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Capsular antigens of gram-negative bacteria Altman, Eleonora 1984

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CAPSULAR ANTIGENS OF GRAM-NEGATIVE BACTERIA  by  ELEONORA ALTMAN B.Sc,  The Hebrew U n i v e r s i t y o f J e r u s a l e m , I s r a e l , 1975  M.Sc., The Hebrew U n i v e r s i t y of J e r u s a l e m , I s r a e l , 1977.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Department of C h e m i s t r y )  We accept t h i s t h e s i s as conforming to t h e r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1984 © E l e o n o r a Altman, 1984  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by the head o f representatives.  my  It is  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department of  CHiSTfl  Y  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T  1Y3  Date  Sefit  written  i i  ABSTRACT  Klebsiella, the of  most  Escherichia  frequently  Salmonella  and  found  saccharides  (K  known  of  antigens)  The  which  relates  whereas  Approximately are  pathogenic  Shigella  lipopolysaccharides,  seventy of  Klebsiella  serotype  K50  Klebsiella  K antigens  for  play  eighty  structure  the is  in  coli,  Salmonella  to  Klebsiella  an i m p o r t a n t  serologically structures  presented  having  a  1  Shigella  Enterobacteria.  mainly  capsular  and  the  0  The  coli  among  classification  antigens  and E.  are  which  capsular  are poly-  role. different  have  been  It  strains  determined.  polysaccharide here.  Klebsiella  is  isolated  unique  five-plus-two'  from  among  repeating  the  unit.  - • 3 ) - p - D - G a l - ( l - > 3 ) - a - D - G l c - ( l - » 4 ) - a - D - G l c A - ( l-»3)-a-D-Man-( l->-2)-a-D-Man-( 1 + 6  1 a-D-Glc 6  1 P-D-Gal Klebsiella  The K a n t i g e n s groups  (A,  comprise coli  bear  B,  L)  acidic  on  of  the  Escherichia basis  of  polysaccharides.  a strong  similarity  to  coli  their The the  K50  can  be  divided  thermolability, extracellular  K antigens  of  into  all  of  A antigens Klebsiella.  three which of  E.  iii  The p r e s e n t i n v e s t i g a t i o n d e s c r i b e s the i s o l a t i o n and the s t r u c t u r a l a n a l y s e s of a c i d i c p o l y s a c c h a r i d e s o b t a i n e d from E s c h e r i c h i a 09:K28(A):H- (K28 a n t i g e n ) and E s c h e r i c h i a c o l i 09:K32(A):H19  coli  (K32  antigen).  ->3)-ct-D-Glc-( l+4)-B-D-GlcA-( l * 4 ) - a - L - F u c - ( 1* 4  2 and 3  •  I  1 B-D-Gal  OAc  Escherichia c o l i  K28  OAc  I 2 -•3 ) - a-D-Glc- (1^4)-a-L-Rha- (l->3)-a-D-Gal-( 1 •* 3 • 1 B-D-GlcA Escherichia c o l i  K32  S p e c i f i c b a c t e r i o p h a g e - b o r n e g l u c a n a s e s were u t i l i z e d to degrade t h e two E s c h e r i c h i a c o l i c a p s u l a r p o l y s a c c h a r i d e s . E. c o l i K32 p o l y s a c c h a r i d e has been degraded  u s i n g a p u r i f i e d <}>32 b a c t e r i o p h a g e w i t h  a - g l u c o p y r a n o s i d a s e a c t i v i t y , w h i l e E. c o l i K28 p o l y s a c c h a r i d e has been degraded  u s i n g crude s o l u t i o n s of the b a c t e r i o p h a g e s <t>28—1 and  tj>28—2  ( b o t h w i t h a - g l u c o p y r a n o s i d a s e a c t i v i t y ) , and the r e s u l t s have been compared.  iv  TABLE OF CONTENTS Page ABSTRACT  '  i  i  TABLE OF CONTENTS  iv  LIST OF APPENDICES  ix  LIST OF TABLES  x  LIST OF FIGURES  x i i  LIST OF SCHEMES  xiv  ACKNOWLEDGEMENTS  xv  PREFACE  xvi1  I.  INTRODUCTION  II.  METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES  11.1  11.2  1  ...  22  I s o l a t i o n and p u r i f i c a t i o n  23  11.1.1  K l e b s i e l l a polysaccharides  25  11.1.2  Escherichia c o l i polysaccharides  26  Sugar a n a l y s i s  26  11.2.1  T o t a l h y d r o l y s i s and m e t h a n o l y s i s  11.2.2  Characterizations of sugars  11.2.3  . . . .  26  and q u a n t i t a t i o n  Carboxyl reduction of a c i d i c polysaccharides  28  28  V  II.2.4  II.3  II. 4  t h e sugars  30  P o s i t i o n of l i n k a g e  31  11.3.1  Methylation analysis  31  11.3.2  G a s - l i q u i d chromatography ( g . l . c . )  11.3.3  Mass-spectrometry  Sequencing  ....  (m.s.)  37  o f sugars  44  Partial hydrolysis  44  11.4.2  P e r i o d a t e o x i d a t i o n and Smith degradation  46  Base-catalyzed degradation  53  D e t e r m i n a t i o n of l i n k a g e  55  11.5.1  Optical rotation  55  11.5.2  N u c l e a r magnetic  resonance  spectroscopy  11.5.3  II.6  33  11.4.1  11.4.3  II.5  D e t e r m i n a t i o n of the c o n f i g u r a t i o n of  11.5.2.1  1  11.5.2.2  13  58  H-n.m.r. s p e c t r o s c o p y C-n.m.r. s p e c t r o s c o p y  58 ....  Other t e c h n i q u e s  71  11.5.3.1  Enzymatic  11.5.3.2  Chromium t r i o x i d e o x i d a t i o n  L o c a t i o n of 0 - a c e t y l group  64  hydrolysis  71 ...  71  73  vi  I I I . GENERAL EXPERIMENTAL CONDITIONS  76  111.1 Paper chromatography  77  111.2 G a s - l i q u i d chromatography and g.I.e.-mass spectrometry 111.3 Gel-permeation  77 chromatography  111.4 O p t i c a l r o t a t i o n  78  and c i r c u l a r d i c h r o i s m  79  111.5 N u c l e a r magnetic resonance  79  111.6 General c o n d i t i o n s  80  111.7 I s o l a t i o n and p u r i f i c a t i o n of the p o l y s a c c h a r i d e s . .  80  111.7.1  K l e b s i e l l a polysaccharides  111.7.2  Escherichia c o l i polysaccharides  80 . . . .  111.8 B a c t e r i o p h a g e p r o p a g a t i o n  IV.  82 83  111.8.1  Tube and f l a s k l y s i s  83  111.8.2  L a r g e - s c a l e p r o p a g a t i o n of the b a c t e r i o p h a g e .  84  STRUCTURAL INVESTIGATION OF K l e b s i e l l a SEROTYPE K50 CAPSULAR POLYSACCHARIDE  86  IV. 1  Abstract  87  IV. 2  Introduction  88  IV.3  R e s u l t s and d i s c u s s i o n  88  IV. 4  Conclusion  100  IV. 5  Experimental  101  vii  V.  STRUCTURAL INVESTIGATION OF E s c h e r i c h i a  coli  CAPSULAR POLYSACCHARIDES  V.l  S t r u c t u r a l i n v e s t i g a t i o n of E s c h e r i c h i a 09:K28(A):H~ (K28 a n t i g e n )  V.2  109  coli  capsular  polysaccharide  110  V.l.l  Abstract  110  V.1.2  Introduction  I l l  V.1.3  R e s u l t s and D i s c u s s i o n  I l l  V.1.4  Conclusion  123  V.1.5  Experimental  125  S t r u c t u r a l i n v e s t i g a t i o n of E s c h e r i c h i a 09:K32(A):H19 (K32 a n t i g e n )  coll  capsular  polysaccharide  133  V.2.1  Abstract  133  V.2.2  Introduction  134  V.2.3  R e s u l t s and d i s c u s s i o n  134  V.2.4  Conclusion  141  V.2.5  Experimental  143  viii  VI.  VII.  BACTERIOPHAGE DEGRADATION OF E s c h e r i c h i a  coli  CAPSULAR POLYSACCHARIDES SEROTYPES K28 and K32  151  VI. 1  Introduction  152  VI. 2  Results  161  VI. 3  Discussion  170  VI. 4  Experimental  174  BIBLIOGRAPHY  179  ix  LIST OF APPENDICES  Appendix I  II  III  Page The known s t r u c t u r e s of the E s c h e r i c h i a 0 antigens  coli  The known s t r u c t u r e s of the E s c h e r i c h i a K antigens  coli  L  H and C-n.m.r. s p e c t r a 13  191  196  204  X  LIST OF TABLES  Table  Page  1.1  1.2  IV.1  IV.2  IV. 3  V. l  V.2  V.3  V.4  V.5  1  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 (K1-K83). Q u a l i t a t i v e a n a l y s i s and chemotype g r o u p i n g . . . .  10  Schematic r e p r e s e n t a t i o n of the a g g l u t i n a t i o n r e s u l t s on which d e f i n i t i o n s of A, B, and L a n t i g e n s a r e based  12  N.m.r. d a t a f o r K l e b s i e l l a K50 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  90  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 K50 p o l y s a c c h a r i d e and d e r i v a t i v e s  95  A n a l y s e s o f a c i d i c o l i g o s a c c h a r i d e s from K l e b s i e l l a K50 p o l y s a c c h a r i d e .  98  H-n.m.r. d a t a f o r E s c h e r i c h i a c o l l K28 polysaccharide  113  l C-n.m.r. d a t a f o r t h e n a t i v e and O - d e a c e t y l a t e d E. c o l i K28 p o l y s a c c h a r i d e  116  M e t h y l a t i o n a n a l y s i s o f E s c h e r i c h i a c o l i K28 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s  118  N.m.r. data f o r E s c h e r i c h i a c o l i K28 o l i g o s a c c h a r i d e s d e r i v e d from p a r t i a l h y d r o l y s i s of the p o l y s a c c h a r i d e  120  3  A n a l y s i s o f the 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 of E s c h e r i c h i a c o l i K28 p o l y s a c c h a r i d e .  .  121  xi  V.6  V. 7  VI. 1  VI.2  VI.3  VI.4  N.m.r. data f o r E s c h e r i c h i a c o l i K32 n a t i v e and O-deacetylated polysaccharides  137  M e t h y l a t i o n a n a l y s i s of E s c h e r i c h i a c o l i K32 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s  138  M e t h y l a t i o n a n a l y s i s and r e d u c i n g end d e t e r m i n a t i o n of E. c o l i K28 o l i g o s a c c h a r i d e i s o l a t e d a f t e r b a c t e r i o p h a g e <J>28—1 d e g r a d a t i o n of E. c o l i K28 polysaccharide  163  D e t e r m i n a t i o n of the degree of p o l y m e r i z a t i o n and the r e d u c i n g end of E. c o l i K28 o l i g o s a c c h a r i d e i s o l a t e d a f t e r b a c t e r i o p h a g e <b28—1 d e g r a d a t i o n of E. c o l i K28 p o l y s a c c h a r i d e  164  P r o t o n assignments (400 MHz) f o r t h e o l i g o s a c c h a r i d e s and r e l a t e d compounds generated by b a c t e r i o p h a g e d e p o l y m e r i z a t i o n of t h e E. c o l i K32 c a p s u l a r p o l y s a c c h a r i d e  169  M e t h y l a t i o n a n a l y s i s o f the reduced f r a c t i o n I I o b t a i n e d a f t e r t h e s e p a r a t i o n of the d e p o l y m e r i z a t i o n p r o d u c t s of E. c o l i K32 p o l y s a c c h a r i d e . . . .  171  xii  LIST OF FIGURES Figure 1.1 1.2 1.3  1.4  1.5  1.6  11.1  11.2  11.3 11.4 11.5 11.6  Page Schematic r e p r e s e n t a t i o n of the G r a m - p o s i t i v e Gram-negative c e l l w a l l of b a c t e r i a  and 3  E l e c t r o n m i c r o g r a p h of a c r o s s - s e c t i o n of E. c o l i 09:K29 a f t e r c o n t r a s t s t a i n i n g w i t h ruthenium r e d . .  5  Schematic diagram of the g e n e r a l s t r u c t u r e of b a c t e r i a l l i p o p o l y s a c c h a r i d e s (LPS)  13  Schematic r e p r e s e n t a t i o n of the s t r u c t u r e s of Na c e t y l n e u r a m i n i c a c i d and 2-keto-3-deoxy-D-manno2-octulosonic acid  15  a) E. c o l i K7 = K56 c r o s s - r e a c t s w i t h a n t i s e r u m t o S. pneumoniae type 3 and type 8. b) E. c o l i K30 c r o s s - r e a c t s w i t h a n t i s e r u m t o S. pneumoniae type 2 .  19  The s t r u c t u r e s of c a p s u l a r p o l y s a c c h a r i d e s o f H. i n f l u e n z a e type b and E s c h e r i c h i a c o l i K100  ...  20  Mass spectrum of a u r o n i c a c i d d e g r a d a t i o n d e r i v a t i v e from K l e b s i e l l a K50 p o l y s a c c h a r i d e ( a ) compared t o the spectrum of a standard d e r i v a t i v e (b) . .  41  Common p r o d u c t s formed on p e r i o d a t e o x i d a t i o n , f o l l o w e d by b o r o h y d r i d e r e d u c t i o n and h y d r o l y s i s of t e r m i n a l and m o n o s u b s t i t u t e d hexoses  48  S e q u e n t i a l p e r i o d a t e o x i d a t i o n and r e d u c t i o n of a l g i n a t e  51  borohydride  R e l a t i o n s h i p between d i h e d r a l angle ( <)>) and c o u p l i n g c o n s t a n t s f o r a- and 6-D-hexoses  61  Schematic r e p r e s e n t a t i o n of d i f f e r e n t r e g i o n s i n the ^H-n.m.r. spectrum of p o l y s a c c h a r i d e s  63  The -^H-n.m.r. s p e c t r a o f n a t i v e ( t o p ) and d e a c e t y l a t e d (bottom) E. c o l i K28 c a p s u l a r p o l y s a c c h a r i d e s  .  65  xiii  11.7  11.8  IV. 1  The c h a r a c t e r i s t i c r e g i o n s f o r resonances of carbon atoms b e l o n g i n g t o d i f f e r e n t monosaccharide residues i n polysaccharides  68  The * C-n.m.r. spectrum of d e a c e t y l a t e d E. c o l i K28 c a p s u l a r p o l y s a c c h a r i d e ...  70  Gel-permeation chromatography of the product o b t a i n e d a f t e r s e l e c t i v e , p a r t i a l h y d r o l y s i s of K l e b s i e l l a K50 p o l y s a c c h a r i d e  96  3  V. l  P a r t i a l s t r u c t u r e o f E. c o l l K28 p o l y s a c c h a r i d e . . .  117  VI.1  Schematic diagram d e m o n s t r a t i n g T bacteriophage  154  the s t r u c t u r e o f  2  VI.2  B a s i c m o r p h o l o g i c a l types of b a c t e r i o p h a g e s the types of n u c l e i c a c i d  with 154  VI.3  The mechanics of i n f e c t i o n by b a c t e r i o p h a g e  VI.4  C a p s u l a t e d E. c o l l K29 exposed t o a m.o.i. ( t h e m u l t i p l i c i t y of i n f e c t i o n ) of 300 phage f o r 8 min a t 37°  158  S e p a r a t i o n of the d e p o l y m e r i z a t l o n p r o d u c t s o f E. c o l i K32 by g e l - p e r m e a t i o n chromatography ( B i o - G e l P-4)  167  VI.5  VI.6  M o l e c u l a r weight d i s t r i b u t i o n of f r a c t i o n I I  ....  ....  156  168  xiv  LIST OF SCHEMES Scheme 11.1  11.2  11.3  Page R e d u c t i o n o f c a r b o x y l i c a c i d 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 reagent  29  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 saccharide  34  K50 p o l y -  The mass s p e c t r a of t h e a c e t a t e s (R = A c ) , methyl e t h e r s (R = Me), and t r i f l u o r o a c e t a t e s (R = COCF ) of a l d i t o l s . Only p r i m a r y fragments a r e shown . . .  39  The A - s e r i e s of fragments f o r the d e g r a d a t i o n o f a d i s a c c h a r i d e methyl g l y c o s i d e  42  Smith d e g r a d a t i o n polysaccharide  52  3  11.4  11.5  11.6  11.7  of K l e b s i e l l a  Uronic a c i d degradation polysaccharide  K50 c a p s u l a r  o f K l e b l s i e l l a K50  L o c a t i o n o f O - a c e t y l s u b s t i t u e n t s a c c o r d i n g t o the de B e l d e r and Norrman procedure  56  74  XV  ACKNOWLEDGEMENTS  I would l i k e t o e x p r e s s my s i n c e r e g r a t i t u d e t o P r o f e s s o r G.G.S. Dutton f o r h i s g u i d a n c e , encouragement and i n t e r e s t throughout the course o f t h i s work. I w i s h t o thank my c o l l e a g u e s and h e l p f u l d i s c u s s i o n s  i n the l a b o r a t o r y f o r t h e i r s u p p o r t  and Dr. E.H. M e r r i f i e l d ( U n i v e r s i t y o f Cape  Town, South A f r i c a ) f o r h i s a s s i s t a n c e  during  the e a r l y s t a g e s o f t h i s  work. Thanks a r e a l s o due t o Dr. S.C. Churms ( U n i v e r s i t y o f Cape Town, South A f r i c a ) f o r g e l - p e r m e a t i o n measurements; Dr. S.O. Chan and the s t a f f of the n.m.r. s e r v i c e and Dr. G. E i g e n d o r f and the s t a f f o f the mass s p e c t r o m e t r y s e r v i c e f o r t h e i r p a t i e n t  assistance.  My s p e c i a l thanks t o Dr. B. Lewis ( C o r n e l l U n i v e r s i t y ) f o r p r o o f reading  of t h i s t h e s i s . I s h o u l d a l s o l i k e t o thank R a n i Theeparajah f o r t y p i n g  thesis. F i n a l l y , my g r a t e f u l thanks to my husband B o r i s f o r h i s encouragement and moral s u p p o r t .  this  xvi  DEDICATED TO THE MEMORY OF  MY LATE FATHER  ISRAEL KATSIN  xv i i  PREFACE  The  t o p i c of t h i s t h e s i s i s concerned w i t h the s t r u c t u r e e l u c i d a -  t i o n 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 .  K l e b s i e l l a and E s c h e r i c h i a c o l i have  many f e a t u r e s i n common and our l a b o r a t o r y has f o r s e v e r a l y e a r s the c a p s u l a r a n t i g e n s  of K l e b s i e l l a .  studied  Now t h a t the s t r u c t u r e s of almost  a l l these 80 K s t r a i n s a r e known we a r e embarking on the e x a m i n a t i o n ofthe c a p s u l a r p o l y s a c c h a r i d e s  of E s c h e r i c h i a c o l i .  This thesis there-  f o r e , d e a l s m a i n l y w i t h s t r u c t u r e s of E s c h e r i c h i a c o l i K28 and K32 capsular polysaccharides.  The s t r u c t u r e of K l e b s i e l l a K50  r i d e was s t u d i e d f i r s t i n order t o become a c q u a i n t e d ogy  of the c a r b o h y d r a t e  polysaccha-  w i t h the m e t h o d o l -  research.  I n the I n t r o d u c t i o n I have attempted t o g i v e a c o n c i s e account of Escherichia c o l i polysaccharides, cal  t h e i r b i o l o g i c a l importance, s e r o l o g i -  c l a s s i f i c a t i o n and s t r u c t u r a l d i v e r s i t y . The Methodology s e c t i o n d e a l s w i t h the standard  s t r u c t u r a l a n a l y s i s together  w i t h more modern ones, such as the use of  n u c l e a r magnetic resonance s p e c t r o s c o p y .  Examples from the s t r u c t u r a l  i n v e s t i g a t i o n of K l e b s i e l l a and E s c h e r i c h i a c o l i polysaccharides  techniques o f  capsular  a r e chosen t o best i l l u s t r a t e the methods used.  Because of t h e i n c r e a s i n g importance of  bacteriophage-associated  g l y c a n a s e s f o r the s t r u c t u r a l a n a l y s i s of b a c t e r i a l s u r f a c e hydrates,  carbo-  a chapter which d e a l s w i t h the c l a s s i f i c a t i o n , s t r u c t u r e and  a p p l i c a t i o n s of b a c t e r i o p h a g e s i s i n c l u d e d . Appendix I c o n t a i n s c o l i 0 antigens. antigens  the l i s t of known s t r u c t u r e s of E s c h e r i c h i a  The l i s t of known s t r u c t u r e s of E s c h e r i c h i a c o l i K  along w i t h the l i t e r a t u r e r e f e r e n c e s  i s i n c l u d e d i n Appendix I I .  1  CHAPTER I  INTRODUCTION  2  I.  INTRODUCTION  N a t u r a l macromolecules c o n t a i n i n g c a r b o h y d r a t e u n i t s a r e o f w i d e spread o c c u r r e n c e i n a l l l i v i n g organisms and i n c l u d e ( a ) p o l y s a c c h a r i d e s as e x c l u s i v e l y c a r b o h y d r a t e polymers; (b) g l y c o p r o t e i n s , glycans,  and p e p t i d o g l y c a n s ;  proteo-  ( c ) g l y c o l i p i d s and l i p o p o l y s a c c h a r i d e s ;  (d) t e i c h o i c a c i d s and r e l a t e d macromolecules c o n t a i n i n g  phosphodiester-  l i n k e d o l i g o s a c c h a r i d e r e p e a t i n g u n i t s ; and ( e ) n u c l e i c a c i d s . Commercially, i n t e r e s t i n polysaccharides  1  has extended from  s t a r c h and c e l l u l o s e i n f o o d , pulp and paper i n d u s t r i e s t o n a t u r a l gums and  mucilages. The u s e f u l n e s s  of most commercial p o l y s a c c h a r i d e s  i s based on  t h e i r c a p a c i t y t o a l t e r the b a s i c p r o p e r t i e s o f water ( e . g . t h i c k e n i n g and  gelling).  Polysaccharides  a l s o p l a y an i m p o r t a n t r o l e i n c o n t r o l l -  i n g the t e x t u r e of foods as w e l l as t h e i r f l a v o r , appearance and c o l o r . They perform as t h i c k e n i n g and s i z i n g agents i n i n d u s t r i a l a p p l i c a t i o n s (the t e x t i l e and paper i n d u s t r i e s ) and as d r i l l i n g f l u i d s i n o i l f i e l d a p p l i c a t i o n s ( e . g . xanthan gum).  2  U n t i l r e c e n t l y , the b i o l o g i c a l f u n c t i o n s of p o l y s a c c h a r i d e s  were  thought t o be l i m i t e d t o s e r v i n g as s t r u c t u r a l polymers and energy reserves.  I t i s now w e l l e s t a b l i s h e d t h a t complex c a r b o h y d r a t e s p l a y an  important r o l e i n b i o l o g i c a l r e c o g n i t i o n as: receptors bacteriocins;  s p e c i f i c surface antigens;  f o r phage and  highly s p e c i f i c receptors i n  e u k a r y o t e s f o r v i r u s e s , b a c t e r i a , hormones and t o x i n s ; and d e t e r m i n a n t s of s e c r e t e d g l y c o p r o t e i n s w i t h i n the c e l l s .  C e r t a i n complex c a r b o -  3  - Capsular Polysaccharide  Capsule  , Capsular Protein Peptidoglycan With Teichoic Acid Polymers  Interior Of Cell  Fig.  Cell Wall  Phospholipid bilayer with various membrane Cytoplasmic proteins, enzymes and Membrane permeases  The  envelope o f the G r a m - p o s i t i v e c e l l  wall  The  envelope o f the Gram-negative c e l l  wall  1.1:  Schematic r e p r e s e n t a t i o n o f the G r a m - p o s i t i v e and the Gram-negative c e l l w a l l o f b a c t e r i a . T.J. Mackie and J . E . McCartney, " M e d i c a l Vol.  From Microbiology",  1: " M i c r o b i a l I n f e c t i o n s " , 13th edn., C h u r c h i l l  L i v i n g s t o n e , Edinburgh, 1978.  4  h y d r a t e s a r e c h e m i c a l messengers and a r e e s p e c i a l l y important  i n regu-  l a t i n g growth, development, r e p r o d u c t i o n , and d i s e a s e r e s i s t a n c e i n plants.  3  The m a j o r i t y of i m m u n o l o g i c a l l y of m i c r o b i a l o r i g i n . shown i n F i g . 1.1.  s i g n i f i c a n t polysaccharides are  A s i m p l i f i e d p i c t u r e of the b a c t e r i a l c e l l i s Outside  the plasma membrane i s the c e l l w a l l which  can be of two g e n e r a l t y p e s : one t h a t has an o u t e r membrane over a peptidoglycan l a y e r (Gram-positive  c e l l ) and one t h a t l a c k s t h e o u t e r  membrane but has a d d i t i o n a l components w i t h i n the p e p t i d o g l y c a n l a y e r (Gram-negative c e l l ) .  The main component i s l i p o p o l y s a c c h a r i d e , which  c o n s t i t u t e s 10-15% o f the d r y c e l l w a l l and e x e r t s both immunogenicity and  f u l l endotoxicity.  The s p e c i e s - s p e c i f i c somatic  polysaccharide  a n t i g e n i n the c e l l w a l l of Gram-negative b a c t e r i a i s c a l l e d t h e b a c t e r i a l 0 antigen. Many b a c t e r i a produce e x t r a c e l l u l a r p o l y s a c c h a r i d e s saccharides). surrounding unattached  They may e x i s t i n the form o f a d i s c r e t e c a p s u l e  the b a c t e r i a l c e l l o r i n the form of a l o o s e s l i m e , t o the c e l l s u r f a c e . * 1  Capsules  "mask" the c e l l w a l l 0  a n t i g e n s and i n t e r f e r e w i t h t h e i r s e r o l o g i c a l d e t e c t i o n . r e c o g n i z e d by t h e I n d i a i n k s t a i n i n g t e c h n i q u e (see F i g . 1.2). Capsules to  (exopoly-  5  They can be  or by e l e c t r o n m i c r o s c o p y  render b a c t e r i a r e s i s t a n t t o p h a g o c y t o s i s and  the a c t i o n o f t h e complement. T h i s c a p s u l a r m a t e r i a l i s a l s o immunogenic and g i v e s r i s e t o  s p e c i f i c a n t i - c a p s u l a r a n t i b o d i e s which r e a c t d i r e c t l y w i t h the encapsulated b a c t e r i a .  5  They were f i r s t found i n e a r l y s e r o l o g i c a l  s t u d i e s o f E. c o l i and the term K a n t i g e n (from the German word  5  F i g I . 2:  E l e c t r o n m l c r o g r a p h of a c r o s s - s e c t i o n of E. c o l i 09:K29 a f t e r c o n t r a s t s t a i n i n g w i t h ruthenium r e d . (From r e f . 5 ) .  6  7  "Kapsel") was i n t r o d u c e d i n 1945 by Kauffmann. p l a y an important  Capsular  polysaccharides  r o l e i n the immune response t o b a c t e r i a l i n f e c t i o n due  to t h e i r l o c a t i o n on the o u t e r s u r f a c e o f the b a c t e r i a and c o n s t i t u t e the p r i n c i p a l a n t i g e n s i n most of the p a t h o g e n i c , Gram-positive  organisms.  Gram-negative and  However, o t h e r a n t i g e n s , such as p r o t e i n s and  l i p o p o l y s a c c h a r i d e s (0 a n t i g e n s ) , can a l s o p l a y a s i g n i f i c a n t r o l e i n the human immune-response t o b a c t e r i a l  infection.  6  The m a j o r i t y 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 a r e h e t e r o g l y c a n s composed o f o l i g o s a c c h a r i d e r e p e a t i n g u n i t s .  T h i s can be shown by mole-  c u l a r weight d i s t r i b u t i o n s t u d i e s and by n u c l e a r magnetic resonance 7  spectroscopy. The acidic.  8  e x t r a c e l l u l a r p o l y s a c c h a r i d e s a r e g e n e r a l l y , but not always,  The a c i d i c component i s most o f t e n a u r o n i c a c i d , a p y r u v i c  a c i d a c e t a l , or a phosphoric  d i e s t e r grouping.  The c a p s u l e c r e a t e s a  s i m p l e , p h y s i c a l b a r r i e r p r o t e c t i n g the u n d e r l y i n g s u r f a c e o f t h e b a c t e r i a and f o r any g i v e n pathogenic  s t r a i n of b a c t e r i a , i t s v i r u l e n c e  i s d i r e c t l y r e l a t e d t o the amount of c a p s u l e .  6  The nomenclature o f b a c t e r i a l p o l y s a c c h a r i d e s i s c o m p l i c a t e d and i s based on the c l a s s i f i c a t i o n of the c o r r e s p o n d i n g b a c t e r i a . Enterobacteriaceae  i s a f a m i l y of Gram-negative, n o n - s p o r u l a t i n g  rods, e i t h e r motile or w i t h p e r i t r i c h o u s f l a g e l l a or non-motile.  They  grow on o r d i n a r y media and ferment g l u c o s e r a p i d l y w i t h o r w i t h o u t gas production.  The f a m i l y i s s u b - d i v i d e d i n t o t r i b e s , genera and s p e c i e s ,  which a r e the fundamental u n i t s of c l a s s i f i c a t i o n . from comparative  9  The system r e s u l t e d  s t u d i e s of the b i o c h e m i c a l r e a c t i o n s g i v e n by  8  relatively  l a r g e numbers of c u l t u r e s of each of the genera.  Kauffmann  has proposed the f o l l o w i n g c l a s s i f i c a t i o n of E n t e r o b a c t e r i a c e a e :  9  Familia Enterobacteriaceae  Genera  Tribes  A.  B.  C.  Eschericheae  Klebsielleae  Proteae  i.  Escherichia  ii.  Shigella  iii.  Salmonella  iv.  Citrobacter  i.  Klebsiella  ii.  Enterobacter  iii.  Hafnia  iv.  Serratia  i .  Proteus  ii.  Morganella  iii.  Rettgerella  iv.  Providencia  Knowledge of the c h e m i c a l s t r u c t u r e 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 i s of g r e a t s i g n i f i c a n c e f o r u n d e r s t a n d i n g  the m o l e c u l a r p r i n c i p l e s of  9  their biological a c t i v i t i e s .  Most of the b a c t e r i a a r e p a t h o g e n i c f o r  man, form s m a l l groups of s e r o l o g i c a l l y typed s p e c i e s and a r e c o n v e n i e n t f o r comparative  immunochemical r e s e a r c h .  be used as human v a c c i n e s  P u r i f i e d p o l y s a c c h a r i d e s can  ( e . g . S t r e p t o c o c c u s pneumoniae) and a con-  s i d e r a b l e amount of r e s e a r c h has been done i n t h i s  Klebsiella  field.  1 0  polysaccharides  The genus K l e b s i e l l a i s composed of t h r e e s p e c i e s : K. pneumoniae, K. ozaenae and K. r h i n o s c h l e r o m a t i s .  1 1  K l e b s i e l l a c u l t u r e s were  classi-  f i e d by 0rskov on t h e b a s i s o f t h e i r K ( c a p s u l a r ) and 0 ( s o m a t i c ) antigens.  1 2 , 1 3  K. pneumoniae i s t h e most important member of t h e f a m i l y .  It is  found i n the r e s p i r a t o r y t r a c t of 5% of normal i n d i v i d u a l s and i s the primary cause o f pneumonia i n 3% o f a l l b a c t e r i a l pneumonias. * 11  Nimmich has r e p o r t e d the q u a l i t a t i v e c o m p o s i t i o n m a t e l y 80 d i f f e r e n t K l e b s i e l l a K s e r o t y p e s ' 1 5  into chemotypes  17  1 6  of the a p p r o x i -  and has c l a s s i f i e d  them  ( s e e Table 1.1).  The s t r u c t u r e s o f seventy 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 a r e known t o d a t e .  V a r i o u s s t r u c t u r a l p a t t e r n s have emerged.  d i v i d e d i n t o four types:  1)  those l a c k i n g u r o n i c a c i d ;  the u r o n i c a c i d i s a component of the main c h a i n ;  These may be 2) those  where  3) those where t h e  u r o n i c a c i d i s i n a s i d e c h a i n ; and 4) those w i t h s i d e c h a i n s of t h r e e units. A d e t a i l e d c o m p i l a t i o n of d i f f e r e n t K l e b s i e l l a s t r u c t u r e s was done by D i F a b i o  1 8  and i s n o t , t h e r e f o r e , r e p e a t e d  i n this thesis.  In  10  TABLE 1.1;  K l e b s i e l l a capsular polysaccharides (K1-K83). Qualitative analysis and P  chemotype grouping  P P , 11 , 15, 25, 27 , 51  GlcA  Gal  Glc  8  GlcA  Gal  Man  20, 2 1 , 2 9 , 4 2 , 43, 66,  GlcA  Gal  Rha  9, 9*,  GlcA  Glc  Man  2, 4, 5 ,  GlcA  Glc  Rha  17, 23, 44, 45, 71  GlcA  Glc  Fuc  GlcA  Gal  Glc  1, 54 P P P P P P 7 , 10, 13 , 26 , 28, 30 , 31 , 33 ,  P  P  74  P  47, 52, 81, 83 24  P  Man  P  3 5 , 39, 4 6 , 50, 59, 60, 61, 62, P  P  GlcA  Gal  Glc  Fuc  16,  GlcA  Gal  Glc  Rha  12 ,  GlcA  Gal  Man  Rha  40, 53,  GlcA  Glc  Man  Fuc  6  GlcA  Glc  Man  Rha  GlcA  Gal  Glc  Man  Fuc  68  GlcA  Gal  Glc  Man  Rha  14 ,  GalA  Gal  Man  3 , 49,  GalA  Glc  Rha  34,  GalA  Gal  Fuc  63  PyrA  Glc  Rha  72  PyrA  Gal  Rha  32  PyrA  Gal  Glc  KetoA  Gal  Glc  GlcA  glucuronic acid  Gal  GalA  galacturonic acid  Man  mannose  PyrA  pyruvic acid  Rha  rhamnose  KetoA  rare uronic acid  Fuc  fucose  Glc  glucose  P  pyruvic a c i d present  Rha  P  58  P  18, 19, 3 6 , 41, 5 5 , 7 0 , P  80  P  P  P  P  P P 64 , 65 P  P  67 57  48  56 22 , 37,  38  galactose  in addition  79  69  ?  11  Appendix I and I I the l i s t of known s t r u c t u r e s of E. c o l i 0 and K antigens i s given.  Escherichia c o l i  polysaccharides  The organism E s c h e r i c h i a c o l i was f i r s t i s o l a t e d from f a e c e s by E s c h e r i c h i n 1885. I t belongs t o 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 whose normal h a b i t a t i s the i n t e s t i n a l t r a c t o f man and a n i m a l s .  1 9  E. c o l i i s  o f t e n found i n human u r i n a r y t r a c t i n f e c t i o n s and i s a s s o c i a t e d w i t h severe i n f a n t i l e d i a r r h e a .  2 0  W i t h i n t h e s p e c i e s , many d i f f e r e n t s e r o t y p e s a r e r e c o g n i z e d .  The  s e r o t y p i n g scheme i s based on the i d e n t i f i c a t i o n of s u r f a c e 'K', s o m a t i c '0' and f l a g e l l a  *H' a n t i g e n s .  The 0 a n t i g e n s a r e t h e r m o s t a b l e  somatic a n t i g e n s ,  h e a t i n g a t 100°, and a r e not d e s t r o y e d by a l c o h o l .  resisting  The term K a n t i g e n  covers a group of e i t h e r envelope o r c a p s u l a r a n t i g e n s which can be d i v i d e d i n t o t h r e e groups (A, B, L ) ( s e e Table 1.2).  S t r a i n s which  c o n t a i n the t h e r m o l a b i l e L o r B a n t i g e n s do not u s u a l l y possess m o r p h o l o g i c a l c a p s u l e s , whereas s t r a i n s w i t h t h e r m o s t a b l e capsulated.  9  A antigen are  The main t e s t s t o demonstrate the presence o f a K a n t i g e n  are 0 i n a g g l u t i n a b i l i t y o f the l i v i n g b a c t e r i a and t h e i r a g g l u t i n a t i o n with K sera. The f i r s t a n t i g e n i c scheme, c o m p r i s i n g 25 0 a n t i g e n s , was e s t a b l i s h e d by Kauffmann i n 1 9 4 7 . been added and a p p r o x i m a t e l y recognized.  9  21  S i n c e t h e n , many 0 a n t i g e n s have  100 'K', 164 '0* and 56 'H' a r e c u r r e n t l y  12  TABLE 1.2;  Schematic presentation of the agglutination  results on  which d e f i n i t i o n s of A, B, and L antigens are based  K type  Antigen  0  preparation  Serum  OK Serum Absorbed culture  by heated  Unabsorbed  at 100° f o r 2 h  L  Live (or formalin treated  B  A  a  _a  +  Boiled (100° f o r 1 h)  +  _c  +  Live  -  -  +  Boiled  +  -  +  Live  -  -  +  Boiled  -  -  +  N e g a t i v e or s i g n i f i c a n t l y lower than t h a t of b o i l e d  b +,  Agglutination  c  No  agglutination  culture  13  0 antigens  0 A n t i g e n s c o n s i s t of t h r e e r e g i o n s : c o r e , and the O - s p e c i f i c p o l y s a c c h a r i d e  i  l i p i d A, an  c h a i n (see F i g .  Core  polysaccharide  oligosaccharide  1.3:  1.3).  r  O-specific  Fig.  oligosaccharide  Lipid  A  Schematic diagram of the g e n e r a l  structure  of b a c t e r i a l l i p o p o l y s a c c h a r i d e s  (LPS).  L i p i d A ( r e g i o n 1) i s b u r i e d i n the o u t e r membrane of b a c t e r i a l c e l l and  i s r e s p o n s i b l e f o r the g e n e r a l e n d o t o x i c  of l i p o p o l y s a c c h a r i d e . fatty acids. The  The  core  2 2  the properties  I t c o n s i s t s of g l u c o s a m i n e , phosphate  and  s t r u c t u r e of l i p i d A has been d e s c r i b e d r e c e n t l y .  2 3  ( r e g i o n 2) i s l i n k e d t o l i p i d A v i a a c a r b o h y d r a t e  component t h a t i s t y p i c a l f o r the LPS  of Gram-negative b a c t e r i a ,  2-keto-3-deoxy-D-manno-2-octulosonic a c i d (KDO). s t r u c t u r e s have been d e s c r i b e d  so f a r .  time t h a t w i l d - t y p e E n t e r o b a c t e r i a c e a e spontaneous S (smooth) —  Five different  core  I t has been known f o r a l o n g growing on agar may  undergo a  > R (rough) m u t a t i o n which i s a s s o c i a t e d  with  14  the disappearance  of the 0 a n t i g e n and l o s s of the p a t h o g e n i c i t y .  A n a l y s i s of the R mutants r e v e a l e d t h a t they l a c k the O - s p e c i f i c c h a i n and c o n s i s t o n l y of the core bound t o l i p i d  A.  2 3  Region 3 i s the s p e c i f i c 0 p o l y s a c c h a r i d e of the LPS of b a c t e r i a l S forms.  I t i s b u i l t up from r e p e a t i n g u n i t s of o l i g o s a c c h a r i d e s which  may c o n t a i n up t o 6-7 sugars. For a l o n g time a l l E. c o l l 0 a n t i g e n s were thought t o c o n t a i n o n l y n e u t r a l p o l y s a c c h a r i d e c h a i n s . recently,  More  LPS were i s o l a t e d t h a t c o n t a i n a c i d i c components such as  g l y c e r o l phosphate, h e x u r o n i c a c i d s , and n e u r a m i n i c  acid.  2 2  The  s t r u c t u r e s of the 0 a n t i g e n s t h a t have been p u b l i s h e d u n t i l now a r e g i v e n i n Appendix I .  K antigens  The K a n t i g e n s a r e c a p s u l a r or envelope a n t i g e n s and can be d e t e c t e d by Immunoelectrophoresis. for  They a r e a l l p o l y s a c c h a r i d e s  two t h a t a r e p r o t e i n s (K88 and K 9 9 ) . . S i n c e these 2 2  capsular  p o l y s a c c h a r i d e s a r e not immunogenic f o r humans and a n i m a l s , they have t o be c h e m i c a l l y m o d i f i e d t o become immunogens.  except  will  F o r such s t u d i e s  knowledge 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 s i s extremely v a l u a b l e . d i f f i c u l t y i n 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 E. c o l i  2 4  The  capsular  p o l y s a c c h a r i d e s a r i s e s from the f a c t , t h a t u n l i k e f o r 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 , the q u a l i t a t i v e c o m p o s i t i o n of the most of the E. c o l l s e r o t y p e s i s not known.  T h i s p l a c e s the r e s e a r c h e r i n a d i f f i c u l t  p o s i t i o n , c o n s i d e r i n g the f a c t t h a t E. c o l i p o l y s a c c h a r i d e s a r e extremely  d i v e r s e and c o n t a i n uncommon and sometimes r a r e sugars.  The  15  most prominent  f e a t u r e of these 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 t h e f r e q u e n t  o c c u r r e n c e of 2-keto-3-deoxy-D-manno-2-octulosonic a c i d (KDO) and a l s o the o c c u r r e n c e of N - a c e t y l n e u r a m i n i c a c i d (NeuNAc o r NANA) (see F i g . 1.4).  N-Acetylmannosaminuronic a c i d (ManNAcA) was found i n t h e K7  antigen.  2 5  H  N-acetylneuraminic acid  2-keto-3-deoxy-D-manno-2-  (NeuNAc o r NANA)  o c t u l o s o n i c a c i d (KDO)  F i g . 1.4:  Schematic  r e p r e s e n t a t i o n of t h e s t r u c t u r e s of  N - a c e t y l n e u r a m i n i c a c i d and 2-keto-3-deoxy-Dmanno-2-octulosonic  acid.  A number o f K a n t i g e n s occur e x c l u s i v e l y i n 0 groups 08, 09 and 0101.  2 2  The K a n t i g e n s of these groups a r e of two t y p e s , one w i t h amino  sugars and one w i t h o u t . m o l e c u l a r weights  Those d e v o i d of amino sugars have h i g h  (3 x 1 0 - 1 0 d a l t o n s ) . 5  6  They a r e p h y s i c a l l y  heterogeneous and become homogeneous a f t e r m i l d a l k a l i  treatment.  2 6  These K a n t i g e n s ( a l l A a n t i g e n s and some B a n t i g e n s ) have a v e r y l o w e l e c t r o p h o r e t i c m o b i l i t y , form t h i c k and c o p i o u s c a p s u l e s and bear a s t r o n g resemblance t o t h e K a n t i g e n s o f K l e b s i e l l a .  2 7  I t i s noteworthy  16  t h a t the 08 and 09 a n t i g e n s antigens.  a r e themselves r e l a t e d t o K l e b s i e l l a 0  Thus the 09 a n t i g e n of E. c o l i i s i d e n t i c a l w i t h the  K l e b s i e l l a 03 a n t i g e n , and the E. c o l i 08 a n t i g e n i s i d e n t i c a l w i t h t h e K l e b s i e l l a ' 05 a n t i g e n . The K a n t i g e n s  5  of a group t h a t c o n t a i n s amino sugars were thought  to be e x t r a c e l l u l a r p o l y s a c c h a r i d e s lipopolysaccharides.  5  E.  but they were found to be  c o l i w i t h these K a n t i g e n s  c o n t a i n two c e l l  w a l l l i p o p o l y s a c c h a r i d e s : an a c i d i c one which i s termed a K a n t i g e n i n a d d i t i o n to a n e u t r a l one ( t h e 08, 09 o r 0101 a n t i g e n ) . l i p o p o l y s a c c h a r i d e s a r e not c a p s u l a r  (K) a n t i g e n s  they were c a l l e d t h e r m o l a b i l e B a n t i g e n s Kauffmann.  The a c i d i c  i n the t r u e sense;  i n the nomenclature of  9  K antigens  o c c u r r i n g i n 0 groups o t h e r than 08, 09 and 0101 a r e  a l l a c i d i c polysaccharides  w i t h r a t h e r low m o l e c u l a r  w e i g h t s (below  50,000 d a l t o n s ) and h i g h e l e c t r o p h o r e t i c m o b i l i t y . ' 5  2 2  Kauffmann has  c a l l e d them t h e r m o l a b i l e L a n t i g e n s , and they were found i n most E s c h e r i c h i a c o l i s t r a i n s i s o l a t e d from p a t h o l o g i c a l m a t e r i a l . c o n t a i n r a r e sugar c o n s t i t u e n t s , such as N - a c e t y l n e u r a m i n i c N-acetylmannosaminuronic a c i d .  They  a c i d or  The K l a n t i g e n , a l s o known as c o l o m i n i c  a c i d , i s a poly-N-acetyl neuraminic a c i d . polymer.  9  2 8  K2 i s a t e i c h o i c a c i d - l i k e  2 4  A l t h o u g h p a r t i a l s t r u c t u r e s have been determined f o r s e v e r a l E. c o l i polysaccharides, published.  r e l a t i v e l y few complete s t r u c t u r e s have been  The s t r u c t u r e s of E. c o l i K42 and K l e b s i e l l a K63 a r e i d e n t i -  c a l and c r o s s - r e a c t s e r o l o g i c a l l y . K20 a n t i g e n s  2 9  The E. c o l l K 3 0  3 0  and K l e b s i e l l a  a r e i d e n t i c a l , and E. c o l i K100 a n t i g e n i s s t r u c t u r a l l y  17  r e l a t e d to the H a e m o p h i l i s i n f l u e n z a e type b c a p s u l a r a n t i g e n . s t r u c t u r e s of the E. c o l i K a n t i g e n s  The  d l  t h a t have been p u b l i s h e d u n t i l  now  are g i v e n i n Appendix I I .  Immunology of polysaccharides  The  polysaccharides  an immune response and  are t r u e immunoantigens i n t h a t they i n d u c e  the g e n e r a t i o n  shown t h a t o n l y a r e l a t i v e l y major s i t e of a n t i b o d y  of s p e c i f i c a n t i b o d i e s .  