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The effect of reproductive condition on sexual behaviour and the olfactory bulb electroencephalogram… Goff, Robert 1979

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STRUCTURAL  STUDIES  CAPSULAR  ON  KLEBSIELLA  POLYSACCHARIDES BY  TIMOTHY EDWARD FOLKMAN B . Sc .'( H o n s . ) , U n i v e r s i t y  A T H E S I S SUBMITTED THE  of A l b e r t a ,  1972  IN P A R T I A L F U L F I L L M E N T  R E Q U I R E M E N T S FOR MASTER  OF  THE DEGREE  OF  SCIENCE  in THE  FACULTY  OF GRADUATE  (Department We  accept to  THE  this  of  STUDIES  Chemistry)  thesis  the required  as  conforming  standard  U N I V E R S I T Y OF B R I T I S H  COLUMBIA  April,1979  ©  Timothy  Edward  Folkman,  1979  OF  In  presenting  an  advanced  the I  Library  further  for  this  thesis  degree shall  agree  scholarly  at theUniversity make  that  purposes  h i s representatives.  of  this  written  of  University  gain  dl\e<mI.S^Yy Columbia  2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Date  k^J^4.l9l f C  of British  Columbia,  f o r reference copying  by t h e Head  i s understood  f o r financial  of British  o f the requirements  forextensive  may b e g r a n t e d It  fulfilment  available  permission.  Department The  i t freely  permission  by  thesis  in partial  shall  that  f  I agree t h a and study.  of this  thesis  o f my D e p a r t m e n t o r  copying  n o t be a l l o w e d  or publication w i t h o u t my  -•-i i ABSTRACT  To d a t e ,  s t u d i e s have  cally  distinct  genic  c h a r a c t e r o f each  considered surrounds  strains  program  well  half  t h e known  The  anti-  reaction i s  Klebsiella  unique.  of the antigens  from  which poly-  strains  In a c o n t i n u i n g  s t r u c t u r e s of these  to e x p l a i n immunological  the structure  exist.  Structures of the capsular  e s t a b l i s h e d ; a l l o f them  in order  bacteria  serologi-  by t h e c a p s u l a r p o l y s a c c h a r i d e  cell.  over  seventy-eight  i n an i m m u n o l o g i c a l  to e l u c i d a t e the chemical  charides for  strain  t o be d e t e r m i n e d  s a c c h a r i d e s from  that  of Klebsiella  the bacterial  have been  revealed  polysac-  responses,  Klebsiella  evidence  serotypes  K17 a n d K 4 4 i s p r e s e n t e d . Methylation of  analysis  the monosaccharide  oxidation-Smith techniques  was u s e d  units.  Uronic  degradation,  employed  to establish  to generate  acid  linkage  degradation,  positions periodate  and p a r t i a l  h y d r o l y s i s were t h e  and s t u d y  o l i g o s a c c h a r i d e s from 1  the  more  nuclear  complex magnetic  configurations  polymer.  E x t e n s i v e u s e was made o f  resonance  spectroscopy  of the o l i g o -  as gas l i q u i d  chromatography,  mass  spectrometry,  gel filtration  and p o l a r i m e t r y have  products  obtained  Although of  a small  structures repeating  from  been  gas l i q u i d  with  a r e found units:  to consist  Methods  chromatographypaper  used  and i d e n t i f y  to i s o l a t e  the various degradative  t h e same  qualitative  C  anomeric  chromatography,  the p o l y s a c c h a r i d e s of K-types  group  H and  and p o l y s a c c h a r i d e s a l i k e .  such  graphy,  to indicate  13  chromato-  techniques.  17 a n d 44 a r e p a r t  composition,  of the f o l l o w i n g  their  dissimilar  the  -  i i i  -  ^-Glcp^L-Rhap^^-GlcAp^L-Rhap^-  K17  L-Rhap  K44  ^0-Glcp^-^O-G lcp^-^D-G 1 c A p ^ - ^ - L - R h a p ^ - L - R h a p - —  3  The l a t t e r pentasaccharide saccharide.  r e p r e s e n t s the repeating unit  a  first  3  example  a  of a  in a Klebsiella  a  linear  capsular  poly-  - iv TABLE  OF  CONTENTS Page  ABSTRACT TABLE  OF  ;  i i  CONTENTS  LIST  OF T A B L E S  LIST  OF  iv v i  FIGURES  ,  v i i  ACKNOWLEDGEMENTS  viii  PREFACE  i x  I  INTRODUCTION  II  METHODS USED IN STRUCTURAL A N A L Y S E S POLYSACCHARIDES II.1 .11.2  2  Isolation Total  OF 5  and P u r i f i c a t i o n .  5  H y d r o l y s i s and C h a r a c t e r i z a t i o n  Component II.2.1  of  Sugars  Total  sugar  6 ratio  7  II.2.2.  11.3  Nuclear magnetic resonance s p e c t r o s c o p y (1H a n d 13c ) A n a l y s i s of L i n k a g e P o s i t i o n f o r Component Sugars  14  11.3.1  Methylation analysis  16  11.3.2  G.l.c. sugars  18  11.3.3 11.3.4 11.4  analysis  of  Mass s p e c t r o m e t r y sugars  8  methylated of  methylated 20  "*"H n . m . r . s p e c t r o s c o p y o f m e t h y l a t e d p o l y s a c c h a r i d e s and o l i g o s a c c h a r i d e s  Sugar Sequence D e t e r m i n a t i o n Acid Degradation  by  24  Uronic 24  -  V  -  Pag_e 11.5  Sugar  Sequence D e t e r m i n a t i o n  Partial  26  Periodate  11.7  Anomeric  11.8  Determination of C o n f i g u r a t i o n of Compon en t S u g a r s B i b l i o g r a p h y f o r S e c t i o n s I and I I  STRUCTURAL  Oxidation Linkage  29  Determination  I N V E S T I G A T I O N OF K l e b s i e l l a  K17 CAPSULAR  IV  Hydrolysis  11.6  11.9 III  from  34  34 37  SEROTYPE  POLYSACCHARIDE  40  111.1  Abstract....  41  111.2  Introduction  42  111.3  Results  43  111.4  Experimental  111.5  Bibliography  and D i s c u s s i o n  59 f o r Section  III  STRUCTURAL I N V E S T I G A T I O N OF K l e b s i e l l a CAPSULAR P O L Y S A C C H A R I D E  74 K44 76  IV.1  Abstract  77  IV. 2  Introduction  78  IV. 3  Results  79  IV. 4  Experimental  IV . 5  Bibliography  APPENDIX:  and D i s c u s s i o n  N.M.R. SPECTRA  93 f o r Section  IV  103 105  - v i LIST  OF  TABLES  Table II.1  111.1  111.2  111.3  111.4  IV.1  IV.2  IV.3  Page C a l c u l a t e d and measured rotations of K l e b s i e l l a oligosaccharides  values f o r s p e c i f i c K17 and K44 p o l y - and :  N.m.r. d a t a o f K l e b s i e l l a K 1 7 c a p s u l a r s a c c h a r i d e and d e r i v e d p o l y - and oligosaccharides Methylation a n a l y s i s of o r i g i n a l polysaccharide  K17  poly44 capsular 48  M e t h y l a t i o n a n a l y s e s o f K17 c a p s u l a r s a c c h a r i d e and d e r i v e d p o l y - and oligosaccharides G.l.c. uronic  35  a n a l y s i s o f K17 p o l y s a c c h a r i d e acid degradation  poly50 after 56  N.m.r. d a t a o f K l e b s i e l l a K44 c a p s u l a r p o l y s a c c h a r i d e and d e r i v e d o l i g o s a c c h a r i d e s  80  Methylation a n a l y s i s of o r i g i n a l K44 c a p s u l a r p o l y s a c c h a r i d e  84  and degraded  M e t h y l a t i o n a n a l y s i s of a c i d i c o l i g o s a c c h a r i d e s i s o l a t e d a f t e r p a r t i a l , a c i d h y d r o l y s i s o f K44 capsular polysaccharide '  88  LIST  v i i OF  FIGURES  Figure 1.1  11.1  11.2  11.3  11.4  11.5  11.6  11.7  11.8  11.9  IV.1  Page Diagramatic r e p r e s e n t a t i o n of a b a c t e r i a l cell with lipopolysaccharide , capsule and slime....  