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Structural studies on Klebsiella capsular polysaccharides Mackie, Keith L. 1977

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STRUCTURAL STUDIES ON KLEBSIELLA CAPSULAR POLYSACCHARIDES by KEITH L. MACKIE B.Sc. (Hons.), Massey University, N.Z., 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES The Department of CHEMISTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY, 1977 EXTERNAL EXAMINER: W.F. DUDMAN C.S.I.R.O., Canberra, A u s t r a l i a © Keith L. Mackie, 1977 In presenting th i s thes i s in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I further agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of this thesis for f inanc ia l gain sha l l not be allowed without my written permission. Department of Chemistry  The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 30th August. 1977 - i -ABSTRACT Eighty-one s e r o l o g i c a l l y d i s t i n c t s t r a i n s of K l e b s i e l l a b a c t e r i a are known. The c a p s u l a r p o l y s a c c h a r i d e s from these b a c t e r i a are t h e i r a n t i g e n i c determinants and i n order to help understand the chemical b a s i s 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 d e t a i l e d chemical s t r u c t u r e s o f these p o l y s a c c h a r i d e s are being determined. The c a p s u l a r p o l y s a c c h a r i d e s i s o l a t e d from K l e b s i e l l a serotypes K32, K36 and K70 are presented here and have been e s t a b l i s h e d u s i n g many d i f f e r e n t chemical t e c h n i q u e s . Methyla-t i o n , p a r t i a l h y d r o l y s i s , p e r i o d a t e o x i d a t i o n and 3 - e l i m i n a t i o n procedures have y i e l d e d a n a l y s a b l e subunits of the p o l y s a c c h a r i d e s E x t e n s i v e use has been made of n.m.r. spectroscopy ("*"H and 13 C), mass spectrometry, g a s - l i q u i d chromatography and g e l f i l t r a t i o n i n the i s o l a t i o n and i d e n t i f i c a t i o n o f the products o b t a i n e d from the v a r i o u s d e g r a d a t i v e techniques. The r e p e a t i n g u n i t s t r u c t u r e s of K32, K36 and K70 are shown to be as f o l l o w s : K32 — D - G a l p ^ L - R h a p ^ L - R h a p ^ i L - R h a p i -" 4\/3" a ~ - 3 = - a A CH^ vCOOH - i i -K36 - D - G a l p ^ L - R h a p ^ L - R h a p ^ L - R h a p ^ 6 D-GlcAp 4 6 1 D-Glcp A CH 3 COOH K70 —g-G1 c A p ^ - L - Rhap^p-L- Rhap^-D-G l c P^pg-G a l p ^ L - R h a p ^ 4\ / 3 CH 3A OOH 50% Some f e a t u r e s of s p e c i a l i n t e r e s t i n these s t r u c t u r e s i n c l u d e : the extreme a c i d l a b i l i t y o f the pyruvate a c e t a l when l i n k i n g h y d r o x y l s on C^ and of a 2 - l i n k e d L-rharanose r e s i d u e (K32, K70); the e x i s t e n c e o f a 3-Lj-rhamnose u n i t i n the s t r u c t u r e of K32; and the presence of the py r u v a t e a c e t a l on o n l y 50% of the l i n e a r , s i x sugar, r e p e a t i n g u n i t s of K70. I t i s a l s o i n t e r e s t i n g t o note t h a t w h i l e K70 and K36 have almost the same q u a n t i t a t i v e composition the chemi c a l s t r u c t u r e s are markedly d i f f e r e n t . An e f f i c i e n t means of i s o l a t i n g l a r g e q u a n t i t i e s o f s i n g l e r e p e a t i n g u n i t s of the K l e b s i e l l a p o l y s a c c h a r i d e s u s i n g glycanase enzymes borne and u t i l i s e d by s p e c i f i c bacteriophage, i s demonstrated. A bacteriophage s p e c i f i c f o r K l e b s i e l l a K32 has been propagated, p u r i f i e d and used to depolymerise K32 p o l y s a c c h a r i d e . A n a l y s i s of the r e s u l t -i n g o l i g o s a c c h a r i d e s has shown the glycanase enzyme to be a a-rhamnosidase which c l e a v e s K32 as shown below. phage CH C COOH The degradation v i a bacteriophage i s a new area of research and the work described here i s only preliminary and as such i s presented as an appendix. - i v -ACKNOWLEDGEMENTS I t has been a rewarding experience to work under P r o f e s s o r G.G.S. Dutton d u r i n g the course of t h i s work. His guidance and encouragement both i n and out of the l a b o r a t o r y have provided i n v a l u a b l e support, and f o r t h i s I g i v e him my s i n c e r e thanks. While i t i s im p o s s i b l e t o thank a l l those other people whose h e l p f u l d i s c u s s i o n s and s e r v i c e s have aided i n the completion of t h i s t h e s i s , I wish to thank i n p a r t i c u l a r , Dr. G.M. Bebault f o r her p a t i e n c e and a d v i c e , and a l s o the other members of the l a b o r a t o r y , v i z . Angela Savage, Dr. A l b e r t o Zanlungo, and Dr. J o f f r e Berry. I g r a t e f u l l y acknowledge the award of a Canadian Commonwealth S c h o l a r s h i p by the Canadian government f o r the d u r a t i o n of t h i s work. My thanks a l s o go to P h y l l i s Moore f o r t y p i n g t h i s t h e s i s . - V -TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS i v TABLE OF CONTENTS v LIST OF FIGURES i x LIST OF TABLES x i i I INTRODUCTION 2 II TECHNIQUES AND METHODS USED IN THE STRUCTURAL ANALYSES OF POLYSACCHARIDES 7 II. 1 Isolation and p u r i f i c a t i o n 8 II. 2 Use of 1H and 1 3 C n.m.r ". 10 II. 2.1 1H n.m.r 10 II.2.2 1 3 C n.m.r 14 11.3 Total hydrolysis and methanolysis 21 11.4 Methylation and methylation analysis 24 11.5 Carboxyl reduction of a c i d i c poly-saccharides 27 II. 6 Periodate oxidation 30 II. 7 P a r t i a l hydrolysis 36 II. 8 B-Elimination 39 II.9 Separation of oligomers obtained from •degradations 45 - v i -Page 11.9.1 Gel chromatography 4 6 11.9.2 S i l i c a gel chromatography 47 11.9.3 Liquid chromatography 4 8 11.9.4 Gas-liquid chromatography 48 11.9.5 Paper chromatography and paper electrophoresis 50 11.10 Structure analysis of p u r i f i e d o l i g o -saccharides 51 11.10.1 Gas-liquid chromatography 53 11.10.2 Mass spectroscopy 58 11.10.2.1 Mass spectroscopy of p a r t i a l l y methylated a l d i t o l acetates .. 62 11.10.2.2 Mass spectroscopy of o l i g o -saccharides . 63 11.10.3 Nuclear magnetic resonance 72 II. 10. 4 Determination of 5- and configuration of a sugar residue . 76 11.11 Immunochemical methods 77 11.12 Bibliography for Sections I and II 80 III THE STRUCTURE OF KLEBSIELLA SEROTYPE K36 POLYSACCHARIDE 8 8 I I I . l Abstract 89 III. 2 Introduction 89 III. 3 Results and Discussion 90 - v i i -Page II I . 4 Experimental 105 III. 5 Bibliography for Section III 118 IV THE STRUCTURE OF KLEBSIELLA SEROTYPE K7 0 CAPSULAR POLYSACCHARIDE .121 IV. 1 Abstract 122 IV. 2 Introduction 122 IV. 3 Results and Discussion 123 IV. 4 Experimental 138 IV. 5 Bibliography for Section IV 151 V THE STRUCTURE OF KLEBSIELLA SEROTYPE K3 2 CAPSULAR POLYSACCHARIDE 153 V. l Abstract 154 V.2 Introduction 154 V.3 Results and Discussion 155 V.4 Experimental 168 V.5 Bibliography for Section V 176 APPENDIX I: BACTERIOPHAGE DEPOLYMERISATION OF KLEBSIELLA K32 CAPSULAR POLYSACCHARIDE 178 Introduction 179 Experimental 181 Discussion 196 References 199 - v i i i -Page APPENDIX I I : THE KLEBSIELLA POLYSACCHARIDES OF KNOWN STRUCTURE 200 APPENDIX I I I : N.M.R. SPECTRA 211 - i x -LIST OF FIGURES  F i g u r e Page I. 1 Diagrammatic r e p r e s e n t a t i o n o f a b a c t e r i a l c e l l w ith l i p o p o l y s a c c h a r i d e , c a p s u l e and slime 3 I I . 1 n.m.r. spectrum of K l e b s i e l l a K36 c a p s u l a r p o l y s a c c h a r i d e 12 13 11.2 C n.m.r. spectrum o f K l e b s i e l l a K36 c a p s u l a r p o l y s a c c h a r i d e 19 11.3 G . l . c . t r a c e o b t a i n e d v i a m e t h y l a t i o n a n a l y s i s o f K l e b s i e l l a K36 26 11.4 Reduction of c a r b o x y l i c a c i d s i n aqueous s o l u t i o n u s i n g c a r b o d i i m i d e reagents 29 11.5 P e r i o d a t e d e g r a d a t i o n o f K l e b s i e l l a K36 .. 32 11.6 Uronic a c i d d e g r a d a t i o n of K l e b s i e l l a K70. 42 11.7 3 - E l i m i n a t i o n d e g r a d a t i o n v i a o x i d a t i o n of a secondary hydroxy1 4 4 11.8 G . l . c . s e p a r a t i o n of mixture of p a r t i a l l y methylated a l d i t o l a c e t a t e s o b t a i n e d from K l e b s i e l l a K32 (pyruvate removed) 55 I I . 9 Degradation pathways f o r permethylated g-glucose d u r i n g e l e c t r o n impact ( e . i . ) mass spectroscopy 66 - x -F i g u r e Page 11.10 Fragmentations d u r i n g e.i.m.s. of per-methylated d i s a c c h a r i d e s v i a the B pathway showing the nature o f the l i n k a g e between two sugar r e s i d u e s 68 11.11 C h a r a c t e r i s t i c fragmentations of per-•methylated o l i g o s a c c h a r i d e a l d i t o l s i n e.i.m.s 69 11.12 The e l e c t r o n impact mass spectrum of a permethylated t r i s a c c h a r i d e a l d i t o l 70 11.13 Mass s p e c t r a of a d i s a c c h a r i d e a l d i t o l o b t a i n e d u s i n g e l e c t r o n impact, chemical i o n i s a t i o n and f i e l d d e s o r p t i o n modes .... 71 11.14 1 3 C n.m.r. spectrum of 6-D-Galp-(1+2)-a-J-Rhap- ( 1 + 2 ) - D - e r y t h r i t o l '. 74 11.15 n.m.r. spectrum of 8-Q-Galp- (1+2)-a-L-Rhap-(1+2)-D-erythritol 75 I I I . l G . l . c . s e p a r a t i o n of p a r t i a l l y methylated a l d i t o l a c e t a t e s o b t a i n e d from: A, n a t i v e K36 p o l y s a c c h a r i d e ; B, degraded K36 p o l y -s a c c h a r i d e 95 V.l "^H n.m.r. spectrum of a-D-Galp- (1+2)-a-Lj-Rhap-(1+3)-B-L-Rhap-(1+3)-1-deoxy-p-e r y t h r i t o l (1) 162 ' - x i -Figure V.2 Scheme for periodate oxidation of K32 capsular polysaccharide AI.1 Correlation of o p t i c a l density and colony concentration for K l e b s i e l l a K32 183 AI.2 .Results of a t y p i c a l bottle l y s i s of K l e b s i e l l a K32 with bacteriophage ^32 186 AI.3 Bacteriophage depolymerisation of K l e b s i e l l a K32 capsular polysaccharide 189 AI.4 Ion exchange chromatography of K l e b s i e l l a K32 oligosaccharides obtained by bacterio-phage depolymerisation. D.E.A.E. Sephadex A25 equilibrated with 0.025 M Tris/HCl buffer was used and eluted with a l i n e a r 0 to 0.3 M NaCl gradient in the same buffer. Fractions were analysed with phenol-sulphuric acid ... 191 AI. 5 """H n.m.r. of neutral repeating unit from phage degraded K32 192 13 AI.6 C n.m.r. of neutral repeating unit from phage degraded K32 193 AI. 7 "^H n.m.r. of a c i d i c repeating unit from phage degraded K32 194 13 AI.8 C n.m.r. of a c i d i c repeating unit from phage degraded K32 195 Page 164 - x i i -LIST OF TABLES Table Page II. 1 • Primary fragments i n the mass spectra of p a r t i a l l y methylated sugars in the form of t h e i r a l d i t o l acetates 64 III. l "*"H n.m.r. data on K l e b s i e l l a K36 capsular polysaccharide and derived oligosaccharides 92 III. 2 Methylation analyses of o r i g i n a l and degraded K36 capsular polysaccharide 96 IV. 1 N.m.r. data for K l e b s i e l l a K70 poly-saccharide and iso l a t e d oligosaccharides . 124 IV. 2 Methylation analyses of native and depyruvalated K l e b s i e l l a K70 capsular polysaccharide 128 V. l Methylation analyses of native and de-pyruvalated K l e b s i e l l a K32 capsular polysaccharide 157 V.2 N.m.r-. data for K l e b s i e l l a K32 capsular polysaccharide and is o l a t e d o l i g o -saccharides 158 INTRODUCTION TECHNIQUES AND METHODS USED IN THE STRUCTURAL ANALYSES OF POLYSACCHARIDES - 2 -I. I n t r o d u c t i o n : B a c t e r i a l E x o p o l y s a c c h a r i d e s . The c e l l s of b a c t e r i a b e l o n g i n g to the f a m i l y E n t e r o -b a c t e r i a c e a e , which i n c l u d e s the genus K l e b s i e l l a , have l i p o p o l y s a c c h a r i d e (L..P.S.) bound and e x t e r n a l t o the c e l l w a l l . The f a c t t h a t reagents such as phenol or e t h y l e n e -d i a m i n e t e t r a a c e t i c a c i d (E.D.T.A.) can e x t r a c t L.P.S. suggests the l i n k a g e between L.P.S. and the c e l l w a l l may be i o n i c r a t h e r than c o v a l e n t . The l i p o p o l y s a c c h a r i d e l a y e r may be enveloped by a f u r t h e r p o l y s a c c h a r i d e l a y e r c a l l e d the e x t r a c e l l u l a r c a p s u l e . Most o f the K l e b s i e l l a s t r a i n s possess heavy c a p s u l e s (K +) but some non-encapsulated v a r i a n t s (K ) can be found. In c u l t u r e s of c a p s u l a t e c e l l s 'slime' i s o f t e n o b t a i n e d due to the gradual r e l e a s e of p o l y s a c c h a r i d e from the capsule, and s i n c e Dudman and Wilkinson"'" have shown t h a t c a p s u l a r and s l i m e p o l y s a c c h a r i d e s are i d e n t i c a l i n chemical . composition, i t may be assumed t h a t these polymers are p h y s i c a l -l y i d e n t i c a l . The name ex o p o l y s a c c h a r i d e p r o v i d e s a g e n e r a l term f o r a l l these forms of b a c t e r i a l p o l y s a c c h a r i d e s found o u t s i d e the c e l l w a l l . F i g u r e 1.1 shows a diagrammatic r e p r e s e n t a t i o n of a b a c t e r i a l c e l l with capsule and s l i m e . V a r i o u s h y p o t h e t i c a l f u n c t i o n s have been suggested f o r b a c t e r i a l e x o p o l y s a c c h a r i d e s . Most of these have i m p l i e d a p r o t e c t i v e f u n c t i o n , such as a g a i n s t d e s i c c a t i o n , a g a i n s t phagocytosis or a g a i n s t bacteriophage ( v i r u s organisms which a t t a c k b a c t e r i a ) . While there may c e r t a i n l y be some p o s s i b i l i t y of the f i r s t two r o l e s being c o r r e c t , the occur-- 3 -.CELL WALL MEMBRANE CYTOPLASM LIPOPOLYSACCHARIDE" CAPSULE SLIME F i g u r e 1.1 Diagrammatic r e p r e s e n t a t i o n of a b a c t e r i a l c e l l with l i p o p o l y s a c c h a r i d e , c a p s u l e and s l i m e . rence of phages capable of i n d u c i n g capsule d e s t r o y i n g enzymes (see Appendix I) suggests t h a t c a p s u l e s f r e q u e n t l y p r e s e n t no r e a l b a r r i e r to phage i n f e c t i o n . E x o p o l y s a c c h a r i d e s may a c t as determinants of s p e c i f i c i t y . T h i s i s c o n s i s t e n t w i t h o t h e r evidence which shows t h a t the exposed components of a s t r u c t u r e a c t as the a n t i g e n i c determinants of the e n t i r e u n i t . Good examples of t h i s are: 2 the blood group antigens , whose end group monosaccharides are the a n t i g e n i c determinants; and the a l l e r g e n i c c o n s t i t -uents of p o l l e n , which are l o c a t e d i n the outer c e l l w a l l of 3 the p o l l e n g r a i n . On the b a s i s of immunochemical t e s t s the genus K l e b s i e l l a has been d i v i d e d i n t o approximately 80 - 4 -4 5 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 ' . These s t r a i n s have been designated K-types as t h e i r s e r o l o g i c a l s p e c i f i c i t y i s derived from the capsular (German; Kapsel) polysaccharide surrounding the b a c t e r i a l c e l l . Polysaccharides are generally considered to be weakly or non-antigenic, i . e . they induce only a weak response by a host's immune system—the system that produces antibodies to combat the invading antigen. This i s probably because polysaccharides, even though they have high molecular weights s i m i l a r to those of strongly antigenic proteins, do not have a d e f i n i t e three dimensional ( t e r t i a r y ) structure. Neverthe-le s s , the repeating nature of the capsular polysaccharide i s somehow "impressive" and does impart some a n t i g e n i c i t y . With an i s o l a t e d repeating unit from a K l e b s i e l l a polysaccharide attached to a peptide " c a r r i e r " , i t has been possible to i s o l a t e antibodies produced by rabbits against t h i s carbo-hydrate unit . When these antibodies were then tested against the i n t a c t native polysaccharide, although the antibodies did show some in t e r a c t i o n , not a l l a c t i v i t y associated with the native, polysaccharide-native antibodies were present. It might therefore be assumed that there i s some contribution from the siz e of the polysaccharide and perhaps from the three dimensional character of the polymer. Antigenic polysaccharides contain s p e c i f i c oligosaccharide units which combine with the antibody i n the immunological reaction and are thus c a l l e d immunodominant s i t e s , antigenic - 5 -determinants, determinants of immunological s p e c i f i c i t y , or haptens. The s t r u c t u r a l composition of the determinant of immunological s p e c i f i c i t y can be determined by hapten 7 8 9 i n h i b i t i o n ' or complement f i x a t i o n i n h i b i t i o n studies using oligosaccharides of known structure. For the l a t t e r method oligosaccharides of known structure are very important and the need for the further developement of synthetic techniques to produce these oligosaccharides remains today. Knowledge of the molecular basis of antigen-antibody in t e r a c t i o n and of the frequent cross-reactions which occur between microorganisms belonging to widely disparate families i s not only of p r a c t i c a l i n t e r e s t , but also has a broader th e o r e t i c a l significance; i t i s capable of revealing s t r i c t r e l a t i o n s between the chemical constitution of certain micro-b i a l antigens and t h e i r immunological s p e c i f i c i t i e s . With this objective i n mind the determination of the detailed structures of 80 K l e b s i e l l a K-types i s presently being undertaken. The determination of the structures of K l e b s i e l l a K-types 32, 36 and 70 i s the p r i n c i p l e contribution of t h i s thesis. 4 5 Nimmich ' has reported the q u a l i t a t i v e compositions of the K-types 1 to 80, and his work has shown that most of the polysaccharides contain D-glucuronic acid or D-galacturonic acid i n combination with g-mannose, g-glucose, g-galactose, and to a lesser extent, with L-rhamnose and L-fucose. Pyruvic 4 5 acid, present as 1-carboxyethylidene acetals, i s known ' to - 6 -be present i n approximately h a l f of the p o l y s a c c h a r i d e s . A l l K l e b s i e l l a p o l y s a c c h a r i d e s c o n t a i n some a c i d i c groups, whether they be u r o n i c a c i d s , pyruvate a c e t a l s or keto a c i d s , and i t i s the o v e r a l l n e g a t i v e charge of these groups which i s thought to impart v i r u l e n c e to the encapsulated b a c t e r i a . The s t r u c t u r e s of approximately 35 K l e b s i e l l a K-types have been determined to date (see Appendix II) and a wide v a r i e t y of s t r u c t u r a l f e a t u r e s are now known to e x i s t i n these p o l y s a c c h a r i d e s . The number of sugars i n the r e p e a t i n g u n i t s ranges between th r e e (K5) and s i x (K18, K28, K36, K52, K70, K81). Uronic a c i d r e s i d u e s , when present, may e x i s t i n the main c h a i n i t s e l f (K5, K70, e t c . ) , as branch p o i n t s ( K l l , K21, e t c . ) , as t e r m i n a l u n i t s i n a s i d e c h a i n (K2, K9, etc.) or as non-terminal u n i t s i n a s i d e c h a i n . In one case (K38) two s i n g l e u n i t s i d e chains are l i n k e d to the same 'main c h a i n ' r e s i d u e , while i n o t h e r s two and three u n i t s i d e chains e x i s t , and i n s t i l l o t h e r s , the s t r u c t u r e s are l i n e a r . To date no o v e r a l l p a t t e r n i s emerging from the p o l y s a c c h a r i d e s t r u c t u r e s . The d i v e r s i t y of s t r u c t u r e s presented by K-types which have the same q u a l i t a t i v e compo-s i t i o n j u s t i f i e s 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 , e.g. compare K-types 18, 36, 55, and 70 which each c o n t a i n g - g l u c u r o n i c a c i d , Dj-galactose, g- glucose and Jj-rhamnose. Furthermore, K l e b s i e l l a K36 and K70 p o l y s a c c h a r i d e s are shown i n t h i s t h e s i s to have completely d i f f e r e n t r e p e a t i n g u n i t s t r u c t u r e s even though they have almost the i d e n t i c a l q u a n t i -t a t i v e composition. - 7 -The existence of a defined s t r u c t u r a l unit that repeats throughout the polysaccharide from any one K l e b s i e l l a sero-type makes these polymers p a r t i c u l a r l y suitable as models for investigation by many d i f f e r e n t techniques. At present studies are being ca r r i e d out to examine the c r y s t a l l i n e conformations' 1 0 and solution conformations"1"'' of these polymers, 12 the behaviour of the polysaccharides as polyelectrolytes , and to examine t h e i r nuclear magnetic resonance c h a r a c t e r i s t i c s A recent developement i n the s t r u c t u r a l elucidation of 14 the K l e b s i e l l a polysaccharides has been the i s o l a t i o n and 15 u t i l i z a t i o n of bacteriophage which have the a b i l i t y to depolymerise the capsular polysaccharide with tremendous precision. Glycanase enzymes (enzymes which cleave g l y c o s i d i c bonds) are produced by the bacteriophage and i t i s these enzymes which degrade the polymer into repeating units and multiples thereof. Preliminary work using the bacteriophage degradation technique i s currently being pursued i n t h i s laboratory (see Appendix I, page 179). The i s o l a t i o n of large quantities of s p e c i f i c "repeat" units made possible by t h i s method w i l l make available excellent model oligosaccharides 13 1 for C and H nuclear magnetic resonance, mass spectrometric, and other studies. II. Techniques and Methods Used i n the Structural Analyses  of Polysaccharides. In order to f u l l y characterise a polysaccharide consist-ing of repeating units, a s t r u c t u r a l study should determine: - 8 -(a) The nature and number of sugar r e s i d u e s and t h e i r r e l a t i v e p r o p o r t i o n s . (b) The p o s i t i o n s of l i n k a g e of the sugar r e s i d u e s . (c) . The sequence of the component sugars. (d) The anomeric c o n f i g u r a t i o n s of the sugars pre s e n t . (e) I f p o s s i b l e , the molecular weight of the p o l y s a c c h a r i d e . Most of the techniques used to achieve these requirements have been a v a i l a b l e to the carbohydrate chemist f o r q u i t e some time and many can be c o n s i d e r e d as standard. I t i s not my o b j e c t i v e to go i n t o these procedures i n d e t a i l as t h i s i n f o r m a t i o n i s r e a d i l y a v a i l a b l e i n g e n e r a l t e x t s , but t o d i s c u s s f e a t u r e s of s p e c i a l i n t e r e s t observed d u r i n g the course of t h i s work and to update the a p p l i c a t i o n s of those techniques f o r which s i g n i f i c a n t improvements have r e c e n t l y been made. While the f o l l o w i n g d i s c u s s i o n w i l l d e a l with p o l y s a c c h -a r i d e s with r e p e a t i n g u n i t s , much of the methodology d e s c r i b e d can be a p p l i e d e q u a l l y w e l l to the i n v e s t i g a t i o n of p o l y -s a c c h a r i d e s with i r r e g u l a r s t r u c t u r e s . II.1 I s o l a t i o n and p u r i f i c a t i o n . S t r a i n s of K l e b s i e l l a b a c t e r i a of known K type were obtained as agar stab c u l t u r e s from Dr. I. 0rskov i n Copenhagen. The b a c t e r i a were p l a t e d out on agar d i s c s s e v e r a l times b e f o r e use, w i t h o n l y s t r o n g growing, sli m e producing c o l o n i e s being p i c k e d . I n n o c u l a t i o n of a s u c r o s e - y e a s t e x t r a c t medium f o r 8h and subsequent i n c u b a t i o n f o r t h r e e or f o u r days on - 9 -t r a y s of s u c r o s e - y e a s t e x t r a c t - a g a r produced a lawn of b a c t e r i a which was h a rvested by simply s c r a p i n g the c e l l s and slime from the agar s u r f a c e . The b a c t e r i a l c e l l s were separated from the slime s o l u t i o n by c e n t r i f u g a t i o n and the c e l l f r e e supernatant was then pre-c i p i t a t e d i n t o e t h a n o l . T h i s p r e c i p i t a t e was d i s s o l v e d i n water and then r e p r e c i p i t a t e d u s i n g 10% h e x a d e c y l t r i m e t h y l ammonium bromide (CETAVLON). The p r e c i p i t a t e , formed by the r e a c t i o n of a c i d i c groups i n the p o l y s a c c h a r i d e w i t h the CETAVLON, was then d i s s o l v e d i n a minimum amount of 4 M sodium c h l o r i d e and then p r e c i p i t a t e d i n t o e t h a n o l or acetone. A f t e r t h i s p r e c i p i t a t e had been d i s s o l v e d i n water the s o l u t i o n was d i a l y s e d f o r two days a g a i n s t running tap water and then l y o p h i l i s e d . I t should be noted t h a t f o l l o w i n g the a d d i t i o n of the 4 M sodium c h l o r i d e to d i s s o l v e the CETAVLON-polysaccharide p r e c i -p i t a t e i t i s not always p o s s i b l e to d i a l y s e d i r e c t l y t h i s s o l u t i o n a g a i n s t tap water as might i n i t i a l l y be thought. In some cases the p r e c i p i t a t e w i l l reform d u r i n g the d i a l y s i s as the sodium c h l o r i d e i s removed. The p r e c i p i t a t i o n i n t o e t h a n o l or acetone before d i a l y s i s prevents t h i s r e p r e c i p i t a t i o n . The K l e b s i e l l a p o l y s a c c h a r i d e s grown and p u r i f i e d i n t h i s manner have been found to be s u f f i c i e n t l y pure f o r s t r u c t u r a l s t u d i e s to be c a r r i e d out. Any major i m p u r i t i e s , such as contaminating n e u t r a l p o l y s a c c h a r i d e s , would become n o t i c e a b l e when techniques such as n u c l e a r magnetic resonance spectroscopy are performed as these methods r e l y h e a v i l y on the homogeneity of the p o l y s a c c h a r i d e s t r u c t u r e . - 10 -II.2 Use of proton magnetic resonance ( H n.m.r.) and carbon magnetic resonance ( C n.m.r.) spectroscopy i n s t r u c t u r a l s t u d i e s . Strong evidence f o r the e x i s t e n c e o f r e g u l a r r e p e a t i n g u n i t s i n K l e b s i e l l a p o l y s a c c h a r i d e s comes from the f a c t t h a t n u c l e a r magnetic resonance s p e c t r a w i t h good r e s o l u t i o n can be obtained on s o l u t i o n s o f the i n t a c t n a t i v e p o l y s a c c h a r i d e . I f a p o l y s a c c h a r i d e , w i t h a molecular weight of approx. 1x10 , d i d not have a r e g u l a r r e p e a t i n g u n i t , meaningful r e s u l t s from n.m.r. would be i m p o s s i b l e to o b t a i n . A r e p e a t i n g u n i t w i l l g i v e the p o l y s a c c h a r i d e an e f f e c t i v e m o lecular weight between 600-1500 (depending on the number of sugar r e s i d u e s i n the r e p e a t i n g u n i t ) as f a r as n.m.r. s t u d i e s are concerned. II.2.1 Proton magnetic resonance. "'"H n.m.r. has been used e x t e n s i v e l y d u r i n g t h i s work and has proven t o be an extremely v a l u a b l e n o n - d e s t r u c t i v e t e c h n i -16 1 "7 que ' . S e v e r a l problems are encountered i n performing a "'"H n.m.r. experiment on a p o l y s a c c h a r i d e . Although dimethyl s u l -phoxide-dg may be used as a s o l v e n t , deuterium oxide (D 20) i s more commonly used. The numerous hydroxyl groups p r e s e n t i n a p o l y s a c c h a r i d e make i t necessary t o exchange the sample with deuterium oxide s e v e r a l times to convert most of the OH groups to OD groups and hence minimise i n t e r f e r e n c e from the temperature s e n s i t i v e HOD s i g n a l . The exchange w i t h deuterium oxide i s u s u a l l y performed by d i s s o l v i n g the p o l y s a c c h a r i d e - 11 -(approximately 15 mg) i n a small volume of 99.9% deuterium oxide and then freeze drying. Following t h i s the sample i s warmed under high vacuum for several hours before the entire process i s repeated. After three or four such treatments the sample i s then dissolved i n 100% deuterium oxide, but even afte r t h i s preparation the OH, OD exchange i s not e n t i r e l y complete and some residual HOD signal i s observed i n the "^H n.m.r. spectrum. As the HOD peak occurs in a s h i f t region of major importance, i t i s necessary to move t h i s peak. It may be shifted u p f i e l d by heating or downfield by cooling or the addition of t r i f l u o r o a c e t i c acid. The HOD peak may also be eliminated by means of Fourier transform (F.T.) "*"H n.m.r. 18 spectroscopy . Use i s made of the longer spin l a t t i c e relaxation time (T^) of the HOD signal as compared to other sugar protons but t h i s technique requires several machine hours to perform. A second problem that i s often encountered i n preparing a polysaccharide sample for "*"H n.m.r. i s that due to the sample v i s c o s i t y . Normally a 1-2% solution of the polysaccharide can be obtained but these solutions are often so viscous that there i s a loss of homogeneity, and hence resolution, when the "^H n.m.r. experiment i s performed. This problem i s somewhat a l l e v i a t e d when the sample i s heated to approximately 95° to move the HOD resonance u p f i e l d but i s not altogether eliminated. It i s possible to use a less concentrated sample but a 1-2% solution i s at the lower l i m i t for continuous wave (c.w.)• « F i g u r e I I . 1 H n.m.r. spectrum of K l e b s i e l l a K36 c a p s u l a r p o l y s a c c h a r i d e . - 13 -"""H n.m.r. and F.T. XH n.m.r. i s o f t e n necessary w i t h more d i l u t e s o l u t i o n s . A technique t h a t i s f i n d i n g more widespread use i s to perform an extremely m i l d a c i d h y d r o l y s i s on the n a t i v e p o l y s a c c h a r i d e and to then do the XH n.m.r. experiment on t h i s p a r t i a l l y depolymerised, and f a r l e s s v i s c o u s , m a t e r i a l . Often, h y d r o l y s i s c o n d i t i o n s can be s e l e c t e d so as t h a t l a b i l e groups, such as pyruvate a c e t a l s or a c e t a t e groups, are not removed but t h i s may not always be the case. Information o b t a i n a b l e by XH n.m.r. i n c l u d e s data on: the presence or absence o f pyruvate a c e t a l s , 6-deoxy sugars and a c e t a t e s ; the number of sugars per r e p e a t i n g u n i t ; the presence of a-D or 3-D anomeric s i g n a l s f o r hexoses and a-Lj or 3-L anomeric s i g n a l s f o r 6-deoxy hexoses, e.g. L-rhamnose. The XH n.m.r. spectrum of K l e b s i e l l a K36 (see F i g u r e I I . 1 , page 12) i s t y p i c a l of those u s u a l l y o b t a i n e d . The spectrum shows the presence of a pyruvate a c e t a l ( s i n g l e t a t T8.41) and moreover, shows i t t o be i n a 1:3 r a t i o w i t h r e s p e c t to the broad peak at T8.70 due to the methyl groups of t h r e e d i f f e r e n t L-rhamnose u n i t s (three d o u b l e t s each with J c , 6Hz). - 5 , 6 In the anomeric r e g i o n (x4.5 - T6.0) three anomeric s i g n a l s appear downfield of x5.0 and can t h e r e f o r e be a s s i g n e d t e n t a -t i v e l y to a - l i n k a g e s , while t h r e e more s i g n a l s appear u p f i e l d of T5.0 and can be a s s i g n e d to 8 - l i n k a g e s . The d i v i s i o n a t T5.0 i s a r b i t r a r y but has been found to be v a l i d i n most cases. The presence of s i x anomeric proton s i g n a l s o v e r a l l i n d i c a t e s K36 c o n s i s t s of a hexasaccharide repeat u n i t . S p i n - s p i n c o u p l i n g - 14 -between the protons a t and C^ of sugars p r o v i d e s v a l u a b l e i n f o r m a t i o n . For 3 - D hexoses (e.g. 3 - D - g l u c o s e , 3-D-galactose) J l 2^s H z wn:'--'-e ^ o r a~U hexoses 0 i s 2-3 Hz. For the 6-deoxy-hexose Jj-rhamnose (6-deoxy-J^-mannose) the a-£-anomeric proton has ^2 Hz while the 3-fj anomeric proton has ^ 1 Hz. While t e t r a m e t h y l s i l a n e i s a good i n t e r n a l standard f o r "*"H n.m.r. i n o r g a n i c s o l v e n t s , i t i s i n s o l u b l e i n D 20 and t h e r e f o r e cannot be used f o r p o l y s a c c h a r i d e samples. Sodium 2,2-dimethyl-2-silapentane-5-sulphonate (D.S.S.) or sodium 3 - t r i m e t h y l s i l y l propionate-2,2,3,3-d^ (T.S.P.) are p o s s i b l e a l t e r n a t i v e s but are d i f f i c u l t to remove from the sample i f the p o l y s a c c h a r i d e i s to be recovered. Acetone (methyl groups g i v i n g a sharp s i n g l e t a t T7.77 r e l a t i v e to i n t e r n a l D.S.S. i n D 20) has proven to be a good i n t e r n a l standard and the s h i f t of the s i n g l e t i s u n a f f e c t e d by v a r i a t i o n s i n temperature. 13 13 II.2.2 Carbon magnetic resonance ( C n.m.r.). 13 19 The f i r s t n.m.r. o b s e r v a t i o n s of C n u c l e i were r e p o r t e d 13 as e a r l y as 1957 but the low n a t u r a l abundance of the C n u c l e i (1.1%) made i t necessary to work with h i g h l y s o l u b l e , low molecular weight m a t e r i a l s . The f i r s t g r e a t breakthrough 13 i n experimental C n.m.r. was the d i s c o v e r y o f wide band 20 proton d e c o u p l i n g i n 1966 . With the f u r t h e r development of i n s t r u m e n t a l techniques, e s p e c i a l l y F o u r i e r t r a n s f o r m n.m.r., 13 C n.m.r. has now become not onl y p r a c t i c a l but a l s o n e a r l y comparable w i t h "Hi n.m.r. i n terms of experimental ease and - 15 -13 q u a l i t y of r e s u l t s f o r most o r g a n i c compounds. The f i r s t C 21 n.m.r. experiments with carbohydrates appeared i n 1969 and s i n c e t h a t time the technique has been a p p l i e d e x t e n s i v e l y to 22-24 . . 