Advances in Carbohydrate Chemistry and Biochemistry Volume 45
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Advances in Carbohydrate Chemistry
and Biochemistry Editors
R. STUART TIPSON DEREK HORTON
Board of Advisors LAURENS ANDERSON STEPHENJ. ANGYAL HANSH. BAER CLINTON E. BALLOU JOHNs. BRIMACOMBE
GUYG. S. DUTTON BENGTLINDBERG HANSPAULSEN NATHAN SHARON ROY L. WHISTLER
Volume 45
ACADEMIC PRESS, INC. Harcourl Brace jovanovich, Publishers
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CONTENTS .............................................
PREFACE .
vii
Burckhardt Helferich. 1887-1982 HERMANN STETTER
..................................
Text . . . . . . . . . . .
1
Francisco Garcia Gonzhlez. 1902-1983 ANToNlO
G6MEZSANCHEZ A N D JOSE FERNANDEZBOLAAOS
Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
F.a.b.-Mass Spectrometry of Carbohydrates
ANNEDELL I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. F.a.b.-Mass Spectrometry: Theory and Practice . . . . . . . . . . . . . . . . . . . . . 111. F.a.b.-M.s. of High-Molecular-Weight Samples . . . . . . . . . . . . . . . . . . . . . . IV . Interpretation of F.a. b.-Mass Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Structure Assignment by F.a.b.-M.s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Future Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20 24 34 41 45 54 71
The Circular Dichroism of Carbohydrates
W . CURTISJOHNSON. JR. 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1. Measuring the Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill . Unsubstituted Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1v. Substituted Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
73 76 78 92
Proton Spin-Lattice Relaxation Rates in the Structural Analysis of Carbohydrate Molecules in Solution PHOTlS DAISA N D ARTHURs. PERLIN
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111. Spin-Lattice Relaxation Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Stereochemical Implications of Relaxation Rates . . . . . . . . . . . . . . . . . . . . . V . Limitations and Relative Merits of Relaxation Methods . . . . . . . . . . . . . . . .
125 128 138 147 163
CONTENTS
vi
"C-Nuclear Magnetic Resonance-Spectral Studies of Labeled Glycophorins
KILIANDILL 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . General Background Information about Glycophorins . . . . . . . . . . . . . . . . . 111. Labeling Studies of Glycophorin A by Way of the Reductive [ ' T I Methylation Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Labeling Studies of Glycophorin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Conclusions and Prognosis for Further Studies . . . . . . . . . . . . . . . . . . . . .
.
170 172 175 195 197
The Chemistry and Biochemistry of the Sweetness of Sugars
CHEAYGKUAILEE 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Stereochemistry of Sweetness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Bitterness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Biochemistry of Sweetness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Methodology of Measurement of Sweet Taste . . . . . . . . . . . . . . . . . . . . . . .
199 201 310
AUTHORINDEXF O R V O L U M E 4 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECT INDEX FOR VOLUME 45 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C1IMUI.ATIVE AUTHORINDEX FOR VOLUMES 41-45 . . . . . . . . . . . . . . . . . . . . . . . . . CUMUI.ATIVE SURJECT INDEX FOH VOLUMES 41-45 . . . . . . . . . . . . . . . . . . . . . . . . .
353 369 394 396
325 349
PREFACE This volume pays tribute to two carbohydrate pioneers. H. Stetter highlights the life and work of Burckhardt Helferich, one of the last of Emil Fischer’s protCgCs and a pioneer in protective-group strategy and glycosidic coupling. The scientific career of Francisco Garcia Gonztilez, presented by his students, A. G6mez-Stinchez and J. Ferntindez-Bolaiios (Seville) provides a sensitive account of the contributions of Spain’s leading carbohydrate chemist whose work emphasized especially the reactions of monosaccharides that generate aromatic heterocycles. In line with the policy of Advances to provide periodic coverage of major developments in physical methodology for the study of carbohydrates, A. Dell (London) here surveys the use of fast-atom-bombardment mass spectrometry in application to carbohydrates. This technique has achieved rapid prominence as the “soft” ionization technique of choice for structural investigation of complex carbohydrate sequences in biological samples. The author’s extensive personal involvement in this field makes her chapter a critical, state-of-the-art overview for the specialist, as well as a valuable primer for the reader unfamiliar with this technique. In contrast to the rapid rise of f.a.b.-mass spectrometry, the use of circular dichroism in the carbohydrate field, here surveyed by W. Curtis Johnson, Jr., (Corvallis, Oregon), has evolved more slowly and been less widely appreciated. Instrumental limitations have hampered the routine use of circular dichroism; most sugars are transparent in the 1000-190-nm wavelength region of c.d. instruments, and the technique has thus invited less use for them than for nucleic acids and proteins, which show strong absorption in this region. However, as Johnson points out, c.d. is a potentially poweful tool for investigation of the stereochemistry of monosaccharides, of intersaccharide linkages, and most importantly, of the secondary structure of polysaccharides. Many valuable applications of this technique evidently lie in the future. The significance of n.m.r. spectroscopy for structural elucidation of carbohydrates can scarcely be underestimated, and the field has become vast with ramifications of specialized techniques. Although chemical shifts and spin couplings of individual nuclei constitute the primary data for most n.m.r.-spectral analyses, other n.m.r. parameters may provide important additional data. I? Dais and A. S. Perlin (Montreal) here discuss the measurement of proton spinlattice relaxation rates. The authors present the basic theory concerning spin-lattice relaxation, explain how reliable data may be determined, and demonstrate how these rates can be correlated with stereospecific dependencies, especially regarding the estimation of interproton distances and the implications of these values in the interpretation of sugar conformations. Specific applications of carbon-13 n.m.r. spectroscopy to the glycophorins, an important family of glycoproteins present in the human erythrocyte membrane, are discussed by K. Dill (Clemson), who demonstrates the value of ”C-n.m.r. spectra for the structural mapping of glycoproteins. vii
viii
PREFACE
Finally, C. K. Lee (Singapore) contributes an extensive article surveying the chemistry and biochemistry of the phenomenon of sweet taste. The sweetness of sugars has been of interest since ancient times, but even now a total understanding of this gustatory response remains elusive. The editors note with regret the passing of Laszl6 Mester on February 23, 1986. Mester worked extensively on hydrazine derivatives of sugars, and he contributed a notable article on the formazan reaction in Volume 13 of this series. Kensington, Maryland Columbus, Ohio August, 1987
R . STUARTTIPSON DEREKHORTON
Advances in Carbohydrate Chemistry and Biochemistry Volume 45
1887-1982
1902-1983
BURCKHARDT HELFERICH
1887-1982
On July 5, 1982, Burckhardt Helferich died in Bonn shortly after his 95th birthday. With his death, we have lost one of the last protagonists of the classical era of carbohydrate chemistry. The son of a Professor of Surgery at the University of Greifswald, Privy Councillor Heinrich Helferich, and his wife, Natalie, Burckhardt Helferich was born on June 10, 1887, in Greifswald. He went to the classical “Gymnasium,” first in Greifswald, and, from 1899, in G e l , where he obtained his “Abitur” leaving-certificate in 1906. He then began the study of geology at the University of Lausanne, being interrupted after only one semester by his call-up for military service. In the autumn of 1907, he started to study chemistry at the Institute chaired by Adolf von Baeyer at the University of Munich, where he passed the first “Verband’s” examination after three semesters. In 1909, he continued his studies at the University of Berlin, gaining his doctorate in 1911 under Emil Fischer. The subject of his doctoral dissertation was “Syntheses. of Certain New Glucosides.” The tremendous personality of Emil Fischer left its mark on Helferich for the rest of his life. In conversation with Helferich, one was often aware of the great veneration he always felt for his tutor and mentor. After his doctoral graduation, Helferich became Emil Fischer’s personal assistant for two years, and, from 1913 onwards, a teaching assistant. Together with Fischer, he published a series of papers on glycoside synthesis during this period. His scientific career was then interrupted by the First World War, in which he served as an officer throughout. In 1919, he resumed his position as assistant in the Berlin Institute and, in 1920, obtained his “Habilitation” with a thesis on the ring-chain tautomerism of y- and 6-hydroxyaldehydes. In 1922, Helferich was called to the position of Departmental Head at the Kaiser Wilhelm Institute for Fibre Chemistry in Berlin-Dahlem. However, he never actually occupied this position, for, in the autumn of that year, he accepted a personal chair in organic chemistry at the University of Frankfurt in the Institute headed by Julius von Braun. 1
Copyright 0 1987 by Academic Ress, Inc. All rights of reproduction in any form reserved.
2
HERMANN STEITER
In 1922, Helferich married Hildegard Kohlrautz. Five children were born of this very happy and harmonious marriage. In the spring of 1925, Helferich accepted the offer of the Chair in Chemistry and the Directorship of the Chemical Institute at the University of Greifswald, becoming the successor to Pummerer. In the summer semester of 1930, he accepted the offer of the Chairmanship of the Chemical Institute at Leipzig as the successor to Hantzsch. The outbreak of the Second World War in 1939 made work at Leipzig extremely difficult. Many students and postgraduates were called up for military service. In the further course of the war, the Institute suffered much damage from bombing. In spite of all these difficulties, Helferich attempted to maintain the functions of the Institute until, finally, in 1945, after 15 very successful years, he was evacuated to the Western Zone by the American forces of occupation. In 1945, Helferich began work as a guest professor at the University of Bonn, where, in 1947, he accepted the offer of the Chair of Chemistry and Directorship of the Bonn Chemical Institute as the successor to Paul Pfeiffer. Although the Bonn Institute had also suffered much war damage, Helferich, with his energy and enthusiasm, was able, within a short space of time, to create the necessary conditions for a resumption of teaching and research. A great personal tragedy for Helferich was the loss of his only son in the war, to be followed by the premature death of his beloved wife. During the course of his academic career, Helferich occupied many important offices. In the academic year 1951-1952, he was Dean of the Faculty of Mathematics and Natural Sciences, and, in the academic year 1954- 1955, Vice-Chancellor of the Rhenish Friedrich Wilhelm University of Bonn. From 1953-1955, he was a member of the council of the Gesellschaft Deutscher Chemiker, and was president of this society from 1956 to 1957. He made a major contribution to the rapid restoration of the scientific and personal links with foreign chemists that had been broken by the war. In 1951, he received the Emil Fischer Medal of the Gesellschaft Deutscher Chemiker, and in 1957, the “Grosse Verdienstkreuz” (Grand Service Cross) of the Federal Republic of Germany. The Technische Hochschule in Stuttgart conferred upon him the honorary doctorate of Dr. ing. h.c. The Saxon Academy of Science and the Leopoldina in Halle, East Germany, elected him an honorary member. His scientific work, which found expression in 328 publications and 16 patents, is characterized by originality and a comprehensive command of experimental method. In his first independent work, Helferich prepared y-hydroxyvaleraldehyde (4-hydroxypentanal) by reduction and ozonolysis of methylheptenone, readily available from citral by a retro-aldol reaction. He was able to show that, similarly to the saccharides, this hydroxyaldehyde exists in
OBITUARY-BURCKHARDT
HELFERICH
3
the cyclic-hemiacetal form. He also observed the same behavior with Shydroxyaldehydes. From these experimental results, Helferich drew a most important conclusion for that period, namely, that, in cyclic-hemiacetal formation by saccharides, the pyranose form must also be considered, as well as the furanose form. Up to that time, chemists had assumed that the cyclic forms of the saccharides existed exclusively in the 1,Cring (furanose) form. Some time later, Haworth and others proved that the majority of carbohydrates adopt the pyranose forms. The results on cyclic-hemiacetal formation by hydroxyaldehydes were communicated in eight papers.' Formation of a cyclic hemiaminal was observed with the corresponding acetamidoalde hydes.* Helferich's work on ethers of triphenylmethanol began as early as his time in Berlin.3 During the Frankfurt period, Helferich showed that use of the triphenylmethyl (trityl) group permits the specific etherification of terminal hydroxyl groups. As cleavage of the trityl group is achieved under mild conditions, this has become one of the most important and specific protecting groups in carbohydrate c h e m i ~ t r yHelferich .~ also extended the use of the trityl protecting group to hydroxy carboxylic acids and amino acids.' This protecting group still plays a major role in peptide synthesis. The use of the trityl protecting group permitted Helferich to carry out the first targeted synthesis of a disaccharide glycoside6 and, finally, that of a free disaccharide, namely, gentiobiose. This work, coauthored with Karl Bauerlein and Friedrich Wiegand,7 was published in 1926, and is still regarded as one of the milestones in carbohydrate chemistry. Originally, the trityl group was removed by hydrogen chloride in methanol. As the was acetyl group at 0 - 1 of the 1,2,3,4-tetra-0-acetyl-6-0-trityl-~-glucose also cleaved under these conditions, an alternative route was chosen, using 2,3,4-tri-O-benzoyl-6-0-trityl-~-glucosyl fluoride. Subsequently, they were able to remove the trityl group very gently by treatment with hydrogen bromide in glacial acetic acid at 0". The route was then open to synthesize (1) B. Helferich, Ber., 52 (1919) 1123-1131, 1800-1812; B. Helferich and 0. Lecher, ibid.,
(2) (3) (4) (5) (6) (7)
54 (1921) 930-935; B. Helferich and M. Gehrke, ibid., 54 (1921) 2640-2647; B. Helferich and T. Malkornes, ibid., 55 (1922) 702-708; B. Helferich and H. Koster, ibid., 56 (1923) 2088-2094; B. Helferich and W. Schafer, ibid., 57 (1924) 1911-1917; B. Helferich and F. A. Fries, ibid., 58 (1925) 1246-1251. B. Helferich and W. Donner, Ber., 53 (1920) 2004-2017. B. Helferich, P. E. Speidel, and W. Toeldte, Ber., 56 (1923) 766-770; B. Helferich, Angew. Chem., 41 (1928) 871-875. B. Helferich and H. Koster, Ber., 57 (1924) 587-591. B. Helferich, L. Moog, and A. Jiinger, Ber., 58 (1925) 872-886. B. Helferich, and J. Becker, Justus Liebigs Ann. Chem., 440 (1924) 1-18. B. Helferich, K. Bauerlein, and F. Wiegand, Justus Liebigs Ann. Chem., 447 (1926) 27-37.
4
HERMANN STETTER
1,2,3,4-tetra-0-acetyl- glucose from 1,2,3 $tetra- 0-acetyl-6- 0-trityl-Dbromide led glucose. Coupling with tetra-0-acetyl-a-D-glucopyranosyl directly to octa-0-acetylgentiobiose. In the following period, numerous diand tri-saccharides were assembled according to the same principle.* During his time at Leipzig, Helferich developed a new procedure for the preparation of phenyl glycosides. This involved the reaction of the peracetate of a reducing sugar with a phenol in the presence of zinc chloride or p-toluenesulfonic acid.' By careful choice of reaction conditions, it is possible to influence the ratio of a and /3 anomers of the corresponding glycosides. The development of a synthesis of ascorbic acid, which was exploited industrially for a time, also occurred during this period." The application of esters of methanesulfonic acid in carbohydrate chemistry were also investigated. A series of significant advantages emerged over the p-toluenesulfonates that had been introduced by Freudenberg." In contrast to the p-toluenesulfonates, it was found possible to introduce several methanesulfonyl groups very readily into a sugar molecule. Numerous halogeno-carbohydrates were accessible by exchange reactions involving the use of methanesulfonates and trityl ethers.I2 The first free fluoro-carbohydrate was also prepared on this basis.I3 The work on the halogeno-carbohydrates also led to the d i s c ~ v e r yof ' ~the so-called "glycoseenes" (1,5-anhydroald-l-enitols). During his Berlin period, Helferich had already begun to examine the glycoside-cleaving enzymes, the glycosidases, and this work was intensified at Leipzig. Particular attention was paid to the emulsin of sweet almonds. Separation of the P-glucosidase from the enzyme mixture was achieved," but this emulsin turned out to be a most complex mixture of enzymes.I6 Much new information was gained about the specificity of the glycosidases, and glycosidases from other sources were also studied."
(8) e.g., B. Helferich and W. Schafer, Jusrus Liebigs Ann. Chem., 450 (1926) 229-236; B. Helferich and H. Rauch, Ber., 59 (1926) 2655-2657; Jusrus Liebigs Ann. Chem., 455 (1927) 168-172. (9) B. Helferich and E. Schmitz-Hillebrecht, Ber., 66 (1933) 378-383. (10) B. Helferich and 0. Peters, Ber., 70 (1937) 465-468; Ger. Pat. 637,448 (29, 10, 1936); Chem. Absrr., 31 (1937) 709'; Br. Pat. 2,068,453 (19,1,1937); Chem. Abstr., 31 (1937) 18259. (11) B. Helferich and A. Gnuchtel, Ber., 71 (1938) 712-718. (12) B. Helferich and M. Vock, Ber., 74 (1941) 1807-1811. (13) B. Helferich and A. Gnuchtel, Ber., 74 (1941) 1035-1039. (14) B. Helferich and E. Himmen, Ber., 61 (1928) 1825-1835. (15) B. Helferich and 0. Lang, J. Prakt. Chem., 132 (1932) 321-334. (16) B. Helferich, Ergeb. Enzymforsch., 7 (1938) 83-104. (17) B. Helfereich, W. Klein, and W. Schafer, Ber., 59 (1926) 79-85; B. Helferich and J. Goerdeler, Ber. Verh. Saechs. Akad. Wiss.Leipzig Math. Phys. K l . , 92 (1940) 75-106.
OBITUARY-BURCKHARDT
HELFERICH
5
During the Bonn period, the work on glycoside and oligosaccharide syntheses was further developed. Replacement of silver oxide by mercuric cyanide in nitromethane constituted a major step forward in the reaction of acetohalogeno-carbohydrates." In this way, it was possible to obtain mainly the a-glycosides. Remarkable progress was also made in the synthesis of N-glycosyl compounds. For example, penta- 0-acetyl-D-glucose was observed to furnish with benzylamine an adduct that was converted into 2,3,4,6-tetra-O-acetyl-~-glucose on exposure to acid. At higher temperatures, the adduct formed tetra-0-acetyl-N-benzyl-D-glucosylamine, from which tetra-0-acetyl-D-glucosylamine was obtained upon catalytic hydrogenoly~is.'~ In the case of 1-amino-1-phenylethane, the reaction also makes possible the separation of racemates as only the dextrorotatory form furnishes a well crystallizing adduct.20 A specific protecting group was discovered for the 1,2-hydroxyl groups in carbohydrates. The protecting group is prepared by cycloaddition of phenanthrenequinone to a glycal, to afford a dioxene. Disaccharide syntheses were carried out with the aid of this protecting group, which is removed by ozonolysis.21 The first peptide syntheses were also initiated in Bonn.22In the later years of his research activity, Helferich concentrated particularly on the chemistry of the sulfonamides. The chemistry of the sultams was of particular interest to him.23 As a spin-off from this work he developed a psychotropic drug, namely, Ospolot. His work on enzymes was continued during the Bonn period. Studies on acid phosphatases were carried out that made a major contribution to our knowledge of these enzymes.24The isolation of a crystalline P-glucosidase from the emulsin of sweet almonds may be regarded as the crowning achievement of his work on glyco~idases.~~ It is not possible to give here a complete appreciation of his research results, impressive as they are in their abundance and scope. In many (18) B. Helferich and K. Wedemeyer, Justus Liebigs Ann. Chem., 563 (1948) 139-145; Chem. Ber., 83 (1950) 538-540. (19) B. Helferich and W. Portz, Chem. Ber., 86 (1953) 604-612. (20) B. Helferich and W. Portz, Chem. Ber., 86 (1953) 1034-1035. (21) B. Helferich and E. von Gross, Chem. Ber., 85 (1952) 531-535. (22) B. Helferich, P. Schellenberg, and J. Ullrich, Chem. Ber., 90 (1957) 700-71 1; B. Helferich and H. Boshagen, ibid., 92 (1959) 2813-2827. (23) B. Yelferich and K. G . Kleb, Justus Liebigs Ann. Chem., 635 (1960) 91-96; B. Helferich, R. Dhein, K. Geist, H. Jiinger, and D. Wiehle, ibid., 646 (1961) 32-44; 45-48. (24) B. Helferich and H. Stetter, Jusrus Liebigs Ann. Chem., 558 (1947) 234-241; 560 (1948) 191-200; Naturwissenschaften, 34 (1947) 278-279. (25) B. Helferich and T. Kleinschmidt, Hoppe-Seyler's 2.Physiol. Chem., 348 (1967) 753-758.
6
HERMANN STETTER
instances, they went far beyond the realms of carbohydrate chemistry, as is shown by a series of papers on inorganic topics.26 Helferich was active in research to almost the very end of his life. His love for experimentation and his enthusiasm for research were the driving forces for his extremely successful scientific work. He also had a supreme gift for infecting his students with his own enthusiasm for chemistry. His lectures, with their masterly experimental demonstrations, were some of the most popular in chemistry. His great personal interest in research was also demonstrated by his twice daily visits to the laboratory benches of his Diploma and Ph.D. students. All who worked with him were impressed by his personality. The benefit of his advice and generous practical assistance was always available to his students and coworkers, and he was an example to them at all times. His students have always regarded it as a special distinction to be able to call themselves Helferich students. Helferich was tall and slender, with blue eyes and darkish-blond hair, and was a very sociable person. An enthusiastic participant in the festivities of the Institute together with his students and colleagues, he also enjoyed the Carnival celebrations, which play such a prominent role in Bonn. He was particularly fond of playing skittles (ninepins) with his Ph.D. students. With Helferich's death, we have lost a great scientist and teacher who had a substantial influence on developments in chemistry. His memory will remain fresh with all those who had the good fortune to be his students and colleagues. The literature references are a selection of his most important publications. A complete list of Helferich's 328 publications and 16 patents is to be found in Chem. Ber., 118 (1985) viii-xix. HERMANN STETTER
(26) e.g., B. Helferich and K. Lang, 2.Anorg. Allg. Chem., 263 (1950) 169-174.
FRANCISCO C A R C ~ AGONZALEZ
1902-1983
The death of Francisco Garcia Gonzilez, Emeritus Professor of Organic Chemistry at the University of Seville, Spain, deprived carbohydrate chemistry of a long-lived and enthusiastic researcher. Professor Garcia Gonzllez was a pioneer in carbohydrate research in Spain, and a leader for many years of an active school of research that has now spread to several universities and research centers in that country. Don Francisco, as he was affectionately called by his students and associates, was born on July 22, 1902, in Fuente Vaqueros, a small village situated in the middle of a rich table-land west of Granada. This is one of the most beautiful areas of Southern Spain, from which Don Francisco, in his boyhood, could see the white city of Granada dominated by the red walls and towers of the Arab palace of Alhambra, and behind, the impressive blue, snow-capped mountains of Sierra Nevada. He belonged to a close-knit family of prosperous farmers, and at one time, his father, Francisco Garcia Rodriguez, was secretary of the village council. His early years were spent in the midst of a large and lively group of relatives which included his great-uncle Baldomero Garcia, a bohemian folk-poet and flamenco-singer of local fame, and his first cousins Federico Garcia Lorca, who was to become of the greatest Spanish poets and playwrights of the present century, and Francisco Garcia Lorca, a future diplomat and Professor of Spanish Literature at Columbia University in New York City. This pleasant atmosphere made Don Francisco's childhood a happy one, and he often spoke of this period of his life and of his family with great affection and pride. Don Francisco's mother, Carmen Gonzilez, died young, and, to complete his primary education, he was sent away to Almeria to study with a private tutor, and, for his secondary education, he attended the State High School in Milaga. During these years, he looked forward to his periodic returns during the holidays to Fuente Vaqueros, where he worked in the fields, performing a variety of different farming chores, especially at harvest time. From these experiences, he developed a taste for farming, and, perhaps more important, an enduring and deep love for Nature. 7
Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
8
A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA~~OS
Don Francisco’s generation was the first in his family to receive a higher education. In 1920, he entered the University of Granada, where he graduated with degrees in Chemistry and Pharmacy in 1925. Upon graduation, he received a scholarship from the same University to do research in organic chemistry under the guidance of Professor Gonzalo Gallas. They studied the addition of hypochlorous acid to a,P-unsaturated ketones, and the displacement reactions of the resulting chlorhydrins with amines. This work was published in Anales de la Sociedad Espaiola de Fisica y Quimica. Granada had at that time a fairly rich cultural and artistic life, especially stimulated by the composer and musician Manuel de Falla, then living in the town, Federico Garcia Lorca, who had already shown signs of his genius, and some other young people of talent who were to become preeminent in Spanish culture. Don Francisco mixed with these circles and from these years, he acquired a genuine taste for music and literature. However, the inadequate research facilities prevailing at the University of Granada at the time convinced him to leave the city and, indeed, Spain. In the summer of 1927, he used his meager savings, earned by private tutoring, to travel to Germany as a tourist and to see what was going on in the field that we now call bio-organic chemistry, a blend of organic chemistry and biochemistry which was particularly appealing to him. After he had spent a short period in Dortmund, learning the rudiments of the German language, he moved to Berlin, where he visited the Chemical Institute of the University and persuaded Professor Heinz Ohle to take him as a student. The research effort to elucidate the mechanism of glycolysis and alcohol fermentation was at a peak in Germany at that time, and, in order to gain some insight into these processes, Ohle had undertaken a systematic investigation of the chemical oxidation of some 0-protected sugar esters. The study of these model systems had led him to suggest that the hexose undergoing the breakdown into two three-carbon compounds was a D-fructose phosphate. Crystalline D-fructose phosphates were needed in order to continue these quantitative oxidation experiments, and Garcia Gonztilez was given the task of studying the phosphorylation of 2,3:4,5-di-O-isopropylidene-P-~fructopyranose. He succeeded in obtaining crystalline tris(2,3:4,5-di-Oisopropylidene-P-D-fructopyranose-1-yl) phosphate, and, by hydrolysis of this triester with sodium hydroxide, the crystalline sodium salt of bis(2,3:4,5di-0-isopropylidene-P-D-fructopyranose-1-yl) phosphate. He also found that the permanganate oxidation of this salt takes place with concomitant, benzilic acid rearrangement, to yield (2-carboxy-2-deoxy-3,4- O-isopropylidene-L-threo-pentonic acid) 5-phosphate; this compound is unstable and is broken down in dilute acid solution into methylglyoxal, glycolic acid, and carbon dioxide.
OBITUARY-FRANCISCO G A R C ~ AGONZALEZ
9
In order to establish the role of the phosphoric ester function in the oxidative splitting of the sugar, he also studied the oxidations of other D-fructose derivatives having, at C-1, polar groups other than phosphate, and established the structures of the products. Likewise, he investigated the activating capacity of an ester group at C-3 of a hexose; for example, he 3found that the oxidation of 1,2-O-isopropylidene-a-~-glucofuranose sulfate gave (1,2-0-isopropylidene-a-D-xylofuranuronicacid) 3-sulfate. These studies were described in three articles coauthored with H. Ohle, published in 1931 issues of the Berichte. During the time that Garcia Gonzilez spent in Ohle’s laboratory, he had a grant from the “Notgemeinschaft der Deutschen Wissenschaft,” and, subsequently, another one from the “Junta de Ampliaci6n de Estudios e Investigaciones Cientificas,” which was at that time the Spanish Government agency to promote scientific research. The latter grant was fairly generous, and, at the going rates of exchange, allowed him to concentrate on his work and to pursue a less ascetic living style. The four years spent in Berlin were decisive in Garcia Gonzilez’s career. His collaboration with Ohle, and the scientific atmosphere prevailing at the Chemical Institute, where such notables as E. Bergmann, H. Pringsheim, and H. 0. L. Fischer’ were still working, made him devoted to carbohydrate chemistry, and this remained his main field of research during the rest of his life. These years were also important from the personal point of view. The German capital was at that time a pole of attraction to many young scientists and artists, people he loved to mix with. He became a close friend of the Hungarian L k l 6 Vargha? then a student of Ohle’s, and shared a room for a period with Francisco Ayala, another young man from Granada, who later became a well-known novelist and taught Spanish Literature at Princeton University, U.S.A. The devaluation of the Spanish peseta in 1931 made him a pauper overnight, and in order to survive, he had to take odd jobs, such as acting as an extra in a play directed by the famous Max Reinhardt at the Deutsches Theater. He finally returned to Spain at the end of 1931. With the experimental results accumulated during his stay in Berlin, Garcia Gonzilez prepared two doctoral dissertations, entitled “New Crystalline Phosphoric Esters of D-Fructose” and “Tests on Some Assumed Phases of Alcoholic Fermentation,” which he presented in order to receive his doctorates in Chemistry and in Pharmacy, respectively, at the University of Madrid in 1932. Armed with these two degrees, he decided to pursue an academic career in his own country. His early training in a provincial (1) For an obituary, see Adu. Carbohydr. Chem., 17 (1962) 1-14. (2) For an obituary, see Adu. Carbohydr. Chem. Biochern., 28 (1973) 1-10.
10
A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA~OS
university, and his long stay abroad, had kept Garcia Gonzilez out of touch with the scientific life in Spain and, particularly, the influential academic circles of Madrid. Fortunately, his work on sugar phosphates and his strong personality impressed a distinguished organic chemist, A. Madinaveitia, who was Professor of Organic Chemistry of the Faculty of Pharmacy at the University of Madrid. Madinaveitia acted as patron of Garcia Gonzilez’s doctoral theses and, more important, gave him the opportunity to work at the National Institute of Physics and Chemistry, recently created in Madrid by the Junta de Ampliacidn de Estudios e Investigaciones Cientificas with the support of the Rockefeller Foundation. The new Institute was quite well equipped, and a select group of young people had gathered there to work under the direction of such internationally reputed scientists as Blas Cabrera (in spectroscopy and electromagnetism) and Enrique Moles (in physical chemistry). Madinaveitia, who was in charge of the Organic Chemistry Section, was mainly interested in the chemistry of natural products and of compounds of pharmaceutical interest, but, instead of integrating Garcia Gonzilez into his research team, he encouraged him to develop his own ideas. Two subjects were appealing to Garcia Gonzilez. The first was the synthesis of the newly discovered vitamin C, but after discussing the project with Madinaveitia, they decided not to investigate it, because it was “too competitive,” and it was known that some strong research teams abroad were working on it. The second subject was the interaction between sugars and ketonic compounds, responsible for the antiketogenic action of the former. Shaff er (1921) had suggested that D-glucose may be antiketogenenic in human metabolism, because it combines with acetoacetic acid, or other ketonic molecules, to form compounds that are more readily oxidized than the unchanged ketonic substances. To test this hypothesis, West ( 1927) caused D-glucose to react with ethyl acetoacetate, and obtained a beautifully crystalline compound that he tentatively formulated as a cyclopropene derivative. To explain the properties of this compound in acid solution, West considered that it was in equilibrium with a 2-C-(glycosyl)acetoacetic ester. Intrigued by these unusual formulations, Garcia Gonzilez set out to study the new compounds, and demonstrated that the condensate of D-glucose and ethyl acetoacetate has the furan structure 1. This compound and its parent acid /COICIHS HC-C
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H H H O C HOH2C-C-C-C/ ‘O’ HOOH H 1
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OBITUARY-FRANCISCO
G A R C ~ AGONZALEZ
11
readily undergo a dehydration reaction; he investigated the structure of the resulting products, and proposed a tentative formulation that he corrected in subsequent work. Garcia Gonzllez also obtained a compound, tentatively formulated as the pyrrole derivative isologous to the furan 1, by the reaction with ethyl acetoacetate. This compound had of 2-amino-2-deoxy-~-glucose been prepared, probably in an impure form, by H. Pauly and E. Ludwig (1922), who hypothesized that the natural pyrrole pigments could be formed in vivo by reaction of amino sugars with a physiological 1,3-dicarbonyl compound. This work was published in the Spanish Andes in 1934. Don Francisco spent two years at the National Institute of Physics and Chemistry, and during that time, he had a scholarship from the University of Madrid. As a consequence of his research achievements, Garcia Gonzllez was elected to the Chair of Organic Chemistry at the University of La Laguna in Tenerife (Canary Islands) at the end of 1934. He spent almost two years there, and he devoted most of this time to organizing a modest research laboratory. There he got his first collaborators (T. Quintero and R. Trujillo), and with their help, he pursued his researches. They obtained evidence of the pyrrole structure of the reaction product of 2-amino-2-deoxy-~-glucose with ethyl acetoacetate, and developed a simple procedure by which to prepare 0-isopropylidene derivatives of D-gluconic acid, starting from its calcium salt. In June of 1936, Don Francisco was appointed Professor of Chemistry of the Medical School of the University of Seville, in CBdiz. His return to Southern Spain, to which he had been looking forward, was intended to be followed by his marriage, planned for that summer, to Amalia Olmedo, a pretty, young school-teacher and musician from Granada, but suddenly, the happy panorama changed. Social and political unrest had been growing in Spain during those months, and in July, the Spanish Civil War broke out, bringing tragedy and ruin to the country and to many families. Academic life stopped, and Don Francisco was conscripted as a pharmacist by the Nationalist Army. His already famous cousin Federico Garcia Lorca and other members of the family were, under the most dramatic circumstances, killed in Granada in the first days of the war; others had to rush into exile, and a period of silent mourning and'sorrow followed. This was somehow relieved by his wedding to Dofia Amalia, which at last could be celebrated in May of 1937. This marriage constituted the beginning of a life-long partnership, marked by the deepest mutual devotion and understanding. In the ensuing years, two sons were born to the Garcia Gonzllezes: Francisco (in 1938, who graduated as a chemist and biochemist, and is Professor of Biochemistry at the Technical University of Madrid), and Bernard0 (in 1940, who became a physicist, and teaches as Professor of Physics at the University of Granada).
12
A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA$JOS
With the end of the war, in 1939, the university activities started again, but new difficulties were ahead. Although never involved in politics, Don Francisco had inherited from his family a liberal and open-minded approach to life and society which was out of tune with that prevailing in the official Spain of those days. This attitude and his detachment from the new regime made him suspected of disloyalty, and provoked an official investigation of his possible political activities and connections. This lasted for two years, after which time a resolution from the judge in charge of the investigation cleared him of all charges. His transfer, in 1943, to the Department of Organic Chemistry of the University of Seville, marked the beginning of a quiet and most fruitful period in his career. The university laboratories in Seville were rudimentarily equipped, but this was compensated for by the dedication and enthusiasm of the small group of students that gathered around him. A new research body, the “Consejo Superior de Investigaciones Cientificas,” was created after the war by the new government, and this organization included an Institute of Chemistry (the “Instituto Alonso Barba”) which had sections in different universities. Garcia Gonzilez headed the Organic Chemistry Section of the University of Seville, and this situation enabled him to pursue his studies more systematically, with some degree of financial support. Several lines of research were developed by Garcia Gonzalez and his collaborators in Seville, all of them dealing with the formation of heterocyclic compounds from monosaccharides. One of these lines was the extension of the reaction of ethyl acetoacetate with D-glucose to other 1,3-dicarbonyl compounds, and to other aldoses (pentoses, hexoses, and heptoses) and to ketoses (D-fructose and D-sorbose). The reaction, originally performed by heating the reactants with zinc chloride, or other metallic salts, in a nonaqueous medium, was also shown to occur under “physiological conditions,” that is, in the absence of catalysts, in water at room temperature and neutral pH. A variety of (alditol-1-yl)furans, 2 and 3, having alditolyl 0
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chains of different lengths and configurations, were thus obtained, and their structures were established. Oxidation with the then-new reagents lead tetraacetate or periodic acid gave a series of furanaldehydes, which were subsequently used as starting materials for the synthesis of furylalanines,
OBITUARY-FRANCISCO G A R C ~ AGONZALEZ
13
furylpyruvic and furylthiopyruvic acids, and other furan derivatives. Such simple a-hydroxyoxo compounds as D-glyceraldehyde, glycolaldehyde, and 1-hydroxy-2-propanone were shown to react with 1,3-dicarbonyl compounds in the same way, yielding furan derivatives. This is, therefore, a general reaction of a-hydroxyaldehydes and a-hydroxyketones, including the monosaccharides, often referred to as the “Garcia Gonzilez reaction,” which provides one of the simplest procedures for the formation of the furan ring. Efforts were made by Garcia GonzAlez and his coworkers to elucidate the mechanism of this reaction. In one of the working hypotheses, it was considered that the aldehydo form of the sugar and the 1,3-dicarbonyl compound undergo an aldol reaction to yield a 2-C-(alditol-l-yl)-l,3-dicarbony1 compound, which is then dehydrated to form the furan. This hypothesis was supported by the isolation of the aldol-addition product of 2,3-0-isopropylidene-~-glyceraldehyde and ethyl acetoacetate. Acid hydrolysis of this compound set free the hydroxyl groups, with concomitant ring-closure to the anticipated furan. These studies, most of them carried out with the valuable collaboration of F. J. L6pez Aparicio, were reviewed in the article “Reaction of Monosaccharides with beta-Ketonic Esters and Related Substances,” published in this S e r i e ~ . ~ A second main subject of research by Garcia Gonzilez, mainly with the collaboration of A. G6mez-Sinchezywas the reaction of amino sugars with 1,3-dicarbonyl compounds. It was also found to be a general reaction of 2-amino-2-deoxyaldoses, 1-amino-1-deoxyketoses, and their N-monosubstituted derivatives, which produces the (alditol-1-y1)pyrroles 4 and 5, respectively. Using 1,3-~yclohexanediones,this reaction provides an easy 0
II
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route to 2- and 3-(alditol-l-yl)-4,5,6,7-tetrahydroindol-4-ones and other indoles, As with the non-nitrogenous monosaccharides, the reaction proceeds through an aldol reaction as the first step, but in this case, the isolation of N-(2-acylvinyl) derivatives of amino sugars, such as 6, which could be
(3) F. Garcia Gonzilez, Adu. Carbohydr. Chem., 11 (1956) 97-143:
14
A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA~IOS
CHZOH
HOQ
O
H
subsequently transformed into the pyrroles, suggests that the aldol reaction could also be an intramolecular process. Aldosyl- and ketosyl-amines, and the N-monosubstituted derivatives, also react with 1,3-dicarbonyl compounds, to yield the pyrroles 5 and 4, respectively; these products are probably formed through p-( N-glycosylamino) a,p-unsaturated esters or ketones, which, in several instances, were isolated. These studies were reviewed in a second article, “Reactions of Amino Sugars with P-Dicarbonyl Compounds,” coauthored with A. G6mez-SlnchezYpublished in this Series? Many (alditol-1-yl) heterocyclic compounds are dehydrated under very mild conditions. This reaction, first observed by West (1927) in the furoic ester 1, was studied in detail by Garcia Gonzllez and his collaborators. The dehydration product of 1 and of its parent acid, were shown (F. Garcia GonzQlezand C. Sequeiros, 1945; F. Garcia Gonzllez, J. L6pez Aparicio, and A. Vlzquez Roncero, 1948) to be 2-C-erythrofuranosylfurans,whose p-Danomeric configuration and formation mechanism were subsequently established (A. G6mez-Slnchez and A. Rodriguez RoldQn, 1972). The reaction was extended to 2- and 3-(alditol-l-yl)pyrroles (4 and 5), the dehydration of the compounds having a pentitolyl chain giving rise to mixtures of C-glyco-furanosyland -pyranosyl derivatives of the heterocycle. On the other hand, other (alditol-1-yl) heterocyclic compounds are more reluctant to undergo the dehydration reaction. For example, 2-( D-urubinotetritol-1-y1)quinoxalineis dehydrated only by the action of strong acid, producing 2-(2-furyl)quinoxaline and 2-(3-hydroxy-2-furyl)quinoxaline (A. G6mez Sinchez, M. Yruela, and F. Garcia Gonzllez, 1954). Another main subject of research of Garcia Gonzllez, with the collaboration of J. Fernlndez-Bolafios, was the reaction of amino sugars with isothiocyanic acid derivatives. 2-Amino-2-deoxy-~-glucose hydrochloride and potassium thiocyanate were shown to give rise to 4-(~-arubino-tetritol-l(4) F. Garcia Gonzilez and A. GBmez SBnchez, Ado. Carbohydr. Chem., 20 (1965) 303-355.
OBITUARY-FRANCISCO G A R C ~ AGONZALEZ
15
yl)-lH-imidazole-2-thiol (7), while the reaction of the amino sugar with alkyl and aryl isothiocyanates produces bicyclic compounds, first considered to be glucopyranoimidazolidine-2-thiones(195 l), but subsequently shown (C. J. Morel et al., 1968; F. Garcia Gonzllez, J. Fernlndez-Bolaiios, et al., 1974) to have structure 8. R’
I
HOCH
I
HCOH
I
HCOH
I
’S
CHiOH 7
8
The reaction of 1-amino-1-deoxyketoses, and their N-alkyl and N-aryl derivatives, with alkyl or aryl isothiocyanates (Huber et al., 1960) was studied in more detail, and new 4 4 alditol- 1-yl)-1-alkyl(aryl)-3-alkyl(ary1)1,3-dihydr0-2H-imidazole-2-thiones were obtained. These compounds were used as starting materials for the synthesis of DL-histidines, ~~-histidine-2thiol, and other imidazole derivatives of biological interest. The results obtained by Garcia Gonzllez and his coworkers attracted interest abroad, and it was a source of great satisfaction for him to receive, in 1953, an invitation from Professor M. L. Wolfrom to write the first of his articles in this Series. A further international recognition as a carbohydrate chemist ensued in 1965, when he was invited to serve as a member of the Editorial Advisory Board of the new international journal Carbohydrate Research. In 1975, he was asked to contribute with a lecture, subsequently p ~ b l i s h e d on , ~ “Synthesis of Polyhydroxyalkyl Heterocycles,” to a Symposium on New Synthetic Methods for Carbohydrates organized by the American Chemical Society to commemorate the 100th anniversary of the Society (New York, 1976). Although Don Francisco was rather reluctant to get involved in the formal activities of academies, he accepted membership in the Royal Academy of Sciences of Spain. He was not an outstanding speaker, but his straightforward manner and direct approach to the subject he was dealing with made (5) F. Garcia Gonzilez, J. Fernindez-Bolafios, and F. J. L6pez Aparicio, ACS Symp. Ser., 39 (1976) 207-226.
16
A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA~OS
his lectures most attractive. As a teacher, he was very conscientious, and conducted his classes in an informal and very relaxed manner which awakened responsiveness in the students. He was able to transmit to his graduate students his interest in the research problem at hand, and shared with them the delights of their experimental successes. His scientific stature made him highly respected; however, his excellent critical sense, which made him speak his mind regardless of the consequences, kept him out of the “corridors of power.” This did not help either his career or the development of his school. It was, therefore, very gratifying to him to see how some of his former students were pursuing research in carbohydrate chemistry in Seville, and in other universities and research centers in Spain, thus assuring the continuity of the task he had initiated. Don Francisco retired from the chair in 1972, but he stayed at the Department for six additional years as Emeritus Professor supervising research work. During his stay in Seville, he went back to farming as a hobby, as he acquired an orange grove on the outskirts of Seville, where he managed the efficient production of bitter oranges, sugar-beets, and potatoes. Although in a comfortable economic situation, he and his wife carried on a simple life-style: he never drove a car, very seldom took a taxi, and walked back and forth to the farm several times a week. He was fond of reading and of classical music and for a time was a member of the board of the Concert Society of Seville. Don Francisco went back to Granada to spend his final years. There, he still kept an interest in a fraction of his father’s land which he had inherited. In 1981, his health visibly declined, and cancer of the intestines was diagnosed. He had to suffer two major operations which were of no avail, and finally he accepted his fate with great fortitude and dignity. Don Francisco spent the following trying months surrounded by his closest relatives, receiving the most devoted care from his wife. He died on November 19, 1983. In recognition of the outstanding services rendered to the University of Seville by Professor Garcia Gonzhlez as an inspiring teacher and investigator, the Government Council of the University decided that the Department of Organic Chemistry that he headed for many years should bear his name. This decision was announced by the Rector of the University during the multitudinous homages paid to Don Francisco on the occasion of the first anniversary of his death, by the University of Seville, and by his former colleagues and students. Don Francisco was a man of many virtues. He was warm and affectionate, and was interested in everything happening around him. He had a quick and penetrating mind, and could perceive almost at first sight the intricacy of a problem or the quality of a person. He was extremely honest, both as
OBITUARY-FRANCISCO G A R C ~ AGONZALEZ
17
a man and as a scientist. It was impossible for all who had the privilege to work with him not to respect, appreciate, and admire him most deeply. ANTCNIOGOMEZ-SANCHEZ JOSE FERNANDEZ-BOLAIQOS*
In addition to those mentioned in the text, Professor Garcia Gonzilez coauthored articles with the following scientists: F. Alcudia Gonzilez, C. Alvirez Gonzilez, F. Ariza Toro, J. Bello GutiCrrez, R. Castro Brzezicki, R. Enriquez Berciano, J. FernPndez Jiminez, J. Fernhndez Garcia-Hierro, J. Fiestas Ros de Ursinos, J. Fuentes Mota, J. Galbis Phrez, J. Gasch G6mez, M. G6mez GuillCn, M. I. Goiii de Rey, A. Heredia Moreno, M. L6pez Artiguez, N. L6pez Partida, R. Maestro Durin, G. Martin Jiminez de la Plata, M. Martin Lomas, D. Martinez Ruiz, M. MenCndez Gallego, S. Muiioz Guerra, M. Ortiz Rizzo, A. Paneque Guerrero, A. M. PCrez de Guzman, M. A. Pradera de Fuentes, M. Repetto JimCnez, L. Rey Romero, J. Rodriguez Gonzhlez, I. Robina Ramfrez, E. Romin Galin, J. Ruiz Cruz, F. Sinchez LaulhC, M.Tena Aldave, and M. Trujillo PCrez-Lanzac.
* The authors express their gratitude to
Professor Francisco Garcia Olmedo, who kindly cooperated in the preparation of this tribute to his father.
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ADVANCES I N CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL. 45
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
BY ANNE DELL Department of Biochemistry. Imperial College of Science and Technology. London SW72A.7, U.K.
I . Introduction ............................. 1. Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . 2. Purpose of This Article ....................... I1. F.a.b.-Mass Spectrometry: Theory and Practice . . . . . . . . . . . . . . 1. Principles of the Technique ..................... 2 . Choice of Supporting Matrix and Matrix Additives . . . . . . . . . . . 3 . Characteristics of F.a.b.-Mass Spectra . . . . . . . . . . . . . . . . . 4 . Sample Purity and the Analysis of Mixtures .............. 5. Choice of Derivatives . . . . . . . . . . . . . . . . . . . . . . . . 6 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. F.a.b.-M.s. of High-Molecular-Weight Samples . . . . . . . . . . . . . . 1. Definition of High Mass . . . . . . . . . . . . . . . . . . . . . . . 2. Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Practical Aspects of Analysis at High Mass . . . . . . . . . . . . . . . 4 . F.a.b. "Mapping" of Permethylated, High-Mass Samples . . . . . . . . . IV. Interpretation of F.a.b.-Mass Spectra . . . . . . . . . . . . . . . . . . . 1. Molecular-Weight Assignment .................... 2. Fragmentation Pathways . . . . . . . . . . . . . . . . . . . . . . . V. Structure Assignment by F.a.b.-M.s. ................... 1. Analysis of Underivatized Samples .................. 2. Analysis of Derivatized Samples ................... 3. Monitoring Chemical and Enzymic Reactions by F.a.b.-M.s. ....... 4. Linkage Assignment . . . . . . . . . . . . . . . . . . . . . . . . . 5. Acyl-Group Location ........................ VI . Applications ............................. 1. Glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Glycoproteins ........................... 3. Bacterial Polysaccharides ...................... 4 . Plant Cell-Wall Polysaccharides . . . . . . . . . . . . . . . . . . . . 5. Cyclic Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . 6. Miscellaneous ........................... VII . Future Developments .........................
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Copyright @ 1987 by Academic Ress Inc. All rights of reproduction in any form reserved.
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ANNE DELL
I. INTRODUCTION 1. Historical Perspective In July 1980, at a Symposium on Soft Ionization Biological Mass Spectrometry held at Imperial College, London, the international mass-spectrometric community was first introduced to a new ionization technique for the analysis of involatile substances that was being developed by Barber and his colleagues at the University of Manchester Institute of Science and Technology.' Within a year, the new technique,* which was christened fast atom bombardment (f.a.b.), was revolutionizing mass-spectrometric studies of b i o p ~ l y m e r s . ~The - ~ reasons for this were simple: for the first time, it was possible to obtain long-lasting spectra of high quality from complex, polar molecules with relative ease; furthermore, the new f.a.b. sources were soon fitted to the high-field, mass spectrometers that had been developed some years earlier for high-mass work: and it was immediately apparent that the f.a.b.-high-field magnet combination would permit the analysis of much larger molecules than had previously been amenable to mass spectr~metry.~" Mass spectrometrists who had been using field desorption (f.d.)9 to define the molecular weights of complex carbohydrates and glycoconjugates were immediately attracted to the new technique." The first f.a.b. experiments' had shown that such small, underivatized oligosaccharides as raffinose gave spectra that were remarkably similar to f.d. spectra but which were much longer lasting. Experimentally, f.a.b. promised to be a much less demanding technique than f.d. The latter required the use of fragile wires, upon which R. S. Bordoli, and R. D. Sedgwick, in H. .R.Moms (Ed.), S o t Ionization Biological Mass Spectrometry, Heyden, London, 1981, pp. 137-152. M. Barber, R. S. Bordoli, R. D. Sedgwick, and A. N. Tyler, J. Chem. SOC., Chem. Commun.,(1981) 325-327. H. R. Moms, M. Panico, M. Barber, R. S. Bordoli, R. D. Sedgwick, and A. N. Tyler, Biochem. Biophys. Res. Commun., 101 (1981) 623-631. D. H. Williams, C. Bradley, G. Bojesen, S. Santikam, and L. C. E. Taylor, J. Am. Chem. SOC.,103 (1981) 5700-5704. H. R. Morns, A. Dell, A. T. Etienne, M. Judkins, R. A. McDowell, M. Panico, and G. W. Taylor, Pure Appl. Chem., 54 (1982) 267-279. K. L. Rinehart, Jr., L. A. Gaudioso, M. L. Moore, R. C. Pandey, J. C. Cook, Jr., M. Barber, R. D. Sedgwick, R. S. Bordoli, A. N. Tyler, and B. N. Green, J. Am. Chem.
(1) M. Barber, (2) (3) (4) (5)
(6)
(7) (8) (9) (10)
SOC.,103 (1981) 6517-6520. H. R. Moms, A. Dell, and R. A. McDowell, Biomed. Mass Specrrom., 8 (1981) 463-473. A. Dell and H. R. Moms, Biochem. Biophys. Res. Commun.,106 (1982) 1456-1462. H.-R. Schulten, In?. J. Mass Spectrom. Ion Phys., 32 (1979) 97-283. M. Linsheid, J. D'Angona, A. L. Burlingame, A. Dell, and C. E. Ballou, Proc. Natl. Acad. Sci. USA, 78 (1981) 1471-1475.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
21
samples were coated, and these had a habit of disintegrating in the high fields of the ion source just as the sample was ionizing. No such problems were encountered with f.a.b., because samples were ionized from a robust, metal surface. In the early stages of carbohydrate f.a.b.-m.s. comparative studies of f.d. and f.a.b. revealed that f.a.b. was the preferred technique.",'* A very striking demonstration of the superiority of f.a.b. was provided by data" obtained from a mycobacterial 0-methyl-D-glucose polysaccharide (MGP; l), the structure of which was being investigated by f.d.-m.s. at the time when f.a.b. was introduced. Some ten years earlier, Ballou and coworkers had proposed for MGP a structure that incorporated 18 glycosyl residues and a unit of glyceric acid.I3 Subsequent n.m.r.-spectral experiments had indicated that an additional glucosyl residue might be present, and it was considered possible that f.d. would resolve the problem by defining the molecular weight of MGP. Unfortunately, MGP had proved totally intractable to f.d.-m.s., and experiments on a smaller relative, namely, AGMGP, produced by enzymically removing the first four glycosyl residues from MGP, were equally unsuccessful. Weak f.d. spectra were eventually obtained from a sample of permethylated MGP. Clusters of ions in the region of m/z 4200 provided the first evidence for the presence of two, rather than one, additional glycosyl residues in MGP. For a short time, these results were very impressive, despite the somewhat scrappy nature of the mass-spectral traces, as no other biological compounds had previously given f.d. spectra above mass 4000.The f.d. data were soon, however, to be overshadowed by those afforded by the new f.a.b. technique. The very first time that MGP and AGMGP were introduced into the f.a.b. source of a high-field-magnet, mass spectrometer they afforded spectra of almost unbelievable quality.1','4~15No derivatization or special sample manipulations were necessary in order to produce spectra that were fully countable right u p to the molecular ions, which, for MGP, were present at rn/z3537 [M+Na]+ and 3553 [M+K]+ in the positive mode and at m / z 3513 [M - HI- in the negative mode. These data defined a molecular weight of 3514 for MGP, and showed that it did, indeed, contain 20 glycosyl residues and that one of the additional residues was methylated.
( 1 1 ) L. S. Forsberg, A. Dell, D. J. Walton, and C. E. Ballou, J. Bid. Chem., 257 (1982) 3555-3563. (12) Y. Kushi, S. Handa, H. Kambara, and K. Shizukuishi, J. Biochem. (Tokyo),94 (1983) 1841-1850. (13) C. E. Ballou, Fure Appl. Chem., 53 (1981) 107-112. (14) A. Dell and C. E. Ballou, Biomed. Mass Spectrom., 10 (1983) 50-56. (15) A. Dell and C. E. Ballou, Carbohydr. Rex, 120 (1983) 95-111.
CO,H CHIOH
I--
!
CHIOH
7
CH20Me7
CH,OMe
\
CHIOH
I
/ H
b
V OH
OCH CH,OH I
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
23
The analysis of MGP and AGMGP was a landmark in the development of f.a.b.-m.s. for the determination of carbohydrate structure. The study highlighted both the advantages and disadvantages of f.a.b.-m.s. and was a guide to the strategies most appropriate for fully exploiting the potential of the technique for the study of unknown compounds. On the positive side, the extraordinary quality of the data that could be obtained from such complex molecules encouraged the immediate application of f.a.b.-m.s. to a wide range of biological problems. Thus, there was no significant lag-time while such relatively trivial standard compounds as di- and tri-saccharides were being exhaustively analyzed. On the negative side, the fragment-ion data from both AGMGP and MGP carried a warning message. Superlicially, the results looked very good indeed; cleavages had obviously occurred at every glycosidic linkage, producing abundant “sequence ions” throughout the spectra. On more detailed analysis, however, it became apparent that these were not true sequence-ions, as many were the products of multiple cleavages and could theoretically have arisen from a variety of parent structures. It was clear that f.a.b. would not be a reliable procedure for sequencing unknown oligosaccharides, particularly those that were branched, unless the problem of ambiguous fragmentation-data could be resolved. In later Sections of this Chapter, current solutions to the problems of sequencing by f.a.b.-m.s. are described. As a general rule, unambiguous sequencing requires the study of derivatives. This is somewhat ironical, when it is considered that the major impetus for the original f.a.b. development was the provision of an ionization method that would not require that samples be derivatized. Derivatization does, however, have a lot to offer in the f.a.b.-m.s. of carbohydrates, as will be seen later. 2. Purpose of This Article
The main purposes of this article are to familiarize those carbohydrate chemists who are not specialists in mass spectrometry with the experimental aspects of f.a.b.-m.s., and to give an indication of the types of problems that can now be solved by using the technique. F.a.b.-m.s. currently plays two major roles in carbohydrate-structure analysis. These are ( i ) molecularweight determination, for which it has superseded f.d.-ms., and, to a large extent, chemical ionization,I6 and ( i i ) sequence assignment, where it has very largely replaced direct-probe, electron-impact mass Spectrometry, particularly for high-molecular-weight compounds. On its own, f.a.b.-m.s. cannot solve the complete structure of a carbohydrate, and it should always be incorporated into experimental programs (16) V. N. Reinhold and S. A. Cam, Mass Spectrom. Rev., 2 (1983) 153-221
24
ANNE DELL
that include the well established methods of methylation analysis, enzymic digestion, chemical degradation, and n.m.r. spectroscopy. Many of these procedures have been the subject of recent review^.'^-'^ 11. F.A.B.-MAss SPECTROMETRY: THEORYAND PRACTICE 1. Principles of the Technique
The main of f.a.b.-m.s. are shown schematically in Fig. 1. The hardware consists of ( i ) an atom gun (or ion gun, see later) which is either mounted on the source housing of the mass spectrometer or, if small enough, inside the housing on the source itself, ( i i ) a sample probe to the end of which is attached a small metal target onto which the sample is loaded, and (iii) suitable source-optics for the efficient extraction of ions into the analyzer of the mass spectrometer. In the f.a.b. experiment, an accelerated beam of atoms (or ions, see later) is fired from the gun towards the target, which has been preloaded with a viscous liquid (referred to as the matrix) containing the sample to be analyzed. When the atom beam collides with the matrix, kinetic energy is transferred to the surface molecules, many of which are sputtered out of the liquid into the high vacuum of the ion source. A significant number of these molecules are ionized during the sputtering process. Thus, gas-phase ions are generated without prior volatilization of the sample. The concept of ionization by means of sputtering processes was not new when Barber and his colleagues began their f.a.b. experiments. Secondaryion mass spectrometry (s.i.m.s.) was a well established technique for analyzing metal surfaces:' and it had also been used to produce spectra from solid organic samples coated on metal surfaces.22 In the s.i.m.s. experiment, beams of high-velocity ions effected ionization by sputtering the sample directly from the solid phase. The resulting mass spectra were weak and transient, and s.i.m.s. had never been used as a routine analytical tool. Barber and coworkers made radical changes to the s.i.m.s. technique. They replaced the ion gun with an atom gun, redesigned the ion source to (17) C. C. Sweeley and H. A. Nunez, Annu. Rev. Biochem., 54 (1985) 765-801. (18) J. F. G. Vliegenthart, L. Dorland, and H. van Halbeek, Adu. Carbohydr. Chem. Biochem., 41 (1983) 209-374. (19) K. Bock and C. Pedersen, Ado. Carbohydr. Chem. Biochem., 41 (1983) 27-66. (20) M.Barber, R. S. Bordoli, G. J. Elliott, D. Sedgwick, and A. N. Tyler, Anal. Chem., 54 (1982) 645A-657A. (21) A. F. Dillon, R. S. Lehrle, and J. C. Robb, Adu. Mass Spectrom., 4 (1968) 477-490. (22) A. Benninghoven, D. Jaspers, and W. Sichtemann, Appl. Phys., 11 (1976) 35-39.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
25
FIG.I.-Schematic Representation of a F.a.b. Source.
make it compatible with high-voltage, double-focusing mass spectrometers of the type used in biopolymer analysis, and finally, and most importantly, they decided to bombard a solution of the sample in glycerol, rather than the solid sample itself. The results were spectacular. F.a.b.-mass spectra lasted for minutes, and sometimes hours, in contrast to seconds in s.i.m.s. experiments. Initially, it was assumed that the use of neutrals rather than ions was an essential factor for producing high-quality spectra. It soon became clear, however, that the glycerol was the real key to the success of f.a.b. When s.i.m.s. experiments were performed with a liquid matrix, the results were identical to those obtained23by f.a.b. Herein, no distinction is therefore made between f.a.b. studies and so-called “liquid s.i.m.s.” experiments. Both positive and negative ions are produced during the sputtering process, and either can be recorded by an appropriate choice of instrumental parameters. Positive ions are the result of protonation, [M+ HI+,or cationization, [M + cation]+, whereas negative ions are preponderantly [M - HI-, but can also be formed by the addition of an anion, that is, [ M +anion]-. The type of pseudomolecular ion produced is governed by the chemical nature of the sample and by the composition of the matrix from which it is ionized. 2. Choice of Supporting Matrix and Matrix Additives
The correct choice of matrix is fundamental to successful f.a.b.-m.s., and the solubility of the sample in the matrix is a prime consideration. Glycerol (2) is the matrix most commonly used in f.a.b. experiments, and it is ideal (23) W. Aberth, K. M. Straub, and A. L. Burlingame, Anal. Chem., 54 (1982) 2029-2034.
ANNE DELL
26
for such polar compounds as underivatized carbohydrates and glycopeptides. It is not, however, a suitable matrix for samples that are very hydrophobic, for example, permethylated or peracetylated derivatives (the latter CH,OH
CH,SH
I CHOH I
CHOH
CH,OH
CHzOH
2
3
I I
give f.a.b. spectra from glycerol if the target is heatedz4but this is a procedure that we no longer recommend), or those that are inclined to form aggregates when dissolved in a polar liquid (for example, glycosphingolipids). For this type of compound, 1-thioglycerol (3) is an excellent matrix. 1-Thioglycerol is, unfortunately, considerably more volatile than glycerol and may evaporate completely before the spectra of high-molecular-weight samples have been fully recorded. If this occurs, more 1-thioglycerol may be added to the target to “revive” the sample, and the complete f.a.b. spectrum is then pieced together from several overlapping scans.z5A mixture of glycerol and 1-thioglycerol may be used in order to ensure that the sample does not dry out completely in the ion source.z6 In the author’s laboratory, it has never been found necessary to resort to matrices other than glycerol or 1-thioglycerol for the analysis of saccharides and glycoconjugates. Nevertheless, alternative matrices are often equally effective, and, in some laboratories, they are preferred. The most widely used include tetraethyleneglycol (4) and its higher-molecular-weight relatives, the poly(ethyleneglyc~l)s,~~~~~ and such basic matrices as N, N ’ bis(2-aminoethy1)ethylenediamine (“triethylenetetramine,” 5), 2,2’iminodiethanol (“diethanolamine,” 6), and 2,2’,2”-nitrilotriethanol (“triethanolamine,” 7) (basic matrices are frequently chosen for negative-ion HO-(CH,),O(CH,),O(CHZ)~~(CH,),-OH 4
H,N-(CH,),NH(CH,),NH(CH,),-NH, 5
HO-CH,-CH,-NH-CH,-CH,-OH 6
(24) A. Dell, H. R. M o m s , H. Egge, H. von Nicolai, and G. Strecker, Carbohydr. Res., 115 (1983) 41-52. (25) J. E. Oates, A. Dell, M. Fukuda, and M. N. Fukuda, Carbohydr. Res., 141 (1985) 149-152. (26) A. Dell, J. E. Oates, H. R. M o m s , and H. Egge, In?. 1. Mass Specrrom. Ion Phys., 46 (1983) 415-418. (27) M. E. Hemling, R. K. Yu, R. D. Sedgwick, and K. L. Rinehart, Jr., Biochemistry, 23 (1984) 5706-5713. (28) K . L. Rinehart, Jr., Science, 218 (1982) 254-260.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
27
f.a.b.).27,29Arita and coworkers3’ introduced a mixed matrix composed of triethanolamine, 1,1,3,3-tetramethylurea (8) and triethylenetetramine for analyzing glycosphingolipids in the negative-ion mode; this gives results similar to those obtained with l-thi~glycerol.~~ HO-CHz-CHz-N-CH,-CH,-OH
I CHZ I
CHZOH 7
H3C
0
II
/
/N-c-N H3C
\
\
CH,
CH,
8
The type of data produced in a f.a.b. experiment is affected by the pH and ionic strength of the matrix. The former may be controlled either by the addition of acids or bases, although, in practice, it is usually preferable to keep the matrix acidic. The ionic strength is partly dictated by the purity of the sample (many biological compounds are still contaminated with salts, even after extensive purification) and partly by exogenous additives. Three additives are especially useful for carbohydrate work. They are as follows. (i) Dilute aq. HCI. Addition of 1 pL of 100 or 200 m M HCl to the matrix improves the quality of the data obtained from certain types of carbohydrate. Both positive and negative studies may be assisted by acidification. In the positive-ion mode, sensitivity is enhanced if basic functional groups are present, for example, the amino group of glycopeptides. The low pH ensures that the sample is “pre-ionized,” and loss of sample by the sputtering of neutrals is minimized.20 Molecules containing several carboxyl groups, for example, gangliosides having three, or four, sialic acid residues3’ or oligogalactosiduronic give excellent, negative f.a.b. spectra when they are in an acidified matrix. The acid protonates the carboxyl groups, and discourages the formation of a plethora of molecular-ion species having various numbers of carboxylate salts. Fig. 2 shows the effect of acid-dosing on the spectrum of an oligogalactosiduronic acid. acetate. In their positive-f.a.b. studies of (ii) Sodium glyc~sphingolipids,~’~~~~~~ Egge’s group routinely add a 0.1% solution of sodium acetate in methanol to the target prior to the addition of the matrix. (29) K. Harada, M. Suzuki, and H. Kambara, Org. Mass Spectrom., 17 (1982) 386-391. (30) M. Arita, M. Iwamori, T. Higuchi, and Y. Nagai, J. Biochem. (Tokyo),93 (1983) 319-322. (31) H. Egge, J. Peter-Katalinic, G . Reuter, R. Schauer, R. Ghidoni, S. Sonnino, and G . Tettamanti, Chem. Phys. Lipids, 37 (1985) 127-141. (32) E. A. Nothnagel, M. McNeil, P. Albersheim, and A. Dell, Plant Physiol., 71 (1983) 916-926. (33) K. R. Davis, A. G . Darvill, P. Albersheim, and A. Dell, Plunt Physiol., 80 (1986) 568-577. (34) H. Egge, J. Dabrowski, and P. Hanfland, Pure Appl. Chem., 56 (1984) 807-819. (35) H. Egge and J. Peter-Katalinic, in A. L. Burlingame (Ed.), Symp. Muss Spectrom., Health and Life Sci., in press.
ANNE DELL
28
IM +3Na-&H 1[M+ZNa-3HIIM+Na-2HI-
IM+&Na-SHl-
[M-HI-
IM+SNa-6HI[M+6Na-7HI‘
A IM-HI-
IM+Na-2Hl-
FIG. 2.-Molecular-ion Regions of the Negative F.a.b. Mass Spectra of AGalA-GalA-GalAGalA-GalA-GalA-GalA. [The upper trace was obtained after loading 1 FL of a solution of the sample in 5 % aq. acetic acid (5 pg/p,L) into glycerol. The lower trace was recorded after the further addition of 1 pL of 100 m M aq. HCI. (AGalA is a dehydrated galacturonic acid residue formed when a lyase cleaves a galacturonic acid polymer; GalA is a galacturonic acid residue.)]
Consequently, the only pseudomolecular ions produced are [M + Na]+ species. These are usually abundant, permitting the o b ~ e r v a t i o nof ~~ molecular ions up to at least 6000. (iii) Ammonium thiocyanate. Dosing with an ammonium salt gives abundant [M + NH,]+ ions in the spectra of some types of permethylated oligosaccharides. Curiously, there is very little effect on the spectra of molecules containing amino sugars, but, for h e ~ o s e ~polymers ~ . ’ ~ and plant cell-wall oligosa~charides,~~ the improvements in sensitivity are dramatic. Some ammonium ions are always present in commercial 1-thioglycerol, but optimal results require a supplement of 1 p L of a 100 mM solution of an ammonium salt. Various salts may be used, but ammonium thiocyanate permits the recording of good negative-ion spectra containing intense signals for [M + SCNI-, and its chaotropic properties probably assist desorption. 3. Characteristics of F.a.b.-Mass Spectra All f.a.b. spectra are characterized by ( a ) abundant, pseudomolecular ions for both the sample and the matrix, and (b) a relatively high level of chemical “noise,” resulting in a signal at every mass number up to the (36) A. Dell, W. S. York, M. McNeil, A. G. Darvill, and P. Albersheim, Carbohydr. Res., 117 (1983) 185-200. (37) L. D. Melton, M. McNeil, A. G. Darvill, P.Albersheim, and A. Dell, Carbohydr. Rex, 146 (1986) 279-305.
F.A.9.-MASS SPECTROMETRY OF CARBOHYDRATES
29
molecular-ion region. The background ions are derived from both the matrix and the sample, and are probably formed from surface molecules that have disintegrated after receiving a direct hit from an accelerated atom; they permit the manual counting of spectra up to m/z -4000, and their appearance is a good guide as to how well the recording is proceeding. If the background signals at m/z > 1000 are weak or nonexistent, it is very unlikely that good molecular-ion signals will be present at high mass. In addition to pseudomolecular i m s and background ions, two other types of signal may be present in the f.a.b. spectrum, namely those of cluster ions and fragment ions. Most cluster ions are matrix-derived. Glycerol, for example, gives peaks at mass numbers corresponding to (92x + 1)+ and (92x - 1)-, where 92 is the mass of glycerol and x is the number of glycerol molecules in the cluster. The most abundant clusters occur below mass 1000, but x can be as high as 15. 1-Thioglycerol gives fewer cluster ions than glycerol and, when present in a mixed matrix with glycerol, it suppresses the glycerol spectrum. The glycerol clusters are absent when the sample is sufficiently surface-active and in high enough concentration to form a complete monolayer at the surface of the matrix. Such dimer ions as [2M+ HI+, [2M+ Na]+, and [2M - HI-,derived from the sample, are an infrequent phenomenon in carbohydrate f.a.b.-m.s.; they are most likely to be found in the spectra of such low-molecular-weight, polar molecules as phosphorylated oligosaccharides. Fragment ions are important for sequencing; they are discussed in Section IV,2. F.a.b. spectra may be recorded with either a data system or an oscillograph. In principle, the method of recording the spectra should not affect the results but, in practice, it does. The oscillograph produces a faithful record of the spectrum, with peak shapes, noise, and any spurious spikes remaining intact and recognizable. In contrast, the computer ignores everything below a pre-set, threshold intensity, and reports all signals as lines. Genuine, but weak, high-mass signals are usually easier to recognize in an oscillographic trace. At m / z >2000, mass assignments differ in the two types of spectra. The data system calibrates the spectrum by using the known, accurate masses (based on C 12.00, H 1.01, 0 15.99, and so on), whereas oscillographic charts are counted manually, with nominal mass assignments given to each of the signals. For an average carbohydrate, nominal masses are lower than calculated masses by about half a mass unit for each 1-thousand mass-unit increment. Pseudomolecular ions do not appear as single, “clean” signals in f.a.b. spectra. Instead, clusters of signals are always present, partly because of the presence of molecules containing the I3Cisotope, the natural abundance of which is 1.1%, and partly because oxidations and reductions can occur in the matrix during the f.a.b. experiment. For example, underivatized
30
ANNE DELL
carbohydrates frequently exhibit an intense “minus 2” signal as a result of oxidation. The most abundant molecular ion is always derived from genuine molecules of the sample, not from those of by-products. However, if the molecule contains more than 90 carbon atoms, the ‘3C-isotope peak is the most intense signal. Fig. 3 shows the type of molecular-ion cluster normally observed in three different regions of the mass spectrum. 4. Sample Purity and the Analysis of Mixtures
The fast-atom beam will desorb only those molecules that are present at the surface of the matrix. Hence, selective ionization occurs from mixtures of compounds having different surface a~tivities.~ If a carbohydrate fails to give a mass spectrum, the most likely cause is contamination with compounds that are more surface-active than the sample. Salts and detergents are frequently present in biological samples, and should be rigorously removed prior to f.a.b.-m.s. Three warning signs that impurities are present are ( i ) intense, sodium-adduct ions for each matrix cluster, ( i i ) a series of signals 44 mass units apart (from detergents), and ( i i i ) a very high level of background signals up to well over 1000 mass units, but no obvious sample or matrix peaks above mass -500. F.a.b.-m.s. is a powerful technique for examining mixtures of carbohydrates. Many examples of such analyses are given in Sections V and VI. Unless the components have very different chemical structures, all will give molecular ions. However, the relative abundance of the ions will not necessarily reflect the relative concentrations of the components. Furthermore, if more than one class of carbohydrate is present, different pseudomolecular ions may be produced for each class. An example of such a phenomenon is given in Fig. 4.
5. Choice of Derivatives Derivatives play an essential part in almost all f.a.b.-m.s. studies of carbohydrates. They facilitate spectral interpretation (see Sections IV-VI), improve sensitivity (see Section 11,6), permit the analysis of salty samples (see later), allow unambiguous sequencing (see Section V,2), confirm the presence of cyclic structures (see Section VI,5), enable spectra to be obtained from very large molecules (see Section 111,4), and help in the location of 0-acylated residues in oligosaccharides (see Section V,5). Fortunately, derivatization for f.a.b.-m.s. makes no special demands on the carbohydrate chemist. The best derivatives are those that have been used for a very long time in carbohydrate work, namely, the per-0-acetyl and the per-0-methyl. Thus, f.a.b.-m.s. can be readily accommodated into existing structural programs.
F.A.B.-MASS SPECTROMETRY O F CARBOHYDRATES
31
FIG.3.-Schematic Representation of the Appearance of Typical, Molecular-ion Clusters. [(a) Near mass 500, (b) near mass 1500, and (c) near mass 3000.1
FIG.4.-Part of the Positive F.a.b.-Mass Spectrum of a Permethylated Sample Containing a Mixture of Man,-,GlcNAc, and Glc,_, . [The high-mannose carbohydrates were obtained by hydrazinolysis of a glycoprotein, followed by N-reacetylation and reduction, and the f.a.b. spectrum shows that only a portion of the molecules were reduced (for an explanation of this, see Section V,3). The glucose polymers are contaminants that were introduced during column purifications. Each of the GlcNAc-containing compounds gives two [M + H]+ molecular-ions, corresponding to the unreduced and reduced molecules, respectively; these are 16 mass units apart. The hexose polymers give [M + NHJ+ and [M + Na]+ molecular-ions. These form pairs 5 mass units apart. Major signals not assigned on the spectrum are 1052 (1084 minus methanol), 1070 (undermethylated 1084), 1248 (I280 minus methanol), 1266 (undermethylated 1280), 1274 (undermethylated 1288), 1280 (Hex,HexNAc+), 1452 (1484 minus methanol), 1470 (undermethylated 1484), 1478 (undermethylated 1492), 1484 (Hex,HexNAc+), 1688 (Hex,HexNAc+), and 1747 (undermethylated 1761). Forthe origin of Hex,-,HexNAc+, see Section IV,2.]
32
ANNE DELL
The choice of derivative is dictated by the exact problem under study. Frequently, it may be necessary to prepare both types of derivative in order to obtain the maximum structural information. The following factors need to be taken into account when choosing the most appropriate derivative. a. Permethylation.-( i ) This gives the smallest increase in the molecular weight of the sample, but it is, experimentally, a “dirty” procedure, and chromatography must be conducted prior to analysis. ( i i ) Most permethylated glycoconjugates fragment very selectively, resulting in a limited number of sequence ions that are easy to assign, but fragmentation may be so selective that not enough sequence information is present. b. Peracety1ation.-( i ) This gives a large increase in molecular weight, but is experimentally “clean,” and spectra can be acquired within 30 min of the start of the acetylation. ( i i ) Fragmentation pathways are less specific than for permethylated samples and may give more structural information, but spectra can be more difficult to interpret. As a general rule, peracetylation is most useful for compounds below M , 2000, particularly those that have been reduced with sodium borohydride and still contain some salt. The best procedure for peracetylation is based on the method of Bourne and Samples are dissolved in 2: 1 (v/v) trifluoroacetic anhydride-acetic acid and the solutions kept for -10 min at room temperature. Reagents are removed under a stream of nitrogen, and a solution of the product in chloroform is washed with water to remove salts, and dried; the peracetylated sample is dissolved in methanol for the f.a.b. analysis. Less widely used, but valuable for particular types of problem, are derivatives prepared by causing a nucleophile to react with the reducing residue of an oligosaccharide. In one very interesting study:’ a strategy was devised for the characterization of oligosaccharides that had been reductively aminated with aniline (9), p-aminoacetophenone (10) or ethyl p-aminobenzoate (11).The chromophore permits sensitive, U.V. monitoring during 1.c. purification, and the nitrogen atom can be readily protonated, resulting in high f.a.b. sensitivity. Oximes constitute another type of reducing-end derivative that have been shown to give good f.a.b. data.26 For example, use of the (pentafluorobenzy1)oxime derivative 12 assisted the analysis of oligogalactosiduronic acids3’ (38) E. J. Bourne, M. Stacey, J. C. Tatlow, and J. M. Tedder, 1.Chem Soc., (1949) 2976-2979. (39) A. Dell, J. E. Oates, and C. E. Ballou in M. A. Chester, D. Heinegard, A. Lundblad, and S. Svensson (Eds.), Glycoconjugates, Roc. Int. Symp. Glycoconjugates, 7th, LundRonneby, Sweden, 1983, pp. 137-138. (40) W. T. Wang, N. C. LeDonne, Jr., B. Ackerman, and C. C. Sweeley, Anal. Biochem., 141 (1984) 366-381.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
33
R-C H=N-O--CHz
c=o
c=o
CH3
OCH1CH3
10
11
I
9
Fs
I
12
6. Sensitivity
Sensitivities achieved in f.a.b. analysis are both operator- and sampledependent. Experienced mass spectrometrists working with well purified samples use between 0.1 and 5 pg of sample when analyzing derivatives, and between 1 and 10 pg when analyzing native compounds. The higher the molecular weight, the greater the quantity of sample needed. Correctly loading the sample into the matrix is one of the most critical steps in f.a.b. analyses. Poor data are inevitable if the sample has been loaded in such a way that it cannot readily be present in high concentration at the surface. Samples should not, therefore, be dried on the target prior to addition of the matrix. In the author’s laboratory, the following experimental protocol has been found to give good results. ( i ) Smear 1-2 pL of glycerol or 1 :1 (v/v) glycerol-thioglycerol on the metal target by using a 5-pL micropipet. ( i i ) Dissolve the sample in either 5% aq. acetic acid (underivatized samples) or methanol (derivatives) to such a concentration that 1 p L contains the desired amount of sample for the f.a.b. experiment. ( i i i ) Using a 5-pL micropipet, carefully blow 1 pL of the sample solution onto the surface of the matrix. ( i u ) Introduce the probe into the high vacuum of the source housing for -30 s in order to pump off most of the solvent (the solvent should not be exhaustively removed, because evaporation of the last traces almost certainly assists in the subsequent f.a.b. analysis). ( u ) Remove the probe in order to check that the volume of the matrix has not significantly decreased. This is necessary because coevaporation of the solvent and the matrix can occur. If necessary, carefully replenish the matrix with a small drop of 1-thioglycerol. ( u i ) Fully insert the probe into the f.a.b. source, until it is about 1 cm from its operating position. ( u i i ) Switch on the high voltages and, while observing the repetitive scanning of a selected region of the spectrum on the visual-display unit, push the probe fully into place. The mass range selected for observation
ANNE DELL
34
will depend on prior knowledge of the type of compounds being examined. It may be possible to select the predicted molecular-ion region, or areas where key fragment-ions are expected. For complete unknowns, it is usually appropriate to choose a region of the spectrum near m / z 1000, as the intensities of background signals at this mass will reflect the likelihood of sample signals being present at higher masses. The appearance of the spectra produced in the first few seconds of atom impact will dictate subsequent steps. If the signals on the oscilloscope are reasonably intense, a full spectrum is recorded immediately; delays result in poorer quality data. If the visual display indicates that the sample is not working well, it may be necessary to retune the source rapidly or to add a little more 1-thioglycerol, or, perhaps, a matrix additive. It may even be necessary to purify the sample by, for example, preparing the per- 0-acetyl derivative. It is now well established that acetylated and permethylated samples can be analyzed at higher sensitivity than their underivatized counterparts, despite the increase in mass upon derivati~ation.’~’~ Minor components in mixtures are often only revealed after derivatization. For example, negative f.a.b.-m.s. of an oligosaccharide mixture isolated from an erythrocyte glycoprotein4’ showed only the major component (see Fig. 5a). After peracetylation, signals from higher-molecular-weight, minor components were clearly present in the spectrum (see Fig. 5b). Similarly, the largest of the cyclic p - ( 1 + 2 ) - g l ~ c a n sand ~ ~ also the largest of the cyclic, Enterobacteriacae-common antigens4’ (see Section VI,5) were detected only after the samples had been permethylated. The higher the molecular weight of the sample, the greater the difference in minimum sample-loadings needed in order to produce spectra from native samples and their derivatives. If M,is significantly >4000, the difference becomes almost infinite, because underivatized polysaccharides and glycoconjugates of such a size rarely give spectra, whereas permethylated samples as large as 20,000 have been shown to be amenable to f.a.b.-m.s. (see Section 111,4). 111. F.A.B.-M.s.
OF
HIGH-MOLECULAR-WEIGHT SAMPLES
1. Definition of High Mass
Ten years ago, it was easy to give a working definition of high mass. At that time, most mass spectrometers had a mass range of less than 1000 at (41) M. Fukuda, A. Dell, J. E. Oates, and M. N. Fukuda, J. Bid. Chem., 259 (1984) 8260-8273. (42) A. Dell, J. E. Oates, C. Lugowski, E. Romanowska, L. Kenne, and B. Lindberg, Curbohydr. Res., 133 (1984) 95-104.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
35
FIG. 5.-(a) Molecular-ion Region of the Negative F.a.b.-Mass Spectrum of a Sample of Oligosaccharides Isolated from the Band 3 Lacto~aminoglycan~' of Human Erythrocytes After Treatment with Endo-0-D-galactosidase. [No significant ions were present above mass 950. The [M - HI- signal corresponds to the composition NeuAc,HexNAc,Hex,. The minor signals at 857, 893, 915, and 937 are adduct ions from contaminating NaCl and have compositions [M+Na-2H]-, [M+NaCl-H]-, [M+Na+NaCI-2H]-, and [ M+ 2 N a+ N aC1 - 3 HI-, respectively.] (b) Molecular-ion Regions of the Negative F.a.b.-Mass Spectrum of the Sample Shown in (a) After Conversion into the Peracetyl Derivative. [The major ions at 1339 (monounderacetylated 1381) and 1381 are afforded by the component observed in the spectrum of the underivatized sample. Molecular ions for minor components are present at 1956, 2243, and 253 1, corresponding to compositions NeuAc,HexNAc,Hex, ,NeuAc,-HexNAc,Hex, , and NeuAc,HexNAc,Hex,, respectively. Each is accompanied by a signal 42 mass units lower, corresponding to one degree of underacetylation.]
full sensitivity, and this was a natural boundary between low- and high-mass work. High-mass m.s. was nonroutine and frequently difficult; few biological molecules had been analyzed at high m a ~ s . 4For ~ practical reasons, it is still useful to differentiate between low- and high-mass experiments. The distinction is somewhat subjective but, for the purposes of this article, it is convenient to consider the upper end of the 3000-4000-mass range as the start of high-mass f.a.b.-m.s. (43) A. Dell and G. W.Taylor, Mass Spectrorn. Rev., 3 (1984) 357-394.
36
ANNE DELL
There are three main reasons for this choice. Firstly, it becomes more and more difficult to obtain recordable, molecular-ion signals from underivatized carbohydrates as their M, increases significantly above 3000. Secondly, the mass spectrometers that have been used in all high-masscarbohydrate studies published at the time of writing this article are not capable of very sensitive analysis above -3800 mass units (see later). Thirdly, at masses >4000, it is usually not practicable to work at the resolution necessary for adjacent peaks to appear as separate signals in the spectrum. To do so would require that the source and collector slits be narrowed to such a degree that there would be an unacceptable loss in sensitivity. Thus, spectra acquired at mass >4000 are usually composed of unresolved clusters. 2. Instrumentation
To date, all high-mass-carbohydrate f.a.b.-m.s. has been conducted by using high-voltage, double-focusing, sector mass spectrometers. Alternative instruments capable of analyzing high-mass ions, such as time-of-flight@ or Fourier-transform mass spectrometers,4’ do not, therefore, fall within the scope of this article. The maximum mass range of a magnetic-sector, mass spectrometer is governed by the fundamental, mass-spectrometric equation m /Z(Y (B 2 R 2 ) /V, where B is the magnetic-field strength, R is the radius of the magnetic sector, and V is the accelerating voltage. High-mass ions can only be focused at high field-strengths or with large-radii magnets, unless they are analyzed at low accelerating voltages. The last option is frequently not practicable, because a lower value of V means lower sensitivity. During the 19703, high-field-magnet mass spectrometers were designed to meet the needs of biopolymer They were fitted with magnets containing pole tips made of Permendur, a high-saturation alloy of cobalt, iron, and vanadium, capable of sustaining magnetic fluxes of up to 2.3 teslas. By the time f.a.b.-m.s. was introduced, two such instruments were commercially available, the Kratos HF-MSSO and the VG Analytical ZAB-HF mass spectrometer, both having a mass range of just over 3000 at maximum accelerating voltage (8 kV). The latter has proved the more popular for carbohydrate f.a.b.-m.s. Operation of the high-field ZAB spectrometer at 7-kV accelerating voltage extends the mass range to nearly 3800 without a significant loss in sensitivity. Above that point, there is a steady decline in sensitivity. When V is lessened (44) R. D. Macfarlane and D. F. Torgerson, Science, 191 (1976) 920-925. (45) C. L. Wilkins and M. L. Gross, Anal. Chem., 53 (1981) 1661A-1668A.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
37
to below 4 kV in order to allow the detection of ions above mass 6600, the resulting sensitivity is very poor indeed. The only successful f.a.b. work performed above 6600 has been in the protein area.46s47 The high-field ZAB and MS50 mass spectrometers have now been superseded by a new generation of extended mass-range instruments that are capable of operation at full sensitivity (that is, at full accelerating voltage) Several laboratories involved in up to masses in e x c e ~ s ~of * *10,000. ~~ carbohydrate work are installing these instruments, and it will be interesting to see what effect this new generation of mass spectrometers will have on the ability to obtain data on compounds having M , values well in excess of 4000.
3. Practical Aspects of Analysis at High Mass Three types of carbohydrate sample have been reported to give data above mass 4000, namely, permethylated polysaccharides,26 permethylated glyco~phingolipids,~~ and naturally acylated forms of a mycobacterial 0methyl-D-glucose polysaccharide.’’ All are hydrophobic, and desorption is probably facilitated by their inability to form strongly hydrogen-bonded aggregates, either with themselves or with the matrix. High-mass samples are usually analyzed at low resolution (see Section III,l), and the resulting spectra contain unresolved clusters having a characteristic, Gaussian appearance (see Fig. 6). Mass assignments are made by using either a mass marker (for chart paper) or a data system. Cesium iodide is normally used for calibrating the mass marker and data system; it gives abundant cluster-ions at intervals of 360 mass units, the mass of CsI, throughout the entire working mass range’ of f.a.b.-m.s. The mass assigned to the centre of each unresolved, sample peak corresponds to its chemical molecular weight. The quality of data recorded from weak, high-mass-ion beams can usually be improved by the use of signal a~eraging.~’The computer software necessary is now available from the mass spectrometer manufacturers. Although signal averaging has not yet been applied to high-mass-carbohy(46) M.Barber, R. S. Bordoli, G. J. Elliott, N. J. Horoch, and B. N. Green, Biochem. Biophys. Res. Commun., 110 (1983) 753-757. (47) A. Bateman, A. Dell, and H. R. Moms,J. Appl. Biochem., 7 (1985) 126-132. (48) J. S. Cottrell, L. C. E. Taylor, and S. Evans, Proc. Meet. Br. Muss Spectrom. Soc, 14rh, 18-21 Sept. 1984, pp. 127-129. (49) B. N. Green and R. S. Bordoli, in S. J. Gaskell (Ed.), Muss Specrromerry in Biomedical Research, Wiley, New York, 1986, pp. 235-250. (50) M. Barber, R. S. Bordoli, A. N. Tyler, J. C. Bill, and B. N. Green, Biomed. Muss Speclrom., 11 (1984) 182-186.
ANNE DELL
38
4660
4700
4740 “/z
FIG. 6 . 4 n e of the Molecular-ion Clusters Obtained from a Sample of Deuteropennethylated, Cyclic p( 1 + 2)-Glucans (see Section VI,5). [All high-mass samples give unresolved clusters of this type if the mass spectrometer is operated at low resolution. The peak is -6 mass units wide at half height. The mass is assigned by using the mass marker, which gives marks every 4 mass units, as shown. The center of the peak corresponds to the chemical molecular weight of an [ M + NH4]+ species.]
drate problems, it will almost certainly play a vital role in this area in the future. 4. F.a.b. “Mapping” of Permethylated, High-Mass Samples From of Band 3, one of the cell-surface glycoproteins of human erythrocytes, a rapid f.a.b. procedure called “mapping” has been devised for analyzing the types of nonreducing structures present in veryhigh-molecular-weight glycoconjugates of the lactosaminoglycan type.z5 These contain long, carbohydrate chains composed of many repeats of the N-acetyllactosamine unit, /&Gal-(1+ 4)-GlcNAc, which may be modified by sialylation, fucosylation, branching, and so on, to afford determinants for a variety of such well recognized antigens as the ABH blood-group system.’* Two properties make this type of molecule an ideal candidate for f.a.b.-m.s. Firstly, as permethylated derivatives, they fragment in a reproducible and predictable way (see Section V,2), to give a diagnostic spectrum or “map” of fragment ions in which is contained information on all of the nonreducing structures present in the intact glycoconjugates. Secondly, good f.a.b. spectra may be obtained on very large molecules indeed. Spectra have been recorded from permethylated lactosaminoglycans as large as 20,000, and it is anticipated that much larger molecules will be amenable to this type of analysis. A tremendous advantage of the mapping procedure for the analysis of high-molecular-weight samples is that it makes no high-mass demands on the instrumentation. Only fragment ions are recorded, and these fall within the high-sensitivity range of high-field instruments, that is, less than 4000 mass units. (51) M. Fukuda, A. Dell, and M. N. Fukuda, J. Bid. Chern, 259 (1984) 4782-4791. (52) T. Feizi, Trends Biochem. Sci., 6 (1981) 333-335.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
39
The following protocol has been proposed2’ for mapping high-molecularweight glycoconjugates. (i) The intact glycoconjugate (or mixture of glycoconjugates) is converted into its permethylated derivative(s). (ii) Positive f.a.b. spectra are acquired in the 3000 mass range by overlapping several shorter scans, using the procedures described in Section 142. (iii) The number of sialic acid, fucose, hexose, or hexosamine residues contributing to each fragment ion is then detefmined by a simple calculation. Each of these residues has a unique mass, and it is possible to assign a unique composition to every fragment ion. Thus, the fundamental, nonreducing structure Hex-HexNAc+ occurs at rn/z 464, and increments of 174, 361, 391, and 449 mass units are added to this for additional fucose, N-acetylneuraminic acid, N-glycolylneuraminic acid, or N-acetyllactosamine moieties, respectively. F.a.b. mapping may be applied to mixtures of glycoconjugates. No prior knowledge of their size, structure, or complexity is needed. Thus, one useful application is the rapid screening of the total lactosaminoglycan fractions isolated from the surface of a cell. The f.a.b. maps are a useful guide to the types of nonreducing structures present, and may reveal the presence of cell-specific antigens. This type of analysis is illustrated in Figs. 7 and 8. Fig. 7 shows part of the f.a.b.-mass spectrum obtained from a lactosaminoglycan sample isolated from chronic myelogenous leukemia (CML) cells.” After an identical analysis, normal granulocytes yielded a sample that gave the spectrum shown in Fig. 8. Most of the fragment ions are obviously present in both spectra, albeit with intensity differences. However, there are two very significant differences. Firstly, the signal at rn/z 999, which is fairly prominent in the spectrum of the CML sample, is very weak indeed in that of the granulocyte sample. Secondly, there is a signal at rn/z 1622 in the CML spectrum that is absent from the spectrum obtained from granulocytes. These ions have compositions NeuAc, Fuc,Hex,HexNAc, and NeuAcl Fuc2Hex2HexNAc2,respectively. Their complete structures, determined by using other methods of carbohydrate analysis, are 13 and 14.
NeuAc a ( 2 + 3) Gal p ( 1 + 4) GlcNAc p( 1 + 3) Gal p ( 1 + 4) GlcNAc p( 1 +
r:.
r:.
Fuc
Fuc
14 (53) M. Fukuda, B. Bothner, P. Ramsamooj, A. Dell, P. R. Tiller, A. Varki, and J. C. Klock, J. Biol. Chem., 260 (1985) 12,957-12,967.
40
ANNE DELL
FIG. 7.-Part of the F.a.b. “Map” of a Lactosaminoglycan Sample Isolated from CML Cells.53 [Signals at 913, 999, 1087, 1261, 1274, 1362, 1448, 1536, 1622, 1710, and 1723 are A,-type ions resulting from cleavage at HexNAc residues. They have the compositions Hex,HexNAc,, NeuAc,Fuc,Hex,HexNAc, , Fuc,Hex,HexNAc,, Fuc,Hex,HexNAc,, NeuAc,Hex,HexNAc,, Hex,HexNAc,, NeuAc,Fuc,Hex,HexNAc,, Fuc,Hex,HexNAc,, NeuAc,Fuc,Hex,HexNAc, , Fuc,Hex,HexNAc, , and NeuAc, Hex,HexNAc, , respectively. The signal at 1029 is an A,-type of ion resulting from cleavage at Hex, and has the composition NeuAc,Hex,HexNAc, . The signal at 1242 is the result of methanol loss from 1274.1
FIG. &-Part
of the F.a.b. “Map” of a Lactosaminoglycan Sample Isolated from Normal
granulocyte^.^^ (See the legend to Fig. 7 for peak assignments.)
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
41
The f.a.b. mapping procedure is applicable to all permethylated polysaccharides and glycoconjugates that fragment readily. Current knowledge indicates that the main requirement is the presence of amino sugar residues at fairly regular intervals. Preliminary work54 suggested that the method could be useful for rapidly defining the type of structure present in bacterial polysaccharides whose repeating units contain amino sugars. OF F.A.B.-MAss SPECTRA IV. INTERPRETATION
1. Molecular-weight Assignment
With the special exception of “maps” (see Section 111,4), all f.a.b. spectra contain one or more pseudomolecular-ion signal(s). These afford the molecular weight of the oligosaccharide or glycoconjugate, and thus define its composition in terms of both the type and number of sugar constituents, the type of aglycon, and the type and number of such substituents as acetyl or sulfate groups. F.a.b.-m.s. cannot distinguish between isomeric monosaccharides, and compositions are given in terms of hexose, pentose, deoxyhexose, hexosamine, uronic acid, and so on. The pseudomolecular-ion region may be complex if several types of ion are present, but the spectra can usually be assigned without difficulty. In the positive-ion mode, four species are commonly present (although not usually in the same sample). They are, in rising order of molecular weight, [M + HI+, [M + NH4]+, [M + Na]+, and [M K]+. If more than one signal is present, the mass differences between the signals (see Fig. 9) usually indicate the nature of the cationizing species. For example, two pseudomolecular-ion signals separated by five mass units must be [ M + NH4]+ and [ M + Na]+. Samples that are heavily contaminated with Na+ or K+, or exist naturally as Na+ or K+ salts, give ions of the type [M - x H + ( x + l)Na]+. The addition of acid to the matrix (see Section II,2) enhances the abundance of [ M + H]+ ions, and this often helps to confirm tentative
+
n [M+HI*
-22--17
I M + N w + [M+Nal+
---+
53-16
[M+KI* [M-H+2Nal*
-c6+
FIG. 9.-Schematic Representation of Molecular-ion Signals That May Be Formed in the Positive-ion Mode, Showing Commonly Observed Mass Differences. (54) A. Dell, unpublished results.
ANNE DELL
42
FIG. 10.-Molecular-ion Region of a Permethylated Glycosphingolipid Sample. [The component having the saturated, 24-carbon fatty acid (Cer2.,:J gives the [M+H]* signal at 1345, and no adduct ion. The component having the unsaturated, 24-carbon fatty acid (Cerz4,) gives a minor [M+H]+ signal at 1343, and a major signal for the 1-thioglycerol-adduct ion at 1451.1
assignments. Some types of carbohydrate frequently form adduct ions with the matrix, giving such signals as [M+ (glycerol), + HI+. These signals are easy to recognize, because they appear at defined mass-increments above the genuine molecular ions. They are rarely more intense than the molecular ions, with one important exception. Glycosphingolipids having an unsaturated, 24-carbon fatty acid group in the ceramide moiety always give [M + 1-thioglycerol H]+ as the dominant, molecular-ion This phenomenon is illustrated in Fig. 10. In the negative-ion mode, [M -HI- is usually observed. Molecules that cannot lose a proton, such as permethylated saccharides, give negative spectra if anions are present in the matrix; for [M+Cl]- or [ M + SCNI-. Underivatized samples do not normally add anions unless the matrix is very and, at such high salt-levels, the data are usually poor. Samples that exist naturally in a salt form at neutral pH retain their Na+ or K+ counter-ion on most of the anionic groups, unless the matrix is acidified (see Section 11,2). If the pK values of the anionic, functional groups are very low, molecular-ion clusters of the general composition [M (x - l)Na - x HI- dominate the spectra, unless strong acids can be added, and this may not be possible if acid-labile groups are present in the sample.
+
+
(55) M. N. Fukuda, A. Dell, J. E. Oates, P. Wu. J. C. Klock, and M. Fukuda, J. Biol. Chem., 260 (1985) 1067-1082. (56) D. Prome, J. C. Prome, G . Puzo, and H. Aurelle, Carbohydr. Res., 140 (1985) 121129.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
43
2. Fragmentation Pathways
In this Section, fragmentation pathways that appear to be common to all classes of polysaccharides and glycoconjugates are d e s ~ r i b e d . ~ ~ ~ ~ ~ * ~ ' * ~ ~ Cleavages that occur only in specific types of molecules, for example, glycosphingolipids, are discussed in Section VI. An arbitrary code-letter is given to each pathway in order to facilitate its citation in later sections. For convenience, a pictorial representation is given for each pathway. These Schemes are speculative, because no rigorous studies, using isotopically labelled compounds, have been conducted to confirm cleavages and hydrogen shifts. Nevertheless, they provide a useful visual guide to fragmentation mechanisms. It should be noted that only the charged product is shown in each scheme. Furthermore, the choice of 4-linked pyranoses was made solely for convenience.
Pathway A Description: glycosidic cleavage to form an oxonium ion; charge retained on nonreducing end; positive-ion mode only; often referred to as A,-type cleavage, because of similarity to one of the cleavages seen in electron impact-mass spectrometry.
G2b0/"1' - y.4 CHiOH
-0
CHzOH
CHZOH
+ H
HO
OH HO
OH
(63) (64) (65)
(66)
OH
K.Yu, M. M. Rapport, and K.Suzuki (Eds.), Ganglioside Structure, Function and Biochemical Potential, Plenum, New York, 1984, pp. 55-63. M. Iwamori, M. Arita, T. Higuchi, Y. Ohashi, and Y. Nagai, Jeol News, 20A (1984) 2-9. M. Arita, M. Iwamori, T. Higuchi, and Y. Nagai, Jeol News, 19A (1983) 2-6. M. Arita, M. Iwamori, T. Higuchi, and Y. Nagai, J. Biochem. (Tokyo),94 (1983) 249-256. M. Iwamori, Y. Ohashi, T. Ogawa, and Y. Nagai, Jeol News, 21A (1985) 10-14. J. P. Kamerling, W. Heerma, J. F. G. Vliegenthart, B. N. Green, I. A. S. Lewis, G. Strecker, and G. Spik, Biomed. Mass Spectrorn., 10 (1983) 420-425. D. Abraham, W.F. Blakemore, A. Dell, M. E. Herrtage, J. Jones, J. T. Littlewood, J. E. Oates, A. C. Palmer, R. Sidebotham, and B. Winchester, Biochem. J., 221 (1984) 25-33. H. Egge, A. Dell, and H.von Nicolai, Arch. Biochem. Biophys., 224 (1983) 235-253. H. Egge, J. Dabrowski, P. Hanfland, A. Dell, and U. Dabrowski, in A. Makita, S. Handa, T. Taketomi, and Y. Nagai (Eds.) New Vistas in Glycolipid Research, Plenum, New York, 1982, pp. 33-40. M. Barber, R. S. Bordoli, R. D. Sedgwick, and J. C. Vickerman, J. Chern. SOC.,Faraday Trans. I , 7 8 (1982) 1291-1296.
(57) H. Egge, J. Peter-Katalinic, and P. Hanfland, in R. W. Leeden, R.
(58) (59) (60) (61) (62)
HO
ANNE DELL
44
Pathway B Description : glycosidic cleavage with a hydrogen transfer; charge retained on reducing end; positive- and negative-ion modes; often referred to as /3-cleavage. -+o*-
-0
/"* + o r - H 0
oo/"* + or -
CHzOH I
HO
+or-H
HO
OH
-+or-
-0
/"* + o r - H
I
0
CH20H I
Pathway D Description: ring cleavage; charge retained on reducing end; ions are 28 mass units heavier than those formed in Pathway B; positive- and negativeion modes.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
4s
-+or-
,--. + o r - H
1
0
-0
HO
OH HO
OH
+ or -
CH2OH I
Pathway E
Description: ring cleavage; charge retained on nonreducing end; infrequent pathway in the positive-ion mode, but a major pathway in the negative mode, because a stable, enolate anion results from loss of the enolic hydrogen atom; ions are 42 mass units higher than those formed in Pathway C. -+or-
CH2OH -0
CHZOH
&o&o,--.+or-H HO
OH HO
OH
V. STRUCTUREASSIGNMENT BY F.A.B.-M.s. 1. Analysis of Underivatized Samples
It is usually desirable to define molecular weights by analyzing underivatized samples, because chemically labile functional groups will then be observed. Unfortunately, underivatized oligosaccharides often give ambiguous sequence-data. The reasons for this may be deduced from a
46
ANNE DELL
consideration of the fragmentation pathways shown in Section IV,2. In an underivatized, unreduced oligosaccharide, pathways B and C are not distinguishable, and it is not possible to establish whether the resulting fragmentions are derived from the nonreducing or the reducing end of the molecule. A further complication is the presence of ions resulting from “double cleavages,” for example, a combination of pathways A and B operating at different glycosidic linkages, which give apparent nonreducing-end sequence ions that are not, in fact, derived from the nonreducing terminus. The abundance and number of fragment ions afforded by an underivatized carbohydrate vary considerably, depending on the structure of the molecule, its purity, the amount of sample loaded, and the nature of the matrix used. Sometimes, no fragment ions are present, despite molecular-ion signals of reasonable quality. Sometimes, one or more linkages are particularly labile. On rare occasions, fragmentation is extensive. A final point to note is that fragmentation pathways may differ according to the nature of the pseudomolecular ion, that is, whether it is [M HI+, [M + Na]+, and so on, because of differing internal energies. In summary, fragmentation data from an underivatized sample need to be interpreted with caution, and the primary objective for analyzing underivatized samples, unless they belong to a well characterized family such as the glycosphingolipids, should be molecular-weight assignment. Sequencing should be carried out by using derivatives.
+
2. Analysis of Derivatized Samples
Per-0-acetyl and per-0-methyl derivatives are used extensively for sequence analysis and for providing molecular-weight information at very high sensitivity. Derivatization resolves the problems of ambiguity referred to in Section V,1, because a true, nonreducing residue will be fully substituted, whereas a nonreducing residue resulting from mass-spectrometric cleavage will carry a free hydroxyl group. Derivatization has the added advantage of directing fragmentation along a limited number of well defined, fragmentation pathways, and the fragmentation of peracetylated and permethylated derivatives is particularly reliable and predictable. The major ions formed from both the peracetyl and the permethyl derivatives are derived from a single, Pathway A cleavage; less abundant ions arise from P-cleavage (Pathway B), together with a Pathway A cleavage farther along the chain. The latter can be readily identified, because they lack an acetyl or methyl group on the nonreducing residue. Thus, the hypothetical sequence M-N-P-Q-R (where the letters refer to fully methylated or acetylated, unspecified sugar residues) is expected to fragment to give sequence ions of composition M+, MN+, MNP+, and MNPQ+, but
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
47
may also give such minor ions as NP+ and NPQ+, in which N bears a free hydroxyl group resulting from a “double cleavage.” This hypothetical sequence is assumed to be hexosamine-free, because a very interesting and important phenomenon is observed when HexNAc residues are present in the sequence. Cleavage then occurs predominantly at each hexosamine Thus, were the hypothetical sequence M-N-HexNAc-Q-R the major fragment ion would be M-N-HexNAc+. In permethylated samples, the HexNAc cleavage may be exclusive, particularly if the molecule is large. In peracetylated samples, it is always the dominant cleavage. Branching patterns can be readily established in N-acetyllactosaminecontaining molecules, because of the specific cleavages that occur at each HexNAc residue. For example, a simple, linear sequence of three lactosamine repeats will give fragment ions of composition Hex,HexNAc:, Hex,HexNAc:, and Hex,HexNAc:, whereas the branched sequence (15) will not give a Hex,HexNAc: fragment-ion. Hex-HexNAc Hex-HexNAc
\
/
Hex-HexNAc-
15
The molecular ions observed in the f.a.b. spectra of peracetylated and permethylated samples are often very important for structural assignment, particularly if f.a.b. spectra were not acquired prior to derivatization. Usually, their interpretation is routine, but the fact that the exact nature of the molecular ion may vary from sample to sample needs to be taken into account. For example, it may be difficult to permethylate some structural types, for example, those containing acetamido and the major molecular-ions from these samples will be one or more multiples of 14 mass units (depending on the number of methyl groups that are absent in the majority of molecules) less than expected. Peracetylated samples frequently lack an acetyl group on the reducing group, and molecular ions of structures 16 and 17 may be present, instead of, or in addition to, 18. It has been observed6’ that peracetylated samples prepared by using acetic anhydrideCH~OAC
RO& > O H + H +
CH~OAC
Ro&>
Ro/QoAc+H+ OAc
OAc 16
CH~OAC
17
(67) C. E. Ballou and A. Dell, unpublished results.
