INTRODUCTION Within the past two decades, the importance of nuclear magnetic resonance (n.m.r.) spectroscopy has enlarged tremendously. Its advantages over other methods in the structural and conformational analysis of such complex molecules as natural products , I and particularly with regard to the still-increasing importance of medical investigations2 using n.m.r. techniques, is abundantly evident. Because destruction of the sample is avoided, different n.m.r. experiments can be performed repeatedly, and
* The authors thank Dr. Karl Dax, Institute of Organic Chemistry, Technical University, Graz,for suggesting that we make such a compilation and for pointing out its potential practical use. ** Present address: Organisch-Chemisches Institut der Universitat, Winterthurer Strasse 190, CH-8057, Zurich, Switzerland. (1) G. Govil and R. V. Hosur, in E. Diehl, E. Fluck, and R. Kosfeld (Eds.), NMR- Basic
Principles and Progress, Vol. 20, Springer Verlag, Berlin, 1982. (2) K. Roth, NMR-Tomography and -Spectroscopy in Medicine, Springer Verlag, Berlin, 1984. 73 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
RENE CSUK AND BRIGITTE I. GLANZER
74
analysis of the spectra is usually convenient, simple, and fast.3This methodology is exceptionally free from artifacts, thus allowing detailed analysis of conformational behavior in solution in comparison with the conformation in the solid state (obtained from X-ray analysis or from a n.m.r. spectrum of the solid4). 11. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
Application of 1H-n.m.r. spectroscopy to such complex molecules as carbohydrate^^.^ in general, and fluorinated monosaccharides7 in particular, soon showed the limits of this methodology, and prompted the development of modifications. Double- and triple-resonance techniques5q6and isotopic labelling of compounds, together with the use of Fourier-transform spectroscopy at very high field-strength now allows acquisition of high-resolution, n.m.r. spectra. Further progress followed the application of two-dimensional* n.m.r. techniques in the carbohydrate field.9 Because of these great improvements, all magnetic nuclei can now be used in n.m.r. analysis of fluorinated monosaccharides,1° including 'H, 13C, 15N,and 19F.Table I gives the relevant, spectroscopic data for these nuclei. TABLEI Comparison of the Relevant Parameters of Different NucleP5 Nuclei
Spin
Natural abundance
H
112 1/2 1/2 112
99.98 1.11 0.37 100
Sensitivity Gyromagnetic ratio FrequencyC Relative Absolute" Rangeb ~~
c
N F
loo0 15.9 1.04 830
~~~
lo00 0.18 0.004
830
20 650 1000 800
2.793 0.702 0.283 2.627
100 25.14 10.13 94.08
a Relative sensitivity: at constant field for equal number of nuclei; absolute intensity: product of relative sensitivity and natural abundance. In parts per million (p.p.m.). For a field strength of 2.3480 tesla.
(3) E. D. Bishop, Annu. Rep. NMR Spectrosc., 1 (1968) 91-134. (4) M. Mehring, Principles of High Resolution NMR in Solids, 2nd edn., Springer Verlag, Berlin, 1983. (5) T. D. Inch, Annu. Rep. NMR Spectrosc., JA (1972) 305-352. (6) L.D. Hall, in W. Pigman and D. Horton (Eds.), The Carbohydrates; Chemistry and Biochemistry, 2nd edn., Vol. IB, Academic Press, New York, 1980, pp. 1299-1326. (7) P. J. Card, J . Carbohydr. Chem., 4 (1985) 451-487. (8) A. Bax, Two-Dimensional Nuclear Magnetic Resonance in Liquids, Reidel, Dordrecht, 1982. (9) L. D. Hall and S. Sukumar, J . Am. Chem. SOC.,101 (1979) 3120-3121. (10) A. A. E. Penglis, Adv. Carbohydr. Chem. Biochem., 38 (1981) 195-285.
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
75
1. 'H-N.m.r. Spectroscopy
a. Chemical Shifts.-Introduction of a fluorine atom into an organic molecule causes a significant shift to low-field of the signals of the geminal hydrogen atoms. However, the practical use of this effect, and its interpretation in terms of structure and conformation, remain small.
b. lH-19F Coupling Constants.-The 'H-19F spin-spin coupling is influenced by steric and electronic influences of substituents, both in close proximity to, or remote from, the coupling nuclei. The stereospecific dependences of fluorine-proton couplings compared to the corresponding 'H-lH couplings are much more sensitive to the steric environment, and the coupling constants are of much greater magnitude. Because IH-19F coupling-constants can be readily extracted from the 19F spectrum without interference of proton-proton coupling-patterns, this additional information facilitates even the interpretation of the proton spectra without the need for fluorine decoupling experiments. Geminal Coupling, 3 ('H, 19F). The sign of geminal coupling constants has been determined" and shown to be absolutely positiveI2 and to occur in secondary, carbohydrate-derived pyranoid derivatives in a range lying between 49 and 59 Hertz (Hz), whereas the furanoid counterparts show coupling of 49-66 Hz; where fluorine is attached to sp-hybridized carbon atoms, couplings of some 80 Hz may be observed. An empirical approachI3 for estimating geminal coupling-constants has been described for a limited class of compounds. Irrespective of whether fluorine is axially or equatorially oriented, an increase (-3 Hz) in 2J results upon changing the vicinal substituent from the axial (a) to the equatorial (e) orientation, whereas changing the fluorine atom from equatorial to axial has little or no effect. An exception to the latter occurs in cases where the substituent is a ring-oxygen or -sulfur atom. Vicinal Coupling, 3J (lH, 19F). The signs14 of vicinal couplings were found to be absolutely po~itive,'~ and their angular dependence parallels*0 that of proton-proton coupling-c~nstants,~~J~ ranging between 0 and (11) L. D. Hall, R. N. Johnson, A. B. Foster, and J. H. Westwood, Can. J . Chem., 49 (1971) 236-240. (12) A. D. Barford, A. B. Foster, J. H. Westwood, L. D. Hall, and R. N. Johnson, Carbohydr. Res., 19 (1971) 49-61. (13) L. Phillips and V. Wray, J . Chem. Soc., Perkin Trans. 2 , (1974) 928-933. (14) L. D. Hall and J. F. Manville, Chem. Znd. (London), (1967) 468-469. (15) L. D. Hall and J. F. Manville, Carbohydr. Res., 9 (1969) 11-19. (16) A. Gaudemer, in H. B. Kagan (Ed.), Stereochernistty: Fundamentals and Methods, Vol. 1, Thieme Verlag, Stuttgart, 1977, pp. 44-136. (17) L. D. Hall, J. F. Manville, and N. S. Bhacca, Can. J . Chem., 47 (1969) 1-17.
76
RENB CSUK AND BRIGITTE I. GLANZER
some 30 Hz (compare, Table 11). The value of J (trans) are found to be larger than J (gauche).These couplings depend on the electronegativity of neighboring substituents. Only a few exceptions to the empirical ranges given in Table I1 are known. trans-Vicinal couplings in the pyranose series depend additionally on the nature of the substituents on the carbon atoms to which the coupled nuclei are themselves bound. The coupling constant decreases as the number of oxygen atoms increases. The factors that control gauche, vicinal coupling depend onlo: the presence of a hydroxyl group trans to the C-C bond bearing the coupling nuclei, the presence of an oxygen atom trans to the fluorine atom by way of the coupling pathway, and also on the number of substituted oxygen atoms on the carbon atoms bearing the coupled nuclei. Long-range Coupling, 4J (*H, '9F). The signs" of these coupling constants vary. The signs for 4J (e,e) in the pyranose series are absolutely positive, whereas 4J (e,a) values are negative; 4J (a,a) are again positive.Ia2O These couplings show the same stereospecificity as compared to their correspondingproton-proton couplings and reach their largest magnitude when a planar M (that is, a coplanar W)relationship is adopted.21 Pyranoses and furanoses both show stereoselectivity in their coupling constants. For the latter, there are possible coupling pathways either via the ring-oxygen atom or exclusively via C-C bonds (compare, Table 111).22 sJ ('H,'9F). Although these long-range coupling-constants have been observed for a few compounds, their practical use remains small. An extended "M" arrangement is anticipated to be the favored pathway for
jJ
Ring form
TABLEI1 (W, '9F) Couplings
Orientation
Coupling constant (in Hz)
Pyranose
trans-diaxial
Furanose
gauche trans cis
21-30 0-18 14-30 2-1s
L. D. Hall, R. N. Johnson, J. Adamson, and A. B. Foster, Can. J . Chem., 49 (1971) 1 18- 123. A. B. Foster, J. H. Westwood, B. Donaldson, and L. D. Hall, Carbohydr. Res., 25 (1972) 228-23 1. A. B. Foster, R. Hems, and L. D. Hall, Can. J . Chem., 48 (1970) 3937-3945. A. B. Foster, R. Hems, L. D. Hall, and J. F. Manville, Chem. Commun., (1968) 158-159. L. D. Hall and P. R. Steiner, Can. J . Chem., 48 (1970) 2439-2443.
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
77
TABLE111 41 (IH,
Ring form Pyranose Furanose
19F) Coupling
Constants Coupling constant (in Hz)
Orientation" e, e e, a a, a trans cis
via via via via
oxygenb C-C bonds' oxygenb C-C bonds'
-4-5 -0- 1 -0.5-2.5 5.5-8 -0.7 1.o-2 - 1.7-2.4
-
" The symbol e stands for equatorial, and a for axial. * Corresponds to a coupling of F-1 to H-4. Corresponds to a coupling of F-1 to H-3.
such couplings, and the couplings reach a maximum when the coupled nuclei are trans-coplanar to the bond that is the midpoint of the coupling pathway.23In addition, an oxygen atom in the coupling pathway leads to increased values as compared to an all-carbon pathway.24 6J (IH,19F). There are only a few examples of this long-range coupling; their use in conformational analysis seems to be negligible. 2. 13C-N.m.r. Spectroscopy
a. Chemical Shifts.-I3C-N.m.r. spectroscopy at natural abundance provides insights on structural and conformational behavior in solution, combined with the advantage of less-crowded and more-readily interpreted spectra. An excellent article on 13C-n.m.r. data for monosaccharides has been published in this Series.25In general, fluorine substitution in carbohydrates results in a significant, low-field shifting of the signal of the adjacent carbon a t o ~ n . ~ ~ , ~ ' b. "C-I9F Coupling Constants.-The measurement of these coupling constants, using proton-broad-band decoupling, is facile, and greatly aids in the interpretation and assignment of the spectra. 'J ( I T , 19F).These coupling constants are of negative sign,28and their magnitude depends on several factors: l o (I) The magnitude increases with (23) L.Phillips and V. Wray, J. Chem. Soc., B , (1971)1618-1624. (24) V. Wray, J . Chern. Soc., Perkin Trans. 2 , (1976)1598-1605. (25) K.Bock and C. Pedersen, Adv. Carbohydr. Chem. Biochem., 41 (1983)27-66. (26) P. KovAC and C. P. J. Glaudemans, J . Carbohydr. Chem., 2 (1983)313-327. (27) P. J. Card, J . Org. Chern., 48 (1983)393-395. (28) K. Bock and C. Pedersen, Acra Chem. Scand., Ser. B , 29 (1975)682-686.
78
RENE CSUK AND BRIGITTE I. GLANZER
increase in the electronegativity of the atoms attached to the coupled carbon atom. (2) There is dependence on the orientation of the fluorine atom under consideration, including remote effects. Secondary deoxyfluoroglycopyranoses exhibit such couplings of -225 Hz, glycosyl fluorides of - 180 Hz, and 6-deoxy-6-fluoroglycopyranoses of 165 Hz. 2J ( 13C, 19F).The sign of these couplings28was found, or assumed, to be positive and their magnitude depends to a much smaller extent on the electronegativity of the atoms attached to the coupled fragment than does that of 'J ( I3C, IyF).However, they are much more sensitive to the orientation of these electronegative substituents with respect to the fluorine atom.I0 jJ ( 13C, 19F).This coupling shows the same angular dependences as the corresponding fluorine-proton or proton-proton counterpart,I6 thus permitting the development of Karplus-type relationship^^^ ; the sign was assumed to be positive.24 The magnitude depends on the electronegativity of substituents attached directly, or adjacent, to the coupled carbon atom; increasing electronegativity causes a small decrease, whereas a large decrease is observed on change from a trans to a gauche orientation of the coupled nuclei. A large increase in the coupling constant is found if the orientation of an electronegative substituent attached to the middle carbon atom of the coupling pathway changes from trans to gauche relative to the fluorine atom, even though the relative positions of the coupled nuclei are held constant.24 4J( uC, 'V). Although there are only a few examples of this long-range coupling, dependences on orientation may be detected. Observable values could only be obtained for equatorially disposed fluorine atoms (except for those in glycosyl fluoride^).^^
-
3. 19F-N.m.r. Spectroscopy a. 19F-Chemical Shifts.-Because the 19F-nucleus shows spectroscopic behavior similar to that of the IH-nucleus (compare Table I) I9Fn.m.r. spectroscopy was used, even at the very beginning of n.m.r. spectroscopy of carbohydrates. I9F-N.m.r. spectroscopy offers some important benefits: the nucleus is at isotopic abundance, the gyromagnetic ratio is high, and the relevant spectroscopic parameters may be readily extracted from the spectra. Importantly, this nucleus is a probe that is very sensitive to changes in the steric and electronic environment.2yIn this way, lyF-n.m.r.spectroscopy must be regarded as a part of (29) L. Phillips and V . Wray, J . Chem. SOC., Perkin Trans. 2, (1972) 223-228.
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
79
the “structural-reporter-group concept” originally developed for IHn. m. r. spectroscopy of oligosaccharides.30 The main difference between 19F-n.m.r.and ‘H-n.m.r. spectra is exemplified by the tremendously enlarged range of chemical shifts. This range reflects the presence of nonbonding electrons on fluorine, in general giving rise to low energy n-r* transitions, and thus the large paramagnetic contributions to the nuclear shielding tensor will cause nuclear deshielding, whereas the diamagnetic contributions are rather small The extensive investigations of single groups of fluorinated carbohydrates has not only provided fundamental information for establishing the structures of the fluorinated derivatives but has also shown I9F to be a very sensitive probe and, as Inch pointed out,5 as an “amplifier” for showing conformational distortions and mobility. The chemical shifts of fluorine atoms are dependent on their site, and their orientation with respect to the carbohydrate ring.29Because only empirical correlations have been made between chemical shifts and stereochemistry, these rules can only be applied to compounds that are closely related structurally to those which have been utilized to establish these correlations.I6 Shifts of I9F although at least one order of magnitude greater than the corresponding IH-values, are therefore generally only indicative, for example, of the chemical environment of fluorine within the molecule. l6 Primary and secondary fluorides may be clearly differentiated; primary resonate at higher field than secondary fluorides; glycosyl fluorides resonate at still lower field. Tertiary fluorides and secondary geminal difluorides resonate still further to low field. The fluorine shifts of a-aldohexopyranosyl fluorides, having an axially oriented fluorine atom, are at higher field than those of the corresponding, equatorially oriented p counterpart^,^^-^^ which, in turn, were found to display temperature-dependent Chemical shifts and chemical-shift differences between pairs of anomers in the monodeoxymonofluoro-D-hexopyranose series were observed to be strongly dependent on the stereochemistry of electronegative substituents in the molecule. In consequence, no a priori distinction between anomers can be made on the sole basis of their respective fluorine shifts.23 The use of fluorine shifts as a probe for structural and conformational (30) J. F. G . Vliegenthart, L. Dorland, and H. Van Halbeek, Adu. Carbohydr. Chem. Biochem., 41 (1983) 209-374. (31) J. B . Lambert and F. G . Riddel (Eds.), The Muftinuclear Approach to N.m.r. Spectroscopy, Reidel, Dordrecht, 1983. (32) L. D. Hall and J . F. Manville, Chem. Ind. (London), (1965) 991-993. (33) L. D. Hall and J. F. Manville, Can. J . Chem., 45 (1967) 1299-1303. (34) L. D. Hall and J. F. Manville, Can. J . Chem., 47 (1969) 361-377.
80
RENE CSUK AND BRIGITTE I. GLANZER
parameters therefore remains of limited value. Furthermore, data reported in the literature need to be viewed with caution because of strong and non-negligible solvent- and temperature-effects on the chemical shifts .35 b. 19F-19F Coupling.-In contrast to IH-I9F and I3C-I9F couplings, I9F-l9F couplings may only be used with caution for stereochemical assignment. Geminal 2J ( I9F, 19F)couplings ranging from -240 to 300 Hz for sp3-hybridized carbon atoms, and some 30-40 Hz for sp2, have been reported. Analysis of 3J ( 19F, I9F) couplings revealed that the angular dependence is more complex than for the corresponding ‘H-IH or IH-l9F couplings. trans-Vicinal were assumed to be more sensitive than gauchevicinal couplings.36These couplings are absolutely negative, and range36 between 10 and 20 Hz. The values of 4J (I9F, I9F) may be of different sign, with J (e,e) being absolutely negative, and J @ , a )and J (a,a)being positive.I7 Although the magnitude of coupling depends on the relative orientation of substituents, there is no enlarging effect of an “M” relationship of the coupled nuclei.21 The values of 5J (I9F, 19F)are of variable sign and are presumed to occur by way of through-bond interaction; their stereospecific dependences seem to be rather complex.I0 4. Other Nuclei
There is little known in the carbohydrate field about coupling of fluorine with nuclei other than ‘H, I3C, or 19F.Tronchet and coworkers37investigated the 2H-19Fcoupling in (2)-and (E)-3-deoxy-3-C-(fluoromethylene)1,2: 5,6-di-O-isopropylidene-a-~-ribo-hexofuranose deuterated at the sp2hybridized fluoromethylene group. The 2J ( 2H, I9F) coupling is, in comparison with the corresponding IH-I9F coupling, significantly lessened (12.4 Hz us. 82 Hz), thus limiting its use and potential significance.
111. TABLES 1. Guide to Use of the Tables
All fluorine chemical-shifts given in this compilation have been converted to the 6 scale, with trichlorofluoromethane (Freon-I 1) as refer(35) H. Giinther, NMR-Spektroskopie, G. Thieme Verlag, Stuttgart, 1973. (36) L. D. Hall, R. N . Johnson, J. Adamson, and A. B. Foster, Chem. Commun., (1970) 463-464. (37) J. M. J. Tronchet, D. Schwarzenbach, and F. Barbalat-Rey, Carbohydr. Res., 46 (1976) 9-17.
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
8I
TABLEIV Conversion Factors Used Throughout this CompilationB Reference material
Shift (to Freon-11) (in p.p.m.)
Reference material
Shift (to Freon-11) (in p.p.m.)
F2 C~HSSO~F CFCI3 CF2CIi C6HsCF3 CFC12-CFCI2 CF$Z02CHX
+422.9 +65.5
CF3C02H CFICCIX CF3COCF3 CFC(OH)2CFj
-78.5 -82.2 -84.6 -92.7 -113.1 - 120.0 - 162.9
0.0
-6.9 -63.9 -67.3 -74.2
FC6H4F-p C6F6
ence, and with positive shifts to high frequency of the reference, using the conversion factors shown in Table IV. Because some authors omit indication of sign or standards, or change conventions, some errors may be present in the tables. Tables V-LVII detail ‘H and I9F shift and coupling information, and Tables LVIII to LXXI incorporate the 13C-n.m.r.data. The data within this compilation are arranged according to the following outline: hexoses prior to pentoses, followed by anhydro sugars, sugar acids and lactones, amino sugars (and their synthetic, N-containing precursors), mono-, di-, and tri-deoxy sugars, branched derivatives, ketoses, polyfluorinated monosaccharides, and, finally, difluorinated amino sugars. Within this compilation, and even within each table, pyranoid derivatives are listed prior to their furanoid counterparts, hexoses prior to pentoses, functionalized prior to deoxy compounds; the arrangement within each “sub-table” is made alphabetically. The tables contain nearly all of the n.m.r. information currently available in the literature. Although there are more fluorinated monosaccharides known, for many of them no n.m.r. data have been reported so far. Throughout the Tables, “S” stands for “solvent”: A for hexadeuterioacetone, B for hexadeuteriobenzene, C for deuteriochloroform, D for deuterium oxide, F for liquid hydrogen fluoride, H for water, M for tetradeuteriomethanol, N for dichlorodideuteriomethane, and s for hexadeuteriodimethyl sulfoxide.
(38) V. Wray, Annu. Rep. NMR Specfrosc., 10B (1980) 5.
RENE CSUK AND BRIGITTE I. GLANZER
82
TABLEV 'H- and I9F-N.m.r. Data for Hexopyranosyl Fluorides
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
J1.z
Jz,~
53.4
54.5
55.6
55.6
56,6
JF,l
JF,Z
JFJ
JF,4
JFJ
Jv.6
JF.6'
6F
S
References
2,3,4,6-Tetra-0-acetyl-o-allopyranosyl fluoride a anomer 5.55 4.79 3.2 3.2 53.5 26.0 p anomer 5.41 5.07 7.3 2.8 52.7 12.0
5.32 3.0
5.12 10.2
4.00 4.6
5.38 2.8
5.05 9.5
3.96
4.20
- 145.5
17"
4.20
- 146.3
17"
2.3
2,3,4,6-Tetra-0-acetyl-~-gdactopyranosy~ fluoride a anomer 5.82 5.27 5.50 2.8 10.0 b2.3 53.0 21.8 p anomer 5.06 5.55 5.09 7.0 10.3 3.4 52.0 12.6 D-Glucopyranosylfluoride a anomer 5.66 3.0 53.2 27.2 p anomer 5.31 6.66 52.0 12.0 2,3,4,6-tetra-O-acetyla anomer 5.74 4.94 5.48 2.7 10.0 9.5 52.8 23.8 p anomer 5.40 5.12 5.20 6.6 9.0 9.0 52.5 12.0
1.8
4.34 5.1
5.41 2.0
3.69 b4.9
5.55
4.18 7.3
%.0
4.06 10.8
4.15 b11.4
-150.1
17,32 a
-141.9
17,32 u
- 146.2
23
-139.1
23
5.14 9.7
4.18 2.0
4.28 4.2
4.12 12.7
- 149.9
14,15, 17,32,33, 39,40"
5.20 9.4
3.92 3.0
4.30 5.0
4.22 12.8
- 137.8
14,15, 17,32,33, 40"
3.60
- 147.5
14,15, 17
2,3-di-O-acetyl-4,6-O-benzylidenea anomer
5.65 3.0 53.0
4.88 9.8 24.6
5.43 9.4
4.30
(continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
83
TABLEV (continued) Compound H-1 H-2 51.2
JFJ
JF,Z
H-3
H-4
H-5
H-6
H-6‘
J3.4
J4.5
55.6
55.6’
J6,6’
JFS
JF,~
JFS
JF,~
JF,6‘
&F
S
References
p anomer 5.55 6.2 53.4
5.02 5.34 4.5 7.8 8.8 11.6 3,4,6-tri-O-acetyl-2-O-methyla anomer
3.60
-138.1
A
14,15, 17
C
41‘
C
41d
2.5 24.4
p anomer 6.4
2,3,4,6-tetra-O-benzoyla anomer 6.19 2.8 53.2
5.64 6.23 5.99 9.6 9.6 9.5 23.7 p anomer 5.26 5.75 6.23 5.84 5.7 8.6 8.6 8.6 51.5 10.3 3,4,6-tri-O-benzoyl-2-O-methyla anomer 5.84 3.65 5.95 5.63 2.7 9.6 9.6 9.6 . 52.0 24.3 p anomer 5.48 3.62 5.70 5.61 6.0 7.8 8.5 8.5 52.4 10.4 2,4,6-tri-O-benzoyl-3-O-methyl(Y anomer 5.70 5.22 4.00 5.63 2.7 9.5 9.5 9.5 53.4 24.3 p anomer 5.63 5.45 3.90 5.60 4.5 6.0 7.5 7.5 51.2 7.5
4.80 4.5
4.74 2.6
5.58 12.2
-148.6
A
17,32
3.87 5.0
4.62 3.6
4.40 11.9
-136.4
B
17,32
4.53 ‘4.3
4.64 ‘3.7
4.42 ‘12.6
-149.9
C
17,32
4.25 5.0
4.65 3.6
4.45 12.3
-135.2
4.32
4.55
4.32
-148.8
Y
C
17,32 (I
B
17,32 (I
4.35 5.0
4.62 5.0
4.55 12.0
-131.8
C
17,32 (1
(continued)
TABLEV (continued) Compound H-1 H-2 Jis J2.3 JFJ
J F ~
H-3
H-4
€I-5
H-6
H-6‘
53.4
J4,s
554
Js.6,
J6.6‘
JFJ
JFC
JFS
JF,~
JF,~’
2,3-di-O-benzoy1-4,6-di-O-methyla anomer 4.06 3.70 3.76 5.9 5.19 5.91 10.0 10.2 9.0 2.8 53.5 23.7 p anomer 5.58 5.2-5.9 3.6 6.0 53.0 6.0 2,3,4,6-tetra-O-benzyl-a5.55 2.68 53.2 25.4 2,3,4,6-tetra-O-methyla anomer 3.60 5.67 3.20 3.55 3.28 3.78 2.7 9.6 10.2 10.2 2.7 2.7 53.5 25.0 p anomer 5.11 6.5 53.0 D - M Q I I I I O ~ ~ I Ifluoride OS~I 2,3,4,6-tetra-O-acetyla anomer 5.66 5.26 5.34 4.18 1.3 10.0 10.0 f4.5 r3.6 48.5 1.5 p anomer 5.45 5.25 5.04 5.04 3.82 4.38 1.9 2.2 5.7 4.7 5.7 5.6 51.2 13.9 3,4,6-tri-O-acetyl-2-0-methyl-a-
6F
S
References
3.70
C
42
3.9
C
42
-149.0
C
43
-149.9
C
44
-138.7
C
44
-138.2
A
17,32, 40
-142.0
C
28,39, 32
c
41
3.60
f13.0 4.22 11.8
2.1
8
0.8
2,3,4,6-tetra-0-benzoyl-a5.84 5.86 5.90 6.22 4.77 4.61 1.1 9.5 e9.5 ‘2.0 ‘4.9 c48.5 ‘1.0 2,3-di-O-benzoy1-4,6-di-O-methyla anomer 5.71 5.69 5.60 3.6 1.6 49.0 1.6
4.49 ‘12.5
4.15
-138.7
C
17,32
C
42
(continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
85
TABLEV (continued)
p anorner 5.75 5.80 5.60 3.7 1.5 3.4 6.9 51.6 12.9 2-O-benzoyl-4,6-di-O-methyl-a5.67 5.47 4.16 3.6 2.0 3.5 8.7 0.5 49.5 3-0-benzoyl-4,6-di-O-methyl-/35.56 1.8 52.8
4.28 3.6 14.6
5.28 6.4
3.5
-
4.1
C
42
-
3.90
C
42
-
3.90
C
42
Ref. 17 reported chemical shifts and coupling constants for solvents A, B, and C. Coupling constant measured in A. J ( F, OMe) = 1.0 Hz.d J (F,OMe) = 1.4 Hz. Coupling constant measured in B. f Coupling constant measured in C. g J (F, OMe) = 0.55 Hz.
(39) C. J. S. Van Rijn, J. D. M. Herscheid, G. W. M. Visser, and A. Hoekstra, Znt. J. Appl. Radiat. Isot., 36 (1985) 111-115. (40)L. D. Hail and J. F. Manville, Chem. Commun., (1968) 37-38. (41) A. B. Foster, R. Hems, J. H. Westwood, and L. D. Hall, Carbohydr. Res., 23 (1972) 316-3 18. (42) c. Pedersen and S. Refn, Acta Chem. Scand., Ser. B, 32 (1978) 687-689. (43) M. Hayashi, S. Hashimoto, and R. Noyori, C h m . Lett., (1984) 1747-1750. (44) G. L. Trainor, J. Carbohydr. Chem., 4 (1985) 545-563.
TABLEVI lH- and '9F-N.m.r. Data for 2-Deoxy-2-Buoro-hexopyranoses and hexopyranosides
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
JI,Z
I23
53.4
JW
J5.6
JW
JW
JFJ
JF,Z
IF3
J F ~
JFJ
Jp.6
JFC,
2-Deoxy-2-Buoro-~-g~a~opyranose a anomer 4.44 4.0 10.5 3.5 0.5 50.5 13.3 2.8 /3 anomer 5.06 7.3 7.5 3.2 3.0 51.5 14.5 2.8
SF
S
-203.1
D
References
45,46, 47 a
-202.9
D
45,46, 47 b
(continued)
RENfi CSUK AND BRIGITTE I. GLANZER
86
TABLEVI (continued) Compound H-1 H-2 Ji,z Jzs JF.~
JF,Z
€5-3
H-4
H-5
H-6
H-6'
J3.4
543
55.6
Js,6*
16.6'
JF.~
Jp.4
JFJ
JFC
JF,~'
1,3,4,6-tetra-O-acetyla anomer 6.48 4.90 5.40 5.31 4.32 10.0 3.@ 3.5 4.0 49.0 11.0 3.5 p anomer 5.10 4.41 5.10 5.34 8.0 9.6 3.5 0.0 4.0 51.1 13.1 2.1 2-Deoxy-2-fluoro-~~g~a~opyranoside methyl p4.65 4.38 9.0 9.0 3.0 52.0 methyl 3,4-O-isopropylidene-6-O-trityl-p4.3 4.24 3.41 1.4 1.45 2.0 47.6 trifluoromethyl 3,4,6-tri-O-acetyl-a5.15 4.17 5.32 5.46 4.35 1.o 3.1 10.5 3.5 0.5 48.5 11.0 3.5 2-Deoxy-2-fluoro-~-glucopyranose a anomer 5.39 4.36 3.32 3.66 9.5 0.5 48.9 14.5 p anomer 4.85 4.05 3.32 1.8 8.8 50.0 14.5 2.5 1,3,4,6-tetra-O-acetyla anomer 6.46 4.64 5.56 5.08 4.03 4.0 9.6 9.6 9.6 2.3 0.5 48.5 12.0 p anomer 5.64 4.33 5.23 4.90 3.11 8.1 8.8 9.6 9.1 4.1 3.0 51.0 14.5 2-Deoxy-2-fluoro-n-glucopyranoside methyl 4,6-di-O-acetyl-3-O-benzyl-p4.42 4.39 3.49-4.34 5.05 3.49 1.7 1.7 9.1 9.1 1.7 52.0
4.10
4.00
References
6F
S
-201.1
c
48,49, 50
-208.1
C
45
-203.9
D
51
C
52
4.33
4.03
-211.0
C
45,46, 5334
4.08
-194.8
D
12,23,24, 47,55,56, 57,58,d
4.08
-194.6
D
23,24,47, 55,56,57, 58,'
4.00 12.5
4.22 12.5
-202.9
C
39,48,49, 50,56,51
4.30 1.6
3.98 12.4
-201.4
C
39,56,57, 58,59
-
4.34
C
57
4.03
(continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
87
TABLEVI (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
J1.2
J23
J3.4
34.5
Js.6
55.6'
J6.6'
JFJ
JF,~
JFJ
JFA
JF,S
JF,~
JF,~
methyl 3-0-acetyl-4,6-0-benzylidene-p4.55 4.28 5.45 4.39 3.49 7.6 8.8 9.9 4. I 1.5 49.0 methyl 4,6-0-benzylidene-3-O-methyl-p 4.49 4.25 3.40-3.88 4.38 3.40 7.9 7.9 10.2 4.5 2.7 51.2 methyl 3-0-benzyl-4,6-0-benzylidene-p4.49 4.34 3.75 4.38 3.40 7.8 7.8 8.0 4.6 15.0 2.W 51.0 phenyl 3,4,6-tri-O-acetyl-P5.13 4.57 5.42 5.10 3.85 4.14 7.7 8.6 9.1 9.7 5.2 2.7 50.0 14.1 3.2 trifluoromethyl 3,4,6-tri-O-acetyl-a3.9 0.5
9.7
9.4 11.5
6F
S
References
3.89
C
57
3.88
C
57.60
3.94
C
57,61
4.31 12.0
C
62
-195.4
C
58
-200.8
D
23,24,47, 55,56,58, 63,64,8
-219.3
D
23,24,47, 55,5633, 63,64,*
9.4
2-Deoxy-2-fluoro-o-mannopyra~1ose a anomer 5.37 4.77 1.95 2.2 30.0 7.6 49.1 p anomer 4.99 4.81 1.0 2.44 20.5 51.3 32.0 1,3,4,6-tetra-O-acetyla anomer 6.09 4.59 5.02 1.9 2.4 10.2 6.3 48.9 27.2 /3 anomer 5.81 4.87 5.07 10.0 1.0 2.4 19.0 51.2 27.0
5.18 9.8
3.88 4.7
4.18 2.2
3.91 12.6
-201.2
C
39,56, 58,63
5.37 10.0
3.81 4.8
4.30 2.3
4.16 12.5
-220.2
C
39,49, 5036, 58,63
3.4
-
4.34
C
63
1,4,6-tri-O-acetyl-3-O-methyla anomer 6.28 4.80 2.0 2.0 7.0 49.0
3.4-4.34 10.0
5.32 10.0
(continued)
RENC CSUK AND BRIGITTE I. GLANZER
88
TABLEVI (continued) Compound H-1 H-2
H-3
H4
H-5
H-6
H-6’
Jiz
Jw
J3.4
J4.5
15.6
J5,v
J6.6,
JFJ
J F ~
JF3
J17.4
JFJ
JF,~
JFC’
p anomer 5.74 4.93 5.26 1.0 2.0 10.0 10.0 19.0 52.0 Methyl 2-deo~2-Buoro-~-maopyraooside 3,4,6-tri-O-acetyI-p4.52 4.82 4.99 5.36 3.54 1.0 2.5 9.9 9.9 17.6 51.0 27.0
&F
S
References
C
63
C
63
C
63
C
63
C
63
5.54 10.0 28.0
C
61
5.34 10.0 26.0
C
61
C
63
-
4.32
4,6,di-O-acetyl-3-O-rnethyl-/34.44 1.0 18.0
4.90 2.0 52.0
3.2-3.7 10.0
5.24 10.0
3-0-acetyl-4,6-O-benzyIidene-P4.57 4.92 5.07 1.0 2.7 10.3 50.0 25.8 20.0 4,6-O-benzylidene-p4.42 4.78 3.24-4.0 1.0 3.0 10.0 20.0 50.0
4.36
3.2-3.7
4.39 4.9
3.38
4.36 6.0
3.24
-
4.18
4.0
3-0-benzoyl-4,6-0-benzyIidenea anomer
4.94 3.0 8.0
3.0
p anomer 4.62 0.0 19.0
5.02 2.5 51.0
3-0-benzyl-4,6-O-benzylidene-p4.40 1.0 19.0
4.78 3.0 51.0
3.24
-
4.44
Tritluoromethyl3,4,6-~-0~ecetyl-2-deoxy.2-fluoro-~~-mannopyranoeide C
58
D
47
1.0 2.5 9.7 9.0 16.0 25.0 ~-Deoxy.~-flnoro-D-t~opyrnnose a anomer
-203.4 12.2
44.0
34.2 (continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
89
TABLEVI (continued)
p anomer 19.7 51.2 32.9 2-Deoxy-2-fluoro-~-tdopyranoride tritluoromethyl3,4,6-tri-O-acetyl-p5.17 4.67 4.99 5.25 4.23 1.0 3.0 3.5 1 .o 16.5 51.0 29.0
-
3.97
-223.5
D
47
-220.0
c
45
Ref. 47 reported a chemical shift of -208.8 a Ref. 47 reported a chemical shift of -208.9 p.p.m. p.p.m. Ref. 48 reported acoupling of 1.2 Hz. Ref. 47 reported a chemical shift of -200.6 p.p.m. Ref. 47 reported a chemical shift of -200.4 p.p.m.fRef. 61 reported a coupling of 4.0 Hz.g Ref. 47 reported a chemical shift of -206.0 p . p m * Ref. 47 reported a chemical shift of -224.5 p.p.m.
(45) J. Adamson and D. M. Marcus, Carbohydr. Res., 22 (1972)257-264.
(46)J. Adamson and D. M. Marcus, Carbohydr. Res., 13 (1970)314-316. (47) M. Diksic and D. Jolly, J . Carbohydr. Chem., 4 (1985)265-271. (48) M. J. Adam, B. D. Pate, J. R. Nesser, and L. D. Hall, Carbohydr. Res., 124 (1983) 215-224. (49) B. I. Glanzer and K. Dax, Eur. Symp. Carbohydr., 3rd, Grenoble, 1985,B.l-24. (50) B. I. Glanzer, Diplomarbeit, Techn. Univ. Graz, 1985. (51) Y. Ittah and C. P. J. Glaudemans, Carbohydr. Res., 95 (1981) 189-194. (52) T. Haradahira, M. Maeda, Y. Yano, and M. Kojima, Chem. Pharm. Bull., 32 (1984) 3317-3319. (53) P. W.Kent, R. A. Dwek, and N. F. Taylor, Tetrahedron, 27 (1971)3887-3891. (54) R.A. Dwek, P. W. Kent, P. T. Kirby, and A. S. Harrison, Tetrahedron Lett., (1970) 2987-2990. (55) G. T. Bida, N. Satyamurthy, and J. R. Barrio, J. Nucl. Med., 25 (1984)1327-1334. (56) G. K. Mulholland and R. E. Ehrenkaufer, J. Org. Chern., 51 (1986) 1482-1489. (57) T. Haradahira, M. Maeda, Y. Kai, H. Omae, and M. Kojima, Chem. Pharm. Bull., 33 (1985)165-172. (58) J. Adamson, A. B. Foster, L. D. Hall, R. N. Johnson, and R. H. Hesse, Carbohydr. Res., 15 (1970)351-359. (59) S. R. Thomas, J. L. Ackerman, J. R. Goebel, M. Davis, J. G. Kereiakes, and Y. Y. Lin, Magn. Reson. Imaging, l(1982) 11-21. (60)S. Levy, E. Livni, D. Elmaleh, and W. Curatolo, J. Chem. Soc., Chem. Commun., (1982)972-973. (61) A. Olesker, A. Dessinges, T. T. Thang, and G. Lukacs, C. R . Acad. Sci. Ser. 2 (Paris), 295 (1982)575-577. (62) J. PacBk, Z.KollnerovB, and M. Cernf, Collect. Czech. Chem. Commun., 44 (1979) 933-941. (63) T.Haradahira, M. Maeda, H. Omae, Y. Yano, and M. Kojima, Chern. Pharm. Bull., 32 (1984)4758-4766. (64)T. Ogawa and Y. Takahashi, J. Carbohydr. Chem., 2 (1983)461-467.
RENI? CSUK AND BRIGITTE I. GLANZER
90
TABLEVII 'H- and '9F-N.m.r. Data for 3-Deoxy-3-fluoro-hexopyr~noses and -hexopyranosides Compound H-1 H-2
H-3
Jw
52.3
J3-4
JF.1
JFZ
JFS
H-4 JF,~
H-5 H-6
H-6'
Js.6
J5,6*
JFJ
J F , ~ JF,~'
References
&F
S
-217.2
A
65
-217.4
C
65
-217.4
A
65
-217.4
A
65,66
-204.3
C
67,68
-200.7
C
67,68
C
69
C
69
D
12,23, 53,70, 71
J6,6'
Methyl 3-deoxy-3-fluoro-~-~opyranoside p anomer 4.48 3.40 4.86 8.0 2.0 2.0 29.0 53.0 4,6-O-isopropylidene-p4.53 3.65 4.97 3.53 8.0 28.0 54.0 27.0 6-O-pivaloyl-p3.42 4.92 3.67 3.86 4.21 4.44 8.0 2.0 6.0 2.0 11.0 30.0 53.0 28.0 6-0-trit yl-/34.62 3.55 4.94 3.80 3.87 3.33 3.50 8.0 1.5 8.0 5.4 2.2 10.0 1.5 53.0 28.0 ~-Deoxy-~-fluoro-~-gPactopyranose 1,2,4,6-tetra-O-acety1a anomer 6.41 5.40 4.86 5.69 4.4 3.95 3.6 10.0 3.6 1.0 4.8 11.4 47.5 6.4 p anomer 5.64 5.38 4.70 5.59 4.24 3.92 8.1 9.5 3.8 0.5 11.5 47.4 5.6 3-Deoxy-3-fluoro-~-gPactopyranos~de methyl 2,4-di-O-benzoyl-6-O-(bromoacetyl)a anomer 5.26 5.54 5.22 5.91 4.25 4.43 4.0 10.0 4.0 1.0 4.0 10.0 48.5 5.5 p anomer 4.60 5.67 4.93 5.87 4.07 4.36 4.36 8.0 10.0 4.0 1.0 6.0 6.0 12.0 47.0 5.0 ~-~eoxy-~-fluoro-~-~ucopyranose a anomer 5.35 3.6 3.9 12.0 53.0 13.6
-195.4
(continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
91
TABLEVII (continued) ~
Compoond H-1 H-2
H-3
H-4
H-5
H-6
H-6'
J1.1
JZJ
J3.4
J~J
Js.6
J5.6,
56.6,
JFJ
JF,Z
JFJ
JFA
JFJ
J F , ~ JF.~'
@ anomer 4.75 7.5 0.5 13.0 52.0 6-phosphate a anorner
3.0
12.0
52.0
SF
-190.5
S
References
D
12,23, 53,70, 71
D
66
D
66
13.0
12.0
p anomer 0.5 13.0 51.0 13.0 1,2,4,6-tetra-O-acetyla anomer 6.31 4.75 4.3 3.2 9.4 9.4 4.0 12.5 52.0 12.5 @ anomer 5.65 5.23 4.60 5.25 3.75 4.29 8.3 8.9 9.2 4.3 2.5 10.0 51.3 12.8 12.8 1.1 1.5 3-Deoxy-3-fluoro-~-~ocopyr~nocl~de benzyl p-
3.9
-200.4
C
4.09 12.5
-196.2
C
20,53
-194.0
D
53
-194.0
C
53
-198.6
C
20
-197.0
D
53,71
-189.3
D
72
-190.8
D
72
20
benzyl 2,4 ,btri-O-acetyl-P15.0 57.5 15.0 methyl 2-O-acetyl-4,6-O-benzylidene-a4.07 5.39 4.53 3.42 2.99 4.08 3.41 7.5 9.3 9.0 10.0 4.7 9.4 10.6 53.0 15.1 1.5 2.0 14.1 Methyl 3-deoxy-3-8uoro-a-~-gulopyranoside
3-Deoxy-3-fluoro-~-idopyranose a anomer 3.0
14.0
51.0
14.0
p anomer 3.0
9.0
46.0
9.0
3.0 (continued)
RENk CSUK AND BRIGITTE I. GLANZER
92
TABLEVII (continued) Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
JIJ
J23
J3,4
J4.s
Js,6
Js.6,
J6,6'
JF,I
JF,Z
Jp.3
JF,~
JF,S
JF,~
Jp.6'
~Deoxy-3-fluoro.~-mannopyranose (I anomer 5.23 4.19 4.75 3.62 1.9 3.5 9.4 5.1 7.1 48.6 /3 anomer 4.91 4.19 4.59 3.62 1.1 3.5 9.4 10.0 5.9 2.4 2.1 7.4 48.0 1.2 1,2,4,6-tetra-O-acetyI-a6.14 5.36 4.91 5.46 3.97 4.28 2.0 3.8 9.6 10.1 5.0 2.5 4.7 48.0 11.4 5.8 1.0 0.0
SF
S
References
4.03
D
73
4.03
D
73
4.13 12.5 1.5
C
73
P. J. Card and G. S. Reddy, J . Org. Chem., 48 (1983)4734-4743. D. G.Drueckhammer and C. H. Wong, J . Org. Chem., 50 (1985)5912-5913. J. S.Brimacombe, A. B. Foster, R. Hems, J. H. Westwood, and L. D. Hall, Can. J . Chem., 48 (1970)3946-3952. J. S. Brimacombe, A. B. Foster, R. Hems, and L. D. Hall, Curbohydr. Res., 8 (1968) 249-250. P. KovBe and C. P. J. Glaudemans, J . Curbohydr. Chem., 4 (1985)613-626. N. F. Taylor, B. Hunt, P. W. Kent, and R. A. Dwek, Carbohydr. Res., 22 (1972) 467-469. P. W. Kent, R. A. Dwek, and N. F. Taylor, Biochem. J . , 121 (1971)1OP-llp. A. B. Foster, R. Hems, J. H. Westwood, and J. S. Brimacombe, Curbohydr. Res., 25 (1972)217-227. M . Cernf, J. DoleMovB, J. MBcovB, J. PacBk, T. Trnka, and M. BudeSlnskf, Collect. Czech. Chem. Commun., 48 (1983)2693-2700. TABLEVIII IH- and W-N.m.r. Data for 4-Deoxy-4-fluoro-hexopyranoses and -hexopyranosides
4-DeoKy.Qfluoro-D-gnladopyranose
1,2,3,6-tetra-O-acetyI-p-
-217.4 8.0 0.8
10.0
2.5 26.5
C
19
1.0 25.0
1.0
1.0 (continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
93
TABLEVIII (continued) ~
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
JIJ
JZJ
J3.4
J4.5
55.6
55.6'
J6.6'
JFJ
JF,Z
JFJ
JFI
JFJ
JP,~
J F , ~
Methyl 4-deoxy-4-8uoro-o-gelPctopyranoside 2,3,6-tri-O-acetyl-a5.01 5.20 5.28 4.92 4.06 4.23 3.50 10.5 2.5 2.5 6.5 7.0 3.7 25.5 51.0 29.0 3.7 2,3,6-tri-O-benzoyl-p8.0 0.5
9.8
2.8 25.0
1.2 50.0
4.31 11.5
SF
S
References
C
49,50 65,74
B
75
C
51
D
76
-219.9
A
65,77
-194.3
D
12,23,78
-195.3
D
12,23
C
12,79
A-M
65,80
-219.7
25.0
6-0-benzoy1-2,3-di-O-benzyl-/3-217.3 27.0 2,3-di-O-benzyl-p4.30 3.69 3.49
51.0
3.44
3.86
3.74 12.0
3.81 4.85 3.91 2.50 6.5 29.0 51.0 30.0 ~Deoxy48uoro-o-~uropyraoose a anomer 5.56 3.5 3.5 15.0 49.0 4.5 p anomer 5.01 7.8 0.0 16.0 49.5 4.5 1,2,3,6-tetra-O-acetyI-p-
3.28 6.5
3.34 9.0
6-0-trityl-a4.41 3.68 3.50"
8.1
4.73
27.0
3.0
2.0
3.0
2.0
9.5
8.8 10.0 2.5 4.4 12.4 14.5 49.5 2.6 1.6 1.5 Methyl 4-deoxy-4-8uoro-~-~ucopyr~oside a anomer 4.47 3.64 4.0 3.15 3.15 3.5 9.0 9.0 9.0 3.5 16.5 50.0 2,3,6-tri-O-acetyl-a4.65 3.42 3.7 5.70 4.10-4.60 9.5 9.5 3.8
-198.0
B
80
(conrinued)
RENfi CSUK AND BRIGITTE I. GLANZER
94
TABLEVIII (continued) Compound
S
H-1
H-2
H-3
H-4
H-5
H-6
H-6’
J1.z
JZJ
53.4
J4s
Js,6
Js.6,
J6.6’
JFJ
JF,Z
JFS
JF,~
JFJ
Jp.6
J17.6’
4.33
4.67
4.67 12.8
-197.6
C
27
3.89 4.34 4.29 3.21 3.65 4.81 4.7 9.9 2.7 8.4 3.6 9.5 15.8 50.2 4.6 1.9 3.3 0.7 2,3-di-O-rnethyl-a4.76 4.30 3.8 8.8 8.8 3.8 15.0 49.5 Methyl 4-deoxy-4-8uoro-~-tdopyrnnoside Q anomer 4.74 4.77
4.23 12.0 1.3
-196.1
C
12,78
2,3,6-tri-O-benzoyl-a5.22 5.18 6.12 4.77 3.5 9.0 9.1 10.0 14.7 51.3
6F
References
6-O-acetyl-2,3-di-O-methyl-ab
C
78
-217.1
A
65
-215.0
C
65
49.0 6-O-trityl-a4.79
4.85 49.0
Ref. 77 reported a coupling of 5.34 Hz. J (F,OMe) = 0.8 Hz.
(74) D. M. Marcus and J. H. Westwood, Curbohydr. Res., 17 (1971) 269-274. (75) A. Maradufu and A. S . Perlin, Curbohydr. Res., 32 (1974) 261-277. (76) T. C. Wong, V. Rutar, J. S. Wang, M. S. Feather, and P. Kov612,J . Org. Chem., 49 (1984) 4358-4363. (77) C. W. Somawardhana and E. G. Brunngraber, Curbohydr. Res., 121 (1983) 51-60. (78) A. B. Foster, R. Hems, and J. H. Westwood, Curbohydr. Res., 15 (1970) 41-49. (79) A. D. Barford, A. B. Foster, J. H. Westwood, and L. D. Hall, Curbohydr. Res., 11 (1%9) 287-288. (80) D. P. Lopes and N. F. Taylor, Curbohydr. Res., 73 (1979) 125-134.
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
95
TABLEIX 'H- and WF-N.m.r. Data for bDeoxy-6-fluoro-hexopyranoses and -hexopyranosides Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
J1J
JZJ
J3.4
J4.5
Js,6
-fs,a.
16.6,
JFJ
JFJ
JFS
Jp.4
JFJ
JF,6
JF.6'
S
References
-229.0 D
81
--229.2 D
81
-230.6 C
81
SF
6-Deoxy-6-Buoro-~-galPctopyranose a! anomer
15.8
46.7
p anomer 18.5
46.6
46.6
I ,2,3,4-tetra-O-acetyl-a-
11.8 48.0 48.0 1,2:3,4-di-O-isopropylidene-a5.55 4.35 4.63 4.27 4.08 4.54 4.58 5.0 2.50 7.90 2.00 6.90 5.2 9.5 1.O 1 .o 13.5 46.2 47.1 Methyl 6-deoxy-6-fluoro-~-gala~opyranoside a! anomer 16.9
46.4
-229.5 C
4431,82
-230.0 D
53,71,81
46.4
2,3,4-tri-O-acetyl-a!-229.0 C 6-Deoxy-6-fluoro-~-glucopyranose a anomer 5.26 3.55 3.75 3.51 3.98 3.8 9.8 8.8 9.8 3.6 0.7 0.0 28.6 p anomer 4.69 3.27 3.50 3.53 3.64 7.8 9.2 2.4 26.0 1,2,3,4-tetra-O-acetyIa! anomer 5.06 5.49 5.13 4.10 6.33 3.8 10.2 9.4 10.2 23.1 p anomer 5.74 5.0 5.38 3.84 7.5 9.6 2.3 21.9
53
4.75 4.66 -231.4 D 1.9 10.8 46.7 48.0
12,23,81,83
4.71 4.70 -230.6 D 3.4 10.6 47.4 47.5
12,23,83
4.44 46.9
4.44 -233.7 C
84
46.9
4.49 4.43 4.2 10.7 47.0 46.8
-233.5 C
84,85,86
(continued)
TABLEIX (continued) Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
115
JZJ
J3,4
J4.s
Js,6
Js,6*
166'
JF,~
JF,Z
JFJ
JF,~
J F ~
JFC
JFC.
References
6F
S
-234.5
C
27,7Ia
-233.1
A
65,1Ib
-234.5
C
44
-233.5
A
65
6-Deoxy-6-tluoro-~-gncopyranoside methyl a anomer
26.2
48.0
48.0
5.0 24.4
4.61 2.0 48.0
4.68 10.0 48.0
/3 anomer
4.23 8.0 2,3,4-tri-O-benzyl-a4.4-4.7 3.55 4.01 9.3
3.55 10.0
3.13
4.4-4.7
28.5
41.5
3.86 5.0 24.0
2.0 48.0
48.0
3.83 4.5 21.0
4.52 2.0 48.0
4.68 10.0 48.0
-236.6
A
65
3.74 4.63 5.0 2.0 24.0 48.0 Methyl 6-deoxy-6-~uoro-~-maopyraaoside a anomer
4.70 10.0 48.0
-233.0
A
65
-232.1
A
65
p-nitrophenyl p5.21 8,O phenyl a anomer 5.51 3.58 3.5 10.0
10.0
3.41 9.0
p anomer 5.03 8.0
3.90 10.0
10.0
25.5
49.0
49.0
4.61 2.0 48.0
4.68 10.0 48.0
-235.0
C
65
4.0 3.12
4.66
4.66
-233.6
C
65
2,3-O-isopropylidene-a4.87 2,3-di-O-methyl-a4.84 3.63 1.5
3.84
47.5
22.0
48.0
48.0
a Ref. 77 reported a chemical shift of -219.4 p.p.m. * Ref. 77 reported a chemical shift of -193.5 p.p.m.
(81) L. Evelyn and L. D. Hall,Curbohydr. Res., 47 (1976) 285-297. (82) S. Colonna, A. Re, G. Gelbard, and E. Cesarotti, J . Chem. Soc., Perkin Trans. 1 , (1979) 2248-2252.
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
97
A. De Bruyn, M. Anteunis, G. Aerts, and C. K. De Bruyne, Acta Cienc. Ind., 4 (1978) 103-107. E. M. Bessell, A. B. Foster, J. H. Westwood, L. D. Hall, and R. N. Johnson, Carbohydr. Res., 19 (1971) 39-48. M. Sharma and W . Korytnyk, Tetrahedron Lett., (1977) 573-576. M. G. Ambrose and R. W. Binkley, J . Org. Chem., 48 (1983) 674-677.
TABLEX IH- and '9F-N.m.r. Data for Hexofuranosyl Fluorides
D-Gdactofuranosyl fluoride 2,3,5,6-tetra-O-acetyla anomer 5.6 3.3 65.0 19.0 6.5 @ anomer 5.74 5.23 5.08 4.49 0.0 1.4 4.5 4.5 58.0 7.3 1.9 2,3,5,6-tetra-O-benzoyl-P6.04 5.67 5.75 4.96 4.0 5.0 59.0 6.5 1.5 D-GIUCO~IU~OSY~ fluoride 2,3,5,6-tetra-O-acetyla anomer 5.99 5.64 5.56 4.60 3.9 3.0 5.2 8.3 1.O 60.8 14.4 p anomer 5.70 5.17 5.47 4.65 0.0 0.0 5.1 9.4 60.3 4.4 5.7 2,3,5,6-tetra-O-benzoyla anomer 5.15 6.22 5.54 6.22 3.7 5.0 6.0 8.6 1.O 60.8 16.5 p anomer 5.99 5.64 6.03 5.19 0.0 0.0 5.2 9.0 60.0 4.4 5.2
4.20 12.0
-122.6
C
87
-126.4
C
87
5.42 4.6
4.35 6.8
6.14 4.5
4.7-4.85 7.0
-123.5
C
87
5.28 2.5
4.56 5.5
4.13 12.4
-138.2
C
88
5.33 2.4
4.63 4.6
4.20 12.5
-119.1
C
88
5.84 2.6
4.97 5.1
4.67 12.3
-136.0
C
88
5.88 2.5
5.00 4.6
4.78 12.3
-118.8
C
88
(continued)
98
RENfi CSUK AND BRIGITTE I. GLANZER TABLEX (continued)
D - M P ~ ~ o ~ fluoride o s ~ I 2,3:5,6-di-O-acetoxonium6.12 6.15 6.34 5.96 5.08 5.20-5.50 0.0 6.5 3.0 6.0 8.5 8.0 56.0 5.0 6.0 2,3,5,6-tetra-O-acetyla anomer 4.15 5.81 5.43 5.66 4.60 5.33 4.60 2.5 5.6 12.5 8.5 2.0 5.2 4.8 64.0 14.0 2.0 p anomer 5.46 4.62 5.80 5.07 5.70 4.47 4.21 2.6 4.5 3.8 5.8 5.1 9.8 12.5 65.8 22.4 7.6 2,3:5,6-di-O-benzoxonium5.4 5.90 6.52 6.50 6.77 6.50 0.0 6.5 3.0 6.0 8.5 55.0 5.0 2,3,5,6-tetra-O-benzoyl-a6.05 5.85 6.19 5.12 5.96 5.04 4.70 0.8 5.4 6.0 8.2 2.5 5.1 12.5 59.9 9.0 2.4 2,3:5,6-di-O-isopropylidenea anomer 4.40 4.11 4.06 5.69 4.77 4.85 4.16 0.0 5.9 3.6 7.7 8.8 6.1 4.5 59.5 6.1 p anomer 5.51 4.71 4.84 4.19 4.47 4.09 3.7 6.2 5.4 7.9 5.5 5.5 66.5 15.3 5.4 2,3,5,6-TebP-0-benzoyl-~~-talofuranosyl fluoride 6.00 5.70 4.99 6.00 4.83 4.73 0.0 3.6 6.0 4.0 6.0 12.0 61.0 2.3 4.5 7.5
F
88
-122.8
C
88
-127.3
C
88
F
88
-121.6
C
88
-130.0
C
89,89a
-126.0
C
89,89a
-114.7
C
87
(87) K. Bock, C. Pedersen, and L. Wiebe, Acta Chem. Scand., 27 (1973) 3586-3590. (88) K. Bock and C. Pedersen, Acta Chem. Scand., 26 (1972) 2360-2366. (89) G. H. Posner and S . R. Haines, Tetrahedron Left., (1985) 5-8. (89a) R. Csuk and A. Vasella, unpublished results.
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
99
TABLEXI 'H- and "F-N.m.r. Data for 2-Deoxy-2-fluoro- and 3-Deo~y-3-fluoro-hexofuranoses Compound H-1 H-2 Jls J2.3
H-3
H-4
H-5
H-6
H-6'
J3.4
54.5
35,6
55.6'
J6.6'
JFJ
JFS
JF,~
JF.5
JF,6
JF.6,
JF,Z
6F
S
References
l-O-Acetyl-3-O-benzyI-2-deoxy-2-8uoro-5,6-O-isopropy~dene-~~-~uco~anose 6.33 5.10 4.28 4.35-4.43 3.97 4.09 -202.8 C 49,50 5.1 5.8 8.6 3.8 2.6 4.5 6.6 51.0 13.0 3-Deoxy-3-fluoro-1,2:5,6~di-0-i90propropy~dene-~~-g~a~ofuranose C 67 4.06- 3.80 -187.9 5.90 4.72 4.82 4.08 4.34 8.0 6.4 0.5 3.3 6.9 6.2 3.9 15.0 51.6 23.7 3-Deoxy-3-fluoro-D-g~ucofurPnose 1,2-O-isopropylidene-a-, 5,dcarbonate .. 4.50 4.50 -207.1 C 20 4.96 6.00 4.72 5.08 4.41 7.5 6.6 3.9 0.7 2.5 5.1 10.8 49.9 29.2 1,2:5,6-di-O-isopropylidene-a4.03 4.12 -208.0 C 20,44, 5.95 4.70 5.01 4.11 4.29 6.1 8.8 5033, 4.8 0.8 2.2 8.3 3.7 66,82,90 10.5 49.0 28.0 1,2-O-isopropylidene-a- 3.60 -209.4 C 20 4.00 5.08 4.11 5.92 4.66 2.0 8.0 3.3 0.5 50.0 28.8 10.5 5,6-phenylboronate-a4.40 -207.8 C 20 4.00 5.84 4.67 5.07 4.21 3.6 0.5 2.0 7.2 10.5 50.0 28.9 3-Deoxy-3-fluoro-~-idofuranose a anomer -199.9 D 72 ~~
(I
20.0
52.0
20.0
p anomer -197.0 28.0b a
50.0
D
14.0b
J (H-I, H-3) = 0.7 Hz. Assignments may have to be reversed.
(90) T. J. Tewson and M. J. Welch, J. Org. Chem., 43 (1978) 1090-1092.
72
RENB CSUK AND BRIGITTE
100
I. GLANZER
TABLEXI1 'H- and I9F-N.m.r. Data for 5-Deoxy-5-fluoro- and 6-Deoxy-6-fluoro-hexofuranosesand -hexofurnnosides Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
J1.2
Jz.3
53.4
54.1
55.6
J5.6
56.6'
JFJ
JF,Z
JFJ
JF,~
JF,S
JF,~
JF,~,
SF
~-~eoxy-5-fluoro-1,2-O-~sopropylidene-~~-glucof~anose 5.84 4.47 4.17 4.17 4.74 3.86 3.65 3.5 2.8 7.9 2.4 5.6 13.1 1.6 47.0 30.0 30.0 5-Deoxy-5-fluoro-1,2-O-isopropylidene-~-~-idof~anose 5.92 4.48 4.20 4.27 4.70 3.82 3.77 3.8 3.5 7.9 3.6 4.6 12.6 3.5 14.0 50.0 23.2 19.0 6-Deoxy-6-fluoro-~-glucofuranose 1,2-O-isopropylidene-a5.95 4.54 4.37 4.10 4.18 4.57 4.67 -233.6 4.0 2.5 8.0 5.0 3.0 10.0 25.0 48.0 48.0 1,2-O-isopropylidene-5-O-benzyl-a-234.0 I ,2:3,5-di-O-methylidene-a-226.6
S
References
C-D
91
C-D
91
A-C
65
92 C
81.93
-213.7
C
81
-229.2
c
81
32.8 48.1 48.1 3,5-O-benzylidene1,2-O-isopropylidene-a36.5 I ,2:3,5-di-O-isopropylidene-ar22.2
47.5
48.0
47.5
48.0
(91) R.Albert, K.Dax,S . Seidl,H.Sterk,and A. E. Stiitz,J . Carbohydr. Chem., 4(1985) 5 13-520. (92) L. D. Hall and P. R. Steiner,Can. J . Chem., 48 (1970)451-458. (93) H.C. Srivastava and V. K. Srivastava,Carbohydr. Res, 60 (1978)210-218.
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
TABLEXI11 'H- and 1 9 F - N . m . r . Data for Pentopyranosyl Fluorides Compound
H-1
H-2
H-3
H-4
H-5
H-5'
J1.2
J2.3
J3,4
J43
54,s'
Js,sf
JFJ
JFJ
JFJ
JFC
JF,~
JF,s,
D-Arabinopyranosylfluoride 3,4-0-acetoxonium-2-O-methyl(Y anomer 5.86 5.9-6.0 4.5-4.6 3.2 55.0 p anomer 4.5-4.6 5.94 5.9-6.0 3.5 52.0 2,3,4-tri-Oacetyl(Y anomer 5.25.35 3.75 4.11 8.0 11.4 2.5 5.3 49.2 5.3 p anomer 5.70 5.30 5.30 5.40 3.69 3.49 2.7 10.4 2.0 1.9 13.1 54.0 25.0 3,4-di-O-acetyl-2-O-methyl-/34.10 3.83 5.79 3.69 5.26 5.38 1.2 1.8 12.8 2.6 10.4 3.5 51.5 24.3 0.0 3,4-0-benzoxonium-2-O-methyla anomer 5.90 6.1-6.25 4.65-4.8 3.0 52.0 p anomer 6.02 6.1-6.25 4.65-4.8 3.5 52.5 2,3,4-tri-O-benzoyl-a5.90 5.71 3.86 4.30 5.53 5.79 3.0 4.5 3.0 4.5 8.3 11.6 49.5 4.3 3,4-di-O-benzoyl-2-0-methyla anomer 4.01 4.37 5.69 5.57 5.55 3.77 7.9 11.3 5.1 3.5 4.3 2.8 49.5 5.7 0.0 ~
6F
S
References
F
94
F
94
-138.4
C
95
-152.8
B
14,95 (I
C
94 b
-137.7
F
94
F
94
B
32,95
C-A
94
(continued)
101
RENE CSUK AND BRIGITTE I. GLANZER
I02
TABLEXI11 (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-5'
J1.2
J2.3
J3,4
J4.5
J4.r
Js,s
JF,~
JF,I
JF,~
J17.4
JFJ
JFJ'
p anomer 6.02 4.10 5.62 5.76 4.34 3.5 2.6 10.2 1.5 2.0 53.0 24.0 0.0 3(or 4)-O-benzoyl-2-O-methyl-p5.66 3.50 4.00 4.30 4.80 10.0 3.5 2.0 2.0 3.5 53.0 25.0 D-Lyxopyranosyl fluoride 2,3,4-tri-O-acetyl-a5.46 5.34.02 2.0 5.0 9.5 49.1 2.0 2,3,4-tri-O-benzoyl-a5.84.35 5.78 1.8 5.3 9.5 48.6 2.0 D-Ribopyranosyl fluoride 2,3,4-tri-O-acetyla! anomer ~
52.1
6F
S
References
4.09 13.2
C-A
94
4.80
C-B
94
3.73
95
-141.1
11.0
4.04 10.0
- 140.7
32,95 n
- 147.7
C
32,95
- 140.4
A
14,15, 32,95
26.3
p anomer 5.60 5.15 5.30 2.6 3.8 2.0 d5.0 49.5 2,3,4-tri-O-benzoyla anomer 5.88 5.35 6.21 3.5 3.1 2.9 53.5 25.3 p anomer 6.05 5.8 5.86 2.0 1.6 4.2 48.0 3.0 D-XylOpymnOSyl fluoride 2,3,4-tri-O-acetyla! anomer 5.67 4.89 5.49 10.0 10.0 2.8 53.5 23.7
5.20 2.0
4.21 3.0
3.92 13.0
(I
5.48 5.5
5.80 2.3
4.05 10.6
4.42 10.9
- 148.1
C
32,95 96
4.56 2.3
4.31 13.4
-139.9
A
32,95 96 a
5.04 6.0
3.97 10.6
3.75 10.6
- 152.1
C
14,15, 95 (1
(continued)
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
103
TABLEXI11 (continued) Compound H-1 H-2
SF
S
References
4.24 12.5
-136.6
A
95,97
4.58 10.8
4.08 10.8
-151.6
C
32,95
4.20 3.5
4.60 13.1
-137.3
C
32,95
H-3
H-4
H-5
H-5’
J1.2
52.3
53.4
54,s
54.5‘
Js,s
JFJ
JF,Z
JFJ
JF.~
JFS
JFS’
4.90 3.1
3.73 4.3
5.50 6.0
5.30 2.5
p anomer 5.46 4.90 5.06 3.5 5.7 5.7 52.1 7.9 2,3,4-tri-O-benzoyla anomer 5.95 5.37 6.18 10.1 2.8 10.2 53.1 23.5 p anomer 5.75 5.30 5.66 2.5 3.5 3.5 49.0 6.0
0
3,4-di-0-benzoyl-2-0-methyla anomer 5.83 2.8 52.2
3.62 9.8 24.5 p anomer 5.59 3.57 3.0 5.1 9.0 51.3
5.95 9.8
5.36 6.0 1.o
4.19 11.0
3.96 11.3
C
94
5.60 5.3
5.28 3.1
4.47 4.2
3.92 12.8
C
94
Values given for other solvents, too. J (H-I, H-5) = 0.5 Hz. J (H-3, H-5a) = 1.3 Hz; J (H-3, H-5e) = 0.9 Hz.* Refs. 14 and 15 reportedJ (H-2, F-I) = 7. I Hz in C, whereas Refs. 32 and 95 reported 3 Hz. J (H-3, H-5’) = 0.7 Hz. L.
(94) S. Jacobsen, S. R. Jensen, and C. Pedersen, Acta Chem. Scand., 26 (1972) 15611568. (95) L. D. Hall and J. F. Manville, Can. J . Chem., 47 (1969) 19-30. (96) B. Coxon, Tetrahedron, 22 (1966) 2281-2302. (97) L. D. Hall and J. F. Manville, Carbohydr. Res., 4 (1967) 512-513.
RENfi CSUK AND BRIGITTE I. GLANZER
104
TABLEXIV *Hand '9F-N.m.r. Data for 2-Deoxy-2-fluoropento-pyranoses and pyranosides ~~~
Compound H-1 H-2
H-3
H-4
H-5
H-5'
J1.2
JZJ
J3,4
545
54,s'
Js,r
JPJ
Jr.2
JFJ
Jr.4
Jr.5
JPJ'
6F
S
References
C
48,49, 50
C
53,98
C
4930
C
539
1,3,4-Tri-0-acetyl-2-deoxy-2-fluoro-~~-arab~nopyranose
6.44 4.0
4.92 5.38-5.46 3.82 4.05 -207.7 10.0 14.0 10.8 3.5 1.5 48.0 Trifluoromethyl3,4-di-O-acetyl-P-~-arnbinopyranoside 5.80 4.90 5.23-5.53 4.13 3.82 -208.0 3.5 9.0 1.5 1.5 13.5 8.0 1.5 "6.0 46.0
1,3,4-Tri-0-acetyl-2-deoxy-2-fluoro-~~-1y~opyranose 5.90 2.4 12.0
4.83 2.4 48.0
5.18
7.2 17.6
4.98-5.09 4.0
4.10 6.0
3.49 12.8
-201.0
Trifluoromethyl3,4-di-O-acetyl-~-~-lyxopyranoside -214.0 3.8 3.8 3.8 8.5 3.0 44.2 2-Deoxy-2-fluoro-~-ribopyranose
2.8
13.1
b
%P2.3
4.1
D
100
7.4
1,3,4-tri-O-acetyla anomer 6.06 4.12 5.48 2.1 2.1 3.5 8.4 46.9 11.7 p anomer 6.23 4.56 5.40 3.2 3.2 6.5 48.0 25.2
5.10 3.5
3.13 6.9
4.06 12.1
-202.9
C
49,50, 101
5.19 3.2
4.03 4.8
3.93 12.9
-202.4
C
101
4.26 3.0
4.58 13.0
A
102
C
49SO
1,3,4-Tri-O-benzoyl-P-~-ribose 6.66 3.0 6.5
5.21 3.0 50.0
6.0 3.0 28.0
5.75 2.0
2-Deoxy-2-fluoro-~-xylopyranose
I ,3,4-tri-O-acetyl-a4.58 4.94d 6.30 4.3 9.8 5.5 48.0 10.3
5.49d 6.3
4.01 11.5
3.80
-195.9
11.1 (continued)
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
I05
TABLEXIV (continued) Compound H-1 H-2
H-3
H-4
H-5
H-5‘
JI,Z
12.3
53.4
54,s
J4,5*
J5.5’
JFJ
JF,Z
Jp.3
JF,~
JF,~
JFJ’
SF
S
References
-214.0
C
53,99
Trifluoromethyl 3,4-di-0-acetyl-cu-~-xylopyranoside 3.8 0.5
9.8 48.0
9.5 1.0
9.5
6.5
10.0
Ref. 98 reported a coupling of 0.0 Hz. Ref. 53 reported J (F, H-1) = 6 and J (F, H-3) = 0.0 Hz. Values also given for other solvents. ,IThe assignments may have to be reversed.
(98) (99) (100) (101)
E. L. Albano, R. L. Tolman, and R. K. Robins, Carbohydr. Res., 19 (1971) 63-70. C. G. Butchard and P. W. Kent, Tetrahedron, 27 (1971) 3457-3463. W. K. Olson, J. Am. Chem. Soc., 104 (1982) 278-286. J. T. Welch and S. Eswarakrishnan, J . Chem. Soc., Chem. Commun., (1985)
186- 188. (102) R. J . Cushley, J . F. Codington, and J. J. Fox, Carbohydr. Res., 5 (1967) 31-35.
TABLEXV IH- and 19F-N.m.r.Data for 3-Deoxy-3-fluoro- and 4-Deoxy-4-fluoro-pentopyranosesand -pentopyranosides Compound H-1 H-2
H-3
H-4
H-5
H-5’
51.2
JZJ
J3.4
54,s
J4.5
J5.s
JFJ
JFJ
JF,~
JF,~
JF,S
JFJ’
6F
S
-202.0
H
53
-200.0
H
53
References
3-Deoxy-3-Buoroxylopyraoose b
13.6 57.0 1,2,4-tri-O-acetyl-~a anomer 6.26 4.0 4.0 p anomer 5.71 5.14 4.57 6.5 7.9 7.9 12.4 49.4
12.6
5.07 8.0 12.4
3.48 5.0
4.18 12.2 4.2
-195.3
C
11
C
11,20
(continued)
RENE CSUK AND BRIGITTE I. GLANZER
106
TABLEXV (continued) Compound H-1 H-2
H-3
H-4
H-5
H-5’
JI,Z
J2,3
53.4
J4,5
J4.s
J5.5’
JFJ
JF,~
JF,~
JF,~
JFJ
JF,Y
Benzyl 3-deoxy-3-fluoro-P-~-xylopyranoside 4.39 3.49 4.23 3.78 3.91 3.25 7.6 8.4 8.4 5.6 10.1 11.3 1.0 13.4 52.4 13.5 5.7 Methyl 4-deoxy-4-fluoro-~-arabinopyranoside a! anomer 4.68 4.74 3.5 50.0 p anomer 4.18 3.66 3.68 4.74 3.61 4.10 7.0 2.0 1.0 3.0 13.0 1.5 31.0 49.0 33.0 26.0
S
SF
References
A
103
-201.5
C
65
-205.9
C
65
For the furanoid counterpart, a chemical shift of - 197.5 was reported. For the furanoid counterpart, a chemical shift of - 194.0 p.p.m. was reported; J (F, H-2) = 13 Hz, J (F, H-3)= 55 Hz, and J (F, H-4)= 13 Hz.
(103) A. De Bruyn, M. J. Anteunis, G. Aerts, and E. Saman, Curbohydr. Res., 41 (1975) 290-294.
TABLEXVI ‘H- and I9F-N.m.r. Data for Pentofuranosyl Fluorides
D-Arabinofuranosyl fluoride 2,3,5-tri-O-benzoyl-a!6.17 5.74 5.75 4.95 4.88 0.9 0.5 4.4 3.3 5.8 58.4 6.4 0.7 1.5 3 ,5-di-O-benzoyl-2-O-methylQ anorner 5.91 4.22 5.45 4.81 4.59 0.5 0.8 3.2 5.4 3.8 59.2 6.8 0.5 1.5 p anomer 5.56 3.86 5.69 4.27 4.54 3.5 6.6 5.4 5.6 3.8 63.8 20.3 6.0
A
104
4.65 11.5
A
94
4.75 11.5
C-B
94
4.73 12.1
-123.8
(continued)
107
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
TABLEXVI (continued) Compound H-1 H-2
H-3
H-4
H-5
J1.2
Jz.3
J3.4
54,s
54,s
H-5' Jw
JF,I
JF.2
JFJ
JF,~
JF.5
JF,s*
SF
2,3,5-tri-O-benzyla anomer 5.78 63.0
S
References
C
105.106
c
105
9.0
p anomer 5.65
-121.0
b
66.0 3.0 D-Lyxofuranosyl fluoride 2,3-acetoxonium-5-O-acetyl-a6.27 6.1-6.6 5.0 4.5-4.8 0.0
F
107
-122.0
c
107
-128.7
C
107
F
107
55.0
2,3,5-tri-O-acetyla anomer
63.0
10.0
0.0
p anomer 67.0
22.5
6.5
2,3-benzoxonium-5-O-benzoyl-a6.58 6.3-6.8 0.0 55.0 2,3,5-tri-O-benzoyla anomer 6.03 5.83 6.09 0.8 5.4 6.0 61.0 8.5 p anomer 6.10 5.48 6.21 3.5 5.8 5.8 67.0 21.5 D-Ribofuranosyl fluoride 2,3,5-tri-O-acetyla anomer
67.7
5.6
5.1-5.3
d
c
5.06 6.0 2.2
4.6-4.8 6.0
-121.0
5.00 6.0 6.0
4.6-4.8 6.0
-133.8
C
107
-133.1
c
107
107 r
20.7
(continued)
RENE CSUK AND BRIGITTE I. GLANZER
108
TABLEXVI (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-5’
Ji.2
52.3
J3,4
J4,s
J4.5’
Jss,
JFJ
JFJ
JFJ
JF,~
JF,S
JFJ
6F
S
References
p anomer 61.8
4.2
2.3
-116.6
C
107
4.51 11.9
-115.8
A
104
4.42 12.4
-115.5
A
104
6.9
2-0-acetyl-3,5-di-O-benzoyl-p5.80 0.5 60.9
5.55
4.7 4.7
5.74 6.9 2.4
4.79 3.5 6.7
4.68 4.6
3-O-acetyl-2,5-di-O-benzoyl-p5.95 0.5 61.6
5.70 5.0 4.5
5.62 5.0 1.7
4.74 4.5 7.9
4.68 5.4
0
5-O-acetyl-2,3-di-O-benzoyl-p6.04 5.81 5.75 0.5 4.9 4.9 61.3 4.9 2.1 2,3,5-tri-O-benzoyla anomer 6.30 5.51 5.86 3.2 6.9 2.3 64.4 20.6 0.0 p anomer 6.18 5.96 6.02 0.5 4.8 5.8 61.5 4.8 2.2 2,3,5-tri-O-benzyla anomer 5.64 3. I 66.0 24.0 p anomer 5.63 63.5 D-Xylofuranosyl fluoride 2,3,5-tri-O-acetyla anorner 5.95 5.13 5.56 3.6 6.5 6.5 61.8 17.2 p anorner 5.63 5.18 5.34 1.0 0.5 5.5 60.5 5.2 0.5
4.82 3.6 7.2
4.61 5.3
4.25 12.1
-115.7
4.93 3.1 1.0
4.76 3.5
4.61 12.1
-133.3
5.11
4.89 5.2
4.69 12.2
-115.9
3.9 6.6
A
104 a
A
104 (1
A
104 a
-133.0
C
106
-116.0
C
106
4.74 4.8 1.4
4.31 4.1
4.13 12.4
-134.8
C
107
4.70 5.3 5.2
4.29 7.1
4.20 11.4
-113.2
C
104
(continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
109
TABLEXVI (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-5’
JIJ
J2.3
J3,4
54.5
JW
JS,Y
JF.1
JF,2
J F ~
JF,~
JFJ
JF,s~
S
SF
References
2,3,5-tri-O-benzoyl-p-
5.96
5.67
5.95 5.8
5.09 4.5-4.8 6.0 6.0 61.5 4.5 6.0 3,5-acetoxoniurn-2-O-rnethyl-/36.03 4.6 5.72 5.72 5.2 0.0 4.0 3.5 61.0 3,5-di-O-benzoyl-2-O-rnethyl-
-118.5
C
107
F
94
4.48
C
94
5.69 4.96 4.70 4.70 5.2 5.5 6.5 62.6 5.9 6.0 3,5-benzoxoniurn-2-O-rnethyl-~6.09 4.85 5.97 5.97 5.4 0.0 0.5 4.2 60.0 4.0
C
94
F
94
a anorner
5.91 3.6
62.5
4.22
6.2 18.0
5.78 6.7
4.98 5.9 1 .o
4.48 5.9
p anomer 5.84
4.13 1.0
a Also reported for a solution in C. Ref. 106 reported the data for the L enantiorner. J (F-1, Me-C+) = 2.90 Hz. d J (F-1, Me-C’) = 2.90 Hz. e.J (H-I, H-3)= 0.6 Hz.
(104) L. D. Hall, P. R. Steiner, and C. Pedersen, Can. J . Chem., 48 (1970)1155-1165. (105) W. Rosenbrook, Jr., D. A. Riley, and P. A. Lartey, Tetrahedron Lett., (1985)3-4. (106)T.Mukaiyama, Y. Hashimoto, and S. I. Shoda, Chem. Lett., (1983)935-938. (107) K.Bock and C. Pedersen, Acta Chem. Scand., Ser. B , 30 (1976)727-732. TABLEXVII
IH-and 19F-N.m.r. Data for 2-Deoxy-2-fluoropento-furanoses and -furanosides Compound H-1 H-2
H-3
H-4
H-5
H-5’
JI,~
52.3
J3.4
54,s
J4,v
Js.5
JFJ
JF.~
JFJ
JF,~
JFJ
JFJ,
2-Deoxy-2-fluoro-~-arabinofuranose Sphosphate, sodium salt a anorner 5.30 4.90 0.0 0.0 9.5 68.0
SF
S
D
References
66 (1
(continued)
RENE CSUK AND BRIGITTE I. GLANZER
I10
TABLEXVII (continued) Compound H-1 H-2
H.3
H-4
H-5
H-5'
51.2
52.3
53.4
54.5
54,s
JW
JFJ
JF,~
JF,~
JF,~
JF,S
JF,~
6F
References
S
p anomer 5.20 4.0 12.0
D
4.80
(1
68.0
1,3-di-O-acetyl-5-0-benzoyl-a5.0 5.24 4.51
6.33
10.0
66
48.0
C
108
A
109
C
108
N
110
C
109
C
108
C
I08
22.0
1,3-di-O-acetyl-5-0-benzyl6.26 0.7 10.5
5.10 49.0
5.25 4.5 22.8
4.47 4.5
3.74
3.74
1-0-acetyl-5-O-benzoyI-3-O-formyl-a-
6.45 5.12 5.45 4-. 0.0 9.5 50.0 22.0 1,3,5-tri-O-benzoyl-a4.76.7 5.4 5.61 49.0
1,3-di-O-benzoyl-5-0-benzyl6.75
5.50
9.0 49.0 5-O-benzo yl-
5.60 3.4 19.5
4.67 5.0
3.86
3.86
a anomer
5.3 0.0 9.5
4.98 0.0 68.0 p anomer 5.28 4.89 3.5 12.0 68.0 1,3,5-tri-O-benzyl-a5.25 5.03 4.36 1.0 2.4 7.3 12.8 51.2 5-0-benzyl5.42 4.87 4.23 3.86 1.6 6.0 9.7 50.0
3.86
3.63
C
111
3.56
3.56
C
109
(continued)
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
III
TABLEXVII (continued) Compound
H-1
H-2
H-3
H-4
H-5 H-5’
Ji.2
J2.3
J3,4
54.5
54,s.
Js,sf
JFJ
JF,Z
JF3
JF,~
JFS
JF,S
SF
Methyl 2-deoxy-2-fluoro-a-~-arabinofuranoside 4.96 4.85 3.70 4.5 1.0 2.6 12.0 52.0 3.5-di-0-benzyl5.09
S
References
A
109
C
112
1.o 12.0
5-0-benzyl5.06 4.82 4.10 3.64 3.64 C 109 I .6 5.5 10.3 51.0 1-0-Acetyl-2-deoxy-2-fluoro-5-0-(methoxymethyl)-a-D-nbofuranose 6.40 5.07 4.29 4.52 3.69 3.72 -216.8 C 49,50 10.1 4.4 5.6 2.4 51.3 4.4 Methyl 3-O-benzoyl-5-O-benzyl-2-deoxy.2-fluoro-&noside 5.08 5.00 5.64 4.71 3.71 3.71 C 1 I3 1.6 5.5 5.5 13.4 49.5 16.5 J (C-S, P) = 8 Hz.
(108) U. Reichrnan, K. A. Watanabe, and J. J. Fox, Carbohydr. Res., 42 (1975) 233-240. (109) J. A. Wright, N. F. Taylor, and J. J. Fox, J . Org. Chem., 34 (1969) 2632-2636. (I 10) C. H. Tann, P. R. Brodfuehrer, S. P. Brundidge, C. Sapino, Jr., and H. G. Howell, J . Org. Chern., 50 (1985) 3644-3647. (111) T. L. Su, R. S. Klein, and J. J. Fox, J. Org. Chem., 47 (1982) 1506-1509. (112) T. L. Su, R. S. Klein, and J. J. Fox, J . Org. Chem., 46 (1981) 1790-1792. (113) J. A. Wright and J. J. Fox, Carbohydr. Res., 13 (1970) 297-300.
RENfi CSUK AND BRIGITTE I. GLANZER
I12
TABLE XVIII 'H- and 19F-N.m.r. Data for 3-Deoxy-3-fluoro-D-pento-furanosesand -furanosides Compound H-1 H-2
H-3
H-4
H-5
H-5'
JIJ
J2.3
53.4
J4.5
54,s
J5.5'
JFJ
JF,~
JFJ
JF.~
JFJ
JF.~'
SF
S
Methyl 2,5-di-O-benzoyl-3-deoxy-3-fluoro-~~-arabinofuranoside -188.0 C
References
53,71
10.0" 7.0" 46.0 3-Deoxy.3-fluoro-1,2-O-isopropyl~dene-5-O-~tolyls~onyl-~-~-~lofuranose 5.86 4.64 4.90 4.70 4.10 -210.6 C 20 3.8 0.5 2.0 7.0 10.5 50.5 25.0 Methyl 2,5-di-O-benzoyl-3-deoxy.3-fluoro-/3-~-xylofuranoside -203.0 C 53,7 1 15.0 56.0 24.0 3-Deoxy-3-fluoro-1,2-O-isopropylidene-~-~-~y~~-pentodi~do-l,4-furanose 6.16 4.76 5.25 4.62 9.67 -205.5 C 114 3.7 0.5 2.6 1.2 10.0 50.0 31.5 The assignments may have to be reversed.
(114) J. M. J. Tronchet, B. Gentile, A. P. Bonenfant, and 0. R. Martin, Helv. Chim. Acfa, 62 (1979) 696-699.
TABLE XIX 'H- and lgF-N.m.r. Data for 5-Deoxy-5-fluoro-~-pento-furanoses and -furanosides Compound H-1 H-2
H-3
H-4
H-5
H-5'
JI,~
52.3
J3.4
54.5
54,s
J5.5,
JF,I
JFJ
J F ~
JF.~
JFJ
JFJ-
6F
S
References
-227.3
D
81
-232.5
C
81
-232.1
C
81
5-Deoxy-5-fluoro-~-ribofuranose 23.5 50.0 Methyl 5-deoxy-5-fluoro-~-ribofuranoside 2,3-di-O-acetyl-
29.5
47.5
50.0
47.5
2,3-O-isopropylidene(Y anorner 30.0
47.5
47.5 (continued)
I 13
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES TABLEXIX (continued) Compound H-2 H-1
SF
S
References
-224.8
C
81
10.5 47.5 47.5 3-0-Benzyl-5-deoxy.5-fluoro-1,2-O-isopropyl~dene-~-~-xy~ofuranose 5.94 4.60 3.97 1.45 4.64 4.58 C 3.4 0.7 3.0 9.0 16.0 42.0 42.0
115
H-3
H-4
H-5
H-5'
Ji,z
52.3
53.4
54.5
J4.5
Js,5*
JF.I
JFJ
JFJ
JF.~
JF.~
JF.~'
p anorner
(115) P. W. Kent and R. C. Young, Tetrahedron, 27 (1971) 4057-4064.
TABLEXX 'H- and I9F-N.m.r. Data for Fluorinated 1,5-Anhydrohexopyranose Derivatives Compound H-2 H-1
H-3
H-4
H-5
H-6
H-6'
JI,Z
JZ,3
53.4
54,5
55.6
Js,~
56.6'
JFJ
JF,Z
JF,~
JF,~
JF,~
JF.6
JF,~
SF
S
References
3,4,6-Tri-0-acetyl-1,5-anhydro-2-deox~2-fluoro-l-C-methyl-~-~-glucopyranose 4.32 4.53 5.23 4.76 3.80 4.19 3.92 C 39 0 5.9 8.8 8.8 9.4 5.0 2.5 12.1 1.5 49.5 12.6 1,5-Anhydro-6-deoxy-6-fluoro-~-ara6~~~-hex-l-enitol 3,4-di-O-acetyl- 1,2-dideoxy6.61 4.90 5.0-5.48 4.15-4.75 5.0-5.48 A 116 b 6.6 3.2 47.0 3,4-di-O-benzyl117 6.39 4.4-5.1 4.1-4.35 3.7-4.1 4.1-4.35 4.4-5.1 -235.4 C 24.6 4-0-benzyl-3-deoxy6.29 4.5-4.8 2.27
47.2
3.47-4.50 29.4
47.2 4.67
48.0
-235.8
C
1 I7
48.0
" M e : 8 = 1 . 3 6 , J ( F , M e ) = 2 . 6 , a n d J ( H - l , M e )=6.9Hz. b J ( H - I , H - 3 ) = 1.2Hz.
(116) M. Sharma and W. Korytnyk, Carbohydr. Res., 83 (1980) 163-169. (117) I. D. Blackburne, P. M. Fredericks, and R. D. Guthrie, Aust. J . Chem., 29 (1976) 381-391.
1 I4
RENE CSUK AND BRIGITTE I. GLANZER TABLEXXI
'H-and 19F-N.m.r.Data for Fluorinated 1,6-AnhydrohexopyranoseDerivatives Compound
H-1 H-2
H-3
H-4
H-5
H-6
H-6'
JI,Z
Jz.3
J3,4
54.5
55.6
55.6
56.6
JF,~
JF,Z
JF,~
JF,~
JF,S
JF,6
JF.6'
6F
S
References
1,6-Anhydro-2-deoxy-2-fluoro-/3-~-glucopyranose 3,4-di-O-acetyl5.51 4.24 5.00
4.64
4.64
C
118,119
17.0 45.0 3-0-acet yl-4-0-benzyl5.51 4.25 5.14
3.25
4.60
C
118,119
3.35 1.4
4.4-4.7
C
I20
45.0 17.0 3,4-di-O-benzyl5.53 4.35 3.6-3.8 I .4 4.9 45.0
3.6-3.8 1.2
3.91 1.3
2-O-Acetyl-1,6-anhydro-4-O-benzyl-3-deoxy-3-fluoro-/3-~-~tropyranose 5.52 1.5
6.5
5.23 9.0 13.0
4.78 4.5 48.0
3.9-3.99
4.65
C
118,119
2,4-Di-0-acetyl-1,6-anhydro-3-deoxy-3-fluoro-/3-~-idopyranose
-202.1 c 0.0 4.5 8.0 0.0 4.5 4.5 1,6-Anhydro-3-deoxy-3-fluoro-/3-~-mannopyranose 5.43 3.83 4.74 4.12 4.63 4.09 3.81 D 1.9 4.5 2.0 2.0 1.2 6.0 1.9 0 26.4 47.1 12.5 0.8 1.4 3.8 2,4-di-O-acetyl5.49 4.15-5.01 5.04 4.65 4.20 3.88 C 1.6 1.8 I .2 5.9 1.9 0.0 12.6 0.0 1.3 3.5 3-~-Acetyl-1,6-anhydro-2,4-dideoxy-/3-~-glucopyranose 4-acetamido-2-fluoro5.52 4.27 4.84 4.10 4.50 4.19 3.80 C 2.5 1.5 1.5 2.5 I .o 6.0 8.0 1.0 43.0 15.0 2-acetamido-4-fluoro5.34 3.99 4.86 4.29 4.56 3.89 3.65 1.5 1.5 1.5 1.5 1.5 5.0 8.0 1.5 15.5 45.0 11.0 1.5 5.0 1,6-Anhydro-2,4-dideoxy.2-fluoro-/3-~-e~~~~o-hexop~anos-3-ulose 4.63 4.20 2.91a 2.40e 4.83 3.75 3.64 N 1.9 5.6 I .3 0.5 4.0 7.8 2.0 49.0 2.0 4.5
8.0 16.0
8.0 50.0
12
4.5 16.5
I
73 h
73
121 c
6,121 P
122
r.Y
For the nonacetylated derivative, a shift of 6F = -198.2 for a solution in D was reported. Also reported, in C. ' J (H-I, H-3)= 1.50, J (H-2, H-4) = 1.0, J (H-3, H-5) = 1.5 Hz. Solvent: 5 : 1 : 1
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
I IS
chloroform-dimethyl sulfoxide-hexafluorobenzene. J (H-I, H-3) = 1.5, J (H-1, H-5) = 1 .O Hz. f J (H-4, H-4') = 17.7 Hz. The symbol a stands for the axial, and e for the equatorial, proton at C-4.
(118) J. Paciik, J. PodeSva, Z. ToCfk, and M. (?ern$, Collect. Czech. Chem. Commun., 37 (1972) 2589-2599. (119) J. Pachk, Z. ToEfk, and M. Cernf, Chem. Commun., (1969) 77. (120) T. Haradahira, M. Maeda, Y. Kai, and M. Kojima, J . Chem. Soc., Chem. Commun., (1985) 364-365. (121) J. Pacak, P. DraSar, D. Stropovit, M. Cern$, and M. BudCSinsk$, Collect. Czech. Chem. Commun., 38 (1973) 3936-3939. (122) J. DoleZalovB, M. Cerny, T. Tmka, and J. Pacak, Collect. Czech. Chem. Commun., 41 (1976) 1944-1953.
TABLEXXII 'H- and 19F-N.m.r.Data for Fluorinated 3,6-Anhydrohexose Derivatives Compound H-2 H-1
H-3
H-4
H-5
H-6
H-6'
J1.2
J2,3
53.4
54,s
55,6
55,6'
56.6'
JF.1
JF,Z
JFJ
JF,~
JFJ
JF,~
Jv.6
6F
S
-138.0
A
References
2-0-Acetyl-3,6~nnhydro-S~O-benzoyl-~-idofuranosyl fluoride a anomer
p anomer 5.72 5.28 5.06 4.72 5.56 4.42 4.06 -119.8 A 0.8 0.5 4.8 1.0 4.0 1.5 10.6 0.5 4.8 1.5 62.3 5.8 3,6-Anhydro-5.deoxy-S-fluoro.1,2-~-isopropylidene-~-~-glucofuranose -207.6 B 1.6" 5.8O 51.6 19.5 3,6-Anhydro-5-deoxy-S-fluoro-~-idofuranose 1 ,2-di-Qacetyla anomer 4.28 4.93 5.14 5.10 4.68 6.12 1.0 0.5 3.0 0.5 4.6 0.5 39.8 10.8 50.6 2.7 0.5 1.0 p anomer 5.08 3.91 4.97 6.34 4.94 4.88 0.5 2.8 0.8 0.5 3.3 4.4 50.6 38.1 8.1 0.5 1.0 2.0 1,2-O-isopropylidene-p4.70 3.59 4.71 4.32 4.59 5.59 0.5 2.8 0.8 0.5 3.2 3.5 38.3 7.5 50.4 0.5 1.5 1.5 a
22
22
92,123
17.9
4.00 11.4 25.5
-186.7
A
92
4.08 11.6 22.3
-190.8
A
92
3.75
-189.7
B
92
11.1
26.1
Assignments may have to be reversed.
(123) R. Csuk and B. I. Glanzer, unpublished results.
RENE CSUK AND BRIGITTE I. GLANZER
116
IH-
TABLEXXIII and I9F-N.m.r. Data for Fluorinated Sugar Acids and Lactones
(D-Glucofuranosylfluoride)urono-6,3-lactone 2,S-di-O-acetyl-P5.91 5.35 5.23 5.36 5.63 0.6 0.5 4.6 6.3 60.1 4.5 0.5 4.4 0.5 2,S-di-O-benzoyl-P6.2 5.7 5.56 5.71 5.98 0.5 0.5 4.6 6.3 60.1 4.0 0.5 4.6 0.3 ~-Deoxy-3-fluoro-~-gluconic acid
-118.3
A
22 a
-118.5
A
22 a
-207.0
D
70
Methyl (methyl 4-deoxy-4-fluoro-cu.~-glucopyranosid)uronate -119.2
M
124
14.7
54.5
5-Deoxy-5-fluoro-1,2-O-isopropylidene-cu-~-glucofuranurono-6,3-lactone 6.08 3.6
4.87 4.90 5.08 5.33 -216.3 C 91 0.6 2.8 4.0 b 3.5 I .3 1.0 48.0 5-Deoxy-5-fluoro-&~-idofuranurono-6,3-lactone 1,2-O-benzylidene6.17 5.09 5.40 5.22 5.09 -199.0 c 125 0.0 4.0 0.0 3.6 0.0 10.0 47.5 I ,2-O-isopropylideneC 91 5.95 4.87 5.10 4.96 4.93 -199.7 3.7 3.3 0.4 1.2 8.7 48.3 Methyl [benzyl 2-(benzyloxycarbonyl)amino-2,3,4-tndeoxy-5-fluoro-~-~-e~~~ro-hex-3enopyranosid]uronate 4.88 4.75 5.75 5.46 B 126 10.0 d 3.5 1 .O 1 .O " J (H-3, H-5) = 0.5 Hz. * J (H-2, H-4) = 0.5 HZ. ' J (H-3, H-5)= 0.4, J (H-2, H-4) = 0.3 Hz. Assignments of H-3 and H-4 may have to be reversed; some values were also given for C as the solvent.
(124) J. Samuel and N. F. Taylor, Carbohydr. Res., 133 (1984) 168-172. (125) R. Albert, K. Dax, U. Katzenbeisser, H. Sterk, and A. E. Stiitz, J. Carbohydr. Chem., 4 (1985) 521-528. (126) J. Kiss, P. C. Wyss, G. Flesch, W. Arnold, K. Noack, and P. Schonholzer, J . Carbohydr. Chem., 4 (1985) 347-361.
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
I 17
TABLEXXIV IH- and UF-N.m.r. Data for Aminodeoxyhexopyranosyl Fluorides (and Their Synthetic N-Containing Precursors) Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
JLZ
JZJ
J3,4
J~J
55.6
5 5 , ~
56.6
JFJ
JF,Z
JFJ
JF.4
JFJ
JF,6
JF,~
S
References
C
I27
M
128
C-M
128
3-Azido-4,6-0-benzyldene-3-deoxy-~~-allopyranosyl fluoride 5.53 8.0 52.0
3.50 3.5 12.5
4.20 3.0 3.4
3.60 9.0
3.99 9.0
3.72 5.0
4.37 9.0
-147.3
2-Acetamido-2-deoxy-~-~-glucopyranosy~ fluoride 5.18 7.8 54.0
11.0 3,4,6-tri-O-acetyl5.39 6.8 53.0 13.0
(127) S. Castillon, A. Dessinges, R. Faghih, G. Lukacs, A. Olesker, and T. T. Thang, J. Org. Chem., 50 (1985) 4913-4917. (128) F. W. Ballardie, B. Capon, W. M. Dearie, and R. L. Foster, Carbohydr. Res., 49 (1976) 79-92.
