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Structural studies and bacteriophage degradation of Klebsiella capsular polysaccharides Lim, Andrew V. S. 1983

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STRUCTURAL STUDIES AND BACTERIOPHAGE DEGRADATION OF KLEBSIELLA CAPSULAR POLYSACCHARIDES by ANDREW V.S. LIM B.Sc. (Hons.), University of Wales I n s t i t u t e of Science and Technology, U.K., 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n 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 A p r i l 1983 © Andrew Vee San Lim, 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f ^ - W e M V S T f t H The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 D a t e r\*>*\\- ^ V NS,%5> 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 have been i s o l a t e d . The capsular polysaccharides which these Gram-negative ba c t e r i a produce are antigenic. 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 a l l 77 str a i n s i s taking place i n t h i s and other l a b o r a t o r i e s . To date, about s i x t y - f i v e structures have been determined. The structure of the capsular antigen i s o l a t e d from K l e b s i e l l a serotype K82 i s presented here and was determined using the techniques of methylation, ^-elimination, periodate oxidation-Smith hydrolysis and p a r t i a l 1 13 hydrol y s i s . Extensive use of n.m.r. spectroscopy ( H and C) was used to e s t a b l i s h the nature of the anomeric linkages of the polysaccharide and of derived poly- and oligo-saccharides obtained through degradative procedures. The K82 polysaccharide was found to comprise of tetrasaccharide repeating units of the 'three plus one' type: +3) B-D-Glcp- (l->3)-a-D-Galp- (l->3)-g-D-Galp- (l-> 4 1 1 B-D-GlcAp The use of bacteriophage-borne glycanases as a technique i n s t r u c t u r a l studies was demonstrated on K l e b s i e l l a K21 polysaccharide. Bacteriophage cj>21 was used to degrade K21 polysaccharide, r e s u l t i n g i n a good y i e l d of the s i n g l e repeating unit. The structure determined f o r t h i s bacteriophage-degraded product i s i n agreement with the previously known structure of K21 polysaccharide, which was determined s o l e l y by chemical methods. i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES v i LIST OF SCHEMES v i i ACKNOWLEDGEMENTS v i i i PREFACE . . i x I INTRODUCTION 2 II METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES = . . 7 I I . 1 I s o l a t i o n and p u r i f i c a t i o n 7 II.2 Sugar analysis 8 11.2.1 Characterization and quantitation of sugars 8 11.2.2 Determination of the configuration (D or L) of the sugars 10 II . 3 P o s i t i o n of linkage 10 11.3.1 Methylation analysis 10 11.3.2 Characterization and quantitation of methylated sugars 11 i v I I . 4 Sugar sequence 151" II.4.1 P a r t i a l hydrolysis 15 II . 4.2 Periodate oxidation and Smith hydrolysis , 16 II.4.3 Uronic acid degradation (g-elimination) . . 18 II . 5 Determination of linkage configurations 21 11.5.1 N.m.r. 21 11.5.1.1 *H n.m.r. 21 13 11.5.1.2 C n.m.r. 24.^, 11.5.2 Other techniques 25 I I I STRUCTURAL INVESTIGATION OF KLEBSIELLA SEROTYPE K82 POLYSACCHARIDE 27 I I I . l Abstract 28 I I I . 2 Introduction 28 I I I . 3 Results and discussion 29 I I I . 4 Experimental 40 IV BACTERIOPHAGE DEGRADATION OF KLEBSIELLA K21 CAPSULAR POLYSACCHARIDE 53 IV. 1 Introduction 54 IV. 2 Results and discussion 56 IV. 3 Experimental 63 V BIBLIOGRAPHY 69 APPENDIX I: K l e b s i e l l a capsular polysaccharides (K1-K83) q u a l i t a t i v e analysis and chemotype grouping . . . . 76 APPENDIX I I : N.m.r. spectra 77 V LIST OF TABLES Table ' Page I I . 1 Three regions of a carbohydrate spectrum 22 I I I . l Sugar analysis of K82 polysaccharide and derived products 31 111.2 N.m.r. data for K l e b s i e l l a K82 capsular polysaccharide and the derived oligosaccharides 32 111.3 Methylation analyses of K82 polysaccharide and derived products 34 IV. 1 Propagation of bacteriophage <j>21 59 IV.2 Methylation analyses of K21 oligosaccharide (PI) from bacteriophage degradation 60 IV.3 N.m.r. data f o r K l e b s i e l l a K21 capsular polysaccharide and the derived oligosaccharides . . . 61 v i LIST OF FIGURES Figure Page I. I Diagramatic representation of the b a c t e r i a l c e l l envelope and the d i f f e r e n t antigens 3 I I . 1 Mass spectra of (a) l , 3 , 5 - t r i - 0 - a c e t y l - 2 , 4 , 6 - t r i - 0 -methyl-D-glucitol and (b) l,5,6-tri-0-acetyl-2,3,4-tri-O-methyl-D-glucitol 14 II I . l C a l i b r a t i o n graph of absorbance versus % 10^ /I0^ . . 37 111.2 Periodate consumption of K82 polysaccharide with respect to time 37 111.3 Separation of K82 p a r t i a l hydrolyzate by gel-permeation chromatography (Bio-gel P-2) 38 IV. 1 Corr e l a t i o n between b a c t e r i a l count and the o p t i c a l density f o r K l e b s i e l l a K21 b a c t e r i a 64 v i i LIST OF SCHEMES Scheme Page 11.1 Methylation analysis of K l e b s i e l l a K82 polysaccharide 12 11.2 Periodate oxidation - Smith hydrolysis of K82 polysaccharide 17 I I . 3 B-elimination of K82 polysaccharide ... 20 v i i i ACKNOWLED GEMENTS I wish to express my g r a t e f u l thanks to Professor G.G.S. Dutton for his help, advice and d i r e c t i o n throughout the en t i r e research and during the preparation of t h i s thesis. My sincere thanks to E. Altman, N. Karunaratne, D. Leek, Dr. E.H. M e r r i f i e l d and Professor H. P a r o l i s f o r t h e i r cooperation and h e l p f u l discussions. I should also l i k e to thank P. Lee Wing, L. P a r o l i s and Professor A.M. Stephen for t h e i r i n t e r e s t . My thanks to the members of the department who have helped i n d i r e c t l y during the course; of t h i s work, v i z . Dr. S.O. Chan and the s t a f f of the n.m.r. service; Dr. G. Eigendorf and the s t a f f of the mass-spectrometry service. I should also l i k e to thank Rani Theeparajah f o r typing t h i s thesis. F i n a l l y , my g r a t e f u l thanks to my wife Mei-Yeng, f or her encouragement and moral support. i x PREFACE* In an effort to familiarize readers who do not work in the particular area of organic chemistry to which this thesis refers, the following explanation of terms used is offered. Fischer projection formulae are used to represent the acyclic modificaton of sugars. Some examples are shown below. Numbering commences from the carbonyl group at the top of the chain (I). Note that D-glucuronic acid (II) differs from D-glucose (I) only CHO 1 2 HOH h OH 3 41— OH 5 - OH 6 C H 2 0 H CHO -OH HCH t-OH -OH COOH CHO -OH I—OH HO — HO -CH. D-glucose D-glucuronic acid L-rhamnose (I) (ID (III) in that C-6 is oxidized to a carboxylic acid group. The C-6 of L-rhamnose (III) is part of a methyl group and is referred to also by another common name,6-deoxy-L-mannose. There are four chiral centers in these six-carbon chains (marked with asterisks in structure III) making i t important to appreciate the spatial arrangement of atoms (configuration) that is implied by these Fischer representations. To simplify the nomenclature ofall the possible isomers (16 for each of I, II, III), X a l l t h o s e h a v i n g t h e h y d r o x y l g r o u p a t t h e h i g h e s t - n u m b e r e d c h i r a l c e n t e r (C-5) p r o j e c t i n g t o t h e r i g h t i n t h e F i s c h e r p r o j e c t i o n f o r m u l a e b e l o n g t o t h e D - s e r i e s , and t h e o t h e r s t o t h e L - s e r i e s . — OH H 0 -CH 2 0H CH 2 0H D - s e r i e s L - s e r i e s P h y s i c a l and c h e m i c a l e v i d e n c e i n d i c a t e s t h a t , i n f a c t , t h e s e s i x - c a r b o n p o l y h y d r o x y a l d e h y d e s e x i s t i n a c y c l i c f o r m . The r i n g c l o s u r e o c c u r s by n u c l e o p h i l i c a t t a c k o f t h e oxygen atom a t C-5 on t h e a l d e h y d i c c a r b o n a t o m , g e n e r a t i n g a new c h i r a l ( a n o m e r i c ) c e n t e r a t C - l . T h i s r e s u l t s i n two a n o m e r s , r e p r e s e n t e d be l ow HO _ J CH 2 0H a-D-glucose ( I V ) HO H \ / I—OH HO— I—OH CH 2 0H 6-D-glucose (V) x i in the "Miens formulae. It 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 limitations with their unequal bond lengths,Haworth developed a perspective method of looking at the six-membered ring (VI and VII). This improvement recognizes that the ring oxygen atom lies behind the carbon chain and that bond lengths are approximately equal. Often in practise regular hexagons are used in Haworth projections; OH OH a-D-glucopyranose e-D-glucopyranose pyran (VI) (VII) (VIII) which he related to such rings at the heterocyclic compound pyran (VIII) and named them pyranoses. Note that hydroxy1 groups not involved in ring formation on the right in Fischer and Tollens formulae point down in the Haworth projections and those on the left point up. Similarly, for aldopyranoses, the group on C-5 points up for D (IX) and down for the L enantiomer (X). It follows, then, that when sugar residues are attached there are two possible configurations, an o - or a e-pyranoside, for each linkage. x i i CH Hi OH HO OH HO OH a-D-rhamnopyranose (IX) a-L-rhamnopyranose (X) 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 (XI), consists of a puckered, six-membered, oxygen-containing carbon ring, with hydroxyl substituents at C-l through C-4, and a hydroxymethyl group at C-5. All substituents on the ring, except for that at C- l , are equatorial. Two isomers (anomers) are possible in relation to the anomeric center (C-l), depending on whether a substituent is axial (a-anomer; XII) HO OH (XI) x i i i or equatorial (e-anomer; X I I I ) , where R = hydrogen, for monosaccharides, and R = another sugar residue, for d i - , o l i g o - , and polysaccharides. Since H-l i s i n a 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 distinguish between them and, thereby, provides invaluable assistance i n assigning anomeric configurations. (XII) (XIII) Haworth projections are most useful and w i l l be used i n this t h e s i s , even though they give no indication of three-dimensional molecular shape. There seems to be l i t t l e j u s t i f i c a t i o n for the use of formulae which depict states of molecules as well as structures, when the true states are often unknown or variable. * Reproduced with the kind permission of T.E. Folkman from his M.Sc. thesis e n t i t l e d "Structural Studies on K l e b s i e l l a Capsular Polysaccharides", University of B r i t i s h Columbia, A p r i l 1979. x i v TO MY PARENTS-IN-LAW Mr. and Mrs. Kok Kim Poh INTRODUCTION 2 I. INTRODUCTION The genus K l e b s i e l l a i s composed of Gram-negative, nonmotile 1 2 b a c t e r i a , of the family Enterobacteriaceae and the t r i b e K l e b s i e l l e a e ' . Seventy-seven s t r a i n s of K l e b s i e l l a have been i s o l a t e d and categorized into three species: K. pneumoniae, K. ozaenae, and K. rhinoschleromatis. K l e b s i e l l a K82 ( s t r a i n CDC 3454-70) was f i r s t i s o l a t e d from a carpet during 3 an epidemiological survey i n the U.S. i n 1970. Dr. Ida 0rskov e t . a l . (Copenhagen), using morphological and biochemical c r i t e r i a , i d e n t i f i e d and c l a s s i f i e d i t as belonging to the species K. pneumoniae. A common feature of Gram-negative organisms i s the occurrence of 4 polysaccharides on the c e l l surface. These polysaccharides may take the form of capsules or they may be an e x t r a c e l l u l a r slime (considered as excreted capsular polysaccharide)~*. K l e b s i e l l a b a c t e r i a are c l a s s i f i e d s e r o l o g i c a l l y on the basis of t h e i r capsular K (German; Kapsel) and somatic 0 (German; ohne Hauch) antigens^' 7 (see Figure 1.1). Since most K l e b s i e l l a b a c t e r i a are heavily encapsulated with the heat stable capsular polysaccha-r i d e , the 0-antigen i s completely shielded. 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. Moreover, the number of 0 types i s small (11) compared with the K types (77). The quantity of capsular polysaccharide produced by organisms has been found to be dependent on culture conditions. For optimal production, g a low nitrogen content of the medium i s e s s e n t i a l . L i t t l e i s known about the function of microbial e x t r a c e l l u l a r polysaccharides though i t has been suggested that they may act as storage material and be a protection against de s i c c a t i o n and predation. 9 In dealing with b a c t e r i a l polysaccharides as antigens , t h e i r immunogenicity ( i . e . t h e i r capacity to induce the formation of antibodies 3 C a p s u l a r p o l y s a c c h a r i d e ( K a n t i g e n ) L i p o p o l y s a c c h a - : r i d e ( 0 a n t i g e n ) O u t e r membrane P e p t i d o g l y c a n l a y e r C y t o p l a s m i c membrane o o o o o o o o o o o a C y t o p l a s m F i g u r e 1 . 1 . D i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e b a c t e r i a l c e l l e n v e l o p e and t h e d i f f e r e n t a n t i g e n s . 