Proton n.m.r. s p e c t r a s h o w i n g the change HOD signal location with temperature  in 10  "''H n . m . r . s p e c t r a o f K 1 7 polysaccharide d e p i c t i n g s i g n a l s o t h e r than anomeric ones the region 64.0 t o 6.0  "^C n.m.r. s p e c t r u m polysaccharide  Methylation analysis polysaccharide  of  Klebsiel la  K17  2  in 12  capsular 15  scheme  for  K17 17  G.l.c. separation of a m i x t u r e of partially methylated a l d i t o l acetates obtained from K l e b s i e l l a K17 polysaccharide  19  Mass s p e c t r a of u r o n i c a c i d d e r i v a t i v e s f r o m K17 and K44  23  Uronic acid degradation polysaccharide  Periodate-Smith polysaccharide  of  degradation  Selective periodate-Smith polysaccharide  degradation  Klebsiella  K44 27  of  Klebsiella  K44 31  degradation  of  K17  Gel f i l t r a t i o n chromatography separation of a mixture of s a c c h a r i d e s obtained from partial h y d r o l y s i s of K l e b s i e l l a K 4 4 p o l y s a c c h a r i d e . . .  33  90  -'/VI11  -  ACKNOWLEDGEMENTS  I  should  G.G.S. D u t t o n thesis  like  whose g u i d a n c e  to a successful  To name o n l y cussions I  wish  Dr.  Stephen,  Savage,  Credit patience  and  sincere  thanks  and u n d e r s t a n d i n g  a f e w o f t h e many p e o p l e aided  appreciation and a l s o  Jose  must  my  support.  Professor  brought  this  the course  t o D r . M.  Vignon,  and M a r c e l  go t o my  whose h e l p f u l  during  to the other  diFabio,  also  to  conclusion.  and s e r v i c e s have  t o show my  A.M.  Angela  to express  wife  members  dis-  of t h i s  D r . A.  Zanlungo  of the laborator  Paulin.  Linda,  work,  f o r her  constant  - ix PREFACE  In  an e f f o r t t o f a m i l i a r i z e r e a d e r s  particular the  area  following  of organic  explanation  Fischer  projection  modification  of sugars.  commences f r o m Note  that  chemistry  group  acid  CHO  OH  —OH  also  (marked  differs  i s oxidized  common  are four  name,  chiral  asterisks  CH.  L-rhamnose (III)  acid  centers  the s p a t i a l arrangement  is  by t h e s e  nomenclature  acid  group  group.  T h e C-6 o f  and i s r e f e r r e d  to  6-deoxy-L-mannose. i n these  in structure  Fischer  ( I ) only  HO—I  of a methyl  appreciate  the  D-glucose  (I).  H0-  to a carboxylic  ( I I I ) i s part  Numberin  OH  ( I D  with  implied  from  COOH  by a n o t h e r There  below.  at the top of the chain  D-glucuronic  C-6  the acycli  I—OH OH  D-glucose (I)  refers,  CHO  OH  L-rhamnose  a r e shown  HOH  3  2  that  to represent  — OH  CH 0H  in  this thesis  CHO  1—OH HO  (II)  i n the  i s offered.  a r e used  Some e x a m p l e s  the carbonyl  D-glucuronic  to which  of terms used formulae  who do n o t w o r k  six-carbon  I I I ) making o f atoms  representations.  of a l l the possible  isomers  chains  i t important to  (conformation)  that  To s i m p l i f y (16 f o r each o f  -  I,  I I , I I I ) , a l l those  numbered Fischer others  chiral  center  projection to  the  X  -  having (C-5)  the  hydroxyl  projecting  formulae  belong  to  Physical  on  the  center  and  right  D-series,  by  aldehydic C-1.  chemical  evidence  nucleophilic carbon  This  atom,  results  OH  H  indicates  exist attack  in a c y c l i c of  generating i n two  that,  the  in  the  and  the  in  fact,  a new  anomers,  h-OH  — —OH  - g l u cose (IV)  HO  0  hOH  $-D - g l u c o s e (V)  at  these ring C-5  (anomeric)  represented  h-OH 0  The  atom  chiral  H  HO  form.  oxygen  C  HO  highest-  L-ser i e s  polyhydroxyaldehydes  occurs  at  the  the  the  HCH  D-series  closure  to  at  L-series.  \—0H  six-carbon  group  below  - xi in  the Tollens  in  having  cetal  two a t t a c h e d  I t should  oxygen  their  the Tollens  unequal  method  bond  of looking  improvement carbon  atoms,  formulae  lengths,  recognizes  chain  and t h a t  bond  lengths  hexagons  pyran  (VIII)  a n d named i n ring  formulae  on  the l e f t  point  on  C-5  It  f o l l o w s , then,  points  up  two p o s s i b l e linkage.  ring  making  i t a hemia-  oxygen  them  atom  i n Haworth  point up. forD that  down  behind the equal.  Often  projections,  pyran (VIII)  Note  on t h e r i g h t  i n t h e Haworth  Similarly,  sugar  configurations,  that  hydroxyl  i n Fischer  groups  and  p r o j e c t i o n s and  f o r aldopyranoses,  ( I X ) a n d down when  lies  This  as t h e h e t e r o c y c l i c compound  pyranoses.  formation  perspective  are approximately  a r e used  rings  a  with  ( V I and V I I ) .  (VII)  he r e l a t e d t o s u c h  ea ch  C - l i s unique  limitations  developed  g-D-glucopyranose  which  Tollens  obvious  the ring  (VI)  involved  have  Haworth  that  a-D-g l u c o p y r a n o s e  are  that  formally  at t h e six-membered  practise regular  not  be n o t e d  carbon. Since  in  formulae.  those  t h e group  f o r the L enantiomer ( X ) ,  residues  are attached  there  an a - o r a 3 - p y r a n o s i d e , f o r  -  -  X11  °xOH  HO OH  HO  a-D-rhamnopyranose (IX)  The to  the  shown  true  chair that  puckered,  a  conformation form  of  hexose,  substituents  group  C-5.  at  at  C-1,  are  All  pyranoid  c y c l o h e x a n e. X - r a y such  six-membered,  hydroxyl  of  as  carbohydrates diffraction  a-D-glucose  (XI),  oxygen - c o n t a i n i n g at  C-1  through  substituents  on  C-4,  the  ring,  a  related  analysis  consists  carbon and  is  ring,  of  has a  with  h y d r o xy m e t h y l  except  for  that  equatorial.  (XI)  Two anomeric  isomers center  (anomers) (C-1),  are  possible  depending  on  in  whether  relation a  to  the  substituent  is  -  axial  (a-anomer;  R = hydrogen, for  for  di-,oligo-,  chemical  equatorial  and  (g-anomer;  polysaccharides. for  provides  the  can  two  invaluable  XIII),where  R = another Since  anomers,  easily  H-l  sugar  is  nuclear  distinguish  in  a  differen  magnetic  between  a s s i s t a n c e in  residue,  them  assigning  configurations.  Haworth  projections  are  thesis,  even  they  dimensional cation  -  m o n o s a c c h a r i d e s , and  spectroscopy  thereby,  anomeric  this  or  environment  resonance and,  XII)  X l l l  for as  though  molecular the  as  well  or  variable.  use  of  structures,  most  shape.  