TABLEXXV 'H- and "F-N.m.r. Data for 3-Amino-2,3-dideoxy-2-fluoro-a-~-altropyranoside Derivatives (and Their N-Containing, Synthetic Precursors) Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
JI,Z
52.3
53.4
J4.5
55.6
5 5 , ~
36.6
JFJ
511.2
JFJ
JF,~
JF,~
JF,~
JF,~
SF
4,6-0-Benzylidene-2,3-dideoxy-2-fluoro-a-~-~tropyranoside methyl 3-amino4.77 4.66 3.57 -189.2 0.2 2.6 2.6 9.8 43.8 11.6 benzyl 3-azido4.90 4.63 4.29 4.09 4.29 3.77 4.29 -190.8 0.0 2.5 4.0 9.5 10.5 10.5 11.5 43.5 11.5 3.0
S
References
C
129
C
127
(continued)
TABLEXXV (continued) Compound
S
References
3.92 10.0
C
128
4.29
C
127
H-1
H-2
H-3
H-4
H-5
J2,3
J3.4
J4,a
$5.6
H-6 Js,~
H-6’
JIS JFJ
JF,Z
JFJ
JF.4
JF,S
JF.~
JF,~
4.17 10.0 2.5
4.11 4.0
4.37 10.0
benzyl 3-benzarnido5.12 4.77 5.14 1.0 4.5 8.5 44.0
6F
56.6‘
Benzyl2,3-dideo~y-2-fluoro-cu-~-altropyranoside 3-azido-6-O-he .~ thvlsulfonvl)4.92 4.67 4.04 4.17 2.0 12.0 43.0 3-benzarnido5.51 4.81 4.34 5.42 5.14 1.0 4.0 44.0 9.5 3-benzamido-6-0-mesyl5.19 4.72 4.97 4.2-4.00 1.0 4.0 1.5 7.5 45.0
4.46
10.0
-
4.60
P
128
4.64
4.49 11.0
C
128
C
128
C
I30
5.5
3-benzarnido-6-O-tos yl-
4.45 4.20 5.06 4.62 4.87 4.03-3.95 1.0 3.0 1.5 11.0 10.0 8.0 45.0 Methyl 3-benzamido-2,3-dideoxy-2-fluoro-cu-~-al~opyranoside 4,6-di-O-acetyl4.92 4.60 5.13 5.18 4.12 4.25 - 193.3 1.5 3.5 4.5 10.0 4.0 4.0 1.5 8.0 45.0 8.6 4,6-di-O-benzoyl5.00 4.67 5.35 5.63 4.40 4.67 4.47 -193.7 1.5 3.6 4.6 9.6 2.6 6.0 12.0 44.0 9.0 2.0 8.0 4,6-O-benzylidene3.87 -191.4 4.85 4.29 5.06 2.5 9.0 1.5 3.0 1.0 8.5 9.0 45.0 Methyl 4,6-0-benzylidene-2,3-dideoxy-2-fluoro-cu-~-al~opyranoside 3-(diallylarnino)- 182.6 4.70 13.0 42.5 18.5 3-(trifluoroacetarnido)4.89 4.66 4.89 1.4 3.0 4.5 7.5 45.0 7.5
4.32 9.8
3.95 4.2
“ J ( H - I , H-3) = 1.2 Hz. b J ( H - l . H-3)
=
4.08 9.8
3.85 9.8
1.5 Hz. ‘J(H-1, H-3)
-191.6
=
1.5 Hz.
‘ I
C
130 b
C
130 c
C
131
C
131
I I9
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
(130) L. Hough, A. A. E. Penglis, and A. C. Richardson, Carbohydr. Res., 83 (1980) 142- 145. (131) D. Picq and D. Anker, J . Carbohydr. Chem., 4 (1985) 113-123.
TABLEXXVI 'H- and 19F-N.m.r. Data for Aminodideoxy-3(or 4)-flUOrO Sugars (and Their N-Containing Synthetic Precursors)
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
JI,~
J2.3
53.4
J4.5
55.6
JS,C
56.6,
JFJ
JFJ
JF,3
JF,4
JF.5
JF.6
JF,C
6F
Methyl 6-azido-3,6-dideoxy-3-fluoro-~-~-allopyranoside 4.52 3.47 4.89 3.61 3.84 3.48 3.48 -217.4 8.5 2.0 2.0 10.0 2.0 29.0 53.0 28.0 4,6-0-Benzylidene-2,3-dideoxy-3-fluoro-a-~-glucopyranoside benzyl 2-acetamido5.05 3.88-3.97 4.78 3.88-3.97 4.28 3.85 10.0 4.0 10.0 10.0 4.0 9.0 60.0 2.0 benzyl 2-azido5.03 3.47 5.03 3.78 3.91 3.78 4.26 -198. 4.0 9.0 9.0 9.0 9.0 4.5 10.0 5.5 9.0 54.5 2.0 methyl 2-benzamido4.89 4.43 4.97 4.05 3.72 4.28 3.87 10.0 3.7 9.8 9.4 9.0 4.8 10.3 3.0 12.1 53.6 12.4 0.7 1.9 Methyl 3,6-di-0-ace:etyl-2,4-dideoxy-4-fluoro-a-~-glucopyranoside 2-acetamido4.67 5.30 4.70 -199.8
S
References
A
65
C
132
C
I27
S
133
P
134
14.7 49.7 2-benzamido4.83 4.42 5.48 4.59 4.42 4.44 -196.9 C I34 3.3 11.3 9.7 9.7 1.8 11.3 13.0 49.7 4.4 3.3 Benzyl 2-acetamido-3-O-ace:etyl-2,4-dideoxy-4-fluoro-6-O-tn:etyl-a-~-g~opyranoside 5.05 -188.2 C 135 4.0 54.0 Methyl 4,6-dideoxy-4-fluoro-a-~-talopyranoside 6-azido4.87 4.70 3.93 3.35 3.68 -216.1 A 65 0.0 5.0 8.0 12.0 51.0 30.0 2.0 (I
(continued)
RENE CSUK AND BRIGITTE I. GLANZER
120
TABLEXXVI (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
Jl,Z
J2,3
J3.4
J4,S
55.6
J5.6'
56.6'
JF,~
JFJ
JFJ
JF.4
JF,S
JF,~
JF.6'
3.68
4.67
3.90
3.37
6-amino4.69 3.64 0.0
32.0
51.0
3.37
6F
S
References
-217.0
A
65
31.0
Data also reported for P as the solvent.
(132) L. H. B. Baptistella, A. J. Marsaioli, J. D. De Souza Filho, G. G. De Oliveira, A. B. DeOliveira, A. Dessinges, S. Castillon, A. Olesker, T. T. Thang, and G. Lukacs, Cnrbohydr. Res., 140 (1985) 51-59. (133) H. H. Baer and A. Jaworska-Sobiesiak, Cnrbohydr. Res., 140 (1985) 201-214. (134) L. Hough, A. A. E. Penglis, and A. C. Richardson, Cnn. J . Chem., 59 (1981) 396-405. (135) M. Sharma and W. Korytnyk, Cnrbohydr. Res., 79 (1980) 39-51. TABLEXXVII 'H- and I9F-N.m.r. Data for Aminodideoxy-6-luoro Sugars
2-Acetamido-l,3,4-tri-O-acetyl-2,6-dideoxy-6-fluoro-~-gda~opyranose -230.8 C
85
18.5 47.5 47.5 Methyl 2,6-dideoxy-6-fluoro-cu-~-gdactopyranoside 2-acetamido15.8
47.5
2-acetamido-3,4-di-O-acetyl4.78 5.16 5.38 1.0 3.5 11.0 3.2
P
134
-238.0
C
134
-234.9
C
134
-236.2
C
134
47.5 4.40
46.0
-235.0
46.0
2-benzamido14.5
46.0
46.0
3,4-di-O-acetyl-2-benzamido4.98 3.5
10.0
5.29 3.5
5.50
1.0 (continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
121
TABLEXXVII (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
J1.2
J2,3
53.4
54,s
55.6
55.6
J6.6,
JF,l
JFJ
JFJ
JF,~
JFJ
JF,6
JF,~
SF
S
-236.2
C
134
-235.2
C
134
-238.0
P
134,136
-238.0
C
85,134
-232.5
C
85
P
85,136
References
2-benzamido-4-0-benzo yl-3-0-benzyl-
5.03 3.3
10.5
3.80 3.2
5.84 1.3
2-benzamido-3-O-benz yl-
14.5 48.5 48.5 2-Acetamido-2,6-dideoxy-6-fluoro-~-glucopyranose a anomer 5.86 5.26 4.18 3.2 2.7 29.3 48.1 48.7 I ,3,4-tri-O-acetyla anomer 6.17 4.44 3.97 4.42 3.5 25.0 48.5 48.5 p anomer
21.8 48.2 48.2 2-Acetamido-2,6-dideoxy-6-fluoro-cu-o-glucopyranoside benzyl 3,4-di-O-benzyl-232.8 5.21 5.02 4.55 3.6 2.5 29.0 47.5 47.5 methyl 3,4-di-O-acetyl-236.1 3.64 4.46 4.14 4.32 5.06 5.16 3.5 10.0 10.0 10.5 23.5 48.5 48.5 methyl 3,4-di-O-methyl-233.4 4.92 4.41 4.95 3.4 2.4 27.5 48.0 48.0 Methyl 2-benzamido-2,6-dideoxy-6-fluoro-a-~-glucopyranoside -239.0 26.0 3,4-di-O-acetyl4.87 4.51 5.40 3.5 10.0 10.0
5.12 10.0
49.5
3.98 23.5
P
134
P
136
A
134
A
134
49.5 4.46
48.5
a
-237.4
48.5 (continued)
RENB CSUK AND BRIGITTE I. GLANZER
I22
TABLEXXVII (continued) Compound H-1 H-2
6F
S
References
-239.1
C
134
-231.8
C
134
A
65
A
65
D
1 I6
-237.3
D
I I6
4.95
-234.1
s
1I6
1,3,4-tri-O-acetyla anomer 5.19 1.6
-234.3
C
85,116
-233.1
C
85,116
H-3
H-4
H-5
H-6
H-6'
J1.2
JZJ
J3,4
54,s
Js,6
55.6'
J6,v
JF.1
JF,~
JFJ
Jp.4
Jp.5
JF,~
JF,6'
3-0-benzyl28.0
49.5
49.5
3-0-benzyl-4-O-mes yl-
22.5 41.4 41.5 Methyl 4,6-dideoxy-6-fluoro-cu-~-glucopyranoside 4-azido4.14 3.50 3.60 4.60 4.60 -234.1 3.5 10.0 26.0 46.0 46.0 4-amino4.1 3.92 4.25 4.33 -233.8 4.0 10.0 4.0 1.5 10.0 28.5 48.0 48.5 2-Amino-2,6-dideoxy-6-fluoro-~-mannopyrmose hydrochloride (I anomer -231.5 21.9
45.9
45.9
p anomer 21.5
41.0
41.0
2-Acetamido-2,6-dideoxy-6-fluoro-~-mannopyranose
25.5
41.2
41.2
p anomer 6.01 1.8 23.8
41.5
41.5
Ref. 85 reported J (H-5, F) = 22.5 Hz.
(136) M. L. Shulman and A. Ya. Khoriin, Carbohydr. Res., 21 (1913) 141-141.
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
123
TABLEXXVIII 'H- and I9F-N.m.r. Data for 3-Amino-2,3,6-trideoxy-2-fluoro-6-halogeno-cr-~altropyranoside Derivatives (and Their N-Containing Synthetic Precursors)
Benzyl 2,3,6-trideoxy-2-fluoro-6-iodo-a-~-altropyranoside 3-azido121 4.88 4.61 3.71 4.06 3.45 3.21 C 3.0 10.5 10.5 2.5 11.0 12.0 44.0 3-benzamido5.20 4.10 4.92 3.99 3.87 3.16 3.32 C 132 1.0 3.5 2.5 10.0 2.0 9.0 11.0 1.5 43.0 2.0 Methyl 4-0-benzoyl-6-bromo-2,3,6-trideoxy-2-fluoro-cr-~-altropyranoside 3-benzamido5.03 4.70 5.30 5.40 4.25 3.61 3.51 C 132,133, 1.5 3.5 4.0 10.0 2.4 8.2 10.9 137 8.0 44.5 9.0 2.0 3-(trifluoroacetamido)C 131 4.99 4.63 5.03 5.38 4.16 3.60 3.5 -204.0 10.8 8.0 1.3 3.0 4.3 10.8 2.5 1.7 44.3 1.7 (137) M. K. G u j a r , V. J. Patil, J. S. Yadav, and A. V. Rama Rao, Carbohydr. Res., 135 (1984) 174-177. TABLEXXIX 'H- and I9F-N.m.r. Data for 3-Amino-2,3,6-trideoxy-2-fluoro-hexopyranoside Derivatives (and Their N-Containing Synthetic Precursors) Compound H-1 H-2 Jls J2.3 JF.1
JF,2
H-3
H-4
H-5
H-6
H-6'
J3c
J4,5
554
J5,6'
J6.6
JFJ
JF.4
JFJ
JF.6
J F . ~
SF
Benzyl 3-benzamido-2,3,6-trideoxy-2-fluoro-cr-~-altropyranoside 4.65 4.12 4.97 3.91 3.83 1.43 2.0 2.0 7.0 8.0 45.0 Methyl 2,3,6-trideoxy-2-fluoro-a-~-altropyranoside 3-amino4.75 4.57 3.45 3.26 3.60 1.34 1.6 3.2 10.0 6.2 10.2 45.5
S
References
C
132
C
133 a
(continued)
RENB CSUK AND BRIGITTE I. GLANZER
124
TABLEXXIX (continued)
Compound
H-1
H-2
H-3
H-4
H-5
H-6
H-6'
JIJ
J2.3
J3,4
J4.5
55.6
J5.6,
564'
JF.1
JF.2
JFJ
JF,~
JFJ
Jw.6
JF,~
5.28 9.8 2.0
4.16 6.3
3.83 9.0
3.76 6.6
SF
S
References
3-benzamido-4-O-benzo y l-
4.92
4.67 5.23 3.2 4.0 8.0 45.3 8.5 3-(trifluoroacetamido)4.84 4.60 4.13 1.5 3.5 4.5 7.8 45.0 9.0 1.5
C
1.32
133 137 0
I .36
C
133 0
2,3,6-Trideoxy-2-fluoro-~-galactopyranose 3-amino-, hydrochloride (I! anomer 5.29 4.15 3.74 3.83 4.21 1.18 -192.2 10.5 3.5 1.0 6.5 3.5 0.0 50.4 11.0 p anomer 4.15 4.40 3.55 3.83 3.76 1.25 -192.4 10.0 3.5 6.5 7.7 3.0 52.0 11.2 I -O-acetyl-4-O-benzoyl-2-fluoro-3-(tnfluoroacetamido)-~5.90 4.70 4.60 5.63 4.13 1.26 10.0 2.0 1.0 7.0 8.0 4.0 45.0
M
131
M
131
C
121
Benzyl2,3,6-trideoxy-2-fluoro-/3-~-g~lactopyr~noside 3-amino4.85 4.77 7.0 8.0 8.0 52.0 3-benzamido4.30-4.60-
3.15 4.0
3-(trifluoroacetamido)4.63 4.37 3.75 7.5 9.0 51.0 9.0
3.78 7.0
1.47
C
127
3.24
2.98 7.0
0.94
B
132
4.29
3.18 7.0
1.33
C
127
C
127
C
133
3.88 3.0
4-O-benzoyl-3-(trifluoroacetamido)4.71 4.57 4.49 5.52 3.90 1.24 7.0 10.0 3.0 7.0 8.0 54.0 5.0 Methyl 2,3,6-trideoxy-2-fluoro-~-galactopyranoside 3-amino-P4.36 4.17 3.02 3.62 3.68 1.35 1.0 6.5 7.6 9.5 3.0 51.0 13.0 3.0 3.3
-193.4
(continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
I25
TABLEXXIX (continued) Compound H-1 H-2
6F
References
H-3
H-4
H-5
J1.z
J2.3
53.4
J4.5
55.6
H-6 H-6' 5 5 , ~ 56.6
JFJ
JF.~
JFJ
JFP
JF,5
JF,6
3.82
4.32 7.0
1.32
C
132
5.65 1.0 2.0
4.05
1.27
c
133,137
3.81 3.0
3.9 6.4
1.26
A
131
3.81
3.90 6.4
1.26
C
133
3-benzamido-P4.50-
JF.6'
3-benzamido-4-O-benzoyI-p4.62 4.55 4.80 7.4 10.7 3.7 3.0 52.9 12.5 3-(trifluoroacetamido)a anomer 4.52 4.45 4.27 7.3 10.0 3.0 51.0 10.0 6.8 P anomer 4.55 4.46 4.30 7.4 10.4 3.1 0.5 50.7 13.5
1.1
S
b
6.4
3.1
"JJH-I, H-3) = 1.5 Hz. Ref. 137 reported couplings: J (H-I, H-2) = 10.5 andJ(H-2, H-3) = 8.0 Hz.
TABLEXXX 'H- and I9F-N.m.r. Data for Amino-2,3,6-trideoxy-2-fluorohexo-pyranosides, -pyranoses, and -pyranosyl Halides Compound H-1 H-2
H-3
H-4
H-5 H-6
51.2
J2.3
J3,4
54,s
55.6
35.6
J64'
JFJ
JF,~
JFJ
JF,~
JF,S
JF.6
JF,~
H-6'
SF
S
References
4-0-Benzoyl-2,3,6-tndeoxy-2-fluoro-3-(tnfluoroacetamido)-~~-gala~opyranosyl bromide 6.68 4.78 4.93 5.63 4.50 1.23 C 127 4.0 10.0 7.0 50.0
6-Azido-QO-benzoyl-2,3,6-tndeoxy-2-fluoro-~-ribo-hexopyranosyl halide chloride a anomer 5.11 4.31 3.36-3.62 C 6.25 4.86 2.31/2.66 3.8 12.0/5.0 11.Y5.0 10.0 5.0 3.0 48.0 p anomer 5.55 4.65 6.5 9.015.0 4.5 48.0
855.0
1.5
2.0/2.88 9.0/5.0 13315.0
5.16 8.0
4.00
3.36-3.62
C
138 a. b
138 o.e
1.0
(continued)
I26
RENE CSUK AND BRIGITTE I. GLANZER TABLEXXX (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6’
Jl.2
J2.3
53.4
J4,5
55.6
Js.6,
56,6’
JFJ
JF,Z
JF$
JF.4
JF,~
JF.6
JF.6,
2.32/2.64
5.14 10.0 1.4
4.28 5.0
2.7
5.16 11.5 1.4
4.05 5.0
2.7
6F
S
References
3.49 13.5
C
138
3.49 13.5
C
a bromide
6.61 3.8
4.65 11.Y4.7 47.5 a iodide 7.05 3.94 11.5/5.0 4.0 2.0 47.5
11.Y5.0
8.3/5.0 2.20/2.57 11.Y5.0
o.d
138 a.e
8.Y5.0 6-Azido-4-O-henzoyl-2,3,6-trideox~2-fluoro-~-ribo-hexopyranose a anomer
5.44 3.8 1.0
4.70 2.2Y2.59 5.02 4.30 3.31-3.50 -190.5 C 12.0/5.0 11.515.0 10.0 48.0 9.0/5.5 1.2 p anomer C 4.93 4.40 1.91/2.81 5.02 3.88 3.31-3.50 -190.2 7.0 11.0/5.0 11.Y5.0 10.0 3.0 49.0 12.0/7.0 1.2 Methyl 6-~do-4-O-henzoyl-2,3,6-trideoxy-2-fluoro-~-~-ribo-hexopyranoside 4.10 4.11 1.47/2.35 4.71 C 7.2 10Y5.2 10Y5.0 3.5 48.0 12.Y7.0
138 a.f
138 a.g
138 ash
Shifts and couplings for axial (a) and equatorial (e) proton, respectively. J (H-3a. H-3e) = 11.5, J (H-I, H-3e) = 0.5 Hz. J ( H - ~ uH-3e) , = 13.5 Hz.d J (H-I, H-5) = 0.3, J (H-I, H-3e) = 1.3, J (H-3a, H-3e)= 11.5Hz.’J(H-l,H-3e) = 1.5Hz.’J(H-l,H-3e)=0.3;J(H-l,OH)= 3.5,J(H-3a,H-3e)= , = 12.0 Hz. 11.5 Hz. g J (H-I, OH) = 5.0, J (H-3a, H-3e) = 12.0 Hz. J ( H - ~ uH-3e) @
(138) T. Tsuchiya, Y.Takahashi, M. Endo, S.Umezawa, and H. Umezawa, J . Carbohydr. Chem., 4 (1985) 587-61 1.
TABLEXXXI ‘H- and 19F-N.m.r. Data for Fluorinated Unsaturated Amino Sugars Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6‘
Jl.2
JZS
J3.4
J4,s
55.6
55.6
J6.6
JF.1
JFJ
JFJ
JF.~
JF,5
Jp.6
JF,6’
6F
S
References
2,3,6-Trideoxy-2-fluoro-a-~-arabino-hex-5-enopyranoside
benzyl 3-azido4.67 4.62 3.11 4.0 7.0 3.5 11.0 47.0 12.5
3.94
4.66
4.44
C
127
(continued)
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
127
TABLEXXXI (continued) Compound H-1 H-2
H-3
H-4
H-5
52.3
J3,4
54.5
55.6
H-6 55.6
H-6'
J1,Z
SF
JFJ
JFJ
JFJ
JF,~
JFJ
JF.6
JF.~
S
References
56,6
benzyl 3-benzamido4.74 4.25 5.10 4.81 5.18 4.90 B 132 1.0 2.5 8.0 45.0 methyl 3-benzamido4.92 4.75 4.68 4.92 4.92 C 132 1.0 3.5 44.0 methyl 3-benzamido-4-0-benzoyl5.03 4.86 5.24 5.98 4.97 4.83 C 132,133 2.0 4.0 4.8 1.5 137 8.0 46.0 8.0 3.3 methyl 4-O-benzoyl-3-(trifluoroacetamido)c 4.33 6.0 -193.7 131 1.7 3.3 3.7 7.3 47.0 7.3 1.7 Methyl 3-benzamido-2,3,4,6-tetradeoxy-2-fluoro-~-~-~~reo-hex-4-enopyranoside 5.11 I .85 C 4.84 4.66 4.77 I37 1.5 3.5 9.0 46.0 Methyl 2-benzamido-3-O-benzyl-2,4,6-trideoxy-6-fluoro-~-~-~~reo-hex-4-enopyr~oside -224.1 C I34 (1
48.0 " J (H-I, H-3)
=
48.0
1.5 Hz.
TABLEXXXII 'H- and IgF-N.m.r. Data for Fluorinated Aminodeoxyhexo-furanosesand -furanosides Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
Ji.2
52.3
534
54.5
55.6
Js,~
J6,6'
JF,I
JFJ
JF.~
JF,~
JF.S
JF,~
JF,~
3,6-Dideoxy-~~-galactofuranosyl fluoride 3-azido-5-0-benzoyl-2-0-benzyl5.83 3.95-4.605.40 6.4 59.3 3-azido-5-O-benzoyl-2-O-methyl5.74 3.95 3.78 4.24 5.36 3.0 6.2 4.2 6.2 60.0 10.5 1.8 ~
S
References
1.41
C
139
1.42
C
139
6F
(continued)
RENfi CSUK AND BRIGITTE I. GLANZER
128
TABLEXXXII (continued) Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6’
51.2
J2J
J3.4
54,s
55.6
55.6’
J6,6#
JFJ
JF,Z
JFJ
JFC
JF,S
JF.6
JF.~
SF
S
References
5-O-benzoyl-2-O-rnethyl-3-(trifluoroacetarnido)5.87 3.92 4.35-4.58 5.42 1.43 C I39 4.0 7.0 60.0 7.0 5-O-benzoyl-2-O-benzyI-3-(tri~uoroacetarnido)5.83 4.04 4.52 4.37 5.39 1.42 C 139 1.O 4.8 2.0 6.6 59.4 7.2 3-Acetamido-2,3,5,6-tetradeoxy-5-luoro-l-O-(4-nitrobenzoyl)-~,~-~~-~~~o-hexofuranose 6.65 2.212.7 4.7 4.13 4.75 1.43 C 140 b 4.9 8.0 2.0 2.7 6.6 24.6 48.7 23.9 Methyl 3-acetamido-2,3,5,6-tetradeoxy-5-luoro-~~~-ri~o-hexofuranoside 5.14 1.7-2.3 4.55 3.86 4.71 1.34 C 140 2.0 3.0 6.4 24.2 24.7 56.0 (1
a
’
J (N-H, H-3) = 9.0 Hz. J (H-2, H-2’) = 14.1 Hz.
(139) A. Hasegawa, M. Goto, and M. Kiso, J . Carbohydr. Chem., 4 (1985)627-638. (140) J. T. Welch, B. M. Svahn, S . Eswarakrishnan, J. P. Hutchinson, and J. Zubieta, Carbohydr. Res., 132 (1984)221-231. TABLEXXXIII ‘H- and IgF-N.m.r. Data for Fluorinated Aminodeoxypentopyranosides Compound H-1 H-2
H-3
H-4
H-5
H-5’
Ji.2
JZJ
J3.4
J4,5
54.5’
Js,s*
JFJ
JFJ
JFJ
JF,~
JF.S
JF,s’
6F
S
Methyl 4-~-~yl-2-amino-2,3-dideoxy-3-fluoro-~~-arabinopyranoside 4.80 3.25 4.65 -198.9 C 10.0 3.9 3.9 10.0 49.1 Methyl 2-amino-2,3,4-trideoxy-3.luoro-f~reo-pentopyranoside (Y-L4.75 2.8 4.5 -187.7 C
3.7 3.7
9.3 11.7
References
129
129 a
51.3
14.117.5
p-D-
4.0 7.3
2.85 9.0 12.0
4.45 9.016.0 50.5
Couplings for the axial and equatorial proton, respectively.
-186.0
C
129 n
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
IH-
I29
TABLEXXXIV and I9F-N.m.r. Data for 2-deoxy-2-halogeno-hexopyranosyl Fluorides
3,4,6-Tri-0-acetyl-2-deoxy-~-galactopyranosyl fluoride 2-bromoa anomer - 145.0
34,141
- 135.6
34
-139.4
34
-130.8
34
50.5 25.1 p anomer 49.7 10.0 2-iodoa anomer
50.0
27.6
p anomer 49.8 10.2 3,4,6-Tri-0-acetyl-2-deoxy-~-glucopyranosyl fluoride 2-bromoa anomer 5.8 2.5 9.0 9.0 9.0 51.5 25.2 /3 anomer
- 144.9
34,40, 141,142, 143,"
- 136.0
15.34, 40
3.94
- 147.6
40,144
3.47
- 138.9
40,144
- 139.8
34,40, 142,143
8.0 9.0 50.3 10.0 2-chloroa anomer 51.2
24.0
p anomer 51.0 10.6 2-iodoa anomer
2.4 50.5
11.0 27.8
b
(continued)
RENB CSUK AND BRIGITTE
I30
I. GLANZER
TABLEXXXIV (continued)
49.9 9.3 3,4,6-Tri-0-acetyl-2-deoxy-~-mannopyranosylfluoride 2-bromoa anomer
C
14,15 34,40 141.143'
C
142,145
-127.8
B
40,144
-118.6
C
144
-116.9
A
34,40, 141,143
-123.2
1.5 50.2
4.0 2.8 p anomer 5.95 I .8 52.0 2-chloroa anomer
9.5
3.92 3.6 49.5 2.0 p anomer
49.4 3.6 2-iodoa anomer
I .5
4.3 51.7 *3.9 p anomer 5.95 I .8 52.0
9.5
d
142
3,4,6-Tri-0-acetyl-2-deoxy-~-talopyranosyl fluoride 2-bromo-a-121.4
C
34
-114.6
C
34
49.8 4.6 2-iodo-a-
50.5
6.3
nJ(H-1,H-3)=0.4Hz.bJ(H-I,H-3)=0.2,J(H-l,H-5)=0.2Hz.LJ(H-2,H-4)=0.3Hz.dRef. reported 4.3 Hz,whereas Ref. 141 reported 1.0 Hz,in addition: J (H-I, H-3)= 0.5,J (H-2, H-4)= 0.5, J (H-1, H-5)= 0.2 Hz.
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
131
(141) J. C. Campbell, R. A. Dwek, P. W. Kent, and C. K. Prout, Carbohydr. Res., 10 (1969) 71-77. (142) K. R. Wood, P. W. Kent, and D. Fisher, J . Chem. Soc., C , (1966) 912-915. (143) L. D. Hall and J. F. Manville, Carbohydr. Res., 8 (1968) 295-307. (144) L. D. Hall and J. F. Manville, Can. J. Chem., 47 (1969) 379-385. (145) J. C. Campbell, R. A. Dwek, P. W. Kent, and C. K. Prout, Chern. Commun., (1968) 34-35.
TABLEXXXV 'H- and IgF-N.m.r. Data for 2-Deoxyhexopyranosyl Fluorides and 2,3-Dideoxy-2-fluoro-hexopyranosides Compound H-1
H-2
H-3
H-4
H-5
H-6
H-6'
J1.z
JZJ
53,4
54,s
55.6
55.6
56.6'
JF,I
JF.Z
JFS
JF,~
JF,S
JF,~
JF,~
4.34 4.5
4.09 -131.1 12.8
2-Deoxy-~-arabino-hexopyranosyl fluoride 3,4,6-tri-O-acetyla anomer 5.08 4.14 5.76 2.43/1.86~ 5.32 9.5 2.0 1.6/2.850 5.2/10.8 51.2 5.0/38.0 9.5 p anorner
6F
S References
C
14,15, 33,34,40 n.6
-125.2
c
40 (I
15.3d10.7e 3,4,6-tri-O-benzoyl-a5.82 2.7512.07~ 5.72 1.5/2.5a 51.1 5.3/38.3 2-Deoxy-D-lyxo-hexopyranosyl fluoride 3,4,6-tri-O-benzoyl-a6.01 2.80/2.0a 5.76 6.00 2.0/2.5a 11 3 6 . 0 3.0 1.0 51.0 3,6-di-O-benzoyl-a5.88 2.0-3.0 5.50 2.0/2.0 11.5/5.5 3.0 52.0 4,6-di-O-benzoyl-a5.93 1.8-2.7 4.2-4.7 5.68 2.012.0 3.0 1.0 52.0
-
4.57
-
-130.1
C
33 a.c
4.75 6.0
4.10-4.70 6.0
4.2-4.8
-
C
I46 n.6
C
146 a
-
4.2-4.7
-
C
146
(continued)
TABLEXXXV (continued) Compound
H-1
H-2
H-3
H-4
H-5
H-6
H-6'
Ji.2
52s
53.4
54.5
55.6
55.6'
56.6'
JFJ
JF,Z
JF.3
JF.4
JF,~
JF,6
JF,~
Methyl 2-deoxy-2-fluoro-~-o-ribo-hexopyranoside 4,6-0- benzylidene-3-deoxy4.10 4.25 1.74/2.3e 2.93 3.05 3.40 7.5 11.3/5.5e 11.5/4.0e 9.0 10.0 4.7 3.5 49.0 11.5a/5.0e 1.5
S References
6F
C
4.09 10.0
The symbol e stands for equatorial, and a for axial. J (H-2e, H-2a) 13.5 Hz." J (H-3a, H-3e) = 11.5 Hz.
138 4.d
=
13.9 Hz. J (H-2e, H-2a)
=
(146) I. Lundt and C. Pedersen, Acta Chem. Scand., 25 (1971) 2749-2756.
TABLEXXXVI
'H-and 'gF-N.m.r. Data for Fluorinated 6-Deoxyhexopyranose Derivatives Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
51.2
52.5
53.4
J4.5
556
Js.6
36,6
JFJ
JF,Z
JF,~
JF,~
JFJ
JF.~
JF,~
6F
S
-119.8
C
150
-208.3
D
147,148
-208.0
D
147,148
-209.9
C
148
-209.9
C
148 *
-197.0
D
149
References
2,3,4-Tri-0-acetyl-6-deoxy.cu-~-mannopyranosyl fluoride 5.45
5.00
-
5.32
3.96 6.0
1.2
49.0
2,6-Dideoxy-2-fluoro-~-galactopyranose a anomer
0.5
49.4
13.0
4.0
p anomer 3.5 51.0 14.5 1,3,4-tri-O-acetyl-P6.42 5.39 4.0 10.5 3.5 0.5 49.5 11.5
4.0 5.33 6.5 3.8
Trifluoromethyl3,4-di-0-acetyl-2,6-dideoxy-cu-~-galactopyranoside 3.9 9.5 3.5 2.0 6.6 0.2 48.5 13.0 3.5 2,6-Dideoxy-2-fluoro-~-glucopyranose a anomer 2.5
50.5
16.0 (continued) 132
TABLEXXXVI (continued)
p anomer -197.2
D
149
C
149
C
149
C
149
0.1 49.5 14.5 1,3,4-tri-O-acetyla anomer
8.0 3.5
p 4.0 0.5
9.5 51.5 anomer
9.5 14.5
9.5
6.5
9.5 49.0
9.5 12.5
9.5
6.5
Trilluoromethyl 3,4-di-O-acetyl-n-~-glucopyranoside 4.0 0.5
9.5 48.0
9.5 12.0
9.5
6.2
b
2,6-Dideoxy-2-fluoro-~-mannopyranose a anomer
51.0 20.1 p anomer
-202.7
D
149
-221.2
D
149
C
149
C
149
30.0
8.0 49.5 30.5 1,3,4-tri-O-acetyl-a-
7.5
49.0
30.0
Trifluoromethyl3,4-di-O-acetyl-2,6-dideoxy-/3-~-mannopyranoside -220.8
0.5 17.0
2.2 50.5
9.8 30.0
8.5 2.0
6.2
“ J (OCF,,F) = 1.25 Hz.* J (OCF,,F) = 0.8 HZ.
(147) W. Korytnyk, S. Valentekovic-Honvath, and C. R. Petrie, 111, Tetrahedron, 38 (1982) 2547-2550. (148) C. G. Butchard and P. W. Kent, Tetrahedron, 35 (1979) 2551-2554. (149) G. C. Butchard and P. W. Kent, Tetrahedron, 35 (1979) 2439-2443. (150) Y. V. Voznyi, Bioorg. Khim., 7 (1981) 239-241.
I33
R E N k CSUK AND BRIGITTE I. GLANZER
134
TABLEXXXVII 'H- and lPF-N.m.r. Data for Fluorinated Hex-L(or 3)-enopyranose Derivatives Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
J1.z
52.3
J3.4
J4.5
J5.6
Js.6,
J6,6
JFJ
JFJ
JFJ
JF,~
JFC
JF,~
JF,~
3-Deoxy-cu-~-erytlrro-hex-2-enopyranosyl fluoride 2,4,6-tn-O-acetyl5.70 5.90 5.40 4.10 4.30 2.0 8.5 56.0 2,4,6-tn-O-benzoyl4.90 6.00 6.25 6.05 4.50 2.0 9.0 56.0 3,4-Dideoxy-~-glycero-hex-3-enopyranosyl fluoride 6-0-acetyl-2-dose a! anomer 5.57 6.29 7.10 4.97 4.40 4.32 5.0 5.0 11.8 10.4 1.7 51.2 5.9 /3 anomer 5.58 6.35 7.18 4.83 4.1-4.6 10.7 3.2 52.0 4.8 6-0-benzoyl-2-dose a! anomer 5.05 4.61 4.55 5.57 6.28 7.12 10.4 1.6 4.8 4.8 12.0 51.0 5.9 p anomer 5.58 6.36 7.13 4.90 4.00-4 S O 10.5 3.0 52.0 4.6 2,3,6-Trideoxy-6-fluoro-cu-~-erythro-hex-2-enopyranoside ethyl 4-0-acetyl5.06 5.85 5.27 4.1-4.40 4.53 2.5 9.6 48.0 48.0 methyl 4-0-acetyl5.00 5.95 5.32 3.75-4.40 4.59 10.2 48.0 48.0 " J (H-I, H-3) H-5) = 2.0 Hz.
=
'
0.7, J (H-3, H-5) = 2.4 Hz. J (H-3, H-5)
=
6F
-141.6
S
References
C
151
C
151
C
151,152 Y
-118.6
C
152 b
-135.2
C
151,152 c
-118.2
C
152
A
153
A
153
2.0 Hz. J (H-1, H-3) = 0.8, J (H-3,
(151) K. Bock and C. Pedersen, Tetrahedron Lerr., (1969) 2983-2986. (152) K. Bock and C. Pedersen, Acra Chem. Scand., 25 (1971) 1021-1030. (153) G. Descotes, J. C. Martin, and Tachi-Dung, Carbohydr. Res., 62 (1978) 61-71.
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
135
TABLEXXXVIII 'H- and I9F-N.m.r. Data for Miscellaneous Fluorinated Hexopyranoside Derivatives
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
Ji.2
J2.3
J3,4
54,s
55.6
5 5 , ~
5 6 , ~
JFJ
JFJ
JF.~
JF,~
JFd
JF.6
JF.6'
SF
S
References
Methyl 4-O-benzoyl-6-bromo-2,3,6-trideoxy-2-fluoro-~-~-r~~~-hexopyranoside 4.54 4.45 1.9312.76e 5.02 3.89 3.47 3.60 C 11.0/5.0 11.0 7.5 48.0 Ethyl 4-0-acetyl-2,3,6-trideoxy-6-fluoro-cu-~-e~thro-hexopyranoside 4.4-4.9 1.68-1.85 4.4-4.9 3.3-4.05 4.45 A 48.0 48.0 Ethyl 4-0-benzyl-2,3,6-trideoxy-6-fluoro-cu-~-e~th~o-hex-2-enopyranoside 6.04 5.67 3.30-4.30 4.57 -233.2 2.4 2.4 48.0 48.0
" J (H-30, H-3e) =
138 a
1.53
C
1 I7
11.O Hz.
TABLEXXXIX 'H- and I9F-N.m.r. Data for 2-Deoxy-2-halogenopentopyranosyl Fluorides
Compound H-1 H-2
H-3
H-4
H-5
H-5'
JIJ
J2.3
53.4
54,s
54.5'
JSd*
JFJ
JFJ
JFJ
JF,~
JFS
JF,I'
SF
S
References
C
141
-126.4
C
34
-121.6
C
34
-126.7
C
34
2-Deoxy-~-arabhopyranosylfluoride 2-bromo-P-
51.2 25.8 3,4-di-O-acetyl-2-bromo-a-
50.5
3.5
3,4-di-O-acetyl-2-iodo-a50.0
5.0
3,4-Di-O-acetyl-2-deoxy-~-lyxopyranosyl fluoride 2-bromoa anomer
50.1
3.5 (continued)
RENB CSUK AND BRIGITTE
136
I. GLANZER
TABLEXXXIX (confinued) Compound H-1 H-2
H-3
H-4
H-5
H-5'
51.2
52.3
J3c
J4.s
54,s
Js,~
JFJ
JFJ
JFJ
JF,~
JF,S
JFF
SF
S
References
p anorner -144.1
34 a
50.8 26.8 2-iodoa anorner -120.4
34
-141.1
34
50.0 3.5 p anomer a
50.8
29.0
3,4-Di-O-acetyl-2-deoxy-~-nbopyranosyl fluoride 2-brornoa anomer
51.3
- 147.6
34
-127.9
34
-141.9
34
- 123.9
34
- 146.5
34
25.1
p anomer 50.4 3.4 2-iodoa anomer
50.7
27.6
p anorner 51.0
6.5
3,4-Di-O-acetyl-2-deox~~-xylopyranosylfluoride
2-bromoa anorner b
50.3
25.1 p anorner - 128.0
51.0
34
9.0 (continued)
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
137
TABLEXXXIX (continued) Compound H-1 H-2
H-3
H-4
H-5
H-5'
11.2
32.3
53.4
54.5
54,s
J5.5'
JF,l
JF,Z
JFS
JF.4
JFJ
JFJ'
SF
S
References
-138.0
C
34
2-iodo(x anomer b
50.8
29.0 p anomer
C
-122.5 51.1
34
9.2
May have to be reversed with
I Y - D - X Y ~ . I,
May have to be reversed with P-D-~Yxo.
TABLEXL 'H- and I9F-N.m.r. Data for 2-DeoxypentopyranosylFluorides Compound H-1
H-2
H-3
H-4
H-5
H-5'
Ji,2
J2.3
J3,4
54,s
543
J5,S
JFJ
JF,~
JFS
JF,~
JF.S
JF.S
SF
S
References
3,4-Di-0-benzoyl-2-deoxy-~-erythro-pentopyranosyl fluoride a anomer 5.70 2.1-2.6 2.0/3.0a 51.0 p anomer 5.53 1.7-2.4 2.112.1 11.0d6.0e 51.0
5.75 3.0
5.45 10.2
4.45 5.2
3.98 11.2
C
154
5.57 3.0
5.50 2.0
3.70 1.0
3.80 13.0
B
154
4.18 10.6
C
4.05 12.5
C
3,4-Di-O-benzoyl-~-threo-pentopyranosyl fluoride a anomer 5.77 2 . 6 Y 2 . 1 0 ~ 5.75 5.46 4.00 2.6/1.8e 50.7 p anomer 5.77 3.2/3.2e
5.0110.8
9.2
10.0
5.5
5.41
5.22 3.0
4.54
0.6
I54 ax
5.0/36.4 2.2-2.8 3.2e
2.5
154 U.d
51.0
" The symbol a stands for axial, and e for equatorial. Hz. ,I J (H-20, H-2e)
J (H-20, H-2e) = 13.0. J (H-2a, H-2e) = 13.8
= 15.0 Hz.
(154) K. Bock and C. Pedersen, Acta Chem. Scand., 25 (1971) 2757-2764.
RENk CSUK AND BRIGITTE I. GLANZER
138
TABLEXLI 'H- and 'PF-N.m.r.Data for Fluorinated DeoxypentofuranoseDerivatives Compound H-1 H-2
H-3
H-4
H-5
H-5'
51.1
J2,3
J3,4
54,s
J4.59
Js,r
6F
S
References
JFJ
JF,Z
JFS
JFA
JFJ
JFJ'
-106.5
C
155
-103.2
C
155
-
C
108
-
N
I10
3,5.Di-O-benzoyl-2-bromo-2-deoxy-~-pentof11ranosy1 fluoride a-D-arabino6.07 4.44 0.8 3.0 61.0 7.9
5.66
4.60
4.90
p-D-XY/O-
6.03
4.42
5.85
5.14 4.71 5.8 5.8 65.0 6.0 6.0 2-Deo~y-2-fluoro-cu-D-~rabinofuranosyl bromide 3-0-acetyl-5-0-benzoyl5.18 4.60 6.45 5.34 0.0 12.0 50.0 23.0 3,5-di-0-benzoyl5.5 4.75 6.67 5.6
4.71
5.0
50.0
(155) K. Bock, C. Pedersen, and P. Rasmussen, Acla Chem. Scand., Ser. B , 29 (1975) 185- 190.
TABLEXLII lH- and "F-N.m.r. Data for 3-C-Branched 3-Fluoro-~-glucofuranoses Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
51.2
52.3
J3,4
J4,s
J5,6
J5.6
56,6
JFJ
JF,Z
JFS
JF,~
JF.~
JF,~
JF,~
3-Deoxy-3-fluoro-~-glucofuranose 3-C-(acetoxymethyl)1,2-O-isopropylidene-a5.88 4.63 3.76 4.0
-
4.15
S
References
C
156 Y
11.0 (continued)
I39
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES TABLEXLII (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
JLZ
J2.3
53.4
J4.5
J5,6
J5.6
J6.6
JFJ
JF,~
JF.~
JF,~
JF,S
JF.6
JF,~
1.2: 5,6-di-O-isopropylidene-a5.88 4.63 3.70 4.0 11.0 6-0-benzoyl1,2-0-isopropylidene-a5.91 4.64 4.55 3.85 3.8 8.0 10.0 25.0 3-C-(ethoxyallyl)1,2-0-isopropylidene-a6.06 4.82 4.10-4.40
-
4.30
S
References
C
156 b
-
4.32
C
156 c
C
157
3.99
C
157
4.06
C
157
C
157
C
157
C
157
C
157
C
157
3.74-3.92
4.0
11.0
I ,2 :5,6-di-O-isopropylidene-a-
6.03 4.0
4.92 14.5
4.75 8.0 27.0
4.21
-
3-C-[(ethoxycarbonyl)(formylirnino)methyl]-1,2: 5.6-
di-0-isopropylidene-a6.02 4.94 4.0 13.0
4.88 6.0 27.0
4.4 6.0
-
3-C-[ethoxy(ethoxycarbonyl)(formylamino)methyl]-1,2: 5,6di-0-isopropylidene-a5.82 4.67 4.89 2.50 3.82 3.5 6.5 14.5 27.5 3-C-(hydroxymethyl)I ,2: 5,6-di-O-isopropylidene-a5.90 4.67 3.60 4.50 4.0 12.0 1,2-0-isopropylidene-a5.90 4.58 3.60 4.20 4.0 12.0 3-C-[ 1,2-di(hydroxyethyl)l-1,2 :5,6-di-O-isopropylidene-a5.88 4.69 3.80 4.60 4.0 12.0 I ,2-O-isopropylidene-3-C-(methoxyallyl)-a6.07 4.83 4.30-4.67 4.16 4.16 3.5 12.0
(continued)
RENE CSUK AND BRIGITTE I. GLANZER
I40
TABLEXLII (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6’
Jt,t
52.3
53,4
54,s
55.6
55.6’
56,6
JFJ
JF,~
Jp.3
JF.~
JFJ
JF,~
JF,~’
I ,2: 5,6-di-O-isopropylidene-3-C-(methoxycarbonyl)-a5.99 4.68 4.64 4.00 4.40 4.0 8.0 15.0 26.0
S
References
C
157
“ J (H-l’, H-1‘3 = 13, J (H-l’, F) = 14 Hz, J (H-I”, F) = 34 Hz; (H-1‘) = 4.84 p.p.m.; (H-1”) = 4.27 p.p.m. J(H-1’. F) = 13 Hz;J(H-I”, F) = 34 Hz;J(H-I’, H-I”) = 13 Hz; (H-1’) = 4.70p.p.m.; (H-1’7 = 4.24 p.p.m. J (H-l’, H-I”) = 13 Hz; J (H-1’, F) = 14 Hz; J (H-I”, F) = 34 Hz; (H-1’) = 4.84 p.p.m.; (H-1’3 = 4.29 p.p.m. L.
(156) A. J. Brink, 0. G. De Villiers, and A. Jordaan, Carbohydr. Res., 54 (1977)285-291. (157) K.Bischofberger, A. J. Brink, and A. Jordaan, J . Chem. Soc., Perkin Trans. I , (1975)2457-2460.
TABLEXLIII ‘H- and I9F-N.m.r. Data for 3-Deoxy-J-C-(monoor di)tluoromethylene-D-hexo(or pento)furanoses
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6’
Jt,2
52.3
53,4
54.5
55.6
55,6’
56,6’
JF,I
JFZ
JF,~
JF,~
JFS
JF.6
JF.6’
SF
S
References
3-Deoxy-3-C-(fluoromethylene)-1,2 :5,6-di-O-isopropylidene-cu-~-hexofuranose ribo-
(2)isomer 5.90 5.06
4.3
1.4
1.5
1.0
( E ) isomer 5.82 5.36 4.0 1.3 0.0 1.3
2.1 82.0 2.0
5.12 3.2 1.0
4.63 7.6
3.36 6.6 1.9
4.00 6.6 0.9
3.76 8.4 0.9
-202.9
3.84
-
4.14
-198.5
C
37 a
C
37 b
82.5
5.5
1.8 82.9
4.87 8.3 2.3
4.42 6.0
1.2 82.0
4.67 6.5 1.7
xylo-
(Z)isomer 5.84 4.93 3.7 0.3 0.3 1.7 (E) isomer 5.81 5.28 3.7 1.0 0.0
1.0
3.78 8.3 1.6
-205.8
0.5
4.00 7.2 0.7
4.42 6.5
4.04 7.1
3.78 8.1
-201.9
0.5
0
0
C
37 c
C
37 d
(continued)
141
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES TABLEXLIII (continued) Compound H-1 H-2 J1.2
J2,3
JF,l
JFJ
H-3
H-4
H-5
H-6
H-6'
J3.4
54,s
554
55.6'
56,6'
JF,~
JFJ
JF,~
JF.~
6F
S
References
3-Deoxy-3-C-(difluoromethylene)-l,2 :5,6-di-0-isopropylidene-cu-o-hexofuranose ribo5.87 4.4 1.4 90.0 xylo5.91 4.0 f0.5 8
5.22 2.6 1.2 5.22
1.0 1 .o
4.86 4.5 3.0 3.0
4.14 6.2 1.0
4.0
3.79 8.0
-247.1 -243.4
C
37
5.5
4.77 7.9 2.8
4.47 6.2 0.5
4.05 7.0 0.2
3.78 8.4 1.7
-244.1 -241.5
C
37
1.o
h
3-Deoxy-3-C-(difluoromethylene)-l,2-0-isopropylidene-(~-o-furanose ribo-hexo5.91 5.28 4.96 3.2 4.3 4.0 1.5 3.2 I 2.8 3.2 erythro-pentodialdo- 1 ,4-furanose 6.04 5.29 5.16 4.1 2.1 '1.0 0.9 3.6 g 2.0 4.0
3.90 2.5 1.2
3.50-3.75 8.5
9.45
-244.4 -241.0
C
158
-245.3 -244.0
C
I58 J
2.1
* (H-3') = 6.83 p.p.rn.; J (H-2, H-4) = 1.4 Hz. (H-3') = 7.01 p,p.rn.; J (H-2, H-4) = 1.3 Hz. (H-3') = 6.82 p.p.m. (H-3') = 6.74 p.p.m. J (H-2, H-4) = 1.2 Hz; J (F, F) = 38 Hz. f Couplings to rrans-oriented fluorine. Y Couplings to cis-oriented fluorine. * J (F, F) = 35.5 Hz. ' J (H-2, H-4)= 1.0 Hz; J (F, F) = 40.8 Hz. J (H-2, H-4) = 1.2 Hz; J (F, F) = 33.8 Hz. J
(158) J. M. J. Tronchet, B. Gentile, J. Ojha-Poncet, G. Moret, D. Schwarzenbach, F. Barbalat-Rey, and J. Tronchet, Carbohydr. Res., 59 (1977) 87-93.
TABLEXLIV 'H- and 'gF-N.m.r. Data for Fluorinated C(or O)-Branched o-Pentopyranose Derivatives Compound H-1 H-2
H-3
H-4
H-5
J1.z
J2,3
J3,4
54.5
54,s'
H-5' Jss,
JF,I
JF,~
JF.~
Jp.4
JF,~
JF,Y
S
1,2-Di-0-acetyl-5-0-benzoyl-2-deoxy-2-fluoro-3-0-formyl-o-arab~ofuranose (Y anomer 6.36 5.71 4.12 4.98 C
References
156
5.5 16.0
(continued)
RENB CSUK AND BRIGITTE I. GLANZER
142
TABLEXLIV (continued) Compound H-1 H-2
H-3
H-4
52.3
53.4
54,5
H-5 54,5'
H-5'
Ji,z
5
References
JFJ
JFJ
JFJ
JF.~
JFJ
JFS,
4.70
C
156
4.70
C
157
4.70
C
156
4.60
C
156
J5.5,
p anomer 6.44
5.35 4.35 3 .O 10.0 21.0 3 4 4Acetoxyrnethyl)-3-deoxy-3-fluoro-~-xylofuranose 5-0-acetyl- 1,2-O-isopropylidene-a5.98 4.64 4.00 4.0 12.0 1.2,5-tri-O-acetyla anomer 6.50 5.46 4.00 5.0
24.0
p anomer 6.10
-
4.10
5.34 14.0
3-Deoxy-3-fluoro-l,2-O-isopropylidene-cu.~-xylofuranose 3-C-(hydroxymethyl)5.92 4.58 4.0 12.0 3-C-(ethoxyallyl)5.95 4.68 4.0 12.0
3.60
-
4.40
C
157
4.30
-
3.81
C
157
5.0
TABLEXLV 'H- and I9F-N.m.r. Data for Miscellaneous, Fluorinated Branched Monosaccharides Compound H-1 H-2
H-3
H-4
H-5
&,a
J3.4
J4.5
J5.6
H-6 55,~
H-6'
JI,~ JF.1
JFJ
JF,~
JF,~
JFS
Jp.6
JF,~'
6F
S
References
56,6'
5(R)-5-C-Acetoxy-6-deoxy-6-fluoro-l,2 :3,4-di-O-isopropylidene-~-~-arabino-hexo-l,5pyranosd-dose 5.55 4.13 3.9 0.0
4.52l' 6.2
4.62"
4.69 46.0
5.13 9.6 46.8
-236.0
C
48
(continued)
N .M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
143
TABLEXLV (continued) ~~______
Compound H-1 H-2 J1t
4
3
JF,l
JF.2
H-3
H-4
H-5
H-6
H-6'
J3.4
J4.5
55.6
55.6'
56.6'
JFS
JF,~
JF.5
JF.6
JF.6'
SF
S
References
~~~
l-O-Acetyl-2,3,5,64etra-O-benzoyl-4-deoxy-4-fluoro-~~-hexofuranose galacto6.56 5.82 6.11 C 159 2.2 4.7 0.0 15.2 4.0 glucoC 159 6.69 5.47 5.88 6.10 5.02 4.72 0.0 0.0 0.0 0.0 7.3 5(S)-5-C-Acetoxy-3,5-O-benzylidene-6-deoxy~0uoro-l,2-O-isopr~pylidenea-D-xylo-hexo1,4-furanosd-dose 6.02 4.66 4.23" 4.62" 4.90 5.13 -240.2 C 48 3.4 0.0 0.0 9.7 45.1 46.7 2,~,4-Tri-~-acetyl-2-fluoro-~-~-ribopyranosyl fluoride 6.47 5.31-5.61 4.22 3.83 -153.0 C 98 2.0 1.5 13.0 -133.0 51.0 0.0 1.5 Trifluorometbyl 2,3,4-tri-0-acetyl-2-fluoro-/3-~-ribopyranos~de C 98 6.44 5.58 5.44 4.26 3.86 -133.0 4.0 2.5 2.5 13.5 6.5 1.2 1-0-Acetyl-2,3,5-trii-O-benzoyl-4-fluoropentofuranose a-L-lyxo-
6.66 3.0 0.0
5.91 5.1 0.8
6.19
4.85
4.65
C
159
17.3
P-D-ribo-
6.61 5.78 6.00 4.78 5.53 C 159 0.0 5.3 1.0 3.9 8.0 4-0-Acetyl-5-deoxy-5-fluoro-1,2-O-isopropylidene-3-O-tosyl-~-~-~~reo-pento-l,4-furanos-4dose 6.08 4.83 5.28 4.48 4.73 -236.1 C 48 3.8 45.7 46.4 a
The assignments may have to be reversed.
(159) R. J. Ferrier and S. R. Haines, J . Chem. SOC.,Perkin Trans. 1 , (1984) 1675-1681.
RENk CSUK AND BRIGITTE I. GLANZER
I44
TABLEXLVI lH- and lgF-N.m.r. Data for Fluorinated Ketopyranose Derivatives Compound H-1 H-1' J3,4 J1.r JF,I
JF,V
H-3
H-4
J4,s
55.6
H-5 55.6
56.6'
H-6
JFJ
JF.~
J F ~
JF,~
H-6'
6F
S
References
C
160
D
161
C
162
JF,~'
l-Deoxy-l-fluoro-2,3 :4,5-di-O-isopropylidene-~-fructopyranose 4.37 4.37 4.25 4.64 4.39 3.74 3.92 -230.1 10.0 8.0 3.0 2.0 12.0 1.5 48.0 48.0 4-Deoxy-4-fluoro-~~-fructopyranose 3.72 3.56 4.06 4.78 4.26 4.03 3.75 11.8 9.75 3.5 1.6 2.0 13.0 1.8 12.9 49.0 6.5 1.3 7.8 4-Deoxy-4-fluoro-1,2-O-~sopropylidene-~-~-sorbopyranose -195.6 5.0 3.0 9.0 46.0 9.0
(160) P. J. Card and W. D. Hitz, J . Am. Chem. Soc., 106 (1984) 53484350. (161) M. BudMnsk$, M. Cern$, J. Dole2alovB, M. Kulhhnek, J. Pacak, and M. Tadra, Collect. Czech, Chem. Commun., 49 (1984) 267-274. (162) M. Sarel-Imber and E. D. Bergmann, Carbohydr. Res., 27 (1973) 73-77.
TABLEXLVII 'H- and '9F-N.m.r. Data for Mono- and Di-fluorinated Ketofuranose Derivatives Compound H-1 H-1'
H-3
H-4
H-5
H-6
54.5
55,6
Js,~
56.6
H-6'
JIJ*
53.4
'JFJ
JFJ
JF,3
JF.4
JF,S
JF,6
JF,~
"JFJ
JFJ-
JFJ
JF,~
JF.5
JF,6
JF,~
SF
S
References
1,3,4,6-Tetra-O-ace~l-~-fructofuranosyl fluoride a anomer 4.2-4.5 1.0
5.55 5.0 7.0
5.00
A.2-4.5-
C
163
d.2-4.5-
C
163
p anomer 4.2-4.5 7.0
5.4-5.7
16.0 6-O-Benzoyl-l-deoxy-l-flnoro-2,3-O-isopropylidene-~-~-f~ctofuranose 4.38-4.6 1 -225.2 C
164 b
47.7 (continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
145
TABLEXLVII (continued)
Methyl 3,4,6-tri-O-benzoyl-l-deoxy-l-fluoro-~-fructofuranoside a anomer 4.68 4.39 5.81 5.60 4 . 4 1 - 4 . 1 7 -230.8 B 164,165 b 2.2 4.9 46.9 p anomer 4.68 4.39 5.81 6.12 -232.3 C-B 164, 6.5 5.5 165” 46.9 6-Deoxy-6-fluoro-2,3-O-isopropylidene-l-O-~tolylsu~onyl-~-~-f~ctofuranose 164,” 4.05-4.95 -226.8 C 18.1
46.2
1,6-Dideoxy-l,6-di~uoro-2,3-O-isopropylidene-~-~-fructofuranose 4.52
4.57
5.0
5.0
C
164, 166,”
-233.1 -230.3
C
164, 165,”
-230.9 -234.8
C-B
164, 165,”
-225.0 -225.5
47.1 17.7
46.4
Methyl 3,4-di-0-acetyl-1,6-dideoxy-1,6-d~uoro-~-fructofuranoside a anomer 4.31
4.31 2.2
5.61 6.4
5.19 3.5
3.94 3.5
4.36
46.4 22.0
41.3
p anomer 4.42 9.6 46.6
4.26 1.3
5.19 7.1
5.64 5.0
3.90 2.4 22.3
4.2-4.34 10.5 46.9
“ The symbols I, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atom. 6F values were taken from Refs. 164 and 165; although no standard was reported, C6F6 had obviously been used. (163) B. Erbing and B. Lindberg, Acta Chem. Scand., Ser. B , 30 (1976) 12-14. (164) R. D. Guthrie, I. D. Jenkins, and R. Yamasaki, Ausf.J . Chem., 35 (1982) 1003-1018. (165) R. D. Guthrie, I. D. Jenkins, and R. Yamasaki, Ausf.J . Chem., 35 (1982) 1019-1029. (166) J. Pacirk, J. HalaSkovir, V. Stepirn, and M. Cvernq, Collecf. Czech. Chem. Commun.,37 (1972) 3646-3651.
RENh CSUK AND BRIGITTE I. GLANZER
I46
TABLEXLVIII 'H- and "F-N.m.r. Data for 2-Deoxy-2-fluorohexopyranosylFluorides Compound H-1 H-2
&'I
H-3
H-4
H-5
H-6
H-6'
J1.t
Ja.3
53.4
54.5
55.6
J5,6'
J6.6'
YF.1
JFJ JFZ
JF,~ JF,~
JF,~
JF,~
JF,~ JF.~
J F , ~ J(F,F)
JF,~ J F ~
'JF,~
m
S
a
References
JF,~
2-Deoxy-2-fluoro-D-gdactopyranosy~ fluoride a anomer 54.3
24.5 48.9 14.6 3,4,6-tri-O-acetyla anomer 5.76 4.68 5.34 2.7 9.7 3.2 52.5 23.0 0.9 48.0 11.0 p anomer
D
-153.0 c-212.0 18.0
C
-142.3 -209.4 14.5
C
147
-150.1 -204.0 20.8
D
47,147, 167
47,147
4.9 5.48
4. 5
4. 5
4.06
1.0
4
,46,53, 54.147
3.5
52.5 14.3 5.0 51.3 14.8 2.5 2-Deoxy-2-fluoro-~-glucopyranosylfluoride (Y anomer 54.1 23.8 0.0 47.7 13.8 3,4,6-tri-O-acetyla anomer 5.80 4.54 5.54 2.9 9.6 9.5 53.3 23.8 0.5 0.0 48.3 12.3 p anomer 5.41 4.46 5.32 6.1 7.9 8.9 1.0 51.7 11.2 4.0 49.0 15.0
-152.2 -211.9 19.6
5.10 9.5 0.0 5.10 9.0 0.0
4.06
-
4.42
-151.5 -204.5 18.8
C
18.36, 58,147, 168,169
4.26
4.20 12.0
-140.3 -200.9 15.8
C
18,36, 147
3.36 5.9
3.21 12.4
-147.5 -222.4 12.7
D
47,167, 170
0.0 0.0
3.89 0.0 0.0
2-Deoxy-2-fluoro-~-mannopyranosyl fluoride
p anomer 5.01
0.0 47.4 17.8
4.41 2.7 2.8d 51.8
3.23 9.7
3.11 9.9
2.95 2.4
30.0 (continued)
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
147
TABLEXLVIII (continued)
3,4,6-tn-O-acetyla anomer 4.06 5.76 4.89 5.27 5.42 4.42 9.0 2.0 2.5 10.0 0.0 2.5 0.0 48.0 0.0 0.0 4.0 48.5 27.0 p anomer 5.51 4.90 5.14 5.29 3.86 4.30 4.30 1.0 2.5 8.5 7.5 7.0 8.0 0.5 0.8 48.5 13.5 '49.0 22.4 2.0 0.5 3,4,6-Tri-0-acetyl-2-deoxy-2-fluoro-~-~-talopyranosyl fluoride 5.33 4.69 5.07 5.19 4.28-3.90 1.0 3.0 3.0 1.0 48.0 6.0 14.5 51.0 25.0
-143.4 -207.2 20.0
C
18,36
-146.4
C
18,36, 58,147, 168,I69
C
45,147
I- 277.0
13.5 -145.8 -218.4 12.8
The symbols I, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atom. Ref. 53 reported a coupling of 20 Hz. Ref. 54 reported a chemical shift of -223 p.p.m. Ref. 47 reported a coupling of 9.8 Hz; additional couplings: J (F, H-4) = 1 Hz, and J (F, H-6) = 1 Hz. 'Ref. 169 reported a coupling constant of 34 Hz. 'Ref. 147 reported a chemical shift of -220.1 p.p.m. (I
(167) N. Satyamurthy, G. T. Bida, H. C. Padgett, and J. R. Barrio, J . Carbohydr. Chem., 4 (1985) 489-512. (168) J. Adamson, A. B. Foster, L. D. Hall, and R. H. Hesse, Chem. Commun., (1969) 309-310. (169) T . Ido, C. N. Wan, J. S. Fowler, and A. P. Wolf, J . Org. Chem., 42 (1977) 23412342. (170) S. G. Withers, I. P. Street, and S. J. Rettig, Can. J . Chem., 64 (1986) 232-236.
TABLE XLIX IH- and '9F-N.m.r. Data for 3(or 4, or 6)-Deoxy-3(or4, or 6)-fluorohexopyranosyI Fluorides
3-Deoxy-3-fluoro-~-glucopyranosylfluoride 2,4,6-tri-O-acetyla anomer 5.73 5.02 4.79 5.29 4.07 4.26 4.14 2.6 9.5 9.5 9.5 12.5 1.0 0.5 52.2 22.6 51.5 13.5 1.0 4.0 14.5 p anomer 4.57 5.31 3.79 4.24 4.21 5.27 5.2 12.4 6.0 7.7 8.5 9.5 51.5 10.8 0.0 0.0 0.0 15.0 51.3 13.5 1.0 2,3,6-Tri-0-acetyl-4-deoxy~4-fluoro-~-galactopyranosy~ fluoride a anomer 2.3
9.9 24.0 2.8 R anomer
2.5
-149.8 -201.1
C
II
-138.8 -196.0 3.0
C
11
C
19
C
19
1 .o
0.5
3.7 25.4
28.0
1.0
1.0
6.7
10.5 2.5 1.0 9.6 2.5 0.8 1.3 25.6 25.2 1.0 4-Deoxy-4-fluoro-~-glucopyranosyl fluoride 2,3,6-tri-O-acetyla! anomer 5.68 4.87 5.6 4.52 4.5 2.8 10.2 8.9 9.5 52.0 23.7 0.8 14.6 48.5 3.0 p anomer 5.37 5.02 5.30 4.65 3.96 4.48 5.8 7.4 8.6 9.4 2.9 4.9 0.8 0.8 0.9 0.6 50.7 10.2 0.5 0.0 15.8 49.5 4.9 1.6 6-Deoxy-6-fluoro-~-glucopyranosyl fluoride 2,3,4-tri-O-acetyla anomer 5.50 5.17 4.03 4.51 5.75 4.94 2.7 9.9 9.4 10.0 2.2 3.5 52.4 23.8 23.9 47.0
1.3 1.0
4.12
-150.0 - 199.5 0.6
C
12
4.24 12.1 0.6 1.9
-137.0 -199.4 3.1
C
12
4.45 10.8
-150.4 -234.8 0.2
C
84
47.0 (continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
149
TABLEXLIX (continued)
p anomer 5.4
4.93
-
5.5
52.7
5.25 9.0
3.95
4.51
4.51
10.4 20.6
46.6
-137.0 -232.2 0.9
C
84
46.6
" The symbols I, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atom.
TABLEL 'H- and I9F-N.m.r. Data for Dideoxydifluorohexo-pyranosesand -pyranosides Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
J1.z
Jz.3
J3,4
54.5
Js,6
55.6'
56.6'
'JFJ
JFJ
JF.~
JF,~
JFJ
JF.6
JF,~
'JF,I
JFJ
JF.3
JF,4
JF,~
JF,6
JF,~
6F1 6F2 J(F,F)
S
-118.8 - 124.3 246.0
s
171
-119.9 -141.4 241 .O
s
171
-192.5 -196.3
A
65,77
-215.0 -234.2
A
References
'
2-Deoxy-2,2-difluoro-~-urubino-hexopyranose (Y
anomer
6.0 p anomer
17.0
6.0 23.0
6.0 20.5
3,6-Dideoxy-3,6-difluoro-~~-allopyranoside methyl 4.50 3.41 8.0 1.3 29.3
4.91 53.7
3.64 10.0 29.3
3.78 4.5 25.6
methyl 2,4-di-O-benzoyl5.08 5.20 5.50 5.33 10.5 8.0 2.0 2.0 1.5 28.0 54.0 27.0
4.44 4.0 24.5
4.61 2.0 47.6 4.73 2.0 47.5
4.64 11.0
b
47.6 4.76 10.5
65
47.5 (continued)
RENE CSUK AND BRIGITTE I. GLANZER
I50
p-nitrophenyl 5.46 3.86 5.01 8.0
3.89 10.0
28.0
52.0
4.15 4.0
5.04 4.0
8.5
28.0
52.0
48.0 4.61 2.0
phenyl a5.63 4.0 3.5 10.0
4.2 29.0
-211.9 -235.5
65
-211.1 -234.5
65
48.0 4.12 10.0
28.0
25.0 46.0 4,6-Dideoxy-4,6-difluoro-~-galactopyranoside methyl a anomer '4.17 4.82 4.0 2.6 0.0 30.6 50.3 30.6 13.5 46.6 p anomer 30.5
4.14 10.0
29.0 28.0
phenyl 5.21
4.61 4.0
51.3 4.96
30.5 13.4
46.7
4.34 1.2
4.55 4.6
46.0
-219.1 -229.9
77
3
172
46.6 - 196.2 -206.2
71
-219.9 -230.1
65
-216.9 -231.9
27,ll
- 196.9 -235.1
65
46.1 4.68 9.5
51.0
43.0
46.0
Methyl 4,6-dideoxy-4,6-difluoro-~-tdopyranoside a anomer
4.88 3.5
3.19
3.83 32.0
4.19 48.6
4.01 32.0 13.8
4.62 2.0 46.6
4.62
d
2.0 46.6
2,3-O-isopropylidene-a25.0
52.0
25.0 25.0
48.0
48.0
6-Deoxy-6,6-difluorogalsetopyranose 1,2,3,4-tetra-O-acetyl-ta anomer
6.39 3.1
5.25 10.7
5.39 3.3
5.65
4.58 5.9
6.02 55.5
6.02
173
54.4 (continued)
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
I5 I
TABLEL (continued)
p anomer 5.76 8.5
5.36 10.7
5.10
5.60
4.00
5.78
5.78
C
I73
C
173, 174
5.5
3.3
55.4
54.4
I ,2:3,4-di-O-isopropylidene-a-~-
5.56 4.9 1.8
4.36
4.65 7.7 1.3
4.36
3.90 6.6
-
5.84 57.5 54.0
10.0
5.5
-131.6 -133.6 298.2
a The symbols I, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atom. Ref. 65 reported chemical shifts of -217.6 and -234.6 p.p.m., respectively. Ref. 172 reported for H-1 = 4.88 p.p.m. Ref. 77 reported chemical shifts of -196.7 and -199.6 p.p.m., respectively. ' Ref. 174 reported data for the corresponding D enantiomer.
(171) J. Adamson, A. B. Foster, and J. H. Westwood, Curbohydr. Res., 18 (1971)345-347. (172) C. W.Somawardhana and E. G. Brunngraber, Carbohydr. Res., 94(1981)cl4-cl5. (173) J. A. May, Jr., and A. C. Sartorelli, J . Med. Chem., 22 (1979)971-976. (174) M.Sharma, I. Kavai, Y.L. Fu, and M. Bobek, Tetrahedron Lett., (1977)3433-3436. TABLELI IH- and I9F-N.m.r.Data for Difluorinated Anhydrohexo-pyranose and -furanose Derivatives Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
6F1
S
31.2
Jz.3
J3,4
54.5
55.6
J6.6'
6F.2
a
VF.1
JFJ
JFJ
Jp.4
JF,S
Js,~ JF,~
JF.~'
J(F,F)
'JFJ
JF,Z
JF,~
JF.~
JF,~
JF.~
JF,~
References
3-O-Acetyl-1,6-anhydro-2,4-dideoxy-2,4-difluoro-~-~-glucopyranose
5.57
4.38
5.13
4.23
4.78
C
17.5 17.5 45.0 1,6-Anhydro-2,4-dideoxy-2,4-difluoro-~-~-ribo-hexo-pyranos-3-ulose hydrate 5.49 4.07 4.23 4.73 4.03 3.75 S 2.2 2.I 1.2 5.9 7.2 2.2 48.0 9.6 9.6 1.0 4.0 2,5-Anhydro-l-deoxy-l,l-d~uoro-~-hexitol
118
45.0
I75 h
arabinoa anomer
5.99 3.0
D
176 (continued)
152
RENE CSUK AND BRIGITTE I. GLANZER TABLELI (continued)
Compound H-1 H-2
H-3
H-4
H-5
H-6
H-6'
JIS
523
53.4
54,s
55.6
53.6
J6.6'
6F1 6F2
'JPJ
JFJ
JFS
JF,4
JFJ
JF,~
Jp.6,
J(F,F)
'JFJ
JFJ
JFJ
JF,~
JF,S
JF,~
&,6*
55.1 3,4,6-tri-O-acetyI5.86 3.6 55.0 ribo5.60 5.40 4.0 54.0 12.0 12.0
-128.2
5.40
4.0
4.0
3,CAnhydro-6,6-di!luoro-~-glucofurnnose 1,2-O-benzylidene-a6.11 4.85 4.92-5.01 4.44 -82.9 3.6 4.0 -76.5 11.0 144.0 7.0 1,2-O-isopropylidene-u5.92 4.70 4.78-4.86 4.39 -82.8 3.6 4.0 -76.4 11.0 144.3 7.0 3,6-Anhydro-6,6-difluoro-l,2-O-isopropylidene-~~-idofuranose 5.92 4.76 4.69 4.95 4.24 -87.0 3.5 0.6 -68.7 3.6 0.7 0.4 3.5 5.8 3.2 150.4 1.2 0.6
S
References
a
C
176
C
177
C
125
C
125
C
125
The symbols I, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atom. J (H-2, H-4) = 1.3 Hz. J (H-I, H-4) = 0.6 Hz; J(H-2, H-4) = 0.6 H z ; J (H-2, H-5) = 0.6 Hz; and J (H-3, H-5) = 0.6 Hz.
(175) J. PacBk, M. BraunovB, D. StropovB, and M. Cerng, Collecr. Czech. Chem. Commun., 42 (1977) 120-131. (176) K. R. Wood and P. W. Kent, J . Chem. SOC., C, (1967) 2422-2425. (177) P. W. Kent and J. E. G. Barnett, Tetrahedron, Suppl., 7 (1966) 69-74.
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
I53
TABLELII 'H- and I9F-N.m.r.Data for 2,6-Dideoxy-2-fluorohexopyranosylFluorides Compound
H-1
H-2
H-3
H-4
H-5
H-6
H-6'
11.2
J2J
J3.4
54.5
J5,6
J5,v
J6,@
%,I
JFJ
JF,~
JF,~
JFJ
JF,~
JF,~
*JFJ
JF,~
JF,3
JP.4
JFJ
Jv.6
JF,~'
m1
m
S
References
0
J(F,F)
2,6-Dideoxy-2-fluoro-~-galactopyranosyl fluoride (Y
anomer
54.4 24.5 0.6 48.2 13.4 3,4-di-O-acetyl(Y anomer 3.0 53.5 0.5
9.5 23.2 48.8 13 anomer
-151.6 -212.2 19.5
D
147
-152.1 -211.9 18.3
C
147, 148b
-142.7 -210.4 15.4
C
147
C
149
C
149
4.2
3.5
2.0
11.3
3.8
6.4
52.8 14.5 4.9 2.9 51.5 13.3 3,4-Di-0-acetyl-2,6-dideoxy-2-luoro-a-~-glucopyranosyl fluoride 3.2 53.5 0.2
9.8 24.0 48.0
9.8
9.8
6.2 19.0
12.5
3,4-Di-0-acetyl-2,6-dideoxy-2-fluoro-~-~-mannopyranosyl fluoride 0.5 48.5 17.0
2.3 4.2 52.0
9.5
7.5
6.2
-148.3 -221.6 13.0
28.0
" The symbols I, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atom. Ref. 148 reported a chemical shift of -148.1 p.p.m. for F-I.
154
RENE CSUK AND BRIGITTE I. GLANZER
TABLELIII
‘Hand l9F-N.m.r.Data for Difluorinated Hex-5-enofuranoses
5,6-Dideoxy-1,2-0-isopropylidenehex-5-enofuranose 6-chloro-5,6-difluoro-3-O-methyl-a-~-xy/o(Z) isomer C 178 5.89 4.62 3.67 4.98 5.47 -91.1 9.0 3.8 3.2 2.0 9.9 (E) isomer 5.73 4.46 3.56 4.85 5.12 -87.3 T 178 3.9 2.9 8.8 2.9 27.8 6,6-difluoro-3-O-methyl-~-~-/yxoC I79 4.91 4.98 -79.0 10.2 -74.0 b 0.3 2.3 35.3 e 0.8 25.2 6,6-difluoro-3-O-methyl-a-~-ribo4.64 4.34 -80.2 c 179 9.8 -76.8 b I .3 1.5 30.9 0.9 23.7 6,6-difluoro-3-O-methyl-a-~-xy/o-79.6 C 179 4.90 4.58 -77.5 9.5 33.5 1.7/1.1 1.6/25.0 3,5,6-Trideoxy-6,6-difluoro-1,2-O-isopropylidene-~-~-erythro-hex-5-enofuranose 5.82 4.74 1.61/2.23 4.84 4.32 -77.0 C 178 3.7 4.6 10.7l4.5 9.1 -77.9 d.e. b 0.8 2.5 34.8 c 1.0 2.4 1,2-~-lsopropylidene-5,6-dideoxy-6,6-difluoro-cu.~-xy~o-hex-5-enofuranose 3-0-benzyl C I79 4.9 4.67 -89.6 9.8 -87.9 b 2.0 2.0 33.4 c 2.2 23.4 3-O-methyl4.90 4.58 -79.6 C 179 9.5 -77.5 b I .7 1.6 33.5 1.1
25.0 (continued)
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
155
TABLELIII (continued)
3-O-benzyl-a4.91
4.67 -89.6 C 179 -87.9 9.8 d 2.0 2.0 33.4 2.2 23.4 Methyl 5,6-dideoxy-6,6-difluoro-2,3-O-isopropylidene-~-~-r~~o-hex-5-enofuranoside 4.97 4.64 4.64 4.90 4.46 -74.2 C 178 10.4 -76.1 1.2 2.8 34.0 1.2 24.0 The symbols I, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atom. Coupling to cis-oriented fluorine atom. Coupling to trans-oriented fluorine atom. J (H3, H-3') = 13.5. e Ref. 178 reported further examples for higher sugars.
(178) J. M. J. Tronchet and A. P. Bonenfant, Helu. Chirn. Acta, 63 (1980) 1644-1653. (179) J. M. J. Tronchet, A. P. Bonenfant, and F. Barbalat-Rey, Carbohydr. Res. 67 (1978) 564-573.
TABLELIV 'H- and I9F-N.rn.r.Data for Difluorinated Pentoses ~~
Compound H-1 H-2
H-3
H-4
H-5
H-5'
sF1
S
JI,~
52.3
J3.4
54.5
J4.5~
JS,Y
m
a
'JF,~
JFJ
JFJ
JF,~
JF,S
JF,V
J(FJ9
'JF.1
JF3
JFJ
JF.4
JF,~
JF,Y
3,4-D~-O-acetyl-2-deoxy.~-fluoro-~-~-arabinopyranosyl fluoride 5.86 4.31 5.50 4.16 3.85 -156.0 3.0 1.5 2.0 13.5 -210.0 53.0 0.5 2.0 2-Deoxy-2-fluoro-D-lyxopyranosy~ fluoride p anorner 3.0 53.0 4.5
3.0 22.0 45.5
3.5
2.5
References
C
53,54, 98
D
99
12.2
(continued)
RENE CSUK AND BRIGITTE I. GLANZER
I56
TABLELIV (continued) Compound H-1 H-2
H-3
H-4
H-5
JIJ
JZJ
J3.4
J4.5
54,s’
H-5’
%,I
JF,Z
JFJ
JF,~
JF.S
JFJ’
‘JFJ
JFJ
JF.~
JF,~
JF,S
JFJ~
sF1
m
S
a
References
J(F,F)
3,4-di-O-acetyla anomer -156.0 -215.0
C
54
-156.0 -215.0 19.0
C
533
-151.0 -206.0
C
98
-152.0 -212.0
C
5334
p anomer 3.6 53.0
3.6 47.0
2.0
12.5
25.0 4.0
3,4-D~-O-acetyl-2-deoxy-2-fluoro-~-~-~bopyranosyl fluoride 5.7Sb 4.9Ib 4.0b 3.67 3.5 10.0 5.0 10.0 24.5 44.0 3,4-Di-O-acetyl-2-deoxy-2-fluoro-~-~-xylopyranosyl fluoride
5.60 3.5 53.5
2,4-Di-O-acetyl-3-deoxy-3-luoro-/3-~-xylopyranosyl fluoride
5.41 5.0 4.65 5.0 4.31 3.72 -137.7 c 11 -195.7 2.7 4.7 4.7 3.0 3.5 12.8 10.4 49.8 7.7 0.2 0.5 0.0 0.0 12.5 45.5 12.5 2.5 0.3 3,5-Dideoxy-3,5-d~uoro-l,2-O-isopropylidene-cu-~-xylofuranose 5.9 5.30 -209.8 T 2033, 4.0 I80 -231.5 1.2 11.0 49.5 29.9 1.4 11.9 46.0 Methyl 5-deoxy-5,5-difluoro-~-ribofuranoside 2,3-O-isopropylidene-PC 181 -129.0 - 129.0 Methyl 2-deoxy-2,2-difluoro-3,4-O-isopropylidene-~~-e~~~r~-pentopyranoside -126.2 C 181 - 146.1 Methyl 4-deoxy-4,4-di8uoro-2,3-O-isopropylidene-/3-~-e~~~r~-pentopyranoside -113.1 C 181 - 115.9 256.5 The symbols I, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atom. The assignments may have to be reversed. J (H-1, H-3)= 0.5 and J (H-3, H-5’) = 0.8 Hz.
(180) A. B. Foster and R. Hems, Carbohydr. Res., 10 (1969) 168-171. (181) R. A. Sharma, I. Kavai, Y. L. Fu, and M. Bobek, Tetrahedron Lett., (1977) 34333436.
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
157
TABLELV 'H- and IgF-N.m.r. Data for Difluorinated Amino Suears Compound
H-l
H-2
H-3
H-4
H-5
H-6
H-6'
sF1
S
J1.z
JZ.3
J3.4
54,s
554
55.6'
5 6 , ~
m
a
'JFJ
JF,Z
JFJ
JF,4
JFJ
JF.6
JF,~
*JFJ
JFJ
JFJ
Jp.4
JFJ
JF,~
JF,~
References
J(F,F)
Methyl 2-acetam~do-2,4,6-trideoxy-4,6-difluoro-~-~-g~actopyranos~de -225.0 P -236.7 33.8 63.0 33.8 13.5 47.3 47.3 3-0-acetyl-222.9 C -227.7 28.1 54.0 28.1 13.5 45.0 45.0 Methyl 2-acetamido-3-O-acetyl-2,4,6-tdeoxy-4,6-difluoro-~-~-glucopyranoside 3.33 -200.4 11.0 8.7 -240.8 13.5 48.4 2.8 24.8 47.3 47.3 Methyl 2-benzamido-2,4,6-tdeoxy-4,6-d~uoro-~-~-g~a~opyranoside 3-0-acetyl-223.4 -237.8 25.9 54.0 25.9 12.4 47.3 47.3 3-0-benzyl-224.0 -236.5 26.4 55.1 26.4 16.9 48.9 48.9 Methyl 2-benzamido-2,4,6-tdeoxy-4,6-difluoro-t~-glucopyranoside 3-0-acetyl5.49 -200.3 10.6 8.7 -240.8 15.8 48.4 1.1 25.9 47.3 47.3 3-0-benzyl-198.1 -240.1
134
134
134 b
134
134
134 b
134
The symbols I, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atom. Partial listing of coupling constants in A is given, too.
RENE CSUK AND BRIGITTE I. GLANZER
158
TABLELVI ‘H-and I9F-N.m.r. Data for Tri- and Tetra-fluorinated Monosaccharides
3,6-Anhydro-5-deoxy.5,6,6-trifluoro-1,2-0-isopropylidene-~-~-glucofuranose 6.00 4.78 4.94 5.07 5.35 -216.8 C 125 b 3.5 0.6 3.4 4.5 -79.3 3.5 6.3 2.0 48.0 -75.7 1.3 1.5 9.0 0.6 5.6 3,6-Anhydro-5-deox~5,6,6-triluoro-1,2-0-isopropylidene-~-~-idofuranose 5.91 4.78 5.07 4.95 5.10 -201.1 c 125 3.6 0.6 3.7 0.6 -86.6 5.6 9.4 47.4 -68.5 0.6 3.6 2.8 0.6 1.4 3,4,6-Trideoxy-3,4,6-trifluoro-c~-~-galactopyranosyI fluoride 5.84 4.21 4.94 5.27 4.57 4.80 4.73 -154.3 M 182 5.4 10.1 2.7 5.2 6.5 9.5 -221.3 d 53.0 25.0 -207.1 2.9 11.0 47.2 -232.5 26.8 51.4 28.1 14.2 47.0 47.0
“ The symbols I, m, h refer to proton-fluorine couplings with increasing number of the corresponding fluorine atoms. J (H-I, H-3) = 0.6; J (H-1, H-5) = 0.4; J (H-2, H-4) = 0.6; J (F-5, F-6) = 12.2; J (F-5, FA’)= 12.2; J (F-6, F-6’) = 140.4 Hz. J (H-1, H-3) = 0.6; J (H-2, H-4) = 0.6; J (H-2, H-5) = 0.6; J (F-5, F-6) = 12.2; J (F-6, F-6’) = 152.6 Hz. d J (F-I, F-3) = 3.7; J (F-3, F-4) = 13.7 Hz. (182) G . H.Klemm,R. J. Kaufman,and R. S . Sidhu,Tetrahedron Lett., (1982)29272930.
TABLELVII IH-and 19F-N.m.r.Data for Acyclic Monosaccharides 2,2’-O-Methylenebis(I-deoxy-l-fluoro-~-glycerol) (C, Ref. 183) H-l = H-I’ = 4.58p.p.m.; J (H-I, H-2) = 5 Hz; J (F, H-I) = J (F, H-1‘) = 45 Hz 2,2‘-O-Methylenebis(3-O-benzoyl-I-deoxy-l-fluoro-~-glycerol) (C, Ref. 183) H-1 = H-I’ = 4.63p.p.m.; J (H-I, H-2)= 4.5 Hz; J (F, H-I) = J (F, H-1’) = 47.5Hz 1,6-Dideoxy-l,6-difluorogalactitol (D,Ref. 184) = J (H-l’, F) = 50 Hz; J (H-2, F) = 18 Hz;6F = -227.6 p.p.m.
J (H-I, F)
(continued)
N.M.R.SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
159
TABLELVII (continued)
Ref. 184) 24 Hz;6F = -232.5 p.p.m.
1,6-Dideoxy-l,6-difluoro-2,3 : 4,6-di-O-isopropylidenegalactitol (s,
J (H-I, F) = J (H-l', F) = 49 Hz;J (H-2, F)
=
I ,2,4,5,6-Penta-O-acetyl-3-deoxy-3-fluoro-~-mannitol (Ref.73) in C in B [J (H,F)1 [J (H,F)1 H-l = 4.55(2.6) J(1,l') 4.63(2.6) H-I'= 4.13(2.2) J( 12) 3.97(2.I ) H-2 = 5.11 (6.8) 5.32(6.8) J( I ' 2) H-3 = 4.85(45.6) 4.87(46.0) J(2,3) H-4 = 5.66(30.I ) J(3,4) H-5 = 5.31 (0.0) 5.50 (1.0) J(43 H-6 = 4.38(0.0) 4.48(0.0) J(5,6) H-6' = 4.16(0.0) 4.17(0.0) J(5,6') J(6,6')
in C
in B
12.5 2.5 4.3 9.3 1.6 7.1 2.6 5.7 12.5
12.4 2.6 4.8 9.3 1.7 7.2 2.7 5.4 12.6
3,4-O-Benzylidene-l,6-dideoxy-l,6-difluoro-2,5-O-methylene-~-mannitol (C, Ref. 183) H-l = H-I'= H-6= H-6'= 4.71 p.p.m.; J (H-I, F) = J (H-l', F) = 47.5Hz. 1,6-D~deoxy-1,6-difluoro-2,5-O-methylene-~-mannito~ (A,Ref. 183) H-l= H-I'= H-6= H-6'= 4.66p.p.m.; J (H-I, F) = J (H-l', F) = 47.5Hz. 3-Deoxy-l-fluoro-3-iodo-l,2 :5,6-di-O-isopropylidene-a-~-xy/o-4-hexulose (B, Ref. 185) H-l= 6.13;H-2= 4.84;H-3= 4.64p.p.m.;J (H-I, H-2)= I : O Hz;J (H-2, H-3)= 10 Hz;J (F, H-I)= 66 Hz;J (F, H-2)= 15 HZ
1,2-Dideoxy-l, I-difluoro-3,4 : 5,6-di-O-isopropylidene-~-arabino-hex-I-enitol (C,Ref. 179)" H-2= 4.39p.p.m.;J(H-2, F) = 1.5 Hz;J(H-2, F') = 23.8Hz;H-3= 4.62p.p.m.; J (H-3, F) = 1.5 Hz;J (H-3, F') = 0.9Hz;J (H-3, H-4)= 9.3Hz;6F-1 = -79.0p.p.m.; 6F-2 = -76.6 p.p.m.; J (F, F')= 32.8Hz " Partial "C-n.m.r. data also given: C-1 = 157.7 p.p.m., J (C, F) = J (C, F') = 293.7 Hz; C-2 = 78.9 p.p.m., J ( C , F) = 24.0 H z ; J ( C , F') = 16.2 Hz; C-3 = 73.2 p . p . m . . J ( C ,F) = 8.7 H z ; J ( C , F') = 0.0 Hz. (183) W. J. Lloyd and R. Harrison,Carbohydr. Res., 26 (1973)91-98. (184) E. M.Acton,M.Keyanpour-Rad,J. E. Christensen,H.H.Tong, R.P.Kwok,and L.Goodman,Carbohydr. Res., 22 (1972)477-486. (185)A.A.Akhrem,N.B. Khripach,and I. A. Mikhailopulo,Carbohydr. Res., 50 (1976) ~6-~8.
RENE CSUK AND BRIGITTE I. GLANZER
I60
TABLELVIII W-N.m.r. Data for Hexopyranosyl Fluorides Compound
c-1
c-2
c-3
c-4
c-5
C-6
JFJ
JFJ
JFJ
JF,~
JF,S
JF,~
2,3,4,6-Tetra-0-scetyl-~-altropyranosyl fluoride a anorner 104.3 67.7 66.2 64.0 66.4 226.7 38.8 0.9 0.5 2.4 p anorner 105.0 67.1 67.7 64.5 74.7 227.4 22.9 1.1 2,3,4,6-Tetra-0-acetyl-~-galactopyranosyl fluoride a anorner 66.9 104.3 67.3 67.3 68.8 228.3 24.1 3.7 p anorner 107.2 69.2 70.1 66.6 71.3 217.3 24.9 1.2 10.5 4.7 D-Glucopyranosyl fluoride a anorner 69.4 108.2 71.9 73.3 75.0 222.2 24.8 2.9 p anorner 110.0 "76.0 "73.6 69.9 "76.9 2.2 211.3 22.1 4.3 2,3,4,6-tetra-O-acetyla anomer 67.I 103.7 69.9 69.2 69.7 228.9 24.3 4.4 p anorner 106.3 71.3 71.9 67.6 72.1 219.4 29.3 8.8 4.46 3,4,6-tri-O-acetyl-2-O-methyl(Y anorner 103.9 78.4 71.0 67.4 69.5 229.9 23.4 4.4 p anorner 108.4 80.3 72.5 71.3 67.7 217.4 24.9 10.6 4.8 2,4,6-tri-O-acetyl-3-O-rnethyia anomer 103.9 72.0 77.7 68.4 70.0 227.4 24.7 4.6 p anorner 106.1 71.0 79.6 67.9 72.1 218.3 27.8 5.9 3.8
s
References
62.0
C
28
63.2 2.1
C
28
61.1
C
28
61.4
C
28
61.O
D
28
61.4
D
24,28
61.0
C
28
61.9
C
24,28
61.3
C
28
61.5
C
28 e
61.4
C
28
62.2
C
28 (continued)
161
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES TABLELVIII (continued)
Compound c-1 c-2 JF,~
JF,~
c-3
c-4
C-5
C-6
JF.3
JFA
JFS
JF,~
D-Mannopyranosyl fluoride 2,3,4,6-tetra-O-acetylQ anomer 104.7 67.6 68.1 65.0 70.8 223.1 39.7 1.8 3.1 p anomer 104.1 67.8 68.4 66.4 72.7 223.6 25.0 5.9 4.4 3,4,6-tri-O-acetyl-2-O-methyl-a104.6 75.9 70.1 64.8 70.0 224. I 35.2 I .o 2.9 2,4,6-tri-O-acetyl-3-O-methyl-a105.0 65.9 76. I 66.4 70.9 221.2 39.6 2.9 2,3,4,6-Tetra-0-acety1-a-~-talopyranosyl fluoride 104.5 64.6 65.4 65.1 65.8 224.7 36.6 2.9
S
References
61.7
C
28
62.7
C
28
61.4
C
28
62.0
C
28
61.8
C
28
The assignments may have to be reversed. Ref. 28 reported a coupling of 2.1 Hz. J (F, OMe) = 1.8
Hz.
TABLELIX W-N.m.r. Data for 2(or J)-Deoxy3(or 3)-fluorohexo-pyranoses and -pyranosides
Compound c-1 (2.2 JFJ
JF.Z
c-3
c-4
C-5
C-6
JF.3
JF,~
JF,S
JF.~
1,3,4,6-Tetra-~-acetyl-2-deoxy-2-fluoro-a-~-gdactopyranose 89.0 84.1 68.2 67.8 68.6 61.0 22.3 191.1 18.9 7.5 Methyl 2-deoxy-2-fluoro-~-gdactopyranoside p anomer 101.2 91.6 71.3 69.3 75.4 60.9 23.2 179.4 17.1 8.5 3,4,6-tri-O-acetyl-p67.7 70.6 61.1 101.6 88.0 71 .O 22.0 186.8 20.8 8.5 2-Deoxy-2-fluoro-~-glucopyranose Q anomer 90.9 91.5 72.4 70.4 72.5 61.6 21.4 185.9 17.5 8.0 1.3
S
References
C
49,50
D
26
C
26
D
24,57 (continued)
RENE CSUK AND BRIGITTE I. GLANZER
I62
TABLELIX (continued) Compound
c-1
c-2
c-3
c-4
c-5
C-6
JF,~
JF,~
JFJ
JF,~
JFb
JF,~
77.3 1.2
61.8
D
2437
70.0
61.8
C
4930
73.6
61.8
D
24,63, 64
77.3
62.0
D
24,63, 64
73.1
61.7
C
49,50
C
64
A
65
A
65
C
186
C
I86
D
26,186
C
26,186
C
69
p anomer 94.7 94.1 75.3 70.6 23.0 183.1 17.3 8.2 I ,3,4,6-tetra-O-acetyl-a88.8 86.6 71.0 67.9 21.8 194.2 19.4 7.5 2-Deoxy-2-fluoro-D-mannopyranose a anomer 92.5 91.6 70.7 68.1 I .4 29.6 172.2 17.5 p anomer 93.6 92.6 73.1 67.9 1.5 15.8 180.0 17.6 1,3,4,6-tetra-O-acetyl-P90.4 86.5 71.3 65.0 15.4 191.2 17.1
s
References
Benzyl3,4,6-tn-O-benzyl-2-deoxy-2.luoro-fi-~-mannopyranoside 97.0 86.9 80.7 15.9 188.0 18.3 Methyl 3-deoxy-3-fluoro-fi-o-allopyranoside 102.0 67.4 93.7 70.3 74.5 62.0 176.0 16.1 2.9 16.I 6-O-pivaloyl-p101.8 67.4 93.4 70.2 72.0 63.7 3.0 17.6 177.9 16.1 2.9 1,2,4,6-Tetra-O-acetyl-3-deoxy-3.luoro-~-gdactopyranose a anomer 89.7 85.4 8.6 192.9 p anomer 91.5 68.9 88.7 66.7 71.5 61.1 11.0 19.5 195.3 17.1 3.0 2.4 Methyl 3-deoxy-3-fluoro-~-gdactopyranoside p anomer 103.2 69.5 93.2 67.0 74.0 60.7 11.0 19.5 183.1 17.1 7.3 3.6 2,4,6-tri-O-acetyl-p101.3 69.7 88.8 66.9 69.8 61.2 11.0 19.5 192.9 17.1 6.1 2,4-di-O-benzoyl-6-0-(bromoacetyl)a anomer 97.7 70.0 85.9 68.8 66.3 63.8 8.6 18.3 192.9 17.1 6.1
(continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
163
TABLELIX (continued)
Compound c-1 c-2
c-3
c-4
C-5
C-6
JFS
JF,~
JF,~
JF.6
67.8 17.6
70.2 6.2
63.4 3.1
C
69
93.5 71.2 96.2 69.1 10.9 16.8 178.6 17.6 p anomer 96.4 73.8 97.8 69.1 12.4 17.2 180.5 17.6 3-Deoxy-3-fluoro-~-mannopyranose a anomer 94.2 68.9 92.3 65.4 7.0 15.0 183.0 18.0 p anomer 93.0 69.5 93.7 65.1 11.0 17.0 183.0 19.0
72.0 7.3
61.4 1.5
D
24
65.8 8.2
61.6 1.9
D
24
72.0 6.0
60.7
D
187
74.8 7.0
60.7
D
187
JF.I
JF.1
S
References
p anomer 101.7 9.5
70.6 18.9
88.9 195.8
3-Deoxy-3-fluoro-~-glucopyranose a anomer
(186) P. KovBt and C. P. J. Glaudemans, Curbohydr. Res., 123 (1983) 326-331. (187) J. R. Rasmussen, S. R. Tafuri, and S. T. Smale, Curbohydr. Res., I16 (1983) 21-29.