OAc 18
ANNE DELL
48
pyridine and those prepared using trifluoroacetic anhydride-acetic acid often differ significantly in the extent of acetal acetylation.
3. Monitoring Chemical and Enzymic Reactions by F.a.b.-M.s. F.a.b.-m.s. is an ideal procedure for monitoring the progress of many of the chemical and enzymic reactions commonly used in carbohydrate chemistry. It can reveal minor by-products, as well as provide data that are often very useful in structural assignments. Some types of reactions, for example, hydrolyses and methanolyses, are conveniently monitored by loading an aliquot of the reaction mixture directly into the matrix. Prior processing is necessary only if the reaction medium is incompatible with f.a.b.-m.s. or if derivatives need to be prepared after the enzymic or chemical digestion. In this Section, several studies that highlight the types of procedures that can be readily monitored by f.a.b.-ms., have been selected for discussion. Other examples are given in Section VI. The complete structure determination of the blood-group B-active glycosphingolipid shown in Fig. l l was achieved by using a combination of m.s. and n.m.r. The composition and sequence were defined by the f.a.b.-mass spectrum of its permethylated derivative (see Fig. 11). Many of the linkage assignments could be determined from n.m.r. spectra of the intact molecule, but some ambiguities were resolved only when n.m.r. spectra were obtained from truncated molecules lacking the two galactose residues on each branch. The strategy designed for removal of these residues was digestion with (Y-D-galactosidasefollowed by Smith degradation. F.a.b.-m.s. proved to be a very useful method for monitoring the progress of each of these reactions. After treatment of the native glycosphingolipid with (Y-Dgalactosidase, the molecular ion of the permethylated derivative shifted to
668 Gal - Gal - G I cNA
1770 7 ‘Gal-GlcNA7
Gal -Gal-GlcNAc\
e
2_872
‘%a1 -GltNAc
668 ‘.i Gal-Gal-GlcNAc
664
I’
;‘,
d
3914
Gal-GltNA,.‘
Gal-Gal - G l r N A a
668
,?(, Gal-GlrNAc -Gal-GlrtCer
q Gal-Gal -GlcNA&
4
658
668
FIG.11.-Sequence of a 25-Sugar Residue Glycosphingolipid Isolated from Rabbit Erythrocyte membrane^.'^ (Cleavage points, and the masses of fragment ions of the permethylated derivative, are shown. No fragment-ions were observed above 4000, because of the poor sensitivity at high mass.)
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
49
46 4 Gal-G I c N A c T 1362 Gal -G lcNAc',\'Gal -GlcNAc2260 y'ha, - G i c N A 7 3158 Gal-GlcNAc Gal-GlcNAz
L64
.\
264
\I!
.";
Gal-GlcNAb
4 64
Gal-GlcNA&
..(
Gal-GlcNAc -Gal -GlciCer L*
658
464
FIG.12.-Sequence of the Glycosphingolipid Shown in Fig. 11, after Enzymic Degradation with cr-D-Galactosidase. (Cleavage points, and the masses of fragment ions of the permethylated derivative, are shown.)
5163, and major fragment-ions appeared at 464, 1362, 2260, and 3158 (see Fig. 12), consistent with the loss of five terminal galactose units. The f.a.b. spectrum also showed, by virtue of a weak signal at 668 for Gal-GalGlcNAc+, that some molecules were incompletely digested. The expected product of the Smith degradation is shown in Fig. 13, together with the predicted fragment-ions for the permethyl derivative. F.a.b.-m.s. confirmed that all of the terminal galactose residues had been removed, because 464 was absent, and 260 had appeared as a strong signal. However, the other predicted fragment-ions shown in Fig. 13 were either absent or of low abundance. Instead, another ion-series was present at m / z 709, 1403,2097, 2971, and 2995, indicating that one of the GlcNAc residues had unexpectedly been degraded during the oxidation with metaperiodate. This information helped in the analysis of the n.m.r.-spectral results. The use of f.a.b.-m.s. to monitor hydrolysis and acetolysis reactions is illustrated by studies67on a yeast high-mannose oligosaccharide containing two phosphoric esters (19). The molecular-ion region of the negative f.a.b. 260
2342
<
G l c N A ~ GltNAc>
260
3_036
Gal-GlcNAc ;Gal- .Glc:;Cer
\/
4
GICNAL 260
FIG.13.-Predicted Sequence of the Smith-Degradation Product of the Glycosphingolipid Shown in Fig. 12. (Predicted Fragment-ions for the permethylated derivative are shown.)
ANNE DELL
50
Man a(1 + 6 ) Man a ( l - 6 ) Man cr(l+6) Man p ( 1 + 4 ) GlcNAc
r:.
Man
t :.
t:.
Man
t:.
Man-P
Man-P
I Man 19
r:. Man r.:
I Man
Man
spectrum of the underivatized molecule showed an [M - HI- signal at 2324, consistent with the expected composition. Following mild, acid hydrolysis, this signal shifted to 2000, confirming that the two mannosyl phosphate linkages had been hydrolyzed. The relative locations of the two phosphate groups were then investigated by partial acetolysis, which is known to effect selective cleavage of the (1 + 6) linkages. The composition of each acetylated product in the acetolysis mixture was determined from the masses of their molecular ions in the f.a.b.-mass spectra. A characteristic molecular-ion was observed at 2192, consistent with the composition Man,HexNAcP, (where P is phosphate), confirming that both phosphate groups are so positioned that they can be retained on a Man6HexNAc fragment produced by partial cleavage of (1 + 6) linkages. Other molecular-ions were present at 1003 (Man,P), 1291 (Man,P), 1578 (Man,HexNAcP), 1579 (Man,P), 1867 (Man,P), 2154 (Man,HexNAcP), 2442 (Man7HexNAcP), 2480 (Man7HexNAcP,), 2688 ( Man8HexNAcP), and 2768 ( Man8HexNAcP2). All of these signals were accompanied by equally prominent signals that were 102 mass units higher. A plausible structure for this “plus 102” species is structure 20. This type of acyclic molecule is known to exist in acetylation reactions. CHzOR P O A c
AcO
doAc + H+
OAc OAc
20
Acetolysis together with f.a.b.-m.s. has been applied to the preliminary screening of glycoproteins for sugar type.68 In this procedure, -100 p,g of intact glycoprotein is subjected to an acetolysis time-course. At suitable intervals, aliquots are removed, quenched with water, and the peracetylated oligosaccharides that have been released from the glycoprotein are extracted into chloroform and analyzed by f.a.b.-m.s. without further purification. The molecular weights of the acetolysis fragments are diagnostic of the type (68) S. Naik, J. E. Oates, A. Dell, G. W. Taylor, P. M. Dey, and J. B. Pridham, Biochem Biophys. Res. Commun., 132 (1985) 1-7.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
51
of glycan present, for example, high-mannose, complex, lactosaminoglycan, and so on. Sialic acid linkages are surprisingly resistant to acetolysis, and the molecular weights of oligosaccharides that still contain sialic acid residues allow the assignment of N-acetyl- or N-glycolyl-neuraminic acid. Methanolysis-f.a.b.-m.s. is probably the most elegant of the conjoint chemical-f.a.b.-m.s. procedures, because the reagents form a perfect solvent for adding the sample to the matrix, the acidic conditions enhance sensitivity, and results can be obtained in a very short time. A study of one of the glycolipids isolated from a human, embryonal Carcinoma cell-line illustrates how useful and how easy the technique can be.69 Earlier work on the lactosaminoglycan-containing glycoproteins of the same cell-line had revealed significant proportions of the disialyl moiety, NeuAc-NeuAc, and it was of interest to establish whether the same structure was present in any of the glycolipids. A novel, disialyl glycolipid was finally isolated, its presence being suggested by a characteristic signal at 1186 in the f.a.b. spectrum of its permethylated derivative. This signal confirmed that a structure of composition NeuAc,Hex,HexNAc, was present at the nonreducing end, but the f.a.b. data did not reveal whether the NeuAc residues were directly linked as NeuAc-NeuAc or whether they were separately attached to the N-acetyllactosamine. In principle, methylation analysis could have solved this problem. In practice, it was easier to use f.a.b.-m.s. As soon as the f.a.b. spectrum had been recorded, a small amount of methanolic HCl was added to the small quantity of sample remaining in the tube (from which aliquots had been taken for the f.a.b. experiment). After warming gently for 15 min at -40°, the mixture was loaded onto the f.a.b. target, and a second f.a.b. spectrum was recorded. The new spectrum contained a much diminished 1186 signal, and a new signal had appeared at 436 that corresponded to a Hex-HexNAc+ fragment-ion containing two free hydroxyl groups. Thus, within 15 min of the first f.a.b. result, it was clear that the glycolipid was not the one being sought, because the two NeuAc residues had to be separately attached in order to generate two hydroxyl groups upon their removal by the methanolic HC1. The final example selected for discussion demonstrates how f.a.b.-m.s. can show what problems may occur during procedures used for isolating or characterizing oligosaccharides. The behavior of N-glycosylically linked glycans during the reaction sequence of hydrazinolysis, N-reacetylation, and reduction with sodium borohydride has been investigated” by using (69) M. N. Fukuda, B. Bothner, K. 0. Lloyd, W. J. Rettig, P. R. Tiller, and A. Dell, 1. Biol. Chem., 261 (1986) 5145-5153. (70) J.-C. Michalski, J. Peter-Katalinic, H. Egge, J. Paz-Parente, J. Montreuil, and G. Strecker, Carbohydr. Res., 134 (1984) 177-189.
ANNE DELL
52
the two model compounds Man,GlcNAc,Asn and Man,GlcNAc( Fuc)GlcNAcAsn. The product mixtures obtained after the reaction sequence was completed were separated by liquid chromatography, and negative-ion f.a.b.-m.s. was used to define the molecular weight of each component. In addition to the desired products, namely, Man,GlcNAcGlcNAcol and Man,GlcNAc( Fuc)GlcNAcol, at least 7 other compounds were formed from each model compound, including unreduced molecules, hydrazones, and the products of osazone-type rearrangement and Wolff -Kishner reduction. 4. Linkage Assignment
In most studies, f.a.b.-m.s. does not reveal linkage positions. There is, however, one well-documented exception where linkage-site-specific fragmentation is observed, namely, permethylated molecules containing HexNAc r e s i d ~ e s . ~ ~ , It ~ ~was , ’ ’ shown earlier (see Section V,2) that such compounds are cleaved specifically at the HexNAc glycosidic linkages, to give abundant A,-type sequence ions. Not mentioned earlier is the fact that these ions may be accompanied by related ions that give information on the substitution pattern of the HexNAc residue. Provided that the sequence ions are <-lo00 mass units, they will fragment further, to give ion 21. The CH20Me
/Q
RO
Ac‘NMe 21
mass value of the daughter ion, compared with the sequence ion from which it is derived, defines the nature of the substituent originally present at 0 - 3 of the HexNAc residue. Thus, a signal 32 mass units (methanol) below the sequence ion shows that 0 - 3 was not substituted in the native molecule, whereas a signal 206 mass units below the sequence ion shows that fucose was linked at 0-3, and so on. The Type 1 [GalP( 1-* 3)GlcNAcI and Type 2 [GalP( 1-* 4)GlcNAcI structures commonly found in glycoconjugates are very easy to distinguish, because only the latter will give prominent “minus methanol” signals 32 mass units below each sequence
(71) P. Hanfland, M. Kordowicz. H. Niermann, H. Egge, U. Dabrowski, J. Peter-Katalinic, and J. Dabrowski, Eur. J. Biochem, 145 (1984) 531-542.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
53
5. Acyl-Group Location
F.a.b.-m.s. of underivatized samples is the procedure of choice for confirming that 0-acyl groups are present in an oligosaccharide or glycoconjugate. The number of molecular ions present in the f.a.b. spectra, and their precise mass values, define the exact number and type of substituent groups. It should, however, be noted that the relative signal-intensities will probably not reflect the relative concentration of each component, because acylated derivatives will give stronger signals than their underivatized counterparts (see Section 11,6). F.a.b.-m.s. has now been used to analyze a variety of acylated molecules, including some containing a~ety1,4~”~ gly~eryl?~ hydroxyb~tanoyl,7~ and long-chain fatty acyl ‘5*75*76 groups. In some of these studies, there were useful fragment-ions that indicated which residues carried the acyl groups. Unfortunately, f.a.b.-m.s. of underivatized samples could not be relied upon to give sufficient, unambiguous sequence-data to make it a recommended, general procedure for assigning acylated residues. This problem has now, however, been solved by the introduction of a derivatization procedure that preserves 0-acyl functional groups and that gives a product that fragments reliably.77 The procedure is none other than one of those recommended earlier for use in sequencing and in cleaning up samples, namely, peracetylation with trifluoroacetic anhydride-acetic acid (see Sections II,6 and V,2). The only change needed is to replace acetic acid with its fully deuterated relative, in order to ensure that natural 0-acetyl groups will be distinguishable from their introduced counterparts. Of course, if the acyl groups are known to be different from acetyl, such a change is not required. The type and number of native acyl groups is then determined from the difference between the calculated molecular weight of a fully per(deuterioacety1)ated molecule and the actual molecular weight given by the f.a.b. data. A similar calculation of the mass of each sequence-ion reveals the exact positions of the acylated residues in the sequence.
(72) A. Dell, G . G . S.Dutton, P.-E. Jansson, B. Lindberg, U. Lindquist, and I. W. Sutherland, Carbohydr. Res., 122 (1983) 340-343. (73) M.-S. Kuo, A. Dell, and A. J. Mort, Carbohydr. Res., 156 (1986) 173-187. (74) M. McNeil, J. Darvill, A. G. Darvill, P. Albersheim, R. van Veen, P. Hooykaas, R. Schilperoort, and A. Dell, Carbohydr. Res., 146 (1986) 307-326. (75) N. Qureshi, K. Takayama, and E. Ribi, J. Bid. Chem., 257 (1982) 11,808-11,815. (76) K. Kamisango, S. Saadat, A. Dell, and C. E. Ballou, J. BioL Chem., 260 (1985) 41 17-4121. (77) A. Dell and P. R. Tiller, Biochem. Biophys. Res. Commun., 135 (1986) 1126-1134.
54
ANNE DELL
VI. APPLICATIONS
1. Glycolipids a. Glycosphingo1ipids.-At one time, glycosphingolipids, especially gangliosides, had a for intractability to f.a.b.-m.s. This was largely because of their tendency to form high-molecular-weight micelles in the presence of water, which meant that glycerol, being hygroscopic, was an unsuitable As soon as alternative matrices were introduced, for example l-thi~glycerol~~ or triethanolamine-l,l,3,3-tetramethylurea,30glycosphingolipids were revealed to be very tractable molecules indeed. Their ceramide component acts as a natural “derivative,” providing a “handle” for the interpretation of fragmentation patterns, and conferring excellent desorbing properties that result in abundant, pseudomolecular ions. Their permethyl derivatives behave even better, and, in fact, the record in carbohydrate f.a.b.-m.s. for the highest-mass molecular-ion obtained from a “real” biological sample (as opposed to a synthetic mixture of hexose polymers26) is held by Egge and coworkers34for their 25-sugar-residue glycosphingolipid (see Fig. 11) which, as the permethyl derivative, gave an [ M + Na]+ signal at 6184. The best f.a.b. strategies for analyzing glycosphingolipids involve the analysis of permethyl derivatives in the positive-ion mode, or underivatized samples in the negative-ion mode, the latter being particularly useful in the study of gangliosides, because it permits the discrimination of positional isomers. Positive-ion f.a.b.-m.s. of underivatized glycosphingolipids is usually not chosen, because it gives only limited sequence-information. Two sets of fragment ions are formed from permethylated glycosphingolipids. Firstly, the carbohydrate chain cleaves at the glycosidic linkages to give A,-type ions (Pathway A, Section IV,2) with the most-abundant ions resulting from HexNAc cleavages (see Fig. 14). Related ions of the type described in Section V,5 will also be formed. The less favored hexosyl cleavages shown in Fig. 14 are more likely to occur if the glycosphingolipid is examined in its protonated form rather than as [M+Na]+. Secondly, characteristic ions are produced from the ceramide moiety. Irrespective of the nature of the molecular ion, cleavage always occurs between the glycan and the ceramide, with the charge remaining on the latter (see Fig. 14), to give a fragment ion or ions whose mass depends on the type of sphingosine base and the type of fatty acid present. [M+ H]+ ions always lose the acyl group, as shown in Fig. 14, to give a prominent fragment-ion whose mass (78) S. Handa, Y. Kushi, H. Karnbara, and K. Shizukuishi, J. Biochem. (Tokyo), 93 (1983) 315-3 18.
F.A.B.-MASS SPECTROMETRY O F CARBOHYDRATES 3 16
a25
1 1’?
NWAC 0 - G a l - 0 - G l c N A c
‘7’7 7 r:,
0-Cal:O-ClcNAc
O-Gal:O-Glc
55
[M+Hlf=2247
i 0 CH ; C H - C H - C H = C H - I C H 2 ~ 2 - C H ~ P[H3 N - CH3 T(b1 0.C -[CH$,cCH3
FIG. I4.-Schematic Representation of the Fragmentation Observed in the Positive F.a.b.Mass Spectrum of a Permethylated Ganglioside Isolated from granulocyte^.^^ [Other glycosphingolipids fragment in a similar way. Major cleavages are shown with solid lines, and minor cleavages with dotted lines. The masses of ions resulting from cleavages (a), (b), and (c) define the type of sphingosine and the type of fatty acid. In this example, (a) is 548, (b) is [M+H]+ minus 238, and (c) is [M+H]+ minus 533.1
difference from the molecular ion defines the carbon-chain length and the state of saturation of the fatty acid. A comparable fragment-ion is not, however, formed from [M + Na]+. Further fragmentation of the ceramide sometimes occurs, for example, cleavage (c) in Fig. 14. The fragmentation behavior of native gangliosides under negative f.a.b. conditions has been thoroughly investigated by Nagai and and by Egge and c o ~ o r k e r s . ~After ‘ * ~ acid ~ ~ ~dosing ~ when necessary (see Section 11,2), all gangliosides give abundant pseudomolecular-ions [ M HI- that frequently constitute the base peak. Lactone formation may occur after acidification, but this does not over-complicate spectral interpretation. Fragment ions are produced by way of Pathways B and C (see Section IV,2). Sequence ions formed by Pathway C are always accompanied by signals two mass units lower, presumably the result of oxidative cleavage. Thus, all nonreducing sequence-ions appear as a characteristic “doublet” that can always be recognized. In contrast, the ions containing the ceramide residue always display the heterogeneity conferred by variations in the sphingosine bases and fatty acids. Positive-ion f.a.b.-m.s. of permethylated derivatives has been used in the following biological studies. ( i ) Characterization of all the major, neutral and acidic glycosphingolipids present in human granulocyte^,^^ human chronic myelogenous-leukemia cellss0 and human embryonal-carcinoma cells.69 (ii) Characterization of blood-group ABH, I, i, Lewis and related glycosphingolipids isolated from human and rabbit erythrocyte ( iii) Characterization of fucose-containing ceramide (79) M.Arita, M.Iwamori, T. Higuchi, and Y. Nagai, 1.Biochem. (Tokyo), 95 (1984) 971-981. (80) M. N. Fukuda, A. Dell, P. R. Tiller, A. Varki, J. C. Mock, and M. Fukuda, J. Biol. Chem., 261 (1986) 2376-2383. (81) H. Egge, M.Kordowicz, J. Peter-Katalinic, and P. Hanfland, J. Biol. Chem., 260 (1985) 4927-4935.
56
ANNE DELL
pentasaccharides isolated from the plasma of blood-group O,Le(a-b-) nonsecretors.82 (iu) Characterization of novel, mannose-containing glyco( u ) Identification of trisalosphingolipids isolated from an sylactosylceramide as a ganglioside in human lung.85 Negative-ion f.a.b.-m.s. has been used to complement the positive f.a.b. data in some of the aforementioned studies, and, as the sole f.a.b. procedure, has been applied to an investigation of the distribution of gangliosides in various rat-tissues,86 and to the analysis of synthetic lysogangli~sides.~~
b. Other Glyco1ipids.-Negative f.a.b.-m.s. was used76to define the sugar sequence and the location of the fatty acid groups in the pyruvic acetalated glycolipids ( 2 2 ) of Mycobacteriurn smegmatis. [ M - HI- Ions were present CH~OR~ HOiC
OH R' = C,, fatty acid R2 = C,, or CL6fatty acid 22
at 1539 and 1511 for molecules containing c16 plus C22fatty acids and C,4 plus CZ2fatty acids, respectively. Two types of fragment ion were observed. Sequence-containing ions were formed by way of Pathway C (see Section IV,2), and additional fragment-ions resulted from loss of an acyl group from the molecular ions. Positive-ion data were also acquired, but these (82) G. Pfannschmidt, J. Peter-Katalinic, M. Kordowicz, H. Egge, J. Dabrowski, U. Dabrowski, and P. HanRand, FEBS Lett., 174 (1984) 55-60. (83) R. G. Dennis, R. Geyer, H. Egge, H. Menges, S. Stirm, and H. Wiegandt, Eur. J. Biochem., 146 (1985) 51-58. (84) R. G. Dennis, R. Geyer, H. Egge, J. Peter-Katalinic, S.-C. Li, S. Stirm, and H. Wiegandt, J. Biol. Chem., 260 (1985) 5370-5375. (85) J.-E. Mansson, H.Mo, H. Egge, and L. Svennerholm, FEBS Lett., 196 (1986) 259-262. (86) M. Iwamori, J. Shimomura, S.Tsuyuhara, and Y. Nagai, J. Biochem. (Tokyo),95 (1984) 76 1-770. (87) S. Neuenhofer, G. Schwarzmann, H. Egge, and K. Sandhoff, Biochemistry, 24 (1985) 525-532.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
57 [M+Nal+= 1762
KMNAcYI-0-
FIG. 15.-Sequence of One of the Oligosaccharides Obtained from the Trehalose-containing Glycolipids of Mycobacferia kansasii. [Cleavage positions in the positive-ion mode (Pathway C, Section IV,2) are shown above the sequence. Cleavage positions in the negative-ion mode (Pathway B, Section IV,2) are shown below the sequence.
were less useful. Abundant [M + NH,]+ ions were produced after ammonium dosing (see Section 11,2), but only one significant fragment-ion was observed, corresponding to loss of the terminal hexose with its attached fatty acid group. F.a.b.-m.s. studies88989 on trehalose-containing glycolipids from Mycobacreria kansasii are included in this Section for convenience, despite the fact that the actual glycolipid was not analyzed. The oligosaccharide chains of these glycolipids may contain xylose (Xyl), fucose (Fuc), 3- O-methylrhamnose (MeRha), and N-acylkanosamine (KanNAcyl) residues, in addition to the aptrehalose-containing tetraglucose ‘‘core.” One example is shown in Fig. 15. Positive and negative f.a.b.-mass spectra were obtained on the underivatized oligosaccharides after the lipids had been released by alkaline hydrolysis. The results defined molecular weights and gave partial sequences. Fig. 15 shows the type of fragmentation observed in this class of molecule. Lipid A is a glucosamine disaccharide which is usually substituted with one or two phosphate groups, two N-acyl groups and three 0-acyl groups, all of the acyl groups being long chain fatty acids. F.a.b.-m.s. can define the overall molecular weight of Lipid A samples and can also assign the fatty acids to the distal and reducing end subunit^.'^^^^ Molecular weights have been obtained using both positive and negative f.a.b.-m.s. Assignment of the fatty acids relies on the characteristic A,-type cleavage at GlcNAc (see Section IV,2) observed in the positive-ion mode. The f.a.b. data from monophosphoryl lipid A’s from Salmonella typhimurium have been reported. The most abundant and highly esterified component gave [M - HI- at 1716 and [M+H]+ at 1718 and corresponds to (23) where X=OH. The corresponding diphosphoryl lipid A (23, X = phosphate) gave [M + H]+ at 1798. (88) S. W. Hunter, P. J. Brennan, and 1. Jardine, Fed. Roc., Fed. Am. SOC.Exp. Biol., 43 (1984) 2036. (89) S . W. Hunter, I. Jardine, D. L. Yanagihara, and P. J. Brennan, Biochemistry, 24 (1985) 2798-2805. (90) N. Qureshi, K. Takayama, D. Heller, and C. Fenselau, J. Biol. Chem., 258 (1983) 12,947- 12.95 1.
Roo
ANNE DELL
58
J --(
Ho O=P-0 \/ HO
NH
NH
I
I
c=o
c=o
CHz
CHI
CH-OR
CH-OR
I
I
I
(CHAo
I
CH3
I
I
I
(CHAI
I
CH3
X = O H or phosphate R = H, lauroyl, hydroxymyristoyl, or myristoyloxymyristoyI 23
Better positive f.a.b. data is recorded if the phosphate groups are converted into their dimethylesters prior to analysis?’ The MGP molecule (1) described in Section I exists in Mycobacreria as polyacylated molecules containing various numbers of acetate, propanoate, isobutanoate, succinate, and octanoate groups. The negative f.a.b.-m.s. properties of several of these substances have been analyzed in detail.” Molecular ions were obtained between m / z 3750 and 4050. The fragmentation pattern was analogous to that exhibited by MGP, with all Pathways described in Section IV,2 being represented. No other carbohydratecontaining compounds have yet given such complex fragmentation patterns, and their f.a.b.-m.s. lability is thus unique so far.
2. Glycoproteins a. N-Glycosylic Cornpounds.+i) Lactosarninog1ycans.-Lactosaminoglycans are composed of long sequences of substituted N-acetyllactosamine residues attached to the Man,GlcNAc, core characteristic of N-linked glycoproteins. These glycans are large and heterogeneous, and thus pose special problems to the analyst. F.a.b.-m.s. has revolutionized their characterization; such complicated structures as that shown in Fig. 16 have now been elucidated, using strategies relying on the f.a.b. mapping procedure described in Section III,4. F.a.b. maps are usually obtained on both the intact lactosaminoglycan, prepared by pronase digestion or by hydrazinolysis, and on the products of exo- and endo-glycosidase digestion. New (91) N. Qureshi, R. J. Cotter, and K. Takayama, J. Microbiol. Methods, 5 (1986) 65-78.
59
F.A.B.-MASS SPECTROMETRY O F CARBOHYDRATES FUC
I I 0
3 N e u N A c o Z - 3 G a l @ l - 4 G l c N A c @ l - 3 G o l ~- I4 G l c N A c @ I - 3 G a l p I - 4 G l c N A c @ I ,
;
r uc
Monal
G o l @ l - 4 G l c N A c @ I-3Ga1~1-4GlcNAc@l-3Gal~1-4GlcNAc@l’
I
\a
1 0
6
3Man@I-4GlcNAcal-4GIcNac NeuNAco2-6Gal@l-4GlcNAc@l-3Ga1~1-4GlcNAcB1-3Gal~1-4GlcNAcBl
- Am
/
‘2Monol G a l @ l - 4 G l c N A c ~ I - 3 G a l ~ 1 - 4 G l c N A -c3~GI a l @ I - 4 G l c N A c B I
9 l o F’JC
7
l o
Fuc
4,
Il a FUC
FIG. 16.-Structure of One of the Major Components of Lactosaminoglycans Found in Human Granulocytes.
nonreducing structures resulting from digestion are immediately apparent from the appearance of diagnostic ions in the f.a.b. map. Several lactosaminoglycans have been fully characterized by using a combined chemical-mass spectrometric strategy based on f.a.b. mapping, methylation analysis, and enzymic digestion. They are the lactosaminoglycan of human Band 3, the erythrocyte anion-transporter, where the fetal5’ and adult4’ glycans contain linear and branched chains, respectively; sialylated fucosylated lactosaminoglycan from human granulocyte^^^.^^; the lactosaminoglycan of human amniotic-fluid f i b r ~ n e c t i n ~the ~ ; branched lactosaminoglycans from PA1 human embryonal-carcinoma cells which are characterized by the presence of novel sialyl a-(2 + 9)-sialyl terminals”; and sialylated fucosylated lactosaminoglycans from chronic myelogenousleukemia cells, which contain the unique structure 14, describeds3 in Section III,4. (ii) Complex Carbohydrates.-Permethylated complex carbohydrates give excellent f.a.b. spectra that are particularly easy to interpret and that contain complete sequence-information. All complex molecules give two sets of A,-type fragment-ions (see Section IV,2) resulting from cleavage of the GlcNAc-mannose linkages and from fission of the chitobiose core. The first set of ions occurs below mass 1500, the second above mass 2000. Minor signals may occur in between. These are derived from p-cleavages, in addition to A,-type cleavage (see Section IV,2) and are only prominent at (92) M. Fukuda, E. Spooncer, J. E. Oates, A. Dell, and J. C. Mock, J. Bid. Chem., 259 (1984) 10,925-10,935. (93) E. Spooncer, M. Fukuda, J. C. Mock, J. E. Oates, and A. Dell, J. Bid. Chem., 259 (1984) 4792-4801. (94) T. Krusius, M. Fukuda, A. Dell, and E. Ruoslahti, J. Bid. Chem., 260 (1985) 4110-41 16. ( 9 5 ) M. N. Fukuda, A. Dell, J. E. Oates, and M. Fukuda, J. Bid. Chem., 260 (1985) 6623-6631.
60
ANNE DELL
high concentrations of the sample. For completeness, they should be assigned, but, in fact, they are not required for sequence analysis. The mass of the molecular ion or ions will confirm the presence or absence of fucose on the chitobiose core. The exact composition of the molecular ion will, however, depend on how the sample was prepared. Complex carbohydrates that are released by hydrazinolysis and subsequently N-reacetylated, reduced and permethylated, sometimes have a variety of structure^'^ at the terminal residue (see Section V,3). Fig. 17 illustrates typical data from a complex oligosaccharide isolated from acute myelogenous leukemia cells?6 Only one nonreducing structure is present, giving the signals at 344 (NeuAc+ minus methanol), 376 ( NeuAc+), 793 (NeuAcGal-GlcNAc+ minus methanol), and 825 (NeuAcGalGlcNAc+). The presence of 793 indicates a 4-linked GlcNAc (see Section V,4). The chitobiose fission-ion is very prominent at 2492, and two sets of molecular ions are present for the non-fucosylated and fucosylated molecules, respectively. The data clearly define the structure as 24. NeuAc-Gal-GlcNAc-Man
\
/
NeuAc-Gal-GlcNAc-Man
Man-GlcNAc-GlcNac
I
f
Fuc
24
Some of the multibranched complex glycans of hen ovomucoid have been analyzed by f.a.b.-m.s. together9' with e.i.-m.s. These glycans exhibit exceptional structural features, because their mannotriose core can be highly substituted, as shown in 25. All samples gave abundant [M+Na]+ ions when analyzed as their permethylated alditols, and this permitted the (96) M. Fukuda and A. Dell, J. Bid. Chem., unpublished results. (97) H. Egge, J. Peter-Katalinic, J. Paz-Parente, G. Strecker, J. Montreuil, and B. Fournet, FEBS Letr., 156 (1983) 357-362.
FIG. 17-Positive F.a.b.-Mass Spectrum of a Complex Oligosaccharide Isolated from Acute Myelogenous Leukemia Cells by Hydrazinolysis Followed by Reduction and Permethylation." {[M+H]+ signals are present at 2769 and 2943 for unreduced NeuAc,Hex,HexNAc, and unreduced NeuAc,Fuc,Hex,HexNAc,; their reduced forms appear at 2785 and 2959, respectively. The signal at 2839 is probably derived from a hydrazone of NeuAc,Hex,HexNAc, (see Section V,3 for an explanation of the molecular-ion data). The major, A,-type ions are assigned in the text. Other signals are assigned as follows: 1464 (combination of p-cleavage and A,-type cleavage; composition NeuAc,Hex,HexNAc;), 1492 (combination of Pathway D cleavage and A,-type cleavage; same composition as 1464), 1668 (compare 1464; composition NeuAc,Hex,HexNAc:), 1696 (compare 1492; same composition as 16681, 1913 (compare 1464; composition NeuAc, Hex,HexNAc:).}
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
61
ANNE DELL
62
GlcNAc p( 1-P 4) ‘Man GlcNAc p( 1 + 2)
a(1 + 3)
’ \
GlcNAc p(1+4)-Man
/
p(1+4)-GlcNAc p ( 1 + 4 ) GlcNAc
GlcNAc p( 1 + 6 ) 25
detection of minor components whose presence was not recognized in n.m.r.spectral and chemical studies. Molecular ions at m / z 983, 1187, 1922, 2167, 2412, 2616, 2657, and 2861 corresponded to compositions Man,.. GlcNAcGlcNAcol, Man,GlcNAcGlcNAcol, GlcNAc4Man,GlcNAcol, GlcNAc4Man3GlcNAcGlcNAcol, GlcNAc,Man,GlcNAcol, GalGlcNac6Man,GlcNAcol, GlcNac,Man,GlcNAcGlcNAcol, and GalGlcNAc,Man,GlcNAcGlcNAcol, respectively. The molecules lacking the terminal GlcNAc of chitobiose were identified by the absence of the fragment ion expected from fission of the chitobiose moiety. Storage products derived from complex glycoproteins have been identified in the brains of Springer spaniels suffering from a progressive nervous disorder.63398F.a.b.-m.s. of the native compounds and their acetylated and permethylated derivatives allowed the assignment of structures 26, 27, and 28. Fuc-GlcNAc-
Asn
26
Gal-GlcNAc-
I
Man-Man-GlcNAc-GlcNAc-
Fuc
I
Asn
Fuc 27
Fuc
I
Gal -GlcNAc-
Man
Gal-GlcNAc-Man
\
/
Man-GlcNAc-GlcNAc-
I
Asn
Fuc
I
Fuc 28
(98) D. Abraham, W. F. Blakemore, A. Dell, M. E. Hentage, J. Jones, J. D. Littlewood, J. E. Oates, A. C. Palmer, R. Sidebotham, and B. G . Winchester, Biochem. Soc. Trans., 12 (1984) 288-289.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
63
(iii) High-D-Mannose Carbohydrates.-The oligosaccharides sensitive to endo-P-N-acetylglucosaminidaseH in the glycoprotein of Friend murine leukemia virus were analyzed by exoglycosidase digestion, by acetolysis, and, after permethylation, by f.a.b.-m.s. and methylation analysis.” Around 85% were found to be high-mannose, and f.a.b.-m.s. gave their exact compositions. The other 15% were of “mixed type,” having an N-acetyllactosamine unit substituted by sialit acid or galactose, in addition to the expected mannose chains. These were readily identified by prominent fragment-ions resulting from cleavage at the GlcNAc residue. The presence of an unusual functional group in the phosphorylated, high-mannose oligosaccharides of slime mold was confirmed by negative f,a.b.-m.s. of an underivatized oligosaccharide released by endo-P-Nacetylglucosaminidase H digestion.”’ A prominent [M - HI-molecular-ion at 1448 was assigned the composition of 1 N-acetylglucosamine, 6 mannoses, and 1 D-mannose 6-phosphate plus an additional 14 mass units, which was subsequently shown by direct chemical ionization-mass spectrometry of a per(deuteriomethy1)ated sample to be a methyl group on the phosphate. The compositions of high-mannose oligosaccharides isolated from urine of animals suffering from genetic or chemically induced mannosidoses have been assigned from their f.a.b.-mass spectra. 101*102 After chromatography, some samples were sufficiently pure to be examined underivatized. Others required conversion into their peracetylated derivatives. F.a.b.-m.s. has contributed to a detailed analysis of the high-mannose carbohydrate chains of prostaglandin endoperoxide synthase from sheep.Io3 Oligosaccharides released by hydrazinolysis were analyzed after N-reacetylation and reduction. The f.a.b. data defined the compositions of the oligosaccharides, and showed that hydrazinolysis had effected a partial fission of the chitobiose core. b. Glycosidic Compounds.-Despite a report to the contrary,lo4f.a.b.-m.s. is preferable to e.i.-m.s. for sequencing the carbohydrates of mucins. The very high abundance of the molecular ions of permethylated samples permits (99) R. Geyer, H . Geyer, H. Egge, H. H. Schott, and S. Stirm, Eur. J. Biochem., 143 (1984) 531-539. (100) C. A. Gabel, C. E. Costello, V. N. Reinhold, L. Kurg, and S. Kornfeld, J. Bid. Chem., 259 (1984) 13,762-13,769. (101) D. J. Abraham, R. Sidebotham, B. G . Winchester, P. R. Dorling, and A. Dell, FEES Lett., 163 (1983) 110-113. (102) D. J. Abraham, P. Daniel, A. Dell, J. E. Oates, R. Sidebotham, and B. G. Winchester, Biochem. J., 233 (1986) 899-904. (103) J. H. G . M. Mustsaers, H. van Halbeek, J. P. Kamerling, and J. F. G. Vliegenthart, Eur. J. Biochem., 147 (1985) 569-574. (104) E. F. Hounsell, M. J. Madigan, and A. M. Lawson, Biochem. J., 219 (1984) 947-952.
64
ANNE DELL
high-sensitivity analysis, and the characteristic cleavages at HexNAc residues facilitate spectral interpretation. The structures of four major and two minor sialylated saccharide-alditols isolated from mucus glycoproteins of human seminal plasma have been established by negative and positive f.a.b.-m.s. of the underivatized saccharide, positive f.a.b.-m.s. of permethylated derivatives, e.i.-m.s., methylation analysis, exoglycosidase digestion, and Cr03 o~idation.’~’ F.a.b.-m.s., n.m.r., and methylation analysis were used to characterize the sialylated carbohydrates derived from salivary-gland mucin glycoproteins of the Chinese swiftlet.lo6 A similar combination of techniques, with the addition of e.i.-m.s., was used in the structural analysis of 9 glycosidically linked, core-region mono-, di-, tri-, and tetra-oligosaccharides of human meconium glycoproteins which express oncofetal antigens.’” These oligosaccharides were obtained by mild hydrolysis with acid, and thus lacked fucose and sialic acid. A detailed study of the 0-linked oligosaccharides present on the surface of normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells has been completed.”* Structures were elucidated by f.a.b.-m.s. after permethylation, and methylation analysis before and after specific exo-glycosidase treatments. Some of the components were shown by f.a.b.-m.s. to be poly( N-acetyllactosaminyl) oligosaccharides, for example, 29. Neu Ac-Gal-GlcNAc-Gal-G1cNAc-Gal-GlcNAc
\ NeuAc-Gal
/
Gal N Acol
29
Human interleukin 2, a 133-residue protein, has been separated into multiple molecular forms by selective immunoaffinity chromatography and chromatofocusing. Most of the heterogeneity has been attributed to variations in glycosylation of the threonine residue in position 3 of the polypep-
(105) F.-G. Hanisch, H. Egge, J. Peter-Katalinic, G . Uhlenbruck, C. Dienst, and R. Fangmann, Eur. J. Biochern., 152 (1985) 343-351. (106) J.-M. Wieruszeski, J.-C. Michalski, J. Montreuil, G . Strecker, J. Peter-Katalinic, H. Egge, H. van Halbeek, J. H. G . M. Mutsaers, and J. F. G. Vliegenthart, in J. F. G . Vliegenthart, J. P. Kamerling and G . A. Veldink (Eds.), Carbohydrates 1984, Abstr. Int. Carbohydr. Symp., 12th, Vonk, Utrecht, July 1-7, 1984, Abstr. A2.26, p. 107. (107) E. F. Hounsell, A. M. Lawson, J. Feeney, H. C. Gooi, N. J. Pickering, M. S. Stoll, S. C. Lui, and T. Feizi, Eur. J. Biochern., 148 (1985) 367-377. (108) M. Fukuda, S. R. Carlsson, J. C. Klock, and A. Dell, J. Bid. Chem., 261 (1986) 12,796- 12,806.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
65
tide hai in.'^^.''^ The N-terminal octapeptides released by tryptic digestion were analyzed by f.a.b.-m.s. Three different molecular ions were obtained, at m / z 778,981, and 1434. The first corresponded to the expected peptide, the second to an additional HexNAc, and the third to an additional trisaccharide unit of composition NeuAc,Hex,HexNAc, . Sequential loss of NeuAc, Hex, and HexNAc by P-cleavages suggested that the NeuAc is linked to Gal and not to GalNAc. In a study of unusual poly(sia1o)glycoproteins from fish eggs, positive f.a.b. spectra have been obtained from underivatized mono-, di, and tri-sialyl oligosaccharides up to five residues in length."' A novel, neutral oligosaccharide present in these glycoproteins has been characterized"* as a-LFuc( 1+ 3)-p-~-GalNAc-( 1 + 3)-P-~-Gal( 1 + 4 ) - P - ~ - G a l1(+ ~ ) - D - G ~ I N A col. The sequence was derived by f.a.b.-m.s. before and after permethylation. Other structural data were afforded by methylation analysis, Smith degradation, deamination, and n.m.r. spectroscopy. 3. Bacterial Polysaccharides
F.a.b.-m.s. has been applied to three types of problem in the bacterial polysaccharide area: assigning compositions (including the number and type of 0-acyl groups), sequencing, and identifying cyclic structures. The last application is covered in Section VI,5. The structure of the mycobacterial methylglucose polysaccharide MGP (1) was revised when its molecular weight was shown to be 3514, 338 mass units higher than had been expected." An enzyme has now been isolated that degrades MGP. F.a.b.-m.s. has helped to characterize the products of its reaction1l3with MGP. The repeating oligosaccharide of the pneumococcal capsular polysaccharide (type 18C) was examined by positive f.a.b.-m.s., using a glycerol matrix dosed with ~a1ts.l'~ Two sets of molecular ions, 74 mass units apart, were present, and these were assigned to a monoacetylated pentasaccharide and its glyceryl glycoside, respectively. F.a.b.-m.s. of the Klebsiellu K54 repeating-unit has conclusively proved that, despite evidence to the contrary, it is not formylated, although acetyl groups are indeed (109) R. J. Robb, R. M. Kutny, M. Panico, H. R. Moms, W. F. DeGrado, and V. Chowdhry, Biochem. Biophys. Res. Commun., 116 (1983) 1049-1055. (110) R. J. Robb, R. M. Kutny, M. Panico, H. R. Morris, and V. Chowdhry, Proc. Natl. Acad. Sci. USA, 81 (1984) 6486-6490. ( 1 11) M. Shimamura, T. Endo, Y. Inoue, S. Inoue, and H. Kambara, Biochemistry, 23 (1984) 317-322. (112) M. Shimamura, T. Endo, Y. Inoue, and S. Inoue, Biochemistry, 22 (1983) 959-963. ( I 13) K. Kamisango, S. Saadat, C. E. Ballou, and A. Dell, Carbohydr. Res., 148 (1986) 309-319. ( 1 14) L. R. Phillips, 0. Nishimura, and B. A. Fraser, Carbohydr. Res., 121 (1983) 243-255.
66
ANNE DELL
present.’, In a comparative of the acidic polysaccharides secreted by different Rhizobium species, a phage has been employed to degrade the polysaccharides. The f.a.b. data gave the first indication that the phage enzyme was an endolyase, rather than a hydrolase, because the mass of the repeating unit was 18 mass units lower then that expected for the intact octasaccharide. The f.a.b. spectra also showed that the repeating unit contained, on average, about one 0-acetyl group, and that some of the repeating units were substituted with a single 0-3-hydroxybutanoyl group. Smith degradation of the capsular polysaccharide from Sfreptococcus pneumoniae Type 9 produced a pentasaccharide derivative whose structure was established by n.m.r. spectroscopy and f.a.b.-m.s. as’’’ a-Glc( 1+ 3)-p-ManNAc(1 -* 4)-p-Glc( 1+ 4)-(~-GlcNAc[ 1-OCH(CH,OH)CH(OH)C02H]. F.a.b.-m.s. of the underivatized sample gave the composition and a partial sequence. The full sequence was afforded by permethyl and peracetyl derivatives. As expected, all major sequence-ions were the result of A,-type cleavage (see Section V,2) at the HexNAc residues, with fragmentation at GlcNAc being particularly prominent. The peracetyl derivative gave additional weak signals for A,-type hexosyl cleavages, and also pcleavages. The structures of the 0-antigen polysaccharides of Klebsiella serotype 0 5 and Escherichia coli serotype 0 8 have been reinvestigated’16 by using n.m.r. spectroscopy, chromium trioxide oxidation, hydrolysis with a specific phage-enzyme, and f.a.b.-m.s. The f.a.b. data showed that the antigen is terminated at the nonreducing end by a methylated residue. Its presence was suggested by the molecular weight of one of the products of phage digestion, and the position of the methylated residue in the sequence was determined from the f.a.b. spectrum of a peracetylated derivative. 4. Plant Cell-Wall Polysaccharides
Negative f.a.b.-m.s. permitted the first complete characterization of plant cell-wall galacturonic acid oligosaccharides that exhibit elicitor activity.32 These molecules are p-( 1+ 4) unbranched homopolymers of galacturonic acid, and molecular-weight data provided sufficient information for a complete structural assignment. Samples that were isolated after partial, acid hydrolysis of soybean cell-walls or citrus pectin gave [M -HI- signals as their major molecular-ions when examined in a glycerol matrix.32 When oligosaccharides were prepared by using an endo(polyga1acturonic acid) (115) C. Jones, 9. Mulloy, A. Wilson, A. Dell, and J. E. Oates, J. Chem. Soc., Perkin Trans I , (1985) 1665-1673. (116) P.-E. Jansson, J. Lonngren, G. Widmalm, K. Leontein, K. Slettengren, S. B. Svenson, G. Wrangsell, A. Dell, and P. R. Tiller, Curbohydr. Res., 145 (1985) 59-66.
F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES
67
lyase, it was found that acid needed to be added to the matrix in order to ensure that multiple molecular ions containing up to 8 or more sodium atoms were converted into one major signal at [M-HI- (see Section II,2).33s"7Spectra were readily obtained on - 5 pg of oligogalactosiduronic acids containing up to 10 residues. Higher-molecular-weight samples were more difficult to analyze, because of their low solubility in suitable solvents. To overcome this problem, these larger oligosaccharides were converted into their (pentafluorobenzy1)oxime derivatives, which yielded both molecular ions and P-cleavage fragment-ions (Pathway B, Section IV,2). The active soybean elicitor was shown32to be a dodecasaccharide from the [M -HI- signal for its di(pentafluorobenzy1)oxime derivative at 2519. Rhamnogalacturonan I1 (RG-11) is a structurally complex, pectic polysaccharide that is present in the primary cell-walls of higher plants. It is composed of 60 glycosyl residues, and is a very complex molecule indeed. For example, on acid hydrolysis, at least ten different monosaccharides are formed, including the novel aceric acid (30), which is the only branched-
OH X =C02H
30
chain, acidic, deoxy sugar that has been identified in Nature."* Aceric acid was first identified in a heptasaccharide obtained when RG-I1 was subjected to mild, acid hydrolysis. Positive and negative f.a.b.-m.s. of the native heptasaccharide defined a molecular weight of 160 for the novel residue, and suggested that it was acidic, because the negative spectrum was considerably more intense than the positive spectrum and the latter contained prominent [M Na]+ and [M K]+ signals, despite the fact that no salts had been added to the matrix. F.a.b.-m.s. of the perdeuteriomethylated derivative,"' together with the results of sugar analysis, yielded the sequence shown in Fig. 18. Cleavages occurred at each glycosidic bond, as shown. Further studies on RG-I1 provided structures for most of the oligosaccharides formed when it is extensively degraded with acid.37 A strategy
+
+
(117) K. R. Davis, A. G . Darvill, P. Albersheim, and A. Dell, Z. Narurforsch., Ted B, 41c (1986) 39-48. (118) M. W. Spellrnan, M. McNeil, A. G . Darvill, P. Albersheirn, and K. Henrick, Carbohydr. Rex, 122 (1983) 115-129. (119) M. W . Spellman, M. McNeil, A. G. Darvill, P. Albersheirn, and A. Dell, Carbohydr. Res., 122 (1983) 131-153.
68
ANNE DELL
198 364 154
948
1128
1 7 tl 7 lM+Na1+=1371 Rha :Aro :Gal f AceA R ha 1Api +7
+
[ L U L
FIG. 18.-Sequence of Heptasaccharide Isolated from RG-11. [Fragment-ions of the deuteropermethylated derivative are shown. Cleavages are all of the A, Type. (Rha = rhamnose, h a = arabinose, AceA = aceric acid, MeFuc = 2-O-methylfucose, and Api = apiose).]
based on the analysis of permethylated oligoglycosyl-alditols was pursued, using a combination of c.i.-m.s., e.i.-m.s., f.a.b.-m.s., and n.m.r. spectroscopy. Because of the inter-laboratory nature of this work, f.a.b.-m.s. was applied only to the molecular-weight determination of the larger oligosaccharides. All f.a.b. analyses were assisted by ammonium dosing (see Section 11,2).
5. Cyclic Polysaccharides Data from f . a . b . - m . ~ . , ~and ~ * ~also'zo * f.d.-m.s., revealed the existence of naturally occurring, large cyclic polysaccharides. The first indication that a molecule may be cyclic comes from its precise molecular-weight determination. Cyclic molecules are 18 mass units less than their linear counterparts. Loss of water may, of course, occur in a number of ways, for example, by dehydration or lactonization, and conclusive evidence for the presence of a cyclic molecule can only be obtained from f.a.b.-m.s. of suitable derivatives, such as the permethyl derivative. Cyclic and dehydrated linear polymers are distinguishable after permethylation, as the cyclic polymer will incorporate one methyl group less than the linear molecule. The p-( 1 + 2)-glucans secreted by Rhizobia and Agrobacteria have been shown to be cyclic.36 Negative f.a.b. spectra of the underivatized glucans contained molecular ions at m / z 2753, 2915, 3077, 3239, 3401,and 3563, corresponding to cyclic polymers varying in size from 17 to 22 residues. After permethylation, the derivatized molecules were subjected to f.a.b.-m.s. with ammonium dosing (see Section 142). Molecular ions were observed at 3499,3693, 3897, 4101, 4305, 4509, 4713, and 4917, and these signals shifted to 3642, 3855, 4068, 4281, 4494, 4707, 4920, and 5133 after perdeuteriomethylation. The molecular weights are those expected for cyclic molecules having 17 to 24 glucosyl units. The very high sensitivity achieved after permethylation is the reason for the observation of components greater than 22 residues. The f.a.b. study also revealed that some degradation had occurred during the permethylation reaction. (120) H. Hisumatsu, A. Amemura, T. Matsuo, H. Matsuda, and T. Harada, 1. Gen. Microbid, 128 (1982) 1873-1879.
F.A.B.-MASS SPECTROMETRY O F CARBOHYDRATES
69
The first heteropolysaccharide for which a cyclic structure has been proposed is the enterobacterial common antigen (ECA), which was shown42 by f.a.b.-m.s. to be a mixture of three cyclic components containing 4, 5 , and 6 repeats, respectively, of a previously characterized trisaccharide (31) in which the GlcNAc residues were known to be partially acetylated at 0-6. CHzOH
CH
v
OH A
B
C 31
The trisaccharide is abbreviated as ABC in the following discussion. Negative-ion f.a.b.-m.s. of ECA showed [M-HI- signals at 3034, 3076, 3118, 3160, and 3202, and at 2427, 2469, 2511, and 2553, indicating that ECA is a mixture of cyclic (ABC)4 and cyclic (ABC)S with different degrees of O-acetylation. F.a.b.-m.s. was then used to monitor a series of reactions, including reduction, hydrolysis, and methyl esterification followed by methanolysis. All of the results were in agreement with cyclic structures. Finally, the f.a.b. spectra of perdeuteriomethylated ECA were investigated. Two major sets of molecular ions were observed, at exactly the mass values predicted for the cyclic molecules. An additional, minor cluster m / z -4400 was also observed, indicating the presence of a small percentage of cyclic (ABC)6 in ECA. Fragmentation pathways A, B, C, and D (see Section IV,2) were all represented in the spectra of perdeuteriomethylated ECA. 6. Miscellaneous
Positive- and negative-ion f.a.b. spectra of sulfated di-, tetra-, and hexasaccharides from chondroitin sulfate have been reported.121The spectra are characterized by multiple molecular-ions containing varying numbers of counter-cations to the sulfate and carboxylate anions, plus fragment ions (121) S. A. Carr and V. N. Reinhold, J. Carbohydr. Chem., 3 (1984) 381-401.
70
ANNE DELL
arising from cleavages on each side of the glycosidic oxygen atom (Pathways B and C, Section IV,2) and fairly prominent losses of sodium sulfite. A positive f.a.b. spectrum of rather poor quality has been reported for a synthetic, sulfated tetrasaccharide having the structure of a heparin fragment.'22 In a study completed during the early development of f.a.b.-m.s., both f.d. and f.a.b. were used to characterize 101 fractions containing neutral oligosaccharides isolated from human milk.64 Samples were examined as their peracetylated alditols. In subsequent work, the structures of two minor acidic oligosaccharides from human milk were i n ~ e s t i g a t e d . 'The ~ ~ permethylated derivatives were analyzed by f.a.b.-ms., and their compositions and sequences were defined by the f.a.b. data. Methylation analysis and partial formolysis were the other principal methods used. The fact that f.a.b.-m.s. can be used to observe cluster ions has been exploited in a study of metal binding to c y c l ~ d e x t r i n s ,and ' ~ ~ in an investigation of the complexes formed between a 3-0-methylmannose dodecasaccharide and alkyltrimethylammonium ions having decyl and hexadecyl as alkyl chains.12' In the latter study, the larger organic cation was shown to form the stronger complex. Cello- and malto-oligosaccharides up to nonasaccharides in the presence of various metal salts'26 have been analyzed by f.a.b.-m.s. The structures of two methyl alduronates obtained by flash hydrolysis of wood chips was deduced by using f.a.b.-ms., n.m.r. spectroscopy, and sugar analy~is.'~' Several groups have analyzed a variety of standard oligosaccharides and glycosides by using m.s.-m.s. technique^'^^'^^^'^^ (for a description of m.s.m.s., see Ref. 131). N9ne of this work gave convincing evidence that m.s.-m.s. is needed, or, indeed, is able, to solve carbohydrate problems, and for this reason, a discussion of m.s.-m.s. is not included herein. Finally, brief mention should made of quantitation. F.a.b.-m.s. is seldom used for quantitation, because the different components in a mixture often (122) M. Petitou, Nouu. Rev. Fr, Hemato/., 26 (1984) 221-226. (123) J.-M. Wieruszeski, A. Chekkor, S. Bouquelet, J. Montreuil, G. Strecker, J. Peter-Katalinic, and H. Egge, Carbohydr. Res., 137 (1985) 127-138. (124) C. Bosso, G. Excoffier, M. R. Vignon, and J. Ulrich, Spectrosc. Inr. J., 3 (1984) 72-80. (125) C. E. Ballou and A. Dell, Carbohydr. Res., 140 (1985) 139-143. (126) C. Bosso, J. Defaye, A. Heyraud, and J. Ulrich, Carbohydr. Res., 125 (1984) 309-317. (127) D. Barnet, C. Bosso, G. Excoffier, and M. R. Vignon, in Ref. 106, Abstr. D8.9, p. 493. (128) S. A. Carr, V. N . Reinhold, B. N. Green, and J. R. Hass, Biomed. Mass Spectrom., 12 (1985) 288-295. (129) G. Puzo, J.-C. Prome, and J.-J. Fournie, Carbohydr. Res., 140 (1985) 131-134. (130) C. Bosso and J. Defaye, in Ref. 106, Abstr. D8.10, p. 494. (131) F. W. McLafferty (Ed.), Tandem Mass Spectrometry, Wiley, New York, 1983.
F.A.9.-MASS SPECTROMETRY OF CARBOHYDRATES
71
desorb at different rates, and relative peak-intensities are therefore timedependent.I2’ Several s t ~ d i e s ~suggested, ~ * ~ ~ however, * ~ ~ ~ that quantitative f.a.b. may sometimes have a part to play in carbohydrate research, especially if the objective is to measure the relative concentrations of closely related compounds. For example, three glycopeptides obtained from fibrinogen, and that had differing sialic acid kontent, were examined as mixtures.13* A linear relationship was observed between ion abundance and molar fraction. VII. FUTUREDEVELOPMENTS The groundwork has now been laid for the exploitation of f.a.b.-m.s. by all carbohydrate chemists involved with the structural analysis of novel compounds. There is no reason why f.a.b.-m.s. should not take its place alongside g.1.c.-m.s., e.i.-m.s., and c.i.-m.s. as a relatively routine, analytical procedure. It is important, however, that f.a.b.-m.s. be integrated into sensible structural ~ t r a t e g i e s , ’and ~ ~ that it be very closely linked to the chemical manipulations that are also being conducted as part of the structural program. The necessary instrumentation should be within the reach of most carbohydrate chemists. Existing mass spectrometers can be readily retrofitted with f.a.b. systems.’34If new mass spectrometers are required, they do not need to be exorbitantly expensive. The majority of carbohydrate problems can be solved by using a 3000 mass-range instrument, because many samples fall within this mass range, and a large proportion of the rest can be brought into it by, for example, phage digestion, hydrolysis, and f.a.b. mapping. Over the past few years, a variety of “small” mass spectrometers have been designed that operate at good sensitivity up to 3000, without the need for expensive, high-field magnets.43 F.a.b.-m.s. can already solve many carbohydrate problems. There is still, however, plenty of scope for improving the technique, and important developments are envisaged in the following key areas over the next few years. ( i ) Improvement in sensitivity, to permit analysis at the picomolar rather than the nanomolar level. Current knowledge suggests that this will require new derivatives that ionize and desorb better than existing derivatives. ( i i ) Improvements in analysis at very high mass, to permit the detection 6f molecular ions from such samples as intact RGII (see Section VI,4) and (132) R. R. Townsend, D. H. Heller, C. C. Fenselau, and Y. C. Lee, Biochemistry, 23 (1984) 6389-6392. (133) A. Dell and M. Panico, in S. J. Gaskell (Ed.), Mass Spectrometry in Biomedical Research, Wiley, New York, 1986, pp. 149-180. (134) M-Scan Ltd. (Ascot, U.K.) specialize in retrofitting all types of mass spectrometers.
12
ANNE DELL
the permethylated lactosaminoglycans (see Section 111,4). It is anticipated that this will necessitate the design of new mass spectrometers that have a better high-mass performance than even the new, extended mass-range instruments described in Section III,2.
ACKNOWLEDGMENTS The author thanks the many FAB users who generously provided reprints and preprints. She is also very grateful indeed lo her collaborators, particularly Professor Clinton E. Ballou, Professor Peter Albersheim, Professor Bengt Lindberg, and Dr. Minoru Fukuda, for introducing her to so many exciting problems, and to Professor Howard R. Moms for his tremendous support. The mass-spectrometery facilities at Imperial College were obtained on grants awarded to Professor Moms by the Science and Engineering Research Council and the Medical Research Council.
ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 45
THE CIRCULAR DICHROISM OF CARBOHYDRATES BY W. CURTISJOHNSON,JR. Department of Biochemistry and Biophysics, Oregon State University, Coroallis, Oregon 97331
I. Introduction..
..........................................................
11. Measuring the Spectrum .................................................. 111. Unsubstituted Carbohydrates ...................... ....................
.................... 1. Monosaccharides .............................. 2. Polysaccharides ... ........................................... 1V. Substituted Carbohydrataes ............................................... 1. Sulfate Derivatives .................................................... 2. h i d e Derivatives.. ................................................... 3. Carboxyl Derivatives.. ................................................. 4. Derivatives Having Mixed Substituents ................................... 5. Non-biological Derivatives .............................................
73 76 78 79 85 92 92 94 102 111 119
I. INTRODUCTION Circular dichroism (c.d.) spectroscopy measures the difference in absorption between left- and right-circularly polarized light by an asymmetric molecule. The spectrum results from the interaction between neighboring groups, and is thus extremely sensitive to the conformation of a molecule. Because the method may be applied to molecules in solution, it has become popular for monitoring the structure of biological molecules as a function of solvent conditions. Commercial instrumentation measures the c.d. of electronic absorption bands in the range of 1000 to 190 nm. Nucleic acids and proteins both have absorption bands in this region, and c.d. has been used extensively to study these molecules. Most sugars are transparent in this region, and so they have been relatively neglected. However, the diverse biological activities of sugars undoubtedly depend on their conformations. Thus, improvements in c.d. instrumentation for the short-wavelength region have stimulated interest in using this powerful technique for investigating the stereochemistry of sugar monomers, the configuration of intersaccharide linkages, the secondary structure of the polymers, and the interaction of sugars with themselves 73
Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
W. CURTIS JOHNSON, JR.
74
CHpOH
CH20H H O G O H
HOG
OH
OH a-D- xy lopyranose
HO@OH
OH HO
a-D-glucopyronose
YOOH
CH20H
HO@OH
a-D-idopyranose CH20H
HO@OHHO
OH
Hb
Q-D-Or0 binopyronose
a-D-0 Itropyranose
a-o-goloctopyranose CH20H
GOH
HO
H
o HO
a-D-1 yxopyronose
a-D-rnonnopyranose CHpOH
HOG
O HC)
Q -D
H
HOO H O O OH
O
H
OH
a-o-gulopyranose
"aoH CH2OH
H
OH
- r i b o pyranose
O
a-D-01 lopyranose
a-D-talopyranose
FIG. l.--a-D-Mdo-pento- and -hexo-pyranoses.
and with other biological molecules. Although the research appears quite promising, work in this area has only begun, so that many problems remain to be solved. In this chapter, the ways in which modern c.d. instrumentation has been used to solve structural problems involving sugars are detailed. The discussion is limited to substituted and unsubstituted pyranoses, and will not cover complexes that can be formed with various chromophores. The structures of the a-D-aldo-pento and -hexo-pyranose monosaccharides are shown in Fig. 1. In all cases, these sugars will be studied as the six-membered-ring tautomer, as shown. The pyranose rings can adopt either of two different chair conformations' called 4C1and 'C4.Pyranoses usually adopt a chair conformation that puts the majority of bulky groups in the equatorial position, so that steric interactions are minimized. The 4Cl(D) conformation and the ring numbering system are shown in formula 1. (1) Eur. 1. Biochem., 111 (1980) 295-298.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
a
75
l
a
1
The orientations of hydroxyl groups for the D-aldohexopyranoses in the 4 Cl(D) conformation are given in Table I. The C-1-0-5 bond is labile in aqueous solution, so that the pyranose exists as an equilibrium mixture of ] p anomer [ 1-hydroxyl the (Y anomer [ 1-hydroxyl group axial for ‘ C 1 ( ~ )and group equatorial for “C,(D)]with other forms. In general, the dissolved monosaccharide is involved in a complex equilibrium involving chair conformations, ring tautomerization, and anomerization. C.d. studies of such unsubstituted sugars are further complicated, because the absorption maxima for these saturated compounds fall below 190 nm. However, these difficulties can be overcome, as will be seen when the c.d. of unsubstituted sugars is discussed. Many biologically important sugars are derivatives having a chromophoric group that absorbs within the range of commercial instrumentation. Not only is the c.d. spectrum of such molecules easier to measure, but the interpretation of the spectrum is simplified, because only the chromophore is involved. Many laboratories have concentrated on the c.d. of such monomers and their polymers, and the results will be discussed. In some cases, derivatives of natural polysaccharides have been synthesized in order to facilitate their study with commercial c.d. instrumentation. Such non-biological derivatives have provided conformational information about the polysaccharide, and they are discussed in Section IV,5.
TABLEI Orientation of Hydroxyl Groups for D-Aldopyranoses in the 4c,(D) Conformation Aldohexose
o-Glucopyranose D-Idopyranose D- Altropyranose D-GakCtOpyranOSe o-Mannopyranose D-GUIOpyraIIOSe D-Allopyranose D-Talopyranose
04
0-3
0-2
16
W. CURTIS JOHNSON, JR. 11. MEASURING THE SPECTRUM
C.d. is a special type of absorption spectroscopy. Thus a c.d. instrument is merely a normal absorption spectrometer with the optics to produce circularly polarized light and the electronics to detect the difference in absorption between left and right polarizations. Fig. 2 gives a block diagram for a typical c.d. instrument. An intense source of light is dispersed by the monochromator. The emerging monochromatic light is first linearly polarized, and the linearly polarized light is then converted into circularly polarized light by a quarter-wave retarder. Modern instruments use an isotropic plate that is stressed electronically to alternately produce light with each type of circular polarization. The light is partially absorbed by the sample, and the transmitted light produces a current in a photomultiplier. The magnitude of this current will have a small alternating-current (AC) component, because the stressed-plate modulator changes the polarization of the light sinusoidally, and the absorption of an asymmetric molecule depends on this polarization. Left- and right-circularly polarized light are produced at the two extremes of the sinusoidal modulation, so that the height of the AC signal as measured by the lock-in detector is a measure of the c.d. This AC signal is very small compared to the average magnitude of the transmitted light, which produces a direct current (DC) in the photomultiplier. To a very good approximation, the difference in absorption (A) between left- (L) and right (R)-circularly polarized light is proportional to the magnitude of the AC signal divided by the magnitude of the DC signal. AL-AR= AA = k(AC/DC)
The absorption of the sample will depend on the pathlength of the cell, 1, and the concentration of the sample, c, just as it does in normal absorption spectroscopy. According to Beer's Law, A(A) = E ( A ) l c ,
where the constant of proportionality, E, called the extinction coefficient, is a function of the wavelength of the incident radiation, A. The standard procedure is to express 1 in cm, and c in mol 1-', so the units of E are 1 cm-' * mol-'. This is the characteristic of the molecule obtained by measuring a normal absorption spectrum. In c.d., the characteristic of the molecule that is seen is the difference in extinction coefficient for the two types of circularly polarized light. Each type of light obeys Beer's Law, so that
-
EJA)-E~(A)=AE(A)=AA(A)/Ic.
Linearly polarized light passing through an asymmetric sample will become
71
THE CIRCULAR DICHROISM OF CARBOHYDRATES
source
+
monochromator
0 polarizer
modulating
retarder
power supply
lock-in
FIG. 2.-Block
J
chart recorder
Diagram of a Circular Dichroism Instrument.
elliptically polarized because of the c.d. Some workers report c.d. as the angle of ellipticity, i,b in degrees. This is related to the difference in absorption by
# = 32.98AA. The characteristic of the molecule is sometimes expressed as the molar ellipticity in deg . d l . mol-' dm-'. This is related to the difference in extinction coefficients by
-
[el = 3298 A&, where the factor of 100 enters for historical reasons. C.d. instruments are usually calibrated for the sign and magnitude of the constant in Eq. (1) by means of an aqueous solution of (+)-locamphorsulfonic acid (CSA). This compound has2 a A& of 2.36 for the c.d. maximum at 290.5 nm. A solution of 1 mg ml-' in a 1-mm pathlength cell has a AA of 1 . 0 2 ~ or a i,b value of 33.5 mdeg. The concentration of this hygroscopic material is readily determined by absorption spectroscopy. Because ~ ( 2 8 5 n m )is 34.5 for the anhydrous material, a solution of 1 mg * ml-' will give an A value of 0.743 in a 5-cm cell. CSA also has a negative c.d. band at 192.5 nm, with a AE of -4.8 for a convenient two-point calibration.
-
(2) G. C. Chen and J. T. Yang, Anal. Leu. 10 (1977) 1195-1205.
78
W. CURTIS JOHNSON, JR.
Because c.d. is measured as the small difference in absorption for the two types of circularly polarized light, these instruments always work at their limits of reliability, and the spectra tend to be “noisy.” The noise is statistical in nature, and proportional to the square root of the amount of light falling on the photomultiplier. As the absorbance of the sample is increased, the c.d. signal is increased proportionally, but the noise simultaneously increases because there is less transmitted light falling on the photomultiplier. An absorbance of 0.87 will give the best signal-to-noise ratio. In practice, 0.4 to 1.0 constitutes a practical range. Too little light falling on the photomultiplier can lead to artifacts. Workers should always measure the total absorbance of cell, solvent, and sample, in order to ensure that it does not exceed 1.0 over the range studied. It goes without saying that solvents should be transparent if the total absorbance is to be kept below 1.0. Furthermore, the wide variety of pathlengths, from 10 cm to 10 pm, that are commercially available to the experimentalist are particularly useful. A solvent that has an absorbance of 1.0 in a 1-mm cell will have an absorbance of only 0.1 in a 100-pm cell. Although the concentration of the sample will have to be increased as the pathlength is decreased, only 100 pL of solution are needed in order to fill the space between the windows in short-pathlength cells. Workers will find 100- and 50-pm cells particularly valuable for work at wavelengths shorter than 200 nm. Relatively long time-constants of 1 to 60 s are used in order to minimize the noise inherent in c.d. spectra. Thus, the scanning rate is low, and as c.d. instruments tend to drift with time, they should be calibrated daily, and baselines should be measured for each sample. CARBOHYDRATES 111. UNSUBSTITUTED Nucleic acids possess chromophoric bases that have absorption bands beginning at 300nm. The amide group of proteins has absorption bands with maxima at 215 and 190nm (and shorter wavelengths). However, unsubstituted sugars are saturated, and their chromophores, the CO and OH groups, have absorption maxima only below 185nm, which is the present limit of commercial c.d. instrumentation. Nevertheless, Listowsky and Englard3 successfully interpreted the long-wavelength tails of the first c.d. bands for a number of monosaccharides. All subsequent c.d. work has used instrumentation capable of making measurements far into the vacuum ultraviolet (u.v.). C.d. spectra of polysaccharides have been measured to 140 nm for films, and to 164 nm for aqueous solutions when extremely thin cells were used. (3) 1. Listowsky and S. Englard, Biochem Biophys. Res. Commun., 3 0 (1968) 329-332.
THE CIRCULAR DlCHROlSM OF CARBOHYDRATES
79
I. Monosaccharides
The pyranoid monosaccharides provide a wide range of asymmetric molecules for study by the c.d. spectroscopist. However, these compounds are not without their difficulties. In aqueous solution, these compounds exist in a complex equilibrium involving the two possible chair conformers of the pyranoses, the furanoses, a and p anomers, and the acyclic form, as well as septanoses for aldohexoses and higher sugars. In order to minimize conformational problems, Nelson and Johnson4 chose D-xylose, D-glucose, and D-galactose for their studies. These sugars preferentially adopt the 4C, conformation of the pyranose form. Furthermore, the kinetics of anomerization are sufficiently slow that it was found possible to measure the c.d. of individual anomers. The results, given in Fig. 3, demonstrated that c.d. spectroscopy is indeed sensitive to differences among the monosaccharides. Fig. 3c shows that the first c.d. band for a-D-galactose is positive, whereas the first c.d. band for P-D-galactose is negative, and considerably more intense. This difference between the cad. spectra of the anomers demonstrates that it is not reasonable to make comparisons between c.d. spectra of pyranoses at anomeric equilibrium. The problems of anomeric equilibrium may be avoided by investigating 2-ketoses. Both a hydroxyl group and a hydroxymethyl group are attached to the anomeric carbon atom in such sugars, and the bulky hydroxymethyl group favors the equatorial position. These authors measured c.d. spectra for three ketoses, the 2-(hydroxymethyl) derivatives of a -L-xylose, a-Dxylose, and a-D-mannose, in aqueous solution. The anomeric hydroxyl group is replaced by a methoxyl group in the methyl glycosides. These compounds have stable ring-structures, so that equilibria among anomers and ring structures is not a problem. There is still an equilibrium between chair conformations, but, if sugars are chosen for which most of the groups are equatorial, one chair conformation will preponderate. Nelson and Johnson’ measured the c.d. spectra of 12 methyl pyranosides in aqueous solutions, and the results for 6 of them are given in Fig. 3. The c.d. spectra of these 21 monosaccharides that were studied contain a wealth of information, although proper analysis of the data is not always obvious. However, c.d.-difference spectra between pairs of sugars that differ at only one carbon atom can be used to simplify the analysis. Each of the chromophores in a monosaccharide (hydroxyl, methoxyl, hydroxymethyl, hemiacetal, and acetal) are symmetric and obtain their c.d. by interaction (4) R. G. Nelson and W. C. Johnson, Jr., J. Am. Chem. SOC.,98 (1976) 4290-4295. (5) R. G. Nelson and W. C. Johnson, Jr., J. Am. Chem. SOC.,98 (1976) 4296-4301.
W. CURTIS JOHNSON, JR.
80
2 I
o Ac -I
-2
0 -I
-2
-3 -4
I
I
I80
X (nml
I
200
I
180
X (nm)
I 200
FIG. 3.-Circular Dichroism Spectra of a (-) and @ ( - - - ) Pyranose Anomers of (a) D-Xylose; (b) D-Glucose; (c) D-Galactose; (d) Methyl D-Xyloside; (e) Methyl D-Glucoside; and ( f ) Methyl D-Galactoside. (Redrawn from Refs. 4 and 5.)
with other groups that are asymmetrically disposed about them. If it is assumed that the principle of pairwise interaction is valid, the c.d. will be given by the sum of pairwise interactions between the groups. C.d.-diff erence spectra then reflect changes in group interactions between the two molecules compared. Fig. 4 shows c.d.-difference spectra that are due to the presence of a hydroxymethyl group in one member of the pair. The pairs of pyranoses have the same configuration about each asymmetric carbon atom that is part of the ring. Three related pairs have the same configuration at the carbon atoms near the hydroxymethyl group and give very similar difference spectra. The similarity indicates that the rotameric distribution for the hydroxymethyl group is similar for each of these pairs. This is to be expected, as all three sugars have 4-hydroxyl groups oriented equatorially, but the relationship is certainly not obvious from the c.d. spectra themselves (see Fig. 3). In contrast, when the configuration near the hydroxymethyl group
THE CIRCULAR DICHROISM OF CARBOHYDRATES I
I
I
180
170
X
I
81
I
190
(nrnl
FIG. 4.-Difference-Circular Dichroism Spectra: a-D-manno- Heptulose minus a - D Tagatose (. . .); a-D-Glucose minusa-D-Xylose (- . - .); P-D-GhJCOSe minusp-D-Xylose (-); a-D-Sorbose minus a-D-Xylose (- - -). (Redrawn from Ref. 4.)
is different (as it is for the fourth pair), the c.d.-difference spectrum is very different. In general, difference spectra are similar when near neighboring groups are similar, but are different when near neighboring groups differ, demonstrating that c.d. is sensitive to configuration and conformation. These similarities reveal that the pyranoses and methyl pyranosides have more in common than is apparent from the c.d. spectra themselves, and where comparisons are possible, that the two types of sugar have the same conformation. Methyl D-pyranoside pairs have difference spectra that are similar to the corresponding pyranose pairs, even though the c.d. spectra are very different. For instance, the two methyl D-glucopyranoside and methyl Dxylopyranoside pairs show difference spectra that are similar to those shown for the pyranoses in Fig. 4, even though no such relationship is obvious from the c.d. spectra themselves. Because so many different chromophores are involved in the c.d. spectrum of a monosaccharide, fundamental interpretation of the spectrum is difficult. On the other hanil, similarities among the various diff erence-spectra having identical conformations in near neighboring groups suggest that a catalog
82
W. CURTIS JOHNSON, JR.
FIG. 5.-Circular Dichroism Fragment-Spectra (from Top to Bottom): Addition of a Hydroxymethyl Group at C-5, with the OH-4 Group Equatorial, Average for Five Pairs of Aldoses; 4-Hydroxyl Group Equatorial to Axial, One Pair of Aldoses; Addition of a Hydroxymethyl Group at C-5, with the 4-Hydroxyl Group Axial for a-D-Aldoses, Average for Two Pairs of Aldoses; and Addition of a Hydroxymethyl Group at C-5, with the 4-Hydroxyl Group Axial for P-D-AIdG. x,Average for Two Pairs of Aldoses. (Redrawn from Ref. 6.)
of c.d. “fragment” spectra might be compiled.6 A c.d. spectrum for a monosaccharide of given configuration and conformation could be predicted by algebraically summing such c.d. fragment-spectra. The c.d. spectra for the 21 monosaccharides that were studied provide the data for taking the first steps in compiling a catalog of fragment spectra. The c.d. spectra of the D-xylose monosaccharides, given in Figs. 3a and 3d, are difference spectra in the sense that the asymmetry of the sugar is due entirely to the functional group on the anomeric carbon atom, C-1. These four c.d. spectra provide the fundamental c.d. fragment-spectra in the catalog. Other examples of c.d. fragment-spectra are given in Fig. 5 for four functional (6) W. C. Johnson, Jr., Curbohydr. Res., 58 (1977) 9-20.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
83
4
A€
2
0
-1
A€
0
-I 180
X
(nm)
200
180
X
200
(nm)
FIG. 6.-Measured Circular Dichroism Spectrum (-), Calculated Circular Dichroism Spectrum (- -), and Fragment Circular Dichroism Spectra (- - -) for the Corresponding DXylose and the Addition of a Hydroxymethyl Group at C-5: (a) a-D-<jlucose; (b) &D-Ghcose; (c) Methyl a-D-Glucopyranoside; and (d) Methyl P-D-Glucopyranoside. (Redrawn from Ref. 6.)
groups. The c.d. spectra of monosaccharides that have already been measured can be computed from a limited number of the fragment spectra in the catalog. As demonstrated in Fig. 6, the agreement between calculated and measured spectra is quite good. The c.d. fragment spectra may also be used to predict the c.d. spectrum of a sugar that has not yet been measured. The c.d. spectra of three monosaccharides are predicted in Fig. 7. These spectra were calculated in two different ways, and the agreement between the calculated spectra is fairly good. C.d. spectra measured for (-)-(S)-2-ethyltetrahydropyran' and ( S ) - ( + ) 1,2,2-trimethylpropyl ethyl ether* in solution demonstrate that the ether chromophore makes a substantial contribution, if not the total contribution, to the long-wavelength band for carbohydrates in aqueous solution. Solvent effects on ( S ) - (+)-1,2,2-trimethylpropyl ethyl ether show that the positions of the long-wavelength bands are sensitive to hydrogen bonding by the solvent,' and thus arise from excitation of the nonbonding pair of electrons. Lowering the temperature for this compound in a hydrocarbon solvent shifts the longest-wavelength band to the blue, testifying to its Rydberg (7) C. Bertucci, R. Lazzaroni, and W. C. Johnson, Jr., Carbohydr. Res., 132 (1984) 152-156. (8) C. Bertucci, R. Lazzaroni, P. Salvadori, and W. C. Johnson, Jr., J. Chem. SOC.,Chem. Commun., (1981) 590-591.
84
W. CURTIS JOHNSON, JR.
FIG. 7.-Predicted (- -) and Fragment (- - -) Circular Dichroism Spectra: P-L-Arabinose Calculated from (a) CY-D-XylOSe, and (b) Methyl P-L-Arabinoside; a-L-Arabinose Calculated from (c) P-D-XylOSe, and (d) Average Calculated for P-L-Arabinose; Methyl a-L-Arabinoside Calculated from (e) Methyl P-D-Xyloside, and (f) Methyl P-L-Arabinoside. (Redrawn from Ref. 6.)
character.'-'' On the other hand, an intrinsic, magnetic-transition dipole is necessary to account for the intensity of the c.d. for oxygen chromophores"p'2 such as hydroxyl groups and ethers, and this is present in a n + u* transition, but not in Rydberg's. The true transition is unE. H. Shaman, 0. Schnepp, P. Salvadori, C. Bertucci, and L. Lardicci, J. Chem. SOC., Chem. Commun., (1979) 1000-1001. M. B. Robin, Higher Excired States of Polyatomic Molecules, Academic Press, New York, 1974, Vol. 1, p. 265. P. A. Snyder and W. C. Johnson, Jr., J. Chem. Phys., 59 (1973) 2618-2628. P.A. Snyder and W. C. Johnson, Jr., J. Am. Chem. SOC.,100 (1978) 2939-2944.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
85
doubtedly a mixture ( n + 3s/ a*) of the two idealized, one-electron assignment~.’~-’~ These assignments are consistent with the c.d. measurements. The c.d. spectra now measured for 21 monosaccharide^^^ together with the difference spectra, provide enough information for specific chromophores to be assigned the c.d. bands observed in the vacuum U.V. First, the configuration of the anomeric carbon atom of the methyl pyranosides is correlated with the sign of the second and third c.d. bands (counting from the longwavelength end of the spectrum). For instance, as may be seen in Fig. 2d, methyl a-D-xylopyranoside has a positive second band and a negative third band, whereas methyl fl -D-xylopyranoside has opposite signs for these two bands. This suggests that the methoxyl group is responsible for the 174-nm band and for at least part of the intensity of the 165-nm band in methyl pyranosides. Many c.d.-diff erence spectra indicate that the hydroxyl chromophore, which hydrogen-bonds well with the aqueous solvent, contributes intensity only below 175nm. The first band in the c.d. spectra of both the pyranoses and the methyl pyranosides is due to the ring-oxygen atom, as Listowsky and Englard3 suggested. This band is not obvious for either a- or fl-D-xylose, but it is significantly red-shifted when a hydroxymethyl group on C-5 or a methoxyl group on C-1 shields the ring-oxygen atom from the hydrogen-bonding ~ o l v e n t . ~The ” sign of the first band is then dependent on the configuration of groups about the ring-oxygen atom, particularly the orientation of the hydroxymethyl group on C-5. This assignment is consistent with assigning the methoxyl group lower energies than the hydroxyl group.
2. Polysaccharides C.d. spectra have now been measured for a number of unsubstituted polysaccharides. The structures for amylose and pustulan are given as formulas 2 and 3, respectively.
:= CHzOH
HO
Ho 2 (13) (14) (15) (16) (17)
Ho’?EL Ho
Hgo3kL Ho
HO
3
J. Texter and E. S. Stevens, J. Chem. Phys., 69 (1978) 1680-1691. J. Texter and E. S. Stevens, J. Chem. Phys., 70 (1979) 1440-1449. J. Texter and E. S. Stevens, J. Org. Chem, 44 (1979) 3222-3225. B. K. Sathyanarayana, E. S. Stevens, and J. Texter, Biopolymers, 24 (1985) 1365-1383. G. A. Segal, K. Wolf and J. J. Diamond, J. Am. Chem. Soc., 106 (1984) 3175-3179.
86
W. CURTIS JOHNSON, JR.
.4
.L
A€ o
-. 2
-.4
I
I 200
I
180
X
I
c
10
(nm)
FIG. 8.-Circular Dichroism Spectra of a Solution of 20 mg of Pustulan mL at Day 0 (. . .), Day 7 (- * . -), Day 17 (- - -), Day 32 (- . -), and Day 38 (-). (Redrawn from Ref. 18.)
The configuration of each anomeric hydroxyl group that forms a polymer linkage is fixed, and the type of linkage has a significant effect on the physical properties of the polymer, as will be seen. Extensive studies have been performed on the (1 + 6)-P-~-glucan(pustulan)I6 and the (1 + 4)-cu-~-glucan(amylase).'* These are linear polysaccharides that may exist as helical polymers in aqueous solution, as demonstrated by c.d. spectros~opy.’~*’~ Characteristic of the helical structure of these glucans is a negative band at 182 nm, a crossover at 177 nm, and a more intensely positive band at shorter wavelengths (see Figs. 8 and 9). Stipanovic and Stevens” monitored the generation of the helical structure of pustulan by observing the formation of gels over a 38-day period. The authors attributed the initial observation of the c.d. band at 190nm (see Fig. 8) to formation of the helical structure, and the blue shift of the band with aging, to aggregation of the helices. Both Na+ and Ca2+ accelerate gelation, with Ca2+ proving to be about twice as effective as Na+. Because pustulan is not charged, the acceleration is presumably attributable to a decrease in the activity of the aqueous solvent. Fresh solutions of pustulan, which are presumably in a random-coil conformation, have a positive c.d. at 180 nm (see Fig. 8). The c.d. spectrum
-
-
(18) D. G. Lewis and W. C. Johnson, Jr., Biopolymers, 17 (1978) 1439-1449. (19) A. J. Stipanovic and E. S. Stevens, Inr. J. Biol. Macromol., 2 (1980) 209-212.
87
THE CIRCULAR DICHROISM OF CARBOHYDRATES
1
Ae
\
/
0 -1
-0
1 ;
‘1
/
I 170
............... 180 .....................190””
I 200
X (nm)
/
-2
FIG. 9.-Comparison Cyclomaltohexaose (-),
of the Circular Dichroism Spectra Observed for Amylose (. . .), and Methyl a-o-Glucopyranoside (- - -). (From Ref. 19.)
of the corresponding dimer, gentiobiose, is similar to that of the fresh solution of pustulan, but more intense. This indicates that some kind of chainlength dependence must exist. Lewis and Johnson’8 compared the c.d. spectra of amylose and cyclomaltohexaose, and showed that amylose is helical in aqueous solution. Cyclomaltohexaose is chromophorically equivalent to amylose, and it is known to assume a pseudohelix having zero pitch, and thus, no helical chirality. The conformation of amylose is clearly different from that of cyclomaltohexaose, as their c.d. spectra are very different (see Fig. 9). The difference in conformation was considered to be a matter of helical chirality. To confirm this, these workers measured the c.d. spectrum of an amylose-lbutanol complex presumed to have the V-form of helical conformation with the 1-butanol complexed in the channel of the helix.” The c.d. spectrum of the complex is identical to that of amylose in aqueous solution. In contrast to pustulan and gentiobiose, the amylose oligomers do not have a chainlength dependence.I8 Fig. 10 shows that the changes in c.d. with chainlength are progressive, with the blue shift of the 165-nm band uncovering the 182-nm band as the chainlength is increased. Pfannemuller and Ziegast” have shown that longer oligomers also fit into this series with (20) W. Banks and C. T. Greenwood, Polymer, 12 (1971) 141-144. (21) B. Pfannemuller and G . Ziegast, in D. A. Brant (Ed.), Solution PropertiesofPolysacchurides, Am. Chem. SOC.Symp. Ser., 150 (1981) 529-548.
W. CURTIS JOHNSON, JR.
88
A€
-31
'
I
170
I 180
X
I
I 190
I
I
200
(nm)
FIG.10.-Circular Dichroism Spectra of Amylose (-), Maltohexaose (- . -), Maltotetraose (- - -), Maltotriose (- . -), and Maltose (. . .) in Aqueous Solution at 10". (From Ref. 19.)
no abrupt change, indicating helix formation. This suggests that the conformation of the interior subunits is approximately the same in the oligomers and in amylose. All three c.d. spectra given in Fig. 9 (of amylose, cyclomaltohexaose, and methyl a-D-glucopyranoside) are due to interior Dglucopyranosyl units. However, the three c.d. spectra are very different from each other. This demonstrates once again the sensitivity of c.d. spectroscopy to differences in the conformation of sugars, probably at the glycosidic oxygen atom in this case. Stevens and coworkers measured the c.d. of a large number of Dg l u c a n ~ . ' ~The . ~ ~results, - ~ ~ given in Table 11, allowed them to reach certain generalizations about the spectra. First, gel formers display a negative band in the 180-190-nm region, as was discussed in detail for amylose and pustulan. They attributed this to local inflexibility in the polysaccharide chain. Second, all D-glucans studied exhibit a c.d. band in the 164-172-nm region. This band is positive for a-linkages and negative for p-linkages in the case of (1-+ 3)-and (1 + 4)-glucans, but uncorrelated with anomeric configuration for the (1+ 6)-glucans. Third, all of the D-glucans showed a (22) A. J. Stipanovic, E. S. Stevens, and K. Gekko, Macromolecules, 13 (1980) 1471-1473. (23) A. J. Stipanovic and E. S. Stevens, in Ref. 21, pp. 303-315. (24) L. A. Buffington, E. S. Stevens, E. R. Moms, and D. A. Rees, In?. J. Biol. Macromol., 2 (1980) 199-203. (25) A. J. Stipanovic and E. S. Stevens, Biopolymers, 20 (1981) 1183-1189.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
89
band in the 145-150-nm region in their film spectra. With the exception of cellulose, this short-wavelength band is opposite in sign to that of the 164-177-nm band. Cellulose has only positive c.d. of strong intensity at short wavelengths, which is probably due to its special structural features in the solid state. TABLEI1 Circular Dichroism Bands for Unsubstituted Polysaccharides
D-Clucan
Branch linkages
Common name
(1+3)-a
-
Pseudonigeran
film
(1+3)-P-
-
Curdlan
gel film
(1+4)-a-
-
Amylose
solution
Form
gel film
(1+4)-a-
-5% (1+6)-a-
Amylopectin
solution film
(1+4)-a-
-15% (1+6)-a-
Glycogen
solution film film
(1 + 4)-p-
-
Cellulose
(1+6)-a-
-5% ( 1 + 3 ) - a -
Dextran
solution film
Pustulan
solution gel
(1+6)-P-
(1+3)-a( 1 + 4)-a( 1 + 6)-a[(I +4)-a-i2
(om)
173 145 1175 172 145 182 1175 184 <175 184 168 150 182 <175 182 166 1150 182 <175 166 157 150 S 177 167 150 <177 184 <175
A%,, +0.82 -0.21 negative -0.42 positive -0.27 positive -0.27 positive --0.1 f0.36 negative -0.27 positive -0.06 +0.33 negative -0.14 positive +0.85 -3.9 negative +0.79 +0.49 negative positive -0.36 positive -0.08
film
180
164 164
f0.09
187 <180 166
-0.06 positive +1.0
-
Nigeran
film
-
Pullulan
solution film
+0.49
90
W. CURTIS JOHNSON, JR.
Texter and Ste~ens’~-’’ investigated the hydroxyl and ether chromophores theoretically, and concluded that each contributes n + a*/3s intensity in the 145-190-nm region. The precise transition wavelength will depend on the chromophore and its conditions, but the 180-190-nm band found for the gel formers is assigned to the ether oxygen atom of the linkage. Sathyanarayana and coworkers’6 calculated the c.d. of these polysaccharides when they are in the region still allowed by their flexibility, and obtained results in good agreement with experiment. Stevens and coworkers used their c.d. on the various D-glucans to assign, tentatively, the bands to specific chromophores. They found that derivatives of these polysaccharides that have all of their hydroxyl groups acetylated still exhibit the 177-nm band. They assigned this band (which occurs at somewhat shorter wavelengths for the helical polymers) to the ether of the acetal chromophore. This assignment is essentially consistent with the results obtained by Johnson and coworkers on unsubstituted monosaccharides. Three related galactomannans have been as films to 140nm. These polysaccharides have a (1 + 4)-P-~-mannopyranosyIbackbone with (1 + 6)-a-~-galactopyranosylsubstituents (see formula 4). H°CH OH
G? HO HO 4
The c.d. spectra were measured for the guar derivative containing 39% of a-D-galactopyranosyl groups; the tara derivative, 25% of a-D-galactopyranosyl; and the carob derivative, 19% of a-D-galactopyranosyl. The c.d. spectrum of guar as a solid film is given in Fig. 11. A positive c.d. band is observed at 169 nm, and a negative band at 145 nm. The intensity of these bands varies linearly with the content of D-galactopyranose, so that it is possible to extrapolate the measurements in order to find the intensities of these two bands for both D-mannan and D-galactan. At 0% of a-D-galactopyranose, the authors22found molar ellipticities of -8,000 and +80,000. At 100% of a-D-galactopyranose, the molar ellipticities are + 14,000 and -33,000. The longer-wavelength band was investigated in aqueous solution, and found to be similar to that observed for the films, but with substantially
THE CIRCULAR DICHROISM OF CARBOHYDRATES
91
.2
Ac
0
-. 2
150
170
160
X
180
190
(nm)
FIG.1 1.-Circular Dichroism Spectrum for a Solid Film of Guar Galactomannan. (Redrawn from Ref. 24.)
greater amplitude. The shorter-wavelength band in aqueous solution was predicted by measuring the optical rotatory dispersion (0.r.d.) and utilizing a Kronig-Kramers transform. The 0.r.d. could be accounted for by the observed band at 169 nm, and a predicted band of opposite sign at 145 nm, as observed in the film spectra. Agarose is closely approximated as the alternating copolymer of (1+ 3)-/3-~-galactopyranoseand (1+ 4)-3,6-anhydro-a-~-galactopyranose (see formula 5).
HoBow
/o
HO
0
5
The polymer is capable of forming left-handed, double helices, and undergoes a sol-gel transition where a network is formed through cooperative association of the helices. Fig. 12 shows the c.d. spectra of agarose in aqueous solution at various temperatures.26 As the temperature is increased, ( 2 6 ) J. N.Liang, E. S. Stevens, E. R. Morns, and D. A. Rees, Biopolyrners, 18 (1979) 327-333.
W. CURTIS JOHNSON, JR.
92 I
I
I
I
b
200
180
X
(nm)
FIG. 12.-Circular Dichroisrn Spectrum of Agarose Solution (1.5% w/v) at (a) 25", (b) 45", (c) 67", and (d) 78". (Redrawn from Ref. 26.)
the positive c.d. band decreases in intensity and shifts to longer wavelengths. This change in c.d. intensity is cooperative, but involves a hysteresis, as Fig. 13 shows. The authors considered that changes in intensity and position of the 180-nm c.d. band characterize helix formation and "melting" of the agarose, whereas the hysteresis monitors helix-helix aggregation. The vacuum-u.v. c.d. of an agarose film measured to 140nm has the positive band, observed in aqueous solution, at 180 nm and a more intense, negative band at 152 nm.
IV. SUBSTITUTED CARBOHYDRATES 1. Sulfate Derivatives
Many naturally occurring sugars have a sulfate group attached to one of the carbon atoms. Because the sulfate group does not have any transitions
THE CIRCULAR DICHROISM OF CARBOHYDRATES
I
20
I 40
I
I
60
80
93
0
T ("C) FIG. 13.-The Intensity of the 188-nm Circular Dichroism Band of Agarose as a Function of Temperature. (Redrawn from Ref. 26)
within the range of commercial instrumentation, these sugars present the same problems that are encountered with unsubstituted sugars. The c.d. spectrum of b-carrageenan, a polymer of sulfate derivatives, has been measuredz7in the vacuum-u.v. region. As shown in formula 6, b-carrageenan is approximated as an alternating copolymer of 4-sulfato-/3-~-galactopyranose and 3,6-anhydro-2-sulfato-c~-~-galactopyranose.
6
The c.d. spectrum of a film of this polymer is shown in Fig. 14. It is similar, but opposite in sign, to the c.d. spectrum of agarose, with the shortwavelength band somewhat red-shifted.
(27) J. S. Balcerski, E. S. Pysh, G. C. Chen, and J. T. Yang, J. Am. Chem. Soc., 97 (1975) 6274-6275.
94
W. CURTIS JOHNSON, JR.
X
(nm)
FIG. 14.-Circular Dichroism of an &-CarrageenanFilm and a Sodium Hyaluronate Film (- - -). (Redrawn from Refs. 27 and 29.)
2. Amide Derivatives Amide derivatives have proved especially useful sugars for study by c.d. spectroscopy. The amide substituent is the same as the chromophore found in proteins, so that its optical properties have been extensively studied both experimentally and theoretically. 2-Acetamido sugars are found in many is glycoproteins. The structure of 2-acetamido-2-deoxy-a-~-glucopyranose given as an example in formula 7.
HO
7
The c.d. spectra of three common 2-acetamido derivatives in aqueous ' - ~ ~ measured such ~ o l u t i o n are ~ * given ~ ~ ~ in Fig. 15. Many l a b ~ r a t o r i e s ~have (28) C. A. Bush, in B. Pullman and N. Goldblum (Eds.), Excited States in Organic Chemistry and Biochemistry. Reidel, Dordrecht, Holland, 1977, pp. 209-220. (29) L. A. Buffington, E. S. Pysh, B. Chakrabarti, and E. A. Balazs, J. Am. Chem. Soc., 99 (1977) 1730-1734. (30) E. A. Kabat, K. 0. Lloyd, and S . Beychok, Biochemistry, 8 (1969) 747-756. (31) A. L. Stone, Biopolymers, 10 (1971) 739-751. (32) J.-P. Aubert, B. Bayard, and M.-H. Loucheux-Lefebvre, Carbohydr. Rer, 51 (1976) 263-268. (33) G . Keilich, Carbohydr. Res. 51 (1976) 129-134. (34) P. L. Coduti, E. C. Gordon, and C. A. Bush, Anal. Biochem., 78 (1977) 9-20. (35) H. R. Dickinson, P. L. Coduti, and C. A. Bush, Carbohydr. Res. 56 (1977) 249-257.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
95
A€
L
I
180
I
I
I
I
200
X
220
I
I
240
(nm)
FIG. 15.-Circular Dichroism of 2-Acetamido-2-deoxy-~-glucopyranose (-), 2Acetamido-2-deoxy-~-galactopyranose (- - -), and 2-Acetamido-2-deoxy-~-mannopyranose (. . .) at Anomeric Equilibrium in Aqueous Solution (redrawn from Ref. 28). and 2-Acetamido2-deoxy-~-glucopyranose(- . -) at Anorneric Equilibrium in Aqueous Solution. (Redrawn from Ref. 29.)
spectra, and all agreed on the features of the long-wavelength band. Observed at -210nm for the anomeric mixtures, this band was assigned to the nr* transition of the amide. It is negative for 2-acetamido-2-deoxy-~glucose and 2-acetamido-2-deoxy-~-galactose,but positive for 2-acetamido2-deoxy-~-mannose.The transition is observed at wavelengths shorter than those typical for a-helical polypeptides, and thus is to be highly solvated. The a anomers of 2-acetamido-2-deoxy-~-glucoseand 2-acetamido-2deoxy-D-galactose have34 the same nr* c.d. bands in 1 : 1 methanol-water at 0" as the anomeric mixtures have in aqueous solution. This indicates that the anomeric configuration has little influence on the n r * c.d. band. However, workers do not agree as to the shape of the c.d. spectrum for these sugars at shorter wavelengths, as Fig. 15 demonstrate^.^^.^^ The correct spectrum still remains an open question, but the intense c.d. band expected at 190 nm for the amide r r * c.d. bands are of opposite sign for the two anomers and nearly cancel in the equilibrium mixture. Thus, differences in the anomeric mixtures could explain differences in the c.d. spectra. The amide rr* c.d. band is obvious for the anomeric mixture from 2-acetamido-
W. CURTIS JOHNSON, JR.
96
I
I
I
I
I
I
I
I
5
4 3 2 1
A€ c -1
-2 -3
-1 /
; I
I
180
200
X
I
220
(nm)
FIG. 16.-Circular Dichroism of Methyl 2-Acetamido-2-deoxy-a-~-galactopyranoside Methyl 2-Acetamido-2-deoxy-/3-~-galactopyranoside (- - -), Methyl 2-Acetamido-2deoxy-a -D-glucopyranoside (. . .), and Methyl 2-Acetamido-2-deoxy-~-~-glucopyranoside (- . -) in Aqueous Solution. (Redrawn from Ref. 38.) (-),
2-deoxy-~-galactose(see Fig. 15) and in the c.d. spectra of the methyl glycopyranosides (see Fig. 16). The c.d. expected from the n r * transition of the amide attached to C-2 of a monosaccharide has been calculated36in the one-electron approximation. The calculated results indicated that the sign of this c.d. band is independent of anomeric configuration, but depends on the relative position of the amide on C-2 and the hydroxyl group on C-3. This is in agreement with the experimental results presented in Figs. 15 and 16. Fig. 16 suggests that, although the n r * band is relatively insensitive to anomeric configuration, the intensity of the r r * band does depend on the orientation of this linkage oxygen atom. As will be seen, the linkage in oligomers can be studied by monitoring the r r * c.d. band of a component 2-acetamido sugar. 2-Acetamido-2-deoxy-~-glucose and 2-acetamido-2-deoxy-~-galactose, as well as their methyl glycopyranosides, have been studied in 1,1,1,3,3,3hexafluoro-2-propanol (HFIP). It was e ~ p e c t e d ’that ~ this solvent might so modify the dihedral angle between the 2-amide and the 3-hydroxyl group as to change the c.d. due to the n r * transition. Indeed, changes in intensity (36) D. Y. Yeh and C. A. Bush, J. Phys. Chem., 78 (1974) 1829-1833.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
I
If -4
I
I
Jl 190
200
I
I
I
I
I
I
I
I
220
230
240
210
X
97
(nm)
FIG. 17.-Circular Dichroism of 2-Acetamido-2-deoxy-~-galactopyranose at Anomerk Equilibrium in Water (-) and in 1,1,1,3,3,3-HexafluoroisopropylAlcohol (- - -). (Redrawn from Ref. 35.)
were observed for the methyl pyranosides and even changes in sign for the pyranoses. Fig. 17 gives the results for 2-acetamido-2-deoxy-~-galactose. The 3-0-methyl derivative of methyl 2-acetamid0-2-deoxy-p-~glucopyranoside has the same c.d. spectrum in water and in fluorinated alcohols. This confirms that solvent binding to the 3-hydroxyl group is important in determining the orientation of the amide relative to the 3substituent. Buffington and Stevens37 measured the c.d. of 2-acetamido-2-deoxy-~glucose as a film cast from HFIP. The spectrum is considerably more intense than that observed by Dickinson and coworkers35 for a solution in HFIP, but shows the same general features shifted somewhat towards the red. This vacuum-u.v. c.d. spectrum (see Fig. 18) has, at 218 nm, an intense, positive band due to the nr*,an intense negative band due to the amide rr* at 200 nm, and a shoulder at 180 nm, but no other significant features down to 145nm. The c.d. spectra of three monosaccharides that have two amide substituents have been measured in the vacuum-u.v. The longwavelength portion of the spectra are in agreement with spectra measured on commercial instruments in earlier ~ o r k . ~These ~ , ~bis(acetamid0) ~ * ~ ~ (37) L. A. Buffington and E. S. Stevens, J. Am. Chem. Soc., 101 (1979)5159-5162. (38) A. Duben and C. A. Bush, Anal. Chem., 52 (1980)635-638. (39) C. A. Bush, A. Duben, and S. Ralapati, Biochemistry, 19 (1980)501-504. (40)T. Y. Shen, J. P. Li, C. P. Dorn, D. Ebel, R. Bugianesi, and R. Fecher, Carbohydr. Res., 23 (1972)87-102.
98
W. CURTIS JOHNSON, JR.
I
l
160
l
I
l
l
200
180
X
I
l
220
l
I
240
(nm)
FIG. 18.-The Circular Dichroism of a 2-Acetamido-2-deoxy-~-glucopyranose Film. (Redrawn from Ref. 37.)
sugars exhibit a low-intensity, positive band in the 240-200-nm region that can be attributed to the amide nr*.The PT* c.d. bands at shorter wavelength have about 10 times the intensity observed for the 2-acetamido sugars. As shown in Fig. 19 for 2-acetamido-1-N-( ~-aspart-4-oyl)-2-deoxy-p-~glucosylamine, exciton coupling between the amide chromophores presumably gives intense c.d. bands corresponding to the splitting of the electrically allowed, m r * transitions, in analogy with amide-amide interactions in proteins. Indeed, calculations on such bis(acetamid0) sugars4' showed that an exciton splitting of the two interacting, m r * transitions is expected. N.m.r. spectra of 2-acetamido-l-N-(~-aspart-4-oyl)-2-deoxy-~-~glucosylamine and the model compound 2-acetamido-1-N-acetyl-2-deoxyP-D-glucosylamine indicated that the amide protons are trans to their respective ring protons.39 The c.d. spectra of these two compounds are almost superposable, with an intense, positive band at 197 nm and an intense, negative band near 180 nm. Calculations of the c.d. expected from interaction of the amide mr* transitions, assuming the trans orientation indicated by the n.m.r. work, are consistent with the c.d. measured. Furthermore, these spectroscopic data are consistent with the proposal that the glucosylated L-asparagine residue in glycoproteins is involved in a type I p-turn involving three other adjacent amino acids in the protein. (41) C. A. Bush and A. Duben, J. Am. Chem. SOC.,100 (1978) 4987-4990.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
180
200
X FIG.
19.-Circular
220
99
240
(nrn)
Dichroism of 2-Acetamido-1-N-(~-aspart-4-oyl)-2-deoxy-~-~-glucosyl-
amine.