give  no  There  formulae when  useful  the  and  will  indication  seems  to  be u s e d of  three-  be l i t t l e  which  depict  states  true  states  are  in  of  often  justifimolecules unknown  I N T R O D U C T I ON  METHODS OF  USED  IN  STRUCTURAL  POLYSACCHARIDES  ANALYSES  -2I  IN TR ODUCTION Bacteria  of  the  Enterobacteriaceae ""well co l i  known (E.  characteristic cultures is  in  of  released  son"'" h a v e  of  saccharide"  such  The of  as  the  producing  a  from  the that  cell the  composition. a bacterial is  a  and  In F i g u r e  to  the  which  S a l m on e l l a  capsular  forming  cell  flora)  bacterial  strains  slime  belong  genera  Klebsiella  some K l e b s i e l l a  shown  chemical  sentation  (gastrointestinal  members  coli).  genus K l e b s i e l l a  an  cell  family  includes and  has  Escherichia  the  notable  polysaccharide. excess  "slime".  of  containing  In  polysaccharide  Dudman  polysaccharide 1.1,  and  are  such  an  Wilkin-  identical  a diagrammatic just  other  repre"exopoly-  shown.  CELL WALL  MEMBRANE CYTOPLASM CAPSULE  UPOPOLYSACCHARIDE  SLIME  Figure  1.1  Diagrammatic r e p r e s e n t a t i o n of  a  bacterial  c e l l with lipopolysaccharide, capsule slime.  and  The surface typed  polysaccharide for  external  solely  envelope  of  on  the  responses.  strains  of  contact. basis  of  the  capsular is  Seventy-eight  K-types  from  been  the  cell  providing  Kleb s i e l l a are  which  K l e b s i e l l a have  designated  -  envelopes  polysaccharide  logical  3  and  to  German  word  it  cause  serologically established  first  serologically  antigens  thought  the  to  is  this  immuno-  distinct date  and  "Kapsel".  and  0rskov  2 Fife-Asbury by  adding  have  K82  recently  to,  and  amended  deleting  the  number  K73,75,76,77,  of  known  strains  and  78  from  the  to  be  mildly  s c h em e . Polysaccharides  are  generally  non-antigenic  but  and  Heidelberger  rabbits.  cross-reactions relationships longing  to  antigenic of  these  studies  antibodies  with  can  widely  capsular of  all  Incidentally,  the  contains  studies  K17  and  and  rides  1 to  81  containing  K44  anti-K  are  known. are  Heidelberger  is  in  study  structures  Thus  structural  et  confirmed  specific  the  this  a result  be-  the  underway.  of  as  to  Immunochemical  until  presented  anti-K47,  sera  microorganisms  is  L-rhamnose,  horses  Unfortunately  assigned  or  The  thesis.  al ^ that of  by  K17  cross-reactistructural  thesis.  Nimmich^'^'^'has K-types  in  families. be  from  species.  predicted  prediction  anti-K19 this  used  K l eb s i e l l a s t r a i n s  of  in  isolated  a l ^ have  polysaccharides  end-groups  with  be  cannot  known of  been  bacterial  disparate  determinant  investigation  vity  thereby  have  et  other  considered  reported  and.has either  found  the them  D-glucuronic  qualitative all  to  acid,  be  composition  acidic  of  polysaccha-  D- ga l a ctu r o n i c  acid,  pyruvic very  acid in  seldom,  D-glucose, L-fucose  form  of  a keto  acid  (K22,37,38).  can  no  L-rhamnose  systematic  structures  number  The n u m b e r  of  of  sugar to  sugar  (per  unit)  repeat  pyruvic  straight  chain  or  unit  three  polymers  diverse. acid,  to  be  has to  the  only  include  and  with  recently  Most  instance,  D-glucose  branched  been  is  some  addition,  a lesser  evident  or  D-galactose,  extent,  in  the  or  from  known  general  unit one  present,  when  in  as  branched  known  chains  date, emerged.  varies  Only  are  poly-  have  (K12,K41,K79). except  the  to  points  repeating  Structures  can  be  but K17  and  unit  capsular  reported  D-galactose, these  serological  can  grouped  their  L-rhamnose  a single  two  importantly,  specific  is  constituents  For  is  to  acetals,  from  acidic combination  to  exist  as  with  one,  two  chains.  Kleb s i e l l a K-types qualitative  40,  seven  acetal.  side  pattern  residues  (K1,K5,K63)  the  and  In  K l eb s i e l l a K - t y p e s  approximately  three  with  1-carboxyethylidene  appear.  Although  which  -  the  D-mannose,  saccharide  4  according  structures  K44 b o t h whereas side  can  contain  K44 i s  chain.  7  8  now  be  structural  reactivity.  and  K45  combined  widely  D-glucuronic and  Originally in  K23  their  be  linear  polysaccharides  that  to  ,  this  at  with  differences  thought  group,  first K17  K17  believed  and  explain  it  K44.  their  -  II  5  -  METHODS USED IN STRUCTURAL ANALYSIS  Techniques  and methods u s e d  OF  POLYSACCHARIDES  in structural  a n a l y s i s of  9 10 polysaccharides  are w e l l e s t a b l i s h e d .  niques are e x p l o i t e d i n order or t o c o n f i r m  i n t e r p r e t a t i o n s from  making u s e o f s m a l l e r Nuclear (~^C),  magnetic  tity and  i n the present  with  of polysaccharide  produced,  on p r o b l e m s  With  this  samples  from Dr. I . jSrskov  f o u n d t o grow w e l l on i n c u b a t i o n  beef  analysis  However, each  solubility,  These c u l t u r e s were s t r e a k e d  from  while  the second  e x t r a c t medium.  degree of a c i d i t y i n mind, the in this  study,  K17 and K44.  of K l e b s i e l l a i n t h e form o f  a t 37 °C.  p l a t i n g were u s e d  Healthy  to i n o c u l a t e a  The b a c t e r i a m u l t i p l i e d p r o f u s e l y  grown on l a r g e  with  polysaccharide  t r a y s of sucrose-yeast After harvesting,  slime  while subse-  extract-agar  t h e b a c t e r i a were  a 1% p h e n o l s o l u t i o n and s e p a r a t e d - containing  stab  single  quently  days.  K17  o u t an a g a r p l a t e s and  i n a w a t e r b a t h a t 37°C f o r 3-4 h o u r s and were  destroyed  13  Klebsiella  shaking  medium f o r t h r e e  times.  Purification  and K44 were r e c e i v e d  colonies  methods  and mass s p e c t r o m e t r y were  encountered with  In S e p t e m b e r , 1977 b a c t e r i a l  cultures.  information  p h y s i c a l c h a r a c t e r i s t i c s s u c h as quan-  to h y d r o l y s i s .  I s o l a t i o n and  tech-  ("''H) and c a r b o n  i s an o u t l i n e o f t h e methods u s e d  emphasis  II.1  classical  investigation.  e x h i b i t s i t s own  following  additional  resonance, both proton  susceptibility  Many modern  q u a n t i t i e s and s h o r t e r  g a s - l i q u i d chromatography  employed K-type  to give  '  from t h e  by u 1 1 r a - c e n t r i f u g a t i o n .  - 6 The  viscous  Firstly with  into  the  s u p e r n a t a n t vi/as p u r i f i e d by  ethanol  and  secondly,  cetyltrimethylammonium  ternary  ammonium c a t i o n  polyanion  dissolving sodium  of  the  to  the  solution  remove t h e  chloride  i s removed d u r i n g  Of  the  produced II.2  two  know t h e the  Hydrolysis  Total  tography  again  cation. of  final  much as and  acid  aid  the  hydrolysis function  with  p r e s e n c e of  repeating  of  a m o l e c u l e of  weight approximating a molecular number o f  weight of  last  insoluble  into  precipitation is s a l t s as  sodium  Lyophilization  of  of  one  Component  a polysaccharide  repeating acid  units  as  after f i r s t  analysis sugar  one  by  600  repeating  must  by  reducing  gas-liquid  t h i s technique  the  relatively  is  1400  unit.  chroma-  also  confirms  simplified  E f f e c t i v e l y , n.m.r. and  the  ratio.  K l e b s i e l l a p o l y s a c c h a r i d e with million.  Sugars  in K l e b s i e l l a  (n.m.r.) s p e c t r o s c o p y  Moreover,  between  sugars i n the  of  T h i s i s somewhat s i m p l i f i e d  total  units  K17  K44.  l e a d s to  desired  t h i s end.  spectra  The  composition.  to  ;  precipitated  Characterization  i s composed o f  y i e l d i n g the  a useful  with  after  i s o l a t e d i n t h i s manner  Nuclear magnetic resonance ;  salt  Finally,  dialysis.  structural analysis  structure  carboxylic  was  polysaccharides  quantitative  K-types.  qua-  completes the p u r i f i c a t i o n .  a b o u t t w i c e as  begin  forms a water i n s o l u b l e  reformation  material  Total To  if  prevent  large  to  dialyzed  water,  (CETAVLON), a l a r g e  a c i d i c polysaccharides."'""'"  required  the  after dissolving in  bromide  which  three p r e c i p i t a t i o n s .  CETAVLON-polysaccharide p r e c i p i t a t e in 4 M  chloride,  ethanol  -  depending  on  molecular "sees" the  - 7 II.2.1  Total  A neutral lyzed  by  sugar  ratio  polysaccharide w i l l  a strong acid,  trifluoroacetic  acid,  monosaccharides  formed.  considerably tively  commonly s u l f u r i c ,  with n e g l i g i b l e  totally  hydro-  h y d r o c h l o r i c or  degradation  of  the  However, u r o n i c a c i d l i n k a g e s a r e  more r e s i s t a n t  under these  n o r m a l l y be  and  will  not h y d r o l y z e  quantita-  conditions. 12  By i s used methyl  a method d e v e l o p e d  in this laboratory,  mainly  the c a r b o x y l i c a c i d m o i e t y .  ester  to e s t e r i f y  i s then  anhydrous methanol, aqueous m e d i a .  reduced  bonds, the r e m a i n d e r ,  methanolysis  now  saccharide  t o be t o t a l l y  acid.  monosaccharides  The  acetylated  h y d r o l y z e d by themselves  to produce a l d i t o l  a p p e a r as t h e i r  reduced  hexoses.  are then  reduced  1  and  x  Uronic acids,  when p r e s e n t ,  D-glucuronic acid,  hexacetate  i s a widely accepted  been r e v i e w e d  sample s i z e s closely  2 |1 t r i f ilu o r o a ce t i c  after  the  f o r example,  above-mentioned  reactions.  G.l.c. has  a l l o w the p o l y -  1  (g.l.c.) analysis.  and  glycosidic  peracetates ^' ^' ^ for gas-liquid  chromatographic  s e r i e s of  saponified in  c l e a v e s most  made s u s c e p t i b l e ,  would a p p e a r as D - g l u c i t o l  The  w i t h sodium b o r o h y d r i d e i n  s i n c e t h e e s t e r would be  Although  methanolysis  stationary  extensively."*"^  i n the microgram  similar  range  p h a s e s was  employed  and  in t h i s  polar silicone  groups,  IOC,  Silar  having  i n the  carbohydrate  field  T h i s method o n l y r e q u i r e s  compounds w i t h e a s e .  SP-2340, a h i g h l y and  tool  performs  A wide s e l e c t i o n study.  c o n t a i n i n g 7 5%  similar  s e p a r a t i o n of of  Included  are  cyanopropyl  c o m p o s i t i o n , which  can  - 8 resolve the a l d i t o l D-galactose  acetates  of L-rrhamnose, D-mannose,  and D - g l u c o s e .  The  use o f a l d i t o l  investigation  acetate d e r i v a t i v e s throughout  i s because they  making q u a n t i t a t i o n e a s i e r .  give rise  t o only  one peak,  Other d e r i v a t i v e s t h a t  t h e a n o m e r i c c e n t e r may r e s u l t  this  preserve  i n two peaks r e p r e s e n t i n g t h e  a- and 8- anomers, f o r i n s t a n c e , t r imet hy 1 s i l y 1 d e r i v a t i v e s . The  fact  that stable,  for g . l . c . research  volatile  c o u l d be c o n s i d e r e d  i n t o high performance  as a c o m p l e m e n t a r y t e c h n i q u e  derivatives  are necessary  disadvantageous liquid  and c o n t i n u e d  chromatography  in carbohydrate  (h.p.l.c.)  chemistry i s  i n ev i t a b l e . II.2.2  Nuclear Proton  magnetic resonance spectroscopy  n.m.r. s p e c t r o s c o p y ,  organic chemist,  ("'"H and  ^C)  an i n d i s p e n s a b l e t o o l  has been u s e d p r o f i t a b l y 17  analysis  f o r the  in the s t r u c t u r a l 18 '  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 . Since being 19 13 i n t r o d u c e d i n 1969 t h e t e c h n i q u e of C n.m.r. has been  applied  e x t e n s i v e l y t o mono- and h i g h e r - s a c c h a r i d e s as seen 20  in  a recent  review.  P o l y s a c c h a r i d e s a r e amenable t o s t r u c t u r a l  a n a l y s i s by "^C n.m.r. s p e c t r o s c o p y ^ practised great  until  success  recently in this  in interpreting  In t h i s  study,  3  but t h i s  method h a s n o t been  laboratory largely  due t o t h e  "''H n.m.r. s p e c t r a .  n.m.r. s p e c t r a were o b t a i n e d  after the  p o l y s a c c h a r i d e underwent m i l d h y d r o l y s i s c o n d i t i o n s (0.4 M TFA,  20 m i n , 95°C) and t h e n  Kleb s i e l l a  was d i s s o l v e d i n D^O  polysaccharides are d i f f i c u l t  solvent.  to d i s s o l v e i n water,  - 9 even as t h e s o d i u m  salt  and  so s l i g h t l y  viscosity  remarkably. and  ever  form,and  s h a r p e n s t h e n.m.r.  size  spectra  f o r ~*"H and "^C n.m.r. r e s p e c t i v e l y .  With o l i g o s a c c h a r i d e s , however, with  as s m a l l  solubility  very  reasonable  as 10 m i l l i g r a m s  "^C n.m.r. A c e t o n e has been its  the polymer  Sample s i z e s were i n t h e r a n g e 20 t o 30 m i l l i g r a m s  80 t o 120 m i l l i g r a m s  obtained  reducing  spectra  f o r b o t h -^"H and  e m p l o y e d as a s t a n d a r d  and t h e f a c t t h a t  were  i t s chemical  because of  shift  i s inde-  pendent o f t e m p e r a t u r e . A l a r g e HOD peak, a l w a y s p r e s e n t change w i t h  D^O,  partially  "'"H n.m.r. s p e c t r a , interfering to s e c u r e HOD by  seen  spectra  t h e 100 MHz  anomeric region  be can  at d i f f e r e n t  t h e whole r e g i o n .  