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Structural investigations and bacteriophage degradations of Klebsiella capsular polysaccharides Savage, Angela V. 1980

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STRUCTURAL INVESTIGATIONS AND BACTERIOPHAGE DEGRADATIONS OF KLEBSIELLA CAPSULAR POLYSACCHARIDES by ANGELA V. SAVAGE .Sc. (Hons), Univers i ty College Galway, Ire land, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1980 © Angela V. Savage, 1980 i In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. I t i s understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my writ ten permission. Department of C \ASUMA/)\ f\M/ The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date b d 6^ i i ABSTRACT Seventy-seven sero log i ca l l y d i f fe rent s t ra ins of K lebs ie l l a are known. The capsular polysaccharides which these bacter ia produce are ant igenic, and in order to understand the chemical basis of serologica l d i f f e ren t i a t i on the structura l invest igat ion of the capsular polysaccharides has been undertaken. To date, f i f t y s i x structures have been determined. The structures of the capsular antigens i so la ted from serotypes K12 and K58 are presented here, along with confirmative data for the structure of K23 and a nuclear magnetic resonance invest igat ion of K70 and i t s spec i f i c degradation products. An e f f i c i en t means of i so l a t i ng large quant i t ies of the . s ingle repeating units of the K lebs ie l l a polysaccharides using glycanase enzymes, borne and u t i l i z e d by spec i f i c bacteriophage, is demonstrated. K lebs ie l l a K21 polysaccharide has been degraded using a highly pur i f i ed bacteriophage ( e-galactopyranosidase a c t i v i t y ) , while K lebs ie l l a K12 and K lebs ie l l a K41(which have s im i la r structures) have both been degraded using a crude solut ion of bacteriophage spec i f i c for K l ebs i e l l a K12 ( 0-galactofuranosidase a c t i v i t y ) , and resul ts compared. A prel iminary invest igat ion of the use of high pressure l i qu i d chromatography in the s t ructura l invest igat ion of hetero-polysaccharides i s included, along with appendices containing i i i compilations of the structures and structura l patterns of the K lebs ie l l a capsular polysaccharides determined to date. K12 K3)-a-D-Galg- (1+2)-3-D-Gal f-(l-^)-a-D-GTcg-(l+3)-a-L-Rhap-(1-|3 1 g-D-Glcp_A 4 1 3-D-Gal£ 6 4 \/ A Me COOH K58 O-Ac 2 ^3)-a-D-Glcp_-(l+4)-B-D-Glcp_A-(M)-a-L-Fucp_-(l-V Me/ \ o O H 3 + 1 a-D-Gal£ i v TABLE OF CONTENTS Page ABSTRACT 1 1 TABLE OF CONTENTS i v LIST OF TABLES V i i i LIST OF FIGURES . x LIST OF SCHEMES x i i ACKNOWLEDGEMENTS xi i i PREFACE x iv I INTRODUCTION 1 II METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES 20 11.1. Iso lat ion and Pur i f i ca t i on 21 11.2. Separation Techniques 22 , . I I . 2 . 1 . Paper chromatography (p.c . ) 22 11.2.2. Paper electrophoresis (p.e.) 23 11.2.3. Gel chromatography (g .c . ) 24 I I . 3 Instrumentation 25 11.3 .1 . Gas- l iquid chromatography ( g . l . c ) . . 25 11.3.2. Mass spectrometry (m.s.) 32 11.3.3. Polarimetry 36 11.3.4. C i r cu la r dichroism (c.d.) 38 11.3.5. Nuclear Magnetic Resonance Spectroscopy • 38 I I . 3 . 5 . 1 . ,1H n.m.r. spectroscopy . . 39 I I .3 .5 .2 C n.m.r. spectroscopy . . 44 I I .4 . Techniques of Structure Determination . . . . 40 11.4.1. Character izat ion of component sugars. 50 11.4.2. Methylation analysis 51 11.4.3. Oxidation 55 V TABLE OF CONTENTS Page 11.4.4. Reduction 57 11.4.5. Base-catalyzed degradation 59 11.4.6. Par t i a l hydrolysis 59 11.4.7. Location of 0-acetyl group 63 III STRUCTURAL INVESTIGATION OF KLEBSIELLA CAPSULAR POLYSACCHARIDES 65 111 . 1 . Structural Invest igat ion of the Capsular Polysaccharide of K lebs ie l l a K12. ABSTRACT . . 66 111.1.1. Introduction 66 111.1.2. Results and discussion 67 111.1.3. Conclusion 76 111.1.4. Experimental 77 111.2. Structural Invest igat ion of the Capsular Polysaccharide of K lebs ie l l a K58.ABSTRACT. . . 83 111.2.1. Introduction . 83 111.2.2. Results and discussion 84 111.2.3. Conclusion 96 111.2.4. Experimental . . . 96 111.3. Confirmation of the Structure of K lebs ie l l a K23 Capsular Polysaccharide 104 111.4. "'H and 1 3 C Spectral Invest igat ion of K l ebs i e l l a K70 Capsular Polysaccharide 108 IV BACTERIOPHAGE DEGRADATION OF KLEBSIELLA CAPSULAR POLYSACCHARIDES K21, K12 AND K41 112 IV. 1. Introduction 113 IV.2. Results 117 IV.2.1. Iso lat ion and pur i f i ca t i on 117 IV.2.2. Conditions of depolymerization. . . . 118 IV.2.3. Pu r i f i ca t i on of analyses of products of depolymerization of K21 118 IV.2.4. Pu r i f i ca t i on and analyses of products depolymerization of K12 and K41. . . 122 IV.3. Discussion 127 vi TABLE OF CONTENTS Page IV. 4. Experimental 129 V. HIGH PRESSURE LIQUID CHROMATOGRAPHY OF CARBOHYDRATES.. 135 V. l . Introduction 136 V.2. Chromatographic Conditions 140 V.3. Results and Discussion 141 V.4. Conclusions 148 V.5. A l ternat ives 150 VI. BIBLIOGRAPHY • . 153 v i i APPENDIX Page I The Known Structures of the K lebs ie l l a Capsular Polysaccharides 174 II Structural Patterns of K lebs ie l l a Capsular Polysaccharides 195 III ]H and 1 3 C n.m.r. Spectra 200 IV Methodology of Bacteriophage Propagation and Polysaccharide Iso lat ion 232 vi . i i LIST OF TABLES TABLE Page I K lebs ie l l a capsular polysaccharides (K1-K83). 'Quantitative analysis and chemotype grouping 6 2 C Methylation analysis of a polysaccharide 53 3 Methylation procedures 53 4 G.I.e. analysis of native and periodate oxidized K12 polysaccharide 68 5 N.m.r. data for K lebs ie l l a K12 capsular polysaccharide and the derived ol igosaccharides 70 6 Methylation analysis of nat ive, and degraded, K lebs ie l l a K12 capsular polysaccharide 73 7 N.m.r. data for K lebs ie l l a K58 capsular polysaccharide and the derived ol igosaccharides 85 8 Methylation analys is of nat ive, and degraded, K lebs ie l l a K58 capsular polysaccharide 89 9 P.m.r. data for K lebs ie l l a K23 capsular polysaccharide 105 10 Methylation analysis of or ig ina l and base degraded K lebs ie l l a K23 capsular polysaccharide 106 II N.m.r. data for K lebs ie l l a K70 capsular polysaccharide and ol igosaccharides i so la ted 109 12 M.m.r. data for K lebs ie l l a 21 capsular polysaccharide and the ol igosaccharides PI and P2 123 13 Determination of degree of polymerization of PI and P2 (K21) and i den t i f i c a t i on of the reducing sugar 124 14, N.m.r. data for Klebsiel la K12 polysaccharide and the phage derived ol igosaccharide 125 15 N.m.r. data for K lebs ie l l a K41 polysaccharide and the phage derived ol igosaccharide 128 LIST OF TABLES Separation of Mono-, Di*- and Trisaccharides using HPLC Separation of products of Smith Degradation using HPLC X LIST OF FIGURES FIGURE Page 1 Diagramatic representation of the bacter ia l ce l l envelope 3 2 Ext race l lu la r polysaccharide 3 3 Cross-reactions of K l ebs i e l l a K12 capsular polysaccharide 12 4 The antibody combining s i t e 13 5 G . l . c . separation of a l d i t o l acetates from periodate oxidation of K lebs ie l l a K12 27 6 G.I.e. separation of products of methylation analysis of K lebs ie l l a K12. 28 7 Degree of polymerization of product of bacteriophage degradation of K lebs ie l l a K21, using g . l . c . 30 8 G . l . c . separation of products of uronic acid degradation of K lebs ie l l a K12. 31 9 Par t ia l hydrolysis apparatus 58 10 G . l . c . separation of the products of methylation analysis of K lebs ie l l a K58 90 11 Morphological grouping of phages according to Bradley 114. 12 Schematic representation of a Type-A pa r t i c l e 114 13 Bacteriophage degradation of K21 polysaccharide 119 14 Separation of products of bacteriophage degradation of K21 polysaccharide 120 15 Electron micrograph of pur i f i ed K21 bacteriophage 121 16 Separation of monosaccharides using HPLC (Column A) 144 17 Separation of monosaccharides using HPLC (Column B) 145 x i LIST OF FIGURES FIGURE Page 18 Separation of d i - and tr isacchar ides using HPLC, with d i f fe rent flow rates 146 19 Separation of some products of Smith degradation, using HPLC 147 20 Growth curve and bacteriophage l y s i s of K lebs ie l l a K21 bacteria 236 x i i LIST OF SCHEMES SCHEME Page 1 Antibody production and the bacter ic ida l react ion 11 2 Phagocytosis 16 3 Primary and secondary fragmentation pattern and mass spectrum of 1,5 Di -O-acety l -2,3,4,6- tetra-0-methyl-Q-glucitol 33 4 Methylation analysis of a polysaccharide 52 5 Select ive oxidation and degradation 56 6 Reduction of a carboxyl ic acid in aqueous so lut ion using a carbodiimide reagent 58 7 Base-catalysed degradation of K lebs ie l l a Kl2 60 8 Par t ia l hydrolysis and pur i f i ca t i on of K lebs ie l l a Kl2 62 9 Base-catalysed degradation of K lebs ie l l a K58 92 10 Periodate oxidation of K lebs ie l l a K58 93 11 Par t ia l hydrolysis of K lebs ie l l a K58 95 12 Block diagram of instrumentation for high pressure l i qu i d chromatography 137 13 Iso lat ion and pur i f i ca t i on of polysaccharide 234 i xi i i ACKNOWLEDGMENTS The st imulat ing d i rec t ion of Professor G.G.S. Dutton and the cheerful support of my col leagues, in par t i cu la r Jose Di Fabio, are gra te fu l l y acknowledged. I wish to thank Robert S t , -P ie r re for the i l l u s t r a t i o n s , Dr. E.H. Mer r i f i e l d for proof reading, and Celine Gunawardene for typing th i s thes i s . I am grateful to MacMillan Bloedel for the award of a graduate scholarship (1977-1978). xiv PREFACE This thesis has been wri t ten in the context of the a c t i v i t i e s of our research,group which are concerned with the determination of the primary structure, along with spec i f i c degradations, of carbohydrate antigens. Many of the methods used in th is f i e l d are considered as standard, and so have been discussed Only b r i e f l y . However, in the l a s t few years there have been considerable advances in instrumental techniques and there are now su f f i c i en t appl icat ions to polysaccharide chemistry reported in the l i t e r a tu re to warrant a review of these methods. Therefore, a more deta i led account of the use of nuclear magnetic resonance spectroscopy 1 13 (both H and C) in the study of polysaccharides i s presented here. I t i s proposed that future theses emanating from th i s group w i l l s im i l a r i l y review gas- l iqu id chromatography, mass spectrometry, and high performance l i qu id chromatography; these techniques are, accordingly, not given special treatment here. In Appendix I I have revised the l i s t of known structures of K lebs ie l l a polysaccharides along with l i t e r a tu re references, compiled by Keith Mackie (Ph.D. 1977). Bacteriophage attack s i t e s , opt ica l ro ta t ions , and x-ray crystal lography references have also been included. In his M.Sc. thesis (1980) Marcel Paul in c l a s s i f i ed the known K lebs ie l l a structures according to the i r structura l patterns. This has been revised and included in Appendix' H with kind permission. XV In the Introduction I have attempted to present my work in a h i s t o r i c a l context and to show i t s relevance to current immunochemical research, although the main body of work i s pr imar i ly chemical in nature. As has been the pract ice with other theses presented by members of our group an explanation of carbohydrate nomenclature4" i s now offered to f am i l i a r i ze readers who are not acquainted with the f i e l d . i Fischer project ion formulae are used to represent the acyc l i c modi f icat ion.of sugars. Some examples are shown below. Numbering commences from the carbonyl group at the top of the chain ( I ) . Note that D-glucuronic acid (II) d i f f e r s from Q-glucose (I) only C H O 1 2 - O H H O - 3 4 - O H 5 ~ O H 6 C H 2 0 H C H O LoH ho-\ - O H - O H C O O H C H O - O H - O H H O -H O -C H . D-glucose ( I ) D-g lucuron ic a c i d ( I I ) L-rhamnose ( I I I ) in that C-6 i s oxidized to a carboxyl ic acid group. The C-6 of ^-rhamnose (II I) is part of a methyl group and is referred to also by another common name, 6-deoxy-L-mannose. XVI There are four ch i ra l centers in these six-carbon chains (marked with aster isks in structure III) making i t important to appreciate the spat ia l arrangement of atoms (configuration) that i s implied by these Fischer representations. To s impl i fy the nomenclature of a l l the possible isomers (16 for each of I , I I , I I I ) , a l l those having the hydroxyl group at the highest-numbered ch i ra l center (C-5) project ing to the r ight in the Fischer project ion formulae belong to the D-series, and the others to the L-ser ies. Physical and chemical evidence indicates that , in fac t , these six-carbon polyhydroxyaldehydes e x i s t . i n a c y c l i c form. The r ing closure occurs by nucleophi l ie attack of the oxygen atom at C-5 on the aldehydic carbon atom, generating a new ch i ra l (anomeric) center at C - l . This resu l ts in two anomers, represented below HCH D-ser ies L -se r ies H OH HO H C t _ 0 H | _ OH HO _ J 0 HO—I 0 I—O H \—0H CH20H o-D-glucose (IV.) CH20H 0-D-glucose (V) \ xvi i in the Tollens formulae. I t should be noted that C-l is unique in having two attached oxygen atoms, formally making i t a hemiacetal carbon. Since the Tollens formulae have obvious l im i ta t ions with t he i r unequal bond lengths, Haworth developed a perspective method of looking at the six-membered r ing (VI and V I I ) . This improvement recognizes that the r ing oxygen atom.l ies behind the carbon chain and that bond lengths are approximately equal. Often in .prac t ice regular hexagons are used in Haworth pro ject ions, OH OH a - D - g l u c o p y r a n o s e p - D - g l u c o p y r a n o s e p y r a n ( V I ) ( V I I ) ( V I I I ) which he related to such r ings at the heterocycl ic compound pyran ( V I I I ) and named them pyranoses. Note that hydroxyl groups not involved in r ing formation on the r igh t in Fischer and Tollens formulae point down in the Haworth project ions and those on the l e f t point up. S im i la r l y , for aldopyranoses, the group on C-5 points up for D(IX') and down for the L enantiomer (X). I t fo l lows, then, that when sugar residues are attached there are two possible conf igurat ions, an a - or a 3-pyranoside, for each l inkage. xvi i i HI OH HO OH HO OH a-D-rhamnopyranose (IX) The true conformation of pyranoid carbohydrates is related to the chair form of cyclohexane. X-ray di f f ract ion analysis has shown that a hexose* such as a-D-glucose (XI) , consists of a puckered, six-membered, oxygen-containing carbon ring with hydroxyl substituents at C-l through C-4, and a hydroxymethyl group at C-5. All substituents on the r ing, except for that at C-l, are equatorial. anomeric center (C- l ) , depending on whether a substituent is axial OH (XI) Two isomers (anomers) are possible in relation to the xi x\ (a-anomer; XII) or equatorial (g-anomer; X I I I ) , where R = hydrogen, for monosaccharides, and R = another sugar residue, for d i - , o l i g o - , and polysaccharides. Since H-l is in d i f fe rent chemical environment for the two anomers, nuclear magnetic resonance spectroscopy can eas i l y d is t inguish between them and, thereby, provides invaluable assistance in assigning anomeric conf igurat ions. Haworth project ions are most useful and w i l l be used in th i s thesis., even though, they give no ind icat ion of three-dimensional molecular shape. There seems to be l i t t l e j u s t i f i c a t i o n for the use of formulae which depict states of molecules as well as st ructures, when the true states are often unknown or var iab le . + The Carbohydrates. Chemistry and Biochemistry Vo l . II B. (Eds.W. Pigman and D. Horton). Academic Press. New York. 809-834 (1972) : t- Reproduced with the kind permission of T.E. Folkman from his M.Sc. thesis en t i t l ed "Structural Studies on K lebs ie l l a Capsular Polysaccharides", Univers i ty of B r i t i s h Columbia, Apr i l 1979. 1 I . INTRODUCTION 2 I . INTRODUCTION Polysacchar ides are ub iqu i tous - they are by f a r the most 1 2 3 abundant biopolymers on e a r t h . Those such as c e l l u l o s e ' and gum 4 5 arab ic ' have been recognized and used by man f o r c e n t u r i e s . More r e c e n t l y the exopolysacchar ide produce by Xanthomonas j u g l a n d i s has become o f economic value in enhanced o i l recovery systems ^ , 8 9 and a l g i n a t e ' , obta ined from c e r t a i n species o f marine algae i s commercia l ly impor tan t as a food a d d i t i v e . 1 0 Research i n t e r e s t s had, f o r many y e a r s , centered on p l a n t po lysacchar ides , and l a t e r , on mucopolysaccharides o f h igher a n i m a l s , ^ but the compara t ive ly recent r e a l i z a t i o n t h a t m ic rob ia l po lysacchar ides are composed o f r e g u l a r repeat ing u n i t s , along w i t h the f a c t t h a t they p l a y an impor tan t r o l e i n fundamental research on the immune r e a c t i o n , have prompted the systemat ic i n v e s t i g a t i o n o f the s t r u c t u r e s o f the exopolysacchar ides o f var ious f a m i l i e s . 1 g M ic rob ia l po lysacchar ides are loca ted on the c e l l sur face and a r e , t h e r e f o r e , o f importance i n the r e c o g n i t i o n and immune response o f a h igher organism to m i c r o b i a l i n f e c t i o n . The po lysacchar ides are e i t h e r an i n t e g r a l p a r t o f the c e l l wa l l - l i popo lysacchar ide (LPS) or occur as a s l ime or capsule as i n the case o f Pneumococcus, Escher ich ia  c o l i , and K l e b s i e l l a (see F i g . 1 ) . "Capsular" po lysacchar ide i s considered to have a d e f i n i t e boundary and t o remain adherent to the c e l l wa l l when suspended in 3 GRAM + POLYSACCHARIDE CAPSULE (K) LIPOPOLYSACCHARIDE COMPLEX (0) PEPTIDOGLYCAN PERIPLASM CELL MEMBRANE Figure 1 Diagramatic representation of the bacter ia l ce l l envelope LPS (0 Antigen) CAPSULE (K Antigen) SLIME Figure 2 Ext race l lu la r polysaccharide. 4 water. The term "slime" i s used to indicate a network of carbohydrate f ibres not d i s t i n c t l y associated with any one bacterium. Dudman and Wilkinson found that capsular and slime polysaccharides are ident i ca l 17 13 in chemical composition. 5 . Due to the high water content of the capsule (99%) only wet preparations, such as India ink f i lms , can give accurate information about the s ize and shape of the capsule or sl ime. When water i s removed from capsules of K lebs ie l l a bacter ia , by means of .ethanol , for v i sua l i za t ion by electron microscopy, the ind iv idual f i b r i l s of the capsule col lapse on one another to form thick project ions. Occasionally there i s some evidence for peripheral l i nk ing of f ib res . This would f i t well the idea of a wel l -def ined capsular edge (see F ig. 2). P r a c t i c a l l y a l l bacter ia l capsules consist of polysaccharides and are associated with pathogenic micro-organisms. The genus K lebs ie l l a i s composed of Gram-negative, nonmotile bacter ia , of the 19 20 family Enterobacteriaceae and the t r ibe K lebs ie l leae ' . I sol ants are i den t i f i ed and c l a s s i f i e d by biochemical reactions into three species: K. pneumoniae, K. ozaenag^ and K. rhinoschleromatis. Two d i s t i n c t antigens are present in encapsulated stra ins of K l ebs i e l l a , one in the capsule (K antigen) and the other in the soma, (see F ig. 1). The capsular antigen was shown to be carbohydrate in nature by p i p o Toenniessen t- ' , t- 1- in 1914. Since most K lebs ie l l a bacter ia are heavi ly encapsulated the 0-antigen i s completely shielded (see Figs. 1 and 2). Consequently, serologica l c l a s s i f i c a t i on i s based so le ly on the i r capsular K-antigens. To date seventy seven sero log i ca l l y d i f fe rent 5 23 24 capsules have been del ineated ' . Many of these micro-organisms are found in healthy carr iers 25 in the upper resp i ratory, i n t e s t i n a l , and genito-urinary t racts Their pathogenicity to man i s wel l known although some stra ins are not tox ic . Acute in fect ion in the lung occasional ly mimics pneumococcal lobar pneumonia. The chronic form resembles tuberculos is . K l ebs i e l l a pneumonia K-types 1, 2, 7 and 8 are the commonest invaders and are responsible for approximately three per cent of a l l bacter ia l pneumonias, 26 occurring when host-resistance i s impaired, for example, in a lcohol ics . Almost a l l s t ra ins of K_. ozaenae are members of K type 4, and are associated with ozena, a f e t i d , catarrhal condit ion of the nose. Infections due to K_. rhinoschleromatis occur rare ly in North America. K lebs ie l l a res i s t s many antimicrobiiC drugs. Streptomycin and chloramphenicol have proved of value in therapy, but the proportion of res is tant s t ra ins , due to mutations, i s increasing s tead i ly . One of the most outstanding features of bacter ia l polysaccharides to become apparent of late i s that they are composed of regular 27 28 repeating units , as shown by molecular weight d i s t r i bu t ion studies^ , 29 and more recently by nuclear magnetic resonance spectroscopy.. A l l are heteropolysaccharides and have proved to be an almost inexhaustible source of novel ol igosaccharides and sugars. 30 31 Nimmich has reported the qua l i t a t i ve composition of the 32 K lebs ie l l e K-types and has also c l a s s i f i e d them into chemotypes (see Table 1). 6 Glucuronic Ac id , Galactose, Glucose 8 P , l l p , 15, 51, 25, 27 p Glucuronic Ac id, Galactose, Mannose 20, 2 1 p , 29 p , 42 p , 43, 66, 74 p Glucuronic Ac id , Galactose, Rhamnose 9, 47, 52, 9*, 81, 83 Glucuronic Ac id, Glucose, Mannose 2, 4, 5 P , 24 Glucuronic Ac id, Glucose, Rhamnose 17, 44, 71, 23, 45 p Glucuronic Ac id, Glucose, Fucose 1, 54 Glucuronic Ac id , 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 , 69 P , 60 Glucuronic Ac id , Galactose, Glucose, Fucose 16, 58 p Glucuronic Ac id, Galactose, Glucose, Rhamnose 18, 19, 12 P ,41, 79, 70 P , 36 p , 55 p. Glucuronic Ac id, Galactose, Mannose, Rhamnose 53, 40, 80 p Glucuronic Ac id, Glucose, Mannose, Fucose 6 P Glucuronic Ac id , Glucose, Mannose, Rhamnose 64 p , 65 p Glucuronic Ac id , Galactose, Glucose, Mannose, Fucose 68 p Glucuronic Ac id , Galactose, Glucose, Mannose, Rhamnose 14 p , 67 Galacturonic Ac id, Galactose, Mannose 3 P , 49, 57 Galacturonic Ac id , Glucose, Rhamnose 34, 48 Galacturonic Ac id , Galactose, Fucose 63 Pyruvic Ac id , Glucose, Rhamnose 72 Pyruvic Ac id , Galactose, Rhamnose 32 Pyruvic Ac id, Galactose, Glucose, Rhamnose 56 Keto Acid, Galactose, Glucose 22, 37, 38 24 K82 has been added but i t s qua l i t a t i ve composition i s not yet known. P- Pyruvic acid present in addit ion Note: K9 and K9*, see Appendices I and II TABLE 1 ; K lebs ie l l a capsular polysaccharides (K1-K83) Quantitat ive analys is and chemotype grouping. 7 All the capsules are acidic, due to the presence of either glucuronic acid, galacturonic acid or a keto acid. In addition pyruvic acid, linked as a ketal may be present and occasionally is the only acidic component (see Table 1). The hexoses Q-glucose, Q-galactose and D-mannose usually occur in the pyranose from - the 33 34 furanosyl form of D-galactose occurs in Kl2 and K 41 . In some strains the 6-deoxyhexoses L-rhamnose and L-fucose are found; non-carbohydrate 0-acetyl and 0-formyl groups may also occur. To date structures have been proposed for fifty five Klebsiella polysaccharides. Various structural patterns (linear, branched, comb-like, etc.) have emerged. These have been tabulated 35 by Paulin (see Appendix II). The number of sugars per repeating unit varies from three to seven .Polysaccharides are capable of greater diversity per unit structure than other types of macromolecules and so i t is not surprising, then, that serological testing has denoted seventy seven different types. The quantity of capsular polysaccharide produced by organisms has been found to be dependant on culture conditions. For 37 optimal production a low nitrogen content of the medium is essential Little is known about the function of microbial extracellular polysaccharides, in contrast to the wealth of detailed knowledge of 3 their composition and structure. This 'imbalance7 has been attributed to the early discovery that the antigenic specificity of bacterial cells was determined by their outermost'components -. invariably polysaccharides. 8 The great majority of polysaccharide - producing micro-organisms appear to be unable to depolymerize or to u t i l i s e the i r own ex t race l l u l a r polysaccharides as carbon sources. The fol lowing functions have, however, been proposed by Dudman. (a) Virulence - protection against serum bacter ic ida l factors and phagocytosis (b) Protect ion against predation (c) Protection against desiccat ion (d) Adhesion in aqueous environments (e) Role in dental cariogenesis (f) Role in ion ic interact ions (g) Role as general barr iers (h) Role in enzyme reactions ( i ) Role in s i l i c on metabolism The f i r s t mentioned w i l l be dealt with l a t e r . The past few years have seen a notable advance in the study of polysaccharide secondary and te r t i a ry st ructure, both in 39 solut ions and gels and by x-ray d i f f r a c t i on . Progress in the l a t t e r f i e l d has been made mainly due to the development of improved 40 c r y s t a l l i z a t i on techniques by Atkins and the concomitant development of sophist icated computer programmes for analysing the 41 d i f f r a c t i on data obtained. In his review of 1979 Atkins offers stereochemical models for eight K lebs ie l l a serotypes - K5, K8, K9, K16, K25, K54, K57 and K63. In the l a s t mentioned case the x-ray d i f f r a c t i on resu l ts help d i f fe ren t ia te between two proposed structures. 