It  s m a l l p o r t i o n of a p o l y s a c c h a r i d e  s p e c i f i c i t y and  was  i s the  t h a t p a r t i s known as the  deter-  minant group. * 1  A determinant group may one  comprise s e v e r a l monosaccharide r e s i d u e s ,  of which c o n t r i b u t e s most to the s p e c i f i c i t y ; t h a t monosaccharide  r e s i d u e i s termed the immunodominant sugar. Kabat  3 2  on an i s o m a l t o s e  The  c l a s s i c a l s t u d i e s of  o l i g o s a c c h a r i d e s e r i e s have shown t h a t  non-reducing t e r m i n a l r e s i d u e c o n t r i b u t e d about 40% and  the next  r e s i d u e s t o g e t h e r about 60% to the t o t a l b i n d i n g energy. i n d i c a t e that i n a l i n e a r polysaccharide  polysaccharide polysaccharides  chain.  The  two  These r e s u l t s  the i m m u n o l o g i c a l  r e s i d e s p r i m a r i l y i n the t e r m i n a l sugar r e s i d u e and  the  specificity  extends a l o n g  the  s i t u a t i o n i s d i f f e r e n t f o r branched  i n which the immunodominant sugars are those which a r e  l o c a t e d on the s i d e c h a i n s .  3 3  C e r t a i n noncarbohydrate groups  f u n c t i o n as a n t i g e n i c d e t e r m i n a n t s .  Jann and W e s t p h a l  2 7  may  have shown t h a t  0 - a c e t y l groups are e s s e n t i a l p a r t s of the determinant r e g i o n s i n some Salmonella  polysaccharides.  18  P y r u v i c a c i d , a t t a c h e d t o a sugar as an a c e t a l was found t o be the immunologic determinant  i n the c a p s u l a r p o l y s a c c h a r i d e s of  K l e b s i e l l a and S t r e p t o c o c c u s  pneumoniae.  34  C r o s s - r e a c t i o n s have been used e x t e n s i v e l y i n immunochemical a n a l y s i s and they can be used t o e s t a b l i s h the s t r u c t u r e s of immunodeterminant  groups.  H e i d e l b e r g e r has used t h i s approach e x t e n s i v e l y and  was a b l e t o p r e d i c t the presence o f some s t r u c t u r a l f e a t u r e s b e f o r e were v e r i f i e d c h e m i c a l l y .  3 5 - 3 8  they  S t u d i e s of c r o s s - r e a c t i o n s of more than  60 c a p s u l a r , t y p e - s p e c i f i c p o l y s a c c h a r i d e s of K l e b s i e l l a w i t h 26 s p e c i f i c types of antipneumococcal  s e r a p e r m i t t e d the assignment of  s e v e r a l s t r u c t u r a l f e a t u r e s such as non-reducing D-glucuronic  terminal residues of  a c i d , L-rhamnose, and D - g a l a c t u r o n i c a c i d and some l i n k a g e s  w i t h i n the p o l y s a c c h a r i d e c h a i n .  The m o l e c u l a r b a s i s of some  c r o s s - r e a c t i o n s i n which E. c o l i 0 and K a n t i g e n s p a r t i c i p a t e has been elucidated  2 2  ( s e e F i g . 1.5).  C a p s u l a r p o l y s a c c h a r i d e s a r e i m p o r t a n t v i r u l e n c e f a c t o r s i n many b a c t e r i a l i n f e c t i o n s i n c l u d i n g those caused by S t r e p t o c o c c u s  pneumoniae,  N e i s s e r i a m e n i n g i t i d i s , Hemophilus i n f l u e n z a e , E s c h e r i c h i a c o l i , S a l m o n e l l a t y p h o s a , K l e b s i e l l a pneumoniae, and S t a p h y l o c o c c u s  aureus.^  The f i r s t c a p s u l a r p o l y s a c c h a r i d e v a c c i n e arose from the e a r l y work on Streptococcus  pneumoniae.  I t i s important  39  t o r e a l i z e , however, t h a t the immunity r e c e i v e d  on r e c o v e r y from i n f e c t i o n by e n c a p s u l a t e d generated vaccines.  by immunization  b a c t e r i a d i f f e r s from t h a t  with p u r i f i e d capsular polysaccharide  I n young c h i l d r e n p o l y s a c c h a r i d e v a c c i n e s g i v e r i s e t o o n l y  IgM a n t i b o d i e s and t h e r e i s no memory response, whereas p r o t e i n v a c c i n e s  19  a)  —  ManNAcA P  Glc — 6 8 OAc  E. c o l i K7 = K56  —  GlcA —  8  Glc  — 8  S. pneumoniae type 3  - GlcA —  Glc —  B  8  Glc —  a  Gal — c  S. pneumoniae type 8  b)  —  Man — j o l l Gal  Gal  3  — B  a  GlcA E. c o l i K30  Rha 1 3 Rha 1 3 Rha 1 4 Glc a 2I l l Glc a  1  a  GlcA S. pneumoniae type 2  Fig.  1.5:  a ) E. c o l i K7 « K56 CROSS-REACTS WITH ANTISERUM TO S. pneumoniae TYPE 3 AND TYPE 8. b) E. c o l i K30 CROSS-REACTS WITH ANTISERUM TO S. pneumoniae TYPE 2  20  induce b o t h IgM and  IgG a n t i b o d i e s and an i m m u n o l o g i c a l memory.  There  H  have been s e v e r a l attempts to overcome t h i s problem by c o n j u g a t i n g saccharides  to a p r o t e i n c a r r i e r .  T h i s approach has been used a l s o i n  0  the development of the v a c c i n e a g a i n s t p a t h o g e n i c E. c o l i and K5 a n t i g e n s  in particular.  organisms e n c o u n t e r e d .  Both a n t i g e n s  The  capsular  antigens and  A p r o p o s a l has been made t o use a c r o s s - r e a c t i n g E.  polysaccharide  polysaccharide  as an a l t e r n a t i v e v a c c i n e . * 1  E. c o l i  1  c r o s s - r e a c t s w i t h H. i n f l u e n z a e type b  (see F i g . 1.6).  H. i n f l u e n z a type b may  w h i c h m e n i n g i t i s i s the most f r e q u e n t  100  polysaccharide  3  1  Rib  f  1  P  ribitol  5  Haemophilus i n f l u e n z a e type b  Fig.  1.6;  The  of  and more s e r i o u s h e a l t h problem. capsular  polysaccharide  because of i t s s t r u c t u r a l r e l a t e d n e s s t o H. i n f l u e n z a e type b. p o s i t i v e r e s u l t s have been o b t a i n e d  coli  capsular  cause s e v e r a l d i s e a s e s ,  Conjugates were prepared w i t h E. c o l i K100  —  and  M e n i n g i t i s of s m a l l c h i l d r e n i s o f t e n caused by  5  of both of these pathogens are i d e n t i c a l , both s t r u c t u r a l l y  Kl  the K l  belong t o the most v i r u l e n t  N. m e n i n g i t i d i s group B and a l s o by E. c o l i K l .  serologically.  poly-  i n animal experiments. * 1  3  Rib  1  f  2  P  ribitol  E. c o l i  First  1  5  K100  s t r u c t u r e s of c a p s u l a r p o l y s a c c h a r i d e s  H. i n f l u e n z a e type b and E s c h e r i c h i a c o l i  of K100.  21  A new  p o t e n t i a l i n p o l y s a c c h a r i d e immunochemistry i s p r o v i d e d by  homogeneous immunoglobulins  t h a t b i n d c a r b o h y d r a t e polymers.  s p e c i f i c immunoglobulin-hapten  Here, the  i n t e r a c t i o n can be s t u d i e d i n d e t a i l . * 1  2  The 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 b a c t e r i o p h a g e d e g r a d a t i o n of b a c t e r i a l s u r f a c e c a r b o h y d r a t e s may  be coupled t o the p r o t e i n  carriers  s e r v i n g as immunogens, r e p r e s e n t a t i v e of the c o r r e s p o n d i n g b a c t e r i a l glycans. * 1  3  B a c t e r i o p h a g e - i n d u c e d d e g r a d a t i o n of 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 d i s c u s s e d i n S e c t i o n VI of t h i s  thesis.  P o l y s a c c h a r i d e s on the s u r f a c e of a m i c r o b i a l c e l l are the s e r o l o g i c a l d e t e r m i n a n t s of t h a t organism and t h e r e f o r e r e p r e s e n t a h i g h l y s p e c i f i c means of i d e n t i f i c a t i o n . approaches,  T h e i r study by c h e m i c a l and g e n e t i c  as w e l l as the i n v e s t i g a t i o n of t h e i r b i o s y n t h e t i c pathways,  are e s s e n t i a l f o r our u n d e r s t a n d i n g of b a c t e r i a l p a t h o g e n i c i t y . In the course of t h i s t h e s i s , the s t r u c t u r e s of K l e b s i e l l a  K50  p o l y s a c c h a r i d e and two E s c h e r i c h i a c o l i p o l y s a c c h a r i d e s , s e r o t y p e  K28  and K32, were i n v e s t i g a t e d .  Two  Escherichia c o l i capsular polysaccha-  r i d e s (K28 and K32) were degraded by t h e i r r e s p e c t i v e b a c t e r i o p h a g e s (<(>28-l, <))28-2 and  <j>32) and the d e g r a d a t i o n p r o d u c t s were c h a r a c t e r i z e d .  22  CHAPTER I I  METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES  23  II.  METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES  An almost i n f i n i t e v a r i e t y o f s t r u c t u r a l types i s p o s s i b l e among polysaccharides. furanose  Each sugar r e s i d u e can e x i s t i n t h e pyranose o r  form, each g l y c o s i d i c l i n k a g e may have t h e oc- o r B - c o n f i g u r a -  t i o n and t h e g l y c o s i d i c l i n k a g e may i n v o l v e s u b s t i t u t i o n o f d i f f e r e n t h y d r o x y l groups i n an a d j a c e n t  sugar r e s i d u e .  Chemical methods o f d e t e r m i n a t i o n o f t h e s t r u c t u r e o f p o l y s a c c h a rides involve: constituents;  1) q u a l i t a t i v e and q u a n t i t a t i v e e s t i m a t i o n o f t h e sugar 2) a n a l y s i s f o r removable s u b s t l t u e n t s ( O - a c e t y l ,  N_-acetyl, phosphate, e t c . ) ; tion;  4) d e t e r m i n a t i o n  3) d e t e r m i n a t i o n  of the l i n k a g e c o n f i g u r a -  o f t h e p o s i t i o n of l i n k a g e ;  5)  determination  of t h e sugar sequence. I t i s obvious t h a t no one s t r u c t u r a l method can g i v e answers t o a l l these q u e s t i o n s .  I n t h e f o l l o w i n g s e c t i o n an attempt i s made t o  d e s c r i b e each o f t h e known s t r u c t u r a l methods t o g e t h e r w i t h some o f i t s limitations.  II. 1  ISOLATION AND PURIFICATION * 1  One o f t h e most i m p o r t a n t t i o n of p o l y s a c c h a r i d e s homogeneous i n d i v i d u a l  and d i f f i c u l t steps i n the i n v e s t i g a -  i s t h e i r p u r i f i c a t i o n and f r a c t i o n a t i o n polysaccharides.  into  24  The s i m p l e s t e x t r a c t i o n methods f o r p o l y s a c c h a r i d e s are t h o s e u s i n g water alone a t v a r i o u s t e m p e r a t u r e s .  Some p o l y s a c c h a r i d e s can be  brought i n t o s o l u t i o n by the use of p o l a r nonaqueous s o l v e n t s , such as d i m e t h y l s u l f o x i d e f o r s t a r c h and glycogen * * or 1  N-oxide f o r c e l l u l o s e .  1 + 5  N-methylmorpholine  1  D i l u t e a l k a l i has been used f o r p o l y s a c c h a r i d e  e x t r a c t i o n s , but one has t o be aware of p o s s i b l e b a s e - c a t a l y z e d degradation.  E x t r a c t i o n of p o l y s a c c h a r i d e s under a c i d i c c o n d i t i o n s i s n o r m a l l y  a v o i d e d due to the p o s s i b l e c l e a v a g e of g l y c o s i d i c bonds.  I n o r d e r to  o b t a i n the c a r b o h y d r a t e p o r t i o n of g l y c o p r o t e i n s , e x t e n s i v e d i g e s t i o n w i t h p r o t e a s e of low s p e c i f i c i t y can be p e r f o r m e d .  1+6  Mucopolysaccha-  r i d e s , l i p o p o l y s a c c h a r i d e s and n u c l e i c a c i d s can be p r e c i p i t a t e d aqueous s o l u t i o n by adding l i q u i d p h e n o l . * 1  from  7  The next s t e p i n v o l v e s r e s o l u t i o n of a p o l y s a c c h a r i d e m i x t u r e i n t o i t s components.  Methods f o r f r a c t i o n a t i o n of p o l y s a c c h a r i d e s  i n t o t h r e e broad c a t e g o r i e s :  1) those based on s e l e c t i v e  fall  precipitation  of p o l y s a c c h a r i d e themselves (by a d d i t i o n of a w a t e r - m i s c i b l e s o l v e n t such as acetone or e t h a n o l * ) or of t h e i r s a l t s ( p r e c i p i t a t i o n of a c i d i c 1  8  polysaccharides w i t h potassium c h l o r i d e , * 1  c u p r i c a c e t a t e or s u l f a t e ,  9  or w i t h c a t i o n i c d e t e r g e n t s such as c e t y l t r i m e t h y l a m m o n i u m bromide (Cetavlon)) *;  2)  5  t h o s e based on f o r m a t i o n of complexes  F e h l i n g s o l u t i o n f o r mannans,  51  (use of  borate f o r galactomannans,  h y d r o x i d e f o r g l u c o - and g a l a c t o m a n n a n s ) ; 53  3)  52  barium  those based on  chromatographic procedures ( g e l f i l t r a t i o n * or m o l e c u l a r s i e v e 5 1  chromatography, chromatography  5 7  55  ).  i o n - exchange  chromatography,  56  affinity  5 0  25  The  p r a c t i c a l problem i s t o e s t a b l i s h the p u r i t y of a p o l y s a c c h a -  r i d e or r a t h e r t o demonstrate the absence of h e t e r o g e n e i t y by as many independent c r i t e r i a as p o s s i b l e .  This includes  demonstration of  constancy i n c h e m i c a l c o m p o s i t i o n (based on sugar r a t i o s ;  spectroscopic  e x a m i n a t i o n by n u c l e a r magnetic resonance s p e c t r o s c o p y ) and p h y s i c a l properties  ( o p t i c a l r o t a t i o n ; m o l e c u l a r weight d e t e r m i n a t i o n by g e l  permeation c h r o m a t o g r a p h y ; 58  electrophoresis ' ). 5 9  II.1.1 Klebsiella p o l y s a c c h a r i d e s ' ' 15  61  6 0  62  A c u l t u r e of K l e b s i e l l a K50, o b t a i n e d from Dr; I d a 0 r s k o v , was grown as p r e v i o u s l y streaked  described. ' 6 1  6 2  B r i e f l y , b a c t e r i a l c u l t u r e was  on agar p l a t e s and grown a t 37° u n t i l l a r g e , i n d i v i d u a l  c o l o n i e s were o b t a i n e d .  B a c t e r i a were grown by i n o c u l a t i o n of b e e f -  e x t r a c t medium and i n c u b a t i o n observed ( u s u a l l y 3-5 h ) .  a t 37° u n t i l a d e f i n i t e growth was  The l i q u i d c u l t u r e thus o b t a i n e d was i n c u b a -  t e d on a t r a y of s u c r o s e - y e a s t e x t r a c t agar f o r 3 days. capsular with  The lawn of  b a c t e r i a produced was h a r v e s t e d and the b a c t e r i a were d e s t r o y e d  1% p h e n o l s o l u t i o n .  The dead c e l l s were spun down by u l t r a - c e n t r i -  f u g a t i o n and t h e p o l y s a c c h a r i d e with ethanol.  was p r e c i p i t a t e d from t h e s u p e r n a t a n t '  The 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 t r e a t e d  with  C e t a v l o n ( c e t y l t r i m e t h y l a m m o n i u m bromide) t o p r e c i p i t a t e t h e a c i d i c polysaccharide.  Further p u r i f i c a t i o n involved  chloride, r e p r e c i p i t a t i o n into ethanol, dialysis. capsular  d i s s o l u t i o n i n 4M sodium  d i s s o l u t i o n i n water and  The d i a l y z e d s o l u t i o n was f r e e z e - d r i e d polysaccharide.  to y i e l d p u r i f i e d  26  II.1.2 Escherichia c o l i polysaccharides  C u l t u r e s of E s c h e r i c h i a c o l i K28 and K32  from Dr. I . I&rskov  (Copenhagen) were grown on M u e l l e r - H i n t o n agar w i t h the a d d i t i o n of sodium c h l o r i d e ( 5 % w/v).  Each t r a y was  l a y e r e d w i t h an  actively  growing l i q u i d c u l t u r e of E. c o l i b a c t e r i a , o b t a i n e d by i n o c u l a t i o n of M u e l l e r - H i n t o n b r o t h w i t h a s i n g l e f r e s h c o l o n y of E. c o l i and f u r t h e r i n c u b a t i o n of the r e s u l t i n g s o l u t i o n f o r 5 h at 37°. l e f t i n the i n c u b a t o r f o r 5 d.  The  The  t r a y s were  p u r i f i c a t i o n procedure was  carried  out as p r e v i o u s l y d e s c r i b e d f o r 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 .  II.2  SUGAR ANALYSIS  II.2.1 Total hydrolysis and methanolysis * 61  Determination  6  3  -  6  7  of the c h e m i c a l c o m p o s i t i o n of a p o l y s a c c h a r i d e  i n v o l v e s an i n i t i a l a c i d h y d r o l y s i s i n t o c o n s t i t u e n t monosaccharides. A l l sugars a r e , to some e x t e n t , degraded by a c i d .  Thus, the c o n d i t i o n s  of h y d r o l y s i s must be c a r e f u l l y chosen and c o n t r o l l e d . ed the advantages and d i s a d v a n t a g e s  Dutton  review-  6 3  i n the use of d i f f e r e n t a c i d s .  H y d r o c h l o r i c a c i d i s commonly employed f o r g l y c o p r o t e i n s . * 6 1  T r i f l u o r o a c e t i c a c i d was  f i r s t used by A l b e r s h e i m and c o - w o r k e r s  6 5  for  h y d r o l y s i s of p l a n t c e l l - w a l l p o l y s a c c h a r i d e s and i t has s i n c e been w i d e l y used f o r h y d r o l y s i s of o t h e r p o l y s a c c h a r i d e s . thus r e a d i l y removed.  It is volatile  A l d o s e - c o n t a i n i n g p o l y s a c c h a r i d e s can  c o m p l e t e l y h y d r o l y z e d w i t h minimum l o s s of sugar w i t h 2M  and  be  trifluoroacetic  27  a c i d at 100°C f o r 6-8  h.  However, o t h e r s u g a r s ,  k e t o s e s i n c l u d i n g s i a l i c a c i d s , and under these c o n d i t i o n s .  such as  2-deoxyaldoses,  anhydro sugars are l a r g e l y d e s t r o y e d  These sugars can be c o m p l e t e l y  r e l e a s e d under  e x t r e m e l y m i l d c o n d i t i o n s , f o r example, s i a l i c a c i d w i t h 0.025-0.05M s u l f u r i c a c i d at 80° methanolysis,  for 1 h .  S i a l i c a c i d s may  6 6  be s t a b i l i z e d a l s o  g i v i n g methyl g l y c o s i d e methyl e s t e r s .  6 7  Incomplete h y d r o l y s i s i s encountered w i t h u r o n i c a c i d s and amlno-2-deoxyglycosidic  linkages.  i s o l a t i o n of u r o n i c a c i d - c o n t a i n i n g o l i g o s a c c h a r i d e s as  The  2-  I n the case of u r o n i c a c i d s , t h a t  might be of an advantage, s i n c e such r e s i s t a n c e to h y d r o l y s i s  hydrolysis  by  permits  partial  products. r e d u c t i o n of a h e x o u r o n i c a c i d to a hexose r e s i d u e p r i o r to  hydrolysis with a water-soluble  carbodiimide  overcomes t h i s d i f f i c u l t y (see l a t e r ) .  6 1  According  sodium  A technique,  l a b o r a t o r y , i n v o l v e s use of m e t h a n o l y s i s polysaccharides.  and  borohydride  developed i n t h i s  for uronic acid-containing  t o t h i s method, the p o l y s a c c h a r i d e  is  f i r s t t r e a t e d w i t h m e t h a n o l i c hydrogen c h l o r i d e w h i c h , t o g e t h e r w i t h  the  c l e a v a g e of g l y c o s i d i c bonds, causes an e s t e r i f i c a t i o n of u r o n i c a c i d residues.  Treatment w i t h sodium b o r o h y d r i d e  i n anhydrous methanol  reduces the u r o n i c e s t e r s to the c o r r e s p o n d i n g m e t h y l g l y c o s i d e s i s then h y d r o l y z e d  by g a s - l i q u i d chromatography  The  mixture  w i t h 2M t r i f l u o r o a c e t i c a c i d  t o g i v e the n e u t r a l sugars which are c o n v e r t e d analyzed  alcohols.  (g.l.c).  of  (TFA)  i n t o a l d i t o l acetates  and  28  11.2.2  Characterization and  The  quantitation of sugars " ** 68  7  c h a r a c t e r i z a t i o n of the sugars formed on h y d r o l y s i s i s the  s t a r t i n g p o i n t i n an i n v e s t i g a t i o n of the s t r u c t u r e of a p o l y s a c c h a r i d e . The  c o n v e n t i o n a l techniques  t h i n layer chromatography  70  i n v o l v e paper c h r o m a t o g r a p h y , ' 6 8  and paper e l e c t r o p h o r e s i s .  c e r t a i n broad c l a s s e s of sugars can be performed cally.  7 2  '  7 1  Analyses  6 9  of  spectrophotometri-  7 3  G a s - l i q u i d chromatographic methods i n v o l v e f o r m a t i o n of s u i t a b l e v o l a t i l e d e r i v a t i v e s (see l a t e r ) .  I n c r e a s i n g l y , h i g h performance l i q u i d  chromatography (HPLC) columns are being developed f o r the s e p a r a t i o n q u a n t i t a t i v e a n a l y s i s of  11.2.3  sugars. * 7l  Carboxyl reduction of a c i d i c polysaccharides  7  The  and  S""  78  r e s i s t a n c e of g l y c o s i d u r o n i c a c i d s to complete h y r o l y s i s  c r e a t e s d i f f i c u l t i e s i n c o m p o s i t i o n a l and s t r u c t u r a l a n a l y s i s of polysaccharides. corresponding  The  best approach i s t o reduce u r o n i c a c i d s to t h e  hexose r e s i d u e s and then to c a r r y out the i n v e s t i g a t i o n on  the c a r b o x y l - r e d u c e d  polysaccharide.  T a y l o r and C o n r a d  a method i n which the a c i d i c p o l y s a c c h a r i d e treated with a water-soluble  carbodiimide  i s then reduced w i t h sodium b o r o h y d r i d e .  7 5  have developed  i n aqueous s o l u t i o n i s  to g i v e O - a c y l i s o u r e a ,  which  Both stages i n t h i s r e a c t i o n  r e q u i r e c a r e f u l pH c o n t r o l (see Scheme I I . 1 ) . The  a l t e r n a t i v e method i n v o l v e s r e d u c t i o n of a  permethylated  p o l y s a c c h a r i d e , u s u a l l y 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  29  RCOOH  + IHR"  IR" H  +  RCOl  pH475  NHR'  NR' E.D.C  RCOCTH  N0BH4 pH 5-7  or C.M.C.  RCH20H  NoBH4  +  RCH  NHR" + H NHR 1  E.D.C.  =  C.M.C.  =  l-ethyl-3-(3-dimethylaminopropyl)carbodiimide l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-_p_-toluene  Scheme  II.1:  Reduction using  sulphonate  of  carboxylic  carbodiimide  acid  reagent  in  aqueous  solution  +  30  or s i m i l a r s o l v e n t . °  T h i s r e a g e n t , however, i s not s u i t a b l e f o r the  r e d u c t i o n of a c i d groups i n u n s u b s t i t u t e d p o l y s a c c h a r i d e s because of t h e i r i n s o l u b i l i t y i n ether-type solvents. may  be formed by treatment  I n t h i s case methyl e s t e r s  of the a c i d i c p o l y s a c c h a r i d e w i t h  diazomethane and then r e d u c t i o n of the e s t e r groups u s i n g sodium borohydride  i n aqueous s o l u t i o n .  7 7  II.2.4 Determination of the configuration (D or L) of  I n g e n e r a l , chromatographic  sugars  s e p a r a t i o n methods and s p e c t r o s c o p i c  a n a l y s e s do not d i s t i n g u i s h between enantiomers.  Enantiomeric  t i a t i o n of sugars can be a c h i e v e d on m i l l i g r a m q u a n t i t i e s by d i c h r o i s m of a l d l t o l a c e t a t e s or the p a r t i a l l y methylated acetates.  7 8 - 8 1  differen-  circular  alditol  7 8  R e c e n t l y , f o r even s m a l l e r amounts of m a t e r i a l and f o r m i x t u r e s of s u g a r s , enantiomers have been d i s t i n g u i s h e d by c o n v e r s i o n to e q u i l i b r i u m m i x t u r e s of g l y c o s i d e s of c h i r a l a l c o h o l s ( e . g . , (+) or (-)-2b u t a n o l or (+)- or ( - ) - 2 - o c t a n o l ) , f o l l o w e d by g . l . c . s e p a r a t i o n of v o l a t i l e d e r i v a t i v e s such as a c e t a t e or t r i m e t h y l s i l y l e t h e r s . t h i s procedure,  7 9  e n a n t i o m e r i c sugars form d i a s t e r e o m e r i c m i x t u r e s  d e r i v a t i v e s whose chromatographic  '  8 0  .  In  of  separations provide a c h a r a c t e r i s t i c  fingerprint. F o r a few sugars enzymatic characterization.  assay can be used f o r e n a n t i o m e r i c  For example, the enzyme D-glucose o x i d a s e i s used f o r  the q u a n t i t a t i v e assay of D-glucose i n a m i x t u r e .  8 1  31  II.3  POSITION OF LINKAGE  II.3.1 Methylation  The  analysis'* >* ~ * 5  technique  2  9t  of m e t h y l a t i o n a n a l y s i s  '  8 2  8 3  i s r o u t i n e l y employed  i n the s t r u c t u r a l c h a r a c t e r i z a t i o n of complex c a r b o h y r a t e s  as a means t o  e s t a b l i s h the l i n k a g e p o s i t i o n s of the c o n s t i t u e n t monosaccharides. T h i s method i s based on the a b i l i t y to s e p a r a t e and c h a r a c t e r i z e the p a r t i a l l y methylated f u l l y methylated  monosaccharides generated  p o l y s a c c h a r i d e , which i s accomplished  l i q u i d chromatography/mass s p e c t r o m e t r y tives.  v i a h y d r o l y s i s of  the  by combined  gas-  of t h e i r a l d i t o l acetate d e r i v a -  The method, however, g i v e s no i n f o r m a t i o n on the sequence or the  anomeric n a t u r e of the l i n k a g e s i n the p o l y s a c c h a r i d e . The aim of m e t h y l a t i o n * * i s to a c h i e v e e t h e r i f i c a t i o n of a l l f r e e 8  h y d r o x y l groups i n the p o l y s a c c h a r i d e . by H a w o r t h  85  t h i s was  a c h i e v e d by repeated  s u l f a t e and sodium h y d r o x i d e . was  In the o r i g i n a l procedure used  The  reaction with  p a r t i a l l y methylated  dimethyl product  obtained  then t r e a t e d w i t h s i l v e r o x i d e i n b o i l i n g m e t h y l i o d i d e , a c c o r d i n g  t o P u r d i e and I r v i n e , P u r d i e ' s method was  8 6  to g i v e f u l l y m e t h y l a t e d  polysaccharide.  c o n s i d e r a b l y improved by Kuhn and c o - w o r k e r s  8 7  who  used N^,N-dimethylformamide as a s o l v e n t i n c o n j u n c t i o n w i t h m e t h y l i o d i d e and s i l v e r o x i d e .  The  s i m p l e s t and most convenient  m e t h y l a t i o n of p o l y s a c c h a r i d e s was was  developed by H a k o m o r i .  f i r s t a p p l i e d to c a p s u l a r p o l y s a c c h a r i d e s by Sandford  method f o r T h i s method  88  and  Conrad.  8 9  The p o l y s a c c h a r i d e i s t r e a t e d w i t h the s t r o n g base sodium m e t h y l s u l f i n y l methanide ( d i m s y l sodium) and m e t h y l i o d i d e i s subsequently  added to  32  effect methylation.  The Hakomori procedure u s u a l l y g i v e s complete  m e t h y l a t i o n i n one s t e p .  I f t h i s i s n o t t h e case complete m e t h y l a t i o n  can be a c h i e v e d u s i n g P u r d i e ' s method, s i n c e a second Hakomori  treatment  would r e s u l t i n B - e l i m i n a t i o n i f t h e p o l y s a c c h a r i d e c o n t a i n s u r o n i c acid.  O-Acyl groups p r e s e n t i n many p o l y s a c c h a r i d e s and g l y c o c o n j u g a t e s  a r e c o m p l e t e l y c l e a v e d under t h e s t r o n g a l k a l i n e c o n d i t i o n s , but p y r u v i c acid acetals are s t a b l e .  S u b s t i t u t i o n o f t h e carbohydrate  O-acyl groups can be determined u s i n g the P r e h m  90  r e s i d u e s by  methylation  procedure,  where t h e p o l y s a c c h a r i d e i s d i s s o l v e d i n t r i m e t h y l phosphate and then methylated  w i t h m e t h y l t r i f l u o r o m e t h a n e s u l f o n a t e and 2 , 6 - d i - ( t e r t -  b u t y l ) p y r i d i n e as p r o t o n scavenger. a c i d s these a r e t r a n s f o r m e d  I f the substrate contains u r o n i c  i n t o methyl e s t e r s .  l a t i o n s " a r e due t o i n c o m p l e t e  Most "undermethy-  d i s s o l u t i o n of a sample.  This  situation  may be improved by c a r e f u l d e - i o n i z a t i o n of a p o l y s a c c h a r i d e ( f o r example, u s i n g A m b e r l i t e IR-120 ( H ) r e s i n ) . +  procedures have been p u b l i s h e d . ' ' * 8 2  8 9  9  Detailed methylation  R e c e n t l y , potassium  s u l f i n y l methanide has been s u c c e s s f u l l y used i n Hakomori tions.  9 2  methyl-  methyla-  Some p o l y s a c c h a r i d e s , such as c e l l u l o s e , a r e i n s o l u b l e i n  d i m e t h y l s u l f o x i d e (DMSO) a l o n e .  R e c e n t l y , N-methylmorpholine N-oxide  (MMNO) has been shown t o d i s s o l v e p o l y s a c c h a r i d e s , so t h a t t h e Hakomori m e t h y l a t i o n can be c a r r i e d out i n MMNO-DMSO m i x t u r e s . * 1  The m e t h y l a t e d  m a t e r i a l i s r e c o v e r e d by d i a l y s i s  5  (polysaccharide)  o r by p a r t i t i o n between water and c h l o r o f o r m ( o l i g o s a c c h a r i d e ) . The completeness o f m e t h y l a t i o n i s checked by i . r .  spectroscopy  (absence o f  h y r o x y l a b s o r p t i o n a t 3600 c m ) o r by a n a l y s i s of t h e methoxyl c o n t e n t . - 1  The  subsequent h y d r o l y s i s i s performed w i t h 2M t r i f l u o r o a c e t i c a c i d on a  33  steam b a t h f o r 16 h.  P o l y s a c c h a r i d e s c o n t a i n i n g u r o n i c a c i d s may  c a r b o x y l - r e d u c e d b e f o r e ( c a r b o d i i m i d e r e d u c t i o n ) or a f t e r aluminum h y d r i d e ) the p e r m e t h y l a t i o n s t e p .  be  (lithium  Scheme I I . 2 i l l u s t r a t e s a  t y p i c a l r e a c t i o n sequence. A r e c e n t r e p o r t by R e i n h o l d and c o - w o r k e r s  9 3  and Gray and c o -  w o r k e r s * d e s c r i b e s a new t e c h n i q u e f o r d e t e r m i n i n g the s t r u c t u r e of 91  p o l y s a c c h a r i d e s , t h a t , p o t e n t i a l l y , has s i g n i f i c a n t advantages over standard methylation a n a l y s i s .  T h i s method i n v o l v e s the i o n i c h y d r o -  g e n a t i o n of a l l g l y c o s i d i c carbon-oxygen bonds i n the f u l l y m e t h y l a t e d p o l y s a c c h a r i d e w i t h t r i e t h y l s i l a n e c a t a l y z e d by boron t r i f l u o r o e t h e r a t e . T h i s r e d u c t i v e c l e a v a g e a f f o r d s a s e r i e s of p a r t i a l l y m e t h y l a t e d a n h y d r o a l d i t o l s , which are s u b s e q u e n t l y a c e t y l a t e d I n s i t u , and a n a l y z e d by g.l.c.-m.s.  I I . 3 . 2 G a s - l i q u i d chromatography  (CUC.)  6 3  '  9 5 - 1 0 6  Gas chromatography i s a s e p a r a t i o n p r o c e s s i n which t h e components to be s e p a r a t e d are v o l a t i l i z e d and d i s t r i b u t e d between a moving gas phase and a s t a t i o n a r y absorbent phase, which may be a s o l i d ( g a s - s o l i d chromatography), or a l i q u i d ( g a s - l i q u i d  chromatography)  adsorbed on an i n e r t s u p p o r t . The c a r b o h y d r a t e d e r i v a t i v e s used i n g a s - l i q u i d  chromatography  must be v o l a t i l e , y e t s t a b l e at the o p e r a t i n g temperature of the column and must not be adsorbed i r r e v e r s i b l y on the s t a t i o n a r y phase. G a s - l i q u i d chromatography of c a r b o h y d r a t e s was f i r s t performed f o r methylated methyl g l y c o s i d e s .  9 5  The d i s c o v e r y of t r i m e t h y l s i l y l  34  KLEBSIELLA K50 POLYSACCHARIDE  Dbose(CH3SCH2"No ) +  2)CH I 3  Ch^OMe MeO }—0,  MeO  MeO OMe  OMe  f 2  ,OMe } 2 — ( r~ , 2 2 MeO ) 0 0 (  H  C H  IJ  OMe CH„OMe S~~OCH  0H  ) — 0  OMe  OMe  2  H MeO> OMe  OMe  1.  2,4,6-OMe3-Galactose  5.  2.  2,4-OMe2~Glucose  6.  3.  2,3-OMe -Glucose  7.  4.  2,4,6-OMe3-Mannose  2  3,4,6-OMe3-Mannose 2,3,4-OMe3-Glucose 2,3,4,6-0Me4-Galactose  0NOBH4 2)Ac 0/Pyr 2  g.l.c.-m.s. r—OAc  — OMe  — OMe  MeO-  AcO—  AcO-  —  |— OAc  OAc  OAc  OAc  — OMe MeO— AcC—  AcOMeO-  — OMe — OAc  — OAc  OMe  — OAc  — OAc  OAc  OMe OAc  —  — OAc  — OMe  OMe  OMe  MeOOMe  MeO —  OAc  | — OAc  OMe  — OMe  MeO— OMe MeO—  Scheme I I . 2 :  OAc  OAc  OAc  OMe  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 K50 p o l y s a c c h a r i d e  35  d e r i v a t i v e s by Sweeley and co-workers  6  i n 1963 has r e v o l u t i o n i z e d t h e  a n a l y s i s o f c a r b o h y d r a t e s by g a s - l i q u i d chromatography. E x t e n s i v e reviews of the a p p l i c a t i o n s of g . l . c . to carbohydrate a n a l y s i s have been p u b l i s h e d by D u t t o n . ' 6 3  9 7  A l t h o u g h 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 a r e r e a d i l y formed and a r e v o l a t i l e , t h e i r obvious d i s a d v a n t a g e i s a m u l t i p l i c i t y o f peaks due t o the i s o m e r i c forms p r e s e n t a t e q u i l i b r i u m . Jones and c o - w o r k e r s  9 8  S e p a r a t i o n s o b t a i n e d by  w i t h complex m i x t u r e s o f a l d i t o l a c e t a t e s a r e  s u p e r i o r t o those o b t a i n e d f o r t h e c o r r e s p o n d i n g m i x t u r e s o f t h e t r i methylsilyl derivatives.  The c a r b o h y d r a t e s p r e s e n t i n g l y c o p r o t e i n s  were q u a n t i t a t i v e l y determined by g . l . c . o f t h e i r a l d i t o l a c e t a t e s  9 9  and  t h i s procedure i s w i d e l y used i n t h e a n a l y s i s o f g l y c o p r o t e i n s and oligosaccharides.  When t h e m i x t u r e i s not t o o complex, a n a l y s i s o f t h e  t r i m e t h y l s i l y l ethers i s p r e f e r r e d , s i n c e the r e a c t i o n r e q u i r e s o n l y 5-15  min.  1 0 0  Jeanes and c o - w o r k e r s  1 0 1  i n v e s t i g a t e d s e v e r a l column  p a c k i n g s , and found t h a t an o r g a n o s i l i c o n e p o l y e s t e r (ECNSS-M) g i v e s good s e p a r a t i o n of a c e t a t e s o f common a l d i t o l s .  However, t h e maximum  o p e r a t i n g - t e m p e r a t u r e o f t h i s column i s r a t h e r low (200°) and SP-2340 (75% c y a n o p r o p y l s i l i c o n e ) i s t h e s t a t i n a r y phase of c h o i c e f o r a n a l y s i s of a l d i t o l a c e t a t e s and i t was used i n t h i s s t u d y .  To determine t h e  degree o f p o l y m e r i z a t i o n o f an o l i g o s a c c h a r i d e and t o i d e n t i f y t h e reducing sugar, p e r a c e t y l a t e d a l d o n o n i t r i l e s are u s e d .  1 0 2  M e t h y l a t i o n a n a l y s i s i s an i m p o r t a n t method i n s t r u c t u r a l p o l y s a c c h a r i d e c h e m i s t r y . G . l . c . o f f e r s t h e b e s t method f o r t h e s e p a r a t i o n and q u a n t i t a t i o n o f t h e m e t h y l a t e d sugars o b t a i n e d on h y d r o l y s i s o f a methylated polysaccharide o r glycoconjugate.  Partially  methylated  36  a l d i t o l a c e t a t e s have been used e x t e n s i v e l y i n the m e t h y l a t i o n a n a l y s i s . One advantage of u s i n g a l d i t o l a c e t a t e s i s t h a t each a l d o s e d e r i v a t i v e w i l l g i v e o n l y one peak on g . l . c . methylated  Another advantage of p a r t i a l l y  a l d i t o l a c e t a t e s i s t h a t the q u a n t i t a t i o n can be performed  w i t h o u t t h e use of response f a c t o r s a n a l y s i s of N-acetamido s u g a r s . methylated  1 0 3  ( w i t h i n ±5%) except  f o r the  The best s e p a r a t i o n s of p a r t i a l l y  a l d i t o l a c e t a t e s a r e o b t a i n e d on medium-polar columns such as  ECNSS-M or OV-225 ( a s i l i c o n polymer c o n t a i n i n g m e t h y l , phenyl and n i t r i l e groups).  OV-225 i s u s u a l l y p r e f e r r e d f o r r o u t i n e work because  of i t s thermal s t a b i l i t y .  A manual on m e t h y l a t i o n a n a l y s i s , g i v i n g  r e l a t i v e r e t e n t i o n times on g a s - l i q u i d chromatography 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 as w e l l as a c o l l e c t i o n of computer-drawn  bar graph mass s p e c t r a o f these d e r i v a t i v e s has been p u b l i s h e d .  9 1  A l b e r s h e i m and co-workers have r e p o r t e d r e l a t i v e r e t e n t i o n times f o r numerous p a r t i a l l y e t h y l a t e d a l d i t o l a c e t a t e s .  They can be used t o  r e s o l v e some of the p o l y s a c c h a r i d e components t h a t a r e c o - e l u t e d as t h e i r p a r t i a l l y methylated The  a l d i t o l acetates.  i d e n t i f i c a t i o n of methylated  a n a l y s i s by g . l . c . may be c o n f i r m e d  1 0 1 +  sugars  o b t a i n e d on m e t h y l a t i o n  by g . l . c . on o t h e r columns and t h e  s u b s t i t u t i o n p a t t e r n can be determined unambiguously by g.l.c.-mass spectrometry The  (see l a t e r ) .  a p p l i c a t i o n o f HPLC ( h i g h performance l i q u i d chromatography)  to carbohydrate  a n a l y s i s was c o n c e n t r a t e d  a n a l y s i s of sugars  i n food and b e v e r a g e s .  o r i g i n a l l y on t h e q u a n t i t a t i v e 1 0 5  R e c e n t l y , A l b e r s h e i m and  co-workers have a p p l i e d HPLC t o the s e p a r a t i o n of m i x t u r e s amounts of p e r a l k y l a t e d o l i g o s a c c h a r i d e a l d i t o l s .  1 0 6  Thus  of s m a l l sequencing  37  complex c a r b o h y d r a t e s by f o r m a t i o n , s e p a r a t i o n and c h a r a c t e r i z a t i o n o f p e r a l k y l a t e d o l i g o s a c c h a r i d e a l d i t o l s has developed i n t o a p o w e r f u l method of s t r u c t u r a l a n a l y s i s .  I I . 3 . 3 Mass  spectrometry ' 8 2  1 0 7 - 1 1 7  Mass s p e c t r o m e t r y (m.s.) has become an i m p o r t a n t and v e r s a t i l e t o o l i n t h e s t r u c t u r a l a n a l y s i s of c a r b o h y d r a t e s . Mass s p e c t r o m e t r y i s based on t h e i o n i z a t i o n of compounds when they are bombarded w i t h a beam of e l e c t r o n s t o form p o s i t i v e m o l e c u l a r i o n s which s u b s e q u e n t l y may break down t o s m a l l e r fragments.  The r e l a -  t i v e peak i n t e n s i t y i s p r o p o r t i o n a l t o the number of i o n s of t h e approp r i a t e m/z (mass-to-charge their  stability.  r a t i o ) value.  The number of Ions depends on  1 0 7  Carbohydrate  d e r i v a t i v e s g i v e weak o r no m o l e c u l a r i o n s on  e l e c t r o n impact ( e . i . ) mass s p e c t r o m e t r y .  M o l e c u l a r weights may more  r e a d i l y be determined by f i e l d i o n i z a t i o n ( f . i . ) , f i e l d d e s o r p t i o n ( f . d . ) or c h e m i c a l i o n i z a t i o n ( c . i . ) t e c h n i q u e s .  1 0 8  '  1 0 9  S i n c e u n d e r i v a t i z e d mono- and o l i g o s a c c h a r i d e s a r e 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 y n o n - v o l a t i l e , they a r e c o n v e r t e d I n t o more v o l a t i l e d e r i v a t i v e s such as methyl o r t r i m e t h y l s i l y l e t h e r s , a c e t a t e s , t r i f l u o r o a c e t a t e s or a l k y l i d e n e d e r i v a t i v e s . The use of combined g.l.c.-mass s p e c t r o m e t r y , i n which the components from the chromatographic  column a r e I n t r o d u c e d d i r e c t l y I n t o  the i o n i z a t i o n chamber of the mass s p e c t r o m e t e r , has l e d t o new methods  38  f o r the q u a l i t a t i v e and q u a n t i t a t i v e lated  sugars.  analysis  of the m i x t u r e s  8 2  A l d i t o l derivatives t i o n of carbohydrate  e x h i b i t the s i m p l e s t p a t t e r n s f o r f r a g m e n t a -  molecular  i o n s . The mass s p e c t r a of the  m e t h y l e t h e r s , and t r i f l u o r o a c e t a t e s of a l d i t o l s d i s p l a y fragments c o r r e s p o n d i n g Scheme I I . 3 ) .  of methy-  acetates,  primary  t o a l l p r i n c i p a l s c i s s i o n s of the compound  (see  1 0 7  A s y s t e m a t i c i n v e s t i g a t i o n of mass s p e c t r a of p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s by L i n d b e r g and co-workers has generalizations.  1.  l e d to the  following  8 2  D e r i v a t i v e s w i t h the same s u b s t i t u t i o n p a t t e r n g i v e v e r y  similar  mass s p e c t r a , t y p i c a l of t h a t s u b s t i t u t i o n p a t t e r n . 2.  The  base peak of the spectrum i s g e n e r a l l y m/z  43 (CH C —  3.  F i s s i o n between a m e t h o x y l a t e d and an a c e t o x y l a t e d carbon i s  3  p r e f e r r e d over f i s s i o n between two a c e t o x y l a t e d 4.  carbons.  When the m o l e c u l e c o n t a i n s two a d j a c e n t m e t h o x y l a t e d c a r b o n s , f i s s i o n between them i s p r e f e r r e d over f i s s i o n between one these and a n e i g h b o r i n g  5.  0).  acetoxylated  carbon.  Secondary fragments are formed from the primary s i n g l e or c o n s e c u t i v e 42), methanol (m/z  w h i c h standard  l o s s of a c e t i c a c i d (m/z  32), or formaldehyde (m/z  T h i s i n f o r m a t i o n was  of  fragments by 60), ketene  (m/z  30).  used t o i d e n t i f y l a b e l l e d compounds f o r  s p e c t r a were not a v a i l a b l e .  