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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), U n i v e r s i t y College Galway, I r e l a n d , 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 t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1980  ©  Angela V. Savage, 1980  i  In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study.  I further agree that permission for extensive copying of  t h i s thesis for s c h o l a r l y purposes may be granted by the Head of my Department or by his representatives.  I t i s understood that copying  or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  Department of  C \ASUMA/)\ f\M/  The U n i v e r s i t y 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 s e r o l o g i c a l l y d i f f e r e n t s t r a i n s of K l e b s i e l l a are known.  The capsular polysaccharides which these bacteria produce  are a n t i g e n i c , and in order to understand the chemical basis of s e r o l o g i c a l d i f f e r e n t i a t i o n the s t r u c t u r a l i n v e s t i g a t i o n of the capsular polysaccharides has  been undertaken.  To date, f i f t y  six  structures have been determined. The structures of the capsular antigens i s o l a t e d from serotypes K12 and K58 are presented here, along with confirmative data f o r the structure of K23 and a nuclear magnetic resonance i n v e s t i g a t i o n o f K70 and i t s s p e c i f i c degradation products. An e f f i c i e n t means of i s o l a t i n g large q u a n t i t i e s of the . s i n g l e repeating units of the K l e b s i e l l a polysaccharides using glycanase enzymes, borne and u t i l i z e d by s p e c i f i c bacteriophage, i s demonstrated.  K l e b s i e l l a K21 polysaccharide has been degraded using  a highly p u r i f i e d bacteriophage ( e-galactopyranosidase a c t i v i t y ) , while K l e b s i e l l a K12 and K l e b s i e l l a K41(which have s i m i l a r structures) have both been degraded using a crude s o l u t i o n of bacteriophage s p e c i f i c f o r K l e b s i e l l a K12 ( 0-galactofuranosidase a c t i v i t y ) , and r e s u l t s compared. A preliminary i n v e s t i g a t i o n of the use of high pressure l i q u i d chromatography i n the s t r u c t u r a l i n v e s t i g a t i o n of heteropolysaccharides i s included, along with appendices containing  iii compilations of the structures and s t r u c t u r a l patterns of the K l e b s i e l l a capsular polysaccharides determined to date.  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  K12  3-D-Gal£  6 4 \/  A Me  COOH O-Ac  2 ^3)-a-D-Glcp_-(l+4)-B-D-Glcp_A-(M)-a-L-Fucp_-(lK58  V  Me  /  \oOH  3 + 1  a-D-Gal£  iv TABLE OF CONTENTS Page ABSTRACT  1 1  TABLE OF CONTENTS  i  LIST OF TABLES  v  Viii  LIST OF FIGURES  .  LIST OF SCHEMES  x xii  ACKNOWLEDGEMENTS  xi i i  PREFACE  xiv  I  INTRODUCTION  1  II  METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES  20  11.1.  I s o l a t i o n and P u r i f i c a t i o n  21  11.2.  Separation Techniques  22  II.2.1. 11.2.2. 11.2.3.  22 23 24  , .  II. 3  Paper chromatography ( p . c . ) Paper electrophoresis (p.e.) Gel chromatography ( g . c . )  Instrumentation 11.3.1. 11.3.2. 11.3.3. 11.3.4. 11.3.5.  25  G a s - l i q u i d chromatography ( g . l . c ) . Mass spectrometry (m.s.) Polarimetry C i r c u l a r dichroism ( c . d . ) Nuclear Magnetic Resonance Spectroscopy •  .  I I . 3 . 5 . 1 . ,1H n.m.r. spectroscopy . . II.3.5.2 C n.m.r. spectroscopy . . II.4.  Techniques of Structure Determination 11.4.1. 11.4.2. 11.4.3.  . . . .  Characterization of component sugars. Methylation analysis Oxidation  25 32 36 38 38 39 44 40 50 51 55  V  TABLE OF CONTENTS Page Reduction Base-catalyzed degradation P a r t i a l hydrolysis Location of 0-acetyl group  11.4.4. 11.4.5. 11.4.6. 11.4.7. III  STRUCTURAL INVESTIGATION OF KLEBSIELLA CAPSULAR POLYSACCHARIDES 111.1.  111.2.  65  Structural Investigation of the Capsular Polysaccharide of K l e b s i e l l a K12. ABSTRACT . . Introduction Results and discussion Conclusion Experimental  111.1.1. 111.1.2. 111.1.3. 111.1.4.  Introduction . Results and discussion Conclusion Experimental  66 66 67 76 77  Structural Investigation of the Capsular Polysaccharide of K l e b s i e l l a K58.ABSTRACT. . . 111.2.1. 111.2.2. 111.2.3. 111.2.4.  IV  57 59 59 63  . . .  83 83 84 96 96  111.3.  Confirmation of the Structure of K l e b s i e l l a K23 Capsular Polysaccharide 104  111.4.  "'H and C Spectral Investigation of K l e b s i e l l a K70 Capsular Polysaccharide 108 1 3  BACTERIOPHAGE DEGRADATION OF KLEBSIELLA CAPSULAR POLYSACCHARIDES K21, K12 AND K41  112  IV. 1.  Introduction  113  IV.2.  Results  117  IV.2.1. IV.2.2. IV.2.3. IV.2.4. IV.3.  I s o l a t i o n and p u r i f i c a t i o n Conditions of depolymerization. . . . P u r i f i c a t i o n of analyses of products of depolymerization of K21 P u r i f i c a t i o n and analyses of products depolymerization of K12 and K41. . .  Discussion  117 118 118 122 127  vi TABLE OF CONTENTS Page IV. 4. V.  VI.  Experimental  129  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.  Alternatives  150  BIBLIOGRAPHY  • .  153  vii  APPENDIX  Page  I  The Known Structures of the K l e b s i e l l a Capsular Polysaccharides  174  II  Structural Patterns of K l e b s i e l l a Capsular Polysaccharides  195  III IV  ]  H and  1 3  C n.m.r. Spectra  Methodology of Bacteriophage Propagation and Polysaccharide I s o l a t i o n  200  232  vi.ii LIST OF TABLES TABLE I 2  Page K l e b s i e l l a capsular polysaccharides (K1-K83). 'Quantitative analysis and chemotype grouping  6  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 f o r K l e b s i e l l a K12 capsular polysaccharide and the derived oligosaccharides  70  6  Methylation analysis of n a t i v e , and degraded, K l e b s i e l l a K12 capsular polysaccharide  73  7  N.m.r. data f o r K l e b s i e l l a K58 capsular polysaccharide and the derived oligosaccharides  85  8  Methylation a n a l y s i s of n a t i v e , and degraded, K l e b s i e l l a K58 capsular polysaccharide  89  9  P.m.r. data f o r K l e b s i e l l a K23 capsular polysaccharide  105  10  Methylation analysis of o r i g i n a l and base degraded K l e b s i e l l a K23 capsular polysaccharide  106  II  N.m.r. data f o r K l e b s i e l l a K70 capsular polysaccharide and oligosaccharides i s o l a t e d  109  12  M.m.r. data f o r K l e b s i e l l a 21 capsular polysaccharide and the oligosaccharides PI and P2  123  13  Determination of degree of polymerization of PI and P2 (K21) and i d e n t i f i c a t i o n of the reducing sugar  124  14,  N.m.r. data f o r Klebsiel l a K12 polysaccharide and the phage derived oligosaccharide  125  15  N.m.r. data f o r K l e b s i e l l a K41 polysaccharide and the phage derived oligosaccharide  128  C  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 b a c t e r i a l c e l l envelope  3  2  E x t r a c e l l u l a r polysaccharide  3  3  Cross-reactions of K l e b s 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 l e b s i e l l a K12  27  6  G.I.e. separation of products of methylation analysis of K l e b s i e l l a K12.  28  7  Degree of polymerization of product of bacteriophage degradation of K l e b s i e l l a K21, using g . l . c .  30  8  G . l . c . separation of products of uronic acid degradation of K l e b s i e l l a K12.  31  9  P a r t i a l hydrolysis apparatus  58  10  G . l . c . separation of the products of methylation analysis of K l e b s i e l l a K58  90  11  Morphological grouping of phages according to Bradley  114.  12  Schematic representation of a Type-A p a 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 p u r i f i e d K21 bacteriophage  121  16  Separation of monosaccharides using HPLC (Column A) Separation of monosaccharides using HPLC (Column B)  144  17  145  xi LIST OF FIGURES FIGURE  Page  18  Separation of d i - and t r i s a c c h a r i d e s using HPLC, with d i f f e r e n t 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 l e b s i e l l a K21 bacteria  236  xii LIST OF SCHEMES SCHEME  Page  1  Antibody production and the b a c t e r i c i d a l reaction  11  2  Phagocytosis  16  3  Primary and secondary fragmentation pattern and mass spectrum of 1,5 D i - O - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 methyl-Q-glucitol  33  4  Methylation analysis of a polysaccharide  52  5  S e l e c t i v e oxidation and degradation  56  6  Reduction of a carboxylic acid i n aqueous s o l u t i o n using a carbodiimide reagent  58  7  Base-catalysed degradation of K l e b s i e l l a Kl2  60  8  P a r t i a l hydrolysis and p u r i f i c a t i o n of K l e b s i e l l a Kl2  62  9  Base-catalysed degradation of K l e b s i e l l a K58  92  10  Periodate oxidation of K l e b s i e l l a K58  93  11  P a r t i a l hydrolysis of K l e b s i e l l a K58  95  12  Block diagram of instrumentation f o r high pressure l i q u i d chromatography  137  13  I s o l a t i o n and p u r i f i c a t i o n of polysaccharide  234 i  xi i i  ACKNOWLEDGMENTS  The stimulating d i r e c t i o n of Professor G.G.S. Dutton and the cheerful support of my colleagues, i n p a r t i c u l a r Jose Di Fabio, are g r a t e f u l l y acknowledged.  I wish to thank Robert S t , - P i e r r e f o r  the i l l u s t r a t i o n s , Dr. E.H. M e r r i f i e l d for proof reading, and Celine Gunawardene f o r typing t h i s t h e s i s . I am grateful to MacMillan Bloedel f o r the award of a graduate scholarship (1977-1978).  xiv PREFACE This thesis has been w r i t t e n i n 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 s t r u c t u r e , along with s p e c i f i c degradations, of carbohydrate antigens.  Many of the methods used in t h i s 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 s u f f i c i e n t a p p l i c a t i o n s to polysaccharide chemistry reported in the l i t e r a t u r e to warrant a review of these methods.  Therefore, a more d e t a i l e d  account of the use of nuclear magnetic resonance spectroscopy 1 (both  13  H and  C) in the study of polysaccharides i s presented here.  I t i s proposed that future theses emanating from t h i s group w i l l s i m i l a r i l y review g a s - l i q u i d chromatography, mass spectrometry, and high performance l i q u i d 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 l e b s i e l l a polysaccharides along with l i t e r a t u r e references, compiled by Keith Mackie (Ph.D. 1977).  Bacteriophage attack s i t e s , o p t i c a l  and x-ray crystallography references have also been included.  rotations, In his  M.Sc. thesis (1980) Marcel Paul in c l a s s i f i e d the known K l e b s i e l l a structures according to t h e i r s t r u c t u r a l patterns. This has been revised and included i n 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 p r i m a r i l y chemical in nature. As has been the p r a c t i c e with other theses presented by members of our group an explanation of carbohydrate nomenclature " i s now offered 4  to f a m i l i a r i z e readers who are not acquainted with the f i e l d . i Fischer projection formulae are used to represent the a c y c l i c m o d i f i c a t i o n . o f sugars.  Some examples are shown below.  Numbering  commences from the carbonyl group at the top of the chain ( I ) . Note that D-glucuronic acid ( I I ) d i f f e r s from Q-glucose (I) only  CHO  CHO  CHO  1  2  -  H O -  -  OH  5  ~  OH  C H  - O H  ho-\  3  4  6  LoH  OH  2  D-glucose (I)  0 H  - O H  - O H  HO  -  - O H  HO  -  COOH  D-glucuronic (II)  CH.  acid  in that C-6 i s oxidized to a carboxylic acid group.  L-rhamnose (III)  The C-6 of  ^-rhamnose ( I I I ) i s part of a methyl group and i s referred to also by another common name, 6-deoxy-L-mannose.  XVI  There are four c h i r a l centers in these six-carbon chains (marked with a s t e r i s k s in structure I I I ) making i t important to appreciate the s p a t i a l arrangement of atoms (configuration) i s implied by these Fischer representations.  that  To s i m p l i f y the  nomenclature of a l l the possible isomers (16 f o r each of I , I I , I I I ) , a l l those having the hydroxyl group at the highest-numbered c h i r a l center (C-5) projecting to the r i g h t in the Fischer p r o j e c t i o n formulae belong to the D-series, and the others to the L - s e r i e s .  HCH  D-series  L-series  Physical and chemical evidence indicates t h a t , in f a c t , these six-carbon polyhydroxyaldehydes e x i s t . i n a c y c l i c form.  The r i n g  closure occurs by n u c l e o p h i l i e attack of the oxygen atom at C-5 on the aldehydic carbon atom, generating a new c h i r a l center at C - l . H  (anomeric)  This r e s u l t s in two anomers, represented below  OH  HO  H  C t_0H  | _ OH HO _ J  HO—I  0  \—0H  I—OH  CH 2 0H  0  o-D-glucose  CH 2 0H  0-D-glucose  (IV.)  (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 i m i t a t i o n s with t h e i r unequal bond lengths, Haworth developed a perspective method of looking at the six-membered r i n g (VI and V I I ) .  This improvement  recognizes t h a t the r i n g oxygen atom.lies behind the carbon chain and t h a t bond lengths are approximately equal.  Often i n . p r a c t i c e regular  hexagons are used in Haworth p r o j e c t i o n s ,  OH  OH  a-D-glucopyranose  p-D-glucopyranose  (VI)  (VII)  pyran (VIII)  which he related to such rings at the heterocyclic compound pyran ( V I I I ) and named them pyranoses.  Note t h a t hydroxyl groups not involved in r i n g  formation on the r i g h t in Fischer and Tollens formulae point down i n the Haworth projections and those on the l e f t point up.  Similarly, for  aldopyranoses, the group on C-5 points up f o r D(IX') and down f o r the L enantiomer (X).  I t f o l l o w s , then, that when sugar residues are attached  there are two possible c o n f i g u r a t i o n s , an a - or a 3-pyranoside, f o r each linkage.  xvi i i  OH  HO  OH  HI  HO  OH  a-D-rhamnopyranose (IX)  The true conformation of pyranoid carbohydrates is related to the chair form of cyclohexane.  X-ray diffraction analysis has  shown that a hexose* such as a-D-glucose ( X I ) , 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 ring, except for that at C-l, are equatorial.  OH (XI)  Two isomers (anomers) are possible in relation to the anomeric center ( C - l ) , depending on whether a substituent is axial  xi x\ (a-anomer; XII)  or equatorial (g-anomer; X I I I ) , where R = hydrogen,  f o r monosaccharides, and R = another sugar residue, f o r d i - , o l i g o - , and polysaccharides.  Since H-l i s i n d i f f e r e n t chemical environment  f o r the two anomers, nuclear magnetic resonance spectroscopy can e a s i l y d i s t i n g u i s h between them and, thereby, provides invaluable assistance in assigning anomeric c o n f i g u r a t i o n s .  Haworth projections are most useful and w i l l be used in t h i s thesis., even though, they give no i n d i c a t i o n of threedimensional molecular shape. There seems to be l i t t l e  justification  f o r the use of formulae which depict states of molecules as well as s t r u c t u r e s , when the true states are often unknown or v a r i a b l e .  + The Carbohydrates. Chemistry and Biochemistry V o 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 e n t i t l e d " S t r u c t u r a l Studies on K l e b s i e l l a Capsular Polysaccharides", U n i v e r s i t y of B r i t i s h Columbia, A p r i l 1979.  1  I.  INTRODUCTION  2  I.  INTRODUCTION  P o l y s a c c h a r i d e s are u b i q u i t o u s - they are by f a r the most abundant biopolymers on e a r t h . arabic  1  Those such as c e l l u l o s e  2 3  '  and gum  4 5 ' 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 e x o p o l y s a c c h a r i d e produce by Xanthomonas j u g l a n d i s has become o f economic value i n enhanced o i l  r e c o v e r y systems  ^,  8 9 and a l g i n a t e  '  , o b t a i n e d from c e r t a i n species o f marine algae  c o m m e r c i a l l y i m p o r t a n t as a food a d d i t i v e .  is  1 0  Research i n t e r e s t s had, f o r many y e a r s , c e n t e r e d on p l a n t p o l y s a c c h a r i d e s , and l a t e r , on mucopolysaccharides o f h i g h e r but the c o m p a r a t i v e l y r e c e n t r e a l i z a t i o n t h a t m i c r o b i a l  animals,^  polysaccharides  are composed o f r e g u l a r r e p e a t i n g u n i t s , along w i t h t h e f a c t t h a t  they  p l a y an i m p o r t a n t r o l e i n fundamental research on the immune r e a c t i o n , have prompted the s y s t e m a t i c i n v e s t i g a t i o n o f t h e s t r u c t u r e s o f the exopolysaccharides of various Microbial  families. 1g  polysaccharides  are l o c a t e d on the c e l l  surface  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 i g h e r organism t o m i c r o b i a l e i t h e r an i n t e g r a l  infection.  p a r t o f the c e l l w a l l  The p o l y s a c c h a r i d e s  - lipopolysaccharide  or occur as a s l i m e o r capsule as i n the case o f Pneumococcus, c o l i , and K l e b s i e l l a (see F i g .  are  (LPS) Escherichia  1).  " C a p s u l a r " p o l y s a c c h a r i d e i s c o n s i d e r e d t o have a d e f i n i t e boundary and t o remain adherent t o t h e c e l l w a l l when suspended i n  3  GRAM + POLYSACCHARIDE CAPSULE (K)  LIPOPOLYSACCHARIDE COMPLEX  (0)  PEPTIDOGLYCAN PERIPLASM CELL MEMBRANE  Figure 1  Diagramatic representation of the b a c t e r i a l c e l l envelope  LPS (0 Antigen) CAPSULE (K Antigen) SLIME  Figure 2  E x t r a c e l l u l a r polysaccharide.  4  water.  The term "slime" i s used to i n d i c a t e a network of carbohydrate  f i b r e s 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 i d e n t i c a l 17 13  i n chemical composition.  5  .  Due to the high water content of the  capsule (99%) only wet preparations, such as India ink  f i l m s , can  give accurate information about the s i z e and shape of the capsule or slime.  When water i s removed from capsules of K l e b s i e l l a b a c t e r i a ,  by means of . e t h a n o l , for v i s u a l i z a t i o n by electron microscopy, the individual f i b r i l s  of the capsule collapse on one another to form  thick p r o j e c t i o n s .  Occasionally there i s some evidence f o r peripheral  l i n k i n g of f i b r e s .  This would f i t well the idea of a w e l l - d e f i n e d  capsular edge (see F i g . 2). P r a c t i c a l l y a l l b a c t e r i a l capsules consist of polysaccharides and are associated with pathogenic micro-organisms.  The genus  K l e b s i e l l a i s composed of Gram-negative, nonmotile b a c t e r i a , of the 19 20 family Enterobacteriaceae  and the t r i b e K l e b s i e l l e a e  '  .  I sol ants  are i d e n t i f i e d 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 s t r a i n s of K l e b s i e l l a , one in the capsule (K antigen) and the other in the soma, (see F i g . 1). The capsular antigen was shown to be carbohydrate in nature by pi  po  Toenniessen -' - - in 1914. Since most K l e b s i e l l a bacteria are heavily t  ,t  1  encapsulated the 0-antigen i s completely shielded (see Figs. 1 and 2 ) . Consequently, s e r o l o g i c a l c l a s s i f i c a t i o n i s based s o l e l y on t h e i r capsular K-antigens.  To date seventy seven s e r o l o g i c a l l y  different  5  23 24 capsules have been delineated ' . Many of these micro-organisms are found in healthy c a r r i e r s 25 in the upper r e s p i r a t o r y , i n t e s t i n a l , and genito-urinary t r a c t s Their pathogenicity to man i s well known although some s t r a i n s are not t o x i c .  Acute i n f e c t i o n i n the lung o c c a s i o n a l l y mimics pneumococcal  lobar pneumonia. The chronic form resembles t u b e r c u l o s i s .  Klebsiella  pneumonia K-types 1, 2, 7 and 8 are the commonest invaders and are responsible for approximately three per cent of a l l b a c t e r i a l pneumonias, 26 occurring when host-resistance i s impaired, for example, in a l c o h o l i c s  .  Almost a l l s t r a i n s of K_. ozaenae are members of K type 4, and are associated with ozena, a  f e t i d , catarrhal condition of the nose.  Infections due to K_. rhinoschleromatis occur r a r e l y in North America. K l e b s i e l l a r e s i s t s many antimicrobiiC drugs. chloramphenicol  Streptomycin and  have proved of value i n therapy, but the proportion  of r e s i s t a n t s t r a i n s , due to mutations, i s increasing s t e a d i l y . One of the most outstanding features of b a c t e r i a l polysaccharides to become apparent of l a t e i s that they are composed of regular 27 28 repeating units , as shown by molecular weight d i s t r i b u t i o n studies^ , and more recently by nuclear magnetic resonance spectroscopy..  29  A l l are heteropolysaccharides and have proved to be an almost inexhaustible source of novel oligosaccharides and sugars. 30 31 Nimmich  has reported the q u a l i t a t i v e composition of the  K l e b s i e l l e K-types and has also c l a s s i f i e d them into chemotypes (see Table 1).  32  6  Glucuronic A c i d ,  Galactose, Glucose  8 , l l , 15, 51, 25, 2 7 P  Glucuronic A c i d , Galactose, Mannose  p  p  20, 2 1 , 2 9 , 4 2 , 43, 66, p  74  p  p  p  Glucuronic A c i d , Galactose, Rhamnose  9, 47, 52, 9*, 81, 83  Glucuronic A c i d , Glucose, Mannose  2, 4, 5 , 24  Glucuronic A c i d , Glucose, Rhamnose  17, 44, 71, 23, 4 5  Glucuronic A c i d , Glucose, Fucose  1, 54  Glucuronic A c i d , Galactose, Glucose, Mannose  10, 28, 39, 50, 59, 61, 62  P  p  7 , 13 , 26 , 30 , 31 , p  p  p  p  p  3 3 , 3 5 , 4 6 , 6 9 , 60 p  p  P  P  Glucuronic A c i d , Galactose, Glucose, Fucose  16, 5 8  Glucuronic A c i d , Galactose, Glucose, Rhamnose  18, 19, 1 2 , 4 1 , 79, 7 0 ,  p  P  36 , p  P  55 . p  Glucuronic A c i d , Galactose, Mannose, Rhamnose  53, 40, 80  Glucuronic A c i d , Glucose, Mannose, Fucose  6  Glucuronic A c i d , Glucose, Mannose, Rhamnose  64 , 65  Glucuronic A c i d , Galactose, Glucose, Mannose, Fucose  68  p  P  p  p  p  Glucuronic A c i d , Galactose, Glucose, Mannose, Rhamnose 1 4 , 67 p  Galacturonic A c i d , Galactose, Mannose  3 , 49, 57  Galacturonic A c i d , Glucose, Rhamnose  34, 48  Galacturonic A c i d , Galactose, Fucose  63  Pyruvic A c i d , Glucose, Rhamnose  72  Pyruvic A c i d , Galactose, Rhamnose  32  Pyruvic A c i d , Galactose, Glucose, Rhamnose  56  Keto A c i d , Galactose, Glucose  22, 37, 38  P  24 K82 has been added but i t s q u a l i t a t i v e composition i s not yet known. P- Pyruvic acid present in addition Note: K9 and K9*, see Appendices I and II TABLE 1 ;  K l e b s i e l l a capsular polysaccharides  (K1-K83)  Quantitative a n a l y s i s 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 furanosyl form of D-galactose occurs in Kl2  34 and K 41  . In some  strains the 6-deoxyhexoses L-rhamnose and L-fucose are found; noncarbohydrate 0-acetyl and 0-formyl groups may also occur. To date structures have been proposed for f i f t y 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 'imbalance has been attributed 7  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 microorganisms appear to be unable to depolymerize or to u t i l i s e t h e i r own e x t r a c e l l u l a r polysaccharides as carbon sources.  The f o l l o w i n g  functions have, however, been proposed by Dudman. (a) Virulence - protection against serum b a c t e r i c i d a l  factors  and phagocytosis (b) Protection against predation (c) Protection against desiccation (d) Adhesion i n aqueous environments (e) Role i n dental cariogenesis (f) Role i n i o n i c  interactions  (g) Role as general b a r r i e r s (h) Role i n enzyme reactions ( i ) Role in s i l i c o n 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 t e r t i a r y s t r u c t u r e , both in 39  solutions and gels and by x-ray d i f f r a c t i o n . Progress i n the l a t t e r f i e l d has been made mainly due to the development of improved c r y s t a l l i z a t i o n techniques by Atkins  40  and the concomitant  development of s o p h i s t i c a t e d computer programmes for analysing the 41 d i f f r a c t i o n data obtained.  