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Studies on bacterial capsular polysaccharides and on a plant gum Di Fabio, Jose Luis 1981

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STUDIES ON BACTERIAL CAPSULAR POLYSACCHARIDES AND ON A PLANT GUM  by JOSE LUIS LDI FABIO B.Sc., U n i v e r s i d a d de l a R e p u b l i c a , Uruguay, 1 9 7 5  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE  FACULTY OF GRADUATE STUDIES (Department o f Chemistry)  We accept t h i s t h e s i s as conforming to the r e q u i r e d  THE  standard  UNIVERSITY OF BRITISH COLUMBIA May, 1 9 8 1  ©  Jose' L u i s D i F a b i o , 1 9 8 1  In  presenting  requirements  this  thesis  f o ran advanced  of  British  it  freely available  agree for  that  i n partial  Columbia,  I agree  that  f o rreference  permission  scholarly  degree  at the University  the Library  shall  and study.  I  f o rextensive  copying  p u r p o s e s may b e g r a n t e d  o r by h i so r h e r r e p r e s e n t a t i v e s .  understood  that  copying  f i n a n c i a l gain  or publication  shall  n o t be a l l o w e d  permission.  Department o f  CH€HJS[«y  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e Vancouver, Canada V 6 T 1W5  Date  i?/7<n  ^ 5 / P f S / / V a *  Columbia  make  further  of this  thesis  b y t h e h e a d o f my  department  for  DF-fi  fulfilment ofthe  I t i s  of this without  thesis my  written  ABSTRACT  The structure of the capsular polysaccharides from K l e b s i e l l a serotype K60 and K26 have been determined using the techniques  of methylation.periodate o x i d a t i o n , p a r t i a l  1  13  hydrolysis, and B-elimination. H- and  ^C-n.m.r. spectros-  copy was used to e s t a b l i s h the nature of the anomeric l i n kages i n both polysaccharides and a l s o i n the oligosaccharides obtained by the d i f f e r e n t degradative  techniques  used. S p e c i f i c hydrolases obtained from bacteriophages were u t i l i z e d to degrade two K l e b s i e l l a  polysaccharides.Larger  quantities of oligosaccharide repeating units can be generated i n t h i s manner.Two bacteriophages, f i r s t one with  060  and  046,the  B-glucosidase a c t i v i t y and the other with  8 -galactosidase activity,were used to degrade the corresponding  polysaccharides according to a new,simplified  procedure. The p u r i f i e d gum exudate from Chorisia speciosa(palo borracho) was studied.The  r e s u l t s from methylation anal-  y s i s , B -elimination and p a r t i a l hydrolysis made possible a tentative assignment f o r an " average structure " of the gum polysaccharide.  i i i  D-G l C £ 1  K60  —^D-Glcrr —^D-GlcpA^—^D-Galp^—2.D-Man£^— 1  &  21 l l D-G1C2  a  2• 1 D-G1C2  P  k 6  D-Gal£ 1[  D-Glcp If  6 D-Glc£  K26  •D-Galj^—^D-GICEA-  -^D-Man£^—-D-ManjA  "  iv TABLE OF CONTENTS Page  ABSTRACT  i i  TABLE OF CONTENTS  iv  LIST OF TABLES  ix  LIST OF FIGURES  xi  LIST OF SCHEMES  xiii  ACKNOWLEDGEMENTS  xiv XV  PREFACE I  INTRODUCTION  1  II  METHODOLOGY OF STRUCTURAL STUDIES 20  ON POLYSACCHARIDES 11.1  The structures of polysaccharides.  11.2  I s o l a t i o n and p u r i f i c a t i o n  11.3  ...  22  11.2.1  K l e b s i e l l a polysaccharides  11.2.2  Gum exudate of Chorisia speciosa  . . .  23 23  Sugar analysis  Zk  11.3.1  Total hydrolysis  Zk  11.3.2  Characterization and quantitation of the sugars . . .  11.3.3  Position of linkage  II.k.1  25  Determination of the configuration of the sugars. . .  TL.h  21  Methylation  26 28  analysis  .28  II.k.1.1  Methylation  procedures .  11.^.1.2  Characterization of the methylated sugars. . . .  28  29  V  11.5  Sequencing  11.5.1  36  o f sugars  36  P a r t i a l hydrolysis  11.5.2 P e r i o d a t e o x i d a t i o n and Smith 38  hydrolysis  11.5.3 D e g r a d a t i o n based on B - e l i m i n a t i o n .  41  11.5.3.1 Base c a t a l y z e d u r o n i c a c i d degradation  43  11.5.3.2 D e g r a d a t i o n preceded by oxidation  44  11.5.4 S e p a r a t i o n o f oligomers obtained ^7  from d e g r a d a t i o n s 11.6  D e t e r m i n a t i o n o f the l i n k a g e s  11.6.1  Optical rotation  11.6.2  Nuclear magnetic  49  49 resonance.  . . .  11.6.2.1 "--H-n.m.r 11.6.2.2 C-n.m.r 1:3  11.6.3 11.7  Other techniques . . . . . . . .  51 57 60  V a r i o u s r e a c t i o n s on o l i g o - and p o l y saccharides  61  II.7.1  Reduction  61  I I . 7.2  Oxidation  64  I I I GENERAL EXPERIMENTAL CONDITIONS 111.1  Paper chromatography  111.2  G a s - l i q u i d chromatography and g.l.c.-m.s.  Gel-permeation  65 66  66  spectroscopy 111.3  51  chromatography  67  vi  .  O p t i c a l r o t a t i o n and c i r c u l a r d i c h r o i s m  111.5  Nuclear magnetic resonance  68  111.6  General  69  111.7  I s o l a t i o n and p u r i f i c a t i o n o f the p o l y -  conditions  69  saccharides 111.7.1 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  . . . .  111.7.2 Gum exudate o f C h o r i s i a s p e c i o s a .  69 70  111.8  Sugar a n a l y s i s  71  111.9  Methylation analysis  72  111.10 Base 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 IV  68  III.4  .  75  STRUCTURAL INVESTIGATIONS OF KLEBSIELLA 77  CAPSULAR POLYSACCHARIDES IV.1  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 serotype  IV.2  K60 c a p s u l a r p o l y s a c c h a r i d e .  78  IV. 1.1  Abstract  78  IV. 1.2  Introduction  78  IV. 1.3  R e s u l t s and d i s c u s s i o n  79  IV. 1.4  Experimental  96  Structural investigation of K l e b s i e l l a serotype K26 c a p s u l a r p o l y s a c c h a r i d e .  IV.3  . .  . .  106  IV. 2.1  Abstract  106  IV. 2.2  Introduction  107  IV.2.3  R e s u l t s and d i s c u s s i o n  107  IV. 2.4  Experimental  121  Bacteriophage  degradation  of 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 K60 and K46  129  VI1  V  VI  IV. 3 . 1  Introduction  129  IV.3.2  Results  132  IV.3.3  Discussion.  152  IV. 3 . 4  Experimental  155  STRUCTURAL STUDIES OF THE GUM EXUDATE OF CHORISIA SPECIOSA.  161  V.l  Abstract  162  V.2  Introduction  162  V.3  R e s u l t s and d i s c u s s i o n  164  V.4  Experimental  172  BIBLIOGRAPHY  177  • • •  V l l l  APPENDIX I  S t r u c t u r a l patterns of K l e b s i e l l a  Page capsular 190  polysaccharides II  The s t r u c t u r e s o f K l e b s i e l l a c a p s u l a r saccharides  I I I H - and ^C-n.m.r. s p e c t r a 1  IV  Uses o f 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  poly195 212 2^5  Ix LIST OF TABLES Page TABLE 1.1  K l e b s i e l l a capsular polysaccharides ( K l K83).Qualitative type  TABLE 1.2  grouping.  Aldobiouronic acids i n K l e b s i e l l a sular  TABLE 1.3  a n a l y s i s and chemo-  cap11  polysaccharides  S u b s i t u t i o n p a t t e r n o f the sugar r e s i dues i n the K l e b s i e l l a c a p s u l a r p o l y -  13  saccharides TABLE 1.1*  Percentage composition glycosidic sular  TABLE 1 . 5  o f a - and B -  l i n k a g e s i n K l e b s i e l l a cap16  polysaccharides  L o c a t i o n o f pyruvate capsular  i n Klebsiella  17  polysaccharides  TABLE IV.1.1 N.m.r. data f o r K l e b s i e l l a K60 c a p s u l a r p o l y s a c c h a r i d e and d e r i v e d p o l y - and 81  oligo-saccharides TABLE IV.1.2 M e t h y l a t i o n a n a l y s i s o f K 6 0 c a p s u l a r p o l y s a c c h a r i d e and d e r i v e d  87  products  TABLE IV.1.3 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 o f K60 p o l y s a c c h a r i d e  9^  TABLE IV.2.1 N.m.r. data f o r K l e b s i e l l a K26 c a p s u l a r p o l y s a c c h a r i d e and d e r i v e d ,poly- and  109  oligo-saccharides TABLE IV.2.2 M e t h y l a t i o n a n a l y s i s o f K26 c a p s u l a r p o l y s a c c h a r i d e and d e r i v e d  116  products  TABLE IV.2.3 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 o f K26 p o l y s a c c h a r i d e TABLE IV.3.1 Propagation  o f bacteriophages  060  and  119  ffo6  13  X  TABLE I V . 3 . 2  D e p o l y m e r i z a t i o n o f K l e b s i e l l a K60 and K46 c a p s u l a r p o l y s a c c h a r i d e s by b a c t e r i o p h a g e s 060 and 046 r e s p e c t i v e l y  134  TABLE I V . 3 . 3 a P.m.r. d a t a f o r K l e b s i e l l a K 6 0 c a p s u l a r p o l y s a c c h a r i d e and the oligomers d e r i v e d from bacteriophage d e g r a d a t i o n  138  TABLE I V . 3 - 3 b N.m.r. ( ^ C ) d a t a f o r K l e b s i e l l a K 6 0 c a p s u l a r p o l y s a c c h a r i d e and the oligomers d e r i v e d from bacteriophage d e g r a d a t i o n TABLE I V . 3 - ^  140  D e t e r m i n a t i o n o f the degree o f 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 o f K 6 0 o l i g o s a c charides  TABLE I V . 3  (P^ and P ) 2  M e t h y l a t i o n a n a l y s i s o f K60 o l i g o s a c c h a r i d e s (P.^ and P ) from bacteriophage d e g r a d a t i o n 2  143  TABLE I V . 3 6a P.m.r data f o r K l e b s i e l l a K46 c a p s u l a r p o l y s a c c h a r i d e and the oligomers d e r i v e d from bacteriophage d e g r a d a t i o n  146  TABLE IV. 3 - 6b N.m.r. ( " ^ O data f o r K l e b s i e l l a K46 c a p s u l a r p o l y s a c c h a r i d e and the oligomers d e r i v e d from bacteriophage d e g r a d a t i o n TABLE I V . 3 > 7  148  D e t e r m i n a t i o n o f the degree o f p o l i m e r i z a t i o n and the r e d u c i n g end o f K46 o l i g o s a c charides  TABLE I V . 3 8  (P^ and Pg)  M e t h y l a t i o n a n a l y s i s o f K46 o l i g o s a c c h a r i d e s (P.^ and Pg) from bacteriophage d e g r a d a t i o n  TABLE V . l  165  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 o f the gum exudate  TABLE V . 3  151  Methylation analysis of Chorisia speciosa gum exudate and d e r i v e d products  TABLE V . 2  150  167  N.m.r. ("Hi) d a t a o f the a c i d i c 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 o f the gum  172  xi LIST OF FIGURES  F i g u r e 1.1  ..Page  Diagrammatic r e p r e s e n t a t i o n o f the b a c t e r i a l c e l l envelope and d i f f e r e n t  F i g u r e 1.2  a)Klebsiella  K5 c r o s s - r e a c t s w i t h  to Pneumococcus I I I ; b ) K l e b s i e l l a r e a c t s w i t h anti-serum  3  antigens anti-serum K26 c r o s s -  to K l e b s i e l l a K l l  6  F i g u r e I I . 1 M.s. o f a) 1 , 2 , 5 - t r i - 0 - a c e t y l - 3 , 4 , 6 - t r i - 0 m e t h y l g a l a c t i t o l and b) l , 2 , 5 - t r i - 0 - a c e t y l 3-0-ethyl-4,6-di-0-methylgalactitol F i g u r e I I . 2 Common products  formed on p e r i o d a t e  33 oxidation  o f t e r m i n a l and monosubstituted hexoses Figure I I . 3  R e l a t i o n s h i p between d i h e d r a l angle(0) and c o u p l i n g constant  Figure I I . 4  for  a - and  e -D-hexoses  5-^  The """H-n.m.r. spectrum o f K l e b s i e l l a K 6 0 56  capsular polysaccharide Figure I I . 5  40  The ^C-n.m.r. spectrum o f K l e b s i e l l a K 6 0 58  capsular polysaccharide F i g u r e IV.1 S e p a r a t i o n o f the a c i d i c  oligosaccharides  from p a r t i a l h y d r o l y s i s o f P^ by gel-permea92  t i o n chromatography F i g u r e IV.2  S e p a r a t i o n o f the a c i d i c  oligosaccharides  from 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 K26 polysaccharide  by gel-permeation  chromato115  graphy Figure IV.3 Attack  o f a bacteriophage  on an encapsulated 131  bacteria F i g u r e IV.4  S e p a r a t i o n o f the d e p o l y m e r i z a t i o n of K60  by gel-permeation  chromatography  F i g u r e I V . 5 S e p a r a t i o n o f the d e p o l y m e r i z a t i o n of K46 by gel-permeation  products 136  products  chromatography  145  xii Figure IV.6  Environment of the g l y c o s i d i c linkage which undergoes enzymic hydrolysis (K60) and the one that does not (K60SH)  Figure IV.?  Growth curve and bacteriophage l y s i s of K l e b s i e l l a K60 bacteria  Figure V . l  One of the possible "average structure" f o r the gum of Chorisia.speciosa  xiii LIST OF SCHEMES Page  Scheme II.1 Methylation analysis of K l e b s i e l l a K26  JO  Scheme I I . 2 Smith degradation of K l e b s i e l l a K26  42  Scheme II.3 Uronic acid degradation of K l e b s i e l l a K26 polysaccharide  45  Scheme II.4 Degradation preceded by oxidation on the K l e b s i e l l a K26 polysaccharide  46  Scheme I I . 5 Reduction of a carboxylic acid i n aqueous solution using a carbodiimide reagent  63  xiv  ACKNOWLEDGMENTS  The  d i r e c t i o n and i n t e r e s t o f Prof. G.G.S. Dutton d u r i n g  t h i s study i s g r a t e f u l l y acknowledged. I wish t o thank  my  c o l l e g u e 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 , s p e c i a l l y Dr. A. Savage,and a l s o the P r o f e s s o r s who v i s i t e d  the l a b o r a t o r y  d u r i n g t h i s time f o r t h e i r h e l p f u l d i s c u s s i o n s . S i n c e r e  thanks  to Dr. E.H. M e r r i f i e l d f o r h i s a s s i s t a n c e and p r o o f - r e a d i n g of t h i s t h e s i s , and a l s o Dr.S.C Churms ( U n i v e r s i t y o f Cape Town) f o r the molecular  weight  determinations.  My s p e c i a l thanks t o my w i f e , E t e l , f o r her encouragement and  support  i n this  venture.  I am g r a t e f u l t o Mac M i l l a n B l o e d e l f o r the award o f a Graduate S c h o l a r s h i p  (1977-1978) and a l s o the U n i v e r s i t y o f  B r i t i s h Columbia f o r the award o f a U n i v e r s i t y Graduate F e l lowship(1980-1981).  XV  PREFACE  One o f the most o u t s t a n d i n g saccharides  i s t h e i r ordered  features of "bacterial poly-  s t r u c t u r e 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 units.These  s u r f a c e carbohydrates  can  be e x t r a c t e d from b a c t e r i a and n o t only s t u d i e d  chemically,  but a l s o used as antigens.Such s t u d i e s a r e very  important  i n the l i g h t o f b a c t e r i a l i n f e c t i o n s and the p r o d u c t i o n o f protective vaccines.Oligosaccharides polysaccharides  can be coupled  obtained  from  these  t o c a r r i e r s and used f o r the  i n d u c t i o n o f a n t i b o d i e s . A p r o l i f i c source  o f enzymes which  depolymerize the c a p s u l a r p o l y s a c c h a r i d e s  into their  oligo-  meric r e p e a t i n g u n i t s are the bacteriophages. The  study on gum exudates has been c a r r i e d out f o r many  years.The main g o a l i s t o f i n d s u b s t i t u t e s f o r the ones a l r e a d y known o f commercial v a l u e , and f o r t h e i r p o t e n t i a l use  i n chemical  taxonomy o f p l a n t s .  1  I .  INTRODUCTION  2  I. -  INTRODUCTION The  outermost s u r f a c e  o f any organism i s o f g r e a t im-  portance i n the i n t e r r e l a t i o n between i t s e l f and i t s e n v i ronment, the and  outermost mediator i s the f i r s t p o r t a l o f e n t r y  the l a s t b a r r i e r t o e x c r e t i o n . I n microorganisms i t must  p l a y an important r o l e i n the r e c o g n i t i o n o f the c e l l by viruses,antibodies,etc. A b a c t e r i a l c e l l w a l l i s composed o f s e v e r a l l a y e r s (see F i g u r e glycan  1.1), i ) the cytoplasmic  membrane,ii) a p e p t i d o -  (murein) l a y e r and i i i ) an outer membrane composed o f  lipopolysaccharides,proteins  and  polysaccharides.  Many b a c t e r i a produce p o l y s a c c h a r i d e s outside  the c e l l wall^". These p o l y s a c c h a r i d e s  form of capsules attached  which a r e l o c a t e d may take the  or they may be an e x t r a c e l l u l a r slime un-  t o the b a c t e r i a l s u r f a c e . M e d i c a l  microbiologists  were f i r s t i n t e r e s t e d i n these macromolecules as they p l a y an important r o l e i n the p a t h o g e n i c i t y p tigens,toxins)  o f the b a c t e r i a ( a n -  .The i n t e r e s t i n c r e a s e d w i t h the i n d u s t r i a l  need o f new g e l l i n g and e m u l s i f y i n g agents (e.g. Xanthomonas campestris^  produces an exopolysaccharide.xanthan gum,which  i s used i n food  industry).  Although the f u n c t i o n o f these s t r u c t u r e s i s n o t w e l l understood,they may be i n v o l v e d ing^  i n one or a l l o f the f o l l o w -  : i)  storage  or r e s e r v e ,  ii)  v i r u l e n c e i p r o t e c t i o n against  i i i ) protection against  predation,  phagocitosis,  Capsular polysaccharide ( K antigen)  Lipopolysaccha-: ride  ( 0 antigen)  Outer membrane  Peptidoglycan layer  Cytoplasmic  oooooooooooa  membrane  Cytoplasm  F i g u r e 1.1 .Diagrammatic  r e p r e s e n t a t i o n o f the b a c t e r i a l  c e l l envelope and the d i f f e r e n t a n t i g e n s .  iv) protection against desiccation, v)  adhesion,  v i ) general b a r r i e r s . The immunological defence system of higher organisms (e.g. mammals) i s based on i t s capacity to recognise structures as foreigners or as part of i t s e l f . I f the foreign structure has the property of inducing the formation of a n t i bodies, i t i s c a l l e d an antigen and the property immunogenicity.After an antibody i s produced i t w i l l react s p e c i f i c a l l y with the antigen that originated i t s production,this i s c a l l e d antigenic specificity.However,there are small  molecules.haptens,  which,by themselves.cannot stimulate antibody synthesis but w i l l combine with the antibody once formed. Bacteria,when introduced into the blood stream of a mammal.will induce t h i s immune response.Certain structures on the c e l l surface of the microorganism are antigens^,e.g. capsular material ( K antigens.protein or polysaccharide i n nature) .lipopolysaccharides ( 0 a n t i g e n s ) . f l a g e l l a (H antigens,prot e i n i n nature). B a c t e r i a l capsular polysaccharides have been recognised as antigens^''''for a long time and most of the pioneering work on quantitative immunochemistry was i n i t i a t e d by Heidelberger R—1 0  and coworkers ~  on pneumococcal capsular polysaccharides and  t h e i r antigenic s p e c i f i c i t i e s . The purpose of the immunochemical analysis of polysaccharides i s to find s p e c i f i c structures i n them,which are the chemical expression of the immunological character.These structures or antigenic determinants  can be a single sugar linked i n  5  a s p e c i f i c manner,an o l i g o s a c c h a r i d e te i n nature  (e.g. p o l y s a c c h a r i d e s  or even non-carbohydra-  with k e t a l - l i n k e d  pyruvate).  There i s some c o n c l u s i v e evidence t h a t charged c o n s t i t u e n t s on polysaccharides  ( u r o n i c a c i d s or other a c i d i c r e s i d u e s ) are  o f t e n p a r t o f the a n t i g e n i c determinant or the immunodominant sugar.One p o l y s a c c h a r i d e  can h a v e ' d i f f e r e n t a n t i g e n i c determi-  nants due t o d i f f e r e n t sugar r e s i d u e s  or d i f f e r e n t  t i o n s o f them.As the b a c t e r i a l p o l y s a c c h a r i d e s oligosaccharide  u n i t s t h a t repeat,the  combina-  a r e formed  antigenic  of  determinants  are expressed many times over. The  same a n t i g e n i c determinant may be present  polysaccharides.As  a consequence,it i s r e c o g n i s e d  i n several by i t s homo-  logous a n t i b o d y i n the d i f f e r e n t p o l y s a c c h a r i d e s . n o  matter i n  what organism they a r e produced.These p o l y s a c c h a r i d e s immunologically (see F i g u r e  r e l a t e d are s a i d  I.2)^®.This  serologically  immunological r e a c t i o n has been used  i n g e n i o u s l y by H e i d e l b e r g e r l o g i c a l determination  to cross-react  t h a t are  and coworkers"*"^"^ f o r the immune^  o f the s t r u c t u r e o f many unknown p o l y -  s a c c h a r i d e s . T h i s approach i s based on the f a c t t h a t the c r o s s r e a c t i o n of a polysaccharide antibodies  o f known chemical s t r u c t u r e w i t h  to a polysaccharide  t u r e may y i e l d  o f u n c e r t a i n o r unknown s t r u c -  i n f o r m a t i o n as t o one or more sugars  contained  i n the unknown and even as t o the p o s i t i o n s a t which the s u gars are l i n k e d ; c o n v e r s e l y , c r o s s - r e a c t i v i t y o f a r i d e o f known composition and l i n k a g e  polysaccha-  ,may be e q u a l l y  infor-  mative. The  Gram-negative f a m i l y E n t e r o b a c t e r i a c e a e  i s o f wide  6 a) Pn I I I  _ 2 D-GlcjpA - — - D-Glcj?  D-GlcpA - — - D-Glcp  K-5  "  D-Manjj  ? —  2  6AC  b) K  11  D-Glcjp - — 2 . D-GlcjpA 6  4  D-Galp  a  / 1 ,'D-Galp !  K 26  —a  D-Galp. ~ — -  D-GICJDA  - — 2 . D-Manp_ - — -  D-Marffi  1| D-Glcp_  6  1| D-GlC£  4|  / 1 'D-Galp ,  F i g u r e 1.2  a) K l e b s i e l l a K 5 c r o s s - r e a c t s w i t h a n t i serum t o Pneumococcus I l l . b ) K l e b s i e l l a  K26  c r o s s - r e a c t s w i t h anti-serum to K l e b s i e l l a K  11.  —  7 i n t e r e s t because i t i n c l u d e s pathogens f o r man and other a n i mals, e.g. Salmonella sp. and S h i g e l l a sp. a r e common cause o f food p o i s o n i n g . Y e r s i n i a p e s t i s i s the cause o f bubonic By combination  plague.  o f m o r p h o l o g i c a l and b i o c h e m i c a l c h a r a c t e r i s t i c s  t h i s f a m i l y has been d i v i d e d i n 5 tribes"""'': E s c h e r i c h i e a e , K l e b s i e l l e a e . P r o t e c e a e . Y e r s i n e a e and Erwinieae which are then s u b d i v i d e d i n s e v e r a l genuses. The genus K l e b s i e l l a  (tribe Klebsielleae  ) i s composed o f  three species,K.pneumoniae,K.ozaenae and K . r h i n o s c l e r o m a t i s . These microorganisms  a r e n o r m a l l y found  i n healthy c a r r i e r s  i n the upper r e s p i r a t o r y , i n t e s t i n a l and g e n i t o - u r i n a r y t r a c t s . They may become pathogenic ble  and have been shown t o be r e s p o n s i -  f o r some r e s p i r a t o r y d i s e a s e s (3$ o f b a c t e r i a l pneumonias).  They can be i s o l a t e d from feces,pus,blood.abscesses.bones  and  j o i n t s " * " ^ . K l e b s i e l l a c u l t u r e s were c l a s s i f i e d s e r o l o g i c a l l y on 17 18 the b a s i s o f t h e i r K ( c a p s u l a r ) and 0 (somatic) a n t i g e n s '* As the number o f 0 types i s s m a l l (11) compared w i t h the K types ( 80) and as the d e t e r m i n a t i o n i s d i f f i c u l t  because  of  the heat s t a b l e K a n t i g e n s . d e t e r m i n a t i o n o f the s e r o l o g i c a l type i s p r i m a r i l y based  on the K determination.The  capsule (K 19 a n t i g e n ) has been demonstrated t o be carbohydrate i n nature . Many immunological r e l a t i o n s h i p s ( c r o s s - r e a c t i o n s ) have been 12 7  demonstrated between the K a n t i g e n s o f K l e b s i e l l a ves and w i t h some K a n t i g e n s o f other b a c t e r i a  themsel-  ( Pneumococci)  13 14 J  *  .As t h i s c r o s s - r e a c t i v i t y i s based  on the r e c o g n i t i o n of  p a r t i a l s t r u c t u r e s o f the a n t i g e n by the antibody or  immune c e l l receptors.knowledge  molecules  o f the s t r u c t u r e o f the  8 carbohydrate  a n t i g e n i s fundamental.For t h i s reason a s t r u c -  t u r a l i n v e s t i g a t i o n of a l l K l e b s i e l l a capsular polysaccharides has been undertaken. Klebsiella  polysaccharides  K l e b s i e l l a polysaccharides,as  most o f the b a c t e r i a l ex20  t r a c e l l u l a r heteropolysaccharides  ,are formed o f o l i g o s a c c h a -  r i d e u n i t s composed o f 3 t o 7 sugar r e s i d u e s . w h i c h g u l a r l y t o b u i l d up the h i g h molecular  weight  repeat r e -  polysaccharide  ( 2*10-' - 2*10? d a l t o n s ) . Q u a l i t a t i v e a n a l y s i s o f the p o l y s a c c h a r i d e s o f the approximately 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 21 22 types was done by Nimmich  '  the c a p s u l a r p o l y s a c c h a r i d e s  .which l e d t o the grouping o f 2  T h i s f i r s t d i v i s i o n i s n o t enough t o e x p l a i n t h e i r immunological The  1.1).  i n t o chemotypes -^(see Table  different  behaviour.  p o l y s a c c h a r i d e s are a l l a c i d i c and t h i s i s due t o  the presence o f u r o n i c a c i d s j D - g l u c u r o n i c a c i d i s the most common and D - g a l a c t u r o n i c The  a c i d i s found i n a number o f s t r a i n s .  capsular polysaccharides  o f K22,  K37 and K38 have been  shown t o c o n t a i n r a r e u r o n i c a c i d s . I n p o l y s a c c h a r i d e s  devoid  o f u r o n i c a c i d s ( K32, K56 and K72 ) the a c i d i t y i s due t o p y r u v i c a c i d t h a t i s l i n k e d as an a c e t a l t o a sugar r e s i d u e . P y r u v i c a c i d a l s o supplements the a c i d c o n t e n t . i n other a c i d containing K l e b s i e l l a polysaccharides.The  uronic  n e u t r a l sugars  found are the common hexoses, D-glucose, D-galactose  and D-  mannose, and the 6-deoxysugars,L-rhamnose and L-fucose.Noncarbohydrate present  s u b s t i t u e n t s as a c e t y l o r f o r m y l may a l s o be  i n a d d i t i o n t o the monosaccharides c o n s t i t u e n t s .  9 GlcA  Gal  Glc  8 ,ll ,15,25,2? ,51  GlcA  Gal  Man  20,21 ,29 ,42 ,43,66,74  GlcA  Gal  Rha  9,9*.47,52,81,83  GlcA  Glc  Man  2,4,5 ,24  GlcA  Glc  Rha  17,23,44,45  GlcA  Glc  Fuc  GlcA  Gal  Glc  1,5^ 7 ,10,13 ,26 ,28,30 ,31 , 33 ,35 ,39,^6 ,50,59.60,  p  p  p  P  P  P  P  P  Man  P  P  P  ,71 P  P  6l,62,69  P  P  P  P  GlcA  Gal  Glc  Fuc  16,58  GlcA  Gal  Glc  Rha  12 ,18,19,36 .41,55 ,70 ,  P  P  P  P  P  79 GlcA  Gal  Man  Rha  40,53,80  GlcA  Glc  Man  Fuc  6  GlcA  Glc  Man  Rha  GlcA  Gal  Glc  Man  Fuc  64 ,65 68  GlcA  Gal  Glc  Man  Rha  14 ,67  GalA  Gal  Man  GalA  Glc  Rha  3 ,49,57 34,48  GalA  Gal  Fuc  63  PyrA  Glc  Rha  72  PyrA  Gal  Rha  32  PyrA  Gal  Glc  KetoA  Gal  p  P  P  P  p  P  P  56  Rha  22,37.38  Glc  GlcA  glucuronic acid  Gal  galactose  GalA  galacturonic  Man  mannose  PyrA  pyruvic acid  Rha  rhamnose  KetoA  rare uronic acid  Fuc  fucose  Glc  glucose  P  pyruvic acid pre-  acid  sent i n a d d i t i o n TABLE I.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 (KI-K83) Q u a l i t a t i v e a n a l y s i s and chemotype grouping.  10  The s t r u c t u r e s of f i f t y - f i v e K l e b s i e l l a  polysaccharides  are known t o date and they may be c l a s s i f i e d a c c o r d i n g s t r u c t u r a l pattern,as  t o the  shown i n Appendix I. I n s p i t e o f the great  d i v e r s i t y o f s t r u c t u r a l patterns,added  t o the p o s s i b l e m u l t i p l e  permutations o f the d i f f e r e n t sugar r e s i d u e s i n the r e p e a t i n g u n i t , t h e r e are c e r t a i n combinations o f sugars acid,see  Table  1.2 ) . s u b s t i t u t i o n s ( s e e Table  linkages  (see Table  1.3) and anomeric  1.4) t h a t a r e p r e f e r r e d :  i ) Aldobiouronic are present  (aldobiouronic  together  acids;D-Glucuronic  a c i d and D-glucose  i n 5 0 s t r a i n s , b u t of the 3 5 s t r u c t u r e s  known t h a t c o n t a i n them,only i n two a r e they combined t o form the a l d o b i o u r o n i c a c i d . D - G l u c u r o n i c  a c i d and D-galactose form  the a l d o b i o u r o n i c a c i d s o f 12 s t r a i n s o f known s t r u c t u r e , a l though GlcA 24 gums  1 4  1 6  G a l and GlcA  a  G a l are v e r y common i n p l a n t  B  ,they a r e not as important  among the K l e b s i e l l a  polysac-  c h a r i d e s . D-Glucuronic a c i d and D-mannose compose the a l d o b i o u r o n i c a c i d s o f 16 s t r a i n s and only two combinations a r e served, GlcA  1 2  Man and GlcA  1 3 J  Man>the f i r s t  ob-  one i s a l s o  24  B  common i n p l a n t gums  .D-Glucuronic a c i d and L-rhamnose a r e  found as the a l d o b i o u r o n i c a c i d i n 9 s t r a i n s and although a l l p o s s i b l e l i n k a g e s have been determined  ( 2 , 3 and 4 r h a m n o s e ) , 8  of the 9 occur as B - g l y c o s i d e s . T h e  aldobiouronic acids  contain-  ing D-glucuronic  as w e l l as  contain-  a c i d and L-fucose  ing D-galacturonic  a c i d are a l s o l i s t e d  P r i o r t o the s y s t e m a t i c saccharides  i n Table  those 1.2.  i d e n t i f i c a t i o n of b a c t e r i a l  poly-  only a few a l d o b i o u r o n i c a c i d s had been i s o l a t e d  As a source o f a l d o b i o u r o n i c a c i d s . c a p s u l a r  polysaccharides  .  1 1  TABLE 1.2  Aldobiouronic acids i n K l e b s i e l l a capsular polysaccharides.  Aldobiouronic Glucuronic  acid  and Glucose Glucuronic  acid  and Galactose  Klebsiella(K)  acid GlcA  acid  Glc  5  2 3 , 5 1  GlcA - — 2 .  3 1 , 5 5 , 8 3  GlcA  l  A  GlcA  l  P  3 4  1a|i  G  a  l  Gal  1 1 , 1 2 , 2 0 , 4 1 , 6 0  Gal  8  -"—Gal  GlcA  1  GlcA  ---r- -  GlcA  and Mannose  4  GlcA - " — - G l c  GlcA  Glucuronic  1  6  B  2  1  _  H  2 5 , 5 9  Gal  2 7  Man  7,28,53,61,62  Man  2,4,13,21,24, 26,30,33,46,64,  74 Glucuronic  acid  Fuc  1 _ 1 1  acid  and Rhamnose  GlcA GlcA GlcA  Galacturonic  acid  A  2  1  l  GlcA  1  G a lA  1  2 a  3  4  B  6 , 5 ^  4  GlcA i - — Fuc  and Fucose Glucuronic  GlcA  Rha  1 , 1 6 , 5 8  9 * , 1 8 , 3 6 , 4 4 , 8 1  Rha  1 7  Rha  4 7  Rha  9 , 7 0  2  Man 1 3 GalA i r — - Man  5 7  a  GalA  1 1 1  a  Fuc  GalA - — - Rha  4 9  63 34,48  12  are,thus,of great  importance.  i i ) S u b s t i t u t i o n p a t t e r n ; The  c o n c l u s i o n s t h a t can  be  drawn from the s u b s t i t u t i o n p a t t e r n o f the monosaccharide r e s i d u e s i n the 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  of known  s t r u c t u r e are the f o l l o w i n g : D-glucose appears mainly as i n - c h a i n r e s i d u e , l i n k e d through p o s i t i o n 3,k t e r m i n a l r e s i d u e ; D-galactose  6 or as a  as an i n - c h a i n r e s i d u e i s almost  exclusively linked at p o s i t i o n 3 mannose does not have any  and  an  ( 30  s t r a i n s out of 3 D ;  D-  particular characteristic.except  t h a t p o s i t i o n 6 i s not commonly s u b s t i t u t e d ; L-rhamnose, l i k e D-mannose does not have any p a r t i c u l a r c h a r a c t e r i s t i c ; f  D-glucuronic  a c i d appears mainly as an i n - c h a i n r e s i d u e  t i t u t e d a t p o s i t i o n k ) or as a t e r m i n a l residue.No c o n c l u s i o n s can be drawn f o r L-fucose  The  acid  polysaccharides.  d i s t r i b u t i o n of a - and B - g l y c o s i d i c l i n k a g e s  found i n 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 the percentage composition l u t i o n s . One  general  and D - g a l a c t u r o n i c  as they are not so common i n the c a p s u l a r iii)  (subs-  would expect  corresponds w i t h  of e q u i l i b r a t e d aqueous sugar so-  thermodynamic s t a b i l i t y to predomi-  nate i n n a t u r a l o c c u r r i n g substances.The 6 anomers o f D-glucose ,D-galactose and D - g l u c u r o n i c  a c i d are more common,as the  b u l k i e r a g l y c o n groups tend to d i s p o s e  themselves i n an equa-  t o r i a l p o s i t i o n . I n the case o f D-mannose and a x i a l h y d r o x y l group a t C-2  L-rhamnose,the  makes the anomeric e f f e c t i n c r e a 26  se i n s i g n i f i c a n c e , t h u s the i v ) Table  1.5  a anomers are more s t a b l e  shows the p r e f e r e n c e  for certain  .  pyruvyla-  ted monosaccharide r e s i d u e s i n the K l e b s i e l l a c a p s u l a r  poly-  13 TABLE 1.3  S u b s t i t u t i o n p a t t e r n o f the sugar r e s i d u e s i n the K l e b s i e l l a c a p s u l a r  polysaccharides K l e b s i e l l a (K)  Substitution pattern of the sugar r e s i d u e a)  terminal  Glucose  18,25,27,28,38,41,46,5-+.  Galactose  60(3) 7.16,52,58,61  Mannose  24,57,62  Rhamnose  17,3^7,53.56.64  Fucose Glucuronic  2,8,9,20,23,27,30,33.51,  acid  55,59,83 Galacturonic b)  48,49  acid  monosubstituted  Glucose  2 3  70 1,2,4(2),6,7,11,13,23,24, 28,31,3^.^.^8,55.58,59, 60,61,64,72  4  2,5,13,17,22,25,26,30,33, 37,44,48,62  6  12,22,23.26,27,37,38,41(2)  51.61 Galactose  2  28  3  8,9.9*.11.12,18,20(2),21, 26,27,31,32,36,38,41,46(2) 47.49(2),51.52,53.55,56(2) 57,62,63,70,74,81,83(2)  14 Mannose  Rhamnose  2  7,21,24,26,28,31,53.57,  3  4,21,24,26,46,59.64  4 6  30,33  2  9,9*(3),18,34(2),36,44, 48,52,53.70,81(2)  3  9,12,17,18,32;34,36,41 44,72(2),81(2)  4 Fucose  Glucuronic a c i d  32,52.70  2 3  6,54,63  4  1,16  2  4,25,63  3 4  7,28,53 5,6,9*,12,16,17,18,36,41, 44,47,52,54.61,64,70,74  Galacturonic acid  2 3 4  c)  disubstitution  Glucose  Galactose  63 p ( b r a n c h i n g or pyruvate )  2,6  16,18  4,6  31 ,36 ,5^,64  2,3  12,41,52,56,60  3,^  Mannose  P  P  8,13 ,22,25,30 ,33 .37. P  P  P  4,6  51,59 11 ,12 ,21 ,26 ,74  2,3  7,20,28,49,53,60,61,62,  P  P  P  74  Rhamnose  P  2,6  2,13  2,3  17,23,36,48  3,^  9,^7.55.83  P  P  15  Fucose  3tk  58  Glucuronic acid  2,4  24,26  3,4  11,21,31,46,60  Galacturonic acid  3,4  3^,57 p  d)  t r i s u b s t i t u t i o n ( double b r a n c h i n g or pyruvate )  Glucose  3,^,6  7 ,27 ,56  Galactose  2,3,4  38  3,^,6  27  Mannose Rhamnose  P  P  P  2,3,4 3,^,6  64 5 ,6 ,30,33,^6  2,3,4  1 ,58  P  P  P  P  P  16 TABLE 1.4  Percentage composition  o f a - and g - g l y c o s i d i c  linkages i n K l e b s i e l l a capsular polysaccharides.  Sugar r e s i d u e  a-linked(%)  B-linked(%)  Glucose  (65)  25  75  Galactose  (5*0  48  52  Mannose  (39)  74  26  Rhamnose  (46)  95  5  Fucose  ( 6)  100  0  (*5)  42  58  ( 5)  100  0  Glucuronic  acid  Galacturonic  acid  ( ) number o f sugar r e s i d u e s c o n s i d e r e d capsular polysaccharides  from a l l K l e b s i e l l a  o f known s t r u c t u r e .  