25-31 monosaccharides and o l i g o s a c c h a r i d e s The p o t e n t i a l of t h i s technique i n determining f e a t u r e s i n p o l y s a c c h a r i d e s t r u c t u r e s has o n l y r e c e n t l y been u t i l i s e d . An i n v e s t i g a t i o n of amylose, a simple homopolymer, has been 24 r e p o r t e d and a l s o a study on heparin, a heteropolymer with 32 33 two d i f f e r e n t component sugars, has been completed ' . The 13 C n.m.r. spectrum of a l i n e a r glucan w i t h (l+4)-a-D and (l+6)-ot-g l i n k a g e s showed t h a t both the sequence and composition 2 8 34 of the s i n g l e monosaccharides c o u l d be determined ' S i m i l a r i n v e s t i g a t i o n s on dextrans of known s t r u c t u r e 13 showed d i a g n o s t i c f e a t u r e s i n the C n.m.r. s p e c t r a f o r a-D-35 (1+2)-, a-D-(1+3)-, or a-g-(1+4)-linkages , and the a s s i g n -ment of the 1 3 C n.m.r. spectrum of a mannan c o n t a i n i n g a l t e r n a t e 3 -(1+3)- and 3~(1+4)-linked g-mannose r e s i d u e s was achieved 3 6 w i t h the a i d of s p e c i f i c d e u t e r a t i o n . Most p o l y s a c c h a r i d e s 13 g i v e w e l l - d e f i n e d C n.m.r. s p e c t r a but i t has proven d i f f i c u l t 13 to completely a s s i g n the C resonances i n a l l but the s i m p l e s t of polymers. The o b j e c t i v e o f the technique i s to p o s t u l a t e chemical s t r u c t u r e on the b a s i s of chemical s h i f t v a l u e s but u n t i l the e x i s t i n g methods f o r the assignment of i n d i v i d u a l resonances are improved, or new s o l u t i o n s to t h i s problem developed, then t h i s o b j e c t i v e w i l l be d i f f i c u l t to achieve f o r complex p o l y s a c c h a r i d e s . - 16 -During the course of t h i s work oligosaccharides obtained by degradative procedures on the K l e b s i e l l a polysaccharides 13 have been examined by C n.m.r. wherever possible, but while these experiments have provided valuable information on the isol a t e d oligosaccharides i t has proven a d i f f i c u l t task to correlate these spectra and the spectra of the in t a c t poly-saccharides. The K l e b s i e l l a polysaccharides have been shown to be well suited to investigation by XH n.m.r. (see section II.2.1) and during the course of t h i s work i t has been shown they are 13 equally well suited to study by C n.m.r. Although the K l e b s i e l l a polysaccharides may contain as many as four or f i v e d i f f e r e n t sugars the precise repeating unit structure of these materials makes i t possible to examine these r e l a t i v e l y complex 13 heteropolymers by C n.m.r. The major d i f f i c u l t y i n preparing a polysaccharide sample 13 for C n.m.r. i s the low s o l u b i l i t y of the material and the v i s c o s i t y of the solution. At best approximately 0.2 M solutions may be obtained and often t h i s figure i s closer to 0.1 M. This low concentration and usually a repeating unit of high molecular weight therefore necessitate a large number of transients (>100,000) being completed on a sample to obtain 13 a reasonable signal to noise r a t i o . As t h i s r a t i o m a C n.m.r. experiment improves as the square root of the number of t o t a l transients, there i s a p r a c t i c a l lower l i m i t to the concentration of solution that can be used. A normal """""c n.m.r. experiment involves pulsing the sample with a short radiofrequency pulse which simultaneously 13 excites a l l the C nuclei. The subsequent precession of 13 the C nuclei i s c a l l e d a free induction decay (F.I.D.) and corresponds to the simultaneous "re-radiation" of a l l the 13 energy absorbed by the C nu c l e i . It takes a f i n i t e time for any one nucleus to return ("relax") to i t s ' normal energy state. Nuclei that do not f u l l y relax before being pulsed again may become saturated and hence w i l l not appear in the 13 C spectrum when a Fourier transformation i s performed on the accumulated F.I.D. signals. For most small organic molecules a pulse delay (time delay between successive pulses) 13 of 2-5 sec i s s u f f i c i e n t to allow most C nuclei to relax but some nu c l e i , e s p e c i a l l y those i n a carbonyl f u n c t i o n a l i t y , may require much longer delays. Experiments have shown that the s p i n - l a t t i c e relaxation rates for the K l e b s i e l l a polysaccharides are very fast, e.g. approximately 20, 000 msec x for the ring carbons of K36 i n a 13 1% solution at 25° . This means that no delay i s required between successive pulses and moreover, the ac q u i s i t i o n time (time for which each F.I.D. i s recorded) for these samples can be as short as 0.2 sec. The normal a c q u i s i t i o n time for most organic molecules i s about 1 sec. It should be r e a l i s e d however, that while these experimental conditions allow greater than f i v e times as many transients to be completed i n the same time as when "usual" conditions are employed, i f some carbons - 18 -do have long relaxation rates then they w i l l only give a p a r t i a l signal or no signal in the f i n a l spectrum. This i n fact occurs and a spectrum of a polysaccharide using an acqu i s i t i o n time of 0.2 sec and no pulse delay w i l l not show any resonances due to carbons present as carbonyls, e.g. as in uronic acids and pyruvate acetals. 13 One further c h a r a c t e r i s t i c feature of C n.m.r. spectra i s worth mentioning. The nuclear Overhauser e f f e c t (N.O.E.) 13 i s always present in proton decoupled C n.m.r. experiments. N.O.E. i s a by-product of the proton decoupling and i s derived 13 from the proton induced relaxation of C nu c l e i . In b r i e f , 13 the decoupled protons i n t e n s i f y the C signal of the carbon to which they are attached. If the dipole-dipole relaxation 13 mechanism were dominant for a l l C nuclei in a sample, a l l signals would have the same integrated i n t e n s i t y i f experimental conditions were chosen such that a l l nuclei were f u l l y relaxing between pulses. In actual fact, the t h e o r e t i c a l N.O.E. 13 enhancement of 2.988 i s not usually observed for a l l C 13 nuclei due to appreciable contributions to the C s p i n - l a t t i c e relaxation from mecha'nisms other than the dipole-dipole mechanism. 13 Hence the integrated i n t e n s i t i e s for C nuc l e i are usually not equal and therefore widely d i f f e r e n t peak heights in a 13 C n.m.r. spectrum are normally observed. 13 The C n.m.r. spectrum of K l e b s i e l l a K36, obtained at 90.5 MHz, i s shown in Figure II.2, page 19 and i s a spectrum 13 which i l l u s t r a t e s well the information C n.m.r. can provide F i e l d ; 90.5 MHz S.W.; 20 KHz N.T.; 50,000 A.T.; 0.2 sec P.W.; 15 usee P.D.; 0 sec ca r b o n y l s (2) 13 anomeric C n u c l e i (6) ' C H 3 . ° f pyruvate CH 3 of rhamnose 175 150 100 50 0 PPm F i g u r e II.2 13 C n.m.r. spectrum of K l e b s i e l l a K36 c a p s u l a r p o l y s a c c h a r i d e . - 20 -in a s t r u c t u r a l investigation. Overall 29 d i f f e r e n t carbon signals can be distinguished (theoretical i s 39). At approxi-mately 175 ppm downfield from T.M.S. two signals a t t r i b u t a b l e 13 to carbonyl C nuclei corresponding to these nuclei i n the uronic acid and pyruvate acetal are observed. At approximately 100 ppm six signals a r i s i n g from anomeric (C^) carbons are apparent and t h i s i s consistent with the structure of K36 consisting of a hexasaccharide repeating unit. In the region 13 usually associated with C nuclei bearing a primary alcohol (60-62 ppm) only one signal i s observed. This signal probably arises from the C g of the D-galactose moiety in K36. In the h i g h f i e l d region (17-25 ppm) three signals a t t r i b u t a b l e to 13 methyl C nuclei appear. The signal at 25 ppm i s from the pyruvate acetal while the two signals at 17 ppm arise from L-rhamnose residues (6-deoxy sugars). It i s probably v a l i d to assume that the N.O.E. on these signals from the L-rhamnose moieties i s equivalent and hence i t could be assumed that three L-rhamnose units ex i s t i n the repeating unit. This i s consistent with the structure of K36 derived by chemical methods. It should be r e a l i s e d that when proton decoupled spectra are run there i s a loss of a l l coupling information. This type of information (J.. „ coupling constants, etc.) has proven to l , z be very useful in "*"H n.m.r. studies. P a r t i a l l y coupled spectra 37 13 can be obtained i n C n.m.r., but the following two factors make t h i s a d i f f i c u l t task with polysaccharides. - 21 -(i) A s i n g l e X J C peak w i l l be s p l i t i n t o two peaks of h a l f the i n t e g r a t e d i n t e n s i t y of the s i n g l e peak. Hence the s m a l l e r s i g n a l s are l e s s d i s t i n g u i s h a b l e from base-l i n e n o i s e . ( i i ) With s i n g l e bond l e n g t h C-H c o u p l i n g N.O.E. e f f e c t s are not observed and hence the approximate three f o l d s i g n a l enhancement i s a l s o l o s t . P a r t i a l l y coupled s p e c t r a on o l i g o s a c c h a r i d e s , which are more s o l u b l e and u s u a l l y c o n t a i n fewer " r e p e a t i n g u n i t " carbons than a p o l y s a c c h a r i d e , have been more s u c c e s s f u l and have been c a r r i e d out wherever p o s s i b l e i n t h i s work. 13 A " s t a t e of the a r t " summary of C n.m.r. of p o l y s a c c h a r i d e s would have to conclude t h a t w hile the technique does p r o v i d e some very u s e f u l i n f o r m a t i o n t h a t i s somewhat complementary to the data o b t a i n e d by XH n.m.r., i n s u f f i c i e n t knowledge of 13 the i n f l u e n c e of c o n f o r m a t i o n a l and s t e r i c e f f e c t s on C chemical s h i f t s does not a l l o w unambiguous assignemt of many s p e c t r a l s i g n a l s . More model s t u d i e s u s i n g o l i g o s a c c h a r i d e s of known s t r u c t u r e are necessary to understand the f i n e r f e a t u r e s of the technique. II.3 T o t a l h y d r o l y s i s and methanolysis. The h y d r o l y s i s of a p o l y s a c c h a r i d e and the subsequent a n a l y s i s of the h y d r o l y s i s products i s o f t e n performed q u a l i t a -t i v e l y . However, the t o t a l h y d r o l y s i s of p o l y s a c c h a r i d e s , p a r t i c u l a r l y those with r e s i s t a n t g l y c o s i d i c l i n k a g e s , i s a more d i f f i c u l t o p e r a t i o n . - 22 -Hydrochloric, sulphuric and t r i f l u o r o a c e t i c acids are commonly used in hydrolysis but the l a s t named has the advantage of being e a s i l y removed under diminished pressure following the hydrolysis. 2 M T r i f l u o r o a c e t i c acid i s usually s u f f i c i e n t to hydrolyse completely a neutral polysaccharide into i t s monomeric sugar units a f t e r 8 hours at 95° and under these conditions the degradation of these monomer units i s normally considered i n s i g n i f i c a n t . Polysaccharides containing uronic acid moieties are very d i f f i c u l t to hydrolyse completely due to the resistance of the uronosyl bond. Normally, t o t a l hydrolysis of these poly-saccharides i s performed on the uronic acid reduced polymer (see Section II.5), but the reduction of uronic acids i n aqueous solution i s not a simple task. 3 8 A method developed i n t h i s laboratory and used during t h i s study, involves the use of methanolysis. The a c i d i c polysaccharide i s f i r s t treated with 3% methanolic hydrogen chloride under reflux for 16 hours. These conditions are s u f f i c i e n t to cleave most g l y c o s i d i c bonds but not a l l uronosyl linkages. During t h i s treatment the methyl ester of the uronic acid i s formed and t h i s can be reduced using sodium borohydride i n anhydrous methanol. It should be noted that sodium borohydride w i l l reduce esters in aqueous solu-39 40 tio n ' but does not proceed i n 100% y i e l d as saponification of the ester i s a competing reaction. Sodium borohydride in anhydrous methanol w i l l reduce methyl esters quan t i t a t i v e l y and i s a clean, simple reaction. When sodium borohydride i s - 23 -d i s s o l v e d i n methanol there i s a reasonably r a p i d r e a c t i o n between the two. As a r e s u l t a s e r i e s of r e d u c i n g s p e c i e s i s produced which are s t r o n g e r r e d u c i n g agents than NaBH^ i t s e l f i n aqueous s o l u t i o n s . These s p e c i e s may be given the g e n e r a l formula shown below Na +(MeO) B~(H) n m where n + m = 4, and i t i s most probable t h a t r e d u c t i o n of the e s t e r to the a l c o h o l i s achieved by these sodium boro-hy d r i d e d e r i v a t i v e s . F o l l o w i n g the r e d u c t i o n a h y d r o l y s i s step i s necessary to c l e a v e those bonds t h a t were u r o n o s y l l i n k a g e s and a l s o to c l e a v e the methyl g l y c o s i d e s which would have been formed d u r i n g the treatment with methanolic hydrogen c h l o r i d e . The p o l y s a c c h a r i d e can then be c o n s i d e r e d f u l l y h y d r o l y s e d and q u a n t i t a t i v e a n a l y s i s of the sugar components by g.l.c.-m.s. as t h e i r a l d i t o l a c e t a t e s may be c a r r i e d out. A u r o n i c a c i d such as D-glucuronic a c i d w i l l appear as a u n i t of D-glucose i n the f i n a l h y d r o l y s a t e and w i l l be i n d i s t i n g u i s h a b l e from other D-glucose u n i t s . However, i f the r e d u c t i o n i n anhydrous methanol i s done u s i n g sodium boro-d e u t e r i d e , then o n l y the D-glucose from D-glucuronic a c i d w i l l be l a b e l l e d a t and can be determined by mass s p e c t r o -scopy . - 24 -II.4 M e t h y l a t i o n and m e t h y l a t i o n a n a l y s i s . M e t h y l a t i o n a n a l y s i s of p o l y s a c c h a r i d e s i s probably s t i l l the most widely used " t o o l " i n the f i e l d today. The data o b t a i n e d from such an a n a l y s i s i n c l u d e i n f o r m a t i o n on the number of sugar r e s i d u e s , the type of sugar r e s i d u e s , the l i n k a g e s between sugars and whether branching occurs i n the p o l y s a c c h a r i d e . A m e t h y l a t i o n a n a l y s i s i s r e l a t i v e l y s t r a i g h t -forward and can be done on m i l l i g r a m q u a n t i t i e s of m a t e r i a l . The a n a l y s i s r e l i e s upon the e t h e r i f i c a t i o n of every f r e e h ydroxyl group i n the p o l y s a c c h a r i d e and t h i s can be achieved by u s i n g s e v e r a l methods. 41 42 (1) Hakomori m e t h y l a t i o n ' - uses methyl s u l p h i n y l anion i n dimethyl sulphoxide as the base f o l l o w e d by t r e a t -ment with methyl i o d i d e . The technique i s by f a r the best and one treatment i s o f t e n s u f f i c i e n t f o r complete m e t h y l a t i o n . 43 (2) Kuhn m e t h y l a t i o n - uses s i l v e r oxide i n N,N-dimethylformamide and methyl i o d i d e . 44 (3) Purdie m e t h y l a t i o n - uses s i l v e r oxide i n methyl i o d i d e . The Hakomori m e t h y l a t i o n , u n l i k e the Kuhn and P u r d i e procedures, cannot be repeated on a p o l y s a c c h a r i d e c o n t a i n i n g a u r o n i c a c i d because the methyl e s t e r of the u r o n i c a c i d can r e a c t with the m e t h y l s u l p h i n y l anion to g i v e a 3 - e l i m i n a -t i o n (see S e c t i o n I I . 8 ) . During t h i s work a l l m e t h y l a t i o n s were performed u s i n g the Hakomori procedure and i f the e t h e r i f i c a t i o n was not deemed to be complete (as determined by examining the h y d r o x y l - 25 -absorbance at 3600 cm in the i n f r a red spectrum) then a Purdie methylation was ca r r i e d out. In a l l cases only one Purdie treatment was necessary to achieve t o t a l e t h e r i f i -cation. • Following the complete methylation of a polysaccharide or oligosaccharide the product i s f u l l y hydrolysed and the p a r t i a l l y methylated monosaccharides produced are i d e n t i f i e d . For methylated material containing no uronic acid moieties t h i s i s r e l a t i v e l y straightforward and t r e a t -ment with 2 M t r i f l u o r o a c e t i c acid at 95° for 16 hours, followed by transformation into a l d i t o l acetates and gas l i q u i d chromatographic (g.l.c.) analysis, w i l l give good r e s u l t s . Those methylated samples that do contain uronic acids are far more re s i s t a n t to acid hydrolysis and treatment with 2 M t r i f l u o r o a c e t i c acid at 95° for 16 hours w i l l not usually cleave a l l the uronosyl bonds. Prior reduction of the uronic acid, which i s present as i t s methyl ester, and then subsequent hydrolysis w i l l a l l e v i a t e t h i s problem. While lithium aluminum hydride in r e f l u x i n g tetrahydro-furan w i l l achieve complete reduction of the ester to the alcohol there are often losses associated with the subsequent work up. The aluminum hydroxide produced aft e r the lithium aluminum hydride has been destroyed tends to adsorb materials, e s p e c i a l l y those with free hydroxyls. On the other hand, reductions using sodium borohydride are r e l a t i v e l y clean and the conditions preferred during t h i s work involve using a - 26 -Column: HIEFF IB ( 6 ' x l / 8 " ) . 3% on Gas Chrom Q (100-120 mesh). Programme: 165° 8 min, 2° per min to 200°. C a r r i e r gas: N^, 20 ml/min. 2,3-GLC _ i i _ i : L_ 165 175 185 195 TEMPERATURE (°C ) F i g u r e II.3 G . l . c . t r a c e o b tained v i a m e t h y l a t i o n a n a l y s i s of K l e b s i e l l a K36. - 27 -l a r g e excess of sodium borohydride i n a mixture of t e t r a h y d r o -f u r a n and ethanol (1:1). The t e t r a h y d r o f u r a n i s necessary to d i s s o l v e completely the methylated p o l y s a c c h a r i d e . Per-methylated a c i d i c p o l y s a c c h a r i d e s t h a t have been reduced i n t h i s manner are f u l l y h y d r o l y s e d u s i n g 2 M t r i f l u o r o a c e t i c a c i d a t 95° f o r 16 hours. A n a l y s i s of the p a r t i a l l y methylated monosaccharides r e l e a s e d a f t e r h y d r o l y s i s i s achieved by g . l . c . and g . l . c -m.s. of these components as a l d i t o l a c e t a t e s (see S e c t i o n s II.10.1 and II.10.2.1). An example of such a g . l . c . s e p a r a t i o n i s shown i n F i g u r e I I . 3 , page 26. The components o b t a i n e d are d e r i v e d from the m e t h y l a t i o n a n a l y s i s of K l e b s i e l l a K36. II.5 Carboxyl r e d u c t i o n of a c i d i c p o l y s a c c h a r i d e s . As has been p o i n t e d out a l r e a d y (Sections II.3 and I I . 4 ) , a c i d i c p o l y s a c c h a r i d e s may present problems d u r i n g s t r u c t u r a l i n v e s t i g a t i o n s . (On the o t h e r hand, the r e s i s t a n c e of u r o n o s y l bonds to h y d r o l y s i s i s somewhat b e n e f i c i a l d u r i n g p a r t i a l h y d r o l y s i s s t u d i e s . See S e c t i o n II.7.) The r e d u c t i o n of the a c i d i c groups can be achieved with ease f o l l o w i n g m e t h y l a t i o n of most p o l y s a c c h a r i d e s , but o f t e n r e d u c t i o n of the na-t i v e m a t e r i a l i s d e s i r a b l e , e.g. to a l t e r p a r t i a l h y d r o l y s i s cleavage p a t t e r n s , or to e a s i e r f a c i l i t a t e p e r i o d a t e o x i d a t i o n . The d i r e c t r e d u c t i o n of n a t i v e p o l y s a c c h a r i d e s w i t h 4 5 4 6 a c i d i c f u n c t i o n a l groups has been achieved ' by f i r s t pro-p i o n a t i n g ( e s t e r i f y i n g ) the f r e e hydroxyl groups i n the p o l y -- 28 -saccharide with propionic anhydride in pyridine and then e s t e r i f y i n g the carboxylic acid groups with diazomethane i n tetrahydrofuran. The f u l l y e s t e r i f i e d material i s then reduced with lithium borohydride in r e f l u x i n g tetrahydro-i furan. Complete reduction can-be achieved by t h i s method but several treatments may have to be carried out. The method r e l i e s upon the lithium borohydride being able to reduce the methyl ester of the carboxylic acid more quickly than the propionate esters of the hydroxyl groups i n the polysaccharide. 47 A more recent technique developed by Conrad and Taylor i s quick and proceeds in good y i e l d . The method involves the use of water soluble carbodiimides and the reaction mechanism i s outlined in Figure II.4, page-29. The i n i t i a l reaction to form the intermediate (I) pro-ceeds rapidly and consumes acid. In t h i s way the reaction can be followed by t i t r a t i o n to pH 4.75 with acid. Formation of (I) i s usually complete within 2 hours. The reduction of the intermediate (I) must be performed under a c i d i c conditions as (I) i s unstable in basic solutions. The reduction i s achieved using sodium borohydride but i s an extremely i n e f f i c i e n t process as the sodium borohydride i s decomposed - 29 -RCOOH , 0 NHR" K NHR" +H II || || —>• RCOC + C + i NHR' , NHR" RCOO + H (I) A c i d i c P o l y -s a c c h a r i d e E.D.C.* or C. M. C. * * RCH2OH Carbodiimide Intermediate Sodium Borohydride pH 5+7 Sodium Borohydride 0 " II RCH NHR" I + 0=C + H I NHR' E.D.C. = l - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) c a r b o d i i m i d e . C.M.C. = l - c y c l o h e x y l - 3 - ( 2 - m o r p h o l i n o e t h y l ) c a r b o d i i m i d e metho-p-toluene sulphonate. F i g u r e II.4 Reduction o f c a r b o x y l i c a c i d s i n aqueous s o l u t i o n u s i n g c a r b o d i i m i d e reagents. (From T a y l o r and Conrad, Bi o c h e m i s t r y , 11, 1383 (1972)). - 30 -r a p i d l y i n a c i d i c c o n d i t i o n s . Vast excesses o f sodium boro-hy d r i d e are used and the r e a c t i o n c o n d i t i o n s are kept a c i d i c by simultaneous t i t r a t i o n w i t h 4 M hydrogen c h l o r i d e . Although sodium cyanoborohydride (NaBH^CN) does not u s u a l l y reduce l a c t o n e s , an attempt to use t h i s reagent to reduce the i n t e r m e d i a t e (I) was made as sodium cyanoboro-hydride i s s t a b l e to a c i d c o n d i t i o n s . No r e d u c t i o n was observed however, even at pH 1.0. II.6 P e r i o d a t e O x i d a t i o n . 48 Since the d i s c o v e r y i n 1928 by Malaprade t h a t sodium metaperiodate c o u l d c l e a v e the carbon-carbon bond between v i c i n a l d i o l s i n aqueous s o l u t i o n , p e r i o d a t e o x i d a t i o n has been used e x t e n s i v e l y by the carbohydrate chemist and i t remains a powerful t o o l f o r s t r u c t u r a l i n v e s t i g a t i o n s today. The product of p e r i o d a t e o x i d a t i o n of a polymer i s termed a 1 p o l y a l d e h y d e ' and i s reduced with NaBH^ to g i v e a ' p o l y o l ' . T h i s d e r i v e d p o l y o l can p r o v i d e c o n s i d e r a b l e i n f o r m a t i o n on the s t r u c t u r e of the polymer. T o t a l h y d r o l y s i s (2 M T.F.A., 95°, 16 hours) of the p o l y o l w i l l c l e a v e a l l g l y c o s i d i c l i n k -ages (except where i n t a c t u r o n i c a c i d s s t i l l e x i s t ) and a c e t a l l i n k a g e s . The h y d r o l y s i s products can be examined q u a l i t a t i v e l y by paper chromatography or q u a n t i t a t i v e l y by 49 g . l . c . The r e l a t i v e p r o p o r t i o n s of the s u r v i v i n g sugar r e s i d u e s and the fragments o b t a i n e d from degraded sugar r e s i d u e s , e.g. g l y c e r o l , e r y t h r i t o l , e t c . , w i l l g i v e i n f o r m a t i o n r e g a r d -- 31 -ing some of the g l y c o s i d i c l i n k a g e s p r e s e n t i n the o r i g i n a l p o l y s a c c h a r i d e . Much more u s e f u l i n f o r m a t i o n can be o b t a i n e d when p a r t i a l h y d r o l y s i s of the d e r i v e d p o l y o l i s c a r r i e d out. 50 Smith and co-workers u t i l i s e d the g r e a t e r s u s c e p t i b i l i t y o f the t r u e a c e t a l l i n k a g e s i n the d e r i v e d p o l y o l as compared to remaining i n t a c t g l y c o s i d i c l i n k a g e s (see F i g u r e I I . 5 , page 32). The a c e t a l l i n k a g e s are i n g e n e r a l approximately 3 4 10 to 10 times more l a b i l e than the g l y c o s i d i c l i n k a g e s . A wide v a r i e t y o f h y d r o l y s i s c o n d i t i o n s has been used f o r t h i s p a r t i a l h y d r o l y s i s . The range extends from 0.005 M 51 a c i d a t 100° f o r 1 hour to 0.5 M a c i d a t room temperature 52 f o r 8 hours , but d u r i n g t h i s work 0.5 M t r i f l u o r o a c e t i c a c i d a t room temperature f o r 16 hours was found to g i v e s a t i s -f a c t o r y r e s u l t s . The Smith d e g r a d a t i o n o f * t h e p o l y o l y i e l d s g l y c o s i d e s of mono, d i and o l i g o s a c c h a r i d e s which can be ana l y s e d to g i v e very u s e f u l s t r u c t u r a l i n f o r m a t i o n , e s p e c i a l l y data on sugar sequences. An example o f the use o f p e r i o d a t e o x i d a t i o n and subsequent Smith d e g r a d a t i o n i s shown (F i g u r e I I . 5 , page 32) u s i n g K l e b s i e l l a K36 as a model. I t s h o u l d be noted t h a t when d u r i n g a Smith d e g r a d a t i o n the t e r m i n a t i n g g l y c o s i d e o r i g i n a t e s from a 2 - l i n k e d sugar r e s i d u e , i . e . from a r e s i d u e t h a t has been c l e a v e d between carbons t h r e e and f o u r , i t i s necessary to i n c l u d e a f u r t h e r r e d u c t i o n step t o reduce the aldehyde group r e l e a s e d a t C-^  d u r i n g the h y d r o l y s i s (see K36 example g i v e n i n F i g u r e I I . 5 , - 32 -CHOH 2 HOOC *= site of periodate attack 1. N a I 0 4 2. NaBK, CH20H HOOC (I) Smith (partial) hydrolysis (II) Na BH, ;H2CH2OH HOCH2CH2OH (I) Total hydrolysis ( H ) N a B H 4 D-galactose 2 x l _ - rhamnose 1-deoxy propanediol glycerol 2xethylene glycol erythronic acid erythritol p yruvic acid 1-deoxy propanediol 2 x ethylene glycol erythritol D-erythronic acid pyruvic acid F i g u r e II.5 P e r i o d a t e degradation of K l e b s i e l l a K36. - 33 -page 32). T h i s i s needed because of the r e l a t i v e d i f f i c u l t y i n working with compounds c o n t a i n i n g aldehyde f u n c t i o n a l i t i e s . P e r i o d a t e o x i d a t i o n i s complicated by both over and under o x i d a t i o n . Over o x i d a t i o n can be minimised by u s i n g d i l u t e s o l u t i o n s of p e r i o d a t e b u f f e r e d a t a c i d i c pH (e.g. pH 4.0) and by keeping the r e a c t i o n mixture i n the dark a t 4°. Incomplete o x i d a t i o n may a r i s e due t o : 1. ' The formation of i n t r a m o l e c u l a r hemiacetals v i a 53 54 the aldehydes generated i n the i n i t i a l p e r i o d a t e o x i d a t i o n ' ; 2. s t e r i c hindrance t h a t l i m i t s the a c c e s s i b i l i t y o f 55 a p a i r o f v i c i n a l d i o l s to p e r i o d a t e ions 3. i o n i c r e p u l s i o n between charged groups, e.g. c a r b o x y l a t e anions, and the a t t a c k i n g p e r i o d a t e molecules During p e r i o d a t e o x i d a t i o n s t u d i e s on K70 and K32, and to a l e s s e r e x t e n t on K36, i t was observed t h a t when the o x i d a t i o n was c a r r i e d out i n an unbuffered medium (hence the pH of the s o l u t i o n was approximately 4.0) the r e l a t i v e l y l a b i l e pyruvate a c e t a l was s l o w l y h y d r o l y s e d and t h i s r e s u l t e d i n f u r t h e r p e r i o d a t e consumption. T h i s 'over o x i d a t i o n ' c o u l d be prevented i n the cases of K70 and K36 but not e n t i r e l y f o r K32. During p e r i o d a t e o x i d a t i o n s t u d i e s on K70 and K36 p o l y -s a c c h a r i d e s an a c e t a l a r i s i n g from the o x i d a t i o n and subsequent r e d u c t i o n o f a 2 - l i n k e d J^-rhamnose sugar (see page 34), was found to be u n u s u a l l y r e s i s t a n t to a c i d h y d r o l y s i s as compared w i t h other s i m i l a r a c e t a l s . T h i s i s even more unusual when - 34 -i t i s r e a l i s e d t h a t the g l y c o s i d i c l i n k a g e s from 6-deoxy sugars are c o n s i d e r e d to be more l a b i l e than the same f o r a 57 normal hexose , and i n the r i n g opened s t a t e the h y d r o l y s i s of the a c e t a l might be c o n s i d e r e d to be more e a s i l y achieved f o r Lj-rhamnose than f o r other 'normal' hexoses, e.g. g-glucos.e, D-galactose. C> 0—SUGAR HOCH^ ^ n 3 H HOCHg-SUGAR" Smith degradation Yesistant' acetal K l e b s i e l l a K36 and K70 s t r u c t u r e s both i n c l u d e p e r i o d a t e s u s c e p t i b l e 2 - l i n k e d L-rhamnose sugars. F o l l o w i n g the Smith degr a d a t i o n (0.5 M t r i f l u o r o a c e t i c a c i d , room temperature, 16 hours), although the expected oligomers were ob t a i n e d v i a g e l f i l t r a t i o n , the y i e l d s of these oligomers were poor. During the g e l f i l t r a t i o n p u r i f i c a t i o n polymeric m a t e r i a l e l u t i n g j u s t a f t e r the v o i d volume was always apparent and t h i s would i n d i c a t e incomplete h y d r o l y s i s d u r i n g the Smith d e g r a d a t i o n . In the case of K36 n.m.r. s p e c t r a of t h i s m a t e r i a l i n d i c a t e d t h a t i t was very s i m i l a r to the t r i s a c c h a r i d e g l y c e r i d e expected (see F i g u r e I I . 5 , page 32), i . e . t h e m a t e r i a l - 35 -con t a i n e d no s i d e c h a i n D - g l u c u r o n i c a c i d or D-glucose, and i s assumed to be a mixture of polymeric m a t e r i a l w i t h the i n - c h a i n 2 - l i n k e d L-rhamnose a c e t a l s t i l l i n t a c t (see below) 3 G a l L _ 3 R h a 1 _ 3 R h a 1 ^ H0CH^ C H 3 H )\ a ^ G a l X ^ R h a ' - ^ R h J - O - C H HOCH2—7 H Ch^ l CHOH Some f u r t h e r support f o r t h i s phenomenon i s shown i n a 5 8 study by Lindberg e t a l . . They compared the r a t e of h y d r o l y s i s of the compounds shown below. HOCH CH2OH HOCH OCH-; CH2OH CH2OH HOCH HOCH II CH30' \ / "OCH3 •H20H CH2OH OCH, OCH-III - 36 -While I and III were found to hydrolyse quite r e a d i l y , II was found to be approximately 10 times more re s i s t a n t to the hydrolysis conditions used. Compound II corresponds to an 1 i n chain' 2-linked hexose(acetylated) following periodate oxidation and NaBH^ reduction. The reason postulated for th i s resistance in II i s the presence of electron a t t r a c t i n g groups i n both the a and 3 positions to the acetal (C-^ ) carbon. However, a l l three compounds (I,II,III) were shown to hydrolyse much more re a d i l y than the parent methyl a-D-glucopyranoside. II.7 P a r t i a l hydrolysis. 57 Capon has comprehensively reviewed the f i r s t order rate constants for the acid catalysed hydrolysis of the glyco-sides of monosaccharides, and several generalisations may be made: (1) furanosides are more l a b i l e than pyranosides, (2) deoxy sugars are more e a s i l y hydrolysed than hexoses, (3) uronic acids are very r e s i s t a n t to hydrolysis (see Section II.2.), (4) amino sugars are more res i s t a n t to hydrolysis than common hexoses, (5) pentopyranosides are more l a b i l e than hexopyranosides, (6) a-glycosidic bonds are usually more l a b i l e than 3 - g l y c o s i d i c bonds, (7) residues present as a sidechain are often more e a s i l y hydrolysed than when present as 'in-chain' residues. - 37 -I t i s t h e r e f o r e l o g i c a l t h a t given a h e t e r o p o l y s a c c h a r i d e t h e r e w i l l be some g l y c o s i d i c bonds t h a t are r e l a t i v e l y r e s i s t a n t to a c i d h y d r o l y s i s and o t h e r s t h a t are c o m p a r a t i v e l y s u s c e p t i b l e . The r e s u l t of t h i s w i l l be t h a t under c e r t a i n c o n d i t i o n s of h y d r o l y s i s ( a c i d c o n c e n t r a t i o n , temperature and l e n g t h of h y d r o l y s i s ) d e f i n e d o l i g o m e r i c s u b u n i t s of the p o l y -s a c c h a r i d e may be produced. For a c i d i c p o l y s a c c h a r i d e s c o n t a i n i n g a u r o n i c a c i d moiety, the r e s i s t a n c e of the u r o n o s y l bond u s u a l l y d i c t a t e s t h a t the a l d o b i o u r o n i c a c i d , and to a l e s s e r extent the a l d o t r i o u r o n i c a c i d , w i l l be produced i n r e l a t i v e l y l a r g e p r o p o r t i o n s . In most cases oligomers o b t a i n e d v i a p a r t i a l h y d r o l y s i s are obtained i n very poor y i e l d as those oligomers produced e a r l y on i n the h y d r o l y s i s are s u b j e c t e d to the a c i d and may be h y d r o l y s e d further.. C o n t r o l l e d , continuous removal of the o l i g o s a c c h a r i d e s as they are produced i s p o s s i b l e u s i n g the 59 apparatus of Galanos et aJ_. but the problems a s s o c i a t e d wxth t h i s apparatus and the l a r g e amounts of s t a r t i n g m a t e r i a l a v a i l a b l e d u r i n g t h i s work (5-10 g) made i t more p r a c t i c a l to accept the poor y i e l d s d u r i n g 'one-pot' p a r t i a l h y d r o l y s i s s t u d i e s and t h i s technique was used throughout. A c e t a l s such as pyruvate a c e t a l have been found to s u r v i v e 6 0 d u r i n g p a r t i a l h y d r o l y s i s i n some cases , w h i l e i n other 61 i n s t a n c e s they have been shown to be very l a b i l e . I t would appear t h a t i f the pyruvate a c e t a l spans carbons four and s i x of a g-hexose sugar i t i s moderately s t a b l e as very l i t t l e - 38 -s t e r i c s t r a i n i s i n v o l v e d . When the a c e t a l spans v i c i n a l -t r a n s p o s i t i o n s of a hexose or 6-deoxy hexose, c o n s i d e r a b l e s t r a i n i s encountered. The v i c i n a l - t r a n s a c e t a l s are very a c i d s e n s i t i v e . The two types of a c e t a l s mentioned are i l l u s t r a t e d below. ^COOH c<Vo 0 HO 4, 6-0-(1-carbc D-glucose During p a r t i a l h y d r o l y s i s s t u d i e s on K70, K36 and K32 no oligomers r e t a i n i n g the pyruvate a c e t a l were i s o l a t e d . The l a b i l i t y of the pyruvate a c e t a l present as 4,6-0-(1-carboxy-e t h y l i d e n e ) - g - g l u c o s e i n K36 i s probably because i t i s t e r m i n a l , and hence very a c c e s s i b l e , on a s i d e c h a i n . In K70, and e s p e c i a l l y K32, the pyruvate a c e t a l was found to be very l a b i l e and treatment with 0.01 M t r i f l u o r o a c e t i c a c i d a t 100° f o r 30 min. c l e a v e d i t completely. During p e r i o d a t e o x i d a t i o n s t u d i e s , which are u s u a l l y c a r r i e d out i n a c i d i c c o n d i t i o n s , i t was necessary to b u f f e r the r e a c t i o n medium at a pH as H,0H >xyethylidene)-H,0H 3,4-0- (1-carboxyethylidene) L-rhamnose - 39 -high as 6.5 to prevent h y d r o l y s i s of the pyruvate a c e t a l s i n K7 0 and K3 2. The p a r t i a l l y h y d r o l y s e d p o l y s a c c h a r i d e m a t e r i a l was normally separated by i o n exchange chromatography i n t o a c i d i c and n e u t r a l f r a c t i o n s u s i n g Dowex 1-X2 i n the formate form. The two f r a c t i o n s were then s u b j e c t e d to g e l f i l t r a t i o n and f r a c t i o n s , a f t e r l y o p h i l i s a t i o n , were monitored by paper chromatography (see S e c t i o n s II.9.1 and I I . 9 . 