TABLELX W-N.m.r. Data for 4(or 6)-Deoxy4(or 6)-fluorohexo-pyranosesand -pyranosides
Compound c-1 c-2
c-3
c-4
JF,~
JFJ
JF.~
JFJ
c-5
4-Deoxy-4-fluoro-~-gdactopyranose a anomer 93.6 69.6 “69.2 91.7 “70.5 2.2 17.6 177.0 17.6 p anomer 97.4 73.1 72.8 90.8 74.8 1.1 18.0 177.7 17.3 Methyl 4-deoxy-4-fluoro-~-galactopyranoside a anomer 100.9 69.6 69.7 91.8 71.9 2.6 18.0 177.4 17.6 p anomer 90.9 75.0 104.9 72.0 73.0 178.2 17.6 17.9
C-6
S
61.3 5.5
D
24
61.1 5.5
D
24
61.5 5.5
D
65,188
61.3 5.1
D
26,188
References
(continued)
RENk CSUK AND BRIGITTE I. GLANZER
164
TABLELX (continued) Compound c-1
c-2
c-3
c-4
c-5
C-6
JFJ
JFJ
JFJ
Jp.4
JF3
JF,~
2,3,6-tn-O-acetyla anomer 97.3 67.9
S
References
“66.7 17.7
87.1 185.3
“68.3 17.7
61.9 5.9
C
50
“70.8 18.3
85.9 185.6
“71.8 17.1
61.4 4.9
C
26
78.9 86.0 73.5 17.7 182.9 17.9 2,3-di-O-benzyl-6-0-trityl-p104.4 78.9 79.0 86.2 72. I 17.1 184.3 17.1 4-Deoxy.4-fluoro-D-glucopyranose a anomer “70.1 93.2 72.2 a72.3 90.5 23.9 1.4 8.3 17.6 179.9 p anomer “72.3 97.1 74.9 “75.0 90.3 24.5 1.4 8.8 18.0 180.0 4-Deoxy.4-fluoro-~-mannopyranose a anomer 69.9 94.0 71.4 68.7 88.5 18.0 176.0 9.0 24.0 p anomer 73.5 93.8 71.7 71.3 88.3 24.0 11.0 18.0 176.0 Methyl 4-deoxy-4-fluoro-a-~-tdopyranoside 102.0 69.9 66.7 90.0 70.0 17.6 16.1 179.0 Methyl 6-deoxy-6-fluoro-~-gdactopyranoside p anomer 104.0 70.6 72.6 68.4 73.4 20.8 7.3 2,3,4-tri-O-acetyl-p101.8 68.6 70.1 66.9 71.3 23.2 6.1 6-Deoxy-6-fluoro-~-glucopyranose a anomer 71.5 93.5 72.6 73.9 69.8 17.2 0.7 6.6 p anomer 75.6 97.3 75.3 76.8 69.7 18.7 6.9
60.9 5.3
D
26,76 1886
61.6 2.4
C
26
61.2
D
24
61.4
D
24
60.5
D
187
60.4
D
187
60.5 7.4
A
65
83.1 164.8
D
26
80.7 171.5
C
26
83.7 167.2
D
24
83.5 167.6
D
24
p anomer 101.7
68.6
2,3-di-O-benzyl-p104.6 79.0
(continued)
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
165
TABLELX (continued) Compound c-1 c-2 JF,l
JFJ
S
References
84.8 168.4
A
27,77
82.3 170.9
A
65,77
82.2 170.6
A
65
82.5 170.6
A
65
82.5 170.6
A
65
83.5 170.6
A
65
82.8 170.6
A
65
83.4 170.6
A
65
c-3
c-4
c -5
C-6
JF.3
JF,~
JF,S
JF,~
Methyl 6-deoxy-6-fluoro-~-glucopyranoside a anomer 102.0 73.7 75.5 71.0 73.0 17.1 6. I /3 anorner 103.8 73.5 76.7 69.0 74.7 7.3 18.3 p-Nitrophenyl6-deoxy-6-fluoro-/3-~-glucopyranoside 73.6 76.9 68.9 75.5 100.5 7.4 17.6 Phenyl6-deoxy-6-fluoro-~-glucopyranoside a anomer 98.5 72.5 74.2 69.4 72.0 4.4 13.2 /3 anomer 101.0 73.7 77.0 69.1 75.2 7.4 17.6 Methyl 6-deoxy-6-fluoro-or-D-mannopyranoside 102.2 72.6 71.5 67.3 72.5 7.4 19.1 2,3-di-O-rnethyl98.9 76.4 81.6 65.4 72.3 7.4 17.6 2,3-O-isopropylidene79.9 76.5 68.6 70.1 99. I 7.3 17.6 * The assignments may have to be reversed.
Ref. 188 reported some data for the a anomer.
(188) J. E. Nam Shin, A. Maradufu, J. Marion, and A. S. Perlin, Carbohydr. Res., 84
(1980) 328-335. TABLELXI UC-N.m.r. Data for Hexofuranogl Fluorides and Deoxyfluorohexofuranoses Compound c-1 c-2 JFJ
JFS
c-3
C-4
c-5
C-6
JFJ
JF.4
JFJ
JF,~
76.7 1.2
67.6 0.7
62.4
D-Glucofuranosyl Ouoride 2,3,5,6-tetra-O-acetyla anomer 106.9 78.2 73.1 234.2 19.6
S
C
References
28 (continued)
RENE CSUK AND BRIGITTE I. GLANZER
166
TABLELXI (continued)
Compound
c-1
c-2
c-3
c-4
c-5
C-6
JF,I
JF,Z
JFS
JF.~
JF,S
JF,6
80.4 2.5
68.2
62.5
C
28
76.8 1.2
67.8 0.9
62.6
C
28
80.1 2.4
68.5
62.8
C
28
81.1
68.7 1.2
62.5
C
28
81.9 2.6
69.3
63.1
C
28
69.8 0.9
78.0 2.1
67.7
62.4
C
28
68.3
77.9 2.4
67.1 1.2
62.2
C
28
C
28
C
89,89a
C
89,89a
C
2530
D
91
D
91
p anomer 111.5 79.0 71.7 228.5 35.4 3,5,6-tri-0-acetyl-2-O-methyla anomer 107.7 86.2 74.0 233.8 18.9 p anomer 112.2 87.0 71.6 226.5 31.4 2,5,6-tri-0-acetyl-3-O-methyla anomer 106.7 77.1 77.7 231.6 19.5 p anomer 112.3 77.8 81.7 227.3 34.5 0.7 D-Mannofuranosyl fluoride
1.5
S
References
(1
3,5,6-tri-O-acetyl-2-O-methyla anomer 112.6 76.0 225.2 36.0 p anomer 106.5 71.6 236.8 20.1
1.5
2,5,6-tri-O-acetyl-3-O-methyl-~62.1 68.4b 78.1 106.7 80.7 66.3b 234.4 20.5 2,3 : 5,6-di-O-isopropylidenea anomer 66.0 72.6 113.7 84.7 78Sb 82.6b 221.5 41.8 p anomer 66.5 73.6 81.1b 107.5 81.2 77.6b 235.6 22.0 1,2 :5,6-Di-O-isopropylidene-3-deoxy-3-fluoro-~-~-glucofuranose 104.8 82.3 93.5 80.4 71.7 66.9 7.4 33.8 183.0 19. I 5-Deoxy-5-fluoro-D-glucofuranose a anomer 97.5 76.4 75.7 76.4 92.0 62.0 28.4 167.7 19.1 p anomer 103.1 81.0 75.3 79.2 92.0 62.3 28.4 167.7 20.5
(continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
167
TABLELXI (continued) Compound c-1 c-2 JFJ
JF,Z
c-3
c-4
c-5
C-6
JFJ
JF,~
JF,S
JF,6
78.8 30.9
91.5 167.6
62.9 19.1
A
91
80.2 17.7
93.6 172.9
61.8 20.0
D
91
77.3 17.7
94.5 170.6
61.8 20.0
D
91
80.7 19.1
94.6 170.6
62.0 22.0
A
91
1,2-O-isopropylidene-a106.1 86.3 74.7
5-Deoxy-5-fluoro-~-idofuranose a anomer 102.6 81.1 75.2 5.8 p anomer 96.7 76.5 75.2 5.8 1,2-O-isopropylidene-p105.7 86.4 75.2 8.8 J (F, OMe)
=
S
0.9 Hz. * The assignments may have to be reversed
TABLELXII W-N.m.r. Data for Fluorinated Pentopyranose Derivatives
2,3,4-Tri-O-acetyl-P~-arabinopyranosyl fluoride 104.6 67.5 68.1 66.4 62.4 C 28 227.1 26.4 2.8 2,3,4-Tri-0-acetyl-~-xylopyranosylfluoride a anomer 103.8 70.3 68.8 68.0 60.1 C 28 228.5 23.4 4.4 p anomer 105.3 67.0 67.1 66.8 60.6 C 28 222.8 36.0 2.8 0.6 3.4 l,3,4-Tr~-O-acetyl-2-deoxy-2-fluoro-&o-arbinopyranose 89.5 84.3 67.8 69.0 62.5 C 49,50 22.1 191.1 18.7 6.8 1,3,4-Tri-~-acetyl-2-deoxy-2-fluoro-cr-~-l~opyranose 89.6 79.6 68.6 67.7 61.8 C 49,50 19.1 192.7 17.6 2.9 1,3,4-Tri-0-acetyl-2-deoxy-2-fluoro-~-ribopyranose a anomer 88.9 84.0 67.2 “65.3 “61.0 C 49,50 20.0 198.1 16.7 (continued)
References
RENE CSUK AND BRIGITTE I. GLANZER
I68
TABLELXII (continued) Compound C-1 C.2 JFZ
JFJ
C-3
C-4
C-5
JFJ
JFA
JFJ
S
References
p anomer 90.8 84.7 66.0 66.1 62.8 C 101 30.5 189.2 18.3 1,3,4-Tri-0-acetyl-2-deoxy-2-fluoro-cu-~-xy~opyrmose 88.8 86.6 70.2 68.3 60.6 C 49,50 20.6 194.4 19.1 7.4 Methyl 4-deoxy-4-fluoro-~-arahinopyranoside a anomer 99.4 67.9 67.5 90.3 60.1 D 65 19.1 173.5 19.1 p anorner 104.6 71.7 72.2 89.4 64.0 C 65 22.0 177.9 20.6 a
The assignments may have to be reversed.
TABLELXIII W-N.m.r. Data for Fluorinated Pentofuranose Derivatives Compound C-1 C-2 JFJ
JFZ
C-3
C-4
C-5
JFS
JF.4
JF.S
o-Arabinofuranosyl fluoride 2,3,5-tri-O-benzoyl-a-~112.7 80.9 76.7 84.4 63.5 225.8 40.3 2,3 ,S-tri-O-benzyl-a-~113.5 224.6 2,3,5-tri-O-benzyl-P-~108.3 228.8 o-Lyxofuranosyl fluoride 5-0-benzoyl-2,3-benzoxonium-a114.2 97.1 95.5 84.3 66.7 229.4 47.1 2,3,5-tri-O-benzoyla anomer 112.3 75.7 70.8 78.3 62.9 1.8 1.8 224.7 37.4
S
References
C
28
C
105,106 a
C
105
F
107
C
28 (continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES TABLELXIII (continued) Compound C-1 C-2 JF.I
J F ~
C-3
C-4
C-5
JFJ
JFR
JF.S
S
References
p anomer 107.1 72.7 68.5 79.0 63.1 C 28 1.2 2.6 236.1 20.5 D-Ribofurnnosyl fluoride 2,3-acetoxonium-5-0-acetyl-P115.5 98.1 97.0 88.5 69.6 F 107 230.9 47.1 2.9 2,3,5-tri-O-benzoyla anomer 28 107.4 71.9 69.6 82.9 63.6 C 234.4 20.5 p anomer 112.4 75.0 71.4 81.3 64.3 C 28 1.5 2.9 0.5 225.5 35.9 2.3,s-tri-0-benzyla anomer C 106 108.5 232.5 p anomer 112.4 C 106 224.0 2,3,S-Tn-O-benzoyl-cu-~-xylofurnnosyl fluoride 112.3 80.2 73.8 81.6 63.2 C 28 228.0 36.8 l-O-Acetyl-2-deoxy-2-fluoro-S-O-( methoxymethyl)-a-~ribofurnnose 94.2 88.1 69.9 84.7 66.7 C 49,50 16.0 200.3 17.0 3.0 3-Deoxy-3-fluoro-1,2-O-isopropylidene-cu-~-~~o-pentodinldo-1,4-furanose 105.9 94.9 197.0 C I I4 182.6 6.9 " Ref.
105 reported data for the
D
enantiomer.
169
RENI? CSUK AND BRIGITTE I. GLANZER
I70
TABLELXIV W-N.m.r. Data for Fluorinated Sugar Acids and Lactones Compound C-1 C-2 JF.l
JF,Z
C-3
C-4
C-5
C-6
JFS
JF,4
JF5
JF.6
References
S
5-Deoxy-5-fluoro-a-~-glucofuranurono-6,3-lactone 1,2-O-isopropylidene107.6 82.7 81.7 76.6 86.2 168.0 C 5.9 14.7 206.9 23.8 5-Deoxy-5-fluoro-&~-idofuranurono-6,3-lactone 1,2-O-benzylidene105.1 82.3 84.5 81.0 87.5 169.0 C 28.2 179.8 20.0 1,2-O-isopropylidene106.5 82.2 85.1 80.0 87.3 169.1 C 28.0 185.3 20.6
91
125 91
TABLELXV W-N.m.r. Data for Fluorinated Amino Sugars and Their N-Containing, Synthetic Precursors
Compound C-1 C-2 JF.1
JF.Z
C-3
C-4
C-5
C-6
JF,3
JF.4
JF.5
JF,6
S
References
3-Azido-4,6-0-benzylidene-3-deoxy-~-~-allopyranosyl fluoride 108.5 76.7 61.4 77.1 64.1 67.8 C 214.9 22.9 9.8 4.6 Benzyl 2-fluoro-cu-D-dtropyranoside 3-azido-4,6-O-benzylidene-2,3-dideoxy96.4 88.0 57.7 75.9 59.3 69.2 C 34.3 174.9 28.9 3-benzamido-2,3,6-trideoxy96.6 87.2 50.3 70.4 65.4 17.4 C 31.0 178.6 26.9 Methyl 2,3,6-trideoxy-2-fluoro-cr-~-altropyranoside 3-amino98.5 89.1 50.6 67.8 64.5 17.6 C 32.0 176.0 24.0
127
I27 132
133
3-(trifluoroacetarnido)-
97.7 86.1 49.9 68.7 64.4 30.2 178.0 28.1 3-benzamido-4-O-benzoyl-6-bromo98.5 86.6 47.0 67.7 67.0 30.7 179.8 28.3
17.4
C
133
31.9
C
133 (conrinued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
I7 I
TABLELXV (continued)
3-benzamido-4-0-benzoyl98.4 86.9 46.9 70.1 62.7 17.5 C 133 30.8 179.2 18.1 8.3 Benzyl 2,3,6-trideoxy-2-fluoro-fi-~-galactopyranoside 3-benzamido100.3 89.6 54.0 72.0 71.8 16.1 B 132 8.1 22.8 187.4 17.8 4-0-benzoyl-3-(trifluoroacetamido)99.8 88.2 53.1 72.2 71.1 16.4 C 127 22.3 189.1 18.7 7.1 Methyl 2,3,6-trideoxy-2-fluoro-/3-~-galactopyranoside 3-amino101.6 92.7 55.6 72.2 71.9 16.3 C I33 23.0 180.4 18.1 8.3 3-(trifluoroacetamido)102.6 89.1 55.5 71.2 72.1 16.4 C 133 22.4 184.0 16.1 7.8 3-benzamido-4-0-benzoyl102.3 88.9 52.7 73.2 70.9 16.4 C I33 21.5 187.5 17.5 7.8 Methyl 3-benzamido-4-O-benzoyl-2,3,6-tndeox~2-fluoro-c~-~-u~u~i~ohex-5-enopyranoside 100.1 86.9 48.5 66.7 149.8 99.5 C 133 31.5 180.1 27.3 Methyl 6~azido-3,6-dideoxy-3-fluoro-fi-~-allopyranoside 102.0 70.4 93.5 68.2 73.8 52.0 A 65 3.4 17.0 177.7 18.0 3.0 0.0 Benzyl 4,6-0-benzylidene-2,3-dideoxy-3-fluoro-c~-~-glucopyranoside 2-azido98.0 62.3 88.7 80.1 62.6 68.8 C 127 8.8 17.4 189.1 17.2 7.6 2-benzamido99.0 54.5 89.5 80.9 63.4 69.2 P 132 9.0 17.1 191.0 16.5 7.5 Methyl 4-azido-4,6-dideoxy.6-fluoro-c~-~-glucopyranoside 100.5 72.8 73.5 61.7 69.2 82.7 A 65 5.9 17.6 172.0 3-Acetamido-2,3,5,6-tetradeoxy-5-fluoro-fi-~~-~i~o-hexofuranose 99.8 40.2 49.6 85.3 91.7 16.3 C 140 20.8 167.2 22.0 Methyl 3-aceta~do-2,3,5,6-tetradeoxy-5-fluoro-~~-~i~o-hexofuranoside a anomer 103.6 39.3 48.4 86.1 90.3 16.8 C 140 43.9 180.7 22.0 (continued)
RENB
I72
CSUK AND BRIGITTE I. GLANZER TABLELXV (continued)
Compound C-1 C-2 JF.1
JF.2
p anomer 105.6 39.3
C-3
C-4
C-5
C-6
JF,3
JF.4
JFJ
JF,6
50.7
88.3 20.7
90.3 180.7
16.8 22.0
S
References
C
140
TABLELXVI W-N.m.r. Data for Branched or Deoxy Fluorinated Monosaccharides
Compound C-1 C-2 JF.1
JF3
C-3
C-4
C-5
C-6
JF,3
JF.4
JF5
JF.6
~
S
References
~~
Methyl 4,6-0-benzylidene-2-deoxy-3-C-[ (ethoxycarbony1)-(fluoromethyl)]-a-~-ribbo-hexopyranoside (I?) isomer 98.2 36.2 71.5 77.2 58.7 69.2 C 189 3.3 19.5 (S)isomer 98.4 33.8 71.0 77.4 58.9 69.1 C 189 1.1 b 1.4 22.0 Methyl [benzyl 2-(benzyloxycarbonyl)amino-2,3,4-trideoxy-5-fluoro-aD-erythro-hex-3-enopyranosid]uronate 94.6 46.9 132.1 122.6 97.3 166.0 B 126 8.4 27.5 224.3 37.4 1-0-Acetyl-2,3,5,6-tetra-O-benzoyl-4-fluoro-~~-g~actofuranose 98.8 79.7 75.5 70.1 62.2 C I59 2.2 0 21.1 35.7 1-0-Acetyl-2,3,5,6-tetra~O-benzoyl-4-fluoro-~-~-glucofuranose 102.0 79.0 77.2 120.7 70.6 62.5 A 159 41.2 232.1 26.4
1-0-Acetyl-2,3,5-tri-O-benzoyl-4-fluoro-a-~-lyxofuranose
C 159 99.4 74.9 74.6 118.2 62.4 2.5 0.0 42.0 232.7 30.2 l-O-Acetyl-2,3,5-tri-O-benzoyl-4-fluoro-~-~-nbofuranose 98.3 73.1 70.1 114.4 62.8 - C 159 0.0 0.0 18.0 237.5 44.4 Methyl 2,4-di-0-benzoyl-6-0-(bromoacetyl)-3-deoxy-3-fluoro-a-~galactopyranosyl chloride 91.1 69.3 85.5 67.9 69.7 62.9 C 69 9.1 18.9 193.1 17.1 4.7 4.0 2,3,4-Tri-0-acelyl-6-deoxy-a-~-glucopymnosyl fluoride 103.8 70.8 69.5 72.7 67.9 17.1 C 28 227.7 24.7 4.6 (continued)
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES TABLELXVI (continued)
Compound C-1 C-2 JF.2
JF,l
C-3
C-4
C-5
C-6
JF,3
JF.4
JFJ
JF.6
S
References
2,3,4-Tri-O-acetyl-6-deox~cu-~-mannopyranosyl fluoride 104.7 67.8 68.1 69.8 68.7 17.1 C 221.3 41.3 2.1 3.1
28
* CO: 167.7 p.p.m.; J (C-F) = 24.4 Hz; CHF (of fluoromethylene): 88.2 p.p.m.; J (C-F) = 194.1 Hz; partial IH-n.m.r. data also given. CO: 167.5 p.p.m.; J (C-F) = 23.8 Hz; CHF (of fluoromethylene): 86.2 p.p.m.; J (C-F) = 186.8 Hz; partial IH-n.rn.r. data also given.
(189) S. Brandange, 0. Dahlman, and L. Morch, J . Am. Chem. Soc., 103 (1981) 4452-4458.
TABLELXVII W-N.m.r. Data for Fluorinated Ketopyranoses and Derivatives
Compound C-1 C-2 JFJ
JFJ
C-3
C-4
C-5
C-6
JFJ
JF.4
JF.5
JF.6
S
References
l-Deoxy-l-fluoro-2,3 :4,5-di-O-isopropylidene-~-fructopyranose 82.2 101.2 69.9 70.2 70.9 61.1 C 160 174.0 21.5 2.0 1.o 4-Deoxy-4-fluoro-~~-fructopyranose 63.6 98.6 66.2 91.6 67.5 62.7 D 161 1.6 9.7 18.3 180.1 16.6 7.0 4-Deoxy-4-fluoro-a-~-sorbopyranose 64.2 99.1 69.8 96.8 68.8 61.8 C 161,190 10.0 17.0 178.0 17.0 10.0 ~-~eox~4-fluoro-1,2-O-~sopropyl~dene-~-~-tagatopyr~ose
72.3
105.4
67.4 26.0
90.7 178.0
64.3 17.0
61.8
C
190
59.9 26.0
C
190
5-Deoxy-5-fluoro-cu-~-sorbopyranose 64.3
98.4
71.0 7.0
73.2 17.0
" An additional coupling, J (F,Me)
90.2 178.0 = 2.9 Hz, is
observed (6 Me
=
24.7).
(190) G. V. Rao, L. Que, Jr., L. D. Hall, and T. P. Fondy, Carbohydr. Res., 40 (1975) 311-321.
173
RENE CSUK AND BRIGITTE I. GLANZER
I74
TABLELXVIII W-N.m.r. Data for Mono- and Di-fluorinated Ketofuranose Derivatives Compound C-1 C-2 JYJ
JF,Z
C-3
C-4
C-5
C-6
JFJ
JF,~
JFJ
JF,~
S
References
C
I63
C
I63
A
I64
A
164
1,3,4,6-Tetra-O-acety~-~-fructofuranosyl fluoride a anomer 61.2 117.5 78.2 83.5 76.7 62.7 1.5 0.2 0.5 28.8 226.8 46.0 p anomer 61.7 113.9 75.0 80.2 74.7 63.6 43.7 232.8 20.9 2.8 0.4 Methyl 1-deoxy-1-fluoro-D-fructofuranoside a anomer 78.6 108.0 81.3 79.4 86.3 62.8 170.9 20.8 p anomer 81.6 102.7 79.1 76.7 83.4 63.7 174.6 19.5 3,4,6-tri-O-benzoyI-p-
C 164 173.3 19.3 6-Deoxy-6-fluoro-2,3-O-isopropylidene-l-O-p-tolyls~onyl-~-~-fructofuranose 68.2 113.9 83.6 74.9 86.7 81.8 C 164 4.9 20.8 170.9 1,6-Dideoxy-1,6-difluoro-2,3-O-isopropylidene-~-~-fructofuranose 81.2 113.3 86.8 75.2 86.7 82.2 C 164 a 172.0 23.2 4.9 20.8 177.0 b Methyl 1,6-dideoxy-1,6-difluoro-~-fructofuranoside a anomer 79.3 107.4 81.9 78.0 82.9 83.5 A 164 172.1 20.8 7.3 19.5 170.9 b p anomer 81.4' 102.9 79.0 75.3 81.3' 83.6 A 164 0 174.5 19.5 7.3 18.3 172.1 b Coupling to fluorine at C-1, Coupling to fluorine at C-6. The assignments may have to be reversed.
N.M.R. SPECTROSCOPY O F FLUORINATED MONOSACCHARIDES
TABLELXIX W-N.m.r. Data for Difluorinated Monosaccharides, Including Anhydro and Unsaturated Derivatives Compound
C-1
C-2
C-3
C-4
C-5
C-6
JF.1
JF,2
JF.3
JF,4
JF.5
JF.6
JF.1
JF.2
JFJ
JF.4
JFJ
JF,6
S
References a
b
3,6-Dideoxy-3,6-difluoro-~-~-allopyranoside methyl 102.1 70.7 93.6 ‘66.3 ‘73.0 83.1 A 65,77 4.3 17.2 177.0 d25.0 7.9 ‘4.3 20.0 170.9 phenyl 65 99.0 70.0 93.6 73.1 65.8 82.5 A 4.4 17.0 177.9 17.6 8.0 2.9 17.6 172.0 Methyl 4,6-dideoxy-4,6-difluoro-~-galactopyranoside (Y anomer 77 102.5 ‘69.9 ‘70.5 92.8 ‘71.5 84.9 A 178.2 6.1 6.1 167.2 p anomer 77 105.1 ‘71.8 ‘72.5 89.8 ‘72.6 82.6 A 180.7 6.1 6.7 167.9 Methyl 4,6-dideoxy-4,6-difluoro-cu-~-talopyranoside 102.0 69.4 65.3 ‘81.9 67.5 %9.6 C 27,77 16.1 ’158.0 22.1 87.4 7.4 17.6 172.0 3,6-Anhydro-6,6-diBuoro-a-~-glucofuranose I ,2-O-benzylidene125 105.1 84.9 84.1 82.1 74.2 128.7 C 6.0 31.0 265.0 26.0 260.0 1,2-O-isopropylidene125 106.1 83.2 83.4 79.6 72.9 127.3 C 1.8 2.0 6.2 30.0 264.0 1.6 25.0 248.0 3,6.Anhydro-6,6-difluoro-1,2-~-isopropylidene-~-~-idofuranose I25 106.1 85.5 86.7 82.9 74.9 130.2 C 2.0 3.0 37.0 259.0 22.0 259.0 5,6-Dideoxy-6,6-difluoro-1,2-O-isopropylidene-u-~-xylo-hex-5-enofuranose 3-0-benzyl73.7 75.1 157.2 C 179 26.2 292.5 8.8 0.0 15.9 289.0 (continued)
175
RENE CSUK AND BRIGITTE I. GLANZER
176
TABLELXIX (continued)
3-O-methyl179 73.4 74.9 157.1 C 27.0 292.4 9.2 0.0 10.8 288.5 5,6-Dideoxy-6,6-diffuoro-l,2-O-isopropylidene-3-O-methyl-~-~-/y~o-hex-5enofuranose 179 73.2 78.6 157.6 C 22.5 290.3 7.5 0.0 16.2 290.3 5,6-Dideoxy-6,6-diffuoro-l,2-O-isopropylidene-3-O-methyl-~-~-ribo-hex-5enofuranose 179 71.8 78.0 158.4 C 25.3 293.9 8.7 0.0 16.5 290.0 6-Chloro-5,6-dideoxy-5,6-difluoro-l,2-O-isopropylidene-3-O-methyl-a-D-~/ohex-5-enofuranose ( E ) isomer 81.8 73.9 101.9 146.3 C 178 104.6 85.1 12.4 306.7 (Z) isomer 178 104.7 85.2 81.90 76.7 101.4 147.0 C 8.3 23.8 301.2 Methyl 5,6-dideoxy.6,6-difluoro-2,3-O-isopropylidene-a-~-ri~o-hex-5-enofuranoside 178 109.6 85.6 79.7 85.4 80.4 156.8 C h 7.4 11.9 20.5 291.5 0.0 17.4 3,5,6-Trideoxy-6,6-diffuoro-l,2-O-isopropylidene-~-~-e~r~ro-hex-5enofuranose 105.2 80.5 39.8 70.8 79.3 157.3 C 178 h 7.8 23.7 288.8 i 0.0 16.5 292.4 a The couplings refer to the “lower”-numbered fluorine atom. The couplings refer to the “higher”-numbered fluorine atom. The assignments may have to be reversed. Ref. 65 reported a coupling of 17.6 Hz. Ref. 65 reported a coupling of 2.9 Hz.f Ref. 77 reported a coupling of 180 Hz (solvent A). * Ref. 77 reported a coupling of 5.5 Hz (solvent A). * Coupling to cis-oriented fluorine atom. Coupling to trans-oriented fluorine atom.
N.M.R. SPECTROSCOPY OF FLUORINATED MONOSACCHARIDES
I77
TABLELXX W-N.m.r. Data for Tri- and Tetra-fluorinated Monosaccharides Compound c-1
c-2
c-3
c-4
c-5
C-6
JFJ
JFJ
JF3
JF.~
JF5
JF,6
JFJ
JFJ
JFJ
JF.~
JFJ
JF,6
JFJ
JFJ
JFJ
JF,~
JF5
JF.6
JFJ
JFJ
JFJ
JF.~
JF,S
JF,6
S
References
3,6-Anhydro-5-deoxy-5,6,6-trifluoro-l,2-O-isopropylidene-cu-~-glucofuranose 106.0 83.8 83.0 77.6 88.1 126.5 C 125 14.0 204.0 266.0 b 4.0 33.0 250.0 b 24.0 22.0 3,6-Anhydro-5-deoxy-5,6,6-trifluoro-l,2-O-isopropylidene-~-~-idofuranose 105.4 86.0 81.7 80.9 89.7 126.7 C 125 L7 3.0 30.0 188.0 258.0 b 42.0 258.0 b 22.0 22.0 3,4,6-Trideoxy-3,4,6-trifluoro-cu-~-galactopyranosyl fluoride 108.95 67.95 89.41 88.09 71.20 81.78 M 182 226.8 53.0 22.7 11.0 22.7 188.7 3.7 e 2.0 18.4 181.7 21.0 b 6.6 21.0 169.3 ' Coupling to fluorine at C-5. Coupling to fluorine at (2-6. Coupling to fluorine at C-I. Coupling to fluorine at C-3. Coupling to fluorine at C-4.
TABLELXXI W-N.m.r. Data for Fluorinated Neuraminic Acid Derivatives Compound
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
JF.1
JF,Z
JF,3
JF,~
JF,S
JF,6
JF.~
JF,~
JF,~
S
References
Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,S-dideo~2-nonulosylfluoride)onate ff-D-glyCer0-aD-gUhCt0165.9 106.9 35.9 68.7 49.0 73.4 67.5 69.9 62.3 C 191 30.0 223.6 25.0 8.6 ff-D-glyCer0-P-D - g U h C t O 165.0 108.0 35.5 68.5 48.6 73.2 29.7 231.0 28.3 N-Acetyl-9-deoxy-9-fluoroneuraminic acid 175.5 96.4 39.1 67.8 53.2 71.5
a
67.4
70.4
62.5
C
191 n
68.5 7.2
69.9 17.8
86.4 164.9
D
Partial 'H-n.m.r. data reported, too. 6F = -235.4 p.p.m.
(191) M. N. Sharma and R. Eby, Carbohydr. Res., 127 (1984) 201-210. (192) M. Sharma and W. Korytnyk, J . Carbohydr. Chem., 1 (1982-1983) 311-315.
192 b
This Page Intentionally Left Blank
ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 46
APPLICATIONS OF PHOTOSENSITIVE PROTECTING GROUPS IN CARBOHYDRATE CHEMISTRY
BY URI ZEHAVI Department of Biochemistry and Human Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel
I. Introduction..
. .. .. . .. . . . . . .... .. .. .. . . .. .. .. . . . . . .. . . ... . .. .. . . . .. . . . . .
11. Hydroxy Functions, Including Dio
1. Protection as Ethers.. . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Protection as Acetals . . . . . . . . . 3. Protection as Esters . . . . . . . . . Amino Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... 1. Protection as Urethans. . . . . . . . 2. Protection as Amides.. . ., , . . , . . , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carbonyl Derivatives .................................................... 1. Protection of Aldehydes and Ketones ..... .............. 2. Protection of Carboxylic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . ... . . . Protection of Phosphoric Esters . . . . . . .. .. .. . .. . . . . Applications to Biological Models .
.
111.
.
IV. V. VI.
179 180 181 182 189 192 192 194 195 195 198 202 203
I. INTRODUCTION The use of selectively removable protecting groups is indispensable in carbohydrate synthesis, being concerned with polyfunctional molecules that bear many sites to be protected. Usually, protecting groups are removed by thermal chemical reactions. However, the photochemical removal of protecting groups normally achieved by thermal reactions, as well as of those specially designed for photochemical cleavage, has beThe development of come a very useful complementary B. Amit, U. Zehavi, and A. Patchornik, Isr. J . Chem., 12 (1974) 103-113. V. N. R. Pillai, Synthesis, (1980) 1-26. R. W. Binkley, Adv. Carbohydr. Chem. Biochem., 38 (1981) 105-193. T. W. Greene, Protecting Groups in Organic Synthesis, Wiley-Interscience, New York, 1981. (5) U. Zehavi, Reactiue Polym., 6 (1987) 189-196. (1) (2) (3) (4)
I79
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
I80
URI ZEHAVI
light-sensitive protecting groups may be considered to be an important outcome of the rapidly growing field of photochemistry; the previous article on carbohydrate photochemistry appeared3 in this Series in 1981. Ideally, a photosensitive protecting group should be simple, selective, and efficient in use. It should be readily introduced by employing accessible reagents, stable under the reaction conditions, and readily removed by irradiation without affecting other parts of the molecule, and the product ought to be easily isolable following the photoremoval of the protecting group. Furthermore, because carbohydrates are chiral themselves, achiral groups are advantageous in carbohydrate modification, avoiding the formation of diastereoisomers. In general, photosensitive protecting groups are removed by photosolvolysis, making use of the different reactivities of the excited and ground states, or by intramolecular, photoactivation reactions. Because most carbohydrates are devoid of longer-wavelength absorbance, an important segment of carbohydrate photochemistry actually deals with what may be termed modifier protecting groups, namely, groups that may be removed, generating the parent functions or, alternatively, may serve in the modification of the molecule (here the saccharide derivative) attached. Relevant examples include the photochemical conversion of esterified carbohydrates (protected hydroxyl function) into deoxy and unsaturated derivative^,^,^-^ a-ketoesters of carbohydrates (protected hydroxyl function) into aldehydes and ketone^,^ and phenylglyoxalyl amides of amino sugars and other amines into the corresponding carbonyl derivatives.8 It is not intended to present in this article a comprehensive treatment, but rather, to illustrate applicable options available to the carbohydrate chemist and biochemist. Protecting groups introduced in other areas of chemistry that can be useful to the carbohydrate chemist will be selectively discussed.
11. HYDROXY FUNCTIONS, INCLUDING DIOLS The selective protection of hydroxyl groups is obviously most frequent in carbohydrate synthesis and, in fact, photosensitive protecting groups have been used to this effect in oligosaccharide synthesis, nucleotide synthesis, and saccharide modification. Here, as well as in other Sections, special attention will be devoted to 2-nitrobenzyl derivatives, whose re(6) R. H. Bell, D. Horton, D. M. Williams, and E. Winter-Mihaly, Carbohydr. Res., 58 (1977) 109-124. (7) T. Kishi, T. Tsuchiya, and S . Umezawa, Bull. Chem. SOC.Jpn., 52 (1979) 3015-3018. (8) U. Zehavi, J . Org. Chem., 42 (1977) 2821-2925.
PHOTOSENSITIVE PROTECTING GROUPS
181
moval in very high yields is based on a photoinduced, intramolecular oxidation-reduction (see Scheme 1) discovered early in this century by Ciamician and Silber.9 CHO
SCHEME 1 .-Photoinduced
Oxidation-Reduction of 2-Nitrobenzaldehyde.
1. Protection as Ethers The protection of alcohols by conversion into light-sensitive ethers is most attractive. Initially, the preparation of 2-nitrobenzyl (nBn) ethers presented a problem, although their potential usefulness as protecting groups, stable under a variety of reaction conditions, that nevertheless are readily photolyzed at >320 nm (see Scheme 2) was recognized immediately. L ~ ~ - ~ ~ RO-CH2+
5
ROH
+ O
02N
SCHEME 2.-Photochemical
H
C
T
ON
Cleavage of 2-Nitrobenzyl Ethers.
2-Nitrobenzyl ethers were employed in the protection of the 2'-hydroxyl groups during the synthesis of ribo-oligonucleotides. These were prepared alongside the 3'-nBn ethers through alkylation with 2-nitrobenzyl chloride of the corresponding 2',3 '-0-stannylene derivative,I2 and more effectively, through alkylation of the 2',3'-diols with 2-nitrophenyl d i a z ~ m e t h a n e 'or ~ .with ~ ~ 2-nitrobenzyl bromide in the presence of sodium hydride in N,N-dimethylformamide. Photoremoval of the protecting (9) E. Ciamician and P. Silber, Ber., 34 (1901) 2040-2046. (10) B. Amit, U. Zehavi, and A. Patchornik, Absrr. Pap. Israel Chem. SOC.Meet., 42nd,
(1972) 60. (11) U. Zehavi, B. Amit, and A. Patchornik, Abstr. Pap. Am. Chem. SOC.Meet., 165rh,
(1973) CARB 11. (12) E. Ohtsuka, S. Tanaka, and M. Ikehara, Nucleic Acid Res., 1 (1974) 1351-1357. (13) D. G. Bartholomew and A. D. Broom, J . Chem. SOC.,Chem. Commun., (1975) 38. (14) E. Ohtsuka, T. Wakabayashi, S. Tanaka, T. Tanaka, K. Oshie, A. Hasegawa, and M. Ikehara, Chem. Pharm. Bull., 29 (1981) 318-327. (15) E . Ohtsuka, S. Tanaka, and M. Ikehara, Chem. Pharm. Bull., 25 (1977) 949-959. (16) E. Ohtsuka, S. Tanaka, and M. Ikehara, Synthesis, (1977) 453-454.
I82
URI ZEHAVI
group was achieved in very high yield (95-100%).'3*'5It should be mentioned that 2'-nBn ethers of nucleotides are substrates of bacteriophage TCinduced, Escherichia coli ribonucleic acid (RNA) ligase (EC 6.5.1.3) in a synthetic, organic-enzymic approach, and that even the simultaneous removal of seven nBn groups in one molecule, following the synthesis, by the triester method, of a protected heptanucleotide, afforded the product in 30% yield.l5J7 Furthermore, synthetic oligonucleotide blocks were connected by the same RNA ligase, culminating in the synthesisla of E. coli tRNA,M"'and of modified tRNA's.I9 The proposed mechanism for the photochemical cleavage of nBn ethers (see Scheme 3) involves a n --f 7 ~ *transition of the nitrogroup and an intramolecular, benzylic hydrogen abstraction by the excited nitro group. Rearrangement leads to a hemiacetal that decomposes to a free alcohol and to 2-nitrosobenzaldehyde that undergoes further thermal and photochemical reactions. Using benzyl esters, difficulties were experienced during the debenzylation (hydrogenolysis) step by many investigators including the author,20 and alternatives were sought. BeMiller and his colleagues, who encountered this problem, particularly with oligosaccharide derivatives which, they assumed, coat the catalyst, offered photolysis in the presence of bromine (see Scheme 4). The yield was as high as 100% where D-glucitol, which is not sensitive to oxidation under the reaction conditions, was the product.21 2. Protection as Acetals
a. G1ycosides.-The photoinduced cleavage of aryl glycosides is a particularly attractive reaction that was c o n d u ~ t e d ~by ~ -utilizing *~ irradiation of wavelength 254 nm. In the context of developing a potential means for using solar irradiation to break lignocellulosic bonds, lower-energy irradi(17) E. Ohtsuka, S. Tanaka, and M. Ikehara, J . Am. Chem. Soc., 100 (1978) 821043213, (18) E. Ohtsuka, S. Tanaka, T. Tanaka, T. Miyake, A. F. Markham, E. Nakagawa, T.
(19) (20) (21) (22) (23) (24) (25)
Wakabayashi, T. Taniyma, S. Nishikawa, R. Fukumoto, H. Uemura, T. Doi, T. Tokunaga, and M. Ikehara, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 5493-5497. E. Ohtsuka, J. Matsugi, T. Jitsuhiro, H. Takashima, S. Aoki, T. Wakabayashi, T. Miyake, and M. Ikehara, Chem. Pharm. Bull, 31 (1983) 513-520. U. Zehavi and A. Patchornik, J . Am. Chem. Soc., 95 (1973) 5673-5677. J. N . BeMiller, R. E. Wing, and C. Y. Meyers, J . Org. Chem., 33 (1968) 4292-4294. G. Tanret, C. R . Acad. Sci., 201 (1935) 1057-1060. L. H. Heidt, J . Am. Chem. Soc., 61 (1939) 2981-2982. T. Yamada, M. Sawada, and M. Taki, Agric. Biol. Chem., 39 (1975) 909-910. W. G . Filby, G. 0. Phillips, and M. G. Webber, Carbohydr. Res., 51 (1976) 269-271.
PHOTOSENSITIVE PROTECTING GROUPS 0-
I83
0I
I
0I
nu-
SCHEME 3.-Proposed Mechanism for the Photochemical Cleavage of 2-Nitrobenzyl Ethers and 2-Nitrobenzyl Glycosides.
RO-CH2
SCHEME 4.-Photochemical
3
hv, Br,
ROH
Cleavage of Benzyl Ethers in the Presence of Bromine.
ation was used in the 1,4-dicyanonaphthalene-sensitizedtransformation of aryl glycosides. High yields of products were reported in a number of instances, starting from phenyl glycosides and leading to free sugars or to methyl glycosides, depending on the reaction conditions26-28(see Scheme 5 ) . (26) J. D. Timpa, M. G. Legendre, G. W. Griffin, and P. K. Das, Curbohydr. Res., 117 (1983) 69-80. (27) H. F. Davis, P. K. Das, G. W. Griffin, and J. D. Timpa, J . Org. Chem., 48 (1983) 5256-5259. (28) J. D. Timpa and G. W. Griffin, Curbohydr. Res., 131 (1984) 185-196.
URI ZEHAVI
184
CH20H
CH2OH
RQph
R’ ‘ O O f ‘ h
R’
6H
OH
OH
bH
R = OH or OMe
A’sOH,
SCHEME5.-Cleavage,
R Z = H or R’=H,
R*=OH
Photosensitized by 1,4-Dicyanonaphthalene (DCN), of Phenyl
Glycosides.
Thus, irradiation of phenyl p-D-glucopyranoside yielded D-glucose (up to 70% yield); the conversion of the same glucoside into methyl a-and pD-glucopyranoside (transglycosylation) may also serve as an example to illustrate the reaction mechanism (see Scheme 6). 2-Nitrobenzyl and 3,4-dimethoxyd-nitrobenzyl (6-nitroveratryl) glycosides of monosaccharides are more stable to acid hydrolysis than are the corresponding benzyl glycosides. On the other hand, they are readily photolyzed at >320 nm to the reducing sugars in very high yields (compare Scheme 7 for an example, and Scheme 3 for a proposed mechaSoon after the introduction of the aforementioned glycosides, a lightsensitive polymer (a modified, cross-linked polystyrene) was synthesized, and it served as the aglycon in developing a polymer-supported, oligosaccharide synthesis.20At the end of the synthesis, a benzylated disaccharide was removed from the polymer by photolysis at >320 nm in very high yield (see Scheme 8), and hydrogenolyzed to isomaltose. The 2-nitrobenzyl linkage between the saccharide derivatives and the polymer provided a stable “hook” during synthesis, and the end product was a free, reducing sugar. Subsequently, a combined organic-enzymic approach was proposed5 for the synthesis of oligosaccharides. By common organic procedures, a (29) U. Zehavi, B. Amit, and A. Patchornik, J . Org. Chem., 37 (1972) 2281-2285. (30) U. Zehavi and A. Patchornik, J . Org. Chem., 37 (1972) 2285-2288.
DCN
hv
'OCN*
CH,OH
+
QPh HO
?
-
'OCN*
+
OCN'
OH
OH
1
I"+
MeOH,-H*
CHzOH HOQMe
+
CH20H
HoQoMe
OH 36 '1.
H
O
G
OH
49 'I. SCHEME 6.-Proposed Mechanism for the Photoinduced, Electron-transfer Reaction of Phenyl p-D-Glucypyranoside with 1.4-Dicyanonaphthalene (DCN) in 1 : 10 Methanol-Acetonitrile. Irradiation at 350 nm was Continued for 72 h.
OMe
OH OMe
CH20H HOO
O
H
+ ocH+OMe
ON OH SCHEME 7.-Photochemical Cleavage of 6-Nitroveratryl p-D-Glucopyranoside.
@I
@I
0
~N@COocH2
, ++oe :
Oeacylation C A 6tta H 6ch , C5H5N ment
NO2
*
HOCH2
OBn
2
1
~
0nO QOCH?
NO2
OBn
NaOEt
OBn
3
4
@I
I
.
Deacylation Elongation
1 + 4
NaOEt
NO2
C ~ H GCsHgN , OBn
OBn
6
5
ooH . @I
6
Release hv c
OBn
NO
en0
H2,PdlC
OH
CHO
OH
OBn
lsomaltose
7
8
SCHEME8.--Synthesis of Isomaltose (8) on a Light-sensitive, Solid Support (@).
PHOTOSENSITIVE PROTECTING GROUPS
187
saccharide derivative is attached, by way of a 2-nitrobenzyl bond, to a polymer where it subsequently serves as an acceptor for glycosyltransferase or transglycosylation reaction^.^'-^^ The nature of the newly formed glycosidic bonds is determined by the enzyme specificity, wherever possible, avoiding, among other things, the complex blocking-group chemistry needed for equivalent chemical synthesis. Irradiation at >320 nm releases from the polymer, whether insoluble or water-soluble, free oligosaccharides in very high yields. A simple illustration of such a sequence carried out with either insoluble 2-aminoethylsubstituted poly(acry1amide) beads3' or with water-soluble, substituted poly(viny1 alcohol)32is presented in Scheme 9; the isolated overall yield of lactose was 29.9% (soluble-polymer approach). The synthesis on lightsensitive polymers facilitates the isolation of products, which is important from the preparative point of view and as a tool for the study of enzymes, permitting efficient comparison of acceptor specificity and being capable of demonstrating de nouo synthesis.
90 . CONH-@
HOCHz
+NO?
+
-
HOCH,
UCHZ it;'
HO
OH
H~N-@
HO
OH
UDP-Ga, D-Galaclosykansferase
~
H2
HOCH, O
HOCHl
~ONH@ O
H
+ CHO
I
OH
SCHEME 9.-Incorporation of D-Galactose into a P-D-Glucopyranosyl Polymer Catalyzed by P-D-Galactosyltransferase (EC 2.4.1.22), Followed by Photochemical Release of Lactose.
(31) U. Zehavi, S. Sadeh, and M. Herchman, Carbohydr. Res., 124 (1983) 23-34. (32) U . Zehavi and M. Herchman, Carbohydr. Res., 128 (1984) 160-164. (33) U . Zehavi and M. Herchman, Carbohydr. Res., 151 (1986) 371-378.
URI ZEHAVI
188
b. Protection of Diols.-2-Nitrobenzylidene derivatives of carbohydrates served in the protection of diols and were photolyzed in the pioneering work of T i W i ~ e s c u . ' Isolation ?~~ and characterization of these derivatives and of their photochemical products were hampered, however, by the inadequate physical techniques available at that time. Subsequently, Collins and his collaborator^^^-^^ developed use of the substituent group as an attractive alternative to benzylidene in the selective protection of glycosides (see Scheme lo).
R'
R5°-co9
R3
H u 4 OH
ON
SCHEME10.-Photochemical Cleavage of 2-Nitrobenzylidene Derivatives.
The preparation of 2-nitrobenzylidene derivatives creates a complication, because, in every case, diastereoisomers (endo and exo) are formed. Irradiation at >290 nm of a mixture of the endo and exo isomers leads to results identical to those from the irradiation of the separate isomers, and, in order to facilitate the isolation and characterization of the photoproducts, the aromatic nitroso groups are further oxidized to the corresponding nitro groups with peroxytrifluoroacetic acid (see Scheme 11). The mechanism proposed for the photochemical reaction is analogous to that discussed in the context of nBn ethers and glycosides (see Scheme 3). However, as only, one 2-nitrobenzylidene group protects two hydroxyl groups, the possibility of regiospecificity occurs in the photochemical
(34) I. TBnBsescu, Bull. Soc. Sci. Cluj, 2 (1924) 1 1 1 ; Chem. A s f r . , 19 (1925) 2932. (35) P. M. Collins and N . N. Oparaeche, J . Chem. Soc., Chem. Commun., (1972) 532-533. (36) P. M. Collins and N . N . Oparaeche, Curbohydr. Res., 33 (1974) 35-46. (37) P. M. Collins and N. N. Oparaeche,J. Chem. Soc., Perkin Trans. I , (1975) 1695-1700. (38) P. M. Collins, N. N . Oparaeche, and V . R . N. Munsinghe, J . Chem. Soc., Perkin Trans. I, (1975) 1700-1706. (39) P. M. Collins and V. R. N . Munsinghe, J . Chem. Soc., Chem. Commun., (1981) 362363.
PHOTOSENSITIVE PROTECTING GROUPS
I89
cleavage. In fact, since a nitrosobenzoate is apparently an intermediate, the hydroxy ester, in which the hydroxyl group is equatorial, is preponderant whenever the orthoester is derived from a vicinal diol on a sixmembered ring. The overall yields in the photochemical reaction (followed by oxidation) are normally very high and often even quantitative. Such groups as acetate, nitrobenzoate, methyl glycoside, anhydro, and ptoluenesulfonate are unaffected by the reaction condition^.^^ 2-Nitrobenzylidene derivatives were successfully utilized in syntheses leading to trisaccharides of biological ~ignificance.~~ OMP
OMe
OMe
SCHEME 1 1 .-Utilization of a 2-Nitrobenzylidene Protecting Group in the Synthesis of Methyl 6-Deoxy-2,3-di-0-~-~-galactopyranosyl-c~-~-galactopyranoside. P-D-Galactopyranosyl (R') Substituents Were Introduced by Koenigs-Knorr Syntheses, and the Photochemical Cleavage, at 350 nm, of the 2-Nitrobenzylidene Groups Proceeded Regiospecifically , Yielding, Following Oxidation, a 95% Yield of the 3-Hydroxy-4-(2-nitrobenzoate) Derivative.
The conversion of benzylidene and ethylidene derivatives into hydroxy benzoates and hydroxy acetates, respectively, following irradiation in acetone, and preferably in the presence of oxygen, was discussed by B i n k l e ~Irradiation .~ is carried out at a shorter wavelength (compared to 2-nitrobenzylidene derivatives) and the yields are significantly lower. 3. Protection as Esters Dimethylthiocarbamates are known to undergo photochemical cleavage, leading, in the case of monosaccharide derivatives, to deoxy sugars and to free alcohols (see Scheme 12).637
URI ZEHAVI
I
MeOH
ROH
SCHEME 12.-Proposed Mechanism for the Photochemical Cleavage of Dimethylthiocarbamates (R’ = NMe2, Routes a and b) and xanthates (R1 = SMe, Route a).
In the particular case of 6-O-(dimethylthiocarbamoyl)-1,2:3,4-di-O-isopropylidene-a-D-galactopyranose, the reported yields of the 6-deoxy and the 6-hydroxy products were 25 and 35%, respectively (see Scheme 13). Xanthates, on the other hand, are photolyzed to yield the corresponding alcohols, and the yield reported, for instance for 1,2:3,4-di-O-isopropylidene-6-O-[(methylthio)thiocarbamoyl]-a-~-galactopyranose (50%, see Scheme 13) is apparently lowered by partial removal of isopropylidene groups under the reaction condition^.^^ Photosensitive, 2-nitrobenzylcarbonate was utilized as a hydroxyl-pro-
9 10
R = OCS- NMe2 R = OH
11 R = H
12
R =OCS-SMe
9
L 10 t 11
12
hv
10
SCHEME13.-Photochemical Cleavage of 6-O-(Dimethylthiocarbamoyl)-l,2 : 3,4-di-0isopropylidene-a-D-galactopyranose(9) and of 1,2 : 3,4-Di-O-isopropylidene-6-O-[(methylthio)thiocarbonyl]-a-D-galactopyranose(U). (40) G . Descotes, A. Faure, B. Kyrczka, and M. N . Bouchu, Bull. Acad. Pol. Sci. Chem., 27 (1979) 173-179.
PHOTOSENSITIVE PROTECTING GROUPS
191
tecting group in penicillin ~hemistry.~' The protecting group is probably removed by a mechanism analogous to that proposed for photocleavage of 2-nitrobenzyl ethers (see Scheme 3), releasing the free alcohol, carbon dioxide, and 2-nitrosobenzaldehyde that undergoes further reactions (see Scheme 14). Ro-CO~CH,
9hv
ROH
+
CO2
+
OCH
4N
ON
SCHEME 14.-Photochemical
Cleavage of 2-Nitrobenzylcarbonates.
p-Toluenesulfonates are photolyzed at <3 10 nm to the parent alcohols, as illustrated in Scheme I5 for a case where a quantitative yield was achieved.42This protection is very convenient and widely ~ t i l i z e d , ~ v ~ ~ - ~ ~ although there are examples of considerably lower yields due to the presence of an additional chromophore in the molecule,51and of p-toluenesulfonate, which is a good leaving-group. Methanesulfonates are also photolyzed, at 254 nm, to the free alcohols in very good (87%) yield^.^ The photochemical removal of nitric esters may be conducted in yields of 92-loo%, although byproducts resulting from ring fission may be expe~ted.~~
Q
Me2C-0
0-CMe2
?ro CH20H
hv
' Me2C-4:
0-CMe2
SCHEME 15.-Photolysis of 1,2 :3,4-D~-O-isopropyl~dene-6-U-p-Tolylsulfonyl-~-~-galactopyranose (R = p-tolylsulfonyloxy) and of 1,2 : 3,4-Di-O-isopropylidene-6-O-nitro-a-~-galactopyranose (R = ONOz). L. D. Cama and B. G. Christensen, J. Am. Chem. Soc., 100 (1978) 8006-8007. S. Zen, S . Tashima, and S. Kot6, Bull. Chem. Soc. Jpn., 41 (1968) 3025. A. D. Bradford, A. B. Foster, and J. H. Westwood, Carbohydr. Res., 13 (1970) 189190. A. D. Bradford, A. B. Foster, J. H. Westwood, L. D. Hall, and R. N. Johnson, Carbohydr. Res., 19 (1971) 49-61. L. Vegh and E. Hardegger, Helv. Chim. Acta, 56 (1973) 2020-2025. W. A. Szarek, R. G. S. Ritchie, and D. M. Vyas, Curbohydr. Res., 62 (1978) 89-103. F. R. Seymour, Carbohydr. Res., 34 (1974) 65-70. R.-A. Borgegrain and B. Gross, Carbohydr. Res., 41 (1975) 135-142. F. R. Seymour, M. E. Slodki, R. D. Plattner, and L. W. Tjarks, Curbohydr. Res., 46 (1976) 189-193. C.-D. Chang and T. L. Hullar, Carbohydr. Res., 54 (1977) 217-230. W. A. Szarek and A. Dmytraczenko, Synthesis, (1974) 579-580. R. W. Binkley and D. J. Koholic, J. Urg. Chem., 44 (1979) 2047-2048.
I92
URI ZEHAVI
111. AMINOFUNCTIONS The protection of amino groups of amino sugars benefits particularly from the use of new blocking groups introduced for peptide synthesis. In this context, light-sensitive urethans and amides that can be utilized for the protection of amino groups in amino sugars are of particular interest in saccharide synthesis and modification.
1. Protection as Urethans The 3,5-dimethoxybenzyloxycarbonylgroup, introduced in 1966 by Chamberlin,s3p54undergoes solvolysis upon irradiation under conditions (254nm) harmful to some amino acid residues, but quite harmless to many carbohydrate derivatives (see Scheme 16). 3,5-Dimethoxy-aa-dimethyl-benzyloxycarbonylconstitutes an improvement over the 3,5-dimethoxybenzyloxycarbonylgroup (see Scheme 16).55,56 The photochemical removal is easier (faster by a factor of 6), proceeding in high yield and, in numerous experiments, in quantitative yields. The group is, however, sensitive to mild acid, a disadvantage on many occasions. On the other hand, it can be photolyzed under conditions that do not affect a benzyloxycarbonyl group. H
OMe RNHZ
+
COz+ HO-C ! 4 0 M e H OMe
I
OMe
CH,
-%
RNH-COz-!-@
I
CH3
YqoMe
OMe
OMe
RNHz + C O z +
/ CH3
OMe
SCHEME16.-Photochemical Removal of 3,5-Dimethoxybenzyloxycarbonyland 3,5-Dimethoxy-a,a-dimethylbenzyloxycarbonylGroups.
J. W. Chamberlin, J . Org. Chem., 31 (1966) 1658-1660. T. Wieland and C. Birr, in H. C. Beyerman, A. van de Linde, and W. M. van den Brink (Eds.), Proc. Eur. P e p f . Symp., 8th, 1966; Peptides, North-Holland Publishing Co., Amsterdam, 1967, pp. 103-106. C. Birr, W. Lochinger, G . Stahnke, and P. Lang, Justus Liebigs Ann. Chem., 736 (1972) 162-172. C. Birr, in Y. Wolman (Ed.), Proc. Eur. Pept. Symp., 13th, 1974: Peptides, Wiley, New York, 1975, pp. 381-384.
PHOTOSENSITIVE PROTECTING GROUPS
I93
3-Nitrophenyloxycarbonyl has not been a successful group in peptide chemistry, as dipeptide products are cyclized to the corresponding diketopiperazine~,~~ but this is not the case with amino acid derivatives. Here, the free amino acids are obtained at >290 nm in very high yields [for example, 89% of L-phenylalanine from 3-(nitrophenyloxycarbonyl)-~phenylalanine] . 6-Nitroveratrylox ycarbonyl and 2-nitrobenzyloxycarbonylgroups were first utilized as light-sensitive blocking-groups for amino acids and peptides.s8 The protecting group is readily removed by mild irradiation at >320 nm, but the yields of the free amino derivatives are lowered by reaction with the aromatic photoproducts (see Scheme 17). However, the yields approach quantitative values when reagents such as hydrazine or semicarbazide hydrochloride, for acids or aldehydes, are included in the reaction mixture. Another way in which to avoid the side reactions that consume the newly formed amino derivative was to develop a similar protecting group, 2,2'-dinitrodiphenylmethyloxycarbonyl, that releases, upon irradiation, a ketone (less reactive), not an aldehyde.
RNH - C O , - CH2
R'
+
CO,
R'
RNH--02-CH
-
NO
02N
RNH,
hv
6
+ OHC NO
SCHEME 17.-Photochemical
Cleavage of 2-Nitrobenzyl (R' = H)and 6-Nitroveratryl
(R' = OMe) Urethans.
The potential usefulness of nBn urethans, or substituted nBn urethans, in syntheses involving amino sugar derivatives was r e c o g n i ~ e d The .~~ compounds are much more stable than benzyloxycarbonyl derivatives to hydrogen chloride in acetic acid, and can be conveniently utilized in a Koenigs-Knorr synthesis leading, probably due to neighboring-group participation, to a p-anomeric product. Inclusion of mineral acids or, (57) T. Wieland, C. Lamperstorfer, and C. Birr, Makromol. Chem., 92 (1966) 277-286. (58) A . Patchornik, B. Amit, and R. B. Woodward, J . A m . Chem. Soc., 92 (1970) 63336335. (59) B . Amit, U . Zehavi, and A. Patchornik, J . Org. Chem., 39 (1974) 192-196.
URI ZEHAVI
194
(8
alternatively, polymeric = cross-linked polystyrene) carriers of aldehyde reagents [@-CH2NH(CH3)CONHNH2and B - N H N H J , during the photoremoval of the protecting groups increases the yields of the free amino derivatives to 95-loo%, and simplifies their isolation. The mechanism suggested for the reaction is analogous to that proposed in Scheme 3, with the difference that the intermediate produced, following the photochemical reaction, decomposes to yield the free amine, carbon dioxide, and 2-nitrosobenzaldehyde (see Scheme 17). 2. Protection as h i d e s
Amino groups are traditionally protected as amides. Sulfonamides were described as light-sensitive protecting-groups for amines. Although irradiation of higher energy (high-pressure mercury lamp) and the presence of sodium borohydride were needed60-62for the removal of p-tolylsulfonyl groups in modest yields (21-65%), 6-methoxy-2-(4-methylphenyl)-4(methylsulfonyl)quinoline has provided a much more attractive option.63 Amines-the authors were particularly interested in nucleotides-are readily protected by using the corresponding chloride, and photocleavage proceeds at 350 nm in yields of up to 96% for aliphatic amines (see Scheme 18).
,R’ c H,SO,
N‘
hv
/
R’
HN
+
so*
+
‘R2
CH3
SCHEME 18.-Photodecomposition ylpheny1)quinoline.
of
(60)0. Hoshino, S . Sawaki, and B . Umezawa, Chem. Pharm. Bull., 18 (1970)182-185. (61)A. Abad, D.Mellier, J. P. PBte, and C. Portella, Tetrahedron Lett., (1971)4555-4558. (62) J. A. Pincock and A. Jurgens, Tetrahedron Lett., (1979) 1029-1030. (63) G.A. Epling and M. E. Walker, Tetrahedron Lett., (1982)3843-3846.
PHOTOSENSITIVE PROTECTING GROUPS
I95
IV. CARBONYL DERIVATIVES 1. Protection of Aldehydes and Ketones Generally, carbonyl derivatives have to be protected during synthesis. In the case of carbohydrate synthesis, this is frequently done through the intramolecular formation of hemiacetals, followed by alkylation or acylation. In the first instance, glycosides are formed. Light-sensitive glycosides were discussed in Section II,2. Photosensitive protecting groups discussed here belong to three different classes: cyclic acetals, thioacetals, and hydrazones. (2-Nitropheny1)ethylene glycol was used to protect simple aldehydes and ketones, as well as some steroid^.^^^^ Acetals were prepared under acid catalysis, leading, in the case of chiral carbonyl compounds to diastereoisomers. The photochemical removal of the protecting group was in several instances complicated by the instability of some carbonyl derivatives to irradiation at 350 nm; otherwise, yields were in the range of 8390% (see Scheme 19). R!
0 1
hv
HO
SCHEME 19.-Proposed Mechanism for the Photochemical Cleavage of (2-Nitropheny1)ethylenedioxy Acetals. (64) J . Herbert and D. Gravel, Can. J . Chern., 52 (1974) 187-189. (65) D. Gravel, J . Herbert, and D. Thoraval, Can. J . Chern.. 61 (1983) 400-410.
URI ZEHAVI
196
Particular attention was given6j to the stability of the protecting group: the (2-nitropheny1)ethylenedioxyacetal of benzophenone was found to be stable under various basic conditions, but to decompose during reflux with sodium hydride in dimethoxyethane, releasing free benzophenone (see Scheme 20).
is-
SCHEME 2O.-F'roposed Acetal by Strong Base.
Mechanism for the Removal of (2-Nitropheny1)ethylenedioxy
The protecting group is considered to be more stable to acid hydrolysis than the phenylethylenedioxy acetal and to possess stability comparable to that of other aromatic nitro derivatives under reducing conditions. Ethylene and 1,2-diphenylethylene dithioacetals derived from different ketones decompose upon irradiation (high-pressure mercury lamp) in the presence of oxygen to the parent ketones (see Scheme 21); the rep ~ r t e dyields ~ , ~ were ~ 57-90%. In an analogous case that may serve as (66) T. Takahashi, C. Y. Nakamura, and J. Y. Satoh, J . Chem. SOC.,Chem. Commun., (1977) 680. (67) 0 . Hoshino, S . Sawaki, and B. Umezawa, Chem. Pharm. Bull., 27 (1979) 538-540.
PHOTOSENSITIVE PROTECTING GROUPS
197
another illustration of the modifier protecting group concept (see Section I), D-galactose diethyl dithioacetal was converted by a similar irradiation, but excluding oxygen, into a mixture of redox and solvolysis products68 (see Scheme 22) rather than into D-galactose and, possibly, oxidation products thereof.
Decomposition of 1 ,ZDiphenylethylene Dithioacetals in
SCHEME 21 .-Photochemical the Presence of Oxygen.
HC(SEtI2 I HCOH
I
HOCH
I
HOCH I HCOH I CHzOH
CHZSEt I HCOH
-
y 3
HCOH
I
I
HOCH
hV
I I
HOCH
+
HCOH
I
CHZOH
HOCH I HOCH I HCOH I CHzOH
+
0
t
CHZOH I HCOH
CH2SEt I
HCOH
I
HOCH I HOCH
I HCOH I
+
HOCH
I
CHzOH
SCHEME 22.-Photochemical
I I
HOCH HCOH I CHzOH
Decomposition of D-Galactose Diethyl Dithioacetal.
N,N-Dimethylhydrazones are photolyzed in the presence of oxygen, under conditions that are assumed to generate singlet oxygen (mediumpressure mercury lamp with Methylene Blue as a sensitizer) in694 8 4 8 % yields (see Scheme 23). (68) D . Horton and J . S. Jewell, J . Org. Chem., 31 (1966) 509-513. (69) E. Friedrich, W. Lutz, H. Eichenauer, and D. Enders, Synthesis, (1977) 893-894.
URI ZEHAVI
198 "Mez
I
R-c-~
SCHEME 23.-Photosensitized ence of Oxygen.
hv, 0 2 Methyiene Blue
RCHO
Decomposition of N,N-Dimethylhydrazones in the Pres-
2. Protection of Carboxylic Acids Ester formation is the main and most efficient means of protecting carboxylic acids. The protection of a carboxylic acid as an amide is infrequent, as its removal normally requires drastic conditions. Most of the work concerned with the use of light-sensitive protecting-groups for carboxylic acids is in peptide synthesis. Carboxylic acids are protected as photosensitive esters, including ester linkages to polymer supports or as (difficult to prepare) photosensitive amides. Many of these techniques may be readily applied to sugar acids. The 2-nitrobenzyl (nBn) group was first used for the protection of carboxylic acids.70The low yield in the photochemical reaction of free acid (17%) was interpreted in terms of formation of the light-absorbing azobenzene-2,2'-dicarboxylic acid, and, indeed, the yields of the photochemical reaction increased dramatically (75-90%) by use of substituted a-phenyl protecting groups,7o or rather, an a-2-nitrophenyl substituted one, and irradiating at a wavelength longer than 320 nm (quantitative yield; see Scheme 24). The mechanism of the reaction is analogous to that proposed in Scheme 3. RC02 -CHR'
-% RC02H
-9)
4 N
SCHEME 24.-Photochemical
+ OCR1
9
ON
Cleavage of 2-Nitrobenzyl Esters.
The photoremoval of nBn groups may be more efficiently effected by including mineral acids or aldehyde reagentP in the reaction mixture. 2Nitrobenzyl esters also served as protecting groups in penicillin chemistry4' and in the protection of carboxyl groups of a triterpene during a Koenigs-Knorr ~ynthesis.~' An important outcome of the study of nBn esters was the development of polymers carrying a-halo-(mostly bromo)-2-nitrobenzyl groups that (70) J. A. Barltrop, P. J. Plant, and P. Schofield, J . Chem. Soc., Chem. Commun., (1966) 822-823. (71) M. Levy, U . Zehavi, M. Naim, R . Evron, and I. Polacheck, Absrr. Pup. Eur. S y m p . Org. Chem., Srh, (1987) 272.
PHOTOSENSITIVE PROTECTING GROUPS
199
served, following the formation of ester bonds to carboxyl groups, in polymer-supported peptide synthesis. The first such polymer was the product of direct nitration of (chloromethyl)ated, cross-linked polystyrene72,73; the polymer suffered from poor swelling and, possibly, also from partial attachment of the carboxy derivative to non-nitrated positions. The release from the polymer, following irradiation, was 53-71%. Improved accessibility, but rather similar photoremoval from the polymer (50-64%), was attained when a 4-(bromomethyl)-3-nitrobenzoylgroup was attached to amino-substituted, cross-linked polystyrene, thus creat~ . ’ ~Fig. 1). ing a reactive p ~ l y m e r ~(see B r - C H 2 F CONH -CH2 O2N’
FIG. I .-a-Bromo-2-nitrobenzyl
(@ = Crosslinked Polystyrene).
Polymer
Condensation steps, reactivity of substituents on the polymer, and photochemical cleavage at 350 nm were dramatically improved (up to 98%) when the insoluble, cross-linked polystyrene was r e p l a ~ e d ’ ~by. ~a~ soluble, modified poly(viny1 alcohol) (see Scheme 25). RCO2- CH2 F
C
O
- -CHz-CHz-OCO
, -(CH?-CHz-O),
02N
WHf NO2
1
OCO
hv
2 RC02H
+
OHC
9-
CO, -(CH2-CHz-O),
ON
SCHEME 25.-Photochemical
-
-CH2-CH2-OCO
*CHO NO
Release of Free Carboxylic Acid from a 2-Nitrobenzyl-
substituted Poly(viny1 Alcohol).
R. B. Merrifield, J . Am. Chem. Soc., 85 (1963) 2149-2154. D. H. Rich and S. K. Gunvara, J . Chem. Soc., Chem. Commun., (1973) 610. D. H. Rich and S. K. Gunvara, J . Am. Chem. Soc., 97 (1975) 1575-1579. E. Giralt, F. Albericio, D. Andreu, R. Erita, P. Martin, and E. Pedroso, An. Quim., Ser. C , 77 (1981) 120-125; Chem. Abstr., 97 (1982) 92,739b. (76) F . 3 . Tjoeng, W. Stakes, S. %-Pierre and R. S. Hodges, Biochim. Biophys. Acfa, 490 (1977) 489-496. (77) F . 4 . Tjoeng, E. K . Tong, and R. S. Hodges, J. Org. Chem., 43 (1978) 4190-4194.
(72) (73) (74) (75)
URI ZEHAVI
200
2,6Dinitrobenzenesulfenic esters were proposed as protecting groups for carboxyl g r o u p ~ . ~The ~ Osolvolysis of only a small variety of esters was studied with a high-pressure mercury lamp, and the yields were 8898% (see Scheme 26). It should be noted, however, that the group is particularly sensitive to hydrolysis and ammonolysis.
NO*
NO2 r
SCHEME26.-Photolysis
1
of 2,4-DinitrobenzenesulfenicEsters.
Esters of 2-(2-azidophenyl)ethyl alcohol are photolyzed under a highpressure mercury lamp to a reactive nitrene intermediate which, following insertion into the alkyl side-chain, undergoes elimination to give the free carboxylic acid (up to 32%) and producing indole.81The photochemical release was somewhat improved (6540%) when 5-azido-4-(hydroxymethyl)- 1-methoxy naphthalene was used (see Scheme 27). Another class of photosensitive protecting groups is that of benzoylphenylmethanol (benzoin) esters82v83 ; esters of 3,5-dimethoxybenzoin are photocyclized to the corresponding benzofuran while releasing the protected carboxylic acid in 87% yield at 366 nm (see Scheme 28). It is pertinent to note, though, that this protecting group has the complication (78) D. H. R. Barton, Y. L. Chow, A. Cox, and G . W. Kirby, Tetrahedron Lett., (1962) 1055- 1057. (79) D. H. R. Barton, Y . L. Chow, A. Cox, and G . W. Kirby, J . Chem. Soc., (1965) 35713578. (80) D. H. R. Barton, T. Nakano, and P. G . Sammes, J . Chem. Soc., C , (1968) 322-327. (81) D. H. R. Barton, P. G. Sammes, and G . G . Weingarten, J . Chem. Soc., C, (1971) 721728. (82) J. C. Sheehan and R. M. Wilson, J . A m . Chem. SOC., 86 (1964) 5277-5281. (83) J. C. Sheehan, R. M. Wilson, and A. W. Oxford, J . Am. Chem. SOC.,93 (1971) 72227228.
PHOTOSENSITIVE PROTECTING GROUPS
OMe
20 I
OM e
OMe
SCHEME 27.-Photocyclization of 2-(2-Azidophenyl)ethyl Esters and 5-Azido-4-(hydroxymethy1)-I-methoxynaphthalene Esters.
that the benzylic carbon atom constitutes another chiral center. 3,4,3',4'Dimethylenedioxybenzoin and 2,3,2',3'-tetramethoxybenzoinare not limited in this way, but the photocyclization yields are lower (75 and 76%). 0
II
RCO
Me0
Me0
SCHEME 28.-Photocyclization of Esters of Benzoin.
Phenacyl esters of benzoic acid, of amino acid derivatives, and of gibberillin A3 undergo photolytic cleavage at >313 nm, giving, in each case, the free carboxylic acid and the corresponding acetophenone (see Scheme 29).84,85 The yields of free carboxylic acids (33-96%) vary considerably, (84) J . C. Sheehan and K . Umazawa, J . Org. Chem., 38 (1973) 3771-3774. (85) E. P. Serebryakov, L. M. Suslova, and V. F. Kucherov, Tetrahedron, 34 (1978) 345351.
URI ZEHAVI
202
depending on the stability of parts other than the protecting group in the irradiated molecules. Should phenacyl esters be used in carbohydrate synthesis, a higher range of yields may most frequently be anticipated, although the use of a-methylphenacyl groups may be complicated by the formation of diastereoisomers.
-
R1
I
RCO2-CHCO+R2
h9
RCO;
+
RCOZH
I
+
R1 ‘CHCO*R’
I
ethanol or 1,4 -dioxane
R1CH2CO*RZ
SCHEME29.-Proposed mechanism for the Photocleavage of 4-Methoxyphenacyl (R’ = H, R2 = OMe) and a-Methylphenacyl (R’ = Me, R2 = H) Esters.
2-Nitroanilides, l-acyl-7-nitroindolines, and N-acyl- 1,2,3,6tetrahydro8-nitroquinolines are readily photolyzed to yield the free carboxylic acids (80-100%) and photoproducts.86-88These nitro derivatives (see Fig. 2) possess higher stability to acid hydrolysis and a lower stability to basic hydrolysis than non-nitrated anilines. Nevertheless, they are unlikely to prove of service to the carbohydrate chemist, unless they can be synthesized from carboxylic acids under milder conditions than those devised to date. R’ RCO?+R.
4N FIG.2.-Nitroanilides
qR’ p RCO
NO2
RCO
NO2
Employed as Protecting Groups for Carboxylic Acids.
V. PROTECTION OF PHOSPHORIC ESTERS Protection of phosphoric esters is particularly required for nucleotide synthesis, where some interesting applications of photosensitive groups, (86) B. Amit and A. Patchornik, Tetrahedron L e f t . , (1973) 2205-2208. (87) B. Amit, D. Ben-Efraim, and A. Patchornik, J . Am. Chem. Soc., 98 (1976) 834-843. (88) B. Amit, D. Ben-Efraim, and A. Patchornik, J . Chem. Soc., Perkin Trans. 1 , (1976) 57-63.
PHOTOSENSITIVE PROTECTING GROUPS
203
including those in biological systems (see Section VI), are to be found. The photosolvolysis of 3-nitrophenyl esters is the first example of the use of a light-sensitive protecting-group that has appeared in the literat ~ r e3-Nitrophenyl . ~ ~ and the more-sensitive 3,5-dinitrophenyl phosphoric esters undergo photohydrolysis (mercury lamp) to a phosphoric derivative and the corresponding n i t r ~ p h e n o l ~an ~ ~example ’; of a synthetic applicationg0is presented in Scheme 30. 2-Nitrobenzyl phosphoric esters, which are photolyzed to the free acids (compare Scheme 3), are used in nucleotide synthesis and are very efficiently cleaved (90-98%) at >320 nm in the presence of a polymeric carbonyl reagent (@-N(CH3)-CONHNH2) to remove the aromatic photoproducts; an example92is given in Scheme 30.
FI
w’
(R’o)~-P-ocH~
:,
(R’OIz - P C I
+ 0
0
%e2
-
hv
__c
0, /O
CMe2
0
0,
/o
CMe2
SCHEME 30.-Synthesis of 2’,3‘-O-Isopropylideneadenosine5’-Phosphate (R’ = 3.5Dinitrophenyl, R2 = Adenin-9-yl) and 2’,3‘-O-Isopropylideneuridine5’-Phosphate (R’ = 2Nitrobenzyl, R2 = Uridin-I-yl).
VI. APPLICATIONS TO BIOLOGICAL MODELS On many occasions, and this is certainly correct for 2-nitrobenzyl derivatives, photocleavable groups can be eliminated under mild conditions (aqueous media, neutral pH, and low temperature). Frequently, at a sufficiently long wavelength, only the bond designated for cleavage is affected by irradiation, so that, whenever the irradiation itself and the photoproducts formed are not harmful to the biological system (such as cells, (89) E. Havinga, R. D. De Jongh, and W . Dorst, R e d . Trau. Chim. Pays-Bas., 75 (1956) 378-384. (90)J. Kirby and A. G. Varvoglis, Chem. Commun., (1967) 406. (91) D. L. Miller and J. Ukena, J . Am. Chem. SOC., 91 (1969) 3050-3053. (92) M. Rubinstein, B. Amit, and A. Patchornik, Tetrahedron Lett., (1975) 1445-1448.
204
URI ZEHAVI
membranes, and proteins), it may be used for photoaffinity labeling,93-95 photo-reversible affinity labeling,96and the rapid formation of effectors in cells and membrane^.^' Examples of the last category often present the quickest means of producing chemical perturbations. Kinetic information has been collected in electrophysiological experiments with photoisomerizable, cholinergic compounds, and in the context of the present article, protected (commonly termed “caged”) nucleotides are frequently photolyzed, by brief, intensive laser irradiation, to the biologically active ones. P3-l-(2-Nitrobenzy1)adenosine 5’-triphosphate and P3-1-(2-nitrophenylethyl)adenosine 5’-triphosphate diffuse into cells and do not affect them. Irradiation cleaves the light-sensitive phosphoric ester bonds (compare, Section V) and releases the high-energy molecule ATP, immediately affecting various cellular s y ~ t e m s . ~In~ al ~similar l fashion, irradiation of 2-nitrobenzyl and 6-nitroveratryl esters of CAMPand cGMP brings about a jump in the intracellular concentrations of these two second m e ~ s e n g e r s *that ~ ~ are J~~ known to trigger cascades of cellular events. (93) W. 9. Jakoby and M. Wilcheck (Eds.), Photolabeling, Methods Enzymol., 46 (1977). (94) J. S. Fedan, G. K. Hogaboom, and J. P. O’Donnell, Biochem. Pharmacol., 33 (1984) 1 167- 1180. (95) H. Bayley and J. V. Staros, in E. F. V. Scriven (Ed.), Azides and Nitrenes, Reactivity and Utility., Academic Press, New York, 1984, pp. 433-490. (96) A. Patchornik, K. Jacobson, and M. P. Strub, Abstr. Pap. FEBS Meet., 16th, Moscow, (1984) 330. (97) H. A. Lester and J. M. Norbonne, Annu. Rev. Biophys. Bioeng., 11 (1982) 151-175. (98) J. N. Kaplan, 9. Forbush, and J. F. Hoffman, Biochemistry, 17 (1978) 1929-1935. (99) J. A. McCray, L. Herbette, K. T. Kihara, and D. R. Trentham, Proc. Nut/. Acad. Sci. U.S.A., 77 (1980) 7237-7241. (100) Y. E. Goldman, M. G. Hibberd, J. A. McCray, and D. R. Trentham, Nature (London), 300 (1982) 701-705. (101) Y. E. Goldman, M. G. Hibberd, and D. R. Trentham, J . Physiol. (London), 354 (1984) 577-604. (102) J. Engels and E. J. Schlaeger, J . Med. Chem., 20 (1977) 907-911. (103) J. M. Nerbonne, S. Richard, J. Margeot, and H. A. Lester, Nature (London), 310 (1984) 74-76.
ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 46
INCLUSION COMPLEXES OF THE CYCLOMALTO-OLIGOSACCHARIDES (CYCLODEXTRINS)
BY RONALDJ. CLARKE,JOHNH. COATES,A N D STEPHEN F. LINCOLN Johnson Laboratories, Department of Physical and Inorganic Chemistry, University of Adelaide, South Australia 5001, Australia 1. Introduction ............................................................ 11. Historical Review. . . . . . . . . . . ............................ 1. Discovery of the Cyclodext ............................ ............. 2. Determination of the Structure of the Cyclodextrins.. ... 3. Formation of the Cyclodextrins from Starch.. ........................... 4. Inclusion Complexes of the Cyclodextrins .............................. 111. Formation of Inclusion Complexes. ...... ........................... 1. Detection of Complex-Formation .......... ................ ..................... 2. Thermodynamics of Complex-Formation.. .. ..................... 3. Kinetics of Complex-Formation . . . . . . . 4. Modified Cyclodextrins ............................................... 5. Chiral Discrimination ....... .......................... IV. Conclusion ............................................................. ............ ...... Addendum to Article 6 . . . . . . . . . . . . . . . . . . .
205 211 211 213 215 217 219 219 220 234 244 247 249 333
I. INTRODUCTION Inclusion complexes are chemical species consisting of two or more associated molecules in which one of the molecules, the “host,” forms or possesses a cavity into which it can admit a “guest” molecule, resulting in a stable association without formation of any covalent bonds. Secondary forces are alone responsible for maintenance of the integrity of all inclusion complexes. Over the past twenty-five years, interest in the physical and chemical properties of inclusion complexes has grown considerably. One of the most important reasons for this is the relevance that inclusion complexes 20s
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
206
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
have to enzyme-substrate and drug-receptor interactions.I4 inclusion complexes have also been found to have wide application in the food and molecular encapsulation of a pharmaceutical i n d u s t r i e ~;~for . ~ ,example, ~ volatile or reactive compound within a suitable host molecule can often lead to a decrease in the susceptibility of the compound to hydrolysis, oxidation, or photolysis. Because they are non-toxic and are able to form complexes with numerous small organic molecules, perhaps the most important of all the compounds capable of acting as host components are the cyclomalto-oligosaccharides (cyclodextrins). The cyclodextrins are cyclic, (1 + 4)-linked oligomers of a-D-glucopyranose, each D-glucopyranosyl residue being in the 4C1conformation. The three most important cyclodextrins are the alpha, beta, and gamma cyclodextrins (Schardinger a-, /3-, and y-dextrins), which respectively consist of six, seven, and eight a-D-glucopyranosyl residues, and are systematically named cyclomaltohexaose, cyclomaltoheptaose, and cyclomalto-octaose. Because of its relative brevity, the term “cyclodextrin” will be used throughout this article. Higher homologs do exist; however, they are difficult to purify, and their complexing ability appears to be poor. l o Cyclodextrins having fewer than six a-D-glucopyranosyl residues are unknown, probably because of steric reasons.1’ As a consequence of the 4C1conformation of the a-D-glucopyranosyl residues and the lack of free rotation about the glycosidic bonds, the compounds are not perfectly cylindrical molecules, but are somewhat cone-shaped, with all of the secondary hydroxyl groups situated at one end of the annulus and all of the primary hydroxyl groups at the other. The cavity is lined by a ring of hydrogen atoms (bonded to C-9, a ring of D-glucosidic oxygen atoms, and another ring of hydrogen atoms (bonded to C-3), thus making the cavity relatively apolar. The shape of the molecule is stabilized by F. Cramer, Rev. Pure Appl. Chem., 5 (1955) 143-164. D. W. Griffiths and M. L. Bender, Adu. Catal., 23 (1973) 209-261. R. J. Bergeron, J . Chem. Educ., 54 (1977) 204-207. M. L. Bender and M. Komiyama, Cyclodextrin Chemistry, Springer-Verlag, Berlin, 1978. ( 5 ) W. Saenger, Angew. Chem., Int. Ed. Engl., 19 (1980) 344-362. (6) J. Szejtli, Cyclodextrins and Their Inclusion Complexes, Akademiai Kiado, Budapest, 1982. (7) I. Tabushi, Acc. Chem. Res., 15 (1982) 66-72. (8) R. Breslow, Chem. Br., (1983) 126-131. (9) Proc. In?. Symp. Cyclodextrins lst, .I.Szejtli (Ed.), Akademiai Kiado, Budapest, 1982. (10) D. French, A. 0. Pulley, J. A. Effenberger, M. A. Rougvie, and M. Abdullah, Arch. Biochem. Biophys., 1 1 1 (1965) 153-160. (11) P. R. Sundararajan and V. S. R. Rao, Carbohydr. Res., 13 (1970) 351-358. (I) (2) (3) (4)
CYCLODEXTRIN INCLUSION COMPLEXES
207
FIG. I.-Chemical Structure and Numbering of the Atoms of Beta Cyclodextrin.
hydrogen bonds between the secondary hydroxyl groups of adjacent Dglucopyranosyl residues. The numbering system and structure of beta cyclodextrin is shown in Fig. 1. The molecular dimensions of the cyclodextrins are given in Table I. The cyclodextrins are produced through the degradation of starch by the enzyme cyclodextrin transglycosylaseI2 (EC 2.4.1.19), which is obtained from the bacterium Bacillus maceruns. The crude starch digests contain alpha, beta, and gamma cyclodextrin, as well as smaller proportions of the higher cyclodextrins and some linear oligosaccharides. The cyclodextrins can be separated by selective precipitation with complexing solvents, and then purified by decomposition of the complex, removal of the guest compound, and crystallization of the cyclodextrin from water. The chemical synthesis of alpha cyclodextrin in 21 steps (and an overall yield of 0.3%) from maltose has been described.I2” The reason that the cyclodextrin transglycosylase occurs only in Bacillus maceruns and a few other thermophilic bacteria has not, as yet, been determined. Nevertheless, it is interesting to speculate about the possible evolutionary advantage that a bacterium might have by possessing the cyclodextrin transglycosylase. Because the cyclodextrins have no end (12) M. Florkin and E. H . Stotz (Eds.), Comprehensive Biochemistry, Vol. 13, Elsevier, Amsterdam, 1973, p. 141. (12a) Y. Takahashi and T. Ogawa, Carbohydr. Res., 164 (1987) 277-296.
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
208
TABLEI Physical Properties of the Cyclodextrin~~ Number of D-ghlCOSYl residues
Name
Molecular weight
Solubility in water (g/lOO mL)
Cavity width (pm)"
6 7 8
alpha cyclodextrin beta cyclodextrin gamma cyclodextrin
972 1 I35 1297
14.5
470-520 600-640 750-830
1.85 23.2
a As measured on Corey-Pauling-Koltun molecular models: the smaller value is for the ring of hydrogen atoms bonded to C-5, and the larger value is for the ring of hydrogen atoms bonded to C-3. The depth of the cyclodextrin cavity is 790-800 pm.
groups susceptible to attack, they are completely resistant towards degradation by beta amylases, and are only slowly degraded by the alpha amylases.6 Thus, bacteria that are capable of producing cyclodextrins can effectively convert starch into a form that is very difficult for all other bacteria to digest. It is possible, then, that bacteria possessing cyclodextrin transglycosylase use the cyclodextrins for the extracellular storage of energy. An important advantage of the inclusion complexes of the cyclodextrins over those of other host compounds, particularly in regard to their use as models of enzyme-substrate complexes, is their ability to be formed in aqueous solution. In the case of clathrates, gas hydrates, and the inclusion complexes of such hosts as urea and deoxycholic acid, the cavity in which the guest molecule is situated is formed by the crystal lattice of the host.' Thus, these inclusion complexes disintegrate when the crystal is dissolved. The cavity of the cyclodextrins, however, is a property of the size and shape of the molecule and hence it persists in solution. In fact, there is evidence that suggests that the ability of the cyclodextrins to form inclusion complexes is dependent on the presence of ~ a t e r . ' ~ J ~ Once an inclusion complex has formed in solution, it can be crystallized; however, in the solid state, additional cavities appear in the lattice, as in the case of the hosts previously mentioned, which enable the inclusion of further guest molecules. As shown in Table I, the space within the cyclodextrin cavity increases with the number of D-glucopyranosyl residues. Thus, as would be expected, the stability of an inclusion complex depends to a large degree on (13) J. L. Lach and T.-F. Chin, J . Pharm. Sci., 53 (1964) 69-73.
(14) B. Siege1 and R. Breslow, J. Am. Chem. Soc., 97 (1975) 6869-6870. (15) J. N. J. J. Lammers, J. L. Koole, and J. Hurkmans, Staerke, 23 (1971) 167-171.
CYCLODEXTRIN INCLUSION COMPLEXES
209
the relative sizes of the cyclodextrin cavity and the portion of the guest molecule to be included. For example, a molecule may be too large to fit within the cavity of alpha cyclodextrin, but might form a stable complex with the (larger) beta cyclodextrin. In solution, the most common cyclodextrin-guest stoichiometric ratio is 1 : 1. In the case of guest molecules that cannot be totally included by a single cyclodextrin molecule, however, a further cyclodextrin molecule may occasionally bind, for example, the complex of Methyl Orange with alpha c y ~ l o d e x t r i n . ~ ~ J ~ It has been found by various workers that certain guest molecules exhibit an enhancement of their excimer18fluorescence band on the addition of gamma cyclodextrin.2&2yThe presence of beta cyclodextrin has also been found to produce a similar effect, although in a smaller number of cases.22,28-30 It has been suggested that this effect indicates one hosttwo guests complexation.20Thus, it seems that the cavities of beta and gamma cyclodextrin are sufficiently large to be able to accommodate two molecules of certain guest compounds simultaneously. Further evidence for one host-two guests complexation has come from the measurement of induced circular d i ~ h r o i s m , ~ the ~ , ~measurement ~ ~ ~ * - ~ ~ of the u.v.-visible (16) F. Cramer, W. Saenger, and H.-C. Spatz, J . Am. Chem. Soc., 89 (1967) 14-20. (17) K. Harata, Bull. Chem. Soc. J p n . , 49 (1976) 1493-1501. produced by collisional interaction between ex(18) Excimers are excited dimers (D*) cited (M*) and unexcited monomers (M), that is,I9 M* + M + D*. (19) J . B. Birks, Photophysics ofAromatic Molecules. Wiley-Interscience, London, 1970, pp. 301-302. (20) A. Ueno, K . Takahashi, and J. Osa, J. Chem. Soc., Chem. Commun.,(1980) 921922. (21) J . Emert, D. Kodali, and R. Catena, J . Chem. SOC., Chem. Commun.,(1981) 758759. (22) N . J . Turro, T. Okubo, and G. C. Weed, Photochem. Photobiol., 35 (1982) 325-329. (23) K . Kano, I . Takenoshita, and T. Ogawa, Chem. Lett., (1982) 321-324. (24) T . Yoruzu, M. Hoshino, and M. Immamura, J . Phys. Chem., 86 (1982) 4426-4429. (25) N . Kobayashi, Y. Hino, A. Ueno, and T. Osa, Bull. Chem. Soc. J p n . , 56 (1983) 1849- 1850. (26) A. Ueno, Y. Tomita, and T. Osa, Chem. Lett., (1983) 1635-1638. (27) N . Kobayashi, R. Saito, H . Hino, Y. Hino, A. Ueno, and T. Osa, J . Chem. Soc., Perkin Trans. 2 , (1983) 1031-1035. (28) R. Arad-Yellin and D. F. Eaton, J . Phys. Chem., 87 (1983) 5051-5055. (29) C. H . Tung, Z. Zhen, and H. J. Xu, J . Phofochem., 32 (1986) 311-317. (30) S. Hamai, Bull. Chem. Soc. J p n . , 55 (1982) 2721-2729. (31) H. Hirai, N. Toshima, and S. Uenoyama, Polym. J . (Tokyo), 13 (1981) 607-610. (32) N. Kobayashi, A. Ueno, and T. Osa, J . Chem. SOC., Chem. Cornmun.,(1981) 340. (33) N . Kobayashi, R. Saito, Y. Hino, A. Ueno, and T. Osa, J . Chem. SOC., Chem. Commun., (1982) 706-707. (34) A . Ueno, Y. Tomita, and T. Osa, J. Chem. Soc., Chem. Commun.,(1983) 976-977. (35) N. Kobayashi, R. Saito, A. Ueno, and T. Osa, Mukromol. Chem., 184 (1983) 837847.
210
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
absorption ~ p e ~ t r ~ m Job's , ~ ~ method ~ ~ ~ of~ continuous ~ , ~ ~ , varia~ ~ ~ ' t i ~ n , ~and ' J ~micro~alorimetry,~'~ as well as additional fluorescence meas u r e m e n t ~It. ~should ~ ~ ~ ~be noted that 2 : 2 complex-formation has also been reported for beta30*43,44 and gamma c y c l o d e ~ t r i n . ~ ~ . ~ ~ - ~ ~ The ability of the cyclodextrins to form one host-two guests complexes has particular significance in the field of catalysis. The facilitation of the association of molecules through the presence of cyclodextrin could lead to an increase in the rate of certain reactions that the two molecules might undergo. Two examples of this have already appeared in the literature. Rideout and Bres10w~~ found that beta cyclodextrin accelerates the DielsAlder reaction of cyclopentadiene with butenone and acrylonitrile by simultaneous inclusion of the two guest molecules. Similarly, Tamaki46 found that 2-anthracenesulfonate forms a one host-two guests inclusion complex with gamma cyclodextrin, and that this complex-formation greatly enhances the photodimerization of the 2-anthracenesulfonate. The majority of reported studies of formation of cyclodextrin inclusion complexes in solution have been mainly concerned with determination of the stability constants by using equilibrium spectroscopic techniques, and the measurement of the enthalpy and entropy changes characterizing the complexation reaction. The aim of much of this work has been to determine the driving force of complex-formation. Despite the amount of research in this area, however, no general agreement has been reached, and
M. Suzuki and Y . Sasaki, Chem. Phurm. Bull., 32 (1984) 832-838. R. J. Clarke, J. H. Coates, and S. F. Lincoln, Curbohydr. Res., 127 (1984) 181-191. R. J . Clarke, J. H. Coates, and S. F. Lincoln, J . Chem. SOC.,Furuduy Trans. 1 , 80 (1984) 3119-3133.
R. J. Clarke, J. H. Coates, and S. F. Lincoln, J . Chem. SOC.,Furuduy Trans. I , 82 (1986) 2333-2343. R. L. Schiller, J. H. Coates, and S. F. Lincoln, J . Chem. SOC.,Furuduy Tmns. I , 80 (1984) 1257- 1266. R. L. Schiller, J. H. Coates, and S. F. Lincoln, J . Chem. Soc., Furuduy Trans. I , 82 (1986) 2123-2132. L.-E. Briggner, X.-R. Ni, F. Tempesti, and I. Wadso, Thermochim. Actu, 109 (1986) 139- 143. A. Ueno, K. Takahashi, Y . Hino, and T. Osa, J . Chem. SOC., Chem. Comrnun., (1981) 194-195. K. Uekama, F. Hirayama, T. Imai, M. Otagiri, and K. Harata, Chem. Phurm. Bull., 31 (1983) 3363-3365. K. Uekama, T. Imai, F. Hirayama, M. Otagiri, and K. Harata, Chem. Pharm. Bull., 32 (1984) 1662-1664. D. C. Rideout and R. Breslow, J . Am. Chem. SOC., 102 (1980) 7816-7817. T. Tamaki, Chem. Lett., (1984) 53-56.
CYCLODEXTRIN INCLUSION COMPLEXES
21 I
several alternative hypotheses have been proposed. A critical review of these hypotheses is presented in Section 111. In order to determine the mechanism of complex-formation, however, kinetic methods must be used. Consider one host-two guests complexation. The two possible mechanisms are dimerization of the guest outside the cyclodextrin cavity followed by inclusion, and dimerization within the cyclodextrin cavity. Equilibrium measurements alone cannot distinguish between these two possibilities. The same is the case for 2 : 2 complexformation, where a larger number of possible mechanisms exist. 11. HISTORICAL REVIEW
1. Discovery of the Cyclodextrins
The first report in the literature of the isolation of a substance recognizable as a cyclodextrin was that of Villiers4’ which appeared in 1891. From digests of Bacillus amylobacter on potato starch, Villiers obtained a small amount (3 g per 1000 g of starch) of a crystalline material, which he named “cellulosine” because of its resemblance in some respects to cellulose. The foundations of c yclodextrin chemistry were laid down, however, in the period 1903-1911 by Schardinger, and, in fact, some of the older literature frequently refers to the cyclodextrins as Schardinger dextrins. During the course of his work on food spoilage, Schardinger’s attention had been drawn to various strains of bacteria that survived the cooking process and that were considered to be responsible for some cases of food poisoning.48Schardinger found49that one of these heat-resistant or “thermophilic” bacteria, which he called strain 11, was able to dissolve starch and form crystalline polysaccharides (“dextrins”). He distinguished two of these crystalline polysaccharides, which he named crystalline dextrin A and crystalline dextrin B. The latter, he observed, was probably identical to the cellulosine of Villiers. The yield of dextrin B, however, was increased approximately ten-fold, which Schardinger explained by referring to Koch’s assertionSothat Villiers had used impure cultures. Schardinger intended to continue his work on these crystalline dextrins in the hope that they might shed some light on the processes of starch synthesis and degradation, or on its composition. However, when, after a long suspension, he again took up work in this direction, the strain used (47) (48) (49) (50)
A. Villiers, C. R . Acad. Sci., 112 (1891) 536-538. F. Schardinger, W e n . Klin. Wochenschr., 16 (1903) 468-474. F. Schardinger, Z . Unrers. Nuhr. Genussm., 6 (1903) 865-880. R. Koch, Jahresber. Giirungsorganism., 2 (1891) 242.