9 In dealing with bacter ia l polysaccharides as antigens ". , two aspects have to be considered. One i s the i r capacity to induce the formation of antibodies in mammals i . e . the i r IMMUNOGENICITY, and the other is t he i r r eac t i v i t y with ant ibodies, i .e . the i r ANTIGENIC SPECIFICITY. Our present knowledge in th is f i e l d i s based to a large extent on the pioneering work of Heidelberger and coworkers at the Rockerfe l ler 45-47 48 Inst i tu te and of Kabat _et _al,. . The former i n i t i a t e d quant i tat ive studies in immunochemistry, while i t i s large ly the work of the l a t t e r from which the concept of the antibody combining s i t e was developed. The aim of the immunochemical analysis of polysaccharide antigens, which combines serologica l and chemical studies, i s to define ol igosaccharide structures within the polysaccharide as chemical expression of i t s immunological character. According to a 49 proposit ion of Staub and Heidelberger the sugar unit that contributes most to the sero logica l s pe c i f i c i t y i s termed the IMMUNODOMINANT sugar. I t may be terminal or within the main chain. Not only the nature of the sugar uni t , i t s substituents ( i f any) and the anomeric configuration of i t s l inkage, but also the pos i t ion to which i t i s l inked may greatly inf luence the antigenic expression of the determinant. In ac id i c polysaccharides, such as those of K lebs ie l l a , the charged constituents are often immunodominant sugars or part of antigenic 50 determinants . Due to the i r repet i t i ve structure, bacter ia l polysaccharides have the same antigenic determinants expressed many times over (see F ig. 3). 10 Recent developments in immunology may offer an explanation of the antigenic properties of these polysaccharides. It is postulated that for the production of antibodies to any immunogen certain cells of the host's immune system have to co-operate (see Scheme 1). The most important cells are the T-lymphocytes (Thymus derived) which recognize and concentrate the antigen and present i t to the B-lymphocytes (bone-marrow derived), which, after further differentiation to plasma cells, produce antibody molecules. Polysaccharides are T-cell independent and also activate the alternate complement pathway. They may be so since their antigenic determinants are close enough together and also numerous enough to react effectively with B-cells. It is worth noting that the antibodies produced are almost exclusively of the lg M type. However, when the antigen is an oligosaccharide linked to a protein carrier (see later), then, T-cell co-operation is required and the antibodies subsequently induced are lg G and IgAas well as lg M. In many polysaccharides from different organisms identical oligosaccharide regions are found. As a consequence of this the same determinant is recognised by its homologous antibody in different polysaccharides. Therefore these polysaccharides, no matter in what organism they are produced, are immunologically related; they cross-c p react serologically (see Figs. 3, 4). 11 OR I : O—D> O—O O—> Cross- l ink ing and Act i vation • [( i ) IgA + IgG + IgM 1 ( i i ) l gM Antibody Production + Complement Inactivated by Polysaccharide Charge CELL LYSIS INFECTION I i Polysaccharide • c - T ce l l receptor -< - B ce l l receptor o = Carr ier Hapten Scheme 1 Antibody production and the bacter ic ida l react ion. 12 KIT 3 a K12 f ^ G a l i - ^ G a l f l - ^ l c ^ R h a M l ^ |_ a — 3 « OL a ' J n 1 GlcA Pn VI f - ^ a l M L a '. '61c-—^Rha^-hib.i.tol - PO, Na Figure 3 K lebs ie l l a K12 capsular polysaccharide cross-reacts with anti-serum to K lebs ie l l a K l l and also with an t i -Pneumococcus VI 13 Recently Heidelberger and colleagues have examined cross-reactions between the capsular polysaccharides of K lebs ie l l a and pneumococcus and subsequently made elegant predict ions on substructures of K lebs ie l l a polysaccharides. Most of the work to date has involved heterogeneous ant ibodies, that i s to say, sera containing antibody populations, that although spec i f i c for one antigen, are made up of d i f f e r i ng molecular species of immunoglobulins. These may bind the same antigenic grouping (hapten)* in d i f fe rent ways, or d i f fe rent immunoglobulins may bind small or large sections of the respective * A hapten i s defined as a small molecule, which, by i t s e l f , cannot stimulate antibody synthesis but w i l l combine with antibody once .(formed. 14 pattern on the polysaccharide chain. A new potent ia l i n polysaccharide immunochemistry i s provided by homogeneous immunoglobulins that bind carbohydrate 57 polymers, . Here, the spec i f i c immunoglobulin - hapten interact ion can be characterized in de t a i l . Thus the d i s c i p l i ne of carbohydrate chemistry can make a real contr ibut ion to the e luc idat ion of the structure of immunoglobulins. In return, carbohydrate chemistry may f ind a tool that can increas ingly be applied to the unraveling of i t s own unsolved problems in the s t ructura l analys is of polysaccharides. The fate of a host which i s invaded by micro-organisms depends on the effect iveness of i t s defence mechanisms. To a large extent these are directed against the surface of the micro-organism.The host defences may be inh ib i ted thus enabling the micro-organisms to mult ip ly to such an extent as to eventual ly damage or k i l l the host. This phenomenon i s ca l led VIRULENCE. The re lat ionsh ip between virulence and capsulation has been long known (Bordet 1897) The virulence of an organism does not depend only on the presence of a capsule and the presence of a capsule does not always confer virulence on a l l s t ra ins of a pathogenic species, but i t has been shown that capsulated, v i ru lent s t ra ins of pathogenic bacter ia generally become less v i ru lent when they lose the i r capsules. The best understood defence reactions with which microbial polysaccharides are known to in teract are those that 15 involve pr imar i ly c i r cu l a t i ng antibody and the complement system, v i z . , the bacter ic ida l react ion and phagocytosis. In the former, the interact ion of serum antibody with i t s antigenic determinant act ivates the complement system (see Scheme 1). P rac t i ca l l y a l l capsular polysaccharides are ac id i c and they inact ivate complement, probably 59 through the i r charge. . A- part of the complement system i s also involved in phagocytosis - the engulfment and k i l l i n g of bacter ia by macrophages and leukocytes (see Scheme 2). The antiphagocytic e f fec t of capsular polysaccharides i s probably not spec i f i c , charge and v i scos i t y of the capsular material being prerequis i tes. •It has been shown that anti-polysaccharide antibodies are protect ive against certa in in fect ions . Heidelberger ^0-62 - j m m u n - j z e c j humans with pneumococcal polysaccharides, providing a s i gn i f i can t degree of protect ion in an epidemic in which the corresponding pneumococci were involved. Protect ive immunization may be performed not only with bacterial vaccines and iso lated polysaccharides, but also with a r t i f i c i a l antigens, thus avoiding the possible tox ic ef fects of bacter ia . The task of producing a r t i f i c i a l antigens in large quant i t ies i s indeed arduous. The fact that monosaccharides are mult i funct ional and may be l inked in e i ther an a or a e conf igurat ion presents an enormous challenge to the synthetic carbohydrate chemist. Consequently, only a very small proportion of the possible number of disaccharides has been synthesised , and although 64 synthetic procedures are progressing rapid ly only a l imi ted 16 INFECTION KILLING AND DIGESTION PHAGOCYTOSIS OF BACTERIUM Scheme 2 Phagocytosis. 17 number of synthetic ol igosaccharides has been rea l i sed ' . An a l te rnat i ve , then, i s to look to the po s s i b i l i t y of obtaining ol igosaccharides from the degration of polysaccharides. fi7 In 1969 a method was described for the hydrolysis of polysaccharides, according to which the ol igosacchar ides, once formed, are separated from the hydrolysis mixture by d i a l y s i s , and are thus protected from 68 further degradation. This technique has been used by Himmelspach et al in 1973 to obtain the tetrasaccharide repeating unit of Salmonella  i l l i n o i s which was then coupled to protein to obtain an a r t i f i c i a l antigen. However the procedure suffers from the fact that acid hydrolysis i s non-speci f ic and many d i f fe rent ol igosaccharides are produced, and subsequent separation may prove d i f f i c u l t . To obtain hydrolysis of polysaccharides at spec i f i c points attent ion has been turned by Stirnv. et al_ ^9-71 t Q d e p 0 i ymer izat ion with the aid of bacteriophage glycanases. By th is method heteropolysaccharides consist ing of repeating units may be spec i f i c a l l y cleaved e i ther by the whole bacteriophage, or the pur i f i ed enzyme, to y i e l d a homologous ser ies of the s ingle repeating unit and 72 mult iples thereof. I t has been pointed out by Dutton that th is i s the only method of obtaining an ol igosaccharide with an in tac t ac id l ab i l e pyruvate group. To obtain the immunogen the haptenic ol igosaccharide i s coupled to an immunologically e f f i c i e n t ca r r i e r prote in. Goebel 73 74 and Avery's method ' of preparing the conjugates via aminophenyl 18 glycosidat ion of the reducing sugar end groups was extensively used and was not improved upon for about three decades, although i t s appl icat ion was almost exc lus ive ly res t r i c ted to mono- and disacchar ides.. 75-77 Newer methods of coupling have no such l im i ta t i on and appear to be suited to v i r t u a l l y any reducing ol igosaccharide and also to any chain length. Microbial polysaccharides have also shown potential in the 78 area of cancer research . I t has been known for more than a century that human malignant growths sometimes undergo regression fol lowing 79 an acute, bacter ia l in fec t ion . Since then extensive studies have been made on noncytotoxic and host-mediated antitumor polysaccharides o n o i from various sources. It has been reported ' that polysaccharide complexes from Klebsiella,among others, were act ive against so l i d tumors. Many questions remain unanswered and the role of the polysaccharides as immunopotentiators i s being espec ia l l y debated. In Chapter II the methodology of the structura l analysis of polysaccharides i s examined. Advances in chemistry are invar iab ly aided by progress in instrumentation. Recent developments are discussed, then, with special reference to n.m.r. spectroscopy. To date the appl icat ion of high performance l i q u i d chromatography to carbohydrate analysis has been l im i ted . Therefore, th is technique i s examined in Chapter V and i t s potent ia l as a tool in the st ructura l e luc idat ion of polysaccharides and in obtaining large quant i t ies of ol igosaccharides i s examined. 19 I f continued advances are to be made in the area of immunochemistry i t i s c lear that co-operation between the chemist and the immunologist i s essent ia l . For the l a t t e r to understand complex immunological react ions, we l l - c l a s s i f i e d antigens are required; and i f the f i e l d i s to expand to include large scale vaccination then, non-toxic immunogens are needed. These two areas ~ structure determination of polysaccharides and the i r degradation; by bacteriophage - - are examined in Chapters III and IV of th is thes is . 20 I I METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES 21 I I . METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES To determine the complete structure of a polysaccharide the const ituent sugars must be i den t i f i ed , and the i r r a t i o , subst i tuents, sequence, l inkage pattern and linkage configurat ion ascertained. The 82 chemistry of polysaccharides has been reviewed by Aspinal l and Stephen and-Aspinall . Recent theses from our group (Paulin 1980, Folkman 1979 and Mackie 1977) have surveyed the methodology of s t ructura l determination of polysaccharides. In the fol lowing section the most widely used 1 13 techniques are discussed b r i e f l y , with special regard to H and C n.m.r. spectroscopy. ' I I . 1. Iso lat ion and Pur i f i ca t i on Samples of K lebs ie l l a bacter ia of spec i f ied serotype and s t ra in number were received as stab cultures from Dr. Ida (|)rskov (Copenhagen). Bacter ia l cultures were streaked on agar plates at 37°C unt i l large, indiv idual capsular colonies were obtained. Bacteria were grown by innoculat ion of beef-extract medium with a s ingle colony for 3h at 37°, with shaking.and subsequent incubation of th is l i qu i d culture on a tray of sucrose - yeast extract - agar for three days. The lawn of capsular bacter ia produced was harvested by scraping from the agar surface, and the bacter ia destroyed with a 1%. phenol so lut ion. The polysaccharide was i so la ted by u l t ra -cent r i fugat ion of th i s so lut ion. The viscous honey-coloured supernatant was prec ip i tated into ethanol. The prec ip i ta te was dissolved in water and 22 84 was treated with CETAVLON (cetyltrimethylammonium bromide) and centrifuged to i so la te only the ac id i c polysaccharide. The CETAVLON-polysaccharide complex was dissolved in 4M sodium ch lor ide , prec ip i tated into ethanol, redissolved in water, and dialyzed against running tap-water for two days. The polysaccharide was i so la ted , as a styrofoam-like mater ia l , by l y oph i l i z a t i on , and was shown to be homogeneous by electrophoresis on ce l lu lose acetate s t r i ps and by nuclear magnetic resonance spectroscopy, (see App. IV). I I . 2. Separation Techniques To obtain information on the const ituent sugars of the polysaccharide i t s e l f and of i.ts.jvdegrada.tion products, the Tatter must be separated, pur i f i ed and then checked for homogeneity. Separation is obtained by the establ ished technique of paper chromatography,by' gel chromatography and paper electrophoresis and by newer techniques such as high pressure l i qu i d chromatography. o r QC I I . 2. 1. Paper chromatography ( :p.c.) ' The great value of th i s technique l i e s in i t ' s a b i l i t y to separate mixtures of monosaccharides^and ol igosaccharides simply, accurately and without de r i va t i za t i on , by employing d i f fe rent solvent systems. For ana ly t i ca l analysis very small amounts of material are needed, while the technique may also be employed on a preparative l e v e l . Ac id ic components may be dist inguished from neutral components by using d i f fe rent solvent systems. Paper chromatography i s used for ident i fy ing constituents 23 87 ei ther as the free sugars or as a l d i t o l s from hydrolysis of the native polysaccharide and of i t s degradation products. Methylated 88 sugars and oligomers may also be iden t i f i ed . P.e. is also used for monitoring contro l led hydrolysis of the polysaccharide and for checking the composition of f ract ions obtained from gel chromatography of mixtures from par t ia l hydrolysis and periodate ox idat ion. When pure oligomers are not obtained by gel chromatography preparative paper chromatography may be employed. Although tedious and time consuming, good resul ts may be obtained, as in the analysis of K58 polysaccharide, where large quant i t ies of the aldobi - , t r i - , and tetrauronic acids were obtained. Sugars are detected with e i ther ( i ) Ag NOg/ ethanol--NaOH / Na 2 S 2 0 3 or. with ( i i ) p-anisidine-hydrochloride in aqueous 1- butanol followed by heating at 110° for 5 m i n .^ I I . 2. 2. Paper electrophoresis ( p . e . ) 8 6 > 9 1 > 9 2 Large ac id i c oligomers move very slowly on paper chromatography. Paper e lectrophores is , however provides an a l ternat ive convenient method for the i r examination. Good separations can be achieved in a number of hours. Electrophoresis involves the migration of charged substances in a conducting so lut ion under the inf luence of an e l e c t r i c f i e l d . Buffer pH conditions are chosen so that the materials to be separated ex i s t in a charged state. The usual coolant used i s kerosene. P.e. may be employed both ana l y t i c a l l y and preparat ively. Sugars are 89 90 detected as for paper chromatography ' . 24 I I . 2. 3. Gel chromatography (g.c.) Gel chromatography or ig inated in 1956 with the work of 93 Lathe and Ruthven , who achieved some degree of separation of t r i - , di - and .monosaccharides on a column of potato starch. 94 The f i e l d has been reviewed by Churms . Also known as gel f i l t r a t i o n , gel-permeation chromatography, or molecular-sieve chroma-tography i t i s based on the decreasing permeabil ity of the three dimensional network of a swollen gel to molecules of increasing s i ze . As the order of e lut ion of a ser ies of s im i la r substances from a gel column is governed large ly by molecular weight, gel chromatography provides a means of determining molecular weights of polymers. This technique has been used extensively for the separation of products of par t ia l hydrolysis and periodate oxidat ion. Both SEPHADEX and BIO-GEL gels have been used in th is study. In cases when the molecular exclusion l im i t of two d i f ferent gels (eg. G-15 and P-2) i s the same,the e lut ion p ro f i l e and the order of e lut ion may not necessar i ly be the same, and th is may be used to advantage to obtain , a clean separation. The pa r t i c l e s ize grade (superfine to coarse) should also be taken into account in choosing a ge l . BIO-GEL has the advantage of being res is tant to bacter ia l contamination since i t i s a synthet ic mater ia l . Wet sephadex (derived by cross l ink ing dextran) should be stored in a so lut ion of 0.1% NaN 3. 25 The material i s eluted with d i s t i l l e d water, or preferably with a buffer eg. water - p y r i d i n e - acet ic acid 1000: 10: 4. Carbohydrate fract ions from the column are f i r s t loca l i zed using 95 the Molisch test . To determine the e lut ion p ro f i l e a quant i tat ive * 96 co lor imetr ic technique such as the phenol-sul fur ic assay may be used, or ind iv idua l f ract ions may be l yoph i l i zed and weighed. R.c. and p.e. are used to invest igate the composition of f rac t ions . Where homogeneity i s not achieved', pu r i f i ca t i on may be obtained with preperative p.c. or p.e. Molecular - weight d i s t r i bu t ion studies of ac id hydrolysis products from K lebs ie l l a K54 exopolysaccharide by Churms and Stephen on BIO-GEL P-10 gave evidence for repeating units in the structure 2 8 9 ' b . . Sephadex LH-20 (G-25 with most of the -OH groups alkylated) may be used successful ly to pur i fy large molecular weight carbohydrate material that i s soluble in organic solvent, eg. permethylated or peracetylated polysaccharide. I I . 3. Instrumentation I I . 3. 1. Gas- l iquid chromatography ( g . l . c . ) Extensive reviews of the appl icat ions of g . l . c . to 97 98 carbohydrates have been published by Dutton (1973 and 1975) ' . Most carbohydrates are not s u f f i c i en t l y vo l a t i l e to be used for g . l . c . and they must therefore be converted into vo l a t i l e compounds. 26 The fact that each monosaccharide may give more than one peak owing to the formation of anomeric der ivat ives has l ed to a search for means to el iminate th is complication. The l a t t e r problems may be surmounted by reduction to the a l d i t o l or by conversion to the corresponding n i t r i l e . Direct acety lat ion then y ie lds vo l a t i l e der ivat ives . Ident i f i ca t ion of the const ituent sugars of an ol igosaccharide or polysaccharide i s achieved by hydro lys is , reduction, acety lat ion and g . l . c . analys is . SP-2340 (75% cyanopropyl s i l i cone) i s the stat ionary phase of choice for analysis of a l d i t o l acetates (see F ig. 5). ECNSS-M (ethylenesuccinate - cyanoethyls i l icone copolymer) may also be used, but the maximum operating temperature i s quite low, and t a i l i n g of peaks may occur. To analyze mixtures from methylation analysis (see la ter) OV-225 (25% phenyl, 25% cyanopropyl, methyl s i l i cone) (see F ig. 6) and OV-17 (50% phenyl, methyl s i l i cone) along with ECNSS-M are used most commonly. A column packed with 0.3% to 0.4% OV-225 on Chromosorb surface modified with high molecular - weight polyethylene 99 glycol has been developed recently to achieve a higher operating temperature, giv ing shorter retention times and neg l ig ib le column bleeding, hence constant retention parameters. OV-225 has also been used to separate d i - and t r i sacchar ide der ivat ives 0V-1 (methyl s i l i cone) i s used for the analysis of permethylated ol igosaccharides. 27 COLUMN : SP-2340 PROGRAMME: 185° 8min, 4°/min to 240° FLOW RATE: ; :20mL/min Figure 5 G . l . c . separation of a l d i t o l acetates from periodate oxidation of K lebs ie l l a K12 28 COLUMN : OV - 225 PROGRAMME : 170° 4min, 2°/min + 200° FLOW RATE 20mL/min. 2,4 Rha Figure 6 G . l . c . separation of products of methylation analysis of K lebs ie l l a K12 29 When d i f f i c u l t y i s encountered in separating permethylated a l d i t o l acetates the tr imethyl s i l y l (TMS) ether der ivat ives 1 0 1 may be used to advantage, employing SE-52 (5% phenyl • methyl s i l i cone) as stat ionary phase. The ease with which the or ig ina l material may be recovered fol lowing der ivat i za t ion makes the use of TMS der ivat ives a t t rac t i ve where the amounts of material are l im i ted . To determine the degree of polymerization of an ol igosaccharide and to ident i fy the reducing sugar the a ldonon i t r i l e method of Morrison 102 (see la te r ) i s used . A ldonon i t r i les give sharp s ingle peaks on g . l . c . and may be analyzed with 0V-17 (see F ig. 7), OV-225 or ECNSS-M. Publicatons by Albersheim et al_ 1 0 3 ' 1 Q 4 ;and. Lindberg et a l 1 2 2 l i s t the re la t i ve retention times for a large number of p a r t i a l l y methylated a l d i t o l acetates. Ident i f i ca t ion of unknowns i s achieved by consideration of the re la t i ve retention time, by co-chromatography with an authentic sample,and by g . l . c . - m.s. (see l a t e r ) . Where uncertainty s t i l l ex is ts the melting point (m.p.) of the sample (from preparative g . l . c . ) may provide i den t i f i c a t i on ( i f the sample i s c r y s t a l l i n e ) . To determine the parent hexose,demethylation ^ 5 and reacety lat ion gives a c r y s ta l l i ne a l d i t o l hexaacetate which may be i den t i f i ed by m.p. or by g . l . c . retention time. Samples co l lected by preparative g . l . c . are used also for mass spectral studies and for c i r cu l a r dichroism measurements (see l a t e r ) . P a r t i a l l y ethylated, p a r t i a l l y methylated additol acetates 30 COLUMN : OV-17 PROGRAMME : 180° 4min, 2°/min to 220° FLOW RATE : 20mL/min. P1 Peracetylated a ldonon i t r i les PAAN Peracetylated Man a l d i t o l - H 1 1 1 r — 1 0 4 8 12 16 20 Time (min) Figure 7 Degree of polymerization of product of bacteriophage degradation of K lebs ie l l a K21, using g . l . c . 31 COLUMN : ECNSS-M PROGRAMME : 165° 4min, 2°/min to 195° FLOW RATE : 20mL/min. 0 A 8 12 16 Time (min) Figure 8 G . l . c . separation of products of uronic acid degradation of K lebs ie l l a K12. 32 are obtained from uronic acid degradation (see l a t e r ) . When compared to a methyl group the ethyl group i s less polar and hence, with polar l i q u i d phases, components containing ethyl groups tend to t ravel more quickly than the i r methyl analogues (see F ig .8) . Conversely, with nonpolar l i qu i d phases retention times are longer. Mass spectrometry i s invaluable in i den t i f i c a t i on of p a r t i a l l y ethylated der ivat ives (see l a t e r ) . For quant i f i ca t ion of peaks molar response factors are taken into considerat ion. It should be noted that phthal ic esters (used extensively as p l a s t i c i s e r s ) may be encountered as contaminants in g . l . c . analyses However, mass spectral data d i f fe ren t ia te the contaminant from sugar der ivat ives . I I . 3. 2. Mass Spectrometry (m.s.) The mass spectral method was f i r s t applied to carbohydrate der ivat ives in 1958 by Reed and coworkers , and since then, i t has become an important and versa t i l e technique in carbohydrate chemistry. Lonngren and Svensson reviewed the f i e l d in 1974 . In th is study m.s. has been used in the analysis of p a r t i a l l y methylated/ ethylated a l d i t o l acetates in order to assign subst i tut ion patterns (see l a t e r , Scheme 3), and in the analysis of ol igosaccharides to determine the sequence of sugars. Mass spectra can be recorded by using any one of several 33 Cr^OAc 117 HCOMe 161 MeOCH JL 205 HCOMe 1 161 1 HCOAc 4 „ „ AcOH—I>I45 I O I « - A c O H - l 6 J J ™ ^ Y " CH^O i0  ' 61 -MeOH ^ 1 2 9 I | - C H 2 C 0 | C Ac I 71  I CH^OMe 4 5 87 100 .43 to 80 H 60 40ti 20 101 45 71 8 | 7 L 1 7 129 |6I 145 J I 40 100 160 m/e 2 0 5 220 Scheme 3 Primary and Secondary Fragmentation Pattern and Mass Spectrum of 1,5-Di-O-acetyl-2,3,4,6-tetra-0-methyl• D-glucitol 34 d i f fe rent systems of instrumentation. The i n l e t system can be e i ther a hot reservoir i n l e t , a d i rec t probe i n l e t , or a g . l . c . i n l e t . The l a t t e r has become increasingly important in the invest igat ion of complex mixtures. Most underivatized mono- and ol igosaccharides are thermally unstable and non-volat i le and therefore must be converted into vo l a t i l e der ivat ives for spectral (m.s.) ana lys is . Stereoisomers of carbohydrate der ivat ives give s imi la r mass spectra, and the small differences in peak in tens i ty sometimes observed do not general ly al low an unambiguous assignment of conf igu-ra t ion . However, consideration of g . l . c . retention time along with mass spectral data aids in assignment. Carbohydrate der ivat ives give weak or no molecular ions on electron impact ( e . i . ) mass spectrometry. Molecular weights may more eas i l y be determined by f i e l d ion izat ion ( f . i . ) ^ ' ^ , f i e l d desorbtion ( f . d . ) ^ 2 - ^ , or chemical ion izat ion ( c . i . ) ^ - ^ techniques. The l a t t e r two techniques are pa r t i c u l a r i l y useful with free sugars and der ivat ives of low v o l a t i l i t y . In th is study mass spectra were obtained by combined g . l . c . - m.s. in the e . i . mode at 70eV for the pa r t i a l l y methylated/ethylated a l d i t o l acetate mixtures, and using a d i rec t probe i n l e t in the e . i . mode at 70 ev for a permethylated ol igosaccharide. 