i n v e s t i g a t i o n on K l e b s i e l l a K50  During  the  structural  c a p s u l a r p o l y s a c c h a r i d e the p o s i t i o n  of  39  H C =0R  ChfeOR  2  HC=OR R=Ac,  C  l  C  2  R=Ac,  m/z  145  R=Me,  m/z  89  m/z  73  R=Me, m/z  45  R=COCF ,  m/z  3  R=COCF , 3  m/z  127  253  HC=OR ROCH  RO=CH HCOR CH2OR C  3  R=Ac,  m/z  217  R=Me,  m/z  133  R=COCF , 3  Scheme  II.3:  m/z  R=Ac,  m/z  362  R=Me,  m/z  222  R=COCF , 3  m/z  HCOR CH2OR  632  C. 4  379  m/z  289  R=Me,  m/z  177  R=COCF , 3  The mass (R=Me), Only  R=Ac,  spectra and  primary  of  the  acetates  trifluoroacetates fragments  are  (R=Ac),  (R=C0CF )  shown.  3  methyl of  m/z  ethers  alditols.  505  40  l i n k a g e between D - g l u c u r o n i c a c i d degradation  f o l l o w e d by l a b e l l i n g w i t h e t h y l i o d i d e .  ted, p a r t i a l l y methylated  The e t h y l a -  a l d i t o l acetate ( l , 5 - d i - 0 - a c e t y l - 3 - 0 - e t h y l -  2,4,6-tri-O—methylmannitol) g.l.c.-m.s.  a c i d and D-mannose was determined by u r o n i c  was o b t a i n e d and a n a l y z e d  by means o f  S e v e r a l masses a r e s h i f t e d by 14 u n i t s as i l l u s t r a t e d i n  Figure I I . 1 . On r e d u c t i o n some p a i r s of methylated  sugars (e.g. a 3-0-methyl-  and a 4-0-methyl hexose) g i v e a l d i t o l s w i t h the same s u b s t i t u t i o n pattern.  The l o s s of i n f o r m a t i o n when sugars a r e reduced t o a l d i t o l s  can be r e a d i l y prevented deuterioborate.  i f the r e d u c t i o n i s performed w i t h sodium  T h i s l a b e l l i n g t e c h n i q u e , when used d u r i n g r e d u c t i o n of  the u r o n i c a c i d r e s i d u e s t o n e u t r a l s u g a r s , p e r m i t s one t o d i s t i n g u i s h the n e u t r a l sugar r e s i d u e s o b t a i n e d  from o t h e r sugar r e s i d u e s p r e s e n t i n  the same p o l y s a c c h a r i d e . G.l.c.-m.s. of p a r t i a l l y m e t h y l a t e d  a l d i t o l a c e t a t e s i s now  w i d e l y used i n c o n j u n c t i o n w i t h the 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 and o t h e r m a t e r i a l s c o n t a i n i n g c a r b o h y d r a t e s .  For materials  h a v i n g o n l y one sugar of any one c l a s s (such as pentose, hexose, o r 6-deoxyhexose), the i d e n t i f i c a t i o n of the components by m.s. i s unambiguous.  108  I f , however, t h e p o l y s a c c h a r i d e c o n t a i n s d i f f e r e n t sugars o f the same c l a s s an i d e n t i f i c a t i o n of the d i f f e r e n t methylated accomplished o n l y i f the r e l a t i v e r e t e n t i o n times relevant a l d i t o l acetates are s u f f i c i e n t l y  sugars can be  (T- v a l u e s ) o f the  different.  A l d o n o n i t r i l e a c e t a t e s a r e s u i t a b l e f o r a n a l y s i s of sugars by g . l . c . and g i v e c h a r a c t e r i s t i c mass s p e c t r a t h a t a r e easy t o i n t e r p r e t .  41  a)  1,5-di-0-acetyl-3-0-ethyl-2,4,6-tri-0-methyl-D-mannitol r-10  Rel. Int. %  | '" I'""T  50  b)  I  1 "—"J  1  |  i  100  i  r  '  "*T—r *  150  I ' I — i — i — i — 1 — i — i — r —  200  250  300  1,5-di-0-acetyl-2,3,4,6-tetra-O-methyl-D-mannitol 10  Rel. i n t . %  5 0 1 6 0 Fig.  II.1:  •  - i4o • ' • '200 ' ' ' '250  Mass spectrum o f a u r o n i c a c i d d e g r a d a t i o n d e r i v a t i v e K50 p o l y s a c c h a r i d e (a) compared t o t h e spectrum o f a standard d e r i v a t i v e (b).  300 from  42  P a r t i a l l y m e t h y l a t e d a l d o n o n i t r i l e a c e t a t e s have been i n v e s t i g a t e d by Kochetkov and c o - w o r k e r s .  The m a s s - s p e c t r o m e t r i c b e h a v i o r  1 1 0  m e t h y l a t e d d i s a c c h a r i d e s having  of p e r -  (1 •*• 2 ) - , (1 -> 4 ) - , and (1 •* 6 ) - l i n k e d  hexose r e s i d u e s has been i n v e s t i g a t e d by Kochetkov and c o - w o r k e r s . I t was found t h a t the f r a g m e n t a t i o n  of both m o i e t i e s of the d i s a c c h a r i d e  f o l l o w s p r i n c i p l e s s i m i l a r t o those f o r p e r m e t h y l a t e d g l y c o s i d e s . nomenclature f o r the d i f f e r e n t f r a g m e n t a t i o n  the d e g r a d a t i o n  1 1 2  '  1 1 3  1 1 1  and m o d i f i e d  later  The nomenclature i s e x e m p l i f i e d f o r  of a d i s a c c h a r i d e m e t h y l g l y c o s i d e (see Scheme I I . 4 ) .  aA| Scheme I I . 4 :  The  s e r i e s of p e r m e t h y l a t e d  g l y c o s i d e s was i n t r o d u c e d by Chizhov and K o c h e t k o v by K o v a c i k and c o - w o r k e r s .  1 1 1  baA,  The A - s e r i e s o f fragments f o r t h e d e g r a d a t i o n  of a  d i s a c c h a r i d e methyl g l y c o s i d e The A - s e r i e s of fragments s e r v e s t o e s t a b l i s h the m o l e c u l a r w e i g h t s of the d i s a c c h a r i d e and i t s component sugar r e s i d u e s . s e r i e s of fragments o b t a i n e d e s t a b l i s h the n a t u r e  by d e g r a d a t i o n  The B-  of r i n g b can be used t o  of t h e l i n k a g e between two sugar  residues.  1 0 8  43  The s e p a r a t i o n and s t r u c t u r a l a n a l y s i s of 21 p e r m e t h y l a t e d s a c c h a r i d e s by g a s - l i q u i d chromatography-mass s p e c t r o m e t r y was  d e s c r i b e d by K a r k k a i n e n .  1 l l  *  tri-  (g.l.c.-m.s.)  The p o s i t i o n of the g l y c o s i d i c l i n k a g e  next to the r e d u c i n g end of s t r a i g h t - c h a i n t r i s a c c h a r i d e s can g e n e r a l l y be e s t a b l i s h e d by m.s.,  whereas d i f f e r e n t i a t i o n of (1 •> 6 ) - and (1 •*• 4 ) -  l i n k a g e s next to the non-reducing end may the s t e r e o c h e m i s t r y of the sugar u n i t s . I n r e c e n t y e a r s , m.s.  r e q u i r e p r e v i o u s knowledge of  l l l +  procedures have found a w i d e r  application  i n the s t r u c t u r e d e t e r m i n a t i o n of o l i g o s a c c h a r i d e s and g l y c o l i p i d s havi n g h i g h m o l e c u l a r w e i g h t s . T h i s has been made p o s s i b l e l a r g e l y as a r e s u l t of i n s t r u m e n t a l developments a l l o w i n g h i g h s e n s i t i v i t y at h i g h mass, and improvements i n sample h a n d l i n g . s p e c t r o m e t r y ( e . i . m.s.)  E l e c t r o n i o n i z a t i o n mass-  of these high-mass substances s u f f e r s , however,  from some l i m i t a t i o n s , namely, the l o s s i n s i g n a l i n t e n s i t y at h i g h mass, which r e s u l t s i n the absence of the m o l e c u l a r i o n and high-mass sequence i o n s .  The method can be improved  by combining e . i .  the r e s u l t s of f i e l d - d e s o r p t i o n ( f . d . ) s t u d i e s . d i f f i c u l t i e s a s s o c i a t e d w i t h f . d . m.s.  m.s.  with  However, the t e c h n i c a l  have l i m i t e d i t s use.  The  development of fast-atom-bombardment mass s p e c t r o m e t r y (f.a.b.m.s.) has been of c o n s i d e r a b l e importance f o r c a r b o h y d r a t e - s t r u c t u r e e l u c i d a tion.  1 1 5  F.a.b.  s p e c t r a are r e l a t i v e l y easy t o a c q u i r e , and  early  r e p o r t s have shown t h a t f.a.b.m.s. i s capable of o b t a i n i n g both molecul a r weights and fragment  data from s m a l l q u a n t i t i e s of p o l a r ,  s u b s t a n c e s , i n c l u d i n g u n m o d i f i e d c a r b o h y d r a t e s and  biological  glycolipids.  1 1 6  '  1 1 7  44  II.4  SEQUENCING OF SUGARS  II.4.1 P a r t i a l  hydrolysis  1 1 8  "  1 2 5  P a r t i a l h y d r o l y s i s f o l l o w e d by c h a r a c t e r i z a t i o n of t h e p r o d u c t ( s ) i s o f t e n used i n s t r u c t u r a l c a r b o h y d r a t e c h e m i s t r y .  The method i s of  p a r t i c u l a r v a l u e when a polymer c o n t a i n s a l i m i t e d number of a c i d - l a b i l e g l y c o s i d i c l i n k a g e s , which may be c l e a v e d w i t h o u t s i g n i f i c a n t h y d r o l y s i s of other g l y c o s i d i c l i n k a g e s .  I t i s advisable, t h e r e f o r e , to perform  some p i l o t experiments i n o r d e r t o determine o p t i m a l p a r t i a l acid hydrolysis. methylation  conditions f o r the  P a r t i a l h y d r o l y s i s may a l s o be combined w i t h  a n a l y s i s of t h e 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 .  Many f a c t o r s  seem t o i n f l u e n c e the r a t e of h y d r o l y s i s , i n c l u d i n g the r i n g s i z e , c o n f i g u r a t i o n , c o n f o r m a t i o n , and p o l a r i t y of the sugar as w e l l as the s i z e and p o l a r i t y of t h e a g l y c o n .  1 1 8  Hence, i t i s o f t e n i m p o s s i b l e t o  point to a s i n g l e f a c t o r that explains the d i f f e r e n c e s i n h y d r o l y s i s r a t e s between two g l y c o s i d e s .  1 1 8  Capon has reviewed the r a t e  constants  f o r the a c i d - c a t a l y z e d h y d r o l y s i s of a l a r g e number of g l y c o s i d e s . In g e n e r a l , f u r a n o s i d e s  are hydrolyzed  1 1 9  more r e a d i l y than p y r a n o s i d e s ,  d e o x y g l y c o p y r a n o s i d e s a r e more a c i d l a b i l e than  glycopyranosides,  and aminosugars and u r o n i c a c i d s a r e r e l a t i v e l y r e s i s t a n t t o h y d r o l y s i s . Graded h y d r o l y s i s of u r o n i c a c i d - c o n t a i n i n g p o l y s a c c h a r i d e s i s o l a t i o n of a c i d i c d i s a c c h a r i d e s  leads to  ( a l d o b l o u r o n i c a c i d s ) and h i g h e r  oligosaccharides. The  e f f e c t of the type of l i n k a g e on the r a t e s of h y d r o l y s i s was  s t u d i e d f o r t h e D-glucose d i s a c c h a r i d e s .  1 1 9  I n a l l c a s e s , except f o r  45  the (1  6 ) - l i n k e d d i s a c c h a r i d e s , the a-D-linkage was  hydrolyzed  than the  polysaccharides,  6-D-linkage.  more r e a d i l y  For d i f f e r e n t types of l i n k a g e s i n  the r a t e s of h y d r o l y s i s seem t o p a r a l l e l the r a t e s of  h y d r o l y s i s of d i s a c c h a r i d e s : (1 •*• 3 ) - l i n k a g e s are h y d r o l y z e d (1 -> 4 ) - and resistant.  f a s t e r than  (1 -> 2 ) - l i n k a g e s w i t h (1 •> 6 ) - l i n k a g e s being most The  r a t e of h y d r o l y s i s depends on the l o c a t i o n of  l i n k a g e w i t h i n the p o l y s a c c h a r i d e  chain.  h y d r o l y s i s of n o n - r e d u c i n g t e r m i n a l and w i t h the main i n - c h a i n b o n d s .  the  There i s a h i g h e r r a t e of s i d e - c h a i n bonds, as compared  1 1 8  A number of a l t e r n a t i v e procedures may  be used f o r the a c i d -  c a t a l y z e d c l e a v a g e of g l y c o s i d i c l i n k a g e s , i n c l u d i n g a c e t o l y s i s , methanolysis, mainly  and m e r c a p t o l y s i s .  The  l a t t e r two  techniques  1 2 0  are used  i n the s t r u c t u r a l a n a l y s i s of the s u l f a t e d p o l y s a c c h a r i d e s  seaweeds.  121  The hydrolysis.  usefulness  of a c e t o l y s i s i s t h a t i t i s complementary t o a c i d  I n the f o r m e r , (1 •*• 6 ) - l i n k a g e s are the most s u s c e p t i b l e t o  a t t a c k ; i n the l a t t e r , they are the l e a s t e a s i l y r u p t u r e d . s  mixture  from  of mono, d i , and h i g h e r o l i g o s a c c h a r i d e s formed on  h y d r o l y s i s may  The  1 2 0  partial  be f r a c t i o n a t e d by a v a r i e t y of chromatographic  p r o c e d u r e s , i n c l u d i n g paper chromatography, g e l - p e r m e a t i o n chromatography, paper e l e c t r o p h o r e s i s , ion-exchange chromatography of a c i d i c o l i g o s a c c h a r i d e s , g a s - l i q u i d chromatography, and h i g h performance l i q u i d chromatography. L i q u i d hydrogen f l u o r i d e has been s u c c e s s f u l l y used i n 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 s Lamport  1 2 2  c o n t a i n i n g amino s u g a r s .  Mort  and  found t h a t hydrogen f l u o r i d e c o u l d c l e a v e sugars from  46  g l y c o p r o t e i n s , l e a v i n g the p e p t i d e moiety i n t a c t .  They a l s o observed a  l a r g e d i f f e r e n c e i n the r a t e of c l e a v a g e of g l y c o s i d i c l i n k a g e s of amino sugars and n e u t r a l sugars i n hydrogen f l u o r i d e a t 0°: the n e u t r a l sugar l i n k a g e s c o u l d be broken w h i l e l e a v i n g those of amino sugars Recently,  Mort  1 2 3  reported  intact.  1 2 2  t h a t i n hydrogen f l u o r i d e a t subzero  t e m p e r a t u r e s , d i f f e r e n t i a l c l e a v a g e of l i n k a g e s of n e u t r a l and a c i d i c sugars can be o b t a i n e d ,  and K n i r e l e t a l .  of amino sugar l i n k a g e s a t 25°. for  production  1 2 1 t  found d i f f e r e n t i a l  The method i s of p a r t i c u l a r advantage  of l a r g e r o l i g o s a c c h a r i d e s  containing 0-acyl  Another way of i s o l a t i n g o l i g o s a c c h a r i d e s  containing  groups.  1 2 5  acid-labile  components, i s t o use the b a c t e r i o p h a g e - i n d u c e d d e p o l y m e r i z a t i o n polysaccharides  cleavage  of  (see l a t e r ) .  I n the p r e s e n t study p a r t i a l h y d r o l y s i s was used i n the s t r u c t u r a l i n v e s t i g a t i o n s of K l e b s i e l l a K50 and E s c h e r i c h i a c o l i polysaccharides. f o r E.  F o r K50, d i ~ , t r i - and t e t r a s a c c h a r i d e s  c o l l K28, an a l d o b i o u r o n i c  K28  were i s o l a t e d ;  a c i d and n e u t r a l d i s a c c h a r i d e  were  isolated.  II.4.2 Periodate  oxidation  1 2 6  "  1 3 8  G l y c o l - c l e a v i n g r e a g e n t s , e s p e c i a l l y p e r i o d i c a c i d and i t s s a l t s , and  l e a d t e t r a a c e t a t e have found widespread a p p l i c a t i o n s i n c a r b o h y d r a t e  chemistry.  Lead t e t r a a c e t a t e  polysaccharides,  1 2 6  had been l i t t l e used i n s t u d i e s on  because they a r e i n s o l u b l e i n the s o l v e n t s  used f o r such o x i d a t i o n s .  generally  47  Treatment of g l y c o l groups w i t h p e r i o d i c a c i d and r e s u l t s i n cleavage  its salts  of carbon bonds and the f o r m a t i o n of two  groups, one m o l e c u l a r  1 2  '  aldehydic  p r o p o r t i o n of p e r i o d a t e b e i n g reduced. I n g e n e r a l ,  open c h a i n g l y c o l s are most r e a d i l y o x i d i z e d , f o l l o w e d by c y c l i c c i s g l y c o l s ; c y c l i c t r a n s - g l y c o l s are more s l o w l y o x i d i z e d , or not o x i d i z e d a t a l l i f f i x e d i n an u n f a v o r a b l e ses).  The  neighboring  r e a c t i v i t y may groups.  Smith and  conformation  ( b i c y c l i c anhydrohexo-  a l s o be a f f e c t e d by the s t e r i c e f f e c t s of  1 2 8  co-workers  1 2 9  have s t u d i e d the products  polysaccharides a f t e r periodate o x i d a t i o n , borohydride hydrolysis.  obtained  reduction  Hexose r e s i d u e s s u b s t i t u t e d i n the 4 - p o s i t i o n g i v e  t o l or t h r e i t o l t o g e t h e r w i t h g l y c o a l d e h y d e ,  w h i l e t e r m i n a l and  s u b s t i t u t e d r e s i d u e s g i v e g l y c e r i t o l and g l y c o a l d e h y d e , r e s i d u e s g i v e g l y c e r i t o l and g l y c e r o s e , and  from and  erythri6-  2-substituted  3-substituted residues  a f f o r d the i n t a c t hexose (see F i g . I I . 2 ) . Where t h r e e a d j a c e n t cleavage  h y d r o x y l groups are p r e s e n t , a double  of the carbon c h a i n o c c u r s w i t h f o r m a t i o n of two  groups, the r e d u c t i o n of two m o l e c u l a r l i b e r a t i o n of one m o l e c u l a r  aldehydic  p r o p o r t i o n s of p e r i o d a t e and  the  p r o p o r t i o n of f o r m i c a c i d .  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 can be, t h e r e f o r e , used f o r l i n k a g e a n a l y s i s , d e t e r m i n a t i o n of degree of p o l y m e r i z a t i o n and c h a i n l e n g t h of polysaccharides  (by measuring the p r o p o r t i o n of f o r m i c a c i d or  hyde r e l e a s e d u s i n g the o x i d a t i o n ) , and degree of b r a n c h i n g . co-workers  1 3 0  formaldeSmith  and  have g i v e n the g e n e r a l procedures used i n the o x i d a t i o n of  polysaccharides.  The  r e a c t i o n i s n o r m a l l y c a r r i e d out i n the dark at  5°, e i t h e r i n d i s t i l l e d water or i n b u f f e r w i t h i n pH 4-5  to a v o i d a c i d  48  T e r m i n a l and monosubstituted  hexoses  Number o f m o l e c u l e s o f 10. 4  P r o d u c t s formed a f t e r o x i d a t i o n , NaBH. r e d u c t i o n 4 and h y d r o l y s i s  CH„OH  :H OH 2  HO  (  \  0-  0  2  (3H0  OH  + (^ O H 2  H0  ( :H OH  OH  2  CH 0-  (]H OH  2  2  :HO  y—o  HO—(  OH  2  + (:H OH 2  HO'  (:H OH  \)H  2  ( :H OH  CH 0H  CH OH  2  2  2  OH 1  — or HO—  —OH  CHO  OH +  CH OH 2  HO  :H OH  OH  CH 0H  /  }  p  0  0-  \>H  HO  CH,OH  (M 0 H 2  HO — — (  \  0-  . 1  J \ F i g . I I . 2:  2  CH„0H  o  HO  CH OH  2  —OH (:H OH 2  Common p r o d u c t s formed on p e r i o d a t e borohydride reduction s u b s t i t u t e d hexoses  OH CH0  3H0  — O H o r HO— CH OH 2  :H OH 2  o x i d a t i o n , f o l l o w e d by  and h y d r o l y s i s o f t e r m i n a l and mono-  49  h y d r o l y s i s and to m i n i m i z e s i d e - r e a c t i o n s i n v o l v i n g n o n - s e l e c t i v e o x i d a tions. Because of the marked d i f f e r e n c e i n s t a b i l i t y between t r u e a c e t a l s and g l y c o s i d e s , i t i s p o s s i b l e by m i l d a c i d h y d r o l y s i s to c l e a v e the a c e t a l l i n k a g e s i n p o l y a l c o h o l s r e s u l t i n g from p e r i o d a t e o x i d a t i o n and b o r o h y d r i d e  r e d u c t i o n of p o l y s a c c h a r i d e s , and  g l y c o s i d i c linkages i n t a c t . This important  to l e a v e  m o d i f i c a t i o n of the  o x i d a t i o n , d e v i s e d by Smith and h i s c o - w o r k e r s ,  1 3 1  s t r u c t u r a l i n f o r m a t i o n on the f i n e s t r u c t u r e of the polysaccharide.  any  gives valuable parent  Depending on the r e l a t i v e l o c a t i o n of the  r e s i s t a n t sugar r e s i d u e s , the d e g r a d a t i o n may  periodate  periodate-  r e s u l t i n formation  of  g l y c o s i d e s of mono- or o l i g o - s a c c h a r i d e s , or degraded p o l y s a c c h a r i d e s , which can be s u b j e c t e d to repeated  Smith  degradation.  A s e l e c t i v i t y i n the a c i d h y d r o l y s i s step i s sometimes d i f f i c u l t t o a c h i e v e , s i n c e h y d r o l y s i s of normal g l y c o s i d i c bonds might  occur  t o g e t h e r w i t h the removal of c l e a v e d fragments. A m o d i f i c a t i o n of Smith h y d r o l y s i s , i n t r o d u c e d by L i n d b e r g  and  co-workers,  m e t h y l a t i o n of the reduced o x i d i z e d p o l y s a c c h a r i d e  1 3 2  the  involves  ("polyalcohol")  t o a c i d h y d r o l y s i s , which ensures complete removal of  prior  glycoaldehyde  fragments from o x i d i z e d r e s i d u e s . Periodate o x i d a t i o n i s complicated tion.  P a i n t e r and L a r s e n  incomplete  1 3 3  '  1 3 , 4  i n c e r t a i n cases due  by b o t h under- and  have demonstrated t h a t the o x i d a t i o n i s to the f o r m a t i o n of i n t e r - r e s i d u e hemi-  a c e t a l s between the aldehyde groups of o x i d i z e d h e x u r o n i c and  the c l o s e s t h y d r o x y l groups of u n o x i d i z e d r e s i d u e s .  p r o t e c t e d through the h e m i a c e t a l  over-oxida-  acid residues Those r e s i d u e s  can be exposed t o o x i d a t i o n by  first  50  s u b j e c t i n g them to b o r o h y d r i d e o x i d a t i o n may  occur due  r e d u c t i o n (see F i g . I I . 3 ) .  to e l e c t r o s t a t i c r e p u l s i o n s between the p e r i o -  date i o n s and weakly a c i d i c groups (-C00H) of a c i d T h i s e f f e c t i s suppressed by adding a s a l t (0.2M Over-oxidation  Incomplete  polysaccharides.  1 3 5  sodium p e r c h l o r a t e ) .  1 3 5  i s m i n i m i z e d i f the r e a c t i o n i s c a r r i e d out i n the  1 3 6  d a r k , at low t e m p e r a t u r e s , w i t h o u t  g r e a t excess of reagent and at  3.6-4.5, so t h a t the f o r m y l e s t e r i n i t i a l l y  pH  formed i s not h y d r o l y z e d  at  a significant rate. D u r i n g the present  study on the s t r u c t u r e of E s c h e r i c h i a c o l i  the p e r i o d a t e o x i d a t i o n conducted on the o r i g i n a l p o l y s a c c h a r i d e temperature and  i n unbuffered  at room  sodium m e t a p e r i o d a t e (NalO^) y i e l d e d a  o v e r - o x i d a t i o n of 3 - l i n k e d oc-galactose, due the rhamnosyl bond (see e x p e r i m e n t a l ) . in buffered periodate  K32  s o l u t i o n (pH 4.5),  46%  t o the p a r t i a l h y d r o l y s i s of  When the o x i d a t i o n was 3-linked galactose  performed  was  recovered q u a n t i t a t i v e l y . R e c e n t l y , k i n e t i c s t u d i e s c a r r i e d out by P a i n t e r and ers  1 3 7  co-work-  have shown t h a t a f t e r a l i m i t e d p e r i o d of o x i d a t i o n the  galactopyranosyl  s i d e groups i n a p o l y s a c c h a r i d e  removed by Smith d e g r a d a t i o n , main c h a i n i n t a c t .  c o u l d be  l e a v i n g the 1 , 4 - l i n k e d  8-D-  selectively  r e s i d u e s i n the  Numerous p o s s i b i l i t i e s f o r s e l e c t i v e o x i d a t i o n  can  be demonstrated by comparison of second-order r a t e c o e f f i c i e n t s f o r p e r i o d a t e o x i d a t i o n of v a r i o u s methyl g l y c o s i d e s . t i o n of the t e r m i n a l a-rhamnosyl r e s i d u e was  1 3 7  first  s t r u c t u r a l i n v e s t i g a t i o n of K l e b s i e l l a s e r o t y p e  demonstrated i n the  K17  A t y p i c a l sequence of p e r i o d a t e o x i d a t i o n and shown i n Scheme I I . 5 f o r K l e b s i e l l a K50  capsular  Selective oxida-  polysaccharide.  1 3 8  Smith h y d r o l y s i s i s  polysaccharide.  0  *  •  12  16 h  An unoxidised doublet, with each unit protected by one oxidised neighbour.  (See curve A)  Fig. I I . 3 :  S e q u e n t i a l p e r i o d a t e o x i d a t i o n (0.025 M NalO^, 20°) and b o r o h y d r i d e r e d u c t i o n of alginate.  At t h e p o i n t s l a b e l l e d R, samples were reduced w i t h sodium b o r o h y d r i d e  and then o x i d i z e d f u r t h e r (curves B and C ) .  From r e f . 133.  52  CH20H Scheme I I . 5 :  Smith d e g r a d a t i o n o f K l e b s i e l l a K50 c a p s u l a r p o l y s a c c h a r i d e  53  II.4.3 Base-catalyzed degradation " ** 139  1  6  I n g e n e r a l , g l y c o s i d i c l i n k a g e s are s t a b l e under a l k a l i n e tions.  However, b a s e - c a t a l y z e d  strongly electron-withdrawing of g l y c o s i d i c l i n k a g e s .  8 - e l i m i n a t i o n s can be i n i t i a t e d  f u n c t i o n a l groups w i t h consequent  Groups t h a t may  condiby cleavage  a c t i n t h i s manner i n c l u d e the  c a r b o n y l groups of r e d u c i n g r e s i d u e s , a c t i v a t e d h e x u r o n i c a c i d d e r i v a t i v e s ( e s t e r s ) , c a r b o n y l groups i n t r o d u c e d by o x i d a t i o n a t s e l e c t e d s i t e s , and s u l f o n e groups formed i n s t r u c t u r a l m o d i f i c a t i o n s .  1 3 9  Reducing sugars undergo a number of competing r e a c t i o n s when t r e a t e d w i t h base.  The  r e a c t i o n involves base-catalyzed 8-elimination  ( n o r m a l l y i n aqueous s o l u t i o n ) w i t h f o r m a t i o n of 3-deoxyhex-2-enopyranoses.  T h i s e l i m i n a t i o n o c c u r s most e a s i l y when t h e r e i s a  s u b s t i t u e n t t o p r o v i d e a good l e a v i n g group.  The  alkaline  3-0-  degradation  of p o l y s a c c h a r i d e s i s known as a " p e e l i n g " r e a c t i o n . Base-catalyzed  degradations  from h e x u r o n i c a c i d r e s i d u e s  occur  when the u r o n i c a c i d i s e s t e r i f i e d and 4 - 0 - s u b s t i t u t e d and r e s u l t i n the 8 - e l i m i n a t i o n of the 4 - 0 - s u b s t i t u e n t w i t h the f o r m a t i o n of hex-4-enopyranosiduronate t h a t developed  residues.  The most w i d e l y used r e a c t i o n sequence i s  by L l n d b e r g and co-workers.  1 1 + 0  ' ** 1  1  The main s t e p s of t h i s d e g r a d a t i o n are o u t l i n e d as f o l l o w s :  54  The u n s a t u r a t e d product w i t h a c i d r e l e a s e s the a g l y c o n w i t h the simultaneous R^OH  i s l a b i l e t o a c i d s and on m i l d h y d r o l y s i s (R^OH) * 11  2  t h a t f u r t h e r y i e l d s the f u r a n  r e l e a s e of the s u b s t i t u e n t s a t 0-2  and 0-3.  i s a s i n g l e r e s i d u e or c h a i n of sugar r e s i d u e s , a second  t i o n r e a c t i o n o c c u r s , and the next sugar may quent m i l d a c i d t r e a t m e n t .  I f R^OH  8-elimina-  be r e l e a s e d on the subse-  i s an a l d o s e s u b s t i t u t e d a t  f u r t h e r d e g r a d a t i o n of the p o l y s a c c h a r i d e w i l l occur d u r i n g the ment w i t h  0-3, treat-  alkali.  In an a l t e r n a t i v e p r o c e d u r e * 11  3  exposed r e d u c i n g groups are s i m u l -  t a n e o u s l y p r o t e c t e d by a c e t y l a t i o n w i t h a c e t i c anhydride i s performed u s i n g the o r g a n i c base (DBU).  When  i f degradation  l,5-diazabicyclo[5.4.0]undec-5-ene  55  L a t e r experiments have shown t h a t , under c o n d i t i o n s n o r m a l l y f o r base d e g r a d a t i o n s ,  used  complete l o s s of u r o n i c a c i d r e s i d u e s occurs and  t h a t the a c i d h y d r o l y s i s i s unnecessary.  11+lt  The n a t u r e of r e s i d u e s r e l e a s e d d u r i n g the d e g r a d a t i o n  i s reveal-  ed by f u r t h e r a l k y l a t i o n w i t h t r i d e u t e r i o m e t h y l i o d i d e or e t h y l i o d i d e , h y d r o l y s i s , and a n a l y s i s of the r e s u l t i n g s u g a r s , as a l d i t o l a c e t a t e s , by g a s - l i q u i d chromatography - mass spectrometry  (g.l.c.-m.s.).  Comparison of t h i s a n a l y s i s w i t h 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 p o l y s a c c h a r i d e r e v e a l s the s i t e of attachment of u r o n i c a c i d units. The d e g r a d a t i v e  sequence developed by Svensson and c o - w o r k e r s  1 1 + 5  i n v o l v e s s e l e c t i v e o x i d a t i o n of t h e exposed h y d r o x y l groups i n t h e methylated  p o l y s a c c h a r i d e ( e i t h e r by b a s e - c a t a l y z e d u r o n i c a c i d  d e g r a d a t i o n or by s e l e c t i v e h y d r o l y s i s of a c i d - l a b i l e l i n k a g e s such as p y r u v a t e ) , a l k a l i n e d e g r a d a t i o n h y d r o l y s i s of e n o l g l y c o s i d i c bonds. with chlorine-DMSO.  glycosidic  and m i l d a c i d  Specific oxidation i s effected  146  Scheme I I . 6 shows a t y p i c a l r e a c t i o n sequence f o r 8 - e l i m i n a t i o n of K l e b s i e l l a K50 p o l y s a c c h a r i d e .  II.5  DETERMINATION OF LINKAGE  II.5.1 O p t i c a l r o t a t i o n * * - * 1  7  1 1  9  O p t i c a l r o t a t i o n can be used t o determine the anomeric c o n f i g u r a t i o n of sugar r e s i d u e s i n o l i g o - and p o l y s a c c h a r i d e s .  56  OOMe  j)H  CH20Me O-feOMe  +  2)NoBH4 $Ac 0/Pyr 2  1,5-di-0-acetyl-3-0-ethyl-2,4,6-tri-O-methylmannitol 1,5-di-0-acetyl-2,3,4,6-te tra-O-methylgalactitol 1,2 , 5 - t r i - 0 _ - a c e t y l - 3 , 4 , 6 - t r i - Q - m e t h y l m a n n i t o l 1.3.5- tri-O^acetyl-2,4,6-tri-O-methylgalactitol 1.5.6- tri-0-acetyl-2,3,4-trl-O-methylglucltol 1,3,5,6-tetra-0-acetyl-2,4-di-0-methylglucitol  Scheme  II.6:  Uronic  acid  degradation  of  Klebsiella  K50  polysaccharide  57  Although carbohydrates  o p t i c a l a c t i v i t y i s one  of the p h y s i c a l p r o p e r t i e s o f  most o f t e n measured, i t p r o b a b l y  understood than any o t h e r .  The  i s more complex and l e s s  s i m p l e s t approach i n v o l v e s a p p l i c a t i o n  of Van't H o f f ' s P r i n c i p l e of O p t i c a l S u p e r p o s i t i o n ,  1 1 + 7  w h i c h proposes  the a d d i t i v i t y of the r o t a t i o n a l c o n t r i b u t i o n s of d i f f e r e n t asymmetric c e n t e r s i n a complex m o l e c u l e . c a b l e to the d e t e r m i n a t i o n  I t was  found by Hudson * 11  the t o t a l m o l e c u l a r The m o l e c u l a r  x  Normally, sodium (589 nm).  r o t a t i o n of p o l y -  c o n t r i b u t i o n s of O-acyl and pyruvate  r o t a t i o n are  and  groups t o  neglected.  where,  [a]  - specific rotation  M.W.  - molecular  weight  the o p t i c a l r o t a t i o n i s measured at the D - l i n e of U s i n g Hudson's I s o r o t a t i o n Rules  one can p r e d i c t  s p e c i f i c r o t a t i o n of o l i g o s a c c h a r i d e s and p o l y s a c c h a r i d e s molecular  of  r o t a t i o n (M) i s d e f i n e d :  M.W.  100  appli-  A p p l i c a t i o n of Hudson's I s o r o t a t i o n  Rules g i v e s i n f o r m a t i o n o n l y on o v e r a l l m o l e c u l a r The  t o be  of c o n f i g u r a t i o n at the anomeric c e n t e r  the f r e e sugars and g l y c o s i d e s .  oligosaccharides.  8  r o t a t i o n v a l u e s of model methyl g l y c o s i d e s .  1 1 + 9  using  the  58  . . f al =  Mi  x 100 r r —  , where,  M.W.  . . . _ . IMi - sum of m o l e c u l a r r o t a t i o n v a l u e s of model m e t h y l glycosides M.W.  - m o l e c u l a r weight of polysaccharide.  II.5.2 Nuclear magnetic resonance spectroscopy  II.5.2.1 ^-n.m.r. s p e c t r o s c o p y P r o t o n magnetic  resonance  1 5 0 - 6 0  s p e c t r o s c o p y o f f e r s to c a r b o h y d r a t e  c h e m i s t s a method both f o r d e t e r m i n i n g the c o n f i g u r a t i o n of unknown c a r b o h y d r a t e s and f o r a s c e r t a i n i n g the c o n f o r m a t i o n s of known c a r b o hydrates i n s o l u t i o n . The fundamental  1 5 0  work of Lemieux and co-workers  i n t r o d u c e d the  s u c c e s s f u l a p p l i c a t i o n of ^-H-n.m.r. s p e c t r o s c o p y to s t r u c t u r a l problems i n the c a r b o h y d r a t e f i e l d .  1 5 1  An i n s t r u m e n t a l development of c o n s i d e r -  a b l e importance to p.m.r. s p e c t r o s c o p y of c a r b o h y d r a t e s has been the i n t r o d u c t i o n of h i g h - r e s o l u t i o n magnets based on s u p e r c o n d u c t i n g solenoids.  1 5 2  The most s i g n i f i c a n t advance i n n.m.r. s p e c t r o s c o p y s i n c e  1964 has been the development of F o u r i e r - t r a n s f o r m t e c h n i q u e s which a f f o r d a l a r g e enhancement i n s e n s i t i v i t y .  1 5 3  The use of a h i g h - f i e l d  s p e c t r o m e t e r i s e s p e c i a l l y b e n e f i c i a l f o r ^-n.m.r. s p e c t r o s c o p y of polysaccharides with regular repeating u n i t s .  Numerous examples of t h i s  59  type of a p p l i c a t i o n a r e found i n s t u d i e s on c a p s u l a r p o l y s a c c h a r i d e s o f Klebsiella.  These p o l y s a c c h a r i d e s and a v a r i e t y of fragments  prepared  from them (by p a r t i a l o r enzymatic h y d r o l y s i s , Smith d e g r a d a t i o n ) were examined by *H- and C-n.m.r. s p e c t r o s c o p y i n c o m b i n a t i o n w i t h c h e m i c a l 13  methods, and unique s t r u c t u r e s were determined.  1 5 t |  H i g h - f i e l d ^-H-n.m.r. s p e c t r o s c o p y has been u t i l i z e d i n d e t e r m i n i n g t h e s t r u c t u r e s of g l y c o p r o t e i n s .  1 5 5  extensively  Two-dimensional  (2-D) homo- and h e t e r o n u c l e a r n.m.r. methods have been used as an a i d i n the assignment  of the p r o t 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 m o i e t i e s o f  glyco-conjugates.  1 5 6  A p p l i c a t i o n s of p.m.r. s p e c t r o s c o p y t o problems of c a r b o h y d r a t e c h e m i s t r y i n v o l v e measurement of f o u r parameters.  (i)  Relative i n t e n s i t i e s of the s i g n a l s The p r o p e r t y t h a t , under proper o p e r a t i n g c o n d i t i o n s , t h e r e l a -  t i v e i n t e n s i t i e s of a b s o r p t i o n s i g n a l s f o r d i f f e r e n t hydrogens a r e e q u a l to the r e l a t i v e numbers of t h e hydrogens p r o d u c i n g t h e s i g n a l s , has been p a r t i c u l a r l y important t o a n a l y t i c a l carbohydrate c h e m i s t r y . number o f anomeric  1 5 7  The  l i n k a g e s , r e l a t i v e amounts of 6-deoxy s u g a r s ,  O - a c e t y l , N - a c e t y l and 1 - c a r b o x y e t h y l i d e n e s u b s t i t u e n t s can be d e t e r m i n ed.  F o r monosaccharides  i t also permits a rapid q u a n t i t a t i v e a n a l y s i s  of the p r o p o r t i o n s o f anomers, i n c l u d i n g furanose and pyranose  (ii)  forms.  Coupling constants N u c l e a r s p i n - s p i n c o u p l i n g c o n s t a n t s a r e d e s i g n a t e d by J and a r e  expressed as h e r t z ( H z ) . When a f i r s t - o r d e r spectrum i s o b s e r v e d , t h e  60  magnitudes of the c o u p l i n g c o n s t a n t s may  be determined d i r e c t l y from t h e  spectrum. The r e l a t i o n s h i p between the v i c i n a l c o u p l i n g c o n s t a n t ( J ) and the d i h e d r a l angle (<t>) between p r o t o n s i s g i v e n a p p r o x i m a t e l y by the Karplus e q u a t i o n .  ^(H^  1 5 8  H) 2  =  {  8.5  cos <)>_0.28,  9.5  cos <()_0-28,  2  2  0° < <t> < 90° 90°  < $ <  180°  The v a l u e s are maximum when the d i h e d r a l angle (<]>) i s 0° o r  180°,  and minimum when i t i s 90°. One  of the most i m p o r t a n t consequences of the K a r p l u s e q u a t i o n i s  t h a t the o r d e r of magnitude of d i a x i a l , a x i a l - e q u a t o r i a l and  diequa-  t o r i a l c o u p l i n g c o n s t a n t s ( J a a , Jae and Jee r e s p e c t i v e l y ) i n a c y c l o hexane r i n g c h a i r system can be p r e d i c t e d and i s i n r e a s o n a b l e agreement w i t h the observed v a l u e s .  T h i s i s shown i n F i g . I I . 4 .  In carbohydrate  c h e m i s t r y , the d e t e r m i n a t i o n of J ( H , H ) v a l u e s has been used t o 3  e s t a b l i s h c o n f i g u r a t i o n as w e l l as c o n f o r m a t i o n a l p r e f e r e n c e s f o r pyranose, furanose and a c y c l i c s u g a r s .  (iii)  Chemical  shift  The c h e m i c a l s h i f t s of p r o t o n s are s t r o n g l y dependent upon s u b s t i t u t i o n a l , o r i e n t a t i o n a l and e l e c t r o n e g a t i v i t y e f f e c t s of n e i g h b o r i n g and d i s t a n t g r o u p s .  1 5 7  The e a r l y o b s e r v a t i o n s t h a t the c h e m i c a l  s h i f t of a p r o t o n depends on i t s environment i n the molecule demonstrated  i n 1958 by Lemieux and c o - w o r k e r s .  1 5 1  was  Thus, e q u a t o r i a l  61  R  Fig.  II.4:  Relationship constants  for  between a-  and  dihedral  angle  g-D-hexoses  (<)>) a n d  2  =  coupling  H,OH  62  r i n g - h y d r o g e n atoms had lower c h e m i c a l s h i f t s than t h e i r counterparts.  axial  The most i m p o r t a n t d i r e c t s h i e l d i n g e f f e c t i n  c a r b o h y d r a t e s i s t h a t of the r i n g - o x y g e n atom, which causes  the  c h a r a c t e r i s t i c , l o w - f i e l d s h i f t of the anomeric hydrogen a t o m . In  1 5 0  the spectrum of an o l i g o s a c c h a r i d e or a p o l y s a c c h a r i d e , t h r e e  main r e g i o n s can be observed:  a) the anomeric  r e g i o n (6 4.5-5.5),  b) the r i n g p r o t o n r e g i o n ( 6 3.0-4.5) and c ) the h i g h f i e l d r e g i o n (6 1.15-2.5) where CH N-acetyl, etc.  3  groups of 6-deoxy s u g a r s , p y r u v a t e s , 0 - a c e t y l ,  can be observed  (see F i g .  II.5).  The c o n f i g u r a t i o n a t the a c e t a l carbon atom of p y r u v i c a c i d a c e t a l s p r e s e n t i n some e x t r a c e l l u l a r b a c t e r i a l p o l y s a c c h a r i d e s has been i n v e s t i g a t e d by Garegg and co-workers.  The c h e m i c a l s h i f t s  f o r the  1 5 9  methyl groups of the p y r u v i c a c i d a c e t a l d i f f e r s i g n i f i c a n t l y  depending  upon whether these groups are a x i a l or e q u a t o r i a l . The r i n g p r o t o n s are u s u a l l y d i f f i c u l t where s p e c i f i c protons r e s o n a t e at lower f i e l d and g a l a c t u r o n i c a c i d , H-5 Two  of f u c o s e , H-2  to a s s i g n except f o r cases (H-5 of g l u c u r o n i c a c i d  of mannose, e t c . ) .  f a c t o r s dominate the a c q u i s i t i o n of h i g h - r e s o l u t i o n H-n x  s p e c t r a of p o l y s a c c h a r i d e s , i n t e r f e r e n c e by exchangeable N-H)  and l i n e broadening of s i g n a l s .  1 5 4  protons  .m.r.  (0-H,  The p r e p a r a t i o n of aqueous  s o l u t i o n s of p o l y s a c c h a r i d e s i n v o l v e s a p r i o r exchange treatment w i t h d e u t e r i u m o x i d e ( p r e f e r a b l y 99.95 atom %) and the use of d e u t e r i u m o x i d e as a s o l v e n t .  N e v e r t h e l e s s a s t r o n g peak due to r e s i d u a l water  signal) i s often obtained. temperature  ( 6 ~4.8  The c h e m i c a l s h i f t of the HOD  p.p.m.) i n t e r f e r e s w i t h the anomeric  i s c l o s e to the H-l s i g n a l of B - g l y c o p y r a n o s y l r e s i d u e s .  (HOD  s i g n a l at room r e g i o n and i t By r a i s i n g the  r i n g protons  N-Ac  H-3a CH of KDO CH o f pyruvate  3  of  3  u>  6(p.p.m. )  Fig. I I . 5 :  Schematic r e p r e s e n t a t i o n o f d i f f e r e n t r e g i o n s i n t h e H-n.m.r. spectrum o f p o l y s a c c h a r i d e s  64  temperature one can d i s p l a c e the HOD s i g n a l u p f i e l d . o f FT t e c h n i q u e s The  There a r e a number  f o r m i n i m i z i n g t h e i n t e r f e r e n c e by the HOD  problem of s i g n a l broadening  polymer protons have l o n g r e l a x a t i o n  signal.  1 6 0  i s l a r g e l y due t o the f a c t t h a t  times.  A substantial  enhancement  i n the q u a l i t y o f most p o l y s a c c h a r i d e *H s p e c t r a can be a c h i e v e d by u s i n g e l e v a t e d temperatures a t 60°-90° or by p e r f o r m i n g a v e r y m i l d h y d r o l y s i s i n o r d e r t o reduce v i s c o s i t y of t h e sample (however, p o s s i b l e l o s s of l a b i l e groups should be c o n s i d e r e d ) .  The *H s i g n a l s o f many  p o l y s a c c h a r i d e s become a p p r e c i a b l y sharper and, when a spectrum i s recorded a t high f i e l d , there i s a corresponding  enhancement i n s i g n a l  separation. Some b a c t e r i a l p o l y s a c c h a r i d e s c o n t a i n O - a c e t y l groups which a r e i r r e g u l a r l y d i s t r i b u t e d a l o n g the p o l y m e r i c c h a i n . anomeric s i g n a l s t o be twinned.  T h i s causes c e r t a i n  Such t w i n n i n g d i s a p p e a r s a f t e r O-de-  a c e t y l a t i o n of the p o l y s a c c h a r i d e g i v i n g r i s e t o a b e t t e r r e s o l v e d trum.  spec-  T h i s i s i l l u s t r a t e d i n F i g . I I . 