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Structural investigation of bacterial capsular polysaccharides Leek, Donald Morley 1982

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STRUCTURAL INVESTIGATION OF BACTERIAL CAPSULAR POLYSACCHARIDES by DONALD MORLEY LEEK B.Sc. (Hons.), McMaster Un i v e r s i t y , 1978 Dip.C.S., Regent College, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1982 <jg) Donald Morley Leek, 1982 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f 0 ( ^ i M . l S ^ " f ^ |  T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 M a i n M a l l V a n c o u v e r , C a n a d a V6T 1Y3 ABSTRACT Eighty s t r a i n s of K l e b s i e l l a b a c t e r i a have been i s o l a t e d and serotyped according to t h e i r capsular polysaccharide antigens (K antigens). A number of research groups have taken part i n an extensive program to determine the chemical structures of these polysaccharide antigens. As part of t h i s continuing program, t h i s t h e s i s includes a s t r u c t u r a l i n v e s t i g a t i o n of the capsular polysaccharide of K l e b s i e l l a K An a c i d i c capsular polysaccharide was i s o l a t e d from K l e b s i e l l a K39 and a p a r t i a l hydrolysis study was conducted. An a c i d i c penta-saccharide was i s o l a t e d from the hydrolysate and studied by ^H- and 13 C-n.m.r., mass spectrometry and methylation analyses. This o l i g o -saccharide was assigned the following structure: 1 2 1 4 1 2 1 ^ GlcA — - Man — - GlcA ±-=- Man Glc~OH 3 a 3 a The r e l a t i o n s h i p of t h i s oligosaccharide to the polysaccharide w i l l be discussed with reference to n.m.r. and methylation analysis studies. Similar studies are being conducted to determine the structures of the E . c o l i capsular polysaccharide antigens. This thesis includes a preliminary study o f E . c o l i K26. i i i TABLE OF CONTENTS Page ABSTRACT \. i i TABLE OF CONTENTS . . : . . ." .' .' . . . . . i i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS . v i i • PREFACE v i i i I. INTRODUCTION 1 II. CHROMATOGRAPHIC AND INSTRUMENTAL METHODS . . . 9 II.1 Paper Chromatography 10 II. 2 Gel Chromatography 12 II. 3 Gas Liquid Chromatography 13 11.4 High-Performance Liquid Chromatography . 16 11.5 Ion-exchange Chromatography 17 11.6 S i l i c a Gel Chromatography 17 11.7 Mass Spectrometry 18 11.8 Nuclear Magnetic Resonance 23 11.9 Polarimetry and C i r c u l a r Dichroism . . . 28 II I . POLYSACCHARIDE STRUCTURAL METHODS 29 I I I . l I s o l a t i o n and P u r i f i c a t i o n 30 III . 2 Sugar Analysis 30 III.'3 Methylation Analysis 33 III.4 P a r t i a l Hydrolysis 37 i v I I I . 5 Uronic Acid Degradation 38 111.6 Periodate Oxidation and Smith Degradation . 39 111.7 Immunochemical Methods 40 IV. A STRUCTURAL INVESTIGATION OF KLEBSIELLA K39 CAPSULAR POLYSACCHARIDE 41 V. A PRELIMINARY STRUCTURAL INVESTIGATION OF ':. E.COLI K26 CAPSULAR POLYSACCHARIDE 70 VI. BIBLIOGRAPHY • 84 APPENDIX I: Q u a l i t a t i v e Sugar Analysis o f the K l e b s i e l l a Capsular polysaccharide. 90 APPENDIX I I : Nuclear Magnetic Resonance Spectra 91 APPENDIX I I I : Mass Spectral Analysis of K39-A1 108 APPENDIX IV: Immunochemical Cross-Reactions of E. c o l i K26 110 APPENDIX V: An Explanation of Carbohydrate Terminology I l l V LIST OF TABLES Page Table 1: N.m.r. study of Al (K39) 46 Table 2: Methylation analyses of Al and A2 . . . 49 Table 3: N.m.r. study of A2 . 55 Table 4: N.m.r. study of K39 capsular polysaccharide 58 Table 5: Methylation analysis of Klebsiella K39 capsular polysaccharide . . . 60 Table 6: Methylation analysis of E. c o l i K26 capsular polysaccharide 72 Table 7: N.m.r. study of Al (K26) 74 v i LIST OF FIGURES Page Figure 1: C e l l wall structure i n gram-negative b a c t e r i a . . 6 Figure 2: Mass spectrometry of p a r t i a l l y methylated a l d i t o l acetates 20 Figure 3: Mass spectral study of a permethylated aldo t r i o u r o n i c acid 22 Figure 4: Comparison of J „ f o r B-D-Glucopyranose . . . . 26 Figure 5: Total sugar r a t i o reaction sequence 31 Figure 6: Methylation analysis of a hypothetical polysaccharide 34 Figure 7: Bio-Gel P-2 chromatography of a c i d i c f r a c t i o n obtained by p a r t i a l hydrolysis 44 Figure 8: Methylation analysis of K39-A1 48 Figure 9: Mass spectrum of K39-A1 as the permethylated a l d i t o l 51 Figure 10: Mass spectrum of K39-A1 as the permethylated a l d i t o l . 52 Figure 11: ^H-n.m.r. anomeric signals for oligosaccharide A l and K39 capsular polysaccharide 57 v i i ACKNOWLEDGEMENTS I would l i k e to thank Dr. G.G.S. Dutton for h i s supervision of t h i s work. Several other people were very kind i n helping me get started on t h i s project. In p a r t i c u l a r s I would l i k e to thank Dr. A.V. Savage, Dr. E.H. M e r r i f i e l d , Dr. J.L. Di Fabio and '. Marcel Paulin. I would also l i k e to thank Dr. G. Eigendorf and the mass spectrometry s t a f f , and Dr. S.O. Chan and the n.m.r. s t a f f f or t h e i r patient assistance. Special appreciation i s extended to Rani Theeparajah for typing t h i s t h e s i s . v i ' i i PREFACE For readers not f a m i l i a r with carbohydrate nomenclature, an explanation of terms, reproduced from the M. Sc. thesis of T.E. Folkman, has been included i n Appendix V. I. INTRODUCTION 2 I. INTRODUCTION A polysaccharide i s a polymeric substance composed of mono-saccharides joined together by g l y c o s i d i c linkages. These carbohydrate polymers are ubiquitous i n nature and have a number of important b i o l o g i c a l functions. Many polysaccharides have commercial value and t h e i r r o l e i n industry continues to expand. Researchers are pursuing s t r u c t u r a l studies i n the hope that the chemical basis for physical and b i o l o g i c a l properties may be better understood. Natural polysaccharides show a wide range of r e l a t i v e complexity with s i g n i f i c a n t v a r i a t i o n s i n such features as the number of d i f f e r e n t sugars, the presence or absence of non-carbohydrate substituents, and the degree of branching. There i s v a r i e t y i n the degree of s t r u c t u r a l r e g u l a r i t y with which organisms produce polysaccharides; for example, some bac t e r i a synthesize complex polymers with p r e c i s e l y defined repeating u n i t s , while plant gums have more random structures. L i s t e d below are several polysaccharides from representative sources. This l i s t i s by no means exhaustive but i t does hi g h l i g h t some important features o f polysaccharide structures. C e l l u l o s e : l / 4 1 \4 Glc Glc Glc~0H This glucose homopolymer i s the main component o f plant c e l l walls. Its importance i n the t e x t i l e and paper products industries i s well known.^ 3 Amylose: Glc Glc~OH Amylose i s an important component of starch and i s a food storage material i n plants. Even though i t i s a glucose homopolymer l i k e c e l l u l o s e , i t s a 1—4 linkages are much more e a s i l y hydrolysed than the 3 1 — 4 linkages of c e l l u l o s e . Heparin: 1 CH 2OS0 3 0S0, NHSCL The polysaccharide shown above was i s o l a t e d from beef lung t i s s u e and consists of a l t e r n a t i n g residues of a-L-idopyranuronic acid 2-sulfate and 2-deoxy-2-sulfamino-a-D-glucopyranose 6-sulfate. Heparin i s a 2 s t r u c t u r a l component o f some animal tissu e s . Gum Tragacanth: 1 4 „ . . 1 4 „ . . 1 GalA GalA — GalA — GalA 0 4 a D 3 p 1 P 1 Xyl Xyl 2 1 Fuc 1 Xyl 2 I 1 Gal 4 Many trees and shrubs exude complex a c i d i c polysaccharides or gums. Because of t h e i r complexity i t i s only possible to propose p a r t i a l or..' 1 3 average structures. 4 Capsular Polysaccharide from K l e b s i e l l a K21 : 3 . 1 3 u 1 2 . , 1 3 „ , 1 GlcA Man Man Gal — ^ a a a 3 a 1 Gal 4 6 \ / CH 3- C- C0 2H Many species o f b a c t e r i a produce a c i d i c polysaccharides that are r e l a t i v e l y complex but have regular repeating u n i t s . The concept of p r e c i s e l y defined oligosaccharide repeating units i n b a c t e r i a l polysaccharides i s now well established by nuclear magnetic resonance,^ p a r t i a l hydrolysis and phage degradation studies. This thesis includes s t r u c t u r a l studies of capsular polysaccha-r i d e s from s t r a i n s of K l e b s i e l l a and Escherichia c o l i b a c t e r i a . The re l a t i o n s h i p of the capsular polysaccharide to the rest of the b a c t e r i a l c e l l i s given i n Figure 1. The capsular polysaccharide has the same chemical composition as the exocellular slime that i s exuded by the bacteria. It i s also known as the K antigen, an antigen being a substance that induces the production of antibodies. (The term K antigen has a precise immunological d e f i n i t i o n but i t i s usually a 9 capsular polysaccharide). The K l e b s i e l l a are Gram-negative b a c t e r i a of the family Enterobacteriaeae, the genus K l e b s i e l l a , and the species K l e b s i e l l a 5 pneumoniae, K l e b s i e l l a ozaenae and K l e b s i e l l a rhinoschleromatis, K l e b s i e l l a may cause i n f e c t i o n s of the r e s p i r a t o r y and urinary t r a c t s . Approximately eighty s t r a i n s , as distinguished by t h e i r K antigens, have been i s o l a t e d and serotyped. Heidelberger has studied the immunochemical reactions of the K l e b s i e l l a capsular polysaccharides.** These polysaccharides were reacted with antibodies prepared using the Pneumococcal polysaccharides, many of which have known chemical structures. The degree of p r e c i p i t a t i o n was measured and conclusions were drawn concerning the r e l a t i o n s h i p between chemical structure and immunochemical r e a c t i v i t y . A number o f research groups have embarked on s t r u c t u r a l studies of the K l e b s i e l l a capsular polysaccharides i n order to complete the understanding of t h i s work. 12 13 Nimmich ' has performed q u a l i t a t i v e analyses of the sugars present i n the K l e b s i e l l a capsular polysaccharides. His r e s u l t s are tabulated i n Appendix I. Along with a c i d i c sugars (e.g., GlcA, GalA) and neutral sugars (e.g., Glc, Gal, Man, Fuc, Rha), pyruvate, a c e t y l , and formyl substituents may also be present. A l l of these polysaccharides contain either an a c i d i c sugar or a pyruvic acid substituent. Di Fabio has tabulated the known structures of K l e b s i e l l a capsular polysaccha-r i d e s . The Escherichia c o l i (E. c o l i ) belong to the family Entero- bacteriaceae, the genus Escherichia and the species E. c o l i . * ^ * E. c o l i was f i r s t i s o l a t e d by Theodor Escherich from feces i n 1885. Most s t r a i n s o f E. c o l i are harmless and occur n a t u r a l l y i n the large bowel of humans and other vertebrates. Some s t r a i n s are pathogenic and cause i n f e c t i o u s diarrhea,.an a f f l i c t i o n that contributes to 6 Cytoplasm oooooooooo Inner Membrane Protein Phospholipid Protein Peptidoglycan Outer Membrane Lipopolysaccharide Protein Phospholipid Protein Capsular • Polysaccharide Figure 1. C e l l wall structure i n Gram-negative bacteria. 7 infant m o r t a l i t y i n underdeveloped areas of the world. 9 (2)rskov and coworkers have reviewed the serology, chemistry and genetics of the 0 and K antigens of the E. c o l i . The K antigens consist almost e x c l u s i v e l y of acid polysaccharides, although K88 i s known to be a protein. Many structures have been published, including one that contains 10 sugars i n a repeating unit (K85) . The review a r t i c l e mentioned above contains the q u a l i t a t i v e sugar analyses of many of the K antigens. Along with a c i d i c , amino, and neutral sugars, threonine (K54) and pyruvate may also be present. S t r u c t u r a l studies of the K antigens continue i n order to further the understanding of the chemical basis for immunology. The s i g n i f i c a n c e of continued studies of b a c t e r i a l polysaccharides goes beyond the immediate applications to b a c t e r i a l immunology. The extensive study of b a c t e r i a l polysaccharides has provided a r i c h supply of oligosaccharides by p a r t i a l hydrolysis and through the use of phage-induced enzymes. A wide range of spectroscopic and chromato-graphic techniques have been applied to these compounds-. Studies of b i o l o g i c a l l y active oligosaccharides have contributed greatly to the understanding of important biomedical problems such as blood-group immunology. As chemists develop t h e i r understanding of the spectros-copic and chromatographic properties of these compounds, i t i s more probable that advanced a n a l y t i c a l techniques w i l l be s k i l f u l l y applied to complex problems inv o l v i n g carbohydrates. Sections II and III complete the introduction to t h i s t h e s i s . Section II describes the "Chromatographic and Instrumental Methods" that are used i n the study of polysaccharides and other carbohydrates. 8 Section I I I , e n t i t l e d "Polysaccharide Structural Methods," includes methods of obtaining oligosaccharides and d e r i v a t i s e d monosaccharides that provide information about the polysaccharides from which they are derived. CHROMATOGRAPHIC AND INSTRUMENTAL METHODS 10 II CHROMATOGRAPHIC AND INSTRUMENTAL METHODS I I . l Paper chromatography Paper chromatography was introduced i n 1944 by Consden and coworkers who used i t to separate amino acids. In 1947 Partridge applied t h i s method to sugars. The arrangement of f i b r e s i n chromato-graphy paper provides a serie s o f channels through which a solvent may flow by c a p i l l a r y action. The paper consists o f highly ordered regions c a l l e d c r y s t a l l i t e s and areas of lesser order c a l l e d the amorphous regions. It i s i n these amorphous regions that bound water acts as a stationary l i q u i d phase. The solute i s p a r t i t i o n e d between t h i s cellulose-water complex and the mobile solvent as i t t r a v e l s through the paper. The paper may be i r r i g a t e d by the descending, ascending, horizontal or r a d i a l methods. The descending mode of 15 paper chromatography was used i n the present study. Paper chromatography i s a simple and e f f e c t i v e method f o r the q u a l i t a t i v e analysis and i s o l a t i o n of mono- and oligosaccharides i n polysaccharide hydrolysates. Excellent separations may be obtained by a judicio u s .choice of solvent system. In t h i s study, the following solvent systems were used f o r the purposes given. Solvent A: Ethyl acetate - ac e t i c acid - formic a c i d - water (18 : 3 : 1 : 4, This solvent system gives r e l a t i v e l y r a pid development of a c i d i c and neutral mono- and oligosaccharides. The r e s o l u t i o n obtained i s not s u f f i c i e n t for the separation of some sugars. Solvent B: 1-Butanol - a c e t i c acid - water ( 2:1 : 1). This solvent system i s used to develop a c i d i c and neutral mono- and oligosaccharides. 