R. J. CLARKE, J. H. COATES, AND S . F. LINCOLN
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had almost completely lost its ability to cause decomposition of ~ t a r c h . ~ ’ Fortunately, in the meantime, Schardinger had isolated a new organisms2 that he had discovered as an accidental contaminant in a nutrient medium. Initially, he named the organism “Rottebazillus I,” because of its rotting action on potato starch, but later, in order to conform to Latin nomenclature, he gave it the currently accepted name of “Bacillus macerans.” Using this new organism, Schardinger found that he was able to prepare crystalline dextrins identical to those he had previously described. Bearing in mind the reactions of his A and B dextrins with iodine, Schardinger proposed the names “crystalline amylose’ ’ and “crystalline amylodextrin. Later, however, Schardinger considered that these names were inappr~priate,~~ and he thus decided upon the less specific names of “crystalline dextrin a” and ‘‘crystalline dextrin p.” At the conclusion of Schardinger’s final paper on the crystalline dexhe summarized his findings on their properties and their formation from starch. The following is a translation of that summary. ( 1 ) Starch paste is changed by specific microbes in such a way that water-soluble substances can be obtained whose behavior is in close chemicophysical relation to the well known dextrins. (2) The amylolytic process caused by one of these microbes (Bacillus macerans) is different under similar external conditions, depending on the type of starch. In the case of potato starch, there is complete solution; for arrowroot, it is almost complete; but, for rice and wheat starch, it is relatively slight. (The first two types of starch are obtained from root sections or rhizomes, the latter two from above-ground fruit. Perhaps the two types of starch have different interior constitutions.) ( 3 ) One part of the dextrins produced is crystalline, another part is amorphous and gumlike. The amount of crystalline rawproduct amounts to 25-30% of the starch used (determined in the case of marantaS4and potato starch). ( 4 ) Crystalline dextrins have hitherto been obtained from potato, maranta, rice, and wheat starch. Moreover, from all the types of starch mentioned, two different types of dextrin were obtained, which are denoted as crystalline dextrin a and p. The major product is the dextrin a.( 5 ) The crystalline dextrins are precipitable from aqueous solution by alcohol, as well as by ether, chloroform, and iodine solution. Copper salts (Fehling solution) are not reduced; yeast (top yeast or bottom yeasts5) produces no fermentation. ( 6 ) The iodine reaction ”
(51) (52) (53) (54)
F. Schardinger, Zentralbl. Bakreriol. Abt. 11, 22 (1909) 98-103. F. Schardinger, Wien. Klin. Wochenschr., 17 (1904) 207-209. F. Schardinger, Zentralbl. Bakreriol. Abr. 11, 29 (191 1) 188-197. Maranta is the scientific name for arrowroot. (55) Top yeast = baker’s yeast; bottom yeast = brewer’s yeast.
CYCLODEXTRIN INCLUSION COMPLEXES
213
offers the simplest means of discrimination between the two crystalline dextrins a and p. In a thin film, crystalline iodo-dextrin a is blue when damp, grey-green when dry; crystalline iodo-dextrin p is brownish (redbrownish) whether damp or dry. Although Schardinger did not propose a structure for his crystalline dextrins, he made several observation^^^ that can now be attributed to their cyclic structure. For example, he discovered their ability to engage in complex-formation: “With various substances, the crystalline dextrins form loose complexes which, like those produced with alcohol, ether, and chloroform, are indeed partly decomposed by water, while the iodine complexes are more stable toward water.” He also found, as previously mentioned, that the crystalline dextrins were nonreducing toward copper salts and nonfermentable by yeast. This last observation he considered was “... the most essential thing that I was able to mention concerning the formation of crystalline dextrins by microbes.” Both of these observations can be explained by the lack of a chain termination. Schardinger brought his work on the crystalline dextrins to a close in 1911. He ended his work with the statement: “I realise that still very many questions remain unsolved. The answer to these I must leave to another, who, owing to more favorable external conditions, can deal with the subject more intensively.” It was to be another twenty-five years before the cyclic nature of Schardinger’s dextrins was recognized.
2. Determination of the Structure of the Cyclodextrins The next major contribution to cyclodextrin chemistry was that of Freudenberg and Jacobi, who developed a method of obtaining pure alpha and beta dextrin~,*~ and in the process also isolated another crystalline dextrin, which they named gamma dextrin. The period between the publication of Schardinger’s last paper and Freudenberg’s work had been relatively fruitless, mainly due to the difficulty in separating the crystalline dextrins and obtaining pure samples. Once this hurdle has been overcome, research into Schardinger’s dextrins accelerated. Credit for the determination of the structure of Schardinger dextrins must also go to Freudenberg and coworkers, who first tentatively proposed5’ a ring structure in 1936. In the next few years, Freudenberg tested his hypothesis, and came to the conclusion58that Schardinger dextrins are cyclic oligosaccharides composed solely of D-glucosyl residues bonded by a-(1 + 4)-glycosidic linkages. In support of this assignment, (56) K. Freudenberg and R. Jacobi, Ann., 518 (1935) 102-108. (57) K. Freudenberg, G. Blomquist, L. Ewald, and K. Soff, Ber., 69 (1936) 1258-1266. (58) K. Freudenberg and M. Meyer-Delius, Ber., 71 (1938) 1596-1600.
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R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
Freudenberg cited the following pieces of experimental evidence: (I) the rate of hydrolysis of Schardinger dextrins in 51% sulfuric acid is too low for there to be any labile p linkages present; (2) enzymic hydrolysis gave no trace of a sugar other than D-glucose; (3) the Schardinger dextrins are n o n r e d ~ c i n gthat , ~ ~is, they do not have a reducing chain-termination; and ( 4 ) methylation studies@ on the Schardinger dextrins gave no products other than 2,3,6-tri-O-methyl-~-glucose. Although Freudenberg had determined the correct chemical structure for the Schardinger dextrins, the number of D-glucosyl residues that he gaves6for the alpha and beta dextrin rings (five and six, respectively) were incorrect. These values he determined from molecular weights obtained by the cryoscopic method, which is known to be sensitive to low-molecular-weight impurities. The correct values of six and seven D-glUCOSy1 residues per molecule were determined by French,61who obtained the molecular weight by X-ray diffraction combined with crystal density measurement. On the basis of these values, French proposed the names “cyclohexaamylose” and “cycloheptaamylose” for Schardinger’s alpha and beta dextrin, respectively. Freudenberg later concurred with French’s results, after studying the X-ray measurements of BorcherP2and also his and Cramer’s optical rotation data.63He also proposed that the gamma dextrin consisted of eight Dglucosyl residues joined by a linkages in a cyclic structure, as in the case of the alpha and beta dextrins. Finally, French and McIntirea studied the periodate oxidation6s of alpha, beta, and gamma dextrin, found that neither formic acid nor formaldehyde was produced, and concluded that all three have a cyclic structure, as earlier proposed by Freudenberg. Also supportedM was the gamma dextrin structure proposed by Freudenberg, on the basis of acid (59) Reducing sugars are normally identified by their ability to reduce metallic salts, for example, Fehling solution. In order to do this, the sugar must have a carbonyl function in the form of a hemiacetal. (60) The methylation technique consists of total methylation of the oligosaccharide, followed by hydrolysis to the partially methylated monosaccharide units, and determination of the unmethylated positions in each unit, these being the positions of linkage between units. (61) D. French and R. E. Rundle, J. Am. Chem. Soc., 64 (1942) 1651-1653. (62) W. Borchert, Z. Nuturforsch., Teil B, 3 (1948) 464-465. (63) K. Freudenberg and F. Cramer, Z. Nuturforsch., Teil B , 3 (1948) 464. (64) D. French and R. L. McIntire, J. Am. Chem. Soc., 72 (1950) 5148-5150. (65) On periodate oxidation, open-chain (1-+4)-linked polysaccharides yield one molecule of formic acid from the nonreducing end and two molecules of formic acid, as well as one molecule of formaldehyde, from the reducing end. (66) D. French, D. W. Knapp, and J. H. Pazur, J. Am. Chem. Soc., 72 (1950) 5150-5152.
CYCLODEXTRIN INCLUSION COMPLEXES
215
hydrolysis experiments and crystallographic data. In keeping with his previously proposed names, French suggested the name “cyclooctaamylose” for the gamma dextrin. The name “cyclodextrin,” which is now most commonly used for the homologous series, seems to have been originated by C ~ - a m e r who , ~ ~ used this name in the title of his Ph.D. dissertation.
3. Formation of the Cyclodextrins from Starch Since the time of Schardinger, one of the most important reasons for studying the cyclodextrins was for the information they might yield on the structure of starch and of the well known blue iodine-starch complex. In fact, the similarity between the iodine-starch reaction and the iodinealpha cyclodextrin reaction was first noted by Schardingers3in 1911, in his final paper on the cyclodextrins. Prior to 1939, however, it was not known whether the cyclodextrins were products of the synthetic metabolism of Bacillus macerans, and therefore, perhaps, quite different from the components of starch, or whether they were formed by a single enzyme and therefore closely related to the starch structure. Then, Tilden and Hudson68announced the discovery of a cell-free enzyme preparation from cultures of Bacillus macerans which had the ability to convert starch into the Schardinger dextrins without the production of maltose, glucose, or any other reducing sugars. They thus concluded that the Schardinger dextrins were either the true components of starch or closely related to such true components. The first to propose a mechanism for the formation of the cyclodextrins was again Freudenberg, who, with Meyer-Delius, suggested that the cyclodextrins are preformed in starch,5sand that they are formed by cleavage of side branches by Bacillus mace ran^^^ (see Fig. 2). Freudenberg and coworkers, however, soon abandoned his proposed starch structure,70 because it did not agree with the usual conceptions regarding the linkage of the D-glucose units; for example, it required certain D-glucose units to be linked to three others. In its place, Freudenberg adopted the screw model of starch first proposed by H a n e ~ , which ~’ represents starch as alinked D-glucose units in a helical arrangement. On the basis of this model, Freudenberg interpreted the formation of the cyclodextrins by F. Cramer, Die Cyclodextrine aus Starke, Dissertation, Heidelberg, 1949. E. B. Tilden and C. S. Hudson, J . Am. Chem. Soc., 61 (1939) 2900-2902. K. Freudenberg, Annu. Reu. Biochem., 8 (1939) 81-112. K. Freudenberg, E. Schaaf, G. Dumpert, and T. Ploetz, Natunvissenschaften, 27 (1939) 850-853. (71) C. S. Hanes, New Phyrol., 36 (1937) 189-239.
(67) (68) (69) (70)
216
R. J. CLARKE, J. H. COATES, AND S . F. LINCOLN
Hypothetical structure of starch
I
Bacillus macerans
II Linear ond cyclic
jo
breakdown products
\
FIG.2.-Freudenberg's Initial Model of Formation of Cyclodextrin.
-
Bacillus macerans L
\ / Helical structure
Starch and
of starch
cyclodextrin
FIG.3.-Freudenberg's Final Model of Formation of Cyclodextrin.
CYCLODEXTRIN INCLUSION COMPLEXES
217
Bacillus macerans amylase as a transglucosylation; that is, he suggested that a winding of the helix is cleaved by the enzyme (see Fig. 3) and, because of the helical arrangement, the first and fifth or sixth D-glucosyl residues are situated close to one another and are able to unite to form rings of five or six D-glucose units. For example, if Glc = a D-glucose or a D-glucosyl residue, the reaction may be written according to the following equations. Glc,
GIc,-~ + alpha cyclodextrin
Glc,
Glc,-,
Glc,
Glc,)-* + gamma cyclodextrin
+ beta cyclodextrin
Thus, Freudenberg concluded that the cyclodextrins are not preformed in starch, but that their formation is made possible by the helicity of the starch chain. Freudenberg’s hypotheses concerning the starch structure (that is, the amylose fraction) and the Bacillus macerans amylase mechanism have been confirmed by X-ray crystallography7* and chromatographic technique^.^^ 4. Inclusion Complexes of the Cyclodextrins
Probably the most important property of the cyclodextrins is their ability to form complexes with a variety of organic and inorganic compounds. This property was discovered by S ~ h a r d i n g e rwho , ~ ~ used the complexes with alcohol, chloroform, and ether as a means of precipitation of his crystalline dextrins, and the complexes with iodine for the discrimination of his alpha and beta dextrins. Freudenberg and coworkers70first interpreted these complexes as being formed by molecular inclusion into the cyclodextrin cavity. He also suggested that hydrophobic forces might be responsible for the binding of molecules within the cavity. The cyclodextrins are not the only molecules capable of forming incluIn contrast to the inclusion complexes (“Einschlus~verbindungen”~~). sion complexes of many other “host” molecules, however, those of the cyclodextrins are able to form in solution as well as in the solid state, a fact which was first recognized by Cra~ner,~~-’* after he had observed that (72) K.Takeo and T. Kuge, Agric. Biol. Chem., 33 (1969) 1174-1 180. (73) D. French, M. L. Levine, E. Norberg, P. Nordin, J. H. Pazur, and G. M. Wild, J . A m . Chem. SOC.,76 (1954) 2387-2390. (74) The term “Einschlussverbindungen” was coined75by W. Schlenk, Jr., in 1951. (75) W. Schlenk, Jr., Forrschr. Chem. Forsch., 2 (1951) 92-145. (76) F. Cramer, Angew. Chem., 64 (1952) 437-447. (77) F. Cramer, Nuturwissenschuften, 38 (1951) 188. (78) F. Cramer, Chem. Eer., 84 (1951) 851-854.
218
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
a number of dyes showed characteristic changes in their absorption spectra in aqueous solutions of the cyclodextrins. Although Freudenberg’s hypothesis that complex-formation occurred by inclusion within the cavity was generally accepted, there was no direct evidence for this, either in solution or in the solid state. Broser and L a u t ~ c h ’had ~ found by spectrophotometric titration that the complexes of a series of dyes with the cyclodextrins in solution obeyed the mass action law with a stoichiometry of 1 : 1. They suggested that association on the outside of the ring might not have a defined stoichiometric composition, and they thus interpreted their results as being consistent with inclusion by cyclodextrin. Their results were not conclusive, however. The first direct evidence for molecular inclusion came from X-ray crystallography. Hybl and coworkerss0 determined the structure of the alpha cyclodextrin-potassium acetate complex by using three-dimensional Xray diffraction data. They found that, in the solid state, the acetate anions are included by the alpha cyclodextrin. In the process, they also found that every D-glucosyl residue of the alpha cyclodextrin is in the 4C1conformation. ‘H-Nuclear magnetic resonance (n.m.r.) spectroscopy provided the first direct evidence of inclusion within the cyclodextrin cavity in solution. Using aromatic “guest” molecules, Demarco and ThakkaP found that, on addition of the guest, the resonances of the hydrogen atoms of the cyclodextrin situated on the inside of the cavity were shifted significantly upfield due to shielding by the aromatic guest. They noted little effect on the resonances of the hydrogen atoms on the exterior of the cyclodextrin annulus. Yamamoto and coworkerssla used two-dimensional, nuclear Overhauser effect experiments (NOESY) to determine the proximity of partic-. ular protons situated on an included p-nitrophenolate ion to particular protons of a host alpha cyclodextrin molecule. The experiments showed cross-peaks connecting the H-3 resonance of alpha cyclodextrin to both meta and ortho proton resonances of the p-nitrophenolate ion, whereas H-5 of the alpha cyclodextrin gave a cross-peak only with the resonance of the meta proton thereof. As a consequence, it was unequivocally confirmed that the p-nitrophenolate ion is, in solution, preferentially included with its nitro group oriented to the narrow end of the alpha cyclodextrin (79) W. Broser and W. Lautsch, Z . Narurforsch., Teil B , 8 (1953) 71 1-722. (80) A. Hybl, R. E. Rundle, and D. E. Williams, J . Am. Chem. Soc., 87 (1965) 2779-2788. (81) P. V. Demarco and A. L. Thakkar, Chem. Commun., (1970) 2-4. (81a) Y. Yamamoto, M. Onda, M. Kitagawa, Y. Inoue, and R . Chcijje, Carbohydr. Res., 167 (1987) c l l - c I 6 .
CYCLODEXTRIN INCLUSION COMPLEXES
219
annulus. Cross-polarization, magic-angle-spinning (c.p .-ma.s .), 13Cn.m.r. spectroscopy was used in studying the structure8Iband molecular dynamics8'" of the cyclodextrins and their inclusion complexes, and it was found81cthat, even in the solid state, the guest molecule undergoes some degree of motion within the cavity of the cyclodextrin molecule. 111. FORMATION OF INCLUSIONCOMPLEXES
1. Detection of Complex-Formation Upon inclusion within the cyclodextrin cavity, a guest molecule experiences changes in its physicochemical properties, as well as changes in its chemical reactivity, due to changes in its environment on its removal from the bulk solution. These changes in behavior have great practical significance; for example, for the stabilization of reactive substances, reduction in volatility, and increase in solubility. In research, however, the changes in physicochemical properties of the guest provide an easy method of detecting inclusion-complex formation. A variety of techniques can be used for studying complex-formation, a number of which are now listed. a. U.v.-Visible Absorption.-Many aromatic organic molecules show changes in their u.v.-visible absorption spectrum on inclusion by cyclod e ~ t r i n . ' ~Generally, .'~ the spectral changes observed are similar to the effects caused by changes in solvent. These changes must be due to a perturbation of the electronic energy levels of the guest, caused either by direct interaction with the cyclodextrin, or by the exclusion of solvating water molecules, or by a combination of these two effects. b. Fluorescence.-When fluorescent molecules in aqueous solution are included by cyclodextrin, fluorescence enhancement usually occurs. This enhancement has been attributed to the reduction in collisional quenching (by water and oxygen) of the fluorescence of the guest upon its fixation within the cyclodextrin cavity.82 c. Circular Dichroism.-The cyclodextrins are chiral molecules as they are composed entirely of D-glucosyl units in a centrosymmetric arrangement. Thus, a chiral guest-molecule exhibits changes in its circular (81b) R. P. Veregin, C. A . Fyfe, R. H. Marchessault, and M. G . Taylor, Carbohydr. Res., 160 (1987) 41-56. (81c) Y. Inoue, F.-H. Kuan, and R. Chfijje, Carbohydr. Res., 159 (1987) cl-cl0. (82) A. Nakajima, Spectrochim. Acta, Part A , 39 (1983) 913-915.
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R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
dichroic spectrum on inclusion due to the interaction of the transition dipoles of the guest with those of the D-glucosyl units of the cyclodext r i n ~Achiral . ~ ~ guest molecules may also often show an induced circular dichroic spectrum upon inclusion, due to this interaction. d. Nuclear Magnetic Resonance.-Upon inclusion of a guest molecule, changes in the chemical shifts of constituent nuclei in both the cyclodextrinE1and the guest molecules4are frequently observed. These changes are due to a change in the environment of the atom concerned; for example, because of anisotropic shielding, interactions between the guest and the cyclodextrin, conformational changes in the cyclodextrin, or expulsion of water from the cavity. e. Acid-Base Titration.-Acidic or basic guest molecules often show apparent changes in their acidity or basicity on inclusion; that is, changes in pK, are observed.85 Depending on the guest molecule used, these changes can be monitored spectrophotometrically or potentiometrically.
f, Solubility.-Substances that are only sparingly soluble in water often show an increase in solubility on complexation with cyclodextrin.86In contrast, the inclusion complex is generally less soluble than the free cycl~dextrin.~~ Although the techniques just considered have been widely used for the development of practical uses of the cyclodextrins, the underlying forces responsible for the stability of the inclusion complexes, as well as the mechanism of their formation, are at present not completely understood. 2. Thermodynamics of Complex-Formation
The standard free-energy decrease associated with formation of cyclodextrin inclusion-complexes is generally due to a negative standard-enthalpy change (AH') accompanying the inclusion process. The standardentropy change (AS") can be either positive or negative, although the majority of guest molecules that have been studied appear to have negative AS' values. (83) M. Kajtar, C. Horvath-Toro, E. Kuthi, and J . Szejtli, Acta Chim. Acad. Sci. Hung., 110 (1982) 327-355. (84) D. D. MacNicol, Tetrahedron Lett., (1975) 3325-3326. (85) K. A. Connors and J. M. Lipari, J . Pharm. Sci., 65 (1976) 379-383. (86) H. Schlenk and D. M. Sand, J . A m . Chem. SOC., 83 (1961) 2312-2320. (87) D. French, M. L. Levine, J. H. Pazur, andE. Norberg, J . A m . Chem. Soc., 71 (1949) 353-356.
CYCLODEXTRIN INCLUSION COMPLEXES
22 I
Values of AHo and AS” for complex-formation between alpha and beta cyclodextrin and a variety of guest molecules are shown in Tables I1 and 111. If AHo is plotted against AS”, a linear relationship is observed, in which AH” and ASo are compensating (see Figs. 4 and 5). The slope of the graph is called the “compensation temperature” or “isoequilibrium TABLEI1 Standard Formation Enthalpies and Entropies of Cyclornaltohexaose Inclusion Complexes (1 :1) Substrate Formic acid Acetic acid Propanoic acid Cyclohexanecarboxylic acid Trimethylacetic acid Adamantenecarboxylic acid Adamantenecarboxylate ion Benzoic acid Benzoate ion 4-Hydroxybenzoic acid 4-Hydroxybenzoate ion 4-Nitrophenol
4-Nitrophenolate ion
4-C yanophenol 4-Cyanophenolate ion 4-Nitrobenzoic acid 3-Hydroxybenzoic acid 3-Nitrophenol
3-Nitrophenolate ion 3-C yanophenol
3-Cyanophenolate ion 3-Nitrobenzoic acid Perchlorate ion Iodide ion Thiocyanate ion Hydroxide ion Acetonitrile
Number in Fig. 4
AH” (kJ.mol-l)
AS” (JK-l.mol-l)
1 2 3 4 5 6 7 8 9
-3.10 -11.21 -5.02 -21.51 -39.50 - 19.7 -23.4 -14.2 -5.0 -42.47 -40.17 - 17.3 -48.53 -25.52 -30.5 -30.5 -30.12 - 18 -46.9 -30 -37.8 -25.5 -28.9 -33.89 -48.12 -37.7 -33.05 -38.5 -25.5 -36.4 -35.1 -26.36 -24.69 -28.5 -20.9 - 10.0
1.3 -18.8 54 -41.84 - 100.00 -43.1 -37.7 -5.4 41.8 -87.4 -75 -35.1 - 105.02 -64.9 -58.6 -63 -56.1 -5.4 -92.0 -37 -62.63 -43.5 -44.4 -69.0 - 110.5 -86.6 -69.9 -83.7 -48.1 -71.1 -77 -56.5 -57.3 -66.5 -37.7 - 19.7
10
11 12 13 14 15 16 17 18 19
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
References 88 88 89 90 90 90 91 91 92 88 89 88 90 90 93 89 94 16 93 16 95
90 90 90. 90 90 94 90 90 90 90 96 96 96 90 97 (continued)
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
222
TABLEI1 (continued) Substrate
Ethanol 2-Propanol 2-Methyl-2-propanol Cyclohexanol 1,CDioxane Phenol
Number in Fig. 4 37 38 39 40 41 42 43
44 Anilinium perchlorate 4-Aminobenzoic acid 3-Methylbenzoic acid Sodium perchlorate Diisopropyl phosphorofluoridate Perchloric acid Hydrocinnamic acid L-Mandelic acid Pyridine L-Tryptophan L-Phenylalanine L-Tyrosine 2-nitro phenol
2-Aminobenzoic acid I -Butanol I-Pentanol I-Hexanol 2,2-Dimethyl-l-propanol
45 46 47 48 49 50 51
52 53 54 55 56 57 58
59 60 61 62
AH" (kJ.mol-') -2.9 -1.7 2.09 -12.6 - 14 -9.62 -12.6 -7.53 -51.5 -48.5 -48.5 -40.6 -3 I -31.4 -31.38 -21 - 10.5 -7.53 -4.6 -4.2 -2.09 - 1.26 -12 - I6 - 19 -12
ASo (JK-l.mol-l) 2.5 7.9 19.2 -7.5 - I4 - 19.2 -15.9 54 - 146 - 109 -117 -96 -88 -63 -46 -25 8 4 63 42 63 88 -2 -5
-8 - 12
References 97 97 97 97 98 97 97 89 89 89 89 89 99 89 89 89 89 89 89 89 89 89 98 98 98 98
(88) R. I. Gelb, L. M. Schwartz, R. F. Johnson, and D. A. Laufer, J . Am. Chem. Soc., 101 (1979) 1869-1874. (89) E. A. Lewis and L. D. Hansen, J. Chem. Soc., Perkin Trans. 2 , (1973) 2081-2085. (90) R. I. Gelb, L. M. Schwartz, B. Cardelino, H. S. Fuhrmann, R. F. Johnson, andD. A. Laufer, J. A m . Chem. Soc., 103 (1981) 1750-1757. (91) R. I. Gelb, L. M. Schwartz, and D. A. Laufer, Bioorg. Chem., 9 (1980) 450-461. (92) M. Komiyama and M. L. Bender, J . A m . Chem. Soc., 100 (1978) 2259-2260. (93) R. I. Gelb, L. M. Schwartz, B. Cardelino, and D. A. Laufer, Anal. Biochem., 103 (1980) 362-368. (94) K. Harata, Bull. Chem. Soc. Jpn., 51 (1978) 2737-2738. (95) K. Takeo and T. Kuge, Staerke, 24 (1972) 331-336. (96) R. I. Gelb, L. M. Schwartz, M. Radeos, and D. A. Laufer, J . Phys. Chem., 87 (1983) 3349-3354. (97) R. I. Gelb, L. M. Schwartz, M. Radeos, R. B. Edmonds, and D. A. Laufer, J. Am. Chem. Soc., 104 (1982) 6283-6288. (98) Y. Matsui and K . Mochida, Bull. Chem. SOC. Jpn., 52 (1979) 2808-2814. (99) C. van Hooidonk and J. C. A. E. Breebaart-Hansen, Recl. Trau. Chim. Pays-Bas, 90 (1971) 680-686.
TABLEI11 Standard Formation Enthalpies and Entropies of Cyclomaltoheptaose Inclusion Complexes (1 :1) Substrate 4-Nitrophenol Phenobarbital" MephrobarbitaP 2-Aminobenzoic acid Thiophenobarbi talc 2-Hydroxybenzoic acid Benzoic acid 5-Heptylbarbituric acid 5-Hexyl-2-thiobarbituric acidd 4-Methylbenzoylacetic acid Amobarbital' 5-Hexylbarbituric acid Thiopentalf Benzoylacetic acid 5-Pentyl-2-thiobarbituricacid Hexobarbitala 3-Chlorobenzoylacetic acid 5-Pentylbarbituric acid 5-Butyl-2-thiobarbituric acid Pentobarbital* C yclobarbital' 4-Hydroxybenzoic acid 4-Aminobenzoic acid 3-Ethylphenyl acetate 5-Propyl-2-thiobarbituricacid 5-Butylbarbituric acid 5-Ethylbarbituric acid 4-Nitrophenolate ion 5-Methylbarbituric acid Phenol 3,4,5-TrimethylphenyI acetate 3-Chlorophenyl acetate I-Butanol 1-Pentanol I-Hexanol 2,2-Dirnethyl-l-propanol C yclohexanol
Number in Fig. 5
AH" (klmol-')
AS" (JK-l.mol-*)
I 2 3 4
-44 -43 -39.7 -35.6 -34.4 -33.5 -32 -32.0 -29.6 - 28 -26.4 -25.9 -25.7 -24 -23.6 -23.5 - 22 -21.0 -20.4 -20.3 -20.2 -20.0 - 19.9 - 19 - 16.4 -15.8
-88 -83 -72 -68.3 -47 -72 -67 -40.8 -34.2 -41 -26.1 -22.8 -21.0 -36 -22.3 - 19.7 - 25 - 12.3 -13.2 -10.2 -15.9 -21.1 - 10.1 - I3 -7.4 -3.2 -0.92 5.06 4.5 29 8 33 33 50 46 21 17
5
6 7 8 9 10
11 12 13 14 15
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
- 15.5 - 14.2
-11.6 -11
- 10 -4 2.9 4.6 0.4 -8.8 - 10.0
References 89 100 100 83 100 83 89 100 100 101 100 100 100 101 100 100
101 100 100 100 100
83 83 102 100 100 100 95 100 89 102 102 98 98 98 98 98
" 5-Ethyl-S-phenyl-2,4,6(IH,3H,5H)-pyrimidinetrione; synonym: 5-ethyl-5-phenylbarbituric acid. 5-Ethyl- I -methyl-5-phenylbarbituric acid. S-Ethyl-5-phenyldihydro-2-thioxo-4,6( lH,5H)-pyrimidinedione; synonym: 5-ethyl-5-phenyl-2-thiobarbituricacid. 5-Ethyl-5-hexyl-2-thiobarbituricacid. 5Ethyl-5-isopentylbarbituric acid. f 5-Ethyl-5-(l-methylbutyl)-2-thiobarbituric acid. 8 5 4 I -Cyclehexen-l-yl)-l,5-dimethylbarbituricacid. 5-Ethyl-S-( I-rnethylbuty1)barbituric acid. ' 5 4 1-Cyclohexen-lyl)-5-ethylbarbituric acid. (100) M. Otagiri, T. Miyaji, K. Uekama, and K. Ikeda, Chem. Pharm. Bull., 24 (1976)
1146-1 154. (101) T. S. Straub and M. L. Bender, J . Am. Chem. Soc., 94 (1972) 8881-8888.
(102) R. L. van Etten, J . F. Sebastian, G. A. Clowes, and M. L. Bender, J. Am. Chem. SOC., 89 (1967) 3242-3253.
224
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
/25
019
-50 54(470F13 I
I
I
I
ASo (J K? macll
FIG.4.-Enthalpy-Entropy Compensation for Alpha Cyclodextrin Inclusion-Complexes. [The guest identification numbers are as given in Table 11.1
10
1
1
I
I
I
I
0
--
- 10
0
8-
Ei -20
7
r 0
-30 -40
-
I
0
0
I 100
ASo (J K;' rnoi')
FIG.5.-Enthalpy-Entropy Compensation for Beta Cyclodextrin Inclusion-Complexes. [The guest identification numbers are as given in Table 111.1
CYCLODEXTRIN INCLUSION COMPLEXES
225
temperature,” and is denoted by T,. The values of T, thus determined for the alpha and beta cyclodextrin complexes are 273 (*14) and 330 (k 13) K , respectively. This correlation implies a common interaction mechanism between the alpha and beta cyclodextrins and the various substrates. Because the substrates have widely differing structures, this mechanism cannot be associated with the structure of the substrate, but rather with common attributes of these systems, that is, the solvent and the host. Compensation effects of this type have frequently been observed in reactions in aqueous solution,’03and have been attributed to changes in the solvation of the participating molecules during the reaction. The values of T, determined are, within experimental error, consistent with the 250-320-K range characteristic of processes dominated by solvation phenomena. lo3 Thus, it seems most likely that changes in the solvation of both the guest molecule and the cyclodextrin play an important role in determining the stability of the resultant inclusion complex. An alternative explanation for the observed enthalpy-entropy compensation has been proposed by Gelb and coworkers ,% who suggested that the cyclodextrin structure is responsible for the compensation. According to Gelb, polar interactions between alpha cyclodextrin and the substrate provide the driving force for the complexation reaction. These interactions then result in torsional constraint of the alpha cyclodextrin structure, reflected in the (usually) substantially negative entropy change. Having found% a T, value of 410 (+15) K, that is, outside the normal range for solvation phenomena, Gelb and coworkers proposed that the complexation process involves no changes in the solvation of the guest molecule or the cyclodextrin. Although Gelb’s suggestions of polar interactions between the guest and the cyclodextrin, and of torsional constraint of the cyclodextrin structure, appear reasonable, his hypothesis that neither the host nor the guest undergoes solvation changes on complexation seems untenable. It can be seen from published crystal structures of inclusion complexes,1o4and also from molecular-model building, that, in the case of the most stable complexes, the fit between substrate and cyclodextrin is very close, providing very little space for any solvating water molecules. Thus, although it may not be the case that all solvating water molecules are removed from the guest and excluded from the cyclodextrin cavity prior to complex-formation, it seems that significant changes in solvation must occur in most cases. This is further supported by the results of Mochida and (103) R. Lumry and S. Rajender, Biopolymers, 9 (1970) 1125-1227. (104) W . Saenger, Proc. In?. Symp. Cyclodextrins, Is?, Budapest, (1981) 141-150.
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R. J. CLARKE, J . H . COATES, AND S. F. LINCOLN
coworker^,^^^ who investigated the effect of inorganic salts on the dissociation of an azo dye-beta cyclodextrin inclusion-complex. They found that increases in the concentration of such salts as phosphate, sulfate, iodate, and fluoride caused a decrease in the apparent dissociation constant of the complex; that is, an increase in the apparent association constant. Mochida attributed this effect to a change in the activity of the water, thus implying its participation in the inclusion reaction. Consider the reaction of a hydrated guest molecule, A(H20), , with a hydrated cyclodextrin, B(H20), . The association can be expressed as follows. A(H,O),
+ B(H2O)y F===
A.B(H,O),+y-, + z H2O
The association constant, K, for this equilibrium is given by where Kappis the apparent association constant, and aH20is the activity of water. Thus, the addition of inorganic salts, provided that they themselves are not included by cyclodextrin, results in a decrease in the activity of water due to hydration of the salts, causing an increase in Kapp. Mochida and coworkersio5found that the cation used also affected the association, the value of K,, increasing with the extent of hydration of the cation; that is, Kapp(K+) < Kapp(Na+) < Kapp(Lit). Now, if it is assumed that water molecules are excluded from the cyclodextrin cavity upon inclusion of a guest, then, in order for a complex to form, solvation of the cyclodextrin-guest complex must be energetically more favorable than solvation of the individual molecules. There are two basic reasons which could account for this, namely, ( I ) an interaction between the guest and the cyclodextrin drives the inclusion process, and (2) the large internal cohesion of water (due to hydrogen bonding) forces the cyclodextrin and the guest together, that is, there is a hydrophobic interaction. Although complex-formation has been observed in nonaqueous solvents, for example, dimethyl s u l f ~ x i d e ,it~ has ~ ~ 'been ~ ~ found that the binding is very weak compared to that in water, thus suggesting the importance of hydrophobic interactions. In a study of the formation of an inclusion complex between beta cyclodextrin and indole,'06" the stability constant in aqueous solution was found, on the addition of formamide, to decrease, accompanied by a (105) K. Mochida, A. Kagita, Y. Matsui, and Y. Date, Bull. Chem. SOC.Jpn., 46 (1973) 3703-3707. (106) W. V. Gerasimowicz and J . F. Wojcik, Eioorg. Chem., I 1 (1982) 420-427. (106a) A. Orstan and J. B. A. Ross, J . Phys. Chem., 91 (1987) 2739-2745.
CYCLODEXTRIN INCLUSION COMPLEXES
227
decrease in the surface tension of water, and to increase on the addition of CaC12 (up to a concentration of 33%), which leads to an increase in the surface tension. These results were interpreted in terms of the decrease in surface area exposed to the solvent on formation of the inclusion complex. Further evidence for hydrophobic interactions comes from the work of Connors and Pendergast,Io7 who observed an inverse relationship between the stability of a complex and the aqueous solubility of the guest. They also observed a direct relationship between the stability of the complex and the partition coefficient of the guest between octyl alcohol and water, which has been suggested as a measure of hydrophobicity.I0*It has also been found that guest molecules having apolar groups favor the inclusion process,1wthus giving further weight to the assignment of complexstability to hydrophobic interactions. Classical, hydrophobic interactions, however, are characterized by a positive entropy of association,II0 in contrast to the cyclodextrin inclusion-complexes, for which the driving force of inclusion is derived primarily from a favorable enthalpy change. Thus, it was suggested by Griffiths and Bender2 that the driving force of inclusion is an example of an “atypical” hydrophobic interaction. The explanation that Griffiths and Bender2 offered to explain the favorable enthalpy change is that the “empty” cyclodextrin contains water molecules that are unable to form their full complement of hydrogen bonds to adjacent water molecules, and thus may be considered to be “enthalpy rich.” The inclusion of a guest molecule would then displace this “high energy” water from the cyclodextrin cavity, leading to a net increase in solvent-solvent hydrogen bonds and a favorable enthalpy of association. It is worth noting, however, that, as the size of the cyclodextrin cavity increases, the energy released per water molecule displaced from the cavity would be expected to decrease, because the energy content of the included water would slowly approach that of the bulk water. The structure of the included water of the crystalline cyclodextrin hydrates has been investigated by Saenger and coworkers’’*-‘I3 using X-ray (107) K. A. Connors and D. D. Pendergast, J . Am. Chem. Soc., 106 (1984) 7607-7614. (108) A. Leo, C. Hansch, and D. Elkins, Chem. Reu., 71 (1971) 525-616. (109) C. van Hooidonk and J . C. A. E. Breebaart-Hansen, R e d . Truu. Chim. Pays-Bus, 91 (1972) 958-964. (110) W. Kauzmann, Adu. Protein Chem., 14 (1959) 1-63. (111) C. Betzel, W. Saenger, B. E. Hingerty, and G. M. Brown, J . A m . Chem. Soc., 106 (1984) 7545-7557. (112) W. Saenger, C. Betzel, B. E . Hingerty, and G . M. Brown, Nurure (London), 296 (1982) 581-583. (113) V. Zabel, W. Saenger, and S. A. Mason, J . Am. Chem. SOC., 108 (1986) 3664-3673.
228
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
and neutron diffraction techniques. Their results showed that the water molecules within the cavity are, in fact, disordered. For example, in the case of the beta cyclodextrin undecahydrate it was found that the 6.13 water molecules situated in the cavity are distributed over eight positions. I I I Saenger and coworkers also found an unusual arrangement of hydrogen bonding within the cyclodextrin cavity. As well as conventional hydrogen bonds, they observed bonds of the type 0-(8H)-(hH)-O. These bonds can be considered to be an average over the two states 0-H-0 0-H-0. In the beta cyclodextrin structure, several of these O-(iH)-(hH)-O hydrogen bonds were found to be interconnected into larger systems. If, in such a system, a hydrogen atom changes position from one state to another, then all of the hydrogen atoms in associated 0-(1H)-(iH)-O bonds must move in a concerted fashion. Thus, Saenger and coworkers introducedII2 the term “flip-flop hydrogen bond” to describe this type of interaction. It was found113that, on decreasing the temperature, an ordering of the water occurs through the transformation of flip-flop hydrogen bonds into those of the conventional type. Thus, Saenger and coworkers113suggested that the water is in a state of dynamic disorder. An additional contribution to the enthalpy of association was proposed by Saenger and coworkers.’14 From an X-ray diffraction study of the crystalline alpha cyclodextrin hexahydrate, they found that one of the Dglucosyl units is rotated into an almost orthogonal position relative to the other five, in order to form hydrogen bonds to the included water, and also that the ring of interglucosidic hydrogen bonds around the rim of the alpha cyclodextrin annulus is d i s r ~ p t e d . ”This ~ unusual arrangement of the D-glucosyl units seems only to occur in the alpha cyclodextrin hexahydrate structure. When alpha cyclodextrin is complexed with other guest molecules, the crystal structures determined show a complete ring of hydrogen bonds, and no rotation of any D-glucosyl units. Thus, it was suggested by Saenger that, in water, the alpha cyclodextrin molecule is in a “strained,” high-energy conformation, and that, when another guest molecule displaces the water, thus forming an inclusion complex, a conformational change of the alpha cyclodextrin molecule occurs, transforming the alpha cyclodextrin structures into an unstrained, “relaxed” state. According to Saenger, then, inclusion-complex formation is accompanied, in the case of alpha cyclodextrin, by relief of strain in the cyclodextrin ring, thus contributing to the enthalpy of association and to the stabil(114) W. Saenger, M. Noltemeyer, P. C. Manor, B. E. Hingerty, and B. Klar, Eioorg. Chem., 5 (1976) 187-195. (1 IS) P. C. Manor and W. Saenger, J . A m . Chem. Soc., 96 (1974) 3630-3639.
CYCLODEXTRIN INCLUSION COMPLEXES
CXC .2H,O
aC.2H20
(tense)
(relaxed) H.0 activated
229
7
OCC. 2H,O .A (tense) substrate on outside
OCC .A (relaxed)
FIG.6.-Schematic Diagram1I4of Saenger’s Theory of Formation of Alpha Cyclodextrin Inclusion-Complexes. [A = guest molecule, HzO* = “activated water,” -0 = 6-hydroxyl group, -0 = 2- and 3-hydroxyl groups. Hydrogen bonds are marked by dashed lines. Saenger defined activated water as incompletely coordinated water molecules which are situated in a “round,” unstrained alpha cyclodextrin cavity and not in the smaller cavity of the alpha cyclodextrin-water complex. Three reaction paths (1,2, and 3) were suggested by Saenger. In path I , the cavity water is directly replaced by the guest. In path 2, the alpha cyclodextrin is first converted into its relaxed state with included, activated water, which is subsequently replaced by the guest. In path 3, the guest binds to the outside of the cavity before inclusion occurs. Adapted from Fig. 4 of W. Saenger, M. Noltemeyer, P. C. Manor, B. Hingerty, and B. Klar, Bioorganic Chemistry, 5 (1976) 187-195.1
ity of the inclusion complex. A scheme of inclusion-complex formation involving both relief of strain energy and release of high-energy water has thus been put forward by Saenger and coworker^"^ (see Fig. 6). In the cases of beta and gamma cyclodextrin, this strain-energy relief-mechanism does not seem to be operative, as the beta and gamma cyclodextrinwater adducts are not ~ t r a i n e d . ’ ~ ~ J ~ ’ (116) K. Lindner and W. Saenger, Angew. Chem., Int. Ed. Engl., 17 (1978) 694-695. (117) J. M. Maclennan and J. J. Stezowski, Biochem. Biophys. Res. Commun., 92 (1980) 926-932.
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R. J. CLARKE, J . H. COATES, AND S. F. LINCOLN
Evidence suggesting a conformational change of alpha cyclodextrin during complex-formation in aqueous solution has been obtained through measurements of optical rotation. Schlenk and Sandx6observed that the optical rotation of alpha cyclodextrin decreases significantly in the presence of various straight-chain alkanoic acids, and that the magnitude of the change increases with the length of the alkanoic acid chain, thus indicating that the effect is due to the interaction of the alpha cyclodextrin with the alkyl chain rather than with the carboxyl group. This shift in optical rotation was attributed by Rees1I8to stabilization of the alpha cyclodextrin cavity by formation of an inclusion complex, and the relief of conformational distortion. Rees, however, did not comment on the nature of the distortion of the alpha cyclodextrin structure in aqueous solution. In order to investigate further the possibility of conformational and solvational changes of the cyclodextrins in aqueous solution, several researchers have used ultrasonic-relaxation technique^.'^^-'^^ In the case of alpha cyclodextrin, two relaxations have been observed.'20-'2'The faster relaxation process has been attributed to a conformational change of the cyclodextrin ring, in accordance with Saenger's ring-strain theory. The slower relaxation process has been attributed to the reorientation of water molecules included in the cavity. Because of its low solubility, beta cyclodextrin has not been studied extensively. It has been found,'20J21however, that gamma cyclodextrin shows a large ultrasonic-absorption effect. A study by Rauh and Knoche12' described the relaxation of gamma cyclodextrin by a narrow distribution of relaxation frequencies. Because the amplitude of the relaxation effect is reduced on complex-formation, and because the calculated value of the reaction volume (AV >9 cm3.mol-l) is much larger than that expected for the rotation of a D-glucosyl unit, they attributed the relaxation effect of gamma cyclodextrin to the reorientation of water molecules in the cavity. They also suggested that the narrow distribution of relaxation frequencies characterizes the disorder of the included water molecules. The results just discussed suggest that it is only alpha cyclodextrin that shows a conformational change on complex-formation, due to the rotation of one of its D-glucosyl units. Whether this conformational change contributes to the stability of the complex, as proposed by Saenger, is, however, debatable. Bergeron and Meeley122showed that methylation of the ( I 18) D. A. Rees, J. Chem. Soc., B , (1970) 877-884. (119) R. P. Rohrbach, L. J . Rodriguez, E. M. Eyring, and J. F. Wojcik,J. Phys. Chem., 81 (1 977) 944-948. (120) S. Kato, H . Nomura, and Y . Miyahara, J . Phys. Chem., 89 (1985) 5417-5421. (121) S. Rauh and W. Knoche, J . Chem. Soc., Faraday Trans. I , 81 (1985) 2551-2559. (122) R. J. Bergeron and M. P. Meeley, Bioorg. Chem., 5 (1976) 197-202.
CYCLODEXTRIN INCLUSION COMPLEXES
23 I
primary hydroxyl groups of alpha cyclodextrin, which they suggested would greatly affect the ease of rotation of a D-glucosyl unit, causes only a slight change in the stability of the inclusion complex. Thus, the conformational change of alpha cyclodextrin on complex-formation may not be a major factor in stabilizing the inclusion complex. Further insights into the differences between the conformations of alpha, beta, and gamma cyclodextrins, and the role of water molecules in stabilizing different conformations have been achieved by c.p.-m.a.s. I3Cn.m.r. studies of hydrated and anhydrous cyclodextrins. 122a~122bWith improvements in technology, it has proved possible to assign, unequivocally, I3C resonances to C-1 and C-4 sites in the cyclic a-(1 + 4)-linked alpha and beta cyclodextrins.122aCertain chemical shifts associated with C-1 and C-4 sites are shifted in the spectrum of hydrated alpha cyclodextrin relative to the remainder, indicating some asymmetry, whereas, in that of hydrated beta cyclodextrin, all of the C-1 resonances are clustered together, as are the C-4 resonances, thus confirming the symmetry of the ring in this case. Cross polarization-dipolar decoupling-magic angle spinning studies122b of both hydrated and anhydrous alpha, beta, and gamma cyclodextrins showed water of hydration to be essential to the symmetry of the beta and gamma cyclodextrin molecules. In addition, these studies revealed a discrepancy between the interpretations of the results of X-ray diffraction s t u d i e ~ I ~on ~ Jhydrated l~ alpha cyclodextrin with respect to the orientations of the HO-6 groups. So far, only hydrophobic interactions have been considered as the source of inclusion-complex stability. Some experimental observations, however, cannot be explained by hydrophobic interactions alone. For example, from a study of a series of substituted phenyl acetates, van Etten and coworkersio2observed an approximately linear relationship between the logarithm of the dissociation constant of the cyclodextringuest complex and the molar refraction of the guest. Because the molar refraction is related to the polarizability, this suggests the participation in the inclusion process of van der Waals forces, that is, permanent dipoleinduced dipole interactions and London dispersion forces. Further support for this comes from the work of Bergeron and coworkers.i23They found that the 4-nitrophenolate anion binds over one hundred times more effectively in the alpha cyclodextrin cavity than 4-nitrophenol, although both guest molecules penetrate the cavity with the nitro group first. This (122a) M. J . Gidley and S . M. Bociek, J . Chmn. SOC., Chem. Commun., (1986) 1223-1226. (122b) I. Fur6, I. P6csik, K. Tompa, R. Teeaar, and E. Lippmaa, Carbohydr. Res., 166 (1987) 21-33. (123) R. J. Bergeron, M. A. Channing, G . J. Gibeily, and D. M. Pillor, J . Am. Chem. Soc., 99 (1977) 5146-5151.
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R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
difference in stability had previously been noted by Connors and L i ~ a r i , ~ ~ who attributed it to the extensive charge delocalization in the 4-nitrophenolate anion. The dependence of the stability of cyclodextrin inclusion on the polarizability of the guest molecule was correctly predicted many years earlier by B r o ~ e r , who ' ~ ~ used the concept to explain the perturbation of the visible absorption spectra of several dye molecules. According to Broser, the fact that the cyclodextrin ring is slightly cone-shaped means that the dipole moments of the hydroxyl groups of the individual D-glucosyl units do not cancel, thus giving the cyclodextrin a resultant dipole moment directed along the cyclodextrin axis, passing through the cavity. This dipole moment produces an electrical field within the cavity, which is thus responsible for the preference of the cyclodextrins for molecules of high polarizability. The dependence of the stability of the complex on the polarizability is a useful concept for explaining differences in the affinity of structurally similar guest molecules for cyclodextrin. In considering guest molecules of significantly different structures, however, steric effects and differences in solvation are usually more important. comUsing molecular orbital calculations, Kitagawa and puted a value for the dipole moment of alpha cyclodextrin by the CND0/2 MO method. The calculated dipole moment is large, 13.5 D, directed from the secondary hydroxyl groups side of the annulus towards the primary hydroxyl groups side. The vector passes through the center of the cavity, making an angle of 153" with its assumed six-fold axis, probably as a consequence of the asymmetry of the alpha cyclodextrin cavity. Thus, in the case of guest molecules having permanent dipole moments, Kitagawa and suggested that dipole-dipole interactions are important in stabilizing the inclusion complex, and also in determining the orientation of the guest within the cyclodextrin cavity. In support of this hypothesis, they showed that, in the case of complexes of alpha cyclodextrin with a number of substituted benzenes, the dipole moment of the guest molecule is so oriented that it is approximately antiparallel to that of the cyclodextrin. The participation of van der Waals forces in inclusion-complex formation is also found to be consistent with crystal structure analyses. Interatomic distances between the guest and the cyclodextrin thus determined are characteristic of van der Waals interactions.lM Hydrogen bonding between the guest and the hydroxyl groups of the cyclodextrin has also (124) W. Broser, Z . Naturforsch., Teil B, 8 (1953)722-729. (124a) M. Kitagawa, H. Hoshi, M. Sakurai, Y. Inoue, and R. ChfijS, Carbohydr. Res., 163 (1987)cl-C3.
CYCLODEXTRIN INCLUSION COMPLEXES
233
been demonstrated crystallographically in certain cases. l04.I~~ The ability of the cyclodextrins to form complexes in aqueous solution with guests incapable of hydrogen bonding, such as rare gases and alkanes,lZhas well as the o b s e r ~ a t i o n ’ ~ that ~ *completely ’~~ methylated cyclodextrins are still able to form stable complexes, suggests, however, that hydrogen bonding between the guest and the cyclodextrin is usually of only minor importance as a driving force for complex-formation. Although, as stated earlier, the stability of cyclodextrin inclusion-complexes is generally due to a favorable change in enthalpy during the inclusion process, it has been found in certain cases, such as the beta cyclo, ~ ~ the predominant driving force for dextrin- 1-alkanol c o m p l e x e ~ that complex-formation is a favorable entropy change. Thus, it has been sugg e ~ t e d ~ that, * J ~ in ~ these cases, the stability of the inclusion complex is due to a classical hydrophobic effect; that is, complex-formation is driven by an increase in entropy resulting from the breakdown of assemblies of ordered water molecules surrounding the apolar, guest molecule. Because the values of AH” and ASo for complex-formation vary over such a wide range, however, it has been proposed by Tabushi and coworker^^^^^^^^ that a ‘‘combined hydrophobic” interaction is responsible for inclusion-complex stability. This combined interaction was presumed to be comprised of the following elemental interactions: (1) van der Waals interaction between the guest molecule and the cyclodextrin cavity; (2) entropy gain due to the destruction of water assembly around the guest molecule; and (3) entropy loss due to “freezing” motional freedom of the guest molecule in the cyclodextrin cavity. Thus, it seems that both the hydrophobic interactions and the van der Waals interactions undoubtedly play a part in inclusion-complex formation, although the relative contribution of each type of interaction may vary with the chemical properties of the guest; this would account for the ability of the cyclodextrins to form complexes with a wide variety of guest molecules. The existence of a close spatial fit between the guest and the cyclodextrin cavity is, however, a necessary requirement for the formation of a stable inclusion-complex.
(125) D. W. Armstrong, T. J. Ward, R . D. Armstrong, and T. E. Beesley, Science, 232 (1986) 1132-1135. (126) F. Cramer and F. M. Henglein, Angew. Chem., 68 (1956) 649. (127) B. Casu, M. Reggiani, and G . ‘R. Sanderson, Carbohydr. Res., 16 (1979) 59-66. (128) W. C. Cromwell, K. Bystrom, and M. R. Eftink, J . Phys. Chem., 89 (1985) 326-332. (129) I. Tabushi, Y. Kiyosuke, T. Sugimoto, and K. Yamamura, J . A m . Chem. Soc., 100 (1978) 916-919. (130) I. Tabushi and Y. Kuroda, Adu. C a d . , 32 (1983) 417-466.
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R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
3. Kinetics of Complex-Formation Although a large number of equilibrium studies of the cyclodextrin inclusion process have been performed, relatively few kinetic investigations have been carried out. Undoubtedly, the reason for this is that the inclusion reaction is rapid, and specialized, rapid-reaction techniques must be used. The most commonly used technique is that of temperatureThis involves a sudden perturbation of jump relaxation. 16,37,38,40,41,131-143 the equilibrium of a guest-cyclodextrin mixture by a rapid increase in the temperature, after which the reaction is monitored (usually spectrophotometrically) as it relaxes to its new equilibrium position. The time constant characterizing this relaxation process (termed the relaxation time, 7)is a function of the rate constants of the reactions being observed and the concentrations of the participating species. Thus, the measurement of the concentration dependence of the relaxation time assists in the assignment of a mechanism to the reaction and in the determination of rate constants. Apart from the temperature-jump technique, other relaxation methods that have been used are those of ultrasonic absorption11g-121-144 and electric-field p u 1 ~ e . Another l~~ technique that has been used for some of the more slowly included guest molecules is that of stopped-flow.142J43,146148 N. Yoshida and M. Fujimoto, Chem. L e t f . , (1980) 231-234. N. Yoshida and M. Fujimoto, Chem. Lett., (1980) 1377-1380. R. P. Rohrbach and J. F. Wojcik, Carbohydr. Res., 92 (1981) 177-181. N. Yoshida and M. Fujimoto, Bull. Chem. Soc. J p n . , 55 (1982) 1039-1045. T. Sano, M. Yamamoto, H. Hirai, andT. Yasunaga, Bull. Chem. Soc. Jpn., 57 (1984) 678-680. (136) H. Nakatani and K. Hiromi, J . Biochem. (Tokyo), 96 (1984) 69-72. (137) A. Orstan and J. F. Wojcik, Carbohydr. Res., 143 (1985) 43-50. (138) K. Taguchi, J . A m . Chem. Soc., 108 (1986) 2705-2709. (139) R. L. Schiller, S. F. Lincoln, and J. H. Coates, J. Chem. Soc., Faraday Trans. 1, 83 (1987) 3237-3248. (140) R. P. Villani, S. F. Lincoln, and J. H. Coates, J . Chem. Soc., Faraday Trans. I , 83 (1987) 2751-2756. (141) S. F. Lincoln, J. H. Coates, and R. L. Schiller, J. Inclusion Phenomena, 5 (1987) 709-7 16. (142) A. Hersey and B. H. Robinson, J. Chem. Soc., Faraday Trans. 1 , 80 (1984) 20392052. (143) A. Hersey, B. H. Robinson, and H. C. Kelly, J . Chem. Soc., Faraday Trans. I , 82 (1986) I27 1- 1287. (144) D. Hall, D. Bloor, K. Tawarah, and E. Wyn-Jones, J . Chem. Soc., Faraday Trans. I , 82 (1986) 2111-2121. (145) M. Sasaki, T. Ikeda, N. Mikami, and T. Yasunaga, J . Phys. Chem., 87 (1983) 5-6. (146) N. Yoshida, A. Seiyama, and M. Fujimoto, Chem. Lett., (1984) 703-706. (147) A. Seiyama, N. Yoshida, and M. Fujimoto, J . Inclusion Phenomena, 2 (1984) 765773. (148) A. Seiyama, N. Yoshida, and M. Fujimoto, Chem. Lett., (1985) 1013-1016. (131) (132) (133) (134) (135)
CYCLODEXTRIN INCLUSION COMPLEXES
235
A technique that has been found useful in studying the inclusion of phosphorescent species is that of phosphorescence decay. 149~150This technique relies on the fact that the included guest molecule is protected by the cyclodextrin from interaction with phosphorescence-quenching agents in the bulk solution. Thus, the presence of cyclodextrin causes an increase in the phosphorescence lifetime of the guest. Turro and c o ~ o r k e r s l ~ ~ showed that the phosphorescence lifetime of halonaphthalenes can be related to the concentrations of beta cyclodextrin and quenching agent (sodium nitrite), as well as the rate constants for the inclusion and quenching reactions. Thus, by measuring the phosphorescence lifetime at various concentrations of quencher and cyclodextrin, it is possible to calculate rate constants for the inclusion reaction. a. One Host-One Guest Complex-Formation.-The majority of kinetic studies reported have been concerned with elucidating the mechanism of 1 : I complex-formation. The most frequently used guest species have been organic dyes, particularly azo dyes, and inorganic anions. In the case of the inclusion of organic dyes, it has been found that steric and charge factors are most important in determining the rate of the inclusion process. It has been ob~erved"j3'~~ that, if the bulkiness of the included group is increased, the rate constants for formation of the inclusion complex, and often its dissociation as well, are decreased. Thus, steric interactions have the effect of slowing the inclusion process. If the guest molecule should be too large, however, it may not fit within the cyclodextrin cavity, and inclusion will not proceed at all. It has been ~ b s e r v e d ' ~ that, , ' ~ ~if~ the ' ~ ~polarity of the included group is increased, assuming no great change in bulkiness, the rate constants of the inclusion reaction decrease. For example, Crarner and coworkersI6 found that, for the inclusion of a series of monosubstituted azo dyes by alpha cyclodextrin, the rate constants decreased as the substituting group was varied in the order: NO2, OH, N(CH3)2,0-. This order would be expected if, according to Cramer, the removal of solvating water molecules from that part of the guest to be included is rate-determining for the complexformation. An alternative explanation, put forward by Hersey and Robinis that charged groups may interact with water in the cyclodextrin cavity, preventing its removal, and hindering access. In contrast to the situation with organic dyes, inorganic anions seem to show no dependence of the rate constants on the degree of solvation. (149) N . J. Turro, J . D. Bolt, Y. Kuroda, and I. Tabushi, Photochem. Photobiol., 35 (1982) 69-72. (150) N . J. Turro, T. Okubo, and C. Chung, J . Am. Chem. Soc., 104 (1982) 1789-1794.
236
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
Rohrbach and coworker^"^ studied the inclusion of the inorganic anions I-, SCN-, Br-, NO;, and C1- by beta cyclodextrin. It was found that the rate constants for complex-formation were very similar for all of the anions, and no correlation between the rate constant and the anionic radius could be found. Thus, in the case of anion complexation, Rohrbach and coworkers suggested that anion desolvation is not rate-determining. These results were supported by those of Hgiland and coworkers,151who measured the partial molar volume change (AVO) during complex-formation between the ions SCN- and I- and alpha and beta cyclodextrin. For the complexes with alpha cyclodextrin, AVO was found to be negative. In the case of beta cyclodextrin, AVO is either zero or only slightly positive. According to Hgiland and coworkers, if anion desolvation occurred on complex-formation, significantly positive values of AVO would be expected. Thus, it appears that inorganic anions may not undergo desolvation on inclusion-complex formation. Unusual behavior was reported119for the complexation of the ions 10; and C10; with beta cyclodextrin. Rohrbach and coworkers found119that these ions show values of the forward rate-constant that approach the diffusion-controlled limit; that is, lo9 to 1Olo dm3.mol-i.s-1,whereas all of the other ions studied show values significantly less than this limit. In order to explain this observation, Rohrbach suggested1Igthat, because of their larger radii, the C10; and 10; ions may be too large to fit into the alpha cyclodextrin cavity, but, instead, may form a complex in which the ions straddle one of the entrances to the cavity. The fact that all of the other inorganic anions studied do not show diffusion-controlled inclusion implies that some other process must be rate-limiting. Because Rohrbach had ruled out desolvation prior to inclusion, he suggested that a conformational change of the inclusion complex may be rate-determining. A conformational change had previously been proposed for alpha cyclodextrin inclusion complexes by Saenger and coworkers1I4on the basis of crystallographic studies (as discussed in Sect. 111,2), and Rohrbach, himself, had observed a conformational change of pure alpha cyclodextrin in aqueous solution by using the ultrasonic-absorption technique. Thus, Rohrbach suggested that the complexation mechanism could be described as K X- + cyclodextrin & X- ... cyclodextrin A X-.cyclodextrin,
where the first equilibrium is achieved very rapidly and may be described as the formation of an encounter complex, and the next step involves the slower, rate-determining, conformational change of the cyclodextrin com(151) H. Hpliland, L. H. Hald, and 0. J. Kvammen, J . Solution Chem., 10 (1981) 775-785.
CYCLODEXTRIN INCLUSION COMPLEXES
237
plex. In the case of beta and gamma cyclodextrin, no direct experimental evidence has as yet been presented to support this mechanism. In the case of interactions with alpha cyclodextrin, however, the situation is somewhat different. Until 1984, all of the stopped-flow and temperature-jump kinetic studies of alpha cyclodextrin inclusion-complex formation were explainable in terms of a single-step, binding mechanism. According to this mechanism, the observed rate constant, kobs, (for stopped-flow) and the reciprocal relaxation time, 1/T, (for temperature-jump) should show a linear dependence on the alpha cyclodextrin concentration. Sano and c o w ~ r k e r s , l ~ ~ however, in the case of the iodide-alpha cyclodextrin interaction, and Hersey and Robinson,'42in the case of various azo dye-alpha cyclodextrin interactions (see Fig. 7), found that certain guest species exhibit a limiting value of kobs and 1 / at ~ high concentrations of alpha cyclodextrin. This behavior can most simply be explained in terms of a mechanism of the type, A
+ cyclodextrin
Ki
k2
A.cyclodextrin
fast
A.cyclodextrin',
k-2 slow
that is, a mechanism similar to the one previously proposed by Rohrbach. For this mechanism, the relationship between kobs and the alpha
0
1
2
3
4
5
FIG. 7.-Variation of k,,, Characterizing the Mordant Yellow 7-Alpha Cyclodextrin Systemi4*with Variation of Total Concentration of Alpha Cyclodextrin. {[Mordant Yellow 71 = 3.0 x mol. dm-'; T = 15.0 (x), 25.0 (o),35.0 (0)"C. Adapted from Fig. 2 of Ref. 142.}
238
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
cyclodextrin concentration (assuming an excess of alpha cyclodextrin) is given by the equation
where [C] is the total concentration of alpha cyclodextrin. Hersey and Robinson also found'42that many guest species that show kinetic behavior apparently explicable in terms of a single-step binding, give a discrepancy between the values of the equilibrium constant determined kinetically and those determined fro equilibrium studies. It was found that the equilibrium constant, deter ined spectrophotometrically, was usually greater than the ratio of the forward and backward rateconstants, determined kinetically. They therefore suggested that this discrepancy could be adequately explained if the two-step mechanism just described was used to interpret the results. A similar proposal has also been made by Hall and coworkers,144who observed a large discrepancy between AVO values for the inclusion of 1-butanol and 1-pentanol by alpha cyclodextrin, calculated from equilibrium-density measurements and kinetic, ultrasonic-absorption data. A study similar to that of Hersey and Robinson has been reported by Seiyama and coworker^.^^^^^^^ From a stopped-flow, kinetic study of the interaction of various azo dyes and some azo dye-metal complexes with alpha cyclodextrin, they observed two kinetic processes. The dependence of the observed rate-constants for these two processes on the alpha cyclodextrin concentration was found to be explainable in terms of a mechanism identical to that proposed earlierI4*by Hersey and Robinson. In the case of the guests used by Seiyama and coworkers, however, values for the rate constants of the binding step could be determined147J4x from the concentration dependence of kobsfor the faster process; thus,
2
kobs
=
kl[Cl
+ k-1,
where [C] is the total concentration of alpha cyclodextrin, which is assumed to be in excess. The reason why Seiyama and coworkers were able to observe the binding step independently of the conformational change is probably that they used azo dyes bulkier than those of Hersey and Robinson, and these would presumably be included more slowly. Further support for the presence of a conformational change has come from the work of Orstan and Wojcik,I3' who also studied azo dye-alpha cyclodextrin complexes by using the temperature-jump technique. The dyes that they investigated showed three different types of behavior. Certain dyes showed a limiting value of I / T at high alpha cyclodextrin concentrations, others showed a linear dependence of 1 / on ~ alpha cyclo-
CYCLODEXTRIN INCLUSION COMPLEXES
239
dextrin concentration, and the third group showed no dependence of I / T on alpha cyclodextrin concentration. The last two types of behavior could arise as limiting cases of the two-step mechanism. Further research by Hersey and using iodide as a competing ion, suggested that the intermediate of the mechanism is associated with the guest’s binding to the inside rather than the outside of the alpha cyclodextrin. The nature of the conformational change is not yet understood. It has been ~ u g g e s t e d ’ ~ that ~J~ it ~is associated with the guest molecule’s inserting farther into the alpha cyclodextrin cavity, and consequent solvational changes. Structural changes in the alpha cyclodextrin and guest may also be occurring. For example, rotation of a D-glucosyl unit of alpha cyclodextrin during complex-formation has been proposed by Saenger and coworkers.’14 Up to now, evidence for the presence of a conformational change during the inclusion process has been presented only for alpha cyclodextrin, and then only in the case of certain guest molecules. Whether the twostep mechanism is generally applicable is not known, because, owing to the nature of kinetic studies, the results are frequently open to more than one interpretation. Nevertheless, sufficient support for the two-step mechanism has appeared to necessitate its consideration in any future kinetic studies. b. Two Hosts-One Guest Complex-Formation.-In the case of guest molecules that cannot be totally included by a single cyclodextrin molecule, it has sometimes been observed that a second cyclodextrin molecule may bind. Thus, various azo d y e ~ , l biphenyl ~ J ~ ~ corn pound^,^^^^^^^ and cinnamates,155-157 which are longer than the depth of a single cyclodextrin molecule, have been found to form 2 : 1 complexes in solution. A crystal structure has also been reported” for the 2 : I complex of Methyl Orange with alpha cyclodextrin. Although two hosts-one guest complex-formation is well established, however, almost no kinetic studies of the process have been reported. The only study which has so far appeared is that of Schiller and coworkers,139who investigated the interaction of the dye (152) A. Harada, M. Fume, and S . Nozakura, Macromolecules, 9 (1976) 705-710. (153) R. I. Gelb, L. M. Schwartz, C. T. Murray, and D. A . Laufer, J . Am. Chem. Soc., 100 (1978) 3553-3559. (154) S. F. Lincoln, A. M. Hounslow, J . H. Coates, and B. G. Doddndge, J . Chem. Soc., Faraday Trans. 1 , 83 (1987) 2679-2703. (155) R. I. Gelb, L. M. Schwartz, and D. A. Laufer, J . Am. Chem. Soc., 100 (1978) 58755879. (156) K . A . Connors and T. W. Rosanske, J . Pharm. Sci., 69 (1980) 173-179. (157) I. M. Brereton, T. M. Spotswood, S. F. Lincoln, and E. H. Williams, J . Chem. Soc., Faraday Truns. 1 , 80 (1984) 3147-3156.
240
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
Pyronine Y (PY) with beta cyclodextrin by using the temperature-jump technique. It was found that the kinetic data could be adequately explained by a mechanism involving the stepwise association of two beta cyclodextrin molecules (C) to the dye. Thus, PY
ki +ce PY.C k-1
and PY.C
k2 +c* PY.C*.
k-2
The association of more than two cyclodextrin molecules to a single guest molecule has also been reported; for example, the complex-formation of the cyclodextrins with long-chain fatty acids.86Although no kinetic studies have been carried out in these cases, stepwise association of the cyclodextrin onto the long guest-chain, like threading pearls onto a string, would seem to be the most likely mechanism. c. One Host-Two Guests Complex-Formation.-As mentioned in the Introduction, it has been found that beta and gamma cyclodextrin are able to include two guest molecules simultaneously, although the number of reported instances of this is much greater in the case of gamma than of beta cyclodextrin, undoubtedly due to the larger cavity size of gamma cyclodextrin. For all of the guests so far studied, the cavity of alpha cyclodextrin seems to be able to include only a single guest molecule. The formation of a one host-two guests inclusion-complex could proceed by way of two main mechanisms: ( I ) dimerization of the guest, followed by inclusion; and (2) stepwise inclusion of two guest molecules. In order to distinguish between these two possibilities, several temperature-jump relaxation kinetic studies have been carried o ~ tusing organic dyes (both anionic and cationic) as the guest species. A similar dependence of the reciprocal relaxation-time on the cyclodextrin concentration has been observed for all cases of one host-two guests complexformation. A rapid rise in I/T is observed at low cyclodextrin concentrations. A maximum is then reached, which is followed by a slower drop in the I/T value as the cyclodextrin concentration increases. A representative example of this behavior is shown in Fig. 8. It has been found that this dependence of I/T on concentration is most adequately explained by the following mechanism, where C = cyclodextrin. A A
+C
+ A.C
ki
k2 k-2
A.C
fast
A2.C
S~OW
,
~
CYCLODEXTRIN INCLUSION COMPLEXES
24 1
FIG. 8.-Variation of I/T Characterizing the Crystal Violet-Gamma Cyclodextrin Systern40 with Variation of Total Concentration of Gamma Cyclodextrin [[Crystal Violet] = 1.5 x mol. dm-'; T = 25°C. Adapted from Fig. 3 of R. L. Schiller, J. H. Coates, and S. F. Lincoln, J . Chem. SOC.Faraday Trans. 1 , 80 (1984) 1257-1266.1
The alternative mechanism, involving dimerization of the guest prior to inclusion, is unable to fit the observed data. Using the method of BernasconiI5*or C z e r l i n ~ k ithe ' ~ ~following equation can be derived, characteriz~ cyclodextrin and guest concentrations for the ing the variation of l / with aforementioned stepwise-inclusion mechanism,
where all concentrations are equilibrium values at the final temperature. All of the dyes studied also dimerize in aqueous solution in the absence of cyclodextrin; thus, 2A
kd
A2
k-d
However, if the dye is of an appropriate size to form a one host-two guests complex with beta or gamma cyclodextrin, it was found that, on addition of the cyclodextrin, dye solutions often show spectral changes (158) C. F. Bernasconi, Relaxation Kinetics, Academic Press, New York, 1976.
(159) G. H. Czerlinski, Chemical Relaxation, Marcel Dekker, New York, 1966.
242
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
characteristic of dimer formation at dye concentrations for which there is a negligible concentration of uncomplexed dimer. Thus, the dye dimer is thermodynamically more stable when included by cyclodextrin. Schiller and coworkers'39have shown that this enhanced stability is due to the fact that the rate constant for the dissociation of a dye monomer from the one host-two guests complex, k-2, is significantly less than the rate constant for dissociation of the dimer, k-d. The rate constants for formation of the complex, k2, and dye dimerization, kd, are not significantly different, and seem to be almost diffusion-controlled. The decrease in the rate of dimer dissociation when included by cyclodextrin is presumably due to changes in the solvation of the dye and to van der Waals interactions between the dimer and the cyclodextrin.
d. Two Hosts-Two Guests Complex-Formation.-In the case of certain guest molecules, such as Methyl Orange, which possess two aromatic groups capable of inclusion, it has been f o ~ n d that ~ ~ a,better ~ ~ fit + to ~ the ~ ~ observed, experimental, temperature-jump kinetic data is obtained if the possibility of a second cyclodextrin's binding to the one host-two guests inclusion complex is considered (see Fig. 9). In these cases, the mechanism can be described by three steps. A A
+C
+ A.C
KI
kz
A.C
fast
A2.C
SIOW
k-2
A2.C + C
K? = A2.C2
fast
FIG. 9.-Schematic Diagram of the Two Hosts-Two Guests Inclusion Mechanism Shown for the Tropaeolin 000 No. 2-Gamma Cyclodextrin System.
CYCLODEXTRIN INCLUSION COMPLEXES
243
The dependence of 1/r on concentration can be explained by an extension to the equation for the two-step mechanism of one host-two guests complex-formation, that is, 1/r =
k2
(
[A] + [C]
+
[A2.C] + l/K3 [C] + [A2.C] + l/K3
1/KI
Thus, the formation of a one host-two guests inclusion-complex seems to proceed by way of the stepwise inclusion of two guest molecules, with the inclusion of the second guest molecule being rate-determining. The formation of a two hosts-two guests complex may then proceed by the rapid addition of a second cyclodextrin molecule. No evidence has been presented that supports dimerization of the guest prior to inclusion. An alternative mechanism for the formation of a two hosts-two guests complex has been suggested. From equilibrium studies of the concentration-dependence of the absorption spectra and excimer fluorescence intensities of naphthalene in the presence of beta cyclodextrin, Hamai30 concluded that the excimer fluorescence is due to a two hosts-two guests complex, formed by the association of two 1 : 1 beta cyclodextrin-naphthalene complexes. Thus, the mechanism can be described as follows. A t C
2A.C
A.C A2.Cl
The same mechanism was also proposed by Herkstroeter and coworkers,160 who carried out absorption and fluorescence measurements on the interaction of 4-pyrenyl butanoate with gamma cyclodextrin. No kinetic studies, however, have yet been performed on these systems in order to verify the proposed mechanism. Because the guest compounds most commonly investigated contain aromatic moieties, it is interesting to consider the size of aromatic group capable of inclusion by the various cyclodextrins and the possible stoichiometries. Clarke and coworkers have studied the interactions of the
(160) W . G . Herkstroeter, P. A. Martic, and S. Farid, J . Chem. SOC., Perkin Trans. 2 , ( 1984) 1453- 1457.
244
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
cyclodextrins with the azo dyes Methyl Orange (1),37 Tropaeolin (2),38 and Roccellin (3).39As these dyes have various sizes of aromatic moieties, the observed stoichiometries of the complexes formed with each cyclodextrin can give information about the suitability of the aromatic groups to each size of cavity. Thus, the cavity of alpha cyclodextrin, which is able to include only a single Methyl Orange molecule, is most suited to the inclusion of a phenyl group. Beta cyclodextrin, which was found to form a one host-two guests complex with Tropaeolin, but only a 1 : 1 complex with Roccellin, seems to be able to include a phenyl group and a naphthyl group simultaneously, but not two naphthyl groups. However, gamma cyclodextrin, which forms a very stable one host-two guests complex with roccellin, is large enough to include two naphthyl moieties. An interesting equilibrium study was performed by Kasatani and coworkers160aon the interaction of a series of cyanine dyes with beta and gamma cyclodextrin, using u.v.-visible spectrophotometric measurements. The results were consistent with the enhancement of dimer formation by inclusion of the dye molecules within the cyclodextrin. As expected from the work of others, such geometrical factor^^"^^ as the size of the aromatic groups and the lengths of the central methine chains strongly influence the occurrence of dimer formation within the cyclodextrin cavity.
4. Modified Cyclodextrins Numerous examples of modifications to the fundamental cyclodextrin structure have appeared in the l i t e r a t ~ r e . ~ The * ~ , aim ~ J ~of~ much of this work has been to improve the catalytic properties of the cyclodextrins, and thus to develop so-called “artificial enzymes. ” Cyclodextrins themselves have long been known to be capable of catalyzing such reactions as ester hydrolysis161J62 by interaction of the guest with the secondary hydroxyl groups around the rim of the cyclodextrin cavity. The replacement, by synthetic methods, of the hydroxyl groups with other functional groups has been shown, however, to improve remarkably the number of reactions capable of catalysis by the cyclodextrins. For example, Breslow and coworkers’63reported the attachment of the pyridoxamine-pyridoxal coenzyme group to beta cyclodextrin, and thus found a two hundred-fold acceleration of the conversion of indolepyruvic acid into tryptophan. (160a) K. Kasatani, M. Ohashi, M. Kawasaki, and H. Sato, Chem. Leu., (1987) 1633-1636. (161) N. Hennrich and F. Cramer, J. Am. Chem. Soc., 87 (1965) 1121-1126. (162) R. L. van Etten, G. A. Clowes, J. F. Sebastian, and M. L. Bender, J . Am. Chem. SOC.,89 (1967) 3253-3262. (163) R. Breslow, M. Hammond, and M. Lauer, J. Am. Chem. SOC., 102 (1980) 421-422.
CYCLODEXTRIN INCLUSION COMPLEXES
245
Some modifications to the cyclodextrin structure have also been found to improve their complexing ability. Casu and coworkers127prepared 2,3,6-tri-O-methyl and 2,6-di-O-methyl derivatives of alpha and beta cyclodextrin. They observed that tri-0-methyl-alpha cyclodextrin shows an almost ten-fold increased stability of the complex with the guest, Methyl Orange, compared with the unmodified alpha cyclodextrin. A possible reason for this increase in stability is that the methyl groups are responsible for an extension of the hydrophobic cavity of the cyclodextrin. Other workers,122however, observed a much smaller enhancement of stability of complexes on methylation of the cyclodextrin, and a decrease in stability has even been reported’40 for the one host-two guests complex of tropaeolin with beta cyclodextrin. Thus, the effect of methylation on the stability of a complex varies with the guest species involved, and cannot be readily predicted. An interesting combination of the approaches just described was achieved by Ikeda and Using imidazolylethyl chloride, the hydrogen atom of the OH-3 group of a single D-glucopyranose unit of 2,6di-0-methylcyclomaltoheptaose was substituted with an imidazolylethyl group. The resulting compound, a partially methylated beta cyclodextrin bearing an imidazolyl substituent, was found to be an effective catalyst for the hydrolysis of p-nitrophenyl acetate. The values obtained of k,,, = 2.67 X lo2 s-I and K, = 2.9 x lo3 m ~ l . d m -for ~ this cyclodextrin derivative at pH 8.2 may be compared with k,,, = 1.1 x lo2 s - I and K, = 0.04 x lo3 m ~ l . d m - ~ for a-chymotrypsin at pH 8.0. Another interesting modification has been the covalent linking of two cyclodextrin molecules. Fujita and c o w o r k e r ~ reported ~ ~ J ~ ~the synthesis of a double gamma cyclodextrin host bridged by a disulfide bond. They found that this new host was capable of including two Methyl Orange or Ethyl Orange molecules. The one host-two guests complex with Methyl Orange was found to be approximately ten thousand times more stable than the corresponding complex with unmodified gamma cyclodextrin. Modifications of this type may be particularly useful for guests having two aromatic portions, such as the azo dyes. Some other interesting modifications of the gamma cyclodextrin structure have been made by the group of Ueno and coworkers. By the (163a) H. Ikeda, R. Kojin, C.-J. Yoon, T. Ikeda, and F. Toda, Chem. Lett., (1987) 14951498.
(163b) V. T. D’Souza, K. Hanabusa, T. O’Leary, R. C. Gadwood, and M. L. Bender, Biochem. Biophys. Res. Commun., 129 (1985) 727-731. (164) K. Fujita, S. Ejima, and T. Imoto, J . Chem. Soc., Chem. Commun., (1984) 12771278. (165) K. Fujita, S. Ejima, and T. Imoto, Chem. Lett., (1985) 11-12.
246
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
FIG.10.-Schematic Diagram of the Space Regulation of the Gamma Cyclodextrin Cavity by an Appended Naphthalene Moiety.
attachment of a naphthyl group to the rim of gamma cyclodextrin, they have o b ~ e r v e d l ~that J ~ the ~ naphthyl group is able to act as a space regulator, narrowing the gamma cyclodextrin cavity so that it can form 1 : 1 complexes with guests that are too small to form complexes with the unmodified host (see Fig. 10). The modification of gamma cyclodextrin by substitution with two naphthyl groups has also been reported by Ueno and c o w o r k e r ~ . In ~ ~this ~ J ~case, ~ it appears that the naphthyl groups promote binding of guest molecules within the cavity by acting as hydrophobic caps, in which the two naphthyl groups are involved in a stacking interaction above the cyclodextrin cavity (see Fig. 11). Many examples of covalently attached hydrophobic caps also exist. In these cases, a sufficiently extended aromatic system is placed across one of the openings of the cyclodextrin by substitution of hydroxyl groups on opposite sides of the cavity170 (see Fig. 12). Although their inclusion properties have not yet been studied, the branched cyclomalto-oligosaccharides prepared by Koizumi and cowork e r ~are~potentially ~ ~ ~interesting. , ~ ~ Branched ~ ~ cyclodextrins were originally isolated by means of high-performance liquid chromatography from the mother liquors of large-scale preparations of unbranched cyclodextrins.170aSubsequently, the reverse action of Pseudomonas isoamylase on mixtures of, for example, maltotriose and beta cyclodextrin was used to which was then sepasynthesize 6-O-a-maltotriosylcyclomaltohepataose, rated and purified by liquid chromatography. This method has been used A. Ueno, Y. Tomita, and T. Osa, J . Chem. SOC., Chem. Commun., (1983) 976-977. A. Ueno, F. Moriwaki, Y. Hino, and T. Osa, J . Chem. Soc., Perkin Trans. 2 , (1985) 921-923. A. Ueno, F. Moriwaki, T. Osa, F. Hamada, and K. Murai, Tetrahedron Lett., (1985) 3339-3342. A. Ueno, F. Moriwaki, T. Osa, F. Hamada, and K. Murai, Bull. Chem. SOC.Jpn., 59 (1986) 465-470. I. Tabushi, K. Shimokawa, N . Shimizu, H. Shirakata, and K. Fujita, J . A m . Chem. SOC.,98 (1976) 7855-7856.
CYCLODEXTRIN INCLUSION COMPLEXES
247
FIG.11.-Schematic of the Interaction of a Guest with Gamma Cyclodextrin Bearing Two Naphthyl Groups. [Ueno and coworkers proposed two possible mechanisms of inclusion, 1 and 2. In 1, one of the naphthyl groups acts as a space regulator. In 2, the two naphthyl groups form a flexible, hydrophobic cap across the cavity of the cyclodextrin. In the cases of the guests that they investigated, Ueno considered that mechanism 2 is predominant.]
successfully to prepare a number of isomers of alpha, beta, and gamma cyclodextrins variously substituted by maltotriose or maltose sidechains.I7Ob
5. Chiral Discrimination As the cyclodextrins are chiral molecules, a racemic mixture of an optically active guest species has the possibility of forming two diastereomers on complexation with cyclodextrin, that is,
(+)-guest (-)-guest
. D( + )-cyclodextrin . D( +)-cyclodextrin.
In principle, these two complexes should show differences in their
U FIG. 12.-Covalent
Capping of Cyclodextrin.
248
R. J. CLARKE, J. H. COATES, AND S. F. LINCOLN
physical properties. Thus, the cyclodextrins may be useful in the resolution of racemates. The first report17' of chiral discrimination by the cyclodextrins was made in 1952. Cramer o b ~ e r v e dthat, ' ~ ~if ~cyclodextrin ~~~ is mixed with a solution of an excess of a racemic guest, one of the optical isomers is preferentially complexed, and thus, the precipitated complex contains an excess of one enantiomer. This was first shown with the esters of mandelic, chlorophenylacetic, and bromophenylacetic acids. Experiments similar to those of Cramer have been carried out by Mikolajczyk and Drabowicz,173-175 who reported the partial resolution of chiral sulfoxides and sulfinates by complexation with cyclodextrin. Benschop and van den Berg176reported partial resolution of isopropyl methylphosphinate and some of its derivatives. Occasionally, very high optical purities are obtained after one precipitation of the complex. For example, Mikolajczyk and Drabowic~"~ reported a 70% purity of the (-)-S-enantiomer of 0isopropyl methylsulfinate. For the majority of guests studied, however, the optical purities obtained are quite low, usually less than 15%. The purities obtained could be improved by repeating the process of complexation, precipitation, and guest extraction. A more efficient method, however, would be the use of column chromatography, in which the cyclodextrin is immobilized as the stationary phase. This method has been used effectively by Hinze and coworkers177for the separation of racemic mixtures of certain amino acids. The column that they prepared consisted of beta cyclodextrin chemically bonded to a spherical silica-gel support. Reactions capable of catalysis by the cyclodextrins also show some degree of chiral selectivity. For example, van Hooidonk and BreebaartHansenI7* found that the (-)-R-isomer of isopropyl methylphosphonofluoridate is hydrolyzed by alpha cyclodextrin much more rapidly than the (+)-S-isomer. Their measured dissociation constants, however, (170a) K. Koizumi, T. Utarnura, M. Sato, and Y. Yagi, Curbohydr. Res., 153 (1986) 55-67. (170b) J . 4 . Abe, N . Mizowaki, S. Hizukuri, K. Koizurni, and T. Utarnura, Carbohydr. Res., 154 (1986) 81-92. (171) F. Crarner, Angew. Chem., 64 (1952) 136. (172) F. Cramer and W. Dietsche, Chem. Eer., 92 (1959) 378-384. (173) M. Mikolajczyk, J. Drabowicz, and F. Cramer, J . Chem. Soc., Chem. Commun., (1971) 317-318. (174) M. Mikolajczyk and J. Drabowicz, Tetrahedron Lett., (1972) 2379-2382. (175) M. Mikolajczyk and J. Drabowicz, J . Am. Chem. SOC., 100 (1978) 2510-2515. (176) H. P. Benschop and G. R. van den Berg, J . Chem. SOC.,Chem. Commun., (1970) 1431-1432. (177) W. L. Hinze, T. E. Riehl, D. W. Arrnstrong, W. DeMond, A. Alak, and T. Ward, Anal. Chem., 57 (1985) 237-242. (178) C. van Hooidonk and J . C. A. E. Breebaart-Hansen, R e d . Trav. Chim. Pays-Bas, 89 (1970) 289-299.
CYCLODEXTRIN INCLUSION COMPLEXES
249
showed that the (+)-,!?-isomer binds more strongly to alpha cyclodextrin than does the (-)-R-isomer. A similar difference in chiral selectivity of binding and catalysis has also been found by Ihara and coworkersf79for the case of the hydrolysis of amino acid nitrophenyl esters by alpha and beta cyclodextrin. Thus, although both catalysis and binding by cyclodextrin show chiral selectivity, the same isomer is not necessarily favored by both processes. Another interesting area of research has been the attempt to improve the chiral selectivity of cyclodextrins by chemical modification. Harata and coworkersfx0showed by X-ray crystallography that L- and D-mandelic acid exhibit different inclusion geometries when included by permethylated alpha cyclodextrin. They suggestedfg0JXf that unmodified cyclodextrins show little chiral discrimination, because of the regular, polygonal shape of the cavity. However, they observed that, after methylation, the cavity becomes distorted, and they thus suggested that this distortion may result in an enhanced chiral selectivity. Armstrong and coworker^,'^^ however, suggested that a differential interaction of the two enantiomers with groups at the mouth of the cyclodextrin may be important for chiral recognition.
IV. CONCLUSION In his review of the cyclodextrins, published in 1957, Frenchlx2predicted that the cyclodextrins would “continue to serve, delight, teach, and intrigue the carbohydrate chemist for many years to come.” This has indeed been found to be the case. Although much progress has been made, and numerous practical applications of the cyclodextrins are now appearing in the literature, the fundamental forces underlying their complexing ability, their mechanism of complex-formation, and the factors affecting their chiral selectivity are still not very well understood. There is, therefore, still much scope for further research in these areas, particularly regarding the nature of conformational changes of the inclusion complex, and the role of water in the inclusion process. It seems that modifications to the cyclodextrin structure will significantly broaden the areas of application of the cyclodextrins. For the optimum design of modifications to suit particular purposes, however, a better understanding of the formation of cyclodextrin inclusion-complexes is still required. (179) Y . Ihara, E. Nakanishi, M. Nango, and J. Koga, Bull. Chem. SOC.Jpn., 59 (1986) 190 1- 1905.
(180) K. Harata, K. Uekama, M. Otagiri, and F. Hirayama, Chem. L e f t . , (1983) 18071810. (181) K. Harata, F. Hirayama, T. Imai, K. Uekama, and M. Otagiri, Chem. L e f t . , (1984) 1549- 1552. (182) D. French, Adu. Curbohydr. Chem., 12 (1957) 189-260.
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ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY, VOL. 46
HYDROLYSIS AND OTHER CLEAVAGES OF GLYCOSIDIC LINKAGES IN POLYSACCHARIDES BY CHRISTOPHER J. BIERMANN Department of Forest Products-Pulp and Paper Science, Oregon State University, Corvallis, Oregon 97331
I. Introduction.
.....................................................
d 0-Linked Carbohydrate Chains. . . . . . . . . . . . . . . . . . . . . . .
111. Methanolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Monosaccharides ................................... ............ 2. Glycoconjugates.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Total Hydrolysis with A c i d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................... 1. Monosaccharides . . . . . . . . . erials . . . . . . . . . . . . . . . 2. Polysaccharides of Wood 3. Glycoconjugates.. . . . . . . . 4. D-Fructans. .. ... . . ... . V. Formolysis and Acetolysis .................................. VI. Enzymic Hydrolysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Reductive Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
.
.
25 1 255 256 251 258 259 259 263 265 269 269 270 27 1
I. INTRODUCTION There are several references dealing with various aspects of the glycosidic linkage, including discussions on mechanisms in carbohydrate chemistry by Capon’ that concentrate on reactions of the anomeric carbon atom, on acid-catalyzed hydrolysis of glycosides by BeMiller,*on various specific degradation reactions of polysaccharides, especially in regard to hydrolysis, acetolysis, periodate oxidation, deamination, and p-eliminat i ~ nand , ~ on the synthesis and cleavage reactions of glycosidic linkage^.^ (1) B. Capon, Chem. Rev., 69 (1969) 407-498. (2) J. N. BeMiller, Adu. Carbohydr. Chem., 22 (1967) 25-108. (3) B. Lindberg, J. Lonngren, and S. Svensson, Adv. Carbohydr. Chem. Biochem., 31 (1975) 185-240. (4) A. F. Bochkov and G. E. Zaikov, Chemistry of the 0-Glycosidic Bond: Formation and Cleauage, Pergamon Press, New York, 1979.
25 I
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
252
CHRISTOPHER J. BIERMANN TABLEI The p H of Aqueous Solutions of Various Acids Acid
Molarity
PH
Hydrochloric sulfuric Trifluoroacetic Acetic
I 0.5
I
0. I 0.3 0.7
1
2.4
This article is a discussion of cleavage of glycosidic linkages useful for the analysis of oligo- and poly-saccharides. Cleavage of glycosidic linkages of larger oligo- and poly-saccharides is necessary in order to determine what monosaccharides compose the larger carbohydrate. Hydrolysis, cleavage of a bond by the addition of the elements of a water molecule, is the most common method for cleavage of glycosidic linkages. Hydrolysis is carried out in aqueous solutions with an acid catalyst, although some special-purpose hydrolyses, such as the liberation of carbohydrate chains from glycoconjugates, require alkaline catalysts. Common acid catalysts are hydrochloric, sulfuric, and trifluoroacetic acids. The relative strengths of these acids are given in Table I. Glycosidic linkages may also be cleaved in other solvents, such as methanol. In this case, water is rigorously excluded, so that the elements of a molecule of methanol are added across the glycosidic linkage to afford the methyl glycosides. Methanolysis is usually catalyzed by the addition of dry hydrogen chloride to the methanol. Other solvents, such as acetic anhydride-acetic acid (acetolysis) and formic acid (formolysis), are occasionally used for special purposes. With any of these methods, there is always the tradeoff between incorhplete cleavage of the glycosidic linkage under relatively mild conditions and decomposition of the liberated monosaccharides under more severe conditions. Fig. 1 shows the liberation of neutral monosaccharides during hydrolysis with 0.5 M sulfuric acid at loo", under reflux, of an algal exopolysaccharide. Even after hydrolysis for 6 hours, additional galactose is being liberated, although some xylose and mannose are lost due to decomposition. Compared to aldohexoses, aldopentoses and deoxy sugars are particularly susceptible to acid decomposition. Uronic and aldonic acids are subject to decomposition by such reactions as decarboxylation, whereas amino sugars are relatively stable, although, if they occur in the N-acetylated form, there is loss of acetyl groups (N-deacetylation) during hydrolysis.
CLEAVAGES OF POLYSACCHARIDE GLYCOSIDIC LINKAGES
-
253
2
fly: 0 W 6
A
v
I
0.5
I
1
I
I
4
2
7
1
6
T i m e (h)
FIG. 1 .-Hydrolysis of Extracellular Polysaccharide Produced by Anabaena 30s-aquae A-37. [Key: 1 , galactose; 2, glucose; 3, mannose; 4, arabinose; 5, rhamnose; 6, ribose, fucose; and 7, xylose.]
To correct for the relatively small amount of decomposition of monosaccharides liberated under the conditions presented in Fig. 1, it is possible to subject a mixture of monosaccharides to identical conditions of hydrolysis and to measure the decomposition compared to that of a mixture not subjected to acid hydrolysis, in order to obtain a correction factor for losses by hydrolysis. Alternatively, in order to measure decomposition, hydrolysis may be continued for an extended period of time, and the concentration of monosaccharide may be extrapolated back to time zero.
254
CHRISTOPHER J. BIERMANN
Once correction factors have been determined for a set of hydrolysis conditions, it is only necessary to recheck the correction factors occasionally. However, as will be shown for the case of acid hydrolysis of pectin and galacturonic acid by 2 M trifluoroacetic acid (CF~COZH), correction factors are only approximate; consequently, it is always important to minimize decomposition. To determine the correction factors, an internal standard is generally added to each of the mixtures and the detector response measured relative to the internal standard. If the ratio of a monosaccharide to the internal standard decreases after hydrolysis of a test mixture of monosaccharides, decomposition has occurred and a recovery factor should be determined. The internal standard must either be resistant to decomposition during hydrolysis or should be added after hydrolysis; it is usually better to add the internal standard after hydrolysis, to be on the safe side. The internal standard must not appear in the samples and must be resolved from other components in the sample, as with any internal standard. In practice, it turns out that the polysaccharides containing only neutral monosaccharides are fairly readily hydrolyzed without more than 10% decomposition of some of the more labile monosaccharides. For several reasons, polymers containing uronic acids or amino sugars are much more resistant to hydrolysis. The presence of carboxyl or amino groups makes hydrolysis of the glycosidic linkages more difficult, and the uronic acids liberated are themselves much more susceptible to degradation, particularly by decarboxylation. Additionally, the amino group in many naturally occurring polysaccharides is acetylated, so that the fate of this acetyl group is also of consequence. Fortunately, many of the hydrolysis procedures developed during the past few years are able to circumvent these problems for many types of samples. The neuraminic acids (also called sialic acids), which occur in the terminal positions of carbohydrate moieties of many glycoproteins and gangliosides, must be hydrolyzed under very mild conditions. For example, hydrolysis with 0.01 M hydrochloric acid for 30 min at 100" causes 20% decomposition of N-acetylneuraminic acid.5aSialic acids are more tolerant to methanolysis; they may also be removed enzymically. It is useful to arrange a discussion of the literature concerning acid hydrolysis by type of substrate, and then to subdivide by type of acid used for a substrate, even though this may mean some overlap. It should also be kept in mind that many of the references for cleavage of glycosidic (5) N. Sharon, Complex Carbohydrates-Their Chemistry, Biosynthesis, and Functions, Addison-Wesley, Reading, 1975, a, p. 54; b, pp. 65-83; c, pp. 84-91.
CLEAVAGES OF POLYSACCHARIDE GLYCOSIDIC LINKAGES
255
linkages of a particular substrate contain methods for the separation (and derivatization, if employed) of the monosaccharides liberated. Many workers use a set of hydrolysis conditions without even referencing the method employed or determining the hydrolysis recoveries. In some cases, such work may be cited if there is not much other literature available for hydrolytic conditions for those types of samples. In most cases, though, literature in which recoveries from hydrolysis or methanolysis are specified will be used; consequently, this article is not comprehensive, but it does contain descriptions of the most comprehensive studies. 11. LIBERATION OF N-
AND
@LINKED CARBOHYDRATE CHAINS
In glycoconjugates, the carbohydrate moieties are linked to the protein portion either by N-glycosylic or glycosidic (0-glycosylic) linkages. Generally, N-acetylglucosamine (GlcNAc) is linked to L-asparagine by an N glycosylic linkage (Asn-GlcNAc). This linkage involves the anomeric hydroxyl group of GlcNAc in the p-pyranose form and the 4-amide of L-aspartic acid. Common glycosidic linkages include xylose and galactose to L-serine, D-mannose and N-acetylgalactosamine to L-serine or Lthreonine, D-galactose to 5-hydroxy-~-lysine,and L-arabinose to 4-hydroxy-L-proline. Except for the last two examples, these linkages are cleaved by dilute alkali,5bp6although the N-glycosylic linkage is somewhat more stable than the glycosidic linkage. For characterization of the monosaccharide constituents of glycoconjugates, the entire glycoconjugate is usually subjected to total hydrolysis with acid. For detailed, structural analysis of the carbohydrate moieties, however, the isolation of the intact carbohydrate moieties from the remaining protein is desirable. Glycosidic linkages having an activating group at the P-position are cleaved by the alkali-catalyzed p-elimination reaction with relatively low concentrations of base (0.1 M OH- at 5 0 7 , whereas N-glycosylic linkages are cleaved by hydrolysis with6 higher concentrations of base at higher temperatures; for example, M OH- at 100”. To prevent decomposition of the liberated oligosaccharides by “peeling” reactions, the reaction is conducted in the presence of a reducing agent.6 Because the monosaccharide unit formerly constituting the reducing end of the oligosaccharide is now reduced, it may be distinguished from the remaining constituents of the oligo~accharide.~ Alkali cleavage of oligosaccharide chains from glycoconjugates in the presence of tritium-labeled or deuterated sodium borohydride identifies the linking monosaccharide for analysis by radioactivity (6) S. Ogata and K. 0. Lloyd, Anal. Biochem., 119 (1982) 351-359. (7) T. P. Mawhinney, J . Chrornatogr., 351 (1986) 91-102.