4 i n mammals) and t h e i r a n t i g e n i c i t y ( i . e . t h e i r r e a c t i v i t y with antibodies) have to be considered. Pioneering work i n these f i e l d s was i n i t i a t e d by Heidelberger e t . a l . ^ and Kabat e t . a l . ^ . The aim of the immunochemical analysis of polysaccharide antigens, which combines s e r o l o g i c a l and chemical studies, i s to define antigenic determinants w i t h i n the polysaccharide as chemical expression of i t s immunological character. These antigenic determinants can be a monosaccharide linked i n a s p e c i f i c manner, an oligosaccharide or even non-carbohydrate i n nature (e.g. polysaccharides with acetates or with k e t a l - l i n k e d pyruvates). In a c i d i c polysaccharides, such as those of K l e b s i e l l a , the charged constituents are often part of 12 antigenic determinants . Due to t h e i r r e p e t i t i v e structure, b a c t e r i a l polysaccharides have the same antigenic determinants expressed many times over. For a quantitative consideration of the antigen-antibody reaction, knowledge of the precise structure of the polysaccharide i s necessary. For t h i s reason, s t r u c t u r a l i n v e s t i g a t i o n of a l l K l e b s i e l l a s t r a i n s have been undertaken. One outstanding c h a r a c t e r i s t i c feature of K l e b s i e l l a capsular poly-saccharides i s that they are composed of oligosaccharide repeating u n i t s , which comprise 3 to 7 sugar residues. The ordered oligosaccharide repeating 13 unit structure has been shown by molecular weight d i s t r i b u t i o n studies , 14 J v . i - ^ • 4 b « • • 1*5,16 . „ , n.m.r. spectroscopy , and X-ray d i f f r a c t i o n . Nimmich has reported the q u a l i t a t i v e composition of most of the K l e b s i e l l a K-types, and categorized them in t o chemotypes'''7 (see Appendix I ) . The polysaccharides are a l l a c i d i c , due to the presence of uronic acids (D-glucuronic acid and D-galactouronic acid) or a keto acid. Hexose sugars commonly found are D-glucose, D-galactose and D-mannose; i n some st r a i n s 6-deoxyhexoses such as L-rhamnose and L-fucose are present. In addition, non-carbohydrate 5 substituents such as acetates and k e t a l - l i n k e d pyruvates may be present. Approximately s i x t y - f i v e K l e b s i e l l a s t r a i n s have been studied and d i f f e r e n t s t r u c t u r a l patterns ( l i n e a r , branched and comb-like) have emerged from the proposed structures. The structure of K l e b s i e l l a K82 capsular polysaccharide was investigated and i t was found to be i n the 18 same chemotype and to have a s i m i l a r structure to that of K8 except for differences i n linkage configurations. Despite the close resemblance between 3 K82 and K8, there i s s t i l l 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 . This fact j u s t i f i e s the d e t a i l e d , precise s t r u c t u r a l study of a l l b a c t e r i a l poly-saccharide antigens. In the course of the research reported i n t h i s t h e s i s , the use of bacteriophage-borne glycanase as a technique i n s t r u c t u r a l studies was demonstrated on K l e b s i e l l a K21 capsular polysaccharide. K21 polysaccharide was depolymerized, to i t s repeating u n i t , i n a good y i e l d , by i t s respective bacteriophage (cj>21). S t r u c t u r a l determination of the repeating u n i t confirmed 19 the previously published structure of K21 METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES 7 I I . METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES In order to understand the chemical bas i s of the a n t i g e n i c i t y of a capsular b a c t e r i a l po lysacchar ide , i t i s important to know i t s p rec i s e s t r u c t u r e . A s t r u c t u r a l study must charac ter ize (a) the nature and proport ions of the sugar const i tuents i n the po lysacchar ide , (b) the l inkage p o s i t i o n s of the sugar residues and any non-carbohydrate subs t i tuent s , i f present , (c) anomeric conf igura t ions , and (d) the sequence of the sugar res idues . In the fo l lowing s e c t i o n s , the techniques used i n t h i s s t r u c t u r a l i n v e s t i g a t i o n are b r i e f l y d i scussed . I I .1 I s o l a t i o n and p u r i f i c a t i o n As i n s tudies on other n a t u r a l polymers, e .g . prote ins and n u c l e i c a c i d s , the f i r s t major task i n polysacchar ide chemistry i s that of obta in ing the m a t e r i a l under i n v e s t i g a t i o n i n a pure form. The p u r i f i c a t i o n process involves two stages: ( i ) the i s o l a t i o n of the polysacchar ide free from low molecular weight mater i a l and other h igh molecular weight m a t e r i a l , and ( i i ) i s o l a t i o n of a s i n g l e , monodisperse, polysacchar ide species . The p u r i t y and homogeneity of the polysaccharide i s o l a t e d can then be checked by ge l chromatography, u l t r a c e n t r i f u g a t i o n , e l e c t r o p h o r e s i s , [ c l ^ measure-ments, sugar a n a l y s i s , n . m . r . spectroscopy e tc . K l e b s i e l l a b a c t e r i a serotypes K21 and K82 were rece ived as stab cu l ture s from Dr. Ida 0rskov (Copenhagen). A c t i v e l y growing b a c t e r i a l cu l tures were propagated by r e p l a t i n g succes s ive ly on agar p la tes at 3 7 ° . Bac ter i a were grown by i n o c u l a t i o n of broth medium wi th a s i n g l e colony for 3 h at 3 7 ° , and then incubated for 3 days on large trays of sucrose-yeast extract agar medium. The lawn o f capsular b a c t e r i a produced was harvested by scraping from the agar surface, d i l u t e d wi th 1% phenol s o l u t i o n to k i l l 8 the b a c t e r i a and ul t r a c e n t r i f u g e d . The viscous supernatant, containing the dissolved polysaccharide, was p r e c i p i t a t e d into 4 : 1 ethanol-acetone mixture. The resultant stringy p r e c i p i t a t e was dissolved i n water and treated with 20 Cetavlon (cetyltrimethylammonium bromide) solution,which s e l e c t i v e l y p r e c i p i t a t e d the a c i d i c polysaccharide. Any neutral polysaccharide present w i l l remain i n solu t i o n and t h i s can be separately i s o l a t e d by d i a l y s i s against running tap water and then l y o p h i l i z a t i o n . The Cetavlon-polysaccharide complex was dissolved i n 4M NaCl solu t i o n , p r e c i p i t a t e d into 4 : 1 ethanol-acetone mixture, redissolved i n water, and dialyzed against running tap water. The l y o p h i l i z e d polysaccharide i s o l a t e d was a styrofoam-like material and was shown to be homogeneous by gel chromatography. II.2 Sugar Analysis II.2.1 Characterization and quantitation of sugars The f i r s t step i n the s t r u c t u r a l study of a polysaccharide i s to determine the nature and proportions of the sugars released on t o t a l h y d r o l y s i s . The h y d r o l y t i c conditions (type and concentration of acid, temperature and time) are important for a qua n t i t a t i v e release of sugars with minimum degradation. Commonly used acids are s u l f u r i c , hydrochloric, formic and 21 t r i f l u o r o a c e t i c . Dutton has reviewed the advantages and disadvantages of these acids and the h y d r o l y t i c conditions used. Aqueous so l u t i o n of 22 t r i f l u o r o a c e t i c acid (2M) has been shown to be the best choice, and i t has the advantage^of^easy removal under diminished pressure. The h y d r o l y t i c rates of g l y c o s i d i c linkages vary greatly (see Section II.4.1). Polysaccharides containing uronic acid residues are more r e s i s t a n t to acid hydrolysis e.g. furanosyl linkages are hydrolyzed 300 x fa s t e r than uronosyl linkages. The presence of electron withdrawing, carboxyl 9 groups s t a b i l i z e s the uronosyl linkages through the h e t e r o c y c l i c oxygen. To hydrolyze a c i d i c polysaccharides a method has been developed i n this 23 laboratory i n v o l v i n g the. use of methanolysis . Treatment of the polysaccha-r i d e with methanolic hydrogen chloride cleaves most g l y c o s i d i c bonds to form the methyl glycosides and at the same time, the methyl esters of the uronic acids. However, some uronosyl linkages could s t i l l remain i n t a c t . Reduction of the uronic ester to i t s corresponding alcohol and subsequent acid hydrolysis ensures complete release of sugar residues. A f t e r the above-mentioned seri e s of reactions, D-glucuronic a c i d , f o r example, would appear as i t s reduced hexose, D-glucose. A l t e r n a t i v e l y , the reduction of a l l carboxyl f u n c t i o n a l i t i e s i n the a c i d i c polysaccharide by d e r i v a t i z a t i o n 24 into carbodiimide derivatives and reduction with sodium borohydride, i s another means of overcoming the r e s i s t a n t uronosyl linkage during acid hydrolysis. The c h a r a c t e r i z a t i o n and quantitation of the sugars released upon hydrolysis of the polysaccharide can be performed by g a s - l i q u i d chromato-graphy on the a l d i t o l acetate derivatives of the sugars. An extensive 21 25 review of t h i s technique has been made by Dutton ' . Paper chromatography i s also used to give a q u a l i t a t i v e analysis. More recently, high performance 26 27 l i q u i d chromatography ' i s used, both q u a l i t a t i v e l y and q u a n t i t a t i v e l y , on underivatized sugars. A l d i t o l acetate derivatives are used throughout th i s i n v e s t i g a t i o n because they give r i s e to only one peak i n the g . l . c . a n a l y s i s , making quantitation easier., Other de r i v a t i v e s that preserve the anomeric center may r e s u l t i n four peaks (a- and 3- pyranosides and a- and 3- furanosides), for instance, t r i m e t h y l s i l y l d e r i v a t i v e s . By comparison of the r a t i o of ne u t r a l sugars only released upon hydrolysis of the a c i d i c polysaccharide, and the r a t i o of neutral sugars 10 and reduced uronic acids, the i d e n t i t y of the uronic acid and the aldobiouronic acid as w e l l as the molar proportions of the component sugars can be deters, mined. II.2.2 Determination of the configuration (D or L) of the sugars The D or L configuration of the sugars can be determined by the following methods: (a) the i s o l a t i o n of the d i f f e r e n t monosaccharides and measurement of t h e i r [a] D» (b) the use of s p e c i f i c oxidases (e.g. D-glucose and D-galactose oxidases) and enzymes, (c) the g . l . c . separation of enantiomers, using a c h i r a l stationary phase or converting the enantiomers in t o diastereomers using c h i r a l reagents and separation on a non-chiral 28 29 phase ' , and (d) the method used i n our laboratory i s to conduct c i r c u l a r 30 dichroism measurements at 213 nm on a l d i t o l acetates, p a r t i a l l y acetylated a l d o n o n i t r i l e s or t h e i r p a r t i a l l y methylated a l d i t o l acetates, where the acetoxy group acts as a chromophore. II.3 P o s i t i o n of linkage II.3.1 Methylation analysis This technique involves the complete e t h e r i f i c a t i o n of the free hydroxyl groups of the sugar residues i n the o l i g o - and poly-saccharides and the i d e n t i f i c a t i o n of the monosaccharides released as t h e i r p a r t i a l l y methylated a l d i t o l acetate (PMAA) de r i v a t i v e s . Methylation analysis gives information on ( i ) number and type of sugars per repeating u n i t , ( i i ) r i n g s i z e , ( i i i ) linkage p o s i t i o n s , (iv) i d e n t i t y of the terminal u n i t ( s ) , branching u n i t ( s ) and (v) the p o s i t i o n of base-stable substituents (e.g. pyruvic acid k e t a l ) . Several methylation procedures have been developed through the years, 11 but the most e f f e c t i v e method i s that developed by Hakomori (sodium methyl-31 32 sulfinylmethanide i n anhydrous dimethyl sulfoxide) ' . Complete methylation i s usually effected i n one treatment (as indicated by i n f r a - r e d spectroscopy). In cases of incomplete methylation, a subsequent Purdie-33 I r v i n e methylation (Ag 20 i n r e f l u x i n g Mel) i s conducted. A second Hakomori methylation i s never conducted on an a c i d i c o l i g o - or poly-saccharide as t h i s w i l l r e s u l t i n 3-elimination (see Section II.4.3). To deduce the i d e n t i t y of the a c i d i c sugar, the methyl ester i s reduced (with LiAlH^) and remethylated. The permethylated carbohydrate i s then hydrolyzed, reduced to i t s a l d i t o l s , and acetylated to give v o l a t i l e p a r t i a l l y methylated a l d i t o l acetate derivatives f or g . l . c . and g.l.c.-m.s. analyses (see Scheme II.1). The unmethylated positions of the sugars represent s i t e s of linkage, except i n the cases of uronic acid residues or residues with pyruvic acid k e t a l attached. Uronic acids can be i d e n t i f i e d by comparison of the methylation r e s u l t s of the a c i d i c polysaccharide with those of the reduced polysaccharide, or the reduced polysaccharide and the corresponding reduced remethylated product. II.3.2 Characterization and quantitation of methylated sugars. The c h a r a c t e r i z a t i o n of p a r t i a l l y methylated monosaccharides released on t o t a l hydrolysis of the permethylated o l i g o - or poly-saccharide i s 34 conveniently performed by paper chromatography . The papers, on develop-ment with _p_-anisidine and heated, give a preliminary i d e n t i f i c a t i o n of the methylated sugars according to t h e i r m o b i l i t i e s (R^ values) and the d i f f e r e n t colours formed. Further q u a l i t a t i v e and quantitative analysis of the methylated sugars can be performed by g . l . c . An extensive review of t h i s 12 K82 POLYSACCHARIDE METHYLATION LiAl H /THF 4 R = OMe CH2R 1. HYDROLYSIS 2. NaBH, REDUCTION 4 3. ACETYLATION 2,4,6-OMe 3-Galactitol 2,6-OMe 2-Galactitol 2,4,6-OMe 3-Glucitol 2,3,4-OMe 3-Glucitol 1-. REMETHYL AT ION 2. HYDROLYSIS 3. NaBH. REDUCTION 4 4. ACETYLATION 2,4,6-OMe 3-Galactitol 2,6-OMe 2-Galactitol 2,4,6-OMe 3-Glucitol 2,3,4,6-OMe^-Glucitol 2,4,6-OMe 3-Galactitol s 1,3,5-tri-0-acetyl-2,4,6-tri-O-methyl-D-galactitol Scheme II.1 Methylation analysis of K l e b s i e l l a K82 polysaccharide 13 21 25 f i e l d has been done by Dutton ' . The i d e n t i f i c a t i o n and quantitation of the methylated sugars, analyzed as t h e i r p a r t i a l l y methylated a l d i t o l acetates, are made by consideration of the r e l a t i v e retention times and 35 36 co-chromatography with authentic samples. Lindberg and Albersheim have provided r e l a t i v e retention times for numerous p a r t i a l l y methylated a l d i t o l acetates as w e l l as molar response f a c t o r s to allow f o r correct quantitation. In instances of c l o s e l y related retention times, unambiguous i d e n t i f i c a t i o n s can be made by analyzing the p a r t i a l l y methylated a l d i t o l acetates (PMAA) on the g.l.c.