17 TABLE 1.5  L o c a t i o n o f pyruvate i n K l e b s i e l l a  capsular  polysaccharides.  L o c a t i o n o f pyruvate  Glucose  ^ 4 6 Glc —  —  Galactose  K l e b s i e l l a (K)  31.36,64 8  3 Glc - r -  4 6 Gal  7,27,56  /x  11,21 a  / 6 Gal — x  12,26,74 p  3 k Gal  Mannose —  Rhamnose  13,30,33  Jt 4 6 3 Man --—  A  2 Rha  Glucuronic acid  4 GlcA 3^2  5,6,46  32,70,72  1,58 B  18  saccharides. As a means of d i s e a s e n i z a t i o n has  prevention  been used by man  been obtained,  f o r a long time.Immunization  i n b a c t e r i a l induced d i s e a s e s  the b a c t e r i a ( b a c t e r i a l v a c c i n e s ) them.Protective immunization was r i a l polysaccharides "^' ^ 2  the haptenic  2  a l s o obtained  by  w i t h the b a c t e antigens ^,where 2  coupled to c a r r i e r  proteins,  the p o s s i b l e t o x i c e f f e c t s of b a c t e r i a .  to the h i g h s p e c i f i c i t y of the antigen-antibody  p u r i f i c a t i o n of antibodies 30  has  by i n j e c t i o n of  or the t o x i n s produced  and w i t h a r t i f i c i a l  o l i g o s a c c h a r i d e was  a v o i d i n g i n t h i s way Due  the phenomenon o f immu-  can be achieved  reaction,  by a f f i n i t y chrom-  31  atography-^ *  J  ,where the haptenic  to a m a t r i x , r e t a i n i n g  oligosaccharide  is  attached  i t s s p e c i f i c b i n d i n g a c t i v i t y to  the  antibody. In e i t h e r case,these haptenic obtained  o l i g o s a c c h a r i d e s must be  i n l a r g e q u a n t i t i e s . S e v e r a l procedures can be used  f o r t h i s purposes a) Synthesis-^ "-^. 2  can  When one  considers  t h a t two  combine t o form t e n d i f f e r e n t d i s a c c h a r i d e s  glucoses  (considering  o n l y the pyranose form),the task i n s y n t h e s i z i n g l a r g e r o l i g o saccharides and  i s enormous.Specific  protections,deprotections  g l y c o s y l a t i o n techniques must be used and  generally  the y i e l d s are  low.  b) P a r t i a l h y d r o l y s i s of the acid hydrolysis i s non-specific,the  polysaccharide-^'-^?.As number o f oligomers  ed are l a r g e which i m p l i e s t e d i o u s p u r i f i c a t i o n s and  obtain-  the y i e l d s  19 are low,Many p o l y s a c c h a r i d e s have a c i d - l a b i l e s u b s t i t u e n t s which i n some cases are.extremely important f o r the immunolo g i c a l a c t i v i t y . T h i s procedure g e n e r a l l y y i e l d s  oligomers  which l a c k these s u b s t i t u e n t s . c) Bacteriophage 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 s - ^ ' ^ 9 . Bacteriophages possess enzymes t h a t s e l e c t i v e l y h y d r o l y z e p o l y s a c c h a r i d e s . The y i e l d  o f o l i g o m e r i c products i s u s u a l l y h i g h  and mainly one or two r e p e a t i n g u n i t s are obtained(see S e c t i o n IV.3)!  In the course o f t h i s T h e s i s , the s t r u c t u r e s o f two 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 , s e r o t y p e K 60 and serotype K 2 6 , were i n v e s t i g a t e d * two 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 ( K 6 0 and K 46) bacteriophages  were degraded by t h e i r r e s p e c t i v e K l e b s i e l l a  ( # 6 0 and 0 46 ) and the d e g r a d a t i o n products  were i s o l a t e d , p u r i f i e d  and c h a r a c t e r i z e d .  20  METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES  21 I I . - METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES.  I I . 1 The s t r u c t u r e s o f p o l y s a c c h a r i d e s . Polysaccharides a)  i n the f o l l o w i n g way:  Homopolysaccharides (based  b)  can be c l a s s i f i e d  on one type  linear  o f sugar)  Heteropolysaccharides (based  branched linear  on two or more sugars)  branched  T h i s c l a s s i f i c a t i o n c o u l d be extended depending on whether the p o l y s a c c h a r i d e  c o n t a i n s r e g u l a r r e p e a t i n g u n i t s or not.A  s t r u c t u r a l study must c h a r a c t e r i z e : a) the nature the  o f the sugar r e s i d u e s and t h e i r p r o p o r t i o n s  in  polysaccharide,  b) the p o s i t i o n o f l i n k a g e , a s the g l y c o s i d i c l i n k a g e may i n volve s u b s t i t u t i o n o f one o f the s e v e r a l h y d r o x y l groups i n the a d j a c e n t sugar r e s i d u e , c) the l i n k a g e c o n f i g u r a t i o n . a s each g l y c o s i d i c l i n k a g e may have the a - or 8 - c o n f i g u r a t i o n , d) the sequence o f the sugar r e s i d u e s , a s  the v a r i o u s s t r u c t u -  r a l u n i t s may be assembled i n many a l t e r n a t i v e sequences. With p o l y s a c c h a r i d e s  consisting of repeating units,the  s t r u c t u r a l study w i l l l e a d t o a d e f i n i t i v e s t r u c t u r e , w h i l e on the other hand,an o v e r a l l sequence cannot be determined i n polysaccharides  l a c k i n g r e p e a t i n g units.The  latter investigation  can o n l y provide i n f o r m a t i o n on an "average s t r u c t u r e " . s u g a r r e s i d u e s present,type  o f end groups.which sugars a r e branch  22 points.nature The  o f some l i n k a g e s . e t c .  f o l l o w i n g d i s c u s s i o n w i l l t r y t o cover s e v e r a l t e c h -  niques o f s t r u c t u r a l e l u c i d a t i o n t h a t a r e a p p l i c a b l e t o both types o f p o l y s a c c h a r i d e s . LQ-LLQ  II.2 I s o l a t i o n and p u r i f i c a t i o n The  7  .  f i r s t major task i n p o l y s a c c h a r i d e  chemistry  i s to  o b t a i n the m a t e r i a l t o be s t u d i e d i n a pure form.This i n v o l v e s a ) i s o l a t i o n o f the carbohydrate m a t e r i a l f r e e from other stances, and b ) i s o l a t i o n o f a s i n g l e p o l y s a c c h a r i d e The  techniques  der t o preserve  salts,amino  i n t a c t the s t r u c t u r e o f the p o l y s a c c h a r i d e as  The  step c o n s i s t s of s e p a r a t i n g the  from low molecular  weight m a t e r i a l  acids,peptides,oligosaccharides,etc.)  h i g h molecular  species.  used should be as m i l d as p o s s i b l e i n o r -  i t e x i s t s i n nature.The f i r s t polysaccharide  sub-  weight m a t e r i a l  (inorganic and other  (proteins,nucleic acid.lignin).  i s o l a t i o n of a s i n g l e polysaccharide  species  presents  g r e a t e r d i f f i c u l t i e s . S e v e r a l procedures have been used,such as f r a c t i o n a l p r e c i p i t a t i o n ^ . s e l e c t i v e p r e c i p i t a t i o n by com+2 42 4? p l e x f o r m a t i o n w i t h Cu or CETAVLON ^ , i o n exchange matography  44  , g e l chromatography  chro-  4*1 .46 ,electrophoresis ,etc.  I n d i c a t i o n o f the presence o f a s i n g l e component by these and  other c r i t e r i a  o f homogeneity ( [ ] . s u g a r a n a l y s i s ) p r o v i -  des  only evidence a g a i n s t h e t e r o g e n e i t y  a  D  and thus the impor -  tance o f s p e c i f y i n g the c r i t e r i a used when d e a l i n g w i t h new polysaccharides. The usedr  f o l l o w i n g i s o l a t i o n and p u r i f i c a t i o n procedures were  23  hn  1 1 . 2 . 1 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 '. Each s t r a i n o f K l e b s i e l l a was i n o c u l a t e d i n b e e f - e x t r a c t medium and incubated a t 3 7 ° u n t i l a d e f i n i t i v e growth was observed; t h i s l i q u i d c u l t u r e was incubated  i n a t r a y of sucrose-  y e a s t e x t r a c t - a g a r f o r three days.The lawn o f 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 , d i l u t e d w i t h aqueous phenol t o k i l l the b a c t e r i a and u l t r a c e n t r i f u g e d . T h e p o l y s a c c h a r i d e was p r e c i p i t a t e d from the supernatant  w i t h ethanol.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 w i t h CETAVLON  (cetyltrimethyl-  ammonium bromide) s o l u t i o n , w h i c h p r e c i p i t a t e d the a c i d i c s a c c h a r i d e . F u r t h e r p u r i f i c a t i o n o f the a c i d i c  poly-  polysaccharide  i n v o l v e d d i s s o l u t i o n i n 4M N a C l . r e p r e c i p i t a t i o n i n t o e t h a n o l , d i s s o l u t i o n i n water and d i a l y s i s . T h e d i a l y z e d s o l u t i o n y i e l d e d a f t e r f r e e z e - d r y i n g the 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 . A c i d i c polysaccharides are normally  easy t o p u r i f y as  they can b e " s e l e c t i v e l y p r e c i p i t a t e d w i t h CETAVLON,any n e u t r a l p o l y s a c c h a r i d e s remaining  i n s o l u t i o n . I n case  o f heavy contam-  i n a t i o n w i t h n e u t r a l p o l y s a c c h a r i d e s t h i s p r e c i p i t a t i o n can be repeated. F u r t h e r p u r i f i c a t i o n can be achieved by anion-exchange chromatography(e.g. d i e t h y l a m i n o e t h y l Sephadex,DEAE) o r by g e l permeation chromatography. LQ h.Q  11.2.2 Gum exudate o f C h o r i s i a s p e c i o s a The  '  .  gum c o l l e c t e d from the t r e e s o f C h o r i s i a s p e c i o s a as  hard nodules was allowed d i s s o l u t i o n i t was heated  t o s w e l l i n water.In order t o h e l p on a steam-bath f o r s e v e r a l hours ,  24 the pH was  kept a t 7 i n order to prevent h y d r o l y s i s . T h e  s o l u t i o n obtained was p r e c i p i t a t e was  p r e c i p i t a t e d with a c i d i f i e d  d i s s o l v e d i n water and  w i t h e t h a n o l was  ethanol.The  a second p r e c i p i t a t i o n  done.This p r e c i p i t a t e y i e l d e d upon d i s s o l u -  t i o n and f r e e z e - d r y i n g the gum II.3  viscous  Sugar a n a l y s i s ^ " 0  6 7  polysaccharide.  .  I I . 3 . 1 Total hydrolysis. The  f i r s t s t e p i n the i n v e s t i g a t i o n o f a p o l y s a c c h a r i d e  the d e t e r m i n a t i o n  is  of the nature of the sugars r e l e a s e d on hydro-  l y s i s . As a c i d causes d e g r a d a t i o n (type of a c i d , c o n c e n t r a t i o n  of sugars,the c o n d i t i o n s  of acid,temperature and  used  time)must  be c a r e f u l l y chosen i n order to o b t a i n a q u a n t i t a t i v e r e l e a s e o f the sugars w i t h a minimum of degradation.Dutton-^ reviewed the advantages and  disadvantages i n the use  of d i f f e r e n t  T r i f l u o r o a c e t i c a c i d (2M)-* has been found to be 1  as i t has  the b e s t  acids. choice,,  approximately the same h y d r o l y t i c s t r e n g t h as HCl(lM)  and HgSO/j, (P.5M) ,does not s i g n i f i c a n t l y degrade sugars under the c o n d i t i o n s n o r m a l l y volatility.it  used  (6-8 h,100°)  and because of i t s  i s e a s i l y removed.  Although t r i f l u o r o a c e t i c a c i d (2M) t e l y a neutral polysaccharide 6 t o 8 hours a t 1 0 0 ° , t h e s e  w i l l break down comple-  i n t o i t s sugar c o n s t i t u e n t s i n  c o n d i t i o n s are not s t r o n g enough to  ensure complete h y d r o l y s i s of g l y c u r o n o s y l l i n k a g e s . A able amount of a l d o b i o u r o n i c a c i d thus s u r v i v e s the  consider-  treatment.  T h i s w i l l cause d i s c r e p a n c i e s i n the sugar r a t i o of the hydrol y z a t e and  i n the polymer.However,incomplete h y d r o l y s i s p r o v i d -  25  es a v e r y u s e f u l i n d i c a t i o n as to the p o s s i b l e composition of the a l d o b i o u r o n i c  acid.  S e v e r a l procedures have been d e v i s e d acid residues  to reduce the  uronic  to n e u t r a l sugars(see S e c t i o n II.7.1).A technique t o  has been developed i n t h i s l a b o r a t o r y ^ d i f f i c u l t i e s . T h e polysaccharide  which overcomes these  i s t r e a t e d w i t h methanolic  hy-  d r o c h l o r i c acid.which c l e a v e s most o f the g l y c o s i d i c l i n k a g e s forming the methyl g l y c o s i d e s and  a t the same time,the methyl  e s t e r s of the u r o n i c acids.Treatment w i t h NaBH^ 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 a l c o h o l s . The  mixture of methyl g l y c o s i d e s i s then h y d r o l y z e d  2M t r i f l u o r o a c e t i c a c i d ( TFA  ) to g i v e the n e u t r a l sugars.  By comparison of the r a t i o of n e u t r a l sugars only upon h y d r o l y s i s o f the a c i d i c p o l y s a c c h a r i d e n e u t r a l sugars and  with  and  released  the r a t i o  of  transformed u r o n i c a c i d s , i t i s p o s s i b l e  to  i d e n t i f y the u r o n i c a c i d as w e l l as the molar p r o p o r t i o n s the sugars  present.  II.3.2 C h a r a c t e r i z a t i o n and Through the years,the  q u a n t i t a t i o n of the  sugars.  c h a r a c t e r i z a t i o n and q u a n t i t a t i o n of  the sugars r e l e a s e d upon 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 veloped from s p e c i f i c r e a c t i o n s and  -  matography-^, to more r e c e n t h i g h pressure  Although the use  has  c r y s t a l l i z a t i o n o f the  r i v a t i v e s , through paper c h r o m a t o g r a p h y - ^ ^ and  graphy and  of  dede-  t h i n - l a y e r chro-  techniques of g a s - l i q u i d chromato-  liquid  chromatography.  of paper chromatography f o r q u a n t i t a t i v e  purposes i n v o l v e s e l u t i o n of the d i f f e r e n t f r a c t i o n s and ther q u a n t i t a t i o n (e.g.colorimetry)  i t i s s t i l l a very  f u r *» r  useful  26  t o o l f o r q u a l i t a t i v e purposes. The volved  development of g a s - l i q u i d chromatography ( g . l . c . ) i n -  the c o n v e r s i o n  t i v e s . An e x t e n s i v e  of the sugars to s u i t a b l e v o l a t i l e  review o f t h i s technique and  deriva-  i t s applications  has been done by Dutton^ '^^.The f i r s t v o l a t i l e d e r i v a t i v e s used 7  were the t r i m e t h y l s i l y l e t h e r s - ^ , w i t h sugar may  the disadvantage t h a t each  g i v e r i s e to f o u r peaks ( a - and B - p y r a n o s i d e s and a -  and 3 - f u r a n o s i d e s ) . T h e chromatograms were s i m p l i f i e d g r e a t l y upon r e d u c t i o n  to the r e s p e c t i v e a l d i t o l s ^  or c o n v e r s i o n  0  to  the  aldononitriles^""". I t must be kept i n mind t h a t ketoses.such f r u c t o s e , g i v e upon r e d u c t i o n two and  alditols  ( g l u c i t o l and  i n t h i s case a more convenient d e r i v a t i v e would be  nonitrile^ (oximes 2  for  as  mannitol) the  aldo-  ketoses).  A wide s e l e c t i o n o f s t a t i o n a r y phases i s now which g i v e a good s e p a r a t i o n  available  of the d i f f e r e n t v o l a t i l e  derivati-  ves. During t h i s i n v e s t i g a t i o n the a l d i t o l a c e t a t e s were used good r e s o l u t i o n , w a s obtained phases; S P - 2 3 4 0 , a  and  w i t h the f o l l o w i n g s t a t i o n a r y  h i g h l y p o l a r s i l i c o n e c o n t a i n i n g 7 5 $ of cyano-  p r o p y l groups,and ECNSS-M,a copolymer of e t h y l e n e g l y c o l s u c c i nate p o l y e s t e r and The  a cyanoethyl s i l i c o n e .  l a t e s t advance i s the use  of h i g h pressure l i q u i d  matography ( H P L C ) ^ ^ ' ^ f o r q u a n t i t a t i v e a n a l y s i s of sugars  chro,a  technique which does not r e q u i r e d e r i v a t i z a t i o n . II.3-3 D e t e r m i n a t i o n o f the c o n f i g u r a t i o n (D or L) of the The D or L c o n f i g u r a t i o n of the sugars was  u n t i l recently  determined by i s o l a t i o n of the d i f f e r e n t monosaccharides measurement of t h e i r o p t i c a l r o t a t i o n ( l a ] ) , D  sugars.  or u s i n g  and  specific  27  oxidases  (e.g. D-glucose oxidase,D-galactose  oxidase).The  latter  method i s not a p p l i c a b l e t o other commonly o c c u r i n g sugars to the absence o f the c o r r e s p o n d i n g A more r e c e n t procedure  due  oxidases.  i s the d e t e r m i n a t i o n of the s i g n  of t h e i r c i r c u l a r d i c h r o i s m c u r v e s ^ m e a s u r e d on s u i t a b l e  deri-  v a t i v e s . These d e r i v a t i v e s . w h i c h can be the a c e t y l a t e d a l d i t o l s or the p a r t i a l l y methylated  a l d i t o l a c e t a t e s , c a n be e a s i l y  l a t e d by p r e p a r a t i v e g a s - l i q u i d chromatography.A  disadvantage  of these d e r i v a t i v e s i s t h a t meso a l d i t o l s , s u c h as are not o p t i c a l l y a c t i v e . H o w e v e r , t h i s c h i r a l p a r t i a l l y methylated  galactitol,  can be overcome by u s i n g  a l d i t o l a c e t a t e s obtained i n , the  m e t h y l a t i o n s t u d i e s . A n o t h e r p o s s i b i l i t y , w h i c h was the course  of t h i s i n v e s t i g a t i o n ' s the use  developed  in  of the p e r a c e t y l a -  ted a l d o n o n i t r i l e s , w h i c h were s y n t h e s i z e d and  then i s o l a t e d i n  a pure form by p r e p a r a t i v e g a s - l i q u i d chromatography (see pendix  iso-  Ap-  IV ).  R e c e n t l y , g ; 1 . c . has been used f o r the s e p a r a t i o n of enant i o m e r s , u s i n g a c h i r a l s t a t i o n a r y phase or c o n v e r t i n g the enant i o m e r s i n t o diastereomers by c h i r a l reagents and a non-chiral phase.Vliegenthart et a l . have used the second  and L i n d b e r g et a l . '  p r i n c i p l e to determine the a b s o l u t e  g u r a t i o n of the sugars»the f i r s t silylated  s e p a r a t i o n on  confi-  author used the t r i m e t h y l -  ( - ) - 2 - b u t y l g l y c o s i d e s and the l a t t e r the a c e t y l a t e d  ( + ) - 2 - o c t y l g l y c o s i d e s , o n c a p i l l a r y columns w a l l - c o a t e d w i t h S E - 3 0 and  SP-1000,respectively.  28 II.4  Position of linkage. A f t e r d e t e r m i n i n g the number and type o f sugar r e s i d u e s  i n the p o l y s a c c h a r i d e , t h e next s t e p i n v o l v e s m e t h y l a t i o n a n a l y s i s t o determine through which p o s i t i o n ( s )  these components  are l i n k e d t o form the polymeric c h a i n . II.4.1 M e t h y l a t i o n a n a l y s i s . This technique r e l i e s on the complete ( m e t h y l a t i o n ) o f the f r e e h y d r o x y l groups dues i n the polymer  etherification o f the sugar  resi-  and the i d e n t i f i c a t i o n o f the p a r t i a l l y  methylated monosaccharides  r e l e a s e d a f t e r cleavage o f a l l the  g l y c o s i d i c bonds i n the p o l y s a c c h a r i d e . II.4.1.1 M e t h y l a t i o n procedures  ~'- . >  S e v e r a l procedures have been developed through the years each o f which i n v o l v e s the treatment of the p o l y s a c c h a r i d e i n s o l u t i o n w i t h a base and an a l k y l a t i n g ( m e t h y l a t i n g ) agent. D i f f e r e n t s o l v e n t s ( HgO,dimethylformamide,dimethylsulfoxide), bases  ( NaOH,Ag 0,CH^S0CH "Na ) and m e t h y l a t i n g agents +  2  2  (CH^I,  CH N ,(CH^JgSO^ ) have been used. 2  2  The methods o f m e t h y l a t i o n g e n e r a l l y used are the Hakomori methylation^'  7 0  and the P u r d i e - I r v i n e m e t h y l a t i o n . T h e 71  i s the more v e r s a t i l e and u s u a l l y complete c h i e v e d w i t h one treatment.The dimethylsulfoxide  m e t h y l a t i o n i s a-  polysaccharide i s dissolved  (DMSO).the base i s sodium methyl  in  sulfinyl-  methanide (DMSO'Na*) and the a l k y l a t i n g agent i s methyl (CH^I  first  iodide  ).The main disadvantage o f t h i s procedure i s t h a t i t i s  not s u i t a b l e f o r more than one treatment on a c i d i c p o l y s a c c h a r i d e s .because  the methyl e s t e r o f the u r o n i c a c i d can undergo  29 72 B -elimination' d u r i n g the base treatment  l e a d i n g t o degrada-  t i o n o f the p o l y s a c c h a r i d e . P u r d i e m e t h y l a t i o n i s used  i n these  cases t o ensure complete m e t h y l a t i o n , a procedure which i n v o l v e s treatment  o f the p o l y s a c c h a r i d e w i t h s i l v e r oxide i n r e f l u x i n g  methyl i o d i d e . F o l l o w i n g the complete m e t h y l a t i o n o f the p o l y s a c c h a r i d e , the product i s f u l l y h y d r o l y z e d . S e v e r a l procedures 2M TFA on a steam-bath f o r 16 hours ensures of a n e u t r a l methylated  are used,but  complete h y d r o l y s i s  p o l y s a c c h a r i d e . T h e presence  of uronic  a c i d s s t a b i l i z e s the u r o n o s y l bonds which are not f u l l y  hydro-  l y z e d under these c o n d i t i o n s . R e d u c t i o n o f the methyl e s t e r w i t h L i A l H ^ (see S e c t i o n II.7.1) overcomes these d i f f i c u l t i e s . The reduced methylated  p o l y s a c c h a r i d e can now be h y d r o l y z e d com-  p l e t e l y or remethylated  p r i o r t o the h y d r o l y s i s .Scheme I I . 1 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. The unmethylated of  p o s i t i o n s o f the sugars r e p r e s e n t s i t e s  l i n k a g e , e x c e p t i n the cases o f u r o n i c a c i d r e s i d u e s or r e s -  idues w i t h pyruvate a t t a c h e d . U r o n i c a c i d s can be i d e n t i f i e d by comparison o f the m e t h y l a t i o n r e s u l t s o f ; t h e a c i d i c r i d e w i t h those o f the reduced  polysaccha-  p o l y s a c c h a r i d e , or the reduced  p o l y s a c c h a r i d e and the corresponding reduced remethylated duct as shown i n the Scheme II.1.The the pyruvate  can be determined  pro-  p o s i t i o n of linkage of  by comparison o f the methyla-  t i o n products o f the o r i g i n a l and s e l e c t i v e l y  depyruvylated  p o l y s a c c h a r i d e s as used f o r the K l e b s i e l l a K 26 p o l y s a c c h a r i d e . II.4". 1.2 C h a r a c t e r i z a t i o n o f the methylated The  sugars.  techniques f o r s e p a r a t i n g and i d e n t i f y i n g the methyl-  30  K26 POLYS ACCHARI DE  l  Methylation  LiAlty / THF  1. Remethylation 2. H  +  2/»6-0Me - Mannose  2,A6-0Me~ Mannose  3,4,6-OMe- Mannose  3A,6-0Me- Mannose  2,4.6-0Me- Galactose  2A6-0Me- Galactose  Z3,4-OMe- Glucose  2.3,A-0Me- Glucose  Z3.6-0Me- Glucose  2,3.6-0Me- Glucose  3  3  3  3  3  22-OMe2 Galactose _  3-0Me - Glucose Scheme I I . 1  3  3  3  3  3  23-0Me- Galactose 2  3,6-OMe- Glucose 2  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 K 26  31  ated sugars have improved  c o n s i d e r a b l y s i n c e the time they were  separated by f r a c t i o n a l d i s t i l l a t i o n o f t h e i r methyl A d s o r p t i o n chromatography,paper phoresis  chromatography  glycosides.  .paper  electro-  , e t c . have been used e x t e n s i v e l y . A l t h o u g h many o f  these methods are seldom used a t p r e s e n t  ,paper chromato-  graphy i s s t i l l a v e r y u s e f u l t o o l i n the i d e n t i f i c a t i o n  of  methylated sugars.By d e v e l o p i n g the papers w i t h _p_-anisidine  76 h y d r o c h l o r i d e or other aromatic amines'  and h e a t . i t i s p o s s i -  b l e to do a primary i d e n t i f i c a t i o n o f the methylated sugars a c c o r d i n g t o the d i f f e r e n t c o l o r s formed as w e l l as from mobilities  their  (shown by t h e i r R^ v a l u e s ) .  With the advent  o f g.I.e.,which  p r o v i d e s a r a p i d and a c -  curate q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s . e f f o r t s have been made t o r e n d e r 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 o f the methylated sugars.An e x t e n s i v e review o f the f i e l d has been done by Dutton 5?.58  < T n e  d e r i v a t i v e s o f c h o i c e d u r i n g the course o f t h i s i n -  v e s t i g a t i o n were the p a r t i a l l y methylated a l d i t o l a c e t a t e s , which can be e a s i l y obtained by r e d u c t i o n o f the methylated a r s w i t h NaBH^ f o l l o w e d by a c e t y l a t i o n w i t h a c e t i c p y r i d i n e . V a r i o u s l i q u i d phases  sug-  anhydride-  (especially,medium p o l a r ones)  are a v a i l a b l e f o r the s e p a r a t i o n o f mixtures o f these d e r i v a t i v e s and depending  on the d i f f i c u l t i e s encountered  on  r a t i o n , s e v e r a l columns may need t o be t r i e d . P u b l i c a t i o n s  sepaby  L i n d b e r g h and Albershein-7® p r o v i d e 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 methylated a l d i t o l a c e t a t e s as w e l l as molar response f a c t o r s which enable c o r r e c t q u a n t i t a t i o n o f the components.  32  In the i n v e s t i g a t i o n o f the K l e b s i e l l a K 2 6 c a p s u l a r p o l y s a c c h a r i d e , the p e r a c e t y l a t e d p a r t i a l l y methylated  aldononitril*.  es were used t o separate 2 , 3 , 4 - a n d 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e (although s e p a r a t i o n of the a l d i t o l a c e t a t e s was  achieved w i t h  a column of S P - 2 3 4 0 ) . T h e s e  d e r i v a t i v e s were e a s i l y prepared  r e a c t i o n of the methylated  sugars w i t h hydroxylamine  r i d e i n p y r i d i n e . f o l l o w e d by treatment w i t h a c e t i c which dehydrates  hydrochloanhydride  the oxime to the n i t r i l e and a t the same time  a c e t y l a t e s the f r e e h y d r o x y l The  by  groups.  i d e n t i f i c a t i o n of an i n d i v i d u a l component can be  c h i e v e d i n almost a l l cases by co-chromatography w i t h  a-  authen-  t i c standards,but by c o u p l i n g the g . l . c . t o a mass spectrometer  (g. I.e.-m.s. ) .assignments  can be made unambiguously. The  f r a g m e n t a t i o n of the p a r t i a l l y methylated  a l d i t o l a c e t a t e s by 79  e l e c t r o n impact  has been e x t e n s i v e l y s t u d i e d ~ y  81 .This t e c h -  nique a l l o w s us to i d e n t i f y the s u b s t i t u t i o n p a t t e r n of the p a r t i a l l y methylated  a l d i t o l a c e t a t e s , b u t i t does not g i v e the  parent i o n mass and cannot  d i s t i n g u i s h between diastereomers,  as mannose.glucose and g a l a c t o s e d e r i v a t i v e s . T h e primary  frag-  ments are formed by the f i s s i o n of i n t e r c a r b o n bonds i n the a l d i t o l c h a i n . T h i s cleavage f o l l o w s a c e r t a i n p r e f e r e n c e bond between carbons one methoxylated  t h a t are methoxylated  : a  i s p r e f e r e d over  and the o t h e r a c e t o x y l a t e d , w h i c h i n t u r n i s  p r e f e r e d over two a c e t o x y l a t e d carbons: >  >  Figure II.1  M.s.  o f (a)  1,2,5-tri-0-acetyl-3»k,6-tri-0-  methyl g a l a c t i t o l  and  (b)  -3-0-ethyl-4,6-di-0-methyl  1,2,5-tri-O-acetyl galactitol.  3k The  primary fragments formed g i v e r i s e t o secondary f r a g -  ments by l o s s o f a c e t i c a c i d ol  (M.W.  (M.W.  60),ketene  (M.W.  42),methan-  3 2 ) or formaldehyde (M.W. 3 0 ) .  T h i s i n f o r m a t i o n was used t o i d e n t i f y s p e c i f i c compounds f o r which standard s p e c t r a  were not a v a i l a b l e . F o r  example,when  u r o n i c a c i d d e g r a d a t i o n was performed on K l e b s i e l l a K 6 0 p o l y s a c c h a r i d e , the  p o s i t i o n where the u r o n i c a c i d was a t t a c h e d was  l a b e l l e d w i t h e t h y l i o d i d e . T h e e t h y l a t e d , p a r t i a l l y methylated a l d i t o l a c e t a t e thus obtained  (  l,2,5-tri-0-acetyl-3-0-ethyl-  4 , 6 - d i - 0 - m e t h y l g a l a c t i t o l ) was i d e n t i f i e d by means o f i t s m.s. and comparison w i t h the c o r r e s p o n d i n g methylated tive  (l,2,5-tri-0-acetyl-3t4,6-tri-0-methyl  galactitol).Several  masses are 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 The  f r a g m e n t a t i o n expected i s shown below:  deriva-  i n F i g u r e II.1.  35  Primary f r a g m e n t a t i o n  CHgOCOCH^  CH ,0C0CHo I2 3  CH-OCH,  CH-0CH CH~ + 2 3 (m/e 203)  CHOCOCH^  I  CHOCOCH^ O  CHgOCH^ (m/e 161)  CHOCH, :H=OCH CH  CHOCOCHI 3 CH 0CH £  3  2  CHgOCOCH^  3  HOCOCH^  3  ;HOCOCH  IH 0CH 2  ;HOCH CH 2  3  CH-OCH+ 3 (m/e 2 4 7 )  3  3  (m/e 219)  Secondary f r a g m e n t a t i o n  CH 0CH 2  CHOCH-  3  CH0C0CH" CH=0CH  -Ac OH  3  II  H  3  H=0CH  3  3  (m/e 101)  (m/e 161)  CH, II  -Me OH  z  -CH C0 2  HOCOCH,  o  -ArOH Z^im  3  CH=0CH CH 2  (m/e  3  203)  CHOCOCH" CH CH=0CH CH 2  (m/e 1 4 3 )  3  (m/e  (m/e 129)  2  4= 0 CH=0CH  H-OCH^  H 0C0CH^ 3 H0C0CH  CH  3  3  87)  36 II.5 Sequencing o f sugars. The  i s o l a t i o n and c h a r a c t e r i z a t i o n o f fragments o f a p o l y -  saccharide  are the keys t o the d e t e r m i n a t i o n  o f the s e q u e n t i a l  arrangement o f the c o n s t i t u e n t monosaccharides i n the polymer. By u t i l i z i n g d i f f e r e n t techniques i t i s p o s s i b l e t o o b t a i n these fragments and by d e t e r m i n i n g the r e l a t i v e p o s i t i o n s o f one  t o the o t h e r , i n an a d d i t i v e manner,is p o s s i b l e t o b u i l d up  the sequence i n the p o l y s a c c h a r i d e  or i n the r e p e a t i n g u n i t .  II.5.1 P a r t i a l h y d r o l y s i s " ^ . 8 2  A considerable  8  amount o f data on r a t e constants  and k i n e t i c  parameters 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 o f g l y c o s i d e s o f 1  82 83 monosaccharides has been presented i n s e v e r a l reviews  ' . J  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 o f h y d r o l y s i s , such as r i n g size,configuration,conformation,polarity  o f the sugar ,  s i z e and p o l a r i t y o f the aglycon.Although data on  polysaccha-  r i d e s are not so e a s i l y a v a i l a b l e . i n f e r e n c e s can be made from the r e s u l t s on the monosaccharide g l y c o s i d e s , a n d  may be sum-  marized as f o l l o w s : i)  furanosides  are more l a b i l e than  pyranosides,  i i ) deoxysugars are more e a s i l y hydrolyzed  than hexoses,  i i i ) pentopyranosides a r e more a c i d l a b i l e than hexopyranosi des, iv)  a - g l y c o s i d e s are g e n e r a l l y more l a b i l e than 3 - g l y c o s i d e s ,  v)  (1-6) l i n k a g e s are more r e s i s t a n t t o a c i d h y d r o l y s i s than  (1-2),(1-3) and (1-4) v i ) residues hydrolyzed  present  linkages,  on s i d e chains  than when present  are o f t e n more e a s i l y  i n the main c h a i n ,  37 v i i ) u r o n i c a c i d s are more r e s i s t a n t to h y d r o l y s i s , v i i i ) aminosugars are more a c i d r e s i s t a n t than common hexoses. In h e t e r o p o l y s a c c h a r i d e s  we  f i n d t h a t some g l y c o s i d i c  l i n k a g e s are more r e s i s t a n t than others,so  t h a t , under c e r t a i n  c o n d i t i o n s of h y d r o l y s i s ( a c i d concentration,temperature time of h y d r o l y s i s ) s e l e c t i v e cleavages polysaccharide polysaccharides  w i l l occur  and  i n the  y i e l d i n g defined oligomeric units.For a c i d i c c o n t a i n i n g u r o n i c a c i d s .there s i s tance of  the  u r o n o s y l bond l e a d s to the accummulation of a l d o b i o u r o n i c and  t o a l e s s e r extent  acid  the a l d o t r i o u r o n i c and/or a l d o t e t r a o -  u r o n i c a c i d fragments.In p o l y s a c c h a r i d e s  w i t h deoxysugars i t  i s d i f f i c u l t to i s o l a t e , b y p a r t i a l h y d r o l y s i s oligomers w i t h deoxyhexosyl bonds.Usually a f t e r a p a r t i a l h y d r o l y s i s , t h e amount of monosaccharides i s high.By c o u p l i n g ' s e v e r a l t i o n techniques  i t i s p o s s i b l e to separate  separa-  the oligomers from  the monosaccharides as w e l l as to i s o l a t e pure o l i g o s a c c h a r i des. The  techniques  most commonly used are paper chromatogra-  phy ,gel-permeation chromatography,paper e l e c t r o p h o r e s i s g a s - l i q u i d chromatography (see S e c t i o n Because a c e t a l s . a c e t a t e s and i n the p o l y s a c c h a r i d e s bonds.it  and  II.5.*0.  formates t h a t may  be  present  are more a c i d l a b i l e than g l y c o s i d i c  i s o f t e n p o s s i b l e to s e l e c t i v e l y remove these sub-  s t i t u e n t s without a f f e c t i n g the r e s t of the  polysaccharide.  P a r t i a l h y d r o l y s i s can a l s o be c a r r i e d out on f u l l y methylated  polysaccharides. A complementary technique i s a c e t o l y s i s - \ a s the 8  r a t e of cleavage of the g l y c o s i d e s i n the two  relative  mechanisms i s  38 reversed.During cleaved,while II.5.2  acetolysis,(1-6)  they  Periodate  a r e t h e most  oxidation  linkages stable  and Smith  are  preferentially  during  acid  hydrolysis.  h y d r o l y s i s ^ " ^ .  86 It salts give  has been  known s i n c e  are capable two  1928  that  a,B-glycols  of cleaving  H  ICv"  H  CHOH  C  ^  to  H  + H„0 + 10 ~  0  ^  J  ^2  When more tions, formic  than acid  two h y d r o x y l groups i s  are i n contiguous  posi-  released,  R,  R,  I CHOH I (j)H0H  I  1  +  210^  +HC0 H + H 0+ 2 I 0 2  R  one o f  the v i c i n a l  h o l , formaldehyde  C H OH  A This  i s  produced,  +  i s  a primary  alco-  HCHQ  I OK  CHO  +  H 0 ?  +  10  R reaction  carbohydrate formation  termined  3  2  hydroxyl groups  2  CHOH  2  CHO  *2 When  1  CHO  CHOH  a  quantitatively  CHO  ^2  as  and i t s  J l 0  +  the  acid  aldehydes:  ?1 C  periodic  has found  chemistry.Since of formic  accurately  an analytical preparative  a wide  acid  range  of applications  the reduction  of periodate  and formaldehyde  on the microscale,periodate  technique,but  procedure.  