256
CHRISTOPHER J. BIERMANN
or mass spectrometry, respectively. The linking amino acid may also be a ~ c e r t a i n e dTwo . ~ ~ studies examined a variety of reduction conditions for isolation of the oligosaccharide A thorough review of carbohydrate-peptide linkages is available.5b
111. METHANOLYSIS Methanolysis is performed with dry halogen chloride in anhydrous methanol at elevated temperatures. It is important to work under anhydrous conditions, as the presence of water sets up an equilibrium between methyl glycosides and the free forms of sugars, leading to very complicated mixtures. Some workers prefer methanolysis to hydrolysis as a method for cleavage of glycosidic linkages, because methanolysis usually causes little degradation of the monosaccharides liberated. On the other side of the coin is the fact that a complex mixture of methyl glycosides is the result. Also, N-acetylated carbohydrates are partially deacetylated; little work has been performed on peracetylation of carbohydrates after methanolysis as a method of analysis; consequently, a separate step for N-reacetylation is necessary. N-Reacetylation with acetic anhydride in the presence of pyridine results in partial acetylation of the hydroxyl groups which requires a second, although milder, methanolysis to r e m o ~ e . ~Typically, ,'~ after methanolysis, samples are made neutral with silver carbonate, N-reacetylated, subjected to mild methanolysis by some workers" (0.1 M hydrogen chloride in methanol for 0.5 h at 65"), dried, and (trimethylsily1)ated. Hydrogen chloride may also be neutralized with Amberlite IRA-400 (HCO?) resinI2or simply evaporated with c o ~ o l v e n t . ~Deamination ~J~ prior to methanolysis is also possible,I3 in which case N-acetylation is no longer a concern. Often, only one or two of the major peaks of a particular monosaccharide are used for quantitation (for simplification), with the assumption that, for a particular set of methanolysis conditions, the ratio of products from a monosaccharide will be ~ 0 n s t a n t .In I ~a study of guar and other plant gums,I2highperformance liquid chromatography of the methyl glycosides without derivation was possible. Perbenzoylation of methyl glycosides has also been accomplished. l 1 (8) J.-R Neeser, Carbohydr. Res., 138 (1985) 189-198. (9) T. Mega and T. Ikenaka, Anal. Biochem., 119 (1982) 17-24. (10) R. E. Chambers and J. R. Clamp, Biochem. J . , 125 (1971) 1009-1018. (11) N . Jentoft, Anal. Biochem., 148 (1985) 424-433. (12) N. W. H. Cheetharn and P. Sirirnanne, Carbohydr. Res., 112 (1983) 1-10. (13) S. Inoue and M. Miyawaki, Anal. Biochem., 65 (1975) 164-174.
CLEAVAGES OF POLYSACCHARIDE GLYCOSIDIC LINKAGES
257
1. Monosaccharides The study of monosaccharides subjected to conditions of methanolysis is considered for two reasons. First, the decomposition of monosaccharides is indicative of the decomposition of monosaccharides liberated during methanolysis; second, the ratio of methyl glycosides of a particular standard monosaccharide is the same for the same monosaccharide released during methanolysis (for a particular set of methanolysis conditions), provided that the concentration of sugars is relatively low. Up to four methyl glycosides (the a and j3 anomers of the pyranoside and furanoside forms) of a particular monosaccharide may be formed; the acyclic dimethyl acetal is an additional possibility. The ratios of methyl glycosides of 10 monosaccharides subjected to methanolysis with M hydrogen chloride for 24 h at 80" has been reported.12 Similar information is also available in another study.'' Mega and Ikenaka9 used methanolysis with 0.8 M methanolic hydrogen chloride at 90" to determine the number of N-glycosylically linked carbohydrate chains and the proportion of glycosidic N-acetylglucosamine (GlcNAc) in glycoproteins. In their study, they employed N-acetylglucosamine having specific linkages. N-Linked GlcNAc of Asn-GlcNAc gave 44% of GlcN after methanolysis for 8 h or longer. The recovery of GlcN itself after methanolysis was 70%, the rest probably being in the form of methyl glycosides. This work showed that methanolysis is capable of cleaving N-glycosylic linkages. Analysis of ovalbumin, flavoprotein, and taka-amylase A glycoproteins by 0.8 M hydrogen chloride in methanol for up to 24 h at 90" showed that 24 h is a reasonable length of time for complete cleavage of the glycosidic linkages. Chambers and Clamplo studied methanolysis of monosaccharides and glycoproteins with various concentrations of hydrogen chloride and various times at 85 and 100". None of the monosaccharides showed any decomposition when subjected to methanolysis employing M hydrogen chloride for 24 h at 85". In 2 M methanolic hydrogen chloride at loo", the percentage decompositions were as follows: hexuronic acids, 15%; xylose, 11%; and N-acetylneuraminic acid, 5%; whereas fucose, hexoses, and aminohexoses remained stable. This is an important reference for the decomposition of monosaccharides subjected to methanolysis with up to 6 M hydrogen chloride in methanol, and for the release of monosaccharides from glycopeptides under various conditions of methanolysis. Analysis of glycopeptides and oligosaccharides of known composition indicated that methanolysis in M hydrogen chloride is complete after 3 h at 85". It is apparent that methanolysis is capable of cleaving glycosidic linkages of glycoproteins with a minimum of decomposition of the monosaccharides liberated.
258
CHRISTOPHER J. BIERMANN
Methanolysis of standard uronic acids has been studied by Inoue and Miyawaki13 in regard to the depolymerization of chondroitin sulfate and dermatan sulfate. These workers found the glucosiduronic linkage to galactosamine to be rather resistant to methanolysis, but that it is more efficiently cleaved after deamination of the amino galactoside, with its conversion into 2,5-anhydrotalose. For iduronic, glucuronic, and mannuronic acids released from a polymer, it was found that the peaks monitored for these acids, relative to an internal standard, increase during the first 8 h of methanolysis ( M hydrogen chloride, 100")and remain constant for up to 20 h of methanolysis. This indicated that 8 h is required for complete methanolysis, and that the monosaccharides liberated are stable to the conditions of methanolysis. 2. Glycoconjugates
In the preceding Section, reference has been made to methanolysis of glycosidic linkages involving peptides. The detailed work of Chambers and Clamplo showed that M methanolic hydrogen chloride for 3 h at 85" is sufficient to release the monosaccharides from glycopeptides and oligosaccharides.IOQuantitation of uronic acids in acid glycosaminoglycans was studied by Inoue and Miyawaki13 by using M methanolic hydrogen chloride for 8-20 h at 100"; whale intestine, beef lung, and umbilical samples were investigated. Jentoftl' examined the standard glycoconjugates sialyllactose, pig submaxillary mucin, and fetuin, and found methanolysis with M methanolic hydrogen chloride containing M methyl acetate to be equally effective for 4 h at 80" or for 16 h at 65". C h a ~ l i nused ' ~ methanolysis for the analysis of carbohydrates in glycoproteins. His method was a variation of the foregoing procedures, with an improvement of using tert-butyl alcohol to remove hydrogen chloride by coevaporation, instead of prolonged trituration with silver carbonate. His method is useful for samples containing uronic acids and lipids. Mononenls studied methanolysis, followed by deamination and reduction with borohydride, for determination of the monosaccharide constituents of glycoconjugates. This method was applied to a lipid-free, protein fraction of rat brain. The composition of lipopolysaccharides (LPS) of bacterial origin have been investigated in two ~ t u d i e s . Both ~ ~ ~ studies '~ used 2 M methanolic hydrogen chloride for 16-24 hours at 85". The method allows analysis of (14) M. F. Chaplin, Anal. Biochem., 123 (1982) 336-341. (15) I. Mononen, Carbohydr. Res., 88 (1981) 39-50. (16) I. Brondz and I. Olsen, J . Chromatogr., 310 (1984) 261-272. (17) K. Bryn and E. Jantzen, J . Chrornatogr., 240 (1982) 405-413; 370 (1986) 103-112.
CLEAVAGES OF POLYSACCHARIDE GLYCOSIDIC LINKAGES
259
carbohydrates, fatty acids, and 3-deoxy-~-manno-2-octu~osonic acid (KDO), which normally occur in LPS samples, as their (trifluoroacety1)ated methyl glycosides or methyl esters.” This method has been applied to differentiation of taxonomically related bacteria. l6 Pyruvated monosaccharides of bacterial origin were studied by Dudman and Lacey,’* using methanolysis with M hydrogen chloride for 16 h at 82”.Silylated methyl glycosides were prepared after N-reacetylation of the amino sugars. IV. TOTALHYDROLYSIS WITH ACID
In Section IV, 1, losses of monosaccharides subjected to conditions of acid hydrolysis are examined. In the remaining Sections, methods of hydrolysis for various types of sample are considered. It should be kept in mind that monosaccharides of polysaccharides exist in an “anhydro” form, as the elements of water are added across the glycosidic linkage during hydrolysis. For example, the molecular weight of glucose is 180; the formula weight for the D-glucosyl units of cellulose, starch, and many other polysaccharides is 162. Thus, 162 grams of pure cellulose will yield 180 grams of D-glucose upon complete hydrolysis if there is no decomposition.
1. Monosaccharides Subjecting monosaccharides to conditions of acid hydrolysis is only of importance in measuring the expected hydrolysis losses during hydrolysis of oligo- and poly-saccharides. Hydrolysis losses may be predicted, based on either the absolute or the relative decomposition of monosaccharides. Absolute decompositions are based on decomposition of monosaccharides. Relative decompositions are based on studies wherein several methods of hydrolysis were applied to the same samples for various lengths of time; in this Section, these are classified under the type of acid that causes the least decomposition (that is the largest yield of monosaccharides liberated), because this acid is usually the one of principal concern in the particular study. a. Sulfuric Acid.-Hydrolysis with sulfuric acid has the drawback that its eventual removal prior to derivatization of the monosaccharides is usually achieved by the use of barium carbonate (or other material which precipitates sulfate and neutralizes the acid), or an ion-exchange resin, or both, followed by filtration. Often, removal of the sulfuric acid is much more laborious than the derivatization itself. However, it is possible to (18) W. F. Dudman and M. J. Lacey, Curbohydr. Res., 145 (1986) 175-191.
260
CHRISTOPHER J. BIERMANN
form the aldononitrile acetates of neutral sugars in the hydrolyzate (for analysis by gas-liquid chromatography) without removal of the sulfuric acid. l9 Fig. 1 shows decomposition of monosaccharides liberated by hydrolysis with 0.5 M H2SO4 of an algal exopolysaccharide. There appeared to be some decomposition of xylose and mannose; however, to obtain accurate rates of decomposition, the hydrolysis should have been continued for a much longer time, as monosaccharides are still being liberated from the polysaccharide and there are not enough data points to extrapolate back accurately to the origin in those cases where there is apparent decomposition. Selvendran and coworkersZostudied hydrolysis of some neutral aldohexoses and aldopentoses with M H2S04for 2, 5, 8, 12, and 18 h at 100". After hydrolysis for 5 h, the recoveries were as follows: galactose, 95%; arabinose, 96.3%; xylose, mannose, and glucose, 98% each; and rhamnose, 100%;after hydrolysis for 18 h, the recoveries were: xylose, 67%; arabinose, 75.3%; rhamnose, mannose, and galactose, 81% each; and of the starting glucose, 82%. Most of the decomposition occurred during the 12 to 18 h of hydrolysis, with the hydrolysis losses at 12 h less than half of those at 18 h. Because hydrolysis of neutral polysaccharides with M H2S04 rarely requires more than 4 h, hydrolysis of neutral polysaccharides with sulfuric acid is an excellent method.
b. Hydrochloric Acid.-Hydrolysis with hydrochloric acid is usually performed in tubes purged with nitrogen and then sealed, because the presence of oxygen increases the decomposition of sugars. Prior to hydrolysis of the human immunoglobulin IgG, Niedermeier2' added monosaccharides, in order to study recoveries of monosaccharides. He found that, in the case of hydrolysis with M hydrochloric acid at loo", 98% of fucose was recovered after 1 h, 91% of the mannose and 95% of the galactose were recovered after 4 h, and over 100% of glucosamine was recovered after 16 h; the same hydrolysis times were used to quantitate each of these monosaccharides of IgG. Torello and coworkersZ2used M hydrochloric acid for hydrolysis of monosaccharides at loo", and found the decomposition to be as follows: galactose, 26%; glucose, 20%; GlcNAc, 14%; and GalNAc, 4%, during the first 4 h of hydrolysis, and little additional decomposition up to 16 h. Because the samples were (19) (20) (21) (22)
G. D. McGinnis, Carbohydr. Res., 108 (1982) 284-292. R. R. Selvendran, J. F. March, and S. G . Ring, Anal. Biochern., 96 (1979) 282-292. W. Niedermeier, Anal. Biochern., 40 (1971) 465-475. L. A. Torello, A. J. Yates, and D. K . Thompson, J . Chrornarogr., 202 (1980) 195-209.
CLEAVAGES OF POLYSACCHARIDE GLYCOSIDIC LINKAGES
261
TABLEI1 Recoveries of Galacturonic Acid Subjected to Treatment with 2 M Trifluoroacetic Acid" Time (h)
0.0 0.5 1 .o 2.0
(Y
Anomer
/3 Anomer
Total
Relative to time = 0 (%)
17.8b 14.8 11.2 6.4
24" 17.3 12.9 7.7
42.1b 32.0 24.1 14.1
100 76 57 34
a Treatment of pectin (containing 79.1% of galacturonic acid) for 1 h at 121" gave 53.5% of galacturonic acid for a net recovery of 67%. The average of duplicate samples is presented. Amounts relative to myoinositol, the internal standard, in arbitrary units.
analyzed as alditol acetates, N-deacetylation was not considered. The authors did not mention whether they purged the sample tubes with nitrogen prior to acid hydrolysis; perhaps, the presence of oxygen accounts for much of the decomposition noted during the first 4 h. Griggs and coworkers23studied hydrolysis with 3 M hydrochloric acid for 3 h at 100"of canine submaxillary mucin (CSM) and CSM mixed with a standard sugar mixture. Sugar recoveries were: 76.0% for fucose, 76.6% for mannose, 84.3% for galactose, 82.5% for N-acetylglucosamine, and 87.7% for N-acet ylgalactosamine. c. Trifluoroacetic Acid.-Hydrolysis with trifluoroacetic acid (TFA) has the advantage that, after hydrolysis, it may be readily removed by evaporation under diminished pressure or with a stream of dry nitrogen. This also constitutes a method for concentrating the sample when high sensitivity is required. Hydrolysis with CF3C02H,like that with hydrochloric acid, is usually conducted in sealed tubes purged with nitrogen in order to lessen any decomposition that might occur in the presence of air or oxygen. Galacturonic acid was treated by B i e r ~ n a n nwith ~~ 2 M CF3C02Hat 121" with the results shown in Table 11. After hydrolysis for 1 h, -57% of the galacturonic acid remained; however, when a sample of pectin was hydrolyzed for 1 h, -67% of the galacturonic acid was recovered. This discrepancy may be explained on the basis of a lower rate of decomposition for the galacturonic acid units while they are in the polysaccharide (pectin), as opposed to being in the free monosaccharide form. (23)L. E.Griggs, A. Post, E. R. White, J. A. Finkelstein, W. E. Moeckel, K. G. Holden, J. E. Zarembo, and J. A. Weisbach, Anal. Eiochem., 43 (1971)369-381. (24) C. J. Biermann, unpublished results.
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CHRISTOPHER J. BIERMANN
Because the galacturonic acid units of pectin spend a significant portion of the time in the polymerized form, there is less decomposition than that of galacturonic acid standards subjected to the same conditions of hydrolysis. For this reason, correction factors for hydrolysis losses, under any conditions, can never be completely accurate; therefore, it is preferable to cleave glycosidic linkages with the minimum of decomposition. Analysis of the rates of decomposition of monosaccharides by 2 M TFA has been accomplished in two s t u d i e ~by ~ ~continuing -~~ hydrolysis of polysaccharides after most of the monosaccharides had been liberated. Albersheim and coworkers26studied the hydrolysis of pinto-bean hypocotyl cell-walls (10 mg/mL) for up to 6 h at 121", although the monosaccharides, except glucose and mannose, had been liberated after 1 h. After 6 h, >50% of the xylose and arabinose and >25% of the galactose, rhamnose, and fucose had been decomposed; however, as most of the monosaccharides are liberated within 1 h, these rates of decomposition are tolerable. Honda and coworkers25studied the hydrolysis of urinary glycoconjugates for up to 15 h at 100". They found that hydrolysis for 6 h gave the maximum yield of aldoses and uronic acids. Recovery of monosaccharides added prior to 6 h of hydrolysis were 93% for GlcN, 94%for Xyl, 95% for Glc, 96% for GlcA, 97% for GalN, 98% for Fuc, 102% for Man and 105% for Gal. The recovery for glucuronic acid is very high, considering how labile this monosaccharide is. This method may be useful for any sample containing uronic acids, such as pectin and some plant gums. Recoveries for monosaccharides added prior to hydrolysis with 4 M hydrochloric acid for 6 h at 100" gave similar yields, although not quite so high on the average. In both cases, the yields are very high for accurate determination of monosaccharide constituents of glycoconjugates. The investigators favored the use of CFJCO~Hfor aldoses and uronic acids, but hydrochloric acid for hexosamines, because polysaccharides containing hexosamines are hydrolyzed with more difficulty. Neeser and S~hweizer*~ studied hydrolysis of neutral sugars with 4 M TFA for 1 h at 125".They used two concentrations of sugars, -0.05 M and -2.5 mM. It was found that, at the higher concentrations, recoveries (25) S. Honda, S. Suzuki, K. Kakahi, A. Honda, and T. Takai, J . Chrornatogr., 226 (1981) 341-350. (26) P. Albersheim, D. J. Nevins, P. D. English, and A. Karr, Carbohydr. Res., 5 (1967) 340-345. (26a) A. Thompson, M. L. Wolfrom, and E. J . Quinn, J . A m . Chem. Soc., 75 (1953) 30033004; A. Thompson, K. Anno, M. L. Wolfrom, and M. Inatome, ibid., 76 (1954) 13091311. (27) J.-R. Neeser and T. F. Schweizer, Anal. Biochem., 142 (1984) 58-67.
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were generally much lower. This may be explained on the basis of reversion products,26athat is, the formation of new glycosidic linkages. Because 4 M TFA is only -50% water, a shift in equilibrium towards the formation of glycosidic linkages becomes favorable, as this reaction produces water. Acid that catalyzes the hydrolysis of glycosidic linkages necessarily also catalyzes the formation of glycosidic linkages. The formation of reversion products is important under any set of hydrolysis conditions when the concentration of sugars is high. By keeping the concentration of monosaccharides relatively low, this problem is averted. At low concentration, yields of monosaccharides were as follows: Rha, 82%; Fuc, 89%; Ara, 86%; Xyl, 72%; xylitol, 88%; 3-O-methylglucose, 97%; Man, 86%; Glc, 97%; Gal, 81%; myo-inositol and GlcN, 100%; GalN, 92%; and ManN, 95%. 2. Polysaccharides of Wood and Other Plant Materials
Hydrolysis of woody materials usually involves the liberation of neutral monosaccharides only, discounting a small amount of 4-0-methylglucuronic acid. From some gums and certainly from polyuronic acids, significant proportions of uronic acids are also liberated. Amino sugars in these substrates have seldom been studied. In addition to the work presented next, Neeser and Schweizerz7 studied hydrolysis of two soluble vegetable fibers by 4 M CF3COZHfor 1 h at 121", and found this to be superior to hydrolysis with 0.5 M HZSO4for 3 h at 100". a. Woody Materials.-Hydrolysis of woody materials with sulfuric acid is usually accomplished by a two-step procedure. In the first step, the sample is swollen with 72% (w/w) H2S04for -1 h at 30", -1.5 h at 25", or -2 h at 20". In the second, the sulfuric acid is diluted to -1-2 M, and the mixture is refluxed for 2 to 5 h. This method is often referred to as Saeman hydrolysis,28although 72% sulfuric acid has been usedz9since 1910. There are many variations of this method.20.z8-35 In some cases, hydrolysis with (28) J . F. Saeman, W. E. Moore, R. L. Mitchell, and M. A. Millett, TAPPI, 37 (1954) 336343. (29) M. L. Laver, D. F. Root, F. Shafizadeh, and J. C. Lowe, TAPPI, 50 (1967) 618-622. (30) J . E. Jeffery, E. V. Partlow, and W. J. Polglase, Anal. Chem., 32 (1960) 1774-1777. (31) J. M. Oades, J . Chromatogr., 28 (1967) 246-252. (32) M. G. Paice, L. Jurasek, and M. Desrochers, TAPPI, 65 (1982) 103-106. (33) J. L. Slavin and J. A. Marlett, J . Agric. Food Chem., 31 (1983) 467-471. (34) A. B. Blakeney, P. J. Harris, R. J. Henry, and B. A. Stone, Carbohydr. Res., 113 (1983) 291-299. (35) R. C. Pettersen, V. H. Schwandt, and M. J. Effland, J . Chromatogr. Sci., 22 (1984) 478-484.
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CHRISTOPHER J. BIERMANN
77% sulfuric acid has been used to obtain better dissolution of wood p ~ l p s . ~ For ~ - ~wood O and fiber samples, the insoluble residue remaining is often weighed; it is attributed to lignin, and is referred to as Klason lignin or acid-insoluble lignin. Selvendran and coworkers20 hydrolyzed plant cell-wall materials by using 2 M CF3C02H for 2 h at 120", and M sulfuric acid for 1,2,5, and 8 h at 100" with (Saeman hydrolysis), or without, a prior 72% H2S04 step for 3 h at 20". They found that Saeman hydrolysis with a 2-h secondary hydrolysis liberated the most monosaccharides, although continuing the secondary hydrolysis for a total of 5 h gave almost identical yields, except for an increased yield of rhamnose and an appreciable decrease of galacturonic acid. A small amount of mannose was produced, possibly from epimerization at C-2 of D-glucose, in the case of Saeman hydrolysis. Hydrolysis by CF3C02Hwas not very effective in liberating D-glucose from cellulose, although the yield of other monosaccharides was similar to that of Saeman hydrolysis. This method has been used by others for the analysis of neutral detergent fiber (dietary fiber).33 Jeffery and coworkers30used 77% H2SO4 followed by 6.6% H2SO4 for 4 h at loo", whereas Laver and coworkers29used 3.0% H2SO4 in their secondary hydrolysis for 4.5 h, which, they claimed, was better than the method of Jeffery and coworkers. Oades3' used 72% H2SO4 followed by 0.5 M H2S04 (-5%) in his study of organic material in soils and peats. Blakeney and coworkers34studied hydrolysis of plant cell-walls with 72% H2S04, followed by secondary hydrolyses with 0.5 M H2S04 for ( a ) 1 h at 121", (b) 2 h at loo", and (c) 3 h a t loo", respectively. The highest recovery of neutral monosaccharides was for condition (c), and it accounted for 93% of the dry weight of cell walls. A few investigators have studied hydrolysis of plant cell-wall material with CF3C02H.25,32,36,37 Generally, the method gives good yields of neutral monosaccharides, except for glucose from cellulose. In their investigation of hydrolysis of wood and pulp samples for the wood sugars Ara, Xyl, Gal, Man, and Glc, using CF3C02H, Paice and coworkers32studied 30, 50, and 80% (vh) CF3C02H for up to 4.8 h at 100". The best conditions were with 80% CF3C02H for 2 h, which gave 87% recovery of monosaccharides. Their method employed 5 mg of material dissolved in 1.6 mL of anhydrous CF3C02H (a good cellulose solvent) for 2 days at room temperature. The acid was then diluted to the desired concentration, and the tubes were sealed under a partial vacuum. (36) T. M. Jones and P. Albersheim, Planr Physiol., 49 (1972) 926-936. (37) G . F. Collings and M. T. Yokoyama, J . Agric. Food Chem., 27 (1979) 373-377.
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b. Gums.-Gums are used in such foods as ice creams, salad dressings, and soft-cheese products, as they bind water, improve flavor and texture, and are thickening agents. Gums are heteropolysaccharides. For example, guar gum is a galactomannan, whereas others may also contain arabinose, xylose, rhamnose, and fucose. Varma and coworkers38studied the neutral monosaccharide components of gums. They hydrolyzed dried gum (2 mg) with 75 mM H2SO4 (1 mL) in a sealed ampoule for 36 h at 95", and claimed that this method was the best compromise for a variety (unspecified) of hydrolysis conditions. In another study, the neutral monosaccharides of tragacanth, arabic, guar, carob, and other gums were i n ~ e s t i g a t e dThe . ~ ~ gum identities and quantities of a variety of prepared foods were also analyzed. Hydrolysis was performed with 0.5 M CF3C02Hfor 4 h at 100". Recovery of gums was determined, based on the addition of known quantities of gums to some food samples. The recovery factors are therefore applicable for the entire method, and not just to sample hydrolysis recoveries. Guaran40was also hydrolyzed with M hydrochloric acid for 24 h at 80".
c. G1ycuronans.-As
already mentioned, hydrolysis with 2 M TFA at
100"may be a useful method for hydrolysis of glycuronans, although it has
not been used specifically for this purpose. D-Mannuronic and L-guluronic acids have been prepared by acid hydrolysis of alginate ~arnples.~' A solution of alginate (50 mg) in 80% H2SO4 (2.5 mL) was kept for 17 h at 20". The H2SO4 was diluted to M , and hydrolysis was conducted for 3 h at 100". The acid was neutralized with calcium carbonate, the suspension filtered, and the filtrate concentrated. Information on the hydrolysis of pectins may be found in a monograph.42 3. Glycoconjugates Because of the numerous methods available for the hydrolysis of various glycoconjugates, including glycoproteins, mucins, glycosaminoglycans, and gangliosides, it is useful to arrange these methods according to the type of acid employed. Some studies compared a particular method of (38) R. Varma, R. S. Varma, and A. H. Wardi, J . Chromatogr., 77 (1973) 222-227. (39) J . F. Lawrence, and J . R. Iyengar, J . Chromatogr., 350 (1985) 237-244. (40) J. Thiem, J. Schwentner, H. Karl, A. Severs, and J. Reimer, J . Chromatogr., 155 (1978) 107-1 18. (41) A. G. I. Voragan, H. A. Schols, J. A. De Vries, and W. Pilnik, J . Chromatogr., 244 (1982) 327-336. (42) M. L. Fishman and J. J. Jen (Eds), Chemistry and Function of Pectins, ACS Symp. Ser., 310 (1986).
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CHRISTOPHER J . BIERMANN
hydrolysis to several other methods; in these cases, the comparison is described under the heading of the central method of such studies. For example, Neeser and S ~ h w e i z e developed r~~ hydrolysis of glycoproteins with 4 M TFA, and compared this method to other available methods; all of their results are described in Section IV,3,c, in order to retain comprehensibility without undue overlap. a. Sulfuric Acid.-Hydrolysis of glycoconjugates with sulfuric acid has not received much attention. Fox and coworkers,43however, tried various conditions for hydrolysis with sulfuric acid, and found M H2S04for 3 h to be useful for hydrolysis of bacterial cell-walls. In this study, the sulfuric acid was neutralized with a 20% solution of N,N-dioctylmethylamine in chloroform.
b. Hydrochloric Acid.-Niedermeier and T ~ r n a n used a ~ ~ 0.5 to 6.0 M hydrochloric acid for 1 to 12 h at 100" for the hydrolysis of human immunoglobulins and bovine submaxillary mucin, and monitored the liberated glucosamine and galactosamine. Hydrolysis with M hydrochloric acid for 10 h at 100" released all of the hexosamines from immunoglobulins, although the glucosamine liberated from bovine submaxillary mucin was only -90% of that liberated by 3 M hydrochloric acid for 3 h, leading the authors to suggest that optimal hydrolysis conditions for each glycoprotein must be determined individually. In an earlier study, Niedermeier2' had used M hydrochloric acid for 1,4, and 10 h at 100" in order to find the mildest conditions required to liberate certain monosaccharides. He found that, for IgM, hydrolysis for 1 h is suitable for galactose, 4 h for mannose and glucosamine, and 10 h for fucose, although, for IgG, hydrolysis for 1 h is suitable for fucose, 4 h for mannose and galactose, and 10 h for glucosamine. The composition of IgM was proportional to the amount hydrolyzed for 1, 2, 3.5, and 5 mg in. 5 mL of M hydrochloric acid, indicating that reversion products are of no consequence at these concentrations. Kannan and coworkers4salso used M hydrochloric acid at 100" for the hydrolysis of neutral glycolipids; 6 h was found sufficient for hydrolysis of galactolipids, but 12 h was required for complete cleavage of the glucoseceramide bond. The use of standard ceramide mono- and di-hexosides subjected to hydrolysis for 12 h gave monosaccharide recoveries of >94% (43) A. Fox, S . L. Morgan, J. R. Hudson, Z. T. Zhu, and P. Y. Lau, J . Chrornatogr., 256 (1983) 429-438. (44) W . Niedermeier and M. Tomana, Anal. Biochern., 57 (1974) 363-368. (45) R. Kannan, P. N . Seng, and H. Debuch, J . Chrornatogr., 92 (1974) 95-103.
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in all cases. Torello and coworkers22used M hydrochloric acid for 1 to 16 h at 100"for the hydrolysis of human cerebral-cortex ganglioside, GM, ; the highest yield of galactose was obtained after 4 h, whereas the highest yield of glucose and GlcNAc was obtained after 12 h. There was appreciable degradation of the liberated monosaccharides under these conditions, although purging the tubes with nitrogen (instead of air) did not significantly affect the yields. By using correction factors, corrected ratios of monosaccharides in various gangliosides were within 10-20% of the theoretical value. Alpenfels and coworkers46studied the hydrolysis of glycoproteins and keratin fibers with I or 2 M hydrochloric acid for various periods of time at 100".These investigators found that the concentration of neutral monosaccharides from hard keratin reached a maximum after hydrolysis with 2 M hydrochloric acid for 2 h at loo", and the yield of the neutral monosaccharides was linear up to 25 mg of hair per mL of hydrochloric acid solution. The latter fact shows that a relatively large amount of protein does not interfere with the analysis of a relatively small amount of carbohydrate. Griggs and coworkersz3studied the hydrolysis of canine submaxillary mucin (CSM) by 0.5, 3, and 6 M hydrochloric acid for 1.5, 3, 4.5, 6, and 24 h at 100". They found that use of 3 M hydrochloric acid for 3 h gives the maximal release of neutral and amino monosaccharides, with the minimal degradation of the monosaccharides liberated, although recoveries of neutral monosaccharides were only 76-88%. Guen-ant and Moss4' also used hydrolysis with 3 M hydrochloric acid, although for 16 h at 75", for the hydrolysis of bacterial cell-walls. Honda and coworkers25used 4 M hydrochloric acid for 6 h at 100" for the hydrolysis of nondialyzable glycoconjugates when determining amino monosaccharides, but preferred 2 M CF3C02Hfor 6 h at 100"when determining the neutral monosaccharides and uronic acids, as these compounds are subject to more-severe degradation by 4 M hydrochloric acid. They obtained complete hydrolysis (with >90% recovery of monosaccharides added prior to hydrolysis) by using these two sets of hydrolytic conditions. c. Trifluoroacetic Acid.-The use of 2 M CF3C02H by Honda and coworkersz5was discussed in Section IV,3,b. Eggert and Jones4*used 2 M (46) W. F. Alpenfels, R. A. Mathews, D. E. Madden, and A. E. Newsom, J . Liy. Chromatogr., 5 (1982) 1711-1723. (47) G . 0. Guerrant and C. W. Moss, Anal. Chem., 56 (1984) 633-638. (48) F. M. Eggert and M. Jones, J . Chromatogr., 333 (1985) 123-131.
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CHRISTOPHER J. BIERMANN
CF3C02Hfor various lengths of time at 105"for the hydrolysis of neutral sugars in glycoproteins. They found 6-10 h of hydrolysis to be ideal for such glycoproteins as proteodermatan sulfate, salivary glycoproteins, and alpha- 1-acid glycoprotein, whereas 4 h of hydrolysis was sufficient for ovalbumin and fetuin; from this, they recommended 8 h of hydrolysis for unknown glycoproteins and, ideally, a 6-, 8-, and 10-h time-course hydrolysis. They also found hydrolysis with CF3C02H to be superior to that with 2 M hydrochloric acid at 105", owing to the decomposition of liberated monosaccharides by hydrochloric acid. Neutralization of the hydrochloric acid after hydrolysis, but prior to freeze-drying, lessened the decomposition somewhat, although there was still more decomposition than in hydrolysis with CF3C02H. 4 M CF3C02Hfor 1 h at 121" for Neeser and S ~ h w e i z e introduced r~~ hydrolysis of glycoproteins. Both neutral and amino sugars were considered. They compared this method to hydrolysis with 0.6 M hydrochloric acid for 4 h at 100" and 3 M hydrochloric acid for 0.75 h at 125". Hydrolysis of fetal-calf-serum fetuin, bovine submaxillary mucin, and horse-radish peroxidase showed hydrolysis with CF3C02H to be superior. d. Other Methods.-Takemoto and coworkers49 used a mixture of equal amounts of 4 M CF3C02Hand 4 M hydrochloric acid for 6 h at 100" for measurement of the neutral and amino monosaccharides of glycoconjugates. Their rationale was that 2.5 M CF3C02H for 4 h at 100" is useful for measurement of the neutral sugars, but not severe enough for the liberation of amino sugars (especially Asp-GlcNAc) from glycoproteins, whereas hydrolysis with 4 M hydrochloric acid for 6 h at 100" is sufficient to release amino sugars from glycoproteins, but decomposes neutral sugars. The authors found that, with their method, amino sugars were completely liberated, even from Asn-GlcNAc, with no decomposition of glucosamine. Neutral sugars were completely liberated, but underwent some decomposition. Correction factors were applied. This method was applied to alpha- 1-acid glycoprotein, submaxillary-gland mucin, and bovine-brain gangliosides. Some investigators have used an ion-exchange resin in the acid form plus a small proportion of acid to hydrolyze g l y c o p r ~ t e i n s . ~ ~ ~ ~ ~ Lehnhardt and WinzlerS0used 0.1-3.0 mg of glycoprotein with 100 p L of a 20% suspension of Dowex 50 X-2 (H+)ion-exchange resin (200-400 mesh) in 0.02 M hydrochloric acid for up to 40 h on a steam bath. This (49) H. Takemoto, S. Hase, and T. Ikenaka, Anal. Biochem., 145 (1985) 245-250. (50) W. F. Lehnhardt and R. J. Winzler, J . Chromatogr., 34 (1968) 471-479. (51) W. H. Porter, Anal. Biochem., 63 (1975) 27-43.
CLEAVAGES OF POLYSACCHARIDE GLYCOSIDIC LINKAGES
269
method, compared to hydrolysis with 0.5 M H2S04 on a steam bath gave much less decomposition of the neutral monosaccharides liberated from orosomucoid. Because mannose was liberated much more slowly than galactose or fucose in both methods, there was some decomposition of them in the case of hydrolysis with sulfuric acid before all of the mannose had been liberated. Hydrolysis with the ion-exchange resin gave complete hydrolysis after 20 h, whereas hydrolysis with sulfuric acid gave complete hydrolysis after 10 h. Porters1 also used this method of hydrolysis with resin in his study of neutral and amino sugars of alpha-1-acid-glycoprotein and bovine luteinizing hormone. 4. D-Fructans
Polymers of D-fructose are important carbohydrate reserves in a number of plants. Inulins and levans are two major types that differ in structure. D-Fructans require only relatively mild conditions for their hydrolysis, for example, levan was qualitatively hydrolyzed5Ia by hot, dilute, aqueous oxalic acid. Permethylated fructans could be hydrolyzeds2 with 2 M CF3C02H for 30 min at 60". Fructan oligosaccharides were hydrolyzed in dilute sulfuric acid (pH 2) at 70" (see Ref. 53) ors4 95" (0.1 M). D-Fructans from timothy haplocorm (where they comprise 63% of the water-soluble carbohydrates) could be hydrolyzeds5 with 0.01 M hydrochloric acid at 98".
V. FORMOLYSIS A N D ACETOLYSIS Formolysis and acetolysis are not common methods for cleavage of glycosidic linkages. They do have some unique applications, however. For instance, methylated polysaccharides are not generally soluble in hot water, and consequently, hydrolysis is best preceded by formolysis under these circumstance^.'^ For example,565 mg of methylated polysaccharide is dissolved in 3 mL of 90%formic acid, and the solution is kept for 2 h at 100". The formic acid is removed by evaporation at 40". The residue is dissolved in 1 mL of 250 mM sulfuric acid and the solution is heated for 12 h at loo", cooled, the acid neutralized with barium carbonate, the (5la) H . Hibbert and R. S. Tipson, J . Am. Chem. Soc., 52 (1930) 2582; H. Hibbert, R. S. Tipson, and F. Brauns, Can. J . Res., 4 (1931) 221-239. (52) C. J. Pollock, M. A. Hall, and D. P. Roberts, J . Chromatogr., 171 (1979) 411-415. (53) A. Heyraud, M. Rinaudo, and F. R. Taravel, Carbohydr. Res., 128 (1984) 311-320. (54) D. D. Wolf and T. L. Ellmore, Crop Sci., 15 (1975) 775-777. (55) M. B. Suzuki, Cun. J . Bor., 46 (1968) 1201-1206. (56) B. Lindberg, Merhods Enzymol., 28 (1972) 178-195.
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CHRISTOPHER J. BIERMANN
suspension filtered, and the filtrate concentrated by evaporation at 40" The formolysis procedure has also been used in the study of gangliosides .49 Acetolysis (using sulfuric acid as a catalyst) with 10 : 10 : 1 acetic anhydride-acetic acid-sulfuric acid for 2-13 h at 40" has been used for selective cleavage of glycosidic linkages in yeast mannan~.~' Selective cleavage of (1 + 6) linkages resulted in mixtures of relatively stable, acetylated oligosaccharides containing (1 + 2) and (1 + 3) linkages. The composition of the oligosaccharide mixture is characteristic of the strain of yeast that is the source of the mannan. Acetolysis (using acetic anhydride and sulfuric acid) has also been employed to form the acetates of cellobiose, cellotriose, and higher cello-oligosaccharides from cellulose, but this reaction is not an analytical method.j8 The applications of acetolysis and of formolysis for hydrolysis of glycopeptides have been compared.59 Both methods, used together, were recommended for analysis of samples containing both neutral and aminodeoxyhexitols.
HYDROLYSIS VI. ENZYMIC Enzymic hydrolysis is a useful tool for the identification of carbohydrate linkages,sc as well as for hydrolysis of the (labile) sialic acids.8 Neesefl developed a method wherein the sialic acids are enzymically hydrolyzed and, simultaneously, enzymically converted into stable 2-amino-2-deoxymannose derivatives. This allows determination of carbohydrate constituents of glycoproteins in a single flask. A number of selective glycosidases are now commercially available (see, for example, Ref. 6). For glycoconjugates containing oligosaccharides that are not released under alkaline conditions, enzymic liberation is possible; the details have been given.5cThe fundamental approach is as follows. Proteolytic digestion is first used, followed by gel filtration to separate the oligosaccharides from the free amino acids and short peptides. Exo-glycosidases may then be used to hydrolyze terminal sugar units from the oligo- or poly-saccharides, in order to elucidate the structure. It is important that the enzymes be pure, so that erroneous results For relatively are not obtained; this had been a problem in the simple oligosaccharides, the use of a few exo-glycosidases soon leaves a compound composed of a single monosaccharide linked to an amino acid or short peptide; this constitutes the carbohydrate-peptide linkage. For (57) T. S. Stewart and C. E. Ballou, Biochemistry, 7 (1968) 1855-1863. (58) M. L. Wolfrom and A. Thompson, Methods Carbohydr. Chem., 3 (1963) 143-150. (59) J. Conchie, A. J. Hay, and J . A. Lomax, Carbohydr. Res., 103 (1982) 129-132.
CLEAVAGES OF POLYSACCHARIDE GLYCOSIDIC LINKAGES
27 I
more information on determination of structure by use of enzymes, an excellent article is available.60
VII. REDUCTIVECLEAVAGE One drawback of methylation analysis of polysaccharides is that the carbon atom involved in the cyclic acetal of a particular monosaccharide is not distinguished from linked positions after hydrolysis of the permethylated polysaccharide. For example, a 4-linked aldohexopyranose gives the same methylation product after acid hydrolysis as a 5-linked aldohexofuranose. By the application of a method that cleaves the glycosidic linkage with the addition of hydride, instead of water, reduction of the anomeric carbon atom is achieved while still maintaining the ring ~ t r u c t u r e . ~If' - this ~ ~ method is applied after permethylation, the linkage position is unequivocally identified. Added advantages of this method are that the anhydroalditol that is generated is stable and, for aldoses, a mixture of anomers is not formed, as there is no chirality at the formerly anomeric carbon atom; however, this means that the configuration of the linkages cannot be determined. The reductive-cleavage step is accomplished with boron trifluoride or trimethylsilyl trifluoromethanesulfonateassisted organosilane reduction. This method has already been applied to structural determination of several complex p o l y ~ a c c h a r i d e s ~and ~-~~ promises to become important. (60)B. V. McCleary and N . K. Matheson, Adv. Curbohydr. Chem. Biochem., 44 (1986) 147-276. (61) D. Rolf and G . R. Gray, J . A m . Chem. Soc., 104 (1982) 3539-3541. (62) D. Rolf and G . R. Gray, Curbohydr. R e s . , 152 (1986) 343-349. (63) J. A. Bennek, M. J . Rice, and G . R . Gray, Curbohydr. Res., 157 (1986) 125-137.
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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 46
AQUEOUS, HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES RELATIVE TO UTILIZATION OF BIOMASS
BY OLOFTHEANDER Swedish University of Agricultural Sciences, S-75007 Uppsala, Sweden
AND
DAVIDA. NELSON
PaciJc Northwest Laboratory, Richland, Washington 99352
I. Introduction. ........................................................... 11. Transformation of Monomeric Saccharides ........................... 1. Aldopentoses, Aldotetroses, and Aldotrioses ..... ............ 2. Hexoses and Alduronic Acids ......................................... 111. Transformation of Polysaccharides I . Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.......................
........................
3. Hemicelluloses and Glycuronans ....................................... IV. Carbohydrate Transformation in the Presence of Amino Compounds . . . . . . . . . V. Carbohydrate Transformation in Chemical Processes, Including Humus Formation. .............................................................
273 275 275 284 295 295 297 305 307 323
I. INTRODUCTION Over the past two decades, considerable interest has been directed toward the conversion of cellulosic biomass (such materials as wood wastes, bagasse, and straw) into useful products, notably fuels. Several procedures, including fermentation, gasification, liquefaction, and pyrolysis, have been commercially applied to carbohydrates with various degrees of success.' In order to use the polysaccharides present in lignocellulosic materials as a substrate in fermentation processes, pretreatments are necessary, such as with steam (under slightly acid conditions) or (1) P. M. Molton and T. F. Demmit, Polym. Plast. Technol. Eng., 11 (1978) 127-157.
273
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
274
OLOF THEANDER AND DAVID A. NELSON
alkali, followed by acid or enzymic hydrolysis. It is difficult to avoid some unwanted carbohydrate transformations in these pretreatments. Gasification is a rather direct and specific process. Liquefaction (hydrothermolysis) and pyrolysis2 are not so specific, as the mixtures obtained by these processes indicate that a complex series of mechanisms is involved. These two thermolytic processes appear related, because a significant number of their products are similar. The mechanism of pyrolysis has received more attention, because of the fundamental work of Shafizadeh3v4and B r ~ i d oHowever, .~ the routes to many of the similar products are not necessarily the same, because of the role of the aqueous solution in liquefaction. Thus, liquefaction may be regarded as a modification of the process of pyrolysis, but the lower temperatures and aqueous conditions present a chemistry rich in overlapping mechanisms and underlying confusion. A modification of the pyrolysis process, developed by Hoppe-Seylefi in 1871, involved the addition of water and alkali to biomass which was converted into oil, gas, water-soluble components, and carbonaceous material.'-" The addition of carbon monoxide and hydrogen in the liquefaction process allowed the production of liquid fuels from Asphalt substitutes have also been prepared from biomass under liquefaction conditions. I8 The research examined in this article will be confined to generally irre(2) P. Tomasik, M. Palasinski, and S. Wiejak, Adv. Carbohydr. Chem. Biochem., in preparation. (3) F. Shafizadeh, Adv. Carbohydr. Chem., 23 (1968) 419-474. (4) F. Shafizadeh and Y. L. Fu, Carbohydr. Res., 29 (1973) 113-122. (5) A. Broido, A. C. Javier-Son, A. C. Quano, and E. M. Barrall, J . Appl. Polym. Sci., 17 (1973) 3627-3635. (6) F. Hoppe-Seyler, Ber., 4 (1871) 15-16. (7) E. Berl and A. Schmidt, Justus Liebigs Ann. Chem., 461 (1928) 192-220. (8) E . Berl, A. Schmidt, and H. Koch, Angew. Chem., 43 (1930) 1018-1019. (9) E. Bed and A. Schmidt, Justus Liebigs Ann. Chem., 493 (1932) 97-123. (10) E. Bed and A. Schmidt, Justus Liebigs Ann. Chem., 496 (1932) 283-303. (11) F. Bergius, Min. J . (London) 163 (1928) 1067-1068. (12) A. H. Weiss, Text. Res. J . , 42 (1972) 526-533. (13) D. V. Gupta, W. L . Kranich, and A. H. Weiss, Znd. Eng. Chem. Process Des. Deu., 15 (1976) 256-260. (14) H. R. Appell, Y. L. Fu, S. Friedman, P. Yavorsky, and I. Wender, U . S . NTZS PB Rep., 1971, No. 203,669, 24 pp. (15) J. C. Cavalier and E. Chornet, Fuel, 56 (1977) 57-64. (16) H. I. Waterman and F. Kortlandt, Recl. Trav. Chim. Pays-Bas, 43 (1924) 691-701. (17) D. C. Elliott, in D. L. Klass and G. H. Emert (Eds.), Fuels from Biomass and Wastes, Ann Arbor Publishers, Ann Arbor, Michigan, 1981, pp. 435-449. (18) J. Donovan, R. Miller, T. Batter, and R. Lottman, EPA Rep. 6092-810242, 1981, NTZS PB 82-1 19082.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
275
versible transformations occurring in aqueous systems at various pH values, at or above 100". The effect of oxidative conditions will usually not be discussed. Most of the processes of the pulping industry will be excluded, except those involving formation of products of low molecular weight. However, reports concerning the combination of carbohydrates with amino acids or amines will be discussed, as these nitrogen compounds may catalyze tautomerization, fragmentations, and certain rearrangements. Furthermore, amino acids or proteins are always present to some extent in all plant raw-materials.
11. TRANSFORMATION OF MONOMERIC SACCHARIDES 1. Aldopentoses, Aldotetroses, and Aldotrioses a. Acidic Conditions.-All aldopentoses form 2-furaldehyde in high yield when exposed to aqueous acid solution at elevated temperature. Such high yields are obtained only if the furaldehyde is removed, usually by distillation, as fast as it is formed.19 D-Xylose is the most effective of the pentoses,20as it can form a nearly quantitative yield of 2-furaldehyde. It is not completely clear why D-xylose has this enhanced ability compared to the other pentoses. However, the stereochemistry of the pentose and other competitive degradation routes apparently play significant roles in the yield of 2-furaldehyde. It has also been reported that 2,5-anhydro-~arabinose readily provides furaldehyde upon warming in an acidic solution.21 The mechanism of pentose dehydration has been a matter of study for several years. The accepted pathway (see Scheme 1) to 2-furaldehyde from a pentose, in this case D-xylose (l),involves the reversible formation of a l ,2-enediol(2) followed by dehydration to the enolic form (3)of a 3-deoxypentosulose, which is further dehydrated to the 3,4-dideoxypent3-enos-2-ulose (4) prior to cyclization22to afford 2-furaldehyde 5. This mechanism, initially suggested by I ~ b e 1 1has , ~ ~been substantiated by later This confirmation required incorporation of deuterium24or trit i u ~ n *into ~ the furaldehyde at various ring positions. However, when (19) C. D. Hurd and L. L. Isenhour, J . A m . Chem. SOC.,54 (1932) 317-330. (20) R. W. Scott, W. E. Moore, M. J. Effland, and M. A. Millett, AnalBiochem., 21 (1967) 68-80. (21) M. Cifonelli, J. A. Cifonelli, R. Montgomery, and F. Smith, J . Am. Chem. Soc., 77 (1955) 121-125. (22) M. S. Feather and J . F. Harris, Adu. Carbohydr. Chem. Biochem., 28 (1973) 161-237. (23) H. S. Isbell, J . Res. Natl. Bur. Stand., 32 (1944) 45-59. (24) M. S. Feather, Tetrahedon Lett., (1970) 4143-4145. (25) M. S . Feather, D. W. Harris, and S. B. Nichols, J . Org Chem., 37 (1972) 1606-1608.
276
OLOF THEANDER AND DAVID A. NELSON
D-xylose was converted into 2-furaldehyde in acidified, tritiated water, no carbon-bound isotope was detected. This suggested that the 1,2-enediol (2) reacted immediately, as otherwise, tritium would have been detected at the aldehydic carbon atom of 2-furaldehyde, as a result of aldoseketose interconversion.26 An acidic dehydration performed with ~ - [ 2 3H]xylose showed that an intramolecular C-2-C- 1 hydrogen transfer had actually occurred.26 Thus, these data indicated that an intramolecular hydride shift is more probable than the previously accepted step involving a 1,2-enediol intermediate.
1
2
3
4
SCHEMEI .-Formation of 2-Furaldehyde from D-Xylose.
Although small proportions of other products are formed when D-XYlose is exposed to rather high acid concentrations, arabinose, lyxose, and ribose form considerably more of alternative products (generally reductic acid) than of 2-furaldehyde under these conditions.20Reductic acid (2,3dihydroxy-2-cyclopenten- 1-one, 47) has been detected as a product after acid exposure of ~ - x y l o s or e ~its ~ major dehydration product, 2-furaldehyde.2xFurther work performed with D-[ l-14C]xyloseand [a-I4C]2-furaldehyde showed that reductic acid having identical label distribution was obtained from both starting materials.29This indicated that a common primary source was involved, probably 2-furaldehyde, as it is readily formed from D-xylose under acidic conditions. D-Xylose and D-arabinose have been treated with a 0.5M acetate buffer (pH 4.5) at re flu^.^^ Besides 2-furaldehyde, some catechols and unique chromones were isolated from the reaction mixture in small proportions. These included 3,8-dihydroxy-2-methylchromone and its precursor, 5,6,7,8-tetrahydro-3,5-dihydroxy-2-methyl-8-oxochromone. A trihydroxy-2-methylchromone was also isolated from the D-xylose reac(26) (27) (28) (29) (30)
D. W. Harris and M. S. Feather, J . Org. Chem., 39 (1974) 724-725. T. Reichstein and R. Oppenauer, Helu. Chim. Acta, 16 (1934) 390-396. Dutch Pat. 61,296 (1948); Chem. Abstr., 42 (1948) 7788. M. S. Feather, J . Org. Chem., 34 (1969) 1998-1999. T. Popoff and 0. Theander, Carbohydr. Res., 27 (1972) 135-149.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
277
tion mixture. A preparative-scale technique using liquid chromatography has been developed for the isolation of 3,8-dihydroxy-2-methylchromone from the D-xylose reaction-mi~ture.~' Several low-molecular-weight aldehydes (formaldehyde, acetaldehyde, and 2-butenal) have also been isolated from D-xylose after acid treatment.32 Both ~-[l-'~C]xylose and ~-[5-'~C]arabinose were exposed to a concen1-Hydroxy-2-propanone (acetrated phosphate buffer solution (pH 6.7).33 tol) was distilled from the heated solution. Radioassay indicated that similar labeling [3-14C]occurred in the acetol from both pentoses, with loss of the configurational difference; thus, a 3-ketopentose or its enediol was suggested as an intermediate. Further work with 3-0- and 6-O-methyl-~glucose and with 1-0-methyl-D-fructose indicated that p-elimination from a 3-ketose or, in the case of a hexose, from a 3-ketose or a 4-ketose, or both, tautomerization of the resulting a-diketone to a P-diketone, and hydrolytic cleavage are essential steps34in the formation of acetol. A bicyclic y-pyranone was isolated in -1% yield from an acidified, refluxed solution of D-erythrose (6).35It was proposed that the compound, 4a,5,6,7a-tetrahydrocyclopenta[b]pyran-4,7-dione (lo), is formed (by way of 9) from two molecules of D-erythrose, namely, an enediol (8) and a dehydrated form (7). Feather and Harris22pointed out that D-erythrose would lose two molecules of water to form an intermediate (7) prior to coupling with the enediol 8 (see Scheme 2) to give 9. D-Erythrose has also been exposed to a boiling solution in pH 4.5 buffer.36Low yields (<0.2%) of a number of products were obtained, as shown in Scheme 3. These included 5-(hydroxymethyl)-2-furaldehyde (ll),2-acetyl-5-(hydroxymethyl)furan (l2),3,4-dihydroxyacetophenone (13), 3,4-dihydroxybenzaldehyde(14), 3,4-dihydroxybenzoic acid (1% 2,3-dihydroxytoluene (16), and 1,2-benzenediol (pyrocatechol) (17). Also detected were formic, hydroxyacetic, and 3-hydroxypropanoic acids. Pyrocatechol seems to be a product formed from all carbohydrates boiled in aqueous solutions at pH 4-10; it may constitute a statistical product arising from retro-aldol and re-aldol reactions. It has been shown3' that the aldol reaction may operate at a pH as low as 4. An aldol reaction G. Lindgren and P.-A. Pernemalm, J . Liq. Chromatogr., 3 (1980) 1737-1742. F. A. H. Rice and L. Fishbein, J . A m . Chem. Soc., 78 (1956) 1005-1009. J. Hayami, Bull. Chem. Soc. Jpn., 34 (1961) 924-926. J. Hayami, Bull. Chem. Soc. Jpn., 34 (1961) 927-932. F. Catala, J . Defaye, P. Laszl6, and E. Lederer, Bull. Soc. Chim. Fr.. (1964) 31823187. (36) 0. Theander and E. Westerlund, Acta Chem. Scund., Ser. 8, 34 (1980) 701-705. (37) D. A. Nelson, P. M. Molton, J. A. Russell, and R. T. Hallen, Ind. Eng. Chem. Product Res. Dev., 23 (1984) 471-475. (31) (32) (33) (34) (35)
OLOF THEANDER AND DAVID A. NELSON
278
CHO
I I
c=o
CH
CHZ
(iHO
7
:
HCOH
I
~
t
HCOH
I CH2OH
CHO
-
CHOH
-3H2,0
~
HCOH
I
c=o
II
I
0
HCOH
!OH
I
I HCOH
6
I II CH 1 fH2
COH
10
CHZOH
I
9
CH2OH
8 SCHEME 2.-Formation of a y-Pyranone from D-Erythrose.
between 2-hydroxyacetaldehyde, also present in the D-erythrose reaction mixture, and D-erythrose could yield a hexose, which might lead to the 5(hydroxymethyl)-2-furaldehyde, as well as to catechol. Glyceraldehyde (~~-2,3-dihydroxypropanal) is readily dehydrated to pyruvaldehyde (2-oxopropanal) in aqueous solutions of hydrochloric or CH3
CHO
I @ C=O
I
HCOH I HCOH
I
H O H 2 C e ! C H0 s
+ H o H 2 c ~ C H o
t
CH2OH
6
OH OH
12
11
w COzH
+
OH 14
&
t
&OH
OH
15 16 SCHEME 3.-Formation of Aromatics from D-Erythrose at pH 4.5.
17
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
279
acetic acid.38 This work also indicated that a true equilibrium does not exist during the isomerization between 1,3-dihydroxy-2-propanone(dihydroxyacetone) and glyceraldehyde. The concentration of dihydroxyacetone decreased more rapidly than that of glyceraldehyde with time, and several unidentified components appeared. Dihydroxyacetone was exposed to boiling aqueous pH 4.5 buffer; the products resulting were considerably more complex than those attribut. ~ ~yields of the twenty compounds isolated able to simple d e h y d r a t i ~ nThe from the mixture ranged from 0.03 to 1.2%. The major products of this and erythro- and threoacidic reaction were 3-hydroxy-2,5-hexanedione, 3,4-dihydroxy-2,5-hexanedione. These apparent reduction products may have been formed by an aldol/retro-aldol/re-aldol sequence involving acetol.
PH
18
PH
16
19
17
OH
20
SCHEME 4.-Formation
21
22
of Aromatics from Dihydroxyacetone in Acid Solution.
The reaction mixture from acidified dihydroxyacetone also included a series of seven di- and tri-hydroxybenzenes (see Scheme 4), namely, 2,3dihydroxytoluene (16), pyrocatechol(17), 3,4-dihydroxytoluene (19), 3 3 dimethyl-l,2-benzenediol(20), 1,2,3-trihydroxybenzene (pyrogallol) (21), 1,2,4-trihydroxybenzene (22), 3,4,5,6-tetrahydroxy-2-methyl-acetophenone, and 2,3-dihydroxy-5,6-dimethyl-p-benzoquinone. Many of these same phenolic compounds are present after hexoses are similarly treated (38) M. Fedorodko and J. Konigstein, Collect. Czech. Chem. Cornmun., 34 (1969) 38813894. (39) T . Popoff, 0. Theander, and E . Westerlund, Acta Chem. Scand., Ser. B , 32 (1978) 1-7.
280
OLOF THEANDER AND DAVID A. NELSON
with acid.'"' Also isolated was a series of four pyranones: 3-hydroxy-6methyl-4H-pyran-4-one (26), 3-hydroxy-2-methyl-4H-pyran-4-one, 3-hydroxy-5-methyl-4H-pyran-4-one, and 3-hydroxy-2,6-dimethyl-4H-pyran4-one. Tentative mechanisms involving cyclization and dehydration were proposed for the formation of the pyranones. Scheme 5 presents an example of the formation of the 3-hydroxy-6-methyl isomer (26).
25
SCHEME 5.-Formation
26
of 3-Hydroxy-6-methyl-4H-pyran-4-one from Dihydroxyace-
tone.
Two hydroxydimethylpyranopyrandiones were also detected. The small proportions of these bicyclic compounds did not allow determination of the exact coupling-position of the pyran rings; that is, [2,3-b] or [3,2-b]. However, the comparatively large proportion of 3-hydroxypyrones in the reaction mixture suggested the [3,2-b], as shown in formulas 27 and 28.
27
28
Substituted acetophenones and a benzofuran were also isolated. The acetophenones may arise in a manner similar to that noted for 2,3-dihydroxyphenyl methyl ketone (2,3-dihydroxyacetophenone)obtained from D-glucuronic acid.30 (40) T. Popoff and 0. Theander, Acta Chem. Scand., Ser. B , 30 (1976)397-402.
28 I
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
b. Basic Conditions.-Although it is quite difficult to separate the various products (and mechanisms) of carbohydrate transformations under acidic or basic conditions, pH 7 was set as an obvious partition value to provide an orderly approach to the research. Aldoses generally undergo benzilic acid-type rearrangements to produce saccharinic acids, as well as reverse aldol (retro-aldol) reactions with /?-elimination, to afford a-dicarbonyl compounds. The products of these reactions are in considerable evidence at elevated temperatures. The conversions of ketoses and alduronic acids, however, are also of definite interest and will be emphasized as well. Furthermore, aldoses undergo anomerization and aldose-ketose isomerization (the Lobry de Bruyn-Alberda van Ekenstein transformation4’) in aqueous base. However, both of these isomerizations are more appropriately studied at room temperature, and will be considered only in the context of other mechanisms. Saccharinic acid formation has been studied for several year^.^^,^^ The four-step reaction proceeds rapidly in alkaline solution because of basic catalysis, particularly in the last two steps. Initially formed is an enediol that can undergo /?-elimination of a functional group, usually a hydroxyl group. The final two steps involve tautomerization to an a,/?-dicarbonyl intermediate followed by a benzilic acid rearrangement. This sequence is shown in Scheme 6 for the formation of the “a-” and “/?”-xylometasaccharinic acids (30) by way of 3-deoxy-~-glycero-pentos-2-ulose (29). CHO
I
HCOH
I I HCOH I CH2OH
HOCH
1
CHOH
-
II COH I
HOCH
1
HCOH
I
CH2OH
2
- CHO
I c=o IH2
HCOH
I
CH2OH
29
CO2H
I I CH2 I HCOH
CHOH
I
CH,OH
30
SCHEME6.-Formation of “a” and “p” Xylornetasaccharinic Acids from D-Xylose in Base.
Five pentosaccharinic acids were formed when D-xylose was treated with calcium hydroxide.43This mixture included 2-C-methyl-~-threonic acid and 2-C-methyl-~-erythronic acid, the structures of which were (41) J. C. Speck, Jr., Adu. Carbohydr. Chem., 13 (1958) 63-103. (42) J. C. Sowden, Adu. Carbohydr. Chem., 12 (1957) 35-79. (43) A. Ishizu, B. Lindberg, and 0. Theander, Acta Chem. Scand., 21 (1967) 424-432.
282
OLOF THEANDER AND DAVID A. NELSON
finally determined. Several C4- and C6-saccharinic acids were also isolated when either D-xylose or D-fructose was exposed to aqueous calcium hydroxide. This result suggested that considerable fragmentation and recombination had occurred. Of considerable interest was the lack of “a”and “p”-D-xylosaccharinic acids in the mixture when M sodium hydroxide was used instead of calcium hydroxide. ~-ribo-Hexos-3-ulosehas also been exposed to both aqueous sodium hydroxide and calcium hydroxide.44The products, principally 3-deoxy-~-threo-pentonicacid, varied depending upon the alkali cation. Calcium ion has also been shown to have a definite effect upon the benzilic acid rearrangen~ent.~~ Because a benzilic acid rearrangement is the last step in the formation of saccharinic acids4*the difference between these results could be due to complexation of hydroxyl groups and alkoxide anions by the divalent calcium ions. Such a complexation apparently promotes isomerization to the xylosaccharinic acid. and, hence, a fragmentation-re~ombination~~ This route has been q u e ~ t i o n e dand , ~ ~yet it provides a rational explanation of the results. ~-[l-’~C]Xylose was subjected to boiling in 4 M aqueous sodium hyd r o ~ i d eThe . ~ ~ resulting mixture contained 2,4-dihydroxybutanoic acid, lactic acid, and D-a,P-xylometasaccharinicacid. The almost uniform distribution of the 14C label among the carbon atoms of 2,4-dihydroxybutanoic acid indicated that this acid is probably formed by the recombination of completely isomerized, two-carbon fragments. Fragmentation of Dxylose occurred mainly at one of the central bonds, C-2-C-3 or C-3-C-4. CH3
I
OH
32
31
A solution of D-xylose in boiling 0.63 M sodium hydroxide gave interesting results49compared with those from a similar reaction30conducted (44) (45) (46) (47)
H. P. Humphries and 0 . Theander, Acta Chem. Scand., 25 (1971) 883-888. D. O’Meara and G. N. Richards, J . Chem. Soc., (1960) 1944-1945. J. Kenner and G. N. Richards, J . Chem. Soc., (1954) 1784-1789. J. C. Sowden, M. G. Blair, and D. J . Kuenne, J . A m . Chem. Soc., 79 (1957) 6450-
6454. (48) J. F. Hams, Carbohydr. Res., 23 (1972) 207-215. (49) I. Forsskbhl, T. Popoff, and 0 . Theander, Carbohydr. Res., 48 (1976) 13-21.
HIGH-TEMPERATURE TRANSFORMATION O F CARBOHYDRATES
283
at pH 4.5. The yield of the products identified was much lower in the basic reaction, and several compounds found at lower pH were not present. Pyrocatechol and four methylated benzenediols (16,19,31, and 32) were isolated from the basic reaction, as well as five acetophenones and a benzaldehyde. It was suggested that 32 might arise from the condensation of butanedione (biacetyl) with pyruvaldehyde. The instability of the catechols is significant at pH >7. Two compounds having strong, caramel-like 1-one odors were isolated; these were 2-hydroxy-3-methyl-2-cyclopenten(33) and 2-hydroxy-3,4-dimethyl-2-cyclopenten-l-one (34). The statistically similar amounts of 33 and 34 present in the alkaline reaction mixtures of D-glucose and D-xylose suggested that fragmentation, probably retro-aldolization, occurs, followed by a recombination of various two-, three-, or four-carbon fragments. Pentoses have also been shown to produce pyrocatechol and 33 in a dilute solution of sodium hydroxide.50Also isolated were five acetophenones, including 13, having methyl-, hydroxy-, dihydroxy-, or trihydroxyphenyl moieties.49
33
34
D-Erythrose undergoes self-aldolization in alkali solution, to form Dgluco-~-glycero-3-octulopyranose by combination of the I ,Zenediol and aldehydo forms.51In weak alkali at 105", syrupy D-erythrose yields Dglycero-tetrulose, P-D-altro-L-glycero-3-octulofuranose, and a-D-gluco-Lgly~ero-3-octulopyranose.~~ At 300" in alkali, the major products from syrupy D-erythrose were 1-5% of butanedione (biacetyl) with smaller proportions of pyrocatechol, 33, 2,5-dimethyl-2,5-cyclohexadiene-1,4dione (2,5-dimethylbenzoquinone), and 2,5-dimethyl-l ,Cbenzenediol (2,5-dimethyIhydroq~inone).~' It was assumed that D-erythrose is reduced to erythritol by a Cannizzaro type of reaction, followed by dehydration of erythritol to form biacetyl. However, very low proportions (<1%) of biacetyl are formed from erythritol compared with D-erythrose itself. Apparently, some other mechanism predominates in the formation of biacetyl. (50) T. Enkvist, Fin. Kern. Medd., 71 (1962) 104-108. (51) R. Schaffer and A. Cohen, J . Org. Chem., 28 (1963) 1929-1930. (52) E. Westerlund, Curbohydr. Res., 91 (1981) 21-30.
284
OLOF THEANDER AND DAVID A. NELSON
Glyceraldehyde (2,3-dihydroxypropanal), acetol, and dihydroxyacetone form 1-5% of biacetyl and a number of other product^,^' including pyrocatechol and 33, after exposure to aqueous alkali at 300". Such trioses as glyceraldehyde and dihydroxyacetone have been shown to form various hexoses by aldol reaction.53Aldolization, followed by retro-aldolization, is undoubtedly a major consideration when three-, four-, and fivecarbon sugars are subjected to elevated temperatures. Differences in thermolysis products, partially quantitative, are noticeable at loo", but, at temperatures near 300", it is quite difficult, if not impossible, to determine if the starting material was a triose, a tetrose, or a pentose. 2. Hexoses and Alduronic Acids a. Acidic Conditions.-Hexoses exposed to acidic conditions produce S-(hydroxymethyl)-2-furaldehyde (11)as the major product.54The rate of formation of 5-(hydroxymethyl)-2-furaldehyde varies considerably among the hexoses. For instance, in aqueous sulfuric acid (2 Mat loo"), the order of reactivity is D-gulose > D-talose > D-mannose > D-galactose > Dglucose > D-altrose. Furthermore, the formation of 11from D-fructose is much faster and of greater yield compared to that from ~ - g l u c o s e . * ~ ~ ~ ~ Initial attempts to use this information in a reasonable mechanism required that the furanose ring of D-fructose remain intact followed by elimination of water to afford the 1,2-enolic form of 2,5-anhydro-~-mannose. Further dehydration and cyclization gave S-(hydroxymethyl)-2-furaldehyde. This mechanism required that D-glucose isomerize to D-fructose prior to entering into the scheme. It was this isomerization that accounted for a lower rate and lower yield. Feather and Harris22properly indicated that 2,5-anhydro-~-mannoseis less reactive than D-fructose and that it has yet to be detected in the reaction mixture. They suggested that the reaction probably proceeds through the formation of acyclic enediols. A prior reporP proposed that 3-deoxyhexosuloses participate in the formation of 11(see Scheme 7). The 3-deoxyhexosuloses were isolated from heated, acid reaction mixtures involving D-fructose and L-sorbose. It was suggested that 3-deoxyhexos-2-ulose (39) underwent reversible equilibrium with 38, its cis form, and was further dehydrated at C-3 and C-4, to form 3,4-dideoxyhex-3-enos-2-ulose (40).This intermediate cyclized to the furaldehyde. Experiments involving treatment of both (53) C. D. Gutsche, D. Redmore, R. S. Bunks, K. Nowotny, H. Grassner, and C. W. Armbruster, J . Am. Chem. SOC., 89 (1967) 1235-1245. (54) F. H. Newth, Adu. Carbohydr. Chem., 6 (1951) 83-106. (55) C. J. Moye and Z. S. Krzeminski, A m ? . J. Chem., 16 (1963) 258-269. (56) E. F. L. J . Anet, Ausr. J. Chem., 18 (1965) 240-248.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
285
HpH CWH 35
J "
36
d 7
HCOH
CWH
J
WH 39
38
H'OH 11
lWH 40
(
cishrans )
SCHEME7.-Formation of 5-(Hydroxymethyl)-2-furaldehyde from D-GhCOSe.
D-glucose and D-fructose in acidified deuterium oxide, and acid conversion of ~-[2-~H]glucose were conducted, in order to determine the importance of 39 as an intermediate from the proportion of deuterium incorporated at C-3 of 5-(hydroxymethy1)-2-f~raldehyde.~~*~~ However, the 2-furaldehyde formed in the reactions contained no deuterium. Thus, an essentially irreversible sequence that involves hexose, 36,38,40, and 11 best explains the acid-catalyzed, dehydration reaction. (57) M. S. Feather and J. F. Hams, Tetrahedron Lert., (1968) 5807-5810. (58) M. S. Feather and J . F. Harris, Carbohydr. Res., 15 (1970) 304-309.
286
OLOF THEANDER AND DAVID A. NELSON
Thermolysis of D-fructose in acid solution provides 11and 2-(2-hydroxyacety1)furan (44) as major products.s9 Earlier work had established the presence of 44 in the product mixtures obtained after acid-catalyzed dehydrations of D-glucose and sucrose.M)Eleven other products were identified in the D-fructose reacti~n-mixture,~~ including formic acid, acetic acid, 2-furaldehyde, levulinic acid, 2-acetyl-3-hydroxyfuran (isomaltol), and 4-hydroxy-2-(hydroxymethyl)-5-methyl-3(2~)-furanone(59). Acetic acid and formic acid can be formed by an acid-catalyzed decomposition of 2-acetyl-3-hydroxyf~ran,~~ whereas levulinic acid is a degradation produ d 2 of 11.2,3-Dihydro-3,5-dihydroxy-6-methyI-4H-pyran-4-one has also been isolated after acid treatment of ~ - f r u c t o s e .The ~ ~ *pyranone ~ is a dehydration product of the pyranose form of I-deoxy-~-erythro-2,3hexodiulose.22In aqueous acid seems to be the major reaction product of the pyranone. The formation of 2-(2-hydroxyacetyl)furan is analogous to the mechanism proposed for furaldehydes from h e x o s e ~ Scheme .~~ 8 shows this mechanism. This reaction was performed in tritiated water ( M H2S04) in order to evaluate the proposed mechanism and its requisite equilibrations." Presuming that the hydroxymethyl carbon atom of 44 corresponds to C-1 of the sugar (in this case, D-glucose), the results indicated that one of the carbon-bound hydrogen atoms of CH20Hwas from an intramolecular source and not from the solvent; this was confirmed by subjecting D[2-3H]glucoseto the same conditions. The position of the specific activity (CHTOH) indicated that the tritium had migrated from C-2 of D-glucose to the carbon atom corresponding to C-1 during the dehydration. The logical step for occurrence of this transfer is during the isomerization of D-glucose to D-fructose; that is, transfer of a hydrogen atom from C-2 of D-glucose to C-1 of ~ - f r u c t o s eThe . ~ ~intramolecular transfer in the conversion of D-glucose into 44 is nearly complete at 100" in M H2S04. The interconversion of aldoses and ketoses by general acid-base catalysis, the Alberda van Eckenstein-Lobry de Bruyn tran~formation,~~ which involves 1,2-enediols, apparently does not apply to this furan formation in
(59) P. E. Shaw, J. H. Tatum, and R. E. Berry, Carbohydr. Res., 5 (1967) 266-273. (60) R. E. Miller and S. M. Cantor, J . Am. Chem. SOC.,74 (1952) 5236-5237. (61) J. E. Hodge and E. C. Nelson, Cereal Chem., 38 (1961) 207. (62) T. M. Reynolds, Adu. Food Res., 12 (1963) 1-52. (63) J. H. Tatum, P. E. Shaw, and R. E. Berry, J . Agric. Food Chem., 15 (1967) 773-775. (64) P. E. Shaw, J . H. Tatum, and R. E. Berry, Carbohydr. Res., 16 (1971) 207-211. (65) E. F. L. J. Anet, Adu. Carbohydr. Chem., 19 (1964) 181-218. (66) D. W . Hams and M. S. Feather, Tetrahedron Lett., (1972) 4813-4816. (67) D. W. Hams and M. S. Feather, Carbohydr. Res., 30 (1973) 359-365.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
I
287
44
CWH 43
SCHEME 8.-Formation of 2-(2-Hydroxyacetyl)furanfrom D-Fructose.
strong acid. The labeling data are more consistent with a concerted mechanism requiring a hydride shift (see Scheme 9). It was found that the intramolecular, C-2 to C-1 hydrogen transfer also occurs during the preparation of 5-(hydroxymethyl)-2-furaldehyde from D-glucose and of 2-furaldehyde from D-xylose.26~-[2-~H]Glucose was converted into an aldehyde that retained 7% of the tritium label. The tritium was located on the aldehyde carbon atom ((2-6). The conversion of ~-[2-~H]xylose into 2-furaldehyde indicated that 13% of the activity of the sugar was retained. Thus, these intramolecular, hydrogen transfers occur during the dehydration reactions involving conversion of aldoses into ketoses.
'lC=cH+ H
HCOH I
I
C=O
I
R 45
I
R
46 SCHEME 9.--Isomerization of Aldose to Ketose with a Hydride Shift.
288
OLOF THEANDER AND DAVID A. NELSON
After the autoclaving (100") of D-glucose solutions, both 5-(hydroxymethyl)-2-furoic acid and 2,5-furandicarboxylic acid were detected.a No attempt was made to remove oxygen from the starting solutions. Although the D-glucose solution was not initially acidic, subsequent experie n ~ indicated e ~ ~ that the final pH was probably -4. Air exposure (mild oxidation) of the mixtures increaseda the two furancarboxylic acids at the expense of 11. Hexuronic acids are decarboxylated in the presence of refluxing, aqueous acid69to form 5, reductic acidz7(2,3-dihydroxy-2-cyclopenten-l-one; 47) and very small amounts of 48 (5-formyl-2-furoic acid).70The yields of these products decrease when dilute acid solutions are used. Most of this work was performed with hydrochloric acid; the use of phosphoric acid For instance, D-galacturonic acid quantitawas not nearly so effe~tive.~' tively lost C 0 2 within 4 h with 3.5 M HCl, whereas only 12 mole% of the COz was recovered after 4 h with 1.2 M H3P04.The yields of C02 from several hexuronic acids (including polygalacturonic acid) were comparatively determined. The acidic decarboxylation of hexuronic acids is bimolecular and dependent on both the hexuronic acid and hydrochloric had established that C-6 of the acid concentration^.^^ Prior labeling hexuronic acid is the source of the COZ.
41
48
The three acid-derived components of the hexuronic acids, namely, 5, 47, and 48, are essentially end products in the reaction.74This is quite surprising, as 48 required only decarboxylation to form 2-furaldehyde. However, the rate of decarboxylation of the furoic acid was <2% of that of D-galacturonic acid under similar conditions. Furthermore, no trace of pentoses was noted in any of the reaction mixtures, which eliminates previous suggestions of their role in the mechanism of 2-furaldehyde for(68) D. G . Durham, C. T. Hung and R. B. Taylor, Int. J . Pharm., I 1 (1982) 31-40. (69) R. W. Herbert, E. L. Hirst, E. G. V. Percival, R. J. W. Reynolds, and F. Smith, J . Chem. Soc., (1933) 1270-1290. (70) E. Stutz and H. Deuel, Helu. Chim. Acta, 39 (1956) 2126-2130. (71) E. Stutz and H. Deuel, Helu. Chim. A c f a , 41 (1958) 1722-1730. (72) D. M. W. Anderson and S . Garbutt, J . Chem. Soc., (1963) 3204-3210. (73) D. M. W. Anderson and S . Garbutt, Talanra, 8 (1961) 605-611. (74) M. S. Feather and J. F. Hams, J . Org. Chem., 31 (1966) 4018-4021.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
289
m a t i ~ nReductic .~~ acid (47) obtained from D-[ l-14C]galacturonicacid was as a mixture of [l-I4C]-and [2-14C]-reducticacid in the ratio e~tablished’~ of 1 :9. This suggested that two different mechanisms may occur during the formation of 47. It has been shown76that higher yields of 47 can be obtained from methyl /3-~-arubino-hexopyranosid-2-ulose and its related ~-ribo-3-ketoisomer (49). Tagging experiments with [1J4C] 49 showed77that C-1 of 49 was lost during its conversion into 47. The C-2 atom of 49 corresponds to C-1 and C-2 of 47, while C-6 of 49 is equivalent to C-4 and C-5 of 47.
49
A 3-deoxyhexosulose analog had been suggested as an intermediate in the formation of 2-furaldehyde from hexuronic acids.72 To assess this mechanism, D-glucuronic acid was converted into 2-furaldehyde in acidic deuterium oxide solution.24No exchange or incorporation of deuterium was observed in any of the positions of furan. This result indicated that the mechanism proceeds without substantial reversible enolization of the intermediates, much in the manner acknowledged for the formation of 11 from hexoses. A mechanism that is consistent with the hexuronic acid conversion was proposed earlier by I ~ b e 1 1 Further .~~ work with tritium labeling confirmed the general mechanism from D-glucuronic acid to 2f ~ r a l d e h y d eThere . ~ ~ were negligible proportions of tritium incorporated at the a-carbon atom in the furan ring. However, conversion of 2-furoic acid into methyl 5-nitro-2-furoate with retention of the activity indicated that tritium was located at C-3 or C-4, or both, of the furan ring. Decarboxylation of L-ascorbic acid in acid solutions has been proposed to involve one of two possible mechani~rns.~~ One pathway required dehydration, decarboxylation, and formation of 2-furaldehyde. The second pathway involved a rearrangement to the 3-keto form followed by a (75) H . Franken, Biochem. J . , 257 (1933) 245-255. (76) 0. Theander, Acta Chem. Scand., 12 (1958) 1897-1905. (77) G . L. Lockhart, M. S. Feather, G. Lindgren, T. Popoff, and 0. Theander, Carbohydr. Res., 79 (1980) 293-297. (78) T. Kurata and Y. Sakurai, Agric. Biol. Chem., 31 (1967) 170-176.
OLOF THEANDER AND DAVID A. NELSON
290
p-keto acid decarboxylation to yield a pentose-1 ,2-enediol which may be converted into 2-furaldehyde. The results of labeling experiments with tritium are consistent only with the second pathway.22The involvement of a pentose-l,2-enediol explains the isolation of pentosuloses during the ascorbic acid dehydration, as the pentosulose would be the more-stable keto form. However, the reaction still proceeds through the enol form. It (50) and 4-hydroxy-5is of interest that 3,8-dihydroxy-2-methylchromone methyL3(2H)-furanone (111) were formed both from ascorbic acid and pentoses after treatment under slightly acid condition^.'^
b3 & b
0 1OH II
0
H
0
II
5Oa
0 5Ob
-665%
-15%
3
OH
0
bl
OH
01
OH
0
5oc
sod
6 5 4 %
12-165%
D-glucose, ~-fructose,~O D-glucuronic a ~ i d , ~ and ~ , ~D-galacturonic ~,~' respectively, were treated with acetate buffer (pH 3.5-4.0) at 96". The total phenolic proportions were 0.3% for hexoses, and 7% for Dglucuronic acid. The major phenols from the hexuronic acids were pyrocatechol, 2,3-dihydroxyacetophenone,and the chromone 50. Two trihydroxy-2-methylchromones were also isolated, as well as precursors of 50 and 2,3-dihydroxyacetophenone.Among the phenolic products from the and 3-hyhexoses, 6,7-dihydroxyphthalide, 2-methylbenzofuran-t,6-diol, droxy-6-(hydroxymethyl)-2-methylchromonemay be mentioned.40Pyrocatechol and pyrogallol were obtained from both the hexoses and hexuronic acids. A reactive phenol such as pyrogallol has been shown to react with an excess of D-glucose to give a C-D-glucosyl Examination of D-glucuronic acid in various buffers (pH 1.9-4.0) at 300" indicated that 2-furaldehyde, catechol, 2,3-dihydroxyacetophenone, and 50 were the major products82after 5 min. Much higher yields of the phenolic compounds were obtained at 300" than at 100". At pH 3.6, the mole% yield of pyrocatechol was 17%, and that of 2,3-dihydroxyacetophenone was 4%. At pH 3.0, the yield of 50 was 6.6 mole%. Labeled 50 was prepared from [l-'3C]pentose (synthesized by a Kiliani reaction of D(79) (80) (81) (81a) (82)
P. Anderegg and H. Neukom, Mitt. Geb. Lebensmiiieluniers. Hyg.. 63 (1972) 81-87. T. Popoff and 0. Theander, Acra Chem. Scand., Ser. B, 30 (1976) 705-710. T. Popoff and 0. Theander, Chem. Commun., (1970) 1576. P.-A. Pernemalm, Acra Chem. Scand., Ser. B , 32 (1978) 72-74. 0. Theander, D. A. Nelson, and R. T. Hallen, Production, Analysis, and Upgrading of Pyrolysis Oils from Biomass, ACS Div. Fuel Chem. Prepts., Vol. 32, No. 2 (1987) 143-148.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
291
erythrose with K[I3C1N)under slightly acid conditions.82aIts I3C-and ‘Hn.m.r. spectra showed a mixture of the doubly labeled species 50a-50d in the proportions indicated. This complex picture might be explained by partial scrambling of the label between C-1 and C-5 in the pentose molecules before these are linked together. Such scrambling may be due to slow passage through a symmetric intermediate (for example, a 3-ketose; compare Ref. 34). If so, the subsequent reaction-steps may follow one single route. b. Basic Conditions.-Formation of saccharinic acids from hexoses has been recognized and extensively i n ~ e s t i g a t e d . The ~ ~ , ~mechanism ~ involves formation of an enediol followed by @elimination of a functional group. The next step requires tautomerization to an a-dicarbonyl intermediate that can undergo benzilic acid rearrange~nent.~~ The initial two steps are the same as those noted for acidic dehydration of hexoses; see Scheme 7. The differences between the acidic and basic reactions obviously lies in the final two steps, where tautomerization to the dicarbonyl intermediate is very rapid under basic condition^.^^ The sequence is quite similar to that shown in Scheme 6, at least for the metasaccharinic acid. The saccharinic acids formed from hexoses have been especially examined because of the relationships of the “a” and ‘‘p” isomers (C-2 epiStructures of saccharinic acids derived from D-glucose are: glucometasaccharinic acid (51), glucoisosaccharinic acid (52), and glucosaccharinic acid (53). The “a-”and “p-”isomers of metasaccharinic acid can reversibly isomerize when exposed to baseg5because of the labile proton at C-2. COzH
I I CHz I HCOH I HCOH I
CHOH
CHzOH 51
C02H
A,CH~OH 1
‘OH CHz
I
HCOH
I
CH20H
52
C02H l/CH3 i\OH HCOH
I
HCOH
I
CHnOH 53
(82a) R. Anderson, E. Olsson, K. Olsson, and 0. Theander, Absrr. Nordic Symp. N M R Spectrosc., 2 n d Uppsala, 1987, p. 26. (83) S. Selman and J. F. Eastman, Q.Rev., 14 (1960) 221-223. (84) A. A. J. Feast, B. Lindberg, and 0. Theander, Acta Chem. Scand., 19 (1965) 11271134. (85) B. Alfredsson and 0 . Samuelson, Suen. Papperstidn., 65 (1962) 690-692.
292
OLOF THEANDER AND DAVID A. NELSON
The presumed intermediates in the formation of D-glucometasacacids were isolated after alkaline charinic86and ~-glucoisosaccharini~~~~~~ treatment of substituted sugars. Respectively, 3-deoxy-~-erythro-hexosulose (39) and 4-deoxy-~-glycero-2,3-hexodiulose (70) were converted into the corresponding saccharinic acids in the presence of calcium hydroxide. The kinetics of the rearrangement of 39 to D-glucometasaccharinic acids in alkali solution were also examined.89In dilute sodium hydroxide, considerable fragmentation of 4-deoxy-~-glycero-2,3-hexodiulose produced glycolic acid, formic acid, and 3,4-dihydroxybutanoic acid.88D-Glucose has been exposed to dilute NaOH (oxygen-free) for up to 50 daysg0at 25". Not only were a-D-glucoisosaccharinic, a-D-glucometasaccharinic, and p-D-glucometasaccharinic acids formed, but also present were formic, glycolic, glyceric, lactic, 2-hydroxybutanoic, and 2-hydroxypentanoic acids. When either D-glucose or D-fructose was kept in 1.2 M KOH for 105 days at 5", a metasaccharinic acid was reported, as well as lactic, glycolic, glyceric, 2,4-dihydroxybutanoic, and 3-deoxypentanoic acids.91 Mass-balance calculations were reported for the acidic components of alkaline-degraded monosaccharide^.^^ These results, based upon the almost identical, final-product compositions from D-glucose, D-fructose, Dmannose, and D-psicose, indicated that alkaline degradation of interconvertible monosaccharides proceeds by way of the same 1.2- and 2,3-enediol anion species. Isomeric c6 acids constitute 4347% of the final acid products. Maximum yields of these acids are obtained at a hydroxyl M when the monosaccharide concentration of between lov3 and concentration exceeds lo-* M. Similar product compositions were noted after alkaline degradation of glyceraldehyde and dihydroxyacetone. This suggests that substantial aldolization occurs prior to formation of the products. Treatment of D-fructose or 1-0-substituted D-fructose with calcium hydroxide produced only the a-D-glucosaccharinic acid (2-C-methyl-~-ribonic acid) and no p-D-glucosaccharhic acid (2-C-methyl-~-arabinonic acid).84However, when D-fructose was treated with sodium hydroxide, the D-glucosaccharinic acids were not detected.43In order to confirm this (86) G . Machell and G. N. Richards, J . Chem. Soc., (1960) 1938-1944. (87) R. L. Whistler and J. N. BeMiller, J . Am. Chem. SOC., 82 (1960) 3705-3707. (88) G. Machell and G . N. Richards, J . Chem. Soc., (1960) 1932-1938. (89) T. Vuorinen, Carbohydr. Res., 127 (1984) 327-330. (90) J. Poloskg, L. RepBSovB, A. PekaroviEovB, and M. Kosik, J . Chromarogr., 320 (1985) 111-118. (91) A. P. G . Kieboom and H. van Bekkum, R e d . Trau. Chim. Pays-Bas, 103 (1984) 1-12. (92) J. M. de Bruijn, A. P. G. Kieboom, and H. van Bekkum, Recl. Trau. Chim. Pays-Bas, 105 (1986) 176-183.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
293
observation, the dicarbonyl intermediate of P-D-glucosaccharinic acid, namely, l-deoxy-~-erythro-2,3-hexodiulose was prepared,93 and subjected to calcium hydroxide or sodium hydroxide, and only traces of the p-glucosaccharinic acid were detected. Instead, the major products were a-D-glucosaccharinic acid and D-erythronic acid. The former was preponderant with calcium hydroxide, and the latter with sodium hydroxide as the base. It was assumed that the asymmetrically substituted C-4 atom adjacent to the dicarbonyl group provides a considerable stereochemical effect to the alkaline reaction. Both ~ - x y l o s and e ~ ~~-sorbose,"however, produced a- and p-D-xylosaccharinic and a- and /I-L-galactosaccharinic acid, respectively, when exposed to calcium hydroxide. Thus, the aasymmetric carbon explanation may require further refinement. Pyruvaldehyde (2-oxopropanal) and acetol (l-hydroxy-2-propanone, hydroxyacetone) are formed in small proportions when D-glucose is exposedg5,%to potassium hydroxide solution at 70-90". Glyceraldehyde (2,3-dihydroxypropanal) was also identified in alkaline solutions of Dglucose by using paper ~hromatography.~~ Acetol had earlier been observed as a product of D-glucose in aqueous Na2C03 .98 The mechanism for the formation of acetol from selectively labeled D-[ 14C]glucosewas i n v e ~ t i g a t e d .Acetol ~ ~ , ~ was distilled from D-glucose in a concentrated K2HP04buffer at pH 6.7. Moreover, acetol was formed at pH 3-1 1. It was conclusively shown that [3-'4C]acetol was formed from D-[ 1-I4C]glu[ l-14C]acetolwas cose as well as from ~ - [ 6 - ' ~ C ] g l u c o sFurthermore, e.~~ formed from ~ - [ 3 -or 4-14C]glucose. The favored mechanism requires isomerization of D-glucose to a 3- and/or 4-ketose (perhaps a 2,3- or 3,4enediol), followed by elimination of the hydroxyl group p to the carbonyl group of the 3-ketose, to yield methyl-a-diketones ( 2 , 3 - d i o ~ o )Isomeri.~~ zation of the methyl-a-diketones to p-diketone (2,4-dioxo) allowed hydrolytic cleavage to an a-ketol (acetol) and pyruvic acid. Studies involving the hydroxide-catalyzed isomerization of D-[ l-13C]mannoseprovided evidence for 3,4-enediol intermediates, as well as for the expected 1,Zenediols. loo Spruce wood or D-galactose under pressure at 100" in NaOH solution A. Ishizu, B. Lindberg, and 0. Theander, Curhohydr. Res., 5 (1967) 329-334. A. Ishizu, K. Yoshida, and N. Yamazaki, Carbohydr. Res., 23 (1972) 23-29. V. Prey and E. Waldmann, Monarsh. Chem., 83 (1952) 65-70. V. Prey, E. Waldmann, H. Berbalk, and E. Ludwig, Monarsh. Chem., 84 (1953) 551562. (97) R. Nodzu and R. Goto, Nippon Kugaku Zasshi, 15 (1940) 209-21 1. (98) 0. Baudisch and H. Deuel, J . A m . Chem. Soc., 44 (1922) 1585-1587. (99) R. Goto, J. Hayami, K. Kudo, and S. Otani, Bull. Chem. SOC.Jpn., 34 (1961) 753-757. (100) M. J. King-Moms and A. S. Serianni, Carbohydr. Res., 154 (1986) 29-36.
(93) (94) (95) (96)
294
OLOF THEANDER AND DAVID A. NELSON
producela' small proportions of 2-hydroxy-5-methylcyclopent-2-en-I-one (54) and triose-reductone (2,3-dihydroxyacrolein) (55). Several other cyclopentenones have been detected after the alkaline treatment of saccharides. For instance, D-fructose refluxed in 50% aqueous NaOH yieldsla233, 34, 2-hydroxy-3 ,S-dimethylcyclopent-2-en- I-one (56), and 3ethyl-2-hydroxycyclopent-2-en1-one (57). Both 33 and 34 have been isolated after alkaline treatment of glucose,49and 33 was detected after base degradation of sucrose. Ia3 Similarly, 33 and 4-hydroxy-2,5-dimethyl3(2H)-furanone (58) were present after liquefaction of sucrose. lo4
C 'Y
54
57
58
Several products were also detected in base-degraded D-fructose solution: acetoin (3-hydroxy-2-butanone; 62), I-hydroxy-2-butanone, and 4hydroxy-2-butanone. Ia2 Three benzoquinones were found in the product mixture after sucrose had been heated at 1 lo" in 5% NaOHIa3;these were 2-methylbenzoquinone, 2,3,5-trimethylbenzoquinone,and 2,5-dimethylbenzoquinone (2,5-dimethyl-2,5-cyclohexadiene-1,4-dione;61). Compound 62 is of considerable interest, as 62 and butanedione (biacetyl; 60) are involved in the formation of 61 and 2,5-dimethyl-l ,Cbenzenediol(63) by a reduction-oxidation pathway.Ia5This mechanism, shown in Scheme 10, will be discussed in a following section, as it has been proposed from results obtained from cellulose. (101) T. Enkvist, B. Alfredsson, M. Merikallio, P. Paakkonen, and 0.Jarvela, Acta Chem. Scand., 8 (1954) 51-59. (102) P. E. Shaw, J. H. Tatum, and R. E. Berry, J. Agric. Food Chem., 16 (1968) 979-982. (103) H. Kato, M. Mizushima, T. Kurata, and M. Fujimaki, Agric. .Biol. Chem., 37 (1973) 2677-2678. (104) P. E. Shaw, J. H. Tatum, and R. E. Berry, J. Agric. Food Chem., 17 (1969) 907-908. (105) D. A. Nelson, S . D. Landsman, and P. M. Molton, Carbohydr. Res., 128 (1984) 356360.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
00 2 H3CCCCH3 II 1 I
2
t
OK
60
-
295
0 b
C
H3C
H
3
It 0 61
61
H3CCCHCH3 0 I1 I
t
OH
t
60
H3C OH
62
SCHEME 10.-Formation
@cH3
63
of 2,5-Dimethylhydroquinonefrom Butanedione.
Catechol and related phenolics 13,16,19, 31, and 32 were also isolated after alkaline treatment of ~ - g l u c o s and e ~ ~sucrose. lo3 Several other substituted acetophenones were isolated.49The mechanism of formation of phenolic compounds from monosaccharides under alkaline conditions has yet to be thoroughly investigated. The similarity in the types of aromatic products from D-glucose and D-xylose indicates the formation of the same C2, C3, or C4 fragments, with subsequent recombination and cyclizat i ~ n Base-catalyzed . ~ ~ aldol reactions are, no doubt, predominant pathways in the initial formation of these aromatic products.
111. TRANSFORMATION OF POLYSACCHARIDES 1. Starch Starch is the most abundant, reserve-energy carbohydrate for higher plants, and is composed of two related polysaccharides, amylose and amylopectin. Amylose is a linear polymer consisting of several hundred D-glucose residues joined by a - ~ -1-4) ( linkages. Amylopectin is a branched polymer having a much higher average molecular weight. Several hundred thousand to several million D-glucosyl residues are joined by a-~-(1+4) and a - ~ - ( 1 + 6bonds. ) The molecular structure of starch has been reviewed.Io6 (106) R. L. Whistler and J. R. Daniel in R. L. Whistler, J. N. BeMiller and E. F. Paschal1 (Eds.), Starch: Chemistry and Technology, Academic Press, Orlando, 1984, pp. 153-
182.