11 Development i s slower than with Solvent A, but the r e s o l u t i o n of oligosaccharides i s increased. This system i s used for preparative separations of oligosaccharides. Solvent C: Ethyl acetate - p y r i d i n e - water ( 8 : 2 : 1 ) . This solvent system gives excellent r e s o l u t i o n of neutral monosaccharides; glucose, galactose and mannose can be separated and i d e n t i f i e d with t h i s system. A c i d i c sugars remain e s s e n t i a l l y at the o r i g i n . Solvent D: 1-Butanol - ethanol - water ( 4 : 1 : 5 ) (upper phase). On mixing the above solvents, two phases appear and the upper phase i s used for paper chromatography. This solvent system gives excellent separation of p a r t i a l l y methylated sugars. This method complements gas l i q u i d chromatography i n the i d e n t i f i c a t i o n of the p a r t i a l l y methylated sugars obtained during methylation analyses of polysaccharides. A number of methods are a v a i l a b l e for v i s u a l i s i n g the chromato-grams. The method i s chosen according to the circumstances.*^ The following methods were used i n t h i s study. Method A: The chromatogram i s successively dipped i n solutions of s i l v e r n i t r a t e , sodium hydroxide and sodium t h i o s u l f a t e . This method i s very s e n s i t i v e and i s u s u a l l y applied to reducing sugars. Method B: p-Anisidine hydrochloride spray.' Although t h i s method i s less s e n s i t i v e than Method A, the colours of the spots obtained 17 may give useful information. Dutton and M e r r i f i e l d used p-anisidine hydrochloride to v i s u a l i s e a s e r i e s of acylated oligosaccharides obtained from a polysaccharide hydrolysate. They showed that the colours varied dramatically according to the presence or absence of acetate and formate. This method i s also used to v i s u a l i s e p a r t i a l l y 12 methylated sugars. Again the colours obtained are s i g n i f i c a n t and imply structural features. This method i s usually applied only to reducing sugars but i t may v i s u a l i s e such sugars as sucrose that have very l a b i l e glycosidic bonds. The chromatographic mobilities of sugars are usually reported as R values, that i s the mobility r e l a t i v e to an appropriate standard. R values have been published for a number of mono= and oligosaccharides i n a variety of solvent systems."^ R values have also been published 18 for a large number of p a r t i a l l y methylated sugars. II.2 Gel Chromatography Gel chromatography (also known as gel permeation chromatography, exclusion chromatography, or molecular sieve chromatography) separates molecules according to t h e i r molecular size. The t y p i c a l r e sin i s a cross-linked polymer with a p a r t i c u l a r range of pore sizes. During the chromatographic experiment, molecules that are larger than the pores pass d i r e c t l y through the column without entering the re s i n . Smaller molecules are retained to a degree dependent on th e i r a b i l i t y to enter the porous resin . I f an appropriate resin i s chosen for a 19 p a r t i c u l a r mixture, excellent separations may be obtained. Gel chromatography has been used extensively for analyt i c a l and preparative separations of carbohydrates. In polysaccharide studies i t has been used to i s o l a t e oligosaccharides from p a r t i a l l y hydrolysed polymers. The resins chosen were designed for elution with aqueous solvents. During a p a r t i a l hydrolysis study of K l e b s i e l l a K53, Paulin was able to separate d i - , t r i - , t e t r a - and pentasaccharides using the 13 r e s i n Bio-gel P-2. Z U During a phage degradation study of K l e b s i e l l a K60, Di Fabio separated saccharides containing 7, 14 and 21 sugars 14 using Bio-Gel P-4. Gel chromatography may also be used to determine the approximate molecular weight of a polymer. Molecular si z e and shape determine the degree of retention on the column. Thus i f the molecular weight of a polysaccharide i s to be measured, the column must be c a l i b r a t e d with polymers of s i m i l a r composition and st r u c t u r e . Gel chromatography gives polysaccharide molecular weights that agree to within 5-10% o f 19 values obtained by other methods. Churms and Stephen used t h i s method to determine molecular weight d i s t r i b u t i o n during p a r t i a l hydrolysis studies of K l e b s i e l l a polysaccharides.^ Sephadex LH-20 i s a l i p o p h i l i c r e s i n that was designed f o r el u t i o n with organic solvents. In t h i s study permethylated polysaccha-rides were p u r i f i e d on LH-20 during methylation analyses. II.3 Gas Liquid Chromatography During s t r u c t u r a l studies of poly- and oligosaccharides, i t i s necessary to separate, i d e n t i f y and quantify the sugars obtained a f t e r t o t a l hydrolysis. It i s also necessary to perform s i m i l a r analyses of the p a r t i a l l y methylated sugars obtained from hydrolysates of permethylated polymers and oligomers. Gas l i q u i d chromatography (g.l.c.) i s an e f f e c t i v e and popular method for accomplishing these obj ectives. Because of t h e i r low v o l a t i l i t i e s carbohydrates must be d e r i v a t i s e d before they can be analysed by g . l . c . Sweeleyand 14 coworkers^* made a s i g n i f i c a n t contribution to t h i s work when i n 1963 they reported the successful g.l.c. analysis of carbohydrates as t h e i r t r i m e t h y l s i l y l (TMS) ethers. TMS d e r i v a t i v e s are prepared r a p i d l y at room temperature i n a s o l u t i o n containing p y r i d i n e , hexa-methyldisilazane and t r i m e t h y l c h l o r o s i l a n e . They applied t h i s method to a number o f monosaccharides and oligosaccharides, up to a t e t r a -saccharide. This method has been used extensively to analyse mixtures 22 of sugars derived 'from b i o l o g i c a l material. Other c y c l i c d e r i vatives of monosaccharides include acetates and t r i f l u o r o a c e t a t e s . Methyl glycosides have been analysed as acetates, t r i f l u o r o a c e t a t e s and methyl ethers. Chromatograms of c y c l i c d e r i v a t i v e s of hexoses and pentoses are complicated by the occurence of a and g anomers and pyranose and furanose r i n g forms. This problem has been remedied by the introduction of a c y c l i c derivatives that give only one peak on a chromatogram. The removal of the anomeric centre i s usually achieved by reduction to the a l d i t o l with sodium borohydride. A l d i t o l s have been analysed as TMS ethers but acetates give better r e s o l u t i o n i f an appropriate l i q u i d phase i s chosen. A l d i t o l s are r e a d i l y acetylated by heating at 100° for 20 minutes i n 1 : 1 p y r i d i n e — 23 a c e t i c anhydride. Anomeric centres may also be removed by conversion of aldoses 24 to a l d o n o n i t r i l e s . Lance and Jones prepared peracetylated aldono-n i t r i l e s (PAAN s) of sugars, v i a t h e i r oximes, by t r e a t i n g them with hydroxylamine hydrochloride i n pyridine at 90° f o r one hour, followed by addition of a c e t i c anhydride and heating for another hour. Recently 25 Chen and McGinnis reported that PAAN derivatives could be prepared 15 i n 10 minutes using 1-methylimidazole as a solvent c a t a l y s t instead o f p y r i d i n e . They also reported excellent g . l . c . separations of common sugars as PAANs. Because PAANs are quickly and simply prepared, and are r e a d i l y separated, i t had appeared that they could replace a l d i t o l acetates as the d e r i v a t i v e s of choice for the analysis of polysaccharide hydrolysates. 73 A recent study, however, has shown that when the d e r i v a t i s a t i o n procedure of Lance and Jones^^ i s used, a s i g n i f i c a n t amount of a c y c l i c d e r i v a t i v e i s produced. For glucose, the actual y i e l d of the PAAN d e r i v a t i v e i s only 70%. No s i m i l a r side-reactions have yet been 25 reported f o r the method of Chen and McGinnis, but a thorough scrutiny o f t h i s method i s required. P a r t i a l l y methylated sugars have been analysed as acetates and 26 TMS ethers, and as the acetates and TMS ethers of methyl glycosides. Again chromatograms are s i m p l i f i e d by using methods that remove the anomeric centre. A l d o n o n i t r i l e acetates give one g.l.c. peak, are 27 r e a d i l y separated, and have int e r p r e t a b l e mass spectra. A l d i t o l acetates, however, have gained the widest popularity. P a r t i a l l y methylated a l d i t o l acetates (PMAAs) give one peak on the chromatogram and show s u f f i c i e n t separation i n most cases. They have been studied extensively by g.l.c./mass spectrometry, and an exhaustive summary of 28 r e l a t i v e retention times and standard mass spectra has been published. Recently a computer program has been developed for the i d e n t i f i c a t i o n 29 o f PMAAs using data obtained by g.l.c./mass spectrometry. Several l i q u i d phases were used during the present s t r u c t u r a l study. SP-2340 was used f o r the separation o f a l d i t o l acetates. Both 16 PMAAs and PAANs were separated on OV-225. HIEFF-1B (diethylene g l y c o l succinate) was used to separate PMAAs. 1,2,5-Tri-0-acetyl-3,4,6-tri-O-methylmannitol and 1,3,5-tri-0-acetyl-2,4,6-tri-0-methylglucitol are not separated on OV-225 or ECNSS-M, but on HIEFF-1B they are separated to a degree s u f f i c i e n t f o r i d e n t i f i c a t i o n by g.I.e./mass spectrometry. II.4 High-Performance Liquid Chromatography (h.p.l.c.) This a n a l y t i c a l technique was not used i n t h i s s t r u c t u r a l study but a discussion i s included here because of i t s p o t e n t i a l i n more advanced approaches to carbohydrate analysis. Several columns are a v a i l a b l e f o r the separation o f underivatised mono- and oligosaccharides. H.p.l.c. has been recommended as a method for routine carbohydrate analysis because one i s able to avoid the d e r i v a t i s a t i o n step required by g.l.c. In polysaccharide,studies h.p.l.c. has been used most c r e a t i v e l y 31 to separate alkylated carbohydrates. Lindberg and coworkers used h.p.l.c. to i s o l a t e a f u l l y methylated oligosaccharide during a uronic 32 33 acid degradation study. Albersheim and coworkers ' have developed a method for the analysis of the p a r t i a l hydrolysis products of permethylated polysaccharides. A f t e r the hydrolysis step, the mixture of p a r t i a l l y methylated oligosaccharides i s reduced and ethylated, thus d e r i v a t i s i n g a l l f r e e hydroxyl groups. The hydrolysate i s then analysed by reversed-phase h.p.l.c. with a d i f f e r e n t i a l refractometer as a detector. A n a l y t i c a l separations have been reported with 20 yg of s t a r t i n g material, and preparative separations with 10 mg. A recent paper reports the use of h.p.1.c./mass spectrometry i n 17 the analysis of derivatised oligosaccharides. 11.5 Ion-exchange Chromatography DEAE-Sephadex, a weakly basic anion exchanger, may be used to 34 33 purify polysaccharides or to check t h e i r purity. An automated ion-exchange system i s available for the analysis of neutral mono-35 saccharides. During p a r t i a l hydrolysis studies of polysaccharides, the separation procedure i s s i m p l i f i e d by an i n i t i a l separation of neutral and acidic sugars. A suitable resin for this purpose i s Bio-Rad AG 1-X2 (formate form), which has quarternary ammonium active s i t e s . After application to the column, neutrals are eluted with d i s t i l l e d 20 water. Acidics are subsequently eluted with 10% formic acid. 11.6 S i l i c a Gel Chromatography Underivatised sugars can be separated by s i l i c a gel t h i n layer 36 chromatography. In polysaccharide structural studies, s i l i c a gel chromatography (thin layer and column) i s most useful i n the i s o l a t i o n 37 of derivatised oligosaccharides. Mackie used s i l i c a gel column chromatography to separate two tetrasaccharides, as the permethylated a l d i t o l s , that could not be separated by gel permeation chromatography. 38 Choy used thin layer chromatography to i s o l a t e p a r t i a l l y methylated oligosaccharides from a permethylated polysaccharide hydrolysate. 39 Van Halbeck and coworkers used short s i l i c a gel columns to purify permethylated oligosaccharide a l d i t o l s for mass spectrometry. The 28 Hakomori methylation i s the standard method by which oligosaccharides are derivatised for mass spectrometry. The present study showed that 1 8 o l i g o s a c c h a r i d e s methylated by t h i s procedure should be p u r i f i e d by t h i n l a y e r o r column chromatography before mass spectrometric a n a l y s i s . II.7 Mass Spectrometry Mass spectrometry (m.s.) i s one of the most powerful t o o l s a v a i l a b l e to the s t r u c t u r a l carbohydrate chemist, e s p e c i a l l y when i t i s combined with g . l . c . Because carbohydrates have low v o l a t i l i e s and low thermal s t a b i l i t i e s , mass s p e c t r a l analyses are u s u a l l y performed with v o l a t i l e d e r i v a t i v e s . When i o n i z e d by e l e c t r o n impact ( e . i . ) the molecular ions o f carbohydrate d e r i v a t i v e s are weak or non-existent. F i e l d i o n i z a t i o n , f i e l d d e s o r p t i o n and chemical i o n i z a t i o n are more l i k e l y to give the molecular i o n , but these techniques have the disadvantage o f being "non-routine" i n most l a b o r a t o r i e s . During polysaccharide s t u d i e s , mass s p e c t r a l analyses are u s u a l l y performed on p a r t i a l l y methylated a l d i t o l acetates and permethylated o l i g o s a c c h a r i d e s . The s a l i e n t features o f t h e i r mass spe c t r a w i l l now be discussed. a) P a r t i a l l y methylated a l d i t o l acetates (PMAAs): The mass spe c t r a o f various monosaccharide d e r i v a t i v e s have been studied. These d e r i v a t i v e s i n c l u d e permethylated g l y c o s i d e s , a l d i t o l acetates and a l d i t o l t r i f l u o r o a c e t a t e s . PMAAs are the products of the methylation a n a l y s i s o f p o l y - and o l i g o s a c c h a r i d e s (see Secti o n I I I . 3 ) . G.l.c./m.s. o f PMAAs has become a r o u t i n e method i n s t r u c t u r a l carbohydrate c h e m i s t r y . 