5 . ) . II.8 3 - E l i m i n a t i o n . The a l k a l i n e d e g r a d a t i o n of methylated p o l y s a c c h a r i d e s c o n t a i n i n g u r o n i c a c i d r e s i d u e s has r e c e n t l y ' been u t i l i s e d to achieve s p e c i f i c cleavages i n such p o l y s a c c h a r i d e s . K i s s 6 4 and A s p i n a l l 6 5 have i n v e s t i g a t e d t h i s r e a c t i o n f o r compounds of low molecular weight and an ex t e n s i o n of t h i s work now allows v a l u a b l e sequence data to be obtained f o r p o l y -s a c c h a r i d e s . The b a s i s f o r the cleavage i s o u t l i n e d on page 4 0. The u r o n i c a c i d r e s i d u e s (eg. I) i n the permethylated polymer c a r r y a good l e a v i n g group a t p o s i t i o n f o u r ; e i t h e r a methoxyl group i f the u r o n i c a c i d i s t e r m i n a l and non-reduci n g , or another sugar r e s i d u e . When t r e a t e d with base the s u b s t i t u e n t a t i s e l i m i n a t e d as a r e s u l t of the r e l a t i v e a c i d i t y of the r i n g proton a t C,-. The s t e r e o c h e m i s t r y of t h i s e l i m i n a t i o n makes i t such t h a t D - g a l a c t u r o n i c a c i d r e s i d u e s should e l i m i n a t e more r e a d i l y than the D-gl u c u r o n i c • i 4. 66 e q u i v a l e n t s - 40 -COOMe COOMe The g - g a l a c t u r o n i c a c i d has the proton a t C,- and the l e a v i n g group a t i n a t r a n s d i a x i a l arrangement. The u n s a t u r a t e d u r o n i c a c i d d e r i v a t i v e produced by the e l i m i n a t i o n (II) i s a c i d s e n s i t i v e as i t c o n t a i n s an enol e t h e r . Treatment under m i l d a c i d i c c o n d i t i o n s t h a t are i n s u f f i c i e n t to c l e a v e i n t a c t g l y c o s i d i c bonds w i l l degrade t h i s unsaturated r e s i d u e (II) to g i v e , u l t i m a t e l y , the furan d e r i v a t i v e (IV). During t h i s a c i d treatment the s u b s t i t u e n t a t of the u r o n i c a c i d i s r e l e a s e d and hence the f u r a n d e r i v a t i v e (IV) i s completely e l i m i n a t e d from the p o l y s a c c h a r i d e . A f u r t h e r 3 - e l i m i n a t i o n r e a c t i o n can take p l a c e i f the 4 s u b s t i t u e n t R i n the u r o n i c a c i d (I) i s another sugar, e.g. rhamnose, as when t h i s sugar i s e l i m i n a t e d the r e s u l t i n g r e d u c i n g terminus t h a t i s r e l e a s e d i s s e n s i t i v e to base. Subsequently t h i s sugar r e s i d u e i s a l s o degraded d u r i n g the b a s e - a c i d treatment. (See page 41.) - 41 -CHO An example of t h i s type of d e g r a d a t i o n i s shown i n F i g u r e II.6, page 42, u s i n g K70 as a s u b s t r a t e . The base used to e f f e c t the e l i m i n a t i o n i s e i t h e r sodium methoxide i n methanol or m e t h y l s u l p h i n y l sodium i n d i m e t h y l -sulphoxide. The l a t t e r was used i n t h i s study. I t i s important to note t h a t the methylated p o l y s a c c h a r i d e must be s c r u p u l o u s l y dry as t r a c e s of water w i l l r e s u l t i n p r e f e r -e n t i a l d e - e s t e r i f i c a t i o n of the e s t e r groups when the base i s added and the B - e l i m i n a t i o n w i l l not proceed where f r e e c a r b o x y l i c a c i d s e x i s t . For t h i s reason the methylated polymers - 42 -^GIcVGalV'Rha^GlcAVRha'-^Rha 1. Sodium Borohydride 2. Ethyl Iodide / A g 20 3 . Hydrolysis | 4-0-ethyl-2,3-di-0-methyhL-rhamnose 3,4-di-O-methyl-L-rhamnose 3,4,6-tri-0-methyl-D-glucose i f 5 - d i -0 - e t h y l-2 t4 f 6-tri-O- methyl- D-galdCtitoI F i g u r e I I . 6 Uronic a c i d d egradation of K l e b s i e l l a K70. were s t i r r e d in the appropriate solvent with small quantities of 2,2-dimethoxypropane and toluene-p-sulphonic acid before the base was added. H 2 0 + MeOH CH3COCH3 .+ MeOH The s e n s i t i v i t y of enol ethers to acid conditions makes the cleavage of these bonds r e l a t i v e l y easy compared to the cleavage of gl y c o s i d i c bonds in methylated polysaccharides. The usual hydrolysis conditions involve the use of either 90% formic acid at 40° for 1 hour or 10% aqueous acetic acid at 100° for 1 hour, but stronger conditions, e.g. 50% aqueous acetic at 100°, overnight, have also been used A further use for thi s a l k a l i n e degradation arises when the uronic acid moiety i s present i n the sidechain of a poly-saccharide structure. Following the degradation, i . e . base and then mild acid treatments, a polymer with only a limited number of free hydroxyls w i l l be produced. Reagents such as ruthenium tetroxide (Ru04) are able to oxidise secondary - 44 --^Ga l^Man 1 -^ M a n ^ G l c V a a a o 12 p uronic acid degradation /3 1 GIcA 3 i G i C CH20CH3 CH2OCH3 acid CH2OCH3 CH2OCH3 CH2OCH3 H.OH + products + 1. NaBD4 2. Methylation (CD3D CH2OCH3l CHgOCH, CH 30\£ij/Vy'CH C'H2OCH3 CH.OCD3 CHOCH3 CHOCH3 C;HDOCD3 F i g u r e I I . 7 B - E l i m i n a t i o n d e g r a d a t i o n v i a o x i d a t i o n o f a secondary h y d r o x y l . - 45 -a l c o h o l s to ketones and these f u n c t i o n a l i t i e s may then be u t i l i s e d i n f u r t h e r 3 - e l i m i n a t i o n s t u d i e s . T h i s i d e a i s e x e m p l i f i e d by the work of Lindberg et. a l . on K l e b s i e l l a K28 (see F i g u r e I I . 7 , page 44). A number of advantages a s s o c i a t e d with these e l i m i n a t i o n s based on u r o n i c a c i d or keto f u n c t i o n a l i t i e s are: (1) the r e a c t i o n s are quick and easy to perform; (2) they g i v e s p e c i f i c cleavages and hence r e s u l t s are u s u a l l y e a s i l y i n t e r p r e t e d ; (3) the s p e c i f i c cleavage d e s t r o y s a u r o n i c a c i d moiety--a moiety which i s normally r e s i s t a n t to degradation; (4) v a l u a b l e sequence i n f o r m a t i o n may be o b t a i n e d . The f a c t t h a t not a l l the r e a c t i o n s proceed i n 100% y i e l d i s a disadvantage, e s p e c i a l l y where a number of sugar r e s i d u e s are s u s c e p t i b l e t o the b a s i c c o n d i t i o n s . II.9 S e p a r a t i o n of o l i g o s a c c h a r i d e s o b t a i n e d from d e g r a d a t i o n s . Degradation techniques i n p o l y s a c c h a r i d e s t r u c t u r a l a n a l y s e s do not g i v e a 100% y i e l d of a s i n g l e oligomer. Often i t i s necessary to separate the d e s i r e d product from unreacted (non-degraded) polymeric m a t e r i a l or from contaminating compounds present as a r e s u l t of s i d e r e a c t i o n s . Depending upon whether methylated or non-methylated oligomers r e q u i r e s e p a r a t i o n or p u r i f i c a t i o n , p r e p a r a t i v e procedures such as paper chromato-graphy, paper e l e c t r o p h o r e s i s , l i q u i d chromatography, g e l chromatography or s i l i c a g e l chromatography may be used. - 46 -II.9.1 Gel chromatography. T h i s technique has been used e x t e n s i v e l y i n t h i s study to separate mixtures of products o b t a i n e d from p a r t i a l h y d r o l y s i s , p e r i o d a t e o x i d a t i o n and bacteriophage d e g r a d a t i o n s . While g e l chromatography of carbohydrates i s w e l l e s t a b l i s h e d , a few p o i n t s are worth n o t i n g . When s e l e c t i n g a g e l to perform a p a r t i c u l a r s e p a r a t i o n , e.g. Sephadex G-15 might be used t o separate a mixture of d i , t r i and t e t r a - s a c c h a r i d e s , i t i s important t o r e a l i s e c e r t a i n i o n i c i n t e r a c t i o n s between the g e l matrix and the carbohydrate molecules are o p e r a t i v e . T h i s i s e s p e c i a l l y so when d i s t i l l e d water i s the e l u a n t and u r o n i c a c i d s are present. For example; i f a mixture of D-glucose and L-rhamnose i s separated on a column of Sephadex G-10, the D-glucose w i l l be e l u t e d a l i t t l e b e f o r e the L-rhamnose, but when the same s e p a r a t i o n i s performed u s i n g B i o - G e l P-2 t h i s order i s r e v e r s e d . T h i s e f f e c t can be put to good use and, f o r example, to separate an a l d o t r i o u r o n i c a c i d composed of a D-glucuronic a c i d u n i t and two L-rhamnose sugars from the r e l a t e d a l d o b i o u r o n i c a c i d Sephadex G-15 would be a b e t t e r g e l to s e l e c t than B i o - G e l P-4. When v o l a t i l e b u f f e r s o l u t i o n s , e.g. water, p y r i d i n e , g l a c i a l a c e t i c a c i d (1000:10:4) are used as e l u a n t s the i o n i c i n t e r a c t i o n s experienced with d i s t i l l e d water alone are negated. S e p a r a t i o n s are then based s o l e l y on molecular s i z e . The m o n i t o r i n g of f r a c t i o n s c o l l e c t e d from a g e l column i s very d i f f i c u l t when carbohydrates are being separated as - 47 -d i f f e r e n t i a l refractometry i s the only continuous detection system that may be used. As the quantities of oligomers that may be separated are often small, t h i s means of detection has only l i m i t e d value. Quantitative c o l o r i m e t r i c techniques 6 8 such as the phenol-sulphuric assay may be used to monitor el u t i o n p r o f i l e s but these techniques are tedious to perform. During t h i s work i t was found to be much easier to f i r s t l y o p h i l i s e the i n d i v i d u a l c o l l e c t e d f r a c t i o n s and to then analyse them by paper chromatography—a technique that uses very l i t t l e material and that can achieve very d e l i c a t e separations where overlapping components occur i n an i n d i v i d u a l f r a c t i o n . I t i s possible to separate carbohydrate mixtures that are soluble i n organic solvents by gel chromatography using Sephadex LH-20. However, t h i s technique has found only l i m i t e d application- and has generally been used to p u r i f y large polymeric material, e.g. permethylated or acetalated poly-saccharides, from small, non-volatile reagents. II.9.2 S i l i c a gel chromatography. This type of procedure may be e f f e c t i v e i n separating r e l a t i v e l y small (hexasaccharide and smaller) d e r i v a t i s e d oligomeric mixtures. An example of t h i s involves the separation of two permethylated tetrasaccharides obtained from p a r t i a l hydrolysis studies on K36 (see Section I I I , page 103). - 48 -II.9.3 L i q u i d chromatography. The r a p i d l y d e v e l o p i n g system of high p r e s s u r e l i q u i d chromatography has only r e c e n t l y been- a p p l i e d to the s e p a r a t i o n 69 of carbohydrates . The s e p a r a t i o n of monosaccharides and o l i g o s a c c h a r i d e s appears promising as the time r e q u i r e d to perform these s e p a r a t i o n s i s s h o r t , v i z . , approximately 15 minutes. I t would seem t h a t t h i s procedure i s w e l l s u i t e d to a n a l y t i c a l s e p a r a t i o n s as as l i t t l e as 20 yg of an i n d i v i d u a l sugar can be d e t e c t e d , but p r e p a r a t i v e s e p a r a t i o n s would be e x c e e d i n g l y t e d i o u s u s i n g small columns and e x c e e d i n g l y c o s t l y i f l a r g e r columns were to be used. One group of workers uses l i q u i d chromatography r o u t i n e l y f o r the s e p a r a t i o n and p u r i f i c a t i o n of permethylated o l i g o s a c c h a r i d e s . II.9.4 Gas l i q u i d chromatography ( g . l . c . ) An e x t e n s i v e review of the a p p l i c a t i o n s of g . l . c . to 71 carbohydrates has been p u b l i s h e d . The technique has been widely used f o r the s e p a r a t i o n of d e r i v a t i s e d monosaccharides but has not developed e x t e n s i v e l y as a t o o l f o r the s e p a r a t i o n of o l i g o s a c c h a r i d e s . Of main concern i s the r a p i d decrease i n v o l a t i l i t y as one progresses from monosaccharides t o d i - , t r i - , o l i g o s a c c h a r i d e s , e t c . O l i g o s a c c h a r i d e s must be t r a n s -formed i n t o v o l a t i l e d e r i v a t i v e s and those d e r i v a t i v e s which may be used are the: (1) t r i m e t h y l s i l y l : (2) t r i f l u o r o a c e t y l ; (3) permethyl. - 49 -The t r i f l u o r o a c e t y l derivatives"are the most v o l a t i l e of these 7 2 derivatives but have been used by only a few workers , whereas t r i m e t h y l s i l y l derivatives have found more widespread applica-t i o n . The ease with which the o r i g i n a l s t a r t i n g material can be recovered following d e r i v a t i s a t i o n makes t r i m e t h y l s i l y l derivatives advantageous where only small amounts of material are available. The separation or p u r i f i c a t i o n of permethylated o l i g o -saccharides by g.l.c. i s becoming more widely used. This has been due to the development of s p e c i f i c degradations of 6 3 methylated polysaccharides , the r e a l i s a t i o n that mass spectro-scopy of permethylated oligosaccharides can give valuable sequence information and evidence for ' v o l a t i l e ' terminal glycosides (see Section II.10.2.2., page 63 ), and the increasing a v a i l a b i l i t y of l i q u i d phases for g . l . c . that are stable at quite high temperatures, v i z . , 250-350°. Methyla-ti o n analysis of polysaccharides i s used extensively and g.l.c. techniques for the analysis of p a r t i a l l y methylated sugars (most often as a l d i t o l acetates) are now very sophisticated. Hence i t i s l o g i c a l that these methyl ether derivatives of oligosaccharides should be subjected to g . l . c -m.s. analysis. Permethylated oligosaccharides are not p a r t i c u l a r l y v o l a t i l e and hence stable columns, such as OV-1, have normally been used. For a permethylated trisaccharide a t y p i c a l reten-t i o n time might be 20 min. at 275° (carrier gas; 20 ml/min.). - 50 -For r e d u c i n g oligomers, a and 8 anomers are not u s u a l l y s e p a r a b l e as the permethylated d e r i v a t i v e s . 7 3 II.9.5 Paper chromatography and paper e l e c t r o p h o r e s i s Since the i n i t i a l use of c e l l u l o s e as an ' i n e r t ' support 7 4 i n a d s o r p t i o n chromatography and the subsequent use of 7 5 f i l t e r paper i n p a r t i t i o n chromatography i t c o u l d be s a i d t h a t paper chromatography has r e v o l u t i o n a l i s e d the study of the s t r u c t u r e of carbohydrate polymers. The gre a t value of the method l i e s i n i t s a b i l i t y to separate the components of complex mixtures o f carbohydrates q u i c k l y , simply and a c c u r a t e l y , and with the expenditure of l e s s than a m i l l i g r a m of m a t e r i a l . Often i t i s p o s s i b l e to deduce many c h a r a c t e r -i s t i c s o f a component by paper chromatography alone. The q u a l i t a t i v e examination of a mixture of o l i g o and monosaccharides i s s t i l l best achieved by paper chromatography. Many d i f f e r e n t s o l v e n t systems may be used and the nature of these e l u t i n g s o l v e n t s o f t e n g i v e s u s e f u l i n f o r m a t i o n r e g a r d -ing the nature of the carbohydrate m a t e r i a l , e.g., i n a b a s i c s o l v e n t a c i d i c components w i l l not move from the base l i n e . Although p r e p a r a t i v e paper chromatography has to a l a r g e extent been superceded by g e l f i l t r a t i o n techniques, there have o f t e n been times when p r e p a r a t i v e paper chromato-graphy would have been a simpler and f a s t e r technique. In 7 6 a r e c e n t s t r u c t u r a l a n a l y s i s o f K l e b s i e l l a K62 , p r e p a r a t i v e - 51 -paper chromatography was used as a tool to i s o l a t e r e l a t i v e l y large quantities of aldobio-, t r i - and tetra-uronic acids. 7 7 7 8 Paper electrophoresis ' provides a convenient means for examining certain carbohydrate oligosaccharides and mono-saccharides and an advantage over conventional paper chromato-graphy i s that good separations can be achieved i n a r e l a t i v e l y short period of time (2-4 hours). Buffer pH conditions are chosen so that the materials to be separated e x i s t in a charged state, e.g. at pH 4.5 g-glucuronic acid w i l l e x i s t in i t s ion i c form and w i l l migrate under an e l e c t r i c p o t e n t i a l to the anode. 11.10 Structure analysis of p u r i f i e d oligosaccharides. To f u l l y characterise an oligosaccharide i t i s necessary to determine several features. These are l i s t e d on page 52 using the oligosaccharide (I), shown below, as an example. 3-g-Galp-(1+3)-a-L-Rhap-(1+3)-a-L-Rhap-(1+2)-glycerol. H ( I ) Feature 1) Quantitative composition 2) Configuration of sugar components 3) Anomeric configuration 4) Positions of linkage between sugars 5) Sequence of sugars Data using (I) as an example Determination technique Gal:Rha:glycerol = 1:2:1 a) g.l.c. of a l d i t o l acetates following t o t a l hydrolysis 1 13 b) H and C n.m.r. Gal, g configuration 2xRha, Jj configuration Gal, 3 -D-2xRha, a-L-Gal, terminal non reducing Rha, in-chain linked at C, (two) As shown i n (I) c i r c u l a r dichroism a) H and C n.m.r. b) o p t i c a l rotation a) methylation analysis b) mass spectroscopy a) mass spectroscopy b) periodate oxidation - 53 -No s i n g l e technique can g i v e a l l t h i s i n f o r m a t i o n and depending on the nature of the o l i g o s a c c h a r i d e d i f f e r e n t methods are chosen to demonstrate c e r t a i n c h a r a c t e r i s t i c s . II.10.1 Gas l i q u i d chromatography. The a p p l i c a t i o n of g . l . c . has two a s p e c t s : (a) the q u a n t i t a t i v e d e t e r m i n a t i o n of sugars and aglycones (b) 'the a n a l y s i s o f p a r t i a l l y methylated sugars. The t o t a l h y d r o l y s i s of an o l i g o s a c c h a r i d e i n i t s u n d e r i v a t i s e d s t a t e (see S e c t i o n I I . 3 . , page 21) and subsequent r e d u c t i o n and a c e t y l a t i o n w i l l g i v e a l d i t o l a c e t a t e s of the component sugars. I f an o l i g o s a c c h a r i d e has been obtained v i a p e r i o d a t e o x i d a t i o n the aglycone, e.g. g l y c e r o l , e r y t h r i t o l , 1-deoxy-e r y t h r i t o l , w i l l a l s o be presen t and can be i d e n t i f i e d by g . l . c , but o f t e n these small fragments are p a r t i a l l y l o s t under reduced p r e s s u r e d u r i n g d e r i v a t i s a t i o n . The a l d i t o l a c e t a t e s of the common hexoses v i z . Jj-rhamnose, D-glucose, D-galactose and D-mannose are separable by g . l . c . Two column systems used i n t h i s work are 3% SP-2340 on Supelcoport 100-120 79 and the 'hybrid' column d e v i s e d by Albersheim e t a l . The q u a n t i t a t i o n as determined by peak i n t e g r a t i o n must be c o r r e c t e d u s i n g molar response f a c t o r s (M.R.F.), but the M.R.F. f o r the hexoses are a l l the same (±2%). The a n a l y s i s of p a r t i a l l y methylated sugars, o b t a i n e d from the h y d r o l y s i s of methylated o l i g o s a c c h a r i d e s or p o l y -s a c c h a r i d e s , has been best achieved by g . l . c . The f i e l d has 71 8 0 81 been e x t e n s i v e l y reviewed ' ' . S e v e r a l types of sugar - 54 -d e r i v a t i v e s may be used, e.g. methyl g l y c o s i d e s , a c e t a t e s , i ^ i - j . i *. 84-86 .. , . . 8 7 a l d i t o l a c e t a t e s and a l d o n o n i t r i l e a c e t a t e s . A s e r i o u s disadvantage of d e r i v a t i v e s p r e s e r v i n g the anomeric c e n t r e i s t h a t a s i n g l e methylated sugar may g i v e r i s e to a t l e a s t two, and p o s s i b l y f o u r , peaks on g . l . c , v i z , a-and 3-pyranosides and a- and 8 - f u r a n o s i d e s . During t h i s study a l d i t o l a c e t a t e d e r i v a t i v e s were used e x c l u s i v e l y as each methylated sugar g i v e s r i s e to o n l y a s i n g l e peak on g . l . c . and q u a n t i t a t i o n of peak areas can be made u s i n g response 8 8 f a c t o r s determined by Albersheim et a l . A n a l y t i c a l s e p a r a t i o n s were c a r r i e d out u s i n g v a r i o u s l i q u i d phases. For d i f f e r e n t mixtures of p a r t i a l l y methylated a l d i t o l a c e t a t e s d i f f e r e n t columns gave optimum s e p a r a t i o n . Good s e p a r a t i o n of the three di-O-methyl-L-rhamnose isomers can be achieved using a column of HIEFF-1B but 2, 3,.4, 6 - t e t r a -O-methyl-g-glucose can not be separated from the 2,4-di-0-methyl-g-rhamnose when they c o - e x i s t i n a mixture. To separate the l a t t e r two compounds columns of OS-138 or OV-17 can be used. Medium-polar columns such as B.D.S., OV-225, S i l a r IOC, and SP-1000 g i v e good s e p a r a t i o n s of t r i m e t h y l hexoses and monomethyl 6-deoxy hexoses, but no one column i s guaranteed of s e p a r a t i n g a l l components i n t h i s r e g i o n . P u b l i -8 9 81 c a t i o n s by Albersheim e t a l . and Lindberg et a l . l i s t the r e l a t i v e r e t e n t i o n times f o r a l a r g e v a r i e t y o f p a r t i a l l y methylated a l d i t o l a c e t a t e s . R e t e n t i o n times are u s u a l l y quoted r e l a t i v e to an i n t e r n a l standard such as l , 5 - d i - 0 -a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l - D - g l u c i t o l . - 55 -Column: HIEFF IB (as i n F i g u r e I I . 3 , page 26). Programme: 160° to 180' at 1° per min. 2,3-Rha 2,4-Rha 3,4-Rha TEMPERATURE (°C) F i g u r e II.8 G . l . c . s e p a r a t i o n of mixture of p a r t i a l l y methylated a l d i t o l a c e t a t e s o b t a i n e d from K l e b s i e l l a K32 (pyruvate removed). - 56 -The i d e n t i f i c a t i o n of an i n d i v i d u a l p a r t i a l l y methylated a l d i t o l a c e t a t e can be achieved i n almost a l l cases by co-chromatography with a u t h e n t i c standards and g.l.c.-m.s. (For mass spectroscopy of p a r t i a l l y methylated a l d i t o l a c e t a t e s see S e c t i o n II.10.2.1.) Where some doubt s t i l l e x i s t s a f t e r these two techniques have been f u l l y exhausted, i t i s p o s s i b l e to p r e p a r a t i v e l y c o l l e c t the compound and to do p h y s i c a l measurements on the sample, i . e . m e l t i n g p o i n t ( i f c r y s t a l l i n e ) , XH n.m.r., d e r i v a t i s a t i o n and then m e l t i n g p o i n t . I t i s u s u a l l y a simple matter t o determine the m e t h y l a t i o n p a t t e r n of a component by mass spectroscopy and any ambiguity then l i e s i n whether the parent sugar i s g a l a c t o s e or glucose, e t c . 90 Demethylation and r e a c e t y l a t i o n g i v e c r y s t a l l i n e hexaacetate d e r i v a t i v e s of D - g l u c i t o l and D - g a l a c t i t o l . An example i l l u s t r a t i n g the use of g . l . c . f o r s e p a r a t i n g a mixture of p a r t i a l l y methylated a l d i t o l a c e t a t e s i s shown i n F i g u r e II.8, page 55. (Also see F i g u r e I I . 3 , page 26). Permethylated K l e b s i e l l a K32 (from which the pyruvate a c e t a l had been p r e v i o u s l y removed) was hydrolysed, reduced and a c e t y l a t e d . The chromatogram reproduced i n F i g u r e II.8, shows good s e p a r a t i o n of the three s u b t l y d i f f e r e n t d i m e t h y l -Jj-rhamnose d e r i v a t i v e s . From the chromatogram i t can be con-cluded t h a t K32 i s composed of a t e t r a s a c c h a r i d e r e p e a t i n g u n i t with the u n i t s being as shown, but no data oh the sequence of these u n i t s are i n f e r r e d by t h i s a n a l y s i s . - 57 -As a r e s u l t of v a r i o u s degradation procedures, e.g. u r o n i c a c i d d e g r a d a t i o n , i t i s p o s s i b l e to i s o l a t e o l i g o s a c c h a r i d e s which c o n t a i n only a l i m i t e d number of f r e e h y d r o x y l groups (the r e s t are u s u a l l y p r e s e n t as methyl e t h e r s ) . These f r e e h y d r o x y l groups o f t e n r e p r e s e n t what were l i n k a g e p o s i t i o n s and the ' l a b e l l i n g ' of these hydroxyls p r i o r to subsequent h y d r o l y s i s , r e d u c t i o n and a c e t y l a t i o n can y i e l d v a l u a b l e i n f o r m a t i o n . Use has been made of CD^I-Ag^O to l a b e l these p o s i t i o n s and i n the f i n a l i n s t a n c e the once f r e e h ydroxyl can be d e t e c t e d i n the m.s. of the p a r t i a l l y methylated a l d i t o l a c e t a t e d e r i v e d from the sugar. However, CD^I i s expensive and a l a b e l l i n g technique used on s e v e r a l o c c a s i o n s d u r i n g t h i s work i n v o l v e d e t h e r i f i c a t i o n with E t l - A g 2 0 . The e t h y l group then p r e s e n t on the once f r e e h y d r o x y l group i s observable i n the d e r i v e d p a r t i a l l y e t h e r i f i e d a l d i t o l a c e t a t e s i n two ways:-(1) The e t h y l group (as compared wi t h a methyl group) i s r e l a t i v e l y l e s s p o l a r and hence with p o l a r l i q u i d g . l . c . phases components c o n t a i n i n g e t h y l groups tend to t r a v e l more q u i c k l y than t h e i r methyl analogues. Conversely, u s i n g l e s s p o l a r l i q u i d phases those components wi t h e t h y l groups are c h a r a c t e r i s e d by having longer r e t e n t i o n times i n comparison wi t h t h e i r methyl analogues. (2) The e t h y l group can be d e t e c t e d by m.s. of the d e r i v e d a l d i t o l a c e t a t e of the sugar c a r r y i n g the once f r e e h ydroxyl group. A c h a r a c t e r i s t i c s h i f t of 14 mass u n i t s i s e a s i l y d e t e c t e d . - 58 -The reader i s r e f e r r e d to the a n a l y s i s of the t e t r a -s a c c h a r i d e i s o l a t e d from the u r o n i c a c i d d e g r a d a t i o n of K l e b s i e l l a K70 and i t s subsequent a n a l y s i s f o l l o w i n g e t h y l a -t i o n (see S e c t i o n IV, page 131) f o r an example of t h i s ' l a b e l l i n g ' technique. II.10.2 Mass spectroscopy. The mass spectrometry (m.s.) of o r g a n i c compounds i s based on fragmentation of o r g a n i c molecules under e l e c t r o n impact, and d i f f e r e n t i a t i o n of the r e s u l t i n g p a r t i c l e s by use of the mass-to-charge r a t i o . In order t o produce a n a l y s a b l e ions o f the compound under i n v e s t i g a t i o n s e v e r a l d i f f e r e n t io n sources can be used and these g i v e r i s e to the d i f f e r e n t modes of m.s., v i z . e l e c t r o n impact ( e . i . ) , chemical i o n i s a -t i o n ( c i . ) , f i e l d i o n i s a t i o n ( f . i . ) , and f i e l d d e s o r p t i o n ( f . d . ) . During t h i s work e . i . , c i . and f . d . mass s p e c t r a were recorded and more d e t a i l e d data on these modes are given below. 91 (1) E l e c t r o n impact . This i s the most widely used mode and i n v o l v e s s u b j e c t i n g the compound under i n v e s t i g a t i o n to a beam of e l e c t r o n s (normally with an energy of 70 eV) and the i n t e r a c t i o n between the e l e c t r o n beam and o r g a n i c compound r e s u l t s i n an energy exchange of around 10 to 20 eV. T h i s i s s u f f i c i e n t to cause i o n i s a t i o n of the molecule as the i o n i s a -t i o n p o t e n t i a l of the m a j o r i t y of o r g a n i c substances ranges between 7 and 12 eV, and i n many cases, causes decomposition to s m a l l e r fragment i o n s . E l e c t r o n impact on the molecule - 59 -u s u a l l y r e s u l t s i n e l i m i n a t i o n of one e l e c t r o n , and, thus, i n the formation of a p o s i t i v e l y charged i o n - - t h e s o - c a l l e d "molecular" or "parent" i o n - - d e s i g n a t e d as M +. The molecular i o n i s subsequently i n v o l v e d i n fragmentation and rearrangement r e a c t i o n s to produce "daughter" i o n s . The abundance of any one i o n i s l a r g e l y dependent on i t s s t a b i l i t y as determined by the usual s t r u c t u r a l f e a t u r e s i n o r g a n i c chemistry: e.g., t e r t i a r y i ons and r a d i c a l s are thermodynamically favoured over secondary and primary ones. 92 93 (2) Chemical i o n i s a t i o n ' . A s e r i o u s shortcoming of e . i . m.s. i s t h a t many types of compounds do not g i v e s t r o n g (or any) s i g n a l f o r the molecular i o n . T h i s i s because d u r i n g the i n i t i a l e l e c t r o n - m o l e c u l e i n t e r a c t i o n many molecules r e c e i v e c o n s i d e r a b l e energy above the i o n i s a t i o n v o l t a g e , and the molecular i o n i s consequently q u i c k l y d estroyed by undergoing one or more bond breaking fragmentations. The chemical i o n i s a t i o n process occurs with a much lower t r a n s f e r of energy, and as a n a t u r a l consequence, the fragmen-t a t i o n process i s m o d i f i e d and g r e a t l y reduced. A q u a s i -molecular i o n , formed by l o s s or g a i n of one hydrogen, i s o f t e n the most prominent i o n i n the spectrum. (See page 71 f o r a comparison of a c i . spectrum and e . i . spectrum.) The c i . m.s. r e s u l t s from the ion-molecule r e a c t i o n t h a t occurs between the primary ions of a high p r e s s u r e r e a c t a n t gas and the low pressure sample gas. Both gases are i n t r o d u c e d i n t o the i o n chamber where they are bombarded by an e l e c t r o n beam but v i r t u a l l y a l l primary i o n i s a t i o n due to the bombardment - 60 -occurs with the r e a c t a n t gas. The i o n i s e d r e a c t a n t gas undergoes ion-molecule r e a c t i o n s with i t s e l f to form a steady-s t a t e plasma which i n t u r n r e a c t s c h e m i c a l l y w i t h the d i l u t e sample vapour. A v a r i e t y of r e a c t a n t gases are used f o r c i . , the most common being methane, isobutane and ammonia. I f methane i s the r e a c t a n t gas the most important ions i n the r e a c t i o n plasma are C H 5 + and C 2 H 5 + . CH 4 + + 2e~ CH 3 + + • H C H * D + CH 3 C 2 H 5 + + H2 CH 4 + e — C H 4 + — C H 4 + + CH 4. CH, + + CH .• 3 4 In the presence of a good proton acceptor, the ions C H * and 5 C 2 H 5 a c t a s B r o n s t e d a c i d s and protonate the sample molecule: C H 5 + + BH B H 2 + + CH 4 These r e a c t i o n s are t y p i c a l of those observed f o r a l c o h o l s , aldehydes and e s t e r s . I f the sample m a t e r i a l i s not a good proton a c c e p t o r the chemical i o n i s a t i o n process w i l l occur as a hydride i o n a b s t r a c t i o n : e.g. For decane C 2 H 5 + + C 1 0 H 2 2 * C 1 0 H 2 1 + + C 2 H 6 - 61 -(3) F i e l d d e s o r p t i o n y q ' y D . F i e l d d e s o r p t i o n i s another technique t h a t p r o v i d e s sample i o n i s a t i o n a t r e l a t i v e l y low energy w i t h r e s u l t a n t reduced fragmentation and much i n c r e a s e d abundance of the parent i o n . In f a c t , i n many f . d . m.s. the molecular i o n i s o f t e n the onl y major peak. In the f . d . source a very high p o s i t i v e e l e c t r i c f i e l d ( i n the range of 7 8 10 -10 volts/cm) i s produced between a t h i n e m i t t e r wire, t h a t i s coated with n e e d l e - l i k e carbon 'whiskers', and the f i r s t s l i t i n the spectrometer. The high e l e c t r i c f i e l d s t r e n g t h induces e l e c t r o n t u n n e l l i n g through a p o t e n t i a l energy b a r r i e r i n the molecule, and the r e s u l t i n g p o s i t i v e i o n i s a c c e l e r a t e d out of the chamber and i n t o the mass spectrometer a n a l y s e r . The sample to be i n v e s t i g a t e d i s put d i r e c t l y onto the e m i t t e r w i r e and u n l i k e e . i . , c i . , and f . i . sources where the sample has to be va p o u r i s e d b e f o r e i t enter s the probe, the sample coated e m i t t e r wire i s put d i r e c t l y i n t o the probe. For very non v o l a t i l e compounds i t i s sometimes necessary to g e n t l y heat the e m i t t e r wire i n order to produce i o n s . The energy a v a i l a b l e f o r f . d . and subsequent e x c i t a t i o n of a molecule i s about 10-13 eV and hence many o r g a n i c mole-c u l e s w i l l have very l i t t l e excess energy i n the parent i o n to cause fragmentation. F r e q u e n t l y q u a s i - m o l e c u l a r ions such as (M+H)+ are observed due to a s u r f a c e r e a c t i o n of the sample with adsorbed water on the e m i t t e r . Carbohydrates i n the u n d e r i v a t i s e d s t a t e are t h e r m a l l y u n s t a b l e and p r a c t i c a l l y non v o l a t i l e , and hence mass s p e c t r a l - 62 -s t u d i e s i n the past have been performed on the more v o l a t i l e d e r i v a t i v e s , such as, methyl e t h e r s , a c e t a t e s and 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 . The r e c e n t advances i n f . i . and f . d . mass spectroscopy do however, now a l l o w mass s p e c t r a l s t u d i e s on the u n d e r i v a t i s e d carbohydrates. During the course of t h i s work mass spectroscopy was employed f o r two d i s t i n c t purposes: the a n a l y s i s o f p a r t i a l l y methylated a l d i t o l a c e t a t e s i n order to a s s i g n m e t h y l a t i o n p a t t e r n s and i n some cases parent sugar i d e n t i t y ; and the a n a l y s i s of o l i g o s a c c h a r i d e s . . 11.10.2.1 A n a l y s i s of p a r t i a l l y methylated a l d i t o l . . 86, 96, 97 a c e t a t e s . The components obtained from m e t h y l a t i o n analyses of p o l y s a c c h a r i d e s , i . e . , p a r t i a l l y methylated a l d i t o l a c e t a t e s , may be r e a d i l y examined by m.s. and i n p a r t i c u l a r , g.l.c.-m.s. The mass s p e c t r a l a n a l y s i s of these compounds i s a r o u t i n e technique performed i n the e . i . mode, and c o n s i d e r a b l e data are a v a i l a b l e on t h e i r fragmentation. The p a r t i a l l y methylated a l d i t o l a c e t a t e s fragment upon i o n i s a t i o n and no molecular ions are seen. The fragmentation pathways f o r a s p e c i f i c compound are d i c t a t e d by the methyla-t i o n p a t t e r n i n the molecule as s c i s s i o n of the carbon-carbon bonds occur with c e r t a i n p r e f e r e n c e s which are o u t l i n e d below. H-C-0CHo H-C-0CH-, H-C-OAc > > H-C-OCH, H-C-OAc H-C-OAc i J i i - 63 -Secondary fragments are derived from the primary fragments by single or consecutive loss of acetic acid (M.W. 60), ketene (M.W. 42), methanol (M.W. 32), or formaldehyde (M.W. 30). On reduction, some pairs of methylated sugars, e.g., 3-0-methyl- and 4-0-methyl-hexose, give r i s e to a l d i t o l acetates with the same su b s t i t u t i o n pattern. This problem can be over-come i f the reduction i s ca r r i e d out with sodium borodeuteride, (see below). CHDOAc CHDOAc I I HC-OAc HC-OAc 1 - — | 2 6 1 CH-.0-CH , o n AcO-CH J x y u j HC-OAc HC-OCH., | 262 |_ J HC-OAC HC-OAC I I CH2OAc CH2-OAc 189 The prominent primary peaks of various methylated a l d i t o l acetates are compiled i n Table I, page 64. I t should be r e a l i s e d that mass spectrometry w i l l not d i s t i n g u i s h between diastereomeric p a r t i a l l y methylated a l d i t o l acetates, e.g., the a l d i t o l acetates of 2,3,4,6-tetra-0-methyl-D-galactose and 2,3,4,6-tetra-0-methyl-D-glucose w i l l give to a l l intents and purposes, i d e n t i c a l mass spectra. II.10.2.2 Analysis of oligosaccharides. Oligosaccharides and monosaccharide glycosides have been • . . ^ 98, 99 ^ . . . . ,100-103 examined by e . i . m.s. as acetate , t r i m e t h y l s i l y l - 64 -Table I I . 