2,3-Bis(acetamido)-2,3-dideoxy-~-dideoxy-~-glucose has39 a single, intense, negative c.d. band at 198 nm. The two amide groups should give rise to a couplet of c.d. bands, but the short-wavelength band is not observed, and the reason for its absence is not yet clear. Kabat and studied amido-substituted sugars in their pioneering work on milk oligosaccharides, blood-group substances, and simpler model compounds. Their work was limited (by the commercial instrumentation then available) to measuring the amide nw* transition at wavelengths longer than 210 nm. Nevertheless, they were able to relate the intensity of this band to configurational features. The lowest intensity was found for oligosaccharides in which the 2-acetamido-2-deoxy-~glucopyranosyl residue is P-linked but unsubstituted. Intermediate intensity was found for oligosaccharides that have the 2-acetamido-2-deoxy-~glucopyranosyl residues substituted at 0 - 3or 0 - 4 by a P-D-galactopyranosyl group, or a-L-fucopyranose linked (1 + 2) to P-D-galactopyranose. The
-
(42) K. 0. Lloyd, S. Beychok, and E. A. Kabat, Biochemistry, 6 (1967) 1448-1454. (43) K. 0. Lloyd, S. Beychok, and E. A. Kabat, Biochemistry, 7 (1968) 3762-3765.
100
W. CURTIS JOHNSON, JR.
highest intensity was observed for oligosaccharides in which the 2acetamino-2-deoxy-~-glucopyranosy~ residue is disubstituted. Aubert and investigated a number of oligosaccharides conand -mannopyranosyl taining 2-acetamido-2-deoxy-~-~-glucopyranosyl residues. Their c.d. measurements to 180 nm showed a negative c.d. band at -210 nm that is due to the amide nr* transition, and a positive c.d. band at -190nm due to the amide mr* transition. Although the spectra of all eight oligosaccharides studied were similar in shape, their magnitudes varied over a wide range. No correlation was found between the magnitude of the c.d. bands observed and the sequence, but the magnitude appeared to depend in a general way on the type of linkage involved. made extensive measurements on oligomers Bush and The c.d. spectra of the P-D-(1 + of 2-acetamido-2-deoxy-~-glucopyranose. 4)- and P-D-( 1 + 6)-linked dimers were measured in both water and HFIP. 1 + 4)-linked dimer in alcohol, the c.d. resemble the Except for the P-D-( average for the monomers in the same solvent, indicating that there is no 1 + 4)-linked dimer interaction between the residues. In contrast, the P-D-( shows a significant change in c.d. from the average of the monomers, suggesting that there may be an intramolecular hydrogen-bond between the 3-hydroxyl group on the reducing residue and the ring-oxygen atom of the nonreducing group. The c.d. spectra for the dimer, tetramer, and hexamer of 2-acetamido-21+ 4) linkages (the chitin series of deoxy-D-glucopyranose having P-D-( oligosaccharides) are shown in Fig. 20 on a per residue basis.34 All three oligomers have a negative nr* c.d. band at 210nm, and a positive m r * band at 193 nm. Although these spectra are similar to those of the monomer, indicating little interaction between the residues, there is a modest increase in intensity with chainlength. The c.d. spectrum of the trimer does not fit nicely into the series. Although the shape is the same, the c.d. is more intense than that observed for the tetramer.28Furthermore, the c.d. spectrum of the polymer (chitin), shown in formula 8, does not fall neatly into the series.
HO H3CCNH
HO
II 0
H3CCNH
I1
0 8
The c.d. spectra of chitin, both in HFIP solution and as a film cast from HFIP, are shown3’ in Fig. 21. Chitin gels have a c.d. spectrum similar to
THE CIRCULAR DICHROISM OF CARBOHYDRATES I
I
190
200
101
1
I
I
I
210
220
230
2
1
A€
0
-1
-2
X (nmI (-)
FIG. 20.-Circular Dichroism of Chitobiose (- * - .), Chitotetraose (- - -), and Chitohexaose in Aqueous Solution. (Redrawn from Ref. 34.)
A€
140
160
180
200
X
220
240
(nm)
FIG.21 .-Circular Dichroism of Chitin in 1,1,1,3,3,3-HexafluoroisopropylAlcohol Solution and as a Film Cast from 1,1,1,3,3,3-HexafluoroisopropylAlcohol (. . .). (Redrawn from Ref. 37).
(-),
102
W. CURTIS JOHNSON, JR.
that of the film in the accessible, longer-wavelength region. In contrast to the oligomers in aqueous solution, there appears to be, in the films and gels, a strong amide interaction that gives rise to the intense r r * transition observed at -200nm. The authors considered that the polymer forms “mats,” as observed by X-ray diffraction and infrared spectroscopy. Intermolecular hydrogen-bonds are formed between the amide substituents, and the amide is probably in the trans orientation so that the chains can form a sheet having each amide acting as both a hydrogen-bond donor and acceptor. This structure appears to be disrupted in the HFIP solvent.
3. Carboxyl Derivatives The c.d. spectra of glycuronic acids have been measured in a number of l a b ~ r a t o r i e s . ~These ~ ~ ~ - sugar ~ ’ derivatives, named after the corresponding aldohexose, have the exocyclic hydroxymethyl group on C-5 replaced by the carboxyl chromophore. The structure of P-D-mannopyranuronic acid is given as an example in formula 9, and the structures of the parent compounds in Fig. 1.
OH
HO 9
Listowsky and coworkers4s presented a particularly nice study of the c.d. of glycuronic acids. They measured D-glucuronic acid and D-galacturonic acid, as well as some methyl glycopyranosiduronic acids, in aqueous solution. Morris and coworkers4’ extended the experimental results to include both anomers of methyl pyranosides of all five naturally occurring glycuronic acids having the D-gluco, D-manno, D-galucto, D-gulo, and L-ido configurations. The c.d. spectrum of methyl a-D-mannopyranosiduronic acid as a function of pH is given in Fig. 22. In all cases, it was found that, in acid solution, the glycuronic acids having an equatorial 4-hydroxyl group exhibit a positive n r * transition at 208 nm and an “anomalous” negative n r * c.d. band at -233 nm. Measurements of the c.d. of D-glucuronic acid in 19: 1 1,Cdioxane-water solution, as well as those of the permethylated Dglucopyranosiduronic acid in water and hexane, demonstrated that the long-wavelength, n r * c.d. band gains its intensity at the expense of the (44) E. J. Eyring and J. T. Yang, Biopolymers, 6 (1968) 691-701. (45) 1. Listowsky, S. Englard, and G. Avigad, Biochemisfry, 8 (1969) 1781-1785. (46) B. Chakrabarti and E. A. Balazs, 1.Mol. Biol, 78 (1973) 135-141. (47) E. R. Moms, D. A. Rees, G. R. Sanderson, and D. Thorn, 1.Chem. SOC., Perkin Trans. 2, (1975) 1418-1425.
THE CIRCULAR DICHROISM OF CARBOHYDRATES I
I
I
.. .. ... ... ..__.. %
200
I
I
I
103
1
:
I
I
I
220
X
I
240
I
260
(nrn)
FIG. 22.-Circular Dichroism of Methyl a-D-Mannopyranosiduronic Acid at Various pH Values. (Redrawn from Ref. 47.)
shorter-wavelength, nr* band. However, D-galacturonic acid exhibits a single nr* band that is almost the same in water and in 19: 1 1,Cdioxanewater solution. These experiments indicated that two species are involved. “Anomalous” nT* c.d. bands having a component of opposite sign at long wavelengths are not peculiar to these sugars, but have been observed for many asymmetric acids and esters. Convincing arguments have been presented for a variety of origins of this effect, and various authors have attributed it to solvation species, conformational species, rotational isomers, and vibronic interactions. At present, the origin of the effect must still be considered an open question. The glyguronic acids having an axial 4-hydroxyl group show only a single, positive nr* c.d. band at -210nm, and that has a greater intensity than the band observed for glycuronic acids having an equatorial 4-hydroxyl group. However, the idoses, which have an axial 4-hydroxyl group when in the 4C,conformation, display a single, positive nr* band at 210 nm, as expected, and probably have a substantial contribution from the ‘C4confor-
W. CURTIS JOHNSON. JR.
104
mer in which the 4-hydroxyl group is equatorial. Of course, the 5-carboxyl group would be axial, not equatorial, in this conformation. The carboxyl chromophore is sensitive to pH, as Fig. 22 shows for the example of methyl a-D-mannopyranosiduronic acid?’ The particular sensitivity of this chromophore to its environment should prove useful in the investigation of uronic polymers. As the acid form is converted into the carboxylate anion, the c.d. spectrum changes significantly. Monitoring of these changes, at a single wavelength, as a function of the pH of the solution yielded a titration curve with pK values of the order of 3.3, in agreement with results derived by other methods. The isodichroic point evident in Fig. 22 demonstrates that two species are involved. Film spectra of D-glucuronic acid and sodium D-glucuronate (see Fig. 23), measured into the vacuum-u.v. region, added to our knowledge of the transitions involved.29The c.d. spectrum of D-glucuronic acid shows both nr* transitions at long wavelength and a c.d. band at 180 nm that is assigned to the carboxyl rr*.In addition to the rr* at 180 nm, sodium D-glucuronate shows two bands at longer wavelengths. Apparently, the carboxylate chromophore also has two c.d. bands of opposite sign that are associated with the n r * transition. The c.d. spectra of these two compounds in aqueous solution have been extended29to 185 nm. The c.d. spectrum of D-glucuronic acid appears to be the same as that measured for the film, but, whereas 1
I
I
I
I
I
I
I
I
I
0
0
AA
0
b I
180
I
200
X
220
240
(nrn)
FIG.23.-Circular Dichroism of (a) D-Glucuronic Acid and (b) Sodium D-Glucuronate as Films. (Redrawn from Ref. 29.)
THE CIRCULAR DICHROISM OF CARBOHYDRATES
105
both c.d. bands for the nn* transition are apparent for sodium D-glucuronate, the TT* c.d. band is now negative. Alginate is a copolymer of a-L-gulopyranuronic and fl -Dmannopyranuronic acid, the sugars being linked (1 + 4) (see formula 10). COzH
,O
10
The two types of residue are arranged in blocks, and in approximately alternating sequences. The negatively charged carboxylate chromophore binds with various cations to provide interesting properties for solutions, gels, and films of this polymer. The c.d. spectra of the alginate polymer have been measured on commercial i n s t r ~ m e n t s ~ ’and - ~ ~a study has been performed on vacuum-u.v. instrument^.^^ C.d. bands at 215 and 203 nm were assigned to the carboxylate nn* transition, and the c.d. band at 180 nm was assigned to the carboxylate m r * . Shorter-wavelength transitions, observed at 169 and 149 nm in film spectra, were assigned to transitions of the sugar in the polymer backbone. Intermolecular association in forming gels and films changes the intensity of both the short- and long-wavelength transition^.^^ The principal spectral changes are attributed to perturbation of the carboxylate chromophore by site-bound cations. Chelation of the divalent cation Ca2+to alginate chains has been studied e x t e n ~ i v e l y The . ~ ~ negative c.d. band at 205 nm for aqueous solutions of sodium poly-( L-gulopyranuronate) (called the m r * band rather than the n?r* band in this early paper) loses negative intensity as Ca2+is added (see Fig. 24). This change is equivalent to gaining positive c.d. intensity for a
-
E. R. Morris, D. A. Rees, D. Thorn, and J. Boyd, Carbohydr. Res., 66 (1978) 145-154. E. R. Morris, D. A. Rees, and G. Young, Carbohydr. Res., 108 (1982) 181-195. D. Thorn, G. T. Grant, E. R. Morris, and D. A. Rees, Carbohydr. Res., 100 (1982) 29-42. R. Seale, E. R. Morris, and D. A. Rees, Carbohydr. Res., 110 (1982) 101-112. E. R. Morris, D. A. Rees, G. Robinson, and G. A. Young, J. Mol. Biol.,138 (1980) 363-374. H. J. Jennings, R. Roy, and R. E. Williams, Carbohydr. Res., 129 (1984) 243-255. E. R. Moms, D. A. Rees, and D. Thorn, Carbohydr. Res., 81 (1980) 305-314. B. Stockton, L. V. Evans, E. R. Moms, and D. A. Rees, I n f . J. Biol. Macromol., 2 (1980) 176-178. B. Stockton, L. V. Evans, E. R. Moms, D. A. Powell, and D. A. Rees, Bof. Mar., 23 (1980) 563-567. J. N. Liang, E. S. Stevens, S. A. Frangou, E. R. Moms, and D. A. Rees, I n f . J. Biol. Macromol., 2 (1980) 204-208.
W. CURTIS JOHNSON, JR.
106
I
-. 8
I
I
I
220
200
X
I
I
240
(nm)
FIG. 24.-Circular Dichroism of Sodium Poly-(L-gulopyranuronate)as a Function of 25% (- . -), 50% (- - -), 75% (- . . -), and 100% (. . .). Stoichiometry of Added Ca2+:0% (-), (Redrawn from Ref. 48.)
band at -207 nm. The stoichiometry, investigated by c.d. and equilibrium dialysis, shows one Caz+bound for each four L-gulopyranuronate residues. This is consistent with dimerization of the chains into an “egg box” structure. Two residues from each of the dimer chains form a nest of oxygen atoms which satisfies the criteria for Ca2+chelation. Similar experiments on alginate indicated occurrence of this type of dimerization for the poly-(Lgulopyranuronate) blocks found in these natural polysaccharides. Furthermore, c.d. and 0.r.d. have been combined to eliminate interference from the poly-(D-mannopyranuronate) and alternating sequences in such studies.49 In a similar manner, c.d. has been used to study gelation of alginates through the addition of various divalent cations.” Monovalent cations have also been studied extensively, and, although the c.d. changes are less spectacular, they indicate association of the poly-( L-gulopyranuronate) blocks.” Finally, competitive inhibition between various polysacAs charides has been used to study specific, intermolecular associati~n.’~ expected, short poly-( L-gulopyranuronate) blocks can abolish alginate gelation, whereas poly-( D-mannopyranuronate) has little effect.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
107
C.d. spectroscopy is now being applied to more complicated polysacacids found in charides. The 3-deoxy-~-manno-2-octulopyranosylonic Escherichia coli LP1092 have been definitely assiglled the 0-D configuration.53 The negative nrr* c.d. band exhibited by this polysaccharide correlates with the negative c.d. of methyl 3-deoxy-a-~-rnanno-2octulopyranosidonic acid rather than the positive c.d. band exhibited by acid. methyl 3-deoxy-~-~-manno-2-octulopyranosidonic The utility of c.d. spectroscopy is well illustrated in its application to C.d. spectra for determine alginate composition and bl~ck-structure.~'~~~-~~ the three types of idealized blocks [poly-( a-L-gulopyranuronic acid), poly(P-D-mannopyranuronic acid), and the alternating polymerIs4 are given in Fig. 25. The c.d. for the alternating polymer is different from the average of spectra for the two homopolymers, showing that the c.d. is sensitive to nearest-neighbor residues. As an additional benefit, this means that the c.d. spectra of natural alginates can be analyzed for the three types of block structure. These workers used a least-squares fitting, together with the
I
!
I
X
(nm)
FIG. ZS.-Circular Dichroism for Blocks of Poly( L-gulopyranuronate) (- -), Poly(omannopyranuronate) (- - -), and Alternating Sequences (- . -) as Found in Alginate. (Redrawn from Ref. 54.)
108
W. CURTIS JOHNSON, JR.
constraint that the sum of the three structures must equal 100%, to analyze alginates from various source^.^^-^^ Because the carboxyl chromophore is sensitive to pH, their solutions were carefully neutralized to pH 7.0. Furthermore, divalents must be strictly excluded, because these ions cause the poly-( a-L-gulopyranuronic acid) blocks to associate and thus change their c.d. spectrum. The analysis of material extracted from the stipes of Alaria esculenta, gathered at Cullercoats on the British east coast, is illustrated in Fig. 26, where the composition is 18% of poly-( P-D-mannopyranuronic acid), 51% of poly-( cu-L-gulopyranuronic acid), and 31Yo of the alternating polymer.55 It is also possible to estimate the proportion of each sugar by dividing the intensity of the negative trough that falls between 218 and 208 nm by the difference between the trough and the peak at -200nm. As long as the overall D-mannopyranuronic acid level is below 60% (corresponding to an all-negative c.d. spectrum), the ratio of D-mannopyranuronic acid to Lgulopyranuronic acid is approximately twice this ratio. The ratio is proportional to the level of D-mannopyranuronic acid when this sugar is at high levels. In this case, the percentage of D-mannopyranuronic acid is -27 times the ratio +40%. Applying these empirical relationships to the c.d.
x
I
I
200
I
220
X
I
I
I
240
(nm)
FIG. 26.-Circular Dichroism of Alginate from the Stipes of Alaria esculenta Gathered at and the Component Analysis for Poly(L-gulopyranuronate)(- - -), Po~Y(DCullercoats (-) mannopyranuronate) (. . .) and Alternating Sequences (- . -). (Redrawn from Ref. 35.)
THE CIRCULAR DICHROISM OF CARBOHYDRATES
109
spectrum of Alaria esculenta (shown in Fig. 25) gives a value of 34% of D-mannopyranuronic acid, in good agreement with values inherent in a block c o m p ~ s i t i o n . ~ ~ The sodium salt of poly(D-galacturonic acid) has a positive c.d. band due to the nm* transition at 208 nm that decreases in amplitude and blue shifts as the polymer gels.58Ravanat and R i n a ~ d conducted o~~ a particularly extensive study on oligo( D-galactopyranuronic acid) in the acid form and as sodium or calcium salts. Dissolved in aqueous solution in the absence of a salt, the c.d. band due to the n r * transition increases somewhat with the degree of polymerization (d.p.) from the dimer through the polymer. As the anionic form is converted into the undissociated form with the addition of hydrochloric acid, the intensity of this c.d. band decreases in all cases. The authors considered that, under these conditions, they observed an acidic form that would be stabilized by hydrogen bonding, and postulated a helix with three-fold screw symmetry, such as has been described for sodium pectinates in the solid state.60The same results are observed if the equilibrium is shifted to the associated form by decreasing the dielectric constant. Additions of sodium hydroxide to afford the sodium salt decrease the intensity of this band and shift it to shorter wavelength^,'^ as shown for the dimer and the polymer in Fig. 27. It may be noted the isodichroic point lies at 198 nm. In contrast, observation of the c.d. with the addition of Ca(OH)*, as a function of d.p., demonstrated that terminal and central units react differently towards Ca2+. This is illustrated in Fig. 27 for the dimer and the polymer. Again, the intensity of the c.d. band decreases as the polymer binds calcium and begins to gel. Results for both salt forms are attributed to a helix having a two-fold screw-symmetry, in analogy with calcium pectates6’ The gelling would then involve a multi-chain association, with crosslinking by the calcium ions to form an “egg box” s t r u ~ t u r e . ~ ~ * s ’ ~ ~ ~ Liang and Stevens6* extended the c.d. spectra of poly(D-galactopyranuronic acid) and the sodium and calcium salts into the vacuum-u.v. region. In addition to the nr* c.d. band at -208 nm, these authors observed a negative band at -170 nm and a positive band at -145 nm for both poly( D-galactopyranuronic acid) and its sodium salt. The c.d. of the calcium salt could be measured only to 170 nm, with the indication of the second negative c.d. band. They observed these two shorter-wavelength bands for (58) G. T. Grant, E. R. Morris, D. A. Rees, P. J. C. Smith, and D. Thorn, FEES Lerr., 32 (1973) 195. (59) G. Ravanat and M. Rinaudo, Biopolymers, 19 (1980) 2209-2222. (60) K. J. Palmer and M. B. Hartzog, J. Am. Chem. Soc., 67 (1945) 2122-2127. (61) C. Sterling, Biochim. Biophys. Acta, 26 (1957) 186-197. (62) J. N. Liang and E. S. Stevens, In?. Biol. Macromol., 4 (1982) 316-317.
W. CURTIS JOHNSON. JR.
110
1.5
A€
1
.5
200
200 220 240
220 240
X
(nm)
FIG. 27.-Circular Dichroism Spectra of Galactopyranuronic Acid for d.p. = 2 (First and Third Panels) and d.p. = 340 (Second and Fourth Panels) as a Function of Percent of Neutralization with NaOH (First and Second Panels) and Ca(OH), (Third and Fourth Panels): 0% (-), 20% (- - -), 40% (- . -), 60% * .), 80% (- . . -), and 100% (- -). (Redrawn from Ref. ( 9
59.)
a number of polysaccharides, and assigned them to ring transitions. In this case, the carboxylate mr* transition probably also contributes to the 170-nm band. The results for a poly(D-galactopyranuronic acid) film are shown in Fig. 28.
AA
140
160
180
200
220
240
X (nm) FIG. 28.-Circular from Ref. 62.)
Dichroism of a Film of Poly(D-galactopyranuronic Acid). (Redrawn
THE CIRCULAR DICHROISM OF CARBOHYDRATES
111
The main component of pectins is poly( D-galactopyranuronic acid) in which the carboxyl chromophore may be esterified to a variable extent. The polymer has a positive c.d. band at -210 nm for the carboxyl, carboxylate, and acetate c h r o m o p h o r e ~ .The ~ ~ *c.d. ~ ~ has been used to investigate the effect of carboxyl ionization, carboxyl esterification, conformational changes, and association, for variations in pH, temperature, and ionic strength.63A linear change wa5 observed upon esterification, indicating that there is no fundamental change in the polysaccharide. On ionization of the carboxyl chromophore, there is a sharp decrease in intensity of the c.d. as the carboxylate anion is formed. Changing the solvent conditions can cause the pectin to gel, and this association gives rise to an increase in the c.d. intensity. Increasing the temperature decreases the association, as witnessed by a decrease in the intensity of the c.d. bands. Gelation by various divalent cations has also been extensively studied by c.d. spectroscopy. 4. Derivatives Having Mixed Substituents
N-Acetylneuraminic acid is a common group in glycoproteins, and it contains both the amide and carboxyl chromophores. As shown in formula 11, this nine-carbon sugar derivative has an equatorial amido group on C-5 and both a hydroxyl group and a carboxyl group on the anomeric carbon atom.
11
The carboxyl chromophore is axial for the a anomer and equatorial for the p anomer. The sugar was studied as the carboxylate anion as it has a (low) p K of 2.6, and the compound is degraded in acidic solution. The c.d. spectrum of this compound contains contributions from the carboxylate nT* at -217 nm, the amide nr* at -210 nm, and the amide w r * at -190 nm. Apparently, all of these bands are positive, giving rise to a c.d. spectrum (see Fig. 29) a maximum at 199 nm and a shoulder at 210 nm. The c.d. spectra of a number of derivatives confirmed these assignments. (63) I. G. Plaschina, E. E. Braudo, and V. 9. Tolstoguzov, Carbohydr. Res., 60 (1978) 1-8. (64) H. R. Dickinson and C. A. Bush, Biochemistry, 14 (1975) 2299-2304. (65) G. Keilich, R. Bossmer, V. Eschenfelder, and L. Holmquist, Carbohydr. Res., 40 (1975) 255-262. (66) L. D. Melton, E. R. Morns, D. A. Rees, and D. Thom, J. Chem. SOC.,Perkin Trans. 2, (1979) 10-17.
W. CURTIS JOHNSON. JR.
112
5
c
-
l
200
220
X
I
240
260
(nrn)
FIG.29.-Circular Dichroism of N-Acetylneuraminate (. . .), and Its Methyl a- (-) and Methyl /3- (- - -) D-Ketopyranosides in Aqueous Solution at -pH 3.2. (Redrawn from Ref. 65.)
The c.d. spectra of a number of other derivatives have been measured as models for the linkages found in o l i g o m e r ~ .Spectra ~ ~ * ~ ~of the methyl aand P-D-ketopyranosides of N-acetylneuraminic acid are given in Fig. 29. They do not differ substantially from the c.d. spectra for the fundamental sugars, except that the a-ketopyranoside has a positive c.d. band of low intensity at 250 nm. These types of bands occur from time to time for still obscure reasons, as discussed for carboxyl derivatives. The c.d. spectra are also available for a few oligosaccharides that contain N-acetylneuraminic a ~ i d . ~ These ~ * ~spectra ~ * depend ~ ~ ~ on ~ the ~ -intersac~ ~ charide linkages and the state of ionization of the carboxyl group, but no systematic scheme has yet been set up to derive configurational information from the c.d. spectra. Of particular interest is the c.d. spectrum of beef ganglioside:' which fully differentiates the amide nr*,amide r r * , and carboxylate nr* c.d. bands. In muramic acid, the 2-hydroxyl group of D-glucose is replaced by an amine group, and a lactic acid group is attached to OH-3. The structure of ~-l-carboxyethyl)-2-deoxy-~-glucopyranose] muramic acid [2-amino-3-0-( is given in formula 12. C H ,OH
qq;.
HO HsC
'H
12
(67) A. L. Stone and E. H. Koludny, Chern. Phys. Lipids, 6 (1971) 274-279. (68) H. I. Jennings and R. E. Williams, Carbohydr. Res., 50 (1976) 257-265.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
113
Listowsky and coworkers showed69that the c.d. of this sugar derivative is due entirely to lactic acid, and confirmed that this chromophore is in the D configuration for muramic acid. N-Acetylmuramic acid, in which the amino group is replaced by an amido group at C-2, has a c.d. spectrum that is roughly a linear combination of the lactic acid in muramic acid and This indicates that the amide the amide in 2-acetamido-2-deoxy-~-glucose. chromophore and the lactic acid chromophore in N-acetylmuramic acid behave independently. Hyaluronic acid is an important biological polysaccharide found in the intracellular matrix of most connective tissues. Fundamentally, it is a linear copolymer of p -D-glucopyranosyluronic acid and 2-acetamido-2-deoxy-PD-glucopyranosyl residues. The D-glucuronic acid is P-D-(1 + 3)-linked to 2-acetamido-2-deoxy-~-glucopyranose, and the 2-acetamido-2-deoxy-~glucopyranose is P-D(1 + 4)-linked to the D-glucopyranuronic acid, to give an alternating copolymer (see formula 13). CO,H
H3CCNH HO
II 0
HO 13
~ t ~ ~ ~ 3 1 , 6first 7 , 7 0measured the c.d. of hyaluronic acid. Chakrabarti and coworker^^^^^'-^^ extensively studied the c.d. of this polysaccharide on commercial instruments, and collaborated with Buffington and Stevensz9in a c.d. study in the vacuum-u.v. region. The c.d. spectra of hyaluronic acid at pH 2.5, and as the hyaluronate anion at pH 6.9, are given in Fig. 30. Only minor differences are observed at the two pH values, although the spectrum of the anion is somewhat more intense than that of the acid. Changes in interaction or configuration must occur on forming the polymer, as the negative band for hyaluronic acid is too intense to be accounted for by averaging the monomer ~pectra.’~ On the other hand, the c.d. bands expected for the acetamido and carboxylate m r * transitions between 180 and 190nm are not observed. (69) (70) (71) (72) (73) (74)
1. Listowsky, G. Avigad, and S. Englard, Biochemistry, 9 (1970) 2186-2189. A. L. Stone, Biopolymers, 7 (1969) 173-188. J . W. Park and B. Chakrabarti, Biopolymers, 16 (1977) 2807-2809. J. W. Park and B. Chakrabarti, Biochim. Biophys. Acra, 544 (1978) 667-675. J. W. Park and B. Chakrabarti, Biopolymers, 17 (1978) 1323-1333. J. W. Park and B. Chakrabarti, Biochim. Biophys. Acra, 541 (1978) 263-269.
114
W. CURTIS JOHNSON, JR.
6t +
-1
-6 O
I b 180
220
200
X
24 0
(nm)
FIG.30.-Circular Dichroism of (a) Hyaluronic Acid at pH 2.5 and (b) Sodium Hyaluronate at pH 6.9. (Redrawn from Ref. 29.)
Cowman and coworker^^^*^^ investigated oligosaccharides featuring both types of linkage found in sodium hyaluronate, in an attempt to ascertain why the two m r * transitions are not observed. An increase in intensity at 210nm was found to result from the conformational changes due to the (1 + 4)-/?-~-glycosidic linkage from 2-acetamido-2-deoxy-/?-~glucopyranose to sodium /?-~-glucopyranosyluronate.~~ In the case of the 190-nm region (see Fig. 31), c.d. spectra of the oligomers showed that the two linkages give rise to W ~ T *c.d. bands of opposite sign, so that there is an accidental cancellation of these spectral contributions, and the expected band is absent for sodium h y a l ~ r o n a t e . ~ ~ Films of sodium hyaluronateZ9have a significantly different c.d. spectrum, as shown in Fig. 14. The intense, negative c.d. band observed at 195nm was assigned to the amide m d ' transition. The authors considered that the amide group participates in an intramolecular hydrogen-bond, to form a solid-state, helical structure, and the resulting decrease in rotational freedom gives rise to the large, negative, rotational strength. (75) M. K. Cowman, E. A. Balms, C. W. Bergmann, and K. Meyer, Biochemistry, 20 (1981) 1379-1385. (76) M. K. Cowman, C. A. Bush, and E. A. Balms, Biopolymers, 22 (1983) 1319-1324.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
115
X (nm) FIG.31.-Circular Dichroism of a Hyaluronic Acid Segment Comprising -18 Disaccharide the Tetrasaccharide Featuring P - D - (+ ~ 3) Linkages for the End Residues (. . .), Units (-), and the Tetrasaccharide Featuring /3-~-(1-4)Linkages for the End Residues (- - -). (Redrawn from Ref. 76.)
The work of Chakrabarti and ~ ~ ~ o r k e showed r ~ ~ that ~ -hyaluronic ~ ~ * ~ ~ , ~ ~ acid in aqueous ethanol solvent at pH 2.5, and the Cu(I1)-hyaluronate complex at pH 6.8, apparently have the same intense, negative, c.d. band observed for the hyaluronate film (see Fig. 14), although spectra had not been measured at <205 nm (see Fig. 32). The transition is readily monitored in solution, and has been found to be cooperative with respect to temperature, solvent composition, and pH. Methyl hyaluronate does not show this c.d. change, confirming that the protonated carboxyl group is involved in the transition for the mixed-solvent system. These workers considered that the four-fold, helical model which allows hydrogen bonding between the amide hydrogen atom and the carboxyl oxygen atom may be the appropriate conformation for all three systems, where helical structures go into an aggregated state. Chondroitin is quite similar to hyaluronic acid, with the exception that the 4-hydroxyl group of the N-acetylglycopyranosylamine is now axial instead of equatorial (see formula 13 for hyaluronic acid). This polysaccharide may be sulfated at C-6 of the N-acetylgalactopyranosylamine, to form chondroitin 6-sulfate. Chondroitin and chondroitin 6-sulfate have (77) N. Figueroa and B. Chakrabarti, Biopolyrners, 17 (1978) 2415-2426. (78) B. Chakrabarti, N. Figueroa, and J. W. Park, in J. D. Gregory and R. W. Jeanloz (Eds.), FTOC.Int. Syrnp. Glycoconjugotes, 4th,(1979) 119-124.
W. CURTIS JOHNSON, JR.
116
A€
FIG. 32.-Circular Dichroism of Hyaluronic Acid in 20% Ethanol at pH 2.5 (-) pH 6.5 (- * -), and the Difference Spectrum (- - -). (Redrawn from Ref. 73.)
and at
similar c.d. spectra in aqueous solution.25.73.15 In the acid form, at pH -2.5, both polysaccharides have a negative c.d. band at 210 nm, and show another negative band that peaks below 170nm. At neutral pH, the uronic acid residue is ionized and, in addition to a negative c.d. band at 210 nm, there
X
(nrn)
FIG. 33.-Circular Dichroism of Chondroitin 6-Sulfate in D20 at p D -7 (- - -) and pD -2.5 (-). (Redrawn from Ref. 25.)
THE CIRCULAR DICHROISM OF CARBOHYDRATES
117
-
is a positive band at 190 nm. The results for chondroitin 6-sulfate'' are shown in Fig. 33. There is little sign of interaction, as the c.d. of the polysaccharides is nearly the same as that of the constituent monosaccharides. Stone's c.d. spectra3' of glycosaminoglycans that contain substituents other than the chromophoric amide group are given in Fig. 34. This pioneering work demonstrated that c.d. spectroscopy could be used to determine the type of sugar polymer, and could probably be used to monitor configuration and conformation. The negative nr* c.d. band at longer wavelengths is common to all of the polymers studied. The shorter-wavelength c.d. band, which is often incompletely measured, depends on the polymer structure. The (1 + 4)-linked amino sugars show a positive m r * amide transition at 190 nm. The (1 + 4)-linked amino sugars have a negative m r * band that generally lies below the limits of the commercial instruments used. Stone
-
w 0
4
2
A€ 0 -2 -4
7I I I I I I I I I I I 200 250
2 0 0 250
200
250
X (nrn)
FIG. 34.-Circular Dichroism of Glycosaminoglycans: (a) Hyaluronic Acid; (b) Heparan Sulfate from Normal Mammalian Tissue; (c) Chondroitin 4-Sulfate; (d) Dermatan Sulfate; (e) Chondroitin 6-Sulfate; (f) Shark Sulfated-Keratan Sulfate (-) and Mammalian Keratan Sulfate (. . .); and ( 9 ) Heparin. (Redrawn from Ref. 31.)
118
W. CURTIS JOHNSON, JR.
and coworker^'^ used these optical properties to monitor glycosaminoglycans in the urine of patients with Hurler’s syndrome, and Chung and EllertonEomonitored the binding of Cu(I1) with heparin by using the c.d. of the amide chromophore. The extracellular polysaccharides from the Xanthornonas species of bacteria are interesting materials that find industrial application. The polymer has a cellulose-like backbone with trisaccharide side-groups on alternate residues. The side groups consist of an a-D-mannopyranosyl residue with a 6-acetate substituent, a p-D-glucopyranuronic acid residue, and a p-Dmannopyranosyl residue with a 4,6-pyruvic acetal substituent on 1/3rd of the residues. Thus, although the cellulose-like backbone absorbs only in the vacuum-u.v. region, the side groups contain acetate and carboxylate chromophores that absorb light within the range of commercial c.d. instruments. The c.d. spectrum of the polysaccharide xanthan from the Curnpestris species (see Fig. 35) confirmed the results of ‘H-n.m.r.-spectral, viscosity, and monochromatic optical-rotation studies.” All of these methods indicated that the polysaccharide forms, in aqueous solution, an ordered structure that can be “melted” to a disordered structure by increasing the temperature. The intensity of the 205-nm, positive c.d. band diminishes linearly with temperature, but the intensity of the 220-nm, negative c.d. band increases with temperature, and this shows a cooperative melting comparable to that disclosed by the optical-rotation studies. There is no dependence on concentration, indicating that only a single chain is involved in forming the ordered structure. Furthermore, the ordered structure is increasingly stable at higher ionic strength. On the basis of all of these physical measurements taken in combination, the authors proposed that the side groups fold down, and interact with the backbone to form the ordered structure. The complex oligosaccharides comprising the T“ and “T” antennae of three glycoproteins have been studied by c.d. spectroscopy.’‘ Five antennae were studied in all, because two of the proteins have two closely related forms. The c.d. for the three fundamental oligosaccharides are given in Fig. 36. These are asparagine-linked glycopeptides, and the intense, conservative c.d. band that results from the interaction between the two amides has already been discussed in Section III,2. Fig. 36 shows that the c.d. spectra
-
(79) A. L. Stone, G . Constantopoulos, S. M. Sotsky, and A. Dekaban, Biochim. Biophys. Acta, 222 (1970) 79-89. (80) M. C. M. Chung and N. F. Ellerton, Biopolymers, 15 (1976) 1409-1423. (81) E. R. Morris, D. A. Rees, G . Young, M. D. Walkinshaw, and A. Darke, J. Mol. Biol., 110 (1977) 1-16. (82) C. A. Bush, V. K. Dua, S. Ralapati, C. D. Warren, G. Spik, G . Strecker, and J. Montreuil, J. Biol. Chem., 257 (1982) 8199-8204.
T H E CIRCULAR DICHROISM OF CARBOHYDRATES
I1
200
I
I
220
I
I
240
I
I
260
I
I
I
I
280
119
3C
X (nm) FIG. 35.-Circular Dichroism of the Xanthan from Xanthomonas campestris at Various Temperatures. (Redrawn from Ref. 81.)
are sensitive to the different types of antennae. By using model oligosaccharides, these workers showed that each of these c.d. spectra is the algebraic sum of the c.d. due to the linkage plus the c.d. of the other chromophores in the system. This means that there are no other interactions between the chromophores in the system, indicating that the antennae have an “open” conformation. 5. Non-biological Derivatives Unsubstituted polysaccharides absorb light only in the vacuum-u.v. region, but derivatives of such polysaccharides can often be synthesized that absorb light within the range of commercial instruments. Bittinger and K e i l i ~ synthesized h~~ carbanilyl derivatives of a number of a-and p-glycans. Derivatives of the P-glycans all displayed a weak, negative c.d. band at -240nm. In contrast, all derivatives of the a-glycans showed a strong, (83) H. Bittiger and G. Keilich, Biopolymers, 7 (1969) 539-556.
120
W. CURTIS JOHNSON, JR.
A€
200
180
X
220
(nm)
FIG. 36.-Circular Dichroism of Antennae Found in Glycoprotein STF-A from Serum Transferin (. . .), OTF-C from Ovotransferin (-), and LTF-D from Lactotransferrin (- - -). (Redrawn from Ref. 82.)
negative c.d. band at -240 nm and a strong, positive c.d. band at -225 nm. Such a c.d. couplet is characteristic of exciton interaction, implying that all of the a glycans studied assume a helical conformation in the 1,4-dioxane solvent used for these studies. An almost planar arrangement was proposed for the p-glycans. Pfannemuller and Bergs4 studied carbanilated and tritylated derivatives of amylose and cellulose in 1,Cdioxane. As shown in Fig. 37, 2,3-di-Ocarbanilylamylose had an especially large exciton interaction between the derivative groups, suggesting a helical structure. The c.d. spectra for 2,3,6-trishowed 0-carbanilylamylose and 2,3-di-O-carbanilyl-6-O-tritylamylose somewhat less-intense exciton bands. In contrast, 6- 0-tritylamylose showed only a weak c.d. band, indicating an unordered structure in 1,Cdioxane. Cellulose was also studied as the 2,3,6-tri-O-carbanilyl and 2,3-di-0carbanilyl-6-0-trityl derivatives. A single, intense c.d. band was observed, suggesting a viable secondary structure without exciton interaction. Acetates have been particularly useful for studies conducted with commercia1 c.d. instruments,'s~25~s5~as and they are usually examined in the par(84) B. Pfannemuller and A. Berg, Makromol. Chem., 180 (1979) 1201-1213. (85) S. Mukherjee, R. H. Marchessault, and A. Sarko, Biopolymers, 11 (1972) 291-301.
(86) S. Mukherjee, A. Sarko, and R. H. Marchessault, Eiopolymers, 11 (1972) 303-314. (87) J. W.-P. Lin and C. Schuerch, 1. Polym. Sci., 10 (1972) 2045-2060. (88) A. Sarko and C. Fischer, Biopolymers, 12 (1973) 2189-2193.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
121
A€
X
(nm)
FIG.37.-Circular bichroism of 2,3-Di-O-carbanilylamylose (-), 2,3,6-Tri-O-carbanilyl(. . .). (Redrawn from Ref. 84.) amylose (- - -), and 2,3-Di-O-Carbanilyl-6-O-tritylamylose
ticularly transparent solvent 2,2,2-trifluoroethanol (TFE). Acetates of xylan, cellulose, dextran, mycodextran, (1 + 6)-cu-~-mannan, and (1 + 6 ) 4 D-galactan show no conformational effects, and assume, presumably, a random coil in solution. However, amylose triacetate shows" two c.d. bands in this region, a positive band at -235 nm and a negative band at -205 nm; this suggests that the amylose assumes a helical conformation. The c.d. spectra of the oligomers confirmed this interpretation. The monomer and dimer display the normal nr* c.d. band expected for the isolated acetate chromophore, whereas the c.d. of the trimer is similar to that of amylose triacetate, indicating acetate-acetate interaction. Furthermore, the c.d. spectrum of amylose triacetate varies with temperature (see Fig. 38), as would be expected for a change in conformation. Films of amylose triacetate have the normal nr* c.d. band of the acetate chromophore, but the c.d. spectrum changes to that for amylose triacetate in solution when the films are annealed to afford a crystalline structure. Crystalline amylose has been found to have a helical conformation. All of these data, taken together, indicate that amylose triacetate in TFE is present in a helical conformation.
122
W. CURTIS JOHNSON,JR. I
.06 -
I
I
I
I
FIG. 38.-Circular Dichroism of Amylose Triacetate in l,l,l-Trifluorethanol at Various Temperatures. (Redrawn from Ref. 88.)
Stipanovic and Stevens25extended the c.d. of acetylated glucans into the vacuum-u.v. region. They studied derivatives for both a- and p - ( l + 3)-, -( 1 + 4)-, and -(1 + 6)-linked glucans in TFE and as films. All show a negative * which is independent of c.d. band near 190nm for the ~ T T transition, configuration and conformation. The expected exciton splitting for the m r * transition of amylose triacetate was not observed. However, the 170nm band observed for films of all of these triacetates did display an exciton splitting, with particularly high intensity bands of opposite sign in the case of cellulose triacetate. Amylose and dextran have been in aqueous solution as the xanthate derivatives. Dextran xanthate has no observable c.d., but amylose xanthate in aqueous solution has a complex c.d. that indicates an organized structure. Benzyl derivatives of (1 + 6)-a-~-glucan,(1 + 6)-a-~-mannan,and (1 + 6)-a-~-galactan have been studied in lY4-dioxane.These derivatives have complex and interesting c.d. spectra due to the m r * transition of the chromophore with resolved vibrational structure.89 However, a conformational interpretation of these interesting spectra is not possible at this time. A carboxymethyl derivative of dextran has been prepared in order to study the effect of charge density, concentration, degree of neutralization, (89) J.-P. Merle and A. Sarko, Carbohydr. Res., 30 (1973) 390-394.
THE CIRCULAR DICHROISM OF CARBOHYDRATES
123
and added salts on the c.d. spectrum.g0 Here, the carboxyl chromophore that is present in a number of biologically important sugars is involved; it has already been discussed in that regard. Because the chromophore herein is far removed from an asymmetric center, the magnitude of the c.d. is lower than for the biological sugars, but the same types of bands are displayed. For the carboxylate anion, there is a single, negative c.d. band at -213 nm due to the nr* transition. For the carboxylic acid at low pH, there are two c.d. bands, one negative, at -207 nm, and one positive, at -227 nm. Both of these bands are probably due to the nr* transition. The author found that an increase in charge density caused an increase in the magnitude of the c.d., with concomitant shift of the c.d. bands to the red. The magnitude of the c.d. was found to be independent of the concentration of the polymer if a salt was present, but there was a concentration dependence in aqueous solution in the absence of a salt. C.d. spectra were recorded for systematic variations in the concentration and type of counter-ion. Ethylene dithioacetals and diethyl dithioacetals have been investigated for a number of monosa~charides.~' The c.d. spectra show one, or two, c.d. band(s) of low intensity between 235 and 250 nm, and a third band of low intensity that peaks below 220 nm. These workers found no overall relationship between the configurational pattern of the monosaccharide and the sign of these bands. However, there does appear to be a correlation between the configurations of C-2, C-3 and C-4 and the sign of the c.d. band that peaks below 220 nm. derivatives linked to the Sallam investigated 2-phenyl-1,2,3-osotriazole anomeric carbon atom for a large number of mon o s a ~ c h a r id e sHe .~ ~showed that the sign of the c.d. band that occurs at -260nm depends on the anomeric configuration. The a-Dor p-Lconfiguration exhibits a positive c.d. band, whereas the p-Dor a- configuration exhibits a negative c.d. band. Nakanishi and coworkers developed a particularly useful system for determining the configuration and conformation of sugars using di-pbromobenzoate derivative^.^^-^^ The strong absorption caused by each of the two benzoate groups attached to the sugar will interact, giving rise to an exciton splitting which results in two intense, c.d. bands of opposite K. Gekko, Biopolymers, 18 (1979) 1989-2003. M. K. Hargreaves and D. L. Marshall, Carbohydr. Rex, 29 (1973) 339-344. M. A. E. Sallam, Curbohydr. Res., 129 (1984) 33-41. N. Harada, H. Sato, and K. Nakanishi, Chem. Commun., (1970) 1691-1693. N. Harada and K. Nakanishi, J. Am. Chem. SOC.,91 (1969) 3989-3991. N. Harada and K. Nakanishi, Circular Dichroic Spectroscopy-Exciton Coupling in Organic and Eioorganic Stereochemistry, Univ. Science Books, Mill Valley, CA, 1982. (96) K. Nakanishi, M. Kuroyanagi, H. Nambu, E. M. Oltz, R. Takeda, G . L. Verdine, and A. Zask, Pure Appl. Chem., 56 (1984) 1031-1048.
(90) (91) (92) (93) (94) (95)
124
W. CURTIS JOHNSON,
JR.
sign. The sign of the long-wavelength band is the sign of the chirality as defined by these workers, and, using their methods can be related to the relative configuration of the two groups or the favored conformation. The difference in intensity between the two extrema of the two c.d. bands can be used to determine the configuration. For a di-p-bromobenzoate, the values in AE units are: 1,2-ee, 62; 1,2-ea, 62; 1,2-aa, 6; 1,3-ee, 0; and 1,3-ea, 16. For tri-p-bromobenzoates, the observed difference in intensity for the two extrema will be the algebraic sum of the three pairwise interactions possible.96Because the extinction coefficients for these sugars are known? concentrations are easily determined. The method can also be used to determine the branching points in o l i g ~ s a c c h a r i d e s . This ~ ~ * ~work ~ demonstrated the power inherent in c.d. spectroscopy for investigating the structure of sugar monomers and polymers.
ACKNOWLEDGMENTS This work was supported by National Science Foundation grant DMB-8415499 from the Biophysics program, and Public Health Service grant GM-21479 from the Institute of General Medical Sciences.
(97) H.-W. Liu and K. Nakanishi, 1. Am. Chem. Soc., 103 (1981) 5591-5593. (98) H.-W. Liu and K. Nakanishi, 1. Am. Chem. SOC.,103 (1981) 7005-7006.
ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY, VOL. 45
PROTON SPIN-LATTICE RELAXATION RATES IN THE STRUCTURAL ANALYSIS OF CARBOHYDRATE MOLECULES IN SOLUTION
BY PHOTISDAIS"AND ARTHURS. PERLIN Department of Chemistry, McGill University, Montreal, Quebec H3C 3G1, Canada
I. Introduction .............................................................
............................................ 11. Theory ... . _ . . ...... .... 1. General Formulation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Separation of the pt, and at,Relaxation Rates.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Evaluation of the p,, and a,,Relaxation Rates, and Molecular Motion . . . . . . . 4. Determination of Interproton Distances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Strongly Coupled Spin-Systems . . . . . . . . . 111. Spin-Lattice Relaxation Measurements
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .
1. Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Analysis of Relaxation Data . . . . . . . . . . . . . ................... 3. Errors and Remedies ................................................... IV. Stereochemical Implications of Relaxation Rates . . . . . . . . . . . . . .......... 1 . Nonselective Spin-Lattice Relaxation Rates. . . ......................... 2. Selective Spin-Lattice Relaxation Rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Limitations and Relative Merits of Relaxation Methods . . . . . . . . . . . . . . . . . . . . . . .
125 128 128 130 136 137 138 138 138 142 145 147 147 159 163
I. INTRODUCTION The proton spin-lattice relaxation-rate (R,) is a well established, nuclear magnetic resonance (n.m.r.) parameter for structural, configurational, and conformational analysis of organic molecules in solution.'-5 As yet, however, its utility has received little attention in the field of carbohydrate chemistry, (1) (2) (3) (4) (5)
C. M. Preston, Ph.D. Thesis, University of British Columbia, B.C., Canada, 1975. L. D. Hall, Chem. SOC.Rev., 4 (1976) 401-420; Chem. Can., 28 (1976) 19-23. L. G. Werbelow and D. M. Grant, Ado. Magn. Reson., 9 (1977) 190-299. R. L. Vold and R. R. Vold, h o g . Nucl. Magn. Reson. Spectrosc., 12 (1978) 79-133. K. F. Wong, Ph.D. Thesis, University of British Columbia, B.C., Canada, 1979.
*Present address: Department of Chemistry, University of Crete, Iraklion, Crete, Greece.
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Copyright @ 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PHOTIS DAIS A N D ARTHUR S. PERLIN
and very few studies have been reported in which relaxation rates were used to extract qualitative or quantitative information, or both, about the geometry of carbohydrate molecules in solution. One obvious reason for this neglect is historical: that is, there was ready access for many years to chemical shifts and coupling constants and, to a lesser degree, nuclear Overhauser enhancement (n.0.e.) values, all with their well established, stereospecific dependencies. Although these parameters provide invaluable information, their validity is usually limited to a qualitative, or, at best, a semi-quantitative description of molecular structure and conformation. Even the universally employed Karplus curve6 entails many ambiguities, because the determination of an accurate, dihedral angle from coupling-constant data requires a knowledge of the magnitude of several factors that can only be approximated. The interpretation of n.0.e. data also becomes complex for molecules characterized by multiple relaxation-pathways, because saturation effects cause secondary population-changes that decrease the magnitude of the primary n.0.e. effect (the so-called “three-spins effect”). Moreover, such factors as large internuclear distances, or failure of the dipole-dipole relaxation mechanism, may cause lower enhancement than expected, leading to erroneous, quantitative conclusions. The second reason is related to the misconception that proton dipolar relaxation-rates for the average molecule are far too complicated for practical use in stereochemical problems. This belief has been encouraged, perhaps, by the formidable, density-matrix calculation^^^^^^-^^ commonly used by physicists and physical chemists for a rigorous interpretation of relaxation phenomena in multispin systems. However, proton-relaxation experiments reported by Freeman, Hill, Hall, and their coworker^^^^^^^-^^ have demonstrated that pessimism regarding the interpretation of proton relaxation-rates may be unjustified. Valuable information of considerable importance for the carbohydrate chemist may be derived for the average molecule of interest from a simple treatment of relaxation rates. (6) M. Karplus, J. Chem Phys., 30 (1959) 11-13; J. Am. Chem Soc., 85 (1963) 2870-2871. (7) R. K. Wangness and F. Bloch, Phys. Reu., 89 (1953) 728-739; F. Bloch, ibid., 102 (1956) 104-135; 105 (1957) 1206-1222. (8) P. S. Hubbard, Rev. Mod. Phys., 33 (1961) 249-264. (9) J. H. Freed and G. K. Fraenkel, J. Chem. Phys., 39 (1963) 326-348. (10) A. G. Redfield, Adu. Magn. Reson., 1 (1965) 1-32. (11) R. M. Lynden-Bell, h o g . Nucl. Magn. Reson. Spectrosc., 2 (1967) 163-204. (12) R. Freeman and S. Wittekoek, J. Magn. Reson., 1 (1969) 238-276. (13) C. W. M. Grant, L. D. Hall, and C. M. Preston, J. Am. Chem. SOC.,95 (1973) 7742-7747. (14) R. Freeman, H. D. W. Hill, B. L. Tomlinson, and L. D. Hall, J. Chem Phys., 61 (1974) 4466-4473. (15) L. D. Hall and H. D. W. Hill, J. Am. Chem. SOC.,98 (1976) 1269-1270.
PROTON SPIN-LATTICE RELAXATION RATES
127
This simple theory"."." is based on the expectation that, to a reasonable degree of approximation, proton-proton, dipolar contributions to the measured spin-lattice relaxation-rate are pairwise additive and decrease as a simple sixth power of the interproton distance. The simplified version of the dipole-dipole mechanism is summarized in the following two equations for spin i coupled intramolecularly with a group of spins j
where
where Ri is the measured spin-lattice relaxation-rate for spin i, pV is the direct relaxation-rate between the pair of spins i and j , y, is the gyromagnetic ratio of spin i, r,, is the internuclear distance between spins i and j , h is Planck's constant divided by 277, and T' (ij)is the rotational correlation-time of the internuclear vector joining spins i and j . Thus, identification of all pairwise, interproton relaxation-contribution terms, pV (in s - ' ) , for a molecule by factorization from the experimentally measured R , values can provide a unique method for calculating interproton distances, which are readily related to molecular structure and conformation. When the concept of pairwise additivity of the relaxation contributions seems to break down, as with a complex molecule having many interconnecting, relaxation pathways, there are reliable separation techniques, such as deuterium substitution in key positions, and a combination of nonselective and selective relaxation-rates, that may be used to distinguish between pairwise, dipolar interactions. Moreover, with the development of the Fourier-transform technique, and the availability of highly sophisticated, n.m.r. spectrometers, it has become possible to measure, routinely, nonselective and selective relaxation-rates of any resonance that can be clearly resolved in a n.m.r. spectrum. The main objectives of this article are: ( i ) to give an account of the simple theory related to spin-lattice relaxation-rates, in a language that is directed, as far as possible, to the practising chemist rather than to the theoretician; (ii) to caution against uncritical use of this simple theory for systems that are strongly coupled, or undergoing anisotropic reorientation, or both; ( i i i ) to introduce the pulse n.m.r. experiments that are used to measure spinlattice relaxation-rates, and to stress the precautions necessary for accurate (16) R. Freeman, S. Wittekoek, and R. R. Emst, J. Chem. Phys., 52 (1970) 1529-1544. (17) J. H . Noggle and R. E. Shirmer, The Nuclear Overhauser Enhancemenf, Academic Press, New York, 1971, pp. 44-75.
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PHOTIS DAIS AND ARTHUR S. PERLIN
measurements; ( i o ) to discuss the types of experiments that reveal stereospecific dependencies of spin-lattice relaxation-rates, along with concrete examples from the literature; and ( 0 ) to assess the reliability of these experiments, and their relative merits as to accuracy and technical simplicity. It will be rewarding for the authors if this article finds interest among carbohydrate chemists, stimulating further experiments in the field. 11. THEORY
1. General Formulation
The process of spin-lattice relaxation involves the transfer of magnetization between the magnetic nuclei (spins) and their environment (the lattice). The rate at which this transfer of energy occurs is" the spin-lattice relaxation-rate ( R l , in s-'). The inverse of this quantity is" the spin-lattice relaxation-time ( T I , in s), which is the experimentally determinable parameter. In principle, this energy interchange can be mediated by several different mechanisms, including dipole-dipole interactions, chemical-shift anisotropy, and spin-rotation interactions. For protons, as will be seen later, the dominant relaxation-mechanism for energy transfer is usually the intramolecular dipole-dipole interaction. A homonuclear spin-system may be excited with radiofrequency (r.f.) pulses that are so intense (in the order of ps), compared to the frequency width of the spectrum, that all resonances are excited essentially uniformly. This is a nonselective excitation. A homonuclear spin-system may also be excited with a relatively weak, r.f. pulse (in the order of ms), in the sense that all components of a given multiplet are inverted at time zero, whereas the other resonances in the spectrum remain essentially unperturbed; this is a selective excitation. The r.f. pulse may be single-selective, that is, there is an inversion of one multiplet in the spectrum, or double-selective, tripleselective, and so on, where two, three, or more separate multiplets in the spectrum are inverted simultaneously while the remaining resonances remain unperturbed. For a multi-spin system, where Z = 1/2, the rate of change of the average magnetization ( M z i )of a spin i coupled by dipole-dipole interactions to a group of spins j is describedI7 by Eq. 3 /
(18) In proton-relaxation experiments, R, values are used extensively, whereas TI values are more frequently reported for 13C relaxation measurements. Although there is no special merit in this preference for "C T, values, the pairwise additivity of relaxation contributions in proton-relaxation experiments is more clearly apparent for the relaxation rates.
PROTON SPIN-LATTICE RELAXATION RATES
129
where the self-relaxation rate pi is given by
(36) Here, Moi is the magnetization of spin i at thermal equilibrium, p9 is the direct, dipole-dipole relaxation between spins i and j , uv is the crossrelaxation between spins i and j , and p? is the direct relaxation of spin i due to other relaxation mechanisms, including intermolecular dipolar interactions and paramagnetic relaxation by dissolved oxygen. Under experimental conditions so chosen that dipolar interactions constitute the dominant relaxation-mechanism, and intermolecular interactions have been minimized by sufficient dilution and degassing of the sample, the quantity p? in Eq. 36 becomes much smaller than the direct, intramolecular, dipolar interactions, that is, (4)
and can be omitted from Eq. 36. Under condition 4, Eq. 3b implies that the self-relaxation rate of spin i is the sum of the pairwise contributions from spin j, although this is true only when total nuclear magnetizations are considered and when the n.m.r. spectrum of the spin system is first-order. For reasons that will be apparent, Eq. 3a is cast in matrix notation as follows.
wherein the various relaxation-relation rates that govern the time evolution of the magnetization of spins i and j are connected with the transition probabilities by (6)
(7)
where W,( i j ) , W,( i j ) , and W,( i j ) are the transition probabilities associated with zero-, single-, and double-quantum transitions, respectively. The relaxation matrix of Eq. 5 is symmetric and contains two classes of elements; the diagonal elements, pi values, which represent the self-relaxation terms, and the off-diagonal elements, aU values, which are the cross-
130
PHOTIS DAIS AND ARTHUR S. PERLIN
relaxation terms between appropriate spins. Because the pi values contain single-quantum transitions, in addition to zero- and double-quantum transitions, as may been seen in Eq. 6, they are a measure of nondipolar as well as dipolar contribution^.'^ However, it is apparent from Eq. 7 that aiivalues are composed only of dipolar contributions. Hence, the determination of aiiprovides a quantitative measure of those dipolar terms and a valuable means for distinguishing the dipolar-relaxation process from the other relaxation mechanisms included in the pC term. The general solution of Eq. 3 or 5 for the evolution of the longitudinal magnetization is multiexponential, and the evaluation of the various relaxation-rates is numerically very difficult. Although progress has been made4*” in lessening the complexity of computing these terms for weakly coupled, heteronuclear-spin systems by use of “normal modes” analysis, a comparable simplification is less likely for most proton spin-systems which, at best, are only pseudo-first-order. Fortunately, the pi and uiiterms in the relaxation matrix of Eq. 5 may be separated by appropriate techniques and, through detailed expressions, can be related to interproton distances.
2. Separation of the pij and uijRelaxation Rates As already noted, the general kinetic equation describing the evolution of the longitudinal magnetization in multispin systems is a multiexponential function of time. A unique definition of spin-lattice relaxation-rates for protons cannot be made, because of the occurrence of nonexponentiality in the recovery curve of the magnetization. Two factors contributing to this behavior of the magnetization bear comment; these are cross-relaxation and cross-correlation. Cross-relaxation effects, represented by the aii term in Eq. 5, are caused by a transfer of magnetization from any neighboring, nonequilibrium spin by way of zero- and double-quantum transitions. The influence of cross-relaxation effects on measured relaxation-rates have been fully evaluated elsewhere. 163’7~21-23Cross-correlation effects arise from the correlated diffusion of vectors between different pairs of protons in the same molecule. This correlation evolves with time, and is described by a correlation function that, by analogy with the autocorrelation function, (19) A single-quantum transition involves one spin only, whereas the zero- and doublequantum transitions involve two spins at the same time. The zero- and double-quantum transitions give rise to cross-relaxation pathways, which provide an efficient mechanism for dipole-dipole relaxation. (20) L. G . Werbelow and D. M. Grant, J. Chem. Phys., 63 (1975) 544-556. (21) 1. D. Campbell and R. Freeman, J. Magn. Reson., 1 1 (1973) 143-162. (22) A. Kalk and H. J. C. Berenden, J. Magn. Reson. 24 (1976) 343-366. (23) 1. D. Campbell, C. M. Dobson, R. G. Ratcliffe, and R. J. P. Williams, J. Magn. Reson., 29 (1978) 397-417.
PROTON SPIN-LATTICE RELAXATION RATES
131
describes the motion of individual vectors. Cross-correlated motions of three or four spins result in a nonexponential recovery of magnetization for both isotropic and anisotropic rotational motion. However, the influence of cross-correlation effects on the magnetization-recovery c ~ r v e ~is~very -*~ small for isotropic motion, as compared to that for molecules rotating anisotropically. The latter are characterized by two or more exponentials, the magnitudes of which depend on the degree of anisotropy. For the proton spin-systems of most organic molecules, the factorization of cross-correlation terms involved in spin-lattice-relaxation experiments is generally very difficult, and need not be considered here; detailed treatments of this subject may be found in Refs. 4, 27, and 28. Problems associated with nonexponential decay of magnetization and cross-correlation effects may be avoided by confining the measurements to those of initial relaxation-rates, RY, following the original suggestion of Freeman and coworkers.'6 The initial relaxation-rate is defined as the relaxation rate obtained immediately after a 180", r.f. pulse. In that case, the population differences in the energy levels associated with a given transition remain the same for all interacting spins, and result in equal relaxation-rates for all lines in a multiplet. In practice, as will be seen later, the determination of the initial relaxation-rate is based on "the initial slope approximation," that is, the initial portion of the recovery curve obtained before a significant build-up of cross-relaxation and cross-correlation effects. The necessary condition for the magnetization-recovery curve to be described by a single rate-constant during the time interval of zero to t is
a y < <1.
(8)
a. Nonselective Relaxation Rates.-For a nonselective excitation under the initial slope condition in Eq. 8, Eq. 5 can be solved. The magnetization recovery is described by the kinetic equation
where, M , , ( t ) specifies the magnetization close to the initial slope, and Mz i ( 0 )specifies the initial condition of the experiment, that is, Mzi(0)= -Moi following the nonselective, 1SO", perturbing pulse. Here, the nonselective (24) P. S. Hubbard, Phys. Rev., 109 (1958) 1153-1158; 1 1 1 (1958) 1746-1747; 128 (1962) 650-658. (25) R. L. Hilt and P. S. Hubbard, Phys. Rev., 134 (1964) A392-A298. (26) L. K. Runnels, Phys. Rev., 134 (1964) A28-A36. (27) L. G . Werbelow and A. G. Marshall, J. Magn. Reson., 1 1 (1973) 299-313. (28) D. M. Grant and L. G . Werbelow, J. Magn. Reson., 21 (1976) 369-371.
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PHOTIS DAIS A N D ARTHUR S. PERLIN
(ns) relaxation-rate is related to the fundamental relaxation rates pi and uii,as follows. Ri(ns)=
c (pii+aii)+p:.
j# i
Furthermore, as will be seen later, under the extreme, narrowing condition, and for isotropic motion, the following simple relationships hold. a,.= f f . . V
JF
p.. = p.. = -2ff.. lJ
JI
V*
Under these conditions, and neglecting external relaxation-interactions according to Eq. 4, the pairwise relaxation-contributions, pii, are evaluated from Eqs. 10-12, provided that an equal number of experimentally determined R ; (ns) values are available. For a weakly coupled, three-spin system, AMX, this is possible, because there are three nonselective relaxation-rate values for three unknown pij values ( P A M , p A X , p M x ) . For a system in which j > 3 proton spins, this analysis cannot be unambiguously applied, because there are j ( j - 1)/2 values of pii to be determined from j measured R{( ns) values. However, under favorable circumstances (see Section IV), depending on the relative disposition of the proton spins in the molecular frame, some pii values may be disregarded. This affords a good estimate of the appropriate pii values, and, hence, information about molecular geometry and conformation. It also is possible to determine pu values from a single, nonselective experiment for more than three spin systems, by combining nonselective relaxation-rates with n.0.e. experiments. According to Noggle and Shirmer,” Eq. 5 can be solved under conditions in which, at time t = 0, spin i is observed with simultaneous saturation of spin j , resulting in the expression
In Eq. 13, the sum over j includes all saturated spins, whereas the sum over
k includes all spins except i and j . Another separation method in using nonselective relaxation-rates is based on deuterium s u b ~ t i t u t i o n ,and * ~ ~utilizes ~~ the fact that the efficiency with which a nucleus contributes to the relaxation of a neighboring nucleus is proportional to the square of the magnetogyric ratio of the donor nucleus, PHD/PHH = 8/3( yD/ yH)2.Because yD/ yH = 116, replacement of that is,29*30 a proton by a deuterium nucleus would be expected to contribute to the (29) K. Akasaka, T. Imoto, and H. Hatano, Chern. Phys. Lett., 21 (1973) 398-400. (30) K. Akasaka, T. Imoto, S. Shibata, and H. Hatano, J. Magn. Reson., 18 (1975) 328-343.
PROTON SPIN-LATTICE RELAXATION RATES
133
relaxation of an adjacent proton by -6%, relative to that of the proton replaced. Thus, a comparison of the proton R,(ns) values for an organic molecule with those of a specifically deuterated analog of it should provide a direct method for separating, and evaluating, individual interproton contributions, the plj. values. The necessary relation is given by3’ pii = 0.6959{R ;( ns)( H - j )
- R f (ns)(D -j ) } ,
(14)
where R f (ns)(H -j) and R i ( n s ) ( D - j ) are the nonselective relaxation-rates for spin i before and after substitution, respectively, at site j. b. Single-selective Relaxation Rates.-For a single-selective experiment, the initial, single-selective, spin-lattice relaxation-rate obtained immediately after the selective, 180’ pulse is defined as
Rf(i)=
c plj.+p:
j#i
under the initial conditions Mzi(0)= -Moi and Mzj = +Moj.The tilde (’) is introduced to designate that only the resonance of spin i has been inverted by the 180” pulse. This leads to a key conclusion, and provides the fundamental means for a quantitative test of the dipole-dipole mechanism. If it is correct to assume that the dipole-dipole interaction is the dominant relaxation-mechanism, that is, that Eq. 4 holds, then a combination of Eqs. 10, 11, 12, and 15 gives the following r a t i ~ ’ ~for ’ ’ ~a weakly coupled spin-system.
Eq. 16 is an extremely useful criterion for examining the extent of dipolar interaction in a multispin system, and gives the relaxation method a major advantage over the n.0.e. method. The equivalent quantitative test for the n.0.e. experiment requires all but the receptor nucleus to be saturated; and this is not readily performed in practice. A deviation from the value of 1.5 indicates that other relaxation mechanisms contribute to the relaxation of spin i. The extent of intramolecular dipole-dipole interactions for spin i is given by”
f
i(
ns) =
2[Ri(ns)-Rf(i)]
R:(i)
I
where f ’ ( n s ) is the fraction of relaxation of spin i arising from the intramolecular dipole-dipole mechanism. For p: = 0, the fraction fi(ns) equals unity. As for nonselective-relaxation experiments, Eq. 15 can only be used for a three-spin system, because, for j > 3, the problem is underdetermined.
134
PHOTIS DAIS AND ARTHUR S. PERLIN
Nevertheless, the overall structural problem can be solved from combined n.0.e. and single-selective relaxation-measurements through the evaluation of individual cross-relaxation terms, uij.According to Noggle and Shirmer,” the n.0.e. value is a function of the cross-relaxation between spins i and j and the relaxation contributions of the neighboring protons to spin i, that is,
c. Double-selective Relaxation Rates.-An obvious extension of the single-selective, pulse experiment is the application of two such pulses simultaneously to two different nuclei. The initial conditions for such an experiment, used to solve Eq. 5 from the initial section of the magnetization-recovery curve, are
M,i(O) = -Moi, Mzj(o)= -Moj
and
MZk(O)= +Mok,
where k values are unperturbed resonances in the spectrum. The initial slope of the recovery curve vs. time defines the double-selective relaxationrates for spins i and j . These are given, respectively, by
and According to these equations, the effect of selectively perturbing the spin states of spins i and j is to isolate the cross-relaxation paths common to these two spins. Combining Eqs. 15 and 19, the individual cross-relaxation terms are readily determined from single-selective and double-selective relaxation-rate measurements, that is, uij= R ;( ;;j ) - R I;(
and
9
(20a)
uji= R { ( (3’) - R { (5’).
(206)
Because it is reasonable to expect that Eq. 11 holds true, a more realistic experimental measure of the cross-relaxation between spins i and j would be UV = {;
R I( ( J’)
+
- R ( 91 [ R(( [j’) - R {( j’)]}.
When only non- and double-selective experiments can be performed, a relationship equivalent to Eq. 21 can be derived from Eqs. 10, 12, 15, and
PROTON SPIN-LATTICE RELAXATION RATES
135
21, namely,
+
uU= ; { [ 3 R f (i j 3 - 2 R f ( n s ) ] [ 3 R ( ([ j i - 2 R ( ( n ~ ) ] } .
(22)
The relative efficiency of the interaction of spins i and j in the system is then defined by"
where fj is the fraction of relaxation that spin i receives from spin j by way of the dipole-dipole mechanism. Clearly, f{Zfj, because, in general, R f (9 f Rj(59. For complete, dipolar relaxation for any two interacting spins in a weakly coupled spin-system,
1 fj=1. j#i
d. Triple-selective Relaxation Rates.-It was mentioned earlier that, for three isolated, mutually relaxing spins, the three pv values can be explicitly calculated from a single set of nonselective relaxation-rates by means of Eqs. 10 and 12. Eq. 10 can be extended to multispin systems, provided that a triple-selective pulse-experiment can be performed, that is, three resonances in the spectrum are simultaneously inverted while the remaining resonances are unperturbed. Thus, for a multispin system i, j, k, 1,. . . , the initial, triple-selective relaxation-rates for spins i, j, and k are defined by
R f ( i iR ) = C
Pjn+u,j.fujk,
(254
n#i
and Equations 25a-c indicate that the effect of selectively perturbing spins i, j, and k is to isolate the cross-relaxation paths common to these three nuclei. Combining Eqs. 10, 12, and 25, the individual cross-relaxation terms are determined from triple-selective and non-selective relaxation-rates according to (26)
where
PHOTIS DAIS AND ARTHUR S. PERLIN
136
3. Evaluation of pii and aijRelaxation Rates, and Molecular Motion For a rigid molecule tumbling isotropically in solution, the quantities pij and uc are given by3’
and where w is the Larmor frequency, rc the distance between spins i and j having gyromagnetic ratios yi and yj, respectively, and T,( ij) is the rotational-correlation time, which describes the orientation of the vector between spins i and j situated in the rigid molecular framework. Equations 28 and 29 reflect several characteristics of the dipole-dipole interactions between spins i and j, as follows. ( a ) The contribution that each nucleus makes to the relaxation of any proton is proportional to the square of its gyromagnetic ratio. Because only protons have a high value of y, intramolecular relaxation will be dominated by interproton interactions, with the nearest protons making the largest contributions. (b) The efficiency of this interproton relaxation depends on the distance between protons, and falls off with the sixth power of the distance; hence, the stereospecific dependencies of the relaxation rates. (c) The relaxation is dependent on the correlation time, T,, which is related to the rate of reorientation of the molecule in solution. (d) It is possible to calculate correlation times from experimental relaxationrates in combination with interproton distances obtained by other techniques, in order to derive information about molecular dynamics. (e) The relative magnitude of the cross-relaxation term depends on the rate of the overall, molecular motion. Quantity uc is negative, and smaller than pu under the extreme narrowing limit (u2?f<< 1). Clearly, where Eqs. 11 and 12 hold, Eqs. 28 and 29 simplify to
Moreover uij,as a function of T,, passes through zero for w27f = (1.118)2, and becomes much larger than pii for w2?f >> 1. In the latter circumstance, corresponding to the diffusion limit22932 for such large molecules as proteins and biopolymers, the rate of the energy transfer between protons becomes much larger than the rate of energy exchange with the lattice. This results in an equalization of the relaxation rates of all protons in the molecule, (31) I. Solomon, Phys. Reo., 99 (1955) 559-565. (32) K. Akasata, J. Magn. Reson., 45 (1981) 337-343.
PROTON SPIN-LATTICE RELAXATION RATES
137
and, hence, to a loss of all stereochemical information. In addition, the relaxation ratio in Eq. 16 is no longer meaningful, because, when molecular motion becomes very slow ( W * T ~>> l), this ratio tends towards zero. This simple relaxation theory becomes invalid, however, if motional anisotropy, or internal motions, or both, are involved. Then, the rotational correlation-time in Eq. 30 is an effective correlation-time, containing contributions from reorientation about the principal axes of the rotationaldiffusion tensor. In order to separate these contributions, a physical model to describe the manner by which a molecule tumbles is required. Complete expressions for intramolecular, dipolar relaxation-rates for the three classes of spherical, axially symmetric, and asymmetric top molecules have been evaluated by Werbelow and Grant,3 in order to incorporate into the relaxation theory the appropriate rotational-diffusion model developed by Woessner.33 Methyl internal motion has been treated in a few instance^,^^'^' by using the equations of Woessner and coworker^'^ to describe internal rotation superimposed on the overall, molecular tumbling. Nevertheless, if motional anisotropy is present, it is wiser not to attempt a quantitative determination of interproton distances from measured, proton relaxationrates, although semiquantitative conclusions are probably justified by neglecting motional anisotropy, as will be seen in the following Section.
4. Determination of Interproton Distances Given the specific, internuclear dipole-dipole contribution terms, pii, or the cross-relaxation terms, uii,determined by the methods just described, internuclear distances, rii, can be calculated according to Eq. 30, assuming isotropic motion in the extreme narrowing region. The values for T=(ij) can be readily estimated from carbon-13 or deuterium spin-lattice relaxationtimes. For most organic molecules in solution, carbon-13 R1 values conveniently provide the motional information necessary, and, hence, the type of relaxation model to be used, for a pertinent description of molecular reorientations. A prerequisite to this treatment is the assumption that interproton vectors and 13C-'H vectors are characterized by the same rotational correlation-time. For rotational isotropic motion, internuclear distances can be compared according to
(33) (34) (35) (36)
D. E. Woessner, J. Chem. Phys., 37 (1962) 647-654. W. M. M. J. BovCe and J. Smidt, Mol. Phys., 26 (1973) 1133-1136; 28 (1974) 1617-1635. R. Rowan 111, J. A. McCammon, and B. D. Sykes, J. Am. Chem. Soc., 96 (1974) 4773-4780. D. E. Woessner, B. S . Snowden, and G. H. Meijer, J. Chem. Phys., 50 (1969) 719-721.
138
PHOTIS DAIS AND ARTHUR S. PERLIN
where the acceptor spin i receives relaxation contributions from the donor spins j and k Eq. 31, derived from Eq. 30, is very useful for solving stereochemical problems, because, if one distance is known, all other distances can be calculated. Eq. 31 has another important feature: it demonstrates that the propagation of experimental errors in relaxation rates through the inverse sixth-root calculation works in favor of the experimentalist. Thus, a 10% error in a pu value is lessened to a 1.7% uncertainty in the calculated interproton distances. In addition, even if a molecule tumbles somewhat anisotropically, Eq. 31 still provides a reasonable measure of interproton distances, because a small anisotropic effect would be effectively diminished to a negligible level.
5. Strongly Coupled Spin-Systems If the foregoing theory is to be applied accurately to a two-spin system, the latter must be weakly coupled, that is, J / S should3’ be <0.1. In strongly coupled spin-systems, serious cross-relaxation effects can arise.37 In such systems, the transition probabilities for the components of a spin multiplet are mixed by the strong coupling, and involve the relaxation parameters of all interacting spins, so that each line in a multiplet shows a different recovery-curve. Therefore, measurements based on initial relaxation-rates would be misleading unless appropriately corrected for this mixing. A simplified treatment of strong coupling in a representative AB spin system was given by Campbell and Freeman,” whereas the density-matrix theory4,’ can be used, in principle, to predict experimental spin-lattice relaxation recovery curves for more-complex n.m.r. spectra. 111. SPIN-LATTICERELAXATIONMEASUREMENTS 1. Experimental Methods
The most popular, and also a very accurate, experimental method for measuring nonselective spin-lattice relaxation-rates is the inversion recovery (180” - t - 90”-AT- PD)NTpulse sequence.38Here, t is the variable parameter, the “little t” between pulses, AT is the acquisition time, PD is the pulse delay, set such that AT+ PD 2 5 x T,, and NT is the total number of transients required for an acceptable signal-to-noise ratio. Sequential application of a series of two-pulse sequences, each using a different pulsespacing, t, gives a series of “partially relaxed” spectra. Values of R , can (37) Ref. 17, pp. 226-232. (38) R. L. Void, J. S. Waugh, M. P. Klein, and D. E. Pheips, J. Chem. Phys.,48 (1968) 3831-3832.
PROTON SPIN-LATTICE RELAXATION RATES
FIG.1.-Diagrammatic
139
Representation of the Recovery of Magnetization along the z-Axis
( M z ) ,from Its Initial Value ( - M o ) to +M,,Following Its Inversion by a 180" Pulse. {The exponential recovery curve shown in [A] depicts the return of magnetization that would be
found in a typical inversion-recovery experiment. The curve in [B] would be obtained from which decreases from an initial value of a three-pulse sequence, and is a plot of (M,- M,), +2M0 to zero at infinite time: in this class of experiment, transitions having shorter relaxationtimes (higher relaxation-rates) decrease most rapidly towards zero intensity. (Reproduced, with permission, from Ref. 39.))
be obtained either directly from the experimental curve of the change of magnetization vs. time (see Fig. lA39),or from the traditional, semilogarithmic plot of In ( M , - M , ) / M , us. time, where M , is the measured intensity at thermal equilibrium. The infinite-intensity value, M,, is obtained in a separate experiment with a pulse spacing that is much larger than the longest relaxation-time. Another method used to measure relaxation rates is the three-pulse (180"- t -900-AT-PD-90°)NT ~equence.~' This method is similar to the inversion recovery, with the addition of a third 90" pulse after the pulse (39) L. D. Hall, C. M. Preston, and J. D. Stevens, Carbohydr. Rex, 41 (1975) 41-52. (40) R. Freeman and H. D. W. Hill, J. Chem. Phys., 54 (1971) 3367-3377.
140
PHOTIS DAIS AND ARTHUR S. PERLIN
delay, so that the deviation from equilibrium ( M , - M , ) can be immediately stored and later transformed. The relaxation rates are obtained as previously, either from the change of the magnetization recovery curve us. time (see Fig. 1B) or from the semilogarithmic plot. With this type of experiment, there is no need to measure the equilibrium intensity. Although it has been claimed4' that this pulse sequence helps to minimize systematic errors due to drifts in instrument resolution, the inversion-recovery method is preferred, mainly for two reasons. One, it is faster than the three-pulse sequence and, second, changes in magnetization can be monitored more readily in the partially relaxed spectra. Because of the need for long waiting-times in the inversion-recovery method, as well as the unavoidably time-consuming procedure of measuring enough points (usually 20-30), other methods of measuring relaxation rates may be preferable, especially for low relaxation-rates. Such methods as saturation-recovery4' (90"- t - 90"-AT- PD)NT and progressive satura t i ~ n [90° ~"~ (~t - 90")NT]pulse sequences, used for I3Cspin-lattice relaxation measurements, can be adapted for measurements in multispin systems. However, both methods suffer from the drawback that 90" pulses cause mixing effects, as will be seen later. Furthermore, if mixing effects do not decay out prior to the monitoring pulse, the resulting spectrum may exhibit phase and intensity anomalies, preventing accurate measurements of relaxation rates. As yet, no appropriate comparison has been made of relaxation parameters obtained by detailed analysis of the recovery curves in inversionrecovery, saturation-recovery, and progressive-saturation experiments on coupled spin systems, and, because there is no guarantee that the last two methods provide reliable results, they should be avoided. To lessen experimental time, the "null-point" method may be employed43 by locating the pulse spacing, tnull, for which no magnetization is observed after the 180"- t - 90" pulse-sequence. The relaxation rate is then obtained directly by using the relationship R , = 0.69/ tnul,.In this way, a considerable diminution of measuring time is achieved, which is especially desirable in measurements of very low relaxation-rates, or for samples that are not very stable. In addition, estimates of relaxation rates for overlapping resonances can often be achieved. However, as the recovery curves for coupled spinsystems are, more often than not, nonexponential, observation of the null point may violate the initial-slope approximation. Hence, this method is best reserved for preliminary experiments that serve to establish the time scale for spin-lattice relaxation, and for qualitative conclusions. (41) J. L. Markley, W. J. Horsley, and M. P. Klein, J. Chem. Phys., 55 (1971) 3604-3605. (42) R. Freeman, H. D. W. Hill, and R. Kaptein, J. Magn. Reson., 7 (1972) 82-98. (43) L. D. Colebrook, and L. D. Hall, Can. J. Chem., 58 (1980) 2016-2023.
PROTON SPIN-LATT’ICE RELAXATION RATES
141
FIG.2.-A. Normal, ‘H-N.m.r. Spectrum of “Asperlin” (1) in Benzene-d, at 400 Mz. B. Representative, ‘H Single-selective Spin-Lattice Relaxation Experiment with “Asperlin”; the H-7 Signal Was Inverted by a Selective, 180”Pulse (-15 ms), t =0.01 s. C. Representative, ‘H Double-selective Spin-Lattice Relaxation Experiment, in which Two Signals, H-5and H-7, Were Inverted Simultaneously by Two Consecutive, Selective, 180” pulses (-15 ms Each) Provided by the Decoupler Channel, t = 0.1 s. (Reproduced from Ref. 44.)
Selective, spin-lattice relaxation-rates are measured by the inversionrecovery technique. A rather weak, 180” pulse of very long duration (1050 ms) inverts a multiplet (single-selective) or two multiplets (double-selective) in the spectrum of asperlin (1; see Fig. 2@) and the recovery of the
1
magnetization after a time, f, is monitored by a nonselective, 90” pulse. In conventional experiments, the selective pulse is provided by the decoupler of the spectrometer. For single-selective pulse experiments, the decoupler frequency is set at the midpoint of the chosen multiplet, whereas, for double-selective inversion experiments, the decoupler frequency is precisely set at the midpoint between the two resonances of interest. An audiooscillator is used to provide a sine wave of frequency equal to one-half of (44) P. Dais and A. S. Perlin, Can. J. Chem., 63 (1985) 1009-1012.
142
PHOTIS DAIS AND ARTHUR S. PERLIN
the chemical-shift separation between the two resonances. L2~16*45-48In performing double-selective experiments, the computer of the spectrometer may be used to control two consecutive, selective, 180”pulses provided by the decoupler, at two resonance frequencies chosen. In this type of experiment, however, there is an important, practical limitation to the selectivity of the frequency with which an individual experiment can be performed. Although the time interval between the two selective pulses is negligible (<200 ns), the duration of the consecutive pulses may be long compared to the relaxation rates, allowing for the occurrence of significant relaxation during excitation. This is particularly important for methylene and methyl protons, which are characterized by high relaxation rates.49 Selective experiments can also be performed by the “tailored excitation” method of Tomlinson and Hill.” The selective pulse is frequency-modulated with a function designed to yield zero effective field at the resonance offset of the neighboring nuclei. Although this technique is especially promising for studies of more-complex spin systems, its use is as yet very limited, in part because the instrumentation needed is not yet commercially available.
2. Analysis of Relaxation Data Relaxation data may be analyzed by two general methods: a twoparameter, linear and a three-parameter, nonlinear, fitting p r ~ c e d u r e . ~The ~ - ’ first ~ method requires an accurate experimental determination of M,,which is both difficult and time-consuming. Furthermore, the (45) P. F. Fagerness, D. M. Grant, K. F. Kuhlmann, C. L. Mayne, and R. 9. Parry, J. Chem. Phys., 63 (1975) 2524-2532. (46) M. F. Augnsteijn, W. M. J. BovCe, S. Emid, A. F. Mehlkopf, and J. Smidt, J. Magn. Reson., 7 (1975) 301-318. (47) D. W. Alderman, J. J. Led, E. J. Pedersen, and N. F. Andersen, J. Magn. Reson., 21 (1976) 77-86. (48) K. Bock, R. Burton, and L. D. Hall, Can. J. Chem., 54 (1976) 3526-3535; 55 (1977) 1045- 1054. (49) P. Dais, T. K. M. Shing, and A. S. Perlin, 1. Am. Chem. SOC., 106 (1984) 3082-3089. (50) B. L. Tomlinson and H. D. W. Hill, 1. Chem. Phys., 59 (1973) 1775-1784. (51) H. Hanssum, W. Maurer, and H. Ruterjans, J. Magn. Reson., 31 (1978) 231-249; H. Hanssum and H. Ruterjans, ibid., 39 (1980) 65-75. (52) R. Crouch, S. Hurlbert, and A. Ragouzeos, J. Magn. Reson., 49 (1982) 371-382. (53) G. H. Weiss and J. A. Ferretti, J. Magn. Reson., 61 (1985) 490-498. (54) D. L. De Fontaine, D. K. Ross, and 9. Temai, J. Magn. Reson., 18 (1975) 276-281. (55) M. Sass and D. Ziessov, J. Magn. Reson., 25 (1977) 263-276. (56) J. Kowalewski, G. C. Levy, L. F. Johnson, and L. Palmer, J. Magn. Reson., 26 (1977) 533-536. (57) A. Ejchart, P. Oleski, and K. Wroblewski, J. Magn. Reson., 59 (1984) 446-451. (58) T. K. Leipert and D. W. Marquardt, J. Magn. Reson., 24 (1976) 181-199. (59) J. Granot, 1. Magn. Reson., 53 (1983) 386-397.
PROTON SPIN-LATTICE RELAXATION RATES
143
calculation, in the form of a semilogarithmic plot, uses (M,,- M,) values, rather than M, values directly, which may be negative for long t pulsespacings due to statistical errors in M,. Finally, semilogarithmic evaluation of the data gives points on one axis, errors in which are not independent of the other axis. The second method has the advantage of incorporating systematic errors, for example, pulse imperfection or missetting, and low signal-to-noise ratio, in the adjustable parameters, and does not require the experimental M,, value because it is treated as an unknown parameter and is not analyzed in detail. Nevertheless, the first method is recommended, as it is much simpler numerically and more appropriate for the initial slope approximation. Also, deviations from linearity due to cross-relaxation effects are clearly evident in the semilogarithmic plot, and are amenable to statistical treatment when slight nonlinearities might be masked by statistical errors.*' The use of an average M, value, obtained from 3 to 4 separate measurements, increases the accuracy of the relaxation rates determined. The initial relaxation-rate, discussed in the preceding Section, is obtained by the initial slope approximation,16 that is, from the initial portion of the magnetization-recovery curve, sufficiently soon to minimize significant cross-relaxation and cross-correlation effects. In the semilogarithmic plot, this corresponds to a straight line, drawn through the experimental points at the initial, linear section of the curve (see Fig. 3). As the error introduced by cross-relaxation depends on the manner in which the straight line is traced through the experimental points, the latter should be sufficient in number to minimize bias in the best fit of the initial section of the recovery curve. Furthermore, the nonexponential recovery should be studied over an extended range, so as to obtain statistically reliable estimates of the relaxation rates, using t values up to successively larger fractions of TI, and to verify that no significant trends occur as more and more long t values are included. These considerations are nicely illustrated in Fig. 3 for the determination of the nonselective relaxation-rates of H-5of 2,3:5,6-di- 0isopropylidene-a-D-mannofuranose60 (2) and H-1' of a deuterated sample 1.92
H
HsC,
1.25
/
H 1.32 (1.37)
0.97
0.94
(0.93)
2 R=H R = D (in parentheses) (60) P. Dais and A. S. Perlin, Can. J. Chem., 60 (1982) 1648-1656.
144
PHOTIS DAIS AND ARTHUR S. PERLIN
0.6 0.0
P
>
s
1.0
s v
c
2.0 0
0.5
1.o
1.5
2.0
2.5
0.6
2.0
o
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
t (s) FIG.3.-A. "Initial Slope Approximation" to Determine the Initial, Nonselective, SpinLattice Relaxation Rate of H-5 of 2,3:5,6-Di-0-isopropylidene-cr-~-mannofuranose (2) in Me,SO-d, Solution. (Points between 0.01 and 1.55 s were selected for tracing the best straight line.) B. The Same as in A for H-l'of a Partially Deuterated Sample of 1,6-Anhydro-~-cellobiose Hexaacetate (3). [Note that the relaxation of H-1' is strongly dependent on the choice of t value. An R:-"( ns) value of 0.24 s-' was obtained from the data points 0s rs 5 s, where a value of 0.18 s-' was obtained from the terminal decay 5 s t s 10 s (see text).]
of 1,6-anhydro-/3-cellobioseh e ~ a a c e t a t e(3). ~ ~The evaluation of the initial R:-'(ns) value (see Fig. 3A) was obtained for t values between 0.1 and HZC-
AcO AcO
0
OAc AcO 3
PROTON SPIN-LATTICE RELAXATION RATES
145
1.5 s (or 0.1 and 1.3 Tl periods). It should be noted that experimental points for t > 1.5 s start deviating from the straight line, which indicates the build-up of significant cross-relaxation effects. For the H-1' resonance, two values were available for Rp-"(ns),one corresponding to the period (0.01 < t < 5 s), 0.24 s, and the second for longer t values ( 5 < t < 10 s), 0.18 s (see Fig. 3B). The second value may be excluded, as it does not conform to the initial slope approximation concept, and also because the RY-"(ns) value, obtained as an initial slope, gives a ratio of 1.5 f0.09 relative to the single-selective R, value.49
3. Errors and Remedies
a. Systematic Errors.-A potentially serious problem in measuring relaxation rates concerns pulse imperfections. Line intensities measured for short t values, which are most useful for the initial slope approximation, are strongly influenced by residual transverse magnetization following an imperfect, 180" initial pulse. The latter, due mainly to effects of resonance offset and spatial inhomogeneity of the r.f. force ( H I ) across the sample, can be compensated for by using composite pulses.61 Another systematic error arises from the monitoring, 90" pulse in an inversion-recovery experiment. In contrast to the initial 180" pulse, which causes only an interchange of all pairs of transitions, the 90" pulse leads to mixing of the populations of all energy 1eve1s40*62 and, hence, of the observed relaxation-rates. For strong coupling, this mixing tends to obscure diff erences in the relaxation rates from multiplet to multiplet in the spectrum, which implies a loss of stereochemical information. An appropriate tool for analysis of mixing effects in strongly coupled spin-systems is the full-density matriX,4,5,62,63unless small flip-angles (10-20") are employed, despite the inherent loss of sensitivity. However, for weakly coupled spin-systems, mixing effects are confined entirely to lines within each m ~ l t i p l e t . 4This .~~~~~ may be an advantage, as differential relaxation of multiplet components is minimized. The initial slope approximation is used here to evaluate the average, initial relaxation-rate of a multiplet by summing up the intensities of each line component. In general, average multiplet relaxation-rates should be measured, whenever possible, by using several different monitoringpulses, in order to verify that the measured relaxation-rate is, indeed, characteristic solely of the multiplet in question. Two additional systematic errors may arise in selective pulse experiments associated with pulse selectivity and relaxation during excitation. Pulse (61) M. H. Levitt and R. Freeman, J. Magn. Reson., 33 (1979) 473-476; R. Freeman, S. P. Kempsell, and M. H. Levitt, ibid., 38 (1980) 453-479; 43 (1981) 65-80. (62) S. Schaublin, A. Hohener, and R. R. Ernst, J. Magn. Reson., 13 (1974) 196-216. (63) R. R. Emst and R. E. Morgan, Mol. Phys., 26 (1973) 49-74.
146
PHOTIS DAIS AND ARTHUR S. PERLIN
selectivity requires r.f. pulses strong enough to irradiate, uniformly, all parts of the multiplet in question, and yet weak enough to avoid significant perturbation of neighboring lines in the spectrum. Although pulse selectivity is not difficult to achieve, a lower limit on pulse strength must be imposed by the magnitude of the measured spin-lattice relaxation-rate in order to avoid relaxation during excitation. If the pulse duration is comparable to the relaxation rate, relaxation during the excitation period may lead to incomplete inversion by the initial 180”pulse and, furthermore, to distortion of the recovery curve obtained by using the monitoring, 90” Although relaxation effects during excitation have not yet been considered in great detail, it is reasonable to expect that, if the spin-lattice relaxationrate is at least -20-50 times lower than the pulse duration, relaxation during the pulses will not introduce a serious problem. The unavoidably long waiting-times required for accurate inversionrecovery experiments place stringent requirements on the static-field homogeneity. This is largely circumvented in modern spectrometers equipped with superconducting magnets, which produce a very stable field. However, when extensive time averaging is required, the procedure described by Mayne and is suggested. According to this technique, the t values are “arrayed” by an appropriate computer-program: that is, instead of accumulating NT transients for a given t value, storing the f.i.d. on the disk, and then moving to the next pulse-spacing, the computer stores a f.i.d. for each t value on the disk, and repeats this process NT times, adding successive f.i.d. values. Thus, slow homogeneity drifts and other systematic errors are averaged more or less equally over spectra at all t values. A potential source of systematic error is the quantity p ? , which may not be negligible, and indicates that relaxation mechanisms other than dipoledipole interactions contribute to the relaxation of the spin in question. Problems arising from intermolecular dipolar contributions, or paramagnetic relaxation due to dissolved oxygen, can be minimized by restricting the relaxation measurements to degassed, dilute solutions in deuterated, preferably inert, solvents. Oxygen may be removed either by bubbling dry nitrogen or helium gas through the sample in the n.m.r. tube or, better, by 5-6 freeze-pump-thaw cycles, after which the tube is sealed under vacuum. Experience has shown that measurements on solutions of 1-3% (w/v) concentration involve only very small intermolecular dipolar contributions that can be safely ignored. However, for a more rigorous treatment of the problem, dilution studies65are recommended. (64) C. L. Mayne, D. W. Alderman, and D. M. Grant, J. Chem. Phys., 63 (1975) 2514-2523. (65) J. Homer and E. R. V. Cedeno, J. Chem. SOC.,Faraday Trans., 79 (1983) 1021-1024; 80 (1984) 375-382.
PROTON SPIN-LATTICE RELAXATION RATES
147
When other relaxation mechanisms are involved, such as chemical-shift anisotropy or spin-rotation interactions, they cannot be separated by application of the foregoing relaxation theory. Then, the full density-matrix formalism should be employed.
b. Random Errors.-Random errors are not crucial in the determination of relaxation rates, and are readily minimized by good experimental methodology and a high-quality spectrometer. To further lessen the influence of random errors in measuring signal intensities, the t values may be “arrayed” in a random manner, whereby long t values are alternated with low t values. A final caution concerns the error introduced into the calculated interproton distances. As this depends on the errors of the measured quantities, it is propagated through the calculations according to Eq. 32 for independent and random namely,
where Sq is the error in the derived quantity, q, which is a function of the measurable quantities xl, x2,. . . ,x, with errors ax,, ax2,. . . ,Sx,, respectively. It is apparent from the foregoing discussion that several precautions are necessary in order to obtain accurate measurements of nonselective and selective relaxation-rates. Under these conditions, and with the availability of the modern Fourier-transform instrumentation, it is now possible to measure relaxation rates with an accuracy of 1-3%. The reward is great: accurate information about the structure and conformation of molecules in the liquid phase, as will be seen in the following section.
IV. STEREOCHEMICAL IMPLICATIONSOF RELAXATIONRATES 1. Nonselective Spin-Lattice Relaxation Rates
The first observations on the stereochemical dependence of spin-lattice relaxation-rates of carbohydrate molecules, beginning in67-701972,provided a general survey of the nonselective relaxation-rates of the anomeric protons of monosaccharide derivatives, oligosaccharides, and some polysaccharides. (66) J. R. Taylor, An Introduction lo Error Analysis, University Science Books, Mill Valley, CA, 1982, pp. 40-74. (67) L. D. Hall and C. M. Preston, Chem. Commun., (1972) 1319. (68) L. D. Hall and C. M. Preston, Carbohydr. Rex, 29 (1973) 322-324. (69) C. M . Preston and L. D. Hall, Carbohydr. Res., 37 (1974) 267-282. (70) L. D. Hall and C. M. Preston, Carbohydr. Res., 49 (1976) 3-11.
TABLEI Nonselective Spin-Lattice Relaxation Rates (s-I) for the Anomeric Protons of Monosaccharides and Derivatives"
-
Monosaccharide
H-la
H-IS
H-IP/H-la
D-Glucose (4) D-Galactose (5) D-Mannose ( 6 ) 2-Deoxy-~-arabino-hexose (7) D-Allose (8) D-Ahrose ( 9 ) L-Rhamnose (10) D-Talose ( I I )
0.23 0.25 0.17 0.26
0.43 0.42 0.55 0.59
1.9 1.7 3.3 2.3
0.14 0.23
0.22 0.34 0.48 0.43
1.6 1.5 3.2 3.3
D-ldose (12) D-XylOSe (13) D-Ribose (14) D-Arabinose (15) D-LyXOSe (16)
0.27 0.18 0.22
0.34 0.30
1.3 1.7 0.7 0.65 2.1
0.15
0.13
0.25
0.14
0.15
0.16 0.29
Derivative
H-la
H-If3
H-I@/H-la
2-acetamido-2-deoxy-~-glucose ( 17) 2-acetamido-2-deoxy-~-mannose (18) methyl D-glucopyranoside (19) methyl-d, D-glucopyranoside (20)
0.23 0.22 0.37 0.21
0.50
0.83 0.63 0.45
2.2 3.8 1.7 2.2
methyl D-xylopyranoside (21) D-glucopyranose pentaacetate (22) D-galactopyranose pentaacetate (23) P- D-galactopyranose penta( acetate-d,) (24) a-D-idopyranose pentaacetate (25)
0.30 0.26 0.30
Degassed solutions, 10% (w/v)in D,O at 42".From Refs. 67-69.
0.83 0.59 0.50
0.38 0.26
2.8 2.3 1.7
TABLEI1 Nonselective Spin-Lattice Relaxation Rates (s-I)''
for the Anomeric Protons of Disaccbarides, Oligosaccharides, and Polysaccharides
Glycosyl group
Clycose residue Compound
-
'o P
Cellobiose (26) Maltose (27) Lactose (28) Melibiose (29) Gentiobiose (30) Sucrose (31) Maltotriose (32) Raffinose (33) Stachyose (34)
H-la
H-1p
H-lp/H-la
H-la
0.48 0.40 0.48 0.38 0.43
0.91 0.91 0.91 0.77 0.83
1.9 2.3 1.9
1.2
2.0
0.91
0.59
1.37
5% (w/v)solutions in D,O at 42".
H-lP/H-la
1.92
2.1 3.0 2.0 2.4 2.0
1.85
1.9 2.3
H-lp
1.69 0.91 1.87 1.16 1.59
Glycosyl residue Polysaccbande
cyclomaltohexaoseb (35) cyclomaltoheptaoseb (36) 2,3,4-tri-O-methyldextran (37) 2,3,6-tri-O-methylamylose (38) 2,3,6-tri-O-methylcellulose (39)
3.2
1% (w/v) solutions in D,O at 42". From Refs. 68 and 70.
H-la
H-1p
3.70 4.35 3.85 4.54 4.00
150
PHOTIS DAIS AND ARTHUR S. PERLIN
The relaxation data for these molecules, listed in Tables I and 11, were analysed in a qualitative manner in order to stress the diagnostic utility of the R,( ns) values for making simple, rapid, configurational assignments. Although there exist, in principle, a rather large number of pairwise interactions possible among the protons in sugar molecules, the inverse sixth-power dependence of intramolecular dipole-dipole interactions effectively diminishes this number to three, stereochemically distinct interactions: vicinal-gauche (vg), vicinal-trans (vt), and 1,3-syn-diaxial (aa). Geminal interactions are also important when carbon atoms bear two or more protons. These pairwise interactions are presented schematically in Fig. 4. With these guidelines, it is understandable why the axially oriented H-lb of the majority of the compounds in Tables I and I1 relaxes faster than the equatorially oriented H-la. It simply receives relaxation contributions from H-3 and H-5, which are syn-axially disposed in the 4C1conformation (see Scheme 1). This is evident from the data for D-allose (8), D-altrose ( 9 ) , and D-idose (12), compared to those of D-glucose (4) through 2-deoxy-~-arabinohexose (7). Removal of one 1,3-diaxial interaction, by inversion of the configuration of (2-3, lowers the relaxation rates of H-lb, and the H-lb : H-la ratio from 1.9 : 1 (D-glucose) to 1.3 : 1 (D-idose). The effectiveness of vicinalgauche interactions in causing relaxation is clearly seen in the differences between the ratios for D-glucose (4) and D-galactose (5) as compared to those for D-mannose (6), L-rhamnose (lo), and D-talose (ll),as a result of the different vicinal dispositions of H-2 and the anomeric protons. The high relaxation-rates for both anomers of 2-deoxy-~-arabino-hexose (7) confirm the importance of these gauche interactions. Long-range interactions between H-4 and the anomeric protons appear to have little or no influence
FIG.4.-The Three Distinct Proton-Proton Relaxation Pathways in a Six-membered Ring in the 4C,Conformation. [Vicinal-Gauche (vg), vicinal-trans (vt), and 1,3-diaxial (aa). Geminal relaxation-pathways are not shown.]
PROTON SPIN-LATTICE RELAXATION RATES
q
H
-
151
%OR
1
OR
H -1
H
4,S, 7,8,13,14,17,19-25
4, S, 13,17,19-25
%OR OR
H
6,7,9,10-12, IS, 16.18
H-1
6,7,10,11,16,18
14
9,12,1s
H-1 15
B
:
R
8.14
&H-1
@-1
H 16
14
IS, 16
S ~ ~ ~ ~ ~ l . - O c c u m of e n Ring c e Protons Expected to Have a Major Influence on the Relaxation Rates ( R , )of the Anomeric Protons (H-1) of Aldopyranoses and Derivatives (4-25, Table I) in the 4C,, or IC4, Conformation.
on the relaxation rates of the latter, as is readily seen from the close similarity in the data for D-glucose (4) and D-galactose (5). Analogous differential trends in the relaxation rates are observed (see Table I) for the remaining monosaccharides and some derivatives, as well as the reducing residues of oligosaccharides (see Table 11), although changes in molecular weight and the presence of substituents moderate direct comparisons. Such qualitative considerations are, however, somewhat more restricted for the aldopentopyranoses (13-16). As these compounds are conformationally inhomogeneous, undergoing rapid interconversion
PHOTIS DAIS A N D ARTHUR S. PERLIN
152
between the ,C, and ‘C, conformations (see Scheme l ) , their relaxation rates are affected by time-averaged contributions of the individual conformers. Nevertheless, some interesting comparisons may be made concerning the relative proportions of the two conformers in the mixture. For instance, the R , ( ns) values for both anomers of D-xylose (13) and that of P-D-lyxose (16p) are consistent with a preponderance of the 4C, form, whereas the diminutions in the relaxation rates for D-ribose (14) and D-arabinose (15) are indicative of a preponderance of the ’ C , conformer for these sugars. An important aspect of the data in Table I1 is the difference between the relaxation rates of the anomeric protons of the reducing (H-1) and nonreducing (H-1’) moieties of the disaccharides. For each molecule, H-1’ relaxes more rapidly than H-1, and this is attributable to the fact that the anomeric proton of the nonreducing moiety receives relaxation contributions from the protons on both sugar rings. Because the extent of such inter-ring interactions depends on the relative proximity of the nonreducing anomeric proton to those of the reducing moiety, quantification of the relaxation rates constitutes a means for determining the orientation of the glycosidic bond. The relaxation data for the anomeric protons of the polysaccharides (see Table 11) lack utility, inasmuch as the R , ( n s ) values are identical within experimental error. Obviously, the distribution of correlation times associated with backbone and side-chain motions, complex patterns of intramolecular interaction, and significant cross-relaxation and cross-correlation effects dramatically lessen the diagnostic potential of these relaxation rates. Stereospecific dependencies of the nonselective relaxation-rates for other protons in a sugar molecule may be seen in the datai3,” for a set of 3,4,6-tri-0-acetyl-l-~-benzoyl-2-deoxy-2-bromo-~-hexopyranose derivatives (40-42), as well as other 2-halogeno derivatives. A comparison of their I ?X
110
n 17 CH,OH
o 11 CHZOH A AcO c o
~
0 17
o 11 5 0
40
B
c 0o7 7 ~ z A AcO
OBCH~OH 0
2
4 A AcOc
OBz
0 29
41
05’
0
0 50
W 0 21
OBz
0 51
42
relaxation data confirms that, for an axially oriented proton, the most important relaxation pathways involve 1,3-diaxial and vicinal-gauche interactions, whereas, for an equatorially oriented proton, only vicinal-gauche interactions are effective relaxation contributors. These characteristics are ( 7 1 ) L. D. Hall and C. M. Preston, Carbohydr. Rex, 27 (1973) 286-288.