4 ^ j 4 1 When a PMAA i s i o n i z e d by e l e c t r o n impact, the molecular i o n 19 i s not observed. The degree of methylation is implied by the g.l.c. retention time and the substitution pattern i s deduced from the mass spectrum. In this case stereoisomers are not differentiated by m.s. Figure 2 illustrates how m.s. can be used to distinguish a 1,3,5-tri-0-acetyl-2,4,6-tri-0-methylhexitol from a 1,2,5-tri-0-acetyl-3,4,6-tri-O-methylhexitol. Note that in the f i r s t case m/e 117 is very 28 strong, but in the second case i t is not observed. Primary fragments are formed by fission between carbon atoms in the a l d i t o l chain. The following scheme demonstrates the relative importance of various modes of fission along the alditol chain. (a) , Cb) .+ t + .+ I • + HC — OMe HC = OMe HC — OMe HC = OMe HC — OMe HC — OMe HC — OAc HC — OAc I I I I (c) | | Cd) | | HC — OMe HC — OMe HC — OAc HC — OAc HC — OAc + .+ + HC = OAc HC — OAc HC = OAc (a) i s favoured over (b); (a) and (b) are favoured over Cc) and (d). Secondary fragments are formed from the primary fragments by the elimination of such compounds as formaldehyde, methanol, ketene and ., 40 acetic acid. 20 1 , 2 , 5 - t r i - O - a c e t y l - 2 , 4 , 6 - t r i - O - m e t h y l h e x i t o l 100 50 -H I. Jill 4™I m/ e 50 100 150 200 250 1 , 2 , 5 - t r i - O - a c e t y 1 - 3 , 4 , 6 - t r i - O - m e t h y l h e x i t o l 1001 50 J 50 100 1 t ' 150 200 T " 250 m/e F i g u r e 2 . Mass s p e c t r o m e t r y o f p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s 21 (b) Permethylated oligosaccharides: Oligosaccharides have been studied extensively, both as permethylated methyl glycosides and as permethylated- a l d i t o l s , and i t has been demonstrated that mass spectrometry can be used to determine 42 the sugar sequence and linkage p o s i t i o n s . Kochetkov and Chizhov, 43 78 followed by Kovacik and coworkers, ' developed a nomenclature f o r 44 the fragment ions of permethylated glycosides. Kovacik and coworkers studied an a l d o t r i o u r o n i c acid by m.s., and t h e i r conclusions were used to in t e r p r e t the mass spectrum of an oligosaccharide i s o l a t e d from K l e b s i e l l a K39. Figure 3 i l l u s t r a t e s some of t h e i r more important r e s u l t s , s p e c i f i c a l l y the o r i g i n s o f the A and J s e r i e s of fragments. These fragments provide information about the sequence o f sugars i n the oligosaccharide. 44 45 Karkkainen ' studied 21 tr i s a c c h a r i d e s by g.l.c./m.s., as the permethylated methyl glycosides and as the permethylated a l d i t o l s . His papers give retention times and d e t a i l e d analyses of the mass spectra. He showed that the linkage p o s i t i o n could be determined by mass spectrometry. Two recent papers demonstrate the use o f mass spectrometry i n the structure determination o f n a t u r a l l y occurring oligosaccharides. 39 During a study of hog submaxillary glycoproteins, Van Halbeek and coworkers i s o l a t e d a serie s of oligosaccharides, up to a pentasaccharide, containing N-acetyl-galactosamine, galactose, fucose, and N-glycoloyl-neuraminic acid. These oligosaccharides were analysed by m.s. as the permethylated oligosaccharide a l d i t o l s , and the spectra obtained were 46 interpreted i n d e t a i l . In another recent paper, Aman and coworkers 22 Figure 3. Mass spectral study of a permethylated a l d o t r i o u r o n i c acid 23 used h.p.1.c./m.s. with chemical i o n i z a t i o n to analyse f u l l y a lkylated oligosaccharides obtained during a p a r t i a l hydrolysis study of a permethylated polysaccharide. II.8 Nuclear Magnetic Resonance (n.m.r.) 13 1 C and H nuclear magnetic resonance have been used extensively and e f f e c t i v e l y i n the structure determination of poly- and o l i g o -saccharides. The fact that K l e b s i e l l a capsular polysaccharides give i n t e r p r e t a b l e n.m.r. spectra i s evidence that they have regular repeating sequences of sugars."' N.m.r. spectroscopy i s e s p e c i a l l y useful i n determining the number of sugars i n t h i s repeating unit and i n deter-mining the anomeric configuration of g l y c o s i d i c linkages. a) "*"H Nuclear magnetic resonance. Derivatised poly- and oligosaccharides have been analysed i n 47 non-aqueous solvents, but i n polysaccharide studies good spectra have been obtained using deuterium oxide, without chemical d e r i v a t i -s a tion.^ Carbohydrates to be studied are repeatedly dissolved i n deuterium oxide and freeze-dried to exchange a l l hydroxylic and a c i d i c protons f o r deuterons. A r e s i d u a l HOD signal i s always observed however, and i t may mask some important anomeric proton s i g n a l s . The HOD s i g n a l may be s h i f t e d u p f i e l d out of the anomeric region by recording the spectrum at high temperature (90-95°C). When-poly-saccharide spectra are run at high temperature the v i s c o s i t y i s reduced and the r e s o l u t i o n i s increased due to the corresponding increase i n T 0. The v i s c o s i t y o f polysaccharides may also be reduced by a very 24 mild acid h y d r o l y s i s , but t h i s method must be used with caution because a c i d - l a b i l e substituents may be removed. The major features- of poly- and oligosaccharide *H-n.m.r. spectra w i l l now be discussed, beginning with those signals that appear at high f i e l d . i ) 6-Deoxyaldohexoses (e.g., Rha and Fuc) give methyl proton signals at approximately 61.3 with = 6 Hz (doublet). I f a 6-deoxyaldohexose i s the reducing sugar of an oligosaccharide, the methyl signal i s s p l i t according to the anomeric r a t i o . i i ) Non-carbohydrate substituents containing methyl groups give d i s t i n c t i v e signals at r e l a t i v e l y high f i e l d . Pyruvate acetals give a methyl signal at 61.6 to 62.1, with the chemical s h i f t being 48 dependent of the stereochemistry. Acetyl groups give a methyl signal at approximately 6 2.2. i i i ) Ring protons and H-6 of aldohexoses give signals that appear i n 49 5( the region 63.0 to 64.5. These signals may be completely assigned, ' but for poly- and oligosaccharides t h i s area of the spectrum i s often too complex f o r complete r a t i o n a l i s a t i o n . Some r i n g proton resonances occur s i g n i f i c a n t l y downfield Of the others. H-2 of a 2-linked mannose may appear as f a r downfield as 64.2, and H-5 of galacturonic acid may 14 appear as f a r downfield as 64.85. iv) The anomeric protons resonate i n the region 64.5 to 55.5. These 25 signals provide information about the anomeric configuration of g l y c o s i d i c linkages, the number of sugars i n the repeating unit of a polysaccharide, and the degree of polymerisation o f an oligosaccharide. The signals i n t h i s region have been e m p i r i c a l l y divided at 65.0: g s i g n a l s , i n general, appear u p f i e l d of t h i s point and a signals down-f i e l d . This d i v i s i o n , however, i s not absolute because H-l of 53 S-galactofuranose has been observed at 65.13. In oligosaccharides the reducing end anomeric protons are u s u a l l y d i s t i n g u i s h a b l e because they integrate to less than one proton, although reducing mannose may ex i s t almost completely i n the a form. The H-l- --H-2 coupling constants (J „) must be taken into account when assigning anomeric signals. According to K a r p l u s , ^ the magnitude o f the coupling constant between two hydrogens on adjacent carbons i s dependent on t h e i r dihedral angle 0: J passes through i > ^  maxima at 6 = 0° and 180° and a minimum at 0 = 90°. An example o f the usefulness o f t h i s r e l a t i o n s h i p i s i l l u s t r a t e d i n Figure 4. v) A formate substituent has been i d e n t i f i e d by a signal at 65.9.^' 13 b) C-Nuclear magnetic resonance. 13 Because of the low natural abundabce o f C, and the low 13 s o l u b i l i t i e s o f polysaccharides, C-n.m.r. studies o f polysaccharides have been aided by the introduction o f pulsed Fourier transform techniques. Polysaccharide samples are dissolved i n deuterium oxide for locking purposes, and spectra may be recorded at elevated temperature to reduce the v i s c o s i t y . Spectra are s i m p l i f i e d through 26 g-D-Glucopyranose OH 6~60° J ~1 Hz g-D-Mannopyrano s e Figure 4. Comparison of J for g-D-Glucopyranose and g-D-Mannopyranose. 27 the a p p l i c a t i o n of broad band proton decoupling. This technique also enhances the signal i n t e n s i t y v i a the nuclear Overhauser e f f e c t (n.O.e.). The n.O.e. i s not uniform over a l l carbons i n the sample, and in t e g r a t i o n i s therefore less useful than i n *H-n.m.r. spectroscopy. The spectra obtained under these conditions show better r e s o l u t i o n than *H-n.m.r. spectra. 13 The major features of C-n.m.r. spectra of poly- and o l i g o -saccharides w i l l now be discussed, beginning with those signals that appear at r e l a t i v e l y high f i e l d . i ) The methyl carbon signals of 6-deoxyhexosyl residues appear at approximately 617. i i ) Methyl carbons of acetate and pyruvate substituents resonate 53 at approximately 630 and 620 re s p e c t i v e l y . The chemical s h i f t of 48 pyruvate i s dependent on i t s stereochemistry. i i i ) Primary alcohol carbons appear at approximately 661 unless they are linkage p o s i t i o n s , i n which case they are s h i f t e d downfield 7-10 ppm. v) Ring carbons, aside from anomeric carbons, appear i n the region 665 to 680. Linkage p o s i t i o n r i n g carbons appear at the lower end o f t h i s region 675 to 680. For oligosaccharides the reducing sugar's linkage p o s i t i o n s i g n a l may be s p l i t i n t o the anomeric r a t i o . 28 v) Anomeric carbons resonate at 693 to 6110 ppm. This region can be e m p i r i c a l l y divided at 6101, with 0 signals appearing downfield o f 6101 and a signals'' u p f i e l d . The reducing end anomeric carbons of oligosaccharides resonate at 693 to 697. II.9 Polarimetry and C i r c u l a r Dichroism The s p e c i f i c r o t a t i o n of a sugar i s dependent on i t s absolute configuration (D or L) and i t s anomeric configuration (a or £5) . The s p e c i f i c r o t a t i o n o f a compound i s given by the following equation: r - i a x 100 [a] = — , £ x c where a i s the measured r o t a t i o n , I i s the length of the sample c e l l i n decimetres, c i s the concentration of the s o l u t i o n i n g/100 ml, and A i s the wavelength of the plane p o l a r i s e d l i g h t . The wavelength that i s u s u a l l y used i s the sodium D l i n e (589 nm). Hudson's Rules of 14 69 i s o r o t a t i o n ' can be used to p r e d i c t the s p e c i f i c rotations of 53 71 poly- and oligosaccharides. Savage and M e r r i f i e l d have tabulated the predicted and measured values for the K l e b s i e l l a polysaccharides. C i r c u l a r dichroism spectroscopy of a l d i t o l acetates, p a r t i a l l y methylated a l d i t o l acetates, and peracetylated a l d o n o n i t r i l e s can be used to determine the absolute configuration of sugars.^>70 POLYSACCHARIDE STRUCTURAL METHODS 30 II I . POLYSACCHARIDE STRUCTURAL METHODS 111.1 I s o l a t i o n and P u r i f i c a t i o n The methods used by our research group for i s o l a t i n g b a c t e r i a l 14 53 capsular polysaccharides have been discussed i n other theses. ' Cultures are grown on agar plates and s i n g l e colonies are then incubated i n a beef-extract broth at 37°C. After several hours the broth becomes tur b i d and i t i s then spread on agar trays containing a high sucrose content. A f t e r several days the b a c t e r i a l slime i s scraped o f f of the agar and centrifuged. The supernatant i s then p r e c i p i t a t e d into ethanol, methanol or acetone. The p r e c i p i t a t e i s dissolved i n water and a c i d i c polysaccharides are then p r e c i p i t a t e d with CETAVLON (cetyltrimethylammonium bromide). This p r e c i p i t a t e i s dissolved i n 2M sodium chloride and p r e c i p i t a t e d into ethanol or methanol. The polysaccharide p r e c i p i t a t e i s then dialysed against running water and freeze-dried. 111.2 Sugar Analysis During the s t r u c t u r a l analysis of a polysaccharide, the i d e n t i o f the sugars present and t h e i r r e l a t i v e amounts ( t o t a l sugar r a t i o ) must f i r s t be determined. A f t e r acid h y d r o l y s i s , paper chromatograph with Solvent C (see Section I I . 1), i s a simple method of i d e n t i f y i n g the neutral sugars present. The t o t a l sugar r a t i o i s usually determined by g . l . c . analysis of the d e r i v a t i s e d sugars.:. A number of acids have been used f o r the hydrolysis step, including aqueous hydrochloric, s u l f u r i c and t r i f l u o r o a c e t i c (TFA) 31 j i D-GlcA ^  L-Rha ^  D-Gal - { Uronic »cids e s t e r i f i e d . Oligo- and aonosaccharides as nethyl glycosides N«BH4/CHjOH W TFA NaBH Ac^O/Pyridine \~ OH HO H I«2OHHC1/1-Methylii«ida:ole OH > OH L OH - OH HO H K OH HO -4 t" OH HO CHjOH CH. " \ ^ O H HO H OH CH,OH CH,OAc I- OAc AcO H ¥- OAc r- OAc CHjOAc CH2OAc V~ OAc r- OAc AcO AcO -4 CH, CHjOAc L.OAC AcO _J AcO J |_OAc CHjOAc Alditol Acetates AcO" CN | - OAc P" OAc OAC CHjOAc i ACjO/l-Methylinidaiole AcO AcO CN t OAc OAc CH. CN L OAc AcO _ AcO -|- OAc CH,OAc PAANs Figure 5. Total sugar ratio reaction sequence a c i d s . " Hough and c o w o r k e r s " ^ s t u d i e d t h e s e t h r e e a c i d s and showed t h a t TFA and s u l f u r i c a c i d g i v e s i g n i f i c a n t l y l e s s d e g r a d a t i o n o f s u g a r s . An a d v a n t a g e o f TFA o v e r s u l f u r i c a c i d i s i t s v o l a t i l i t y ; i t i s e a s i l y removed f rom t h e h y d r o l y s a t e u n d e r r e d u c e d p r e s s u r e . When s t u d y i n g p o l y s a c c h a r i d e s c o n t a i n i n g u r o n i c a c i d s , i t i s d i f f i c u l t t o o b t a i n an a c c u r a t e t o t a l s u g a r r a t i o due t o t h e r e s i s t a n c e o f u r o n o s y l bonds t o a c i d h y d r o l y s i s . I n t h i s s t u d y o f K l e b s i e l l a K54, C o n r a d and coworkers"*"* showed t h a t t h e g l u c u r o n o s y l bond i s h y d r o l y s e d much more s l o w l y t h a n t h e g l y c o s i d i c bonds o f n e u t r a l s u g a r s . To overcome t h i s p r o b l e m , t h e c a r b o x y l g roups o f u r o n i c a c i d s a r e u s u a l l y r e d u c e d t o p r i m a r y a l c o h o l s . A d i s a d v a n t a g e o f s u c h a r e d u c t i o n i s t h a t g l u c u r o n i c a c i d , f o r e x a m p l e , i s no l o n g e r d i s t i n g u i s h a b l e f rom g l u c o s e . T h i s p r o b l e m c a n be r e m e d i e d by u s i n g a d e u t e r a t i n g r e d u c i n g agen t ( e . g . sod ium b o r o d e u t e r i d e ) and a n a l y s i n g t h e h y d r o l y s a t e by g . l . c . / m . s . S e v e r a l methods have been d e v e l o p e d f o r t h e c a r b o x y l r e d u c t i o n o f u r o n i c a c i d s . The p o l y s a c c h a r i d e may be t r e a t e d w i t h p r o p i o n i c a n h y d r i d e i n p y r i d i n e t o c o n v e r t a l l o f t h e h y d r o x y l g roups t o p r o p i o n a t e e s t e r s . The u r o n i c a c i d c a r b o x y l g roups a r e t h e n e s t e r i f i e d w i t h 56 d i a z o m e t h a n e and r e d u c e d w i t h l i t h i u m b o r o h y d r i d e . I n a n o t h e r i m p o r t a n t m e t h o d , u r o n i c a c i d s a r e e s t e r i f i e d w i t h a c a r b o d i i m i d e and 57 r e d u c e d w i t h sod ium b o r o h y d r i d e . I n t h e p r e s e n t s t u d y , p o l y - and o l i g o s a c c h a r i d e s were f i r s t m e t h a n o l y s e d i n r e f l u x i n g 3% m e t h a n o l i c h y d r o g e n c h l o r i d e . A f t e r s u c h a t r e a t m e n t , a l l c a r b o x y l g roups a r e c o n v e r t e d t o m e t h y l e s t e r s and t h e p r o d u c t i s a m i x t u r e o f o l i g o - and m o n o s a c c h a r i d e s as m e t h y l 33 glycosides. The esters are then reduced by treatment with sodium borohydride i n anhydrous methanol. A f t e r carboxyl reduction the polysaccharide can be completely depolymerised by hydrolysis with 2M TFA at 95°C for 8 h o u r s . 