-m.s. The fragmentation of the PMAAs by 37 electron impact has been extensively studied . This technique allows one to i d e n t i f y the s u b s t i t u t i o n pattern of the PMAA, e.g. Figure II.1 depicts the differences i n the mass spectra of 1,5,6-tri-0-acetyl-2,3,4-tri-O-methyl-D-glucitol and 1,3,5-tri-O-acetyl-2,4,6-tri-0-methyl-D-glucitol. However, the parent ion mass i s not indicated nor can the diastereomeric galactose, glucose,and mannose de r i v a t i v e s be distinguished. The primary fragments are formed by C-C bond f i s s i o n i n the a l d i t o l chain and t h i s cleavage follows a c e r t a i n p r e f e r e n t i a l order: CHOMe v CHOMe v CHOAc I > 1 > I CHOME ' CHOAc ' CHOAc a bond between carbons that are methoxylated i s preferred over one methoxy-lated and the other acetoxylated, which i n turn i s preferred over two .-.„••. • acetoxylated carbons. Secondary fragments are subsequently formed from the primary ones by sing l e or consecutive eliminations of formaldehyde (m/e 30), methanol Cm/e 32), ketene (m/e 42), a c e t i c acid (m/e 60), methyl acetate (m/e 74), methoxymethyl acetate (m/e 104), or acetoxymethyl acetate (m/e 132). 100 - 1 75 50 25 H 14 (a) T 50 100 150 200 T 250 50 25 (b) 50 100 150 200 250 Figure II .1 Mass spectra of (a) 1,3,5-tri-0-acetyl-2,4,6-tri-0-methyl-D - g l u c i t o l and (b) l,5,6-tri-0-acetyl-2,3,4-tri-0-methyl-D - g l u c i t o l . 15 I I . 4 Sugar sequence The i s o l a t i o n and c h a r a c t e r i z a t i o n of o l i g o s a c c h a r i d e fragments are the major keys to e l u c i d a t i n g the sequence of sugars i n the polys a c c h a r i d e and a l s o to making assignments i n the n.m.r. spectra. By usin g d i f f e r e n t fragmentative techniques, these o l i g o m e r i c fragments can be obtained and c h a r a c t e r i z e d a d d i t i v e l y to b u i l d up a pol y s a c c h a r i d e sequence. Lindberg 38 and coworkers have reviewed the v a r i o u s s p e c i f i c degradation techniques on p o l y s a c c h a r i d e s . II.4.1 P a r t i a l h y d r o l y s i s Many f a c t o r s a f f e c t the r a t e of h y d r o l y s i s of sugars, such as r i n g s i z e , c o n f i g u r a t i o n , conformation, p o l a r i t y , s i z e and nature of the non-39 carbohydrate s u b s t i t u e n t s . Capon has reviewed the r a t e constants f o r the a c i d c a t a l y z e d h y d r o l y s i s of a wide v a r i e t y of g l y c o s i d e s and inf e r e n c e s from these data may be as l i s t e d below. (These data could a l s o be extended to p o l y s a c c h a r i d e s ) . ( i ) The r e l a t i v e degree of s t a b i l i t y of the va r i o u s sugars to a c i d h y d r o l y s i s isi, as-tabulated: Degree of s t a b i l i t y to Type of sugars a c i d h y d r o l y s i s 2-deoxy-2-amino sugars A u r o n i c a c i d s hexopyranoses pentopyranoses hexofuranoses pentofuranoses 6-deoxy sugars, 2-keto-3-deoxy -D-manno-octonic a c i d 2-deoxy sugars, s i a l i c a c i d s di-deoxy sugars 16 ( i i ) non-reducing terminal and side-chain g l y c o s i d i c bonds are more e a s i l y hydrolyzed than the main in-chain g l y c o s i d i c bonds, ( i i i ) (1-6) linkages are more r e s i s t a n t to a c i d hydrolysis than (1-2) and (1-4) linkages, (1-3) linkages are generally the l e a s t r e s i s t a n t , (iv) a-glycosides are generally more l a b i l e than 3-giycosides. By ..varying acid type and concentration, along with temperature and reaction time, an optimum y i e l d of oligomeric fragments may be i s o l a t e d i f the hydrolysis of the polysaccharide i s arrested before i t s completion. The mixtures of mono, d i - , and higher oligosaccharides thus formed may then be fractionated by d i a l y s i s , gel chromatography, preparative paper chromatography and paper electrophoresis. One s i g n i f i c a n t drawback of obtaining oligomeric fragments v i a p a r t i a l hydrolysis i s that non-carbohydrate substituents l i k e acetals and acetates, which are more acid l a b i l e than g l y c o s i d i c bonds, are cleaved during the process. To obtain oligomeric fragments with these non-carbohydrate substituents i n t a c t , s p e c i f i c bacteriophage depolymerization of capsular polysaccharides i s employed (see Section IV). II.4.2 Periodate oxidation and Smith hydrolysis The uses of the c l a s s i c a l periodate oxidation, which cleaves the C-C bond between v i c i n a l d i o l s , are two-fold. One i s an a n a l y t i c a l technique using small amounts of material and d i l u t e periodate solutions to maintain s e l e c t i v e oxidation. The periodate consumption can be monitored spectro-40 photometrically and the r e s u l t indicates the number of periodate s e n s i t i v e sugars i n a polysaccharide. Except for 1-6 linked, in-chain sugars, one mole of periodate i s consumed for every o x i d i z a b l e sugar i n a repeating 17 K82 POLYSACCHARIDE I CARBODIIMIDE REDUCTION CH0R CH.R 2.0 R_J-1Q R = OH (CH 2OH) 2 NaBH, REDUCTION 4 1. 0.5 M TFA r . t . 48 h 2. NaBH, REDUCTION 4 R R CH2OH H OH + CH2OH CH2OH CH2OH Scheme II.2 Periodate oxidation - Smith hydrolysis of K82 polysaccharide 18 unit . The number of periodate r e s i s t a n t sugars i n the polysaccharide should be consistent with the methylation data. The second and more important use i s a fragmentative technique conducted on larger q u a n t i t i e s . The sugar residues i n the polysaccharide which are not oxidized by periodate are released from the oxidized residues by Smith hydrolysis as mono-, o l i g o - or poly- saccharides a f t e r some 41, 42 chemical modification. Smith hydrolysis i s based on the differ e n c e i n s t a b i l i t y towards acid between the g l y c o s i d i c bond and the a c y c l i c a c e t a l. The a c y c l i c a c e t a l linkages, which r e s u l t from the oxidized sugar residues, being more acid l a b i l e are s e l e c t i v e l y hydrolyzed (see Scheme II.2). 43 Painter has shown that various sugar residues are oxidized at d i f f e r e n t rates; c i s - being oxidized f a s t e r than t r a n s - d i o l ; the terminal residue, being more accessible, i s more r a p i d l y oxidized than the in-chain residues; and due to e l e c t r o n i c repulsions uronic acids are very slowly oxidized. ' 44 A recent technique, the ' s e l e c t i v e Smith degradation , e x p l o i t s these facts to s e l e c t i v e l y oxidize and hydrolyse polysaccharides to oligomeric fragments. II.4.3 Uronic acid degradation (g-elimination) This f u n c t i o n a l l y - s e l e c t i v e reaction can be used as a technique to degrade a c i d i c polysaccharides to defined oligosaccharide fragments. A 45 review on the 3-elimination reaction has been compiled by Kiss . The mechanism for t h i s reaction i s as outlined: 19 On methylation of an a c i d i c polysaccharide, the carboxylic f u n c t i o n a l i t y of the uronic acid i s e s t e r i f i e d , and t h i s being electron-withdrawing increases the a c i d i t y of the r i n g proton at C-5. On treatment with base (sodium methyl-s u l f i n y l methanide), the proton at C-5 i s removed followed by the elimination, from the g-position, of the leaving group, R^O, to give the hex-4-enopyranosyl 46 uronate ( I I ) . A s p i n a l l and R o s e l l have observed that the base treatment w i l l cleave the g l y c o s y l uronic linkage, without a f f e c t i n g i n t a c t g l y c o s i d i c bonds, to expose the hydroxyl group to which the uronic residue was attached. This free hydroxyl group can then be l a b e l l e d by a l k y l a t i o n with methyl iodide, e t h y l iodide or trideuteromethyl iodide. The 8 - e l i m i n a t i o n reaction 20 METHYLATED K82 POLYSACCHARIDE CH2R CH2R = OMe CH 3SCH 2 Na + 2. Mel 3. DIALYSIS CH R _CH_R 2 l 2 1. HYDROLYSIS 2. NaBH. REDUCTION 4 3. ACETYLATION 2,4,6-OMe 3-Galactitol 2,4,6-OMe 3-Glucitol 2 MOLES 1 MOLE 2,4,6-OMe 3-Galactitol = 1,3,5-tri-0-acetyl-2,4,6-tri-O-methyl-D-galactitol Scheme II.3 g-elimination of K l e b s i e l l a K82 polysaccharide 21 was used to determine the p o s i t i o n of the uronosyl linkage 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 on K82 polysaccharide (see Scheme II.3). II.5 Determination of linkage configurations II.5.1 Nuclear magnetic resonance (n.m.r.) N.m.r. spectroscopy i s now fi 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 . Current advances i n instrumentation i n recent years have increased tremendously the v e r s a t i l i t y of t h i s f i e l d . Extensive reviews on the a p p l i c a t i o n of n.m.r. to carbohydrate chemistry 47-54 have been w r i t t e n , but the information given here r e f e r s only to the d i r e c t a p p l i c a t i o n of n.m.r. 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 o l i g o - and poly- saccharides. The spectra of the capsular polysaccharides from the K l e b s i e l l a s e r i e s can be a r b i t r a r i l y divided i n t o three regions (see Table II.1). The fac t that these polysaccharides, although of molecular weights >10 , give i n t e r p r e t a b l e spectra i s a good i n d i c a t i o n of t h e i r regular, repeating-unit structures. 47-49 II.5.1.1 Proton magnetic resonance (p.m.r.) In the s t r u c t u r a l studies of carbohydrates, useful information can be derived from the following n.m.r. parameters: (a) Chemical s h i f t . The chemical s h i f t of a proton i n a molecule i s dependent on i t s chemical and magnetic environment, and these are c l o s e l y r e l a t e d to the s u b s t i t u t i o n a l , o r i e n t a t i o n a l and electronegative e f f e c t s of the neighbouring groups.., In the anomeric region (see Table I I . 1) , a d i v i s i o n at 6 5.0 was a r b i t r a r i l y accepted whereby signals appearing u p f i e l d TABLE II. 1 Three regions of a carbohydrate spectrum 1 13 a Region H C p.p.m.— Anomeric 4.5-5.5 9 3 - 1 1 0 >5.0 (J 1-3 Hz) <101 i , z <5.0 (Jl 2 7-9 Hz) >101 Ring 3.0 - 4.5 60 - 85 H-2 Man ) \ 4.0 - 4.5 H-5 GlcA J 2°C 7 5 ± 5 2°C (linked) 8 0 - 8 5 l 0 c 60 - 65 High f i e l d 1.0 - 2.5 15 - 30 N-acetate -2.03 -21 0-acetate -2.15 -21 CH 3 (6-deoxyhexose) -1.33 -17 CH^ (pyruvic acid ketal) -1.4 - -1.6 a x i a l CH 3 -18 equatorial CH -26 C of C = 0 group (acetate, pyruvate and uronic acid) appear -170 p.p.m. 23 of i t are assigned to 3-linkages ( a x i a l protons) and those signals appearing downfield are assigned to a-linkages (equatorial protons). In a polysaccharide spectrum, the number of anomeric signals and t h e i r corres-ponding i n t e g r a l s i n d i c a t e the number of sugar residues per repeating u n i t ; furthermore the linkage configurations (a orCB) can be determined from the combined measurements of the chemical s h i f t and coupling constant (see l a t e r ) . For the .•complex r i n g proton region, assignments are d i f f i c u l t ; solutions have been proposed by H a l l ^ ~ \ More recently, homonuclear two-56 dimensional J-n.m.r. has been used to s i m p l i f y spectra of mono- and disaccharides, by separating the e f f e c t s of chemical s h i f t s and s c a l a r coupling. In the high f i e l d region, the methyl resonances of L-rhamnose, L-fucose, acetate and pyruvate can be detected. (b) Coupling constant (J value). The r e l a t i o n s h i p between the three-3 ' bond v i c i n a l coupling constant ( J) and the dihedral angle (<j>) between the protons i s given approximately by the Karplus^ 7 equation: 8.5 c o s 2 * - 0.28 0°<(|> < 90° 9.5 cos 2* - 0.28 90 °<<t><180° The values are maximum when <j> i s 0° or 180°, and minimum when i t i s 90°. Although the coupling constant i s dependent on other parameters (such as e l e c t r o n e g a t i v i t y , angle s t r a i n , bond length, etc.) i t i s very useful for the assignment of t r a n s - d i a x i a l protons (cj> = 180°, g-linked, J 7-9 Hz);, and e q u a t o r i a l - a x i a l or e q u a t o r ia l-equatorial protons (cj> = 60°, a-linked, J l-3'i.:.Hz). These J values can be applied to hexoses l i k e D-glucose, D-galactose, D-glucuronic acid and D-galactouronic acid. Due to the equatorial proton at C-2, L-rhamnose and D-mannose have a d i f f e r e n t set of J values (a-anomer, ^ = 2. Hz, g-anomer, ^ = 1 Hz). 