In his review of 1979 Atkins  offers  stereochemical models f o r eight K l e b s i e 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 o n r e s u l t s help d i f f e r e n t i a t e between two proposed s t r u c t u r e s .  9  In dealing with b a c t e r i a l polysaccharides as antigens two aspects have  to be considered.  ".  ,  One i s t h e i r capacity to induce the  formation of antibodies in mammals i . e .  t h e i r IMMUNOGENICITY, and the  other i s t h e i r r e a c t i v i t y with antibodies, i . e .  t h e i r ANTIGENIC SPECIFICITY.  Our present knowledge in t h i s f i e l d i s based to a large extent on the pioneering work of Heidelberger and coworkers at the R o c k e r f e l l e r Institute  45-47  and of Kabat _et _al,.  48  .  The former i n i t i a t e d  q u a n t i t a t i v e studies in immunochemistry, while i t i s l a r g e l y 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 s e r o l o g i c a l and chemical s t u d i e s , i s to define oligosaccharide structures w i t h i n the polysaccharide as chemical expression of i t s immunological character.  According to a  49 proposition of Staub and Heidelberger  the sugar unit that contributes  most to the s e r o l o g i c a l s p e 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 u n i t , i t s substituents ( i f any) and the anomeric  configuration of i t s linkage, but also the p o s i t i o n to which i t  is  linked may greatly influence the antigenic expression of the determinant. In a c i d i c polysaccharides, such as those of K l e b s i e l l a , t h e charged constituents are often  immunodominant sugars or part of antigenic  50 determinants  .  Due to t h e i r r e p e t i t i v e s t r u c t u r e , b a c t e r i a l  polysaccharides have the same antigenic determinants expressed many times over (see F i g . 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 i t s homologous antibody in different polysaccharides.  Therefore these polysaccharides, no matter in what  organism they are produced, are immunologically related; they crossreact serologically  c p  (see Figs. 3, 4).  11  O—D> O—O O—> OR  I: Cross-linking and Acti vation  [  •  ( i ) IgA + IgG + IgM  1  (ii)lgM  + Complement  Antibody Production  Inactivated by Polysaccharide Charge  CELL LYSIS  INFECTION  Ii •c  Polysaccharide -  -< o Scheme 1  Antibody production and the bactericidal reaction.  T cell  receptor  B cell  receptor  = Carrier Hapten  12  KIT  K12  3  a  f^Gali-^Galfl-^lc^RhaMl^ a — 3« OL ' J  |_  a  n  1 GlcA  Pn VI  Figure 3  f - ^ a l M'61c-—^Rha^-hib.i.tol L '. a  - PO, Na  K l e b s i e l l a K12 capsular polysaccharide cross-reacts with anti-serum to K l e b s i e l l a K l l and also with a n t i Pneumococcus VI  13  Recently Heidelberger and colleagues  have examined  cross-reactions between the capsular polysaccharides of K l e b s i e l l a and pneumococcus and subsequently made elegant predictions on substructures of K l e b s i e l l a polysaccharides. Most of the work to date has involved heterogeneous antibodies, that i s to say, sera containing antibody populations, that although s p e c i f i c f o r one antigen, are made up of d i f f e r i n g molecular species of immunoglobulins. These may bind the same antigenic grouping (hapten)*  in d i f f e r e n t ways, or d i f f e r e n t  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 potential i n polysaccharide immunochemistry i s provided by homogeneous immunoglobulins that bind carbohydrate 57 polymers,  .  Here, the s p e c i f i c immunoglobulin - hapten i n t e r a c t i o n  can be characterized in d e t a i l .  Thus the d i s c i p l i n e of carbohydrate  chemistry can make a real contribution to the e l u c i d a t i o n of the structure of immunoglobulins.  In r e t u r n , carbohydrate chemistry  may f i n d a tool that can i n c r e a s i n g l y be applied to the unraveling of i t s own unsolved problems i n the s t r u c t u r a l a n a l y s i s of polysaccharides. The fate of a host which i s invaded by micro-organisms depends on the effectiveness 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 i n h i b i t e d thus enabling the micro-organisms to m u l t i p l y to such an extent as to eventually damage or k i l l the host. This phenomenon i s c a l l e d VIRULENCE. The r e l a t i o n s h i p 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 r a i n s of a pathogenic species, but i t has been shown that capsulated, v i r u l e n t s t r a i n s of pathogenic b a c t e r i a generally become less v i r u l e n t when they lose t h e i r capsules. The best understood defence reactions with which microbial  polysaccharides are known to i n t e r a c t are those that  15 involve p r i m a r i l y c i r c u l a t i n g antibody and the complement system, v i z . , the b a c t e r i c i d a l reaction and phagocytosis.  In the former, the  i n t e r a c t i o n of serum antibody with i t s antigenic determinant activates the complement system (see Scheme 1).  Practically all  capsular  polysaccharides are a c i d i c and they i n a c t i v a t e complement, probably 59 through t h e 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 bacteria by macrophages and leukocytes (see Scheme 2).  The antiphagocytic  e f f e c t of capsular polysaccharides i s probably not s p e c i f i c , charge and v i s c o s i t y of the capsular material being p r e r e q u i s i t e s . •It has been shown that anti-polysaccharide antibodies are protective against c e r t a i n i n f e c t i o n s .  Heidelberger ^0-62 - j  m m u n  -j  z e c  j  humans with pneumococcal polysaccharides, providing a s i g n i f i c a n t degree of protection in an epidemic in which the corresponding pneumococci were involved. Protective immunization may be performed not only with bacterial vaccines and i s o l a t e d polysaccharides, but also with a r t i f i c i a l antigens, thus avoiding the possible t o x i c e f f e c t s of b a c t e r i a . The task of producing a r t i f i c i a l antigens i n large q u a n t i t i e s i s indeed arduous. The fact that monosaccharides are m u l t i f u n c t i o n a l and may be l i n k e d in e i t h e r an a or a e configuration presents an enormous challenge to the synthetic carbohydrate chemist. Consequently, only a very small proportion of the possible number of disaccharides has been synthesised synthetic procedures are progressing r a p i d l y  , and although 64  only a l i m i t e d  16  INFECTION  KILLING OF  Scheme 2  AND DIGESTION BACTERIUM  Phagocytosis.  PHAGOCYTOSIS  17 number of synthetic oligosaccharides has been r e a l i s e d  '  .  An a l t e r n a t i v e , then, i s to look to the p o s s i b i l i t y of obtaining oligosaccharides from the degration of polysaccharides. fi7  In 1969 a method was described  f o r the hydrolysis of polysaccharides,  according to which the o l i g o s a c c h a r i d e s , 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 n o n - s p e c i f i c and many d i f f e r e n t oligosaccharides are produced, and subsequent separation may prove d i f f i c u l t . To obtain hydrolysis of polysaccharides at s p e c i f i c points attention has been turned by Stirnv. et al_ ^9-71 d p iymerization t Q  with the aid of bacteriophage glycanases.  e  0  By t h i s method  heteropolysaccharides c o n s i s t i n g of repeating units may be s p e c i f i c a l l y cleaved e i t h e r by the whole bacteriophage, or the p u r i f i e d enzyme, to y i e l d a homologous series of the s i n g l e repeating unit and 72 multiples thereof.  I t has been pointed out by Dutton  that t h i s  i s the only method of obtaining an oligosaccharide with an i n t a c t a c i d l a b i l e pyruvate group. To obtain the immunogen the haptenic oligosaccharide i s coupled to an immunologically e f f i c i e n t c a r r i e r p r o t e i n . and Avery's method  Goebel  73 74 ' of preparing the conjugates via aminophenyl  18 glycosidation of the reducing sugar end groups was extensively used and was not improved upon for about three decades, although i t s a p p l i c a t i o n was almost e x c l u s i v e l y r e s t r i c t e d to mono- and d i s a c c h a r i d e s . . 75-77 Newer methods of coupling  have no such l i m i t a t i o n and appear to  be suited to v i r t u a l l y any reducing oligosaccharide and also to any chain length. Microbial polysaccharides have also shown potential in the 78 area of cancer research . I t has been known f o r more than a century that human malignant growths sometimes undergo regression following 79 an acute,  bacterial infection  .  Since then extensive studies have  been made on noncytotoxic and host-mediated antitumor polysaccharides o n  from various sources.  It has been reported  o i  '  that polysaccharide  complexes from Klebsiella,among others, were a c t i v e against s o l i d tumors.  Many questions remain unanswered and the role of the  polysaccharides as immunopotentiators i s being e s p e c i a l l y debated. In Chapter II the methodology of the s t r u c t u r a l of polysaccharides i s examined.  analysis  Advances in chemistry are i n v a r i a b l y  aided by progress i n instrumentation.  Recent developments are discussed,  then, with special reference to n.m.r. spectroscopy.  To date the  a p p l i c a t i o n of high performance l i q u i d chromatography to carbohydrate analysis has been l i m i t e d . Therefore, t h i s technique i s examined in Chapter V and i t s potential as a tool in the s t r u c t u r a l  elucidation  of polysaccharides and in obtaining large q u a n t i t i e s of oligosaccharides i s examined.  19 I f continued advances are to be made in the area of immunochemistry i t i s c l e a r that co-operation between the chemist and the immunologist i s e s s e n t i a l .  For the l a t t e r to understand  complex immunological r e a c t i o n s , w e 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 t h e i r degradation; by bacteriophage - - are examined i n Chapters III and IV of t h i s t h e s i s .  20  II  METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES  21  II.  METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES To determine the complete structure of a polysaccharide the  constituent sugars must be i d e n t i f i e d , and t h e i r r a t i o , s u b s t i t u e n t s , sequence, linkage pattern and linkage configuration ascertained. The 82 chemistry of polysaccharides has been reviewed by Aspinall and Stephen and-Aspinall  .  Recent theses from our group (Paulin 1980, Folkman 1979  and Mackie 1977) have surveyed the methodology of s t r u c t u r a l of polysaccharides.  determination  In the following 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. ' II.  1.  I s o l a t i o n and P u r i f i c a t i o n Samples of K l e b s i e l l a bacteria of s p e c i f i e d serotype and  s t r a i n number were received as (Copenhagen).  stab cultures from Dr. Ida (|)rskov  B a c t e r i a l cultures were streaked on agar plates at  37°C u n t i l l a r g e , i n d i v i d u a l capsular colonies were obtained. Bacteria were grown by innoculation of beef-extract medium with a s i n g l e colony f o r 3h at 37°, with shaking.and subsequent incubation of t h i s l i q u i d culture on a tray of sucrose - yeast e x t r a c t - agar for three days.  The lawn of capsular bacteria produced was harvested by  scraping from the agar surface, and the bacteria destroyed with a 1%. phenol s o l u t i o n . of t h i s s o l u t i o n .  The polysaccharide was i s o l a t e d by u l t r a - c e n t r i f u g a t i o n The viscous honey-coloured supernatant was  p r e c i p i t a t e d into ethanol.  The p r e c i p i t a t e was dissolved i n water and  22 was treated with CETAVLON  84  (cetyltrimethylammonium bromide) and  centrifuged to i s o l a t e only the a c i d i c polysaccharide. The CETAVLONpolysaccharide complex was dissolved in 4M sodium c h l o r i d e , p r e c i p i t a t e d into ethanol, redissolved in water, and dialyzed against running tapwater f o r two days.  The polysaccharide was i s o l a t e d , as a styrofoam-like  m a t e r i a l , by l y o p h i l i z a t i o n , and was shown to be homogeneous by electrophoresis on c e l l u l o s e acetate s t r i p s and by nuclear magnetic resonance spectroscopy, (see App. IV). II.  2.  Separation Techniques To obtain information on the constituent sugars of the  polysaccharide i t s e l f and of i.ts.j degrada.tion products, the Tatter must v  be separated, p u r i f i e d and then checked f o r homogeneity.  Separation  i s obtained by the established technique of paper chromatography,by' gel chromatography and paper electrophoresis and by newer techniques such as high pressure l i q u i d chromatography. QC  o r  II.  2. 1.  Paper chromatography ( p.c.) :  '  The great value of t h i s technique l i e s in i t ' s a b i l i t y to separate mixtures of monosaccharides^and oligosaccharides simply, accurately and without d e r i v a t i z a t i o n , by employing d i f f e r e n t solvent systems.  For a n a l y t i c a l analysis very small amounts of material are  needed, while the technique may also be employed on a preparative l e v e l . A c i d i c components may be distinguished from neutral components by using d i f f e r e n t solvent systems. Paper chromatography i s used for i d e n t i f y i n g constituents  23  e i t h e r as the free sugars or as a l d i t o l s  87  from hydrolysis of the  native polysaccharide and of i t s degradation products.  Methylated  88 sugars and oligomers may also be i d e n t i f i e d  . P.e. i s also used f o r  monitoring c o n t r o l l e d hydrolysis of the polysaccharide and f o r checking the composition of f r a c t i o n s obtained from gel chromatography of mixtures from p a r t i a l hydrolysis and periodate o x i d a t i o n .  When pure  oligomers are not obtained by gel chromatography preparative paper chromatography may be employed.  Although tedious and time consuming,  good r e s u l t s may be obtained, as in the analysis of K58 polysaccharide, where large q u a n t i t i e s of the aldobi - , t r i - , and tetrauronic acids were obtained. NaOH / N a S 0 2  2  3  Sugars are detected with e i t h e r ( i ) Ag NOg/ e t h a n o l - or. with  ( i i ) p-anisidine-hydrochloride  in  aqueous 1- butanol followed by heating at 110° f o r 5 m i n . ^ I I . 2. 2.  Paper electrophoresis  (p.e.) > 8 6  9 1  >  92  Large a c i d i c oligomers move very slowly on paper chromatography.  Paper e l e c t r o p h o r e s i s , however provides an  a l t e r n a t i v e convenient method f o r t h e i r examination.  Good  separations can be achieved in a number of hours. Electrophoresis involves the migration of charged substances i n a conducting s o l u t i o n under the influence 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 e x i s t in a charged s t a t e .  The usual coolant used i s kerosene. P.e.  may be employed both a n a l y t i c a l l y and p r e p a r a t i v e l y . 89 90 detected as f o r paper chromatography ' .  Sugars are  24 I I . 2. 3.  Gel chromatography ( g . c . ) Gel chromatography o r i g i n a t e d 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 s t a r c h . 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 chromatography i t i s based on the decreasing permeability of the three dimensional network of a swollen gel to molecules of increasing size.  As the order of e l u t i o n of a series of s i m i l a r substances  from a gel column i s governed l a r g e l y by molecular weight, gel chromatography provides a means of determining molecular weights of polymers. This technique has been used extensively f o r the separation of products of p a r t i a l hydrolysis and periodate o x i d a t i o n . SEPHADEX and BIO-GEL gels have been used in t h i s study. when  Both  In cases  the molecular exclusion l i m i t of two d i f f e r e n t gels  (eg. G-15 and P-2) i s the same,the e l u t i o n p r o f i l e and the order of e l u t i o n may not n e c e s s a r i l y be the same, and t h i s may be used to advantage to obtain  , a clean separation.  The p a r t i c l e s i z e grade  (superfine to coarse) should also be taken into account in choosing a gel.  BIO-GEL has the advantage of being r e s i s t a n t to b a c t e r i a l  contamination since i t i s a synthetic m a t e r i a l .  Wet sephadex  (derived by c r o s s l i n k i n g dextran) should be stored i n a s o l u t i o n 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 - a c e t i c acid 1000: 10: 4.  Carbohydrate f r a c t i o n s from the column are f i r s t l o c a l i z e d using 95 the Molisch t e s t . To determine the e l u t i o n p r o f i l e a q u a n t i t a t i v e * 96 c o l o r i m e t r i c technique such as the p h e n o l - s u l f u r i c assay may be used, or i n d i v i d u a l f r a c t i o n s may be l y o p h i l i z e d and weighed. are used to i n v e s t i g a t e the composition of f r a c t i o n s .  R.c. and p.e. Where homogeneity  i s not achieved', p u r i f i c a t i o n may be obtained with preperative p.c. or p.e. Molecular - weight d i s t r i b u t i o n studies of a c i d  hydrolysis  products from K l e b s i e l l a K54 exopolysaccharide by Churms and Stephen on BIO-GEL P-10 gave evidence f o r repeating units in the structure  2 8 9  ' .. b  Sephadex LH-20 (G-25 with most of the -OH groups  alkylated)  may be used s u c c e s s f u l l y to p u r i f y large molecular weight carbohydrate material that i s soluble in organic solvent, eg. permethylated or peracetylated polysaccharide.  II.  3.  Instrumentation  II.  3. 1.  G a s - l i q u i d chromatography  (g.l.c.)  Extensive reviews of the applications 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 e n t l y v o l a t i l e to be used f o r g . l . c . and they must therefore be converted into v o l a t i l e compounds.  26 The f a c t that each monosaccharide may give more than one peak owing to the formation of anomeric d e r i v a t i v e s has l e d means to eliminate t h i s complication.  to a search f o r  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 a c e t y l a t i o n then y i e l d s  volatile  derivatives. I d e n t i f i c a t i o n of the constituent sugars of an oligosaccharide or polysaccharide i s achieved by h y d r o l y s i s , reduction, a c e t y l a t i o n and g . l . c . a n a l y s i s . SP-2340 (75% cyanopropyl s i l i c o n e ) i s the s t a t i o n a r y phase of choice f o r analysis of a l d i t o l acetates (see F i g . 5).  ECNSS-M (ethylenesuccinate - cyanoethylsilicone  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 l a t e r ) OV-225 (25% phenyl, 25% cyanopropyl, methyl s i l i c o n e ) (see F i g . 6) and OV-17 (50% phenyl, methyl s i l i c o n e ) 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, g i v i n g shorter retention times and n e g l i g i b l e column bleeding, hence constant retention parameters. been used to separate d i - and  OV-225 has also  trisaccharide derivatives  (methyl s i l i c o n e ) i s used for the analysis of permethylated oligosaccharides.  0V-1  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 l e b s i e 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 l e b s i e 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 trimethyl s i l y l  (TMS)  ether d e r i v a t i v e s  1 0 1  may be used to advantage, employing SE-52 (5% phenyl • methyl s i l i c o n e ) as s t a t i o n a r y phase.  The ease with which the o r i g i n a l material may  be recovered f o l l o w i n g d e r i v a t i z a t i o n makes the use of TMS d e r i v a t i v e s a t t r a c t i v e where the amounts of material are l i m i t e d . To determine the degree of polymerization of an oligosaccharide and to i d e n t i f y the reducing sugar the a l d o n o n i t r i l e method of Morrison 102 (see l a t e r ) i s used  .  A l d o n o n i t r i l e s give sharp s i n g l e peaks on  g . l . c . and may be analyzed with 0V-17 (see F i g . 7 ) , OV-225 or ECNSS-M. Publicatons by Albersheim et al_  1 0 3  '  1 Q 4 ;  and. Lindberg et  al  1 2 2  l i s t the r e l a t i v e retention times f o r a large number of p a r t i a l l y methylated a l d i t o l acetates.  I d e n t i f i c a t i o n of unknowns i s achieved  by consideration of the r e l a t i v e 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 e x i s t s the melting point (m.p.) of the sample (from preparative g . l . c . ) may provide i d e n t i f i c a t i o n ( 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  r e a c e t y l a t i o n gives a c r y s t a l l i n e a l d i t o l hexaacetate which may be i d e n t i f i e d by m.p. or by g . l . c . retention time. Samples c o l l e c t e d by preparative g . l . c . are used also for mass spectral studies and f o r c i r c u l a r dichroism measurements (see l a t e r ) . P a r t i a l l y e t h y l a t e d , 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 l d o n o n i t r i l e s  PAAN Peracetylated alditol  Man  -H  1  0  4  Figure 7  1 8  1 12  r— 16  1 20  Time (min)  Degree of polymerization of product of bacteriophage degradation of K l e b s i e 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  Figure 8  G . l . c . separation of products of uronic acid degradation of K l e b s i e l l a K12.  Time (min)  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 r a v e l more q u i c k l y than t h e i r methyl analogues (see F i g . 8 ) . nonpolar l i q u i d phases retention times are longer.  Conversely, with Mass spectrometry  i s invaluable in i d e n t i f i c a t i o n of p a r t i a l l y ethylated d e r i v a t i v e s (see l a t e r ) . For q u a n t i f i c a t i o n of peaks molar response factors  are  taken into consideration. It should be noted that phthalic 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 f e r e n t i a t e the  contaminant from sugar d e r i v a t i v e s .  II.  3.  2.  Mass Spectrometry (m.s.)  The mass spectral method was f i r s t applied to carbohydrate d e r i v a t i v e s in 1958 by Reed and coworkers  , and since then, i t  has become an important and v e r s a t i l e technique i n carbohydrate chemistry.  Lonngren and Svensson reviewed the f i e l d i n 1974  .  In  t h i s study m.s. has been used i n the analysis of p a r t i a l l y methylated/ ethylated a l d i t o l acetates i n order to assign s u b s t i t u t i o n patterns (see l a t e r , Scheme 3 ) , and in the analysis of oligosaccharides to determine the sequence of sugars. Mass spectra can be recorded by using any one of several  33  Cr^OAc HCOMe  117  MeOCH J ™ ^ JL Y" H i0Me HCOMe 1| 1 HCOAc HCOAc 4 „ „ CH^OMe  I O I « - A c O H - l161 6J CH^O I | 71  100  205  AcOH—I>I45  '161 61 - M e O H ^ 1 2 9 -CH C0 I 2  I  4  87  5  .43  80 H 101  60 to  40ti 4 5 20  40 Scheme 3  7 129  |6I 145  71  8  |  205  7  L1  100  J  m/e  I  160  220  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 f e r e n t systems of instrumentation.  The i n l e t system can be  e i t h e r a hot r e s e r v o i r i n l e t , a d i r e c t probe i n l e t , or a g . l . c . inlet.  The l a t t e r has become i n c r e a s i n g l y important i n the  i n v e s t i g a t i o n of complex mixtures.  Most underivatized mono- and  oligosaccharides are thermally unstable and n o n - v o l a t i l e and therefore must be converted i n t o v o l a t i l e d e r i v a t i v e s f o r spectral analysis.  (m.s.)  Stereoisomers of carbohydrate d e r i v a t i v e s give s i m i l a r  mass spectra, and the small differences i n peak i n t e n s i t y sometimes observed do not generally allow an unambiguous assignment of configuration.  However, consideration of g . l . c . retention time along with  mass spectral data aids i n assignment. Carbohydrate d e r i v a t i v e s give weak or no molecular ions on electron impact ( e . i . ) mass spectrometry.  Molecular weights may  more e a s i l y be determined by f i e l d i o n i z a t i o n ( f . i . ) ^ ' ^ , desorbtion ( f . d . ) ^ techniques.  2 -  ^ , or chemical i o n i z a t i o n  field  (c.i.)^ ^ -  The l a t t e r two techniques are p a r t i c u l a r i l y useful with  free sugars and d e r i v a t i v e s of low v o l a t i l i t y .  In t h i s study  mass spectra were obtained by combined g . l . c . - m.s. in the e . i . mode at 70eV f o r the p a r t i a l l y methylated/ethylated a l d i t o l acetate mixtures, and using a d i r e c t probe i n l e t in the e . i . mode at 70 ev f o r a permethylated oligosaccharide. 121 122 Considerable data  '  are now a v a i l a b l e 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 i n our laboratory.  35  As an example most of the signals in the mass spectrum of 1 , 5 - d i - 0 - a c e t y l - 2,3,4,6 - t e t r a - 0 - methyl-D-glucitol can be accounted for from primary and secondary fragmentation (see Scheme 4). Vliegenthart and coworkers have shown that there are c h a r a c t e r i s t i c differences between the fragmentation patterns of hexopyranosides and hexofuranosides  123  A l d o n o n i t r i l e acetates (which have the advantage of molecular asymmetry) are s u i t a b l e for analysis by g . l . c - m.s. and give c h a r a c t e r i s t i c mass spectra that are easy to i n t e r p r e t ,  119 124 ' .  Oligosaccharides have been examined by e . i . - m . s . as t h e i r acetate  1 2 5  the l a s t  '  1 2 6  ,  TMS,  1 2 7  -  1 3 0  and methyl ether  proving most expedient.  1 3 1  "  1 3 4  derivatives,  The nomenclature used by  135 Chizov and Kochetkov  f o r the d i f f e r e n t fragmentation series of  permethylated glycosides, modified, as suggested by Kovacik and coworkers  , i s now standard.  In t h i s work a t r i s a c c h a r i d e  glyceride was obtained on periodate oxidation of K12.  The sequence  of sugars was r e a d i l y ascertained by d i r e c t probe> e . i . - m . s . of i t s permethylated d e r i v a t i v e since the constituent moieties were, f o r t u i t o u s 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  ions;^'^  the fragments observed make i t possible to determine the nature of the constituent monosaccharides but not the i n t e r - s u g a r linkages.  36  However, conversion of the reducing sugar to the corresponding a l d i t o l with a deuterated hydride d i s t i n g u i s h e s between 3 - or 4 - and 2- or 5- linkages r e s p e c t i v e l y . P a r t i a l hydrolysis of methylated polysaccharides, followed by reduction and remethylation with trideuteriomethyl iodide gives a mixture of oligosaccharide alditol.s that can be analyzed by g . l . c . m.s.  -  The CD^ groups occupy positions to which a sugar residue i s  l i n k e d in the o r i g i n a l polysaccharide. This technique  has been used  i n s t r u c t u r a l studies of the 1ipopolysaccharide from K l e b s i e l l a n  n  0-group 9  137  Mass spectrometry has become i n c r e a s i n g l y s i g n i f i c a n t i»n the s t r u c t u r a l e l u c i d a t i o n of b i o l o g i c a l l y important glycoconjugates 1 qg  since q u a n t i t i e s of material are very l i m i t e d . Egge and coworkers have made many contributions to t h i s r a p i d l y developing area of carbohydrate chemistry. II. 3 . 3 .  Polarimetry Assignment of the anomeric configuration of a s p e c i f i c  g l y c o s i d i c linkage i n a poly- or  oligosaccharide may be accomplished  by nuclear magnetic resonance spectroscopy (see l a t e r ) . However, ambiguities may a r i s e f o r some sugars, depending on the linkage pattern and ring s i z e .  In these instances polarimetry, using 143  Hudson'srules of i s o r o t a t i o n may be employed to advantage.  144  '  Since K l e b s i e l 1 a polysaccharides have been shown to consist of  37  repeating units of hexoses and t h e i r d e r i v a t i v e s the t h e o r e t i c a l value f o r the molecular r o t a t i o n can be found, to a f i r s t approximation, by summation of the molecular contribution of each component.  I t i s assumed that the 0_-acetyl and pyruvic acid ketal  groups present make n e g l i g i b l e contributions to the t o t a l molecular rotation. •Application of Hudson'srules gives information only on the o v e r a l l molecular r o t a t i o n of the poly - or oligosaccharide.. However, the i n d i v i d u a l g l y c o s i d i c linkage configurations may be deduced i f the rotations of a series of oligomers, eg. d i - , t r i - , and tetrasaccharides from the repeating unit are observed. The s p e c i f i c r o t a t i o n of the polysaccharide i s  calculated  from the equation  M  D  M r s . *  =  M  where  1  0  0  o  [M]p^ i s the summation of the i n d i v i d u a l rotations and M  the molecular weight of the repeating u n i t . The experimental s p e c i f i c r o t a t i o n i s  r  -i  _  a  - _JJ n  x 100 1 x c_  where  i s the polarmeter reading 1 i s the c e l l length i n dm,  and  c_is the concentration i n g/100 ml.  Q  is  38 Merri f i e l d  145  has compared the t h e o r e t i c a l and observed  values f o r a number of K l e b s i e l l a polysaccharides and found very l i t t l e discrepancy in most instances.  He also found that the  e f f e c t of temperature change i s n e g l i g i b l e .  His compilation i s  included i n Appendix 1.  I I . 3. 4.  C i r c u l a r dichroism ( c . d . )  I  AC  The configuration (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 t h e i r methylated d e r i v a t i v e s , where the acetoxy group acts as a chromophore  The method i s well suited to analysis of samples  obtained by preparative g . l . c , of material are required.  since only milligram q u a n t i t i e s  Configurational assignments are made  a f t e r comparison with data from authentic samples. ,In the case of D-glucose and D-galactose s p e c i f i c oxidases are a v a i l a b l e (Worthington Biochemical Corporation).  This method  was employed in the i n v e s t i g a t i o n of K58 to determine that the galactose was of the D-configuration, since the c . d . method i s inapplicable  I I . 3. 5.  to the meso-galactitol  hexaacetate.  Nuclear Magnetic Resonance Spectroscopy 1 Both  H and  13 C n.m.r,. spectroscopy have been used  extensively in t h i s work.  The f a c t that i n t e r p r e t a b l e spectra can  be obtained by these two methods on polysaccharides with molecular  39 weights of the order of 10 repeating u n i t s .  indicates t h a t the structures have regular  Spectra of the native and permethylated polysaccharides  and of degradation products were analyzed, i n d i c a t i n g 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 r m l y established as the most widely used technique  f o r the s t r u c t u r a l , c o n f i g u r a t i o n a l , and  conformational analysis of carbohydrates and t h e i r d e r i v a t i v e s . The observations of Arnold and coworkers  148  and Gutowski and Hoffman  149  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  u n t i l 1957 that the f i r s t ^H n.m.r. spectra of carbohydrates were reported by Lemieux and coworkers  150  .  In a c l a s s i c paper i n 1958  151  these authors showed the e f f e c t s of configuration and conformation on the chemical s h i f t s and coupling constants  of acetylated sugars. This  work was extended to free sugars in fifi s o l u t i o n by Lenz and Heeschen in 1961 and the observation that g l y c o s i d i c linkage protons resonate 153 downfield of the r i n g protons was made in 1963 by van der Veen who also succeeded i n c o r r e l a t i n g the s p l i t t i n g s , observed for the anomeric hydrogen atoms, with the glycoside c o n f i g u r a t i o n . review of the f i e l d appeared in 1964 by Hall  The f i r s t  1 ^4  155 Progress in the intervening years has been rapid  due to  (a) the development of superconducting solenoid spectrometers with higher magnetic f i e l d s and resonance frequencies (b) the improved performance of radio - frequency c i r c u i t s (c) the development of  152  40  the Fourier - transform n.m.r, method and (d) decoupling and multinuclear a b i l i t i e s along with (e) advancements i n data systems eg. m u l t i t a s k i n g and queuing c a p a b i l i t i e s .  To date many reviews of t h i s 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 i m i t a t i o n s inherent with increase in molecular s i z e and complexity, i n p a r t i c u l a r when attention i s turned to polysaccharides; solutions to these problems 159 have been proposed by Hall  .  More r e c e n t l y , homonuclear two-  dimensional J n.m.r. has been used mono - and  to s i m p l i f y complex spectra of  d i s a c c h a r i d e s , by separating the e f f e c t s of chemical  shifts  and s c a l a r coupling. The ^H n.m.r. spectrum of a polysaccharide provides information on the number of sugars present i n the repeating unit and indicates the presence of 6-deoxysugars  0 - A c e t y l , 0-formyl ^ a n d 1-carboxya  ethylidene acetal substituents are also recognised T 3b.>c 6  _ j  n  e  anomeric nature of the linkages (a or p ) may be d i f f e r e n t i a t e d f o r 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 i b u t a b l e to four anomeric centres  -:three  ct( 6>5 ppm, J-j <4) and one e( 6<5 ppm, J-| >6) l i n k e d pyranoses. 2  2  Care should however, be exercised when furanosyl sugars are present (from g . l . c . - m.s. data). Spectrum Nd.T shows the anomeric signal f o r the  41  g - g a l a c t o f u r a n o s y l unit at 6 5.13 ( 6>5). Ring protons may resonate in the s o - c a l l e d "anomeric r e g i o n " , depending on the r i n g 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 Garegg and coworkers  1 7 1  s i z e and  1 6 9 , 1 7 0  .  have demonstrated that the differences  in chemical s h i f t s obtained f o r stereoisomenc pairs of a c e t a l i c CH 172 groups from pyruvic acid  3  and related acetals are of s u f f i c i e n t  magnitude to make possible the unequivocal determination of the stereochemistry of the a c e t a l s . For oligosaccharides, the degree of polymerization may be c a l c u l a t e d from the r a t i o of the i n t e g r a l of the anomeric protons from the reducing sugar («and 3) to those from the non-reducing sugars (see Spectrum No. 26) High r e s o l u t i o n n.m.r. spectra contain, i n addition to chemical s h i f t values, coupling constants  and area i n t e g r a t i o n s , two  f u r t h e r sets of nuclear parameters: the spin-spin (T^) and s p i n - l a t t i c e (Tj) r e l a x a t i o n times  1 7 3 - 1 7 5  _  since the l a t t e r show a number of  s t e r e o s p e c i f i c dependencies they provide a useful basis f o r configurational assignments.  Another very useful a p p l i c a t i o n of  r e l a x a t i o n studies l i e s i n the removal of the unwanted residual peaks of deuterated solvents - i n t h i s study of HOD - when high temperature f a c i l i t i e s are u n a v a i l a b l e ' (see Spectrum No. 3). The l i t e r a t u r e contains few ^H n.m.r. i n v e s t i g a t i o n s of b a c t e r i a l polysaccharides.+  These assignments are made a f t e r  +see, however, references in Appendix I from Dutton e t _ a j _ et al and reference 176 from Perl in et a l .  a n c  ' Joseleau  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 refined 177 and extended the increment rules of Lemieux  f o l l o w i n g analysis  of parameters obtained i n the 300-MHz. ^H - n.m.r. spectra of 178 D -glucose, D-mannose, D-galactose, and t h e 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 glucotrioses  and of various d i - and . t r i s a c c h a r i d e s  containing  182 L-rhamnose  have been described.  spectra of some mucopolysaccharides  1  Perl i n has published 220 MHz -ic  ^H n.m.r.. examination of carbohydrate d e r i v a t i v e s soluble in organic solvents have advantages over the use of aqueous s o l u t i o n s : there i s no need for proton exchange, i f the hydroxyl groups are d e r i v a t i z e d and no residual HOD peak, therefore ambient temperature i s s u f f i c i e n t and also solvent e f f e c t s may be employed to advantage. Vliegenthart and coworkers have described the complete i n t e r p r e t a t i o n I  of  I  H n.m.r. spectra of solutions of permethylated a- and &-  D-glucose  184 and galactose  and of mannose and of the 6-deoxy analogues of  mannose, glucose and galactose  "I  o c  , (with a view to use in methylation 186 analysis) and of permethylated disaccharides . The chemical s h i f t s of anomeric protons of the methyl ethers of various disaccharides 187  were reported in 1972 by Minnikin  but the complete i n t e r p r e t a t i o n  of ^H n.m.r. spectra of permethylated o l i g o - and polysaccharides . i s yet to be achieved some valuable information may be  provided. For  go  43 example i n the s t r u c t u r a l analysis of K l e b s i e l l a Kl2 comparison of the spectra of the permethylated polysaccharide and of the 3 - e l i m i n a t i o n product (see l a t e r ) , indicates that the two side-chain sugars removed were  3-linked. Stephen et al_ have used lanthanide s h i f t reagents in  n.m.r.  studies on f u l l y methylated aldohexopyranosides and t h e i r 6-deoxy analogues  100  and on the permethyl ethers of galactose  1  00  .  TMS d e r i v a t i v e s have been used to determine the number of 190 hydroxyl groups in a molecule  and to determine the configuration 191 of the g l y c o s i d i c linkages i n an oligosaccharide The solvent of choice f o r underivatized poly- and  n.m.r. spectroscopy of  oligosaccharides i s D2O.  To eliminate  interference in the spectrum by the numerous hydroxyl groups present, a number of exchanges are made with 99.7% D2O, followed by l y o p h i 1 i z a t i o n and heating under vacuum. The sample i s then dissolved in 99.9% DgO and any residual HOD i s s h i f t e d away from the anomeric region (to-6 4.18 ,90°) when the spectrum i s run at elevated temperature. A l t e r n a t i v e l y , a r e l a x a t i o n type (T-|) experiment may be performed whereby the HOD signal i s n u l l e d .  This may be e s p e c i a l l y successful  with non-viscous oligosaccharides.  Sample sizes of polysaccharides  are generally of the order of 1-2%, while l a r g e r samples may be employed with oligosaccharides.  I f the polymeric sample i s extremely  viscous p a r t i a l depolymerization with acid may improve the sharpness of the spectrum.  44  Because samples are of low concentration spectra are generally run in the FT mode.  Where sample s i z e i s minimal a 5mm  sample tube with a c y l i n d r i c a l semi-micro volume c a v i t y may be used to advantage. Spectra are usually run with an i n t e r n a l 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 v o l a t i l e (hence i t  may be r e a d i l y removed from the sample) and i t s chemical s h i f t i s v i r t u a l l y unaffected by v a r i a t i o n s i n temperature. Stephen et a l have recommended caution in the use of some reference systems f o r  I I . 3. 5. 2.  13  1 192 H n.m.r. spectroscopy of carbohydrates  C n.m.r. spectroscopy  The p r o l i f e r a t i o n of studies on the  13  C spectra of carbohydrates 13  during the past few years a t t e s t s to the f a c t that  C n.m.r. i s  acquiring a s t a t u s , not only as a useful adjunct to ^H n.m.r. spectroscopy, but one characterized by i t s own unique c o n t r i b u t i o n s .  As in ^H  n.m.r. progress i n instrumentation has been r a p i d , with concomitant increase in the number of a p p l i c a t i o n s ~. s t r u c t u r a l e l u c i d a t i o n , configurational and conformational analyses, detection of i m p u r i t i e s , 193 194 and analysis of mixtures Perlin  '  .  The f i e l d has been reviewed by  ' . Not s u r p r i s i n g l y glucose has been one of the e a r l i e s t and 197-200 most extensively studied of the carbohydrates . Previous 1 9 5  1 9 6  45  l3 assignments of natural abundance , C n.m.r. chemical s h i f t s of monoand  disaccharides have been re- evaluated by use of a d i f f e r e n t i a l  isotope  r  (DIS) technique  2f)l  202 13 , Gorinhas assigned the C signals, of the more common  sugars and t h e i r methyl glycosides - the l a t t e r proving most expedient in approximating the chemical s h i f t s of g l y c o s i d i c carbons i n o l i g o 203 and polysaccharides.  Chizhov et aT_  here extended these data to  include a l l the methyl ethers o f methyl (methyl a-D-glucopyranosid) uronates, since uronic acid residues are frequently encountered in n a t u r a l l y occuring polysaccharides. 13 Vliegenthart and coworkers have interpreted  C spectra  "I O O  of permethylated a- and p- glucopyranoses 185 mannopyranoses and 6- deoxy sugars  ]  04  , galactopyranoses  with a view to i n t e r p r e t i n g  methylation analysis data (see l a t e r ) , in conjunction with data.  n.m.r.  13 The e f f e c t of O - a l k y l a t i o n 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 f e c t of the t r i c h l o r o a c e t y l i 1 4 . • i_ • 206,207 . . group on glucopyranose and gentiobiose ' , and Seymour et al 13 have examined the C spectra.of compounds containing the 208 c  3 - fructofuranosyl group 13 C n.m.r. data of monosaccharides and t h e i r d e r i v a t i v e s have been extended to d i - , o l i g o - , and polysaccharides. Usui et al 209 210 have studied the glucobioses , and glucotrioses and glucans Di - and t r i s a c c h a r i d e s containing galactose have been examined 211 by Cox et a]_  , and those containing glucose, galactose and  ,  46  rhamnose by L a f f i t t e et a l  212  and Colson and King  213  .  Hough et al  214  have extended t h e i r analysis of permethylated carbohydrates to include disaccharides. 215 Gagnaire e_t aj_  determined the spectrum of a - c e l l o b i o s e  octaacetate and compared i t to the spectrum of c e l l u l o s e t r i a c e t a t e . 71  These data have been extended by Capon  fi  et, a l  to include spectra of  peracetylated a - c e l l o t r i o s e , a - c e l l o t e t r a o s e and a-cellopentaose 217 In 1971 Dorman and Roberts  demonstrated the a p p l i c a b i l i t y  13 of  C- N.M.R. spectroscopy to the study of oligosaccharides. Boyd and 218  Turvey  have i d e n t i f i e d spectra of oligosaccharides derived from 219  a l g i n i c a c i d , and Kochetkov et_ al_  have applied the technique in  the s t r u c t u r a l study of complex oligosaccharides. Perl in has reviewed the c h a r a c t e r i z a t i o n 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 e a r l y 217 13 as 1970 Dorman and Roberts - extended a C survey of oligosaccharides to include a b r i e f i n v e s t i g a t i o n of amylose and c e l l u l o s e 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 following year  used the technique to assign completely two  cyclodextrins and several l i n e a r glucans by comparison with spectra of glucobioses and g l u c o t r i o s e s . 222 In 1975 Gorin  commented on the methodology of assigning  signals of a mannan containing a l t e r n a t e (l->3) and (1+4) linked  47  3 - D - mannopyranose residues.  The 12 signals were assigned by  preparation of D - mannans from s p e c i f i c a l l y deuterated D-glucoses and observation of a - and g - deuterium isotope - e f f e c t s . Dextrans  ' ' and levans.  have been studied by Seymour et a l ,  as have galactomannans by Grasdalen and P a i n t e r ^ -  Glycosidically  substituted and free C-5 groups have been d i s t i n g u i s h e d , and d i f f e r e n t 225 types of linkages have been i d e n t i f i e d by Joseleau et al_.  in a  13 C study of two arabinans. Methyl and acetyl substituent e f f e c t s on C chemical s h i f t s have been determined on a - and g -  (1-+3) and  (l->4) l i n k e d polysaccharides by Gagnaire et al_ 1 13 As i n H n.m.r., very few C n.m.r. spectral data of 227 heteropolysaccharides have been published. P e r l i n e t aj_ in 1972 13 gave evidence for a biose repeating unit f o r heparin using and i n 1979  C n.m.r.  made a conformational study of the polymer.  Variations with respect to the presence and l o c a t i o n of sulphate groups 228 in agar and some carrageenans were shown i n a study by Hamer et The spectrum of s p e c i f i c a l l y l a b e l l e d (  1q  C)nigeran - a regular, a l t e r 13 nating copolysaccharide having differences i n C signal i n t e n s i t i e s 229. has been assigned by B o b b i t e t a]_ . 230 In 1977 Dutton et a_ delineated the diagnostic potential 13 of  C n.m.r. spectroscopy in the s t r u c t u r a l e l u c i d a t i o n of K l e b s i e l l a  polysaccharides composed of three to s i x sugar residues and carrying 0acetyl and 1-carboxyethylid.ene substituents. Since then many K l e b s i e l l a 13 polysaccharides have been c h a r a c t e r i z e d , using by our group.  C n.m.r.  spectroscopy  Assignments have been made on the basis of spectral data  48 obtained from degradation products of the polysaccharides, from methyl 231 g l y c o s i d e s , and from synthesized oligosaccharides. 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 f o r the 3 (equatorial) 1  anomer of glucose than f o r the a ( a x i a l ) anomer (169 Hz) other than the anomeric centre the magnitude of  is  go  .  For nuclei  substantially  smaller, and there i s an o v e r a l l tendency for d i r e c t coupling to 13 decrease with an increase in s h i e l d i n g of the C nucleus. In 1979 232 F r i e b o l i n et a]_  1 used  J ( C - l , H) coupling constants to i d e n t i f y  the anomeric configuration of some polysaccharides and t h e i r methyl d e r i v a t i v e s . To gain fu r t h e r i n s i g h t into the a r c h i t e c t u r e o f the gel 233 network of some branched 3 (l->3) linked 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 - C couplings i n spectra of o l i g o s a c c h a r i d e s . Bock and Hall in 1975 showed the p r a c t i c a l relevance of 13 1 3  2 3 4 9  T-j measurements in obtaining e f f i c i e n t F.T.  C spectra : the time  i n s o l u successive t i o n . T-j values f o r polysaccharides havethan beenf i v e T-j i n tlecules e r v a l between 90° pulses should be no less mo 9Q C  O OOU  periods to prevent saturation of resonances. Coupling and T-j experiments reported f o r bovine nasal c a r t i l a g e and a gel-forming glucan , provide i n s i g h t into the microdynamics of the motion of carbohydrate giving information on the molecular motion and the o v e r a l l conformation r e s p e c t i v e l y . T-| measurements on branched - chain polysaccharides  49 made by Gorin and Mazurek  236  show that these values can be useful in  d i s t i n g u i s h i n g resonances of side-chains from those of the main chain. 13 in 50%  C spectra of underivatized carbohydrates are generally run  to give a deuterium lock and with acetone as i n t e r n a l 13  standard.  