121 122 Considerable data ' are now avai lab le on the fragmentation pattern of p a r t i a l l y methylated a l d i t o l acetates, and a bank of standard mass spectra i s maintained in our laboratory. 35 As an example most of the s ignals in the mass spectrum of 1,5-d i -0-acety l - 2,3,4,6 - te t ra -0 - methyl-D-glucitol can be accounted for from primary and secondary fragmentation (see Scheme 4). Vl iegenthart and coworkers have shown that there are charac te r i s t i c differences between the fragmentation patterns of 123 hexopyranosides and hexofuranosides A ldonon i t r i l e acetates (which have the advantage of molecular asymmetry) are sui table for analysis by g . l . c - m.s. and give 119 124 charac te r i s t i c mass spectra that are easy to interpret , ' . Oligosaccharides have been examined by e . i . -m.s . as the i r acetate 1 2 5 ' 1 2 6 , TMS, 1 2 7 - 1 3 0 and methyl ether 1 3 1 " 1 3 4 der ivat ives , the las t proving most expedient. The nomenclature used by 135 Chizov and Kochetkov for the d i f fe rent fragmentation ser ies of permethylated glycosides, modif ied, as suggested by Kovacik and coworkers , i s now standard. In th is work a t r isacchar ide glyceride was obtained on periodate oxidation of K12. The sequence of sugars was readi ly ascertained by d i rec t probe> e . i . -m.s . of i t s permethylated der ivat ive since the constituent moieties were, fo r tu i tous l y ,a hexose, a deoxyhexose, a pentose and glycerol (see l a t e r ) . Mass spectra obtained from permethylated d i - and t r i -saccharides by the f . i . technique show strong molecular i o n s ; ^ ' ^ the fragments observed make i t possible to determine the nature of the constituent monosaccharides but not the inter-sugar l inkages. 3 6 However, conversion of the reducing sugar to the corresponding a l d i t o l with a deuterated hydride dist inguishes between 3 - or 4 - and 2- or 5- l inkages respect ive ly. Par t ia l hydrolysis of methylated polysaccharides, followed by reduction and remethylation with tr ideuteriomethyl iodide gives a mixture of ol igosaccharide alditol.s that can be analyzed by g . l . c . -m.s. The CD^  groups occupy posit ions to which a sugar residue i s l inked in the o r ig ina l polysaccharide. This technique has been used in structura l studies of the 1ipopolysaccharide from K lebs ie l l a n n 1 3 7 0-group 9 Mass spectrometry has become increasingly s i gn i f i c an t i»n the structura l e luc idat ion of b i o l og i ca l l y important glycoconjugates 1 q g since quant i t ies of material are very l im i ted . Egge and coworkers have made many contr ibutions to th i s rapid ly developing area of carbohydrate chemistry. I I . 3 . 3 . Polarimetry Assignment of the anomeric configuration of a spec i f i c g lycos id ic l inkage in a poly- or ol igosaccharide may be accomplished by nuclear magnetic resonance spectroscopy (see l a t e r ) . However, ambiguities may ar ise for some sugars, depending on the l inkage pattern and r ing s i ze . In these instances polarimetry, using 1 4 3 1 4 4 Hudson'srules of i sorotat ion may be employed to advantage. ' Since K lebs ie l1 a polysaccharides have been shown to consist of 37 repeating units of hexoses and the i r der ivat ives the theoret ica l value for the molecular rotat ion can be found, to a f i r s t approximation, by summation of the molecular contr ibut ion of each component. I t i s assumed that the 0_-acetyl and pyruvic acid ketal groups present make neg l ig ib le contr ibutions to the tota l molecular ro ta t ion . •Application of Hudson'srules gives information only on the overal l molecular rotat ion of the poly - or ol igosaccharide.. However, the ind iv idua l g lycos id ic l inkage configurations may be deduced i f the rotat ions of a ser ies of oligomers, eg. d i - , t r i - , and tetrasaccharides from the repeating unit are observed. The spec i f i c rotat ion of the polysaccharide is calculated from the equation M D = M r s . * 1 0 0  Mo where [M]p^ is the summation of the ind iv idual rotations and MQ i s the molecular weight of the repeating uni t . The experimental spec i f i c rotat ion i s r -i _ a n x 100 - _JJ 1 x c_ where i s the polarmeter reading 1 i s the ce l l length in dm, and c_is the concentration in g/100 ml. 38 145 Merri f i e l d has compared the theoret ica l and observed values for a number of K lebs ie l l a polysaccharides and found very l i t t l e discrepancy in most instances. He also found that the ef fect of temperature change i s neg l ig ib le . His compilation i s included in Appendix 1. I AC I I . 3. 4. C i rcu lar dichroism (c.d.) The configurat ion (D or L) of a sugar may be determined conveniently by c .d. measurements a t 213 nm on a l d i t o l acetates, o r the i r methylated der ivat ives , where the acetoxy group acts as a chromophore The method i s well suited to analysis of samples obtained by preparative g . l . c , since only mil l igram quant i t ies of material are required. Configurational assignments are made af ter comparison with data from authentic samples. ,In the case of D-glucose and D-galactose spec i f i c oxidases are ava i lab le (Worthington Biochemical Corporation). This method was employed in the invest igat ion of K58 to determine that the galactose was of the D-configuration, since the c .d. method is inappl icable to the meso-galactitol hexaacetate. I I . 3. 5. Nuclear Magnetic Resonance Spectroscopy 1 13 Both H and C n.m.r,. spectroscopy have been used extensively in th is work. The fact that interpretable spectra can be obtained by these two methods on polysaccharides with molecular 39 weights of the order of 10 indicates that the structures have regular repeating units . Spectra of the native and permethylated polysaccharides and of degradation products were analyzed, ind icat ing that information from the two techniques i s very often complementary. I I . 3. 5. 1. ^H. n.tri.r. spectroscopy Proton magnetic resonance i s now f i rmly establ ished as the most widely used technique for the s t ruc tu ra l , conf igurat iona l , and conformational analysis of carbohydrates and the i r der ivat ives. The 148 149 observations of Arnold and coworkers and Gutowski and Hoffman that the chemical s h i f t of a proton depends on the precise chemical environment of that proton were made in 1951. However, i t was not unt i l 1957 that the f i r s t H^ n.m.r. spectra of carbohydrates were 150 151 reported by Lemieux and coworkers . In a c l a s s i c paper in 1958 these authors showed the ef fects of configurat ion and conformation on the chemical sh i f t s and coupling constants of acetylated sugars. This 152 work was extended to free sugars in fifi so lut ion by Lenz and Heeschen in 1961 and the observation that g lycos id ic l inkage protons resonate 153 downfield of the r ing protons was made in 1963 by van der Veen who also succeeded in corre lat ing the s p l i t t i n g s , observed for the anomeric hydrogen atoms, with the glycoside conf igurat ion. The f i r s t 1 ^ 4 review of the f i e l d appeared in 1964 by Hall 155 Progress in the intervening years has been rapid due to (a) the development of superconducting solenoid spectrometers with higher magnetic f i e lds and resonance frequencies (b) the improved performance of radio - frequency c i r cu i t s (c) the development of 40 the Fourier - transform n.m.r, method and (d) decoupling and mult inuclear a b i l i t i e s along with (e) advancements in data systems eg. mult i tasking and queuing capab i l i t i e s . To date many reviews of th is ever expanding r' i j i . . , . . . 82, 83, 156-160 f i e l d have been published The technique,howeve^suffers from a number of l im i ta t ions inherent with increase in molecular s ize and complexity, in par t i cu lar when attent ion i s turned to polysaccharides; solut ions to these problems 159 have been proposed by Hall . More recent ly, homonuclear two-dimensional J n.m.r. has been used to s impl i fy complex spectra of mono - and disacchar ides, by separating the ef fects of chemical sh i f t s and scalar coupling. The H^ n.m.r. spectrum of a polysaccharide provides information on the number of sugars present in the repeating unit and indicates the presence of 6-deoxysugars 0-Acety l , 0-formyl ^ a a n d 1-carboxy-ethylidene acetal substituents are also recognised T63b.>c _ j n e anomeric nature of the linkages (a or p ) may be d i f fe rent ia ted for both pyranosyl ^ and furanosyl ^ sugars, from consideration of the chemical s h i f t (S.) along with the value of the coupling constant (J) between H-l and H-2(J ] 2 ) . Spectrum No. 10 (App. I l l ) shows the presence-of'a 6-deoxy sugar, a 1-carboxyethylidene, and an 0-acetyl group in equimolar proportions, along with signals a t t r ibutab le to four anomeric centres -:three ct( 6>5 ppm, J-j 2<4) and one e( 6<5 ppm, J-| 2>6) l inked pyranoses. Care should however, be exercised when furanosyl sugars are present (from g . l . c . - m.s. data). Spectrum Nd.T shows the anomeric signal for the 41 g-galactofuranosyl unit at 6 5.13 ( 6>5). Ring protons may resonate in the so-ca l led "anomeric region", depending on the r ing s ize and linkage pattern of the sugar 1 6 6 ~ 1 6 8 . Spectrum No. 1 shows H-2 and H-3 of galactofuranosyl unit at 6 4.3-4.5 ppm 1 6 9 , 1 7 0 . Garegg and coworkers 1 7 1 have demonstrated that the differences in chemical sh i f t s obtained for stereoisomenc pairs of aceta l i c CH 3 172 groups from pyruvic acid and related acetals are of su f f i c i en t magnitude to make possible the unequivocal determination of the stereochemistry of the aceta ls . For ol igosaccharides, the degree of polymerization may be calculated from the ra t io of the integra l of the anomeric protons from the reducing sugar («and 3) to those from the non-reducing sugars (see Spectrum No. 26) High resolut ion n.m.r. spectra contain, in addit ion to chemical sh i f t values, coupling constants and area integrat ions, two further sets of nuclear parameters: the spin-spin (T^) and sp in - l a t t i c e (Tj) re laxat ion times 1 7 3 - 1 7 5 _ since the l a t t e r show a number of s tereospec i f i c dependencies they provide a useful basis for configurat ional assignments. Another very useful appl icat ion of re laxat ion studies l i e s in the removal of the unwanted residual peaks of deuterated solvents - in th is study of HOD - when high -temperature f a c i l i t i e s are unava i lab le ' (see Spectrum No. 3). The l i t e ra tu re contains few H^ n.m.r. invest igat ions of bacter ia l polysaccharides.+ These assignments are made a f ter +see, however, references in Appendix I from Dutton e t _ a j _ a n c ' Joseleau et al and reference 176 from Perl in et a l . 42 consideration of the spectra of the degradation products eg. the backbone polymer, and d i - , t r i - , and tetrasaccharides, or of •--methyl glycosides of monosaccharides. De Bruyn et aj_ have ref ined 177 and extended the increment rules of Lemieux fol lowing analysis of parameters obtained in the 300-MHz. H^ - n.m.r. spectra of 178 D -glucose, D-mannose, D-galactose, and the i r methyl glycosides, 179 of the glucose disaccharides, and of rhamnose, the methyl glycoside and a di saccharide The H^ n.m. n. spectra of glucobioses and 181 glucotr ioses and of various d i - and .tr isacchar ides containing 182 L-rhamnose have been described. Perl in has published 220 MHz 1 -ic spectra of some mucopolysaccharides H^ n.m.r.. examination of carbohydrate der ivat ives soluble in organic solvents have advantages over the use of aqueous so lut ions: there i s no need for proton exchange, i f the hydroxyl groups are der ivat ized and no residual HOD peak, therefore ambient temperature i s su f f i c i en t and also solvent ef fects may be employed to advantage. Vl iegenthart and coworkers have described the complete interpretat ion I I go of H n.m.r. spectra of solut ions of permethylated a- and &- D-glucose 184 and galactose and of mannose and of the 6-deoxy analogues of "I o c mannose, glucose and galactose , (with a view to use in methylation 186 analys is) and of permethylated disaccharides . The chemical sh i f t s of anomeric protons of the methyl ethers of various disaccharides 187 were reported in 1972 by Minnikin but the complete interpretat ion of H^ n.m.r. spectra of permethylated o l igo - and polysaccharides . i s yet to be achieved some valuable information may be provided. For 43 example in the st ructura l analysis of K lebs ie l l a Kl2 comparison of the spectra of the permethylated polysaccharide and of the 3 - e l iminat ion product (see l a t e r ) , indicates that the two side-chain sugars removed were 3 - l i n k e d . Stephen et al_ have used lanthanide sh i f t reagents in n.m.r. studies on f u l l y methylated aldohexopyranosides and the i r 6-deoxy 100 1 0 0 analogues and on the permethyl ethers of galactose . TMS der ivat ives have been used to determine the number of 190 hydroxyl groups in a molecule and to determine the configuration 191 of the g lycos id ic l inkages in an ol igosaccharide The solvent of choice for n.m.r. spectroscopy of underivatized poly- and ol igosaccharides i s D2O. To el iminate interference in the spectrum by the numerous hydroxyl groups present, a number of exchanges are made with 99.7% D2O, followed by lyophi1 izat ion and heating under vacuum. The sample is then dissolved in 99.9% DgO and any residual HOD is sh i f ted away from the anomeric region (to-6 4.18 ,90°) when the spectrum i s run at elevated temperature. A l te rna t i ve l y , a re laxat ion type (T-|) experiment may be performed whereby the HOD signal i s nu l led. This may be espec ia l ly successful with non-viscous ol igosaccharides. Sample sizes of polysaccharides are general ly of the order of 1-2%, while larger samples may be employed with ol igosaccharides. I f the polymeric sample i s extremely viscous par t ia l depolymerization with acid may improve the sharpness of the spectrum. 44 Because samples are of low concentration spectra are general ly run in the FT mode. Where sample s ize i s minimal a 5mm sample tube with a cy l i nd r i ca l semi-micro volume cavity may be used to advantage. Spectra are usual ly run with an internal standard of acetone. However, i n i t i a l spectra of an unknown polysaccharide should be run without acetone, since a substituent 0-acetyl group may be masked by the standard. Acetone has the advantages of being vo l a t i l e (hence i t may be readi ly removed from the sample) and i t s chemical sh i f t i s v i r t u a l l y unaffected by var iat ions in temperature. Stephen et a l have recommended caution in the use of some 1 192 reference systems for H n.m.r. spectroscopy of carbohydrates 13 I I . 3. 5. 2. C n.m.r. spectroscopy 13 The p ro l i f e ra t i on of studies on the C spectra of carbohydrates 13 during the past few years attests to the fact that C n.m.r. i s acquir ing a status, not only as a useful adjunct to H^ n.m.r. spectroscopy, but one characterized by i t s own unique contr ibut ions. As in H^ n.m.r. progress in instrumentation has been rap id , with concomitant increase in the number of appl icat ions ~. structura l e luc idat ion , configurat ional and conformational analyses, detection of impur i t ies , 193 194 and analysis of mixtures ' . The f i e l d has been reviewed by Per l in 1 9 5 ' 1 9 6 . Not surpr i s ing ly glucose has been one of the ea r l i e s t and 197-200 most extensively studied of the carbohydrates . Previous 45 l 3 assignments of natural abundance , C n.m.r. chemical sh i f t s of mono-and disaccharides have been re- revaluated by use of a d i f f e ren t i a l isotope 2f)l 202 13 (DIS) technique , Gorin- has assigned the C signals, of the more common sugars and the i r methyl glycosides - the l a t t e r proving most expedient in approximating the chemical sh i f t s of g lycos id ic carbons in o l i go -203 and polysaccharides. Chizhov et aT_ here extended these data to include a l l the methyl ethers of methyl (methyl a-D-glucopyranosid) uronates, since uronic acid residues are frequently encountered in natura l ly occuring polysaccharides. 13 Vl iegenthart and coworkers have interpreted C spectra "I O O ] 0 4 of permethylated a- and p- glucopyranoses , galactopyranoses 185 mannopyranoses and 6- deoxy sugars with a view to interpret ing methylation analysis data (see l a t e r ) , in conjunction with n.m.r. data. 13 The e f fec t of O-alkylat ion on the C n.m.r. spectra of 204 methyl pentafuranosides has been determined by Gorin and by 205 Ishido . Vignon has studied the e f fec t of the t r i ch lo roacety l i 1 4. • i_ • 206,207 . c . , group on glucopyranose and gentiobiose ' , and Seymour et al 13 have examined the C spectra.of compounds containing the 208 3 - fructofuranosyl group 13 C n.m.r. data of monosaccharides and the i r der ivat ives have been extended to d i - , o l i go - , and polysaccharides. Usui et al 209 210 have studied the glucobioses , and glucotr ioses and glucans Di - and tr isacchar ides containing galactose have been examined -211 by Cox et a]_ , and those containing glucose, galactose and 46 212 213 214 rhamnose by La f f i t t e et a l and Colson and King . Hough et al have extended the i r analysis of permethylated carbohydrates to include disaccharides. 215 Gagnaire e_t aj_ determined the spectrum of a -ce l lob iose octaacetate and compared i t to the spectrum of ce l lu lose t r i ace ta te . 71 fi These data have been extended by Capon et, a l to include spectra of peracetylated a - c e l l o t r i o se , a -ce l lotetraose and a-cellopentaose 217 In 1971 Dorman and Roberts demonstrated the app l i c ab i l i t y 13 of C- N.M.R. spectroscopy to the study of ol igosaccharides. Boyd and 218 Turvey have i den t i f i ed spectra of ol igosaccharides derived from 219 a l g i n i c ac id , and Kochetkov et_ al_ have applied the technique in the structura l study of complex ol igosaccharides. Perl in has reviewed the character izat ion of carbohydrate 13 polymers by C n.m.r. spectroscopy. As expected, homopolymers have been among the f i r s t carbohydrate polymers to be studied. As ear ly 217 13 as 1970 Dorman and Roberts - extended a C survey of ol igosaccharides to include a b r i e f invest igat ion of amylose and ce l lu lose acetate. 220 In 1973 Jennings and Smith determined the composition and 13 sequence of a glucan containing mixed linkages by C n.m.r. and the 221 fol lowing year used the technique to assign completely two cyclodextr ins and several l i near glucans by comparison with spectra of glucobioses and glucotr ioses. 222 In 1975 Gorin commented on the methodology of assigning s ignals of a mannan containing al ternate (l->3) and (1+4) l inked 47 3 - D - mannopyranose residues. The 12 signals were assigned by preparation of D - mannans from spec i f i c a l l y deuterated D-glucoses and observation of a - and g - deuterium isotope - e f fec ts . Dextrans ' ' and levans. have been studied by Seymour et a l , as have galactomannans by Grasdalen and P a i n t e r ^ - G lycos id i ca l l y subst i tuted and free C-5 groups have been d ist inguished, and d i f fe rent 225 types of l inkages have been i den t i f i ed by Joseleau et al_. in a 13 C study of two arabinans. Methyl and acetyl substituent ef fects on C chemical sh i f t s have been determined on a - and g - (1-+3) and (l->4) l inked polysaccharides by Gagnaire et al_ 1 13 As in H n.m.r., very few C n.m.r. spectral data of 227 heteropolysaccharides have been published. Per l in et aj_ in 1972 13 gave evidence for a biose repeating unit for heparin using C n.m.r. and in 1979 made a conformational study of the polymer. Variat ions with respect to the presence and locat ion of sulphate groups 228 in agar and some carrageenans were shown in a study by Hamer et 1 q The spectrum of s pe c i f i c a l l y labe l led ( C)nigeran - a regular, a l t e r -13 nating copolysaccharide having differences in C signal i n tens i t i e s -229. has been assigned by Bobb i t e t a]_ . 230 In 1977 Dutton et a_ delineated the diagnostic potent ia l 1 3 of C n.m.r. spectroscopy in the structura l e luc idat ion of K lebs ie l l a polysaccharides composed of three to s i x sugar residues and carrying 0-acetyl and 1-carboxyethylid.ene subst i tuents. Since then many K lebs i e l l a 13 polysaccharides have been character ized, using C n.m.r. spectroscopy by our group. Assignments have been made on the basis of spectral data 48 obtained from degradation products of the polysaccharides, from methyl 231 glycosides, and from synthesized ol igosaccharides. 13 Routine C spectra are run with complete proton decoupling. However, valuable information may be obtained from the coupled spectrum. 13 A smaller C-l-H-1 coupling (161 Hz) i s found for the 3 (equatorial) 1 g o anomer of glucose than for the a(ax ia l ) anomer (169 Hz) . For nuclei other than the anomeric centre the magnitude of i s substant ia l l y smaller, and there i s an overal l tendency for d i rec t coupling to 13 decrease with an increase in sh ie ld ing of the C nucleus. In 1979 232 1 Fr iebo l in et a]_ used J ( C - l , H) coupling constants to ident i fy the anomeric conf igurat ion of some polysaccharides and the i r methyl der ivat ives . To gain further ins ight into the architectureof the gel 233 network of some branched 3 (l->3) l inked glucans Saito et al_ made 13 a C n.m.r. study of sodium hydroxide - induced conformation changes. 13 C 2-D J spectroscopy has been used to f a c i l i t a t e the measurement of H^ - 1 3 C couplings in spectra of o l i g o s a c cha r i d e s . 2 3 4 9 Bock and Hall in 1975 showed the pract ica l relevance of 13 T-j measurements in obtaining e f f i c i e n t F.T. C spectra : the time interva l between successive 90° pulses should be no less than f i ve T-j periods to prevent saturat ion of resonances. Coupling and T-j experiments provide ins ight into the microdynamics of the motion of carbohydrate mo lecules in so lu t ion . T-j values for polysaccharides have been 9 Q C O O O U reported for bovine nasal ca r t i l age and a gel-forming glucan , giv ing information on the molecular motion and the overal l conformation respect ive ly . T-| measurements on branched - chain polysaccharides 49 236 made by Gorin and Mazurek show that these values can be useful in d is t inguish ing resonances of side-chains from those of the main chain. 13 C spectra of underivatized carbohydrates are general ly run in 50% to give a deuterium lock and with acetone as internal 13 standard. Chemical sh i f t s of the C nuclei of carbohydrates and der ivat ives encompass most regions of the 200 pp, range covered by organic compounds. S imp l i f i ca t ion of the spectrum by proton decoupling, together with the resolut ion of less than 0.1 ppm afforded by present instruments usual ly ensures an excel lent overa l l separation of resonance s ignals even for highly complex molecules or mixtures. Peak areas, which are highly sens i t ive to the re laxat ion properties of 13 the various C nuc le i , and to the extent of Overhauser enhancement, may be ysed i f comparisons of the integrated in tens i t i e s are based on s ignals representing the same class of carbon (such as the anomeric centres in a polysaccharide). Spectra should be run at elevated temperature, 196 when poss ib le, to reduce l i ne broadening. Resonances of simple sugars are dist inguishable for the most part in terms of carbonyls from uronic acids and pyruvic ac id (180 ± 6 ppm), anomeric carbons (100 ± 8 ppm), secondary carbons (75 ± 5 ppm), primary carbons (65 ± 5 ppm), methyl groups from ()-acetyl substituents (30 ± 3 ppm) and from 6-deoxy sugars and pyruvate substituents (20 ± 5 ppm). Variat ions within each class are associated with changes in r ing s i ze , conf igurat ion, conformation and subst i tu t ion. For example in the spectrum of K12 (No.2) the signal due to the B - galactofuranosyl residue appears at 108.39 ppm while that 50 due to the g - galactopyranosyl residue appears at 106.99 ppm, and the sn'gnal due to the a - galactopyranosyl residue occurs at 99.43 ppm, Resonances due to the anomeric carbons at reducing centres occur upf ie ld of the corresponding glycosidic centres. For example in the disaccharide 1 from K12 (No.6) the signal due to g - galactose •-occurs at 96.98 ppm and that due to the a anomer at 93.01 ppm. I I . 4. Techniques of Structure Determination I I . 4. 1. Characterization of component sugars. Klebsie l la polysaccharides are heteropolysaccharides, made up of regular repeating un i ts . To iden t i f y the const i tuent sugars, and to determine the r a t i o s , various techniques are employed. The most informative f i r s t analysis is made with paper chromatography; the polysaccharide is hydrolyzed completely with acid and, using various solvent systems, hexoses, 6-deoxy hexoses and acidic sugars are i d e n t i f i e d . G . l . c . analysis of the corresponding a l d i t o l acetates is used to determine the quant i ta t ive composition. I t is of prime importance, then, to hydrolyze a l l the glycosidic l inkages, and to minimize degradation of monosaccharides. The rates of hydrolysis of in terg lycos id ic linkages vary 237 great ly ; 6-deoxy sugars and furanosyl bonds hydrolyze eas i l y , while uronic acid residues are most res is tant . This problem is 238 overcome with a technique developed in th is laboratory. Treatment of the polysaccharide with methanolic hydrogen chloride cleaves most glycosidic bonds, leaving some uronosyl linkages i n tac t . 51 At the same time the methyl ester of the uronic acid is formed and 239 240 can be reduced using sodium borohydride ' in anhydrous methanol. Subsequent hydrolysis with 2M tr i f luoroacetic acid (TFA) ensures complete hydrolysis. Theoretically, for each free sugar five forms are possible (a - and 3 - pyranoses, a - and 3 - furanoses, and l inear). Reduction, then, of C-l to the alcohol, simplifies the si tuation, and subsequent acetylation yields volat i le derivatives for g. l .c. analysis. The use of HPLC to determine the quantitative composition of the polysaccharide is investigated in Section V. 1 13 As evinced in Sec. I I .3 .5 . H and C n.m.r. spectroscopy also give, information on the constituent sugars, and their substituents ( i f any), along with the relative numbers of a - and 3 - linkages. Constituent analysis of degradation products of the polysaccharide is performed in a similar manner. 