24 (c) Relative i n t e n s i t y of the s i g n a l s . The r e l a t i v e i n t e n s i t i e s of the d i f f e r e n t hydrogens are equal to the r e l a t i v e amounts of hydrogen producing the s i g n a l s . Consequently, a quantitative analysis of the r a t i o of a- to 3- linkages, number of 6-deoxy sugars, acetates, pyruvates etc. present i n the spectrum can be conducted. For oligosaccharides, the anomeric proton signals for the reducing end can be distinguished from signals of the other protons because i t displays the e f f e c t of mutarotation, showing two separate signals for the a- and 0- anomers. II.5.1.2. C n.m.r. spectroscopy Many methods have been developed that increase the s e n s i t i v i t y 13 of n a t u r a l abundance C-n.m.r.; such as pulse-Fourier transform n.m.r. 58 spectroscopy, and proton broad band decoupling which e f f e c t s the c o l l a p s i n g of spin multiplets i n t o s i n g l e t s and subsequently produces the nuclear 59 Overhauser e f f e c t that enhances the s i g n a l s , etc. More recently, advances 13 i n C 2-D n.m.r. spectroscopy have greatly s i m p l i f i e d complex carbohydrate fc 56,60,61. spectra 13 The C-n.m.r. spectrum of a polysaccharide contains u s e f u l information rel a t e d to the f i n e structure of the molecule, and l i k e the p.m.r. spectrum, t h i s too can be categorized i n t o three regions (see Table II.1). However unlike p.m.r., the main parameter used for assignment i s the chemical s h i f t . In the anomeric region, an a r b i t r a r y d i v i s i o n at 101 p.p.m. was accepted, but contrary to p.m.r. the a-anomeric carbons appear u p f i e l d of the 3-anomeric carbons due to a s h i e l d i n g e f f e c t . I t has been found that increased 13 s h i e l d i n g of a C nucleus i s accompanied by a decrease i n the s h i e l d i n g of 13 1 62 the appended proton, i . e . C and H s h i f t s are affected i n v e r s e l y . The anomeric carbons of free sugars (reducing end) appear u p f i e l d , i n the 25 region 93 - 97 p.p.m. In the r i n g carbon region, the signals due to the carbons of primary alcohols are very d i s t i n c t i v e at 60 - 65 p.p.m., which can be d i f f e r e n t i a t e d as linked or non-linked by t h e i r chemical s h i f t s (non-linked, 60 - 62 p.p.m., when linked, they are s h i f t e d 7 - 1 0 p.p.m. downfield). The signals due to the carbons of secondary alcohols appeared at 75 ± 5 p.p.m., but on O-glycosylation and or O-alkylation, the carbon(s) involved i s s u f f i c i e n t l y deshielding (by 7 - 11 p.p.m.) as to produce a s i g n a l w e l l separated from the other ri n g carbons (80 ±5 p.p.m.) This i s c a l l e d the 'a-effect'. However carbons immediately adjacent to that carbon w i l l be s l i g h t l y 54 shielded (1 - .2 p.p.m.), and t h i s i s the g-effect. These a- and g- s h i f t s 13 have been used i n assignments of C signals of o l i g o - and poly- saccharides and consequently d e l i n e a t i n g the s t r u c t u r a l sequence. In the high f i e l d region, the methyl groups are d i s t i n c t l y separated and can be e a s i l y a t t r i b u t e d to 6-deoxysugars (-17 p.p.m.), acetate (~21 p.p.m.) and pyruvate. I t has been shown that the stereochemistry of a pyruvic acid 63 k e t a l can be d i f f e r e n t i a t e d by the chemical s h i f t of the methyl group II.5.2 Other techniques O p t i c a l r o t a t i o n has been used to d i s t i n g u i s h between enantiomers. Based on Hudson's Isorotation R u l e s t h e configuration of s p e c i f i c linkages can be deduced from the measured o p t i c a l rotations. The use of chromium t r i o x i d e o x i d a t i o n 6 ^ on peracetylated o l i g o - and poly- saccharides i s another technique of determining the stereochemistry of the g l y c o s i d i c linkages. . The peracetylated hexopyranosides with aglycone occupying equatorial positions (g-linked) are oxidized p r e f e r e n t i a l l y to those with 66 a x i a l aglycones (d-linked). The c l a s s i c a l technique of enzymic hydrolysis 26 can be used to determine g l y c o s i d i c linkage configurations. This procedure depends mainly on the a v a i l a b i l i t y of enzymes having the proper s p e c i f i c i t y f o r a- and g- g l y c o s i d i c linkages. In K82, the D-GlcA^^D-Gal linkage was confirmed by the use of g-D-glucuronidase. 21 I I I STRUCTURAL INVESTIGATION OF KLEBSIELLA SEROTYPE K82 POLYSACCHARIDE 2 8 I I I . STRUCTURAL INVESTIGATION OF KLEBSIELLA SEROTYPE K82 POLYSACCHARIDE I I I . l ABSTRACT The structure "of the capsular polysaccharide from K l e b s i e l l a K82 has been studied by using the techniques of methylation, g-elimination, periodate oxidation-Smith hydrolysis and p a r t i a l h ydrolysis. N.m.r. 1 13 spectroscopy ( H and C) was used to e s t a b l i s h the nature of the anomeric linkages of the polysaccharide and of derived poly- and o l i g o - saccharides obtained through degradative procedures. The polysaccharide has been shown to comprise of tetrasaccharide repeating units of the 'three plus one' type: -*3) g-D-Glcp- (1+3)-a-D-Galp- (1+3)-g-D-Galp- (l-> 4 T 1 g-D-GlcAp III.2 Introduction 6 7 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 0rskov ' into approximately 77 serotypes, based on t h e i r antigenic, capsular polysaccharides. Nimmich'''^'^6 has analyzed q u a l i t a t i v e l y the polysaccharides from most st r a i n s and grouped them into chemo types'''7, K82 polysaccharide was found to belong to the chemotype containing glucose, galactose and glucuronic a c i d . (see Appendix I ) . K82 does not react with any antiserum against previously e s t a b l i s h K antigens 7. K82 antiserum gives capsular quellung with K8 and K20, but at such low t i t r e l e v e l s that absorptions have not been ca r r i e d out. As part of our program to cor r e l a t e chemical structure with immunological response, we now report on the e l u c i d a t i o n of the structure 29 of the K82 polysaccharide. III.3 Results and discussion Composition and n.m.r. spectra • K l e b s i e l l a K82 b a c t e r i a was grown on an agar medium and the a c i d i c capsular polysaccharide 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. The product was monodispersed by gel-permeation chromatography (M = 2x10 ) and had [a]p +31°, which compares w e l l with the calculated value of +32° 64 using Hudson's Rule of Is o r o t a t i o n . Paper chromatography of the acid hydrolyzate of the polysaccharide showed the presence of galactose, glucose and an aldobiouronic acid. Conversion of the sugars released to t h e i r a l d i t o l acetates, followed by g . l . c . a nalysis gave galactose and glucose i n the r a t i o 1.5 : 1. Further t o t a l sugar analyses, conducted on the carboxyl-reduced polysaccharide and carbodiimide-reduced polysaccharides, gave the data as shown i n Table I I I . l . These r e s u l t s i n d i c a t e that K82 polysaccharide contains galactose, glucose and glucuronic acid residues i n the r a t i o s of 2 : 1 : 1 , and that the polysaccharide consists of a tetrasaccharide repeating unit. The glucose and glucuronic acid were shown to be of the D configuration 30 by the c i r c u l a r dichroism curves of the corresponding a l d i t o l acetates The configuration of the galactose was shown to be D by the c i r c u l a r dichroism curve of the 2,4,6-tri-O-methyl d e r i v a t i v e obtained from the methylation a n a l y s i s . 1 13 The H and C-n.m.r. spectra of the K82 polysaccharide, which had to be m i l d l y depolymerized i n order to reduce the v i s c o s i t y , further substantiate that the repeating unit i s a tetrasaccharide (see Table III.2). The n.m.r. spectrum indicated (a) that three of the sugars are 3-linked and one a-linked, (b) the absence of deoxy-sugars, acetates, and pyruvic 30 acid k e t a l s , and (c) unsubstituted 0-6 posit i o n s of the hexoses (as indicated by the strong s i g n a l at 61.5 p.p.m.). The broad downfield s i g n a l at 6 4.50 was a t t r i b u t e d to the H-5 of the B-D-glucuronic acid. More precise assignment of the signals was achieved a f t e r studying the n.m.r. 1 13 spectra (both H and C) of the r e s i d u a l polysaccharide obtained a f t e r Smith degradation, and the various oligosaccharide fragments obtained a f t e r p a r t i a l hydrolysis (see l a t e r and Table I I I . 2 ) . Methylation analysis Methylation of the K82 polysaccharide, carbodiimide-reduced poly-saccharide, and remethylation of the carboxyl-reduced polysaccharide, followed by hy d r o l y s i s , d e r i v a t i z a t i o n as a l d i t o l acetates, and g.l.c.-m.s. analysis, gave the values shown i n Table III.3, columns I-IV. These r e s u l t s i n d i c a t e that (a) the polysaccharide consists of a tetrasaccharide repeating u n i t , (b) the branch-point is. a g a l a c t o s y l residue linked at C-3 and C-4, and (c) the acid i s D-glucuronic acid and i t appeared as a terminal, non-reducing residue. To determine the l o c a t i o n of the glucuronic acid and the length of the side-chain the permethylated polysaccharide was 6 7 subjected to a base-catalyzed, uronic acid degradation ( B-elimination) using sodium methylsulfinylmethanide. The product was d i r e c t l y alkylated with methyl iodide and the residue i s o l a t e d was hydrolyzed, reduced and acetylated. The g.l.c.-m.s. analysis (Table III.3, column V) showed 2,4,6-tri-0-methylgalactose and 2,4,6-tri-0-methylglucose i n the r a t i o 2 : 1. This r e s u l t showed that the polysaccharide consists of a 3-linked hexose backbone with the terminal glucuronosyl residue linked at C-4 of the branch galactose. 31 TABLE III.1 SUGAR ANALYSIS OF K82 POLYSACCHARIDE AND DERIVED PRODUCTS Sugars— (as a l d i t o l acetates) Mole r a t i o I XI I l l IV A4 A3 A2 Galactose 1.5 1.0 1.09 2.2 1.08 1.0 1.0 Glucose 1.0 0.83 1.0 1.0 1.0 1.94 0.87 Glycerol - - 0.15s- - -— Using SP-2340 column programmed f o r 195° for 4 min, 2°/min to 260°. — I, o r i g i n a l a c i d i c polysaccharide; I I , carboxyl-reduced polysaccharide; I I I , carbodiimide-reduced polysaccharide; IV, p o l y o l from periodate oxidation; A4, A3 and A2, carboxyl-reduced a c i d i c oligosaccharides obtained from p a r t i a l h y d r o l y s i s ; — Quantitation of g l y c e r o l t r i a c e t a t e has been shown to be inaccurate, due to i t s high v o l a t i l i t y . TABLE III . 2 N.M.R. DATA FOR K l e b s i e l l a K82 CAPSULAR POLYSACCHARIDE AND THE DERIVED OLIGOSACCHARIDES Compound- 1. H-n.m.r. data 13 C-n.m.r. data b J l . 2 * (Hz) Integral (H) Assignment— p.p.m.— A • f Assignment— GlcA-i-^Gal~OH 5.29 3 0.30 a-Gal-OH 103.5 g-GlcA p 4.75 8 1.00 g-GlcA 93.4 a-Gal~OH A2 4.61 8 0.70 g-Gal-OH 4.50 1.00 H-5 (GlcA) i 3 Glc-r-Gal~OH 172.3 C=0 (GlcA) $ 4 i g 5.30 3 0.65 a-Gal~OH 105.4 g-Glc i GlcA 4.97 8 1.00 -.'g-GlcA 103.3 g-GlcA A3 4.69 8 0.35 g-Gal-OH 97.5 g-Gal-OH 4.65 8 1.00 6-Glc 93.4 a-Gal~0H 13 1 3 G1C——Gal Gal~ OH 5.31 3 1.00 a-Gal " 4 a g 4.95 8 1.00 B-GlcA 1 GlcA 4.77 8 1.00 g-Gal-OH A4 4.66 8 1.00 g-Glc Glc . Gal G a l a c t i t o l 5.5 s 1.00 a-Gal B 4 a B 4.73 8 1.00 B-GlcA 1 GlcA 4.54 8 1.00 B-Glc A4 (reduced) 4.50 b 1.00 H-5 (GlcA) 3 1 3 1 3 1 — ^ l c ^ - ^ G a l - ^ - ^ G a l — 5.40 s 1.00 a-Gal 105.3 B-Glc p a p 4.75 8 1.00 3-Gal 105.15 6- Gal 4.71 8 1.00 B-Glc 100.35 a-Gal SD 62.0 C-6 of hexoses K82 capsular polysaccharide 5.40 s a-Gal 171.3 C=0 (GlcA) 5.30 s a-Gal~OH 105.31 B-Glc (mildly hydrolyzed) 4.96 8 1.00 B-GlcA 103.3 B-GlcA 4.71 8 3-Gal 105.02 3-Gal • 2.4 3-Gal~0H 99.8 a-Gal 4.64 8 - B-Glc 61.5 C-6 of hexoses 4.50 b H-5 (GlcA) — For the o r i g i n of compounds A2, A3, A4 and SD, see text. See Appendix I I for reproductions of the spectra. — Chemical s h i f t r e l a t i v e to i n t e r n a l acetone; <5 2.23 downfield from sodium 4,4-dimethy1-4-silapentane-1-c d sulfonate (D.S.S.). — Key: b = broad, unable to assign accurate coupling constant, s = s i n g l e t . — For example, B~Gal = proton on C-l of B-linked D-Gal residue and Gal~0H = terminal, reducing ga l a c t o s y l residue. — Chemical s h i f t i n 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 f d 13 D.S.S. — As for —, but for anomeric C n u c l e i . TABLE III.3 METHYLATION ANALYSES OF K82 POLYSACCHARIDE AND DERIVED PRODUCTS 3. Methylated sugar— (as a l d i t o l acetate) ^c III* Mole i v * V^ VI* V I I - V I I I -2,3,4,6-Glc 19 23 23 36 2,3,4-Glc 22 25 - 30 2,4,6-Glc 32 24 28 25 34 34 2,4,6-Gal 37 28 30 25 66 66 10 2,5,6-Gal 15 2,6-Gal 31 26 23 27 27 34 - 2,3,4,6-Glc = l,5-di-0-acetyl-2, 3,4,6-•tetra-O-methylglucitol, etc. — Values are corrected by use of the e f f e c t i v e , carbon-response factors given by Albersheim e t . a l . 36 c Using ECNSS-•M column programmed for 180° for 4 min and 2°/min to 200 d Using SP-1000 column at 200° isothermal. e — Using ECNSS-M column at 170° isothermal. I, o r i g i n a l a c i d i c polysaccharide; I I , reduction of uronic ester; I I I , remethy-l a t i o n a f t e r reduction of uronic ester; IV, carbodiimide-reduced capsular polysaccharide; V, product from ^ e l i m i n a t i o n and remethylation; VI, product from Smith degradation; VII, methylated and reduced tetrasaccharide; VIII, methylated and reduced trisaccharide. 35 Periodate oxidation - Smith hydrolysis Periodate oxidation of the carbodiimide-reduced K82 polysaccharide was terminated a f t e r 100 h. See Figures I I I . l and III.2. A t o t a l of two moles of periodate per repeating unit was consumed, and th i s agreed with the t h e o r e t i c a l value. The p o l y o l recovered, a f t e r d i a l y s i s , was analyzed as i t s a l d i t o l acetates (see Table I I I . l , column IV). Galactose, glucose and g l y c e r o l appeared i n the r a t i o s 2 : 1 : 0.15, i n d i c a t i n g that one of the glucosyl residue (glucuronic acid) has been oxidized. The product SD, recovered a f t e r Smith hydrolysis was a polysaccharide (as indicated by d i a l y s i s and paper chromatography). The n.m.r. data (Table III.2) indicated three anomeric protons, one a- and two 8-linkages. The signals at 6 4.96 ( J 1 „ 8 Hz) and <5 4.50 which appeared i n the o r i g i n a l polysaccharide have disappeared. This indicated that the glucuronic acid i s B - l i n k e d and the broad s i g n a l at 6 4.50 can be a t t r i b u t e d to H-5 of the 68 glucuronic acid. Other studies have demonstrated that the n.m.r. s i g n a l of H-5 of D-glucuronic acid can appear down-field, approaching the anomeric region (6 4.5). Methylation analysis of SD (Table III.3, column VI) showed 2,4,6-tri-O-methylgalactose and 2,4,6-tri-O-methylglucose i n the r a t i o 2:1, which i s i d e n t i c a l to the r e s u l t from B-elimination (Table III.3, column V). Hence by two d i f f e r e n t techniques, K82 polysaccharide was shown to have a 3-linked hexose backbone with the terminal glucuronic acid linked to C-4 of the branch galactose. P a r t i a l hydrolysis Two possible structures (A or B) are consistent with the r e s u l t s thus f ar discussed. 