Chemical s h i f t s of the  C nuclei of carbohydrates and  d e r i v a t i v e s encompass most regions of the 200 pp, range covered by organic compounds. S i m p l i f i c a t i o n of the spectrum by proton decoupling, together with the r e s o l u t i o n of less than 0.1 ppm afforded by present instruments usually ensures an e x c e l l e n t o v e r a l l separation of resonance s i g n a l s even for highly complex molecules or mixtures. Peak areas, which are highly s e n s i t i v e to the r e l a x a t i o n properties of 13 the various  C n u c l e i , and to the extent of Overhauser enhancement,  may be ysed i f comparisons  of the integrated i n t e n s i t i e s are based on  signals representing the same class of carbon (such as the anomeric centres in a polysaccharide). Spectra should be run at elevated temperature, 196 when p o s s i b l e , to reduce l i n e broadening. Resonances of simple sugars are d i s t i n g u i s h a b l e f o r the most part i n terms of carbonyls from uronic acids and pyruvic a c i d (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).  Variations within each class are associated  with changes i n r i n g s i z e , c o n f i g u r a t i o n , conformation and s u b s t i t u t i o n . 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 u p f i e l d of the corresponding glycosidic centres. disaccharide 1 from K12 (No.6)  the signal  For example in the  due to g - galactose •-  occurs at 96.98 ppm and t h a t due to the a anomer at 93.01 ppm.  I I . 4.  Techniques of Structure Determination  I I . 4. 1.  Characterization of component sugars. K l e b s i e l l a polysaccharides are heteropolysaccharides, made  up of regular repeating u n i t s .  To i d e n t i f y the constituent 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 a c i d i c sugars are identified.  G . l . c . analysis o f the corresponding a l d i t o l  is used to determine the q u a n t i t a t i v e composition.  acetates  I t is of prime  importance, then, to hydrolyze a l l the glycosidic linkages, and to minimize degradation of monosaccharides. The rates of hydrolysis of i n t e r g l y c o s i d i c linkages vary 237 greatly  ;  6-deoxy sugars and furanosyl bonds hydrolyze e a s i l y ,  while uronic acid residues are most r e s i s t a n t .  This problem is 238  overcome with a technique developed in t h i s laboratory. Treatment of the polysaccharide with methanolic hydrogen chloride cleaves most g l y c o s i d i c bonds, leaving some uronosyl linkages i n t a c 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 trifluoroacetic acid (TFA) ensures complete hydrolysis. Theoretically, for each free sugar five forms are possible (a - and 3 - pyranoses, a - and 3 - furanoses, and linear). Reduction, then, of C-l to the alcohol, simplifies the situation, and subsequent acetylation yields volatile derivatives for g.l.c. analysis. The use of HPLC to determine the quantitative composition of the polysaccharide is investigated in Section V. 1 As evinced in Sec. I I . 3 . 5 .  H and  13 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 identity 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 Table 2).  0_-acetyl group (see Scheme 4 and  POLYSACCHARIDE i) base ii) M e l  ii) r e d u c t i o n CHO—Cry)H  iii) acetylation 11  r-OAc  r-OAc  -OAc -OAc MeO-OAc AcO-OMe -OAc  -OAc -OAc -OAc -OAc  •-OMe A  -OMe C  r-OAc  -OMe B  r  OAc -OMe  MeO-OMe -OAc D  <  gi c - - m s Scheme 4  Methylation Analysis of a Polysaccharide.  53 METHYLATION ANALYSIS Methylation pattern i s i d e n t i f i e d by: (a) Retention time on g . l . c . (b) Mass-spectrum from g . l . c .  m.s.  Methylation analysis gives information on: (i) (ii) (iii) (iv)  number of sugars per repeating u n i t , ring s i z e . linkage p o s i t i o n s and l o c a t i o n , pyruvate s u b s t i t u t i o n . 6-Deoxy Hexose  Hexose OMe. 0Me~ OMeOMeT  Location terminal in-chain terminal .+ pyruvate, or branch i n - c h a i n + pyruvate, or doubly branched  0Me o OMe: OMe' 1  Methylation at p o s i t i o n 5 i n d i c a t e s a furanose sugar TABLE 2  YEAR 1903 1915 1926 1934 1955 1964 1966 1975 1980 1980 TABLE "3  METHYLATION ANALYSIS OF A POLYSACCHARIDE.  NAME 242 243 Haworth 244 Menzies Muskat 246 Kuhn' ,247 Hakomori .248 Gros 249 Arnap 250 Prehm 251 Finne Purdie  245  SOLV.  BASE.  ME.REAGENT  CH I  Ag 2 0  CH I  (CH3)2C0  Na0H/H 2 0  Me2S04  H0O/CH3I  T10H .  CH I  NH  K/Na  CH I  Ag 2 0  CH I  3  3  HC0N(CH ) 3  2  (CH3)2S0 CH 2 C12  3  3  3  CH 3 S0CH Na 2  CH 2 C1 2  3 K2C03  (CH30)3P0 (CH3)2S0  METHYLATION PROCEDURES  3  B F  CH I 3  CH N 2  2  CF3S0 CH  3  2,^6 DTBP  CF3S0 CH  3  rBuo  CH I  3  3  3  54  The procedure involves treatment of the polysaccharide, in s o l u t i o n , with base and a methylating agent. Table 3 indicates various P47  procedures.  The most v e r s a t i l e method i s t h a t developed by Hakomori  Usually complete e t h e r i f i c a t i o n i s r e a l i z e d with one treatment (as evinced by i n f r a - r e d ( i . r . ) spectroscopy).  I f t h i s i s not the case 242  complete methylation can be achieved by a subsequent Purdie reaction since a second Hakomori treatment would r e s u l t in 3 - e l i m i n a t i o n (see l a t e r ) i f the polysaccharide i s a c i d i c . To deduce the i d e n t i t y of the a c i d i c sugar the methyl e s t e r i s reduced with l i t h i u m aluminum hydride in oxolane  (tetrahydrofuran)  and then re-methylated. Methylated material i s recovered e i t h e r by d i a l y s i s followed by l y o p h i l i z a t i o n i n the case of polysaccharides, or by e x t r a c t i o n with chloroform f o r oligomers.  Subsequent h y d r o l y s i s , reduction  the r e s u l t i n g sugars to the a l d i t o l s , and a c e t y l a t i o n y i e l d s d e r i v a t i v e s f o r g . l . c . - m.s. a n a l y s i s .  of  volatile  Comparison of data from the  n a t i v e , the uronic acid reduced and the depyruvulated materials y i e l d s valuable: information (see Tables 6 , 8 ) . Various s t a t i o n a r y phases may be used i n 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.  I d e n t i f i c a t i o n i s made by consideration of r e l a t i v e retention times, co-chromatography with authentic samples, and mass-spectral data.  pep  55  I I . 4. 3.  Oxidation Two main types of oxidation are used in s t r u c t u r a l  of polysaccharides .  The c l a s s i c a l periodate reaction  investigations  253-256  cleaves  the carbon-carbon bond between v i c i n a l d i o l s , while reaction of a  .  257 selected alcohol group  with e i t h e r t r i f l u o r o a c e t i c a c i d , or c h l o r i n e ,  along with dimethyl s u l f o x i d e 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 i s a polyaldehyde, which i s then reduced with sodiumi "borohydride.The t o t a l hydrolysis product of the derived polyol may be examined q u a l i t a t i v e l y by paper chromatography or q u a n t i t a t i v e l y by g . l . c .  258  , a f t e r d e r i v a t i z a t i o n to the a l d i t o l  nrQ  ace tates.'Al-ternati vely, a mild Smith hydrolysis  OCA  '  , whereby  only the true acetal linkages are cleaved and the g l y c o s i d i c linkages are l e f t i n t a c t , gives glycosides of mono- or o l i g o s a c c h a r i d e s .  These  products, a f t e r separation and p u r i f i c a t i o n , are investigated by 13 and  C n.m.r. spectroscopy, and methylation„ and m.s. a n a l y s i s of the i n t a c t  methylated oligomer to give information on the sequence and linkage p C "I  patterns of residues. A s e l e c t i v e oxidation , using periodate, under c o n t r o l l e d conditions, has been used to advantage, f o r example 262 to o x i d i z e p r e f e r e n t i a l l y a terminal residue The second type of oxidation has only been employed quite recently in s t r u c t u r a l i n v e s t i g a t i o n s of polysaccharides.  Selected  26 3 alcohol groups may be derived, f o r example,  by mild hydrolysis of  a methylated polysaccharide to remove only the pyruvic acid k e t a l .  56  POLYSACCHARIDE +PYRUVATE i) m e t h y l a t i o n jj) mild hydrolysis [-Glcp-]n  Scheme 5  S e l e c t i v e Oxidation and Degradation.  57  Oxidation gives a residue containing ketone/aldehyde  functionalities  which may then be degraded with base (see Scheme 5). Another residue with  a  free hydroxyl group i s then exposed i n the chain and the series  of reactions may be repeated.  The newly exposed hydroxyl groups may 1  be l a b e l l e d with e i t h e r EtI  or CD^I.  1  3  H and  C n.m.r. spectroscopic  analysis along with g . l . c . - m.s. analysis gives information on the anomeric nature of linkages and the linkage patterns of the products. In the i n v e s t i g a t i o n of K12 a t r i s a c c h a r i d e glyceride was obtained by periodate o x i d a t i o n , while in K58 the terminal  galactose  was s i m i l a r l y o x i d i z e d , leaving the i n t a c t polymeric backbone. A s e l e c t i v e functional group oxidation was not used i n t h i s study. I I . 4. 4.  Reduction I t may be deemed expedient to perform reactions on the uronic -  acid reduced polysaccharide, f o r example to a l t e r hydrolysis patterns or to f a c i l i t a t e periodate o x i d a t i o n .  A technique developed recently  264 by Taylor and Conrad  involves the use of water-soluble carbodiimides  and sodium borohydride (see Scheme 6).  The product i s recovered by  d i a l y s i s and l i y o p h i l i z a t i o n . 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 t h e r with l i t h i u m aluminium  hydride or with calcium borohydride ^ i n tetrahydrofuran (THF). To transform 26  the reducing sugar of a methylated oligosaccharide to the a l d i t o l , sodium borohydride i n  THF: ... ethanol (1:1) may be used, and to reduce free  58  11 NHR  RCOOH 41  II  +  RCOC  II  1 NHR  RCOO"H  11  I II  C  +  NHR  n  +  l  NHR XS NaBH, P  NaBH;  RCH 0H 2  5-7  NHR11  0 RCH  H  +  I  0=C  +H  n  I  NHR Scheme 6  Reduction of Carboxylic acid i n aqueous s o l u t i o n using a carbodiimide reagent.  Rubber Tubi ng  Polysaccharide Solution (acid or H 0 2  Steam bath dialysis tubing  Glass tubing Figure 9  HYDROLYZE COOL P a r t i a l Hydrolysis Apparatus  DIALYZE  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 s p e c i f i c degradation of polysaccharides has been an OCC. Of,~7  area of keen i n t e r e s t recently  '  .  In t h i s study a base-catalyzed  263 271 3 - elimination  ~  reaction was used in the s t r u c t u r a l  gations of both K12 and K58.  investi-  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 l o c a t i o n (whether in the backbone or side-chain) and i t s point of attachment. 272 The base used i s the m e t h y l s u l f i n y l 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 l a b e l l e d with ethyl iodide  . The reaction product i s characterized 274  1 13 I I . 4. 6. PCa rn.m.r. t i a l Hydrolysis by H and spectroscopy and by g . l . c . - m.s. a n a l y s i s . / H  I s o l a t i o n of fragments from p a r t i a l hydrolysis i s a major key to e l u c i d a t i n g the sequence of sugars in the polysaccharide and 1 also to making assignments i n the  H and  13 C n..m.r. spectra.  237 Capon  has reviewed the f i r s t order rate constants f o r the  acid catalyzed hydrolysis of the glycosides and these data may be  60  1) DMSO" N a  f  2) E t l / A g 0 2  ABC  [Rha,  Glc.Galp]-  D  hOMe O^OMe  Scheme 7  Jn  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 oligosaccharides may be obtained. The following generalisation may be madejfuranosidie and deoxy sugars are more l a b i l e than the corresponding pyranosidic hexoses, which are i n turn more l a b i l e than uronic acid residues. To minimise further degradation of oligosaccharides, once formed, an apparatus s i m i l a r to that described by Galanos et al_ ^ was employed in the i n v e s t i g a t i o n of K12.  (see F i g . 9)  After separation and  p u r i f i c a t i o n (see Scheme 8) a neutral and an a c i d i c dissacharide were characterized by n.m.r. spectroscopy. In the i n v e s t i g a t i o n of K58 the aldobi - , aldotetraouronic  acids were obtained. 1  incrementally, by  H and  aldotri-and  Characterization of each,  13 C n.m.r. spectroscopy permitted the  assignment of the spectra of the i n t a c t polysaccharide. To demonstrate conclusively the linkage positions of the pyruvic acid  ketal of K58, a very mild acid hydrolysis was performed  on the native polysaccharide i n which the ketal evinced by  n.m.r. spectroscopy).  was removed (as  Subsequent methylation analysis  (see Table 8) indicated the p o s i t i o n s of attachment.  In addition  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 v e r i f i e d the p o s i t i o n s of linkage (see Table 8).  62  OH  OH  F 0.1 M  TFA  95°  16 h  Dialysis  A-B Scheme 8  P a r t i a l hydrolysis and p u r i f i c a t i o n of K l e b s i e l l a K12  E-D  63  I I . 4. 7.  Location of O-acetyl group An O-acetyl substituent in the polysaccharide may be detected  1 by  13 H and  C n.m.r. spectroscopy.  The r a t i o of the i n t e g r a t i o n of  the O-acetyl CHg peak to the peaks in the anomeric region w i l l i f s u b s t i t u t i o n occurs on every repeating u n i t .  indicate  A sharp n.m.r.  peak i n d i c a t e s that the group occurs a t a d i s c r e t e p o s i t i o n i n each repeating u n i t , (see Spectrum No. 21). The O-acetyl group may be located by the method of de Belder 275 and Norman.  .  With t h i s procedure a l l the free hydroxyl groups are  blocked with methyl vinyl ether. The base l a b i l e O-acetyl group i s removed and replaced with a stable methyl group.  The protecting  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 a r i s e i f (a) a l l the free hydroxyl groups are not protected (methylation at more than one p o s i t i o n ) , (b) the 0_-acetyl group i s not removed with base (no methylation) or (c) the O-acetyl group i s removed at the protecting stage and replaced with methyl v i n y l ether (no methylation). An a l t e r n a t i v e 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 trimethyl 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 trifluoro-methanesulfonate as methylating agent.  64  In t h i s work the O-acetyl substituent in K l e b s i e l l a K58 polysaccharide was located by comparing g . l . c . - m.s. data from 275 the a n a l y s i s , by the method of de Belder and Norman, of the native and de-acetylated polysaccharide.  of samples  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 l e b s i e l l a K12 capsular polysaccharide has been investigated  by the techniques of methylation, Smith degradation - periodate o x i d a t i o n , uronic acid degradation and p a r t i a l h y d r o l y s i s , i n conjunction with 1  13  H-n.m.r. at 20 MHz.  spectroscopy at 100 and 220 HMz and  C-n.m.r.  spectroscopy  The structure has been found to consist of the hexa-  saccharide repeating unit shown, having a D-galactofuranosyl the branch point.  unit at  A galactofuranosyl residue has only previously been  found, in t h i s s e r i e s , in the polysaccharide from K l e b s i e 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  III. I . I .  Introduction: 23 The genus K l e b s i e l l a has been c l a s s i f i e d by (prskov  into  approximately 80 serotypes, based on t h e i r a n t i g e n i c , capsular 30 31 polysaccharides.  Nimmich  '  has analyzed q u a l i t a t i v e 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 a c i d .  As part of  our continuing i n v e s t i g a t i o n of the r e l a t i o n s h i p between primary, chemical structure and immunological a c t i v i t y , we now report on the e l u c i d a t i o n of the structure of the K12 polysaccharide. This structure i s in agreement with the predictions made by Heidelberger and coworkers, based on the cross-reactions of the polysaccharide with anti-pneumococcal  and a n t i - K l e b s i e l l a  of the occurrence of a 1,3-a l i n k e d L-rhamnosyl residue (non-reducing) 4,6-0-(l-carboxyethylidene)-D-galactosyl  55  sera, and of a 54  group  in the repeating u n i t . I I I . 1. 2.  Results and discussion Composition andn.-m.r.^ spectra K l e b s i e l l a K12 bacteria were grown on an agar medium, and  the capsular polysaccharide i s o l a t e d was p u r i f i e d by one p r e c i p i t a t i o n with Cetavlon.  As described in Sec. I I . 1 . the product had [ a ]  Q  + 24.2°.  Paper chromatography of an a c i d hydrolyzate of the polysaccharide showed the presence of glucose, galactose, glucuronic acid and rhamnose.  Carboxyl-reduced K12 polysaccharide was hydrolyzed, and  the presence of glucose, galactose and rhamnose i n the r a t i o of 2:3:1 was determined by g a s - l i q u i d chromatography ( g . l . c . ) of t h e 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 configuration by c i r c u l a r dichroism ( c . d . ) 147 measurements of the derived a l d i t o l acetates  .  Subsequently  a r a b i n i t o l pentaacetate was s i m i l a r l y shown to have the L c o n f i g u r a t i o n ,  68  TABLE 4 G.L.C. ANALYSIS OF NATIVE AND PERIODATE OXIDIZED^- POLYSACCHARIDES.  Sugars (as a l d i t o l  acetates)  * c Column C(SP 2340)  Mole %  Glycerol  0.09  11.7-  Erythri tol  0.26  12.4-  Threitol  0.33  9.9^-  Rhamnose  0.45  Arabinose  0.65  Galactose  0.95  51.7  21.7  Glucose  1.00  31.0  -  17.3  21.8 22.5  — On carboxyl-reduced polysaccharide. — Retention time r e l a t i v e to g l u c i t o l  hexaacetate.  — Programmed at 180° f o r 8 min, and then 4° per min to 240°. — I , native polysaccharide, uronic acid reduced. 11,periodate o x i d a t i o n , reduction, and t o t a l hydrolysis of the carboxyl reduced polysaccharide. — Some loss of v o l a t i l e components during d e r i v a t i z a t i o n .  69  i n d i c a t i n g that the galactofuranose unit from which i t was derived by loss of C-6 on oxidation has the D c o n f i g u r a t i o n .  An acid hydrolyzate  of the polysaccharide gave a p o s i t i v e reaction with  D-galactostat  reagent,*thus confirming the D configuration of the galactose. The 220 MHz, ^H-n.m/r.. spectrum of the polysaccharide,  after  mild hydrolysis to lower the v i s c o s i t y , showed a sharp s i n g l e t at 6 1.66 i n d i c a t i v e of a 1-carboxyethylidene group.  This signal was  present in a 1:1 r a t i o with a doublet at 6 1.34 a t t r i b u t a b l e to the I  methyl group of rhamnose  CO  1CA  '  .  Six d i s c e r n i b l e signals were  observed i n the anomeric region, at 65.22 (1H, (1H, J  }  6Hz).  2  3Hz), 65.13  The  13  (2H  2  2 H z ) , 6 4.66  (l.H ^  ^ weak), _65.16 2  8 H z ) , 6 4.48  C - n . m . r , spectrum of the polysaccharide  2 0 2  '  2 3 1  (T;H ^  > 2  (150 mg/2 ml)  showed high f i e l d peaks at 17.57 p.p.m. (rhamnose CH ) and 22.13 p.p.m. 3  (pyruvate CH^).  In the anomeric region f i v e sionals in the r a t i o  1:1:2:1:1 were seen at 108.39, 106.99, 102.64, 99.43 and 97.18 p.p.m. 13 I n t e r p r e t a t i o n 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 e d residues, while a - l i n k e d and three  C-n..m.r.  data i n d i c a t e d three  p - l i n k e d 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 linkage. F u l l assignments are shown in Table 5. * A c o l o r i m e t r i c enzymic reagent (Worthington Biochem.Co.)  TABLE 5 N.M.R. DATA FOR K l e b s i e l l a K12 CAPSULAR POLYSACCHARIDE -AND THE DERIVED OLIGOSACCHARIDES.  -i Compound5.30  T,2 (Hz) 2  4.76  7.,5  4.65  8  6GTcA ^  P  Gal-OH (1) 'X,  J  TO  H-n.m. r. • data Integral Assignment(H) 0.6 a-Gal ,~OH GlcA . 1  c  0.4  3-Gal~0H  E-D  Glc^Rha-OH (2) A-B  C-n.-m.r, data p . p . m . - Assignment104.46  p-Gl cA  96.98  B- Gal~ OH  93.01  a-Gal~0H  61.79  C-6 of Gal  5.15  s  0.6  a-Rha~0H  96.41  a-Glc  5.10  b  0.6  a-Glc  96.13  a-Glc  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  3  CH of Rha  17.76  CH of Rha  ( 5, ) J  3  £  £  3  6  Rha^Gal—Ara^glycerol Ct  B-C-D-A  Ot  Ot  (3)  106.93  Ara -  103.10  Rha  a-Gal  99.14  Gal  CH of Rha  17.46  CH of Rha  5.19  1  1  a-Rha  5.10  2  1  a-Ara-  5.05  1  1  1.30  4  3  3  :  3  TABLE 5 Contd.  • G a l £ ^ a l f-^rGl c ^ R h a 3  C-D-A-B E F  pyruvate  186.47  C-6 of 3-GlcA  a-Rha  108.39  3-Galf  5.22  s  5.16  3  1  a-Glc  106.99  3-Gal£  5.13  2  a-Galp  102.64  a-Rha + 3-GlcA  5.13  2  2  3-Galf  99.43  a-Gal  4.66  8  1  3-GlcA  97.18  a-Glc  4.48  6  1  3-Gal£  85.71  C-2  4.3-4.5 b  2  H-2, H-3 3-Galf  84.24  C-3 of 3-Galf  1.66  s  3  CHg of acetal  83.00  C-4  1.34  6  3  CH o f Rha  66.44  C-6 of a-Glc  63.85  C-6 of 3-Galf  61.77  C-6 of a-Gal  22.13  CH of acetal  17.57  CH of Rha  3  Galf GlcA  3  3  — For the o r i g i n of compounds 1 - 3, see t e x t . See Appendix I I I f o r reproductions of the spectra. b ^ — Chemical s h i f t r e l a t i v e to internal acetone; 6;2.23 downfield from sodium 4 , 4 - d i m e t h y l - 4 - s i l a p e n t a n e - l sulfonate (D.S.S.). — Key: b = broad, unable to assign accurate coupling constant, s= s i n g l e t . — For example, a-Gal = Proton on C-l of a-1inked D - Gal residue. %  — Chemical s h i f t in p.p.m. downfield from Me,Si, r e l a t i v e to i n t e r n a l acetone; 31.07 p.p.m. downfield from D.S.S. f d 13 — As for —, but for anomeric C nuclei. -2- This g l y c o s i d i c atom resonates as two doublets,because of the anomeric e q u i l i b r i u m of the reducing u n i t .  -  72  Methylation of o r i g i n a l Methylation  2 4 1  polysaccharide >  2 4 2  '  2 4 7 0  f Kl2 polysaccharide, followed  by reduction of the uronic e s t e r , h y d r o l y s i s , d e r i v a t i z a t i o n 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 v e pyranose sugars and one furanose, namely, galactofuranose, which constitutes a branch point (see Table 6).  These data also i n d i c a t e  that the (1-carboxyethylidene) group i s l i n k e d 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 e s t a b l i s h i n g that the uronic acid i s glucuronic a c i d . Base-catalyzed degradation To determine the location of the glucuronic a c i d , the methylated polysaccharide was subjected to a base-catalyzed degradati 273 and was then d i r e c t l y ethylated  .  The i s o l a t i o n of a polymeric,  degraded product indicates that the uronic acid i s in the side chain (see Scheme 7).  On 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 . -m.s.  the compounds shown in Table 6 were obtained, i n d i c a t i n g that the glucuronic acid i s attached to 0-3 of the galactofuranosyl u n i t , and that the only other sugar i n the side chain i s a 4,6-0(l-carboxyethylidene)-D-galactose group.  The  - n.m.r.•spectrum  indicated the absence of two 8- linkage signals in the anomeric region a t t r i b u t a b l e to the sugars of the side chain.  73 TABLE 6 METHYLATION ANALYSIS OF NATIVE, AND DEGRADED, K l e b s i e l l a K12 CAPSULAR POLYSACCHARIDE  Methylated sugarslas a l d i t o l acetates)  * . Column A - Column B^ (OV-225)  (ECNSS-M )  Mole % II  III  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  2,3,6-Glc  2.05  1.63  5,6-Gal  2.28  1.75  15.88  2,3-Glc  3.30  2.31  13.38  2,3-Gal  3.42  2.40  12.10  3,5,6-Gal 5  25.24 31.10 13.11 15.74  1.46  25.82  -2,4-Rha = 1 , 3 , 5 - t r i - 0 - a c e t y l - 2 , 4 - d i - 0 - m e t h y l - L - r h a m i n i t o l ________ ^-Retention time r e l a t i v e to that of the a l d i t o l acetate d e r i v a t i v e of 2,4-Rha. ^Programme: 180° f o r 4 min and then 2° per min to 200°. ^Programme: 165° f o r 4 min, and then 2° per min to 200°. —Values corrected by using e f f e c t i v e carbon response f a c t o r s ^ . —I o r i g i n a l polysaccharide methylated and uronic e s t e r reduced, column B. II as i n I but remethylated, column A. I l l a f t e r uronic acid degradation and e t h y l a t i o n . -S-l , 2 , 4 - T r i - 0 - a c e t y l - 3 - 0 - e t h y l - 5 , 6 - d i - 0 - m e t h y l - g a l a c t i t o l .  74  Partial  hydrolysis A sample of K12 polysaccharide in the f r e e - a c i d form was  hydrolyzed f o r lOh with 0.1M t r i f 1 u o r o a c e t i c a c i d in an apparatus s i m i l a r to that described by Galanos and c o l l e a g u e s ^ , y i e l d i n g a mixture of oligomers and monosaccharides, which was separated with AG-1 X2 ion exchange r e s i n i n t o a c i d i c and neutral f r a c t i o n s (see Scheme 8.').  