PROTON SPIN-LATTICE RELAXATION RATES
153
reflected in the reciprocal effects of the contribution of H-1 to the relaxation of H-2 and H-3. The relaxation rate of H-5 is influenced less, because its dominant relaxation pathway is by way of the 6,6’-protons. The latter relax most rapidly, reflecting the greater effectiveness of the geminal interaction. Qualitative relaxation-studies have also been reported for an extensive series of derivatives of inositols,’ pentopyran~ses,~’1,6-anhydro-P-~hexopyrano~es:~f u ~ a n o s e sand , ~ ~septan~ses.~’ In all instances, the experimentally determined R , ( ns) values reflect the anticipated geometry. For the furanose derivative^^^ especially, they provide a better means for distinguishing between epimeric pairs than the relatively ambiguous interpretation of coupling-constant data. From these studies of sugars and related molecules, it is possible to derive empirical rules whereby R,( ns) values can be used as the basis for configurational assignments. These rules are illustrated graphically in Fig. 5, where the relative relaxation-efficiency between two protons is plotted as a function of the interproton distance. It should be noted that comparisons of individual R , ( n s ) values, either among the series in Tables I and 11, or within a set of related sugar molecules, presupposes that the sugars all have the same rotational-correlation times. Also, as the rate of molecular motion in solution depends mainly on temperature, concentration, and solvent, these factors need to be evaluated before conclusions may be derived. Although several, scattered, relaxation data are available for sugar molecules as a function of these three variables, led to a systematic study of a series of 1,6-anhydro-P-~-hexopyranoses~~ the conclusion that comparisons between different molecules are justified only with data obtained at the same temperature, in the same solvent, and at the same standardized concentration. Proton nonselective relaxation-rates combined with several other relaxation techniques have also been reported76 for a series of mono- and di-saccharides in aqueous solution, in an attempt to elucidate specific solute-solvent interactions and the extent of hydration of these sugars. Moreover, it seems possible to determine, and compare, relaxation rates, even if they are obtained at different provided that the measurements are confined to the extreme narrowing region. Although nonselective relaxation-rates have been interpreted in terms of configuration and conformation, they do not readily provide information (72) (73) (74) (75) (76)
L. Evelyn and L. D. Hall, Carbohydr. Rex, 100 (1982) 55-61. K. Bock, L. D. Hall, and C. Pedersen, Can. J. Chem., 58 (1980) 1916-1922. K. Bock, L. D. Hall, and C. Pedersen, Can. J. Chem., 58 (1980) 1923-1928. J. M. Berry, L. D. Hall, and J. D. Stevens, unpublished results. A. Suggett, S. Ablett, and P. J. Lillford, J. Solution Chem., 5 (1976) 17-31; A. Suggett, ibid., 5 (1976) 33-46.
PHOTIS DAIS AND ARTHUR S. PERLIN
154
2 .o
3.0
4.0
s.0
FIG. 5.-Plot of the Relative Relaxation Efficiency between Two Protons uersus the Inverse Sixth Power of Their lnternuclear Separation. (Derived from measurements of Dreiding stereomodels; reproduced. with permission, from Ref. 43.)
about the exact spatial arrangement of the protons involved in relaxation. That is, the simplified form of Eq. 31, which is concerned with an isolated, three-spin system having pairwise additivity in relaxation rates, seems to break down for sugar molecules, wherein many interconnecting relaxation pathways exist. This failure is evident, for example? when the relaxation ratios of the various protons of 1,6-anhydro-~-cellobiose hexaacetate (3; see Table 111) are converted into interproton distances (see Table IV) by using, as a known parameter, the distance between the geminal protons of the anhydro ring.49 That is, these values differ appreciably from those obtained from crystallographic data. Some simplified assumptions for extracting interproton relaxation-contributions from a single set of nonselective relaxation-rates have been made for a tetrachlorotetra-0-mesyl-galucro-sucrosederivative77 (43). By con-
(77) J. M . Berry, L. D. Hall. D. W. Welder, and K . F. Wong, ACSSymp. Ser., 87 (1979) 30-49.
TABLE111 Spin-Lattice Relaxation Rates (in s-') of 2~,4,6,2'~-Hexa-O-acetyl-1,6-anhydro-4-O-~-~-glucopyranosyl-~-~-glucopyranose (3)" and Its Partially Deuterated Analog (3')" Experiment
H-1
H-2
H-3
H-4
H-5
H-6ab
H-6afb
H-1'
H-2'
H-3'
H-4'
H-5'
H-6'b
H-6'b'
1.56
0.36 0.38
0.44 0.47 1.07
0.54 0.52 0.96
0.88 0.96 1.09
0.72 0.74 1.03
1.49
1.49 1.66 1.12
0.17 0.51 1.51
0.74 0.50 1.51
1.64 1.10 1.49
1.64 1.11 1.48
3 Nonselective 400 MHz 200 MHz' 200/400 MHz
1.05
0.48
0.53 0.55 1.04
0.51
0.99 1.11 1.12
1.56 1.63 1.04
1.05
Yd
Nonselective Single-selective R:(ns)/R;( i)' Doub!e selective RA!, R,(L 4')
2)
Rl(Z,): R d ? , 27 R,(4', 6'b')
R,($',f'b) R,(5', 6'b')
0.83 0.55 1.51
0.62 0.64 0.56
0.50 0.33 1.51
0.24 0.16 1.50
0.40 0.60 0.58 0.60
0.5 1 0.57
1.15 0.62 0.535
1.19 1.12
Degassed 0.05 M solutions of 3 and 3 in acetone-d,. Notations a and b refer to the geminal protons of the glucopyranosyl group and 1,6-anhydroglucopyranoseresidue, respectively. Signals for H-I, 2, 4, 6a', and 6'b were unresolved. Signals for H-2, 3, 4, 6a,a', 2' and 3' were eliminated (290%) through deuteration. The estimated statistical error in the various relaxation rates was better than * 5 % , which yields a *60?' error in the relaxation rate ratio. The reproducibility in the various relaxation measurements was better than * 5 % . From Ref. 49.
TABLEIV Interproton Distances (A) for 2,3,4,6,2',3'-Hexa-O-acetyl-1,6-anbydro4-
O-~-glucopyrano~l-~-~-glucopyranose (3)
Protons
(ii)
1,2
r,J from X-ray
2.98'
diffraction data r,J from R ; ( n s ) data r,J from R ; ( i )
3.04'
2.22
29
3,4
43
2.91" 2.90" 2.97' 2.85' 2.95' 3.08' 2.49 2.51 2.25
1,5
1,4'
1,5'
4',5'
3',4'
4',6'b'
5',6'b
5',6'b'
2',3'
2.51* 2.30' 2.23
2.21' 2.57' 2.01
4.06" 3.98' 2.08
2.47' 2.29d 2.14
2.49b 2.35d 2.25
2.33' 2.44d 1.90
2.19' 2.49d 1.96
2.63b 2.73d 1.96
2.41' 2.53' 2.80d 2.29d 2.32 2.58
2.35
2.42 (*0.06) 2.39 (zt0.04) 2.30 (*0.08)
2.22 (h0.03) 2.46
2.21 (*0.08) 2.25 (k0.05)
2.78 (*0.09)
2.20
2.69 (*0.10) 3.31J (*0.20)
(*0.05)
and n.0.e. datae rij from R ( i)
and Rf(i,f) data'
2.37 (*0.08) 2.40 (*0.12)
'
3.29 (*0.20) 3.51 (k0.15) 3.31 (*0.15)
2.50 (*0.02) 2.49 (*0.03) 2.39 (*0.06) 2.63l (*0.07)
(*0.05)
2.80 (*0.07)
(*0.06) 2.36 (*0.05)
1',2'
6'b, 6'b'
1.74' 1.72d
1.82 (*0.05) 1.80 (*0.07)
Interproton distances of p-cellobiose (see Ref. 49); error *0.01 A. Interproton distances of 1,6-anhydro-p-~-glucopyranose (see Ref. 49); error *0.01 A. Interproton distances of p-cellobiose octaacetate (see Ref. 49); error *0.05 A. Interproton distances of 2,3,4-tri-O-acetyl-l,6-anhydro-~-~-glucopyranose (see Ref. 49); error k0.05 A. Error calculations based on the errors of the measured quantities in Eqs. I8 and 21. Interproton distances calculated from the relaxation parameters of the methylene protons.
PROTON SPIN-LATTICE RELAXATION RATES
157
3.5 1.4
0 43
sidering nearest-neighbor interactions, namely, vicinal-gauche, vicinal- trans, and 1,3-diaxial interactions and, for the anomeric proton, the inter-ring contributions, it was possible to apply the additivity rule of pairwise, relaxation contributions for estimating the p i j values corresponding to the nearest-neighbor interactions already mentioned. The interproton distances calculatedlwere then used to determine the conformations of the D-galactopyranosyl and D-fructofuranosyl 'moieties. However, this method of extracting individual, interproton relaxation-contributions suffers from serious limitations; for example, it ignores non-neighboring interactions, and has not been tested for other complex molecules. For more-accurate work on molecular stereochemistry, it is necessary to utilize reliable separation-techniques for extracting the pij values from the measured nonselective relaxation-rates. One such technique is deuterium substitution at specific sites in a molecule, leading to a decrease of the relaxation rate of the receptor proton. This effect is very clearly seen in Table I for the anomeric protons of the methyl D-glucopyranosides (19) and P-D-galactose pentaacetate (23P), as compared to the relaxation rates of their deuterated analogs (20 and 24, respectively). By utilizing Eqs. 14 and 30, and selectively deuterating the 2,3:5,6-di-O-isopropylidene-a-~anomeric hydroxyl group of mannofuranose (2), interproton distances for the H-1,OH and H-2,0H pairs of protons were calculated60to be 2.23 (k0.03) and 3.32 (~t0.27)A, respectively. These distances, when compared with computer-simulated distances obtained by rotating the OH group about the C-1-0 bond, indicated that, in its favored orientation, the 0 - H bond forms a dihedral angle of -60" with the H-1-C-1 bond. Depicted in projection formula 2a, this favored conformation is confined to the first quadrant of the circular
"-'73°-4 c-2 2a
PHOTIS DAIS AND ARTHUR S. PERLIN
158
motion of the OH group. The specific deuterium-substitution method has proved to be very useful for determining the solution geometry of 1,2,3,4-tetra-O-(t~deuterioacetyl)-~-~-arabinopyranose~' (44), and the OCOCD3
0R@
OCOCD,
II
CD,CO OCoCD3
44. R,R'=H 44b R,R'=D 4 4 ~R = H , R ' = D 44d R = D , R ' = H
configuration at the quaternary carbon atom of the acetal ring of the em (45) and endo (46) diastereoisomers of 1,2-0-( I-methoxyethylidene) A
c CH,OAc O w
' R r R'
AcO
45a 45b 46s 46b
R = CH,, R1 = OCH, R = CH,, R' = OCH,Ph R = OCH, ,R1 = CH, R = OCH,Ph, R' = CH,
and 1,2-0-(1-benzyloxyethylidene) derivatives of 3,4,6-tri-0-acetyl-P-Dmann~pyranose,'~as well as for qualitative evaluations of relaxation contributions in glycosides and disac~harides."~~~ Also, the relative configurations of E (47) and 2 (48) isomeric elimination products obtained from 0benzylaldoses with borohydride in 2-propanol were deduced'' from R , ( ns)
,OAc
\I:( H I
Ac? BnOH2C\ ,c\
c1
C---H
c,
I
H
C/'O
I
H OBnlH
I ,C---H
H A
C6Hs
47
(78) (79) (80) (81)
Ac? BnOH2C,;,C,
OCH2CsHs
I
, C
I
cA c / \
I OBn I
H
H 48
H T'.-OAc H (D)
L. D. Hall, K.F. Wong, W. F. Hull, and J. D. Stevens, Chem Commun., (1979) 953-955. P. Dais, T. K. M. Shing, and A. S . Perlin, Carbohydr. Res., 122 (1983) 305-313. J. M.Berry, L. D. Hall, D. W. Welder, and K. F. Wong, Carbohydr. Rer, 54 (1977) c22-c24. V. S. Rao and A. S. Perlin, J. Org. Chem, 47 (1982) 3265-3269.
PROTON SPIN-LATTICE RELAXATION RATES
159
values obtained, following specific deuteration in the vicinity of the double bonds. It is worth noting that, for such compounds as 47 and 48, spin-spin coupling cannot be used for determining the configurations of the alkene, in contrast to compounds containing vic alkenic protons. The second separation method involves n.0.e. experiments in combination with non-selective relaxation-rate measurements. One example6' concerns the orientation of the anomeric hydroxyl group of molecule 2 in Me2S0 solution. By measuring nonselective spin-lattice relaxation-rates and n.0.e. values for OH-1, H-1, H-2, H-3, and H-4, and solving the system of Eq. 13, the various pij values were calculated. Using these and the correlation time, T,, obtained by I3C relaxation measurements, the various interproton distances were calculated. The distances between the ring protons of 2, as well as the computer-simulated values for the H-1,OH and H-2,OH distances was commensurate with a dihedral angle of 60 f 30" for the H-1-C-1-OH array, as had also been deduced by the deuterium-substitution method mentioned earlier. 2. Selective Spin-Lattice Relaxation Rates
One of the major advantages offered by single- and double-selective spin-lattice relaxation-rates is information about dipolar contributions in relaxing proton nuclei. The extent of dipolar contributions can be determined either by comparing non-selective and single-selective relaxationrates by means of Eqs. 16 and 17, or comparing single- and double-selective relaxation-rates by using Eq. 23. This was demonstrated with great success in the first pioneering ~ t u d i e s ' ~and, - ' ~ in particular, the R f ( n s ) / R , ( 9 ratio was established as an extremely important, and rapid, quality-control parameter. Single-selective relaxation-rates alone cannot be used satisfactorily to determine interproton distances beyond those of a three-spin system, suffering from the same drawback as the nonselective relaxation-rates, although, as already noted, they are useful in combination with n.0.e. values. Perhaps more effective is a combination of single- and doubleselective experiments for the separation of the various relaxation contributions and the determination of interproton distances. One example, wherein both methods were used, concerned49 the geometry of the disaccharide 1,6-anhydro-P-cellobiosehexaacetate (3) in acetone-d6 solution and, more specifically, the determination of the conformation of its glucosidic bond. Table 111 summarizes the various relaxation rates for the protonated compound and its partially deuterated analog (3') (see footnote to Table 111), the latter having been synthesized in order to decrease the experimental time, as well as the complexity. Intermolecular, dipolar contributions were
160
PHOTIS DAIS AND ARTHUR S. PERLIN
kept to a minimum by conducting the relaxation measurements on degassed, dilute solutions. Other, competing, relaxation mechanisms were found to be absent, as shown by the ratio (-1.5: 1) of the non-selective to singleselective relaxation-rates for each proton. Problems associated with crossrelaxation and cross-correlation effects were avoided by confining measurements to initial relaxation-rates, although some variations (-20%) in the relaxation rates of individual lines in multiplets of the two strongly coupled protons, H-1 and H-2, were observed. Motional anisotropy was not a factor, as I3C Tl measurements indicated that compound 3 reorients isotropically, with an average rotational correlation-time of 40.6 f 0.15 ps. Under these conditions, Eqs. 18, 21, and 30 could be used to calculate interproton distances, which are tabulated in Table IV. These may be compared with the crystallographic data for P-cellobiose and its octaacetate, and 1,6anhydro-P-D-glucopyranoseand its triacetate, shown in the same table. Clearly, the internuclear distances obtained from single- and double-selective experiments are in good agreement with the crystallographic data, reflecting the 4C1and C4conformations adopted by the D-glucopyranose and the anhydro-D-glucopyranose,respectively. The most probable conformation of the glucosidic bond of 3 is reflected in the H-1-H-4' and H-1-H-5' distances, which may be compared with the computer-generated distances as a function of torsional angles and 9 (see 3a and 3b). However, within 0 - 5 p C - 2
c-4'
LH-1 3a
-3'
C-5@,
c-1
\*-H-4' 3b
the limits of experimental error (-*0.2 A), computer simulation shows that several sets of C#J and 9 angles are consistent with the n.m.r. data. These conformations are depicted in Fig. 6 in the form of a "conformational map" wherein the region of each of the allowed conformations is enclosed by a solid line, corresponding to a range for C#J of -50-+50°, and, for 9,of 190-250" (or 10-70"). Included among the allowed conformations are those derived from coupling constants.49 Single- and double-selective relaxation-rates, together with n.0.e. experiments, have been used to examine the configuration and conformation of asperlin (1) in benzene solution.44 Comparing experimental distances for the proton pairs H-4,H-7 and H-5,H-7 with those obtained from molecular models, it was possible to confirm earlier evidence that the oxirane ring is trans, and also to show that, of the two possible diastereoisomeric forms (49a and 49b), the data are more fully compatible with structure 49a, the
PROTON SPIN-LATTICE RELAXATION RATES
161
+ (degrees) FIG.6.-Allowed Rotational Conformation, Enclosed by Solid Line, of the Glucosidic Bond of 1,6-Anhydro-P-cellobioseHexaacetate (3). Calculated as a Function of the H-1,H-4‘ and H-1,H-5‘ Interatomic Distances. (Reproduced from Ref. 49.)
6 R 7 S isomer. However, this proposal is tentative, because X-ray diffraction has shown8* that another specimen of “asperlin,” possessing a different crystalline form, has structure 49b. It should be noted that 1 tumbles somewhat anisotropically, with D ( ( / D I= 1.3, as deduced from 13Crelaxation measurements. If, however, the anisotropic motion of 1 were not properly corrected for, the largest error in the measurement of its interproton distances would not exceed 4 % .
49a
49b
Other ~ t u d i e s ’ ~ , on ~ ~ ,carbohydrates, ~~” employing single- and doubleselective experiments, have been made on compound 44, and the two (82) K. Fukayama, Y. Katsube, A. Noda, T. Hamasaki, and Y. Hatsuda, Bull. Chem. SOC. Jpn., 51 (1978) 3175-3181. (83a) Ref. 5 , pp. 221-235.
162
PHOTIS DAIS AND ARTHUR S. PERLIN
anomeric forms of methyl 2,3,4,6-tetra-O-(trideuterioacetyl)-a-~-glucopyranoside-2,3,4,6,6'-d5(50 and 51). The conformations of the methyl groups of the latter two compounds, determined from the data for the H-l,CH3 and H-5,CH3 pairs of protons, appear to be consistent with the presence of the so-called exo-anomeric effect, summarized in Fig. 7 for the a- and the /3-anomeric forms, respectively. Finally, triple-selective experiments were performed83bfor compound 44. a Anomer (50)
0-5
A
4 =
soo
ex o - an m r i c ef f ect
c-2
B
s-anomeric ef f ect
FIG.7.-A. The Orientation of the Methyl Group of 50, Including the Projection Viewed along the 0-H-C-1 bond. [For & = 50", the computer-simulated values of rICH1and rSCH,are 2.88 and 2.85 A, respectively. These are to be compared with the values of 2.86k0.06 and 3.49 f 0.15 obtained, respectively, from single- and double-selective experiments, using acetoned6 as the solvent.] B. The Same as Above, but for 51. [For 4 = 55", the computer-simulated values of r,CH, and rSCHlare 2.82 and 4.37, respectively. The values obtained from single- and double-selective experiments, respectively, are 2.82 f 0.10 and 4.05 f 0.84, using acetone-d, as the solvent. (Reproduced, with permission, from Ref. 82.) (83b) Ref. 5, pp. 102-134.
PROTON SPIN-LATTICE RELAXATION RATES
163
However, the relaxation contributions obtained from Eq. 22 were not satisfactorily compared with those obtained from specific, deuterium-substitution experiments and single- and double-selective relaxation-rates. Moreover, the errors estimated for the triple-pulse experiments were very much larger than those observed for the other techniques. This point will be discussed next. V. LIMITATIONS AND RELATIVEMERITSOF RELAXATION METHODS
From the previous discussion, it is clear that relaxation experiments constitute a very powerful tool for investigation of the structure and conformation of carbohydrate molecules in solution. However, the nature of the individual problem may determine which relaxation experiment should be chosen in order to extract interproton distances to the desired accuracy of <*0.2 A. Although the limitations and relative merits of all of the various relaxation methods have not yet been systematically studied, accumulated experience provides some direct knowledge about the range of errors associated with relaxation experiments. As already noted, although nonselective relaxation experiments are simpler and easier to perform than selective experiments, they cannot, alone, be used to solve complex structural problems. The same is true for singleselective relaxation-rates. Simplification is possible for these because, depending on the relative disposition of the spins in the molecular framework, some pU values may be disregarded, so that the remaining, unknown pU values match the number of the experimentally measured R,( ns) values. Nevertheless, this simplified method often leads to interproton distances of low accuracy. Another consideration is the fact that selective experiments require good selectivity conditions, which are limited by the magnitude of the relaxation rates of the inverted spins and the relative separation of the resonances in question across the n.m.r. spectrum. Low relaxation rates, as compared to the pulse duration, cause relaxation during excitation, leading to erroneous results. This may be seen in Table IV for the r4,5,, r5'6'b, and r5,6rb,distances of 3 calculated from the relaxation parameters of the fast-relaxing methylene protons on C-6. A second limitation of selective experiments involves off-resonance fields generated by the selective pulse. Because the selective pulse is normally arranged so that the chemical shift of the spin to be irradiated is at the midpoint of the bandwidths4 of the pulse, any resonance that is within * A v / 2 Hz of the pulse (84) The frequency bandwidth, in Hz, of a weak, selective pulse is approximately given by A v = 1 / ~ where ~ , T~ is the pulse duration in ms.
164
PHOTIS DAIS A N D ARTHUR S. PERLIN
would be expected to experience an off -resonance field. The ensuing perturbation of neighboring resonances introduces systematic errors in the measured selective-relaxation rates, which are propagated in the calculated interproton distances. Combinations of non-selective and/or single-selective relaxation-rates, or both, with n.0.e. values may conveniently be performed with reliable results, especially when other methods seem impractical. However, these experiments are time-consuming, as they entail the determination of a rather large number of experimental values. Moreover, the n.0.e. parameters carry their own systematic and random errors, which are magnified in the calculation of interproton distances. The deuterium-substitution method requires specific deuteration at a strategic position, which, in many cases, may be inconvenient or impractical. Also, this technique is valid only when the relaxation rates obtained after deuterium substitution are at least 5% enhanced, relative to the relaxation rates of the unsubstituted compound, and it requires that, for a meaningful experiment, the following condition3' be satisfied. { R f ( ns)(H - j ) - R f (ns)(D - j ) } 2 0.05Rf( ns)( H - j )
(33)
tt t 0
5
10
15
20
25
fii/pik
FIG.8.-Plot of the Ratio of Interproton Distances, r i k / r i j , as a Function of the Inverse Ratio of Interproton, Dipolar Relaxation Contributions, p i j / p i k , for a Molecule Which Is Tumbling Isotropically. (Reproduced, with permission, from Ref. 5.)
PROTON SPIN-LATTICE RELAXATION RATES
165
A general factor that limits the range of validity of all relaxation experiments is the relative efficiency of the existing, interconnecting relaxationpathways between protons. Interproton distance, when corresponding to a very efficient relaxation-pathway, is determined with high accuracy. However, distant protons are characterized by inefficient dipolar interactions, which result in poorly estimated distances. This "dynamic range" limitation may be seen by examining Eq. 31. If pik<
Interprotoo distances'
Relaxation rates 1. Double- and single-selective 2. Nonselective 3. Single-selective 4. Triple- and nonselective
r1.2
r1,3
2.29 f0.04 2.29 f0.02 2.29 f0.02 2.27 f0.07
2.99 f 0.22 3.02 f0.09 2.93 f 0.07 3.4*1.4
Calculation 1. Dreiding stereomodels 2. Computer simulationb
2.28 f0.02 2.21
2.92 f0.02 2.90
Calculated using rz,3= 1.80 A, this being the value obtained by both methods of calculation. Input parameters: bond lengths, C-H 1.10 and C-C 1.54A; bond angles 109.5". dihedral angles, 0" and 120". From Refs. 87 and 88. (85) L. D. Hall and K. F. Wong, Chem. Cornmun., (1979) 951-953. (86) Ref. 5 , pp. 60-90. (87) D. B. Davies, h o g . Nucl. Magn. Reson. Specrrosc., 12 (1978) 135-953. (88) H.D. Ludeman, E. Westhof, and 0. Roder, Eur. J. Biochem., 49 (1974) 143-150.
166
PHOTIS DAIS AND ARTHUR S. PERLIN Cl"C1
52
system. It is apparent from Table V that the rI2 distance calculated by various relaxation methods is in excellent agreement with that obtained from stereomodels and computer simulation. For all experiments, the r13 distance is less accurate than r12, and, indeed, gives the lowest accuracy in all of the double- and triple-selective pulse-experiments. This is due to the large dynamic range involved in the H-1 and H-3 interactions, due to the r-6 dependence. Indeed, the H-1 and H-3 interaction is a very small fraction of that between H-2 and H-3 (p23= 17.5pI3), and also weaker than that' between the latter pair of protons and H-1 and H-2 (pz3=4.0pl2). In addition to the dynamic-range problem, off -resonance field-effects contribute to the low accuracy of the r13values obtained from the double- and triple-relaxation rate experiments mentioned before. A second example comparing the relative merits of the various relaxation methods concerns the more complex carbohydrate molecule 44. Three relaxation-methods were employed in order to determine interproton distances, namely, deuterium substitution, and two combinations of doubleand single-selective, and triple- and non-selective, pulse experiments.78982 The results of these calculations are summarized in Table VI, along with the interproton distances in A obtained independently from a neutrondiffractionstudy; the calculated ratios of distances were normalized on the assumption, from the neutron-diffraction data, that r5.,5e = 1.80 A. It is apparent from Table VI that the deuterium-substitution method is superior to the others, as it provides an explicit determination of almost all interproton distances in the molecule. Although dynamic-range limitations can be observed for some interproton distances, the overall good agreement between the two complete sets of distances suggests that 44 in benzene-d6 favors the same conformation ' C , as it does in the solid state. The failure of the selective experiments to match the data of the neutron-diffraction study, and the large systematic errors involved in the calculated distances, are caused mainly by off -resonance field-effects on neighboring resonances. Finally, it is worth while to consider the applicability of these relaxation methods to molecules having flexible conformations. Examples given in the previous Section demonstrated that relaxation rates are able to define either the most probable conformation of a flexible molecular segment, or to specify a range of allowed conformations from the total number of confor-
TABLEVI Interproton Distances (A) for 44, Calculated from the Specific, Interproton Dipole-Dipole Relaxation Contributions, and Assuming that rsaSc = 1.8 Protons
(4i) r,j from R ; ( n s ) values and deuterium substitution" r,j from double- and singIe-selectiveb r,j from triple- and non-selectiveb r,j from neutron diffraction'
2,5a
2,5e
3.65d (rtO.38)
3.97
3.99
3,Sa
3,Se
2.68 3.33 (k0.07) (k0.32)
2.64
3.81
45a
45c
248d (k0.04)
2.38
2.53
1,Se
3.9e
(kI.6)
4.06
13
23
294
3,4
(*0.07)
3.33e (k0.32)
3.19 (*0.41)
3.33' (rt0.32)
2.43 (*0.04)
2.39 (*0.08)
(ztO.64)
3.37 (*1.08) 3.08
2.60 (k0.03) 3.81
2.42 (*0.11) 2.45
1,2
2.43e
2.49
5a,Se
3.31
3.81
1.80
Error calculations based on a 5 % error in the measured R, values. Calculated from Eq. 31 and the p,, values given in Ref. 85. Estimated error of ltO.01 A. Only the average value could be determined for t h e e protons, because their resonances were not separately resolved for the isotopomers 44c and 446. Calculations based on the assumption that plSa = pz,4 = p3.5c= P , , ~ .From Refs. 78 and 85.
168
PHOTIS DAIS AND ARTHUR S. PERLIN
mations, to within k0.2 A of the calculated distances. However, quantitative interpretation of the interproton distances in terms of molecular conformation should, in general, include a statistical treatment over possible conformers. Such a treatment involves the computation of the average relaxation contributions, pii, between spins i and j that are located, respectively, in the rigid and flexible parts of the molecule, as a function of a torsion angle, whose magnitude determines the number of conformations in equilibrium. The various relaxation contributions, which are determined by a discrete set of torsional angles, each of which corresponds to a particular conformer, must be weighted by using population fractions obtained from other sources, for example, coupling constants. Hence, the torsion angles that give the best agreement between calculated and experimental pii values correspond to the most probable conformations of the flexible molecular segment. Alternatively, instead of a discrete set of torsion-angle values, angular continuous distributions can be used, derived from Boltzmann statistics of potential-energy curves. This type of statistical analysis has been successfully applied to proton nonselective relaxati~n-rates*~-~' and n.0.e. value^*^*^^ for a large number of nucleosides and nucleotides, although not to other classes of flexible carbohydrate molecules, or to the treatment of selective spin-lattice relaxation-rate data.
(89) C. Chachaty, T. Zemb, G. Langlet, and T. D. Son, Eur. J. Biochem., 62 (1976) 45-53. (90) C. F. G. C. Geraldes, H. Santos, and A. V. Xavier, Con. 1. Chem, 60 (1982) 2976-2983. (91) R. E. Shirmer, J. P. Davies, J. H. Noggle, and P. A. Hart, J. Am. Chem. Soc., 94 (1972) 2561-2572.
ADVANCES I N CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY, VOL. 45
13C-NUCLEAR MAGNETIC RESONANCE-SPECTRAL STUDIES OF LABELED GLYCOPHORINS
BY KILIAN DILL Deparimeni of Chemistry, Clemson University, Clemson, South Carolina 29631 I. Introduction ............................................................ 11. General Background Information about Glycophorins
. . . . . . . . . . . . . . . . . . .. .. .
1. Description of Glycophorins A, B, and C . . . . . . . ............ .. 2. Carbohydrate Structure and Amino Acid-Sequence Data for Glycophorin A, and for What Is Known about Glycophorin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Mode of Display of the MN Blood-group Determinants by Glycophorin A. A Current Controversy . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Labeling Studies of Glycophorin A by Way of the Reductive, ['3C]Methylation Technique ........................................................... 1. Description of Method: Its Specificity, Usefulness, and Possible Drawbacks 2. Methods Used for Assignment of the I3C Labels. Distinction between Labels on Lysine and on an N-Terminal Amino Acid.. . . . . . . . . . . . . . . . . . 3. Work with Intact Glycophorins Derived from Heterozygous, and Homozygous, Red-blood Cells ...................................................... 4. Work with Glycopeptides Derived from Glycophorin A, and with Some Related Peptides and Glycopeptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. pH-Titration Results Involving Reductively ["CIMethylated Glycophorin, Glycophorin Glycopeptides, and Related Peptides and Glycopeptides . . . . . . . . . . 6. pH-Titration Studies Involving Mono[13C]methylated Glycopeptides and Peptides Related to the N-Terminus of Glycophorins.. . . . . . . . . . . . . . . . . . . . . . . . 7. Implications of These Results in Regard to the Display of the MN Blood-group Determinants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Labeling Studies of Glycophorin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Relationship of the Results to Those Obtained for Glycophorin A . . . . . . . . . . 2. pH-Titration Studies of Glycophorin B. Relationship of the Results to Those Obtained for Glycophorin AN and Glyco-octapeptide AN . . . . . . . . . . . . . . . . . . V. Conclusions, and Prognosis for Further Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. .
169
170 172 172 172 175 175 175 177 178 186 187 192 194 195 195 196 197
Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.
170
KILIAN DILL
I. INTRODUCTION The family of glycophorins comprises the predominant, transmembrane sialoglycoproteins found in the human, erythocyte membrane.’-’ This glycoprotein family is known to consist of glycophorins A, B, and C, which had earlier been identified as periodic acid-Schiff reagent (PAS) bands 1, 2, and 3, respectively, on the basis of their sodium dodecyl sulfate (SDS) electrophoresis patterns.” However, subsequent work, in conjunction with higher-resolution electrophoresis,7*’indicated that there are more than three PAS bands, and that these result from glycophorins in various monomeric and dimeric forms. It is known that glycophorin A constitutes -75% of the sialoglycoproteins,4.7” and that glycophorins B and C are only minor components. Because of this fact, glycophorin A has been extensively studied; the detailed compositions of the various glycophorins are given in Section 11. Glycophorin A appears to serve a variety of functions on the red-cell membrane, and has been implicated in several red-cell disorders. Because it extends from the external environment of the cell into the cell cytoplasm, it is considered to constitute a receptor for malarial p a r a ~ i t e s , Iinfluenza ~-~~~ l e ~ t i n s , ~ * ~and ” ~ ”Portuguese ’ man-of-war toxin.I8 Many of these receptor functions are attributable to the carbohydrate composition of these (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (15a) (16) (17) (18)
V. T. Marchesi and H. Furthmayr, Annu. Rev. Biochem., 45 (1976) 667-698. R. J. Juliano, Biochim. Biophys. Acra, 300 (1973) 341-378. M. Tomita and V. T. Marchesi, Proc. Narl. Acad. Sci. USA, 72 (1975) 2964-2968. H. Furthmayr, M. Tomita, and V. T. Marchesi, Biochem. Biophys. Res. Commun., 65 (1975) 113-121. H. Furthmayr, J. Supramol. Struct., 7 (1977) 121-134. H. Furthmayr, Narure (London), 271 (1978) 519-524. H. Furthmayr, J. Supramol. Srrucr., 9 (1978) 79-95. V. T. Marchesi, Semin. Hematol., 16 (1979) 3-20. J. W. Owens, T. J. Mueller, and M. Morrison, Arch. Biochem. Biophys., 204 (1980) 241-254. T. L. Steck, J. Cell Biol., 62 (1974) 1-19. M. Jungery, D. Boyle, T. Patel, G. Pasvol, and D. J. Weatherall, Nature (London), 301 (1983) 704-705. G . Pasvol, M. Jungery, D. J. Weatherall, S. F. Parsons, D. J. Austee, and M. J. A. Turner, Lancer, (1982) 947-950. G. Pasvol and R. J. M. Wilson, Br. Med. Bull., 38 (1982) 133-141. C. A. Facer, Bull. soc. Pathol. Exot., 76 (1983) 463-469. C. A. Facer, Trans. R Soc. Trop. Med, Hyg., 77 (1983) 524-530. M. E. Perkins, Mol. Biochem. Parasitol., 10 (1984) 67-78. J. Petryniak, B. Petryniak, K. Wasniowska, and H. Krotkiewski, Eur. J. Biochem., 105 (1980) 335-341. Z. Drzeniek, H. Krotkiewski, D. Syper, and E. Lisowska’, Carbohydr. Res., 120 (1983) 3 15-321. D. C. Lin and D. A. Hessinger, Biochem. Biophys. Res. Commun., 91 (1979) 761-769.
'3C-N.M.R.-SPECTRAL STUDIES O F GLYCOPHORINS
171
glycoproteins. Glycophorins A and B are also responsible for the display of the M N and Ss blood-group determinant^,^-'"^*^^ which are related to the differences in amino acid sequence found in glycophorins AM and AN, and glycophorins BS and B'. Removal of some of the carbohydrate residues of glycophorin A results in the exposure of the T and T N antigens. These antigens have been found on the surface of human carcinoma cells,21-26and the TN antigen on the surface of erythrocytes has also been correlated with such hematological disorders as leucopenia and thrombopenia.21*22Furthermore, removal of the N-acetyl-a-D-neuraminicacid (sialic acid) groups from the glycophorin molecules of the red-cell membrane apparently causes premature aging of the red-blood ell.^'-'^ Hence, it has been postulated that glycophorin A plays a role in the aging mechanism of the red-blood cell. Glycophorin A is also considered to be involved in the binding of such important biological metal-ions as Ca2+ and Mg2+ to the red-cell membrane.30-32This phenomenon may be important, because it has now been shown that glycophorin is a crucial component in the cytoplasmic, Ca2+induced, morphological changes observed in red blood cells.33 This may result from the fact that glycophorin interacts with the proteins that create the red-cell, cytoskeleton ~ t r u c t u r e . ~ ~ - ~ ~ ~ (19) W. Dahr, W. Gielen, K. Beyreuther, and J. Kriiger, Hoppe-Seyler's 2.Physiol. Chem., 361 (1980) 145-152. (20) W . Dahr, K. Beyreuther, H. Steinbach, W. Gielen, and J. Kriiger, Hoppe-Seyler's Z. Physiol. Chem., 361 (1980) 895-906. (21) J. P. Cartron, G . Andreu, J. Cartron, G . W. G. Bird, C. Salmon, and A. Gerbal, Eur. J. Biochem., 92 (1978) 111-119. (22) J. P.Cartron and A. T. Nurden, Nature (London), 282 (1979) 621-623. (23) W. Dahr, G . Uhlenbruck, H. H. Gunson, and M. van der Hart, Vox Sung., 28 (1975) 249-252. (24) G . F. Springer, P. R. Desai, and I. Banataola, J. Natl. Cancer Inst., 54 (1975) 335-339. (25) L. Limas and P. Lange, Cancer, 46 (1980) 1366-1373. (26) G . F. Springer, M. S. Murthy, P.R. Desai, and E. F. Scanlon, Cancer, 45 (1980) 2949-2954. (27) G . Perret, D. Bladier, R. Vassy, and P. Cornillot, Comp. Biochem. Physiol, A, 69 (1981) 59-63. (28) I. Kahane, E. Ben-Chetrit, A. Shifter, and E. A. Rachmilewitz, Biochem. Biophys. Acta, 596 (1980) 10-17. (29) G. Perret, D. Bladier, L. Gattegno, and P. Cornillot, Mech. Ageing Deu., 12 (1980) 53-63. (30) C. Long and B. Mouat, Biochem. J., 123 (1971) 829-836. (31) R. Prohaska, T. A. W. Koerner, Jr., I. M. Armitage, and H. Furthmayr, J. Biol. Chem., 256 (1981) 5781-5791. (32) M. E. Daman and K. Dill, Carbohydr. Res., 1 1 1 (1983) 205-214. (33) R. A. Anderson and R. E. Lovrien, Nature (London), 292 (1981) 158-161. (34) E. A. Nigg, C. Bron, M. Girardet, and R. J. Cherry, Biochemistry, 19 (1980) 1887-1893. (35) S. E. Lux, Semin. Hernatol, 14 (1979) 51-52. (35a) R. A. Anderson and R. E. Lovrien, Nature (London),307 (1984) 655-658.
172
KILIAN DILL
11. GENERALBACKGROUND INFORMATION
ABOUT
GLYCOPHORINS
1. Description of Glycophorins A, B, and C
Because glycophorin A is the preponderant c ~ m p o n e n tof~ the . ~ ~trans~~ membrane sialoglycoproteins (-75%), it has been the most extensively studied. It has been shown that the molecular weight of glycophorin A is -31,000, and that it contains 131 amino acid residues (see later).' Of the molecule having a molecular weight of 31,000, 60% is carbohydrate. The carbohydrates are found in 15 0-linked tetra saccharide^,',^' which are composed of D-Gal, D-GalNAc, and D-NeuAc, and in one complex, Nlinked oligo~accharide.'*~~ Glycophorin B is the next major component of the sialogly~oproteins.~~~~~~ It is known that glycophorin B lacks the complex, N-linked, oligosaccharide chain, but it probably contains the 15 0-linked tetra saccharide^.^ Moreover, it has been f o ~ n d ~that , ~the~ first ' ~ 26~ amino ~ ~ acid residues of glycophorin B are identical in sequence to that of those in glycophorin AN. However, it is also known that glycophorin B has a shorter, amino acid chain (by -35 amino acid residues) near the C-terminus, and, therefore, that it does not contain an extended, cytoplasmic ~ e g m e n t . ~ Very little is known about the minor component, glycophorin C. It is considered to be smaller than glycophorin A, again near the C - t e r m i n ~ s . ~ Glycophorin C appears7 to have the same carbohydrate content as glycophorin A, and it therefore probably contains the same types of oligosaccharide chains. Furthermore, glycophorin C seems to contain tryptophan, which is not present in any of the other glycophorin specie^.^
2. Carbohydrate Structure and Amino Acid-Sequence Data for Glycophorin A, and for What Is Known about Glycophorin B The amino acid sequences for glycophorins AM and AN (see Refs. 8 and 19) are presented in Fig. 1. It may be clearly seen that residues 1-70 of
these glycoproteins extend into the cell exterior. The hydrophobic portion of the glycoprotein, residues 71-92, appear to be imbedded in the phospholipid membrane, and residues 93- 13 1 protrude into the cell cytoplasm.' It may be noted that there appear to be two amino acid sequences for glycophorin; one contains Leu and Glu at amino acid sequence positions 1, and 5 , respectively, whereas the other contains Ser and Gly at amino
(36) H. Furthrnayr and V. T. Marchesi, Methods Enzymol, 96 (1983) 268-280. (37) E. Lisowska, M. Duk, and W. Dahr, Carbohydr. Res., 79 (1980) 103-1 13. (38) H. Yoshirna, H.Furthrnayr, and A. Kobota, J. Bid. Chem., 255 (1980) 9713-9718.
'3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS
Membrane
External
173
Internal
FIG. I.-Amino Acid Sequence for Glycophorins AM and AN. [Diamonds represent points of 0-glycosylation. The single N-glycosylation point occurs at Asn-26. The data were obtained from Refs. 8 and 19.1
acid sequence positions 1, and 5, respectively. These glycoproteins are known as glycophorins AN and AM,respectively. The diamonds in Fig. 1 represent the point of 0-glycosylation by tetrasaccharide 1. Asn-26 indicates the point of N-glycosylation by the complex oligosaccharide 2. a - ~ - N e u A c - (+ 2 3)-P-D-Gal-(1+ 3 ) - a - ~ - G a l N A c - ( l 3)-Ser(Thr) + 6
t D-NeuAc
(I-
1
The first 26 amino acid residues of glycophorin B are identical in ~ e q u e n c e 'to ~ *those ~ ~ in glycophorin AN. It apparently also has' the same 0-linked tetrasaccharides found in glycophorin A, but lacks the N-linked, complex oligosaccharide. The heterogeneity in amino acid sequence of glycophorin B at amino acid position 29 (Met/Thr) of the sequence results in the display of the Ss blood-group antigens.
P-D-Gal-( 1 t 4)-P-[>-GlcNAc-(1 --t 2 ) - a - ~ - M a nI 6 I
T
-~-NeuAc-2
(I
3
p - ~ - G a l -1(+ 4 ) - p - ~ - G l c N A c -1(+ 2)-(1-[>-Man-(1 + 6 ) - P - ~ - M a n1- (+ 4 ) - & ~ - G l c N A c - (1 + 4 ) - p - ~ - G l c N A c -1(+ N4)-Asn 6
T
a-~-NeuAc-2
4
T
P-D-GIcNAc-1 2
i
IX-L-FUCI
I3C-N.M.R.-SPECTRAL STUDIES O F GLYCOPHORINS
175
3. Mode of Display of the MN Blood-group Determinants by Glycophorin A. A Current Controversy In 1927, Landsteiner and L e ~ i n discovered e~~ a new blood-group system called MN. Since that time, a large number of research groups have investigated the mode of display of the blood-group determinants. Springer and coworker^^^-^^ postulated that the M and N antigenic difference lies in the quantity, and linkage differences, of N-acetyl-a -D-neuraminic acid groups found in glycophorins AM and AN. Furthmayr and coworker^^-^*^^ have conclusively shown that amino acid differences exist at positions 1 and 5 in the amino acid sequence of pure glycophorins AM and AN, and they postulated that these differences, especially at residue 1, are largely responsible for the display of the MN antigens. Several other research groups investigating the mode of display of the MN blood-group determinants have focused on chemical and enzymic modification of glycophorins. In order to establish which amino acid residues are important for the display of the M N determinants, Lisowska and coworker^^^.^ monitored the serological activity of glycophorin A as a function of the chemical modification of the amino groups on the glycoprotein. They found that the amino groups, especially the N-terminal NH2, are crucial for the display of the M N determinants. Furthermore, as a result of the chemical modification^^^*^* of amino groups and a-D-NeuAc groups of glycophorin A, it was postulated that the interactions of the carboxylic groups of the a-D-NeuAc groups with the charged 6-amino group of one of the lysine residues may be crucial for the display of the MN blood-group determinants. 111. LABELING STUDIESOF GLYCOPHORIN A BY WAY OF THE REDUCTIVE, [ 13C]METHYLATIONTECHNIQUE 1. Description of Method: Its Specificity, Usefulness, and Possible Drawbacks
As already pointed out, the three units that have been implicated in the display of 'the M N blood-group antigens by glycophorins AM and AN are (39) (40) (41) (42) (43) (44)
(45) (46) (47) (48)
K. Landsteiner and P. Levine, Proc. SOC.Exp. Biol. Med., 24 (1927) 600-602. G. F. Springer, H. Tegtmeyer, and S. V. Huprikar, Vox Sang., 22 (1972) 325-343. G. F. Springer and R. Desai, Carbohydr. Res., 40 (1975) 183-192. R. Desai and G . F. Springer, J. Immunogenef., 7 (1980) 149-155. G. F. Springer and R. Desai, J. Biol. Chem., 257 (1980) 2744-2746. H. Furthmayr, M. N. Metaxas, and M. Metaxas-Buhler, Proc. Narl. Acad. Sci. USA, 78 (1981) 631-635. E. Lisowska and M. Duk, Eur. J. Biochem., 54 (1975) 469-474. E. Lisowska and M. Duk, Eur. J. Biochem., 88 (1978) 247-252. W. Dahr, G. Uhlenbruck, and H. Knoll, J. Zmmunogenef., 2 (1975) 87-100. W. Ebert, J. Metz, and D. Roelcke, Eur. J. Biochem., 27 (1972) 470-472.
176
KILIAN DILL
the lysine residues, the N-terminal amino acid groups, and the carbohydrate residues. The lysine residues and N-terminal amino acid groups contain primary amino groups which may be specifically 13C-labeled48aby using the reductive [13C]methylationt e c h n i q ~ e .Therefore, ~ ~ - ~ ~ this technique may place into several positions in the molecule isotopic labels which then act as biological probes. The reaction scheme for placing 13C-labelsinto the 6-amino groups of lysine residues and the 2-amino groups of N-terminal amino acid groups is depicted in Scheme 1, which shows the reductive [13C]methylation of the amino groups. M~no['~C]methylation of the amino groups can occur when limited amounts of formaldehyde are employed in the reaction. It may be noted that Scheme 1 shows the amino groups in the neutral rather than the protonated form; this is because the reaction occurs only when the amino species is neutral. Conditions may then be employed for introducing labels, whereby 13Clabels may only be introduced into the N-terminal amino acid groups and not the lysine residues. In Scheme 1, NaCNBH, was used as the reducing agent rather than NaBH,. There are several reasons for this choice: NaCNBH, is considerably more stable under slightly acidic and neutral conditions, which are used most often for this reaction.49s53 NaCNBH, has been shown to be a more efficient reducing agent for the reductive-methylation reaction. NaCNBH, appears to be the less reactive as regards the possible modification of the protein; NaBH, has been shown to reduce disulfide bonds and may cleave peptide NaCNBH,, on the other hand, is a much milder reducing agent. It does not cleave disulfide bonds, and also does not reduce aldehydes or ketones at neutral pH. There appear to be several possible side-reactions which may produce some unwanted products. [ 13C-]Formaldehydeis reduced to [ '3C]methanol to a small extent, but this reaction product can be removed by dialysis of (48a) This technique actually results in isotopic substitution, not isotopic labeling. According to IUPAC recommendations, the products should be designated as (I3C)methylated. Throughout this article, the labeling designator [I3C] is used, but it is to be understood that essentially complete replacement of 12C by I3C is implied. (49) N. Jentoft and D. G. Dearborn, J. Biol. Chem., 254 (1979) 4359-4365. (49a) A. D. Sherry and J. Teherani, J. Biol. Chem., 258 (1983) 8663-8669. (50) J. E. Jentoft, N. Jentoft, T. A. Gerken, and D. G. Dearborn, J. Bid. Chem., 254 (1979) 4366-4370. (51) J. E. Jentoft, T. A. Gerken, N. Jentoft, and D. G. Dearborn, J. Biol. Chem., 256 (1981) 231-236. (52) T. A. Gerken, J. E. Jentoft, N. Jentoft, and D. G. Dearborn, J. Bid. Chem., 257 (1982) 2894-2900. (53) J. W. Marsh, A. Nahum, and D. F. Steiner, Int. J. Pepr. Prorein Res., 22 (1983) 39-49. (54) A. M. Crestfield and S. Moore, J. B i d . Chem., 238 (1963) 622-627.
1I7
13C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS
H
O R
II I ---C-CH- NH,
H
\ 'T=O /
NaCNBH,
0 R
1
I l l
/
---C-CH -N
3
~
~
~
\ "CH,
SCHEME 1.-Reaction Scheme for the Total, Reductive [13C]Methylation of the 6-Amino Group of Lysine, and the 2-Amino Group of an N-terminal Amino Acid Group.
the reacted protein. Reductive methylation may also occur with secondary amines, and therefore, in theory, the peptide backbone is susceptible to monomethylation; however, this has been shown to be a considerably minor reaction, if it occurs at all.49 Another real possibility is inter- or intramolecular cross-linking of proteins with formaldehyde, to form methylene bridges.49This reaction constitutes a real drawback, because it may produce anomalous results; however, it has been detected in only one instance,49a where a reducing agent was present. The main drawback in introducing 13C-labelsinto proteins is the possibility of modifying the structure of the protein. Apparently, in the studies thus far conducted using the reductive methylation technique, the structures of several proteins have only been altered m i r ~ i m a l l y . ~ Therefore, ~-'~ as there appear to be only very little gross structural changes in the protein, although some minor local changes may occur, this technique appears to be well suited for specifically introducing I3C-labels into a protein molecule.
2. Methods Used for Assignment of the ''C Labels. Distinction between Labels on Lysine and on an N-Terminal Amino Acid After the introduction of I3C-labels into the protein or glycoprotein molecule, the ability to assign the resonances to specific carbon atoms is essential. In the case of glycophorin (see Fig. l ) , it may readily be seen that 5 lysine residues and 1 N-terminal amino acid (per species) can be reductively di[13C]methylated. This could theoretically lead to 6 resonances (or possibly more, if chemical-shift nonequivalence is observed for the dimethyl species) in the I3C spectrum of methylated glycophorin A. However, in most cases, the N6,N6-di['3C]methyllysine resonances all occur near, or at, the same frequency. It is then necessary to be able at least to assign, or
178
KILIAN DILL
differentiate, the N6,N6-di[13C]methyllysine resonances and the N 2 , N 2 di[13C]methyl N-terminal resonances. This specific assignment can be made by using the partial-methylation technique, pH-titration studies, and enzymic or chemical cleavage of peptide bonds. Each of these techniques is discussed next. a. Partial-methylation Technique.-Scheme 1 shows that the reductivemethylation reaction proceeds when the amino group is in the neutral state. The pK, difference between an N-terminal 2-amino group (pK, -7) and a lysine 6-amino group (pK, -10) is -3 pK, units. Therefore, if a reductivemethylation reaction at pH 7.0 is undertaken with a limited amount of formaldehyde, the 2-amino group will be methylated first. Only after the addition of much more formaldehyde would the lysine amino groups be methylated (see Section 111,3). b. pH-Titration Results.-From previous w~rk,~’-’~ it has been established that, in the I3C-n.m.r. spectrum, the resonances of N6,N6-di[13C]methyllysine “normally” move downfield as the pH is increased, whereas the N 2 , N 2 di[13C]methylated, N-terminal resonances “normally” move upfield. N-terminal amino acids However, those of the N2,N2-mono[’3C]methylated also move downfield when the pH is increased, but they resonate -10 p.p.m. upfield of those of the dimethyl species. These results indicate that this method may be used, with caution, to determine the nature of the [13C]methyl label. c. Enzymic Digestion and Chemical Cleavage of the G1ycoprotein.-This method provides a unique and unequivocal method for assigning the I3C resonances to specifically [13C]methylatedresidues. This usually means that either a tryptic or chymotryptic digestion of the glycoproteins must be undertaken, and the peptide fragments must then be isolated. Another method would be to fragment peptide bonds chemically with cyanogen bromide. This reagent is known to cleave the peptide bond at a methionine residue, resulting in the conversion of the methionine left on the N-terminal peptide into homoserine. All of these methods have been used to identify, and gain structural information about, the reductively [13C]methylated glycophorins. 3. Work with Intact Glycophorins Derived from Heterozygous, and Homozygous, Red-blood Cells
In order to show that the structure of glycophorin A is not greatly perturbed by the reductive methylation reaction, circular dichroism (c.d.)
'3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS
J
I
I
I
I
I
200
210
220
230
240
250
179
Wavelength (nm)
FIG.2.-C.d. Spectra of Glycophorin A (-) and Reductively Methylated Glycophorin A (---),in H,O at pH 7.0. [Reproduced from Ref. 60, by permission ofthe publishers, Butterworth & Co (Publishers) Ltd. @ 1984.1
spectra of virgin and reductively methylated glycophorin A are presented in Fig. 2. Clearly, the c.d. curves of the unmodified and modified proteins are almost identical. Because c.d. is a good tool for determining the overall structure of protein^,'^ the indications are then that the gross structure of the glycophorin had not substantially been altered as a result of reductive methylation. Fig. 3 shows a portion of the aliphatic region of the proton-decoupled, 13 C-n.m.r. spectra of virgin glycophorin ANand fully reductively ['3C]methylated glycophorins AM, AN,and A M N (see Ref. 56). These glycophorins were isolated from homozygous (NN, MM), and heterozygous (MN), red-blood cells. In Figs. 3B, 3C, and 3D, it may readily be seen that a prominent resonance occurs at 44.1 p.p.m. downfield for all three spectra. On the basis of the evidence cited later, this resonance has been assigned residues to the 10 methyl carbon atoms of the 5 N6,N6-di[13C]methyllysine of these related glycoproteins. The resonances upfield of the large lysine
(55) D. Freifelder, Physical Biochemistry, Applications to Biochemistry and Molecular Biology, Freeman, San Francisco, 1982. (56) R. E. Hardy, R. L. Batstone-Cunningham, and K. Dill, Arch. Biochern. Biophys., 222 (1983) 222-230.
180
KILIAN DILL
A
44.1
FIG.3.-A Portion of the Aliphatic Region of the Proton-decoupled, l3C-N.rn.r. Spectra of Native Glycophorin A (in H 2 0 at 30") and Fully Reductively [13C]Methylated Glycophorins AM,AN,and AMN(in H 2 0 at 30"), at 22.5 MHz. [Taken from Ref. 56. Time-domain data were accumulated in 8192 addresses, with a recycle time of 1 s (except for A, where 2 s was used). A digital broadening of 2.8 Hz was applied: (A) 1.9 mM virgin glycophorin A, at pH 6.5, after 50,000 accumulations; (B) 1.6 mM fully reductively ['3C]methylated glycophorin AM, at pH 8.5, after 12,015 accumulations; (C) 1.6 mM fully reductively [13C]methylated glycophorin AN, at pH 7.3, after 14,208 accumulations; (D) 1.5 mM fully reductively ["Clmethylated glycophorin AMN,at pH 7.2, after 12,815 accumulations.]
%-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS
181
resonance must then be assigned to the N2,N2-di[”C]rnethylated, Nterminal amino acid residue^.'^-^^ In the spectrum of fully reductively [13C]methylated glycophorin AN, the resonance at 42.8 p.p.m. must correspond to the N2,N2-di[13C]methylated, N-terminal amino acid residue. The ratio of the integrated intensities of the N2,N2-di[’3C]methylLeu resonance to the N2,N2-di[13C]methyllysineresonances is 5 : 1, as expected. The integration values determined were valid, because the recycle times of spectra in Figs. 3B, 3C, and 3D were twice the spin-lattice relaxation-times ( TIvalues) of those of the di[13C]methyl carbon atoms, and also because the n.0.e. values of the N6,N6-di[13C]methyland N2,N2-di[L3C]methylcarbon atoms were eq~ivalent.’~ The spectrum of fully reductively [ 13C]methylated glycophorin AM is much more complex; it appears to have a sharp resonance at 43.3 p.p.m. and a broad resonance at 42.8 p.p.m., which have been attributed to N 2 , N 2 di[’3C]methylserine carbon atom^.'^-^^ The resonance at 43.3 p.p.m. exhibits “normal,” pH behavior for an N2,N2-di[’3C]methyl specie^,'^-'^ whereas the broad resonance appears to titrate “normally” when the sample is only partially methylated and the titration is performed62 at -50”. The broad, “minor” component is lost when various glycopeptides are isolated.59960 On heating the sample at 80” at pH 5.0, the broad component is considerably sharpened. The “minor” component had previously been a t t r i b ~ t e d ’ ~to- ~ ~ another structural state of glycophorin AM. It is known that it does not result64 from the loss of carbohydrate residues or oligosaccharide chains near the N-terminus of glycophorin AM, and also that the proportion of the “minor” component is enhanced when the sample is deglycosylated before it is reductively [13C]methylated.Experiments involving the reductive 13 C-methylation of intact erythrocyte~,6~* as well as the reductive 13C-
(57) R. L. Batstone-Cunningham, R. E. Hardy, M. E. Daman, and K. Dill, Biochim. Biophys. Acfa, 746 (1983) 1-7. (58) R. L. Batstone-Cunningham, R. E. Hardy, and K. Dill, I n f . J. Biol. MacromoL, 5 (1983) 3 14-317. (59) M. E. Daman, R. L. Batstone-Cunningham, R. E. Hardy, and K. Dill, Inf. J. Bid. Macromol., 5 (1983) 371-373. (60) R. E. Hardy, R. L. Batstone-Cunningham, M. E. Daman, A. M. Holbrooks, and K. Dill, In(. J. Bid. Macromol., 6 (1984) 103-107. (61) K. Dill, R. E. Hardy, R. L. Batstone-Cunningham, M . E. Daman, B. Ferrari, and A. A. Pavia, Carbohydr. Res., 128 (1984) 183-191. (62) R. D. Carter, R. E. Hardy, and K. Dill, Inr. J. Bid. Macromol., 6 (1984) 164-166. (63) R. D. Carter, J. R. Brooks, and K. Dill, Biochim. Biophys. Acfa, 190 (1984) 285-287. (64) R. D. Carter, R. E. Hardy, H. K. Lannom, K. Dill, B. Ferrari, and A. A. Pavia, Int. 1. Bid. Macrornol., 6 (1984) 348-352. (65a) R. D. Carter, H. K. Lannom, and K. Dill, Biochirn. Biophys. Acra, 845 (1985) 396-402.
182
KILIAN DILL
FIG.4.-pH-Dependence of the Carbon-13 Resonances for the Di['3C]methylamino Groups of Glycophorin AMN.[A, B, and C correspond to the identically labeled peaks in Fig. 3D. Taken from Ref. 56.1
methylation of glycophorins that had been treated with pyrylium aminoblocking indicated that this "minor" component results from the head-to-head cross-linking of two glycophorin AMmolecules by way of the two N-terminal L-serine residues. This minor component may relate to the structure of glycophorin AM in solution; there is reason to believe that it may result from cross-linking of the glycophorin AM during the reductivemethylation The specific assignments of the resonance to the N6,N6-di['3C]methyllysine and N2,N2-di['3C]methylleucine and -serine residues were based on the results of three experiments: pH-titration studies of these resonances, partial-methylation studies of glycophorins AMNand AM,and the isolation of glycophorin glycopeptide (for this portion, see Section 111,4). The pH-titration results for the resonances of fully reductively [ I3C]methylated glycophorin AMNare shown in Fig. 4. Clearly, the large resonance (at 44.1 p.p.m.) moves downfield as the pH is increased, with a pK, of >lo. The resonance for N2,N2-di['3C]methylleucine moves upfield as the pH is increased, with a pK, of 7.4. The major resonance of the N-terminal N2,N2-di[13C]methylserine residue behaves unusually as the pH is increased. It may readily be seen that the dimethylated lysine species and (6Sa.b) K. Dill, R. D. Carter, and A. R. Katritzky, Int. J. Bid. MacrornoL, 8 (1987) 318-320.
I3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS
183
the dimethylated N-terminal species may be differentiated on the basis of pH studies. Further evidence for these assignments was educed with the aid of partial-methylation studies, the results of which are shown in Figs. 5 and 6. Partial-methylation studies of glycophorins AMNand AM,using limited proportions of formaldehyde, are depicted. Clearly, the resonances pertaining to the N-terminal dimethyl resonances appear first in the 13C-n.m.r.
p.p.m.
from
Me4Si
FIG. 5.-Proton-decoupled, "C-N.m.r. Spectra (at 22.5 MHz) of the Partial-reductive, [13C]MethylationStudies of 1.5 mM Glycophorin A M N in H20at 30".[Spectra of methylated samples were recorded at a sample pH of -7.3, and typically required 10.000-30,000 accumulations. Time-domain data were accumulated in 8192 addresses, with a recycle time of 1 s. A digital broadening of 2.8 Hz was applied during the processing of the data. The n.m.r. traces in this Figure relate to the following ratios of forma1dehyde:glycophorin in the reductive methylation reaction: 0.0, 2.0, 3.0, 4.6, 5.4, and 50. The peak at 34.5 p.p.m. represents monomethylated lysine. (Taken from Ref. 56.)]
-
184
KILIAN DILL
I
I
91
Qo
I . 3
I
aa
-
46
40
p.p.m.
p.p.m.
from
a6
from M e 4 9
Me4Si
FIG. 6.-Proton-de~oupled,'~C-N.m.r. Spectra (at 22.5 MHz) of the Partial-reductive, ["CIMethylation Studies of 1.2 mM Native Glycophorin AM in H,O at 30" and of -1.5 mM Deglycosylated Glycophorin AM in H 2 0 at 30".[Spectra of methylated samples were recorded at a sample pH of 7.3,and typically required 15,000-60,000accumulations. Time-domain data were collected in 8192 addresses, with a recycle time of 1 s. A digital broadening of 2.8Hz was applied during the processing of the data. The peak at 34.5p.p.m. in the spectra of traces (A) and (B) represents monomethylated lysine. (A) Native glycophorin AM; the n.m.r. traces in the Figure relate to the following molar ratios of formaldehyde: glycophorin in the reductivemethylation reaction: 2,0, 8.0,14.0,24.0, and 74.0. (B) Deglycosylated glycophorin AM; the n.m.r. traces in the Figure relate to the following molar ratios of formaldehyde :glycophorin in the reductive-methylation reaction: 2.0, 4.0,8.0,16.0,and 75.0.Reproduced from Ref. 58, by permission of the publishers, Butterworth & Co (Publishers) Ltd. @ 1983.1
spectrum. The N6,N6-di["C]methyllysine resonances appear in the spectrum only after larger proportions of formaldehyde are added. The peak at -35 p.p.m. in these spectra represents N6-mono[ '3C]methyllysine. The work dealing with the reductive [ 13C]methylation studies of glycophorin glycopeptides (see later) fully corroborated these assignments. In order to obtain a preliminary view on how the oligosaccharides may influence the structure about the N-terminus, the 13C-n.m.r.spectra of the N-terminal proteins of glycophorins AMNand AM in various degrees of glycosylation were recorded; see Figs. 7 and 8.
'3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS
185
n 4.0
7.3
9.0
FIG.7.-A Portion of the Aliphatic Region of the Proton-decoupled, '3C-N.m.r. Spectra (at 22.5 MHz) of Fully Reductively ['3C]Methylated Glycophorin AMN,Asialoglycophorin AMN, and Deglycosylated Glycophorin AMN,at pH Values of 4.0, 7.3, and 9.0, Respectively. [Time-domain data were accumulated in 8192 addresses, with a recycle time of 1 s. A digital broadening of 3.0 Hz was applied to the data. All glycophorin samples were -1.2 m M (in H,O), and required 10,000-20,000 accumulations. (Taken from Ref. 57.)]
I3C-MN Native
1, 4.0
7.3
9.0
FIG.8.-A Portion of the Aliphatic Region of the Proton-decoupled, I3C-N.m.r. Spectra (at 22.5 MHz) of Fully Reductively ['3C]Methylated Glycophorin AM, Asialoglycophorin AM, and Deglycosylated Glycophorin AM, at pH Values of 4.0, 7.3, and 9.0, Respectively. [Timedomain data were accumulated in 8192 addresses, with a recycle time of 1 s. A digital broadening of 3.0 Hz was applied to all data. All glycophorin samples were 1.2 m M (in H,O), and required 10,000-20,000 accumulations. Reproduced from Ref. 58, by permission of the publishers, Butterworth & Co (Publishers) Ltd. @ 1983.1
-
186
KILIAN DILL
On a gross, structural basis, it would appear that removal of the (Y-DNeuAc groups does not substantially perturb the structure about the Nterminus. However, deglycosylation of almost the entire molecule seems to have a profound effect; this structural effect about the N-terminus appears to be heavily associated with glycophorin AM (see Fig. 6 and laters8). 4. Work with Glycopeptides Derived from Glycophorin A, and with Some Related Peptides and Glycopeptides
One way in which to determine whether one part of the molecule may influence the structure about the N-terminus, or whether the assignments of the [13C]methyl resonances in the 13C-n.m.r.spectra of fully reductively [13C]methylatedglycophorins AMand ANare correct is to isolate the various glycophorin glycopeptides that have been produced by enzymic or chemical means. Fig. 9 shows a portion of the aliphatic region of the I3C-n.m.r. spectra of full reductively [13C]methylated glycophorins AM and AN, fully reductively [13C]methylatedtryptic glycopeptides from glycophorins AMand AN, and fully reductively [13C]methylated N-terminal glyco-octapeptides AM and AN. The tryptic glycopeptides were obtained by treatment of glycophorins AMand AN with tryp~in.~’ They contain a mixture of glycopeptides having amino acid residues 1-31 and 1-39. In both cases, three of the five lysine residues have been removed, and the I3C-n.m.r. spectra are indicative of this fact. The ratio of the integrated intensities of the N 6 ,N6di[ 13C]methyllysine residues to those of the N-terminal N2, N2di[13C]methylated residues is 2: 1, as expected. The I3C-n.m.r. spectrum of the fully reductively [ 13C]methylated tryptic glycopeptide AMindicated that this species still appears to contain a “minor” structural component. The AM and ANglyco-octapeptides were obtained by treatment of reductively [13C]methylated, intact or tryptic glycopeptides AM and AN with cyanogen bromide. This procedure cleaves the peptide bond on the Cterminal side of the protein, and converts methionine into homoserine.60 These glyco-octapeptides should contain no lysine residues, and this was confirmed by the spectra of the [ 13C]methylated glyco-octapeptides AMand AN shown in Fig. 9C. The resonances for NZ,N2-di[13C]methylleucine and N 2 ,N2-di[’3C]methylserine residues of the glyco-octapeptides resonate at the same position as their counterparts in the spectra of the reductively [13C]methylated, intact glycophorins AM and AN, except that some of the resonances are now narrower, as expected, due to the decrease in the size of the molecule. It should be noted that the “minor” component of glycophorin AM has now been lost. This result again indicated that it may result from a cross-linking of the glycoprotein.
I3C-N.M.R.-SPECTRAL STUDIES O F GLYCOPHORINS LYS 44.1
187
LYs 44.1
FIG.9.-A Portion of the Aliphatic Region of the Proton-decoupled, "C-N.m.r. Spectra (at 22.5 MHz) of Fully Reductively [13C]Methylated Glycophorins AM and AN, Tryptic Glycophorin Glycopeptides AM and AN, and Glycophorin Glyco-octapeptides AM and AN (All in H,O at 30"). [Time-domain data were accumulated in 8192 addresses, with a recycle time of 1-1.5 s. A digital broadening of 2.8 Hz was applied. (A) 1.6 mM reductively [13C]methylated glycophorin ANat pH 7.3, after 14,208 accumulations, and 1.5 mM reductively [13C]methylated glycophorin AM at pH 7.3 after 17,847 accumulations; (B) 1.6 mM reductively [13C]methylated, tryptic glycopeptides AN at pH 7.2 after 6600 accumulations, and 2.0 mM reductively [13C]methylated, tryptic glycopeptides AM at pH 7.4 after 6999 accumulations; (C) 0.5 mM reductively ['3C]methylated glyco-octapeptide AN at pH 7.2 after 27,948 accumulations, and 0.5 mM reductively [13C]methylated glyco-octapeptide AM at pH 7.2 after 31,985 accurnulations. Reproduced from Ref. 60 by permission of the publishers, Butterworth & Co (Publishers) Ltd. @ 1984.1
The pH dependence of these various glycopeptides should probably provide some insight as to their structural surroundings; such information is provided, and discussed, in Section 111,s. 5. pH-Titration Results Involving Reductively [ "CIMethylated Glycophorin, Glycophorin Glycopeptides, and Related Peptides and Glycopeptides
In order further to determine whether the reductive, ['3C]methylation technique possibly perturbs the structure of these glycoproteins and glycopeptides, natural-abundance '3C-n.m.r.-spectral data were obtained for unmodified, and for reductively ['3C]methylated, compound 10 (for struc-
KILIAN DILL
188
TABLEI Selected, I3C-N.m.r., Chemical-shift Data" for Glycopentapeptide 10 and Its Reductively ['3C]Methylated Derivative6' Chemical shift
Carbon atom
Glycopeotapeptide
Reductively ['3C]methylated glycopentapeptide
1'
100.6 99.9 99.3 51.2 70.0 69.2 72.8 62.6 23.5 54.9 56.4 68.5 58.8 58.4 77.0 78.8 19.5 19.1 44.8
100.6 99.9 99.2 51.2 70.0 69.6 72.6 62.7 23.6 54.8 60.1 68.5 60.5 58.8 58.5 77.1 79.0 19.6 19.2 -b
2'
5' 6' CH,(Ac-2') Ser C-2
Ser C-3
Thr C-2 Thr C-3
Thr C-4 Gly C-2
Not all of the spectral data are given. The "C-enriched-methyl resonance of the di["C]methylSer residue overlaps with this resonance.
ture; see Table 11). These data are tabulated in Table I. From the chemicalshift data, it was apparent that no great structural perturbation of the glycopentapeptide had occurred as a result of the reductive ['3C]methylation. One way in which to probe the structural surroundings of a protein is to monitor the pH behavior of specific carbon sites of the 13C probes. pHtitration studies, of given resonances, had previously been used for probing of the protein structure, because they are known to provide information concerning electrostatic (salt-bridging) interactions in the protein, neighboring group-ionizations, and local environment^.^^'^^'^' (66) L. R. Brown, A. DeMarco, R. Richarz, G. Wagner, and K. Wiithrich, Eur. 1.Biochem., 88 (1978) 87-95. (67) J. S. Cohen, L. J. Hughes, and J. B. Wooten, in J. S. Cohen (Ed.), Magnetic Resonance in Biology, Wiley, New York, 1983, pp. 130-247.
TABLEI1
13C Chemical-shift Data and Titration Data" for the N-Terminal Di['3C]methylamino Groups of Glycophorins, Glycophorin Glycopeptidg and Related Peptides and Glycopeptides Titration parameters
Compoundb
PK
Intact glycophorin AN (3) Intact glycophorin AM (4) Glyco-octapeptide AN (5) Glyco-octapeptide AM (6) Asialoglyco-octapeptide AN (7) Asialoglyco-octapeptide AM (8) Glycogho:in N! ( 9 ) Ser-Sy-Tty-Tkr-Gly (10) Ser-Sef-Th;-Th;-Glu (11) Leu-Ser-Thr-Thr-Glu (12) Leu-Ser-Thr-Thr-Glu (13) Ser-Ser-Thr-Thr-Glu (14) Ser-Ser-Thr-Thr-Gly (15) Leu-Ser-Thr-Thr-Gly (16) (16) D20f Leu-Ser-Thr-Asn-Glu (17) Ser-Ser-Ser (18) (18) D 2 0 J Ser-Ala (19) Ser-Gly (20) (20) D20' Ser-Tyr (21) Ser-Met (22) Szr (23) Ser (24) Hse (25) ThrCOOCH, (26) Thr-Val-Leu (27) Tlk-Thr (28) Ala-Ser (29) Val-Thr (30) Val-Gln (31) Val-Gly (32)
7.4, 7.7, 7.4, 7.8, 7.4, 7.5, 7.4, 7.4, 7.4, 7.g8 7.5, 7.4, 7.1, 9.3, 8.7, 9.3,
Hill coefficient (nr 0.97 0.85 0.80 0.87 1.13 0.88 2.00 1.66 1.08 1.35 0.93 1.41 0.86 -
Ad
6,
Chemical shift'
0.52
42.4 42.3 42.3 42.3 42.1 42.1 43.0 43.1 42.2 42.1 42.1 43.0 43.0 43.2 42.8 42.3 42.4 42.1 42.0 41.9
42.7 43.3 42.7 43.3 42.6 43.3 42.7 43.1 43.1 42.5 42.6 43.1 43.2 42.6 42.5 42.6 43.1 43.1 43.0 43.1 43.1 42.7 43.1 43.3 43.3 43.8 42.7 42.3 42.9 42.6 42.8 42.7 43 .O
-
0.87 0.73 0.74
0.59 0.56 0.59 0.62 0.68 0.12 0.13 0.65 0.50 0.71 0.12 0.12 0.18 0.46 0.47
-
-
-
8.4, 8.0, 7.g3 8.0,
1.05 0.91 0.81 0.98
0.20 0.83 1.03 1.13
-
a Titration data for the intact glycophorins and some related glycopeptides were taken from Ref. 64. * Indicates point of 0-glycosylation by an a-D-GalNAc group. ' Indicates point of 0-glycosylation by an a-D-Gal group. In cases where A is very small, the titration parameters may be error-prone (especially n ) . A represents the chemical-shift difference between the di['3C]methylamino group in the protonated and nonprotonated forms. Chemical shifts are given for the resonances at pH 7.3. In the case where the resonance titrates as a function of pH, the chemical-shift value given is determined from the theoretical fit of the data. Titration of some samples was performed in D,O, with a limited number of titration points.
'
KILIAN DILL
190
Because of the potential of gaining structural information for reductively [13C]methylatedglycophorins AM and AMby pH-titration studies, the pHtitration behavior of reductively, di[’3C]methylated glycophorins AM and AN, and 28 related, reductively di[13C]methylated glycoproteins, glycopeptides, and peptides in H 2 0 and D20were investigated. These results are presented in Table 11, and Figs. 10 and 11. The pH-titration data for the N-terminal N2,N2-[13C]dimethylamino species were analyzed for the best pK, values and Hill coefficients ( n ) by using the following equation. A~o~(PK-PH) + 10n(pK-pH)’
In this case, ST is the best, theoretical, I3C chemical-shift value, SB is the chemical shift of the di[ ‘3C]methylamino group in the nonprotonated form, and A is the chemical-shift difference between the di[ 13C]methylamino group in the protonated and nonprotonated forms. The best fit was obtained when Ci [ST(i)- 6,,b~(i)]~ was minimized; Sobs is the chemical-shift value observed at that given pH value.
2
3
4
5
6
7
8
9
10
11
12
PH FIG.10.-pH-Dependence of the 13C Resonances for the Di[”C]methylated N-Terminal Amino Groups of Tri-L-Ser(A, 18). Glyco-octapeptide AN ( 0 , 5 ) ,and Asialoglyco-octapeptide AN (0, 7). (Taken from Ref. 61.)
'3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS
191
44
42
2
3
4
5
6
7
8
9
1
0
1
1
1
2
PH FIG. 11.-pH-Dependence of the 13C Resonances for the Di[13C]methylated N-Terminal Amino Groups of Glyco-octapeptide AM (0,6), Asialoglyco-octapeptide AM (0,S), and glycopentapeptide 10. (Taken from Ref. 61.)
Several results are quite apparent from the data shown in Table 11. It is evident from the pentapeptide model compounds that substitution of amino acid residues at positions 4 and 5 does not significantly affect the structure about the N-terminus. This observation corroborated earlier work6* from agglutination-inhibition assays, which demonstrated that the nature of the amino acid at position 4 of the peptide (or glycopeptide) is not a requirement for specificity. The results also showed that glycosylation at amino acid positions 2, 3, and 4 appears to influence the structure about the N-terminus. The structural influence of neighboring glycosylation had been observed by Dill and coworkers.69 Therefore, if the structure about the N-terminal NH2 group is primarily responsible for the display of the MN blood-group antigens, the carbohydrate residues of the glycoprotein must play a role in the antigenicity. This phenomenon may be enhanced when bulkier oligosaccharide units (1) (68) J. P. Cartron, B. Ferrari, M. Huet, and A. A. Pavia, Exp. Clin. Imrnunogener., 1 (1984) 112-1 16. (69) K. Dill, R. E. Hardy, M. E. Daman, J. M. Lacombe, and A. A. Pavia, Curbohydr. Rex, 108 (1982) 31-40.
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KILIAN DILL
are attached to amino acid positions 2, 3, and 4 of the glycoproteins, especially in glycophorin AM. Many of these results were corroborated, as discussed in the next Section, dealing with N2-mono[13C]methylspecies. The results in Table I1 also indicated that, from the I3C-n.m.r. titration data for the glycoprotein and glycopeptide species that contain an N 2 ,N2di[13C]methylSerresidue, all exhibit unusual pH behavior, and A is either very small, or zero. This may be explained on the basis that some unusual steric or hydrogen-bonding phenomena are associated with the serine residues. In fact, a number of possible hydrogen-bondings schemes have been associated with serine (and threonine) resid~es.''-'~ One that is particularly relevant to the present case deals with a strained, internal hydrogenbond between N2-H and 0-4. The results in Table I1 suggest that the titrations of the N-terminal N2,N2-di['3C]methylserine residues are influenced by the solvent ( H 2 0 or D,O); compound 18 titrates (A+0.12) in H20, and also titrates in D20, but then A is zero. These results indicate that, in glycophorin AM,a weak, internal hydrogen-bond may influence the structure about the N-terminus.
6. pH-Titration Studies Involving M~no['~C]methylatedGlycopeptides and Peptides Related to the N-Terminus of Glycophorins Another way in which to gain structural information concerning the N-terminal residue of glycophorins AM and AN is to study the N-terminal, m~no['~C]methyl derivatives; these are produced by using limited amounts of [ '3C]formaldehyde. There are distinct differences between the N2,N2di[13C]methylamino and N2-mono['3C]methylamino species: ( i ) a significant, chemical-shift difference exists between the N-terminal dimethyl ; of the 13Cresonances and monomethyl species (43 and 34 ~ . p . m . )(~i i~) all of the N-terminal dimethyl species move upfield as the pH is increased (if they move at all), whereas all of the I3C resonances of the N-terminal, monomethyl species move downfield as the pH is increased64; and ( i i i ) A for the N-terminal monomethyl species tends to be much larger than that for the N-terminal dimethyl species.64 Point ( i i i ) would tend to indicate that it may be more advantageous to study the N-terminal monomethyl species. However, because of allowable protein concentrations, detection limits on available instruments, and technical difficulties, it has thus far (70) R. E. London, J. M. Stewart, R. Williams, J. R. Cann, and N. A. Matwiyoff, J. Am. Chem. Soc., 101 (1979) 2455-2469. (71) A. Aubry, N. Ghermani, and M. Marraud, Int. J. Pept. Protein. Ree, 23 (1983) 113-122. (72) M. Marraud and A. Aubry, Int. 1. Pept. Protein Rex, 23 (1983) 123-133. (73) D. Peters and J. Peters, J. Mol. Srrucr., 90 (1982) 305-320. (74) D. Peters and J. Peters, 1. Mol. Struct., 90 (1982) 320-334.
'3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS
193
been found difficult to study I3C-labeled, intact glycophorin, N-terminal monomethyl labels. Table I11 gives the titration parameters for the I3C resonances of the N-terminal m~no['~C]methyl labels of a variety of peptides and glycopeptides related to the N-terminus of glycophorins AM and AN. The titration TABLE111 '3C Chemical-shift Data and Titration Data for the N-Terminal M~no['~C]methylamino Groups of Glycophorin-related Glycopeptides and Peptidesu Titration parameters Hill coefficient Compound"
PK,
(n)
A*
6,
Chemical shift'
Ser s::-T$-~$-cI~ (11) Leu-Ser-Thr-Thr-Glu (12) Leu-Ser-Thr-Thr-Glu (13) Ser-Ser-Thr-Thr-Glu (14) Ser-Ser-Thr-Thr-Gly (15) Leu-Ser-Thr-Thr-Gly (16) (16) D20d Leu-Ser-Thr-Asn-Glu (17) Ser-Ser-Ser (18) (18) D,Od Ser-Ala (19) Ser-Gly (20) (20) D20d Ser-Tyr (21) Sy-Met (22) Ser (24) Hse (25) ThrCOOCH, (26) Thr-Val-Leu (27) fir-Thr (28) Ala-Ser (29) Val-Thr (30) Val-Gln (31) Val-Gly (32)
7.80 7.8, 8.1, 7.7, 7.7, 8.1, 8.22 8.1, 7.53 7.7, 7.9, 7.9,
0.97 1.05 1.00 0.93 1.04 1.09 0.97 1.02 1.02 1.12 0.98 1.04 0.95 1.00 1.09 1.14 1.03 0.76 0.92 1.16 1.13 0.98 1.07 1.12
-1.57 -1.28 -1.19 -1.51 -1.37 -1.13 -1.19 -1.19 -1.49 -1.49 -1.43 -1.49 -1.55 -1.32 -1.49 -1.31 -1.19 -0.98 -1.44 -1.19 -1.56 -1.19 -1.07 -1.01
34.6 34.2 34.2 34.5 34.5 34.2 34.1 34.2 34.4 34.3 34.4 34.5 34.4 34.2 34.5 34.4 34.7 35.2 34.9 34.9 34.1 35.0 34.9 34.8
33.7 33.1 33.2 33.4 33.5 33.2 33.1 33.2 33.5 33.2 33.2 33.3 33.1 33.2 33.4 33.1 33.5 34.8 34.0 34.1 33.7 34.0 33.8 33.8
8.0, 7.7, 7.7, 8.9, 9.6, 7.14 7.4, 7.5, 8.43 8.0, 8.1, 8.4,
* Indicates a point of 0-glycosylation by an a-D-GalNAc group. Indicates the point of 0-glycosylation by an a-D-Gal group. A represents the chemical-shift difference between the mono[ 13C]methylamino group in the protonated and nonprotonated forms. Chemical shifts are given for the resonances at pH 7.3. In the case where the resonance titrates as a function of pH, the chemical-shift value given is determined from the theoretical fit of the data. Titration of some samples was performed in D,O, with a limited number of titration points.
194
KILIAN DILL
results seem to be in agreement with the conclusions drawn from the N-terminal di[ "Clmethylated species discussed in Section 111,5, although some of the results are much clearer. The data in Table 111 also indicate that titration of the N-terminal, mon~['~C]rnethyl label may be influenced by the nature of the adjacent, amino acid residue. 7. Implications of These Results in Regard to the Display of the MN Blood-group Determinants It has already been indicated that three functional groups on glycophorins AMand ANare involved in the display of the MN blood-group determinants: the lysine residues, the N-terminal amino groups, and the carbohydrate residues. From the work in the preceding Sections, and from the data given in the next Section, a number of facts concerning the structures of the MN blood-group determinants may be obtained. It may definitely be concluded that one (or more) of the lysine residues plays a small role in determining the N-terminal structure of the glycophorins. This may best be judged by the work on glycophorin AM (see the preceding) and glycophorin BN (see later), and supports earlier work indicating that the lysine residues may be important in the display of the MN blood-group determinants. The other two functional groups that play a crucial role in the make-up of the MN blood-group determinants are the carbohydrate residues and amino residues 1 and 5 in the protein sequence of glycophorins AM and AN.Various chemical modifications and enzyme digests, along with hemaglutination assays, led to the conclusion that the N-terminal NH2 group and the a-D-NeuAc groups are indeed important. However, it must be pointed out that the various immunoglobulins and lectins may be specific for the N-terminal amino group or the carbohydrate residues. Certain evidence indicates that structural differences do exist between glycophorins AMand AN that lack the a-D-NeuAc groups, and also that these may not necessarily involve the N-terminal amino g r o ~ p s . ' ~ - ~ ~ The results concerning the N-terminal structures of glycophorins AMand AN were based on the labels that were placed on the crucial, N-terminal amino group, and they clearly showed that the amino acid residues at position 5 may play only a minor, if any, role in determining the structure of the MN blood-group determinants. The carbohydrate residues appear (75) W. J. Judd, P. D. Issitt, B. G. Pavone, J. Anderson, and D. Aminoff, Transfusion, 19 (1979) 12-18. (76) Y.Ochiai, H. Furthrnayr, and D. M. Marcus, J. ImmunoL, 131 (1983) 864-868. (77) W.L. Bigbee, M. Vanderleen, S. S. N. Fong, and R. H. Jensen, MoL Immunof., 20 (1983) 1353-1362.
'-'C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS
195
to play a crucial role in the structure about the N-terminus; the disaccharide may be more important than the cy-D-NeuAcgroups. The hydrogen bonding that may occur intra- or inter-molecularly in glycophorin AM also appears to be particularly important. IV. LABELINGSTUDIESOF GLYCOPHORIN B 1. Relationship of the Results to Those Obtained for Glycophorin A
As mentioned earlier, glycophorin B carries the N and the Ss blood-group antigens. It is known that the first 26 residues of the amino acid sequence are identical to those in the N-terminal portion of glycophorin AN. Moreover, relative to glycophorin A, it has a shortened amino acid chain, comprising -35 amino acid residues at the C-terminus. It is also known to contain -4 lysine residues. Fig. 12 shows a portion of the aliphatic region (30-50 p.p.m.) of the proton-decoupled, 13C-n.m.r.spectra63of fully reductively [I3C]methylated glycophorin AMNand glycophorin BN. Glycophorin B was isolated from heterozygous, red-blood cells, and was then separated from glycophorin AMNby gel-filtration chromatography on A m m ~ n y x - L o . ~ Clearly, . ~ ~ the
A
B
FIG. 12.-A Portion of the Aliphatic Region of the Proton-decoupled, "C-N.m.r. Spectra (at 22.5 MHz) of Fully Reductively Methylated Glycophorin AMN and Glycophorin BN, in H,O at 30". [Time-domain data were accumulated in 8192 addresses, with a recycle time of 1 s. A digital broadening of 3.0 Hz was applied: (A) 1.5 mM reductively, ['-'C]methylated glycophorin AMN, at pH 7.2, after 12,815 accumulations; (B)0.3 mM reductively, [*-'C]methylated glycophorin BN, at pH 7.1, after 67,874 accumulations. (Taken from Ref. 63.)]
KILIAN DILL
196
spectrum of glycophorin BN is considerably different from that of glycophorin AMN,indicating that the separation of glycoproteins was successful. The spectrum of glycophorin BN lacks the resonance of N 2 , N 2 di['3C]methylserine that is associated with glycophorin AM. However, it does contain the resonance at 42.8 p.p.m. that is associated with N 2 , N 2 di['3C]methylleucine, which is expected, because glycophorin BN contains leucine as N-terminal amino acid residues. In order to determine the number of lysine residues present in glycophorin B, the resonance for N6,N6-di['3C]methyllysine (44.1 p.p.m.) was integrated relative to the resonance for N2,N2-di[13C]methylleucine (42.8 p.p.m.). In this case, no differential Tl values or n.0.e. values should be observed for these resonances. A value of 3-4 lysine residues was obtained from integration studies63;higher accuracy could not be achieved because of the signalto-noise of the spectrum. However, these results corroborated the earlier finding that glycophorin B contains a smaller number of lysine residues than glycophorin A. 2. pH-Titration Studies of Glycophorin B. Relationship of the Results to Those Observed for Glycophorin AN and Glyco-octapeptide A N
Fig. 13 shows the effects of pH on the N-terminal N2,N2-di[13C]methylleucine resonances of glycophorin AN, glyco-octapeptide AN, and glycophorin BN. The titration parameters are listed in Table 11. The titration of N2,N2-di['3C]methylleucine of glycophorin B N resembles that of N 2 , N 2 -
43.2
-
I
I
I
I
I
I
I
I
-
c
fc-
42.8
-
-
.O 42.6
-
-
5
0
k
L
0
42.4
-
42.2
-
I 4
I
5
I
6
I
7
I
8 9 PH
I
1
I
I
0
1
1
FIG. 13.-pH-Dependence of the I3C Resonances for N-Terminal di[13C]methylleucine of Glycophorin AN, Glycophorin BN, and N-Terminal Glyco-octapeptide AN [(GO(N)]. (Taken from Ref. 63.)
I3C-N.M.R.-S PECTRAL STUD1ES 0 F G LYCOPHORI NS
197
di['3C]methylleucine of glycophorin AN and glyco-octapeptide AN. In fact, the titration curve for the methylated glycophorin BN is more similar to that of ['3C]methylated glyco-octapeptide AN than to that of [ I3C]methylated intact glycophorin AN. The results just mentioned have several implications. One is that some other portion of the intact glycophorin AN molecule seems to influence the structure of glyco-octapeptide AN, which results in different titrationpatterns for the N-terminal N2,N2-di['3C]methylleucineresidues of reductively methylated, intact glycophorin ANand glyco-octapeptide AN.Another interesting feature of Fig. 13 is that the titration curve for the N 2 , N 2 di[13C]methylleucine residue of reductively [ I3C]methylated glycophorin BNresembles that of the N2,N2-di[13C]methylleucineresidue of the reductively ['3C]methylated glyco-octapeptide AN more than that of reductively [I3C]methylated, intact glycophorin AN, indicating that some portion of the glycophorin B molecule does not influence the structure about the Nterminus which the intact glycophorin AN molecules seem to have. This difference about the N-terminus may result from the fact that glycophorin A severely aggregates in solution. For glycophorin B, on the other hand, the mode, and, perhaps, the degree, of aggregation may be slightly different.
V. CONCLUSIONS, A N D PROGNOSIS FOR FURTHER STUDIES The possible role that the lysine residues, N-terminal amino acid residues, and carbohydrate residues may play in the display of the MN blood-group determinants by glycophorins AM, AN, and BN has been investigated. Assuming that the N-terminal amino acid in each of these glycoproteins plays a prominent role in the display of the M N blood-group determinant, 13 C labels were placed on the N-terminal amino acid residues of the glycoproteins. As a result of the work, a number of conclusions may be drawn concerning the display of the MN blood-group determinants. (i) A portion of the glycoprotein, not near the N-terminus (possibly lysine residues) may influence the structure about the N-terminus. (ii) Amino acid substituents at position 4 or 5 of the glycophorin AM and AN sequence play only a little, or no, role in determining the structure about the N-terminal amino acid residue. (iii) Titration studies of the various glycopeptides indicated that the carbohydrate residues, especially D-Gal and D-GalNAc appear to influence the structure about the N-terminus significantly. There are a number of studies that need to be made in order to provide further information about the structure of the N-terminal amino acid residues. One crucial set of experiments revolves around determination of the exact structure of the minor component observed in reductively
198
KILIAN DILL
[ '3C]methylated glycophorin AM;work in this laboratory indicates that it results from head-to-head dimerization of glycophorin AMmolecules. This phenomenon could be studied by isolation of the cross-linked product (intact, or enzymically digested). These studies could provide information about the nature of the glycophorin A aggregates in aqueous solution.
ACKNOWLEDGMENTS The author thanks Alicia Brown for typing this article. He also acknowledges the financial support of DHEW (BRSG grant), the Research Corporation, and the South Carolina affiliate of the American Heart Association.
ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY. VOL. 45
THE CHEMISTRY AND BIOCHEMISTRY OF THE SWEETNESS OF SUGARS
BY CHEANG-KUAN LEE Department of Chemistry, National University of Singapore, Kent Ridge, Singapore 051 1
I. Introduction ............................................................ 11. Stereochemistry of Sweetness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Early Theories ....................................................... 2. Fundamental Structural Requirements for Sweetness . . . . . . . . . . . . . . . . . . . . . . 3. Sweetness-Structure Relationship for Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Bitterness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The AH,B Concept for Bitterness.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Lipophilicity and Bitterness. . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Bitterness-Sweetness Relationships . . . . . . . . . . . . . .................... IV. Biochemistry of Sweetness . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The Sensory System .................................................. 2. The Peripheral Mechanisms in Taste. 3. Taste-receptor Binding . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The Quality of Sweetness.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Methodology of Measurement of Sweet Taste.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
199 201 202 207 238 310 312 318 320 325 325 326 328 339 349
Science at its best provides us with questions, not absolute answers. Norman Cousins (1976)
I. INTRODUCTION Although always a matter of popular fascination, the sense of taste was once only a subject of academic interest, but it is now of great practical and economic importance. Deep esthetic enjoyment is experienced through the sense of taste; we eat food, not nutrition, and food will preferably be eaten only if it is palatable and attractive. In civilized countries, it is far from true that a hungry man will eat anything. Commercial interest in the sensory properties of foods during the past decade has led to a great increase in basic knowledge of the phenomenon. 199
Copyright @ 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
200
CHEANG-KUAN LEE
However, as Lord Zuckerman’ pointed out “our understanding of taste physiology (and of flavour technology) is abysmal.” Sweetness is a gustatory response evoked by most sugars, and is relished by man as well as by many other organisms. The desire for sweetness appears to be universal and undisputed. Today, sugar, or one of its synthetic substitutes, appears in more articles of the diet than any other food ingredient, except, perhaps, common table salt.2 Sweetness is a quality that defies definition, but whose complexity can be appreciated merely by examining the molecular structures of those compounds that elicit the sensation. They come in all molecular shapes and sizes, and they belong to such seemingly unrelated classes of compounds as aliphatic and aromatic organic compounds, amino acids, peptides and proteins, carbohydrates, complex glycosides, and even certain inorganic salts. The phenomenon of sweetness has been of interest from the time of the Ancient Greeks. Theophrastus (372-287 B.C.), who succeeded Aristotle at the Lyceum in Athens, wrote a review3of the subject in his book De Sensibus. Since then, many attempts have been made4 to correlate chemical structure with sweet taste, but most of them are of limited value. These will, therefore, be discussed only briefly in the present article. A major advance in the evolution of taste theory came with understanding that the primary event in the initiation of a taste response involves the interaction of a stimulant molecule with a receptor located at the taste-cell plasma-membrane. Further progress in our understanding of the phenomenon, although slow, has been significant. Considerable interest in the mechanism of taste perception was stimulated by the Shallenberger AH,B hypothesis proposed’ in 1967. A comprehensive discussion will be provided of the influence of structure on the taste of sugars and sugar derivatives, and the effect of the structure of some high-intensity, noncarbohydrate sweeteners on taste will also be considered where it helps to elucidate the mechanism involved. Because many sweet compounds also taste bitter, a brief risumC of the role of structure on bitterness will also be incorporated. The strengths and deficiencies of the various hypotheses will be highlighted, and critically interpreted.
(1) Lord Zuckerman, British Nutrition Foundation Annual Lunch, 1975. (2) R. M. Pangborn, in G . E. Inglett (Ed.), Symposium: Sweeteners, AVI, Westport, CT, 1974, pp. 23-44. (3) Theophrastus, D e Sensibus, G . M. Stratton (Ed.), Bonset-Schippers, Amsterdam, 1964. (4) R. W. Moncrieff, The Chemical Senses, 3rd edn., CRC Press, Cleveland, Ohio, 1967. (5) R. S. Shallenberger and T. E. Acree, Nature, 216 (1967) 480-482.
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201
One reason for the seemingly slow progress of understanding is the interdisciplinary nature of sweetness research.6 The conclusions that can be drawn, from, for example, physiological and psychophysical experimentation¶ must be related to what is known of the structural chemistry of the stimulus and how it may interact at the molecular level. All too often, it is not appreciated that one particular line of experimentation cannot be viewed in isolation, but must relate to other disciplines. Only by fully understanding all of the associated events leading to sweetness perception shall we understand the mechanism of sweetness perception itself. The theories that attempt to describe the initial event in sweet-taste stimulation will be discussed, as will some of the practical attempts to isolate sweet-receptor molecules. Relevant, behavioral data will be examined, particularly where the effects of molecular structure on behavior responses are being evaluated. The evaluation of sweet compounds by taste panels is crucial to the development of worthwhile structure-taste relationships. Many structuretaste studies have employed dubious, taste-panel techniques. Therefore, a critical examination of structure-taste data in the light of this observation is relevant, as are recommendations for a consistent approach in utilizing taste techniques.* Observations that have been made will be critically discussed in the light of their relevance to other approaches that attempt to elucidate mechanistic features of this complex phenomenon. No claim is made for a complete coverage of the literature, but an attempt has been made to collate all relevant information and to discuss the results that best illustrate the principles involved. OF SWEETNESS 11. STEREOCHEMISTRY
Interest in the chemical senses dates back to the earliest records of philosophic speculation. Theophrastus3 referred to the work of Democrites, who suggested that “Sweetness consists of atomic figures that are rounded and not too small; wherefore, it quite softens the body by its gentle action, and unhesitatingly makes its way throughout. Yet it disturbs the other savours, for it slips in among the other atomic figures and leads them from their accustomed ways and moistens them.” This is probably the earliest attempt to explain the phenomenon of sweetness. We have progressed a (6) M. G . Lindley, in G. G. Birch and K. P. Parker (Eds.), Sugar: Science and Technology, Applied Science, London, 1979, pp. 403-413.
* In this article, all numerical comparisons of relative sweetness are given on a molar basis.
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CHEANG-KUAN LEE
long way since then, but our world is so predominantly visual that we tend to belittle the importance of the chemical senses, perhaps the oldest and most widely distributed of the senses in both vertebrates and non-vertebrates. This is evident from the fact that, despite the naked exposure of the taste buds in the tongue of man and other vertebrates, as well as on the fins of fishes, we have not been able to learn more about how a taste-receptor cell functions than just that it responds to chemical stimuli and transmits impulses to the brain in its associated nerve fiber. Sadly, it is probably true that our modern understanding of the phenomenon of sweetness, although more completely described and more accurately measured, is only a little nearer to an absolute explanation. 1. Early Theories
The most comprehensive attempt to relate chemical structure to taste is that of C ~ h n In . ~his classic book Die organischen Geschmacksrofe, published in 1914, he proposed that all sweet and bitter stimulants contain more than one functional group in their molecules. A multiplicity of hydroxyl groups gave sweetness, and these and a-amino acid groups and other sweet-eliciting or “sapophoric” groups were termed “glucogenes.” He also noted that these groups usually occur in pairs (see Table I). Cohn also pointed out’ that the increase of molecular weight as a homologous series is ascended is often accompanied by a gradual change in the taste of the members from sweet to bitter; for example, for the glycols, ethylene glycol, sweet; trimethylene glycol, sweet; 1,2-propyIene glycol, sweetish; tetramethylene glycol, less sweet; and hexamethylene glycol, bitter. It was suggested that the size of the sapophoric, taste-inducing group is important in lower members of the series. In the higher members, however, having larger molecules, that group is now relatively small and becomes less significant. By this, Cohn probably implied steric effects attributable to the rest of the molecule. He also noted the diminished solubility of the higher members (tastelessness eventually resulting when the compound becomes insoluble) but he was, however, unable to arrive at a hypothesis relating the changes in taste to the chemical structure. Following the success of the chromophore-auxochrome theory of dyeing during the latter half of the 19th century, Oertly and Myers’ proposed that a sweet-tasting substance must possess an “auxogluc” and a “glucophore.” This is an extension of Cohn’s observation that sapophoric groups occur in pairs. The glucophore was defined as a group of atoms having the power to form a sweet compound when combined with any, otherwise tasteless, ( 7 ) G . Cohn, Die Organischen Geschmackmfe, Siemenroth, Berlin, 1914. (8) E. Oertly and R. G . Myers, J. Am. Chem. SOC.,4 (1919) 855-867.
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CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
TABLEI Cohn’s Sapophoric Groups Group (OH),
Category
Taste
polyhydroxy
sweet
a-amino acids
sweet
=N--O-CH,CO,H
oximacetic acids
sweet
-N =N+= N-
azides
sweet
=N-OH
oximes
sweet
-CN
nitriles
sweet
a-ketocarboxylic acids
sweet-bitter
polynitro
bitter
nitrosulfonic acids
bitter
tertiary amines
bitter
quaternary ammonium salts
bitter
betakes
bitter
-SH
thiols
bitter
-S-
sulfides
bitter
-s-s-
disulfides
bitter
thioamides and thioureas
bitter
NH2
/
-C
\
CO, H
0
\/ I C
‘C02H
/
-N
\ I
S CS of
II
-C
-NH-
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CHEANG-KUAN LEE TABLEI1
Oertly and Myers' Auxogluc and Glucophore Components of Sweet-tasting Compounds Compound
Formula
Aux ogI uc
Glucophore -CH-CH-
I
-H
I
OH O(H) -CH3
-CH-CH,
I
I
-CH,CHJ
OH OH -CH-C02H
-CH,CH,CH,
I
NH
0 +/
-CH,-O-N
\0-
/
-C
H34 -CH,OH
\ CL H3.n
I I
OH
H2.m
c-c-
I 1
I
CH3-CH-
x,
X"
--CH2--CH,OH OH
I
-(CH2),-CHzOH Ethylene glycol
H2C-CH-
H2C-CH,
I
I
1
Glycerol
1
I
HO OH OH D-Glucose
I
I
I
I
I
H
/
HO OH
H H OHH H2C-C-C-C-C-C
I
-CHZOH
H2C-CH-
H2C-CH-CH2
I
-H
I
HO OH
HO OH
/
0
0
I
HOHO O H H OH \ H
-c-c
Glycine
H2C-CO2H
-CH--CO,
NH2
NH2
Chloroform
CHCI,
I
-CHZOH
I
OH \ H
I
-CCI,
H
-H
-H
0 Ethyl nitrate
H3C-CH2-0-N
+ /
No-
-CH3
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
205
auxogluc. Restricting their efforts to sweet aliphatic compounds, they identified six glucophores and nine auxoglucs (see Table 11). The theory succeeded in bringing some order to the mass of empirical data, but it was inadequate in many respects. In particular, it could not account for the sweetness of such nonhydroxylated, but intensely sweet, compounds as saccharin (1,2-benzisothiazolin-3-one 1,l-dioxide) and dulcin [( p ethoxypheny1)ureal. Furthermore, it offered no consideration of the effects of stereoisomerism. For example, many compounds, such as amino acids, are sweet in one optically active form, and less sweet, tasteless, or even bitter in the other. Therefore, it would appear that taste is dependent not only upon the nature and number of groups in the molecule, but also their molecular geometry. All of the sweet compounds that contain Cohn’s sapophoric groups and Oertly and Myer’s glucophores contain hydrogen as an auxogluc. Substitution of this hydrogen atom often affects the taste. This led Kodama’ to propose the “vibratory hydrogen” hypothesis. Kodama’s vibratory hydrogen atom was viewed as being a form of tautomerism, and the transposition of the hydrogen atom to afford the different tautomeric forms would result in “electronic vibrations.” He therefore concluded that the taste of organic compounds is due to electronic vibrations of the sapophoric hydrogen atom. The taste of various amino acids, sugars, and aliphatic nitro compounds was studied, and it was concluded that the distance over which this hydrogen atom migrates, to give a second tautomeric form, determines the sweetness. In the case of saccharin, the sweetness was explained as due to two tautomeric forms.
Kodama’ proposed three additional rules governing the relation between taste and structure. These were (1) optical isomers have different tastes; (2) substitutions always affect the taste; and (3) the taste of electrolytes is due to the sum of the tastes of the molecular electrolyte, the anions, the cations, and complex ions. The taste of saccharin was further studied,” and it was found that (1) the alkaline-earth-metal salts are sweet, whereas the heavy-metal salts are astringent; (2) the sweet taste is lost if the sulfimide ring is cleaved, or if (9) S. Kodama, J. Tokyo Chem. SOC.,41 (1920) 495-534. (10) A. F. Hollerman, Red. Trav. Chim. Pays-Bas, 42 (1923) 839-845.
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CHEANG-KUAN LEE
the sulfimide nitrogen atom is alkylated or acylated; (3) substitution in the benzene ring diminishes the sweet taste, and introduces a bitter taste; and (4) the substitution of a halogen atom on C-6, that is, para to the carbonyl group, results in a steady decrease in sweetness, with concomitant increase in bitterness. The effect of halogen increased with its atomic weight. These early successes generated considerable interest in the relationships between taste and chemical composition. Attempts were made to relate various physical, chemical, and physicochemical properties of compounds to their sweet taste. Beck” reported that the relative sweetness of sugars could be correlated with the “contraction coefficient,” that is, the ratio of the sum of the atomic volumes to the molecular volume of the sugar. In a closely related series of compounds, such as a group of sugars, some relationship seems to exist between sweet taste and s~lubility.’~*’~ In acids and esters, a decrease in taste was observedI4 to accompany an increase in molecular weight. The taste of isomeric esters and acids was, however, irregular. In such compounds as the halogenated aminonitrobenzenes, the melting points correlated excellently with s ~ e e t n e s s . ’This, ~ however, fails with other groups of sweet compounds. The melting point of a crystal or the solubility of such compounds as sugars, is indicative of nonbonded, chemical interactions, but the theory of the existence of weak, nonbonded, chemical interactions was just becoming established at the time when these observations were made. Barral and Rand6 however, stated that there was no general law by which the taste of any compound could be predicted. Finizi and Colona” came to the same conclusion in the late 1930’s, after a very critical survey of the taste of aromatic compounds. They stated that sweet taste depends not on any factor, such as a sapophoric group, but on the entire chemical structure of the particular compound. These statements were of particular significance, because this was the first acknowledgment of the fact that the earlier theories of sweetness were gross oversimplifications of an extremely complex situation.