1 . Primary Fragments i n the Mass Spe c t r a o f P a r t i a l l y Methylated Sugars i n the Form of T h e i r A l d i t o l A c e t a t e s . P o s i t i o n m/e Of CH 3 45 59 89 117 131 161 175 189 203 205 217 233 Pentoses 2 (4) X 3 X 2,3(3,4) X X 2,4 X X 2,5 X X X 3,5 X X X 2,3,4 X X 2,3,5 X X X Hexoses 2(5) X 3 (4) X 6 X 2,3 X 2,4(3,5) X X 2,5 X 2,6 X ' X 3,4 X 3,6 X X X 4,6 X X 5,6 X X 2,3,4 X X X X 2,3,5 X X 2,3,6 X X X 2,4,6 X X X X 2,5,6 X X X 3,4,6 X X X 2,3,4,6 X X X X 2,3,5,6 X X X X 6-Deoxyhexoses 2 X 3 X X 4 X 2,3 X X 2,4 X X X 3,4 X X 2,3,4 X X X X 2,3,5 X X X X X X X X X X - 65 -and permethylated d e r i v a t i v e s 1 1 1 4 ± u b # while the s p e c t r a of a c e t a t e d e r i v a t i v e s are somewhat complex due to the f a c t t h a t an a c e t o x y l group can be e l i m i n a t e d i n f o u r d i f f e r e n t modes, and 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 s u f f e r from the d i s -advantage of i n c u r r i n g a l a r g e i n c r e a s e i n mass over the parent sugar, the on l y inconvenience of permethylated d e r i v a -t i v e s i s t h a t they are more d i f f i c u l t t o make. Since i t i s common p r a c t i c e to permethylate o l i g o s a c c h a r i d e s d u r i n g s t r u c t u r a l s t u d i e s i t i s n a t u r a l t h a t they be analysed by mass spectrometry. Since fragmentation of permethylated o l i g o s a c c h a r i d e s proceeds i n an analogous manner to t h a t o f permethylated mono-sa c c h a r i d e g l y c o s i d e s , i t i s convenient to look c l o s e l y a t the d e g r a d a t i o n pathways i n the l a t t e r b e f o r e c o n s i d e r i n g the more complex o l i g o s a c c h a r i d e s . Using permethylated g-glucose as an example, F i g u r e I I . 9 , page 66, o u t l i n e s the most important d e g r a d a t i o n pathways f o r the monosaccharide d e r i v a -104 t i v e . The nomenclature employed by Kochetkov and Chizhov i s used throughout. The M +-ion i s a l s o degraded i n t o the H-and K - s e r i e s of fragments having low masses. The s e r i e s A-K r e p r e s e n t fundamental modes of fragmentation f o r a l l g l y c o s i d e s . S u b s t i t u e n t s , or such m o d i f i c a t i o n s as the i n t r o d u c t i o n of deoxy groupings, can a l t e r the r e l a t i v e importance of p a r t i c u l a r 107 s e r i e s of fragments. Uronic a c i d d e r i v a t i v e s f o l l o w the same major breakdown pathways. - 66 -F i g u r e I I . 9 Degradation pathways of permethylated D-glucose d u r i n g e l e c t r o n impact ( e . i . ) mass spectroscopy. (From J . Lonngren and S. Svensson. Adv. Carbohyd. Chem. Biochem., 2 9 , 4 2 ( 1 9 7 4 ) ) . - 67 -For permethylated o l i g o s a c c h a r i d e s the nomenclature f o r d e g r a d a t i o n pathways i s a l i t t l e more complex and i s i l l u s t r a t e d below f o r the degradation of a d i s a c c h a r i d e methyl g l y c o s i d e . baA, In t h i s scheme baA^ denotes t h a t the i o n has been formed by cleavage of r i n g b f o l l o w i n g pathway A, and i s s u b s t i t u t e d w i t h r i n g a. The A s e r i e s of fragments serves to e s t a b l i s h the molecular weight of the o l i g o s a c c h a r i d e and i t s component sugar r e s i d u e s . The B s e r i e s of fragments o b t a i n e d by degrada-t i o n of r i n g b can be used to e s t a b l i s h the nature of the l i n k a g e between the two sugar r e s i d u e s (see F i g u r e 11.10, page 68 ) . For o l i g o s a c c h a r i d e a l d i t o l s , which o f t e n a r i s e from p e r i o d a t e o x i d a t i o n degradations, the mass s p e c t r a l fragmenta-t i o n s are somewhat simpler. The non-reducing sugar i s i d e n t i f i e d by the A s e r i e s of fragments, and the a l d i t o l p a r t by a fragment assumed t o be produced by the D sequence of fragmentation (see F i g u r e 11.11, page 69 ) . To i l l u s t r a t e the i n f o r m a t i o n o b t a i n a b l e by e . i . m.s. of an o l i g o s a c c h a r i d e a l d i t o l the spectrum of a component obtained - 68 -F i g u r e 11.10 Fragmentations d u r i n g e.i.m.s. of permethylated d i s a c c h a r i d e s v i a the B pathway — s h o w i n g the nature of the l i n k a g e between two sugar r e s i d u e s . - 69 -© CHjOMe MeO MeO CH 2OMe CH,OMe HCOMe I CILOMe CHjOMe I HCOMe I HCOMe v ^ 1 S^ O - ^ - C H © HCOMe I CH,OMe CH,OMe HCOMe I HCOMe ®CH I HCOMe I CH,OMe F i g u r e II.11 C h a r a c t e r i s t i c fragmentations of permethylated o l i g o s a c c h a r i d e a l d i t o l s i n e.i.m.s. from K l e b s i e l l a K70 by a u r o n i c a c i d d egradation (see S e c t i o n IV, page 130) i s shown i n F i g u r e 11.12, page 70. Some of the major fragments are a s s i g n e d . For more d e t a i l e d i n f o r m a t i o n r e g a r d i n g e . i . m.s. of permethylated o l i g o s a c c h a r i d e s the 108 reader i s r e f e r r e d to p u b l i c a t i o n s by Moore and Waight , , v.. , , . 109-111 and Karkkainen E . i . m.s. can be c o n s i d e r e d a r o u t i n e technique i n most l a b o r a t o r i e s but c i . m.s. and f . d . m.s. are modes l e s s w idely F i g u r e 1 1 . 1 2 The e l e c t r o n impact mass spectrum of a permethylated t r i s a c c h a r i d e a l d i t o l . - 71 -100 80 60 to 20 FIELD DESORPTION T6T 200 300 400 500 600 100i 80 R E L . so INT. % 401 20 100 260 CHEMICAL IONISATION 30~0~ tOO 500 600 100r 80 60 to 20 ELECTRON IMPACT 100 200 300 tiT m / e 500 600 F i g u r e I I . 13 Mass spectra of a disaccharide a l d i t o l obtained using electron impact, chemical ionisation and f i e l d desorption modes. - 72 -a v a i l a b l e to most chemists. During the l a t e r stages of the p r e p a r a t i o n of t h i s t h e s i s i t was p o s s i b l e to have some s p e c t r a run u s i n g these 'more g e n t l e ' methods on a non-routine b a s i s . Shown i n F i g u r e 11.13, page 71, are the e . i . , c i . and f . d . s p e c t r a of a permethylated o l i g o s a c c h a r i d e w i t h the s t r u c t u r e shown below: CH 2OMe CH 2OMe CHOMe 187<H219)! 361*-(393) MW=556 147 11.10.3 Nuclear magnetic resonance. The use of n.m.r. as an i n v e s t i g a t i v e t o o l has been d i s -cussed i n terms of p o l y s a c c h a r i d e s i n S e c t i o n I I . 2 . The technique has a l s o been used e x t e n s i v e l y d u r i n g t h i s work to c h a r a c t e r i s e o l i g o s a c c h a r i d e s . Often o n l y small q u a n t i t i e s of pure oligomers are o b t a i n e d from d e g r a d a t i v e s t u d i e s and t h i s n o n - d e s t r u c t i v e technique g i v e s v a l u a b l e i n f o r m a t i o n . For XH n.m.r. as l i t t l e as 2 mg may be s u f f i c i e n t t o g i v e a good spectrum but f o r X^C n.m.r. as much as 15 mg may be necessary to g i v e a reasonable s i g n a l to n o i s e r a t i o . There i s no problem w i t h v i s c o s i t y when working with s m a l l o l i g o -s a c c h a r i d e s and D 20 exchange can be performed much more r e a d i l y . The data t h a t can be o b t a i n e d from n.m.r. s t u d i e s are q u i t e e x t e n s i v e and t h i s i s e x e m p l i f i e d u s i n g the d i s a c h a r i d e g l y c o s i d e shown on page 73. - 73 -3-g-Galp- (1- 2)-a-L-Miap- ( 1 + 2 ) - C j - e r y t h r i t o l . Shown i n F i g u r e s 11.14 and 11.15, pages 74, 75, are both the "^H n.m.r 13 and C n.m.r. s p e c t r a of thxs u n d e r i v a t i s e d oligomer. The "^H n.m.r. spectrum shows the presence of one 6-deoxy sugar (L-rhamnose), and two non-reducing anomeric s i g n a l s . Moreover, the chemical s h i f t and c o u p l i n g c o n s t a n t s of these anomeric protons i n d i c a t e the presence of an a-L-rhamnose moiety and a 13 B-g-hexose ( g a l a c t o s e ) . The C n.m.r. spectrum, a l t h o u g h not 13 i n t e g r a t e d , shows s i g n a l s from 16 C n u c l e i o v e r a l l . T h i s immediately i s a good i n d i c a t i o n f o r a fo u r - c a r b o n fragment 13 as the aglycone on the 'reducing' terminus. A l s o i n the C n.m.r. spectrum, s i g n a l s from a 6-deoxy methyl group (6 17.35 p.p.m.), three primary hydroxyls (61.75, and 63.19 p.p.m.), two ' l i n k a g e ' carbons (80.26, 80.94 p.p.m.) and two non-reducing anomeric s i g n a l s (100.19, 105.75 p.p.m.) can be C 2 Rha C3 erythritol C 6 of Gal C-j,C^of erythritol S.W. 2000 Hz AT. 1.023 sec RW. 16Lisec RD. 0 sec C L 58,000 61.75 63.19 acetone 31.07 C 6 of Rha 17.35, —1 F i g u r e 11.14 C n.m.r. spectrum of 8-g-Galp- (1+2)-a-^-Rhap- (1+2) - g - e r y t h r i t o l . (See page 73.) HOD dc-Rha Solvent D 2 0 Temp. 90° S.W. 500 Hz (acetone) T 7.77 CH 3 of Rha \T8.69 I S  F i g u r e 11.15 H n.m.r. spectrum of B-D_-Galp- (1+2) -a-L-Rhap- (1+2) - g - e r y t h r i t o l (See page 73.) - 76 -observed. (From the chemical s h i f t s of the anomeric carbon n u c l e i s i g n a l s i t i s u s u a l l y s a f e to assume t h a t s i g n a l s to h igher f i e l d of 102 p.p.m. are from a - l i n k a g e s w h i l e those to lower f i e l d are from 8 - l i n k a g e s . For t h i s d i s a c c h a r i d e 13 37 g l y c o s i d e a p a r t i a l l y coupled C spectrum confirmed t h i s assignment: 8-D-Gal, J-, 162 Hz; a-L-Rha, J - , 1 7 2 Hz.) C-H ~ JC-H The evidence f o r the oligomer being a non-reducing d i s a c c h a r i d e , comprising a hexose sugar and a 6-deoxy hexose sugar, w i t h a f o u r carbon fragment on the r e d u c i n g terminus i s q u i t e s u b s t a n t i a l . The evidence f o r the g l y c o s i d e p o r t i o n i s of p a r t i c u l a r i n t e r e s t as these s m a l l fragments can o n l y be 'observed' by h y d r o l y s i s s t u d i e s on the u n d e r i v a t i s e d oligomer and subsequent paper chromatography or g . l . c , or by mass spectroscopy. For the l a t t e r technique c i . m.s. on the permethylated d e r i v a t i v e or f . d . m.s. on the u n d e r i v a t i s e d sample would y i e l d molecular i o n s , but these analyses can not be c o n s i d e r e d ' r o u t i n e ' a t t h i s time. II.10.4 Determination of D- or L - c o n f i g u r a t i o n of a sugar r e s i d u e . The D- or L - c o n f i g u r a t i o n of i n d i v i d u a l sugars can be determined by t h e i r s p e c i f i c o xidases (e.g., g - g l u c o s i d a s e and D - g a l a c t o s i d a s e ) or by the s i g n of t h e i r c i r c u l a r dichroisrn curves as measured on s u i t a b l e d e r i v a t i v e s . For the l a t t e r , measurements at 213 my on a l d i t o l a c e t a t e s or p a r t i a l l y methylated a l d i t o l a c e t a t e s i n a c e t o n i t r i l e gave s p e c t r a which were - 77 -compared with those o b t a i n e d from a u t h e n t i c s a m p l e s 1 1 ^ . These components were r o u t i n e l y c o l l e c t e d by p r e p a r a t i v e g . l . c . 11.11 .Immunochemical methods. When an a n t i g e n i s i n j e c t e d i n t o an animal, e.g. a horse, the immune system of t h a t organism i s s t i m u l a t e d i n t o producing a n t i b o d i e s to c o u n t e r a c t the 'invading' a n t i g e n . The b a c t e r i a l p o l y s a c c h a r i d e s p r o v i d e a r i c h source of a n t i g e n i c m a t e r i a l , and s i n c e they are o f t e n the p r i n c i p a l a n t i g e n i c determinants of the parent micro-organisms, the c o r r e s p o n d i n g a n t i s e r a are a l s o f r e q u e n t l y a v a i l a b l e . Hence the c r o s s - r e a c t i o n of a p o l y s a c c h a r i d e of unknown or u n c e r t a i n chemical s t r u c t u r e with a n t i b o d i e s to a p o l y s a c c h a r i d e of known s t r u c t u r e may y i e l d i n f o r m a t i o n as to one or more sugars c o n t a i n e d i n the unknown and even as to the p o s i t i o n s a t which the sugars are l i n k e d . Conversely, c r o s s - r e a c t i v i t y of a p o l y s a c c h a r i d e of known s t r u c t u r e w i t h a n t i b o d i e s to a p o l y s a c c h a r i d e of unknown com-p o s i t i o n and l i n k a g e may be e q u a l l y i n f o r m a t i v e . 113 H e i d e l b e r g e r has developed methods u t i l i s i n g the p r e c i p i t a t i o n r e a c t i o n s of antibody ( p r e c i p i t i n ) and a n t i g e n . T h i s immunochemical r e a c t i o n i s c a l l e d the p r e c i p i t i n t e s t . H e i d e l b e r g e r et a l _ . 1 1 4 1 1 6 have examined the c r o s s r e a c t i o n s between approximately 60 of the s e r o l o g i c a l l y d i s t i n c t K l e b s i e l l a K-types 1+80 and a n t i s e r a to some s e l e c t e d Pneumo-coccus p o l y s a c c h a r i d e s . The degree of c r o s s - r e a c t i o n , as measured by the amount of p r e c i p i t a t i o n , i s an i n d i c a t i o n of - 78 -the degree of s i m i l a r i t y of the structure of the unknown to the structure of the polysaccharide used to e l i c i t the immune serum for the t e s t . A series of p r e c i p i t i n tests has to be performed to deduce the fin e p a r t i a l structure of the unknown. Cross reactions, when cle a r cut in t e r p r e t a t i o n i s possible, can y i e l d s t r u c t u r a l information that could take months to obtain by purely organic chemical means. However, the complete structures of many of the Pneumococcus poly-saccharides are not known and some strongly p o s i t i v e cross reactions between Pneumococcus antisera and K l e b s i e l l a polysaccharides cannot be interpreted f u l l y . Many examples ex i s t where t h i s ' s e r o l o g i c a l ' technique has given useful information in determining a K l e b s i e l l a structure but unfortunately few p o s i t i v e reactions were observed for the K-types studied i n t h i s work, v i z . K-types 32, 36 and 70. For c l a r i t y , however, an example using K l e b s i e l l a K5 i s outlined below: 117 K5 polysaccharide p r e c i p i t a t e d antisera to Pneumo-114 coccus type III and Pneumococcus type VIII polysaccharides The structures of these Pneumococcus polysaccharides are not completely known but the p a r t i a l structures are +3)-8-g-GlcAp-(1+4)-8 (?)-D-Glcp-(1+ Pneumococcus I I I 1 1 8 +4) -B-rJ-GlcApj- (1+4) -8-D-Glcp- (1+4) -cx-D Glcp-(1+4)-a-g-Galp-(1+ Pneumococcus V I I I 4 0 - 79 -A common f e a t u r e of these two Pneumococcus p o l y s a c c h a r i d e s i s the presence of the c e l l o b i o u r o n o s y l r e s i d u e s i . e . 8-g-GlcAp-(1+4)-D-Glcp 114 I t was suspected t h a t the r e p e a t i n g u n i t f o r K l e b s i e l l a K5 would encompass t h i s s t r u c t u r a l f e a t u r e and t h i s was i n f a c t found to be the case when the s t r u c t u r a l d e t e r m i n a t i o n of K5 was completed. The s t r u c t u r e of K l e b s i e l l a K5 i s shown below. • 4 ) -0-D-GlcAp- d+4) -B-p-Glcp- (1+3)-a-D-Manp-(1 " 2 " 6 ~ 4 \ / OAc C CE^ \ : O O H As more i n f o r m a t i o n on the K l e b s i e l l a p o l y s a c c h a r i d e s becomes a v a i l a b l e , and b e a r i n g i n mind the d i v e r s i t y of these s t r u c t u r e s determined to date, i t w i l l be most l i k e l y p o s s i b l e to deduce the f i n e r d e t a i l s o f many of the complex, and to date incomplete, Pneumococcus p o l y s a c c h a r i d e s t r u c t u r e s . - 80 -11.12 Bibliography for Sections I and II. 1. W.F. Dudman and J.F. Wilkinson, Biochemical J., 62, 289 (1956). 2. E.H. Kabat, Blood Group Substances, Their Chemistry and Immunochemistry, New York, Academic Press, Inc., 1956. 3. T. Hubscher and A.H. Eisen, Internat. Arch. Allergy and Applied Immunology, 42_, 466 (1972) . 4. W. Nimmich, Z. Microbiol. Immunol., 154, 117 (1968). 5. W. Nimmich, Acta B i o l . Med. 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Nimmich, Immunochem. , 13_, 67 (1976). 117. G.G.S. Dutton and M.T. Yang, Can. J. Chem., 5_0, 2382 (1972). 118. R.E. Reeves and W.F. Goebel, J. B i o l . Chem., 139, 511 (1941). - 88 -I I I . THE STRUCTURE OF K l e b s i e l l a SEROTYPE K36 ' CAPSULAR POLYSACCHARIDE - 89 -I I I . l A b s t r a c t K l e b s i e l l a K36 c a p s u l a r p o l y s a c c h a r i d e has been i n v e s t i -gated u s i n g m e t h y l a t i o n , Smith-periodate o x i d a t i o n and p a r t i a l h y d r o l y s i s t echniques. The s t r u c t u r e was shown to c o n s i s t of a hexasaccharide repeat u n i t as shown below. The anomeric c o n f i g u r a t i o n s of the sugar u n i t s were determined by perform-1 1 3 i n g H and C n.m.r. spectroscopy on i s o l a t e d oligomers o b t a i n e d d u r i n g the d e g r a d a t i v e s t u d i e s on the i n t a c t p o l y -s a c c h a r i d e . +3)-3-D-Galp- (1+3) -a-Jj-Rhap- (1+3)-a-L-Rhap- (1+2) -a-L-Rhap- (1 2" " ~ 1 3-g-GlcAp 4 1 B-g-Glcp pyruvate III.2 I n t r o d u c t i o n The b a c t e r i a l genus K l e b s i e l l a i s d i v i s i b l e i n t o a p p r o x i -mately 80 d i f f e r e n t serotypes which are d i s t i n g u i s h e d by the 1 2 s t r u c t u r e of t h e i r c a p s u l a r p o l y s a c c h a r i d e s . Nimmich ' has analysed q u a l i t a t i v e l y the p o l y s a c c h a r i d e from each s t r a i n 3 w hile H e i d e l b e r g e r and Nimmich have summarized the s t r u c t u r e s of the c a p s u l a r p o l y s a c c h a r i d e s which have been determined t o date. - 90 -Many of the s t r a i n s so f a r examined possess capsules of d i f f e r e n t q u a l i t a t i v e compositions. There are, however, s e v e r a l l a r g e groups of K l e b s i e l l a b a c t e r i a which have c a p s u l e s w i t h the same q u a l i t a t i v e a n a l y s i s but w i t h d i f f e r e n t s e r o l o g i c a l r e a c t i o n s . One such group i n c l u d e s the s t r a i n s of K-types K12, K36, K45, K55 and K70 whose ca p s u l e s are composed of the sugars D-glucuronic a c i d , D-galactose, D-glucose and L-rhamnose. In an attempt to e x p l a i n these s e r o l o g i c a l d i f f e r e n c e s on a s t r u c t u r a l b a s i s , t h i s group i s now being examined i n d e t a i l and the s t r u c t u r e f o r K-type K36 here presented r e p r e s e n t s the f i r s t s t r a i n examined i n t h i s s e r i e s . I I I . 3 R e s u l t s and D i s c u s s i o n . Composition and n.m.r. s p e c t r a . K l e b s i e l l a K36 p o l y s a c c h a r i d e was prepared on an agar medium and p u r i f i e d by one p r e c i p i t a t i o n with C e t a v l o n . The p'roduct had [a] -56°. E l e c t r o p h o r e s i s showed the m a t e r i a l to be homogeneous. The 1H n.m.r. spectrum of the p o l y s a c c h a r i d e i n D^O a t 90° (see Appendix I I I , spectrum No. 1) showed a sharp 4 s i n g l e t at T 8.41 i n d i c a t i v e of a pyruvate a c e t a l . T h i s s i g n a l was p r e s e n t i n a r a t i o of 1:3 with the 6-deoxy s i g n a l due to rhamnose at T 8.67. In the anomeric r e g i o n f i v e d i s c e r n a b l e s i g n a l s were observed a t T 4.76, 2H, J , 9 broad; x 4.98, IH, J± 2 2 Hz; T 5.13, IH, 2 7 Hz; x 5.30, IH, - 91 -2 7 * 5 H z ' T 5.50, IH, J 1 2 broad. These chemical s h i f t s 5 6 indicate ' that the repeating unit contains six monosaccharide units, three of which are a-linked and three 8-linked. More precise assignment of these signals was achieved aft e r study-ing the XH n.m.r. spectra of the oligosaccharides obtained by p a r t i a l hydrolysis; see below and Table I I I . l , page 92. 1 13 The H n.m.r. analysis was confirmed by C n.m.r. spectro-scopy of the polysaccharide (160 mg/2 ml) which showed high f i e l d peaks at 17.6 p.p.m. (rhamnose CH^) and 25.5 p.p.m. (pyruvate CH^); in the anomeric region only f i v e signals could be distinguished, at 101.4, 101.8, 102.7, 103.9 and 105.1 p.p.m. Two other signals were att r i b u t a b l e to the C-6 carbons of hexoses and a further two signals at 173.2 and 174.2 p.p.m. were due to the carboxyl groups of the pyruvate acetal and the glucuronic acid (see Appendix II I , spectrum No. 2). I t 1 13 i s thus clear that H and C spectra provide complementary 7 information . The two sets of data show the K36 polysaccharide to be composed of three units of rhamnose, two of hexose, one uronic acid, one pyruvate acetal and having three a- and three 3 - g i y c o s i d i c linkages. Acid hydrolysis of the polysaccharide showed by paper chromatography the presence of glucose, galactose, glucuronic acid and rhamnose. Carboxyl reduced K36 was hydrolysed and the presence of g-glucose, D-galactose and Lj-rhamnose in the r a t i o of 2:1:3 was confirmed by gas l i q u i d chromatography (g.l.c.) of the i r a l d i t o l acetates. The configurations of Table I I I . l Data on K l e b s i e l l a K36 C a p s u l a r P o l y s a c c h a r i d e and D e r i v e d O l i g o s a c c h a r i d e s Compound Repeating u n i t o f compound T (Hz) R a t i o i n t e g r a l s Proton assignment 1 2 3 GlcA-^—Rha-OH 4 GlcA^^-Rha^^-Rha-OH 5 G l c ^ - ^ G l c A i ^ R h a i - ^ R h a - O H ~ P 8 a 6b 1 -Rha-2 3 1 G: .cA a 1 Gal^^-Rha^—^-Rha^—^glycerol ~ P a a ^ J 1 3 1 2 2 R h a — — R h a — - g l y c e r o l 5.37 (7) 1 6-GlcA 4.63(1.8) 0.6 a-Rha-OH 5.17(S) 0.4 8-Rha-OH 8.70(J C r 6Hz) 3 CH 3 of Rha 5.34 (7) 1 8-GlcA 4.65(2) 1 a-Rha 4.89(2) 0.6 a-Rha-OH 5.13 (S) 0.4 6-Rha-OH 8.70(J C _ 5, 6 6Hz) 6 CH 3 of Rha 5.19 (7.5) 1 8-Glc 5.35 (7) 1 3-GlcA 4.63(2b) 1 a-Rha 4.89 (1.8) 0.6 a-Rha-OH 5.13(S) 0.4 6-Rha-OH 8.70(J C . b, b 6Hz) 6 CH 3 of Rha 5. 21(7.5) 1 6-Gal 5.32 (7) 1 3-GlcA 4. 71(2b) 1 a-Rha 4.91 (2) 0.6 a-Rha-OH 5.13(S) 0.4 8-Rha-OH 8.70 (J_ . 5 / o 6Hz) 6 CH 3 of Rha 5.34(7.5) 1 3-Gal 5.01 (1.8) 1 a-Rha 4.92(1.8) 1 a-Rha 8.70(J C -b, 6 6Hz) 6 CH 3 of Rha 4.94(2) 1 a-Rha 5.03(2) 1 a-Rha 8 . 7 0 ( J 5 / 6 6Hz) 6 CH 3of Rha c o n t- ' r Table I I I . l - continued Compound Repeating u n i t of compound T R a t i o Proton ( H ) i n t e g r a l s • assignment 3 1 3 1 3 1 3 1 ^ a l V ^ R h a ^ - i R h a — R h a — „ a a 6 B 1' GlcA 4 6 1 Glc pyr 5.50(7 b) 1 5. 35(7) 1 5.15(7.5) 1 5. 02(b) 1 4.84(b) 2 8.41 (S) 3 8.67(J C , b) 9 B-Gal B-GlcA B-Glc a-Rha (three) CH, of pyruvate CH 3 of Rha S h i f t s quoted r e l a t i v e to acetone i n t e r n a l standard (x7.77). J c o u p l i n g c o n s t a n t s i n Hz are given i n b r a c k e t s . S i n g l e t s are d e s i g n a t e d as S and where i t was not p o s s i b l e to get an accurate J t h i s i s i n d i c a t e d w i t h b. - 94 -the g l u c i t o l hexaacetate and r h a m n i t o l pentaacetate, as determined by c i r c u l a r d i c h r o i s m ( c d . ) , were shown to be g- and j j - r e s p e c t i v e l y , while t h a t of the g a l a c t o s e was a l s o g- based on the c d . of the p a r t i a l l y methylated 2 , 4 , 6 - t r i -O-methyl-D-galactitoi t r i a c e t a t e o b tained d u r i n g m e t h y l a t i o n s t u d i e s (see l a t e r ) . M e t h y l a t i o n of o r i g i n a l and autohydrolysed p o l y s a c c h a r i d e s . M e t h y l a t i o n " ^ of K36 p o l y s a c c h a r i d e and subsequent reduc-t i o n w i t h l i t h i u m aluminum hydride, h y d r o l y s i s , d e r i v a t i s a t i o n 1 1 1 2 as a l d i t o l a c e t a t e s and g.l.c.-m.s. a n a l y s i s ' i n d i c a t e d t h a t K36 i s composed of a hexasaccharide repeat u n i t (Table I I I . 2 , column I, page 96 ). The presence of a monomethyl rhamnose r e s i d u e i s a t t r i b u t a b l e to a branch p o i n t , but the absence of any t e r m i n a l r e s i d u e ( e i t h e r t e t r a m e t h y l hexose or t r i m e t h y l rhamnose) i n d i c a t e s the pyruvate a c e t a l must be p r e s e n t on a t e r m i n a l s i d e c h a i n sugar (see F i g u r e I I I . l , page 95 , f o r g . l . c . t r a c e ) . A sample of K l e b s i e l l a K36 p o l y s a c c h a r i d e was auto-h y d r o l y s e d a t pH 2.2 overnight., A "*"H n.m.r. spectrum of the recovered n o n - d i a l y z a b l e polymer showed the absence of pyruvate a c e t a l and of r e d u c i n g protons, c o n s i s t e n t with a product having s t i l l a h i g h degree of p o l y m e r i s a t i o n (see Appendix I I I , spectrum No. 3). In the anomeric r e g i o n s i g n a l s observed had the same chemical s h i f t as f o r the unde -graded K36 p o l y s a c c h a r i d e but the s i g n a l a t T 5.13, J 1 0 7 Hz H I E F F - 1 B A 2,3-GLC 2,4-RHA 4-2,4,6-GAL RHA 3,4-RHA | I65 165 175 I85 195 TEMPERATURE (°C) B OV-17 2,4-RHA 3,4-RHA 2,3,4-GLC-f- 2,4,6-GAL 4-RHA 2,3,4,6-Gld 2,3-GLC 175 175 185 195 205~ TEMPERATURE (°C) Ul Figure I I I . l G.l.c. separation of p a r t i a l l y methylated a l d i t o l acetates obtained from: A, native K36 polysaccharide; B, degraded K36 polysaccharide. For column d e t a i l s see Table III.2, page 96. - 96 -Table III.2 Methylation Analyses of Origi n a l and Degraded K36 Capsular Polysaccharide Methylated (as A l d i t o l Sugars 3 Acetates) T b Mole % C ,d Column (HIEFF A 6 IB) Column Bf (OV 17) Column C g (OV 225) I II III 3,4-Rha 0.87 0.79 0. 90 16. 6 17. 0 20.9 2,4-Rha 1. 00 0. 91 0.96 16. 3 19.7 20. 6 2,3,4,6-Glc 1. 00- 1. 00 1.00 10. 6 8.4 4-Rha 1.45 1.24 1.40 16.9 20.0 19. 0 2,4,6-Gal 1.77 1. 59 1.68 20.3 ) 17. 5 25.0 2,3,4-Glc 1.77 1. 59 1.77 \ 6.2 2,3-Glc 2. 32 2.00 2.56 30.0 7.5 7.4 "3,4-Rha = 1,2,5-tri-0-acetyl-3,4,-di-O-methyl-L-rhamnitol, etc. Retention time r e l a t i v e to the a l d i t o l acetate deri v a t i v e of 2,3,4,6-tetra-O-methyl-D-glucose. I, o r i g i n a l polysaccharide, column A; II, degraded polysaccharide (see text for d e t a i l s ) , column B; III , degraded polysaccharide, column C. Values corrected using 'Effective carbon response' molar-response 13 e factors . Programme; 165° for 8 minutes and than 2° per minute to 200°. "^Programme; 175° for 8 minutes and then 2° per minute to 210°. ^Programme; 180° for 8 minutes and then 2° per minute to 200°. - 97 -d i d not i n t e g r a t e as a complete proton; the reason f o r t h i s i s e x p l a i n e d below. M e t h y l a t i o n of t h i s degraded m a t e r i a l and r e d u c t i o n w i t h l i t h i u m aluminum hydride f o l l o w e d by d e r i v a t i s a t i o n and g . l . c -m.s. a n a l y s i s (Table I I I . 2, columns I I , I I I , page 96 ) gave a complex mixture o f components which c o u l d not be separated completely on any one g . l . c . column. A n a l y s i s o f the mixture on columns of OV 17, OV 225 and HIEFF IB (see F i g u r e I I I . l , page 95) enabled a l l peaks to be r e s o l v e d . The presence of a t e r m i n a l glucose u n i t demonstrates t h a t the pyruvate a c e t a l i s a t t a c h e d to t h i s r e s i d u e i n the o r i g i n a l p o l y s a c c h a r i d e . The r e l a t i v e molar r a t i o s o f 2,3,4-tri-O-methyl-D-glucose and 2 , 3 , 4,6-tetra-O-methyl-D-glucose i n d i c a t e t h a t the D-gluc-u r o n i c a c i d r e s i d u e i s a l s o p r e s e n t i n the s i d e c h a i n and t h a t the t e r m i n a l g l u c o s i d i c bond i s being p a r t i a l l y c l e a v e d (^40%) d u r i n g the a u t o h y d r o l y s i s ; t h i s i s c o n s i s t e n t w i t h the XH n.m.r. spectrum. P e r i o d a t e O x i d a t i o n . 14 P e r i o d a t e o x i d a t i o n of K36 p o l y s a c c h a r i d e proceeded r a p i d l y with 3 moles of p e r i o d a t e being consumed per mole of repeat u n i t a f t e r 25 hours ( t h e o r e t i c a l = 3 moles). The con-sumption g r a d u a l l y i n c r e a s e d to a value of 4.2 moles per mole of repeat u n i t over a f u r t h e r 4 8 hours. By XH n.m.r. of the sodium borohydride reduced p e r i o d a t e product i t was e v i d e n t t h a t p a r t of t h i s slow i n c r e a s e was due to the h y d r o l y s i s o f the pyruvate a c e t a l a t the r e a c t i o n pH of 3.0. - 98 -Smith hydrolysis"""" 1 of the sodium borohydride reduced p e r i o d a t e product (3 mole uptake) u s i n g 0.5 ^ t r i f l u o r o a c e t i c a c i d a t room temperature f o r 16 hours and subsequent r e d u c t i o n again with sodium borohydride y i e l d e d a mixture which was r e s o l v e d by g e l chromatography (Bio-Gel P-4). A s i g n i f i c a n t l y l a r g e amount of polymeric m a t e r i a l was e l u t e d s h o r t l y a f t e r the v o i d volume f o l l o w e d by an o l i g o s a c c h a r i d e d e r i v a t i v e , oligomer 1.. T h i s incomplete h y d r o l y s i s , u s i n g t r i f l u o r o a c e t i c a c i d to e f f e c t the Smith d e g r a d a t i o n , of a p o l y o l d e r i v e d from 16 these c a p s u l a r p o l y s a c c h a r i d e s was observed f i r s t d u r i n g 17 s t u d i e s on K18 and has been noted subsequently i n the case 18 of K55. There are e a r l i e r r e p o r t s on incomplete h y d r o l y s i s 19 20 21 a t t r i b u t e d to u r o n i c ' a c i d or e s t e r s u l f a t e groups, but i t i s p a r t i c u l a r l y s u r p r i s i n g i n the th r e e b a c t e r i a l examples c i t e d t h a t i n each case i t i s an o x i d i s e d rhamnose u n i t which i s r e s i s t a n t to the a c i d c o n d i t i o n s used. Oligomer 1 ([a] -60°) was o b t a i n e d pure and, a f t e r h y d r o l y s i s and paper chromatography, was shown to c o n t a i n rhamnose,galactose and g l y c e r o l . XH n.m.r. of 1 (see Appendix I I I , spectrum No. 4) i n d i c a t e d (Table .111.1, page 92) the presence of th r e e non-reducing anomeric s i g n a l s ; one 6 hexose s i g n a l at x 5.34 (J.. „ 7.5 Hz) a t t r i b u t a b l e to the D-galactose and two f u r t h e r s i g n a l s a t x 5.01 (J, „ 1.9 Hz) and x 4.92 13 ( J ^ 2 1>8 Hz) due to two a-J^-rhamnose s i g n a l s . C n.m.r. of 1^  (see Appendix I I I , spectrum No. 5) gave a spectrum w i t h 21 carbon s i g n a l s which i n d i c a t e d t h a t i n a d d i t i o n to the - 99 -t h r e e hexose sugars a three carbon fragment ( g l y c e r o l ) was present on the 'reducing' terminus. Attempted a c e t o l y s i s of 1 i n an e f f o r t to remove the small aglycone ( g l y c e r o l ) 2 2 p r e f e r e n t i a l l y was u n s u c c e s s f u l . Smith p e r i o d a t e d e g r a d a t i o n of 1 and g e l chromatography of the r e s u l t i n g m a t e r i a l y i e l d e d oligomer 2 ([a] -67°) which gave o n l y rhamnose and g l y c e r o l on h y d r o l y s i s . In the "^H n.m.r. spectrum (see Appendix I I I , spectrum No. 6) the o n l y anomeric s i g n a l s corresponded to two a-L-rhamnose protons (non-reducing) a t T 4.94 (J, „ 2 Hz) and T 5.03 (J, „ 1, 2. i., 2. 2 Hz) . M e t h y l a t i o n a n a l y s i s of £ gave 2 , 3, 4 - t r i - 0 - m e t h y l - L -rhamnose and 2,4-di-0-methyl-L-rhamnose (the v o l a t i l e 1,3-di-O-methylglycerol d e r i v a t i v e was l o s t under reduced p r e s s u r e d u r i n g work up). Mass s p e c t r a of f u l l y methylated 2 u s i n g e l e c t r o n impact ( e . i . ) , chemical i o n i s a t i o n ( c i . ) and f i e l d 23 d e s o r p t i o n (f.d.) modes a l s o confirmed the presence of two 6-deoxy hexoses and a g l y c e r o l moiety. A parent peak a t m/e 482 was o b t a i n e d i n the f . d . spectrum. The c i . spectrum a l s o gave a parent peak (M-l) a t m/e 481 and the o r i g i n s of other major peaks i n the spectrum are shown on page 100. The s t r u c t u r e of 2 i s thus e s t a b l i s h e d as a-L-Rhap- (1+3)-a-L-Rhap- (1+2) - g l y c e r o l (2) and t h a t of 1 as B-D-Galp-(1+3)-a-L-Rhap-(1+3)-a-L-Rhap-(1+2)-g l y c e r o l (1) - 100 -The m e t h y l a t i o n a n a l y s i s of the o r i g i n a l . a n d autohydrolyzed polymers i n d i c a t e s t h a t the s i d e c h a i n i s the d i s a c c h a r i d e 1 4 u n i t D-Glc —g— D - G l c A — and i t i s the glucose u n i t t h a t c a r r i e s the 4,6-0-(1-carboxyethylidene) a c e t a l . I t i s thus now p o s s i b l e to deduce t h a t the g l y c e r o l fragment i n 1 above o r i g i n a t e d from a 2 - s u b s t i t u t e d J^-rhamnose r e s i d u e . Hence, the backbone of K36 i s e s t a b l i s h e d as + 3) -3-D-Galp- (1+3) -a-L-Rhap- (1+3 ) -a-L-Rhap- (1+2)-a-Jj-Rhap- (1+ Attachment of s i d e c h a i n Having determined the sequence of the t e t r a s a c c h a r i d e backbone, the nature of the s i d e c h a i n and t h a t the sugar on - 101 -which branching occurs y i e l d s 4-0-methyl-L-rhamnose i n the me t h y l a t i o n a n a l y s i s , the only remaining problem i s to i d e n t i f y which of the t h r e e rhamnose u n i t s c o n s t i t u t e s the branch p o i n t . T h i s was achieved by examination of the products of p a r t i a l h y d r o l y s i s . 