Preparative, paper electrophoresis of the a c i d i c f r a c t i o n  gave an aldobiouronic acid (1) which, a f t e r hydrolysis and paper chromatography, was shown to c o n s i s t 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 v e r i f i e d that the side chain linkage is  3 (see Table 5 ) .  The structure of the aldobiouronic a c i d (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 p u r i f i c a t i o n on paper chromatography, gave a disaccharide (A-B 2) which, a f t e r hydrolysis and paper chromatography, was shown to consist of 1 glucose and rhamnose.  The  1  3  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 the polymer, the carboxyl-reduced polysaccharide periodate. After 90h. the consumption of oxidant moi of repeating u n i t .  264 256  was oxidized with ., was 5.4 moi per  The t h e o r e t i c a l consumption i s 5 moi i f  the (1-carboxyethylidene) group remains i n t a c t ; the higher consumption indicates loss of some of the  ketal. groups.  Total hydrolysis of  75  the polyol obtained a f t e r sodium borohydride reduction, followed by d e r i v a t i z a t i o n as the a l d i t o l acetates, gave the g . l . c . shown in F i g . 5 (see Table 4 ) .  separation  The low proportion of t h r e i t o l  consistent with some loss of the pyruvic acid ketal .  is  Smith  253 hydrolysis  of the p o l y o l , followed by sodium borohydride reduction,  y i e l d e d a mixture of oligosaccharides 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 i n agreement with these data (see Table 5). To determine the sequence of sugars in 3, the oligosaccharide was permethylated 242 by the Purdie method  and the product p u r i f i e d 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 i n k e d to the hexose, not to the pentose.  The source  of some pertinent fragments i s i l l u s t r a t e d 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 established as having the a-L-Rha£-(l+3)-a-D-Gal£-(U2)-a-L-Araf-(l+l)-glycerol structure  | 0 - C H - •CHOMe2  MeO  m/e  OMe 189  583!  627!  "CHgOMe  76  I I I . 1. 3  Conclusion I t thus follows that K l e b s i e l l a K12 capsular polysaccharide  had the f o l l o w i n g s t r u c t u r e .  A f t e r the r e a l i z a t i o n , from the methylation g . l . c . - m . s . data, 1 that a furanosyl residue was present, the were more e a s i l y i n t e r p r e t e d .  13 H- and  C-n.m.r.  spectra  In the former, the 3 - Galf anomeric  s i g n a l appears at 6 5.13, the region normally a t t r i b u t e d to a-1inked 13 pyranoses. In the . C spectrum, however, the anomeric signal occurs i n the unambiguous 3-linkage region at 108.39 p.p.m. (see Table 6 ) . I t i s i n t e r e s t i n g to note that the only other K l e b s i e l l a 169 polysaccharide reported  to have a furanosyl u n i t , K41 (see App.I)  has a very s i m i l a r structure i n which the terminal ethylidene )-B-D-galactopyranosyl ;  4,6-0-(1-carboxyr  group i s replaced by 3-D-Glcp_-(l-*-6)-a-  D-Glcp_-. As expected,no c r o s s - r e a c t i o n occurs between these two polysaccharides, because the sidechain i s usually the immunodominant  77  group.  Cross-reaction does, however, occur with a n t i - K l l " ^ which  has a 4,6-0-1(1-carboxyethylidene)-a-D-galactopyranose side chain 55 (see App.I) and with anti - Pn-VI  276  which has an in-chain-a-D-Glcp_-  (l->3)-a-L-Rha£- u n i t . I I I . 1. 4.  Experimental General methods —  Concentrations were c a r r i e d 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. pyridine - a c e t i c a c i d - water ( 5 : 2 :  The buffer used contained  743, v / v ) , pH 5.3.  Strips  of Whatman No. 1 paper (77 cm x 20 cm) were used f o r a l l runs, with a p p l i c a t i o n of 25 - 50 mA f o r 1% h.  Descending paper chromatography  was c a r r i e d out using Whatman No.l paper.  The following solvent system  (v/v) were used: (]_) f r e s h l y prepared 2:1:1 1-butanol - - a c e t i c acid - water, and {_) 8:2:1 ethyl acetate — pyridine - - water.  Sugars and  oligosaccharides were detected, a f t e r electrophoresis and a f t e r descending, paper chromatography, with an a l k a l i n e s i l v e r n i t r a t e . 24 reagent  .  A n a l y t i c a l g . l . c . separations were performed with a Hewlett  Packard 5700 instrument f i t t e d with dual f l a m e - i o n i s a t i o n detectors. An Infotronics CRS-100 e l e c t r o n i c i n t e g r a t o r was used to measure peak areas.  Separations were performed in s t a i n l e s s - s t e e l columns  (1.8 m x 3 mm) with a c a r r i e r - g a s flow-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) f o r 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 i o n i s a t i o n current of 100 pA and an ion-source temperature of 200°. "'H n.m.r.' spectra were recorded on e i t h e r 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 t h e r a  Varian CFT-20 or a Bruker WP-80 temperature. and A. Lee ('H  1 3  C 20.1 MHz  instrument at ambient  Additional spectra were obtained courtesy of Dr. A.A. Grey : HR-220 MHz, 90°) and Dr. Michel  250 MHz, 90° and  1 3  Vignon ( H : Cameca ]  C : Cameca 62.87 MHz, 8 0 ° ) .  C i r c u l a r dichroism ( c . d . ) spectra were recorded on a Jasco J20 automatic recording spectropolarimeter with a quartz c e l l of path length 0.01 cm. Optical rotations 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 spectrophotometer. Preparation and p r o p e r t i e s . — A culture of K l e b s i e l l a Kl2 (313) was obtained from D.I. (|)rskov (Copenhagen).  The polysaccharide  was i s o l a t e d as described, in section II.1 and showed [ a ]  Q  + 24.2°  (c_ 1, water). Analysis of constituent sugars. —  Methanolysis of a sample  (20 mg) of K12 polysaccharide with 3% methanolic hydrogen chloride  79  and subsequent treatment with sodium borohydride in anhydrous methanol reduced the uronic e s t e r .  Hydrolysis with 2M t r i f l u o r o a c e t i c a c i d  (TFA) overnight at 95° followed by reduction (NaBH^) and a c e t y l a t i o n gave g a l a c t i t o l hexaacetate, g l u c i t o l hexaacetate and rhamnitol pentaacetate i n the r a t i o 3:,2;::1 (column £ , programmed at 180° for 8 min and then 4°/min to 240°).  C i r c u l a r dichroism ( c . d . ) of the  l a t t e r two components i s o l a t e d by preparative g . l . c , showed p o s i t i v e and negative curves, r e s p e c t i v e l y , confirming that glucose has the D configuration and rhamnose the L c o n f i g u r a t i o n .  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 r a b i n i t o l  pentaacetate.  This was confirmed by the p o s i t i v e 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  conditions, followed by  242 a Purdie  treatment y i e l d e d a product that showed no hydroxyl  absorption i n the i . r .  spectrum.  This material was reduced overnight  with sodium borohydride in'oxolane (THF) and ethanol (1:1 v / v ) . A portion of t h i s product  was hydrolyzed with 2M t r i f l u o r o a c e t i c  acid f o r 16 h at 95°; the r e s u l t i n g mixture was reduced with sodium borohydride and the product acetylated.  G.l.c  - m.s. gave the  r e s u l t s shown in Table 6. Another portion of the material (reduced uronic  ester)  was remethylated under Purdie conditions f o r 2 days, and d e r i v a t i z e d for g . l . c . - m.s. giving the compounds shown in Table 6.  80  Uronic acid degradation^""'— A s o l u t i o n of c a r e f u l l y d r i e d , methylated polysaccharide (100 mg) and £ - t o l u e n e s u l f o n i c a c i d (a trace) in 19:1 dimethyl sulfoxide - 2,2-dimethoxypropane (20 mL) was prepared in a serum v i a l which was then sealed with a rubber cap. The v i a l was flushed with n i t r o g e n , and the s o l u t i o n was s t i r r e d f o r 3h.  Sodium methylsulphinylmethanide (2M) in dimethyl s u l f o x i d e (10 mL)  was then added with the a i d of a syringe, and the s o l u t i o n was s t i r r e d a t room temperature overnight.  A f t e r external cooling to 10°, ethyl  iodide (3 mL) was added slowly using a syringe  .  The s o l u t i o n was  s t i r r e d f o r a further 30 min., excess of ethyl iodide was removed using a r o t a r y evaporator, and the s o l u t i o n was dialyzed overnight against tap water.  After l y o p h i l i z a t i o n the product (65 mg) was  p u r i f i e d by p r e c i p i t a t i o n with petroleum ether (30°- 60°), y i e l d i n g 60 mg of polymeric m a t e r i a l .  Subsequent hydrolysis 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 in Table 6.  P a r t i a l hydrolysis —  A sample of K l e b s i e l l a K12  polysaccharide was exchanged to the f r e e - a c i d form with Amberlite IR-120 (H+) ion-exchange r e s i n , and l y o p h i l i z e d .  This material  (lg)  was dissolved in water (100 mL pH 3.2) and was then auto-hydrolyzed on a steam bath f o r 16 h i n an apparatus s i m i l a r to that described by Galanos e_t al_  .  Very l i t t l e hydrolysis occurred; therefore the  s o l u t i o n was made 0.1M i n TFA, and the reaction continued f o r a f u r t h e r 16 h.  A f t e r removal of TFA the products (700 mg) were  separated into neutral and a c i d i c f r a c t i o n s by using AG-IX2 ion  81  exchange r e s i n .  Portions (200 mg) of the a c i d i c f r a c t i o n were  separated by gel chromatography on a column (100 x 2.5 cm) of Sephadex G-25, which was i r r i g a t e d with a buffer (1000:10:4 v/v water - - pyridine - - a c e t i c a c i d , ) 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 l e c t r o p h o r e s i s . A component 1 with -GlcA 0.69 was obtained pure (30 mg) and was shown on t o t a l hydrolysis to consist of glucuronic 1 acid and galactose (E-D). the presence of a g -  13  H- and  C-n.m.r. spectroscopy indicated  l i n k e d glucuronic acid and a reducing  galactose (see Table 5 ) .  Portions (200 trig) of the neutral  fraction  were separated by gel chromatography on a column (100 x 2.5 cm) of B.iogel P-2.  I r r i g a t i o n with the same b u f f e r , at a flow rate of  10 mL/h, l y o p h i l i z a t i o n of the f r a c t i o n s , and examination by. paper, chromatography (solvent 2) revealed that separation was not complete. D  P u r i f i c a t i o n of a component with - G l c 0.61 by paper chromatography then y i e l d e d compound 2 (30 mg) which on hydrolysis was shown to 1 13 c o n s i s t 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 i n order to achieve  complete reduction.  Reduced, capsular polysaccharide (200 mg) was  82  dissolved i n water (40 mL) to which 0. IM sodium metaperiodate (40 m L) was then added.  The solution-was s t i r r e d in the dark at 3° and 256  periodate consumption was monitored spectrophotometrically  .  After  three days,consumption had reached 5.4 molecules per repeating u n i 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 s o l a t e d by d i a l y s i s and l y o p h i l i z a t i o n . A portion (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 y c e r o l , a t e t r o s e , rhamnose, arabinose and galactose.  Conversion of the hydrolysis products i n t o the corresponding  a l d i t o l acetates gave the g . l . c . r e s u l t s 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 p u r i f i e d by paper chromatography. H- and 13 C- n.m.r. data are shown, i n . Table 5. The mass spectrum of the 242 permethylated (Purdie  method) oligomer showed s i g n i f i c a n 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  III.  2  STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDE OF KLEBSIELLA SEROTYPE K58 ABSTRACT K l e b s i e l l a K58 capsular polysaccharide has been investigated  by the techniques of methylation, Smith degradation - periodate o x i d a t i o n , uronic a c i d degradation and p a r t i a l h y d r o l y s i s , 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 u n i t . A uronic a c i d residue bearing a 1-carboxyethylidene moiety has previously been found, i n t h i s s e r i e s , only i n the polysaccharide from K l e b s i e l l a K l .  •3)-a-D-Glc£-(l->4)  -p-D-Glc£A-(l-»4)-a-L-FuC£-(l2  A  Me  III.2.1.  'I 0-Ac COOH  a-Q-Gal£  n  Introduction 23 The genus K l e b s i e l l a has been c l a s s i f i e d by (prskov  into  approximately 80 serotypes, based on t h e i r a n t i g e n i c , capsular 30 31 polysaccharides.  Nimmich  '  has q u a l i t a t i v e l y analyzed  the  polysaccharide from each s t r a i n ; K58 was found to contain glucose, galactose, fucose, glucuronic a c i d and pyruvic a c i d .  In a d d i t i o n ,  K58 was shown to contain one O-acetyl group per repeating u n i t . As  84  part of our continuing i n v e s t i g a t i o n of the r e l a t i o n s h i p between primary, chemical structure and immunological a c t i v i t y we now report on the e l u c i d a t i o n of the structure of K58.  III.2.2.  Results and Discussion Composition and n.m.r.. spectra K l e b s i e l l a K58 b a c t e r i a were grown on an agar medium, and  the capsular polysaccharide i s o l a t e d was p u r i f i e d by one p r e c i p i t a t i o n with Cetavlon as described in Sec. I I . 1 .  The product had [ a ]  D  + 19.0°.  Paper chromatography of an acid hydrolyzate of the polysaccharide showed the presence of glucose, galactose, glucuronic acid and fucose.  Carboxyl-reduced K58 polysaccharide was hydrolyzed,  and the presence of glucose, galactose and fucose i n the r a t i o of 2:1:1 was determined by g a s - l i q u i d chromatography ( g . l . c . ) of t h e 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 c u 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 configuration by the p o s i t i v e reaction of D-galactostat reagent with an a c i d hydrolyzate of the polysaccharide. The 220-MHz ^H - n. m. r. spectrum of the polysaccharide showed sharp s i n g l e t s at 62.17 and 61.64 and a doublet a t 61.33 i n the approximate r a t i o of 1:1:1.  These were assigned to methyl groups  of 0-acetate, 1-carboxyethylidene and fucose, r e s p e c t i v e l y ^2-164^ Four d i s c e r n i b l e signals were observed i n 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 l e b s i e l l a K58 CAPSULAR POLYSACCHARIDE AND THE DERIVED OLIGOSACCHARIDES 13  ^H-n. m.r. data  Compound—  eb  J  T,2 (Hz)C  Intergral (H)  Assignment-  - C-n.m.r.  data  p.p.m.—  Assignment— B-GlcA  5.26  3  .5  a-Fuc~OH  103.91  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  CH of a-Fuc~OH  23.7  CH of  1.29  6.5  CH of B-Fuc-OH  16.3  CH of fucose  1.64  s  0.3  CH of  5.46  3.5  1  a-Glc  5.26  3  .6  4.61  7  1.8  4.57  7  1.64  s  1.33  6  1.29  6  GlcA^-iFuc~OH  Glc'^GlcA^fuc-OH Ct  (2) a.  p  3  3  3  0.4  3  3  3  1-carboxyethylidene  3  1-carboxyethylidene 103.94  B-GlcA  a-Fuc~OH  99.5  a-Glc  6-Fuc-OH  97.16  B-Fuc~0H  B-GlcA  93.3  a-Fuc~0H  CH of 1-carboxyethylidene  61.2  C-6 of Glc  CH of a-Fuc~0H  23.7  CH of 1-carboxyethylidene  CH of B-Fuc-OH  16.25  CH of fucose  3  3  3  3  3  TABLE 7 Contd.  GIC^GICAM-FUC-OH I Gal  (3)  5.45  35  5.29  3  ;  4.60  8  4.55  7  1.64  s  .9. 1.6  1.7 0.4  a.  1.33  6.5  1.29  6.5  a-Glc  104.0  B-GlcA  a-Fuc~0H  99.7  a-Gal  a-Gal  99.5  a-Glc  B-Fuc~0H  97.2  3-Fuc~0H  3-GlcA  93.3  a-Fuc~0H  CH of 1-carboxyethylidene  61.07  C-6 of Glc  62.59  C-6 of Gal  CH of  Fuc-OH  23.7  CH of 1-carboxy ethylidene  CH of  Fuc~0H  16.25  3  3  3 • 3  3  CH of fucose 3  ••^GlcLiGlcAMFucl a  3  Me  2 /  \ X  p  C00H  (10%) (4)  2 I  u  O-Ac  5.44  2  a-Glc  104.0  5.27  b  a-Fuc  100.3  4.57  8  B-Gl cA  99.7  1.64  s  CH, of 1-carboxyethylidene  61.3  CH of fucose  23:5  1.33  3  (3-G1 cA a-Fuc a-Glc C-6 of Glc CH o f 1-carboxy e t h y l i dene CH of fucose 3  16.1  0  TABLE 7  Contd. s  1  2-0-Ac  5.25  s  0.9  a-Fuc  a  5.16  s  1  a-Gal  4.53  8  1  3-GlcA  2.17  s  2  CHg of acetate  1.33  6  3  CH of fucose  5.45  2  1  a-Glc  104.5  B-GlcA -  b  2  a-Fuc  101.2  a-Fuc  a-Gal  99.5  a-Glc  1  Gal +0Ac (5) ^Ic^-^lc/vLiFuc  V  Me  3  2-0-Ac  5.18 5.13  1 COOH Gal 1  + O-Ac  :  3  9  4.59  8  1  3-GlcA  97.5  a-Gal  2.17  s  3  CH of acetate  62.5  C-6 o f Gal  1.64  s  3  ChL of 1-carboxye t h y l i dene  61.4  C-6 of Glc  CH o f fucose  29.98  CH o f acetate  23.4  CH o f 1-carboxyethylidene CH o f fucose  (6) a.  a-Gl c  5.35  ^Gl c - ^ G l c / V ^ F u c —  1.33  3  3  16.0  3  3  3  -For o r i g i n of compounds I see t e x t . * - Chemical s h i f t r e l a t i v e to i n t e r n a l acetone; 62.23 downfield from sodium 4,4-dimethyl-4silapentane-l-sulfonate (D.S.S. ).S ..b = broad, unable to assign accurate coupling constant, s - s i n g l e t . For example, a-Gal = proton on C-l of a - l i n k e d D-Gal residue, e. Chemical s h i f t i n p.p.m. downfield from Me4Si, r e l a t i v e to i n t e r n a l acetone; 31.07 p.p.m. downfield from D.S.S. lAs f o r ^L, but f o r anomeric 13c n u c l e i . £ Spectrum recorded on Brucker WP-80 (20.1 MHz) using 5 mm tube with semi-micro volume c y l i n d r i c a l cavity (6mm x 4.2 mm), i l 10% o f 1-carboxyethylidene remaining a f t e r Smith hydrolysis. *See Appendix I I f o r reproductions o f the spectra.  88  and 84.59 ( I H , ^  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 i n t e n s 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 i n the r a t i o 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 atoms of hexoses. Both the  13  H n. m.r.and  C data indicate the presence  of three a - l i n k e d and one g - l i n k e d residues.  Assignments were made  a f t e r n.m.r. -spectral i n v e s t i g a t i o n of oligosaccharides i s o l a t e d from p a r t i a l hydrolysis and periodate oxidation (see l a t e r ) . Methylation of o r i g i n a l polysaccharide Methylation  242 247 ' of K58 polysaccharide, followed by  reduction of the uronic e s t e r , h y d r o l y s i s , d e r i v a t i z a t i o n 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 u n i t s .  The sugars  are in the pyranoid form with fucose c o n s t i t u t i n g a branch point (see Table 8 ) . Analysis of a re-methylated sample of the reduced product showed the formation of 6-0_-methylglucose, thus establishing t h a t the uronic acid i s glucuronic acid. remethylated polysaccharide (to remove the  Mild hydrolysis of t h i s 1-carboxyethylidene  group), followed by methylation, showed on g . l . c . - m.s. a n a l y s i s , the formation of 2 , 3 , 6 - t r i - 0 - m e t h y l  glucose i n d i c a t i n g that the  1-carboxyethyl idene;residue i s linked at fj-2 and 0-3 of the glucuronic acid residue;  t h i s was confirmed by methylation analysis of a  sample of autohydrolyzed native polysaccharide (see  Fig....10).  TABLE 8 METHYLATION ANALYSIS OF NATIVE, AND DEGRADED K l e b s i e l l a K58 CAPSULAR POLYSACCHARIDE  Methylated sugars(as a l d i t o l acetates)  T* Column A i Column B£ (OV-225) (ECNSS-M)  2,3,4-Fuc  0.80  -  2,3-Fuc  0.82  0.92  -  0.9  2,4-Fuc  0.89  -  2,3,4,6-Gal  1.00  2-Fuc  4-0Et, 2-Fuc  Mole %e II  II  III  IV 4  V  30 38  2  2  2  2  1.00  28  28  29  23  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  6-Glc  2.17  -  Glc  2.78  3.4  -  VI  -  43  21  3 19  5  20  2,3,4-Fuc = 1 , 5 - d i - 0 - a c e t y l - 2 , 3 , 4 - t r i - 0 - m e t h y l - L - f u c i t o l  30  19  etc.  — Retention time r e l a t i v e to that of the a l d i t o l acetate d e r i v a t i v e of 2 , 3 , 4 , 6 - G a l . — Programme: 180° for 4 min and then 2°per min to 200°. -  Programme: 160° f o r 4 min and then 4° per min to 200°.  — Values corrected by using e f f e c t i v e carbon response f a c t o r s , ^ and adjusted to the nearest integer f - I, o r i g i n a l polysaccharide, methylated and uronic ester reduced.II,as i n I. but re-methylated. I l l , as i n II,then hydrolyzed to remove 1-carboxyethylidene moiety and remethylated. IV,autohydrolyzed polysaccharide, methylated, and uronic ester reduced. V, Smith degradation product, methylated, and uronic ester reduced. VI,product of uronic acid degradation, and e t h y l a t i o n . 1  6  90  ~N  1  1  1  1  0  4  8  12  16  1 20  Time (min)  Figure 10 G . l . c . separation of products of methylation analysis of K l e b s i e l l a K58 = IV, —  - III  (see Table 8 ) .  91 Base-catalyzed  degradation  To determine the l o c a t i o n o f the u r o n i c a c i d , the m e t h y l a t e d p o l y s a c c h a r i d e was s u b j e c t e d t o b a s e - c a t a l y z e d d e g r a d a t i o n , 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 g o s a c c h a r i d e  i n d i c a t e s t h a t the u r o n i c a c i d i s i n the backbone. and d e r i v i t i z a t i o n , f o r g . l . c .  - m.s.,  On h y d r o l y s i s ,  the compounds shown i n  Tables  were o b t a i n e d i n d i c a t i n g t h a t g l u c u r o n i c a c i d i s a t t a c h e d t o 0-4 o f fucose.  Loss o f some glucose suggests t h a t i t  i s linked to the  g l u c u r o n i c a c i d i n the backbone (see Scheme 9 ) . :  Periodate  Oxidation The n a t i v e p o l y s a c c h a r i d e consumed 1.8 moles o f  periodate  255 per r e p e a t i n g u n i t < i n 10 h, y i e l d i n g , a f t e r sodium b o r o h y d r i d e 253 r e d u c t i o n and Smith h y d r o l y s i s a polymeric product ( 4 ) . Reduction <\>  o f t h e u r o n i c a c i d f o l l o w e d 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  g.l.c.  showed t h e presence o f glucose and fucose i n the r a t i o  1  13 H and  C n . m . r . spectroscopy o f t h e Smith -  degradation  (4) showed the presence o f two a - l i n k e d and one indicating that,  the o x i d i z e d t e r m i n a l  for  2:1  product  B- l i n k e d s u g a r s ,  galactose i s a - l i n k e d  (see  Table 7 , Scheme 1 0 ) . M e t h y l a t i o n o f a p o r t i o n o f the Smith - d e g r a d a t i o n f o l l o w e d by r e d u c t i o n o f t h e u r o n i c e s t e r , and d e r i v a t i z a t i o n g.l.c.  - m.s.  product, for  , 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 i d e chain g a l a c t o s e i s l i n k e d t o 0-3 o f f u c o s e .  92  MeO Scheme 9  Base-catalysed degradation of K l e b s i e l l a K58  93  P-0 Scheme 10  Periodate Oxidation of K l e b s i e l l a K58  94  Autohydrolysis A sample of K58 polysaccharide in the f r e e - a c i d form was autohydrolyzed at 95° for 3 h and then d i a l y z e d .  1  H«n...m.r.  spectroscopy of the product (5) showed the absence of  1-carboxyethylidene.  Methylation of t h i s material followed by reduction of the uronic ester and d e r i v a t i z a t i o n for g . l . c . - m.s. gave the r e s u l t s in Table 8  i n d i c a t i n g that the 1-carboxyethylidene group i s attached  to 0-2 and 0-3 of the glucuronic acid residue.  Partial  Hydrolysis A sample of K58 polysaccharide was hydrolyzed with 0.5M H^^SO^  at 95° for 30 min y i e l d i n g a mixture of oligosaccharides.  Preparative  paper chromatography gave an aldobiouronic a c i d (1) an a l d o t r i o u r o n i c acid (2) and an aldotetraouronic acid (3). H and  C spectroscopic  data (see Table 7) indicated that the glucuronic a c i d i s 3 - l i n k e d , and the glucose i s a-1inked and confirmed that the side chain galactose is a-linked.  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 d i s c r e e t p o s i t i o n i n each repeating u n i t . In order to locate the O-acetyl group K58 was treated with methyl vinyl ether in the presence of an acid c a t a l y s t , and the product was then subjected to methylation a n a l y s i s  .  However complete  95  Scheme 11  Partial Hydrolysis of Klebsiella  K58  96  blocking of a l l OH groups was not r e a l i z e d , but comparison of data from analysis of the native polysaccharide and from a s i m i l a r analysis of a deacetylated sample indicated that the 0-acetyl group was linked to 0-2 of fucose. III.  2.3.  