( 1 1 ) G.Beck, Wien. Chem. Zrg., 46 (1943) 18-22. (12) N. E. Loginov, Pishch. Promsr., 1 (1941) 32.
(13) H. T. Andersen, M. Funakoshi, and Y. Zotterman, in Y. Zotterman (Ed.), Oljacrion and Tasre, I, Pergamon, Oxford, 1963, pp. 177-192. (14) Y. Renqvist, S h n d . Arch. Physiol., 38 (1919) 97-201; 40 (1920) 117-124. (15) J. J. Blanksma, W. J. van den Broek, and D. Hoegen, Recl. Trau. Chim Pays-Bas, 65 (1946) 329-332. (16) F. Barral and A. Ranc, Rev. Sci., 56 (1918) 712-723. (17) C. Finizi and M. Colonna, Garz. Chim. Iral., 68 (1938) 132-142.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
207
2. Fundamental Structural Requirements for Sweetness
By 1938, one fact was clearly established. Sweet compounds, unlike salty and sour compounds, are found in all classes of chemical compounds, including such inorganic salts as beryllium (“glucinium”) and lead salts. They are also found among compounds of all molecular shapes and sizes, and stereochemical changes may result in a very dramatic change in the taste, as seen in the gustatory differences between enantiomorphs. Kaneko” pointed out that stereo-structure was an obvious clue to this behavior. He emphasized that, whereas D-amino acids are usually sweet, the L enantiomers are generally tasteless or bitter; their tastes are not related to their dextro- or levo-rotatory power, but rather to the result of certain molecular configurations. A series of a-amino acids was synthesized, and the tastes were studied.” The results (see Table 111) showed that the D-amino acid of the general formula RR’C(CH2)C02H,will taste sweet only when R is a hydrogen atom or any homolog of the methyl group, and R is smaller than a propyl group. The great difference in taste between the D- and L-amino acids clearly confirmed that taste depends not only on the groups present but also on their arrangement in space. The importance of configuration in the sugar series’’ in determining the differences in taste was also recognized, but, in this class of compounds, there are several asymmetric centres in the molecule, each of which may or may not affect the overall taste-properties. Early studies” implicating the configuration of carbon atoms bearing hydroxyl groups (of sugars) with taste properties led to the generalization that the a is a more effective stimulant than the @ anomer. However, the results2’ obtained since then definitely show that this generalization is not only a gross oversimplification, but is basically untrue, in that the anomeric configuration alone does not directly affect the sweetness of the molecule. For example, @-D-glucose is perceived to be significantly sweeter than a-D-glucose when the crystals are allowed to dissolve in the mouth,21 whereas @-lactose is nearly twice as sweet as a-lactose.2”22It was e ~ t a b l i s h e dthat ~ ~ it is only because of changes in the bitterness of sugar molecules resulting from a change in the anomeric configuration that the overall sweetness of a sugar molecule might in turn be enhanced or depressed. T. Kaneko, Nippon Kagaku Zasshi, 59 (1938) 433-439; 60 (1939) 531-538. A. R. Lawrence and L. N. Ferguson, Narure, 183 (1959) 1468-1471. G. G. Birch, Crif.Rev. Food Sci. Nufr., 8 (1976) 57-95. R. S. Shallenberger and T. E. Acree, in Handbook of Sensory Physiology. IV; Chemical Senses, 2; Tasre, Springer Verlag, Berlin, 1971, pp. 221-277. (22) R. S. Shaltenberger, 1. Food Sci., 28 (1963) 584-589. (23) G . G. Birch, N. D. Cowell, and R. H. Young, 1.Sci. Food Agric., 23 (1972) 1207-1212. (18) (19) (20) (21)
208
CHEANG-KUAN LEE
TABLEI11 Taste of D- and L-Amino Acids” Taste Amino Acids Me
L
Form
D
Form
NH,
\c/ Me’
\CO,H
Et
NH, \C /
tasteless-bitter
sweet
tasteless- bitter
sweet
tasteless-bitter
sweet
tasteless-bitter
sweet
trace bitter
trace bitter
sweet
tasteless
sweet
sweet
sweet
sweet
bitter
bitter
-\CO,H
Et’
Me
NH, \C/
Ph
NH, \C/
H” \CO,H Me
NH, \C/
H’
\CO,H
Me
NH,
\ / C Et’
\CO,H
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
209
Lawrence and Ferguson” also studied a number of physical properties of sweet-tasting compounds in an attempt to find the common property responsible for their sweet taste. They reported that all of the sweet-tasting compounds that they studied had structures that formed hydrogen bonds with water. They also studied the melting points of wet, sweet compounds, working on the theory that the wet melting-point depression, as compared to the dry melting-point, is a method of detecting intermolecular hydrogenbonding in the presence of intramolecular h y d r ~ g e n - b o n d sIn . ~ some ~ cases, limited trends were observed. The influence of surface tension was also studied, as it was argued that surface tension might affect the penetration of the molecule into the taste buds, and thus affect the overall taste. The latter property would also indicate whether an enzymic reaction was involved, and, if it was a redox enzyme-system, the ease of reduction or oxidation of a compound might have a bearing on its taste. However, on the whole, no relationship could be derived between these properties and the sweetness of a compound. a. Receptor Mechanism of Taste.-A major advance in the stereochemical basis of taste came with the understanding that the primary event in the initiation of taste involves the interaction of a stimulant molecule with a receptor located at the taste-cell plasma-membrane. As early as 1848, it had been suggested that sensory receptors transduce only one sensation, independent of the manner of stimulation. Behavioral experiments2’ tend to support this theory. In 1919, Renqvist14 proposed that the initial reaction of taste stimulation takes place on the surface of the taste-cell membrane. The taste surfaces were regarded as colloidal dispersions in which the protoplasmic, sensory particles and their components were suspended in the liquor or solution to be tested. The taste sensation would then be due to adsorption of the substances in the solution, and equal degrees of sensation would correspond to adsorption of equal amounts. Therefore, the rate of adsorption of taste stimulants would be proportional to the total substances adsorbed. The phenomenon of taste differences between isomers was partly explained by the assumption that the mechanism of taste involves a three-dimensional arrangement; for example, a layer of fatty acid floating on water would have its carboxylic groups anchored in the water whereas the long, hydrocarbon ends would project upwards.
(24) L. N. Ferguson, J. Chem. Educ., 9 (1958) 436-444. (25) R. Bernard, in D. A. Denton and J. P. Coghlan (Eds.), OIfaction and Taste, V, Academic Press, New York, 1975, p. 68.
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CHEANG-KUAN LEE
Lasareff26put forward a chemical theory in which each receptor was responsive to only a single taste, and that applied stimuli caused the decomposition of a material within the cell. This decomposition produced ions which then excited the nerve endings in the papillae, the concentration of the ionized products determining the magnitude of the neural activity. There are, however, a number of criticisms of these theories. Beidler27 argued that, as Renqvist14 had assumed that the magnitude of response is proportional to the amount of stimulant adsorbed per unit time, it is evident that, at equilibrium, the net velocity of adsorption is zero. It would follow that taste intensity should be zero, and the receptors completely adapted. However, Beidler showed28that the receptors do not adapt completely, but reach a steady level of response that is consistent for the duration of stimulation. Therefore, he concluded that human taste-adaption is dominated by events in the central nervous system, and not by the peripheral receptor. The same facts also prove Lasareffs assumption to be incorrect, as his experimental data also depended on a change in adaption that is not seen at the receptor level. The speed with which taste stimulation occurs, coupled with the fact that stimulation with toxic substances does no damage to the receptors, led Beidler29to suggest that taste stimulus need not enter the interior of the taste cell in order to initiate excitation. Because a taste cell has been shown to be sensitive to a number of taste qualities, and to a large number of chemical ~ t i m u l i , ~he” ~and ~ his coworkers concluded that a number of different sites of adsorption must exist on the surface of the cell. Therefore, they assumed that taste response results from adsorption of chemical stimuli to the surface of the receptor at given receptor sites. This adsorption is described by a monomolecular reaction similar to that assumed by Renqvi~t,’~ Lasareff,26and H a h r ~but , ~ ~with a difference. From the fact that each type of chemical-stimulus compound has a unique level of saturation of the taste receptor, it was concluded32that the magnitude of the response is dependent on the initial reaction with the receptor, and not on other, subsequent receptor-reactions that are common to all types of receptor stimulation. Therefore, it was assumed that the magnitude of neural response is directly proportional to the number of sites filled, the maximum response occurring when all of the sites are filled. Beidler29derived a fundamental (26) (27) (28) (29) (30) (31) (32)
P. Lasareff, Pjluegers Arch. Gesamte Physiol. Menschen Tiere, 194 (1922) 293-297. L. M. Beidler, Prog. Biophys. Biophys. Chem., 12 (1962) 107-151. L. M . Beidler, J. Neurophysiol, 16 (1953) 595-607. L. M. Beidler, J. Gen. Physiol., 38 (1954) 133-139. K. Kimura and L. M. Beidler, Am. J. Physiol., 187 (1956) 610-615. H. Tateda and L. M. Beidler, J. Gen. PhysioL, 47 (1964) 479-486. H. Hahn, Klin. Wochschr., 15 (1936) 933; Physiol. Absfr., 22 (1937) 212.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
211
equation, analogous to the Michaelis-Menten equation employed to study enzyme-substrate reactions, relating the magnitude of response to the concentration of the saporous compound, namely,
C / R = C / R,+ 1/ K R , , where C is the concentration, R is the magnitude of response, R , is the maximum response, and K is the equilibrium constant. The law of mass action has been successfully applied to many drug dose-response relationships since the early work of Clark.33The systematic relation between the dose of a drug and the magnitude of its response is based on three assumption^^^: (1) response is proportional to the level of receptor occupancy (occupancy theory), (2) one drug molecule combines with one receptor site, and (3) a negligible fraction of total drug is combined with the receptors. These assumptions must also apply to Beidler’s equation. But how does the mere adsorption of a chemical stimulus to the surface of a receptor stimulate the fiber innervating the taste cells? A plausible e ~ p l a n a t i o nwas ~ ~ that there would be a difference in the concentration of ionic constituents between the cell interior and exterior when adsorption occurs. It was shown36that the cells of taste buds are normally electrically charged, the interior being negative with respect to the environment, as are most receptors. When an electrolyte or nonelectrolyte is adsorbed on the receptor surface of the taste bud, a slight change in spatial configuration of the receptor molecule may result, such that a hole is formed that is large enough for certain ionic species (probably K+) contained within the cell to escape to the exterior, thus decreasing the potential across the receptor membrane. This could stimulate the innervating fiber, either by chemical or electrical means, such that the frequency of nerve impulses generated would be proportional to the magnitude of receptor depolarization?6 It was further proposed3’ that nonionic stimuli were adsorbed to the microvillus membrane by way of hydrogen bonds, as thermodynamic calculations showed that the binding energy is of the order of only -8 kilojoules per mole, which is about the magnitude of that for hydrogen-bond formation. The events that follow the initial adsorption of the taste stimulus to the receptor matrix of the taste-cell membrane are conformational changes in (33) A. J. Clark, The Mode of Action of Drugs on Cells, Williams and Wilkins, Baltimore, 1933. (34) A. Goldstein, L. Aronow, and S . M. Malman, Principles of Drug Action, 2nd edn., Wiley, New York, 1974. (35) L. M. Beidler, Annu. Rev. Physiol., 12 (1961) 363-388. (36) K. Kimura and L. M. Beidler, J. Cell. Comp.Physiol., 58 (1961) 131-140. (37) L. M. Beidler, in Ref. 13, pp. 133-148.
CHEANG-KUAN LEE
212
A
I
B
I C
FIG. 1 .-Diagrammatic Representation of the Three Steps in the Taste-cell Transdu~tion.'~ Step 1, interaction of stimulus (S) with membrane-bound receptor (R) to form stimulusreceptor complex (SR); step 2, conformational change (SR) to (SR)', brought about by interaction of S with R (this change initiates a change in plasma-membrane conformation of taste cells, probably below the level of the tight junction); and step 3, conformational changes of the membrane result in lowered membrane resistance, and the consequential influx on intracellular ionic species, probably Na+. This influx generates the receptor potential which induces synaptic vesicular release to the innervating, sensory nerve, leading to the generator potential.
the receptor rnatri~.~' These initiate changes in the cell-membrane structure at sites distant from the receptor matrix and changes in ionic permeability of the taste-cell plasma-membranes, resulting in generation of the receptor potential. The receptor potential presumably then initiates the release of synaptic vesicles that generate the neural-spike potential3'" (see Fig. 1). Sat0 and Beidler's has been questioned by various investigators. It was that the correlation of concentration with response reflects some aspect of the neuromechanism, rather than receptor binding, but, in general, the theory has been widely a~cepted.~'.~' Dzendolet4* pointed out that the property common to all sweet compounds is that of being a proton acceptor. Based on electrophysiological (38) J. G. Brand, in J. H. Shaw and G. G. Roussos (Eds.), Sweeteners and Dental Caries, Information Retrieval, Washington, D.C., 1978, pp. 13-32. (38a) T. Sato and L. M. Beidler, J. Gen. Physiol., 66 (1975) 735-763. (39) J. D. Watson, Molecular Biology ofthe Gene, 3rd edn., Benjamin, Menlo Park, CA, 1976. (40) S. Price and J. A. DeSimone, Chern. Senses Flaoor, 2 (1977) 427-433. (41) D. R. Evans and D. Mellon, Jr., J. Gen. PhysioL, 45 (1962) 487-500. (42) F. Dzendolet, Percept. Psychophys., 2 (1967) 519-520.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
213
data obtained by Beidler,28he suggested that the initial taste-response is induced as the receptor sites are evacuated, and not at the initial adsorption stage, that is, it is one of dissociation (removal of a proton from the gustatory receptor-sites) rather than of adsorption. The various sweetnesses of compounds was viewed as resulting from the distribution of electrons in a molecule as a function of substitution of various groups, and it was considered that inductive and electrostatic effects alter the facility with which a proton is exchanged. However, Shallenberger21.43argued that the ease with which compounds of differing sweetness exchange protons is probably not sufficiently different to account for their varied sweetness. The energy required to exchange the proton of a very sweet compound, such as chloroform, was not consistent with the calculated activation energy of the sweet-taste response, unless the proton of chloroform was also hydrated. Furthermore, the Dzendolet concept also did not need as strict a steric interpretation as does the AH,B proposal (see Section 11,2,b), and investigators are now well aware that steric features are probably the most critical features in structure-sweet-taste activity relationships. In essence, Dzendolet's hypothesis is of such a general nature that no predictive power can be gained by its application. Shallenberger and Acree21thus suggested that the mechanism could best be described as a proton exchange between the receptor site and the sweet-tasting compound. That the initial event of taste stimulation takes place on the cell surface of the taste receptor is now universally accepted. In addition, accumulated evidence strongly suggests that taste-bud stimulation is extracellular in nature. For example, (1) the sweet-taste response is both rapid and reversible, (2) the intensely sweet proteins monellin" and t h a ~ m a t i ncould ~ ~ not possibly penetrate the cell, because of their size, and (3) miraculin, the taste-modifying glycoprotein, having a molecular weight of 44,000 would also be too large to penetrate the taste ell.^^,^' Understanding of receptors was further extended by Kaneko's earlier findings" and, subsequently, those of Solms and coworkers? that certain (43) R. S. Shallenberger, in G. Ohloff and A. Thomas (Eds.), Gustation and Olfaction, Academic Press, New York, 1971, pp. 126-132. (44) J . A. Morris and R. H. Cagan, Biochim. Biophys. Acra, 261 (1972) 114-122; R. H. Cagan, Science, 181 (1973) 32-35; R. H. Cagan and J. A. Moms, Proc. SOC.Exp. Bid. Med., 152 (1976) 635-640. (45) 2. Bohak and S.-L. Li, Biochim. Biophys. Acta, 427 (1976) 153-170; H. van der We1 and K. Loeve, Eur. J. Biochem., 31 (1972) 221-225. (46) (a) K. Kurihara, Y. Kurihara, and L. M. Beidler, in Olfaction and Tasre, Proc. Int. Symp., 3rd, 1968, Rockefeller University Press, New York, 1969, pp. 450-469; (b) K. Kurihara and L. M. Beidler, Science, 161 (1972) 1241-1243; (c) Nature, 222 (1961) 1176-1179. (47) J. Solms, L. Vuataz, and R. H. Egli, Experientia, 21 (1965) 692-694.
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CHEANG-KUAN LEE
D-amino acids are sweet whereas their L enantiomers are not. Therefore, it appears that the receptor is chiral in nature. The tastes of D and L sugars are, however, not significantly but this does not cast any doubt on those findings, as the spatial barrier does not come into play, because of the relatively small size of sugar molecules, and they could, therefore, interact equally effectively (see Section 11,2,a). In the case of amino acids, Lawrence and Ferguson” showed that R in RR’C(NH,)CO,H must not be larger than an ethyl group, which means that the spatial area is only a short distance from the carbon atom carrying the AH,B unit. This will be discussed in Section II,3,a.
b. The AH,B Hypothesis.-The bulk of the evidence of the early work suggested that the initial excitation of the taste receptor by sweet-tasting compounds involves neither enzymic nor strong chemical forces. Rather, the forces operative appeared to be rather weak bonds, although clearly dependent on chemical structure4; this has since been c ~ n f i r r n e d . “ ~ - ~ ~ However, none of the early theories offered a unified, structural answer as to why some compounds taste sweet. As early as 1943, ReinickeS3had observed that the sweetness of sucrose is due to at least “two oxygen tetrahedra in para position with two intervening carbon tetrahedra.” By this, he was probably referring to ethylene glycol, which is known to be rather The dominating physical property of sugar molecules is their hydrogenbonding capacity. With a clearer understanding of hydrogen-bond theorySsss6Shallenberger” suggested that hydrogen bonding might explain the various sweetness of sugars. He noted that the different sweetnesses encountered in the sugar series appears to be related to the degree to which the hydroxyl groups of the sugars might participate in intramolecular hydrogen-b~nding.~~.~~ Vicinal hydroxyl groups, as in an ethylene glycol unit, have conformational attributes that can be described by using acyclic orientational notation (48) (49) (50) (51) (52) (53) (54) (55) (56) (57) (58)
R. S. Shallenberger, T. E. Acree, and C. Y. Lee, Nature, 221 (1969) 555-556. F. R. Dastoli and S. Price, Science, 154 (1966) 905-907. R. H. Cagan, Biochim. Biophys. Acta, 252 (1971) 199-206. C. K. Lee, S. E. Mattai, and G. G. Birch, J. Food. Sci., 40 (1975) 390-393. M. G. J. Beets, Structure-Actioity Relationship in Human Chernoreception, Applied Science, London, 1978. R. Reincke, Zuckerindustrie, 1 (1943) 79-82. C. J. Carr, F. F. Beck, and J. C. Karantz, Jr., 1. Am. Chem. SOC.,58 (1936) 1394-1395. L. Pauling, The Nature of the Chemical Bond, and Structure of Molecules and Crystals, IBH Publishers, Oxford, 1960. G. C. Pimentel and A. L. McClellan, The Hydrogen Bond, Freeman, San Francisco, 1960. R. S. Shallenberger, New Sci., 23 (1964) 569-570; Agric. Sci. Rev., 2 (1964) 11-20. R. S. Shallenberger, Roc. Symp. Front. Food Res., Cornell University, 1966, 45-62.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
eclipsed (0”)
gauche (60”)
FIG. 2.-Some
anticlinal (120”)
215
antiperiplanar (180”)
Orientations of Rotamers of a n a-Glycol.
(see Fig. 2). The stereochemical arrangement of vicinal hydroxyl groups is important as some a-glycol groups are incapable of eliciting the sweet sensation. For hydrogen-bond formation,5s*56a hydrogen atom must be attached, through a single covalent bond, to an electronegative atom, A (such as oxygen or nitrogen), and must have a second electronegative atom, B (such as oxygen or nitrogen), or an electronegative center (such as an unsaturated carbon-to-carbon bond), within a distance of 250-280 pm. According to Pauling,ss hydrogen atoms of alcohols and of sugar hydroxyl groups may participate in an intramolecular hydrogen-bond when the oxygen-oxygen distance lies between 285 and 251 pm. The distance between the centers of vicinal oxygen atoms in sugar a-glycol groups was calculated by Reevess9 (see Table IV). Thus, in the eclipsed orientation (which is normally only encountered in certain derivatives of the furanose forms of sugars), the interatomic, oxygen-oxygen distance is well within the intramolecular hydrogen-bonding distance, so that a “short” bond is formed that has appreciable covalent character.” The anticlinal arrangement, on the other hand, has an oxygen-oxygen distance well beyond that for hydrogen-bond formation.60 In the pyranoses in the chair conformations, the vicinal hydroxyl groups can exist only in the anticlinal or gauche orientations. The gauche arrangement of a-glycols is normally encountered as diequatorial or axialTABLEIV The Oxygen-Oxygen Distance of a-Glycol Units5’ Projected angle (degrees)
Distance (pm)
0 60 120 180
25 1 286 345 371
(59) R. E. Reeves, J. Am. Chem. Soc., 71 (1949) 2116-2119. (60) R. S. Shallenberger, T. E. Acree, and W. E. Guild, J. Food Sci, 30 (1965) 560-563.
216
CHEANG-KUAN LEE
equatorial dispositions. The oxygen-oxygen distance is 286 pm, and this is just beyond the distance at which an intramolecular hydrogen-bond may form. L. P. Kuhn6‘ pointed out that, if hydroxyl groups of an a-dihydroxy compound are sufficiently close, they will form an internal hydrogen-bond. H
RC-
CR
For compounds in which the O-H...O distance is <330pm, the infrared spectra of these compounds in CC14 solution showed multiple OH absorption-bands. Kuhn6’ found that the difference in the wavenumber of free hydroxyl absorption and the intramolecularly bonded hydroxyl absorption (Av)was directly proportional to the OH.-.O distance, and that it indicated the relative strength of the bond. In the sugar series, in addition to the vicinal hydroxyl groups forming hydrogen bonds, some hydroxyl groups on a sugar ring are also able to bond to the ring-oxygen atom, as in a-D-galactose (1) and a-D-mannose6’ (2) (see Fig. 3). Shallenberger2’ determined the A v values for various sugars (see Table V), and reported that there was a very good correlation between the Av values and the relative sweetness of those sugars. This led him to the conclusion that sugar sweetness varies inversely with the degree to which sugar hydroxyl groups are able to hydrogen-bond intramolecularly. Shallenberger did not discuss why a-D-glucose, which is much sweeter than a-Dgalactose, has a larger Av value (205 cm-I) than the latter (170 cm-I). Similarly, the Av value for P-D-glucose is about the same as that for a-D-mannose, and yet the sweetnesses are vastly different. However, Shallenberger stressed that intramolecular hydrogen-bonding may not be the only factor controlling sweetness, and that the Av value for P-D-glucose seemed to be too large to be a measure of intramolecular hydrogen-bonding alone. In fact, later work by Shallenberger and L i n d l e ~showed ~~ that, in certain cases, intramolecular hydrogen-bonding can enhance sweetness (see Section 11,3,a,ii). When Shallenberger” proposed that sweetness varies inversely with the extent of intramolecular hydrogen-bonding, he pointed out that “while this (61) L. P. Kuhn, J. Am. Chem. Soc., 74 (1952) 2492-2499. (62) A. B. Foster, Annu. Rev. Biochem., 30 (1961) 45-69. (63) C. Nieman, Zucker Suesswaren Wirtsch., 11 (1958) 465-467. (64) R. S. Shallenberger and M. G . Lindley, Food Chem., 2 (1977) 145-153.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
o/
217
‘\
H
HO
O CHiOHH 0
Ho OH
m
OH
1
2
FIG. 3.-Intrarnolecular Hydrogen-bonding in D-Galactose (1) and D-Mannose (2).
concept was the major basis of the argument presented, the role of intermolecular .hydrogen-bonding cannot be excluded.” Building upon that knowledge, Shallenberger and Acree’ proposed, in 1967, that vicinal hydroxyl groups of sugars in the staggered glycol orientation formed the saporous unit (that is, the chemical unit responsible for the sweetness) of sugars. They further suggested that, if the saporous unit was viewed as an AH,B unit (the separation between AH, the proton donor, and B, the proton acceptor, is -250 to 400 pm, the optimum being 300 pm), as is usually used to define and describe hydrogen bonding, that must also be the saporous unit for the other sweet compounds. Therefore, it was concluded that the initial mechanism of the sweet-taste response must arise by a simultaneous, intermolecular hydrogen-bonding between the saporous unit of the compound and a complementary, AH,B system on the receptor (see Fig. 4).
TABLEV Infrared Hydroxyl Absorption Bands and Hydrogen-bonding Strength ( A u cm-’) for Various Sugars and Their Relative Sweetness” Relative sweetness Bonded OH
Au
(cm-’)
(cm-I)
(cm-I)
Solution’
“Crystal”
3520 3570 3410 3545 3380 3550 3530 3460 -
3400 3395 3315 3340 3210 3340 3360 3380 3180
120 175 95 205 170 210 170 80 300
100- 175 100 40-79
180 100 74 82 32 32 16 32 1
Free O H Sugar
P-D-Fructose Sucrose a-D-Ghcose P-D-Glucose a-D-Galactose a-D-Mannose a-Lactose P-Lactose Raffinose a
From Ref. 63.
218
CHEANG-KUAN LEE
Receptor site
FIG. 4.-Shallenberger’s AH,B System2’
This proposal went some way towards unravelling the tangle of information that had for many years faced chemists interested in structure-sweetness relationships, that is, why seemingly unrelated compounds having such diverse chemical structure should possess this taste property. The AH,B concept seems the most plausible approach to the problem, as it selects a simple feature and neatly embraces all earlier theories about sweetness. It contains Cohn’s idea7 that saporous groups occur in pairs. It also encompasses Oertly and Myers’s concept’ of auxogluc and glucophore. Kodama’s “vibratory” hydrogen’ can also be related to the AH moiety, and Tsuzuki’s “resonance” energy65 can be equated with the change in the acidity and, therefore, the sweetness of the compound when substitution is carried out. The significance of Warfield’s “taste couples,” consisting66 of an acidic proton and a neighboring, unshared electron pair (Pauling’s hydrogen-bond theory was then still in its infancy) is now clear. More important, it is possible to identify an AH,B system in sweet compounds of various chemical classes. The question remaining was whether or not the AH,B system of these compounds had the steric requirements dictated by the gauche glycol group. Indeed, in all cases, sweet-tasting compounds were found6’ to have an AH,B system with an AH proton to B distance of -300pm. Furthermore, in most cases, the AH,B system of other sweet-tasting compounds was found to be not as structurally complicated, or as subject to extensive conformational variation, as for sugars. The importance of the configuration and conformation of the saporous group cannot be overstressed. This is demonstrated by the correct prediction that P-L-arabinopyranose tastes sweet because of its similar configuration (65) Y . Tsuzuki, J. Chem. Soc. Jpn., Ind. Chem. Sect., 51 (1948) 32-40. (66) R. B. Warfield, Abstr. Pap. Am. Chem. SOC.Meer., 126 (1954) 1 5 ~ . (67) R. S. Shallenberger and T. E. Acree, Carbohydr. Res., 1 (1966) 495-497.
CHEMISTRY A N D BIOCHEMISTRY O F SWEETNESS
219
TABLEVI Typical Hydrogen-bond LengthsType of bond
Type of compound
AH * .* B distance (pm)
OH...O OH...O OH...O NH...O NH...N
carboxylic acid primary alcohol water urea, peptides, amfdes ammonia, amines
260 270 280 290 310
to that of a-D-galactopyranose; in fact, both were found to have the same relative sweetness.48Related to the conformation is the directional influence of the hydrogen bond on the stimulant molecule. This is more important than its binding strengthY3’as the energy liberated by the formation of a hydrogen bond between polar functional groups compensated the free energy required to remove first any solvating hydrogen atoms associated with the polar functional groups through similar hydrogen-bonds.
The net gain in energy is, however, not many times larger than that of most hydrophobic interactions,68 but the strength of hydrogen bonding is dependent on the spatial geometry of the A-H bond and the electron-rich center interacting with the electron-deficit hydrogen atom (see Table VI). Therefore, the more the A-H bond is directed towards the receptor element, the stronger will be the hydrogen bond.69 The bond angles resulting from the interaction of different functional groups vary with the nature of the functional groups them~elves.’~ NH..
202m
OH
(68) S. Bernhard, The Structure and Functions ofEnzymes, Benjamin, New York, 1968. (69) A. Korolkavas, Essentials of Molecular Pharmacology, Wiley-Interscience, New York, 1970. (70) J. Donahue, in A. Rich and N. Davidson (Eds.), Structural Chemistry and Molecular Biology, Freeman, San Francisco, 1968, pp. 443-465.
220
CHEANG-KUAN LEE
The strong, directional influence of the hydrogen bond has a major effect on the positioning of the stimulant molecule within a receptor site, causing the formation of weaker, non-covalent interactions (for example, hydrophobic bonding) at other locations in the molecule to be more or less favorable. Thus, the strength or effectiveness of these neighboring interactions will be markedly governed by the directional influence of the stronger hydrogen bond, as the magnitude of such forces as Van der Waals (London dispersion) forces is inversely proportional to the 6th power of the distance between the interacting species. This may explain why some molecules, such as drug antagonists, can bind to a receptor without producing much biological response.69The importance of these weaker, non-covalent interactions in enhancing biological response, including sweetness, is discussed in Section II,2,c. By identifying the most acidic proton in a number of sweet-tasting compounds, Shallenberger and A ~ r e e ~proposed .~' possible locations for the AH,B systems of these compounds (see Fig. 5 ) . The hypothesis could also account for differences in the relative sweetness of sugars by simple consideration of the way in which configurational differences might affect hydrogen bonding. The sweetness of D-galactose (l), which is only about half that of D-glucose, was attributed to an intramolecular hydrogen bonding between the 4-hydroxyl group and the oxygen atom of the pyranose thereby greatly restricting a glycol moiety from participating in the interaction with the receptor. D-Mannose (2) is less sweet than D-glucose, because its axial 2-hydroxyl group is also similarly sterically disposed to bond to the ring-oxygen atom62(see Fig. 3). Evidence of intramolecular hydrogen-bonding was obtained from infrared spectra (see Table V). Furthermore, it was concluded that the lack of very distinct OH absorption bands for D-galactose, and the shift towards the lower wavenumbers, are attributable to fairly strong hydrogen-bonding. This was similarly observed for D-mannose and Further support for this hypothesis was provided by the change in sweetness of D-glucose and D-galactose solutions at elevated temperature^.^^^" The sweetness of D-galactose was observed to increase twice as fast as that of D-glucose until, finally, the sweetness of the two sugars was virtually equal. Presumably, intramolecular hydrogen-bonds were severed at elevated temperature, with a resulting increase in sweet taste, suggesting that the data and the concept were thermodynamically sound.s8 However, one factor that Shallenberger failed to take into account was the effect of temperature on mutarotation. (71) R. S. Shallenberger and T. E. Acree, J. Agnc. Food Chem., 17 (1969) 701-703. (72) J. S. Brimacornbe, A. B. Foster, M. Stacey, and D. H. Whiffen, Tetrahedron, 4 (1958) 35 1-360.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
p- o-Fructopyranose
Saccharin
Cyclamic acid B
221
Chloroform
L-Alanine AH
AH\oAo H @NH= B CH
I
Ph 2-Amino-4-nitrobenzenes
.
Aspartame
( L-Aspartoyl-L-phenylalaninemethyl
FIG. 5.-The
Perillartine ester)
AH,B System in Various Sweet-tasting compound^.^^^'
Although the change in the position of pyranose-pyranose equilibria with variations in temperature is only slight, the effect on pyranose-furanose and it has been shown that the position equilibria may be of the galactose equilibrium containing pyranose and furanose forms is also shifted markedly with temperature change.75This could partly account for the decrease in relative sweetness of D-fructose with increasing temperature, reported by Tsuzuki and Y a m a ~ a k i . The ~ ~ thermal mutarotation b y gas-liquid chromatography of the perof ~ - f r u c t o s e , ’ ~monitored *~~ (73) (74) (75) (76)
R. S. Shallenberger and G. G. Birch, Sugar Chemisrry, AVI, Westport, CT, 1975. R. S. Shallenberger, Pure Appl. Chem., 50 (1978) 1409-1420. T. E. Acree, R. S. Shallenberger, and L. R. Mattick, Carbohydr. Res., 6 (1968) 498-502. Y. Tsuzuki and J. Yamazaki, Biochem. Z., 323 (1953) 525-531.
222
CHEANG-KUAN LEE
,i 90 80
70 2 60 al
u
n
2
50 40
30 20
10
10
20
30
40
50
60
70
$0
90
Temperature (T)
FIG.6.-Thermal
Mutarotation of o - F r ~ c t o s e . ’ ~
(trimethylsilyl)ethers, is shown in Fig. 6. A second factor that Shallenberger failed to consider was the effect of temperature on intermolecular hydrogen-bonding. If the temperature is sufficiently high to sever intramolecular hydrogen-bonds, it should follow that intermolecular hydrogen-bonding between the compound and receptor should be equally diminished. There is little doubt that Shallenberger’s AH,B hypothesis is the most plausible concept in the explanation of the initial stimulation of the sweettaste receptor. However, it was unfortunate that the evidence was accrued largely with the aid of reducing sugars, which, in solution, equilibrate between many isomers, so that it is not possible to relate total gustatory response to any one particular stereochemical ~ t r u c t u r e It ~ ~is- also ~ ~ not (77) G. G. Birch, C. K. Lee, and E. J. Rolfe, J. Sci. Food Agric., 21 (1970) 650-653. (78) C. K. Lee, Ph.D. Thesis, University of Reading, 1973. (79) G. G. Birch and C. K. Lee, in G . G . Birch, L. F. Green, and C. B. Coulson (Eds.), Sweerness and Sweefeners, Applied Science, London, 1971, pp. 95-1 11.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
223
possible to compare the taste of two sugars that have attained equilibrium, as the proportion of different isomers present at equilibrium is unlikely to be the same in each c a ~ e . * ~ * ’Furthermore, ~-~’ even in a monosaccharide, there are present a number of different a-glycol groups, each satisfying the structural requirements of Shallenberger’s AH,B system. Assuming that a system of binding as postulated by Shallenberger is operative, the question is: which glycol unit(s) of the sugar constitute(s) the sweet-eliciting grouping(s). Extensive and systematic studies by Lee,78 Lindley,” and Birch” conclusively identified the AH,B units responsible for sweetness in selected sugars (see Section 11,3). c. The Hydrophobic Bonding Concept.-As early as 1917, it was found” that an odorous substance must be soluble in water and in oil. Thus, the middle members of a homologous series, such as butyl alcohol and pentyl alcohol, have strong odors, whereas lower members, such as methanol, ethanol, and propanol (which are not readily oil-soluble), and higher members, such as stearyl and lauryl alcohol (which are water-insoluble), are odorless. It was postulated” that an odorant coming in contact with the olfactory region must first dissolve in the watery mucus and then, later, in the lipoid tissue. In the study of sweetness, early workers had tried to correlate sweetness with lipoid solubility. Lawrence and Ferguson” attempted to correlate the surface tension of solutions of organic compounds with their taste because this property might affect the penetration of the molecule into the taste-bud cells and thereby govern the taste response. The great dependence of taste on hydrophobicity was quantitatively demonstrated by Deutsch and Hansch.82 From the bulk of the evidence available, theys2 reasoned that the mechanism of sweet-taste stimulation is probably not very different from the mode of action of drugs. In a pharmacological structure-activity relationship, the biological system is so complex that it makes the search for a quantitative relationship between biological response and molecular structure by means of the classical methods of kinetics and thermodynamics nearly i m p ~ s s i b l e .Only ~ ~ an empirical approach, in which a quantitative response-parameter can be simultaneously compared with various physical properties for a series of related drugs, can possibly be successful. The fundamental problem is to discover which substituents produce the most advantageous effects in the biological profile of a series. Normally, the purpose of introducing a substituent is to alter the physicochemical (80) (81) (82) (83)
M. G . Lindley, Ph.D. Thesis, University of Reading, 1974. E. L. Backman, J. Physiol. Pathol. Gen., 17 (1917) 1-4; Physiol. Abstr., 2 (1917) 497. E. W. Deutsch and C. Hansch, Nature, 211 (1966) 75. C. Hansch, Encycl. Inf. Encyclo. Pharmacol., Secr., 5 , 1 (1973) 75-165.
224
CHEANG-KUAN LEE
characteristics of the parent system, and it is these changes that are associated with the altered biological response. Hansch and c o ~ o r k e r s ~ suggested ~-~’ an approach based upon the search for quantitative relationships between quantifiable response-parameters and combinations of factors that might be presumed to play a part in the interaction between the drug and the biological system.
+
Response, = system, drugj.
(1)
Thus, the biological activity of a drug molecule could be expressed as a function of its electronic, hydrophobic, and steric properties, and one or more such factors as hydrogen bonding or polarizability (x) which might be involved. Response,
= f(hydrophobic)
+ f(e1ectronic) + f(steric) + f(x) + e.
(2)
If the free-energy-related parameters for electronic (a), hydrophobic (P), and steric ( E s ) factors are defined, Eq. 2 gives the well known, free-energy relationship: log (Response)c =a(log P)’+b(log P ) + c u + d E , + e ,
(3)
where log(Response)c is a measure of the biological activity at the concentration, C, which results in a defined standard response, such as that obtained from acute toxicity (LD,,)or from inhibition data (Iso). The term containing log P quantifies the hydrophobic properties of the molecules in such a way that biological activity is parabolically related to hydrophobicity. The hydrophobic constant is defined as log P J P , ( = T ) , where P, is the experimentally determined partition coefficient of the test substance (x) between 1-octanol and water, relative to that of a reference compound, n. The term containing u is the Hammett constant, an expression of electronic character which, by definition, relates the electronic properties of the test substance to those of the reference. The term E, represents a measure of the steric effects of x relative to the reference compound n, and the coefficients a, b, c, d, and e are then determined by regression analysis, using least-squares methods. Thus, Eq. 3 becomes log (Response), = aT2+ b.rr + c u + dE, + e.
(4)
(84) C. Hansch, in E. J. Ariens (Ed.), Drug Design, Vol. 1, Academic Press, New York, 1971, pp. 271-337. (85) W. P. F’urcell, G. E. Bass, and J. M. Clayton, Strategy in Drug Design: A Guide to Biological Activity, Wiley-Interscience, New York, 1973. (86) M . S. Tute, Adv. Drug Res., 6 (1971) 1-77. (87) W. van Valkenburg, Adv. Chem. Ser., 114 (1972) 1-304.
225
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS TABLEVII Correlation of Relative Sweetness of 2-Substituted 5-Nitroaoilines with Hammett Constant (a)and Hydrophobicity” ( m )
-0CH3 -0CHZCH3 -OCH,CH,CH, -H -CH, -F -CI -Br -1
-0.27 -0.24 -0.24 0 -0.17 +0.06 +0.23 +0.23 +0.28
-0.02 +0.48 +0.98 0 +0.56 +0.14 +0.71 +0.86 +1.20
2.519 3.146 3.669 1.602 2.519 1.602 2.602 2.903 3.097
2.192 2.942 3.746 1.729 2.942 1.845 2.451 2.693 3.149
Deutsch and Hansch” applied this principle to the sweet taste of the 2-substituted 5-nitr0anilines.’~’~~ Using the data available (see Table VII), the calculated regression Eqs. 5-7 (using the method of least squares) optimally expressed the relationship between relative sweetness (RS), the Hammett constant, a, and the hydrophobic-bonding constant, V. log R S = 1 . 2 1 4 ~ +1.970 r = 0.766;
s = 0.476
log R S = 1.610~+1.831a+1.729
r = 0.936;
(6)
s = 0.282
log RS = 0.11 9 ~+’ 1 . 4 8 5 ~ 1 . 8 4 8+ ~ 1.742 r = 0.936;
(5)
(7)
s = 0.308
The results (see Table VII) clearly showed that log RScalcand the experimentally determined values, log RS,,, , correlated very well. Eq. 6 suggests a high dependence of relative sweetness on the hydrophobic-binding constant, V, (confirming Lawrence and Ferguson’s observation that sweetness increases with basicity’’) and the Hammett constant, a, (a substituent’s electron-withdrawing power), accounting for the findings9 by Tsuzuki and coworkers that compounds having the largest resonance energies taste sweetest; this energy is an index of a compound’s intramolecular, electron delocalization. The v value is essentially a partition coefficient indicative of a compound’s hydrophobic nature. Thus, an important prerequisite for (88) J. J. Blanksma and D. Hoegen, Red. Trav. Chim. Pays-Bas, 65 (1946) 333-337. (89) Y. Tsuzuki, S. Kato, and H. Okazaki, Kagaku, 24 (1954) 523-524.
226
CHEANG-KUAN LEE
a compound to taste sweet is a high hydrophobic character, or the ability to form hydrogen bonds with water. Once the compound is in solution, its sweetness is determined by favorable partitioning onto the receptor. The latter step was viewed as being dependent on hydrophobic bonding, degree of polarity, distance of charge separation in the molecule, electron density and steric effects. Sugars have at a particular point in the molecule (a), very low sweetness compared with certain artificial sweeteners, even though they have high solubility in water. It was concluded that this is due to their weak attachment to the receptors; that is, they possess little hydrophobic character.82 The high sweetness of saccharin is partly attributed to the hydrophobic character of the benzene ring. As expected, the sweetness of saccharin and its CaZ+or Na+ salts decreases if the dissociation is inhibited by increasing the concentration in water, or by addition of calcium or sodium ions.9o Similarly, any substitution on the isothiazole nucleus that causes loss of solubility also causes loss of sweetness.” Deutsch and Hansch” pointed out that the sweetness of 2-amino-4-nitroand 4-amino-2-nitro-benzene derivatives is dependent on the positions relative to the 1-substituent and the resulting effect on the electrons of the ring. Favorable charge-distribution occurs when an electron-donating group is para to the nitro group (as in 3), or when an electron-withdrawing group is para to the amino group (as in 4) (see Fig. 7 and Table VIII). With these types of compounds, Ferguson and Childers9’ suggested that sweetness is associated with the molecules having flatness and a charge distribution as found in a para-disubstituted benzene, with an electrondonating or an electron-withdrawing group, and a “third group off -center.”
For example:
An extension of this concept to other classes of sweet compounds is, however, lacking. (90) Y. Magidson and S. W. Gorbatschow, Ber., 56 (1923) 1810-1817. (91) G. Harnor, Science, 134 (1961) 1416-1417. (92) L. N. Ferguson and L. G . Childers, J. Org. Chem., 25 (1960) 171-175.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
o”N+o
227
N H 2
3
4
FIG.7.-Relative Sweetness of Derivatives of 3-Arninonitrobenzene.8’ When R is an electrondonating group, 3 is sweet and 4 is not sweet. When R is an electron-withdrawing group, 4 is sweeter than 3.
TABLEVIlI Relative Sweetness of Aminonitrobenzene Derivatives’’ Relative sweetness
-0CH3 -CH, - Br -CO,H -SO,NHz a
Compound 3
Compound 4
167 298 715 25 -
tasteless tasteless tasteless 120h sweet
Compared to sucrose as unity.
Ref. 88.
Eq. 6, proposed by Deutsch and Hansch,82 indicates that the saporous group involved in the generation of sweet taste consists of an area for hydrophobic bonding coupled with an area for electronic bonding, a conclusion that is closely similar to Shallenberger’s AH,B concept. Shallenberger and AcreesS2’.”suggested that the AH,B unit responsible for sweetness in these aminonitrobenzene derivatives is the combination of the nitro group and the activated ortho proton. This assignment has been criticized (see later). A modification of the Deutsch and Hansch equation was proposed by M ~ F a r l a n d . ’He ~ argued that, in order to develop a theory of drug action, a reasonable model containing most of the implicit assumptions that are fundamental to that theory must first be considered. How certain drugs act depends on the probability that, at some initial state, the drug molecule will go through three stagesg3:(1) the drug reaches the receptor by passive (93) J. W. McFarland, B o g . Drug Res., 15 (1971) 123-147.
228
CHEANG-KUAN LEE
transport, that is, it is applied to some external region, and, by a “random walk,” it crosses a number of cell membranes to arrive in the neighborhood of the receptor; (2) the drug binds to the receptor to form a drug-receptor complex; and (3) the drug-receptor complex may undergo some chemical reaction leading to a biological response. It was further proposed93 that, for particular cases, some of these steps are not necessarily critical or limiting. Such a mechanism is found in a probability argument. By also taking into account the group dipole-moment and the polarizability of the substituent, regression analysis gave an equation which shows a very significant correlation accounting for 98% of the variance in the data. ~ 0.45p2+ ( a, - a”)+ 1.66, log R S = 1.31T - 1 . 0 8 +
r = 0.998;
s = 0.149
where p is the group dipole-moment of the substituent, and aH is the polarizability of 3-nitroaniline and a x ,that of a 2-substituted 5-nitroaniline. However, the model is defined such that the terms T,a, p, and a may all be measures of different types of receptor-sweetener binding. These all reflect the probability of that event’s occurring, while, at the same time, the probability of the sweetener’s reaching its receptor, and that of the receptor complex’s undergoing the response-eliciting reaction, may both be unity.94 The sole criterion for the sweet-taste response would thus require the formation of the proper sweetener-receptor complex. The validity of such a hypothesis has yet to be pr0ved.9~ Further studies of the 2-substituted 5-nitroanilines, conducted by Kier and coworker^,^^-'^^ searched for a linear combination of structural variables that describes a line, plane, or surface that separates the molecule classes in the optimum manner. They found that sweetness correlated very well with the substituent polarizability-constants for the 2-substituent, implicating the involvement of the 2-substituents in dispersive-binding interactions at the receptor. This is in agreement with the results of H a n ~ c hand ~~ M ~ F a r l a n dThe . ~ ~ correlation equation was not, however, reported. (94) G . A. Crosby, G. E. DuBois, and J. E. Wingard, Jr., in Ref. 84, Vol. 5,1979, pp. 215-310. (95) L. B. Kier and L. H. Hall, Molecular Connectivity in Chemistry and Drug Research, Academic Press, New York, 1976. (96) L. B. Kier, L.H. Hall, W. J. Murray, and M. Randic, 1.Pharm. Sci.,64(1975) 1971-1974. (97) L. H. Hall, L. B. Kier, and W. J. Murray, J. Pharm. Sci., 64 (1975) 1974-1977. (98) W. J. Murray, L. H. Hall, and L. B. Kier, J. Pharm. Sci., 64 (1975) 1978-1981. (99) L. B. Kier and W. J. Murray, J. Med. Chem., 18 (1975) 1272-1274. (100)L. B. Kier, W. J. Murray, M. Randic, and L. H. Hall, J. Pharm. Sci.,65 (1976) 1226-1230. (101) W. J. Murray and L. B. Kier, J. Med. Chem., 19 (1976) 573-578. (102) L. B. Kier and L. H. Hall, J. Pharm. Sci., 65 (1976) 1806-1809.
229
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
developed an interesting concept termed "molecular Kier and connectivity," in which the molecular-connectivity index, x, was defined as n
x = c ck=cl/-,
(9)
k
k=l
where C is the connectivity of each bond k, and d is the degree of connectivity assigned to each non-hydrogen atom, this being equal to the number of bonded, non-hydrogen atoms. The values 1/are for the atoms i and j , which make up this bond, and the connectivity index, 'x, is obtained as the sum of the bond connectivities. In molecules containing heteroatoms, the d values were considered to be equal to the difference between the number of valence electrons ( E ") and the number of hydrogen atoms (hi).Thus, for an alcoholic oxygen atom, d = 1, and d " = 5 . The valence connectivity-index, 'xu can then be calculated; the use of 'xu removes redundancies that can occur through the use of Ix alone. The calculation of connectivity indices Ix and 'xufor the case of two isomeric heptanols is as follows. (a) 2,2,3-Trimethyl-l-butanol
lC1-
1/43
3c3
1/J(3X4)
1
1
4C-2C2
ilJ3
1c1
1/4(4x2)
(1/42)(l/JlO)"
105
1/44
1c1
'x = 3.504,
'xu= 3.466
(b) 3,4-Dimethyl-l-pentanol 1c1
1c1-
1/43
I 3c3-
1/49
1/J6
3C3-2C2-
I
1/44
2c2
(1/42)(I/lO)"
105
l/J3
1c1 I
x " =3.289
Kier and coworkers found that the molecular connectivity-index and such molecular properties as polarizability,96 molecular volume,'00 and partition coefficients between water and octanoIi0' show very good correlation. Because all of these properties could be correlated with biological activity,
230
CHEANG-KUAN LEE
as shown by Hansch and other workers, it should be possible to correlate x with biological properties. Kier found that this was applicable to such activities as enzyme inhibition, barbiturate activity, and tadpole narcosis.99 In addition, Kier and Hall9' reported that a high correlation ( r = 0.953, s = 0.222) was obtained for the relative sweetness of the 2-substituted 5-nitroanilines. The concept of molecular connectivity was modified by Daniel and Whistler1o3in their study of the sweetness of sugars and alditols, by insertion of terms to characterize chiral centers and by summation of appropriate molecular subgraphs. They calculated a third-order connectivity index, 3x,, for various simple sugars, glycosides, methyl ethers, and alditols, and found these to bear a direct relationship to sweetness intensity. For those sugars that did not fall into a known order and ratio of sweetness, it was argued that this could probably be remedied by using additional "xm values. Carbohydrate analogs having atoms other than carbon, hydrogen, and oxygen, and those having the more-flexible furanoid ring-system, gave poor correlations. It was also predictedlo3that this could be overcome with some refinement and reinforcement of its theoretical foundation. Although it is possible to "order all sugars in known order and ratio of sweetness," this does not seem to have any predictive value. The concept of molecular connectivity is particularly attractive, because of the simplicity with which x can be computed. However, this simplicity may be a disadvantage. Crosby and coworkers94 argued that the Hansch and that extended by M ~ F a r l a n dused ~ ~ various, distinct parameters presumed to be important for drug action. Thus, when a correlation equation is obtained, consideration of the significance of the various terms gives a hint of the mechanism of the action of the drug. Consequently, if the 7 ~ ' term of Eq. 7 is found to be significant, transport of the stimulant molecule may produce a major effect on the activity, whereas if p and a are significant, binding may be important; and, if a is significant, factors relating to intrinsic activity may be critical. The molecular-connectivity approach, however, considers only a single parameter, which embodies all of the parameters in the Hansch and McFarland analyses and, therefore, does not allow the synthetic chemist to sort out the factors responsible for a~tivity.'~ Of interest is a nonmathematical approach to drug design proposed by T o p l i s ~ "and ~ Topliss and Martin."' This concept is based on the fundamental approach of the Hansch concept, namely, that a particular sub(103) J. R. Daniel and R. L. Whistler, Cereal Chem., 59 (1982) 92. (104) J. G. Topliss, J. Med. Chem., 15 (1972) 1006-1011. (105) J. G. Topliss and Y. C. Martin, in Ref. 84, Vol. 5, 1975, pp. 1-21.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
23 1
stituent may modify activity relative to that of the parent compound by virtue of resulting changes in hydrophobic, electronic, and steric effects. The Topliss approach is of great interest, as it presents a stepwise selection for the synthesis of new analogs of an active, leading compound, each step being designed to maximize the changes needed in synthesizing the most potent compounds in the series as early as possible. The concept has yet to be applied to sweetener design. d. The Tripartite Concept.-The vast amount of data accumulated clearly establishes the validity of the Shallenberger AH,B concept. However, sucrose and D-fructose, the two sweetest simple sugars, rank rather low in sweetness compared to such sweeteners as saccharin and cyclamates, even though the sugars possess a number of a-glycol groups, each satisfying the Shallenberger requirement of geometrically suitable AH,B systems. From a number of independent studies, it appears that a third binding-site in a molecule is necessary for a potent, sweet-taste response. For example, Shallenberger and suggested the existence of a barrier somewhat removed from the AH unit of the receptor. Kaneko's work18on the sweetness of amino acids similarly suggested the presence of such a barrier. The investigations of Deutsch and Hansch8* and M ~ F a r l a n don ~ ~a series of intensely sweet, 2-substituted 5-nitroanilines clearly showed the existence of a hydrophobic-bonding area. Furthermore, the observation that some D-amino acids are sweet, whereas the L enantiomers are not, indicates a stereoselective receptor,'8*106-108 and, hence, that there are at least three binding-sites on the molecule. Kierio9determined the favored conformations of some of these acids (5-8) by molecular-orbital calculations (see Fig. 8), and found that all of the examples studied showed a common, conformational preference for the H:N-CH-COO- moiety having an N - 0 distance of 260 pm, agreeing with the A-to-B dimension postulated by Shallenberger and Acree.'.'' The difference in the sweetness intensity of these compounds clearly implicates the participation of a third factor, which must lie within the side-chain functionality. Molecular orbital calculations"0 and nuclear magnetic resonance studies'" revealed that C-2 in indole and related compounds,"' the phenyl (106) H. Stone, in T. Hayashi (Ed.), Olfaction and Taste, II, Pergamon, London, 1967, pp. 289-306. (107) C. P. Berg, Physiol. Rev., 33 (1953) 145-189. (108) J. Solms, J. Agric. Food Chem., 17 (1969) 686-688. (109) L. B. Kier, J. Phorm. Sci., 61 (1972) 1394-1397. (110) A. Szent-Gyorgyi and I. Isenberg, Proc. Nut/. Acud. Sci. U.S.A., 46 (1965) 1334. ( 1 1 1 ) J. P. Green and J. P. Malrieu, Proc. Narl. Acud. Sci. U.S.A., 54 (1965) 659. (112) R. Foster and C. A. Fyfe, J. Chem. Soc., B, (1966) 926-929.
232
CHEANG-KUAN LEE
eH H
H
co;
H
5
6
D-Tryptophan (35 x sucrose)
D-Phenylalanine (7 x sucrose)
7
8 D-LeUCine (6 x sucrose)
D-Histidine (7 x sucrose)
FIG. 8.-Favored Conformation of Some D-Amino Acid Zwitterions as Indicated by Molecular-orbital Calculations.'og
~ i n g , " ~ *and " ~ C-4 of i m i d a ~ o l e "were, ~ or had high potential for localized charge-transfer, susceptible to electrophilic attack (see Fig. 9). All of these positions exist in identical positions relative to the zwitterion moiety, suggesting that a common, third site in these compounds involves an electronrich position capable of undergoing electrophilic attack, engaging in localized charge-transfer, or participating in some type of nonspecific interaction involving the electron component, probably the result of short-range dispersion-forces. Theoretical calculations of the favored conformation of Dleucine, which is moderately sweet, showed that the favored orientation of the methyl side-chain possesses the same relationship to the -CHCO; moiety as that in three other amino acids (see Fig. 8). Thus, it was concluded that the third binding site probably involves a dispersion interaction at the
FIG. 9.-Centers
of Electron-rich Density in D-Amino Acids.'o9
(1 13) K. Fukui, C. Nagata, and A. Imamura, Science, 132 (1960) 87-89. (114) J. Crow, 0. Wasserman, and W. C. Holland, J. Med. Chem., 12 (1969) 764-766. (1 15) B. Pullman and A. Pullman, Quantum Biochemistry, Wiley-Interscience, New York, 1963.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
AH
233
-550 pm
FIG. 10.-The AH,B,X Glucophore of Sweet-tasting C o ~ n p o u n d s . ' ~ ~
receptor. The poor dispersion binding-ability of the methyl group probably explains the lower sweetness of leucine. Therefore, it appears that the active participation of the AH,B pair in the interaction with the taste receptor suffices to impart a basal level of sweetness, such as in sucrose, glycine, D-alanine, and ethylene glycol, but a third binding-feature is required for attainment of intense sweetness. Calculations by Kier'" located the third dispersion bond (x)at -350 pm from AH and -550 pm from B (see Fig. 10). Examinationlog of several classes of intense sweeteners showed the existence of potential x sites whose relation to the AH,B unit is identical to the Kier proposal (9-13) (Fig. 11). In the 2-substituted 5-nitroanilines, the hydrophobic binding-group (the C-2 substituent) postulated by Deutsch and Hansch82 is clearly the third dispersion binding-group specified by Kier.'09 Thus, Shallenberger"6*"7 viewed this site as the hydrophobic site, y, rather than as a dispersion bonding. A comparable, functional-group relationship explaining the odorous properties of enantiomers had earlier been proposed by Shallenberger,'18 and, after re-examining numerous sugars, Shallenberger and ( 1 16) R. S. Shallenberger, Deu. Food Sci., Proc. Int. Congr. Food Sci Techno/., 5th, 1978, pp. 360-366. ( 1 17) R. S. Shallenberger, Zuckerindustrie, 104 (1979) 121-124. (118) R. S. Shallenberger, in G . G. Birch, J. G. Brennan, and K. J. Parker (Eds.), Sensory Properties of Foods, Applied Science, London, 1977, pp. 91-100.
CHEANG-KUAN LEE
234
-360 pm I
,
B A0H
-360 pm
10 Saccharin
9
Cyclamic acid
-360 pm
OMe 11
12
13
Perillaldehyde oxime
Nitroanilines
Neohesperidin dihydrochalcone
FIG. 11.-Identity of the Third Structural Feature Comprising the Postulated Glucophore in Some Sweet-tasting molecule^?^
L i n d l e ~proposed ~~ an oblique, planar, stereogeometric arrangement between AH,B and y that is strikingly similar to Kier's tripartite glucophore, with distance and direction parameters as shown in Fig. 12. A similar y site for other sweeteners, such as the sweet dipeptides, has also been p r o p o ~ e d , " ~ *and ' * ~ it appears that the location of y in relation to AH,B is directional rather than positional. The interesting feature of the Kier glucophore is that the tripartite grouping of AH,B, y is dissymmetric, as the distance parameters describe a scalene triangle not superposable upon its mirror Shallenberger and L i n d l e ~had ~ ~previously reported that, unlike the amino acids, there is no significant difference in the sweet taste of sugar enantiomers; this would be expected if the tripartite requirement for sweet taste is correct. The structural requirement that AH,B, and y shall describe a scalene triangle is, in fact, (119) A. van der Heijden, L. P. B. Brussel, and H. G. Peer, Food Chem., 3 (1978) 207-211. (120) L. B. P. Brussel, H. G . Peer, and A. van der Heijden, Z.Lebensm. Unters.-Forsch.,159 (1975) 337-343.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
235
6
\
\
-740pm
FIG. 12.-Lo~ation"~ of the Third Binding-site in Nitroanilines (X), Sugars (y), and Dipeptide Sweeteners ( 8 ) . [Distances given in pm.]
the simplest chiral-form when considered in two dimensions. Such a triangle, ABC, possessing three unequal sides, being one-sided in two dimensions, is not superposable upon its mirror image ACB. However, such a chiral specification is nonexistent in Euclidean space, because rotation in space leads to superposability and, therefore, equivalence. of model sugars and their derivatives established that the 3- and 4-hydroxyl groups constitute the AH,B unit proposed by Shallenberger and Acree,' the 4hydroxyl group being the principal proton-donor, and the oxygen atom of the 3-hydroxyl group, the principal proton-acceptor (see Section 11,3). This being the case, the methylene carbon atom (C-6) appears to be an unambiguous choice for a hydrophobic site. This is supported by the Lemieux effect,I2' where the -CH20H group of the aldopyranoses in a relatively (121) R. U. Lemieux and J . T. Brewer, in H. S. Isbell (Ed.), Carbohydrates in Solution, Adu. Chem. Ser., 117 (1973) 121-146.
236
CHEANG-KUAN LEE
(AH),
(AH),
p- D-Glucopyranose FIG. 13.-Superpositioning of
p- L-Glucopyranose
p-D-and 0-L-Glucopyranose over the Same Receptor Site.
nonpolar environment was shown to assume a position in space such that it may intramolecularly hydrogen-bond to the oxygen atom of the 4-hydroxyl group. It may thus be seen (in Fig. 13) that a D or L sugar could be equally positioned over the same receptor site, keeping in mind that the receptor is chiral in two dimensions, whereas the D and L sugars are chiral in three dimensions; the AH,B, y of D- and L-glucose are alternatively directed away from the receptor sites in one case, and towards them in the other. The situation is different for amino acids, as is shown in Fig. 14. The D-amino acid can be positioned over the receptor, whereas the L enantiomer cannot make a tripartite fit. Although the Shallenberger and Kier concepts are sufficient to explain the stereochemical fit at the receptor that is ultimately responsible for the sweetness, they suffersome obvious limitation^.^^ The major disappointment is the complete absence of any predictive value. Almost any organic comNH2 0
\/
0
Nyc/
\
k-Asparagine ( tastelea.)
FIG. 14.-Superpositioning of
p p a m g i n s (.met)
D-
and L-Asparagine over the Same Receptor Site.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
237
pound possessing an OH, NH, or CH bond (AH) and some center of electron density, such as an electronegative atom (for example, oxygen, nitrogen, and sulfur), or a region of unsaturation (B) and a hydrophobic region ( y ) , should be sweet, as it should possess an AH,B,y glucophore. Clearly, this is not the case, for there are numerous examples where even minor structural modifications in compounds having the prescribed glucophore often result in a drastic change in the taste. Furthermore, the assigned AH units of some compounds, for example, the nitroanilines and perillaldehyde oxime, are not capable of forming energetically significant hydrogen-bonds, let alone strong hydrogen-bonds. The Shallenberger and Kier concepts also make the assumption that all classes of sweet-tasting substances bind to a common receptor. The difficulty in locating a common, essential glucophore in many classes of sweeteners strongly suggests the participation of different recept01-s.~~ Electrophysiological results, also, have often shown that fibers of gustatory nerves respond to more than one type of chemical stimulus, although it has been suggested that each fiber generally responds best to a single-quality stimulus, and to others to only a lesser degree.122Furthermore, evidence from work on a also seemed to show the presence of strain of mutant Drosophilu different receptors for different sugars, result^'^^,'^^ strongly suggesting that there are two receptor sites for different groups of monosaccharides. These molecules bind either to different receptor proteins or to different regions of the same receptor Furthermore, too much weight is given to the assumption that compounds in a related series will be effecting stimulation through the same AH,B,y system, although this may-be true in most cases. Thus, if (in humans) different classes of sweeteners act by binding to different receptors, it may be meaningless to search for a “common essential glucophore.” The interpretation of the experimental data obtained by researchers in the field is much to be desired. There is great tendency to invoke the great escape-route termed steric hindrance whenever results are acquired that d o not quite fit any theory, and there is a great reluctance to admit that there are many data that do not fit the present theories. Furthermore, the shortage of accurate and appropriate data for most sensory work must cast doubt on some of the conclusions reached. There are very few quantitative data available from which to refine the arguments and hypotheses.
(122) (123) (124) (125)
M. Frank, J. Gen. Physiol., 61 (1973) 588-618. K. Isono and T. Kikuchi, Nature, 248 (1974) 243-244. I. Shimada, A. Shiraishi, H. Kijima, and H. Morita, J. Znsecr Physiol., 20 (1974) 605-613. T. Kikuchi and 1. Shimada, Kagaku No Ryoiki, 30 (1976) 510-517.
238
CHEANG-KUAN LEE
3. SweetnessStructure Relationship for Sugars The relative taste-intensities of carbohydrates are low, or, at most, moderate, compared to those of other classes of sweeteners. If the unidimensional, AH,B concept of Shallenberger and Acree’ is accepted, it is possible to rationalize the low sweetness-level of sugars, as the third dispersion bindingsite, proposed by Kier”’ and assigned to the C-6 methylene group in 1 7 ~ 1 1 8is capable of only weak, nonbonded interaction. pyranose However, sugars contain several glycol units, each conformationally favorable for sweetness induction. Through a series of methodical studies utilizing deoXy,~8,19~126 and other derivative^,^^*^^-^^.'^^-'^^ the identity of the AH,B units responsible for the sweetness of carbohydrates and their derivatives was identified. a. Simple Sugars.-When determining relative-sweetness values, sucrose is the reference compound usually employed, and its sweetness is arbitrarily taken to be unity, or is assigned a score of 100. However, even the taste of sucrose has been described as “mixed,” although sweetness is the dominant taste. NeumannI2’ reported that, near the threshold concentration, it tastes both sweet and bitter-sour, whereas at threshold concentrations, such tactual sensations as bitter, medicinal, chemical, sour, salty, lemon, and peppermint were described. 130*131 Whether or not these tastes emanated from the water used as solvent is open to question. Often, the sweetness of simple sugars is reported in the literature without any indication of anomeric specification, although the values recorded in some cases refer to an equilibrated solution. In other instances, they refer to some unstated, partially mutarotated solution. Even when an equilibrated solution is used, complications still arise because of the uncertainty of the effect of the concentration of an individual isomer at the receptor site. Furthermore, the taste and intensities of some of these isomers are not known. Consequently, Shallenberger and Acree2’*22 suggested the use of crystalline sugars, arguing that the anomeric form was known, so that the effective concentration of an individual sugar at the receptor site would be high, as the sugar identity would not be complicated by mutarotation reactions. This argument seems justified, as oral response
(126) (127) (128) (129) (130) (131)
G. G. Birch and C. K. Lee, J. Food Sci., 39 (1974) 947-949. M. G. Lindley and G. G. Birch, J. Sei Food Agric., 26 (1975) 117-124. G. G . Birch, C. K. Lee, and M. G. Lindley, Staerke, 27 (1975) 51-56. F. J. Neuman, Abstr. Doctoral Diss., The Ohio State University, 43 (1944) 115-121. S. D. Beck, Biol. Bull., 110 (1956) 219-228. C. P. Richter and K. H. Campbell, Am. J. Physiol., 128 (1939-40) 291-297.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
239
for sweet taste occurs132in -0.5 s, whereas electrophysiological studies'33 showed that the interval between initial response of the receptors and the report of a reaction is 0.02-0.06 s. However, such parameters as crystal size and lattice energy, which govern the rate of dissolution of the sugar in the saliva, may cause errors (owing to a concentration effect) by the taster's confusing the speed of stimulation with the intensity of taste.77The results reported in the literature for reducing sugars may thus be expected to be inconsistent. (i). Aldopyranose~.-Studies'~ showed that, in general, the relative sweetnesses of a-D-aldopyranoses are higher than those of the P-D-anomers. Tsuzuki and M ~ r i reported ' ~ ~ that the sweetness of P-D-glucopyranose is only 67% of that of a-D-glucopyranose. The higher intensity of sweetness of a-D-glucopyranose was likewise reported by Pangborn and Gee,135who also found that a-D-galactopyranose is sweeter than its /3 anomer. It is known that a-D-mannopyranose is sweet, whereas p-D-mannopyranose is bitter, with only a trace of ~ w e e t n e s s . ~These ' ~ ~ ~observations ~ ~ ~ ~ ' ~ suggest ~ involvement of the anomeric hydroxyl group in taste stimulation. Birch and coworkers,~3.~~8.~37-~39 however, proposed that the 1,2-glycol unit of sugars is not (even weakly) involved in the generation of sweetness. The configuration of the anomeric center was found to be important in inducing a bitter response. Both the a- and p-anomeric configurations can produce the bitter response, but the /3 configuration seemed much more likely to do The lower sweetness of @-D-glucosidescompared to the a anomers appears to be due to depression of sweetness by b i t t e r n e ~ s , ~ ~ and this may occur to the extent that the bitterness of an aglycon may completely eradicate the sweetness of the sugar residue (see Section 11,4,c). By using mixtures of sucrose and quinine sulfate, Birch and coworkersz3 were able to determine the depression of sweetness by bitterness, and vice versa. The effect of either additive on the other is to depress the taste intensity according to the expression
AT=klogC/C,,, where A T is the depression in the sweet or bitter taste, C is the concentration (132) F. Kiesow, in H. L. Hollingworth and A. T. Poffenberger (Eds.), The Sense of Taste, Moffat, Yard, New York, 1911. (133) C. Pfaffmann, J. Neurophysiol., 18 (1955) 429-440. (134) Y. Tsuzuki and K. Mori, Nature, 174 (1954) 458-459. (135) R. M. Panborn and S. C. Gee, Nature, 191 (1961) 810-811. (136) R. G. Steinhardt, A. D. Calvin, and E. A. Dodd, Science, 135 (1962) 367-368. (137) G. G. Birch and A. R. Mylvaganam, Nature, 260 (1976) 632-634. (138) G. G. Birch and M. G . Lindley, J. Food ScL, 38 (1973) 665-667. (139) G. G. Birch and C. K. Lee, J. Food Sci., 41 (1976) 1403-1407.
240
CHEANG-KUAN LEE TABLEIX “True” Sweetness of Methyl a- and f3-D-GlucopyranosideU
Molarity isosweet with M sucrose
Molarity isobitter with quinine sulfate
“True” molarity isosweet with M sucrose (after correction for bitterness)
4.35
-
4.35
5.95
5000
4.89
Methyl a-D-glucopyranoside Methyl p-D-glucopyranoside
of the additive producing the depression, C,,, is the maximum concentration having no effect on the other, and k is a constant for the class of compounds under test. By this method, assuming that k is constant for methyl glycosides, it was found that the true concentration of methyl P-D-glucopyranoside equivalent in sweetness to a 10.35% solution of methyl a-D-glucopyranoside is 11.62%. This is sufficiently small to be possibly due to experimental error (see Table IX), showing that there is no difference in the sweetness, and thus indicating that the anomeric center is not directly involved in the sweetness response. Support for this result was obtained from the taste of 1,5-anhydrohexitols, which, only for purposes of comparison, can be reparded as “l-deoxyaldopyranoses.” 1,5-Anhydro-~-glucitol(that is, the incorrectly named “1deoxy-D-glucopyranose”) (14), 1,5-anhydro-~-mannitol (“1-deoxy-Dmannopyranose” or “2-deoxy-~-fructopyranose”)(15), and 1,5-anhydro-~galactitol (“1-deoxy-D-galactopyranose”)(16) are all purely sweet, without any trace of Furthermore, the complete absence of bitterness of 1,5-anhydromannitol (16) clearly indicates that the anomeric CHZOH
HO OH 14
CHlOH H HO O
W
=
piHloH OH HO
15
Hh CHZOH
HO
OH
16
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
241
hydroxyl group is not essential for sweetness, but is probably necessary for a bitter taste response ‘ 267128p138(see Section 11,4,c). It might be anticipated that the primary hydroxyl group would be of great significance, because of the bulky nature of the 5-hydroxymethyl group and its steric projection out of the plane of the ring. However, like the 1-hydroxyl group, the 6-hydroxyl group does not seem to play any significant does not possess part in sweet-taste s t i m u l a t i ~ n . ’ ~ p’ ~D-Fructopyranose ~”~~ a free 6-hydroxyl group, and P-L-arabinopyranose is as sweet as CK-Dg a l a c t ~ p y r a n o s e .Similarly, ~~ the pentopyranoses (and their methyl glycopyranosides), L-rhamnose, and D-quinovose and its methyl CK-Dglycopyranoside (see Table X) all possess some sweetness. The cyclitols, for example, the inositols and quercitols, are also sweet78v80”40 (see Section 11,3,h). Also, neohesperidin dihydrochalcone (13) retains its sweet taste qualitatively and quantitatively when the 6-hydroxyl group of the Dglucopyranosyl residue is methylated,141‘143giving 17, although subsequent results indicated that the presence of the sugar portion is not a requirement for sweetness of this class of compounds.1u3145 CH,OR
H3qH
HO
13 17
R = H; neohesperidin dihydrochalcone R = CH3; 6“-0-Methylneohesperidin dihydrochalcone
Thus, it appears that the stereospecific part of the sugar ring, in terms of glycol arrangements, must reside77 within the hydroxyl groups on C-2, -3, and -4. G. G. Birch and M. G. Lindley, J. FoodSci., 38 (1973) 1179-1181. R. M. Horowitz and B. Gentili, J. Agric. Food Chem., 17 (1969) 696-700. R. M. Horowitz and B. Gentili, in Ref. 79, pp. 69-80. R. M. Horowitz and B. Gentili, in H. W. Schultz, R. F. Cain, and R. W. Wrolstad (Eds.), Symposium on Foods: Carbohydrates and Their Role, AVI, Westport, CT, 1969, pp. 253-268; in Ref. 2, pp. 182-193. (144) G. E. DuBois, G. A. Crosby, and P. Saffron, Science, 195 (1977) 397-399. (145) G. E. DuBois, G. A. Crosby, R. A. Stephenson, and R. E. Wingard, Jr., 1.Agric. Food Chem., 25 (1977) 763-771.
(140) (141) (142) (143)
TABLE X Taste Properties of Reducing Monosaccharides and Their GIycosided3 Free sugar Aldose a-D-Glucopyranose P-o-Glucopyranost a-D-Galactopyranose P- D-Galadopyranose a-D-Mannopyranose P- D-Mannopyranose P-D-Fructopyranose a-L-Sorbopyranose B-L-Lyxose 8-D-Arabinopyranose a-LArabinopyranose P-~Arabinopyranose~~ a-D-Xylopyranose P- D-Xylopyranose a-L-Rhamnose ~-~-Rharnnose~~ a-D-Quinovose a-D-Glucofuranose: P-D-Glucofuranose a
S = sweet. B =bitter.
“1-Deoxy”
Bb
S”
S
B
o o
s s
o o
S S
O
S S
O 0
S
B
B
o o o o
{s s s
o o
S
tr
0
BB =very bitter.
s
o
S
0
tr = trace.
S d
B
BB BB
0
B B
0
0
BB BB
0
9
B
O O
BBc BB
0 0
O t
r
S
S
S B
s s
S
B
B
S
o o B
O O
S
S
B
s s s s s s
S S
B
B
S
O B
hnyl glycoside
Butyl glycoside
Ethyl glycoside
S S
Pheoyl glycoside
Propyl glycoside
Methyl glycoside
B
0
BB BB
0
0
S S
O O
O t
O r
B r O
S S tr S S tr
B B tr B B 0
t
r
B
tr
0
S
0
t
tr
B
O
B
O
B
O
B
B
0
BB
0
BB
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
243
TABLEXI Taste of Methyl 4,6-0-Methylene-~-hexopyranosides'~~ Sugar a-gluco
p an~rner'~~ a-galacto
a-manno a-alrro
Sweetness'
Bitternessb
0.27 0.3 0.33 0.23 0.17
3.0 2.8 2.0 3.0 3.0
a Sweetness of 3% sucrose= 1.00. caffeine = 2.0.
Bitterness of 0.05%
Studies on the taste of some of the acetals of methyl a-D-ghcopyranoside and a,a-trehalose indicated that the 2,3-a-glycol arrangement in the gluco configuration cannot by itself produce sweetness. For example, 4,6,4',6'tetra-0-methyl-aptrehalose and some methyl 4,6-0-methylene-oh e x o p y r a n ~ s i d e s(see ' ~ ~ Table XI) are only faintly sweet, and methyl 4,6-diO-methyl-cy-~-glycopyranoside'~~ is completely devoid of sweet taste, clearly giving support to this proposal. Goodwin and reported that, in the cyclohexanediols, vicinal syn-clinal (gauche) hydroxyl groups are incapable of inducing sweetness; this has also been reported by Birch and L i n d l e ~ . ' ~Nevertheless, ' the acetals and methyl derivatives gave detectable sweetness (in the presence of intense bitterness) (see Table XI). Thus, it seems that the mere presence of two gauche-oriented hydroxyl groups in cyclic molecules does not induce a sweet taste, but, in the presence of a third electronegative, functional group (for example, the ring-oxygen atom), properly spaced (-350-550 pm), they may be able to produce ~ w e e t n e s s . ' ~ ~ The intense bitterness is probably the result of a high lipophilic character of the molecule (discussed in Section 11,4,c). Therefore, by elimination, 3- and 4-hydroxyl groups appear to be highly significant for sweetness. This conclusion was also reached by Evans'48 (working on the taste preference of the blowfly) and R. M. Horowitz and ~ ~ ~ ~ i l i l 4 1 (working -143 on the dihydrochalcone glycosides). Later studies on the taste of d e o ~ y ~ ~and , ' *methylated80*127 ~ sugars showed that it is, in fact, the 3-hydroxyl group that is of greatest importance in eliciting the sweet-taste response (see Section 11,3,b). (146) J. C. Goodwin and J. E. Hodge, Carbohydr. Rex, 28 (1973) 213-219; J. E. Hodge, J. C. Goodwin, and K. A. Warner; Abstr. Pap. Int. Symp. Carbohydr. Abstr. Chem., 6th, Madison, Wisconsin, 1972, p. 25. (147) J. C. Goodwin, J. E. Hodge, E. C. Nelson, and K.A. Warner, J. Agric. Food Chem., 29 (1981) 929-938. (148) D. R. Evans, in Ref. 13, pp. 165-176.
TABLEXI1 Comparison of Sweetness between Trehalose Derivatives and the Corresponding Monosaccharide Analogs by Paired Comparison ~
Total
Obs.
Calc.
No. correct in excess of expectancy in ( I values
20 20 40
45 50 47.5
50 50 50
0.447 0 0.316
No No No
% Correct
Test
Number correct
Number incorrect
Significant difference
a,a-Trehalose us. methyl a-D-glucopyranoside 1 2
Total
9 10 19
11 10 21
4,4-Dideoxy-a,a-trehalose us. methyl 4-deoxy-a-D-xylo-hexopyranoside 1
2
Total
10 10 20
6 6 12
16 16 32
62.5 62.5 62.5
50 50 50
1.ooo 1.000 1.414
No No No
24 24 48
54.1 58.3 56.3
50 50 50
0.408 0.816 0.866
No No No
a,a’-g&c?o-Trehalose us. methyl a-D-galactopyranoside 1 2
Total
13 14 21
11
10 21
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
245
TABLE XI11 Determination of Threshold by Triangular T e d M Threshold concentration ( m M )
Significance level (YO)
Methyl a-D-glucopyranoside a,a-Trehalose
42 43
0.2
Methyl 4-deoxy-a-~-xylo-hexopyranoside 4,4'-Dideoxy-a,a- trehaiose
61 59
0.2
Sugars
If the 3- and 4-hydroxyl groups constitute the Shallenberger AH,B unit, it is possible that a disaccharide (such as a,a-trehalose) and methyl (Y-Dglucopyranoside may possess the same molar sweetness. By a ranking procedure, and using trained panelists in a Paired Comparison Test, it was, indeed, found that isomolar solutions of these two sugars are equisweet, 149,150 indicating that only half of the trehalose molecule is actually involved in the interaction with the receptor site, the other D-glucosyl group being excluded, presumably for steric reasons. Tables XI1 and XI11 present some of the results for the sugars studied.149It is, however, uncertain, owing to insufficient information, whether there is a minimum size that a stimulus molecule must have in order to cause such steric hindrance to binding that not even one sugar residue would be able to bind to the receptor. Results obtained from studies on the effects of aglycons on the taste of g l y c o ~ i d e s ' ~ ~ and D-glucose s y r ~ p s ' ~seem ' to indicate that this size is large; this was supported by studies on aspartame (the L-aspartoyl dipeptide sweetener 119,120,152-156 18) and the 4-0-(carboxyalkyl)- (19) and 4-0(sulfoalky1)-dihydrochalcone(20) sweeteners. 1447145*157*158However, it is not G. G. Birch, N. D. Cowell, and D. Eyton, J. Food Technol., 5 (1970) 277-280. C. K. Lee and G. G. Birch, J. Sci. Food Agric., 26 (1975) 1513-1521. M. W. Kearsely, S. Z. Dzidzic, G. G. Birch, and P. D. Smith, Staerke, 32 (1980) 244-247. R. H.Mazur, J. M. Schlatter, and A. H. Goldkamp, J. Am. Chem. Soc., 91 (1969) 2684-2691. R. H. Mazur, J. A. Reuter, K. A. Swiatek, and J. M. Schlatter, J. Med. Chem., 16 (1973) 1284- 1289. Y. Ariyoshi, Agric. Biol. Chem., 40 (1976) 892-893. Y. Ariyoshi, Agric. Biol. Chem., 44 (1980) 943-945. Y. Ariyoshi, in J. C. Boudreau (Ed.), Food Taste Chemistry, ACS Symp. Ser., 115 (1979) 133-148. G. A. Crosby, G. E. DuBois, and R. E. Wingard, Jr., in C. M. Apt (Ed.), Flavour: Its Chemical, Behavioral and Commercial Aspecrs, Westview Special Studies in Science and Technology, 1977, pp. 51-66. (158) G. E. DuBois, G. A. Crosby, and P. Saffron, Synth. Commun., 7 (1977) 44-46.
246
CHEANG-KUAN LEE
I8 Aspartame
0~c(cH2).0
Me
CH2(CHz)z0 T O i N a + ( K + ) d o M e
HO’
HO
0 19
(when n = 3, the sweetness = 308 x that of sucrose)
HO
0
20 (sweetness = 496 x sucrose)
certain if the different classes of sweetener bind to the same receptor-sites. For the larger sweet compounds, it appears that a somewhat vague 3dimensional-receptor attribute was proposed. It seems that the AH,B, y sites do not lie on the membrane “surface,” but are contained in a “narrow” cleft. 1 5 9 ~ 1 6 0 Comparison of the taste of isomolar solutions of a number of monosaccharides and related, reducing disaccharides has also been reported.16’ There was no significant difference in the sweetness in those cases where the disaccharide has an a linkage (see Table XIV), possibly indicating that the (nonreducing) glycosyl end is involved in binding to the receptor protein; this, however, does not appear to be justified on the basis of the evidence. Nevertheless, if the ring hydroxyl groups constitute the AH,B system, any disturbance of this in the reducing sugar residue is most likely to preclude it from interacting with the “sweet” receptor-site. In comparing the sweetness of cellobiose and D-glucose and that of lactose and D-galactose, the (159) F. Lelj, T. Tancredi, P. A. Temussi, and C. Toniolo, J. Am. Chem. SOC.,98 (1976) 6669; in A. Loffet (Ed.), Proc. Eur. Pepride Symp., M h , Editions UnivenitC de Bruxelles, Brussels, 1979, pp. 585-590. (160) Y. Ariyoshi, Absrr. Pap. Am. Chem. Soc/Chem. SOC.Jpn., Joint Congr., Hawaii, April, 1979. (161) C. K. Lee, Food Chem., 2 (1977) 95-105.
TABLEXIV
Comparison of Sweetness between Reducing Disaccharides and Monosaccharides" by Paired Comparison T e d M on Sugar Solutions
Number incorrect
Total
Obs.
Calc.
No. correct in excess of expectancy in values
9 10 19
24 24 48
62.5 62.5 60.4
50 50 50
1.200 0.800 1.443
17 16 33
7 8 15
24 24 48
70.8 66.7 68.8
50 50 50
2.000 1.633 2.598
15 14 29
9 10 19
24 24 48
62.5 58.3 60.4
50 50 50
1.200 0.800 1.443
Cellobiose us. D-glUCOSe 1 16 2 16 Total 32
8 8 16
24 24 48
66.7 66.7 66.7
50 50 50
1.633 1.633 1.633
% Correct
Number correct
Test
Significant difference
~~
Melibiose us. D-galactose 1 15 2 14 Total 29 Lactose
0s.
D-galactose
1 2 Total
Maltose
US.
1 2 Total
a
No significant difference
Significant difference P 0.05
D-glUCOSe
Assuming that the monosaccharides are the sweeter.
No significant difference
Significant difference P 0.05
248
CHEANG-KUAN LEE
monosaccharides were judged to be sweeter in both cases. Furthermore, melibiose was found to be sweeter than lactose on an equimolar basis. Significantly, both cellobiose and lactose are p-linked disaccharides, suggesting that the lower sweetness response may be due to a tendency for the P-linkage to act on the “bitter” receptor-site. As has been discussed earlier, /3 anomers of sugars have difficulty in binding to sweet-receptors. For example, p-D-mannose appears to be very bitter, with only a trace of sweet taste.’36 Similarly, gentiobiose ( 6 - 0 - P - ~ glucopyranosyl-D-glucopyranose)has a fairly intense, bitter taste. Examples of other P-linked sugars being less sweet than the corresponding a anomers are seen in the erythritol and glycerol glycosides’61 (see Section 11,3,f). Evidence that this phenomenon is not unique to perception by humans comes from studies on the taste receptors of insect^.'^^-'^^ These have shown that, in sugar analogs, a linkages are more effective than /3 linkages in stimulating blowfly receptors. It was s u g g e ~ t e d ’ ~ *that *’~~ the /3 linkage probably hinders the molecule from binding to the receptor. (ii) Ketopyranoses.-In contrast to the D-aldopyranoses, which normally exist in the 4C, conformation, the D-ketopyranoses, like most Laldopyranoses, adopt the C4 conformation. D-Fructose is a prominent member of this group, and it is the sweetest of the naturally occurring sugars. It normally exists in the P-pyranose form (21), and 21 is the only
’
21
crystalline form that has been reported. Because its mutarotation consists mainly of a conversion to generate some of the P-furanose form, there is no reliable information regarding the sweetness of a-D-fructopyranose, although Pangborn and Gee135reported that it is the sweeter anomer of the sugar. This report was, however, probably a result of confusion regarding the anomeric configuration of the material being evaluated. The equilibrium (162) V. G. Dethier, The Physiorogy of Insect Senses, Methuen, London, 1963. (163) V. G. Dethier, in R. G . Grenell and L. J. Mullins (Eds.), Molecular Structure and
Functional Activity ofNerue Cells, American Institute of Biological Sciences, Arlington, VA, 1956, pp. 1-35. (164) V. G. Dethier and Y. M. Arab, J. Taiwan Pharm. Assoc., 2 (1958) 153-161. (165) W. W. Pflumm, in D. Schneider (Ed.), Olfaction and Taste, I y Wissenschafftliche Verlagsgesellschaft, Stuttgart, 1972, pp. 346-370. (165a) A. Allerhand, Pure Appl. Chem., 41 (1975) 255-259.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
249
composition of D-fructose in water is -2% of a-pyranose, 65% of ppyranose, 5% of a-furanose, and 23% of p - f ~ r a n o s e ,as ’ ~calculated ~~ from n.m.r.-spectral data. The taste of D-fructose has been widely studied, and numerous relativesweetness intensity-scores have been assigned to it. Shallenberger and Acree” reported that the “crystalline solid” is 1.8 times as sweet as sucrose. Verstraeten’66 claimed that it is 8 times as sweet, but this must have been a misinterpretation of a statement made by E. G. V. P e ~ c i v a I . ’ ~ ~ By application of first-order, kinetic equations, B. Anderson and DegnI6’ claimed that an equilibrated (25”) aqueous solution of D-fructose contains 31.56% of p-D-fructofuranose and 68.44% of P-D-fructopyranose. N.m.r. however, showed that, at equilibrium, a solution of D-fructose contains p-D-fructopyranose, p-D-fructofuranose, a-D-fructofuranose, and a trace of a-D-fructopyranose; the distribution of these isomers was shown by gas-liquid chr~matography”~ to be 76, 19.5, and 4%, respectively. Based on Anderson and Degn’s result,’68 Shallenberger’73 reasoned that, as 0.68 x 1.8 = 1.22 (which approximates the reported sweetness of mutarotated ~ - f r u c t o s e ~ the ~*~ furanose ~), form(s) must possess very little sweetness. When Shallenberger and coworkers6’ attempted to explain the sweetness of P-D-fructopyranose, they intuitively assigned the anomeric 2-hydroxyl group as AH and the oxygen atom of the 2-(hydroxymethyl) substituent as B. This assignment has since been supported by Lindley and It was shown that 1,5-anhydro-~-mannitol(15, “2-deoxy-~-fructopyranose”) and p-D-arabinopyranose (22) (in both of which, one of the AH or B units
@:
HO OH
22
is missing) are much less sweet than p-D-fructopyranose. Furthermore, methyl P-D-fructopyranoside, whose C-2 substituents cannot function as (166) L. M. J. Verstraeten, Adu. Carbohydr. Chem., 22 (1967) 229-305. (167) E. G. V. Percival, Structural Carbohydrate Chemistry, 2nd edn., Maclehose, Glasgow, 1962. (168) B. Anderson and H. Degn, Acta Chem. Scand., 16 (1962) 215-220. (169) D. Dodrell and A. Allerhand, J. Am. Chem. SOC.,93 (1971) 2779-2781. (170) A. S. Perlin, P. C. M. Hewe du Penhoat, and H. S. Isbell, Adu. Chem. Ser., 117 (1973) 39-50. (171) S. J. Angyal and G. S. Bethell, Aus?. 1. Chem., 29 (1976) 1249-1265. (172) Refs. 73 and 74. (173) R. S. Shallenberger, in Ref. 79, pp. 42-50.
250
CHEANG-KUAN LEE
the AH unit, is also considerably less sweet than P-D-fructopyranose. Additional support was found in the difference in the relative sweetnesses of P-D-fructopyranose and a-~-sorbopyranose’~~ (23).
The latter sugar is the C-5 epimer of P-D-fructopyranose and it is only half as sweet as P-D-fructopyranose, suggesting the participation of the 5-hydroxyl group in sweet-taste stimulation by these compounds. Inspection of molecular models shows that, in P-D-fructopyranose, the axial 2- and 5-hydroxyl groups are both sterically disposed to intramolecularly hydrogenbond with the ring-oxygen atom. In a-L-sorbopyranose, hydrogen bonding between the ring-oxygen atom and the equatorial, 5-hydroxyl group is not possible. Thus, Lindley and proposed that the efficiency of the anomeric 2-hydroxyl group and the 2-(hydroxymethyl) oxygen atom to function as the AH,B unit, and consequently, the intensity of the sweet taste, depends on the extent to which the axial 2-hydroxyl group is involved in hydrogen bonding with the ring-oxygen atom. Lindley and further proposed that the axial 5-hydroxyl group in P-D-fructopyranose forms the stronger hydrogen-bond, leaving the 2-hydroxyl group free to exert maximum interaction with the “sweet” receptor. If the argument is valid, any factor preventing the formation of intramolecular hydrogen-bonding between the 5-hydroxyl group and the ring-oxygen atom will cause a decrease in the sweetness of p-D-fructopyranose, as hydrogen bonding can then occur between the ring-oxygen atom and the anomeric hydroxyl group. Alternatively, any modification that is more effective in preventing hydrogen bonding between the 2-hydroxyl group and the ring-oxygen atom than the presence of an axial 5-hydroxyl group, will enhance the sweetness intensity. This proposal appears to be capable of explaining some of the previous findings. Tsuzuki and Y a m a ~ a k reported i~~ that the relative sweetness of P-D-fructopyranose decreases dramatically with increasing temperature; this had also been reported by Lee and coworker^.^' It was suggested76that the loss in sweetness is caused by a shift in the mutarotational equilibrium. However, the loss is too great to be accounted for by this shift, so that Lindley and attributed it to the weakening of the hydrogen bond between the ring-oxygen atom and the 5-hydroxyl group, resulting in a slight loss of freedom of the anomeric 2-hydroxyl group.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
25 1
Support for this postulate was obtained from infrared studies of these sugars. The i.r. spectrum of P-D-fructopyranose shows a well defined band in the free-hydroxyl stretching-region (at -3500 cm-'), whereas that of a-L-sorbopyranose shows no such absorption band (see Fig. 15). p-DArabinopyranose, which possesses the same trans-diaxial hydroxyl groups at C-2 and C-5 as P-D-fructopyranose, also has a sharp peak in the same region. Because arabinose does not have a hydroxymethyl group at the anomeric center to participate in the putative AH,B system, it does not have the sweetness of P-D-fructopyranose. Furthermore, the i.r. spectra of the methyl glycosides of the three sugars show peaks attributed to free hydroxyl
p-2-Arabinopyranose
d -L--Sorbopyranose -
p-PFructopyranose
3600
3400
3200
7000
Wavenumber (cm-')
FIG. lfi.-Hydroxyl
Absorption Bands in the Infrared Spectra of Three Free Sugars.'*'
252
CHEANG-KUAN LEE
absorption, indicating that the free hydroxyl bands at -3500 cm-’ in the spectra of the reducing sugars must be due to the axial anomeric groups. Therefore, the anomeric hydroxyl group and the hydroxymethyl oxygen atom of P-D-fructopyranose probably constitute the AH,B system, and the lower sweetness intensity of cY-L-sorbopyranose may be due to the 2hydroxyl group’s participation in hydrogen bonding with the ring oxygen atom. A report174on the taste properties of 6-thio-P-~-fructopyranose also supports the hypothesis. The thio analog possesses a sulfur atom in the sugar ring instead of an oxygen atom, and this substituent makes the 2-hydroxyl group unable to participate in intramolecular hydrogen-bonding owing to the inability of the ring-sulfur atom to act as a proton acceptor, but it is free to interact fully with the “sweet” receptor as the proton donor. Lindley and found that 6-thio-P-~-fructopyranoseis, indeed, sweeter than P-D-fructopyranose. The taste of 5-deoxy-~-threo-2-hexulose (“5-deoxyfructose” or “5-deoxysorbose”), reported by Martin and coworkers175has cast some doubt on Lindley and Birch’s proposed mechanism of a proton-releasing effect on the 2-hydroxyl group by competitive hydrogen-bonding of the 5-hydroxyl group with the ring-oxygen atom.’27The 5-deoxy sugar was reported to be much sweeter than L-sorbose, and nearly as sweet as D-fructose. Martin and coworkers argued175that the large difference in the sweetness between D-fructose and L-sorbose cannot arise only from a different degree of hydrogen bonding between the 2-hydroxyl group and the ring-oxygen atom. They suggested that “the equatorial 5-hydroxyl group exerts some detrimental influence on the saporous unit, possibly of a steric nature, which could preclude a correct alignment of the sugar on the taste receptor.” Although it is difficult to comprehend how this can occur with such a small group as a hydroxyl group, there does not seem to be an alternative explanation at present. An interesting compound having a structure analogous to that of Dfructopyranose is 1,3-dihydroxy-2-propanone(“dihydroxyacetone,” 24); HO
HoH2c+ CHzOH
0
OH
24
(174) M. G. Lindley, R. S. Shallenberger, and R. L. Whistler, J. FoodSci., 41 (1976) 575-577. (175) 0. R. Martin, S.-L. Korppi-Tomola, and W. A. Szarek, Can. J. Chem., 60 (1982) 1857- 1862.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
253
this, like glyceraldehyde and glycolaldehyde, exists as a crystalline dime,.. 176,177 In this form, it possesses two AH,B units of the type found in P-D-fructopyranose. However, it is only slightly Birch” suggested that this is presumably because, being symmetrical, both “anomeric” hydroxyl groups must have the same capacity for hydrogen bonding to ring-oxygen atoms, but this explanation is now in doubt, based on the results of Martin and c o ~ o r k e r s . ” However, ~ there are two lipophilic, methylene groups (in the 1 and 4 positions), and these, like the two AH,B systems, probably promote the occurrence of bilateral interaction, thereby depressing the frequency of sweetness-generating, unilateral interactions. Glyceraldehyde in the dimeric form also has a sweet taste,179but the monomer is tasteless.”’ It is probable that the two hydroxyl groups form a weak AH,B unit in a manner similar to the behavior of some of the deoxy sugars (see Section 11,3,b). The taste properties of various other ketoses, including 2-pentuloses, 2-hexuloses, and 2-octuloses, have also been studied’78 (see Table XV) to determine the significance of the 2-hydroxyl-2-hydroxymethyl arrangement of 2-ketopyranoses. Some of these, for example, a-D-tagatose (25), (Y-Dgluco-2-heptulose (26), a-~-manno-2-heptulose (27), a-~-talo-2-heptulose (28), a-D-altro-2 - heptulose (29), and D-glycero-a-~-gluco-2 -octulose (M), HO-
H
HO
CHiOH O
HO
CH2OH
W
H
CHZOH : W
CHZOH
Ho OH
CHZOH
OH
25
OH 27
26
HOCH2 \CAH HO‘ HOCH~OH CHZOH
HO
OH 28
H
O
mCH2OH
HO
OH 29
Hob CHZOH
HO
HoOH
30
unlike D-fructopyranose, have the 4C,( D) or ’C2( D) conformation. All of the 2-ketoses tested were consistently sweeter than, or of about the same (176) (177) (178) (179) (180)
H. 0. L. Fischer and M. Milbrand, Ber., 57 (1924) 707-712. B. Arrequin Lazano and J. Taboada, J. Chromatogr. Sci., 8 (1970) 187-191. C. K. Lee and G. G. Birch, J. Pharm. Sci., 65 (1976) 1222-1227. H. J. H. Fenton and H. Jackson, J. Chem. Soc., 75 (1899) 575-581. The Merck Index 8th edn., Merck, Rahway, NJ, 1968.
CHEANG-KUAN LEE
254
TABLEXV Taste Properties of Ketose~"~
Sugars
Sweetness"
Bitterness
S S
0 0 0
PI
ro1
2-Hexuloses L-Sorbopyranose D- Fructopyranose D-Tagatose [ 1,s-Anhydro-mannitol ("2-deoxy-~-fructopyranose")] 2-Heptuloses D-gluco-Heptulose D-manno-Heptulose D-lalo-Heptulose D-altro-Heptulose (sedoheptulose) Sedoheptulosan L-gluco- Heptulose L-galaeto-Heptulose L-allo-Heptulose 1-Deoxy-D-manno-heptulose D-altro-Heptulose 7-Deoxy-~-altro-heptulose 7-Deoxy-~-galactoheptulose
ss
S S S
ss ss S
S S S
0-tr S
2-Octuloses D-glycero-L-gluco-Octulose D-glycero-L-gulo-Octulose Disaccharides Maltulose Lactulose Palatinose Turanose a
S
0 B B
S S
0 0
S S S
0 0 0 0
ss
S = sweet, SS =very sweet, tr = trace of sweetness. * B =bitter.
sweetness as, the corresponding aldoses and none were bitter."* Whether or not the 1,2-glycol unit constitutes the AH,B system probably depends on the overall conformation of the sugar. Possibly, like P-D-fructopyranose, the 1,2-glycolunit does function as the glucophore in those molecules having the C,(L)conformation, such as ru-~-gluco-2-heptulose(31),a-~-gluc0-2heptulose (31), a-~-gaZucto-2-heptulose(32), cu-~-allo-2-heptulose(33), OH H HO O H e i H 2 0 H OH 31
OH HOH2C@zHzOH HO OH
32
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
33
255
34
and ~-glycero-a-~-gluco-2-octulose (34).This is probably not true for those having the 'C2 conformation, because, for example, l-deoxy-~-rnanno-2heptulose is not significantly different in sweetness from ~-rnanno-2hept~lose.'~'Birch" thus suggested that inversion of the sugar ring to the 2 C, conformation appears to cause a corresponding "inversion of polarization" of the molecule on the taste receptor and, in turn, a correspondingly greater sweetness. Direct evidence for this concept has yet to be obtained. The sweetness of sedoheptulosan (35) is, however, puzzling, as it has no anomeric hydroxyl group present to function as a proton donor. HZC&;"?OH HO O H
35
Taste studies of reducing disaccharides containing a reducing ketose residue have also been c ~ n d u c t e d ' ~(see ' Table XV). In contrast to the aldoses,l5' results obtained with palatinose (36), maltulose (37), lactulose
HO HO 36
-0
37
(38), and turanose (39) implicated the involvement of the anomeric hydroxyl group and the 1-hydroxyl group as the AH,B system. These sugars were all found to be considerably sweeter than their corresponding aldose analogs. It is particularly significant that turanose (3-O-cr-~-glucopyranosyl-~-fructopyranose) (39) is nearly as sweet as sucrose. Apart from indicating the importance of the 1,2-glycol grouping, as in D-fructopyranose, the 3hydroxyl group of the D-fructopyranose ring does not seem to be critical
256
HoBvoH H=q CHEANG-KUAN LEE
CHzOH
HOCHZ 0
HO
HO
CHZOH
HOh 9 - O
0
39
38
for the sweetness of D-fructose, in accord with the explanation for the sweetness of D-fructose already discussed. (iii) Furanoses and Furanoid Compounds.-Little systematic work has been conducted on the sweetness-structure relationships of furanoses. Generally, furanoid rings of sugars are considered to be less stable than pyranoid rings. In a (hypothetical) planar, furanoid ring, vicinal hydroxyl substituents are in the eclipsed orientation when they are cis, and describe a dihedral angle of 120" when they are trans.'" It is well recognized that the furanose ring is not planar, but exists in either a twist or an envelope conformation182 (see Fig. 16). The vicinal hydroxyl groups may approach either the eclipsed or the anti orientations. As has already been discussed, eclipsed, and anti, vicinal hydroxyl groups are not expected to elicit the sweet taste. Thus, Shallenberger'73 suggested that P-D-fructofuranose would be expected to have very little sweetness. The taste of this sugar has yet to be confirmed, but, significantly, Lindley is practically devoid and coworkers174found that 5-thio-P-~-fructofuranose of sweet taste. However, methyl P-D-glucofuranoside (40) and some of the
40
Twist
Envelope
FIG. I6.-Two Conformations of a Furanoid Ring. (181) J. W. Green, Ado. Carbohydr. Chem., 21 (1966) 95-142. (182) L. D. Hall, Chem. Ind. (London),(1963) 950; Ado. Carbohydr. Chem., 19 (1964) 51-93.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
257
1,4-anhydroalditols, for example, 1,4-anhydro-~-glucitol(41), 1P-anhydro(U), D-mannitol (42), 1,4-anhydro-~-xylitol(43)and 1,4-anhydro-~-ribitol
Ho'c
HOCHZ H
HOCH2 H
.
O
\ I
Y 41 Y
o
H
42
HocwoH Ho HO
43
44
are all sweet, and without a trace of b i t t e r n e ~ s . ~ Some ~ * ~ ~of* these ' ~ ~ compounds have vicinal hydroxyl groups that could function as AH,B units, but others do not. However, in all cases, the combination of a hydroxyl group and the ring-oxygen atom satisfies the distance condition of the Shallenberger AH,B requirement. Therefore, it is probable that the ringoxygen atom in these compounds is involved in the interaction with the of methyl 3,6taste receptor. This is supported by the sweet anhydro-a-D-glucofuranoside(45), 3,6-anhydro-a-~-glucofuranosyl 3,6anhydro-a-~-glucofuranoside~~*~~~ (46), 1,4 :3,6-dianhydro-~-glucitol~~~ (47),and 1,4 :3,6-dianhydro-~-mannito1'8~ (48).These structures do not
6H R=OMe 46 R = 0-repeating unit 41 R = H 45
48
(183) C. K. Lee and G. G. Birch, J. Food Sci., 40 (1975) 784-787. (184) G. G. Birch, C. K. Lee, and A. C. Richardson, Curbohydr. Res., 19 (1971) 119-121. (184a) R. K. Dinda, 1. T. Beck, W. A. Szarek, G. W. Hay, E. R. Ison, and D. Vayas, Can. 1 Physiol. Phurm.,60 (1982) 652-654. (184b) M. L. Wolfrom and A. Thompson, J. Am. Chem. SOC.,68 (1946) 791-793. (184c) W. C. Austin and F. L. Humoiler, 1. Am. Chem. SOC.,56 (1934) 1153. (184d) R. S . Shallenberger, Food Chem., 12 (1983) 89-107. (185) T. Y. Shen, Methods Curbohydr. Chem., 2 (1963) 191-192.
CHEANG-KUAN LEE
258
contain any a-glycol grouping having the required oxygen-oxygen, interorbital distance of 280-400 pm. The 5-hydroxyl group is, however, situated at a distance of 260-280 pm from the ring-oxygen atom (see Section 11,3,e). (iv) L Sugars.-Shallenberger and coworkers48suggested that the sweetness of the enantiomeric amino acids was caused by the presence of a spatial barrier behind the receptor AH,B unit, so the two-space geometry of the non-sweet isomer simply could not be placed over the receptor site. There is, however, very little difference in the sweetness of the enantiomeric sugars. Using the magnitude scaling procedure, Shallenberger and associates48 showed that the panelists were unable to distinguish the sweetness of seven pairs of enantiomeric sugars (see Table XVa). Unfortunately, other similar studies on differential sweetness of enantiomeric sugars are lacking in the literature, although Dinda and reported that the sweetness of both the D and L form of sucrose is qualitatively indistinguishable. Other, even less quantative, statements on the taste of L sugars have been reported. Wolfrom and Thompson'84b stated that L-fructose is "very sweet," and Austin and Hum01ler,'*~~ that L-allose and L-altrose are sweet. The reason why there is no difference in sweetness of enantiomeric sugars, and not in that of amino acids, was suggested by Shallenberger"6."7,'84d as being due to the fact that the glucophores of sugars are doubly chiral, with AH,B situated on separate carbon atoms; those of amino acids, on the other hand, are singly chiral, with AH,B located upon a single chiral center. Therefore, in spite of the spatial barrier, any vicinal pair of hydroxyl groups acting as AH,B could be positioned over an AH,B receptor site. Even with the inclusion of the third, y function, the primary AH,B and also the y function of sugar enantiomers can be equally positioned over a tripartite, but diastereomeric, receptor site. The sweet glucophores of the sugar enanTABLEXVa
Relative Sweetness of Sugars Arabinose Xylose Glucose Rhamnose Mannose Galactose gluco- Heptulose Fructose Ref. 22.