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 s from p a r t i a l h y d r o l y s i s P a r t i a l h y d r o l y s i s of K36 p o l y s a c c h a r i d e (2 M t r i f l u o r o -a c e t i c a c i d , 3 h, 95°) and s e p a r a t i o n o f a c i d i c and n e u t r a l components by i o n exchange y i e l d e d a mixture of a c i d i c oligomers which were separated by g e l permeation chromato-graphy (Bio-Gel P-4) . Three pure oligomers (3^ £, 5) were obtained i n d e c r e a s i n g q u a n t i t i e s . Compound 3^  [ a ] D -12°, by 1H and 1 3 C n.m.r. (see Tab l e I I I . l , page 92) was shown to c o n t a i n one non-reducing 3 anomeric s i g n a l and two r e d u c i n g anomeric s i g n a l s a t t r i b u t a b l e to a 6-deoxy hexose (see Appendix I I I , s p e c t r a No.'s. 7, 8). Me t h y l a t i o n , l i t h i u m aluminum hydride r e d u c t i o n , h y d r o l y s i s , and g . l . c . o f the a l d i t o l a c e t a t e s gave 2,3,4-tri-0-methyl-g-glucose and 3 , 4-di-O-methyl-Jj-rhamnose. The s t r u c t u r e of 3 i s thus e s t a b l i s h e d as 5-E)-GlcAp- (1+2) -L-Rhap (3) Compound 4, [ a ] D -31.3°; 1R n.m.r. (see Table I I I . l , page 92) i n d i c a t e d i n the anomeric r e g i o n the presence of one 8 non-- 102 -r e d u c i n g s i g n a l , one a non-reducing s i g n a l , and two s i g n a l s due to a r e d u c i n g 6-deoxy hexose (see Appendix I I I , s p e c t r a No.'s 9, 10). M e t h y l a t i o n of £ and subsequent l i t h i u m aluminum hy d r i d e r e d u c t i o n , h y d r o l y s i s and d e r i v a t i s a t i o n as a l d i t o l a c e t a t e s gave, i n a d d i t i o n to the two components obt a i n e d from .3, 2 , 4-di-O-methyl-L-rhamnose. Compound £ i s t h e r e f o r e e s t a b l i s h e d as B-D-GlcAp-(1+2)-a-L-Rhap-(1+3)-L-Rhap (4) Compound £ was ob t a i n e d i n on l y very s m a l l q u a n t i t y . "'"H n.m.r. (see Table I I I . l , page 92) i n d i c a t e d the presence o f two non-reducing 3 anomeric s i g n a l s , one a non-reducing anomeric s i g n a l , and two s i g n a l s a t t r i b u t a b l e to a r e d u c i n g 6-deoxy hexose (see Appendix I I I , spectrum No. 11). Methyla-t i o n a n a l y s i s (as d e s c r i b e d f o r compounds 3^  and 4_) y i e l d e d 2,3,4,6-tetra-O-methyl-g-glucose, 2,3-di-O-methyl-D-glucose, 2,3-di-O-methyl-L-rhamnose and 3,4-di-O-methyl-L-rhamnose i n equal p r o p o r t i o n s . Having e s t a b l i s h e d the s t r u c t u r e of i t i s p o s s i b l e to a s s i g n the s t r u c t u r e of !5 as 3-g-Glcp-(1+4) -3-g-GlcAp-(1+2)-a-L-Rhap-(1+3)-L-Rhap (5) A p a r t i a l h y d r o l y s i s of K36 u s i n g 0.3 M t r i f l u o r o a c e t i c a c i d f o r 3 hours at 95° was a l s o performed. A f t e r s e p a r a t i o n of a c i d i c and n e u t r a l components the a c i d i c compounds were - 103 -separated by g e l permeation chromatography. S u c c e s s f u l r e s o l u t i o n over the e n t i r e range of oligomers, i . e . r e s i d u a l polymeric m a t e r i a l to monomers, was not achieved i n i t i a l l y but a f t e r removal of most 'polymeric 1 f r a c t i o n s and r e -chromatography of the s m a l l e r oligomers (Bio-Gel P-4) good s e p a r a t i o n was achieved. In a d d i t i o n to compounds .3 and 4 a s m a l l amount of another oligomer j5 was o b t a i n e d . A "*"H n.m.r. spectrum of 6^  (see Appendix I I I , spectrum No. 12) i n d i c a t e d the presence of s i x sugar components. Reduction of £ with sodium b o r o d e u t e r i d e and subsequent m e t h y l a t i o n gave two components (R 0.7 3 and R_, 0.57) which — r — r were separated on s i l i c a g e l ( e t h y l a c e t a t e : e t h a n o l , 92:8). The component with R p 0.73 was analysed and e s t a b l i s h e d as being i d e n t i c a l to the permethylated d e r i v a t i v e of 5. The compound with R^ 0.57 was analysed s i m i l a r l y and y i e l d e d 2,3,4-tri-O-methyl-D-glucose, 2,3,4,6-tetra-0-methyl-g-galactose, 4-0-methyl-L-rhamnose and 1,2,4,5-tetra-0-methyl-g-rhamnitol. Again, the l a t t e r component was monodeuterated at . The s t r u c t u r e of t h i s component (6b) i s t h e r e f o r e shown to be -D-Galp-(1+2)-a-L-Rhap-(1+3)-L-Rhap 3 ' 1 B-D-GlcAp (6b) - 104 -Hence the i s o l a t e d oligomer £ was not one pure hexasaccharide as o r i g i n a l l y thought but was a mixture of two t e t r a s a c c h a r i d e s which c o e l u t e d i n g e l permeation chromatography and had i d e n t i -c a l R F values on paper chromatography with the two s o l v e n t systems used. The i s o l a t i o n of f i v e d i f f e r e n t y e t compatible oligomers from K l e b s i e l l a K36 i s i n agreement with the s t r u c t u r e being as shown below. The cleavage p a t t e r n of t h i s p o l y s a c c h a r i d e on p a r t i a l a c i d h y d r o l y s i s i s markedly i n f l u e n c e d , as i n d i c a t e d by the very low y i e l d s of any oligomers w i t h the g a l a c t o s y l bond i n t a c t , by the l i a b i l i t y of the th r e e l i n k e d g a l a c t o s y l bond. Of the K l e b s i e l l a s t r u c t u r e s known to t h i s time K28, having a s i x sugar repeat u n i t w i t h a 2 u n i t s i d e c h a i n of a 24 t e r m i n a l g l u c o s e and a non t e r m i n a l g l u c u r o n i c a c i d , i s the c l o s e s t analogue to K36. 3 •g-Galp^^^-Rhap^^L-Rhap^-^L-Rhap-3 1 D-GlcAp 4 -3 C00H r 1 C D Glcp - 105 -III.4 Experimental General Methods Descending paper chromatography was c a r r i e d out u s i n g Whatman No. 1 paper and the f o l l o w i n g s o l v e n t systems (v/v) were used: (A) F r e s h l y prepared 1 - b u t a n o l - a c e t i c a c i d -water (2:1:1); (B) e t h y l a c e t a t e - p y r i d i n e - water (8:2:2). 25 Chromatograms were developed u s i n g s i l v e r n i t r a t e . Con-c e n t r a t i o n s were c a r r i e d out under reduced p r e s s u r e a t bath temperatures which d i d not exceed 40°. A n a l y t i c a l g . l . c . s e p a r a t i o n s were performed u s i n g a Hewlett Packard 5700 instrument f i t t e d with dual flame i o n i s a t i o n d e t e c t o r s . An I n f o t r o n i c s CRS-100 e l e c t r o n i c i n t e g r a t o r was used to measure peak areas. S t a i n l e s s s t e e l columns 1/8 i n c h o u t s i d e diameter were used with a c a r r i e r gas flow r a t e of 20 m£/min. Columns used were (A) 3% of HIEFF IB on Gas Chrom Q (100/ 120 mesh); (B) 3% of OV 17 on the same support; (C) 3% of OV 225 on the same support; (D) 0.2% of p o l y e t h y l e n e g l y c o l s u c c i n a t e , 0.2% of p o l y e t h y l e n e g l y c o l a d i p a t e , 0.4% of XF 1150 on the same support. A l l columns were 6 f e e t i n l e n g t h . P r e p a r a t i v e g . l . c . was performed u s i n g an F and M model 720 instrument with d u a l thermal c o n d u c t i v i t y d e t e c t o r s . Columns (6 f e e t by 1/4 inch) analogous to those used f o r a n a l y t i c a l s e p a r a t i o n s were used. G.l.c.-m.s. was c a r r i e d out u s i n g a Micromass 12 instrument f i t t e d with a Watson-Biemann s e p a r a t o r . S p e c t r a were recorded at 70 eV w i t h an i o n i s a t i o n c u r r e n t of 100 uA and an i o n source temperature of 200°. Other e l e c t r o n - 106 -impact m.s. were run on a MS 902 instrument w h i l e chemical i o n i s a t i o n s p e c t r a (of o l i g o s a c c h a r i d e d e r i v a t i v e s ) were recorded on a F i n n i g a n 3200 quadrapole mass spectrometer. "^H n.m.r. s p e c t r a were run on a V a r i a n XL-100 instrument. Samples run i n D 20 were exchanged and f r e e z e d r i e d three or four times i n 99.9% D^O and then f i n a l l y d i s s o l v e d i n 100% D 20. Acetone (T 7.77, measured a g a i n s t aqueous sodium '2,2-dimethyl-2-silapentane-5-sulfonate (DSS)) was used as an i n t e r n a l standard. S p e c t r a were recorded a t approximately 90°. Spectra of methylated d e r i v a t i v e s were run u s i n g CDCl^ as s o l v e n t w i t h an i n t e r n a l Me^Si standard. 13 C n.m.r. s p e c t r a were ob t a i n e d u s i n g a V a r i a n CFT-20 i n s t r u -ment and were run a t ambient temperature i n 50% D 20 u s i n g acetone (31.07 p.p.m. from DSS) as i n t e r n a l standard. C i r c u l a r d i c h r o i s m s p e c t r a were recorded on a Jasco J20 automatic r e c o r d i n g s p e c t r o p o l a r i m e t e r . O p t i c a l r o t a t i o n s were measured at 23 ± 2° on a Perkin-Elmer model 141 p o l a r i m e t e r u s i n g a 10 cm c e l l . IR s p e c t r a were recorded u s i n g a P.E. 457 s p e c t r o -photometer. Gel permeation chromatography was c a r r i e d out u s i n g a column (2.5 x 120 cm) of B i o - G e l P-4 (minus 400 mesh). The column was i r r i g a t e d with water a t a flow r a t e of a p p r o x i -mately 7 ml/h. F r a c t i o n s (1-2 ml.) were c o l l e c t e d , f r e e z e d r i e d and chromatographed on paper. P r e p a r a t i o n and p r o p e r t i e s of K36 c a p s u l a r p o l y s a c c h a r i d e . A c u l t u r e of K l e b s i e l l a K36 (8306) was o b t a i n e d from Dr. I. 0rskov, Copenhagen, and was grown on a medium of 8 g - 107 -NaCl, 4 g K2HPC>4, 1 g MgS0 4'7H 20, 2 g CaCC>3, 120 g sucrose and 8 g Bacto yeast extract, i n 4 £ of water f o r 4 days. The c e l l s were harvested and d i l u t e d to 1600 ml with water con-t a i n i n g 1% phenol. T h i s s o l u t i o n was then c e n t r i f u g e d f o r 6 hours a t 30,000 r.p.m. i n a Beckman T4 zo n a l r o t o r . A f t e r t h i s time the c l e a r supernatant was separated and co n c e n t r a t e d to approximately 400 ml. Crude p o l y s a c c h a r i d e , o b t a i n e d by p r e c i p i t a t i o n i n t o e t h a n o l (2 I), was r e d i s s o l v e d i n 400 ml. of water, p r e c i p i t a t e d with 10% Cetavlon, r e d i s s o l v e d i n 4 M NaCl (500 ml.) and then d i a l y s e d a g a i n s t tap water o v e r n i g h t . L y o p h i l i z a t i o n of t h i s s o l u t i o n y i e l d e d 10 g of the p o l y s a c c h a r i d e , [ a ] Q -56° (c 3.4, water). C a r b o x y l - r e d u c t i o n o f the n a t i v e p o l y s a c c h a r i d e . A sample of the p o l y s a c c h a r i d e was reduced u s i n g the g procedure d e s c r i b e d by T a y l o r and Conrad . Two treatments were r e q u i r e d to achieve complete r e d u c t i o n as estimated by t i t r a t i o n . One 'treatment' i s d e s c r i b e d as f o l l o w s . The p o l y s a c c h a r i d e (approximately 1 g) i n the Na s a l t form was d i s s o l v e d i n approximately 100 ml of d i s t i l l e d water and the pH a d j u s t e d to 4.75 by the a d d i t i o n of 0.1 M HCl. A ten f o l d molar excess (over the t o t a l moles of c a r b o x y l groups a v a i l a b l e f o r r e d u c t i o n i n the p o l y s a c c h a r i d e ) o f 1 - c y c l o h e x y l -3 - ( 2 - m o r p h o l i n o e t h y l ) c a r b o d i i m i d e metho-p-toluene sulphonate (8 g) was added as the s o l i d . As the r e a c t i o n proceeded the pH began to r i s e and t h i s was compensated f o r by t i t r a t i o n to - 108 -pH 4.7 5 with the 0.1 M HCl. A f t e r approximately 5 hours the pH remained almost constant i n d i c a t i n g the c o u p l i n g r e a c t i o n was complete. A t o t a l of 14.8 ml of 0.1 ^ HCl had been con-sumed. NaBH^ (120 ml of a 2 M s o l u t i o n ) was then added w h i l e the pH was kept between 5 and 7 by the simultaneous a d d i t i o n o f 4 ^ HCl. During t h i s p a r t of the procedure o c t a n o l was used as an antifo a m i n g agent and a g e n t l e stream of a i r was d i r e c t e d a t the r e a c t i o n s u r f a c e to a l s o reduce foaming. The 'reduced 1 s o l u t i o n was d i a l y s e d a g a i n s t running tap water f o r two days and subsequently l y o p h i l i s e d . Sugar and m e t h y l a t i o n a n a l y s i s o f n a t i v e p o l y s a c c h a r i d e . H y d r o l y s i s o f a sample (20 mg) of carboxyl-reduced K36 with 2 ^ t r i f l u o r o a c e t i c a c i d a t 95° o v e r n i g h t and subsequent d e r i v a t i s a t i o n of the l i b e r a t e d monosaccharides as a l d i t o l a c e t a t e s gave Jj-rhamnitol pentaacetate, D - g a l a c t i t o l hexaacetate (m.p. 168°) and g - g l u c i t o l hexaacetate (m.p. 99°) i n the r a t i o of 2.98:1.00:2.01. (Column D; programmed a t 120° f o r 8 minutes and then l°/min t o 200°.) C i r c u l a r d i c h r o i s m of the rh a m n i t o l MeCN pentaacetate showed ^213 ~ 1*12 and the g l u c i t o l hexaacetate MeCN e2±2 + x*^3* C i r c u l a r d i c h r o i s m of 2,4,6-tri-0-methyl-D-g a l a c t i t o l t r i a c e t a t e o b t a i n e d from the t o t a l h y d r o l y s i s of methylated K36, was shown t o be p o s i t i v e . M e t h a n o l y s i s of K36 p o l y s a c c h a r i d e u s i n g r e f l u x i n g 3% methanolic hydrogen c h l o r i d e o v e r n i g h t y i e l d e d methyl a-L-rhamnopyranoside; (m.p. 108 - 110°). - 109 -M e t h y l a t i o n of K36 u s i n g Hakomori c o n d i t i o n s f o l l o w e d by a subsequent P u r d i e treatment y i e l d e d a product which showed no h y d r o x y l a b s o r p t i o n i n the i . r . spectrum. The Hakomori m e t h y l a t i o n was performed as f o l l o w s . The p o l y s a c c h a r i d e (^ 1 g ) , i n the ' f r e e a c i d ' form, was c a r e f u l l y d r i e d under a heat lamp and h i g h vacuum o v e r n i g h t . In a 250 ml round bottom f l a s k f i t t e d with a rubber serum cap and c o n t a i n i n g a s t i r r e r bar, the p o l y s a c c h a r i d e was d i s s o l v e d i n 50 ml of d r i e d and d i s t i l l e d d imethyl sulphoxide. The f l a s k was f l u s h e d with dry n i t r o g e n . Methyl s u l p h i n y l anion (20 ml of a 2 M s o l u t i o n ) was then added and the r e a c t i o n l e f t to pro-ceed with s t i r r i n g f o r approximately 6-8 hours. A f t e r t h i s time a s m a l l p o r t i o n of the r e a c t i o n mixture was removed and t e s t e d w i t h triphenylmethane to ensure excess methylsulphiny1 anion was present. Methyl i o d i d e (10 ml) was then added w h i l e the r e a c t i o n was kept a t 4° i n an i c e bath. A f t e r 0.5 hours the s o l u t i o n was d i a l y s e d a g a i n s t running tap water f o r approximately 20 hours and l y o p h i l i s e d . Y i e l d ; 900 mg. The Purdie m e t h y l a t i o n was performed as f o l l o w s . The m a t e r i a l o b tained from the Hakomori treatment (above) was d i s s o l v e d i n 15 ml of methyl i o d i d e and the s o l u t i o n was r e f l u x e d f o r 4 8 hours, d u r i n g which time 4 x 0.5 g of Ag 20 was added. The Ag^O was then separated by c e n t r i f u g a t i o n and r e f l u x e d i n 150 ml of CHCl^ f o r 3 hours and removed by c e n t r i f u g a t i o n again. The combined supernatants (Mel and CHCl^) were evaporated and y i e l d e d 550 mg of the f u l l y methylated p o l y s a c c h a r i d e . The methylated p o l y s a c c h a r i d e , as shown by i . r . and "*"H n.m.r. - 110 -(CDCl^)f c o n t a i n e d i m p u r i t i e s and was p u r i f i e d by p r e c i p i t a -t i o n i n t o petroleum ether (30-60°). The p u r i f i e d methylated m a t e r i a l was reduced w i t h l i t h i u m aluminum h y d r i d e i n r e f l u x i n g t e t r a h y d r o f u r a n o v e r n i g h t and, f o l l o w i n g h y d r o l y s i s with 2 M t r i f l u o r o a c e t i c a c i d a t 95° f o r 8 hours, the mixture was reduced with sodium borohydride and then a c e t y l a t e d . G . l . c . (column A; programmed a t 160° f o r 4 minute and then 1° per minutes to 190°) and m.s. of the c o l l e c t e d components allowed t h e i r assignment as i n column I, Table I I I . 2 , page 96. M e t h y l a t i o n a n a l y s i s of degraded p o l y s a c c h a r i d e . A u t o h y d r o l y s i s a t pH 2.2 f o r 16 hours a t 95° gave a very s o l u b l e polymer of K36 which, a f t e r d i a l y s i s a g a i n s t running tap water o v e r n i g h t and l y o p h i l i z a t i o n , was examined by "*"H n.m.r. ( D 2 0 ' 90°, see Appendix I I I , spectrum No. 3). The degraded m a t e r i a l was methylated (as above) and subsequently reduced w i t h l i t h i u m aluminum hy d r i d e i n r e f l u x i n g t e t r a h y d r o -f u r a n o v e r n i g h t . H y d r o l y s i s , r e d u c t i o n and a c e t y l a t i o n , as performed on the n a t i v e p o l y s a c c h a r i d e , f o l l o w e d by g . l . c . a n a l y s i s showed the presence of seven components. (See columns II and I I I , Table I I I . l , page 9 2 ) . A column of OV 17 (column B; programmed at 170° f o r 4 minutes and then l°/min to 190°) achieved complete s e p a r a t i o n of 2,3,4,6-tetra-0-methyl-D-glucose and 2,4-di-O-methyl-L-rhamnose, w h i l e the s e p a r a t i o n of 2 , 3 , 4-tri-O-methyl-D-glucose and 2 , 4 , 6 - t r i - O - m e t h y l - r j -g a l a c t o s e was achieved on OV 225 (column C; programmed at 170° - I l l -f o r 8 minutes and then 2° per minute to 200°). P e r i o d a t e O x i d a t i o n . Capsular p o l y s a c c h a r i d e (1 g) was d i s s o l v e d i n 250 ml of 27 a s o l u t i o n o f NaI0 4 (0. 05 Mj and NaC10 4 (0.2 M) . The pH of t h i s s o l u t i o n was 2.7. The s o l u t i o n was kept i n the dark at 4°. A f t e r 20 hours 3 moles of p e r i o d a t e per repeat u n i t had been consumed ( t h e o r e t i c a l = 3), r i s i n g to 4.5 moles a f t e r 172 hours. F o l l o w i n g the a d d i t i o n of et h y l e n e g l y c o l (2 ml) a f t e r 25 hours, r e d u c t i o n w i t h sodium borohydride, d i a l y s i s , ^ d e i o n i s a t i o n , l y o p h i l i z a t i o n and removal of borate, the pro-duct was h y d r o l y s e d (0.5 M t r i f l u o r o a c e t i c a c i d ) a t room temperature o v e r n i g h t . A f t e r removal of the a c i d and sub-sequent r e d u c t i o n w i t h sodium borohydride the m a t e r i a l (2 x 175 mg) was a p p l i e d to the top of a B i o - G e l P-4 column (2.5 x 120 cm). E l u t i o n w i t h water gave a range of polymeric products appearing soon a f t e r the v o i d volume (blue dextran) f o l l o w e d by pure oligomer 1 which had R Q 1 c 1-0 ( s o l v e n t A). Oligomer 1, 35 mg, had [a]_ -60.7° (c 2.5, water) and 1 13 was examined both by H n.m.r. ( D2^' 90°) and C n.m.r. spectroscopy (see Appendix I I I , S p e c t r a No. 1s 4,5). H y d r o l y s i s of 1 (0.5 M t r i f l u o r o a c e t i c a c i d , 4 hours, 95°) showed by paper chromatography the presence o f g a l a c t o s e , rhamnose and g l y c e r o l . P e r i o d a t e o x i d a t i o n of 1 (12 mg) i n 0.05 M NaI0 4 a t 4° ov e r n i g h t and subsequent a d d i t i o n of ethylene . g l y c o l , r e d u c t i o n - 112 -wi t h sodium borohydride, Smith h y d r o l y s i s (0.5 M t r i f l u o r o -a c e t i c a c i d a t room temperature overnight) and r e d u c t i o n w i t h sodium borohydride y i e l d e d m a t e r i a l which was a p p l i e d to the top of a B i o - G e l P-4 column (2.5 x 120 cm). Component _2 was i s o l a t e d (4 mg) having R n 0.73 ( s o l v e n t A) and [a] -67° — GJ-C D (c 0.35, water). XH n.m.r. of 2 showed anomeric s i g n a l s a t T 4.94, IH, 2 2 Hz andx 5.03, IH, 2 2 H z (see Appendix I I I , spectrum No. 6). H y d r o l y s i s of a sma l l p o r t i o n of oligomer 2 gave o n l y L-rhamnose and g l y c e r o l by paper chromato-graphy. M e t h y l a t i o n of _2 y i e l d e d 2.5 mg of the permethylated d e r i v a t i v e having R„ 0.38 on t . l . c . ( e t h y l a c e t a t e ) . M.s. of — r t h i s d e r i v a t i v e i n the f . d . mode gave major peaks a t m/e 481, 482, 483 and 484 c o r r e s p o n d i n g to M-l, M, M+l and M+2 r e s p e c t i v e l y . Chemical i o n i s a t i o n (methane) m.s. of t h i s same d e r i v a t i v e gave a spectrum which showed, i n t e r a l i a , the f o l l o w i n g f r a g -ments ( r e l a t i v e i n t e n s i t i e s i n b r a c k e t s ) : 88(11), 99(12), 125(27), 127(29), 129(10), 149(10) , 159 (11) , 189(100), 190(12), 205(49), 233(9), 363(49), 364(10), 391(18), 481(2). The peak at 391 i s a t t r i b u t e d to a d i o c t y l p h t h a l a t e i m p u r i t y . H y d r o l y s i s of the permethylated d e r i v a t i v e of 2 (2 M t r i f l u o r o a c e t i c a c i d , 6 hours, 90°) and subsequent d e r i v a t i s a t i o n y i e l d e d 2 , 3 , 4 - t r i -0-methyl-L-rhamnose and 2,4-di-0-methyl-L-rhamnose i n equal amounts. The d i m e t h y l g l y c e r o l fragment was too v o l a t i l e t o be i s o l a t e d d u r i n g work up. - 113 -P a r t i a l h y d r o l y s i s of p o l y s a c c h a r i d e . K36 (1 g) was hyd r o l y s e d f o r 3 hours at 95° i n 2 M t r i f l u o r o a c e t i c a c i d . A f t e r removal of the a c i d by ev a p o r a t i o n w i t h s e v e r a l p o r t i o n s of water, the mixture was n e u t r a l i s e d (NaOH) and then a p p l i e d to the top of a column (2 x 20 cm) of Dowex 1-X2 (formate form). The column was then washed with 1 £ of d i s t i l l e d water. The a c i d i c components were e l u t e d w i t h 10% HCOOH and a f t e r e v a p o r a t i o n to dryness t h i s m a t e r i a l weighed 350 mg. Paper chromatography ( s o l v e n t B) showed no n e u t r a l monosaccharides were prese n t i n the mixture. Gel chromatography (Bio-Gel P-4; 2 x 175 mg) of the n e u t r a l i s e d (NaOH) oligomers d i d not gi v e good s e p a r a t i o n of the mixture of components. S e l e c t e d f r a c t i o n s c o n t a i n i n g s m a l l oligomers were rerun (Bio-Gel P-4) y i e l d i n g pure a l d o b i o u r o n i c a c i d 3^  (60 mg) , a l d o t r i o u r o n i c a c i d £ (35 mg) , and an a c i d i c t e t r a s a c c h a r i d e 5 (4 mg). The a l d o b i o u r o n i c a c i d 3 (R^, 1.0, s o l v e n t A) showed ~ - G l c [ a ] D -12° (c 1.2, w a t e r ) 2 8 . 1H n.m.r. (D 20, 90°) showed anomeric s i g n a l s a t T 5.37, IH, J 7 Hz; x 4.63, 0.6 H, 1, z 2 1.8 Hz; and x 5.17, 0.4 H, s i n g l e t (see Appendix I I I , 13 s p e c t r a No.'s 7,8). The C n.m.r. spectrum showed two anomeric s i g n a l s ; one 104.9 p.p.m. dow n f i e l d from T.M.S. a t t r i b u t a b l e to the of the g - g l u c u r o n i c a c i d and another a t 93.7 p.p.m. corresponding t o the of the re d u c i n g a-^-rhamnose. No resonances f o r e i t h e r the redu c i n g 3-L-rhamnose or the Cg 29 of a hexose were observed. Hakomori m e t h y l a t i o n of 3_ y i e l d e d permethylated a l d o b i o u r o n i c a c i d which on t . l . c . had R 0.80 - 114 -( e t h y l a c e t a t e ) . Reduction o f t h i s compound with l i t h i u m aluminum hy d r i d e i n t e t r a h y d r o f u r a n y i e l d e d a compound wit h R„ 0.32 on t . l . c . ( e t h y l a c e t a t e : e t h a n o l , 9:1). Subsequent h y d r o l y s i s (2 M t r i f l u o r o a c e t i c a c i d a t 95° f o r 6 hours), r e d u c t i o n and a c e t y l a t i o n y i e l d e d two components as t h e i r a l d i t o l a c e t a t e s i n a 1:1 r a t i o c orresponding to 2 , 3 , 4 - t r i -O-methyl-p-glucose and 3,4-di-0-methyl-L-rhamnose ( g . l . c . column A). G.l.c.-m.s. confirmed the assignment o f the above components. The a l d o t r i o u r o n i c a c i d 4 (R„. 0.88, s o l v e n t A) showed ~ - G l c [ a ] D -31° (c 1.1, w a t e r ) 3 0 . 1H n.m.r. (D 20, 90°) showed anomeric s i g n a l s a t x 5.34, IH, J 1 2 7 Hz; x 4.65, IH, J 1 2 2 Hz; x 4.89, 0.6 H, J 1 2 1.8 Hz; and x 5.13, 0.4 H, s i n g l e t . 13 The C n.m.r. spectrum d i s p l a y e d s i g n a l s i n the anomeric r e g i o n a t 105.2 p.p.m., 101.6 p.p.m. and two s i g n a l s (94.7 p.p.m., 94.1 p.p.m.) a t t r i b u t a b l e to the a and 8 r e d u c i n g anomeric carbons o f rhamnose (see Appendix I I I , spectrum No.*s 9,10) . Hakomori m e t h y l a t i o n of £ y i e l d e d permethylated a l d o t r i o -u r o n i c a c i d having R 0.70 on t . l . c . ( e t h y l a c e t a t e ) . Reduc-— r t i o n w i t h l i t h i u m aluminum h y d r i d e i n t e t r a h y d r o f u r a n y i e l d e d the c o r r e s p o n d i n g product having R„ 0.27 on t . l . c . ( e t h y l — £ a c e t a t e : e t h a n o l , 9:1). H y d r o l y s i s (2 ^  t r i f l u o r o a c e t i c a c i d at 95° f o r 6 hours), r e d u c t i o n and a c e t y l a t i o n y i e l d e d the a l d i t o l a c e t a t e s corresponding to 2,3,4-tri-0-methyl-g-glucose, 3 , 4-di-O-methyl-Jj-rhamnose and 2, 4-di-0-methyl-L :-rhamnose, ( g . l . c . column A), i n the r a t i o 1:1:1. G.l.c.-m.s. confirmed the m e t h y l a t i o n p a t t e r n of the above components. - 115 -Compound 5 (R~n 0.64, s o l v e n t A) gave a ±R n.m.r. ~ -Glc spectrum (see Appendix I I I , spectrum No. 11) having anomeric resonances a t T 5.35, IH, 2 7 H z ' T 5.19, IH, 2 7.5 Hz; x 4.63, IH, 2 2 Hz; T 4.89, 0.6 H, 2 1.8 Hz; and x 5.13, 0.4 H, s i n g l e t . M e t h y l a t i o n of _5 y i e l d e d the permethylated d e r i v a t i v e having R„ 0.7 5 on t . l . c . ( e t h y l a c e t a t e : e t h a n o l , 92:8). Reduction of t h i s m a t e r i a l w i t h l i t h i u m aluminum h y d r i d e i n t e t r a h y d r o f u r a n gave a compound w i t h R p 0.37 on t . l . c . (chloroform:acetone, 2:1). H y d r o l y s i s and c o n v e r s i o n to a l d i t o l a c e t a t e s (same as f o r compounds 1 and 2) y i e l d e d f o u r components i n a 1:1:1:1 r a t i o . G . l . c . (column B) and g.l.c.-m.s. i d e n t i f i e d these as being the a l d i t o l a c e t a t e s of 2,3,4,6-tetra-O-methyl-D-glucose, 2,3-di-0-methyl-g-glucose, 3 , 4-di-O-methyl-Jj-rhamnose and 2 , 4-di-O-methyl-L-rhamnose. Compound 5 i s t h e r e f o r e B-g-Glcp-(1+4)-3-g-GlcAp-(1+2)-a-L-Rhap- (1+3) -a-L-Rhap. A p a r t i a l h y d r o l y s i s of K36 (200 mg) u s i n g 0.3 M t r i f l u o r o -a c e t i c a c i d at 95° f o r 3 hours was c a r r i e d out. Treatment of the h y d r o l y z a t e as d e s c r i b e d f o r the preceding p a r t i a l h y d r o l y s i s y i e l d e d , i n a d d i t i o n to components 3^, £ and 15 (above) , a component £ (8 mg; R G l c 0.64, s o l v e n t A) which was i n i t i a l l y thought to be a hexasaccharide from i t s "'"H n.m.r. spectrum (see Appendix I I I , spectrum No. 12). Compound J5 was reduced w i t h l i t h i u m b o r o d e u t e r i d e and then methylated. Two components, w i t h R p 0.73 (compound 6a) and R^ 0.57 (compound 6b) on t . l . c . ( e t h y l a c e t a t e : e t h a n o l , - 116 -92:8) were o b t a i n e d . Column s e p a r a t i o n on s i l i c a g e l y i e l d e d 6a (4.5 mg) and 6b (2.5 mg). Reduction of 6a with l i t h i u m aluminum hy d r i d e i n t e t r a -hydrofuran gave a compound wit h R 0.37 on t . l . c . (chloroform: — r acetone, 2:1). H y d r o l y s i s r e d u c t i o n , a c e t y l a t i o n and g . l . c . s e p a r a t i o n (column B) showed 6a to be i d e n t i c a l to permethylated compound 15. G . l . c . and g.l.c.-m.s. gave the a l d i t o l a c e t a t e s c o r r e s p o n d i n g to 2,3-di-O-methyl-D-glucose, 2,4-di-0-rnethyl-Jj-rhamnose and 2 , 3 , 4 , 6-tetra-0-methyl-g-glucose, together with 3 - 0 - a c e t y l - l , 2 , 4 , 5 - t e t r a - O - m e t h y l - L - r h a m n i t o l . (Some of the l a t t e r component, monodeuterated a t C^, was l o s t d u r i n g d e r i v a t i s a t i o n . ) Reduction of 6b with l i t h i u m aluminum hydride i n t e t r a -hydrofuran gave a component with R„ 0.30 on t . l . c . (chloroform: — r acetone, 2:1). H y d r o l y s i s r e d u c t i o n and a c e t y l a t i o n f o l l o w e d by g.1.c./g.1.c.-m.s. (column A) gave peaks corresponding to the a l d i t o l a c e t a t e s of 2,3,4-tri-0-methyl-g-glucose, 4-0-methyl-L—rhamnose and 2,3,4,6-tetra-0-methyl-g-galactose, together with 3 - 0 - a c e t y l - l , 2 , 4 , 5 - t e t r a - O - m e t h y l - L - r h a m n i t o l . The r a t i o of the f o u r components was 1:1:1:0.5 with 50% of the v o l a t i l e tetra-0-methyl-L-rhamnose d e r i v a t i v e , mono-deuterated at C-^ , being l o s t under vacuum d u r i n g work up. Compound 6b i s thus 'the permethylated d e r i v a t i v e of the t e t r a s a c c h a r i d e as shown on the f o l l o w i n g page. - 117 -D-Gal———L-Rha———Jj-Rhamnitol 2 1 g-GlcA A n a l y s i s of the XH n.m.r. spectrum of compound 6 ( t a k i n g i n t o account t h a t the XH n.m.r. spectrum of compound 5 has al r e a d y been obtained) allowed the assignment (as shown i n Table I I I . l , page 92 ) of the resonances and l i n k a g e s of t h i s branched t e t r a s a c c h a r i d e . - 118 -III . 5 B i b l i o g r a p h y f o r S e c t i o n I I I . 1. W. Nimmich, Acta B i o l . Med. Ger., _26, 397 (1971). 2. W. Nimmich, Z. M i c r o b i o l . Immunol., 154, 117 (1968). 3. M. H e i d e l b e r g e r and W. Nimmich, Immunochemistry, 13, 67 (1976). 4. P.A.J. G o r i n and T. Ishikawa, Can. J . Chem., 4_5, 521 (1967). 5. Y.M. Choy, G.G.S. Dutton, A.M. Stephen and M.T. Yang, A n a l . L e t t . , 5, 675 (1972). 6. G.M. Bebault, Y.M. Choy, G.G.S. Dutton, N. F u n n e l l , A.M. Stephen and M.T. Yang, J . B a c t e r i o l . , 113, 1345 (1973). 7. J.M. Berry, G.G.S. Dutton, L.D. H a l l and K.L. Mackie, Carbohyd. Res., 5_3, C8 (1977). 8. R.L. T a y l o r and H.E. Conrad, Biochemistry, 11, 1383 (1972). 9. G.M. Bebault, J.M. Berry, Y.M. Choy, G.G.S. Dutton, N. F u n n e l l , L.D. Hayward and A.M. Stephen, Can. J . Chem., 51, 324 (1973). 10. S. Hakomori, J . Biochem. (Tokyo), 5_5, 205 (1964). 11. H. B j o r n d a l , B. Lindberg and S. Svensson, Carbohyd. Res., 5, 433 (1967). 12. H. B j o r n d a l , C.G. H e l l ' e r q v i s t , B. Lindberg and S. Svensson, Angew. Chem. Int . Ed. E n g l . , 9_, 610 (1970). 13. D.P. Sweet, R.H. Shapiro and P. Albersheim, Carbohyd. Res. , 4_0, 217 (1975) . 14. G.W. Hay, B.A. Lewis and F. Smith, Methods Carbohyd. Chem., 5, 357 (1965). I.J. Goldstein, G.W. Hay, B.A. Lewis and F. Smith, Method; Carbohydr. Chem., 5, 361 (1965). M.T. Yang, Ph.D. Thesis, University of B r i t i s h Columbia, June 1974. G.M. Bebault, G.G.S. Dutton, K.L. Mackie and A.V. Savage, Abstracts Papers 172nd ACS Meeting, San Francisco, C a l i f o r n i a , Aug., 1976, CARB. 30. G. G.S. Dutton and K.B. Gibney, Carbohyd. Res., 2_5, 99, (1972). H. O. Bouveng, Acta Chem. Scand., 1_9, 953 (1965). G.O. A s p i n a l l , M.J. Johnston and R. Young, J. Chem. S o c , 2701 (1965). P.G. Johnson and E. Per c i v a l , J. Chem. Soc. (C), 906 (1969). Y.C. Lee and C E . Ballou, Biochemistry, 4_, 257 (1965) . J. Lonngren and S. Svensson, Advan. Carbohyd. Chem. and Biochem., 29_, 42 (1974). M. Curvall, B. Lindberg, J. Lonngren and W. Nimmich, Carbohyd. Res., 42_, 95 (1975). W.E. Trevelyan, D.P. Proctor and J.S. Harrison, Nature, 166, 444 (1950). E.L. H i r s t and E. Per c i v a l , Methods Carbohyd. Chem., 5, 287 (1965). J.E. Scott and R.J. Harbinson, Histochemie, 1_4, 215 (1968) M.T. Yang. Unpublished re s u l t s from t h i s laboratory. L.D. H a l l and L.F. Johnson, Chem. Comm., 509 (1969). - 120 -M. C u r v a l l , B. Lindberg, J . Lonngren and W. Nimmich, Carbohyd. Res., 4_2, 73 (1975). - 121 -IV. THE STRUCTURE OF K l e b s i e l l a SEROTYPE K7 0 CAPSULAR POLYSACCHARIDE - 122 -IV.1 A b s t r a c t Using the techniques of m e t h y l a t i o n a n a l y s i s , u r o n i c a c i d d e g r a d a t i o n , p a r t i a l h y d r o l y s i s and p e r i o d a t e o x i d a t i o n the s t r u c t u r e o f the c a p s u l a r p o l y s a c c h a r i d e from K l e b s i e l l a serotype K70 has been i n v e s t i g a t e d . Nuclear magnetic reson-ance was used e x t e n s i v e l y to c h a r a c t e r i s e fragments o b t a i n e d as a r e s u l t of the v a r i o u s d e g r a d a t i o n procedures. The e x i s t e n c e of a l i n e a r hexasaccharide repeat u n i t as shown below, wi t h a pyruvate a c e t a l attached to a 2 - l i n k e d L-rhamnose r e s i d u e on every second r e p e a t i n g u n i t , has been demonstrated. +4)-$-p-GlcAp-(1+4)-a-L-Rhap-(1+2)-a-L-Rhap-(1+2)-a-D-Glcp-(1+3)-3-D-Galp-(1+2)-a-L-Rhap-(1+ IV.2 I n t r o d u c t i o n Of the 81 d i f f e r e n t K l e b s i e l l a serotypes the s t r u c t u r a l a n a l y ses o f approximately 30 of the c a p s u l a r p o l y s a c c h a r i d e s produced by these b a c t e r i a have been r e p o r t e d . K l e b s i e l l a K70 has been shown to c o n t a i n g l u c u r o n i c a c i d , g a l a c t o s e , glucose and rhamnose and i s one of 11 s e r o l o g i c a l l y d i f f e r e n t 1 2 K-types having t h i s same q u a l i t a t i v e composition ' . We r e p o r t here the r e s u l t s of our s t r u c t u r a l i n v e s t i g a t i o n o f t h i s p o l y s a c c h a r i d e . (50%) C H 3 C O ~ H - 123 -IV.3 Results and Discussion 3 The polysaccharide, i s o l a t e d as previously described (see Section III.4, page 106), had [a] -43° (c 2.8, water). Proton magnetic resonance (XH n.m.r.) indicated the presence 4 5 of one pyruvic acid acetal per 12 sugar residues ' (see Appendix II I , spectrum No. 13). In the anomeric region (x 4.5 - x 5.5) six proton signals were observed while a nine proton doublet at x 8.7, due to the CH^ groups of 6-deoxy sugars, was also apparent (see Table IV..1, page 124). Carbon magnetic resonance ( C n.m.r.) information was i n agreement with the x H n.m.r. data (see Appendix II I , spectrum No. 14), and in addition, indicated the presence of two hexose sugars; (two signals were observed between 60-62 p.p.m. in d i c a t i v e of the signals from Cg of hexose sugars). The XH 13 and C n.m.r. spectra therefore indicate the presence of three rhamnose residues and two hexose residues. Knowing the q u a l i t a t i v e composition of K70 capsular polysaccharide i t can be deduced that the remaining sugar residue i n the repeating unit must be a glucuronic acid unit. Hydrolysis of K70 and paper chromatography of the hydroly-sate indicated the presence of glucose, glucuronic acid, galactose and rhamnose. Progressive hydrolysis of the poly-saccharide, monitored by paper chromatography, did not show the release of any one sugar p r e f e r e n t i a l l y and indicated K70 was probably a l i n e a r polysaccharide. Methanolysis of K l e b s i e l l a K7 0, reduction with sodium borohydride in dry TABLE IV.1 N.M.R. Data f o r K l e b s i e l l a K70 P o l y s a c c h a r i d e and I s o l a t e d O l i g o s a c c h a r i d e s 5 1 13 H C b e d e f Compound x (J, „) ,int e g r a l , a s s i g n m e n t p.p.m. assignment 1, 2. GlcA^^Rha-OH 4.89 (1.8 Hz), 0. 6H, a-Rha-OH 103.70 B-GlcA 8 a 5.14 (S),0.4H, 8-Rha-OH 94.55 a-Rha-OH 5.28 (8 Hz), IH, 8-GlcA 93.23 8-Rha-OH 8.70,(J 5 6 6 Hz),3H, CH 3 of Rha 17.82 CH 3 of Rha Glc^-^al^-^Rha-OH 4.59 (1.8 Hz), 0. 6H, a-Rha-OH 105.51 8 - G a l g 4.85 (3.5 Hz), IH, a-Glc 104.77 5.13 (S), 0.4H, 8-Rha-OH 96.33 a-Glc 5.34 (7*), IH, 8-Gal 93.92 a-Rha-OH 8.70, ( J c , 6 Hz),3H, CH- of Rha 93. 59 8-Rha-OH _> b j 17.56 CH 3 o f Rha TABLE IV.1 Continued 1 2 1 2 Gal.Q Rha e r y t h r i t o l p. a 4.78 (1.8 Hz), IH, a-Rha (16 s i g n a l s o v e r a l l ) 5.43 (7.5 Hz), IH, B-Gal 105. 75 B-Gal 8.70,(J 5 6 6 Hz),3H, CH 3 of Rha 100. 19 a-Rha 63. 19 j C, of Gal 6 61. 77 [ C^ o f e r y t h r i t o l 61. 74 ) C^ of e r y t h r i t o l 17. 35 CH 3 of Rha 1 2 Gal fi g l y c e r o l 5.45 (7.5 Hz), B-Gal (9 s i g n a l s o v e r a l l ) 103.33 B-Gal 62.44 61. 90 61. 78 ( C 6 of Gal i , c 3 o f g i y c e r o 1 TABLE VI.1 Continued N a t i v e K70 P o l y s a c c h a r i d e 4.78 (S*), IH, a-Rha 4.90 (S*), 2H\ /a-Rha > /a-Rha 5.03 (S*), i n ) (a-Glc 5.23 (7 Hz), IH, 8-GlcA 5.45 (7 Hz*), IH, 8-Gal 8.41 (S) , 1.5H, CH 3 of pyruvate 8.70,(J 5 6 6 Hz),9H, CH 3 of Rha 105.7 103. 8 102. 9 101.7 100. 9 95.7 62. 20 61.30 17. 