296
OLOF THEANDER AND DAVID A. NELSON
The acid hydrolysis of starch produces D-glucose, as well as the degradation products of D-glucose: 5-(hydroxymethyl)-2-furaldehyde,levulinic acid, and formic acid.Io7However, both levulinic acid and formic acid can Although the be obtained from 5-(hydro~ymethyl)-2-furaldehyde.'~*~'~ hydrolysis of malto-oligosaccharides has not been studied, investigations of gluco-oligosaccharides (cellobiose through cellohexaose) suggests that the rate of hydrolysis may be more related to solubilities than to chemical Representative rate-constants of the acid hydrolysis of starch, amylose, amylopectin, and maltose have been tabulated.'" Under anaerobic conditions, the alkaline hydrothermolysis of amylose with 0.5 M NaOH at 100" virtually ceased112after 20 hours. Approximately 60% of the amylose had been degraded. This produced 23% of Dglucoisosaccharinic acid, 35% of formic acid, and 6% of lactic acid, based on the total acid fraction. The degradation involved the P-alkoxycarbonyl mechanism which will be discussed more completely for cellulose. Alkaline hydrothermolysis with calcium hydroxide favors formation of 73% of D-glucoisosaccharinic acid at loo", and production of formic acid and lactic acids is depressed. Both glycolic acid and 3 ,4-dihydroxybutanoic acid (2-deoxytetronic acid) were detected as alkaline-hydrothermolytic products with both bases. The difference between these results appears to be due to a catalytic effect on the benzilic acid rearrangement by calcium ions. Maltose showed a similar, but more dramatic, difference with 89% of'D-glucoisosaccharinic acid formed by the calcium hydroxide as compared to 13% of the acid from NaOH solution^."^ Neither basic degradation was complete, owing to the stopping or stabilization reaction (formation of D-glucometasaccharinic acid on the terminal units of the amylose chains). The (branched) amylopectin is generally considered more alkali-stable than amylose. Amylopectin produced a very small proportion of glucoisosaccharinic acid after exposure to M KOH at 25" for nearly 2 months.'I4 -( However, glycogen, which has a similar structure, but more a - ~ 1+6) ( bonds are prebonding, undergoes the peeling process where a - ~ -1+4) (107) J. N. BeMiller, in R. L. Whistler and E. F. Paschal1 (Eds.), Starch: Chemistry and Technology (1st. ed.), Vol. 1, Academic Press, New York, 1965. (108) C . H. G. Hands and F. R. Whitt, J . Soc. Chem. Znd. London, 66 (1947) 415-416. (109) T. Takahashi, Nippon Nogei Kagaku Kaishi, 20 (1944) 553-556; Chem. Abstr., 42 (1948) 8166. (110) K . Freudenberg and G. Blomqvist, Ber., 68 (1935) 2070-2082. (111) M. L. Wolfrom and J. C. Dacons, J . Am. Chem. Soc., 74 (1952) 5331-5333. (112) G . Machell and G. N. Richards, J . Chem. Soc.. (1958) 1199-1204. (113) G . Machell and G. N. Richards, J . Chem. SOC., (1960) 1924-1932. (114) C. J . Stacy, J. F. Foster, and S. R. Erlander, Makromol. Chem., 17 (1956) 181-188.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
297
valent. 1 1 5 ~ 1 1 The 6 glucoisosaccharinic acid that was released may have many of the branched structures attached to it; that is, a stopping reaction with glucoisosaccharinic acid as the terminal unit. Amylopectin has been shown to have a multibranched structure similar to that of g1y~ogen.l~~ Thus, analogous chemistry should be applicable for both polysaccharides. Because of the extensive branching of amylopectin, cleavage at the branch points can rapidly lower the molecular weight of the polymer. However, the glucoisosaccharinic acid terminal units and, no doubt, glucometasaccharinic acid units (from the stopping reaction) provide an alkali-stability to a still large polysaccharide compared to amylose. 2. Cellulose
Cellulose is the most abundant organic molecule found in Nature. Approximately 40-45% of the dry weight of wood from most sources is cellulose. However, it constitutes 98% of cotton fiber. Although cellulose appears to be a simple, linear polymer containing as many as lo4 p-Dglucopyranosyl residues joined by (1+4)-glycosidic linkages, its secondary and higher-order structure and consequent structural intricacy have yet to be completely defined.lls Owing to intra- and inter-molecular hydrogen-bonding, the cellulose molecules are aggregates in elementary fibrils and microfibrils (bundles) having highly crystalline order which alternate with less-ordered regions. Because of its particular structure, cellulose is insoluble in most solvents and has low accessibility to aqueous acids and dissolved enzymes. Acid hydrolysis (saccharification) of cellulose in a homogeneous reaction system introduces a random cleavage of the glucosidic linkages containing hemiacetal and hydroxyl terminal groups. D-Glucose may be obtained in yields of >95% under suitable acidic condition^."^ Acid hydrolysis is quite dependent upon pH, and the rate of hydrolysis is appreciable, even below loo", if the acid concentration is high. For instance, cellulose forms a homogeneous solution in 51% sulfuric acid at 18", and the rate constant for hydrolysis under these conditionsIZ0is 5.1 X M. R. Stetten and H. M. Katzen, J . A m . Chem. SOC., 83 (1961) 2912-2918. R. L. Whistler and J. N . BeMiller, Arch. Biochem. Biophys., 98 (1962) 120-123. D. J. Manners, Adu. Carbohydr. Chem., 17 (1962) 371-430. 0. Theander, in R. P. Overend, T. A. Milne, and L. K. Mudge (Eds.), Fundamenfals of Thermochemical Biomass Conversion, Elsevier Applied Science Publishers, New York, 1985, pp. 35-60. (119) B. Philipp, Pure Appl. Chem., 56 (1984) 391-402. (120) S. A. Rydholm, Pulping Processes, Interscience, New York, 1965, pp. 124-127.
(115) (116) (117) (118)
OLOF THEANDER AND DAVID A. NELSON
298
lo-' s-1. Several rate-studies were performed in 8 0 4 6 % phosphoric acid, as it provided a lower rate of hydrolysis than that of sulfuric acid.I2' Similar work was also performed in concentrated hydrochloric acid.'22 The kinetics of cellulose hydrolysis in dilute acid (0.4-1.6% H2S04) at 170- 190" has been in~estigated.'~~ Further acid-hydrolysis studies were performed at 75-80" with 0.5 M H2S04 with cellulose and c e l l ~ b i o s e . ~ ~ ~ The results of this work suggested that hydrolysis is initially confined to the amorphous regions of cellulose. After their depletion, degradation begins upon the ends of the crystallites. Other systems used for cellulose hydrolysis include trifluoroacetic acid,'25and mixtures of acetic acid, acetic anhydride, N,N-dimethylformamide, and sulfuric acid.'26 Cellobiose was used as a model for cellulose under acid-hydrolysis condition^.'^^ This investigation used 0.01 A4 H2SO4 and 1.O% cellobiose to evaluate the first-order kinetics of the hydrolysis of cellobiose to D-glucose. The results of an Arrhenius diagram indicated that the activation energy for this hydrolysis was 133 kJ.mo1-I. Temperatures from 160 to 220" were examined during the hydrolysis of cellobiose.
R- 0 Q - R -
R-0
OH
64
QC1. OH
R-0
4,
t
L+H20 R - Q
OH
OH
65 66 67 SCHEME 11 .-Mechanisms for the Acid-catalyzed Hydrolysis of Cellulose.
The mechanism of the acid-catalyzed hydrolysis of cellulose is based on that normally expected for an acetal12*(see Scheme 1 1 ) . This involves formation of a conjugate acid by protonation of either of the acetal oxygen atoms at C-1, and formation of a carbonium ion, followed by stabilization of the product by heterolysis of a participating water molecule. The carE. C. Sherrard and A . W. Froehlke, J . Am. Chem. Soc., 45 (1923) 1729-1734. F. Bergius, Br. Pat. 315,198 (1928); Chem. Absrr., 24 (1929) 1739. J. F. Saeman, Ind. Eng. Chem., 37 (1945) 43-52. A. Sharples, Trans. Faraday SOC., 53 (1957) 1003-1013. D. Fengel and G . Wegener, in R. D. Brown, Jr., and L. Jurasek (Eds.), Hydrolysis of Cellulose: Mechanisms of Enzymatic and Acid Catalysis, Adu. Chem. Ser., 181 (1979) 145-158.
K. Garves, in Ref. 125, pp. 159-165. 0 . Bobleter, W . Schwald, R. Concin, and H. Binder, J. Carbohydr. Chem., 5 (1986) 387-399.
L. P. Clermont and A. Schwartz, Pulp Pap. Mag. Can., 53 (1952) 142-143.
H'
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
299
bonium ion may be either cyclic or acyclic, depending upon the primary site of p r o t o n a t i ~ n .It' ~was ~ suggested that a hydronium ion may partially protonate both oxygen atoms of the acetal by forming a six-membered ring. The rate of the acidic cleavage of cellulose, compared to that of smaller glycosides, is lower by one or two orders of magnitude, because of the tertiary structure of cellulose and the limited accessibility of the acetal groups. Aldobiouronic acid linkages in polysaccharides are known to be more stable towards acid hydrolysis than ordinary glycosidic linkages; strong evidence suggested that polar and conformational effects are operative. I3O The rates of hydrolysis of cellobiose, pseudocellobiouronic acid (carboxyl group in the reducing unit), and cellobiouronic acid were studied as models for the oxidation of the primary hydroxyl groups of cellulose to carboxyl groups. The rates of hydrolysis of the two former compounds were almost identical; whereas, the rate of hydrolysis of cellobiouronic acid is considerably lower. I 3 I Introduction of carbonyl groups into cellulose, and other polysaccharides, by bleaching has a large effect on hydrolysis.I3*Keto groups at C-2 or C-3 in cellulose increases alkali depolymerization by @-elimination at elevated temperatures. The oxidation of primary hydroxyl groups to aldehydo groups increases the depolymerization of cellulose for similar Experiments conducted at 80" indicated that @-eliminationwas also responsible for the depolymerization of cellulosic material under neutral or slightly acid conditions; this occurred down to at least pH 3.5. However, at pH 1.2 and 80", acid-catalyzed hydrolysis of the glycosidic bonds of oxidized cellulose occurred, because of the presence of the 6-aldehydo group.133 Cellulose is readily hydrolyzed in water at >170" at a rate that depends on the hydrogen-ion concentration, in the range between pH 5 and 8. Because acids from the degradation of D-glucose are produced when cellulose is exposed to high temperature, the pH of the aqueous mixture drops in the absence of buffers. Earlier work'35J36had indicated that ether-soluble products could be obtained by heating cellulose in (129) B. Philipp, V . Jacopian, F. Loth, W. Hirte, and G . Schulz, in Ref. 125, pp. 127-143. (130) 0.Theander, in W. Pigman, D. Horton, and J . D. Wander (Eds.), The Carbohydrates, Vol. IB, Academic Press, New York, 1980, pp. 1013-1099. (131) I. Johansson, B. Lindberg, and 0. Theander, Acta Chem. Scand., 20 (1963) 20192024. (132). 0. Theander, Tappi, 48 (1965) 105-110. (133) A. E. Luetzow and 0. Theander, S u m . Papperstidn., 77 (1974) 312-318. (134) B. B. Mithel, G . H . Webster, and W. H. Rapson, Tappi, 40 (1957) 1-4. (135) P. A. Bobrov, J . Appl. Chem. USSR, 6 (1933) 1105-1110. (136) H. Tropsch, Brennst. Chem., 5 (1924) 288-289.
300
OLOF THEANDER AND DAVID A. NELSON
water at 275". In the absence of any added acid or base, the major products of the hydrothermolysis of cellulose after 2.5 min at 300" were 30% of 11, 13% of 2-furaldehyde (3,and >30% of saccharides [including Dglucose, cellobiose, D-mannose, D-fructose, and 1,6-anhydro-fi-~-glucopyranose (levoglucosan)] .37 These results with cellulose are somewhat similar to those obtained by using'37 a flow system (rapid removal of products) for 5 min at 264". The major products were 42.6% of D-glucose, 12.1% of 11, 4.8% of cellobiose, 5.2% of D-fructose, and 1.0% of Dxylose. The presence of 1,6-anhydro-fi-~-glucopyranose~~ could indicate the lack of homogeneity within the hydrothermolytic reactor, and a concurrent pyrolytic process. The pyrolytic degradation of cellulose involves the initial formation of levoglucosan without first converting the cellulose into D-glucose.'38A lack of homogeneity may also have been the source of 40% of levoglucosan when cellulose was treated with superheated steam at diminished pressures.'39 It should be noted that levoglucosan is also f ~ r m e d ' ~after ~ J ~ the ' alkaline hydrolysis of cellobiitol at 170". Furthermore, levoglucosan is also present after the alkaline hydrolysis of phenyl fi-~-glucosides.'~~ Rapid degradation of cellulose in the range of 250 to 350" also produces 5-methyl-2-furaldehyde, levulinic acid, biacetyl, and a methylfuranone. 143 Acid hydrolysis of cellulosic materials that include some hemicellulose, produces D-xylose, D-glucose, and cellobiose, as well as 11, 2-furaldehyde (3,levulinic acid, formic acid, and acetic acid.'@ In order to lessen the contamination due to hemicellulose, acid hydrolysis is generally performed in two steps: dilute sulfuric acid (1%) at 80-120" followed'45by 520% sulfuric acid at 180". The initial stage removes most of the pentoglycans (pentosans). The effect of alkaline solutions upon carbohydrates has been known for a considerable time, but the complexity of the reaction mixtures was not initially recognized as resulting from a combination of alkaline degrada(137) 0. Bobleter, R. Niesner, and M. Rohr, J . Appl. Polym. Sci., 20 (1976) 2083-2093. (138) D. Gardiner, J . Chem. Soc., (1966) 1473-1476. (139) Ya. A. Epshtein, 0. P. Golova, and L. I. Durynina, Izu. Akad. Nauk SSSR, Otd. Fiz. Mar. Khim. Nauk, (1959) 1126-1127. (140) E. Dryselius, B. Lindberg, and 0. Theander, Acta Chem. Scand., 11 (1957) 663-667. (141) R. E. Brandon, L. R. Schroeder, and D. C. Johnson, in A. F. Turbak (Ed.), Cellulose Technology Research, ACS Symp. Ser., 10 (1975) 125-146. (142) C. M. McCloskey and G . H . Coleman, J . Org. Chem., 10 (1945) 184-192. (143) R. Krishna, M. R. Kallury, C. Ambridge, T. T. Tidwell, D. G. B. Boocock, F. A. Agblevor, and D. J. Stewart, Carbohydr. Res., 158 (1986) 253-261. (144) H. E. Grethlein, Biorechnol. Bioeng. Symp., 5 (1975) 303-318. (145) A. E. Humphrey, in Ref. 125, pp. 15-53.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
301
tion and oxidation r e a c t i ~ n s . Once ~ ~ ~the J ~ importance ~ of the exclusion of oxygen was realized, great strides were made toward understanding the chemistry of the alkaline degradation of cellulose.14s The initial interaction between cellulose and metal hydroxides appears to be salt formation occurring at the reducing, terminal, D-glucose unit (enediol form).149Because a tautomeric shift is needed in order to form the enediol from an aldehyde, salt formation is usually relegated to the reducing terminal units of cellulose. The initial suggestion*50concerning alkali attack only at the reducing end of cellulose has received considerable experimental s ~ p p o r tHowever, . ~ ~ ~ ~ above ~ ~ 170", ~ dilute sodium hydroxide attacks cellulose at random midpoints, as well as at the reducingterminal end.153J54Thus, all the newly formed terminal groups are available for cleavage (peeling). The random cleavage, or alkaline scission,lS5may occur by intramolecular d i ~ p l a c e m e n tor ' ~by ~ the addition of the hydroxyl ion.157 Below 170", the stepwise-peeling mechanism follows the theory of /3alkoxycarbonyl e l i m i n a t i ~ nScheme .~~ 12 represents the sequential process involving the elimination of D-glucoisosaccharinic acid (52) as the terminal unit of alkali-exposed cellulose. IS1 Most of the mechanistic work was performed with hydrocellulose (cellulose pretreated with hot, dilute hydrochloric acid), in order to provide a uniform and highly crystalline starting-material. The intermediate 70 has been isolated from model systems, including 4-O-methyl-~-ghcose and maltose, as well as the polysaccharides cellulose and a m y l o ~ e Alkaline . ~ ~ ~ reactions performed at 100" with calcium hydroxide double the formation of 52 from cellulose, compared to those performed with NaOH. A study of cellobiose exposed to dilute NaOH at 90" provided an isolated ketodisaccharide having a (146) (147) (148) (149)
(150) (151) (152) (153) (154) (155)
(156) (157)
J. U. Nef, Justus Liebigs Ann. Chem., 403 (1914) 204-383. W. L. Evans, Chem. Rev.. 31 (1942) 537-560. R. L. Whistler and J. N. BeMiller, Adv. Carbohydr. Chem., 13 (1958) 289-329. J . A. Rendleman, Jr., Ionization of Carbohydrates in the Presence of Metal Hydroxides and Oxides, Adv. Chem. Ser., 117 (1973) 51-69. G . F. Davidson, J . Text. Inst., 25 (1934) ~ 1 7 4 - ~ 1 9 6 . G. N. Richards and H. H. Sephton, J . Chem. Soc., (1957) 4492-4499. G. Machell and G . N . Richards, J . Chem. Soc., (1957) 4500-4506. W. M. Corbett and G. N. Richards, Sven. Papperstidn., 60 (1957) 791-794. 0. Samuelson, G. Grangord, K. Jonsson, and K. Schramm, Sven. Papperstidn., 56 (1953) 779-784. G. N . Richards, in N . M. Bikales and L. Segal (Eds.), High Polymers, Vol. 5 , WileyInterscience, New York, 1971, pp. 1007-1014. J . Janson and B. Lindberg, Acta Chem. Scand., 13 (1959) 138-143. R. J. Femer, W. G. Overend, and A. E. Ryan, J . Chem. Soc., (1965) 3484-3486.
302
OLOF THEANDER AND DAVID A. NELSON CHZOH
CHO
I
HCOH
I HOCH I HC-O+Glc)n
I I CH,OH
HCOH
CHzOH
I 1 HOCH I HC-O+Glc), c=o
I
HCOH
I
c= 0
I
I
HCOH
I
+
H ;:I I CHzOH
CHzOH 'O
68
CHO
I c= 0 I
HOCH
I HC-O+Glc),I HCOH I CHzOH
n
\
69
CHz
I I CHZOH
HCOH
52
SCHEME 12.-The Stepwise-peeling Mechanism Involving Cellulose.
(1 4)-glycosidic bond.15gThis lends support to the formation of 69, shown in Scheme 12. At higher temperatures, the products become more complex, and an increasing proportion of acids, such as formic, lactic, and acetic, is formed.'51 The formation of 7-8% of formic acid from cellulose is probably due to degradationg8of the reaction intermediates of 70. This degradation is accompanied by formation of glycolic acid (hydroxyacetic acid) and 3,4-dihydroxybutanoic acid. A marked difference in the product pattern has been reported for the treatment of cellobiose with either NaOH or NaHC03.159 The formation of 3-deoxy-2-O-(hydroxymethyl)pentonicacids (52) from cellobiose is less important in sodium hydrogencarbonate solution than in sodium hydroxide, while the relative amounts of 2-deoxytetronic, 3-deoxypentonic, and 3 ,Cdideoxypentonic acids are much larger. According to Scheme 12, the alkaline peeling of cellulose should continue until the entire polymer is degraded. However, cellulose dissolves partially, but not completely, in hot alkali, and this remaining polysaccharide contains an increased carboxyl content.I6O Thus, a second reaction is occurring that competes with the step-wise, peeling procedure. (158) H. Sihtola and L. Laamanen, C e / / u / .Chem. Techno/., 3 (1969) 3-8. (159) L. Lowendahl and 0. Samuelson, Acta Chem. Scund., Ser. B , 30 (1976) 691-694. (160) 0 . Samuelson and A. Wennerblom, Suen. Pupperstidn., 57 (1954) 827-830.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
303
This stopping or stabilization reaction with cellulose was presumed to involve a saccharinic acid rearrangement of the reducing group on the terminal D-glucose residue.I6l This mechanism was confirmed by the formation of D-glucometasaccharinic acid, which still remained attachedIs2 as the terminal unit (73).The stopping reaction is presented in Scheme 13. CHO
I HCOH I HOCH I HC-O+Glc), I HCOH
CHO
COZH
L O
CHOH
I I + HC--O+Glc)n I HCOH I CH2
CHZOH
68
72
I I
CHZ
I
HC-WGlc),
I
HCOH
I
CH,OH 73
SCHEME 13.-The Stopping Reaction Involving Cellulose.
The terminal D-glucometasaccharinic acid residue blocks further peeling; however, the rate of peeling is much greater than that of the stopping reaction. Thus, several hundred D-glucosyl residues are removed by peeling prior to stabilization by the terminal metasaccharinic acid residue.IS5 An increase in temperature favors increase in the rate of the stopping reaction over peeling, as the activation energies are 100 kJ.mol-' for peeling, and 134 kJ.mol-' for the stopping reaction.'62 Although D-glucometasaccharinic acid is the most abundant terminal unit of cellulose after high temperature (170") alkaline exposure, a large proportion (23%) of 2-C-methylglyceric acid terminal units has also been identified.'@ The methylglyceric terminal unit may be formed by the Lobry de Bruyn-Alberda van Ekenstein rearrangement of D-glucose, to provide a 3-keto group, followed by a retro-aldol reaction which allowed loss of hydroxyacetaldehyde. The remaining terminal unit can then eliminate a P-hydroxyl group at C-1, to afford an unstable 2,3-diketose which should undergo benzilic rearrangement to 2-C-methylglyceric acid. A minor proportion (1%) of 2-C-methylribonic acid was also observed as a component of the terminal units. It had previously been tentatively identified.150 (161) H. Richtzenhein and B. Abrahamsson, Suen. Pappersridn., 57 (1954) 538-541. (162) D. W. Haas, B. F. Hrutfiord, and K. V. Sarkanen, J . Appl. Polym. Sci., 1 I (1967) 587600. (163) M. H. Johansson and 0. Samuelson, Carbohydr. Res., 34 (1974) 33-43.
304
OLOF THEANDER AND DAVID A. NELSON
It had been suggested that many of the products identified as derived from the alkaline degradation of cellulose may be in question, due to varying proportions of lignin present.'@ However, much work over the ensuing 30 years has shown that this potential problem is moot. In fact, many of the aromatic products usually assumed to be due to the lignin are, in fact, products of cellulose d e g r a d a t i ~ n For . ~ ~ instance, cresols and various other alkylphenols have been identified'65J66by mass spectrometry as products of alkaline hydrothermolysis at 300".A considerable number of other low-molecular-weight products were also identified in the acetone-soluble fraction of hydrothermolyzed cellulose. These included furans, cyclopentenones, ketones, and alcohols. Mechanisms for the forand phenol from cellulose mation of 2,5-dimethyl-2-cyclopenten-l-one were proposed, based on small fragment~.'~' Although gas-liquid chromatography-mass spectrometry (g.1.c.-m.s.) is an excellent tool for the identification of minute amounts of products in such complex mixtures as those observed after the hydrothermolysis of cellulose, it must be combined with isolation, or other techniques. For instance, 2-methoxyphenol (guaiacol) had been tentatively identified by g . l . ~ . - m . s .;' ~however, ~ derivatization experiments could not confirm its presence.la This also emphasized the difficulty of working with mixtures containing more than 250 components, all in small amounts. To avoid this problem, caused by long reaction times, much shorter heating and quench periods were used for hydrothermoly~is.~~ This lessened the number of products considerably. After 5 min in 0.1 M Na2C03 at 300°,the major products from cellulose (33). Howwere catechol and 2-hydroxy-3-methyl-2-cyclopenten-l-one ever, neither was present in proportions of > l .5%. Other isolated produ c t from ~ ~ ~the alkaline hydrothermolysis of cellulose included acetic acid, acetol, biacetyl, acetoin, 2,3-pentanedione, 2,5-hexanedione, phenol, 4-methylphenol, 16, 19, and 3-methyl-2-cyclopenten-1-one. Of considerable interest was the detection of 2,5-dimethylbenzoquinone (61), 2,5-dimethyl-1,Cbenzenediol(63), acetoin (62), and butanedione (60)in mixtures form hydrothermolyzed cellulose.~OsThe results from experiments performed at 300" with [ l-*3C]acetoinsuggested that two molecules of biacetyl combined to form 61 and 63, as equal distribution was observed in four positions. This implied that a symmetrical pre(164) W. Funasaka and C. Yokokawa, Nippon Kagaku Zasshi, Ind. Chem. Sect., 56 (1953) 34-36. (165) P. M. Molton, R. K. Miller, J. A. Russell, and J. M. Donovan, in D. L. Klass (Ed.), Biomass as a Nonfossil Fuel Source, ACS Symp. Ser., 144 (1981) 137-162. (166) P. M. Molton, R. K. Miller, J. M. Donovan, and T. F. Demmitt, Carbohydr. Res., 75 (1979) 199-206. (167) R . K. Miller, P. M. Molton, and J. A. Russell, in Ref. 17, pp. 451-459. (168) D. A. Nelson, J. A . Russell, and P. M. Molton, in Ref. 118, pp. 1039-1050.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
305
cursor, such as biacetyl, was needed prior to an aldol reaction-cyclization. A reduction-oxidation mechanism is apparently involved which provides 63 and 62 after the bimolecular combination of biacetyl to form 61 (see Scheme 10).Confirmation of the intermediacy of 61 was achieved by combining 61 with acetoin under aqueous alkaline conditions; a yield well in excess of the theoretical, based upon acetoin alone, was obtained.
3. Hemicelluloses and Glycuronans The polysaccharides of hemicelluloses are related to those of cellulose, as they possess @-(1 + 4) linkages between the monosaccharide units. The hemicellulose polysaccharides are quite heterogeneous, because they can contain hexoses, pentoses, and, occasionally, alduronic acids. In general, they are composed of only 100-200 sugar units."* Galactoglucomannans are the major constituents (20%) of softwood hemicellulose. This polysaccharide contains D-glucopyranosyl and D-mannopyranosyl residues coupled by p-(1 j . 4) bonding, as well as some solitary (Y-Dgalactopyranosyl groups bonded to 0-6. Moreover, there are present 510% of arabinoglucuronoxylans (xylans or pentosans) that contain @-( 1 j . 4)-linked D-xylopyranose, side-chain substituted arabinofuranose, and 40-methyl-a-D-glucuronic acid. Hardwood hemicellulose contains glucuronoxylan (15-30%) as the main component, with a @-(I + 4)-linked Dxylose polymer backbone similar to that of softwoods. The bonding between 4-O-methyl-a-~-glucuronicacid and D-xylose is through 0-2 of the D-xylose, but only with - 10% of the available pentoses. Some 70% of the 0 - 2 or 0-3 atoms of D-xylose are acetylated. The structural considerations regarding hemicellulose have been reviewed. 169 Xyloisosaccharinic acid [2,4-dihydroxy-2-(hydroxymethyl)butanoic acid] is one of the major, alkaline-degradation products of wood xylan, in particular, that of birch. I7O The disaccharide, 2-O-~-xylopyranosyl-~arabinose, which was isolated as a hydrolysis product of corn-cob hemicellulose, is readily degraded at 100" in 15 mM Ca(OH), to acidic products, primarily saccharinic acids. Xylan oligosaccharides from corn-cob hemicellulose produced 2,4-dihydroxy-2-(hydroxymethyl)butanoic acid172when exposed to 0.02 M Ca(OH), at 25". However, it was noted that the xylan, itself, was stable at 100" in M NaOH. The major acidic component of the hemicellulose fraction of slash pine (Pinus elliotti) after acid hydrolysis was identified as 4-O-methyl-~-glucuronic (169) R. (170) E. (171) R. (172) R.
E. Timell, Adu. Carbohydr. Chem., 20 (1965) 409-483. S. Sjostrom, Tappi, 60 (1977) 151-154. L. Whistler and W. M. Corbett, J. Am. Chem. Soc., 77 (1955) 3822-3823. L. Whistler and W. M. Corbett, J. Am. Chem. Soc., 78 (1956) 1003-1005.
306
OLOF THEANDER AND DAVID A. NELSON
acid. 173 Exposure of this acid to calcium hydroxide solution produces a dibasic acid analogous to the monobasic D-glucoisosacchannic acid. The kinetics of the alkaline degradation ( M NaOH at 130-190”) of eight model compounds related to (4-~-methyl-D-g~ucurono)-D-xy~an has been r e ~ 0 r t e d . The I ~ ~ model compounds had no reducing groups, and the ratedetermining step in the alkaline degradation was the hydrolysis of the glycosidic bonds. The rate constants for the glucuronoxylans were greater by one order of magnitude than those for the corresponding pentose models. Several other models, including ~-arabino-(4,0-methyI-~-glucurono)-D-xylan, were examined under alkaline conditions. 175 Initially, Larabinose and 4-0-methyl-~-glucuronicacid were eliminated. The first step in the elimination of 4-0-methyl-~-glucuronicacid was the loss of the 4-methoxyl group and the formation of a 4,5-double bond. The reducingend residues were transformed into 3-deoxyaldonic end units by the stopping reaction. There are many other polysaccharides in lignocellulosic and “biomass” materials that are of technical interest, but they lie outside the scope of this chapter. One important type of polysaccharide is pectin, found in agricultural side-products. For instance, sugar beet, citrus, and potato pulp contain pectins. This complex group of polysaccharides has D-galacturonic acid as the principal back-bone constituent. L-Rhamnosyl groups seem to be included at intervals in the D-galacturonic acid backbone, and such other neutral sugars as L-arabinose, D-xylose, D-galactose, and Lfucose may also be present as side chains. Neutral galactans, arabinans, and arabinogalactans are often associated with these acidic polymers. Some of the carboxyl groups in the galacturonan chain are present as methyl esters. That substitution makes pectin solutions thermally sensitive to depolymerization by 0-elimination. Pectins, also like pentosans, can be transformed into 2-furaldehyde, in good yield, by aqueous acid at elevated temperatures. As discussed in Section II,2,a, alduronic acids and glycuronans are decarboxylated in hot aqueous acid. Stoichiometric proportions of carbon dioxide were produced when D-galacturonic acid, 2-0(4-0-methyl-a-~-glucopyranosyluronic acid)-D-xylose, and purified D-galacturonan (pectic acid) were boiled in concentrated hydriodic acid. 176 Many marine algae are rich in glycuronans. One of the more technically important materials is alginic acid, composed of D-mannuronic and L(173) R. L. Whistler and G . N. Richards, J . A m . Chem. Soc., 80 (1958) 4888-4891. (174) I. Simkovic, A. Ebringerovti, J. Hirsch, and J. Konigstein, Carbohydr. Res., 152 (1986) 131-136. (175) I. Sirnkovic, J. Alfoldi, and M. Matolova, Carbohydr. Res., 152 (1986) 137-141. (176) 0. Theander and P. Aman, Swed. J . Agric. Res., 9 (1979) 97-106.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
307
guluronic acids. The chromone 50 has been isolated'77 from alginic acid that had been autoclaved at 160".
IV. CARBOHYDRATE TRANSFORMATION I N THE PRESENCE OF AMINOCOMPOUNDS The reaction between sugars and amino compounds, usually termed the Maillard reaction, has been extensively studied,178and has been the subject of three recent symposia.178a*Initial interest in this type of reaction was directed toward explanations of melanoidin formation and its role in food chemistry. Further work involved the degradation encountered during the handling of food, especially dehydrated products. Hence, the reaction is also known as nonenzymic browning or, simply, browning. Because of interest in biomass utilization and in those model reactions which appear applicable in explaining the formation of products, and in improving their yields, this Section will be generally limited to reports that involve the chemistry of the Maillard reaction at elevated temperatures. The factors affecting the Maillard reaction include temperature, time, moisture content, concentration, pH, and nature of the r e a c t a n t ~ . l It ~~~~l has been shown that, out of 21 amino acids, glycine, lysine, tryptophan, and tyrosine provide the most intense browning when exposed to five saccharides, especially a-lactose. 18* The Maillard reaction is also responsible for the decreased availability of lysine in proteinaceous foods. 1 8 3 ~ 1 8 4 The kinetics of the Amadori-compound formation from D-glucose and Llysine, as well as melanoidin formation, has been examined.'85 The socalled Amadori compounds are formed at an exponential rate, while showing pseudo-first-order disappearance of L-lysine in the presence of (177) K. Aso, Nippon Nogei Kagaku Kaishi, 10 (1934) 1189-1200. (178) G . P. Ellis, Adu. Carbohydr. Chem., 14 (1959) 63-134. (178a) C. Ervksson (Ed.), Maillard Reactions in Food, Prog. Food Nutr. Sci., Vol. 5 , Pergamon Press, Oxford, 1981. (178b) G. R. Waller and M. S. Feather (Eds.), The Maillard Reaction in Foods and Nutrition, ACS Symp. Ser. 215, Washington, DC, 1983. (178c) M. Fujimaki, M. Namiki, and H. Kato (Eds.), Amino-Carbonyl Reactions in Food and Biological Systems, Dev. in Food Sci. 13, Elsevier, Amsterdam, 1986. (179) L. M. Benzing-Purdie, J. A. Ripmeester, and C. I . Ratcliffe, J . Agric. Food Chem., 33 (1985) 31-33. (180) H . E. Nursten, Food Chem., 6 (1981) 263-277. (181) G . Vernin, Parfums Cosmet. Aromes, 32 (1980) 77-89. (182) S. H. Ashoor and J. B. Zent, J . Food Sci., 49 (1984) 1206-1207. (183) R. E. Feeney, G . Blankenhorn, and H. B. F. Dixon, Adu. Protein Chem., 29 (1975) 135-203. (184) J. Mauron, in Ref. 178a, pp. 5-35. (185) C. M. Lee, B. Sherr, and Y.-N. Koh, J . Agric. Food Chem., 32 (1984) 379-382.
OLOF THEANDER AND DAVID A. NELSON
308
an excess of D-glucose, Formation of the Amadori compounds will be explained. The initiating reaction between aldoses and amines, or amino acids, appears to involve'78 a reversible formation of an N-substituted aldosylamine (75); see Scheme 14. Without an acidic catalyst, hexoses form the aldosylamine condensation-product in 80-90% yield. An acidic catalyst raises the reaction rate; and yet, too much acid rapidly promotes the Amino acids act in an autocatformation of l-amino-l-deoxy-2-ketoses.'78 alytic manner, and the condensation proceeds even in the absence of additional acid. A considerable number of glycosylamines have been prepared by heating the saccharides and an amine in anhydrous ethanol in the presence of an acidic c a t a 1 y ~ t . N.m.r. l~~ spectroscopy has been used to show that primary amines condense with D-ribose to give D-ribopyranosylamines. 188 CHO
I
HCOH
1HOCH I HCOH I HCOH I
CHpOH
NHR
I I
HCOH
-
CH,OH
- HpO
-
+HpO
HOQNHR
I
HCOH
OH
I
~H,OH 35
74
75
SCHEME 14.-Forrnation of N-Substituted Aldosylarnine by the Carbonyl-Arnine Reaction. ,
The next step of the Maillard sequence involves the conversion of the N-substituted aldosylamine (74 75) into a 1-amino-1-deoxy-2-ketose (79) by the Amadori rearrangement (see Scheme 15).189J90 Amino acids react with ketoses to form 2-amino-2-deoxyaldoses by the Heyns rearrangement,191which is closely allied to the Amadori rearrangement. As already mentioned, these rearrangements are acid-catalyzed, and involve prototropic shifts. An alternative pathway, which invokes a carbonium (186) J. E. Hodge, J . Agric. Food Chem., 1 (1953) 928-943. (187) G . P. Ellis and J. Honeyrnan, Adv. Carbohydr. Chem., 10 (1955) 95-168. (188) C. Chavis, C. de Gourcy, F. Durnont, and J.-L. Irnbach, Carbohydr. Res., I13 (1983) 1-20.
(189) J . E. Hodge, Adu. Carbohydr. Chem., 10 (1955) 169-205. (190) T. M. Reynolds, Adu. Food Res., 14 (1965) 168-283. (191) H. H. Baer, Fortschr. Chem. Forsch., 3 (1958) 822-910.
HIGH-TEMPERATURE TRANSFORMATION O F CARBOHYDRATES NHR
I
+
t
HC - OH2
74
' Ht -
H'
I HCOH I HOCH I HCOH I HCOH I
CH2OH
76
309
CH=NHR
d 7
I HCOH I HOCH I HCOH I HCOH I
CHNHR
10,
- HC
+
I
HOCH
I
HCOH
v -
I
HCOH
I
CH20 H
CYOH
n
78
SCHEME 15.-Mechanism
CHrNHR
I c=o I HOCH I HCOH I HCOH I
CHpOH
79
of the Amadori Rearrangement.
ion on C-1 of 77, rather than the iminium ion, has also been proposed.'92 The Amadori rearrangement has been demonstrated with primary and secondary a m i n e ~ ,as ' ~well ~ as with amino acids.'94The aminodeoxyketoses (79) formed with primary amines can react with a second aldose molecule. This allows a further Amadori rearrangement, and formation6s of a diketosamine (81). The diketosamine undergoes enolization, followed by a p-elimination, to afford 3-deoxyhexos-2-ulose and l-amino-ldeoxyket~se'~'(see Scheme 16). Cleavage of di-(l-deoxy-D-fructos-1y1)glycine in deuterium oxide failed'" to show incorporated deuterium in (39). Thus, it the 3-methylene group of 3-deoxy-~-erythro-hexos-2-ulose was suggested that a 1,2-hydride shift occurs, in preference to enolization, for the formation of 39, but further examination of the deuterium experiments, monitored by n.m.r. spectroscopy, indicated incorrect spectral as'signments, and confirmed that enolization was most probable during this reaction.'97 Electron-spin resonance (e.s.r.) spectra with characteristic hyperfine structure have been recorded during the initial stages of the Maillard reaction between various sugar and amino c o r n p o ~ n d s . The ' ~ ~ products responsible for the spectra appear to be N,N'-disubstituted pyrazine radical cations. The pyrazine derivatives are assumed to be formed by the bimolecular condensation of two- and t h r e e - c a r b ~ nenaminol '~~~ compo(192) A. Gottschalk, in A. Gottschalk (Ed.), Glycoproteins, 2nd edn., Vol SB, Elsevier, Amsterdam, 1972, pp. 141-157. (193) J. E. Hodge and C. E. Rist, J . Am. Chem. Soc., 74 (1952) 1494-1497. (194) K. Heyns and H. Breuer, Chem. Ber., 91 (1958) 2750-2762. (195) E. F. L. J. Anet, A m . J. Chem., 13 (1960) 396-403. (196) G. Fodor and J.-P.Sachetto, Tetrahedron Lett., (1968) 401-403. (197) E. F. L. J. Anet, Tetrahedron Lett., (1968) 3525-3528. (198) M. Namiki and T. Hayashi, in Ref. 178a, 81-91; and Ref. 178b, 21-46. (198a) T. Hayashi and N. Namiki, in Ref. 178c, 29-38.
3 10
OLOF THEANDER AND DAVID A. NELSON
fHO c=o
I
c=o
I
R
I
I
HOCH
CHZ
I
HCOH HCC H ,'"
II CoH I HOCH I
CHZNHR
I
'i="
/
HOCH
HCOH
I
CH,OH
I I
HCOH
79
39
HCOH
I
R
I
CHzOH
I
CHZOH
% so
I I HCOH I CHZOH HCOH
t
I
CHz'
'CH2
I
I
c=o
c=o
I I HCOH I HCOH I CHZOH
I
HOCH
HOCH
I
,IoH I
H OH
CHZOH
81
SCHEME16.-Mechanism
for the Cleavage of Diketosarnines.
nents involving the amino reactant. Product concentrations during the preliminary stages of the Maillard reaction suggested a sequence in which a glycosylamine was initially formed, followed by partial cleavage to reducing fragmentation. It was shown that N,N'-dialkylpyrazones, or mixtures of glycolaldehyde with amino compounds were highly active in free radical formation as well as in browning. Production of C2 and C3 sugar fragments in a D-glucoselp-alanine system was negligible in acidic mixture but increased with pH in a manner parallel to the increase in browning.198a The results indicated that the proposed pathway plays an important role in the initial stages of browning in the Maillard reaction under neutral and alkaline conditions. (39) can be Scheme 16 shows that 3-deoxy-~-erythro-hexos-2-ulose formed by the cleavage of a diketosamine. Such an intermediate has already been implicated in the formation of S-(hydroxymethy1)-2-furaldehyde (see Scheme 7). Interest in 39 and its formation is directed towards its role in the production of two major groups of products observed in
HIGH-TEMPERATURE TRANSFORMATION O F CARBOHYDRATES
3I I
hydrothermolyzed biomass. These are the oxygen and nitrogen heterocyclic compounds. Flavor and aroma technologies have been concerned with volatile nitrogen and oxygen heterocycles produced by the Maillard reaction. Although several of these compounds may be present in hydrothermolyzed biomass, the extensive literatureIw will not be covered. Only that research which can be related to general mechanisms involving formation of the heterocycles will be discussed. During the course of the Amadori rearrangement, the 1 ,Zenolamine intermediate (82) can rearrange to form 3-deoxyhexos-2-ulose (39) under aqueous conditions while releasing the amine or, more importantly, the amino acid200(see Scheme 17). The amino acid can undergo decarboxylt
I I
HCOH
I I
CHO
hoH CH
COH
I
COH HOCH
CH=NRR'
t Hi-
*
-Ha
HCOH CH20H 82
SCHEME 17.-Formation ment.
I I HCOH I
HCOH
CH20H
i- H20
CHO
I c=o
I
I!
CH
-a
A 7
HLOH
I I
HCOH CH20H
I I HCOH 1 CH2
HCOH
I
CH20H
83 38 39 of 3-Deoxyhexo-2-dose by Way of the Amadon Rearrange-
ation to an aldehyde by the Strecker degradation. In fact, isovaleraldehyde, formed from L-leucine, has been used as an early indication of the Maillard reaction during thermal processes.2o0Not only does decarboxylation of the amino acid occur, but the amino group may be transferred to a carbonyl compound which is thereby reduced.201The carbonyl compound may contain an a-dicarbonyl grouping, such as 3-deoxy-~-erythro-hexos2-ulose (39). Cyclization of the resulting amino saccharide (for instance, derived from D-fructose and L-alanine in slightly acidic solution at reflux), or some of its dehydration products, yields various heterocyclic components,202such as the acylpyrroles, 5-methylpyrrole-2-carboxaldehyde(84) (199) S. Fors, in Ref. 178b, pp. 185-286. (200) K . Eichner and M. Ciner-Doruk, in Ref. 178a, pp. 115-135. (201) T . Nyhammar, K. Olsson, and P.-A. Pernemalm, in Ref. 178b, pp. 71-82. (202) P. E . Shaw and R. E. Berry, J. Agric. Food Chem., 25 (1977) 641-644.
OLOF THEANDER AND DAVID A. NELSON
312
f,="
84
85
87
86
39
88
SCHEME 18.-Mechanism for Acylpyrrole formation from 3-Deoxyhexos-2-dose by the Strecker Degradation.
and 2-acetylpyrrole (86) (see Scheme 18). Methylpyridinols, such as 6methyl-3-pyridinol (87), 2-methyl-3-pyridinol(88), and the acylpyrroles, 84 and 86, were obtained by refluxing D-glucose and glycine under acidic The results of labelling experiments with conditions (pH 2, 3, and 6).201,203 D-( l-13C)glucosewere consistent with the proposed mechanism for the formation of 84, shown in Scheme 18. However, little or no 86 was formed after 3-deoxy-~-erythro-hexos-2-ulose was subjected to the same acidic refluxing conditions. A mechanism that does not require 39, or its derivatives, as an intermediate has been proposed.203Elimination of the 2-hydroxyl group of D-glucose to afford the enamine (91) of 2-deoxy-~-urubino-hexose is assumed to occur by lactonization of the imine formed from glycine and D-glucose. A related, but less-direct route to 91 was also proposed. Although 2-deoxy-~-urubino-hexosewas not detected in the reaction mixtures, it was assumed that this was due to much faster pelimination involving dehydration of the enamine 91 compared to its hydrolysis. This process, leading to the formation203of 86 and 88, is presented in Scheme 19. As Scheme 19 indicates, the reaction between glycine (89) and D-glucose is quite dependent upon a number of sequential steps, any one of which can be misdirected to another product. This complexity was found (203) T. Nyhammar, K. Olsson, and P.-& Pernemalm, Acra Chem. Scand., Ser. B, 37 (1983) 879-889.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
3 I3
H3N CHzCO; CH=NCH2COF
89
I
t
CHO
I I
HCOH HOCH HlOH HCoH
I
- HCoH
HojH HCOH I I
HCOH
CHNH,
CH=NH
CH,NHz
CHzNH2
CH
CH
CH
CH
I I HOCH I - I HCOH
I
HCOH
I
I
I
CH HCOH
I
HCOH
I
CH2OH
CH zOH
CHzOH
90
91
92
I II
Amadori
CH
Steps
C=O
-
I
I
HCOH
I
CH20H
93
I II
- I
fl
86
CH c=o
I I
88
c=o CH3
94
CHzOH
35
SCHEME 19.-Mechanism for Formation of 2-Acetylpyrrole and 2-Methyl-3-pyridinol.
in the reaction between glycine and D-glucose in slightly acidic, aqueous solution. The products included D-fructose, furans, pyrroles, pyridines, phenols, carboxylic acids, and lactones.zMIt is notable that metasaccharinic acids (isolated as lactones), normally formed under alkaline conditions, were formed in acid in the presence of glycine. The pyrroles formed included not only 86 and 88, but also 2-formyl-5-(hydroxymethyl)1-methylpyrrole (98), 2-formyl-5-methylpyrrole-1-acetic acid, 2-formyl-5(hydroxymethy1)pyrrole-1-acetic acid (96), and 6-formyl-3,4-dihydro-3oxopyrrolo[1,2-a]pyrazine-2(1H)-acetic acid (97). Compounds 96 and a7 are of interest, as a bicyclic heterocycle had been formed from the original pyrrole by addition of a second molecule of glycine (see Scheme 20). The structures of 2-formyl-5-methylpyrrole- 1-acetic acid and 96 have been confirmed by synthesis.z052-Formyl-5-(hydroxymethyl)-l-methylpyrrole (98) had previously been observed as a product of the interaction of glycine and D-fructose.zMSeveral other bicyclic compounds were isolated from the reaction of D-glucose with glycineZMor methylamine,z07including furans, pyrroles, pyridines, and a pyrogallol derivative. The reaction between L-rhamnose and ethylamine producedz0*l-ethyl2-formyl-5-methylpyrrole and 2-acetyl-1-ethylpyrrole. Pyrroles such as 2formyl-5-(hydroxymethyl)-l-methylpyrrole (98) have also been detected (204) K. Olsson, P.-A. Pernemalm, and 0. Theander, Acta Chem. Scand., Ser. B , 32 (1978) 249-256. (205) K. Olsson and P.-A. Pernemalm, Acfa Chem. Scand., Ser. B , 32 (1979) 125-132. (206) H. Kato, H. Sonobe, and M. Fujimaki, Agric. Biol. Chem., 41 (1977) 711-712. (207) K. Olsson, P.-A. Pernemalm, T. Popoff, and 0. Theander, Acra Chem. Scand., Ser. B, 31 (1977) 469-474. (208) H. Kato, H. Shigematsu, T. Kurata, and M. Fujimaki, Agric. Biol. Chem., 36 (1972) 1639-1642.
3 I4
OLOF THEANDER AND DAVID A. NELSON
H'OH LOH
39
+ 89
+
How*cHo 96
SCHEME20.-Formation
97
of a Dihydropyrrolopyrazine.
as products of the reaction of D-glucose with methylamine in dilute acetic a ~ i d .The ~ ~anhydro ~ - ~ dimer ~ of 98, formed by dehydration of the 5hydroxymethyl group, was also observed.207An analogoue of 98 was obtained from the reaction of D-glucose and L-lysine.2'0*211 This product, 6-[2-formyl-5-(hydroxymethyl)pyrrol1-yllnorleucine (99), was formed in 2.1% yield at pH 4.5and 100". Only the 6-amino group was involved in the formation of this Maillard reaction-product. Several other compounds, such as 84 and 86-88 were present, but they represented utilization of the a-amino
98
99
100
(209) G. R. Jurch and J. H. Taturn, Curbohydr. Res., I5 (1970) 233-239. (210) R. K. Miller, K. Olsson, and P.-A. Pernernalrn, Acta Chem. Scund., Ser. B , 38 (1984) 689-694. (211) H. Kato, T. Nakayarna, S . Sugirnoto, and F. Hayase, Agric. BidChern., 46 (1982) 2599-2600.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
3 I5
Aqueous solutions of L-proline and D-glucose have been exposed to a temperature of 150" in order to prepare a series of unique products that included the pyrrolidine ring of p r ~ l i n e . ~ 'Of ~.~ considerable '~ interest was214the formation of 2,3,6,7-tetrahydro-7-methylcyclopent[b]azepin8(lH)-one (100). It was shown that cyclic a-dicarbonyl compounds (in equilibrium with enolones) appeared to act as precursors, in conjunction with L-proline, by way of a Strecker degradation to afford the azepines. It was suggested that the cyclic a-dicarbonyl compounds are formed during the Maillard reaction by an aldol reaction from glycolaldehyde, pyruvaldehyde, and other components, produced by retro-aldol cleavage of D-glucose. A series of cyclic enolones, such as 2-hydroxy-3,4-dimethyl-2-cyclopenten-1-one 34, were heated with L-proline or pyrrolidine, to gain evidence as to the mechanism. Several products, based on the 2-( 1-pyrrolidinyl)-2-~yclopenten1-one structure, were isolated. Furthermore, the concentration of the cyclopent[b]azepines was increased by three orders of magnitude when the cyclic enolones were substituted for saccharides and combined with L-proline. Studies were also conducted with hydroxy-L-proline and D-glucose or ~ - r h a m n o s e , *as~ ~ well as Larabinose and D-erythrose,215 in water at 100- 150". Hydroxy-L-proline combines with a-dicarbonyl compounds, forming iminium carboxylate intermediates that are decarboxylated and dehydrated to 1-pyrrolyl-2-oxo product^.^'^ For example, pyruvaldehyde, 2,3-butanedione and 1,2-cyclopentanedione, respectively yielded 1-acetonylpyrrole, 3-(l-pyrrolyl)-2butanone and 2-(1-pyrrolyl)cyclopentanone.No pyrazines were observed in the hydroxy-L-proline reaction-mi~tures.~'~ Alkylated pyrazines are generally formed when aqueous solutions of saccharides plus amino acids are heated at reflux. These products have and several been implicated as potent odorants in cooked roasted food D-Glucose or D-fructose in combination with Lasparagine or L-glutamic acid was examined2I7for total pyrazine content after dissolution in aqueous diethylene glycol at 120". Labelling experiments with D-[1-14C]glucoseand L-alanine or. L-asparagine indicated that (212) R. Tressl, K. Griinewald, and B. Helak, in P. Schreier (Ed.) Flavour 81, Weurman Symp., 3rd, Walter de Gruyter, Berlin, 1981, pp. 397-416. (213) R. Tressl, B. Helak, H. Koppler, and D. Rewicki, J . Agric. Food Chem., 33 (1985) 1132-1137. (214) R. Tressl, K. G. Griinewald, E . Kersten, and D. Rewicki, J. Agric. Food Chem., 33 (1985) 1137-1 142. (215) R. Tressl, K . G. Griinewald, E . Kersten, and D. Rewicki, J . Agric. Food Chem., 34 (1986) 347-350. (216) P. Marce and D. Hadziyev, Can. Znst. Food Sci. Technol. J . , 10 (1977) 272-279. (217) P. E. Koehler, M. E. Mason, and J. A. Newell, J. Agric. Food Chem., 17 (1969) 393396.
OLOF THEANDER AND DAVID A. NELSON
316
TABLEI Pyrazines Formedfn from the Reaction of D-Glucose(G1c) with Glycine(G1y) at 300" and pH 4.5 Yield (wt. % of total product) Glc: Gly(1 M :2 M ) 1omin
Pyrazine
Smin
MethylDimethyl- (2,5- and 2,6) 2,3-Dimethyl- and ethylTrirnethylTetrarnethylOther C,-alkyl-
0.30 0.80 0.34 2.18 0.61 0.10
0.33 0.85 0.39 2.21 0.57 0.09
Glc: Gly(1 M :1 M) Smin 10 min 0.25 0.38 0.11 0.30 0.05 0.03
0.30 0.48 0.13 0.41 0.06 0.04
the carbohydrate was the prime source of the carbon atoms in the product, and that the amino acid furnished only the nitrogen atom to the pyrazines. Fragmentation of the hexoses into C2-C4 units prior to pyrazine incorporation seems a most probable mechanism. The proportions of methyl- and dimethyl-pyrazine formed by D-glucose with various amino acids has been L-Asparagine produced the highest proportions of these two pyrazines, whereas glycine gave very little alkylpyrazine in the aqueous diethylene glycol system. The yields of alkylpyrazines increased with the addition of equimolar amounts of sodium hydroxide to the saccharide-amino acid solutions. Acetaldehyde and aqueous ammonia produced large proportions of unsubstituted pyrazine. However, when acetaldehyde reacted with L-asparagine, methyl- and dimethylpyrazines were also produced. The reaction of glycine and D-glucose at 300" was investigated in buffered acid.219The results concerning the formation of pyrazines are presented in Table I. The additional glycine, in the molar ratio of 2 : 1 with D-glucose, provided a higher proportion of pyrazines compared with stoichiometric reactions of the two components. This is particularly evident for the formation of trimethylpyrazine. Other components identified in these reaction mixtures were 5, 11,17, and 23.
(218) P. E. Koehler and G . V. Odell, J . Agric. Food Chem., 18 (1970) 895-901. (219) D. A. Nelson, R. T. Hallen, and 0. Theander, Absfr. Pap. Norfhwest Regional Meet. A m . Chem. SOC.,41st, Portland, Oregon, 1986, Abstr. 297.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
3 17
Several hexoses and pentoses were refluxed in 8 M aqueous ammonia.220The pentoses gave slightly larger yields of pyrazines than did the hexoses, but the types of pyrazines from both groups of saccharides were similar. Several possible pathways of formation for the pyrazines have been suggested, using various a-amino carbonyl intermediates.220*221 Scheme 21 shows the formation of 2-methylpyrazine (104) from two proposed fragments. The formation of the necessary carbonyl groups of the small fragmented compounds was assumed to be due to a retro-aldol reaction.220A mechanism has also been advanced for the formation of pyrazines by ammonia addition to cyclopentenone derivatives, and a subsequent condensation with a-amino carbonyl compounds.222
101
102
103
104
SCHEME21 .-Formation of 2-Methylpyrazine from Saccharide Fragments.
Studies have been conducted with the products obtained from refluxing A series of indoaqueous solutions of D-glucose with ~-phenylalanine.~~~ lines was identified; these included 3-phenylindoline (105), l-phenethylindoline (106), and 2-(indolin-l-yl)-3-phenylpropanoic acid (107). Several aldehydes formed by the Strecker degradation were also identified. The D-glucose-L-phenylalanine reaction was further in 1-octanol at 100-160". Several pyrroles were identified among the low-molecularweight products. The combination of DL-cysteine with D-galactose, in refluxing methanol containing a small proportion of pyridine and water, provided225the diastereomers (2S,4S and 2R,4R) of 2-(~-gulucto-hydroxypentityl)-4thiazolidinecarboxylic acid (108). The diastereomers can be separated by recrystallization, and then cleaved with acid, to yield resolved D- or Lcysteine. An investigation was conducted concerning the reaction be(220) T. Shibamoto and R. A. Bernhard, J . Agric. Food Chem., 25 (1977) 609-614. (221) T. Shibamoto and R. A. Bernhard, Agric. Biol. Chem.. 41 (1977) 143-153. (222) T. Shibamoto and R. A. Bernhard, J . Agric. Food Chem., 26 (1978) 183-187. (223) G . Westphal and E. CieSlik, Nuhrung, 26 (1982) 765-776. (224) G . Westphal and E. CieSlik, Nuhrung, 27 (1983) 55-62. (225) J. Martens and K. Drauz, Justus Liebigs Ann. Chem., (1983) 2073-2078.
OLOF THEANDER AND DAVID A. NELSON
318
105
tween L-cysteine and 4-hydroxy-2,5-dimethyl-3(2H)furanone(58) in water at 160".226Two thiophenes, 3-methyl-2-(2-oxopropyl)thiophene(109) and 2-methyl-3-propanoylthiophene(110), as well as 2,4-hexanedione, were the major volatile products. A mechanism was proposed for the formation of 109 and 110 via 2,4-hexanedione and 2-mercaptoacetaldehyde, both of which are present in the reaction mixture.
4 : Y HrioH
0
HO H
'I
H OH
I
QCkCCY CY
108
109
CWH
QCY
ccwcy
a
110
The formation of oxygen-containing heterocyclic compounds is also a consequence of the Maillard reaction. Amines and amino acids have a catalytic effect upon the formation of 2-furaldehyde (5),la65-(hydroxymethyl)-2-furaldehyde ( l l ) , I a 6 2-(2-hydroxyacetyl)furan (44),207and 4-hydroxy-5-methyl-3(2H)-furanone (111) (see Ref. 214). This catalytic effect can be observed with several other non-nitrogenous products, including m a l t 0 1 . ~The ~ ~ amino acid or amine catalysis has been attributed to the transient formation of enamines or immonium ions,Is3or the 1,2-2,3 enolization of carbohydrate^.^'^ Of interest is the detection of N-(Zfuroyl(226) C.-K. Shu, M. L. Hagedorn, and C.-T. Ho, J . Agric. Food Cbem., 34 (1986) 344-346. (227) J. E. Hodge, B. E. Fisher, and E. C. Nelson, Proc. Am. Soc. Brew. Cbem., (1963) 8492.
HIGH-TEMPERATURE TRANSFORMATION OF CARBOHYDRATES
3 I9
methyl)-L-alanine in acidic aqueous solutions of D-fructose and L-alanine after heating228at 90". It was assumed that formation of this compound followed a route somewhat similar to that of 2-(2-hydroxyacetyl)furan (44);that is, indicating an initial 2,3-enolization of D-fructose, followed by elimination of water (Scheme 8).59
111
The formation of 5-(hydroxymethyl)-2-furaldehyde(11)from Amadori compounds (79) such as 1-deoxy-1-p-toluidino-D-fructosein deuterium oxide has been described.229Although the product of this reaction is the same, namely, 11, as that obtained from the acidic dehydration of Dglucose, the pathway is not quite the same. Yields of 11from 79 are much higher than those from D-glucose, and the conditions for the reaction are considerably milder when using the Amadori compounds. There is apparently considerable equilibration between the 1-amino-1-deoxy-2-ketose (79) and its 1,Zen01 (82), and between 3-deoxyhexos-2-ulose (39) and its enol (38) during the dehydrations (see Scheme 7).230The weak acid, usually acetic acid, favors these equilibria more than does a strong acid. Thus, due to the presence of the amine substituent, 79 undergoes 1,2enolization to 82 much more readily than does a corresponding, unsubstituted sugar (D-glucose to 36, for instance). The intermediates (82,83, and 39) are more stable in weak acid, which increases the possibility of their equilibration with tautomers and the incorporation of solvent protons. Once 3-deoxyhexos-2-ulose (39) is formed, the sequence shown in Scheme 7 can contribute, until the final dehydration to 11.Similar results concerning the equilibrations were obtained with the Amadori products of pentoses and hexuronic acids, using tritiated water and deuterium oxide.231In these reactions, distribution of the respective isotopic label was determined for labeled 2-furaldehyde (5). The pentose-derived 5 contained 14% of deuterium at C-3, 5% at C-4, and none at C-5, whereas the hexuronic acid-derived 5 contained 50, 44, and 7% at C-3, C-4, and C-5, (228) K . Heyns, J. Heukeshoven, and K. H. Brose, Angew. Chem., Int. Ed. Engl., 7 (1968) 628-629. (229) M. S. Feather and K. R. Russell, J . Org. Chem., 34 (1969) 2650-2652. (230) M. S. Feather, in Ref. 178a, pp. 37-45. (231) K . B. Hicks and M. S. Feather, Carbohydr. Res., 54 (1977) 209-215.
320
OLOF THEANDER AND DAVID A. NELSON
respectively. Thus, considerable equilibration occurs prior to the formation of 5 , in contrast to that observed for unsubstituted pentoses and hexuronic acids. It had been suggested that 4-hydroxy-5-methyl-3(2H)-furanone(111) can be formed from a pentose and an amine by way of an Amadori compound and its 2,3-enoli~ation.~~~ Moreover, 111 has also been preacid pared from l-deoxy-l-(dibenzylamino)-~-arabino-2-hexulosuron~c (112), which was synthesized from dibenzylamine and D-glucuronic This indicates that two different Amadori compounds can undergo 2,3-enolization in weak acids) to afford 111. In strong acid, both Amadori compounds undergo 1,2-enolization to afford 2-furaldehyde. Three Amadori compounds were exposed to weak and strong acidic conditions; these were 112, l-(benzylamino)-l-deoxy-~-arabino-2-hexulosuronic acid, and 1-(benzy1amino)-1-deoxy-~-threo-2-pentulose.~~~ All three compounds produced 2-furaldehyde in strong acid, although the highest yield Howwas obtained from l-(benzylamino)-l-deoxy-~-threo-2-pentulose. ever, only 112 produced the furanone 111 under mildly acidic conditions. This confirms that, when the Amadori compounds are almost completely protonated, 1,2-enolization occurs, and 2-furaldehyde is formed. Under mildly acidic conditions, the (less-protonated) amino group allows 2,3enolization, and formation of 111,to predominate. Compound 112,a tertiary amine, is the least basic and, thus, the least protonated in mild acid. l-14C]-arabLabeling experiments with 1-deoxy-l-(dibenzylamino)-~-[ ino-2-hexulosuronic acid [ 1-14C] 112 indicated that the 14C label corresponded to the 5-methyl group of 111 (see Ref. 234). This is also consistent with a l-deoxy-2,3-dicarbonyl intermediate (115),and indicates that 111 is a decarboxylation product (see Scheme 22). The precise step entailing decarboxylation has not yet been determined. The carboxyl group could be carried through to ring closure (furanone formation). Such a step would provide a 2-carboxylate which is a p-keto acid subject to ready decarboxylation. The labeling information and the initial steps of the mechanism in Scheme 22 are also consistent with the formation of 111 from D-[ l-I4C]ribose and a secondary amine.232 3-Hydroxy-2-methyl-4H-pyran-4-one (maltol) and 2-acetyl-3-hydroxyfuran (isomaltol) are also products of the Maillard reaction.227Both have a considerable history, due to their early detection in beer and breads. A mechanism based upon the pyranose form of a methyl-a-dicarbonyl inter(232) H. G. Peer, G. A. M. van den Ouweiand, and C. N . deGroot, R e d . Trau. Chim. PaysBas, 87 (1968) 101 1-1016. (233) K . B. Hicks, D. W. Hams, M. S . Feather, and R. N. Loeppky, J . Agric. Food Chem., 22 (1974) 724-725. (234) K . B. Hicks and M. S. Feather, J . Agric. Food Chem., 23 (1975) 957-960.
HIGH-TEMPERATURE TRANSFORMATION O F CARBOHYDRATES CHZNRR'
I c=o I
HOCH
I
HCOH
I
HCOH
I
C02H
-
CHZNRR'
CHZ
COH
COH
I
II
COH
I I HCOH I HCOH
COZH
112
SCHEME 22.-Mechanism
CH3
I1
1 I
C=O
,
-
I
c=o
I
C=O
I I
HCOH
HCOH
HCOH
HCOH
I
I CO,H
32 I
-co, -111
I
COZH
1l3 114 115 for the Formation of 4-Hydroxy-S-methyl-3(2H)-furanone.
mediate for maltol formation has been suggested.227Other products of the Maillard reaction, which apparently require a similar mechanism, include 4-hydroxy-S-methy1-3(2H)-furanone(lll),2,3-dihydro-3,5-dihydroxy-6methyl-4-pyranone, and a c e t y l f ~ r m o i nThe . ~ ~ ~reaction between L-lysine and D-glucose affords 2-furanmethanol and 2,3-dihydro-3,5-dihydroxy-6methyl-4-pyranone, as well as several pyrroles and D-fructose-L-lysine condensation products.2" 5-(Hydroxymethyl)-2-furaldehyde(11)and several pyrroles were detected236in the reaction mixture after D-glucose and butylamine were refluxed in aqeuous solution at 95". Several other non-nitrogenous products have been identified as products of the Maillard reaction. These include butanol, butanone, butanedione, and ~ e n t a n e - 2 , 3 - d i o n eas ~ ~well ~ ~ , ~as dihydroxyacetone, glyceraldehyde, and ~ - e r y t h r o s eObviously, .~~~ the same products are present after mild acidic or basic degradation of carbohydrates. Thus, the necessity of an amine or amino acid in the mechanism of their formation is uncertain. Dihydroxyacetone forms dimeric ketosylamines when it reacts with primary amines at low temperatures .238 However, the reaction of dihydroxyacetone with amino acids apparently generates pyruvaldehyde (23) as an intermediate for several products, including allomaltol (5-hydroxy2-methyl-4-pyranone). In contrast to other amino acids, glycine reacts with dihydroxyacetone to yield a preponderance of butanedione. (235) J. E. Hodge, F. D. Mills, and B. E. Fisher, Cereal Sci. Today, 17 (1972) 34-40. (236) F. Hayase and H. Kato, Agric. B i d . Chem., 49 (1985) 467-473. (236a) F. Hayase and H. Kato, in Ref. 178c, pp. 39-48. (236b) G. MacLeod and J. M. Ames, in Ref. 178c, pp. 263-272. (237) T. Severin, J. Hiebl, and H. Popp-Ginsbach, Z. Lebensm. Unters. Forsch., 178 (1984) 284-287. (238) K. Heyns, U. Sage, and H. Paulsen, Curbohydr. Res., 2 (1966) 328-337.
322
OLOF THEANDER AND DAVID A. NELSON
An effort has also been made to determine the structure of products providing coloration in the Maillard reaction prior to melanoidin formation. The reaction between D-xylose and isopropylamine in dilute acetic acid produced2392-(2-furfurylidene)-4-hydroxy-5-methyl-3(2H)-furanone (116). This highly chromophoric product can be produced by the combination of 2-furaldehyde and 4-hydroxy-5-methyl-3(2H)-furanone (111) in an aqueous solution containing isopropylammonium acetate."O The reaction between D-xylose and glycine at pH 6 , under reflux conditions, also prod u c e ~116. ~ ~ Other ] chromophoric analogs may be present, including 117,
@IH 117
118
because 116 can be readily condensed wit.. 2 - f ~ r a l d e h y d e.. ~chromo~~ phoric furfurylidene-P-pyranone (US), similar to the furanone 116, was isolated after D-xylose had been heated with glycine in aqueous methanol.242It was proposed that 118 could be formed in the manner suggested for the pyranone (120) obtained from D-glucose and glycine (see Scheme 23). Under acidic reaction conditions, N-substituted 1-arnino-l-deoxy-Dfructose (79) loses its amino component (see Scheme 17) to yield 39, which can form both 11 and 2-hydroxy-6-(hydroxymethyl)-3(2H,6H)-
b
Howcc -
-W
CH$H
'19
t
HO CW ,H )(O .
__+
"YF
0cy
H
O
M
~
C
H
\ / 39
SCHEME23.-Formation (239) (240) (241) (242)
11 120 of a Furfurylidene-P-pyranonein the Presence of Methanol.
T. Severin and U. Kronig, Chem. Mikrobiol. Technol. Lebensm., 1 (1972) 156-157. F. Ledl and T. Severin, 2. Lebensm. Unters. Forsch., 167 (1978) 410-413. H. E. Nursten and R. O'Reilly, in Ref. 178b, pp. 103-121. F. Ledl, J. Hiebl, and T. Severin, 2. Lebensm. Unters. Forsch., 177 (1983) 353-355.
HIGH-TEMPERATURE TRANSFORMATION O F CARBOHYDRATES
323
pyranone (119). Compounds 11 and 119 then condense together, to give 120.
V. CARBOHYDRATE TRANSFORMATION IN CHEMICAL PROCESSES, INCLUDING HUMUSFORMATION
In some chemical processes, including natural humus formation, nonenzymic carbohydrate transformation, in aqueous media, into noncarbohydrate products is important. Improvements can probably be made in the yield and economy of certain compounds, including furans and phenolics, from carbohydrates. These compounds can be sources for various technical products, such as solvents, pesticides, plastics and other polymers, liquid fuels, and asphalt substitutes. 2-Furaldehyde (5) is an example of a carbohydrate-based compound of technical importance for various products. A promising use of the related 5-(hydroxymethyl)-2-furaldehydethat saves up to 40% of the phenol in resins has been reported.243Some phenolic products discussed in this Chapter might also be useful compounds, if further research can improve their yields and processing. Production of liquid fuels and other product mixtures by thermal liquefaction, particularly from the abundant lignocellulosic biomass sources, is an interesting alternative to total gasification or fermentation processes. Further research in processes, and a better understanding of the chemical reactions involved, might improve yields and quality by lessening inevitable degradation and recombination of reactive intermediates formed during liquefaction. The liquefaction process has considerable relation to the important, but much slower, formation of humus from plant materials in Nature. Early studies of sphagnum mosses and peat samples of different ages strongly indicated that the humic part, increasing with age, mainly originated from carbohydrates2@and not from lignin or other polyphenolic components which amounted to only -1% in the original mosses.245However, lignin may be the more important source of humus during the humification of wood and other lignocellulosic sources. The dried mosses contain up to 85% of polysaccharides, with high contents of pentoses and uronic acids. Under slightly acidic conditions, pentoses and uronic acids are converted into enones and phenols, which readily react to form dark-colored polyacid mers. 246 The presence of very reactive ~-lyxo-5-hexosulopyranuronic (243) (244) (245) (246)
H. Kock, F. Kranse, R. Steffen, and H. U. Woelk, Staerke, 35 (1983) 304-313. 0. Theander, Acta G e m . Scand., 8 (1954) 989-1000. 9. Lindberg and 0. Theander, Acta Chem. Scand., 6 (1952) 311. 0. Theander, in S. S. Stivala, V. Cresenzi, and I. C. M. Dea (Eds.), Recent Deuelopments in Industrial Polysaccharides, Gordon and Breach Science Publishers, New York, 1987, pp. 50-61.
324
OLOF THEANDER AND DAVID A. NELSON TABLEI1 Accelerated Aging of Filter Papers Impregnated with Model Compounds" Model compoundb
Brightness (% Scan)
None Methyl p-D-glucoside Cellobiose 5-(Hydroxymethyl)-2-furaldehyde(11) D-Ghcuronic acid Reductic acid (47)
86.8 85.9 83.5 74.3 37.4 19.4
L? The model compounds were exposedz4 for 16 h at 80" to 80% relative humidity. Proportion: (1.8 mmol per mmol of cellulose.)
residues,247together with D-galacturonic acid, in mosses supports the proposed importance of carbohydrate transformation in natural humus formation processes.II8 The presence of amino compounds is also important in these transformations, both as catalysts for carbohydrate degradation and as contributors to the chemical constituents of the humus.248 Dark-brown, water-insoluble polymers analyzed as lignin by the conventional, gravimetric, sulfuric acid-lignin method were isolated in large amounts, in addition to low-molecular acyclic and heterocyclic compounds in the Maillard reaction-systems containing D-glucose-glycineZM and ~-glucose-rnethylamine~~~ at pH 4.5 and 96". In thermal food-processes, formation of the favorable aroma compounds from carbohydrate transformation is a positive factor, but formation of off-flavors by over-processing, the loss of valuable amino acids and proteins, and the browning reaction often occurring are unwanted. In various cellulose processes, color formation is also a problem, often with lignin as the main cause. However, it has also been shown that carbohydrate transformation, either via partial hydrolysis of polysaccharides or the peeling reaction, is a factor contributing to yellowing of cellulosic materials by aging.246This is particularly important for bleached products and in sulfate (kraft) pulping. Table I1 provides some examples from accelerated aging of filter papers impregnated with aqueous solutions (pH 5.5) of different model compounds .246 A nonreducing carbohydrate, such (247) T. J. Painter, Curbohydr. Res., 124 (1983)c18-c21. (248) L. M. Benzing-Purdie, M. V. Cheshire, B. L. Williams, G. P. Sparling, C. I. Ratcliffe, and J. A. Ripmeester, J . Agric. Food Chem., 34 (1986)170-176.
HIGH-TEMPERATURE TRANSFORMATION O F CARBOHYDRATES
325
TABLEI11 Treatment" of Cellulose"plus Additives under Sulfate-Pulping Conditions Additive
Brightness of resulting pulp (70scan)
None Milled wood lignin Birch xylan Spruce glucomannan
80.1 65.6 69.5 72.2
Cellulose, 4 parts, and additive, 1 part.
as methyl-p-D-glucoside, and a reducing glucosaccharide, such as cellobiose, have very little effect on the color formation. The major acid-conversion product from hexoses, namely, 5-(hydroxmethyl)-2-furaldehyde (ll), provides a significant contribution to the color, but the conversion products from D-glucuronic acid, particularly reductic acid (47) contribute considerably more color. Furthermore, enones and phenols formed by carbohydrate transformation yield dark-blue or black complexes in the presence of ferric ions. The effect of color production from carbohydrate transformation in an alkaline pulping process, such as kraft pulping, is illustrated in Table 111. Cotton linters, which originally had a brightness of 90.5%, were treated for 4 h at 180"with kraft liquor, with and without additives.246Brightness was measured on paper made from the resulting, washed pulp. Sulfur dioxide and hydrogen sulfite solutions are the major additives for the prevention of discoloration of foods, but these additives are also important in some pulping processes. The treatment of cotton linters under the conditions of sulfite pulping, pH 2.1-8.0 and 135-160", for 2 h alone or with addition of birch xylan, L-arabinose, or D-glucuronic acid, respectively,245effectively stopped color formation in all of these experiments. As a comparison, cotton linters, alone or with birch xylan, Larabinose, or D-glucuronic acid, respectively, were treated with acetate buffer, pH 4.5,for 2 h at 160". In these experiments, the brightness respectively decreased to 84.7, 50.4, 35.8, and 18.3%. Most likely, the color-stopping reaction noted in food processing or sulfite pulping occurs through the formation of sulfohexosuloses that are further transformed into heat-stable sulfonic acids. 13032493250
(249) B. Lindberg, J. Tanaka, and 0. Theander, Acta Chem. Scand., 18 (1964) 1164-1170. (250) 0. Theander, in Ref. 178a, pp. 471-476.
326
OLOF THEANDER AND DAVID A. NELSON
There has been an increasing interest in the utilization of renewable resources for producing ethanol and other fermentation products. Such lignocellulosic materials as straw, bagasse, and wood waste have been used as fermentation sources, in addition to materials rich in sucrose and starch. The lignocellulosic materials are much more difficult to fractionate and hydrolyze without losses of carbohydrates and incomplete hydrolysis. It is not within the scope of the present Chapter to discuss the considerable variation of pretreatment and hydrolysis procedures that are being examined in this field. However, it is generally impossible to avoid some degradation by acidic pretreatments, such as steaming, or by final acid hydrolysis. Thus, in particular, if these procedures involve the (more reactive) pentoses and uronic acid components from hemicellulose polysaccharides and pectins, the acidic procedures promote the formation of furans, enones, and phenolics that can lead to carbohydrate losses and browning. Furthermore, there is a risk of formation of inhibiting products in substantial concentrations that can disturb the following fermentation process. Catechols, for instance, are frequent carbohydrate transformation-products that react readily with proteins and are known enzyme inhibitors. Sucrose solutions that had been heated for up to 8 h at 121" had an inhibitory effect on a number of bacteria and yeasts.2s1It was also shown2'* that the antimicrobial activity formed in heated neutral solutions of D-glucose and D-fructose was the result of di- or trivalent phenols, several of which were the same as those previously isolated246from heattreated sugar solutions. Inhibition of bacterial growth by Maillard reaction-products has also been reported.253 ACKNOWLEDGMENTS The authors thank the U.S. Department of Energy, Office of Basic Energy Sciences, for support of this effort under contract DE-AC06-76RLO 1830, as well as the Northwest College and University Association for Science (NORCUS).
(251) D. C. Wilson and H. D. Brown, Food Techno/., 7 (1953) 250-256. (252) T. Suortti, Z . Lebensm. Unters. Forsch., 177 (1983) 94-96. (253) H. Einarsson, B. E. Snygg, and C. Eriksson, J . Agric. Food Chem., 31 (1983) 10431047.
ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY,
VOL. 46
ADDENDUM TO ARTICLE 3: REFERENCES PUBLISHED A m E R 1986 (ADDED AT PROOF STAGE)
BY KEVINB. HICKS GENERALAREA
SPECIFIC TOPIC AND REFERENCE Simple and complex carbohydrate~~~’ Laser-based refractive index det e c t o r ~ “Diol” , ~ ~ ~ silica gel phases,333Monoclonal antibodybased stationary phases334
Review articles Instrumentation and stationary phases Separations Neutral mono-and di-saccharides Ionic mono- and di-saccharides
Simple, neutral oligosaccharides Simple, ionic oligosaccharides
Kestoses and n y ~ t o s eFermen,~~~ tation-derived sugar Ascorbic acid-2-pho~phates,~~~ Sugar phosphates,338Inositol phosphate^^^^.^^^
Gluco-olig~saccharides,~~~~~~~~~ Polysialic turonic
Oligogalac-
(331) K. Kakehi and S. Honda, J. Chromatogr., 379 (1986) 27-55. (332) D. J. Bornhop, T. G. Nolan, and N. J. Dovichi, J. Chrornatogr., 384 (1987) 181-187. (333) M. Abbou and A.-M. Siouffi, J. Liq. Chromatogr., 10 (1987) 95-106. (334) J. Dakour, A. Lundblad, and D. Zopf, Anal. Eiochern., 161 (1987) 140-143. (335) P. C. Ivin and M. L. Clarke, J . Chromatogr., 408 (1987) 393-398. (336) R. A. Lazarus and J. L. Seymour, Anal. Eiochem., 157 (1986) 360-366. (337) G. L. Moore and R. M. Fishman, J . Chromatogr., 419 (1987) 95-102. (338) A. V. Smrcka and R. G. Jensen, Plant Physiol., 86 (1988) 615-618. (339) J. A. Shayman and D. M. BeMent, Eiochem. Eiophys. Res. Commun., 151 (1988) 114-122. (340) K. A. Wreggett and R. F. Irvine, Eiochem. J . , 245 (1987) 655-660. (341) G. Bonn, J. Chrornatogr., 387 (1987) 393-398. (342) K. Koizumi, T. Utamura, Y. Kubota, and S. Hizukuri, J. Chromatogr., 409 (1987) 396-403. (343) P. C. Hallenbeck, F. Yu, and F. A. Troy, Anal. Eiochem., 161 (1987) 181-186. (343a) K. B. Hicks and A. T. Hotchkiss, Jr., J. Chrornatogr., 441 (1988) 382-386. 327
328
KEVIN B. HICKS
Complex, neutral oligosaccharides Complex, ionic oligosaccharides Applications Analysis of food carbohydrates Analysis of carbohydrates in biomass conversion processes Compositional analysis of carbohydrate polymers Structural and sequence analysis of carbohydrates
Cyclic o l i g o ~ a c c h a r i d e s , ~ ~ ~ - ~ ~ ~ ~ ~ ~ N-linked o l i g o s a ~ c h a r i d e s , ~ ~ ~ J ~ ~ Heparin fragments349
Nanogram detection of sugars,35o L a c t ~ l o s ePectins343a ,~~~ Sugars, oligosaccharides, and degradation p r o d ~ ~ t s , ~ ~ ~ , ~ ~ Pectins343a Proteoglycan-derived carbohyd r a t e ~Lipopolysac,~~~ ~ h a r i d e sG, ~l y~c~o p r ~ t e i n s , ~ ~ ~ Food P o l y s a ~ c h a r i d e s ~ ~ ~ ~ ~ ~ ~ ~ N-Linked oligosa~charides,~~~~~~~ Degree of polymerization of neutral oligosaccharides,357 Degree of methylation and acetylation of pectin,358“High mannose” oligosa~charides~~~
(344) M. Benincasa, G. P. Cartoni, F. Coccioli, R. Rizzo, and L. P. T. M. Zevenhuizen, J. Chromatogr., 393 (1987) 263-271. (345) H. W. Frijlink, J. Visser, and B. F. H. Drenth, J. Chromatogr., 415 (1987) 325-333. (346) K. Koizumi, Y. Kubota, T. Utamura, and S. Horiyama, J. Chromatogr., 368 (1986) 329-337. (347) S . Hirani, R. J. Bernasconi, and J. R. Rasmussen, Anal. Eiochem., 162 (1987) 485492. (348) N. Tomiya, M. Kurono, H. Ishihara, S. Tejima, S. Endo, Y. Arata, and N. Takahashi, Anal. Biochem., 163 (1987) 489-499. (349) Y. Guo and H. E. Conrad, Anal. Eiochem., 168 (1988) 54-62. (350) R. A. Femia and R. Weinberger, J . Chromatogr., 402 (1987) 127-134. (351) I. Martinez-Castro, M. M. Calvo, and A. Olano, Chromatographia, 23 (1987) 132136. (352) E. Burtscher, 0. Bobleter, W. Schwald, R. Conch, and H. Binder, J . Chromatogr., 390 (1987) 401-412. (353) A. G . J. Voragen, H. A. Schols, M. F. Searle-Van Leeuwen, G. Beldman, and F. M. Rombouts, J. Chromatogr., 370 (1986) 113-120. (354) L. S. Lohmander, Anal. Eiochem., 154 (1986) 75-84. (355) I. W. Sutherland and A. F. D. Kennedy, Appl. Enuiron. Microbiol., 52 (1986) 948950. (356) M. Takeuchi, S. Takasaki, N. Inoue, and A. Kobata, J. Chromatogr., 400 (1987) 207-2 13. (357) M. Takagi, Y.Daido, and N. Morita, Anal. Sci., 2 (1986) 281-285. (358) A. G. J. Voragen, H. A. Schols, and W. Pilnik, Food Hydrocolloids, 1 (1986) 65-70. (359) S. Natsuka, S. Hase, and T. Ikenaka, Anal. Biochem., 167 (1987) 154-159.
ADDENDUM TO ARTICLE 3
Preparative I.c.
Special aspects Detectability and accuracy Post-column detection methods
Pre-column derivatives Combined I.c. techniques Future trends
329
14C-Labeledoligosaccharides, sugars, and sugar degradation products,341T r e h a l u l ~ s eGen,~~~ eral methods for mono- and disaccharides and sugar acids,361 Kestoses and n y ~ t o s e , ~Chitin ” oligosa~charides~~~ Laser-based refractive index det e c t ~ ~ -Cuprammonium ,~~* reagent ,333,363 4-Aminobenzoic acid reagent,350Indirect detection methods for cyclodexand sugar phosphates338 Reversible derivatization using 2amin~-pyridine~~ Direct coupling of 1.c. to f.a.b.m.s. for analysis of oligosaccharides365-367 Capillary I.c. of sugars,332Affinity separations of oligosaccharide~~~~
(360) D. Cookson, P. S. J. Cheetham, and E. B. Rathbone, J. Chromatogr., 402 (1987) 265-272. (361) K. B. Hicks, S. M. Sondey, and L. W. Doner, Carbohydr. Res., 168 (1987) 33-45. (362) K. B. Hicks, Methods Enzymol., 161B (1988) 410-416. (363) D. B. McKay, G. P. Tanner, D. J. Maclean, and K. J. Scott, Anal. Eiochem., 165 (1987) 392-398. (364) G. R. Her, S. Santikam, V. N. Reinhold, and J. C. Williams, J . Carbohydr. Chem., 6 (1987) 129-139. (365) P. Boulenguer, Y. Leroy, J. M. Alonso, J. Montreuil, G. Ricart, C. Colbert, D. Duquet, C. Dewaele, and B. Fournet, Anal. Eiochem., 168 (1988) 164-170. (366) Y. Ito, T. Takeuchi, D. Ishii, M. Goto, and T. Mizuno, J. Chromatogr., 391 (1987) 296-302. (367) S. Santikam, G. R. Her, and V. N. Reinhold, J . Carbohydr. Chem., 6 (1987) 141154.
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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 46
ADDENDUM TO ARTICLE 4
BY RENB CSUKA N D BRIGETTEI. GLANZER Additional n.m.r.-spectral data have been become available since about mid-1986. Further data for hexopyranosyl fluoride^,'^^^^ as well as for 2deoxy-2-fluoro-hexopyranoses and -hexopyranosides,200-2'0 have been reported by several groups, whereas only a few articles dealt with pentopyranose2@'or hexo(or pento)furanose analogs2@'*211 of these compounds. or C-4,213as well as at Monosaccharides fluorinated at C-3207,209,212 (193) I. P. Street and S. G. Withers, Can. J . Chem., 64 (1986) 1400-1403. (194) J. Thiem and M. Wiesner, Synthesis, (1988) 124-127. (195) S. J. F. Macdonald and T. C. McKenzie, Tetrahedron Lett., (1988) 1363-1366. (196) J. Thiem, M. Kreuzer, W. Fritsche-Lang, and H.-M. Deger, Ger. Offen. DE3528654A1 (1987). (197) P. KovBC, H. J. C. Yeh, G. L. Jung, and C. P. J. Glaudemans, J. Carbohydr. Chem., 5 (1986) 497-512. (198) M. Kreuzer and J. Thiem, Carbohydr. Res., 149 (1986) 347-361. (199) P. KovBC, H. J. C. Yeh, and G. L. Jung, J . Curbohydr. Chem., 6 (1987) 423-439. (200) K. Dax, B. I. Glanzer, G. Schulz, and H., Vyplel, Carbohydr. Res., 162 (1987) 13-22. (201) M. Tada, T. Matsuzawa, K. Yamaguchi, Y.Abe, H. Fukuda, M. Itoh, H. Sugiyama, T. Ido, and T. Takahashi, Carbohydr. Res., 161 (1987) 314-317. (202) R. W. Binkley, M. G. Ambrose, and D. G. Hehemann, J . Carbohydr. Chem., 6 (1987) 203-2 19. (203) F. Oberdorfer, W. E. Hull, B. C. Traving, and W. Maier-Borst, Int. J . Radiat. Appl. Instrum., Purr A , 37 (1986) 695-701. (204) P. KovBC, Carbohydr. Res., 153 (1986) 168-170. (205) M. Diksic and D. Jolly, Carbohydr. Res., 153 (1986) 17-24. (206) P. Di Raddo and M. Diksic, Curbohydr. Res., 153 (1986) 141-145. (207) P. KovBC, H. C. Yeh, and C. P. J. Glaudernans, Carbohydr. Res., 169 (1987) 23-34. (208) W. A. Szarek, G. W. Hay, B. Doboszewski, and M. M. Perlmutter, Carbohydr. Res., 155 (1986) 107-118. (209) B. Doboszewski, G. W. Hay, and W. A. Szarek, Can. J . Chem., 65 (1987) 412-419. (210) A. Luxen, N. Satyamurthy, G. T. Bida, and J . R. Barrio, I n t . J . Rudiar. Appl. Instrum., Purr A , 37 (1986) 409-413. (211) H. G. Howell, P. R. Brodfuehrer, S. P. Brundidge, D. A. Benigni, and C. Sapino, Jr., J. Org. Chem., 53 (1988) 85-88. (212) G. W. J. Fleet, J. C. Son, and A. E. Derome, Tetrahedron, 44 (1988) 625-636. (213) F. Latif, A. Malik, and W. Voelter, Justus Liebigs Ann. Chem., (1987) 617-620. 33 1 Copyright 8 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
332
RENE CSUK AND BRIGETTE I. GLANZER
C-6,20932'4 anhydro sugars,199,208~215~216 branched monosaccharide^,^^^ fluorinated amino sugars,194,218-225 difluorinated monosaccharide^,'^^,^^^^^"^^^ and fluorinated monosaccharide phosphates,23b234and p h o ~ p h o n a t e s ~ ~ ~ have been described. Further progress has been achieved in the synthesis and n.m.r.-spectral analysis of fluorinated avermectin B1,,236tylonolide.237and neuraminic acid (214) J. R. Durrwachter, D. G. Drueckhammer, K. Nozaki, H. M. Sweers, and C.-H. Wong, J. Am. Chem. Soc., 108 (1986) 7812-7818. (215) J. Doleialovfi, M. Cerng, T. Tmka, and J. Pacfik, Collect. Czech. Chem. Commun., 41 (1976) 1944-1953. (216) D. J. Baillargeon and G. S. Reddy, Carbohydr. Res., 154 (1986) 275-279. (217) K. Bischofberger, R. H. Hall, A. Jordaan, and G. R. Woolard, S. Afr. J. Chem., 33 (1980) 92-94. (218) C. Bosso, J. Defaye, A. Domard, A. Gadelle, and C. Pedersen, Carbohydr. Res., 156 (1986) 57-68. (219) R. L. Thomas, S . A. Abbas, C. F. Piskorz, and K. L. Matta, Carbohydr. Res., 175 (1988) 158-162. (220) R. L. Thomas, S. A. Abbas, and K. L. Matta, Carbohydr. Res., 175 (1988) 153-157. (221) D. Picq, I. Drivas, G. Carret, and D. Anker, Tetrahedron, 41 (1985) 2681-2690. (222) M. Sharma, G. G. Potti, 0. D. Simmons, and W. Korytnyk, Carbohydr. Res., 163 (1987) 41-51. (223) R. Faghih, F. C. Escribano, S. Castillon, J. Garcia, G. Lukacs, A. Olesker, and T. T. Thang, J . Org. Chem., 51 (1986) 4558-4564. (224) L. H. B. Baptistella, A. J. Marsaioli, P. M. Imamura, S. Castillon, A. Olesker, and G. Lukacs, Carbohydr. Res., 152 (1986) 310-315. (225) D. Picq and D. Anker, Carbohydr. Res., 166 (1987) 309-313. (226) J. P. Praly and G. Descotes, Tetrahedron Lett., (1987) 1405-1408. (227) S.-H. An and M. Bobek, Tetrahedron Lett., (1986) 3219-3222. (228) Y. Hanzawa, K. Inazawa, A. Kon, H. Aoki, and Y. Kobayashi, Tetrahedron Lett., (1987) 659-662. (229) A. Dessinges, F. C. Escribano, G. Lukacs, A. Olesker, and T. T. Thang, J. Org. Chem., 52 (1987) 1633-1634. (230) P. Le Markchal, C. Froussios, and R. Azerad, Biochimie, 68 (1986) 1211-1215. (231) E. M. Bessell and P. Thomas, Biochem. J . , 131 (1973) 77-82. (232) T. Nakada, I. L. Kwee, G. A. Rao, and C. B. Conboy, Biochem. Arch., 1 (1985) 163166. (233) T. Nakada and I. L. Kwee, Biochem. Arch., 2 (1986) 53-61. (234) S. G. Withers, D. J. MacLennan, and I. P. Street, Carbohydr. Res., 154 (1986) 127144. (235) G. M. Blackburn and M. J. Parratt, J . Chem. Soc., Perkin Trans. 1 , (1986) 1425-1430. (236) C. Bliard, F. C. Escribano, G. Lukacs, A. Olesker, and P. Sarda, J. Chem. Soc., Chem. Commun.. (1987) 368-370. (237) S. Kageyama, T. Onoda, T. Tsuchiya, S. Umezawa, and H. Umezawa, Carbohydr. Res., 169 (1987) 241-246. (238) K. Okamoto, T. Kondo, and T. Goto, Bull. Chem. Soc. J p n . , 60 (1987) 631-636. (239) M. Imoto, N. Kusunose, Y. Matsuura, S. Kusumoto, and T. Shiba, Tetrahedron Lett., (1987) 6277-6280. (240) M. Sharma, C. R. Petrie, IIIrd, and W. Korytnyk, Carbohydr. Res., 175 (1988) 25-34.
ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 46
ADDENDUM TO ARTICLE 6 BY RONALD J. CLARKE, JOHNH. COATES,AND STEPHENF. LINCOLN In this Addendum, reference will be made to a selection of publications in order to indicate current trends to readers. (a).-Preparation of cyclodextrin derivatives; substitution at a secondary hydroxyl group of the cyclodextrin annulus. Murakami and cowora new and convenient method for the regioselective k e r ~ described ’~~ tosylation of the 2-hydroxyl groups of alpha, beta, and gamma cyclodextrin by means of a cyclic tin intermediate. The method is based on the reaction of dibutyltin oxide with 1 ,2-diols to form five-membered dibutylstannylidene derivatives. Useful yields of the 2-0-tosyl derivatives of the cyclodextrins were obtained. (b).-Kinetic studies of the mechanism of complex-formation between alpha cyclodextrin and a series of hydroxyphenylazo derivatives of naph’ ~ ~ out an extensive, thalenesulfonic acids. Yoshida and F ~ j i m o t ocarried stopped-flow, kinetic study of a series of five of the aforementioned compounds, modified by substitution with groups of various charges and sizes, interacting with alpha cyclodextrin under pH conditions where the phenolic OH group was undissociated. These experiments were then repeated under pH conditions where the phenolic OH groups were ionized. The results were interpreted in terms of an “associative interchange” mechanism. An intermediate species was postulated, with its formation involving included water being displaced from the cyclodextrin cavity and partial collapse of the water structure around the incipient guest. Changes of slope of the Arrhenius plots for the forward rate-constants of the reactions of the phenolate species were interpreted in terms of structural changes of the reactants, or changes of the rate-determining steps in the lower-temperature range of the reactions investigated, or both. (c).-Fluorescence enhancement by means of cyclodextrin inclusion complexes. This has been examined by several groups of workers in a (183) T. Murakami, K. Harata, and S. Morimoto, Tetrahedron Lett., (1987) 321-324. (184) N . Yoshida and M. Fujimoto, J . Phys. Chem., 91 (1987) 6691-6695.
333 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
334 RONALD J. CLARKE, JOHN H. COATES, AND STEPHEN F. LINCOLN
variety of systems. Kano and coworker^^^^ studied the binding sites of pyrene and related compounds, and chiral excimer formation, within the cavities both of cyclodextrins and branched cyclodextrins. Using circular dichroism and circularly polarized fluorescence, they showed that the dimer of pyrene that was included within gamma cyclodextrin had lefthanded chirality, whereas the 1,3-dinaphthylpropane dimer had righthanded chirality when included therein. These dimers were believed to be bonded to the relatively hydrophobic, primary-hydroxyl side of the cyclodextrin annulus, because the analogous pyrene dimer formed in the 6-0a-maltosyl-gamma cyclodextrin cavity (that was capped on the primary hydroxyl side) exhibited right-handed chirality. Agbaria and GillIa6studied excimer fluorescence arising from extended 2,5-diphenyloxazole-gamma cyclodextrin aggregates, in which the inclusion of pairs of phenyl groups from different 2,S-diphenyloxazole (PPO) molecules within a common gamma cyclodextrin annulus resulted in both excimer fluorescence and the formation of a staggered, linear array of PPO molecules linked by phenyl groups overlapping within gamma cyclodextrin molecules. The use of cyclodextrins to enhance the fluorescence of fluorophores used in analytical procedures is exemplified in an article by Sanchez and coworkers. The ligand, benzyl 2-pyridyl ketone-2-pyridyl hydrazone may be used in a fluorescence assay for gallium. They found that, when this ligand was used in the presence of beta cyclodextrin, the fluorescence observed was linear over the gallium concentration range of 0.8-800 ng-mL-', with very much increased selectivity against competing cations. Presumably, the increased selectivity can be ascribed to the steric constraints imposed on the ligand by inclusion within the beta cyclodextrin annulus. (4.-Studies of the diastereomeric complexes formed between cyclodextrin molecules and chiral molecules. Harata and coworkersIE8determined the crystal structures of the inclusion complexes of (S)- and ( R ) mandelic acid with hexakis(2,3,6-tri-O-methyl)-alphacyclodextrin. The crystal structures showed that an induced conformational change in the alpha cyclodextrin molecule resulted in distinctly different geometry for each of the diastereoisomers. (185) K. Kano, H. Matsumoto, Y. Yoshimura, and S. Hashimoto, J . Am. Chem. SOC.,110 (1988) 204-209. (186) R. A. Agbaria and D. Gill, J . Phys. Chem., 92 (1988) 1052-1055. (187) F. G . Sanchez, M. H. Lopez, and J. C. M. Gomez, Fresenius Z . Anal. Chem., 328 (1987) 499-500. (188) K. Harata, K. Uekama, M. Otagiri, and F. Hirayama, Bull. Chem. SOC. J p n . , 60 (1987) 497-502.
ADDENDUM TO ARTICLE 6
335
The inclusion modes of flurbiprofen with beta cyclodextrin and with heptakis(2,3 ,ti-tri-O-methyl)-beta cyclodextrin have been studied by Imai and coworkers.lS9 They showed that, although the Cotton effects in the circular dichroic spectra induced by beta cyclodextrin in R ( - ) and S ( + ) flurbiprofen are identical, those induced by heptakis(2,3,6-tri-O-methyl)beta cyclodextrin differ from each other and from those induced by beta cyclodextrin. C.p.-m.a.s. 13C-n.m.r. experiments showed that the cyclodextrin ring is probably more distorted in the flurbiprofen inclusion complex with methylated beta cyclodextrin than in that with beta cyclodextrin. (el.-The use of cyclodextrins bonded to solid supports as chromatographic media for the resolution of isomers and of enantiomers. This is becoming quite widespread (see, for example, a review by ArmstrongIgo). Alpha cyclodextrin molecules bonded to silica beads (5 p m diam.) by seven-atom-long, ether-linkage-containing, polymethylene spacergroupsI9l have been used to separate a number of racemic compounds into their constituent enantiomers, including racemic tryptophan, phenylalanine, and tyrosine. Similar materials incorporating beta instead of alpha cyclodextrin have been used to separate the optical isomers of scopolamine, cocaine, homatropine, and atropine. 192 ( f ) . - A novel application of cyclodextrins in liquid membranes. This has been demonstrated by Armstrong and Jin.193Enantiomeric o r isomeric enrichment was shown to occur when racemic mixtures, or mixtures of isomers, were allowed to diffuse from a nonaqueous phase through an aqueous liquid membrane containing a dissolved cyclodextrin into a second nonaqueous phase. The degrees of enrichment were considered to be encouraging, and it was foreshadowed that efforts will be made to develop this method for practical separations of enantiomers and of isomers in the future.
(189) T. Imai, M. Otagiri, H. SaitB, and K. Uekarna, Chem. Phurm. Bull., 36 (1988) 354359. (190) D. W. Armstrong, Anal. Chem., 59 (1987) 84A-91A. (191) D. W. Armstrong, X. Yang, S. M. Han, and R. A. Menges, Anal. Chem., 59 (1987) 2594-2596. (192) D. W. Armstrong, S. M. Han, and Y. 1. Han, Anal. Biochem., 167 (1987) 261-264. (193) D. W . Armstrong and H. L. Jin, Anal. Chem., 59 (1987) 2237-2241.