This  and 270 MHz i n s t r u m e n t s  II.1, spectra  obtained  can be o b s e r v e d  a t 65.0.  be t e n t a t i v e l y  g-linkages. interesting  Meaning  assigned  sidelight  on t h a t  temperature since  (see Figure  portion of spectra  the whole  can n o m i n a l l y  signals downfield  general  (95°C)  interference.  to ^-linkages  K17 f o l l o w s t h i s  tem-  However, as can be  the anomeric region that  the  and t h e r e -  whose a m b i e n t  at h i g h  without  taken  was a c c o m p l i s h e d  much more c o n f i d e n c e  "*"H n.m.r. s p e c t r o s c o p y  divided  placed  temperatures thus observing  areas of the anomeric region  be i n t e r p r e t e d w i t h  In  to*acetone. Since the  a r e 32°C and 23°C r e s p e c t i v e l y .  i n Figure  of the  i s t e m p e r a t u r e d e p e n d e n t , a d v a n t a g e was  piecing together  peratures  ex-  obscures the anomeric region  64.5 t o 5.5 r e l a t i v e  s i g n a l at separate  using  can  peak  due t o i n c o m p l e t e  o f 65.0  and t h o s e u p f i e l d t o  r u l e b u t e xh i b i ts an o t h er  II.2).  Normally  emphasis i s  between  64.5 t o 65.5 b u t i f  - 10 -  80°C HOD 4. 25 6 Figure  II.1  90°C HOD 4.126  P r o t o n n.m.r. s p e c t r a s h o w i n g t h e change i n HOD signal  location  with  temperature  i n the anomeric  64. 0 t o 6.0. A.  270 MHz s p e c t r u m , U n i v e r s i t y  o f B.C.  B.  100 MHz s p e c t r u m , U n i v e r s i t y  o f B.C.  C.  250 MHz s p e c t r u m , C.N.R.S.,  D.  220 MHz s p e c t r u m ,  University  Grenoble,  France  of Toronto  region  - 11 one  e x t e n d s o b s e r v a t i o n up t o  64.0  additional  information Q  may  be g l e a n e d from  s i g n a l s at  64.5-4.0 may  to c a r b o n - 5 ( H-5) neutral  the r e c o r d i n g .  sugars.  First  diagnose e i t h e r  o b s e r v e d i n K45 , a hydrogen  of u r o n i c a c i d s or o t h e r r i n g p r o t o n s of 21 2 2 I t has been shown ' t h a t H-5 o f and  3 - g a l a c t u ron i c a c i d s p r o d u c e s a "''H n.m.r. s i g n a l 64.40-4.75 w i t h  t h e a-anomer a l w a y s f u r t h e r  i n f e r e n c e then, H-5 upfield  bonded  downfield.  o f D - g l u c u r o n i c a c i d s would  s i n c e the average d e s h i e l d i n g  o v e r g l u c o p y r a n o s e i s about 0.26  effect 22  p.p.m.  between By  appear  slightly  of gal actopyranose  K17,  which  contains  an a - D - g l u c u r o n i c a c i d  component, g i v e s s i g n a l s a t 64.60 and  4.51,  be due  one o f w h i c h may  evidence f o r t h i s f i r s t l y sugar r a t i o five  t o H-5  of the u r o n i c a c i d .  comes from t h e f a c t  y i e l d s a rhamnose-glucose  r a t i o o f 3:2  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  5 of the 6 a n o m e r - l i k e s i g n a l s Secondly, the proton s i g n a l instead signal  of the n o r m a l at  3-glucose)  64.51  doublet.  in Figure  (C-2) and  and D-mannose p r o d u c e  to p u l l  a p p e a r s t o be a  o f 10 Hz  a t 64.51  triplet that  (generally  shows two  the  7-QBz f o r  couplings of  I I . 2 , other signals also  between 64.0-4.5 i n t h e s p e c t r u m  effect  accounting f o r only  7Hz.  As can be seen  number two  indicating  I t i s also noteworthy  64.60 shows a c o u p l i n g  approximately  total  i n t h e "*"H n.m.r. s p e c t r u m .  at  whereas the s i g n a l  that the  Other  o f K17.  P r o t o n s on  appear  carbon  c a r b o n number t h r e e (C-3) o f L-rhamnose 25 s i g n a l s i n the range  o f b o n d i n g on C-2  and/or  these s i g n a l s downfield  C-3  63.80-3.9 5.  The  as i n a p o l y s a c c h a r i d e  towards the anomeric  region  tends  - 12 -  "*"H n.m.r. s p e c t r a o f K17 p o l y s a c c h a r i d e signals 64.0  other  t o 6.0.  t h a n a n o m e r i c ones  i n the region  Shown a t a r r o w s .  A.  A t 80°C, s h o w i n g s i g n a l  a t 64.51.  ii.  A t 25°C, s h o w i n g  a t 64.29.  signal  depicting  - 13 p e r h a p s as much a s +0.4 p.p.m. that the  n.m.r. s i g n a l  H-2 p r o t o n  point linked  stated before,  to  other  than  in  polysaccharide The  are  a t 64.29 f o r K17 may a r i s e  of L - r h a m n o s e , where an L-rhamnose u n i t  a branching As  I n d i c a t i o n s are, t h e r e f o r e ,  at ring  structural  identifying  may be a u s e f u l d i a g n o s t i c t o o l  p r o p o r t i o n of deoxy  i n the region u p f i e l d  i . e . approximately  61.30.  sugars  from t h e a c e t o n e  Another  component s u g a r s  o f ''"H n.m.r. s i g n a l s  analysis.  p r e s e n c e and r e l a t i v e  evident  o c c u r s as  p o s i t i o n s 2 and 3.  the assignment  anomeric protons  from t h e  standard  f e a t u r e which a i d s i n  i stheir  spin-spin coupling  cons-  21 tants and  especially  3 - D - g a l a c t o s e a r e of t h e o r d e r  a-anomers J , =  2-3 Hz.  9  with  i n the anomeric r e g i o n .  a  -L-anomeric  proton  a lower  O  3-D-glucose  T  7-9 Hz w h e r e a s f o r t h e i r  F o r L-rhamnose t h e s i t u a t i o n having  J , ^ 1 Hz.  J ^  However, c h e m i c a l  9  i s reversed  2 Hz and t h e B-L-an omer i c  J, 1  proton  ^ ^  shift  i s more  1> ^  important normally  diagnostically difficult  because these  values  a r e s m a l l and  t o measure.  "^C n.m.r. s p e c t r o s c o p y  h a s been u s e d t h r o u g h o u t  this  work f o r t h e p o l y s a c c h a r i d e s and f o r o l i g o s a c c h a r i d e s o b t a i n e d from  degradation  s t u d i e s as w e l l .  a c e t o n e as s t a n d a r d As  giving  Samples were r u n i n D^O  a reference  with  peak a t 31.07 p.p.m.  a complement t o "*"H n.m.r., i n f o r m a t i o n on t h e number o f  monosaccharides i n the repeating  unit  of a K l e b s i e l l a  polysac-  c h a r i d e , t h e c o n f i g u r a t i o n a t t h e a n o m e r i c c e n t e r s and t h e 13 p r e s e n c e o f deoxy s u g a r s can be o b t a i n e d from C n.m.r.  - 14 experiments. ambient is  26  The d i f f e r e n c e b e i n g  101 p.p.m. may  3-linked  caution  t o a- and  gation  n.m.r.  that  "^C  were run a t  s o l v e n t re,S;On.an'ces  a dividing line  around  s i g n a l s appearing  down-  s u g a r s and t h o s e u p f i e l d a - l i n k e d  must be e x e r c i s e d  when a p p l y i n g  B-L-rhamnose l i n k a g e s , as seen  this  sugars.  general  i n the i n v e s t i -  o f K17. B o r k and c o w o r k e r s  constants ever,  Generally,  be drawn s u c h  represent  However, rule  spectra  t e m p e r a t u r e s i n c e no i n t e r f e r e n c e from  e x p e c t e d as i n  field  that  can be a p p l i e d  27  have shown  to a s s i g n  l o s s of s i g n a l s t r e n g t h  number  of peaks, already  preclude samples.  