i n a d d i t i o n . i t  i n and  c a n a l l be d e i s  invaluable  c a n be used  as  39 When of  used  as  an  polysaccharide  a n a l y t i c a l  is  oxidized  oxidation  being  periodate  and  i f  formed.Over-,and  any  is  plicate the  monitored  the  release  and  at  low  repulsions  charide  in  i f  certain  sugar 91  hemiacetals and  problem i t  the  of  formic  free is  the  hydroxyl  overcome  by  subjecting  oxidation  is  hydrolysis  the  gives  r e s i s t a n t  tent  with  the  possible  the  by  the  may  the  periodate,the  and/or may  occur  due  com-  working  i n  solutions to  of  electrothe  polysac-  a f f e c t s  both  periodate  ions  hindrance  formation  however  and  the  of  formaldehyde  by  ions  to  amount  consumption  buffered  d i o l s  known  of  minimized  periodate  dialdehydes  groups  i n  a d d i t i o n  r e p u l s i o n  them  to  very  sugars  products of  of  intramolecular  requisite  polysaccharide  are  of  not  (NaClO^)  be  exposed  the  products  i n  polysaccharide  data.As  formed  that  and  and  an  used  some  of  oxidized  by  oxidation  of  as the  a  the  released  number should  upon  of be  periodate  monosubstituted  is  to  p e r i o consis-  II.2  periodate  shows  consumed  hexoses.  fragmentation  sugar  inhib-  protected  example.Figure  moles  f i r s t  reduction.When  information.The  the  oxida-  which  substrate.The  borohydride  oxidation is  salts  can  by  polysaccharide.The  the  of  generated  u s e f u l  terminal  periodate  the of  hemiacetals)  methylation  oxidation  n i q u e , the  acid  with  7  complete.analysis  date  When  is  v i c i n a l  through  7  e l e c t r o s t a t i c  f i r s t  the  under-oxidation  residues,and  (with  upon  measuring  ,a  92 '  7  of  residues  the  by  oxidation  between  87 88  solution  acidic;furthermore,steric  a c c e s i b i l i t y  tion  a  temperatures  periodate.Incomplete  the  i n  results.Overoxidation*^  dark  s t a t i c  technique  residues and  can  be  techi n  a  se-  40  Products  T e r m i n a l a n d monosubstituted  formed a f t e r o x . and  hexoses  hydrolysis CHnOH *-  '  CHO  h-OH + I HO  C^OH  OH  CHUOH  j  CHO  1  hOH • CH^DH CHJOH  CH^DH  —OH  - O H  -OH  HO-  CH-OH  IHJOH  CHJDH HjOH  V-C—  CH^H HO—/  *  cr  CHjOH  HO—/  CHO  CHJOH  CHO  UoH + h-OH  \ — 0 -  CHO HOH  tHJDH CH-DH  CH^H QL  F i g u r e I I . 2 . Common p r o d u c t s tion  f o r m e d on p e r i o d a t e o x i d a -  o f 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.  41  parated  from the o x i d i z e d r e s i d u e s as mono-,oligo- or p o l y -  s a c c h a r i d e d e r i v a t i v e s a f t e r some chemical m o d i f i c a t i o n s . I f , a f t e r oxidation with periodate,the  aldehydes generated are  reduced t o a l c o h o l s w i t h sodium borohydride,the ( p o l y o l ) thus obtained  polyalcohol  i s c o n s t i t u t e d by g l y c o s i d i c l i n k a g e s 03  as w e l l as by a c y c l i c a c e t a l groupings.Smith  olf  degradation ^ 7  i s based on the d i f f e r e n t s t a b i l i t y towards a c i d of the g l y c o s i d i c bond and an a c y c l i c acetal.The  l a t t e r hydrolyzes  much  f a s t e r . T h e p o l y o l i s t r e a t e d w i t h m i l d a c i d and the products obtained,comprising;  a) s m a l l fragments d e r i v e d from o x i d i z e d ,  r e s i d u e s , o r b) g l y c o s i d e s o f mono- or o l i g o - s a c c h a r i d e s (the aglycons  being the s m a l l fragments mentioned b e f o r e ) and p o l y -  s a c c h a r i d e s d e r i v e d from non-oxidized  fragments are i s o l a t e d  and p u r i f i e d by d i f f e r e n t s e p a r a t i o n techniques I I . 5 . 4 ) and c h a r a c t e r i z e d by c o n v e n t i o n a l  (see S e c t i o n  procedures.Based  on the d i f f e r e n c e s i n the r a t e s o f o x i d a t i o n of v i c i n a l  diols  ( c i s being o x i d i z e d f a s t e r than t r a n s ) , t h e f a c t t h a t t e r m i n a l r e s i d u e s are more a c c e s s i b l e t o p e r i o d a t e uronate anions tend t o r e p e l l the p e r i o d a t e  ions and t h a t  i o n s . i t i s pos-  s i b l e to s e l e c t i v e l y oxidize c e r t a i n residues  i n the p o l y s a c -  c h a r i d e ^ , doing what may be c a l l e d a " s e l e c t i v e Smith degradation" . 9 6  Scheme I I . 2 shows the Smith degradation K 26  of K l e b s i e l l a  polysaccharide.  II.5.3 Degradations based on g - e l i m i n a t i o n ' 7  .  S e v e r a l groups ( a l k o x y l , h y d r o x y l , e t c . ) i n the B - p o s i t i o n to an e l e c t r o n withdrawing group,such as c a r b o n y l , c a r b o x y l i c  kz  1. NalO^ 2. (CH OH) 3. NaBH 2  2  4  Z>. 0.5MTFA r.t. 24h 5. NaBty  Scheme 11,2  Smith  d e g r a d a t i o n o f K l e b s i e l l a K 26  polysaccharide.  *3 ester,amide or s u l f o n e are e l i m i n a t e d on treatment w i t h "base. The  presence of a hydrogen atom i n the <*-position to  these  groups i s e s s e n t i a l . T h e s e e l e c t r o n withdrawing groups can i n t r o d u c e d i n t o the p o l y s a c c h a r i d e s  by e s t e r i f i c a t i o n  be  of  c a r b o x y l i c a c i d s , o r by o x i d a t i o n of a l c o h o l s to c a r b o n y l compounds .Treatment w i t h base w i l l y i e l d an unsaturated r e s i d u e which i s very a c i d l a b i l e and  thus can be  sugar  selective-  l y c l e a v e d under m i l d a c i d c o n d i t i o n s . Q O  II.5-3*1  Base c a t a l y z e d u r o n i c a c i d  On m e t h y l a t i o n acid  "i  np  degradation  of an a c i d i c p o l y s a c c h a r i d e , t h e  carboxylic  ( u r o n i c a c i d ) i s e s t e r i f i e d , a n d on subsequent treatment  w i t h base,a 3 - e l i m i n a t i o n w i l l o c c u r . e l i m i n a t i n g the methoxyl or sugar r e s i d u e attached and  forming  to C-4  of the u r o n i c a c i d r e s i d u e  an hex-4-enopyranosyl u r o n a t e . l t has been observed  t h a t the base treatment w i l l c l e a v e the g l y c o s y l u r o n i c age  1 0 0  , exposing  r e s i d u e was  link-  the h y d r o x y l group to which the u r o n i c a c i d  attached.This  f r e e h y d r o x y l group can be  by a l k y l a t i o n w i t h methyl i o d i d e , e t h y l i o d i d e or  labelled  trideuterio-  methyl i o d i d e . When the r e s i d u e a t t a c h e d  to C-4  of the u r o n i c a c i d i s a  sugar, a second B - e l i m i n a t i o n can take p l a c e because o f the exposure of the r e d u c i n g end  to the b a s e ; i f the aldose  s u b s t i t u t e d a t p o s i t i o n 3»the d e g r a d a t i o n  is  can continue further.  In order to a v o i d t h i s problem,the exposed r e d u c i n g ends can be p r o t e c t e d by u s i n g bases of low n u c l e o p h i l i c i t y , a n d by a c e t y l a t i o n with a c e t i c  anhydride  1 0 1  .  Comparison o f the methylated sugars r e l e a s e d upon hydro-  l y s i s o f the 3 - e l i m i n a t e d p o l y s a c c h a r i d e w i t h the m e t h y l a t i o n a n a l y s i s o f 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 e n v i r o n ment s u r r o u n d i n g  the u r o n i c a c i d r e s i d u e s , i . e . the sugar r e -  sidue t o which the a c i d i s l i n k e d and the sugar r e s i d u e tached  t o p o s i t i o n C-4 o f the a c i d  at-  ( i f any).An example o f t h i s  procedure u s i n g K l e b s i e l l a K 26 i s shown i n Scheme I I . 3 • I I . 5 . 3 . 2 Degradation  preceded by o x i d a t i o n - ^ " ^ . 1 0  By u s i n g s e l e c t i v e procedures methylated  1 0  i t i s possible to obtain  p o l y s a c c h a r i d e s w i t h a l i m i t e d number o f f r e e  h y d r o x y l groups: s e l e c t i v e removal o f a c e t a l groups,from mination  eli-  o f u r o n i c a c i d s , b y removal o f a c i d l a b i l e sugar r e -  s i d u e s , e t c . The f r e e h y d r o x y l groups o f the m o d i f i e d  polysac-  c h a r i d e s can then be s e l e c t i v e l y o x i d i z e d t o c a r b o n y l groups (see S e c t i o n I I . 7 . 2 ) which can be the s u b s t r a t e f o r an a l k a line  degradation. T h i s procedure was used on the K 26  a c i d i c p o l y s a c c h a r i d e was reduced r i d e by the carbodiimide A f t e r methylation,the  polysaccharide.The  t o the n e u t r a l p o l y s a c c h a -  procedure (see S e c t i o n I I . 7 . 1 )  methylated  reduced,acetal-linked  pyru-  v i c a c i d was s e l e c t i v e l y removed by h y d r o l y s i s . e x p o s i n g f r e e h y d r o x y l groups a t C-4 and C-6 o f the t e r m i n a l  galactose.The  g a l a c t o s e was then o x i d i z e d (see S e c t i o n I I . 7 - 2 ) t o the d i c a r b o n y l d e r i v a t i v e . d e g r a d e d w i t h base and removed by m i l d h y d r o l y s i s . The product was rernethylated,showing t e r m i n a l g a l a c t o s e was l i n k e d t o the C-4 o f Scheme I I . 4 ) .  t h a t the  glucose (see  Scheme II.3 ^  Uronic a c i d d e g r a d a t i o n of K l e b s i e l l a K 26 polysaccharide.  46  Reduced K26 Fblysaccharide Methylation  1. 50V. CKjCOOH 2.0x.(DMSO/TFAA)  1. Base 2. 50% Ch^COOH 3. Methylation  1.5 - O A c - 2,3.4 6-OMe4-Glucitol 2  4  1.3,5-OAc -2A5-OMe - Mannitol 3  3  12,5-0^- 3.4.6 - 0Me - Mannitol 3  U5-OAc -2A.6-OMe -Galactitol 3  Scheme I I . 4  3  2NaBfy  t56-OAc "-2,3.4-OMe Glucitol  lAc^O/Py  1.^5-OAc -3.6-OMe -Glucitol  3  3  A  2  Degradation preceded by o x i d a t i o n On the K l e b s i e l l a K 26 p o l y s a c c h a r i d e .  47  II.5.4 S e p a r a t i o n o f oligomers obtained from Normally,the  degradations  d e g r a d a t i o n techniques d e s c r i b e d g i v e  1 0 6  "  1 0 9  mix-  t u r e s o f o l i g o m e r s . t o g e t h e r w i t h some unreacted polymeric mat e r i a l and contaminants  from the side r e a c t i o n s . T h e d e s i r e d  oligomers must be separated and p u r i f i e d , t h e s e p a r a t i o n p r o cedures depending  o f the products.methylated  or non-methyl-  a t e d , b a s i c or a c i d i c , e t c . T h e techniques normally used are paper chromatography,paper e l e c t r o p h o r e s i s , g e l - c h r o m a t o g r a p h y , g a s - l i q u i d chromatography and more r e c e n t l y h i g h pressure liquid  chromatography.  Paper c h r o m a t o g r a p h y  1 0 6 , 1 0 7  used e x t e n s i v e l y i n the f i e l d  . P a p e r chromatography has been o f carbohydrate c h e m i s t r y . I t has  the a b i l i t y t o separate components o f complex mixtures r a t e l y and simply.The  accu-  p o s s i b i l i t y of using d i f f e r e n t solvent  systems g i v e s u s e f u l i n f o r m a t i o n on the nature o f the carbohydrate m a t e r i a l , e . g . a c i d i c oligomers do not move i n b a s i c s o l v e n t s . T h i s procedure Charged oligomers  can be used on a p r e p a r a t i v e s c a l e .  ( a c i d i c or b a s i c ) o f h i g h molecular  weights move v e r y s l o w l y on p a p e r , i n which case paper  electro-  p h o r e s i s can p r o v i d e an a l t e r n a t i v e convenient method o f sepa r a t i n g them.It i s f a s t e r and can a l s o be c a r r i e d out on a preparative  scale.  Gel chromatography ^" 1 0  chromatographic  1 1 0  . G e l chromatography i s a l i q u i d  method which separates molecules  primarily  a c c o r d i n g t o d i f f e r e n c e s i n molecular dimensions.Solutes are e l u t e d i n the order o f d e c r e a s i n g molecular s i z e s . T h i s  field  .  48  has been reviewed by Churms been produced,hydrophobic  . D i f f e r e n t types  of g e l s have  and h y d r o p h i l i c g e l s which are used  w i t h o r g a n i c and aqueous s o l v e n t s r e s p e c t i v e l y , w i t h d i f f e r e n t exclusion  limits,etc.  G e l chromatography has been used i n carbohydrate  chemistry  109 f o r molecular-weight  determination  ,and f o r s e p a r a t i o n  of  products of p a r t i a l h y d r o l y s i s , p e r i o d a t e o x i d a t i o n , e t c . A - s e p a r a t i o n based upon molecular s i z e d i f f e r e n c e s alone i s o f t e n not enough t o o b t a i n a completely pure p r o d u c t . T h i s means i t must be used s e q u e n t i a l l y w i t h other techniques such as i o n exchange chromatography,paper chromatography,For example, by 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 of K 6 0 a f t e r degradation,mixtures  of a c i d i c t r i s a c c h a r i d e s and  Smith  tetrasac-  c h a r i d e s were obtained by g e l chromatography which had t o be p u r i f i e d by paper  chromatography.  Gas-liquid chromatography tility  1 1 1  "  1 1 6  . The decrease i n v o l a -  on going from mono- to d i - and h i g h e r o l i g o - s a c c h a r i -  des i s the main disadvantage w i t h u s i n g t h i s technique f o r such s e p a r a t i o n s . A l t h o u g h s u i t a b l e d e r i v a t i v e s can be duced , t r i m e t h y l s i l y l and permethyl  pro-  ethers,trifluoroacetyl,  i t has not reached the p o i n t of e x c e l l e n c e . L i q u i d phases f o r g . l . c . are a v a i l a b l e t h a t are s t a b l e to h i g h  temperatures  ( 2 5 0 - 3 0 0 ° ) . T h i s a l l o w s s e p a r a t i o n of oligomers up to t e t r a s a c c h a r i d e s i n reasonable p e r i o d s ( 3 0 - 4 0 minutes a t The c o u p l i n g of g . l . c t o m.s.  250°).  i s an important t o o l i n the  s t r u c t u r a l s t u d i e s of p o l y s a c c h a r i d e s as shown i n s e v e r a l publications  1 1 5  '  1 1 6  .  49 117-119 High p r e s s u r e l i q u i d chromatography has  recently  found i n c r e a s i n g  i s t r y . I t has  This  a p p l i c a t i o n i n carbohydrate chem-  the advantage over the  other techniques of a high  r e s o l u t i o n power as w e l l as speed of s e p a r a t i o n . l t used i n the  separation  technique  has  of methylated o l i g o s a c c h a r i d e s  cent p u b l i c a t i o n by A l b e r s h e i m  119  s h o w s the use  been and  a re-  of HPLC i n the  sequencing of g l y c o s y l r e s i d u e s of complex carbohydrates. II.6 D e t e r m i n a t i o n of the l i n k a g e s . The  assignment of anomeric c o n f i g u r a t i o n  c o s i d i c linkages the  analysis  mers. The dative  i n a p o l y s a c c h a r i d e i s accomplished  of t h e i r o l i g o m e r i c  s u b - u n i t s and  d i f f e r e n t techniques used may  (enzymic h y d r o l y s i s  non-degradative  to s p e c i f i c g l y -  be  derived  poly-  c l a s s i f i e d as  or chromium t r i o x i d e  ( o p t i c a l r o t a t i o n and  through  degra-  oxidation),or  n u c l e a r magnetic r e -  sonance ). II.6.1 O p t i c a l  rotation  O p t i c a l r o t a t i o n has enantiomorphs and it  1 2 0  "  1 2 3  .  been used to d i s t i n g u i s h between  as a c r i t e r i o n of p u r i t y and  can be as c h a r a c t e r i s t i c as a m e l t i n g p o i n t  i d e n t i t y since i n the  case  of  carbohydrates. The  specific rotation  ( [ a ] ^ ) of a compound i s d e f i n e d  as:  X  [a)  where,  { =  .«*100 1 x c  a  i s the  observed r o t a t i o n  1  i s the  length  c  i s the  c o n c e n t r a t i o n of the  of the  sample tube  (dm)  solution  (g/lOOmL)  50  The  t  is  the  X  is  the  molecular  £  _  temperature wavelength  rotation  of  M  (  the is  )  polarized  defined  l i g h t  as  [ al x M . W .  100 Normally,the line.although presence  of  wavelength  when  and  a  between  at  a c t i v i t y  better  rules  used  rotations  o p t i c a l  Several ship  wavelength  have  and  the  the  can  w e l l  D-line  be  physical been  structure  is  are  proposed  that  is  sodium  very  demostrated  constant  o p t i c a l  known  at  D-  small,the shorter  obtained.  show  the  a c t i v i t y , s u c h  r e l a t i o n as,Van't  120 Hoff's  Rule  of  Optical  Superposition  .Hudson's  Rule  of  Iso-  121 rotation sons  ,etc.They  are  made  Hudson's  on  give  closely  satisfactory related  Isorotation  Rules  results  when  compari-  substances.  can  be  applied  to  predict  spe-  122 c i f i c  rotations  consideration and  the  of of  molecular  oligosaccharides  the  i n d i v i d u a l  rotations  glycosides) NI = z M . l  where  of  NI. l  polysaccharides  g l y c o s i d i c model  is  component  and  the  linkages  compounds  molecular  sugars  as  i n  involved  (e.g.  methyl  rotation  the  by  methyl  of  the  g l y c o s i -  des . [ ]  -  a  M x  100  M.W. These ing  the  equations  are  configuration  of  useful  for  s p e c i f i c  calculating linkages  from  and the  determinmeasured  51  optical rotations. 124— 148 II.6.2 Nuclear magnetic resonance  (n.m.r.)  Current advances i n i n s t r u m e n t a t i o n conducting  solenoids.spectrometers  (development o f super-  w i t h h i g h e r magnetic  e t c . ) have i n c r e a s e d enormously the progress  i n this  fields,  field.An  e x t e n s i v e review w i t h a l l the i n f o r m a t i o n a v a i l a b l e on carbohydrates  i s a c o n s i d e r a b l e task.The i n f o r m a t i o n t h a t w i l l be  g i v e n here r e f e r s o n l y t o the d i r e c t a p p l i c a t i o n o f n.m.r. s t u d i e s on s t r u c t u r a l a n a l y s i s o f 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 . The although  f a c t t h a t the p o l y s a c c h a r i d e s o f K l e b s i e l l a ,  o f h i g h molecular weight ( ~ 1 0 ) , g i v e i n t e r p r e t a b l e 6  s p e c t r a a r i s e s from t h e i r r e g u l a r s t r u c t u r e s to the complex s t r u c t u r e s o f the p l a n t little  -  polysaccharides.where  i n f o r m a t i o n can be obtained. 1  II.6.2.1 P r o t o n magnetic resonance ( The  .compared  >t  127—133 H-n.m.r.)  ~  J J  .  a p p l i c a t i o n o f ^H-n.m.r. t o problems i n carbohydrate  chemistry  i n v o l v e s the measurement o f s e v e r a l n.m.r. param-  eters : i)  Chemical s h i f t . T h i s parameter depends upon the e n v i r o n -  ment o f the p r o t o n i n the molecule,and i s s t r o n g l y dependant on the s u b s t i t u t i o n a l , o r i e n t a t i o n a l and e l e c t r o n e g a t i v i t y e f f e c t s o f the n e i g h b o u r i n g  groups.In the spectrum o f 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  proton r e g i o n ( 6 3 - 0 - ^ . 5 ) and c) the h i g h f i e l d s p e c i f i c groups can be observed 6-deoxy sugars.pyruvates  region,where  such as methyl groups from  and a c e t a t e s . W i t h  some e x c e p t i o n s ,  52  a g e n e r a l r u l e i s t h a t a x i a l l y o r i e n t e d protons resonate a t higher f i e l d  than the c h e m i c a l l y s i m i l a r e q u a t o r i a l l y o r i e n t e d  p r o t o n s . S e v e r a l e m p i r i c a l r u l e s have been proposed  t o c a l c u ->  l a t e chemical s h i f t s o f protons. An a r b i t r a r y d i v i s i o n a t <$ 5-0 was accepted which d i v i d e s the anomeric r e g i o n i n t w o , s i g n a l s appearing d o w n f i e l d of 65.O are a s s i g n e d t o a - l i n k a g e s ( p r o t o n e q u a t o r i a l l y o r i e n t e d ) and s i g n a l s appearing u p f i e l d are a s signed t o 6 - l i n k a g e s ( p r o t o n a x i a l l y oriented).When t h i s i s applied to a polysaccharide spectrum.it i s p o s s i b l e to determine the number o f a - and B - l i n k a g e s p r e s e n t , b u t no f u r t h e r assignment o f p a r t i c u l a r g l y c o s i d i c bonds can be comparison  w i t h s p e c t r a o f oligomers i s o l a t e d . F o r the r i n g  protons.assignments been observed  are more d i f f i c u l t  t o make,although i t has  t h a t s p e c i f i c protons may resonate a t lower  ( 6 4 . 0 - 4 . 5 ) ( H - 2 o f mannose r e s i d u e s , H - 5  fields  done,unless  of glucuronic  and g a l a c t u r o n i c a c i d r e s i d u e s , e t c ) . a n d can be as low as 1  6 4.85  p O  .In the h i g h f i e l d r e g i o n , p a r t i c u l a r groupings can  be d e t e c t e d , i n c l u d i n g the methyl groups o f L-rhamnose or L129 f u c o s e . a c e t a t e and pyruvate.Garegg  and coworkers  7  have ob-  served t h a t the d i f f e r e n c e s i n c h e m i c a l s h i f t o f stereoisomer i c p a i r s o f a c e t a l i c CH^- groups o f pyruvate are g r e a t enough to  be used f o r u n e q u i v o c a l d e t e r m i n a t i o n o f t h e i r stereochem-  istry. i i ) C o u p l i n g c o n s t a n t . T h i s parameter.for  v i c i n a l hydrogens,  bears a r e l a t i o n s h i p t o the t o r s i o n angle^3°(dihedral angle, 0  ) b e i n g a u s e f u l t o o l f o r the d e t e r m i n a t i o n o f c o n f i g u r a t i o n  and/or conformation o f c a r b o h y d r a t e s . K a r p l u s - ^ 1  1  demonstrated  53 t h i s r e l a t i o n s h i p and gave an e m p i r i c a l e q u a t i o n which enables us t o c a l c u l a t e c o u p l i n g constants.The the d i h e d r a l 1 3 2  ,Although  angle(0)  v a l u e s are maximum when  i s 0 or 180°,and minimum when i t i s 9 0 ° .  the c o u p l i n g c o n s t a n t depends on a v a r i e t y o f  parameters b e s i d e s the t o r s i o n angle  ( e l e c t r o n e g a t i v i t y of  s u b s t i t u e n t s , H ( l ) - C ( l ) - C ( 2 ) and C ( l ) - C ( 2 ) - H ( 2 ) bond a n g l e s , e t c ) it  i s very u s e f u l f o r the assignment o f t r a n s - d i a x i a l  protons  ( 0=180°) and e q u a t o r i a l - a x i a l or e q u a t o r i a l - e q u a t o r i a l pro-tons  ( gauche conformation  stant  ,0=60°)  as a l a r g e c o u p l i n g con-  7 - 9 Hz and a s m a l l e r c o u p l i n g c o n s t a n t  1-3 Hz i s ob-  t a i n e d r e s p e c t i v e l y . T h i s i s shown i n F i g u r e I I . 3 i i i ) R e l a t i v e i n t e n s i t y o f the s i g n a l s . U n d e r proper o p e r a t i n g conditions,the r e l a t i v e  i n t e n s i t i e s of the d i f f e r e n t  hydro-  gens are e q u a l t o the r e l a t i v e amounts o f hydrogen p r o d u c i n g the s i g n a l s . T h i s a l l o w s a r a p i d q u a n t i t a t i v e a n a l y s i s o f the r a t i o of a - t o B-linkages.number  of 6-deoxy s u g a r s . a c e t a t e s ,  p y r u v a t e s . e t c . p r e s e n t i n the spectrum.For o l i g o s a c c h a r i d e s , the anomeric proton s i g n a l s f o r the r e d u c i n g end can be d i s t i n g u i s h e d from s i g n a l s o f the other protons because i t d i s p l a y s the e f f e c t o f mutarotation,showing  two separate  f o r the a - and B-anomers.Anomeric e q u i l i b r i a  signals  ( r a t i o a / B )can  a l s o be c a l c u l a t e d . S e v e r a l problems a r e encountered n.m.r. experiment  i n p e r f o r m i n g a proton  on a p o l y s a c c h a r i d e . F i r s t l y , a l l the protons  from the f r e e h y d r o x y l groups are interchanged by deuterium. T h i s exchange i s done by s u c c e s i v e d i s s o l u t i o n s o f the sample i n 99.7% D 0 and f r e e z e - d r y i n g . A f t e r h e a t i n g the sample under o  54  Figure I I . 3  R e l a t i o n s h i p between d i h e d r a l angle (0)  and  c o u p l i n g c o n s t a n t f o r <* -and 8-D-hexoses. ( R=H,OH  and R-,= H,etc. )  55  vacuum f o r s e v e r a l h o u r s . i t i s d i s s o l v e d i n 99-9% D^O.Despite the exchanges,a r e s i d u a l HOD  peak i s always found which ap-  pears i n the anomeric r e g i o n of the spectrum.In minate t h i s i n t e r f e r e n c e s e v e r a l procedures i n c r e a s e o f the temperature,that fields  ( 6 4.1-4.2).addition  order to  eli-  have been d e v i s e d ,  s h i f t s the HOD  peak t o upper  of a c i d ( t r i f l u o r o a c e t i c  acid)that  s h i f t s the peak d o w n f i e l d , or a r e l a x a t i o n timeCl^) type periment which e l i m i n a t e s the HOD a t h i g h e r temperatures  ex-  peak --^. Recording the s p e c t r a 1  ( 90-95°) not o n l y e l i m i n a t e s the  ference o f HOD,but a l s o h e l p s t o reduce  inter-  problems a s s o c i a t e d  w i t h the v i s c o s i t y o f the sample(loss of r e s o l u t i o n ) . A l e s s v i s c o u s sample can be prepared by performing a v e r y m i l d l y s i s o f the p o l y s a c c h a r i d e . T h e problem a s s o c i a t e d w i t h procedure  i s the p o s s i b i l i t y of l o s s of l a b i l e  hydrothis  groups(acetate,  pyruvate,etc.). F i g u r e I I . 4 shows the """H n.m.r. spectrum  of K l e b s i e l l a  K60 p o l y s a c c h a r i d e . l t has been recorded w i t h acetone n a l standard  ( 6 2 . 2 3 ) . T h e spectrum  as  inter-  exhibits 6 signals i n  anomeric r e g i o n , 4 i n the a - and 2 i n the B -region.From  the  the  i n t e g r a t i o n data,one of the a - l i n k a g e s i g n a l s corresponds  to  two p r o t o n s . F u r t h e r i n f o r m a t i o n can be obtained from the coupl i n g c o n s t a n t s , t h e p o l y s a c c h a r i d e i s known to c o n t a i n 4 g l u coses, 1 g l u c u r o n i c a c i d , l g a l a c t o s e and  1 mannose per r e -  p e a t i n g u n i t , a n d none of the B - l i n k a g e s are a s s o c i a t e d w i t h the mannosyl r e s i d u e , a s the c o u p l i n g c o n s t a n t i n t h i s should be 2-3 linked.  case  Hz.The c o n c l u s i o n i s t h a t mannose must be a  -  vip;iire  II.4  The  capsular  H-n.m.r. spectrum o f K l e b s i e l l a polysaccharide.  K60  57 II.6.2.2 Carbon magnetic resonance (  13 \127 134-148 ^C-n.m.r.) '' . J  Many methods have been developed t h a t i n c r e a s e the s e n s i tivity  of n a t u r a l abundance "^C-n.m.r.,including p u l s e - F o u r i e r 134  transform  n.m.r. spectroscopy.proton  broad band d e c o u p l i n g  J  which e f f e c t s the c o l l a p s i n g of s p i n m u l t i p l e t s i n t o s i n g l e t s and as a s i d e e f f e c t produces the n u c l e a r  Overhauser e f f e c t ^ 13 t h a t enhances the s i g n a l s , e t c . In r e c e n t y e a r s , -^C-n.m.r. has 136-141 found e x t e n s i v e  use  use  i n the study of carbohydrates  of low molecular  of ^ C  s p e c t r a of polymeric  by c o r r e l a t i o n w i t h  .The  J  weight model compounds f o r a n a l y s i s  s p e c t r a of p o l y s a c c h a r i d e s  i s generally  of  valid.Assignment  carbohydrates i s g e n e r a l l y done  the s p e c t r a of t h e i r o l i g o m e r i c  subunits  as w e l l as w i t h monosaccharide g l y c o s i d e s , o l i g o s a c c h a r i d e s related  and  polysaccharides ** " ^. 1  2  1  13 The  main parameter used f o r assignment of the  i s the chemical  y l and  main r e g i o n s  c a r b o x y l groups (around 170  ( 93-HO  spectra  s h i f t . I n a spectrum of an o l i g o s a c c h a r i d e  a polysaccharide,four  primary a l c o h o l s ( 6 0 - 8 5 ppm)  and  or  can be observed:a) carbonppm),b) anomeric carbons  ppm),c) the remainder of the r i n g carbons and  the  d) methyl groups of 6-deoxy  sugars.acetates.pyruvates.The most u s e f u l r e g i o n i s t h a t  of  the anomeric carbons.As a g e n e r a l r u l e , t h e s e carbons are strongly deshielded and  (7-10  v  ppm)  through g l y c o s i d e  formation  the C - l resonance of an a x i a l isomer i s s h i e l d e d  relati-  ve to t h a t of i t s e q u a t o r i a l isomer.With these g e n e r a l i z a t i o n s i n mind, g l y c o s i d i c l i n k a g e s may  be a s s i g n e d  p o s i t i o n r e l a t i v e to an a r b i t r a r y d i v i s i o n a t 101  by  their  ppm,signals  58  Figure I I . 5  The^c-n.m.r. spectrum o f K l e b s i e l l a K60 capsular  polysaccharide.  59  appearing u p f i e l d r e p r e s e n t - l i n k e d sugars a  ( a x i a l anomers)  and s i g n a l s appearing d o w n f i e l d r e p r e s e n t 3 - l i n k e d sugars (equatorial isomers) ^.The 1  anomeric carbons o f f r e e  ( r e d u c i n g end) appear u p f i e l d , i n the r e g i o n 9 3 - 9 7  sugars  ppm.  Very d i s t i n c t i v e are the s i g n a l s due t o the carbons o f primary a l c o h o l g r o u p s ( 6 0 - 6 5 ppm),which can be d i f f e r e n t i a ted as l i n k e d or n o n - l i n k e d by t h e i r chemical s h i f t s (nonl i n k e d , 6 0 - 6 2 ppm;when l i n k e d , t h e y are s h i f t e d 7-10 ppm downfield). Carbons i n v o l v e d i n g l y c o s i d i c bonding t o the anomeric p o s i t i o n o f the a d j a c e n t r e s i d u e are i n most-eases  sufficient-  l y d e s h i e l d e d as t o produce s i g n a l s w e l l separated from the other r i n g carbons (~80  ppm).  Methyl groups are d i s t i n c t l y separated and can be e a s i l y a t t r i b u t e d t o 6-deoxysugars ( ~ 1 7 ppm),acetate (~21 pyruvate  (~24  ppm)and  ppm).It has been shown t h a t the s t e r e o c h e m i s t r y  of an a c e t a l i c l i n k e d pyruvate  can be d i f f e r e n t i a t e d by the  148 chemical s h i f t of the methyl group 13 1 The c o u p l i n g constants  (  J  C - H ) may be used t o a s s i g n  anomeric c o n f i g u r a t i o n , h o w e v e r , l o s s  of s i g n a l s t r e n g t h due t o  s p l i t t i n g and the a d d i t i o n a l number of peaks p r e c l u d e the use of c o u p l i n g constants f o r p o l y s a c c h a r i d e a n a l y s i s . The  ^C  s p e c t r a o f 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  were r e c o r d e d w i t h p r o t o n decoupling.Samples were d i s s o l v e d i n DgO and acetone  was used as i n t e r n a l standard  (31.07  ppm).  As the s p e c t r a had t o be r u n a t room t e m p e r a t u r e . d i f f i c u l t i e s w i t h v i s c o s i t y were overcome by m i l d h y d r o l y s i s o f the p o l y -  60  saccharide. F i g u r e II.5 shows the "^CJ-n.m.r. spectrum(proton  decoup-  l e d ) o f the K60 p o l y s a c c h a r i d e which had undergone m i l d hydrol y s i s . F i v e s i g n a l s can be observed i n the anomeric responding t o seven carbons  region cor-  ( 4 6 - l i n k e d and 3 « - l i n k e d ) . I n -  tense s i g n a l s are observed a t  61.5 ppm c o r r e s p o n d i n g t o  s e v e r a l f r e e C-6 carbons. II.6.3  Other techniques.  Among the d e g r a d a t i v e techniques used f o r d e t e r m i n i n g the s t e r e o c h e m i s t r y o f the g l y c o s i d i c l i n k a g e s only two w i l l  be  considered: a) Enzymic hydrolysis ** 1  -9,15  °.This technique was c o n s i d e r e d t o  be,together w i t h the s p e c i f i c r o t a t i o n s , t h e c l a s s i c a l method of e s t a b l i s h i n g the nature o f the g l y c o s i d i c l i n k a g e s . l t  is  based on the s u s c e p t i b i l i t y o f c e r t a i n l i n k a g e s t o the hydrol y t i c a c t i o n o f s p e c i f i c enzymes(glycosidases  or g l y c o h y d r o -  l a s e s ) . T h i s procedure depends mainly on the a v a i l a b i l i t y  of  enzymes having the proper s p e c i f i c i t y f o r <* - and 8 - g l y c o s i d i c l i n k a g e s . P u r e enzymes having the proper s p e c i f i c i t y have, however,not always been a v a i l a b l e . l t should be emphasized the r a t e o f l i b e r a t i o n o f a monosaccharide u n i t from  that  differ-  ent s u b s t r a t e s by s p e c i f i c g l y c o s i d a s e v a r i e s . d e p e n d i n g on the l e n g t h and s e q u e n t i a l arrangement o f the o l i g o s a c c h a r i d e chain.The  c o n c l u s i o n drawn i s t h a t e i t h e r a n e g a t i v e or a  p o s i t i v e r e s u l t must be c a r e f u l l y examined. b) Chromium t r i o x i d e o x i d a t i o n . l t has been observed t h a t chromium t r i o x i d e i n a c e t i c a c i d r a p i d l y o x i d i z e s p e r a c e t y l a t e d  61  hexopyranosides i n which the aglycone occupies  an e q u a t o r i a l  p o s i t i o n ; t h e p e r a c e t y l a t e d hexopyranosides w i t h aglycons cupying  a x i a l p o s i t i o n s are o x i d i z e d more slowly.These  s u l t s l e a d to a g e n e r a l method f o r d i s t i n g u i s h i n g  a  ocre-  - and s _  p y r a n o s i d i c l i n k a g e s i n g l y c o s i d e s , o l i g o s a c c h a r i d e s and  poly-  saccharides ^"*"'" ^. 1  II.7  1  V a r i o u s r e a c t i o n s on o l i g o - a n d p o l y - s a c c h a r i d e s .  II. 7 . 1  Reduction - **" ^ 1  Reduction  5  1  of c a r b o x y l groups i n a c i d i c carbohydrates  be c a r r i e d out a t d i f f e r e n t stages d u r i n g a s t r u c t u r a l s i s of a  can analy-  polysaccharide:  i ) i n the t o t a l sugar r a t i o , i n order to o b t a i n a n e u t r a l o l i g o or p o l y s a c c h a r i d e  so t h a t i t can be e a s i l y  i i ) d u r i n g the m e t h y l a t i o n  hydrolyzed,  a n a l y s i s , f o r the i d e n t i f i c a t i o n  of  the s u b s t i t u t i o n p a t t e r n of the u r o n i c a c i d , i i i ) p r i o r to p e r i o d a t e  o x i d a t i o n , i n order to a v o i d e l e c t r o s -  t a t i c r e p u l s i o n s between p e r i o d a t e ions and carbohydrate  which leads to  under-oxidation.  Depending on the s u b s t r a t e o l i g o m e r i c or polymeric)  the p o l y a n i o n i c  (methylated  or non-methylated,  s e v e r a l procedures can be  followed.  