3 7 The derivatives a v a i l a b l e f o r g . l . c . analysis have been discussed i n Section II.3. A l d i t o l acetates and peracetylated aldono-n i t r i l e s (PAANs) are the derivatives of choice. The problems associated with PAANs are discussed i n Section II.3. Figure 5 i l l u s t r a t e s the reaction sequence for the t o t a l sugar r a t i o of a hypothetical poly-saccharide. III.3 Methylation Analysis The methylation analysis provides more information than any other s i n g l e method used i n the s t r u c t u r a l analysis of polysaccharides. The steps i n the analysis are: the methylation of a l l hydroxyl groups, acid hydrolysis, and the analysis of the p a r t i a l l y methylated sugars released. In the case of a c i d i c polysaccharides, carboxyl reduction may precede the acid hydrolysis; otherwise depolymerisation may be incomplete due to the resistance o f uronosyl linkages to acid hydrolysis.' The type.^of information provided by the methylation of a hypothetical polysaccharide i s i l l u s t r a t e d i n Figure 6. Before the introduction of the Hakomori method i n 1964, several procedures were used to methylate polysaccharides. The most 58 important methods w i l l now be discussed. In 1903 Purdie and Irvine reported that sucrose could be methylated using methyl iodide as an a l k y l a t i n g agent and s i l v e r oxide as a c a t a l y s t . This technique has 34 — D-GlcA D-Gal — L-Rha 1 D-Glc C02H CH2OH > — 0 - HQ H 3CSCH 2 /DMSO CH 3I LiAlH^/THF 2M TFA CH2OH CH2OMe MeO, H > 0 H \0H / H ' 0 H H,OH OMe OMe MeO OMe CH2OMe MeO OMe 7 H,OH OMe 35 NaBH. Ac^O/Pyridine CH2OAc OMe MeO OAc I— OAc CH2OAc 2,3-Glc CH2OAc OAc AcO ~f MeO OAc CH2OMe 4,6-Gal CH2OAc OMe ACO AcO OMe CH. 2,3-Rha CH2OAc h- OMe MeO" OMe OAc CH 2OCH 3 2,3,4,6-Glc V G.1.c./m.s. Figure 6. Methylation analysis of a hypothetical polysaccharide. 36 often been used to complement other procedures that have achieved only 59 p a r t i a l methylation. In 1915 Haworth reported that carbohydrates could be methylated using dimethyl sulphate i n aqueous sodium hydroxide. In 1955 Kuhn showed that the Purdie method could be improved as a method for methylatihg polysaccharides by using dimethyl formamide as - 6 0 a solvent. Today the most important methylation method f o r poly- and oligosaccharides i s that reported by Hakomori i n 1964.^ ''" He showed that g l y c o l i p i d s and polysaccharides could be completely methylated using methyl iodide as an a l k y l a t i n g agent, the methylsulfinyl carbanion as a base and dimethyl sulfoxide as a solvent. The reaction takes place i n two steps: 1. R — OH + CH 3 — SO — CH 2" Na + > R — 0~ Na + + CH 3 — SO — 2. R — 0~ Na + + CH 3I — • R — 0 — CH 3 + Nal Most polysaccharides should be completely methylated i n one treatment. Completeness of methylation i n indicated by the absence of i n f r a r e d absorption at 3600 cm *. Unfortunately t h i s method does not lend i t s e l f to successive treatments of polysaccharides containing uronic acids. On e s t e r i f i c a t i o n , H-5 of a hexuronic acid becomes a c i d i c and the polysaccharide may be degraded i n the presence of m e t h y l s u l f i n y l 64 carbanion, e s p e c i a l l y i f the uronic acid i s linked at C-4. In the present study, the uronic esters of permethylated poly-and oligosaccharides were reduced with l i t h i u m aluminum hydride i n tetrahydrofuran. Hydrolysis was effected i n 2M TFA on a steam bath. 37 P a r t i a l l y methylated sugars obtained by hydrolysis of permethylated polysaccharides have been separated by column chromatography ( c e l l u l o s e , 3 s i l i c a g e l , charcoal) and by paper chromatography. Today the most important method for quantitative and q u a l i t a t i v e analysis i s g.l.c./m.s. of the p a r t i a l l y methylated a l d i t o l acetate d e r i v a t i v e s . Lindberg and coworkers have published a booklet giving the d e t a i l s of the methylation procedure. This p u b l i c a t i o n includes standard g.l.c. retention times 28 and standard mass spectra f o r p a r t i a l l y methylated a l d i t o l acetates. III.4 P a r t i a l Hydrolysis During the s t r u c t u r a l analysis of a polysaccharide, i t i s useful to i s o l a t e oligosaccharides from the polymer by p a r t i a l h y d r o l y s i s . Data obtained from oligosaccharides (e.g., n.m.r., methylation analyses) can be used to help interpret the corresponding data obtained from the polysaccharide. The sugar sequence of a polysaccharide can be determined by the analysis of a series of oligosaccharides obtained by p a r t i a l hydrolysis. Wolfrom and coworkers determined the f i r s t - o r d e r rate constants f o r eight glucose disaccharides. They showed that the rate of hydrolysis i s dependent on both the linkage p o s i t i o n and the anomeric 6 3 configuration. More important, however, i n the study of b a c t e r i a l polysaccharides, i s the resistance of uronosyl bonds to acid h y d r o l y s i s . Therefore the hydrolysis of a polysaccharide i s not a completely random process, and i f proper conditions are chosen c e r t a i n o l i g o -saccharides w i l l accumulate i n the hydrolysate. It i s almost always possible to i s o l a t e an aldobiouronic acid from an a c i d i c polysaccharide, 38 but the acid concentration, temperature and reaction time should be chosen to give the maximum y i e l d of higher oligomers. Oligomers up to pentasaccharides have been i s o l a t e d from K l e b s i e l l a p o l y s a c c h a r i d e s . ^ ^ ' ^ Af t e r the hydrolysis step, the i s o l a t i o n of oligosaccharides presents an i n t e r e s t i n g separation problem. Neutral and a c i d i c components can be separated by ion exchange chromatography. The present study employed Bio-Rad AG 1X-2, a r e s i n with quarternary ' ammonium active s i t e s . With the r e s i n i n the formate form, neutral sugars are eluted with water and a c i d i c s with 10% formic acid. Oligo-saccharides may be i s o l a t e d from each f r a c t i o n by gel permeation chromatography,^ paper chromatography,*^ or paper electrophoresis."' 3 S i l i c a gel chromatography may be used to i s o l a t e d e r i v a t i s e d o l i g o -37 saccharides. A f t e r i s o l a t i o n oligosaccharides can be analysed by the methods of sugar a n a l y s i s , methylation analysis, n.m.r. and mass spectrometry. Some workers have studied the products obtained by p a r t i a l h ydrolysis of permethylated polysaccharides. Extra information i s provided by the loc a t i o n of hydroxyl groups obtained on hydrolysis of g l y c o s i d i c linkages. Choy used t h i s method i n his study of K l e b s i e l l a 38 32 46 K56. Albersheim ' and coworkers have developed a method by which a permethylated polysaccharide i s p a r t i a l l y hydrolysed and the oligosaccharide products are i s o l a t e d by h.p.l.c. III.5 Uronic Acid Degradation (8-elimination) Many b a c t e r i a l polysaccharides contain hexopyranosyluronic acid residues. If such a polysaccharide i s permethylated, the methyl uronate-39 residues have a good leaving group at C-4 (either methoxyl or another sugar residue); treatment with the methylsulfinyl anion w i l l cause 3-elimination with i n i t i a l removal of a proton from C-5. Mild a c i d i c hydrolysis r e s u l t s i n complete degradation of the uronate residue. This sequence of reactions has been discussed i n d e t a i l . ^ According 66 to A s p i n a l l the hydrolysis o f the uronate residue w i l l occur on treatment with the methylsulfinyl anion. It i s this.-3-elimination "o reaction that eliminates the p o s s i b i l i t y of successive Hakomori methylations of uronic acid containing polysaccharides. Uronic acids i n the polysaccharide may be located by l a b e l l i n g (with trideuteriomethyl or ethyl groups) the hydroxyl groups l i b e r a t e d by uronic acid degradation. This method may also be used 68 to i s o l a t e a l k y l a t e d oligosaccharides. III.6 Periodate Oxidation and Smith Degradation Periodic a c i d and i t s s a l t s o x i d a t i v e l y cleave 1,2-diols to give two aldehydes, and 1,2,3-triols to give two aldehydes and formic acid. Under proper conditions these reactions are considered to be quantitative. By measuring the amount o f periodate consumed by a polysaccharide, and by determining which sugars have been oxidised, some of the r e s u l t s of the methylation analysis can be confirmed. The Smith degradation o f a polysaccharide (periodate oxidation, sodium borohydride reduction, and c o n t r o l l e d acid hydrolysis) can be used to i s o l a t e glycosides o f mono- and oligosaccharides. Oligo-saccharides i s o l a t e d by t h i s procedure provide information about the 3 sequence o f sugars i n the polysaccharide. 40 III.7 Immunochemical Methods Heidelberger and coworkers have prepared antibodies using, as antigens, the Pneumococcal capsular polysaccharides, many of which have known chemical structures. The degree of r e a c t i o n between an antigen and antibody i s measured by determining the amount of p r e c i p i t a t e produced on mixing. Thus i t i s possible to predict s t r u c t u r a l features of an unknown polysaccharide, having determined i t s r e a c t i v i t y with the Pneumococcal anti-sera. Cross r e a c t i v i t y has been determined • 11 72 f o r many of the K l e b s i e l l a polysaccharides. ' 41 A STRUCTURAL INVESTIGATION OF KLEBSIELLA K39 CAPSULAR POLYSACCHARIDE 42 IV A STRUCTURAL INVESTIGATION OF KLEBSIELLA K39 CAPSULAR POLYSACCHARIDE. ABSTRACT: An a c i d i c capsular polysaccharide was i s o l a t e d from K l e b s i e l l a K39. This polysaccharide was p a r t i a l l y hydrolysed and an unusual pentasaccharide, containing two uronic acid residues, was i s o l a t e d and assigned the following structure. 1 2 1 4 1 2 1 3 GlcA =-=- Man — - GlcA Man Glc~OH 3 a 3 a This pentasaccharide was studied by sugar analysis, methylation 1 13 analysis, H- and C-n.m.r., and by mass spectrometry of the permethylated oligosaccharide a l d i t o l . The r e l a t i o n s h i p of t h i s oligomer to the polysaccharide w i l l be discussed with reference to methylation and n.m.r. studies. RESULTS AND DISCUSSION: Is o l a t i o n and Sugar Analysis The K l e b s i e l l a K39 capsular polysaccharide was i s o l a t e d and p u r i f i e d by the methods described i n Section I I I . l and i n the Experimental section. Molecular weight analysis by gel chromatography (Dr. S.C. Churms, U n i v e r s i t y of Capetown) showed that the product moved as one band with a molecular weight of 560,000. According to 43 the q u a l i t a t i v e analysis of Nimmich (see Appendix 1), t h i s polysaccharide i s composed of glucuronic acid, glucose, mannose, and galactose. A sample of the polysaccharide was methanolysed, carboxyl reduced, and hydrolysed. The hydrolysate was analysed by g . l . c . a f t e r preparation of the PAAN d e r i v a t i v e s , giving Man/Glc/Gal = 1.0/2.2/0.8. The problems associated with PAAN derivatives have been discussed i n Section II.3. Hassell used equimolar mixtures of sugars to prepare 74 molar response f a c t o r s . These response factors were used to correct the data obtained from K39, and therefore the errors associated with PAANs should have been reduced. P a r t i a l Hydrolysis In a preliminary study a sample of K39 polysaccharide (470 mg) was hydrolysed i n 1M TFA f o r 5% hours on a steam bath. A c i d i c s and neutrals were separated by ion exchange chromatography on Bio-Rad AG 1-X2. The a c i d i c f r a c t i o n was analysed by gel chromatography on Bio-Gel P-2. The e l u t i o n p r o f i l e i s i l l u s t r a t e d i n Figure 7. This chromatogram shows that one a c i d i c component i s obtained i n excess o f a l l others. A sample of t h i s compound was analysed by 100 MHz "^ H-n.m.r. i n deuterium oxide at 95°C. The spectrum gave three a anomeric signals at 65.20, 65.26, and 65.47 and a multiplet of 6 signals between 64.59 and 64.73 (see Spectrum 1). A sample of t h i s compound was treated with sodium borodeuteride to convert a l l reducing sugars to a l d i t o l s . This reduced sample was studied by 100 MHz "'"H-n.m.r. at 90°C (Spectrum 2) and by 400 MHz "^H-n.m.r. at ambient temperature Fractions collected (2.5 mis/fraction) 45 (see Spectrum 3 and Table 1). The spectra c l e a r l y show two signals i n the a region (65.27 and 65.37) and two signals i n the 3 region (64.51 and 64.59). This compound, designated A l , i s therefore believed to be a pentasaccharide. •In a further study a sample of K39 polysaccharide (700 mg) was hydrolysed i n 1M TFA f o r 7 hours. Aci d i c s were separated from neutrals by ion exchange chromatography on Bio-Rad AG 1-X2. The a c i d i c f r a c t i o n was chromatographed on paper with Solvent B (see Section II.1) Three bands were extracted from the paper and they were designated A l , A2 and A3. The analysis of these components w i l l now be discussed. Analysis of Al This compound, [ a ] n + H ° (£_ 0.184, water), had a chromatographic mobility on Whatman No. 1 paper of R„ n 0.076 (Solvent B). A sample GI C was hydrolysed i n 3M TFA for 20 hours on a steam bath. The neutral sugars released were analysed by g.l.c. as the a l d i t o l acetate deriva-t i v e s and the chromatogram gave Man/Glc = 1.56/1. Another sample was methanolysed, carboxyl-reduced, and hydrolysed. The products were analysed by g.l.c. as the a l d i t o l acetates giving Glc/Man = 1.47/1. 