241 I I . 4. 2. Methylation Analysis. This technique has proved invaluable for the elucidation of ring size and of the position of linkage between the sugar residues. In addition the number of sugars in the repeating unit may be ascertained, along with the identi ty of the terminal uni.t(s), branching unit(s) and the position of base-stable substituents (eg. pyruvic acid ketal ) > but not generally, of the base-labile 0_-acetyl group (see Scheme 4 and Table 2). POLYSACCHARIDE i) base ii) Me l ii) reduct ion CHO—Cry)H iii) acetylation 11 r-OAc r-OAc r-OAc r OAc -OAc -OAc -OMe -OAc MeO- -OAc MeO--OAc AcO- -OAc -OMe -OMe -OAc -OAc -OAc •-OMe -OMe -OMe A B < C D gi c- - m s Scheme 4 Methylation Analysis of a Polysaccharide. 53 METHYLATION ANALYSIS Methylation pattern i s i den t i f i ed by: (a) Retention time on g . l . c . (b) Mass-spectrum from g . l . c . m.s. Methylation analysis gives information on: ( i ) ( i i ) ( i i i ) ( iv) number of sugars per repeating unit , r ing s i ze . l inkage posit ions and locat ion, pyruvate subst i tu t ion . Hexose OMe. 0Me~ OMe-OMeT 6-Deoxy Hexose 0Meo OMe: OMe' 1 Location terminal in-chain terminal .+ pyruvate, or branch in-chain + pyruvate, or doubly branched Methylation at posi t ion 5 indicates a furanose sugar TABLE 2 METHYLATION ANALYSIS OF A POLYSACCHARIDE. YEAR 1903 1915 1926 1934 1955 1964 1966 1975 1980 1980 NAME Purdie 242 Haworth 243 244 Menzies Muskat 2 4 5 246 Kuhn' Hakomori .248 ,247 Gros Arnap Prehm Finne 249 250 251 SOLV. BASE. ME.REAGENT CH3I Ag 2 0 CH3I (CH 3 ) 2 C 0 Na0H/H20 Me 2 S 0 4 H0O/CH3I T10H . CH3I NH3 K/Na CH3I HC0N(CH 3) 2 Ag 2 0 CH3I (CH 3 ) 2 S 0 CH3S0CH2Na CH3I CH2C12 B F 3 CH 2N 2 CH 2 C 1 2 K 2 C 0 3 CF 3 S 0 3 CH 3 (CH 3 0 ) 3 P 0 2,^ 6 DTBP CF 3 S 0 3 CH 3 (CH 3 ) 2 S 0 r Buo CH3I TABLE "3 METHYLATION PROCEDURES 54 The procedure involves treatment of the polysaccharide, in so lu t ion , with base and a methylating agent. Table 3 indicates various P 4 7 p e p procedures. The most versa t i l e method i s that developed by Hakomori Usually complete e the r i f i c a t i on i s rea l i zed with one treatment (as evinced by in f ra- red ( i . r . ) spectroscopy). I f th is i s not the case 242 complete methylation can be achieved by a subsequent Purdie reaction since a second Hakomori treatment would resu l t in 3 - e l iminat ion (see la ter ) i f the polysaccharide i s ac id i c . To deduce the ident i t y of the ac id i c sugar the methyl ester i s reduced with l i th ium aluminum hydride in oxolane (tetrahydrofuran) and then re-methylated. Methylated material i s recovered e i ther by d i a l y s i s followed by l yoph i l i z a t i on in the case of polysaccharides, or by extract ion with chloroform for oligomers. Subsequent hydro lys is , reduction of the resu l t ing sugars to the a l d i t o l s , and acety lat ion y ie lds vo l a t i l e der ivat ives for g . l . c . - m.s. analys is . Comparison of data from the nat ive, the uronic acid reduced and the depyruvulated materials y ie lds valuable: information (see Tables 6 , 8 ) . Various stat ionary phases may be used in the g . l . c . analysis (see Section I I .3 .1 . ) of p a r t i a l l y methylated a l d i t o l acetates. Ident i f i ca t ion i s made by consideration of re la t i ve retention times, co-chromatography with authentic samples, and mass-spectral data. 55 I I . 4. 3. Oxidation Two main types of oxidation are used in structura l invest igat ions 253-256 of polysaccharides . The c l a s s i ca l periodate react ion cleaves the carbon-carbon bond between v i c ina l d i o l s , while reaction of a . 257 selected alcohol group with e i ther t r i f l uo roace t i c ac id , or ch lor ine, along with dimethyl sul fox ide and t r i ethyl amine gives a:product." which can then be subjected to a base-catalyzed degradation (see Fig.12). The product of the former is a polyaldehyde, which i s then reduced with sodiumi "borohydride.The tota l hydrolysis product of the derived polyol may be examined qua l i t a t i ve l y by paper chromatography 258 or quant i ta t ive ly by g . l . c . , a f te r der ivat i za t ion to the a l d i t o l nrQ O C A ace tates. 'Al-ternati vely, a mild Smith hydrolysis ' , whereby only the true acetal l inkages are cleaved and the g lycos id ic l inkages are l e f t i n tac t , gives glycosides of mono- or o l igosacchar ides. These products, af ter separation and pu r i f i c a t i on , are invest igated by 13 and C n.m.r. spectroscopy, and methylation„ and m.s. analys is of the in tact methylated oligomer to give information on the sequence and l inkage p C "I patterns of residues. A se lec t ive oxidat ion , using periodate, under contro l led condit ions, has been used to advantage, for example 262 to ox id ize p re fe rent ia l l y a terminal residue The second type of oxidation has only been employed quite recently in structura l invest igat ions of polysaccharides. Selected 26 3 alcohol groups may be derived, for example, by mild hydrolysis of a methylated polysaccharide to remove only the pyruvic acid ke ta l . 56 POLYSACCHARIDE +PYRUVATE i ) m e t h y l a t i o n j j ) m i l d h y d r o l y s i s [-Glcp-]n Scheme 5 Select ive Oxidation and Degradation. 57 Oxidation gives a residue containing ketone/aldehyde func t iona l i t i e s which may then be degraded with base (see Scheme 5). Another residue with a free hydroxyl group i s then exposed in the chain and the ser ies of reactions may be repeated. The newly exposed hydroxyl groups may 1 1 3 be labe l led with e i ther EtI or CD^I. H and C n.m.r. spectroscopic analysis along with g . l . c . - m.s. analysis gives information on the anomeric nature of l inkages and the l inkage patterns of the products. In the invest igat ion of K12 a t r isacchar ide glyceride was obtained by periodate ox idat ion, while in K58 the terminal galactose was s im i l a r l y ox id ized, leaving the in tac t polymeric backbone. A se lec t ive functional group oxidation was not used in th i s study. I I . 4. 4. Reduction It may be deemed expedient to perform reactions on the uronic -acid reduced polysaccharide, for example to a l t e r hydrolysis patterns or to f a c i l i t a t e periodate oxidat ion. A technique developed recently 264 by Taylor and Conrad involves the use of water-soluble carbodiimides and sodium borohydride (see Scheme 6). The product is recovered by d i a l y s i s and l i yoph i l i za t ion . Two treatments may be necessary to achieve complete reduction . During the course of methylation analysis the uronic ester must be reduced. This may be achieved e i ther with l i th ium aluminium hydride or with calcium borohydride 2 6^ in tetrahydrofuran (THF). To transform the reducing sugar of a methylated ol igosaccharide to the a l d i t o l , sodium borohydride in THF: ... ethanol (1:1) may be used, and to reduce free 58 RCOOH 41 RCOO"H+ + NHR II C II NHR 11 1 n NHR I II RCOC + l NHR 11 XS NaBH, P H 5-7 0 NaBH; NHR I 11 RCH20H RCH + 0=C I +Hn NHR Scheme 6 Reduction of Carboxylic acid in aqueous solut ion using a carbodiimide reagent. Polysaccharide Solution (acid or H20 Steam bath Glass tubing Rubber Tubi ng HYDROLYZE COOL DIALYZE d ia l y s i s tubing Figure 9 Par t ia l Hydrolysis Apparatus 59 sugars to the corresponding a l d i t o l s sodium borohydride in water i s used. In the analysis of the constituent sugars the methyl ester of the uronic acid i s reduced with sodium borohydride in anhydrous methanol. I I . 4. 5. Base-catalyzed degradation The spec i f i c degradation of polysaccharides has been an OCC. Of,~7 area of keen interest recently ' . In th is study a base-catalyzed 263 271 3 - e l iminat ion ~ reaction was used in the structura l i n ve s t i -gations of both K12 and K58. The reaction i s performed on the methylated polysaccharide to achieve degradation of the uronic acid residue (see Scheme 7) and hence to determine i t s locat ion (whether in the backbone or side-chain) and i t s point of attachment. 272 The base used i s the methylsul f inyl anion. Joseleau has suggested the use of the potassium instead of the sodium counter-ion. The point of attachment of the uronic acid residue i s then d i r e c t l y 273 labe l led with ethyl iodide . The reaction product i s characterized 1 13 by H and C n.m.r. spectroscopy and by g . l . c . - m.s. analys is . 274 I I . 4. 6. Par t i a l Hydrolysis / H Iso lat ion of fragments from par t ia l hydrolysis i s a major key to e luc idat ing the sequence of sugars in the polysaccharide and 1 13 also to making assignments in the H and C n..m.r. spectra. 237 Capon has reviewed the f i r s t order rate constants for the acid catalyzed hydrolysis of the glycosides and these data may be 60 1 ) DMSO" N a f 2) E t l / A g 2 0 A B C [ R h a , G l c . G a l p ] -D hOMe O^OMe J n Scheme 7 Base-catalysed degradation of Klebsiella K12 61 extended to polysaccharides. By varying acid type and concentration, along with temperature and the reaction time, an optimal y i e l d of ol igosaccharides may be obtained. The fol lowing general isat ion may be madejfuranosidie and deoxy sugars are more l ab i l e than the corresponding pyranosidic hexoses, which are in turn more l a b i l e than uronic acid residues. To minimise further degradation of ol igosacchar ides, once formed, an apparatus s im i la r to that described by Galanos et al_ ^ (see F ig . 9) was employed in the invest igat ion of K12. After separation and pur i f i ca t i on (see Scheme 8) a neutral and an ac id i c dissacharide were characterized by n.m.r. spectroscopy. In the invest igat ion of K58 the aldobi - , a ldotr i -and aldotetraouronic acids were obtained. Character izat ion of each, 1 13 incremental ly, by H and C n.m.r. spectroscopy permitted the assignment of the spectra of the in tac t polysaccharide. To demonstrate conclusively the l inkage posit ions of the pyruvic acid ketal of K58, a very mild acid hydrolysis was performed on the native polysaccharide in which the ketal was removed (as evinced by n.m.r. spectroscopy). Subsequent methylation analysis (see Table 8) indicated the posit ions of attachment. In addit ion a mild hydrolysis was performed on the f u l l y methylated material in order to remove the pyruvate ketal , and the product was re-methylated. G . l . c . analysis of the product ve r i f i ed the posit ions of l inkage (see Table 8). 62 OH OH F 0.1 M T F A 9 5 ° 16 h D i a l y s i s A - B E-D Scheme 8 Par t i a l hydrolysis and pur i f i ca t i on of K lebs ie l l a K12 63 I I . 4. 7. Location of O-acetyl group An O-acetyl substituent in the polysaccharide may be detected 1 13 by H and C n.m.r. spectroscopy. The ra t io of the integrat ion of the O-acetyl CHg peak to the peaks in the anomeric region w i l l indicate i f subst i tut ion occurs on every repeating uni t . A sharp n.m.r. peak indicates that the group occurs at a d iscrete pos i t ion in each repeating uni t , (see Spectrum No. 21). The O-acetyl group may be located by the method of de Belder 275 and Norman. . With th is procedure a l l the free hydroxyl groups are blocked with methyl v inyl ether. The base l ab i l e O-acetyl group i s removed and replaced with a stable methyl group. The protect ing groups are removed with acid and the product i s analyzed by g . l . c . -m.s., as the a l d i t o l acetates of the constituent sugars. Problems ar ise i f (a) a l l the free hydroxyl groups are not protected (methylation at more than one pos i t i on) , (b) the 0_-acetyl group i s not removed with base (no methylation) or (c) the O-acetyl group i s removed at the protect ing stage and replaced with methyl v iny l ether (no methylation). An a l ternat ive i s to methylate the native polysaccharide 250 under conditions which do not remove the 0_-acetyl group. Prehm has described a procedure (see Table 3),using tr imethyl phosphate as solvent ( less electron - donor a c t i v i t y ) , 2, 6 d i - ( t e r t - butyl) pyridine as proton scavenger,and tr if luoro-methanesulfonate as methylating agent. 64 In th is work the O-acetyl substituent in K lebs ie l l a K58 polysaccharide was located by comparing g . l . c . - m.s. data from 275 the ana lys is , by the method of de Belder and Norman, of samples of the native and de-acetylated polysaccharide. 65 STRUCTURAL INVESTIGATION OF KLEBSIELLA CAPSULAR POLYSACCHARIDE SEROTYPES K12, K58, K23 and K70 66 I I I . I. STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDE _OF KLEBSIELLA SEROTYPE K12. ABSTRACT K lebs ie l l a K12 capsular polysaccharide has been invest igated by the techniques of methylation, Smith degradation - periodate ox idat ion, uronic acid degradation and par t i a l hydro lys is , in conjunction with 1 13 H-n.m.r. spectroscopy at 100 and 220 HMz and C-n.m.r. spectroscopy at 20 MHz. The structure has been found to consist of the hexa-saccharide repeating unit shown, having a D-galactofuranosyl unit at the branch point. A galactofuranosyl residue has only previously been found, in th is ser ies , in the polysaccharide from K lebs ie l l a K41. -^3)-a-p-Gal£-(1^2)-B-D-Galf-(1^6)-a-D,-Glcp_-(l->3)-a-L-Rhap-(l-» , _ 3 1 B-D-GlcjiA; 4 1 B-D-Galp_ pyruvate I I I . I . I . Introduction: 23 The genus Klebsiel l a has been c l a s s i f i e d by (prskov into approximately 80 serotypes, based on the i r ant igenic , capsular 30 31 polysaccharides. Nimmich ' has analyzed qua l i t a t i ve l y the poly-saccharide from each s t r a i n ; K12 was found to contain glucose, 67 galactose, rhamnose, glucuronic acid and pyruvic ac id. As part of our continuing invest igat ion of the re lat ionsh ip between primary, chemical structure and immunological a c t i v i t y , we now report on the e luc idat ion of the structure of the K12 polysaccharide. This structure i s in agreement with the predict ions made by Heidelberger and coworkers, based on the cross-react ions of the polysaccharide with anti-pneumococcal and an t i -K l ebs i e l l a sera, 55 of the occurrence of a 1,3-a l inked L-rhamnosyl residue and of a 54 (non-reducing) 4,6-0-( l-carboxyethyl idene)-D-galactosyl group in the repeating uni t . I I I . 1. 2. Results and discussion Composition andn.-m.r.^ spectra K l ebs i e l l a K12 bacter ia were grown on an agar medium, and the capsular polysaccharide i so lated was pur i f i ed by one prec ip i ta t ion with Cetavlon. As described in Sec. I I . 1 . the product had [ a ] Q + 24.2°. Paper chromatography of an acid hydrolyzate of the poly-saccharide showed the presence of glucose, galactose, glucuronic acid and rhamnose. Carboxyl-reduced K12 polysaccharide was hydrolyzed, and the presence of glucose, galactose and rhamnose in the rat io of 2:3:1 was determined by gas- l iqu id chromatography ( g . l . c . ) of the i r a l d i t o l acetates (see Table A). Rhamnose was shown to be of the L configuration and glucose of the D configurat ion by c i r cu l a r dichroism (c.d.) 147 measurements of the derived a l d i t o l acetates . Subsequently arab in i to l pentaacetate was s im i l a r l y shown to have the L conf igurat ion, 68 TABLE 4 G.L.C. ANALYSIS OF NATIVE AND PERIODATE OXIDIZED -^ POLYSACCHARIDES. Sugars * c Column C-(SP 2340) Mole % (as a l d i t o l acetates) Glycerol 0.09 11.7-Erythr i to l 0.26 12.4-Threi to l 0.33 9.9^-Rhamnose 0.45 17.3 21.8 Arabinose 0.65 22.5 Galactose 0.95 51.7 21.7 Glucose 1.00 31.0 -— On carboxyl-reduced polysaccharide. — Retention time re la t i ve to g luc i to l hexaacetate. — Programmed at 180° for 8 min, and then 4° per min to 240°. — I , native polysaccharide, uronic acid reduced. 11,periodate ox idat ion, reduction, and tota l hydrolysis of the carboxyl reduced polysaccharide. — Some loss of vo l a t i l e components during der i va t i za t ion . 69 ind icat ing that the galactofuranose unit from which i t was derived by loss of C-6 on oxidation has the D conf igurat ion. An acid hydrolyzate of the polysaccharide gave a pos i t ive react ion with D-galactostat reagent,*thus confirming the D configuration of the galactose. The 220 MHz, ^H-n.m/r.. spectrum of the polysaccharide, af ter mild hydrolysis to lower the v i s cos i t y , showed a sharp s ing le t at 6 1.66 ind ica t ive of a 1-carboxyethylidene group. This signal was present in a 1:1 ra t io with a doublet at 6 1.34 at t r ibutab le to the I CO 1CA methyl group of rhamnose ' . Six d iscern ib le s ignals were observed in the anomeric region, at 65.22 (1H, ^ weak), _65.16 (1H, J} 2 3Hz), 65.13 (2H 2 2Hz) ,6 4.66 (l.H ^ 2 8 H z ) , 6 4.48 (T;H ^ > 2 6Hz). The 1 3 C-n .m. r , spectrum of the polysaccharide 2 0 2 ' 2 3 1 (150 mg/2 ml) showed high f i e l d peaks at 17.57 p.p.m. (rhamnose CH3) and 22.13 p.p.m. (pyruvate CH^). In the anomeric region f ive s ionals in the ra t io 1:1:2:1:1 were seen at 108.39, 106.99, 102.64, 99.43 and 97.18 p.p.m. 13 Interpretat ion of these p.m.r. and C-n.m.r. data i n i t i a l l y caused some d i f f i c u l t y , as the p.m.r. data suggested four a-1inked and 13 two 3 - l i n k ed residues, while C-n..m.r. data indicated three a- l inked and three p- l inked residues. This problem was resolved when the methylation data showed the presence of a furanosyl sugar (see 1ater). The signals at 63.85 p.p.m. and 61.77 p.p.m. were assigned to the C-6 carbon atoms of hexoses and the signal at 66.44 p.p.m. to a C-6 carbon involved in a l inkage. Ful l assignments are shown in Table 5. * A co lor imetr ic enzymic reagent (Worthington Biochem.Co.) TABLE 5 N.M.R. DATA FOR K lebs ie l l a K12 CAPSULAR POLYSACCHARIDE -AND THE DERIVED OLIGOSACCHARIDES. -i TO Compound- H-n.m. r. • data C-n.-m.r, data 6 - J T ,2 Integral Assignment- p.p.m.- Assignment-(Hz) c (H) GTcA ^ Gal-OH (1) P 'X, E-D Glc^Rha-OH (2) A - B R h a ^ G a l — A r a ^ g l y c e r o l (3) Ct Ot Ot B-C-D-A 5.30 2 0.6 a-Gal ,~OH 104.46 p-Gl cA 4.76 7. ,5 1 GlcA . 96.98 B- Gal~ OH 4.65 8 0.4 3-Gal~0H 93.01 a-Gal~0H 61.79 C-6 of Gal 5.15 s 0.6 a-Rha~0H 96.41 a - G l c £ 5.10 b 0.6 a-Glc 96.13 a - G l c £ 5.08 b 0.4 a-Glc 94.52 a+B-Rha~0H 4.88 s 0.4 g-Rha~0H 61.12 C-6 of Glc 1.30 6 ( J 5 , 6 ) 3 CH3of Rha 17.76 CH3 of Rha 5.19 1 1 a-Rha 106.93 Ara -5.10 2 1 a-Ara- : 103.10 Rha 5.05 1 1 a-Gal 99.14 Gal 1.30 4 3 CH 3 of Rha 17.46 CH3 of Rha TABLE 5 Contd. • 3 G a l £ ^ a l f-^rGl c ^ R h a -C-D-A-B E F pyruvate 5.22 5.16 5.13 5.13 4.66 4.48 s 3 2 2 8 6 4.3-4.5 b 1.66 s 1.34 6 1 2 1 1 2 3 3 a-Rha a-Glc a-Galp 3-Galf 3-GlcA 3-Gal£ H-2, H-3 3-Galf CHg of acetal CH 3 of Rha 186.47 C-6 of 3-GlcA 108.39 3-Galf 106.99 3-Gal£ 102.64 a-Rha + 3-GlcA 99.43 a-Gal 97.18 a-Glc 85.71 C-2 Galf 84.24 C-3 of 3-Galf 83.00 C-4 GlcA 66.44 C-6 of a-Glc 63.85 C-6 of 3-Galf -61.77 C-6 of a-Gal 22.13 CH 3 of acetal 17.57 CH 3 of Rha — For the o r ig in of compounds 1 - 3, see text . See Appendix III for reproductions of the spectra. b ^ % — Chemical sh i f t re la t ive to internal acetone; 6;2.23 downfield from sodium 4,4-dimethyl-4-s i lapentane- l -sulfonate (D.S.S.). — Key: b = broad, unable to assign accurate coupling constant, s= s ing le t . — For example, a-Gal = Proton on C-l of a-1inked D - Gal residue. — Chemical sh i f t in p.p.m. downfield from Me,Si, re la t i ve to internal acetone; 31.07 p.p.m. downfield from D.S.S. f d 13 — As for —, but for anomeric C nuc le i . -2- This g lycos id ic atom resonates as two doublets,because of the anomeric equi l ibr ium of the reducing unit . 72 Methylation of or ig ina l polysaccharide Methylation 2 4 1 > 2 4 2 ' 2 4 7 0 f Kl2 polysaccharide, followed by reduction of the uronic ester , hydro lys is , der ivat i za t ion as 103 121 a l d i t o l acetates, and g . l . c . - m.s. analysis ' indicated that K12 i s composed of a hexasaccharide repeating unit with f i ve pyranose sugars and one furanose, namely, galactofuranose, which const i tutes a branch point (see Table 6). These data also indicate that the (1-carboxyethylidene) group i s l inked to 0-4 and 0-6 of a (terminal) galactopyranosyl group. Analysis of a re-methylated sample of the reduced product showed the formation of 2,3,6-tri-0_-methylglucose and the disappearance of the 2,3-di-0-methylether, thus estab l ish ing that the uronic acid i s glucuronic ac id . Base-catalyzed degradation To determine the locat ion of the glucuronic ac id , the methylated polysaccharide was subjected to a base-catalyzed degradati 273 and was then d i r ec t l y ethylated . The i so l a t i on of a polymeric, degraded product indicates that the uronic acid i s in the side chain (see Scheme 7). On hydro lys is , and der i va t i za t i on , for g . l . c . -m.s. the compounds shown in Table 6 were obtained, ind icat ing that the glucuronic acid i s attached to 0-3 of the galactofuranosyl un i t , and that the only other sugar in the side chain i s a 4,6-0-(l-carboxyethyl idene)-D-galactose group. The - n.m.r.•spectrum indicated the absence of two 8- l inkage signals in the anomeric region at t r ibutab le to the sugars of the side chain. 73 TABLE 6 METHYLATION ANALYSIS OF NATIVE, AND DEGRADED, K lebs ie l l a K12 CAPSULAR POLYSACCHARIDE Methylated sugars- * . Mole % -las a l d i t o l acetates) Column A-(OV-225) Column B^  (ECNSS-M ) I I I I I 2,4-Rha 1.00 1.00 20.02 18.17 22.48 2,4,6-Gal 1.88 1.55 21.66 21.88 26.46 2,3,4-Glc 2.05 1.63 16.96 25.24 2,3,6-Glc 2.05 1.63 31.10 5,6-Gal 2.28 1.75 15.88 13.11 2,3-Glc 3.30 2.31 13.38 2,3-Gal 3.42 2.40 12.10 15.74 3,5,6-Gal 5- 1.46 25.82 -2,4-Rha = 1,3,5-t r i -0-acety l -2,4-d i -0-methy l-L - rhamin i to l ________ ^-Retention time re la t i ve to that of the a l d i t o l acetate der ivat ive of 2,4-Rha. ^Programme: 180° for 4 min and then 2° per min to 200°. ^Programme: 165° for 4 min, and then 2° per min to 200°. —Values corrected by using e f fec t ive carbon response f a c t o r s ^ . —I o r ig ina l polysaccharide methylated and uronic ester reduced, column B. II as in I but remethylated, column A. I l l a f te r uronic acid degradation and ethy la t ion. -S-l ,2 ,4-Tr i -0-acety l -3-0-e thy l -5 ,6-d i -0-methy l -ga lac t i to l . 74 Par t ia l hydrolysis A sample of K12 polysaccharide in the f ree-ac id form was hydrolyzed for lOh with 0.1M t r i f1uoroacet ic ac id in an apparatus s im i la r to that described by Galanos and co l leagues^, y i e ld ing a mixture of oligomers and monosaccharides, which was separated with AG-1 X2 ion exchange res in into ac id i c and neutral f ract ions (see Scheme 8.'). Preparative, paper electrophoresis of the ac id i c f rac t ion gave an aldobiouronic acid (1) which, a f ter hydrolysis and paper chromatography, was shown to consist of glucuronic acid and galactose. 1 13 H- and C-n.m/r. spectroscopy indicated that the reducing galactose was now in the pyranose form and ve r i f i ed that the side chain linkage i s 3 (see Table 5 ) . The structure of the aldobiouronic ac id (E-D 1) i s thus 3-D-GlcpA-(l+3)-D-GaljD~0H. ... Separation of the mixture of neutral oligomers by gel chromatography (Bio-gel P-2), followed by pur i f i ca t i on on paper chromatography, gave a disaccharide (A-B 2) which, af ter hydrolysis and paper chromatography, was shown to consist of 1 1 3 glucose and rhamnose. The H- and C- n.m.r. spectra are consistent with the structure a-D-Glcp-(l->3)-L-Rhap~0H. Periodate oxidation of the carboxyl-reduced polysaccharide To determine the sequence of the sugars in the backbone of 264 the polymer, the carboxyl-reduced polysaccharide was oxidized with 256 periodate. After 90h. the consumption of oxidant ., was 5.4 moi per moi of repeating unit . The theoret ica l consumption i s 5 moi i f the (1-carboxyethylidene) group remains in tac t ; the higher consumption indicates loss of some of the ketal. groups. Total hydrolysis of 75 the polyol obtained af ter sodium borohydride reduct ion, followed by der ivat i zat ion as the a l d i t o l acetates, gave the g . l . c . separation shown in F ig. 5 (see Table 4 ) . The low proportion of th re i to l i s consistent with some loss of the pyruvic acid ketal . Smith 253 hydrolysis of the po lyo l , followed by sodium borohydride reduction, y ie lded a mixture of ol igosaccharides which was separated by gel chromatography. Oligomer 3 was obtained pure by preparative paper chromatography and was shown to consist of rhamnose, galactose, 1 13 arabinose, and glycerol in equal proportions. H- and C-n.m.r. spectra were in agreement with these data (see Table 5). To determine the sequence of sugars in 3, the ol igosaccharide was permethylated 242 by the Purdie method and the product pur i f i ed by g . l . c . on OV-1 and examined by electron-impact, mass spectrometry. Detection of peaks at m/e 189 and 393, among others, indicated that the deoxyhexose i s l inked to the hexose, not to the pentose. The source of some pertinent fragments i s i l l u s t r a t ed below. The-anomeric •• 1 13 nature of the linkages was determined by H- and C-n.m.r. spectroscopy. Oligomer (B-C-D-A 3) i s thus establ ished as having the structure a -L-Rha£-( l+3)-a-D-Gal£-(U2)-a -L-Araf-( l+l)-g lycerol MeO OMe m/e 189 | 0 - C H 2 - •CHOMe- "CHgOMe 583! 627! 76 I I I . 1. 3 Conclusion I t thus fol lows that K lebs ie l l a K12 capsular polysaccharide had the fol lowing structure. After the rea l i za t ion, from the methylation g . l . c . -m.s . data, 1 13 that a furanosyl residue was present, the H- and C-n.m.r. spectra were more eas i l y interpreted. In the former, the 3 - Galf anomeric s ignal appears at 6 5.13, the region normally at t r ibuted to a-1inked 13 pyranoses. In the . C spectrum, however, the anomeric signal occurs in the unambiguous 3- l inkage region at 108.39 p.p.m. (see Table 6). I t i s in terest ing to note that the only other K l ebs i e l l a 169 polysaccharide reported to have a furanosyl un i t , K41 (see App.I) has a very s im i l a r structure in which the terminal 4,6-0-(1-carboxyr ethylidene ;)-B-D-galactopyranosyl group i s replaced by 3-D-Glcp_-(l-*-6)-a-D-Glcp_-. As expected,no cross-react ion occurs between these two polysaccharides, because the sidechain i s usual ly the immunodominant 77 group. Cross-reaction does, however, occur with an t i -K l l "^ which 276 has a 4,6-0-1(1-carboxyethylidene)-a-D-galactopyranose side chain 55 (see App.I) and with anti - Pn-VI which has an in-chain-a-D-Glcp_-(l->3)-a-L-Rha£- unit . I I I . 