The r e d u c i n g agents most commonly used are NaBH^ and  LiAlH^  as w e l l as some of t h e i r d e r i v a t i v e s . NaBH; . T h i s r e d u c i n g agent may 4  and  i t i s the reagent  be used i n aqueous s o l v e n t s ,  used f o r c o n v e r t i n g the f r e e sugars to  a l d i t o l s . T h e disadvantage i s t h a t i t does not reduce a c i d s . T h i s problem can"be.avoided by c o n v e r t i n g the a c i d s i n t o e s -  62  t e r s which can then he reduced w i t h NaBH^ t o the corresponding alcohols  1 5 6  .Methyl  e s t e r s may be formed by treatment o f the  a c i d i c polysaccharide  w i t h methanolic h y d r o c h l o r i c a c i d or  w i t h diazomethane.With the f i r s t p r o c e d u r e . g l y c o s i d i c  linkages  are c l e a v e d a t the same time.modifying the p o l y s a c c h a r i d e . T h i s technique i s used e x t e n s i v e l y i n t h i s l a b o r a t o r y f o r the t o t a l sugar r a t i o . T h e second procedure must be repeated  i n order t o  ensure complete e s t e r i f i c a t i o n . T h e use o f NaBH^ t o reduce e s t e r s has the disadvantage t h a t h y d r o l y s i s o f the sodium borohydride  produces h y d r o x y l  thus d e c r e a s i n g  i o n s which can s a p o n i f y the e s t e r s ,  the y i e l d o f r e d u c t i o n . T h i s d i f f i c u l t y can be  overcome w i t h the use of Ca(BU^)2*  as the s o l u t i o n s o f a l k a l i -  ne b o r o h y d r i d e s a r e n e a r l y n e u t r a l soluble i n tetrahydrofuran  1 5 7  . N a B H ^ and C a ( B H ^ ) are 2  and they a r e a l s o used i n the r e -  d u c t i o n o f permethylated a c i d i c T a y l o r and C o n r a d  1 5 6  oligosaccharides.  have used a water s o l u b l e  carbodiimi-  de to a c t i v a t e the c a r b o x y l i c a c i d which can then be reduced w i t h NaBH^ a c c o r d i n g  t o the r e a c t i o n mechanism o u t l i n e d i n  Scheme I I . 5 . LiAlH^.Although  t h i s reducing  agent i s powerful enough t o r e -  duce c a r b o x y l i c a c i d s i t s use i s l i m i t e d t o s u b s t r a t e s i n ether-type saccharides  solvents.Permethylated  soluble  a c i d i c o l i g o - and p o l y -  are e a s i l y reduced w i t h L i A l H ^ i n oxolane.Reduc-  t i o n of a c y l a t e d p o l y s a c c h a r i d e s  which are s o l u b l e i n t h i s  type o f s o l v e n t i s n o t s u i t a b l e , a s e s t e r s are reduced f a s t e r than the c a r b o x y l i c a c i d w i t h the subsequent of the p o l y s a c c h a r i d e . A  insolubilization  m o d i f i c a t i o n i n v o l v e s the treatment  63  RCOOH  R'  R"  I  N  Rcocr  H  II  C  H  +  II  II  pH=A,75  N  *N H n  0  +  RC-O-C  NH  I  R"  NaBK pH-5-7 R NH C=0  0  R-CH 0H  II  NH  R-C-H  2  I  R" Scheme  II.5  Reduction solution  of  a  using  carboxylic a  acid  carbodiimide  i n  aqueous  reagent.  64  of the a c y l a t e d ane  (propionylated)  polysaccharide  w i t h diazometh-  t o make the methyl e s t e r of the c a r b o x y l i c a c i d s which are  reduced f a s t e r than the p r o p i o n i c e s t e r s w i t h LiBH^ however r e q u i r e s s e v e r a l treatments i n order  D  .This  t o achieve  com-  p l e t e r e d u c t i o n . A n a l t e r n a t i v e procedure i n v o l v e s the use o f diborane.a reagent which reduces c a r b o x y l i c a c i d s i n p r e f e r ence t o e s t e r groups II.7.2  J 7  .  Oxidation..  Two main types o f o x i d a t i o n may be used 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 of polysaccharides.The  first  i n v o l v e s the cleavage  of the carbon-carbon bond o f v i c i n a l d i o l s by reagents such as periodate  or l e a d t e t r a a c e t a t e and has a l r e a d y been d e s c r i b e d  (see S e c t i o n I I . 5 . 2 ) . T h e second i n v o l v e s the o x i d a t i o n o f s e l e c t i v e hydroxyl Aspinall  groups t o c a r b o n y l  or c a r b o x y l  groups.  has used the o x i d a t i o n o f primary a l c o h o l s  to c a r b o x y l i c a c i d s w i t h 0^ and P t as c a t a l y s t , i n order t o obtain stable uronosyl  bonds.As shown b e f o r e , 3 - e l i m i n a t i o n i s  an important r e a c t i o n 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 s o f p o l y s a c c h a r i d e s and the s u b s t r a t e s carbonyl  f o r these r e a c t i o n s are mainly  compounds.Several procedures have been developed  through the years by which a l c o h o l s can be o x i d i z e d t o carbonyIs.Oxidizing  agents such as RuO^ have been used as w e l l as  the s e r i e s based on d i m e t h y l s u l f o x i d e , w i t h acetic a c i d  1 (  Cl^^,P^,0^  Z  ,  ^ , t r i f l u o r o a c e t i c anhydride ( T F A A ) ^ and carbo-  diimide'*'^.During  1  the course o f t h i s work,the DMSO/TFAA p r o -  cedure was used on the K 2 6 p o l y s a c c h a r i d e .  65  GENERAL EXPERIMENTAL CONDITIONS  66 III.-  GENERAL EXPERIMENTAL CONDITIONS.  111.1  Paper chromatography. Paper chromatography was  performed  by the descending  meth-  od u s i n g Whatman No.l 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  (18:3slt4)  B)  ethyl acetate:pyridine:water  (  8:2:1  )  C)  1 - b u t a n o l - a c e t i c acid-water  (  2:1:1  )  D)  1-butanol-ethanol-water  ( 4:1:5, upper phase )  Chromatograms were developed w i t h a l k a l i n e s i l v e r or by h e a t i n g a t 110°  f o r 5-1°  minutes a f t e r b e i n g  nitrate  sprayed  w i t h j 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 descending method u s i n g Whatman 3MM  c a r r i e d out by  the  paper and s o l v e n t ( C ) ( un-  l e s s otherwise stated).The r e l e v a n t s t r i p s were c u t out e l u t e d w i t h water f o r 6 hours.The aqueous s o l u t i o n s were  and fil-  t e r e d ,concentrated and f r e e z e - d r i e d . 111.2  G a s - l i q u i d chromatography and g.l.c.-mass  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 Hewlett-Packard  spectrometry. with a  5700 instrument f i t t e d w i t h d u a l f l a m e - i o n i -  a a t i o n d e t e c t o r s . A n I n f o t r o n i c s CRS-100 e l e c t r o n i c i n t e g r a t o r was  used to measure the ./peak areas. S t a i n l e s s - s t e e l columns  ( 1.8mx3mm) were used w i t h a c a r r i e r - g a s f l o w - r a t e of 20mL per minute.The f o l l o w i n g packing m a t e r i a l s were used: (a) 3% of SP-2340 on S u p e l c o p o r t  (100-120 mesh)  (b) 5% o f ECNSS-M on Gas Chrom Q  (100-120 mesh)  (c) 3% of 0V-225 on Gas Chrom Q  (100-120 mesh)  67 (d) 5% of SP-1000 on Gas Chrom Q (e) 5% o f SE-52  on Chromosorb W  (100-120 mesh) ( 60- 80 mesh).  The temperature programs used w i t h each column r e s p e c t i v e l y were (unless otherwise s t a t e d ) t  (a) 195° f o r 4 min.,2°/min , 260° f o r 32 min., (b) isothermal a t 1 7 0 ° , o r 160° f o r 4 min. Z°/min , 1 9 0 ° f o r f  32 min., (c) i s o t h e r m a l a t 1 7 0 ° , o r 180° f o r 4 min.,2°/min  ,230° f o r  32 min., (d) i s o t h e r m a l a t 220°, (e) i s o t h e r m a l a t 1 7 0 ° . 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 instrument f i t t e d w i t h thermal c o n d u c t i v i t y det e c t o r s . S t a i n l e s s - s t e e l columns ( 1.8mx6.3mm) were used w i t h the 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  packing was used: ( f ) 5% o f S i l a r 10 C on Gas Chrom Q (100-120 mesh). The temperature was programmed from 210° a t 4 ° / min t o 250°, and l e f t i s o t h e r m a l f o r 30 minutes. G^l.c.-m.s. a n a l y s i 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 e d 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 o f 100 u A and an i o n source temperature o f 200°. III.3  Gel-permeation  chromatography.  P r e p a r a t i v e g e l - p e r m e a t i o n chromatography  was performed  u s i n g columns (2.5x 100cm) o f B i o - G e l P-2 or B i o - G e l P-4 (both  400 mesh).The c o n c e n t r a t i o n o f the samples a p p l i e d t o  68  the columns ranged  from 40-100 mg/mL.Columns were i r r i g a t e d  with water-pyridine-acetic acid 8 mL/hour.Fractions  (500:5:2)  a t a flow r a t e o f  ( 2 - 3 mL) were c o l l e c t e d , f r e e z e - d r i e d ,  weighed and a f t e r o b t a i n i n g an e l u t i o n p r o f i l e . c h r o m a t o g r a phed on paper. 111.4  O p t i c a l r o t a t i o n and c i r c u l a r d i c h r o i s m .  O p t i c a l r o t a t i o n s were measured from aqueous s o l u t i o n s a t 20 ± 3 ° on a Perkin-Elmer model 141 p o l a r i m e t e r w i t h a 10 cm cell. 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 r e c o r d e d on a Jasco J20 automatic r e c o r d i n g s p e c t r o p o l a r i m e t e r w i t h a q u a r t z  cell  0 . 3 mL c a p a c i t y and a path l e n g t h o f 0.1 cm.Compounds were  of  d i s s o l v e d i n h i g h p u r i t y a c e t o n i t r i l e and the s p e c t r a r e c o r d ed i n the range 210-240 nm. 111.5  Nuclear magnetic  resonance.  Proton magnetic resonance  s p e c t r a were r e c o r d e d on V a r i a n  XL-100,Bruker WP-80,or Bruker WH-400 i n s t r u m e n t s . S p e c t r a were r e c o r d e d a t temperatures  o f 9 0 ± 5 ° and acetone was used as an  i n t e r n a l s t a n d a r d . A l l v a l u e s are g i v e n r e l a t i v e t o t h a t o f i n t e r n a l sodium-4,4-dimethyl-4-silapentanesulfonate  taken as  0 .Samples were prepared by d i s s o l v i n g i n D^O and f r e e z e - d r y ing  2 - 3 times from DgO s o l u t i o n s . ^c-n.m.r. s p e c t r a were recorded on a V a r i a n CFT-20 spec-  trometer a t ambient temperature.Samples were d i s s o l v e d i n D 0 and acetone was used as i n t e r n a l standard. o  69  111.6  General conditions.  The  I.R.spectra of methylated d e r i v a t i v e s were r e c o r d e d  on a Perkin-Elmer model 4 5 7 spectrophotometer.The used was  carbon  solvent  tetrachloride.  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 t o r y evaporator of 4 0 ° .  i n vacuo a t a bath temperature  Ion-exchange chromatography f o r s e p a r a t i o n o f a c i d i c n e u t r a l oligomers was  performed  on a column ( 2.0x28cm)  Bio-Rad AG-1-X2 (formate form,200-400 mesh).The n e u t r a l t i o n was  and of frac-  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 . De^-ionizations were c a r r i e d out w i t h Amber l i t e  IR-120(H ) +  resin. 111.7  I s d l a t i o n and p u r i f i c a t i o n o f the p o l y s a c c h a r i d e s .  III.7-1 K l e b s i e l l a polysaccharides. The f o l l o w i n g media 1.  were used to grow the b a c t e r i a :  B e e f - e x t r a c t medium  5 g of  Bactopeptone  3 g of Bacto beef e x t r a c t 2 g o f NaCl 1  2. Sucrose-yeast e x t r a c t - a g a r  L of 75 5  2  g of Sucrose g of Bacto y e a s t e x t r a c t  37-5g  5  H0  o f agar  g of NaCl  2 . 5 g of KH P0^ 2  0.625g  of MgS0^.7H 0 2  1 . 2 5 g of CaSO^ 2.5  L of  H0 2  70  Samples o f K l e b s i e l l a b a c t e r i a o f serotypes K 6 0 and K 2 6 were r e c e i v e d as s t a b c u l t u r e s from  Dr.I.0rskov(Copenhagen).  They were s t r e a k e d on agar p l a t e s a t 37°.An i n d i v i d u a l c o lony was i n n o c u l a t e d on b e e f - e x t r a c t medium and b a c t e r i a were grown f o r 3 hours a t 37° w i t h continuous liquid  c u l t u r e was incubated  shaking.This  on a t r a y o f s u c r o s e - y e a s t ex-  t r a c t - a g a r f o r three days.The lawn o f c a p s u l a r b a c t e r i a produced was harvested by s c r a p i n g the agar s u r f a c e and the bact e r i a k i l l e d w i t h 1% phenol s o l u t i o n . T h e p o l y s a c c h a r i d e was separated The  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 < ( 3 0 0 0 0 rpm).  v i s c o u s s o l u t i o n o f p o l y s a c c h a r i d e was p r e c i p i t a t e d  with  e t h a n o l (3 volumes).The p r e c i p i t a t e was d i s s o l v e d i n the minimum amount o f water and r e p r e c i p i t a t e d w i t h a s a t u r a t e d s o l u t i o n of C E T A V L 0 N  (cetyltrimethylammonium  f u g a t i o n y i e l d e d the p r e c i p i t a t e d a c i d i c  bromide).Centri-  polysaccharide.The  C e t a v l o n - p r e c i p i t a t e d complex was d i s s o l v e d i n kM NaCl,rep r e c i p i t a t e d into ethanol l y z e d a g a i n s t running The  (3 v o l . ) and the p r e c i p i t a t e d i a -  t a p water a f t e r d i s s o l u t i o n i n water.  d i a l y z e d s o l u t i o n o f p o l y s a c c h a r i d e was f r e e z e - d r i e d  a f t e r a l l the s a l t had been removed. III.7.2 The  Gum exudate from C h o r i s i s s p e c i o s a . crude gum,which was obtained as l i g h t y e l l o w  was allowed  nodules,  t o s w e l l i n water f o r 2k h o u r s . I t was then  on a steam-bath f o r 8 hours.The pH was kept constant  heated  around  7 i n order t o a v o i d 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 . A f t e r c e n t r i f u g a t i o n , t h e s o l u t i o n was poured i n t o a c i d i f i e d  ethanol  71 ( 4 v o l s . ) . T h e p r e c i p i t a t e d m a t e r i a l was d i s s o l v e d i n water and f r e e z e - d r i e d . III.8 The  Sugar a n a l y s i s . i d e n t i f i c a t i o n and q u a n t i t a t i o n o f the sugars  present  i n p o l y - and o l i g o - s a c c h a r i d e s was done by g . l . c . o f the d e r i ved a l d i t o l acetates.Samples  (2-10 mg) were h y d r o l y z e d w i t h  2M TFA on a steam-bath (time o f h y d r o l y s i s : p o l y s a c c h a r i d e s 8 hours and o l i g o s a c c h a r i d e s 4 hours).The d i s t i l l a t i o n w i t h water.Part  TFA was removed by co-  o f the h y d r o l y z a t e was used f o r  paper chromatography.while the r e s t was d i s s o l v e d i n H" 0(5mL) 2  and reduced w i t h NaBH^ (20 mg).After  3 hs.,the excess NaBH^  was decomposed w i t h IR-120 ( H ) r e s i n , f i l t e r e d , c o n c e n t r a t e d t o +  dryness and c o d i s t i l l e d w i t h three p o r t i o n s o f methanol (5mL). The a l d i t o l s were a c e t y l a t e d w i t h a c e t i c a n h y d r i d e - p y r i d i n e ( 1:1, 2mL) on a steam-bath f o r one hour and the excess r e agents were removed by c o d i s t i l l a t i o n w i t h e t h a n o l and water. The a l d i t o l a c e t a t e s were d i s s o l v e d i n c h l o r o f o r m and i n j e c ted i n t o the g.l.c.The  column used was (A) and the tempera-  t u r e program as i n d i c a t e d b e f o r e . Samples c o n t a i n i n g u r o n i c a c i d s were t r e a t e d i n the f o l lowing way before h y d r o l y s i s : Samples o f 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 ( 2 - 1 0  mg)dried  i n vacuo were t r e a t e d w i t h Jfo HC1 i n methanol on a steam-bath f o r 8 hours.The a c i d was n e u t r a l i z e d w i t h PbCO^or Ag C0^,cen2  t r i f u g e d and concentrated t o dryness.The  r e s i d u e was d i s s o l v e d  i n anhydrous methanol ( 5 m L ) and NaBH^ (20 mg) was added.The r e a c t i o n was allowed t o proceed a t room temperature  for 8  72  hours.The excess NaBH^ was decomposed w i t h IR-120 ( H ) r e s i n , +  f i l t e r e d , c o n c e n t r a t e d t o dryness and c o d i s t i l l e d w i t h three p o r t i o n s (5mL) o f methanol.The samples thus obtained were t r e a t e d as d e s c r i b e d before  ( i . e . h y d r o l y s i s . r e d u c t i o n and  acetylation). III.9  Methylation analysis.  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 and o l i g o s a c c h a r i d e s was c a r r i e d out by the Hakomori procedure  ,and i n cases where  complete m e t h y l a t i o n was not achieved w i t h t h i s method,a subsequent m e t h y l a t i o n by the P u r d i e - I r v i n e procedure was performed.Methylation  was c o n s i d e r e d t o be;complete when the  product was devoid o f h y d r o x y l a b s o r p t i o n i n the i n f r a r e d spectrum  (3000-3200 cm" ). 1  Method A ( f o r p o l y s a c c h a r i d e s ) A d r i e d sample (100-300 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 ved w i t h s t i r r i n g and h e a t i n g a t 60° i n anhydrous d i m e t h y l sulfoxide  (15-^5 mL) under Ng.After c o o l i n g t o room tempera-  t u r e , 2M sodium  methylsulfinylmethanide  (10-15 mL) was added  to the s o l u t i o n . A f t e r 4 hours o f s t i r r i n g , t h e s o l u t i o n was f r o z e n and methyl i o d i d e ( 5 - 1 ° mL) was added dropwise.It was then l e f t s t i r r i n g a t room temperature  f o r I f hours before  the excess methyl i o d i d e was removed by r o t a t o r y e v a p o r a t i o n and the r e a c t i o n mixture was d i a l y z e d o v e r n i g h t a g a i n s t r u n n i n g tap water.The contents o f the bag were f r e e z e - d r i e d t o y i e l d the methylated  polymer.  I f m e t h y l a t i o n was incomplete,the p o l y s a c c h a r i d e was f u r t h e r methylated  partially  methylated  u s i n g the Purdie and  73  I r v i n e procedure  as d e s c r i b e d below:  The d r i e d sample (100-200 mg) o f p a r t i a l l y methylated  poly-  s a c c h a r i d e was d i s s o l v e d i n d r y methyl i o d i d e ( 1 0 - 1 5 mL) and refluxed with s i l v e r The s i l v e r  ( I ) oxide  ( 2 0 0 - 3 0 0 mg) f o r 1 - 3 days.  oxide and s i l v e r s a l t s were washed twice with:  CHCl^ a f t e r c e n t r i f u g a t i o n . T h e c h l o r o f o r m e x t r a c t s were combined and the s o l v e n t was removed by r o t a t o r y e v a p o r a t i o n . T h i s procedure was repeated u n t i l no h y d r o x y l a b s o r p t i o n was detected i n the i . r . spectrum. Method B ( f o r o l i g o s a c c h a r i d e s ) The m e t h y l a t i o n procedure  i s as above,except f o r the s o l u b i - .  l i z a t i o n and r e c o v e r y o f the product.A saccharide foxide  sample of d r i e d  (1-10 mg) was d i s s o l v e d i n anhydrous d i m e t h y l s u l -  ( 1 - 3 mL).with s t i r r i n g under N . Sodium m e t h y l s u l f i n y l 2  methanide (2M, 1 - 3 mL) was added and the s o l u t i o n at room temperature iodide  oligo-  f o r 2 hours before being  stirred  frozen.Methyl  ( 1 - 3 mL) was added and was then allowed to s t i r a t  room temperature  f o r 1 hour.The excess methyl i o d i d e was r e -  moved i n vacuo and the product was r e c o v e r e d by adding f o u r volumes o f water and e x t r a c t i n g three times w i t h one h a l f volume o f chloroform.The  combined c h l o r o f o r m e x t r a c t s were  washed w i t h water ( 3 times) and the s o l v e n t removed by r o t a t o r y e v a p o r a t i o n . R e s i d u a l DMS0 was removed i n vacuo by h e a t i n g w i t h an i . r . lamp. H y d r o l y s i s o f permethylated  p o l y s a c c h a r i d e s and o l i g o -  s a c c h a r i d e s was c a r r i e d out w i t h 2M TFA on a steam-bath(time of h y d r o l y s i s , 16 hours f o r a p o l y s a c c h a r i d e , a n d 6 hours f o r  74 an o l i g o s a c c h a r i d e ) , t h e a c i d was  then removed by  codistilla-  t i o n w i t h water.Samples of the h y d r o l y z a t e were s p o t t e d on paper,and chromatographed i n s o l v e n t (D).The p a r t i a l l y methy l a t e d sugars were i d e n t i f i e d by d e v e l o p i n g the papers 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,and for  5 - 1 0 minutes.The r e s t of the h y d r o l y z a t e was  sodium borohydride  ( 4 0 - 6 0 mg)  sodium borohydride was the s o l u t i o n was  with  heating  reduced  with  i n water ( 5 - 1 ° mL).Residual  decomposed w i t h I R - 1 2 0 ( H ) r e s i n , +  f i l t e r e d , c o n c e n t r a t e d t o dryness and c o d i s -  t i l l e d w i t h three p o r t i o n s of methanol ( 5 m D . T h e p a r t i a l l y methylated anhydride-pyridine  resulting  a l d i t o l s were a c e t y l a t e d w i t h a c e t i c  (Is 1 , 2 mL)  on a steam-bath f o r 1 h o u r , f o l -  lowing which the excess reagents were removed by  codistilla-  t i o n w i t h e t h a n o l and water. A n a l y s i s of the p a r t i a l l y methylated was  performed  a l d i t o l acetates  by g . l . c . u s i n g columns ( b ) , ( c ) and  i d e n t i t y of each component i n the mixture was  (d). The  confirmed  by  g. 1 . c. -m. s. . The u r o n i c methyl  e s t e r s i n a c i d i c p o l y s a c c h a r i d e s and  o l i g o s a c c h a r i d e s were converted i n t o the c o r r e s p o n d i n g ars  sug-  p r i o r t o h y d r o l y s i s i n the f o l l o w i n g manners  Method A ( f o r p o l y s a c c h a r i d e s ) A sample of permethylated  m a t e r i a l ( 3 - 5 0 mg) mL),LiAlH^  was  dissolved  mg)  was  i n anhydrous oxolane  (3-10  and the r e a c t i o n was  l e f t t o s t i r a t room temperature  (3°-50  added for  6 h o u r s . P o l y s a c c h a r i d e s were r e f l u x e d f o r the same p e r i o d . The excess L i A l H  k  was  destroyed by dropwise  a d d i t i o n of ,  75 ethanol,the p r e c i p i t a t e formed was d i s s o l v e d i n 4 volumes o f 10% HCl.and the s o l u t i o n was then e x t r a c t e d w i t h CHCl^ ( 3 * 1 volume).The combined c h l o r o f o r m e x t r a c t s were c o n c e n t r a ted  t o dryness. I . r . s p e c t r o s c o p y of the sample d i s s o l v e d i n  CCl^  showed complete r e d u c t i o n when no c a r b o n y l a b s o r p t i o n  was d e t e c t e d and h y d r o x y l a b s o r p t i o n was  observed.  Method B ( f o r o l i g o s a c c h a r i d e s ) A d r i e d sample ( 1 - 5 mg) of permethylated  ( 5 mL) and t r e a t e d w i t h NaBH^  d i s s o l v e d i n anhydrous oxolane (20 mg) and C a C l  2  o l i g o s a c c h a r i d e was  f  (20 mg),the C a ( B H ^ )  2  generated  the r e d u c i n g agent used.The r e a c t i o n was s t i r r e d hours a t room temperature,and centrifuged.Amberlite  i n s i t u was during 8  the s o l u t i o n was f i l t e r e d or  IR-120 ( H ) r e s i n w a s +  pose the excess borohydride,the  added t o decom-  s o l u t i o n was f i l t e r e d , c o n -  c e n t r a t e d t o dryness and c o d i s t i l l e d w i t h three p o r t i o n s ( 5 m L ) of methanol.Reduction  was checked  III.10 Base c a t a l y z e d u r o n i c a c i d Permethylated  by i . r . as above. degradation.  p o l y s a c c h a r i d e ( 3 0 - 5 0 mg) was c a r e f u l l y  dried i n vacuo,dissolved i n dimethylsulfoxidei2,2-dimethoxypropane ( 1 9 : 1 , 1 5 mL) w i t h j D - t o l u e n e s u l f o n i c a c i d s t i r r e d under N (2M)  2  f o r 1 hour.Sodium m e t h y l s u l f i n y l m e t h a n i d e  i n d i m e t h y l s u l f o x i d e ( 1 5 mL) was added and the s o l u t i o n  was l e f t s t i r r i n g a t room temperature ing  (1-2 mg)and  the r e a c t i o n mixture.methyl  overnight.After freez-  iodide (or e t h y l  iodide)(lOmL)  was added and the s o l u t i o n allowed t o s t i r a t room temperat u r e f o r l i hour.The excess methyl i o d i d e was removed by r o t a t o r y e v a p o r a t i o n , t h e s o l u t i o n was d i l u t e d w i t h H 0 ( 4 v o l . ) ?  76  and  then e x t r a c t e d w i t h CHCl^ ( 3 x 1 5 mL).The product was  h y d r o l y z e d and the sugars r e l e a s e d were analyzed as d e s c r i bed p r e v i o u s l y f o r the m e t h y l a t i o n a n a l y s i s .  77  STRUCTURAL INVESTIGATIONS OF KLEBSIELLA CAPSULAR POLYSACCHARIDES  78  IV.1  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 serotype K 60 capsular polysaccharide.  IV.1.1 Abstract. Non-linear capsular polysaccharides of K l e b s i e l l a bacteria usually have a single side chain per repeating unit, less commonly two side chains attached to the same sugar.The capsular polysaccharide from K l e b s i e l l a serotype K 60 i s unique i n having three side chains i n the heptasaccharide r e peating unit shown.The structure,including the configuration of the g l y c o s i d i c linkages,was established mainly by charact e r i z a t i o n of the oligosaccharides obtained by p a r t i a l hydrol y s i s of both,the o r i g i n a l capsular polysaccharide,and the polymer r e s u l t i n g from the removal,by Smith degradation,of the side chains.  D-Glc£ -—-- D-GlcpA - — 2 . D-Galjj - — 2 D-Manp - — B  4f  ll  D-Glcp_  ll  ll  D-Glcp_  D-Glcp_  IV.1.2 Introduction. The capsular polysaccharides of K l e b s i e l l a that are composed of D-glucuronic acid,D-glucose,D-galactose  and D-man-  nose comprise the largest chemogroup i n this genus with  20  strains represented,half of which incorporate pyruvic acid as an a c e t a l into the polysaccharide.The  capsular polysaccharide  from K l e b s i e l l a serotype K 60 discussed here,represents a novel s t r u c t u r a l pattern i n this s e r i e s .  79 IV.1.3 R e s u l t s and d i s c u s s i o n . Composition and n.m.r. s p e c t r a . K l e b s i e l l a K 60 b a c t e r i a was  grown on an agar medium and  was  p u r i f i e d by one  the c a p s u l a r p o l y s a c c h a r i d e •  p r e c i p i t a t i o n w i t h CETAVLON.The  product  moved as a s i n g l e band on e l e c t r o p h o r e s i s on c e l l u l o s e te and  aceta-  had[ a ] +58°,which compares w e l l w i t h the c a l c u l a t e d D  value o f + 4 7 ° u s i n g Hudson's Rule of I s o r o t a t i o n . T h e molecul a r weight of the p o l y s a c c h a r i d e was matography to be 8.1x of 1100  determined by g e l c h r o -  10^ daltons.The  which corresponds  e q u i v a l e n t weight  was  to one u r o n i c a c i d per seven sugar  residues. Paper chromatography of 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 showed the presence o f mannose.glucose.galactose . g l u c u r o n i c a c i d . g l u c u r o n o l a c t o n e acid.These of Nimmich  and an a l d o b i o u r o n i c  r e s u l t s were i n c o n t r a s t to the a n a l y t i c a l where fucose and not g a l a c t o s e was  data  detected.Acid  h y d r o l y s i s of the c a r b o x y l reduced p o l y s a c c h a r i d e and  con-  v e r s i o n to a l d i t o l a c e t a t e s gave mannose.galactose and g l u cose i n a r a t i o 1:1:5. The cose was dichroism  c o n f i g u r a t i o n of mannose and  determined to be D by measurements of the (cd.)  ated d e r i v a t i v e i s o l a t e d The recorded  circular  of the a l d i t o l a c e t a t e s ; g a l a c t o s e was  ned a l s o a D c o n f i g u r a t i o n by c d .  glu-  assig-  measurements of a methyl-  subsequently.  "'"H-n.m.r. spectrum of the n a t i v e p o l y s a c c h a r i d e i n DgO  a t 90° w i t h acetone as i n t e r n a l  Appendix III,spectrum  was  standard(see  NaD.The spectrum e x h i b i t s s i x d o u b l e t s  i n the anomeric r e g i o n : 6 5,47  (J^  2  2Hz,2H)j 6 5.37  (J-j^  2  2Hz,  80  1H)|«5.04 ( J  1  >  2  7Hz,lH);  IH) and <$ 4.54 ( J  2 1  <5  4.85 ( J  1 > 2  7Hz,lH);6 4.70 ( J  1  (  2  7Hz,  7Hz,lH).From the values of the chemical  s h i f t s and coupling constants, 3 -anomeric linkages and 4 B a  anomeric linkages were assigned f o r the heptasaccharide ing unit.No deoxy-sugar,0-acetyl  repeat-  or acetal-linked pyruvic acid  could be detected.As mannose i s present i n the polysaccharide, i t was assigned an a-linkage,as the 6 -signals exhibit large coupling constants (7Hz) which do not correspond  to B - l i n k e d  mannose. The  1  3c-  ,  n # m < r >  spectrum of K 60(see Appendix III,spec.No2)  corroborates the r e s u l t s obtained by "'"H-n.m.r. spectroscopy. Six signals appear i n the anomeric region at 99.56;100.60; 102.44;103.07;104.02 and 104.23 ppm.The s i g n a l at 99-56 corresponded to two anomeric carbons.Several signals around 61.51 ppm are attributable to sugar residues that are not linked at position 6. Precise assignment  n  1 a f t e r studying  of the anomeric signals was achieved  H- and  -^C-n.m.r. spectra of oligosaccharides  and polysaccharides obtained by s e l e c t i v e degradative techniques, see Table I V . 1.1 Methylation analysis. Methylation followed by carboxyl reduction,hydrolysis and conversion into a l d i t o l acetates gave the r e s u l t s shown inTablelV.1.2,column  I,while further  methylation a f t e r carboxyl reduction gave the data presented i n column II.These r e s u l t s show that the polysaccharide cons i s t s of a heptasaccharide repeating unit,with three terminal glucose residues and one unit each of mannose.galactose  TABLE IV.1.1 N.M.R. DATA FOR KLEBSIELLA K60 CAPSULAR POLYSACCHARIDE AND DERIVED POLY- AND OLIGOSACCHARIDES H-n.m.r. Compound  £l 2 (Hz) f  G l c A 2—2 Gal-OH  GlcA-—^Gal-OH 2  5.30 4.74 4.60  2 8 7  Integral  Assignment  No.  proton  0.4 1.0 0.6  5-38  Spectrum  -OH  3-GalG l c A  "T  3-Gal  OH  3-^al-  -OH  6  1 Glc  8  GlcA-  4.66  8  Glc— 3-Gal 2  GlcA-—'hal-—^Man-OH B  4.70  a  Glc-—^-LCA-I—^al-OH  5.32  2  1.0  5.20  2  4. 94  2  4.78  7  0.5 0.5 1.0  5.28  2  0.4  4.80  7  1.0  OH B  3-Gal a  3-Man  OH a  3-Man—OH GlcA 3-Gal  OH a  Glc-—  8  TABLE  IV.1.1  (cont.)  GlcA-—^Gal-—-Wn-—-<Jlc-0H B  . A  Glc  a  a  Glc-OH a  l  —-Glc-—-<UcA-—-<Jal-—-ManB  B  a  3-Gal——OH B  1.0  — - G l c - — - k l l c A - — - G a l - — - M a n B 4 2 « 2  1| lc  1.0  2-Man-  0.6  3-Glc-  1.0 0.4  Glc- 6 M 3-Glc- -OH  1.0  3-Man-  1.0  polysaccharide  1 G  0.6  1.6  Man  N  3-GlcA  1.4  5  B  1.0  G:  1.0  a a  B  3-Gal- aa  3-Glc- "g~  1.0  3-GlcA  2.0  3-Man2 a Glc-  1.0  -OH  3-Gal-  a  a  TABLE IV.1.1 (cont.)  K60 c a p s u l a r p o l y s a c c h a r i d e  5.04 4.85  4.70 4.54  7 7 7 7  1.0  3-Glc-  1.0  Glc—  1.0  Glc—  1.0  3-GlcA4  ^C-n.m.r.  Compound  A  l  Chemical s h i f t  104.50 97.04 93.08  A  2  A3  Assignment  GlcA— 3 - G a l — — OH 3-Gal  a  104.21  GlcA-  102.46  Glc—  OH  92.87  3-Gal—OH  104.54  GlcA—  101.40  3-Gal—  Spectrum  TABLE IV.1.1 (cont.) 9^.82  3-Man—OH  94.30  3-Man  OH  104.35  3-GlcA-  103.52  Glc-  97.04  3-Gal—OH  93.00  3-Gal  OH  a  104.51 101.51 101.35 96.76 93-04 102.54  GlcA  8  3-Gal a  3-Man a  3-Glc—OH 3-Glc OH G l c  a  —  99.85 96.80  2-Man--—  93.07  3-Glc  3- G l c — O H OH  104.19  3-GlcA-  103.34  3-Glc-  101.42  3-Gal-  a  3-Man a  TABLE IV.1.1 (cont.)  K60 c a p s u l a r p o l y s a c c h a r i d e  104.23  ^3-GlcA 4  B  Glc  3-GIC-I-  103.0? 102.44 100.60 99.56  ~ Glc—  G  c  3-Man— 2 3-Gal—  {  i  l  ?  a  a  The numerical 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 ; the a  or B t o the c o n f i g u r a t i o n o f the g l y c o s i d i c bond or the anomer i n the case o f  a t e r m i n a l reducing sugar.Thus,  3-Gal—-—  r e f e r s t o the anomeric proton o f a 3 - l i n -  ked g a l a c t o s y l r e s i d u e i n the a c o n f i g u r a t i o n . T h e absence o f a numerical p r e f i x i n d i c a t e s a terminal,non-reducing r e s i d u e . ( i n ^C-n.m.r.spectra,the The chemical s h i f t s are r e l a t i v e to i n t e r n a l acetones ^C-n.m.r. a t 31.0? p.p.m.  nuclei)  "hi-n.m.r a t 6 2.23 »and  86  and g l u c u r o n i c a c i d being the b r a n c h i n g One  may.therefore.write  two  g e n e r a l s t r u c t u r e of the K 60  A)  O  O  I  I  Glc  B)  O  points.  p o s s i b l e p a t t e r n s f o r the  polysaccharide:  3Glc  I  Glc  Glc  Glc  Glc  O  —  Glc 3  I  Glc  where  r e p r e s e n t s galactose.mannose and g l u c u r o n i c a c i d .  In e i t h e r c a s e . i t i s c l e a r t h a t o n l y the three t e r m i n a l g l u cose r e s i d u e s are s u s c e p t i b l e t o p e r i o d a t e o x i d a t i o n . P e r i o d a t e o x i d a t i o n . P e r i o d a t e o x i d a t i o n was  carried  on the sodium s a l t of the s t a r t i n g m a t e r i a l as w e l l as on carboxyl-reduced  the  ( c a r b o d i i m i d e procedure) p o l y s a c c h a r i d e . I n  both cases,the  consumption of p e r i o d a t e reached  (4.85  moles of p e r i o d a t e per mol  and 4.95  out  a plateau  of r e p e a t i n g u n i t ,  r e s p e c t i v e l y ) i n 140 h.Reduction of the polyaldehyde  and  ox-  i d a t i o n o f the p o l y a l c o h o l s thus obtained caused the consumpt i o n o f a f u r t h e r 0.? The  and  0.9  moles of oxidant r e s p e c t i v e l y .  t h e o r e t i c a l value i s 6 moles of p e r i o d a t e per mole of  TABLE IV.1.2 METHYLATION ANALYSIS OF K60 CAPSULAR POLYSACCHARIDE AND DERIVED PRODUCTS. Methylated (as a l d i t o l  Mole % -  R e l a t i v e r e t e n t i o n times  sugars -  OV-225  SP-1000  170°  170°  220°  ECNSS-M  acetates)  I °-  II  III  IV  2,3,4,6 -  Glc  1.00  1.00  1.00  42  48  —  41  2,4,6  -  Glc  1.85  1.83  1.68  16  16  28  25  2,4,6  -  Man  1.91  -  1.82  —  30  —  2,4,6  Gal  2.12  -  1.91  —  —  24  —  4,6  Man  3.13  2.96  2.60  13  13  —  18  4,6  Gal  3.50  3.26  2.82  14  12  —  —  2,6  Glc  3.50  3.26  2.72  —  11  —  2,4  Glc  4.75  -  -  —  —  18  — —  2  Glc  7.92  -  15  —  —  —  —  —  —  16  3?4,6 -  -  Gal -  2,3,4,6-Glc  d  7.61  -  0.92  l , 5 - d i - 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c i t o l , e t c . - Values were  c o r r e c t e d by use of 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  ejt a l .  — I , o r i g i n a l capsular polysaccharide: II,remethylation a f t e r reduction of uronic e s t e r 1 I I I , polymer P O-methylgalactose.  1  : IV, product  from B - e l i m i n a t i o n . - 3 - 0 - e t h y l -  4,6- d i -  88  polysaccharide. The carboxyl-reduced  product was s u b j e c t e d t o a m o d i f i e d  Smith d e g r a d a t i o n , t h a t i s , p e r i o d a t e o x i d a t i o n , b o r o h y d r i d e r e d u c t i o n , m e t h y l a t i o n , h y d r o l y s i s under m i l d r i d e r e d u c t i o n and e t h y l a t i o n  conditions,borohyd-  H y d r o l y s i s o f the product  before the f i r s t  borohydride  r e d u c t i o n y i e l d e d mannose.galac-  tose and glucose  (as a l d i t o l a c e t a t e s ) i n the r a t i o of 1:1:2.  