6 which shows the ^H-n.m.r.  s p e c t r a of E. c o l i K28 p o l y s a c c h a r i d e ( a f t e r a u t o h y d r o l y s i s , 95°, o v e r n i g h t ) and the same p o l y s a c c h a r i d e a f t e r O - d e a c e t y l a t i o n (0.01M NaOH, 23°,  overnight).  standard  Both s p e c t r a were r e c o r d e d w i t h acetone as an i n t e r n a l  ( 6 2.23).  I I . 5 . 2 . 2 Carbon-13 n.m.r.  spectroscopy  1 5 9  '  1 6 1 - 1 7 2  Carbon-13 n u c l e a r magnetic resonance ( C-n.m.r.) has proved t o 13  be a p o w e r f u l t e c h n i q u e , y i e l d i n g i n f o r m a t i o n on c o m p o s i t i o n , and c o n f o r m a t i o n  of the p o l y s a c c h a r i d e s .  sequence  U s i n g the F o u r i e r t r a n s f o r m  (FT) method, i t a l l o w s s p e c t r a of p o l y s a c c h a r i d e s t o be o b t a i n e d  using  65  Fig. II.6:  The  H-n.m. r . s p e c t r a (400 MHz,  deacetylated  (bottom) E. c o l i K28  95°  ) of n a t i v e (top)  capsular  and  polysaccharides  66  o n l y t h e i r n a t u r a l abundance spectroscopy  13  C atoms; i t complements ^-H-n.m.r.  i n t h a t i t g i v e s b e t t e r s i g n a l s e p a r a t i o n owing t o t h e  wider range of c h e m i c a l  shifts  involved.  1 6 1  I n many c a s e s , i n p a r t i c u l a r when d e a l i n g w i t h complex m o l e c u l e s such as p o l y s a c c h a r i d e s ,  the amount of i n f o r m a t i o n o b t a i n a b l e  from 1_ H  n.m.r. s p e c t r a i s l i m i t e d compared t o t h a t r e v e a l e d by technique  i s r a p i d and n o n d e s t r u c t i v e  1 3  C-n.m.r.  1 6 2  The  and can be used on r e l a t i v e l y  s m a l l amounts o f m a t e r i a l . Most of the s t u d i e s t o date have been concerned w i t h the p r o t o n decoupled  spectra.  1 6 1  However, a p r o t o n - c o u p l e d 1 3  C - H coupling constants  t h a t may be u s e f u l f o r t h e assignment o f  1  anomeric c o n f i g u r a t i o n .  spectrum c o n t a i n s i n f o r m a t i o n about the  I n pyranosides  1 6 3  Cl-Hl coupling  1  J[ CH(1)] i s 1 3  l a r g e r when H - l i s e q u a t o r i a l (~170 Hz) than when H - l i s a x i a l (~160 Hz).  A l a r g e number o f hexopyranose d e r i v a t i v e s and t h e i r  v a l u e s have been examined by Bock and Pedersen.  1 6 1  *'  1  J[ CH(1)] 1 3  1 6 5  The method o f a n a l y s i s o f C-n.m.r. s p e c t r a of p o l y s a c c h a r i d e s 13  i s t o a l a r g e e x t e n t based on the comparison of the resonances Of t h e i n d i v i d u a l carbon atoms of the p o l y s a c c h a r i d e w i t h those of the p r e v i o u s l y assigned m o n o s a c c h a r i d e tuents.  1 6 7  '  1 6 8  The c h e m i c a l  1 6 2  '  1 6 6  and o l i g o s a c c h a r i d e  s h i f t s o f the monosaccharides a r e s i m i l a r  to those of t h e monosaccharide u n i t s w i t h i n the p o l y s a c c h a r i d e for substituent effects. any  consti-  except  These e f f e c t s produced by t h e attachment o f  s u b s t i t u e n t t o a sugar moiety cause an i n c r e a s e i n c h e m i c a l  s h i f t of  the carbon d i r e c t l y i n v o l v e d i n the l i n k a g e ; t h i s i s u s u a l l y accompanied  67  by a decrease o f s m a l l e r magnitude cal  (sometimes an i n c r e a s e ) i n t h e chemi-  s h i f t s of t h e n e i g h b o r i n g ( 3 - c a r b o n s .  161  The s e n s i t i v i t y of carbon-13 c h e m i c a l s h i f t s towards changes i n s u b s t i t u t i o n p e r m i t s l o c a t i o n of such s u b s t i t u e n t s as a c e t a t e , malonate, phosphate, o r s u l f a t e groups.  I n t r o d u c t i o n of an a c y l group onto  oxygen  causes a s m a l l (1.5-4 p.p.m.) d o w n f i e l d s h i f t of t h e oc-carbon atom. However, as O j - a c y l a t i o n causes t h e s i g n a l o f t h e (3-carbon atom t o s h i f t u p f i e l d (1-5 p.p.m.), t h e c u m u l a t i v e e f f e c t of s e v e r a l a c y l groups may be d i f f i c u l t t o p r e d i c t .  An a l t e r a t i o n i n r i n g s i z e i s a l s o accom-  1 6 2  p a n i e d by a change of c h e m i c a l s h i f t s ; t h u s , f u r a n o s e s have c h e m i c a l s h i f t s d o w n f i e l d from those of t h e p y r a n o s e s .  Sometimes, an immediate  i d e n t i f i c a t i o n may be made when v e r y l o w - f i e l d s i g n a l s a t 6 = 107 p.p.m. or more a r e p r e s e n t , f o r example, f o r p - g a l a c t o f u r a n o s i d e and a - a r a b i n o furanoside.  1 6 9  The s i g n a l s i n C-n.m.r. s p e c t r a o f p o l y s a c c h a r i d e s a r e known t o 13  d i s t r i b u t e i n groups each o f which o c c u p i e s s t r i c t l y d e f i n i t e  regions.  The t y p i c a l resonance r e g i o n s u s e f u l f o r the f i r s t o r d e r a n a l y s i s of  t h e C-n.m.r. spectrum a r e shown i n F i g . I I . 7 . 13  1 7 0  They i n c l u d e :  a)  c a r b o n y l groups from u r o n i c a c i d , p y r u v i c a c i d , N- and 0 - a c e t y l  substi-  t u e n t s (175 ± 6 p.p.m.);  c)  b) anomeric carbons (100 ± 8 p.p.m.);  secondary carbons (75 ± 5 p.p.m.);  d)  p r i m a r y carbons (65 ± 5 p.p.m.);  e) m e t h y l groups from 0 - a c e t y l and N - a c e t y l s u b s t i t u e n t s (20-28 p.p.m.), p y r u v a t e (18-26 p.p.m.) and 6-deoxy sugars (~15-16 p.p.m.). The c h e m i c a l s h i f t s f o r t h e R- and S-forms of t h e p y r u v a t e d i f f e r s i g n i f i c a n t l y b e i n g ~18 p.p.m. f o r t h e a x i a l m e t h y l groups and ~26 p.p.m. f o r the e q u a t o r i a l .  1 5 9  The anomeric c o n f i g u r a t i o n s of KDO  AcO  CO  C  f  1  HCOR  CI  HCN  HCOH  I I T  CCH,  AcN  "| CH2OH  I I  B a g  00  I— 175  Fig.  II.7:  l  r~"  —r~  r_  110  The c h a r a c t e r i s t i c monosaccharide  80  100  regions  residues  in  for  70  resonances  polysaccharides  60  of  carbon  r~ 50  atoms  -n  r  40  belonging  —1~  20  to  different  0  p.p.m.  69  residues  can be e s t a b l i s h e d by C-n.m.r. s p e c t r o s c o p y . 13  The C - l  resonance i s s e n s i t i v e t o anomeric c o n f i g u r a t i o n b e i n g 6 = 174.8 p.p.m. f o r t h e p-anomeric form and 6 = 176.5 p.p.m.  f o r the a-anomer.  171  In  the case of N - a c e t y l n e u r a m i n i c a c i d (NANA) t h e d i f f e r e n c e between chemic a l s h i f t s o f C - l i n a- and B-forms i s s m a l l e r , b e i n g 6 = 175.9 p.p.m. f o r t h e 8-anomeric form and 6 = 174.6 p.p.m. f o r the oc-anomer. Quantitative ted  13  d a t a cannot be s a t i s f a c t o r i l y o b t a i n e d  172  from i n t e g r a -  C-n.m.r. s p e c t r a , because of s a t u r a t i o n phenomena and n u c l e a r  Overhauser e f f e c t s .  However, i f s p e c t r a a r e measured under s u i t a b l e  c o n d i t i o n s and i f i n t e g r a l s ( o r peak h e i g h t s )  of s i g n a l s from carbon  atoms c a r r y i n g t h e same number of hydrogen atoms a r e compared, i t i s possible to obtain rather accurate information amounts of components i n a m i x t u r e .  about t h e r e l a t i v e  1 6 2  An i m p o r t a n t l i m i t i n g f a c t o r i n t h e C-n.m.r. t e c h n i q u e i s t h e 13  s i g n a l - t o - n o i s e r a t i o obtained  i n the spectra.  l a r g e sample t u b e s , and i n c r e a s e d  concentration  a larger signal-to-noise r a t i o (s/n).  A h i g h - f i e l d instrument, of t h e sample r e s u l t i n  I f , however, a l i m i t e d amount of  compound i s a v a i l a b l e , i t may be advantageous t o use a s m a l l e r  probe-  insert.  material  1 6 2  In contrast  t o ^-H-n.m.r., p a r t i c l e s o r u n d i s s o l v e d  make v e r y l i t t l e d i f f e r e n c e t o t h e q u a l i t y of t h e s p e c t r a .  1 6 1  F i g . I I . 8 shows t h e C-n.m.r. spectrum ( p r o t o n decoupled) of t h e 13  E. c o l i K28 d e a c e t y l a t e d  polysaccharide.  Three s i g n a l s can be observed  i n t h e anomeric r e g i o n c o r r e s p o n d i n g t o f o u r anomeric carbons (2 8 - l i n k ed and 2 a - l l n k e d ) . group o f f u c o s e .  The s i g n a l a t 16.1 p.p.m. corresponds t o t h e C H  3  acetone  capsular  polysaccharide  71  II.5.3 Other techniques  11.5.3.1  Enzymatic h y d r o l y s i s  1 3 9  *  1 7 3  A l l glycosidases are s p e c i f i c for the sugar unit undergoing hydrolysis and for i t s anomeric configuration. polysaccharide (or other glycoconjugate) non-reducing terminal u n i t s .  Exoglycosidases  act on  substrates by the removal of  These enzymes, however, can approach only  to within a limited distance of other structural features such as other linkages or branch points. Thus, the oligosaccharides, generated by p a r t i a l hydrolysis or bacteriophage factory r e s u l t s .  degradation usually give more s a t i s -  Incubation with glycosidases i s usually performed i n  buffer at 37° at the appropriate pH for varying periods of time (sometimes as long as 6-7 d a y s ) .  In contrast, endo enzymes are not l i m i t -  1 7 3  ed by action pattern and can cleave unbranched regions of both external and internal c h a i n s . bacteriophages  11.5.3.2  1 3 9  Highly s p e c i f i c endoglycanases are derived from  (see Section VI).  Chromium t r i o x i d e o x i d a t i o n Angyal and James  17<t  1 7 4  *  - 1 7 6  showed that a f u l l y acetylated aldopyrano-  side, i n which the aglycon occupies an equatorial position i n the most stable chair form (generally the B-anomer) i s readily oxidized when treated with chromium trioxide i n acetic a c i d .  The anomer with an a x i a l  aglycon (generally the o-form) Is only slowly oxidized.  72  OR where, R = a l k y l group or sugar residue T h i s r e a c t i o n has t i o n of sugar r e s i d u e s  been used t o determine the anomeric  i n o l i g o - and  a c e t y l a t e d m a t e r i a l and trioxide i n acetic acid.  polysaccharides.  vatives.  The  Sugar a n a l y s i s of the o r i g i n a l m a t e r i a l  However, s u b s t i t u t i o n i n o l i g o - and  and  have been o x i d i z e d .  method i s v a l i d f o r g l u c o - , g a l a c t o - , manno-, and  the c o n f o r m a t i o n a l  fully  an i n t e r n a l s t a n d a r d are t r e a t e d w i t h chromium  the o x i d i z e d product show which sugar r e s i d u e s The  1 7 5  configura-  polysaccharides  e q u i l i b r i u m of o - f u c o s y l and  oc-rhamnosyl  xylo- derimay  alter  residues  thus making them s u s c e p t i b l e t o o x i d a t i o n . I n some s i t u a t i o n s , i t may  be p o s s i b l e t o I s o l a t e o l i g o m e r s a f t e r  r e d u c t i o n of the o x i d i z e d p r o d u c t . the K l e b s i e l l a type 37 c a p s u l a r  T h i s method was  polysaccharide.  capsular  polysaccharide.  was  8-1inked.  used to d e t e r m i n e  acid i n Escherichia c o l i  Sugar a n a l y s i s performed on the  product showed the d i s a p p e a r a n c e of g l u c u r o n i c  of  1 7 6  I n t h i s study chromium t r i o x i d e o x i d a t i o n was the anomeric c o n f i g u r a t i o n of g l u c u r o n i c  used i n s t u d i e s  K32  oxidized  a c i d thus p r o v i n g  that i t  73  II.6  LOCATION OF O-ACETYL GROUPS161-177-180  Some p o l y s a c c h a r i d e s w i t h l a b i l e O-acyl groups occur n a t u r a l l y . The removal of such s u b s t i t u e n t s may a l t e r p h y s i c a l and immunological p r o p e r t i e s of these p o l y s a c c h a r i d e s . are extremely  S i n c e some of these a c e t a t e groups  l a b i l e and can be e a s i l y removed, they c r e a t e c e r t a i n  d i f f i c u l t i e s i n o b t a i n i n g unambiguous e v i d e n c e f o r t h e i r  original  location. The f i r s t and s i m p l e s t method t o use i s n u c l e a r magnetic resonance s p e c t r o s c o p y  (II.5.2).  P r o t o n n u c l e a r magnetic resonance o f  the n a t i v e a c e t y l a t e d p o l y s a c c h a r i d e would show a s i n g l e t a t 6 = 2.15-2.28 due t o t h e presence o f an O-acyl group. a c e t a t e group can cause a d d i t i o n a l c h e m i c a l protons;  1 7 7  however, t h e t o t a l c u m u l a t i v e  Presence o f t h e  s h i f t s of the n e i g h b o r i n g  e f f e c t may be d i f f i c u l t t o  predict. 13  C-n.m.r. s p e c t r o s c o p y  i s more i n f o r m a t i v e i n t h i s case, and  comparison of t h e C-n.m.r. s p e c t r a of a c e t y l a t e d and d e a c e t y l a t e d 13  polysaccharides group  1 6 1  can o f t e n p r o v i d e very u s e f u l i n f o r m a t i o n on t h e 0_-acyl  and sometimes unambiguous e v i d e n c e f o r i t s l o c a t i o n ( f o r  example, when present  on C-6 o f h e x o p y r a n o s e ) .  Spectrophotometry  25  1 7 8  and gas c h r o m a t o g r a p h i c  1 7 9  methods can be  used f o r 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 o f t h e 0 - a c e t y l groups. C h e m i c a l methods f o r l o c a t i n g these s u b s t i t u e n t s i n c l u d e p e r i o d a t e o x i d a t i o n (see S e c t i o n I I . 4 . 2 ) , which i n some cases i d e n t i f i c a t i o n o f sugar r e s i d u e s c o n t a i n i n g t h e a c e t a t e group. methylation  9 0  permits Prehm  ( s e e S e c t i o n I I . 3 . 1 . 1 ) and the method of de B e l d e r and  74  Norrman,  l b U  which i n v o l v e s the c o n v e r s i o n of u n s u b s t i t u t e d h y d r o x y l  groups t o m e t h o x y e t h y l a c e t a l s  on r e a c t i o n w i t h methyl v i n y l e t h e r (see  Scheme I I . 7 ) , f o l l o w e d by b a s e - c a t a l y z e d d e - O - a c e t y l a t i o n and methylation.  Scheme I I . 7 :  L o c a t i o n of 0-acetyl s u b s t i t u e n t s according t o the de B e l d e r and Norrman p r o c e d u r e  H y d r o l y s i s of t h e m o d i f i e d p o l y s a c c h a r i d e then g i v e s sugar d e r i v a t i v e s l a b e l l e d w i t h O-methyl groups a t t h e s i t e s o r i g i n a l l y occupied by 0-acetyl substituents.  T h i s method g i v e s the most s a t i s f a c t o r y  results*,  however, p o s s i b i l t y of " u n d e r p r o t e c t i o n " should be c o n s i d e r e d i n t h e case of t h e p o l y s a c c h a r i d e s .  O l i g o s a c c h a r i d e s b e a r i n g 0 - a c e t y l groups  o b t a i n e d by b a c t e r i o p h a g e - i n d u c e d d e g r a d a t i o n of p o l y s a c c h a r i d e s (see  75  S e c t i o n V I ) or by p a r t i a l h y d r o l y s i s u s i n g hydrogen f l u o r i d e ( s e e S e c t i o n I I . 4 . 1 ) g i v e more r e l i a b l e  results.  I n t h i s study the p o s i t i o n of an C H a c e t y l the methyl v i n y l e t h e r p r o c e d u r e . confirmed  group was l o c a t e d us  The r e s u l t s o b t a i n e d were f u r t h e r  by ^-H- and C-n.m.r. f i n d i n g s ( f o r d e t a i l e d procedure see  Section IV).  13  76  CHAPTER I I I  GENERAL EXPERIMENTAL CONDITIONS  77  III  HI.l  GENERAL EXPERIMENTAL CONDITIONS  PAPER CHROMATOGRAPHY  Paper chromatography was performed by the descending method 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:  A)  e t h y l a c e t a t e : a c e t i c a c i d : f o r m i c acid:water  B)  e t h y l acetate:pyridine:water  C)  1-butanol:acetic  D)  1 - b u t a n o l : e t h a n o l : w a t e r (4:1:5, upper phase)  acid:water  (18:3:1:4)  (8:2:1) (2:1:1)  Chromatograms were developed w i t h a l k a l i n e s i l v e r n i t r a t e o r by h e a t i n g a t 110° f o r 5-10 min a f t e r s p r a y i n g w i t h j > - a n i s i d i n e h y d r o c h l o r i d e i n aqueous  1-butanol.  P r e p a r a t i v e paper chromatography was c a r r i e d out by t h e descendi n g method u s i n g Whatman 3MM paper and s o l v e n t C ( u n l e s s stated). h.  The r e l e v a n t s t r i p s were c u t o u t and e l u t e d w i t h water f o r 6  The aqueous s o l u t i o n s were f i l t e r e d , c o n c e n t r a t e d  III.2  otherwise  and f r e e z e - d r i e d .  GAS-LIQUID CHROMATOGRAPHY AND G.L.C.^IASS SPECTROMETRY  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 H e w l e t t P a c k a r d 5700 i n s t r u m e n t  f i t t e d with dual f l a m e - i o n i z a t i o n detectors.  S t a i n l e s s - s t e e l columns (1.8 m x 3 mm) were used w i t h a c a r r i e r - g a s n i t r o g e n f l o w - r a t e o f 20 mL/min.  The f o l l o w i n g p a c k i n g m a t e r i a l s and  78  programs were used ( u n l e s s o t h e r w i s e Supelcoport  stated):  (A) 3% of SP-2340 on  (100-120 mesh), programmed from 195° f o r 4 m i n , and then a t  2°/min t o 260°;  (B) 5% o f ECNSS^l on Gas Chrom Q (100-120 mesh),  i s o t h e r m a l a t 170°, or programmed from 180° f o r 4 min, and then a t 2°/min t o 200°;  (C) 3% of OV-225 on Gas Chrom Q (100-120 mesh),  i s o t h e r m a l a t 170°, o r programmed from 180° f o r 4 min,  and then a t  2°/min t o 230°. A l l q u a n t i t a t i v e data present l e a s t three  a r e based on t h e average of a t  runs.  P r e p a r a t i v e g . l . c . was c a r r i e d out w i t h a F & M model 720 d u a l column i n s t r u m e n t  f i t t e d w i t h thermal  conductivity detectors.  S t a i n l e s s - s t e e l columns (1.8 m x 6.3 mm) were used w i t h c a r r i e r - g a s helium  f l o w - r a t e o f 60 mL/min.  The f o l l o w i n g p a c k i n g m a t e r i a l s and  programs were used ( u n l e s s o t h e r w i s e Supelcoport  stated):  (D) 3% of SP-2340 on  (100-120 mesh), programmed from 195° t o 240° a t 2°/min; ( E )  3% o f 0V-225 on Gas Chrom Q (100-120 mesh), programmed from 180° t o 230° a t 2°/min. G.l.c.-m.s. a n a l y s e s were performed w i t h a V.G. Micromass 12 instrument  f i t t e d w i t h a Watson-Biemann s e p a r a t o r .  S p e c t r a were r e c o r d -  ed a t 70 eV w i t h an i o n i z a t i o n c u r r e n t of 100uA and an i o n s o u r c e a t 200° ( u n l e s s o t h e r w i s e  III.3  stated).  GEL-PERMEATION CHROMATOGRAPHY  Preparative gel-permeation  chromatography was performed u s i n g a  column (2.5 x 100 cm) of B i o - G e l P-4 (400 mesh).  The c o n c e n t r a t i o n of  79  the sample a p p l i e d t o t h e column was 100 mg/mL.  The column was  i r r i g a t e d w i t h w a t e r : p y r i d i n e : a c e t i c a c i d (500:5:2) a t a f l o w r a t e o f 10.5 and  mL/hour.  F r a c t i o n s of 2 mL were c o l l e c t e d , f r e e z e - d r i e d , weighed  an e l u t i o n p r o f i l e was o b t a i n e d .  III.4  OPTICAL ROTATION AND CIRCULAR DICHROISM  O p t i c a l r o t a t i o n s were measured on aqueous s o l u t i o n s a t 20°±3° on a P e r k i n - E l m e r  model 141 p o l a r i m e t e r w i t h a 1 dm c e l l  C i r c u l a r d i c h r o i s m s p e c t r a ( c . d . ) were recorded  (5 mL).  on a Jasco  J-500A  a u t o m a t i c 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 w i t h a q u a r t z c e l l of 0.3 mL c a p a c i t y and a p a t h l e n g t h o f 0.1 cm o r 0.2 cm. dissolved i nacetonitrile  Compounds were  ( s p e c t r o s c o p i c grade) and t h e s p e c t r a  recorded  i n t h e range o f 190-260 nm.  III.5  NUCLEAR MAGNETIC RESONANCE  P r o t o n magnetic resonance s p e c t r a were r e c o r d e d  on a V a r i a n  XL-100, Bruker WP-80, N i c o l e t - O x f o r d H-270, o r Bruker WH-400 instruments.  S p e c t r a were r e c o r d e d  a t a temperature o f 90°±5° and  acetone was used as an i n t e r n a l s t a n d a r d . r e l a t i v e l y t o t h a t o f i n t e r n a l sodium s u l f o n a t e taken as 0.  A l l values are given  4,4-dimethyl-4-silapentane-  Samples (10-20 mg) were prepared by d i s s o l v i n g i n  D 0 and f r e e z e - d r y i n g 2-3 times from D 0 s o l u t i o n s . 2  2  Tubes o f 5 mm i n  diameter were used. 13  C-n.m.r. s p e c t r a were r e c o r d e d  on a B r u k e r WP-80 o r B r u k e r  WH-400 s p e c t r o m e t e r a t ambient t e m p e r a t u r e .  Samples (30-50 mg) were  80  dissolved i n D 0 2  and acetone was used as an i n t e r n a l s t a n d a r d .  Tubes o f  5 o r 10 mm i n d i a m e t e r were used.  111.6  GENERAL CONDITIONS  The I.R. s p e c t r a of m e t h y l a t e d d e r i v a t i v e s were r e c o r d e d on a P e r k i n - E l m e r model 710B s p e c t r o p h o t o m e t e r i n carbon t e t r a c h l o r i d e (spectroscopic grade). A l l s o l u t i o n s were c o n c e n t r a t e d on a r o t a r y e v a p o r a t o r i n vacuo at a b a t h temperature of 40°. Ion-exchange  chromatography  f o 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  o l i g o s a c c h a r i d e s was performed on a column (2.0 x 28 cm) of Bio-Rad AG-1-X2 (formate form, 200-400 mesh).  The n e u t r a l f r a c t i o n was e l u t e d  w i t h water and the a c i d i c w i t h 10% f o r m i c a c i d . f o r p u r i f i c a t i o n of p e r m e t h y l a t e d  Sephadex LH-20 was used  o l i g o - and p o l y s a c c h a r i d e s .  D e - i o n i z a t i o n s were c a r r i e d out w i t h A m b e r l i t e I R - 1 2 0 ( H ) r e s i n . +  111.7  III.7.1  ISOLATION AND PURIFICATION OF THE POLYSACCHARIDES  K l e b s i e l l a polysaccharides  The f o l l o w i n g media were used t o grow the b a c t e r i a .  1.  B e e f - e x t r a c t medium  5 g of Bactopeptone  ("nutrient broth")  3 g Bacto beef e x t r a c t 2 g of NaCl  81  1 L of H 0 2  2.  Sucrose-yeast extract-agar  75 g of s u c r o s e 5 g of Bacto y e a s t  extract  37.5 g of agar 5 g of NaCl 2.5 g of KH PO 2  l+  0.625 g of MgSO^H^O 1.25 g of CaS0  1+  2.5 L of H 0 2  A sample of K l e b s i e l l a b a c t e r i a s e r o t y p e K50 was r e c e i v e d s t a b c u l t u r e from Dr I . 0 r s k o v (Copenhagen). s t r e a k e d on agar p l a t e s inoculated  and i n c u b a t e d a t 37°.  i n beef-extract  w i t h continuous s h a k i n g .  The b a c t e r i a were An i n d i v i d u a l c o l o n y was  medium and b a c t e r i a were grown f o r 5 h a t 37° This l i q u i d culture  (50 mL) was i n c u b a t e d on a  t r a y (86 cm x 46 cm) of s u c r o s e - y e a s t e x t r a c t - a g a r  f o r 3 d.  c a p s u l a r b a c t e r i a produced was h a r v e s t e d by s c r a p i n g surface,  as a  and the b a c t e r i a were d e s t r o y e d w i t h  The lawn of  from the agar  1% p h e n o l s o l u t i o n .  The  p o l y s a c c h a r i d e was s e p a r a t e d from the c e l l s by u l t r a c e n t r i f u g a t i o n (30,000 rpm). volumes). and  The v i s c o u s s u p e r n a t a n t was p r e c i p i t a t e d i n t o e t h a n o l (3  The p r e c i p i t a t e was d i s s o l v e d  r e p r e c i p i t a t e d with a saturated  (cetyltrimethylammonium bromide).  i n the minimum amount o f w a t e r  (10%) s o l u t i o n of C e t a v l o n The p r e c i p i t a t e d a c i d i c p o l y s a c c h a -  r i d e was i s o l a t e d by c e n t r i f u g a t i o n .  I t was d i s s o l v e d  i n 4M N a C l ,  r e p r e c i p i t a t e d i n t o e t h a n o l and the 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  solution  d i a l y z e d a g a i n s t r u n n i n g t a p water f o r 2 d.  The d i a l y z e d  82  of p o l y s a c c h a r i d e was u l t r a c e n t r i f u g e d and the supernatant  was f r e e z e -  dried.  III.7.2  Escherichia c o l i  polysaccharides  Stab c u l t u r e s of E s c h e r i c h i a c o l i b a c t e r i a K28 and K32 were o b t a i n e d from Dr. I . 0 r s k o v  (Copenhagen).  The f o l l o w i n g media were used t o grow the b a c t e r i a .  1)  Bacto M u e l l e r H i n t o n B r o t h , dehydrated ( D i f c o )  2)  M u e l l e r H i n t o n agar (BBL) w i t h a d d i t i o n of NaCl 0.5%  (w/v).  The s t r e a k e d p l a t e s were i n c u b a t e d a t 37° o v e r n i g h t .  The  b a c t e r i a were c u l t u r e d as d e s c r i b e d f o r the K l e b s i e l l a  polysaccharides  (see S e c t i o n I I I . 7 . 1 ) and were grown i n t h r e e s m a l l t r a y s (30 x 50 cm) u s i n g 1.5 L of M u e l l e r H i n t o n agar and 5 g NaCl per t r a y . f i l l e d w i t h water and used as a h u m i d i t y  source.  One t r a y was  1 8 1  Each t r a y was l a y e r e d w i t h an a c t i v e l y growing l i q u i d c u l t u r e of E. c o l i b a c t e r i a , o b t a i n e d by i n o c u l a t i o n of 50 mL of M u e l l e r  Hinton  b r o t h w i t h a s i n g l e f r e s h c o l o n y of E. c o l i and f u r t h e r i n c u b a t i o n of the r e s u l t i n g s o l u t i o n f o r 5 h a t 37°.  The t r a y s were l e f t i n the i n c u -  b a t o r f o r 5 d and then the s l i m e was c o l l e c t e d .  The p u r i f i c a t i o n  procedure was c a r r i e d out as d e s c r i b e d f o r the K l e b s i e l l a polysaccharide.  83  III.8  BACTERIOPHAGE PROPAGATION  III.8.1  Tube and f l a s k  lysis  The b a c t e r i o p h a g e s <J>28—1, <J>28-2, and <j>32 were i s o l a t e d  from  sewage ( c o u r t e s y of Dr. S. S t i r m , F r e i b u r g , Germany) and propagated on t h e i r host s t r a i n s i n n u t r i e n t b r o t h by tube and f l a s k  a)  Tube l y s i s .  lysis.  An a c t i v e l y growing b a c t e r i a l c u l t u r e of E s c h e r i c h i a  c o l i was o b t a i n e d by s u c c e s s i v e r e p l a t i n g s on agar p l a t e s . An a c t i v e l y growing b a c t e r i a l c u l t u r e (0.5 mL) was i n o c u l a t e d w i t h 6 x 5 mL of s t e r i l e n u t r i e n t b r o t h and a f t e r 1 h of i n c u b a t i o n 0.5 mL of a b a c t e r i o p h a g e - c o n t a i n i n g s o l u t i o n was added t o each t e s t tube a t 30 min intervals.  C o n t i n u e d i n c u b a t i o n r e s u l t e d i n g r a d u a l c l e a r i n g of t h e  c l o u d y s o l u t i o n due t o c e l l l y s i s .  A f t e r 4-5 h of i n c u b a t i o n a few  drops of c h l o r o f o r m were added t o each tube t o prevent b a c t e r i a l  growth  and the c o n t e n t s of the f i r s t t h r e e and l a s t t h r e e t e s t tubes were combined.  The b a c t e r i a l d e b r i s was then sedimented by c e n t r i f u g a t i o n  and t h e phage s o l u t i o n s were a n a l y z e d by t h e "plaque assay t e c h n i q u e " . The t e c h n i q u e was based on a s e r i e s of s u c c e s s i v e d i l u t i o n s of phage by t r a n s f e r r i n g 0.3 mL p o r t i o n s of phage t o 2.7 mL p o r t i o n s of d i l u e n t ( n u t r i e n t b r o t h ) , thus making 1 0 ~ , 10~ , 2  original solution. b a c t e r i a l "lawn".  h  1 0 ~ and 1 0 ~ d i l u t i o n s of 6  8  One drop from each d i l u t i o n was then p l a c e d on a (The "lawn" was p r e p a r e d by i n o c u l a t i o n of 2 mL  l i q u i d medium w i t h an a c t i v e l y growing c o l o n y and i n c u b a t i n g c u l t u r e f o r 4-5 h.  this  An agar p l a t e , p r e v i o u s l y d r i e d i n t h e i n c u b a t o r a t  84  37° was covered w i t h t h i s l i q u i d c u l t u r e , l e f t f o r 15 min and the e x c e s s of l i q u i d was removed.  I n c u b a t i o n f o r 1 h gave a s t a b l e " l a w n " ) .  p l a t e was then i n c u b a t e d a t 37° o v e r n i g h t . (normally d i l u t i o n s 10  -tt  The  A t h i g h phage c o n c e n t r a t i o n s  , 1 0 ~ , and 1 0 ) i n d i v i d u a l phage p a r t i c l e s 5  - 6  c o u l d not be d i s t i n g u i s h e d , but a t s u i t a b l e d i l u t i o n s ( 1 0  - 7  and 1 0 ~ ) 8  i n d i v i d u a l plaques surrounded by h a l o s ( n o t always) c o u l d be e a s i l y counted.  The t i t e r of the b a c t e r i o p h a g e s o l u t i o n was then c a l c u l a t e d .  number o f plaques Bacteriophage titer  x  dilution  drop s i z e  ( i n plaque forming u n i t s (P.F.U.) per mL)  b)  Flask l y s i s .  T h i s t e c h n i q u e was e s s e n t i a l l y the same as  d e s c r i b e d f o r the tube l y s i s , except t h a t l a r g e r amounts of b a c t e r i o p h a g e c o u l d be produced.  An o v e r n i g h t a c t i v e l y growing  culture  (5 mL) was added t o 6 x 50 mL o f n u t r i e n t b r o t h and a f t e r 1.5 h o f i n c u b a t i o n 5 mL o f b a c t e r i o p h a g e s o l u t i o n i s added t o each f l a s k a t 30 min i n t e r v a l s . tube l y s i s .  The procedure was then c o n t i n u e d as d e s c r i b e d f o r the  A f l a s k l y s i s t y p i c a l l y y i e l d e d 300 mL of phage s o l u t i o n  w i t h a t i t e r of 1 0  III.8,2  1 0  P.F.U./mL  Large-scale propagation o f the bacteriophage  B a c t e r i o p h a g e <J>32 was propagated fermentor model M i c r o f e r m , New Brunswick  i n a 14 L fermentor j a r , Scientific.  The f o l l o w i n g  85  c o n d i t i o n s were used: t e m p e r a t u r e , 37°.  a e r a t i o n , 5 L.p.m.; a g i t a t i o n , 500 r.p.m.;  N u t r i e n t b r o t h (10 L) was a u t o c l a v e d i n the fermentor  j a r f o r 40 min a t 121°.  The s t e r i l e medium was p l a c e d i n a fermentor  and a g i t a t e d f o r 15 min, then i t was i n o c u l a t e d w i t h 450 mL of a c t i v e l y growing b a c t e r i a l c u l t u r e and s t i r r e d f o r 1 h. of  9.6 x 1 0  1 0  To t h i s s o l u t i o n a t o t a l  P.F.U. i n 400 mL n u t r i e n t b r o t h was added.  A f t e r 2.5 h of  i n c u b a t i o n 400 mL of CHC1 was added t o prevent b a c t e r i a l growth. 3  The  b a c t e r i a l d e b r i s was removed by c o n t i n u o u s c e n t r i f u g a t i o n u s i n g a h i g h speed C e p a - S c h n e l l Model LE b e n c h - s c a l e c e n t r i f u g e c o o l e d w i t h t a p water and o p e r a t e d a t 38,000 r.p.m., and w i t h a f l o w r a t e of ~250 mL/min.  86  CHAPTER I V  STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDE OF K l e b s i e l l a SEROTYPE K50  87  IV  STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDE OF K l e b s i e l l a SEROTYPE K50.  IV.1  ABSTRACT  The s t r u c t u r e o f t h e 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 K50 has been determined by u s i n g t h e t e c h n i q u e s  of m e t h y l a t i o n , p e r i o d a t e  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 and 8 - e l i m i n a t i o n .  N.m.r.  spectros-  copy (*H and C ) was used t o e s t a b l i s h the n a t u r e of the anomeric l i n k 1 3  ages.  The p o l y s a c c h a r i d e i s comprised o f r e p e a t i n g u n i t s of the h e p t a -  s a c c h a r i d e shown and i s t h e one of 19 c a p s u l a r p o l y s a c c h a r i d e s t h a t a r e composed o f D - g l u c u r o n i c residues.  a c i d , D-galactose,  D-glucose, and D-mannose  I t has t h e o n l y known " f i v e - p l u s - t w o " r e p e a t i n g u n i t ; t h e  s t r u c t u r e of t h e 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 K50 i s , t h e r e f o r e , unique i n t h i s  series.  -•3 ) - 8-D-Gal-(1 ->-3 ) - a-D-Glc- ( 1 -»4 ) - a-D-GlcA- (1 -»-3 ) - a-D-Man- (1 -»-2 ) - ct-D-Man- (1 6 t 1 a-D-Glc 6 + 1 8-D-Gal  88  IV.2  INTRODUCTION  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 s e r o t y p e K50 i s  composed of D - g l u c u r o n i c  a c i d , D-glucose, D - g a l a c t o s e ,  and D-mannose  r e s i d u e s , and i s one o f t h o s e , from almost twenty s t r a i n s , h a v i n g t h e same q u a l i t a t i v e c o m p o s i t i o n .  1 5  I d e n t i t y of q u a l i t a t i v e a n a l y s i s ,  however, bears no r e l a t i o n s h i p t o t h e s t r u c t u r a l p a t t e r n o f t h e p o l y s a c c h a r i d e , and e x a m i n a t i o n  of the c a p s u l a r m a t e r i a l from K l e b s i e l l a K50  has shown i t t o have a unique s t r u c t u r e i n t h i s s e r i e s . c a p s u l a r p o l y s a c c h a r i d e s based on a h e p t a s a c c h a r i d e  Several  repeating u n i t are  known, but t h i s i s t h e f i r s t i n s t a n c e where such a u n i t i s o f the "5 + 2" type. Furthermore, i n those c a p s u l a r p o l y s a c c h a r i d e s i n which the u r o n i c a c i d r e s i d u e i s p a r t of the main c h a i n , o n l y s i n g l e - u n i t s u b s t i t u e n t s have p r e v i o u s l y been found. K50  lateral  The p o l y s a c c h a r i d e of s e r o t y p e  i s t h u s , on two c o u n t s , unique among the some s i x t y s t r u c t u r e s now  known i n t h i s s e r i e s .  The e x p e r i m e n t a l evidence  on which t h i s s t r u c t u r e  i s based i s summarized n e x t .  IV.3  RESULTS AND DISCUSSION  The  i s o l a t i o n and p u r i f i c a t i o n of the p o l y s a c c h a r i d e were  a c h i e v e d as d e s c r i b e d i n S e c t i o n I I I . 7 . 1 .  1 5 , 6 1 , 6 2  s a c c h a r i d e o b t a i n e d a f t e r one p r e c i p i t a t i o n w i t h  The a c i d i c p o l y cetyltrimethylammonium  bromide had t a ] +102°, which compares w e l l w i t h the c a l c u l a t e d v a l u e o f D  +96° u s i n g Hudson's Rules o f I s o r o t a t i o n .  1 4 8  The p o l y s a c c h a r i d e was  89  shown t o be homogeneous by g e l - p e r m e a t i o n 10  6  chromatography w i t h  = 3.2 x  daltons. Paper chromatography of an a c i d h y d r o l y z a t e o f the p o l y s a c c h a r i d e  showed g a l a c t o s e , g l u c o s e , g l u c u r o n i c a c i d , mannose and an a l d o b i o u r o n i c acid.  A c i d h y d r o l y s i s of c a r b o x y l - r e d u c e d  polysaccharide  7 5  and  c o n v e r s i o n t o a l d i t o l a c e t a t e s gave mannose, g a l a c t o s e , and g l u c o s e i n the molar r a t i o s of 2.0:2.4:3.0.  Glucose was proved t o be of t h e D-con-  f i g u r a t i o n by c i r c u l a r d i c h r o i s m (c.d.) measurements made on g l u c i t o l hexaacetate.  G a l a c t o s e and g l u c o s e were a s s i g n e d the D c o n f i g u r a t i o n  from c.d. measurements made on p a r t i a l l y methylated The  derivatives.  7 9  ^H-n.m.r. spectrum of p a r t i a l l y h y d r o l y z e d p o l y s a c c h a r i d e was  recorded i n D 0 a t 90° w i t h acetone as i n t e r n a l s t a n d a r d 2  (see Appendix  I I I , Spectrum NO. 1 ) . The spectrum e x h i b i t s 7 s i g n a l s i n t h e anomeric region: (1H);  6 5.46 (1H); 6 4.70 ( ^  2  6 5.29 (1H);  6 5.24 (1H);  8 Hz, 1H) and 6 4.49 ( J  x 2  6 5.06 (1H);  6 5.01  8 Hz, 1H). From t h e  v a l u e s of the c h e m i c a l s h i f t s and c o u p l i n g c o n s t a n t s , 5 a- and 2 6-anomeric l i n k a g e s were a s s i g n e d . pyruvate  No deoxy-sugar, 0_-acetyl o r  c o u l d be d e t e c t e d .  The  13  C-n.m.r. spectrum confirmed  these r e s u l t s (see Appendix  I I I , Spectrum No. 2 ) . F i v e s i g n a l s appear i n t h e anomeric r e g i o n a t 104.04;  103.97; 103.17; 101.30 and 99.16 p.p.m.  and 99.16 p.p.m. each corresponded  t o two anomeric  The s i g n a l s a t 104.04 carbons.  P r e c i s e assignment of t h e anomeric s i g n a l s was a c h i e v e d  after  comparison of t h e *H- and C-n.m.r. s p e c t r a o f t h e p o l y s a c c h a r i d e w i t h 13  the c o r r e s p o n d i n g  s p e c t r a 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 a f t e r  partial  TABLE IV.1 N.M.R. DATA FOR K l e b s i e l l a K50 POLYSACCHARIDE AND THE DERIVED OLIGOSACCHARIDES Compound  -^H-N.m.r. data _-g  "1,2 (Hz) 1 3  GlcA-^-Man-OH Al  1 3  1 2  GlcA-^-Man-^an-OH A2  C-N.m.r. data  13  Integral  Assignment  P.p.m  Assignment  proton  4.93  s  0.4  -Man-p-OH  5.19  s  0.6  -Man- -OH  5.34  2.5  1.0  GlcA-  4.93  2  0.3  2 —Man- -OH  5.08  2  0.7  -Man- -OH  5.32  2.5  3  3  -Mar  101.93  GlcA—a  GlcA2.0  93.81  2 —Man-  3  103.4  2 —Man-  5.37  13  1 2  1 3  GlcA-^Ian-j-Man-^-Gal-OH A3  3  4.65  0.8  -Gal-  5.08  1  -Man-  3  -OH  3  103.1  —Man-  101.34  GlcA-  Compound*  ^-H-N.m.r. data  C-N.m.r. data  13  Jj 2  Integral  (Hz)  proton  5.19  Assignment  3  -  0  P.p.m.  2  3  -Gal-j-OH  97.16  -Gal-p-OH  GlcA——  95.41  2 -Man——Gal-o-OH  95.10  2 -Man-^-Gal-^-OH  93.04  -Gal——OH  OC  5.31  Assignment  2 -Man—  u  3  1 2  13  CHOH  I  Gal-p-€lc-^-OCH  4.65  8  0.9  Gal—p-  104.11  Gal—p-  99.62  -Glc—  CH 0H 2  3  £H1 5.20  3 1 3 Ik 1 3 1 2 1 -Gal—s-Glc—_<;I A——Han—r-Man—< x a a — a T  4  1  —Glc———  Gal-  C  1  4.49  3 -Gal  104.04  Glc  6 1  Gal  3  -Gal-  S50  4.70  Gal-  103.97  -Man-  p  Compound  C-N.m.r. data  H-N.m.r. data  13  l  •V  (Hz)  5.01  Integral proton  P.p.m.a  Assignment  6  2-Man103.17  5.06  3-Glc-  5.24  3-Mar 101.30  5.29  Assignment  6-Glc  -Glc  4  -GlcA2  5.46  99.16  -Glc/  -Mar  3  -Glc 3  -Gal-  1 3  1 H  1 3  1 2  1  c „ GlcA „ Man _ Man—— a a  Glc  3  4.51  3  103.83  -Gal-  -Gal3  Degraded K50  -Man-  2  5.02  -Man-  103.16  Glc-  3  -Gal5.27  3  101.30  —ManGlc-  t  -GlcA-  a  C-N.m.r.  ^H-N.m.r . data  Compound  (Hz)  5.51  13  Integral proton  1  Assignment-  0  4 -GlcA—— a  P.p.m.  99.16 98.80  data • Assignment  6  2 -Man 3 - G l c —a—  For t h e source o f A l , A2, A3, and SHI, see t e x t . Chemical s h i f t r e l a t i v e t o i n t e r n a l a c e t o n e ; 6 2.23 d o w n f i e l d from sodium 4 , 4 - d i m e t h y l - 4 - s i l a p e n t a h e - l - s u l f o n a t e (DSS). The n u m e r i c a l p r e f i x i n d i c a t e s the p o s i t i o n i n which the sugar i s s u b s t i t u t e d ; t h e a o r B, t h e c o n f i g u r a t i o n of t h e g l y c o s i d i c bond, o r the anomer i n t h e case o f a ( t e r m i n a l ) r e d u c i n g - s u g a r r e s i d u e . Thus 3 - G a l — — r e f e r s to the anomeric p r o t o n o f a 3 - l i n k e d g a l a c t o s y l r e s i d u e i n t h e a-anomeric c o n f i g u r a t i o n . The absence of a n u m e r i c a l p r e f i x i n d i c a t e s a ( t e r m i n a l ) n o n r e d u c i n g group. C h e m i c a l s h i f t i n p.p.m. d o w n f i e l d from Me^Si, r e l a t i v e t o i n t e r n a l acetone; 31.07 p.p.m. d o w n f i e l d from DSS. but f o r C n u c l e i . 1 3  See t e x t .  As i n c ,  94  h y d r o l y s i s and of degraded p o l y s a c c h a r i d e a f t e r s e l e c t i v e h y d r o l y s i s (see Table  IV.1).  Methylation analysis M e t h y l a t i o n a n a l y s i s of the K50  p o l y s a c c h a r i d e , f o l l o w e d by  h y d r o l y s i s , d e r i v a t i z a t i o n as a l d i t o l a c e t a t e s , and g.l.c.-m.s. s i s , gave the v a l u e s shown i n Table IV.2, column I .  analy-  These r e s u l t s  i n d i c a t e d t h a t the p o l y s a c c h a r i d e c o n s i s t s of a h e p t a s a c c h a r i d e  repeat-  i n g u n i t h a v i n g a branch on g l u c o s e , w i t h g a l a c t o s e as the t e r m i n a l group.  By r e d u c t i o n of the methylated  p o l y s a c c h a r i d e (see column I I ) ,  the p r o p o r t i o n of 2,4,6-tri-O-methylmannose was methylglucose  was  i n c r e a s e d and  2,3-di-0-  formed, i n d i c a t i n g t h a t g l u c u r o n i c a c i d i s s u b s t i t u t e d  at 0_-4, and t h a t i t i s l i n k e d t o mannose. M e t h y l a t i o n - e t h y l a t i o n a n a l y s i s of the product  o b t a i n e d by b a s e - c a t a l y z e d d e g r a d a t i o n of  the  u r o n i c a c i d showed the presence of 3-0-ethyl-2,4,6-tri-0-methylmannose d e r i v e d from e t h y l a t i o n at 0-3  of the mannose of the a l d o b i o u r o n i c a c i d  (see column I I I ) .  Selective partial hydrolysis Treatment of K50 p o l y s a c c h a r i d e w i t h v e r y d i l u t e a c i d , and s i s of the products  against d i s t i l l e d water, afforded a non-dialyzable  p o l y m e r i c m a t e r i a l and a d i a l y z a t e . The as was  dialy-  d i a l y z a t e contained  galactose,  shown by paper chromatography and g . l . c . (as a l d i t o l a c e t a t e ) .  