1 13 H- and C-n.m.r. spectra were recorded and the r e s u l t s are given i n Table 1. *H-n.m.r. confirmed that t h i s compound was the previously i s o l a t e d pentasaccharide. The 400 MHz *H-n.m.r. spectrum, recorded at ambient temperature, showed s i x d i s t i n c t signals i n the anomeric region (see Spectrum 4). Three of these signals (54.57, 64.66, 64.62) are assigned to 6 anomers on the basis of t h e i r chemical 46 Table 1: N.M.R. STUDY OF A l A. 400 MHz H-n.m.r. at ambient temperature; a) A l , b) Al af t e r reduction with NaBD^. a) Chemical S h i f t of Anomeric ^H's(S) J1)2 (Hz) Integral Number of "^ H's Assignment 4.57 4.62 4.66 5.15 5.23 5.47 sin g l e t 4 broad s i n g l e t 1 1 0.55 1 0.45 1 B-GlcA g-GlcA B-Glc~0H a-Man a-Glc~0H a-Man b) 4.51 4.58 5.27 5.37 si n g l e t s i n g l e t 6-GlcA B-GlcA a-Man a-Man 200 MHz 1 3C-n.m.r. of Al Chemical S h i f t 13 Integral 13, of Anomeric C 1 s(6) . Number of C's Assignment 92.98 96.74 100.19 100.92 102.31 102.98 0.42 0.58 1 1 1 1 a-Glc~0H g-Glc-OH a-Man a-Man g-GlcA B-GlcA 47 s h i f t s . These signals have r e l a t i v e l y large coupling constants (8 Hz) and can therefore be assigned to 6-glucopyranosyl or B-glucurono-pyranosyl residues. The signal at 4.62 i s assigned to a reducing sugar on the basis of i t s i n t e g r a l . The signals at 65.15 and 65.47 are assigned to a-mannopyranosyl residues. The signal at 65.23 i s assigned to a reducing sugar and the broadening of the signal at 65.47 indicates that i t i s adjacent to the reducing sugar. 13 The C-n.m.r. spectrum o f A l (see Table 1 and Spectrum 5) shows six signals i n the anomeric region, two of which are assigned to the reducing sugar. Five signals between 678.48 and 683.03 are assigned to the linkage p o s i t i o n carbons. The signals at 683.03 and 680.46 are assigned to the linkage p o s i t i o n carbon of the reducing sugar. A sample of Al was studied according to the methylation analysis procedure described i n Section I I I . A sample of the oligosaccharide was methylated by the Hakomori procedure, and a portion of the product was hydrolysed and analysed by g . l . c . as the a l d i t o l acetate d e r i v a t i v e s . The r e s u l t s are given i n column I of Table 2 and chromatogram "a" of Figure 8. 3,4,6-Man and 2,4,6-Glc were observed. Another portion of the methylated product was treated with l i t h i u m aluminum hydride, hydrolysed and analysed by g . l . c . as the a l d i t o l acetate d e r i v a t i v e s . The r e s u l t s are given i n column II of Table 2 and chromatogram "b" of Figure 8. Two add i t i o n a l peaks (2,3,4-Glc and 2,3-Glc) were observed on the chromatogram, i n d i c a t i n g that Al contains two a c i d i c sugars, one of which i s at the non-reducing terminus. Analysis by g.l;c./m.s. determined the methylation patterns of the PMAAs,3,4,6-Man and 2,4,6-Glc were not completely separated by g . l . c , but the mass spectra on 48 Gas l i q u i d chromatography Column: HIEFF-1B Temperature Program: 165°C f o r 8 min, increase by 2°/min to 200°C. C a r r i e r Gas: N 2, 20 mls/min a) PMAAs obtained without carboxyl reduction 3,4,6-Man 2,4,6-Gle b) PMAAs obtained with carboxyl reduction 3,4,6-Man 2,4,6-Glc 2,3,4-Glc 2,3-10 20 (min) 10 20 (min Mass Spectrum at "A": Mass Spectrum at "B": m/e 0, "0 base peak m/e % base peak 43 97 43 100 45 38 45 31 71 19 71 12 87 38 87 14 99 14 101 20 101 16 117 37 129 100 129 35 161 35 161 16 189 24 Figure 8. Methylation analysis of K39-A1 49 Table 2: METHYLATION ANALYSES OF Al AND A2 P a r t i a l l y Methylated Mole % C cl A l d i t o l Acetates T b Id II III IV 1,3,4,5,6-Man6 0.42 10.9 1,2,4,5,6-Glc'e 0.43 6.0 3,4,6-Man 1.82 i ( 94.0 33.5 > 100 > 55.6-2,4,6-Glc 1.87 ) ) 2,3,4-Glc 2.08 23.3 30.4 . 2,3-Glc 3.06 21.1 25.3 e.g., 3,4,6-Man = 1,2,5-tri-0-acetyl-3,4,6-tri-0-methylmannitol. The PMAAs l i s t e d are consistent with g.l.c. and mass spectral data. b G.l.c. retention time r e l a t i v e to 2,3,4,6-Glc on 3% HIEFF-1B, temperature program: 165°C f or 8 min, increase 2°/min to 200°C. Published molar response factors were used where applicable. No such response factors are a v a i l a b l e for pentamethylaalditol acetates. ^ I, A l methylated and hydrolysed without carboxyl reduction; II, Al methylated, carboxyl reduced and hydrolysed; I I I , Al reduced with NaBD^, methylated and hydrolysed; IV, A2 reduced with NaBD4, methylated, carboxyl reduced and hydrolysed. Monodeuterated at C-1. 50 the leading and t a i l i n g edges of the peak were s u f f i c i e n t l y d i f f e r e n t to i d e n t i f y both PMAAs (see Figure 8 and Figure 2). Another sample of Al was treated with sodium borodeuteride to convert the reducing sugar to the a l d i t o l . The sample was then methylated, hydrolysed, and analysed by g.l.c./m.s. as the a l d i t o l acetates (see column I I I , Table 2). 1,.2,4,5.,6-Glc and 3,4,6-Man were observed. No standard spectra of the acetates of pentamethyl h e x i t o l s are a v a i l a b l e but the structure of 1,2,4,5,6-Glc was confirmed by fragment ions at m/e 90 and m/e 206. These ions are assigned the following structures: ,HC = OMe ' HC = OMe' m/e 90 CHDOMe HCOAc m/e 206 HCOMe I CHDOMe The mass spectrum i s tabulated i n the Experimental section. The y i e l d of 1,2,4,5,6-Glc i s lower than expected. Other workers have attr i b u t e d t h i s phenomenon to losses during work-up due to the 75 v o l a t i l i t y of pentamethyl sugars and t h e i r d e r i v a t i v e s . This experiment confirms that the reducing glucose i s 3-linked. The analyses of Al discussed so f a r have given only p a r t i a l information about the sugar sequence. Mass spectrometry of permethy-lated oligosaccharides i s an established method for sequencing 51 cn cn r-. cn M M . cn cn < en SI St < o CO (Ti 8) SI St 5) 5) co VO _01 < Xi -to-o l * St cn s i SI m « vo 3 > l I I I II I II II St st -CO St Ul St J- u> S i St CM < X) r < T2L CN < CD T 3 St Ul -10 cn < £ ^ «3 ui 4 St SI CM n I I I I I I I I I I VO Ol CM S | Ul Ul Ul cS % -I S s t SI ca -M S | SI t- ui Cl Ul 4 - 3 co <M_ I I I I I I I II S | St S | St i 9 0) It 4 St S | CM St St St St St CD VO •» SI est S| SI ta si si si VO ^ CM S | CO at VO St I CM C0 2CH 3 H3CO \ OCH3 aA (m/e 233) -MeOH aA2(m/e 201) I -MeOH aA3(m/e 169) OCH. C H 2 ° C H 3 | H 3 C 0 \ ? C H 3 baAjCm/e 437) -MeOH baA2(m/e 405) -MeOH baA7(m/e 373) cabA^(m/e 655) -MeOH cabA ?(m/e 623) H3CO \ O C H 3 dabcA 1(m/e 859) -MeOH dabcA ?(m/e 827) CH2OCH3 H3CO-HC=0 CHDOCH, r- OCH, o H H 3C0 \ OCH3 l-OCH, r OCH. CH2OCH3 cdeJ (m/e 500) -HC02CH3 deA1(m/e 440) MeOH deA2(m/e 408) CHDOCH, LOCH. 0 -4 l-OCH, H-OCH C H 2 O C H 3 M (m/e 1111) Figure 10. Mass spectrum of K39 - Al as the permethylated a l d i t o l 53 oligosaccharides. A sample of Al was treated with sodium borodeuteride o to convert the reducing sugar to the a l d i t o l . It was then methylated by the Hakomori procedure. A sample of the product was applied to a s i l i c a gel t h i n - l a y e r plate and eluted with ethyl acetate. The permethylated oligomer had R^ . 0.36 but a s i g n i f i c a n t amount of material remained at the o r i g i n . The product was i s o l a t e d by preparative t . l . c . and gave an i n f r a r e d spectrum that confirmed i t s i d e n t i t y as a permethylated oligosaccharide a l d i t o l . In p a r t i c u l a r absorption maxima -1 -1 were observed at 1750 cm (ester carbonyl stretch) and at 1105 cm , 1080 cm * (ether s t r e t c h ) . This compound was analysed by electron impact mass spectrometry with an electron beam energy of 20 eV. The mass spectrum i s tabulated i n Figure 9 and the i n t e r p r e t a t i o n i s given i n Figure 10. The spectrum was interpreted according to the p r i n c i p l e s described i n Section II.7. Other d e t a i l s regarding the mass spectrum of t h i s compound are given i n Appendix I I I . On the basis of these data, the following structure i s proposed for A l . 1 2 1 4 1 2 1 3 GlcA ^-^ Man GlcA — - Man — Glc~0H 3 a " 0 a Analysis of A2: This compound moved s l i g h t l y f a s t e r than Al on Whatman No. 1 paper ( R Q 1 ( , 0.12, Solvent B) . The *H-n.m.r. spectrum (see Table 3 and Spectrum 6) shows that A2 i s a tetrasaccharide containing four of the 54 sugars found i n A l , but without the reducing glucose. On reduction with sodium borohydride the signal at 65.35 i s removed from the spectrum (see Spectrum 7),, i n d i c a t i n g that the reducing sugar i s mannose. It i s i n t e r e s t i n g to note that 94% of the reducing mannose exists as the a anomer. The r e s u l t s of the methylation analysis are given i n Table 2. A2 was reduced with sodium borodeuteride, methylated, carboxyl reduced, hydrolysed and analysed by g.l.c./m.s. The presence of 1,3,4,5,6-Man confirms that the reducing mannose i s 2-linked. Standard mass spectra are not ava i l a b l e f o r the a l d i t o l acetates of pentamethyl h e x i t o l s but 1,2,4,5,6-Man was i d e n t i f i e d by fragment ions at m/e 162 and m/e 130. These ions are assigned the following structures: + + HC = OMe HC = OMe I -MeOH COAc II CHD m/e 161 m/e 130 The mass spectrum i s tabulated i n the Experimental section. 3,4,6-Man, 2,3,4-Glc and 2,3-Glc were also observed as expected. On the basis of these data, the following structure i s proposed for A2. HCOAc CHDOMe GlcAp — Manp — GlcAp — - Manp~0H B a B 55 Table 3: N.M.R. STUDY OF A2 A. 400 MHz *H-n.m.r. at ambient temperature; a) A2, b) A2 a f t e r reduction with NaBD.. a) Chemical S h i f t of Anomeric ^H's(6) J l , 2 ^ Integral Number of "*"H's Assignment 4.55 4.60 4.93 5.15 5.35 si n g l e t s i n g l e t s i n g l e t 1 • 1 0.06 1 0.94 B-GlcA B-GlcA B-Man-OH a-Man a-Man-OH b) 4.55 4.59 5.11 si n g l e t B-GlcA B-GlcA a-Man 20 MHz 13C-n.m.r. of A2 Chemical S h i f t 13 of Anomeric C's(6) Integral 13 Number of C's Assignment 93.30 100.80 102.37 103.04 a-Man~OH a-Man B-GlcA B-GlcA 56 Analysis of A3 When i r r i g a t e d with Solvent B, t h i s compound has a chromatographic mobility of Rri 0.42 on Whatman No. 1 paper. A sample was hydrolysed o l C i n 2M TFA f o r 16 hours on a steam bath. A sample of the hydrolysate • was chromatographed on paper with Solvent C along with glucose, mannose, and galactose. The chromatogram showed that the neutral sugar released during hydrolysis was galactose. N.m.r. Study A sample of K39 capsular polysaccharide was p a r t i a l l y depolymerized by hydrolysis i n 0.02M TFA on a steam bath f o r 10 minutes. It was then dialysed against running' water and freeze-dried. This sample was analysed by 400 MHz *H-n.m.r. at 90°C (see Spectrum 8). Figure 11 compares the anomeric proton signals of t h i s polysaccharide with s i m i l a r signals i n the *H-n.m.r. spectrum of the pentasaccharide A l . In the polysaccharide spectrum, the H-l signals of the a-mannopyranosyl residues (65.20 and 65.45) are c l e a r l y distinguished. A B signal i s observed at 6 4.95 and a number of unresolved B signals appear between 64.53 and 64.74. The i n t e g r a l units of the various signals appear below the spectrum. The t o t a l number o f i n t e g r a l units i s 95. Assuming that 14 units account f o r one sugar residue, then the pentasaccharide Al accounts f o r 70 of the 95 i n t e g r a l units a t t r i b u t e d to anomeric s i g n a l s . 1 13 The H-n.m.r. and C-n.m.r. data are tabulated i n Table 4. 13 (The sample analysed by C-h.m.r. was p a r t i a l l y depolymerised by the 57 6 5 . 4 5 6 5 . 2 0 Integral: 14, 15 , 11 , 55 Figure 11. 1H-n.m.r. anomeric signals f o r oligosaccharide Al (top), and K39 capsular polysaccharide (bottom) 58 Table 4: N.M.R. STUDY OF K39 CAPSULAR POLYSACCHARIDE. A. 400 MHz H-n.m.r. at 90°C Chemical S h i f t of Anomeric *H's(6) Integral Assignment 4.76-4.53 4.95 (J 5.20 5.45 1,2 8 Hz) 55 11 15 14 B-GlcA(x2) ,-3-linked B-Glc B-Glc (terminal) a-Man a-Man 100.6 MHz 13 C-n.m.r. at 60°C. Chemical S h i f t 13 of Anomeric C;s(6) 100.15 100.91 101.30 102.42 102.60 Integral 5.3 7.0 6.9 6.2 16.5 Assignment a-Man a-Man 3-linked B-Glc B-GlcA -GlcA, B-Glc (terminal) 59 same method). Not a l l of the sugar residues indicated by the sugar analysis could be accounted f o r i n the n.m.r. spectra; therefore the assignments are not complete. In p a r t i c u l a r some of the 3 signals may ar i s e , i n part, from galactosyl residues. A '''H-n.m.r. spectrum was also recorded without an i n i t i a l h y d r o l y s i s , and without acetone as a standard (see Spectrum 9). No major differences were observed i n the anomeric region of the spectrum. Traces of acetate (62.18) and pos s i b l y pyruvate (61.48) were also observed. Methylation Analysis Three methylation analysis experiments were performed: I, K39 methylated and hydrolysed without carboxyl reduction; I I , K39 methylated, carboxyl reduced and hydrolysed; I I I , K39 hydrolysed f o r 3 hours i n 0.02M TFA, followed by d i a l y s i s and freeze-drying, methylation, carboxyl reduction and hy d r o l y s i s . The products were analysed by g. l . c . and g.l.c./m.s. as the a l d i t o l acetate d e r i v a t i v e s (see Table 4). These experiments show some s i m i l a r i t i e s to the methylation analysis of A l . In experiment II , 3,4,6-Man, 2,4,6-Glc and 2,3-Glc are observed as i n the analysis of A l . 2,3,4,6-Glc i s observed i n a l l three cases i n d i c a t i n g the presence of terminal glucopyranosyl residues. In experiment I I I , the r e l a t i v e amounts of 2,3,4,6-Glc and 2-Glc decreased, with a corresponding increase i n 2,3-Glc. These r e s u l t s indicate that the side chain containing the terminal glucopyranosyl residue i s • linked to the 3-position of a 4-linked glucuronopyranosyl residue. 