50 s i x unassigned anomeric C s i g n a l s ;C, of Gal I 6 C, of Glc b CH 3's of Rha Foot n o t e s : For o r i g i n of o l i g o s a c c h a r i d e s 1,2,5, and 6 see t e x t . Chemical s h i f t taken r e l a t i v e t o i n t e r n a l acetone; T 7.77 downfield from D.S.S. cS»singlet. Those values marked w i t h an a s t e r i s k cl were broad s i g n a l s . e.g., a-Rha=proton on C-^ of £-Rha r e s i d u e which i s a - l i n k e d (Gal=D-Gal) . e C h e m i c a l s h i f t quoted as p.p.m. downfield from T.M.S. r e l a t i v e to i n t e r n a l acetone; 31.07 p.p.m. from D.S.S. f As f o r d but f o r anomeric 1 3 C n u c l e i . gTwo value s are giv e n f o r 1 3 C 1 of 8-Gal as the chemical s h i f t o f t h i s carbon atom i s a f f e c t e d by a- and 8 - e q u i l i b r i u m of the r e d u c i n g Rha r e s i d u e . 105.51 = 8-Gal(a-Rha), 104.77 = 8-Gal(g-Rha). - 127 -methanol and then hydrolysis, yielded a mixture of sugars which was shown by paper chromatography to contain only glucose, galactose and rhamnose. Reduction and acetylation to convert t h i s mixture to a l d i t o l acetates and gas chromato-graphic (g.l.c.) analysis confirmed the presence of the above three sugars in the proportions 31:17:52. This r e s u l t indicated that K70 contained glucose, glucuronic acid, galactose and rhamnose residues in the r a t i o of 1:1:1:3 respectively, and that the polysaccharide consisted of a hexasaccharide repeat unit. The glucose and rhamnose were shown to be of D- and L- configuration respectively by the 7 c i r c u l a r dichroism curves of the a l d i t o l acetates. The configuration of the galactose was shown to be p- by the c i r c u l a r dichroism curve of the 2,4,6-tri-0-methyl deriva-t i v e obtained from methylation analysis. 8 9 Methylation analysis ' of the native polysaccharide and of a sample of K7 0 which had been autohydrolysed at pH 2.2 for 16 hours, confirmed the existence of a hexa-saccharide repeat unit (see Table IV.2, page 128). The concomitant increase of 3,4-di-O-methyl-L-rhamnose and loss of L-rhamnose afte r the pyruvate acetal had been removed, located the acetal on a 2-linked L-rhamnose and confirmed the existence of t h i s substituent on every second repeat unit of K l e b s i e l l a K70. P a r t i a l a c i d i c hydrolysis of K70 was performed using 0.5 ^ t r i f l u o r o a c e t i c acid at 95° for 45 minutes. Follow-ing separation of a c i d i c and neutral material by ion exchange - 128 -TABLE IV.2 M e t h y l a t i o n Analyses of N a t i v e and Depyruvalated  K l e b s i e l l a K70 Capsular P o l y s a c c h a r i d e Methylated sugars Mole % (as a l d i t o l a c etates) T — -3,4-Rha 0.89 22. 6 33.1 2,3-Rha 1.00, 17. 0 16.9 3,4,6-Glc 1. 50 17.2 17.3 Rha 1. 58 10. 2 2,4,6-Gal 1. 67 16. 6 17.3 2,3-Glc (from D-GlcA) 2.40 16.3 15.3 a D e p y r u v a l a t e d =K10 p o l y s a c c h a r i d e autohydrolysed a t pH 2.2 f o r 16 hours a t 95°. 3,4-Rha = 1 , 2 , 5 - t r i - 0 - a c e t y l - 3 , 4 - d i - 0 - m e t h y l - L - r h a m n i t o l , e t c . C R e t e n t i o n time of p a r t i a l l y methylated a l d i t o l a c e t a t e s , r e l a t i v e t o 1 , 5 - d i - 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l - p - g l u c i t o l , on a column of 3% of HIEFF IB on Gas-Chrom Q (100-120 mesh) programme at 160° f o r 8 min. and then 2° per min. to 190°. dColumn I; n a t i v e p o l y s a c c h a r i d e . Column I I ; depyruvalated p o l y s a c c h a r i d e . - 129 -chromatography each f r a c t i o n was analysed using a Bio-Gel P-4 gel permeation column. From the a c i d i c f r a c t i o n a pure oligomer 1 with [ a ] D -30° was i s o l a t e d 1 0 . The XH 'and 1 3 C n.m.r. spectra were in agreement with 1 being an aldobio-uronic acid (see Table IV.1, page 124, and also Appendix III, spectrum No.'s 15, 16). Compound 1 was reduced with lithium borodeuteride and then methylated. Subsequent reduction with lithium aluminum hydride, hydrolysis and 9 11 g.l.c.-m.s. ' analysis of the liberated p a r t i a l l y methylated monosaccharides as a l d i t o l acetates, gave components confirm-ing the structure of 1 as being B-D-GlcAp-(1+4)-L-Rhap (1) The neutral material obtained from the p a r t i a l hydrolysis was also separated on a Bio-Gel P-4 column. Oligomer 2 with 1 13 [ a ] D +10° was iso l a t e d and from the H and C n.m.r. spectra (see Table IV.1, page 124, and also Appendix III, spectra 13 No.'s 17, 18) was thought to be a trisaccharide. The C n.m.r. spectrum of 2 was of p a r t i c u l a r i n t e r e s t i n that the 1 13 terminal reducing sugar (easily recognisable in H and C n.m.r. as a rhamnose residue) was af f e c t i n g the chemical s h i f t of the non-terminal (middle) anomeric carbon. Hence, the C^ signals from the reducing rhamnose at 93.92 p.p.m. (a) and 93.59 p.p.m. (8) influenced the anomeric carbon on the middle sugar so that i t gave two signals at 105.51 and 104.77 p.p.m. The chemical s h i f t (approximately 105 p.p.m.) - 130 -of t h i s double signal allowed i t to be assigned to a B-hexose. Reduction of 2 with lithium borodeuteride and subsequent methylation yielded a product which was hydrolysed, reduced, 9 11 acetylated and analysed by g.l.c.-m.s. ' . I d e n t i f i c a t i o n of the three components observed (see Experimental) established 2 as being a-D-Glcp-(1+3)-B-D-Galp-(1+2)-L-Rhap (2) A sample of K l e b s i e l l a K7 0 from which the pyruvate acetal had been s e l e c t i v e l y removed by autohydrolysis, was per-12 methylated and then subjected to a uronic acid degradation This technique can provide valuable information in sequencing a l i n e a r polysaccharide, even though the B-elimination reactions may not go to completion. After treatment with methylsulphinyl anion in dimethyl sulphoxide and subsequent mild acid treat-ment to cleave the enol ethers formed during the elimination reactions, the mixture of products was reduced with sodium borohydride in dry methanol. Methylation of t h i s reduced mixture with Purdie's reagents and chromatography on s i l i c a gel f a c i l i t a t e d the i s o l a t i o n of a component 3a which, by "*"H n.m.r., was shown to have signals at x 4.88 (2H, J 1 „ broad) and x 5.08 (IH, ^ 3 H z ) i - n the anomeric region (see Appendix II I , spectrum No. 19). The mass spectrum of 3a (e.i.) was in agreement with that expected for a t r i -saccharide alditol"'""'". The origins of some pertinent fragments are outlined i n the accompanying diagram. It i s perhaps - 131 -worthy of note t h a t besides the fragments aA^, baA^, cbaA^, by which the sequence of 3a can be determined, the main fragments arose from the c J ^ , b c J ^ and abcJ^ i o n s . These l a t t e r , fragments are i n agreement with the i n t e r g l y c o s i d e l i n k a g e s being as shown CrLOMe I 2 CHOMe I CH l CHOMe 1 5 7 — ( 1 8 9 ) HOMe CH2OMe 2 3 5 9 11 H y d r o l y s i s of 3a and a n a l y s i s by g.l.c.-m.s. ' of the a l d i t o l a c e t a t e s gave peaks corre s p o n d i n g to 1,2,4,5,6-p e n t a - O - m e t h y l - g - g a l a c t i t o l , 2,3,4-tri-O-methyl-L-rhamnose, 3, 4-di-0-methyl-L =-rhamnose and 3,4,6-tri-O-methyl-g-g l u c o s e . E t h y l a t i o n of the sodium borohydride reduced u r o n i c a c i d d e g r a d a t i o n product gave a component 3b and a n a l y s i s of t h i s m a t e r i a l as d e s c r i b e d f o r the permethylated product (3a) gave 1 , 5 - d i - 0 - e t h y l - 2 , 3 , 6 - t r i - 0 - m e t h y l - g - g a l a c t i t o l , 4-0-ethyl-2, 3-di-O-methyl-I^-rhamnose, 3, 4-di-O-methyl-L-rhamnose and 3,4,6-tri-O-methyl-D-glucose. Bearing i n mind t h a t the sequence of compound 2 (obtained from p a r t i a l - 132 -hydrolysis) i s already known i t i s possible to write the structure of 3a as the permethylated d e r i v a t i v e of a-L-Rhap- (1+2)-a-L-Rhap- (l->2)-a-g-Glcp- (1+3)-D-galactitol. The 4-0-ethyl-2,3-di-O-methyl-L-rhamnose obtained from 3b indicates the terminal rhamnose was linked through the hydroxyl on C-4 i n the polysaccharide. The sequence of reactions in the B-elimination of K70 i s outined in Figure II.6, page 42. The r e s u l t s obtained from p a r t i a l hydrolysis and uronic acid degradation studies are s u f f i c i e n t to e s t a b l i s h the repeating unit of K7 0 as being that shown below +4)-8-D-GlcAp-(1+4)-a-L-Rhap-(1+2)-a-L-Rhap-(1+2)-a-Q-Glcp- (1+3) -B-g-Galp- (1+2)-a-L-Rhap (l-> The pyruvate moiety, shown to occur on only 50% of the repeating units by methylation analysis and "^H n.m.r. of K70, i s present as an acetal spanning the hydroxyls on and of a 2-linked rhamnose. The presence of two 2-linked rhamnoses in the repeating unit made i t necessary to locate the pyruvate more prec i s e l y . This was done using periodate •A 4 - • 1 3 oxidation When K l e b s i e l l a K70 polysaccharide was reacted with 14 sodium periodate a plateau value was obtained correspond-ing to the consumption of six moles of oxidant per true repeat unit (taken as twelve sugar u n i t s ) . This value i s - 133 -three moles below the t h e o r e t i c a l value. However, follow-ing reduction of the polyaldehyde product, d i a l y s i s , and treatment again with the periodate solution, a further 4.2 moles of periodate was consumed. (This value rose to 5 moles a f t e r 156 hours.) These r e s u l t s indicate that hemi-15 acetal formation i s occurring during the i n i t i a l periodate treatment. By "'"H n.m.r. i t was shown that the twice periodate-oxidised polyalcohol contained only a trace of the pyruvate acetal and i t was assumed t h i s had been cleaved by hydrolysis at the reaction pH of 4.2. Another sample of K l e b s i e l l a K7 0 was subjected to oxidation in a buffered solution (pH 5.0) of periodate and after 24 hours the product was reduced with sodium borohydride and the polyalcohol was oxidised again using the same conditions. The polyalcohol derived from t h i s sequence of reactions was then methylated and following re-duction with lithium aluminum hydride and hydrolysis, the 9 11 p a r t i a l l y methylated sugars were analysed by g.l.c.-m.s. ' as t h e i r a l d i t o l acetates. Only 1,2,3,4,5-penta-0-acetyl-L-rhamnitol and 1,3,5-tri-0-acetyl-2,4,6-tri-O-methyl-Q-g a l a c t i t o l were found to be present i n a 1:2 r a t i o . This r e s u l t indicated the pyruvate acetal was s t i l l present on the doubly oxidised polymer and that complete periodiate oxidation had been achieved. The polyalcohol from the f u l l y 16 oxidised polymer was then subjected to a Smith hydrolysis and reduced with sodium borohydride. The mixture of products was separated by gel permeation chromatography giving three - 134 -pure oligomers; £, £ and (5. Compound £ showed [ a ] D -16° and XH n.m.r. spectroscopy (see Table IV.1, page 124, and also' Appendix I I I , spectra No.'s 20, 21) revealed signals a t t r i b u t a b l e to anomeric protons at T 4.72 (IH, 2 1-8 Hz) and x 5.43 (IH, J 1 2 7.5 Hz). Hydrolysis of £ showed by paper chromatography the presence of galactose, rhamnose and two other components which were indistinguishable from erythronic acid and erythronolactone. Reduction of 4_ with sodium borohydride gave an oligomer which was chromato-graphically indistinguishable from oligomer !5 and some unchanged s t a r t i n g material. Methylation of £ gave a product which had a strong absorbance at 1750 cm x in the. i . r . spectrum. The mass spectrum of permethylated £ using the 17 chemical i o n i s a t i o n ( c i . ) mode was consistent with the structure given. The source of some pertinent fragments i s i l l u s t r a t e d : Oligomer 4 i s therefore shown to be B-g-Galp-(1+2)-a-L-Rhap-(1+3)-L-erythronic acid. (4) .- 135 -Compound 5, had t a ] D +4.5° and the 1H n.m.r. spectrum showed s i g n a l s a t x 4.78 (1H, J „ 2 Hz) and x 5.43 (IH, J J-1 2 1/2 13 7.5 Hz) i n the anomeric r e g i o n . In the C n.m.r. spectrum 16 s i g n a l s o v e r a l l were observed; two s i g n a l s i n the anomeric r e g i o n a t 105.75 and 100.19, and thr e e s i g n a l s i n the r e g i o n (60-63 p.p.m.) a s s o c i a t e d with the C-6 of hexose sugars (see Table IV.1, page 124, and a l s o Appendix I I I , s p e c t r a No. 's 22,23). H y d r o l y s i s of _5 showed by paper chromatography the presence of rhamnose, g a l a c t o s e and e r y t h r i t o l . Methyla-t i o n of _5 y i e l d e d a product which upon h y d r o l y s i s and con-v e r s i o n to a l d i t o l a c e t a t e s gave components corresponding to 2,3,4,6-tetra-O-methyl-D-galactose and 3,4-di-0-methyl-L-rhamnose. The mass spectrum o f permethylated !5 i n the c i . mode was c o n s i s t e n t w i t h the s t r u c t u r e shown below. Some p e r t i n e n t fragments are i n d i c a t e d : Compound 5 i s t h e r e f o r e e s t a b l i s h e d as being 3-g-Galp- ( 1 + 2 )-a-L-Rhap- ( 1 + 2 )-D-erythritol (5) - 136 -13 Compound 6, [a]_ +3.7° was shown by J'JC n.m.r. to c o n t a i n ~ D nine carbons o v e r a l l . In the anomeric r e g i o n one s i g n a l at 103.33 p.p.m. was apparent and th r e e s i g n a l s between 60-63 p.p.m. were observed. In the XH n.m.r. spectrum o n l y one anomeric r e g i o n (see Table IV.1, page 124, and a l s o Appendix I I I , s p e c t r a No.'s 24, 25). H y d r o l y s i s o f (5 showed by paper chromatography the presence of g a l a c t o s e and g l y c e r o l . M e t h y l a t i o n of (5 and subsequent h y d r o l y s i s gave a component corres p o n d i n g to 2,3,4,6-tetra-0-methyl-p-galactose as i t s a l d i t o l a c e t a t e on g.l.c.-m.s. The mass spectrum of per-methylated £ ( e . i . ) was c o n s i s t e n t w i t h the s t r u c t u r e shown below. Some p e r t i n e n t fragments are i n d i c a t e d : s i g n a l a t x 5.45 (IH, J 1,2 7.5 Hz) was apparent i n the Me H20Me H :H20Me 1 8 7 — 219 (220) 3 0 7 — (338)M f 337 M-1 + Oligomer 6 i s thus e s t a b l i s h e d as being 8-D-Galp-(1+2)-glycerol (6) - 137 -The i s o l a t i o n of oligomers £, !5, 6 i s compatible with the l i n e a r sequence of K70 shown previously. Compound 5^  res u l t s from the spontaneous la c t o n i s a t i o n of periodate oxidised D-glucuronic acid residues during the double oxida-tion-reduction procedure used and the subsequent p a r t i a l reduction of t h i s lactone with sodium borohydride r e s u l t s in the reduction of the acid group to a primary alcohol. E f f e c t i v e l y , some of the D-glucuronic acid residues give r i s e to erythronic acid and some give r i s e to e r y t h r i t o l , either of which are found as terminating glycosides i n £ and _5. The survival of the 2-linked rhamnose through the periodate oxidation indicates that the pyruvate acetal was attached to t h i s sugar during the oxidation. The evidence presented for K l e b s i e l l a K70 i s consistent with the structure being as shown below. Because of 1 13 the good resolution obtained in H and C n.m.r. studies of K70, i t i s probable that the pyruvate acetal i s d i s t r i -buted evenly throughout the polysaccharide and i s therefore present on every second repeat unit. +4)-8-D-GlcAp- (1+4) -cx-L-Rhap- (1+2)-a-L-Rhap- (1+2)-a-g-Glcp-(1+3)-B-Q-Galp-(1+2)-a-^-Rhap(1 4 \ / 3 CH 3 (50%) - 138 -IV.4 Experimental  General methods Instrumentation used was the same as p r e v i o u s l y d e s c r i b e d (see S e c t i o n I I I . 4 , page 105). For descending paper chroma-tography the f o l l o w i n g s o l v e n t systems (v/v) were used: (A) (2:1:1) f r e s h l y prepared 1 - b u t a n o l - a c e t i c acid-water; (B) (8:2:2). e t h y l a c e t a t e - p y r i d i n e - w a t e r . A n a l y t i c a l g . l . c . s e p a r a t i o n s were performed u s i n g s t a i n l e s s s t e e l columns (1/8 i n c h x 6 fe e t ) w i t h a c a r r i e r gas flow r a t e of 20 ml/ min. Columns used were (A) 0.2% of p o l y e t h y l e n e g l y c o l s u c c i n a t e , 0.2% of p o l y e t h y l e n e g l y c o l a d i p a t e , 0.4% of XF 1150 on Gas Chrom Q (100/120 mesh); (B) 3% of HIEFF IB on the same support. Analogous columns (1/4 i n c h x 6 fe e t ) were used f o r p r e p a r a t i v e g . l . c . s e p a r a t i o n . P r e p a r a t i o n and p r o p e r t i e s of K l e b s i e l l a K70 c a p s u l a r p o l y s a c c h a r i d e . A c u l t u r e of K l e b s i e l l a K70, obt a i n e d by c o u r t e s y of Dr. I. 0rskov, was grown and i s o l a t e d as d e s c r i b e d p r e v i o u s l y 3 f o r K l e b s i e l l a K36 . The p o l y s a c c h a r i d e showed [a] -43° D (c 2.8, water). The "''H n.m.r. spectrum of K70 was recorded on a s o l u t i o n of 20 mg/ml i n D 20 a t approximately 90° and 13 the C n.m.r. spectrum was recorded on a s o l u t i o n of 150 mg/ml i n 50% D„0 a t 40°. - 139 -Sugar analysis. 3 This was performed as described before , v i z . methanolysis, reduction with sodium borohydride in anhydrous methanol, hydrolysis, reduction and acetylation. The a l d i t o l acetates derived from rhamnose, galactose and glucose were separated by g. l . c . (column A, programmed from 120° to 190° at 1° per minute), and found to be present in the r a t i o of 31:17:52 respectively. Preparative g. l . c . gave g a l a c t i t o l hexaacetate (m.p. 166-168°) and g l u c i t o l hexaacetate (m.p. 99°). C i r c u l a r MeCN dichroism of the rhamnitol pentaacetate showed ^213 ~ 1*52, MeCN and of the g l u c i t o l hexaacetate showed ^213 + 1«83. By comparison with authentic standards t h i s confirmed the L-and D- configurations of the two sugars respectively. The configuration of the galactose residue was determined by examining the c i r c u l a r dichroism curve of l , 3 , 5 - t r i - 0 -acetyl-2,4,6-tri-0-methyl-galactitol, obtained during MeCN methylation analysis. This component showed ^213 + u - z ^ and by comparison with an authentic standard, established the galactose as being i n the D-configuration. 8 9 Methylation analysis. ' A sample of K l e b s i e l l a K70 which had previously been passed through a column of Amberlite IR-120(H+) was g methylated by the Hakomori procedure. Methylation was found to be incomplete and treatment with s i l v e r oxide in methyl iodide (Purdie) was required to obtain a product - 140 -showing no absorbance at 3600 cm in the i . r . spectrum. A XH n.m.r. spectrum of t h i s material showed a broad doublet at T 8.76 (due to the CH^ of rhamnose) which integrated as 9 protons and a sharp si n g l e t at x 8.43 (due to the CH^ of the pyruvate a c e t a l ) . These two signals were in the r a t i o of 6:1. In the anomeric region signals were observed at x 4.81,. 2H, J „ broad; T 5.05, 2H, J , „2Hz; T 5.27, IH, J± 2 7 Hz; and x 5.52, IH, J1 2 7 Hz-Reduction of a sample of methylated K70 with lithium aluminum hydride in reflux i n g tetrahydrofuran overnight yielded a product with no carbonyl absorption (1750 cm i) in the i . r . . Hydrolysis with 2 M t r i f l u o r o a c e t i c acid at 95° overnight and subsequent reduction with sodium boro-hydride followed by acetylation in pyridine-acetic anhydride yielded a mixture of p a r t i a l l y methylated a l d i t o l acetates. 9 11 This mixture was analysed by g.l.c.-m.s. ' . The a l d i t o l acetates of 3,4-di-O-methyl-L-rhamnose, 2,3-di-O-methyl-L-rhamnose, L-rhamnose, 3,4,6-tri-O-methyl-D-glucose, 2,4,6-tri-0-methyl-g-galactose and 2,3-di-0-methyl-g-glucose were i d e n t i f i e d in the r a t i o of 1.5:1:0.5:1:1:1. (See Table IV.2, page 128 for exact r a t i o s and column used.) A sample of K l e b s i e l l a K7 0 which had been autohydrolysed on a steam-bath for 16 hours at pH 2.2 was methylated as described for the native K70 polysaccharide. In the XH n.m.r. spectrum the sin g l e t at T 8.43 was absent. Reduction with lithium aluminum hydride, hydrolysis, reduction with - 141 -sodium borohydride and a c e t y l a t i o n as f o r the non-degraded, methylated m a t e r i a l y i e l d e d a mixture of p a r t i a l l y methylated 9 11 a l d i t o l a c e t a t e s which were analysed by g.l.c.-m.s. ' The same components as o b t a i n e d from methylated n a t i v e K70 were i d e n t i f i e d w i t h the e x c e p t i o n t h a t no L-rhamnose was present. The r a t i o s of the f i v e components were 2:1:1:1:1. (See Table IV.2, page 128 f o r exact r a t i o s and g . l . c . column used.) The concomitant l o s s of the 1 , 2 , 3 , 4 , 5 - t e t r a - 0 - a c e t y l -L-rhamnitbl and g a i n i n the amount of 1 , 2 , 5 - t r i - 0 - a c e t y l -3,4-di-methyl-L-rhamnitol i n d i c a t e s t h a t the pyruvate r e s i d u e was p r e s e n t i n the n a t i v e p o l y s a c c h a r i d e as an a c e t a l span-n i n g p o s i t i o n s t h r e e and four of a 2 - l i n k e d rhamnose. P a r t i a l a c i d i c h y d r o l y s i s . K l e b s i e l l a K70 p o l y s a c c h a r i d e (500 mg) was h y d r o l y s e d w i t h 0.5 M t r i f l u o r o a c e t i c a c i d a t 95° f o r 45 minutes. A f t e r removal of the a c i d by s e v e r a l s u c c e s s i v e e v a p o r a t i o n s w i t h water, the m a t e r i a l was d i a l y s e d a g a i n s t tap water o v e r n i g h t . The n o n d i a l y s a b l e m a t e r i a l (250 mg) was l y o p h i l i s e d and s u b j e c t e d to the same treatment as above, i . e . h y d r o l y s i s and d i a l y s i s . The d i a l y s a b l e m a t e r i a l from the h y d r o l y s e s (400 mg) was then separated i n t o n e u t r a l and a c i d i c components u s i n g Dowex 1-X2 (formate form) r e s i n . The a c i d i c components were n e u t r a l i s e d with sodium hydroxide and then a p p l i e d to the top of a B i o - G e l P-4 column (1.8 x 100 cm) which was subsequently i r r i g a t e d with - 142 -water at a flow r a t e o f 4 ml/h. F r a c t i o n s (1-2 ml) were c o l l e c t e d , l y o p h i l i s e d and analysed by paper chromatography. A major component C l , 50 mg) w i t h R Q ^ c 1.0 (s o l v e n t A) and [ o c ] D -30° (c 1.3, water) was obt a i n e d . "*"H n.m.r. of 1 gave s i g n a l s a t t r i b u t a b l e to anomeric protons a t x 5.28, IH, j ' 1 2 8 Hz; x:4.89, 0.6H, J 1 2 1.8 Hz; and x 5.14, 0.4H, s i n g l e t . A 6 Hz doublet (3 protons) a t x 8.70 was a l s o apparent (see Appendix I I I , spectrum No. 15). T h i s spectrum i s c o n s i s t e n t w i t h 1, being an a l d o b i o u r o n i c a c i d . w i t h a 13 r e d u c i n g rhamnose u n i t . In the C n.m.r. spectrum (see Appendix I I I , spectrum No. 16) a nonreducing s i g n a l a t 103.70 p.p.m. and 2 re d u c i n g s i g n a l s a t 94.55 p.p.m. and 94.23 p.p.m. were observed i n the anomeric r e g i o n . No s i g n a l a t t r i b u t a b l e to the CR of a hexose was observed w h i l e D a s i g n a l a t 17.82 p.p.m. (CH^ of 6-deoxy sugar) was apparent. Reduction of .1 w i t h l i t h i u m b o r o d e u t e r i d e and subsequent m e t h y l a t i o n , h y d r o l y s i s (2 M t r i f l u o r o a c e t i c a c i d , 95°, overnight) r e d u c t i o n w i t h sodium borohydride and a c e t y l a t i o n y i e l d e d two components i d e n t i f i e d by g.l.c.-m.s. (column B) as 1 , 5 , 6 - t r i - 0 - a c e t y l - 2 , 3 , 4 - t r i - O - m e t h y l - D - g a l a c t i t o l and 4-0 - a c e t y l - l , 2 , 3 , 5 - t e t r a - O - m e t h y l - L - r h a m n i t o l . The l a t t e r component was monodeuterated at . (Some of the t e t r a m e t h y l r h a m n i t o l d e r i v a t i v e was l o s t under d i m i n i s h e d p r e s s u r e d u r i n g work up.) The n e u t r a l oligomers from the p a r t i a l h y d r o l y s i s of K70 were separated by g e l chromatography u s i n g B i o - G e l P-4 - 143 -also. Besides obtaining large quantities of monosaccharides, an oligomer (12 mg) with 0.50 (solvent A) and [ot] +10° (c 1.2, water) was isola t e d . A XH n.m.r. spectrum of 2 (D20, 90°) gave signals i n the anomeric region as follows: x 5.34, IH, J 7 Hz (broad); x 4.85, IH, J 0 3.5 Hz; x 4.59, 0.6 H, J± 2 1.8 Hz and x 5.13, 0.4 H, 2 s i n g l e t . A doublet at x 8.70, 6 Hz, integrating as three protons was also observed (see Appendix II I , spectrum 13 No. 17). In the C n.m.r. spectrum (see Appendix III, spectrum No. 18), besides a signal at 17.56 p.p.m. a t t r i -butable to the CH^ of a 6-deoxy sugar, f i v e signals assigned to anomeric carbons were observed. Two signals from the of hexoses were also apparent (refer to Discussion and Table IV.1, page 124, for the assignment of the signals in the anomeric region). Compound 2 was reduced with lithium borodeuteride and subsequently methylated. Hydrolysis with 2 M t r i f l u o r o -acetic acid, reduction with sodium borohydride and acetyla-t i o n yielded a l d i t o l acetates corresponding to 2,3,4,6-tetra-0-methyl-D-glucose and 2,4,6-tri-O-methyl-D-galactose together with 2-0-acetyl-l,3,4,5-tetra-O-methyl-L-rhamnitol. G.l.c. and g.l.c.-m.s. were performed using column B. Some (^50%) of the tetra-0-methyl-rhamnitol derivative, monodeuterated at C1, was l o s t under diminished pressure during.work up. - 1 4 4 -Uronic A c i d D e g r a d a t i o n . ± Z A sample of K l e b s i e l l a K70 which had been autohydrolysed at pH 2.2 f o r 16 hours a t 95° (pyruvate a c e t a l removed) was methylated and c a r e f u l l y d r i e d . T h i s m a t e r i a l (140 mg) was then d i s s o l v e d i n 20 ml of a mixture of d i m e t h y l s u l p h o x i d e and 2,2-dimethoxy-propane (19:1). To t h i s s o l u t i o n 5 mg of t o l u e n e - p - s u l p h o n i c a c i d was added and the mixture was s t i r r e d under n i t r o g e n f o r 2 hours. M e t h y l s u l p h i n y l anion (2 M, 15 ml) was then added and the r e a c t i o n was s t i r r e d o v e r n i g h t . F o l l o w i n g the a d d i t i o n of 50% aqueous a c e t i c a c i d to a d j u s t the pH to 6.0 the s o l u t i o n was e x t r a c t e d with c h l o r o f o r m (3 x 25 ml). The combined c h l o r o f o r m e x t r a c t s were back e x t r a c t e d w i t h water (25 ml). The methylated m a t e r i a l o b t a i n e d from the c h l o r o f o r m e x t r a c t i o n was then t r e a t e d with 10% aqueous a c e t i c a c i d a t 95° f o r 1 hour. Removal of the a c e t i c a c i d was achieved by l y o p h i l i s a -t i o n . The degraded m a t e r i a l was then reduced w i t h sodium borohydride i n dry methanol o v e r n i g h t to g i v e a component !3 and t h i s reduced product was d i v i d e d i n t o two equal p o r t i o n s . One p o r t i o n was methylated u s i n g methyl i o d i d e and s i l v e r o xide. P u r i f i c a t i o n on s i l i c a g e l ( e t h y l acetate) y i e l d e d component 3a (7 mg) with R„ 0.15 ( e t h y l a c e t a t e ) . "'"H n.m.r. of 3a (CDCl^) r e v e a l e d s i g n a l s i n the anomeric r e g i o n a t x 4.88 (2H, J 1 2 broad) and x 5.08 (IH, 2 3 Hz). Two doublets (6 Hz each) at x 8.72 and x 8.76, each i n t e g r a t -i n g as t h r e e protons were a l s o apparent (see Appendix I I I , - 145 -spectrum No. 19). E l e c t r o n impact m.s. of 3a showed, i n t e r  a l i a , the f o l l o w i n g peaks: m/e 88(45), 101(28), 157(29), 189(100), 235(26), 295(19), 331(9), 363(9), 499(4), 535(3), 567(2) and 673(4). H y d r o l y s i s of 3a (2 M t r i f l u o r o a c e t i c a c i d , 95°, 6 hours) and g.l.c.-m.s. a n a l y s i s of the a l d i t o l a c e t a t e d e r i v a t i v e s of the p a r t i a l l y methylated sugars gave peaks corr e s p o n d i n g to 1,2,4,5,6-penta-0-methyl-p-galactitol, 2,3,4-tri-O-methyl-L-rhamnose, 3,4-di-O-methyl-L-rhamnose and 3, 4 , 6-tri-O-methyl-D-glucose. A second p o r t i o n of 3, was e t h y l a t e d u s i n g e t h y l i o d i d e and s i l v e r oxide and f o l l o w -i n g p u r i f i c a t i o n on s i l i c a g e l ( e t h y l acetate) y i e l d e d a component 3b (6.2 mg) w i t h R p 0.19 ( e t h y l a c e t a t e ) . H y d r o l y s i s of 3b and a n a l y s i s by g.l.c.-m.s. as a l d i t o l a c e t a t e s gave peaks corr e s p o n d i n g to 4-0-ethyl-2,3-di-0-methyl-|j-rhamnose 1 , 5 - d i - 0 - e t h y l - 2 , 4 , 6 - t r i - O - m e t h y l - D - g a l a c t i t o l , 3,4-di-O-methyl-L-rhamnose and 3,4,6-tri-O-methyl-D-glucose. P e r i o d a t e O x i d a t i o n of K70 p o l y s a c c h a r i d e . A sample (400 mg) of n a t i v e p o l y s a c c h a r i d e was d i s s o l v e d i n 100 ml of a s o l u t i o n of NaI0 4 (0.05 M) and NaC10 4 (0.2 M). The pH of t h i s s o l u t i o n was 4.2. The r e a c t i o n was allowed to proceed a t 4° i n the dark and the p e r i o d a t e consumption was f o l l o w e d by removing 5 ml a l i q u o t s -which 18 were analysed by the M u l l e r - F r i e d b e r g e r method. P e r i o d a t e consumption reached a p l a t e a u v a l u e a f t e r 26 hours of 6 moles per repeat u n i t of K l e b s i e l l a K70 ( t h e o r e t i c a l ; 9 - 146 -moles). Following the addition of ethylene g l y c o l , d i a l y s i s , reduction with sodium borohydride and l y o p h i l i s a t i o n , the modified polysaccharide was subjected to a second oxidation as described above. A further 4.2 moles of periodate per repeat unit of K70 was consumed afte r 20 hours and t h i s value gradually increased to 5.0 moles after 156 hours. Conversion of t h i s material to the polyalcohol (as above) yielded 250 mg. of a non-dialysable polymer which did not contain any pyruvate acetal (as shown by "'"H n.m.r.). Periodate oxidation of K70 polysaccharide (1 g) was also performed using a solution of NalO^ (0.05 M) , NaClC>4 (0.2 Mj and buffered at pH 5.0 with a sodium acetate buffer. After 24 hours the reaction was stopped by the addition of ethylene g l y c o l and af t e r d i a l y s i s overnight against running tap water, the material was reduced with sodium borohydride. Excess hydride was destroyed by the addition of acetic acid and the pH of the solution was adjusted to 6.5. D i a l y s i s and l y o p h i l i s a t i o n yielded 950 mg of a polymeric material. Treatment again with NaIC>4 and NaC104 at pH 5.0 as described above and subsequent work up yielded 7 00 mg of a polymer which was considered to have undergone complete periodate oxidation. A sample of the periodate oxidised product was methylated by the Hakomori procedure and then reduced using lithium aluminum hydride in r e f l u x i n g tetrahydrofuran overnight. Subsequent hydrolysis (2 M t r i f l u o r o a c e t i c acid, 95°, over-- 147 -n i g h t ) , r e d u c t i o n and a c e t y l a t i o n y i e l d e d a mixture of a l d i t o l a c e t a t e s which were analysed by g.l.c.-m.s. (column A ) . The a l d i t o l a c e t a t e s o f L-rhamnose and 2 , 4 , 6 - t r i - 0 -methyl-D-galactose were found to be present i n the r a t i o of 1:2. The remainder of the p e r i o d a t e o x i d i s e d m a t e r i a l was then s u b j e c t e d to a Smith h y d r o l y s i s u s i n g 0.5 M t r i f l u o r o -a c e t i c a c i d a t room temperature o v e r n i g h t and reduced wi t h sodium borohydride. The mixture o f products was then a p p l i e d t o the t o p of a B i o - G e l P-2 column (160 x 2.5 cm) which was i r r i g a t e d with d i s t i l l e d water. Three pure oligomers (£, 5^, 6) were i s o l a t e d . Compound £ (60 mg) , R„, 0.92 ( s o l v e n t A), had [ a ] ^ -16° (c 2.1, water). In the XH n.m.r. spectrum s i g n a l s a t t r i b u t a b l e to anomeric protons were observed a t x 4.78, IH, J, „ 1.8 Hz and x 5.43, IH, J 2 7.5 Hz (see Table IV.1, page 124, and Appendix I I I , 13 spectrum No. 20). The C n.m.r. spectrum of £ (see Appendix I I I , spectrum No. 21), was not completely c o n s i s t e n t with the proposed s t r u c t u r e f o r £ due to the very slow r e l a x a t i o n of the t e r m i n a l e r y t h r o n i c a c i d moiety. H y d r o l y s i s of 4 (2 M t r i f l u o r o a c e t i c a c i d , 95°, 6 hours) and paper chromato-graphy ( s o l v e n t A) r e v e a l e d the presence of rhamnose, g a l a c t o s e and two f u r t h e r components w i t h m o b i l i t i e s e q u i v a l e n t to e r y t h r o n o l a c t o n e and e r y t h r o n i c a c i d r e s p e c t i v e l y . Methyla-t i o n of 4_ gave a component which showed *a st r o n g absorbance at 1750 cm 1 i n the i . r . spectrum. The mass spectrum of - 148 -permethylated £ ( c i . ) showed, i n t e r a l i a , the f o l l o w i n g peaks: m/e 111(64), 155(17), 186(15), 187(99), 219(55), 303(17), 361(100), 362 (96), 539(13),. 568 (2), 569 (3. 0) and 570(1.5). Reduction of permethylated 4, wit h sodium borohydride i n dry methanol and subsequent h y d r o l y s i s , r e d u c t i o n and a c e t y l a -t i o n gave a mixture of a l d i t o l a c e t a t e s which were analysed by g.l.c.-m.s. The a l d i t o l a c e t a t e s of 2,3,4,6-tetra-0-methyl-D-galactose and'3,4-di-O-methyl-L-rhamnose i n equal amounts were i d e n t i f i e d . (The v o l a t i l e di-p-methyl e r y t h r i t o l d e r i v a t i v e was l o s t under reduced p r e s s u r e d u r i n g work up.) Oligomer !5 (60 mg) , R G^ C 0.96 (s o l v e n t A), had [ a ] D +4.5° (c 1.5, water). In the 1H n.m.r. spectrum (D 20, 90°) s i g n a l s were observed i n the anomeric r e g i o n a t T 4.78, IH, J 1 2 2 Hz and x 5.43, IH, J± 2 7 Hz. A doublet (6 Hz) a t T 8.70 i n t e g r a t i n g as three protons was a l s o p r e s e n t (see 13 Appendix I I I , spectrum No. 22). In the C n.m.r. spectrum (see Appendix I I I , spectrum No. 23) 16 s i g n a l s o v e r a l l were observed. Two s i g n a l s (105.75 p.p.m. and 100.19 p.p.m.) were assig n e d as a r i s i n g from anomeric carbons w h i l e three s i g n a l s appeared i n the r e g i o n a s s o c i a t e d w i t h the of 13 hexoses (60 to 62 p.p.m.). The C n.m.r. spectrum i s i n agreement with an oligomer comprising a hexose sugar, a 6-deoxy sugar and a four-carbon aglycone. H y d r o l y s i s of 5 (2 M t r i f l u o r o a c e t i c a c i d , 95°, 4 hours) r e v e a l e d g a l a c t o s e , rhamnose and e r y t h r i t o l by paper chromatography ( s o l v e n t A). M e t h y l a t i o n of 5 y i e l d e d a product w i t h R^ 0.23 ( e t h y l - 149 -acetate). The mass spectrum ( c i . ) of permethylated f> showed, inter a l i a , the following peaks: m/e 71(65), 88(55), 101(49), 103(40), 112(100), 133(75), 145(39), 155(68), 163(75), 186(60), 220(99), 307(64), 337(30), and 338 (6). Hydrolysis of permethylated J5 and subsequent d e r i v a t i s a t i o n yielded a l d i t o l acetates corresponding to 2,3,4,6-tetra-O-methyl-g-galactose and 3,4-di-O-methyl-L-rhamnose. The v o l a t i l e tri-0-methyl e r y t h r i t o l derivative was l o s t under vacuum during work up. Compound £ (20 mg) , 1*14 (solvent A), had [a] +3.7° (c 0.8, water). In the XH n.m.r. spectrum (D2 0' 90°) only one signal at x 5.45, IH, J 1 ^ 7.5 Hz, was 13 observable i n the anomeric region. In the C n.m.r. spectrum nine signals were observed o v e r a l l . The presence of one signal (103.33 p.p.m.) i n the anomeric region and three signals between 60-62 p.p.m. are i n agreement with £ being a hexose sugar linked through C^ to a three carbon fragment (see Appendix III, spectra No.'s 24, 25). Hydrolysis (2 ^ t r i f l u o r o a c e t i c acid, 95°, 4 hours) of (5 and paper chromatography (solvent A) showed the presence of galactose and g l y c e r o l . Methylation of 6 gave a compound with R „ 0.34 (ethyl acetate) and the mass spectrum ( c i . ) of t h i s component showed, inter a l i a , the following peaks: m/e 71(65), 75(24), 88 (55), 101(49), 103(40), 112 (100), 127 (33), 133(75), 145(37), 155(68), 163(75), 186(60), 220(99), 307(64), and 337(30). Hydrolysis of t h i s permethylated derivative yielded - 150 -on l y 1,5-di-0-acetyl-2,3,4,6-tetra-O-methyl-D-galactose a f t e r d e r i v a t i s a t i o n as a l d i t o l a c e t a t e s . Once again, the v o l a t i l e di-0-methyl g l y c e r o l moiety was l o s t d u r i n g work up. - 151 -IV.5 B i b l i o g r a p h y f o r S e c t i o n IV. 1. W. Nimmich, A c t a B i o l . Med. Ger., 26_, 397 (1971). 