Examination  of the n o n - d i a l y z a b l e p o r t i o n by g e l - p e r m e a t i o n  graphy showed the polymer to be e x t e n s i v e l y depolymerized IV.1), and t h i s was  chromato(see F i g .  a t t r i b u t e d to the a c i d l a b i l i t y of the 3 - s u b s t i t u t e d  95  TABLE IV.2  METHYLATION ANALYSES OF K l e b s i e l l a  K50 POLYSACCHARIDE AND DERIVATIVES  T  Methylated sugars (as a l d i t o l acetates)  Mole %  a  Column B  I  C  b  II  III  (ECNSS-M)  0.87  ..-  2,3,4,6-Gal  1.00  19.8  14.5  3,4,6-Man  1.42  12.9  10.1  2,4,6-Man  1.49  2,4,6-Gal  1.59  15.8  14.5  2,3,4-Glc  1.66  26.7  15.2  2,4-Glc  2.52  16.0  11.1  4.4  2,3-Glc  2.59  -  15.5  -  2,3,4,6-Man  6  _  .  .  16.5 23.5  25.6 8.7  18.4  a  8.9 20.9  b R e l a t i v e r e t e n t i o n t i m e , r e f e r r e d t o 2,3,4,6-Gal as 1.00.  Values are  c o r r e c t e d by use of t h e e f f e c t i v e , carbon-response f a c t o r s g i v e n by Albersheim 200°. III,  ^ I,  et a l .  1 0 3  C  Programmed f o r 4min a t 160°, and then a t 2°/min t o  o r i g i n a l polysaccharide;  u r o n i c acid-degraded  tri-O-methylmannitol,  etc.  I I , carboxyl-reduced  polysaccharide.  polysaccharide;  2,3,4,6-Man = 3 - 0 - e t h y l - 2 , 4 , 6 -  96  Fig. IV.1:  G e l - p e r m e a t i o n chromatography  of the product obtained  a f t e r s e l e c t i v e , p a r t i a l h y d r o l y s i s o f K l e b s i e l l a K50 polysaccharide.  ( B i o - G e l P-300 column 85 x 1.5 cm,  M NaCl e l u a n t , f l o w - r a t e 3 mL/h).  Courtesy of  Dr. S.C. Churms, Cape Town, South A f r i c a  97  8-D-galactopyranosyl  u n i t , even under these m i l d c o n d i t i o n s .  n.m.r. spectrum of t h i s m a t e r i a l l a c k e d one s i g n a l (6 4.7)  The  *H-  corresponding  t o a 6 - l i n k a g e , but i n t e g r a t i o n of the s i g n a l s f o r anomeric p r o t o n s u n s a t i s f a c t o r y due to the d e p o l y m e r i z a t i o n (see Table I V . 1 ) .  was  Methyla-  t i o n a n a l y s i s of the n o n - d i a l y z a b l e m a t e r i a l showed t h a t 2,3,4,6-tetra-O-methylglucose  l a r g e l y r e p l a c e d the  corresponding  g a l a c t o s e compound.  P a r t i a l hydrolysis P a r t i a l h y d r o 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 w i t h a c i d f o l l o w e d by s e p a r a t i o n of the a c i d i c and exchange chromatography. rides.  was  the n e u t r a l f r a c t i o n s by i o n -  The n e u t r a l f r a c t i o n c o n t a i n e d monosaccha-  The a c i d i c f r a c t i o n c o n t a i n e d t h r e e a c i d i c o l i g o s a c c h a r i d e s .  the b a s i s of paper chromatography, t h e i r n . m . r . - s p e c t r a l data (see  On  Table  I V . 1 ) , and m e t h y l a t i o n a n a l y s i s (see Table I V . 3 ) , the s t r u c t u r e s of these compounds were shown to be as  M A2 A3  The  follows.  a-GlcA-( 1 ->3 ) -Man ct-GlcA-(l+3)-a-Man-(l->2)-Man a-GlcA-( l->-3)-a-Man-( l * 2 ) - c r t ! a n - ( l + 3 ) - G a l  a l d o t e t r a o u r o n i c a c i d (A3) 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  had p r e v i o u s l y been i s o l a t e d from o t h e r 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 , namely K21, K26 and  K74.  98  TABLE IV.3  ANALYSES OF ACIDIC OLIGOSACCHARIDES FROM K l e b s i e l l a K50 POLYSACCHARIDE  Oligosaccharide  Paper chromatography  Sugar a  Methylation c analysis  b analysis (molar proportions)  GlcA  Man  Man  Glc  GlcA  Man  Man  Glc  GlcA  Al  A2  A3  glucosyluronic  (GlcA)  < > J  -  (1.5)  2,4,6-Man ( 0 . 7 )  (1)  3,4,6-Man (1.0)  Man  (1.75)  2,4,6-Gal (1.0)  Man  Gal  (1.2)  2,4,6-Man ( 0 . 7 )  d  Gal  Glc  (1)  3,4,6-Man ( 0 . 8 )  d  (GlcA)  (GlcA)  d  c . a l d i t o l acetates, neutral acetates. As R a t i o s a r e low, due t o i n c o m p l e t e h y d r o l y s i s of t h e  S o l v e n t s A and B. sugars o n l y .  (molar proportions)  15  linkage.  As a l d i t o l  99  Periodate  oxidation  Periodate  o x i d a t i o n of c a r b o x y l - r e d u c e d p o l y s a c c h a r i d e , 7 5  ed by m e t h y l a t i o n ,  Smith h y d r o l y s i s , r e m e t h y l a t i o n ,  1 8 2  follow-  and h y d r o l y s i s ,  gave a m i x t u r e t h a t was found, by g . l . c . a n a l y s i s of t h e a l d i t o l acetates,  t o c o n t a i n d e r i v a t i v e s o f 2,3,4,6-tetra-O-methylmannose,  2 , 3 , 4 , 6 - t e t r a - O - m e t h y l g a l a c t o s e , and 2 , 4 , 6 - t r i - O - m e t h y l g l u c o s e , i n d i c a t i n g that the galactose the branch p o i n t . in i t ,  i n the main c h a i n i s 3 - l i n k e d t o g l u c o s e ,  The f o l l o w i n g s t r u c t u r e may, t h e r e f o r e be proposed;  t h e c o n f i g u r a t i o n o f three  l i n k a g e s has y e t t o be determined.  -•3 )-? - G a l - ( 1 -»3 )-? - G l c - ( 1 +4 )-cc-GlcA-( 1+3 )-<x-Man-( 1 -»2 ) - a-Man-( 1 + 6 + 1 ?-Glc 6 + 1 B-Gal  Smith d e g r a d a t i o n of t h e o r i g i n a l K50 p o l y s a c c h a r i d e oligosaccharide,  gave an  a n a l y s i s o f w h i c h showed i t t o be  COOH  I  HCOH  I  Gal-(l-»3)-Glc-0CH  I  CH OH 2  and  i t ' s '•H-n.m.r. spectrum i n d i c a t e d t h e presence of one a and one 8  linkage.  Because t h e two monosaccharides t h e r e i n e x h i b i t s i m i l a r  coupl-  100  i n g - c o n s t a n t s ; no d e f i n i t i v e assignment c o u l d be made by such s p e c t r o s copy.  Incubation w i t h a 8-D-galactosidase  caused cleavage  s i d a s e , n e g a t i v e ) o f g a l a c t o s e , thus d e m o n s t r a t i n g g a l a c t o s e has t h e (3 c o n f i g u r a t i o n .  (a-D-galacto-  t h a t the i n - c h a i n  S i x of the seven anomeric l i n k a g e s ,  i n c l u d i n g both of the 8 s i g n a l s , h a v i n g been p o s i t i v e l y a s s i g n e d , i t f o l l o w s t h a t the s i d e c h a i n must be a t t a c h e d t o t h e main c h a i n by an a - g l y c o s i d i c bond.  IV.4  CONCLUSION  From the sum of these e x p e r i m e n t s , capsular polysaccharide  the complete s t r u c t u r e o f t h e  from K l e b s i e l l a s e r o t y p e K50 may be w r i t t e n  -•3 ) - 8-D-Gal -(1 -»3 ) - cHD-Glc - (1 -»4 ) - a-D-Gl c A- (1 +3 ) - a-D-Man- (1>2)-ct-D-Man- ( 1 -> 6 t 1 a-D-Glc 6 + 1 0-D-Gal  I t i s c o n s i s t e n t w i t h the q u a l i t a t i v e ed by N i m m i c h .  15  The s t r u c t u r e o f the K50 p o l y s a c c h a r i d e i s unique  among t h e K l e b s i e l l a K a n t i g e n s unit.  analysis originally report-  i n having a " f i v e - p l u s - t w o " repeating  101  IV.5  EXPERIMENTAL  General methods The i n s t r u m e n t a t i o n used f o r n.m.r., g . l . c ,  g.l.c.-m.s.  i n f r a r e d , c.d., and measurements of o p t i c a l r o t a t i o n has been d e s c r i b e d i n S e c t i o n I I I . Paper chromatography, g a s - l i q u i d chromatography, and ion-exchange chromatography were performed as d e s c r i b e d i n S e c t i o n I I I .  Preparation and properties of K50 capsular A c u l t u r e of K l e b s i e l l a K50, o b t a i n e d  polysaccharide  from Dr. I d a 0 r s k o v  (Copenhagen), was grown as d e s c r i b e d i n S e c t i o n I I I . 7 . 1 . i s o l a t e d polysaccharide  (3.4 g) had [ a ]  2 5  1 5  '  6 1  '  6 2  The  +102° ( c 0.095, w a t e r ) .  average m o l e c u l a r weight was determined by g e l chromatography  The  (courtesy  of Dr. S.C. Churms, U n i v e r s i t y of Cape Town, South A f r i c a ) t o be 3.2 x 10  6  d a l t o n s . N.m.r. s p e c t r o s c o p y  ( *H and C ) was performed on t h e 1 3  o r i g i n a l K50 p o l y s a c c h a r i d e , but much b e t t e r s p e c t r a were o b t a i n e d treatment  after  of t h e p o l y s a c c h a r i d e w i t h 0.01M t r i f l u o r o a c e t i c a c i d d u r i n g 2  h a t 95°, i n order t o lower t h e v i s c o s i t y .  The p r i n c i p a l s i g n a l s and  t h e i r assignments f o r t h e *H- and C - n.m.r. s p e c t r a a r e r e c o r d e d i n 1 3  Table I V . 1 .  Hydrolysis of the polysaccharide H y d r o l y s i s of a sample (20 mg) of K50 p o l y s a c c h a r i d e w i t h 2M t r i f l u o r o a c e t i c a c i d (TFA) o v e r n i g h t a t 95°, removal of t h e a c i d by repeated  coevaporation  w i t h w a t e r , f o l l o w e d by paper chromatography  ( s o l v e n t s A and B, see S e c t i o n I I I . l ) , showed t h e presence of D-mannose,  102  D - g a l a c t o s e , D-glucose, D - g l u c u r o n i c a c i d and an a l d o b i o u r o n i c a c i d . H y d r o l y s i s of c a r b o x y l - r e d u c e d p o l y s a c c h a r i d e  7 5  w i t h 2M t r i f l u o r o a c e t i c  a c i d o v e r n i g h t a t 95°, f o l l o w e d by r e d u c t i o n w i t h NaBH^ and a c e t y l a t i o n w i t h 1:1 a c e t i c a n h y d r i d e - p y r i d i n e a f f o r d e d t h e a l d i t o l a c e t a t e s mannose, g a l a c t o s e , and g l u c o s e which were i d e n t i f i e d by g . l . c .  1 8 3  of  (column  A, see S e c t i o n I I I . 2 ) and found t o be p r e s e n t i n t h e r a t i o s of 2.0:2.4:3.0.  Circular dichroism measurements Glucose was proved t o be of t h e D - c o n f i g u r a t i o n by c i r c u l a r d i c h r o i s m (c.d.) measurements made on g l u c l t o l h e x a a c e t a t e .  Galactose  and mannose were a s s i g n e d D c o n f i g u r a t i o n from c.d. measurements made on p a r t i a l l y methylated d e r i v a t i v e s . p r e p a r a t i v e l y i n column D. s e p a r a t e d i n column E.  7 8  A l d i t o l a c e t a t e s were s e p a r a t e d  P a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s were  F o r e x p e r i m e n t a l d e t a i l s see S e c t i o n I I I . 2 and MeCN  Section III.4.  The f o l l o w i n g v a l u e s o f & 2\3  a c e t a t e s o f : g l u c i t o l , +1.3;  e  were o b t a i n e d f o r t h e  2 , 3 , 4 , 6 - t e t r a - 0 _ - m e t h y l g a l a c t l t o l , +0.7;  and 2 , 4 , 6 - t r i - O - m e t h y l m a n n i t o l , +0.64. Methylation analysis The c a p s u l a r p o l y s a c c h a r i d e (297 mg) i n the f r e e - a c i d  form,  o b t a i n e d by p a s s i n g t h e sodium s a l t through a column of A m b e r l i t e IR-120 ( H ) r e s i n , was d i s s o l v e d i n d r y d i m e t h y l s u l f o x i d e (20 mL) and +  methylated  8 8  by treatment w i t h 10 mL d i m e t h y l s u l f i n y l a n i o n f o r 4 h, and  then w i t h 15 mL m e t h y l i o d i d e f o r 1 h. The product (316 mg), r e c o v e r e d  103  a f t e r d i a l y s i s a g a i n s t tap w a t e r , had been c o m p l e t e l y m e t h y l a t e d h y d r o x y l a b s o r p t i o n i n the i . r . A sample (27.8 mg)  spectrum).  of t h i s m a t e r i a l was  hydrolyzed with  t r i f l u o r o a c e t i c a c i d , reduced w i t h sodium b o r o h y d r i d e , 1:1  g . l . c . and g.l.c.-m.s. i n columns B and C (see Table C a r b o x y l r e d u c t i o n of the f u l l y methylated (10 mL)  2M  acetylated with  a c e t i c a n h y d r i d e - p y r i d i n e , and a n a l y z e d as a l d i t o l a c e t a t e s  L i A l H ^ i n anhydrous oxolane  (no  by  IV.2, column I ) .  p o l y s a c c h a r i d e (103 mg)  with  at room temperature o v e r n i g h t ,  h y d r o l y s i s of the product w i t h 2M t r i f l u o r o a c e t i c a c i d , f o l l o w e d by r e d u c t i o n of sugars w i t h sodium b o r o h y d r i d e , a l d i t o l s w i t h 1:1 methylated  and a c e t y l a t i o n of the  a c e t i c a n h y d r i d e - p y r i d i n e gave a m i x t u r e of p a r t i a l l y  a l d i t o l a c e t a t e s which was  a n a l y z e d by g . l . c . and  g.l.c.-m.s.  i n columns B and C (see Table IV.2, column I I ) .  Uronic acid  degradation ** 1  A sample (76 mg)  2  of m e t h y l a t e d  K50 p o l y s a c c h a r i d e was  t o g e t h e r w i t h a t r a c e of _p_-toluenesulfonic a c i d , was dimethyl sulfoxide-2,3-dimethoxypropane was  then s e a l e d .  (20 mL)  D i m e t h y l s u l f i n y l a n i o n (5 mL)  r e a c t f o r 16 h at room temperature,  dissolved i n  under N was  2  and, 19:1  i n a f l a s k that  added and a l l o w e d to  when e t h y l i o d i d e (7 mL)  The  s o l u t i o n was  was  i s o l a t e d by p a r t i t i o n between water and c h l o r o f o r m .  g.l.c.-m.s.  dried  s t i r r e d f o r 1 h, and the m e t h y l a t e d ,  was  degraded  added. product  H y d r o l y s i s and  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 showed the  presence of 3-0-ethyl-2,4,6-tri-0-methylmannose (see Table IV.2, column III).  104  Selective, p a r t i a l h y d r o l y s i s  i B H  A s o l u t i o n of K50 p o l y s a c c h a r i d e (500 mg) i n 0.01M TFA (50 mL) was heated on a steam b a t h f o r 12 h.  The a c i d was removed and t h e  product was d i a l y z e d a g a i n s t d i s t i l l e d water (1 L ) , t o a f f o r d a p o l y m e r i c m a t e r i a l (250 mg).  Paper chromatography o f t h e d i a l y z a b l e  frac-  t i o n showed g a l a c t o s e , c o n f i r m e d by g . l . c . as g a l a c t i t o l h e x a a c e t a t e . M e t h y l a t i o n a n a l y s i s o f t h e p o l y m e r i c m a t e r i a l and g . l . c . (column C) i n d i c a t e d the presence of 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e i n s t e a d of t h e 2 , 3 , 4 - t r i - 0 - m e t h y l g l u c o s e found o r i g i n a l l y .  P a r t i a l hydrolysis A s o l u t i o n o f K50 p o l y s a c c h a r i d e (710 mg) i n 1M TFA (50 mL) was heated f o r 5 h on a steam b a t h .  A f t e r removal o f the a c i d by r e p e a t e d  c o e v a p o r a t i o n w i t h w a t e r , an a c i d i c and a n e u t r a l f r a c t i o n were s e p a r a t e d on a column o f Bio-Rad AG1-X2 ( f o r m a t e form) ion-exchange resin.  The a c i d i c f r a c t i o n (198 mg) was s e p a r a t e d by p r e p a r a t i v e paper  chromatography ( s o l v e n t C ) , t o g i v e 51.5 mg o f a pure a l d o b i o u r o n i c a c i d ( A l ) , 19.1 mg of a pure a l d o t r i o u r o n i c a c i d ( A 2 ) , and 27.7 mg o f a pure a l d o t e t r a o u r o n i c acid (A3).  Paper chromatography o f t h e n e u t r a l  f r a c t i o n showed g l u c o s e , g a l a c t o s e and mannose. The a n a l y s e s performed on each o l i g o s a c c h a r i d e were as f o l l o w s , (a)  Paper chromatography.  A c i d i c o l i g o s a c c h a r i d e s were t r e a t e d w i t h 2M  TFA o v e r n i g h t , and t h e h y d r o l y z a t e s were examined by paper chromatography ( s o l v e n t s A and B ) .  (b) Sugar a n a l y s i s .  The h y d r o l y z a t e s were  then reduced w i t h NaBH^, and the a l d i t o l s were a c e t y l a t e d w i t h 1:1 acetic anhydride-pyridine.  The a l d i t o l a c e t a t e s o b t a i n e d were a n a l y z e d  105  by g . l . c . i n column A.  (c)  Methylation a n a l y s i s . A l l methylations  were conducted by the method of H a k o m o r i .  88  D r i e d samples of 5-6  mg were  d i s s o l v e d i n 1 mL anhydrous d i m e t h y l s u l f o x i d e , t r e a t e d w i t h 1 mL d i m e t h y l s u l f i n y l a n i o n f o r 2 h and then w i t h 2 mL m e t h y l i o d i d e f o r 1 h. The m i x t u r e s were d i l u t e d w i t h water and e x t r a c t e d w i t h c h l o r o f o r m (4 x 10 mL). mL)  The  combined e x t r a c t s were back e x t r a c t e d w i t h water (3 x  and evaporated  to dryness  h y d r o l y z e d w i t h 2M TFA,  and a n a l y z e d  g.l.c.-m.s. i n columns B and The  under reduced p r e s s u r e .  10  The p r o d u c t s were  (as a l d i t o l a c e t a t e s ) by g . l . c .  and  C.  r e s u l t s o b t a i n e d f o r each o l i g o s a c c h a r i d e are g i v e n i n Table  IV.3, and the n.m.r. d a t a , i n Table  IV.1.  Carbodiimide reduction of capsular p o l y s a c c h a r i d e A sample of K50  polysaccharide (Na  s a l t , 502 mg)  +  75  was d i s s o l v e d  i n 80 mL water. 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_t o l u e n e s u l f o n a t e (CMC, pH was  maintained  2.2  at 4.75  g) was  added.  As the r e a c t i o n proceeded, the  by t i t r a t i o n w i t h 0.1  a l l o w e d to proceed f o r at l e a s t two h.  N HC1.  The  When the consumpton of  h y d r o c h l o r i c a c i d ceased, an aqueous s o l u t i o n of 2M sodium (12 g/150 was  mL H 0) 2  maintained  of 2 - o c t a n o l  added s l o w l y .  between 5-7  The  by t i t r a t i o n w i t h 4M HC1.  r e d u c t i o n was  borohydride  The pH rose r a p i d l y t o 7.0  ( a n t i f o a m i n g agent) was  amount of foam. m i x t u r e was  was  r e a c t i o n was  and i t  A drop  added p e r i o d i c a l l y to c o n t r o l the  completed i n one h, and the r e a c t i o n  s t i r r e d f o r an a d d i t i o n a l 30 min.  a g a i n s t tap water d u r i n g 24 h, c o n c e n t r a t e d  The m i x t u r e was d i a l y z e d  and f r e e z e - d r i e d .  A second  106  treatment was  c a r r i e d out.  recovered a f t e r  A t o t a l of 412.5 mg of the product  was  freeze-drying.  A sample of the reduced p o l y s a c c h a r i d e (10 mg) was h y d r o l y z e d o v e r n i g h t w i t h 2M t r i f l u o r o a c e t i c a c i d on a steam b a t h and the sugars i n the h y d r o l y z a t e were c o n v e r t e d i n t o a l d i t o l a c e t a t e s , g . l . c . a n a l y s i s of which showed m a n n i t o l , g a l a c t i t o l and g l u c i t o l h e x a a c e t a t e s i n a r a t i o 2.0:2.4:3.0, i n d i c a t i n g complete r e d u c t i o n of u r o n i c a c i d .  Periodate oxidation of carboxyl-reduced K50  polysaccharide  C a r b o d i i m i d e - r e d u c e d K50 p o l y s a c c h a r i d e (46.9 mg) mL) was mixed w i t h a s o l u t i o n (5 mL) kept i n the dark at 4°.  of 0.1M  NalO^ and 0.4M  The p e r i o d a t e consumption was  a l i q u o t s by the F l e u r y - L a n g e m e t h o d  1 8 5  (20  NaClO^, and  f o l l o w e d on 1 mL  and reached a p l a t e a u a f t e r 6 d  (7.62 moles of p e r i o d a t e per mole of p o l y s a c c h a r i d e ) . (10 mL) was added, the p o l y a l d e h y d e was  i n water  Ethylene g l y c o l  reduced t o the p o l y a l c o h o l w i t h  NaBH^, the base n e u t r a l i z e d w i t h 50% a c e t i c a c i d , and the s o l u t i o n  was  d i a l y z e d o v e r n i g h t , and f r e e z e - d r i e d to y i e l d the p o l y a l c o h o l (50.9 which was m e t h y l a t e d by Hakomori p r o c e d u r e .  8 8  O n e - t h i r d of the m e t h y l a -  t e d product was h y d r o l y z e d w i t h 50% a c e t i c a c i d f o r 90 min, and remethylated.  then  The p r o d u c t was h y d r o l y z e d , and the sugars were  c h a r a c t e r i z e d as a l d i t o l a c e t a t e s by g . l . c . i n column B. of the m a t e r i a l was a steam b a t h .  mg)  The  remainder  h y d r o l y z e d w i t h 2M t r i f l u o r o a c e t i c a c i d o v e r n i g h t on  C o n v e r s i o n of the p a r t i a l l y m e t h y l a t e d sugars i n t o  a l d i t o l a c e t a t e s and g . l . c . 2,4,6-tri-O-methylmannose,  t h e r e o f i n column B, showed the presence o f 2 , 4 , 6 - t r i - O - m e t h y l g a l a c t o s e , and  2,4-di-0-methylglucose i n equimolar p r o p o r t i o n s .  107  Smith d e g r a d a t i o n P o l y s a c c h a r i d e (295 mg) t i o n (28 mL)  of 0.1M  NaI0  i n water  and 0.4M  4  NaC10 , and kept i n the dark a t 4°. 4  A f t e r 95 h, e t h y l e n e g l y c o l (10 mL) was d i a l y z e d o v e r n i g h t , the p o l y a l d e h y d e was NaBH^ (1 g ) , the base was s o l u t i o n was which was  (100 mL) was mixed w i t h a s o l u -  added. reduced  The s o l u t i o n  was  to the p o l y a l c o h o l w i t h  n e u t r a l i z e d w i t h 50% a c e t i c a c i d , and  the  d i a l y z e d , and f r e e z e - d r i e d , to y i e l d p o l y a l c o h o l (258  s u b j e c t e d t o Smith h y d r o l y s i s w i t h 0.5M  f o r 20 h at room temperature.  trifluoroacetic  mg) acid  Paper chromatography ( s o l v e n t C) of the  p r o d u c t s showed g l y c e r o l and t h r e e o l i g o s a c c h a r i d e s . The  chromatograms  o b t a i n e d i n d i c a t e d t h a t the Smith h y d r o l y s i s was not complete, but mg of a pure, a c i d i c o l i g o m e r (SH^) was w a t e r ) and R„  t  0.55  ( s o l v e n t C).  Sugar a n a l y s i s of the product  i s o l a t e d , [ a ] ^ +59° (c_ 0.08, 5  N.m.r. d a t a are g i v e n i n Table  88  IV.1.  (by g . l . c , as the a l d i t o l a c e t a t e s ) gave  g a l a c t o s e and g l u c o s e i n the r a t i o of 1:1. method  49.6  M e t h y l a t i o n by the Hakomori  gave the p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s c o r r e s p o n d i n g t o  2,3,4,6-tetra-0-methylgalactose  and  2,4,6-tri-O-methylglucose.  Enzymatic h y d r o l y s i s The a c i d i c product pH 7.3,  (SH^, 2.3 mg) was  and a s o l u t i o n of 8 - D - g a l a c t o s i d a s e  Sigma) i n 0.1  mL of the same b u f f e r , was  (1.1 mg,  added.  t e d f o r 3 d a t 37°; t h e n , the r e a c t i o n was acetic acid.  d i s s o l v e d i n 1 mL of b u f f e r , from E.  coll,  The m i x t u r e was  incuba-  t e r m i n a t e d by a d d i t i o n of  The p r o d u c t , i s o l a t e d by l y o p h i l i z a t i o n , was  paper chromatography ( s o l v e n t B ) , which showed the presence  50%  examined by of  108  g a l a c t o s e , t h e i d e n t i t y of which was confirmed  by g . l . c . as g a l a c t i t o l  hexaacetate. I n a c o n t r o l experiment,  compound SH^ and m e l l b i o s e were  separately incubated w i t h a-D-galactosidase  (pH 4 ) . G a l a c t o s e  was  r e l e a s e d from m e l i b i o s e ( a - D - g a l a c t o p y r a n o s y l - ( l ^ - 6 ) - D - g l u c o s e ) , but not from SH,.  109  CHAPTER V  STRUCTURAL INVESTIGATION OF Escherichia c o l i CAPSULAR POLYSACCHARIDES  no  V.1  STRUCTURAL INVESTIGATION OF ESCHERICHIA COLI 09:K28(A):H~ (K28  V.l.l  ANTIGEN) CAPSULAR POLYSACCHARIDE.  ABSTRACT  The  s t r u c t u r e of t h e c a p s u l a r p o l y s a c c h a r i d e from E s c h e r i c h i a  c o l i 09:K28(A):H~ (K28 a n t i g e n ) has been determined by u s i n g t h e techniques N.m.r.  o f m e t h y l a t i o n , p e r i o d a t e 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 .  spectroscopy  (*H and C ) was used t o e s t a b l i s h the n a t u r e of 1 3  the anomeric l i n k a g e s . was  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 0 - a c e t y l groups  done s p e c t r o p h o t o m e t r i c a l l y .  The l o c a t i o n of the 0 - a c e t y l group was  determined u s i n g m e t h y l v i n y l e t h e r as a p r o t e c t i v e r e a g e n t . The  p o l y s a c c h a r i d e i s comprised of r e p e a t i n g u n i t s of t h e t e t r a -  s a c c h a r i d e shown ( t h r e e - p l u s - o n e t y p e ) w i t h 70% of t h e f u c o s y l r e s i d u e s b e i n g 0 - a c e t y l a t e d a t e i t h e r 0-2 o r 0-3. -•3 ) - a-D-Glc-(1 -»-4 ) - 8-D-Glc A- (1 -»-4 ) - a-L-Fuc-( 1 -> 4 2 or 3  •  1 8-D-Gal  I  OAc  I l l  V.1.2  INTRODUCTION  The K a n t i g e n s o f E. c o l i can be d i v i d e d i n t o t h r e e groups (A, B, L),  a l l o f which comprise a c i d i c p o l y s a c c h a r i d e s .  D i s t i n c t i v e features  of A a n t i g e n s a r e t h a t they occur o n l y t o g e t h e r w i t h 08 and 09 a n t i g e n s , t h a t they form t h i c k c a p s u l e s and t h a t they a r e f r e e of amino  sugars.  2 7  The e x t r a c e l l u l a r A a n t i g e n s of E. c o l i bear a s t r o n g s i m i l a r i t y t o t h e K antigens of K l e b s i e l l a .  2 7  The p r e s e n t i n v e s t i g a t i o n i s thus a l o g i c a l  e x t e n s i o n and d e s c r i b e s t h e i s o l a t i o n and s t r u c t u r a l a n a l y s i s of an a c i d i c p o l y s a c c h a r i d e o b t a i n e d from E. c o l i 09:K28(A):H~.  V.1.3  RESULTS AND DISCUSSION  Isolation  and characterization  A c u l t u r e o f E s c h e r i c h i a c o l i K28 was grown on M u e l l e r  Hinton  agar and t h e i s o l a t i o n and p u r i f i c a t i o n o f t h e p o l y s a c c h a r i d e were a c h i e v e d as d e s c r i b e d i n S e c t i o n I I I . 7 . 2 .  The p u r i f i e d product  obtained  a f t e r C e t a v l o n p r e c i p i t a t i o n had [ o t ] -18.2° w h i c h compares w e l l w i t h D  the c a l c u l a t e d v a l u e o f -19.3° u s i n g Hudson's R u l e s o f I s o r o t a t i o n , * 1 1  and was shown t o be heterogeneous by g e l - p e r m e a t i o n  8  chromatography,  c o n t a i n i n g two components w i t h m o l e c u l a r weight 9 x 1 0 d a l t o n s ( 3 0 % by 6  w e i g h t ) and 450,000 d a l t o n s (70% by w e i g h t ) ; weight M^was 3 x 1 0 d a l t o n s . 6  molecular  The p o l y s a c c h a r i d e was found t o be  homogeneous a f t e r m i l d a l k a l i treatment a well-known phenomenon f o r E.  t h e average  (M = 350,000 d a l t o n s ) . T h i s i s w c o l i K a n t i g e n s a s s o c i a t e d w i t h 0 groups  112  08, 09 and 0101. C a p s u l a r (K) a n t i g e n s b e l o n g i n g t o t h i s group form v e r y v i s c o u s aqueous s o l u t i o n s . the v i s c o s i t y d r a s t i c a l l y .  1 8 6  '  Treatment w i t h d i l u t e a l k a l i 1 8 7  reduces  These f i n d i n g s i n d i c a t e the presence  of i n t e r - c h a i n e s t e r l i n k a g e s between c a r b o x y l i c groups of h e x u r o n i c a c i d c o n s t i t u e n t s and h y d r o x y l groups o f sugar r i n g s . presence  1 8 6  '  1 8 7  The  of a c e t a t e groups c o u l d a l s o c o n t r i b u t e t o the f o r m a t i o n o f  aggregates,  s i n c e the removal of a c e t a t e y i e l d s a homogeneous  polysaccharide. The K28 p o l y s a c c h a r i d e i s composed of D-glucose, D - g l u c u r o n i c a c i d and L - f u c o s e .  D-galactose,  I t does not c o n t a i n D - g a l a c t u r o n i c a c i d  and D-mannose as was e a r l i e r t h o u g h t .  1 8 8  The presence  of g l u c o s e ,  g a l a c t o s e , f u c o s e , g l u c u r o n i c a c i d and an a l d o b i o u r o n i c a c i d i n the a c i d h y d r o l y z a t e of the p o l y s a c c h a r i d e was observed  by paper chromatography.  D e t e r m i n a t i o n of the n e u t r a l sugars as the a l d i t o l a c e t a t e s gave f u c o s e , g a l a c t o s e and g l u c o s e i n t h e r a t i o s of 0.45:1:0.76. ed p o l y s a c c h a r i d e  7 5  The c a r b o x y l - r e d u c -  gave f u c o s e , g a l a c t o s e and g l u c o s e i n the r a t i o s o f  1:1.1:1.58, i n d i c a t i n g t h a t the u r o n i c a c i d i s g l u c u r o n i c a c i d .  JH-N.m.r. spectroscopy The  ^-H-n.m.r. spectrum of the E. c o l i K28 p o l y s a c c h a r i d e ( s e e  Appendix I I I , spectrum No.  ) i n d i c a t e d the r e p e a t i n g u n i t t o be a  t e t r a s a c c h a r i d e and t o c o n t a i n 50% of 0 - a c e t y l groups ( s e e Table V . l ) . The  s p e c t r o p h o t o m e t r i c 0 - a c e t y l d e t e r m i n a t i o n showed t h a t 70% o f t h e  p o l y s a c c h a r i d e was a c e t y l a t e d w i t h one a c e t y l group per repeat The  unit.  spectrum e x h i b i t s a s i g n a l a t 6 = 1.3 which a r i s e s from the C H  group of L - f u c o s e .  Two s i n g l e t s a t 6 2.15 and 6 2.18 a r e due t o the  3  113  TABLE V . l ^H-N.M.R. DATA FOR ESCHERICHIA COLI K28 POLYSACCHARIDE  Polysaccharide  ^H-N.m.r. data Integral proton  l,2 (Hz)  J  Native,  2.0  5.41  Native  after  autohydrolysis (100°, o v e r n i g h t )  4.93  b  4.86  b  4.48  }  Assignment  a-Glc a-Fuc  1.0  B-GlcA  b  1.0  8-Gal  4.44  b  1.0  2.18  s  2.15  s  1.30  b  -1  5.41  ring  proton  CH  3  of 0 - a c e t y l  3.0  CH  3  o f a-Fuc  1.0  or G l c  1.0  a-Fuc  5.39  s  4.94  8.0  4.83  8.0  4.48  8.0  1.0  8-Gal  4.42  8.0  1.0  ring  2.18  s  2.15  s  1.4  CH  1.30  6.0(J  }  } 5  6  )  >1  3.0  8-GlcA  3  proton of 0_-acetyl  CH of a-Fuc 3  114  Native,  after  carbodiimide reduction  Deacetylated  5.43  s  5.37 4.95  s b  4.85  b  4.47  b  2.20  s  2.16  s  1.31  b  5.41  }  2.0  }  0.76  a-Glc a-Fuc 6-Glc B-Gal  2.0  ring }  0.6  proton  CH  3  of 0 - a c e t y l  3.0  CH  3  of a-Fuc  s  1.0  a-Glc  5.39  s  1.0  a-Fuc  4.83  8.1  1.0  6-GlcA  4.48  6..  1.0  8-Gal  4.42  6.  1.0  ring  1.30  b  3.0  CH  3  proton o f a-Fuc  Chemical s h i f t r e l a t i v e t o i n t e r n a l acetone; 6 2.23 d o w n f i e l d from sodium 4,4-dimethyl-4-silapentane-l-sulfonate  (D.S.S.).  ^ Key:  to a s s i g n a c c u r a t e c o u p l i n g c o n s t a n t ;  s = singlet.  b = broad, unable  115  presence o f OCOCH groups. 3  The presence o f t w i n s i g n a l s can be a t t r i b u -  t e d t o t h e l o c a t i o n o f 0 - a c e t y l groups on both 0-2 and 0-3 p o s i t i o n s o f fucose.  I n t h e spectrum of d e a c e t y l a t e d E. c o l i K28 p o l y s a c c h a r i d e  those s i g n a l s were absent. detected.  I n t h e anomeric r e g i o n f o u r s i g n a l s were  The s i g n a l a t 6 4.48 r e p r e s e n t s B - l i n k e d g a l a c t o s e .  s i g n a l s a t 6 4.86 and 6 4.93 belong t o t h e 8 - g l u c u r o n i c acd.  Two The t w i n n -  i n g i s a t t r i b u t e d t o t h e presence o f 0 - a c e t y l groups on t h e a d j a c e n t fucose, s i n c e , a f t e r d e a c e t y l a t i o n , i t disappeared s i g n a l a t 6 4.83. protons  giving rise to a  A broad s i g n a l a t 6 5.41 r e p r e s e n t s t h e two anomeric  of a-L-fucose and o-D-glucose.  G e n e r a l l y , t h e *H-n.m.r.  spectrum was n o t w e l l r e s o l v e d due t o t h e extreme v i s c o s i t y of t h e s o l u tion.  The q u a l i t y o f t h e spectrum was improved a f t e r a u t o h y d r o l y s i s o f  the p o l y s a c c h a r i d e (100°, o v e r n i g h t ) .  A good spectrum was a l s o o b t a i n e d  a f t e r d e a c e t y l a t i o n o f t h e p o l y s a c c h a r i d e due t o t h e decrease i n viscosity.  13  C-N.m.r.  spectroscopy  I n t h e C-n.m.r. s p e c t r a o f t h e n a t i v e and 0_-deacetylated K28 13  p o l y s a c c h a r i d e s (see Appendix I I I , S p e c t r a No. 12) t h e s i g n a l s a r i s i n g from CH C0 (21.39 p.p.m.) and CH C=0 (175.62 p.p.m.) were absent i n t h e 3  3  spectrum of t h e d e a c e t y l a t e d p o l y s a c c h a r i d e (see Table V.2).  The  s i g n a l s f o r anomeric carbons i n the d e a c e t y l a t e d p o l y s a c c h a r i d e were o n l y s l i g h t l y changed i n p o s i t i o n (see Table V.2).  Carbons 2 and 3 o f  the f u c o s y l r e s i d u e e x h i b i t e d d o w n f i e l d s h i f t s (A6 = 2.9 p.p.m. f o r C-2 and A6 = 2.67 p.p.m. f o r C-3) i n t h e n a t i v e p o l y s a c c h a r i d e due t o t h e presence o f a c e t a t e .  Once t h e a c e t a t e was removed the s i g n a l s f o r C-2  116  TABLE V.2 C-N.M.R. DATA FOR THE NATIVE AND O-DEACETYLATED E. COLI K28 POLYSACCHARIDE 13  Native polysaccharide a  p.p.m.  102.79  polysaccharide  p.p.m.  Assignment  C-1 6-GlcA  103.91  C-1 B-GlcA  C-1 B - G a l  102.81  C-1 B - G a l C-1 a-Fuc  Assignment  }  99.30  C-1 a-Fuc  99.26  C-1 a-Glc  76.94  C-3 B-GlcA  77.97  C-3 B - G l c A  76.89  C-4 a-Fuc  76.88  C-4 a-Fuc  75.91  C-5 B - G a l  75.93  C-5 B - G a l  74.34  C-3 a-Glc  74.40  C-3 a-Glc  73.74  C-4 B-GlcA  73.74  C-4 B-GlcA  73.46  C-2 B-GlcA  73.33  C-4 B - G a l  72.27  C-4 a-Fuc  72.21  C-4 a-Fuc  72.14  C-3 a-Fuc  69.47  C-3 a-Fuc  72.10  C-2 a-Fuc  69.19  C-2 a-Fuc  69.11  unassigned  67.57  unassigned  62.28  C-6 a - G l c , 8-Gal  62.26  C-6 a - G l c ,  21.39  CH  3  of  acetate  CH  3  of  a-Fuc  16.02 15.97  a  O-Deacetylated b  Chemical  acetone; comparison  99.32  }  73.43  C-1 a-Glc  C-2 B-GlcA  {  16.12 16.06  C-4 p-Gal  }  p-Gal  C H o f a-Fuc 3  s h i f t i n p.p.m. d o w n f i e l d from Me^Si, r e l a t i v e t o i n t e r n a l 31.07 p.p.m. d o w n f i e l d from DSS.  ^ The assignments  w i t h t h e l i t e r a t u r e v a l u e s , see r e f .  166.  were made by  117  and C-3 carbons of fucose s h i f t e d u p f i e l d and a r e i n agreement w i t h t h e literature values.  These d a t a , t o g e t h e r w i t h ^-n.m.r. f i n d i n g s ,  1 6 6  suggest t h a t C H a c e t y l  groups a r e d i s t r i b u t e d between 0-2 and 0-3 o f t h e  oc-L-fucosyl r e s i d u e s .  The presence o f 0 - a c e t y l on 0-3 o f fucose can  e x p l a i n t h e s p l i t t i n g o f t h e anomeric s i g n a l a s s i g n e d t o acid.  B-D-glucuronic  A m o l e c u l a r model ( D r e i d i n g ) shows t h a t o n l y i n t h i s case i s  t h e r e a p o s s i b i l i t y o f i n t e r m o l e c u l a r I n t e r a c t i o n between H - l o f g l u c u r o n i c a c i d and t h e c a r b o n y l group of the a c e t a t e ( s e e F i g . V . l ) .  Fig.  V . l :  Partial  structure  of  E.  coli  K28  polysaccharide  These c o n c l u s i o n s were r e i n f o r c e d by the r e s u l t s of p e r i o d a t e o x i d a t i o n o f the n a t i v e and d e a c e t y l a t e d p o l y s a c c h a r i d e s and the p o s i t i o n s of the 0 - a c e t y l groups were determined by e t h y l a t i o n ( s e e l a t e r ) .  Methylation analysis M e t h y l a t i o n o f t h e K28 p o l y s a c c h a r i d e , f o l l o w e d by h y d r o l y s i s , d e r i v a t i z a t i o n as 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 , gave t h e v a l u e s shown i n T a b l e V.3, column I . These r e s u l t s i n d i c a t e d t h a t t h e p o l y s a c c h a r i d e c o n s i s t s 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 h a v i n g a branch on g l u c o s e , w i t h g a l a c t o s e as the t e r m i n a l group.  By r e d u c t i o n  118  TABLE V.3  METHYLATION ANALYSIS OF Escherichia c o l i K28 POLYSACCHARIDE AND DERIVED PRODUCTS  3,  Mol %  Methylated sugar  I  C  b  - • II  III  IV  V  (as a l d i t o l acetate) 2,3,4-Fuc  -  -  -  42  -'  2,3-Fuc  23  28  26  -  21  2,3,4,6-Glc  -  -  -  20  -  2,3,4,6-Gal  37  22  24 .  -  -  2,4,6-Glc  -  -  -  38  -  2,3,6-Glc  -  -  18  -  -  2,6-Glc  40  26  31  -  79  2,3-Glc  -  23  -  -  -  a  2,3,4-Fuc=l,5-di-C^-acetyl-2,3,4-tri-0-methylfucitol, e t c .  Values a r e  c o r r e c t e d by use o f the e f f e c t i v e , carbon-response f a c t o r s g i v e n by Albersheim  et a l .  1  0  3  u s i n g an OV-225 column, programmed from 180° f o r 4 m i n ,  and then a t 2°/min t o 230°.  c  I , o r i g i n a l polysaccharide;  I I , compounds  from L i A l H ^ r e d u c t i o n of m e t h y l a t e d E. c o l i K28; I I I , c a r b o d i i m i d e reduced p o l y s a c c h a r i d e ; polysaccharide; polysaccharide.  V,  I V , p r o d u c t from Smith d e g r a d a t i o n  of the o r i g i n a l  p r o d u c t from p e r i o d a t e o x i d a t i o n of d e a c e t y l a t e d  119  of the m e t h y l a t e d p o l y s a c c h a r i d e  (see Table V.3,  p r o p o r t i o n of 2,3-di-O-methylfucose was glucose was 0-4,  and  column I I ) , the  i n c r e a s e d , and  2,3-di-O-methyl-  formed, i n d i c a t i n g t h a t g l u c u r o n i c a c i d i s l i n k e d t h r o u g h  t h a t i t i s j o i n e d to f u c o s e .  imide-reduced p o l y s a c c h a r i d e methylglucose,  M e t h y l a t i o n a n a l y s i s of  carbodi-  showed the presence of 2 , 3 , 6 - t r i - 0 -  7 5  d e r i v e d from r e d u c t i o n of the c a r b o x y l group of  g l u c u r o n i c a c i d (see T a b l e V.3,  the  column I I I ) .  P a r t i a l hydrolysis P a r t i a l h y d r o 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 w i t h a c i d f o l l o w e d by s e p a r a t i o n of the a c i d i c and  the n e u t r a l f r a c t i o n s by i o n -  exchange chromatography.  The  n e u t r a l f r a c t i o n contained  and  The  a c i d i c f r a c t i o n contained  a disaccharide (Nl).  acid ( A l ) .  On the b a s i s of t h e i r n.m.r.  t h e i r methylation  a n a l y s i s (Table V.5),  was  monosaccharides an  aldobiouronic  s p e c t r a l data (Table V.4)  and  the s t r u c t u r e s of these  compounds were shown to be as f o l l o w s :  Al  8-GlcA-(1^4)-Fuc  Nl  B-Gal-(l->4)-Glc  Periodate oxidation Smith d e g r a d a t i o n methylation  of the n a t i v e p o l y s a c c h a r i d e ,  and h y d r o l y s i s showed the presence of  f u c o s e , 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e and Table V.3,  column I V ) .  followed  by  2,3,4-tri-O-methyl-  2,4,6-tri-O-methylglucose  These r e s u l t s show t h a t t e r m i n a l g a l a c t o s e  the g l u c u r o n i c a c i d were c o m p l e t e l y  (see and  o x i d i z e d thus i n d i c a t i n g the absence  120  TABLE V.4  N.M.R. DATA FOR Escherichia c o l l K28 OLIGOSACCHARIDES DERIVED FROM PARTIAL HYDROLYSIS OF THE POLYSACCHARIDE  Compound  *H-N.m.r. data "1,2 (Hz)  GlcA-^Fuc-OH P Al  Gal^Glc-OH P Nl  Assignment  C-N.m.r. data c d P.p.m. Assignment  4-Fuc  103.89  13  Integral proton  5.24  2.7  0.3  4.63  5.4  0.7  4.54  8.0  1.0  GlcA-  4.29  q  1.0  H-5 o f Fuc  1.33 1.29  6.75 6.75  5.23  4  0.4  4-Glc-  4.67  7  0.6  4-Gle  96.62  4-Glc- -OH  4.45  7.5  1.0  Gal  92.68  4-Glc-  } 3.0  OH a 4-Fuc—r-OH 8  CH CH  3 3  B  16.23  C H o f Fuc  97.04  3  103.83 G a l -  p  4 , 4 - d i m e t h y l - 4 - s i l a p e n t a n e - l - s u l f o n a t e (D.S.S.). the p o s i t i o n  93.11  P 4-Fuc-s-OH P 4-Fuc- -0H  o f 8-Fuc-OH o f a-Fuc-OH  Chemical s h i f t r e l a t i v e t o i n t e r n a l acetone; indicates  G1CA-T-  P  -0H  6 2.23 d o w n f i e l d from sodium ^ The n u m e r i c a l p r e f i x  i n which the sugar i s s u b s t i t u t e d ;  t h e a o r 8, t h e  c o n f i g u r a t i o n o f t h e g l y c o s i d i c bond, o r t h e anomer i n the case o f a (terminal) reducing-sugar residue.  Chemical s h i f t i n p.p.m. d o w n f i e l d  from Me^Si, r e l a t i v e t o i n t e r n a l acetone; 31.07 p.p.m. d o w n f i e l d from D.S.S. ^ As i n c, but f o r C 1 3  nuclei.  121  TABLE V.5  ANALYSIS OF THE OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF Escherichia c o l l K28 POLYSACCHARIDE  Oligosaccharide  Al  Nl  Sugar analysis  As a l d i t o l acetates (molar proportions)  Methylation analysis  As a l d i t o l acetates (molar proportions)  Fuc  1.0  Glc(GlcA)  0.8  Gal  1.0  2,3,4,6-Gal  0.95  Glc  0.9  2,3,6-Glc  1.00  122  of an 0 - a c e t y l group on e i t h e r of them. h y d r o l y z e d d u r i n g the Smith d e g r a d a t i o n methylglucose, linkage.  The f u c o s y l l i n k a g e was g i v i n g r i s e t o 2,3,4,6-tetra-O-  p r o v i n g t h a t fucose and g l u c o s e a r e engaged i n a 1+3  The p r o p o r t i o n of 2 , 3 , 4 - t r i - O - m e t h y l f u c o s e  found i n d i c a t e s  t h a t 70% of t h e fucose s u r v i v e d p e r i o d a t e o x i d a t i o n .  This  suggests t h a t 70% of t h e f u c o s y l l i n k a g e s a r e O - a c e t y l a t e d  result a t 0-2 o r 0-3  and a r e thus p r o t e c t e d a g a i n s t o x i d a t i v e d e g r a 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 d e a c e t y l a t e d p o l y s a c c h a r i d e f o l l o w e d by m e t h y l a t i o n and h y d r o l y s i s showed t h a t o n l y 25% of the fucose had s u r v i v e d (see Table V.3, column V ) . The incomplete  o x i d a t i o n of fucose  c o u l d be e x p l a i n e d on t h e b a s i s o f s t e r i c h i n d r a n c e  and p o s s i b l e  hemiacetal  formation.  1 3 3  Quantitative determination of Q-acetyl groups The  percentage of 0 - a c e t y l groups p r e s e n t  i n E. c o l i K28  p o l y s a c c h a r i d e was determined spectrophotomet r i c a l l y .  