36 3 1 3 1 3 1 — Glc Gal Gal — 1' GlcA 3 1 3 1 3 1 — Gal Gal — - Glc — l 1 GlcA To determine the sugar sequence of the backbone, oligomeric fragments of the polysaccharide must be generated.;' From the s t r u c t u r a l studies conducted 69 on K36 polysaccharide , i t was demonstrated that 3-linked galactosyl bonds are very l a b i l e to acid hydrolysis. Based on t h i s f a c t , h y d r o l y t i c conditions were monitored to. s e l e c t i v e l y cleave the l a b i l e 3-linked galactosyl linkages to give defined oligomeric fragments. Monitoring by paper chromatography of a progressive, acid hydrolysis of a small amount of native polysaccharide revealed that optimal production of oligosaccharides occurred a f t e r treatment for lh h with IM TFA on a steam-bath. P a r t i a l , acid hydrolysis of a larger quantity of the native polysaccharide, followed by separation on an ion-exchange column, gave a n e u t r a l and an a c i d i c f r a c t i o n . Paper chromatography of the neutral f r a c t i o n showed mainly galactose and glucose, and an i n s i g n i f i c a n t amount of a disaccharide, which was not further analyzed. Separation of the a c i d i c f r a c t i o n by gel-permeation chromatography gave two main f r a c t i o n s , 1 and 2 (see Figure I I I . 3 ) . Paper electrophoresis and paper chromatography of f r a c t i o n 1 showed i t to be polymeric. Further separation by paper chromatography of f r a c t i o n 2 y i e l d e d three pure oligomers, A2, A3 and A4. Compound A2 (R . 0.44), on t o t a l sugar analysis gave glucose and Gl c galactose i n the r a t i o 0.87 : 1 (see Table I I I . l ) , and the n.m.r. spectrum (Table III.2) indicated i t to be an aldobiouronic acid. Enzymic hydrolysis of A2 using B-glucuronidase gave a p o s i t i v e r e s u l t , confirming that the 37 hours 50 100 Figure III.2 Periodate consumption by K82 polysaccharide with respect to time. 38 Weight i n mg 50 100 150 E l u t i o n volume (mL) Figure III.3. Separation of K82 p a r t i a l hydrolyzate by gel-permeation chromatography (Bio-gel P-2) 39 uronosyl linkage has a B configuration. Based on t h i s evidence, the structure of A2 i s as shown i n Table III.2. Compound A3, (R . 0.33), Glc on t o t a l sugar analysis gave glucose and galactose i n the r a t i o ~2 : 1 (Table I I I . l ) . Methylation of A3, and subsequent reduction with l i t h i u m aluminum hydride, 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 as a l d i t o l acetates, gave products corresponding to 2,3,4,6-tetra-O-methylglucose, 2,3,4-tri-O-methyl-glucose and 2,6-di-0-methylgalactose (see Table II I . 3 ) . On the basis of thi s methylation analysis and the n.m.r. s p e c t r a l data, the structure and anomeric configurations of A3 are deduced as shown i n Table III.2. Compound A4 (RQ-^ 0-2), on t o t a l sugar analysis gave glucose and galactose i n the r a t i o 1 : 1 (Table I I I . l ) . Methylation analysis (as described f o r A3) yielded 2,3,4,6-tetra-O-methylglucose, 2,3,4-tri-O-methyl-glucose, 2,4,6-tri-O-methylgalactose, 2,5,6-tri-0-methylgalactose and 2,6-di-0-methylgalactose i n the proportions as shown i n Table III.3. The presence of 2,4,6- and 2,5,6-tri-O-methylgalactose, whose proportions integrated to one galactose u n i t , indicated that A4 has a terminal, reducing galactosyl residue. The presence of 2,5,6-tri-O-methylgalactose, which can be equated to a galactofuranose residue linked at C-3, i s explained as follows. In DMSO, during the Hakomori methylation, the 3-linked reducing galactose can interconvert from the pyranoid to the furanoid configuration; the l a t t e r , due to less s t e r i c and e l e c t r o n i c i n t e r a c t i o n s , i s more favour-a b l e ^ . On the basi s of t h i s methylation analysis and n.m.r. s p e c t r a l data, the structure assigned to A4 i s as shown i n Table III.2. To determine the anomeric configuration of the branch g a l a c t o s y l residue, A4 was reduced with sodium borohydride, exchanged with IR 120 H + r e s i n and c o d i s t i l l e d (4X) with methanol to remove the borate formed. A f t e r several exchanges with D o0, the reduced A4 was n.m.r. analyzed. From the n.m.r. data of reduced 40 A4 i n Table III.2, the branch g a l a c t o s y l residue was deduced to be a-linked. Conclusion. The structure of the capsular polysaccharide from K l e b s i e l l a serotype K82 i s thus based on the tetrasaccharide repeating unit shown. This structure i s consistent with the biochemical analysis reported by 0rskov and Fife-Asbury, that the K82 antiserum gives capsular quellung with K8 and K20. This i s probably due to the non-reducing, terminal GlcA - Gal residue which i s common i n a l l three K types. A comparison of the p.m.r. spectrum of K8 (see Appendix II) to K82 showed i t to be s i g n i f i c a n t l y d i f f e r e n t , thus i n d i c a t i n g the differences i n t h e i r structures and j u s t i f y i n g t h e i r 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 . III.4 EXPERIMENTAL General methods. Solutions were concentrated at bath temperatures not exceeding 40°. Frozen solutions were obtained using a dry i c e - acetone mixture and l y o p h i l i z e d on a Unitrap II freeze-dryer. Deionizations were performed on a column of Amberlite IR-120 (H +) r e s i n . O p t i c a l rotations were measured on aqueous solutions at 23 - 25°C on a Perkin-Elmer model 141 polarimeter with a 10 cm c e l l . C i r c u l a r dichroism spectra ( c d . ) were recorded on a Jasco J20 automatic recording spectropo.larimeter with a quartz c e l l of 0.3 mL capacity and a path length of 0.1 cm. Compounds were dissolved i n spectroscopic grade a c e t o n i t r i l e and the spectra recorded i n the range 210 - 240 nm. The i n f r a r e d ( i . r . ) spectra of methylated derivatives were recorded on a Perkin-Elmer model 457 spectro-photometer. The solvent used was spectroscopic grade carbon t e t r a c h l o r i d e . A n a l y t i c a l paper chromatography was performed by the descending method 41 using Whatman No 1 paper and the following solvent systems: (1) 18 : 3 : 1 : 4 et h y l acetate - a c e t i c acid - formic acid - water, (2) f r e s h l y prepared 2 : 1 : 1 1-butanol - a c e t i c acid - water, (3) 8 : 2 : 1 e t h y l acetate -p y r i d i n e - water and (4) upper phase of 4 : 1 : 5 1-butanol - ethanol -water. Preparative paper chromatography was carried out using Whatman No. 3 paper and e i t h e r solvent system 1 or 2. A n a l y t i c a l paper e l e c t r o -phoresis was conducted on a Savant high voltage (5KV) system (model LT-48A). Kerosene was the coolant and the b u f f e r system used was 5 : 2 : 743 pyridine -a c e t i c acid - water, pH 5.3. Whatman No. 1 paper (77 cm x 20 cm) was used for a l l runs, with current a p p l i c a t i o n of 25 - 50 mA. Chromatograms were ei t h e r developed with a l k a l i n e s i l v e r n i t r a t e ^ or by heating at 110° for 10 min a f t e r being sprayed with p-anisidine hydrochloride 7*' i n aqueous 1-butanol. Sugars and oligosaccharides were detected by these methods. Preparative gel-permeation chromatography was performed using columns (2.5 x 100 cm) of Bio-Gel P-2 (400 mesh). The void volume of the column and the e f f i c i e n c y of packing were determined using blue dextran (0.2%). The concentration of the samples applied to the column ranged from 40-100 mg/mL. Eluant used was 500 : 5 : 2 water - pyridine - a c e t i c a c i d at a flow-rate of 10 mL/h. Fractions (2.5 mL) were c o l l e c t e d , freeze-dried, weighed and the e l u t i o n p r o f i l e was obtained. Sephadex LH-20 was used to p u r i f y large molecular weight carbohydrate material that i s soluble i n organic solvent e.g. permethylated o l i g o - and poly- saccharides. A n a l y t i c a l g . l . c . separations were performed on a Hewlett-Packard 5700 instrument f i t t e d with dual flame-ionization detectors. An Infotronics CRS-100 e l e c t r o n i c integrator was used to quantify the peak areas. S t a i n l e s s -s t e e l columns (1.8 m x 3 mm) were used with a nitrogen carrier-gas flow-rate of 20 mL/min. The columns used were: (A) 3% of SP-2340 on Supelcoport 42 (100-120 mesh), programmed for 195° for 4 min, 2°/min to 260°, (B) 5% of ECNSS-M on Gas Chrom Q (100-120 mesh), isothermal at 170° or programmed at 180° for 4 min, 2°/min to 190°, (C) 3% of OV-225 on Gas Chrom Q (100-120 mesh), isothermal at 170° or programmed at 180° f o r 4 min, 2°/min to 190°, and (D) 5% of SP-1000 on Gas Chrom Q (100-120 mesh), isothermal at 220°. Preparative g . l . c . was performed with a F and M model 720 dual column instrument f i t t e d with thermal conductivity detectors, and a helium carrier-gas flow-rate of 60 mL/min. S t a i n l e s s - s t e e l columns (1.8 m x 6.3 mm) used were : (E) 3% of SP-2340 on Supelcoport (100-120 mesh), programmed from 200 -260° at 4°/min, and (F) 5% of OV-225 on Supelcoport (100-120 mesh), programmed from 190 -260° at l°/min. G.l.c.-m.s. was performed with a Micromass 12 instrument f i t t e d with a Watson-Biemann separator and a helium carrier-gas flow-rate of 25 mL/min. Spectra were recorded at 70 eV with an i o n i z a t i o n current of 100 uA and an ion-source temperature of 200°. The columns used f o r the separation were (B), (C), and (D). Proton magnetic resonance spectra were recorded on a Bruker WH-400 instrument. Spectra were recorded e i t h e r at ambient or elevated temperatures (95°±5) and acetone (2.23 p.p.m.) was used as an i n t e r n a l standard. A l l values are given r e l a t i v e to that of external sodium-4,4-dimethy1-4-sila-pentanesulfonate taken as 0. Samples were prepared by d i s s o l v i n g i n D^O and freeze-drying 3 times from D^O solutions. Carbohydrate samples (10-20 mg) 13 were dissolved i n D 20 and submitted i n 5 mm diameter n.m.r. tubes. C-n.m.r. experiments were conducted on Bruker WH-400, Bruker WP—80, or Varian CFT-20 instruments. A l l spectra were recorded at ambient temperature and acetone was used as the i n t e r n a l standard (at 31.07 p.p.m.). Samples were dissolved i n the minimum of D 20 and submitted i n n.m.r. tubes of diameter sizes 5 mm or 10 mm. 43 Preparation and properties of K l e b s i e l l a K82 capsular polysaccharide. The media ..used,were: ( i ) nutrient agar: bactopeptone (5 g) , bactobeef extract (3 g), NaCl (2 g) and agar (15 g) per l i t r e of water. The nutrient medium without agar i s referred to as broth. Agar plates were made by pouring the s t e r i l e , l i q u i d nutrient agar into P e t r i dishes (8.5 cm diameter) and allowing i t to set at room temperature. ( i i ) Sucrose-yeast extract-agar medium: sucrose (75 g), NaCl (5 g), yeast extract (5 g), K 2HP0 4 (2.5 g), MgS04-7H20 (0.62 g), CaC0 3 (0.5 g) and agar (37.5 g) i n 2.5 l i t r e s of water. S t e r i l i z a t i o n of glassware and nutrient media were performed i n an American S t e r i l i z e r model 57-CR for 20 min at 121° and 15-20 p . s . i . A culture of K l e b s i e l l a K82 b a c t e r i a was obtained from Dr. Ida 0rskov (WHO International Escherichia Center, Copenhagen). Actively-growing colonies of t h i s bacterium were propagated by r e - p l a t i n g several times on to agar plates i a s i n g l e colony being selected each time the b a c t e r i a were to be plated. Growth overnight at 37° was s u f f i c i e n t , a f t e r which the agar plate can be kept i n the r e f r i g e r a t o r . Actively-growing K l e b s i e l l a K82 b a c t e r i a , which were cultured for ~4 h i n broth (100 mL) at 37°, were poured on to a s t e r i l e , sucrose-yeast extract-agar medium (in a metal tray 60 x 40 cm) and l e f t to incubate for 3 days at room temperature. The b a c t e r i a l lawn produced was harvested by scraping the agar surface and the b a c t e r i a k i l l e d with 1% phenol s o l u t i o n . A f t e r constant s t i r r i n g for ~48 h at 4°, u l t r a c e n t r i f u g a t i o n (for 4 h at 15° on a Beckmann L3-50 u l t r a c e n t r i -fuge using rotor 35 at 31000 r.p.m. or 80000 g) separated the polysaccharide from the dead b a c t e r i a l c e l l s . The viscous supernatant, containing the dissolved polysaccharide, was p r e c i p i t a t e d into 4 : 1 ethanol - acetone mixture. The resultant stringy p r e c i p i t a t e was dissolved i n water and 44 20 treated with Cetavlon (cetyltrimethylanunonium bromide). The Cetavlon-polysaccharide complex was dissolved i n 4M_J NaCl s o l u t i o n , p r e c i p i t a t e d i n t o 4 : 1 ethanol - acetone mixture, redissolved i n water, and dialyzed against running tap water (3 days). The l y o p h i l i z e d polysaccharide (6 g) i s o l a t e d was a styrofoam-like material and had [a]^ +31° (c 0.29, water). Analysis by gel chromatography (courtesy of Dr. S.C. Churms, U n i v e r s i t y of Cape Town, S. A f r i c a ) showed i t to be homogeneous with an average molecular weight of 6 1 1 3 2 x 10 Daltons. N.m.r. spectroscopy ( H and C) was performed on the o r i g i n a l polysaccharide, but bet t e r spectra were obtained a f t e r mild t r e a t -ment of the polysaccharide with 0.5M t r i f l u o r o a c e t i c acid f o r 30 min on a steam-bath. The p r i n c i p a l signals and t h e i r assignments f or both the 13 and C-n.m.r. spectra are recorded i n Table III.2 (See Appendix I I for the reproduction of a l l n.m.r. spectra). Sugar analysis Hydrolysis of a sample (25 mg) of K82 polysaccharide with 2M t r i -f l u o r o a c e t i c acid (TFA) overnight on a steam-bath, removal of the acid by c o d i s t i l l a t i o n s with water (4X) followed by paper chromatography (solvent 1), showed three major spots of glucose, galactose and an aldo-biouronic acid (R . 