1. 4. Experimental General methods — Concentrations were carr ied out under diminished pressure at bath temperatures not exceeding 40°. Paper electrophoresis was performed on a Savant high voltage (5 Kv) system (model LT - 48A) with kerosene as coolant. The buffer used contained pyridine - acet ic ac id - water ( 5 : 2 : 743, v/v) , pH 5.3. Str ips of Whatman No. 1 paper (77 cm x 20 cm) were used for a l l runs, with appl icat ion of 25 - 50 mA for 1% h. Descending paper chromatography was carr ied out using Whatman No.l paper. The fol lowing solvent system (v/v) were used: (]_) f resh ly prepared 2:1:1 1-butanol - - acet ic acid - -water, and {_) 8:2:1 ethyl acetate — pyridine - - water. Sugars and ol igosaccharides were detected, a f ter electrophoresis and after descending, paper chromatography, with an a lka l ine s i l v e r n i t ra te . 24 reagent . Analyt ica l g . l . c . separations were performed with a Hewlett Packard 5700 instrument f i t t e d with dual f lame-ionisat ion detectors. An Infotronics CRS-100 e lec t ron ic integrator was used to measure peak areas. Separations were performed in s ta in less -s tee l columns (1.8 m x 3 mm) with a carr ier-gas f low-rate of 20 mL/min. Columns used were (A) 3% of OV-225 on Gas Chrom Q (100-120,mesh); (B) 5% of. ECNSS-M on the same support; and (C.) 3% of SP-2340 on Supelcoport (100-1.20 mesh). Analogous columns (1.8 m x 6.3 mm) were used, along 78 with a column of 5% of OV-1 on Gas Chrom Q (100-120 mesh) for preparative g . l . c . separations. G. l . c . -m.s . was performed with a Micromass 12 instrument f i t t e d with a Watson-Biemann separator. Spectra were recorded at 70 eV with an ion isat ion current of 100 pA and an ion-source temperature of 200°. "'H n.m.r.' spectra were recorded on e i ther a Varian XL-100 instrument at 90°, or a (Nicolet/Oxford Instruments) H-270 at 13 ambient temperature. C n.m.r.. spectra were recorded on e i ther a Varian CFT-20 or a Bruker WP-80 1 3 C 20.1 MHz instrument at ambient temperature. Addit ional spectra were obtained courtesy of Dr. A.A. Grey and A. Lee ('H : HR-220 MHz, 90°) and Dr. Michel Vignon ( ]H : Cameca 250 MHz, 90° and 1 3 C : Cameca 62.87 MHz, 80°). C i rcu lar dichroism (c.d.) spectra were recorded on a Jasco J20 automatic recording spectropolarimeter with a quartz ce l l of path length 0.01 cm. Optical rotat ions were measured at 23 ±2° on a Perkin Elmer model 141 polarimeter, with a 10-cm c e l l . Infrared spectra were recorded using a Perkin-Elmer 457 spectrophoto-meter. Preparation and p r ope r t i e s .— A culture of K lebs ie l l a Kl2 (313) was obtained from D.I. (|)rskov (Copenhagen). The polysaccharide was i so la ted as described, in section II.1 and showed [ a ] Q + 24.2° (c_ 1, water). Analysis of const ituent sugars. — Methanolysis of a sample (20 mg) of K12 polysaccharide with 3% methanolic hydrogen chlor ide 79 and subsequent treatment with sodium borohydride in anhydrous methanol reduced the uronic ester. Hydrolysis with 2M t r i f l uo roace t i c ac id (TFA) overnight at 95° fol lowed by reduction (NaBH^) and acety lat ion gave ga lac t i t o l hexaacetate, g luc i to l hexaacetate and rhamnitol pentaacetate in the ra t io 3:,2;::1 (column £, programmed at 180° for 8 min and then 4°/min to 240°). C i r cu la r dichroism (c.d.) of the l a t t e r two components i so lated by preparative g . l . c , showed pos i t ive and negative curves, respect ive ly , confirming that glucose has the D configurat ion and rhamnose the L conf igurat ion. The configuration of galactose was deduced to be D from the negative c .d . of the pentaacetate of i t s oxidation product, namely, a rab in i to l pentaacetate. This was confirmed by the pos i t ive action of D - Galactostat (Worthington Biochemical Co.) on the hydrolysis product of the polysaccharide. Methylation of the native polysaccharide. — Methylation 247 of K12 polysaccharide under the Hakomori condit ions, followed by 242 a Purdie treatment y ie lded a product that showed no hydroxyl absorption in the i . r . spectrum. This material was reduced overnight with sodium borohydride in'oxolane (THF) and ethanol (1:1 v/v). A portion of th is product was hydrolyzed with 2M t r i f l uo roace t i c acid for 16 h at 95°; the resu l t ing mixture was reduced with sodium borohydride and the product acetylated. G . l . c - m.s. gave the resul ts shown in Table 6. Another portion of the material (reduced uronic ester) was remethylated under Purdie conditions for 2 days, and der ivat ized for g . l . c . - m.s. giv ing the compounds shown in Table 6. 80 Uronic acid degradation^""'— A solut ion of care fu l l y dr ied, methylated polysaccharide (100 mg) and £-toluenesulfonic ac id (a trace) in 19:1 dimethyl sulfoxide - 2,2-dimethoxypropane (20 mL) was prepared in a serum v ia l which was then sealed with a rubber cap. The v ia l was flushed with ni trogen, and the so lut ion was s t i r r ed for 3h. Sodium methylsulphinylmethanide (2M) in dimethyl sul foxide (10 mL) was then added with the aid of a syr inge, and the so lut ion was s t i r r ed at room temperature overnight. Af ter external cool ing to 10°, ethyl iodide (3 mL) was added slowly using a syringe . The solut ion was s t i r r ed for a further 30 min., excess of ethyl iodide was removed using a rotary evaporator, and the so lut ion was dialyzed overnight against tap water. After l yoph i l i za t i on the product (65 mg) was pur i f i ed by prec ip i ta t ion with petroleum ether (30°- 60°), y i e ld ing 60 mg of polymeric mater ia l . Subsequent hydrolysis and der iva t i za t ion for g . l . c . - m.s. gave the resul ts in Table 6. Pa r t i a l hydrolysis — A sample of K l ebs i e l l a K12 polysaccharide was exchanged to the f ree-ac id form with Amberlite IR-120 (H+) ion-exchange res in , and l yoph i l i zed . This material ( lg) was dissolved in water (100 mL pH 3.2) and was then auto-hydrolyzed on a steam bath for 16 h in an apparatus s im i l a r to that described by Galanos e_t al_ . Very l i t t l e hydrolysis occurred; therefore the solut ion was made 0.1M in TFA, and the reaction continued for a further 16 h. After removal of TFA the products (700 mg) were separated into neutral and ac id i c f ract ions by using AG-IX2 ion 81 exchange res in . Portions (200 mg) of the ac id i c f ract ion were separated by gel chromatography on a column (100 x 2.5 cm) of Sephadex G-25, which was i r r i ga ted with a buffer (1000:10:4 v/v water - - pyridine - - acet ic ac id ,) at a flow rate of 10 mL/h. Separation of components was poor. Fractions containing components with —Glc >0.2 (solvent J_) were combined, and separated by preparative, D paper e lectrophoresis. A component 1 with -GlcA 0.69 was obtained pure (30 mg) and was shown on tota l hydrolysis to consist of glucuronic 1 13 acid and galactose (E-D). H- and C-n.m.r. spectroscopy indicated the presence of a g - l inked glucuronic acid and a reducing galactose (see Table 5 ) . Portions (200 trig) of the neutral f ract ion were separated by gel chromatography on a column (100 x 2.5 cm) of B.iogel P-2. I r r i ga t ion with the same buffer, at a flow rate of 10 mL/h, l yoph i l i za t i on of the fract ions, and examination by. paper, chromatography (solvent 2) revealed that separation was not complete. D Pur i f i ca t i on of a component with -G lc 0.61 by paper chromatography then y ie lded compound 2 (30 mg) which on hydrolysis was shown to 1 13 consist of glucose and rhamnose (A-B). H- and C- n.m.r. spectroscopy indicated the presence of an a-1inked glucose and a (reducing) rhamnose (see Table 5 ) . Periodate oxidation of carboxyl-reduced polysaccharide. — A sample of the polysaccharide was reduced by the procedure of Taylor 264 and Conrad ; two treatments were required in order to achieve complete reduction. Reduced, capsular polysaccharide (200 mg) was 82 dissolved in water (40 mL) to which 0. IM sodium metaperiodate (40 m L) was then added. The solution-was s t i r r ed in the dark at 3° and 256 periodate consumption was monitored spectrophotometrically . After three days,consumption had reached 5.4 molecules per repeating uni t . Ethylene glycol (10 mL) was then added. After s t i r r i n g for a further 30 minutes the mixture was dialyzed overnight against running tap water, and the product reduced with sodium borohydride. The polyol was i so la ted by d i a l y s i s and l yoph i l i z a t i on . A port ion (5 mg) of the polymeric product was hydrolyzed with 2M TFA overnight at 95°. Paper chromatography (solvent 2) then showed the presence of g l yce ro l , a tetrose, rhamnose, arabinose and galactose. Conversion of the hydrolysis products into the corresponding a l d i t o l acetates gave the g . l . c . resu l ts shown in Table 4. Smith hydrolysis (0.5M TFA overnight at room temperature) of the polyol gave a mixture which was separated on Biogel P-2. An oligomer (B-C-D-A) R 1 3 (—Glc 0.46 solvent 1) was pur i f i ed by paper chromatography. H- and 1 3 C- n.m.r. data are shown, in . Table 5. The mass spectrum of the 242 permethylated (Purdie method) oligomer showed s i gn i f i can t peaks at 627, 583, 553, 540, 527, 467, 393, 375, 361, 290, 289, 273, 272, 260, 259, 217, 189, 187 and 103. Total hydrolysis of the oligomer (2M TFA, 90°, 16 h) gave galactose, rhamnose, arabinose and glycerol by paper chromatography (solvent 2). G . l . c . analysis of the derived a l d i t o l acetates showed that the constituents were present in equimolar proportions. The anomeric nature of a l l the linkages was shown to be 1 13 a by H- and C- n.m.r. spectroscopy (see Table 5) . 83 I I I . 2 STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDE OF  KLEBSIELLA SEROTYPE K58 ABSTRACT K lebs i e l l a K58 capsular polysaccharide has been invest igated by the techniques of methylation, Smith degradation - periodate ox idat ion, uronic acid degradation and par t i a l hydrolys is , in conjunction with 1 13 H-n..m.r. spectroscopy at 100 and 220 MHz, and C-n.m.r.. spectroscopy at 20 MHz. The structure has been found to consist of the tetrasaccharide repeating unit shown, with one O-acetyl group per repeating unit . A uronic acid residue bearing a 1-carboxyethylidene moiety has previously been found, in th is ser ies , only in the polysaccharide from K lebs ie l l a K l . •3)-a-D-Glc£-(l->4) -p-D-Glc£A-( l -»4 ) -a-L-FuC£-( l -A Me COOH 2 'I 0-Ac a-Q-Gal£ n I I I . 2 . 1 . Introduction 23 The genus K lebs ie l l a has been c l a s s i f i ed by (prskov into approximately 80 serotypes, based on the i r ant igenic , capsular 30 31 polysaccharides. Nimmich ' has qua l i t a t i ve l y analyzed the polysaccharide from each s t r a i n ; K58 was found to contain glucose, galactose, fucose, glucuronic acid and pyruvic ac id . In add i t ion, K58 was shown to contain one O-acetyl group per repeating uni t . As 84 part of our continuing invest igat ion of the re lat ionship between primary, chemical structure and immunological a c t i v i t y we now report on the e luc idat ion of the structure of K58. I I I . 2 .2 . Results and Discussion Composition and n.m.r.. spectra K lebs ie l l a K58 bacter ia were grown on an agar medium, and the capsular polysaccharide i so lated was pur i f i ed by one prec ip i ta t ion with Cetavlon as described in Sec. I I . 1 . The product had [a] D + 19.0°. Paper chromatography of an acid hydrolyzate of the poly-saccharide showed the presence of glucose, galactose, glucuronic acid and fucose. Carboxyl-reduced K58 polysaccharide was hydrolyzed, and the presence of glucose, galactose and fucose in the rat io of 2:1:1 was determined by gas- l iqu id chromatography ( g . l . c . ) of the i r a l d i t o l acetates. Fucose was shown to be of the L configuration and glucose of the D configuration by c i r cu l a r dichroism (c.d.) 147 measurements of the derived a l d i t o l acetates . Galactose was shown to be of the D configurat ion by the pos i t ive reaction of D-galactostat reagent with an acid hydrolyzate of the polysaccharide. The 220-MHz H^ - n. m. r. spectrum of the polysaccharide showed sharp s ing lets at 62.17 and 61.64 and a doublet at 61.33 in the approximate ra t io of 1:1:1. These were assigned to methyl groups of 0-acetate, 1-carboxyethylidene and fucose, respect ively ^2-164^ Four d iscern ib le s ignals were observed in the anomeric region, at 65.45 (1H, J- 2 2Hz), 65.18 (TH, J , 2 2Hz), 65.13 (1H, J , 2 2Hz) TABLE 7 N.M.R. DATA FOR K lebs ie l la K58 CAPSULAR POLYSACCHARIDE AND THE DERIVED OLIGOSACCHARIDES Compound— eb- J T , 2 C (Hz)-^H-n. m.r. data Intergral Assignment-(H) 13 - C-n.m.r. data p.p.m.— Assignment— GlcA^-iFuc~OH 5.26 3 .5 a-Fuc~OH 103.91 B-GlcA P 4.59 10 1.7 3-Fuc~0H 97.07 B-Fuc~0H (1) 4.54 6.5 3-GlcA 93.16 a-Fuc~0H 1.33 6.0 3 CH 3 of a-Fuc~OH 23.7 CH 3 of 1-carboxy-ethylidene 1.29 6.5 CH 3 of B-Fuc-OH 16.3 CH 3 of fucose 1.64 s 0.3 CH 3 of 1-carboxy-ethylidene G l c ' ^ G l c A ^ f u c - O H Ct p 5.46 3.5 1 a-Glc 103.94 B-GlcA 5.26 3 .6 a-Fuc~OH 99.5 a-Glc 4.61 7 1.8 6-Fuc-OH 97.16 B-Fuc~0H (2) a. 4.57 7 B-GlcA 93.3 a-Fuc~0H 1.64 s 0.4 CH 3 of 1-carboxy-ethylidene 61.2 C-6 of Glc 1.33 6 3 CH 3 of a-Fuc~0H 23.7 CH 3 of 1-carboxy-ethylidene 1.29 6 CH 3 of B-Fuc-OH 16.25 CH 3of fucose TABLE 7 Contd. GIC^ GICAM-FUC-OH I Gal (3) a. 5.45 5.29 4.60 4.55 1.64 3 ;5 3 8 7 s .9. 1.6 1.7 0.4 1.33 1.29 6.5 6.5 3 • ••^GlcLiGlcAMFucl a 3 2 p \ / Me XC00H (10%) (4) 2 u I O-Ac 5.44 5.27 4.57 1.64 1.33 2 b 8 s a-Glc 104.0 a-Fuc~0H 99.7 a-Gal 99.5 B-Fuc~0H 97.2 3-GlcA 93.3 CH 3 of 1-carboxy-ethylidene 61.07 62.59 CH 3 of Fuc-OH 23.7 CH 3 of Fuc~0H 16.25 a-Glc 104.0 a-Fuc 100.3 B-Gl cA 99.7 CH, of 1-carboxy-ethylidene 61.3 CH 3 of fucose 23:5 16.1 B-GlcA a-Gal a-Glc 3-Fuc~0H a-Fuc~0H C-6 of Glc C-6 of Gal CH 3 of 1-carboxy ethyl idene CH 3 of fucose (3-G1 cA a-Fuc a-Glc C-6 of Glc CH 3 of 1-carboxy ethy l i dene CH0 of fucose TABLE 7 Contd. ^Gl c - ^G l c / V ^ F u c — 2-0-Ac a 1 Gal +0Ac (5) ^ I c ^ - ^ l c / v L i F u c V 3 1 Me COOH 1 2-0-Ac Gal + O-Ac (6) a. 5.35 5.25 5.16 4.53 2.17 1.33 5.45 5.18 5.13 4.59 2.17 1.64 1.33 s s s 8 s 6 2 b 8 s s 1 : 0.9 1 1 2 3 1 2 1 3 3 a-Gl c a-Fuc a-Gal 3-GlcA CHg of acetate CH3 of fucose a-Glc a-Fuc a-Gal 3-GlcA CH3 of acetate ChL of 1-carboxy-e thy l i dene CH3 of fucose 104.5 B-GlcA9-101.2 a-Fuc 99.5 a-Glc 97.5 a-Gal 62.5 C-6 of Gal 61.4 C-6 of Glc 29.98 CH3 of acetate 23.4 CH3 of 1-carboxy-ethylidene 16.0 CH3 of fucose -For o r ig in of compounds I - see t e x t . * - Chemical s h i f t re la t i ve to in ternal acetone; 62.23 downfield from sodium 4,4-dimethyl-4si lapentane-l-sulfonate (D.S.S.).S..b = broad, unable to assign accurate coupling constant, s-s ing let . For example, a-Gal = proton on C-l of a - l i nked D-Gal residue, e. Chemical s h i f t in p.p.m. downfield from Me4Si, re la t i ve to internal acetone; 31.07 p.p.m. downfield from D.S.S. lAs for ^L, but for anomeric 13c nucle i . £ Spectrum recorded on Brucker WP-80 (20.1 MHz) using 5 mm tube with semi-micro volume cy l indr ica l cavity (6mm x 4.2 mm), i l 10% of 1-carboxyethylidene remaining a f t e r Smith hydrolysis. *See Appendix I I fo r reproductions of the spectra. 88 and 84.59 ( IH, ^ 2 8Hz). The , J C spectrum of the p o l y s a c c h a r i d e ^ ' ^ ' showed signals of equal in tens i t y at 16.0 p.p.m. (fucose CH^), 23.4 p.p.m. (1-carboxyethylidene CH )^ and 29.98 p.p.m. (acetate CH^). In the anomeric region four signals in the ra t io 1:1:1:1 at 104.5, 101.2, 99.5 and 97.5 p.p.m. were observed (see Table 7 ) . The signals at 61.4 and 62.5 p.p.m. were assigned to the C-6 1 13 atoms of hexoses. Both the H n. m.r.and C data indicate the presence of three a - l i nked and one g- l i nked residues. Assignments were made a f t e r n.m.r. -spectral invest igat ion of oligosaccharides iso lated from pa r t i a l hydrolysis and periodate oxidation (see l a t e r ) . Methylation of o r ig ina l polysaccharide 242 247 Methylation ' of K58 polysaccharide, followed by reduction of the uronic ester , hydrolys is, der iva t iza t ion as the 103 121 a l d i t o l acetates, and g . l . c . - m.s. analysis ' indicated that K58 is composed of tetrasaccharide repeating un i ts . The sugars are in the pyranoid form with fucose const i tu t ing a branch point (see Table 8 ) . Analysis of a re-methylated sample of the reduced product showed the formation of 6-0_-methylglucose, thus establ ishing that the uronic acid is glucuronic acid. Mild hydrolysis of th is remethylated polysaccharide ( to remove the 1-carboxyethylidene group), followed by methylat ion, showed on g . l . c . - m.s. analysis, the formation of 2 ,3 ,6- t r i -0-methy l glucose ind ica t ing that the 1-carboxyethyl idene;residue is l inked at fj-2 and 0-3 of the glucuronic acid residue; th is was confirmed by methylation analysis of a sample of autohydrolyzed native polysaccharide (see Fig....10). TABLE 8 METHYLATION ANALYSIS OF NATIVE, AND DEGRADED K lebs ie l l a K58 CAPSULAR POLYSACCHARIDE Methylated sugars- T* Mole %e (as a l d i t o l acetates) Column A i Column B£ I I II III IV V VI (OV-225) (ECNSS-M) 2,3,4-Fuc 0.80 - 4 2,3-Fuc 0.82 0.92 30 4-0Et, 2-Fuc - 0.9 38 2,4-Fuc 0.89 - - 2 2 2 2 2,3,4,6-Gal 1.00 1.00 28 28 29 23 43 2-Fuc 1.14 1.23 22 23 25 19 4 2,4,6-Glc 1.31 1.45 25 25 25 26 36 2,3,6-Glc 1.39 - 3 19 2,3-Glc 2.05 - 3 21 6-Glc 2.17 - 19 Glc 2.78 3.4 20 5 30 19 - 2,3,4-Fuc = 1,5-d i -0-acety l -2,3,4-t r i -0-methy l -L- fuc i to l etc . — Retention time re la t i ve to that of the a l d i t o l acetate der ivat ive of 2,3,4,6-Gal . — Programme: 180° for 4 min and then 2°per min to 200°. - Programme: 160° for 4 min and then 4° per min to 200°. — Values corrected by using ef fect ive carbon response fac to r s , 1 ^ 6 and adjusted to the nearest integer f - I, o r ig ina l polysaccharide, methylated and uronic ester reduced.II,as in I. but re-methylated. I l l , as in II,then hydrolyzed to remove 1-carboxyethylidene moiety and remethylated. IV,auto-hydrolyzed polysaccharide, methylated, and uronic ester reduced. V, Smith degradation product, methylated, and uronic ester reduced. VI,product of uronic acid degradation, and ethy la t ion . 90 ~N 1 1 1 1 1 0 4 8 12 16 20 Time (min) Figure 10 G . l . c . separation of products of methylation analysis of K lebs ie l l a K58 = IV, — - III (see Table 8) . 91 Base-cata lyzed degradat ion To determine the l o c a t i o n o f the u ron ic a c i d , the methylated po lysacchar ide was sub jec ted to base-cata lyzed degrada t ion , and was 273 then d i r e c t l y e t h y l a t e d . The i s o l a t i o n o f an o l i gosacchar ide i n d i c a t e s t h a t the uron ic ac id i s i n the backbone. On h y d r o l y s i s , and d e r i v i t i z a t i o n , f o r g . l . c . - m.s . , the compounds shown i n T a b l e s were obta ined i n d i c a t i n g t h a t g lucu ron ic ac id i s a t tached t o 0-4 o f fucose. Loss o f some glucose suggests t h a t i t i s l i n k e d to the g lucu ron ic ac id i n the backbone :(see Scheme 9 ) . Per iodate Ox ida t ion The n a t i v e po lysacchar ide consumed 1.8 moles o f per ioda te 255 per repea t ing u n i t < i n 10 h, y i e l d i n g , a f t e r sodium borohydr ide 253 reduc t ion and Smith h y d r o l y s i s a polymer ic product ( 4 ) . Reduction <\> o f the u ron ic a c i d fo l lowed by t o t a l h y d r o l y s i s and d e r i v a t i z a t i o n f o r g . l . c . showed the presence o f glucose and fucose i n the r a t i o 2:1 1 13 H and C n.m.r . spectroscopy o f the Smith - degradat ion product (4) showed the presence o f two a - l i n k e d and one B- l i n k e d sugars , i n d i c a t i n g t h a t , the o x i d i z e d te rmina l galactose i s a - l i n k e d (see Table 7 , Scheme 10) . Methy la t ion o f a p o r t i o n o f the Smith -degrada t ion p roduc t , fo l l owed by reduc t ion o f the u ron ic e s t e r , and d e r i v a t i z a t i o n f o r g . l . c . - m.s. , gave the r e s u l t s shown i n Table 8 i n d i c a t i n g t h a t the s ide chain galactose i s l i n k e d to 0-3 o f fucose. 92 MeO Scheme 9 Base-catalysed degradation of K lebs ie l l a K58 93 P-0 Scheme 10 Periodate Oxidation of K lebs ie l l a K58 94 Autohydrolysis A sample of K58 polysaccharide in the f ree-ac id form was autohydrolyzed at 95° for 3 h and then dia lyzed. 1H«n...m.r. spectroscopy of the product (5) showed the absence of 1-carboxyethylidene. Methylation of th is material followed by reduction of the uronic ester and der ivat i za t ion for g . l . c . - m.s. gave the resu l ts in Table 8 ind icat ing that the 1-carboxyethylidene group i s attached to 0-2 and 0-3 of the glucuronic acid residue. Par t i a l Hydrolysis A sample of K58 polysaccharide was hydrolyzed with 0.5M H^ S^O^  at 95° for 30 min y ie ld ing a mixture of ol igosaccharides. Preparative paper chromatography gave an aldobiouronic ac id (1) an a ldotr iouronic acid (2) and an aldotetraouronic acid (3). H and C spectroscopic data (see Table 7) indicated that the glucuronic ac id i s 3 - l i nked , and the glucose i s a-1inked and confirmed that the side chain galactose is a - l inked. Comparison of spectral data of (2) and (4) indicated that the fucose i s a-1inked(see Scheme 11). . Location of the O-acetyl group. — The H^ n.m.r. spectrum of the native polysaccharide showed a sharp acetate peak at 6 2.17 suggesting that the group appears in a discreet posi t ion in each repeating uni t . In order to locate the O-acetyl group K58 was treated with methyl vinyl ether in the presence of an acid ca ta lys t , and the product was then subjected to methylation analys is . However complete 95 Scheme 11 Partial Hydrolysis of Klebsiella K58 96 blocking of a l l OH groups was not rea l i zed , but comparison of data from analysis of the native polysaccharide and from a s im i la r analysis of a deacetylated sample indicated that the 0-acetyl group was l inked to 0-2 of fucose. I I I . 2.3. Conclusion I t thus fol lows that K lebs ie l l a K58 capsular polysaccharide has the fol lowing structure. +3) - a- D- Gl C£- (1 +4) - (3- D- Gl C£A- (1 +4) - a- L- Fuc£- (1 + 3 2 3 X • 0-Ac Me XOQH a -D-Gal£ A uronic acid residue bearing a 1-carboxyethylidene group has only previously been found, in th i s se r ies , in the polysaccharide from K lebs ie l l a K l 2 7 7 . (see note P. 102). I I I . 2. 4. Experimental General methods. — Concentrations were carr ied out under diminished pressure at bath temperatures not exceeding 40°. The equipment for m.s., n.m.r. spectroscopy, g . l . c , and g . l . c . - m.s. was the same as that used in the invest igat ion of K lebs ie l !a Kl2 polysaccharide (see Sec I I I . l ) Paper electrophoresis was performed on 1 ' a Savant high voltage (5 KV) system (model LT - 48A) with kerosene as coolant. The buffer used contained pyridine - acet ic acid - water (5:2:743, v/v) pH 5.3. Str ips of Whatman No. 1 paper (77 cm x 20 cm) were used for a l l runs, with appl icat ion of 25-50 mA 97 for 1% h. For descending paper chromatography the fo l lowing solvent system (v/v) were used: .