Conclusion I t thus follows that K l e b s i e l l a K58 capsular polysaccharide  has the following s t r u c t u r e . +3) - a- D- Gl C£- (1 +4) - (3- D- Gl C£A- (1 +4) - a- L- Fuc£- (1 + 3  X  Me  2  3 0-Ac a -D-Gal£  •  XOQH  A uronic acid residue bearing a 1-carboxyethylidene group has only previously been found, in t h i s s e r i e s , i n the polysaccharide from K l e b s i e l l a K l I I I . 2. 4.  2 7 7  .  (see note P. 102).  Experimental General methods. —  Concentrations were c a r r i e d out under  diminished pressure at bath temperatures not exceeding 40°. equipment for m.s., n.m.r. spectroscopy, g . l . c ,  The  and g . l . c . - m.s.  was the same as that used in the i n v e s t i g a t i o n of K l e b s i e l ! a Kl2 polysaccharide (see S e c  I I I . l ) Paper electrophoresis was performed  on ' a Savant high voltage (5 KV) system (model LT - 48A) with 1  kerosene as coolant.  The buffer used contained pyridine - a c e t i c  acid - water (5:2:743, v/v) pH 5.3.  S t r i p s of Whatman No. 1 paper  (77 cm x 20 cm) were used f o r a l l runs, with a p p l i c a t i o n of 25-50 mA  97  f o r 1% h. For descending paper chromatography the f o l l o w i n g solvent system (v/v) were used:  .(1_) f r e s h l y prepared 2:1:1 1-butanol - a c e t i c  acid - - water, (2J 8:2:1 ethyl acetate - pyridine - - water, and (_3) 18:3:1:4 ethyl acetate - a c e t i c acid - formic acid - water. Sugars and oligosaccharides were detected with an a l k a l i n e s i l v e r n i t r a t e 21 reagent  . A n a l y t i c a l g . l . c . separations were performed i n s t a i n l e s s -  steel columns (1.8 x 3 mm) with a c a r r i e r - g a s flow-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 f o r preparative g . l . c . Preparation and properties. —  separations. A culture of Klebsiel1 a K58 (636/52)  was. obtained from Dr. I. (j)rskov (Copenhagen).  The polysaccharide  was i s o l a t e d as previously described, (see Sec. I I . l ) and showed [a]  D  + 1 9 . 0 ° (c 1, water).  Analysis of constituent sugars. —  Methanolysis of a sample (20 mg)  of K58 polysaccharide with 3% methanolic hydrogen chloride and subsequent reduction with sodium borohydride in anhydrous methanol reduced the uronic e s t e r .  Hydrolysis with 2M t r i f l u o r o a c e t i c acid  overnight at 95°, followed by reduction (NaBH^), and a c e t y l a t i o n , gave g a l a c t i t o l hexaacetate, g l u c i t o l hexaacetate, and f u c i t o l pentaacetate in the r a t i o 1:2:1 min and then 4°/min to 240°).  (column C_, programmed at 180° f o r 8 C i r c u l a r dichroism ( c . d . ) of the  l a t t e r two components i s o l a t e d by preparative g . l . c ,  showed  98  p o s i t i v e and negative c . d . curves r e s p e c t i v e l y , confirming that glucose has the D configuration and fucose the L c o n f i g u r a t i o n . Galactose was shown to be of the D configuration by the p o s i t i v e action 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 the Hakomori  247  conditions, followed by a Purdie  242  treatment y i e l d e d  a product that showed no hydroxyl absorption in i t ' s i . r .  spectrum.  This material was reduced overnight with sodium borohydride i n 1:1 (v/v) oxolane (THF) and ethanol. A portion of the product was hydrolyzed with t r i f l u o r o a c e t i c acid (2M) f o r 16 h at 95°, and the mixture was reduced with sodium borohydride and then a c e t y l a t e d . G . l . c . - m.s. gave the r e s u l t s shown in Table 8. Another portion of the reduced, uronic ester material was re-methylated under the Purdie conditions f o r 2 days, and d e r i v a t i z e d f o r g . l . c . - m.s. giving the compounds shown i n Table 8. A portion (10 mg) of the re-methylated material was hydrolyzed with 90% formic acid f o r 30 min at 95° to remove the 1-carboxyethylidene group.  Methylation under the Purdie conditions f o r 2 days,  and d e r i v a t i z a t i o n for g . l . c . - m.s. gave the r e s u l t s shown in Table 8.  Uronic acid degradation  —  A s o l u t i o n of c a r e f u l l y d r i e d , methylated  polysaccharide (100 mg) and p_-toluenesulfonic acid (a trace) in 19:1  99  dimethyl s u l f o x i d e and 2,2-dimethoxypropane (20 mL) was prepared i n a serum v i a l which was:sealed with a rubber cap.  The v i a l was flushed  with dry- n i t r o g e n , and the s o l u t i o n was s t i r r e d for 3 h. Sodium methylsulphinylmethanide (2M) i n methyl sulfoxide (10 mL) was then added with the a i d of a syringe, and the s o l u t i o n was s t i r r e d overnight at room temperature.  After cooling to 10°, ethyl iodide  (3 mL) was added slowly, using a syringe  273  Following the addition of water, the e t h y l a t e d , degraded product was i s o l a t e d by p a r t i t i o n between chloroform and the aqueous s o l u t i o n . Hydrolysis of the i s o l a t e d product was performed with 2M t r i f l u o r o a c e t i c a c i d ; g . l . c . - m.s. analysis of the a l d i t o l acetate d e r i v a t i v e s y i e l d e d peaks corresponding to 4 - 0 - e t h y 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 l e b s i e l l a K58 capsular polysaccharide  (200 mg) was dissolved in water (25 mL), to which a s o l u t i o n (25 mL) of 0.1M sodium metaperiodate was then added.  The s o l u t i o n was  s t i r r e d i n the dark at 3° and periodate consumption was monitored (Fleury-Lange method)  , a f t e r 10 h consumption had reached 1.8  molecules per repeating u n i t . Ethylene glycol (10 mL) was then added, and, a f t e r s t i r r i n g f o r a f u r t h e r 30 minutes the mixture was dialyzed overnight against running tap-water, and the product was reduced with sodium borohydride. d i a l y s i s and l y o p h i 1 i z a t i o n .  The polyol was i s o l a t e d by  Smith hydrolysis (0.5M TFA overnight  TOO  at room temperature) gave a polymeric product.  A portion (5 mg)  of t h i s 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  glucose and fucose i n the r a t i o 2:1.  H and  13 C n.m.r. spectral  data are shown i n Table J. Methylation analysis of a portion (20 mg) of the Smithdegradation product, as f o r the native polysaccharide, gave the r e s u l t s shown in Table 8 . Autohydrolysis —  A sample of K l e b s i e l l a K58 polysaccharide was  exchanged to the f r e e - a c i d form with Amberlite IR-120 (H*) i o n exchange r e s i n , and the s o l u t i o n was l y o p h i l i z e d .  A portion of  t h i s material (100 mg) was dissolved in 5 mL ^ 0 (pH 3.0) and was introduced to a sealed length of standard c e l l u l o s e d i a l y s i s tubing. This was autohydrolyzed in 100 ml ^ 0 at 95° f o r 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  e a r l i e r gave the r e s u l t s shown i n Table 8 .  P a r t i a l Hydrolysis —  A sample (300 mg) of the native polysaccharide  was hydrolyzed with 0.5M H^SO^ f o r 30 min at 95°.  The products were  separated by preparative paper chromatography (solvent 3). Compound  101  1  (35 mg) with  0.63 and [ a ] - 2 0 . 0 ° (c_ 1, water) was shown to be D  1 the aldobiouronic acid by 2 (30 mg) with 3 (10 mg) with  H and  C n.m.r. spectroscopy.  Compound  0.32 and [ a ]  D  + 16.0° (C 1, water) and compound  0.12 and [ a ]  n  + 38.0° (c 0.5, water) were s i m i l a r l y  u IC  'M  13  U  —  shown to be the a l d o t r i o u r o n i c and aldotetraouronic acids r e s p e c t i v e l y (see Table 7 ) . Deacetylation Deacetylation was achieved by treatment of a s o l u t i o n of the native polysaccharide (200 mg) i n water (25 m L) with excess of sodium borohydride for 3h, at room temperature, with s t i r r i n g . D i a l y s i s and l y o p h i l i z a t i o n y i e l d e d 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 i n l y ether proved d i f f i c u l t , giving small amounts of methylated sugars other than 2-ONe fucose, on g . l . c . - m . s . a n a l y s i s , the 275 0-acetyl l o c a t i o n procedure of de Belder and Norman  was performed  on both the native polysaccharide, and a deacetylated sample. Reaction conditions were i d e n t i c a l in both cases. The e n t i r e reaction was c a r r i e d out in a sealed v i a l , flushed with nitrogen.  A s o l u t i o n of c a r e f u l l y d r i e d 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 r o z e n (-60 ) and methyl v i n y l e t h e r (ca 3 mL) was i n t r o d u c e d from a gas b o t t l e t o the r e a c t i o n vessel v i a . a hypodermic syringe.  The s o l u t i o n was brought t o room temperature and s t i r r e d  f o r 3 hr.  Two f u r t h e r p o r t i o n s o f methyl v i n y l  e t h e r were  similarly  i n t r o d u c e d , a f t e r which t i m e t h e r e a c t i o n m i x t u r e had a r e d / y e l l o w colour.  Methylsulfinyl  anion i n d i m e t h y l s u l f o x i d e  (3 mL) was  i n t r o d u c e d and the r e a c t i o n m i x t u r e s t i r r e d f o r a f u r t h e r 30 min. Methyl i o d i d e  (2 mL) was added t o the cooled m i x t u r e which was then  stirred for Ih.  Dialysis  ( 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  m i x t u r e o f d e r i v a t i z e d p o l y s a c c h a r i d e and p o l y m e r i c methyl ether.  vinyl  Pure p o l y s a c c h a r i d e was e l u t e d froma column o f Sephadex  LH-20 w i t h methanol. Since the 0 - a c e t y l  s u b s t i t u e n t c o u l d n o t be on the  g l u c u r o n i c a c i d t h i s f u n c t i o n a l i t y was not reduced.  The p o l y s a c c h a r i d e  was h y d r o l y z e d w i t h t r i f l u o r o a c e t i c a c i d (2M), f o r 16h a t 9 5 ° , and the m i x t u r e was reduced w i t h sodium b o r o h y d r i d e , 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 p o l y s a c c h a r i d e and 1 : 1 f o r the d e a c e t y l a t e d (g.l.c.  polysaccharide  - 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 - a c e t y l  group i s  attached to 0 - 2 o f fucose.  Note added i n  proof.  Dr. J..M. F o u r n i e r ( I n s t i t u t P a s t e u r , P a r i s ) has shown t h a t , a l t h o u g h K l e b s i e l l a K58 c a p s u l a r p o l y s a c c h a r i d e i s n o t v i r u l e n t m i c e , immunization w i t h t h e p o l y s a c c h a r i d e does p r o v i d e  for  protection  103  against i n f e c t i o n with the v i r u l e n t K l e b s i e l l a Kl capsular 277 polysaccharide.  This i s in agreement with the s i m i l a r i t i e s  the structures of the two polysaccharides (see App.I).  in  104  III. 3  CONFIRMATION OF THE STRUCTURE OF KLEBSIELLA K23 CAPSULAR POLYSACCHARIDE. 2 78 Structural investigation  of K l e b s i e 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 i d e - c h a i n , 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 i n the backbone was 3 - l i n k e d and that the rhamnosyl residue was a-linked.  The anomeric nature of the side-chain residues could not,  however, be demonstrated conconclusively. To obtain t h i s information the methylated polysaccharide was subjected to a 3 - e l i m i n a t i o n 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 i n k e d glucuronic acid residue (see Table  9).  By deduction the glucosyl residue i n the side-chain  105  JMLE_9„'  P.M.R.  :  DATA FOR K l e b s i e l l a K23 CARSULAR POLYSACCHARIDES  Compound  6-  Integral  Assignment  Methylated,  5.21  1  a-Rha  Native  5.08  1  a-Glc  Polysaccharide.  4.52  1  3-Glc  4.34  1  3-GlcA  1.31  3  Methylated/ethylated,  5.24  1  a-Rha  degraded  5.10  1  a-Glc  polysaccharide.  4.55  1  3-Glc  1.31  3  CH of Rha  1.21  3  CH of ethyl  b  CH of Rha. 3  3  3  - C h e m i c a l s h i f t r e l a t i v e to i n t e r n a l acetone; 6 2.23 downfield from sodium 4,4-dimethyl -4-si1apentane-1-sulfonate ( D . S . S . ) . Spectra were run in CDC1 at 270MHz and ambient temperature. 3  - a - R h a = proton on C-l of a - l i n k e d l-Rha residue, e t c .  106  TABLE 10  METHYLATION ANALYSIS OF ORIGINAL AND BASE DEGRADED K l e b s i e l l a K23 CAPSULAR POLYSACCHARIDE.  Methylated sugars(as a l d i t o l acetates)  T-  IMole %-  IIMole %£  4-Rha  0.91  26.0  34.1  2,3,4,6-Glc-  0.72  —  32.4  2,3,4-Glc  1.13  48.1  2,4,6-Glc  1.00  25.9  — 4-Rha = 1,2,3,5-tetra-0-acetyl-4-0-methylrhamnitol  33.5  etc.  — Retention time r e l a t i v e to a l d i t o l acetate of 2,4,6-tri-O-methyl-Dglucose on OV-225. — I , o r i g i n a l polysaccharide, methylated and uronic ester reduced. — I I , degraded polymer obtained a f t e r g-el imi nation. — Values are corrected by use of the e f f e c t i v e carbon response factors given by Albersheim et a l . — 1,5-Di-0-acetyl-6-C^-ethyl-2,3,4-tri-0-methylglucitol  107  is a-linked. The product was d i r e c t l y ethylated  106  , thus l a b e l l i n g  the p o s i t i o n of attachment of the glucuronic acid residue.  Hydrolysis,  103 121 and g . l . c . - m.s.  '  analysis of the ethylated product showed  that only the glucuronic acid residue had been removed and v e r i f i e d that i t was attached to 0-6 of the side-chain glucosyl  residue  (see Table 10). I t thus follows that the K l e b s i e l l a K23 polysaccharide has the s t r u c t u r e ~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 e s s e n t i a l l y the same as those used i n the i n v e s t i g a t i o n of the structure capsular polysaccharide (see Section 111,1).  of K l e b s i e l l a Kl2  108  III. 4  ]  H AND  1 3  C SPECTRAL INVESTIGATION.OF K l e b s i e l l a K70  CAPSULAR POLYSACCHARIDE The structure of the capsular polysaccharide of K l e b s i e l l a 279 K70 has been shown .  to c o n s i s t of a l i n e a r hexasaccharide repeating  unit having a 1-carboxyethylidene attached to a 2-linked a-L-rhamnosyl residue i n every second repeating unit as shown: - R h a p j ^ l c A p ^ R h a p j - ^ - R h a £ ^h^c_^2  A  B  C  D 1  In that i n v e s t i g a t i o n  H and  some assignments were made.  E  Galp_ F  L  n  13 C n.m.r. spectroscopy were used, and However, the complete assignment of a l l  the signals i n the anomeric region proved d i f f i c u l t , since four a - l i n k e d residues were present, three of which were due to rhamnosyl residues. The polysaccharide has since been degraded by M e r r i f i e l d 298 using bacteriophage, to give an oligosaccharide corresponding to two repeating u n i t s .  Another s p e c i f i c degradation by Mort 323 whereby  the native polysaccharide was cleaved at the uronic acid residue, using l i t h i u m in ethylamine, with concomitant loss of that residue, 1 13 produced a pentasaccharide. penta - and  H and  hexasaccharide now allow  C spectral i n v e s t i g a t i o n s of the 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  l-A-B-C-D-E-F-]  J 5.22  n  (1)  1  1>2  (Hz)  s  i  IJ  Assignment—  Integral 1  a Rha C  105..7  1  103. 8  1  B Gal B GlcA  F B  5.10  s  2  a Rha A+D  4.97  s  1  a Glc  102..9  1  a  Rha  C  7  1  3 Glc E .  101.,7  1  Rha  A  4.55  7  1  B Gal F  100.,9  1  Rha  D  1.59  s  1.5  CH,of acetal  95.,7  1  a a a  Glc  E  1.30  6*  9  CH of Rha  62.,2  1  C-6 Gal  61.,3  1  17.,5  3  C-6 Glc C-6 Rha  105.64  1  B Gal  F  4.77  A-B-C-D-E-F-AW-C (2)  13 Data C n.m.r. d Integral Assignmentp.p. m—  Vn.m.r. Data  Compound^  3  -E^F - OH 5.28  2  0.7  5.23  2  1.8  a  5.10  s  4  4.98  s  4.80  1  E  Gal OH.F  1  a  .1 Rha C + C  103.88  2  B GlcA  B + B  a Rha A+AUD+D'  102.79  2  Rha  C+ C  2  a Glc E + E  101.7  1  a a  Rha  A  7  2  B GlcA B+B  100.98  2  a Rha  4.58  4  0.3  B GaVOH F  4.40  7  1  6 Gal F  1.30  6*  9  1  1  1  CH of Rha 3  99.7  1  a  Rha  98.61  1  a  Glc  97.28  0.6  B Gal-OH  95.77  1  a Glc  94.80  0.4  a Gal-OH  61.78  C-6 Gal  61.36  C-6 Glc  17.6  C-6 Rha  D+ D .1  TABLE 11 Contd.  5.23  2  0.8 1  5.10  3  1  5.32  D-E-F-A-OH (3)  2  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  1  a Rha  D  4.98  2  1  a Glc  E  101.0  4.80  s  0.2  B Rha-OH A  95.7  E  B Gal  1 0.6  a Glc  93.9  a Rha-OH  A  93.6  0.4  B Rha-OH  A  4.55W  4  4.58  4  1.30  &a  0.4 9  F  CH of Rha 3  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 s h 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 s h i f t in p.p.m., downfield from Me.Si, relative 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,- . overlapping doublets centred at 61.30. h - A small amount of B-GlcA was not eliminated. -The chemical s h i f t of this proton is affected by the a,B equilibrium of the reducing Rha residue. fi  in  Acknowledgments I wish to thank Dr. I. (j)rskov (Copenhagen) f o r the K l e b s i e l l a cultures used, Dr. A.A. Grey (Toronto) f o r 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) f o r a sample of degraded K70. The i s o l a t i o n and i n i t i a l i n v e s t i g a t i o n of K l e b s i e l l a Kl2 polysaccharide were performed by C h r i s t i a n e M a r t e l .  112  BACTERIOPHAGE DEGRADATION OF K l e b s i e l l a CAPSULAR POLYSACCHARIDES K21, K12 and  K41.  113 IV.  1.  Introduction  The f i e l d of medicinal microbiology became well - established i n the period between 1880 and 1900 with the i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of many animal diseases.  of the causative agents of both human and  In 1892, Iwanowski and B e i j e r i n c k while indepen-  dently studying the tobacco mosaic disease, and Pasteur while carrying out studies on r a b i e s , recognised the causative agents to be " f i l t e r a b l e substances", which the l a t t e r termed " v i r u s e s . "  280  Some years l a t e r , i n 1915 and 1917 virus i n f e c t i o n s in bacteria were also described.  Twort and d ' H e r e l l e demonstrated  independantly that cultures of b a c t e r i a l c e 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 u n t i l the electron microscope was  developed that the morphological character of viruses was e l u c i d a t e d . Today, bacteriophages (phages, designated  are the best  characterized and studied group of v i r u s e s , since t h e i r propagation and manipulation has proven t e c h n i c a l l y much e a s i e r than equivalent studies on other types of v i r u s e s .  281  Phages, which are quite d i f f e r e n t from other virus types, i n that they tend to be s t r u c t u r a l l y more complex, are grouped 282 according to the morphological c l a s s i f i c a t i o n of Bradley (see  F i g . 11). For type A (see F i g . 12) • the head, which i t s e l f  i s composed of repeating i d e n t i c a l protein monomers, has b a s i c a 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 o f a Type-A phage p a 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 h e l i c a l l y arranged protein molecules, encased by a c o n t r a c t i l e sheath, also composed of protein monomers. attached the t a i l  The plate contains small pins to which are  spikes.  283 a  Type B i s s i m i l a r to type A, sheath.  but without the c o n t r a c t i l e  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  filamentous.  Bacteriophage which T.yse encapsulated bacteria often form plaques, surrounded by large haloes that continue to spread a f t e r growth has ceased (see App. IV).  Within the haloes the 283b c  b a c t e r i a have l o s t t h e i r capsules.  I t has been long known that,  generally, the formation of these haloes i s due to the production of enzymes during phage i n f e c t i o n .  These enzymes d i f f u s e from the  plaque and catalyze the hydrolysis of the bacterial capsules.  A  wide v a r i e t y of enzymic a c t i v i t i e s , c a t a l y z i n g d i f f e r e n t degradation reactions of host surface, polysaccharides may be associated with 284 b a c t e r i a l virus p a r t i c l e s . So f a r esterases ( s a p o n i f i c a t i o n of 0-acetyl s u b s t i t u e n t s ) , 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 addition to whole v i r u s , and that the n  p u r i f i e d spikes exert the same g l y c o s i d i c 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 p a r t i c l e i n f e c t s a susceptible h o s t i t causes that c e l l to l y s e , with the concomitant release of a c h a r a c t e r i s t i c number of newly formed phage p a r t i c l e s .  The phases  of the cycle include the f o l l o w i n g : (i)  absorption of the phage p a r t i c l e s to the susceptible host  (ii)  i n j e c t i o n of v i r a l DNA (or RNA) into the host  (iii)  r e p l i c a t i o n of the phage n u c l e i c acid and synthesis of phage p r o t e i n , 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 t e s on the b a c t e r i a l c e l l .  There i s considerable  evidence that r e c i p r o c a l charges on the phage t a i l and on the receptor s i t e s of c e l l s are involved in the formation of e l e c t r o s t a t i c bonds during attachment. Phage are r e l a t i v e l y easy to i s o l a t e from almost any 287 b a c t e r i a l environment, f o r example, sewage  .  Those a c t i v e  on exopolysaccharide producing b a c t e r i a l s t r a i n s are generally exopolysaccharide s p e c i f i c ; non - capsulate or non - slime producing mutants are r e s i s t a n t to the phages.  One common feature of a l l  phage, a c t i v e on exopolysaccharides, i s that the baseplates, as seen i n the electron microscope, are provided with spikes, and that no t a i l f i b r e s are seen.  The spikes appear as hollow tubes  117 (12.5 nm i n K l e b s i e l l a phage 11).  286  Stirm and Rieger - Hug,  288  employing seventy four s e r o l o g i c a l l y d i f f e r e n t K l e b s i e l l a t e s t s t r a i n s , tested the host range of f i f t y f i v e K l e b s i e l l a bacteriophage. The v i r a l depolymerases proved to be very s p e c i f i c (33 not crossr e a c t i n g , 18 c r o s s - r e a c t i n g with one, 2 with two, 1 with three and 1 with four heterologous polysaccharides.  § 12 cross-reacts with  34 K41,  which i s i n agreement with the structure proposed f o r K12  capsular polysaccharide. Depolymerization of a polysaccharide using bacteriophage y i e l d s products corresponding to a s i n g l e repeating unit of the polysaccharide, and multiples thereof, with l a b i l e 0 - a c e t y l (pyruvate) groups i n t a c t .  2 8 9 b  2 8 9 a  and 1-carboxyethylidene  These may then be used f o r (a) the  preparation of synthetic antigens,  7 7  ( b ) d e t a i l e d examination by nuclear  magnetic resonance spectroscopy, and (c) the study of conformations i n 290 solution.  The degradation of K l e b s i e l l a K21 capsular polysaccharide  using p u r i f i e d <)j 21 p a r t i c l e s , and the degradation of K l e b s i e l l a Kl2 and K41 using a crude <|) 12 suspension are presented here, and r e s u l t s compared in terms of e f f i c i e n c y of depolymerization and y i e l d s of oligosaccharides. IV.  2.  IV.  2. 1.  RESULTS I s o l a t i o n and p u r i f i c a t i o n Both < > j 21 and § 12 were i s o l a t e d from sewage, and stock  suspensions in broth were obtained by the confluent l y s i s method. The bacteriophage were propogated on t h e i r host s t r a i n s , K l e b s i e l l a K21  118  and 12 r e s p e c t i v e l y , to a volume of v l . 5 L . . (see App.IV) p u r i f i e d by p r e c i p i t a t i o n with poly (ethylene g l y c o l ) 6000  <|) 21 was 291  ,  followed by isopycnic c e n t r i f u g a t i o n , and was shown by electron microscopy (see F i g . 15) to belong to Bradley Type B.  IV. 2.2.  282  Conditions of depoTymerization The p u r i f i e d capsular polysaccharide from K l e b s i e l l a  K21  292  was dissolved in buffered s a l i n e , and the depolymerization 293  was followed v i s c o m e t r i c a l l y and by assay of the reducing power (see F i g . 13) which became constant a f t e r 24 h. The depolymerizations of K l e b s i e l l a K12 and K41 capsular polysaccharides were c a r r i e d out i n separate, crude,.broth suspensions of <[) 12 p a r t i c l e s and followed v i s c o m e t r i c a l l y . Although the v i s c o s i t y of both solutions decreased dramatically during the f i r s t 3 h:.  