Ref. 184b.
D
uersus L Sugars
D-Form
L-Form
5.2 4.6 5.4 4.6 4.9 5.6 5.2 1.O- 1.8"
5.6 4.4 6.0 6.5 5.0 6.0 6.6
very sweetb
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
259
tiomers are two-space and three-space homotopic, whereas those of the amino acids are two-space configurational diasterotopic and three-space conformationally d i a s t e r e ~ t o p i c . ’As ~ ~shown ~ in Fig. 14, AH,B and y of D-glucose is directly superpositioned upon an AH,B and y receptor site, and, in the case of L-glucose, this occurs merely by inverting the sugar molecule.
b. Deoxy Sugars.-Assuming that such a system of binding as was postulated by Shallenberger and Acree’ is operative in sugars, there are a number of different a-glycol groupings, each satisfying the structural requirements of the Shallenberger AH,B system. The most direct method of verifying Shallenberger’s hypothesis and locating the AH,B system is systematically to eliminate an oxygen atom at different carbon atoms in the sugar ring, and assess the effect on taste. By such a process, it might be possible to eliminate superfluous hydroxyl groups and identify the saporous function(s). A series of mono- and di-deoxy derivatives of methyl a-D-glycopyrano86,187 and a,a - t r e h a l ~ s e were ~ ~ ” synthesized ~~ and tested. As expected, the taste of each dideoxy-a,a-trehalose derivative was found to be only marginally different from that of the corresponding methyl a-Dglucopyranoside derivative (see Table XVI). The taste of these monodeoxy derivatives was either sweet or trace sweet, or, in some cases, trace bitter. The dideoxy derivatives of the methyl glycoside and the corresponding tetradeoxy-a,a-trehalose analogs, on the other hand, were never sweet, and always bitter.126If the Shallenberger AH,B system can be identified with a particular a-glycol grouping within these hexopyranosyl units, then at least some of the dideoxy derivatives should exhibit some sweetness. For example, methyl 2-deoxy-a-~-arabino-hexopyranoside, methyl 4-deoxy-a-~-xylohexopyranoside, and the corresponding tetradeoxy-a,a-trehalose analogs are all sweet (see Table XVI). Therefore, by inference, methyl 2,6-dideoxy-aD-arabino-hexopyranoside, methyl 4,6-dideoxy-a-~-xylo-hexopyranoside, and the corresponding a,a-trehalose analogs should have some sweetness, as no configurational change has occurred at the hydroxylated carbon atoms in the deoxy derivatives. It was suggestedlZ6that this lack of sweetness might be attributable to the dideoxyhexopyranosyl molecule’s aligning itself differently from the parent sugar on the taste receptor owing to the newly created hydrophobic sites (-CHz- groups). This was also observed in the cyclohexanepolyols’40 and the methylated sugar^.^^*'^^ It is now generally (186) M. Haga, M. Chonan, and S. Tejima, Carbohydr. Res., 16 (1971) 486-491. (187) B. T. Lawton, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 14 (1970) 255-258; 15 (1970) 397-402. (188) G. G. Birch, C. K. Lee, and A. C. Richardson, Carbohydr. R e x , 36 (1974) 97-109.
260
CHEANG-KUAN LEE
TABLEXVI Taste Properties of Deoxyglycosides’” Glycoside Methyl 2-deoxy-a-~-arabino-hexopyranoside Methyl 2-deoxy-a-~-ribo-hexopyranoside Methyl 3-deoxy-a-~-arabino-hexopyranoside Methyl 3-deoxy-a-D- ri60-hexopyranoside Methyl 4-deoxy-a- D-xylo-hexopyranoside Methyl 6-deoxy-a- D-gluco-hexopyranoside Methyl 2,6-dideoxy-a-~-arabino-hexopyranoside Methyl 2,6-dideoxy-a-~-ribo-hexopyranoside Methyl 3,6-dideoxy-a-~-arabino-hexopyranoside Methyl 4,6-dideoxy-a-~-xy/o-hexopyranoside 2-Deoxy-a-~-ribohexopyranosyl 2-deoxy-a-~-ribohexopyranosided 3-Deoxy-a-~-arabino-hexopyranosyl 3-deoxy-a-~-arabinohexopyranosided 3-Deoxy-a-~-ribo-hexopyranosyl 3-deoxy-a-~-ribohexopyranosided 4-Deoxy-a-~-xy/o-hexopyranosyl 4-deoxy-a-~-xy/ohexopyranosided 2,6-Dideoxy-a-~-xy/o-hexopyranosyl 2,6-dideoxy-a-~-xy/ohexopyranosided 2,6-Dideoxy-a-~-ribo-hexopyranosyl 2,6-dideoxy-a-~-ribohexopyranosided 3,6-Dideoxy-a-~-arabinohexopyranosyl 3,6-dideoxy-a-~arabino-hex~pyranoside~ 4,6-Dideoxy-a-~-xylo-hexopyranosyl 4,6-dideoxy-a-~-xy/ohexopyranosided 2,3-Dideoxy-a-~-erylhro-hexopyranosyl 2,3-dideoxy-a-~eryrhro-hexopyranosided
Sweetness’
Bitternessb
S S tr tr S S 0 0 0 0
tr tr 0‘ 0 0 tr
BB BB
B B
S
tr
tr
0
tr
0
S
0
S
tr
0
BB
0
B
0
B
0
B
a S =sweet, tr = trace sweet, 0 = no sweet taste. B = bitter, BB = very bitter, tr = trace bitter, 0 = n o bitter taste. Some panelists reported trace bitterness. (1 -P 1)-Linked disaccharide.
accepted57that the presence of two hydrophobic centers drastically disturbs the orientation pattern of the molecules, thereby preventing interactions between “sweet” AH,B unit(s) of the molecules and the “sweet” receptor site(s) and favoring other orientations which result in a collection of “bitter” interactions. It has been suggested that the 4-hydroxyl group is uniquely important in eliciting the sweet-taste response.77The taste of the monodeoxy sugars (see Table XVI), however, showed a marked decrease in sweetness when the 3-hydroxyl group was eliminated, thus suggesting the immense importance of the 3-hydroxyl group in the sweetness of the parent sugar. It is, however,
26 1
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
TABLEXVII Possible AH,B Unit in Deoxybexopyrsnosides That May Be Involved in Sweet-taste ~ t i m u ~ a t i o n ~ ~ Taste Compound 2-Deoxy 3-Deoxy 4-Deoxy 6-Deoxy
AH,B System
Sweet
Bitter
S
tr 0 0 tr
tr
S S
3e,4e
2eJe
la,2e
OrJa
Or,6e
+ +
+ + +
+ + + +
+ + +
+ +
not clear whether it functions as the AH or the B moiety. The importance of the 3-hydroxyl group was also reported by Goodwin and c o ~ o r k e r s . ' ~ ~ This parallels the results obtained by BarnettIE9for the binding of sugars to carrier protein during intestinal transport. The sweetness and complete lack of bitterness of the 1,5-anhydroalditols has already been mentioned (see Section II,3,a and Table X). The 6-hydroxyl group has been shown to play no part in sweet-taste stimulation (see Section 11,3,a,i), and the sweet taste of the 6-deoxy derivatives (see Table XVI) clearly confirms this. Consequently, the trace sweetness of the 3-deoxy analogs (see Table XVI) is likely to be due to the la,2e-hydroxyl groups,52 implying that this a-glycol grouping must constitute a sweet AH,B unit of low effectiveness. Furthermore, as both 3 e,4e hydroxyl groups (the 2-deoxya-D-hexopyranosides) and the combination of 2e,3e and 1a,2e hydroxyl can produce considerable groups (the 4-deoxy-a-~-hexopyranosides) sweetness (see Table XVII), the order of effectiveness of sweetness stimulation must follow the sequence 3e,4e > 2e,3e > la,2e. c. Methyl Ethers of Sugars.-An alternative method of locating the Shallenberger AH,B unit is by selectively substituting the hydrogen atom of a hydroxyl group with a suitable blocking-group. Such a procedure is not the most ideal, as these groups may confer a taste of their own which would thus complicate the taste assessment. The increase in the size of the substituent or the number of substituents also often causes a decrease in the solubility of the product, eventually resulting in tastelessness. The drop in sweetness of alkyl 2-amino-4-nitrophenyl ethers is the result of this, and compounds higher in the series than the butoxy derivative could not be studied, owing to their in~olubility.~ Therefore, Lindley and B i r ~ h * ~ used *~*' (189) J. E. G. Barnett, in G. G. Birch and L. F. Green (Eds.), Molecular Strucrpre and Functions of Food Carbohydrate, Applied Science, London, 1973, pp. 216-234.
262
CHEANG-KUAN LEE
TABLEXVIII Taste Properties of Methyl Ethers of Glycosides'*' Glycoside Methyl a-D-glucopyranoside 2-0-methyl3-0-methyl4-0-methyl6-0-methyl2.3-di-0-methyl3,4-di-O-methyl4,6-di-O-methyl-
Sweetness"
Bitternessb
S S S S 0 0 0
tr tr tr tr
qa-Trehalose 2,2'-di-O-methyl3,3'-di-O-methyI4,4'-di-O-methyI6,6'-di-O-methyl2,3,2',3'-tetra-O-methyl4,6,4',6'-tetra-O-methyISucrose'92 4-0-methyl6'-0-methyl6,6'-di-O-methyl4,6'-di- 0-methyl4.6-di-0-methyl1',6'-di-O-methyl-
B B B tr tr tr tr B B
S
OC
S
0 tr tr tr tr
ss ss Sd Sd
a S = sweet, SS =very sweet, tr = trace sweet, 0 = no sweetness. B =bitter, tr = trace bitter, 0 = n o bitterness. 70% agreement among panelists. 80% agreement among panelists. All other values: 90-100°/~ agreement among panelists.
the smallest of the alkyl groups, forming the methyl ethers. The taste properties127of the methyl derivatives of methyl a-D-glucopyranoside and a , c ~ - t r e h a l o s eagain * ~ ~ ~emphasized ~~ the almost complete analogy between the two sets of compounds as regards their taste (see Table XVIII). All of the mono-0-methylated hexopyranosides are sweet, with only a trace of bitterness. The bitterness is more pronounced in the methyl glycoside derivatives, and this was attributed to the presence of two lipophilic centers (-OMe) in the methyl glycosides and only one in the a,a-trehalose ana10gs.l~~ The sweet taste of the 3-0-methyl derivatives seems to conflict with the results obtained with deoxy sugars. However, the sweet taste of these (190) C. K. Lee and M. G. Lindley, Carbohydr. Res., 63 (1978) 277-282.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
263
derivatives was rationalized as being due to the 0 - 3 atom’s acting as the proton-acceptor function, B, in the Shallenberger AH,B unit, with either the 2- or the 4-hydroxyl group acting as AH, the 4-hydroxyl group being preferred for reasons discussed earlier. The taste properties of the di- 0-methylhexopyranosyl derivatives, like those of the corresponding deoxy sugars, are never sweet, and always bitter. As with the deoxy sugars, this is possibly the result of increased lipophilicity of the molecule. In sucrose, however, the presence of two methyl groups on the D-glucopyranosyl or the D-fructofuranosyl group does not seem to cause any marked bitterness (see Table XVIII).191*’92 The taste of mono- 0-methylsucrose derivativeslg1 again substantiates some of the conclusions already put forward. 4-0-Methylsucrose (49) was found to be considerably less sweet than sucrose. This, together with the extremely low sweetness of “galacto-sucrose” (50) (which has an axial M
e
Q,
O
Hoq
q
HOCH~OH
HO
HO
HOCHz 0
HO
0
H
O
C
CHZOH H0
U CH20H
HO 49
50
4-hydroxyl group in the hexopyranosyl moiety) indicates the considerable importance of the 4-hydroxyl group of the D-glucopyranosyl group with regard to sweetness. However, as sucrose is much sweeter than either methyl a-D-glucopyranoside or a,a-trehalose, the interaction of the Dglucopyranosyl group with the taste receptor cannot be solely responsible for the sweetness of sucrose. The crystal and molecular structure of sucrose has been studied by Brown and Levy.’93Neutron-diff raction studies showed that the 4-hydroxyl group is not involved in any hydrogen bonding. The 1’-hydroxyl group is, however, intramolecularly hydrogen-bonded to the 0 - 2 atom, so that the two glycosyl groups in sucrose are held together in a fixed inclination in the approach to the receptor site.’93 This is analogous to Shallenberger and Acree’s6’ rationalization of the higher sweetness-intensity of D-glucopyranose com(191) M. G . Lindley, G. G . Birch, and R. Khan, J. Sci. Food Agric., 27 (1976) 140-144. (192) M. G. Lindley, G. G. Birch, and R. Khan, Carbohydr. Rex, 43 (1975) 360-365. (193) G. M. Brown and H. M. Levy, Science, 19 (1963) 921-923.
CHEANG-KUAN LEE
264
pared to D-galactopyranose, where the 6-hydroxyl group of the former is sterically disposed to bond to the 4-hydroxyl group, particularly in a hydrophobic environment (the Lemieux eff ectI2'), so that the 4-hydroxyl group is now rigidly fixed, and, at the same time, the acidity of the 4-hydroxyl proton is enhanced. In D-galactose, such a bonding accentuates the ability of the 4-hydroxyl proton to bond to the ring-oxygen atom, thereby limiting its ability to serve as AH. However, it is not certain which hydroxyl groups of sucrose act as the AH,B system. 6'-0-Methyls~crose'~~ (51) was found to have a sweetness close to that of sucrose, indicating that the 6'-hydroxyl group plays little or no part in the overall sweetness of su~rose.'~'Similarly, the 6-hydroxyl group does not appear to be important either, as 6,6'-di-O-methylsucrose (52) is also very sweet. The 1'-hydroxyl group, on the other hand, is H
:
-
H
CH20Me Z q
HO
HO
HO
52
51
important, as shown by the lowered sweetness of 1',6'-di-0-methylsucrose (53). This hydroxyl group probably acts as a hydrogen donor in its interacCHzOH
HO 53
tion with the receptor, so that substitution of the 1'-hydroxyl group makes the substituent no longer capable of functioning as a hydrogen donor. Therefore, the intramolecular hydrogen-bond between the 1'-hydroxyl hydrogen atom and the 2-hydroxyl oxygen atom that is seen in sucrose no longer exists. Consequently, the molecule will not be able to approach the
CHEMISTRY A N D BIOCHEMISTRY O F SWEETNESS
265
receptor site in a fixed geometry, as it does in sucrose. Therefore, it appears that both of the sugar rings of sucrose are involved in the interaction with the receptor site, and this is again evident for the chlorodeoxysucrose derivatives (see Section 11,3,d). d. Deoxyha1osucroses.-Although chemical modification of simple sugars always results in products that are either sweet, bitter-sweet, or bitter,20.77.78.80.128 no sugar derivative had shown a sweetness intensity substantially greater than those of the parent sugars until, in 1976, Hough and P h a d n i ~ reported '~~ the synthesis of 4,6-dichloro-4,6-dideoxy-a-~-galactopyranosyl 1,6-dichloro-l,6-dideoxy-~D-fructofuranoside (54) and found CI
HO 54
it to possess a sweetness several hundred times that of sucrose. Since then, various other chlorinated derivatives of sucrose have been ~ y n t h e s i z e d , ' ~ ~ " ~ ~ and these have sweetnesses comparable to, or several orders of magnitude greater than, that of saccharin, but without the unpleasant after-taste associated with saccharin'97 or neohesperidin dihydrochalconeL44.157.198*'99 (see Table XIX). It is puzzling that the chlorine and other halogen substituents are not known to enhance the sweetness of other sugars, such as methyl a-Dglycopyranosides, a,a-trehalose, maltose, or lactose.200On the contrary, all of the deoxyhalo sugar derivatives tasted7' were extremely bitter. The high sweetness of the deoxyhalosucroses is clearly inexplicable in terms of either (194) L. Hough and S. P. Phadnis, Nature, 263 (1976) 800-801. (195) L. Hough and R. Khan, Trends Biol. Sci., 3 (1978) 61-63. (196) M. R. Jenner, in C. K. Lee (Ed.), Developments in Food Carbohydrates, 2, Applied Science, London, 1981, pp. 91-143. ( 197) K. M . Beck, in T. E. Furia (Ed.), CRC Handbook of Food Additives, 2nd edn., CRC Press, Boca Raton, FL, Vol. 2, 1980, pp. 125-185. (198) G. A. Crosby and T. E. Furia, in Ref. 197, pp. 187-227. (199) G . A. Crosby and R. E. Wingard, Jr., in C. A. M. Hough, K. J. Parker, and A. J. Vlitos (Eds.), Deuelopmenrs in Sweeteners, 1, Applied Science, London, 1979, pp. 135-164. (200) C. K . Lee, Developments in Food Carbohydrates, 2, Applied Science, London, 1981.
CHEANG-KUANLEE
266
TABLEXIX Relative Sweetness of Deoxyhalo Derivatives of S u ~ r o s e ' ~ ~ * ~ ~ Sugar
Sucrose 1'-Chloro1'-deoxysucrose 4-Chloro-4-deoxy-galacto-sucrose 6-Chloro-6-deox ysucrose
6'-Chloro-6'-deoxysucrose
4,1'-Dichloro-4,1 '-dideoxy-galacro-sucrose 1',4'DichIoro-l',4'-dideoxysucrose 1',6'-Dichloro-1',6'-dideoxysucrose 6,6'-Dichloro-6,6'-dideoxysucrose 4,1',6'-Trichloro-4,1',6'-trideoxy-ga/acto-sucrose 4,1',4'-Trichloro-4,1',4'-trideoxy-ga/acto-sucrose 4,4',6'-Trichloro-4,4',6'-trideoxy-galacto-sucrose 1',4',6'-Trichloro-t',4',6'-trideoxysucrose 4,6,1',6'-Tetrachloro-4,6,1'6'-tetradeoxysucrose 4,6,1',6'-Tetrachloro-4,6,1',6'-tetradeoxy-galacto-sucrose 4,1',4',6'-Tetrachloro-4,1',4',6'-tetradeoxy-ga/ac~o-sucrose 4-Chloro-4-deoxya-D-galactopyranosyl 1,4,6-trichloro1,4,6trideoxy-P-D-sorbofuranoside 4'-Bromo-4,1',6'-trichloro-4,1',4',6'-tetradeoxy-ga/acto-sucrose 4,1',4',6'-Tetrabromo-4,1',4',6'-tetradeoxy-galaclo-sucrose 1',4',6'-Tribromo-4-chloro-4,1',4',6'-tetradeoxy-galacro-sucrose 4,1',6'-Trichloro-4'-iodo-4,1',4',6'-tetradeoxy-galacto-sucrose 1',4',6'-TrichIoro-4,1',4',6'-tetradeoxy-4-fluoro-ga~acto-sucrose 4,1',6'-Trichloro-4,1',4',6'-tetradeoxy-4'-fluoro-galacto-sucrose
Relative sweetness 1
20 5
bitter 20 120"
3500 76 not sweet 650" 220 160 100 100 200 2000 200 3000
7500 30
7000 200 1000
See Ref. 196.
the D-glucopyranosyl or D-fructofuranosyl residues alone.20' Probably, intramolecular hydrogen-bonding holds the two monosaccharide residues in a fixed i n ~ l i n a t i o n , 'such ~ ~ that the AH,B,y system(s), which may span both of the sugar units,202locks perfectly on the complementary AH,B,y system(s) of the receptor site. Clearly, the sweetness is due to a complex interplay of several types of nonbonded interaction. Some of the derivatives203-204 (see Table XIX) are even sweeter than thaumatin on a weight basis:05 indicating that, if the AH,B,y concept is correct, the AH,B,y unit(s) (201) G. G. Birch and C. K.Lee,in Ref. 199,pp. 165-186. (202) C . K.Lee, World Reu. Nufr. Diet, 33 (1979)142-197. (203) C. K.Lee,Br. Pat.2,088,855A (1982). (204) G. Jackson,M.R.Jenner,R.A. Khan,C.K.Lee,K. S. Mufti,G . D.Patel,and E.B. Rathbone, Br. Pat. 8,211,427A (1982). (205) J. D.Higginbottom,in Ref. 199,pp. 87-124.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
261
in these halogenated sucrose analogs must constitute the ideal glucophore for sweetness. The relationship between structure and enhancement of sweetness in chlorodeoxysucroses was studied by Hough and Khan.195Examination of a range of mono-, di-, tri-, and tetra-chlorodeoxy derivatives of sucrose and galacto-sucrose suggested that C-4, C-l’, and C-6’ appear to be important in the enhancement of sweetness when substituted with chloro substituents. The increase in sweetness is substantial if a combination of two of these hydroxyl groups is replaced; for example, the 4,l’-dichloride (55) and the 1’,6’-dichloride (56) were reported to be respectively 120 and 76 times
c1
“q CHzOH
HO
I
HO
HO 55
56
sweeter than sucrose, compared to only 4 times for 4-chloro-4-deoxygalacro-sucrose and 20 times for 1’- or 6’-chlorodeoxysucrose (57 and 58, H
CHzOH O q
H
\
q
HO
HO
HO
HO 57
58
respectively). The combined effect of chlorine at all three positions, as in “4,1’,6’-trichloro-4,1’,6’-trideoxy-galacto-sucrose” (59),produces asweetness 650 times that of sucrose.’96In all cases where the 6-hydroxyl group of the aldopyranosyl group is replaced by a chlorine atom, a decrease in the
268
CHEANG-KUAN LEE
59
sweetness of the compound is observed. For example, 4,6-dichloro-4,6dideoxy-a-D-galactopyranosyl 1,6-dichloro-l,6-dideoxy-~-~-fructofuranoside (53) is only -200 times as sweet as sucrose (see Table XVIII), Hough and Khani95 concluded that the 1-chloro substituent on the D-fructofuranosyl group acts as the B unit, and the AH was assigned to the equatorial group of the aldopyranosyl residue. From an inspection of molecular models, it was propo~ed'~'that the intense enhancement of sweetness is due to two hydrophobic locking-sites, namely, the axial chloro substituent on C-4 of the favored chair conformation of the aldopyranosyl (see Fig. 17a) and the C-6' substituent of the D-fructofuranosyl group (see Fig. 17b). These two centers must work in concert in order to produce intense sweetness, as exhibited by 4,1',6'-trichloro-4,1',6'-trideoxy-galactosucrosei99(59). The dimensions of the AH,B,y units proposed by Hough and Khan195 (see Fig. 17) approximate very closely to the interorbital dimensions of the Kier tripartite system109(see Fig. 12). However, the discovery that replacement of the 4'-hydroxyl group of the D-fructofuranosyl group by a halogen atom produces derivatives that are very much sweeter than the 4,1',6'-
FIG. 17.-AH,B,y
Systems of Chlorodeoxys~croses.'~~
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
269
t r i ~ h l o r i d e ' ~(59) ~ * ~shows ~ ~ some of the shortcomings of the Hough and Khan arguments. An explanation of the sweetness of these derivatives has yet to be expounded. It is, however, interesting that 4-chloro-4-deoxy-cu-~(60) galactopyranosyl 1,4,6-trichloro-l,4,6-t~deoxy-~-~-sorbofuranoside has only one-tenth of the sweetness of the D-fructose isomer 61.
c1
c1
CH2OH
CH2OH HO
HO
clcu CHZCl
OH
60
c1 61
e. Anhydro Sugars.-It is quite possible that the changes in ring size and conformation may, by affecting molecular geometry, also cause changes in the taste of the corresponding sugar. The ' C , conformation of sugars is not normally found for the more common sugars in the free state. However, molecules locked in the ' C, conformation can be obtained by intramolecular elimination of a molecule of water under suitable conditions to form anhydro derivatives. The taste properties of some of these sugars have been reportedI6' (see Table XX). The taste properties of sugars in the ' C , conformation cannot be strictly compared with those in the ,C1 conformation, because their alignment on the taste receptor might be different. The 1,6-anhydro-P-~-hexopyranoses were all found to be bitter-sweetlE3 (see Table XX,i). As all of the 1,6anhydro derivatives have in common a free 4-hydroxyl group, it is possible that this group may function with the 2- or 3-hydroxyl group, or the ring-oxygen atom, as the AH,B system. However, it is not apparent which particular combination constitutes the AH,B system. The 3-hydroxyl group, which was shown to be uniquely important in the ~ " ~ not ~ seem to have sweetness of sugars in the ,C1 c ~ n f o r m a t i o n , ' ~does the same importance in the alternative chair conformation. For example, 1,6-anhydro-P-~-glucopyranose (62)and its 2-O-methyl derivative (63), 1,6-anhydro-P-~-galactopyranose (67), 1,6-anhydro-P-~-mannopyranose (65), 1,6-anhydro-P-~-talopyranose (66), and the 1,6-anhydro-2-deoxy-Parabino- (64) and -lyxo-hexopyranoses (68) are all sweet, although the 3-hydroxyl group is only -260 pm from the anhydro-ring oxygen atom (see Table XXI), and consequently, this hydroxyl group would probably be
CHEANG-KUAN LEE
270
TABLEXX Taste Properties of Anhydro Sugars'83 Sugar i. Anhydrohexopyranoid compounds 1,6-Anhydro-P-~-hexopyranose p-D-glUCOp- D-gahCt0p-~mannop-D-altrop-D-allo-
p- D-gUlOp-o-ta10p-D-ido2-~-methyl-p-~-gluco2-deoxy-p-~-ribo2-deoxy-P- D-arabino2-deoxy-p-D- iyxoMethyl 3,6-anhydro-a-~-hexopyranoside a-D-glUC0-
4-chloro-4-deoxy-a-~-galacto4-deoxy-a-~-xyio3,6-Anhydro-a-~-glucopyranosyl 3,6-anhydro-a-Dglucopyranoside 3,6-Anhydro-4-chloro-4-deoxy-a-~-galactopyranosy1 3,6anhydro-4-chloro-4-deoxya-D-galactopyranoside 3,6-Anhydro-4-deoxy-a-~-xylohexopyranosyl 3,6anhydro-4-deoxy-a-~-xylohexopyranoside
ii. Anbydrohexofuranoid compounds Methyl 3,6-anhydro-a-~-glucofuranoside 3,6-Anhydro-a-~-glucofuranosyl 3,6-anhydro-a-~glucofuranoside 1,4:3,6-Dianhydro-~-glucitol 1,4:3,6-Dianhydro-~-mannitol a-D-Glucopyranosyl 1,4:3,6-dianhydro-p-~-fructofuranoside a-D-Glucopyranosyl 3,6-anhydro-P- D-fructofuranoside 3,6-Anhydro-a-~-glucopyranosyl 3,6-anhydro-p-~-fructofuranoside 3,6-Anhydro-a-~-glucopyranosyl 1,4:3,6-dianhydro-p-~fructofuranoside
Sweetness"
Bitternessb
S S S S S S S S tr tr tr tr
B B B B B B B B B BB BB BB
0
0' B
0 0
B
S
0
0
B
0
B
S
tr
S S S
tr tr tr
S S
tr tr
0'
tr-B
0
tr-B
" S =sweet, tr = trace sweetness, 0 = n o sweetness. B =bitter, BB = very bitter, tr = trace bitterness, 0 = no bitterness. ' Some panelists reported trace sweetness-bitterness.
27 1
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
H0HDoH Hogo
H2C-
Ho@
HO
R
62 R = O H 63 R = O M e 64 R = H
HO
R 65
66
67 R = O H 68 R = H
intramolecularly hydrogen-bonded to the ring-oxygen atom, and would not be expected to elicit the sweet taste.21 Thus, the sweetness of 62 and some of the other 1,6-anhydrides may be caused by (i) an AH,B system consisting of the 2- or 4-hydroxyl group and the ring-oxygen atom, where the interorbital distance is 280-400 pm, or (ii) the two axial hydroxyl groups on C-2 and C-4 where, in 1,6-anhydro-P-~-gIucopyranose, the interorbital distance, as determined by X-ray studies,206is 330 pm. This assumption is supported by studies on cis-diaxial 1,3-diols. cis-1,3-Cyclohexanediol was, somewhat surprisingly, reported to be The observed conformation of cis-1,3-cyclohexandediol is not, however, that chair in which the hydroxyl groups are in the diequatorial arrangement, as might be expected from simple energy considerations, but rather that in which they are cis-diaxial, where they are stabilized by intramolecular hydrogen bonding.208Similar stabilization has been observed in methyl 2-deoxy-a-~-ribohexopyranoside.209 Beetss2 cautioned that the sweetness of sweet compounds, even within a series of closely related analogs, is not necessarily due to the same AH,B unit. Consequently, the presence of the 4-hydroxyl group in all of these 1,6-anhydrides does not imply that that group is involved in the interaction with the sweet-taste receptor-site in all cases. Similarly, the trace sweetness detected in all 3-substituted 1 , 6 - a n h y d r i d e ~excludes '~~ the 3-hydroxyl group as a potential AH function in only those derivatives. Beets" identified the (67) as the 3possible AH,B systems for 1,6-anhydro-/3-~-galactopyranose and 4-hydroxyl groups, and for the D-mannopyranose derivative (65) as the 2- and 3-hydroxyl groups. For the other 1,6-anhydrides, the AH,B unit may be a combination of the hydroxyl groups on C-2 and C-3, and C-3 the 2,4-diaxial and C-4, although, in 1,6-anhydro-/3-~-allopyranose, hydroxyl groups are, as proposed by Lee and B i r ~ h , "probably ~ responsible (206) Y . Park, H. S. Kim, and G . A. Jeffrey, Acta Crystallogr., Sect. B, 27 (1971) 220-227. (207) H. Lindemann and H. Baumann, Ann., 477 (1929) 78-85. (208) S. N. Vinogradov and R. H. Linell, Hydrogen Bonding, Van Nostrand-Reinhold, New York, 1971. (209) R. U. Lemieux and S. Levine, Can. J. Chem., 42 (1963) 1473-1480.
TABLEXXI Interatomic Oxygen-Oxygen Distances (pm) in l,6-Anhydro-B-D-hexopyranosesa
0-2...0-3 0-2...0-4 0-3...0-4 0-2...O-5 0-3...0-5 0-4...0-5
0-2...0-1 O-3...0-1 0-4...0-1
glue0
aUo
altro
manno
galact0
talo
360 330' 360 290 360 290 360
260b 330 260 290 390 290 360 430 430
290' 430
260 430 360 360 360 290 280 260 430
360 430 260b 290b 360 360 360 260 390
260 470 260' 360 360 360 280h 260 390
260 430
Measured by using Dreiding stereomodels.
260b 360 390' 290
280 420 430
Remeasured distances (compare Ref. 166).
260 430 290 290' 390b 360 360 420 390
X-Ray crystallographic data.210
290 470 290 360 390' 360
280' 420 390
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
273
TABLEXXII Interatomic Oxygen-Oxygen Distances (pm) in 3,CAnhydro Sugar Derivatives
0-2...0-4 0-2..3ugar-ring 0 0-4...sugar-ring 0 O-S..wgar-ring 0 0-2...anhydro-ring 0 0-4..,anhydro-ring 0 a
240 280 270
360 350
340
330
280 340
280 260
-
-
Measured by using Dreiding stereo-models.
for the sweetness, whereas, for the other anhydrides in the series, the combination of an axial hydroxyl group and a ring-oxygen atom was proposed, although it was not stated which axial hydroxyl group and which ring-oxygen atom were involved. An analogous system exists in methyl 3,6-anhydroaldohexopyranosides (see Table XX). Molecules of the 3,6-anhydro type cannot be strictly compared with those of the 1,6-anhydro type, where ring distortion causes the 0-2-0-4 cis-diaxial, interorbital distance to be 330pm. In the 3,6anhydrides, the distortion at C-3 caused by formation of the anhydro ring2” shortens the 0-2-0-4distance’83 to -240 pm (see Table XXII). A strong, intramolecular hydrogen-bond between such hydroxyl groups would be expected, so that sweetness would be lost, as was found for methyl 3,6anhydro-a-D-glucopyranoside (69),3,6 : 3’,6’-dianhydro-a,a-trehaIose(70), and 3,6-anhydro-a-~-glucopyranosyl3,6-anhydro-P-~-fructofuranoside (73) (see Table XX), Interestingly, 3,6-anhydro-a,a-trehalose(71) (Y-Dglucopyranosyl 3,6-anhydro-fi-~-fructofuranoside(73), and (Y-Dglucopyranosyl 1,4:3,6-dianhydro-/3-~-fructofuranoside (74) (see Table XX) are distinctly sweet, which seems to indicate that, in these disaccharide molecules, only one half of the disaccharide actually interacts with the taste receptor, as reported by Birch and coworker^.'^^ (210) G . G . Birch, C. K. Lee, and A. C. Richardson, Carbohydr. Rex, 16 (1971) 235-238.
CHEANG-KUAN LEE
214
H:q CHzOH
HO 69 70 71 72
I
OH
R=H R = repeating unit R = D-glucopyranosyl R = D-fructofuranosyl
Hzce H2w CHZOH
I
HO
CH2
0
73
74
Another interesting observation was that, although the methyl 3,6-anhydrohexopyranosides are tasteless, the methyl 3,6-anhydrohexofuranosides, for example, methyl 3,6-anhydro-a-~-glucofuranoside ( 4 9 , and 1,4:3,6dianhydro-D-glucitol (47), 1,4:3,6-anhydro-~-mannitol(48), and 3,6anhydro-a-D-glucofuranosyl3,6-anhydro-a-~-glucofuranoside (46) are all sweet (see Table XX). Their structures do not possess any pair of hydroxyl groups having the required oxygen-oxygen distance and stereochemistry specified by Shallenberger and Acree.s However, the 5-hydroxyl oxygen atom is separated from the ring-oxygen atom by an interorbital distance of -280pm (see Table XXII) and, consequently, this could operate as the AH,B unit responsible for the sweet taste of these compounds.183 The 2-hydroxyl oxygen atom is also within the prescribed distance from the ring-oxygen atom, and presumably may also function as an AH,B unit, but of lower effectiveness, because of the greater deviation from the optimum A to B distance proposed. f. G1ycosides.-Birch and coworkers149produced evidence that only one half of a disaccharide molecule actually binds to the taste-receptor site, the other half being excluded, presumably for steric reasons. If this is so, then a series of glycosides (which are conformationally stable, and not subject to mutarotational complications) having an increasing size of aglycon would constitute a useful model for studying the critical size beyond which the molecule would be rendered tasteless. The taste characteristics of a series of glycosides having increasing chainlength and size of aglycon (see Table X) were examined by Birch and Lindley.I3*The results showed that, as the aglycon is increased in molecular weight, the intensity of bitterness increases, indicating that, far from being excluded, the molecule in fact binds to a receptor site, presumably with increasing strength, as the lipophilicity of the aglycon is increased. Thus, whereas all methyl glycosides examined are either sweet or bitter-sweet (see
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
275
TABLEXXIII Taste Properties of Alditol Clycosides'61 ~~
0-Glycosylalditol 4-O-~-D-GlUCOpyranOSyl-D-glUCitOl (maltitol) 4-O-a-~-Galactopyranosyl-~-glucitol (lactitol) 4-O-P-~-Glucopyranosyl-~-glucitol (cellobiitol) 6-O-a-D-Ga~actopyranosy~-~-gh~cito~ (melibiitol) 2-O-a-~-Glucopyranosylglycerol
2-O-P-D-GlUCOpyranOSylglyCerOl
2-O-a-~-Galactopyranosylglycerol 2-O-a- D-Glucopyranosyl-D-erythritol 2-0-P-D-Glucopyranosyl-D-erythritol 2-O-a- D-Galactopyranosyl-D-erythritol 3-O-P- D-Galactopyranosyl-D-arabinitol 4-O-P-D-Mannopyranosyl-~-erythritol
Sweetness"
Bitternessb
S(63)' S(34)' tr( 11)' tr(9)c
0 0 0 0 0 0 0 0 0 0 tr B
tr tr tr tr tr tr tr Od
S = sweet, tr = trace sweet, 0 =no sweetness. * B =bitter, tr = trace bitter, 0 = no bitterness. Values in parentheses are relative sweetness-intensities compared to sucrose ( = 100). Some panelists reported trace sweetness.
Table X), sweetness is not normally observed for ethyl glycosides, and does not occur at all for sugars having larger aglycons; these are either bitter or very bitter.'38 The taste properties of glycosides having polyhydric aglycons have also been studiedl6' (see Table XXIII). Just as with the alkyl, phenyl, and benzyl glycosides listed in Table X, increase in the chain length of the polyhydric aglycon does not sterically exclude the molecule from the receptor site. In fact, sweetness appears to increase with increase in chain-length. Thus, the glycerol-1-yl and erythritol- 1-yl glycosides appear to have only trace sweetness, whereas maltitol and lactitol are definitely sweet. It is, however, not certain which hydroxyl groups constitute the AH,B system, but the glycosyl group appears to have a strong influence in determining the state. For like P- D-mannopyranose, example, 4- 0-/3-~-mannopyranosyl-~-erythritol, is bitter, and lactitol, melibiitol, and 3-O-~-~-ga~actopyranosy~-~-arabinito~ are much less sweet than maltitol.161 Maltitol has an "anomalously" high sweetness compared with other, related alcohols studied. Birch and KearsleyZ1l suggested this may be due to an intramolecular, hydrogen-bonding interaction between the sugar residue and the polyhydric aglycon in a manner similar to that shown in Fig. 18. Such a system of hydrogen bonding would increase the acidity of (211) G.G.Birch and M. W. Kearsley, Staerke, 29 (1977)348-352.
276
CHEANG-KUAN LEE
H-?\
FIG. 18.-Structure of Maltitol, Showing Multiple Hydrogen-bonding.’” [Key: possible hydrogen-bond.] acidic; ---,
*,
more
the 4-hydroxyl proton and, at the same time, hold it in an orientation favorable for enhancing sweetness. Therefore, if the intramolecular hydrogen-bond is ruptured, the sweetness of maltitol would be expected to decrease and should eventually approach that of maltose. A significant decrease in the sweetness of maltitol was observed151with increasing temperature, in accord with this conclusion. Lactitol was reported by Lee16’ to be sweeter than lactose. Subsequent studies1519212213confirmed that the alcohol is the sweeter. It was proposed that the greater sweetness might be due to a diminution in the hydrogen bonding between the 4-hydroxyl proton of the D-galactosyl group and the ring-oxygen atom (the consequence of the Lemieux effect’”), owing to the competitive binding of the polyhydric aglycon with the D-galactosyl ringoxygen atom (see Fig. 19). This, in turn, lessens the hydrogen bonding of the 4-hydroxyl proton to the ring-oxygen atom, and enables it to function more effectively with the 3-hydroxyl group as the AH,B system (assuming that these hydroxyl groups constitute the Shallenberger AH,B glucophore), and, consequently, lactitol is sweeter than 1 a ~ t o s e . The l ~ ~ lower sweetness of lactose compared with cellobiose can similarly be attributed to the configuration of C-4 of the (nonreducing) glycosyl group. Because of the equatorial position of this hydroxyl group in cellobiose, hydrogen bonding between the 4-hydroxyl proton and the ring-oxygen atom is absent. The intramolecular hydrogen-bonding between the 4-hydroxyl oxygen atom and the 6-hydroxyl proton now makes the 4-hydroxyl group a better protondonor and, at the same time, fixes its position in its approach to the receptor Site.i17,1i8
(212) K. Hayashibara and K. Sugimoto, US. Pat. 3,973,050, 1976. (213) J. A. van Velthuijsen, J. Agric. Food Chem., 27 (1979) 630.
CHEMISTRY AND BIOCHEMISTRY O F SWEETNESS
FIG. 19.-Possible
211
Hydrogen-bonding in La~titol.'~' [Key: - - -, possible hydrogen-bond.]
g. (1+ 2)-Linked Disaccharides.-It is particularly interesting that many intensely sweet, naturally occurring carbohydrates are glycosides of (1+ 2)-linked disaccharides. However, it is not certain whether the sugar moiety actually participates directly in the generation of sweetness, or whether this moiety merely helps in imparting water solubility while maintaining the hydrophilic-lipophilic balance. Of this class of naturally occurring, sweet compounds, the flavanone glycosides found in citrus fruits have achieved considerable interest, owing to the systematic studies of Horowitz and Gentili141-143 (see Fig. 20). In terms of taste, the flavanone glycosides can be divided into two groups. The first group consists of compounds that are bitter. These are glycosides
Group 1 R = neohesperidosyl (2-O-n-~-rhamnopyranosyl-P-~-glucopyranosyl) R' =OH, R2= H (naringin) R' = OMe, R2= OH (neohesperidin) R' = OMe, R2= H (poncritin) R' = R2=O H (neoeriocitrin) Group 2 R = P-rutinosyl (6-O-~-~-rhamnopyranosyl-P-~-glucopyranosyl) R' =OH, R2= H (naringenin rutinoside) RL= OMe, R2 = OH (hesperidin) RL= OMe, R2= H (isoiakuranetin rutinoside) RL= R2= OH (eriocitrin) FIG. 20.--Some
Flavanone Glycosides.
278
CHEANG-KUAN LEE
of the disaccharide P-neohesperidose (2-O-a-~-rhamnopyranosyl-~-~glucopyranose). The second group comprises glycosides of the disaccharide these are all rutinose (6- 0-a-L-rhamnopyranosyl-/3-~-glucopyranose); tasteless. Thus, the only difference between the two groups is that the a-L-rhamnopyranosyl group is attached to the 2-hydroxyl group of the D-glucopyranosyl residue of the bitter compounds, and to the 6-hydroxyl group of that in the tasteless compounds. 141-143214 The conversion of the bitter flavanone glycosides into those of the corresponding chalcones by alkali-catalyzed fission of the pyrone ring, and of the dihydrochalcone glycosides by hydrogenation thereof (see Scheme 1)
Chalcone
Dihydrochalcone SCHEME1.-Conversion of a Flavone Glycoside into the Corresponding Chalcone and Dihydrochalcone Analogs.
(214) R. M. Horowitz and B. Gentili, Tetrahedron, 19 (1963) 773-782.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS H
H
279
H
Hydrophobic part of aweetener 0 1
I .o 7
Hydrophobic s i t e s
I
-__-_
Hydrophilic aitea
C=O
I,
0 t "Probe"
z
Critical AH,B or cationic s i t e (for intenee meet-reeponse)
Hydrogen bonding aitea (beat f i t t e d by) 2-~lycosyl-~-~glaco~ide~
FIG. 21.-Schematic Diagram of the Proposed Types of Bonding of Sweet Glycosides to the Taste-bud Receptor-sites.*"
generally produces intense sweetness. For example, naringin dihydrochalcone has about the same sweetness intensity as saccharin, whereas neohesperidin dihydrochalcone is 20 times as ~ ~ e e t ~ as ~ 'saccharin. - ' ~ ~ The , ~ ~ ~ tasteless flavanone rutinoside, on the other hand, yields only tasteless dihydrochalcone g l y c o ~ i d e s . ' ~ ~ The importance of the (1 + 2)-linked disaccharide moiety in eliciting the sweet taste of the dihydrochalcone glycosides was stressed by Hodge and Inglett.2'5 It was proposed that the AH,B units are located in this moiety (see Fig. 21), although the free disaccharide (neohesperidose) is only slightly sweet; rutinose, on the other hand, is tasteless.215The structural features most directly involved in the sweet-taste stimulation of the dihydrochalcones appear to be the 3- and 4-hydroxyl groups. If this is so, it would be anticipated that modification of these two hydroxyl groups, either by epimerization or substitution, would have a marked effect on the taste. However, van Niekerk and Koeppen2I6 reported that the 2-O-a-~-rhamnopyranosyl-P- D-galactopyranoside of naringin dihydrochalcone has about the same sweetness as naringin dihydrochalcone. Similarly, hesperitin dihydrochalcone-4'-yl P-~-galactopyranoside'~~~~~~ was found to be 1.5-2.0 times (215) J. E. Hodge and G. E. Inglett, in Ref. 2, pp. 216-234. (216) D. M. van Niekerk and B. H. Koeppen, Experientia, 28 (1972) 123-124. (217) R. M. Horowitz and B. Gentili, U.S. Pat. 3,826,856 (1974).
CHEANG-KUAN LEE
280
sweeter than the D-glucose analog, hesperitin dihydrochalcone-4'-yl p-Dxyloside is twice as sweet as the 4'-yl p-D-glucoside analog;" and the p-D-sophorosyldihydrochalconesare tasteless."* It is known that the naturally occurring dihydrochalcone glycosides phloridzin (75) and glycyphillin (76), which are 2-yl glycosides, possess little or no sweetness,'43whereas phyllodulcin (77), which is not a glycoside, has the same taste properties as the dihydrochalcones: it is -400 times sweeter than sucrose.219 OH
I 5 R = P-D-glucopyranosyl R = a-L-rhamnopyranosyl
11
76
It thus appears that the taste of the dihydrochalcones is not solely controlled by the sugar moiety, and subsequent s t u d i e ~ ' ~ * 'confirmed ~~*'~~*~~~ this. The flavanone 78 and the non-glycosidic dihydrochalcone 81 are intensely sweet. Furthermore, replacement of the bulky glycosyl residue by ~ ~ substituents did not significantly carboxyalkyl'44 (82) or ~ u l f o a l k y l '(83)
R
@
Me
HO
0
I8 R = H 19 R = O H 80 R=O(CH,),SO,Na
Ro@
Me
HO 81 82 83 83n 83b 83c 83d
0 R=H R=(CH,),CO; R = (CH,),, SO;
n= 1 n=2 n=3 n=4
(218) M. Yamato, K. Hashigaki, E. Honda, K. Sato, and T. Koyama, Chem. Pharm. Bull., 25 (1977) 695-699. (219) S. Esaki, S. Kamiya, and F. Konishi, Agric. Bid. Chem., 38 (1974) 1785-1790; 39 (1975) 1385-1389; 40 (1976) 1887-1888. (220) L. Farkus, M. Nogradi, A. Gottsegen, and S. Antus, Ger. Pat. 2,258,304 (1973).
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
281
change the taste properties. Although the disaccharide portion does not contribute to the taste response, it can have a detrimental effect, and, in some cases, can totally destroy the sweetness. The spatial disposition of the disaccharide group relative to the aromatic unit in hesperidin dihydrochalcone is most certainly the cause of the tastelessness of this compound. Early studies on the relationship between taste and structure, conducted by Horowitz and Gentili,’42s143.2’7 demonstrated that the structural elements of the aromatic unit are the important features for inducing the sweet-taste response of the dihydrochalcones. With regard to the B-ring, it was observed that (i) a B-ring hydroxyl group is necessary, but does not guarantee sweetness (see Fig. 22,i); (ii) the absence of a hydroxyl group will definitely cause tastelessness or bitter-sweetness; (iii) for sweetness in alkoxy-hydroxy compounds disubstituted in the B-ring the combination of 4-alkoxy-3hydroxy substituents produces intense sweetness, whereas the 3-alkoxy-4hydroxy isomer is tasteless (see Fig. 22,ii); and (iv) taste is lost if three adjacent groups are present on the ring (see Fig. 22,iii). It was concluded143that, as a minimum condition, the receptor has sites that respond to hydroxyl, alkoxyl, and the glycosyl (or adjacent) parts of the molecule (R) (see Fig. 23). The orientation of the B-ring is critical. It must be in quasiaxial orientation for active interaction with the r e ~ e p t o r . ” ~
i.
@OH very sweet
very sweet
bitter
BoMe H?
ii.
very sweet
very sweet
tasteless
tasteless
HO iii. &Me
Meo&Me tasteless FIG.22.-Structure-Sweetness drochalcone Sweeteners.
tasteless Relationship (With Regard to B-Ring Substitution) in Dihy-
CHEANG-KUAN LEE
282
(iv)
(iii) FIG. 23.-Postulated
Dihydrochalcone-ReceptorC o m p l e x e ~ . ’ ~ ~
For example, the rigid, planar flavone 84 is completely tasteless. Phyllodulcin (77)and the flavanones 79 and 80, on the other hand, can exist in a conformational equilibrium between a “planar” form (with the B-ring in the quasiequatorial disposition) and a “bent” form (with the B-ring assuming a quasiaxial orientation). Fig. 23,i presents the “best binding” to the receptor site. OH
HO
0 84
Several studies, however, conclusively showed that the intensity of the sweet taste depends strongly upon the number of hydroxyl groups in the ~-.i~~.1“,145.157 For example, compound 85 and its sodium salt, which have only one hydroxyl group in the A-ring, are less sweet than the 2,6-dihydroxy compound. Furthermore, there is also a requirement for an ortho-hydroxy ketone system in ring A, as compound 86 is tasteless, and because it had already been shown that the alkoxy-hydroxy substituents on ring B are also
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
283
OH
85 R = O H 86 R = H
essential for sweetness, it appears that there are two potential, AH,B units in the dihydrochalcones and in phyllodulcin. Inspection of molecular models shows that these hydrogen donor-acceptor units in the dihydrochalcones and in phyllodulcin are essentially superposable, and a comparison of the interorbital distances showed that the center of one of the AH,B units is separated from the individual elements of the other AH,B unit by almost the exact distance specified by Kier"' (see Fig. 24). It appears that the Kier third site for dihydrochalcone sweeteners may actually be a second AH,B unit. It also appears that the two binding centers must work in concert to produce the intensely sweet taste-response. The concept of dual AH,B,y units could explain some of the taste properties of the dihydrochalcones and phyllodulcin. Binding would be expected to be stronger and, therefore, would persist longer, leading to a lingering taste, as is found for the dihydrochalcone sweeteners. However, in such compounds as saccharin and perillartine, there is only one binding system, and yet these too have a bitter after-taste. Crosby and coworker^'^' suggested that there may be different receptors for molecules having two AH,B units compared with those with only one. Alternatively, it is possible that a single, common receptor-site might require two molecules of perillartine or saccharin working in concert to produce the intense sweet taste, whereas, for the dihydrochalcones, only one is required. The existence of dual receptor-units has been indicated in studies conducted by Morita and Shirashi221on the stimulation of the labella sugar receptors of the fleshfly by mono- and di-saccharides. The results seemed to demonstrate that a 1 : 1 complex is formed between sucrose and the receptor site, whereas, with such monosaccharides as D-glucose and D-fructose, a 2 : 1 complex is formed. The receptor site has to be occupied simultaneously at the two subunits for taste excitation to occur. Whether or not the hydroxyl group and the rnethoxyl group can function as an AH,B unit is debatable. Hodge222suggested that the substituents on (221) H. Morita and A. Shirashi, J. Gen. PhysioL, 52 (1968) 559-583. (222) J. E. Hodge, Absrr. Pap. Am. Chem. SOC.Meet., 166 (1973) 118.
i.
Dihydrochalcone
ii.
Phyllodulcin
Interatomic distancea (pm)
(i)
(ii)
AIBl A1X
280
280
350
360
BIX
590
580
A2B2
260
250
A1A2
390
390
640
640
B1% AP2 B1B2
FIG.24.-Proposed
380
360
600
6 40
AH,B,X Systems of (i) Dihydrochalcone and (ii) P h y l l ~ d u l c i n . ' ~ ~
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
285
the B-ring are incapable of acting as an AH,B system. Yamamoto and coworkers223 however, showed that 2-[(3-hydroxy-4-methoxyphenyl)ethyllbenzene (87) is sweet. OH
87
Although it is now assumed that the sweetness of the glycosidic and nonglycosidic dihydrochalones is due to the same AH,B unit(s) in the dihydrochalcone moiety, this may not necessarily be valid. For example, there is still some doubt that the sugar moiety contributes to the taste response,222apart from exerting steric factors. None of the (carboxyalky1)and (sulfoalky1)-dihydrochalcones tested have a sweetness intensity comparable to that of neohesperidin dihydrochalcone or some of the other sweet glycosidic dihydrochalc~nes.~"~'~~~~~~ More experimental support is required; specifically, the effect of further variations of the sugar moiety should be investigated. The structure-sweetness relationships of other naturally occurring, sweet glycosides have received very little attention. In most cases, virtually no systematic, structural modifications have been reported. (i) Stevioside.-Ste~ioside~~~ (88) and rebaudoside A (Ref. 226) (89) are found in the leaves of the Paraguayan shrub, Stevia rebaudiana Bertoni. In both compounds, the aglycon steviol carries two glycosyl groups, a p-Dglucopyranosyl group (at the carboxylic end) and a P-sophorosyl group (in the case of stevioside) and a 3-O-p-~-glucopyranosyl-P-sophorosyl group (in the case of rebaudoside A). Reports relating to the sweetness of stevioside are conflicting.224Briedel and LavielleZZSreported that it has 10 times the sweetness of sucrose, whereas Schultz and Pilgrim227ascertained a value of 280 times. C r ~ s b yhowever, , ~ ~ ~ reported that, on a weight basis, its taste potency is only 160 times. It has a significant liquorice off-taste. Rebaudoside A has a sweetness of 190 times that of sucrose. (223) M. Yamato, K. Sato, K. Hashigaki, M. Oki, and T. Koyama, Chem. Pharm. Bull., 22 (1974) 475-479. (224) G . A. Crosby, Cri?. Rev. Food Sci. Nurr., (1976) 297-323. (225) M. Briedel and R. Lavielle, C. R. Acad. Sci.,192 (1931) 1123-1125; J. Pharm. Chim., 14 (1931) 99-113, 154-161; Bull. SOC.Chim. Biol., 13 (1931) 636-655; C. R. Acad. Sci, 193 (1931) 72-74. (226) H. Kohda, R. Kasai, K. Yamasaki, K. Murakami and 0. Tanaka, Phyrochemistry 15 (1976) 981-983. (227) H. Schultz and J. J. Pilgrim, Food Res., 22 (1957) 206-213.
rS-'" CHEANG-KUAN LEE
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Me 'CORl
Compound Steviol
R'
R2
-OH
-OH
CHiOH
H:=Y How CHiOH
"o-HO o/ OH Stevioside (88) Sweetness = 300 x sucrose
HO
OH
CH2OH
CHpOH H HOO
W
HO
o
/
H
OCHaOH W '
'
OH Rebaudioside A (89) Sweetness = 190 x sucrose
HO OH
The only information available on taste-structure relationships is that the D-glucopyranosyl group seems to be essential for sweetness, as steviol sophoroside is not sweet." (ii) 0sladin.-Osladin (90), also a bisglycoside, is isolated from the It was reported to have a rhizomes of the fern Polypodium vulgare L.228*229 sweetness intensity 3000 times that of sucrose. However, the glycoside carries a physiologically active aglycon. Because of this, and its presence in polyploid rhizomes, and also because of the difficulty in isolation, the prospects of its being developed as a commercial sweetener seem very doubtful. (228) J. Jibza and V. Herout, Collect. Czech. Chem. Commun., 32 (1967) 2867-2874. (229) J. Jibza, L. Dolejs, V. Herout, and F. $om, Tetrahedron Lett., (1971) 1329-1332.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
287
H:w b I
HO
OH
90 Osladin
The steroidal aglycon carries a neohesperidosyl group at one end and an a-L-rhamnopyranosyl group at the other.
(iii) G1ycyrrhizin.-Glycyrrhizin or glycyrrhizic acid [ (3p,20p)-20carboxfl-1 1-oxo-30-norolean-12-en-3-yl2 - 0 - ( p-D-glucopyranosyluronic acid)-a-D-glucopyranosiduronicacid] (91) is the sweet principle of liquorice root, Glycyrrhiza glabra L. The molecule consists of a triterpenoid aglycon, namely, glycyrrhetic acid, glycosidically linked to 2- 0-( P-D-glucopyranosyluronic acid)-a-D-glucopyranuronicacid. It has a relative sweetness 50 times that of sucrose,230*231 with a persisting, liquorice after-taste, and it
H
i
91 Glycyrrhizic acid
91a Glycyrrhetic acid
(230) M. R. Cook, Flavour I d , 1 (1970) 831-832. (231) B. Crammer and R. Ikan, Chem. SOC.Rev., 6 (1977) 431-465.
288
CHEANG-KUAN LEE
appears to have a synergistic effect when mixed with sucrose, the sweetness potency being doubled. It is now generally accepted that the hydrophilic-lipophilic balance of a molecule is one of several important factors in determining the intensity of sweetness. Thus, hydrophilic and hydrophobic binding-parts on a compound bind to hydrophilic and hydrophobic binding-sites of the receptor, as shown215in Fig. 25 (for N-formylkynurenine, 92). Hodge and Inglett2I5 proposed that, in addition to the hydrophilic and hydrophobic sides, another hydrophobic function, designated the “polar probe,” is a necessary structural feature, without which, a compound is devoid of sweet taste. The polar probe may be a sugar or a phenolic radical possessing the Shallenberger AH,B function, or it may simply be an anionic center, such as the 20-carboxylate anion of ammonium glycyrrhizinate or the phenolate anion of naringin dihydrochalcone (see Table XXIV). In neohesperidin dihydrochalcone and phyllodulcin, the 3’-hydroxyl group and the 4’-methoxyl group on the B-ring were proposed. Interestingly, DuBois and proposed that this part is one of the AH,B units. It was suggested that the “probe” functions by binding, either by hydrogen bonding or by ionic forces, to a more specific type of receptor, which, by electron transfer or electrostatic-charge displacement, triggers the message of sweetness to the brain.*19 Evidence supporting this concept is, however, lacking, although a similar hypothesis had been proposed’46 for the action
“Ionic probe”
FIG.25.-Proposed Hydrophilic and Hydrophobic Interaction of N-Formylkynurenine (92) with Receptor Sites.”’
TABLEXXIV Structural Features of Intensely Sweet C l y c o s i d e ~ ~ ’ ~ Glycoside
Disaccharide
Hydrophobic part
Stevioside Glycyrrhizin
2-O-P-D-g~ucopyranosy~-~-D-g~ucopyranoside steviol 2-0-( /3-D-glucosyluronic acid)-P- D-glucoglycyrrhetic acid
Osladin Neohesperidin dihydrochalcone
siduronic acid 2-O-a-~-rhamnosyl-~-~-glucopyranoside 2-O-cr-~-rhamnosyl-p-~-glucopyranoside
Naringin dihydrochalcone
2-O-a-L-rhamnosyl-P-~-glucopyranoside
polypodosaponin substituted phloroglucinol,
“Polar probe”
p- D-glucopyranose --C02H
0-L-rhamnopyranose phenyl OH and -0Me
--C2H4
substituted phloroglucinol, --C&
phenyl OH
CHEANG-KUAN LEE
290
of m i r a c ~ l i n ,a~glucoprotein ~ ~ - ~ ~ ~ isolated from the berries of a West African shrub, Synsepalum dulcijcum. Furthermore, based on Hodge and Inglett’s proposal for the sweetness of N-formylkynurenine (92)(see Fig. 25) (35 x sucrose235),the sweetness of glykergenic acid (93)(lOOOx which is a structural analog of tryptophan (94)(35x sucrose), would be difficult
92
93
94
to rationalize, because, if the carboxylic acid group is the “probe,” there will be no functional group to serve as the AH,B system in the last two compounds. Also, the carboxylic group is too far from the methylamino group to be identified as the AH,B unit, in analogy to the NH,, C02.H pair in tryptophan or other amino acids. A comparative study by beet^'^*^^' of molecular models of tryptophan (94),N-forrnulkynurenine (93),and glykergenic acid (93)revealed that, not only are the three molecules structurally very similar, but all three can readily assume a conformation in which the distance between the carboxylic acid group and the second nitrogen atom is within the prescribed distance of an AH,B system. Thus, the three compounds will have the same AH,B unit in combination with a third hydrophobic feature, situated as shown in Fig. 10. h. Cyclitols.-Cyclohexanepolyols resemble the sugars, except for the absence of a ring-oxygen atom. Therefore, if diequatorial or gauche arrangements of a-glycol groupings can produce sweetness in ring structures, the cyclohexane polyols should provide an interesting model for taste study. These molecules have the added advantage of the absence of a hemiacetal function, so that mutarotational isomerization cannot occur in aqueous solution. (232) G . E. Inglett, B. Dowling, J. J. Albrecht, and F. A. Hoglan, 1.Agric. Food Chem, 13 (1965) 284-287. (233) G . E. Inglett and J. C. Findlay, Abstr. Pap. Am. Chem. Soc. Meet., 154 (1967) A-075; J. Food Sci, 34 (1969) 408-41 1. (234) J. N. Brouwer, H. van der Wel, A. Francke, and G. J. Henning, Nature, 220 (1968) 313-314. (235) J. W. Finley and M. Friedrnan, J. Agric. Food Chem., 21 (1973) 33-34. (236) A. Hofrnann, Helu. Chim. Acta, 55 (1972) 2934-2940. (237) M. G . J. Beets, in Ref. 118, pp. 71-90.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
29 1
Studies by Birch and Lindley14' indicated that molecules of cyclohexane bearing one, two, or three hydroxyl substituents are always bitter and never sweet, whereas those containing five or six hydroxyl groups are always sweet and never bitter; this is contrary to Meiser's finding,238in 1899, that trans- 1,2-cyclopentanediol is sweet, since confirmed by Goodwin and cow o r k e r ~ . ' ~ Using ~ . ' ~ ~a trained panel, the diol, together with cis- and trans1,3 - and 1,4-~yclohexanediols(the interorbital oxygen-oxygen spacing is between 420 and 490 pm) were found to be sweet, but with a very strong bitter taste. Birch" argued that it is possible that, at the extreme low-response level, the panelists might have been confused by a tactile response, akin to sweetness, that is presumed to occur at the tip of the tongue. However, the taste panelists employed by Goodwin and coworkers had been trained to detect very low levels of sweetness in the presence of bitterness, and their results'43 showed that the diols are distinctly sweet. Many other ringcompounds derived from sugars, and containing no (or, at most, two) hydroxyl groups, have also been reported to have a sweet taste.239 The strong bitterness of the diols again emphasized the importance of hydrophobicity in this taste quality. The only tetrahydric alcohol tested, cyclohexane-1,2/4,5-tetrol,gave no response either way.14' The pentahydric alcohols, on the other hand, are all free from bitterness, but only (*)viboquercitol (95, 96) is distinctly sweet. Both (+)-protoquercitol (97) and (-)-viboquercitol(96) have only a trace of sweetness. Similarly, the hexahydric alcohols are all devoid of bitterness, and as epi-inositol (98) is an analog of P-D-mannose, Birch and Lindley14' suggested that the lack of bitterness must be due to absence of a ring-oxygen atom.
HO 95
" HOO
m 96
O HO
H
H O HO W 97
OH
H HO
O
M OH 98
The absence of taste for cyclohexane-l,2/4,5-tetrol is puzzling, as it contains AH,B systems. This behavior was attributed to the presence of (238) W. Meiser, Ber., 32 (1899) 2049-2061. (239) J. E. Hodge, in H. W. Schultz, E. A. Hay and L. M. Libbey (Eds.), Chemistry and Physiology of Flavors, AVI, Westport, CT, 1967.
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CHEANG-KUAN LEE
AH,B units at "opposite" ends of the molecule, just as in the case of 1,3-dihydroxy-2-propanone(see Section 11,3,a,ii), so that the molecule cannot align itself correctly on the receptor ~urface.'~' Odorant molecules have been observed to behave similarly.52The poor polarization of the taste receptor is enhanced by the combined effect of (a) the absence of a ring-oxygen atom, and hence a polar center, and ( b ) the presence of hydrophobic, methylene groups. It is also interesting that, although the (*)-viboquercitol mixture is sweet, (-)-viboquercitol (96) has only trace sweetness. Therefore, (+)-viboquercitol (95) would be expected to have a fairly strong, sweet taste, probably close to that of sucrose or ~ - f r u c t o s eit~has ~ ; not, however, yet been obtained in pure form to permit testing of the speculation. The sweetness of the cyclohexanepentols may be due to participation of the methylene group in the same way as for D-fructopyranose, so long as it does not interfere with the alignment of the AH,B system(s). Beets,52 however, suggested that a methylene unit may play a totally passive role. Its hydrophobicity may prevent the unfavorable, sweetness-depressing, bilateral interactions by controlling molecular orientations at the receptor site, so that the molecules are forced to complex with the "sweet" receptor sites by means of a single pair of hydroxyl groups. If this is the case, the AH,B unit responsible for the sweetness must be situated at a specific distance from the methylene group, for example, as governed by the Kier proposal.'09 Thus, the AH,B unit cannot be adjacent to the methylene group. If it is correct that the methylene group behaves as proposed, the cyclohexanehexols would be less sweet than the pentols. Four of the seven hexols tested were, indeed, found to have only trace ~weetness.'~' An ideal model for testing the postulate would be the pseudosugars of McCasland240(see Fig. 26). These are true analogs of the hexopyranoses, lacking the ring-oxygen atom. Some have been conducted on the taste of pseudo-P-DL-glucose and pseudo-a-DL-galactose, which have, indeed, been found to be sweet, the relative sweetness of pseudo-P-DLglucose being half, and that of pseudo-a-DL-galactose, 40-50%, that of sucrose (at 10% c ~ n c e n t r a t i o n ) . It ~ ~appears '~ that the sweetness of a pseudo sugar is the same as that of the related true sugar. Because the relative sweetness of a D sugar is the same as that of its L enantiomer,"8 it may be assumed that the same would apply to the pseudo sugars. As with the thio (240) G. E. McCasland, S. Furuta, and L. J. Durham, J. Org. Chem., 31 (1966) 1516-1521; G. E. McCasland, M. 0. Naumann, and L. J. Durham, ibid., 31 (1966) 3079-3089. (241) M. Rudrum and D. F. Shaw, 1. Chem. SOC.,(1965) 52-57. (241a) S. Ogawa, M. Ara, T. Kondoh, M. Saitoh, R. Masuda, T. Toyokuni, and T. Suami, Bull. Chem. SOC.Jpn., 53 (1980) 1121-1124. (241b) T. Suami, S. Ogawa, and T. Toyokuni, Chem. Lett., (1983) 611-612.
CHEMISTRY AND BIOCHEMISTRY O F SWEETNESS HO
293
H?
HO
HO OH
OH falo (1,2,3,4/5)
galacto (1,2,3/4,5)
HO OH manno (1,3,4/2,5)
FIG. 26.--Some Pseudosugars.
sugars, the ring-oxygen atom is clearly not essential for sweetness, and may even be replaced by a methylene group without any lessening of the sweetness. i. Aldito1s.-That a variety of non-ionized, aliphatic alcohols, such as ethylene glycol, glycerol, erythritol, and various acyclic polyols, are sweet has been known for some time. showed that the sweetness of these can be satisfactorily explained by applying the concepts of the Shallenberger hydrogen-bonding theory. Spectroscopic measurements of ethylene glycol indicated that considerable intramolecular hydrogen-bonding exists,61 suggesting that steric and dipolar repulsion of the hydroxyl groups are more than outweighed by the energy gained in the formation of the hydrogen bond. The conformation possessing the gauche orientation, having an 0-0 distance of 280-290 pm, is, from energy considerations, the most likely to be the favored arrangement. The sweetness of erythritol could be rationalized on the basis that the two terminal, a-glycol groups have the gauche orientation, owing to 1,3-diaxial, non-bonded interactions. Like the polymethylene hydrocarbons, acyclic carbohydrates and alditols tend to adopt the planar, zigzag conformation wherein all of the carbon atoms lie in the same plane.2419242 However, non-bonded interactions between “parallel” hydroxyl groups in the 1,3 positions (syn-axial interaction) are the most highly destabilizing, and, in xylitol and ribitol, this (242) M. J. Aroney, R. J. W. LeFavre, and J . D. Saxby, J. Chem. Soc., (1966) 414-416.
CHEANG-KUAN LEE
294
HO HO +OH
A /
HO =-axial
interaction c
FIG. 27.-syn-Axial
Interaction in Xylitol.
-L-(-)-Arabinitol
Xylitol Ribitol
0
3600
3400
3200
3000
Wavenumber (cm 1 ) ~
FIG. 28.4nfrared Spectra of Three Pentit01s.I~'
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
295
interaction introduces considerable strain into the planar zigzag conformers, which is relieved by turning around the C-3-C-4 bond243(see Fig. 27). Xylitol is the sweetest of the alditols thus far examined. Inspection of a molecular model shows that the oxygen-oxygen distance between the four pairs of vicinal groups in xylitol is 290-300 pm, so that they are all ideally suited to function as AH,B systems. As expected, xylitol shows a strong, free-hydroxyl absorption-peak at 3440 cm-' in its infrared spe~trurn,'~' indicating the presence of free hydroxyl groups. On the other hand, ribitol and arabinitol show no such peak191 (see Fig. 28), indicating strong intramolecular hydrogen-bonding. Among the hexitols, n.m.r. and X-ray crystallographic data243showed that galactitol and D-mannitol exist as planar zigzag conformers. Solutions of the others contain appreciable proportions of sickle and other bent carbon-carbon chain conformers.2433244 The low sweetness of the hexitols16' could possibly be due to increased hydrogen-bonding due to increased chain-length. j. Other Sweeteners.-To exemplify the validity of the Shallenberger and Kier concepts, some nonsugar sweeteners are discussed. It will be evident that all of these examples have present a hydrophobic center optimally situated from the AH,B unit, and it is fairly obvious that it is this circumstance that makes these classes many times sweeter than the sugars.
(i) Hernandu1cidin.-A systematic search through ancient Mexican botanical literature carried out by Compadre and coworkers24' for new plant sources of intensely sweet substances resulted in the isolation of a sweet sesquiterpene of the bisbolane class, namely, hernandulcin [6-( 1(W), from the hydroxy- 1,5-di~ethylhex-4-enyl)-3-methylcyclohex-2-enone] leaves and flowers of the plant Lippia dulcis Trev. The compound was reported to have a sweetness of 1250 times that of sucrose, but with some bitterness, as well as off- and after-tastes. Taste studies carried out by Compadre and coworkers245suggested that the 1-carbonyl and the 1'-hydroxyl groups are essential for the sweetness, as reduction of the carbonyl group to an alcohol, or acetylation of the 1'-hydroxyl group, causes loss of the sweet taste. These functional groups are -260 pm apart in the favored conformation. They can, therefore, functior, as the AH,B unit (99). The configuration of C-1' also appears to be
-
(243) D. Horton and J. D. Wander, Curbohydr. Res., 10 (1969) 279-288. (244) N. Azarinia, G. A. Jeffrey, H. S. Kim, and Y. J. Park, Abstr. Pap. Joint ACSICIC. cO& (1970) CARB-26. (245) C. M. Cornpadre, J. M. Pezzuto, A. D. Kinghorn, and S. K. Karnath, Science, 227 (1985) 417-419. (245a) E. M. Acton and H. Stone, Science, 193 (1976) 584-586.
296
CHEANG-KUAN LEE
important, as the C-l’-( R)isomer of (*)-hernandulcin, namely, (*)-epihernandulcin (loo),does not appear to possess any sweetness. Examination
99
100
of molecular models reveals that this hydroxyl group in epi-hernandulcin could occupy a position >350 pm from the 1-carbonyl group, thus making it an ineffective, primary AH function. If the assignment of the 1’-hydroxyl group and the 1-carbonyl group as the AH,B glucophore is correct, it is then very likely that the third ( 7 ) site is located at C-4’, as inspection of a molecular model shows that this carbon atom could be in a position which approximates very closely the Kie? or Shallenberger and L i n d l e ~tripartite ~~ glucophore. This could be readily confirmed by structural-modification studies. The structural simplicity of hernandulcin and its intense sweet taste offer an excellent model for further studies on the structure-sweetness relationship. (ii) 0ximes.-It has long been known that the a-syn-oxime of perillaldehyde (11) is intensely sweet. The sweetness of the crystalline compound was to be about twice that of sucrose. In solution, the sweet taste appears to be very much more intense (370 x sucrose).
11 Perillaldehyde oxime
For oximes, the relationship between the sweet and bitter tastes and their structures was systematically studied by Acton and BY modifying the terpene moiety and keeping the oxime group intact, it was ( 2 4 6 ) E. M. Acton, M. A. Leaffer, S. M. Oliver, and H. Stone, J. Agric. Food Chem., 18 (1970)
1061- 1068.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
297
established that a double bond conjugated with the oxime is essential for sweet taste. There is little doubt that the AH,B unit responsible for the sweetness of oximes is located in the oxime part of the molecule. However, the oxime group itself can hardly function as the AH,B system. KierIog suggested that the nitrogen atom of the oxime functions as the B part, and the AH was assigned to the C-6-H (see ll), as the latter is suitably positioned from the groupings that might act as the B and y units. However, this C-H group is certainly incapable of functioning as a proton donor; the mere existence of AH,B groups disposed in the correct positions does not guarantee that a molecule will elicit a sweet taste, and assignments cannot be made on the basis of just one or two compounds! Beetsz4' suggested that the oxime function undergoes hydration prior to interaction, to afford the glycol-like structure 99a, which has the freedom to assume the favorable, staggered conformation. Evidence supporting this concept is the bitterness of the anti-oxime (100a), which, for steric reasons, cannot be readily hydrated. The third structural feature, in the sense proposed by Kier,Iog would then be either of the activated, ring-methylene groups,5znot the para substituent proposed by Kier.'@
lOOa
99a
Beetsz4' suggested that the bitterness of oximes is due to oxime-isonitroso tautomerism, a system analogous to the tautomerism of thioamides (see Section 11,4,b). 0-H
0
N
N
I
H-C
I
R
I
. - c H
/:\ '
/ R
(iii) Su1famates.-The two most well known sweet compounds in this class are cyclamate (9) and saccharin (10). Considerable structural modifications have been recorded, and their tastes ascertained. (247) M. G. J. Beets, in C. J. Cavalito (Ed.), Structure-Acrioity Relationships, Vol. 1, Section 5, International Encyclopedia of Pharmacology and Therapeutics, Pergamon, Oxford, 1973, pp. 225-295.
CHEANG-KUAN LEE
298
-360 Dm
9
10
Cyclamic acid
Saccharin
Moncrief4 summarized the work of Cohn' and the information in the early literature. As early as 1923, it was known" that rupture of the heterocyclic ring, as well as substitution of the imino hydrogen atom, results in the loss of sweetness. Thus, o-carboxybenzenesulfonamide and N-alkyl derivatives of saccharin are tasteless. This loss of sweetness would be expected, as the NH group is the only proton-donor function available in the molecule. Replacement of the sulfonyl group by a carbonyl group gave" the tasteless, phthalimide analog (101). On the other hand, if the carbonyl groups are replaced by sulfonyl groups, to produce benzodithioimideZz6 (102),
102
101 (tasteless)
(sweet/bitter)
sweetness is retained; however, the compound also possesses a strong, bitter after-taste. Thus, Shallenberger' assigned the AH,B unit to an NH-SO2 moiety, and this is supported248by the sweet taste of compound 103, as this compound would have the same AH,B pair as saccharin (10). As discussed earlier, the third hydrophobic site for intense sweetness was
103 (sweet) (248) C. Runti, Bull. SOC.Pharm. Bordeaux, 101 (1962) 197-201.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
299
a~signed’~’ to C-4. Later research has mainly resulted in confirmation of these basic facts. Structurally related to saccharin are the oxathiazinone dioxides (104). Clauss and synthesized a series of these compounds, and demonstrated that they possess intense sweetness. Acesulfame-K, the 2,2-dioxide potassium salt of 3,4-dihydro-6-methyl-1,2,3-0xathiazin-Cone (104) has a sweetness intensity 130 times that of sucrose.
104
R’= Me, R2 = H (Acesulfame)
The second sweetener in this class is cyclamate ( 9 ) . As with saccharin, the presence of a free, imino hydrogen atom is essential for s w e e t n e s ~ , ~ ~ ” ~ ~ ~ and the AH,B unit was assigned to the NH-S03 gro~ping.~.” Considerable structural modifications have since been reported, and numerous homologs and analogs of this sulfamate have been synthesized.251-256 Benson and S ~ i l l a n reported e ~ ~ ~ that although N-methylation of N-isobutyl- and N-cycloheptyl-sulfamate gave products that did not retain the sweetness of the unmethylated compounds, indicating the importance of the imino hydrogen atom, the retention of this hydrogen atom is not a necessary condition for sweetness in those analogs that contain sulfur acid (105), heterocycles. For example, tetrahydro-2H-thiopyran-4-sulfamic its 1.1-dioxide (106), and their N-ethyl, N-butyl, and (2-hydroxyethyl)
105
106
K. Clauss and H. Luck, Ger. Pat. 2,228,423 (1973). K. Clauss and H. Jensen, Angew. Chem., Inf. Ed. Engl., 12 (1973) 869-876. L. F. Audrieth and M. Sveda, J. Org. Chem., 9 (1944) 89-101. C. A. Benson and W. J. Spillane, J. Med. Chem., 19 (1976) 869-872. C. Nofre and F. Pautet, Bull. SOC.Chim. Fr., 3-4 (1975) 686-688. B. Unterhalt and L. Boschemeyer, Z. Lebensm. Unters.-Forsch., 147 (1971) 153-155; 149 (1973) 227-229.
F. F. Blicke, H. E. Millson, Jr., and N. J. Doorenbos, J. Am. Chem. Soc., 76 (1954) 2498-2499.
K. M. Beck and A. W. Weston, U.S. Pat. 2,785,195 (1957).
300
CHEANG-KUAN LEE
derivatives are all sweet. Benson and Spillane252argued that the more crucial part of the NH-SO; group is the SO; moiety. Thus, modification of this part of the sulfamate function destroys sweetness. For example, N-cyclohexylsulfamyl chloride was found to be tasteless.252Consequently, Benson and S ~ i l l a n e ~suggested ’~ that the NH group does not function as the AH unit. Furthermore, the SO;, rather than the -SO2- considered by Shallenberger and Acree,’.’’ appears to be the B center. It was further that substitution of a methyl group at C-1 of either cyclohexyl- or cycloheptyl-sulfamate destroys the sweetness of these compounds. N-(1-Adamantyl)sulfamate (107), which possesses an imino hydro-
107
gen atom, for example, is not sweet, and, significantly, every sweet sulfamate compound known contains the =CHN( R)SO; structure.252Therefore, it was concluded that the presence of at least one hydrogen atom a to the sulfamate function is important for sweetness. It was proposed that the a-hydrogen atom, not the imino hydrogen atom, acts as the donor proton of the AH,B unit. This suggestion was criticized by Crosby and c~workers:~ because tastelessness of adamantyl analogs is more likely to be due to steric hindrance adjacent to the -NHSO; unit than to the absence of a hydrogen atom on the tertiary a-carbon atom. Furthermore, if the Shallenberger concept is valid, the hydrogen donor-acceptor system depends on the presence of an acidic hydrogen atom for noncovalent bonding. The ability of the -CH group [in -CHN(R)SO;] to serve as a hydrogen donor is debatable. The same argument applies to Shallenberger and Acree’s7’ selection of the nitro group and the aromatic ortho proton as the hydrogen donor-acceptor unit for the alkoxynitroanilines. Allerhand and von Schleyerzs7reported that there must be three strong, electron-withdrawing groups before intermolecular, aromatic C-H hydrogen-bonding can be observed. In the alkoxyaminonitrobenzenes, the o-aminoalkoxy groups would appear to be a more logical choice for the AH,B system, because of their similarity to hydrogen donor-acceptor systems present in other sweeteners.
(257) A. Allerhand and P. von Schleyer, J. Am. Chem. SOC.,85 (1963) 1718-1723.
poT CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
0
301
0 0
Saccharin: 0 = 59"
FIG. 29.-Newman s~lfamates.2~~
Cyclamate: 0 = 60"
N-( 1-Methylcyclohexyl)sulfamate: 0 = -0"
Projection along the N-S Bond for Saccharin and Two Cyclohexyl-
Three-dimensional, X-ray analysis of cyclamate, N-( 1-methylcyclohexyl)sulfamate, and s a c ~ h a r i nshowed ~ ~ ~ *that ~ ~the ~ sulfamate anion exists in the syn-clinal orientation (8 -60") between the NH and SO bonds, with the imino hydrogen atom and sulfonate oxygen atom 280pm apart (see Fig. 29). This is consistent with the Shallenberger and Acree' proposal. The necessity of an optimal torsion-angle of 60" in the aminosulfonate group for interaction with the receptor site probably explains the loss in sweetness of molecules wherein the carbon atom on the nitrogen atom is sterically hindered. The presence of a methyl group on the first carbon atom of either an alkyl chain or a cycloalkyl group would transform the syn-clinal (8 -60") orientation into a syn-periplanar (8 -0") orientationz6' resulting in a decrease in, or loss of, sweet taste, as the SO and the NH groups are now eclipsed, as may be seen for N-(1-methylcyclohexyl)sulfamate (see Fig. 29). Studies on the size of the group bonded to the nitrogen atom have also been performed.260 Sweetness occurs when the alkyl chains have three or four aligned carbon atoms. Higher or lower homologs are often not sweet. Similarly, with cycloalkyl substituents, sweetness is not detected when the size of the cycloalkyl ring is less than five, or greater than nine, carbon atoms. Measurement of the lengths of the groups [by using a Corey-PaulingKoltum (CPK) space-filling, atomic model] showed that sweetness is observed when the length of the group is >500 and <700 pm, and the width, <800 pm. This implies that this portion of the molecule is involved in some form of interaction with the receptor site, presumably by way of hydrophobic binding. Because those compounds having a carbon length of less than five lack sweetness, these groups appear to lack hydrophobic interaction. The (258) L. Peraldo-Bicelli, Roc. Symp. Sulphamic Acid, Elecrromer. Appl., Milan (1966) 19; B. E. Cain and N. Y. Kanda, Z. Krisrallogr. Mineral., 135 (1972) 253-260. (259) Y. Okaya, Acra Crystallogr., Sect. B, 25 (1969) 2257-2265. (260) F. Pautet and C. Nofre, Z. Lebensm. Unters.-Forsch., 166 (1978) 167-170.
302
CHEANG-KUAN LEE
outer limit of 700 pm seems to indicate the presence of a spatial barrier on the sweet-taste receptor located at -700 pm from the NH interaction point.260This is in agreement with the length of the side chain reported by Brussel and coworkers120for amino acids. Re-examination of the sulfamates261’262 showed that, with those analogs in which the substituent on the -NHSO; group contains carbon and hydrogen only (carbosulfamates), the use of the length ( x ) and width ( z ) dimensions only was insufficient to describe the spatial geometry of the substituent. For example, 2,3-dimethylcyclohexyl-, 3,3,5-trimethylcyclohexyl-, cis-myrantyl-, and 2-adamantyl-sulfamate, the lengths of whose substituents all lie between 500 and 700 pm, with a width of <800 pm, are not sweet. Spillane and McClinchey261 measured the height ( y ) of the substituent, and defined a parameter V, which is the product xyz; V is not the molecular volume, but the volume generated when the group is rotated through 360”. It was found that almost all of the sweet sulfamates in this class fall within a certain region of a plot of the length, x (pm) us. V (nm’). Almost all of the non-sweet analogs fall outside this region.262Other 2dimensional plots of Vus. height, or width, did not give as high a correlation between sweet and non-sweet molecules. The limiting values of V appear to lie between -0.090 and 0.250 nm3. Thus, Spillane262suggested that those analogs having substituent groups with lengths of 720 pm and V = 0.250 nm3 are too large to fit onto the receptor site, and therefore cannot be “locked” thereon. Consequently, the mechanism for initiating the sweet-taste stimulus cannot operate. Those having substituent groups for which x > 720 pm, or V>0.250nm3, or both, are also too large, or too long, or both, to be accommodated at the binding site. On the other hand, a value of x of <520 pm and a V value of C0.250 nm3 will also not cause sweetness, as these will result in a loose fit, and the compounds cannot be properly locked onto the receptor site. This approach did not seem to be as satisfactory for those sulfamates having heteroatom substituents (hetero-sulfamates). Spillane262suggested that the various electronic effects of the hetero-atoms probably introduce an additional variable that is apparently absent, or constant, for the carbosulfamates. Because molecular connectivity correlates structure with molecular volume and electronic effects, Spillane262included molecular connectivity, ‘xu(computed for the entire molecule, RNHSO;) to the four variables, x, y, z, and V , and applied the statistical technique of lineardiscrimination analysis to 33 heterosulfamates (10 sweet, 23 not sweet). A correlation of >80% was obtained for the x, z, ‘x”subset; 5 of the 33 (261) W. J. Spillane and G. 0. McClinchey, J. Pharm. Sci., 70 (1981) 933-935. (262) W. J. Spillane, Chem. Ind. (London), (1983) 16-22.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
303
compounds were misclassified. Five other 3-, 4-, and 5-variable subsets were found to misclassify 6 of the compounds, the same compounds being misclassified by the subsets examined. Spillane262was, however, not able to establish any common factor(s) that might be responsible for this consistent misclassification of thcse compounds. (iv) Nitroanilines-The sweetness of 3-nitroaniline has been known for a long time. I t s 2- and 4-isomers are, however, tasteless. Since the discovery by Blanksma and van der W e ~ d e nthat ~ ~certain ~ 2-substituted 5-nitro’~~~-~~~ anilines are intensely sweet, many structural m o d i f i c a t i o r ~ s ’ ~have been performed. The structure-sweetness relationship of these compounds has been well studied, 19*82*94*267and it was these compounds that Deutsch and Hansch8’ used in order to examine the dependence of sweetness on the Harnmett constant, a, and hydrophobicity. Kier’s tripartite-bonding conceptlW was also derived from studies conducted on these compounds. Although the 4-substituted 5-nitroanilines are generally found to be bitter or tasteless, the 4-carboxyl derivative was, surprisingly, found to be 120 times as sweet as sucroses2 (compare the 2-carboxyl derivative, which is only 25 times as sweet as sucroses8).The superior sweetness of the 4-carboxyl derivative suggested that the AH,B system must be represented by the combination of the nitro group and this (ortho) carboxyl group. The lack of taste of methyl esters of both acids appears to support” this conclusion. Further evidence that a carboxyl substituent on a benzene ring is capable of functioning as a suitable proton-donor in combination with an acceptor group in the ortho position was obtained from the sweet taste (150 times that of sucrose at a concentration below 0.2%)7 of 2-(4-methoxybenzoy1)benzoic acid. Tinti and coworkers268acompared the sweetness of the derivatives of 2-propoxy-5-nitroanilines in which the nitro group was replaced by a C N or a COY substituent. They found that the cyano derivative is sweet (although the sweetness is only one-third that of the nitro derivative), whereas the carboxyl derivative is not; this seemed to suggest that the nitro group may not be the B unit of the AH,B system, as assigned by both Shallenberger (263) J. J. Blanksma and P. W. M. van der Weyden, Red. Trau. Chim. Pays-Bas, 59 (1940) 629-632. (264) J. J. Blanksma and D. Hoegen, Recl. Trau. Chim. Pays-Bas, 65 (1946) 343-347. (265) J. J. Blanksma and P. G . Fohr, Red. Trau. Chim. Pays-Bas, 65 (1946) 711-721. (266) P. E. Verkade, C. P. van Dijk, and W. Meerbug, Red. Trau. Chim. Pays-Bas, 65 (1946) 346-360. (267) A. R Lawrence and L. N. Ferguson, J. Org. Chem., 25 (1960) 1220-1224. (268) J. Kirimura, A. Shimizu, A. Kimizuka, T. Ninimiya, and N. Katsuya, J. Agric. Food Chem., 17 (1969) 689-695. (268a) J. M. Tinti, D. Durozard, and C. Nofre, Natunvissenschafren, 67 (1980) 193-194.
304
CHEANG-KUAN LEI
and Acree' and Kier.'" However, this is only one example, and it would be desirable to investigate whether analogs in which the nitro group has been replaced by such other acceptors as carbonyl and halogen are still sweet. At the same time, whether the proton that is ortho to the nitro group represents the AH unit could also be confirmed by testing those derivatives in which the position(s) ortho to the nitro group carry no hydrogen atom.52 Tinti and coworkers268b*c also synthesized, and measured the sweetness of, a variety of analogs and derivatives of suosan (107a), including those
Suosan 107a
with the general structure X-C6H4-Y (see Table XXIVa). They proposed that the sweetness of these compounds is the result of interaction through three groups: ( i ) a hydrogen-bond donor, AH unit, ( i i ) a negatively charged B unit which binds to the receptor by ionic interaction, and ( i i i ) a hydrogenbond-acceptor unit, D, the spatial distance between these three units being: AH-B, 250; AH-D, 550; and B-D, 600 pm (see 107a). This oblique, planar, stereogeometric arrangement also resembles the direction parameters proposed by Kier'" and Shallenberger and L i n d l e ~ , 6and ~ both the distance and direction parameters proposed by van der Heijden and coworkers' l9 (see Fig. 13). The AH unit was assigned to the carboxyl group, as the sweet taste of compounds 2 and 3 in Table XXIVa is lost when the carboxyl group is absent or when it is converted into the methyl ester (compound 4 in Table XXIVa). It was suggested268' that this group mainly interacts with its counterpart on the receptor by ionic interaction. The absence of sweet taste (268b) J. M. Tinti, C. Nofre, and D. Durozard, Nufunvissenschuffen, 68 (1981) 143. (268c) J. M. Tinti, C. Nofre, and A. M. Peytavi, Z.Lebensm. Unters.-Forsch., 175 (1982) 266-268.
CHEMISTRY AND BIOCHEMISTRY O F SWEETNESS
305
TARLEXXIV"
Taste Properties of Compounds of the Structure X--C,H,-Y Compound No. I I
3 4 5 6 7
X 9
10
II I:! 13
14 15 16 17
18 19
20 21 22 23 24 25 ('
Y
X
-NO2 -NOZ -NO, -NOZ -NO, -NO2 - NO, -NOZ -NOZ -NO2 -NO, -NO, -NO, -NO2 -NO, -NO, -NO2 -CN -CN -CN -CN -CN -CN -CN
- NH-CO- NH-CHZ--CH2-CO, -NH-CO-NH-CH2-CH, -NH-CO-NH, -NH-CO-NH-CH2-CH,-CO2CH,
-NH-CO-NH-CH~-CO~ -NH-CO-NH-CH,-CH2-CHzCO,
- NH -CO-
N H -CH2-CH,-SO, -NH-CO-NH-CH=CH-CO, -NH-CO-O-CH,-CH2-CO, - N H --CO-CH,--CH2-CH2--CO, - N H -CSN H -CH,-CH2-CO, -CH2-CO-NH-CH,-CH,-CO, -0-CONH-CH,-CHZ-CO, - N H - NH-CO-CH2-CH2--CO, -CO-NH-NH-CH,-CH,-COz -NH-CO-CH(NHCOCF,)-CH2CH,CO, -NH-CO-CH(NHCOCF,)-CH,-COZ - NH-CO- N H -CH,-CH2-CO, -NH-CO-NH-CH2-C02 -NH-CO-CH( NHCOCF,)-CH2-CHZ-COZ -NH-CO-CH(NHCOCF,)-CH2-Co,
Taste"
Relative sweetness
S T
700
T T S T
-
T T T T S B S
T T S S S S
-NH-CO-CH(NHZ)-CH2-CH?-COi
S S T
-NH-CO-CH(NH,)-CH,-CO; -NH-CO-NH-CO-CH2-CH,-COT -NH-CO-NH-CH2-CH2-COI
S S T
-
-
10 -
2400 -
20
-
I000 I00 450 30 3000 3000 -
I2 450
-
B, bitter; S , sweet; and T, tasteless.
for compound 7 in Table XXIVa, where the -COT group is replaced by a -SO; group is, however, difficult to explain, although Tinti and coworkersZhXC attributed this to the bulkiness of the sulfonyl substituent. The B unit was assigned to the N H group closer to the -COZ group. Molecular models show that, in suosan, the -COT group is situated at a distance of -250 pm from this vicinal N H group. Furthermore, its replacement by an oxygen atom (compound 9 in Table XXIVa) or a CH, group (compound 10 in this Table) causes loss of the sweet taste. The aniline N H group cannot be the hydrogen-bond donor, as its replacement by an oxygen atom (compound 13 in this Table) did not eliminate the sweet taste. The bitterness of compound 12 in this Table may be due to increase in lipophilicity of the molecule (see Section 11,b). The ureido carbonyl group is not the
306
CHEANG-KUAN LEE
B (hydrogen-bond acceptor) unit, as it is also present in many of the derivatives that are not sweet. Its presence in these derivatives promotes the cis orientation of the amide (or thioamide) bond formed with the aniline amino group, as it is known, from infrared and n.m.r. studies,268dthat a bulky substituent on an amide (or thioamide) favors the cis orientation, owing to the steric interaction between the bulky group and the carbonyl oxygen atom. Such an arrangement will bring the AH,B system into the correct spatial separation from the D unit. It was proposed by Tinti and coworkers that the third hydrogen-bondacceptor unit, D, is the nitro substituent (compounds 1, 5, 11, 13, 16, and 17, in Table XXIVa) or the cyano group (compounds 18-21, 23, and 24 in the Table), the unit being 550 pm from AH and 600 pm from B. Although this may account for the sweet taste of these compounds, this tripartite system cannot explain the sweetness of most compounds that are sweet but which do not possess a negatively charged group, or D group, that can behave like a lone-electron-pair donor. (v) Dipeptide Esters.-Peptide fractions obtained by enzymic hydrolysis of proteins are frequently bitter. Dipeptides containing acidic and basic amino acids having small alkyl groups, or aromatic amino acids, are normally either tasteless, or have a very weak taste.268Furthermore, dipeptides consisting of sweet amino acids are normally almost tasteless, and blocking of terminal functions, for example, by acetylation of the amino group or methylation of the carboxyl group, always increases the intensity of bitterness, often269by a factor of ten. Thus, the accidental discovery by Mazur and coworkers’52that the methyl ester of the dipeptide L-aspartoyl-L-phenylalanine, that is, aspartame (18), is 160 times as sweet as sucrose27ocame as s ~ ~ also ~ ~ ’ ~been a big surprise. Since then, ~ - a s p a r t o y l - ~ - a l a n i n a m i d ehave found to be sweet. The more-stable N-(~-aspartoyl)-l,l-diaminoalkanes~~~~ (unlike the dipeptide esters, these are very stable to hydrolysis and cannot be cyclized to form the tasteless diketopiperazine) were synthesized, and these also possess intense sweetness, the methylcyclopentyl derivative being almost as sweet as aspartame (18). (268d) C. N. Rao, K. G. Rao, A. Goel, and D. Balasubramanian, J. Chem. Soc., A, (1971) 3077-3083. (269) T. Matoba and T. Hata, Agric. Biol. Chem., 36 (1972) 1423-1431. (270) M. R. Cloninger and R. E. Baldwin, Science, 170 (1970) 81-82. (270a) M. Sukehiro, H. Minematsu, and K. Noda, Seikarsu Kagaku, 11 (1977) 9-16; Chem. Absrr., 87 (1977) 168,407h. (270b) T. M. Brennan and M. E. Hendrick, US. Pat. 4,411,925 (1983). (270c) M. Verlander, W. D. Fuller, and M. Goodman, Eur. Pat. Appl. 0128654 (1984); W. D. Fuller, M. Goodman, and M. Verlander, J. Amer. Chem. SOC.,107 (1085) 5821-5822.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
3 07
NH b
18 Aspartame
Since that discovery, many studies of this compound have been made. It has been found that the L-phenylalanine moiety can be replaced by a variety of amino acids without loss of the sweet taste. Replacement of the L-aspartic acid moiety by other amino acids, on the other hand, always produces bitter pep tide^.^^' Therefore, it appears that derivatives of the 1-carboxyl group of L-aspartic acid may possibly be sweet when certain structural requirements are satisfied. The R' group of 108 must preferably be a small group, such as the C0,Me group of aspartame (18), and R2 must be a large, hydrophobic group, such as the benzyl group of aspartame. The length and size of the ester groups also influence the s ~ e e t n e s s . ' ~ ~ -The ' ~ ~intense .~~' sweetness of the dipeptide a-L-aspartoyl-D-alanine isopropyl ester (108, R' = CH,, R2= C02Me) supports this h y p o t h e ~ i s , 'confirmed ~~ by Fujino and who also reported that a number of esters of L-aspartoylaminomalonic acid (109) are intensely sweet (see Table XXV).The fenchyl
Rl 108
C02R 109
methyl diester was reported to have a sweetness of 22,000-32,000 times that of sucrose, and to be without any bitterness; this extremely high value has not been confirmed by other workers. It was proposed272that the primary amino group and the free carboxyl group function as the AH,B unit responsible for sweetness, with the large, hydrophobic group (R2 of 108) acting (271) R. H. Mazur, A. H. Goldkamp, P. A. James, and J. M. Schlatter, J. Med. Chem., 13 (1970) 1217-1221. (272) M. Fujino, M. Wakimusu, K. Tanaka, H. Aoki, and N. Nakajima, Natunvissenschaften, 60 (1973) 351.
308
CHEANG-KUAN LEE
TABLEXXV Relative Sweetness of L-AspartoylaminomaIonic Acid derivative^^'^ R substituent
Relative sweetness
Cyclopentyl Cyclohexyl trans-2-Methylcyclohexyl Fenchyl
300-600 550-880 5450-7300 22,200-32,200
as the Kier third feature. The sweetness of these compounds is, however, difficult to rationalize. In particular, it is hard to imagine that the fenchyl group can fit into the deep, narrow cleft where, it was proposed, the receptor site is situated,’563159 instead of the benzyl group. Because of the flexibility of the molecule, aspartame (18) can have nine possible conformations, arising from the combination of two rotameric series, namely, D I , DII,and DIlland FI, Frr, and Fill (see Fig. 30). Lelj
DI I
FIG. 30.-Possible
DIII
Projection Formulas”6 of Isomers of Aspartame (18).
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
309
FIG.31.-The Two Possible Conformations of Aspartame (18) Interacting with the Receptor Site."' [Key: 0, carbon; 0 , oxygen; 0, nitrogen; and 0,hydrogen.]
and coworkers159studied these conformations in aqueous solution at pH 3.511.7 by n.m.r. spectroscopy and by theoretical calculations. They deduced that the sweet taste of 18 is due to the FIDII or FIIDII conformer (see Fig. 3 1), as these conformations have space-filling properties similar to those of leucine. However, van der Heijden and argued that, in the FIDII conformation (see Fig. 31,i), the space-filling structure of the side chain (the benzyl group) cannot have any influence on the sweetness response, because if the NH: and CO; groups are attached to the receptor site, the space-filling group falls exactly in line with the projections of the rest of the molecule (see Fig. 31,i), Brussel and coworkers"' found that the length of the side chain is a limiting factor for perceived sweetness, suggesting that the FI,DII conformation is the more likely conformer to interact with the receptor site (see Fig. 344). This is in agreement with the findings that the length and size of the ester groups152-156.271 and those of the space-filling groups 120*152-154,156 influence the sweetness of all sweet-tasting dipeptide esters.
CHEANG-KUAN LEE
310
Using molecular models, Brussel and coworkers12’ observed that all of the sweet esters examined have side chains with lengths of 480-880 pm and a volume 20.030 nm3. An excellent correlation between the logarithms of the relative sweetness and the side-chain volume was found. For example, in the L-aspartoylaminomalonic esters2’* (109), the size of the space-filling group increases from -0.070nm3 for the cyclopentyl ester to about -0.013 nm3 for the fenchyl ester (see Table XXV). Beets,52however, argued that this interpretation seems difficult to accept unless the assumption is made that the growing bulk of the side chain prevents interaction with “bitter” sites, so that the increasing sweetness is actually a result of the diminishing masking bitterness. Even then, it is impossible to account for the extremely high sweetness of the esters of L-aspartoylaminomalonic acid. 111. BITTERNESS
Bitter taste, like sweet taste, is exhibited by a very diverse group of chemical compounds. Because the bitter taste is generally regarded as unpleasant, and because it is often associated with such compounds as those alkaloids and glycosides that are harmful to man, detailed studies have been minimal. There is thus very little information available from which to deduce the chemical grouping common to those compounds eliciting the bitter response. Just as a multiplicity of hydroxyl groups is normally related to sweetness, or -C=Smultiple nitro groups and the sulfur atom in the -S-Slinkage have been associated with bitter taste. Thus, it was observed4 that a compound containing three nitro groups, such as picric acid, is usually bitter, and that those with two nitro group may be bitter. Compounds having structure A are also frequently bitter,273and it was deduced that the bitterness of the acyl-thiocarbamide class of compounds is to structure B. CH20H
I
C-NO2 A
H S
I
II
-N---C--NH,
B
Such a grouping would be expected to exhibit tautomerism, as in 110, 111, and 112, and Beets” concluded that the presence either of the tautomeric groups 110 and 111 or the mesomeric group 112 is a sufficient condition for bitterness. The bitterness of the thio analog (102) of saccharin is presumably attributable to the same phenomenon, as it exists in tautomeric forms and has no hydrogen-donor group.52 (273) L. Henry, C. R. Acad. Sci., 121 (1895) 213. (274) A. L. Fox, Proc. Narl. Acad. Sci. V.S.A., 18 (1932) 115-120.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
110
111
31 1
112
The taste sensation of arylthiocarbamides may hold the key to understanding of bitter taste. For example, (p-ethoxyphenyl)thiourea,which is generally considered to be exceedingly bitter, is tasteless to some people (taste blindness).274Taste-blind persons are not, however, insensitive to the taste of other bitter compounds, such as quinine. It appears that the bittertaste response can be elicited by chemical groupings having chemical properties with more than one set of “common” features. Therefore, there is probably more than one bitter-taste quality”; the chemical feature common to one of them is contained in structure B, proposed by A. L. The mechanism for the initial chemistry of bitterness is not well understood. Dastoli and coworkers27’ reported the isolation and purification of a bitter-sensitive protein from porcine tongue. This appeared to be localized at the back of the tongue, the area presumed to be the most sensitive to bitterness. The protein appeared to interact with bitter-tasting substances in a manner directly proportional to the bitterness of the compounds.275 Price argued that, The result was, however, strongly criticized by S. based on the data reported, the values of 1/K (K being the rate constant) differed from the taste threshold, by a factor of 100 times the threshold concentration for caffeine, to over 6500 times for brucine (see Table XXVI). Therefore, even if allowances for species differences are made, it is unlikely that the protein in question is the physiological receptor for bitter-taste TABLEXXVI Taste Threshold of Bitter Compounds, and Equilibrium Constants of Binding of Bitter Compounds by a “Bitter-sensitive” Protein276 Compound Quinine. HCI Brucine . HCI Naringin Caffeine
( 1 / ~ ) ~(mM) ’~
3.9 4.6 5.1 7.8
Threshold c o n ~ e n t r r t i o n( M ~ ~) ~ ~ ~ ~ ~ 3 x 10-5 7 x lo-’
7 x 10-~
(275) F. R. Dastoli, D. V. Lopiekes, and A. R. Doig, Nature, 218 (1968) 884-885. (276) S. Price, J. Agric. Food Chem. 17 (1969) 709-711. (277) C. ffaffmann, in J. Field (Ed.), Handbook ofPhysiology, Vol. 1, American Physiological Society, Washington, D.C., 1959, pp. 587-633. (278) F. M. Scholl and J. C. Munch, J. Am. Pharm. Assoc., 26 (1937) 127-129.