1 7 8  I t was found  t h a t 72-74% of t h e n a t i v e p o l y s a c c h a r i d e was O - a c e t y l a t e d . groups c o u l d be e a s i l y removed by m i l d a l k a l i  treatment.  The a c e t a t e These f i n d i n g s  are i n a v e r y good agreement w i t h the r e s u l t s o b t a i n e d a f t e r o x i d a t i o n o f t h e a c e t y l a t e d and d e a c e t y l a t e d p o l y s a c c h a r i d e s .  periodate Partial  removal of 0 - a c e t y l groups d u r i n g the h i g h temperature n.m.r. can be e x p l a i n e d on the b a s i s of t h e l a b i l i t y of the 0 - a c e t y l groups l o c a t e d on the f u c o s y l r e s i d u e s .  123  L o c a t i o n o f Pj-acetyl g r o u p s  1 8 0  O - A c e t y l groups i n t h e p o l y s a c c h a r i d e were l o c a t e d  1 3  by r e a c t i o n  w i t h m e t h y l v i n y l e t h e r and an a c i d i c c a t a l y s t , f o l l o w e d by e t h y l a t i o n a n a l y s i s of t h e p r o d u c t .  I t was found t h a t e i t h e r 0-2 or 0-3 o f the  a - L - f u c o s y l r e s i d u e s i s a c e t y l a t e d but no 2 , 3 - d i - O - e t h y l f u c o s e  was  obtained.  V.. 1.4  CONCLUSION  From the sum o f t h e s e e x p e r i m e n t s the complete s t r u c t u r e o f t h e capsular polysaccharide  from E s c h e r i c h i a c o l i K28 may be w r i t t e n .  +3 ) - <x-D-Glc-( 1 ->4)- 8-D-GlcA-( 1+4)-a-L-Fuc~( 1 -> 4 2 or 3 1 8-D-Gal  OAc  T h i s s t r u c t u r e c l o s e l y resembles t h a t o f the c a p s u l a r a n t i g e n s coli K27  1 8 6  '  1 8 9  from E_.  and i s o f the same s t r u c t u r a l p a t t e r n as t h a t o f t h e  capsular polysaccharide  from K l e b s i e l l a  K54.  8 9 , 1 9 0  124  +4) - o-Glc-(1+4)-a-GlcA-(1+3)-a-Fuc-(1+ 3 + 1 a-Gal E. c o l i K27  +3)-S-Glc-(l-»4)-a-GlcA-(l+3)-a-Fuc(l+ 4 2  •  I  1 8-Glc  OAc  K l e b s i e l l a K54  125  V.l. 5  EXPERIMENTAL  General methods The  i n s t r u m e n t a t i o n used f o r i n f r a r e d and n.m.r.  spectroscopy,  g . l . c , and g.l.c.-m.s., c.d., and measurements o f o p t i c a l r o t a t i o n has been d e s c r i b e d i n S e c t i o n I I I . S p e c t r o p h o t o m e t r i c made by u s i n g a P e r k i n - E l m e r  measurements were  552A UV/VIS spectrophotometer.  Paper  chromatography, g a s - l i q u i d chromatography, and ion-exchange chromatography were performed as d e s c r i b e d i n S e c t i o n I I I .  Preparation and properties of E. c o l i K28 capsular polysaccharide A c u l t u r e of E. c o l i K28 was o b t a i n e d from Dr. Ida 0rskov (Copenhagen).  The b a c t e r i a were f i r s t grown on s u c r o s e - r i c h medium as  previously described for K l e b s i e l l a capsular polysaccharides.  The  r e s u l t s were n o t s a t i s f a c t o r y and the y i e l d of p o l y s a c c h a r i d e was v e r y poor.  I n o r d e r t o f i n d s u i t a b l e growth c o n d i t i o n s s i x d i f f e r e n t media  were t r i e d :  (1) t r y p t i c a s e soy agar (BBL);  (3) n u t r i e n t b r o t h ( D i f c o ) ; (5)  beef h e a r t i n f u s i o n  (Difco);  (4) n u t r i e n t b r o t h + y e a s t e x t r a c t ( D i f c o ) ;  (Difco);  ( 6 ) M u e l l e r H i n t o n agar  Sodium c h l o r i d e improves t h e growth of E. p r e p a r a t i o n o f a l l s i x media (0.5% w/v). i n c u b a t e d a t 37° o v e r n i g h t .  (2) L u r i a broth  coli  1 9 1  (BBL).  and was used i n  The s t r e a k e d p l a t e s were  The best r e s u l t s were o b t a i n e d on M u e l l e r  H i n t o n agar and i t was l a t e r used as a growth medium w i t h the a d d i t i o n of a s m a l l amount of NaCl (0.5% The  w/v).  p u r i f i c a t i o n procedure was c a r r i e d out as d e s c r i b e d i n  Section III.7.2.  Yield:  a c i d i c p o l y s a c c h a r i d e ~ 400 mg, n e u t r a l p o l y -  126  s a c c h a r i d e ~ 100 mg  (per b a t c h ) .  Three d i f f e r e n t batches of the  p o l y s a c c h a r i d e were grown. The  m o l e c u l a r weight of the p o l y s a c c h a r i d e was  permeation chromatography ( c o u r t e s y of Dr. S.C. Cape Town, South A f r i c a ) .  determined by g e l -  Churms, U n i v e r s i t y of  The n a t i v e p o l y s a c c h a r i d e was  shown to be  heterogeneous, but became homogeneous a f t e r m i l d a l k a l i treatment  (M  = w  350,000 d a l t o n s ) .  N.m.r. s p e c t r o s c o p y  ( l H and  o r i g i n a l and d e a c e t y l a t e d K28 p o l y s a c c h a r i d e . the *H- and  1 C3  13  C)  The  was  performed on  the  principal signals in  n.m.r. s p e c t r a and t h e i r assignments are r e c o r d e d i n  Tables V . l and V.2 r e s p e c t i v e l y .  Deacetylation of polysaccharide The p o l y s a c c h a r i d e was o v e r n i g h t a t room temperature. water and f r e e z e - d r i e d .  The  d i s s o l v e d i n 0.01M The  product was  NaOH and  stirred  dialyzed against  completeness of d e a c e t y l a t i o n was  tap  checked  by *H-n.m.r. s p e c t r o s c o p y .  Hydrolysis of the polysaccharide H y d r o l y s i s of a sample (4 mg) t r i f l u o r o a c e t i c a c i d (TFA)  of K28  polysaccharide with  2M  f o r 18 h at 95°,  removal of the a c i d  by  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 w a t e r , f o l l o w e d by paper chromatography ( s o l v e n t s 1 and 2) showed f u c o s e , g l u c o s e , g a l a c t o s e , g l u c u r o n i c a c i d , and an a l d o b i o u r o n i c a c i d . carboxyl-reduced  Q u a n t i t a t i v e sugar a n a l y s i s of the  p o l y s a c c h a r i d e was  performed, and the a l d i t o l  acetates  of f u c o s e , g l u c o s e , and g a l a c t o s e were i d e n t i f i e d by g . l . c . (column  A)  127  and  found t o be p r e s e n t  i n the r a t i o s of 1:1.1:1.58.  (column D), f o l l o w e d by measurements of the c i r c u l a r spectra,  7 8  showed the g l u c i t o l h e x a a c e t a t e  and  the f u c i t o l p e n t a a c e t a t e  was  assigned  oxidase  Preparative g . l . c . dichroism  to be of the D c o n f i g u r a t i o n ,  to be of the L c o n f i g u r a t i o n .  Galactose  the D c o n f i g u r a t i o n by the p o s i t i v e a c t i o n of D - g a l a c t o s e  (Worthington  polysaccharide.  Biochem. Co.)  on the h y d r o l y s i s product  of  the  1 9 2  Methylation a n a l y s i s The  capsular polysaccharide  (60 mg)  i n the f r e e - a c i d form,  o b t a i n e d by p a s s i n g the sodium s a l t through a column of A m b e r l i t e (H ) +  r e s i n , was  methylated  d i s s o l v e d i n dry d i m e t h y l s u l f o x i d e (6 mL)  by the Hakomori p r o c e d u r e .  The  8 8  d i a l y s i s a g a i n s t tap w a t e r , was a b s o r p t i o n i n the i . r .  spectrum).  I t was  and  product, recovered  not c o m p l e t e l y  methylated  after  (hydroxyl  dissolved i n chloroform  s u b j e c t e d t o P u r d i e m e t h y l a t i o n * w i t h m e t h y l i o d i d e and s i l v e r y i e l d e d a f u l l y methylated  p o r t i o n of t h i s product  (5 mg)  was  polysaccharide  h y d r o l y z e d w i t h 2M  a c i d , the sugars were reduced w i t h sodium b o r o h y d r i d e , a c e t y l a t e d w i t h 1:1  a c e t i c anhydride  i n column C (see Table V.3, methylated  polysaccharide  —  column I ) .  (12.1 mg)  and  oxide.  81  T h i s treatment  IR-120  (57 mg).  A  trifluoroacetic the a l d i t o l s were  p y r i d i n e , and a n a l y z e d  by g . l . c .  C a r b o x y l r e d u c t i o n of the  fully  w i t h L i A l H ^ i n anhydrous o x o l a n e  at room temperature o v e r n i g h t , h y d r o l y s i s of the product w i t h  2M  t r i f l u o r o a c e t i c a c i d , f o l l o w e d by r e d u c t i o n of sugars w i t h sodiumborohydride,  and a c e t y l a t i o n of the a l d i t o l s w i t h 1:1  p y r i d i n e gave a m i x t u r e  of p a r t i a l l y m e t h y l a t e d  acetic  anhydride-  a l d i t o l acetates which  128  was  a n a l y z e d by g . l . c . and g.l.c.-m.s. i n column C (see Table  column I ) .  The n e u t r a l p o l y s a c c h a r i d e o b t a i n e d by  reduction  was  mg)  7 5  carbodiimide  a l s o s u b j e c t e d to m e t h y l a t i o n a n a l y s i s . A sample  of c a r b o d i i m i d e - r e d u c e d  s u l f o x i d e (1 mL)  p o l y s a c c h a r i d e was  and m e t h y l a t e d  by treatment  d i s s o l v e d i n dry  w i t h 1 mL  a n i o n f o r 4 h, and then 2 mL m e t h y l i o d i d e f o r 1 h. r e c o v e r e d a f t e r d i a l y s i s a g a i n s t tap w a t e r , was t r i f l u o r o a c e t i c a c i d o v e r n i g h t at 95°, ted  V.3,  converted  (9.4  dimethyl  dimethylsulfinyl  The  product  hydrolyzed with into partially  2M methyla-  a l d i t o l a c e t a t e s and a n a l y z e d , w i t h the r e s u l t s shown i n Table  V.3,  column I I I .  Carbodiimide reduction of the capsular p o l y s a c c h a r i d e A sample of K28 i n 20 mL water.  +  s a l t , 36.2  mg)  was d i s s o l v e d  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-  t o l u e n e s u l f o n a t e (CMC, pH was  polysaccharide (Na  maintained  0.4  at 4.75  g) was  added.  As the r e a c t i o n proceeded, the  by t i t r a t i o n w i t h 0.1  a l l o w e d t o proceed f o r at l e a s t two h.  N HC1.  The  r e a c t i o n was  When the consumption of  h y d r o c h l o r i c a c i d c e a s e d , an aqueous s o l u t i o n of 2M sodium (1 g/15  mL H 0) 2  maintained  was  added s l o w l y .  between 5-7  The  r e d u c t i o n was  borohydride  The pH r o s e r a p i d l y to 7.0  by t i t r a t i o n w i t h 4M HC1.  ( a n t l f o a m i n g agent) was foam.  75  A drop of  and i t was 2-octanol  added p e r i o d i c a l l y t o c o n t r o l the amount of completed i n one h.  The m i x t u r e was d i a l y z e d  a g a i n s t tap water d u r i n g 24 h, c o n c e n t r a t e d and f r e e z e - d r i e d . A second treatment  was  c a r r i e d out.  p o l y s a c c h a r i d e (11.1 mg)  was  Yield:  32.3 mg.  A sample of the reduced  h y d r o l y z e d o v e r n i g h t w i t h 2M  trifluoro-  129  a c e t i c a c i d a t 95°, and the h y d r o l y z a t e was c o n v e r t e d a c e t a t e s as b e f o r e .  G.l.c.  into  alditol  a n a l y s i s was conducted i n column A.  P a r t i a l hydrolysis The K28 p o l y s a c c h a r i d e (514 mg) was d i s s o l v e d i n 125 mL of 0.01M t r i f l u o r o a c e t i c a c i d (TFA) and the s o l u t i o n was heated steam b a t h .  f o r 32 h on a  A f t e r removal of the a c i d by s u c c e s s i v e e v a p o r a t i o n s  with  w a t e r , an a c i d i c and a n e u t r a l f r a c t i o n were s e p a r a t e d on a column o f Bio-Rad AG1-X2 ion-exchange r e s i n .  The a c i d i c f r a c t i o n was s e p a r a t e d by  p r e p a r a t i v e paper chromatography ( s o l v e n t 3 ) , t o g i v e 45.7 mg o f a pure a l d o b i o u r o n i c a c i d ( A l ) . Paper chromatography of the n e u t r a l f r a c t i o n showed f u c o s e , g l u c o s e , g a l a c t o s e and a n e u t r a l d i s a c c h a r i d e (Nl^), which was  i s o l a t e d by p r e p a r a t i v e paper chromatography ( s o l v e n t 3) t o g i v e 21  mg o f N l .  P.m.r. s p e c t r a l d a t a a r e r e c o r d e d  ( T a b l e V.4) f o r each  o l i g o s a c c h a r i d e and a n a l y s e s were performed as f o l l o w s : analysis.  ( a ) Sugar  The a c i d i c o l i g o s a c c h a r i d e was t r e a t e d w i t h 3% HC1 i n anhy-  drous methanol f o r 18 h on a steam bath.  The methyl e s t e r methyl g l y c o -  s i d e o b t a i n e d was reduced w i t h sodium b o r o h y d r i d e  i n anhydrous methanol,  f o l l o w e d by h y d r o l y s i s w i t h 2M TFA, r e d u c t i o n t o the a l d i t o l s , and a c e t y l a t i o n w i t h 1:1 a c e t i c a n h y d r i d e — p y r i d i n e . o b t a i n e d were a n a l y z e d by g . l . c . i n column A. r i d e was h y d r o l y z e d , and a n a l y z e d s i m i l a r l y , Compound N l was m e t h y l a t e d g i v e n i n Table V.5.  The a l d i t o l  acetates  The n e u t r a l o l i g o s a c c h a (b) M e t h y l a t i o n a n a l y s i s .  by the method of H a k o m o r i  8 8  and the r e s u l t s  130  Periodate oxidation A s o l u t i o n o f K28 p o l y s a c c h a r i d e (21.5 mg) i n water (10 mL) was mixed w i t h 0.03M NalO^ (10 mL) and s t i r r e d i n the dark a t room t u r e (23°) f o r 6 d.  tempera-  A f t e r e t h y l e n e g l y c o l (2 mL) was added, t h e  p o l y a l d e h y d e was reduced t o t h e p o l y a l c o h o l w i t h NaBH^, t h e base was n e u t r a l i z e d w i t h 50% AcOH, t h e s o l u t i o n was d i a l y z e d o v e r n i g h t , and f r e e z e - d r i e d t o y i e l d t h e p o l y a l c o h o l (15 mg).  A p o r t i o n (4.2 mg) was  h y d r o l y z e d wth 2M TFA o v e r n i g h t on a steam b a t h and c o n v e r t e d i n t o a l d i t o l acetates.  A n a l y s i s by g . l . c . i n column A showed f u c i t o l , g a l a c -  t i t o l and g l u c i t o l i n the r a t i o s o f 0.72:0.10:1.  The remainder o f the  m a t e r i a l was t r e a t e d w i t h 0.5M TFA f o r 48 h a t room temperature. product (10.7 mg) was m e t h y l a t e d by t h e Hakomori p r o c e d u r e , w i t h 2M TFA o v e r n i g h t on a steam bath and c o n v e r t e d i n t o acetates.  8 8  The  hydrolyzed  alditol  G . l . c . a n a l y s i s , conducted i n column B showed t h e presence o f  2 , 3 , 4 - t r i - 0 - m e t h y l f u c o s e , 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e and 2 , 4 , 6 - t r i 0-methylglucose i n the r a t i o s o f 0.70:0.35:0.65.  These r e s u l t s  indicate  t h a t d u r i n g t h e Smith 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 of the f u c o s y l l i n k a g e has o c c u r r e d . P e r i o d a t e o x i d a t i o n of d e a c e t y l a t e d p o l y s a c c h a r i d e was performed similarly.  A f r a c t i o n of t h e p o l y a l c o h o l p r o d u c t (1.6 mg) was a n a l y z e d  f o r c o n s t i t u e n t sugars and t h e remainder of t h e m a t e r i a l was m e t h y l a t e d by t h e Hakomori p r o c e d u r e .  8 8  Conversion of the p a r t i a l l y methylated  sugars i n t o a l d i t o l a c e t a t e s , and g . l . c .  t h e r e o f i n column B, showed  the presence of 2 , 3 - d i - 0 - m e t h y l f u c o s e and 2 , 6 - d i - 0 - m e t h y l g l u c o s e i n t h e r a t i o s 0.25:1.00.  131  Quantitative determination of 0,-acetyl g r o u p s  1 7 8  O - A c e t y l groups were determined s p e c t r o p h o t o m e t r i c a l l y . r e a c t i o n of 0 - a c e t y l groups w i t h h y d r o x y l a m i n e I n a l k a l i hydroxamic a c i d was  employed.  The hydroxamic a c i d was  f o r m a t i o n of a c o l o r e d complex w i t h F e  3 +  The  to form  measured by  i n acid solution.  the  To 1 mL  of  s o l u t i o n which c o n t a i n e d 40 ug of the E. c o l i K28 p o l y s a c c h a r i d e i n 0.001M sodium a c e t a t e b u f f e r 2 mL  of a f r e s h l y prepared  2M hydroxylamine h y d r o c h l o r i d e - 3 . 5 M temperature 1 mL  s o l u t i o n was  added.  of 12% h y d r o c h l o r i c a c i d was  0.37M FeCl »6H 0 i n 0.1N 3  NaOH was  2  HC1.  swirled rapidly.  purple-brown s o l u t i o n was spectrophotometer.  1:1 m i x t u r e  A f t e r 2 min at room  added f o l l o w e d by 1 mL  D u r i n g the a d d i t i o n of each reagent The absorbance at 540 nm of the  measured i n a P e r k i n - E l m e r  w a s  the  resulting  the  c a l c u l a t e d from a s t a n d a r d curve prepared  0.004M a c e t y l c h o l i n e c h l o r i d e i n 0.001M sodium a c e t a t e pH  Location of Q-acetyl g r o u p s  using  4.5.  1 8 0  E. c o l i K28 p o l y s a c c h a r i d e (17.6 mg)  and p_-TsOH (5 mg) were d r i e d  o v e r n i g h t under vacuum and d i s s o l v e d i n dry d i m e t h y l s u l f o x i d e (10 M e t h y l v i n y l e t h e r (3 mL) was r e a c t i o n m i x t u r e was 4 h.  added to the f r o z e n s o l u t i o n , and  The  the r e s i d u e was  The  was  c l e a r , r e d s o l u t i o n o b t a i n e d was  Sephadex LH-20 column (58 cm x 1.5 slight suction).  mL).  the  brought to room temperature and a l l o w e d t o s t i r f o r  Then a second p o r t i o n of m e t h y l v i n y l e t h e r (3 mL)  i n a s i m i l a r manner.  of  552A UV/VIS  After correcting for non-specific color,  q u a n t i t y of 0 - a c e t y l  of  product was  cm)  introduced  p l a c e d on a  and e l u t e d w i t h acetone ( w i t h  c o l l e c t e d and c o n c e n t r a t e d .  used f o r e t h y l a t i o n a c c o r d i n g t o the Hakomori  H a l f of  132  procedure.  8 8  The p r o d u c t , a dark red-orange o i l , was d i a l y z e d  (cutoff  3,500) o v e r n i g h t a g a i n s t t a p water and e x t r a c t e d w i t h c h l o r o f o r m .  The  p o r t i o n o f e t h y l a t e d , methyl v i n y l e t h e r p r o t e c t e d p o l y s a c c h a r i d e was h y d r o l y z e d w i t h 2M TFA o v e r n i g h t a t acetates.  G . l . c . a n a l y s i s , conducted  showed the presence  95° and c o n v e r t e d i n t o  alditol  i n column B (210° i s o t h e r m a l )  o f 2-0-ethy1fucose ( 1 6 . 9 % ) , 3 - 0 - e t h y l f u c o s e  f u c o s e ( 1 2 . 7 % ) , g a l a c t o s e ( 2 5 . 1 % ) , and g l u c o s e ( 2 1 . 6 % ) .  (23.8%),  These r e s u l t s  were confirmed by g.l.c.-m.s., w h i c h was performed on a KRATOS MS80RFA i n s t r u m e n t , u s i n g DB-225 c a p i l l a r y column (150° f o r 1 min, and then 10°/min t o 210°).  133  V.2  STRUCTURAL INVESTIGATION OF E s c h e r i c h i a c o l l 09:K32(A):H19 (K32 ANTIGEN) CAPSULAR POLYSACCHARIDE  V.2.1  ABSTRACT  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 E s c h e r i c h i a c o l i 09:K32(A):H19 (K32 a n t i g e n ) has been determined by u s i n g t h e techniques  of m e t h y l a t i o n , c a r b o x y l r e d u c t i o n and Smith  degradation.  The n a t u r e of the anomeric l i n k a g e s was e s t a b l i s h e d by u s i n g H - and 1  13  C-n.m.r. s p e c t r o s c o p y ,  and was f u r t h e r confirmed  o x i d a t i o n of the f u l l y a c e t y l a t e d p o l y s a c c h a r i d e .  by chromium t r i o x i d e The l o c a t i o n of  0 - a c e t y l groups was determined u s i n g m e t h y l v i n y l e t h e r as a p r o t e c t i v e reagent. The polymer was found t o c o n s i s t of the 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 shown ( t h r e e - p l u s - o n e being O-acetylated  t y p e ) w i t h h a l f of the L-rhamnosyl  a t 0-2. OAc  I  2 ->3)-<x-D-Glc-( l+4)-oc-L-Rha-( l+3)-a-D-Gal-( l-> 3 1 B-D-GlcA  residues  134  V.2.2  INTRODUCTION  E s c h e r i c h i a c o l i s e r o t y p e K32  i s of i n t e r e s t i n t h i s  series,  b e i n g a s p e c i f i c host f o r t h r e e d i f f e r e n t E. c o l i c a p s u l a r b a c t e r i o phages <p28—1, <(>31 and  <J>32.  193  Because of the i n c r e a s i n g importance of  s t u d i e s on the s u b s t r a t e s p e c i f i c i t y of the bacteriophage-borne  enzymes,  the complete s t r u c t u r a l a n a l y s i s of E. c o l i p o l y s a c c h a r i d e i s needed. Although  the c o m p o s i t i o n of the E. c o l i K32  i t s s t r u c t u r e i s not. t h i s genus, we now  capsule i s known,  I n c o n t i n u a t i o n of our c h e m i c a l e x a m i n a t i o n  r e p o r t the p r i m a r y  22  of  s t r u c t u r e of E s c h e r i c h i a c o l l  K32.  V.2.3  RESULTS AND  DISCUSSION  A c u l t u r e of E s c h e r i c h i a c o l i K32 was a g a r , and the a c i d i c p o l y s a c c h a r i d e was w i t h cetyltrimethylammonium  bromide.  grown on M u e l l e r  p u r i f i e d by one  The  Hinton  precipitation  polysaccharide obtained  had  [<x]p +80.9° w h i c h compares w e l l w i t h the c a l c u l a t e d v a l u e of +71.5° u s i n g Hudson's Rules of I s o r o t a t i o n . by g e l - p e r m e a t i o n with dilute a l k a l i s i g n i f i c a n t l y (M  w  i l + 8  I t was  chromatography w i t h M^ =9 x 1 0  shown t o be homogeneous 6  daltons.  Treatment  d i d not reduce the v i s c o s i t y of the p o l y s a c c h a r i d e = 6 x 10  6  daltons).  Paper chromatography of an a c i d h y d r o l y z a t e of the  polysaccharide  showed g a l a c t o s e , g l u c o s e , g l u c u r o n i c a c i d , rhamnose and an a l d o b i o uronic acid.  Determination  of the n e u t r a l sugars as the  alditol  135  a c e t a t e s gave rhamnose, g a l a c t o s e , and g l u c o s e 0.7:1.0:1.5.  The c a r b o x y l - r e d u c e d  i n the r a t i o s o f  p o l y s a c c h a r i d e gave rhamnose,  g a l a c t o s e , and g l u c o s e i n the r a t i o s o f 0.9:1.0:1.9, i n d i c a t i n g t h a t t h e uronic acid i s glucuronic acid.  The g l u c o s e was proved t o be of the D  and rhamnose t o be of t h e L c o n f i g u r a t i o n by c i r c u l a r measurements  78  made on t h e a l d i t o l a c e t a t e s .  dichroism  G a l a c t o s e was a s s i g n e d the  D c o n f i g u r a t i o n by the p o s i t i v e a c t i o n o f D - g a l a c t o s e o x i d a s e h y d r o l y s i s product  1 9 2  on t h e  of the polysaccharide.  N.m.r. spectroscopy The H-n.m.r. spectrum o f the E. c o l i K32 p o l y s a c c h a r i d e ( s e e 1  Appendix I I I , spectrum No. 2 0 ) i n d i c a t e d the r e p e a t i n g u n i t t o be a t e t r a s a c c h a r i d e and t o c o n t a i n 0.5 0 - a c e t y l groups.  The spectrum  e x h i b i t s a s i g n a l a t 6 = 1.34 which a r i s e s from the CH L-rhamnose. groups.  3  group o f  The s i g n a l a t 6 = 2.18 I s due t o the presence of 0C0CH  3  I n the anomeric r e g i o n s i x p r i n c i p a l s i g n a l s can be d e t e c t e d .  The s i g n a l a t 6 = 4.73 r e p r e s e n t s f u r t h e r confirmed polysaccharide  (3-linked g l u c u r o n i c a c i d which was  by chromic a c i d o x i d a t i o n o f the f u l l y a c e t y l a t e d  (see l a t e r ) .  The s i g n a l a t 6 = 5.52 belongs t o t h e  a-rhamnosyl r e s i d u e b e a r i n g an 0 - a c e t y l group, and i t s h i f t s t o 6 = 5.25 on the d e a c e t y l a t i o n o f the p o l y s a c c h a r i d e .  T h i s suggests t h a t t h e  0 - a c e t y l group i s l o c a t e d on ()-2 o f the a-rhamnosyl r e s i d u e . at 6 = 5.11 belongs p r o b a b l y O-deacetylation  5.20.  t o a-Glc and i t remains unchanged a f t e r  o f the p o l y s a c c h a r i d e .  and 6 = 5.16 d i s a p p e a r  The s i g n a l  The twinned s i g n a l s a t 6 = 5.19  on d e a c e t y l a t i o n g i v i n g r i s e t o a s i n g l e t a t 6 =  However, the d e f i n i t i v e assignment o f a-Glc and a-Gal cannot be  136  made on t h e b a s i s o f t h e ^-n.m.r. s p e c t r a l d a t a (see Table V . 6 ) . I n t h e C-n.m.r. s p e c t r a of t h e n a t i v e and O - d e a c e t y l a t e d E. 13  c o l i K32 p o l y s a c c h a r i d e s  (see Appendix I I I , S p e c t r a No. 21 and 23) t h e  s i g n a l a r i s i n g from CH^CO (21.18 p.p.m.) was absent i n t h e spectrum o f the d e a c e t y l a t e d p o l y s a c c h a r i d e . were changed. downfield s h i f t polysaccharide.  The p o s i t i o n s of t h e anomeric s i g n a l s  Carbon 1 o f t h e a-rhamnosyl r e s i d u e e x h i b i t e d a d e f i n i t e due t o t h e presence o f t h e a c e t a t e i n t h e n a t i v e However, t h e unambiguous assignment o f t h e anomeric  carbons c o u l d n o t be made (see T a b l e V.6).  '  Methylation 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 t h e E. c o l i K32 p o l y s a c c h a r i d e , by h y d r o l y s i s , d e r i v a t i z a t i o n as a l d i t o l  followed  a c e t a t e s , and g.l.c.-m.s.  a n a l y s i s , gave t h e v a l u e s shown i n T a b l e V.7, column I . These r e s u l t s i n d i c a t e t h a t t h e p o l y s a c c h a r i d e c o n s i s t s of a t e t r a s a c c h a r i d e r e p e a t ing  u n i t h a v i n g a branch on rhamnose w i t h g l u c u r o n i c a c i d as t h e  t e r m i n a l group.  By r e d u c t i o n of t h e m e t h y l a t e d p o l y s a c c h a r i d e ( s e e  column I I ) , the p r o p o r t i o n o f 2-0-methylrhamnose was i n c r e a s e d , and 2,3,4-tri-0_-methylglucose  was formed, i n d i c a t i n g t h a t g l u c u r o n i c a c i d i s  t e r m i n a l , and t h a t i t i s l i n k e d t o rhamnose. carbodiimide-reduced p o l y s a c c h a r i d e tetra-O-methylglucose,  7 5  Methylation a n a l y s i s of  showed the presence o f 2,3,4,6-  d e r i v e d from r e d u c t i o n of the c a r b o x y l i c group o f  the g l u c u r o n i c a c i d (see Table V.7, column I I I ) .  137  TABLE V.6 N.M.R. DATA FOR Escherichia c o l i K32 NATIVE AND POLYSACCHARIDES Compound  native  O-DEACETYLATED  ^-N.m.r.. data A* Integral (H)  Assignment  C-N.m.r,. data . P.p.m.* Assignment  5.52  a-Rha  173.85  COOH of B-GlcA  102.98 }  8-GlcA a-Rha a-Gal  1.0  13  1  (with 0-Ac) 5.25  0.8  5.19 1  a-Rha  c  1.8  a-Gal  1.9  a-Glc  1.1  unknown o r i g i n  5.16 5.11 5.05 5.02 4.73 ( J  , I  C  95.97  a-Glc  21.18 17.99  CH CH  3 3  C  of 0-Ac of a-Rha  1.8  B-GlcA  2.18  3.0  CH  3  of 0-Ac  1.34  6.1  CH  3  of a-Rha  5.52  0.2  a-Rha (with 0-Ac)  102.97  B-GlcA  5.25  0.8  a-Rha  101.68 }  a-Rha^ a-Gal  5.20  0.9  a-Gal  C  96.66  a-Glc  5.11  1.0  a-Glc  C  18.01  CH  =8Hz)  1  2  9  3-deacetylated  4.73 (J, =8Hz) 0.9 2ll8 0.5 1.34 3.2 2  C  3  C  of a-Rha  B-GlcA CH CH  3  3  of 0-Ac of a-Rha  Chemical s h i f t r e l a t i v e to internal acetone; 6 2.23 for ^-H-n.m.r. and 31.07 p.p.m. for C-n.m.r. downfield from sodium 4,4-dimethyl-4-silapentane-l-sulfonate (D.S.S.). The assignment was made by comparison with l i t e r a t u r e values, see r e f . 166. A d e f i n i t e assignment could not be made; the assignments of a-Glc and a-Gal are tentative. 3  13  138  TABLE V.7  METHYLATION ANALYSIS OF ESCHERICHIA COLI K32 POLYSACCHARIDE AND DERIVED PRODUCTS  T  Methylated sugar (as a l d i t o l acetate)  Mol %  a  Column B  I  C  b  II  d  III  IV  (ECNSS-M)  2,3-Rha  0.99  e  ,  42.7  ~.  2,3,4,6-Glc  1.00  -  -  26.5  -  2,3,4,6-Gal  1.24  -  -  -  17.5  2-Rha  1.55  32.5  16.4  26.5  10.3  2,4,6-Glc  1.96  53.6  38.7  23.5  29.3  2,4,6-Gal  2.26  13.6  25.7  23.5  -  2,3,4-Glc  2.49  19.2  R e l a t i v e r e t e n t i o n t i m e , r e f e r r e d to 2,3,4,6-Glc as 1.00. c o r r e c t e d by use o f t h e e f f e c t i v e , carbon-response Albersheim e t a l .  1  0  3  C  I s o t h e r m a l ; 170°.  I I , r e d u c t i o n of u r o n i c e s t e r ; IV,  product  I,  e  Values  f a c t o r s g i v e n by  o r i g i n a l polysaccharide;  I I I , carbodiimide-reduced  from Smith d e g r a d a t i o n .  di-O-methylrhamnitol, e t c .  d  b  polysaccharide;  2,3-Rha = 1 , 4 , 5 - t r i - 0 - a c e t y l - 2 , 3 -  139  Chromium t r i o x l d e o x i d a t i o n  1 7 a  The anomeric nature of the glucuronic acid was confirmed by chromium trioxide oxidation of the f u l l y acetylated polysaccharide, followed by sugar analysis.  8-Linked residues should be oxidized under  these conditions, but the corresponding a-linked residues should be resistant.  Determination, as a l d i t o l acetates, of the sugars obtained  from the chromium trioxide oxidation gave rhamnose, galactose, and glucose i n the molar r a t i o s of 0.9:1.0:0.5 indicating that D-glucuronic acid i s B-linked. The following two structures are consistent with the results obtained so f a r .  +3)-o-Glc-(l+3  or 4)-a-Rha-(l->-3)-a-Gal-(l+ 4 or 3 + 1 + 0-Ac B-GlcA  +3)- a-Gal-(1+3 or 4)-a-Rha-(l+3)-a-Glc-(1+ 4 or 3 + 1 + 0-Ac B-GlcA  B  140  Periodate oxidation  P e r i o d a t e o x i d a t i o n of the o r i g i n a l E. c o l i K32  polysaccharide  f o l l o w e d by h y d r o l y s i s and g . l . c . a n a l y s i s of the a l d i t o l a c e t a t e s rhamnose, g a l a c t o s e and g l u c o s e  i n the molar r a t i o s of  gave  1.0:0.5:1.0,  i n d i c a t i n g t h a t p a r t i a l h y d r o l y s i s of the rhamnosyl bond w i t h f u r t h e r degradation  of g a l a c t o s e had o c c u r r e d .  on the c a r b o d i i m i d e - r e d u c e d  polysaccharide  sodium a c e t a t e b u f f e r pH 4.5. the o x i d i z e d and b o r o h y d r i d e and glucose  The o x i d a t i o n was then 7 5  i n the presence of  repeated 0.1M  G . l . c . a n a l y s i s , as a l d i t o l a c e t a t e s , o f reduced product gave rhamnose, g a l a c t o s e ,  i n the r a t i o s of 0.9:1.0:1.0.  These r e s u l t s a r e c o n s i s t e n t  w i t h the concept t h a t o n l y one t e r m i n a l r e s i d u e  (glucuronic acid) i s  oxidized.  Smith degradation P e r i o d a t e o x i d a t i o n of the o r i g i n a l p o l y s a c c h a r i d e , methylation, mixture  Smith h y d r o l y s i s , r e m e t h y l a t i o n ,  f o l l o w e d by  and h y d r o l y s i s , gave a  t h a t was found, by g . l . c . a n a l y s i s of the a l d i t o l a c e t a t e s , t o  c o n t a i n d e r i v a t i v e s o f 2,3-di-0-methylrhamnose, 2,3,4,6-tetra-O-methylg a l a c t o s e , 2-0-methylrhamnose, and 2 , 4 , 6 - t r i - 0 - m e t h y l g l u c o s e V.7, column I V ) . These r e s u l t s i n d i c a t e t h a t the g l u c o s e c h a i n i s 4 - l i n k e d t o rhamnose, the b r a n c h p o i n t . was h y d r o l y z e d  (see T a b l e  i n t h e main  The rhamnosyl l i n k a g e  d u r i n g the Smith h y d r o l y s i s g i v i n g r i s e t o  2,3,4,6-tetra-  C H m e t h y l g a l a c t o s e , p r o v i n g t h a t the rhamnose i n the main c h a i n i s l i n k e d t o the g a l a c t o s e and not the g l u c o s e . 2,4,6-tri-O-methylgalactose  The u n u s u a l i n s t a b i i t y of the  was n o t i c e d throughout the s t r u c t u r a l  141  i n v e s t i g a t i o n of the E. c o l i K32 methylation analyses  p o l y s a c c h a r i d e , e s p e c i a l l y i n the  of the o r i g i n a l , a c i d i c  L o c a t i o n of the  fl-acetyl  polysaccharide.  group  S i n c e the complete b l o c k i n g of the p o l y s a c c h a r i d e w i t h m e t h y l v i n y l e t h e r proved d i f f i c u l t , the 0 - a c e t y l l o c a t i o n procedure of B e l d e r and N o r r m a n a f t e r bacteriophage saccharide  1 8 0  was  performed on the o l i g o s a c c h a r i d e , o b t a i n e d  depolymerization  (see S e c t i o n V I ) .  of the E. c o l i K32  capsular poly-  E t h y l a t i o n of the p r o t e c t e d o l i g o s a c c h a -  r i d e , f o l l o w e d by h y d r o l y s i s , c o n v e r s i o n t o the a l d i t o l  acetates,  g.l.c.-m.s. a n a l y s i s of the m i x t u r e of p a r t i a l l y e t h y l a t e d  V.2.4  t h a t the O - a c e t y l group i s a t t a c h e d to 0-2  and  alditol  a c e t a t e s gave a r a t i o of 2-0-ethylrhamnose to rhamnose of 4.5:1 ing  de  indicat-  of rhamnose.  CONCLUSION  The  sum  of these experiments demonstrates t h a t 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 E s c h e r i c h i a c o l i 09:K32(A):H19 i s based on the 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 shown w i t h h a l f of the L-rhamnosyl  142  OAc  I  2 +3 ) - cc-D-Glc-( 1+4)-o-L-Rha-( 1+3)-cc-D-Gal-( 1+ 3 • 1 B-D-GlcA  E. c o l l  r e s i d u e s b e i n g O - a c e t y l a t e d a t 0-2. Klebsiella K82;  1 9 t f  K32  The s t r u c t u r e resembles t h a t o f  i t has the same s t r u c t u r a l p a t t e r n ( " t h r e e - p l u s - o n e "  t y p e ) and a l a t e r a l p - D - g l u c o s y l u r o n i c a c i d group as a t e r m i n a l u n i t . I n common w i t h K l e b s i e l l a K 5 5 , the  1 9 5  a-L-rhamnose i s a branch p o i n t w i t h  C H a c e t y l group p r e s e n t on p o s i t i o n 0-2 of L-rhamnose.  The s t r u c t u -  r a l p a t t e r n s of the two p o l y s a c c h a r i d e s a r e , however, d i f f e r e n t .  OAc  I +3)-8-Glc-( 1 +3)-a-Gal-( 1 +3)- 8-Gal-( 1 * 4 t 1 8-GlcA  Klebsiella  K82  2 ->3)-8-Glc-( l->4)-<r-Rha-( l-> 3 + 1 a-Gal 3 t 1 oc-GlcA Klebsiella  K55  143  V.2.5  EXPERIMENTAL  General methods The i n s t r u m e n t a t i o n used f o r n.m.r., g . l . c , i n f r a r e d , c.d., and measurements of o p t i c a l r o t a t i o n has been d e s c r i b e d i n S e c t i o n I I I . G.l.c.-m.s.  was performed w i t h the NERMAG R10-10 i n s t r u m e n t .  The  c a p i l l a r y columns used were : (F) DB-225, programmed from 195° f o r 8 min, and t h e n 4°/min t o 220°, (G) SE-30, programmed from 70° f o r 1 min, and then 10°/min t o 250°.  Paper chromatography, g a s - l i q u i d  chromato-  graphy, and ion-exchange chromatography were performed as d e s c r i b e d i n Section I I I .  Preparation  and properties of E. c o l i K32  polysaccharide  A c u l t u r e of E s c h e r i c h i a c o l i K32, o b t a i n e d from Dr. I . 0rskov, (Copenhagen), was grown on M u e l l e r H i n t o n agar as d e s c r i b e d i n S e c t i o n III.7.2. ~130  Yield:  a c i d i c p o l y s a c c h a r i d e ~660 mg, n e u t r a l p o l y s a c c h a r i d e  mg. The i s o l a t e d a c i d i c p o l y s a c c h a r i d e had  [or.]  25  +80.9 (c 0.147,  w a t e r ) , and a n a l y s i s by g e l - p e r m e a t i o n chromatography S.C.  ( c o u r t e s y of Dr.  Churms, Cape Town, South A f r i c a ) showed i t t o be homogeneous, w i t h  an average m o l e c u l a r weight of 9 x 1 0 (*H and  1 3  C)  6  daltons.  N.m.r.  spectroscopy  was performed on the o r i g i n a l and the d e a c e t y l a t e d  polysaccharide.  The p r i n c i p a l s i g n a l s i n the *H- and  13  C-n.m.r.  s p e c t r a and t h e i r assignments a r e r e c o r d e d i n T a b l e V.6.  K32  144  Deacetylation of polysaccharide 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 0.01M  o v e r n i g h t at room t e m p e r a t u r e . water and  freeze-dried.  The  The  product  was  d e a c e t y l a t i o n was  NaOH and  stirred  dialyzed against  tap  almost complete (as  judged from ^-H-n.m.r. spectrum) and 90% of the 0 - a c e t y l groups were removed.  A n a l y s i s of the d e a c e t y l a t e d p o l y s a c c h a r i d e by  chromatography showed i t t o be homogeneous w i t h M  Hydrolysis of the  = 6 x 10  6  daltons.  polysaccharide  H y d r o l y s i s of a sample (3 mg) 2M t r i f l u o r o a c e t i c a c i d (TFA) successive evaporations  w  gel-permeatlon  of E. c o l i K32  f o r 18 h at 95°,  polysaccharide  with  removal of the a c i d by  w i t h w a t e r , f o l l o w e d by paper chromatography  ( s o l v e n t s A and B ) , showed g a l a c t o s e , g l u c o s e , g l u c u r o n i c a c i d , and rhamnose.  N e u t r a l sugars were q u a n t i t a t i v e l y determined by g . l . c . as  t h e i r a l d i t o l acetates. sample (6 mg)  of K32  The  ing  overnight.  IR-120 ( H ) +  3  neutralized with  w i t h methanol.  The  sample was  t r i f l u o r o a c e t i c a c i d f o r 18 h at 95°  overthe  and  removed by  then h y d r o l y z e d w i t h  and the a l d i t o l a c e t a t e s were  and i d e n t i f i e d by g . l . c . (column A ) .  Preparative g.l.c.  (column D), f o l l o w e d by measurements of the c i r c u l a r  stirr-  Amberlite  r e s i n , and the b o r i c a c i d , as m e t h y l b o r a t e , was  co-evaporation  prepared  2  i n anhydrous methanol (4 mL)  excess of NaBH^ was  a  i n methanol (4 mL)  w i t h P b C 0 , removing P b C l , t r e a t i n g  w i t h NaBH^ (50 mg) The  reduced by r e f l u x i n g  p o l y s a c c h a r i d e w i t h 3% HC1  n i g h t , n e u t r a l i z i n g the HC1 d r i e d product  u r o n i c a c i d was  dichroism  2M  145  spectra,  showed the g l u c i t o l h e x a a c e t a t e  and the r h a m n i t o l p e n t a a c e t a t e  t o be of the D c o n f i g u r a t i o n ,  t o be of the L c o n f i g u r a t i o n .  Methylation a n a l y s i s The  p o l y s a c c h a r i d e (29.8 mg),  converted  i n t o the f r e e a c i d form  by p a s s i n g the sodium s a l t through a column of A m b e r l i t e IR-120 r e s i n , was  d i s s o l v e d i n dry d i m e t h y l s u l f o x i d e (4 mL)  by treatment  w i t h 3 mL  d i m e t h y l s u l f i n y l a n i o n f o r 4 h,  methyl i o d i d e f o r 1 h. tap w a t e r , was i.r.  spectrum).  The  not c o m p l e t e l y m e t h y l a t e d I t was  and then 6 mL  of  against  d i s s o l v e d i n c h l o r o f o r m and s u b j e c t e d to P u r d i e  y i e l d e d a f u l l y methylated was  8 8  ( h y d r o x y l a b s o r p t i o n i n the  This  81  (5 mg)  +  methylated  product, recovered a f t e r d i a l y s i s  m e t h y l a t i o n * w i t h m e t h y l i o d i d e and s i l v e r o x i d e .  product  and  (H )  p o l y s a c c h a r i d e (15.2 mg).  treatment  A p o r t i o n of  h y d r o l y z e d w i t h 2M t r i f l u o r o a c e t i c  acid.  The  this  sugars  were reduced w i t h sodium b o r o h y d r i d e , and the a l d i t o l s were a c e t y l a t e d w i t h 1:1  a c e t i c a n h y d r i d e - p y r i d i n e , and a n a l y z e d by g . l . c . i n ,  column B (see T a b l e V.7,  column I ) .  l a t e d p o l y s a c c h a r i d e (10.2 mg)  C a r b o x y l r e d u c t i o n of f u l l y methy-  w i t h L i A l H ^ i n anhydrous oxolane  at room temperature o v e r n i g h t , h y d r o l y s i s of the product w i t h  (5  mL)  2M  t r i f l u o r o a c e t i c a c i d , f o l l o w e d by r e d u c t i o n of sugars w i t h sodium b o r o h y d r i d e , and a c e t y l a t i o n of the a l d i t o l s w i t h 1:1 a c e t i c p y r i d i n e gave a m i x t u r e of p a r t i a l l y m e t h y l a t e d was  a n a l y z e d by g . l . c .  a l d i t o l a c e t a t e s which  (column B) and g.l.c.-m.s.  G . l . c . a n a l y s i s d a t a f o r the p a r t i a l l y m e t h y l a t e d shown i n Table V.7,  anhydride-  (column F and  G).  a l d i t o l acetates  are  column I I . The n e u t r a l p o l y s a c c h a r i d e o b t a i n e d  carbodiimide r e d u c t i o n  7 5  was  a l s o s u b j e c t e d to m e t h y l a t i o n a n a l y s i s .  by A  146  sample (8.1 mg)  of c a r b o d i i m i d e - r e d u c e d  d r y d i m e t h y l s u l f o x i d e (1.5 mL)  p o l y s a c c h a r i d e was  and m e t h y l a t e d  dissolved i n  by the treatment  with  1.5  mL d i m e t h y l s u l f i n y l a n i o n f o r 4 h, and then 3 mL methyl i o d i d e f o r 1 h. The  p r o d u c t , r e c o v e r e d a f t e r d i a l y s i s a g a i n s t tap w a t e r , was  w i t h 2M t r i f l u o r o a c e t i c a c i d o v e r n i g h t a t 95°, the a l d i t o l s were a c e t y l a t e d w i t h 1:1 Subsequent a n a l y s i s by g . l . c . gave the r e s u l t s p r e s e n t e d  hydrolyzed  reduced w i t h NaBH^ and  acetic anhydride-pyridine.  (column B) and g.l.c.-m.s. (column F)  i n Table V.7,  column I I I .  Carbodiimide reduction of capsular p o l y s a c c h a r i d e  75  A sample of E. c o l i K32 p o l y s a c c h a r i d e (Na"!" s a l t , 50.15 d i s s o l v e d i n 30 mL water ( i n i t i a l pH 5.4).  As the r e a c t i o n proceeded, the pH was  t i t r a t i o n w i t h 0.1N  HC1.  The  r e a c t i o n was  The  The  pH was  r e d u c t i o n was  maintained  between 5-7  completed i n one h.  (CMC,  g)  was  maintained  0.5  a t 4.75  by  a l l o w e d to proceed f o r 2 h.  An aqueous s o l u t i o n of 2M sodium b o r o h y d r i d e slowly.  was  l-Cyclohexyl-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_-toluenesulfonate added.  