Total  C-H  coupling  anomeric c o n f i g u r a t i o n .  due t o s p l i t t i n g  about  t h e use of c o u p l i n g  13  that  30 i n d e c o u p l e d  constants  for  How-  and t h e a d d i t i o n a l "^C  spectra,  polysaccharide  a s s i g n m e n t o f even a n o m e r i c c a r b o n  s i g n a l s has 28  proven and  difficult.  other  data  Klebsiella  polysaccharides  o f K17 i s i l l u s t r a t e d  Klebsiella anomeric  capsular  centers  discussed  e x i s t but assignments.  in Figure  polysaccharides.  e x i s t s with  this  on m e t h y l  No p r o b l e m  spectra  A "^C  n.m.r.  of  in detecting  as i n ^"H n.m.r.  five  spectra  earlier.  determining  a polysaccharide  t h e number  the next  step  accomplished  through methylation  Sugars  and t y p e s o f s u g a r involves  residues  a n a l y s i s o f where  t h e s e c o m p o n e n t s a r e l i n k e d t o form t h e p o l y m e r i c is  glycosides  insufficient  II.3, typical  A n a l y s i s o f L i n k a g e P o s i t i o n f o r Component After  in  material 29  a r e a v a i l a b l e t o make a b s o l u t e  spectra  II.3  Some r e f e r e n c e  chain.  This  and s u b s e q u e n t c h a r a c t e r i z a t i o n  Field; S.W.j N.T., A.T.; P.W.; P.D.;  20 MHz 6 KHz 357,900 0.5 sec 18 qsec 0 sec  C-6 of /rhamnose (~CHg)  5 anomeric carbons  acetone I  C-6 of ^glucose (-CH 0H)  C-6 of glucuronic acid | (-C00H)  175 Figure I I . 3  150 C n.m.r. s p e c t r u m  2  100 of K l e b s i e l l a  ppm K17 c a p s u l a r  polysaccharide.  - 16 of products  by  gas-liquid  -  chromatography  and  gas-liquid  chromatography-mass spectrometry  (g.1.c.-m.s.)  Information  occurs  included on  on  whether b r a n c h i n g  in methylation  t h e number and  II.3.1  Methylation Methylation  order -OH  types  to achieve  absorption  as  confirmative  components  data  present.  analysis  of  carbohydrates  i s a well  complete m e t h y l a t i o n  (obtained  was  sufficient  from p a r t i a l  known p r o c e s s .  (judged  at 3500cm ^ i n t h e i n f r a r e d  Hakomori p r o c e d u r e ^ ' ^ saccharides  in a polysaccharide i s  a n a l y s i s as w e l l of sugar  techniques.  by  the  absence  spectrum) a  in this  study  h y d r o l y s i s , see  In of  single  of  oligo-  section 32  II.5),  but  f o r the n a t i v e p o l y s a c c h a r i d e  methylation  was  necessary.  have r e s u l t e d i n  3 - e l i m i n a t i o n s i d e r e a c t i o n s (see  r e d u c e d to a l d i t o l s  the  t h e C-6  by  then  alditol  function prior position  as w e l l  the  acetylated.  unmethylated  a C-6  Due  illustrates  derivative these  to r e d u c t i o n o f alditol  as l i n k a g e s i t e s  The  peak i n t h e  can  will  the  acetate  easily  be  the  be  the  identified  carboxyl-  disappearance  the appearance of a  g.l.c.  workup  would be a c e t y l a t e d .  r e a c t i o n s on  result  II.4)  o f l i n k age,, excep t i n  t o h y d r o l y s i s and  a c e t y l a t e d d e r i v a t i v e peak and  methylated  The  sites  residue.  same s e q u e n c e o f  reduced p o l y s a c c h a r i d e .  section  hydrolyzed,  d e r i v a t i v e of a u r o n i c a c i d moiety repeating  would  t h e m a t e r i a l was  represent  c a s e of a u r o n i c a c i d  carboxyl  The  and  Purdie  A second Hakomori m e t h y l a t i o n  Following methylation,  p o s i t i o n s of the  a subsequent  tracing.  p o i n t s i n a r e a c t i o n scheme (page  Figure 17).  C-6 II.4  of  - 1 7 -  ^GIcAL^Rha ^1  0  „ 3  a  Rha CH OH 2  1. C H S 0 C H ~ N a / C H I 4  3  3  OH O H  W  CH 0 3  in THF / EtOH  NaBH^ OCH  (1:1)  3  CH^OH  CH^OC^  1. A c i d  hydrolysis  2. N a B H ^ CH 0  OCH  3  3  CH-OAc I 2 H-C-OCH,  CH-OAc l 2 H-C-OAc  0-C-H  H-C-OAc  H-C-OCH, I 3 CH 0-C-H  (A)  CH  3  (B)  Methylation  3  AcO-C-H  CH OAc 2  (C)  analysis  H-C-OCH .H - C - O C H  H. - i -. O A. c  3  H-C-OAc  3  2  3  H-C-OAc 2  II.4  2  H-C-OAc CH 0-6-H  AcO-C-H  CH OAc  CHJ)Ac I 2 H~C~OCH  CH OAc  H - C - O A c C H j O - C.- H C'H OCH  Figure  3. AC2O / P y r i d i n e  3  3  CH-jO-fJ-H AcO-C-H  .AH.  (D)  scheme  3  f o r K17  CH-  (E) polysaccharide.  - 18 An i n t e r e s t i n g the m e t h y l a t i o n The m e t h y l a t e d reduced with furan  observation  was made i n t h i s work  a n a l y s i s o f K17 p o l y carboxyl  ( 1 : 1 ) a t room  and o l i g o s a c c h a r i d e s .  groups of the p o l y s a c c h a r i d e  sodium b o r o h y d r i d e  i n anhydrous  temperature.  during  Methylated  were  easily  ethanol-tetrahydroacidic oligosac-  c h a r i d e s , h o w e v e r , were n o t s u c c e s s f u l l y r e d u c e d i n t h i s manner but  required  reaction with l i t h i u m  tetrahydrofuran.  Normally  because the l a t t e r  aluminum h y d r i d e  the f i r s t  results  reaction i s preferred  i n aluminum h y d r o x i d e  being  w h i c h t e n d s to a b o s r b any m a t e r i a l w h i c h h a s f r e e groups present. methylated at t i m e s  Therefore  alditol  formed  hydroxyl  to q u a n t i t a t i v e l y recover  acetates  partially  from o l i g o s a c c h a r i d e s i t was  t o e x t r a c t t h e m a t e r i a l from  t h e method o f D u t t o n  in refluxing  and S m i t h " ^ .  aluminum h y d r o x i d e  necessary by  K44 was t h e n t r e a t e d i n t h e  same way w i t h no p r o b l e m . II.3.2 G;l.c.  a n a l y s i s of methylated  Reviews^' ^  a r e a v a i l a b l e on t h e a p p l i c a t i o n o f g . l . c .  to  the a n a l y s i s o f p a r t i a l l y  of  choice  in this  capabilities will  study  sugars,  liquids  rhamnitol acetates  phase.  acetates  Systems  the r e s o l v i n g  One s t a t i o n a r y p h a s e  can be  t h e r e s o l v i n g power o f s e v e r a l analyzed.  o f K17 n a t i v e p o l y s a c c h a r i d e mixture  acetates.  a l l p o s s i b l e c o m b i n a t i o n s o f methy-  e x e r c i s i n g  a l m o s t any m i x t u r e  separated  alditol  were made t o e x p l o i t  separate  b u t by  Methylation easily  methylated  of a certain l i q u i d  not adequately  lated  sugars  p r o v i d e d an  o f t r i - , d i - and mono-methyl a t e d L -  and t r i - and d i - m e t h y l a t e d D - g l u c i t o l  (see Figure  11. 5 ) .  Columns c o n t a i n i n g OV-17, OS-138  - 19 2,3,4-RHA  Column:  0V-17(1.8m 3%  Program:  x 3 mm)  o n G a s C h r o m Q.  175° 8 m i n , 2°/min t o 210°C.  