A f t e r the f i r s t m e t h y l a t i o n , h y d r o l y s i s of p a r t o f the m a t e r i a l and c o n v e r s i o n i n t o a l d i t o l a c e t a t e s y i e l d e d 2 , 4 , 6 - t r i - 0 methylglucose,4,6-di-0-methylmannose,2,6-di-0-methylglucose and 4 , 6 - d i - 0 - m e t h y l g a l a c t o s e  as expected.After  the f i n a l s t e p  ( e t h y l a t i o n ) . h y d r o l y s i s and c o n v e r s i o n i n t o a l d i t o l y i e l d e d 2,4,6-tri-O-methylglucose  acetates  and 3 other products  that  although they could not be separated and i d e n t i f i e d , c o r r e s p o n d ed to monoethyl-dimethylhexoses from g l u c o s e . g a l a c t o s e and mannose.These r e s u l t s showed t h a t the s t r u c t u r a l p a t t e r n o f the r e p e a t i n g u n i t must" be  O Glc  where  Glc  3Glc  I  Glc  r e p r e s e n t s mannose,galactose and g l u c u r o n i c a c i d .  Reduction  o f the product from p e r i o d a t e o x i d a t i o n o f the  sodium s a l t o f K 60 p o l y s a c c h a r i d e f o l l o w e d by m i l d h y d r o l y sis  (Smith degradation)  y i e l d e d a p o l y s a c c h a r i d e ( P, ) com-  89  posed o f D-glucose,D-galactose,D-mannose and 1 a c i d i n equimolar  amounts.The  (see Appendix I I I , s p e c . 6 and two a - and  two  B-anomeric  D-glucuronic  13 H- and  7) of  ^C-n.m.r. spectrum showed the presence  l i n k a g e s ( s e e TablelV.1.1)  and  of  the  m e t h y l a t i o n a n a l y s i s ( s e e TablelV.1.2,column I I I ) demonstrated t h a t P^ i s a l i n e a r p o l y m e r . l t f o l l o w s from these t h a t of the three l a t e r a l glucose u n i t s , o n e B - l i n k e d , a n d t h a t the s i d e c h a i n s are g l u c u r o n i c a c i d and  t o 0-2  data  i s a - a n d two  j o i n e d t o 0-4  of both the D-mannose and  are  of the the  D-  galactose u n i t s . P a r t of the methylated  polysaccharide  p r i o r to c a r b o x y l r e d u c t i o n and  ( P ) was 1  separated  hydrolyzed  by ion-exchange  chromatography i n t o a c i d i c and n e u t r a l f r a c t i o n s . T h e a c i d i c f r a c t i o n was  t r e a t e d w i t h 3% methanolic  e s t e r s formed were reduced  HC1  and  the methyl  with NaBH^ihydrolysis  c o n v e r s i o n i n t o a l d i t o l a c e t a t e s showed on g . l . c . methylglucose  and  by  2,4-di-0-  2,4,6-tri-0-methylgalactose.This indicated  t h a t the a l d o b i o u r o n i c a c i d i s A p a r t i a l s t r u c t u r e may  -1  followed  G l c - — 2 . GlcA - — 2  4|  1 Glc  now  G  a  l  2" 1 Glc  GlcA — — 2 be  -2  Gal .  elaborated:  Man  a  1 Glc  w i t h the p r o v i s o t h a t the sequence of the main c h a i n i s unknown ( r e l a t i v e p o s i t i o n of mannose and anomeric c o n f i g u r a t i o n s o f almost  g l u c o s e ) as are  a l l the linkages.These  the pro-  90  blems may be r e s o l v e d by i s o l a t i o n and c h a r a c t e r i z a t i o n o f suitable  oligosaccharides.  P a r t i a l h y d r o l y s i s . I n e f f e c t , t h e r e are two p o l y s a c c h a r i des  o r i g i n a l K 6 0 capsular  t o be s t u d i e d : t h e  polysaccharide  which i s h i g h l y branched,and the l i n e a r polymer P^ by p e r i o d a t e  degradation.  a) O r i g i n a l c a p s u l a r  p o l y s a c c h a r i d e . P a r t i a l h y d r o l y s i s o f the  s t a r t i n g m a t e r i a l gave two a c i d i c o l i g o s a c c h a r i d e s together  obtained  (A^ and Ag)  w i t h a n e u t r a l one (N^J.On the b a s i s o f t h e i r n.m.r.  s p e c t r a l data (see TablelV.1.1) and t h e i r m e t h y l a t i o n (see TablelV.1.3)the  analyses  s t r u c t u r e s o f these compounds were shown  to be: A,  GlcA - — 2 . G a l  ;  A  0  GlcA - — 2 . Q i a  •3 1 Glc  N  l  Glc - —  2  B  Man - — 2  G  i  c  a  Comparison o f the s p e c t r a l data charide  and P  e r a l glucose  1  o f the o r i g i n a l  has a l r e a d y demonstrated t h a t o f the three  u n i t s one i s a - and two a r e B -linked.The  two are shown d i r e c t l y , b y the s t r u c t u r e s o f A those l i n k e d to g a l a c t o s e t h i r d glucose  polysac-  must be  2  lat-  latter  and N^,to be  and mannose{by e l i m i n a t i o n , t h e  a - l i n k e d t o 0 - 4 o f the g l u c u r o n i c a c -  i d r e s i d u e , whence i t f o l l o w s t h a t one may now w r i t e :  91  1—2  —2-  GlcA - — 2 ° 4 3 1 Glc  a  Gal ^ — 2 2 1 Glc  B  Man 2  -— a  1 Glc  There are two anomeric l i n k a g e s unassigned,those of the g a l a c t o s e and  the i n - c h a i n glucose r e s i d u e ; a c c o r d i n g to the  s p e c t r a of the o r i g i n a l polysaccharide,one e  i s a and  the  other  .  b) P o l y s a c c h a r i d e P^  . 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  yielded several acidic  P  1  o l i g o s a c c h a r i d e s . S e p a r a t i o n by gel-per-  meation chromatography (see F i g u r e IV.1)  and f u r t h e r p u r i f i c a -  t i o n of each f r a c t i o n by paper chromatography a f f o r d e d 2 pure a l d o t r i o u r o n i c a c i d s ( A^ and A^ t e t r a o u r o n i c a c i d s ( A^  ) and a mixture of two  aldo-  ) .  Nuclear magnetic resonance data i n c o n j u n c t i o n w i t h anal y t i c a l and m e t h y l a t i o n data(see  TablelV.1.3).showed  these  compounds to be: A~  GlcA - — 2  3  Ai, A  e  Gal - — 2 .  B  Glc - — 2  M  a  n  a  GlcA - — 2  Gal  i s a 3 to 1 mixture of GlcA - — 2  G  a l -—2  5  B  and  Man  -—2 a  G  l c -—2  GlcA - — 2  B  B  G  M  a  n  a  1—2.  G l c  a  al  From these r e s u l t s , t h e s t r u c t u r e of K 6 0 c a p s u l a r p o l y s a c c h a r i d e can unambiguously be assigned  as:  Figure  IV.1  Separation  of the a c i d i c  oligosaccharides  from p a r t i a l h y d r o l y s i s of P.^ by g e l permeation chromatography  ( Bio-Gel  P-2)  93  _1  Glc 1  2 GlcA  -2 G a l - - - - - Man  4  1  *  a  1  2  A  6  1  Glc  Glc  Glc T h i s s t r u c t u r e was  2  f u r t h e r confirmed  by a  base-catalyzed  u r o n i c a c i d degradation,where the u r o n i c a c i d and n a l glucose a t t a c h e d to i t s p o s i t i o n 0-4 p o s i t i o n 0-3 of g a l a c t o s e was C o n c l u s i o n . The  the  termi-  were degraded,  subsequently  and  ethylated.  experiments d e s c r i b e d above l e a d to the  c o n c l u s i o n t h a t the s t r u c t u r e of the c a p s u l a r  polysaccharide  of K l e b s i e l l a serotype K 60 i s based on the r e p e a t i n g u n i t  —2  D-Glcp - — 2 . D-GlcpA - — 2  J. i  R  1  B  2 |  D-Manp.  2|  A  ll  D-Glc£  The  D-Galp - — 2  D-Glcp.  ll  D-Glcp  s t r u c t u r e i s of a unique p a t t e r n i n t h i s s e r i e s of  b a c t e r i a l p o l y s a c c h a r i d e having three separate per r e p e a t i n g unit.Based  s i d e chains  on the s p e c t r a l data of the  s a c c h a r i d e s i s o l a t e d . i t was  IV.1.1).  oligo-  then p o s s i b l e to a s s i g n the  n a l s i n the s p e c t r a of the o r i g i n a l p o l y s a c c h a r i d e Table  A  (see  sig-  TABLE IV.1.3 ANALYSIS OF THE OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF K60 POLYSACCHARIDE. Oligosaccharide  [a J  D  (water) A  A  ±  2  A^  A^  A^  +12  ~  + 49  +10  +79-4  Sugar a n a l y s i s (as  Methylation analysis  alditol  (as a l d i t o l  acetates)  acetates)  (1.0) - G a l (1.0)  Glc(GlcA)  (1.0  2,3.4  Gal  (1.0  2,4,6  Glc (GlcA)  (1.0  Glc Gal  (1.0 (1.0  2 , 3 , 4 , 6 - Glc (1.0) 2,3.4 - Glc (1.0) 1,4,5,6- G a l - —  Glc(GlcA)  (1.0  Gal Man  (1.0 (1.0  Glc(GlcA)  (1.0  Glc  (1.0  Gal  (1.0  Glc(GlcA)  (1.0  Glc  (1.0 (1.0 (1.0  Gal Man  2,3.4 2,4,6 2,4,6  - Glc  - Glc (1.0) - G a l (1.0) - Man (0.9)  2 , 3 , 4 , 6 - Glc (1.0) 2,4,6 - G a l (1.0) 2,4 Glc (0.9) 2 , 3 , 4 , 6 - Man (0.3) 2,4,6 - Glc (1.2) 2,4,6 - Man (1.0) 2,4,6 2,3,4 2,4  - G a l (1.3) - Glc (1.0) Glc  (0.3)  TABLE IV.1.3  (cont.)  +31  Glc  (2.0)  1.2,4,5.6  Gl<£~  Man  (1.0)  2,3,4,6  Glc  (1.0)  3.^.6  Man  (1.0)  a b — and — The o l i g o s a c c h a r i d e s were reduced  p r i o r to methylation.  96  IV.1.4  Experimental.  G e n e r a l 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.I.e.-m-s..infrared,c.d.,and measurements o f o p t i c a l has been d e s c r i b e d p r e v i o u s l y tography, gas - l i q u i d  rotation  (see S e c t i o n I I I ) . P a p e r chroma-  chromatography,gel-permeation and i o n - e x -  change chromatography  were performed as d e s c r i b e d i n S e c t i o n  III. P r e p a r a t i o n and p r o p e r t i e s o f K 6 0 c a p s u l a r p o l y s a c c h a r i d e . A culture of K l e b s i e l l a K 6 0 ( 4 4 6 3 / 5 2 )  was obtained from Dr.  I.jtfrskov,Copenhagen,and was grown by the procedure 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 i o n and p u r i f i c a t i o n o f the p o l y s a c done as d e s c r i b e d i n S e c t i o n I I I . 7 . 1 . ( y i e l d : 8g  c h a r i d e was  df polysaccharide: from 1 2 . 5 1 o f medium). The product h a d [ a ] +58°  D  ( c 0.33.water).The p u r i t y o f the p o l y s a c c h a r i d e was  checked by e l e c t r o p h o r e s i s . u s i n g a 1% s o l u t i o n on c e l l u l o s e (Sepraphore 1 1 1 , 1 5 x 2 . 5 cm) a t 300V f o r 9 0 min.  acetate s t r i p s  and then development  i n e i t h e r A l c i a n Blue i n c i t r a t e - b u f f e r e d  e t h a n o l (pH 4 ) , o r p e r i o d a t e - S c h i f f r e a g e n t ^ ? . T h e p o l y s a c c h a 1  r i d e had a M i 810.000 d a l t o n s and was monodisperse  according  w  to g e l - p e r m e a t i o n chromatography. The ^H-n.m.r. spectrum o f K 6 0 p o l y s a c c h a r i d e i n D 0 a t 2  9 0 ° , r e v e a l e d s i g n a l s c o r r e s p o n d i n g t o 7 anomeric protons a t 6 5.^7 (  2  H  . i J  6 4 , 8 5 (1H,J ^1 2 ?  H z  ) (  f  2  1 ( 2  s e e  T a  2Hz)| 6 5 . 3 7 - ( l H , J  1  2  2 H z ) } 6 5-04 ( 1 H , J  7 H z ) ; 6 4 . 7 0 (1H,J  1  2  7Hz)  °le  IV.1 f o r assignments  1 > 2  7Hz) j  and 6 4 . 5 ^ (IH, ).The "^C spectrum  showed s i x s i g n a l s i n the anomeric r e g i o n a t 1 0 4 . 2 3 , 1 0 4 . 0 2 , 103.07,102.44,100.60  and 9 9 . 5 6 p.p.m. w i t h the s i g n a l a t  97  99*56  p.p.m. b e i n g twice the h e i g h t of any  of the other  s i g n a l s . S e v e r a l s i g n a l s between 6 1 . 1 - 6 2 . 2 due  to C - 6  five of  hexose u n i t s were a l s o present (see T a b l e l V . 1 . 1 f o r a s s i g n ment). 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 . H y d r o l y s i s of a sample (20 mg)  of K 6 0 p o l y s a c c h a r i d e w i t h 2M  overnight at 9 5 ° ,  TFA  removal of the a c i d by s u c c e s s i v e evaporations f o l l o w e d by paper chromatography ( s o l v e n t s  w i t h water ,  (A) and  (B)),  showed D-mannose,D-galactose,D-glucose,D-glucuronolactone , D-glucuronic was  a c i d and  an a l d o b i o u r o n i c acid.Sugar  performed as p r e v i o u s l y d e s c r i b e d  t o l a c e t a t e s of mannose.galactose and by g . l . c .  of 1 : 1 : 5 .  glucose were  Preparative g.l.c.  lowed by measurements of the c d . mannitol  (see page 7 1 ).The  (column (a) .program (a) ) and  in a ratio  and  g l u c i t o l hexaacetates  analysis aldi-  identified  found to be  present  (column ( f ) ) f o l -  s p e c t r a showed both the to be  of the D c o n f i g u r a -  tion. Methylation analysis.The  capsular polysaccharide  (300  mg)  i n the f r e e a c i d form (obtained by p a s s i n g the sodium s a l t through a column of Amberlite ved  i n 3 0 mL  procedure  IR-120 (H ) +  resin).was  dissol-  of anhydrous DMS0 and methylated by the Hakomori  (see page 7 2 ) . M e t h y l a t i o n was  incomplete  as shown  by h y d r o x y l a b s o r p t i o n i n the i . r . spectrum.A subsequent die  (see page 7 3 ) treatment a f f o r d e d complete  (yield,272  mg).Carboxyl-reduction  Pur-  methylation,  of the f u l l y methylated  p o l y s a c c h a r i d e w i t h L i A l H ^ i n anhydrous oxolane  (see page 7k \  98 hydrolysis  (of a p o r t i o n ) w i t h 2M  a l d i t o l a c e t a t e s gave a mixture t o l a c e t a t e s which was and  TFA.and c o n v e r s i o n  into  o f p a r t i a l l y methylated  analyzed by g . l . c .  aldi-  on columns ( b ) , ( c )  (d) and by g.1.c.-m.s.(see TablelV.1.2.column I).The  methylated  and  carboxyl-reduced  f u r t h e r methylated  polysaccharide  by the Hakomori method ;and  ( 3 0 mg)  a f t e r hydro-  l y s i s . r e d u c t i o n and a c e t y l a t i o n , a n a l y s i s of the methylated  was  partially  a l d i t o l a c e t a t e s by g . l . c . and g.l.c.-m.s.  on  column (d) showed the replacement of 2-0-methylglucose 2,6-di-0-methylglucose(see  by  TablelV.1.2,column I I ) . P r e p a r a t i -  ve g . l . c . on column ( f ) ( 2 1 5 ° i s o t h e r m a l ) a f f o r d e d a sample of 4 , 6 - d i - 0 - m e t h y l g a l a c t i t o l a c e t a t e whose p o s i t i v e curve  i n d i c a t e d t h a t g a l a c t o s e has  Carbodiimide  i n 100 mL  of  the D - c o n f i g u r a t i o n .  ( N a s a l t , 1 . 0 2 g) was +  dissol-  water.l-Cyclohexyl-3-(2-morpholinoethyl)-  carbodiimide metho-p_-toluenesulfonate T h i s corresponds  K 60.  r e d u c 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  A sample of K 6 0 p o l y s a c c h a r i d e ved  cd.  ( CMC,4.0 g) was  added.  to t e n times the e q u i v a l e n t of c a r b o x y l i c  a c i d i n the p o l y s a c c h a r i d e , b a s e d  on one g l u c u r o n i c a c i d r e -  sidue per sequence.As the r e a c t i o n proceded, the pH was  main-  t a i n e d a t 4 . 7 5 by t i t r a t i n g when necessary w i t h hydrogen c h l o r i d e (0. 10N)  solution.When the consumption of HC1  ceased  ( 6 . 7 0 mL).approximately two hours l a t e r , a n aqueous s o l u t i o n of sodium borohydride was  (2M) was  minimized by a continuous  added slowly.Bubble flow of a i r blowing  f a c e of the s d l u t i o n . A drop of 1-octanol was  formation on the s u r -  added p e r i o d i -  c a l l y to c o n t r o l the amount of foam.At the same time the  so-  99  dium borohydride was  added,the pH o f the s o l u t i o n was  t a i n e d a t 6 . 5 w i t h HC1 l u t i o n was  ( 4 . O K ) . A t o t a l of 3 0 0 mL  main-  of NaBH^ so-  added over a p e r i o d o f two hours.The s o l u t i o n  was  c o n c e n t r a t e d and d i a l y z e d a g a i n s t t a p water d u r i n g 48 h.  and  f r e e z e - d r i e d . A second  treatment was  t i o n o f HC1(0.1 N )was 2.80 was  mL.A  c a r r i e d out.The consump-  t o t a l o f 780 mg  o f product  recovered a f t e r drying. A sample o f the reduced  l y z e d o v e r n i g h t w i t h 2M  TFA  p o l y s a c c h a r i d e (10 mg)  was  hydro-  on a steam-bath and a f t e r  conver-  s i o n o f the sugars i n t o a l d i t o l a c e t a t e s , g . 1 . c . showed n i t o l , g a l a c t i t o l and g l u c i t o l hexaacetates  in a ratio  man1:1:5,  i n d i c a t i n g complete r e d u c t i o n o f the u r o n i c a c i d s . E s t i m a t i o n of the e q u i v a l e n t weight de from the consumption o f HC1 r e t i c a l , 1148  of the p o l y s a c c h a r i -  g i v e s a v a l u e o f 1100  ( theo-  ).  P e r i o d a t e o x i d a t i o n o f carboxyl-reduced K 6 0 p o l y s a c c h a r i d e .A sample ( 6 5 mg) c h a r i d e was (0.1 M,10  of the carboxyl-reduced K 6 0 p o l y s a c -  d i s s o l v e d i n 1 5 mL  mL)  was  of water.A s o l u t i o n of NalO^  added.The r e a c t i o n was  allowed t o proceed  a t 4° i n the dark.The p e r i o d a t e consumption was 168 1 mL  a l i q u o t s by the Fleury-Lange  p l a t e a u a f t e r 140 h.  de  and reached  a  ( 4 . 9 5 moles o f p e r i o d a t e p e r mole o f  polysaccharide).Ethylene g l y c o l dehyde was  method  f o l l o w e d on  ( 3 mL)  was  added,the p o l y a l -  d i a l y z e d overnight.reduced w i t h sodium b o r o h y d r i -  (0.2 g ) . n e u t r a l i z e d w i t h 5 0 $ a c e t i c a c i d , 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 the p o l y a l c o h o l ( 4 5 mg).This m a t e r i a l was  f u r t h e r o x i d i z e d w i t h p e r i o d a t e (20 mL  of 0 . 0 5 M  NalO^).  100 P e r i o d a t e consumption was constant a f t e r ?2 h. (0.95 moles of p e r i o d a t e per mole o f p o l y s a c c h a r i d e ) . R e - i s o l a t i o n gave the p o l y a l c o h o l (35 mg). A sample o f the p o l y a l c o h o l (5 mg) 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 on a steam-bath.Reduction  and a c e t y l a t i o n  gave (on g . l . c . ) 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  hexaaceta-  tes i n a r a t i o 1:1:2.The r e s t o f the m a t e r i a l (30 mg) was methylated by the Hakomori procedure.One t h i r d o f the methylated product 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 on a steambath tconversion  o f the p a r t i a l l y methylated  p a r t i a l l y methylated  sugars i n t o the  a l d i t o l a c e t a t e s and g . l . c . on column  ( d ) , showed the presence  of 2,4,6-tri-0-methylglucose,4,6-  di-0-methylmannose,2,6-di-0-methylglucose  and 4 , 6 - d i - 0 - m e t h y l -  g a l a c t o s e i n equimolar p r o p o r t i o n s . T h e r e s t o f the methylated product was t r e a t e d w i t h $0% a c e t i c a c i d f o r one hour on  a  steam-bath.The a c i d was removed by s e v e r a l e v a p o r a t i o n s w i t h water,the  product was d i s s o l v e d i n dioxane-ethanol(3«l»10 mL)  and reduced w i t h NaBH^ (50 mg).After  e l i m i n a t i o n o f the NaBH^  i n the u s u a l manner,the m a t e r i a l was d r i e d and then e t h y l a t e d by the Hakomori p r o c e d u r e . H y d r o l y s i s , r e d u c t i o n and a c e t y l a t i o n gave a mixture o f p a r t i a l l y methylated methylated  and 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 which c o u l d not be s e -  parated by g . l . c . on any o f the columns a v a i l a b l e . F r o m the r e t e n t i o n times i t was concluded t h a t the mixture was composed  of 3-or 4-0-ethyl-2,6-di-0-methylglucose,2,4,6-tri-0-  methylglucose  and the 2-or 3 - 0 - e t h y l - 4 , 6 - d i - 0 - m e t h y l  t i v e s o f both mannose and g a l a c t o s e .  deriva-  101 P e r i o d a t e o x i d a t i o n of K 60 c a p s u l a r p o l y s a c c h a r i d e . A s o l u t i o n o f K 6 0 p o l y s a c c h a r i d e (1.0 g) i n water ( 1 5 0 mL) mixed w i t h 0.1M was  was  NaClO^ ( 1 5 0 mL).The r e a c t i o n  NalO^ and 0.4M  allowed to proceed a t 4° i n the dark.The p e r i o d a t e con-  sumption and r e l e a s e of formic a c i d were f o l l o w e d on 1 mL a l i q u o t s by the Fleury-Lange  method and t i t r a t i o n a g a i n s t  0.001N NaOH,respectively.Periodate  consumption and formic a c -  i d p r o d u c t i o n reached a p l a t e a u a f t e r 140 h.  ( 4 . 8 moles of  p e r i o d a t e and 1 . 7 moles of formic a c i d per mole of p o l y s a c charide ) .Ethylene g l y c o l (10 mL) was was  added,the  polyaldehyde  d i a l y z e d overnight,reduced w i t h NaBH^ ( 1 . 5 g) n e u t r a l i z e d  w i t h 5 0 % a c e t i c a c i d , 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 (680 mg).This m a t e r i a l was periodate  ( 200  consumption was  mL  of 0.05M NalO^ and  constant a f t e r 7 0 h.  the  further oxidized with 0.2M  NaClO^ ).Periodate  ( 0 . 7 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 ) . R e - i s o l a t i o n gave the p o l y a l c o h o l (  660  mg).  T h i s product was  hydrolyzed  (TFA,  0.5M  ) a t room temper-  ature f o r 24 h.Paper chromatography i n s o l v e n t (A) showed the presence  of one mobile  compound i d e n t i f i e d , b y comparison  w i t h a standard as g l y c e r o l , a n d a polymeric a f f o r d e d 5 5 0 mg of P  1  productjdialysis  of polymeric m a t e r i a l ( P ) . T o t a l h y d r o l y s i s  and examination  1  on paper ( s o l v e n t s (A) and  (B)) showed  D-mannose,D-galactose,D-glucose and D - g l u c u r o n i c acidjan a l d o b i o u r o n i c a c i d was  a l s o present.Sugar  v i o u s l y described,gave  (g.1.c..column (a)) 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 hexaacetates  a n a l y s i s , a s pre-  i n a r a t i o of It 1:2  ,where one  102  of the g l u c i t o l u n i t s i s d e r i v e d from the g l u c u r o n i c a c i d . M e t h y l a t i o n o f P-^ ( 2 5 mg) by the Hakomori procedure and one Purdie treatment  a f f o r d e d a f u l l y methylated  polysaccharide  w i t h no h y d r o x y l a b s o r p t i o n i n the i n f r a r e d . T h e f u l l y methylated m a t e r i a l (10 mg) was reduced  with LiAlH^,hydrolyzed  2M TFA f o r 10 h,reduced w i t h NaBH^ and a c e t y l a t e d . T h e l y methylated  a l d i t o l a c e t a t e s were analyzed  with  partial-  by g . l . c . and  g.l.c-m.s. on columns (b) and (d) w i t h the r e s u l t s g i v e n i n T a b l e l V . 1.2,column I I I . A p o r t i o n (10 mg) o f the methylated 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 f o r 5 h and separ a t e d i n t o a c i d i c and n e u t r a l components on ion-exchange r e sin  (Bio-Rad  methanolic  AGl-X2).The a c i d i c f r a c t i o n was r e f l u x e d i n 3%  HC1,reduced w i t h NaBH^ i n anhydrous methanol,  h y d r o l y z e d w i t h 2M TFA f o r 3 h,reduced w i t h NaBH^ and a c e t y l ated.The r e s u l t s obtained by g . l . c .  i n column (b) showed the  of 2,4,6-tri-O-methylgalactose  presence  and 2,4-di-0-methyl-  glucose i n a r a t i o of 1.0:1.3. The  ^H-n.m.r. spectrum o f P^ showed s i g n a l s s i g n a l s a t  6 5 . 3 3 (1H,J and 6 4 . 7 7  1  2  (1H,J  2Hz)»6 5 . 2 7 ( 1 H , J 1  2  7Hz)  1  2  7Hz)  ( 1 C ) , 1 0 3 . 3 4 ( I C ) , and 101,42 (2C)p.p.m  i n the anomeric region,and 76.60  2Hz);6 4.83 ( 1 H , J  (see spectrum N o . 6 ) . T h e ^ ^ g p e c t r u - i  showed s i g n a l s a t 1 0 4 . 1 9  and  1 ( 2  four s i g n a l s at 84.11,83.02,80.13  p.p.m. due t o C - 3 o f the f o u r hexoses.For a s s i g n -  ments see Table  IV.1.1  P a r t i a l hydrolysis of K 6 0 capsular p o r t i o n o f K 60 p o l y s a c c h a r i d e  polysaccharide.A  ( 5 ° 0 mg) was d i s s o l v e d i n TFA  (0.5M,100 mL) and the s o l u t i o n was r e f l u x e d f o r 3 h.The a c i d  103  was removed by evaporation,and  a c i d i c and n e u t r a l  were separated on a column o f Bio-Rad tion  components  AGl-X2.The a c i d i c  frac-  ( 1 3 0 mg) was separated on B i o - G e l P - 2 t o g i v e 2 5 mg  pure a l d o b i o u r o n i c a c i d ( A ) and 1 0 mg of pure  of  aldotriouroriic  1  acid ( A ) . 2  The a l d o b i o u r o n i c a c i d A  had R ^ . ^ 0 . 2 8 ( s o l v e n t  1  [ a ] + 1 2 ° ( c 1.05,water).Sugar  a n a l y s i s showed g l u c i t o l  D  u r o n i c a c i d ) and g a l a c t i t o l hexaacetates The a l d o t r i o u r o n i c a c i d A examination  had R Q I C ' ^ 0  2  (A)) and  1  i n equimolar (solvent  (from amounts.  (A)) and  on paper f o l l o w i n g h y d r o l y s i s i n d i c a t e d ( s o l v e n t s  (A) and (B)) g l u c o s e . g a l a c t o s e and an a l d o b i o u r o n i c a c i d . A c i d A  2  was reduced w i t h aqueous NaBH^,methylated,and the e s t e r  f u n c t i o n was reduced w i t h L i A l H ^ i n anhydrous l y s i s and g . l . c . a n a l y s i s  oxolane.Hydro-  ( column (b)) showed a component  w i t h R^ 0.8 t o g e t h e r w i t h peaks c o r r e s p o n d i n g t o 2 , 3 , 4 , 6 tetra-O-methylglucose  (R^ 1 . 0 ) a n d 2 , 3 , 4 - t r i - 0 - m e t h y l g l u c o s e  (R_-t 1 . 9 ) . M a s s spectrometry showed was  -  t h a t the f a s t e s t  component  l,4,5,6-tetra-0-methylgalactitol. The n e u t r a l f r a c t i o n was separated on B i o - G e l P - 2 t o g i v e  i n a pure s t a t e , a t r i s a c c h a r i d e ( s o l v e n t (A)) a n d [ ] + 3 1 ° ( c a  D  ( N 3 0 mg) l t  M e t h y l a t i o n a n a l y s i s o f reduced  0  c  gave  in a ratio l t 2 .  N-^ gave (columns (c) and (d))  l,2,4,5,6-penta-0-methylglucitol  (R_ 0.42, ( d ) ) , 2 , 3 , 4 , 6 - t e t r a t  ( R ^ . 1 . 0 0 ) and 3 , ^ , 6 - t r i - 0 - m e t h y l r a a n n o s e ( R ^ . l . 6 6 )  The n.m.r. data f o r A^,A IV. 1 . 1  E(ji '32  0 . 3 2 , w a t e r ) . A n a l y s i s of  ( g . l . c . ) m a n n i t o l and g l u c i t o l hexaacetates  O-methylglucose  having  2  and  are presented i n Table  104  P a r t i a l hydrolysis of polysaccharide  P^ from  o x i d a t i o n . A sample o f P^ (350 mg) was h y d r o l y z e d  periodate w i t h IM TFA  (75 mL,lh) and the r e s u l t i n g o l i g o s a c c h a r i d e s were  separated  i n t o a c i d i c and n e u t r a l f r a c t i o n s on a column o f Bio-Rad AG1X2.The a c i d i c f r a c t i o n was separated  by g e l chromatography on  a column o f B i o - G e l P-2. Three f r a c t i o n s were c o l l e c t e d (see F i g u r e IV.1) corresponding  t o ( l ) t h e - a l d o b i o u r o n i c - a c i d ( 4 3 mg), ( 3 0 mg),and (3) a mixture  (2)a mixture o f t w o , t r i s a c c h a r i d e s of two t e t r a s a c c h a r i d e s Fraction  2  ( 3 2 mg).  was p u r i f i e d by paper chromatography(solvent  (C)) and two pure a l d o t r i o u r o n i c a c i d s were i s o l a t e d t A ^ and A^) O l i g o s a c c h a r i d e A^ (7 mg) had [ a ]+49°(c 0.7,water) and D  -Wldobi 6 5-32 and  0  ,  6  ( s o l v e n t ( C ) ) . P r o t o n n.m.r. showed s i g n a l s a t  7  (lH,J  6 4.78  1 > 2  2 H z ) , 6 5.20 (0.5H,J  (1H,J  1  2  7 H z  ^  ±  2  2 H z ) , 64.94 (0.5H,J ^ 2Hz) ±  2  i n d i c a t i n g onea-and one B - l i n k a g e  a r e d u c i n g mannose (low c o u p l i n g constant  and  o f the r e d u c i n g  s i g n a l ) . O l i g o s a c c h a r i d e A^ (14 mg) had [ a] ^+I0°(c 1.1,water) and Rgidoiji °.45  ( s o l v e n t (C)).Sugar  a n a l y s i s of t h i s material,  as p r e v i o u s l y described,showed on g . 1 . c . . g a l a c t i t o l and g l u c i t o l hexaacetates i n a r a t i o 1»2.Methylation a n a l y s i s gave 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e , 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 a p p r o x i m a t e l y equimolar amounts. Proton n.m.r. showed s i g n a l s a t 6 5.28 ( 0 . 4 H , J (m J t  1  2  7Hz),  6 4.77 ( 1 H , J 1  2  1  2  2 H Z ) , <5 4.80  7Hz) and 64.64 ( 0 . 6 ^ ^ 7Hz). 2  13 From the it  -T spectrum o f the mixture o f a l d o t r i o u r o n i c a c i d s  i s p o s s i b l e t o a s s i g n s i g n a l s t o both  comparison t o other  oligosaccharides,by  oligosaccharide spectra  (see spec.NoJLO  105  and T a b l e l V . 1.1 f o r assignments ). F r a c t i o n 3 was (C)) and  a p a r t i a l l y p u r i f i e d t e t r a s a c c h a r i d e was  (A^,18 mg)  having[a]  this material,as  Methylation  D  isolated  + 7 9 ° ( c 1.7»water).Sugar a n a l y s i s  of  g l u c i t o l hexaacetates i n a r a t i o Is 1:2.  a n a l y s i s of the  (column  (solvent  p r e v i o u s l y described,showed on g . l . c . mannit-  o l , g a l a c t i t o l and  g.l.c.  p u r i f i e d by paper chromatography  reduced  oligosaccharide  gave  on  (b)),l,2,4,5.6-penta-0-methylglucitol,l,2,4,5»6-  penta-0-methylgalactitol,2,3.4,6-tetra-0-methylmannose,2,4,6tri-O-methylglucose,2,4,6-tri-0-methylmannose,2,4,6-tri-Om e t h y l g a l a c t o s e , 2 , 3 . 4 - t r i - 0 - m e thy l g l u c o s e and  2,4-di-0-methyl-  glucose i n a r a t i o of 0 . 3 : 1 . 0 : 0 . 3 : 0 . 3 : 1 . 0 : 1 . 0 : 1 . 0 : 0 . 3 .showi n g t h a t i t i s a mixture of two ratio 3*1  -The  the  2  13  1  2  2 H z ) and 6 4 . 7 7 ( 1 . 6 H , J  C-n.m.r. spectrum a t and  signments  c o u l d be made f o r both  <5 5 . 3 1 ( - H ,  broad)  and  93-04 p.p.m.From the r e l a t i v e i n t e n s i t i e s . a s oligosaccharides.  a c i d d e g r a d a t i o n . M e t h y l a t e d and  K 6 0 polysaccharide  was  as d e s c r i b e d  (see p a g e 7 5 ).The  before  subjected  of the p r o d u c t . r e d u c t i o n  shown i n T a b l e l V . 1.2column I I .  and  carefully dried  to a u r o n i c  c a r r i e d out w i t h e t h y l i o d i d e i n s t e a d lysis  l f 2  a  104.51,104.28,103.48,101.51,101.35,  96,76,94.7  Uronic  acids i n  "'"H-n.m.r. spectrum showed s i g n a l s a t  J ^ 2Hz), 6 5.25(1.4H,J 1  aldotetrauronic  a c i d degradation  d i r e c t a l k y l a t i o n was o f methyl i o d i d e . H y d r o -  a c e t y l a t i o n gave r e s u l t s  106 IV.2  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 K l e b s i e l l a serotype  K 26  capsular polysaccharide. IV.2.1 A b s t r a c 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  K l e b s i e l l a K 26 has been determined u s i n g the techniques o f methylation,periodate 1  13  e l i m i n a t i o n . H- and t a b l i s h the nature  o x i d a t i o n , p a r t i a l h y d r o l y s i s and B -  ^C-n.m.r. s p e c t r o s c o p y was used t o e s -  o f the anomeric l i n k a g e s and t o i d e n t i f y  o l i g o s a c c h a r i d e s obtained by the d i f f e r e n t degradative niques  tech-  used.  The p o l y s a c c h a r i d e i s shown t o comprise the heptasacchar i d e r e p e a t i n g u n i t below.  —2  D-Galp_  1  2 B  D-GlcpA  41 * a  11 D-Gl C £  61 II  D-GL  41 1  D-i  1 _a 1  D-Manp.  1 a  2 D-Manp. - — a  10?  I V . 2 . 2 Introduction. K l e b s i e l l a serotype K 2 6 i s one  of 1 7 s t r a i n s whose cap-  s u l a r p o l y s a c c h a r i d e s are composed of D - g l u c u r o n i c actose,D-glucose  and D-mannose.Eight of these  have 1-carboxyethylidene  s u b s t i t u e n t s and  the s t r u c t u r e s of K l e b s i e l l a K 7 K^ ^ 1  are known. The  l 6 9  ,K13  and K69  remain to be  i n t h i s subgroup  °,K30  presented  1 7 1  ,K31  and  1 7 2  polysaccha-  here;the  serotype  examined.  T h i s polymer i s shown to be based on a repeating unit  polysaccharides  s t r u c t u r e of K l e b s i e l l a K26  r i d e , a member of t h i s subgroup,is K35  1 7  acid,D-gal  heptasaccharide  ( f o u r p l u s three type) a n d , i n t h i s r e s p e c t , i s  s i m i l a r to the p o l y s a c c h a r i d e i s o l a t e d from K ^ l ^ ^ . I n the l a t t e r case.however,the D - g l u c u r o n i c s i d e - c h a i n and  a c i d r e s i d u e i s i n the  the branch p o i n t i s a u n i t of  D-galactofuran-  ose.The s t r u c t u r e of the 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 is,therefore,unique i n this  K26  series.  I V . 2 . 3 R e s u l t s and d i s c u s s i o n . Composition  and n.m.r. s p e c t r a . The  f i c a t i o n of the p o l y s a c c h a r i d e was described.The  p u r i f i e d product  i s o l a t i o n and  conducted as p r e v i o u s l y  obtained a f t e r C e t a v l o n  c i p i t a t i o n had [ a ] + 8 0 ° , w h i c h compares w e l l to the D  value o f  +68°  pre-  calculated  u s i n g Hudson's Rule of I s o r o t a t i o n . T h e mole-  c u l a r weight of the p o l y s a c c h a r i d e was chromatography to be 1 x 1 0 ? d a l t o n s j t h e was  puri-  determined by g e l e q u i v a l e n t weight  650.  Paper chromatography of an a c i d h y d r o l y z a t e of the  poly-  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 acid,mannose  108 and  an a l d o b i o u r o n i c a c i d . D e t e r m i n a t i o n  of the  a l d i t o l a c e t a t e s , o f the carboxyl-reduced  sugars,as  polysaccharide  gave  i n a r a t i o of 1 . 0 j l . 0 t l . 5  mannose.galactose and glucose  •  Glucose and mannose proved to be of the D - c o n f i g u r a t i o n c i r c u l a r dichroism t e s . Galactose  and  (c.d.)measurements of the a l d i t o l g l u c u r o n i c a c i d were a l s o assigned  by  acetathe  D-  c o n f i g u r a t i o n by c d .  measurements of p a r t i a l l y methylated  derivatives isolated  subsequently.  The  ^H-n.m.r. spectrum of the n a t i v e p o l y s a c c h a r i d e  recorded  i n D^O  was  a t 9 0 ° w i t h acetone as i n t e r n a l standard.The  spectrum e x h i b i t s the presence of seven anomeric protons responding  to  f o u r a - and  carboxyethylidene  three B - l i n k a g e s ; a l s o one  a c e t a l per r e p e a t i n g u n i t was  "^H-n.m.r. of the depyruvylated  polysaccharide  stants  to the  showed t h a t protons  g - l i n k a g e s e x h i b i t l a r g e c o u p l i n g con-  (?Hz) which do not correspond  (see spectrum N o l 3 , a n d Table confirmed  1-  detected.The  both mannoses should be a - l i n k e d as the anomeric corresponding  cor-  to B - l i n k e d mannoses  IV. 2.1). The  the proton r e s u l t s and  ^C-n.m.r. spectrum  from the chemical  shift  of  the methyl group of the a c e t a l ( 2 5 - 7 p.p.m.) i t i s p o s s i b l e to a s s i g n the R c o n f i g u r a t i o n to the a c e t a l assignment  of the s i g n a l s was  achieved  carbon.Precise  a f t e r studying  ^H-  -^C-n.m.r. s p e c t r a o f o l i g o s a c c h a r i d e s obtained by  se-  13 and  l e c t i v e degradative  techniques  (see Table  IV.2.1)  M e t h y l a t i o n a n a l y s i s . M e t h y l a t i o n of K26  polysaccharide  f o l l o w e d by r e d u c t i o n of the u r o n i c e s t e r , h y d r o l y s i s , d e r i v a t i z a t i o n as 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  TABLE  IV.2.1  N.M.R. DATA FOR KLEBSIELLA K26 CAPSULAR POLYSACCHARIDE AND DERIVED POLY- AND OLIGOSACCHARIDES . H-n.m.r. Compound  Integral  =-1,2  -Man-OH a  A-  2.5  5.20  2  1.0 0.6 0.4  4.92  GlcA-—-Man-—-Man-OH a  5.32  a  5-37  2  5.34  2.5  5.18 4.94  2 2 2  5.34  2.5  5.29  2  5.17  2  5.07  2  0.3 1.0  4.64  7  0.7  5.08  GlcA-—-Wan-—^arr-—-<Gal-OH  }  1  2.0 0.3 0.7 0.3 2.0  Spectrum No.  proton  (Hz) GlcA-  Assignment  GlcA-— a  3-Man  OH  14  a  3-Man-r-OH 3-ManGlcA16  unknown 3-Man  OH a  3-Man—r-OH GlcA 3-Man 3-Gal 2 -Man3 - Gal-  a  OH -OH  18  TABLE IV.2.1  (cont.)  