(1_) f reshly prepared 2:1:1 1-butanol - acet ic acid - - water, (2J 8:2:1 ethyl acetate - pyridine - - water, and (_3) 18:3:1:4 ethyl acetate - acet ic acid - formic acid - water. Sugars and ol igosaccharides were detected with an a lka l ine s i l v e r n i t ra te 21 reagent . Ana ly t i ca l g . l . c . separations were performed in s ta in less -steel columns (1.8 x 3 mm) with a carr ier-gas f low-rate of 20 mL/min. Columns used were (A) 3% of OV-225 on Gas Chrom Q (100 - 120 mesh); (B) 5% of ECNSS-M on the same support; and (C) 3% of SP-2340 on Supelcoport (100-120 mesh). Analogous columns (1.8 m x 6.3 mm) were used for preparative g . l . c . separations. Preparation and propert ies. — A culture of Klebsiel1 a K58 (636/52) was. obtained from Dr. I. (j)rskov (Copenhagen). The polysaccharide was i so la ted as previously described, (see Sec. I I . l ) and showed [a] D +19.0° (c 1, water). Analysis of constituent sugars. — Methanolysis of a sample (20 mg) of K58 polysaccharide with 3% methanolic hydrogen chlor ide and subsequent reduction with sodium borohydride in anhydrous methanol reduced the uronic ester . Hydrolysis with 2M t r i f l uo roace t i c acid overnight at 95°, followed by reduction (NaBH^), and acety la t ion, gave ga lac t i to l hexaacetate, g luc i to l hexaacetate, and fuc i t o l pentaacetate in the ra t io 1:2:1 (column C_, programmed at 180° for 8 min and then 4°/min to 240°). C i rcu lar dichroism (c.d.) of the l a t t e r two components i so la ted by preparative g . l . c , showed 98 pos i t i ve and negative c .d . curves respect ive ly , confirming that glucose has the D configuration and fucose the L conf igurat ion. Galactose was shown to be of the D configuration by the pos i t ive act ion of D -Galactostat (Worthington Biochemical Company) on the hydrolysis product of the polysaccharide. Methylation a n a l y s e s . — Methylation of K58 polysaccharide under 247 242 the Hakomori condit ions, followed by a Purdie treatment y ie lded a product that showed no hydroxyl absorption in i t ' s i . r . spectrum. This material was reduced overnight with sodium borohydride in 1:1 (v/v) oxolane (THF) and ethanol. A portion of the product was hydrolyzed with t r i f l uo roace t i c acid (2M) for 16 h at 95°, and the mixture was reduced with sodium borohydride and then acetylated. G . l . c . - m.s. gave the resul ts shown in Table 8. Another port ion of the reduced, uronic ester material was re-methylated under the Purdie conditions for 2 days, and der ivat ized for g . l . c . - m.s. giv ing the compounds shown in Table 8. A portion (10 mg) of the re-methylated material was hydro-lyzed with 90% formic acid for 30 min at 95° to remove the 1-carboxy-ethylidene group. Methylation under the Purdie conditions for 2 days, and der ivat i za t ion for g . l . c . - m.s. gave the resul ts shown in Table 8. Uronic acid degradation — A solut ion of care fu l l y dr ied, methylated polysaccharide (100 mg) and p_-toluenesulfonic acid (a trace) in 19:1 99 dimethyl sul foxide and 2,2-dimethoxypropane (20 mL) was prepared in a serum v ia l which was:sealed with a rubber cap. The v ia l was flushed with dry- n i trogen, and the so lut ion was s t i r r ed for 3 h. Sodium methylsulphinylmethanide (2M) in methyl sulfoxide (10 mL) was then added with the aid of a syr inge, and the solut ion was s t i r r ed overnight at room temperature. After cool ing to 10°, ethyl iodide 273 (3 mL) was added slowly, using a syringe Following the addit ion of water, the ethylated, degraded product was iso lated by par t i t i on between chloroform and the aqueous so lut ion. Hydrolysis of the i so la ted product was performed with 2M t r i f l uo roace t i c ac id; g . l . c . - m.s. analysis of the a l d i t o l acetate der ivat ives y ie lded peaks corresponding to 4-0-ethy l-2, 0-methylfucose, 2,3,4,6-tetra-0-methylgalactose and 2 ,4 ,6 - t r i - 0 -methylglucose (see Table 8 ) . Periodate Oxidation. — K lebs ie l l a K58 capsular polysaccharide (200 mg) was dissolved in water (25 mL), to which a solut ion (25 mL) of 0.1M sodium metaperiodate was then added. The solut ion was s t i r r ed in the dark at 3° and periodate consumption was monitored (Fleury-Lange method) , af ter 10 h consumption had reached 1.8 molecules per repeating uni t . Ethylene glycol (10 mL) was then added, and, a f ter s t i r r i n g for a further 30 minutes the mixture was dialyzed overnight against running tap-water, and the product was reduced with sodium borohydride. The polyol was i so la ted by d ia l y s i s and lyoph i1 izat ion. Smith hydrolysis (0.5M TFA overnight TOO at room temperature) gave a polymeric product. A port ion (5 mg) of th is material was hydrolyzed (2M TFA overnight at 95°); paper chromatography (solvent 2) then showed the presence of glucose and fucose, and the absence of galactose. Analysis of the constituent sugars of the Smith-degradation product was performed as for the native polysaccharide. G . l . c . analysis showed the presence of 1 13 glucose and fucose in the ra t io 2:1. H and C n.m.r. spectral data are shown in Table J. Methylation analysis of a portion (20 mg) of the Smith-degradation product, as for the native polysaccharide, gave the resu l ts shown in Table 8 . Autohydrolysis — A sample of K lebs ie l l a K58 polysaccharide was exchanged to the f ree-ac id form with Amberlite IR-120 (H*) ion-exchange res in , and the so lut ion was l yoph i l i zed . A portion of th i s material (100 mg) was dissolved in 5 mL ^ 0 (pH 3.0) and was introduced to a sealed length of standard ce l lu lose d i a l y s i s tubing. This was autohydrolyzed in 100 ml ^ 0 at 95° for 3 h. Loss of the 1-carboxyethylidene group was shown to be complete by ^H.n.m.r. spectroscopy. Methylation analysis of the product, as described ea r l i e r gave the resul ts shown in Table 8 . Pa r t i a l Hydrolysis — A sample (300 mg) of the native polysaccharide was hydrolyzed with 0.5M H^SO^ for 30 min at 95°. The products were separated by preparative paper chromatography (solvent 3). Compound 101 1 (35 mg) with 0.63 and [a] D -20.0° (c_ 1, water) was shown to be 1 13 the aldobiouronic acid by H and C n.m.r. spectroscopy. Compound 2 (30 mg) with 0.32 and [ a ] D + 16.0° (C 1, water) and compound 3 (10 mg) with 0.12 and [ a ] n + 38.0° (c 0.5, water) were s im i l a r l y 'M u IC U — shown to be the a ldotr iouronic and aldotetraouronic acids respect ively (see Table 7). Deacetylation Deacetylation was achieved by treatment of a so lut ion of the native polysaccharide (200 mg) in water (25 m L) with excess of sodium borohydride for 3h, at room temperature, with s t i r r i n g . D ia lys is and l yoph i l i za t i on y ie lded a product with no acetyl peak at 62.17 in the p.m.r. spectrum. Location of 0-acetyl group Since complete blocking of the polysaccharide with methyl v in ly ether proved d i f f i c u l t , giv ing small amounts of methylated sugars other than 2-ONe fucose, on g . l . c . -m.s . ana lys i s , the 275 0-acetyl locat ion procedure of de Belder and Norman was performed on both the native polysaccharide, and a deacetylated sample. Reaction conditions were ident ica l in both cases. The ent i re react ion was carr ied out in a sealed v i a l , flushed with nitrogen. A so lut ion of ca re fu l l y dr ied polysaccharide (50 mg) and jp_- toluenesulfonic acid (20 mg) in dimethylsulfoxide (20 mL) was prepared in a serum v i a l , which was sealed and flushed with nitrogen. 102 The s o l u t i o n was f rozen (-60 ) and methyl v i n y l e t h e r (ca 3 mL) was in t roduced from a gas b o t t l e to the r e a c t i o n vessel v i a . a hypodermic s y r i n g e . The s o l u t i o n was brought to room temperature and s t i r r e d f o r 3 hr . Two f u r t h e r po r t i ons o f methyl v i n y l e ther were s i m i l a r l y i n t r o d u c e d , a f t e r which t ime the r e a c t i o n mix tu re had a r e d / y e l l o w co lou r . M e t h y l s u l f i n y l anion i n d i m e t h y l s u l f o x i d e (3 mL) was in t roduced and the r e a c t i o n mix tu re s t i r r e d f o r a f u r t h e r 30 min. Methyl i od ide (2 mL) was added t o the cooled mix tu re which was then s t i r r e d f o r I h . D i a l y s i s ( o v e r n i g h t ) and l y o p h i l i z a t i o n gave a mix tu re o f d e r i v a t i z e d po lysacchar ide and polymer ic methyl v i n y l e the r . Pure po lysacchar ide was e l u t e d froma column o f Sephadex LH-20 w i t h methanol. Since the 0 -ace ty l s u b s t i t u e n t could not be on the g lucu ron ic ac id t h i s f u n c t i o n a l i t y was not reduced. The polysacchar ide was hydro lyzed w i t h t r i f l u o r o a c e t i c ac id (2M), f o r 16h a t 95° , and the mix tu re was reduced w i t h sodium borohydr ide , and then a c e t y l a t e d . The r a t i o o f 2-0Me fucose t o fucose o f 19 : 1 f o r the n a t i v e po lysacchar ide and 1 : 1 f o r the deacety la ted po lysacchar ide ( g . l . c . - m.s. a n a l y s i s ) i n d i c a t e s t h a t the 0 -ace ty l group i s a t tached to 0 - 2 o f fucose. Note added i n p roo f . Dr. J..M. Fourn ier ( I n s t i t u t Pasteur , Pa r i s ) has shown t h a t , a l though K l e b s i e l l a K58 capsular po lysacchar ide i s not v i r u l e n t f o r mice, immunizat ion w i t h the po lysacchar ide does prov ide p r o t e c t i o n 103 against in fect ion with the v i ru lent K lebs ie l l a Kl capsular 277 polysaccharide. This is in agreement with the s im i l a r i t i e s in the structures of the two polysaccharides (see App.I). 104 I I I . 3 CONFIRMATION OF THE STRUCTURE OF KLEBSIELLA K23 CAPSULAR  POLYSACCHARIDE. 2 78 Structural invest igat ion of K lebs ie l l a K23 polysaccharide by the techniques of methylation analysis and Smith degradation:indicated that the structure consisted of a tetrasaccharide repeating unit with a two-sugar s ide-chain, as shown: ^3)-D-Glcp_-(l->3) -L-Rha p_ 2 I 1 D-GlcAp (1+6)Q-Glc p Comparison of H^ n..m.r. spectra of the native polysaccharide 253 and the polysaccharide obtained on Smith degradation (the two side-chain sugars were removed) showed that the glucosyl residue in the backbone was 3 - l i n k ed and that the rhamnosyl residue was a- l inked. The anomeric nature of the side-chain residues could not, however, be demonstrated conconclusively. To obtain th is information the methylated polysaccharide was subjected to a 3-el iminat ion reaction , whereby, only the terminal glucuronic acid residue was degraded. Comparison of the H^ n.m.r. spectra of the methylated polysaccharide and this product demonstrated the absence of a signal in the anomeric region at 6 4.34, corresponding to loss of a 3- l inked glucuronic acid residue (see Table 9 ) . By deduction the glucosyl residue in the side-chain 105 JMLE_9„' : P.M.R. DATA FOR K lebs ie l l a K23 CARSULAR POLYSACCHARIDES Compound 6 - Integral Assignment Methylated, 5.21 1 a -Rha b Native 5.08 1 a -G lc Polysaccharide. 4.52 1 3-Glc 4.34 1 3-GlcA 1.31 3 CH 3 of Rha. Methylated/ethylated, 5.24 1 a-Rha degraded 5.10 1 a -G lc polysaccharide. 4.55 1 3-Glc 1.31 3 CH 3 of Rha 1.21 3 CH 3 of ethyl -Chemica l sh i f t r e l a t i ve to internal acetone; 6 2.23 downfield from sodium 4,4-dimethyl -4-si1apentane-1-sulfonate (D.S.S.) . Spectra were run in CDC13 at 270MHz and ambient temperature. -a - Rha = proton on C-l of a - l i nked l-Rha residue, etc. 106 TABLE 10 METHYLATION ANALYSIS OF ORIGINAL AND BASE DEGRADED K lebs ie l l a K23 CAPSULAR POLYSACCHARIDE. Methylated sugars- T- I - I I -(as a l d i t o l acetates) Mole %- Mole %£ 4-Rha 0.91 26.0 34.1 2 ,3 ,4 ,6-G lc- 0.72 — 32.4 2,3,4-Glc 1.13 48.1 2,4,6-Glc 1.00 25.9 33.5 — 4-Rha = 1,2,3,5-tetra-0-acetyl-4-0-methylrhamnitol etc. — Retention time re la t i ve to a l d i t o l acetate of 2,4,6-tr i-O-methyl-D-glucose on OV-225. — I, o r ig ina l polysaccharide, methylated and uronic ester reduced. — I I , degraded polymer obtained af ter g-el imi nation. — Values are corrected by use of the e f fec t ive carbon response factors given by Albersheim et a l . — 1,5-Di-0-acetyl-6-C^-ethyl-2,3,4-tr i-0-methylglucitol 107 i s a - l i nked . 106 The product was d i r e c t l y ethylated , thus l abe l l i ng the pos i t ion of attachment of the glucuronic acid residue. Hydrolys is, 103 121 and g . l . c . - m.s. ' analysis of the ethylated product showed that only the glucuronic acid residue had been removed and ve r i f i ed that i t was attached to 0-6 of the side-chain glucosyl residue (see Table 10). I t thus follows that the K lebs ie l l a K23 polysaccharide has the structure ~3)- 3-D-Glcp_ -( l->3)-a-L-Rhap_-(l~ " 2 1 3-D-GlcAp_-(l+6)a-Q-GlC£ n The experimental conditions were essent ia l l y the same as those used in the invest igat ion of the structure of K lebs ie l l a Kl2 capsular polysaccharide (see Section 111,1). 108 I I I . 4 ] H AND 1 3 C SPECTRAL INVESTIGATION.OF K lebs ie l l a K70  CAPSULAR POLYSACCHARIDE The structure of the capsular polysaccharide of K lebs ie l l a 279 K70 has been shown . to consist of a l i near hexasaccharide repeating unit having a 1-carboxyethylidene attached to a 2-l inked a-L-rhamnosyl residue in every second repeating unit as shown: - 2 Rhap j^ l cAp^Rhap j -^-Rha£ ^ h^c_^- Galp_ L A B C D E F n 1 13 In that invest igat ion H and C n.m.r. spectroscopy were used, and some assignments were made. However, the complete assignment of a l l the s ignals in the anomeric region proved d i f f i c u l t , since four a- l inked residues were present, three of which were due to rhamnosyl residues. The polysaccharide has since been degraded by Mer r i f i e l d 298 using bacteriophage, to give an ol igosaccharide corresponding to two repeating uni ts . Another spec i f i c degradation by Mort 323 whereby the native polysaccharide was cleaved at the uronic acid residue, using l i th ium in ethylamine, with concomitant loss of that residue, 1 13 produced a pentasaccharide. H and C spectral invest igat ions of the penta - and hexasaccharide now allow a more complete assignment of a l l the signals in the anomeric region of the native polysaccharide. TABLE 11 N.M.R. DATA FOR Klebsiella K70 POLYSACCHARIDE AND OLIGOSACCHARIDES ISOLATED Compound^ Vn .m.r. Data 13 I J C n.m.r. Data J 1 > 2 (Hz) Integral Assignment— p.p. d m— Integral Assignment-l-A-B-C-D-E-F-]n 5.22 s i 1 a Rha C 105. .7 1 B Gal F 5.10 s 2 a Rha A+D 103. 8 1 B GlcA B 4.97 s 1 a Glc E 102. .9 1 a Rha C (1) 4.77 7 1 3 Glc E . 101. ,7 1 a Rha A 4.55 7 1 B Gal F 100. ,9 1 a Rha D 1.59 s 1.5 CH,of acetal 95. ,7 1 a Glc E 1.30 6* 9 CH3 of Rha 62. ,2 1 C-6 Gal 61. ,3 1 C-6 Glc 17. ,5 3 C-6 Rha A-B-C-D-E-F-AW-C1-(2) -E^F1- OH 5.28 5.23 5.10 4.98 4.80 4.58 4.40 1.30 2 2 s s 7 4 7 6 * 0.7 a Gal OH.F1 1.8 a Rha C + C 4 a Rha A+AUD+D'1 2 a Glc E + E 2 B GlcA B+B1 0.3 B GaVOH F1 1 6 Gal F 9 CH3of Rha .1 105.64 103.88 102.79 101.7 100.98 99.7 98.61 97.28 95.77 94.80 61.78 61.36 17.6 1 2 2 1 2 1 1 0.6 1 0.4 B Gal B GlcA a Rha a Rha a Rha a Rha a Glc B Gal-OH a Glc a Gal-OH C-6 Gal C-6 Glc C-6 Rha F B + B C + C A D + D .1 TABLE 11 Contd. D-E-F-A-OH 5.32 2 0.8 5.23 2 1 (3) 5.10 3 1 4.98 2 1 4.80 s 0.2 4 . 5 5 W 4 0.4 4.58 4 1.30 &a 9 a Rha-OH.A 105.5 1 B-Gal F a Rha C 104.7 0.2^ B GlcA B a Rha D 103.1 0.8 a Rha C a Glc E 101.0 1 a Rha D B Rha-OH A 95.7 1 a Glc E B Gal F 93.9 0.6 a Rha-OH A 93.6 0.4 B Rha-OH A CH3 of Rha 61.7 C-6 Gal 61.2 C-6 Glc 17.5 C-6 of Rha - For structures of (1), (2), (3) see text. -Chemical sh i f t relative to internal acetone; 62.23 downfield from sodium 4,4-dimethyl-4-silapentane - 1-sulfonate (D.S.S.) - a-Rha=proton on C-l of a-linked L - Rha residue etc. ^Chemical sh i f t in p.p.m., downfield from Me.Si, relat ive to internal acetone; 31.07 p.p.m. from D.S.S. e c 13 ^As for - but for anomeric C nuclei. ^5-singlet 3-Value for J,- fi. overlapping doublets centred at 61.30. h - A small amount of B-GlcA was not eliminated. -The chemical sh i f t of this proton is affected by the a,B equilibrium of the reducing Rha residue. i n Acknowledgments I wish to thank Dr. I. (j)rskov (Copenhagen) for the K lebs ie l l a cultures used, Dr. A.A. Grey (Toronto) for recording the 220 MHz H^ n.m.r. spectra at high temperature, Dr. M. Vignon 13 (Grenoble) for recording C spectra at high temperature, and Dr. A. Mort (Charles F. Kettering Research Laboratory, Ohio) for a sample of degraded K70. The i so la t ion and i n i t i a l invest igat ion of K lebs ie l l a Kl2 polysaccharide were performed by Chr ist iane Marte l . 112 BACTERIOPHAGE DEGRADATION OF K lebs ie l l a CAPSULAR POLYSACCHARIDES K21, K12 and K41. 113 IV. 1. Introduction The f i e l d of medicinal microbiology became well - establ ished in the period between 1880 and 1900 with the i den t i f i c a t i on and character izat ion of many of the causative agents of both human and animal diseases. In 1892, Iwanowski and Bei jer inck while indepen-dently studying the tobacco mosaic disease, and Pasteur while carrying out studies on rabies, recognised the causative agents to be 280 " f i l t e r a b l e substances", which the l a t t e r termed "v i ruses ." Some years l a t e r , in 1915 and 1917 virus infect ions in bacter ia were also described. Twort and d'Herel le demonstrated independantly that cultures of bacter ia l ce l l s could be infected with and destroyed by f i l t e r a b l e agents that were subsequently termed "bacteriophages". I t was not unt i l the electron microscope was developed that the morphological character of viruses was elucidated. Today, bacteriophages (phages, designated are the best characterized and studied group of v i ruses, since the i r propagation and manipulation has proven techn ica l l y much eas ier than equivalent 281 studies on other types of v iruses. Phages, which are quite d i f fe rent from other virus types, in that they tend to be s t ruc tu ra l l y more complex, are grouped 282 according to the morphological c l a s s i f i c a t i on of Bradley (see F ig. 11). For type A (see F ig . 12) • the head, which i t s e l f i s composed of repeating ident ica l protein monomers, has bas i ca l l y 114 2-DNA 2-DNA 1 -DNA 1-RNA 1-DNA Morphological grouping of phages according to Bradley COATPROTEIN HEAD DNA COLLAR TAIL CONTRACTILE SHEATH BASE PLATE SPIKES Schematic representation of a Type-A phage pa r t i c l e . 115 an icosahedral structure and contains doubly stranded DNA. The phage t a i l consists of a tube which i s made up of he l i c a l l y arranged protein molecules, encased by a cont rac t i le sheath, also composed of protein monomers. The plate contains small pins to which are 283 a attached the t a i l spikes. Type B i s s im i l a r to type A, but without the cont rac t i le sheath. Type C contains a baseplate and spikes, but no t a i l . Type D consists only of a head, with a capsomere on each apex, type E consists of a head, without capsomeres, and type F:is fi lamentous. Bacteriophage which T.yse encapsulated bacter ia often form plaques, surrounded by large haloes that continue to spread af ter growth has ceased (see App. IV). Within the haloes the 283b c bacter ia have l os t the i r capsules. I t has been long known that, ' general ly, the formation of these haloes i s due to the production of enzymes during phage in fec t ion . These enzymes di f fuse from the plaque and catalyze the hydrolysis of the bacter ia l capsules. A wide var iety of enzymic a c t i v i t i e s , cata lyz ing d i f fe rent degradation reactions of host surface, polysaccharides may be associated with 284 bacter ia l v irus pa r t i c l e s . So far esterases (saponi f icat ion of 0-acetyl subst i tuents) , glycanases, and lyases have been 285a recognized . It has been shown that the depolymerases responsible for the formation of haloes are in fact free phage spikes 285b,c,d^ p r o c j u c e c j - j n addit ion to whole v i rus , and that the pur i f i ed spikes exert the same g lycos id ic a c t i v i t y . The enzymic 116 a c t i v i t y has been found to be associated with a subunit (m.w. 62,500.) of the v i r a l spike (m.w. 155,000). When a phage pa r t i c l e in fects a susceptible host i t causes that ce l l to l y se , with the concomitant release of a charac te r i s t i c number of newly formed phage pa r t i c l e s . The phases of the cycle include the fo l lowing: ( i ) absorption of the phage par t i c les to the susceptible host ( i i ) in jec t ion of v i r a l DNA (or RNA) into the host ( i i i ) rep l i ca t ion of the phage nucle ic acid and synthesis of phage prote in, and ( iv) phage maturation and release. The f i r s t step occurs by attachment of the phage t a i l to receptor s i tes on the bacter ia l c e l l . There i s considerable evidence that rec iprocal charges on the phage t a i l and on the receptor s i tes of c e l l s are involved in the formation of e l ec t ros ta t i c bonds during attachment. Phage are r e l a t i v e l y easy to i so la te from almost any 287 bacter ia l environment, for example, sewage . Those act ive on exopolysaccharide producing bacter ia l s t ra ins are general ly exopolysaccharide spec i f i c ; non - capsulate or non - slime producing mutants are res is tant to the phages. One common feature of a l l phage, act ive on exopolysaccharides, is that the baseplates, as seen in the electron microscope, are provided with spikes, and that no t a i l f ib res are seen. The spikes appear as hollow tubes 117 286 288 (12.5 nm in K lebs ie l l a phage 11). Stirm and Rieger - Hug, employing seventy four se ro log i ca l l y d i f fe rent K lebs ie l l a test s t ra ins , tested the host range of f i f t y f i ve K lebs ie l l a bacteriophage. The v i ra l depolymerases proved to be very spec i f i c (33 not cross-react ing, 18 cross-react ing with one, 2 with two, 1 with three and 1 with four heterologous polysaccharides. § 12 cross-reacts with 34 K41, which i s in agreement with the structure proposed for K12 capsular polysaccharide. Depolymerization of a polysaccharide using bacteriophage y ie lds products corresponding to a s ingle repeating unit of the polysaccharide, and mult ip les thereof, with l ab i l e 0 - a c e t y l 2 8 9 a and 1-carboxyethylidene (pyruvate) groups in tac t . 2 8 9 b These may then be used for (a) the preparation of synthet ic antigens, 7 7 ( b ) deta i led examination by nuclear magnetic resonance spectroscopy, and (c) the study of conformations in 290 so lut ion. The degradation of K lebs ie l l a K21 capsular polysaccharide using pur i f i ed <j) 21 pa r t i c l e s , and the degradation of K l ebs i e l l a Kl2 and K41 using a crude <|) 12 suspension are presented here, and resul ts compared in terms of e f f i c i ency of depolymerization and y ie lds of ol igosaccharides. IV. 2. RESULTS IV. 2. 1. Iso lat ion and pu r i f i ca t i on Both <j> 21 and § 12 were iso lated from sewage, and stock suspensions in broth were obtained by the confluent l y s i s method. The bacteriophage were propogated on the i r host s t ra ins , K lebs ie l l a K21 118 and 12 respect ive ly , to a volume of v l . 5 L . . (see App.IV) <|) 21 was 291 pur i f i ed by prec ip i ta t ion with poly (ethylene g lycol) 6000 , followed by isopycnic centr i fugat ion, and was shown by electron 282 microscopy (see F ig . 15) to belong to Bradley Type B. IV. 2.2. Conditions of depoTymerization The pur i f i ed capsular polysaccharide from K lebs ie l l a 292 K21 was dissolved in buffered sa l ine , and the depolymerization 293 was followed v iscometr ica l ly and by assay of the reducing power (see F ig. 13) which became constant af ter 24 h. The depolymerizations of K lebs ie l l a K12 and K41 capsular polysaccharides were carr ied out in separate, crude,.broth suspensions of <[) 12 par t i c les and followed v iscometr ica l ly . Although the v i scos i ty of both solut ions decreased dramatical ly during the f i r s t 3 h:. the reactions were allowed to continue for 48 h. IV. 2. 3. Pu r i f i ca t i on and analyses of products of depolymerization of K21. The lyoph i l i zed depolymerization mixture was desalted on a 95 column of Sephadex Gl0. The carbohydrate f rac t ion was l yoph i l i zed , redissolved in Tr is HC1 buffer, and the solut ion added to a column of DEAE - Sephadex A25. The e lut ion pattern i s shown in F ig . 14, where PI represents the s ingle repeating unit of the polysaccharide, P2 the double repeating un i t , and P3 polymeric mater ia l . PI and P2 were separately desalted (Sephadex G10), and examined by " ' H n.m.r. 119 Galactose equivalents [n\ Mole/ml) Figure 13 (a) Decrease in v ' iscosity, and (b) increase in reducing power of K21 polysaccharide so lu t ion, on incubation with bacteriophage. 120 ~1 1 1 — 50 100 150 Elution volume (ml) Figure 14 Separation of products of bacteriophage degradation of Klebsie l la K21 121 Figure 15 Electron Micrograph of pur i f i ed K21 bacteriophage, negatively stained. X=122,500 Courtesy of Dr. H. Chanzy C.E.R.M.A.V. (Grenoble). Bradley 2 8 2 Type B. 122 spectroscopy (see Table 12). Several spurious peaks were observed at low f i e l d , but, after passage through a column of Amberlite IR-120 (H+) res in gave the spectra shown in App. . I I I . H and C spectra demonstrate that PI i s a hexasaccharide corresponding to one repeat ing-unit , with galactose as the reducing residue, and P2 i s composed of two repeating uni ts . The two ol igosaccharides were analyzed by gas- l iqu id chromatography, using 102 294-297 the method of Morrison, ' whereby the ra t io of acetylated a ldonon i t r i l e to acetylated a l d i t o l i s determined. The results(see F ig . . 7, Table 13), confirmed that PI i s a hexasaccharide and that P2 i s the dimer. The mobi l i ty of PI in paper chromatography was R Glc 0-045, and in paper e lectrophores is , PI and P2 had R g ^ 0.75 and 0.85, respect ive ly . IV. 2. 4. Pu r i f i ca t i on and analyses of the products of depolymerization of Kl2 and K41. The reaction mixtures were dialyzed (M.W. Cutoff = 3,500) against water, and the dia lysates were l yoph i l i zed . Preparative paper chromatography y ie lded products free of most broth contaminants. Only baseline carbohydrate spots were observed. Further pu r i f i c a t i on , by passage through a column of Amberlite IR-120 (H+) res in gave products which were examined by n.m.r. spectroscopy. The resul ts shown in Table 14 show that the product of depolymerization of K12 polysaccharide with <j> 12 i s a hexasaccharide, corresponding to a s ingle repeating unit of the polysaccharide. This 123 TABLE 12 N.M.R. DATA FOR Klebsiella K21 CAPSULAR POLYSACCHARIDE AND THE OLIGOSACCHARIDES PI AND P2 OA/ Compound 1 H- n.m.r. data-K21 polysaccharide 292 Oligosaccharide PI Oligosaccharide P2 5.48 5.30 5.25 5.08 4.88 1.55 5.49 5.34 5.30 5.10 4.66 1.52 5.46 5.31 5.27 5.06 4.87 4.64 1.53 Integral (H) 1 2 1 1 3 1 2.4 1 0.6 3 1 2.3 1 0.5 0.2 1.5 Assignment a-GlcA a-Gal a-Man a-Man 3-Gal acetal a-GlcA a-Gal a-Man a-Gal-OH a-Man B-Gal-OH acetal a-GlcA a-Gal a-Man a-Gal-OH B-Gal 6-Gal-OH acetal Chemical shi f t relative to internal acetone; 62.23 downfield from sodium 4,4-dimethyl-4-silapentane-l-sulfonate (D.S.S.) Chemical shifts are recorded only for anomeric protons and those of the 1-carboxyethylidene acetal. 124 TABLE 13 DETERMINATION OF DEGREE OF POLYMERIZATION OF PI AND P2 (K21) AND IDENTIFICATION OF THE REDUCING SUGAR Acetylated Relative Retention time Moles %-der ivat ive of 0V-17- PI P2 Mannononitrile 0.67 2.0 2.0 Glucononi t r i le 0.72 0 .91- 0 .93-Galactononi t r i le 0.75 0.98 1.5 Ga lact i to l 1.00 0.94 0.47 — 3% OV-17 on Gas Chrom Q (100-120 mesh) programmed at 180° for 4 min and then 2°/min to 220°. — Values corrected „ using molar response factors . — Due to incomplete reduction of uronic ac id . TABLE 14 P .M.R. DATA FOR K lebs ie l la K12 CAPSULAR POLYSACCHARIDE AND THE PHAGE DERIVED OLIGOSACCHARIDE. Compound— 1 H-n.m.r. data J l , 2 (Hz) Integral Assignment— ^Gl c ^ h a ^ G a l p j -^a l f -3 i pyruvate 5.22 s 1 a-Rha 5.16. 3 1 a-Glc 5.13 2 2 a-Ga l£ 5.13 2 B-Galf 4.66 8 1 B-GlcA 4.48 6 1 B-Galp_ 4.3-4.5 b 2 H-2, H-3 B-Galf 1.66 s 3 CH^ of acetal 1.34 6 3 CH 3 of Rha TABLE 14 ( C o n t d . . ) Compound- 1 H-n.m.r. data b J1.2 (Hz) Integral (H) Assignment— Gl c ^ R h a ^ G a l p^-?€al ~0H a a ^ a 5.26 b .6 a-Rha 3 g 5.20 b .6 a-Gal~0H 1 5.16 s .6 a-Glc G' cA 5.09 b 1 a-Gal 4 g 4.61 b 1 g-GlcA PI 1 4.52 8 .4 g-Gal~0H 4.48 6 (1) g-Gal 6 4 1.66 b CH^  of acetal pyruvate 1.34 b CH3 of Rha r o - For the origin of compound PI see text. See Appendix I I I for reproductions of the spectra -Chemical shi f t relative to internal acetone; 62.23 downfield from sodium 4,4-dimethyl-4-silapentane-l - Key: b = broad, unable to assign accurate coupling constant, s = singlet. - For example, a-Gal = Proton on C-l of a-linked Q - Gal residue. 127 was confirmed by g . l . c . analys is (see above). In contrast, the product of the depolymerization of K41 polysaccharide with (j) 12, was shown to be an ol igosaccharide corresponding to two repeating units of the polysaccharide. Confirmatory evidence was provided by g . l . c . analysis (see Table 15) IV. 3. DISCUSSION THe ultimate aim of our research group, in degrading polysaccharides using bacteriophage, is to obtain pure ol igosaccharides, in as high a y i e l d as poss ib le. In pur i fy ing the bacteriophage, as for <j) 21, the volume of the phage suspension i s reduced by a factor of p 10 , but with a concomitant loss of ^ 75% of the phage. The e f f i c i ency of depolymerization i s , however, very high, giv ing a good y i e l d of the s ingle repeating uni t . Pure ol igosaccharides may also be obtained with a crude solut ion of bacteriophage, thereby a l lev iat ing the time consuming and expensive (CsCl) pur i f i ca t ions using u l t racentr i fuges. In the degradations of Kl2 and K41, the y ie lds of the s ingle repeating un i t were low (for K41 only the double repeating uni t was obtained). This can be improved upon by using a higher t i t r e 298 pf crude phage pa r t i c l e s . D ia lys i s of the products, using tubing with a low exclusion l im i t , i s an e f f i c i en t method of separating the s ingle and double repeating units from larger mater ia l . However, i f a TABLE 15 N.M.R.. DATA FOR Klebsiella K41 CAPSULAR POLYSACCHARIDE AND THE PHAGE DERIVED OLIGOSACCHARIDE Compound— ^H-n.m.r. data ^C-n.m.r. data 6— Integral Assignment- p.p.m.