2. W. Nimmich, Z. M i c r o b i o l . Immunol., 154, 117 (1968). 3. G.G.S. Dutton and K. Mackie, Carbohyd. Res., 55, 49 (1977). 4. Y.M. Choy, G.G.S. Dutton, A.M. Stephen and M.T. Yang, An a l . L e t t . , 5_, 675 (1972). 5. G.M. Bebault, Y.M. Choy, G.G.S. Dutton, N. F u n n e l l , A.M. Stephen and M.T. Yang, J . B a c t e r i o l . , 113, 1345 (1973). 6. J.M. Berry, G.G.S. Dutton, L.D. H a l l and K.L. Mackie, Carbohyd. Res., 53_, C8 (1977). 7. G.M. Bebault, J.M. Berry, Y.M. Choy, G.G.S. Dutton, N. F u n n e l l , L.D. Hayward and A.M. Stephen, Can. J . Chem., 51, 324 (1973). 8. S. Hakomori, J . Biochem. (Tokyo), 5_5, 205 (1964). 9. H. B j o r n d a l , C.G. H e l l e r q v i s t , B. Lindberg and S. Svensson, Angew. Chem. I n t . Ed. E n g l . , 9, 610 (1970). 10. B. Lindberg, J . Lonngren, J.L. Thompson and W. Nimmich, Carbohyd. Res., 25_, 49 (1972). 11. J . Lonngren and S. Svensson, Advan. Carbohydr. Chem. Biochem., 2J3, 41 (1974). 12. B. Lindberg, J . Lonngren and J.L. Thompson, Carbohyd. Res., 28, 351 (1973). 13. G.W. Hay, B.A. Lewis and F. Smith, Methods Carbohyd . Chem., 5, 357 (1965). 14. J.E. S c o t t and R.J. Harbinson, Histochemie, IA, 215 (1968). - 152 -15. T. Painter and B. Larsen, Acta Chem. Scand., 2_4, 813 (1970). 16. I.J. Goldstein, G.W. Hay, B.A. Lewis and F. Smith, Methods Carbohyd. Chem., 5, 361 (1965). 17. O.S. Chizhov, V.I. Kadentsev and A.A. Solov'yov, J. Org. Chem., 41, 3425 (1976). '18. E. Muller and 0. Friedberger, Ber., 35, 2652 (1902). - 153 -V. THE STRUCTURE OF KLEBSIELLA SEROTYPE K3 2 CAPSULAR POLYSACCHARIDE - 154 -V.1 Abstract The capsular polysaccharide from K l e b s i e l l a K32 has been studied using methylation, periodate oxidation and p a r t i a l hydrolysis techniques. The polysaccharide i s shown to com-prise a four sugar repeating unit as shown below. Features of i n t e r e s t i n t h i s structure include the presence of a 3-linked J^-rhamnose sugar, and the extreme acid l a b i l i t y of the pyruvate acetal. N.m.r. has been used extensively to est a b l i s h the nature of the anomeric linkages and to i d e n t i f y oligosaccharides obtained by the various degradative techniques used. +3 ) -a-D-Galp (1+2) -a-^-Rhap- (l->3) -8-L-Rhap- (1+4 ) -a-4 \ ? L-Rhap(l+ C CE^ \ : O O H V. 2 Introduction 1 2 Nimmich ' , i n a q u a l i t a t i v e analysis of the capsular polysaccharide from K l e b s i e l l a K32, found the presence of only galactose, rhamnose, and pyruvic acid. The absence of any uronic acid indicates that as a capsular antigen the polysaccharide r e l i e s on the pyruvic acid acetal for i t s o v e r a l l negative charge and hence i t s virulence. K l e b s i e l l a 3 4 K types 56 and 72 have been investigated previously and the present study completes the s t r u c t u r a l analysis of t h i s t r i o of K l e b s i e l l a serotypes i n which pyruvic acid i s the only acid component. - 155 -V.3 Results and Discussion Composition and n.m.r. spectra. Isolation and p u r i f i c a t i o n of the polysaccharide were 5 carried out as previously described . One Cetavalon p r e c i -p i t a t i o n was performed. The p u r i f i e d material had tct] D +113°. XH n.m.r. of the polysaccharide i n deuterium oxide showed the presence of four anomeric protons and also indicated that three 6-deoxy sugars (rhamnose residues) and one pyruvate acetal were present per repeating u n i t ^ ' ^ a ' ^ ^ . Acid hydrolysis of the polysaccharide and paper chromatography of the hydrolysate indicated the presence of rhamnose and galactose. Quantitation as a l d i t o l acetates proved these two sugars to be present in the proportions 72:28 respectively. The rhamnose was shown to belong to the L-series by c i r c u l a r dichroism g measurements on the a l d i t o l acetate while the galactose was confirmed as being in the Q-configuration by the action of D-galactostat on the free sugar. Methylation of o r i g i n a l and p a r t i a l l y depyruvalated poly- saccharide . Complete methylation of K32 proved to be d i f f i c u l t . Due to the l a b i l i t y of the pyruvate acetal (see later) the poly-saccharide could not be converted to the free acid form without the loss of a substantial proportion of the acetal. Methyla-tion of the polysaccharide was hindered by the poor s o l u b i l i t y - 156 -i n d i m e t h y l s u l f o x i d e of the n a t i v e m a t e r i a l i n the sodium 9 s a l t form. Two s u c c e s s i v e Hakomori treatments and a sub-sequent P u r d i e " ^ m e t h y l a t i o n were necessary to achieve a product which showed no hydroxyl absorbance i n the i n f r a 11 12 red. M e t h y l a t i o n a n a l y s i s ' of the n a t i v e p o l y s a c c h a r i d e and m a t e r i a l t h a t had been converted to i t s f r e e a c i d analogue by passage through ion-exchange r e s i n (Table V . l , page 157, columns I and II) i n d i c a t e d t h a t K l e b s i e l l a K32 i s composed of a l i n e a r t e t r a s a c c h a r i d e r e p e a t u n i t comprising t h r e e L-rhamnose r e s i d u e s ( l i n k e d through p o s i t i o n s two, t h r e e , and f o u r r e s p e c t i v e l y ) and one D-galactose r e s i d u e ( l i n k e d through p o s i t i o n t h r e e ) . The p y r u v i c a c i d u n i t i s present as an a c e t a l spanning p o s i t i o n three and f o u r of the two-linked g-rhamnose r e s i d u e . S e p a r a t i o n of the p a r t i a l l y methylated a l d i t o l a c e t a t e s c o r r e s p o n d i n g to the 2,3-, 2,4-, and 3,4-di-O-methyl-L-rhamnose d e r i v a t i v e s was achieved by gas l i q u i d chromatography u s i n g a p o l a r l i q u i d phase of HIEFF-1B (see Table V . l , page 157 f o r d e t a i l s ) . 14 P e r i o d a t e o x i d a t i o n P e r i o d a t e o x i d a t i o n of n a t i v e K32 p o l y s a c c h a r i d e pro-ceeded r a p i d l y and a f t e r 40 hours a n a l y s i s of the o x i d i s e d polymer (see Experimental) i n d i c a t e d complete o x i d a t i o n . The o x i d a t i o n was performed i n 0.05 M sodium p e r i o d a t e and was b u f f e r e d a t pH 6.5 to minimise h y d r o l y s i s of the pyruvate a c e t a l . However, the a n a l y s i s of the polymer a f t e r 40 hours - 157 -Table V . l M e t h y l a t i o n Analyses of Native and D e p y r u v a l a t e d 3 K l e b s i e l l a K32 Capsular P o l y s a c c h a r i d e Methylated s u g a r b T C Mole % d , e I I 3,4-Rha 0.88 - 15.1 2.3- Rha 0.91 26.8 25.6 2.4- Rha 1.00 27.0 23.3 Rha 1.53 21.6 8.2 2,4,6-Gal 1.61 24.7 28.0 "Conversion of the n a t i v e p o l y s a c c h a r i d e i n t o the ' f r e e a c i d ' form by passage through IR-120(H +) r e s u l t e d i n approximately 50% of the pyruvate a c e t a l groups being removed. 3,4-Rha = 3,4-di-O-methyl-L-rhamnose, e t c . R e t e n t i o n time of c o r r e s -ponding a l d i t o l a c e t a t e r e l a t i v e to 1,5-di-0-acetyl-2,3,4,6-t e t r a - 0 - m e t h y l - g - g l u c i t o l . Column used was 3% of HIEFF-IB on Gas Chrom Q (100-120 mesh) programmed from 160° to 190° at 1° per min. Column dimensions: 6' x 1/8". ^ F i g u r e s are c o r r e c t e d u s i n g molar response f a c t o r s given by Albersheim 13 e e t a l . I, n a t i v e p o l y s a c c h a r i d e ; I I , depyruvalated p o l y -s a c c h a r i d e . Table V.2 N.M.R. Data f o r K l e b s i e l l a K32 Capsular P o l y s a c c h a r i d e and I s o l a t e d O l i g o s a c c h a r i d e s . a Hn.m.r. C n.m.r. Compound r j / b ( J 1 „) C,integral,assignment^ p.p.m.e assignment^ l , z. 1 2 1 3 1 3 Ga l ^ ^ R h a ^ ^ - R h a ^ ^ - l - deoxy- D-a a 3 = e r y t h r i t o l 4.81 (1.8 Hz) , IH, a-Rha 4.92 (3.5 Hz), IH, a-Gal 5.21 (1 Hz), IH, 3-Rha 8.69 (J_ , 6 Hz), 6H, 5/6 8.79 (6 Hz), CH 3 of Rha's 3H, CH 3 of 1-deoxy-D - e r y t h r i t o l (22 s i g n a l s o v e r a l l ) 100.41) 100.35 98.42 62. 06 61.76 17.95^ 17. 55 17.36 J C, of Gal b C 4 of 1-deoxy-g - e r y t h r i t o l ^CH3 of Rha's ICH 3 of 1-deoxy-D - e r y t h r i t o l h-1 CO 1 3 Rha—£— 1-deoxy-D-erythrito1 p 2 or l b 5.24 (1 Hz), IH, 3-Rha 8.69 (J_ , 6 Hz), 3H, CH, of Rha 8.79 (6 Hz), 3H, CH 3 of 1-deoxy-D - e r y t h r i t o l (10 s i g n a l s o v e r a l l ) 100.69 3-Rha 62.10 C 4 of 1-deoxy-D = e r y t h r i t o l CH 3 of Rha CH., of 1-deoxy-D - e r y t h r i t o l Table V.2 Continued 1 2 Gal g l y c e r o l Ga l ^ ^ R h a ^ - R h a ^ ^ h a - OH a a 8 4.83 (3 Hz), a-Gal 4.80 4. 87 4. 94 5.12 5. 25 8.69 8.79 (1.8 Hz), IH, a-Rha (1.8 Hz), 0.6H, a-Rha-OH (3 Hz), IH, a-Gal (1 Hz), 0.4H, 8-Rha-OH (1 Hz), IH, B-Rha (9 s i g n a l s o v e r a l l ) 98.89 a-Gal 61.28^ 62. 01 62.28 J (Cr of Gal 6 (c^,C 3 of g l y c e r o l I n s u f f i c i e n t m a t e r i a l ( J5,6 6 (6 Hz) , Hz), 6H, 3H, CH., of Rha's CH 3 of 1-deoxy-g-e r y t h r i t o l 3_ .1 2 n, 1 3„, 1 4^ u 1 — G a l Rha Rha R h a — a 3 \ / 4 a g a C • / \ - + CH 3 COO Na NATIVE K3 2 POLYSACCHARIDE 4.8 0 (2 Hz), IH, a-Rha 4.93 (b), 2H, a-Gal and a-Rha Incomplete 5.25 (S), IH, 3-Rha (S), 3H, CH 3 o: ( J c , 6 Hz), 9H, CH 0 of Rha's D , b j 8.41 ), f a c e t a l 8.70 For o r i g i n o f o l i g o s a c c h a r i d e s 1, 2, 3 and 4 see t e x t . Chemical s h i f t taken r e l a t i v e r^j fsj c d to i n t e r n a l acetone; T 7.77 downfield from D.S.S. b=broad, S = s i n g l e t . e.g. a-Rha= (continued over page) Table V.2 Continued proton on C^ of L-Rha r e s i d u e which i s a - l i n k e d . (Gal=g-Gal). e C h e m i c a l s h i f t quoted as p.p.m. downfield from T.M.S. r e l a t i v e to i n t e r n a l acetone; 31.07 p.p.m. f e 13 from T.M.S. As f o r , but f o r anomeric . C n u c l e i . CTl o I - 161 -o x i d a t i o n at t h i s pH i n d i c a t e d t h a t some (15%) of the pyruvate a c e t a l s had i n f a c t been removed. The d e r i v e d p o l y o l was 15 s u b j e c t e d to a Smith d e g r a d a t i o n and the r e s u l t i n g mixture of o l i g o s a c c h a r i d e s was separated u s i n g g e l chromatography. Three c h r o m a t o g r a p h i c a l l y pure components, 1, 2, 3, were i s o l a t e d . 1 13 Component 1. (120 mg) showed [a] +102°. H and C n.m.r. (see Appendix I I I , spectrum No. 26) s t u d i e s i n d i c a t e d 1 t o be a t r i s a c c h a r i d e g l y c o s i d e c o n s i s t i n g of one g a l a c t o s e r e s i d u e ( a-linked), two rhamnose r e s i d u e s (one a-linked and the other 3 - l i n k e d ) , and 1 - d e o x y e r y t h r i t o l (see Table V.2, page 158 f o r data) . The XH n.m.r. spectrum of 1. i s shown (see F i g u r e V . l , page 162) and c l e a r l y demonstrates the presence of th r e e non-reducing anomeric proton s i g n a l s . The s i g n a l at T 5.21 wit h J.^ ^ 1 Hz i s from the anomeric proton of the 8 - l i n k e d ^-rhamnose r e s i d u e and i s e a s i l y d i s t i n g u i s h -able from the anomeric proton s i g n a l (x 4.81, J, 9 1.8 Hz) l , ^ of the a-linked L-rhamnose r e s i d u e . The f u l l assignment of the spectrum appears i n Table V.2, page 158. F i e l d d e s o r p t i o n 16 1 "7 mass spectrometry ' of 1 gave peaks a t m/e 561, 583 and 599 cor r e s p o n d i n g t o (M+l) +, (M+Na) + and (M+K) + r e s p e c t i v e l y . P e r i o d a t e o x i d a t i o n of 1 and subsequent r e d u c t i o n , Smith d e g r a d a t i o n and g e l f i l t r a t i o n y i e l d e d a component l b , w i t h 1 13 [a]Q +104°. H and C n.m.r. i n d i c a t e d l b i s composed of one rhamnose r e s i d u e ( 8-linked) and a d e o x y e r y t h r i t o l fragment. (See Table V.2, page 158, and Appendix I I I , s p e c t r a No.'s 27, CH 2 0H T4.81 (0 1 > 2 i .eHz) C H 2 O H Acetone Internal Standard (T777) a-Rha a-Gal /3-Rha CH3S Of two [T8.67 ^ L - r h a m n o s e IT 8.69 A (8.79 CH^of 1-deoxy-D erythr itol 5.0 5.2 8.0 a2 a4 8.6 8.8 T F i g u r e V . l 4.8 1H n.m.r. spectrum of a-D-Galp-(1+2)-a-L-Rhap-(1+3)-8-L-Rhap-(1+3)-1-deoxy-D - e r y t h r i t o l . (1) " • to - 163 -28, f o r n.m.r. d a t a ) . H y d r o l y s i s of a s m a l l p o r t i o n of l b and paper chromatography of the h y d r o l y s a t e gave two compon-ents i n d i s t i n g u i s h a b l e from rhamnose and 1 - d e o x y e r y t h r i t o l . F i e l d d e s o r p t i o n mass spectrometry of l b gave peaks at m/e 253, 275 and 291, c o r r e s p o n d i n g to (M+l) +, (M+Na) + and (M+K) + r e s p e c t i v e l y . The s u r v i v a l of the d e o x y e r y t h r i t o l (which i s o b t a i n e d i n i t i a l l y from p e r i o d a t e a t t a c k a t a four l i n k e d L-rhamnose res i d u e ) d u r i n g the p e r i o d a t e o x i d a t i o n of o l i g o s a c c h a r i d e 1 i n d i c a t e s t h a t the l i n k a g e to t h i s t e r m i n a t i n g g l y c o s i d e must be to p o s i t i o n t h r e e . The s t r u c t u r e of component l b i s t h e r e f o r e e s t a b l i s h e d as being B-L-Rhap-(1+3)-1-deoxy-D-erythritol (lb) The s t r u c t u r e of o l i g o s a c c h a r i d e 1 can t h e r e f o r e be w r i t t e n as a-Q-Galp-(1+2)-a-L-Rhap-(1+3) -B-L-Rhap-(1+3)-1 - d e o x y - D - e r y t h r i t o l (1) O l i g o s a c c h a r i d e 2 (20 mg), obtained from the p e r i o d a t e o x i d a t i o n of the n a t i v e p o l y s a c c h a r i d e , was i n d i s t i n g u i s h a b l e from component l b (above) i n every r e s p e c t . O l i g o s a c c h a r i d e 3 (20 mg) , a l s o obtained from the p e r i o -date o x i d a t i o n 'of the n a t i v e p o l y s a c c h a r i d e , showed [ a ] ^ +118°. - 164 -CHPOH CH2OH 85 7c i. IO4 (15% of pyruvate acetal cleaved) II. NaBH4 111. Smith degradation 15% CH2OH CHoOH n.NaBH4 .. iii.Smith degradation lb = 2 F i g u r e V.2 Scheme f o r p e r i o d a t e o x i d a t i o n of K32 c a p s u l a r p o l y s a c c h a r i d e . Hydrolysis and paper chromatography revealed the presence of 1 13 only galactose and g l y c e r o l . H and C n.m.r. studies (see Table V.2, page 158, and Appendix III , spectrum No. 29 for data), were in agreement with 3, comprising one D-galactose moiety a-linked to a gl y c e r o l fragment. F i e l d desorption mass spectrometry of _3 gave peaks at m/e 255, 277 and 293, corresponding to (M+l) +, (M+Na)+ and (M+K)+ respectively. The structure of oligosaccharide 3 i s therefore established as being a-D-Galp- (l->2)-glycerol {3) The r e s u l t s of the periodate oxidation studies on K l e b s i e l l a K32 are summarised i n Figure V.2, page 164. I d e n t i f i c a t i o n and characterisation of components 1, l b , 2 t~^> and 3^ establishes the tetrasaccharide repeating structure of the polysaccharide and also locates the pyruvate acetal. P a r t i a l hydrolysis. Although the structure of K32 capsular polysaccharide can be deduced from the periodate oxidation data .' (above) , supporting evidence was sought using the technique of p a r t i a l hydrolysis. Mild acid hydrolysis of K32 native polysaccharide resulted i n very non-specific cleavage of the gly c o s i d i c linkages. As a r e s u l t a large number of very similar o l i g o -saccharides was simultaneously released. Examination by paper chromatography of a progressive acid hydrolysis of a - 166 -s m a l l amount of n a t i v e p o l y s a c c h a r i d e i n d i c a t e d t h a t maximum o l i g o s a c c h a r i d e p r o d u c t i o n o c c u r r e d a f t e r 9 hours treatment at 95° with 0.03 M t r i f l u o r o a c e t i c a c i d . A l t e r n a t i v e l y , u s i n g the p o l y s a c c h a r i d e i n i t s ' f r e e a c i d ' form (pH 3.0) maximum o l i g o s a c c h a r i d e p r o d u c t i o n o c c u r r e d a f t e r 18 hours a u t o h y d r o l y s i s at 95°. Using the l a t t e r c o n d i t i o n s a l a r g e r sample of K32 was h y d r o l y s e d and then d i a l y s e d a g a i n s t a f i x e d volume of water. The d i a l y s a b l e m a t e r i a l was examined by g e l chromato-graphy and a pure o l i g o s a c c h a r i d e £ (4 mg) was o b t a i n e d . XH n.m.r. (see Appendix I I I , spectrum No. 30) i n d i c a t e d £ to be a t e t r a s a c c h a r i d e c o n t a i n i n g three L-rhamnose r e s i d u e s , one of which was r e d u c i n g (see Table II f o r XH n.m.r. data and i n t e r p r e t a t i o n ) . Reduction of £ w i t h sodium b o r o d e u t e r i d e and subsequent m e t h y l a t i o n a n a l y s i s y i e l d e d the a c e t y l a t e d d e r i v a t i v e s of 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l - g - g a l a c t i t o l , 3,4-di-O-methyl-L-rhamnitol, 2,4-di-O-methyl-L-rhamnitol and, from the r e d u c i n g terminus of £, 1,2,3,5-tetra-O-methyl-L-rhamnitol. These components were ob t a i n e d i n approximately equal amounts but some of the l a s t named component, monodeuterated a t C^, was l o s t under reduced pressure d u r i n g d e r i v a t i s a t i o n . In l i g h t of the sequence of o l i g o s a c c h a r i d e 1, o b t a i n e d by p e r i o d a t e o x i d a t i o n the s t r u c t u r e of the r e d u c i n g t e t r a -s a c c h a r i d e 4 may be w r i t t e n as shown below a-g-Galp-(1+2)-a-L-Rhap-(1+3)-B-L-Rhap (l+4)-L-Rhap (4) During the i s o l a t i o n of £ by g e l f i l t r a t i o n , d i f f i c u l t i e s i n s e p a r a t i n g t h i s o l i g o s a c c h a r i d e from many other c h e m i c a l l y s i m i l a r components were encountered. T h i s r e s u l t e d i n an extremely low y i e l d of p u r i f i e d £. The t e t r a s a c c h a r i d e does, however, support the data o b t a i n e d from p e r i o d a t e o x i d a t i o n s t u d i e s . From the s t u d i e s r e p o r t e d above i t i s deduced t h a t the r e p e a t i n g - u n i t s t r u c t u r e f o r K l e b s i e l l a K32 c a p s u l a r p o l y -s a c c h a r i d e i s as shown below. +3)-a-D-Galp- (1+2)-a-L-Rhap- (1+3)-3-L-Rhap-4 \ } (1+4)-a-L-Rhap-(1+ cr C H 3 ' \ : O O H The e x i s t e n c e of a 3 - l i n k e d L-rhamnose sugar has been proposed i n Pneumococcus type II polysaccharide"'" 8'"'"^ and i n Pneumococcus 20 type XXVII p o l y s a c c h a r i d e , but we b e l i e v e t h i s to be the f i r s t u nequivocal evidence f o r the e x i s t e n c e of such a l i n k a g e . 4 I f L-rhamnose i s assumed to e x i s t i n the C-^  conformation as a pyranose sugar, then i t might be assumed t h a t a 3 - l i n k e d L-rhamnose r e s i d u e would be more s u s c e p t i b l e to a c i d h y d r o l y s i s than the comparable a - l i n k e d s t r u c t u r e . However, the i s o l a t i o n of an o l i g o s a c c h a r i d e (e.g., 4J by p a r t i a l h y d r o l y s i s i n which the 3 _ L _ r h a m n o s e l i n k a g e i s i n t a c t , suggests t h a t t h i s may not always be the case. - 168 -The pyruvate acetal in K32 i s perhaps the most acid sensitive acetal examined to date in the K l e b s i e l l a poly-saccharides. Part of the reason for t h i s s u s c e p t i b i l i t y to acid hydrolysis i s that the acetal i s spanning trans-diequatorial v i c i n a l hydroxyls. Only as a r e s u l t of a large degree of d i s t o r t i o n of the sugar ring can these two hydroxyl groups be brought into a reasonably planar orientation that i s necessary for the formation of t h i s type of acetal. The existence of a pyruvate acetal attached to the 3- and 4-poritions of a L-rhamnopyranosyl residue has been noted 4 21 previously ' . Pyruvate acetals spanning v i c i n a l trans-diequatorial hydroxyls (0-2 and 0-3) of a 4-linked D-22 glucuronosyl residue , and the same positions of a D-23 galactosyl residue , have been demonstrated. In a l l these cases the acetal has been found to be e a s i l y removed under mild acid conditions. V.4 Experimental General Methods. Equipment for m.s., n.m.r., g. l . c . and g.l.c.-m.s. was the same as i n the investigation of K l e b s i e l l a K36 poly-5 saccharide . F i e l d desorption mass spectrometry was performed using an A.E.I. M.S. 902 mass spectrometer equipped with an e . i . / f . i . / f . d . source. The columns used for g.l . c . separations were. (A) 3% of HIEFF-1B on Gas Chrom Q (100-120 mesh) and (B) 0.2% of polyethylene g l y c o l succinate, 0.2% of polyethylene - 169 -g l y c o l a d i p a t e , 0.4% of XF-1150 on the same support. For descending paper chromatography the f o l l o w i n g s o l v e n t systems (v/v) were used: (A) f r e s h l y prepared 2:1:1 1 - b u t a n o l - a c e t i c acid-water; (B) 8:2:2 e t h y l a c e t a t e - p y r i d i n e - w a t e r . P r e p a r a t i o n and p r o p e r t i e s of K32 c a p s u l a r p o l y s a c c h a r i d e . T h i s was performed as p r e v i o u s l y d e s c r i b e d 5 . The i s o l a t e d p o l y s a c c h a r i d e showed [ot] D +113° (c 3.8, water). "*"H n.m.r. spectroscopy was performed on the p o l y s a c c h a r i d e i n the sodium s a l t form and r e v e a l e d s i g n a l s i n the anomeric r e g i o n t h a t i n t e g r a t e d as f o u r protons r e l a t i v e to the s i g n a l s a t x 8.41 (3H, s i n g l e t ) and x 8.70 (9H, ^ approx. 6 Hz). For the assignment of the anomeric proton s i g n a l s see Table V.2, page 158. Sugar and m e t h y l a t i o n a n a l y s i s of n a t i v e and p a r t i a l l y d e p yruvalated p o l y s a c c h a r i d e s . Hydrolysis- of a sample of n a t i v e K32 p o l y s a c c h a r i d e with 2 M t r i f l u o r o a c e t i c a c i d f o r 6 hours a t 95° and subsequent d e r i v a t i s a t i o n of the l i b e r a t e d monosaccharides as a l d i t o l a c e t a t e s gave peaks corre s p o n d i n g to r h a m n i t o l p e n t a c e t a t e and g a l a c t i t o l hexaacetate i n the p r o p o r t i o n s 72:28 r e s p e c t i v e l y (Column B; programmed at 120° f o r 8 min and then at l°/min to 200°). P r e p a r a t i v e g . l . c . gave g a l a c t i t o l hexaacetate (m.p. 168°) and r h a m n i t o l p e n t a a c e t a t e . C i r c u l a r d i c h r o i s m MeCN of the l a t t e r component showed £„, -1.20 and by comparison - 170 -w i t h a u t h e n t i c standards confirmed the sugar as being i n the L - c o n f i g u r a t i o n . The c o n f i g u r a t i o n of the g a l a c t o s e was shown to be D_ by the a c t i o n of g - g a l a c t o s t a t on a p o r t i o n of the f r e e sugar i s o l a t e d by g e l f i l t r a t i o n of a h y d r o l y s e d sample of the p o l y s a c c h a r i d e . M e t h y l a t i o n of K3 2 c a p s u l a r p o l y s a c c h a r i d e i n the sodium 9 s a l t form was performed u s i n g the Hakomori procedure . D i f -f i c u l t y was encountered i n d i s s o l v i n g the p o l y s a c c h a r i d e i n d i m e t h y l s u l f o x i d e and a g i t a t i o n i n a s o n i c a t o r f o r two days at room temperature was needed to achieve complete s o l u t i o n . Two s u c c e s s i v e Hakomori m e t h y l a t i o n s were performed and these were f o l l o w e d by a Purdie"*"^ treatment u s i n g s i l v e r oxide i n methyl i o d i d e . The f i n a l product showed no absorbance at 3600 cm 1 i n the i . r . spectrum and had [ c t ] D +10° (c 2.2, c h l o r o f o r m ) . H y d r o l y s i s of t h i s m a t e r i a l u s i n g 2 M t r i f l u o r o -a c e t i c a c i d a t 95° f o r 16 hours and subsequent d e r i v a t i s a t i o n as a l d i t o l a c e t a t e s gave a mixture of components which was analysed by g.l.c.-m.s. as d e t a i l e d i n Table V . l , column I. A sample of K32 c a p s u l a r p o l y s a c c h a r i d e t h a t had been passed through a column of Amberlite IR 120 (H +) was shown by "'"H n.m.r. to have l o s t approximately 70% of the pyruvate a c e t a l groups. M e t h y l a t i o n of t h i s p a r t i a l l y d e pyruvalated m a t e r i a l i n the f r e e a c i d form proceeded without c o m p l i c a t i o n and the subsequent m e t h y l a t i o n a n a l y s i s r e s u l t s are shown i n Table V . l , page 157, column I I . - 171 -P e r i o d a t e O x i d a t i o n . A sample (616 mg) of K l e b s i e l l a K32 c a p s u l a r p o l y s a c -c h a r i d e i n the sodium s a l t form (with a f u l l complement of pyruvate) was d i s s o l v e d i n 11 of a phosphate b u f f e r a t pH 6.5. The b u f f e r was 0.05 M i n NalO^. The s o l u t i o n was s t i r r e d a t 4° i n the dark f o r 40 hours a f t e r which time ethylene g l y c o l (10 ml) was added. D i a l y s i s of the s o l u t i o n a g a i n s t tap water o v e r n i g h t was f o l l o w e d by r e d u c t i o n with sodium borohydride (1 g ) . The s o l u t i o n was then n e u t r a l i s e d w i t h g l a c i a l a c e t i c a c i d , d i a l y s e d o v e r n i g h t and l y o p h i l i s e d to y i e l d 610 mg of the d e r i v e d p o l y o l . A small p o r t i o n (10 mg) of t h i s p o l y o l was h y d r o l y s e d w i t h 2 t r i f l u o r o a c e t i c a c i d and by paper chromatography ( s o l v e n t B) the present of 1-d e o x y e r y t h r i t o l , g a l a c t o s e and rhamnose was confirmed. Reduc-t i o n and a c e t y l a t i o n of t h i s h y d r o l y s a t e gave a mixture of a l d i t o l a c e t a t e s which was shown by g.l.c.-m.s. (column B) to c o n t a i n components corres p o n d i n g to rhamnose and g a l a c t o s e i n the p r o p o r t i o n s 63:37 r e s p e c t i v e l y ( t h e o r e t i c a l 66:33). T h i s r e s u l t i n d i c a t e d o x i d a t i o n was complete. Smith h y d r o l y s i s of the p o l y o l u s i n g 0.5 ^ t r i f l u o r o a c e t i c a c i d a t room temperature o v e r n i g h t was f o l l o w e d by removal of the a c i d by e v a p o r a t i o n w i t h s u c c e s s i v e p o r t i o n s of water and then r e d u c t i o n w i t h sodium borohydride i n water o v e r n i g h t . A f t e r the u s u a l workup, l y o p h i l i s a t i o n y i e l d e d 520 mg of m a t e r i a l which was i n v e s t i g a t e d u s i n g g e l f i l t r a t i o n chromato-- 172 -graphy. The material (3 x 150 mg) was applied to a column of Bio-Gel P-4 (2.5 x 160 cm) which was i r r i g a t e d with d i s t i l l e d water at a flow rate of approximately 7 ml/h. Fractions (2 ml) were collected, l y o p h i l i s e d i n d i v i d u a l l y , and examined by paper chromatography (solvent A). Three pure components, 1, 2 and .3, with RQ^ values of 1.15, 1.75 and 1.14 respectively (solvent A) were is o l a t e d . Oligosaccharide 1, 120 mg, had [a]' +102° (c 3.2, water) and was examined by XH and X^C n.m.r. spectroscopy (see Table V.2, page 158, and Appendix III, spectrum No. 26 for d e t a i l ) . Hydrolysis of 1 (2 M t r i f l u o r o a c e t i c acid at 95° for 6 hours) revealed the presence of rhamnose, galactose, and 1-deoxyerythritol by paper chromatography (solvent A). The mass spectrum (f.d.) of 1 gave peaks, inter a l i a , at m/e 561 (36) , 562 (12), 583 (100), 584 (34), 585(17), 599(21) and 600(22). The major peaks at m/e 561, 583 and 599 correspond to (M+l) +, (M+Na)+, and (M+K)+ respectively. Methylation of a small portion of 1 yielded a product which upon hydrolysis with 2 ^ t r i f l u o r o a c e t i c acid at 95° for 16 hours and subsequent d e r i v a t i s a t i o n was shown by g.l.c.-m.s. (column A) to contain the a l d i t o l acetates of 2,3,4,6-tetra-0-methyl-D-galactose, 3,4-di-0-methyl-L-rhamnose, and 2,4-di-O-methyl-L-rhamnose i n equal proportions. The v o l a t i l e tri-O-methyl-l-deoxy-D-erythritol component was l o s t under reduced pressure during work up. Periodate oxidation of 1 (30 mg) with 0.05 M sodium periodate for 24 hours and sodium borohydride reduction in - 173 -the u s u a l manner gave a product which was h y d r o l y s e d w i t h 0. 5 |? t r i f l u o r o a c e t i c a c i d a t room temperature f o r 16 hours (Smith h y d r o l y s i s ) . The h y d r o l y s a t e was i n v e s t i g a t e d u s i n g g e l chromatography and was a p p l i e d to a B i o - G e l P-4 column (2.5 x 160 cm) and the column was i r r i g a t e d w i t h d i s t i l l e d water. A pure component l b (7 mg) was i s o l a t e d and showed [al +104° (c 0.78, water). ^ and 1 3 C n.m.r. data f o r l b are recorded i n Table V.2, page 158, and Appendix I I I , s p e c t r a No.'s 27, 28. H y d r o l y s i s of l b and paper chromato-graphy ( s o l v e n t B) of the h y d r o l y s a t e gave components i n d i s t i n g u i s h a b l e from a u t h e n t i c rhamnose and 1-deoxy-e r y t h r i t o l samples. The mass spectrum of l b (f.d.) gave peaks, i n t e r a l i a , at m/e 253(100), 254(24), 275(30) and 291(11). The peaks at m/e 253, 275, and 291 correspond to (M+l) +, (M+Na) + and (M+K) + r e s p e c t i v e l y . O l i g o s a c c h a r i d e l b i s t h e r e f o r e shown to be B-L-Rhap- (1+3)-1-deoxy-D-e r y t h r i t o l , and 1. i s e s t a b l i s h e d as being a-p_-Galp-(1+2) -a-L-Rhap-(1+3 ) - B-IrRhap-(1+3)-1-deoxy-D-erythritoi. O l i g o s a c c h a r i d e 2 (20 mg) was i d e n t i c a l to component l b i n every r e s p e c t . Component _3 (20 mg) showed [ct] D +117° (c 0.4, water). The mass spectrum (f.d.) of 3_ gave peaks, i n t e r a l i a , at m/e 255(29), 277(17), and 293(100). These peaks c o r r e s -pond to (M+l) +, (M+Na) + and (M+K) + r e s p e c t i v e l y . The "'"H 13 and C n.m.r. s p e c t r a of j3 are d e t a i l e d i n Table V.2, page 158, and Appendix I I I , spectrum No. 29. H y d r o l y s i s - 174 -of a small p o r t i o n of 3^  and subsequent paper chromatography (s o l v e n t A) of the h y d r o l y s a t e showed two components i n d i s -t i n g u i s h a b l e from a u t h e n t i c samples of g a l a c t o s e and g l y c e r o l . Component 3^  was c h r o m a t o g r a p h i c a l l y i d e n t i c a l to an a u t h e n t i c sample of a - D - G a l p - ( 1 + 2 ) - g l y c e r o l . P a r t i a l H y d r o l y s i s . K l e b s i e l l a K32 (0.5 g) was autohydrolysed a t pH 3.0 f o r 18 hours on a steam bath. T h i s m a t e r i a l was then d i a l y s e d a g a i n s t a f i x e d volume of water and l y o p h i l i s a t i o n y i e l d e d 175 mg of d i a l y s a b l e o l i g o s a c c h a r i d e s c o n t a i n i n g a t l e a s t s i x d i f f e r e n t components, as i n d i c a t e d by paper chromato-graphy ( s o l v e n t A). Gel chromatography u s i n g a column (2.5 x 170 cm) of Sephadex G10 w i t h a flow r a t e of 10 ml/h and subsequent l y o p h i l i s a t i o n o f f r a c t i o n s and examination by paper chromatography r e v e a l e d t h a t the s e p a r a t i o n of the mixture was poor. However, c a r e f u l examination d i d allow the i s o l a t i o n of a smal l amount of a pure oligomer £ (4 mg). "'"H n.m.r. of £ (see Table V.2, page 158, and Appendix I I I , • spectrum No. 30 f o r d e t a i l ) i n d i c a t e d the presence of one D-galactose r e s i d u e ( a - l i n k e d ) and three L-rhamnose r e s i d u e s (one a - l i n k e d , one B - l i n k e d and one r e s i d u e r e d u c i n g ) . O l i g o s a c c h a r i d e 4_ was reduced with NaBD^ and subsequently methylated. The permethylated d e r i v a t i v e was h y d r o l y s e d w i t h 2 ^ t r i f l u o r o a c e t i c a c i d at 95° f o r 8 hours, reduced, a c e t y l a t e d and analysed by g.l.c.-m.s. (column A). P a r t i a l l y methylated a l d i t o l a c e t a t e s c o r r e s p o n d i n g to 2,3,4,6-tetra-O-methyl-Dj-galactose, 3 , 4-di-O-methyl-L-rhamnose, 2,4-di-O-methyl-L-rhamnose and 1,2,3,5-tetra-O-methyl-L-rhamnitol were i d e n t i f i e d . (The l a t t e r component was deuterated at - 176 -V.5 B i b l i o g r a p h y f o r S e c t i o n V. 1. W. Nimmich, Acta B i o l . Med. Ger. , 2_6, 397 (1971). 2. W. Nimmich, Z. M i c r o b i o l . Immunol., 154, 117 (1968). 3. .M. Choy and G.G.S. Dutton, Can. J . Chem., 51., 3021 (1973) . 4. Y.M. Choy and G.G.S. Dutton, Can. J . Chem., 52_, 684 (1974) . 5. G.G.S. Dutton and K.L. Mackie, Carbohyd. Res., 55, 49 (1977). ' 6. P.A.J. Gorin and T. Ishikawa, Can. J . Chem., 4_5, 521 (1967). 7. (a)Y.M. Choy, G.G.S. Dutton, A.M. Stephen and M.T. Yang, Anal . L e t t . , 5, 675 (1972). (b)G.M. Bebault, Y.M. Choy, G.G.S. Dutton, N. F u n n e l l , A.M. Stephen and M.T. Yang, J . B a c t e r i d . , 113, 1345 (1973) . 8. G.M. Bebault, J.M. Berry, Y.M. Choy, G.G.S. Dutton, N. F u n n e l l , L.D. Hayward and A.M. Stephen, Can. J . Chem., 51, 324 (1973). 9. S. Hakomori, J . Biochem. (Tokyo), 55, 205 (1964). 10. E.L. H i r s t and E. P e r c i v a l , Methods Carbohyd. Chem., 5, 287 (1964). 11. H. B j o r n d a l , B. Lindberg and S. Svensson, Carbohyd. Res., 5, 433 (1967) . 12. H. B j o r n d a l , C.G. H e l l e r q v i s t , B. Lindberg and S. Svensson, Angew. Chem. I n t . Ed. E n g l . , 9, .610 (1970). - 177 -13. D.P. Sweet, R.H. Shapiro and P. Albersheim, Carbohyd. Res., 40, 217 (1975). 14. G.W. Hay, B.A. Lewis and F. Smith, Methods Carbohyd. Chem., 5, 357 (1965) . 15. I . J . G o l d s t e i n , G.W. Hay, B.A. Lewis and F. Smith, Methods Carbohyd. Chem., 5_, 361 (1965). 16. H. Krone and H.D. Beckey, Org. Mass Spectrom., 5_, 983 (1971). 17. J . Moore and E.S. Waight, Org. Mass Spectrom., 9_, 903 (1974). 18. 0. Larm, B. Lindberg, S. Svensson and E.A. Kabat, Carbohyd. Res., 22,, 39 (1972). 19. 0. Larm, B. Lindberg and S. Svensson, Carbohyd. Res., 31., 120 (1973). 20. L.G. Bennett and C.T. Bishop, Can. J . Chem., 5_5, 8 (1977). 21. G.G.S. Dutton and K.L. Mackie, Carbohyd. Res., i n p r e s s . 22. C. E r b i n g , L. Kenne, B. Lindberg, J . Lonngren and I. S u t h e r l a n d , Carbohyd. Res., 50, 115 (1976). 23. J.Y. Lew and M. H e i d e l b e r g e r , Carbohyd. Res., 52, 255 (1976). 