mg)  (1.3 g/15  mL H 0) 2  was  by t i t r a t i o n w i t h 4M  The m i x t u r e was  added  HC1.  dialyzed against  tap water f o r two days, c o n c e n t r a t e d and f r e z e - d r i e d . Then a second treatment was  was  c a r r i e d out s i m i l a r l y .  A t o t a l of 47.2 mg  product  recovered a f t e r f r e e z e - d r y i n g . A sample of the c a r b o d i i m i d e - r e d u c e d  was  of the  7 5  p o l y s a c c h a r i d e (2.4  h y d r o l y z e d o v e r n i g h t w i t h 2M t r i f l u o r o a c e t i c a c i d (TFA)  b a t h and the h y d r o l y z a t e of sugars was  converted  mg)  on a steam  into a l d i t o l acetates,  g . l . c . a n a l y s i s of w h i c h (column A) showed r h a m n i t o l  pentaacetate,  147  g a l a c t i t o l h e x a a c e t a t e and g l u c i t o l h e x a a c e t a t e i n r a t i o s o f 0.7:1.0:1.70, i n d i c a t i n g 70% r e d u c t i o n .  Chromium t r i o x i d e o x i d a t i o n  1 7 5  A sample (16.1 mg) o f 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  formamide (5 mL), and t r e a t e d w i t h a c e t i c a n h y d r i d e (1 mL) and p y r i d i n e (1 mL) o v e r n i g h t  a t room t e m p e r a t u r e .  The a c e t y l a t e d m a t e r i a l (22.9 mg)  was r e c o v e r e d by d i a l y s i s and f r e e z e - d r y i n g , and d i s s o l v e d i n a c e t i c a c i d (1 mL).  The a c e t i c a c i d s o l u t i o n was t r e a t e d w i t h  t r i o x i d e (50 mg) a t 50° f o r 1 h. t i o n between c h l o r o f o r m trifluoroacetic analyzed  The m a t e r i a l was r e c o v e r e d by p a r t i -  and w a t e r .  acid overnight,  chromium  The product was h y d r o l y z e d  w i t h 2M  c o n v e r t e d i n t o a l d i t o l a c e t a t e s and  by g . l . c . i n column A.  Periodate oxidation A s o l u t i o n o f K32 p o l y s a c c h a r i d e  (42.5 mg) i n water (10 mL) was  mixed w i t h 0.03M NalO^ (10 mL) and s t i r r e d i n t h e dark a t room temperat u r e (23°).  The r e a c t i o n was c a r r i e d out f o r 6 d.  g l y c o l (2 mL) was added t o t e r m i n a t e  After  ethylene  the r e a c t i o n , t h e p o l y a l d e h y d e was  reduced t o t h e p o l y a l c o h o l w i t h NaBH^, t h e base was n e u t r a l i z e d w i t h 50% a c e t i c a c i d , and t h e s o l u t i o n was d i a l y z e d o v e r n i g h t y i e l d e d the p o l y a l c o h o l (28.35 mg). w i t h 2M TFA o v e r n i g h t  and f r e e z e - d r i e d t o  A p o r t i o n (3.4 mg) was  a t 95° and c o n v e r t e d i n t o a l d i t o l  hydrolyzed  acetates.  A n a l y s i s by g . l . c . i n column A showed r h a m n i t o l , g a l a c t i t o l and g l u c i t o l i n the r a t i o s o f 1.0:0.5:1.0, i n d i c a t i n g t h a t p a r t i a l d e g r a d a t i o n g a l a c t o s e had o c c u r r e d .  Periodate  of the  o x i d a t i o n was then r e p e a t e d on t h e  148  carbodiimide-reduced  polysaccharide'  and low temperature (4°)  53  u s i n g 0.1M  sodium a c e t a t e b u f f e r  i n o r d e r to a v o i d p a r t i a l h y d r o l y s i s of the  rhamnosyl bond and o v e r - o x i d a t i o n of the g a l a c t o s e . A sample of the c a r b o d i i m i d e - r e d u c e d d i s s o l v e d i n 0.1M NalO^ (6 mL)  p o l y s a c c h a r i d e (15.7 mg)  sodium a c e t a t e b u f f e r (pH 4.5), mixed w i t h 0.015M  and s t i r r e d i n the dark at 4°.  A l i q u o t s (0.1 mL)  withdrawn p e r i o d i c a l l y , and d i l u t e d 250 times w i t h w a t e r . absorbances  1 8 5  Perkin-Elmer reached  was  were  The  a t 223 nm of the r e s u l t i n g s o l u t i o n s were measured i n a  552A UV/VIS spectrophotometer.  a p l a t e a u a f t e r ~10  days.  The  p e r i o d a t e consumption  E t h y l e n e g l y c o l (2 mL)  decompose the excess of p e r i o d a t e , the p o l y a l d e h y d e sodium b o r o h y d r i d e , the s o l u t i o n was  the base was  was  was  added t o  reduced w i t h  n e u t r a l i z e d w i t h 50% a c e t i c a c i d ,  and  d i a l y z e d and l y o p h i l i z e d to y i e l d the p o l y a l c o h o l .  p o r t i o n of t h i s p o l y a l c o h o l (7.2 mg) t r i f l u o r o a c e t i c a c i d o v e r n i g h t at 95° into a l d i t o l acetates.  was  hydrolyzed with  and  A  2M  converted  A n a l y s i s by g . l . c . i n column A showed r h a m n i t o l ,  g a l a c t o s e , and g l u c o s e i n the r a t i o s of 0.9:1.0:1.0.  Smith degradation A sample of the p o l y a l c o h o l (6.1 mg) o x i d a t i o n of the K32 f o x i d e (3 mL)  p o l y s a c c h a r i d e was  and m e t h y l a t e d  obtained a f t e r periodate  d i s s o l v e d i n dry d i m e t h y l  by treatment  sul-  w i t h 2 mL of d i m e t h y l s u l f i n y l  a n i o n f o r 4 h, and then 4 mL of m e t h y l i o d i d e f o r 1 h.  The  product  was  r e c o v e r e d by p a r t i t i o n between water and c h l o r o f o r m , and i t s i . r . spectrum showed complete m e t h y l a t i o n (no h y d r o x y l a b s o r p t i o n i n the spectrum).  I t was  then s u b j e c t e d to the Smith h y d r o l y s i s by  i.r.  treatment  149  w i t h 50% a c e t i c a c i d f o r 90 min at 95°. e v a p o r a t i o n w i t h water and  A c e t i c a c i d was  the dry r e s i d u e was  t i o n by the Hakomori p r o c e d u r e .  8 8  s u b j e c t e d to  The methylated  and the  hydrolyzed partially  sugars were c o n v e r t e d i n t o a l d i t o l a c e t a t e s and a n a l y z e d  g . l . c . i n column B (see Table V.7,  co-  remethyla-  product was  w i t h 2M t r i f l u o r o a c e t i c a c i d (TFA) o v e r n i g h t at 95°, methylated  removed by  by  column IV) and g.l.c.-m.s. I n column  E.  Location of the Q-acetyl g r o u p A sample (5 mg)  1 8 0  of the o l i g o s a c c h a r i d e o b t a i n e d a f t e r b a c t e r i o -  phage d e g r a d a t i o n of the E. c o l i K32 p o l y s a c c h a r i d e ( f r a c t i o n I I ) was d r i e d t o g e t h e r w i t h a t r a c e of j v - t o l u e n e s u l f o n i c a c i d and then d i s s o l v e d i n dry d i m e t h y l s u l f o x i d e (4 mL).  M e t h y l v i n y l e t h e r (3 mL) was  to a f r o z e n s o l u t i o n , and the r e a c t i o n m i x t u r e was a l l o w e d to s t i r f o r 4 h. (3 mL)  was  obtained.  brought to 23°  The c l e a r red s o l u t i o n  was  p l a c e d on a Sephadex LH-20 column (16 cm x 2 cm)  and  e l u t e d w i t h acetone ( w i t h s l i g h t s u c t i o n ) . and the r e s i d u e was  The product was  s u b j e c t e d to e t h y l a t i o n .  d i m e t h y l s u l f o x i d e (4 mL)  I t was  concentrated  d i s s o l v e d i n dry  and t r e a t e d w i t h 2 mL of d i m e t h y l s u l f i n y l  a n i o n f o r 4 h, and then w i t h 3 mL of e t h y l i o d i d e f o r 1 h. The product was  and  Then a second p o r t i o n of methyl v i n y l e t h e r  i n t r o d u c e d i n a s i m i l a r manner. I t was  added  dark-red  e x t r a c t e d w i t h c h l o r o f o r m and p u r i f i e d on a Sephadex LH-20  column (14.5 cm x 2 cm) d a r k - r e d o i l , was  by e l u t i o n w i t h methanol.  The p r o d u c t , a  h y d r o l y z e d w i t h 2M t r i f l u o r o a c e t i c a c i d o v e r n i g h t a t  95° and c o n v e r t e d i n t o a l d i t o l a c e t a t e s . column A, programmed from 195°  G . l . c . a n a l y s i s , conducted  f o r 8 min, and then at 4°/min to 260°,  in  150  showed the presence of 2-0-ethylrhamnose ( 4 3 % ) , rhamnose ( 9 . 6 % ) , g a l a c t o s e ( 1 6 . 4 % ) , and g l u c o s e ( 3 0 . 8 % ) . g.l.c.-m.s.  (column F ) .  These r e s u l t s were confirmed by  CHAPTER VI  BACTERIOPHAGE DEGRADATION OF Escherichia c o l l CAPSULAR POLYSACCHARIDES K28 and K32  152  VI.  BACTERIOPHAGE DEGRADATION OF E s c h e r i c h i a c o l i CAPSULAR POLYSACCHARIDES K28 and K32.  VI.1  INTRODUCTION  L i k e many o t h e r organisms, b a c t e r i a a r e s u b j e c t t o i n f e c t i o n by a range of v i r u s e s o r v i r u s - l i k e p a r t i c l e s which f a l l n a t u r a l l y i n t o two p h y s i o l o g i c a l l y s e p a r a t e groups, b a c t e r i o p h a g e s and b a c t e r i o c i n s . B a c t e r i o p h a g e s (<)>) a r e t r u e v i r u s e s , i n f e c t i n g t h e i r h o s t s and m u l t i p l y i n g w i t h i n them.  The members of t h e second group d i f f e r i n t h a t they do  not m u l t i p l y i n the c e l l a f t e r i n f e c t i n g i t ,  but o n l y k i l l  it.  B a c t e r i o c i n s may be d e f i n e d as a n a t u r a l c l a s s of h i g h l y s p e c i f i c  anti-  biotics. The f i r s t account o f t h e b a c t e r i o p h a g e was p u b l i s h e d by Twort i n 1915.  He demonstrated  t h a t t h e c u l t u r e s of b a c t e r i a l c e l l s c o u l d be  i n f e c t e d w i t h and d e s t r o y e d by f i l t e r a b l e agents t h a t were s u b s e q u e n t l y termed b a c t e r i o p h a g e s . Two y e a r s l a t e r , d ' H e r e l l e i n d e p e n d e n t l y i s o l a t e d a d y s e n t e r y b a c t e r i o p h a g e , c h a r a c t e r i z i n g i t as an u l t r a m i c r o s c o p i c p a r a s i t e of b a c t e r i a , and g i v i n g i t the name " b a c t e r i o p h a g e " , which means " b a c t e r i a - e a t e r " . A l t h o u g h b a c t e r i o p h a g e s (phages) were t h e l a s t major group o f v i r u s e s t o be r e c o g n i z e d , today they a r e t h e best c h a r a c t e r i z e d and studied.  The reasons f o r t h a t l i e i n the f a c t t h a t p r o p a g a t i o n and  m a n i p u l a t i o n of phages has proven t e c h n i c a l l y much e a s i e r than e q u i v a l e n t s t u d i e s on t h e o t h e r types of v i r u s e s .  153  Many d i f f e r e n t s t r a i n s o f phages have been i s o l a t e d and characterized  s i n c e they were f i r s t demonstrated  i n 1915. P r o b a b l y  each  b a c t e r i a l s t r a i n i s s u s c e p t i b l e t o s e v e r a l d i f f e r e n t types of phage. Phages a r e r e l a t i v e l y easy t o i s o l a t e from almost any b a c t e r i a l e n v i r o n ment i n which a number of c l o s e l y r e l a t e d b a c t e r i a l s t r a i n s c o e x i s t ( e . g . , t h e g a s t r o i n t e s t i n a l t r a c t , sewage).  I f a sample of sewage  f i l t r a t e i s mixed w i t h a growing c u l t u r e of an e n t e r i c b a c t e r i u m and spread on a p l a t e , t h e e n s u i n g c u l t u r e w i l l show growth of the organism on the p l a t e i n t e r r u p t e d by s m a l l zones of c l e a r i n g , which a r e termed plaques.  Each plaque r e p r e s e n t s the p r o p a g a t i o n of a s i n g l e phage  p a r t i c l e i n the growing lawn of b a c t e r i a l c e l l s and i s analogous  t o an  i s o l a t e d b a c t e r i a l colony. Morphologically  phages a r e q u i t e d i s t i n c t from o t h e r v i r u s types  i n t h a t they tend t o be s t r u c t u r a l l y more complex.  B a c t e r i o p h a g e s have  been i n t e n s e l y s t u d i e d by many d i f f e r e n t t e c h n i q u e s , but one of the most s i g n i f i c a n t c o n t r i b u t i o n s t o our knowledge of these v i r u s e s has been made by e l e c t r o n m i c r o s c o p y .  The m o r p h o l o g i c a l c l a s s i f i c a t i o n of the  b a c t e r i o p h a g e s was i n t r o d u c e d by B r a d l e y .  1 9 6  The phage head ( s e e F i g . V I . 1 ) c o n t a i n s the v i r u s n u c l e i c a c i d . I n phage the n u c l e i c a c i d i s u s u a l l y i n the form of d o u b l e - s t r a n d e d DNA. The head has b a s i c a l l y an i c o s a h e d r a l repeating  structure.  I t i s composed o f  i d e n t i c a l p r o t e i n c h a i n s and w i l l v a r y i n s i z e a c c o r d i n g t o  the s t r a i n , a p p r o x i m a t e l y 50 nm i n d i a m e t e r . complex i n s t r u c t u r e than i s the head.  The phage t a i l  A l a y e r of h e l i c a l l y  p r o t e i n m o l e c u l e s forms the i n n e r tube of the t a i l .  i s more arranged  The tube i s encased  by the c o n t r a c t i l e s h e a t h , which extends from the c o l l a r t o the end  154  Head  F i g . VI.1:  Schematic diagram d e m o n s t r a t i n g bacteriophage.  2-DNA  F i g . VI.2t  2-DNA  2-DNA  the s t r u c t u r e o f T  1-DNA  1-KNA  1-DNA  Basic morphological types of bacteriophages w i t h the t y p e s o f n u c l e i c a c i d (from r e f . 1 9 6 ) .  155  plate.  The sheath p l a y s an important r o l e i n t h e i n f e c t i v e p r o c e s s by  f o r c i n g t h e phage n u c l e i c a c i d through t h e h o l l o w t a i l i n t o the host bacterium.  The p l a t e c o n t a i n s s m a l l p i n s t o which a r e a t t a c h e d s i x  v e r y l o n g , f i n e t a i l f i b e r s by which phage p a r t i c l e s a t t a c h to the c e l l w a l l s of s u s c e p t i b l e host  cells.  themselves  1 9 7  The b a c t e r i a l v i r u s e s e x h i b i t a g r e a t d i v e r s i t y of forms but i t i s p o s s i b l e t o d i v i d e them i n t o s i x b a s i c m o r p h o l o g i c a l types (see F i g . VI.2).  The f i r s t f o u r groups ( w i t h t a i l s ) a r e unique t o the b a c t e r i a l  viruses.  Groups E and F a r e d i f f e r e n t ; they resemble many p l a n t ,  a n i m a l , and i n s e c t  viruses.  1 9 6  When a phage p a r t i c l e i n f e c t s a s u s c e p t i b l e host i t causes c e l l to l y s e . following:  that  The phases of the l y t i c c y c l e (see F i g . VI.3) i n c l u d e t h e  1 9 7  (i)  a d s o r p t i o n of the phage p a r t i c l e s t o the s u s c e p t i b l e h o s t  (ii)  i n j e c t i o n of v i r a l DNA ( o r RNA) i n t o the host  (iii)  r e p l i c a t i o n of the phage n u c l e i c a c i d and s y n t h e s i s of the phage protein  (iv)  phage m a t u r a t i o n and r e l e a s e  A d s o r p t i o n i s v e r y host s p e c i f i c and depends on the presence i n the b a c t e r i a l c e l l w a l l of p r e c i s e r e c e p t o r s i t e s .  A d s o r p t i o n of a  phage t o i t s r e c e p t o r on the c e l l i s , i n most i n s t a n c e s , f o l l o w e d by p e n e t r a t i o n of the n u c l e i c a c i d through t h e c y t o p l a s m i c m e m b r a n e . Bacteriophages  a c t i v e on e x o p o l y s a c c h a r i d e - p r o d u c i n g  s t r a i n s are generally exopolysaccharide s p e c i f i c ,  1 9 9  198  bacterial  n o n - c a p s u l a t e or  156  Fig.  VI.3:  The  Mechanics of I n f e c t i o n by  Bacteriophage  A.  Free phage.  B.  Phage a t t a c h e s to c e l l w a l l w i t h f i b r e s , base p l a t e c l o s e c o n t a c t w i t h o u t e r l a y e r s of c e l l w a l l .  in  C.  Sheath c o n t r a c t s and c e n t r a l c o r e i s pushed through c e l l w a l l and DNA t r a n s f e r b e g i n s .  the  D.  T r a n s f e r of DNA completed. Phage head i s now e a r l y events of phage growth c y c l e b e g i n .  and  empty  (From T.J. Mackie and J.E. McCartney, " M e d i c a l M i c r o b i o l o g y " , V o l . 1, " M i c r o b i a l I n f e c t i o n s " , 13th C h u r c h i l l L i v i n g s t o n e , E d i n b u r g h , 1978).  edn.,  157  non-slime  p r o d u c i n g mutants are r e s i s t a n t t o the phages.  Morphological  e x a m i n a t i o n of e x o p o l y s a c c h a r i d e - s p e c i f i c phages has r e v e a l e d t h a t most of them belong t o group C, t h e i r b a s e - p l a t e s are p r o v i d e d w i t h the s p i k e s and no t a i l f i b r e s are  seen.  A t t a c k of b a c t e r i o p h a g e s  1 9 8  on e x o p o l y s a c c h a r i d e - p r o d u c i n g b a c t e r i a  i s o f t e n r e v e a l e d by o c c u r r e n c e of h a l o s around the c l e a r plaques i n the lawn of c a p s u l a t e d b a c t e r i a . decapsulated.  W i t h i n the h a l o the b a c t e r i a l lawn i s  1 9 8  These h a l o s a r e , a t l e a s t p a r t l y , the r e s u l t of d i f f u s i o n  of a phage-induced enzyme which h y d r o l y z e s the c a p s u l e w i t h o u t the b a c t e r i a .  As shown by Bayer e t a l . ,  v i s u a l i z e d under the e l e c t r o n microscope  2 0 0  such phages can  on t h e i r way  killing  be  from the o u t e r  s u r f a c e of the b a c t e r i a l c a p s u l e to the c e l l w a l l underneath (see F i g . VI.4). The b a c t e r i o p h a g e - a s s o c i a t e d enzymes may to  be c l a s s i f i e d  according  the type of r e a c t i o n they c a t a l y z e , and a c c o r d i n g t o the genus of the  respective b a c t e r i a l host.  Most v i r a l  few l y a s e s have a l s o been found.  p e n e t r a s e s are h y d r o l a s e s , but a  The h y d r o l a s e s are e i t h e r  glycanases  ( g l y c o s i d e h y d r o l a s e s ) or they are " d e a c e t y l a s e s " which c l e a v e o f f acetyl s u b s t i t u e n t s .  2 0 1  To d a t e , v i r u s - a s s o c i a t e d enzymes of  these  types have m a i n l y been s t u d i e d u s i n g phages f o r b a c t e r i a b e l o n g i n g t o the f a m i l y of E n t e r o b a c t e r i a c e a e , and w i t h i n the f a m i l y to the genera E s c h e r i c h i a c o l i , K l e b s i e l l a , S a l m o n e l l a , S h i g e l l a and In  an e x t e n s i v e s t u d y ,  2 0 2  Proteus.  55 d i f f e r e n t K l e b s i e l l a  bacteriophages  were t e s t e d f o r t h e i r enzymic a c t i o n on 74 d i f f e r e n t ( a c i d i c ) 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 ( s e r o t y p e s K1-K72, K74 may  be summarized as  follows.  2 0 2  and K80).  The  results  158  F i g . VI.4:  C a p s u l a t e d E. c o l i K29 exposed t o a m.o.i. ( t h e m u l t i p l i c i t y o f i n f e c t i o n ) of 300 phage f o r 8 min a t 37°. V i r u s p a r t i c l e s can be seen on the o u t e r membrane s u r f a c e . One v i r u s p a r t i c l e (upper l e f t c o r n e r ) has a p p a r e n t l y r e l e a s e d most of i t s DNA. (From r e f . 2 0 0 ) .  159  160  (i)  The K l e b s i e l l a v i r u s - a s s o c i a t e d g l y c a n a s e s were found t o be v e r y s p e c i f i c , 33 c r o s s - r e a c t i n g w i t h none, 18 w i t h one, two w i t h two, and one each w i t h 3 or 4 of the 73 h e t e r o l o g o u s  (ii)  I n most cases cleavage  polysaccharides;  o c c u r r e d on e i t h e r s i d e of the sugar u n i t  c a r r y i n g the n e g a t i v e charge, but r e d u c i n g g l u c u r o n i c a c i d s a r e not produced; (iii)  Most o f t e n , the r e d u c i n g end sugar formed i s s u b s t i t u t e d a t p o s i t i o n 3;  (iv)  I n the m a j o r i t y of c a s e s , 6 - g l y c o s i d i c l i n k a g e s a r e h y d r o l y z e d ;  (v)  I n most p o l y s a c c h a r i d e s which a r e a c t e d upon by s e v e r a l phage enzymes, the same g l y c o s i d i c bonds a r e s p l i t by the d i f f e r e n t agents.  The b a c t e r i o p h a g e - a s s o c i a t e d  glycanases  i s o l a t i o n of o l i g o s a c c h a r i d e fragments. components, b a c t e r i o p h a g e  a l l o w the p r e p a r a t i v e  W i t h some a c i d - l a b i l e  d e g r a d a t i o n may be the o n l y method f o r t h e  i s o l a t i o n of r e p e a t i n g u n i t o l i g o m e r s .  2 0 3  Bacteriophage  degradation  may  be used as a complement t o o t h e r methods f o r the p a r t i a l d e g r a d a t i o n of b a c t e r i a l polysaccharides. repeating u n i t oligomers  The l a r g e - s c a l e a c c e s s i b i l i t y of these  i s a l s o of advantage f o r a n a l y s e s of n u c l e a r  magnetic resonance s p e c t r o s c o p y .  When coupled  to s u i t a b l e p r o t e i n  c a r r i e r s , b a c t e r i a l s u r f a c e o l i g o s a c c h a r i d e s of two or more r e p e a t i n g u n i t s may serve as immunogens, r e p r e s e n t a t i v e of the c o r r e s p o n d i n g bacterial  glycans.  Although  2 0 1  a number of h i g h l y s p e c i f i c "K b a c t e r i o p h a g e s "  found f o r E s c h e r i c h i a c o l i c a p s u l a r s t r a i n s ,  1 9 3  have been  the enzymic a c t i o n of  161  these phages has been s t u d i e d t o a much l e s s e r e x t e n t .  The i n t e r a c t i o n  between the c a p s u l a t e d E s c h e r i c h i a c o l i s t r a i n of s e r o t y p e K29 and a c a p s u l e K 2 9 - s p e c i f i c b a c t e r i o p h a g e has been s t u d i e d , u s i n g v i r u s  adsorp-  t i o n k i n e t i c s and i m m u n o l o g i c a l methods i n c o m b i n a t i o n w i t h e l e c t r o n microscopy.  2 0 0  R e c e n t l y , the c a p s u l e - d e g r a d i n g enzymic a c t i v i t y of two  E. c o l i b a c t e r i o p h a g e s (<t>92 and <t>1.2) has been  tested.  2 0 4  '  2 0 5  The r e s u l t s of the d e g r a d a t i o n of two E s c h e r i c h i a c o l i c a p s u l a r p o l y s a c c h a r i d e s (K28 and K32) w i t h t h e i r r e s p e c t i v e b a c t e r i o p h a g e s ((j)28-l and (p28—2, and $32, r e s p e c t i v e l y ) a r e p r e s e n t e d h e r e .  VI.  RESULTS  E s c h e r i c h i a c o l i b a c t e r i o p h a g e s were i s o l a t e d from sewage ( c o u r t e s y of Dr. S. S t i r m , F r e i b u r g , Germany). The b a c t e r i o p h a g e s have been c h a r a c t e r i z e d by S t i r m and Freund-MOlbert and t h e i r morphology i s k n o w n .  1 9 3  Phage <p28—1 belongs  to B r a d l e y group A, and phages $28-2 and <b32 belong t o B r a d l e y group C. Most of the b a c t e r i o p h a g e s t h a t a r e capable of i n f e c t i n g e n c a p s u l a t e d E n t e r o b a c t e r i a c e a e belong t o B r a d l e y group C ,  2 0 6  and  bacteriophage-borne  enzymatic a c t i v i t y seems t o be a s s o c i a t e d w i t h s p i k e s t r u c t u r e s .  D e p o l y m e r i z a t i o n w i t h b a c t e r i o p h a g e s $28-1 and <t>28—2 The b a c t e r i o p h a g e s $28-1 and $28-2 were propagated  on t h e i r h o s t  s t r a i n E s c h e r i c h i a c o l i K28 u s i n g n u t r i e n t b r o t h as a medium. t i o n was c o n t i n u e d on an i n c r e a s i n g s c a l e u n t i l the crude c o n t a i n e d a t o t a l of ~ 1 0  1 3  Propaga-  lysates  p l a q u e - f o r m i n g u n i t s , an amount s u f f i c i e n t t o  162  degrade one gram of the p o l y s a c c h a r i d e . c o l i K28  c a p s u l a r p o l y s a c c h a r i d e was  of b a c t e r i o p h a g e s  d>28—1 and  <j>28-2,  The  Z [ )1  depolymerization  208  respectively.  then c o n c e n t r a t e d ,  a g a i n s t d i s t i l l e d water. repeated  The  I t was  process  IR-120 ( H ) +  d i a l y z a t e was  of E. c o l i K28  bacterial  and the c o n c e n t r a t e  dialyzed  of c o n c e n t r a t i o n and d i a l y s i s  t r e a t e d w i t h ion-exchange r e s i 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 a f t e r  p o l y s a c c h a r i d e w i t h phage <J>28—1 was  was The  Amberlite  carbohydrate  m a t e r i a l was  gel-permeation South A f r i c a ) .  o b t a i n e d . I t was  into  Only base l i n e  i s o l a t e d and examined by  chromatography ( c o u r t e s y of Dr. S.C. The  depolymerization  then s e p a r a t e d  pure components by p r e p a r a t i v e paper chromatography.  Churms, Cape Town,  r e s u l t s showed t h a t the i s o l a t e d m a t e r i a l was  a  of h i g h o l i g o s a c c h a r i d e s w i t h the average m o l e c u l a r weight M  daltons.  method  to  t h r e e times and f r e e z e - d r i e d .  The m i x t u r e  2100  allowed  s i x t i m e s , the d i a l y z a t e s were combined and c o n c e n t r a t e d .  concentrated  mixture  E.  conducted w i t h the crude s o l u t i o n s  proceed f o r t h r e e days, c h l o r o f o r m b e i n g added to prevent growth; the m i x t u r e was  of  1 0 2  The  degree of p o l y m e r i z a t i o n was  determined by  and by m e t h y l a t i o n a n a l y s i s on the m i x t u r e  of  o l i g o s a c c h a r i d e - a l d i t o l s (see T a b l e VI.1 and T a b l e V I . 2 ) .  w  =  Morrison's  corresponding They  i n d i c a t e d t h a t the average l e n g t h of the o l i g o s a c c h a r i d e i s 20 sugars or 5 r e p e a t i n g u n i t s and t h a t the g l u c o s y l r e s i d u e i s a r e d u c i n g  sugar.  T h i s i s i n agreement w i t h ^-H-n.m.r. f i n d i n g s which show the presence of two s i g n a l s at 6 = 4.67 = 4 Hz) c o r r e s p o n d i n g  p.p.m. ( J , o  =  ^ ** ^ z  to the B - g l u c o s y l and  aTU  *  a t  °  =  P«P« « ( ^ 1 2 m  a-glucosyl residues  163  TABLE VI.1  METHYLATION ANALYSIS AND THE REDUCING END DETERMINATION OF E. c o l l K28 OLIGOSACCHARIDE ISOLATED AFTER BACTERIOPHAGE $28-1 DEGRADATION OF E. c o l i K28 POLYSACCHARIDE  Methylated a sugars (as a l d i t o l )  T  b  Column B  Mole %° b  (ECNSS-M)  acetates)  1,2,5,6-Glc  0.8  15.3  2,3-Fuc  1.14  12.9  2,3,4,6-Gal  1.24  71.7  f  1,2,5,6-Glc = 3 , 4 - d i - 0 - a c e t y l - l , 2 , 5 , 6 - t e t r a - 0 - m e t h y l g l u c i t o l , e t c . R e l a t i v e r e t e n t i o n time r e f e r r e d t o 2,3,4,6-Glc as 1.00. Values a r e c o r r e c t e d by use of t h e e f f e c t i v e , c a r b o n response f a c t o r s g i v e n by A l b e r s h e i m e t a l .  1  0  3  R a t i o s a r e l o w , due t o i n c o m p l e t e h y d r o l y s i s of the g l u c o s y l u r o n i c l i n k a g e (2,3-Fuc) and h i g h v o l a t i l i t y of t h e d e r i v a t i v e  (1,2,5,6-Glc).  164  TABLE VI.2  DETERMINATION OF THE DEGREE OF POLYMERIZATION AND THE REDUCING END OF E. c o l i K28 OLIGOSACCHARIDE ISOLATED AFTER BACTERIOPHAGE <J»28-1 DEGRADATION OF E. c o l i K28 POLYSACCHARIDE  Peracetylated d e r i v a t i v e of  Mole % Column C (OV-225)  a  Fucononitrile  0.35  24.8  Glucononitrile  1.00  39.9  Galactononitrile  1.08  30.0  Glucitol  1.20  5.2  I s o t h e r m a l a t 230°.  165  respectively. borohydride  Both s i g n a l s d i s a p p e a r e d  a f t e r r e d u c t i o n w i t h sodium  due t o the c o n v e r s i o n of the r e d u c i n g sugar i n t o the  alditol. The m o l e c u l a r weight d i s t r i b u t i o n of the n o n - d i a l y z a b l e p o r t i o n gave a m i x t u r e of h i g h o l i g o s a c c h a r i d e s w i t h the average weight  = 4500.  molecular  These r e s u l t s i n d i c a t e t h a t o n l y p a r t i a l  d e p o l y m e r i z a t i o n had o c c u r r e d . Depolymerization  of E. c o l i K28 c a p s u l a r p o l y s a c c h a r i d e w i t h  <t>28-2 gave s i m i l a r r e s u l t s .  C o n f i r m a t i o n of the r e d u c i n g end was  o b t a i n e d by M o r r i s o n ' s m e t h o d , to  the a l d i t o l  1 0 2  whereby the o l i g o s a c c h a r i d e i s reduced  and a f t e r h y d r o l y s i s , the f r e e sugars a r e c o n v e r t e d  the p e r a c e t y l a t e d a l d o n o n i t r i l e s w i t h the r e d u c i n g end b e i n g i n t o the p e r a c e t y l a t e d a l d i t o l . glucitol,  into  converted  The r e s u l t s showed the presence of  g l ' u c o n o n i t r i l e , g a l a c t o n o n i t r i l e and f u c o n o n i t r i l e  indicating  t h a t the g l u c o s y l l i n k a g e was c l e a v e d by b a c t e r i o p h a g e a c t i o n .  Depolymerization with bacteriophage <p32 The b a c t e r i o p h a g e  4>32 was propagated on i t s h o s t  strain  E s c h e r i c h i a c o l i K32 u s i n g n u t r i e n t b r o t h as a medium. When the phage c o n c e n t r a t i o n reached f u r t h e r propagated i n a fermentor Bacteriophage  2 0 1  ensure s t e r i l i t y .  1 0  P.F.U./mL i t was  (see S e c t i o n I I I . 8 . 2 ) .  <j>32 was p u r i f i e d by p r e c i p i t a t i o n w i t h  g l y c o l 6000 (10% w / v ) . volatile buffer  ~10  2 1 3  polyethylene  The d e p o l y m e r i z a t i o n was c a r r i e d out i n a  * f o r two days i n the presence of some c h l o r o f o r m t o The d e p o l y m e r i z a t i o n m i x t u r e was d i a l y z e d a g a i n s t  166  d i s t i l l e d water o v e r n i g h t .  The d i a l y s i s was  repeated t w i c e more.  d i a l y z a t e s were combined, c o n c e n t r a t e d and f r e e z e - d r i e d . The p o r t i o n was  s e p a r a t e d by g e l - p e r m e a t i o n  B i o - G e l P-4..  gel-permeation  The  second f r a c t i o n was  chromatography ( c o u r t e s y of Dr.  South A f r i c a ) f o r m o l e c u l a r weight  distribution.  Three  f u r t h e r examined by  S.C.  Churms, Cape Town,  I t showed t h a t the  f r a c t i o n c o n s i s t e d m a i n l y of an o c t a s a c c h a r i d e (M Fig.  dialyzable  chromatography on a column of  The e l u t i o n p a t t e r n i s shown i n F i g . VI.5.  f r a c t i o n s were o b t a i n e d .  The  = 1600 d a l t o n s ) (see  VI.6).  Analysis of the depolymerization products of E. c o l i  K32  polysaccharide F r a c t i o n s I , I I and I I I were examined by H-n.m.r. s p e c t r o s c o p y . i  F r a c t i o n I c o n t a i n e d h i g h l y p o l y m e r i c m a t e r i a l , and the spectrum of t h i s f r a c t i o n was  v e r y s i m i l a r t o t h a t of the n a t i v e a c e t y l a t e d p o l y -  saccharide.  The presence  1  of an o c t a s a c c h a r i d e was not obvious from the  H-n.m.r. spectrum of the second f r a c t i o n .  However, a f t e r sodium b o r o -  h y d r i d e r e d u c t i o n of t h a t f r a c t i o n the ^-n.m.r. r e s o l v e d , and was  s i m i l a r t o the spectrum of the d e a c e t y l a t e d E.  K32 p o l y s a c c h a r i d e .  The  comparison of two s p e c t r a d i d not permit  assignment of the r e d u c i n g end other p r i n c i p a l s i g n a l s ) . as was  judged  spectrum became b e t t e r  the  (due t o the p o s s i b l e o v e r l a p p i n g w i t h  F r a c t i o n I I I had a low carbohydrate  from i t s ^-H-n.m.r. spectrum.  content  I t r e v e a l e d the presence  an o c t a s a c c h a r i d e w i t h a low a c e t a t e content (30% by n.m.r.). 1  coli  H-n.m.r. data of a l l t h r e e f r a c t i o n s i s summarized i n Table  The VI.3.  of  Fig.  V I . 5:  Separation  of  chromatography  the  depolymerization  (Bio-Gel  P-4)  products  of  E.  coli  K32  by  gel-perme  168  1600 ©  801  70-  E l u t e d volume (mL) Molecular weight d i s t r i b u t i o n  Mol. wt.  F i g . VI.6:  % by wt.  mol. %  4400  13  6  3300  11  7  2700  18  14  1600  58  73  Molecular weight d i s t r i b u t i o n of f r a c t i o n II (Bio-Gel P-10 column 52 x 1.5 cm, M NaCl eluant, flow-rate 20mL/h). Courtesy of Dr. S.C Churms, Cape Town, South A f r i c a .  TABLE VI.3  PROTON ASSIGNMENTS (400 MHz) FOR THE OLIGOSACCHARIDES AND RELATED COMPOUNDS GENERATED IN BACTERIOPHAGE DEPOLYMERIZATION OF THE E. c o l i K32 CAPSULAR POLYSACCHARIDE.  Fraction IA (p.p.m.)  Integral (H) 1.2  Fraction I I A (p.p.m.)  5.45  0.4  5.51 5.48 5.44  5.24  1.0  5.24  5.3  5.18 5.15  5.51  5.18 5.14  }  i  4.72(8Hz) 2.19 2.16 1.35  i  1.3 2.2  5.05 5.02  A (p.p.m.)  Integral (H)  1.0  5.23  5.2  5.17 5.14  1.5  4.71(8Hz) } 2.0 4.68(8Hz)  2.6  2.17(8Hz)  5.0  1.35(6Hz) } 6.5 1.33(6Hz)  Fraction I I I A (p.p.m.)  }  1.0  5.25  3.0  5.19 5.16 5.11  5.10 , '  C  5.52 5.47  0.6 0.4 0.6  5.11  5.10 5.05 5.03  }  Integral (H)  Fraction II ( R )  5.07  0.4  4.66(8Hz)  1.0  2.7 1.32  6.0  i  >  Integral  Assignment^  (H) 0.25  i '  a-Rha  1.0 |  2.0  }  1.0  5.05  0.5  4.72(8Hz)  2.0  2.19(8Hz)  1.5  1.35(6Hz)  6.3  a-Rha w i t h 0-acetyl  a-Gal a-Glc unassigned  j  6-GlcA CH of 0-acetyl 3  i  '  C H of a-Rha 3  and I I I see i t e x t . k Chemical s h i f t r e l a t i v e t o i n t e r n a l F o r t h esource of F r : I , I I a c e t o n e ; 6 2.23 d o w n f i e l d from sodium 4 , 4 - d i m e t h y l - 4 - s i l a p e n t a n e - l - s u l f o n a t e (DSS). c d F r a c t i o n I I a f t e r r e d u c t i o n w i t h sodium b o r o h y d r i d e . A d e f i n i t e assignment c o u l d not be made, the assignments of a-Gal and a-Glc are t e n t a t i v e . a  170  M e t h y l a t i o n of the reduced  f r a c t i o n I I , f o l l o w e d by h y d r o l y s i s ,  d e r i v a t i z a t i o n as 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 gave v a l u e s shown i n Table VI.4. The d a t a demonstrate t h a t the b a c t e r i o p h a g e enzyme i s an <x-Dg l u c o s i d a s e and t h a t the g l u c o s e i s p r e s e n t a t the r e d u c i n g end of t h e o l i g o s a c c h a r i d e which i s comprised  of two r e p e a t i n g u n i t s .  I t also  shows t h a t the b a c t e r i o p h a g e - b o r n e  enzyme does not have a " d e a c e t y l a s e "  a c t i v i t y , s i n c e a c e t a t e - b e a r i n g o l i g o s a c c h a r i d e s were i s o l a t e d a f t e r the b a c t e r i o p h a g e d e g r a d a t i o n of E. c o l i K32 p o l y s a c c h a r i d e .  VI.3  DISCUSSION  The main purpose of the b a c t e r i o p h a g e work c a r r i e d out on E s c h e r i c h i a c o l i bacteriophages  $28-1, cb28—2 and <j>32 was t o o b t a i n  o l i g o s a c c h a r i d e s r e p r e s e n t i n g s u b u n i t s of the p o l y s a c c h a r i d e s degraded, w i t h the l a b i l e 0 - a c e t y l s u b s t i t u e n t s p r e s e n t as i n the o r i g i n a l p o l y saccharide. and  13  These o l i g o s a c c h a r i d e s c o u l d be then s t u d i e d by ^-H-n.m. r .  C-n.m.r. s p e c t r o s c o p y i n o r d e r t o l o c a t e the a c e t a t e p o s i t i o n .  However, t h i s aim has not been a c h i e v e d . The b a c t e r i o p h a g e d e g r a d a t i o n of the p o l y s a c c h a r i d e was out u s i n g two methods. was propagated  carried  A c c o r d i n g t o the f i r s t method, the b a c t e r i o p h a g e  on an i n c r e a s i n g s c a l e u n t i l a t o t a l of ~ 1 0  1 3  P.F.U. was  o b t a i n e d and a f t e r d i a l y s i s a c o n c e n t r a t e d crude s o l u t i o n of the b a c t e r i o p h a g e was used d i r e c t l y f o r the d e p o l y m e r i z a t i o n . procedure  2 0 8  was developed  K l e b s i e l l a bacteriophages.  This  i n our group and gave e x c e l l e n t r e s u l t s w i t h However, use of t h i s method f o r b a c t e r i o -  1.71  TABLE VI.4  METHYLATION ANALYSIS OF THE REDUCED FRACTION I I OBTAINED AFTER THE SEPARATION OF THE DEPOLYMERIZATION PRODUCTS OF E. c o l i K32 POLYSACCHARIDE  Methylated (as a l d i t o l  sugars  T  acetates)  Mole %°  b  Column B  b  (ECNSS-M)  1,2,4,5,6-Glc  0.35  2,4-Rha  1.01  11.9  2,3,4,6-Gal  1.55  28.6  2-Rha  1.96  35.5  2,4,6-Glc  2.26  19.1  4.9  d  1,2,4,5,6-Glc = 3 - 0 - a c e t y l - l , 2 , 4 , 5 , 6 - p e n t a - 0 - m e t h y l g l u c i t o l , e t c . R e l a t i v e r e t e n t i o n time r e f e r r e d t o 2,3,4,6-Glc as 1.00. V a l u e s a r e c o r r e c t e d by use of the e f f e c t i v e , carbon-response f a c t o r s g i v e n by A l b e r s h e i m  et a l .  1  0  3  R a t i o s o f c e r t a i n sugars a r e low, due t o incomplete  h y d r o l y s i s of t h e  g l u c o s y l u r o n i c l i n k a g e (2,4-Rha) and h i g h v o l a t i l i t y o f the d e r i v a t i v e (1,2,4,5,6-Glc).  172  phages $28-1  and  $28-2 d e g r a d a t i o n of E. c o l i K28 p o l y s a c c h a r i d e d i d n o t  g i v e s a t i s f a c t o r y r e s u l t s . Only p a r t i a l d e p o l y m e r i z a t i o n was S i n c e these b a c t e r i o p h a g e s  achieved.  form v e r y s m a l l " h a l o s " , i t i s p o s s i b l e t h a t  they have low enzymic a c t i v i t y , and do not produce o l i g o s a c c h a r i d e s corresponding  t o one or two  r e p e a t i n g u n i t ( s ) of the p o l y s a c c h a r i d e .  B e a r i n g t h i s i n mind, the second method was phage $32.  I t was  used f o r b a c t e r i o -  p u r i f i e d by p r e c i p i t a t i o n w i t h p o l y e t h y l e n e  6000 p r i o r t o the d e p o l y m e r i z a t i o n .  The  d e g r a d a t i o n was  glycol  done i n  " v o l a t i l e " b u f f e r i n o r d e r t o a v o i d p o s s i b l e l o s s of the a c t i v i t y the d e p o l y m e r i z a t i o n .  The  r e s u l t s o b t a i n e d were more e n c o u r a g i n g ,  s m a l l amount of the o c t a s a c c h a r i d e (two r e p e a t i n g u n i t s ) b e a r i n g 0 - a c e t y l group was  and  a  an  obtained.  A l l three bacteriophages a-D-glucosidase  during  activity.  The  ($28-1, $28-2 and bacteriophages  $32)  $28-1  and  exhibit $28-2 c l e a v e d  the same g l y c o s y l l i n k a g e ,  ->3)-oc-D-Glc-( 1->4)-8-D-G1CA-( l+4)-<x-L-Fuc-( 1-+ 4 1 8-D-Gal  $28-1, $28-2 E. c o l i  K28  a l t h o u g h m o r p h o l o g i c a l l y they belong to the d i f f e r e n t B r a d l e y $28-1  (group A ) and  expected.  groups, -  $28-2 (group C ) , and d i f f e r e n t enzymic a c t i v i t y  1  was  93  173  I n the case o f b a c t e r i o p h a g e <p32 oc-glucosidase  a c t i v i t y was q u i t e  unexpected, s i n c e the rhamnosyl l i n k a g e i s the most l a b i l e one ( t o acid). •>3 ) - a-D-Glc-(1 ->4) - a-L-Rha-(1 +3 ) - a-D-Gal-(1 -> 3 <J>32  1 B-D-GlcA  E. c o l i K32  Here a g a i n the tendency o f the b a c t e r i o p h a g e t o produce a l i n e a r  struc-  t u r e can be n o t i c e d . A n o t h e r i n t e r e s t i n g aspect of t h i s work i s the f a c t b a c t e r i o p h a g e s $28-1 and $32 g i v e c r o s s - a d s o r p t i o n  that  w i t h the b a c t e r i a l  s t r a i n of E s c h e r i c h i a c o l i K32, but not the phage $ 2 8 - 2 .  1 9 3  Comparison of the s t r u c t u r e s o f E. c o l i K28 and E. c o l i K32 suggests c e r t a i n s i m i l a r i t y i n t h e i r s t r u c t u r a l p a t t e r n . s t r u c t u r e s are of a "three-plus-one" composition i s s i m i l a r . structures are d i f f e r e n t .  Both  t y p e , and t h e i r q u a l i t a t i v e  However, d e s p i t e these s i m i l a r i t i e s the two T h i s suggests t h a t the b a c t e r i o p h a g e  n i z e s only a small p o r t i o n of the polysaccharide  chain.  recog-  The a c t i o n o f  the b a c t e r i o p h a g e $28-1 on t h e b a c t e r i a l s t r a i n o f E s c h e r i c h i a c o l i K32 has y e t t o be d e t e r m i n e d .  174  VI. 4  EXPERIMENTAL  The b a c t e r i o p h a g e s  d>28—1, $28-2 and  $32 were r e c e i v e d from Dr.  S t i r m ( F r e i b u r g , Germany) and propagated on t h e i r host s t r a i n s .  S.  For  e x p e r i m e n t a l d e t a i l s see S e c t i o n I I I . 8 .  Paper chromatography, gas-  l i q u i d chromatography and g e l - p e r m e a t i o n  chromatography were performed  as d e s c r i b e d i n S e c t i o n I I I . P r e p a r a t i v e paper chromatography performed by the descending The  was  method u s i n g s o l v e n t C (see S e c t i o n  I n s t r u m e n t a t i o n used f o r n.m.r., g . l . c . and I n f r a r e d has  d e s c r i b e d i n S e c t i o n I I I . G.l.c.-m.s. was  III.l).  been  performed on the NERMAG R10-  10 i n s t r u m e n t u s i n g c a p i l l a r y column of DB-225, programmed from 195°  for  8 min, and then 4°/min t o 220°.  I n c u b a t i o n o f E. c o l i K28 p o l y s a c c h a r i d e w i t h P u r i f i e d , c a p s u l a r p o l y s a c c h a r i d e (100 mg) K28 was  d i s s o l v e d i n 10 mL  containing 2 x 10  1 3  of d i s t i l l e d water.  