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AUTHOR INDEX Numbers in parentheses are footnote reference numbers and indicate that an author’s work is referred to although his name is not cited in the text. Armstrong, D. W., 233, 248 Ashoor, S. H.,307 Aso, K., 307
A Abad, A., 194 Abbot, S. R., 24, 38(37) Abe, J.-I., 247(170b), 248 Abraharnsson, B., 303 Abusabah, E. K. E., 62(279), 63 Acevedo, 0. L., 28 Ackerman, J. L., 86(59), 87(59), 89 Acton, E. M., 159 Adam, M. J., 86(48), 89, 104(48), 142(48), 143(48) Adamson, J., 76, 80, 89, 146(168), 147, 151 Ahderinne, R., 36 Aitzetmuller, K., 23, 38, 52(31) Akhrem, A. A., 159 Albano, E. L., 104(98), 105, 143(98), 155(98) Albericio, F., 199 Albersheim, P., 20, 40, 41, 42, 54, 57, 59(165), 60(165), 262, 264 Albert, R., 100, 116, 152(125), 158(125), 166(91), 167(91), 170(91, 125), 175(129, 177(125) Alfiildi, J., 306 Alfredsson, B., 291, 294 Alpenfels, W. F., 267 Aman, P., 57, 306 Ambrose, M. G., 95(86), 97 Amit, B., 179, 181, 184, 188(1), 193, 198(58), 202, 203 Anderegg, P., 290 Anderson, A. W., 34, 39, 54(150) Anderson, D. M. W., 288 Anderson, S., 34 Anderson, R., 291 Anet, E. F. L. J., 284, 286, 309 Angyal, S. J., 25, 26 Anker, D., 117(129), 118, 119, 128(129) Annison, G., 54 Anteunis, M.,95(83), 97, 106 Antonopoulos, C. A., 37, 5 5 , 60(130), 65(253) Appell, H. R., 274 Arad-Yellin, R., 209
B Baenziger, J. U., 42, 45, 46, 48(193), 60(168) Baer, H. H., 119(133), 120, 123(133), 124(133), 125(133), 127(133), 170(133), 171(133), 308 Baker, J. O., 57, 70(267a) Ballardie, F. W., 117, 118(128) Ballou, C. E., 270 Baptistella, L. H. B., 119(132), 120, 123(132), 124(132), 125(132), 127(132), 170(132), 171(132) Barbalat-Rey, F., 80, 140(37), 141, 155 Barford, A. D., 75, 86(12), 90(12), 91(12), 93(12), 94, 95(12), 148(12) Barker, H. M., 67 Barker, P. E., 62(278), 63 Barker, S. A., 25, 39(44) Barltrop, J. A., 198 Bamett, J. E. G., 152 B a t h , H. G., 67, 68(290, 291), 69 Bartholomew, D. G., 181, 182(13) Barton, D. H. R., 200 Batley, M., 68 Baudisch, O., 293 Bauman, W.C., 24 Baust, J. G., 19, 21, 23(9), 24, 32(39), 33(39), 41(39), 59(39), 60(39), 64(39) Bax, A., 74 Bayley, H.,204 Beebe, J. M., 51 Bell, R. H.,180, 189(6) BeMiller, J. N., 182, 251, 292, 296, 297, 301 Bender, H., 44 Bender, M. L., 206, 221(92), 222, 223, 227(2), 244, 245, 248 Ben-Efraim, D., 202 Bennek, J. A., 271 Benschop, H. P., 248
137
338
AUTHOR INDEX
Benzing-Purdie, L. M., 307, 325 BOOS,K.-S., 31 Bergeron, R. J., 206, 230, 23 I , 245( 122) Borchert, W., 214 Bergh, M. L. E., 33, 42, 43(88), 45, 46, Borgegrain, R.-A., 191 65(170) Bouquelet, S., 45 Bergius, F., 274, 298 Bradford, A . D., 191 Bergmann, E. D., 144 Brandange, S., 172(189), 173 Berl, E., 274 Brandes, W. B., 51, 64(233) Bernasconi, C. F., 241 Brandon, R. E., 300 Bernhard, R. A . , 317 Braunovh, M., 151(175), 152 Berry, R. E., 311 Breebaart-Hansen, J. C. A. E., 222, 227, Bessell, E. M., 95(84), 97, 148(84), 149(84) 248 Bethell, G. S., 25 Brereton, I. M., 239 Betzel, C., 227, 228(111) Breslow, R.,206, 208, 210, 244 Beveridge, R. J., 25 Breuer, H . , 309 Bhacca, N. S., 75, 79(17), 80(17), 82(17), Briggner, L.-E., 210 83(17), 84(17) Brimacombe, J. S., 90(67, 68), 91(72), 92, Bida, G. T., 86(55), 87(55), 89, 146(167), 99(67, 72), I14(72) 147 Brink, A . J., 138(156), 139(156), 140, Bienkowski, M. J., 48, 62(216) 141(156), l42( 156) Biermann, C. J., 261 Brinkman, U. A. T., 67, 68 Binder, H., 23 Brobst, K. M., 25, 51 Binkley, R. W., 95(86), 97, 179, 180(3), 191 Brodfuehrer, P. R.,110(110), 111, 138(110) Birks, J. B., 209 Broido, A . , 274 Birks, J. W., 67 Brondz, I., 258 Birr, C., 192, 193 Brons, C., 24, 51(38), 52(38), 64(38) Bisehofberger, K., 139(157), 140, 142(157) Broom, A. D., 181, 182(13) Bishop, E. D., 74 Brose, K. H., 319 Blackburne, I. D., 113, 135(117) Broser, W., 218, 219(79), 232 Blair, M. G., 282 Brown, H. D., 326 Blake, J. D., 34 Brown, R. D., Jr., 39, 54 Blakeney, A. B., 263, 264(34) Briiller, W., 39 Blanken, W. M., 33 Brunngraber, E. G., 93(77), 94, 96(77), Blankenhorn, G., 307, 319(183) 149(77), 150(77), 151, 165(77), 175(77) Blaschek, W., 52, 53(244) Brunt, K., 21, 27(23), 36 Blomquist, G., 213, 296 Bryn, K., 258, 259(17) Bloor, D., 238(144) Buchberger, W., 65 Blumberg, K., 29 Buckee, G. K., 51 Bobleter, O., 26, 52, 53, 66(247), 298, 300 BuddSinskL, M., 144, 173(161) Bobrov, P. A , , 299 Bueno, M. P., 51 Bochkov, A. F., 251 Burns, I. W., 21, 23(26) Bociek, S. M., 231, 233(122a) Burtscher, E., 26, 53(54) Bock, K., 77, 78(28), 97(87), 98, 107(107), Bush, C. A., 29, 42, 43(174), 59(174) 108(107), 109, 134, 137, 138, 166(25), Butchard, C. G., 104(99), 105, 132(148, 168(107), 169(107) 149), 133, 153(148, 149) Boehm, J. C., 28 Bystrom, K., 233 Boersma, A . , 42, 43(171), 46, 59(199), 61 Bolt, J. D., 235 C Bonenfant, A. P., 112, 154(178), 155, 169(114) Callaert-Deveen, B., 68 Bonn, G., 20, 26, 39(13), 53, 54, 66(247) Cama, L. D., 191, 198(41)
AUTHOR INDEX Campbell, J. C., 129(141), 130(141), 131, 135(141) Cantor, S. M., 286 Capon, B., 117, 118(128), 251 Caputo, A. G., 38 Card, P. J., 74, 77, 92, 93(92), 94(92), 96(27), 100(65), 106(65), I19(65), 120(65), 122(65), 144, 149(65), 150(27), 162(65), 163(65), 164(65), 165(27), 168(65), 171(65), 173(166), 175(27) Cardelino, B., 221(90), 222, 225(90) Cardon, P., 46 Cam, P. W., 72 Carr, S. A., 56, 69 Castillon, S., 117, I18(127), 119(127), 120, l23( l27), 124(127), 1 2 3l27), l26( l27), 170(l27), I7 I ( 127) Casu, B., 233, 245(127) Catala, F., 277 Cavalier, J. C., 274 Cerny, M., 87(62), 89, 92, 114(73), 144, 145, 152, 159(73), 173(161) Chamberlin, J. W., 192 Chambers, R. E., 256, 258(10) Chan, L., 21, 61(28) Chang, C.-D., 191 Channing, M. A,, 231 Chaplin, M. F., 258 Chavis, C., 308 Cheetham, N. W. H., 19, 21, 29, 32(5), 33, 39(29), 41, 54, 62(19), 63, 256, 257(12) Chekkor, A., 45 Cheng, J. C.-Y., 28 Cheshire, M. V., 325 Chester, T. L., 71 Chin, T.-F., 208, 219(13) Chornet, E., 274 Chow, Y. L., 200 Christensen, B. G., 191, 198(41) Chung, C., 235 Churms, S. C., 18, 72(1) Ciamician, E., 181 CieSlik, E., 317 Cifonelli, J. A,, 275 Cifonelli, M., 275 Ciner-Doruk, M., 311 Claessens, H. A , , 29, 39(64), 59(64), 70(64) Clamp, J. R., 256, 258(lO) Clamp, P. I., 42, 57(167), 60(167) Clarke, M. L., 34, 35(98)
339
Clarke, R. J., 209(37), 210, 234(37, 38). 242(37, 38). 244(37, 38, 39) Clermont, L. P., 298 Clowes, G. A., 244 Coates, J. H., 209(37, 38, 39), 210, 234, 239, 242(37, 38), 244(37, 38, 39), 245(140) Codington, J. F., 104(102), I05 Cohen, A., 283 Coleman, G. H., 300 Coles, E., 56, 69 Collings, G. F., 264 Collins, P. M., 188, 189(38, 39) Colonna, S., 95(82), 96, 99(82) Comin, J. H., 13 Conchie, J., 48, 270 Connors, K. A., 220, 227, 232(85), 239 Conrad, H. E., 48, 54, 5 5 , 62(216) Cook, J. C., 70 Corbett, W. M., 301, 305 Corran, P. H., 38 Costello, D. E., 42 Couperwhite, I., 54 Covey, T. R., 69, 70(318) Cox, A., 200 Coxon, B., 102(96), 103 Cramer, F., 206, 208(1), 209, 214, 215, 217, 219(16), 221(16), 233, 234(16), 235(16), 239(16), 244 Cromwell, W. C., 233 Crowther, J. B., 69, 70(318) Csuk, R., 98, 115, 166(89a) Cummings, L., 25, 30(46) Cushley, R. J., 104(102), 105 Czerlinski, G. H., 241
D Dacons, J. C., 296 Dahlin, C. E., 46 Dahlman, O., 172(189), 173 D’Amboise, M., 23, 67(34) Damon, C. E., 51 Daniel, P. F., 68 Darvill, A. G., 29, 42, 49(63), 54, 57 Das, P. K., 183 Date, Y.,226 Davidson, G. F., 301 Davis, H. F., 183 Davis, M. A. F., 40
340
AUTHOR INDEX
Davis, W. A., 50 Dawson, R., 67 Dax, K., 86(49), 89, 93(49), 99(49), 100, 104(49), 111(49), 116, 161(49), 162(49), 166(91), 167(49, 91), 168(49), 169(49), 170(91) Day, W. R., 29, 39(60), 60(60),70(60) de Bruijn, J. M., 292 De Bryn, A., 95(83), 97, 106 Defaye, J., 277 De Feudis, D. F., 68 de Gourcy, C., 308 de Groot, C. N., 320 De Jongh, R. D., 203 Delaney, S. R., 37, 48, 55 Dell, A., 44,70 Demarco, P. V., 218 Demmit, T. F., 273 Descotes, G., 134(153), 135, 190 Dessinges, A., 87(61), 88(61), 89, 117, 118(127), 119(127), 120, 123(127), 124(127), 125(127), 126(127), 170(127), 171(127) Dethy, J.-M., 68 Deuel, H., 288, 293 Deulofeu, V., If, 13 De Villiers, 0. G., 138(156), 139(156), 140, 141(156), 142(156) Dewaele, C., 58, 61(275) Diaz, S., 36 Dick, J., 66 Dietsche, W., 248 Dijkgraaf, P. J. M., 34, 35(99) Diksic, M., 85(47), 86(47), 87(47), 89 D i r k , J. M. H., 34 Dixon, H.B. F., 307, 319(183) Dmytraczenko, A., 191 DoleZ6lova, J., 92, 114(73), 144, 159(73), 173(161) Donaldson, B., 76, 92(19), 148(19) Doner, L. W., 33, 35, 51, 63(241) Donovan, J., 274 Dorland, L., 42, 43, 46(172), 79 Drabowicz, J., 248 Drauz, K., 317 Dreux, M., 66 Dreyer, R. N., 61 Drueckhammer, D. G., 90(66),91(66), 92, 99(66), 109(66), 11N66) Dryselius, E., 300
D’Souza, V. T., 245 Dua, V. K., 29, 42, 43(174), 44, 47(62), 48(62), 59(61), 60(61, 62), 65(61) Duang, E., 35 Dudman, W., 57, 259 Durham, D. G., 288 Dwek, R. A., 86(54), 89, 92, %(53), 99(53), 104(53), 105, 112(53), 131, 133, 146(53), 155(53), 156(53)
E Eastman, J. F., 291 Eaton, D. F., 209 Ebringerovh, A., 306, 321(174) Eby, R., 177 Egge, H., 44 Eggert, F. M., 56, 68(263), 267 Ehrenkaufer, R. E., 86(56), 87(56), 89 Eichner, K., 311 Einarsson, H., 326 Ejima, S., 245 El Khadem, H. S., 295 Elliott, D. C., 274 Ellis, G. P., 307, 308 Ellmore, T. L., 269 Emert, J., 209 Enami, K., 67 Engels, J., 204 Engfeldt, B., 32(92), 33, 55, 56(92), 65(253) Enkvist, T., 283, 294 Epling, G. A., 194 Epshtein, Ya. A., 300 Erbing, B., 144(163), 145, 174(163) Eriksen, P. B., 48, 55(212), 57(212) Eriksson, C., 326 Escott, R. E. A., 67 Eswarakrishnan, S., 104(101), 105 Evans, W. L., 301 Evelyn, L., 95(81), 96, 100(81), 112(81), 113(81) Ewald, L., 213
F Faillard, H., 36 Faure, A., 190 Feast, A. A. J., 291 Feather, M. S., 275, 276, 277(22), 284(22), 285, 286, 288, 289, 291(22), 319, 320 Fedan, J. S., 204
AUTHOR INDEX Fedoroilko, M., 279 Feeney, R. E., 307, 319(183) Fengel, D., 298 Ferrier, R. J., 143, 172(159), 301 Fiedler, H., 36 Filby, W. G., 182 Finley. J. W., 35 Fishbein, L., 277 Fisher, B. E., 318, 320(227), 321 Fishman, M. L., 265 Fitt, L. E., 25, 39(50) Florkin, M., 207 Fodor, G., 309 Foltz, A. K., 50 Forbush, B., 204 Fors, S., 311 ForsskBhl, I., 282, 294(49), 295(49) Foster, A. B., 75, 76, 80, 83(41), 84(41), 85, 86(12), 87(58), 89, 90(12), 91(12), 92, 93(12), 94, 95(12), 97, 99(20), 105(11, 20), 112(20), 114(72), 146(18), 147, 148(11, 12), 149(84), 151, 155(11), 156, 191 Foster, J. F., 296 Foucault, A., 31 Fox, A., 266 Franken, H., 289 FranzCn, L.-E., 57 Freed, J. H., 20, 43, 47(16) Frei, R. W., 67, 68 French, D., 206, 214, 217, 220, 249 Freudenberg, K., 213, 214, 215, 217(70), 296 Friday, D., 24, 32(39), 33(39), 41(39), 59(39), 60(39), 64(39) Friedrich, E., 197 Froehlke, A. W., 298 Fu, Y. L., 274 Fujimaki, M., 313 Fujimoto, M.,234 Fujita, K.,245 Funasaka, W., 304 Fur6, I., 231 Furue, M., 239 Fyfe, C. A,, 219
G Gabel, C. A., 42 Gacesa, P., 33, 34(%), 54(%)
341
Galensa, R., 68 Galitzer, S. J., 51 Games, D. E., 69 Gandelman, M. S., 67 Garbutt, S., 288 Gardiner, D., 300 Carves, K.,298 Gaudemer, A., 75, 78(16), 79(16), 82(16) Geeraert, E., 21, 58(24), 61(24) Geigert, J., 34 Gelb, R. I., 221(88, 90, 91, 92), 222, 225(90), 239 Gentile, B., 112, 141, 169(114) Gerasimowicz, W. V., 226 Gibeily, G. J., 231 Gidley, M. J., 231, 233(122a) Giersch, C., 36 Gilpin, R. K.,51 Giralt, E., 199 Glad, M., 31, 72 Glanzer. B. I., 86(49), 87(49), 89, 93(49), 104(49), 111(49), 115, 161(49), 162(49), 167(49), 168(49), 169(49) Glaudemans, C. P. J., 77, 89, 92, 161(26), 162(186), 163, 164(26) Goldman, Y. E., 204 Golik, J., 57, 68(266) Golova, 0. P., 300 Goso, K., 44 Goto, M., 127(139), 128 Goto, R., 293 Gottschalk, A., 309 Goulding, R. W., 26, 70(53), 71(53) Govil, G., 73 Grangord, G., 301 Gravel, D., 195, 196(65) Gray, G. R., 271 Green, E. D., 46 Greene, T. W., 179 Grethlein, H. E., 300 Gridley, M. J., 40 Griffin, G . W., 183 Griffiths, D. W.,206, 227(2), 244(2) Griggs, L. E., 261, 267(23) Grimble, G. K.,67 Gross, B., 191 Griin, M.,35 Griinewald, K., 315 Guerrant, G.O., 267 Gum, E. K.,Jr., 39
AUTHOR INDEX
342
Giinther, H., 80 Gupta, D. V., 274 Gurjar, M. K., 123, 124(137), 125(137), 127(137) Gurr, E., 55 Gurwara, S. K., 199 Guthrie, R. D., 144(164), 145, 174(164) Gutsche, C. D., 284 Guyon, F., 31 H Haas, D. W., 303 Hacksell, U . , 28 Hadfield, A. F., 61 Hadziyev, D., 315 Hagedorn, M. L., 318 Hagemeier, E., 31 Haines, S. R.,98, 143, 166(89), 172(159) HalaSkovB, J., 145 Hald, L. H., 236 Hall, D., 234, 238(144) Hall, L. D., 74, 75, 76, 79, 80, 82(14, 15), 83(14), 85, 86(48), 89, 90(67), 92, 93(79), 94, 95(81), 96, 97, 99(67), 100, IOl(14, 32), 102(14, 32), 103, 106(104), 108(104), 109, 112(81), 113(81), I15(92), 129(34, 143), 130(34, 143, 144). 131, 142(48), 143(48), 146(18), 147, 148(84), 149(84), 173 Hall, M. A., 269 Hallen, R. T., 290, 316 Hamada, F., 246 Hamai, S., 209, 210(30), 243(30) Hammond, M., 244 Hanabusa, K., 245 Hanai, T., 39 Hands, C. H. G., 296 Hanes, C. S., 215 Haney, C. A,, 69 Hansch, C., 227 Hansen, L. D., 221(89), 222, 223(89) Hansson, L., 31, 72 Harada, A., 239 Harada, K., 31 Haradahira, T., 86(52), 89, 162(63) Harata, K., 209, 222, 249 Hardegger, E., 191 Hams, J. F., 275, 276, 277(22), 282, 284(22), 285, 286, 288, 289(74), 291(22), 320
Harris, P. J., 263, 264(34) Harrison, R., 159 Hart, G. W., 43, 47(182) Hartford, C. G., 50 Hase, S., 57, 68, 268, 270(49) Hasegawa, A., 127(139), 128 Hashimoto, Y., 107(105), 108(105), 109 Hassler, W., 25, 39(50) Hatt, B. W., 25, 39(44) Havinga, E., 203 Havlicek, J., 38 Hay, A. J., 48, 270 Hayami, J., 277, 291(34), 293 Hayase, F., 314, 321 Hayashi, M., 84(43), 85 Hayashi, T., 309 Heidt, L. H., 182 Hemminki, K., 28 Hems, R.,76, 83(41), 84(41), 85, 90(67), 91(20), 92, 93(78), 94, 99(20), 105(20), 112(20), 1 l4(72), 156 Henderson, D. E., 36, 67(118) Henderson, S. K., 36, 67(118) Hendrix, D. L., 19, 23(9), 24, 32(39), 33(39), 41(39), 59(39), 60(39), 64(39) Henglein, F. M., 233 Hennrich, N., 244 Herbert, J., 195, 196(65) Herbert, R. W., 288 Herbette, L., 204 Herchman, M., 187, 188 Herkstroeter, W. G., 243 Herscheid, J . D. M., 85, 86(39), 87(39), I13(39) Hersey, A., 234, 237(142), 238(142), 239( 142) Heyns, K., 309, 319, 321 Heyraud, A., 29, 32, 38, 39, 40(68), 57, 269 Hibberd, M. G., 204 Hibbert, H., 269 Hicks, K. B., 19, 23(7), 25, 33, 35, 51, 61(89), 62(89), 63(241), 319, 320 Hiebl, J., 321, 322, 323(242) Himmel, M. E., 57, 70(267a) Hingerty, B. E., 227, 228(111) Hino, Y.,209, 210, 246 Hinze, W. L., 248 Hirai, H., 209, 210(31) Hirano, D. S., 34 Hirayama, F., 210, 249 Hiromi, K., 234
AUTHOR INDEX Hirst, E. L., 288 Hirter, P., 69 Hitz, W. D., 144, 173(160) Hjerpe, A., 32(92), 33, 37, 55, 56(92), 65(253) Hoagland, P. D., 57 Hodge, J. E., 286, 308, 309, 318, 321 Hogaboom, G. K., 204 HZiland, H., 236 Hokse, H., 44 Honda, S., 30, 31, 32, 33, 34(71), 54(71), 56(80), 66(81), 67, 68(81), 70(78), 71(80), 262, 264(25), 267(25) Hooghwinkel, G. J. M., 45 Hoppe-Seyler, F., 274 Horton, D., 180, 189(6), 197 Horvath-Toro, C., 220, 223(83) Hoshi, H., 232 Hoshino, M., 209 Hoshino, O., 194, 196 Hosur, R. V., 73 Hough, L., 118(130), 119, 120, 121(134), 122(134), 127(134), 157(134) Hounslow, A. M., 239 Howard, D. R., 42 Hrutfiord, B. F., 303 Hudson, C. S., 215 Huebner, A. L., 25, 39(49), 51(49), 64(49) Huekeshoven, J., 319 Hughes, D. E., 33, 65(87) Hughes, S., 65 Hull, S. R., 42, 68 Hullar, T. L., 191 Humphrey, A. E., 300 Humphries, H. P., 282 Hung, C. T., 288 Hunt, B., 90(70), 91(70), 92, 116(70) Hupe, K.-P., 18 Hurd, C. D., 275 Hybl, A,, 218 I
Ido, T., 146(169), 147 Ihara, Y., 249 Ikeda, H., 245 Ikeda, T., 234, 245 Ikehara, M., 181, 182 Ikenaka, T., 57, 256, 268, 270(49) Imai, T., 249 Inch, T. D., 74, 79(5)
343
Innis, D. P., 71 Inoue, M., 67 Inoue, S . , 256, 258(13) Inoue, Y., 219 Irlam, G. A., 62(279), 63 Isbell, H. S., 275, 281(23), 301(23) Isenhour, L. L., 275 Ishizu, A,, 281, 292(43), 293 Ito, Y., 70 Ittah, Y., 86(51), 89, 93(51) Iverson, J. L., 51 Iwata, S., 38 Iyengar, J. R., 265 J
Jacobi, R., 213 Jacobsen, S., 101(94), 102(94), 103, 106(94), 109(94) Jacobson, K., 204 Jacopian, V., 299 Jakoby, W. B., 204 JakovljeviC, J. B., 38, 39(137) James, H., 19, 21, 23(9), 24, 32(39), 33(39), 41(39), 59(39), 60(39), 64(39) James, P., 27 Jansen, H., 67 Janson, J., 301 Jantzen, E., 258, 259(17) Javier-Son, A. C., 274 Jaworska-Sobiesiak, A., 119(133), 120, l23( l33), l24( l33), 12%l33), 127(133), l70( l33), l71( 133) Jeanloz, R. W., 19, 44(19), 59(19), 62(19) Jeffrey, J. E., 263, 264(30) Jen, J. J., 265 Jenkins, I. D., 144(164), 145, 174(164) Jensen, S. R., 101(94), 102(94), 103, 106(94), 109(94) Jentoft, N., 56, 68(261), 256, 257(11), 258(1 I ) Jeon, I. J., 51, 63(238) Jewell, J. S., 197 Jitsuhiro, T., 182 Johansson, I., 299 Johansson, M. H., 303 John, M., 39, 54(151) Johncock, S. I. M., 51 Johnson, D. C., 19, 65 Johnson, L., 19, 20(12), 32(12), 68(12) Johnson, R. N., 75, 76, 80, 86(12), 87(58),
344
AUTHOR INDEX
89, 90(12), 91(12), 93(12), 94(12), 97, 105(11), 146(18), 147(18), 148(11, 12). 149(84), 155(11) Jolly, D., 85(47), 86(47), 87(47), 88(47), 89 Jones, A. D., 21, 23(26) Jones, J. K. N., 25 Jones, M., 56, 68(263), 267 Jones, T. M., 264 Jordaan, A., 138(156), 139(156), 140, 141(156), 142(156) Jurasek, L., 263, 264(32) Jurch, G. R., 314 Jurgens, A., 194
K Kabat, E. A., 43 Kagita, A., 226 Kahle, V., 38, 71(135) Kainuma, K., 38 Kajtar, M., 220, 223(83) Kallury, M. R., 300 Kannan, R., 266 Kano, K., 209 Kaplan, J. N., 204 Karim, K. A., 36 Kasatani, K., 244 Kato, A., 70, 71 Kato, H., 294, 313, 314, 321 Kato, S., 230, 234(120) Katzen, H. M., 297 Katzenbeisser, U., 116, 152(125), 158(125), l70( 129, l75( 125). 177(125) Kaufmann, R. J., 158, 177(182) Kauzmann, W., 227 Kavai, I., 151, 156 Keeling, P. L., 27 Kenner, J., 282 Kent, P. W., 86(54), 89, 92, 95(53), 99(53), 104(53), 105, 112(53), 131, 133, 146(53), 151(176), 152, 155(53), 156(53) Kersten, E., 315 Keyanpour-Rad, M., 159 Khorlin, A. Ya., 121(136), 122 Khripach, N. B., 159 Kieboom, A. P. G., 292 KingMorris, M. J., 293 Kingsbury, W. D., 28 Kirby, G. W., 200
Kirby, J., 203 Kirkland, J. J., 18 Kishi, T., 180, 191(7) Kiss, J., 116, 172(126) Kitagawa, M., 232 Kiyosuke, Y.,233, 244(130) Klein, A., 46 Klein, R. S., 110(111), 111 Klemm, G. H., 158, 177(182) Knapp, D. W., 214 Knoche, W., 230, 234(121) Knudsen, P. J . , 48, 55(212), 57(212) Kobayashi, N., 209, 210(25) Koch, R., 211 Kock, H., 323 Kodali, D., 209 Kodama, C., 68 Koehler, P. E., 315, 316 Koholic, D. J., 191 Koizumi, K., 44,45(191a), 57(189), 247(370b), 248 Kojin, R., 245 KSllnerov6, Z., 87(62), 89 Komiyama, M., 206, 221(92), 222 Konami, Y., 46 Konigstein, J., 279 Konishi, T., 31, 67 Koole, J. L., 208 Koops, J., 64 Koppen, P. L., 33,42,43(170), 45,46, 65(170, 202) Koppler, H., 315, 319(213) Kortlandt, F., 274 Korytnyk, W., 95(85), 97, 113, 120, 121(85), 122(85), 133, 146(147), 147(147), 153(147), 177 Kosakai, M., 55, 68(256) KovBE, P., 77, 92, 161(26), 162(186), 163, 164(26) Kracht, W. R., 20, 53(19) Kranich, W. L., 274 Kranse, F., 323 Krawczyk, S. H., 28 Krishna, R., 300 Kronig, U,, 322 Krzeminski, 2. S., 284 Kuan, F.-H., 219 Kucherov, V. F., 201 Kudo, K., 293 Kuge, T., 217, 221(95), 222
AUTHOR INDEX Kumanotani, J., 21, 33, 56, 57(262), 59(25), 62(25), 70 Kumar, N., 28, 69(57e) Kuo, J. C., 66 Kurata, T., 289, 313 Kurosu, Y.,33 Kuster, B. F., 23, 29, 50(29), 66(29), 67(29) Kyrczka, B., 190
L Laakso, E. I., 35 Laamanen, L., 302 Lacey, M. J., 259 Lach, J. L., 208, 219(13) Ladisch, M. R., 21, 25, 39, 53(22), 54(22) Lafosse, M., 66 Laine, R. A., 42 Lake, B. G., 35 Lambert, J. B., 79 Lamblin, G., 42, 43(171), 46, 59(199), 61 Lammers, J. N. J. J., 208 Lamperstorfer, C., 193 Lamport, D. T. A., 56 Landsman, S. D., 294, 304(105) Lang, P., 192 Larsson, P.-O., 72 Lau, J. M., 20, 57(18) Laude, D. A., 69 Lauer, M., 244 Laufer, D. A., 239 Lautsch, W., 218, 219(79) Laver, M. L., 263, 264(29) Lawrence, J. F., 265 Ledger, P. W., 42, 46(176) Ledl, F., 322 LeDonne, N. C., Jr., 57, 69(270) Lee, C. M., 307 Lee, G. J.-L., 37, 55 Lee, R. E., Jr., 19, 21, 23(9), 24, 32(39), 33(39), 41(39), 59(39), 60(39), 64(39) Lee, R. W.-K. 69 Legendre, M. G., 183 Leger, M., 37, 55(133) Lehnhardt, W. F., 268 Leo, A., 227 Leonard, J. L., 31 Leroy, Y.,46, 62(198) Lester, H. A., 204 Levine, M. L., 217, 220
345
Levy, M., 198 Levy, S., 87(60), 89 Lewis, E. A., 221(89), 222, 223(89) Lim, P. C., 25, 26(48), 33(48), 34(48), 35(48) Lincoln, S. F., 209(37, 38, 39), 210, 234, 239, 242(37, 381, 244(37, 38, 39), 245(140) Lindberg, B., 144(163), 145, 174(163), 251, 269, 281, 291, 292(43), 293, 299, 300, 301, 323, 325 Lindgren, G., 277, 289 Lindner, K., 229, 231(116) Liniere. F., 29 Lipari, J. M., 220, 232(85) Little, M. R., 57, 66(265) Liu, D.-W., 55 Liu, H.-W., 57, 68(266) Livni, E., 87(60), 89 Lloyd, K. O., 255, 256(6) Lloyd, W. J., 159 Lochinger, W., 192 Lochmiiller, C. H., 69 Lockhart, G. L., 289 Loeppky, R. N., 320 Loewus, F., 35 Lomax, J. A., 270 Long, D. E., 51 Lonngren, J., 251 Lopes, D. P., 93(80), 94 Lowendahl, L., 302 Luetzow, A. E., 299 Lumry, R., 225 Lundt, I., 131(146), 132 Lutz, w . , 197
M McCleary, B. V., 271 McCloskey, C. M., 300 McCray, J. A., 204 McGinnis, G. D., 28, 260 Machell, G . , 292, 296, 301, 303(152) McIntire, R. L., 214 McLaughlin, H., 40, 41(154) Maclennan, J. M., 229 McNeil, M., 20, 29, 40, 42, 49(63), 57, 58(63) McNicholas, P. A., 68 MacNicol, D. D., 220
346
AUTHOR INDEX
Macrae, R . , 50, 66 Maeda, M., 86(52), 89, 162(63) Majors, R . E., 67, 68(290, 291), 69 Manius, G. J., 21, 30(27) Manners, D. J., 297 Manor, P. C., 228, 229(114), 239(114) Manville, J. F., 75, 76, 79, 82(14, 15). 83(14, IS), 85, 101(14), 102(14), 103, 129(15, 34), 130(14, 15, 34), 131, 135(34), 136(34), 137(34) Maradufu, A., 94, 163(188), 165 Marce, P., 315 March, J. F., 260, 263(20), 264(20) Marcus, D. M., 85(45), 89, 94, 146(45), 147(45) Marcy, A. D., 52, 53(243) Margeot, J., 204 Markham, A. F., 182 Marks, C., 34 Marlett, J. A., 52, 53(242), 263, 264(33) Marsaioli, A. J., 119(132), 120, 123(132), l24( l32), 1 2 3132). 127(132), l70( 132), 171(132) Martens, J., 317 Martic, P. A., 243 Martin, J. C., 134, 135 Mason, M. E., 315 Matheson, N. K., 271 Mathews, R. A., 267 M a t h , S. A., 21, 61(28) Matolova, M., 306 Matsugi, J., 182 Matsui, Y., 222, 223(98), 226, 233(98) Mauro, D., 36 Mauron, J., 307 Mawhinney, T. P., 255 May, J. A,, Jr., 150(173), I51 Meeley, M. P., 230, 245(122) Mega, T., 256 Mehring, M., 74 Mellier, D., 194 Mellis, S. J., 42, 45, 46(168), 48(193), 60(168) Merkel, K. E., I9 Merrifield, R. B., 199 Meyer-Delius, M., 213, 215(58) Mikes, O., 40 Mikolajczyk, M., 248 Miller, D. L., 203 Miller, R . , 274
Miller, R. E., 286 Miller, R. K., 304, 314 Milligan, L. P., 53, 54(246), 65(246) Mills, F. D., 321 Mithel, B. B., 299 Miyaji, T., 223 Miyake, T., 182 Miyawaki, M., 256, 258(13) Mizowaki, N., 247(370b), 248 Mizushima, M., 294 Mochida, K., 222, 223(98), 226, 233(98) Molton, P. M., 273, 277, 283(37), 284(37), 294, 300(37), 304 Mononen, I., 258 Moody, W., 62(280), 63 Moore, W. E., 263, 275, 276(20) Mopper, K., 19, 20(12), 32(12), 33, 67, 68( 12) Moret, G., 141 Morgan, S. L., 266 Moriwaki, F., 246 Moriyasu, M., 70, 71 Mort, A. J., 56 Moschel, R. C., 28 Moss, C. W., 267 Moye, C. J., 284 Mukaiyama, T., 107(105), 108(105), 109 Mulholland, G. K., 86(56), 87(56), 89 Munsinghe, V. R. N., 188, 189(38, 39) Murai, K., 246 Murata, K., 37, 55(128, 129) Murray, C. T . , 239
N Naim, M., 198 Nakajima, A,, 219 Nakakuki, T., 38 Nakamura, C. Y.,196 Nakanishi, E., 249 Nakano, T., 200 Nakatani, H., 234 Nakayama, T., 314, 321(211) Namiki, M., 309 Nam Shin, J. E., 163(188),, 165 Narasimhan, S., 42, 57(167), 60(167) Narui, T., 38 Natowicz, M., 42, 46 Nebinger, P., 48, 57(214) Neeser, J.-R., 256, 262, 263(27), 270(8)
AUTHOR INDEX Nef, J. U.,301 Nelson, D. A,, 277, 283(37), 284(37), 290, 294, 300(37), 304, 316 Nelson, E. C., 286 Neukom, K., 290 Nevins, D. J., 262 Newth, F. H., 284 Ng Ying Kin, N. M. K., 44, 45(186), 46(186), 48(186), 64(186) Ni, X.-R., 210 Nichols, S. B., 275 Niedermeier, W., 260, 266 Niesner, R., 39, 300 Nikolov, Z. L., 23, 32(90), 33, 38, 39(137), 41 Nishimoto, S. K., 42, 46(176) Nodzu, R., 293 Noel, D., 23, 39, 67(34) Noltemeyer, M., 228, 229(114), 239(114) Nomura, H., 230, 234(120) Norbonne, J. M., 204 Nordin, P., 67 Nothnagel, E. A,, 40 Nursten, H. E., 307, 322 Nyhammar, T., 311, 312 0 Oades, J. M., 263 Odell, G. V., 316 Ogata, S., 255, 256(6) Ogawa, T., 87(64), 89, 161(64), 207, 209 Ohashi, M., 244 Ohlson, S., 31, 72 Ohtsuka, E., 181, 182 Ojha-Poncet, J., 141 Okada, Y., 44, 57(189, 190) Okubo, T., 209, 235 Olesker, A., 87(61), 88(61), 89 Olieman, C., 24, 51(38), 52, 64 Olsen, I., 258 Olson, W. K., 104(100), 105 Olsson, E., 291 Olsson, K., 311, 312, 313, 314 O’Meara, D., 282 Onda, M., 218 Oparaeche, N. N., 188, 189(38, 39) Oppenauer, R., 276, 288(27) O’Reilly, R., 322 Orstan, A., 226, 234, 238(137)
347
Osa, J . , 209, 210, 246, 247(169) Oshima, R., 21, 33, 56, 57, 59(25), 62(25), 70 Osman, S. F., 57, 69(269) Ossowski, P., 41 Otagiri, M., 223, 249 Otani, S., 293 Ototani, N., 68 Overend, W. G., 301
P PacBk, J., 87(62), 89, 144, 145, 152, 173(161) Pachla, L. A , , 35 Paice, M. G., 263, 264(32) Painter, T. J., 324 Palasinski, M., 274 Pallasch, G., 55 Palmer, J. K., 51, 54, 64(233) Parente, J. P., 43, 46, 51, 60(179), 62(198), 63(229) Parrish, F. W., 51, 63(241) Partlow, E. V., 263, 264(30) Patchornik, A., 179, 181, 182, 184, 188(1), 193, 198(58), 202, 203, 204 Pate, B. D., 86(48), 89, 104(48), 142(48), 143(48) Patil, V. J., 123, 124(137), 125(137), l27( 137) Patrick, D. W., 20, 53(19) Paulsen, H., 321 Pav, J. W., 55 Pecina, R., 20, 26, 39(13), 53(13), 54(13) Pedersen, C., 77, 78(28), 84(42), 85, 97(87), 98, 101(94), 102(94), 103, 106(94), 107(107), 108, 109, 131(146), 132, 134, 137, 138, 166(25), 168(107), 169(107) Peer, H. G., 320 Pendergast, D. D., 227 Penglis, A. A. E., 74, 75(10), 76(10), 77(10), 80(10), 118(130), 119, 120, 121(l34), 122(l34), l27( 134), l57( 134) Percival, E. G. V., 288 Perlin, A. S., 94, 163(188), 165 Pernemalm, P.-A., 277, 290, 312, 313, 314 Pettersen, R. C., 33, 52(83), 53(83), 63(83), 64(83), 263 Pettit, B. C., Jr., 51 Phillip, B., 297, 299
348
AUTHOR INDEX
Phillips, G. O., 182 Phillips, L., 75, 77, 78, 79(23, 29), 82(23), 86(23, 24), 87(23, 24), 90(23), 91(23), 93(23), 94(23), 95(23), 160(24), 162(24), 163(24), 164(24) Picq, D., 117(129), 118, 119, 128(129) Pillai, V. N. R., 179 Pincock, J. A., 194 Pirisino, J. F., 23, 25(30), 50(30), 51 Pittet, A. O., 25 Plant, P. J., 198 Plattner, R. D., 191 Pocsik, I., 231 Podolsky, D. K., 45, 46(196) Polacheck, I., 198 Pollock, C. J., 269 Poloskq, J., 292 Polta, J. A., 19 Popoff, T., 276, 279, 280, 282, 289, 290, 294(49), 295(49), 313 Popp-Ginsbach, H., 321 Porsch, B., 19, 24(10), 33(10), 38, 59(10, 143) Porter, W. H., 268 Posner, G. H., 98, 166(89) Post, A,, 261, 267(23) Praznik, W., 39 Preston, J. F., 40, 41(160) Prey, V.,293 Prince, S., 28 Prout, C. K., 129(141), 130(141, 145), 131, 135(141) Pulley, A. O., 206
Q Que, L., Jr., 173
R Rabel, F. M., 38 Radeos, M., 221(96, 97), 222, 225(96) Rajakyla, E., 28, 34 Rajender, S., 225 Rao, G. V., 173 Rao, V. S. R., 206 Rasmussen, J. R., 163, 164(187) Rasmussen, P., 138 Rauh, S., 230, 234(121) Raupp, D. L., 33, 51(85)
Re, A., 95(82), 96, 99(82) Reddy, G. S., 90(65), 92, 93(65), 94(65), 96(65), 100(65), 106(65), 119(65), 120(65), 122(65), 149(65), I50(65), 162(65), 163(65), 164(65), 165(65), 168(65), 17l(65), 175(65) Redmond, J. W., 68 Redmore, D., 284 Rees, D. A., 230 Refn, S., 84(42), 85 Reggiani, M., 233, 245(127) Reichman, U., 110(108), 111, 138(108) Reichstein, T., 276, 288(27) Reilly, P. J., 23, 33, 41 Reinhold, V. N., 56, 57(260), 69, 70 Rendleman, J. A., Jr., 301 RepBSovA, L., 292 Rewicki, D., 315, 319(213) Reynolds, D. L., 35 Reynolds, T. M., 286, 308 Rice, F. A. H., 277 Rice, M. J., 271 Rich, D. H., 199 Richard, S., 204 Richards, G. N., 62(280), 63, 282, 296, 301, 302(151), 303(152, 155). 306 Richardson, A. C., 118(130), 119, 120, 121( I 341, 122(1341, l27(134), l57( 134) Richmond, M. L., 27 Richtzenhein, H., 303 Riddel, F. G., 79 Rideout, D. C., 210 Riehl, T. E., 248 Riley, D. A., 107(105), 108(105), 109 Rinaudo, M., 32, 38, 39, 269 Ripmeester, J. A., 307, 325 Rist, C. E., 309 Ritchie, R. G. S . , 191 Robards, K., 32 Robinson, B. H., 234, 237(142), 238(142), 239(142) Robinson, J., 67 Rocklin, R. D., 30, 65(72) Rodriguez, L. J., 230 Rohrbach, R. P., 230, 234, 236(119) Rojas, R. R., 24, 32(39), 33(39), 41(39), 59(39), 60(39), 64(39) Rolf, D., 271 Romeo, T., 40, 41(160) Root, D. F., 263, 264(29)
AUTHOR INDEX Rosanske, T. W., 239 Rosenbrook, W., 107(105), 108(105), 109 Rosenfelder, G., 56, 68(264), 69(264) Ross, J. B. A., 226 Roth, K., 73 Rubinstein, M., 203 Rundle, R. E., 214, 218 Russell, K. R., 319 Rutar, V., 94, 164(76) Rydholm, S. A., 297
S Sachetto, J.-P., 309 Sadeh, S., 187 Saeman, J. F., 263, 298 Saenger, W., 206, 209, 225, 227, 228, 229, 232(104), 233(104) Sage, U., 321 Saito, R., 209 Sakurai, Y.,289 Salemis, P., 57, 66(267) Salimi, S. L., 35 Samarco, E. C., 51, 63(229) Sammes, P. G., 200 Samuel, J., 116 Samuelson, O., 31, 38, 291, 301, 302, 303 Sand, D. M., 220, 230(86), 240(86) Sandberg, A.-S., 36 Sanger, M. P., 56 Sano, T., 234 Sarel-Imber, M., 144 Sartorelli, A. C., 150(173), 151 Sasaki, M., 234 Sasaki, Y.,209(36), 210 Sato, M., 248 Satoh, J. Y.,196 Satyamurthy, N., 86(55), 87(55), 89, 146(167), 147 Sawada, M., 182 Sawaki, S., 194, 196 Schaaf, E., 215, 217(70) Schaffer, R., 283 Schardinger, F., 211, 212, 215(53), 217(53) Schauer, R., 36, 37(119, 120, 121, 122) Schiller, R. L., 210, 234, 239(139), 242(139), 245(140) Schlabach, T. D., 67 Schlaeger, E. J., 204 Schlenk, H., 220, 230(86), 240(86)
349
Schlenk, W., Jr., 217 Schmidt, J., 39, 54(151) Schmit, A. S., 19, 44(19), 59(19), 62(19) Schofield, P., 198 Schols, H. A., 265 Scholz, N., 36, 37(119) Schroeder, L. R., 300 Schwald, W., 52, 53(245), 298 Schwandt, V. H., 33, 52(83), 53(83), 63(83), 64(83), 263 Schwartz, A., 298 Schwartz, L. M., 221(88, 90,91, 92), 222, 225(90), 239 Schwarzenbach, D., 80, 140(37), 141, 154(178), 155 Schwarzenbach, R., 20, 38 Schweizer, T. F., 262, 263(27), 266(27), 268(27) Schwentner, J., 265 Scobell, H. D., 25, 51, 64(232) Scott, R. W., 275, 276(20) Sebastian, J. F., 223 Seidl, S., 100, 116(91), 166(91), 167(91), 170(91) Seiyama, A., 234, 238(147, 1481, 245( 148) Seki, T., 35 Seldin, D. C., 37, 55(131), 60(131) Selman, S., 291 Selvendran, R. R., 260, 263(20), 264(20) Seng, P. N., 266 Seno, N., 37, 55(131), 60(131) Sephton, H. H., 301 Serebryakov, E. P., 201 Serianni, A. S., 293 Seshadri, R., 28, 69(57e) Severin, T., 321, 322 Seymour, F. R., 191 Shafizadeh, F., 274 Sharma, M., 95(85), 97, 113, 120, 121(85), 122(85), 151, 156, 177 Sharon, N., 254, 256(5), 270(5) Sharp, J. K., 41, 42, 59(165), 60(165) Sharples, A., 298 Shaw, P. E., 51, 65(234), 286, 294, 311, 3 19(59) Sheehan, J. C., 200, 201 Sherblom, A. P.. 46 Sherr, B., 307 Sherrard, E. C., 298
350
AUTHOR INDEX
Shibamoto, T., 317 Shigematsu, H., 313 Shimizu, N., 246 Shimokawa, K., 246 Shoemaker, S. P., 54 Shu, C.-K., 318 Shukla, A. K., 36, 37(119, 120, 121, 122) Shulman, M. L., 121(136), 122 Sidhu, R. S., 158, 177(182) Siegel, B., 208, 226(14) Sihtola, H., 302 Silber, P., 181 Silver, H. K. B., 36 Simkovic, I., 306, 321(174) Sirimanne, P., 19, 29, 32(5), 33, 62(19), 63, 256, 257(12) Sjostrom, E. S., 305 Slavin, J. L., 52, 53(242), 263, 264(33) Slodki, M. E., 39, 60(149), 191 Sloman, K. G., 50 Smale, S. T., 163, 164(187) Smiley, K . L., 39, 60(149) Snyder, L. R., 18 Somawardhana, C. W., 93(77), 94, 96(77), 149(77), 150(77), 151, 165(77), 175(77) Sondey, S. M., 19, 21(8), 22(8), 23(7, 8), 2381, 61(8), 62(8), 71(8) Sonobe, H., 313 Sowden, J. C., 281, 282, 291(42) Speck, J. C., Jr., 281 Spellman, M. W., 20, 57(17) Spiro, M. J., 33 Spotswood, T. M., 239 Squire, A., 33, 34(96), 54(96) Srivastava, H. C., 100 Srivastava, V. K., 100 Stacy, C. I., 296 Stahnke, G., 192 Staines, W., 199 Staros, J. V., 204 Steele, E. M 5 1 Steffen, R., 323 Steiner, P. R., 76, 100. 106(104), 108(104), 109, I15(22), 116(22) Stetten, M. R., 297 Stewart, T. S., 270 Stezowski, J. J., 229 Stikkelman, R. M., 36 Stotz, E. H., 207 Straub, T. S., 223
Strecker, G., 43, 60(179) Street, I. P., 146(170), 147 Stroh, J. G., 70 Stutz, E., 288 Su, T. L., 110(111), 111 Sugii, A., 31 Sugimoto, S., 314, 321(211) Sukumar, S., 74 Sundararajan, P. R., 206 Suortti, T., 326 Suslova, L. M., 201 Suzuki, M., 209(36), 210, 269 Suzuki, S., 30, 31, 33(71), 34(71), 54(71), 56(80), 67, 70(78), 71(78, 80), 262, 264(25), 267(25) Svahn, B. M., 128, 171(140), 172(140) Svensson, S., 251 Swiedler, S. J., 20, 43, 47(16) Symanski, E. V., 33, 61(89), 62(89) Syngg, B. E., 326 Szarek, W. A,, 191 Szejtli, J., 206, 208(6), 209, 220, 223(83)
T Tabushi, I., 206, 233, 235, 244(130), 246 Tafuri, S. T., 163, 164(187) Taguchi, K., 234 Takahashi, K., 209, 210 Takahashi, M., 33, 67(87) Takahashi, T., 196, 296 Takahashi, Y., 87(64), 89, 125(138), 126, 132(138), 135(138), 161(64), 207 Takai, N., 70 Takazano, I., 48, 55(217) Takemoto, H., 68, 268, 270(49) Takenoshita, I., 209 Takeo, K., 217, 221(95), 222 Takeuchi, T., 70 Tamaki, T., 210 Tnnnkn, J , , 325 l'anaka, M., 27 Tanuka. S . , 181. 182 lanaka, T., 181, 182 Tanaka, Y., 48, 55(217) TBnBsescu, I., 188 Tang, P. W., 20, 56(15), 57. 68(15, 272) T a m , C. H., 110(110), 111, 138(110) Tanret, G., 182 Tashima, S., 191
35 I
AUTHOR INDEX Tatum, J . H . , 286, 294, 314, 319(59) Taylor, A. F., 67 Taylor, C., 41 Taylor, N. F., 86(53), 89, 90(53), 92, 93(80), 94, 95(53), 99(53), 104(53), 105(53), l10(109), 1 I I, 112(53), 116, 146(53), 155(53), 156(53) Teng, G., 29 Tesarik, K., 38, 71(135) Tewson, T. J., 99 Thakkar, A. L., 218 Thawait, S ., 62(278), 63 Theander, O., 276, 277, 279, 280, 28 1, 282, 289, 290, 291, 292(43), 293(43), 297, 299, 300, 305(118), 306, 313, 316, 323, 324, 325, 326 Thibault, J.-F., 40 Thiem, J., 265 Thomas, S. R., 86(59), 87(59), 89 Thompson, A., 262, 263(26a), 270 Thoraval, D., 195, 196(65) Tieckelmann, H., 37, 55 Tilden, E . B., 215 Timbie, D. J., 51 Timmell, R. E., 305 Timpa, J. D., 183 Tipson, R. S., 269 Tjioe, T. T., 36 Tjoeng, F.-S., 199 Tokola, R. A,, 35 Tolman, R. L., 104(98), 105, 143(98), 155(98) Tomana, M., 266 Tomasik, P., 274 Tomita, Y.,31, 246 Tong, E. K., 199 Torello, L. A., 260, 267(22) Toshima, N., 209, 210(31) Trainor, G. L., 84(44), 85, 95(44), 96(44), 99(44) Tressl, R., 315 Tronchet, J . M. J., 80, 112, 140(37), 141, 154(178), 155, 169(114) Tropsch, H., 299 Trugo, L. C., 66 Tsao, C. S., 29, 35, 36(66) Tsao, G. T., 21, 25, 34, 39, 53(22), 54(22) Tscherne, R. J . , 21, 30(27) Tsuchiya, T., 125(138), 126, 132(138), 135(138), 180, 191(7)
Tsuji, T., 46 Tsunehisa, S., 46 Tung, C. H., 209 Turco, S. J., 43, 68 Turro, N . J., 209, 235 U
Uekama, K., 210, 249 Ueno, A., 209, 210, 246, 247(169) Uenoyama, S., 209, 210(31) Ukena, J., 203 Umazawa, K., 201 Umezawa, B., 194, 196 Umezawa, S., 180, 191(7) Utamura, T., 247(170b), 248
V Valent, B., 42, 59(165), 60(165) Valentekovic-Horwath, S., 132(147), 133, 146(147), 147(147), 153(147) van Bekkum, H., 292 Van Damme, F., 31 van den Berg, G. R., 248 van den Berg, J. H. M., 68 van den Eijnden, D. H., 33, 42, 43(88, 170), 45, 46, 65(170, 202) Van den Ouweland, G. A. M., 320 Van Der Aalst, M. J. M., 51 van Eikeren, P., 40, 41(154) van Etten, R. L., 223, 244 van Halbeek, H., 42, 43, 46(172), 79 van Hooidonk, C., 222, 227, 248 van Riel, J. A. M., 52 Van Rijn, C. J. S., 85, 86(39), 87(39), I13(39) Varki, A., 36 Varma, R., 265 Varma, R. S., 265 Varvoglis, A. G., 203 Vasella, A., 98, 166(89a) Vegh, L., 191 Veregin, R. P., 219 Verhaar, L. A. T., 23, 29, 34, 50(29), 51, 66(29), 67(29) Vernengo, M. J., 13 Vernin, G., 307 Verzele, M., 21, 31, 58, 61(24) Vestal, M. L., 69
352
AUTHOR INDEX
Villani, R. P., 234, 245(140) Villiers, A., 211 Vliegenthart, J. F. G., 79 Voragen, A. G. J., 29, 33(67), 34(67), 40(67), 41, 54(67), 265 Voyksner, R. D., 69 Voznyi, Y. V., 132(150), 133 Vrfitnq, P., 30, 39(69), 40, 67, 68 Vuorinen, T., 292 W
WagstafTe, P. J., 51 Wakabayashi, T., 181, 182 Waldmann, E., 293 Walker, G. W., 41 Walker, M. E., 194 Wall, R. A., 25 Walters, D. G., 35 Walther, H. J. 69 Wan, C. N., 146(169), 147 Wang, W. T., 57, 69(270) Ward, T. J., 233, 248 Warren, C. D., 19, 44(19), 59(19), 62(19) Warthesen, J. J., 38, 51(145) Watanabe, K. A., 110(108), 111, 138(108) Watanabe, N., 67 Waterman, H. I., 274 Webster, G. H., 299 Wegener, G., 298 Weingarten, G. G., 200 Weiss, A. H., 274 Welch, J. T., 104(101), 105, 128, 171(140), 172(140) Welch, M. J., 99 Wennerblom, A., 302 Wentz, F. E., 52, 53(243) Westerlund, E., 279, 283 Westphal, G., 317 Westwood, J. H., 75, 76, 83(41), 84(41), 85, 86(12), 90(12), 91(12), 92, 93(12), 94, 95(12), 97, 99(67), 105(1I), 114(72), 148(11, 12), 149(84), 151. 155(11) Wetzel, D., 36 Wheals, B. B., 23 Wheaton, R. M., 24 Whistler, R. L., 292, 297, 301, 305, 306 White, A. R., 54 White, C. A., 38 White. P. C.. 23
Whitelaw, M., 32 Whitt, F. R., 296 Wiebe, L., 97(87), 98 Wieland, T., 192, 193 Wieruszeski, J.-M., 43, 45 Wilcheck, M., 204 Williams, D. M., 180, 189(6) Williams, J. M., 20, 56( 1 3 , 57, 68(15) Wills, R. B. H., 65 Wilson, C. W., 51 Wilson, D. C., 326 Wilson, R. M., 200 Wimalasiri, P., 65 Wing, R. E., 182 Winsauer, K., 65 Winzler, R. J., 268 Withers, S. G., 146(170), 147 Wnukowski, M., 51, 67(235) Woelk, H. U., 323 Wojcik, J. F., 226 Wolf, D. D., 269 Wolfe, L. S., 44,45(186), 46(186), 48(186), 64(186) Wolfrom, M. L., 262, 263(26a), 270, 295, 296 Wong, C. H., 90(66), 91(66), 92, 99(66), 109(66), 1 lO(66) Wong, T. C., 94, 164(76) Wood, K. R., 151(176), 152 Wood, R., 25, 30(46) Woodward, R. B., 193, 198(58) Woollard, D. C., 27, 51(57) Wray, V., 75, 77, 78, 79(23), 81, 82(23), 8 W 3 , 241, 87(23, 241, 90(23), 91(23), 93(23), 94(23), 95(23), 160(24), 162(24), 163(24), 164(24) Wright, J. A., 110(109), 111 Wu, A. M., 43 Wyss, P. C., 116, 172(126)
Y Yagi, Y., 248 Yamada, T., 182 Yamaguchi, Y., 35 Yamamoto, M., 234 Yamamoto, Y., 218 Yamazaki, N., 293 Yang, M. T., 53, 54(246), 65(246) Yates, A. J., 260, 267(22) Yeransian, J. A., 50
AUTHOR INDEX Yeung, E. S., 66 Yokokawa, C . , 304 Yokoyama, M. T., 264 Yokoyama, Y., 37, 55(128, 129) Yoruzu, T., 209 Yoshida, K., 293 Yoshida, N., 234 Yosizawa, Z., 55 Young, M . , 29, 36(66)
353 Z
Zaikov, G . E., 251 Zehavi, U . , 179, 180, 181, 182, 184, 187, 188, 193, 198 Zen, S., 191 Zent, J. B . , 307 Zhen, Z., 209 Zygmunt, L. C., 51, 63(228)
SUBJECT INDEX A Acetaldehyde, 321 Acetals, protection as, 182-189 Acetic acid cyclodextrin inclusion complexes with, 22 1 liquid chromatography, 53-54 pH of aqueous solutions of, 252 Acetoin formation, 294 from hydrothermolyzed cellulose, 304305 Acetol formation, 293 high-temperature transformation, in alkali conditions, 284 Acetolysis, for cleavage of glycosidic linkages, 269-270 Acetonitrile, cyclodextrin inclusion complexes with, 221 N-Acetyl-9-deoxy-9-fluoroneuraminic acid, I3C-n.m.r. data for, 177 2-Acetyl- 1-ethylpyrrole, formation of, 3 133 14 N-Acet ylglucosamine glycosidic, in glycoproteins, 257 linked to L-asparagine, N-glycosylic linkage, 255 2-Acetyl-3-hydroxyfuran, 320-32 1 2-Acetylpyrrole, formation of, 312313 1-O-Acetyl-2,3,5-tri-O-benzoyl-4-deoxy-4fluoropentofuranose -, a-L-lyxo-, IH-and I9F-n.m.r. data for, 143 -, p - D - r i b , 'H- and "F-n.m.r. data for, 143 a-Acid glycoprotein, oligosaccharides, liquid chromatography separation, 4243 Acid glycosaminoglycans, quantitation of uronic acids in, 258 Acylpyrrole formation, from 3-deoxyhexos-2-ulose, by Strecker degradation, 311-312 Adamantenecarboxylate ion, cyclodextrin inclusion complexes with, 221
Adamantenecarboxylic acid, cyclodextrin inclusion complexes with, 221 Adriamycin analogs, 28 Aldaric acids, analytical high-performance liquid chromatography, 33-35 Aldehydes, protection of, 195-198 Alditols, liquid chromatography, 33 pre-column derivatization procedures, 68 Aldonic acid acid decomposition, 252 analytical high-performance liquid chromatography, 33-34 ultraviolet-absorbances of, 65 Aldopentoses acid decomposition, 252 high-temperature transformation, 275284 Aldose isomerization of, to ketose, with hydride shift, 287 liquid chromatography methods for analysis of, 33 transformations, basic conditions, 28 1 Aldotetroses, high-temperature transformation, 275-284 Aldotrioses, high-temperature transformation, 275-284 Aldulosonic acids, analytical high-performance liquid chromatography, 34 Alduronic acid decarboxylation, 306 high-temperature transformation acidic conditions, 284-291 basic conditions, 291-295 Alginic acid, 307 oligomers, liquid chromatography, 40 D- Allop yranoside -, methyl 6-azido-3,6-dideoxy-3-fluoro-
P-
"C-n.m.r. data for, 171 'H- and I9F-n.m.r. data for, 119 -, methyl 3-deoxy-3-fluorop anorner, IH-and IgF-n.m.r. data for, 90 -, 4,6-O-isopropylidene-p-, 'H- and IyF-n.m.r. data for, 90 -, 6-O-pivaloyl-p-, 'H- and I9F-n.m.r. data for, 90 354
SUBJECT INDEX
355
-, 6-O-trityl-P-, 'H- and I9F-n.m.r. -, 4,6-O-benzylidene-2,3-dideoxy-2fluoro-adata for, 90 -, methyl 3-deoxy-3-fluoro-P-, benzyl 3-azido-, 'H- and 19F-n.m.r. data for, 117 13C-n.m.r.data for, 162 -, 6-O-pivaloyl-P-, T - n . m . r . data -, benzyl 3-benzamido-, 118 -, methyl 3-amino-, 'H- and I9Ffor, 162 Allopyranosyl fluoride n.m.r. data for, 117 -, D-, 3-azido-4,6-O-benzylidene-3-, methyl 3-benzamido-2,3-dideoxy-2deoxy-Pfluoro-aW-n.m.r. data for, 170 -, 4,6-O-benzylidene-, IH- and I9F'H- and 19F-n.m.r. data for, 117 n.m.r. data for, I18 -, 2,3,4,6-tetra-O-acetyl-~-, IH- and -, 4,6-di-O-acetyl-, 'Hand I9F19F-n.m.r. data for, 82 n.m.r. data for, 118 Allose, liquid chromatography methods for -, 4,6-di-O-benzoyl-, 'H- and I9Fanalysis of, 33 n.m.r. data for, 118 Alpha-I-acid glycoprotein, hydrolysis, with -, methyl 4-O-benzoyl-6-bromo-2,3,6trifluoroacetic acid, 268 trideoxy-2-fluoro-aAlternative chair conformation, 7 -, 3-benzamido-, 'H- and 19F-n.m.r. D-Altropyranoside data for, 123 -, 3-amino-2,3-dideoxy-2-fluoro-a-, -, 3-(trifluoroacetamido)-, 'H- and derivatives I9F-n.rn.r. data for, 123 'H- and I9F-n.rn.r.data for, 117-1 18 -, methyl 4,6-O-benzylidene-2,3-diN-containing, synthetic precursors, deoxy-2-fluoro-a'H- and I9F-n.m.r. data for, 117-, 3-(diallylamino)-, 'H- and I9FI I8 n.m.r. data for, 118 -, 3-amino-2,3,6-trideoxy-2-fluoro-6-, 3-(trifluoroacetamido)-, 'H- and halogeno-a-, derivatives '9F-n.m.r. data for, 118 IH- and I9F-n.m.r. data for, 123 -, methyl 2,3,6-trideoxy-2-fluoro-aN-containing synthetic precursors, 'H-, 3-(trifluoroacetamido)and '9F-n.m.r. data for, 123 I3C-n.m.r. data for, 170 -, benzyl 3-benzamido-2,3,6-trideoxy-2IH- and I9F-n.m.r. data for, 124 fluoro-a-, 'H- and 19F-n.m.r. data -, 3-aminofor, 123 I3C-n.m.r. data for, 170 -, benzyl 2,3-dideoxy-2-fluoro-aIH- and I9F-n.m.r. data for, 123 -, 3-azido-6-O-(methylsulfonyl)-, IH-, 3-benzamido-4-0-benzoyland I9F-n.m.r. data for, 118 13C-n.m.r. data for, 171 -, 3-benzamido-, IH- and I9F-n.m.r. 'Hand 19F-n.m.r. data for, data for, 118 124 -, 3-benzamido-6-O-mesyI-, 'H- and -, 3-benzamido-4-O-benzoyl-6I9F-n.m.r. data for, 118 bromo-, I3C-n.m.r. data for, 170 -, 3-benzamido-6-O-tosyI-, 'H- and I9F-n.m.r. data for, 118 D-Altropyranosyl fluoride, 2,3,4,6-tetra-O-, benzyl 2-fluoro-a-, 3-benzamidoacetyl2,3,6-trideoxy-, I3C-n.m.r.data for, a anomer, 13C-n.m.r.data for, 160 170 p anomer, V-n.m.r. data for, 160 -, benzyl 2-fluoro-2,3,6-trideoxy-6-iodo-Altrose, liquid chromatography methods afor analysis of, 33 -, 3-azido-, 'H- and 19F-n.m.r. data Amadori compounds for, 123 2,3-enolization in weak acids, 320 -, 3-benzamido-, 'H- and 19F-n.m.r. formation of, 307-308 data for, 123 Amadori rearrangement, 3 11
356
SUBJECT INDEX
formation of 3-deoxyhexo-2-dose by way of, 311 mechanism of, 308-309 Amides, protection as, 194 4-(Amidosulfonylmethyl)-6-methoxy-2-(4methylphenyl)quinoline, photodecomposition of, 194 Amines, catalytic effect, on formation of products from Maillard reaction, 318321 Amino acids, catalytic effect, on formation of products from Maillard reaction, 318-321 2-Aminobenzoic acid, cyclodextrin inclusion complexes with, 222-224 4-Aminobenzoic acid, cyclodextrin inclusion complexes with, 222-224 . p-Aminobenzoic hydrazide, for enhancing detectability of carbohydrates, 67 Amino compounds, carbohydrate transformation in presence of, 307-323 Amino-containing sugars, Onodera's work on, 3 Aminodeoxyhexofuranoses, fluorinated, IH-and I9F-n.m.r. data for, 127128 Aminodeoxyhexofuranosides, fluorinated, IH-and 19F-n.m.r. data for, 127-128 Aminodeoxyhexopyranosyl fluorides 'H- and 19F-n.m.r. data for, 117 N-containing, synthetic precursors, IHand 19F-n.m.r. data for, 117 Aminodeoxypentopymosides, fluorinated, IH-and 19F-n.m.r. data for, 128 Aminodideoxy-3(or 4)-flUOrO sugars IH- and I9F-n.m.r. data for, 118 N-containing synthetic precursors, IHand 19F-n.m.r. data for, 119 Aminodideox y-6-fluoro sugars, IH-and 19F-n.m.r. data for, 120-122 Amino sugars amino groups of, protection of, 192194 difluorinated, 'H- and 19F-n.m.r.data for, 157 fluorinated, W-n.m.r. data for, 170 fluorinated unsaturated, 'H-and I9Fn.m.r. data for, 126-127 N-containing, synthetic precursors, I T n.m.r. data for, 170
Amino-2,3,6-trideoxy-2-fluorohexopyranoses, IH-and 19F-n.m.r.data for, 125-126 3-Amino-2,3,6-trideoxy-2-fluoro-hexopyranoside derivatives 'H-and I9F-n.m.r. data for, 123-125 N-containing synthetic precursors, IHand I9F-n.m.r. data for, 123-125 Amino-2,3,6-trideoxy-2-fluorohexopyranosides, IH- and 19F-n.m.r. data for, 125-126 Amino-2,3,6-trideoxy-2-fluorohexopyranosyl halides, 'H-and I9F-n.m.r. data for, 125-126 Amobarbital, cyclodextrin inclusion complexes with, 223, 224 Amylopectin, 295 high-temperature transformation of, 296297 Amylose, 295 alkaline hydrothermolysis of, 296 Anabaena flosaquue, extracellular polysaccharide, hydrolysis of, 253, 260 Anhydro-4-O-benzyl-3-deoxy-3-fluoro-~Daltropyranose, 2-O-acetyl-l,6-, 'H- and I9F-n.m.r. data for, 114 2,5-Anhydro-l-deoxy-l, 1-difluoro-Dhexitol -, arubino-, IH-and 19F-n.m.r.data for, 151 -, ribo-, IH-and 19F-n.m.r. data for, 152 -, 3,4,6-tri-O-acetyl-, IH- and I9Fn.m.r. data for, 152 1,5-Anhydro-6-deoxy-6-fluoro-~-uru binohex-1-enitol -, 3,4-di-O-acetyl-l ,Zdideoxy-, 'H- and I9F-n.m.r. data for, 113 -, 4-O-benzyl-3-deoxy-, IH-and I9Fn.m.r. data for, 113 -, 3,4-di-O-benzyl-, IH- and I9F-n.m.r. data for, 113 Anhydro-3-deoxy-3-fluoro-P-~-idopyranose, 2,4-di-O-acetyl-l,6-, IH-and 19F-n.m.r. data for, 114 1,6-Anhydr0-2,4-dideoxy-2-fluoro-P-~erythro-hexopyranos-3-ulose,'H-and 19F-n.m.r. data for, 114 Anhydroglucose, liquid chromatography, 53-54
SUBJECT INDEX
357
Anhydrohexofuranose derivatives, diArabinopyranose fluorinated, 'H- and I9F-n.m.r. data -, D-,1,3,4-tri-O-acetyl-2-deoxy-2for, 151-152 fluoro-pAnhydrohexopyranose derivatives, diW-n.m.r. data for, 167 fluorinated, IH-and I9F-n.m.r. data IH-and 19F-n.m.r.data for, 104 for, 151-152 -, 3-deoxy-3-fluoro-p-, IH-and I9F1,5-Anhydrohexopyranose derivatives, n.m.r. data for, 105 fluorinated, IH-and 19F-n.m.r. data D- Arabinopyranoside -, methyl 4-0-allyl-2-amino-2,3-difor, 113 I ,6-Anhydrohexopyranose derivatives, deoxy-3-fluoro-p-, IH-and I9Ffluorinated, IH- and 19F-n.m.r. data n.m.r. data for, 128 for, 114 -, trifluoromethyl 3,4-di-O-acetyl-P-, 3,dAnhydrohexose derivatives, fluoriIH-and I9F-n.m.r. data for, 104 nated, IH-and I9F-n.m.r. data for, L-Arabinopyranoside, methyl 4-deoxy-4I I5 fluoroAnilinium perchlorate, cyclodextrin inclua anomer sion complexes with, 222, 224 Wn .m .r. data for, 168 'H- and 19F-n.m.r. data for, 106 Anno, K., 9 P anomer 2-Anthracenesulfonate, inclusion complex 13C-n.m.r.data for, 168 with gamma cyclodextrin, 210 IH- and I9F-n.m.r. data for, 106 D-Apiose, 14 Arabinitol, liquid chromatography methods D-Arabinopyranosyl fluoride -, 3,4-O-acetoxonium-2-O-methyl-, IHfor analysis of, 33 and 19F-n.m.r.data for, 101 o-Arabinofuranoside IH-, 1,2-di-O-acetyl-5-O-benzoyl-2-deoxy- -, 3,4-O-benzoxonium-2-O-methyl-, and I9F-n.m.r. data for, 101 2-fluoro-3-0-formyl-, 3(or 4)-O-benzoyl-2-O-methyl-p-,'Ha anomer, IH- and 19F-n.m.r. data for, and 19F-n.m.r.data for, 102 141 -, 2-deoxyp anomer, IH- and I9F-n.m.r. data for, -, 2-bromo-p-, 'H-and 19F-n.m.r. 142 data for, 135 -, methyl 2,5-di-O-benzoyl-3-deoxy-3-, 3,4-di-O-acetyl-2-bromo-a-, 'Hfluoro-a-, IH- and I9F-n.m.r. data and 19F-n.m.r. data for, 135 for, 112 -, 3,4-di-O-acetyl-2-iodo-a-,IH-and -, methyl 2-deoxy-2-fluoro-a19F-n.m.r. data for, 135 -, 5-O-benzyl-, IH-and I9F-n.m.r. -, 3,4-di-O-acetyl-2-deoxy-2-fluoro-~-, data for, 11 1 IH-and I9F-n.m.r. data for, 155 -, 3,5-di-O-benzyl-, IH-and I9Fn.m.r. data for, 111 -, 3,4-di-O-acetyl-2-O-methyl-p-, 'HIH-and I9F-n.m.r. data for, 11 1 and I9F-n.m.r. data for, 101 Arabinofuranosyl fluoride -, 3,4-di-O-benzoyl-2-O-methyl-, IHand I9F-n.m.r. data for, 101-102 -, 3,5-di-O-benzoyl-2-0-methyl-, I3C-, 2,3,4-tri-O-acetyl-, IH- and I9Fand I9F-n.m.r. data for, 106 -, 2,3,5-tri-O-benzoyl-a-~-,n.m.r. data n.m.r. data for, 101 for, 106, 168 -, 2,3,4-tri-O-acetyl-P-, W-n.m.r. data for, 161 -, 2,3,5-tri-O-benzyl-, 'H- and I9Fn.m.r. data for, 107 -, 2,3,4-tri-O-benzoyl-a-, 'H- and I9F-, 2,3,5-tri-O-benzyl-a-~-, T-n.m.r. n.m.r. data for, 101 data for, 168 Arabinose -, 2,3,5-tri-O-benzyl-P-~-, I3C-n.m.r. -, D-, high-temperature transformation, acidic conditions, 216-211 data for, 168
SUBJECT INDEX
358
determination of enantiomeric form, 66 -, L-, in plant cell-wall hydrolyzates, liquid chromatography analysis, 55 liquid chromatography of, 33, 52-53 Aryl glycosides, photoinduced cleavage of, 182-183 Ascorbic acids -, L-, decarboxylation of, in acid solution, 289-290 liquid chromatography, 35-36 ultraviolet absorbances of, 65 Aspidosperma australe, 13 5-Azido-4-(hydroxymethyl)-1methoxynaphthalene esters, photocyclization of, 201 6-Azido-4-O-benzoyl-2,3,6-trideoxy-2fluoro-D-ribo-hexopyranosyl halide a bromide, IH- and 19F-n.m.r. data for, 126 chloride, 'H- and I9F-n.m.r. data for, 125 a iodide, 'H- and '9F-n.m.r. data for, 126 2-(2-Azidophenyl)ethyl alcohol, 200 2-(2-Azidophenyl)ethyl esters, photocyclization of. 201
B Bacillus macerans, 2 12, 215-2 17 cyclodextrin transglycosylase, 207 Bacteria, thermophilic, 21 I Bacterial polysaccharides complex, structural and sequence analysis of, 57 extracellular, liquid chromatography fractionation, 49 Benzoate ion, cyclodextrin inclusion complexes with, 221 Benzoic acid, cyclodextrin inclusion complexes with, 221, 223, 224 Benzoin, esters of, photocyclization of, 20 1 Benzoquinones, formation, 294 Benzoylacetic acid, cyclodextrin inclusion complexes with, 223, 224 Benzoyl-phenylmethanol (benzoin) esters, as protecting group, 200-201 I-(Benzylamin0)-l -deoxy-~-rhreo-2-hexulose, 320 I -(Benzylamino)- I-deoxy-~-nrabino-2hexulosuronic acid, 320
Benzyl ethers, photochemical cleavage of, in presence of bromine, 182-183 Benzyl glycosides, preparative liquid chromatography, 60 Biomass-conversion processes, carbohydrates in, analysis of, 52-54 Biomass materials, polysaccharides in, 306 Blood-group gl ycoproteins, oligosaccharides, liquid chromatography separation, 43 a-Bromo-2-nitrobenzyl polymer, 199 Browning, 307, 324 Bufadienolides, 13 Bufo, 13 Butanedione, 321 formation of 2,5-dimethylhydroquinone from, 294-295 from hydrothermolyzed cellulose, 304305 I-Butanol, cyclodextrin inclusion complexes with, 222-224 2-rert-Butylanthraquinone, for enhancing detectability of carbohydrates, 67 5-Butylbarbituric acid, cyclodextrin inclusion complexes with, 223, 224 5-Butyl-2-thiobarbituric acid, cyclodextrin inclusion complexes with, 223, 224 C
Carbohydrate anomers, separation of, 7071 Carbohydrate photochemistry, 180 Carbohydrate polymers, compositional analysis of, 54-57 Carbohydrates N-reacetylation, after methanolysis, 256 separation on cation-exchange columns, mechanisms, 26 sequence. liquid chromatography methods for determining, 57-58 structure, liquid chromatography methods for determining, 57-58 Carbonyl-amine reaction, formation of N substituted aldosylamine by, 308 Carbonyl derivatives, photosensitive protecting groups, 195-202 Carboxylic acids free, photochemical release of, from a 2-
SUBJECT INDEX
359
nitrobenzyl-substituted poly(viny1 oligosaccharides, liquid chromatography alcohol), I99 separation, 42-43 protection of, 198-202 sialylated oligosaccharides, fractionaK-Carrageenan, oligomers, liquid chromation, 46 tography, 40 Chemical processes, carbohydrate transCartilage, glycosaminoglycans, analysis, 56 formation in, 323-326 Catechol, 295 Chitin as enzyme inhibitor, 326 hydrolyses, neutral, N-acetylated anaCathepsin, isolation of carbohydrates logs from, liquid chromatography, from, on analytical-scale columns, 60 41 Cathepsin-B, glycopeptides, liquid chromaoligosaccharides, liquid chromatography, tography separation, 48 39-40 Cathepsin-D, glycopeptides, liquid chroma- 3-Chlorobenzoylacetic acid, cyclodextrin tography separation, 48 inclusion complexes with, 223, 224 Cellobiose 6-Chloro-5,6-dideoxy-5,6-difluoro1,2-0alkaline degradation, product pattern, isoprop ylidene-3-0-meth yl-a-D-xylo302 hex-5-enofuranose hydrolysis of, 299 -, isomer, 13C-n.m.r.data for, 176 liquid chromatography methods for -, (2)isomer, I3C-n.m.r. data for, 176 analysis of, 33 3-Chlorophenyl acetate, cyclodextrin Cellobiouronic acid, hydrolysis of, 299 inclusion complexes with, 223, 224 Cellobiulose, liquid chromatography methChondroitin, disaccharides, liquid chromaods for analysis of, 33 tography, 37 Cello-oligosaccharides, liquid chromatogra- Chondroitin sulfate phy, 39 composition, analysis, 55 peak-area analyses, 64 oligosaccharides from, sulfation patpreparative, 60 terns, 49 Cellulases, mode of action, analysis of, 54 structural and sequence analysis of, 57 Cellulose Chondroitin 6-sulfate, conformational acid-catalyzed hydrolysis of, mechanism inversion, 7 of, 298-299 "C-n. m. r. spectroscopy acid hydrolysis of, 297-298 chemical shifts, 77 alkaline degradation of, products, 304 "C-r~.m.r.-~~F coupling constants, 77-78 alkaline peeling of, 301-303 'J ( I T , I9F),77-78 high-temperature transformation of, 2J ( I T , I9F), 78 alkaline conditions, 300-305 ("C, I9F),78 hydrothermolysis of, 299-300 4J ( I T , I9F),78 liquid chromatography analysis, 39 Coformycin analogs, 28 non-fermentable oligosaccharides, liquid Copper bis(phenanthroline), for enhancing chromatography methods for, 52 detectability of carbohydrates, 67 stopping reaction involving, 302-303 Corn syrup, oligosaccharides, liquid chrostructure, 297 matography analysis, 39 Cellulosic materials, yellowing of, by Cotton fibers, liquid chromatography aging, 324-325 analysis, 52 Cellulosine, 21 1 Crystalline dextrins, 211-213 Ceruloplasmin Cuprammonium, for enhancing detectabilglycopeptides, liquid chromatography ity of carbohydrates, 67 separation, 48 Curacin, 14 isolation of carbohydrates from, on o-Curacose, 14 analytical-scale columns, 60 Curamycin, 14
(a
360
SUBJECT INDEX
Curamycose, 14 Cyanoacetamide, for enhancing detectability of carbohydrates, 67 3-Cyanophenol, cyclodextrin inclusion complexes with, 221 4-Cyanophenol, cyclodextrin inclusion complexes with, 221 3-Cyanophenolate ion, cyclodextrin inclusion complexes with, 221 4-Cyanophenolate ion, cyclodextrin inclusion complexes with, 221 Cyclic acetals, as photosensitive protecting groups, 195 Cyclic adenosine monophosphate, 2nitrobenzyl and 6-nitroveratryl esters of, irradiation of, 204 Cyclic guanosine monophosphate, 2nitrobenzyl and 6-nitroveratryl esters of, irradiation of, 204 Cyclobarbital, cyclodextrin inclusion complexes with, 223, 224 Cyclodextrin alpha, 206 chemical synthesis of, 207 conformational change of, during complex-formation, 230-23 1 dipole moment, 232 D-glucosyl units, 228 inclusion-complexes, enthalpy-entropy compensation for, 224 strained, high-energy conformation, 228 beta, 206 chemical structure, 207 inclusion-complexes, enthalpy-entropy compensation for, 224 numbering of atoms of, 207 partially methylated, 245 branched, 246 I F c.p.-m.a.s. studies of, 231 cavity, water of, 227-228 chiral discrimination by, 247-249 covalent capping of, 247 2,6-di-O-methyl derivative, 245 discovery of, 211-213 enthalpy of association, 228 facilitation of association of molecules through presence of, 210 formation of, from starch, 215-217 further research in, 249
gamma, 206 modified, 245-246 space regulation of cavity by a p pended naphthalene moiety, 246 substitution with two naphthyl groups, 246-247 D-glucosyl residues, 214 liquid chromatography separation, 44-45 modified catalytic properties, 244-247 c h i d selectivity of, 249 complexing ability, 245 molecules, covalent linking of two, 245 periodate oxidation of, 214-215 physical properties of, 208 properties, 206 reactions capable of catalysis by, c h i d selectivity, 248-249 reactions catalyzed, 244 separation, 207 structural and sequence analysis of, 57 structure of, determination of, 213-215 -, tri-0-methyl-a-, 245 ultrasonic-relaxation techniques for, 230 Cyclodextrin inclusion complexes, 205-250 acid-base titration, 220 advantage of, 208 with azo dyes, 235, 239 with biphenyl compounds, 239 with cinnamates, 239 circular dichroism, 219-220 complex-formation detection of, 219-220 enthalpy-entropy compensation, 221225 favorable entropy change in, 233 hydrophobic interactions in, 233 kinetics of, 234-244 in nonaqueous solvents, 226 solvation changes on complexation, 225-226 standard-enthalpy change accompanying, 220-223 standard-entropy change, 220-223 standard free-energy decrease associated with, 220 thermodynamics of, 220-234 conformational change, 236, 238-239 cross-polarization, magic-angle-spinning, 13C-n.m.r. spectroscopy, 219
36I
SUBJECT INDEX crystal structure analyses, 232-233 with Crystal Violet, 241 with cyanine dyes, 244 discovery of, 217 electric-field pulse relaxation, 234 fluorescence, 219 formation of, 210-211 kinetics, 237-238 IH-nuclear magnetic resonance spectroscopy, 218 hydrophobic interactions, 226-227 with inorganic anions, 235-236 isoequilibrium, 221-225 with long-chain fatty acids, 240 with Methyl Orange, 242-244 nuclear magnetic resonance, 220 one-host-one guest complex-formation,
235-239 one host-two guests complexation, 209-
210,240-243 mechanisms, 21I with Methyl Orange, 245 with organic dyes, 235,240-242 phosphorescence decay, 235 properties, 217-218 with Pyronine Y,240 with Roccellin, 244 Saenger's theory of formation, 229 single-step, binding mechanism, 237 solubility, 220 stability, 208-209 dependence on polarizability of guest molecule, 232 stopped-flow kinetic studies, 234,237-
238 temperature, 225 temperature-jump relaxation kinetic studies, 234,237-238,240 with Tropaeolin, 242,244 two-dimensional, nuclear Overhauser effect experiment on, 218-219 two hosts-one guest complex formation,
239-240 two hosts-two guests complex-formation, 242-244 two-step mechanism, 238-239 ultrasonic absorption relaxation, 234 ultraviolet-visible absorption, 219 van der Waals forces in, 231-233 X-ray crystallography, 218
Cyclodextrin transglycosylase, 207-208 Cyclohexanecarboxylic acid, cyclodextrin inclusion complexes with, 221 Cyclohexanol, cyclodextrin inclusion complexes with, 222-224 Cyclomaltoheptaose, 206 Cyclomaltohexaose, 206 Cyclomaltohexaose inclusion complexes, standard formation enthalpies and entropies of, 221-223 Cyclomalto-octaose, 206 Cyclomalto-oligosaccharides.See also C yclodextrins branched, 246 liquid chromatography separation, 44-45 Cyclopentenones, formation, 294 Cyclosophoroses liquid chromatography separation, 44-45 preparative liquid chromatography, 60 L-Cysteine, and a-dicarbonyl compounds, reaction between, 318 DL-Cysteine, with D-galactose, products from, 317-318 D
Dansylhydrazones, liquid chromatography, pre-column derivatization procedures,
68,68 1-Deoxy-1-(dibenzylamino)-~-arabino-2hexulosuronic acid, 320 3-Deoxy-3-C-(mono or di)fluoromethyleneD-hexo(or pento)furanoses, 'H-and 19F-n.m.r. data for, 140-141 6-Deoxy-6,6-d~uorogalactopyranose -, 1,2:3,4-di-O-isopropylidene-a-~-, 'Hand 19F-n.m.r. data for, 151 -, 1,2,3,4-tetra-O-acetyI-~-, IH-and 19F-n.m.r. data for, 150-151 2-Deoxy-2,2-difluoro-~-ara bino-hexopyranose a anomer, 'Hand I9F-n.m.r. data for,
149 p anomer, IH-and 19F-n.m.r. data for, 149 2-Deoxy-2-fluoro-~-arab~nofuranose -, l-O-acetyl-5-0-benzoyl-3-O-formyla-,IH- and 19F-n.m.r. data for, 110 -, 5-0-benzyl-, IH-and I9F-n.m.r. data for, 110
362
-,
SUBJECT INDEX
3-Deoxy-a-~-erythro-hex-2-enopyranosyl 1,3-di-O-acetyl-5-O-benzoyl-a-, 'Hfluoride and I9F-n.m.r. data for, I10 -, 2,4,6-tri-O-acetyl-, 'H- and I9F-, 1,3-di-O-acetyl-5-O-benzyl-, 'H- and n.m.r. data for, 134 I9F-n.m.r. data for, I10 -, 2,4,6-tri-O-benzoyl-, 'H- and I9F-, 1,3-di-O-benzoyl-5-O-benzyl-, 'Hand I9F-n.m.r. data for, 110 n.m.r. data for, 134 4-Deoxy-~-glycero-2,3-hexodiulose, 292 -, Sphosphate, sodium salt, 'H- and 6-Deoxyhexopyranose derivatives, fluori19F-n.m.r. data for, 109 nated, 'H- and '9F-n.m.r. data for, -, 1,3,5-tri-O-benzoyl-(y-, 'H- and I9F132-133 n.m.r. data for, 110 -, 1,3,5-tri-O-benzyl-c-, 'H- and I9F3-Deoxy-~-erythro-hexosulose, 292 3-Deoxy-~-erythro-hexos-2-ulose, forman.m.r. data for, 110 2-Deoxy-2-fluoro-a-~-arabinofuranosyl tion, 310-311 bromide 3-Deoxyhexos-2-ulose, formation of, 3 11 -, 3-O-acetyl-5-O-benzoyl-, 'H- and 6-Deoxylglucose, liquid chromatography I9F-n.m.r. data for, 138 methods for analysis of, 33 -, 3,5-di-O-benzoyl-, 'H- and I9F-n.m.r. Deoxy-2-octulosonic acids, liquid chromadata for, 138 tography, 36-37 5-Deoxy-5-fluoro-a-~-g~ucofuranurono-6,3Deoxypentofuranose derivatives, fluorilactone, 1,2-O-isopropylidene-, I3Cnated, 'H- and I9F-n.m.r. data for, 138 n.m.r. data for, 170 2-Deoxypentopyranosy1 fluorides, 'H- and Deoxyfluorohexofuranoses, "C-n.m.r. data 19F-n.m.r. data for, 137 Deoxy sugars for, 165-167 acid decomposition, 252 2-Deoxy-2-fluorohexopyranosyl fluorides, 'H- and I9F-n.m.r. data for, 146-147 Liquid chromatography, 31, 33 3(or 4, or 6)-Deoxy-3(or 4, or 6)-fluorohex- Dermatan sulfate, composition, analysis, 55 opyranosyl fluorides, IH- and I9FDeulofeu, Venancio n.m.r. data for, 148-149 5-Deoxy-5-fluoro-p-~-idofuranurono-6,3- academic career, 12 awards and honors, 14 lactone -, 1,2-O-benzylidenebook, 13 Doctoral Thesis, 11 I3C-n.m.r. data for, 170 at E. R. Squibb & Sons, 12, 14 IH- and 19F-n.m.r. data for, 116 editorial work, 14 'H- and 19F-n.m.r. data for, 116 education, 11 -, 1,2-O-isopropylidene"C-n.m.r. data for, 170 family, 11 publications, 13 'H- and I9F-n.m.r. data for, 116 research, 11-13 5-Deoxy-5-fluoro- 1,2-O-isopropylidene-a~-g~ucofuranurono-6,3-lactone, 'HDextrans branching patterns of, 41 and 19F-n.m.r.data for, 116 3-Deox y-3-fluoroxylop yranose liquid chromatography analysis, 39 'H- and '9F-n.m.r. data for, 105 3,6-Dideoxy-3,6-difluoro-P-~-allopyrano-, 1,2,4-tri-O-acetyl-~-,IH- and I9Fside n.m.r. data for, 105 -, methyl 2-Deoxy-2-halogenohexopyranosyl fluoI3C-n.m.r. data for, 175 rides, 'H- and I9F-n,m.r. data for, 'H- and 19F-n.m.r. data for, 149 I 129 -, methyl 2,4-di-O-benzoyl-, IH- and I9F-n.m.r. data for, 149 2-Deoxy-2-halogenopentopyranosylfluorides, 'H- and I9F-n.m.r. data for, -, p-nitrophenyl, 'H- and 19F-n.m.r. data for. 150 135- 137
SUBJECT INDEX
363
formation of pyranones from, 280 -, phenyl formation of substituted acetophenones I3C-n.m.r. data for, 175 from, 280 IH-and I9F-n.m.r. data for, 150 high-temperature transformation, in 1,6-Dideoxy- 1,6-difluoro-2,3:4,6-di-Oalkali conditions, 284 isopropylidenegalactitol,'H- and I9Freaction of, with amino acids, 321 n.m.r. data for, 159 I ,6-Dideoxy-l,6-difluoro-galactito1, IH-and 2,3-Dihydroxyacetophenone,290 1,2-DihydroxyacroIein. See Triose-reducI9F-n.m.r. data for, 158 tone Dideoxydifluorohexopyranoses, 'H- and 3,8-Dihydroxy-2-methylchromone, formaI9F-n.m.r. data for, 149-151 tion, 290-291 Dideoxydifluorohexopyranosides, 'H- and 6,7-Dihydroxyphthalide, 290 I9F-n.m.r. data for, 149-151 Diisopropyl phosphorofluoridate, cyclodex5,6-Dideoxy-6,6-difluoro1,2-O-isopropylitrin inclusion complexes with, 222, dene-a-D-xylo-hex-Senofuranose 224 -, 3-O-benzyl-, I3C-n.m.r. data for, 175 -, 3-O-methyl-, 13C-n.m.r.data for, 176 Diketosamines, mechanism for cleavage of, 309-311 2,3-Dideoxy-2-fluoro-hexopyranosides, IH3,5-Dimethoxy-a,a-dimethylbenzyloxycarand 19F-n.m.r. data for, 131-132 bony1 groups, photochemical removal 2,6-Dideoxy-2-fluorohexopyranosyl fluoof, 192 rides, IH- and I9F-n.m.r. data for, 153 3,4-Dideoxy-~-g~ycero-hex-3-enopyranosyl3,5-Dimethoxybenzyloxycarbonylgroup, photochemical removal of, 192 fluoride 2.5-Dimethyl-I ,4-benzenediol, from hy-, 6-O-acetyl-2-ulose, 'H- and I9Fdrothermolyzed cellulose, 304-305 n.m.r. data for, 134 2,5-Dimethylbenzoquinone -, 6-O-benzoyl-2-ulose, IH-and 19Fformation, 294 n.m.r. data for, 134 from hydrothermolyzed cellulose, 3045,6-Dideoxy- 1,2-O-isopropylidenehex-5305 enofuranose photo6-chloro-5,6-difluoro-3-O-methyI-a-3,4,3',4'-Dimethylenedioxybenzoin, cyclization of, 201 D-xylo-,'H- and 19F-n.m.r. data for, N,N-Dimethylhydrazones, 197 154 -, 6,6-difluoro-3-O-methyl-a-D-ribo-, photosensitized decomposition of, in presence of oxygen, 198 IH- and 19F-n.m.r. data for, 154 -, 6,6-difluoro-3-0-methyl-f3-~-xylo-, 2.2-Dimethyl- I-propanol, cyclodextrin inclusion complexes with, 222-224 IH- and I9F-n.m.r. data for, 154 -, 6,6-difluoro-3-O-methyl-a-~-xyb,Dimethylthiocarbamates, photochemical cleavage, 189- 190 IH-and 19F-n.m.r. data for, 154 6-O-(Dimethylthiocarbonyl)-1,2:3,4-di-0Difluorinated hex-5-enofuranoses, IH- and isopropylidene-a-D-gdactopyranose, 19F-n.m.r. data for, 154 photochemical cleavage of, 190 Dihydropyrrolopyrazine, formation of, 3 14 2,4-Dihydroxy-2-(hydroxymethyl)butanoic 2,4-Dinitrobenzenesulfenicesters photolysis of esters, 200 acid, 305 as protecting groups for carboxyl Dihydroxyacetone, 321 groups, 200 formation of aromatics from, in acid 3.5-Dinitrophenyl phosphoric esters, phosolution, 279-280 tohydrolysis, 203 formation of benzofuran from, 280 Diols, protection of, 188-189 formation of hydroxydimethylpyranoI ,4-Dioxane, cyclodextrin inclusion compyrandiones from, 280 plexes with, 222, 224 formation of 3-hydroxy-6-methyl-4H1,2-Diphenylethylene dithioacetals, 196 pyran-4-one from, 280
-.