Glc-—^lc-OH N-  Gal —^Glc^-^lc-OH 1  N,  3  0.4  6-Glc—OH  8  0.6  6-Glc-j-OH  8  1.0  3  0.4  6-Glc  7.5  0.6  6-Glc—OH  7-5  0.4  4-Glc ^Glc  7.5  0.6  4-Glc~%Jlc—£—OH  7-5  1.0  20  GlcOH  1  7  OH  22  °  G a l —  p 3  Gal-—^GlcA-—\a.n-—^Gly  2  1.0  3-Man—  7  1.0  G a l —  -^Gal-—^GlcA-—^Man-—^an 1  a  1 Glc  11  Glc H Gal  a  a  2-GlcA a  SH  6 Zj.  1.0  2.0  a  J 2-Glc  {  4  6-Glc-  2  1.0  3-Man-  2  1.0  2-Man-  7.5  1.0  Gal-  7.5  1.0  4-Glc-  7.5  1.0  3-Gal-  a a  et a  H  23  TABLE IV.2.1 (cont.)  1 2 1 - ^ G a l i — ^ G l c A — 2Man-—%Ian— 3  2.0  J 4-GlcA  5.28  1.0  5.08  1.0  I 6-Glc V a 3- Man  4.7-4.5  3-0  1.65  3-0  5.50  ^  ll Glc 4 1 Gal 6 4  H"C 3  X  a  2-Man  a  G a l — 4- G l c — 3- G a l C30 H CH~—C 3 I 2  C0 H 2  13 C-n.m.r.  Compound  Chemical s h i f t  Assignment  No.  (P.P.m.) A  1  101.^2 94. 82 9^-33 102.86  Spectrum  GlcA 3-Man 3-Man 3-Man  a  3 ci  OH OH  15  TABLE I V . 2 . 1 (cont.) 101.36  A  3  93.56  2-Man—^-0H  93.35  2-Man  103.06 101.33 97.23  x  saccharide  GlcA  a  3-Gal—---OH  95-11  2-Man-=—^Gal-  103.50  92.95  K26 c a p s u l a r p o l y -  3-M?  2-Marr-—-Gal-  96.81  SH  OH a  95.38  93.10  N  GlcA  105.41  3-Gal G l c  —  6 - G l c — OH 6-Glc  -Man3-Man  100.22  2-GlcA-  -^Q  3  7  0  103.24  OH  Gal-  100.92  105.41  OH  a  a  TABLE IV.2.1  (cont.)  101.00  6-Glc—  100.76  2-GlcA— 4 2-Man— CH^—C  99.87 25.77  C0 H 2  ( R confign. )  114 the values shown i n Table IV.2.2 .column I.These r e s u l t s i n d i c a t e 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 heptasaccharide r e p e a t i n g u n i t w i t h a branch on the g l u c u r o n i c a c i d i t h e  ter-  m i n a l g l y c o s y l r e s i d u e i s a u n i t of g a l a c t o s e which has a c a r b o x y e t h y l i d e n e group present as an a c e t a l spanning 0-6.Methylation  and  of the carboxyl-reduced p o l y s a c c h a r i d e (see  Table IV.2.2,columnII) showed the disappearance methylglucose  0-4  1-  and the f o r m a t i o n of  3-0-  of the  3.6-di-0-methylglucose  c o n f i r m i n g the g l u c u r o n i c a c i d as the branch  point.Removal  of the m o d i f i e d a c e t a l ( r e d u c t i o n , m e t h y l a t i o n ) and a t i o n (Table IV.2.2,column I I I ) confirmed  remethyl-  the l o c a t i o n of  t h i s r e s i d u e by the f o r m a t i o n of 2,3»4,6-tetra-0-methylg a l a c t o s e and the disappearance  of  2,3-di-0-methylgalactose.  P a r t i a l h y d r o l y s i s . P a r t i a l a c i d 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 was  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  n e u t r a l f r a c t i o n s by ion-exchange chromatography.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 monosaccharides and a d i s a c c h a r i d e (N^)f which were separated by gel-permeation  chromatography.The  a c i d i c f r a c t i o n contained three a c i d i c o l i g o s a c c h a r i d e s (A^, Ag and A^) which were a l s o separated by gel-permeation matography (see F i g u r e IV.2  ).On  chro-  the b a s i s of t h e i r n.m.r.  s p e c t r a l d a t a (see Table IV.2.1) and t h e i r a n a l y t i c a l  and  m e t h y l a t i o n data (see Table IV.2.3) the s t r u c t u r e s of these compounds were shown to be: A,  GlcA - — 2 .  A  GlcA - — ^  0  -•  a  a  Man M a n  I—2 a  M  a  n  115  tetra-  25  5  50 Elution  Figure  IV.2  Separation  of a c i d i c  volume  oligosaccharides  from 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 K26 p o l y s a c c h a r i d e  by g e l - p e r m e a t i o n  chromatography ( B i o - G e l P-2).  (mL)  TABLE IV.2.2 METHYLATION ANALYSIS OF K26 CAPSULAR POLYSACCHARIDE AND DERIVED PRODUCTS. Methylated (as a l d i t o l  sugars  -  acetates)  Mole % °-  R e l a t i v e r e t e n t i o n times — SP-2340- ECNSS-M 170°  OV-225 170°  I ^  II  Ill  1.19 1.82  —  — '  14.7  12.4  1.90  17. 8  13.7 16.0  13.3 14.0  2.29  2.03  15.2  15.3  12.2  2.50  2.26  15. k  17.5  16.8  1.70  2.50  2.26  16.3  17.2  - Glc  2.04  4.40  3.70  —  17.9 11.2  2,3  - Gal  2.24  5.64  4.70  11.4  3  - Glc  —  9.49  7.^0  11.5  2,3,^,6 - G a l  1.14  1.25  3.^.6  - Man  2,4,6  - Man  1.47 1.47  1.95 2.08  2,4,6  - Gal  2,3.^ 2,3.6  - Glc  1.51 1.62  - Glc  3.6  8.4  —  11.7  — —  - and - , as i n Table IV.1.2 .- R e l a t i v e r e t e n t i o n times r e f e r r e d t o 2,3,4,6-Glc as 1.00. - Programmed a t 160° f o r 4 min and then 2 % > i n to 230°. - I , o r i g i n a l c a p s u l a r p o l y s a c c h a r i d e ; I I . m e t h y l a t i o n o f the carbodiimide reduced c a p s u l a r I I I , methylation  o f the d e a c e t a l a t e d methylated  polysaccharide;  reduced c a p s u l a r p o l y s a c c h a r i d e .  117  A  0  GlcA - — 2  M  a  I—A  n  a  j  N, Glc - — - Glc 1 6 The a l d o t e t r a o u r o n i c  M  a  1—1  n  a  Gal  a  a c i d (A^) obtained  from p a r t i a l hy-  d r o l y s i s has been p r e v i o u s l y i s o l a t e d from other capsular polysaccharides,  Klebsiella  K 7 4 ' ^ ; gentiobiose  ^l^^and  (N^)  1  has been a l s o i s o l a t e d by p a r t i a l h y d r o l y s i s from K ^ l  1  ^ and  Two p o s s i b l e s t r u c t u r e s are c o n s i s t e n t w i t h the r e s u l t s thus f a r o b t a i n e d , e i t h e r  2li) 4(2) G  l  c  A  I_Ji  M  a  n  A or B :  1_1  a  M  a  n  G  a  a  l  1_1  a  V or  214) GlcA -—a 4(2j 1 Glc  6  1| Glc  41 11  Gal  v  M  a  n  1—2  M  a  n  1_2  l  c  1 _ £ de B  1 Gal  B  G  Gal  I-  118  In order t o determine reduced  the p o s s i b l e s t r u c t u r e , t h e c a r b o x y l  p o l y s a c c h a r i d e was methylated,the  s e l e c t i v e l y removed by m i l d a c i d  m o d i f i e d a c e t a l was  treatment and the f r e e hy-  d r o x y l groups thus obtained were oxidized ^(DMSO/TFAA) l6  to  the d i c a r b o n y l d e r i v a t i v e . T h i s product was s u b j e c t e d t o a l k a l i n e d e g r a d a t i o n and rernethylated.The f o r m a t i o n o f 2 , 3 . 4 , 6 tetra-O-methylglucose methylglucose  of 2 , 3 . 6 - t r i - 0 -  and the disappearance  i n d i c a t e d t h a t the s t r u c t u r e o f K 2 6 p o l y s a c c h a -  r i d e i s B. 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 Smith h y d r o l y s i s and r e d u c t i o n o f the product gave an oligomer which on the b a s i s o f the n.m.r. and m e t h y l a t i o n analysis,was shown t o be s  G a l - — - GlcA - — 2 . Man - — - G l y c e r o l 3  a  a  T h i s r e s u l t i s c o n c l u s i v e evidence i n favour o f s t r u c t u r e B, but s t i l l and  leaves two anomeric l i n k a g e s unassigned  ( one a  the other 3 ) S e l e c t i v e a c i d h y d r o l y s i s . T r e a t m e n t o f the p o l y s a c c h a r i d e  with very d i l u t e a c i d  (0.01M T F A , 9 5 ° » 5 h ) and d i a l y s i s a g a i n s t  d i s t i l l e d water a f f o r d e d a n o n - d i a l i z a b l e polymeric m a t e r i a l and a d i a l y z a t e . The d i a l y z a b l e m a t e r i a l was shown by paper chromatography to be composed o f g a l a c t o s e , a d i s a c c h a r i d e ( i d e n t i c a l t o N.^) and a t r i s a c c h a r i d e  ( N ) . 0 n the b a s i s o f the n.m.r. s p e c t r a l 2  data  ( see Table IV.2.1) and a n a l y t i c a l and m e t h y l a t i o n a n a l -  ysis  (see Table I V . 2 . 3 ) the s t r u c t u r e o f N  0  was shown to be:  TABLE I V . 2 . 3 ANALYSIS OF THE OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF K26 POLYSACCHARIDE Oligosaccharide (water)  Sugar a n a l y s i s (as  Methylation analysis  alditol  (as a l d i t o l  acetates)  (Molar p r o p o r t i o n s )  + 64  c  Man  (1)  G l c ( G l c A ) (1)  + 79'  +106  c  Man  (2)  N,  + 8.8° + 22  v  (Molar p r o p o r t i o n s )  2,3,4 -Glc ( 1) 2,4,6 -Man ( 0.9) 2,3,4 -Glc ( 1)  G l c ( G l c A ) (1)  2,4,6 -Man ( 1) 3,4,6 -Man ( 0.9)  Gal  (1)  Man  (2)  2,3,4 2,4,6 3,4,6 2,4,6  G l c ( G l c A ) (1)  N-  acetates)  -Glc ( 1) -Man ( 1) -Man ( 0.9) -Gal ( 0.7)  2,3,4, 6 -Glc (1) 2,3,4 -Glc (0.9)  Glc  Gal  (1)  Glc  (2)  2,3,4, 6 - G a l (1) 2,3,6 -Glc (1) 2,3,4 -Glc  (0.9)  120  N  Gal — - Glc  - Glc  1  0  The  s t r u c t u r e of' N  2  demonstrates t h a t the t e r m i n a l g a l a c -  tose u n i t has the 8 - c o n f i g u r a t i o n and thus,the  side chain  is  a - l i n k e d t o the g l u c u r o n i c a c i d . M e t h y l a t i o n a n a l y s i s of the polymeric  m a t e r i a l gave 3»^-di-0-methylglucose which i s d e r i -  ved from the g l u c u r o n i c a c i d , a f t e r removal o f the s i d e c h a i n attached The  t o p o s i t i o n 4. sum o f these  experiments permits  t u r e o f the p o l y s a c c h a r i d e  t o be w r i t t e n , b u t c o n f i r m a t i o n was  made by c a r r y i n g out a b a s e - c a t a l y z e d On m e t h y l a t i o n  the complete s t r u c -  o f the degraded  uronic acid  degradation.  product.hydrolysis.conversion  i n t o a l d i t o l a c e t a t e s and g . l . c . a n a l y s i s . i t was p o s s i b l e t o observe the f o r m a t i o n ved  from m e t h y l a t i o n  o f 2,3»4,6-tetra-Q-methylmannose d e r i a t p o s i t i o n 3 o f the mannose o f the a l d o -  b i o u r o n i c acid.A decrease was observed i n the amount o f 2 , 3 . 4 tri-O-methylglucose  due t o the d e g r a d a t i o n  o f t h i s sugar on  l i b e r a t i o n and exposure t o the base. Conclusion.  The s t r u c t u r e o f the c a p s u l a r  from K l e b s i e l l a serotype  polysaccharide  K26 i s thus based on the heptasac-  c h a r i d e r e p e a t i n g u n i t shown.This s t r u c t u r e i s c o n s i s t e n t 21 w i t h the a n a l y s i s r e p o r t e d by Nimmich  and w i t h the s e r o l o 12  g i c a l c r o s s - r e a c t i o n observed w i t h K l e b s i e l l a K21 t e r i s due t o u n i t s present  .The l a t -  4,6-0-(l-carboxyethylidene)-D-galactopyranosyl i n both polymers.  121  IV.2.4  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 . 1 . c . - m . s . , i n f r a r e d , c . d . and measurement 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 p r e v i o u s l y ( S e c t i o n I I I ) . P a p e r phy, g a s - l i q u i d chromatography,gel-permeation and  chromatograion-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 . P r e p a r a t i o n and  p r o p e r t i e s of K26 c a p s u l a r  A c u l t u r e of K l e b s i e l l a K 26 0rskov,Copenhagen,and was  (5884) was  polysaccharide.  obtained  from Dr.  grown by the procedure d e s c r i b e d i n  S e c t i o n I I I . 7 . 1 (page 69 ).The  p o l y s a c c h a r i d e was  isolated  p u r i f i 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 . Y i e l d 12 s a c c h a r i d e from 1 2 . 5  I.  L of medium.The product  g of p o l y -  had[al +80 D  water).The p u r i t y of the p o l y s a c c h a r i d e was  and  (cO.25,  determined by g e l -  permeation chromatography and an average molecular  weight of  n  1 x i O ' d a l t o n s was The DgO  obtained.  ^"H-n.m.r. spectrum on the o r i g i n a l p o l y s a c c h a r i d e  a t 90° r e v e a l e d s i g n a l s c o r r e s p o n d i n g  tons a t  6 5.50  (2H,s), 6 5.28  between 6 4. 7 and 4 . 5 there was  (lH,s), $5.08  corresponding  a s i g n a l corresponding  t o 7 anomeric (lH,s) and  to 3H.At<5 I . 6 5  in  pro-  signals  (3H,s)  to the methyl group o f an  a-  c e t a l l i n k e d p y r u v i c acid.On removal of the a c e t a l by m i l d hydrolysis obtained  ( 0.1M  TFA,30 min.,95°) a b e t t e r H-spectrum  (see Table  1  IV.2.1 and  was  spectrum N 0 J . 3 ) • ^C-n.m.r.spec-  trum showed 6 s i g n a l s i n the anomeric r e g i o n a t 105.41,103.70, 103.24,101.00,100.76 and  99.87 P.P.m. w i t h the s i g n a l a t IO3.7  b e i n g twice the h e i g h t o f any  o f the other f i v e s i g n a l s . A s i g -  n a l a t 25.77P.P-ni. corresponds t o the pyruvate  (TablelV.2.1)  122  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 . H y d r o l y s i s of a sample of K26  (20 mg)  w i t h 2M  TFA  overnight at 95°.removal  of the a c i d  by s u c c e s i v e e v a p o r a t i o n s w i t h water .followed by paper chroma-, tography  ( s o l v e n t ( A ) and  glucose,D-glucuronic  (B)) showed D-mannose,D-galactose,D-  a c i d and an a l d o b i o u r o n i c acid.The  t i t a t i v e sugar a n a l y s i s of the carboxyl-reduced was  performed as p r e v i o u s l y d e s c r i b e d  polysaccharide  (see page 7 1 ) . T h e  t o l a c e t a t e s of mannose.galactose and glucose were by g . l . c . and  found  quan-  aldi-  identified  t o be p r e s e n t i n a r a t i o o f 1.0:1.0:1.5 .  P r e p a r a t i v e g . l . c . f o l l o w e d by measurement of the c d . showed both m a n n i t o l and g l u c i t o l hexaacetates  t o be  spectra  of the  D-configuration. Methylation analysis.The capsular polysaccharide i n the f r e e a c i d form through i n 40 mL  (obtained by p a s s i n g the sodium  a column o f Amberlite of d r y DMS0 and was  cedure (see page 7 2 ).The  IR-120(H )resin)  product  o f the f u l l y methylated anhydrous oxolane  ( 3 0 0 mg).recovered  after  and  (no  spectrum).Carboxyl-reduction  polysaccharide  ( 9 0 mg)  (see page 7k ).hydrolysis  with LiAlH^ i n  w i t h 2M  c o n v e r s i o n i n t o a l d i t o l a c e t a t e s gave a mixture l y methylated  salt  by the Hakomori p r o -  d i a l y s i s a g a i n s t tap water,showed complete m e t h y l a t i o n h y d r o x y l a b s o r p t i o n i n the i . r .  a l d i t o l a c e t a t e s which was  g.l.c.-m.s. on columns (b) and  of  TFA  and  partial-  analyzed by g . l . c  (c) (see Table  IV.2.2,  column I).A good s e p a r a t i o n o f 2,3»^-tri-0-methylglucose (R 1.62)and 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e t  mg)  was d i s s o l v e d  +  methylated  (290  ( R ^ l . 7 0 ) was  w i t h column (a) programmed a t 160° f o r 8 min.  and  obtained then a t  123  Z°/min up t o 230° f o r 32 min.They were a l s o c h a r a c t e r i z e d as the 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 of the a l d i t o l s 1?0°  ).From p r e p a r a t i v e g . l . c .  (column ( e ) ,  (column ( f ) 2 1 5 ° ) f  methylgalactose and 3-0-methylglucose  2,3-di-O-  were i s o l a t e d . T h e g a l a c -  t i t o l d e r i v a t i v e showed a p o s i t i v e c d .  curve i n d i c a t i n g t h a t  the g a l a c t o s e has the D - c o n f i g u r a t i o n , a n d the g l u c i t o l d e r i c u r v e , c o n f i r m i n g the 3-0-methyl-  v a t i v e , showed a n e g a t i v e c d . D-glucitol  pentaacetate . 6 5  26.  Carbodiimide r e d u c t i o n o f c a p s u l a r p o l y s a c c h a r i d e K A sample of K26 p o l y s a c c h a r i d e ( Na*salt,640 i n 150  mL  o f HgO.  the r e a c t i o n proceeded,the  ( CMC  pH was  ,4.0  ceased  (7-50  s o l u t i o n of NaBH^ (2M) was  mL)  g) was  added.As  maintained a t 4 . 7 5  when necessary w i t h hydrogen c h l o r i d e  sumption o f HC1  was d i s s o l v e d  l-Cyclohexyl-3-(2-morpholinoethyl)-carbodi-  imide metho-p_-toluenesulfonate  ing  mg)  by  titrat-  (0.ION).When the  con-  a f t e r 2 hours,an aqueous  added slowly.Bubble  formation  was  minimiffiized by a continuous f l o w o f a i r blowing on the s u r f a c e of  the s o l u t i o n . A drop of 1-octanol was  added p e r i o d i c a l l y to  c o n t r o l the amount o f foam.At the same time the NaBH^ was ded, the pH of the s o l u t i o n was  s t a b i l i z e d a t 6.5  w i t h HC1  A t o t a l o f 300 mL o f sodium borohydride s o l u t i o n was a p e r i o d of two hours.The f i n a l s o l u t i o n was  ad(4M).  added over  concentrated  and  d i a l y z e d a g a i n s t tap water d u r i n g 48 h and f r e e z e - d r i e d . I n a second  treatment  t o t a l o f 520 mg  the consumption of HC1(0.10 N)was 2.30 o f reduced  p o l y s a c c h a r i d e was  A sample of the reduced  p o l y s a c c h a r i d e (15  mL.A  obtained. mg)  l y z e d w i t h 2M TFA and a f t e r c o n v e r s i o n o f the sugars  was  hydro-  liberated  124  i n t o a l d i t o l a c e t a t e s , g . 1 . c . 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 i n a r a t i o of 0 . 9 5 : 1 . 0 0 : 1 . 4 5  i n d i c a t i n g almost  com-  p l e t e r e d u c t i o n of the u r o n i c a c i d s . E s t i m a t i o n of the e q u i v a l e n t weight of the  polysaccharide  g i v e s a value of 6 5 0  from the consumption of HC1  (theoretical,  640). A sample of the carboxyl-reduced was  was  (250  by the Hakomori procedure to g i v e 2 0 0 mg  methylated  l y methylated  polysaccharide  product.Part  of the methylated  mg)  of  material ( 2 5  fulmg)  h y d r o l y z e d and g . l . c . a n a l y s i s of the p a r t i a l l y methylIV.2.2,column II  ated a l d i t o l a c e t a t e s i s shown i n Table r e s t of the product was a c i d d u r i n g 9 0 min.  heated  on a steam-bath w i t h 50? a c e t i c o  1-methoxyisopropyl-  i n order t o remove the  idene group.The excess  a c e t i c a c i d was  P a r t of the d e a c e t a l a t e d product Hakomori p r o c e d u r e . A n a l y s i s  .The  evaporated  ( 2 0 mg)  was  w i t h water.  remethylated  by  of the h y d r o l y z a t e as a l d i t o l  t a t e s gave on g . l . c . the r e s u l t s shown i n Table  ace-  IV.2.2,column  III. P a r t i a l hydrolysis.The K 2 6 polysaccharide d i s s o l v e d i n 2M  TFA  ( 7 5 mL)  and  the s o l u t i o n was  ( 1 . 0 g)  was  heated  on a  steam-bath f o r 2 h . 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 w i t h water,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 separated The  on a column of ion-exchange r e s i n  acidic fraction  g i v e 8 2 mg  was  (A ) 2  and  AG1-X2).  on B i o - G e l P - 2  separated  of a pure a l d o b i o u r o n i c a c i d  aldotriouronic acid nic acid  ( 4 3 0 mg)  (Bio-Rad  ( A ) , 4 2 mg 1  to  o f a pure  1 2 5 nig of a pure a l d o t e t r a o u r o -  (A~).The n e u t r a l f r a c t i o n showed on paper chromato-  125  graphy g l u c o s e . g a l a c t o s e and mannose and de  (N^ , 3 0 mg)  which was  a l s o separated  a neutral disaccharion B i o - G e l P-2.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,a) sugar sis  (see S e c t i o n III,page ?1 ),b) m e t h y l a t i o n a n a l y s i s (see  S e c t i o n III,page 7 3  ).The  r e s u l t s obtained  c h a r i d e are g i v e n i n Table IV.2.3 ed i n TablelV.2.1  P a r t of the methylated ( 5 0 mg)  saccharide  a c e t i c anhydride  and n.m.r. data are  present-  was  deacetalated polysaccharide.  and d e a c e t a l a t e d carboxyl-reduced d i s s o l v e d i n CHgClg  ( 1 . 6 mL)  was  poly-  ( 5 mL).Trifluoro-  d i s s o l v e d i n CHgClg (10 mL)  cooled t o -60°. D i m e t h y l s u l f o x i d e  (1.1 mL)  i n CHgClg  and  (10 mL )  added t o the TFAA s o l u t i o n over a p e r i o d of 10 min.The  mixture  was  s t i r r e d d u r i n g 10 minutes and  s o l u t i o n was -65°-After  allowed  t o warm up to room tem-  over a 10 minute  the  added min.  dichloromethane  washed a f u r t h e r three times w i t h water.The o r -  g a n i c l a y e r was  d r i e d w i t h anhydrous Na S0^ and 2  removed by evaporation.The ( 1 5 mg)  showed on i . r .  P a r t of the product converted  was  p e r i o d . A f t e r 10  three volumes of water were added and  fraction  polysaccharide  a f t e r 40 minutes t r i e t h y l a m i n e (4 mL)  i n p o r t i o n s (1 mL)  s o l u t i o n was  the  added c a r e f u l l y , k e e p i n g the temperature below  10 minutes i t was  perature and  was  f o r each o l i g o s a c -  .  O x i d a t i o n of the methylated  was  analy-  ( 5 mg)  was  carbon two  the s o l v e n t  tetrachloride soluble  carbonyl absorptions.  h y d r o l y z e d w i t h 2M  TFA  and  i n t o a l d i t o l a c e t a t e s j g . 1 . c . i n d i c a t e d the d i s a p -  pearance o f 2 , 3 - d i - 0 - m e t h y l g a l a c t o s e The r e s t of the product  (10 mg)  was  which had been o x i d i z e d . d i s s o l v e d i n CH,C1  0  and  126 sodium ethoxide s o l u t i o n i n e t h a n o l (1M,1.5 mL) was added . The s o l u t i o n was s t i r r e d d u r i n g 1\ h. a t room temperature  after  which i t was n e u t r a l i z e d w i t h h y d r o c h l o r i c a c i d , c o n c e n t r a t e d andcheated on a steam-bath d u r i n g t h r e e h o u r s . A f t e r e x t r a c t i o n w i t h chloroform,the product thus obtained was remethylated by the Hakomori p r o c e d u r e . H y d r o l y s i s and c o n v e r s i o n i n t o  alditol  a c e t a t e s showed on g . l . c . the appearance o f 2 , 3 , ^ » 6 - t e t r a - 0 methylglucose  and disappearance  of  2,3i6-tri-0-methylglucose.  Periodate o x i d a t i o n . A s o l u t i o n o f K26 p o l y s a c c h a r i d e ( 1 . 0 g) i n water (150 (150  mL) was mixed w i t h 0.1M NalO^ and 0.4M NaClO^  mL).The s o l u t i o n was kept i n the dark a t 4 ° . A f t e r 240 h  (consumption glycol  o f p e r i o d a t e 5.2  moles per mole o f K 2 6 ) . e t h y l e n e -  (10 mL) was added.The polyaldehyde was d i a l y z e d  over-  night,reduced w i t h NaBH^ (1 g) t o the p o l y a l c o h o l , n e u t r a l i z e d w i t h 20% a c e t i c a c i d , d i a l y z e d and f r e e z e - d r i e d t o y i e l d the p o l y a l c o h o l (600  mg).Eart o f the p o l y a l c o h o l (300  t r e a t e d w i t h 0.5M TFA f o r 20 h a t room temperature  mg) was ,the a c i d  was removed by s u c c e s s i v e evaporations w i t h water,and the product was reduced w i t h NaBH^.The excess NaBH^ was destroyed w i t h IR-120 ( H ) r e s i n and borate was removed by e v a p o r a t i o n w i t h +  methanol.Paper chromatography o f the products showed ( s o l v e n t (C)) g l y c e r o l , e r y t h r i t o l and/or t h r e i t o l and an oligomer  with  a R^^O. 28. H y d r o l y s i s w i t h 2M TFA o v e r n i g h t and paper chromatography o f the h y d r o l y z a t e i n s o l v e n t (A) showed g l y c e r o l , e r y t h r i t o l and/or t h r e i t o l , m a n n 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 acid.Sugar a n a l y s i s o f the carboxyl-reduced product as a l d i t o l a c e t a t e s showed on g . l . c . the presence o f  12? g l y c e r o l , t h r e i t o l , e r y t h r i t o l m a n n i t o l , g a l a c t i t o l and t  The r a t i o of the h e x i t o l s was of the m a t e r i a l was  glucitol.  1.0s1.0s1.1 r e s p e c t i v e l y . T h e r e s t  separated i n a column of B i o - G e l P-2.The  e l u t i o n p r o f i l e obtained i n d i c a t e d t h a t the Smith d i d not go to completion,but  25 mg  hydrolysis  of a pure oligomer  (SH)  was  isolated. SH had [ a ]  D  +  67°  (c 3,water) and R  G a l  0.28  ( solvent ( C ) )  {  n.m.r. data are g i v e n i n TablelV.2.1 .Sugar a n a l y s i s of the carboxyl-reduced product as a l d i t o l a c e t a t e gave on g . l . c . , glycerol,mannose.galactose  and glucose i n a r a t i o of l s l s l s l .  M e t h y l a t i o n a n a l y s i s of the oligomer gave the f o l l o w i n g part i a l l y methylated  a l d i t o l acetates $  galactose,2,4,6-tri-0-methylmannose  2,3,4,6-tetra-0-methyland  3»4-di-0-methylglucose.  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 . K26 p o l y s a c c h a r i d e (500 was  d i s s o l v e d i n 0.01M  TFA  f o r 7 hours.The a c i d was  (70 mL)  and heated on a steam-bath  removed and the product was  dialyzed  a g a i n s t 1L of d i s t i l l e d water.A polymeric m a t e r i a l (410 and a d i a l y z a t e  ( 80 mg)  mg)  were obtained.Paper  mg)  chromatography  of the d i a l y z a b l e f r a c t i o n showed p y r u v i c a c i d . g a l a c t o s e , a disaccharide  ( i d e n t i c a l to N-^) and a t r i s a c c h a r i d e  T h i s t r i s a c c h a r i d e was  (Ng) .  i s o l a t e d by p r e p a r a t i v e paper  chromato-  graphy ,-yield 25 mg,[a] +22° (c 2.5, water) .Sugar a n a l y s i s i n D  d i c a t e d glucose and g a l a c t o s e i n a r a t i o of 2 s i . M e t h y l a t i o n h y d r o l y s i s , r e d u c t i o n and t r i m e t h y l s i l y l a t i o n of the a l d i t o l s gave ( g . l . c . column  (e)),2,3,4,6-tetramethylgalactose,2,3,6-  t r i - 0 - m e t h y l g l u c o s e and  2,3,4-tri-0-methylglucose.  M e t h y l a t i o n a n a l y s i s o f the polymeric m a t e r i a l , ( g . 1 . c  128 column (c) and g . 1 . c . - m . s . ) i n d i c a t e d the presence o f 3>^-di-0methylglucose i n l i e u o f the 3-0-methylglucose  found o r i g i n a l -  ly. Uronic a c i d degradation.A sample o f methylated K26 p o l y s a c c h a r i d e (60 mg) was c a r e f u l l y d r i e d and then s u b j e c t e d t o a 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 (see page 75  ).Methyl  i o d i d e was the a l k y l a t i n g agent u s e d . H y d r o l y s i s and g . l . c o f the a l d i t o l a c e t a t e d e r i v a t i v e s  gave  2,3»4,6-tetra-0-methyl-  mannose,3»^»6-tri-0-methylmannose,2,4,6-tri-0-methylgalactose, 2 , 3 . 4 - t r i - 0 - m e t h y l g l u c o s e , 2 , 3 , 6 - t r i - 0 - m e t h y g l u c o s e and 2 , 3 - d i O-methylgalactose i n the r a t i o 1.0:1.0:0.9:0.4:1.0:1.0 .  129  IV.3  Bacteriophage  d e g r a d a t i o n of 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  K 60 and K 46J"  88  IV.3.1 Introduction. L i k e many other o r g a n i s m s , b a c t e r i a are s u b j e c t t o i n f e c t i o n by a range o f v i r u s e s c a l l e d b a c t e r i o p h a g e s . V i r a l i n f e c t i o n o f b a c t e r i a has been known s i n c e 1 9 1 5 and s i n c e the e a r l y i n v e s t i g a t i o n s , t h e h i g h s p e c i f i c i t y f o r b a c t e r i a l host was observed. T h i s i s r e l a t e d t o the r e c o g n i t i o n o f s p e c i f i c r e c e p t o r s on the c e l l s u r f a c e of the b a c t e r i a which can be:  flagella,pili,  c a p s u l e s , l i p o p o l y s a c c h a r i d e s , t e i c h o i c a c i d - p e p t i d o g l y c a n com plexes,surface proteins,etc.Phages  v a r y w i d e l y i n s i z e and i n  shape. They were c l a s s i f i e d by B r a d l e y " ^ 1  8  a c c o r d i n g to t h e i r mor-  phological differences. The d i f f e r e n t steps i n a v i r a l  i n f e c t i o n can be summarized  i n the f o l l o w i n g c y c l e : i) ble  a d s o r p t i o n o f the phage p a r t i c l e s onto the s u s c e p t i -  host, i i ) i n j e c t i o n of the v i r a l DNA  (or RNA)  i n t o the host,  i i i ) r e p l i c a t i o n of the phage n u c l e i c a c i d and phage prot e i n s a t the expense of the m e t a b o l i c processes iv)  phage m a t u r a t i o n  l y s i s of the host  of the host,  and r e l e a s e which r e s u l t s i n the  cell.  The i n j e c t i o n of the n u c l e i c a c i d i n t o the host c e l l needs the attachment o f the phage t o the c y t o p l a s m i c membrane.Al -. though i t was c o n s i d e r e d t h a t c a p s u l a t e b a c t e r i a were g e n e r a l l y phage r e s i s t a n t . b a c t e r i o p h a g e s can be i s o l a t e d f o r many cap-  130  s u l a t e s p e c i e s of b a c t e r i a . A f t e r the r e c o g n i t i o n and b i n d i n g of the phage t o the e x o p o l y s a c c h a r i d e , t h e through  the e x o p o l y s a c c h a r i d e  phage must f i n d  i t s way  l a y e r . A t t a c k of bacteriophage  on  e x o p o l y s a c c h a r i d e producing b a c t e r i a i s o f t e n r e v e a l e d by the fact that a halo  1 7 9  h a l o i s formed s u r r o u n d i n g the p l a q u e . W i t h i n  , b a c t e r i a are decapsulated,which  this  can come from the r e -  s u l t of the d i f f u s i o n of a phage-induced enzyme which  hydro-  l y s e s the capsule without k i l l i n g the b a c t e r i a . T h i s enzyme a c t i v i t y w i t h h y d r o l y s i s of the capsule allows the movement of the phage v e r t i c a l l y and l a t e r a l l y , a l l o w i n g the phage t o r e a c h the c y t o p l a s m i c membrane (see F i g u r e IV.3).Many other tic activities  .catalyzing d i f f e r e n t degradation r e a c t i o n s of  host s u r f a c e p o l y s a c c h a r i d e s may be a s s o c i a t e d w i t h virus  enzyma-  bacterial  particles. The ease of i s o l a t i o n , m a n i p u l a t i o n and p r o p a g a t i o n of the  bacteriophages depolymerize  has p r o v i d e d a p r o l i f i c  source  b a c t e r i a l polysaccharides.These  the e x o p o l y s a c c h a r i d e s y i e l d products  o f enzymes which enzymes.acting  on  corresponding to s i n g l e  r e p e a t i n g u n i t s o f the p o l y s a c c h a r i d e as w e l l as m u l t i p l e s thereof.The  c o n d i t i o n s o f d e p o l y m e r i z a t i o n a r e such t h a t l a b i l e  s u b s t i t u e n t s . w h i c h by other d e g r a d a t i v e techniques  ( p a r t i a l hy-  1R1 T R? d r o l y s i s ) are l o s t , s u r v i v e t h i s treatment * .This a f f o r d s a v a l u a b l e procedure f o r o b t a i n i n g oligomers which can be used 39  i n numerous ways as, a) p r o d u c t i o n o f s y n t h e t i c a n t i g e n s ^ , 7  b) s u b s t r a t e f o r n.m.r. s t u d i e s , c) source o f new o l i g o s a c c h a rides, etc. A l a r g e number o f these v i r u s e s have been i s o l a t e d r e c e n t -  131  Bacteriophage  Li po polysaccharide  j Cell membrane  Figure  IV.3  Attack  of  bacteria.  a  bacteriophage  on  an  encapsulated  132 l y by S t i r m and co-workers -^,which are s p e c i f i c f o r depoly18  m e r i z i n g the c a p s u l a r p o l y s a c c h a r i d e produced by the host strain  ( Klebsiella  ) and i n some cases other c a p s u l a r g l y c a n s  of r e l a t e d s t r u c t u r e . The r e s u l t s o f the d e g r a d a t i o n o f two 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 ( K 60 and K 46 riophages  )with t h e i r r e s p e c t i v e bacte-  ( 0 60 and 0 46 . r e s p e c t i v e l y ) are presented  here.  IV.3.2 R e s u l t s . K l e b s i e l l a bacteriophages were i s o l a t e d from sewage and propagated  on t h e i r host s t r a i n s u s i n g n u t r i e n t b r o t h as medi-  um. Propagation was continued on an i n c r e a s i n g s c a l e u n t i l the  13 crude  l y s a t e s contained a t o t a l o f ~ 1 0  J  plaque-forming  units,  an amount s u f f i c i e n t t o degrade one gram of polysaccharide.The crude l y s a t e s were concentrated t o one f o u r t h o f t h e i r volume and d i a l y z e d a g a i n s t r u n n i n g tap water f o r 2 days to remove low molecular weight m a t e r i a l s from b r o t h and c e l l  lysates.  The n o n - d i a l y z a b l e f r a c t i o n was concentrated t o a convenient volume and was added t o a s o l u t i o n o f the 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 i n water.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 at  37° d u r i n g 2 d . A f t e r d e p o l y m e r i z a t i o n , t h e s o l u t i o n was  c e n t r a t e d and 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 ( 3  con-  times).The  combined d i a l y z a t e s were concentrated and f r e e z e - d r i e d , y i e l d ing  the crude mixture  o f oligomers which were f u r t h e r p u r i f i e d  by treatment w i t h Amberlite  IR^120 ( H ) r e s i n . T a b l e IV.3.2 +  shows the c o n d i t i o n s used f o r depolymerization,and  yields for  the d e g r a d a t i o n o f K60 and K46 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 -  133  TABLE IV. 3^1 PROPAGATION OF BACTERIOPHAGES 060 AND 046. 046  060 Test tube  lysis;  Titre  (PFU/mL)-  Volume ( mL ) T o t a l 0 (PFU) Small f l a s k Titre  (PFU/mL)  T o t a l 0 (PFU)  Titre  10  2.0 x 10  14  10  6  ,11 1.8 x 10-- -  1.2x10  10  1.0 x 10  1  11  lysis;  Volume ( mL )  Wash b o t t l e  1.3 * 10  6.3 * 10  10  200  100 ,12 6.3 x l O  2.0 x 10  2.8 x 10 10  l.Ox  A f c  12  lysis; (PFU/mL)  Volume ( mL ) T o t a l 0 (PFU)  — PFU : plaque forming u n i t  10  1000  900 2.5x  10  10  1 3  1 . 0 * 10 1 3  134  TABLE-IV.3.2 DEPOLYMERIZATION OF KLEBSIELLA K60 AND K46 CAPSULAR POLYSACCHARIDES BY BACTERIOPHAGES 060 AND 046 RESPECTIVELY.  06OK6Q  046K46  400  1000  )  Volume of phage (I) -  ( mL  Titre  (PFU/mL)  Volume o f phage ( I I ) -  ( mL )  2.8 * l O  1 0  l.Oxio  300  150  Weight o f p o l y s a c c h a r i d e ( g )  1.0  0.83  T o t a l volume  ( mL )  450  300  Yield  ( % )  76  78  of oligomers  -Volume o f phage from the wash b o t t l e  l y s i s . - Volume of phage  (I) a f t e r p u r i f i c a t i o n by d i a l y s i s a g a i n s t running and  concentration.  1 0  t a p water,  135  rides with bacteriophage  060 and 046 respectively.  Analysis of the products of depolymerization of K60.The p u r i f i e d products of K 6 0 depolymerization were added to a c o l umn of Bio-Gel P-4 and eluted with water-pyridine-acetic acid (500*5:2).The  P  1  e l u t i o n pattern i s shown i n Figure IV.4,where  represents the repeating unit of the polysaccharide,P the 2  dimer of P^ and P^ represents several higher oligomeric products. Components P^ and P  2  were analyzed by n.m.r. spectros-  copy and methylation analysis. The ^C-n.m.r. of P.^ (see Table IV. 3 . 3 a ) that i t i s an heptasaccharide  demonstrates  corresponding to one repeating  unit.By comparison with the ^ C spectrum of the o r i g i n a l polysaccharide (see Table I V . 3 . 3 a ) the s i g n a l at 103.07 p.p.m..assigned to a  —2. Glcp_ —g— .disappeared,indicating that the  enzyme i s a B -glucosidase.The  signals corresponding to the  anomeric carbons of the reducing end  (96.74 and  9 3 . 0 3 p.p.m.)  confirmed the glucose as the reducing end.The^H-n.m.r.( see Table I V . 3 . 3 b ) i s i n agreement with t h i s results,as the doub l e t at 6 5.04,corresponding to the B-glucose i n the backbone of the o r i g i n a l  polysaccharide.disappeared.  