60 Table 4: METHYLATION ANALYSIS OF KLEBSIELLA K39 CAPSULAR POLYSACCHARIDE. P a r t i a l l y Methylated cL A l d i t o l Acetates Mole % II III 2,3,4,6-Glc 3,4,6-Man 2,4,6-Glc 2,4,6-Gal 2,3-Glc 6 2-Glc 1.00 1.82 1.87 2.05 3.06 3.68 28.1 8.5 12.0 24.6 1 51.3 j 38.2 8.7 12.9 15.6 1177 46.6 8.0 25.3 8.4 e.g., 2,3,4,6-Glc = 1,5-tri-0-acetyl-2,3,4,6-tetra-O-methylglucitol b G.l.c. retention time r e l a t i v e to 2,3,4,6-Glc on 3% HIEFF-1B, temperature program: 165°*;for 8 min, increase 2°/min to 200°C. c 76 Integrals were corrected with published molar response factors. ^ I, K39 methylated and hydrolysed without carboxyl reduction; I I , K39 methylated, carboxyl reduced and hydrolysed; I I I , K39 hydrolysed f o r 2 hours i n 0.02M TFA followed by d i a l y s i s and freeze-drying, methylation, carboxyl reduction and hydrolysis. G.l.c./m.s. also detected 2,4-Glc on the leading edge of t h i s peak. 61 Conclusions On the b a s i s o f the experiments d e s c r i b e d , i t i s proposed that the capsular polysaccharide o f K l e b s i e l l a K39 contains the f o l l o w i n g pentasaccharide u n i t i n i t s r e p e a t i n g sequence o f sugars: — - GlcAp_ — - Manp — — GlcAp -1—^- Man£ — - Glcp - — B a B a B These experiments also i n d i c a t e the presence of a s i d e chain w i t h a te r m i n a l B-glucopyranosyl residue. The s i d e chain i s attached to the 3- p o s i t i o n o f one of the glucuronopyranosyl r e s i d u e s . To t h i s date, no other published K l e b s i e l l a capsular p o l y s a c c h a r i d e has had two u r o n i c a c i d residues i n the rep e a t i n g u n i t o f sugars. I t was not p o s s i b l e to l o c a t e the gala c t o s e i n the s t r u c t u r e o f the polys a c c h a r i d e . D i f f e r e n t batches o f K39 had d i f f e r e n t galactose contents, and par t or a l l o f the galactose may be present as an im p u r i t y . The immunochemical st u d i e s o f Heidelberger and Nimmich i n d i c a t e d that K39 shares s t r u c t u r a l f eatures w i t h K l and K31. A l l three s t r u c t u r e s have a 3- l i n k e d B-glucopyranosyl residue. A l l three s t r u c t u r e s a l s o have glucuronopyranosyl r e s i d u e s , although the degree o f s u b s t i t u t i o n and the anomeric c o n f i g u r a t i o n v a r i e s . I t i s probable t h a t these two s t r u c t u r a l f eatures p l a y a r o l e i n the antigen-antibody r e a c t i o n . 62 EXPERIMENTAL General Methods Descending paper chromatography was performed using the solvent systems and v i s u a l i s a t i o n methods described i n Section II.1. G.l.c. analyses were performed on a Hewlett Packard 571OA gas chromatograph f i t t e d with dual flame i o n i z a t i o n detectors. G.l.c./ m.s. analyses were performed on a Micromass 12 instrument with an electron beam energy of 70 eV. N.m.r. spectra were recorded on Bruker WP-80, Bruker WH-400 and Varian XL-100 spectrometers. Samples were dissolved i n deuterium oxide and acetone was included as a standard. Mass spectra o f a permethylated oligosaccharide were recorded on a Kratos MS50. Is o l a t i o n and P u r i f i c a t i o n An authentic culture of K l e b s i e l l a K39 was obtained from Dr. I. 0rskov, Copenhagen, and grown on f i v e agar t r a y s . Each tray contained agar composed of the following materials: 5g NaCI, 2.5g K 2HP0 4, 0.62g MgS02-7H20, 0.5g CaC0 3, 75g sucrose, 5g Bacto yeast extract, 37.5g agar, 2.5L water. Af t e r 3 days the b a c t e r i a l slime was scraped o f f of the trays and the so l u t i o n (600 ml) was made 1% in phenol. Insoluble material was removed by u l t r a c e n t r i f u g a t i o n at 30,000 r.p.m. for 3 hours. The supernatant was mixed with 2.4L of acetone-methanol (3:1) to p r e c i p i t a t e the polysaccharide component. 63 The p r e c i p i t a t e was dissolved i n water and the a c i d i c polysaccharides were p r e c i p i t a t e d with 10% Cetavlon. The p r e c i p i t a t e was dissolved i n 2M sodium chloride (500 ml) and p r e c i p i t a t e d again by mixing with 2L of acetone-methanol (1250:750). The p r e c i p i t a t e was dissolved i n water and dialysed against running tap water for 2 days. The contents of the.dialysis bag were freeze-dried giving 1.5g of polysaccharide, [a]p--3° (c 0.064, water). The molecular weight was determined to be 560,000 by gel chromatography on Sepharose 4B (courtesy of Dr. S.C. Churms, University of Capetown). Sugar Analysis A sample (20 mg) of K39^capsular polysaccharide was dissolved i n water, passed through a column of Amberlite IR-120(H +), and freeze-dried. It was then refluxed i n 3% methanolic hydrogen chloride for 20 hours. The so l u t i o n was neutralized with lead carbonate, f i l t e r e d , concentrated and dried under vacuum. The sample was dissolved i n methanol, sodium borohydride was added, and the solution was allowed to s i t overnight. The methanol was removed by evaporation and the residue was dissolved i n water. The s o l u t i o n was a c i d i f i e d with Amberlite IR-120(H +), f i l t e r e d and concentrated. Borates were removed by c o d i s t i l l a t i o n with methanol. The methanolysis and reduction steps were repeated. The sample was then hydrolysed i n 2M TFA overnight on a steam bath. PAAN de r i v a t i v e s were prepared 25 by the method of Chen and McGinnis. G.l.c. analysis on 3% OV-225 (isothermal at 220°C) gave Man/Glc/Gal = 1.0/2.2/0.8. 64 Preparation of A l d i t o l Acetates In t h i s study, both sugars and p a r t i a l l y methylated sugars were analysed by g . l . c . as the a l d i t o l acetate d e r i v a t i v e s . With minor va r i a t i o n s these d e r i v a t i v e s were prepared using the following method. Hydrolysates were dissolved i n water (-2 ml) and 2 drops of 10% NH^ were added. Sodium borohydride was added and the so l u t i o n was allowed to s i t for 1 - 3 hours. The so l u t i o n was a c i d i f i e d with IR-120(H +), f i l t e r e d and concentrated by evaporation. Borates were removed by c o d i s t i l l a t i o n with methanol. The a l d i t o l s were acetylated with a c e t i c anhydride-pyridine (1:1) on a steam bath f o r 30-60 minutes. The residual a c e t i c anhydride was destroyed by adding methanol or ethanol. The so l u t i o n was concentrated and pyridine was removed by c o d i s t i l l a t i o n with water. P a r t i a l Hydrolysis The K39 capsular polysaccharide was a c t u a l l y prepared several times. This preliminary p a r t i a l hydrolysis study was a c t u a l l y performed on an e a r l i e r batch. A sample of K39 polysaccharide (470 mg) was hydrolysed in 1M TFA (80 ml) for 5*5 hours. The hydrolysate was concentrated by evaporation under reduced pressure, and was then applied to a column of Bio-Rad AG 1-X2. Neutral sugars were eluted by washing the column with water. A c i d i c s were eluted with 10% formic acid. The a c i d i c f r a c t i o n was concentrated and applied to a column (96 x 2.2 cm) of Bio-Gel P-2. The mixture was eluted with water-pyridine-acetic acid (1000:10:4) at a rate of 5 ml/hour and c o l l e c t e d i n 2.5 ml 65 f r a c t i o n s . The samplest were c o l l e c t e d i n tared test tubes which were then freeze-dried and reweighed to give the chromatogram i n Figure 7. In a further study K39 polysaccharide (700 mg) was hydrolysed i n 1M TFA (90 ml) f o r 7 hours on a steam bath. The hydrolysate was concentrated and neutrals were separated from a c i d i c s as described above. The a c i d i c f r a c t i o n (290 mg) was applied to Whatman 3MM chromato-graphy paper and i r r i g a t e d with Solvent B (see Section II. 1) f o r 4 days. Each day the papers were removed from the tank and dried to enhance r e s o l u t i o n . S t r i p s were cut from the sides of the papers and v i s u a l i s e d by Method A (see Section II. 1). Three bands were cut from the paper and extracted with water. The extracts were concentrated and freeze-dried. giving Al (80 mg) , A2 (36 mg) , and A3 (16 mg). A n a l y t i c a l paper chromatography showed that A2 was not pure. A2 was applied to Whatman No. 1. paper and i r r i g a t e d with Solvent B for 4 days with; drying each day. A2 (15 mg) was extracted from the chromatogram. Analysis of A l A portion (less than 1 mg) of Al was hydrolysed i n 3M TFA on a steam bath f o r 20 hours. The hydrolysate was concentrated and the neutral sugars released were converted to the a l d i t o l acetates which were analysed by g.l . c . on 3% SP-2340 with a nitrogen flow rate of 20 ml/minute. Temperature program: 195°C f o r 4 min; increase by 2°/minute to 265°C. This analysis gave Man/Glc = 1.56/1. Another portion (3 mg) of Al was methanolysed and carboxyl reduced by the method already described, followed by hydrolysis i n 66 2M TFA for 21 hours. The hydrolysate was analysed by g.l.c. on SP-2340 a f t e r conversion to the a l d i t o l acetates. This analysis gave Glc/Man = 1.47/1. A sample (12 mg) of Al was methylated by the Hakomori procedure. The sample was dissolved i n DMSO (2 ml) i n a sealed f l a s k , which was then evacuated with nitrogen. 2M di m e t h y l s u l f i n y l sodium (2 ml) was added and the so l u t i o n was s t i r r e d f o r 50 minutes. The solu t i o n was frozen s o l i d and methyl iodide (5 ml) was added, followed by s t i r r i n g for one hour. The methyl iodide was evaporated o f f with a stream of nitrogen and water was added. The so l u t i o n was extracted with chloroform and the combined extracts were extracted with water. The chloroform phase was concentrated! and the r e s i d u a l DMSO was removed with a vacuum pump and heat lamp. One t h i r d of the residue was hydrolysed i n 2M TFA on a steam bath f o r 17 hours, and the a l d i t o l acetates were prepared as previously described. The remainder of the residue was dissolved i n tetrahydrofuran, l i t h i u m aluminum hydride was added, and the mixture was refluxed for 3 hours. The excess li t h i u m aluminum hydride was destroyed by adding 90% ethanol (10 ml) followed by water (10 ml). The mixture was concentrated to dryness and washed with chloroform. The chloroform washings were concentrated to dryness and hydrolysed i n 2M TFA on a steam bath f o r 12 hours. The hydrolysate was concentrated and a l d i t o l acetates were prepared. The p a r t i a l l y methylated a l d i t o l acetates obtained were analysed by g. l . c . on 3% HIEFF-1B. Temperature program: 165°C for 8 minutes, increase by 2°/minute to 200°C. G.l.c./m.s. and comparison with standard mass spectra indicated the presence of 3,4,6-Man, 2,4,6-Glc, 2,3,4-Glc and 67 2,3-Glc. Another sample (4.3 mg) of Al was dissolved i n water, sodium borodeuteride was added, and the s o l u t i o n was allowed to s i t for 2^ hours. The so l u t i o n was a c i d i f i e d with Amberlite IR-120(H +) and the borates were removed by c o d i s t i l l a t i o n with methanol. The r e s u l t i n g 28 oligosaccharide a l d i t o l was methylated by the Hakomori procedure. The product was hydrolysed i n 2M TFA for 14 hours on a steam bath. A l d i t o l acetates were prepared and analysed by g.l.c./m.s. 3,4,6-Man was i d e n t i f i e d by comparison with a standard spectrum. A standard spectrum was not a v a i l a b l e f or 1,2,4,5,6-Glc and the m/e values obtained are therefore l i s t e d below ( r e l a t i v e abundances i n brackets): 43 (100), 44 (24), 45 (88), 46 (16), 58 (34), 59 (28), 60 (40), 71 (24), 75 (30), 85 (18), 87 (18), 88 (16), 89 (20), 90 (66), 100 (16), 101 (80), 114 (16), 206 (22). For analysis by mass spectrometry, a sample (7.7 mg) of Al was converted to the oligosaccharide a l d i t o l by the sodium borodeuteride reduction already described. It was then methylated by the Hakomori procedure and i s o l a t e d by preparative s i l i c a gel t . l . c . An i n f r a r e d spectrum showed no hydroxyl absorption at 3600 cm 1 . Absorption maxima were observed at 2900 cm 1 (C-H s t r e t c h ) , 1750 cm 1 (C = 0 s t r e t c h ) , and 1105 cm"1, 1080 cm 1 (ether s t r e t c h ) . This permethylated oligosaccharide a l d i t o l was analysed by electron impact mass spectrometry with electron beam energies of 20 eV and 70 eV. Analysis of A2 A2 (7.6 mg) was converted to the oligosaccharide a l d i t o l by 68 the sodium borodeuteride reduction already described. It was then methylated, carboxyl reduced, and hydrolysed by the same methods as A l . The products were analysed by g.l.c./m.s. as the a l d i t o l acetates. 3,4,6-Man, 2,3,4-Glc and 2,3-Glc were i d e n t i f i e d by comparison with 28 standard mass spectra. Analysis o f 1,3,4,5,6-Man by g.l.c./m.s. gave ions with the following m/e values ( r e l a t i v e abundances i n brackets) : 43 (100), 45 (89), 46 (46), 59 (30), 71 (19), 72 (30), 73 (19), 75 (24), 87 (14), 88 (32), 89 (31), 101 (51), 102 (25), 103 (25), 114 (13), 130 (55), 133 (14), 141 (43), 162 (34), 187 (14). Methylation Analysis A sample (42 mg) of K39 capsular polysaccharide was p u r i f i e d by a further Cetavlon p r e c i p i t a t i o n . The r e s u l t i n g polysaccharide was passed through a column of Amerlite IR-120(Fif) to convert i t from the sodium s a l t form to the acid form. The freeze-dried product (35 mg) was dissolved i n DMSO (7 ml) under nitrogen with heating to approximately 50°C. 2M d i m e t h y l s u l f i n y l sodium (5 ml) was added and the solution was s t i r r e d f o r 3 hours at room temperature. The s o l u t i o n was frozen s o l i d and methyl iodide (12 ml) was added. The i c e bath was removed and the so l u t i o n was s t i r r e d f o r 40 minutes. Methyl iodide (2 ml) was added and the so l u t i o n was s t i r r e d f o r another 20 minutes. The r e s i d u a l methyl iodide was removed with a stream of nitrogen, water was added, and the product was dialysed against running water overnight. The contents of the. d i a l y s i s bag were extracted with chloroform and the combined extracts were concentrated 69 and dried. Infrared analysis of the methylated polysaccharide showed no hydroxyl absorption at 3600 cm * and methylation was assumed to be complete. The i n f r a r e d spectrum also showed a strong ester carbonyl absorption at 1750 cm *. A short column of Sephadex LH-20 (swollen i n ethanol) was prepared. The permethylated polysaccharide was dissolved i n chloroform, applied to the column and eluted with ethanol-chloroform (2:1). The fast moving material was c o l l e c t e d and concentrated. A t h i r d of t h i s material was hydrolysed i n 2M TFA overnight on a steam bath. A l d i t o l acetates were prepared f o r g . l . c . analysis. The remaining permethylated polysaccharide was carboxyl reduced with l i t h i u m aluminum hydride i n tetrahydrofuran for 3 hours r e f l u x i n g and overnight at room temperature. The carboxyl reduced polymer was i s o l a t e d from the reaction mixture as described i n the analysis of A l . It was then hydrolysed i n 2M TFA for 17 hours on a steam bath and a l d i t o l acetates were prepared for g . l . c . analysis. Column: 3% HIEFF-1B. Temperature program: 165°C for 8 minutes followed by an increase of 2°/minute to 200°C. A sample (254 mg) of K39 capsular polysaccharide was hydrolysed i n 0.02M TFA for a t o t a l of 3 hours on a steam bath. The hydrolysate was dialysed against running water and freeze-dried. A methylation a n a l y s i s , with carboxyl reduction, was performed as previously described. 