364
SUBJECT INDEX
photochemical decomposition of, in presence of oxygen, 197 Disaccharides in dairy products, liquid chromatography separation, 52 ionic, high-performance liquid chromatography, 33-37 large-scale preparative liquid chromatography, 62 neutral, analytical high-performance liquid chromatography, 32 preparative liquid chromatography, 60 sulfated, liquid chromatography, 37 Doisy, E.A., 12
Europium salts, for enhancing detectability of carbohydrates, 67
F Fagara COCO, 13 y-Fagarine, 13 Fast-atom-bombardment ionization, 70 Fermentation, 323 Fetuin hydrolysis. with trifluoroacetic acid,
268 methanolysis, 258 sialylated oligosaccharides, fractionation, 46 E Fibronectin oligosaccharides, liquid chromatography Endo-glucanases, mode of action, analysis separation, 42-43 of, 54 sialylated oligosaccharides, fractionaEnones, formation of, 326 tion, 46 Enzymic hydrolysis, of glycosidic linkages, Flip-flop hydrogen bond, 228 270-271 3-F~uoro-~-g~ucofuroses, 3-C-branched, D-Erythrose, 321 IH- and 19F-n.m.r. data for, 138-140 formation of aromatics from, 277-278 I9F-n.m.r. spectroscopy formation of y-pyranone from, 277-278 19F-chemicalshifts, 78-80 high-temperature transformation of, in I9F-I9F coupling, 80 alkali solution, 283 Food processing, color-stopping reaction Escasany, Irene, 12 in, 324-325 Esters, protection as, 189-191 Foods Ethanol carbohydrates in cyclodextrin inclusion complexes with, analysis of, 50-52 222, 224 soluble, liquid chromatography methfermentation sources, 326 ods for, 51 Ethers, protection as, 181-182 discoloration of, 324-325 5-Ethylbarbituric acid, cyclodextrin incluFormaldehyde, 321 sion complexes with, 223, 224 Formic acid, 296 Ethylenediamine, for enhancing detectabilcyclodextrin inclusion complexes with, ity of carbohydrates, 67 221 Ethylene dithioacetals, 196 liquid chromatography, 53-54 1 -Ethyl-2-formyl-5-methylpy1role, formaFormolysis, for cleavage of glycosidic tion of, 313-314 linkages, 269-270 3-Ethyl-2-hydroxycyclopent-2-en-l-one, 2-Formyl-5-(hydroxymethyl)-l-methylpyrformation, 294 role, 314 3-Ethylphenyl acetate, cyclodextrin incluformation of, 312-314 sion complexes with, 223, 224 2-Formyl-5-(hydroxymethyl)pyrrole-1 Ethyl 2,3,6-trideoxy-6-fluoro-ci-~-erythroacetic acid, formation of, 312-314 hexopyranoside 6,[2-Formyl-5-(hydroxymethyl)pyrrol-l-, 4-O-acetyl-, 'H- and 19F-n.m.r. data yllnorleucine, formation of, 314 for, 135 2-Formyl-5-methylpyrrole-l-acetic acid, -, 4-O-benzyl-, 'H- and I9F-n.m.r. data formation of, 312-314 for, 135 2H,5H-6-Formyl-3-oxopyrrolo[ 1 ,2-a]pyra-
SUBJECT INDEX
365
zin-4-acetic acid, formation of, 312D-Fructose 314 and L-alanine, products from, 319 D-Fructans, hydrolysis, 269 alkaline degradation of, 292 D-Fructofuranose base-degraded solution, products, 294 -, 6-0-benzoyl- I-deoxy-l-fluoro-2,3-0in food, liquid chromatography separaisopropylidene-p-, IH-and I9Ftion, 52 n.m.r. data for, 144 formation of 2-(2-hydroxyacetyl)furan -, 6-deoxy-6-fluoro-2,3-O-isopropylifrom, 286-287 dene- 1-0-p-tolylsulfonyl-pheated neutral solutions of, antimicrobial 13C-n.m.r.data for, 174 activity formed in, 326 IH- and I9F-n.m.r. data for, 145 phenols from, 290 -, I,6-dideoxy-l,6-difluoro-2,3-0thermolysis of, in acid solution, prodisopropylidene-pucts, 286 13C-n.m.r. data for, 174 Fucose, liquid chromatography methods IH- and 19F-n.m.r. data for, 145 for analysis of, 33 D-Fructofuranoside D-Fucose, 4-O-methyl-. See D-Curacose -, methyl l-deoxy-l-fluoroFukumi, H., 6 a anomer, 13C-n.m.r.data for, 174 2-Furaldehyde p anomer, l3C-n.m.r. data for, 174 -, 5-(hydroxymethy1)-,323 -, 3,4,6-tri-O-benzoyl-p-, 13C-n.m.r. formation of, 318 data for, 174 from pentoses, 275-276 -, methyl 3,4-di-O-acetyl-l,6-dideoxy- liquid chromatography, 53-54 1,bdifluorotechnical importance, 323 a anomer, IH- and 19F-n.m.r. data for, D-Furanose 145 -, 3-deoxy-3-C-(difluoromethylene)-1,2p anomer, 'H- and I9F-n.m.r. data for, O-isopropylidene-a145 -, ribo-hexo-, IH- and 19F-n.m.r. data -, methyl 1,6-dideoxy-l ,6-difluorofor, 141 a anomer, 13C-n.m.r. data for, 174 -, erythro-pentodialdo-1,4-furanose, p anomer, W-n.m.r. data for, 174 IH- and I9F-n.m.r. data for, 141 -, methyl 3,4,6-tri-O-benzoyl-l-deoxy- Furans I-fluorofrom carbohydrates, 323 a anomer, IH- and 19F-n.m.r. data for, formation of, 326 145 Furfurylidene-P-pyranone p anomer, IH-and 19F-n.m.r. data for, chromophoric, 322 145 formation of, in presence of methanol, Fructo-oligosaccharides, liquid chromatog322-323 raphy, 39 2-Furoic acid, conversion of, into methyl D-Fructopyranose 5-nitro-2-furoate, 289 -, 4-deoxy-4-fluoro-pN-(2-Furoylmethyl)-~-alanine, 3 19 W-n.m.r. data for, 173 2-(2-Furylidene)-4-hydroxy-5-methyl-3(2iY)IH- and I9F-n.m.r. data for, 144 furanone, 322 -, 1-deoxy-l-fluoro-2,3:4,5-di-O-isopropylideneW-n.m.r. data for, 173 IH- and 19F-n.m.r. data for, 144 G Fructose large-scale preparative liquid chromatog- Galactitol, liquid chromatography methods for analysis of, 33 raphy, 62 liquid chromatography methods for l-yl)-4-thiazolidinecarboxy2-(~-Galactitoltic acid, diastereomers, 317-318 analysis of, 33
366
SUBJECT INDEX
f3 anomer, IH- and 19F-n.m.r. data for, D-Galactofuranose 95 -, I-O-acetyl-2,3,5,6-tetra-O-benzoyl-4-, 1,2:3,4-di-O-isopropylidene-a-, IHfluoro-p-, I3C-n.m.r. data for, 172 and lyF-n.m.r. data for, 95 -, 3-deoxy-3-fluoro- 1,2:5,6-di-O-isopro-, 1,2:3,4-di-O-isopropylidene-6-Opylidene-a-, 'H- and 19F-n.m.r. data [(methylthio)thiocarbonyl]-a-, phofor, 99 tochemical cleavage of, 190 D-(;alactofuranosyl fluoride -, I ,2:3,4-di-O-isopropylidene-6-O-, 2,3,5,6-tetra-O-acetyl-, 'H- and I9Fnitro-a-, photolysis of, 191 n.m.r. data for, 97 -, I ,2:3,4-di-O-isopropylidene-6-O-p-, 2,3,5,6-tetra-O-benzoyl-a-, IH- and l9F-n.m.r. data for, 97 tolylsulfonyl-a-, photolysis of, 191 -, 1,2,4,6-tetra-O-acetyl-3-deoxy-3L-Galactofuranosyl fluoride fluoro-, 3,6-dideoxy-pa anomer, I3C-n.m.r. data for, 162 -, 3-azido-5-O-benzoyl-2-O-benzyl-, p anomer, "C-n.m.r. data for, 162 'H- and 19F-n.m.r. data for, 127 top yranose -, 3-azido-5-O-benzoyl-2-O-methyl-,~-Galac -, 2,6-dideoxy-2-fluoro'H- and I9F-n.m.r. data for, 127 a anomer, IH- and I9F-n.m.r. data for, -, 5-O-benzoyl-2-O-benzyl-3-(triI32 fluoroacetamido)-, IH- and I9Ff3 anomer, IH- and 19F-n.m.r. data for, n.m.r. data for, 128 I32 -, 5-O-benzoyl-2-O-methyl-3-(tri-, 1,3,4-tri-O-acetyl-p-, 'H- and I9Ffluoroacetamido)-, 'H- and I9Fn.m.r. data for, 132 n.m.r. data for, 128 -, 2,3,6-trideoxy-2-fluoroGalactoglucomannans, 305 -, 1-0-acetyl-4-0-benzoyl-2-fluoro-3Galactonic acids, analytical high-perfor(trifluoroacetamido)-p-, 'H- and mance liquid chromatography, 34 I9F-n.m.r. data for, 124 D-Galactop yranose -, 3-amino-, 'H- and '9F-n.m.r. data -, 2-acetamido-l,3,4-tri-O-acetyl-2,6for, 124 dideoxy-6-fluoro-, IH- and 19F-n.m.r. D-Galactop yranoside data for, 120 -, benzyl 3,4,6-tri-O-benzyl-2-deoxy-2-, 2-deoxy-2-fluorofluoro-p-, "C-n.m.r. data for, 161 a anomer, 'Hand 19F-n.m.r. data for, -, 2-deoxy-2-fluoro85 -, methyl p-, 'H- and "F-n.m.r. data p anomer, 'H- and I9F-n.m.r. data for, for, 86 85 -, methyl 3,4-O-isopropylidene-6-0-, 1,3,4,6-tetra-O-acetyl-, IH- and trityl-p-, IH- and IyF-n.m.r. data I9F-n.m.r. data for, 85-86 for, 86 -, 3-deoxy-3-fluoro-, 1,2,4,6-tetra-O-, trifluoromethyl 3,4,6-tri-O-acetyIacetyl-, 'Hand 19F-n.m.r. data for, a-, 'H- and I9F-n.m.r. data for, 86 90 -, 3-deoxy-3-fluoro-, methyl 2,4-di-O-, 4-deoxy-4-fluoroa anomer, I3C-n.m.r. data for, 163 benzoyl-6-O-(bromoacetyl), IH- and 19F-n.m.r. data for, 90 p anomer, I3C-n.m.r. data for, 163 -, 1,2,3,6-tetra-O-acetyl-p-, IH-and -, 4,6-dideoxy-4,6-difluoro'9F-n.m.r. data for, 92 -, methyl, 'H- and '9F-n.m.r. data -, I ,3,4,6-tetra-O-acetyl-2-deoxy-2for, 150 -, phenyl a-,'H- and I9F-n.m.r. data fluoro-a-, 13C-n.m.r. data for, 161 -, 6-deoxy-6-fluorofor, 150 -, methyl 2-acetamido-2,4,6-trideoxya anomer, 'H- and I9F-n.m.r. data for, 95 4.6-difluoro-a-
SUBJECT INDEX
-,
3-O-acetyl-, IH- and I9F-n.m.r. data for, 157 IH- and 19F-n.m.r. data for, 157 -, methyl 2-benzamido-2,4,6-trideoxy4,6-difluoro-a-, 3-O-acetyl-, 'H- and I9F-n.m.r. data for, 157 -, 3-O-benzyl-, IH- and 19F-n.m.r. data for, 157 -, methyl 2-deoxy-2-fluorop anomer, 13C-n.m.r.data for, 161 -, 3,4,6-tri-O-acetyl-p-, I3C-n.m.r. data for, 161 -, methyl 3-deoxy-3-fluorop anomer, 13C-n.m.r.data for, 162
367
-, 2-acetamido-, IH- and I9F-n.m.r. data for, 120 -, 2-acetamido-3,4-di-O-acetyl-, IHand I9F-n.m.r. data for, 120 -, 2-benzamido-, IH- and I9F-n.m.r. data for, 120 -, 2-benzamido-4-O-benzoyl-3-0benzyl-, 'H- and 19F-n.rn.r. data for, 121 -, 2-benzamido-3-O-benzyl-, 'H- and I9F-n.m.r. data for, 121 -, 3,4-di-O-acetyl-2-benzamido-, 'Hand I9F-n.m.r. data for, 120 L-Galactopyranoside -, benzyl 2,3,6-trideoxy-2-fluoro-p-, 2,4-di-O-benzoyl-6-O-(bromoace-, 3-amino-, 'H- and I9F-n.m.r. data ty1)-, I3C-n.m.r.data for, 162-163 for, 124 -, 2,4,6-tri-O-acetyl-P-, "C-n.m.r. -, 3-benzamidodata for, 162 13C-n.m.r.data for, 171 -, methyl 4-deoxy-4-fluoroIH- and I9F-n.m.r. data for, 124 a anomer, 13C-n.m.r.data for, 163 -, 4-O-benzoyl-3-(trifluoroace(3 anomer, I3C-n.m.r. data for, 163 tamido)-, 6-O-benzoyl-2,3-di-O-benzyl-p-, I3C-n.m.r. data for, 171 IH- and 19F-n.m.r. data for, 93 'H- and I9F-n.m.r. data for, 124 -, 2,3-di-O-benzyl-p-, I3C-n.m.r.data -, 3-(trifluoroacetamido)-,'H- and for, 164 I9F-n.m.r. data for, 124 -, 2,3-di-O-benzyl-6-O-trityl-p-, I3C-, methyl 6-deoxy-2,3-di-O-p-~-galacton.m.r. data for, 164 pyranosyl-a-, synthesis of, utiliza-, 2,3-di-O-trityl-P-, IH- and I9Ftion of 2-nitrobenzylidene protecting n.m.r. data for, 93 group in, 188-189 -, 2,3,6-tri-O-acetyl-, I3C-n.m.r.data -, methyl 2,3,6-trideoxy-2-fluoro-pfor, 164 -, 3-amino-, I3C-n.m.r.data for, 171 -, 2,3,6-tri-O-acetyl-u-, 'H- and 19F-, 3-benzarnido-4-O-benzoyl-, I3Cn.m.r. data for, 93 n.m.r. data for, 171 -, 2,3,6-tn-O-benzoyl-p-, IH- and -, 3-(trifluoroacetamido)-,I3C-n.m.r. 19F-n.m.r. data for, 93 data for, 171 -, methyl 6-deoxy-6-fluoro-, methyl 2,3,6-trideoxy-2-fluoroa anomer, 'H- and I9F-n.m.r. data for, -, 3-amino-p-, IH- and 19F-n.rn.r. 95 data for, 124 P anomer, 13C-n.m.r. data for, 164 -, 3-benzamido-p-, 'H- and I9F-, 1,2,3,4-tetra-O-acetyI-a-, IH- and n.m.r. data for, 125 I9F-n.m.r. data for, 95 -, 3-benzamido-4-O-benzoyl-p-, IH-, 2,3,4-tri-O-acetyl-P-, I3C-n.m.r. and 19F-n.m.r. data for, 125 data for, 164 -, 3-(trifluoroacetamido)-, 'H- and -, 2,3,4-tri-O-acetyl-a-, IH- and I9F19F-n.m.r. data for, 125 n.m.r. data for, 95 -, trifluoromethyl 3,4-di-O-acetyl-2,6-, methyl 4,6-dideoxy-4,6-difluorodideoxy-a-, 'H- and I9F-n.m.r. Q anomer, I3C-n.m.r.data for, 175 data for, 132 p anomer, I3C-n.m.r.data for, 175 L-Galactopyranos yl bromide, 4-0-benzoyl-, methyl 2,6-dideoxy-6-fluoro-a2,3,6-trideoxy-2-fluoro-3-(trifluoroacet-
368
SUBJECT INDEX
Gasification, 273-274, 323 amido)-a-, IH- and I9F-n.m.r. data for, 125 Gentiobiose, liquid chromatography methods for analysis of, 33 D-Galactopyranosyl chloride, methyl 2,4Glucans, cyclic, 60 di-O-benzoyl-6-0-(bromoacety1)-3Glucitol, liquid chromatography methods deoxy-3-fluoro-a-, W-n.m.r. data for, for analysis of, 33 172 D-Glucofuranose Galactopyranosyl fluoride -, 1-0-acetyl-2,3,5,6-tetra-O-benzoyl-4-, 2-deoxy-2-fluoro-~fluoro-p-, W-n.m.r. data for, 172 a anomer, IH- and I9F-n.m.r. data for, -, 3,6-anhydro-5-deoxy-5-fluoro1,2-0I46 -, 3,4,6-tri-O-acetyl-, 'H- and t9Fisopropylidene-a-, IH- and I9Fn.m.r. data for, 115 n.m.r. data for, 146 -, 3,6-anhydro-5-deoxy-5,6,6-trifluoro-, 2,6-dideoxy-2-fluoro-~a anomer, IH- and I9F-n.m.r. data for, 1,2-0-isopropylideneI3C-n.m.r. data for, 177 153 -, 3,4-di-O-acetyl-, 'H- and 19FIH- and I9F-n.m.r. data for, 158 n.m.r. data for, 153 -, 3,6-anhydro-6,6-difluoro-, 2,3,4,6-tetra-O-acetyI-~-, I ,2-0-benzylidene-a-, IH- and I9Fa anomer, 13C-n.m.r.data for, 160 n.m.r. data for, 152 p anomer, Wn.m.r. data for, 160 -, 1,2-0-isopropylidene-a-, IH- and 'H- and 19F-n.m.r. data for, 82 19F-n.m.r.data for, 152 -, 2,3,6-tri-0-acetyl-4-deoxy-4-fluoro-D- -, 3,6-anhydro-6,6-difluoro-aa anomer, IH- and I9F-n.m.r. data for, -, 1,2-0-benzylidene-, Wn.m.r. 148 data for, 175 p anomer, IH- and 19F-n.m.r.data for, -, 1,2-0-isopropylidene-, "C-n.m.r. 148 data for, 175 -, 3,4,6-tri-O-acetyl-2-deoxy-~-, 3-deoxy-3-fluoro-, 2-bromo-, IH- and f9F-n.m.r.data -, 3-C-(acetoxymethyl)-I ,2-0-isoprofor, 129 pylidene-a-, IH- and i9F-n.m.r. -, 2-iodo-, IH- and I9F-n.m.r. data data for, 138 for, 129 -, 6-0-benzoyl- 1,2-0-isopropylidene-, 3,4,6-trideoxy-3,4,6-trifluoro-a-~a-,IH- and I9F-n.m.r. data for, I3C-n.m.r. data for, 177 I39 'H- and I9F-n.m.r. data for, 158 -, 3-C-[1,2-di(hydroxyethyl)]-1,2:5,6Galactose di-0-isopropylidene-a-, IH- and -, DI9F-n.m.r. data for, 139 in plant cell-wall hydrolyzates -, 1,2:5,6-di-O-isopropylidene-a-, IHand I9F-n.m.r. data for, 99, 138, liquid chromatography analysis, 55 139 determination of enantiomeric form, 66 liquid chromatography, 33, 52-53 -, 1,2:5,6-di-O-isopropylidene-3deoxy-3-fluoro-a-, I3C-n.m,r. data D-GalaCtOSe diethyl dithioacetal, photofor, 166 chemical decomposition of, 197 Galacturonic acid -, 1,2:5,6-di-O-isopropylidene-3-Cacid hydrolysis of, correction factors, (methoxycarbony1)-a-, IH- and 254 19F-n.m.r.data for, 140 -, D-, 3-C-(ethoxyallyl)-1,2-0-isopropyliin mosses, 324 dene-a-, IH- and I9F-n.m.r. data phenols from, 290 for, 139 treated with trifluoroacetic acid. recover3-C-[(ethoxycarbonyl)(formylies of, 261-262 imino)methyl]-1,2:5,6-di-0-
-.
SUBJECT INDEX
369
and I9F-, 2,5-di-O-benzoyl-p-, 'H- and I9Fisopropylidene-a-, 'Hn.m.r. data for, 139 n.m.r. data for, I16 Glucoisosaccharinic acid -, 3-C-[ethoxy(ethoxycarbonyl)(formylamino)methyl]-l,2:5,6-di-O- formation of, 292 structure, 291 isopropylidene-a-, IH- and 19FGlucometasaccharinic acid, structure, 291 n.m.r. data for, 139 -, 3-C-(hydroxymethyl)-1,2:5,6-di-O- -, D-,formation of, 292, 303 D-Gluconic acid isopropylidene-a-, 'H- and I9Fanalytical high-performance liquid chron.m.r. data for, 139 matography, 34 -, 1,2-O-isopropylidene-a-, 3-deoxy-3-fluoro-, 'Hand I9F-n.m.r. 5,6-carbonate, 'H- and I9F-n.m.r. data for, 116 data for, 99 Gluco-oligosaccharides, a-(I+4)-linked, 'H- and 19F-n.m.r.data for, 99, 139 -, I ,2-O-isopropylidene-3-C-(methox- liquid chromatography analysis, 3840 yally1)-a-, IH- and 19F-n.m.r.data D-GlUCOpyranOSe for, 139 -, 2-acetamido-2,6-dideoxy-6-flu0r0-, 5,6-phenylboronate, IH- and I9Fa anomer, IH- and 19F-n.m.r. data for, n.m.r. data for, 99 121 -, 5-deoxy-5-fluoro-, 1,3,4-tri-O-acetyl-, 'H- and I9Fa anomer, I3C-n.m.r. data for, 166 n.m.r. data for, 121 p anomer, I3C-n.m.r. data for, 166 -, 3-0-acetyl- 1,6-anhydro-2,4-dideoxy-, 1,2-O-isopropylidene-a-, ITn.m.r. data for, 167 P-, 2-acetamido-4-fluoro-, 'H- and I9F-, 6-deoxy-6-fluoron.m.r. data for, 114 -, 3,5-O-benzylidene-1,2-O-isopropy-, 4-acetamido-2-fluoro-, 'H- and I9Flidene-a-, IH-and I9F-n.m.r. data for, 100 n.m.r. data for, 114 2,4-difluoro-P-, 'H- and 19F-n.m.r. data -, 1,2:5,6-di-O-isopropylidene-a-, IHfor, 151 and I9F-n.m.r. data for, 100 -, 1,2:3,5-di-O-methylidene-a-, IH-, 1,6-anhydro-2-deoxy-2-fluoro-pand I9F-n.m.r. data for, 100 -, 3-O-acetyl-4-O-benzyI-,IH- and -, 1,2-O-isopropylidene-a-, IH- and 19F-n.m.r. data for, 114 19F-n.m.r.data for, 100 -, 3,4-di-O-acetyl-, 'H- and I9F-, 1,2-O-isopropylidene-5-O-benzyln.m.r. data for, I14 a-,'H- and 19F-n.m.r.data for, -, 3,4-di-O-benzyl-, 'H- and 19F100 n.m.r. data for, I14 D-Glucofuranosyl fluoride -, 1,5-anhydro-2-deoxy-2-fluoro1-C-, 2,3,5,6-tetra-O-acetylmethyl-a-, 3,4,6-tri-O-acetyl-, 'H13C-n.m.r. data for, 165 and 19F-n.m.r. data for, 113 IH-and I9F-n.m.r. data for, 97 -, 2-deoxy-2-fluoro-, 2,3,5,6-tetra-O-benzoyl-, 'H- and I9Fa anomer n.m.r. data for, 97 I3C-n.m.r. data for, 162 -, 3,5,6-tri-U-acetyl-2-O-rnethyl-, I3C'H-and I9F-n.m.r. data for, 86 n.m.r. data for, 166 p anomer -, 2,5,6-tri-O-acety1-3-O-methyl-, 13C"C-n.m.r. data for, 162 n.m.r. data for, 166 'H- and I9F-n.m.r. data for, 86 (D-Glucofuranosyl fluoride)urono-6,3-, 1,3,4,6-tetra-O-acetyl-, 'H- and lactone I9F-n.m.r. data for, 86 -, 2,5-di-O-acetyl-p-, 'H- and I9F-n.m.r. -, 1,3,4,6-tetra-O-acetyl-a-, 'H-and data for, 116 IgF-n.m.r. data for, 162
370
SUBJECT INDEX
-, 3-deoxy-3-fluoroa anomer 4hu-n.r. data for, 163 'H- and 19F-n.m.r. data for, 90 p anomer Wn.m.r. data for, 163 IH- and I9F-n.m.r. data for, 91 -, 6-phosphate, IH- and 19F-n.m.r. data for, 91 -, 1,2,4,6-tetra-O-acetyl-, 'H-and I9F-n.m.r. data for, 91 -, 4-deoxy-4-fluoroa anomer 13C-n.m.r.data for, 164 IH- and I9F-n.m.r. data for, 93 p anomer W-n.m.r. data for, 164 IH- and I9F-n.m.r. data for, 93 -, 1,2,3,6-tetra-O-acetyl-p-, 'H- and I9F-n.m.r. data for, 93 -, 6-deoxy-6-fluoroa anomer I3C-n.m.r. data for, 164 IH- and 19F-n.m.r. data for, 95 p anomer I3C-n.m.r. data for, 164 IH- and I9F-n.m.r. data for, 95 -, 1,2,3,4-tetra-O-acetyl-, IH- and 19F-n.m.r. data for, 95 L-Glucopyranose -, 2,6-dideoxy-2-fluoroa anomer, IH- and I9F-n.m.r. data for, 132 p anomer, IH- and 19F-n.m.r.data for, 132 -, 1,3,4-tri-O-acetyl-, 'H- and I9Fn.m.r. data for, 133 n-Glucopyranoside -, 2-acetamido-2,6-dideoxy-6-fluoro-a-, benzyl 3,4-di-O-acetyl-, IH- and 19F-n.m.r. data for, 121 -, methyl 3,4-di-O-acetyl-, 'H-and I9F-n.m.r. data for, 121 -, methyl 3,4-di-O-methyl-, IH- and 19F-n.m.r.data for, 121 -, 4,6-O-benzylidene-2,3-dideoxy-3fluoro-u-, benzyl 2-acetamido, 'H- and I9Fn.m.r. data for, 119 -, benzyl 2-azidoW-n.m.r. data for, 171
IH- and I9F-n.m.r. data for, I19 -, benzyl 2-benzamido-, T-n.m.r. data for, 171 -, methyl 2-benzamido-, IH- and I9Fn.m.r. data for, 119 -, 2-deoxy-2-fluoro-, methyl 4,6-di-O-acetyl-3-O-benzylp-, IH- and I9F-n.m.r. data for, 86 -, methyl 3-O-acetyl-4,6-O-benzylidene-p-, IH- and 19F-n.m.r. data for, 87 -, methyl 3-O-benzyl-4,6-O-benzylidene-P-, IH- and I9F-n.m.r. data for, 87 -, methyl 4,6-0-benzylidene-3-0methyl-p-, IH- and 19F-n.m.r. data for, 87 -, phenyl 3,4,6-tri-O-acetyl-p-, IHand I9F-n.m.r. data for, 87 -, trifluoromethyl 3,4,6-tri-O-acetyla-,IH- and I9F-n.m.r. data for, 87 -, 3-deoxy-3-fluoro-, benzyl p-, IH- and 19F-n.m.r. data for, 91 -, benzyl 2,4,6-tri-O-acetyl-p-, 'Hand I9F-n.m.r. data for, 91 -, methyl 2-O-acetyl-4,6-O-benzylidene-p-, IH- and I9F-n.m.r. data for, 91 -, 6-deoxy-6-fluoro-, methyl, 'H- and 19F-n.m.r. data for, 96 -, p-nitrophenyl p-, IH- and 19Fn.m.r. data for, 96 -, phenyl, IH- and I9F-n.m.r. data for, 96 -, methyl 2,3,4-tri-O-benzyl-a-, 'Hand I9F-n.m.r. data for, 96 -, methyl 2-acetamido-3-U-acetyl-2,4,6trideoxy-4,6-difluoro-a-,IH- and I9F-n.m.r. data for, 157 -, methyl 4-azido-4,6-dideoxy-6-fluoroa-,I3C-n.m.r. data for, 171 -, methyl 2-benzamido-2,6-dideoxy-6fluoro-a-, 3-O-benzyl-, IH- and IgF-n.m.r. data for, 122 -, 3-O-benzyl-4-O-mesyl-, IH- and 19F-n.m.r. data for, 122 -, 3,4-di-O-acetyl-, IH- and I9Fn.m.r. data for, 121
SUBJECT INDEX
37 1
-, methyl 2-benzamido-2,4,6-tndeoxy-, 3,4,6-tri-O-acetyl-, IH- and I9F4,6-difluoro-an.m.r. data for, 117 -, 3-O-acetyl-, 'H- and 19F-n.m.r. -, 2-deoxy-2-fluorodata for, 157 Q anomer, 'H- and 19F-n.m.r.data for, -, 3-O-benzyl-, 'H- and 19F-n.m.r. 146 data for, 157 -, 3,4,6-tri-O-acetyl-, IH- and I9F-, p-nitrophenyl 6-deoxy-6-fluoro-, l3Cn.m.r. data for, 146 n.m.r. data for, 165 -, 3-deoxy-3-fluoro-, 2,3,6-tri-O-acetyl-, -, methyl 4-deoxy-4-fluoroIH- and I9F-n.m.r. data for, 148 -, 6-0-acetyl-2,3-di-O-methyl-a-, 'H-, 4-deoxy-4-fluoro-, 2,3,6-tri-O-acetyl-, and I9F-n.m.r. data for, 94 IH- and 19F-n.m.r. data for, 148 Q anomer, 'H- and I9F-n.m.r. data for, -, 6-deoxy-6-fluoro-, 2,3,4-tri-O-acetyl-, 93 IH- and 19F-n.m.r. data for, 148149 -, 2,3-di-O-methyl-a-, IH- and I9F-, 2,3-di-O-acetyl-4,6-0-benzylidene-, n.m.r. data for, 94 -, 2,3,6-tri-O-acetyl-a-, IH- and I9F'H- and 19F-n.m.r. data for, 82 -, 2,3-di-O-benzoy1-4,6-di-O-methyl-, n.m.r. data for, 93 IH- and 19F-n.m.r. data for, 83-, 2,3,6-tri-O-benzoyl-a-, 'H- and 84 I9F-n.m.r. data for, 94 -, methyl 6-deoxy-6-fluoro-, 2,3,4,6-tetra-O-acetyla anomer, 13C-n.m.r.data for, 165 I3C-n.m.r. data for, 160 f3 anomer, 13C-n.m.r.data for, 165 'H- and 19F-n.m.r. data for, 82 -, methyl 3,6-di-O-acetyl-2,4-dideoxy-, 2,3,4,6-tetra-O-benzoyI-, IH- and I9F4-flU01-0-an.m.r. data for, 83 -, 2-acetamido-, 'H- and 19F-n.m.r. -, 2,3,4,6-tetra-O-benzyl-a-, IH- and I9F-n.m.r. data for, 84 data for, 119 -, 2-benzamido-, IH- and 19F-n.m.r. -, 2,3,4,6-tetra-O-methyl-, IH- and I9Fdata for, 119 n.m.r. data for, 84 -, methyl 4,6-dideoxyd-fluoro-a-, 3,4,6-tri-O-acetyl-2-deoxy-, 4-amino-, 'H- and 19F-n.m.r.data -, 2-bromo-, IH- and I9F-n.m.r. data for, 122 for, 129 -, 4-azido-, IH- and 19F-n.m.r. data -, 2-chloro-, 'H- and I9F-n.m.r. data for, 122 for, 129 -, phenyl p-, photoinduced, electron-, 2-iodo-, IH- and I9F-n.m.r. data transfer reaction of, with 1,4-difor, 129-130 cyanonaphthalene, 184-185 -, 3,4,6-tri-O-acetyl-2-0-methyl-, phenyl 6-deoxy-6-fluoroI3C-n.m.r. data for, 160 a anomer, I3C-n.m.r. data for, 165 IH and 19F-n.m.r.data for, 83 p anomer, 13C-n.m.r.data for, 165 -, 2,4,6-tri-O-acetyl-3-0-methyl-, I3CL-Glucopyranoside, trifluoromethyl 3,4-din.m.r. data for, 160 0-acetyl-a-, 'H-and 19F-n.m.r.data -, 3,4,6-tri-O-benzoyl-2-0-methyl-, IHfor, 133 and 19F-n.m.r. data for, 83 D-Glucopyranosyl fluoride -, 2,4,6-tn-O-benzoyl-3-O-methyl-, IHQ anomer and 19F-n.m.r. data for, 83 13C-n.m.r. data for, 160 L-Glucopyranosyl fluoride 'H- and I9F-n.m.r. data for, 82 -, 3,4-di-O-acetyl-2,6-dideoxy-2-fluorof3 anomer a-,IH- and 19F-n.rn.r. data for, 153 13C-n.m.r. data for, 160 'H-and I9F-n.m.r. data for, 82 -, 2,3,4-tri-O-acetyI-6-deoxy-c~-, I3Cn.m.r. data for, 172 -, 2-acetamido-2-deoxy-pp-D-Glucopyranosyl polymer, catalyzed by IH- and 19F-n.m.r. data for, 117
372
SUBJECT INDEX
p-D-galactosyltransferase, incorporation of D-galactose into, 187 Glucosaccharinic acid -, a-D-, formation, 292-293 structure, 291 Glucose liquid chromatography methods for analysis of, 33 liquid chromatography separation, 52-53 D-Glucose alkaline degradation of, 292 with L-asparaghe, pyrazines formed from, 316-317 degradation products of, 296 in food, liquid chromatography separation, 52 formation of S-(hydroxymethyl)-2-furaldehyde from, 284-285 formation of Amadori compounds from, 307-308 heated neutral solutions of, antimicrobial activity formed in, 326 Maillard reaction between glycine and, 309 phenols from, 290, 295 with L-phenylalanine, products obtained from, 317 in plant cell-wall hydrolyzates, liquid chromatography analysis, 55 reaction of with glycine, pyrazines formed, 315316 with methylamine in dilute acetic acid, 3 14 D-Glucose oligosaccharides, a-( 1-4)linked, liquid chromatography analysis, 37-40 Glucosiduronase -, p-, isolation of carbohydrates from, on analytical-scale columns, 60 -, p-D-, oligosaccharides, liquid chromatography separation, 42-43 D-Glucuronic acid, phenols from, 290 Glycans, complex, glycoprotein-derived, reversed-phase chromatography, 4344 Glyceraldehyde, 321 dehydrated to pyruvaldehyde, acidic conditions, 278-279 high-temperature transformation, in alkali conditions, 284
Glycocalicin, sialylated oligosaccharides, fractionation, 46 GIycoconjugates acid hydrolysis of, 265-269 methanolysis, 258-259 neutral and amino monosaccharides of, measurement of, 268 nondialyzable, by hydrochloric acid, 267 Glycolipids, hydrolysis with hydrochloric acid, 266 GIycopeptides N-acylated, liquid chromatography separation, 48 complex, ionic, liquid chromatography, 45-49 liquid chromatography separation, 4748 methylated, liquid chromatography separation, 48 prechromatographic purification of, 20 preparative liquid chromatography, 60 sialylated, liquid chromatography separation, 48 Glycoprotein carbohydrates, compositional analysis of, 56 glycosylation site(@, 47-48 hydrolysis of with hydrochloric acid, 267 with ion-exchange resin in acid form, 268-269 methanolysis, 257 for analysis of carbohydrates in, 258 structure, liquid chromatography analysis, 46-47 Glycosaminoglycans, 7 composition, analysis, 55 isolation of carbohydrates from, on analytical-scale columns, 60 GIycosaminoglycuronans oligosaccharides, liquid chromatography separation, 48 sulfated oligosaccharides, chromatographic separation of, 49 Glycosidases, 270 Glycoside linkages, methanolysis, 259 Glycosides large-scale preparative liquid chromatogw h y , 62
SUBJECT INDEX liquid chromatography, pre-column derivatization procedures, 68 photosensitive protecting groups, 182187 preparative liquid chromatography, 60 Glycosidic linkages, 250 acetolysis, 252, 269-270 with activating group at p-position, cleavage, 255 cleavage, 250-25 1 liberation of N- and 0-linked carbohydrate chains, 255-256 enzymic hydrolysis, 270-271 formolysis, 252, 269-270 hydrolysis correction factors, 254 internal standard, 254 liberation of neutral monosaccharides during, 252-253 recoveries from, 255 methanolysis, 252 deamination prior to, 256 recoveries from, 255 reductive cleavage, 271 synthesis, 250 total hydrolysis with acid, 259-269 Glycosylation sites isolation, 47-48 separation of, 47-48 N-Glycosylic linkage, cleavage, 255 Glycuronans decarboxylation, 306 high-temperature transformation of, 305307 hydrolysis, 265 in marine algae, 307 Guanosine derivatives, diastereoisomeric, 28 Guaran, hydrolysis, 265 Guatambuine, 13 o-Gulopyranoside, benzyl 2-acetamido-3O-acetyl-2,4-dideoxy-4-fluoro-6-0trityl-a-, 'H- and 19F-n.m.r. data for, 119 Gums, hydrolysis of, 263, 265
H Hayashi, H., 7 Hemicellulose, 300 hardwood, 305
373
high-temperature transformation of, 305307 non-fermentable oligosaccharides, liquid chromatography methods for, 52 polysaccharides, 305 softwood, 305 Heparin isolation of carbohydrates from, on large-scale columns, 62 structural and sequence analysis of, 49, 57 sulfated disaccharides, liquid chromatography, 37 Heparin sulfate, composition, analysis, 55
Heptonic acids, D-glyCerO-D-gUlO-, analytical high-performance liquid chromatography, 34 5-Heptylbarbituric acid, cyclodextrin inclusion complexes with, 223, 224 Hexa-O-p-D-glucopyranosyl-D-glucitols isomeric, liquid chromatography separation, 41-42 preparative liquid chromatography, 60 2,4-Hexanedione, formation of, 318 1-Hexanol, cyclodextrin inclusion complexes with, 222-224 D-nrubino-Hex- 1-enitol, 1,2-dideoxy-I , 1-
difluoro-3,4:5,6-di-O-isopropylidene-, IH- and 19F-n.m.r. data for, 159
~-ery?hro-Hex-5-enofuranose,3,5,6-trideoxy-6,6-difluoro-l,2-O-isopropylidene-a']C-n.m.r. data for, 176 'H- and 19F-n.m.r. data for, 154 ~-lyxo-Hex-5-enofuranose,5 ,6-dideoxy-6,6difluoro-1,2-O-isopropylidene-3-0methyl-p-, Y h . m . r . data for, 176 ~-ribo-Hex-5-enofuranose,5 ,6-dideoxy6,ddifluoro- I ,2-O-isopropylidene-Omethyl-a-, lIC-n.m.r. data for, 176 ~-xy~o-Hex-5-enofuranose, 1,2-o-isopropylidene-5,6-dideoxy-6,6-difluoro-3-0methyl-a-, IH- and 19F-n.m.r. data for, 154 ~-ribo-Hex-5-enofuranoside,methyl 5,6dideoxy-6,6-difluoro-2,3-O-isopropylidene-a-
374
SUBJECT I N D E X
IT-n.m.r. data for, 176 -, xylo-, IH- and I9F-n.m.r. data IH- and 19F-n.m.r. data for, 154 for, 141 -, 3-deoxy-3-C-(fluoromethylene)Hex-2(or 3)-enopyranose derivatives, fluorinated, IH- and I9F-n.m.r. data 1,2:5,6)-di-O-isopropylidene-afor, 134 -, ribo-, 'H- and I9F-n.m.r. data ~-erylhro-Hex-2-enopyranoside,2,3,6for, 140 -, xylo-, 'H- and '9F-n.m.r. data trideoxy-6-fluoro-afor, 140 -, ethyl 4-0-acetyl-, IH- and 19F-n.m.r. -, 2-deoxy-2-fluoro-, 'H- and 19F-n.m.r. data for, 134 data for, 99 -, methyl 4-0-acetyl-, IH- and I9F-, 3-deoxy-3-fluoro-, IH- and 19F-n.m.r. n.m.r. data for, 134 data for, 99 ~-threo-Hex-4-enopyranoside,methyl 3benzamido-2,3,4,6-tetradeoxy-2-fluoro- -, 5-deoxy-5-fluoro-, 'H- and 19F-n.m.r. data for, 100 a-,IH-and 19F-n.m.r. data for, -, 6-deoxy-6-fluoro-, IH- and I9F-n.m.r. 127 data for, 100 ~-threo-Hex-4-enopyranoside,methyl 2benzamido-3-0-benzyl-2,4,6-trideoxy- -, D,L-ribo-, 3-acetamid0-2,3,5,6-tetradeoxy-S6-fluoro-p-, 'H- and I9F-n.m.r. data for, 127 fluoro-p-, "C-n.m.r. data for, ~-arabinp-Hex-5-enopyranoside 171 -, methyl 3-benzamido-4-0-benzoyl-, 3-acetamido-2,3,5,6-tetradeoxy;52,3,6-trideoxy-2-fluoro-(~-,I3C-n.m.r. fluoro- 1-044 nitrobenzoy1)-u,pdata for, 171 IH- and 19F-n.m.r.data for, 128 -, 2,3,6-trideoxy-2-fluoro-2-a-, methyl 3-acetamido-2,3,5,6-tetra-, benzyl 3-azido-, 'H- and 19F-n.m.r. deoxy-5-fluoro-p-, IH- and I9Fdata for, 126 n.m.r. data for, 128 -, benzyl 3-benzamido-, IH- and I9F- Hexofuranosides n.rn.r. data for, 127 -, 6-deoxy-6-fluoro-, 'H- and I9F-n.m.r. -, methyl 3-benzamido-, IH- and I9Fdata for, 100 n.m.r. data for, 127 -, 5-deoxy-5-fluoro-, IH- and I9F-n.m.r. -, methyl 3-benzamido-4-0-benzoyl-, data for, 100 'H- and I9F-n.m.r. data for, 127 -, methyl 3-acetamido-2,3,5,6-tetra-, methyl 4-0-benzoyl-3-trifluoroacedeoxy-5-fluoro-~,~-ribotamido-, IH- and I9F-n.m.r. data a anomer, W-n.rn.r. data for, 171 for, 127 p anomer, "C-n.m.r. data for, 128, Hexobarbital, cyclodextrin inclusion com172 plexes with, 223, 224 D-xylo-Hexo-l,4-furanos-5-ulose, S(S)-S-CHexodiulosonic acid, 2,5-D-lhreO-, 34 acetoxy-3,5-0-benzylidene-6-deoxy-6Hexofuranoses fluoro-l,2-O-isopropylidene-ar-, IHDand 19F-n.m.r. data for, 143 -, 1-0-acetyl-2,3,5,6-tetra-O-benzoyl-Hexofuranosyl fluorides 4-deoxy-4-fluoro-p13C-n.m.r.data for, 165-167 'H- and I9F-n.m.r. data for, 97-98 -, galacto-, 'H-and I9F-n.rn.r. data for, 143 Hexop yranose -, gluco-, 'H- and I9F-n.m.r. data -, 6-azido-4-0-benzoyl-2,3,6-trideoxy-2for, 143 fluoro-D-ribo-, 3-deoxy-3-C-(difluoromethylene)(Y anomer, IH- and I9F-n.m.r. data for, 1,2:5,6-di-O-isopropylidene-a126 -, ribo-, 'H- and 19F-n.m.r. data p anomer, 'H- and 19F-n.m.r. data for, for, 141 126
SUBJECT I N D E X -, 2-deoxy-2-fluoro-, 'H- and 19F-n.m.r.
375
-, 2-deoxy-~-/yxodata for, 85-89 -, 3,6-di-O-benzoyl-a-, 'H- and IyF-, 3-deoxy-3-fluoro-, 'H- and 19F-n.m.r. n.m.r. data for, 131 data for, 90-92 -, 4,6-di-O-benzoyl-a-, 'H- and I9F-, 4-deoxy-4-fluoro-, IH- and i9F-n.m.r. n.m.r. data for, 131 data for, 92-94 -, 3,4,6-tri-O-benzoyl-a-, 'H- and -, 6-deoxy-6-fluoro-, IH- and I9F-n.m.r. 19F-n.m.r. data for, 131 data for, 95-96 13C-n.m.r. data for, 160-161 Hexopyranoside derivatives, fluorinated, 'H- and '9F-n.m.r. data for, 82-85 'H- and '9F-n.m.r.data for, 135 Hexosamines, liquid chromatography, preHexopyranosides column derivatization procedures, 68 -, D-riboHexose -, methyl 6-azido-4-O-benzoyl-2,3,6- -, 2-acetamido-2-deoxy-, liquid chrotrideoxy-2-fluoro-p-, IH- and I9Fmatography methods for analysis of, n.m.r. data for, 126 33 -, methyl 4-O-benzoyl-6-bromo-2,3,6- -, D-, 2-acetamido-2-deoxy-, 31 trideoxy-2-fluoro-P-, 'H- and IyF-, 2-deoxy-urubino-, liquid chromatogn.m.r. data for, 135 raphy methods for analysis of, 33 -, methyl 4,5-O-benzylidene-2-deoxy- -, 4-O-(dichloroisoeverninyl)-2,63-C-[(ethoxycarbonyl)- (fluorodideoxy-D-urubino-. See Curacin me thyl)]-(~-~-ribo-hexopyranoside high-temperature transformation ( R ) isomer, "C-n.m.r. data for, 172 acidic conditions, 284-291 ( S ) isomer, I3C-n.m.r. data for, 172 basic conditions, 291-295 -, 2-deoxy-2-fluoro-, 'H- and IyF-n.m.r. saccharinic acids formed from, 291 data for, 85-89 L-nrubino-Hexo- 1 ,5-pyranos-5-ulose, 5(R)-, 3-deoxy-3-fluoro-, 'H- and I9F-n.m.r. 5-C-acetoxy-6-deoxy-6-fluoro-1,2:3,4data for, 90-92 di-0-isopropylidene-(3-,'H- and I9F-, 4-deoxy-4-fluoro-, 'H- and IyF-n.m.r. n.m.r. data for, 142 data for, 92-94 D-lyxo-5-Hexosulopyranuronic acid, in -, 6-deoxy-6-fluoro-, 'H- and iyF-n.m.r. mosses, 323-324 data for, 95-96 1,3,-dideoxy-l-fluoro-3~-xy~o-4-Hexulose, Hexopyranosid-2-dose iodo- 1,2:5,6-di-O-isopropylidene-a-, -, ~-ribo-3-keto,reductic acid forma'H- and I9F-n.m.r. data for, 159 tion from, 289 Hexulosonic acid -, methyl P-D-urubino-, reductic acid -, ~-urubino-2-,analytical high-perforformation from, 289 mance liquid chromatography, 34 D-ribo-Hexo-pyranos-3-ulose hydrate, 1,6-, D-XylO-5-, analytical high-perforanhydro-2,4,-dideoxy-2,4-difluoro-P-, mance liquid chromatography, 34 'H- and 19F-n.m.r. data for, 151 -, ~-xylo-2-,analytical high-perforHexopyranosyl fluorides mance liquid chromatography, 34 -, 2-deoxy-, 'H- and i9F-n.m.r. data Hexuronic acids for, 131-132 decarboxylation, acidic conditions, 288-, 2-deoxy-~-urubino289 -, 3,4,6-tri-O-acetylformation of 2-furaldehyde from, 289 (Y anomer, 'H- and I9F-n.m.r. data ultraviolet-absorbances of, 65 for, 131 5-Hexylbarbituric acid, cyclodextrin inclup anomer, IH- and I9F-n.m.r. data sion complexes with, 223, 224 for, 131 5-Hexyl-2-thiobarbituric acid, cyclodextrin -, 3,4,6-tri-O-benzoyl-a-, 'H- and inclusion complexes with, 223, 224 "F-n.m.r. data for, 131 Heyns rearrangement, 308
376
SUBJECT I N D E X
High-mannose oligosaccharides, sizefractionation of, 62 High-performance affinity chromatography,
18 High-performance liquid chromatography,
17-72 alkylated (reversed-phase) silica gels,
27-30 amine-modified silica gels, 23 aminopropyl-bonded phase columns, maintenance of, 24 aminopropyl-bonded silica gels, for preparative purposes, 59 analytical separation, 32-49 anion-exchange resins and silica gels, 30 automated fraction-collectors, 23 bonded-phase silica cartridges, 19 Cls-bonded silica columns, for preparative purposes, 59 boronic acid substituted silica gel, 31 cartridge-type filtration-units, 20 cation-exchange resin columns, 24-27 applications, 26 calcium-form, 26-27,50-52 hydrogen-form, 25-26,50 lead-form, 26,50-51 maintenance, 27 for preparative purposes, 59 silver-form, 26,50 Cls-bonded silica-gel columns, 28-29 column design, 21 column ovens, 22 column-packing equipment, 21-22 data systems, 22-23 degassing instruments, 19 detectors, 22 diol-modified silica gel, 31 equipment for preserving column-life,
19-20 filters, 20 fittings, 20 guard columns, 19 injectors, 20-21 instrumentation, 18-23 ion-exchange resins, 31 in plastic cartridges, 19 mini-columns, 19-20 phenyl-bonded phase, 28 post-column reaction-modules, 22 pre-columns, 19,27
preparative, solvent-delivery system, 19 refractive-index detectors, 18 reversed-phase columns, 24,29 reversed-phase silica-gel phases, 28 silica saturator columns, 19 solvent-delivery systems, 18-19 stationary phases, 23-31 switching valves, 20 Hirano, S., 4,6,7 Hirase, Susumu, 1, 9 Histocompatability antigens, oligosaccharides, liquid chromatography separation, 43 ‘H-N.m.r. spectroscopy, 59 chemical shifts, 75 geminal coupling, 2J (IH, 19F),75 IH-l9F coupling constants, 75-77 long-range coupling 4J, (IH,I9F),76-77 long-range coupling sJ, (IH, I9F), 76-77 long-range coupling 6J, (IH, I9F),77 structural-reporter-group concept, 79 vicinal coupling, 3J (IH, I9F), 75-76 Houssay, B., 12 Human cerebral-cortex ganglioside, hydrochloric acid hydrolysis, 267 Humus formation, carbohydrate transformation in, 323-326 Hyaluronate oligosaccharides, liquid chromatography separation, 48-49 Hyaluronic acid composition, analysis, 55 structural and sequence analysis of, 57 Hydrazones, as photosensitive protecting groups, 195 Hydrochloric acid hydrolysis of glycoconjugates with, 266 hydrolysis of polysaccharides with, recoveries of monosaccharides after, 260-261 pH of aqueous solutions of, 252 Hydrocinnamic acid, cyclodextrin inclusion complexes with, 222,224 Hydrolysis, for cleavage of glycosidic linkages, 252 Hydroxide ion, cyclodextrin inclusion complexes with, 221 2-Hydroxy-6-(hydroxymethyI)-3(2H,6H)pyranone, 323 3-Hydroxy-6-(hydroxymethyl)-2-methylchromone, 290
SUBJECT I N D E X
377
L-Idofuranose 2-(2-Hydroxyacetyl)furan, 3 18 -, 3,6-anhydro-5-deoxy-5-fluoroformation, 285-287 -, 1,2-di-O-acetyl-, IH- and I9F4-Hydroxybenzoate ion, cyclodextrin n.m.r. data for, 115 inclusion complexes with, 221 -, 1,2-O-isopropylidene-p-, 'H- and 2-Hydroxybenzoic acid, cyclodextrin 19F-n.m.r.data for, 115 inclusion complexes with, 223, 224 -, 3,6-anhydro-5-deoxy-5,6,6-trifluoro3-Hydroxybenzoic acid, cyclodextrin 1,2-O-isopropylidene-pinclusion complexes with, 221 Y-n.m.r. data for, 177 4-H ydroxybenzoic acid, cyclodextrin IH- and 19F-n.m.r. data for, 158 inclusion complexes with, 221, 223, -, 3,6-anhydro-6,6-difluoro-I ,2-0224 I-Hydroxy-2-butanone, formation, 294 isopropylidene-p"C-n.m.r. data for, 175 3-Hydroxy-2-butanone, 321 4-Hydroxy-2-butanone, formation, 294 IH- and I9F-n.m.r. data for, 152 2-Hydroxy-3,5-dimethylcyclopent-2-en1-, 5-deoxy-5-fluoroone, formation, 294 a anomer, %-n.m.r. data for, 167 4-Hydroxy-2,5-dimethyl-3(2H)-furanone, p anomer, I3C-n.m.r. data for, 167 formation, 294, 318 -, I ,2-O-isopropylidene-p-, 13CHydroxyl groups, photosensitive protectn.m.r. data for, 167 ing groups, 180-191 5-deoxy-5-fluoro-1,2-O-isopropylidene2-Hydroxy-5-methylcyclopent-2-en-I-one, p-, IH- and 19F-n.m.r. data for, 100 formation, 294 L-Idofuranosyl fluoride, 2-0-acetyl-3,65-(Hydroxymethyl)-2-furaldehyde,296, anhydro-5-0-benzoyla anomer, IH-and 19F-n.m.r. data for, 318 115 effect on color formation, under sulfatepulping conditions, 325 p anomer, IH- and '9F-n.m.r. data for, formation of, 284-285, 287 115 from Amadori compounds, 319-320 L-Idopyranose, 3-deoxy-3-fluoroa anomer, IH- and I9F-n.m.r. data for, liquid chromatography, 53-54 4-Hydroxy-5-methyl-3(2H)-furanone, 3 18 91 p anomer, IH- and I9F-n.m.r. data for, formation, 290-291, 321 from Amadori compound and its 2,391 enolization, 320 IgD 3-Hydroxy-2-methyl-4H-pyran-4-one, 320isolation of carbohydrates from, on analytical-scale columns, 60 32 1 Hydroxy-L-proline, and D-glucose or Loligosaccharides, liquid chromatography separation, 42-43 rhamnose, high-temperature transformation of, 315 sialylated oligosaccharides, fractionation, 46 I-Hydroxy-2-propanone, 32 1 I-Hydroxy-2-propanone, formation, 293 IgM isolation of carbohydrates from, on analytical-scale columns, 60 oligosaccharides, liquid chromatography I separation, 42-43 Ilex paraguariensis, 13 D-Idofuranose Immunoglobulins -, 3-deoxy-3-fluoroglycopeptides, liquid chromatography a anomer, IH- and I9F-n.m.r. data for, separation, 48 99 hydrolysis with hydrochloric acid, p anomer, IH- and 19F-n.m.r. data for, 99 266
378
SUBJECT I N D E X
Inclusion complexes, 205-206 Indolines, formation of, 317 2-(Indolin-l-yl)- I-phenylpropanoic acid, 317 Inoue, Yoshiyuki, 3 Insulin, I 1 Inulins, hydrolysis, 269 Iodide ion, cyclodextrin inclusion complexes with, 221 Iseki, N., 9 Isomaltol. See 2-Acetyl-3-hydroxyfuran Isomalto-oligosaccharides, preparative liquid chromatography, 60 Isomaltose liquid chromatography methods for analysis of, 33 synthesis of, on light-sensitive, solid support, 184, 186 Isomaltotriose, liquid chromatography separation, 41 2',3'-O-Isopropylideneadenosine5'-phosphate, synthesis of, 203 Isopropylidene-a-D-glucofuranose -, I-0-acetyl-3-0-benzyl-2-deoxy-2fluoro-5,6-0-, IH- and I9F-n.m.r. data for, 99 -, S-deoxy-S-fluoro-I,2-0-, IH- and I9Fn.m.r. data for, 100 2',3'-O-lsopropylideneuridine5'-phosphate, synthesis of, 203 Isovaleraldehyde, formed from L-leucine, 311
K Karasawa, I., 3 Kashimura, N., 6, 7 Keratin fibers, hydrolysis of, with hydrochloric acid, 267 Ketofuranose derivatives, mono- and difluorinated "C-n.m.r. data for, 174 IH- and I9F-n.m.r. data for, 144-145 Ketones, protection of, 195-198 Ketopyranose derivatives, fluorinated, IHand I9F-n.m.r. data for, 144 Ketopyranoses, fluorinated, 13C-n.m.r. data for, 173 Ketose disaccharides, large-scale preparative liquid chromatography, 62
Ketoses, liquid chromatography methods for analysis of, 33 Kitaoka, S.,3-4 Kojibiose, liquid chromatography methods for analysis of, 33 Komano, T., 4, 6, 7
L Lactones fluorinated I3C-n.m.r. data for, 170 IH- and IgF-n.m.r. data for, 116 ultraviolet-absorbances of, 65 Lactose in food, liquid chromatography separation, 52 liquid chromatography methods for analysis of, 33 Lactulose, liquid chromatography methods for analysis of, 33 Laminarabiose, liquid chromatography methods for analysis of, 33 D-Laudanine, 13 Leucrose, liquid chromatography methods for analysis of, 33 Levans, hydrolysis, 269 Levulinic acid, 296 Licopodium sururus, 13 Lignin, source of humus, 323 Lignocellulosic materials fermentation products, 326 polysaccharides in, 306 Lipopolysaccharides, bacterial, methanolysis, 258-259 Liquefaction, 273-274, 323 Liquid chromatography, 17-18 accuracy, 63-64 combined techniques, 69-70, 72 detectability, 63-69 direct-detection methods, 65-66 electrochemical detectors, 65 flame-ionization detectors, 65-66 future trends in, 71-72 mass detectors, 65-66 polarimetric detectors, 66 post-column derivatization methods, 6668 pre-column derivatization methods, 6869
379
SUBJECT INDEX
preparative, 58-63, 71-72 in analytical-scale equipment, 59-61 general aspects of, 58-59 in large-scale equipment, 61-63 sample recovery from columns, 59 refractive index detection, 64-65 stationary phases, 71 triple-pulsed amperometry on platinum or gold electrodes, 65 ultraviolet detectors, 65 Liquid chromatography-mass spectrometry, 69-70 Liquid chromatography-n.m.r. spectrosCOPY,69-70 Lobry de Bruyn-Alberda van Ekenstein transformation, 281 Of D-glUCOSe, 303 Lysosomal-storage disorders, urine in liquid chromatography, 44 sialylated oligosaccharides, liquid chromatography analysis, 45-46 L-Lyxofuranose, I-O-acetyl-2,3,5-tri-Obenzoyl-4-fluoro-a-, W-n.m.r. data for, 172 D-LyXOfUranOSyl fluoride -, 2,3-acetoxonium-5-O-acetyl-p-, IHand I9F-n.m.r. data for, 107 -, 2,3-benzoxonium-5-0-benzoyl-a-, 'H-and 19F-n.m.r. data for, 107 -, 5-O-benzoyl-2,3-benzoxonium-a-, W-n.m.r. data for, 168 -, 2,3,5-tri-O-acetyl-, 'H-and I9Fn.m.r. data for, 107 -, 2,3,5-tri-O-benzoylI3C-n.m.r. data for, 168-169 IH-and I9F-n.m.r. data for, 107 D-Ly XOpyranOSe, 1,3,4-tri-O-acetyl-2deoxy-2-fluoro-a-, 'H- and I9F-n.m.r. data for, 104 D-Lyxopyranoside, trifluoromethyl 3,4-di0-acetyl-p-, IH- and 19F-n.m.r. data for, 104 D-LyXOpy~anOSylfluoride
-, 3,4-di-O-acetyl-2-deoxy-, 2-bromo-, IH-and I9F-n.m.r. data for, 135 -, 2-iodo-, 'Hand I9F-n.m.r. data for, 136 -, 2,3,4-tri-0-acetyl-ar-,'H-and I9Fn.m.r. data for, 102
-, 2,3,4-tri-O-benzoyl-a-, IH-and I9Fn.m.r. data for, 102
M Maillard reaction, 311, 318 chromophoric products, 322 at elevated temperatures, 307 factors affecting, 307 non-nitrogenous products, 3 18-321 products of, 320 inhibition of bacterial growth by, 326 Maltol. See 3-Hydroxy-2-methyl-4H-pyran4-one Malto-oligosaccharides linear, preparative liquid chromatography, 60 liquid chromatography large-scale preparative, 61-63 peak-area analyses, 64 Maltose in food, liquid chromatography separation, 52 liquid chromatography methods for analysis of, 33 6-O-a-Maltotriosylcyclomaltoheptaose, 246 Maltulose, liquid chromatography methods for analysis of, 33 L-Mandelic acid, cyclodextrin inclusion complexes with, 222, 224 Mannitol -, D-, 3,4-O-benzylidene-1,6-dideoxy-1,6difluoro-2,5-O-methylene-, 'Hand I9F-n.m.r. data for, 159 -, 1,6-dideoxy-I,6-difluoro-2,5-0methylene-, 'H-and I9F-n.m.r. data for, 159 -, 1,2,4,5,6-penta-O-acetyl-3-deoxy3-fluoro-, 'Hand I9F-n.m.r. data for, 159 liquid chromatography methods for analysis of, 33 D-Mannofuranosyl fluoride -, 2,3:5,6-di-O-acetoxonium-, 'H- and I9F-n.m.r. data for, 98 -, 2,3:5,6-di-O-benzoxonium-, 'H-and I9F-n.m.r. data for, 98
380
SUBJECT INDEX
p anomer, 'H- and 19F-n.m.r. data for, -, 2,3:5,6-di-O-isopropylidene133 13C-n.m.r. data for, 166 -, I ,3,4-tri-O-acetyl-a-, IH- and I9FIH- and 19F-n.m.r. data for, 98 n.m.r. data for, 133 -, 2,3,5,6-tetra-O-acetylD-Mannopyranose, hydrochloride I3C-n.m.r. data for, 166 -, 2-amino-2,6-dideoxy-6-fluoroIH- and I9F-n.m.r. data for, 98 a anomer, IH- and 19F-n.m.r. data for, -, 2,3,5,6-tetra-0-benzoyl-a-, 'H- and 122 I9F-n.m.l'. data for, 98 p anomer, 'H- and IgF-n.m.r. data for, -, 3,5,6-tri-O-acetyl-2-O-methyI-~-, "C122 n.m.r. data for, 166 D-Mannop yranoside Mannonic acids, analytical high-perfor-, methyl 2-deoxy-2-fluoromance liquid chromatography, 34 -, 3-0-acetyl-4,6-0-benzylidene-p-, D-Mannop yranose 'H- and I9F-n.m.r. data for, 88 -, 2-acetamido-2,6-dideoxy-6-fluoroIH- and I9F-n.m.r. data for, 122 -, 3-0-benzoyl-4,6-0-benzylidene-, 'H- and 19F-n.m.r. data for, 88 -, 1,3,4-tri-O-acetyl-, IH- and I9F-, 3-0-benzyl-4,6-0-benzylidene-p-, n.m.r. data for, 122 'H- and I9F-n.m.r. data for, 88 -, 1,6-anhydro-3-deoxy-3-fluoro-p-, 2,4-di-O-acetyl-, IH- and 19F-, 4,6-O-benzylidene-P-, IH- and 19Fn.m.r. data for, 114 n.m.r. data for, 88 IH-and I9F-n.m.r. data for, 114 -, 4,6-di-O-acetyl-3-0-methyl-p-, IH-, 2-deoxy-2-fluoroand I9F-n.m.r. data for, 88 a anomer -, 3,4,6-tri-O-acetyl-P-, IH- and I9F"C-n.m.r. data for, 162 n.m.r. data for, 88 IH- and I9F-n.m.r. data for, 87 -, methyl 6-deoxy-6-fluorop anomer a anomer, IH- and 19F-n.m.r. data for, I3C-n.m.r. data for, 162 96 IH- and I9F-n.m.r. data for, 87 13C-n.m.r. data for, 165 -, 1,2,4,6-tetra-O-acetyl-a-, IH- and -, 2,3-di-O-methyl-, "C-n.m.r. data I9F-n.m.r. data for, 92 for, 165 -, 1,3,4,6-tetra-O-acetyl-p-, I3C-, 2,3-di-O-methyl-a-, 'H- and I9Fn.m.r. data for, 162 n.m.r. data for, 96 -, 1,3,4,6-tetra-O-acetyl-, IH- and -, 2,3-O-isopropylidene-, "C-n. m.r. data for, 165 19F-n.m.r. data for, 87 -, 1,4,6-tri-O-acetyI-3-O-methyl-, 'H-, 2,3-O-isopropylidene-a-, IH- and and I9F-n.m.r. data for, 87-88 "F-n.m.r. data for, 96 -, trifluoromethyl 3,4,6-tri-O-acetyI-2-, 3-deoxy-3-fluorodeoxy-2-fluoro-p-, IH- and I9Fa anomer n.m.r. data for, 88 IT-n.m.r. data for, 163 L-Mannopyranoside, trifluoromethyl 3,4-diIH- and I9F-n.m.r. data for, 92 O-acetyl-2,6-dideoxy-p-, IH- and I9Fp anomer n.m.r. data for, 133 IT-n.m.r. data for, 163 D-Mannopyranosyl fluoride IH- and I9F-n.m.r. data for, 92 -, 2-O-benzoyl-4,6-di-O-methyl-a-, 'H-, 4-deoxy-4-fluoroand I9F-n.m.r. data for, 85 a anomer, I3C-n.m.r. data for, 164 -, 3-0-benzoyl-4,6-di-0-methyl-p-, IHp anomer, I3C-n.m.r. data for, 164 and 19F-n.m.r. data for, 85 L-Mannopyranose -, 2-deoxy-2-fluoro-, 2,6-dideoxy-2-fluorop anomer, 'H- and I9F-n.m.r. data for, a anomer, 'H- and 19F-n.m.r. data for, 146 133
SUBJECT INDEX -, 3,4,6-tri-O-acetyl-, IH- and I9F-
38 1
liquid chromatography, pre-column derivatization procedures, 68 n.m.r. data for, 147 -, 2,3-di-O-benzoyl-4,6-di-O-methyl-, Methyl (5-acetamido-4,7 8,9-tetra-O-acetyl3,5-dideoxy-2-nonulosyl fluoride)onate 'H- and I9F-n.m.r. data for, 84-85 -, 2,3,4,6-tetra-O-acetyl-, a-D-glyCerO-a-D-gUlUCtO-, 13C-n.m.r. data for, 177 I3C-n.m.r. data for, 161 IH- and 19F-n.m.r. data for, 84 -, a-D-glyCerO-p-D-gUlUCtO-, I3C-n.m.r. data for, 177 -, 2,3,4,6-tetra-O-benzoyl-a-, IH- and I9F-n.m.r. data for, 84 Methyl (methyl 4-deoxy-4-fluoro-a-~-, 3,4,6-tri-O-acety1-2-deoxyglucopyranosid)uronate, IH-and 19Fn.rn.r. data for, 116 -, 2-bromo-, IH- and 19F-n.m.r. data Methylation technique, 214 for, 130 5-Methylbarbituric acid, cyclodextrin -, 2-chloro-, 'H- and 19F-n.m.r.data inclusion complexes with, 223, 224 for, 130 2-Methylbenzofuran-5,6-diol, 290 -, 2-iodo-, IH- and I9F-n.m.r. data 3-Methylbenzoic acid, cyclodextrin inclufor, 130 sion complexes with, 222, 224 -, 2,4,6-tri-O-acetyl-3-U-methyl-a-, I3C2-Methylbenzoquinone, formation, 294 n.m.r. data for, 161 -, 3,4,6-tri-O-acetyl-2-O-methyl-a- 4-Methylbenzoylacetic acid, cyclodextrin inclusion complexes with, 223, 224 I3C-n.m.r. data for, 161 Methyl [benzyl2-(benzyloxycarbonyl) IH- and 19F-n.m.r. data for, 84 amino-2,3,4-trideoxy-5-fluoro-a-~L-Mannopyranosyl fluoride erythro-hex-3-enopyranosid]uronate -, 3,4-di-O-acetyl-2,6-dideoxy-2-fluoroW-n.m.r. data for, 172 p-, 'H- and I9F-n.m.r. data for, 153 IH- and 19F-n.rn.r.data for, 116 -, 2,3,4-tri-O-acetyl-6-deoxy-aMethyl 5-deoxy-5,5-difluoro-~-ribofurano13C-n.m.r. data for, 173 side IH-and I9F-n.m.r. data for, 132 -, 3-O-benzyl-a-, 'H- and I9F-n.m.r. Mannose data for, 156 D-, 2,3-O-isopropylidene-p-, IH-and I9Falkaline degradation of, 292 n.m.r. data for, 156 -, 2,6-di-O-methyl-. See Curamycose Methyl 3-deoxy-3-fluoro-a-~-gulopyranoin plant cell-wall hydrolyzates, liquid side, IH- and 19F-n.m.r. data for, chromatography analysis, 55 91 determination of enantiomeric form, 66 Methyl 2-deoxy-2-fluoro-P-~-ribo-hexopyliquid chromatography, 33, 52-53 ranoside, 4,6-0-benzylidene-3-deoxy-, Marenzi, A.D., 13 Marini-Bettolo, G., 13 IH- and 19F-n.m.r. data for, 132 Methyl 5-deoxy-5-fluoro-~-ribofuranoside Mass spectrometers, 69 -, 2,3-di-O-acetyl-, 'H- and 19F-n.m.r. Masuda, F., 6, 7 data for, 112 Mate, 13, 14 -, 2,3-O-isopropylidene-, 'H- and 19FMatsushima, Y.,9 n.m.r. data for, 112-113 Melanoidin, formation, 307 l-deoxyMelibiose, liquid chromatography methods 2,2'-O-Methylenebis(3-O-benzoyl1-fluoro-L-glycerol),IH- and 19F-n.m.r. for analysis of, 33 data for, 158 Mephrobarbital, cyclodextrin inclusion Z,Z'-O-Methylenebis(1-deoxy-l-fluoro-~complexes with, 223, 224 glycerol), IH-and 19F-n.m.r. data for, Methanesulfonates, photolysis of, 191 158 4-Methoxyphenacyl, photocleavage of, 4-O-Methyl-~-glucuronicacid, 306 201-202 2-C-Methylglyceric acid, 303 N-(p-Methoxypheny1)glycosylamines,
-
3
382
SUBJECT I N D E X
Methyl glycosides, 29 high-performance liquid chromatography of, 256 perbenzoylation of, 256 preparative liquid chromatography, 60 Methylglyoxal, liquid chromatography, 5354 Methyl rnaltosides, large-scale preparative liquid chromatography, 62 3-Methyl-2-(2-oxopropyl)thiophene,formation of, 318 a-Methylphenacyl, photocleavage of, 201202 2-Methyl-2-propano1,cyclodextrin inclusion complexes with, 222, 224 2-Methyl-3-propanoylthiophene, formation of, 318 2-Methylpyrazine, formation of, 317 2-Methyl-3-pyridino1,formation of, 312, 313 6-Methyl-3-pyridin01,3 12 5-Methylpyrrole-2-carboxaldehyde,formation of, 312 Milk, human complex, neutral oligosaccharides, liquid chromatography, 44 oligosaccharides, preparative liquid chromatography, 60 sialylated oligosaccharides, liquid chromatography analysis, 45-46 Misaki, A., 9 Miyazaki, N., 6 Moffatt, J.G., 6 Monosaccharides absolute or relative decomposition of, 259 acid hydrolysis, losses, 259 acyclic, IH- and I9F-n.m.r. data for, 158- 159 branched, I3CC-n.m.r. data for, 172-173 deoxy fluorinated, 13C-n.m.r.data for, 172- I73 fluorinated, n.m.r. spectroscopy of, 73178 fluorinated branched, 'H- and IgF-n.m.r. data for, 142-143 formation of phenolic compounds from, 295 high-performance liquid chromatography, 31
ionic, high-performance liquid chromatography, 33-37 large-scale preparative liquid chromatography, 62 methanolysis, 257-258 neutral, analytical high-performance liquid chromatography, 32-33 partially methylated, structural and sequence analysis of, 57 preparative liquid chromatography, 60 pyruvated, of bacterial origin, studied using methanolysis, 259 structural and sequence analysis of, 57 tri- and tetra-fluorinated I3C-n.m.r. data for, 177 IH-and 19F-n.m.r.data for, 158 Mosses, formation of humus from, 323324 Much hydrolysis, by hydrochloric acid, 266267 isolation of carbohydrates from, on analytical-scale columns, 60 methanolysis, 258 oligosaccharides, liquid chromatography separation, 42-43 sialylated oligosaccharides, fractionation. 46
N Neuraminic acid derivatives, fluorinated, I3C-n.m.r. data for, 177 Neuraminic acids hydrolysis, 254 liquid chromatography, 36-37 Neutral sugars, hydrolysis of, 262-263 Nigerose, liquid chromatography methods for analysis of, 33 Nitroanilides, as protecting groups for carboxylic acids, 202 2-Nitrobenylidene derivatives, photochemical cleavage of, 188 2-Nitrobenzaldehyde, photoinduced oxidation-reduction of, 181 3-Nitrobenzoic acid, cyclodextrin inclusion complexes with, 221 4-Nitrobenzoic acid, cyclodextrin inclusion complexes with, 221
SUBJECT INDEX 2-Nitrobenzyl, photochemical cleavage of, I93 P3-1-(2-Nitrobenzyl)adenosine 5’-triphosphate, 204 2-Nitrobenzylcarbonates, photochemical cleavage of, 190-191 2-Nitrobenzyl esters, photochemical cleavage of, 198 2-Nitrobenzyl ethers, 181-182 photochemical cleavage, 18I - I83 proposed mechanism for, 183 2-Nitrobenzyl glycosides photochemical cleavage of, 183 proposed mechanism for, 183 2-Nitrobenzyl group, for protection of carboxylic acids, 198 2-Nitrobenzylidene derivatives in protection of diols, 188 utilized in syntheses leading to trisaccharides of biological significance, 189 2-Nitrobenzyloxycarbonyl group, photochemical cleavage of, 193 2-Nitrobenzyl phosphoric esters, in nucleotide synthesis, 203 2-Nitrophenol, cyclodextrin inclusion complexes with, 222-224 3-Nitropheno1, cyclodextrin inclusion complexes with, 221 4-Nitrophenol, cyclodextrin inclusion complexes with, 221 3-Nitrophenolate ion, cyclodextrin inclusion complexes with, 221 4-Nitrophenolate ion, cyclodextrin inclusion complexes with, 221, 223, 224 3-Nitrophenyl esters, photosolvolysis of, 203 P3-l-(2-Nitrophenylethyl)adenosine5’triphosphate, 204 (2-Nitrophenyl)ethylenedioxy acetals, 195 removal of, by strong base, proposed mechanism for, 196 (2-Nitropheny1)ethylene glycol, 195 (p-Nitrophenyl)hydrazones, liquid chromatography, pre-column derivatization procedures, 68 3-Nitrophenyloxycarbonyl, 193 6-Nitroveratryl p-D-ghcopyranoside, photochemical cleavage of, 184-185
383
6-Nitroveratrylox ycarbonyl group, photochemical cleavage of, 193 6-Nitroveratryl urethans, photochemical cleavage of, 193 Nuclear magnetic resonance spectroscopy advantages of, 73-74 comparison of relevant parameters of different nuclei, 74 of fluorinated monosaccharides, 74 other nuclei, 80 two-dimensional techniques, 74 Nucleoside antibiotics, diastereoisomeric pyrimidine, 28 Nucleoside derivatives, high-performance liquid chromatography, 28 Nucleosides isomeric 2’-deoxy-C-, 28 synthesis of, 6
0 Ochiai, H., 4 Ocotea puberula, 13 Ocotein, 13 Oligoglycosiduronic acids, liquid chromatography, 40-41 Oligosaccharides N-acetylated or deoxy sugar-containing, 29 acidic, animal-derived, large-scale preparative liquid chromatography, 62 chitin-derived, 29 cleavage of glycosidic linkages, 252 complex determining sequence of glycosyl residues in, 58 preparative liquid chromatography, 60 complex (and cyclic), neutral, liquid chromatography, 41-45 complex, ionic, liquid chromatography, 45-49 containing 2-acetamido-2-deoxyhexose units, ultraviolet-absorbances of, 65 cyclic, liquid chromatography separation, 44-45 gl ycoprotein-derived liquid chromatography, 42-43 structural.and sequence analysis of, 57 glycosaminoglycan-derived, liquid chro-
384
SUBJECT INDEX
matography, pre-column derivatization procedures, 68 from glycosaminoglycans, ultravioletabsorbances of, 65 high-mannose, preparative liquid chromatography, 60 hyaluronic acid-derived, liquid chromatography separation, 48 ionic, of animal origin, preparative liquid chromatography, 60 linear, structural and sequence analysis of, 57 liquid chromatography, peak-area analyses, 64 microbial, preparative liquid chromatography, 60 neutral of animal origin, preparative liquid chromatography, 60,62 structural and sequence analysis of, 57 peralkylated, prechromatographic purification of,20 phosphorylated fractionation, 46 preparative liquid chromatography, 60 plant-derived large-scale preparative liquid chromatography, 62 liquid chromatography, 41 sialic acid-containing large-scale preparative liquid chromatography, 62 preparative liquid chromatography, 60 sialylated liquid chromatography separation, 4546 ultraviolet-absorbances of, 65 simple, ionic, liquid chromatography separation of, 40-41 simple, neutral, liquid chromatography analysis, 37-40 simple and complex, liquid chromatography , pre-column derivatization procedures, 68 starch-derived, 29 sulfated preparative liquid chromatography, 60 uronic acid-containing, large-scale preparative liquid chromatography, 62
synthesis of, combined organic-enzymic approach, 184-187 Olivacine, 13 Ombuin, 13 Onodera, Akifumi, 9 Onodera, Fumi, 9 Onodera, Koji, 9 Onodera, Konoshin accomplishments, 4-5 awards and honors, 8-9 baccalaureate graduation thesis, 3 childhood, 2 climbing in Himalayas, 5 editorial work, 8 family, 1-9 fields of research, 6 founder of Japanese Society of Carbohydrate Research, 9 interest in mountain climbing, 2 investigation of synthetic procedure for nucleosides, 6 W.D. thesis, 3 scientific research, 3-4, 5-8 study of biochemistry, 2-3 as teacher, 5 work for Agricultural Chemical Society, 8 work for Biochemical Society of Japan, 8 work habits, 5 work in Laboratory of Biological Chemistry, 3 Onodera, Mizuyo, 9 Onodera, Yukari, 3, 9 Orosomucoid glycopeptides, liquid chromatography separation, 48 isolation of carbohydrates from, on analytical-scale columns, 60 sialylated oligosaccharides, fractionation, 46 Ovalbumin glycopeptides, liquid chromatography separation, 48 hydrolysis, with trifluoroacetic acid, 268 isolation of carbohydrates from, on analytical-scale columns, 60 oligosaccharides, liquid chromatography separation, 42-43
SUBJECT INDEX
sialylated oligosaccharides, fractionation, 46 Ovarian-cyst glycoproteins, oligosaccharides from, liquid chromatography separation, 42-43, 60 Ovomucoid glycopeptides, liquid chromatography separation, 48 isolation of carbohydrates from on analytical-scale columns, 60 on large-scale columns, 62 oligosaccharides, liquid chromatography separation, 43 sialylated oligosaccharides, fractionation, 46
385
-, 5-deoxy-5-fluoro-o-, 'H- and 19Fn.m.r. data for, 112-1 13 L-threo-Pento-1,4-furanos-4-ulose, 4-0acetyl-5-deoxy-5-fluoro-1,2-0-isopropylidene-3-O-tosyl-P-, 'H- and I9Fn.m.r. data for, 143 Pentofuranosides -, 2-deoxy-2-fluoro-, 'H- and I9F-n.m.r. data for, 109-1 I I -, 3-deoxy-3-fluoro-~-,IH- and I9Fn.m.r. data for, I12 -, 5-deoxy-5-fluoro-~-,'H- and I9Fn.m.r. data for, 112-113 Pentofuranosyl fluorides -, 3,5-di-O-benzoyl-2-bromo-2-deoxy-~-, a-D-urubino-, 'H- and I9F-n.m.r. P data for, 138 -, p-D-Xylo-, 'H- and I9F-n.m.r. data Palatinose, liquid chromatography methods for, I38 'H- and 19F-n.m.r. data for, for analysis of, 33 106- I09 Panose, liquid chromatography separation, Pentopyranose derivatives, fluorinated 41 I3C-n.m.r. data for, 167-168 Pectic acid, oligomers, liquid chromatograC(or 0)-branched, IH- and 19F-n.m.r. phy, 40 Pectin, 306, 326 data for, 141-142 Pentopyranoses acid hydrolysis of, correction factors, 254 -, 2-deoxy-2-fluoro-, 'H- and I9F-n.m.r. galacturonic acid units of, hydrolysis data for, 104-105 losses, 261-262 -, 3-deoxy-3-fluoro-, 'H- and 19F-n.m.r. non-fermentable oligosaccharides, liquid data for, 105-106 -, 4-deoxy-4-fluoro-, IH- and I9F-n.m.r. chromatography methods for, 52 2,4-Pentanedione, for enhancing detectabildata for, 105-106 Pentopyranoside ity of carbohydrates, 67 I-Pentanol, cyclodextrin inclusion com-, 2-deoxy-2-fluoro-, 'H- and 19F-n.m.r. plexes with, 222-224 data for, 104-105 Pentobarbital, cyclodextrin inclusion com-, 3-deoxy-3-fluoro-, IH- and 19F-n.m.r. plexes with, 223, 224 data for, 105-106 D-xylo-Pentodialdo-l,Cfuranose -, 4-deoxy-4-fluoro-, IH- and I9F-n.m.r. -, 3-deoxy-3-fluoro-I ,2-0-isopropylidata for, 105-106 dene-a-, n.m.r. data for, 169 -, methyl 2-amino-2,3,4-trideoxy-3-, 1,2-0-isopropylidene-3-deoxy-3fluoro-threofluoro-a-, IH- and 19F-n.m.r.data -, a - ~ - 'H, and 19F-n.m.r. data for, for, 112 128 Pentofuranose derivatives, fluorinated, I3C-, p-D-, IH- and 19F-n.m.r. data for, n.m.r. data for, 168-169 128 Pentofuranoses L-eryrhro-Pentopyranoside -, 2-deoxy-2-fluoro-, 'H- and I9F-n.m.r. -, methyl 2-deoxy-2,2-difluoro-3,4-0data for, 109-1 11 isopropylidene-p-, IH- and 19F-, 3-deoxy-3-fluoro-~-,'H- and I9Fn.m.r. data for, 156 n.m.r. data for, I12 -, methyl 4-deoxy-4,4-difluoro-2,3-0-
386
SUBJECT INDEX
isopropylidene-p-, IH- and I9Fn.m.r. data for, 156 Pentopyranosyl fluorides
Phenylisocyanates, liquid Chromatography, pre-column derivatization procedures, 68 -, 3,4-di-O-benzoyl-2-deoxy-~-erythro-Phosphoric esters, protection of, 202-203 a anomer, 'Hand I9F-n.m.r. data for, Phosphorus pentaoxide, oxidation and 137 polymerization of sugars with, 6 p anomer, IH-and I9F-n.m.r. data for, Phytolncca dioica, 13 137 Pinus elliotti. See Slash pine IH-and t9F-n.m.r. data for, 101-103 Plant cell-wall materials, hydrolysis, 264 Pentose Plant cell-walls -, 2-deoxy-erythro-, liquid chromatogcomplex, acidic oligosaccharides from, raphy methods for analysis of, 33 fractional liquid chromatography difluorinated, IH-and I9F-n.m.r. data methods, 49 for, 155 liquid chromatography analysis, 52 Pentose dehydration, mechanism of, 275 Plant cell-wall sugars, preparative liquid Pentulose, erythro-2-, liquid chromatograchromatography, 60 phy methods for analysis of, 33 Plant fiber, liquid chromatography analy5-Pentylbarbituric acid, cyclodextrin inclusis, 52 sion complexes with, 223, 224 Plant oligosaccharides, preparative liquid 5-Pentyl-2-thiobarbituric acid, cyclodextrin chromatography, 60 inclusion complexes with, 223, 224 Plant polysaccharides Perbenzoates, liquid chromatography, precomplex, structural and sequence analycolumn derivatization procedures, 68 sis of, 57 Perbenzoylated sugars, prechromatomonosaccharide composition of gas-liquid chromatography analysis, graphic purification of, 20 Per-p-bromobenzoates, liquid chromatogra54 liquid chromatography separations, phy, pre-column derivatization proce54-55 dures, 68 Pogonopus tubulosus, 13 Perchlorate ion, cyclodextrin inclusion Poly(galactur0nic acid) lyases, mode of complexes with, 221 action, analysis of, 54 Perchloric acid, cyclodextrin inclusion complexes with, 222, 224 Polysaccharide-degradingenzymes, mode of action, liquid chromatography Periodate, for enhancing detectability of carbohydrates, 67 analysis, 53-54 Polysaccharide hydrolyzates, isolation of, Pernaphthoates, liquid chromatography, pre-column derivatization procedures, on large-scale columns, 62 68 Poly saccharides 1-Phenethylindoline, 3 17 complex, determining sequence of glycoPhenobarbital, cyclodextrin inclusion syl residues in, 58 complexes with, 223, 224 glycosidic linkages, cleavages of, 251Phenol, cyclodextrin inclusion complexes 272 with, 222-224 high-temperature transformation of, 295Phenolics 307 from carbohydrates, 323 mono- and di-saccharides enzymically formation of, 326 released from, structural and seL-Phenylalanine, cyclodextrin inclusion quence analysis of, 57 complexes with, 222, 224 reductive-cleavage, 27 1 Phenyl glycosides, cleavage, photosensiPolysaccharide side-chains, structural and tized by 1,4-dicyanonaphthalene, 184 sequence analysis of, 57 3-Phenylindoline, 3 17 Polyuronic acids, hydrolysis of, 263
SUBJECT INDEX Propanoic acid, cyclodextrin inclusion complexes with, 221 2-Propanol, cyclodextrin inclusion complexes with, 222, 224 5-Propyl-2-thiobarbituric acid, cyclodextrin inclusion complexes with, 223, 224 Protecting groups light-sensitive, 180 modifier, 180 photochemical removal of, 179-180 photosensitive, application to biological models, 203-204 Proteodermatan sulfate, hydrolysis, with trifluoroacetic acid, 268 Pseudocellobiouronic acid, hydrolysis of, 299 Pseudocidamine, racemic, 13 Pseudocorydine, 13 Psicose -, D-, alkaline degradation of, 292 liquid chromatography methods for analysis of, 33 Pyranoses -, 2(or 3)-deoxy-2(or 3)-fluorohexo-, I3C-n.m.r. data for, 161-163 -, 4(or 6)-deoxy-4(or 6)-fluorohexo-, I3C-n.m.r. data for, 163-165 Pyranosides -, 2(or 3)-deoxy-2(or 3)-fluorohexo-, 13C-n.m.r. data for, 161-163 -, 4(or 6)-deoxy-4(or 6)-fluorohexo-, W-n.m.r. data for, 163-165 Pyrazine alkylated, formation of, 315 formation, pathways of, 317 Pyridine, cyclodextrin inclusion complexes with, 222, 224 Pyrocatechol, 290 Pyrogallol, 290 Pyrolysis, 273-274 Pyruvaldehyde, 321 formation, 293 R
Reducing sugars, 214 Reductic acid effect on color formation, under sulfatepulping conditions, 325 formation of, 288
387
as product after acid exposure of Dxylose, 276 Reichstein, T., 13 Rhamnogalacturonan 11, oligosaccharides, liquid chromatography fractionation, 49 Rhamnose determination of enantiomeric form, 66 liquid chromatography methods for analysis of, 33 liquid chromatography separation, 53 L-Rhamnose and ethylamine, reaction between, 313 in plant cell-wall hydrolyzates, liquid chromatography analysis, 55 Ribitol, liquid chromatography methods for analysis of, 33 D-Ribofuranose -, 1-O-acetyl-5-O-(methoxymethyl)-a-, IH- and I9F-n.m.r. data for, 111 -, I-0-acetyl-2-deoxy-2-fluoro-5-0(methoxymethy1)-a-, 13C-n.m.r.data for, 169 -, I-0-acetyl-2,3,5-tri-O-benzoyl-4&oxy-4-fluoro-p-, 13C-n.m.r. data for, 172 -, 5-deoxy-5-fluoro-, IH-and I9F-n.m.r. data for, 112 D-Ribofuranosyl fluoride -, 2,3-acetoxonium-5-O-acetyl-p-, Wn.m.r. data for, 169 -, 2-O-acetyl-3,5-di-O-benzoyl-p-, IHand I9F-n.m.r. data for, 108 -, 3-O-acetyl-2,5-di-O-benzoyl-p-, IHand 19F-n.m.r.data for, 108 -, 5-0-acetyl-2,3-di-O-benzoyI-p-, 'Hand 19F-n.m.r. data for, 108 -, 2,3,5-tri-O-acetyl-, IH-and I9Fn.m.r. data for, 107 -, 2,3,5-tri-O-benzoylI3C-n.m.r. data for, 169 'H- and I9F-n.m.r. data for, 108 Ribonic acids, analytical high-performance liquid chromatography, 34 D-Ribop yranose -, 2-deoxy-2-fluoroa$-, 'Hand 19F-n.m.r.data for, 104 -, 1,3,4-tri-O-acetyl-, IH- and I9Fn.m.r. data for, 104 -, 1,3,4-tri-O-acetyl-2-deoxy-2-fluoro-
388
SUBJECT I N D E X
Slime-mold gl ycoprotein, oligosaccharides, liquid chromatography separation, 4243 Ribonuclease B, glycopeptides, liquid Sodium perchlorate, cyclodextrin inclusion chromatography separation, 48 complexes with, 222, 224 Ribonucleic acid ligase, bacteriophage T4Solanum glaucophyllum, 14 induced, Escherichia coli, 182 Sophorose, liquid chromatography methRibo-oligonucleotides, synthesis of, 181 ods for analysis of, 33 D-Ribopyranosyl fluoride -, 3,4-di-O-acetyl-2-deoxy-2-bromo-, Sorbopyranose -, DIH- and 19F-n.m.r.data for, 136 -, 4-deoxy-4-fluoro-a-, I3C-n.m .r. -, 3,4-di-O-acetyl-2-deoxy-2-fluoro-a-, data for, 173 'H- and I9F-n.m.r. data for, 155 -, 4-deoxy-4-fluoro-1,2-O-isopropyli-, 2,3,4-tri-O-acetyl-, 'H- and I9Fand I9F-n.m.r. data dene-p-, 'Hn.m.r. data for, 102 for, 144 -, 2,3,4-tri-O-acetyl-2-Buoro-p-, IH-, L-, 5-deoxy-S-fluoro-a-, W-n.m.r. and I9F-n.m.r. data for, 143 data for, 173 -, 2,3,4-tri-O-benzoyl-, IH- and I9FSorbose, liquid chromatography methods n.m.r. data for, 102 for analysis of, 33 D-Ribose, 1,3,4-tri-O-benzoyl-p-, 'H- and Sordelli, A., 11 IgF-n.m.r. data for, 104 Starch, 295 S acid hydrolysis of, 296 screw model of, 215 Saccharides Stoppani, A.O.M., 13 from glycoproteins, liquid chromatograStrecker degradation, 311-312, 315, 317 phy separation, 56-57 Streptomyces curacoi, 14 monomeric, high-temperature transforN-Substituted aldosylamine mation of, 275-295 conversion, into l-amino-l-deoxy-2acidic conditions, 275-280 ketose, 308-309 basic conditions, 281-284 formation of, 308 Saccharinic acid, formation, 281-282, 291 Sucrose Saeman hydrolysis, 263-264 in food, liquid chromatography separaSalivary glycoproteins, hydrolysis, with tion, 52 trifluoroacetic acid, 268 liquefaction, 294 Saurine, 13 phenols from, 295 Sauroxine, 13 Sucrose derivatives, 0-methylated, largeSchardinger dextrins. See Cyclodextrins scale preparative liquid chromatograSchima liukiuensis, 3 phy, 62 Sialic acids Sugar acids, fluorinated high-performance liquid chromatogra'lC-n.m.r. data for, 170 'H- and '9F-n.m.r. data for, 116 phy, 31 hydrolysis, 254, 270 Sugar anomers, separation of, 70 liquid chromatography, pre-column Sugar nucleotides, conformations of, 7 derivatization procedures, 68 Sugar phosphates, liquid chromatography separation of, 36 methanolysis, 254 ultraviolet-absorbances of, 65 Sugars determination of enantiomeric form, 66 Sialyllactose, methanolysis, 258 Simple sugars, liquid chromatography, premutarotation rates of, determination, 71 column derivatization procedures, 68 Slash pine, hemicellulose fraction of, 306 pyranose anomers, separation, 70-71 a anomer, '3C-n.m.r. data for, 167
p anomer, 13C-n.m.r.data for, 168
SUBJECT INDEX Sulfite pulping, color-stopping reaction in, 324-325 Sulfuric acid hydrolysis of glycoconjugates with, 266 hydrolysis of polysaccharides with, monosaccharide recoveries after, 259-260 pH of aqueous solutions of, 252 Suzuki, Bunsuke, 2-3
T D-Tagatopyranose, 4-deoxy-4-fluoro-1,2-0isopropylidene-p-, I3C-n.m.r. data for, 173 Tagatose, liquid chromatography methods for analysis of, 33 D-Talofuranosyl fluoride, 2,3,5,6-tetra-Obenzoyl-a-, 'Hand 19F-n.m.r. data for, 98 D-Tdopyranose -, 2-deoxy-2-fluoroa anomer, 'H- and 19F-n.m.r.data for, 88-89 p anomer, 'H-and I9F-n.m.r. data for, 89 D-Talopyranoside -, 2-deoxy-2-fluoro-, trifluoromethyl 3,4,6-tri-O-acetyl-P-, IH-and I9Fn.m.r. data for, 89 -, methyl 4-deoxy-4-fluoro-a"C-n.m.r. data for, 164 'H- and 19F-n.m.r. data for, 94 -, 6-O-trityl-a-, IH- and I9F-n.m.r. data for, 94 -, methyl 4,6-dideoxy-4,6-dilluoro-aW-n.m.r. data for, 175 'Hand 19F-n.m.r. data for, I50 -, 2,3-0-isopropylidene-a-, 'H- and 19F-n.m.r. data for, 150 -, methyl 4,6-dideoxy-4-fluoro-a-, 6-amino-, 'H-and I9F-n.m.r. data for, 120 -, 6-azido-, IH- and 19F-n.m.r. data for, 119 D-Talopyranosyl fluoride -, 2,3,4,6-tetra-O-acetyl-a-, W-n.m.r. data for, 161
-, 3,4,6-tri-O-acetyl-2-deoxy-
389
-, 2-bromo-a-, IH-and 19F-n.m.r. data for, 130 -, 2-iodo-a-, 'H-and I9F-n.m.r. data for, 130 -, 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-p, 'H- and 19F-n.m.r. data for, 147 1,3,4,6-Tetra-O-acetyl-~-fructofuranosyl fluoride a anomer I3C-n.m.r. data for, 174 IH- and I9F-n.m.r. data for, 144 p anomer T-n.m.r. data for, 174 IH- and 19F-n.m.r. data for, 144 2,3,6,7-Tetrahydro-7-methylcyclopent[b]azepin-8(lti)-one, 3 15 2,3,2',3'-Tetramethoxybenzoin,photocyclization of, 201 Tetrazolium Blue, for enhancing detectability of carbohydrates, 67 Thin-layer chromatography, 28 Thioacetals, as photosensitive protecting groups, 195 Thiocyanate ion, cyclodextrin inclusion complexes with, 221 Thiopental, cyclodextrin inclusion complexes with, 223, 224 Thiophenes, formation of, 318 Thiophenobarbital, cyclodextrin inclusion complexes with, 223, 224 Thyroglobulin, sialylated oligosaccharides, fractionation, 46 p-Toluenesulfonates, photolysis of, 191 Trehaiose, liquid chromatography methods for analysis of, 33 Trifluoroacetic acid hydrolysis of glycoconjugates with, 267268 hydrolysis of polysaccharides with, monosaccharide recoveries after, 261-263 pH of aqueous solutions of, 252 Trimethylacetic acid, cyclodextrin inclusion complexes with, 221 2,3,5-Trimethylbenzoquinone,formation, 294 3,4,5-Trimethylphenyl acetate, cyclodextrin inclusion complexes with, 223, 224
390
SUBJECT I N D E X
Triose-reductone, formation, 294 L-Tryptophan, cyclodextrin inclusion complexes with, 222, 224 Tubulosine, 13 Tumor-cell antigens, glycopeptides, liquid chromatography separation, 48 Tumor-cell glycoproteins, sialylated oligosaccharides, fractionation, 46 Tumor cells, oligosaccharides, liquid chromatography separation, 42-43 Turanose, liquid chromatography methods for analysis of, 33 L-Tyrosine, cyclodextrin inclusion complexes with, 222, 224
-, (4-O-methyl)-~-glucurono)-,model compounds related to, alkaline degradation, 306 Xylanases, mode of action, analysis of, 54 Xylans liquid chromatography analysis, 39 oligosaccharides, 305 wood, alkaline-degradation products of, 305 Xylitol, liquid chromatography methods for analysis of, 33 Xylobiose, liquid chromatography methods for analysis of, 33 D-Xylofuranose -, 3-C-(acetoxymethyl)-3-deoxy-3U fluoro-, 5-0-acetyl- 1,2-O-isopropylideneUmezawa, S., 9 a-,IH- and IgF-n.m.r. data for, Urethans, protection as, 192-194 142 Urinary glycoconjugates, hydrolysis, 262 -, 1,2,5-tri-O-acetyl-, 'H- and I9FUronic acid n.m.r. data for, 142 acid decomposition, 252 -, 3-O-benzyI-5-deoxy-5-fluoro-1,2-Oliquid chromatography isopropylidene-a-, 'H- and 19Fanalytical, 33-34 n.m.r. data for, 113 large-scale preparative, 62 -, 3-deoxy-3-fluoro- 1,2-O-isopropylipre-column derivatization procedures, dene-a68 -, 3-C-(ethoxyallyl)-, 'H- and I9Fmethanolysis of, 257-258 n.m.r. data for, 142 -, 3-C-(hydroxymethyl)-, 'H- and V I9F-n.m.r. data for, 142 -, 3,5-dideoxy-3,5-difluoro-l,2-0Virus glycoproteins isopropylidene-a-, IH-and lyFglycopeptides, liquid chromatography n.m.r. data for, 156 separation, 48 -, 1,2-O-isopropylidene-3-deoxy-3isolation of, on analytical-scale columns, fluoro-5-O-p-tolylsulfonyl-a60 'H- and lyF-n.m.r. data for, 112 D-Xylofuranoside W -, methyl 3-O-benzoyl-5-O-benzyl-2deoxy-2-fluoro-p-, 'H- and 19FWieland, H., 12 n.m.r. data for, 11 I Wolfrom, M.L., 4 -, methyl 2,5-di-O-benzoyl-3-deoxy-3Wood pulps, liquid chromatography analyfluoro-a-, 'H- and I9F-n.m.r. data sis, 52 for, 112 Woody materials, hydrolysis of, 263-264 D-Xylofuranosyl fluoride X -, 3,5-acetoxonium-2-0-methyl-p-, IHand 19F-n.m.r. data for, 109 Xanthates, photochemical cleavage of, 190 -, 3,5-benzoxonium-2-O-methyl-p-, 'HD-Xyh and I9F-n.m.r. data for, 109 -, ~-arabino-(4-O-methyl-~-glucurono)-, -, 3,5-di-O-benzoyl-2-O-methyl-, 'H306 and I9F-n.m.r. data for, 109
391
SUBJECT I N D E X
-, 2,3,5-tri-O-acetyl-, IH- and I9Fn.m.r. data for, 108 -, 2,3,5-tri-O-benzoyl-a-, I3C-n.rn.r. data for, 169 -, 2,3,5-tri-O-benzoyl-P-, IH- and I9Fn.m.r. data for, 109 Xyloisosaccharinic acid, 305 Xylo-oligosaccharides, preparative liquid chromatography, 60 D-Xylopyranose, 1,3,4-tri-O-acetyl-2deoxy-2-fluoro-aI3C-n.m.r. data for, 167, 168 'H- and I9F-n.m.r. data for, 104 D-Xylopyranoside -, benzyl 3-deoxy-3-fluoro-P-, IH- and I9F-n.m.r. data for, 106 -, trifluoromethyl 3,4-di-O-acetyl-a-, IH- and I9F-n.m.r. data for, 105 2-O-~-Xylopyranosyl-~-arabinose, 305 D-Xylopyranosyl fluoride -, 2-deoxy-2-fluoro-, P anomer, 'H-and 19F-n.m.r. data for, 155 -, 3,4-di-O-acetyl-2-deoxy-, 2-bromo-, 'H- and I9F-n.m.r. data for, 136 -, 2-iodo-, IH- and 19F-n.m.r.data for, 137 -, 2,3,4-tri-O-acetyl-, IH- and I9Fn.m.r. data for, 102-103 -, 2,3,4-tri-O-benzoyI-, IH- and I9Fn.m.r. data for, 103
-, 3,4-di-O-acetyl-2-deoxy-2-fluoro-a-, 'H- and 19F-n.m.r.data for, 155
-, 2,4-di-O-acetyl-3-deoxy-3-fluoro-p-,
'H-and I9F-n.m.r. data for, 155 -, 3,4-di-O-benzoyl-2-O-methyl-, 'Hand 19F-n.m.r. data for, 103 -, 2,3,4-tri-O-acetyla anomer, I3C-n.m.r. data for, 167 p anomer, l3C-n.m.r. data for, 167 Xylose, liquid chromatography, 33, 52-53 D-Xylose formation of a- and P-xylometasaccharinic acids from, in base, 281282 formation of 2-furaldehyde from, 275276 formation of saccharinic acids from, 281-282 high-temperature transformation acidic conditions, 276-277 basic conditions, 281-283 in plant cell-wall hydrolyzates, liquid chromatography analysis, 55 Y Yajima, T., 6 Yamada, Yukari, 3 Yamakawa, T., 9 Yoshimura, Juji, 1, 9 Yoshizawa, Z., 9
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