— Integral Assignment— 6 f i l c L 3 R h a L 3 G a l i Z G a l f I a a a 3 I — ' G l c i ^ l c ^ G l c A B a 5.48 5.22 5.17 5.12' 5.12 4.63 4.52 1.34 a-Glc a-Rha a-Glc a-Gal£ B-Galf B-GlcA B-Glc CH3 of Rha 109 105.5 104.8 104.45 101.55 101.05 99.5 19.7 B-Galf B-Glc 6-GlcA a-Rha a-Gal a-Glc a-Glc CH3 of Rha P2 n = 2 1 1.3 2.5 1 1.2 a-Glc a-Rha a-Gal~0H a-Glc a-Gal£ B-Galf B-GlcA B-Glc B-Gal~0H CH3 of Rha. 107.2 103.44 102.64 101.7 100.24 100.10 99.96 97.2 91.10 17.65 0.5 1 1 0.5(?) 1 1 1 0.2(?) 0.3(?) B-Galf B-Glc B-GlcA a-Rha a-Gal a-Glc a-Glc B-Gal~0H a-Gal~0H CH3 of Rha. - For the origin of compound P2, see text. See Appendix I I I for reproduction of the spectra. -Chemical sh i f t relative to internal acetone; 62.23 downfield from sodium 4,4-dimethyl-4-silapentane-l-sulfonate (D.S.S.) - For example, a-Gal = Proton on C-l of a-linked Q - Gal residue. -Chemical sh i f t in p.p.m. downfield from Me.Si, relative to internal acetone; 31.07 p.p.m. downfield from D.S.S. e c 13 - As for —, but for anomeric C nuclei. 129 crude phage solut ion in both i s employed, then lowM-W. material from the broth contaminates the ol igosaccharides. The a l ternat ive then, is f i r s t to reduce the volume of phage suspension, (by l yoph i l i z a t i on , or by rotary evaporation at < 4 0 ° ) 2 9 8 and d i a l y s i s of th i s so lut ion to remove broth contaminants. Low M.W. products may then be eas i l y i so lated by d i a l y s i s , and separated, e i ther by preparative paper chromatography, or by gel permeation chromatography. IV. 4. EXPERIMENTAL For a l l phage work the standard procedures given by Adams were used. § 12 and (j) 21 were o r i g i na l l y iso lated from Freiburg sewage, and were propogated on the i r respective host stra ins by tube and bott le lyses (see App. IV). Pu r i f i ca t i on 4 21 l ysa te , which had a t i t r e of 7.5 x lO^PFU/mL (1.5L), was centrifuged at 5,000 g (20 min) and made 0.5 JJ in NaCl. 10% w/v of poly (ethylene glycol) 6000 was added slowly to the supernatant and after storage at 4° for 48 h v the phage par t i c les were sedimented at 20,000 (g.) (30 min). The supernatant was shown to contain 0.15% of phage. 130 The pellets were taken up in PBS (20 mL), using a syringe (22g) and the solution was centrifuged at 5,000g (20 min). The supernatant was then centrifuged at 100,000 g for 4hand the pellets were taken up in a minimum of PBS (4 mL). The concentrated virus-suspension was further purif ied by isopycnic centrifugation in linear CsCl gradients as follows: 32.3g of CsCl was dissolved in 32.1 mL0.1% of Tris HC1 ( p = 1.63 g/mL) and 6.5g of CsCl in 31.1 mL ( p = 1.13 g/mL). The two solutions were mixed using a three channel per ista l t ic pump into two disposable cellulose nitrate tubes (capacity 38.5 mL) for a Beckman SW 27 rotor. The phage suspension in PBS (2x2mL) was loaded carefully onto each gradient, and was centrifuged at 86,000 g for 1.5 h. An opalescent band ( p = 1.445 g/mL) was then clearly visible and was withdrawn using a syringe with new needle, and dialyzed against PBS to remove CsCl. Conditions of depolymerization of K21 Purified capsular polysaccharide (250 mg) from Klebsiella K21 was dissolved in PBS (100 mL) and to this solution was added a total of 2.5 x 10 1 2 plaque forming units (PFU) in 30 mL of PBS. A sample of the reaction mixture was transferred to an Ostwald viscometer which, together with the flask containing the bulk of the solution, was placed in a bath at 37°. The viscosity of the solution was determined periodically and at the same time aliquots were withdrawn and analyzed for reducing power by comparison with 131 293 a standard curve based on galactose. Pur i f i ca t i on and separation of depolymerized K21 mater ia l . Portions of the crude l yoph i l i zed depolymerization mixture (2 x 1.5 g) were desalted using a column of Sephadex G10 (100 cm x 19.5 cm 2). The column was eluted with buffer (water - - pyridine - -g lac ia l acet ic ac id , 1000:10:4, pH 4.5) at a flow rate of 25 mL/h and carbohydrate material was loca l i zed using the Molisch tes t . This material (220 mg) was then applied to the top of a DEAE - A25 2 Sephadex column. The column 25 x 5 cm, was packed in 0.5M Tris/HCl buffer (pH 7.2) and then equi l ibrated with 0.025^ Tris/HCl buffer; at least 10 column volumes of the l a t t e r buffer are required to achieve equl ibrat ion as determined by performing conduct iv i ty measurements. The material was applied as a solut ion in 0.025M Tris/HCl(2 mL). The column was eluted (10 mL/h) with 0.025^ Tris/HCl (140 mL) and a l inear sa l t gradient (from 0 to 0.35M NaCl) was then begun. Fractions (2 mL) were co l lected and examined using the phenol-su l fur ic acid assay. The e lut ion p ro f i l e i s shown in F ig . 14. Analyses of depolymerization products The p.m.r. spectra (XL-100, 90°) of products PI and P2 showed several spurious peaks (buffer?) at low f i e l d , but these were el iminated by passage through a column of Amberlite IR-120 (H ) 13 ion - exchange res in . The C spectrum of PI was in agreement with 132 the 1H n.m.r. spectrum.(§ee App. I I I ) . The degree of polymerization of the products (the ra t io of acetylated a ldonon i t r i l es to acetylated a l d i t o l ) was determined 102 using the method of Morrison. The ol igosaccharide (5 mg) was reduced with sodium borohydride (excess) for 3 h (reducing sugar converted to a l d i t o l ) . The glucuronic acid was then reduced v i a , the methyl ester 2 9 9 by methanolysisand reduction (see Sec. I I .4 .1 . ) The residue was hydrolyzed with t r i f l uo roace t i c acid (2MJ at 95° for 16 h, and evaporated to dryness. The aldoses were converted to the oxime by heating at 95° for 15 min with 5% hydroxylammonium chlor ide in pyridine (0.2 mL/mg of aldose). After coo l ing, acet ic anhydride (0.2 mL/mg of aldose) was added and heating was continued for a further 30 min to dehydrate the oxime to the n i t r i l e and to acetyl ate the free hydroxyl groups. G . l . c . analysis gave the resul ts shown in Figure 7 and Table 13. Paper electrophoresis was performed on a SAVANT high voltage 5kV) system (model LT-48A) with kerosene as coolant. The buffer used contained pyridine - - acet ic acid - - water (5:2:743, v /v) , pH 5.3. St r ips of Whatman no. 1 paper (77 cm x 20 cm) were used with a current of 100 mA for 4 h. For descending paper chromatography f resh ly prepared 2:1:1 1 butanol - - acet ic acid - - water was used. 133 Conditions of depolymerization of K12 and K41. The conditions of depolymerization of Kl2 and K41 capsular polysaccharides, using the bacteriophage propagated on K12 were i den t i c a l . Pur i f i ed capsular polysaccharide (200 mg) was dissolved in the crude broth suspension (20 mL) of <|> 12 10 9 PFU/mL). The reaction mixture was transferred to an Ostwald viscometer which, was placed in a bath at 37°. The v i scos i ty of the so lut ion was determined pe r i od i ca l l y , and was shown to decrease substant ia l l y in both cases, with in 3 h. After 48 h the so lut ion was transferred to a sealed portion of d i a l y s i s tubing (M.W. cutoff = 3,500) and dialyzed against three portions (200 mL) of d i s t i l l e d water. The dia lysates were reduced to dryness and broth contaminants were removed by preparative paper chromatography using f resh ly prepared 2:1:1 1-butanol - - acet ic acid - - water. The y i e l d of the s ingle repeating unit of Kl2 was 10 mgs and of the double repeating unit of K41, 20 mgs. The degree of polymerization of the products was determined as for the products of K21 § 21. 134 Acknowledgments I wish to thank Dr. S. Stirm (Giessen) formerly of II Max Planck - Ins t i tu t fur Immunobiologie .Freiburg, Germany for a g i f t of the bacteriophages, Dr. P. Salisbury for his help and patience in sharing f a c i l i t i e s , and Mark Vagg for mechanical assistance. 1.35 V HIGH PRESSURE LIQUID CHROMATOGRAPHY OF CARBOHYDRATES 136 V. 1. Introduction Although c lass i ca l column l i qu i d chromatography has been an ef fect ive separation method since the beginning of th is century, i t i s s t i l l characterized by low column e f f i c i enc ies and long separation times. . However, in the l as t decade i t has been recognised that column e f f i c iency and speed of separation could be improved by several orders of magnitude i f materials of very small pa r t i c l e s ize are used as column packings. Such columns give a high theoret ica l plate number which decreases only s l i g h t l y with flow ve loc i t y . As a r e su l t , a high resolv ing power and speed of separation can be achieved. Since such columns require a high pressure for the i r operation, th is modern version of column l i qu i d chromatography i s often ca l led high-pressure l i qu i d chromatography (HPLC). 3 0 i The fundamental instrumentation necessary for HPLC separations consists of (see Scheme 12) (a) a solvent reservo i r , (b) a pump capable of giv ing flow against moderately high back pressures (6,000 psi) with a recycle capab i l i t y (c) an in jec t ion head (d) a column, f i t t ed with a pre-column, (e) a detector ( for carbohydrate ana lys i s , usual ly a thermostated re f rac t ive index detector) and (f) a .recorder/integrator/ data system. The basis of chromatographic separation i s the d i s t r i bu t ion (or par t i t ion) of sample components between two phases which are 301 immiscible. The interact ions between the molecules of the mobile and stat ionary phases determine the degree of sorption of par t i cu la r substances and also the effect iveness or s e l e c t i v i t y of the separations. 137 INJECTOR PRE-COLUMN PUMP s Recycle SOLVENT COLUMN T i i DETECTOR System Contro l ler Recorder Integrator 1 FRACTION COLLECTOR DATA MODULE Scheme 12. Block Diagram of Instrumentation for High Performance Liquid Chromatography 138 Gas chromatography separations are based on vapour pressure. L iquid chromatography separations are based on s o l u b i l i t y . With the l a t t e r the composition of the mobile phase, along with the type of packing mater ia l , i s of prime importance in obtaining separation. Research to date on the appl icat ion of HPLC to carbohydrate analysis has focused on the quant i tat ive analysis of sugars in foods 302-304 and beverages . The main sugars of interest here are glucose, fructose and sucrose. Since three d i f fe rent types of sugars (hexose, ketose, disaccharide) are involved, the technique has enjoyed some success in routine ana lys i s , thus replacing paper chromatography (which i s time consuming) and gas- l iqu id chromatography (which requires de r i va t i za t i on) . As yet , the appl icat ion of HPLC in the structura l analysis of heteropolysaccharides has been l i m i t e d 3 2 4 , although the technique shows much promise. The fol lowing areas of app l i c ab i l i t y are apparent. On an ana l y i t i c a l l e ve l : ( i ) Constituent analysis of the native polysaccharide. ( i i ) Constituent analysis of the products of Smith degradation. ( i i i ) To monitor the par t i a l hydrolysis of polysaccharides. ( iv) Analysis of the products of base-catalyzed degradation. (v) Determination of the molecular weight and the homogeneity of polysaccharides. 139 On a preparative l e ve l : (v i) To obtain ol igosaccharides from par t i a l hydrolysis and from Smith degradation. ( v i i ) To pur i fy and separate the products of bacteriophage degradation. ( v i i i ) To obtain methylated/ethylated ol igosaccharides from based-catalysed degradation (of the methylated polysaccharide).. ( ix) To separate methylated ol igosaccharides obtained from methylation of the products of par t ia l hydro lys is . (x) To separate methylated/ethylated ol igosaccharides obtained on ethylat ion of the products of par t ia l hydrolysis of the methylated polysaccharide. The inherent problems are: (a) The d i f f i c u l t y of separating c lose ly related compounds e.g. glucose, galactose.and mannose,or e ry th r i t o l and th re i to l [ for ( i ) and (11)1. (b) The presence of ac id ic sugars - i t i s not possible to separate these on amine - bonded columns [ for ( i ) , ( i i ) , ( i i i ) , and ( v i i ) ] . In th i s study the retention times and molar response factor are measured for (1) the neutral sugars occuring in K lebs ie l l a capsular polysaccharides, (2) some products of Smith degradation (3) a number of disaccharides and a t r i sacchar ide, and the appl icat ion of HPLC in the analysis of K lebs ie l l a capsular polysaccharides i s 140 discussed. V. 2. Chromatographic Conditions The following equipment was used: Waters Associates ALC 201 l iquid chromatograph equipped with M6000A pump, U6K Universal (septumless) injector, R401 di f ferent ial refracto-meter, thermostated at 40° using a Brinkmann Instruments Landa K-2/R circulating water bath,and Waters Model 730 Data Module (printer/ plot ter/ integrator) . Column A: Waters Carbohydrate Analysis (10y) 3.9mm I..D. x 30cm thermostated at 40° with a home-made stainless steel water jacket connected to a circulating water bath. Column B: Stainless steel 4mm x 24cm, with Swagelok end f i t t i n g s . The column was slurry packed with 5p Lichrosorb SI60 (Merck) in methanol - water (9:1). For both columns a small pre-column f i l l e d with Lichrosorb S160 was also used. Solvents: Acetonitr i le, HPLC grade (BDH), and water, glass d is t i l l ed . Eluant, Column A: Acetonitri le water(80:20, 85:15 v/v) . Column B was modified with 500 ml acetonitr i le : water (4:1) containing 0.1% of HPLC amine modifier I (NATEC, Hamburg, G .F .R . ) 3 0 5 > 3 0 6 Thereafter the eluant (acetonitr i le - water, 4:1 or 9:1 v/v) contained 0.01% amine modifier. 141 Sugars were dissolved in water, f i l t e r e d through 0.5 yin Mi H i pore f i l t e r s , and between 5 and 20 y 1 of 5% solutions were injected using a 25 u l Waters Gas Tight Syringe. V. 3. Results and Disscussion ( i ) Separation of monosaccharides (Column A and Column B). I t is possible to get baseline separation for d i f fe rent classes of sugars, e.g. rhamnose fructose and glucose (see Table 16, - F ig. 16). Separation of the hexoses, mannose, glucose and galactose (See Fig.17) i s poor, and because the molar response factors to re f rac t ive index of galactose and mannose are low, these peaks appear as shoulders on the glucose. ( i i ) Separation of d i - and tr isacchar ides It i s possible to ident i fy mono-,di-and t r i sacchar ides, based on the re la t i ve retention times (see Table 16, F ig. 18). Disaccharides with d i s s im i l a r structures e.g. ce l lob iose (Glc Glc) and mel ib iose (Gal Glc) give a good separation, whereas disaccharides with c lose ly re lated structures eg. ce l lob iose and m'al.tose (Glc Glc) do no t . a Comparison of the change in flow rate from 2 mL/min to 3 mL/min (see F ig . 18) shows that good separation i s s t i l l obtained with the shorter retention times. The retention times were longer on Column A than on Column B for the same flow rate and solvent ra t i o . TABLE 16 SEPARATION OF MONO- DI- AND TRISACCHARIDES BY HPLC COLUMN A- COLUMN B-M.R.F.- 2 mL/min (80 : 20) C 2 mL/min (80 : 20)- : SUGAR R.I. Time(mins) Re l .R .T . - Time(mins) Rel. R. Rhamnose 1.4 1.84 0.46 1.5 0.51 Fucose 1.5 2.27 0.56 1.75 0.6 Arabinose 0.7 2.69 0.67 2.05 0.7 Fructose 1.2 3.0 0.75 2.2 0.76 Mannose 0.35 3.8 0.95 2.6 0.9 Glucose 1.0 4.0 1.0 2.8 1.0 Galactose 0.3 4.2 1.05 3.1 1.07 Sucrose 0.53 6.6 1.65 4.3 1.5 Ce l l i b iose 0.53 9.0 2.25 5.5 1.90 Maltose 0.55 9.5 2.37 5.65 1.94 Mel 1ibiose 0.26 13.0 3.25 7.53 2.62 Raffinose 0.34 20.7 5.18 11.33 3.93 — For column spec i f icat ions see Chromatographic conditions (V.2). — Molar response factor to ref ract ive index. r d — Acetoni t r i e : water (v/v) - W i t h 0.01% amine modif ier I added. — Retention time re la t i ve to that of glucose. TABLE 17 SEPARATION OF PRODUCTS OF SMITH DEGRADATION USING HPLC. COLUMN A- COLUMN B^ -SUGAR M.R. F.-R.I. 2 mL/min (85 Time(mins) : 15)^ e Rel. R.T.-2 mL/min (90 Time(mins) : 1 0 ) ^ Rel. R.T. (H 20) - 0.86 0.07 0.95 0.08 Ethylene Glycol - 1.10 0.12 1.15 0.1 Glycerol 0.7. 1.88 0.15 1.85 0.15 Rhamnose 1.4 2.6 0.30 3.09 0.25 Ery thr i to l 0.7 2.8 0.32 3.16 0.26 Threitol 0.7 2.8 0.32 3.2 0.27 Arabinose 0.7 4.3 0.51 6.4 0.53 Gl ucose 1.0 8.36 1.0 12.0 1.0 — For column spec i f icat ions see Chromatographic conditions (V.2). — Molar response factor to ref ract ive index. — Aceton i t r i l e : water (v.v) — With 0.01% amine modif ier I added. — retention time re la t i ve to that of glucose. 144 • R T 8 . 9 4 H20 1 . 8 8 glycerol 2 . 6 1 rhamnose 3 . 3 9 fucose 5 . 4 4 fructose 8 . 3 6 glucose 00 00 • C O L U M N A S O L V E N T 85=15 P R E S S U R E 4 7 8 8 . 8 p s i C H A R T 1 . 8 8 C r V H I N FLOW 2 . 8 8 M L / M I N Figure 16 Separation of Monosaccharides using HPLC (Column A) 145 IX" r-I d P E A K WIDTH see.eee N O I S E R E J E C T I O N 1 9 9 A R E A R E J E C T I O N x. CD CO ro o u. \ X. o CD CE> at <x o CD * —I o <r •-• CL O cu 0) cu to to to o o CL) o c +-> to c • r-o O E jQ (O c o (0 03 r— CM x: S- fO Ol L i . L U U . u. U . - J o (NI s o> CM rw tx> <C CM ID CD r-i CD L U CTi " k £ —< Ct r w "Xi m <r h- •<-< ^ - CM in rw n- in CM •xi CD ro rw '-i in CM CM CM CM •X> t -"X> z 4k LU z> Z — I => o a . x LU <X> CO CO 00 CD in in ^ CD • CD CD CD CD co in <X» - LU <fcCQ _J in ct cr Z CO _ J E OK: • co a_ r> ui z 0 f— CD •—• ~* CM CM « <t r-z t— CD CD CD CD CD CD Z CD CD CD CD CD CD 3 CD CD CD CD CD CD O CD CD CD CD CD CD Z . CD —* CD in Ch CD O <t • • • • • • Q£ <x> >x> •X* CO <I CM rw O I O N * in CM Z CD ro <x» r w • + <r — in CM CM CM rt 1— CM ro to D C t < r o ~> a. co CJ Ul <r ui o Figure 17 Separation of monosaccharides using HPLC (Column B) 146 F L O W 5 8 9 . 8 0 0 1 0 0 3 . 0 0 M L / M I N P E A K W I D T H N O I S E R E J E C T I O N A R E A R E J E C T I O N S O L V E N T 80: 20 C O L U M N B H20 glucose Cellobiose Melibiose Raffinose 2 . 0 0 M L / M I N P E A K W I D T H N O I S E R E J E C T I O N A R E A R E J E C T I O N Figure 18 Separation of d i - and tr isaccharides using HPLC, with d i f f e ren t f low-rates. J U L . 2 2 , 1 9 8 9 2 1 = 3 7 4 7 P R E S S U R E 3 8 8 8 . 8 S A M P L E # 3 3 C O L U M N B C H A R T 1 . 8 8 C M / M I N RUN # 9 4 S O L V E N T 90=10 FLOW 2 . 0 8 M L / M I N C A L C #8 OPR ID= 1 E X T E R N A L S T A N D A R D Q U A N T I T A T I O N AMOUNT RT A R E A 1 4 8 . 3 3 7 8 8 8 . 8 1 1 4 8 3 3 7 E H L -2 8 8 1 3 . 7 8 8 8 8 8 . 9 5 2 8 8 1 3 7 8 2 F H2O 6 6 0 8 2 . 9 8 8 8 8 1 . 1 5 6 6 0 8 3 4 0 3 F ethylene glycol 9 5 9 5 4 . 7 8 8 8 8 1 . 8 5 9 5 9 5 5 4 2 6 L glycerol 6 5 6 7 3 . 7 8 8 8 8 3 . 1 6 6 5 6 7 4 1 9 8 L 1 r y th r i to l T O T A L 2 4 8 6 7 3 . 8 8 8 8 8 148 ( i i i ) Separation of products of Smith degradation. ' ' Glycerol and e ry th r i t o l are well separated from each other and from the solvent (H^O) peak. Ethylene glycol has a very :short retent ion time (see Fig.19) and therefore, samples containing th is compound should.be injected in a solvent composition ident i ca l to the eluant. E ry th r i to l and th re i to l co-chromatograph (see Table 17) with solvent 85:15 and have very s im i la r retention times for solvent 90:10. The retention time of glucose with solvent 90:10 i s r e l a t i v e l y long (12 mins.) V. 4. Conclusion HPLC has the potent ia l to become an important technique in the st ructura l analysis of heteropolysaccharides. The two columns described here give s imi la r resu l ts . Column B has, however, the advantage of being less expensive (since i t i s home-packed) and a l so , the separations may be extended to a preparative scale. In th is case a larger pa r t i c l e s ize of s i l i c a would have to be used to decrease the back-pressure generated. Column A i s avai lable only as a prepacked column suitable for ana ly t i ca l work. Either column could be used in determining the rat io of sugars in a polysaccharide and in its., degradation products. Although the separation of mannose, glucose and galactose i s poor, these three sugars do not necessar i ly occur in the same polysaccharide.--* Since ac id i c sugars (uronic acids) may not be applied to the 149 (amino-bonded) column an ind icat ion of the number of sugars in the repeating unit may be obtained as follows 238 (a) Total hydrolysis of the po ly- , .o l igosacchar ide. (b) Separation into neutral and ac id i c f ract ions (AG-1X2). (c) Analysis of the neutral sugars on HPLC (d) Total hydrolysis of the reduced polysaccharide (v ia . carbodiimide 264) 0 r reduced ol igosaccharide (v ia . the methyl ester)238,239 (e) Analysis of sugars on HPLC (f) Comparison of data from (c) and (e). The advantage of th is method over gas- l iqu id chromatography l i e s in the absence of the need for d e r i v i t i z a t i o n . The major products obtained from par t i a l hydrolysis of an ac id i c polysaccharide are ac id i c (aldobi - a ldot r i - and aldotetrauronic ac ids) . Hence columns A and B could be used only in the analysis of the minor, neutral oligomers obtained. On a preparative l e v e l , th i s technique shows potential in obtaining pure neutral ol igosaccharides. Separation of the products of Smith degradation into neutral and ac id i c components may give some/all of the fo l lowing: (a) neutra l : g l yce ro l , e r y t h i t o l , t h r e i t o l , ethylene, g l yco l , o l igosacchar ides, polysaccharide. (b) ac id i c : erythronic ac id , threonic ac id , o l igosacchar ides, polysaccharides. Analysis of (a) on an ana lyt ica l level would provide useful information, and could be extended to a preparative scale. 150 V. 5. Al ternat ives As denoted above, the main problems encountered in the use of columns A and B are ( i ) the poor separation of mannose, glucose and galactose and ( i i ) ac id i c sugars may not be separated. 307 To overcome ( i ) Adam has reported the use of a 308 r ad i a l l y compressed S i l i c a (10 urn) column (Waters Assocs.) which el iminates voids and channels in the packed bed, thereby giving higher e f f i c i enc i e s . Although not quite basel ine, the separation of mannose, glucose and galactose are much improved, and resu l ts are reproducible. 309 Barton et aj_ report excel lent separation of rhamnose, xylose, arabinose and glucose, and adequate separation of glucose and galactose using a Micromeremetics Micros i l amine bonded phase column, in the analysis of hydrolyzates of plant ce l l 310 wall f i be r . Meagher and Furst have used a y bondapak -carbohydrate (Waters) column with ace ton i t r i l e - water (85 : 15) in the analysis of carbohydrates in rat urine. The second solut ion to ( i ) i s the use of an ion - exchange 311 type column, using water or ethanol as the eluant.Lawrence has + + reported the use of the resins Aminex A-6 Li , ZC225 XI and Technicon Type S to achieve separation of mannose, glucose and galactose, but retention times are up to four hours. 151 312 Durrum Chemical Co. have reported the separation of mannose, fucose, galactose, xylose and glucose (in that order) on. a column of Durrum type DA - X4 (25 cm) in 2.5 hours. Scobell 313 et_ al_ report excel lent separation of arabinose, galactose, glucose, melibiose and melezitose on Aminex A-5 CA + + , at 85° in 32 mins. 314 In 1975 Linden and Lawhead suggested the use of HPLC to separate ol igosacchar ides, on a micro Bondapak (Waters) 315 column, and in 1978 Ladisch et al^  used the ion - exchange res in AG 50W - X4Ca + + to separate the oligomers celloheptaose through glucose wi th in 30 min, using water as eluant. These 316 authors have also described a packing procedure and have commented on the theory of rapid l i qu i d chromatography at moderate pressures, using water as eluant. 317 Belue has used a column of Poras i l A to separate polyhydric alcohols (from Smith degradation) using methyl ethyl ketone - water - acetone (85 : 10 : 5) as eluant. McGinnis and 318 Fang have separated subst i tuted carbohydrates on a column of 10 ym s i l i c a , Part i s i 1 10 (Whatman) using ace ton i t r i l e - water (90 : 10) as eluant. One of the more in terest ing appl icat ions of HPLC in carbohydrate analysis has been the recent (1980) use of a Dupont TM 320 Zorbax 0DS column by Albersheim and coworkers to separate peralkylated ol igosaccharides. These were generated by successive 152 par t ia l acid hydro lys is , reduction and ethylat ion of a permethylated, complex carbohydrate. By th i s technique,the structure of a nonasaccharide derived from xyloglucan, a structura l polymer of plant c e l l -wa l l s , was e luc idated. 321 Doner and Hicks have determined retention times, capacity factors and re l a t i ve responses to re f rac t i ve index detection for over fo r ty pentoses,hexoses, d i - and t r i sacchar ides , and a l d i t o l s on two bonded phase s i l i c a columns and on a cation ++ 322 (Ca ) exchange column. Scrobel l et a]_ describe the preparation and operation of "second generation" s i l v e r form cation exchange res in columns that outperform the equivalent calcium form by a factor of two with respect to time, resolut ion and the number of oligomers seperated. The seperation of ol igosaccharides obtained by phage degradation of K lebs ie l !a K2 polysaccharide was performed by Stirm ejt a ] / 7 using a y-Bondapak-NHg column with 2% formic ac id as eluant. Acknowledgments Jewish to thank Dr. D. 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Varma, R.S. Varma and A.H. Wardi. J . Chromatog. 77, 222-227, (1973). 