178 -BACTERIOPHAGE DEPOLYMERISATION OF KLEBSIELLA K32 CAPSULAR POLYSACCHARIDE. - 179 -APPENDIX I. Bacteriophage D e p o l y m e r i s a t i o n o f K l e b s i e l l a K32 Capsular P o l y s a c c h a r i d e I n t r o d u c t i o n . An e f f i c i e n t method f o r s p e c i f i c a l l y degrading p o l y -s a c c h a r i d e s i n t o more e a s i l y analysed subunits, and i n p a r t i c u l a r f o r the K l e b s i e l l a p o l y s a c c h a r i d e s i n t o r e p e a t i n g u n i t s , has been a c h a l l e n g e to workers i n the f i e l d of p o l y -s a c c h a r i d e s t r u c t u r a l d e t e r m i n a t i o n f o r many years. I f such s p e c i f i c cleavages c o u l d be achieved then many problems, v i z . v i s c o s i t y , a c c e s s i b i l i t y , s o l u b i l i t y , a s s o c i a t e d w i t h h a n d l i n g the undegraded polymers and i n determining t h e i r d e t a i l e d s t r u c t u r e c o u l d be e l i m i n a t e d . Low y i e l d , non s p e c i f i c techniques such as p a r t i a l h y d r o l y s i s have i n the past been used e x t e n s i v e l y to o b t a i n o l i g o s a c c h a r i d e s which are needed to e l u c i d a t e the d e t a i l e d s t r u c t u r e (sequence data i n p a r t i c u l a r ) of many p o l y s a c c h a r i d e s , but i n s e v e r a l i n s t a n c e s the i s o l a t i o n of a d e f i n i t e subunit, e.g. a repeat-i n g u n i t i n the K l e b s i e l l a p o l y s a c c h a r i d e s , would p r o v i d e a f a r e a s i e r and simpl e r means t o a s t r u c t u r a l d e t e r m i n a t i o n . A method whereby high y i e l d s of o l i g o s a c c h a r i d e s can be obtained by s e l e c t i v e fragmentation o f some K l e b s i e l l a c a p s u l a r p o l y s a c c h a r i d e s has r e c e n t l y been developed"''. The method r e l i e s on the s p e c i f i c i t y of enzymes, born and u t i l i s e d by bacteriophage, t h a t are capable of de p o l y m e r i s i n g K l e b s i e l l a - 180 -p o l y s a c c h a r i d e s . Although i t was o r i g i n a l l y thought t h a t c a p s u l a t e b a c t e r i a were g e n e r a l l y phage r e s i s t a n t , b a c t e r i o -phage have now been i s o l a t e d f o r s e v e r a l d i f f e r e n t 2 species,, i n c l u d i n g the genus K l e b s i e l l a . In many cases a c h a r a c t e r i s t i c f e a t u r e of phage i n f e c t i o n when an a c t i v e l y growing 'lawn' of b a c t e r i a i s i n f e c t e d by a r e l a t i v e l y s m a l l number of phage, i s the formation of plaques surrounded by 3 l a r g e c l e a r haloes . The haloes comprise an area i n which p o l y s a c c h a r i d e depolymerase enzymes, induced i n phage i n f e c t e d b a c t e r i a , d e s t r o y c a p s u l a r m a t e r i a l without a f f e c t i n g b a c t e r i a l v i a b i l i t y . S p e c i f i c bacteriophage, i . e . bacteriophage t h a t show depolymerase a c t i v i t y w i t h a p a r t i c u l a r K l e b s i e l l a serotype, can be prepared f o r the m a j o r i t y of the 81 serotypes i n con-s i d e r a b l e amounts u s i n g l y s i s techniques, p r e c i p i t a t i o n 1 4 with p o l y e t h y l e n e g l y c o l and i s o p y c n i c c e n t r i f u g a t i o n ' . T h i s , together w i t h the f a c t t h a t the phage w i l l depolymerise the c e l l f r e e e x o p o l y s a c c h a r i d e by i t s e l f , makes a v a i l a b l e a powerful and s e l e c t i v e cleavage t o o l . The bacteriophage d e g r a d a t i o n of K l e b s i e l l a K32 i s presented here. T h i s work r e p r e s e n t s some of the i n i t i a l experiments c a r r i e d out i n t h i s l a b o r a t o r y u s i n g bacteriophage and s i n c e the completion of the d e p o l y m e r i s a t i o n of K32 other p o l y s a c c h a r i d e s have been s i m i l a r l y examined wi t h very encouraging r e s u l t s . - 181 -Experimental General techniques and media. Most of the techniques used are described in d e t a i l by Stirm et a l . Phosphate-buffered physiological saline (P.B.S.) was made up using 8.5 g NaCl, 1.76 g Na 2HP0 4•12H 20, and 0.1 g KH2PC>4 in 1 £ of water. "Standard" l i q u i d broth or medium contained 5 g Bactopeptone, 3 g Bacto beef extract, and 2 g NaCl per l i t r e of water. "Standard" agar plates were made using a solution of "standard" l i q u i d broth to which 15 g of agar per l i t r e had been added; 8.5 cm d i s -posable p l a s t i c plates were used. Bacteriophage and bacteria. The bacteriophage used was K l e b s i e l l a bacteriophage No. 32, o r i g i n a l l y i s o l a t e d from sewage^ and kindly given to us by Dr. S. Stirm, Max Planck-Institut fur Immunbiologie, Freiburg, Germany. The host for t h i s bacteriophage i s K l e b s i e l l a 6837 (K32) and the slime polysaccharide was isol a t e d from t h i s bacterium as previously described. (See procedure for K l e b s i e l l a K36, Section III.4, page 106). Correlation of o p t i c a l density and bacteria concentration. A f l a s k of l i q u i d medium was innoculated with a culture of a c t i v e l y growing K l e b s i e l l a K32 bacteria and vigorously aerated at 37°. Aliquots were removed at 30 minute i n t e r v a l s , — 5 — 8 appropriately d i l u t e d (10 to 10 ) with l i q u i d medium and - 132 -a small quantity (0.1 ml) of the di l u t e d solution was incubated on an agar plate for 12-16 hours. Individual b a c t e r i a l colonies could then be counted. The o p t i c a l density of the a c t i v e l y growing b a c t e r i a l solution was recorded at each 30 minute i n t e r v a l . A plot of o p t i c a l density versus the logarithm of the number of colonies i s shown in Figure A I . l , page 183. Preparation of bacteriophage ( i fj) . (a) Tube l y s i s . An active b a c t e r i a l culture of K l e b s i e l l a K32 was obtained by successive replatings on agar plates. 7x5 ml of s t e r i l e l i q u i d medium was then innoculated with the bacteria by the addition of 0.5 ml of an a c t i v e l y growing l i q u i d K32 b a c t e r i a l culture. These seven test tubes were incubated at 37° and at 30 minute in t e r v a l s the tubes were innoculated with 0.5 ml of a solution of l i q u i d medium con-taining ip32. Continued incubation resulted in the f i r s t few tubes changing from the cloudy solution associated with a c t i v e l y growing K32 bacteria to a clear solution ( l y s i s ) . After the l a s t tube had cleared the incubation was continued for 30 minutes and then a few drops of CHCl^ was added to the tube and the mixture was shaken well. A phage " t i t r e " on the solution was performed by successively d i l u t i n g a small volume (0.1 ml) of the clear l i q u i d with l i q u i d medium and then applying approximately 0.03 ml of these d i l u t i o n s to a 'lawn' of a c t i v e l y growing K l e b s i e l l a K32. (The lawn of K32 was prepared by innoculating 2 ml of l i q u i d medium with an a c t i v e l y growing colony of K l e b s i e l l a K32 and incubating t h i s - 183 -LOG. COLONIES/Ml. F i g u r e AI.1 C o r r e l a t i o n of o p t i c a l d e n s i t y and colony c o n c e n t r a t i o n f o r K l e b s i e l l a . K 3 2 . -- 184 -culture for 3 hours. An agar plate, previously dried for approximately 1 hour in the incubator at 37°, was covered with th i s l i q u i d culture, l e f t for 5 minutes and then the excess l i q u i d was removed. Incubation for 3 0 minutes gave a stable 'lawn' of K l e b s i e l l a K32.) Individual bacteriophage were observed as clear spots (approximately 0.3 cm. i n diameter) on the b a c t e r i a l lawn after incubation for 16 hours. At high phage concentrations i n d i v i d u a l phage could not be _ g distinguished but at more suitable d i l u t i o n s , e.g. 10 to 10 , i n d i v i d u a l 'haloes' could be e a s i l y counted. As a r e s u l t of a single tube l y s i s of t h i s nature an assay yielded 9 10 plaque forming units (P.F.U.) per ml. of medium in the l a s t tube to completely clear. (b) Small f l a s k l y s i s . T h i s technique i s e s s e n t i a l l y the same as t h a t d e s c r i b e d f o r the tube l y s i s . As l a r g e r volumes of l i q u i d medium can be used the o v e r a l l t o t a l of b a c t e r i o -phage can be i n c r e a s e d even though the phage t i t r e per ml. may not be s i g n i f i c a n t l y h i g h e r . In a t y p i c a l s m a l l f l a s k l y s i s 50 ml. s o l u t i o n s of K32 c u l t u r e s were i n n o c u l a t e d with 9 1.5 ml. of a phage s o l u t i o n c o n t a i n i n g 10 P.F.U./ml (from tube l y s i s ) . In an analogous manner to t h a t d e s c r i b e d f o r the tube l y s i s , t i t r a t i o n of the f i n a l f l a s k t o completely c l e a r gave a t i t r e of 1 . 2 x l 0 1 0 P.F.U./ml. (c) B o t t l e l y s i s . Three 300 ml. bottles each containing - 185 -250 ml of an a c t i v e l y growing l i q u i d c u l t u r e o f K32 were v i g o r o u s l y a e r a t e d a t 37°. A small amount of a s i l i c o n a ntifoam agent (Dow antifoam FG-10 emulsion) was added to each. The o p t i c a l d e n s i t y of each f l a s k was monitored and at a p p r o p r i a t e o p t i c a l d e n s i t y readings ( c a l c u l a t e d such t h a t the r a t i o of t o t a l bacteriophage to t o t a l b a c t e r i a l c o l o n i e s was approximately 3:1) a l i q u o t s of l i q u i d phage c u l t u r e s were added and the o p t i c a l d e n s i t y m o n i t o r i n g continued. A sub-sequent drop i n o p t i c a l d e n s i t y i n d i c a t e d l y s i s had o c c u r r e d . The r e s u l t s of a t y p i c a l b o t t l e l y s i s are shown i n F i g u r e AI.2, page 186. A b o t t l e l y s i s might t y p i c a l l y y i e l d 400 ml of a s o l u t i o n w i t h a t i t r e of 3 . 0 x l 0 1 0 P.F.U./ml. (d) One l i t r e f l a s k l y s i s . In an analogous manner to t h a t d e s c r i b e d f o r the b o t t l e l y s i s three one l i t r e f l a s k s , each c o n t a i n i n g 600 ml of l i q u i d medium, were i n n o c u l a t e d with K32 b a c t e r i a , aerated and incubated to a p p r o p r i a t e o p t i c a l d e n s i -t i e s , and then bacteriophage s o l u t i o n s were added. A t y p i c a l r e s u l t of such a l y s i s might y i e l d 1400 ml of a phage s o l u t i o n with a t i t r e of 3 x l 0 1 0 P.F.U./ml. Bacteriophage c o n c e n t r a t i o n . A s o l u t i o n (1400 ml) of l i q u i d medium c o n t a i n i n g 3 x l 0 ± 0 13 P.F.U./ml (4.2x10 P.F.U. i n t o t a l ) was c e n t r i f u g e d a t 5000 g f o r 15 minutes j u s t p r i o r to the a d d i t i o n of 160 g of p o l y e t h y l e n e g l y c o l . T h i s s o l u t i o n was l e f t a t 4° f o r - 186 -F i g u r e AI.2 R e s u l t s of a t y p i c a l b o t t l e l y s i s of K l e b s i e l l a K32 with bacteriophage ^32. - 187 -thr e e days. The f i n e misty grey p r e c i p i t a t e t h a t formed d u r i n g t h i s p e r i o d was separated by c e n t r i f u g a t i o n a t 20,000 g f o r 30 minutes. The p r e c i p i t a t e was then suspended i n p h y s i o l o g i c a l l y b u f f e r e d s a l i n e (P.B.S.) with the a i d of a s y r i n g e and 20 gauge hypodermic. C e n t r i f u g a t i o n at 5000 g f o r 15 minutes removed a l a r g e amount of a dense p r e c i p i t a t e which upon t i t r a t i o n was shown to c o n t a i n approximately 1% of the t o t a l plaque forming u n i t s . The m i l k y supernatant from t h i s slow speed c e n t r i f u g a t i o n was then c e n t r i f u g e d a t 100,000 g f o r 1 hour and a s o l i d phage p e l l e t was o b t a i n e d . (The c l e a r supernatant was subsequently shown to c o n t a i n l e s s than 1% of the t o t a l P.F.U.) The phage p e l l e t was resuspended i n P.B.S. (10 ml) but appeared inhomogeneous. A low speed c e n t r i f u g a t i o n (30 minutes, 5000 g) gave a p r e c i p i t a t e (which was subsequently shown to c o n t a i n l e s s than 1% of the t o t a l P.F.U.) and a m i l k y white supernatant. At t h i s p o i n t the bacteriophage were present i n 12 ml of a P.B.S. s o l u t i o n w i t h a t i t r e of 1 . 8 x l 0 1 2 P.F.U./ml. ( 2 . 4 x l 0 1 3 P.F.U. i n t o t a l ) . P u r i f i c a t i o n of bacteriophage. The bacteriophage were p u r i f i e d by i s o p y c n i c c e n t r i f u g a -t i o n i n a l i n e a r CsCl g r a d i e n t . In two c e l l u l o s e n i t r a t e tubes f o r a Beckman S.W.27 r o t o r (tube c a p a c i t y 38.5 ml each), two l i n e a r g r a d i e n t s were formed s i m u l t a n e o u s l y u s i n g a three channel p e r i s t a l t i c pump. The two s o l u t i o n s used to form the g r a d i e n t were made up as f o l l o w s ; 32.35 g Cs C l - 188 -i n 32.14 ml of T r i s HCl, and 6.47 g of C s C l i n 31.15 ml of T r i s HCl. S u f f i c i e n t space was l e f t i n the S.W.27 tubes to l o a d approximately 7 ml of phage suspended i n P.B.S. onto each g r a d i e n t . The loaded g r a d i e n t s were spun at 86,000 g f o r 1.5 hours. An o p a l e s c e n t band (lowest band) was c l e a r l y v i s i b l e a f t e r t h i s time and t h i s was c o l l e c t e d by p i e r c i n g the c e l l u l o s e n i t r a t e tube from the s i d e u s i n g a s y r i n g e and new hypodermic needle. The r e f r a c t i v e index of t h i s m a t e r i a l c o r r e l a t e d w i t h a d e n s i t y of 1.44 g/ml. and was d i a l y s e d a g a i n s t P.B.S. to remove the C s C l . E l e c t r o n microscopy. The c o n d i t i o n s and s t a i n i n g techniques used f o r e l e c t r o n . . . . 5 microscopy were those d e s c r i b e d by S t i r m and Freund-Molbert . The e l e c t r o n micrographs c l e a r l y showed the i c o s a h e d r a l b a c t e r i o -phage head and the s p i k e s a t t a c h e d d i r e c t l y to the head. T h i s morphology has been c l a s s i f i e d by B r a d l e y as type C. C o n d i t i o n s of d e p o l y m e r i s a t i o n . 800 mg. of K l e b s i e l l a K32 c a p s u l a r p o l y s a c c h a r i d e was d i s s o l v e d i n 400 ml of P.B.S. (pH 7.0) and to t h i s s o l u t i o n was added a t o t a l of l x l O 1 3 P.F.U. i n 30 ml of P.B.S. The mixture was incubated at 37° f o r a t o t a l of 4 8 hours. At v a r i o u s time i n t e r v a l s (see F i g u r e AI.3, page 189) the v i s c o s i t y of the mixture was measured u s i n g an Ostwald v i s c o s i m e t e r (a p o r t i o n of the d e p o l y m e r i s i n g r e a c t i o n mix-t u r e was l e f t i n the v i s c o s i m e t e r a t 37°). At the same time a l i q u o t s of the r e a c t i o n mixture were removed and t e s t e d TIME (h) F i g u r e AI.3 Bacteriophage d e p o l y m e r i s a t i o n of K l e b s i e l l a K32 c a p s u l a r p o l y s a c c h a r i d e . - 190 -f o r 'reducing power' u s i n g K 3[Fe(CN)g] . L-Rhamnose was used to e s t a b l i s h a standard curve. The changes i n v i s c o s i t y and red u c i n g power are recorded i n F i g u r e AI.3, page 189. P u r i f i c a t i o n and s e p a r a t i o n of depolymerised m a t e r i a l . P o r t i o n s of the crude l y o p h i l i s e d d e p o l y m e r i s a t i o n mixture (2x1.5 g) were d e s a l t e d u s i n g a column of Sephadex G10 2 (100 cm x 19.5 cm ). The column was e l u t e d with a water-p y r i d i n e - g l a c i a l a c e t i c a c i d (1000:10:4) b u f f e r (pH 4.5) at a flow r a t e of 25 ml/h and carbohydrate m a t e r i a l was 8 l o c a l i s e d u s i n g the M o l i s c h t e s t . P a r t of the d e s a l t e d m a t e r i a l (430 mg) was d i a l y s e d a g a i n s t 3 x 11 of d i s t i l l e d water and l y o p h i l i s a t i o n y i e l d e d 64.5 mg (15%) of n o n - d i a l y s a b l e m a t e r i a l and 350 mg of d i a l y s a b l e m a t e r i a l t h a t was c o n s i d e r e d to c o n t a i n no oligomers c o n t a i n i n g more than approximately 12 sugar r e s i d u e s . The d i a l y s a b l e m a t e r i a l (320 mg) was then a p p l i e d to the top of a D.E.A.E.-A25 Sephadex column. (The column, 90x1.5 cm, was packed i n 0.5 M T r i s / H C l b u f f e r (pH 7.2) and then e q u i l i b r a t e d w i t h 0.025 M T r i s / H C l b u f f e r — a t l e a s t 10 column volumes of the l a t t e r b u f f e r are r e q u i r e d to achieve e q u i l i b r a t i o n as determined by performing c o n d u c t i v i t y measurements.) The m a t e r i a l was a p p l i e d as a s o l u t i o n i n 0.025 M T r i s / H C l (2 ml). The column was e l u t e d (10 ml/h) with 0.025 M T r i s / H C l (140 ml) and a l i n e a r s a l t g r a d i e n t (from 0 to 0.35 M NaCl) was then begun. F r a c t i o n s (2 ml) 2.5 TUBE NUMBER ( 2 ml fractions ) F i g u r e AI.4 Ion exchange chromatography of K l e b s i e l l a K32 o l i g o s a c c h a r i d e s o b t a i n e d by bacteriophage d e p o l y m e r i s a t i o n . D.E.A.E. Sephadex A25 e q u i l i b r a t e d w i t h 0.025 ^ T r i s / H C l b u f f e r was used and e l u t e d w i t h a l i n e a r 0 to 0.3 ^ NaCl g r a d i e n t i n the same b u f f e r . F r a c t i o n s were analysed w i t h phenol-s u l p h u r i c a c i d . F i g u r e AI.5 H n.m.r. of n e u t r a l r e p e a t i n g u n i t from phage degraded K32. RKa-1 34 2 0 2 J • 5 1 0 1 • 07 2 4 6 2 0 0 9 • 9 1 0 0 • 44 3 4 7 19 70 • 0 98 9 50 4 2 5 189 1 • 1 94 • 55 5 1 6 1 8 3 6 o 3 94 • 3 1 6 28 16 72 a 4 83 • 62 7 1 7 1 6 6 5 a 2 8 3 • 26 a 3 7 1 6 2 7 • 0 8 1 • 34 9 4 2 1 5 3 6 • 4 76 • 32 1 0 74 14 57 • 0 72 • 84 1 1 24 1 4 4 9 o 2 72 C 4 5 1 2 54 1 4 4 0 • 2 72 • 0 1 1 3 60 14 3 6 • 2 7 1 8 0 1 4 7 1 1 4 2 8 • 6 7 1 • 4 2 1 5 35 1 4 2 0 « 5 7 1 • 0 2 1 6 4 5 1 4 0 9 • 3 70 • 46 I 7 58 1 4 0 3 • 4 70 • 1 7 1 8 ) 0 5 1 4 0 0 • 5 70 • 02 1 9 38 13 94 • 0 6 9 • 70 20 4 4 133 1 • 6 69 • 08 2 1 2 3 1 3 5 2 • 2 67 • 60 2 2 37 1 2 3 5 • 5 6 1 • 7-7 2 3 . 4 8 62 1 e 7 3 1 • 0 8 ^ 2 4 8 9 35 1 * 4 1 7 • 5 7 2 5 6 1 3 4 9 • 1 1 7 • 4 5 71 :• . i i l " of. ot 7 - f " | - 3*CH3ofRha j F i g u r e AI.6 1 J c n.m.r. of n e u t r a l r e p e a t i n g u n i t from phage degraded K32. 1 2 3 2 1 6 8 0 8 1 0 8 • 4 4 2 3 3 2 0 3 7 • 0 1 0 1 • 8 4 3 2 8 2 0 19 • 0 1 0 0 • 9 5 4 3 6 1 9 8 1 • 5 9 9 9 0 7 5 - 2 1 1 8 8 9 • 4 9 4 • 4 6 6 1 6 1 8 e 4 • 7 9 4 9 2 3 . 7 2 3 16 7 0 9 1 8 3 9 5 0 8 1 3 16 6 3 • 0 8 3 • 1 4 9 3 1 16 3 3 • 5 8 1 9 6 7 1 0 2 8 15 3 7 • 1 7 6 9 8 5 1 1 2 8 1 5 2 4 • 0 7 6 9 2 0 I 2 3 2 1 4 9 5 • 1 7 4 9 7 5 1 3 3 5 1 4 5 5 9 5 7 2 9 7 7 1 4 1 5 1 4 4 6 9 8 7 2 0 3 3 1 5 5 3 14 3 4 • 5 7 1 9 7 2 1 6 9 1 1 4 2 7 • 9 7 1 9 3 9 1 7 2 6 14 19 • 3 7 0 9 9 6 1 8 6 8 1 4 0 0 • 6 7 0 9 0 3 1 9 2 7 1 3 9 1 • 7 6 9 9 5 8 2 0 3 3 1 3 8 0 • 2 6 9 9 0 1 2 1 2 3 13 5 1 • 0 6 7 9 5 4 2 2 3 0 1 2 4 3 • 9 6 2 9 1 9 2 3 2 4 1 2 4 2 • 0 6 2 9 1 0 2 4 1 ! 9 1 2 0 5 • 6 6 0 • 2 8 2 5 1 5 6 2 1 • 6 3 1 9 0 8 2 6 3 0 4 7 0 • 1 2 3 9 5 0 2 7 2 7 3 6 1 • 4 1 8 9 0 7 2 8 6 0 3 5 0 • 4 1 7 • 5 2 W 7(: . . - V -4-CAJ, ^dooU i 3 4 CH (atebne) 3|.ol F i g u r e AI.8 1 3 c n . m . r . of a c i d i c r e p e a t i n g u n i t from phage degraded K32, - 196 -were co l l e c t e d and examined using the phenol-sulphuric acid 9 assay . The elution p r o f i l e for t h i s column separation i s shown i n Figure AI.4, page 191. The peaks are la b e l l e d 1+9. The n.m.r. spectra of peak 3 are shown in Figure AI.5, AI.6, pages 192 and 193, and those of peak 7 in Figures AI.7, AI.8, on pages 194 and 195. Other spectra were recorded for peaks 1, 2, and 6, but are not shown here. Discussion The growing of 4>32 in order to u t i l i s e the s p e c i f i c depolymerising action of the glycanase enzyme liberated by the bacteriophage proceeded as expected and afte r 4-6 weeks s u f f i c i e n t p u r i f i e d bacteriophage were propagated to degrade approximately 800 mg of capsular polysaccharide that had previously been is o l a t e d . The depolymerisation of the poly-saccharide proceeded rapid l y and was e s s e n t i a l l y complete afte r 3-4 hours. The separation of the oligosaccharides obtained as a re s u l t of the bacteriophage action was not as clear cut as hoped for. D i a l y s i s of the carbohydrate material (after desalting on G10) resulted i n only 15% of the oligosaccharides remaining i n the d i a l y s i s bag. To a f i r s t approximation t h i s was taken to mean that 15% of the o r i g i n a l polysaccharide had a degree of polymerisation (D.P.) greater than 12; i . e . , the majority (85%) of material was of lower D.P. than 12. The p r o f i l e shown i n Figure AI.4 as a r e s u l t of the D.E.A.E.-Sephadex separation i s complex. Much of the carbo-- 1 9 7 -hydrate m a t e r i a l d i d not bind to the support (peaks 1 + 5 ) and "^H n.m.r. examination of these components confirmed the absence of any pyruvate a c e t a l . The n e u t r a l o l i g o s a c c h a r i d e s were thus separated s o l e l y a c c o r d i n g t o s i z e ( r a t h e r than charge) on the column. The pyruvate a c e t a l i n K l e b s i e l l a K32 i s very l a b i l e (see s e c t i o n V . 3 , page 1 6 8 ) , and was probably l o s t d u r i n g the d e s a l t i n g of the d e p o l y m e r i s a t i o n mixture. T h i s d e s a l t i n g was performed on a Sephadex G - 1 0 column e q u i l i b r a t e d w i t h a w a t e r - p y r i d i n e - g l a c i a l a c e t i c a c i d ( 1 0 0 0 : 1 0 : 4 ) b u f f e r (pH 4 . 5 ) . Those components (peaks 6 + 9 ) e l u t e d by the l i n e a r s a l t g r a d i e n t d i d c o n t a i n some pyruvate a c e t a l groups (as e s t a b l i s h e d by "'"H n.m.r.). However, component 6 , shown by "*"H n.m.r. to be a t e t r a s a c c h a r i d e , d i d not c o n t a i n i t s f u l l complement of pyruvate and hence t h i s must have been l o s t d u r i n g d e s a l t i n g of peak 6 a f t e r the D.E.A.E.-Sephadex s e p a r a t i o n or d u r i n g p r e p a r a t i o n of the "'"H n.m.r. sample. A l s o , the appearance of spurious peaks i n the n.m.r. s p e c t r a of some of the components suggests t h a t a f u r t h e r c o m p l i c a t i o n i n t h i s s e p a r a t i o n may be pres e n t . The n.m.r. s p e c t r a shown i n F i g u r e s A I . 5 + 8 i n d i c a t e t h a t the glycanase enzyme r e s p o n s i b l e f o r de p o l y m e r i s i n g K32 c a p s u l a r p o l y s a c c h a r i d e i s a a-rhamnosidase. The component 3 r e p r e s e n t s a s i n g l e r e p e a t i n g u n i t of K32 c a p s u l a r p o l y -s a c c h a r i d e without the pyruvate a c e t a l and component 7 the same u n i t with the a c e t a l . (The l a t t e r component a l s o has an unknown impurity.) By examination of n.m.r. s p e c t r a of - 198 -these two components i t i s possible to locate the point of phage cleavage more s p e c i f i c a l l y as shown below. I/J 3 2 The l a b i l i t y of the pyruvate acetal in K32 complicates the f i n a l separation of the s p e c i f i c a l l y depolymerised pro-ducts obtained from the action of ij;32. In another case i n t h i s laboratory using K l e b s i e l l a K21, where t h i s complication did not arise, very good res u l t s were obtained and from 1 g of polysaccharide 200 mg of the single repeating unit of K21 and 200 mg of the double repeating unit were obtained"*"^. 3^ ..1 2_. 1 3_. 1 4„. 1 —Ga 1 Rha Rha—75—Rha— a pyr - 199 -APPENDIX I. References 1. H. Thurow, H. Niemann and S. Stirm, Carbohyd. Res., 41," 257 (1975) . 2. H. R a e t t i g , B a k t e r i o p h a g i e , Gustav F i s c h e r V e r l a g , S t u t t g a r t , 1958 and 1967. 3. - W. B e s s l e r , E. Freund-Molbert, H. Knlifermann, C. Rudolph, H. Thurow and S. St i r m , V i r o l o g y , 56, 134 (1973). 4. K.R. Yamamoto, B.M. A l b e r t s , R. Benzinger, L. Lawhorne and G. T r e i b e r , V i r o l o g y , _40, 734 (1970) . 5. S. S t i r m and E. Freund-Molbert, J . V i r o l . , 8, 330 (1971). 6. D.E. Bradley, B a c t e r i o l . Rev., 3_1, 230 (1967). 7. T. Imoto and K. Yag a s h i t a , Agr. B i o l . Chem., 35, 1154 (1971). 8. H. M o l i s c h , Monatsh. Chem., 7, 198 (1886). 9. M. Dubois, K.A. G i l l e s , J.K. Hamilton, P. Rebers and F. Smith, A n a l . Chem., 2_8, 350 (1956). 10. A. Savage, unpublished r e s u l t s . - 200 -APPENDIX I I - THE KLEBSIELLA POLYSACCHARIDES OF KNOWN STRUCTURES. - 201 -APPENDIX I I . The K l e b s i e l l a P o l y s a c c h a r i d e s of Known S t r u c t u r e (as of June 30, 1977) . A l l sugars are D- except f o r L-rhamnose and L-fucose. Pyr = pyruvate a c e t a l . K-type S t r u c t u r e ( r e f e r e n c e s a t end) K l ^ . 1 C / A V F u c l^ l c lF 3\/2 pyr K2 3_n 1 4 M 1 4 _ 1 — G l c - ^ - M a n - y - G l c — a I 1 GlcA K5 — G l c A n Glc—g—Man—„ 3 /r \ I A 3 2 M 6 \ / 4 ' pyr OAc K6 3 1 4 1 4 1 -±G1 cip^Ma n =^G 1 c — a I 1 Glc K7 3_n ,1 2 M 1 2„ 1 3_n 1 —GlcA—^—Man Man G l c — ^ 8 3 i a a -> < - ? •50% 1 Gal 6\/4 ' pyr 50% K8 3 1 3 1 3 1 -^Glc 1 Xy±Ga 1 a i 1 GlcA - 202 -K9 3„ ..1 3„. 1 3„. 1 2_. 1 —Gal—-—Rha—-—Rha—-—Rha a 8 1 GlcA a a K l l 3 1 3 1 3 1 , a 1 ' Gal 6 \ / 4 pyr K13 3 1 4 1 4 1 - :^lc—TT—Man—TT—Glc a i 1 GlcA 4 3 i Gal 4\/3 pyr a K16 3„, 1 4_, ,1. 4_ 1 -Glc GlcA—pj—Fuc—— a p a 3 1 Gal K18 -Gal 1 4„, 1 3 VI J 3 a 1 Rha 2 8 1 GlcA 4 a 1 G: -C a -Rha-- 203 -K20 2 M 1 3. ,1 —Man——<3a 1—Q-a 3 a Gal +OAc 3 1 GlcA (not l o c ated) K21 3 , .cA 4 a 1 Gal 1 3 M 12., 1 3. .1 Man Man Gal—75-a a a 6 6 \ / 4 pyr K22 - ^ G a l ^ G l c ^ T T a t 1 Glc a XA XA K23 —Rha G l c — a c 3 1 GlcA a K24 2_n ,1 3„ 1 2 • • 1 3_n 1 -GlcA Man Man Glc—75-a a a 3 3 1 Man - 204 -3 1 4 1 K25 — ^ a r V ^ l c - ^ n -4 8 1 GlcA 2 8 1 G] .c K28 2 1 3 -=Gal a _. 1 2 M 1 3_, 1 Man Man Glc—? a a 8 1' GlcA 3 i 8 1' G K32 3 1 2 1 3 1 4 1 -^Ga 1—-Rha—-Rha^V=-Rha— a , . a 8 a A/ 3 pyr K36 3 1 3 1 3 1 2 1 -^Gal ±V 1Rha x-^-Rha ±-^Rha— 8 „ i a a a 1 GlcA 4 8 1' Glc A/« pyr K37 - ^ G a l ^ G l c ^ P p a I 1 G] 6 1 YA YA COOH — Q CH-t ' 3 K0H H — C — ( COOH 0-- 205 -DPA 2 K38 6_, 1 3_ ,1 4 —Glc—TT—Gal 6 ^ a 8 1 Glc DPA = 3-deoxy-L-glycero-pentulosonic~acid K41 — ^ l c ^ ^ R h a i - ^ a l ^ G a l f — a a 3 ^ a 1 GlcA 4 a 1 Glc 6 8 1 Glc 3 1 4 1 K47 ^ a l ± - l R h a — 3 3 , a 1 GlcA 4 1 Rha K52 3 1 2 1 4 1 3 1 4 1 -^ Ga l i ^ R h a ^ G 1 cA-^r—Ga l ^ - R h a ^ -1 Gal - 206 -K54 1 4 . ,1 3 B 1 c— 5 —GlcA F u c — B a a K55 3_n 1 3 — G l c OAc 2 8 ? T a a 1 Gal 3 a 1 GlcA K56 3 1 3 1 3 1 3 1 J-G IcArKSa l ^ V ^ a l^V^Ga 1 — 6 V 4 pyr a 1 1 Rha K57 3 1 3 1 2 1 —Gal—77—GalA^ 1——Man—— a a a 1 Man K59 3_n 1 3. .1 2 M 1 3 M 1 Glc——Gal——Man——Man-1 GlcA 6! OAc a 6! OAc a (dotted l i n e s i n d i c a t e OAc's not) (on a l l r e s i d u e s ) - 207 -K62 2 1 3 1 3 1 4 1 8 n i a 6 a a 1 Man 3 1 ^ 1 ^ 1 K6 3 ^ a l A i — i F u c — G a l — a a a 4 1 4 1 2 1 2 1 3 1 ? 1 K7 0 — ^ I c A ^ ^ R h a ^ - ^ R h a i - ^ l c ^ - ^ G a l i ^ R h a — 8 a a a 8 . . a 4\ /3 v / 50% pyr 3 1 3 1 2 1 3 1 8 a . . a a V 3 pyr 2 1 3 1 4 1 ? 1 3 1 3 1 K 8 1 — - R h a ^ - R h a ^ - ^ G l c A - ^ R h a i — - R h a ± - ^ G a l ^ a a 8 a a ( - 208 -APPENDIX II. References. Kl C. Erbing, L. Kenne, B. Lindberg, J. Lonngren and I. Sutherland, Carbohyd. Res., 50, 115 (1976). K2 L.C. Gahan, P.A. Sandford and H.E. Conrad, Biochemistry, 6, 2755 (1967). K5 G.G.S. Dutton and M.T. Yang, Can. J. Chem., 50, 2382 (1972). G.G.S. Dutton and M.T. Yang, Can. J. Chem., 51, 1826 (1973). K6 S. Stirm, et al_. , unpublished. K7 G.G.S. Dutton, A.M. Stephen and S.C. Churms, Carbohyd. Res. , 38.' 225 (1974) . K8 I.W. Sutherland, Biochemistry, 9_, 2180 (1970). K9 B. Lindberg, J. Lonngren, J.L. Thompson and W. Nimmich, Carbohyd. Res., 25, 49 (1972). K l l H. Thurow, Y.M. Choy, N. Frank, H. Niemann and S. Stirm, Carbohyd. Res., 41, 241 (1975). K13 H. Niemann, et al_. , unpublished. K16 A.J. Chakraborty, H. F r i e b o l i n , H. Niemann and S. Stirm, . Carbohyd. Res., in press. K18 G.G.S. Dutton, K.L. Mackie and M.T. Yang, unpublished r e s u l t s . K20 Y.M. Choy and G.G.S. Dutton, Can. J. Chem., 51, 3015 (1973). B. Whitehouse, 449 Thesis, U.B.C., 1976. K21 Y.M. Choy and G.G.S. Dutton, Can. J. Chem., 51, 198 (1973). K22 H. Niemann, et a l . , unpublished. K23 G.G.S. Dutton and M. Stephenson, unpublished r e s u l t s from t h i s laboratory. - 209 -K24 Y.M. Choy, G.G.S. Dutton and A.M. Zanlungo, Can. J . Chem., 51, 1819 (1973). K25 H. Niemann, et a_l. , unpublished r e s u l t s . K28 M. C u r v a l l , B. Lindberg, J . Lonngren and W. Nimmich, Carbohyd. Res., 42, 95 (1975). K32 G.M. Bebault, G.G.S. Dutton, N. F u n n e l l and K.L. Mackie, unpublished r e s u l t s . K36 G.G.S. Dutton and K.L. Mackie, Carbohydr. Res., 5_5, 49 (1977) . K37 B. Lindberg, et a_l. , unpublished r e s u l t s . K38 B. Lindberg, B. Samuelson and W. Nimmich, Carbohyd. Res., 30, 63 (1973). K41 J.P. J o s e l e a u , et al_. , unpublished r e s u l t s . K47 H. B j o r n d a l , B. Lindberg, J . Lonngren, W. Nimmich and K. R o s e l l , Carbohyd. Res., 27_, 373 (1973). K52 H. B j o r n d a l , B. Lindberg, J . Lonngren, M. Meszaros, J.L. Thompson and W. Nimmich, Carbohyd. Res., 31, 93 (1973). K54 (a) P.A. Sandford and H.E. Conrad, Biochemistry, 5^, 1508 (1966). (b) H.E. Conrad, J.R. Bamburg, J.D. Epley and T.J. Kindt, Biochemistry, 5_, 2808 (1966) . K55 G.M. Bebault and G.G.S. Dutton, unpublished r e s u l t s . K56 Y.M. Choy and G.G.S. Dutton, Can. J . Chem., 51, 3021 (1973). K57 J.P. Kamerling, B. Lindberg, J . Lonngren and W. Nimmich, Acta Chem. Scand., (B) 29, 593 (1975). - 210 -K59 B. Lindberg, unpublished r e s u l t s . K62 G.G.S. Dutton and M.T. Yang, Carbohyd. Res., i n pre s s . K63 J.P. J o s e l e a u , et a l . , unpublished r e s u l t s . K70 G.G.S. Dutton and K.L. Mackie, Carbohyd. Res., i n p r e s s . K72 Y.M. Choy and G.G.S. Dutton, Can. J . Chem., 5_2, 684 (1974) K81 M. C u r v a l l , B. Lindberg, J . Lfinngren and W. Nimmich, Carbohyd. Res., 42, 73 (1975). APPENDIX I I I . N.M.R. SPECTRA. K3b Polysaccharide Solvent 3^0 , S.w S"oo Hz _iJ.U . .J.,LLj .J_ l ' i -J_iJ--U^-Li- I I 1 I 1 -L_L I I LJL I I I I I I I I I I t I I I Spectrum No. 1. ^\Sb PoljSaccKaridle (see. R<y , jxxje fl) '5 150 Spectrum No. 2, 100 I I to I W 50 0 PPm Spectrum No. 3. Spectrum No. 4. I I 5.03 —glycerol Solvent 3)_jO TemJ. :1o' .<=,.w. 5oo 7.77 i i ' I I I i i l i i i i I i J i l i i , i i I i i , i i l i I ! I I I I I I I 1 to h-1 ^8.70 l • i i I i i ' i I i i i i i i i i i I i i ' I i i i i I i i i i I i i i i i i i ' I i i ' i I i i Spectrum No. 6 K3b S l c M h < H i Spectrum No. 7. <=>.W. AT. Rui. P-D -C . T . tf-OOO Kj! : ' oas sec;: 0 XA.SCC. i • 10 Sec j 5^000'.. • 1 1 1 1 f i: i 17.63 p.p.m. I 1 . . . ; . . . I ; • • . . . : . . . 1 I Spectrum No. 11. Spectrum No. 12. •: •.!'! Y I tot-. :;' i , ' i n= i i j '•' || V i 40'. ij 501 K38T .£<d^Rhi-^Rha~oH f ty <ilcR i : I I 10 qitL^llcfll-'Rha^Rha-oH i Temp. a1S° ! s.w. SOO H i . i i I OJ I. I J 78.70 T L X LJ_LJ_ I I I I ' I I I ! I ' I I I I I I I I I I I I I < I I I I I I I I I I I KK) PoWccKari^e.. Solve ni Temp D40 ft 15° 50O tfi. 102.9 S5.7 105.7 172.9 I Spectrum No. 14. i KK) Pol s.w. ft.T. P-W. P.T>. NT. kjSaccKarCde. /f.000 Hi I.OB Sec. 0 >^«c. 2>o*,0oo. Temj>. ft<?5' S.w. 5"0O H Spectrum No. 15. N'- u . Pf m -l 79 2 0 7 7 - 9 1 0 3 . 8 9 2 5 S 9 4 . 6 3 3 2 8 • - 9 4 - 2 6 4 5 1 8 2.. 1 4 5 2 7 : • : . ; 8 1 . 7 0 6 79 7 6 . 1 9 7 . 2 9 7 5 . 5 6 9 84 : : . : 7 4 - 30 3 3 : : • . ; 7 3 . 69 IJO 7 a . 2 a 1 1 5 7 : - - . • 7 1 . 7 7 1 ? 3 1 i : -• . " 7 1 . 28 1 3 53 I ' :. 7 0 . 8 9 1 <*. 5 0 : -J - . . 6 7 . 5 4 1 r 10 0 3 1 . 0 7 i r- 5 3 . - 1 7 . 7 8 0 K70 : 1 -v •')' 31.07 i P. S.W. Aooo A-T. "1.053 ittT 10yu.sec. P.W. C T . to to ;Spectrum ; No. 16. Spectrum No. 17. Spectrum No. 18. sec] ~£5\,00O. M VD C% flu Spectrum No. 19. Spectrum No. 20. ,£5Z -1 «D i O X ^ W 3 fj H v/) <r o- o , i c ^ i n o o j o i n c j o c n r M o r r c ^ o r n +t i o c r r ' c u c - r - c - c - c - c - v o i D v o r o — T, — o rr o in s* if. n c ^ O ' ^ c i n t f — c- r\i o n r> vo «o f. — c r~ rr. rv q — c ui vr- i f ^ «? <r? m r j c i vo n c ~ — — • - ^. . cr. rr. — o; cr. c to r j rv r- rvj <3 n rv rv f.. p.. p-, c c c r^  — r- r> * i r — rv — r». r; «c c t> cr c. c — rv r. «? in 1^  • T5.43 Solvent 1)J0 SW. 5 00 Hi. J U L J U J i'i I I I I I I I I J I I I I, I i I I I I I I I I I I I I I I I I I I I I I I I I I I ,l I I I I I I I I i r i i i i r . i i i I ; : i L— - J i I ' i : I ' I i ! 1 1 i I I I ! I I I I I I I I I I 1 I I I I I I I I I I I ! I I I ! I I I I I [ I I I I I I I • I • I I [ ! I . I ! 1 rg.70 L_ L Spectrum No. 22, - 234 -= £ • e n o »> — VP 8 ^ > c-4 -3 t-; 3 s 8 o m o i : I . _• !••• mffi'dvooi — vonip'qcoinir/aiawt-^r-in o c> c c r r~ r> c- c- r- r- c- vr v.1" u> \c \i> ir> m — i-" o" u~ c f*' c ^ n- ?^ r- r r, t*^  o LI c " ^ — c — o — r~ vr V3 C\J — — c <T «J n m o i <j - c i f ii" r ^ " c r r OJ p' C PI f ir r> ( V f V — — — — — — — . — . c- o r c i r r « < , p ^ O ' S f - ' O d i ' c r\i c «- <-• i " T" r~ i " i r i r t r IP — IP vr IP — — rr- vr to n r- ,^ i " r- rr o- c — c ci n in vr r- cT C - 235 -—: _ <lio •-1 4 4 2 0 6 5 .' 5 1 0 3 • 3 3 2 4 5 I 5 3 4 . H 3 1 • 7 4 3 54' 15 1 3 . 7 7 5 • 9 8 <a 5 3 1 17 1 . 2 7 3 • 5 8 5 5 1 14 3 7 . 3 7 1 • • 8 6- 5 0 . 1 3 3 0 . 7 6 9 • 5 3 7 5 5 1 2 4 3 . 9 6 2 • a 4 3 6 9 1 2 3 3 . 1 6 1 • 9 0 9 7 5 1 2 3 5 . 7 6 1 • 7 0 1 0 1 1 -32 1 . 6 3 1 • 0 7 6o i f >; • X\J\) : r - v ! : . r r : 1 $ 0 ' : A .T. 1:: 0.5 4ec . P.W.i': : l5yU.Sec :. :i | . C-T".!:';: .360 ,0 0 0 : : i'! •;'. '. . ; i : ; :::; i: .' ;-•)•" j:.; j. : • • ; ; • ' - j Hi!;!'.': ^rl:' • . ' : ; . j '!". i ;*i ' . !-j f i - j . ... ... , , I I : ' ' ! : t 31.07 U > Spectrum No. 25, 98.*»2 1 5 5 2 0 0 3 • 3 1 0 0 • 4 1 2 3 4 2 0 0 7 • 0 1 0 0 • 3 5 3 4 0 1 3 5-3 • 4 9 8 • 4 2 4 3 9 1 5 9 3 a 2 8 4 • 6 5 5 . 3 9 1 5 2 7 • B • 8 1 • 3 9 6 4 1 1 ~> 3 3 o 7 6 • 7 7 7 4 7 1 4 5 6 0 1 7 2 • 3 0 3 5 5.2 1 4 " 3 • 5 7 2 • 6 7 9 15 . 1 4 4 3 • 0 7 2 • 1 4 1 0 5 0 1 4 3 1 0 9 7 1 • 7 4 •1 1 . 4 5 1 4 3 0 • p 7 1 • 5 4 1 2 4 3 1 4 0 1 7 7 0 • 4 8 • 1 3 1 0 0 14 0 0 • 7 7 0 • 0 3 1 4 4 1 1 3 3 1 • G 6 9 fl OB 1 5 4 0 13 5 1 e 3 6 7 • 7 1 1 6 3 7 1 2 4 1 0 3 6 2 • 0 6 1 7 3 9 12 3 5 • 4 6 1 • 7 6 1 0 ' 2 6 3 5 9 • 2 1 7 0 9 5 1 9 3 5 3 5 1 1 1 7 • 5 5 2 0 3 7 3 4 7 • 3 1 7 • 3 6 r:.TU|2'|b7pif . : i h < ; • i i • : 1 : . i 1. AT. . •.;!•:::: ! P.w. P.D 0 CT. l S , 8 * o ; ; • i : : : • ..-.ii:! f ISJ to Spectrum No. 26. Spectrum No. 27. 1 • 2 * 0 1 4 • 0 10 0 • 6 9 c 2 7 1 C O S • 3 4 • 3 2 -> 2 3 1 4 7 1 • 9 7 3 • 5 9 <1 6 0 1 4 5 7 e 3 7 2 • 3 9 5 2 0 1 3 3 3 e 4 7 1 • 5 7 6 ° 3 1 3 3 5 • 6 7 • 7 6 7 3 3 1 2 4 2 • J 6 2 • 1 0 n 1 3 6 2 1 • 4 3 1 • 0 7 9 1 9 3 5 1 • 0 1 3 0 0 5 1 0 2 1 3 "v 1 • 0 1 7 • 5 4 S A J J . f>x C-T-JAopo HI JiiiHM-,000 •i •!•»'• I-f :T'U[.U' i to I 31.07 Spectrum No. 28. 1 1 ? 1 9 7 7 . 9 3.8 0 0 9 p 1 5 1 "i ) 2 . 6 7 9 • 6 3 -3 ? 0 1-13 0. 0 7 1 0 3 9 fl 2 3 1 7 0 • 3 0 5 2 7 1-10 3 . A • 7 0 • 1 7 6 1 9 1 3 3 7 . 5 5 9 • 3 7 1 2 3 . 1 .? 4 5 . 7 62 • 2e 8 ? 5 1 ? 1 ~> . 2 6 2 • 0 1 1 9 <Wj 0 1 9 ;> 3 1 ? 2 5 . -3 2 1 . 7 3 6 1 2 1 • • 2 0 0 6 •'!:• s.w;; 2.000 ^ • ArT.i P.W.; •.. i: P.D. s i 31.07 :rr:. ft:.: I 'i: Spectrum No. 29. 0< OC R Solent D A0 Tonp. a go' S.o-. 50O Hi. i :i : ; .1 i'i T8.69, ro 

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