Carrier gas: NL. 2 0 ml/min.  2,4-RHA U-RHA  I  2,3,6-GLC  II  2,3-GLC  l  ~ i —  175  175  ~185  TEMPERATURE  Figure  I I . 5 'G.l.c.  i—  195  (°C)  s e p a r a t i o n of a m i x t u r e  methylated  alditol  Kleb s i e l l a  K17 p o l y s a c c h a r i d e .  of p a r t i a l l y  acetates obtained  from  and  ECNSS-M  used  a l l gave  predominantly  peratures  good  20  -  s e p a r a t i o n s , however  because of e x c e l l e n t  o f 37 5 C .  A particularly  U  0V-17  stability  difficult  degradation  experiments  by  the i n i t i a l  reducing  HIEFF-1B, reason. other  For  K44  2,3,6of  on  only  where  good  adequate  lated  alditol  derivatives data  (see  section  II.3.3  Mass  Although  factors  spectrometry  pattern  from  times  study  before on  0U-17.  polar  compounds and  35  and  column  in  Albersheim partially  identification was  possible  made by  mass  o f peak  determined  by  of methylated  return isomers  et a l  g.l.c.  from  retention  spectrometry  areas  was  done  et a1 .  to confirm alditol  in particular,  36  methy-  sugars  methylated  r e c o r d i n g s and,  of  Albersheim  (m.s.) i s employed  of p a r t i a l l y  g.l.c.  and  a c e t a t e s of  A medium  f o r numerous  Quantitation  spectrometry  methylation  et a l  c o n f i r m a t i o n was  II.3.3).  response  indicated  retention  only  stated  elutes  alditol  acid  role.  Lindbergh  acetates.  alone,  Mass  by  i n the present  time  applying  s e p a r a t i o n of these  a better  Publications relative  possible  in reverse order  resolve  between  uronic  s e p a r a t i o n of di-0-methyl-L-rhamnose  played  tem-  2,4-di-0-  f o r the above  2,4,6-tri-O-methyl-D-glucose.  OV-17  provide  could not  after  compound  and  to  c o l u m n s o f 01/-17  again  column  and  I I . 4 ) was  on  the e t h y l a t e d  t h e 01/-17 c o l u m n and  chosen  the HIEFF-1B  OV-225 g a v e  gave  temperature  with the former Interestingly  the  (see section  up  separation  2 , 3 , 4-1 r i - 0 - m e t h y l - 1, 5 - d i - 0 - a c e t y l - L - r h a m n i t o l methyl-3-0-ethyl-1,5-di-0-acetyl-L-rhamnitol  was  the  acetates through  application  - 2>1 technique.  o f g.l.c.-m.s.  now a v a i l a b l e o n derivatives  C o n s i d e r a b l e data i s  fragmentation of these  and t h i s  laboratory  carbohydrate  a l s o keeps a supply o f standard  mass s p e c t r a on hand. 38 Swedish  workers  fragmentation  have made s y s t e m a t i c i n v e s t i g a t i o n s o f  patternsof partially  G e n e r a l i z a t i o n s made by them w i l l in  this  study.  provided  Uronic acid  methylated  a l d i t o l acetates.  be i l l u s t r a t e d  by e x a m p l e s  d e g r a d a t i o n and s u b s e q u e n t  d i - O - m e t h y 1 - 0 - e t h y 1-L-rhamnose i s o m e r s  from  ethylation  K17 and  K44 m e t h y l a t e d p o l y s a c c h a r i d e s . First will lated  o f a l l i t s h o u l d be m e n t i o n e d  not d i s t i n g u i s h alditol  t h a t mass s p e c t r o m e t r y  between d i a s t e r e o m e r i c p a r t i a l l y  acetates.  methy-  T h e r e f o r e , t h e s e d e r i v a t i v e s o f 2,3,4-  t r i - 0 - m e t h y 1-L-rhamnose ( 6 - d e o x y - L - m a n n o s e ) and 2 , 3 , 4 - t r i - O methyl-L-fucose mass  rather,  primary  Cleavage  i m p a c t m.s. does n o t y i e l d fragments  of carbon-carbon  preferred  shown  t h e same  spectrum. Electron  is  (6-deoxy-L-gal actose) give e s s e n t i a l l y  between  carbon  bonds a d j a c e n t t o m e t h o x y l a t e d  atoms. carbons  t o t h o s e a d j a c e n t t o a c e t o x y l a t e d c a r b o n s , as  below.  — g — O C H  H  o c c u r by f i s s i o n  a m o l e c u l a r i o n peak,  C •l  3  OCH, 3  Secondary  fragments  - 22 then o r i g i n a t e  from  ments by l o s s o f one o r more o f t h e f o l l o w i n g : (M.W.30), m e t h a n o l  (M.W.32), k e t e n e  primary  frag-  formaldehyde  (M.W.42), o r a c e t i c  acid  (M . W . 60 ) . When s t a n d a r d compounds a r e u n a v a i l a b l e , in u r o n i c a c i d  d e g r a d a t i o n s t u d i e s , mass s p e c t r a may be  interpreted using this observe  information.  s t r o n g p e a k s from  of t h e 2 - 0 - e t h y l isomer.  primary Although  p r e s e n t i n t h e mass s p e c t r u m rance of a d d i t i o n a l distinguish below.  as may o c c u r  Thus, one would  fragments  expect to  m / e l 3 1 and m / e l 7 5  these fragments  are also  of the 3-0-ethyl isomer,  appea-  p e a k s a t m / e l l 7 and m/el89 s e r v e s t o  t h e s e compounds.  Primary  f r a g m e n t a t i o n i s shown  Mass s p e c t r a o f t h e s e compounds i s d e m o n s t r a t e d i n  Figure I I . 6 . H C-0Ac  hUC-OAc  o  HC-0CH ChU o  HC-OCH  131 175 175 131  ChUO-CH 3 i AcO-CH  AcO-CH CH  HC-0CH„CH, I 2 3  3  139  175 131  (a)  23 -  3-0-ethyl-2^-di-0-methyl45-di-0-acetyl-L-rhamnitol  754'  REL.  50H  INT.  % 25^  1 ^njtni I ijimjiXf I  ' llll|llll|IUl|u  muiiyn|«it^inHim['iH»"['i' ntfwyntniiimt""!" li|l'liyiii|iirt|B>t|*iii|«i»|iiii|iiii[iiiipiii|iiH|"iittiii|»i»|Hi<|iiii|iiiijnntwmii |  50  100  150  200  inuiinni[iiii|iiit| im[iJii|i] 11^ 250  100-1 (b)  2-0-ethyl-3.4-di-0-methyl-1,5-di-0-acetyl-L-rhamnitol  75^  50H  25-  ii|i»|iii|ii^iiinuinn>oiiiipii  50  150  200  of u r o n i c acid  degradation  100  250  M/E Figure  II.6  Mass  spectra  from  K 1 7 ( a ) and  K44(b).  derivatives  - 24 II.3.4  "'"H n.m.r. s p e c t r o s c o p y and  thylated  has been done on p e r m e t h y 1 a t e d  polysaccharides on m e t h y l a t e d  Preliminary  observations  o f algaef"'"'^ however, g e n e r a l l y , bacterial in this  f o r n.m.r. s p e c t r o s c o p y  saccharides. solvents, ference  polysaccharides  study  of methylated  p o l y - and o l i g o -  Some a d v a n t a g e s a r e t h a t by e m p l o y i n g  such as d e u t e r a t e d  of the kind  Loss o f homogeneity  i s scarce.  i n d i c a t e a promising  chloroform  organic  or a c e t o n i t r i l e ,  inter-  from t h e l a r g e HOD peak e x p e r i e n c e d i n  "'"H n.m.r. s p e c t r a o f n a t i v e p o l y s a c c h a r i d e s  native  and p a r t i a l l y  m o n o s a c c h a r i d e s and d i s a c c h a r i d e s ^ ' ^ and on perme-  information  future  polysaccharides  oligosaccharides  Some r e s e a r c h methylated  of methylated  due t o h i g h  polysaccharides  viscosity  p o s e s no b a r r i e r  i s n o t a problem.  as i n w a t e r s o l u b l e  f o rmethylated  material.  Q u a n t i t i e s u s e d were i n t h e r a n g e o f 25 mg f o r m e t h y l a t e d polysaccharides  and l e s s f o r m e t h y l a t e d  oligosaccharides.  