295. F.R. Seymour, E.C.M. Chen- and S.H. Bishop. Carbohydr. Res., 73, 19-45 (1979). 296. T.P. Mawhinney, M.S. Feather, G.J. Barbero and R.C. Martinez, Anal. Biochem., 101_, 112-117 (1980). 297. C.-C. Chen and G.D. McGinnis submitted to Div. Carb. Chem. Am. Chem. Soc. Chem. Congress, San Francisco, (1980). 298. E.H. Me r r i f i e l d , personal communication. 299. G.G.S. Dutton and M.-T, Yang, Carbohydr. Res., 59, 179-192,(1977). 172 300. Instrumentation for High-Performace :1 iquid Chromatography Journal of Chromatography Library 1_3, Ed. J .F .K. Huber, E lsev ier S c i en t i f i c Publ ishing Company (1978). 301. High Speed Liquid Chromatography Chromatographic Science 6_ P.M. Rajcsanyi and E. Rajcsanyi, Marcel Dekker, Inc. New. York (1975). 302. J . S . Hobbs and J.G. Lawrence, J . Sc i . Fd. A g r i c , 23 45-51 (1972). 303. Edward C. Conrad and James K. Palmer. Food Technology 84-92 (1976). 304a. J .K. Palmer and W.B. Brandes. J . Agr. Food. Chem., 22, 709-712 (1974). 304b. J.K. Palmer Ana*. Letters 8, 215-224 (1975). 305. K. A izetmul ler , M. Bohrs and E. Arzberger, J . High Res. Chromatogr. Commun. 2, 589, (1979). 306. K. A i tzetmul ler , Journal of Chromatography, 156, 354-358 (1978). 307. M.J. Adam, personal communication. 308. Waters Associates, Technical bu l l e t i n F99 (1979). 309. F.E. Barton, W.R. Windham and D. Burdick, submitted to Div. Carbohydr. Chem., Am. Chem. Soc. Chem. Congress San Francisco (1980). 310. R.B. Meagher and A. Furst. J . Chromatog. 1V7, 211-215 (1976). 311. J.G. Lawrence,. Chi mi a 29, 367-373 (1975). 312. Durrum. Resin Report (1972). 313. H.D. Scobel l , K.M. Brobst, and E.M. Steele,Cereal Chem. 54, 905-917 (1977). 314. J .C. Linden and C L . Lawhead. J . Chromatog. 105, 125-133 (1975). 315. M.R. Ladisch, A.L. Huebner and G.T. Tsao, J . Chromatog. 147, 185-193 (1978). 173 316. M.R. Ladisch and G.T. Tsao. J . Chromatog. 166, 85-100, (1978). 317. G.P. Belue, J . Chromatog. TOO, 233-235 (1974). 318. G.D.McGinnis and D. Fang, J . Chromatog. 130, 181 (1977). 319. G.D.M. McGinnis and P. Fang, J . Chromatog. 153, 107-114 (1978). 320. B.S. Valent, A.G. Da r v i l l , M. NcNei l , B.K. Robertsen and P. Albersheim, Carbohydr. Res., 79, 165-192, (1980). 321. L.W. Dover and K.B. Hicks,.submitted to Div. Carbohydr. Chem., Am. Chem. Soc. Chem. Congress. San Francisco (1980). 322. H.D. Scobell and K.M. Brobst, submitted to Div. Carbohydr. Chem. Am. Chem. Soc. Chem. Congress. San Francisco (1980). 323. A. Mort, Carbohydr. Res., submitted for pub l i ca t ion. 324. E.A.Kabat.Personal communication from M. Heidelberger. 174 APPENDIX I The Known Structures of the K lebs ie l l a Capsular Polysaccharides (as of July 1, 1980) 175 K-typeJ- X-Ray-la) '(b) (c) [a] D Structure-References at end Kl -85 o 6-3\/2 pyr 1 : K2 +79' ^ G l c ^ l a n ^ G l c -1 3 . a 1 GlcA & a K3 K4 +9(f Gal A, Gal, Man, Pyr(J) -•^ Gl c ^ G l c A ^ M a n ^ G l c V a a a p K5 •45° -^IcAMsicVManl 2 B 6 \ / 4 OAc pyr 1 K6 +46° -^•Fuc l^Glc-^^lan^GlcA^ t 6 V 4 I pyr K7 +41T -^ Gl cAl^Man^Man^Gl c^Gl c-1 &3 a a 1 4 Gal .1/. pyr. K8 ^ a l l ^ G a l l l l GlcA 176 K-type ti? X-Ray [ a ] Q (a) (b) (c) Structure 9 K9^ •17' 3 1 3 1 3 1 2 1 -^al-L^Rha-L^Rha—Rha— a A a a a ,11 GlcA K9* -5° — ^ l c A ^ ^ h a ^ ^ a l ^ R h a l - ^ R h a — 8 a a a a K10 Kl 1 +106' GlcA, Gal, Glc, Man (c )-3 1 3 1 3 1 - ^ I c - ^ l c A - ^ a l — 8 8 a 1 a , Gal V 6 V 4 pyr K12 +24' -^G l c l^Rha i^Ga l l^Ga l f l a a a — 1 p 1 GlcA 41 1' Gal 6 1/4 pyr K13 +45' 3 1 4 1 4 1 - ^ l c — -^lan-!—tGlc-1 1 1 8 a a 1 GlcA 4 3 1 . ^ 3 Gal T _ 2 > Pyr. 177 K-type <|) X-Ray [ a ] Q (a) (b) (c) Structure K14 GlcA, Ga l , Glc, Man, Rha. (L) 8 K15 K16 +65' GlcA, Gal , Glc. (S.S.) - ^ G l c ^ G l cA^Fuc^— n OL B Oi 1 Gal 8 K17 +30° - ^ - G l c A ^ R h a ^ G l c ^ R h a -p a a 3 i 1 1 Rha K18 +771 Jal 1 4, 1 1 Rha 2 t 1 GlcA 4 c 1 Glc K19 GlcA, Gal , Glc , Rha(J)-178 K-type (a) (b) X-Ray (c) Structure K20 +941 - ^ l a n ^ G a l -31 a I a 1 Gal +OAc 3 1 GlcA 1 K21 K22 +i30 u - ^ I c A ^ M a n ^ M a r v ^ G a l -a a a 1 Gal 6\/4 Pyr 3 n . J 4„,J 4 a 1 Gl c 6 a 1 XA p I XA .= K23 +28' —Rha—^Glc— a 1 Gl 6 1 GlcA K24 7 1 3 1 ? 1 3 1 + 7 0 ° - ^ l c A - ^ a n - L - ^ l a n - L ^ G l c J - ^ T ' 3 a a a B 1 Man I 179 K-type <j> X-Ray [ a ] D (a) (b) (c) Structure K25 -41' -^allAlcl 1 GlcA 2 \ 1 Glc 1 K26 +80 L - G1 c A -^^ Ma n—Ma n^Ga 1 -2 I a 4 1 Gal 6 \ / 4 pyr a a 180 K-type <|) X-Ray [a] n (a) (b) (c) D Structure K29 GlcA, Gal , Man, pyr. (N) 8 K30 +16L GlcA 1 4 1 4 1 4 1 - ^ a n - ^ l a n - ^ l c -6 1 a 1 T ? T Gal +0Ac— 4V.3 K31 • ^ G l c ^ G l c A ^ G a l l 3 41 a a 11 Man 21 l l Glc 6\/ 4 pyr K32 +113° - ^ a l ^ R h a ^ - ^ h a ^ R h a ^ — a \ / a 8 a 4 \ / 3 pyr r K33 +221 GlcA OAc o a 6 4 1 4 1 4 1 ^ l a n - ^ a n - ^ l c - ' a B 1 Gal pyr 181 K-type (a) (b) X-Ray (c) [ a ] n Structure K34 +21" 3 1 ? 1 3 1 3 1 ? 1 ^ h a - ^ h a - ^ 1 c - ^ a l A-L-^ha— a a 8 a a 1 Rha K35 GlcA, Gal , Glc , Man ( L ) -K36 •56l -^ Ga l ^ R h a i - ^ R h a ^ R h a -GlcA 4 8 1 Glc pyr 182 K-type f (a) (b) X-Ray [a] (c) D Structure K38 +28' DPA 2L i G l d ^ G a l l ^ a l l 3 i 1 Glc DPA = 3-deoxy-L-glycero-pentulosonic acid K39 K40 GlcA, Gal , Glc , Man (D) 8 GlcA, Gal , Man, Rha, Pyr. (C) 8 K41 +231 -^Gl c^Rha -^Ga l ^-?Gal f -a a a 1 GlcA 4 1 G 6 1 Glc K42 K43 K44 +4L GlcA, Gal , Man, pry (N) GlcA, Gal , Man. (N)— 8 -^1 c ^ R h a ^ G l c ^ G l cA-^Sha-— & a a 3 a 183 K-type <() X-Ray . - ..[a] D . (a) (b) (c) Structure K45 GlcA, Glc, Rha, pyr. (D) 8 K46 +1161 - ^ a l ^ a l ^ G1 a P 4 1 3 1 cA-^Man- 1— a a Glc^Man pyr K47 •46 0* - ^ a l ^ R h a -r i GlcA 4| 1 Rha K48 +23^  •^ G-l clr^Rha^Gl c^-^ha— p £ | a a a 1 Gal A K49 +1521 -^Gal^Man^Gal- 1 a a l l Gal A — OAC 2or4 184 K-type 4> "(a) (b) X-Ray [ a ] n (c) D Structure K50 GlcA, Gal, Glc, Man (D) 8 K51 -^ Gall-^ Gal-1 a 4 i a 1 GlcA K52 -^ alV^Rha-V^1 cA^ -^ Gal V^RhaV 2' 1 Gal K53 +2' -^ Gl c A l ^ a r v ^ - ^ a n ^ G a l ^ R h a — p 3 . a a B a 1 Rha K54 K55 -28' +90' 6 n c I ^ l c A l l F u c i _ B a a 1 Glc t OAc 2 ^ G l c l W 1' Gal 3 1 T GlcA 185 K-type 4 x " R a y ["] n (a) (b) (c) D Structure K56 +79' •^Gl c - ^ G a l - M G a l ^ G a l -6 4 pyr 1 Rha K57 +104' -^Ba l l^Ga lA^Man l 1 l l Man K58 +19' -^Glc^Gl CAMFUC-3 \ / 2 pyr 1 Gal K59 +26' 3 1 3 1 2 1 3 1 •^ Gl c - ^ a l - ^ a n - ^ a n -1 GlcA 6 • OAc 6 t i OAc K60 K61 +58' +56' (dotted l ines indicate OAc's not on a l l residues) —^Gl c ^ G l cA^Ga l^Man - 1 T 4 a V Glc 21 e a 21 r Glc 1 Glc •^Gl cA^Man^-^Gl c^-#Gl c-1 3 3 1 Gal 186 K-type 4 x " R a y (a) (b) (c) .["Ii Structure K62 11 +60' ? 1 3 1 1 1 4 1 cA^-^Man-^al-VGl c-— 1 Man K63 +133' - ^ a l A ^ F u c ^ a l - 1 a a a Rha 11 • • a T K64 +28° — ^ I c A ^ M a n ^ G l c ^ M a n l -a a 3 £i a 3 T K65 K66 K67 K68 K69 K70 GlcA, Glc, Man, Rha, Pyr (A.M.S.)-GlcA, Gal, Man (N)— GlcA, Gal , Glc, Man, Rha (L) 8 GlcA, Gal, Glc, Man, Fuc, Py r ( L ) -GlcA, Glc, Gal, Man, Pyr ( I . S . ) -4 1 4 1 2 1 -*G1 cAJ^LRha-L^Rha-L-» 8 a a ? 1 3 1 2 1 -^1 c-L^Gal-L/Rha— * \ ! 3 50% I pyr 1187 K-type f X-Ray [a] Q (a) (b) (c) Structure K71 -45° -Rha-Rha-Rha-RHa:- 3 Glc i i i i i r i GlcA Glc K72 -54° - ^ I c ^ R h a ^ R h a - ^ R h a -a i I a a 4\ / 3 pyr 1 ? K73-L=- = Aerobacter aerogenes K74 +67' 3 1 ? 1 2 1 - ^ G a l - ^ a n - ^ a n — a a r ; Gl cA 4 1 1 Gal 6\/4 pyr K75^- = K68 = K46 K77^ = K39 K78^- = K15 K79 GlcA, Gal, Glc, Rha (p)-188 K-type <|) X-Ray [ a] ^ Structure (a) (b) (c) K80 GlcA, Gal, Man, Rha, Pyr. K81 -52° — ^ h a ^ R h a ^ l c A ^ R h a i ^ R h a i ^ G a K 8 2 12 , 13 a a B a a K83 +89° - _ i G a l i_4 Rha 1 6 3 a 1 a Gal 3 a 1 GlcA 189 Footnotes 1 2 3 4 5 6 7 Serotyping by Qrskov. Structure Ref. (a) Bacteriophage degradation Ref. (b) X-Ray crysta l lographic study Ref. (c) Rotations at Na-D l i ne except where noted. Data compiled by E.H. Me r r i f i e l d . A l l sugars are D except for rhamnose and fucose which are I Rotation at 578 nm. Bacteriophage attack s i t e . D. Rieger - Hug and S. St irm. Virology in press. Q Under invest igat ion by J = J .P . Joseleau C = A . J . Chakraborty L = B. Lindberg S.S. = S. Stirm N. = W. Nimmich D. = G.G.S. Dutton A.M.S. = A.M. Stephen I.S. = I. Sutherland Q This serotype has been invest igated in two laborator ies , and two d i f fe rent structures have been proposed; denoted K9, K9* ^ OAc group located on every th i rd repeating un i t . ^ Rotation measured on methylated polysaccharide. 1 ? G. (prskov and M.A.Fife - Ashbury Internat. J . Systematic B a c t e r i d . , 27_ 386 (1977). 13 No quant i tat ive ana lys is . Not assigned to a research group. 190 APPENDIX I BIBLIOGRAPHY Kl (a) C. Erbing, L. Kenne, B. Lindberg, J . Lonngren and I. Sutherland, Carbohydr. Res., 50 115-120 (1976). K2 (a) L.C. Gahan, P.A. Sandford and H.E. Conrad, Biochemistry. K2 (b) H. Geyer, S. Stirm and K. Himmelspach. Med. M ic rob io l . Immunol., 165, 271-288 (1979). K4 (a)( i )E .H. Me r r i f i e l d , Ph.D. Thesis U. Cap Town (1978). ( i i ) S . C . Charms and A.M. Stephen, Carbohydr. Res., 35_, 73 (1974). K5 (a)( i )G.G.S. Dutton and M.T. Yang, Can. J . Chem., 50, 2382-2384, (1972). ( i i )G .G .S . Dutton and M.T. Yang, Can. J . Chem., 5]_, 1826-1832 (1973). K5 (c) E.D.T. Atk ins , D.H. Isaac and H.F. Elloway, in Microbiol Polysaccharides and Polysacchariases. [Eds. R.C.W. Berkeley, G.W. Gooday and D.C. Elwood] 161-189. Academic Press London (1979). K6 (a) U. E l sasser -Be i le , H. Fr iebo l in and S. St i rm, Carbohydr. Res. , 65, (245-249) 1978. K7 (a) G.G.S. Dutton, A.M. Stephen and S.C. Churms, Carbohydr. Res., 38, 225-237 (1974). K8 (a) I.W. Sutherland, Biochemistry, 9, 2180-2185 (1970). K8 (b) I.W. Sutherland, J . Gen. Microb io l . 94, 211-216 (1976). K8 (c) E.D.T. Atk ins, D.H. Isaac and H.F. Elloway, in Microbiol Polysaccharides and Polysacchariases. [Eds. R.C.W. Berkeley, G.W. Gooday and D.C. Elwood]. 161-189. Academic Press London (1979). K9 (a) B. Lindberg, J . Lonngren, J .L . Thompson and W. Nimmich, Carbohydr. Res., 25, 49-57 (1972). K9* (a) S.C. Churms, E.H. Mer r i f i e l d and A.M. Stephen, S. A f r . J . S c i . 76, (1980) 233-234. K9 (c) E.D.T. Atk ins , D.H. Isaac and H.F. Elloway, in Microbiol Polysaccharides and Polysacchariases. [Eds. R.C.W. Bermeley, G.W. Gooday and D.C. Elwood]. 161-189. Academic Press London (1979). 191 a) H. Thurow, Y.M. Choy, N. Frank, H. Niemann and S. St irm, Carbohydr. Res., 4V, 241-255 (1975). b) W. Bess ler, E. Freund-Molbert, H. Knufermann, C. Rudolph, H. Thurow and S. St irm. Virology 56, 134-151 (1973). a) G.G.S. Dutton and A.V. Savage, Carbohydr. Res. 83, (1980) b) G.G.S. Dutton and A.V. Savage, unpublished resu l t s . a) H. Niemann, N. Frank, and S. St irm, Carbohydr. Res. 59_, 165-177 (1977). b) H. Niemann, H. Bei lharz and S. St irm. Carbohydr. Res. 60, 353-366 (1978). a) A . J . Chakraborty, H. F r i ebo l i n , H. Niemann and S. St irm, Carbohydr. Res. 59, 523-530 (1977). c) E.D.T. Atk ins , D.H. Isaac and H.F. Elloway, in Microbiol Polysaccharides and Po lysacchar ides . [Eds. R.C.W. Berkeley, G.W. Gooday and D.C. Elwood]. 161-189. Academic Press, London (1979). a) G.G.S. Dutton and T.E. Folkman. Carbohydr. Res. 80, 147-161 (1980). a) G.G.S. Dutton, K.L. Mackie and M.T. Yang, Carbohydr. Res. 65, 251-263 (1978). b) G.G.S. Dutton, A.V. Savage and M. Vignon, Can. J . Chem. in press. a)( i )Y .M. Choy and G.G.S. Dutton, Can. J . Chem., 51_ 3015-3020 (1973). a) ( i i ) B . Whitehouse, 449 Thesis, UBC, 1976. b) H. Thurow, H. Niemann, C. Rudolph and S. St irm. Virology 58, 306-309 (1974). a)( i )Y .M. Choy and G.G.S. Dutton, Can. J . Chem. 5J_, 198-207 (1973). a) ( i i )Y.M. Choy and G.G.S. Dutton, Carbohydr. Res. 21_, 169-172 (1972). b) G.G.S. Dutton, K.L. Mackie, A.V. Savage, D. Rieger-Hug and S. St irm. Carbohydr. Res. 83, (1980). 192 [a) H. Niemann and S. St i rm, unpublished resu l t . [c) G.G.S. Dutton, M. Stephenson, K.L. Mackie, and A.V. Savage Carbohydr. Res. 66, 125-131 (1978). [a) Y.M. Choy, G.G.S. Dutton and A.M. Zanlungo, Can. J . Chem., 5 1 , 1819-1825 (1973). [b) H. Thurow, N. Niemann, C. Rudolph and S. St irm. Virology 58, 306-309 (1974). [a) H, Niemann, B. Kwiatkowski, 0. Estphal and S. St irm. J . B a c t e r i d . , 130, 366-374 (1977). [c) E.D.T. Atk ins, D.H. Isaac and H.F. Elloway, in Microbiol Polysaccharides and Polysacchariases. [Eds. R.C.W. Berkeley, G.W. Gooday and D.C. Elwood]. 161-189. Academic Press London (1979). [a) J . L . DiFabio and G.G.S. Dutton, unpublished resu l t . [a) S.C. Churms, E.H. Mer r i f i e l d and A.M. Stephan, Carbohydr. Res. 8 1 , 49-58 (1980). [a) M. Curva l l , B. Lindberg, J . Lonngren and W. Nimmich, Carbohydr. Res., 42, 95-105 (1975). [a) B. Lindberg, F. Lindh, J . Lonngren and I.W. Sutherland. Carbohydr. Res., 70, 135-144 (1979). [a) C.C. Cheng, S.L. Wong, and Y.M. Choy, Carbohydr. Res., 73_, 169-174 (1979). >) G.M. Bebault, G.G.S. Dutton, N. Funnell and K.L. Mackie, Carbohydr. Res., 63, 183-192 (1978). [b) G.G.S. Dutton, K.L. Mackie, A.V. Savage, D. Rieger-Hug and S. St irm. Carbohydr. Res. [a) B. Lindberg, F. Lindh, J . Lonngren and W. Nimmich, Carbohydr. Res. 70, 135-144 (1979). [a) J-P. Joseleau, personal communication. [a) G.G.S. Dutton and K.L. Mackie, Carbohydr. Res., 55_, 49-63. (1977). [a) B. Lindberg, B. L indquist , J . Lonngren and W. Nimmich, Carbohydr. Res., 49, 411-417 (1976). 193 K38 (a) B. Lindberg, B. Samuelson and W. Nimmich, Carbohydr. Res.. 30, 63-70 (1973). K41 (a) J-P. Joseleau, H. Lapeyre, M. Vignon and G.G.S. Dutton Carbohydr. Res., 67_, 197-212 (1978). K41 (b) J-P. Joseleau and A.V. Savage, unpublished resu l t . K44 (a) G.G.S. Dutton and T.E. Folkman, Carbohydr. Res. 78, 305-315 (1980). K46 (a) G.G.S. Dutton and K. Okutani, Carbohydr. Res., in press. K47 (a) H. Bjorndal , B. Lindberg, J . Lonngren, W. Nimmich and K. Rose l l , Carbohydr. Res., 27, 272-278 (1973). K48 (a) J -P. Joseleau, personal communication. K49 (a) J -P. Joseleau, and F. Michon, personal communication. K51 (a) A.K. Chakraborty and S. St irm, Abst. Int. Symp. Carbohydr Chem., 9th, London, 439-440 (1978). K52 (a) H. Bjorndal , B. Lindberg, J . Lonngren, M. Meszaros, J . L . Thompson and W. Nimmich, Carbohydr. Res., 3J_, 93-100, (1973). K53 (a) G.G.S. Dutton and M. Paul in , Carbohydr. Res., in press. K54 (a)( i )P .A. Sandford and H.E. Conrad, Biochemistry, 5_, 1508-1516, (1966). K54 (a)( i i ) H.E. Conrad, J.R. Bamburg, J.D. Epley and T . J . Kindt, Biochemistry, 5, 2808 (1966). K54 (b) I.W. Sutherland. Biochem. J . 104, 278-285 (1967). K54 (c) E.D.T. Atk ins , D.H. Isaac and H.F. Elloway, in Microbiol Polysaccharides and Po lysacchar ides . [Eds. R.C.W. Berkel G.W. Gooday and D.C. Elwood]. 161-189. Academic Press London (1979). K55 (a) G.M. Bebault and G.G.S. Dutton, Carbohydr. Res., 64, 199-213 (1978). K56 (a) Y.M. Choy and G.G.S. Dutton, Can. J . Chem., 51_, 3021-3026 (1973). K57 (a) J .P . Kamerling, B. Lindberg, J . Lonngren and W. Nimmich, Acta Chem. Scand., (B) 29, 593 (1975). 194 c) E.D.T. Atk ins, D.H. Isaac and H.F. Elloway, in Microbiol Polysaccharides and Polysacchariases. [Eds. R.C.W. Berkeley, G.W. Gooday and D.C. Elwood]. 161-189. Academic Press London (1979). a) G.G.S. Dutton and A.V. Savage, Carbohydr. Res. 83_, (1980). a) B. Lindberg, J . Lonngren, U. Ruden, and W. Nimmich, Carbohydr. Res., 42, 83-93 (1975). a) G.G.S. Dutton and J .L . DiFabio, Carbohydr. Res., in press. a ) ( i )A .S . Rao, N. Roy and W. Nimmich, Carbohydr. Res. 67, 449-456 (1978). a ) ( i i ) A . S . Rao, N. Roy and W. Nimmich, Carbohydr. Res.76 , 215-224 (1979). _ a) G.G.S. Dutton and M.T. Yang, Carbohydr. Res., 59, 179-192 (1977). a) J .P . Joseleau and M-F. Marais, Carbohydr. Res. 7_7, 183-190 (1979). b) E.H. Me r r i f i e l d , unpublished resu l t . c) E.D.T. Atk ins, D.H. Isaac and H.F. Elloway, in Microbiol Polysaccharides and Polysacchariases. [Eds. R.C.W. Berkeley, G.W. Gooday and D.C. Elwood]. 161-189. Academic Press London (1979). a) E.H. Mer r i f i e l d and A.M. Stephen, Carbohydr. Res. 74, 241-257 (1979). a) G.G.S. Dutton and K.L. Mackie, Carbohydr. Res., 62, 321-335 (1978). b) G.G.S. Dutton and E.H. Me r r i f i e l d , unpublished resu l t . a) E.G. Mer r i f i e l d and A.M. Stephen, unpublished resu l t . a) Y.M. Choy and G.G.S. Dutton, Can. J . Chem., 52, 684-687 (1974). a) G.G.S. Dutton and M. Paul in Carbohydr. Res., in press. a) M. Curva l l , B. Lindberg, J . Lonngren and W. Nimmich, Carbohydr. Res., 42, 73-82 (1975). a) B. Lindberg and W. Nimmich, Carbohydr.Res.48,81-84 (1976) 195 APPENDIX II Structural Patterns of K lebs ie l l a Capsular Polysaccharides 196 Key : = Uronic Acid 0 = Neutral sugar (X) = 3-deoxy-L-glycero-pentulosonic acid [X] = 4-0 [(s)- l-carboxyethyl ] -D-glucuronic ac id <(X^  = 2R, 3R-hex-4-enopyranosyluronic acid Pyruvate and acetate omitted A. Uronic acid absent - 0 - 0 - 0 - 0 - - 0 - 0 - 0 - 0 - - 0 - 0 - 0 -K32, K72 1 n K56 K38 - 0 - 0 - - 0 - 0 -0 0 [X] K37 ^ * M K22 B. Uronic acid in chain a) l i near - X - 0 - 0 -K l , K5, K63 - X - 0 - 0 - 0 K4, K6 - X - 0 - 0 - 0 - 0 K9*, K44 - X - 0 - 0 - 0 - 0 - 0 K70, K81 197 branch point on uronic acid i ) s ingle unit side chain - X - 0 - 0 - X - 0 - 0 - 0 I I 0 0 K l l , K57 K21, K24 i i ) two unit side chain - X - 0 - 0 - X - 0 - 0 - 0 . f I 0 0 1 I 0 K31 0 K46 i i ) three unit side chain iv) plus branch points neutral sugars - X - 0 - 0 - 0 I 0 0 0 K26 - X - 0 - 0 - 0 I I I ; 0 0 0 K60 branch not on uronic ac id X - o - 0 - - - X - 0 - 0 - 0 - - X - 0 - 0 0 0 0 K16, K54 K7, K61, K62 K52, K53 - X - 0 - 0 - 0 - - X - 0 - 0 I I K17 0 K58 0 double branch not on uronic acid 0 I - X - 0 - 0 - 0 -I K64 0 198 C. Uronic acid in side chain a) s ingle unit side chain - 0 - 0 - 0 - - 0 - 0 - 0 - 0 -X X K2, K8, K9, K59 b) two s ingle unit side chains - 0 - 0 - 0 - 0 - 0 X 0 exact locat ions of side chains not determined c) two s ingle units side chains forming a double branch 0 0 1 I - 0 - 0 - 0 - - 0 - 0 - 0 - 0 I ' I X X K30, K33 K27 d) two unit side chain i ) uronic acid terminal - 0 - 0 -0 I X K20, K23, K51, K55 i i ) uronic acid non-terminal - 0 - 0 - - 0 - 0 - 0 - - 0 - 0 - 0 - 0 I I I X X X I I I 0 0 0 K25, K47 Kl3, K74 Kl2, K28, K36 199 6;) Three unit side chain i ) uronic acid non-terminal 0 I 0 0 - 0 - 0 I X I 0 0 - 0 - 0 K18 K41 Note: K9 has been invest igated in two d i f fe rent laborator ies , and two d i f fe rent structures have been proposed. These are denoted K9 and K9* 200 APPENDIX III 13 H and C n.m.r. spectra K12 polysaccharide HOD "*H n.m.r. 220 MHz, 90°C Spectrum No. 1 K12 polysaccharide 13 I J C n.m.r. 20MHz, amb. temp. 102.64 I 108.39 106.99 99.43 1—r Spectrum No. 2 Kl2 Compund (3) Rha ^ — ^ Gal 1 2 Ara ^—glycerol 1 a a a H n.m.r. 100 MHz, amb.temp. Spectrum No. 3 K12 Compound (3) Rha — Gal — Ara ^ -Glycerol a a a 13 r C n.m.r. 20 MHz, amb. temp. 106.93 99.14 103.10 Spectrum No. 4 K-12 Compound (1) GlcA ^-g3- Gal~0H ^3C n.m.r. 20MHz, amb. temp 104.46 I 1 I 1 I 1 I 1 I 1 I i — I — r Spectrum No.6 Kl2 Compound (2) Spectrum No. 8 J _ J L__l I I 1,1 I L_l i l l I I I L—L Spectrum No. 10 K58 polysaccharide C n.tn. r. Spectrum No. 11 K58 polysaccharide (5) pyruvate removed n.m.r. 100 MHz, 90°C 5.35 i i T [ I i i i" i Spectrum No. 12 I i i i i i i i j i I J i i i i i i I I I 1 1 1 1 1 1 I—j—U_l—(—I ! —J 1— L Spectrum No. 13 K58 Compound (4) •f^Glc^GlcA ^ F u c — w 100.3i 104 Pyr 1 3 I JC n.m.r. 20 MHz, amb. temp. 99.7 i — i — i — i — i — i — i — r Spectrum No. 14 4.54 Spectrum No. 15 K58 Compound (1^ ) GlcA 1 ^ Fuc~0H 1 3C n.m.r. 20 MHz, amb. temp. 97.07 Spectrum No. 16 K59 Compound (2) Glcl^lcA^W-OH a p 1 3 C n.m.r. 20 MHz, amb. temp. K58 Compound (3) G l c ^ G l CA1/FUC~0H a 3 3 a 1 Gal n.m.r. 100 MHz, 90°C 5.29 Spectrum No GlcA-^l cA-l-^ Fuc-OH Gal 62.59 61.07 Il 6.25 r o r o o Spectrum No. 21 1.93 1.62 1.33 1 1 ' I 1 Spectrum No. 22 K70 (p70 Compound PI n.m.r. 80 MHz, 95°C n 5.10 4.80 4.98| i/ 5.23 5.28 4.58 J 1 • I Spectrum No. 24 K70 pO Compound PI I 0 C n.m.r. Spectrum No. 25 ro ro K21 $21 Compound PI Gl cA^^an^-^an^Ga l ~0H a a a "*H n.m.r. Gal 100 MHz, 90°C Pyr • I I I I I 1 1 1 1 1 1 1 SDectrum No. 26 Acetone 2.23 1.55 ro ro i I I I l' • i I • l I ' 1 1 1 1 1 ' K21 4>21 Compound PI G l c A ^ l a n — M a n ^ G a l ~0H a a a 1 Ga V Pyr 101.37 103.04 ^3C n.m.r. 20MHz, amb. temp 101.18 Spectrum No. 27 25.96 PO PO 31.07 I I I I I I I I I I I I I I I I I I I Spectrum No. 28 Kl 2 <j) 1 2 PI H^-n.m.r. 400 MHz, 90° K41 <|> 12 P2 A c e t o n e 1 3 I J C n.m.r. 20 MHz amb. temp. I I I I ! i I I I I ! I I 1 I I Spectrum No. 31 232 METHODOLOGY OF BACTERIOPHAGE PROPAGATION AND POLYSACCHARIDE ISOLATION APP.IV 233 Media and Buffers "Standard" l i qu i d broth or medium contained 5g Bactopeptone, 3g Bacto beef extract,and 2g NaCl per l i t r e of water. "Standard" agar plates were made using a so lut ion of "standard" l iquid broth to which 15g of agar per l i t r e had been added. 8.5 cm disposable pet r i plates were used. Ac t ive ly growing cultures of K lebs ie l l a bacter ia (50 mL) were grown, in 100cm x 300cm t rays , on a medium of sodium chlor ide (8g), calcium carbonate (2g), sucrose (120g), and Bacto yeast extract (8g) in 2L of water for three days. (See Scheme 13 ) . Phosphate-buffered sa l ine (PBS) pH=7 was made up using 8.5g NaCl, 1.76g Na 2HP0 4 .H 20, and O.lg KH 2P0 4 in IL of water. T r i s HC1 (pH=7.5) was made up using 82 mL HC1, 12.1g T r i s (hydroxymethyl aminomethane),5g NaCl, Ig NH^Cl i n IL of water. Core l la t ion of opt i ca l density with bacter ia l count. A f lask of l i qu i d medium was inoculated with a cul ture of ac t i ve ly growing K lebs i e l l a K21 bacter ia and vigorously aerated at 37° . A l iquots were removed at 30 minute i n t e r va l s , appropriately d i luted (10 to 10" ) with l i q u i d medium and a small quantity (0.1 mL) of the d i lu ted solut ion was incubated on an agar plate for 12-16 hours. Individual bacter ia l colonies could then be counted. The bacter ia l count core l la tes with opt ica l dens i ty. 234 STANDARD AGAR BEEF-EXTRACT SUCROSE, YEAST-EXTRACT, AGAR STREAK 37° 16h INOCULATE 37° 3h GROW CENTRIFUGE EtOH PRECIPITATE ^ 5 ' PREC -EtOH PITATE 4 / H20 do DISSOLVE 30,000 r.p.m. — — 3-5h -f -25° 3d HARVEST 1% PHENOL DILUTE H20 10% CETAVLON DISSOLVE -4 do* PRECHPITATE 5,000 r.p.m. 20 min. ? s 4M NaCl DISSOLVE CENTRIFUGE AN" H20 DIALYZE FREEZE-DRY Scheme 13 Iso lat ion and Pur i f i ca t i on of Polysaccharide. 235 Bacteriophage Propogation (a) Tube l y s i s . An act ive bacter ia l cu l ture of K lebs ie l l a K21 was obtained by successive replat ings on agar p lates. 7x5 mL of s t e r i l e l i qu i d medium was then inoculated with the bacter ia by the addit ion of 0.5 mL of an ac t i ve ly growing l i q u i d K32 bacter ia l cu l ture. These seven test tubes were incubated at 37° and at 30 minute interva ls the tubes were inoculated with 0.5 mL of a so lut ion of l i qu i d medium containing <j)2V. Continued incubation resulted in the f i r s t few tubes changing from the cloudy solut ion associated with ac t i ve ly growing K21 bacter ia to a c lear solut ion ( l y s i s ) . After the l a s t tube had cleared the incubation was continued for 30 minutes and then a few drops of CHC1^ was added to the tube and the mixture was shaken we l l . A phage " t i t r e " on the so lut ion was performed by successively d i l u t i ng a small volume (0.1 mL) of the c lear l i qu i d with l i qu i d medium and then applying approximately 0.03 mL of these d i luat ions to a ' lawn' of ac t i ve ly growing K lebs ie l l a K21. (The lawn of K21 was prepared by inoculat ing 2 mL of l i qu id medium with an ac t i ve ly growing colony of K lebs ie l l a K21 and incubating th i s culture for 3 hours.. An agar p la te , previously dried for approximately 1 h in the incubator at 37°, was covered with th i s l i qu i d cu l ture , l e f t for 5 minutes and then the excess l i qu i d was removed. Incubation for 30 minutes gave a stable ' lawn' of K lebs ie l l a K21.) Individual bacteriophage were observed as c lear spots (approximately 0.3 cm in diameter) on the bacter ia l lawn af ter incubation for 16 hours. At high phage concentrations ind iv idual phage could not be dist inguished but at more sui table d i l u t i on s , 236 Figure 20 Growth Curve -of Klebsiella , and bacteriophage lysis K21 bacteria. 237 u 1 [ 1 e.g. 10" to 10" , ind iv idual 'haloes' could be eas i l y counted. As a g resu l t of a s ingle tube l y s i s of th is nature an assay yie lded 10 plaque forming units (PFU) per mL of medium in the l a s t tube to completely c lea r . (b) Small f lask l y s i s . This technique i s essent ia l l y the same as that described for the tube l y s i s . As larger volumes of l i qu i d medium can be used the overal l to ta l of bacteriophage can be increased even though the phage t i t r e per mL. may not be s i gn i f i c an t l y higher. In a typ ica l small f lask l y s i s 50 mL: solut ions of K21 cultures were .inoculated with 1.5 mL of a phage solut ion containing 10 9 PFU/mL (from tube l y s i s ) . In an analogous manner to that described for the tube l y s i s , t i t r a t i o n of the f i n a l f lask to completely c lear gave a t i t r e of 1 .2x l0 1 0 PFU/mL. 250 mL of an ac t i ve ly growing l i qu i d culture of K32 were vigorously aerated at 37°. A small amount of a s i l i c on antifoam agent (Dow antifoam FG-10 emulsion) was added to each. The opt ica l density of each f lask was monitored and at appropriate opt ica l density readings (calculated such that the ra t io of tota l bacteriophage to tota l bacter ia l colonies was approximately 3:1) a l iquots of l i qu i d phage cultures were added and the opt ica l density monitoring continued. A subsequent drop in opt ica l density indicated l y s i s had occurred. The resul ts of a typ ica l bot t le l y s i s are shown in Figure 20. A bott le l y s i s might t yp i ca l l y y i e l d 400 mL of a so lut ion with a t i t r e of 3 . 0 x l 0 1 0 PFU/mL. 238 (c) One l i t r e f lask l y s i s . In an analogous manner to that described for the bot t le l y s i s three one l i t r e f l a sks , each containing 600 mL of l i qu i d medium, were inoculated with K bacter ia , aerated and incubated to appropriate opt ica l dens i t i es , and then bacteriophage solut ions were added. A typ ica l resu l t of such a l y s i s might y i e l d 1400 mL of a phage solut ion with a t i t r e of 3 x l 0 1 0 PFU/mL. 

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