0.41). The sugars released were reduced (sodium Glc borohydride i n water, f or 45 min) and the mixture made neutral with IR-120 (H +) r e s i n , f i l t e r e d , evaporated to dryness and c o d i s t i l l e d with portions of methanol (5 mL). Ac e t y l a t i o n ( 1 : 1 a c e t i c anhydride - pyridine) f o r 1 h on a steam-bath under anhydrous conditions, followed by g. l . c . analysis (column A) showed the a l d i t o l acetates of galactose : glucose i n the r a t i o 1.5 : 1 (Table I I I . l , column 1). A sample of K82 polysaccharide (20 mg), dried i n vacuo and under an 45 i . r . lamp, was treated with methanolic hydrogen chl o r i d e (3%) and refluxed overnight on a steam-bath under anhydrous conditions. The excess acid was neutralized with lead carbonate, and the mixture was centrifuged to remove the resultant lead chloride p r e c i p i t a t e . The supernatant was concentrated to dryness and the residue obtained was reduced with sodium borohydride i n anhydrous methanol. A f t e r 1 h, the mixture was made neutral with Amberlite IR-120 (H +) cation-exchange r e s i n , and f i l t e r e d , and the f i l t r a t e concentrated to dryness and c o d i s t i l l e d with three portions of methanol (5 mL) i n order to remove the borate ion. The residue was further hydrolyzed with 2M TFA on a steam-bath overnight. The TFA was removed by c o d i s t i l l a t i o n with water, and paper chromatography of the residue (in solvent 1) indicated two major spots of glucose and galactose. A f a i n t spot (R A 0.4) was ( j l C a t t r i b u t e d to the aldobiouronic acid. The sugars released were reduced with aqueous sodium borohydride s o l u t i o n for 45 min at room temperature. A f t e r the usual workup, the a l d i t o l s were acetylated (1 : 1 a c e t i c anhydride -pyridine mixture) for 1 h under anhydrous conditions. Excess a c e t i c anhydride was neutralized with ethanol; the pyridine was removed by c o d i s t i l l a t i o n with four portions of water. The a l d i t o l acetates dissolved i n chloroform were analyzed by g . l . c . using column A (See Table I I I . l , column I I ) . Samples for c d . analysis were obtained by preparative g . l . c . (column E). By comparison with authentic standards"^, the c d . curves of the a l d i t o l acetates corresponding to glucose and glucuronic acid (reduced to glucose) showed these sugars to have the D configuration. The configuration of the ga l a c t o s y l residue was determined by examining the c d . curve of the 2,4,6-tri-O-methylgalactose as the a l d i t o l acetate obtained during methylation analysis, and separated by preparative g . l . c . (Column F). By comparison 46 with an authentic standard, the galactose was shown to be of the D configuration. 24 Carbodiimide reduction of K82 polysaccharide A sample of K82 polysaccharide (H + form, 1.05 g) was dissolved i n water (100 mL) and the resultant pH was 3.75. l-Cyclohexyl-3-(2-morpholino-e t h y l ) - carbodiimide metho-p-toluenesulfonate (CMC, 8 g) was added. This corresponds to about ten times the equivalent of carboxylic acid i n the polysaccharide, based on one glucuronic acid residue per tetrasaccharide repeating u n i t . As the reaction proceeded (with consumption of hydrogen io n s ) , the pH was maintained at 4.75 by t i t r a t i n g with hydrochloric acid s o l u t i o n (0.1M). When the consumption of acid ceased, approximately three hours l a t e r , an aqueous s o l u t i o n of sodium borohydride (2M) was added dropwise. Foaming was c o n t r o l l e d by the addition of a few drops of 1-octahol. A t o t a l of 300 mL of sodium borohydride s o l u t i o n was added over a period of two hours. The pH of the s o l u t i o n was maintained between 6.5 - 7.0 with hydrochloric acid (4M) throughout the base addition. A f t e r concentration (to a smaller volume), the polysaccharide s o l u t i o n was dialyzed against running tap water (3 days). The l y o p h i l i z e d product, on d i s s o l u t i o n i n water (100 mL), indicated a pH of about 7. A second carbodiimide-reduction treatment was c a r r i e d o u t , ( y i e l d = 1.03 g). A sample of the reduced polysaccharide (30 mg) was hydrolyzed over-night with 2M TFA on a steam-bath, followed by sodium borohydride reduction and a c e t y l a t i o n to i t s a l d i t o l acetates. The g . l . c . analysis indicated that the reduction of the glucuronic acid was complete (See Table I I I . l , column "." 1 I I I ) . 47 Methylation analysisT""' ~"~ A sample of K82 polysaccharide (Na + s a l t , 300 mg) was converted into i t s free acid form by passage through a column of Amberlite IR-120 (H +) cation-exchange r e s i n . The l y o p h i l i z e d product, a f t e r further drying i n vacuo and under i . r . lamp, was dissolved with s t i r r i n g i n anhydrous dimethylsulfoxide (DMSO, 30 mL) under nitrogen atmosphere. Sodium methyl-sulfinylmethanide (2M, 10 mL) was added to the s o l u t i o n v i a a syringe and l e f t to s t i r f o r 3 h. The reaction mixture was frozen and methyl iodide (15 mL) was added v i a a syringe to the nitrogen sealed system. When the i n i t i a l murky reaction mixture was transformed to a c l e a r , pale-yellow s o l u t i o n (1*2 h), the excess methyl iodide was evaporated o f f . The methylated polysaccharide was recovered by d i a l y s i s (m.w. cut o f f 13,500) against running tap water overnight and the contents of the bag freeze-dried. Further drying i n vacuo and under i . r . lamp, followed by i . r . spectroscopic analysis indicated incomplete methylation (hydroxyl absorptions at 3625 cm ^ -1 33 and 3200 - 3500 cm ). A subsequent Purdie-Irving methylation was conducted. The dried sample of p a r t i a l l y methylated polysaccharide was dissolved i n anhydrous methyl iodide (15 mL) and refluxed with dried s i l v e r ./(I) oxide (300 mg) for 5 days. The s i l v e r oxide and s i l v e r s a l t s were washed twice with chloroform a f t e r c e n t r i f u g a t i o n . The chloroform extracts were combined and the solvent removed by evaporation. The dried product, on i . r . analysis showed no hydroxyl absorptions, i n d i c a t i n g complete methylation. Methylated K82 polysaccharide (60 mg) was hydrolyzed with 2M TFA overnight on a steam-bath. A f t e r the removal of excess acid by c o d i s t i l l a t i o n s with methanol, paper chromatography of the hydrolyzate (solvent 4, developed with p-anisidine hydrochloride) revealed 4 spots: 2,4,6-tri-O-methylglucose 48 ( T T M G t 0.77); 2,4,6-tri-O-methylgalactose ( T T M G 0.57); 2,6-di-O-methyl-galactose ( T T M G 0.47); and the aldobiouronic acid ( G l c A — Gal, 0.13). Subsequent reduction with sodium borohydride, followed by a c e t y l a t i o n with a c e t i c anhydride - p y r i d i n e , yielded a mixture of p a r t i a l l y methylated, a l d i t o l acetates which was analyzed by g . l . c . and g.l.c.-m.s. (column B) (See Table III.3, column I ) . A separate portion of the methylated polysaccharide (120 mg) was reduced with l i t h i u m aluminum hydride i n r e f l u x i n g oxolane overnight. Ethanol was added to decompose the excess l i t h i u m aluminum hydride, the p r e c i p i t a t e formed was dissolved i n hydrogen chloride s o l u t i o n (10%), and the product recovered by chloroform extraction (3X). The dried product, on i . r . a n a l y s i s , showed no carbonyl absorption. Hydrolysis of t h i s material (50 mg) with 2M TFA overnight on a steam bath, and subsequent reduction with sodium borohydride, followed by a c e t y l a t i o n with a c e t i c anhydride -pyridine gave on g . l . c . and g.l.c.-m.s. analyses the data i n Table III.3, column I I . Methylated, carboxyl-reduced polysaccharide (60 mg) was 32 remethylated by the Hakomori procedure and the product hydrolyzed with 2M TFA (22 h). Paper chromatography of the hydrolyzate (solvent 4, developed with p - a n i s i d i n e hydrochloride) indicated the appearance of 2,3,4,6-tetra-0-methylglucose (T 1.0, pink). The conversion of these sugars to t h e i r a l d i t o l acetates, followed by g . l . c . and g.l.c.-m.s. analyses gave the r e s u l t s i n Table III.3, column I I I . Methylation of the carboxyl-reduced K82 polysaccharide (30 mg), the polysaccharide recovered a f t e r the carbodiimide reduction, by the Hakomori procedure, followed by 2M TFA hydrolysis and conversion to i t s a l d i t o l acetates gave on g . l . c . and g.l.c.-m.s. analyses the data i n Table I I I , TMG = 2,3,4,6-tetra-O-methylglucose. 49 column IV. 67 Uronic acid degradation Methylated K82 polysaccharide (80 mg), dried i n vacuo and under i . r . lamp, was dissolved i n 19 : 1 DMSO - 2,2-dimethoxypropane (20 mL) mixture with a trace of p-toluenesulfonic acid and s t i r r e d under nitrogen atmosphere. Sodium methylsulfinylmethanide (15 mL) was added and the reaction mixture l e f t s t i r r i n g at room temperature f o r 18 h. Methyl iodide was added to the frozen mixture and a f t e r s t i r r i n g f o r 1.5 h, the excess methyl iodide was evaporated o f f . The methylated, degraded product was recovered by p a r t i t i o n between chloroform and water (3 x 15 mL). The dried product was hydrolyzed with 2M TFA and the sugars released were analyzed as described previously for the methylation analysis (See Table III.3, column V). 40 41 42 Periodate oxidation and Smith-hydrolysis ' of carbodiimide-reduced K82 polysaccharide. The consumption of periodate may be followed spectrophotometrically by monitoring the decrease i n absorbance of periodate ion (10 ^  ) with a correction f o r absorbance of iodate ion (10 3 )• Solutions of 10^ /IO^ , each of 0.015M, were made i n varying molar proportions. Aliquots (0.1 mL) of these solutions were withdrawn and d i l u t e d with water to 25 mL. The absorbances of the r e s u l t i n g solutions were measured i n a G i l f o r d spectro-photometer model 240 at 223 nm' (See Figure I I I . l ) . A sample of the carbodiimide-reduced K82 polysaccharide (26 mg) was dissolved i n water (5 mL). Sodium metaperiodate s o l u t i o n (0.03 M, 5 mL) was added, r e s u l t i n g i n a s o l u t i o n of periodate concentration of 0.015 5Q m o l e s / l i t r e . The reaction was conducted at room temperature and i n the dark. Aliquots (0.1 mL) were withdrawn p e r i o d i c a l l y and d i l u t e d 250 times with water. The absorbances of the r e s u l t i n g solutions were determined and the r e s u l t s compared with those of the standard 10 ^ /IO^ solutions. The periodate consumption reached a plateau a f t e r about 100 h (See Figure III. 2 ) , and approximately 0.08 mmoles 10^ had been consumed. This i s equivalent to 2 moles of 10^ per mole of repeating u n i t . Ethylene g l y c o l (0.1 mL) was added to n e u t r a l i z e the excess periodate, and the polyaldehyde was reduced with sodium borohydride, neutralized with a c e t i c acid (50%), dialyzed and l y o p h i l i z e d to y i e l d the polyalcohol (13.5 mg) . A sample of the polyalcohol (5.7 mg) was hydrolyzed with 2M TFA overnight, the product was reduced, and the a l d i t o l s were acetylated. The a l d i t o l acetates of galactose, glucose and g l y c e r o l were analyzed by g . l . c . (column A) and found to be present i n the r a t i o s of 2 : 1 :0.15 (See Table I I I . l , column IV). A s o l u t i o n of carbodiimide-reduced K82 polysaccharide (270 mg) i n water (150 mL) was mixed with 0.2M sodium metoperiodate s o l u t i o n (150 mL) and kept i n the dark at room temperature. A f t e r 125 h, the excess periodate was destroyed by the ad d i t i o n of ethylene g l y c o l (5 mL). A f t e r d i a l y s i s (2 days), the l y o p h i l i z e d polyaldehyde i n water (50 mL) was reduced to the p o l y o l with sodium borohydride, neutralized with a c e t i c acid (50%), dialyzed and freeze-dried. A second treatment was conducted, and a f t e r s i m i l a r workup the polyalcohol was recovered ( y i e l d 205 mg). A sample of the polyalcohol (150 mg) was subjected to Smith hydrolysis with 0.5M TFA for 28 h at room temperature. The freeze-dried product (105 g), recovered a f t e r d i a l y s i s , was a polysaccharide. The n.m.r. data indicated three anomeric s i g n a l s ; f o r d e t a i l s , see Table III.2. A sample of the Smith hydrolyzed polysaccharide (30 mg) was methylated by the Hakomori procedure, 51 hydrolyzed with 2M TFA on a steam-bath overnight, reduced and acetylated to i t s p a r t i a l l y methylated a l d i t o l acetates. The r e s u l t s obtained by g . l . c . analysis i n column D showed the presence of 2,4,6-tri-O-methyl-galactose and 2,4,6-tri-0-methylglucose i n the r a t i o s 2 : 1 (See Table III.3, column VI). P a r t i a l hydrolysis The K82 polysaccharide (530 mg) was hydrolyzed using IM TFA on a steam-bath f o r 1% h. A f t e r removal of the TFA by successive evaporations with water, the mixture was separated on a column of Bio-Rad AG1-X2 (formate) ion-exchange r e s i n to give a neutral (-170 mg) and an a c i d i c f r a c t i o n (330 mg). The neutral f r a c t i o n was eluted with d i s t i l l e d water and the a c i d i c f r a c t i o n with formic acid (10%). The a c i d i c f r a c t i o n was applied onto a column of Bio-Gel P-2 and the e f f l u e n t c o l l e c t e d i n f r a c t i o n s (2.5 mL). These were freeze-dried, weighed and the e l u t i o n p r o f i l e obtained (see Figure III.3) showed two main f r a c t i o n s , 1 and 2. F r a c t i o n 1, which was eluted immediately a f t e r the void volume (320 mL, blue dextran) was shown to be polymeric by paper chromatography. Further separation of f r a c t i o n 2 by paper chromatography (solvent 1), gave an aldobiouronic acid A2 (15 mg, R - 0.44), a pure a l d o t r i o u r o n i c acid A3 (32 mg, R n 0.33) G l c C j l C and an aldotetraouronic acid A4 (24 mg, RQ^ c 0.2). Paper chromatography of the neutral f r a c t i o n showed predominantly galactose, glucose and an i n s i g n i f i c a n t amount of a neutral disaccharide (R n 0.43). G l C The analyses performed on each oligosaccharide were as follows, (a) Sugar an a l y s i s . A c i d i c oligosaccharides were treated with 3% methanolic hydrogen chloride for 8 h on a steam bath. The methyl ester obtained was reduced with sodium borohydride i n anhydrous methanol, followed 52 by hydrolysis with 2M TFA, sodium borohydride reduction to the a l d i t o l s , and a c e t y l a t i o n with 1 : 1 a c e t i c anhydride - p y r i d i n e . Data for the g . l . analyses (column A) are given i n Table I I I . l . (b) Methylation analysis 32 A l l methylations were conducted by the method of Hakomori (the a c i d i c oligosaccharides being reduced with l i t h i u m aluminum hydride i n anhydrous oxolane a f t e r methylation), hydrolysis with 2M TFA, sodium borohydride reduction and a c e t y l a t i o n to t h e i r p a r t i a l l y methylated a l d i t o l acetates, which were analyzed by g . l . c . and g.l.c.