7 0 V. A PRELIMINARY STRUCTURAL INVESTIGATION OF E. COLI K26 CAPSULAR POLYSACCHARIDE 7 1 ABSTRACT The capsular polysaccharide of E. c o l i K26 has been isolated and a preliminary structural investigation has been performed. The polysaccharide has been studied by the methods of sugar analysis, methylation analysis, partial hydrolysis and "'"H-n.m.r. RESULTS AND DISCUSSION Isolation and Sugar Analysis The capsular polysaccharide of E. c o l i K26 was prepared and isolated by the methods described in the Experimental section. This material gave a specific rotation of -30°. A molecular weight analysis by gel chromatography (courtesy of Dr. S.C. Churms, University of Capetown) showed that the polysaccharide moved as one band on 7 Sepharose 4B with a molecular weight of 10 .. According to 0rskov 9 and coworkers, this polysaccharide is composed of rhamnose, galactose and glucuronic acid. The presence of these sugars was confirmed by hydrolysis and paper chromatography as described in the Experimental section. A sample of K26 capsular polysaccharide was methanolysed, carboxyl reduced, hydrolysed and analysed by g.l.c. as the PAAN derivatives. The chromatogram gave Rha/Gal/Glc = 3.0/2.9/1.0. Methylation Analysis Two methylation analysis experiments were performed: I, K26 72! Table 5. METHYLATION ANALYSIS OF E. COLI K26 CAPSULAR POLYSACCHARIDE. P a r t i a l l y Methylated A l d i t o l A c e t a t e s E T b I d Mole %° II 2,3,4-Rha 0.48 12.6 7.8 2,4-Rha 0.94 51.2 54.5 2,4,6-Gal 1.89 27.2 20.9 2-Glc 3.66 9.0 16.7 e.g., 2,3,4-Rha = 1,5-di-O-acetyl-2,3,4-tri-O-methylrhamnitol. G.l.c. r etention time r e l a t i v e to 2,3,4,6-Glc on 3% OV-225. Temperature program: 190°C for 16 minutes, followed by an increase of 2°/minute to 230°C. 7 6 Integrals were corrected using published molar response f a c t o r s . I, K26 polysaccharide methylated and hydrolysed without carboxyl reduction; I I , K26 polysaccharide methylated, carboxyl reduced, and hydrolysed. 73 polysaccharide methylated and hydrolysed without carboxyl reduction; I I , K26 polysaccharide methylated, carboxyl reduced and hydrolysed. The r e s u l t s are given i n Table 5. Both experiments ind i c a t e the presence of 3-linked rhamnosyl residues and 3-linked galactosyl residues. The presence of 2-Glc i n experiment I cannot be explained given the present knowledge o f the polysaccharide. A f t e r carboxyl reduction the amount of 2-Glc increases, i n d i c a t i n g that the glucuronosyl residues are substituted at the 3 and 4-positions. The nature o f the substituents i s unknown. Not a l l of the galactose that was indicated by the sugar analysis could be accounted f or i n the methylation analysis. It i s therefore assumed that some o f the galactose i s present as an impurity. P a r t i a l Hydrolysis A sample of K26 capsular polysaccharide was hydrolysed i n 0.5M TFA on a steam bath f o r 4 hours. Neutrals and a c i d i c s were separated by ion exchange chromatography on Bio-Rad AG 1-X2. The a c i d i c f r a c t i o n was chromatographed on paper and a compound Al ( R Q ^ c 0.79, Solvent B) was i s o l a t e d . The neutral f r a c t i o n was chromatographed on paper and a compound NI ( R Q c 1-9, Solvent C) was i s o l a t e d . Analysis of Al 1 13 Al was analysed by H- and G-n.m.r. The r e s u l t s are given i n Table 6, Spectrum 10 and Spectrum 11. These data i n d i c a t e that Al i s an aldobiouronic acid c o n s i s t i n g of a i i 3-glucuronosyl residue linked 74 Table 6. N.M.R. STUDY OF Al A. B. Also: 400 MHz ^H-n.m.r. at 90°C Chemical S h i f t of Anomeric ^H'sOS) J l , 2 ^ Integral Number of ^H' s Assignment 4.71 8 .0.6 B-GlcA linked to a-Rha-OH 4.73 8 0.4 B-GlcA linked to B-Rha-OH 4.86 si n g l e t 0.4 B-Rha-OH 5.12 2 0.6 a-Rha-OH Also: 1.30 1.31 7 CJ5,6> 8 ( J 5 f 6 D 0.6 x 3 0.4 x 3 CH 3 of a-Rha-CH 3 of B-Rha-20 MHz 13C-n.m.r. at 35°C Chemical S h i f t 13 of Anomer C's(6) Integral 13 Number of C's Assignment 94.49 0.3 B-Rha-OH 94.98 0.7 a-Rha-OH 104.98 1 B-GlcA 17.54 81.44 83. 58 1 0.8 0.2 CH 3 of~Rha OH C-3 of a-Rha-OH C-3 of B-Rha-OH 75 to a reducing rhamnose. A sample of Al was methanolysed, carboxyl reduced, hydrolysed and converted to the PAAN der i v a t i v e s . GH.c. analysis gave Rha/Glc = 1.4/1. A sample of Al was methylated by the Hakomori procedure and hydrolysed i n 2M TFA. Paper chromatography of the hydrolysate showed that the neutral sugar released was a dimethyl rhamnose (R-. , 1.04, to 2,3,6-Glc Solvent D). The hydrolysate was reduced with sodium borohydride and acetylated. G.l.c. analysis on 3% OV-225 (isothermal at 190°C) gave a retention time of 0.92 r e l a t i v e to 2,3,4,6-Glc. G.l.c./m.s. analysis gave a mass spec-rum that was consistent with l,3,5-tri-0-acetyl-2,4-28 di-O-methylrhamnitol. Al has a s p e c i f i c r o t a t i o n of [ a ] n -27° which i s consistent with 37 a S-D-glucuronosyl residue linked to a reducing L-rhamnose. Al i s therefore assigned the following structure: D - GlcA — L - Rha~0H 6 Analysis of NI Because of i t s mobi l i t y , NI was assumed to be a rhamnose containing disaccharide, but i t was shown to be unaffected by acid hydrolysis. A *H-n.m.r. spectrum (Spectrum 12) was recorded at 90°C. The spectrum showed carbohydrate r i n g proton signals between 63.2 and 64.0. Also observed were anomeric proton signals at 64.58 (J = 7 Hz) and 65.19 (J = 3 Hz) i n the r a t i o 66 : 34. The anomeric signals i n d i -cate that NI i s a monosaccharide. The most unusual feature of t h i s 76 spectrum i s a group of signals between 67.61 and 68.39. NI i s there-fore assumed to contain an unsaturated moiety. (A ^ H-n.m.r. spectrum was also recorded at ambient temperature. See Spectrum 13). A sample of the polysaccharide was p u r i f i e d by a further Cetavlon p r e c i p i t a t i o n and a ^H-n.m.r. spectrum was recorded. There was no evidence that NI was present and i t was assumed to be an impurity. Nuclear Magnetic Resonance A "''H-n.m.r. spectrum of E. c o l i K26 capsular polysaccharide was recorded at 90°C (see Spectrum 14). Too l i t t l e i s known about K26 for an adequate i n t e r p r e t a t i o n but te n t a t i v e assignments of anomeric protons have been made. Coupling constants and approximate i n t e g r a l s are given i n brackets. A signal at 65.07 ( s i n g l e t , 2H's) i s assigned to a-rhamnosyl residues. A signal at 54.'87 ( s i n g l e t , IH) i s . assigned to a 3-rhamnosyl residue, although the signal i s broader than 77 expected. A signal at 64.72 (J^ 2 - 7 Hz, IH) i s assigned to a g-glucuronosyl residue. A signal at 61.31 i s assigned to the methyl protons of rhamnosyl residues. Conclusions Dr. M. Heidelberger of the New York University Medical Center studied the cross-reactions of E. c o l i K26 with the anti-sera to a number of Pneumococcal K antigens. His r e s u l t s are given i n Appendix IV. He at t r i b u t e d some o f the cross-reactions to the pos s i b l e presence of 3-linked L-rhamnosyl residues. The presence of 3-linked 77 rhamnosyl residues has been confirmed by the methylation analysis, and by the i s o l a t i o n of an aldobiouronic acid by p a r t i a l h y d r o l y s i s . 78 EXPERIMENTAL I s o l a t i o n arid P u r i f i c a t i o n An authentic culture of E. c o l i K26 was incubated i n broth and then grown on agar trays f o r 3 days (the agar was prepared as described i n Section IV). The b a c t e r i a l slime was scraped o f f of the trays and the so l u t i o n was made 2% i n phenol. C e l l u l a r debris was removed by u l t r a c e n t r i f u g a t i o n at 30,000 r.p.m. for 4 hours. The supernatant (570 ml) was added to methanol (1800 ml) but no p r e c i p i t a t e appeared. On addition of acetone (1000 ml), the polysaccharide p r e c i p i t a t e d out of sol u t i o n . The a c i d i c capsular polysaccharide was i s o l a t e d by two Cetavlon p r e c i p i t a t i o n s , as described i n Section IV. After d i a l y s i s and freeze-drying the y i e l d was 660 nig. t a ] n "30° (c 0.13, water). A molecular weight analysis by gel chromatography (courtesy of Dr. S.C. Churms, Uni v e r s i t y of Capetown) showed that the polysaccharide moved as one band on Sepharose 4B, and that the molecular weight was 7' 10 . Sugar Analysis and Methylation Analysis A portion (2.5 mg) of K26 capsular polysaccharide was hydrolysed i n 2M TFA for 17 hours on a steam bath. The hydrolysate was chromato-graphed on Whatman No. 1 paper with Solvent C, along with xylose, glucose, mannose, galactose, fucose, and rhamnose as standards. Galactose and rhamnose were observed along with traces of mannose and glucose. Some u n i d e n t i f i e d materials were also observed. A sample 79 (11 mg) of polysaccharide was methanolysed by r e f l u x i n g i n 3% methanolic hydrogen c h l o r i d e , reduced with sodium borohydride and hydrolysed i n 2M TFA as described i n Section IV. The hydrolysate was chromatographed on Whatman No. 1 paper with Solvent C. Rhamnose, galactose and a dark spot due to glucose were observed, thus proving that glucuronic acid i s present.-The portion of polysaccharide used f o r the following sugar and methylation analyses was p u r i f i e d by a further Cetavlon p r e c i p i t a t i o n . A sample (34 mg) of K26 polysaccharide was passed through a column of Amberlite IR-120(H +) and freeze-dried. A sample (28 mg) was set aside f o r methylation analysis and the remainder was refluxed i n 3% methanolic hydrogen chloride and reduced with sodium borohydride i n methanol as described i n Section IV. The methanolysis and reduction were repeated. The products were then hydrolysed i n 2M TFA overnight on a steam bath. Water and TFA were removed by evaporation under reduced pressure. PAAN der i v a t i v e s were prepared by the method of 25 Chen and McGinnis. The i n t e g r a l s were corrected with molar c o r r e c t i o n 74 f a c t o r s . G.l.c. analysis on 3% OV-225 (isothermal at 220 C) gave Rha/Gal/Glc = 3.0/2.9/1.0. A sample (28 mg) of K26 polysaccharide was dissolved i n DMSO (5 ml), 2M d i m e t h y l s u l f i n y l sodium (3 ml) was added, and the so l u t i o n was s t i r r e d f o r one hour. The s o l u t i o n was frozen s o l i d and methyl iodide (5 ml) was added a f t e r removal of the dry ice/acetone bath. Af t e r s t i r r i n g f o r one hour, the residual methyl iodide was removed with a stream of nitrogen. Chloroform and water were added and the mixture was dialysed against running water. The contents of the 80 d i a l y s i s bag were extracted with chloroform, concentrated and p u r i f i e d by chromatography on Sephadex LH-20. (The r e s i n was swelled i n chloroform and eluted with chloroform). An in f r a r e d spectrum of the product showed no hydroxyl absorption at 3600 cm * and a carbonyl absorption at 1740 cm *. Methylation was assumed to be complete. A t h i r d of t h i s material was hydrolysed i n 2M TFA f o r 16 hours on a steam bath and a l d i t o l acetates were prepared f o r g.l . c . a n a lysis. The remaining methylated polysaccharide was carboxyl reduced with l i t h i u m aluminum hydride i n r e f l u x i n g tetrahydrofuran for 3 hours. The resi d u a l l i t h i u m aluminum hydride was destroyed with ethanol. 10% hydrochloric acid was added to dissolve the aluminates, and the so l u t i o n was extracted with chloroform. A f t e r concentrating and drying, an in f r a r e d spectrum showed no carbonyl absorption. This material was hydrolysed i n 2M TFA overnight on a steam bath and concentrated under reduced pressure. A sample of the hydrolysate was chromatographed on Whatman No. 1 paper with Solvent D and v i s u a l i s e d with p-anisidine hydrochloride. The chromatogram indicated the presence of a trimethyl rhamnose (R„ „ , 1.19, green), a dimethyl rhamnose (R„ _ , Z,o,D-blC Z , J , D - ( J 1 C 1.05, green), a trimethyl galactose (R^ 3 ^ _ Q^ c 0.89, pink), and a monomethyl glucose (R„ „ . 0.35, brown). A l d i t o l acetates were Z,O,O-blC prepared and analysed by g . l . c . on 3% OV-225 (190°C f o r 16 minutes followed by an increase of 2°/minute to 230°C). The PMAAs obtained from the carboxyl reduced product were analysed by g.l.c./m.s. on 3% HIEFF-1B (165°C f o r 8 minutes followed by an increase of 2°/min to 200°C). 81 P a r t i a l Hydrolysis A sample (415 mg) of K26 capsular polysaccharide was dissolved i n 0.5M TFA (70 ml) and heated on a steam bath f o r 4 hours. Water and TFA were removed by evaporation under reduced pressure. Neutrals and ac i d i c s were separated by ion exchange chromatography on Bio-Rad AG 1 - X2 (formate form). The hydrolysate was applied to the column and washed with water (250 ml) to give the neutral f r a c t i o n (266 mg a f t e r concen-t r a t i o n and freeze-drying). The column was then washed with 10% formic acid (50 ml), 20% formic acid (50 ml), and 10% formic acid (100 ml) to give the a c i d i c f r a c t i o n (107 mg a f t e r concentration and freeze-drying) . The a c i d i c f r a c t i o n was chromatographed on Whatman 3MM paper with Solvent B. One component (designated Al) had approximately the same mobility as galactose. This material was extracted from the paper and chromatographed on Whatman No. 1 paper with Solvent C (a basic solvent) overnight, and then f o r 7 hours with Solvent B (an a c i d i c solvent). Under these conditions Al moves slower than a neutral monosaccharide, and was therefore assumed to be a c i d i c , but the chromatogram showed that further p u r i f i c a t i o n was necessary. Al was applied to Whatman 3MM paper and i r r i g a t e d with Solvent C f or 19 hours and Solvent B f or 16 hours. Al was extracted from the paper and gave a y i e l d of 22 mg a f t e r freeze-drying. r Q 1 c 0.79 (Solvent B). [ct] D -27° (cV0.090, water). A slower moving component was also i s o l a t e d from the i n i t i a l chromatogram but ^ H-n.m.r. showed that i t was not pure enough for 82 unambiguous analysis. A portion (44 mg) of the neutral f r a c t i o n was chromatographed on Whatman 3MM paper with Solvent C. A component, designated NI, was i s o l a t e d from the chromatogram and gave a y i e l d of 15 mg a f t e r freeze-.':' drying. R G l c 1 - 9 (Solvent C). A sample of NI was hydrolysed i n 2M TFA on a steam bath overnight. Chromatography on Whatman No. 1 paper with Solvent C showed that NI was not hydrolysed. Analysis of Al A sample of A l (2.5 mg) was methanolysed i n 3% methanolic hydrogen chl o r i d e , carboxyl reduced with sodium borohydride i n methanol, and hydrolysed i n 2M TFA overnight on a steam bath. PAAN derivatives 25 were prepared by the method of Chen and McGinnis. Analysis by g.l . c . on 3% OV-225 (isothermal at 220°C) gave Rha/Glc = 1.4/1. Another sample of Al (7.6 mg) was methylated by the Hakomori procedure (DMSO (0.8 ml)';, 2M dimethyl sul f i n y l sodium (0.8 ml), methyl iodiee (1 ml)). A portion of the methylated product was hydrolysed i n 2M TFA for 17 hours on a steam bath. A sample of the hydrolysate was chromatographed on Whatman No. 1 paper with Solvent D and sprayed with p-anisidine hydrochloride. A greem spot appeared at R^ 3 6-Glc 1-04 and was assumed to be a dimethyl rhamnose. Two slower moving a c i d i c components were also observed and were bright pink i n colour. The hydrolysate was analysed by g.l . c . and g.l.c./m.s. as the a l d i t o l acetates. On 3% OV-225 (isothermal at 190°C) a component was observed with a retention time of 0.92 r e l a t i v e to 2,3,4,6-Glc. The mass 83 spectrum of t h i s compared well with a standard spectrum of a 1,3,5-tri- -0-acetyl-2,4-di-O-methylhexitol. VI BIBLIOGRAPHY 85 BIBLIOGRAPHY R.D. Guthrie, Introduction to Carbohydrate Chemistry, Oxford University Press, London (1974). L. Ayotte, E. Mushayakarara, and A.S. P e r l i n , Carbohydr. Res., 87, 297 (1980). G.O. A s p i n a l l , Polysaccharides, Pergamon Press, Toronto, (1970). Y.M. Choy and G.G.S. Dutton, Can. J. Chem., 51, 198 (1973). G.M. Bebault, Y.M. Choy, G.G.S. Dutton, N. Funnell, A.M. Stephen, and M.T. Yang, J . B a c t e r i d . , 1345 (1973). S.C. Churms and A.M. Stephen, Carbohydr. Res., 35_, 73 (1974). G.G.S. Dutton, K.L. Mackie, A.V. Savage, D. Rieger-Hug, and S. Stirm, Carbohydr. Res., 84, 161 (1980). W.F. Dudman and J.F. Wilkinson, Biochem. J . , 62, 289 (1956). I. 0rskov, F. 0rskov, B. Jann, and K. Jann, B a c t e r i o l o g i c a l Reviews, 667 (1977). A.L. Smith, P r i n c i p l e s of Microbiology, The CV. Mosby Company, Toronto, (1981). M. Heidelberger and W. Nimmich, Immunochem., 1_3, 67 (1976). W. Nimmich, Z. Med. Mikrobiol. Immunol., 154, 117 (1968), c i t e d i n reference 14. W. Nimmich, Acta. B i o l . Med. Ger. , 26., 397 (1971), c i t e d i n reference 14. J.L. Di Fabio, Ph.D. Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1981. J.A. Dean, Chemical Separation Methods, Van Nostrand Reinhold Company, New York, 1969. 86 16. L. Hough and J.K.N. Jones, Methods i n Carbohydrate Chemistry, Volume I, 21 (1962). 17. G.G.S. Dutton and E.H. M e r r i f i e l d , Carbohydr. Res., 103, 107 (1982). 18. F. Smith and R. Montgomery, The Chemistry of Plant Gums and Mucilages, Reinhold Publishing Company, New York, (1959). 19. S.C. Churms, Adv. Carb. Chem. Biochem., 2_5, 13 (1970). 20. G.G.S. Dutton and M. Paulin, Carbohydr. Res., 87, 107 (1980). 21. C C . :Sweeley, R. Bentley, M. Makita, and W.W. Wells, J. Am. Chem. Soc. , 85_, 2497 (1963) . 22. J.E. Karkainen, E.O. Haahti, and A.A. Lehtonen, Anal. Chem., 38, 1317 (1966). 23. G.G.S. Dutton, Adv. Carb. Chem. Biochem., _28, 11 (1973). 24. D.G. Lance and J.K.N. Jones, Can. J. Chem., 45_, 1995 (1967). 25. C C . Chen and CD. McGinnis, Carbohydr. Res., 90, 127 (1981). 26. G.G.S. Dutton, Adv. Carb. Chem. Biochem., 30, 9 (1974). 27. B.A. Dimitriev, L.V. Backinowsky, O.S. Chizhov, and N.K. Kotchetkov, Carbohydr. Res., 1£, 432 (1971). 28. P. Jansson, L. Kenne, H. Liedgren, B. Lindberg, and J . Lonngren, Chej. Commun. (University of Stockholm), No. 8, (1976). 29. J.A. Lomax and J . Conchie, J . Chromatography, 236, 285 (1982). 30. CD. McGinnis and P. Fang, Methods i n Carbohydrate Chemistry, Volume VIII, 33, (1980). 31. M. C u r v a l l , B. Lindberg, and J. Lonngron, Carbohydr. Res., 42, 73 (1975). 32. B.S. Valent, A.G. D a r v i l l , M. McNeill, B.K. Robertsen, and P. Albersheim, Carbohydr. Res., 79, 165 (1980). 87 33. B.K. Robertsen, P. Aman, A.G. D a r v i l l , M. McNeill, and P. Albersheim, Plant Physiol., 67, 389 (1981). 34. R.C.E. Guy, M.J. Howe, M. Stacey, and M. Heidelberger, J . B i o l . Chem., 242, 5106 (1967), c i t e d i n : 0. Larm and B. Lindberg, Adv. Carb. Chem. Biochem.,33, 295 (1976). 35. J.F. Kennedy and J.E. Fox, Methods i n Carbohydrate Chemistry, Volume VIII, 3, (1980). 36. R.E. Wing and J.N. BeMiller, Methods i n Carbohydrate Chemistry, Volume VI, 42, (1972). 37. K.L. Mackie, Ph.D. Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1977. 38. M.Y.M. Choy, Ph.D. Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1973. 39. H. Van Halbeek, L. Dorland, J . Haverkamp, G.A. Veldink, J.F.G. Vliegenthart, B. Fournet, G. Rica r t , J . Mo n t r e i u i l , W.D. Gathmann, and D. Aminoff, Eur. J. Biochem., 11_8, 487 (1981). 40. J . Lonngren and S. Svensson, Adv. Carb. Chem. 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Res., 103, 107 (1982). 53. A.V. Savage, Ph.D. Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1980. 54. L. Hough, J.V.S. Jones, and P. Wusteman, Carbohydr. Res., 21, 9 (1972). 55. H.E. Conrad, J.R. Bamburg, J.D. Epley and T.J. Kindt, Biochemistry, _5, 2808 (1966). 56. G.G.S. Dutton and S. Kabir, Anal. Lett., 4_, 95 (1971). 57. R.L. Taylor and H.E. Conrad, Biochemistry, U_, 1383 (1972). 58. T. Purdie and J.C. Irvine, J. Chem. Soc. (London), 83, 1021 (1903). 59. W.M. Haworth, J . Chem. Soc. (London), 107, 8 (1915). 60. R. Kuhn, H. Trischmann, and I. Low, Angew. Chem., 67_, 32 (1955), c i t e d i n reference 62. 61. S. Hakomori, J . Biochemistry (Tokyo), _55, 205 (1964). 62. H. Bjorndal, C.G. H e l l e r q v i s t , B. Lindberg, and S. Svensson, Angew. Chem. Internat. E d i t . , 9_, 610 (1970). 63. B. Capon, Chem. Rev., 69, 407 (1969). 64. T. Folkman, M.Sc. Thesis, University of B r i t i s h Columbia, 1979. 65. B. Lindberg, J . Lonngren, and J.L. Thompson, Carbohydr. REs., 28, 351 (1973). 66. G.O. A s p i n a l l and K.G. Ro s e l l , Carbohydr. Res., 57, C23 (1977). 89 67. G.O. A s p i n a l l , T.N. Krishnamurthy, W. Mitura, and M. Funabashi, Can. J . Chem., 53, 2182 (1975). 68. M. C u r v a l l , B. Lindberg, and J. Lonngren, Carbohydr. Res.,42, 73 (1975). 69. C S . Hudson, J . Am. Chem. Soc. , 31, 66 (1909). 70. G.M. Bebault, J.M. Berry, Y.M. Choy, G.G.S. Dutton, N. Funnell, L.D. Hayward, and A.M. Stephen, Can. J . Chem., 51_, 324 (1973). 71. E.H. M e r r i f i e l d , Ph.D. Thesis, U n i v e r s i t y of Capetown, 1978. 72. G.G.S. Dutton and M: Heidelberger, J. Immunol., I l l , 857 (1973). 73. R.H. Furneux, Xlt h International Carbohydrate Symposium, Abstracts, 11-28 (1982). 74. G. H a s s e l l , Chemistry 449 Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1982. 75. H. Bjorndal, B. Lindberg, J . Lonngren, M. Meszaros, J.L. Thompson, and W. Nimmich, Carbohydr. Rgs., 31_, 93 (1973). 76. D.P. Sweet, R.H. Shapiro, P. Albersheim, Carbohydr. Res., 40, 217 (1975). 77. T.E. Folkman, M.Sc. Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1979. 78. V. Kovacik, S. Bauer, J. Rosik and P. Kovac, Carbohydr. Res., 8_, 282 (1968). 90 APPENDIX I: Q u a l i t a t i v e Sugar Analysis of the K l e b s i e l l a Capsular Polysaccharide. Glucuronic Acid, Galactose, Glucose 8 P, l l p , 15, 51, 25, 27p Glucuronic Acid, Galactose, Mannose 20, 2 1 p , 2 9 p , 42 p, 43, 66, 74p Glucuronic Acid, Galactose, Rhamnose 9, 47, 52, 9*, 81, 83 Glucuronic Acid, Glucose, Mannose 2, 4, 5 P , 24 Glucuronic Acid, Glucose, Rhamnose 17, 44, 71 Glucuronic Acid, Glucose, Fucose 1, 54 Glucuronic Acid, Galactose, Glucose, Mannose 10, 28, 39, 50, 59, 61, 62, 7 p , 13 p, 26 p, 30 p, 31 p, 33 p, 35 P, 46 p, 69p , 60 Glucuronic Acid, Galactose, Glucose, Fucose 16, 58p Glucuronic Acid, Galactose, Glucose, Rhamnose 18, 19, 23, 41, 79, 12p, 36 p, 45 p, 55 p, 70P Glucuronic Acid, Galactose, Mannose, Rhamnose 53, 40, 80p Glucuronic Acid, Glucose, Mannose, Fucose 6 P Glucuronic Acid,- Glucose, Mannose, Rhamnose 64 p, 65p Glucuronic Acid, Galactose, Glucose, Mannose, Fucose 68p Glucuronic Acid, Galactose, Glucose, Mannose, Rhamnose 14p, 67 Galacturonic Acid, Galactose, Mannose 3 P , 49, 57 Galacturonic Acid, Glucose, Rhamnose 34, 48 Galacturonic Acid, Galactose, Fucose, Rhamnose 63 Pyruvic Acid, Glucose, Rhamnose 72 Pyruvic Acid, Galactose, Rhamnose 32 Pyruvic Acid, Galactose, Glucose, Rhamnose 56 Keto Acid, Galactose, Glucose 22, 37, 38 12 K82 has been added but its qualitative composition is not yet known. P- Pyruvic acid present in addition APPENDIX I I N u c l e a r M a g n e t i c Resonance S p e c t r a 93 5.0 102 S r -103 108 Appendix III The main purpose f o r the m.s. analysis of K39-A1 as the permethylated a l d i t o l was to d i s t i n g u i s h between the following two possible structures: 1 2 Man — GlcA 1 2 Man 1 3. Glc~ OH i 5 a i I a 1 4 GlcA — Man 1_ _2 •Man 1. 3 Glc~ OH In t h e i r study of a permethylated a l d o t r i o u r o n i c acid, Kovacik and 43 coworkers-,: showed that the A-series fragments should be intense enough to provide unambiguous sequence information. Structure X gives the following m/e values f o r the A-se r i e s : 437 (baAp; 405 (baA^), 373 (baA^). Structure Y gives the following m/e values for the A-series: 451 (baA 'p , 419 (baA^) ; 387 (baA ). A spectrum was recorded with an electron beam energy of 20 eV. The m/e values mentioned above are given i n the following l i s t with the r e l a t i v e abundances obtained given i n brackets: 373 (46), 387 (18), 405 (100), 419 (52), 437 (14), 451 (18). Therefore: Sum of the abundances.for structure X Sum of the abundances f or structure Y A spectrum was recorded with an electron beam energy of 70 eV. The 109 m/e values mentioned above are given i n the following l i s t with the r e l a t i v e abundances given i n brackets: 373 (59), 387 (22), 405 (100), 419 (49), 437 (20), 451 (18). Therefore: Sum of the abundances for structure X = 2.0 Sum of the abundances for structure Y On the basis of these data, structure X i s preferred. Further studies of the permethylated a l d i t o l of K39 - Al could include a reduction of the esters to primary alcohols with a deuterating reducing agent, followed by r e a l k y l a t i o n . M.s. analysis of the product could provide more information regarding the o r i g i n of the fragment ions mentioned above, and thus remove any ambiguity i n the sugar sequence determination. 110 APPENDIX IV: Immunochemical Cross-Reactions of E. c o l i K26. I l l APPENDIX V : An Explanation of Carbohydrate Terminology In an e f f o r t to f a m i l i a r i z e readers who do not work in the p a r t i c u l a r area of o rganic chemistry to which t h i s t h e s i s r e f e r s , the f o l l o w i n g exp lana t ion of terms used i s o f f e r e d . F i s c h e r p r o j e c t i o n formulae are used to represent the a c y c l i m o d i f i c a t i o n of sugars. Some examples are shown below. Numberin commences from the carbonyl group at the top of the chain ( I ) . Note that D - g l u c u r o n i c a c i d (I.I) d i f f e r s from D-glucose (I) only CHO * -OH * —OH HO— * HO— * C! I 3 L-rhamnose ( I I I ) in that C-6 i s o x i d i z e d to a c a r b o x y l i c a c i d group. The C - 6 of L-rhamnose ( I I I ) i s part of a methyl group and i s r e fe r red to a lso by another common name, 6-deoxy-L-mannose. There are four c h i r a l centers in these s ix -ca rbon chains (marked with a s t e r i s k s in s t r u c t u r e I I I ) making i t important to apprec ia te the s p a t i a l arrangement of atoms that i s i m p l i e d by these F i s c h e r r e p r e s e n t a t i o n s . To s i m p l i f y the nomenclature of a l l the p o s s i b l e isomers (16 for each of ,CHD 2 ^-OH H O H 3 4 j—OH 5 —OH CH^OH CHO — OH H 0—H — OH — UH C00H D-glucose ( I ) D - g l u c u r o n i c ac id ( I D 112 I , I I , I I I ) , a l l those having the hydroxyl group at the h ighes t -numbered c h i r a l center ( C - 5 ) p r o j e c t i n g to the r i g h t in the F i s c h e r p r o j e c t i o n formulae belong to the D - s e r i e s , and the o thers to the L - s e r i e s . —OH H0-CH 20H CH20H D - s e r i e s L - s e r i e s P h y s i c a l and chemical evidence i n d i c a t e s tha t , in f a c t , these s ix -ca rbon polyhydroxyaldehydes e x i s t in a c y c l i c form. The r i n g c lo su re occurs by n u c l e o p h i l i c a t tack of the oxygen atom at C-5 on the a ldehyd ic carbon atom, genera t ing a new c h i r a l (anomeric) center at C-1. This r e s u l t s in two anoiners, represented below CH20H H O H CH2-0H ct-D -g lu c ose (IV) 6-D -g lucose ( V ) 113 in the T o l l e n s formulae. It should be noted that C-1 i s unique in having two at tached oxygen atoms, fo rmal ly making i t a hemia-c e t a l carbon. Since the T o l l e n s formulae have obvious l i m i t a t i o n s with t h e i r unequal bond l e n g t h s , Haworth developed a pe r spec t ive method of l o o k i n g at the six-membered r i n g (VI and V I I ) . This improvement recognizes that the r i ng oxygen atom l i e s behind the carbon chain and that bond l eng ths are approximately equa l . Often in p r a c t i s e r egu la r hexagons are used in Haworth p r o j e c t i o n s , which he r e l a t e d to such r ings as the h e t e r o c y c l i c compound pyran ( V I I I ) and named them pyranoses . Note that hydroxyl groups not i n v o l v e d i n r i ng formation on the r i gh t i n F i s c h e r and To l l ens formulae point down i n the Haworth p r o j e c t i o n s and those on the l e f t po in t up. S i m i l a r l y , for a ldopyranoses , the group on C - 5 p o i n t s up for D (IX) and down for the L enantiomer (X) , I t f o l l o w s , then, that when sugar res idues are at tached there are two p o s s i b l e c o n f i g u r a t i o n s , an a - or a 8 - p y rano s i de, for each l i n k a g e . a-D - glucopyranose 3-D-glucopyranose (VI) (VI I ) pyran (VI I I ) 114 HO OH a-D-rhamnopyranose ct-L-rhamnopyranose (IX) (X) The t rue conformation of pyranoid carbohydrates i s r e l a t to the cha i r form of cy c l oh e xan e. X-ray d i f f r a c t i o n a n a l y s i s h shown that a hexose, such as a-O-glu cose ( X I ) , c o n s i s t s of a puckered, six-membered, oxygen-conta in ing carbon r i n g , with hydroxy l s u b s t i t u e n t s at C-1 through C-4, and a hydroxymethyl group at C - 5 . A l l s u b s t i t u e n t s on the r i n g , except for that at C-1 , are e q u a t o r i a l . Two anomeric i som er cen t e r s (anomers) are p o s s i b l e i n r e l a t i o n to the ( C-1), depending on whether a subs t i tuen t i s 115 a x i a l (a-anomer; X I I ) or e q u a t o r i a l ((3-anomer; X 111 ),where R = hydrogen, for monosaccharides,and R = another sugar r e s idue , for d i - , o l i g o - , and p o l y s a c c h a r i d e s . S ince H - l i s i n a d i f f e r e n chemical environment for the two anomers, nuc lea r magnetic resonance spectroscopy can e a s i l y d i s t i n g u i s h between them and, thereby, p rov ides i n v a l u a b l e a s s i s t ance in a s s ign ing anomeric c o n f i g u r a t i o n s . ( X I I ) ( X I I I ) Haworth p r o j e c t i o n s are mo>st usefu l and w i l l be used in t h i s t h e s i s , even though they g ive no i n d i c a t i o n of th ree -dimensional molecu la r shape. There seems to be l i t t l e j u s t i f i -ca t ion for the use of formulae which depic t s ta tes of molecules as wel l as s t r u c t u r e s , when the t rue s ta tes are often unknown or v a r i a b l e . Reproduced from t'he M.Sc. t h e s i s o f T.E. Folkman ( U n i v e r s i t y of B r i t i s h Columbia, 1979). -

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