CHEANG-KUAN LEE
312
stimuli, or that it can be considered a convenient model approximating the properties of the receptor(s). 1. The AH,B Concept for Bitterness
Kubota and K ~ b examined o ~ ~ a~series of diterpenes of the Isodon species, and suggested that the bitterness of these diterpenes could be explained by using the AH,B concept that had been employed to explain sweetness. It was, however, proposed that the AH-B distance is -150 pm, compared to -300pm for sweetness. Such a distance would probably cause strong, intramolecular hydrogen-bonding. Therefore, whereas sugar sweetness is restricted by intramolecular hydrogen-bonding, such bonds seem to be a prerequisite for the bitter taste. Studies on the bitterness of other compounds'399280-282 do not, however, support this model. The bitter amino acids and sugars, for example, do not possess an AH,B unit of this dimension. As already mentioned, there is some evidence suggesting that there is more than one type of bitter-taste quality2' and receptor.283If this is the case the diterpenes probably interact with a receptor showing a steric requirement different from that involved with the other classes of compounds. As with sweetness, an enantiomeric difference in response is observed in the bitter-taste quality. Isodonal (113) and dihydroisodonal (114) are bitter, 0
.H
113
114
whereas the corresponding stereoisomers, trichodonin (115) and dihydrotrichodonin (116), in which the 11-hydroxyl group is in the p orientation, are tasteless. For molecules involved in a bipartite interaction with a receptor site, a difference in the taste of enantiomers is only possible if at least one (279) T. Kubota and I. Kubo, Nature, 223 (1969) 97-99. (280) H.Wieser and H. D. Belitz, 2.Lebensm. Unters.-Forsch.,159 (1975) 65-72; 160 (1976) 383-392. (281) C. K. Lee, Stoerke, 29 (1977) 204-209. (282) P. C. Schober, P. W. Bowers, and S.E. Smith, J. Phorm. PharmacoL, 30 (1978) 111-112. (283) M. J. Hall, L. M. Bartoshuk, W. S. Cain, and J. C. Stevens, Nature, 253 (1975) 442-443.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
313
1 I6
115
other feature of the molecular profile is involved in the interaction. This may frequently be an inert, structural detail having a simple, morphological function in the interaction, or it may, in some cases, be a polar or a polarizable moiety capable of active participation in the interaction in a sense analogous to that postulated by Kier for sweet taste. Systematic studies to test this concept have yet to be conducted. One criticism of the Kubota and Kubo p r o p o ~ a l ” ~is that, as with sweetness, some of the AH,B units assigned d o not seem capable of forming energetically significant hydrogen-bonds, let alone strong ones. It has also been suggested that the AH-B distance is too short.”3 However, even if the concept is accepted, together with the inclusion of a third actively participating feature, as suggested by Beets,” it is still not possible to account for the bitterness of numerous other compounds. For example, quinine (117), isohumulone (118), and the lupoxes-A (119; see Ref. 284) HO
B
HO”
AH
OMe 117
1I8
119
contain functional groups that could serve as AH,B systems in the sense proposed by Kubota and Kubo, but, in such bitter stimulants as limonine (120) and nomilin (121), there are sterically adjacent polar features, but none of these can be reasonably assumed to serve as a bitter AH,B unit. These molecules contain a large number of oxygen atoms, but none of them (284) S .
R. Palamand and J. M. Aldenhoff, J. Agric. Food
Chem., 21 (1973) 535-543.
314
poo@$/$j CHEANG-KUAN LEE
Q
H
H
121
120
is present as a proton-donor group, such as an alcoholic or enolic hydroxyl group. In contrast to this group of bitter compounds, found in citrus, the sesquiterpenes possess only a single lactone group, and this seems to suffice for bitterness. Germacrolide (122) and many analogous, naturally occurring,
122
C-15 lactones are examples of this class; the structure consists mainly of a lipophilic profile. These may, perhaps, interact as for the tautomeric form discussed earlier (see Section 11,4). Belitz and coworker^^^^*^*^ studied a large number of bitter-tasting amino acids, their esters, and N-acyl derivatives, and found that a monopolar (electrophilic), hydrophobic structure is a sufficient condition for bitterness of these compounds. In bipolar, hydrophobic compounds, the overall steric properties determine whether they are sweet, bitter, bitter-sweet, or tasteless. In general, the structural requirement for sweetness is the presence of two groups, an electrophilic group (p') and a nucleophilic group (p-), having the appropriate spatial arrangement. An apolar group (a) is not essential, but is important for the intensity of taste. For bitter compounds, only one polar group, an electrophilic group (p+), and an apolar group (a) are required. In rectangular coordinates, the polar groups (p') and (p-) would occupy the positions shown in Fig. 32. By superimposing probable conformers of a 2-amino-1-carboxylic acid on the proposed system, as in Fig. 32, Belitz and coworker^^^^^^^^ were able to indicate positions that are allowed and (285) H. D. Belitz, W. Chen, H. Jugel, R. Treleano, H. Wieser, J. Gasteiger, and M. Marsili, in J. C. Boudreau (Ed.), Food Taste Chemistry, ACS Symp. Ser., 115 (1979) 93-131.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
315
*Y
7
(p-)m
"'
; ,
>'\ - - - - - - - CR, Ri 2
-t
*x
+z
FIG. 32.-Fixation dinates.*"
of Sweet and Bitter Compounds (Amino Acids) in Rectangular Coor-
forbidden for sweet compounds (see Fig. 33). For example, D-norleucine, which is sweet, could occupy the positions (p')(p-)a(+x, *y, +z) (see Fig. 34,i), but L-norleucine (which is bitter), in the stretched conformation, cannot occupy such positions, because of forbidden positions (see Fig. 32,ii and iii). It is, however, difficult to rationalize the enantiomeric-response difference by using this concept of one polar group and one apolar group. For L-norleucine, which is bitter, it was proposed that the apolar group occupies forbidden positions285(see Fig. 34,ii). However, it can also occupy
t -- - - -
--- -
- -
.
..
I
\
, \-
- -- - - .
FIG.33.-Fixation of Sweet and Bitter Compounds (Amino Acids) in Rectangular Coordinates2$' [Key: 0 , allowed positions for sweetness; 0, forbidden positions for sweetness.]
Y
\
-
&-Norleucine (bitter) (p+)(p-)s(+x, w, +z) allowed
FIG. 34.-Fixation of D-Norleucine (Sweet) and L-Norleucine (Bitter) in Rectangular Coordinates in Allowed and Forbidden Positions.285
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
317
I'
--d
p Jr Type 4 :
lvl
lype 5
bitter
IYII Type6
bitter
hitter
FIG. 34a.-Schematic Representation of the Structural Parameters for Sweet and Bitter Taste.**'" [n,/e, and n,/e, represent the systems of the stimulant and the receptor respectively.]
allowed positions, (p')a(+x, *y, +z), after 60" rotation about the y-axis (see Fig. 34,iii). Similarly, rotation about other axes is possible. In general, Belitz and C O W O ~ ~ expanded ~ ~ S ~ Shallenberger ~ ~ * ~ ~ and ~ ~ Acree's concepts to include all nucleophilic-electrophilic systems, instead of confining it to only those potential, polar, contact groups capable of forming hydrogen bonds. They also expanded Kier's third sitelm by proposing the existence of a hydrophobic pocket having a nucleophilic-electrophilic system at the receptor level, rather than a restricted, hydrophobic contact. Using this model, they classified the sweet stimulants into six types (see Fig. 34a). Compounds containing a suitable nucleophilic-electrophilic (285a) H. D. Belitz, W. Chen, H. Jugel, W. Stempfl, R. Treleano, and H. Wieser, in P. Schreier (Ed.), Flaoour '81, Walter de Gruyter, Berlin, 1981, pp. 741-755.
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system are sweet (Type 1, see Fig. 34a). Whether a compound is sweet (Type 2), bitter-sweet (Type 3), or bitter (Type 4) depends on the particular steric arrangement of the nucleophilic-electrophilic system and the apolar groups in the molecule. Types 5 and 6 are for bitter compounds. These require suitable apolar and polar groups; the latter may be an electrophilic (Type 5 ) or a nucleophilic (Type 6) group. Furthermore, the presence of bipolar group excludes the possibility that the compound will be bitter (Types 3 and 4). In essence, this hypothesis is very similar to that proposed by Beetss2 (see Section 111,3). 2. Lipophilicity and Bitterness
The importance of lipophilicity to bitterness has been well established, both directly and indirectly. The importance of partitioning effects in bitterness perception has been stressed by Rubin and coworkers,286and Gardner287demonstrated that the threshold concentration of bitter amino acids and peptides correlates very well with molecular connectivity (which is generally regarded as a steric parameter, but is correlated with the octanolwater partition coefficientg8).Studies on the surface pressure in monolayers of lipids from bovine, circumvallate papillaeza8also indicated that there is a very good correlation between the concentration of a bitter compound that is necessary in order to give an increase in the surface pressure with the taste threshold in humans. These results and the observations of others289 suggested that the ability of bitter compounds to penetrate cell membranes is an important factor in bitterness perception. In the sugar series, when a sugar molecule is chemically modified to increase its lipophilicity, by, for example, esterification, etherification, or g l y c ~ s i d a t i o n , ' ~this ~ * ~frequently ~' causes an increase in the bitterness. It is well established that sugar acetates are bitter,77*281 and some similar evidence has been reported for the methyl ethers.I2' The bitterness of glycosides seems to increase with increasing length of the carbon chain of the a g l y ~ o n(see ' ~ ~Table X). It has also been observed that bitterness begins to appear in certain monodeoxyhexopyranosyl structures, and that dideoxyhexopyranosides are distinctly bitter, and devoid of sweet taste.lz6 The cyclohexanepolyols also demonstrate this trend; the pentols and hexols are definitely sweet and devoid of bitterness, but the diols are distinctly bitter, with little or no sweetness.Ia It was suggested'26 that the lack of sweetness of the dideoxy sugars is due to increased lipophilicity, resulting in an (286) (287) (288) (289)
T. R. Rubin, F. Griffin, and R. Fischer, Nature, 195 (1962) 362-363. R. J. Gardner, Chem. Senses Hauor, 4 (1979) 275-286. N. Koyama and K. Kurihara, Biochim Biophys. Acra, 288 (1972) 22-26. J. G. Brand, B. R. Zeeberg, and R. H. Cagan, Znr. J. Neurosci., 6 (1976) 237-278.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
319
alignment of the molecule on the receptor site that differs from that of the parent sugar. This concept appears to be in agreement with the postulate52 that the presence of two lipophilic centers drastically changes the composition of the orientation pattern of the molecules, so that the “sweet” AH,B unit(s) of the molecule are prevented from interacting with the “sweet” receptor site, and favor other orientations, resulting in a collection of “bitter” interactions. Beets52also suggested that it may be possible that a tripartite arrangement involving a methylene group, similar to Kier’s postulate for intense sweetness, is responsible for the bitterness of deoxy derivatives. The bitterness of amino acids has also been related to the hydrophobicity of their side chains280*28’*290; this hydrophobicity, particularly in those amino acids having purely hydrophobic side-chains (as measured from solubility factors291),is directly proportional to the intensity of the bitterness.292 Similarly, the bitterness of peptides is caused by the hydrophobic action of the side chains of amino Peptides having a high “average hydrophobicity” (>5.4 W/amino acid residue) are generally bitter, whereas those of low, average hydrophobicity are not. This generalization does not, however, hold true for many peptides containing glycine; these have average hydrophobicity, but are Although the average hydrophobicity is generally considered to reflect lipoid solubility, Beets52 suggested that it may be a measure of the availability of a particular group for hydrogen bonding to the receptor site. However, in general, the thresholds (expressed as the logarithm of the reciprocal of threshold concentration in mol/liter) and hydrophobicity are, significantly, linearly related ( r = 0.88, significant at the p‘ <0.001 Gardner294reported that excellent, quantitative relationships also exist between the thresholds of amino acids and peptides and the connectivity indices (‘x”).These relationships are of the same order of significance as the relationship between thresholds and hydrophobicity, but they are applicable to a wider range of compounds. Further supporting evidence for the importance of lipophilicity in bitter response is provided by the taste of isohumulone (118), the principal, bitter-tasting component of beer, and some of its derivatives. Isohumulone can exist in both cis and trans forms. Clarke and Hilderbrand2” reported that the cis form, having a partition coefficient of 0.78, is more bitter than (290) K. H. Ney, Z. Lebensm. Unters.-Forsch., 147 (1971) 64-68; 149 (1972) 321-323. (291) C. Tanford, J. Am. Chem. Soc., 84 (1960) 4240-4247. (292) J. C. Boudreau, in G. Charalambous and G. E. Inglett (Eds.), Hauor of Food and Beuerages, Academic Press, New York, 1978, pp. 231-246. (293) K. H. Ney, in Ref. 285, pp. 149-173. (294) R. J. Gardner, J. Sci. Food Agric., 31 (1980) 23-30. (295) B. J. Clarke and R. P. Hilderbrand, J. Inst. Brew., 71 (1972) 26-29.
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the trans form, whose partition coefficient is 0.64, although other reports296 indicated that they have similar bitterness. A more convincing example is p-isohumulone, the reduced form of isohumulone. The introduction of a hydroxyl group makes p-isohumulone more lipophobic than the parent compound, and, consequently, it is less bitter.297Furthermore, just as for the cis- and trans-isohumulones, cis-p-isohumulone would be expected to have a higher partition coefficient than the trans form, and this was reported to be the case.298 Reduction of the isoprenyl groups of isohumulone and p-isohumulone to tetrahydro- and hexahydro-isohumulone, respectively, increases the lipophilicity of the resultant products. A corresponding increase in their bitterness was observed.297A similar increase in bitterness was reported282 for hydroquinine following reduction of the vinylic double bond of quinine (117). Because the stereochemistry of the quinines does not have any significant effect on the bitterness, this change must be due to change in the relative l i p ~ p h i l i c i t yof~ the ~ ~ compound. 3. BitternessSweetness Relationships
That the sweet and bitter responses are intimately associated is clear from the results of gustatory studies of all of the conformationally defined sugars and of other organic compounds. If a carbohydrate has any taste at all, this is invariably sweet, bitter-sweet, or bitter. Chemical modification may alter the taste of a sweet compound so that the product is bitter-sweet or bitter, and it is now generally agreed that the two basic tastes may each be a feature of a single compound. It appears, therefore, that the interactions of these polyfunctional stimulants involve two different sets of receptor sites, representing sweet and bitter modalities.20*23952*299 It may be safely assumed that each of these sets of receptor locations is structurally somewhat heterogeneous, and that it occupies a specific area of the sensory epithelium, with a characteristic, possibly very complex, t~pology.’~ Because knowledge of this taste modality is still minimal, the topological structure of the taste modality is pure speculation. One such speculation was offered by Beets.52 To simplify his reasoning, Beetss2 assumed the topology to refer to a two-dimensional area of the sensory (296) J. S. Hough, D. E. Briggs, and R. Stevens, Malting and Brewing, Chapman and Hall, London, 1971. (297) P. H. Todd, P. A. Johnson, and L. R. Worden, Tech. Q. Master Brew. Assoc. Am., 9 (1972) 30-32. (298) M . Verzele and A. Khokher, 1. Inst. Brew., 73 (1967) 255-257. (299) D. G. Guadagni, V. P. Maier, and J. G. Turnbaugh, J. Sci. Food Agric., 25 (1974) 1349- 1354.
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epithelium, although the topological structure of the modality is undoubtedly very complex, extending into space and time; the arguments will, however, still be fundamentally valid. If the topologies of the areas representing the sweet modality (S) and the bitter modality (B) are represented by a circle and a hexagon (see Fig. 35), the sensory epithelial area that will be able to complex with a specific structure of the stimulant molecules, to produce the sweet taste, is distributed over the areas SB and SB (area of overlap; see Fig. 35,i), whereas that capable of forming interaction complexes contributing to the bitter modality is distributed over the areas SB
i
ii purely sweet
iii purely b i t t e r
V
sweet with a secondary bitter taste
iv b i tter-sweet
vi no response
FIG. 35.-Diagrammatic Representation of the Topologies of the Areas Representing the Sweet (Circle) and Bitter (Hexagon) M~dalities.'~
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CHEANG-KUAN LEE
and SB. Thus, the area of overlap, SB, contains a variety of representatives of the sweet and bitter collections of receptor sites. This being the case, a purely sweet molecule may be expected to interact with structurally suitable features distributed over the areas SB and SB (see Fig. 35,ii); interactions in the area of overlap SB do not produce a secondary bitter note, but are essential for the generation of the sweet taste. A bitter taste can be produced only when suitable features in the area of the overlap and in the whole of the bitter area (SB) interact, as in Fig. 35,iii (pure bitter), Fig. 35,iv (bitter-sweet), and Fig. 35,v (sweet with a secondary bitter taste). Therefore, as long as the interactions are distributed over the whole area representing a modality, that modality will be identified by higher centers, provided that its intensity is not subliminal. If interactions are distributed over a part of a modal area (see Fig. 35,vi), identification of the modality will not result, so that no response will be produced. Interactions in the bitter or in the sweet area have a low efficiency, or they may be effective but involve only a small fraction of the sites. This can only affect the intensity of the generated response. As has already been discussed, the 3- and 4-hydroxyl groups in aldohexopyranosyl structures constitute the most effective AH,B systems for sweet taste, whereas the 1-hydroxyl group does not appear to play any part. The distinctly bitter taste of p-D-mannose, which differs from p-Dglucose (which is devoid of bitterness”) only in the attachment of the 2-hydroxyl group, suggests that the anomeric-oxygen atom in the p-D configuration interacts with the 2-hydroxyl group to elicit the bitter response of p - ~ - m a n n o s e .The ’ ~ ~involvement of the anomeric-oxygen atom implies that the ring-oxygen atom also functions in a chemical manner, to elicit the bitter response, as the anomeric-oxygen atom can have no chemical existence ’ ~ does ~ not prove except by reason of the ring-oxygen atom per ~ e . This that the ring-oxygen atom actually binds to the taste receptor, but indicates that its presence is essential. Therefore, in bitter-sweet sugar molecules there appears to be a “sweet end” and a “bitter end,” and it is possible that such molecules are “polarized” on the taste receptors so that one end of the molecule binds to sweet receptor(s) while the other is attached to bitter receptor-sites (see Fig. 36). Alternatively, these molecules may distribute themselves, some on sweet receptors (see Fig. 3 7 3 and some on bitter receptors (see Fig. 374). Theoretically, an interaction of the type shown in Fig. 36 can only occur on the membranes of receptor cells carrying sites belonging to both types of modality, these being separated by a sufficiently small distance, not greater than the length of the molecule.52 Consequently, such interactions, if they occur at all, can only take place in the area of overlap (SB; see Fig. 35,i). However, two areas representing different informational modalities
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
323
FIG.36.-Binding of a Bitter-sweet Molecule to a Sweet Receptor- and a Bitter Receptor-site.
can only overlap to a limited extent, and increasing overlap necessarily results in a gradual blurring of the informational identity of such modalities, until they become identical at total overlap. Because such interactions can occur only in an area of overlap, no gustatory response can be generated from an interaction pattern consisting exclusively, or even mainly, of the type represented in Fig. 36. Therefore, Beets52argued that any taste response will be the result of the type shown in Fig. 37, or of interactions of the type shown in Fig. 36 in combination with an excess of interactions of the type shown in Fig. 37. To distinguish between these possibilities, Birch and Myl~aganam’~’ studied the effect of the sweetness of sucrose and the bitter-sweetness of methyl a-D-mannopyranoside on the bitterness of quinine sulfate, and the effect of the bitterness of quinine sulfate and methyl a-D-mannopyranoside on the sweetness of sucrose. A significantly diminished sweetness-response
(i)
(ii)
FIG. 37.-Binding of One Molecule of a Bitter-sweet Compound to (i) a Sweet Receptor, and of a Second Molecule of the Same Compound to (ii) a Bitter Receptor.
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CHEANG-KUAN LEE
was observed with methyl a-D-mannopyranoside, but not with sucrose, after presaturation of the human tongue with quinine sulfate solution. When the tongue was presaturated with sucrose, the bitterness of the D-mannoside was found to be significantly lowered, whereas that of quinine sulfate was not affected. These results strongly suggested that the bitter-sweet D-mannoside was simultaneously polarized on a sweet-receptor site and an adjacent, bitter-receptor site (see Fig. 38). Beets” argued that a necessary condition for the validity of this conclusion is, obviously, that the paired sites can only operate as a unit, and that no effective contribution to the sweet- or bitter-taste response can be obtained by the separate interaction of either site with the sweet (see Fig. 37,i) or the bitter end (see Fig. 37,ii) of the glycoside molecule. This restriction immediately assumes the existence of two different types of sweet-receptor site, one type producing sweetness by interaction with a single set of structural features in the stimulant, while the second type can only produce a response in cooperation with an adjacent site. This would mean that the sweetness of predominantly sweet molecules and that of sweet-bitter molecules is produced by different mechanisms, and similarly, for bitter and bitter-sweet molecules. The alternative, more generalized concept, as shown in Fig. 37, does not impose such a strong restriction, as it does not require that sweetness or bitterness is attributable to summation of identical interactions with identical sites, but with a pattern of contributions arising from a multiple collection of sites distributed over an epithelial area having a characteristic topology. The collection is assumed to comprise many structurally related types of membrane features having the ability to accommodate sweet or bitter molecules with various efficiencies. Some of these, such as those in the area SB (see Fig. 35,i), function separately, while others, like those in the area of overlap SB, operate in pairs. Beets52stressed that this does not mean that there is necessarily a major difference between the subsets of “sweet” sites in the areas SB and SB (or bitter sites in areas SB and SB), but only that the areas SB and SB contain no, or hardly any, bitter sites, or sweet sites, respectively. Consequently, bilateral interactions
\ CH2OH y & oHk i:t t se ro R y i t t e r ~
“sweet end”
\
end”
HO
FIG. 38.-“Polarization” of Bitter-Sweet Molecules on Taste receptor^.'^'
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
325
of the type shown in Fig. 36 cannot occur in SB or SB. Therefore, if this generalized concept is accepted, the bitter and sweet modalities are related by a topographical overlap of their interaction patterns.
IV. BIOCHEMISTRY O F SWEETNESS Taste-modality recognition is a function of the cells of the taste buds. Perception of the sensation is a result of complex processes in the brain. The biological events that are discussed are those that occur, or are suggested as occurring, in taste-receptor cells, beginning at the instant when the taste-stimulus molecule interacts with the cell, until the membrane of the receptor cell is polarized. These are peripheral events. However, our knowledge of the peripheral mechanisms in taste perception is not sufficiently complete to provide a detailed, biophysical explanation of this phenomenon. Nevertheless, several stages in this explanation have been hypothesized, and some are demonstrable. Studies on the biochemistry of the taste system should take into account results obtained at other levels, such as electrophysiological recordings and, particularly, behavioral responses to taste stimuli. The term “sweetness” should strictly be used only in studies conducted on humans, because the description of taste modality is a verbal response. It is usually concluded that positive behavioral responses in animals, that is, preferences, or electrophysiological response to a stimulus compound that is known to be sweet to man, are due to the sweet taste. This may not necessarily be true in some cases, because behavioral or electrophysiological response may result from other taste modalities. It is, therefore, critical that comparative aspects be carefully interpreted. 1. The Sensory System
Taste and smell, unlike sight and sound, are not induced by any known waves in the air, but are caused by the presence of certain specific, chemical compounds by interaction with certain receptor-sites. The power of reaction to chemical stimuli has evolved from the diffused, and confused, chemotactic sensitivity of the primitive protozoa through to man, where specialized organs have been developed. The function of a sensory system is to select suitable modalities from the multitude presented by the environment, and translate them into corresponding modalities of sensory information that are then projected and processed into the various parts and finally submitted to the central processing-unit, the brain. A working hypothesis of the mechanism by which the taste system senses chemical compounds is that macromolecules that are
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part of the gustatory receptor-cells are able to interact with particular gustatory-stimulus molecules. Thus, the initiating event is the arrival of a stimulus molecule with particular structural details and its interacting effectively with a site of the receptor area, or that, within a very short period of time, a number of such effective interactions with a group of related sites take place. This interaction is assumed to be a binding interaction, initiating a specific message about the quality and concentration of a stimulus. The nerve impulses thus initiated are transmitted to the central nervous system for further processing. The whole sensory process consists of three different extensions in time and space.” The first is that stimulation of the receptor area does not normally produce a single peripheral, information-pattern, but continues for some time. The result is that a rapid succession of such patterns is generated, and transmitted to the sensory system. In reality, it is probably not possible to isolate a single, peripheral pattern from this succession, as the latter can be assumed to be produced as a single block of information extending semi-continuously over the period of stimulation. Beets52 suggested that, for a clearer comprehension, it is necessary to assume the generation of a single signal at the periphery, and to concentrate on the fate of this signal, bearing in mind that it does not travel alone, but as the smallest element of an information pattern. The second is that the initiated signal is the smallest building-element of a peripheral information-pattern extending into the space in which it happens to be generated; the nature of the dimension of this space depends on the sensory process involved. The third is that the signal, or, rather, the causal sequence of its successors, travels through the various parts of the sensory system, undergoing various projecting and processing operations, until it ends its life at the central processing unit.” 2. The Peripheral Mechanisms in Taste
The mechanisms by which the taste (and also the olfactory) system senses chemical compounds is assumed to occur by way of a chemoreceptory system that interacts effectively with a broad, structural variety of stimulant molecules, by means of a receptor epithelium consisting of the mosaic of adjacent, peripheral membranes of many receptor cells, exposed to a medium carrying stimulus molecules. A receptor cell is conveniently and, for our present purpose, sufficiently defined as a cell equipped to interact, according to some mechanism, with stimulus molecules, to convert the effect of this interaction into a signal, and to project this signal into the system.’’ The taste receptor is thus a differentiated, epithelial cell synaptically contact-
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
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ing sensory nerve-fiber(s). A collection of these cells and other, presumably supporting, cells forms the multicellular structure known as the taste bud. The taste receptors allow for the collection of only a small amount of information, enabling us to distinguish the taste modalities, namely, sweet, bitter, saline, or acid. Responses to the true taste-receptors are produced with a high degree of probability over relatively simple pathways, containing few fibers. The cortical centers for tastes, if present at all, are Effective, stimulus molecules in at least one type of chemoreception are those which have the solubility or volatility to enable them to approach the receptor area. Such molecules are found in all structural classes of chemical compounds, carrying all possible functional groups, and differing widely in chemical reactivity and stability. This implies that a single receptor-system is equipped to interact rapidly and effectively with this wide variety of molecules, and to produce distinct, informational patterns which permit an amazing degree of chemoreceptory discrimination. A chemical, interaction mechanism, with the ability to achieve all of this, must indeed be very complex. It clearly cannot be an enzymic mechanism, as that would require the presence of a multivalent, enzymic system having the versatility and ability to convert in some way not only most of the molecules that have ever been synthesized, but also those which the imagination of a chemist could design.52 Stimulus molecules approach the receptor area in a random distribution. Therefore, there cannot be a homogeneous distribution of chemical or enzymic processing capabilities over the area, as this would produce a chaotic mass of information. The capabilities of such precise chemoreceptory discrimination that we observe can only arise from an ordered system in such a way that specific reaction-types would be localized, or at least be concentrated in specific areas of the e p i t h e l i ~ m . ’ ~ There has, so far, been no report of the generation of fragments, or conversion products, of stimulus molecules. We are, therefore, left with the only remaining possibility, namely, that chemoreceptory interaction is a reversible, physical contact between a stimulus molecule and a location on the epithelium, resulting in the generation of an energy effect. This conclusion removes the speculative reputation of the concept of “receptor site,” and gives it its proper position in real it^.'^ Its function in the process may have many facets and may even be questionable, but its existence is undeniable, and the physical contact between a stimulus molecule and the membrane defines a receptor site having dimensions that are not too different from those of the accommodated molecule.52 Furthermore, the receptor site must have a specific function in the information-generating process. This (300) J. Z. Young, The LiJe of Mammals, Clarendon Press, Oxford, 1957.
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CHEANG-KUAN LEE
is clear because the structural information contained in the molecule can be transferred to the membrane only by means of some site-selecting process; a specific, stimulus molecule interacts effectively only with one group of sites, perhaps less with a second group and not at all with the remainder. Thus, the receptor site in chemoreception may be defined” as “a location of molecular dimensions on the peripheral membrane of the receptor cell with the ability to accommodate in a reversible complex any single member of a class of structurally related stimulus molecules.’’ 3. Taste-receptor Binding
and biochemical a. “Sweet-sensitive Protein.”-Neurophysiological experiments both point to the existence of several steps in taste-receptor functioning for transduction of the chemical message. The first step involves the binding of the stimulus to the taste-receptor cell-membrane at the apex of the taste (see Fig. 1). Attempts to detect this interaction of the stimulus with the receptor have generally taken the form of attempting to measure the putative, receptor-stimulus complex.30’ Dastoli and Price49 reported the extraction and purification of a “sweet-sensitive protein” that interacts specifically with sweet compounds. From changes in refractive index of the protein on mixing with several sugars and saccharin, binding constants of the sugars and of saccharin to the protein-containing fraction were calculated, but Cagan” criticized this reported specificity of “binding,” as it did not correspond well with the data on relative sweetness of the sugars and saccharin in humans, nor did the data correspond with the taste-preference data on bovines. The conclusion that the purified protein is a specific-receptor protein, and the many conclusions and assumptions, have since been shown to be in error. Experiments by Koyama and Kurihara,288Price and Osfretsova and coworker^^^^ and Price304 showed that the “sweet-sensitive protein” is very heterogeneous. Furthermore, proteins that were extracted from circumvallate and fungiform papillae, which contain taste buds, were shown288(by disc-gel electrophoresis) also to be present in the epithelium lacking taste buds, so that they are not unique to taste buds, and, therefore, probably without any special function in the taste-sensing mechanism. (301) R. H. Cagan, in H. L. Sipple and K. W. McNutt (Eds.), Sugars in Nutrition, Academic Press, New York, 1974, pp. 19-36. (302) S. Price and R. M. Hogan, in Ref. 46(a), pp. 397-403. (303) I. B. 0. Osfretsova, E. Kh. Sataryan, and R. N. Etingof, Proc. Acad. Sci. USSR, 223 (1975) 1484-1487. (304) S. Price, N a m e , 241 (1974) 54-55.
329
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
.loo
150
.90 100
2
50
0
hcross D.GlucoseD-Fructose LactoBe Biochemical
FIG. 39.-Binding Bovines?''
r
%Cross D-GlucoseD-hUctoee
.80
-70
1 x
Lactose
Behavorial
of Sugars to Taste Papillae versus Behavioral Taste-preferences of
b. Binding of Taste Stimulus to Taste Receptors.-Studies similar to that of Dastoli and Price were reported by Hiji and coworker^^^^^^^^ using rat tongue. It was demonstrated that the affinity of various sugars to these protein fractions paralleled the magnitude of the electrophysiological response of the chorda tympani nerve, which innervates the anterior twothirds of the tongue. In addition to this, Price276 showed that protein preparations from other parts of the animal body do not have any affinity for sweet compounds. However, in the light of later findings,2B8,302-304 no arguments in favor of the specialized receptor-site can be constructed from these results. Cagan301sought to confirm these results, paying special attention to control preparations lacking taste buds, in order to check the specificity of the effect. 14C-Labelledsugars were bound to homogenates of epithelium, or to intact, rat-tongue epithelium, but the results obtained were not reproducible, probably due to the combined effect of the small proportion of taste-bud cells and the relatively large amount of nonspecific binding to the rest of the tissue3'' that occurs. The specific binding of taste-stimulus compounds, and correlations between the biochemical results obtained and the behavioral responses, have also been studied (see Fig. 39). Direct measurement of the binding of (305) Y. Hiji, N. Koboyashi, and M. Sato, Comp. Biochem. Physiol., B, 39 (1971) 367-375; Kumamoto Med. J., 20 (1969) 137-139; 22 (1969) 104-107. (306) Y. Hiji and M. Sato, in Ref. 165, pp. 221-225.
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CHEANG-KUAN LEE
sweet-tasting compounds to bovine taste-papillae and nontaste-papillae appeared to show that the posterior region of the tongue, where most of the taste buds are localized in the circumvallate papillae,307are somewhat more sensitive to sugars than is the anterior area,3o8v3w where the fungiform papillae (which contain only a small percentage of taste buds3") are located, although stimulation of the calf tongue with sugars gave poor electrophysiological responses from the two nerves that innervate the tongue. Studies5' on the binding of l4C-labe1led sugars to homogenates of bovine tastepapillae and nontaste-papillae indicated that sugars bind more extensively to taste-bud papillae homogenates. In addition, sucrose, D-fructose, and D-glucose, which are sugars highly preferred by cows,31obind to tastepapillae homogenates to a greater extent than does l a c t o ~ e , ~which " is a poor s t i m ~ l a t o r . ~The ~ ' character of the binding to circumvallate papillae also appeared to differ from that to the control, fungiform papillae, as binding to circumvallate papillae could be inactivated by heating the tissue preparation prior to the binding a ~ s a y ~ (see ' , ~ Fig. ~ ~ 40). The temperaturestability of this binding was also in agreement with the results of behavioral and electrophysiological experiments, that is, the binding was reasonably temperature-insensitive, although it could be inactivated by prolonged heating. By measuring the dissociation of the labelled, stimulus molecule in a medium containing a low concentration of the stimulus compound, and by displacing the bound, labelled molecule with a large excess of unlabelled molecule added to the complex,312the binding was also found to be reversible. The binding specificity of D-[ L4C]glucoseby the taste-papillae membranes, compared to that of control membranes isolated from epithelial tissue, has been confirmed in two s t u d i e ~ . ~One ~ ~ inherent . ~ ' ~ problem in the approach is that the stimuli, primarily carbohydrate sweeteners, are not ideal model compounds to use, as they are not active at low concentrations and do not show sufficiently high binding-constants. The use of other stimulus compounds that are at least several hundred times sweeter than sucrose, such as saccharin, dihydrochalcone sweeteners, dipeptide sweeteners, stevioside, perillartine and other sweet oximes, the 2-substituted 5-nitroanilines, and (307) M. R. Kare, in M. J. Swenson (Ed.), Duke's Physiology of Domestic Animals, Cornell University Press, Ithaca, NY, 1970, pp. 1160-1 185. (308) R. A. Bernard, Am. J. Physiol., 206 (1964) 827-835. (309) F. R. Bell and L. R. Kitchell. J. Physiol. (London), 183 (1966) 145-149. (310) M. R. Kare and M. S. Ficken, in Ref. 13, pp. 285-297. (311) R. H.Cagan, in Y. Katsuki, M.Sato, S. F. Takaji, and Y.Oomura (Eds.), Food Intake and Chemical Senses, Japan Scientific Societies Press, Tokyo, 1977, pp. 113-138. (312) J. M. Krueger and R. H.Cagan, J. Biol. Chem., 251 (1976) 88-97. (313) C. K. L. Lum and R. I. Henkin, Biochim. Biophys. Acta, 421 (1976) 380-394.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
33 1
500
400
300
3
-4 0
8
200
1oc
control
0 0
10
20
30
Heating t i m e (min)
FIG. 40.-Inactivation of Binding of ['4C]Sucrose to Taste Papillae by Heating in Boiling water."'
332
CHEANG-KUAN LEE TABLEXXVII Comparison of Relative Sweetness relative to
Compound
Mol. wt.
20,000 1 Thaumatin 10,700 2 Monellin 3 Neohesperidin dihydrochalcone 642 4 4,6,1',6'-Tetrachloro4,6,1',6'-tetradeoxygalacto-sucrose 418 310 5 Aspartame (18) 6 6,1',6'-Trichloro-6,1',6'399 trideoxysucrose 24 1 7 Sodium saccharin 200 8 Calcium cyclamate
10% Sucrose (wt. basis)
Thaumatin (molar basis)
3000 3000
113
1 x 100 2.4 x lo2
1000
1/100
9.ox 105
200 200
11720 11970
3.0 x lo6 4.0 x lo6
100
1/1500 1/1600 1/6000
3 . 0 10' ~ 2.0 x 108 1.ox lo8
150 50
1
Immunoreactivity"
Molar ratios of each substance required to give equivalent displacement of '251-labeled thaumatin from antibody in coated tubes.
even the sweet proteins monellin and thaumatin, would be desirable, but, apart from one study on thaumatin, such biochemical studies have yet to be reported. C. A. M. Hough and Edwardson314reported that, in the rabbit, thaumatin can raise antibodies which can cross-react with a wide variety of nonprotein, sweet substances. All of the sweet substances tested, including unlabelled thaumatin, caused significant displacement of antibody-bound 1251-labelled thaumatin (see Table XXVII); these did not produce similar interference when tested at much higher concentrations in radioimmunoassays for luteinizing hormone and corticotrophin. When the immunoreactivity and relative sweetness of these compounds were compared (see Fig. 41), an excellent correlation (correlation coefficient, 0.9606; P < 0.001) was obtained. Hough and Edwardson314 suggested that this could possibly provide a method for in uitro measurement of relative sweetness, and for studying the stimulus-taste-receptor interaction-mechanism. The study has so far been only preliminary, and several questions have yet to be answered. Firstly, sucrose, which is a relatively poor sweetener, was reported to cause displacement of 1251-thaumatinfrom the antibody at high concentrations (314) C. A. M. Hough and J. A. Edwardson, Nature, 271 (1978) 381-383; J. Physiol (London), 275 (1978) 7 4 ~ .
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
5 1 4 m
4
!i3 ‘
i
2
3
4
5
6
333
/ 7
8
9
1
0
log (immnnoreactirity)
FIG. 41 .-Regression Table XXVI.
A n a l y ~ i s ”of ~ Relative Sweetness of Compounds as Numbered in
(50-100 mg/mL). Although this was explained as being due to nonspecific binding, because of the high concentration, it is important to confirm this with other relatively poor sweeteners. Secondly, it would be interesting to know whether structurally related, nonsweet compounds, for example, 6”-0-methylneohesperidindihydrochalcone and methylated monellin, will cross-react with the isolated antisera or not. Furthermore, it is surprising that the study did not include 4,1’,6’-trichloro-4,1’,6‘-trideoxy-guluctosucrose (TGS), as, all too often, exceptions are conveniently excluded. of the three-dimensional structure of thaumatin, it was In a reported that, not only do antibodies raised against thaumatin cross-react with m ~ n e l l i n but , ~ ~antibodies ~ raised against monellin also cross-react with thaumatin, suggesting that there is some structural similarity between portions of the two sweet-protein molecules. Earlier had shown that there is a limited homology in the amino acid sequence in the two proteins. Five tripeptides in monellin have their counterparts in thaumatin, (314a) A. M. de Vos, M. Hadata, H. van der Wel, H. Krabbendam, A. F. Peerdeman, and S. H. Kim, Proc. Natl. Acad. Sci. U.S.A., 82 (1984) 1406-1409. (314b) R. B. Iyengar, P. Smits, F. van der Ouderaa, H. van der Wel, J. van Brouwershaven, P. Ravestein, G. Richters, and P. D. van Wassenaar, Eur. J. Biochem.,96 (1979) 193-204.
334
CHEANG-KUAN LEE
in residues 94-96, 100-102, 101-103, 118-120, and 128-130, but it is not certain whether one of these homologous tripeptides (or any combinations of them) is responsible for the sweet taste. However, all of these 5 regions are well exposed, so that it is very likely that they are responsible for the immunological cross-reactivity, and their conformation may be important for sweet-receptor binding.314aThis is further supported by the finding that, when these proteins are bound to their respective cross-reacting antibodies, the sweetness disappears. It will be interesting to compare the threedimensional structures of thaumatin and monellin when that of the latter is finally determined. It is interesting that the stimulus compounds used in the study differ widely in their molecular structures, and yet they all interact with antibodies to thaumatin. It is, therefore, probable that a single receptor-structure responds to all sweet stimuli,315 there being a variation in the relative effectiveness of sweet stimuli across individual nerve-fibers, and the characteristics of all receptor sites do not appear to be i d e n t i ~ a l . ~Earlier '~ electrophysiological studies of single primary, afferent taste-neurons uniformly agreed that individual fibers very often have multiple sensitivities, and that individual, gustatory receptors are part of the receptive field of more than one afferent fiber.317-320 We have yet to learn how these interact, and the nature of their excitatory, or possible inhibitory, relations, or both. The binding specificity seems to suggest that the receptor for sweet taste is in the non-soluble phase, presumably membrane-bound. It also appears that this membrane-bound moiety is present either uniquely, or at least to a greater extent, in tissue containing taste buds. The correlation between the binding results and the behavioral experiments in the same animal gives support to the hypothesis that peripheral receptors are responsible for the specificity of the sweet-taste stimulation. This had not been rigorously shown earlier. This idea of peripheral control is in agreement with the earlier studies of O a k l e ~ ~ on~ cross ' regeneration of the sensory nerves in taste. (315) M. G. Lindley, in G. G . Birch and K. J. Parker (Eds.), Sugar: Science and Technology, Applied Science, London, 1978, pp. 403-414. (316) C. Pfaffman, M. Frank, L. M. Bartoshuk, and T. C. Shell, in J. M. Sprague and A. N.
(317)
(318) (319) (320) (321)
Epstein (Eds.), Progress in Psychobiology, Physiology and Psychology, Academic Press, New York, 1976, pp. 1-19. C. Pfaffman, J. Cell. Comp. Physiol., 17 (1941) 243-258; J. Neurophysiol., 18 (1955) 429-440; in G. E. W. Wolstenholme and J. Knight (Eds.), Taste and Smell, Churchill, London, 1970, pp. 31-50. M. J. Cohen, S. Hagiwara, and Y. Zotterman, Acta Physiol. Scand., 33 (1955) 316-332. I. Y. Fishman, J. Cell. Comp. Physiol., 49 (1957) 319-334. H. Ogawa, M. Sato, and S. Yamashita, J. Physiol. (London), 199 (1968) 323-340. B. Oakley, J. Physiol. (London), 188 (1976) 353-371.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
335
He reported321that sensory neural-activity is a function of the region of innervation, and not of the nerve innervating that area. The nature of the transductive event and the mechanism underlying it are more obscure than those of the peripheral receptive event. M o o ~ e r ~ ~ ~ and Mooser and L a m b ~ t demonstrated h~~~ that protein-modifying reagents can inhibit the integrated chorda tympani electrophysiological responses to using sulfhydryl-complexing reagent groups, in taste stimuli. A which time-to-inhibition and ether-water partition coefficients were measured, suggested that the sulfhydryl blockers act at sites within the membrane, or even in the cell. The use of carboxyl-complexing on the other hand, indicated that the reagents act at sites at the cell curface. After treatment with these reagents, stimulation by sodium chloride, sucrose, and hydrochloric acid was inhibited. It was suggested that a transduction step was inactivated in each case, because, firstly, the kinetics of inactivation were independent of the concentration of sodium chloride used to pre-adapt the electrophysiological response, and independent of the degree of receptor-cell stimulation; and, secondly, the protein-modifying reagents inhibit . responses due to all modalities (sweet, salty, and sour) tasted. However, these results do not preclude the possibility that other noncompetitive events may be responsible for the inactivation. For example, the complexing could decrease the binding of the stimulus for the active receptor-site. Also, the kinetics of inactivation of the preadapted sodium chloride response may be complicated by the possibility that the reagents inhibit their own response, in addition to the response of the stimuli. c. A Model for the Sweet-taste Receptor.-There is now sufficient evidence to assume that the receptor is most probably proteinaceous in nature. Firstly, the sweet taste is inhibited by SH and proteolytic enzymes,325 and, secondly, there is a suppression of recovery of sweet taste after inhibition by cycloheximide, an inhibitor of protein synthesis.326The nature of the conformational change of the taste-receptor protein has been sug. ~is~ known ~ that the reversible change of Gramgested by R. F i s ~ h e r It positive to Gram-negative staining-behavior, which occurs in wool and bacterial membranes, involves a change from a P-pleated sheet to an a-helical conformation of the protein. F i s ~ h e thus r ~ ~ proposed ~ that the initial step in taste stimulation might be due to the affinity of the stimulus (322) (323) (324) (325) (326) (327)
G. Mooser, J. NeurobioL, 7 (1976) 457-468. G. Mooser and N . Larnbuth, J. NeurobioL, 8 (1977) 193-206. A. Norna and Y. Hiji, Jpn. J. PhysioL, 23 (1972) 393-402. Y. Hiji, Nature, 256 (1975) 427. Y. Hiji and J. Ito, Comp. Biochem. PhysioL, A, 58 (1977) 109-113. R. Fischer, in Ref. 43, pp. 198-200.
336
CHEANG-KUAN LEE
molecule for an a-helical region of a protein. Such interaction then causes a transition to a P-pleated-sheet conformation, or to a random coil. Desorption of the stimulus molecule then allows transformation to the original, helical structure. Such a change is possible, although the intramolecular hydrogen-bonds are of major importance in the formation of the a-helical, resting state, as proper interaction between the side chains projecting outward from the coil is also necessary for stabilization of the protein conformation. Optical rotatory dispersion and circular dichroism studies of a wide range of membranes appeared to support the foregoing concept. The studies328 showed that a major percentage of the protein has the a-helical conformation, the remainder existing largely as a random coil. From model, interaction studies between various amino acid side-chains and some 2-substituted hitroanilines (12), Kier329suggested that a tryptophan residue may be the hydrophobic binding-region of the sweet-taste receptor-site. It is interesting that tryptophan is known to stabilize the a-helical conformation of prot e i n ~ Therefore, .~~~ in the nitroaniline sweeteners, it has been speculated that the receptor may be located on an a-helix in close proximity to a tryptophan re~idue.9~ Similarly, other classes of sweeteners may also bring about an identical conformational change of the same receptor through interaction with other side-chain substituents. This model is attractive, in that it rationalizes the diversity of structure occurring among sweet compounds, and illustrates the role of multiple receptor-sites which may, when bound to different classes of sweet compounds, yield a common biological response.94 d. Sweet-taste Inhibitors: Gymnemic Acids and Ziziphim-The interaction of taste-stimulus molecules with the membrane of a single cell can be expected to be a very highly selective process. A population of such molecules must, because of their polarity, be assumed to approach the potential sites on the epithelium in a highly homogeneous orientationpattern. This orientation pattern is, however, of little importance, because, for most molecules, it is presumed that a physical contact between their pair, or triplet, of polar groups and a complementary set at the suitable membrane-locations is established prior to the interaction, which defines the orientation in the interaction c o m p l e ~ .Thus, ~ ~ *when ~ ~ molecules having the correct stereochemistry approach the locations possessing polar features in conformations that are complementary to those of the molecules, very (328) R. Harrison and G. G. Lunt, Biological Membranes, Wiley, New York, 1975. (329) L. M. Kier, in G . Benz (Ed.), Structure-Activity Relationships in Chemoreception, IRL, Washington, D.C., 1976, pp. 101-108. (330) A. L. Lehninger, Biochemistry, Worth, New York, 1970.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
337
effective and stable interaction-complexes, with high energy effects, are formed. Structurally different molecules will interact very weakly, or hardly at all, with suitable locations. Gymnemic acids and ziziphins, from extracts of the leaves of Gymnema syluestre,have long been known to be inhibitors of sweet taste in human331-333 and other vertebrates.334s335 These compounds are closely related, triterpene saponin g l y ~ o s i d e s . In ~ ~humans, ~ - ~ ~ ~the sweet sensation of all the sweet compounds tested is depressed, or completely eliminated, for up to several Studies3" on the binding of [ ''C]sucrose to circumvallate and filiform (control) papillae of the bovine after treatment of the homogenates with gymnemic acid suggested that gymnemic acid acts at a step subsequent to the initial binding of a stimulus molecule to the receptor. This appears to be in agreement with behavioral data on humans. The presence of sucrose or saccharin during treatment with gymnemic acid does not alter the sweet taste of the sweetener, suggesting that there is no competition between gymnemic acid and sweet compounds for the same r e ~ e p t o r - s i t e It . ~would ~~ have been expected that, as the binding by the acid is much the stronger, as seemed likely from the persistence of its even in the presence of a sweet compound, gymnemic acid could still be effective, because it would tend to remain bound. It has been proposed340 that the mechanism(s) of action of gymnemic acids and ziziphins is a biphasic, model-membrane penetration-process. The model suggested that the modifier molecules interact first with the receptor-cell plasma-membrane surface. It was postulated that this initial interaction involves a selective effect on taste perception, including the transduction and quality specification of the sweet stimuli, and selective depression of sweetness perception. Following the initial interaction, the modifier molecules interact with the membrane-lipid interior to produce a general disruption of membrane function and a nonselective effect on taste
(331) R. M. Warren and C. Pfaffman, J. Appl. Physiol., 11 (1959) 367-378. (332) H. Diamant, B. Oakley, L. Strom, C. Wells, and Y. Zotterman, Acta Physiol. Scand., 64 (1965) 67-74. (333) L. M. Bartoshuk, G. P. Dateo, D. J. Vandenbelt, R. L. Buttrick, and L. Long, Jr., in Ref. 46(a), pp. 436-444. (334) B. Anderson, S. Landgren, L. Olssen, and Y. Zotterman, Acta Physiol. Scand., 21 (1950) 105-109. (335) J. R. Faull and B. P. Halpern, Physiol. Behau., 7 (1971) 903-907. (336) W. Stocklin, J. Agric. Food Chem., 17 (1969) 704-708. (337) J. E. Sinsheimer, G. S. Rao, and H. M. McIlhenny, J. Pharm. Sci., 59 (1970) 622-628. (338) L. M. Kennedy and B. P. Halpern, Chem. Senses Hauor, 5 (1980) 123-147. (339) R. P. Warren, R. M. Warren, and M. G. Weninger, Nature, 223 (1969) 94-95. (340) L. M. Kennedy and B. P. Halpern, Chem. Senses Flavor, 5 (1980) 149-158.
338
CHEANG-KUAN LEE
perception. The model appears to be able to account for available chemical, physiological, and psychophysical data, but L. M. Kennedy and H a l ~ e r n ~ ~ ’ emphasized that this does not necessarily prove correctness of the model, because the experimental data were obtained from experiments that were not designed to, and did not, test the model directly. However, the biphasic model makes explicit predictions that can be tested in future experiments. The nature of the initial interaction between the modifier molecules and the taste-receptor membranes is still uncertain. This surface interaction could involve any of several po~sibilities.~~’ Binding of modifier molecules could be to classical, “lock and key” sweet-receptor sites, thus blocking access of sweet-stimulus molecules to these sites. However, Cagan’s findings3” suggested that the site of action of gymnemic acid is elsewhere than at the binding site of sweet molecules. Alternati~ely?~’binding of the modifier molecules may be to “accessory sites,” leading to interference in the “mutual molding” of sweet-stimulus molecules with sweet receptors, or in amplification-eff ector functions of other membranes. In addition, or alternatively, the taste modifier could change membrane-surface physical chemistry that may be involved in the stimulation-transduction process. This fascinating approach needs further, well planned studies. Studies on the initial, selective-surface interactive-phase of taste-modifier action could give a better understanding of the taste-receptor mechanism of the specification of stimulus quality. Blocking of the sweetness responses with the modifier compounds, and probing of the psychophysical phenomena of, or the neurophysiological coding for taste modifier, or both, may reveal which of the two phases of taste-modifier action is functional at the time when the measurements are made. If the biphasic model proposed proves to be the mechanism of action of the gymnemic acids and ziziphins, it is probable that at least one process involved in the specification of a sweet stimulus must be located at the surface of the taste-receptor cellmembrane.340 It is surprising that, in view of the widespread interest in studying various aspects of the mechanism of taste by observing the effects of naturally occurring taste-modifiers, no similar effort has been made to examine synthetic taste-inhibitors. Crosby and coworkersg4suggested that selected, tasteless analogs of dihydrochalcones, dipeptides, or saccharin could be synthesized, and evaluated for their ability to inhibit the sweet taste of sweet compounds, in order to obtain information regarding the specificity of the receptor. For example, does a tasteless dipeptide that inhibits the taste of sweet dipeptides also block the taste of cyclamate, saccharin, or the dihydrochalcones? Such studies could identify various classes of sweeteners that act at a common receptor, or at different receptors. Furthermore, the inhibitory effect caused by altering various molecular parameters within a
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
339
single class should provide valuable information regarding the influence of each molecular parameter on the receptor. e. Sweet-taste Modifier: Miracu1in.-Another taste modifier is miraculin, a glycoprotein of molecular close to 44,000. When the tongue is treated with this glycoprotein, subsequent exposure to acid causes the sour acid to taste sweet. It was suggested341that the mechanism of action involves a conformational change of the taste-receptor membrane by the acid, so as to allow the arabinosyl or xylosyl residues of the bound (to the receptor membrane) miraculin molecule to stimulate the sweet-receptor site in the normal fashion. There is no direct, experimental evidence to support this concept, but it is consistent with the psychophysical observations.342It should, nevertheless, be interesting to test this by studying the activity of the molecule after removing, and replacing, the glycosyl residues. 4. The Quality of Sweetness
A century ago, F i ~ proposed k ~ ~ ~the concept of four primary tastes, namely, sweet, salty, sour, and bitter. It has since been found that taste sensations are not describable by a single collection of discrete primaries. Electrophysiological studies of afferent taste-units in the chorda tympani and glossophyrangeal nerves have revealed that a continuous spectrum of gustation may be based on these four taste elements. Furthermore, the intensities of the “tastes” that we commonly experience are due not only to gustatory sensations but also to tactile, hot and cold, and, above all, olfactory sensations. The complexities of taste studies are such that, unless one of the taste modalities is singled out for study, there is very little hope of success. Although some neural fibers respond to sweet-stimulus compounds placed on the tongue, others do not. This pattern of sensitivity is often a very complicated one. The fibers often respond to more than one, sometimes even to all, of the four taste modalities. Very rarely does a fiber respond specifically to only “sweet” or “salty” substances. Furthermore, other fibers may have an entirely different spectrum of sensitivities, and may respond strongly to one sweetener and very weakly to another. Pfaffmann344reported how two fibers, one having one pattern of sensitivity to taste, and the other, a different pattern, can signal two different taste-qualities, even though (341) Y. Kurihara, Life Sci., Part I , 8 (1969) 539-543; Y. Kurihara and L. M. Beidler, Nature, 222 (1969) 1176-1179. (342) L. M. Bartoshuk, in Ref. 118, pp. 5-26. (343) A. Fick, Lehrbuch der Anatomie and Physiologie der Sinnesorgane, Schaunburg, Lahr, Germany, 1864. (344) C. Pfaffmann, Am. PsychoL, 14 (1959) 225-232.
340
CHEANG-KUAN LEE
neither is specifically sensitive to either taste alone. Working with an array of sensitive, single neural-units, each with its own pattern of responses to stimulus chemicals, E r i c k ~ o n ~reported ~’ that the mechanism by which the brain may conceivably reproduce our unique taste-sensations does not depend upon single responses from one tuned neuron. It in fact relies on a complex pattern of responses obtained from an entire array of responding neurons. However, despite the complexity of the pattern, the taste may still be perceived as a coherent sensation. Perceptually, the single unitary sensation can be broken down, and the perception of sweetness can be analyzed in respect to its fine differences. For example, a taster presented with a range of equisweet-taste materials (determined by subjective measurements) will attempt to detect differences among the substances, and expand the “unitary” sensation of sweet. In taste- and flavor-difference testing, when judgments are treated as distances between points in a “subjective space,” the technique of multidimensional scaling346provides an estimate of where those points lie in space. Distances between points then correspond to the subjective estimates of taste (or flavor) difference originally collected by experimental procedures. The presence of “side-tastes,” not only sour, salty, or bitter, but also slight variants in sweetness, that accompany sweetness must be considered when the supposed unitary percept of sweetness is analyzed. When the taster is instructed to concentrate on the nodal point “sweetness,” a significantly more-complex sensation may be found than in situations where sweet is opposed to sour, salty, or bitter as a taste description. a. Time-Intensity Relationship in Sweetness.-Sensory responses to sugars and other stimuli can be categorized in terms of (1) sensitivity of discrimination, (2) perceived strength or intensity, and (3) affective or hedonic behavior. There is a fourth sensory attribute, that of duration or persistence of the sensation, and the time-intensity studies of Dubois and co~orkers,34~ Larson-Powers and P a n g b ~ r n ? ~Swartz ’ and F ~ r i a and , ~ ~Birch ~ and coworker^'^^*^^^ clearly established the importance of the temporal properties of synthetic sweeteners. Support of this concept may also be found in Kier’slog modification of the Shallenberger and Acree hyp~thesis.’.~’ This inclusion of a third hydrophobic site was intended to represent the distribution of molecules between (345) R. P. Erickson, in Ref. 13, pp. 205-213. (346) R. N. Shepard, Psychometrika, 27 (1962) 219-246. (347) G. E. DuBois, G . A. Crosby, R. A. Stephenson, and R. E. Wingard, Jr., J. Agric. Food Chem., 25 (1977) 763-772. (348) N. Larson-Powers and R. M. Pangborn, J. Food Sci., 43 (1978) 41-46. (349) M. L. Swartz and T. E. Furia, Food Technol. (Chicago), 31 (1977) 51-55, 67. (350) G. G . Birch, Z . Lafymer, and M. Hollaway, Chem. Senses Flauor, 5 (1980) 63-78.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
341
the hydrophilic, aqueous environment of the oral fluid and the hydrophobic environment of the taste-receptor membrane. For sugars, this would make little or no contribution to the differing sweetness as they are essentially hydrophilic molecules that can be adequately distinguished from one another purely on the basis of AH,B systems. If this third site in the sweet glucophore does represent the partition of stimulus molecules between aqueous and hydrophobic environments, it might be anticipated that it could affect the temporal qualities of taste, that is, the rate of approach and accession to, and vacation of, receptor sites. The traditional approach adopted when comparing the sweetness of two compounds consists of tasting pairs of samples, one member containing a fixed concentration of the standard, the others containing variable concentrations of the comparison sweetener. The percentage of responses selecting the fixed concentration of the standard (usually sucrose) as the sweeter, is plotted against the concentrations of the experimental sweetener. From the regression line calculated, the concentration of the experimental compound that intersects the 50% response level is taken to represent the equivalent sweetness.351 This technique gives valid estimates of relative sweetness if the relationship is linear, or if the data can be converted into functions which will give a linear relationship with concentration. However, the method becomes inappropriate if there is synergism between the test compound and the standard, for example, sucrose and cyclamate, resulting in a parabolic relationship in which the line approximates, but does not cross, the predesignated, 50%-response criterion.348Difficulties were also found when comparing saccharin and sucrose,348 because of the faster increase in bitter taste than in sweet taste with increasing concentration. In addition, the sweetness and bitterness of saccharin appear to persist longer than would the taste of sucrose. The lingering after-taste characteristic is also found with neohesperidin dihydrochalcone (13) and the sulfoalkyl analogs347(81).The lingering sweetness can affect subsequent evaluations, and it was reported that intensity values for nonlingering sweeteners are lower if they are tasted after, rather than before, a dihydrochalcone sample. When such a problem is encountered in the direct comparison of sweeteners that differ quantitatively, qualitatively, and temporally, the comparison can be made by using the perceived taste-intensity us. time procedure. These time-intensity effects are illustrated in Fig. 42 by a plot of perceived intensity us. time? curve A being given by a stimulus molecule, such as sucrose, which exhibits rapid taste onset and cutoff, and curve B approximates the behavior of most dihydrochalcone sweeteners. (351) A. T. Cameron, The Sense of Taste and the Relative Sweetness of Sugars and Other Sweet Substances, Sugar Res. Found, Sci. Rep. Ser., No. 9, 1947.
CHEANG-KUAN LEE
342
,--, I
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Time
FIG.42.-Perceived Taste-intensity versus Time, for Type A and Type B Sweetener.347[Type A, rapid taste onset and cut-off; type B, slow taste-onset and persistent after-taste.]
One important aspect in the time-intensity plots is a plateau of maximum (see Fig. 43). Sugars generally intensity, which is c~ncentration-dependent~~~ resemble other sweeteners in exhibiting an increase in absolute sweetnessintensity with increasing temperature. However, they seem to show a corresponding decrease in persistence (see Fig. 44). This property of sugars might probably be due to their lack of a y dispersion site, making them unable to take advantage of increased temperature to persist in their effects at receptors (as with other sweetener^).^'^ Thus, after the appropriate number of sites are filled, the remaining sugar molecules are simply carried away rapidly by diffusion. The taste properties of the sulfoalkyl dihydrochalcones (83a-d)appear to support this argument.347The lingering, sweet after-taste of approximately equi-intense solutions of these derivatives was found to increase significantly with increase in the length of the sulfoalkyl chain. Thus, sulfomethyl dihydrochalcone (83a)was found to linger less than the sulfoethyl derivative (83b),which lingered less than the sulfopropyl derivative (83c),which, in turn, lingered less than the sulfobutyl derivative (83d). The increase in lingering on proceeding from 83a to 83d is accompanied by an increase in taste intensity. The only change in this series appears to be a regular increase in hydrophobic character. Deutsch and HanschE2had proposed that the increase in sweetness accompanying the increase in (352) G . G . Birch, in P. Koivistoinen and L. Hyvonen (Eds.), Carbohydrate Sweeteners in Food and Nurririon, Applied Science, London, 1980, pp. 61-76.
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
5
343
10
Time (s)
FIG. 43.-Diagrammatic Plots of Reaction Time, High-intensity Plateau, and Overall Persistence-time at Different Concentrations of Sucrose.352
hydrophobic character is the result of increased partitioning from the saliva phase onto the presumably more-hydrophobic, receptor phase. The results of DuBois and coworkers347appeared to suggest that the increase in the hydrophobicity of the sulfoalkyl dihydrochalcones also increases the molecular partitioning into all kinds of oral, hydrophobic material. Thus, only after increasingly longer periods of time does the concentration of glucophore at the receptor reach an equilibrium concentration sufficient to elicit a response. This hypothesis appears to be in accord with the observation that delayed onset is not found when very concentrated solutions of dihydrochalcones are tested, this presumably being due to all of the oral, hydrophobic phase, including the less-accessible receptor-sites, being rapidly saturated with the glucophore, thereby resulting in rapid onset.347 If this argument is correct, it would be expected that equimolar solutions
Tmprattur
FIG. 44.-Temporal
Tcmparetwa
Differences in Sweetener, Caused by y Dispersion Site?52
344
CHEANG-KUAN LEE
of 83a-d will exhibit perceived intensity us. time curves which will show increased, perceived intensities, as measured by the maxima of the curves (see Fig. 45). If this is the case, the total magnitudes of the taste responses, as measured by the integrals of the curves, may be essentially identical for 83a-d, but taste intensities actually measured, as determined from curve maxima, will appear to give the impression that taste intensity increases in the order 83d-a. Experimental data obtained347showed this to be the case, and the product of perceived taste-intensity and time can be used as a reliable measure of overall, gustatory re~ponse.’~’ The “degree of sweetness” of sweet molecules has been universally determined by one of three methods, namely, (1) the classic method of threshold evaluation of the lowest concentration at which the stimulus is detected, (2) the traditional methods of category scaling, wherein the panellist selects numbers from a limited scale, for example, 1-9, to match gradations in perceived stimulus intensity, and (3) magnitude estimation, where a panellist can quantify the perceived sweetness in such a way that the ratios of numbers that the panellist assigns can be compared to ratios of actual concentration, the subjective measuring-technique of “magnitude estimation.” The degree of sweetness that is universally reported in the literature refers to how many times more, or less, efficient than sucrose is a particular stimulus molecule at eliciting the sweetness response, and, thus, by measuring the ratio of concentrations of sucrose and other sweeteners
Time
FIG. 45.-Hypothetical drochal~ones.~~’
Curves for Perceived Intensity uersus Time, for (Su1foalkyl)dihy-
CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS
345
at the same intensity of sweetness, the so-called degree of sweetness is derived. Moskowitz and Chandler353pointed out that this is an erroneous concept, and the psychophysical studies347-350 of time and intensity factors clearly show that overall gustatory response is probably determined by both of these factors; indeed, the intensity factor alone, as often reported by taste panellists, is probably affected by time. Such considerations should be borne in mind in future studies, both in the search for new sweetening agents and in the study of the sweetness-activity relationship. The intensity-time studies of sweetness have been extended to simple, two-component, sugar systems by Munton and The intensity-time characteristics of pure and binary mixtures of 9 different, sweet stimuli were examined. When the “effective concentrations” of each stimulus in a binary mixture were calculated, and compared to actual concentrations present, it was found that, irrespective of the proportions of the two stimuli, one compound in each mixture appeared to be completely dominant, the other being in a very low “effective concentration”; that is, it is actually present at an effective concentration far higher than is actually possible. This suggests that the effective concentrations may reflect real concentrations of a single molecular species in the micro-environment of the receptor. Munton and Birch thus suggested that, if this is true, and if taste chemoreception operates by the two-stage, “orderly queue” m e c h a n i ~ m(see ~ ~mechanism ~.~~~ 3, given later), one type of molecule may be completely favored over another at the receptor site, for conformational or steric reasons, and thus dominate the observable taste properties. b. The “Queue” Hypothesis in Sweet Taste.-When intensity and persistence of any sweetener are plotted as a function of concentration, curves of the type shown in Fig. 46 are ~ b t a i n e d . ~ ~Persistence O - ~ ~ ~ appears to continue to increase at concentrations above which saturation intensity occurs. This does not seem to be explicable on the basis of a single, molecular event in sweet-taste c h e r n ~ r e c e p t i o nThe . ~ ~ relationship ~ between intensity, persistence, and onset time must depend on molecular structure, but very little is known concerning the actual binding of the stimulus molecule to the receptor, except that the molecule appears to be polarized, or aligned and very slight structural changes in a particular way, on the in the stimulus molecule probably alter this alignment.’26What then, is the mechanism whereby the stimulus molecule is presented to, or arrives at, the receptor? Birch and coworker^^^'^^^^ suggested that this may clearly be (353) H. R. Moskowitz and J . W. Chandler, in Ref. 118, pp. 141-212. (353a) S. L. Munton and G . G. Birch, J. Theor. Bid., 112 (1985) 539-551. (354) G. G . Birch, in J. Le Magnen and P. MacLeod (Eds.), Proc. Int. Symp. Olfnction Tasfe 6th, Paris, France, IRL, London, 1977, pp. 27-32.
346
CHEANG-KUAN LEE
Concentration of atimulue molecule FIG.46.-Schematic Effect of Concentration of Persistence Time (TJ, Subjective Intensity (Si), and Onset Time (To) of Sucrose Sweeteners.352
related to temporal factors. The very existence of the three factors appears to suggest that the mechanism responsible for onset time and persistence in sapid molecules may differ from the intensity-governing mechanism at the level of the ion-channel. Separate mechanisms, therefore, probably exist to account for the time and intensity factors, the intensity being governed by the Shallenberger concept of “fit” by hydrogen bonding, and persistence, by a localized concentration of stimulus molecules at the receptor.352 Birch and coworkers350studied the time-intensity interrelationships for the sweetness of sucrose and thaumatin, and proposed three thematically different processes (see Fig. 47). In mechanism ( l ) , the sweet stimuli approach the ion-channel, triggering site on the taste-cell membrane, where they bind, open the ion-channel (ionophore), and cause a flow of sodium and potassium ions into, or out of, the cell. Such a mechanism would correspond to a single molecular event, and would thus account for both time and intensity of response, the intensity of response being dependent on the ion flux achieved while the stimulus molecule binds to the ionophore,
(3-($$ Na' K'
diffusion
~
ionophore
e
diffusion
uB a
opening of ionophore causer intensity and persistence
Na'
diffusion
K '
a
g Na'
K+
-KALA=mqueue CBUSCS persktence
cyclic open-shut process at ionophore causes intensity
FIG. 47.-Three
Possible Courses of Chemoreceptive Events in Sweet-taste C h e m o r e c e p t i ~ n . ~ ~ ~
348
CHEANG-KUAN LEE
and the persistence related to the time during which the binding of the molecule continues. Therefore, this mechanism seems unlikely, from evidence of separate effects on persistence and intensity, and, especially, the rather constant reaction-time over the range of concentration studied also did not show any evidence (see Fig. 46). Neurophysiological of significant change in reaction time with concentration. In mechanism (2), it was proposed3” that the molecules first bind reversibly to a nonspecific part of the membrane, in order to achieve a localized concentration of stimuli. This would account for persistence; however, it was argued3” that, on vacating this nonspecific area, by random diffusion to the ionophore trigger-mechanism, each molecule “unlocks” the ionchannel by stereochemical fit, causing ion flow and an elemental depolarization. This process would then cause release of the stimulus compound, and closure of the ion channel, which would then be ready to repeat the process with another stimulus molecule. The intensity of response could thus be explained by the rapid occupation and evacuation of the binding site, with concurrent rapid opening and closing of the ion channel. Such a process is not, however, consistent with the plateau of maximum intensity, shown in Figs. 42 and 45. Furthermore, the reaction time in such a mechanism should be based largely on the diffusion rate, in which case, it would be expected that concentration-dependence would be larger than the rather constant relationship that was actually observed (see Fig. 46). In mechanism (3), Birch and proposed that the stimulus molecules pass into an irreversible, “orderly queue” approaching the ionophore. The ionophore trigger-mechanism is the same as in mechanism (2), that is, rapid occupation and vacation resulting from an effective “fit” of the molecule. The persistence was equated to the physical length of the queue, and is thus separated from (although geared to) the intensitydetermining mechanism of the ionophore. It was suggested3” that the alignment of stimulus molecules as they approach the ionophore mechanism is the consequence of the queue, and the time needed for a particular molecule to cross the queue accounts for the onset time. suggested that, in this mechanism, the hitherto-ignored dimension of taste chemoreception, namely, the spatial function and its effects on the temporal quality of sweetness had been taken into account. This type of multiple, sequential interactions of stimuli with the receptor site, in which a number of discrete, geometrically determined, binding-site interactions occur as a result of dispersion and dipole forces, had earlier been described by K a f l ~ a . ~ ~ ~ Whether or not this mechanism really exists will be determined by reliable (355) L. A. Marowitz and B. P. Halpern, Chem. Senses Flauor, 2 (1977) 457-463. (356) W. A. Kafka, Coll09. Ges. Biol. Chem., 25 (1974) 257-278.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
349
elucidation of taste chemoreceptive-mechanisms by biochemical techniques, and by the outcome of psychophysical experiments with taste modifiers of defined conformation. V. METHODOLOGY O F MEASUREMENT OF SWEETTASTE Much of our present day knowledge of sweetness intensity, both at the threshold level, where taste begins, and above the threshold level, derives from the application of psychophysical techniques. It is now evident that the psychophysical procedure used measure separate aspects of sweetness perception. Hedonic responses cannot be predicted from intensity of discrimination data, and vice versa. The taste-panel evaluation of sweetness is of fundamental importance in the development of worthwhile structuretaste relationships. Therefore, it is vital that the appropriate psychophysical method and experimental procedure be adopted for a particular objective of investigation. Otherwise, false conclusions, or improper inferences, or both, result. This situation results from the failure to recognize that individual tests measure separate parameters of sensory behavior. It is not uncommon that the advocates of a specific method or procedure seldom
I
I
S t i d w concentration
I I
"Thremhold"
Acceptance/rejection
-; -1 '
I I
Perceived i n t e n s i e
Preference
___ -
__I
I : I I I 1 1 I I I 1
Category ecale Batio scale ee of liking
I
(
1
1
b-r_lI!j
Ranks
Category scale Ratio scale
Analytical
I I
Affective
FIG. 48.-Diagrammatic Representation of Independent and Inter-related Responses to Sensory Stimuli, Emphasizing the Distinction between Analytical and Affective judgment^.'^'
350
CHEANG-KUAN LEE
acknowledge the limitations of their techniques. Therefore, there should be a consistent approach in utilizing taste techniques in sweetness research, as only then will a more critical and constructive approach be attained, and results be meaningful and comparable. A simplistic compilation of distinct sensory behaviors, and their interrelationship^,^^^ is shown in Fig. 48. Basically, there are four major types of measures that are used in taste intensity measurements: (a) threshold measures or estimates of the physical level at which the sensation of sweetness begins, (b) equal-sweetness matches between a sugar and other sweeteners, (c) category or rating scales, and (d) ratio scales. Each method has found its adherents and uses, and each possesses specific advantages and defects that indicate its use for one application, but contraindicate its use for another. These methods and their applications have been critically analyzed and reviewed,353.357-364 and it is, therefore, superfluous to deal with the topic here. It is, perhaps, appropriate at this point to make some comments concerning intensity of sweetness. It has already been mentioned that this value is commonly reported in the literature as a multiple of the sweetness of sucrose, this being estimated by the ratio of concentrations of sucrose and the sweetener at the same intensity of sweetness. Moskowitz and Chandler3” suggested that this is an erroneous concept, because what the figure actually represents is that, at a lower concentration, the molecule is a more efficient stimulant. Secondly, comparisons are commonly made on a weight basis, but, unless the compounds being compared have the same molecular weight, such results are worthless until they have been recalculated to a molar basis.358The initial mechanism of sweetness is chemical in nature, as has now been universally agreed, and intensities expressed on a molar basis are meaningful when the effects of structural modification on the sweetness potency are considered. Furthermore, data obtained by comparison with one concentration, for example, with 1% sucrose solution, cannot be extrapolated to higher levels, because the dependence of intensity on concentration may not necessarily (357) (358) (359) (360) (361)
R. M. Pangborn, in Ref. 352, pp. 87-110. J. H. Geller and R. S. Tipson, Arch. Biochem., 12 (1947) 319-322. R. H. Moskowitz, Percept. Psychophys., 7 (1970) 315-318; 8 (1970) 40-42. R. H. Moskowitz, in Ref. 301, pp. 37-64. R. H. Moskowitz, in J. H. Shaw and G. G. Russos (Eds.), Sweetness and Dental Caries, IRL, London, 1978, pp. 41-74. (362) R. H. Moskowitz, Crit. Rev. Food Sci. Nurr., 9 (1977) 41-79. (363) R. H. Moskowitz, in J. M. Weiffenbach (Ed.), Taste and Development. The Genesis of Sweet Preference, U.S. Dept. Health, Educ., Welfare Publ. No. (NIH) 77-1067, Bethesda, M, 1977, pp. 282-294. (364) R. H. Moskowitz, in M. R. Kare and 0.Maller (Eds.), The ChemicalSenses in Nutrition, Academic Press, New York, 1977, pp. 71-98.
CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS
351
be proportional. The uselessness of such extrapolations was illustrated by The Crosby and coworkers? using data cited for 2-propoxy-5-nitroaniline. relative sweetness of this compound had been reported*" to be 4100. Therefore, the concentration of this substance which is equisweet with 1 % sucrose would be 1/4100 or 2.44 x It has beenshown by M o s k o ~ i t z ~ ~ ~ that the logarithm of the total taste-intensity (I), and that of the sweetness intensity (RS), is linearly related to the logarithm of the concentration (C), that is,
I or RS = kC". Therefore, for 1OO/ sucrose, and using M o s k o ~ i t z ' sdata ~ ~ ~( k = 56, n = 1.4), the relative sweetness, RS( 1 YO) = k, C" = 56(
1p4= 56.
Because the 1 % sucrose solution is equisweet with the propoxy analog, therefore, 56= kpCn, where the concentration of the sweetener is 2.44 x Crosby and coworkers94 assigned a value of n = 0.5 for the propoxy analog, this value being chosen as it was considered to be realistic, relative to the result obtained for the less-intense sweetener ( n = 0.6). Thus, 56 = kp(2.44x 10-4)0.5, and therefore,
kp = 3590.
If the concentration of sucrose is increased to 10'3'0, a commonly used level, the intensity of this sucrose solution may be calculated as being RS(1OYo) = 56(
1410.
Therefore, for an equisweet solution of the propoxy analog, 1410 = 3590C0.5, and C = 0.154. Thus, the increase in concentration needed to match the effect of a 10-fold increase in sucrose concentration is 0.154/2.44 x lop4= 630-fold. Crosby and coworkers94suggested that this is close to the situation that occurs with saccharin, and it is doubtless responsible for the differences in perceived sweetness intensity (200-700 times that of sucrose) for sensory determinations conducted at different concentrations.
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AUTHOR INDEX FOR VOLUME 45 Numbers in parentheses are footnote reference numbers and indicate that an author’s work is referred to although his name is not cited in the text.