the  reactions were allowed to continue f o r 48 h. IV. 2. 3.  P u r i f i c a t i o n and analyses of products of depolymerization of K21. The l y o p h i l i z e d depolymerization mixture was desalted on a 95  column of Sephadex Gl0.  The carbohydrate f r a c t i o n  was l y o p h i l i z e d ,  redissolved in T r i s HC1 buffer, and the s o l u t i o n added to a column of DEAE - Sephadex A25.  The e l u t i o n pattern i s shown in F i g . 14,  where PI represents the s i n g l e repeating unit of the polysaccharide, P2 the double repeating u n i t , and P3 polymeric m a t e r i a 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 s o l u t i o n , on incubation with bacteriophage.  120  ~1  50  1  100  1 —  150  Elution volume ( m l ) Figure 14  Separation of products of bacteriophage degradation of K l e b s i e l l a K21  121  Figure 15  Electron Micrograph of p u r i f i e d K21 bacteriophage, negatively stained. X=122,500 Courtesy of Dr. H. Chanzy C.E.R.M.A.V. (Grenoble). Bradley Type B. 2 8 2  122 spectroscopy (see Table 12).  Several spurious peaks were observed at  low f i e l d , but, a f t e r passage through a column of Amberlite IR-120 (H ) r e s i n gave the spectra shown in App. . I I I . +  H and  C spectra demonstrate that PI i s a hexasaccharide  corresponding to one repeating-unit, with galactose as the reducing residue, and P2 i s composed of two repeating u n i t s .  The two  oligosaccharides were analyzed by g a s - l i q u i d chromatography, using 102 the method of Morrison,  294-297 '  whereby the r a t i o of acetylated  a l d o n o n i t r i l e to acetylated a l d i t o l i s determined. F i g . . 7,  Table 13), confirmed that PI i s a hexasaccharide and that P2  i s the dimer. R  The results(see  The m o b i l i t y of PI i n paper chromatography was  G l c 0-045, and i n paper e l e c t r o p h o r e s i s , PI and P2 had R g ^ 0.75  and 0.85, r e s p e c t i v e l y . IV. 2. 4.  P u r i f i c a t i o n 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 d i a l y s a t e s were l y o p h i l i z e d .  Preparative  paper chromatography y i e l d e d products free of most broth contaminants. Only baseline carbohydrate spots were observed.  Further p u r i f i c a t i o n ,  by passage through a column of Amberlite IR-120 (H ) r e s i n gave products +  which were examined by n.m.r. spectroscopy. The r e s u l t s shown in Table 14 show that the product of depolymerization of K12 polysaccharide with < > j 12 i s a hexasaccharide, corresponding to a s i n g l e 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/  1 H- n.m.r. dataIntegral Assignment (H)  Compound  K21 polysaccharide 292  5.48 5.30 5.25  Oligosaccharide PI  1  a-GlcA  2  a-Gal a-Man  5.08  1  a-Man  4.88  1  3-Gal  1.55  3  acetal  5.49  1  a-GlcA  5.34 5.30  a-Gal 2.4  a-Man a-Gal-OH  5.10 4.66 1.52 Oligosaccharide P2  5.46 5.31 5.27 5.06 4.87 4.64 1.53  a-Man 1  B-Gal-OH  0.6 3 1  acetal  2.3  a-GlcA a-Gal a-Man  1  a-Gal-OH  0.5  B-Gal  0.2 1.5  6-Gal-OH acetal  Chemical s h i 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 d e r i v a t i v e of  Relative Retention time  Moles %-  0V-17-  PI  P2  Mannononitrile  0.67  2.0  2.0  Glucononitrile  0.72  0.91-  0.93-  Galactononitrile  0.75  0.98  1.5  Galactitol  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 f a c t o r s . — Due to incomplete reduction of uronic a c i d .  TABLE 14 P.M.R. DATA FOR K l e b s i e l l a K12 CAPSULAR POLYSACCHARIDE AND THE PHAGE DERIVED OLIGOSACCHARIDE.  1 H-n.m.r. data  Compound—  ^Gl c ^ h a ^ G a l p j - ^ a l f 3  i  pyruvate  Assignment—  l,2 (Hz)  Integral  5.22  s  1  a-Rha  5.16.  3  1  a-Glc  5.13  2  5.13  2  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 of Rha  J  2  a-Gal£ B-Galf  3  TABLE 14  (Contd..)  1H-n.m.r. data  Compoundb  Gl c ^ R h a ^ G a l p^-?€al ~0H a  a  ^ a  PI 6  3 g  1.2 (Hz)  J  Integral (H)  Assignment—  5.26  b  .6  a-Rha  5.20 5.16  b s  .6  a-Gal~0H  .6  a-Glc  1 G' cA 4 g  5.09  b  1  a-Gal  4.61  b  1  g-GlcA  1  4.52  8  .4  4.48  6  (1)  1.66  b  CH^ of acetal  1.34  b  CH of Rha  4  pyruvate  ro  g-Gal~0H g-Gal  3  - For the origin of compound PI see text. See Appendix I I I for reproductions of the spectra -Chemical shift 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 . a n a l y s i s (see above). In contrast, the product of the depolymerization of K41 polysaccharide with (j) 12, was shown to be an oligosaccharide 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, i s to obtain pure oligosaccharides, i n as high a y i e l d as p o s s i b l e .  In p u r i f y i n g the bacteriophage, as  f o r <)j 21, the volume of the phage suspension i s reduced by a f a c t o r of p  10 , but with a concomitant loss of ^ 75% o f the phage.  The  e f f i c i e n c y of depolymerization i s , however, very high, g i v i n g a good y i e l d of the s i n g l e repeating u n i t . Pure oligosaccharides may also be obtained with a crude s o l u t i o n of bacteriophage, thereby a l l e v i a t i n g the time consuming and expensive (CsCl) p u r i f i c a t i o n s using u l t r a c e n t r i f u g e s . In the degradations of Kl2 and K41, the y i e l d s of the single repeating u n i t were low ( f o r K41 only the double repeating u n i t was obtained).  This can be improved upon by using a higher t i t r e  pf crude phage p a r t i c l e s .  298  D i a l y s i s of the products, using tubing with a low exclusion l i m i t , i s an e f f i c i e n t method of separating the s i n g l e and double repeating u n i t s from l a r g e r m a t e r i a 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 6—  6  f i l c  L3  R h a  a  L3  G a l  a  iZ a  G a l f  3I  Glci^lc^GlcA B  a  P2 n =2  —  I '  Integral  ^C-n.m.r. data  Assignment-  p.p.m.—  Integral  Assignment—  5.48  a-Glc  109  B-Galf  5.22  a-Rha  105.5  B-Glc  5.17  a-Glc  104.8  6-GlcA  5.12'  a-Gal£  104.45  a  5.12  B-Galf  101.55  a-Gal  4.63  B-GlcA  101.05  a-Glc  4.52  B-Glc  99.5  a-Glc  1.34  CH of Rha  19.7  CH of Rha  3  -Rha  3  1  a-Glc  107.2  0.5  B-Galf  1.3  a-Rha  103.44  1  B-Glc  a-Gal~0H  102.64  1  B-GlcA  a-Glc  101.7  0.5(?)  a-Rha  a-Gal£  100.24  1  a-Gal  B-Galf  100.10  1  a-Glc  B-GlcA  99.96  1  a-Glc  B-Glc  97.2  0.2(?)  B-Gal~0H  B-Gal~0H  91.10  0.3(?)  a-Gal~0H  CH of Rha.  17.65  2.5 1 1.2  3  CH of Rha. 3  - For the origin of compound P2, see text. See Appendix I I I for reproduction of the spectra. -Chemical s h i f t relative to internal acetone; 62.23 downfield from sodium 4,4-dimethyl-4-silapentane-l-sulfonate - For example, a-Gal = Proton on C-l of a-linked Q - Gal residue. -Chemical s h 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.  (D.S.S.)  129 crude phage s o l u t i o n in both i s employed, then lowM-W. material from the broth contaminates the oligosaccharides. The a l t e r n a t i v e then, i s f i r s t to reduce the volume of phage suspension, (by l y o p h i l i z a t i o n , or by rotary evaporation at < 40°)  2 9 8  and d i a l y s i s of t h i s s o l u t i o n to remove  broth contaminants.  Low M.W. products may then be e a s i l y i s o l a t e d by d i a l y s i s , and separated, e i t h e r 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 n a l l y i s o l a t e d from Freiburg  sewage, and were propogated on t h e i r respective host s t r a i n s by tube and b o t t l e lyses (see App. IV).  Purification 4 21 l y s a t e , which had a t i t r e of 7.5 x lO^PFU/mL ( 1 . 5 L ) , was centrifuged at 5,000 g (20 min) and made 0.5 JJ in NaCl.  10%  w/v of poly (ethylene g l y c o l ) 6000 was added slowly to the supernatant and a f t e r storage at 4° f o r 48 h were sedimented at 20,000 (g.) (30 min). to contain 0.15% of phage.  v  the phage p a r t i c l e s  The supernatant was shown  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 purified 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 p e r i s t a l t i c 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 a standard curve based on galactose.  293  P u r i f i c a t i o n and separation of depolymerized K21 m a t e r i a l . Portions of the crude l y o p h i l i z e d depolymerization mixture (2 x 1.5 g) were desalted using a column of Sephadex G10 (100 cm x 19.5 cm ).  The column was eluted with buffer (water - - pyridine - -  2  g l a c i a l a c e t i c a c i d , 1000:10:4, pH 4.5) at a flow rate of 25 mL/h and carbohydrate material was l o c a l i z e d using the Molisch t e s 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 e q u i l i b r a t e d with 0.025^ Tris/HCl buffer; at l e a s t 10 column volumes of the l a t t e r buffer are required to achieve e q u l i b r a t i o n as determined by performing conductivity measurements. Tris/HCl(2 mL).  The material was applied as a s o l u t i o n in 0.025M The column was eluted (10 mL/h) with 0.025^  Tris/HCl (140 mL) and a l i n e a r s a l t gradient (from 0 to 0.35M NaCl) was then begun.  Fractions (2 mL) were c o l l e c t e d and examined  using the p h e n o l - s u l f u r i c acid assay.  The e l u t i o n p r o f i l e i s shown  in F i g . 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 eliminated by passage through a column of Amberlite IR-120 (H ) ion - exchange r e s i n .  The  13  C spectrum of PI was in agreement with  132 the H n.m.r. spectrum.(§ee App. I I I ) . 1  The degree of polymerization of the products (the r a t i o of acetylated a l d o n o n i t r i l e s to acetylated a l d i t o l ) was determined 102 using the method of Morrison.  The oligosaccharide (5 mg) was  reduced with sodium borohydride (excess) f o r 3 h (reducing sugar converted to a l d i t o l ) . the methyl ester  2 9 9  The glucuronic acid was then reduced v i a ,  by methanolysisand reduction (see Sec.  II.4.1.)  The residue was hydrolyzed with t r i f l u o r o a c e t i c acid (2MJ at 95° f o r 16 h, and evaporated to dryness.  The aldoses were converted to  the oxime by heating at 95° for 15 min with 5% hydroxylammonium c h l o r i d e in pyridine (0.2 mL/mg of aldose).  After c o o l i n g , a c e t i c  anhydride (0.2 mL/mg of aldose) was added and heating was continued f o r a f u r t h e r 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 r e s u l t s  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 - - a c e t i c acid - - water (5:2:743, v / v ) , pH 5.3. S t r i p s of Whatman no. 1 paper (77 cm x 20 cm) were used with a current of 100 mA for 4 h. f r e s h l y prepared 2:1:1  For descending paper chromatography  1 butanol - - a c e t i c 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 identical. P u r i f i e d 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 s c o s i t y of the s o l u t i o n was  determined p e r i o d i c a l l y , and was shown to decrease s u b s t a n t i a l l y in both cases, w i t h i n 3 h. A f t e r 48 h the s o l u t i o n was transferred to a sealed portion of d i a l y s i s tubing (M.W. c u t o f f = 3,500) and dialyzed against three portions (200 mL) of d i s t i l l e d water.  The d i a l y s a t e s were reduced  to dryness and broth contaminants were removed by preparative paper chromatography using f r e s h l y prepared 2:1:1 1-butanol - - a c e t i c acid - - water. The y i e l d of the s i n g l e 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 - I n s t i t u 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 l a s s i c a l column l i q u i d chromatography has been  an e f f e c t i v e separation method since the beginning of t h i s century, i t i s s t i l l characterized by low column e f f i c i e n c i e s and long separation times. . However, in the l a s t decade i t has been recognised that column e f f i c i e n c y and speed of separation could be improved by several orders of magnitude i f materials of very small p a r t i c l e s i z e are used as column packings.  Such columns give a high t h e o r e t i c a l plate number which  decreases only s l i g h t l y with flow v e l o c i t y .  As a r e s u l t , a high  resolving power and speed of separation can be achieved.  Since such  columns require a high pressure f o r t h e i r operation, t h i s modern version of column l i q u i d chromatography i s often c a l l e d high-pressure l i q u i d chromatography (HPLC). 30i  The fundamental instrumentation necessary f o r HPLC separations consists of (see Scheme 12) (a) a solvent r e s e r v o i r , (b) a pump capable of giving flow against moderately high back pressures (6,000 psi) with a recycle c a p a b i l i t y (c) an i n j e c t i o n head (d) a column, f i t t e d  with a  pre-column, (e) a detector ( f o r carbohydrate a n a l y s i s , usually a thermostated r e f r a c t i v e index detector) and (f) a .recorder/integrator/ data system. The basis of chromatographic separation i s the d i s t r i b u t i o n (or p a r t i t i o n ) of sample components between two phases which are 301 immiscible.  The i n t e r a c t i o n s between the molecules of the mobile  and stationary phases determine the degree of sorption of p a r t i c u l a r substances and also the effectiveness or s e l e c t i v i t y of the separations.  137  INJECTOR  PUMP  PRE-COLUMN  s  Recycle  COLUMN  ii  T  DETECTOR  SOLVENT  System C o n t r o l l e r Recorder Integrator  Scheme 12.  1  FRACTION COLLECTOR  DATA MODULE  Block Diagram of Instrumentation f o r High Performance Liquid Chromatography  138  Gas chromatography separations are based on vapour pressure. L i q u i d 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 m a t e r i a l , i s of prime importance i n obtaining separation. Research to date on the a p p l i c a t i o n of HPLC to carbohydrate analysis has focused on the q u a n t i t a t i v e analysis of sugars in foods 302-304 and beverages  .  fructose and sucrose.  The main sugars of i n t e r e s t here are glucose, Since three d i f f e r e n t types of sugars (hexose,  ketose, disaccharide) are involved, the technique has enjoyed some success i n routine a n a l y s i s , thus replacing paper chromatography (which i s time consuming) and g a s - l i q u i d chromatography (which requires d e r i v a t i z a t i o n ) . As y e t , the a p p l i c a t i o n of HPLC in the s t r u c t u r a l of heteropolysaccharides has been l i m i t e d shows much promise.  3 2 4  analysis  , although the technique  The following areas of a p p l i c a b i l i t y are  apparent. On an a n a l y i t i c a l  level:  (i)  Constituent analysis of the native polysaccharide.  (ii)  Constituent analysis of the products of Smith degradation.  (iii)  To monitor the p a r 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 v e l : (vi)  To obtain oligosaccharides from p a r t i a l hydrolysis and from Smith degradation.  (vii)  To p u r i f y and separate the products of bacteriophage degradation.  ( v i i i ) To obtain methylated/ethylated oligosaccharides from basedcatalysed degradation (of the methylated polysaccharide).. (ix)  To separate methylated oligosaccharides obtained from methylation of the products of p a r t i a l  (x)  hydrolysis.  To separate methylated/ethylated oligosaccharides obtained on e t h y l a t i o n of the products of p a r t i a 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 l o s e l y related compounds e . g . glucose, galactose.and mannose,or e r y t h r i t o l and t h r e i t o l [ f o r ( i ) and (11)1.  (b)  The presence of a c i d i c sugars - i t i s not possible to separate these on amine - bonded columns [ f o r ( i ) , ( i i ) , ( i i i ) , and (vii)]  .  In t h i s study the retention times and molar response f a c t o r are measured f o r (1) the neutral sugars occuring in K l e b s i e l l a capsular polysaccharides, (2) some products of Smith degradation (3) a number of disaccharides and a t r i s a c c h a r i d e , and the a p p l i c a t i o n of HPLC in the analysis of K l e b s i e l l a capsular polysaccharides i s  140 discussed.  V.  2.  Chromatographic Conditions The following equipment was used:  Waters Associates ALC 201 liquid chromatograph equipped with M6000A pump, U6K Universal (septumless) injector, R401 differential refractometer, thermostated at 40° using a Brinkmann Instruments Landa K-2/R circulating water bath,and Waters Model 730 Data Module (printer/ plotter/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: distilled.  Acetonitrile, HPLC grade (BDH), and water, glass  Eluant, Column A:  Acetonitrile water(80:20, 85:15 v/v).  Column B was modified with 500 ml acetonitrile : water (4:1) containing 0.1% of HPLC amine modifier I (NATEC, Hamburg, G . F . R . )  305  >  306  Thereafter the eluant (acetonitrile - 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 i n j e c t e d 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 i s possible to get baseline separation for d i f f e r e n t  classes of sugars, e . g . rhamnose fructose and glucose Table 16,  - F i g . 16).  (see  Separation of the hexoses, mannose, glucose  and galactose (See Fig.17)  i s poor, and because the molar response  factors to r e f r a c t i v e index of galactose and mannose are low, these peaks appear as shoulders on the glucose. (ii)  Separation of d i - and t r i s a c c h a r i d e s I t i s possible to i d e n t i f y mono-,di-and t r i s a c c h a r i d e s ,  based on the r e l a t i v e retention times (see Table 16, F i g . 18). Disaccharides with d i s s i m i l a r structures e.g. c e l l o b i o s e (Glc  Glc) and melibiose (Gal  Glc) give a good  separation, whereas disaccharides with c l o s e l y r e l a t e d structures eg. c e l l o b i o s e and m'al.tose (Glc  Glc) do n o t . a  Comparison of the change i n flow rate from 2 mL/min to 3 mL/min (see F i g . 18) shows that good separation i s  still  obtained with the shorter retention times. The retention times were longer on Column A than on Column B f o r the same flow rate and solvent r a t i o .  TABLE 16 SEPARATION OF MONO- DI- AND TRISACCHARIDES BY HPLC  COLUMN A SUGAR  M.R.F.R.I.  COLUMN B-  2 mL/min (80 : 2 0 ) Time(mins) Rel.R.T.C  2 mL/min (80 : 2 0 ) 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  Cellibiose  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 s p e c i f i c a t i o n s see Chromatographic conditions (V.2). — Molar response f a c t o r to r e f r a c t i v e index. r d — A c e t o n i t r i e : water (v/v) - W i t h 0.01% amine modifier I added. — Retention time r e l a t i v e to that of glucose.  TABLE 17 SEPARATION OF PRODUCTS OF SMITH DEGRADATION USING HPLC.  COLUMN A SUGAR  M.R. F.R.I.  COLUMN B^-  2 mL/min (85 : 15)^ Time(mins) Rel. R.T.e  2 mL/min (90 : 1 0 ) ^ Time(mins) Rel. R.T.  -  0.86  0.07  0.95  0.08  -  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  Erythritol  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  (H 0) 2  Ethylene Glycol  — For column s p e c i f i c a t i o n s see Chromatographic conditions (V.2). — Molar response f a c t o r to r e f r a c t i v e index. — A c e t o n i t r i l e : water (v.v) — With 0.01% amine modifier I added. — retention time r e l a t i v e to that of glucose.  144  RT 8.94  •  1.88 2.61 3.39 5.44 8.36  H  20  glycerol rhamnose fucose fructose glucose  COLUMN  A  SOLVENT  85=15  PRESSURE CHART FLOW  4788.8psi  1.88  CrVHIN  2.88  ML/MIN  00  00  •  Figure 16  Separation of Monosaccharides using HPLC (Column A)  145 Id  IX" r-  see.eee 199  PEAK WIDTH NOISE REJECTION AREA R E J E C T I O N  x. CD CO  ro  cu 0) cu to to to o o CL) o c +-> to c o O E jQ (O c (0 03  CD *  CM x: S- fO Ol  <r •-• o  CL O  u.  • rr—  o  —I o  Li.  u.  LU U .  o>  U.  - J  tx>  CM rw <C CM ID CD CD L U CTi " k £ —< Ct r w m <r h- • < < ^- CM in rw n- in CM •xi CD r o rw in CM CM CM CM  r-i  "Xi  \  X. o  '-i  o  s  CD CE>  (NI  a.  x  •X> t "X> 4k LU  z  at  Z  <x  LU  z> —I  => o  <X>  z 0  «  o  <t r-  CD  z  in in ^ CD • CD CD CD CD co in <X»  - LU <fcCQ  in ct  CO _ J E  O Q£  Figure 17  — f CD •—• ~*  CM CM  CD CD CD CD CD CD 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 <t • • • • • • t— Z  •X*  < x > > x >in I ON* CD ro — in <x»  <I O Z  <r 1— to  CM  CO  CM rw CM  CM CM CM r w rt •+  ro  _J  cr Z  OK:  • co a_ r> ui D C t < r o ~> a. co CJ  CO CO 0 0  <r ui  Ul  Separation of monosaccharides using HPLC (Column B)  o  146  FLOW  3.00 PEAK  589.800  NOISE  100  AREA  S O L V E N T 80: COLUMN  20  ML/MIN WIDTH REJECTION REJECTION  H0 2  glucose Cellobiose Melibiose Raffinose  B  2.00  ML/MIN  PEAK WIDTH NOISE REJECTION AREA REJECTION  Figure 18  Separation of d i - and trisaccharides using HPLC, with d i f f e r e n t f l o w - r a t e s .  JUL. 22, 1989 21=37 PRESSURE 3888.8 S A M P L E #33 COLUMN B EXTERNAL  STANDARD  CHART RUN  1.88  CM/MIN  FLOW  #94  SOLVENT  2.08 CALC  90=10  OPR  ID=  H2O  ML/MIN  #8 1  QUANTITATION  AMOUNT  TOTAL  47  RT  AREA  148.33788  8.81  148337  28813.78888  8.95  28813782  EHL F  66082.98888  1.15  66083403  F  95954.78888 65673.78888  1.85 3.16  95955426 65674198  L  248673.88888  L  -  ethylene glycol glycerol 1rythritol  148 (iii)  Separation of products of Smith degradation.  '  '  Glycerol and e r y t h r i t o l are well separated from each other and from the solvent (H^O) peak.  Ethylene glycol has a  very :short retention time (see Fig.19) and therefore, samples containing t h i s compound should.be injected in a solvent composition i d e n t i c a l to the eluant. co-chromatograph  E r y t h r i t o l and t h r e i t o l  (see Table 17) with solvent 85:15 and have  very s i m i l a r retention times f o r 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 p o t e n t i a l to become an important technique in the s t r u c t u r a l analysis of heteropolysaccharides. columns described here give s i m i l a r r e s u l t s .  The two  Column B has, however,  the advantage of being less expensive (since i t i s home-packed) and a l s o , the separations may be extended to a preparative s c a l e . In t h i s case a l a r g e r p a r t i c l e s i z e of s i l i c a would have to be used to decrease the back-pressure generated.  Column A i s a v a i l a b l e only  as a prepacked column s u i t a b l e for a n a l y t i c a l work. Either column could be used in determining  the r a t i o of  sugars i n a polysaccharide and in its., degradation products. Although the separation of mannose, glucose and galactose i s poor, these three sugars do not necessarily occur i n the same polysaccharide.--* Since a c i d i c sugars (uronic acids) may not be applied to the  149  (amino-bonded) column an i n d i c a t i o n of the number of sugars i n the repeating unit may be obtained as follows 238 (a)  Total hydrolysis of the p o l y - , . o l i g o s a c c h a r i d e .  (b)  Separation i n t o neutral and a c i d i c f r a c t i o n s (AG-1X2).  (c) (d)  Analysis of the neutral sugars on HPLC Total hydrolysis of the reduced polysaccharide ( v i a . carbodiimide 264) r reduced oligosaccharide ( v i a . the methyl ester)238,239 0  (e)  Analysis of sugars on HPLC  (f)  Comparison of data from (c) and (e). The advantage of t h i s method over g a s - l i q u i d  chromatography l i e s i n the absence of the need f o r d e r i v i t i z a t i o n . The major products obtained from p a r t i a l hydrolysis of an a c i d i c polysaccharide are a c i d i c (aldobi - a l d o t r i - and aldotetrauronic acids).  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 , t h i s technique shows potential i n obtaining pure neutral oligosaccharides. Separation of the products of Smith degradation into neutral and a c i d i c components may give some/all of the f o l l o w i n g : (a)  n e u t r a l : g l y c e r o l , e r y t h i t o l , t h r e i t o l , ethylene, g l y c o l , o l i g o s a c c h a r i d e s , polysaccharide.  (b)  a c i d i c : erythronic a c i d ,  threonic a c i d ,  o l i g o s a c c h a r i d e s , polysaccharides. Analysis of (a) on an a n a l y t i c a l level would provide useful information, and could be extended to a preparative s c a l e .  150  V.  5.  Alternatives  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 ) a c i d i c sugars may not be separated. 307 To overcome ( i ) Adam  has reported the use of a 308  r a d i a l l y compressed S i l i c a (10 urn) column  (Waters Assocs.)  which eliminates voids and channels i n the packed bed, thereby giving higher e f f i c i e n c i e s .  Although not quite baseline, the  separation of mannose, glucose and galactose are much improved, and r e s u l t s are reproducible. 309 Barton et aj_  report e x c e l l e n t separation of  rhamnose, xylose, arabinose and glucose, and adequate separation of glucose and galactose using a Micromeremetics M i c r o s i l amine bonded phase column, i n the analysis of hydrolyzates of plant c e l l 310 wall f i b e r .  Meagher and Furst  have used a y bondapak -  carbohydrate (Waters) column with a c e t o n i t r i l e - water (85 : 15) in the analysis of carbohydrates i n rat urine. The second s o l u t i o n 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  Durrum Chemical Co.  312  have reported the separation of  mannose, fucose, galactose, xylose and glucose (in that order) on. a column of Durrum type DA - X4 (25 cm) i n 2.5 hours.  Scobell  313 et_ al_  report e x c e l l e n t separation of arabinose, galactose,  glucose, melibiose and melezitose on Aminex A-5 C A , at 85° i n 32 mins. ++  In 1975 Linden and Lawhead  314  suggested the use of  HPLC to separate o l i g o s a c c h a r i d e s , on a micro Bondapak  (Waters)  315 column, and in 1978 Ladisch et al^ r e s i n AG 50W - X4Ca  ++  through glucose w i t h i n  used the ion - exchange  to separate the oligomers celloheptaose 30 min, using water as eluant. 316  authors have also described  These  a packing procedure and have  commented on the theory of rapid l i q u i d chromatography at moderate pressures, using water as eluant. 317 Belue has used a column of P o r a s 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 substituted carbohydrates on a column  of 10 ym s i l i c a , P a r t i si 1 10 (Whatman) using a c e t o n i t r i l e - water (90 : 10) as eluant. One of the more i n t e r e s t i n g a p p l i c a t i o n s of HPLC i n carbohydrate analysis has been the recent (1980) use of a Dupont TM 320 Zorbax 0DS column by Albersheim and coworkers to separate peralkylated oligosaccharides.  These were generated by successive  152  p a r t i a l acid h y d r o l y s i s , reduction and e t h y l a t i o n of a permethylated, complex carbohydrate.  By t h i s technique,the structure of a  nonasaccharide derived from xyloglucan, a s t r u c t u r a l polymer of plant c e l l - w a l l s , was e l u c i d a t e d . 321 Doner and Hicks  have determined retention times,  capacity f a c t o r s and r e l a t i v e responses to r e f r a c t i v e index detection f o r over f o r t y pentoses,hexoses, d i - and t r i s a c c h a r i d e s , 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. Scrobell et a]_ describe the preparation and operation of "second generation" s i l v e r form cation exchange r e s i n columns that outperform the equivalent calcium form by a f a c t o r of two with respect to time, r e s o l u t i o n and the number of oligomers seperated. The seperation of oligosaccharides obtained by phage degradation of K l e b s i e l ! a K2 polysaccharide was performed by Stirm ejt a ] /  7  using a y-Bondapak-NHg column with 2% formic a c i d as eluant.  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Res., submitted for p u b l i c a t i o n .  324.  E.A.Kabat.Personal communication from M. Heidelberger.  J . Chromatog. 130, 181 (1977). J . Chromatog. 153,  174  APPENDIX I The Known Structures of the K l e b s i e l l a Capsular Polysaccharides (as of July 1, 1980)  175 K-typeJla) Kl  X-Ray'(b)  [a]  Structure-  D  (c)  References at end -85  o6  3\/2 pyr K2  +79'  1:  ^Glc^lan^Glc& a 3 . a 1 GlcA 1  Gal A, Gal, Man, Pyr(J)  K3 K4  +9(f  K5  •45°  •^Gl c ^ G l c A ^ M a n ^ G l a a a  2  +46°  cV p  -^IcAMsicVManl B  OAc  K6  -  6\/4 pyr  1  -^•Fucl^Glc-^^lan^GlcA^  t6V4 I pyr  K7  +41T  -^Gl c A l ^ M a n ^ M a n ^ G l c^Gl c&3 a  1  a 1 Gal K8  4  ^ a l l ^Gall  ll GlcA  .1/. pyr.  176 K-type  ti?  X-Ray  (a)  (b)  (c)  9  K9^  [ ] a  Q  •17'  Structure  3 13 13 12 1 -^al-L^Rha-L^Rha—Rha— a  a  A  a  a  ,11  GlcA K9*  -5°  8  a  a  a  a  GlcA, Gal, Glc, Man (c )-  K10 Kl 1  —^lcA^^ha^^al^Rhal-^Rha—  +106'  3 13 13 1 -^Ic-^lcA-^al— 8  8  1  a  a,  Gal  6  V  V 4  pyr K12  +24'  -^Glcl^Rhai^Gall^Galfl a  a  a  —  1  GlcA 41 1' Gal 6  1/4 pyr  K13  +45'  3 14 14 1 - ^ l c — -^lan- — Glc-  1  !  t  8  1  1  a  a  1 GlcA 4  3  .^3 Gal T _ 2 > Pyr.  1  1 p  177  K-type  <|)  X-Ray  (a)  (b)  (c)  [a]  Structure  Q  K14  GlcA, G a l , G l c , Man, Rha. (L)  K15  GlcA, G a l , Glc.  K16  +65'  (S.S.) 8  -^Gl c ^ G l cA^Fuc^—  n  B  OL  Oi  1 Gal K17  K18  +30°  +77  1  -^-GlcA^Rha^Glc^Rhap a a i  1  Jal  3  1 Rha  1 4,  1  1 Rha 2  t  1 GlcA 4 c  1 Glc K19  GlcA, G a l , G l c , R h a ( J ) -  8  178 K-type (a) K20  (b)  X-Ray (c)  Structure  -^lan^Gal31  +94  1  1  a  Ia  1 Gal 3  +OAc  1 GlcA K21  +i30  u  -^IcA^Man^Marv^Gala  a  a  I  1 Gal  6\/4 Pyr  4„,J  3n.J  K22  4  a  p  1 Gl c 6  XA .=  a  1 XA K23  +28'  —Rha—^Glc— a 1  Gl 6 1 GlcA K24  + 7 30 ° T  '  7  1 3  1 ?  1L 3  1J  -^lcA-^an- -^lan- ^Glc -^ a  1 Man  L  a  a  I  B  179 K-type  <j>  (a)  (b)  K25  X-Ray [ a ]  Structure  D  (c) -41'  -^allAlcl 1  \  GlcA 2  1  1 Glc K26  +80  L  - G1 c A^-^Ma n—Ma n ^ G a 1 2I a a a  4 Gal 1  6\/4 pyr  180 K-type (a)  <|) (b)  X-Ray (c)  Structure  [a]  n D  GlcA, G a l , Man, pyr. (N) 8  K29  K30  +16  L  GlcA 1 4 14 14 1 -^an-^lan-^lc61  a  1 Gal  T?T +0Ac—  4V.3 K31  •^Glc^GlcA^Gall 3 41  a  11  a  Man 21  ll Glc 6\/ 4 pyr K32  +113°  a  o  K33  +22  4  \ / a  4\/3 pyr  GlcA 1  r  -^al^Rha^-^ha^Rha^— 8  OAc  a  6  1 4  1 4  a  B  1  ^lan-^an-^lc-' 1 Gal pyr  a  181  K-type (a) K34  X-Ray (b)  [a]  Structure  n  (c) +21"  3  1  ?  1  3  1  1 ?  3  a  a  a  8  1 Rha GlcA, G a l , G l c , Man ( L ) -  K35  K36  •56  l  1  ^ h a - ^ h a - ^ 1 c - ^ a l A-L-^ha—  -^Ga l ^ R h a i - ^ R h a ^ R h a -  GlcA 4  8  1 Glc pyr  a  182  K-type (a)  f (b)  X-Ray  [a]  (c)  D  Structure  DPA 2L  K38  +28'  iGld^Gall^all i  3  1 Glc DPA = 3-deoxy-L-glyceropentulosonic acid  K39  GlcA, G a l , G l c , Man (D) 8  K40  GlcA, G a l , Man, Rha, Pyr. (C) 8  K41  +23  1  -^Gl c ^ R h a - ^ G a l ^-?Gal f a  a  a  1 GlcA 4 1 G 6 1 Glc K42  GlcA, G a l , Man, pry (N) 8  K43  GlcA, G a l , Man. (N)—  K44  +4  L  -^1 c ^ R h a ^ G l c ^ G l c A - ^ S h a - — &  a  a  3  a  183  K-type . (a)  <() (b)  X-Ray . - ..[a]  Structure  D  (c) GlcA, Glc, Rha, pyr. (D) 8  K45 K46  +116  1  -^al^al^G a  1 3 1 cA-^Man- — 1  1  a  P 4  a  Glc^Man pyr  K47  •46  0*  r  -^al^Rhai  GlcA 4| 1 Rha  K48  +23^  •^G-l c l r ^ R h a ^ G l c ^ - ^ h a — p  £  |  a  a  1 Gal A  K49  +152  1  -^Gal^Man^Gala  1  a  ll Gal A — OAC 2or4  a  184 K-type "(a)  4> (b)  X-Ray [ a ] (c)  n  Structure  D  GlcA, Gal, Glc, Man (D)  K50  -^Gall-^Gal-  8  1  K51  a i 4  a  1 GlcA  -^alV^Rha-V^ cA^-^Gal V^RhaV  K52  1  2' 1 Gal K53  +2'  -^Gl c A l ^ a r v ^ - ^ a n ^ G a l ^ R h a — p . a a B a 3  1 Rha K54  -28'  6 I^ ll i_ n c  t  l c A  B  1 Glc  OAc 2 K55  +90'  ^GlclW 1' Gal 31  T GlcA  F u c  a  a  185  K-type (a) K56  4 (b)  x  "  y (c) R a  Structure  ["] n D  +79'  •^Gl c - ^ G a l - M G a l ^ G a l 6  4  1 Rha  pyr  K57  K58  +104'  -^Ball^GalA^Manl  1 +19'  ll Man  -^Glc^Gl 3  CAMFUC-  \/  2  1 Gal  pyr K59  +26'  3 13 12 13 1 •^Gl c - ^ a l - ^ a n - ^ a n 6 6 t 1 GlcA  •  i  OAc  OAc  (dotted l i n e s i n d i c a t e OAc's not on a l l residues) K60  K61  +58'  +56'  —^Gl c ^ G l c A ^ G a l ^ M a n 21 ea 21 4 a V 1 Glc Glc Glc •^Gl cA^Man^-^Gl c^-#Gl c3 3 1 Gal 1  T  r  1  186  K-type (a) K62  4 (b)  " y (c)  x  R a  Structure  .["Ii  +60'  11  ?  13 1 1 14 1 c A ^ - ^ M a n - ^ a l - V G l c-— 1 Man  K63  +133'  -^alA^Fuc^ala  1  a  a  Rha  T K64  +28°  11  •• a  —^IcA^Man^Glc^Manla  a  3 £i  a  3  T  K65  GlcA, Glc, Man, Rha, Pyr ( A . M . S . ) -  K66  GlcA, Gal, Man (N)—  K67  GlcA, G a l , Glc, Man, Rha (L) 8  K68  GlcA, Gal, Glc, Man, Fuc, P y r ( L ) -  K69  GlcA,  K70  Glc, Gal, Man, Pyr  4 14 12 1 -*G1 cA ^ Rha- ^Rha- -» J  L  L  L  a  8  a  ? 13 12 1 -^1 c-L^Gal-L/Rha—  1  *\! 3  50%  I  pyr  (I.S.)-  187 K-type  f  X-Ray  (a)  (b)  (c)  K71  [a]  Structure  Q  -45°  -Rha-Rha-Rha-RHa:i i i  r  GlcA K72  -54°  Glc  -^Ic^Rha^Rha-^Rhaa  1? K73- =-  i  I  4\/ 3 pyr  a  a  = Aerobacter aerogenes  L  K74  Glc  3  i i i  +67'  3 1 ? 12 1 -^Gal-^an-^an—  r;  a  a  Gl cA 41  1 Gal 6\/4 pyr K75^-  = K68 = K46  K77^  = K39  K78^-  = K15  K79  GlcA, Gal, Glc, Rha  (p)-  188  K-type  <|)  X-Ray  (a)  (b)  (c)  [ a] ^  K80 K81  K 8 2  Structure  GlcA, Gal, Man, Rha, Pyr. -52°  —^ha^Rha^lcA^Rhai^Rhai^Ga a  a  B  12,13  K83  +89° -  _i  G a l  i _ 4 Rha 1 6 3 a 1  a  Gal  3  a  1 GlcA  a  a  189 Footnotes 1 2 3 4  5 6 7  Q  Serotyping by Qrskov.  Bacteriophage degradation Ref. (b) X-Ray c r y s t a l l o g r a p h i c study  Ref. (c)  Rotations at Na-D l i n e except where noted. Data compiled by E.H. M e 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. Stirm. Virology in press. Under i n v e s t i g a t i o n by J C L S.S. N. D. A.M.S. I.S.  Q  Structure Ref. (a)  = = = = = = = =  J . P . Joseleau A . J . Chakraborty B. Lindberg S. Stirm W. Nimmich G.G.S. Dutton A.M. Stephen I. Sutherland  This serotype has been investigated in two l a b o r a t o r i e s , and two d i f f e r e n t structures have been proposed; denoted K9, K9*  ^ OAc group located on every t h i r d repeating u n 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 . , 13  27_ 386 (1977).  No q u a n t i t a t i v e a n a l y s i s . 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 i c r o b i o l . Immunol., 165, 271-288 (1979).  K4  ( a ) ( i ) E . H . M e 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. A t k i n s , 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 s a s s e r - B e i l e , H. F r i e b o l i n and S. S t i r m , 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. M i c r o b i o l . 94, 211-216 (1976).  K8  (c)  E.D.T. A t k i n s , 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. M e r 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. A t k i n s , D.H. Isaac and H.F. Elloway, i n 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. Stirm, Carbohydr. Res., 4V, 241-255 (1975).  b)  W. B e s s l e r , E. Freund-Molbert, H. Knufermann, C. Rudolph, H. Thurow and S. Stirm. 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 r e s u l t s .  a)  H. Niemann, N. Frank, and S. Stirm, Carbohydr. Res. 59_, 165-177 (1977).  b)  H. Niemann, H. Beilharz and S. Stirm. Carbohydr. Res. 60, 353-366 (1978).  a)  A . J . Chakraborty, H. F r i e b o l i n , H. Niemann and S. Stirm, Carbohydr. Res. 59, 523-530 (1977).  c)  E.D.T. A t k i n s , D.H. Isaac and H.F. Elloway, i n Microbiol Polysaccharides and P o l y s a c c h a r i d e s . [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. 147-161  a)  G.G.S. Dutton, K.L. Mackie and M.T. Yang, Carbohydr. Res.  b)  Carbohydr. Res. 80,  (1980).  65, 251-263 (1978).  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, U B C , 1976. b)  H. Thurow, H. Niemann, C. Rudolph and S. Stirm. 58, 306-309  Virology  (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. Stirm. Carbohydr. Res. 83, (1980).  192 [a)  H. Niemann and S. S t i r m , unpublished r e s u 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. Stirm. Virology 58, 306-309 (1974).  [a)  H, Niemann, B. Kwiatkowski, 0. Estphal and S. Stirm. J . B a c t e r i d . , 130, 366-374 (1977).  [c)  E.D.T. A t k i n s , 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 r e s u l t .  [a)  S.C. Churms, E.H. M e r r i f i e l d and A.M. Stephan, Carbohydr. Res. 8 1 , 49-58 (1980).  [a)  M. C u r v a 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. Stirm. 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 i n d q u i s t , 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 r e s u 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. B j o r n d a l , B. Lindberg, J . Lonngren, W. Nimmich and K. R o s e 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. Stirm, Abst. Int. Symp. Carbohydr Chem., 9 t h , London, 439-440 (1978).  K52  (a)  H. B j o r n d a l , 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. P a u l i n , 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.  K54  (c)  E.D.T. A t k i n s , D.H. Isaac and H.F. Elloway, i n Microbiol Polysaccharides and P o l y s a c c h a r i d e s . [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).  Biochem. J . 104, 278-285 (1967).  194  c)  E.D.T. A t k i n s , 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. M e r r i f i e l d , unpublished r e s u l t .  c)  E.D.T. A t k i n s , 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. M e r 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. M e r r i f i e l d , unpublished r e s u l t .  a)  E.G. M e r r i f i e l d and A.M. Stephen, unpublished r e s u l t .  a)  Y.M. Choy and G.G.S. Dutton, Can. J . Chem., 52, 684-687 (1974).  a)  G.G.S.  a)  M. C u r v a 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)  Dutton and M. Paulin Carbohydr. Res., in press.  195  APPENDIX II Structural Patterns of K l e b s i e l l a Capsular Polysaccharides  196 =  Key :  0 = Neutral sugar  Uronic Acid  (X)  = 3-deoxy-L-glycero-pentulosonic  acid  [X]  = 4-0 [(s)-l-carboxyethyl ] -D-glucuronic a c i d  <(X^ = 2R, 3R-hex-4-enopyranosyluronic acid Pyruvate and acetate omitted A.  Uronic acid absent  - 0 - 0 - 0 - 0 K32,  - 0 - 0 - 0 - 0 -  K72  1  n  K38  K56  - 0 - 0 -  - 0 - 0 -  0  0 [X] K37  B.  - 0 - 0 - 0 -  ^  *  K22 M  Uronic acid i n chain a)  linear - X- 0 - 0 K l , K5, K63  - X - 0 - 0 - 0 K4, K6  - X - 0 - 0 - 0 - 0 - 0 K70, K81  - X - 0 - 0 - 0 - 0 K9*, K44  197 branch point on uronic acid i)  s i n g l e unit side chain - X- 0 - 0  - X - 0 - 0 - 0  I  I  0  0  K l l , K57 ii)  K21, K24  two u n i t side chain - X- 0 - 0 .  - X - 0 - 0 - 0  I  f  0  0  1 ii)  0 K31 three u n i t side chain  I  0 K46 i v ) plus branch points neutral sugars  - X - 0 - 0 - 0  I  0 - X - 0 - 0 - 0  0 0  I  0  K26  I  0  I  0  ;  K60  branch not on uronic a c i d X - o - 0 K16, K54  - - X- 0 - 0 - 0 -  0  0 K7, K61, K62  - X - 0 - 0 - 0 -  I  K58  double branch not on uronic acid 0  I - X - 0 - 0 - 0 K64  0 K52, K53  - X - 0 - 0  0  K17  -  I  0  - X- 0 - 0  I  0  198 C.  Uronic acid in side chain a) s i n g l e unit side chain - 0 - 0 - 0 -  - 0 - 0 - 0 - 0 -  X K2, K8,  X  K9, K59  b) two s i n g l e unit side chains - 0 - 0 - 0 - 0 - 0 X  0  exact locations of side chains not determined  c) two single units side chains forming a double branch 0  0 I  1  - 0 - 0 - 0 -  d)  - 0 - 0 - 0 - 0  I  'I  X  X  K30, K33 two unit side chain i)  K27  uronic acid terminal - 0 - 0  -  0  I  X K20, K23, K51, K55 ii)  uronic acid non-terminal - 0 - 0 -  I  X  - 0 - 0 - 0 X  I  - 0 - 0 - 0 - 0  I  X  I  I  I  0  0  0  K25, K47  K l 3 , K74  Kl2, K28, K36  199  6;)  Three unit side chain i)  uronic acid non-terminal  0  0 - 0 -  I  I  0-0-0  X I 0  0  K18 Note:  0  K41  K9 has been investigated in two d i f f e r e n t l a b o r a t o r i e s , and two d i f f e r e n t structures have been proposed. These are denoted K9 and K9*  200  APPENDIX III 13 H and  C n.m.r. spectra  K12 polysaccharide "*H n.m.r. 220 MHz, 90°C  Spectrum No. 1  HOD  K12 polysaccharide 13 I J  C n.m.r.  20MHz, amb. temp.  I  108.39  102.64  106.99  1—r Spectrum No. 2  99.43  Kl2 Compund (3) Rha ^ — ^ Gal Ara ^—glycerol 1 a a a H n.m.r. 100 MHz, amb.temp. 1  2  Spectrum No. 3  K12 Compound (3) Rha —  a  Gal —  13 C n.m.r.  a  Ara ^-Glycerol a  r  20 MHz, amb. temp.  106.93  99.14 103.10  Spectrum No. 4  K-12 Compound (1) GlcA ^-g - Gal~0H 3  ^ C n.m.r. 3  20MHz, amb. temp  104.46  I  1  I  1  I  Spectrum No.6  1  I  1  I  1  iI— I — r  Kl2 Compound (2)  Spectrum No. 8  J_J  L__l  I I  1,1  I L_l  Spectrum No. 10  ill  I I I  L—L  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  Spectrum No.  i I  13  J  i i i  i i i I I I 111  1 1 1 I—j—U_l—(—I  !—J  1—  L  K58 Compound (4) •f^Glc^GlcA ^ F u c —  w  Pyr 100.3i  13 C n.m.r. 20 MHz, amb. temp. IJ  104 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 13  C n.m.r.  20 MHz, amb. temp.  97.07  Spectrum No. 16  K59 Compound (2)  Glcl^lcA^W-OH a  p  13 C n.m.r. 20 MHz, amb. temp.  K58 Compound (3) G l c ^ G l CA1/FUC~0H 3  a  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  ro ro o  1.93  Spectrum No. 21  1.62  1.33  1  1  '  I  1  Spectrum No. 22  K70 (p70 Compound PI n.m.r. 80 MHz, 95°C  n4.80  5.10  4.98| i/  4.58  5.23 5.28  J  1  •  Spectrum No. 24  I  K70 pO I 0  Compound PI  C n.m.r.  ro ro  Spectrum No. 25  K21  $21  Compound PI  Gl c A ^ ^ a n ^ - ^ a n ^ G a l ~0H a a a "*H n.m.r. Gal  Acetone 2.23  100 MHz, 90°C  Pyr  ro ro  1.55  • I  I I I I  1  1  SDectrum No. 26  1  1  1  1  1  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 ^ C n.m.r. 3  1 Ga  20MHz, amb. temp  V  Pyr  101.18  101.37 25.96  103.04  31.07  Spectrum No. 27  PO PO  I  I  I I  I  I  I  Spectrum No. 28  I I  I I  I  I  I  I  I  I I I  Kl 2  <j) 1 2  PI  ^H-n.m.r.  400 MHz, 90°  K41 < > | 12 P2  A  13 C n.m.r. 20 MHz amb. temp. I J  I  I  I  I  Spectrum No. 31  !  i  I  I  I  I  !  I  I  1  I  I  c  e  t  o  n  e  232  METHODOLOGY OF BACTERIOPHAGE PROPAGATION AND POLYSACCHARIDE ISOLATION APP.IV  233 Media and Buffers "Standard" l i q u 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 s o l u t i o n of " s t a n d a r d " l i q u i d broth to which 15g of agar per l i t r e had been added. 8.5 cm disposable  petri  plates were used. A c t i v e l y growing cultures of K l e b s i e l l a bacteria (50 mL) were grown, i n 100cm x 300cm t r a y s , on a medium of sodium c h l o r i d e (8g), calcium carbonate (2g), sucrose (120g), and Bacto yeast extract (8g) in  2L of water f o r three days.  (See Scheme 13 ) .  Phosphate-buffered s a l i n e (PBS) pH=7 was made up using 8.5g NaCl, 1.76g Na HP0 .H 0, and O.lg KH P0 i n IL of water. 2  4  2  2  4  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. C o r e l l a t i o n of o p t i c a l density with b a c t e r i a l count. A f l a s k of l i q u i d medium was inoculated with a c u l t u r e of a c t i v e l y growing K l e b s i e l l a K21 bacteria and vigorously aerated at 37° . A l i q u o t s were removed at 30 minute i n t e r v a l s , appropriately d i l u t e d (10  to 10" ) with l i q u i d medium and a small quantity  (0.1 mL) of the  d i l u t e d s o l u t i o n was incubated on an agar plate f o r 12-16 hours. Individual b a c t e r i a l colonies could then be counted. count c o r e l l a t e s with o p t i c a l  density.  The b a c t e r i a l  234 SUCROSE, EXTRACT,  BEEF-EXTRACT  STANDARD AGAR  STREAK  INOCULATE  37° 16h  37° 3h  YEASTAGAR  25° 3d  GROW  HARVEST  30,000 r.p.m.  ——  3-5h  f  1% PHENOL  -  -  DILUTE  CENTRIFUGE  H0  EtOH  2  DISSOLVE  PRECIPITATE  PRECHPITATE 5,000 r.p.m. 20 min.  -4  ^5 '  10% CETAVLON  ? s 4M NaCl  -EtOH  do* CENTRIFUGE  DISSOLVE  PREC PITATE  4 /  H0 2  AN" H0 2  do DISSOLVE Scheme 13  DIALYZE  FREEZE-DRY  I s o l a t i o n and P u r i f i c a t i o n of Polysaccharide.  235 Bacteriophage Propogation (a) Tube l y s i s .  An a c t i v e b a c t e r i a l c u l t u r e of K l e b s i e l l a  K21 was obtained by successive replatings on agar p l a t e s . 7x5 mL of s t e r i l e l i q u i d medium was then  inoculated with the bacteria by the  addition of 0.5 mL of an a c t i v e l y growing l i q u i d K32 b a c t e r i a l culture.  These seven t e s t tubes were incubated at 37° and at 30  minute i n t e r v a l s the tubes were  inoculated with 0.5 mL of a  s o l u t i o n of l i q u i d medium containing <j)2V.  Continued incubation  resulted in the f i r s t few tubes changing from the cloudy s o l u t i o n associated with a c t i v e l y growing K21 bacteria to a c l e a r s o l u t i o n (lysis).  A f t e r the l a s t tube had cleared the incubation was  continued f o r 30 minutes and then a few drops of CHC1^ was added to the tube and the mixture was shaken w e l l . A phage " t i t r e " on the s o l u t i o n was performed by successively d i l u t i n g a small volume (0.1 mL) of the  c l e a r l i q u i d with l i q u i d medium and then applying approximately  0.03 mL of these d i l u a t i o n s to a 'lawn' of a c t i v e l y growing K l e b s i e l l a K21.  (The lawn of K21 was prepared by  inoculating 2 mL of l i q u i d  medium with an a c t i v e l y growing colony of K l e b s i e l l a K21 and incubating t h i s c u l t u r e f o r 3 hours.. An agar p l a t e , previously dried f o r approximately 1 h in the incubator at 37°, was covered with t h i s l i q u i d c u l t u r e , l e f t f o r 5 minutes and then the excess l i q u i d was removed.  Incubation f o r 30 minutes gave a stable 'lawn' of  K l e b s i e l l a K21.)  Individual bacteriophage were observed as c l e a r  spots (approximately 0.3 cm in diameter) on the b a c t e r i a l lawn a f t e r incubation f o r 16 hours. At high phage concentrations  individual  phage could not be distinguished but at more s u i t a b l e d i l u t i o n s ,  236  Figure 20  Growth Curve - , and bacteriophage lysis of Klebsiella K21 bacteria.  237  e . g . 10"  u  1 [ 1  to 10"  , individual  'haloes' could be e a s i l y counted. As a g  r e s u l t of a s i n g l e tube l y s i s of t h i s nature an assay yielded 10 plaque forming units (PFU) per mL of medium i n the l a s t tube to completely c l e a r . (b)  Small f l a s k l y s i s .  This technique i s e s s e n t i a l l y the  same as that described f o r the tube l y s i s . As larger volumes of l i q u i d medium can be used the o v e r a l l t o t a l of bacteriophage can be increased even though the phage t i t r e per mL. may not be s i g n i f i c a n t l y higher.  In a t y p i c a l small f l a s k l y s i s 50 mL: solutions of K21  cultures were  .inoculated with 1.5 mL  of a phage s o l u t i o n  containing 10 PFU/mL (from tube l y s i s ) .  In an analogous manner to  9  that described f o r the tube l y s i s , t i t r a t i o n of the f i n a l f l a s k to completely c l e a r gave a t i t r e of 1 . 2 x l 0  10  PFU/mL. 250 mL of an  a c t i v e l y growing l i q u i d culture of K32 were vigorously aerated at 37°. A small amount of a s i l i c o n antifoam agent (Dow antifoam FG-10 emulsion) was added to each.  The o p t i c a l density of each f l a s k  was monitored and at appropriate o p t i c a l density readings  (calculated  such that the r a t i o of t o t a l bacteriophage to t o t a l b a c t e r i a l colonies was approximately 3:1)  a l i q u o t s of l i q u i d phage cultures were added  and the o p t i c a l density monitoring continued. A subsequent drop i n o p t i c a l density indicated l y s i s had occurred.  The r e s u l t s of a t y p i c a l  b o t t l e l y s i s are shown i n Figure 20. A b o t t l e l y s i s might t y p i c a l l y y i e l d 400 mL of a s o l u t i o n 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 l a s k l y s i s .  In an analogous manner to  that described f o r the b o t t l e l y s i s three one l i t r e f l a s k s , each containing 600 mL of l i q u i d medium, were  inoculated with K  b a c t e r i a , aerated and incubated to appropriate o p t i c a l d e n s i t i e s , and then bacteriophage solutions were added.  A t y p i c a l r e s u l t of such  a l y s i s might y i e l d 1400 mL of a phage s o l u t i o n with a t i t r e of 3xl0  1 0  PFU/mL.  

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