Confirmation of the reducing end and of the degree of polymerization (D.P.) of the repeating unit was obtained by 184 the method of Morrison  .whereby the oligosaccharide i s r e -  duced to the a l d i t o l and a f t e r hydrolysis,the free sugars are converted into the peracetylated aldononitriles;the reducing end i s converted into the peracetylated a l d i t o l . R e s u l t s are shown i n Table IV.3.k. and indicate that  glucitol.mannono-  136  Figure  IV.4  Separation of (  K60  by  Bio-Gel  of  the  depolymerization  gel-permeation P-4)  products  chromatography  137  n i t r i l e . g l u c o n o n i t r i l e and g a l a c t o n o n i t r i l e are present i n a r a t i o o f 1:1:4:1 ;before h y d r o l y s i s , t h e g l u c u r o n i c a c i d  resi-  due was converted i n t o a g l u c o s y l r e s i d u e by the normal p r o cedure  (methanolysis-s.odium  borohydride  r e d u c t i o n ) .Methyla-  t i o n o f P ^ , r e d u c t i o n w i t h L i A 1 H ^ , h y d r o l y s i s and c o n v e r s i o n i n t o the a l d i t o l a c e t a t e s showed on g . l . c . the r e s u l t s  indi-  cated i n Table I V . 3 . 5 . By comparison w i t h the m e t h y l a t i o n an a l y s i s o f the o r i g i n a l p o l y s a c c h a r i d e , 2 , 3 - d i - 0 - m e t h y l g l u c o s e c o r r e s p o n d i n g t o the g l u c u r o n i c a c i d r e s i d u e a p p e a r s , i n s t e a d of the 2 - 0 - m e t h y l g l u c o s e . i n d i c a t i n g t h a t the l i n k a g e c l e a v e d by the bacteriophage  —2-  born-enzyme i s :  2.  D - G 1 C £  D-GlcpA  1  f and  that P  1  i s the f o l l o w i n g r e p e a t i n g u n i t o f K60:  D-Glc£  4 1 — D-Glc£A — -2- D-Gal£ 2  ——2. nD-Man£ _ 2'  1  D-G l C £  A n a l y s i s o f Pg by n.m.r. s p e c t r o s c o p y and  DD-GlC£  ll  D-GlC£  (see T a b l e s I V . 3 . 3 a  I V . 3 . 3 b f o r r e s u l t s and assignments ) confirmed  the r e -  s u l t s obtained from the a n a l y s i s o f P^, showing the s i g n a l a t 103.04 p.p.m. ( i n c-n.m.r l3  ) and a t 5 4.99  ( i n ^-n.m.r. )  TABLE IV.3.3a P.M.R. DATA FOR KLEBSIELLA K6Q CAPSULAR POLYSACCHARIDE AND THE OLIGOMERS DERIVED FROM BACTERIOPHAGE DEGRADATION. A  Compound  Ji  2  (Hz)  -2C]LcA  -2G1C-  —Avian— a 2  1  4  I5  a  2 1  l Gl -C  5M  3  Glc  4  1  P  a  2  •Avian  2  a  3  3  Glc  Glc  Glc  1  1  1  n  t  e  g  r  a  l  1  Glc  Glc-OH  Assignment  proton  2.0  a  /  G  1  3-Man-  2  5-37  2  1.0  3-Gal-  5.04  1.0  3-Glc-  1.0 1.0 1.0  Glc-  4.54  7 7 7 7  5.^9  s  1.0  5.46  s  1.0  4.85 4. 70  G]LcA  2  I  5.^5  s  5.28  s  4.87  7  1.0  a  Glc3-GlcA-  4  {  Glc3-Man—  2  a  3-Gal  2  0.3 1.0  ° —  *  3-Glc a  Glc-r-  OH  TABLE IV.3.3a  P  2  (cont.) 4.75 4.64  7  G l c —  7  1.0 0.7  3- Glc-g—OH  4.45  7  1.0  4- GlcA--—  5^9 5.46  s  2.0  s  2.0  5-45  s  2.0  /  G 1 C  —  3-Gal— 2  a  5.28  s  4.99 4.74  8  0.3 1.0  8  2.0  G l c —  4.70  8  2.0  G l c —  4.66  8  4.45  8  0.7 2.0  3-Glc—OH 3 cA  3-Glc  OH  3-Glc—  jT — 1.4-GlcA  TABLE IV.3.3b N.M.R. ( C 1 3  ) DATA FOR KLEBSIELLA K60 CAPSULAR POLYSACCHARIDE AND THE OLIGOMERS  DERIVED FROM BACTERIOPHAGE DEGRADATION. Chemical s h i f t  Compound  Assignment  (p.p.m.)  — - G l c - — - G l c A - : — - k l a l - — 2Man* 4  104.23  4  . Glc-  B  ll Glc  1 G  3-GlcA-  l l  Glc  103.0?  3-Glc-  102.44  Glc-  100.60  Glc-  99-56  3-Man2 3-Gal-  r  l  104.29  {  4-GlcAGlc—  102.31 100.54 99.67  Glc— Glc-  3-Man— 2 a  TABLE IV.3.3b  (cont.) 3-Glc 3-Glc  OH B  OH a  P  4-GlcA-  *  4-GlcA3  2x  Glc— 3-Glc-  2x  Glc—— 1 4 Glc^AjlcAB  a  }  1 4 Glc^-^GlcAa  2x3-Man 2  a  2 x 3 -Gal  2  a  3-Glc—OH 3-Glc  OH a  - see Table IV.3.3a . - see text.  142  TABLE IV.3.4  DETERMINATION OF THE DEGREE OF POLYMERIZATION AND THE REDUCING END OF K6Q OLIGOSACCHARIDES  Peracetylated derivative  of  ( P^ AND Pg ).  Relative  retention  time  Mole % Pj  P  2  OV-225 Mannononitrile  1.00  15  15  Glucononitrile  1.2?  56  64  Galactononitrile  I.38  14  14  Glucitol  1.60  15  7  - i s o t h e r m a l a t 210°  143  TABLE IV.3.5 METHYLATION ANALYSIS OF K60 OLIGOSACCHARIDES ( Pj^ AND Pg ) FROM BACTERIOPHAGE DEGRADATION.  P a r t i a l l y methylated  Relative retention  a l d i t o l acetates -  time  Mole % P-  L  Pg  OV-225 ~  2,3,4,6 - G l c  1.00  46.7  2,4,6  - Glc  1.83.  10.0  13-3  4,6  - Man  2.96  12.9  1^.0  4,6  - Gal  3-26  15.2  15-1  2,3  - Glc  4.63  15.3  7.4  2  - Glc  7.61  —  5.7  -  2,3,4,6 - G l c  l,5-di-0-acetyl-2,3,4,6-tetra-0-methylglucitol,  e t c . — Values were c o r r e c t e d by use o f carbon-response g i v e n by Albersheim e t a l . ^ — i s o t h e r m a l a t 170°. 7  factors  144 corresponding  3 1 3 — ^ G l c — r - * GlcA 3 4  to the l i n k a g e  M e t h y l a t i o n a n a l y s i s as w e l l as the D.P. P  2  IV. 3 . 4  t o be a dimer of P^^ (see Tables  1  —  d e t e r m i n a t i o n showed and  IV. 3 . 5 , r e s p e c t -  ively). A n a l y s i s o f the products p u r i f i e d products  o f d e p o l y m e r i z a t i o n o f K46.The  from the d e p o l y m e r i z a t i o n  of K l e b s i e l l a  K46  c a p s u l a r p o l y s a c c h a r i d e were e l u t e d from a column of B i o - G e l P-4  with water-pyridine-acetic acid  (500:5*2).The  e l u t i o n pat-  t e r n i s shown i n F i g u r e IV.5.where P^ r e p r e s e n t s the r e p e a t i n g u n i t of the K46  polysaccharide,P  oligomers.Components P^ and  P  2  2  a dimer of P-p and  P^  higher  were analyzed by n.m.r. spec-  t r o s c o p y and m e t h y l a t i o n a n a l y s i s . The  ^C-n.m.r. spectrum of P-^ ( see Table IV. 3 . 6 a ) , i n d i -  cates t h a t i t i s a hexasaccharide i n g u n i t of the K46  corresponding  polysaccharide.By  to one  repeat-  comparison w i t h the  spec-  trum of the o r i g i n a l p o l y s a c c h a r i d e  (see Table I V . 3 . 6 a )  s i g n a l a t 1 0 3 , 1 6 , w h i c h was  to a 3 g a l a c t o s y l r e s i d u e ,  assigned  the  d i s a p p e a r e d , i n d i c a t i n g t h a t the enzyme i s a 3 g a l a c t o s i d a s e . The  anomeric carbons o f the r e d u c i n g end  ( 9 7 . ^ 4 and  93.11)  con-  f i r m e d g a l a c t o s e as the r e d u c i n g sugar.The ^H-n.m.r. spectrum (see Table I V . 3 . 6 b ) i s i n agreement w i t h these r e s u l t s . T h e r e d u c i n g end b e i n g the g a l a c t o s e , a s w e l l as the D.P.of were confirmed Table  IV.3-7.  by the method o f M o r r i s o n . R e s u l t s  are shown i n  G a l a c t i t o l and g l u c o n o n i t r i l e were found  r a t i o of 1 : 1 which i s the value expected u n i t ; g l u c u r o n i c a c i d was  not reduced  f o r one  i n this  P^  in a  repeating  case.Methylation  145  F i g u r e IV.5  S e p a r a t i o n o f the d e p o l y m e r i z a t i o n of K46 by gel-permeation (Bio-Gel  P-4)  products  chromatography  TABLE IV.3.6a P.M.R. DATA FOR KLEBSIELLA K46 CAPSULAR POLYSACCHARIDE AND THE OLIGOMERS DERIVED FROM BACTERIOPHAGE DEGRADATION. Compound  2  (Hz)  ^Ian—-GlcA-—-Man-—-kjal-" cAex  a  -Gal--  B  Man !hP  3'  6  Integral  Assignment  proton  5-29  1.0  3-GlcA  5.20  1.0 1.0 2.0  3-Gal  5.05 4.88  4 a  3-Man 3-Gal-f 3-Man—  {  l l  Glc  r  6  V  1.0  4.62 1.47  4 6 Glc-  3.0  CH, C0 H 2  G cA-—-Man-=—-Gal-—-Gal-OH a a a 4  3' 1  Glc  6  /  4-GlcA—  1 3-Gal—  5.21 Man 5>P  1.3  5-31  5.20  2 2  1.0  5.06  s  4.71  s  1.0 1.0  3-Gal  { 3-Gal  a  3-Man- aa 3-ManP  Gal  a  OH  Gal-r-OH B  TABLE I V . 3 . 6 a  (cont.) 4.66  3-Gal  0.7  4.65  1.0  1.52  3.0  OH B  Glc—sCH-—C 3 i C0 H 2  P  2  5-30  2. 3  4-GlcA-  <  3-GlcA4 k  3 - G a l - -OH  5.19  2.  0  3-Gal-  5.06  2.  0  3-Man-  1.0  3-Man - KJlcA 4 6 ' 1  p  4.85  V  1.0  '  3-Man-^GlcA  4.69 3.7  4.65 1.53  3-0  1.52  3-0  2 x  C  H 3  -  (  [  C0 H 2  - From ref.188  TABLE IV.3.6b N.M.R. ( C 1 3  ) DATA FOR KLEBSIELLA K46 CAPSULAR POLYSACCHARIDE AND THE OLIGOMERS  DERIVED FROM BACTERIOPHAGE DEGRADATION.  Compound  Chemical s h i f t  Assignment  (p.p.m.)  -2G: c A - — ^ M a n - — h a l - — A l a l — 4  a  a  a  g  l Man ;>P 6 4^, 3 3  a  103.16  3-Gal  100.77  3-glcA-  3  — . ~ 100.16  3  — 6 / 3-Ms... Glc4 6 W  ^  e  v  r 97.16 96.16  Glc  25.40  3-Man 3-Gal ° a CH.,—C 3 i C0 H 2  b P,  101.33 101.00 100.37  Glc 3 4-GlcA— a 3-Man—4 6 B  V  TABLE IV.3.6b (cont.)  97.1^ 95.97 \ 95.81j  {  3-Man a  3-Gal-r-OH p  3-Gal  93.11  3-Gal—OH  25.43  CH~—C 3 1 C0 H 2  P  2  103.35 101.15  3-Gal— 3- G l c A — 4 4- G l c A — a  100.20  2x G l c - r — 2x3-Man—— 4 6 V B  96.92 96. 80  (  2x3-Man a  3-Gal-^-OH  93.10  2x3-Gal  25.34  3-Gal  OH  2 * 0H —C I C0 H o  3  2  150  TABLE IV.3.7 DETERMINATION OF THE DEGREE OF POLYMERIZATION AND THE REDUCING END OF K46 OLIGOSACCHARIDES ( P  Relative  Peracetylated derivative of  2  AND Pg ).  Mole %  retention  time OV-225 -  Mannononitrile  1.00  34.0  38.0  Gluc ononi t r i l e  1.27  21.8  20.6  Galactononitrile  1.38  22. 0  30.9  Galactitol  1.51  22.2  10.5  - as i n Table  IV.3.4  151  TABLE IV.3.8 METHYLATION ANALYSIS OF K46 OLIGOSACCHARIDES ( P FROM BACTERIOPHAGE  ±  AND Pg )  DEGRADATION.  P a r t i a l l y methylated  Relative retention  a l d i t o l acetates -  time OV-225  Mole % — P  -  1 n  P.  x  2  2,3,4,6 - Glc  1.00  17.9  2,4,6  - Man  1.91  16.0  16.1  2,4,6  - Gal  2.05  32.3  33.0  2,3  - Glc  4.63  15.9  16.8  2  - Man  5.70  17.9  8.1  2  - Glc  7.61  -,- and -  as i n Table  IV.3.5  —  17.8  8.2  152 a n a l y s i s of P^^ showed on g . l . c . the r e s u l t s i n d i c a t e d i n Table IV.3.8. By comparison  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 na-  t i v e p o l y s a c c h a r i d e , 2,3-di-0-methylglucose the g l u c u r o n i c a c i c appeared  corresponding to  i n s t e a d of the  2-0-methylglucose  from p r e v i o u s l y . T h i s i n d i c a t e d t h a t the l i n k a g e c l e a v e d by the bacteriophage borne-enzyme of  —2  D-Galp  1  . ^ D-GlcpA  is:  -—  f and P^ i s the f o l l o w i n g  k D-Glc£ ~a  hexasaccharide:  6  D-Man£  D-Glc£A  D-Man£  M e t h y l a t i o n a n a l y s i s as w e l l as the D.P. P  2  D-Galp_  1  d e t e r m i n a t i o n of  confirmed i t to be the dimer of P^^ (see Tables IV. 3.7  IV.3.8). Nuclear magnetic resonance IV.3.6a and of  D-Galp. -2  P  and  spectroscopy (see Tables  IV.3.6b ) confirmed the r e s u l t s from the a n a l y s i s  r  IV.3.3  Discussion.  The main purpose  o f the bacteriophage work c a r r i e d out by  our r e s e a r c h group i s to o b t a i n l a r g e amounts of pure of the p o l y s a c c h a r i d e degraded  subunits  ( one r e p e a t i n g u n i t or/and  the  dimer),with the a c i d or base l a b i l e 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 s a c c h a r i d e . I n order to a v o i d the  expensive  153  and time consuming p u r i f i c a t i o n of the phage,several  possibil-  i t i e s have been i n v e s t i g a t e d which would y i e l d i n a s h o r t e r per i o d of time the amount of phage necessary f o r the d e g r a d a t i o n (~10 to  phages/g of p o l y s a c c h a r i d e ) and i n such c o n d i t i o n s as  1 3  s i m p l i f y the p u r i f i c a t i o n of the d e g r a d a t i o n  b e s t procedure,developed  products.The  up to t h i s moment,is the one used i n  the course of the p r e s e n t i n v e s t i g a t i o n . A c c o r d i n g to t h i s method.the bacteriophage i s propagated  i n b r o t h as u s u a l  D  till  the amount of phage i s s u f f i c i e n t t o degrade the d e s i r e d  a-  mount of p o l y s a c c h a r i d e . T h e crude phage s o l u t i o n i s d i a l i z e d e x h a u s t i v e l y to remove a l l low molecular weight m a t e r i a l s i n stead of p u r i f y i n g the phage 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 186 ene g l y c o l 6000  and  isopycnic centrifugation.This  solution,  a f t e r c o n c e n t r a t i o n , i s the source of bacteriophage f o r the degradation.The  d e g r a d a t i o n i s c a r r i e d out i n d i s t i l l e d  a v o i d i n g the use of PBS  water,  (buffered s a l i n e ) , a s t h i s s a l i n e  me-  dium means more steps i n the p u r i f i c a t i o n . A f t e r d e g r a d a t i o n , d i a l y s i s a f f o r d s the oligomers.The a f a c t o r of f o u r when we  time has been reduced  by  compare the two procedures,and there  i s l i t t l e d i f f e r e n c e i n the q u a l i t y and q u a n t i t y of products obtained. In  order t o demonstrate  phage-borne enzyme, 060 ed by removal  was  the s p e c i f i c i t y of the b a c t e r i o used on the p o l y s a c c h a r i d e o b t a i n -  of the three t e r m i n a l g l u c o s y l groups from  p o l y s a c c h a r i d e by Smith d e g r a d a t i o n . The v i r a l enzyme was tally due  i n a c t i v e on the Smith degraded  to changes on the environment  K60 to-  polysaccharide (K60SH),  of the g l y c o s i d i c l i n k a g e  F i g u r e IV. 6  Environment of the g l y c o s i d i c  linkage  which undergoes enzymic h y d r o l y s i s ( K 6 0 ) and the one t h a t does not (K60SH)  155  which undergoes enzymic h y d r o l y s i s (see F i g u r e IV.6) S p e c i f i c h y d r o l a s e s were used on both r e p e a t i n g u n i t s o f K60 and K 4 6 i n order t o i s o l a t e s m a l l e r o l i g o s a c c h a r i d e s , b u t no a c t i v i t y was observed.The enzymes used were a - and s - g l u c o s i d a s e s which probably are a c t i v e towards s h o r t c h a i n s o f sugar r e s i d u e s such as d i - or t r i - s a c c h a r i d e s and have v e r y little IV.3.4  or no a c t i o n on l o n g e r chains '''. 18  Experimental.  Bacteriophage  p r o p a g a t i o n and d e p o l y m e r i z a t i o n . 0 6 0 and  0 4 6 were i s o l a t e d from sewage and propagated  on the host  s t r a i n s u s i n g n u t r i e n t b r o t h as medium a c c o r d i n g t o the s t a n dard procedures  of virology.Three successive  propagations  u s i n g i n c r e a s i n g amounts o f b a c t e r i a l c u l t u r e s and b a c t e r i o phage were necessary  to obtain s u f f i c i e n t v i r u s p a r t i c l e s  with  which t o degrade the c o r r e s p o n d i n g c a p s u l a r p o l y s a c c h a r i d e s . This involved: a) tube l y s i s ; 4 mL o f n u t r i e n t b r o t h , 0 . 5 mL o f b a c t e r i a l c u l t u r e , 0 . 5 mL o f bacteriophage  solution;  b) s m a l l f l a s k l y s i s ; 4 8 mL o f n u t r i e n t b r o t h , 1 mL o f b a c t e r i a l c u l t u r e and 1 mL o f bacteriophage  solution  (from a );  c) wash b o t t l e l y s i s ; 200 mL o f n u t r i e n t b r o t h , 10 mL o f b a c t e r i a l c u l t u r e and 2 5 mL o f bacteriophage  solution  (from b ) .  In the l a t t e r c a s e , i n order t o c o n t r o l the p r o p a g a t i o n o f phage,the b a c t e r i a l growth was monitored  by o p t i c a l d e n s i t y  ( see F i g u r e IV.7).When the r e a d i n g s o f o p t i c a l d e n s i t y were appropiate  ( 0.5-0.6)  the bacteriophage  s o l u t i o n was added.  One hour a f t e r l y s i s had occurred a few drops o f c h l o r o f o r m  156  0.2 x 10  9  BC/mL  0.8 x 10  9  BC/mL  1.2 x 10  9  BC/mL  1.8x  9  BC/mL  10  2.4 x l O  9  BC/mL  ( BC = b a c t e r i a l colonies )  Phage t i t r e : 3 x 10 10 (P.F.U /mL)  Time (h)  F i g u r e IV.7  Growth c u r v e — , and bacteriophage l y s i s .... of K l e b s i e l l a K 6 0 b a c t e r i a .  157  were added i n order to prevent f u r t h e r b a c t e r i a l growth.Cent r i f u g a t i o n y i e l d e d a c l e a r phage s o l u t i o n which was  then  t i t r a t e d to determine the amount o f phage.Table I V . 3 . 1 shows the c o n c e n t r a t i o n s obtained a f t e r each s t e p . A volume o f bacteriophage  s o l u t i o n c o n t a i n i n g the amount T O  of v i r u s r e q u i r e d to degrade the p o l y s a c c h a r i d e ( 1 0 7 of p o l y s a c c h a r i d e ) was to one  g  concentrated by e v a p o r a t i o n i n vacuo  t h i r d , a n d d i a l y z e d a g a i n s t running tap water  for  2 d a y s . A f t e r c o n c e n t r a t i o n to h a l f the volume,these phage s o l u t i o n s were added to the p o l y s a c c h a r i d e s o l u t i o n s i n d i s t i l l e d water.The d e p o l y m e r i z a t i o n was 3 0 hours.Chloroform  ( 3 mL)  was  3 7 ° for  c a r r i e d out a t  added to a v o i d b a c t e r i a l growth.  I s o l a t i o n and p u r i f i c a t i o n of d e g r a d a t i o n products.When the d e p o l y m e r i z a t i o n was to a s m a l l volume ( 5 0 mL) ter  ( 1 L ) . T h i s procedure  over,the  s o l u t i o n was  and d i a l y z e d a g a i n s t d i s t i l l e d was  r e d i s s o l v e d i n HgO  was  added.The mixture was  and  t h i s procedure  was  obtained.The  was  ( 5 0 mL)  to dryness.  and I R - 1 2 0  resin  solution  p u r i f i e d oligomers were obtained i n t h i s  separated on B i o - G e l P-4  way.  of o l i g o s a c c h a r i -  shown i n Table I V . 3 . 2  A p o r t i o n o f the d e p o l y m e r i z a t i o n product  lected.  +  repeated u n t i l a c o l o r l e s s  des, volumes used,etc.are  .The  (H )  The  s t i r r e d , f i l t e r e d and f r e e z e - d r i e d  The amount o f p o l y s a c c h a r i d e d e g r a d e d . y i e l d  67  wa-  repeated three times.The three  d i a l y z a t e s were c o l l e c t e d and concentrated s o l i d was  concentrated  . (5  0 0  mg)  was  column u s i n g c o n d i t i o n s as i n page  e l u t i o n p r o f i l e was  obtained and f r a c t i o n s were c o l -  158  A n a l y s i s o f K60 d e g r a d a t i o n products. Two f r a c t i o n s were ( 3  collected:  0  0  nig) and P  2  (14-5 mg),the r e s t  corresponded  to h i g h e r oligomers. P  1  had an [ a] + 6 l ° ( c D  0.7,water)(calculated  + 6 2 ° ).For  n.m.r. data see Table I V . 3 . 3 a and I V . 3 . 3 b ( s i g n a l s and a s s i g n ment) and s p e c t r a N ° 2 5 and 2 6 . D e t e r m i n a t i o n o f the r e d u c i n g end and P.P. A sample o f P  1  (lOmg)  was d i s s o l v e d i n HgO ( 5 mL) and NaBH^ ( 1 5 mg) was added.After s t i r r i n g f o r 2 h , the excess sodium borohydride was decomposed as u s u a l . The d r i e d reduced  o l i g o s a c c h a r i d e was r e f l u x e d i n yfo  methanolic HC1 o v e r n i g h t . A f t e r n e u t r a l i z a t i o n w i t h Ag^CO^.and e v a p o r a t i o n o f 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 , t h e u r o n i c e s t e r s were reduced w i t h NaBH^ ( 1 5 mg) i n anhydrous methanol (5mL). H y d r o l y s i s was e f f e c t e d w i t h 2M TFA on a steam-bath f o r 3 h and  the excess TFA was removed by e v a p o r a t i o n s w i t h water.A  solution  ( 0 . 5 mL) o f 5 % hydroxylamine  was added and heated hydride  hydrochloride i n pyridine  on a steambath f o r 1 5 m i n u t e s . A c e t i c an-  ( 0 . 5 mL) was added t o the c o o l e d s o l u t i o n which was  then heated  f o r one hour on a steam-bath. G . l . c . o f the mix-  t u r e o f 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 was done on column ( c ) i s o t h e r m a l l y a t 210°.Results a r e shown i n Table  IV.3.4.  M e t h y l a t i o n a n a l y s i s ^ P^ (10 mg) was methylated procedure.Reduction  by the Hakomori  w i t h L i A l H ^ , h y d r o l y s i s , r e d u c t i o n and a c e t -  y l a t i o n gave a mixture  o f p a r t i a l l y methylated  a l d i t o l acetates  which was separated and i d e n t i f i e d by g . l . c . and g.l.c.-m.s. R e s u l t s are shown i n Table I V . 3 . 5 .  159  Enzvmic h y d r o l y s i s , a) B - g l u c o s i d a s e . P-j^ (40 mg) was d i s s o l v e d i n a c e t a t e b u f f e r (pH 5 . 3 ) and B - g l u c o s i d a s e (NBC)(8 mg) was added and the s o l u t i o n incubated a t 3 7 ° . S a m p l e s were taken a t d i f f e r e n t times  (up t o a week) but no glucose was d e t e c t e d by-  paper chromatography;the enzyme was however a c t i v e on e e l l o b i o s e . b) a - g l u c o s i d a s e . P^ ( 1 5 mg)was d i s s o l v e d i n water(5mL) and  a - g l u c o s i d a s e (Type I,from  Yeast,SIGMA Chem.Com.)(1 mg)  was added.Incubated a t 3 7 ° . S a m p l e s were taken a t d i f f e r e n t i n tervals  (up t o 5 days) b u t no g l u c o s e was d e t e c t e d by paper  chromatography:the enzyme was a c t i v e on maltose. P  2  had a n [ a l + 4 5 ° D  (c 0 . 6 , w a t e r c a l c u l a t e d  +56°).N.m.r.  s p e c t r o s c o p y data are shown i n T a b l e s I V . 3 - 3 a and I V . 3 . 3 b and spectrum NO.26. D e t e r m i n a t i o n  o f r e d u c i n g end and D.P.was done  as f o r P^ and the r e s u l t s are shown i n Table I V . 3 - 4 .  The r e -  s u l t s o f m e t h y l a t i o n a n a l y s i s (as f o r P^) a r e i n d i c a t e d i n Table I V . 3 - 5 • A n a l y s i s o f K46 d e g r a d a t i o n products. Two f r a c t i o n s were c o l l e c t e d from g e l s e p a r a t i o n P^ (190 mg) and Pg (150 mg),the r e s t corresponded P  1  to higher  had a n [ a ] + 6 9 ° D  oligomers.  (c 1 . 3 , water c a l c u l a t e d + 7 2 ° ) .N.m.r.  s p e c t r o s c o p y data are shown i n T a b l e s I V . 3 • 6 a spectra N o 2 7  and 28.Pg had[<*]  D  + o >  and I V . 3 - 6 b and  0 ° ( c0.5,water c a l c u l a t e d  + 6 4 ° ).N.m.r. s p e c t r a l data are shown i n T a b l e s I V . 3 . 6 a and I V . 3 . 6 b and spectrum  No29.  The d e t e r m i n a t i o n o f the r e d u c i n g end and D.P.of P^ and P^ was done by d i r e c t h y d r o l y s i s o f the reduded o l i g o s a c c h a -  160 ride.conversion ditol.Results  i n t o the  peracetylated  shown i n Table I V . 3 - 7 .  of the g . l . c . are  ation analysis  of  and  shown i n Table I V . 3 . 7 Enzvmic h y d r o l y s i s .  P^  Pg  (as f o r K60  ( 1 3 mg)  was  5.3)  was  l e f t a t room temperature and  ent  i n t e r v a l s (up to one  B-glucosidase  chromatography.  Methyl-  p r o d u c t s ) gave r e s u l t s  .  (pH  and  a l d o n o n i t r i l e s and a l -  (NBC,  dissolved 3 m g ) was  i n acetate  buffer  added.The s o l u t i o n  samples were taken a t  week).No glucose was  differ-  d e t e c t e d by  paper  161  V .  STRUCTURAL STUDIES OF THE GUM OF CHORISIA SPECIOSA  EXUDATE  162  V.-  STRUCTURAL STUDIES OF THE  GUM  EXUDATE OF CHORISIA  SPECIOSA. V.1  Abstract. The  p u r i f i e d gum  racho ) was  exudate from C h o r i s i a s p e c i o s a  s t u d i e d . l t c o n t a i n s L-arabinose  D-mannose (1),D-galactose  (9),D-glucuronic  of D-xylose.The r e s u l t s from m e t h y l a t i o n t i o n and  acid  (3) and  traces  a n a l y s i s , 8-elimina -  p a r t i a l h y d r o l y s i s make p o s s i b l e a t e n t a t i v e a s s i g n polysaccharide.  Introduction. The  and  bor-  ( 1),L-rhamnose (2),  ment f o r the " average s t r u c t u r e " of the gum  V.2  (Palo  exudate gums from the bark and  shrubs may  fruit  of many t r e e s  be produced f r e q u e n t l y a t s i t e s of i n j u r y to  the p l a n t or even spontaneously.Many of these gums have found commercial a p p l i c a t i o n s such as, gum and  other A c a c i a gums, tragacanth  ( S t e r c u l i a urens),etc.Chemical gum  exudates has been c a r r i e d  a r a b i c ( A c a c i a Senegal)  ( A s t r a l g u s sp.)  examination of  .karaya  gum  polysaccharide  out f o r many years.Much of the  present knowledge of the chemical  composition  of p l a n t gums 189  can be found i n monographs by, G.0.Aspinall  1 9 0  ,  and  F.Smith and R.Montgomery  G.O.Aspinall  t u r e s of the p o l y s a c c h a r i d e  and  A.M.Stephen  .The s t r u c -  components of gums are a l l h i g h l y  complex.In s p i t e of the f a c t t h a t newer a n a l y t i c a l have been d e v e l o p e d , i t i s s t i l l v e r y d i f f i c u l t d e t a i l e d s t r u c t u r e of these  191  ,  techniques  to unravel  the  polysaccharides.  C e r t a i n s t r u c t u r a l p a t t e r n s have been  shown  to p r e v a i l .  163 Stephen based on t h i s a c l a s s i f i c a t i o n o f the gum exudates. Three types were p r o p o s e d  1 9 2  and each can be d e s c r i b e d  briefly  i n the f o l l o w i n g way: Type A , c o n s i s t i n g o f a h i g h l y branched core o f D-galactopyranos y l residues,mutually  j o i n e d through  with residues of D-glucuronic ose attached  B 1-3 and B 1-6 l i n k a g e s ,  acid.L-rhamnose and/or L - a r a b i n -  t o i t ; Type B , c o n s i s t i n g o f an i n t e r i o r c h a i n of  D-galacturonic  a c i d and L-rhamnose r e s i d u e s i n v a r y i n g r e l a t i v e  p r o p o r t i o n s and arrangments,with some other sugars present i n outer chains;and  Type C , c o n s i s t i n g o f a c h a i n o f D-xylose r e -  s i d u e s w i t h branches o f L-arabinose,D-xylose  and D-glucuronic  acid residues. Aspinall classified ars forming  the gum exudates depending on the sug-  the i n t e r i o r chains o f the p o l y s a c c h a r i d e s  into,  I) the g a l a c t a n , I I ) the glucuronomannan, I I I ) the g a l a c t u r o norhamnan, IV) the x y l a n and other minor groups. Group I I ) , as w e l l as group I ) , i s i n c l u d e d i n the Type A o f Stephen's c l a s s i f i c a t i o n , the reason b e i n g t h a t although i n t e r i o r c h a i n o f a l t e r n a t i n g D-glucuronic residues,the  these gums have an a c i d and D-mannose  outer chains are from the g a l a c t a n  type.Several  examples are found among the gums from Prunus sp. The  s t r u c t u r e o f the gum exudates has been s t u d i e d f o r  t h e i r p o t e n t i a l use i n chemical  taxonomy o f p l a n t s  c o n c l u s i o n s up t o the moment are t h a t although  1 9 2  , b u t , the  i t i s not so  c l e a r a t the l e v e l o f o r d e r s , w i t h i n a genus the d i f f e r e n t  spe-  c i e s have gums o f the same type w i t h many s t r u c t u r a l f e a t u r e s i n common but n o t completely  identical  (e.g. A c a c i a gums).  164 C h o r i s i a s p e c i o s a S t . H i l . i s a l a r g e t r e e from the genus 193 Bombacaceae o r i g i n a l l y from T r o p i c a l South America -\ I t has 194 7  t y p i c a l b u l g i n g trunks a l l o w i n g f o r water storage  y  .When the  trunk s u f f e r s i n j u r y , a gum exudes.apparently  t o h e a l wounds.  Although  1 9 5  the l e a f mucilage has been a n a l y z e d  ,no  information  i s a v a i l a b l e about the gum.Due t o the r e l a t i o n s h i p o f Bombacaceae t o S t e r c u l i a c e a e , as they both belong  t o the order o f the  M a l v a l e s . i t i s therefore o f i n t e r e s t to analyze  the composi -  t i o n o f t h i s gum. V.3 R e s u l t s and d i s c u s s i o n . The  gum p o l y s a c c h a r i d e from C h o r i s i a s p e c i o s a S t . H i l . ,  a f t e r p u r i f i c a t i o n by p r e c i p i t a t i o n w i t h e t h a n o l had[ a ] + l 8 ° . D  By g e l chromatography i t was shown t o c o n s i s t o f two f r a c t i o n s a) 1 . 0 5 * IC- d a l t o n s 5  (80#) and b) 4 x 1 0 ^ d a l t o n s  l y s i s w i t h 2M TFA y i e l d e d L-arabinose  (0. 9)*L-rhamnose  D-mannose (1.0),D-galactose  D-glucuronic  (?.8),and  Hydro-  (20#).  (1.8),  a c i d (2.8)  i n the p r o p o r t i o n s shown,together w i t h t r a c e s o f x y l o s e . M e t h y l a t i o n a n a l y s i s o f the gum gave the r e s u l t s shown i n Table V.l,column I. P a r t i a l h y d r o l y s i s o f the gum y i e l d e d monosaccharides, n e u t r a l and a c i d i c o l i g o s a c c h a r i d e s ( N^.Ng.N^ and A-^.Ag r e s p e c t i v e l y ) and a polymeric m a t e r i a l (P^).The monosaccharides r e l e a s e d were arabinose.rhamnose and galactose.The  oligosac-  c h a r i d e s were i s o l a t e d by p r e p a r a t i v e paper chromatography a f t e r s e p a r a t i o n i n t o a c i d i c and n e u t r a l components by ion-exchange chromatography.The r e s u l t s o f the analyses  on each  TABLE V . l METHYLATION ANALYSIS OF CHORISIA SPECIOSA GUM EXUDATE AND DERIVED PRODUCTS • d Mole % R e l a t i v e r e t e n t i o n times Methylated sugars IV III II £ SP-1000 2,3.^  -Rha  0.4?  0.46  10. 9  4.3  2,3.4  -Ara  0.57  0.59  5.4  6.0  2,3+3.4 2,3  3-6  0.84  -Rha -Ara  2,3.4,6-Gal  V  0.95  1.00  2.8  1.2  1.00  1.00  4.8  34.0  4.1  —  40.9  42.6  35.0  —  —  3.^.6  -Man  1.32  1.44  2,4,6  -Man  1.33  1.63  —  2,4,6  -Gal  1.39  1.69  7.8  12.5  1.2  31.5  33-3  2,3.4  -Glc  1.49  2.10  5.2  —  8.8  —  ——  2,3,4  -Gal  1.93  2.10  28.8  5-2  —  —  4,6  -Man  1.93  2.27  —  2.0  2,3  -Glc  2.08  2.98  13.6  —  2,4  -Gal  2.20  3.56  10.0  9-2  2  -Gal  2.44  4.00  2.5  4  -Gal  2.57  4.24  2.3  as i n Table IV. 1 . 2  hydrolysis.  1  5-9  —  37.8  . - Programmed a t 180° f o r 4 min,then a t 2 % i i n  i s o t h e r m a l f o r 3 2 min . - I , o r i g i n a l gum exudate; I I I , m e t h y l a t i o n of P  21.8  4.0  I I , p r o d u c t from  7.4  18.5 —  to 2 3 0 ° ,  s -elimination;  ;IV,product A from Smith h y d r o l y s i s ; V , p r o d u c t B from Smith  22. 8  8.9 —  166  oligosaccharide  are g i v e n  i n Table V.2,and i n d i c a t e d the  fol-  lowing s t r u c t u r e s : Gal  l  N  N  N  1  3  Gal  s 1 6 Gal Gal  2  Gal  3  I  1  6  3  B  Gal  and A A  GlcA -y -  Gal  GlcA ^ y -  Gal  1  1  6  6  £  No mannose was i n t e g r a l p a r t of the  detected  e i t h e r as the  ( d e r i v e d from the g l u c u r o n i c  confirmed by  of two  the  i s o l a t i o n and  a c i d i c oligosaccharides  They were i d e n t i f i e d A~ 3  A., 4  The  a c i d and  2,3-di-0re-  i n f a c t com-  mannose  residues.This  characterization  (TableV.3)  from p a r t i a l h y d r o l y s i s of  P^.  as GlcA - — -  Man  B  GlcA ^ — B  Man  -—-  GlcA  1  —-  a  r e s u l t s from m e t h y l a t i o n a n a l y s i s and  l y s i s suggested the  analysis  acid).These  s u l t s suggested t h a t the polymeric m a t e r i a l was posed of a l t e r n a t i n g g l u c u r o n i c  gluc-  carboxyl-reduced  P^ i n d i c a t e d mainly 3,4,6-tri-0-methylmannose and  was  (s)  a n a l y s i s gave mannose.galactose and  V.l,column I I I ) on the methylated and  methylglucose  Hl-n.m.r.  a t 6 5.40  and  a c i d i n a r a t i o of 3 - 9 s 1 . 0 : 4 . 1 .Methylation  uronic (Table  (J]_ 2 ®^z)  an  i t s presence  had[a ]^+20°,while the  spectrum showed s i g n a l s a t 6 4 . 5 3 ratio.Sugar  f r e e sugar or as  oligosaccharides,suggesting  i n the polymeric m a t e r i a l .  in a 1:1  Gal  Man  B  p a r t i a l hydro-  following s t r u c t u r a l features  of the  gum:  TABLE V . 2 ANALYSIS OF THE OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF THE GUM EXUDATE. Oligosaccharide  [a ] D (water) +13°  -3.0'  -36°  -8°  N-  + 40°  Sugar a n a l y s i s (as  Methylation analysis  alditol  (as a l d i t o l  acetates)  Glc(GlcA)  (1.0)  2,3.4  -  Glc  Gal  (1.0)  2,3,4  -  Gal ( 0 . 5 )  2,3.5  -  Gal ( 0 . 3 ) Glc  Glc(GlcA)  (1.0)  2,3,4  -  Gal  (2.0)  2,3.4  -  + 14  Glc(GlcA)  (1.0)  2,3.4  -  Glc  Man  (1.0)  3,4,6  -  Man ( 0 . 9 )  Glc(GlcA)  (1.0)  2,3,4  -  Glc  Man  (1.0)  3,4,6  -  Man ( 1 . 9 )  2,3  -  Glc  Gal  Gal  Gal  (1.0)  (1.0)  (0.9)  - Gal(l.O)  2,3,4,6 -  Gal ( 0 . 9 ) - Gal(l.O)  2,3.4,6  2,3.5 + 22  (1.0)  2,3,5  2,3,4  N,  (1.0)  Gal ( 1 . 5 ) - Gal ( 0 . 3 )  2,4,6  N,  acetates)  Gal ( 0 . 5 ) - Gal ( 0 . 3 )  -  2,3,4,6  - Gal(l.O)  2,4,6  -  Gal ( 1 . 0 )  2,3,4  -  Gal  (0.8)  168 a) a backbone of a l t e r n a t i n g g l u c u r o n i c a c i d and mannose r e sidues l i n k e d i n the f o l l o w i n g manner, 4 1 ? — - GlcA ±-r^- Man 6  b) 6S%  1 4 -—a  1 ? GlcA ^ B  Man  1  -— a  (approx.) of the mannosyl r e s i d u e s from branch p o i n t s  a t p o s i t i o n 0-3  ,  c) the branches c o n s i s t of a g a l a c t a n framework of mainly 3 (1-6)  l i n k e d g a l a c t o s e r e s i d u e s which may  a t p o s i t i o n 0-3  a l s o be s u b s i t u t e d  ,  d) the main t e r m i n a l non-reducing sugar i s L-rhamnose. The  permethylated  gum  was  subjected to a 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 gave the r e s u l t s shown i n Table V . l , column II,from which,the f o l l o w i n g c o n c l u s i o n s may  be drawn:  a) the m a j o r i t y of the L-rhamnose r e s i d u e s are l i n k e d to o f g l u c u r o n i c a c i d and  0-4  are thus degraded d u r i n g the r e a c t i o n  w i t h base, b) the i n c r e a s e i n the amount of 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g a l a c tose i s due tached  to the d e g r a d a t i o n  to p o s i t i o n 0-6  c) the disappearance  of u n i t s o f g l u c u r o n i c a c i d a t -  of the g a l a c t o s e , of 4,6-di-0-methylmannose and  appearance  o f 2,4,6-tri-0-methylmannose i s i n accordance w i t h the s t r u c ture of the backbone.The s m a l l amount of trimethylmannose found  i n the h y d r o l y z a t e  i s due  to incomplete  degradation  with  base. The  gum  consumed 9.8  i d a t i o n w i t h 0.1M  mmoles of NalO^ per gram ,upon  NalO,, s o l u t i o n , a s expected  ox-  from the methyl-  169 a t i o n r e s u l t s . S m i t h h y d r o l y s i s o f the p o l y o l and p r e c i p i t a t i o n w i t h e t h a n o l y i e l d e d a product  (A) which upon m e t h y l a t i o n and  m e t h y l a t i o n a n a l y s i s gave the r e s u l t s shown i n Table umn IV. The presence  V.l,col-  o f l a r g e amounts o f 4,6-di-0-methylman-  nose was e x p l a i n e d by the r e s i s t a n c e t o h y d r o l y s i s o f the r e sidue o f the o x i d i z e d g l u c u r o n i c a c i d ^ . T r e a t m e n t 19  0.1M  of A with  TFA on a steam-bath f o r an hour y i e l d e d a product  (B)which  upon m e t h y l a t i o n and m e t h y l a t i o n a n a l y s i s (Table V.l,column V) showed an i n c r e a s e i n the p r o p o r t i o n of 2,4,6-tri-O-methylmannose compared to 4,6-di-0-methylmannose.The r e s u l t s o f the Smith h y d r o l y s i s i n d i c a t e d t h a t p e r i o d a t e r e s i s t a n t g a l a c t o s e r e s i d u e s a r e a t t a c h e d d i r e c t l y t o the backbone a t 0-3 o f the mannose r e s i d u e s . The age  a n a l y s i s o f C h o r i s i a s p e c i o s a gum i n d i c a t e s an "aver-  s t r u c t u r e " t h a t can be r e p r e s e n t e d as i n F i g u r e V.1 .This  p o s s i b l e s t r u c t u r e s a t i s f i e s the a n a l y t i c a l evidence,but r e presents only one o f the many t h a t have the same c h a r a c t e r i s t i c . A l l must c o n t a i n the backbone o f D - g l u c u r o n i c a c i d and D-  4 mannose,and the s i d e chains ended i n Rha some arabinose  1 6 GlcA  Gal — ,  or g a l a c t o s e fragments.The s i d e chains  offer  many a l t e r n a t i v e s . There i s a c l e a r d i f f e r e n c e w i t h the r e l a t e d  Sterculia ^ 1 9  gum.Those gums have D - g a l a c t u r o n i c acid,rhamnose and g a l a c t o s e i n the c e n t r a l backbone,and i n them,D-glucuronic a c i d may be present as a minor component. I t would be i n t e r e s t i n g to extend  the present study t o  other C h o r i s i a s p e c i e s , a s w e l l as to other members of the Bom-  TABLE V.3 N.M.R. ( H) DATA OF ACIDIC OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF THE GUM. 1  j.  