A Aberth, W., 25 Ablett, S., 153 Abraham, D. J., 43, 56(63),62(63),63 Ackerman, B., 32 Acree, T. E., 200, 207, 213(21), 214, 215, 216, 218(16),219(48), 220, 221(5, 71), 227, 231(48),235, 238, 241(48), 249(60), 258(48),259, 263, 271(21), 274, 292(48),298(5),299(5, 71), 300, 301, 304, 311(21),312(21), 340 Acton, E. M., 295, 296(245a) Akasaka, K., 132, 133(30),136, 164(30) Albersheim, P., 27, 28, 32(32),34(36), 43(36),53, 66(32, 74), 67(32, 33, 37), 68(36) Albrecht, J. J., 290 Aldenhoff, J. M., 313 Alderman, D. W., 142, 146 Allerhand, A., 248, 249(165a), 300, 349(257) Amemura, A., 68 Aminoff, D., 194 Andersen, H. T., 206 Andersen, N. F., 142 Anderson, B., 249 Anderson, J., 194 Anderson, R. A., 171 Andersson, B., 337 Andreu, G., 171 Angyal, S. J., 249 Antus, S., 280 Aoki, H., 307, 308(272),310(272) Ara, M., 292 Arab, Y. M., 248 Arita, M., 27, 43, 54(30),55(58-SO), 71(79) Ariyoshi, Y.,245, 246, 307(154-156), 308(156), 309(154-156) Armitage, I. M., 171 Aroney, M. J., 293
Aronow, L., 211, 212(34) Arrequin Lazano, B., 253 Aubert, J.-P., 94, 100 Aubry, A., 192 Audrieth, L.F., 299 Augnstejn, M. F., 142 Aurelle, H., 42, 70(56) Austee, D. J., 170 Austin, W. C., 257, 258 Avigad, G., 102, 113 Azarinia, N., 295
B Backman, E. L., 223 Balasubramanian, D., 306 Balazs, E. A., 94, 95(29), 102(29), 104(29), 113(29,46), 114(29), 115(76),116(75) Balcerski, J. S.,93, 94(27) Baldwin, R. E., 306 Ballou, C. E., 20, 21, 32, 37(15),47, 49(67), 53(15), 56(76), 58(15),65(11), 70, 71(125) Banataola, I., 171 Banks, W., 87 Barber, M., 20, 24(1, 2), 27(20), 30(3),37, 43 Barnet, D., 70 Barnett, J. E. C., 261 Barral, F., 206 Bartoshuk, L. M., 312, 334, 337, 339 Bass, G. E., 224 Bateman, A., 37 Batstone-Cunningham, R. L., 179, 180(56),181(56),182(56),183(56), 184(58),185(57, 58), 186(58,59, 60), 188(61),190(61),191(61) Bauerlein, K., 3 Baumann, H., 271 Bayard, B., 94, lOO(32)
353
354
AUTHOR INDEX, VOLUME 45
Beck, F. F., 214 Beck, G., 206 Beck, I. T., 257, 258(184a) Beck, K. M., 265,299 Beck, S . D., 238 Becker, J., 3 Beets, M. G. J., 214, 261(52), 271, 286(52),290, 292, 297(52), 303(52), 304(52),310, 313, 318, 319(52), 320(52),321(52), 322(52),323, 324, 326(52),327(52), 328(52),336(52) Beidler, L. M., 210, 211, 212, 213, 337(46),339(46) Belitz, H. D., 312, 314(280),315(280, 285), 316(285), 317, 319(280, 285) Bell, F. R., 330 Ben-Chetrit, E., 171 Benninghoven, A., 24 Benson, C. A., 299, 300 Berenden, H. J. C., 130, 136(22) Berg, A,, 120, 121(84) Berg, C. P., 231 Bergmann, C. W., 114, 116(75) Bergmann, E., 9 Bernard, R., 209 Bernard, R. A., 330 Bernhard, S., 219, 336(68) Berry, J. M., 153, 154, 158, 161(77) Bertucci, C., 83, 84(7, 8) Bethell, G . S., 249 Beychok, S., 94, 99(30), 112(30) Beyreuther, K., 171, 172 (19,20), 173(19, 20) Bigbee, W. L., 194 Bill, J. C., 37 Birch, G. G., 207, 214, 221, 222, 223(77, 79), 238(20, 79), 239(23, 77, 128), 240(23, 79, 128), 241(77,80, 126, 128, 138), 242(138), 243(126, 127), 244(150),245(138), 247(150), 249(73),250(51, 127), 251(127), 252(127),253, 254(178), 255(150, 178), 257(79, 138), 259(126, 140), 260(77, 126, 127), 261(127),262(127), 263, 264(191, 192), 265(20, 77, 128), 266, 269(126, 183), 270(183), 271(183),272(210), 273(149, 183), 274(183),275(138), 276(151, 211), 291, 292(79, 140), 293(191),294(191), 295(191),312(139), 318(77, 126, 127,
138, 139, 140), 320(20, 23), 322(139), 323, 324(137), 340, 342, 343(352), 344(137), 345(126, 137, 201, 350, 352), 346(352), 347(350), 348(350) Bird, G. W. G., 171 Bittiger, H., 119 Bladier, D., 171 Blakemore, W. F., 43, 52(63), 62(63) Blanksma, J. J., 206, 225(15), 227(88), 303(15, 88) Blicke, F. F., 299 Bloch, F., 126 Bock, K., 24, 142, 153 Boshagen, H., 5 Bohak, Z., 213 Bojesen, G., 20 Bordoli, R. S . , 20, 24(1, 2), 27(20), 30(3), 37,43 Boschemeyer, L., 299,300(254) Bossmer, R., 111, 112(65) Bosso, C., 70 Bothner, B., 39, 40(53), 51, 52(69),55(69) Boudreau, J. C., 319 Bouquelet, S., 70 Bourne, E. J., 32 BovBe, W. M. M. J., 137, 142 Bowers, P. W., 312, 320(282) Boyd, J., 105, 106(48) Boyle, D., 170 Bradley, C., 20 Brand, J. G., 212, 219(38), 318 Braudo, E. E., 111 Brennan, P. J,, 57 Brennan, T. M., 306 Brewer, J. T., 235, 276(121) Briedel, M., 285 Briggs, D. E., 320 Brimacombe, J. S., 220 Bron, C., 171 Brooks, J. R., 181, 195(63), 196(63) Brouwer, J. N., 290, 339(234) Brown, G. M., 263, 266(193) Brown, L. R., 188 Brussel, L. P. B., 234, 245(119, 120), 302, 304(119), 309(119), 310 Bruton, R., 142 Buffington, L. A., 88, 90(24), 91(24), 94, 95(29), 97, 98(37), 100(37), 101(37), 102(29), 104(29), 113(29), 114(29) Bugianesi, R.; 97
AUTHOR INDEX. VOLUME 45 Burlingame, A. L., 20, 25 Bush, C. A., 94, 95(28,34), 96(38), 97(34, 35),98(39), 99(39), lOO(28, 34, 35), 101(34), 108(35), 111, 112(64), 114, 115(76), 118, 120(82) Buttrick, R. L., 337 C Cagan, R. H., 213,214,318,328, 329(301), 330(50, 301), 331(301), 337(301), 338(301) Cain, B. E., 301, 315(258) Cain, W. S., 312 Calvin, A. D., 239, 248(136) Cameron, A. T., 341, 345(351) Campbell, I. D., 130, 138, 143(21) Campbell, K. H., 238 Cann, J. R., 192 Carlsson, S. R., 64 Carr, C. J., 214 Carr, S. A., 23, 69, 70 Carter, R. D., 181, 182, 188(64),189(64), 192(64), 193(94), 195(63), 196(63) Cartron, J., 171 Cartron, J. P., 171, 191 Cedeno, E. R. V., 146 Chachaty, C., 168 Chakrabartl, B., 94, 95(29), 102(29), 104(29),113(29, 46), 114(29), 115, 116(73) Chandler, J. W., 345, 350(353) Chekkor, A., 70 Chen, G. C., 77, 93, 94(27) Chen, W., 314, 315(285), 316(285), 3 17(285), 3 19(285) Cherry, R. J,, 171 Childers, L. G., 226 Chonan, M., 259 Chowdhry, V., 65 Chung, M. C. M., 118 Clark, A. J., 211 Clarke, B. J., 319 Clauss, K., 299 Clayton, J. M., 224 Cloninger, M. R., 306 Coduti, P. L., 94, 95(34), 97(34, 35), lOO(34, 35), 101(34), 108(35) Cohen, J. S., 188 Cohen, M. J., 334
355
Cohn, G., 202, 214(7), 218, 298, 303(7) Colebrook, L. D., 140, 154(43) Colonna, M., 206 Compadre, C. M., 295 Constantopoulos, G., 118 Cook, J. C., Jr., 20 Cook, M. R., 287 Cornillot, P., 171 Costello, C. E., 63 Cotter, €3. J., 58 Cottrell, J. S., 37 Cowell, N. D., 207, 239(23), 240(23), 245, 273(149), 274(149), 320(23) Cowman, M. K., 114, 115(76), 116(75) Crammer, B., 287 Crestfield, A. M., 176 Crosby, G. A., 228, 230, 234(94) 236(94), 237(34), 245(144, 145), 265(144, 157), 268(199), 280(144, 145, 158), 281(157), 282(144, 145, 157), 283, 284(145), 285(144, 145), 300, 303(94), 336(94), 338, 340, 341(347), 342(347), 343(347), 344(347), 345(347), 351 Crouch, R., 142 Crow, J., 232, 237(114) D Dabrowski, J., 27, 28(34), 34(34), 37(34), 43(34), 48(34), 52(34), 54(34), 55(34, 71), 56 Dabrowski, U., 43, 52, 55(71), 56 Dahr, W., 171, 172(19, 20), 173(19, 20), 175 Dais, P., 141, 142, 143, 144(49), 145(49), 153(49), 154(49), 155(49), 156(49), 158, 159(49, 60), 160(44, 49), 161(49) Daman, M. E., 171, 181, 185(57), 186(59, 60), 188(61), 190(61), 191(61) D’Angona, J., 20 Daniel, J. R., 230 Daniel, P., 63 Darke, A., 118, 119(81) Darvill, A. G., 27, 28, 34(36), 43(36), 53, 66(74), 67(33, 37), 68(36) Darvill, J., 53, 66(74) Dastoli, F. R., 214, 311, 328 Dateo, G. P., 337 Davies, D. B., 165, 168(78) Davies, J. P., 168
356
AUTHOR INDEX. VOLUME 45
Davis, K. R., 27, 67(33) Dearborn, D. G., 176, 177(49,50-52), 178(50-52) Defaye, J., 70 DeFontaine, D. L., 142 Degn, H., 249 DeGrado, W. F., 65 DeKaban, A., 118 Dell, A., 20, 21, 26, 27, 28(26), 32(26, 32), 34(26, 36), 35(41), 36(7, 43), 37(5, 15, 26), 38(25, 41), 39(25), 40(53), 41, 42(26), 43(24, 26, 36), 47(26, 42, 64), 49(67), 50, 51, 52(63, 69), 53(15, 42), 54(26), 55(55, 69), 56(76), 58(15), 59(41), 60, 62(63), 63, 64,65(11), 66(32, 72, 74), 67(32, 33, 37), 68(36, 42), 69(42), 70(64), 71(43, 125) DeMarco, A., 188 Dennis, R. G., 76 Desai, P. R., 171, 175 DeSimone, J. A., 212 Dethier, V. G., 248 Deutsch, E. W., 223, 224(82), 225, 226(82),227, 231, 233, 303(82), 342 deVos, A. M., 333, 334(314a) Dey, P. M., 50 Dhein, R., 5 Diamant, H., 337 Diamond, J. J., 85 Dickinson, H. R., 94, 97(35), 100(35), 108(35),111, 112(64) Dienst, C., 64 Dill, K., 171, 179, 180(56), 181(56), 182(56),183(56), 184(58),185(57, 58), 186(58,59, 60), 188(61,64), 189(64),190(61), 191(161),192(64), 193(64),195(63), 196(3) Dillon, A. F., 24 Dinda, R. K., 257, 258 Dobson, C. M., 130 Dodd, E. A., 239, 248(136) Dodrell, D., 249 Doig, A. R., 311 Dolejs, L., 286 Donahue, J., 219 Donner, W., 3 Doorenbos, N. J., 299 Dorland, L., 24
Dorling, P. R., 63 Dorn, C. P., 97 Dowling, B., 290 Drzeniek, Z., 170 Dua, V. K., 118, 120(82) Duben, A., 96(38), 97, 98(39), 99(39) DuBois, G. E., 228, 230(94), 234(94), 236(94), 237(94), 245(144, 145), 265(144, 157), 280(144, 145, 158), 281(157),282(144, 145, 157), 283(157), 284(145), 285(144, 145), 300(94), 303(94), 336(94), 338(94), 340, 341(347), 342(347), 343, 344(347),345(347), 351(94) Duk, M., 172, 175 Durham, L. J., 292 Durozard, D., 303, 304 Dutton, G. G. S., 53, 66(72) Dzendolet, F., 212 Dzidzic, S. Z., 245, 276(151)
E Ebel, D., 97 Ebert, W., 175 Edwardson, J. A., 332, 333(314) Egge, H., 26, 27, 28(26, 34), 32(26), 34(26, 34), 37(26, 34), 42(26, 35), 43(24, 26, 31, 34), 47(26, 64), 48(34, 35), 51, 52(34), 54(26, 34), 55(31, 34, 35, 71), 56, 60(70), 63, 64, 70(64) Egli, R. H., 213 Ejchart, A., 142 Ellerton, N. F., 118 Elliott, G. J., 24, 27(20), 37 Emid, S., 142 Endo, T., 65 Englard, S., 78, 85, 102, 113 Erickson, R. P., 340 Ernst, R. R., 127, 130(16), 131(16), 142(16), 143(16), 145, 146(16) Esaki, S., 280, 288(219) Eschenfelder, V., 111, 112(65) Etienne, A. T., 20, 37(5) Etingof, R. N., 328, 329(303),330(303) Evans, D. R., 212, 243, 248(148), 328(41) Evans, L. V., 105, 107(55, 56), lOS(55, 56), 109(55)
AUTHOR INDEX. VOLUME 45
Evans, S., 37 Evelyn, L., 153 Excoffier, G., 70 Eyring, E. J., 102 Eyton, D., 245, 273(149), 274(149)
F
Facer, C. A., 170 Fagerness, P. F., 142 Fangmann, R., 64 Farkus, L., 280 Faull, J. R., 337 Fecher, R., 97 Feeney, J., 64 Feizi, T., 38, 64 Fenton, H . J. H., 253 Ferguson, L. N., 207,209, 214,223, 225(19), 226, 227(19), 303(19) Fernirndez-Bolafios, J., 15 Fenselau, C. C., 57, 71 Ferrari, B., 181, 188(61, 64), 189(64), 190(61), 191(61), 192(64), 193(64) Ferretti, J. A., 142 Fick, A., 339 Ficken, M. S., 330 Figueroa, N., 115 Findlay, J. C., 290 Finizi, C., 206 Finley, J. W., 290 Fischer, C., 120, 122(88) Fischer, H. 0. L., 253 Fischer, 0. L., 9 Fischer, R., 335 Fisher, R., 318 Fishman, I. Y., 334 Fong, S. S. N., 194 Forsberg, L. S., 21, 65(11) Foster, A. B., 216, 220(62) Foster, R., 231 Fournet, B., 60 Fournie, J.-J., 70 Fox, A. L., 310, 311(274) Fraenkel, G. K., 126 Francke, A., 290, 339(234) Frangou, S. A., 105, 109(57) Frank, M., 237, 334 Fraser, B. A., 65
357
Freed, J. H., 126 Freeman, R., 126, 127(14), 130(16), 131, 133(14), 138, 139, 140(40), 142(12, 16), 143(16, 21), 145(40), 146(16), 159(14) Freifelder, D., 179 Friedman, M., 290 Fries, F. A., 3 Fujino, M., 307, 308(272), 310(272) Fukayama, K., 161, 162(82), 166(82) Fukuda, M., 26, 34, 35(41), 38(25, 41), 39(25), 40(53), 42, 55(55), 59(41, 51), 60,64 Fukuda, M. N., 26, 34, 35(41), 38(25, 41), 39(25), 42, 51, 52(69), 55(55, 69), 59(41, 51) Fukui, K., 232, 237(113) Fuller, W. D., 306 Funakoshi, M., 206 Furia, T. E., 265, 340, 345(349) Furthmayr, H., 170, 171(5-7), 172(4, 6, 7), 173(7), 175, 194, 195(4, 36) Furuta, S., 292 Fyfe, C. A., 231
Gabel, C. A., 63 Garcia Gonzirlez, F., 13, 15 Gardner, R. J., 318, 319, 320(287) Gasteiger, J., 314, 315(285), 316(285), 317(285), 319(285) Gattegno, L., 171 Gaudioso, L. A., 20 Gee, S . C., 239, 248 Gehrke, M., 3 Geist, K., 5 Gekko, K., 88, 90(22), 123 Geller, J. H., 350 Gentili, B., 241,243, 277, 278(141-143), 279(141-143, 214), 280(143, 217), 281, 282(142), 291(143) Geraldes, C. F. G. C., 168 Gerbal, A., 171 Gerken, T. A., 176, 177(50-52), 178(50-52) Geyer, H., 63 Geyer, R., 56,63 Ghermani, N., 192
AUTHOR INDEX, VOLUME 45
358
Ghidoni, R., 27, 43(31), 55(31) Gielen, W., 171, 172(19, 20), 173(19, 20) Girardet, M., 171 Gniichtel, A., 4 Goel, A., 306 Goerdeler, J., 4 Goldkamp, A. H., 245, 306(152), 307(152), 309(152, 271) Goldstein, A., 211, 212(34) Gbmez Sinchez, A,, 14 Goodman, M., 306 Goodwin, J. C., 243, 261, 269(146), 271(146), 288(146), 291 Gooi, H. C., 64 Gorbatschow, S. W., 226 Gordon, E. C., 94, 95(34), 97(34), 100(34j, lOl(34) Gottsegen, A., 280 Granot, J., 142 Grant, C. W. M., 126, 152(13), 159(13) Grant, D. M.,125, 126(3), 130, 131, 137, 142, 146 Grant, G. T., 105, 106(50), 109, l l l ( 5 0 ) Green, B. N., 20, 37, 43, 70 Green, J. W., 256 Green, J. P., 231 Greenward, C. T., 87 Griffin, F., 318 Gross, M. L., 36 Guadagni, D. G., 320 Guild, W. E., 215, 249(60) Gunson, H. H., 171
H Hadata, M., 333, 334(314a) Haga, M., 259 Hagiwara, S., 334 Hahn, H., 210 Hall, L. D., 125, 126, 127(14), 133(14, 15), 135(15), 139, 140, 142, 147, 148(67-69), 149(68, 70), 152(13), 153, 154(43), 158, 159(13, 14, 15), 161(77, 78), 165, 166(78), 167(78, 85), 256 Hall, L. H., 228, 229(96, loo), 230, 318(98) Hall, M. J., 312 Halpern, B. P., 337, 338(340), 348
Hamasaki, T., 161, 162(82), 166(82) Hamor, G., 226 Handa, S., 21, 54( 12) Hanfland, P., 27, 28(34), 34(34), 37(34), 43(34), 48(34), 52(34), 54(34), 55(34, 71), 56 Hanisch, F.-G., 64 Hansch, C., 223, 224, 225, 226(82), 227, 228, 230, 231, 233, 303(82), 342 Hanssum, H., 142 Harada, K., 27 Harada, N., 123 Harada, T., 68 Hardy, R. E., 179, 180(56), 181(56), 182(56), 183(56), 184(58), 185(57, 58), 186(58, 59, 60), 188(61, 64), 189(64), 190(61), 191(61), 192(64), 193(64) Hargreaves, M. K., 123 Harrison, R., 336 Hart, P. A., 168 Hartzog, M. B., 109 Hashigaki, K., 280, 285 Hass, J. R., 70 Hata, T., 306 Hatano, H., 132, 133(30), 164(30) Hatsuda, Y., 161, 162(82), 166(82) Hay, G. W., 257, 258(184a) Hayashibara, K., 276 Heernia, W., 43 Helferich, B., 3, 4, 5, 6 Heller, D. H., 57, 71 Hemling, M. E., 26, 27(27), 54(27), 71(27) Hendrick, M. E., 306 Henkin, R. I., 330 Henning, G. J., 290, 339(234) Henrick, K., 67 Henry, L., 310 Herout, V., 286 Herrtage, M. E., 43, 52(63), 62(63) Herve du Penhoat, P. C. M., 249 Hessinger, D. A., 170 Heyraud, A., 70 Higginbottom, J. D., 266 Higuchi, T., 27, 43, 54(30), 55(58-60), 71(79) Hiji, Y., 329, 335 Hilderbrand, R. P., 319 Hill, H. D. W., 126, 127(14), 133(14, 15),
AUTHOR INDEX, VOLUME 45 135(15), 139, 140(40), 142, 145(40), 159(14, 15) Hilt, R. L., 131 Himmen, E., 4 Hisumatsu, H., 68 Hodge, J. E., 243, 261(146), 269(146), 271(146), 279, 283, 285(222), 288(146, 215), 289(215), 291(146, 147) Hoegen, D., 206, 225(15), 227(88), 303(15, 88) Hofmann, A., 290 Hogan, R. M., 328, 329(302) Hoglan, F. A., 290 Hohener, A., 145 Holbrooks, A. M., 181, 186(60) Holland, W. C., 232, 237(114) Hollaway, M., 340, 345(350), 346(350), 347(350), 348(350) Hollerman, A. F., 205, 298(10) Holmquist, L., 111, 112(65) Homer, J., 146 Honda, E., 280 Hooykaas, P., 53, 66(74) Horoch, N. J., 37 Horowitz, R. M., 241, 243, 277, 278(141-143), 279(141-143, 214), 280(143, 217), 281, 282(142), 291(143) Horsley, W. J., 140 Horton, D., 295 Hough, C. A. M., 332, 333(314) Hough, J. S., 320 Hough, L., 265, 266(195), 267, 268 Hounsell, E. F., 63, 64 Hubbard, P. S., 126, 131 Huet, M., 191 Hughes, L. J., 188 Hull, W. F., 158, 161(78), 166(78), 167(78) Humoller, F. L., 257, 258 Hunter, S. W., 57 Huprikar, S. V., 175 Hurlbert, S., 142
I Ikan, R., 287 Imamura, A., 232, 237(113) Imoto, T., 132, 133(30), 164(30)
359
Inglett, G. E., 279, 288(215), 289(215), 290 Inoue, S., 65 Inoue, Y., 65 Isbell, H. S., 249 Isenberg, I., 231 Ison, E. R., 257, 258(184a) Isono, K., 237 Issitt, P. D., 194 Ito, J., 335 Iwamori, M., 27, 43, 54(30), 55(58, 59, 60), 56, 71(79) Iyengar, R. B., 333
J Jackson, G., 266, 269(204) Jackson, H., 253 James, P. A., 307, 309(271) Jansson, P.-E., 53, 66(72) Jardine, I., 57 Jaspers, D., 24 Jeffrey, G. A., 271, 295 Jenner, M. R., 265, 266,267(196), 269(204) Jennings, H. J., 105, 107(53), 108(52), 112 Jensen, H., 299 Jensen, R. H., 194 Jentoft, J. E., 176, 177(50-52), 178(50-52) Jentoft, N., 176, 177(49, 50-52), 178(50-52) Jibza, J., 286 Johnson, L. F., 142 Johnson, P. A., 320 Johnson, W. C., Jr., 79, 80(45), 81(4), 82, 83(6),84(6, 7, 8), 85(4, 5), 86, 87, 120(18) Jones, C., 66 Jones, J., 43, 52(63), 62(63) Jones, J. K. N., 259 Judd, W. J., 194 Judkins, M., 20, 37(5) Junger, A., 3 Junger, H., 5 Jugel, H., 314, 315(285), 316(285), 317(285),319(285) Juliano, R. J., 170 Jungery, M., 170
360
AUTHOR INDEX, VOLUME 45 K
Kabat, E. A., 94, 99, 112(30) M a , W. A,, 348 Kahane, I., 171 Kalk, A,, 130, 136(22) Kamath, S. K., 295 Kambara, H., 21, 27, 54(12), 65 Kamerling, J. P., 43, 63 Kamisango, K., 53, 56(76), 65 Kamiya, S., 280, 288(219) Kanda, N. Y., 301, 315(258) Kaneko, T., 207,213,231 Kaptein, R., 140 Karantz, J. C., Jr., 2-14 Kare, M. R., 330 Karplus, M., 126 Kasai, R., 285, 298(226) Kato, S., 225 Katritzky, A. A., 182 Katsube, Y., 161, 162(82), 166(82) Katsuya, N., 303, 306(268) Kearsely, M. W., 245, 275, 276(151, 211) Keilich, G., 94, 97(33), 111, 112(65),119 Kenne, L., 34, 47(42), 53(42), 68(42), 69(42) Kennedy, L. M., 337,338(340) Khan, R., 263, 264(191, 192), 265, 266(195),267, 268, 269(204), 293(191),294(191), 295(191) Khokher, A., 320 Kier, L. B., 228, 229(96, 100, 101), 230(99),231, 232(log), 233(109),238, 268, 283, 292(log), 296, 297, 299(109),303(109), 304, 317, 318(98), 340 Kier, L. Y., 336 Kiesow, F., 239 Kijima, H., 237 Kikuchi, T., 237, 264(125) Kim, H. S., 271,295 Kim, S. H., 333, 334(314a) Kimizuka, A., 303, 306(268) Kimura, K., 210, 211 Kinghom. A. D., 295 Kirimura, J., 303, 306(268) Kitchell, L. R., 330 Kleb, K. G., 5 Klein, M. P., 138, 140 Klein, W., 4 Kleinschmidt, T., 5
Klock, J. C., 39, 40(53), 42, 55(55), 59, 64 Knoll, H., 175 Kobota, A., 172 Koboyashi, N., 329 Kodama, S., 205, 218 Koeppen, B. H., 279 Koerner, T. A. W., Jr., 171 Koster, H., 3 Kohda, H., 285, 298(226) Koludny, E. H., 112, 113(67) Kondoh, T., 292 Konishi, F., 280, 288(219) Kordowicz, M., 52, 55(71), 56 Kornfeld, S., 63 Korolkavas, A., 219 Korppi-Tomola, S. L., 252, 253(175) Kowalewski, J., 142 Koyama, N., 318, 328, 329(288) Koyama, T., 280,285 Krabbendam, H., 333, 334(314a) Krotkiewski, H., 170 Kriiger, J., 171, 172(19, 20), 173(19, 20) Krueger, J. M., 330 Krusius, T., 59 Kubo, I., 312, 313 Kubota, T., 312, 313 Kuhlmann, K. F., 142 Kuhn, L. P., 216, 293(61) Kuo, M. S., 53 Kurg, L., 63 Kurihara, K., 213, 318, 337(46), 339(46) Kurihara, Y., 213, 328, 329(288), 337(46), 339(46) Kuroyanagi, M., 123, 124(96) Kushi, Y., 21, 54(12) Kutny, R. M., 65
L Lacombe, J. M., 191 Lafymer, Z., 340, 345(350), 346(350), 347(350), 348(350) Lambuth, N., 335 Landgren, S., 337 Landsteiner, K., 175 Lang, K., 6 Lang, O., 4 Lange, P., 171 Langlet, G., 168 Lannom, H. K., 181, 188(64), 189(64), 192(64), 193(64)
AUTHOR INDEX, VOLUME 45 Lardicci, L., 84 Larson-Powers, N., 340, 341(348), 345(348) Lasareff, P., 210 Lavielle, R., 285 Lawrence, A. R., 207,209,214,223, 225(19), 227(19), 239(19), 303(19) Lawson, A. M., 63, 64 Lawton, B. T., 259 Lazzaroni, R., 83, 84(7, 8) Leaffer, M. A., 296 Lecher, O., 3 Led, J. J., 142 LeDonne, N. C., Jr., 32 Lee, C. K., 214, 222, 223(77-79), 235(78), 238(78, 79), 239(77, 128), 240(78, 79, 128), 241(77, 78, 128), 243(78, 126), 244(150), 245, 246, 247(150), 248(161), 250, 254(178), 255(150, 178), 257(78, 79), 258(48), 259(78, 126), 260(77, 126), 262, 265(77, 78, 128), 266, 269(126, 183, 203, 204), 270(183), 271(183), 272(210), 273(183), 274(183), 275(161), 276, 292(79), 293(161), 295(161), 312(139), 318(77, 126, 139, 281), 322(139), 345(126, 201) Lee, Y. C., 71 Lee, C. Y.,214, 219(48), 231(48), 292(48) LeFavre, R. J. W., 293 Lehninger, A. L., 336 Lehrle, R. S., 24 Leipert, T. K., 142 Lelj, F., 246, 308(159), 309(159) Lemieux, R. U.,235,271,276 Leontein, K., 66 Levine, P., 175 Levine, S., 271 Levitt, M. H., 145 Levy, C. C., 142 Levy, H. M., 263, 266(193) Lewis, D. C., 86, 87, 120(18) Lewis, I. A. S., 43 Li, J. P., 97 Li, S.-C., 56 Li, S.-L., 213 Liang, J. N., 91, 92(26), 93(26), 105, 109(57), llO(62) Lillford, P. J., 153 Limas, L., 171 Lin, D. C., 170
361
Lin, J. W.-P., 120 Lindberg, B., 34, 47(42), 53(42), 66(72), 68(42), 69(42) Lindemann, H., 271 Lindley, M. C., 201, 223, 234, 235(80), 238(64, BO),239(128), 240(128), 241(80, 128, 138), 242(138), 243(80, 127), 245(138), 249, 250(127), 251(127), 252(127), 256, 257(138), 259(140), 260(80, 127), 261(80, 127), 262(80, 127), 263, 264(191, 192), 265(80, 128), 274, 275(138), 291, 292(140), 293(191), 294(191), 295(191), 296, 304, 318(127, 138, 140), 334 Lindquist, U., 53, 66(72) Linell, R. H., 271 Linsheid, M., 20 Lisowska, E., 170, 172, 175 Listowsky, I., 78, 85, 102, 113 Littlewood, J. T., 43, 52(63), 62(63) Liu, H.-W., 124 Lloyd, K. O., 51, 52(69), 55(69), 94, 99(30), 112(30) Lonngren, J., 66 Loeve, K., 213 Loginov, N. E., 206 London, R. E., 192 Long, C., 171 Long, L., Jr., 337 L6pez Aparicio, F. J., 15 Lopiekes, D. V., 311 Lord Zuckerman, 200 Loucheux-Lefebvre, M.-H., 94, lOO(32) Lovrien, R. E., 171 Luck, H., 299 Ludeman, H. D., 165, 168(88) Lugowski, C., 34, 47(42), 53(42), 68(42), 69(42) Lui, S. C., 64 Lum, C. K. L., 330 Lunt, C. C., 336 Lux, S. E., 171 Lynden-Bell, R. M., 126 M McCammon, J. A., 137 McCasland, C. E., 292 McClellan, A. L., 214, 215(56) McClinchey, G . O., 302
362
AUTHOR INDEX, VOLUME 45
McDowell, R. A., 20, 36(7), 37(5) McFarland, J. W., 227, 228(93),230, 231 Macfarlane. R. D.. 36 McIlhenny; H. M:, 337 McLafferty, F. M., 70 McNeil, M., 27, 28, 32(32), 34(36), 43(36), 53, 66(32, 74), 67(32, 37), 68(36) Madigan, M. J., 63 Magidson, Y.,226 Maier, V. P., 320 Malkomes, T., 3 Malman, S. M., 211, 212(34) Malrieu, J. P., 231 Mansson, J.-E., 56 Marchesi, V. T., 170, 172(4, 8), 173(8), 195(4, 36) Marchessault, R. H., 120, 122(85,86) Marcus, D. M., 194 Markley, J, L., 140 Marowitz, L. A., 348 Marquardt, D. W., 142 Marraud, M., 192 Marsh, J. W., 176, 177(53) Marshall, A. G., 131 Marshall, D. L., 123 Marsili, M., 314, 315(285), 316(285), 317(285),319(285) Martin, 0. R., 252,253 Martin, Y. C., 230 Masuda, R., 292 Matoba, T., 306 Matsuda, H., 68 Matsuo, T., 68 Mattai, S. E., 214, 250(5l) Mattick, L. R., 221 Matwiyoff, N. A., 192 Maurer, W., 142 Mayne, C. L., 142, 146 Mazur, R. H., 245, 306, 307(152, 153), 309(152, 153, 271) Meerbug, W., 303, 351(266) Mehikopf, A. F., 142 Meijer, G. H., 137 Meiser, W., 291 Mellon, D., Jr., 212, 328(41) Melton, L. D., 28, 67(37), 111, 112(66) Menges, H., 56 Merle, J.-P., 122 Metaxas, M. N., 175
Metaxas-Buhler, M., 175 Metz, J., 175 Meyer, K., 114, 116(75) Michalski, J.-C., 51, 60(70), 64 Milbrand, M., 253 Millson, H. E., Jr., 299 Minematsu, H., 306 Mo, H., 56 Moncrieff, R. W., 200, 214(4), 261(4), 298 Montreuil, J., 51, 60(70), 64, 70, 118, 120(82) Moog, L., 3 Moore, M. L., 20 Moore, S., 176 Mooser, G., 335 Morgan, R. E., 145 Mori, K., 239 Morita, H., 237,283 Morris, E. R., 88, 90(24), 91(24), 92(26), 93(26), 102, 103(47), 105(47), 106(48-51), 107(51, 54-56), 108(54-56), 109(49, 51, 55, 57), 111(50),112(66), 118, 119(81) Morris, H. R., 20, 26, 28(26), 30(3), 32(26), 34(26), 36(7), 37(5, 26), 42(26), 43(24, 26), 47(26), 54(26), 65 Morris, J. A., 213 Morrison, M., 170 Mort, A. J., 53 Moskowitz, H. R., 345,350(353), 351 Mouat, B., 171 Mueller, T. J., 170 Mufti, K. S., 266, 269(204) Mukherjee, S., 120, 122(85, 86) Mulloy, B., 66 Munch, J. C., 311 Munton, S. L., 345 Murakami, K., 285, 298(226) Murray, W. J., 228, 229(96, 100, 101), 230(99),318(98) Murthy, M. S., 171 Mustsaers, J. H. G. M., 63, 64 Myers, R. G., 202,218 Mylvaganam, A. R., 239, 323, 324(137), 340(137), 344(137), 345(137)
N Nagai, Y., 27, 43, 54(30), 55(55-60), 56, 71(79)
AUTHOR INDEX, VOLUME 45 Nagata, C., 232, 237(113) Nahum, A,, 176, 177(53) Naik, S., 50 Nakajima, N., 307, 308(272), 310(272) Nakanishi, K., 123, 124(96), 124 Nambu, H., 123, 124(96) Naumann, M. 0..292 Nelson, E. C., 243, 291(147) Nelson, R. G., 79, 80(4, 5), 81(4),85(4, 5) Neuenhofer, S., 56 Neuman, F. J., 238 Ney, K. H., 319 Nieman, C., 216, 217(63), 239(63), 249(63) Niermann, H., 52, 55(71) Nigg, E. A., 171 Ninimiya, T., 303, 306(268) Nishimura, O., 65 Noda, A., 161, 162(82), 166(82) Noda, K., 306 Nofre, C., 299, 301, 302(260), 303, 304, 305(268c) Noggle, J. H., 127, 130(17), 134, 138(17), 168 Nogradi, M., 280 Noma, A., 335 Nothnagel, E. A,, 27, 32(32), 66(32), 67(32) Nunez, H. A., 24 Nurden, A. T., 171
0 Oakley, B., 334, 335(321),337 Oates, J. E., 26, 28(26), 32(26), 34(26), 35(41), 37(26), 38(25, 41), 39(25), 42(26), 43(26), 47(26, 42), 50, 52(63), 53(42), 54(26), 55(55), 59(41), 62(63), 63, 66, 68(42), 69(42) Ochial, Y.,194 Oertley, E., 202, 218 Ogawa, H., 334 Ogawa, S., 292 Ogawa, T., 43 Ohashi, Y., 43 Okaya, Y., 301 Okazaki, H., 225 Oki, M., 285 Oleski, P., 142 Oliver, S. M., 296
363
Olssen, L., 337 Oltz, E. M., 123, 124(96) Osfretsova, I. B. O., 328, 329(303), 330(303) Owens, J. W., 170
P Palamand, S. R.,313 Palmer, A. C., 43, 52(63), 62(63) Palmer, K. J., 109 Palmer, L., 142 Pandey, R. C., 20 Pangborn, R. M., 200,239, 248,340, 341(348), 344(348), 350 Panico, M., 20, 30(3), 37(5), 65, 71 Park, J. W., 113, 115(71-74), 116(73) Park, Y.J., 271, 295 Parry, R. B., 142 Parsons, S . F., 170 Pasvol, G., 170 Patel, G. D., 266, 269(204) Patel, T., 170 Pauling, L., 214, 215(55) Pautet, F., 299, 301, 302(260) Pavia, A. A., 181, 188(61, 64), 189(64), 190(61), 191(61), 192(64),193(64) Pavone, B. G., 194 Paz-Parente, J., 51, 60(70) Pedersen, C., 24, 153 Pedersen, E. J., 142 Peer, H. G., 234, 245(119, 120), 302(120), 304(119), 309(119, 120), 310 Peerdeman, A. F., 333, 334(314a) Peraldo-Bicelli, L., 301, 315(258) Percival, E. G. V., 249 Perkins, M. E., 170 Perlin, A. S., 141, 142, 143, 144(49), 145(49), 153(49), 154(49), 155(49), 156(49), 158, 159(49, 60), 160(44, 49), 161(49),249 Perret, G., 171 Peter-Katalinic, J., 27, 42(35), 43(31), 48(35), 51, 52, 55(31, 35, 71), 56, 60(70), 64, 70 Peters, D., 192 Peters, J., 192 Peters, O., 4 Petitou, M., 70
364
AUTHOR INDEX, VOLUME 45
Petryniak, B., 170 Petryniak, J., 170 Peytavi, A. M., 304, 305(268c) Pezzuto, J. M., 295 Pfaffmann, C., 239, 311, 334,337, 339 Pfannemiiller, B., 87, 120, 121(84) Pfannschmidt, G., 56 Pflumm, W. W., 248 Phadnis, S. P., 265 Phelps, D. E., 138 Phillips, L. R., 65 Pickering, N. J., 64 Pilgrim, J. J., 285 Pimentel, G. C., 214, 215(56) Plaschina, I. G., 111 Portz, w., 5 Powell, D. A., 105, 107(56), 108(56) Preston, C. M., 125, 126(1), 139, 147, 148(67-69), 149(68, 70), 152(13), 153(l), 159(13) Price, S., 212, 214, 311, 328, 329(302, 304) Pridham, J. B., 50 Pringsheim, H., 9 Prohaska, R., 171 Prome, D., 42, 70(56) Prome, J. C., 42, 70(56) Pullman, A., 232 Pullman, B., 232 Purcell, W. P., 224 Puzo, G., 42, 70(56) Pysh, E. S., 93, 94(27), 95(29), 102(29), 104(29),113(29), 114(29)
Q Qureshi, N., 53, 57(75), 58
R Rachmilewitz, E. A., 171 Ragouzeos, A., 142 Ralapati, S., 97, 98(39), 99(39), 118, 120(82) Ramsamooj, P., 39, 40(53) Ranc, A., 206 Randic, M., 228, 229(96, 100) Rao, C. N., 306
Rao, G. S., 337 Rao, K. G., 306 Rao, V. S., 158 Ratcliffe, R. G., 130 Rathbone, E. B., 266, 269(204) Rauch, H., 4 Ravanat, G., 109, llO(59) Ravestein, P., 333 Redfield, A. G., 126 Rees, D. A., 88, 90(24), 91(24), 92(26), 93(26), 102, 103(47), 105(47), 106(48-51), 107(51, 54-56), 108(54-56), 109(49, 51, 55, 57), 111(50), 112(66), 118, 119(81) Reeves, R. E., 215 Reincke, R., 214 Reinhold, V. N., 23, 63, 69, 70 Renqvist, Y., 206, 209, 210 Rettig, W. J., 51, 52(69), 55(69) Reuter, G., 27, 43(31), 55(31) Reuter, J. A., 245, 307(153), 309(153) Ribi, E., 53, 57(75) Richardson, A. C., 257, 259,272(210), 273 Richarz, R., 188 Richter, C. P., 238 Richters, G., 333 Rinaudo, M., 109, llO(59) Rinehart, K. L., Jr., 20, 26, 27(27), 54(27), 71(27) Robb, J. C., 24 Robb, R. J., 65 Robin, M. B., 84 Robinson, G., 105 Roder, O., 165, 168(88) Roelcke, D., 175 Rolfe, E. J., 222, 223(77), 239(77), 241(77), 260(77), 265(77), 318(77) Romanowska, E., 34, 47(42), 53(42), 68(42), 69(42) Ross, D. K., 142 Rowan, R., 111, 137 Roy, R., 105, 107(53), 108(53) Rubin, T. R., 318 Rudrum, M., 292, 293(241) Runnels, L. K., 131 Runti, C., 298 Ruoslahti, E., 59 Ruterjans, H., 142
AUTHOR INDEX, VOLUME 45 S Saadat, S., 53, 56(76), 65 Saffron, P., 241, 245(144), 265(144), 280(144, 158), 282(144), 285(144) Saitoh, M., 292 Sallam, M. A. E., 123 Salmon, C., 171 Salvadori, P., 83, 84(8) Sanderson, G. R., 102, 103(47), 105(47) Sandhoff, K., 56 Santikarn, S., 20 Santos, H., 168 Sarko, A., 120, 122(85, 86, 88) Sass, M., 142 Sataryan, E. Kh., 328, 329(302), 330(303) Sathyanarayana, B. K., 85, 90 Sato, H., 123 Sato, K., 280, 285 Sato, M., 329, 334 Sato, T., 212 Saxby, J. D., 293 Scanlon, E. F., 171 Schafer, W., 3, 4 Schaublin, S., 145 Schauer, R., 27, 43(31), 55(31) Schellenberg, P., 5 Schilperoot, R., 53, 66(74) Schlatter, J. M., 245, 306(152), 307(152, 153),309(152, 153, 271) Schmitz-Hillebrecht, E., 4 Schnepp, O., 84 Schober, P. C., 312, 320(282) Scholl, F. M., 311 Schott, H. H., 63 Schuerch, C., 120 Schulten, H.-R., 20 Schultz, H., 285 Schwarzmann, G., 56 Seale, R., 105, 106(51), 107(51), 109(51) Sedgwick, R. D., 20, 24(1, 2), 26, 27(20, 27), 30(3), 43, 54(27), 71(27) Segal, G. A., 85 Shallenberger, R. S., 200, 207, 213, 214, 215, 216, 217(22), 218, 219(48), 220(58), 221(5, 71), 222(74), 223(22), 227, 231, 233, 234(116, 117), 235, 238(64, 118), 239(21), 241(48), 249(73, 74), 252, 256(174), 251, 258,
365
259(184d), 260(57), 263, 271(21), 274, 276(117, 118), 292(48), 296, 298, 299(5, 71), 300, 301, 304, 311(21), 312(21), 313(173), 322(57), 340 Sharman, E. H., 84 Shaw, D. F., 292,293(241) Shell, T. C., 334 Shen, T. Y., 97, 257 Shepard, R. N., 340 Sherry, A. D., 176, 177(49a) Shibata, S., 132, 133(30), 164(30) Shifter, A,, 171 Shimada, I., 237, 264(125) Shimamura, M., 65 Shimizu, A., 303, 306(268) Shimomura, J., 56 Shing, T. K. M., 142, 144(49), 145(49), 153(49), 154(49), 155(49), 156(49), 158, 159(49), 160(49), 161(49) Shiraishi, A., 237, 283 Shirmer, R. E., 127, 130(17), 134, 138(17), 168 Shizukuishi, K., 21, 54(12) Sichtermann, W., 24 Sidebotham, R., 43, 52(63), 62(63), 63 Sinsheimer, J. E., 337 Slettengren, K., 66 Smidt, J,, 137, 142 Smith, P. D., 245, 276(151) Smith, P. J. C., 109 Smith, S. E., 312, 320(282) Smits, P., 333 Snowden, B. S., 137 Snyder, P. A., 84 Solms, J., 213, 231 Solomon, I., 136 Son, T. D., 168 Sonnino, S., 27, 43(31), 55(31) Sorm, F., 286 Sotsky, S. M., 118 Speidel, P. E., 3 Spellman, M. W., 67 Spik, G., 43, 118, 120(82) Spillane, W. J., 299, 300, 302, 303 Spooncer, E., 59 Springer, G . F., 171, 175 Stacey, M., 32, 220 Steck, T. L., 170 Steinbach, H., 171, 172(20), 173(20)
366
AUTHOR INDEX, VOLUME 45
Steiner, D. F., 176, 177(53) Steinhardt, R. G., 239, 248(136) Stempfl, W., 317 Stephenson, R. A., 241, 245(145), 280(145), 282(145), 284(145), 285(145),340, 341(347), 342(347), 343(347),344(347), 345(347) Sterling, C., 109, 112(61) Stetter, H., 5 Stevens, E. S., 85, 86, 87(19),88(19), 90(16, 22, 24), 91(24), 92(26), 93(26), 97, 98(37), 100(37), 101(37),105, 109(57),110(62), 116(25), 117(25), 120(25),122 Stevens, J. C., 312 Stevens, J. D., 139, 153, 158, 161(78), 166(78),167(78) Stevens, R., 320 Stewart, J. M., 192 Stipanovic, A. J., 86, 87(19), 88(19, 22), 116(25),117(25), 120(25), 122 Stirm, S., 56, 63 Stocklin, W., 337 Stockton, B., 105, 107(55, 56), l08(55, 56), 109(55) Stoll, M. S., 64 Stone, A. L., 94, 112(31), 113, 117, 118 Stone, H., 231, 295, 296(245a) Straub, K. M., 25 Strecker, G., 26, 43(24), 51, 60(70), 64, 70, 118, 120(82) Strom, L., 337 Suami, T., 292 Suggett, A., 153 Sugimoto, K., 276 Sukehiro, M., 306 Sutherland, I. W., 53, 66(72) Suzuki, M., 27 Sveda, M., 299 Svennerholm, L., 56 Svenson, S . B., 66 Swartz, M. L., 340, 345(349) Sweeley, C. C., 24, 32 Swiatek, K. A., 245, 307(153),309(153) Sykes, B. D., 137 Syper, D., 170 Szarek, W. A., 252, 253(175), 257, 258(1844, 259 Szent-Gyorgyi, A., 231
T Taboada, J., 253 Takayama, K., 53, 57(75), 58 Takeda, R., 123, 124(96) Tanaka, K., 307, 308(272), 310(272) Tanaka, O., 285, 298(226) Tancredi, T., 246, 308(159), 309(159) Tanford, C., 319 Tateda, H., 210 Tatlow, J. C., 32 Taylor, G. W., 20, 35, 36(43), 37(5), 50, 71(43) Taylor, J. R., 147 Taylor, L. C. E., 20, 37 Tedder, J. M., 32 Tegtmeyer, H., 175 Teherani, J., 176, 177(49a) Tejima, S., 259 Temussi, P. A., 246, 308(159), 309(159) Ternal, B., 142 Tettamanti, G., 27, 43(31), 55(31) Texter, J., 85, 90(16) Theophrastus, 200, 201 Thom, D., 102, 103(47), 105(47), 106(48, 50), 107(54), 108(54), 109, 111(50), 112(66) Thompson, A., 257, 258 Tiller, P. R., 39, 40(53), 51, 52(69), 53, 55(69), 66 Tinti, J. M., 303, 304, 305 Tipson, R. S., 350 Todd, P. H., 320 Toeldte, W., 3 Tolstoquzov, V. B., 111 Tomita, M., 170, 172(4), 195(4) Tomlinson, B. L., 126, 127(14), 133(14), 142, 159(14) Toniolo, C., 246, 308(159), 309(159) Topliss, J. G., 230 Torgerson, D. F., 36 Townsend, R. R., 71 Toyokuni, T., 292 Treleano, R., 314, 315(285), 316(285), 317(285), 319(285) Tsuyuhara, S., 56 Tsuzuki, Y., 221, 225, 239, 250 Turnbaugh, J. G., 320 Turner, M. J. A., 170
367
AUTHOR INDEX, VOLUME 45 Tute, M. S., 224 Tyler, A. N., 20, 24(2), 27(20), 30(3), 37 U
Uhlenbruck, G., 64, 171, 175 Ullrich, J., 5 Ulrich, J., 70 Unterhalt, B., 299, 300(254)
V van Brouwershaven, J., 333 Vandenbelt, D. J,, 337 van den Broek, W. J., 206, 225(15), 303(15) van der Hart, M., 171 van der Heijden, A,, 235, 245( 119, 120), 302( 120), 304, 309(120), 310(120) Vanderleen, M., 194 van der Ouderaa, F., 333 van der Wel, H., 213,290,333, 334(314a), 339(234) van der Weyden, P. W. M., 303 van Dijk, C. P., 303, 351(266) van Halbeek, H., 24, 63, 64 van Niekerk, D. M., 279 van Valkenburg, W., 224, 225(87), 227(87) van Veen, R., 53,66(74) van Velthuijsen, J. A., 276 Van Wassenaar, P. D., 333 Vargha, LBszI6, 9 Varki, A., 39, 40(53), 55 Vassy, R., 171 Vayas, D., 257, 258(184a) Verdine, G. L., 123, 124(96) Verkade, P. E., 303, 351(266) Verlander, M., 306 Verstraeten, L. M. J., 249, 272(166) Verzele, M., 320 Vickerman, J . C., 43 Vignon, M . R., 70 Vinogradov, S. N., 271 Vliegenthart, J. F. G., 24,43, 63, 64 Vock, M., 4 Vold, R. L., 125, 126(4), 130(4), 131(4), 138(4), 145(4)
Vold, R. R., 125, 126(4), 130(4), 131(4), 138(4), 145(4) von Gross, E., 5 von Nicolai, H., 26, 43(24), 47(64), 70(64) von Schleyer, P., 300, 349(257) Vuataz, L., 213
W
Wagner, G., 188 Wakimusu, M., 307, 308(272), 310(272) Walkinshaw, M. D., 118, 119(81) Walton, D. J., 21, 65(11) Wander, J. D., 295 Wang, W. J., 32 Wangness, R. K., 126 Warner, K. A., 243, 261(146), 271(146), 288(146), 291(146, 147) Warren, C. D., 118, 120(82) Warren, R. M., 337 Warren, R. P., 337 Wasniowska, K., 170 Wasserman, O., 232,237(114) Watson, J. D., 212 Waugh, J. S., 138 Weatherall, D. J., 170 Wedemeyer, K., 5 Weiss, G. H., 142 Welder, D. W., 154, 158, 161(77) Wells, C., 337 Weninger, M. G., 337 Werbeiow, L. G., 125, 126(3), 130, 131, 137 Westhof, E., 165, 168(88) Weston, A. W., 299 Whiffen, D. H., 220 Whistler, R. L., 230, 252, 256(174) Widmalm, G., 66 Wiegand, F., 3 Wiegandt, H., 56 Wiehle, D., 5 Wieruszeski, J.-M., 64, 70 Wieser, H., 312, 314(280), 315(280, 285), 316(285), 317(285), 319(280, 285) Wilkins, C. L., 36 Williams, D. H., 20 Williams, R., 192
368
AUTHOR INDEX. VOLUME 45
Williams, R. E., 105, 107(53),108(53), 112 Williams, R. J. P., 130 Wilson, A., 66 Wilson, R. J . M., 170 Winchester, B., 43, 52(63), 62(63) Winchester, B. G., 63 Wingard, J. E., Jr., 228, 230(94),234(94), 236(94), 237(94), 241, 245(145), 265(157),268(199),281(157), 282(145, 157), 283(157),284(145), 285(145), 300(94),303(94),336(94), 338(94),340, 341(347),342(347), 343(347),344(347),345(347),351(94) Wittekoek, S., 126, 127, 130(16),131(16), 142(12, 16), 143(16), 146(16) Woessner, D. E., 137 Wolf, K., 85 Wolfrom, M. L., 257, 258 Wong, K. F., 125, 126(5), 138(5),145(5), 146(5),154, 158, 161(5, 77, 78), 163(5),164(5), 165(5), 166(78), 166(78,85) Wooten, J. B., 188 Worden, L. R., 320 Wrangsell, G., 66 Wrobleski, K., 142 Wu, P., 42, 55(55) Wiithrich, K., 188
X Xavier, A. V., 168
Y Yamasaki, K., 285, 298(226) Yamashita, S., 334 Yamato, M., 280, 285 Yamazaki, J., 221, 250 Yanagihara, D. L., 57 Yang, J. T., 77, 93, 94(27), 102 Yeh, D. Y., 96 York, W. S., 28, 34(36), 43(36),68(36) Yoshima, H., 172 Young, G. A., 105, 106(49),109(49), 118, 119(81) Young, J. Z., 327 Young, R. H., 207, 239(23),240(23), 320(23) Yu, R. K., 26, 27(27), 54(27), 71(27)
2 Zask, A., 123, 124(96) Zeeberg, B. R., 318 Zemb, T., 168 Ziegast, G., 87 Ziessov, D., 142 Zotterman, Y., 206,334,337
SUBJECT INDEX FOR VOLUME 45 A
Aceric acid, 67 Acesulfame-K, 299 Acetates, circular dichroism, 120-122 Acetohalogeno carbohydrates, 5 Acetol ysis and f.a.b.-mass spectrometry, application to preliminary screening of glycoproteins for sugar type, 5051 monitoring with f.a.b.-mass spectrometry, 49 Acetylated molecules, f.a.b.-mass spectrometry, 53 Acetylgentiobiose, octa-0-, Helferich’s work on, 4 N-Acetylmuramic acid, circular dichroism, 113 N-Acetylneuraminic acid circular dichroism, 111, 112 methyl a-and P-D-ketopyranosides, circular dichroism, 112 Acid phosphatases, Helferich’s work on, 5 Acute myelogenous leukemia cells complex oligosaccharide, f.a.b.-mass spectrometry, 60-61 0-linked oligosaccharides, f.a.b.-mass spectrometry, 64 Acyl-thiocarbamide, bitterness, 310 N-(1-Adamantyl)suIfamate, taste properties, 300 Agarose, circular dichroism, at various temperatures, 91-93 AGMGP, f.a.b.-mass spectrometry, 2123 Agrobacteria, P-(1-2)-glucans, f.a.b.mass spectrometry, 68 AH,B hypothesis. See Sweetness, AH,B concept Alanine D-,sweetness, 233 L-, AH,B system in, 221 Alaria esculenta, stipes, material from, circular dichroism, 108-109 Alditols, sweetness-structure relationship, 293-295
Aldohexopyranosides, methyl 3,6-anhydro-, sweetness-structure relationship, 273 Aldopentopyranoses, proton spin-lattice relaxation rates, 151 Aldopyranoses anomeric protons, ring protons expected to have major effect on proton relaxation rates, 151 D-,sweetness-structure relationship, 248 sweetness, and structure, 239-248 Aldoses, circular dichroism fragmentspectra, 82 Alginate from A. esculenta stipes, circular dichroism, 108-109 alternating sequences, circular dichroism, 107-108 chelation of Ca2+to, circular dichroism, 105-106 circular dichroism, 105 composition and block-structure, circular dichroism, 107 Allopyranose D-, orientation of hydroxyl groups for, in 4c1(D) conformation, 75 ff-D-, 74 -, 1,6-anhydro-p-~-,sweetnessstructure relationship, 271 Allose D-
nonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation rates, 150 L-, sweetness-structure relationship, 258 Altropyranose D-, orientation of hydroxyl groups for, in 4 c 1 ( D ) conformation, 75 ff-D-, 74 Altrose D-
nonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation rates, 150 369
370
SUBJECT INDEX, VOLUME 45
L-, sweetness-structure relationship,
258 Amide derivatives, circular dichroism, 94-102 p-Aminoacetophenone, effect on f.a.b. sensitivity, 32 Amino acids bitterness, and hydrophobicity of side chains, 319 D-
centers of electron-rich density, 232 zwitterions, favored conformations, 232 D- and Lsuperpositioned over same receptor site, 236 sweetness, 214,231 taste of, 207-208 sweet and bitter, fixation in rectangular coordinates, 314-317 3-Aminonitrobenzene, derivatives, sweetness, 226-227 2-Amino-4-nitrobenzene, sweetness, AH,B system in, 221 Amino sugars (1+4)-linked, circular dichroism, 117 reaction with p-dicarbonyl compounds, 13-14 reaction with isothiocyanic acid derivatives, 14-15 Ammonium thiocyanate, addition to matrix for f.a.b.-mass spectrometry, 28 Amylopectin, circular dichroism, 89 Amylose carbanilated derivatives, circular dichroism, 120-121 circular dichroism, 85-89 Amylose-1-butanol complex, circular dichroism, 87 Amylose triacetate, circular dichroism, 121-122 Amylose xanthate, circular dichroism, 122 1,5-Anhydroald-l-enitols.See Glycosenes Anhydroalditols, 1,5-, sweetness-structure relationship, 261
1,6-Anhydro-p-cellobiosehexaacetate nonselective relaxation rates of H-1’, 143-145 proton spin-lattice relaxation, 154, 159 Anhydrohexofuranoid compounds, taste properties, 270 Anhydro sugars, sweetness-structure relationship, 269-274 Aniline, effect on f.a.b. sensitivity, 32 Arabinitol infrared spectrum, 294 sweetness-structure relationship, 295 Arabinopyranose 0-D-, 74 a - ~ -taste , properties, 242 p-D-
sweetness-structure relationship, 249,251 taste properties, 242 -, 1,2,3,4-tetra-O-(trideuterioacety1)interproton distances, 167 proton spin-lattice relaxation, 158, 166
8-Lsweetness, 218-219 sweetness-structure relationship, 241 taste properties, 242 Arabinose D-
nonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation rates, 152 a-L-, circular dichroism, predicted and fragment spectra, 84 p-L-,circular dichroism, predicted and fragment spectra, 84 Aryl-thiocarbamide, taste properties, 311 Ascorbic acid, synthesis, Helferich’s work on, 4 Asparagine, D- and L-, superpositioned over same receptor site, 236 Aspartame possible conformations, 308-309 and interaction with receptor site, 309 relative sweetness, 332
371
SUBJECT INDEX, VOLUME 45 sweetness, 306-307 AH,B system in, 221 sweetness-structure relationship, 245 L-Aspartoyl-D-alaninamides, taste properties, 306 a-L-Aspartoy1-D-alanine isopropyl ester, taste properties, 307 L-Aspartoyl-aminomalonicacid, esters, taste properties, 307-308, 310 N-(L-Aspartoy1)-1,l-diaminoalkanes,taste properties, 306 t-Aspartoyl-L-phenylalaninemethyl ester. See Aspartame Asperlin in benzene solution, configuration and conformation, 160-161 proton spin-lattice relaxation rates, 141 Auxogluc, 202-205, 218
B Bacterial polysaccharides, f.a.b.-mass spectrometry, 65-66 Baeyer, Adolf von, 1 Band 3 lactosaminoglycans f.a.b. mapping, 38-41 f.a.b.-mass spectrometry, 35, 59 Bauerlein, Karl, 3 Beef ganglioside, circular dichroism, 112 Beer's Law, 76 1,2-Benzisothiazolin-3-one1,l-dioxide. See Saccharin Bergmann, E., 9 Bis(acetamid0) sugars, circular dichroism, 98 N,N'-Bis(2-aminoethyI)ethylenediamine, as matrix for f.a.b.-mass spectrometry, 26 Bitterness, 310-325 AH,B concept for, 312-318 anomeric hydroxyl group essential for, 240-241 and configuration of anomeric center, 239 initial chemistry, 311 and lipophilicity, 243, 318-320 and nitro groups, 310 role of structure in, 200
structural parameters for, schematic representation, 315-317 Bitterness-sweetness relationships, 320325 Bitter-sensitive protein equilibrium constants of binding of bitter compounds, 311 from porcine tongue, 311 taste threshold of bitterness, for various compounds, 311 Bitter-sweet molecules binding to receptors, 322-324 polarization on taste receptors, 324 receptor locations, 320-322 topographical overlap of interaction patterns on taste receptors, 323325 Blood group determinants MN display, 171, 191, 194-195, 197 display by glycophorin A, 175 functional groups with crucial role in, 194 Ss, 173 display, 171 Blood group O,Le(a-b-), 56 Braun, Julius von, 1 Bromobenzoates di-p-, circular dichroism, 123-124 tri-p-, circular dichroism, 123-124 Brucine . HCI, taste properties, 311
L
Cabrera, Blas, 10 Caffeine, taste properties, 311 Calcium cyclamate, relative sweetness, 332 (+)-10-Camphorsulfonic acid, aqueous solution, calibration of c.d. instruments with, 77 Carbohydrates 1,2-hydroxyl groups, protecting group, Helferich's work on, 5 high-D-mannose, f.a.b.-mass spectrometry, 63 Carbon-13 isotope, effect on f.a.b. spectra, 29
372
SUBJECT INDEX, VOLUME 45
Carboxyl derivatives, circular dichroism, 102- 111 &-Carrageenan,circular dichroism, 93-94 Cellobiitol, sweetness-structure relationship, 275-276 Cellobiose p-, proton spin-lattice relaxation, 160 nonselective spin-lattice relaxation rates, 149 sweetness, comparison to D-glucose, 247-248 Cellobiose hexaacetate, 1,6-anhydro-p-, allowed rotational orientation of glucosidic bond, 160-161 Cellulose acetate, circular dichroism, 121 circular dichroism, 89, 120-121 Ceramide, pentasaccharides, fucosecontaining, characterization, 55-56 Cesium iodide, for calibration of mass marker and data system in f.a.b.mass spectrometry, 37 Chitin, circular dichroism, 100-101 Chitobiose, circular dichroism, 101 Chitohexaose, circular dichroism, 101 Chitotetraose, circular dichroism, 101 Chlorodeoxysucroses, AH,B,y systems of, 268 Chloroform, sweetness, AH,B system in, 22 1 Chondroitin, circular dichroism, 115116 Chondroitin sulfate, f.a.b.-mass spectrometry, 69-70 Chondroitin 4-sulfate, circular dichroism, 117 Chondroitin 6-sulfate, circular dichroism, 115-117 Chorclu tympani response to taste stimuli, proteinmodifying agents that inhibit, 335 taste units in, 339 Chronic myelogenous leukemia cells glycosphingolipids, 55 lactosaminoglycans f.a.b.-mass spectrum, 39-40 sialylated fucosylated, f.a.b.-mass spectrometry, 59 0-linked oligosaccharides, f.a.b.-mass spectrometry, 64
Circular dichroism, 73-124 artifacts, 78 instrumentation, 73, 76-78 calibration, 77, 78 principles, 73, 76 spectra, predicted, 83-84 spectrum measurement, 76-78 noise in, 78 of substituted carbohydrates, 92-124 of unsubstituted carbohydrates, 78-92 Circular dichroism-difference spectra, 80-81 Circular dichroism fragment-spectra, 8183 Complex carbohydrates, f.a.b.-mass spectrometry, 59-62 Curdlan, circular dichroism, 89 Cyclamate sweetness, 231 sweetness-structure relationship, 297303 three-dimensional x-ray analysis of, 301 Cyclamic acid sweetness, AH,B system in, 221 third structural feature comprising postulated glucophore in, 234 Cyclic polysaccharides, f.a.b.-mass spectrometry, 68-69 Cyclitols, sweetness-structure relationship, 241,290-293 Cyclodextrins, metal binding to, f.a.b.mass spectrometry, 70 Cyclohexanediol, cis-l,3-, sweetnessstructure relationship, 271 Cyclohexanepentols, sweetness, 290293 Cyclomaltoheptaose, nonselective spinlattice relaxation rates, 149 Cyclomaltohexose, circular dichroism, 87
D 5-Deoxyfructose. See Hexulose, 5-deoxy~-threo-2Deoxyglycosides, taste properties, 259260
SUBJECT INDEX, VOLUME 45 Deoxyhalosucroses, sweetness-structure relationship, 265-269 Deoxyhexopyranosides, possible AH,B unit involved in sweet-taste stimulation, 261 5-Deoxysorbose. See Hexulose, 5-deoxyo-threo-2Deoxy sugars, sweetness-structure relationship, 259-261 Dermatan sulfate, circular dichroism, 117 Dextran acetate, circular dichroism, 121 carboxymethyl derivative, circular dichroism, 122-123 circular dichroism, 89 Dextran xanthate, circular dichroism, 122 Diethanolamine. See 2,2’-Iminodiethanol Diethyl dithioacetals, circular dichroism, 123 Dihydrochalcone glycosides, taste properties, 278-285 Dihydrochalcone-receptor complexes, 281-282 Dihydrochalcones proposed AH,B,X systems of, 283-285 sulfoalkyl sweetness-structure relationship, 245 taste properties, 342 sulfobutyl, taste properties, 342-344 sulfoethyl, taste properties, 342-344 sulfomethyl, taste properties, 342-344 -, 4-O-(carboxyaikyl)-, sweetnessstructure relationship, 245 Dihydroisodonal, taste properties, 312 Dihydrotrichodonin, taste properties, 312 Dihydroxyacetone. See Propanone, 1,3dihydroxy-2-
373
Dipeptide sweeteners, location of third binding-site in, 234-235 Disaccharide glycoside, synthesis, Helferich’s work on, 3 Disaccharides (1+2)-linked, sweetness-structure relationship, 277-290 anomeric protons, nonselective spinlattice relaxation rates, 149-152 proton spin-lattice relaxation rates, 152 reducing, sweetness, comparison to monosaccharides, 246-247 Diterpenes, of Zsodon species, bitterness, 312 Drug action, 224 theory of, 227-228 Drug design, nonmathematical approach, 230-231 Drug dose-response relationships, 211 free-energy relationship, 224 parameters, 224 Dulcin, sweetness, 205
E
Electron impact mass spectrometry, 23, 43, 63, 71 Emulsin, of sweet almonds, Helferich’s work on, 4-5 Enterobacterial common antigen f.a.b.-mass spectrometry, 34 perdeuteriomethylated, f.a.b.-mass spectrometry, 69 structure, on f.a.b.-mass spectrometry, 69 Erythritol, sweetness-structure relationship, 248, 293 Erythrocyte glycoprotein, permethylated, f.a.b.-mass spectrometry, 34-35 2-C-Erythrofuranosylfurans,14 Di-0-isopropylidene-P-D-fructopyranose,Escherichia coli, serotype 0 8 , 0-antigen polysaccharides, f.a.b.-mass spec2,3:4,5-, phosphorylation, Garcia trometry, 66 Gonziilez’ work on, 8 Dimethyl-1-pentanol, 3,4-, molecularEscherichia coli LP1092, polysacconnectivity index, correlation with charides, circular dichroism, 107 biological activity, 229-230 (p-Ethoxyphenyl)thiourea,taste properDi-0-methylhexopyranosylderivatives, ties, 311 sweetness, 263 (p-Ethoxypheny1)urea. See Dulcin Dipeptide esters, sweetness-structure Ethyl p-aminobenzoate, effect on f.a.b. relationship, 306-310 sensitivity, 32
SUBJECT INDEX, VOLUME 45
374
Ethylene dithioacetals, circular dichroism, 123 Ethylene glycol sweetness, 214,233 sweetness-structure relationship, 293 Ethyltetrahydropyran, (-)-(S)-2-, circular dichroism, 83
F Fast atom bombardment-mass spectrometry, 19-72 acyl-group location by, 53 analysis of mixtures, 30-31 applications, 54-71 background ions, 29 of bacterial polysaccharides, 65-66 choice of derivatives, 30-32 cluster ions, 29, 70 of cyclic polysaccharides, 68-69 experimental protocol, 33-34 fragmentation pathways, 43-45 Al-type cleavage, 43, 57, 59, 66 8-cleavage, 44, 46, 59, 67 double cleavages, 46-47 pathway A, 43, 46, 54, 69 pathway B, 44, 46, 55, 69, 70 pathway C, 44,46, 55,69, 70 pathway D, 44-45, 69 pathway E, 45 fragment ions, 29, 39, 46 future developments, 71-72 of glycolipids, 54-58. See also specific glycolipid of glycoproteins, 58-65 of glycosidic compounds, 63-65 hardware, 24 high-mass definition, 35-36 instrumentation, 36-37 practical aspects, 37-38 of high-molecular-weight samples, 3441 history, 20-23 linkage assignment by, 52 mapping of permethylated, high-mass samples, 38-41 protocol, 39 mass assignments, 37
mass range selected for observation, 33-34 matrix, 24-25 acidified, 27, 42 choice of, 25-27 effect of addition of ammonium thiocyanate, 28 effect of addition of dilute aq. HCI, 27-28 effect of addition of sodium acetate, 27-28 ionic strength, 27 loading sample into, 33 PH, 27 matrix additives, 27-28 molecular-ion clusters, 29-31 molecular ions, 53 of peracetylated and permethylated samples, 47-48 molecular-weight assignment with, 41-42,45 in molecular weight determination, 23 and minimum sample-loadings, 3334 monitoring chemical and enzymic reactions by, 48-52 negative ion mode, 25, 42 of N-glycosylic compounds, 58-63 peracetylated samples, 30, 32-35, 46 permethylated samples, 30-32, 34, 38, 46 HexNAc cleavage, 47, 52 of plant cell-wall polysaccharides, 6668 principles, 24-25 pseudomolecular ions, 25, 28-29, 4142 produced by analysis of mixtures, 30 types produced, 25 quantitation with, 70-71 role in carbohydrate-structure analysis, 23 sample purity, 30 sensitivity, 33-35 in sequence assignment, 23 sequence ions, 23 sequencing by, 23 signal averaging, 37-38 source, 25
SUBJECT INDEX, VOLUME 45
375
spectra (Y-D-, sweetness-structure relationship, characteristics, 28-30 248 interpretation, 41-45 p-Drecording, 29 AH,B system, 221,252 structure assignment by, 45-53 infrared spectrum, 251 analysis of derivatized samples, 46sweetness 48 comparison to a-L-sorbopyranose, analysis of underivatized samples, 250 45-46 effect of temperature, 250 types of problems solved by, 23 sweetness-structure relationship, of underivatized samples, 53 241,249 FernPndez-Bolafios, J., 14 taste properties, 242 Fibronectin, human amniotic fluid, -, 3-O-a-~-glucopyranosyl-~-. See lactosaminoglycan, f.a.b.-mass specTuranose trometry, 59 -, 6-thio-p-~-,taste properties, 252 Field desorption, 20-21 Fructopyranoside, methyl p-D-,sweetFischer, Emil, 1 ness-structure relationship, 249Fischer, H.O.L., 9 250 Flavanone glycosides Fructose conversion into corresponding chalDcone and dihydrochalcone anabinding to taste papillae, 329-330 logs, 278-279 solution, distribution of ketotaste properties, 277-278 pyranose isomers in, 249 Fluoro carbohydrates, 4 sweetness, 231 N-Formylkynurenine and temperature, 221-222 interaction with receptor sites, 288 sweetness-structure relationship, sweetness, 290 248,256 Friend murine leukemia virus, glycoprotaste, 249 tein, f.a.b.-mass spectrometry, 63 thermal mutarotation, 221-222 Fructofuranose in water, equilibrium composition, p-D-,sweetness-structure relationship, 248-249 256 p-D-,sweetness, effect of infrared -, 5-thio-p-~-,sweetness-structure hydroxyl absorption bands and relationship, 256 hydrogen-bonding strength, 217 Fructofuranoside L-, sweetness-structure relationship, -, 3,6-anhydro-a-~-glucopyranosyl 258 1,4:3,6-dianhydro-p-~-,taste prop- Furan derivatives, Garcia GonzPlez’ erties, 270,273 work on, 12-13 -, 3,6-anhydro-a-~-glucopyranosyl Furanoid compounds, sweetness-struc3,6-anhydro-p-~-,taste properties, ture relationship, 256-258 270,273 Furanoid ring, conformations, 256 -, 4,6-dichloro-4,6-dideoxy-a-~-galac-Furanose derivatives, proton spin-lattice topyranosyl 1,6-dichloro-l,6relaxation rates, 153 dideoxy-p-D-, sweetness, 265,268 Furanoses, sweetness-structure relation-, @-D-ghCOpyranOSyl1,4:3,6-dianhyship, 256-258 dro-p-D-, taste properties, 270, 273 Furans, Garcia GonzBlez’ work on, 10-, a-D-glucopyranosyl 3,6-anhydro-p11,12 D-, taste properties, 270, 273 Furylalanines, 12 Fructopyranose Furylpyruvic acid, 13 D-, taste properties, 254 Furylthiopyruvic acid, 13
SUBJECT INDEX, VOLUME 45
376 G
Galactan, a-D-, (1+6)acetate, circular dichroism, 121 benzyl derivative, circular dichroism, 122 Galactitol sweetness-structure relationship, 295 -, 1,5-anhydro-~-,sweetness-structure relationship, 240 Galactomannans, circular dichroism, 90 Galactop yranose D-
-, 2-acetamido-2-deoxy-, circular dichroism, 95-96 orientation of hydroxyl groups for, in 4c1(D) conformation, 75 sweetness, 264 a-D-, 74 sweetness, 219 sweetness-structure relationship, 239,241 taste properties, 242 p-D-
sweetness-structure relationship, 239 taste properties, 242 -, 2-acetamido-2-deoxy-, circular dichroism, 97 -, 1,6-anhydro-p-DAH,B systems for, 271 sweetness-structure relationship, 269 D-Galactopyranose pentaacetate, nonselective spin-lattice relaxation rates, 148 P-D-Galactopyranose penta(acetate-&), nonselective spin-lattice relaxation rates, 148 Galactopyranoside, 3,6-anhydro-4-chloro4-deoxy-cr-~-galactopyranosyl3,6anhydro-4-chloro-4-deoxy-c~-~-, taste properties, 270 Galactopyranosybarabinitol, 3-0-p-~-, taste properties, 275 Galactopyranosyl-D-erythritol,2-0-a-D-, taste properties, 275
Galactopyranosylglycerol, 2-0-(Y-D-, taste properties, 275 Galactopyranuronic acid, circular dichroism, 109-110 Galactose D-
a and p pyranose anomers, circular
dichroism, 79-80 intramolecular hydrogen bonding in, 2 16-2 17 nonselective spin-lattice relaxation rates, 148, 150 proton spin-lattice relaxation rates, 150-151 sweetness, 220 a-D-,sweetness, effect of infrared hydroxyl absorption bands and hydrogen-bonding strength, 216217 Galactose pentaacetate, p-D-,proton spin-lattice relaxation, 157 Galacturonic acid, D-, circular dichroism, 102-103 Galacturonic acid oligosaccharides, plant cell-wall, f.a.b.-mass spectrometry, 66-67 Gangliosides, f.a.b.-mass spectrometry, 55-56 Garcia GonzAlez, Francisco, 7-17 coauthors, 17 doctoral dissertations, 9 doctorates, 9 early life, 7 education, 7-8 family, 7 marriage, 11 research, 8- 15 Garcia GonzAlez reaction, 13 Gentiobiose circular dichroism, 87 nonselective spin-lattice relaxation rates, 149 sweetness-structure relationship, 248 synthesis, Helferich’s work on, 3 Germacrolide, taste properties, 314 Glossopharyngeal nerves, taste units, taste perception by, 339 Glucans /3-(1+2)-, f.a.h.-mass spectrometry, 68
SUBJECT INDEX, VOLUME 45 acetylated, circular dichroism, 122 cyclic p-( 1-+2)-,f.a.b.-mass spectrometry, 34 D-, circular dichroism, 88-90 (Y-D-, (1+6)-, benzyl derivative, circular dichroism, 122 deuteropermethylated, cyclic p( 1+2)-, molecular ion clusters obtained from, in f.a.b.-mass spectrometry,
37-38 Glucitol -, 1,4-anhydro-~-,sweetness-structure relationship, 257 -, 1,5-anhydro-~-,sweetness-structure relationship, 240 -, 1,4:3,6-dianhydro-~sweetness-structure relationship, 257,274 taste properties, 270 Glucofuranose a-D-, taste properties, 242 p-D-,taste properties, 242 Glucofuranoside -, 3,6-anhydro-a-~-glucofuranosyl 3,6-anhydro-a-~sweetness-structure relationship, 257,274 taste properties, 270 -, methyl 3,6-anhydro-a-~taste properties, 270 sweetness-structure relationship, 257,274 -, methyl p-D-,sweetness-structure relationship, 256 Glucogenes, 202 Gluconic acid, D-, 0-isopropylidene derivatives, 11 Glucophore, 202-205,218 Glucopyranose D-
orientation of hydroxyl groups for, in 4c1(D) conformation, 75 -, 2-acetamido-2-deoxycircular dichroism, 98 oligomers, circular dichroism, 100
74 sweetness-structure relationship, 239 taste properties, 242
ff-D-,
377
-, acetamido-2-deoxy-, circular dichroism, 94-95 p-D-
and P-L-, superpositioned over same receptor site, 236 sweetness-structure relationship, 239 taste properties, 242
-, 2,3,4,6,2',3'-hexa-O-acety1-1,6anhydro-4-O-~-glucopyranosylinterproton distances, 156 spin-lattice relaxation rates, 155 -, 1,6-anhydro-p-~proton spin-lattice relaxation, 160 sweetness-structure relationship, 269,271 D-Glucopyranose pentaacetate, nonselective spin-lattice relaxation rates, 148 Glucopyranoside -, 3,6-anhydro-a-~-glucopyranosyl 3,6-anhydro-a-~-,taste properties, 270 -, methyl 3,6-anhydro-a-~-,taste properties, 273 -, methyl a-Dacetals, taste of, 243 methyl derivatives, sweetness, 262 methyl ethers, taste properties, 262 mono- and di-deoxy derivatives, sweetness-structure relationship, 259-260 sweetness, 263 sweetness-structure relationship, 241,244-245 sweetness threshold, determination by triangular test, 245 true sweetness of, 240 -, methyl p-D-, true sweetness of, 240 -, methyl-& D-, nonselective spinlattice relaxation rates, 148 Glucopyranosyh-erythritol -, 2-0-a-D-, taste properties, 275 -, 2-0-p-D-, taste properties, 275 Glucopyranosylgl ycerol -, 2 - 0 - a - D - , taste properties, 275 -, Z-O-p-D-, taste properties, 275 Glucose D-
p pyranose anomers, circular dichroism, 79-80
a and
378
SUBJECT INDEX, VOLUME 45
antiketogenic action, Garcia Gonzh-, tetra-0-acetyl-N-benzyl-, 5 lez’ work on, 10 p-Dbinding to taste papillae, 329-330 -, 2-acetamido-l-N-acetyl-2-deoxy-, nonselective spin-lattice relaxation n.m.r., 98 rates, 148, 150 -, 2-acetamido-l-N-(~-aspart-4-oyl)proton spin-lattice relaxation rates, 2-deoxy150-151 circular dichroism, 98-99 reaction of ethyl acetoacetate with, n.m.r., 98 10-12 Glucosyl fluoride, D-, 2,3,4-tri-O-bensweetness, 220 zoyl-6-0-trityl-, 3 sweetness-structure relationship, Glucuronic acid, D-, circular dichroism, 259 102, 104-105 syrups, taste, effect of aglycons on, Glycans 245 a-and @-, carbanilyl derivatives, circu-, 2-acetamido-2-deoxylar dichroism, 119-120 circular dichroism, 97-98 f.a.b.-mass spectrometry, 58 nonselective spin-lattice relaxation N-glycosylically-linked,behavior rates, 148 during reaction sequence of hy-, 2-amino-bdeoxy-, reaction proddrazinolysis, 51-52 uct with ethyl acetoacetate, Glyceraldehyde, D-, reaction with 1,3 pyrrole structure, 10-11 dicarbonyl compounds, 13 -, 2,3-bis(acetamido)-2,3-dideoxy-~- Glycerol dideoxy-, circular dichroism, 99 cluster ions, 29 -, 1,2,3,4-tetra-O-acetyl-,4 as matrix for f.a.b.-mass spectrometry, -, 1,2,3,4-tetra-O-acetyyl-6-O-trityl-, 25-26 3 sweetness-structure relationship, -, 2,3,4,6-tetra-O-acetyyl, 5 293 -, penta-0-acetyl-, 5 Glycerol glycosides, sweetness-structure a - ~ - sweetness, , 207 relationship, 248 effect of infrared hydroxyl absorpGlycine, sweetness, 233 tion bands and hydrogen-bondGlycoconjugates ing strength, 216-217 f.a.b.-mass spectrometry, fragmentation p-0-,322 pathways, 43 sweetness, 207 mixtures, f.a.b. mapping, 39 effect of infrared hydroxyl absorpGlycogen, circular dichroism, 89 tion bands and hydrogen-bonda-Glycol, rotamers, orientations of, 215ing strength, 216-217 216 -, minus p-D-xylose, differenceGlycolaldehyde, reaction with 1,3-dicarcircular dichroism spectra, 81 bony1 compounds, 13 L-, sweetness-structure relationship, GI ycolipids 259 f.a.b.-mass spectrometry, 54-58 p-Glucosidase, Helferich’s work on, 5 pyruvic acetylated, f.a.b.-mass specGlucosides trometry, 56-57 WD-, sweetness-structure relationship, trehalose-containing, f.a.b.-mass spec239 trometry, 57 p-D-,sweetness-structure relationship, a-Glycol units, oxygen-oxygen distance of, 215-216 239 Glucosylamine GIycophorin D glycopentapeptide 10, ’3C-n.m.r. chemical shift data, 188 -, tetra-0-acetyl-, 5
SUBJECT INDEX, VOLUME 45 labeled, 13C nuclear magnetic resonance, 169-198 N-terminal di[13C]methylaminogroups '3C chemical shift data, 189-192 titration data, 189 N-terminal structure effect of carbohydrate residues, 197 effect of hydrogen bond, 192 effect of neighboring glycosylation, 191-192 role of lysine residues in determining, 194 N-terminus, peptides related to, pHtitration studies, 192-194 properties, 170 reductively [Wlmethylated, pH titration studies, 187-192 Glycophorin A, 170 amino acid residues, 172 amino acid sequence data, 172-173 carbohydrate structure, 172-173 I3C labels chemical cleavage of glycoprotein for assigning, 178 enzymic digestion for assigning, 178 on lysine and N-terminal amino acid, distinction between, 177178 methods used for assignment, 177178 partial-methylation assignment technique, 178 pH-titration, 178 deglycosylation, structural effects, 184-186 dimethylated N-terminal species, differentiation by p H studies, 182-183 display of MN blood group determinants, 175 fully reductively [Wlmethylated, 13Cn.m.r. spectra, 186-187 functions, 170-171 intact, from heterozygous and homozygous RBC, 178-186 labeling, reductive [Wlmethylation technique, 175-195 drawbacks, 177
379
method, 175-177 NaCNBH3 as reducing agent, 176177 reaction scheme, 176-177 side-reactions, 176-177 specificity, 177 molecular weight, 172 native and reductively methylated, aliphatic region of proton-decoupled 1%-n.m.r. spectra, 179-182 N-terminal proteins, 13C-n.m.r. spectra in various degress of glycosylation, 184-186 partial methylation studies, 182-184 pH titration studies, 182 as red cell receptor, 170 virgin and reductively methylated, circular dichroism studies, 178179 Glycophorin A" hydrogen bonding in, 195 N-terminal structure, 194 reductively methylated, minor component, 181, 182, 186, 197-198 Glycophorin AN. See Glycophorin A Glycophorin B, 170 amino acid residues, 172-173 function, 171 labeling studies of, 195-197 relationship to results obtained for glycophorin A, 195-196 pH titration studies, 196-197 relationship to results for glycophorin AN and glyco-octapeptide AN, 196-197 Glycophorin C, 170 carbohydrate content, 172 properties, 172 Glycophorin glycopeptide, 182 isolation, 186 N-terminal di[13C]methylamino groups chemical shift data, 189-192 titration data, 189 pH dependence, 187 production, 186 reductively [13Clmethylated,pH titration studies, 187-192 Glycophorin-related glycopeptides, mono[Wlmethylated, pH-titration studies, 192-194
SUBJECT INDEX, VOLUME 45
380
Glycoprotein LTF-D, from lactotransferrin, antennae, circular dichroism,
120 Glycoprotein OTF-C, from ovotransferrin, antennae, circular dichroism,
120 GIycoproteins acetolysis and f.a.b.-mass spectrometry, as screening method, 50-51 complex, storage products derived from, f.a.b.-mass spectrometry of,
62 f.a.b.-mass spectrometry, 58-65 Y and T antennae, circular dichroism studies, 118-120 Glycoprotein STF-A, from serum transferrin, antennae, circular dichroism, 120 Glycosaminoglycans, circular dichroism,
117-118 Glycosenes, 4 Glycosidases, Helferich’s work on, 4, 5 Glycosides alditol, sweetness-structure relationship, 275-276 bitterness, 318 ethyl, sweetness-structure relationship, 275 methyl, sweetness-structure re1at’ionship, 274 methyl ethers, taste properties, 262 sweet bonding to taste-bud receptor sites,
279 structural features, 289 sweetness-structure relationship, 274-
276 taste, effect of aglycons on, 245 Glycosphingolipids 25-sugar residue, f.a.b.-mass spectrometry, 54 25-sugar residue isolated from rabbit erythrocyte membrane sequence of, 48 after enzymic degradation with aD-galactosidase, 49 Smith-degradation product, predicted sequence, 49 in blood group ABH, I, i, Lewis characterization, 55
blood group B-active digestion with a-D-galactosidase followed by Smith degradation,
48 structure determination of permethylated derivative, by f.a.b.-mass spectrometry, 48 f.a.b.-mass spectrometry, 27-28, 54-56 molecular ion species, 42 in human chronic myelogenous leukemia cells, characterization, 55 in human embryonal-carcinoma cells, characterization, 55 in human granulocytes, characterization, 55 mannose-containing, from insect, characterization, 56 permethylated, f.a.b.-mass spectrometry, 37,54 N-Glycosyl compounds, Helferich’s work on, 5 Glycuronic acids, circular dichroism,
102-105 Glycyphillin, taste properties, 280 Glycyrrhetic acid, 287 Glycymhiza glabra L., 287 Glycyrrhizic acid, 287 Glycyrrhizin structural features, 289 taste properties, 287-290 Clykergenic acid, sweetness, 290 Gbmez-Sinchez, A., 13-14 Granulocytes normal, f.a.b. map of lactosaminoglycan sample from, 39-40 0-linked oligosaccharides, f.a.b.-mass spectrometry, 64 permethylated ganghoside from, f.a.b.mass spectrometry, fragmentation observed in, 54-55 Guar galactomannan, solid film, circular dichroism, 90-91 Gulopyranose D-, orientation of hydroxyl groups for, in 4 c l ( D ) conformation, 75 a-D-,
74
Gyrnnema syluestre, sweet-taste inhibitors from, 337 Gymnemic acids, as sweet-taste inhibitors, 336-339
SUBJECT INDEX, VOLUME 45
H Halogeno carbohydrates, 4 2-Halogeno derivatives, nonselective relaxation-rates, stereospecific dependencies, 152 Hammett constant, 224-225,303 Helferich, Burckhardt, 1-6 awards, 2 career, 1-2 education, 1 marriage, 2 publications and patents, 6 research, 2-6 Hen ovomucoid, complex glycans, f.a.b.mass spectrometry, 60-62 Heparan sulfate, circular dichroism, 117 Heparin binding of Cu(I1) with, monitoring with circular dichroism study, 118 circular dichroism, 117 Heptulose -, L-allo-, taste properties, 254 -, D-UhO-, taste properties, 254 -, a - D - U h O - 2 ; sweetness-structure relationship, 253-254 -, 1-deoxym-manno-, taste properties, 254-255 -, 7-deoxy-~-altro-,taste properties, 254 -, 7-deoxy-~-gaZacto-,taste properties, 254 -, L-galacto-, taste properties, 254 -, L-gluco-, taste properties, 254 -, a-D-gluco-2-, sweetness-structure relationship, 253-254 -, CY-D-mUnnV-, minus a-D-tagatose, difference-circular dichroism spectra, 81 -, cY-D-mUnn0-2-, sweetness-structure relationship, 253-254 -, a-D-talo-e-, sweetness-structure relationship, 253-254 Hernandulcidin, sweetness-structure relationship, 295-296 Hesperitin dihydrochalcone-4’-yI p-Dgalactopyranoside, taste properties, 279 Hesperitin dihydrochalcone-4’-yl p-Dxyloside, taste properties, 280
381
Hexafluoro-2-propanol, 1,1,1,3,3,3-, as solvent for circular dichroism studies, 96-97 Hexitols, sweetness-structure relationship, 295 HexNAc cleavages, 54 HexNAc residues, permethylated molecules containing, Iinkage-site-specific, 52 Hexofuranosides, methyl 3,6-anhydro-, sweetness-structure relationship, 274 Hexopyranose -, 1,6-anhydro-P-~proton spin-lattice relaxation, 153 interatomic oxygen-oxygen distances, 272-273 sweetness-structure relationship, 269 -, P-D-allO-, taste properties, 270 -, p-D-dtrO-, taste properties, 270 -, 2-deoxy-/3-~-arabino-,taste properties, 270 -, 2-deoxy-P-~-lyxo-,taste properties, 270 -, 2-deoxy-p-~-ribo-,taste properties, 270 -, P-D-galaCtO-, taste properties, 270 -, p-D-glUC0-, taste properties, 270 -, p-D-gulo-, taste properties, 270 -, p-D-ido-, taste properties, 270 -, p-D-manno-, taste properties, 270 -, p-D-talo-, taste properties, 270 -, 2-O-methyl-P-~-gluco-,taste properties, 270
-, 1,6-anhydro-2-deoxy-~-arabino-, sweetness-structure relationship, 269 -, 1,6-anhydro-2-deoxy-P-lyxo-, sweetness-structure relationship, 269 Hexopyranose derivatives, proton spinlattice relaxation rates, 153 Hexopyranoside -, 3,6-anhydro-4-deoxy-a-~-xylohexopyranosyl 3,6-anhydro-4deoxy-a-D-xylo-, taste properties, 270 -, methyl 3,6-anhydro-a-~4-chloro-4-deoxy-a-~-galacto-, taste properties, 270
382
SUBJECT INDEX, VOLUME 45
4-deoxy-a-~-xylo-,taste properties, 270 a-D-gluco, taste properties, 270 -, methyl 2-deoxy-a-~-ribo-,sweetness-structure relationship, 271 -, methyl 4-deoxy-a-~-xylo-,sweetness threshold, determination by triangular test, 245 Hexose, 2-deoxy-~-arabinoproton spin-lattice relaxation rates, 150 nonselective spin-lattice relaxation rates, 148 Hexulose, 5-deoxy-~-threo-2-,taste properties, 252 2-Hexuloses, sweetness-structure relationship, 253-254 High mass, definition, 34-36 Histidine D-, favored conformation, 232 DL-, 15 Histidine-2-thiol, DL-, 15 Hurler’s syndrome, 118 Hyaluronic acid in aqueous ethanol solvent at pH 2.5, circular dichroism, 115-116 circular dichroism, 113-115, 117 Hydrogen bond, directional influence, in sweetness, 219-220 Hydrogen-bond lengths, 219 Hydrolyses, monitoring with f.a.b.-mass spectrometry, 48,49,50 Hydroquinine, bitterness, 320 H ydroxyaldehydes LY-, 13 D-, Helferich’s work on, 3 a-Hydroxyketones, 13 2-[ (3-Hydroxy-4-methoxyphenyl)-ethyl]benzene, sweetness, 285 y-Hydroxyvaleraldehyde, Helferich’s work on, 2-3 I Idopyranose D-, orientation of hydroxyl groups for, in 4c1(D) conformation, 75 a-D-, 74 Idopyranose pentaacetate, a-D-,nonselective spin-lattice relaxation rates, 148
Idose, Dnonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation rates, 150 2,2’-Iminodiethanol, as matrix for f.a.b.mass spectrometry, 26 Influenza virus, red cell receptor, 170 Inositol, sweetness-structure relationship, 241 Inositol derivatives, proton spin-lattice relaxation rates, 153 Interleukin 2, Ea.b.-mass spectrometry, 64-65 Isodonal, taste properties, 312 Isohumulone bitterness, 319-320 taste properties, 313 isopropylidene-a-D-glucofuranose3sulfate, 1,2-0-, oxidation, Garcia GonzBlez’ work on, 9 Isopropylidene-a-D-mannofuranose,
2,3:5,6-di-Ononselective relaxation rates of H-5, 143-144 proton spin-lattice relaxation, 157 Isothiocyanates, reaction with amino sugars, 14-15
K Karplus curve, 126 Keratan sulfate mammalian, circular dichroism, 117 shark sulfated, circular dichroism, 117 Ketopyranoses, sweetness-structure relationship, 248-256 Ketoses circular dichroism, 79 taste properties, 253-255 Klebsiella, serotype 0 5 , 0-antigen polysaccharides, f.a.b.-mass spectrometry, 66 Klebsiella K54 repeating-unit, f.a.b.-mass spectrometry, 65,66 Kratos HF-MS50 spectrometer, 36-37
L Lactitol possible hydrogen bonding in, 276277
SUBJECT INDEX. VOLUME 45 sweetness-structure relationship, 275276 Lactosaminoglycans f.a.b.-mass spectrometry, 58-59 permethylated, f.a.b.-mass spectrometry, 38 Lactose a-,sweetness, 207 effect of infrared hydroxyl absorption bands and hydrogen-bonding strength, 217 p-, sweetness, 207, 220 effect of infrared hydroxyl absorption bands and hydrogen-bonding strength, 217 binding to taste papillae, 329-330 nonselective spin-lattice relaxation rates, 149 sweetness, 276 comparison to D-galaCtOSe, 247 Lactulose, taste properties, 254-255 Lectins, red cell receptor, 170 Lemieux effect, 264 Leucine, D-, favored conformation, 232 Leucopenia, 171 Limonine, taste properties, 313 Lipid A, f.a.b.-mass spectrometry, 57 Lippia dulcis Trev., 295 Ldpez Aparicio, F.J., 13 Lupoxes-A, taste properties, 313 Lysogangliosides, analysis, 56 Lyxopyranose, (Y-D-,74 Lyxose D-, nonselective spin-lattice relaxation rates, 148 p-L-,taste properties, 242 M Madinaveitia, A., 10 Malarial parasites, red cell receptor, 170 Maltitol structure, 276 sweetness-structure relationship, 275276 Maltose nonselective spin-lattice relaxation rates, 149 sweetness, comparison to D-ghcose, 247-248
383
Maltotriose, nonselective spin-lattice relaxation rates, 149 Maltulose, taste properties, 254-255 Mannan, a-D-, (1+6)-, acetate, circular dichroism, 121 -, (1+6)-, benzyl derivative, circular dichroism, 122 Mannitol D-, sweetness-structure relationship, 295 -, 1,5-anhydrosweetness-structure relationship, 240-241 taste prdperties, 254 -, 1,4-anhydro-~-,sweetness-structure relationship, 257 -, 1,5-anhydro-~-,sweetness-structure relationship, 240, 249 -, 1,4:3,6-dianhydro-~sweetness-structure relationship, 257,274 taste properties, 270 Mannopyranose D-
orientation of hydroxyl groups for, in 4 c ~ ( Dconformation, ) 75 -, 2-acetamido-2-deoxy-, circular dichroism, 95 (Y-D-, 74 sweetness-structure relationship, 239 taste properties, 242 p-D-
sweetness-structure relationship, 239 taste properties, 242 -, 3,4,6-tri-O-acetyl, 1,2-0-(l-benzyloxyethylidene) derivatives, proton spin-lattice relaxation, 158 -, 3,4,6-tri-O-acetyl, 1,2-0-(1methoxyethylidene) derivatives, proton spin-lattice relaxation, 158 -, 1,6-anhydro-p-~-,sweetnessstructure relationship, 269 Mannopyranosyl-D-erythritol, 4 - 0 - p - ~ - , taste properties, 275 Mannopyranuronic acid, p-D-,structure, 102
384
SUBJECT INDEX, VOLUME 45
Mannose D-
intramolecular hydrogen bonding in, 216-217 nonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation rates, 150 sweetness, 220 -, 2-acetamido-2-deoxy-, nonselective spin-lattice relaxation rates, 148 a-D-
2-(hydroxymethyl) derivatives, circular dichroism, 79 sweetness, effect of infrared hydroxyl absorption bands and hydrogen-bonding strength, 216-217 p-D-
bitterness, 322 sweetness-structure relationship, 248 Mannosidoses, 63 Mass spectrometer extended mass-range, 37 Fourier-transform, 36 future developments, 71-72 high-voltage, double-focusing, sector, 36 magnetic-sector, maximum mass range, 36 time-of-flight, 36 Mass spectrometry, high mass, 35 Mass spectrometry-nmr spectroscopy, 70 Meconium glycoproteins, expressing oncofetal antigens, tab.-mass spectrometry, 64 Melibiitol, sweetness-structure relationship, 275 Melibiose nonselective spin-lattice relaxation rates, 149 sweetness, comparison to D-galactose, 247 Metal salts, cello- and malto-oligosaccharides in presence of, f.a.b.-mass spectrometry, 70 Methanesulfonic acid, esters, Helferich’s work on, 4
Methanolyses, monitoring with f.a.b.mass spectrometry, 48 Methanolysis-f.a.b.-mass spectrometry, 51 Methyl 2-acetamido-2-deoxy-~-~-galactopyranoside, circular dichroism, 96 Methyl 2-acetamido-2-deoxy-a-~-glucopyranoside, circular dichroism, 96 Methyl 2-acetamido-2-deoxy-~-~-glucopyranoside, circular dichroism, 96 in water and fluorinated alcohols, 97 Methylamylose, 2,3,6-tri-O-, nonselective spin-lattice relaxation rates, 149 Methyl a-L-arabinoside, circular dichroism, predicted and fragment spectra, 84 Methylcellulose, 2,3,6-tri-0-, nonselective spin-lattice relaxation rates, 149 N-(1-Methylcyclohexyl)sulfamate,threedimensional x-ray analysis of, 301 Methyl 3-deoxy-a-~-manno-2-octu~opyranosidonic acid, circular dichroism, 107 Methyl 3-deoxy-~-~-manno-2-octu~opyranosidonic acid, circular dichroism, 107 Methyldextrantaose, 2,3,4-tri-0-, nonselective spin-lattice relaxation rates, 149 Methyl D-galactoside, a and p pyranose anomers, circular dichroism, 80 Methyl D-ghcopyranoside circular dichroism-difference spectra, 81 nonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation, 157 Methyl a-o-glucopyranoside, circular dichroism, 87-88 0-Methyl-D-glucose polysaccharide f.a.b.-mass spectrometry, 21-23, 58, 65 molecular weight, 21 mycobacterial, f.a.b.-mass spectrometry, 37 structure, 21-22 Methyl D-ghcoside, a and p pyranose anomers, circular dichroism, 80 Methyl glycopyranosides, circular dichroism, 96 Methyl glycopyranosiduronic acids, circular dichroism, 102
SUBJECT INDEX, VOLUME 45
385
Methyl glycosides, circular dichroism,
Monosaccharides anomeric protons, nonselective spinMethyl a-D-mannopyranosiduronicacid, lattice relaxation rates, 148, 150 circular dichroism, 102-104 circular dichroism, 78-85 3-0-Methylmannose dodecasaccharide, isomolar solutions, and related reduccomplex with alkyltrimethylammoing disaccharides, comparison of nium ions having decyl and hexas-weetness, 246-247 decyl as alkyl chains, f.a.b.-mass 2-phenyl-1,2,3-osotriazole derivatives, spectrometry, 70 circular dichroism, 123 Methyl 4,6-O-methylene-~-hexopyrano- Mucus glycoproteins, sialylated sacsides, taste, 243 charide-alditols, structures, 64 Methyl pyranosides, circular dichroism, Muramic acid, circular dichroism, 112-
79
85
113
Methylsucrose Mycobacterium kansasii, glycolipids, -, 4-0-, sweetness-structure relationf.a.b.-mass spectrometry, 57 ship, 263 Mycobacterium smegmatis, glycolipids, -, 6'-0-, sweetness, 264 f.a.b.-mass spectrometry, 56-57 -, 1',6'-di-0-, sweetness, 264 Mycodextran, acetate, circular dichroism, -, 6,6'-di-O-, sweetness, 264 12 1 Methyl 2,3,4,6-tetra-O-(trideuterioacetyl)-a-~-glucopyranoside-2,3,4,6,6'ds, anomeric forms, proton spinN lattice relaxation, 162 Methyl D-xylopyranoside, circular diNaringin, taste properties, 31 1 chroism-difference spectra, 81 Naringin dihydrochalcone Methyl a-D-xylopyranoside, circular structural features, 289 dichroism, 85 sweetness, 279 Methyl P-D-xylopyranoside, circular Neohesperidin dihydrochalcone dichroism, 85 after-taste, 265, 341 Methyl D-xyloside, a and /3 pyranose relative sweetness, 332 anomers, circular dichroism, 80 structural features, 289 MGP. See 0-Methyl-D-glucose polysacsweetness, 279 sweetness-structure relationship, charide Milk, human, oligosaccharides, f.a.b.24 1 mass spectrometry, 70 taste properties, 288 Miraculin, 290 third structural feature comprising sweetness, 213 postulated glucophore in, 234 as sweet-taste modifier, 339 /3-Neohesperidose, taste properties, 278Molecular connectivity, 229-230 279 Molecular-connectivity index, 229, 319 Nigeran, circular dichroism, 89 third-order, 230 2,2',2"-Nitrilotriethanol, as matrix for Molecular orbital calculations, 231 f.a.b.-mass spectrometry, 26-27 Moles, Enrique, 10 Nitroanilines Monellin location of third binding-site in, 234relative sweetness, 332 235 sweetness, 213 of structure X-CBH4-Y, taste properthree-dimensional structure, 333-334 ties, 304-306 Monodeoxy sugars, taste properties, sweetness-structure relationship, 303-
260 Mono-0-methylsucrose derivatives, taste properties, 263
306 third structural feature comprising postulated glucophore in, 234
386
SUBJECT INDEX, VOLUME 45
Oligogalactosiduronic acid, f.a.b.-mass 5-Nitroanilines spectrum 2-propoxyeffect of acid-dosing, 27-28 derivatives, sweetness, 303-304 effect of (pentafluorobenzy1)oxime relative sweetness, 351 derivative, 32 2-substituted Oligosaccharides binding to sweet-taste receptor, 336 anomeric protons, nonselective spinsweetness, 228-231,233 lattice relaxation rates, 149-152 correlation with Hammett constant reductively aminated with aniline, and hydrophobicity, 225 f.a.b.-mass spectrometry, 33 sweetness-structure relationship, underivatized, unreduced, f.a.b.-mass 303 spectrometry, fragmentation pathNitrobenzene derivatives ways, 46 2-amino-4-, sweetness, 226 yeast high-mannose, hydrolysis and 4-amino-2-, sweetness, 226 acetolysis, monitoring with f.a.b.Nomilin, taste properties, 313 mass spectrometry, 49-50 Norleucine Optical rotatory dispersion, 91 D-,taste properties, 315-316 Osladin L-, taste properties, 315-316 structural features, 289 Nuclear magnetic resonance of 2-acetamido-l-N-acety1-2-deoxy-P-~- taste properties, 286-287 Ospolot, 5 glucosylamine, 98 of 2-acetamido-1-N-(~-aspart-4-0~1)-2- Oximes effect on f.a.b.-mass spectrometry deoxy-P-D-glucosylamine, 98 sensitivity, 32 I3C, of labeled glycophorins, 169-198 sweetness-structure relationship, 296study of electron-rich positions in 297 sweet compounds, 231-232 Nuclear Overhauser enhancement, 126, P ,168 in combination with nonselective PA1 human embryonal carcinoma cells, lactosaminoglycans, f.a.b.-mass relaxation-rate measurements, 159 spectrometry, 59 combined with proton spin-lattice Palatinose, taste properties, 254-255 relaxation, 164-167 Pectins, circular dichroism, 111 single- and double-selective relaxaPentitols, infrared spectra, 294 tion-rates with, 160 Pentopyranose derivatives, proton spinlattice relaxation rates, 153 0 2-Pentuloses, sweetness-structure relationship, 253 Octulopyranosylonic acids, 3-deoxy-~Peracetylation, with trifluoroacetic anhymanno-2-, circular dichroism, 107 Octulose dride-acetic acid, 53 Perillaldehyde oxime -, D-glycero-a-D-ghco-2-, sweetnesssweetness-structure relationship, 296 structure relationship, 253-254 third structural feature comprising -, D-glyCerO-L-ghCO-, taste properties, 254-255 postulated glucophore in, 234 Perillartine, sweetness, AH,B system in, -, D-glycero-L-gulo-, taste properties, 22 1 254 2-Octuloses, sweetness-structure relaPhenylalanine, D-,favored conformation, 232 tionship, 253-254 Phenylethane, 1-amino-1-, Helferich’s Odorous substances, 223 Ohle, Heinz, 8-9 work on, 5
SUBJECT INDEX, VOLUME 45 Phenyl glycosides, preparation of, Helferich's work on, 4 Phloridzin, taste properties, 280 Phyllodulcin proposed AH,B,X systems of, 283-
285 taste properties, 280, 282-283, 288 Plant cell-wall polysaccharides, f.a.b.mass spectrometry, 66-68 Pneumococcal capsular polysaccharide, f.a.b.-mass spectrometry, 65 Poly(D-ga~actopyranuronicacid), circular dichroism, 109-110 Poly-(L-gulopyranuronate),blocks, circular dichroism, 107-108 Poly-(a-L-gulopyranuronicacid), circular dichroism, 108 Poly(sialo)glycoproteins,f.a.b.-mass spectrometry, 65 Pol y-( D-mannopyranuronate) blocks, circular dichroism, 107-108 interference in circular dichroism studies of CaZ+chelation of alginates, elimination of, I06 Poly-(P-D-mannopyranuronic acid), circular dichroism, 108 Poly(ethyleneglycol), as matrix for f.a.b.mass spectrometry, 26 Polypodium vulgare L., 286 Polysaccharides anomeric protons, nonselective spinlattice relaxation rates, 149-152 circular dichroism, 78, 85-92 f.a.b.-mass spectrometry, fragmentation pathways, 43 non-biological derivatives, circular dichroism, 75, 119-124 permethylated, f.a.b.-mass spectrometry, 37 Portuguese man-of-war toxin, red cell receptor, 170 Pringsheim, H., 9 Propanone -, 1,3-dihydroxy-2-, taste-structure relationship, 252-253 -, 1-hydroxy-2-, reaction with 1,3 'dicarbonyl compounds, 13 Prostaglandin endoperoxide synthase, high-mannose carbohydrate chains, f.a.b.-mass spectrometry, 63
387
Proton spin-lattice relaxation rates, 125-
168 analysis of data, 142-145 applicability to molecules with flexible conformations, 166-168 as basis for configurational assignments, 153 carbon-13, 137 combinations of nonselective and/or single-selective relaxation-rates,
164-167 cross-correlation effects, 130-131, 143 cross-relaxation effects, 130-131, 138,
143 density-matrix theory, 138 deuterium, 137 deuterium substitution, 158, 164, 166 effect on rate of receptor proton,
157-158 dipolar interactions, dynamic range limitation, 165-166 dipole-dipole interaction, 128 dipole-dipole mechanism, simplified version of, 127 double-selective inversion experiments, 141-142 double-selective relaxation rates, 134-
135, 159 exo-anomeric effect, 162 experimental methods for, 138-142 failure to provide information on spatial arrangement of protons,
153-154 for homonuclear spin-system, 128 initial relaxation-rate, 143 definition, 131 internuclear distances, calculation,
137-138 interpretation of, 126-127 interproton distances, 163 calculation, 127 determination, 137-138 error introduced into, 147 quantitative interpretation in terms of molecular conformation, 168 and relative efficiency of relaxation pathways between protons,
164-165 inversion recovery ( 18Oo-t-90"-ATPD)NTpulse sequence, 138-141
388
SUBJECT INDEX, VOLUME 45
inversion-recovery experiments requirements for static-field homogeneity, 146 systematic error in, 145 limitations, 163-165 magnetization-recovery curve, 130131 measurements, 138-147 methods, comparison of relative merits of, 165-166 for multispin system, 128-129 nonselective relaxation experiments, 163 nonselective relaxation rates, 128, 131-133 extraction of p l j values from, 157158 statistical analyses, 168 stereochemical implications, 147159 pairwise additivity of relaxation contributions, 127-128 problems from intermolecular dipolar contributions, 146 from paramagnetic relaxation due to dissolved oxygen, 146 progressive-saturation [90"-(t-9Oo)NT1 pulse sequence, 140 random errors, 147 relative relaxation-efficiency between two protons, as function of interproton distance, 153-154 pit and uu definition, 129-130 evaluation, 136-137 and molecular motion, 137 separation, 130-135 saturation-recovery (90"-t-9Oo-ATP D ) N T sequence, 140 selective pulse experiments, systematic errors in, 145-146 selective relaxation rates, 163 stereochemical implications, 159163 single-selective relaxation rates, 133134, 159, 163 stereochemical implications of, 147163 in strongly coupled spin-systems, 138
systematic errors, 145-147 tailored excitation experiments, 142 theory, 128-138 general formulation, 128-130 three distinct proton-proton relaxation pathways in six-membered ring in 4C1conformation, 150 three-pulse ( 180"-t-9O0-AT-PD90")~Tsequence, 139-140 triple-selective relaxation rates, 135, 163 (*)-Protoquercitol, taste properties, 291 Pseudo-a-DL-galactose, 292 Pseudo-p-DL-glucose, 292 Pseudonigeran, circular dichroism, 89 Pseudosugars, taste properties, 292-293 Pullulan, circular dichroism, 89 Pustulan, circular dichroism, 85-87, 89 Pyranose rings 4c1(D) conformation, 74-75 chair conformations, 74 Pyranoses circular dichroism, 85 a-D-aldo-hexo-, 74 D-aldohexo-, orientations of hydroxyl groups for, 75 a-D-aldo-pento-, 74 Pyrroles, Garcia Gonzilez' work on, 1314
Q Quercitols, sweetness-structure relationship, 241 Quinine, taste properties, 313 Quinine . HCI, taste properties, 311 Quinovose D-, sweetness-structure relationship, 24 1 a-D-, taste properties, 242 Quinoxaline -, 2-(2-furyl)-, 14 -, 2-(3-hydroxy-2-furyl)-, 14 -, 2-(D-arabino-tetritol-l-yl)-, 14
R Raffinose f.a.b.-mass spectrometry, 20 nonselective spin-lattice relaxation rates, 149
SUBJECT INDEX, VOLUME 45 sweetness, effect of infrared hydroxyl absorption bands and hydrogenbonding strength, 217 Rebaudoside A, taste properties, 285286 Red blood cell, role of glycophorin A in, 170- 171 Resonance energy hypothesis, of sweetness, 218 Rhamnogalacturonan I1 f.a.b.-mass spectrometry, 67-68 heptisaccharide isolated from, sequence of, 67-68 Rhamnose L-
nonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation rates, 150 sweetness-structure relationship, 24 1 CX-L-, taste properties, 242 p-L-,taste properties, 242 Rhizobia p-( 1-+2)-glucans,f.a.b.-mass spectrometry, 68 acidic polysaccharides, f.a.b.-mass spectrometry, 66 Ribitol infrared spectrum, 294 sweetness-structure relationship, 295 -, 1,4-anhydro-~-,sweetness-structure relationship, 257 Ribopyranose, (Y-D-, 74 Ribose, Dnonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation rates, 152 Rutinose, taste properties, 278-279 S
Saccharides, cyclic-hemiacetal formation by, Helferich’s work on, 3 Saccharin after-taste, 265, 341 and sucrose, comparison of, 341 sweetness, 205, 226,231 AH,B system in, 221 early explanations of, 202-206
389
sweetness-structure relationship, 297303 thio analog, bitterness, 310 third structural feature comprising postulated glucophore in, 234 three-dimensional x-ray analysis of, 301 Salmonella typhimurium, monophosphoryl lipid A, f.a.b.-mass spectrometry, 57-58 Sapophoric groups, 202-203,218 Secondary-ion mass spectrometry, 24-25 Sedoheptulosan, taste properties, 254255 Sensory system function, 325-326 Septanose derivatives, proton spin-lattice relaxation rates, 153 Shallenberger AH,B hypothesis. See Sweetness, AH,B hypothesis Slime mold, high-mannose oligosaccharide, f.a.b.-mass spectrometry, 63 Smell, and sensory system, 325 Sodium D-glUCUrOnate, circular dichroism, 104-105 Sodium hyaluronate, circular dichroism, 113-114 Sodium poly-(L-gulopyranuronate),chelation of Ca2+to, circular dichroism, 105-106 Sodium saccharin, relative sweetness, 332 Sophorosyldihydrochalcones,p-D-,280 Sorbofuranoside, 4-chloro-4-deoxy-a-~galactopyranosyl 1,4,6-trichloro1,4,6-trideoxy-p-~-,sweetness, 269 Sorbopyranose L-, taste properties, 254 (Y-L-
sweetness-structure relationship, 250-252 taste properties, 242 Sorbose, a - D - , minus a-D-XylOSe, difference-circular dichroism spectra, 81 Spin-lattice relaxation time, 128 Stachyose, nonselective spin-lattice relaxation rates, 149 Steoia rebaudiana Bertoni, 285 Stevioside structural features, 289 taste properties, 285-286
390
SUBJECT INDEX, VOLUME 45
Streptococcus pneumoniae type 9, capamino-substituted, circular dichroism, sular polysaccharide, Smith degra99-100 3,6-anhydro derivatives, interatomic dation product, f.a.b.-mass spectromoxygen-oxygen distances, 273 etry, 66 Sucrose p anomers, binding to sweet-receptors, binding to taste papillae, 329-330 248 inactivation by heating in boiling circular dichroism spectrum, 75 water, 330-331 D- and Ldeoxyhalo derivatives, relative sweetrelative sweetness, 258 ness, 266-268 superpositioned over same receptor site, 236 galactodeoxyhalo derivatives, relative di-p-bromobenzoate derivatives, circusweetness, 266-268 lar dichroism, 123-124 sweetness-structure relationship, L-, sweetness-structure relationship, 263 258-259 nonselective spin-lattice relaxation location of third binding-site in, 234rates, 149 235 sweetness, 214,231,233,238 methyl ethers, sweetness-structure effect of infrared hydroxyl absorprelationship, 261-265 tion bands and hydrogen-bondrelative sweetness, differences in, ing strength, 217 hypothesis about, 220 time-intensity relationships for, 346 simple, structure, and sweetness, 238sweetness-structure relationship, 263 259 tetrachlorotetra-0-mes yl-galactostructure, and sweetness, 238-310 in ICI conformation, taste properties, derivative, proton spin-lattice relaxation, 154-157 269 -, l‘$‘-di-O-methyl-, taste properties, in 4Clconformation, taste properties, 262 269 -, 4,6-di-O-methyl-, taste properties, sweetness, 230 contraction coefficient, 206 262 -, 4,6’-di-O-methyl-, taste properties, effect of infrared hydroxyl absorption bands and hydrogen-bond262 -, 6,6’-di-O-methyl-, taste properties, ing strength, 216-217 and hydrogen bonding, 214-217 262 taste, importance of configuration in, -, 4-0-methyl, taste properties, 262 -, 6’-0-methyl, taste properties, 262 207 -, 4,6,1‘,6’-tetrachloro-4,6,1’,6’-tetra- Sulfamates hetero-, sweetness-structure relationdeoxy-golacto-, relative sweetness, 332 ship, 302 -, 6,1‘,6’-trichloro-6,1’,6’-trideoxy-, sweetness-structure relationship, 297relative sweetness, 332 303 -, 4,1’,6’-trichloro-4,1‘,6’-trideoxy- Sulfamic acid, tetrahydro-2H-thiopyrangalacto-, 333 4-, 299 relative sweetness, 266-268 Sulfate derivatives, circular dichroism, Sugar acetates, bitterness, 318 92-94 Sugar derivatives, having mixed substiSulfonamides, Helferich’s work on, 5 tuents, circular dichroism, 111-119 Sultams, Helferich’s work on, 5 Sugar methyl ethers, bitterness, 318 Suosan, sweetness, 304 Sugars. See also Anhydro sugars; Deoxy Sweet compounds, as proton acceptors, sugars 212-213
SUBJECT INDEX, VOLUME 45 Sweeteners nonsugar, sweetness-structure relationship, 295 structure, 317-318 Sweetness, 200 3- and 4-hydroxyl-groups’ role in, 243245 3-hydroxyl group’s role in, 260-261 4-hydroxyl group’s role in, 260 6-hydroxyl group’s role in, 261 AH,B concept, 200-201, 213-223, 231, 246,257-261,264,271,283-285, 292,297-299,303-307,322,341 AH,B system, 217-218 in various compounds, 221 AH,B units, 238 and anomeric configuration, 207 biochemistry of, 325-349 chemoreception, possible courses of events in, 346-349 comparisons, between two compounds, 341 degree of, methods of determining, 344-345 depression by bitterness, 239-240 early theories of, 202-206 fundamental structural requirements for, 207-237 hydrophobic bonding concept, 22323 1 initial mechanism of, 350 intensity, 350 differences in sensory perception of, at different concentrations, 350351 intensity us. time curves, 344 and lipoid solubility, 223 measurement, methodology, 349-351 perception, 201 plateau of maximum intensity, concentration dependence of, 342-343 quality of, 339-349 queue hypothesis of, 345-349 receptor, initial stimulation, 222 receptors participating in, 237 role of structure in, 200 saporous groups involved in, 227 and sensory system, 325-326 side-tastes with, 340 stereochemistry, 201-310
391
steric features in, 213 structural parameters for, schematic representation, 315-317 and structure of sugars, 238-310 and temperature, 221-222, 250, 342 time-intensity relationship in, 340345 tripartite concept, 231-237 Sweet-sensitive protein, 328 Sweet-taste inhibitors, 336-339 Sweet-taste modifiers, 339 initial reaction with taste receptor membranes, 338 Sweet-taste receptor, model, 335-336 Sweet-tasting compounds AH,B,X glucophore of, 233 y-sites, 233-234 third structural feature comprising postulated glucophore in, 233-234 Synsepalum dulcificum, 290
T Tagatose D-, taste properties, 254 (Y-D-, sweetness-structure relationship, 253 Talopyranose D-, orientation of hydroxyl groups for, in 4c1(D) conformation, 75 (Y-D-, 74 -, 1,6-anhydro-p-~-,sweetnessstructure relationship, 269 Talose, Dnonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation rates, 150 T antigen, 171 Taste and chemical composition, early explanations of, 206 and hydrophobicity, 223 perception effect of structure on, 200-201 mechanism, 200 peripheral mechanisms in, 326-328 receptor mechanism for, 209-214 factors determining response of, 210-211 sense, 199
392
SUBJECT INDEX, VOLUME 45
and sensory system, 325 and structure, early explanations of, 205 Taste blindness, 311 Taste-cell transduction, steps in, 211212 Taste couples, 218 Taste intensity, measurements, types of measures used for, 350 Taste receptor, 326-328 binding of taste stimulus to, 329-330 us. behavioral responses, 329-330, 334 binding to, 328-339 membranes, initial interaction with taste modifier molecules, 338 in sweetness, 237 Taste theory, evolution of, 200-201 Tetraethyleneglycol, as matrix for f.a.b.mass spectrometry, 26 1,1,3,3-Tetramethylurea,as matrix for f.a.b.-mass spectrometry, 27 Thaumatin antibodies, cross-reactivity, 333-334 immunoreactivity, and relative sweetness, 332 relative sweetness, 332 sweetness, 213 time-intensity relationships for, 346 three-dimensional structure, 333-334 Theophrastus, 200,201 1-Thioglycerol cluster ions, 29 as matrix for f.a.b.-mass spectrometry, 26-28,54 Thrombopenia, 171 TNantigen, 171 p-Toluenesulfonates, 4 Trehalose a,a-
3,6-anhydro-, sweetness-structure relationship, 273 3,6:3',6'-dianhydro-, sweetnessstructure relationship, 273 4,4-dideoxy-, sweetness, comparison to methyl 4-deoxy-a-~-rylohexopyranoside, 244 4,4'-dideoxy-, sweetness threshold, determination by triangular test, 245
methyl derivatives, sweetness, 262 methyl ethers, taste properties, 262 mono- and di-deoxy derivatives, sweetness-structure relationship, 259-260 sweetness, 263 comparison to methyl a-D-glucopyranoside, 244 sweetness-structure relationship, 243 sweetness threshold, determination by triangular test, 245 a,a'-galacto-, sweetness, comparison to methyl a-D-galactopyranoside, 244 Trehalose derivatives, sweetness, comparison to corresponding monosaccharide analog, by paired comparison test, 244-245 3,4,6-Tri-O-acetyl- 1-0-benzoyl-2-deoxy2-bromo-~-hexopyranosederivatives, nonselective relaxation-rates, stereospecific dependencies, 152 Trichodonin, taste properties, 312 Triethanolamine. See 2,2',2"-Nitrilotriethanol Triethanolamine-l,1,3,3-tetramethylurea, as matrix for f.a.b.-mass spectrometry, 54 Triethylenetetramine. See N,N'-Bis(2aminoethy1)ethylenediamine Trimethyl-1-butanol, 2,2,3-, molecularconnectivity index, correlation with biological activity, 229-230 Trimethylpropyl ethyl ether, (S)-( +)1,2,2-, circular dichroism, 83 Trisalosylactosylceramide, identification, 56 Trityl protecting group, Helferich's work on, 3 Tryptophan D-, favored conformation, 232 as hydrophobic binding-region of sweet-taste receptor, 336 Turanose, taste properties, 254-255 V Vargha, LBsz16, 9 VG Analytical ZAB-HF mass spectrometer, 36-37
SUBJECT INDEX, VOLUME 45 (*)-Viboquercitol, sweetness, 291-292 (-)-Viboquercitol, taste properties, 291292 Vibratory hydrogen hypothesis, of sweetness, 205, 218 Vitamin C, synthesis, Garcia GonzBlez’ work on, 10 W
Wiegand, Friedrich, 3 Wolfrom, M.L., 15
X Xanthan, circular dichroism, 118-119 Xanthomonas, extracellular polysaccharides, circular dichroism, 118119 Xylan acetate, circular dichroism, 121 Xylitol infrared spectrum, 294 sweetness-structure relationship, 295 syn-axial interaction on, 294 -, 1,4-anhydro-~-,sweetness-structure relationship, 257
393
Xylopyranose a-D-, 74 taste properties, 242 p-D-,taste properties, 242 Xylose D-
a and p pyranose anomers, circular
dichroism, 79-80 monosaccharides, circular dichroism, 82 nonselective spin-lattice relaxation rates, 148 proton spin-lattice relaxation rates, 152 (Y-D-, 2-(hydroxymethyl) derivatives, circular dichroism, 79 p-D-, proton spin-lattice relaxation rates, 152 a-L-, 2-(hydroxymethyl) derivatives, circular dichroism, 79
2 Ziziphins, as sweet-taste inhibitors, 336339
CUMULATIVE AUTHOR INDEX FOR VOLS. 41-45* A J., The Composition of ANGYAL,STEPHEN Reducing Sugars in Solution, 42, 15-68 ANTONAKIS, KOSTAS,Ketonucleosides, 42, 227-264
B
DEY,PRAKASH M. and BRINSON, KEN, Plant Cell-Walls, 42, 265-382 DEY,PRAKASH M. See u k o , Pont Lezica, Rafael DILL, KILIAN,l3C-Nuc1ear Magnetic Resonance-Spectral Studies of Labeled Glycophorins, 45,169-198 DILL, KILIAN,BERMAN, ELISHA, and PAVIA,AND&, Natural-abundance, 13C-Nuclear Magnetic ResonanceSpectral Studies of Carbohydrates Linked to Amino Acids and Proteins, 43, 1-49 DORLAND, LAMBERTUS. See Vliegenthart, Johannes F. G
BARRETO-BERGTER, ELIANA and GORIN, PHILIPA. J., Structural Chemistry of Polysaccharides from Fungi and Lichens, 41,67-103 BERMAN, ELISHA. See Dill, Kilian BOCK,KLAUSand PEDERSEN, CHRISTIAN, Carbon- 13 Nuclear Magnetic ResoF nance Spectroscopy of Monosaccharides, 41,27-66 FERNhNDEZ-BOLAROS, Jose hl. See BOCK,KLAUS,PEDERSEN, CHRISTIAN, and G6mez-S5nchez, Antonio PEDERSEN, HENRIK, Carbon-13 Nuclear Magnetic Resonance Data for G Oligosaccharides, 42, 193-225 GARC, HARI C. and JEANLOZ, ROGERW., BRINSON, KEN. See Dey, Prakash M Synthetic N- and 0-Glycosyl Derivatives of L-Asparagine, C L-Serine, and L-Threonine, 43, 135-201 CASU,BENITO,Structure and Biological G6MEZ-ShNCHE2, ANTONIO, and Activity of Heparin, 43, 51-134 FERNhNDEZ-BOLAROS, JOSG M. [Obituary of1 Francisco Garcia D ConzLlez, 45, 7-17 GORIN,PHILIPA. J. See Barreto-Berger, DAIS,PHOTIS,and PERLIN,ARTHURS., Eliana Proton Spin-Lattice Relaxation Rates in the Structural Analysis of Carbohydrate Molecules in Solution, H 45, 125-168 HAY,GEORGE W. See Szarek, Walter A DALEO,GUSTAVO R. See Pont Lezica, Rafael DELL,ANNE,F.a.b.-Mass Spectrometry of I Carbohydrates, 45, 19-72 INOKAWA, SARUBO. See Yamamoto, DELMER,DEBORAH P., Biosynthesis of Hiroshi Cellulose, 41, 105-153
* Starting with Volume 30, a Cumulative Author Index covering the previous 5 volumes will be published in every 5th volume. That listing the authors of chapters in Volumes 1-29 may be found in Volume 29. 394
CUMULATIVE AUTHOR INDEX FOR VOLS. 41-45
J JEANLOZ, ROGERW. See Garg, Hari G JEFFREY,GEORGEA. and SUNDARALINGAM,MUTI-AIYA, Bibliography of Crystal Structures of Carbohydrates, Nucleosides, and Nucleotides for 1979 and 1980; Addenda and Errata for 1970-1978; and Index for 19351980,43,203-421 JENNINGS, HAROLD J., Capsular Polysaccharides as Human Vaccines, 41, 155-208 JOHNSON, W. CURTIS,JR., The Circular Dichroism of Carbohydrates, 45, 73- 124
395
PERLIN,ARTHURS. See Dais, Photis PONTLEZICA,RAFAEL,DALEO,GUSTAVO R., and DEY,PRAKASH M., Lipidlinked Sugars as Intermediates in the Biosynthesis of Complex Carbohydrates in Plants, 44, 341-385 S
SHIBAEV, VLADIMIR N., Biosynthesis of Bacterial Polysaccharide Chains Composed of Repeating Units, 44, 277-339 SMIRNOVA, GALINAP. See Kochetkov, Nicolai K See Szarek, Walter A STACEY, MAURICE. STETTER,HERMANN, [Obituary of] K Burckhardt Helferich, 45, 1-6 SUNDARALINGAM, MUTTAIYA. See JefFrey, KAJI,AKIRA,L-Arabinosidases, 42, George A 383-394 WALTERA., STACEY, MAURICE, KOCHETKOV, NICOLAIK. and SMIRNOVA, SZAREK, of] and HAY, GEORGE W., [Obituary GALINAP., Glycolipids of Marine John Kenyon Netherton Jones, 41, Invertebrates, 44,387-438 1-26 KOENIG,JACKL. See Mathlouthi, Mohamed V
L LEE, CHEANG-KUAN, The Chemisty and Biochemistry of the Sweetness of Sugars, 45, 199-351 M MATHESON, NORMAN K. See McCleary, Barry V MOHAMEDand KOENIG, MATHLOUTHI, JACKL., Vibrational Spectra of Carbohydrates, 44, 7-89 MCCLEARY, BARRY V. and MATHESON, NORMAN K., Enzymic Analysis of Polysaccharide Structure, 44, 147276 MCGINNIS,GARYD., [Obituary of] Fred Shafizadeh, 44, 1-6 P PAVIA,ANDMA. See Dill, Kilian PAZUR,JOHNH., [Obituary 04 Dexter French, 42, 1-13 PEDERSEN, CHRISTIAN.See Bock, Klaus PEDERSEN, HENRIK. See Bock, Klaus
VANHALBEEK, HERMAN. See Vliegenthart, Johannes F. G F. G., VLIEGENTHART, JOHANNES DORLAND, LAMBERTUS, and VAN HALBEEK, HERMAN, High Resolution, 'H-Nuclear Magnetic Resonance Spectroscopy as a Tool in the Structural Analysis of Carbohydrates Related to Glycoproteins, 41, 209-374
w WITCZAK, ZBIGNIEW J., Monosaccharide Isothiocyanates and Thiocyanates: Synthesis, Chemistry, and Preparative Applications, 44,91-145
Y YAMAMOTO, HIROSHIand INOKAWA, SABURO, Sugar Analogs Having Phosphorus in the Hemiacetal Ring, 42,135-191 YOSHIMURA, JUJI, Synthesis of Branched-chain Sugars, 42,69-134
CUMULATIVE SUBJECT INDEX FOR VOLS. 41-45* A
L-Arabinosidases, 42, 383-394
B Bacterial polysaccharides, chains composed of repeating units, biosynthesis of, 44,277-339 Bibliography, of crystal structures of carbohydrates, nucleosides, and nucleotides for 1979 and 1980; addenda and errata for 1970-1978; and index for 1935-1980,43,203-421 Biochemistry, and chemistry of the sweetness of sugars, 45, 199-351 Biosynthesis, of bacterial polysaccharide chains composed of repeating units, 44, 277-339 of cellulose, 41, 105-153
C
Capsular polysaccharides, as human vaccines, 41, 155-208 Carbohydrates. See ulso, Cellulose, Monosaccharides, Oligosaccharides, Polysaccharides, Sugars bibliography of crystal structures of, 43,203-421 circular dichroism of, 45, 73-124 complex, in plants, lipid-linked sugars as intermediates in the biosynthesis of, 44,341-385 f.a.b.-mass spectrometry of, 45, 19-72 linked to amino acids and proteins, natural-abundance L3C-nuclear magnetic resonance-spectral studies of, 43, 1-49
molecules in solution, proton spinlattice relaxation rates of, in the structural analysis of, 45, 125-168 related to glycoproteins, high-resolution 'H-nuclear magnetic resonance spectroscopy of, 41,209-374 vibrational spectra of, 44, 7-89 Carbon-13, nuclear magnetic resonance data for oligosaccharides, 42, 193-225 nuclear magnetic resonance spectroscopy of monosaccharides, 41,2766 Cellulose, biosynthesis of, 41, 105-153 Chemistry, and biochemistry of the sweetness of sugars, 45, 199-351 Circular dichroism, of carbohydrates, 45, 73-124 Composition, of reducing sugars in solution, 42, 1568 Crystal structures, of carbohydrates, nucleosides, and nucleotides, bibliography of, 43, 203-421
E Enzymic analysis, of polysaccharide structure, 44, 147276
F F.a.b.-mass spectrometry, of carbohydrates, 45, 19-72 French, Dexter, obituary of, 42, 1-13 G Garcia Gonzhlez, Francisco, obituary of, 45, 7-17
* Starting with Volume 30, a Cumulative Subject Index covering the previous 5 volumes will be published in every 5th volume. That listing the chapters in Volumes 129 may be found in Volume 29. 396
CUMULATIVE SUBJECT INDEX FOR VOLS. 41-45 Glycolipids of marine invertebrates, 44, 387-438 H Helferich, Burckhardt, obituary of, 45, 1-6 Heparin, structure and biological activity of, 43, 51-134 High-resolution, 'H-nuclear magnetic resonance spectroscopy, as a tool in the structural analysis of carbohydrates related to glycoproteins, 41, 209-374
J Jones, John Kenyon Netherton, obituary of, 41, 1-26
K Ketonucleosides, 42,227-264 L Lipid-linked sugars, as intermediates in the biosynthesis of complex carbohydrates in plants, 44,341-385 M
Monosaccharide isothiocyanates and thiocyanates, synthesis, chemistry, and preparative applications, 44, 91-145 Monosaccharides, nuclear magnetic resonance spectroscopy of, 4 1 , 2 7 4 6 N
Natural-abundance, 13C-nuclearmagnetic resonance studies, of carbohydrates linked to amino acids and proteins, 43, 1-49 L3C-Nuclearmagnetic resonance-spectral studies, of labeled glycophorins, 45, 169-198
397
Nucleosides, keto-, 42, 227-264 Nucleosides and nucleotides, bibliography of crystal structures of, 43,203-214 0 Obituary, of Dexter French, 42, 1-13 of Francisco Garcia Gonzilez, 45, 717 of Burckhardt Helferich, 45, 1-6 of John Kenyon Netherton Jones, 41, 1-26 of Fred Shafizadeh, 44,l-6 Oligosaccharides, nuclear magnetic resonance data for, 42,193-225 P Plants, cell walls of, 42, 265-382 Polysaccharides, bacterial, biosynthesis of, 44,277-339 capsular, as human vaccines, 41, 147208 enzymic analysis of structure of, 44, 147-276 from fungi and lichens, structural chemistry of, 41,67-103 Proton spin-lattice relaxation rates, in the structural analysis of carbohydrate molecules in solution, 45, 125-168 S Shafizadeh, Fred, obituary of, 44, 1-6 Structural chemistry, of polysaccharides from fungi and lichens, 41,67-103 Structure, and biological activity of heparin, 43, 51-134 Sugar analogs, having phosphorus in the hemiacetal ring, 42, 135-191
398
CUMULATIVE SUBJECT INDEX FOR VOLS. 41-45
Sugars, chemistry and biochemistry of the sweetness of, 45, 199-351 reducing, composition of, in solution, 42, 15-68 Sweetness of sugars, chemistry and biochemistry of the, 45, 199-351 Synthesis, of branched-chain sugars, 42,69134
Synthetic N- and 0-glycosyl derivatives, of L-asparagine, L-serine, and Lthreonine, 43, 135-201
V Vaccines, capsular polysaccharides as human, 41, 147-208 Vibrational spectra, of carbohydrates, 44, 7-89