-m.s. (See Table I I I . 3 ) . The n.m data for each oligosaccharide are given i n Table III.2. Enzymic hydrolysis (a) g-glucosidase. Compound A4 (3 mg) was dissolved i n acetate bu f f e r (2 mL, pH 7.0) and g-glucosidase (SIGMA, 1 mg) was added and the s o l u t i o n incubated at 37° for 2 days. No glucose was detected by paper chromatography (solvent 1); the enzyme was however a c t i v e on c e l l o b i o s e . (b) g-galactosidase. Compound A4 (3 mg) was used and the procedure followed as f o r (a). No galactose was detected by paper chromatography; the enzyme was active on lactose. (c) g-glucuronidase. Compound A2 (2 mg) was dissolved i n acetate b u f f e r (4 mL, pH 7.0) and g-glucuronidase (SIGMA, 1 mg) was added and the so l u t i o n incubated at 37° for 1 day. Glucuronic acid and galactose were detected by paper chromatography (solvent 1); the enzyme was a c t i v e on phenolphthalein mono -g-glucuronic acid. 5 5 IV. BACTERIOPHAGE DEGRADATION OF K l e b s i e l l a K21 CAPSULAR POLYSACCHARIDE 54 IV. BACTERIOPHAGE DEGRADATION OF K l e b s i e l l a K21 CAPSULAR POLYSACCHARIDE IV.1 Introduction Bacteriophages, usually abbreviated to 'phages' (and designated by the Greek l e t t e r <j>), are viruses that i n f e c t b a c t e r i a . They are s t r u c t u r a l l y more complex than other v i r u s types and are grouped according to the morphological c l a s s i f i c a t i o n of Bradley 7*. Phages demonstrate s p e c i f i c i t y regarding the species of b a c t e r i a they w i l l attack and may have ei t h e r a broad or r e s t r i c t e d host range. This s p e c i f i c i t y depends on the presence i n the c e l l w a l l of s p e c i f i c receptor s i t e s which can be: f l a g e l l a , p i l i , capsules, lipopolysaccharides, etc. When a phage p a r t i c l e i n f e c t s and m u l t i p l i e s i t s e l f w ithin a susceptible host, they lyse the c e l l by causing the production of an enzyme, lysozyme, that attacks the murein of the c e l l w a l l , weakening i t so that i t bursts and l i b e r a t e s the phages within. The four phases of a v i r a l i n f e c t i o n are: ( i ) adsorption of the phage p a r t i c l e onto the susceptible host, ( i i ) i n f e c t i o n of the v i r a l DNA (or RNA) into the host, ( i i i ) r e p l i c a t i o n of the phage n u c l e i c acid and phage protein at the expense of the metabolic processes of the host, ( i v ) phage maturation and release which r e s u l t s i n the l y s i s of the host c e l l . Phage p a r t i c l e s carrying host surface carbohydrate degrading enzyme 72 a c t i v i t i e s occur i n great v a r i e t y ; polysaccharide deacetylases , glycanases 74 75 and lyases ' have been described. The enzymes act either upon c e l l w a l l or capsular glycans, and they are generally s p e c i f i c for one or a few of these substrates. Phages which lyse encapsulated b a c t e r i a often form plaques, surrounded by large halos that continue to spread a f t e r growth has ceased. Within the halos, the b a c t e r i a have l o s t t h e i r capsules. The formation of these halos i s due to the production of enzymes during phage 55 i n f e c t i o n , which d i f f u s e from the plaque and catalyse the hydrolysis of the b a c t e r i a l capsules. Plaque size.and morphology are c h a r a c t e r i s t i c of a given phage type and thus are often useful i n d i s t i n g u i s h i n g d i f f e r e n t phages. S t r u c t u r a l studies conducted on the capsular polysaccharides of the genus K l e b s i e l l a have revealed polymers b u i l t up of repeating u n i t s , and ex h i b i t i n g many d i f f e r e n t s t r u c t u r a l patterns. For most of these capsular 7 6 polysaccharides, there e x i s t s a s p e c i f i c phage containing an endoglycanase Each enzyme i s capable of hydrolyzing a p a r t i c u l a r capsular polysaccharide into oligosaccharides corresponding to one (or more) repeating units. Since the pure, capsular polysaccharides are obtainable i n gram q u a n t i t i e s , t h i s technique of using the appropriate phage also makes a v a i l a b l e the corresponding oligosaccharides. Moreover, t h i s i s the only method of obtaining an oligosaccharide repeating unit with i t s a c i d - or b a s e - l a b i l e . non-carbohydrate substituents (such as acetate and k e t a l - l i n k e d pyruvate) i n t a c t 7 7 . This affords a valuable procedure for obtaining oligomers that 7 8 may subsequently be used ( i ) for production of synthetic antigens , ( i i ) as substrates f o r d e t a i l e d n.m.r., mass-spectral, and x-ray d i f f r a c t i o n studies, ( i i i ) i n the study of conformations i n so l u t i o n , and (iv) as a source of complex and novel oligosaccharides, etc. 76 Recently, Stirm and Rieger-Hug 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 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 bacteriophages. The v i r a l depolymerases were found to be very s p e c i f i c (33 cross-reacting with none, 18 with one, 2 with two, and 1 each with three or four heterologous polysaccharides). From t h e i r work, the following general conclusions can be made: ( i ) i n most cases cleavage occurs on either side of the Ca) sugar unit carrying the negative charge(s), but 56 reducing glycuronic acids are not produced; ( i i ) most often, the reducing end sugar formed i s l i n k e d at C-3; ( i i i ) i n most cases, 3 - g l y c o s i d i c linkages are hydrolyzed; (iv) i n most polysaccharides which are acted upon by several phage enzymes, the same g l y c o s i d i c bonds are s p l i t by the d i f f e r e n t agents. 79 The capsular polysaccharide from K l e b s i e l l a K21 has been shown to comprise a . pyruvylated pentasaccharide repeating u n i t : —K3)-ct-D-GlcAp-(l—>3)-a-D-Manp-(l—*-2)-a-D-Manp-(l—*-3)-B-D-Galp-(1-*-4 1 1 a-D-Galp 6\/4 pyr The use of bacteriophage-borne glycanase as a technique i n s t r u c t u r a l studies was demonstrated on t h i s polysaccharide. The method of Dutton 80 and co-workers , which has been shown to give high y i e l d s of depolymerized products, was employed i n the bacteriophage depolymerization of K21 polysaccharide. IV.2 Results and discussion Bacteriophage depolymerization of K21 polysaccharide. <J)21 was o r i g i n a l l y i s o l a t e d from Freiburg sewage and propagated on i t s host s t r a i n , K l e b s i e l l a K21. The r e s u l t s of the phage assays from tube l y s i s , f l a s k l y s i s and wash-bottle l y s i s are as tabulated i n Table IV.1. A f t e r d i a l y s i s for 2 days and concentration, the cj) s o l u t i o n was added to. a s o l u t i o n of p u r i f i e d K21 capsular polysaccharide. The reaction mixture was incubated (37°) f o r a t o t a l of 48.h, a f t e r which i t appeared s i g n i f i c a n t l y less viscous, an i n d i c a t i o n that depolymerization has occurred. The 57 depolymerized products formed were separated from the polysaccharide-phage mixture by d i a l y s i s and p u r i f i e d by passage through a cation-exchange column of IR 120 (H +) r e s i n . Further separation by preparative paper chromatography gave the s i n g l e repeating-unit, PI (191 mg, 38% y i e l d ) . Analysis of PI 20 PI had an [a]n +49° (c 0.36, water; ealc. 52°) and R n 1 0.15 i n u Glc paper chromatography. The constituent sugars analyzed as t h e i r a l d i t o l acetates, gave Man, Gal and Glc i n the r a t i o s 2.0 : 1.92 : 0.91. Methylation analyses of PI and the uronic ester reduced PI, de r i v a t i z e d as t h e i r p a r t i a l l y methylated a l d i t o l acetates, gave the values shown i n Table IV.2. The methylation data i n d i c a t e (a) that the acid i s glucuronic acid linked at C-4; (b) the glucuronic acid i s linked to C-3 of a mannosyl residue, (c) the pyruvic acid k e t a l i s linked to C-4 and C-6 of a galactosyl residue; and (d) PI i s a pentasaccharide with a terminal, reducing galactose (revealed as such by the presence of 2,4,6- and 2,5,6-tri-0-methylgalactose which cumulatively integrate to one hexose u n i t ; for explanation see Section III.3 under p a r t i a l h y d r o l y s i s ) . 1 13 The n.m.r. spectra ( H and C) of PI (see data i n Table IV.3) also substantiate a pyruvylated pentasaccharide corresponding to a single repeating unit. By comparison with the proton spectrum of K21 polysaccharide, the s i g n a l at 6 5.57 (assigned to the branch glucuronic acid) has been 3 s h i f t e d u p f i e l d to 6 5.47; and the s i g n a l at 6 4.90 (assigned to a —^-Galp—) p disappeared to give two corresponding signals at 6 4.63 ((3 anomer) and <5 5.28 (a anomer), thus showing that the cf)21 glycanase has g-galactosidase 13 a c t i v i t y . S i m i l a r l y , i n the C spectrum of PI, the signals corresponding to the anomeric carbons of the reducing end (93.09 and 97.17 p.p.m.) 58 confirmed the galactose as the reducing end. Hence, the structure of P], i s the pyruvylated pentasaccharide: a-D-Galp- (1—*4)-a-D-GlcAp- (1—*-3)-a-D-Manp- (1—>2)-a-D-Manp- (l-*3)-D-Galp~0H 6\/4 pyr This structure i s i n agreement with the preliminary work conducted by A. Savage e t . a l . N.m.r. study of PI The absolute configuration of the pyruvic acid k e t a l i n K21 poly-saccharide i s the R-form as determined, from the chemical s h i f t of i t s methyl group, i n the n.m.r. spectra of PI (see Table IV.3). This confirmed 63 63 the e a r l i e r f i n d i n g by Garegg e t . a l . . I t has been shown that the chemical s h i f t s f o r the methyl groups of the pyruvic acid k e t a l d i f f e r s i g n i f i c a n t l y and depend upon whether these groups are a x i a l or equatorial. 13 The d i f f e r e n c e i s e s p e c i a l l y pronounced i n C spectra, the chemical s h i f t s being -18 p.p.m. for the a x i a l methyl groups and ~26 p.p.m. for the equatorial. The proton spectrum of PI showed a region of three signals 2 3 (a-Galp +pyr, —:.-.Marip — , and — Galp~OH) overlapping at about 6 5.30. The res o l u t i o n of these overlapping resonances was achieved by conducting a sodium borohydride reduction on the reducing galactose of PI (see Table IV.3). The 2-linked mannosyl residue which i s linked to the reducing galactose (in 13 PI), i s now s h i f t e d u p f i e l d to <5 5.24; correspondingly i n the C spectrum, i t i s s h i f t e d downfield to 100.33 p.p.m. (from 95.4 p.p.m.). In contrast, the s i g n a l due to the terminal, non-reducing pyruvylated galactose remained at <5 5.33. This resonance s h i f t e f f e c t , which i s observed when the 59 reducing end i s converted (by reduction) to i t s a l d i t o l , can be a useful 84 aid i n delineating the s t r u c t u r a l sequence of an oligosaccharide Autohydrolysis of PI followed by paper chromatography of the hydrolyzate showed the presence of an oligosaccharide and pyruvic acid. Separation of the oligomeric f r a c t i o n by paper chromatography, followed by ^H-n.m.r. analysis of the pure product showed that i t i s depyruvylated PI (see Appendix I I ) . This a c i d l a b i l i t y of the pyruvic acid k e t a l i s i n 85 d i r e c t contrast to the r e s u l t of Gorin and Ishikawa , who were able to hydrolyze the glycoside- methyl 4,6-0-(l-carboxyethylidene)-a-D-galactoside without cleavage of the k e t a l ; but agrees with the r e s u l t of Choy and Dutton TABLE IV.1 PROPAGATION OF BACTERIOPHAGE $21 II I I I T i t r e (p.f.u./mL)— 9 5 x 10 1.25 x 10 10 Volume ( mL) 30 105 750 To t a l <f> (p.f.u.) 5.25 x 10 11 9.37 x 10 12 I, test-tube l y s i s ; I I , small f l a s k l y s i s ; I I I , wash-bottle l y s i s ; p.f.u. = plaque forming unit 60 TABLE IV.2 METHYLATION ANALYSES OF K21 OLIGOSACCHARIDE (PI) FROM BACTERIOPHAGE DEGRADATION b c P a r t i a l l y methylated R^- Mole %— a l d i t o l acetates— I II 2,4,6-Gal 1.94 0.43 0.45 2,5,6-Gal 1.77 0.57 0.55 2,3-Gal 3.62 1.0 1.0 2,4,6-Man 1.84 0.45 1.0 3,4,6-Man 1.65 1.0 1.0 2,3-Glc 3.40 - 1.0 — 2,4,6-Gal = l , 3 , 5 - t r i - 0 - a c e t y l - 2 , 4 , 6 - t r i - 0 - m e t h y l g a l a c t i t o l , etc. — Relative to 2,3,4,6-Glc on column SP-1000, isothermal at 220°. — Values were corrected by use of e f f e c t i v e carbon-response factors 22 given by Albersheim e t . a l . ; I, methylated PI; I I , methylated and reduced P i . TABLE IV.3 N.M.R. DATA FOR K l e b s i e l l a K21 CAPSULAR POLYSACCHARIDE AND THE DERIVED OLIGOSACCHARIDES Compound— H-n.m.r. data ..C-n.m.r. data Integral Assignment p.p.m.