Compound  Integral  Assignment  Spectrum  proton  5.27  2  0.4  6- • G a l — -OH  B  4.61  8  6- • G a l — -OH  l  4.52  8  0.6 1.0  5.25 4.87 4.52  2  0.4  6--Gal „ -OH  7  0.6 1.0  6--Gal  1.0  6--Gal  G l c A — ^ G a l - 'OH 1  A  No.  G l c A ^ - ^ G a l - —^Gal-OH 1  B  A  B  2  G l c A - — ^ a n - OH B  A  3  GlcA-^-^ManB  A  4  H  ^GlcA-^-^-Man-OH a  B  30  GlcA-g  -OH  g  GlcA—g—  4.42  7 8  5.30  s  0.8  2--Man  4.99  s  2--Man  B  -OH a  4.55  8  0.2 1.0  5.40  s  1.0  -Man— 2-  5.29 4.98  s  2 - M a n — -OH  s  0.8 0.2  4.53 4.49  8 8  1.0 1.0  GlcA—  -OH  B  GlcA—  31  I  a  a  2 -Man—— -OH B  4 -GlcA-  3~  1  32  171  ^GA-— 4M— 2  GA: g l u c u r o n i c a c i d  R : rhamnose  M : mannose  A i arabinose  G : galactose  F i g u r e V.1  One o f the p o s s i b l e "average s t r u c t u r e s " f o r the gum o f C h o r i s i a s p e c i o s a ,  (Some o f the c o n f i g u -  r a t i o n s o f the g l y c o s i d i c l i n k a g e s are t e n t a t i v e ) .  172 bacaceae,to  f u r t h e r e s t a b l i s h the chemical r e l a t i o n s h i p of  these p l a n t s . V.4  Experimental. G e n e r a l methods.The a n a l y t i c a l techniques and  instrumen-  t a t i o n used i n t h i s study have been a l r e a d y d e s c r i b e d i n S e c t i o n III. P u r i f i c a t i o n of the gum. indicated i n Section I I I . 7 . 2 .  The gum  ( 5 . 0 g) was  (Yield 3 . 4 g).  M o l e c u l a r weight determination.A sample was gel-permeation chromatography and was f r a c t i o n s , one of 5f w  FL  1 . 0 5 x l 0  t r e a t e d as  5  shown to c o n s i s t of  daltons  w 4 X IC/* daltons (20%) .  analyzed by  (80%)  H y d r o l y s i s of the gum.A sample of the gum  two  and the other of  ( 3 0 mg)  was  heated w i t h 2M TFA on a steam-bath.After 4 h o f h y d r o l y s i s a sample was was  taken and a f t e r removal  of the a c i d , t h e h y d r o l y z a t e  examined by paper chromatography i n s o l v e n t (A).Rhamnose,  a r a b i n o s e . g a l a c t o s e and s e v e r a l o l i g o s a c c h a r i d e s were d e t e c t e d . A f t e r 4 8 h hydrolysis.rhamnose.arabinose,mannose,galactose g l u c u r o n i c a c i d were d e t e c t e d on p.c.  and  (solvent (A)).Conversion  of the n e u t r a l sugars i n t o a l d i t o l a c e t a t e s and g . l . c . gave rhamnose,arabinose,mannose and.galactose i n the r a t i o of 1.9:0.9:1.0:6.7  .Sugar a n a l y s i s , a s d e s c r i b e d before (see S e c t i o n  I I I . 8 ) , o n the gum  showed rhamnose.arabinose,mannose.galactose  and glucose i n the r a t i o o f 1 . 8 : 0 . 9 : 1 . 0 : 7 . 8 : 2 . 8 . M e t h y l a t i o n a n a l y s i s . T h e gum the Hakomori procedure  ( 1 5 0 mg)  was  methylated  f o l l o w e d by a s i n g l e Purdie  by  treatment  173  ( see S e c t i o n I I I . 9). The a b s o r p t i o n i n the i . r .  product  (125 nig) showed no  spectrum.Methylation  m a t e r i a l gave the p r o p o r t i o n of methylated Table V.l,column  hydroxyl  a n a l y s i s of t h i s sugars shown i n  I.  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 . P a r t of the permethylated  gum  ( 5 ° mg)  was  s u b j e c t e d to 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 as i n S e c t i o n III.10 . H y d r o l y s i s of the product and m e t h y l a t i o n a n a l y s i s of the sugars r e l e a s e d gave the r e s u l t s shown i n Table V.l,column P a r t i a l h y d r o l y s i s . The gum 1M TFA  II . (450  mg)  was  on steam-bath f o r one hour.The a c i d was  v a p o r a t i o n w i t h water and (220 mg)  hydrolyzed with removed by  the product separated i n t o  and n e u t r a l (160 mg)  e-  acidic  components on a column of B i o -  Rad AG1-X2. Two  f r a c t i o n s , A^  (40 mg)  and A  2  (25 mg)  were i s o l a t e d  by paper chromatography from the a c i d i c f r a c t i o n . R e s u l t s of the a n a l y s i s o f these a c i d i c o l i g o s a c c h a r i d e s are g i v e n i n Table V.2.  The  "Hi-n.m.r.  a t 65.27 ( 0 . 4 H , J  1  2  2HZ),  spectrum  6 4.61  ( l . O H . J ^ 8 H z ) , while t h a t of A 2  (0.4H,J and  1>2  6 4.42  A^ was  2Hz),  6 4.87  (1.0H,J  1  2  ( O ^ H . J ^ 8 H z ) and 6 2  showed s i g n a l s a t 6  2  (0.6H,J  8Hz).  o f A^ showed s i g n a l s  6  lj2  7 H z ) , 6 4.52  (see Table V . 3  4.52 5-25  (1.0H,J  lf2  7Hz)  and Spectrum N°30).  i d e n t i f i e d as 6-0-( R -D-glucopyranosyl u r o n i c a c i d ) - D -  g a l a c t o s e and  the i d e n t i t y confirmed by co-chromatography w i t h  an a u t h e n t i c sample. A  2  was  i d e n t i f i e d as 6-0-( g-D-glucopyran-  o s y l uronic a c i d ) - 6 - 0 - ( 8 -D-galactopyranosyl)-D-galactose.  174  ( 1 0 mg),Ng ( 1 5 mg) and  Three n e u t r a l o l i g o s a c c h a r i d e s ,  ( 5 mg) were i s o l a t e d by p r e p a r a t i v e paper chromatography from the n e u t r a l f r a c t i o n . A n a l y s i s from these i n Table  V . 2 , i n d i c a t e d the s t r u c t u r e s shown belows  N-^ 3 - 0 - ( 3 N  2  oligosaccharides,given  6-0-(  -D-galactopyranosyl)-D-galactose, B-D-galactopyranosyl)-D-galactose,  N^ 3-0-( B-D-galactopyranosyl)-6-0-(  g  -D-galactopyranosyl)-D-  galactose. A sample o f the gum  ( 2 5 0 mg) was t r e a t e d w i t h  I M TFA on  a steam-bath f o r 1-f h.The a c i d was removed and the r e s i d u e d i s s o l v e d i n water ( 1 0 mL) and d i a l y z e d f o r 7 2 h a g a i n s t l e d water (l.OL).The +20°  non-dialyzable  6 4 . 5 3 (J^  2  distil-  m a t e r i a l ( 5 0 mg) h a d [ a ] ^  (c 1 . 7 » w a t e r ) and the "hi-n.m.r. spectrum i n DgO  s i g n a l s a t 6 5 - 4 0 and  was  recorded  ^Hz) i n a l s l r a t i o . S u g a r  a n a l y s i s gave mannose,galactose and g l u c u r o n i c a c i d i n a r a t i o of 3 - 9 s 1 . 0 : 4 . 1 . M e t h y l a t i o n Table  a n a l y s i s gave the r e s u l t s shown i n  V.l,column I I I . H y d r o l y s i s o f t h i s m a t e r i a l ( 2 0 mg)  with  2M TFA on a steam-bath f o r 7 h showed by p.c. ( s o l v e n t ( A ) ) , galactose,mannose.glucuronic a c i d , t h e a l d o b i o u r o n i c a c i d A^ , the a l d o b i o u r o n i c a c i d A^ and other h i g h e r  oligomers.A^ ( 6 mg)  was i s o l a t e d by paper chromatography and analyzed Table  V . 2 . The ^H-n.m.r. spectrum (see Table  as shown i n  V . 3 and spectrum  N ° 3 D showed s i g n a l s a t 6 5 . 3 0 ( 0 . 8 H , s ) , <S 4 . 99 ( 0 . 2 H , s ) and 6 4.55  (I.OH.JJL  2  8 H z ) . A ^ was i d e n t i f i e d as 2 - 0 - ( 6 -D-gluco-  p y r a n o s y l u r o n i c acid)-D-mannose.Another  oligosaccharide,A^  ( 5 mg) was i s o l a t e d by paper chromatography from the hydro-  175  l y z a t e and analyzed as shown i n Table V.2. The H-n.m.r. 1  spec-  trum (see Table V . 3 and spectrum N0.32) showed s i g n a l s a t 6 5.40 ( l . O H . s ) , 6 5 . 2 9 ( 0 . 8 H . s ) , s 4 . 9 8 (0.2H,s), (l.OH.J^  2  8Hz) and <5 4 . 4 9 ( 1 . 0 H , J  1 2  5 4.53  8Hz).A^ was hydrolyzed  w i t h 2M TFA f o r 4 h and p.c. o f the h y d r o l y z a t e showed the a l d o b i o u r o n i c a c i d Aj,mannose  and g l u c u r o n i c a c i d . T h i s oligomer was  i d e n t i f i e d as the dimer o f A^. P e r i o d a t e o x i d a t i o n . A s o l u t i o n o f the gum (200 mg) i n  H0 2  (100 mL) was t r e a t e d w i t h 0.1M NalO^ (100 mL) f o r 9 6 h a t 4 ° i n the dark.The p e r i o d a t e consumption was f o l l o w e d by the F l e u r y Lange method.The f i n a l consumption o f p e r i o d a t e was 9 . 8 mmoles per gram o f the gum.Ethyleneglycol  (10 mL) was added,the p o l y -  aldehyde was d i a l y z e d overnight,reduced  w i t h NaBH^ (1.0 g ) ,  n e u t r a l i z e d w i t h 5 ° % a c e t i c a c i d , d i a l y z e d and then f r e e z e d r i e d t o y i e l d the p o l y a l c o h o l (120 mg).This product  ( 5 mg) was  h y d r o l y z e d w i t h 2M TFA on a steam-bath o v e r n i g h t and the sugars present i n the h y d r o l y z a t e found  (detected by p.c.  i n solvent(A))were  to be g a l a c t o s e and mannose.Quantitation (by g . l . c . ) o f  the sugars as a l d i t o l a c e t a t e s gave g a l a c t i t o l and m a n n i t o l i n the r a t i o o f 2.8:1.0 . Smith h y d r o l y s i s o f the p o l y a l c o h o l (100 mg) was c a r r i e d out w i t h 0 . 7 5 M TFA d u r i n g 20 h a t room temperature.The a c i d was removed by e v a p o r a t i o n and the r e s i d u e a f t e r i n water ( 3 mL) was p r e c i p i t a t e d w i t h e t h a n o l  dissolution  ( Y i e l d o f A,  2 5 mg).Methylation on p a r t o f t h i s m a t e r i a l ( 5 mg) gave the r e s u l t s shown i n Table V.l,column IV .The r e s t o f the p r e c i pitate  (20 mg) was heated w i t h 0.1M TFA on a steam-bath f o r  1  176  one h o u r . A f t e r removal of the a c i d by e v a p o r a t i o n the r e s i d u e (B) was  methylated  by the Hakomori procedure  and a f t e r hydro-  l y s i s . r e d u c t i o n and a c e t y l a t i o n gave the r e s u l t s shown i n Table V.l,column V.  177  VI.  BIBLIOGRAPHY  178  BIBLIOGRAPHY 1.  " Surface Carbohydrates Sutherland).Academic  2.  J.A.  of the P r o k a r y o t i c C e l l  " (ed.I.  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Uronic a c i d i n c h a i n , a) l i n e a r . _ X -  0 - 0 -  K l , K5,  K63  - X - 0 - 0 - 0 -  K4,  _ x  K6  0 - 0  K9*,  _ X - 0 - 0 - 0 - 0 - 0 K70,  -  K81  -  K44  192  b) branch p o i n t on u r o n i c  acid  i ) s i n g l e u n i t side chain _ X - 0 - 0 -  - X - 0 - 0 - 0 -  I  I  0  0  Kll, ii)  K5?  K21, K24  two u n i t s i d e c h a i n  - X - 0 - 0 -  - X - 0 - 0 - 0 -  1  I  0  0  1  I  0  0  K31 i i i ) three u n i t s i d e c h a i n  K46  - X - 0 - 0 - 0 -  I 0  I  0  K26  I  0 i v ) p l u s branch p o i n t s on n e u t r a l sugars X - 0 - 0 - 0 -  I  |  |  0  0  0  K60  c) branch not on u r o n i c _ X - 0 - 0 -  I  0 K58  acid  - x - O - O - O -  - X - 0  I  I  0 K7, K61, K62  0 K52,  193 • - x - 0 - 0 -  •.  - X - 0 - 0 - 0 -  I  I  0  0  K16, K54 d)  double  Kl?  branch  not  on  uronic  acid  0 - X - 0 - 0 - 0 -  I  0 Uronic a)  acid  single  i n  side  chain  unit  side  chain  _ 0 - 0 - 0 -  I  I  X  X  K2, b)  _ o - 0 - 0 - 0 -  two  K8 single  K9t unit  side  K59  chain  - 0 - 0 - 0 - 0 - 0 -  I  I  X  0  K  c)  two  single  1  . 0 - 0 - 0 -  location  chains  r , j  units  0  exact  side  chain  forming  a  not  I  - 0 - 0 - 0 - 0 -  I  I  X  X  K30.K33  K27  side  determined  double  0  of  branch  194  d) two u n i t s i d e c h a i n i)  uronic acid terminal - 0 - 0 -  I K20, K23, K51. K55  0  X  i i ) uronic a c i d non-terminal - 0 - 0 -  - 0 - 0 - 0 -  I  - 0 - 0 - 0 - 0 -  I  X  X  I  0 K25, K47  I X  I  I  0 K13. K74  0 K12, K28, K36  e) three u n i t s i d e c h a i n i ) uronic a c i d non-terminal - 0 - 0 - 0 -  - 0 - 0 - 0 - 0 -  I  I  0  X  1 X  I  I 0  I  0  0  K18  K4l  195  APPENDIX I I THE STRUCTURES OF KLEBSIELLA CAPSULAR POLYSACCHARIDES  196  Structure  K-type Kl  1 man— 3  K2  a  1 G].cA K4  -2G1C—^GlcA-—^Man^—2(ji I_ c  P  K5  -AucA 1  4  2  A  V  1  1  B  OAc  K6  A  -2-Fuc-—^Glc-—^anl—^1 A— C  a  B  B  a  Gal ll  K7  P  4 6—2<jlcA-—^Mar^—^an^—^Glc-—^Glc—a  GlcA 11  K8  -^Glc-—^Gal-—^Gal— B  B  a  197 GlcA  K9  -2ca  K9* "  -^GlcA —^Rha —^Gal —^Rha —^Rha -  1-—2-Rha-——^Rha— 1  p  <4  1  G  a  1  1  l  ^ II Kll  -Ailc—-SlcA^—halGal 1  P  4 GlcA 1 a  K12 K13  3  -Ailc-—2 l—2-Gal-^—^alf— •<Jalf— Rha  a  a  a  -^Glc-—^Man —^-Glc— 3 3 1  II  GlcA 4|  P C 3 Gal Gal 1  K16  1 4 1 4 1 c^—^GlcA^—^Fuc—  1  198 ha  K17  J ^  K18  ^al —-^Glc —2Rha—  l  c  A  l _ ^  R  h  l ^ G l  a  a  p  1  c  1 ^ 2 •Rha— a  i  ll  Rha 2  ll  GlcA  ll  Glc GlcA 1 3 Gal  + OAc  11  2 -=Man' 3  K20  P-Cl G a l * 1  -4IcA-—Aflanl—^an^—hal— 4  K21  a  <x> 1  6 Glc 11 4 K22  -3d  a  a  0  B  199  K23  'Rha-—HJlc2  ll  Glc 6 e l GlcA Man 11  K24  Man-  -^GlcA-  -^Man-—2Glc-  Glc 1 2 GlcA 11  K25  P C £ Gal ^ 1 4 Glc  11  6 Glc 1 4  K26  1  -AncA^  2l« l an  £war>i 2. kJalMan^  200 Glc  11  P  A.  K27  Gal-  kSal  1 GlcA -^al-—^anl—^an-  K28  1  —  W  il GlcA 31 Glc  P C S  K30  -  Gal  —%an—  OAc /i  l  6  —Glc—  ll GlcA  P C S  G  L  C  2 Man 1 GlcA-1—1 Gal-  K31  P  K32  - 2 G al-  A  ^Rha 1  k Rha :  201  P C S  K33  Gal  ^ 11  OAc  -^Vlan—^Vlan^—^Glc—  ll  GlcA Rha  11  K34  -^Rha-—-Rha-—Ajicl—2^ 1 — ^ R h a —  K36  -\al-—2 l—2-Rha —^Rha—  a1 A  1  Rha  s  2  ll  GlcA 4|  P C S  Glc  ® 1  6  6 Glc 1  a  K37  -4 a l l - W -  202  (X) 2  K38  ^  l  c  l ^  a  l  l _ 4  a  l  l _  ll Glc  Glc 1 6 G 1 41 GlcA  K4l  -^Glc^—iRha^—*Gal^-^Galf^-  K44  -^lcA^-^Rha^-2-Rha^—-2G1C^-^G1C^-  a  a  8  a  B  GlcA  11  K45  Rha-—-Rha-—2. . Rha  Glc 1 3' Man  K46  ;"^P  -^Gal-—2(j il—^GlcA^—^Mana  a  + 0AC  203  •Rha—  K47  a  ll GlcA  1 Rha K48  'Rha-1  3QJ^^1  _1 =Glc-  3T»I  2TM—1  Rha-  a  a  ll GalA GalA 11 %an  K49  + 0Ac  x  ;  Gal—  a  a  GlcA 1 a  6 Glc 1 a  K51 K52  a  Gal—  a  1 1 2 -^Gal-—-Rha' T  I*  GlcA-  Ajal-—-Rha—  ll  Gal Rha  11  K53  - 3 Q IcA  1  2 Man-  1 2 1 Gal-—-Rha—  204  Glc  + 0F  11 K54  -  K55  4  + 0Ac 1 4 lc^-r^GlcA-  -^Fuci-  1  Auc^-r^Rha— +0Ac  ll Gal 3 1 GlcA  P K56  ,4 6-  Gal-^ G a l ^  Gal-  11 Rha Man  11 GalA——^an -  K57  1  a  K58  •Auc 1  *G l c A 1 P  a  1 4 F u c —a 3 a  1 Gal  GlcA  11 K59 -  -^Glc  1 _ 1 Gal^  +0Ac ^ a n  1  1 - ^ Mana  a  205  Glc 1  -2G1C^—^GlcA^—^Gal-—2jvi l_ a n  2  2  a  8  6  1  1  Glc  Glc  a  Gal 11 1  2;  -^lcA^-^Man^—'Glc^—*Glc8  a  8  a  -^GlcA^—harr—2Gal^-^Glc—  ll  Man  +0Ac + 0F  —2<jalA——^Fuc-—2<j l_ al  Rha 11 -A}lcA-—^anl—2-Glc^—^Man— a  a  8  £  zr 1  PC£  G L C  -\lcA^—-Rha^—2-Rha^—2-Glc-—^Gal^&  —Rha  GlcA  a  Rha  a  Rha  Glc  a  Rha  ^Glc—  206  K72  A.  -^Glc^-r^ha-—-Rha-—2  R h a  l_  P C £ Gal 1 4!  GlcA  K?4 K81 K83  1 2  -*Gal^-  ct  1  a  •^Rha —^Rha^-^lcA^^Rha --2Rha --^Gal 1  1  -^Gal-—-Rha—  ll  Gal 3  1  GlcA  1  1  20?  The  other p o s s i b l e s t r u c t u r e i s :  -^-GlcA —-Rha-—-Rha-—Ajail—lRha— 1  P  a  a  a  a  T h i s s t r u c t u r e i s l i k e K33 except OAc i s present  every  other r e p e a t i n g u n i t . T h i s s t r u c t u r e has been re-examined by E.H. M e r r i f i e l d ; Formate l o c a t e d on p o s i t i o n 4 o f the t e r m i n a l and  Glucose,  acetate on p o s i t i o n 2 o f Fucose.  Acetate  on p o s i t i o n 2 o f Rhamnose.  Acetate  on p o s i t i o n 6 o f Mannose,but not on a l l r e s i d u e s .  Acetate  l o c a t e d on p o s i t i o n 2 o f G a l a c t u r o n i c  acid,and  Formate on p o s i t i o n 4 o f the same sugar r e s i d u e .  Pyruvate on every other r e p e a t i n g u n i t .  Tentative structure.  208 APPENDIX I I  BIBLIOGRAPHY  Kl  C. Erbing, L. Kenne, B. Lindberg, J . Lonngren and I. Sutherland, Carbohydr. R e s . , £ 0 , ( 1 9 7 6 ) 1 1 5 - 1 2 0 .  K2  L.C. Gahan, P.A. Sandford and H.E. Conrad, Biochemistry, 6,(1967)  K4  2755-2767.  i ) E.H. M e r r i f i e l d , Ph.D. Thesis, U. of Cape Town ( 1 9 7 8 ) i i ) S . C . Churms and A.M. Stephen, Carbohydr. R e s . , 3 5 , (1974) 7 3 .  K5  i ) G.G.S. Dutton and M.T. Yang, Can. J . C h e m . , £ 0 , ( 1 9 7 2 ) 2382-2384.  ii)G.G.S. Dutton and M.T. Yang, Can. J . C h e m . , £ 1 , ( 1 9 7 3 ) 1826-1832  K6  U. E l s a s s e r - B e i l e , H. F r i e b o l i n , and S. Stirm, Carbohydr. Res.,6£,(1978)  K7  245-249.  G.G.S. Dutton, A.M. Stephen, and S.C. Churms, Carbohydr. Res.,28,(1974)225-237.  K8  I.W. Sutherland, Biochemistry, 9_, ( 1 9 7 0 ) 2 1 8 0 - 2 1 8 5 .  K9  B. Lindberg, J . Lonngren, J.L.Thompson, and W. Nimmich, Carbohydr. R e s . , 2 £ , ( 1 9 7 2 )  K9*  49-57-  S.C. Churms, E.H. M e r r i f i e l d , and A.M. Stephen, S. Afr. J. S c i . , 2 6 , ( 1 9 8 0 ) 2 3 3 - 2 3 4 .  Kll  H. Thurow, Y.M. Choy, N. Frank, H. Niemann , and S. Stirm Carbohydr. Res.,41,241-255  K12  (1975).  G.G.S. Dutton and A.V. Savage, Carbohydr. R e s . , 8 2 , ( 1 9 8 0 ) 351-362.  K13  H. Niemann, N. Frank, and S. Stirm, Carbohydr. Res.,59. (1977)165-177.  K16  A.J. Chakraborty, H. F r i e b o l i n , H. Niemann, and S. Stirm, Carbohydr.  K17  Res.,£2,(1977)523-530.  G.G.S. Dutton and T.E. Folkman, Carbohydr. R e s . , 8 0 , ( 1 9 8 0 ) 147-161.  209  K18  G.G.S. Dutton, K.L. Mackie, and M.T. Yang,Carbohydr. Res.,6£,(1978)  K20  251-263.  Y.M. Choy and G.G.S. Dutton, Can. J . C h e m . , £ 1 , ( 1 9 7 3 ) 3015-3020.  K21  i ) Y.M. Choy.and G.G.S. Dutton,Can. J . C h e m . , £ 1 , ( 1 9 7 3 ) 198-207.  ii)Y.M. Choy and G.G.S. Dutton,Carbohydr. Res.,21,(1972) 169-172.  K22  H. Niemann and S. Stirm, unpublished r e s u l t s .  K23  G.G.S. Dutton, M. Stephenson, K.L. Mackie,and A.V. Savage Carbohydr. R e s . , 6 6 , ( 1 9 7 8 ) 1 2 5 - 1 3 1 .  K24  Y.M. Choy, G.G.S. Dutton, and A.M. Zanlungo, Can. J . Chem., £1,(1973)1819-1825.  K25  H. Niemann, B. Kwiatkowski, 0. Westphal, and S. Stirm, J . Bacteriol..130.(1977) 3 6 6 - 3 7 4 .  K26  J.L. Di Fabio and G.G.S. Dutton, Carbohydr. Res., i n press.  K27  S.C. Churms, E.H. M e r r i f i e l d , and A.M. Stephen,Carbohydr. Res.,81,(1980)49-58.  K28  M. C u r v a l l , B. Lindberg, J . Lonngren and W. Nimmich, Carbohydr. Res.,42,  K30  B. Lindberg, F. Lindh, J . Lonngren and I.W. Sutherland, Carbohydr.  K31  (1975)95-105-  Res.,20,(1979)135-144.  C C . Cheng, S.L. Wong, and Y.M. Choy, Carbohydr. Res., 22.(1979)169-174.  K32  . G.M. Bebault, G.G.S. Dutton, N. Funnel and K.L.Mackie, Carbohydr.  K33  Res.,63,(1978)183-192.  B. Lindberg, F. Lindh, J . Lonngren and W. Nimmich, Carbohydr. Res.,22,(1979)135-144.  K34  J.P. Joseleau, personal communication.  K38  B. Lindberg, B. Samuelson, and W. Nimmich,Carbohydr. Res., 20.(1973)63-70.  210 K4l  J.P. Joseleau, M. Lapeyre, M. Vignon, and G.G.S. Dutton, Carbohydr.  K44  Res.,62,(1978)197-212.  G.G.S. Dutton and T.E. Folkman, Carbohydr. R e s . , 2 8 , ( 1 9 8 0 ) 305-315.  K46  G.G.S Dutton and K. Okutani, Carbohydr.  Res.,86,(1980)  259-271.  K47  H. Bjorndal, B. Lindberg, J . Lonngren, W. Nimmich and K. R o s e l l , Carbohydr.  Res.,22,(1973)272-278.  K48  J.P. Joseleau, personal communication.  K49  J.P. Joseleau, personal communication.  K51  A.K. Chakraborty and S. Stirm, Abst. Int. Symp. Carbohydr. Chem., 9 t h , London, (1978)4-39-440  K52  H. Bjorndal, B. Lindberg, J . Lonngren, M. Meszaros, J.L.  Thompson and W. Nimmich, Carbohydr. Res.,31,  (1973)  K53  93-100.  G.G.S. Dutton and M. Paulin, Carbohydr. Res.,82,(I98O) 107-117.  K54  i ) P.A. Sandford, J.R. Bamburg, J.D. Epley and T.J. Kindt, Biochemistry,£,(1966) 2808. i i ) P . A . Sandford and H.E. Conrad, Biochemistry,£,(1966) 1508-1516.  K55  G.M. Bebault and G.G.S. Dutton, Carbohydr. Res.,64, (1978)  K56  199-213.  Y.M. Choy and G.G.S. Dutton,Can. J . Chem.,51.(1973) 3021-3026.  K57  J.P- Kamerling, B. Lindberg, J . Lonngren and W. Nimmich, Acta Chem. Scand.,(B),2^,(1975) 5 9 3 -  K58  G.G.S Dutton and A.V. Savage, Carbohydr. Res.,84.(1980) 297-305.  K59  B. Lindberg, J . Lonngren ,U. Ruden and W. Nimmich, Carbohydr. R e s . , 4 2 , ( 1 9 7 5 )  83-93.  211 K60  G.G.S. Dutton and J . L . D i Fabio,Carbohydr. R e s . . 8 7 . (1980) 1 2 9 - 1 3 9 .  K61  i ) A.S.Rao, N. Roy and W. Nimmich, Carbohydr. R e s . , 6 7 , (1978) 4 4 9 - 4 5 6 . i i ) A . S . Rao, N. Roy and W. Nimmich, Carbohydr. R e s . , 7 6 , ( 1 9 7 9 ) 215-224  K62  G.G.S. Dutton and M.T. Yang, Carbohydr.  Res.,£2,(1977)  179-192. K63  J.P. J o s e l e a u and M.F. M a r a i s , Carbohydr.  Res,22»(1979)  183-190. K64  E.H. M e r r i f i e l d (1979)  K?0  and A.M. Stephen,Carbohydr. R e s . , 7 4 .  241-257.  G.G.S. Dutton and K.L. Mackie, Carbohydr.  Res.,62,(1978)  321-335. K71  E.H. M e r r i f i e l d  and A.M. Stephen, u n p u b l i s h e d r e s u l t s .  K72  Y.M. Choy and G.G.S. Dutton, Can. J . C h e m . , £ 2 , ( 1 9 7 4 ) 684-687.  K74  G.G.S. Dutton and M. P a u l i n , Carbohydr. R e s . , 8 2 , ( 1 9 8 0 ) 119-127.  K81  M. C u r v a l l ,  B. L i n d b e r g , J . Lonngren, arid W. Nimmich,  Carbohydr. R e s . , 4 2 , ( 1 9 7 5 ) 7 3 - 8 2 . K83  B. L i n d b e r g and w. Nimmich, Carbohydr. R e s . , 4 8 , ( 1 9 7 6 ) 81-84.  212  APPENDIX I I I AND C-N.M.R. SPECTRA 13  K'60 polysaccharide 1  Spectrum  No.1  H n.m.r.  220 MHz, 90 C i  Acetone  i  2.23  !  K 60 polysaccharide  Spectrum No.2  n.m.r. 20 MHz, amb.temp. acetone  31 .07  ro H  102.M+  K 60 Compound A  Spectrum  1  2 Gal~0H  GlcA -  HOD  H n.m.r.  100 MHz, 90°C .  I  I.ii.i I , i  L  i  I  I I I I I  i i i i  I I  acetone  I I I 1 I I I I It II I I I I I I Ii i i  i  i  —U-l _L_L  No.3  K 60  Compound A  GlcA-!  Spectrum No>  1  ^ Gal^OH B  ^C n.m.r. 20 MHz, amb.temp.  acetone 31 . 0 7  I ' I  1  I  1  I  1  1  I  1  1  1  I  1  I  1  I  1  i  '  i  '  i  ' i  I  K 60 Compound N  Spectrum No. 5  1  Glcv/OH  Glc ^rr- Man  13 C  n.m.r*  20 MHz. amb.temp.  102.54  99.85  96.80 83  93.07  K  60,  P  hal-  [ -JGICAJ 8 1„ H  Spectrum  polysaccharide  1  2 -  ^Glc—]  Man  a  a  B  n  n.m.r.  100 MHz, 90°C  5.27  5.33  No.6  K 60, P-| p o l y s a c c h a r i d e y  Z  Spectrum  No.?  n.m.r.  2 0 MHz, amb.temp.  acetone 31 .07  101 . 4 2  Spectrum No. 11  K 60 , Compound GlcA-} ^  H  ^QalB  ^MarJ a  2G1C~0H a  n.m.r.  100 MHz,  90° C  5. 5.31  K 60, Compound A^  Spectrum No.12  ^ C n.m.r. 2 0 MHz, amb.temp.  ro ro 101.51 101.35  104.51  96.67 I  I  93  1  K 26 , depyruvylated  polysaccharide  Spectrum No. 1 3  1  H  n.m.r  400 MHz  , water n u l l acetone 2.23  4.51 5.^9  5.rTs-01 4 . 6 3 [ 4 . 4 5  K 26,Compound  GlcA - — ^ Man~OH  1H n.m.r. a  100 MHz, 90°C  5.32 5.20  4.92  »)0M^  -J  1 1 1 1 1  1  1 1 1 1  I I I I  ' '_i J 1 1 1  i rJ  ,  1 1  L_l  K 26,Compound  Spectrum No.15  GlcA - — ^ Man~OH 13C J  ° n.m.r.  20 MHz, amb.temp.  acetone 31.07  K 26,Compound  GlcA - — ^ a  M  a  n  A  Spectrum No.l?  2  1—?. Man^OH a  n.m.r. 2 0 MHz , amb.temp. 102.86  101.36  acetone 31.0?  93.56 93-35  i ' i  1  i ' I  1  I  1  I  r-r-' | i |  ro ro vo  Spectrum No.19  K 26, Compound Aj GlcA - — 2 .  M  a  n  1—2  a  M  a  n  1—1 Gal~OH  a  a  13 -^C n.m.r. 20 MHz , amb.temp.  '  I  '  I  1  I  1  l  1  l  1  I  1  I  1  1  1  I  1  I  1  I  r  K 26, Compound N Glc — Glc~OH 1 H n.m.r. 8  100 MHz ,90°C  Spectrum No.21  K 26 .Compound 1^ Glc - — - Glc~OH B  1 3' rC  n.m.r.  2 0 MHz , amb.temp.  61.60  acetone 31*0?  ro  103.50  96.81  92.95  K 26,Compound N  Spectrum No.22  2  Gal - — - Glc - — - Glc~OH *H n.m.r. 400 MHz , amb.temp.  HOD  4.46  acetone 2.23  4.5 5.24  4.67*  I  Spectrum No.23  Spectrum No. 24  K 26, Compound SH Gal  GlcA — ^ Man — - Gly 1  8  a  a  ^C n.m.r. 20 MHz ,amb.temp.  acetone 31.07  0 6 0 K60, Compound X  Spectrum No.25  ^C n.m.r.  20 MHz,amb.temp.  acetone 31.07  0 6 0 K60 , Compound P  n -^C n.m.r. 20 MHz  ,amb.temp.  2  Spectrum NoZ6  046 X  K46,Compound  ?  Spectrum No.2?  1  H n.m.r.  400 MHz, water n u l l acetone 2.23  ro  V>)  1.52  046  K46,Compound  P  x  Spectrum No.28  -T n.m.r. 20 MHz  ,amb.temp.  ro -po  100.3? 101.00 101.33  97.14 95/ 97 95 81 :  93.11  acetone 31-0?  046 X  K46,Compound  P  Spectrum N 0 . 2 9  2  H n.m.r.  400 MHz, water n u l l  acetone 2.23  5.19 5.30  4.85 5.06  4.65  4.69  C. speciosa, Compound A  Spectrum No.31  GlcA - — - Man~OH 1 H n.m.r. 8  400 MHz, water n u l l  acetone 2.23  5.30  J  Spectrum No.32  C. speciosa, Compound GlcA - — - Man - — - GlcA - — - Man~OH ,  3  H n.m.r.  a  3  acetone  400 MHz, water n u l l  2.23  HOD  245  APPENDIX I V USES OF  PERACETYLATED  ALDONONITRILES  246  USES OF PERACETYLATED ALDONONITRILES  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 have been known s i n c e 1 8 9 3 . They were f i r s t used  i n s y n t h e s i s , a ) the Wohl degradation" ' 1  (pentoses a r e obtained from the p e r a c e t y l a t e d h e x o n o n i t r i l e s ) , b) f o r m a t i o n o f 1 - a m i n o - l - d e o x y a l d i t o l s ,etc.The  synthesis of  these d e r i v a t i v e s has been s t u d i e d and the b e s t c o n d i t i o n s observed were the treatment  o f the aldose w i t h hydroxylamine  hy-  d r o c h l o r i d e i n p y r i d i n e and then a c e t y l a t i o n and d e h y d r a t i o n done a t the same time w i t h a c e t i c anhydride  a t h i g h tempera -  ture-^ ( i n the case o f g l u c o s e . i t has been observed temperature  a cyclic derivative i s preferentially  As d e r i v a t i v e s o f a n a l y t i c a l i n t e r e s t , t h e  t h a t a t low formed).  ,the t r i m e t h y l -  4 silylated  oximes  and Jones-* used  were f i r s t used  for g.l.c.  separations.Lance  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 f o r g . l . c . s e -  p a r a t i o n o f the methyl  ethers o f D - x y l o s e . S e v e r a l s t a t i o n a r y  phases have been employed s i n c e 1 9 7 1 t o improve the s e p a r a t i o n  6—8 of  the PAAN ( 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 ) ~ . The g . l . c . r e t e n t i o n times and g.l.c.-m.s. f o r the a c e t y l 9 6 1 ated a l d o n o n i t r i l e s o f methyl e t h e r s o f mannose and glucose ' 7  have been reported.The  methylated  sugars i n a m e t h y l a t i o n a-  n a l y s i s can now be c o m p l e t e l y c h a r a c t e r i z e d and i d e n t i f i e d by u s i n g g.l.c.-m.s. o f the d e r i v e d a l d o n o n i t r i l e s and a l d i t o l ac e t a t e s . A l l methyl e t h e r s o f mannose can be separated by g . l . c . as a l d o n o n i t r i l e s  ( see Table l ) . I n the course o f t h i s i n v e s -  t i g a t i o n , 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 o f the methyl were a l s o used as means o f a n a l y z i n g the m e t h y l a t i o n  ethers  products  247 TABLE 1 RELATIVE G.L.C. RETENTION-TIMES OF PERACETYLATED ALDONONITRILES OF METHYL ETHERS OF D-GLUCOSE AND D-MANNOSE. M e t h y l ether —  Retention  times(on  5% o f  butanediol succinate) — D-Glucose  D-Mannose  2 , 3 , ^ , 6-tetra-  0.94  1.00  2,4,6  -tri-  1.45  1.59  2,3.6  -tri-  2.00  1.65  3.4,6  -tri-  1.85  1.89  2,3,4  -tri-  2.00  2.03  2,6  -di-  2.28  4,6  -di-  — —  2,3  -di-  3-50  2.55  3,6  -di-  —  2.85  2,4  -di-  2.84  3.16  3,4  -di-  3-58  3.68  -  2,3,4,6-tetra  nonitrile.etc.  :  2.45  5^0-acetyl-2,3,4,6-tetra-0-methyl-D-gluco-  - Relative to 5 - 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l -  mannononitrile.Data  from r e f e r e n c e s  9) and 1 0 ) .  TABLE 2 RELATIVE G.L.C. RETENTION TIMES OF PERACETYLATED ALDONONITRILES OF METHYL ETHERS OF SUGARS.  M e t h y l ether -  R e l a t i v e r e t e n t i o n times OV-225 3% -  2,3,4,6 2,4,6 2,4,6 2,4,6 2,3,4 2,3,6 3,4,6 4,6 4,6 3,6 2,3 2,3 2  -  - Glc - Glc - Man - Gal - Glc - Glc - Man - Man - Gal - Glc - Glc - Gal — Glc  1.00 °1.39 1.59 1.65 1.85 1. 98 1.98 2.20 2.44 2.59 2.81 2.81 3.02  2,3,4,6 - Glc : 5-0-acetyl-2,3,4,6-tetra-0-methylglucononitrile.  nitrile. -  Programmed a t 1 6 5 ° f o r 4 min,then a t 2°/min t o 220°,  i s o t h e r m a l f o r 32 min. - 6.6 min .  249  e s p e c i a l l y t o separate the 2,3,4-and ses  2,3,6-tri-0-methylgluco-  (see Table 2 ) . Ketoses  can a l s o be c h a r a c t e r i z e d and separated by g . l . c .  and g.l.c.-m.s. as the p e r a c e t y l a t e d o x i m e s . 11  In g e n e r a l , t h e PAAN have been used f o r examining n e u t r a l 12 13 sugars from p o l y s a c c h a r i d e s . g l y c o p r o t e i n s .mucopolysaccharides  13  .and products r e s u l t i n g from Smith d e g r a d a t i o n  14  .Another  use f o r these d e r i v a t i v e s , i s the d e t e r m i n a t i o n o f the degree o f p o l y m e r i z a t i o n o f 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 as w e l l as the i d e n t i f i c a t i o n o f the r e d u c i n g end.The g e n e r a l proce dure,known as the M o r r i s o n procedure -*, i n v o l v e s the r e d u c t i o n 1  w i t h NaBH^ o f the r e d u c i n g e n d , h y d r o l y s i s , a n d treatment h y d r o l y z a t e w i t h hydroxylamine lowed by a c e t i c anhydride.The  o f the  hydrochloride i n pyridine f o l f r e e sugars a r e converted  into  the PAAN and the a l d i t o l i n t o the a l d i t o l a c e t a t e . A f t e r g . l . c . s e p a r a t i o n and q u a n t i t a t i o n , i t i s p o s s i b l e t o determine the r a t i o o f f r e e s u g a r / r e d u c i n g end which g i v e s the D.P.By i d e n t i f i c a t i o n o f the a l d i t o l a c e t a t e , t h e r e d u c i n g end i s d e t e r mined. A l d i t o l a c e t a t e s have been used f o r c d . measurements ^, 1  to determine ars  the a b s o l u t e c o n f i g u r a t i o n o f t h e i r parent  sug-  ( D or L ).Sugars whose a l d i t o l s are meso compounds  ( g a l a c t o s e , x y l o s e , e t c . ) c a n n o t be s t u d i e d , a s they do not show cd.  a c t i v i t y . T h e 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 , a s they keep  the c h i r a l i t y o f the parent sugars and chromophores a r e pres e n t , show c d . a c t i v i t y and can be used t o determine s o l u t e c o n f i g u r a t i o n o f the parent sugars.They  the ab-  can be e a s i l y  250  TABLE 3 CIRCULAR DICHROISM  Peracetylated  OF THE PERACETYLATED ALDONONITRILES.  Configuration  aldononitriles  S i g n o f the c.d. curve  Arabinose  L  -  Arabinose  D  +  Fucose  L  -  Fucose  D  +  Galactose  D  +  Glucose  D  +  Mannose  D  +  251  prepared and s e p a r a t e d by p r e p a r a t i v e g . l . c . R e s u l t s o f t h i s i n v e s t i g a t i o n are g i v e n i n Table 3 « Experimental. P r e p a r a t i o n o f the p e r a c e t y l a t e d The f r e e  aldononitriles.  sugars ( 5 - 1 0 mg) a r e d i s s o l v e d i n hydroxylamine  hydrochloride i n pyridine f o r 1 5 min.The s o l u t i o n  ( 5 ^ , 1 mL) and heated on a steam-bath  i s cooled t o room temperature and a c e t i c  anhydride ( 1 mL) i s added and heated f o r 1 h on a steam-bath. Water ( 5 - 1 0 mL) i s added and the PAAN a r e e x t r a c t e d w i t h CHCl^. A f t e r removal o f the s o l v e n t , t h e samples a r e ready f o r g . l . c . S e p a r a t i o n o f the PAAN by g . l . c . The PAAN d e r i v e d from the f r e e sugars were s e p a r a t e d on a column o f 3 % O V - 2 2 5 on Gas Chrom Q and the temperature used was 2 1 0 ° isothermal.The PAAN d e r i v e d from methylated sugars were separated on the same column,with the temperature programme 1 6 5 ° f o r 4 min,then 2 % i i n  t o 2 2 0 ° f o r 3 2 min.  C i r c u l a r d i c h r o i s m measurements o f the PAAN. Samples o f the PAAN i s o l a t e d by p r e p a r a t i v e g . l . c . on O V - 2 2 5 3% ( i s o t h e r m a l 2 1 0 ° ) were d i s s o l v e d i n a c e t o n i t r i l e and the  c d . curves were measured.  252  BIBLIOGRAPHY 1.  A. Wohl, Ber.,26,(1893) 7 3 0 .  2.  C.H. Winestock and J.W.E. P l a u t , J . Org. Chem.,26,(1961) 4456-4462.  3.  V. D e u l o f e u , P. Cattaneo, and G. M e n d i v e l z a , J . Chem. S o c , (1934) I 4 7 r l 4 8 .  4.  C C . Sweeley, R. B e n t l e y , M. M a k i t a , and W.W. J . Amer. Chem. S o c , 8 5 , ( I 9 6 3 )  Wells,  2497T2507.  5.  D.G. Lance and J.K.N. Jones, Can. J . Chem. ,45_, (1967)1995^8.  6.  B.A. D m i t r i e v , L.V. Backinowsky,  O.S. Chizhov, B.M. Z o l o -  t a r e v , and N.K. Kochetkov, Carbohydr. R e s . , 1 2 , ( 1 9 7 1 ) 4 3 2 - 5 . 7.  J . Szafranek, C D . P f a f f e n b e r g e r , and E.C. Horning, Anal. Lett.,6,(1973)479-493.  8.  R. Varma, R.S. Varma, and A.H. Wardi, J . Chromatog.,77. (1973) 222-227.  9.  F.R. Seymour, R.D. P l a t t n e r , and M.E. S l o d k i , Res.,44,(1975)  Carbohydr.  181-198.  10. F.R. Seymour, M.E. S l o d k i , R.D. P l a t t n e r , a n d A. Jeanes, Carbohydr. Res. , £ 2 , (1977)  153-166.  11. F.R. Seymour, J . E . S t o u f f e r , and E.CM. Chen, Carbohydr. Res. ,83_, (1980) 201-242. 12. R. Varma, R.S. Varma, W.S. A l l e n , a n d A.H. Wardi,J. Chromat o g . ,86.(1973)205r210. 1 3 a . T . P . Mawhinney, M.S. F e a t h e r , G.J. Barbero,and J.R. M a r t i nez, A n a l . Biochem..101.(1980) 112-117. 13b.R. Varma and R.S. Varma, J . Chromatog.,128,(1976) 4 5 r 5 2 . 14. J.K. B a i r d , M.J. Holroyde, and E.C. E l l w o o d , Carbohydr. Res.,22,(1973) 15.  464^467.  I.M. M o r r i s o n , J . Chromatog.,  108, (1975) 36lr-364.  253  G.M. Bebault, J.M. Berry, Y.M. Choy, G.G.S. Dutton, N. Funnel,L.D. Hayward, and A.M. Stephen, Can. J . Chem., £1.(1973)  324.326.  

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