P.F.U. was  concentrated  bacteriophages  from E s c h e r i c h i a c o l i  A phage $28-1  solution  to a s m a l l volume p r i o r t o  the d e p o l y m e r i z a t i o n , d i a l y z e d a g a i n s t tap water f o r t h r e e days and c o n c e n t r a t e d a g a i n t o 100 mL. a phage s o l u t i o n (100 mL) x 10  1 3  plaque-forming  A p o l y s a c c h a r i d e s o l u t i o n was  mixed w i t h  i n a n u t r i e n t b r o t h , c o n t a i n i n g a t o t a l of  u n i t s (P.F.U.) and i n c u b a t e d f o r 48 h a t 37°.  s i g n i f i c a n t drop i n the v i s c o s i t y of the o r i g i n a l p o l y s a c c h a r i d e t i o n was  n o t i c e d a f t e r approximately  3 h of i n c u b a t i o n .  2.0 A  solu-  175  P u r i f i c a t i o n and  separation of depolymerlzed material  The r e s u l t i n g crude d e p o l y m e r i z a t i o n m i x t u r e was 250 mL of d i s t i l l e d water. d i a l y s i s bag was  The procedure was  changed a f t e r each t i m e .  dialyzed against  repeated s i x times and  the  The d i a l y z a t e s were combined,  c o n c e n t r a t e d to a s m a l l volume on a r o t a r y e v a p o r a t o r and exchanged t h r e e times on A m b e r l i t e I R - 1 2 0 ( H ) r e s i n .  The r e s u l t i n g s o l u t i o n  +  freeze-dried.  The d e g r a d a t i o n p r o d u c t s were i s o l a t e d by p r e p a r a t i v e  paper chromatography i n s o l v e n t C. was  was  o b t a i n e d (21.7 mg).  Only b a s e l i n e c a r b o h y d r a t e m a t e r i a l  The content of a d i a l y s i s bag was  w i t h A m b e r l i t e I R - 1 2 0 ( H ) ion-exchange +  exchanged  r e s i n and f r e e z e - d r i e d .  It yiel-  ded 92.5 mg of the n o n - d i a l y z a b l e m a t e r i a l . In a s i m i l a r way a d e g r a d a t i o n of E s c h e r i c h i a c o l i K28 u s i n g crude b a c t e r i o p h a g e $28-2 s u s p e n s i o n was  done.  The b a c t e r i o p h a g e  d a t i o n was not complete and y i e l d e d o n l y h i g h o l i g o m e r s as was  degra-  judged  by  m o l e c u l a r weight d i s t r i b u t i o n of a d i a l y z a b l e f r a c t i o n (see S e c t i o n VI.2).  P u r i f i c a t i o n of the bacteriophage Bacteriophage  $32 was propagated  $32 i n a fermentor (see S e c t i o n  I I I . 8 . 2 ) to g i v e 9 L of a c l e a r s o l u t i o n w i t h the t i t e r P.F.U./mL.  10  1 0  From t h i s p r e p a r a t i o n 2.5 L was p u r i f i e d by p r e c i p i t a t i o n  with polyethylene g l y c o l 6 0 0 0 The s o l u t i o n was  2 0 9  i n the presence of NaCl (73.1 g, 0.5M).  then p l a c e d i n a c o l d room and kept f o r 42 h a t 4°.  v e r y f i n e p r e c i p i t a t e of a b a c t e r i o p h a g e appeared beaker.  1.6 x  I t was  p r e c i p i t a t e was  at the bottom of a  spun down i n a c e n t r i f u g e a t 1400 r.p.m. r e d i s s o l v e d i n 50 mL of 10 mM  (pH 7.1)  The  tris(hydroxy-  A  176  methyl)methylamine ( T r i s ) - h y d r o c h l o r i d e b u f f e r which c o n t a i n e d NaCl.  I n o r d e r to remove p o l y e t h y l e n e g l y c o l the m i l k y and  phage s o l u t i o n was temperature (23°)  opalescent  f o r 18 h and then at 4° f o r an a d d i t i o n a l 24 h.  phage s o l u t i o n was  changed once d u r i n g the d i a l y s i s .  then spun at low speed 1200  t i t e r was  P.F.U. ( f o r 50 mL  mM  d i a l y z e d a g a i n s t the same b u f f e r f i r s t at room  The T r i s - H C l b u f f e r was  bacteriophage  10  2.5 x 1 0  1 1  8  P.F.U./mL.  phage p r e c i p i t a t i o n was  and assayed.  P.F.U./mL or a t o t a l of 1.25  of phage s o l u t i o n ) .  a t i t e r of 7.2 x 1 0  r.p.m.  The  x  The  The 10  assay of the s u p e r n a t a n t  1 3  gave  T h i s r e s u l t shows t h a t the b a c t e r i o -  not complete and t h a t some a c t i v i t y was  p r e s e n t i n the s u p e r n a t a n t .  phage was  a d j u s t e d t o pH 7.2  still  then d i a l y z e d a g a i n s t  v o l a t i l e b u f f e r which c o n t a i n e d 0.05M ammonium carbonate ammonium a c e t a t e and was  The  and  w i t h 50% a c e t i c  0.1M acid.  2 0 4  Depolymerization of the capsular polysaccharide from E. c o l i K32  by bacteriophage 4>32  E. c o l i K32  p o l y s a c c h a r i d e (195 mg)  was  d i s s o l v e d i n 15 mL  v o l a t i l e b u f f e r and to t h i s s o l u t i o n a t o t a l of 0.85 mL  of a v o l a t i l e b u f f e r was  37°,  added.  The m i x t u r e was  c h l o r o f o r m b e i n g added t o prevent  d e p o l y m e r i z a t i o n m i x t u r e was  x 10  P.F.U. i n 50  1 3  kept f o r 48 h a t  b a c t e r i a l growth.  The  t r a n s f e r r e d i n t o a d i a l y s i s bag  d i a l y z e d a g a i n s t d i s t i l l e d water o v e r n i g h t . The  of a  d i a l y s i s was  and repeated  t w i c e more, and the d i a l y z a t e s were combined and f r e e z e - d r i e d .  The  r e s i d u e was  r e d i s s o l v e d i n d i s t i l l e d water and f r e e z e - d r i e d a g a i n .  p r o c e s s was  repeated  s e v e r a l times u n t i l the weight of the sample  This  177  remained unchanged (114.2 mg).  The  contents  f r e e z e - d r i e d s e p a r a t e l y to y i e l d 260.5  of the d i a l y s i s bag  was  mg.  Separation of the depolymerized material by gel-permeation chromatography The  crude d i a l y z a b l e m a t e r i a l (114.2 mg)  o f B i o - G e l P-4  (400 mesh) and e l u t e d at 10.5  each) were c o l l e c t e d and F i g . VI.1  freeze-dried.  (see S e c t i o n V I . 2 ) .  p r o d u c t s were o b t a i n e d : - tubes 15-21  (20 mg);  The  was  mL/h.  p l a c e d on a column F r a c t i o n s (2  e l u t i o n p r o f i l e i s shown i n  Three f r a c t i o n s of the  f r a c t i o n I - tubes 9-14  (17.4  degradation mg);  weight d i s t r i b u t i o n of f r a c t i o n I I showed the presence of  polysaccharide  fraction II  f r a c t i o n I I I - tubes 22-34 (22.75 mg).  o c t a s a c c h a r i d e , which r e p r e s e n t e d  mL  Molecular  an  a double r e p e a t i n g u n i t of E. c o l i  K32  (see S e c t i o n V I . 2 ) .  Determination of the reducing end  and  the degree of  depolymerization A sample of o l i g o s a c c h a r i d e (10 mg) and to t h i s NaBH^ (15 mg)  was  of sodium b o r o h y d r i d e  removed by treatment  ion-exchange r e s i n . 3% m e t h a n o l i c  HC1  was The  added.  was  dissolved i n H 0  with Amberlite  IR-120(H ) +  refluxed i n  After n e u t r a l i z a t i o n with Ag C0 , 2  and  3  e v a p o r a t i o n of the s o l v e n t a f t e r c e n t r i f u g a t i o n , the u r o n i c e s t e r reduced w i t h NaBH^ i n anhydrous methanol (5 mL). was  The  removed by c o - e v a p o r a t i o n s  was  reduced m a t e r i a l  h y d r o l y z e d w i t h 2M t r i f l u o r o a c e t i c a c i d on a steam bath  and the excess a c i d was  mL)  A f t e r s t i r r i n g f o r 2 h, the excess  d r i e d , reduced o l i g o s a c c h a r i d e was  overnight.  (5  2  overnight  with water.  A solu-  178  t i o n (0.5 mL) o f 5% hydroxylamine h y d r o c h l o r i d e i n p y r i d i n e was then added and the r e a c t i o n m i x t u r e was heated on a steam b a t h f o r 15 min. A c e t i c a n h y d r i d e (0.5 mL) was added t o the c o o l e d s o l u t i o n which was heated on a steam b a t h f o r 1 h.  The m i x t u r e of p e r a c e t y l a t e d a l d o n o -  n i t r i l e s and p e r a c e t y l a t e d a l d i t o l a c e t a t e s was i s o l a t e d by p a r t i t i o n between water and c h l o r o f o r m .  G.l.c.  a n a l y s i s was performed on column  C i s o t h e r m a l l y a t 230°.  Methylation analysis and reducing end determination The o l i g o s a c c h a r i d e (5 mg) was reduced w i t h NaBH^ p r i o r t o methyl a t i o n by the Hakomori p r o c e d u r e .  8 8  The reduced m a t e r i a l was d i s s o l v e d  i n d r y d i m e t h y l s u l f o x i d e (2 mL) and m e t h y l a t e d by treatment w i t h 1 mL of d i m e t h y l s u l f i n y l a n i o n f o r 2 h, and then 2 mL o f methyl i o d i d e f o r 1 h.  The m e t h y l a t e d product was r e c o v e r e d by p a r t i t i o n between water and  chloroform.  I t was then h y d r o l y z e d w i t h 2M t r i f l u o r o a c e t i c a c i d on a  steam b a t h o v e r n i g h t , reduced w i t h NaBH^ and a c e t y l a t e d w i t h 1:1 a c e t i c anhydride-pyridine.  A m i x t u r e of p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s  was a n a l y z e d by g . l . c .  (column B, i s o t h e r m a l l y 170°) and g.l.c.-m.s.  (see Table VI.1 and Table V I . 4 ) .  179  CHAPTER VII  BIBLIOGRAPHY  180  VII  BIBLIOGRAPHY  1  G.O. A s p i n a l l , i n G.O. A s p i n a l l ( E d . ) , "The P o l y s a c c h a r i d e s " , V o l . 1, Academic P r e s s , New Y o r k , 1982, p. 1.  2  P.A. Sandford and J . B a i r d , i n G.O. A s p i n a l l ( E d . ) , "The P o l y s a c c h a r i d e s " , V o l . 2, Academic P r e s s , New York, 1982, pp. 411-490.  3  P. A l b e r s h e i m , A.G. D a r v i l l , M. M c N e i l , B.S. V a l e n t , J.K. S h a r p , E.A. N o t h n a g e l , K.R. D a v i s , N. Yamazaki, D.J. G o l l i n , W.S. Y o r k , W.F. Dudman, J.E. D a r v i l l , and A. D e l l , i n 0. C i f e r r i and L. Dure I I I ( E d s . ) , " S t r u c t u r e and F u n c t i o n of P l a n t Genomes", V o l . 63, NATO ASI S e r i e s , Plenum P r e s s , New Y o r k , 1982, pp. 293-312.  4  C.T. B i s h o p and H.J. J e n n i n g s , i n G.O. A s p i n a l l ( E d . ) , "The P o l y s a c c h a r i d e s " , V o l . 1, Academic P r e s s , New York, 1982, pp. 291330.  5  K. Jann, B. Jann, P r o g . A l l e r g y , 33 (1983) 53-79.  6  H.J. J e n n i n g s ,  7  S.C. Churms and A.M. Stephen,  8  Y.M. Choy, G.G.S. D u t t o n , A.M. Stephen, and M.-T. Yang, L e t t r . , 5 (1972) 675-681.  9  F. Kauffmann, "The B a c t e r i o l o g y of E n t e r o b a c t e r i a c e a e " , Munksgaard, Copenhagen, 1966.  Adv. Carbohydr. Chem. Biochem., 41 (1983) 155-208. Carbohydr. Res., 35 (1974) 73-86. Anal.  10  R.M. K r a u s e ,  J . I n f e c t . P i s . , 135 (1977) 318-329.  11  P.R. Edwards, W.H. Ewing, " I d e n t i f i c a t i o n of E n t e r o b a c t e r i a c e a e " , 3rd edn., Burgess P u b l i s h i n g Company, M i n n e a p o l i s , 1972.  12  I . 0rskov,  13  I . 0rskov and M.A. F i f e - A s b u r y , B a c t e r i o l . , 27_ (1977) 386.  14  H.R. Morgan, i n R. Dubos and J.G. H i r s c h ( E d s . ) , " B a c t e r i a l and M y c o t i c I n f e c t i o n s of Man", J.B. L i p p i n c o t t Company, P h i l a d e l p h i a , 1965, pp. 610-648.  15  W. Nimmich,  Z. Med. M i c r o b i o l . Immunol., 154 (1968) 117-131.  16  W. Nimmich,  A c t a B i o l . Med. Ger., 26 (1971) 397-403.  A c t a P a t h o l . M i c r o b i o l . Scand., 38 (1956) 375-384. I n t e r n a t . J . Systematic  181  17  W. Nimmich,  Z . A l l g . M i c r o b i o l . , 19 ( 5 ) (1979) 343-347.  18  J.L. D i Fabio, 1981.  19  E.M. Cooke, " E s c h e r i c h i a c o l i and Man", C h u r c h i l l L i v i n g s t o n e , London, 1974.  20  F. Kauffmann,  21  A.H. L i n t o n , " M i c r o b e s , Man and A n i m a l s " , A W i l e y - I n t e r s c i e n c e , New York, 1982.  22  I . 0 r s k o v , F. 0 r s k o v , B. Jann, and K. Jann, 41_ (1977) 667-710.  23  0. W e s t p h a l , K. Jann, K. Himmelspach, 9-39.  24  K. Jann, B. Jann, M.A. Schmidt, and W.F. Vann, (1980) 1108-1115.  25  F.-P. T s u i , R.A. B o y k i n s , and W. Egan, 263-271.  26  K. Jann, B. Jann, K.F. S c h n e i d e r , F. 0 r s k o v , and I . 0 r s k o v , Eur. J . Biochem., 5_ (1968) 456-465.  27  K. Jann and 0. W e s t p h a l , i n M. S e l a ( E d . ) , " M i c r o b i a l p o l y s a c c h a r i d e s " , V o l . 3, "The A n t i g e n s " , Academic P r e s s , New York, 1975, pp. 1-125.  28  E . J . McGuire and S.B. B r i n k l e y ,  29  H. Niemann, A.K. C h a k r a b o r t y , H. F r i e b o l i n , and S. S t i r m , B a c t e r i o l . , 133 (1978) 390-391.  30  A.K. C h a k r a b o r t y , H. F r i e b o l i n , and S. S t i r m , (1980) 971-972.  31  W. Egan, F.-P. T s u i , R. Schneerson, and J.B. R o b b i n s , Chem., ( i n p r e s s ) .  32  E.A. K a b a t ,  33  C.E. B a l l o u , P.N. L i p k e , and W.C. Raschke, (1974) 461-467.  34  M. H e i d e l b e r g e r , W.F. Dudman and W. Nimmich, (1970) 1321-1328.  Ph.D. T h e s i s , U n i v e r s i t y of B r i t i s h C o l u m b i a ,  J . Immunol., 57 (1947) 71.  B a c t e r i o l . Rev.,  P r o g . A l l e r g y , 33 (1983)  J . B a c t e r i o l . , 143  Carbohydr. Res., 102 (1982)  B i o c h e m i s t r y , 3_ (1964) 247-251. J.  J . B a c t e r i o l . , 141 J. Biol.  J . Immunol., 84 (1960) 82-85. J . B a c t e r i o l . , 117 J . Immunol., 104  182  35  M. H e i d e l b e r g e r ,  Res. Immunochem. Immunobiol., 3_ (1978) 1-40.  36  M. H e i d e l b e r g e r and W. Nimmich, 1344.  37  M. H e i d e l b e r g e r and G.G.S. D u t t o n , 859.  38  M. H e i d e l b e r g e r and W. Nimmich,  39  M. H e i d e l b e r g e r , C.M. MacLeod, J . J . K a i s e r , and B. R o b i n s o n , Exp. Med., 83 (1946) 303-320.  40  H.J. J e n n i n g s and C. L u g o w s k i ,  41  R. Schneerson, J.E. R o b b i n s , C. Chu, A. S u t t o n , G. S c h i f f m a n , and W.F. Vann, Prog. A l l e r g y , 33 (1983) 144-158.  42  C.P.J. Glaudemans, 346.  43  H. Geyer, K. Hiramelspach, B. K w i a t k o w s k i , S. S c h l e c h t , and S. S t i r m , Pure & A p p l . Chem., _55 (1983) 637-653.  44  H.W. Leach and T.J. Schoch,  45  J.-P. J o s e l e a u , G. Chambat, and B. Chumpitazi-Hermoza, Carbohydr. Res., 90 (1981) 339-344.  46  R.G. S p i r o ,  47  0. Westphal and K. Jann, 83-91.  48  R.L. W h i s t l e r and J.L. S a n n e l l a , (1965) 34-36.  49  T.J. P a i n t e r ,  50  J.K.N. Jones and R.J. S t o o d l e y , 36-38.  51  J.E. S c o t t ,  Methods Carbohydr. Chem., _5 (1965) 38-44.  52  A.O. P i t t e t ,  Methods Carbohydr. Chem., _5 (1965) 3-5.  53  H. M e i e r ,  54  K.A. G r a n a t h ,  55  Y.C. Lee and R. Montgomery, 28-34.  J . Immunol., 109 (1972) 1337J . Immunol., I l l  (1973) 857-  Immunochemistry, 13 (1976) 67-80. J.  J . Immunol., 127 (1981) 1011-1018.  Adv. Carbohydr. Chem. Biochem., 31_(1975) 313-  C e r e a l Chem., 39 (1962) 318-327.  Methods Carbohydr. Chem., 7_ (1976) 185-190. Methods Carbohydr. Chem., 5_ (1965) Methods Carbohydr. Chem., _5  Methods Carbohydr. Res., 5_ (1965) 98-100. Methods Carbohydr. Chem., _5 (1965)  Methods Carbohydr. Chem., 5^ (1965) 45-46. Methods Carbohydr. Chem., _5 (1965) 20-28. Methods Carbohydr. Chem, 5_ (1965)  183  56  H. Neukon and W. K u e n d i g , 14-17.  Methods Carbohydr. Chem., 5_ (1965)  57  I . J . G o l d s t e i n and C.E. Hayes, (1978) 127-340.  58  S.C. Churns,  59  D.H. N o r t h c o t e ,  60  M. B r e e n , H.G. W e l n s t e i n , L . J . B l a c k , M.S. B o r c h e r d i n g , and r.A. S i t t i g , Methods Carbohydr. Chem., _7 (1966) 101-115.  61  G.G.S. D u t t o n and M.-T. Yang,  Can. J . Chem., 51_ (1973) 1826-1832.  62  K. O k u t a n i and G.G.S. D u t t o n ,  Carbohydr. Res., 86_ (1980) 259-271.  63  G.G.S. D u t t o n ,  64  R.G. S p i r o ,  65  P., A l b e r s h e i m , D.J. N e v i n s , P.D. E n g l i s h and A. K a r r , Res., 5 (1967) 340-345.  66  J . F . C o d i n g t o n , K.B. L I n s l e y and C. S i l b e r , Chem., 1_ (1976) 226-232.  67  V.N. R e i n h o l d ,  68  L. Hough,  69  K. Macek, i n I.M. H a i s and K. Macek ( E d s . ) , "Paper Chromatography", Academic P r e s s , New Y o r k , 1963.  70  R.E. Wing and J.N. B e M i l l e r , Methods Carbohydr. Chem., 6_ (1972) 42-59.  71  H. W e i g e l , Adv. Carbohydr. Chem., 18_ (1963) 61-97.  72  Z. D i s c h e ,  73  D. A m i n o f f , W.W. B i n k l e y , R. S c h a f f e r and R.W. Mowry, i n W. Pigman and D. H o r t o n ( E d s . ) , "The C a r b o h y d r a t e s " , 2nd edn., V o l . 2B, Academic P r e s s , New Y o r k , 1970, pp. 739-807.  74  G.D. M c G i n n i s and P. Fang, 33-43.  75  R.L. T a y l o r and H.E. Conrad,  76  M. Abdel-Akher and F.Smith, 1038.  Adv. Carbohydr. Chem Biochem., 35  Adv. Carbohydr. Chem. Biochem., 25 (1970) 13-51. Methods Carbohydr. Chem., _5 (1965) 49-53.  Adv. Carbohydr. Chem. Bichem., 28 (1973) 11-160.  Methods Enzymol., 28 (1973) 3-43. Carbohydr.  Methods Carbohydr.  Methods Enzymol., 25 (1972) 244-249.  Methods Carbohydr. Chem., 1_ (1962) 21-31.  Methods C a r b o h y d r a t e Chem, 1_ (1962) 477-514.  Methods Carbohydr. Chem., 8_ (1980) B i o c h e m i s t r y , 11_ (1972) 1383-1388. Nature (LOndon), 166 (1950) 1037-  184  77  J.K.N. Jones and M.B. P e r r y , 2793.  78  G.M. B e b a u l t , J.M. B e r r y , Y.M. Choy, G.G.S. D u t t o n , N. F u n n e l l , L.D. Hayward, and A.M. Stephen, Can. J . Chem., 51 (1973) 324-326.  79  K. L e o n t e i n , B. L i n d b e r g , and J . LOnngren, (1978) 359-362.  80  G.J. G e r w i g , J.P. K a m e r l i n g , and J.F.G. V l i e g e n t h a r t , Carbohydr. Res., 77. (1979) 1-7. J.H. P a z u r , Methods Enzymol., _9 (1966) 82-87.  81  J . Am. Chem. S o c , 79 (1957) 2787-  Carbohydr. R e s . , 62  82  H. B j O r n d a l , C.G. H e l l e r q v i s t , B. L i n d b e r g , and S. Svensson, Angew. Chem. I n t . Ed. E n g l . , 9^ (1970) 610-619.  83  B. L i n d b e r g ,  84  E.L. H i r s t and E. P e r c i v a l , 287-296.  85  W.N. Haworth,  86  T. P u r d i e and J.C. I r v i n e ,  87  R. Rutin, H. Trischmann, and I . Lew,  88  S.I. Hakomori,  89  P.A. S a n d f o r d and H.E. Conrad,  90  P. Prehm,  91  P.-E. Jannsson, L. Kenne, H. L i e d g r e n , B. L i n d b e r g , and J . LcSnngren, Chem. Commun., U n i v . Stockholm, JJ (1976).  92  B.K. R o b e r t s e n , P. Aman, A.G. D a r v i l l , M. M c N e i l , and P. A l b e r s h e i m , P l a n t P h y s i o l . , 67 (1981) 389-400.  93  V.N. R e i n h o l d , E. C o l e s , and S.A. C a r r , (1983) 1-18.  94  J.U. Bowie, P.V. T r e s c o n y , and G.R. Gray, (1984) 301-307.  95  A.G. M c l n n e s , D.H. B a l l , F.P. Cooper, and C.T. B i s h o p , Chromatogr., _1 (1958) 556-557.  96  C.C. Sweeley, R. B e n t l e y , M. M a k i t a , and W.W. S o c , 85 (1963) 2497-2507.  Methods Enzymol., 28 (1972) 178-195. Methods Carbohydr. Chem., _5 (1965)  J . Chem. S o c , 107 (1915) 8. J . Chem. S o c , 83 (1903) 1021-1037. Angew. Chem. , 67 (1955) 32.  J . Biochem. (Tokyo)., 55 (1964) 205-208. B i o c h e m i s t r y , _5 (1966)  1508-1516.  Carbohydr. Res., 78 (1980) 372-374.  J . Carbohydr. Chem., 2, Carbohydr. Res., 125  Wells,  J. J . Am. Chem.  185  97  G.G.S. D u t t o n ,  Adv. Carbohydr. Chem. Biochem., 30 (1974) 9-110.  98  S.W. Gunner, J.K.N. J o n e s , and M.B. P e r r y , (1961) 1892-1899.  99  W.F. Lehnhardt and R.J. W i n z l e r , 479.  Can. J . Chem., 39  J . Chromatogr., 34 (1968) 471-  100  V.N. R e i n h o l d ,  Methods Enzymol., 25_(1972) 244-249.  101  J.S. Sawardeker, J.H. S l o n e k e r , and A.R. J e a n e s , (1965) 1602-1604.  102  I.A. M o r r i s o n ,  103  D.P. Sweet, R.H. S h a p i r o , and P. A l b e r s h e i m , Carbohydr. Res., 40 (1975) 217-225.  104  D.P. Sweet, P. A l b e r s h e i m , and R.H. S h a p i r o , (1975) 199-216.  105  E.C. Conrad and J.K. P a l m e r ,  106  M. M c N e i l , A.G. D a r v i l l , P. Aman, L.-E. F r a n z e n , and P. A l b e r s h e i m , Methods Enzymol., 83 (1982) 3-45.  107  N.K. Kochetkov and O.S. C h i z h o v , Methods Carbohydr. Chem., 6^ (1972) 540-554.  108  J . LOnngren and S. Svensson, Adv. Carbohydr. Chem. Biochem., 29_ (1974) 41-106.  109  D.C. Dejongh, i n W. Pigman and D. H o r t o n ( E d s . ) , "The C a r b o h y d r a t e s " , 2nd edn. , V o l IB, Academic P r e s s , New Y o r k , 1981, pp. 1327-1353.  110  B.A. D m i t r i e v , L.V. Backinowsky, O.S. C h i z h o v , B.M. Z o l o t a r e v , and N.K. Kochetkov, Carbohydr. Res., _19 (1971) 432-435.  111  N.K. Kochetkov and O.S. C h i z h o v , Adv. Carbohydr. Chem. Biochem., 21 (1966) 39-93.  112  V. K o v ^ c i k , S. Bauer, J . R o s f k , and P. Kova'c', (1968) 282-290.  113  V. Kov£cik, S. Bauer, and J . R o s f k , 291-294.  114  J . Karkka'inen,  115  L.S. F o r s b e r g , A. D e l l , D.J. W a l t o n , and C.E. B a l l o u , Chem., 257 (1982) 3355-3363.  A n a l . Chem., 37  J . Chromatogr., 108 (1975) 361-364.  Carbohydr. Res., 40  Food Technology, (1976) 84-92.  Carbohydr. Res. , j5  Carbohydr. Res., 8 (1968)  Carbohydr. Res. , 17 (1971) 1-10. J. Biol.  186  116  A. D e l l , H.R. M o r r i s , H. Egge, and H. von S t r e c k e r , Res., 115 (1983) 41-52.  Carbohydr.  117  A. D e l l and C.E. B a l l o u ,  118  J.N. B e M i l l e r ,  119  B. Capon,  120  R.D. G u t h r i e and J.F. McCarthy, 22 (1967) 11-23.  121  E. P e r c i v a l and R.H. M c D o w e l l , " C h e m i s t r y and Enzymology o f M a r i n e A l g a l P o l y s a c c h a r i d e s " , Academic P r e s s , New Y o r k , 1967.  122  A . J . Mort and D.T.A. Lamport,  123  A . J . Mort and W.D. Bauer,  124  Yu. A. K n i r e l , E.V. V i n o g r a d o v , A.S. Shashkov, B.A. D m i t r i e v , and N.K. Kochetkov, Carbohydr. Res., 112 (1983) C4-C6.  125  A.J. Mort,  126  A.S. P e r l i n ,  127  H.O. Bouveng and B. L i n d b e r g , 53-89.  128  E.F. G a r n e r , I . J . G o l d s t e i n , R. Montgomery, Chem. Soc., 80 (1958) 1206-1208.  129  M. A b d e l - A k h e r , J.K. H a m i l t o n , R. Montgomery, Am. Chem. S o c , 74 (1952) 4970-4971.  130  G.W. Hay, B.A. L e w i s , and F. S m i t h , (1965) 357-361.  131  I . J . G o l d s t e i n , G.W. Hay, B.A. L e w i s , and F. Smithy h y d r . Chem., 5 (1965) 361-370.  Methods Carbo-  132  B. L i n d b e r g , J . LBnngren, W. Nimmich, and U. Rude"n, Scand., 27 (1973) 3787-3790.  A c t a Chem.  133  T. P a i n t e r and B. L a r s e n ,  A c t a Chem. Scand., 24 (1970) 813-833.  134  M.F. I s h a k and T. P a i n t e r ,  Carbohydr. Res., 64 (1978) 189-197.  135  J.E. S c o t t and R.J. H a r b i n s o n ,  136  L. Hough, Methods Carbohydr. Chem., _5 (1965) 370-377.  Carbohydr. Res., 120 (1983) 95-111.  Adv. Carbohydr. Chem. Biochem., 22 (1967) 25-108.  Chem. Rev., 69 (1969) 407. Adv. Carbohydr. Chem. Biochem.,  A n a l . Biochem., 82 (1977) 289-309.  J . B i o l . Chem., 257 (1982)  Carbohydr. Res., ^ ( 1 9 8 3 )  1870-1875.  221-232.  Adv. Carbohydr. Chem., jL4_(1959) 9-61. Adv. Carbohydr. Chem., 15 (1960) and F. S m i t h ,  J . Am.  and F. S m i t h , J .  Methods Carbohydr. Chem., _5  H i s t o c h e m i e , 14 (1968) 215-220.  187  137  K.M. Aalmo, M.F. I s h a k , and T. P a i n t e r , C3-C7.  Carbohydr. Res., 63 (1978)  138  G.G.S. D u t t o n and T.E. Folkman, 161.  139  G.O. A s p i n a l l , i n G.O. A s p i n a l l ( E d . ) , "The P o l y s a c c h a r i d e s " , V o l . 1, Academic P r e s s , New York, 1982, pp. 35-131.  140  B. L i n d b e r g , J . LOnngren, and Svensson, Biochem., 3i_ (1975) 185-240.  141  B. L i n d b e r g and J . LOnngren, 142-148.  142  B. L i n d b e r g , J . LOnngren, and J.L. Thompson, (1973) 351-357.  143  G.O. A s p i n a l l and A.S. C h a u d h a r i , Can. J . Chem., 53 (1975) 21892193.  144  G.O. A s p i n a l l and K.-G. R o s e l l , C26.  145  L. Kenne, J . LOnngren, and S. Svensson, (1973) 3692-3698.  146  E . J . Corey and C.U. Kim, T e t r a h e d r o n L e t t . ,  147  J.H. Van't H o f f , "The Arrangement o f Atoms i n Space", Longmans, Green and Co., London, 1898.  148  C S . Hudson,  149  E.H. M e r r i f i e l d ,  150  L.D. H a l l ,  151  R.U. Lemieux, R.K. K u l l n i g , H.J. B e r n s t e i n , and W.G. S c h n e i d e r , Am. Chem. S o c , 80 (1958) 6098-6105.  152  B. Coxon,  153  R.R.Ernst,  154  A.S. P e r l i n and B. Casu, i n G.O. A s p i n a l l (Ed.) "The P o l y s a c c h a r i d e s " , V o l . 1, Academic P r e s s , New Y o r k , 1982, pp. 133-193.  155  J.F.G. V l i e g e n t h a r t , L. D o r l a n d , and H. van H a l b e e k , hydr. Chem. Biochem., 41 (1983) 209-374.  Carbohydr. Res., 80 (1980) 147-  Adv. Carbohydr. Chem.  Methods Carbohydr. Chem., ]_ (1976)  Carbohydr. R e s . , 28  Carbohydr. Res., 57 (1977) C23A c t a . Chem. Scand., 27 (1973) 919-922.  J . Am. Chem. S o c , 31_ (1909) 66-86. Ph.D. T h e s i s ,  University  o f Cape Town, 1978.  Adv. Carbohydr. Chem., 19 (1964) 51-93. J.  Adv. Carbohydr. Chem. Biochem., 27 01972) 7-83. Adv. Magn. Resonance, 1_ (1966) 1-135.  Adv.  Carbo-  188  156  J.H. P r e s t e g a r d , T.A.W. K o e r n e r , J r . , P.C. Demou, and R.K. Yu, J . Am. Chem. S o c . , 104 (1982) 4993-4995.  157  G. Kotowycz and R.U. Lemieux,  158  M. K a r p l u s ,  159  P . J . Garegg, P.E. J a n s s o n , B. L i n d b e r g , F. L i n d h , and J . LOnngren, Carbohydr. Res., 78 (1980) 127-132.  160  S.L. P a t t and B.D. Sykes,  161  H.J. Jennings and I . C P . S m i t h , 39-50.  162  K. Bock and C. P e d e r s e n , 27-66.  163  J.-P. J o s e l e a u , F. M i c h o n , and M. V i g n o n , (1982) 175-185.  164  K. Bock and C. P e d e r s e n , 293-297.  J . Chem. S o c , P e r k i n T r a n s . 2, (1974)  165  K. Bock and C. P e d e r s e n , 258-264.  A c t a . Chem. Scand., S e r . B, 29 (1975)  166  P.A.J. G o r i n and M. Mazurek,  167  P. C o l s o n and R.R. K i n g ,  168  J.H. Bradbury and G.A. J e n k i n s , 126.  169  P.A.J. G o r i n ,  170  B.A. D m i t r i e v ,  171  A.K. B h a t t a c h a r j e e , H.J. J e n n i n g s , and C P . Kenny, 17_ (1978) 645-651.  172  H.J. J e n n i n g s and A.K. B h a t t a c h a r j e e , 105-112.  173  R.G. S p i r o ,  174  S.J. A n g y a l and K. James,  175  J . Hoffman, B. L i n d b e r g , and S. Svensson, (1972) 661-666.  Chem. Rev., 73 (1973) 669-698.  J . Chem. Phys., 30_ (1959) 11-15.  J . Chem. Phys., 56 (1972) 3182-3184. Methods Enzymol., 50 (1978)  Adv. Carbohydr. Chem. Biochem., 41 (1983) Carbohydr. Res•, 101  Can. J . Chem., 53 (1975) 1212-1223  Carbohydr. Res., 47 (1976) 1-13. Carbohydr. Res., JL26_(1984) 125-  Adv. Carbohydr. Chem. Biochem., 38 (1981) 13-104. Pure & A p p l . Chem., 55 (1983) 655-670. Biochemistry,  Carbohydr. Res., 55 (1977)  Methods Enzymol., 1_ (1966) 26-52. A u s t . J . Chem., 23 (1970) 1209-1221. A c t a . Chem. Scand., 26  189  176  B. L i n d b e r g , B. L i n d q v i s t , J . LOnngren, and W. Nimmich, Carbohydr. Res., 58 (1977) 443-451.  177  V.S. Rao and A.S. P e r l i n ,  178  E.A. Rabat and M.M. Mayer, " E x p e r i m e n t a l Immunochemistry", 2nd edn., C.C. Thomas, S p r i n g f i e l d , I L . , 1961, pp. 493-495.  179  I . Fromme and H. B e i l h a r t z ,  180  A.N. de B e l d e r and B. Norrman,  181  BBL Manual o f P r o d u c t s and L a b o r a t o r y P r o c e d u r e s , P.A. Rhode ( E d . ) , 5th edn., BBL, D i v i s i o n of B e c t o n , D i c k i n s o n and Company, M a r y l a n d 21030, U.S.A., 1973, p. 126.  182  M. C u r v a l l , B. L i n d b e r g , and J . LOnngren, (1975) 95-105.  183  G.G.S. Dutton and A.V. Savage, 362.  184  J . L. D i F a b i o and G.G.S. D u t t o n , 298.  Can. J . Chem., 6l_ (1983) 2688-2694.  A n a l . Biochem., 84 (1978) 347-353. Carbohydr. Res., 8^ (1968) 1-6.  Carbohydr. R e s . , 42  Carbohydr. Res., 83 (1980) 351Carbohydr. Res., 92 (1981) 287-  185. R.D. G u t h r i e , Methods Carbohydr. Chem., ± (1962) 445-447. 186  K. Jann, B. Jann, and K.F. S c h n e i d e r , 456-465.  187  D. Hungerer, K. Jann, B. Jann, F. J . Biochem., 2_ (1967) 115-126.  188  B.B. W i l e y and H.W.  189  A.K. C h a k r a b o r t y ,  190  P.A. S a n d f o r d , J.R. Bamburg, E.D. E p l e y , and T.J. K i n d t , B i o c h e m i s t r y , _5 (1966) 2808-2817.  191  R.E.W. Hancock, Department o f M i c r o b i o l o g y , U n i v e r s i t y Columbia, p e r s o n a l communication.  192  W o r t h i n g t o n Enzyme Manual, W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n , F r e e h o l d , New J e r s e y , U.S.A. 07728, 1972, p. 21.  193  S. S t i r m and E. Freund-MOlbert,  194  G.G.S. D u t t o n and A.V.S. L i m , Carbohydr. Res., 123 (1983) 247257.  Scherp.  E u r . J . Biochem., 5^ (1968)  0rskov, and I . 0rskov, E u r .  Can. J . M i c r o b i o l . , A_ (1958) 505-516.  Macromol. Chem., 183 (1982) 2881-2887.  of B r i t i s h  J . V i r o l . , 8 (1971) 330-342.  190  195  G.M. B e b a u l t and G.G.S. D u t t o n , 213.  Carbohydr. Res., 64 (1978) 199-  196  D.B. B r a d l e y ,  197  J . L e v y , J.J.R. C a m p b e l l , T.H. B l a c k b u r n , " I n t r o d u c t o r y M i c r o b i o l o g y " , John W i l e y & s o n s , i n c . , New Y o r k , 1973.  198  A.A. L i n d b e r g , i n I.W. S u t h e r l a n d ( E d . ) , " S u r f a c e C a r b o h y d r a t e s o f the P r o k a r i o t i c C e l l " , Academic P r e s s , New York, 1977, pp. 289356.  199  S. S t i r m ,  200  M.E. B a y e r , H. Thurow, and M.H. B a y e r , 95-118.  201  H. Geyer, K. Himmelspach, B. K w i a t k o w s k i , S. S c h l e c h t , and S. S t i r m , Pure & A p p l . Chem., 55 (1983) 637-653.  202  D. Rieger-Hug and S. S t i r m ,  203  G.G.S. D u t t o n , K.L. M a c k i e , A.V. Savage, D. Rieger-Hug, and S. S t i r m , Carbohydr. Res., 84 (1980) 161-170.  204  B. K w i a t k o w s k i , B. Boschek, H. T h i e l e , and S. S t i r m , (1982) 697-704.  J . V i r o l . , 43  205  B. K w i a t k o w s k i , B. Boschek, H. T h i e l e , and S. S t i r m , (1982) 367-374.  J . V i r o l . , 45  206  D. R i e g e r , E. Freund-MOlbert, and S. S t i r m , 859-864.  207  W. B e s s l e r , E. Freund-MOlbert, H. Kntffermann, C. Rudolph, H. Thurow, and S. S t i r m , V i r o l o g y , 56 (1973) 134-151.  208  G.G.S. D u t t o n , J . L . D i F a b i o , D.M. Leek, E.H. M e r r i f i e l d , J.R. Nunn, and A.M. Stephen, Carbohydr. Res., 9T_ (1981) 127-138.  209  K.R. Yamamoto, B.M. A l b e r t s , R. B e n z i n g e r , L. Lawthorne, and G. T r e i b e r , V i r o l o g y , 40 (1970) 734-744.  B a c t e r i o l . Rev., 31 (1967) 230-314.  J . V i r o l . , 2 (1968)  1107-1114. V i r o l o g y , 94 (1979)  V i r o l o g y , 113 (1981) 363-378.  J . V i r o l . , 17 (1976)  191  APPENDIX I  THE KNOWN STRUCTURES OF THE Escherichia c o l l 0 Antigens (as of August 1, 1984)  192  APPENDIX I  E. c o l l 0 antigens  —  06  3  14 .  1 4  13  Man — —  Man - r - GlcNAc  8  1  GalNAc  8  —  a  a  'I? Glc  3  07  —  a  lk  , 1 2  13  GlcNac  Qui 4Nac p  Man  1  Gal — a  a  f  3  Rha  Quip4NAc=4-acetamido-4,6-dideoxy-D-glucopyranose  3  08  —  12  Man a  3  09  —  13  Man a  —  a  Rha  16  a  . 3  •I. GlcNAc  Man  —  a  GlcNAc a  a  1  13  Glc a  1  12  Man a  Gal  — a  12  Man  Ik  2  018ac  Man a  12  Man  1  12  Man  — a  193  020  —  Gal  1 2  1  Rha  3  —  025  3  FucNAc  13  a  a i  1 3  1  GlcNAc - 5 - G l c — B 6 P  1  Glc  055  Gal  1 3  GalNAc  1 6  GlcNAc 3 1  P  Gal 2  a  Col  Col=3,6-dideoxy-L-xylopyranose  069  3  —  1 2  GlcNAc - 5 - Rha 8  1 2  a  1  1 2  Rha  Gal a  — a  194  3  075  - GlcNAc  13  a  Gal  i+  1  1 k  -  Rha  1  —  Man  —  086  Gal  GalNAc  GalNAc  I  I  Fuc  Glc  Col 1  0111  GlcNAc  1 h  Glc  1 i+  Gal  6  Col  0114  3  —  GlcNAc  1 4 a  13 1 k 1 Qui 3N - 5 - R i b . - 5 - G a l — r P I P f P P  C=0  I  AcHNCH  } N-acetyl-L-serine  CH 0H 2  where Qui 3N = 3-amino-3,6-dideoxy-B-D-glucopyranose  195  References 06  V. VSisa"nen-Rhen, J . E l o , E. VSisa'nen, A. Stftonen,  I.  0rskov, F. 0rskov, S.B. Svenson, P.H. Ma*kela\ and T.K. Korhonen,  I n f e c t . Immun., 43 (1984) 149.  07  V.L. L'vov, A.S. Shashkov, B„A. D m i t r i e v , N.K. Kochetkov, B. J a n n , and K. J a n n , Carbohydr. R e s . , 126 (1984) 249-259.  08  K. Reske and K. J a n n ,  09  P. Prehm, B. Jann, and K. J a n n , 53-56.  018ac  D.S. Gupta, B. Jann, and K. J a n n , 12th, 1984 A b s t r a c t , p. 373.  I n t . Symp. Carbohydr. Chem.,  020  U.N. V a s i l i e u and I.Y. Z a k h a r o v a , 199-206.  B i o o r g . Chem., 2 (1976)  025  L. Kenne and B. L i n d b e r g ,  055  B. L i n d b e r g , F. L i n d h , J . LOnngren, A.A. L i n d b e r g , and S.B. Svensson, Carbohydr. Res., 97 (1981) 105-112.  069  C. E r b i n g , L. Kenne, B. L i n d b e r g , G. Naumann, and W. Nimmich, Carbohydr. Res., 56 (1977) 371-376.  075  C. E r b i n g , L. Kenne, B. L i n d b e r g , and S. Hammarstrtfm, Carbohydr. Res., 60 (1978) 400-403.  086  G.F. S p r i n g e r ,  0111  K. E k l i n d , P . J . Garegg, L. Kenne, A.A. L i n d b e r g , and B. L i n d b e r g , I n t . Symp. Carbohydr. Chem., 9 t h , 1978 A b s t r a c t , p. 493.  0114  B.A. D m i t r i e v , V.L. Lvov, N.V. Tochtamysheva, A.S. Shashkov, N.K. Kochetkov, B. Jann, and J . Jann, E u r . J . Biochem. ( i n p r e s s ) .  E u r . J . Biochem., 31_ (1972) 320-328. E u r . J . Biochem., 67 (1976)  Carbohydr. Res., 122 (1983) 249-256.  Ann. N.Y. Acad. S c i . , 169 (1970) 134-152.  196  APPENDIX I I  THE KNOWN STRUCTURES OF THE Escherichia c o l l K Antigens (as of August 1, 1984)  197  APPENDIX I I  E. c o l l K antigens  8 2 — NANA5Ac —  a  E. c o l i Kl  —  P  k  Gal  1 2  a  Gly  1(3)  -—(-P —  5  Gal  f  2n  J  12 c  Gly  a  1(3)  x  E. c o l i K2  —  4  14 1 GlcA — — GlcNAc — P a E. c o l i K5  2 12 17 2 — Rib. — 5 - Rib. - 5 - KDO f 8 f P a  or —  —  3 2  Rib.  |  Rib E. c o l i 6a  17  —r-  f  KDO  P  f  E. c o l i K6  )  n  2 —  P  198  ManNAcA ^J  —  1  P  G l c ±K8 6J OAc  E. c o l i K7 and K56  Rha  Rha  a  KDO  a  -  7 / 8  OAc E. c o l i K12 and K82  —  3  Rib  2  17  ^j- KDO  f  ^  OAc E. c o l i  — GalNAc  K13  a  KDO 8  \ P  OAc E. c o l i  — GlcNAc  KDO  a E. c o l i  — Rib  K14  f  p K15  KDO  OAc E. c o l i  K20  199  — Rlb  KDO --p2  f  E. c o l i  K23  E. c o l i  K27  Gal  — Glc B  G l c A -^A F U C 2 or 3 I  1  Gal  OAc E. c o l i  — Man - - - G l c -M" i  3  P  a  K28  G l c A -M" 8 k 1  Glc  a  -M-  Man  pyr E. c o l i  K29  —  V ll  G a l  ip.  a  GlcA -^p - G a l 3  E. c o l i  K30  Gal  200  —  Gal —  Glc —  GlcA  E. c o l i  Rha —  Rha  —  K31  ?Ac  -  V  G l c  fa V  Gal  GlcA E. c o l i  -  Glc a  3  K32  GlcA ^ P V  E. c o l i  —  Gal  a  3  a a +  0  Ac  K33  G a l A - ia- - Fuc a3  E. c o l i  —  Fuc 31 l l Gal  2  i  K42  1  "  Gal - 0 - P - 0 — ^2 Fru  +  E. c o l i  OH °  A c  +  °  P r  K52  GlcA --^- G l c A - - - Man - - - Man - - - GlcNAc - — or 1 1 2 2  t  L  6  L  3  L  3  R h a  CO i l l  K85  Man — 1 Rha  Man -^-- GlcNAc 3  —  201  —  GlcA - i r FucNAc  GlcNAc -^-- G a l 6  ll  j  6  Glc E.  —  NANA5Ac E.  —  Rib  f  ^  c o l i K87  a  NANA5Ac - -r a 2  c o l i K92  ribitol —  E.  2-OAc  0  c o l i K100  T  0 II 1  -  —  202  References Kl  E . J . McGuire and S.B. B i n k l e y ,  B i o c h e m i s t r y , 3_ (1964) 247-251.  K2  K. Jann, B. Jann, and A.M. S c h m i d t , J . B a c t . , 143 (1980) 1108-1115.  K5  W.F. Vann, M.A. Schmidt, B. Jann, and K. Jann, Biochem., 116 (1981) 359-364.  K6  P. Messner and F.M. Unger, (1980) 1003-1010.  K6a  H.J. J e n n i n g s , K.-G. R o s e l l , and K.G. Johnson, 105 (1982) 45-56.  K7=K56  F.-P. T s u i , R.A. B o y k i n s , and W. Egan, (1982) 263-271.  Eur. J .  Biochem. B i o p h y s . Res. Commun., 96,  K12=K82 M.A. Schmidt, B. Jann, and K. J a n n , (1982) 69-74.  Carbohydr. Res.,  Carbohydr. Res., 102  FEMS M i c r o b i o l . L e t t . , 14  K13  W.F. Vann and K. J a n n , I n f e c t . Immun., 25 (1979) 85-92. W.F. Vann, T. Soderstrom, W. Egan, F.-P. T s u i , R. Schneerson, I . 0 r s k o v , and F. 0 r s k o v , I n f e c t . Immun., 39 (1983) 623-929.  K14  B. Jann, P. Hofmann, and K. Jann, (from K. Jann and B. J a n n , Prog. A l l e r g y , 33 (1983) 53-79.  K15  W. Vann, u n p u b l i s h e d r e s u l t s . (From K. Jann and B. J a n n , Prog. A l l e r g y , 33 (1983) 53-79).  K20.K23 W.F. Vann, T. Soderstrom, W. Egan, F.-P. T s u i , R. Schneerson, I . 0 r s k o v , and F. 0 r s k o v , I n f e c t . Immun., 39 (1983) 623-629. K27  K. Jann, B. Jann, K.F. S c h n e i d e r , F. 0 r s k o v , and I . 0 r s k o v , Eur. J . Biochem., 5_ (1968) 456-465. A.K. C h a k r a b o r t y , Macromol. Chem., 183 (1982) 2881-2887.  K28 K29  E. Altman and G.G.S. D u t t o n , Carbohydr. Res., ( i n p r e s s ) . Y.-M. Choy, F. Fehmel, N. Frank, and S. S t i r m , J . V i r o l . , 16 (1975) 581-590.  K30  A.K. C h a k r a b o r t y , H. F r i e l b o l i n , 141 (1980) 971-972.  K31  K. Jann, u n p u b l i s h e d r e s u l t s . (From I . 0 r s k o v , F. 0 r s k o v , B. Jann, and K. Jann, B a c t e r i o l . Rev., 41 (1977) 667-710.  and S. S t i r m ,  J. Bacteriol.,  203 K32  E. A l t m a n ,  unpublished r e s u l t s .  K33  B.A. L e w i s ,  K42  H. Niemann, A.K. C h a k r a b o r t y , H. F r i e b o l i n , and S. S t i r m , B a c t e r i o l . , 133 (1978) 390-391.  K52  P. Hofmann, B. Jann, and K. J a n n , 12th, 1984 A b s t r a c t s , p. 367.  K85  K. Jann, B. Jann, F. 0rskov, and I . 0rskov,  unpublished r e s u l t s . J.  I n t . Symp. Carbohydr. Chem.,  Biochem. Z.,  346 (1966) 368-385. K87  L. T a r c s a y , B. Jann, and K. Jann, 505-514.  E u r . J . Biochem., 23, (1971)  K92  W. Egan, T.-Y. L u i , D. Dorow, J.S. Cohen, J.D. R o b b i n s , E.C. G o t s c h l i c h , and J.B. R o b b i n s , B i o c h e m i s t r y , 16 (1977) 3687-3692.  K100  W. Egan, F.P. T s u i , R. Schneerson, and J.B. R o b b i n s , Chem., ( i n p r e s s ) .  J. Biol.  204  APPENDIX I I I  X  H AND C-n.m.r. SPECTRA 13  Spectrum No. 1  j i 4.0  1  1  '  •  1  »  '  '  T 3.0  K50  Polysaccharide  Spectrum No.  C—n.m.r. 100.6  MHz,  90°  99.16  100  2  K50 Degraded  Spectrum No. 3  Polysaccharide  C-n.m.r. 20.1 MHz, ambient temp.  ho O  103.83  To  K50 P o l y s a c c h a r i d e Compound A3 13  GlcA  12  Man  a H-n•m.r. 100 MHz,  13  Man a  Gal^OH a  90°  5.31  Spectrum No.  4  K50 P o l y s a c c h a r i d e Compound Aj£ Gl c A ^ — ^ l a n ^ M a n ^ G a l ^ O H 13  C-n.m  r;  20.1 MH;;, a n b i e n t  :emp  _L. 101.  7f  103 i  !9 -\t  41 i .95 i o ,93 o/  J  I  100  L  Spectrum No. 6  K50 P o l y s a c c h a r i d e Compound A2 13  GlcA  1 2  Man a  ManMDH a  1„ H-n.m. r .  100 MHz, 90°  5.37  5.32  -i—i—i—i—|—i—i—I—i—I—I—i—I—I  |  i  5.0  i  r  -I—i  |  I—I—i—I—[—i—i—I—I  2.0  |  i  I i  r  100  Spectrum No. 8  K50 P o l y s a c c h a r i d e Compound GlcA^-Alan^OH  „  H-n.m.r. 100 MHz,  90°  ?7o  JTo  K50 P o l y s a c c h a r i d e Compound SHI \/\f\j J  Spectrum No. 9  r  C0 H 2  CHOH  1 3  1 2 I  Gal—r-Glc OCH 8 a | CH OH 13  0  C—n.m.r. 400 MHz, ambient temp.  4.65  T" 5.0  i' 3.0  K50 P o l y s a c c h a r i d e Compound SHI  T V  Spectrum No. 10  CH0H  104.11  Gal-Lr^GlcrL-^OCH 6 a | CH 0H 2  H-n.m.r. 100.6 MHz, ambienjt temp, 99.62  acetone 31.07  1— 100  E. c o l l  Spectrum No. 12  K28 P o l y s a c c h a r i d e  13_ C-n.m. r . 100.6 MHz, ambient temp.  acetone 31.07  to I—  16.02 ,15.97  I'OO  90  8b  70  AO  30  20  1  E. c o l i K28 D e a c e t y l a t e d  Spectrum No.  Polysaccharide  15  L—n. m. r •  100.6 MHz, ambient temp. acetone 31.07  K3 i—  0  1  -r—  5.0  1  "  1  4.0  1—  3.0  1—  2.0  E. c o l i K28 Compound A^  Spectrum No. 17  1- 4  GlcA——Fuc^OH ~^C—n. m. r . 20.1 MHz, 95  c  iL03J8A  -97 04~ •r~  i...  •ti 3:. to  .100  90  80  E. c o l i K28 Compound N l Gal-V^lc OH p %  H-n.m. r. 4.00 MHz,  ambient temp.  |  .  !  !  ! I . ;  II  ;  i I  1  !  .:  i i : . j :  .  '  '  .  j i I  •  i  5.23  — i —  - i —  4.0  3.0  223  E. c o l i K32 ^H-n.m.r. 400 MHz,  95°  Polysaccharid  E. c o l l K32 D e a c e t y l a t e d  Polysaccharide  Spectrum No . 22 1 .34  *H-n.m.r. 400 MHz,  95°  acetone 2.23  — i —  5.0  4  2.0  Spectrum No.  E. c o l i K32 D e a c e t y l a t e d C—n.m.r.  Polysaccharide  acetone 31.07  100.6 MHz, ambient temp.  18.01  23  E. c o l l K28/bacteriophage <p28—1 ^H-n.m.r. 400 MHz, 95°  4.0  5.0 r  E. c o l i K28/bacteriophage <j>28-l  Spectrum No. acetone 2.23  _575  ,~  '  :  ATO  2.0  r  25  E. c o l i K32/bacteriophage Fraction I  1„ H-n.m.r. 400 MHz, 95°  $32  E.  coli  K32/bacteriophage  Fraction  <j>32  Spectrum No.  II  1.12  ^H-n.m.r.  400 MHz, 95°  5.0  4.0  2.0  27  

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