— Assignment— (Hz) (H) 5.47 3 1 a-GlcA 103.03 3-Man — a 5.31 5.28 3 I » a-Gal + pyr 2- Man — •a 3- Gal — OH a 101.3 100.9 97.17 a-GlcA a-Gal + pyr 3-Gal sr OH p 5.03 3 1 3-Man — a 95.4 2-Man — a 4.63 8 0.6 3-Gal sr OH p 95.3 2-Man — a 1.52 s 3 CH 3 of pyr 93.09 61.88 25.81 3-Gal — OH a C-6s of hexoses CH 3 of pyr 5.47 3 1 a-GlcA 102.68 3-Man — a 5.33 3 1 a-Gal+ pyr 101.20 a-GlcA 5.24 s 1 2-Man — a 101.12 a-Gal+ pyr 5.08 s 1 3-Man — a 100.33 2-Man — a 1.58 s 2.6 CH 3 of pyr 61.88 25.78 C-6s of hexoses CH 3 of pyr K21 5.57 b 1 a-GlcA 5.26 b 2 a-Gal+-pyr 2-Man — a 5.05 b 1 3-Man — 4.90 b 1 3-Gal -y 1.46 s 3 CH 3 of pyr For the o r i g i n of compounds PI and PI (reduced), see text. See Appendix II f o r reproductions of the spectra. Chemical 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-silapentane-1-sulfonate (D.S.S.). Key: b = broad, s = s i n g l e t . Key: + pyr = with pyruvate; 3-Gal —— = proton on C-l of 0 3-linked D-Gal residue and Gal-OH = p terminal, reducing galactosyl residue. Chemical s h i f t i n 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. d 13 As for —, but for anomeric C n u c l e i . 63 IV.3 EXPERIMENTAL General methods The b a c t e r i a l lawn required for phage assay was prepared as follows. A nutrient agar p l a t e was dried, upside down and l i d o f f , at 37° i n an incubator f o r 1 h. Actively-growing b a c t e r i a l culture (2 mL) was pipetted on to the agar surface, and a f t e r 15 min at 37°, excess l i q u i d was drained o f f . To e s t a b l i s h the b a c t e r i a l lawn, the plate was then incubated at 37°, f i r s t with the l i d p a r t i a l l y on, then upside down, for 1 h. The determination of phage concentration (phage assay) was performed as follows. Phage suspension (0.3 mL) was d i l u t e d t e n - f o l d by adding to s t e r i l e broth (2.7 mL). An a l i q u o t (0.3 mL) of the resultant s o l u t i o n was further d i l u t e d ten-fold (in a s i m i l a r manner), and the process repeated on subsequent solutions u n t i l a d i l u t i o n range of 10 ^ - 10 ^ was obtained. One small drop was added from each d i l u t e d phage s o l u t i o n , by means of a s t e r i l e p i p e t t e drawn to a f i n e t i p on to the growing b a c t e r i a l lawn at po s i t i o n s near the perimeter of the p l a t e . A f t e r overnight incubation at 37°, plaques (clear spots) were observed. At the highest d i l u t i o n , s i n g l e c l e a r spots marked the p r o l i f e r a t i o n of i n d i v i d u a l phage p a r t i c l e s . A c a l c u l a t i o n based on the volume of the phage s o l u t i o n applied (measured by c a l i b r a t i n g the p i p e t t e f o r drop volume), the number of plaques and the d i l u t i o n gave the count of plaque-forming units (p.f.u.) per mL of undiluted phage suspension. The r e l a t i o n s h i p of c e l l count per mL to o p t i c a l density read at 545 nm i n a densitometer was determined i n the o p t i c a l density range 0.3 0.9 as follows. A f l a s k of broth (100 mL) was inoculated with a culture of K21 b a c t e r i a and vigorously aerated at 37°. A drop of s t e r i l i z e d Dow antifoam FG-10 emulsion was added to prevent excessive foaming. Aliquots 64 1 1 — 0.5 1.0 Absorbance (545 nm) Figure IV.1 C o r r e l a t i o n between b a c t e r i a l count and the o p t i c a l density f o r K l e b s i e l l a K21 b a c t e r i a 65 —5 —'8 (3 mL) were removed at 30 min i n t e r v a l s , appropriately d i l u t e d (10 to 10 ) with broth and a small quantity (0.2 mL) from each of the d i l u t e d solutions was incubated on an agar plate overnight. Individual colonies were then counted and the c o r r e l a t i o n between o p t i c a l density and b a c t e r i a l count pl o t t e d (see Figure IV,1). Three successive propagations using increasing amounts of b a c t e r i a l cultures and bacteriophages were necessary to obtain s u f f i c i e n t v i r u s p a r t i c l e s with which to degrade the K21 capsular polysaccharide. (a) Tube l y s i s . An ac t i v e K21 b a c t e r i a l colony was picked up i n t o s t e r i l e broth (5 mL) and incubated at 37° u n t i l the b a c t e r i a l culture turned-turbid (4 h). S t e r i l e broth ( 5 x 4 mL) i n culture test-tubes was then inoculated with the b a c t e r i a l culture (0.5 mL) and further incubated. At 30 min i n t e r v a l s , consecutive tubes were inoculated with phage-suspended broth s o l u t i o n (0.5 mL). Continued incubation resulted i n the f i r s t few tubes changing from the turbid s o l u t i o n , to a clear s o l u t i o n ( b a c t e r i a l l y s i s ) . A f t e r the l a s t tube has cleared (about 5 h a f t e r the f i r s t phage i n o c u l a t i o n ) , chloroform (1 mL) was added to a l l the tubes and the mixtures shaken. Combinations of tubes that cleared, and those that did not, followed by c e n t r i f u g a t i o n yielded c l e a r phage solutions which were assayed to determine the phage concentrations, (b) Small f l a s k l y s i s . This technique i s s i m i l a r to the tube l y s i s except that larger volumes are used. S t e r i l e broth (5 x 48 mL) i n Erlenmeyer fl a s k s (125 mL) was inoculated with K21 b a c t e r i a l culture (1 mL) and incubated at 37°. At 30 min i n t e r v a l s , <f>21 so l u t i o n (1 mL, from tube l y s i s or otherwise) was consecutively added to the f l a s k s . Continued incubation and workup were performed as i n (a). (c) Wash-bottle l y s i s . Three wash-b o t t l e s were set up, each containing s t e r i l e broth (200 mL), and inoculated with actively-growing K21 b a c t e r i a l culture (10 mL). Growth was maintained 66 ait 37° with vigorous aeration, e f f l u e n t a i r being passed through aqueous phenol to decontaminate i t . A few drops of Dow antifoam FG—10 emulsion was added to each wash-bottle. O p t i c a l density was monitored, and when a value was reached that corresponded to the appropriate number of c e l l s i n r e l a t i o n to the quantity of phage to be expended (see Figure IV.1), the $21 suspension from (b) was added to each of the three wash-bottles. For optimum phage propagation, experience indicated that the phage to b a c t e r i a l c e l l r a t i o should be approximately 3:1. When the o p t i c a l density had f a l l e n to near i t s blank value (about 3 h), i n d i c a t i n g complete b a c t e r i a l l y s i s , work up and phage assay followed the usual pattern. Bacteriophage depolymerization of K21 capsular polysaccharide $21 was o r i g i n a l l y i s o l a t e d from Freiburg sewage, and propagated on 81 K l e b s i e l l a K21 b a c t e r i a according to the standard procedures of Adams Successive $21 propagations using tube l y s i s , small f l a s k l y s i s and wash-82 13 b o t t l e l y s i s were performed (see Table IV.1). Stirm had shown that 10 bacteriophages are needed to degrade 1 g of the corresponding b a c t e r i a l 12 capsular polysaccharide. Bacteriophage $21 (5.6 x 10 p.f.u.) was used to 80 depolymerize K21 polysaccharide (500 mg), by a technique that was recently developed i n t h i s laboratory. $21 s o l u t i o n (450 mL, 1.25 x 1 0 ^ p.f.u. per mL) was concentrated by evaporation i n vacuo and dialyzed (cut o f f 3500) against tap water (2 days). A f t e r concentration, the phage s o l u t i o n was added to K21 polysaccharide (500 mg; prepared by Dr. A. Savage according to the procedure described i n III.4) s o l u t i o n . The depolymerization was conducted i n a water-bath at 37° f o r 24 h, a f t e r which another portion of dialyzed phage s o l u t i o n (6.25 x 1 0 ^ p.f.u.) was added. Further incubation for another 24 h resulted i n a less viscous reaction mixture, i n d i c a t i n g 67 that depolymerization has occurred. Paper chromatography (solvent 2) of the crude r e a c t i o n mixture indicated the presence of an oligosaccharide. The reaction mixture was concentrated, and dialyzed (cut o f f 3500) against d i s t i l l e d water (3X). The d i a l y z a t e c o l l e c t e d each time was concentrated, subjected to IR 120 (H +) cation-exchange treatment and freeze-dried. Paper chromatography (solvent 2) of the product indicated an oligosaccharide and some higher molecular weight polymeric material at the o r i g i n . Separation by preparative paper chromatography i n solvent 2 gave a pure f r a c t i o n of the s i n g l e repeating unit, PI (191 mg, y i e l d 38%). The double repeating-unit (P2) which was at the o r i g i n , was a c c i d e n t a l l y discarded. T o t a l sugar r a t i o (PI) Dried PI (15 mg) was methanolyzed with 3% methanolic hydrogen chloride on a steam-bath overnight. The methyl ester obtained was reduced with sodium borohydride i n anhydrous methanol, followed by hydrolysis with 2M TFA, sodium borohydride reduction to the a l d i t o l s , and a c e t y l a t i o n with 1 : 1 a c e t i c a n h y d r i d e - pyridine mixture. G.l.c. analysis (column A) of the a l d i t o l acetates gave Man, Gal and Glc i n the r a t i o s 2.0 : 1.92 : 0.91. Methylation analysis 31 32 PI (17 mg) was methylated by the Hakomori procedure ' . A portion of the methylated product (8 mg) was hydrolyzed with 2M TFA on a steam-bath overnight, reduced with sodium borohydride (45 min), and acetylated to give a mixture of p a r t i a l l y methylated a l d i t o l acetates (see Table IV.2, column I ) . The remaining methylated product (8 mg) was reduced with l i t h i u m aluminum hydride i n anhydrous oxolane followed by hydrolysis with 2M TFA, and d e r i v a t i z a t i o n to t h e i r p a r t i a l l y methylated a l d i t o l acetates. The r e s u l t 68 of the g.1.c.-m.s. analysis are as shown i n Table IV.2, column I I . N.m.r. study of PI 1 13 The p r i n c i p a l n.m.r. data ( H and C) for K21 polysaccharide and derived oligosaccharides are as shown i n Table IV.3. A solu t i o n of PI (23 mg i n 2.5 mL H^O) was reduced with sodium borohydride (45 min), and subjected to cation-exchange with IR 120 (H +) r e s i n u n t i l the pH was a c i d i c . The eluant obtained was concentrated to dryness, and the borate formed removed by c o - d i s t i l l a t i o n with methanol (4X). A f t e r several exchanges with D^O, the reduced PI was submitted for n.m.r. analysis. A s o l u t i o n of PI (50 mg i n 3 mL H^O, pH 3.7) was heated on a steam-bath f o r 18 h. A f t e r treatment with IR 120 (H +) r e s i n , the eluant was l y o p h i l i z e d . Paper chromatography (in solvent 1) of the product showed, on development with p-anisidine hydrochloride, a depyruvylated PI and the loss of some terminal galactose. When viewed under a u.v. lamp, the paper showed a blue fluorescent streak which i s associated with the free pyruvic acid. Separation by paper chromatography (solvent 2) gave a pure depyruvylated PI. ''H-n.m.r. analysis of the product gave a spectrum which was i d e n t i c a l to that of PI, except that the h i g h - f i e l d s i g n a l at <5 1.52 (attributed to the methyl of the pyruvic acid ketal) has disappeared. See Appendix II for the reproduction of a l l n.m.r. spectra. V. BIBLIOGRAPHY 70 V. BIBLIOGRAPHY I. 0rskov, Bergey's Manual of Determinative Bacteriology, 8th Ed., (1974) 321-324. 2. P.R. Edwards and W.H. Ewing, " I d e n t i f i c a t i o n of Enterobacteriaceae", Burgess Publishing Company, Minneapolis, 1972. 3. I. 0rskov and M.A. Fife-Asbury, Int. J . Syst. B a c t e r i o l . , 27, (1977) 386-387. 4. R.C'.:W. Berkeley, G.W. Gooday and D.C. Ellwood, M i c r o b i a l poly-saccharides and polysaccharases, Academic Press, London (1979). a) A. 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Chem., ;45, (1967) 521-530. 15 APPENDICES 76 GlcA Gal Glc GlcA Gal Man GlcA Gal Rha GlcA Glc Man GlcA Glc Rha GlcA Glc Fuc GlcA Gal Glc Man GlcA Gal Glc Fuc GlcA Gal Glc Rha GlcA Gal Man Rha GlcA Glc Man Fuc GlcA Glc Man Rha GlcA Gal Glc Man Fuc GlcA Gal Glc Man Rha GalA Gal Man GalA Glc Rha GalA Gal Fuc PyrA Glc Rha PyrA Gal Rha PyrA Gal Glc Rha KetoA Gal Glc 8 p f l l p , l 5 . 2 5 , 2 7 P , 5 1 , , 82 2 0 , 2 1 P , 2 9 P , ^ 2 P , 4 3 , 6 6 , 7 ^ P 9 . 9 * ^ 7 . 5 2 , 8 1 , 8 3 2 , 4 , 5 P , 2 4 17 .23,^.^5 P .71 1.54 7 P , 1 0 , 1 3 P . 2 6 P , 2 8 , 3 0 P . 3 1 P , 3 3 P . 3 5 P . 3 9 . ^ 6 P , 5 0 , 5 9,60, 6 l , 6 2 , 6 9 P 16 .58 P 1 2 P,18 , 1 9 , 3 6 P.41 , 5 5 P , 7 0 P , 79 40,53,80 p . 6 P 6 4 P , 6 5 P 6 8 P 1^P,67 3 P ,^9,57 3^,48 63 72 32 56 22,37,38 GlcA glucuronic acid Gal galactose GalA galacturonic acid Man mannose PyrA pyruvic acid Rha rhamnose KetoA rare uronic acid Fuc fucose Glc glucose P pyruvic acid sent i n addition /Appendix I ~ : K l e b s i e l l a capsular polysaccharides (K1-K83) Qualitative analysis and chemotype grouping. 1 1 APPENDIX II N.m.r. Spectra 5 r 4 r K82, Compound A3 Spectrum No. 3 Spectrum No. 4 acetone 2.23 K82, Compound A4 (reduced) 1 3 1 3 Glc — — Gal G a l a c t i t o l 1' GlcA HOD H n.m.r. 400 MHz, 95° Spectrum No. 5 •'' laclatone'•" 2.23 K82, Compound SD 3 1 3 1 3 1 — Glc — ~ Gal Gal ~ Spectrum No. 6 acetone 2.23 H n.m. r. 400 MHz, 95° K82, Compound SD _3 G l c I_3 1_ C n.m.r. 20 MHz, amb. temp Spectrum No. 7 acetone 31.07 oo 4> K82 polysaccharide (mildly hydrolyzed) H n.m.r. 400 MHz, 95° 4.96 -4.70 Spectrum No. 8 acetone '2.23 ZJJ K82 polysaccharide (mildly hydrolyzei L n.m.r. 20 MHz, amb. temp. ' i ' i ' i • i ' i I I I I r Spectrum No. 9 K8 polysaccharide 3 3 , 1 3 — Glc — — Gal 4 a 1 GlcA p a *H n.m.r. 400 MHz, 95° Spectrum No. 10 HOD K21 $21 Compound PI K H nmr 400 MHz, water n u l l Spectrum No. 11 K21 <j>21 Compound PI (depyruvylated) H n.m.r. 400 MHz, water n u l l Spectrum No. 12 oo .-'•i  -i'l K21 $21 compound PI (reduced) H n.m.r. 400 MHz, 95° Spectrum No. 13 K21 polysaccharide H n.m. r. 400 MHz, 95° Spectrum No. 14 K21 q>21 compound P I ( r e d u c e d ) C n . m . r . 20 MHz, amb. temp. 101.12 101.20: 100.33 S p e c t r u m No. 15 K21 $21 Compound PI C n.m.r. 20 MHz, amb. temp. 101.3 103.03 100.9 95.4 97.17 95.3 .93 Spectrum No. 16 

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