One p o i n t t o remember i s t h a t s a m p l e s must be p u r i f i e d in  order  to o b t a i n  reliable  results.  Dialysis  methylated  polysaccharides  methylated  oligosaccharides yielded light  stead  o f pure white  a small s i l i c a  a thin  samples.  By p a s s i n g  extraction for brown p r o d u c t s i n impure p r o d u c t s  g e l column u s i n g a s o l v e n t m i x t u r e ,  chloroform-methanol on  and c h l o r o f o r m  i n t h e case o f  (95:5),  determined  l a y e r chromatographic p l a t e ,  initially should  through  f o r instance  from  provide  values sufficient  pu r i f i c a t i o n . II.4  Sugar S e q u e n c e D e t e r m i n a t i o n Specific  degradation  by U r o n i c A c i d  of polysaccharides  Degradation  h a s been an a r e a  - 25 43 of keen i n t e r e s t r e c e n t l y . such as u r o n i c  44 '  Function a l l y - s e l e c t i v e  acid degradation  employed  in this  methods,  study,  provide  45 solutions to d i f f i c u l t associated quick  these  specific  result  t o degrade i s d e s t r o y e d ,  give  Some a d v a n t a g e s  e l i m i n a t i o n s are, r e a c t i o n s are  and easy t o p e r f o r m , t h e u r o n i c  difficult  for  with  s t r u c t u r a l problems.  a c i d moiety  and s p e c i f i c  that  The b a s i s  i s o u t l i n e d as below.  COOCH  COOCH-  3  3  +  R 0H,R 0H J  OCH/O^COOCH  J  Y  i  )~0R  rs  1  Z  cleavages  e a s i l y ^ i n t e r p r e t e d sequence i n f o r m a t i o n .  cleavage  2  normally  1  •+ R V  3  £=0  /  H  y  H0  3  (IV)  + R QH 1  OR' (III)  Methylated with  polysaccharides  from K17 and K44 were t r e a t e d  sodium m e t h y l s u l f i n y l m e t h a n i d e  a c t i n g a s base i n d i m e t h y l 45  sulfoxide.  Normally the Lindberg  treatment to e f f e c t f i n a l uronic  residue  (II).  method  degradation  c a l l s f o r mild  acidic  of t h e h e x - 4 - e n o - p y r a n o s y 1 -  However, A s p i n a l l e t a l have  reported^  t h a t t r e a t m e n t w i t h t h e above b a s e r e s u l t s i n c o m p l e t e l o s s o f the  hexuronic  acid residue  i s unnecessary. operation",  ( I I ) and s u b s e q u e n t a c i d  These w o r k e r s a l s o s u b m i t t h a t  methylated  polysaccharide  hydrolysis  in a "single 45 i s t r e a t e d w i t h base  - 26 and  then,without  isolation,  alkylated  t o which  uronic acid  l a b e l the s i t e ( s )  These m o d i f i c a t i o n s were a p p l i e d  with ethyl  iodide to  r e s i d u e s were a t t a c h e d .  with success i n the present  i n v e s t i g at i o n . It place  s h o u l d be m e n t i o n e d  i f the substituent  i s another resulting and if  sugar.  is  When t h e a c i d i c  r e d u c i n g end i s a l s o  i t happens t o be a n o t h e r  polysaccharide. further  performed  from  both  Exposed  sugar,  not occur.  during  the analysis  such a c o n s t i t u e n t immediately  of a c e t i c  when t h e e l i m i n a t i o n anhydride.^  identified.  such as 3 - d e o x y - h e x - 2 further re-  s u g a r s and d i s a p p e a r a n c e o f  provides evidence f o r placing  a d j a c e n t t o t h e degraded  i t i n sequence  hexuronate r e s i d u e .  these p o i n t s f o r the 8 - e l i m i n ation  Sugar Sequence D e t e r m i n a t i o n from or graded  acid  of p o l y s a c c h a r i d e s .  Partial Hydrolysis  h y d r o l y s i s i s a well-known  t h e d e t e r m i n a t i o n of sugar  certain  Products  of K44.  Partial  tion  which,  These a c i d - l a b i l e u n i t s a r e d e s t r o y e d  of m e t h y l a t e d  II.7 illustrates  environment  can be p r o t e c t e d  ( r e s i d u e A on page 27) i s formed  will  for  i s eliminated the  c o n t i n u e s t o degrade t h e  reducing sugars  i n the presence  component  e l i m i n a t e s t h e C-3 s u b s t i t u e n t  d e g r a d a t i o n by a c e t y l a t i o n  action  II.5  sugar  s i d e s o f t h e bond c l e a v a g e can t h u s be  enopyranose,  reaction  d e g r a d a t i o n can t a k e  s e n s i t i v e to the basic  However, when a b a s e - s t a b l e u n i t ,  Figure  further  on C-4 of t h e u r o n i c a c i d  in a chain-type reaction  from  that  s e q u e n c e and a n o m e r i c  O l i g o s a c c h a r i d e s , produced  c o n d i t i o n s of h y d r o l y s i s  (acid  method configuraunder  c o n c e n t r a t i o n , temperature  27  ^Glc^GtcL-^GlcA —^Rhal—^Rha — (X j9 cx c* 1  CH OCH  CH-0CH-  •o  -o.  9  COOCH, CH.O/—0  OCH.  OCH  1  OCH  CH OCH 2  ?  -o  3  2. Ethyl Iodide 3  OCH CH 2  OCH  CH 0  BASE  1.  CH OCH~  3  CHJVT~° 3/cH  CH_0/T~0  3  3  CH 0  OCK  3  H  T  0  0CH CH 2  3  +  2-0-ethyl-3.£-di-0-methyl-L-rhamnose 2,4-di-O-methyl-L-rhamnose 2A6-tri-0-methyl-D-glucose  Figure II.7  Uronic  acid  d e g r a d a t i o n of K l e b s i e l l a  poly saccharide.  K44  H  3 OCH.  - 28 and  length  o f h y d r o l y s i s ) , can be c h a r a c t e r i z e d by  and  n.m.r. s t u d i e s  i n order  s t r u c t u r e of the t o t a l work w i t h lished  Klebsiella  by i s o l a t i n g Hydrolysis  ring  t o e l u c i d a t e t h e more complex  molecule.  This  i s e s p e c i a l l y so i n  where t h e r e p e a t i n g only  two o r t h r e e  of p o l y s a c c h a r i d e s  s i z e and c o n f o r m a t i o n  methylation  u n i t may be  estab-  oligosaccharides.  i s a f f e c t e d by t h e t y p e ,  of the monosaccharide r e s i d u e s , the  anomeric c o n f i g u r a t i o n of the g l y c o s i d i c l i n k a g e s , the p o s i t i o n of l i n k a g e such as ( (1*2 ) , (1*3) , (1*4)  and ( 1 * 6 ) ) , t h e p r e s e n c e  of f u n c t i o n a l g r o u p s , and t h e i n t e n s i t y  of i n t e r -  molecular  f o r the a c i d  interactions.  Rate c o n s t a n t s  and i n t r a catalyzed 48  h y d r o l y s i s of a l a r g e although  data  v a r i e t y of g l y c o s i d e s  on p o l y s a c c h a r i d e s  have been  are mainly  reviewed,  unpublished  and  empirical. Inferences For  instance,  resistant  t h e g l y c o s i d i c bond o f u r o n i c  t o h y d r o l y s i s so t h a t  structural acid  can be made, h o w e v e r , and a r e g e n e r a l l y  have been i s o l a t e d .  reducing  acid i s extremely  i n the m a j o r i t y  studies aldobiouronic  accepted  of K l e b s i e l l a  a c i d and u s u a l l y a l d o t r i o u r o n i c  A g l y c o s i d i c l i n k a g e a t a non-  end i s h y d r o l y z e d  more r a p i d l y t h a n  others.  This  fact  i s made u s e o f i n a n a l y s i s o f K17 where t h e s i n g l e u n i t Lrhamnose s i d e  chain  was c l e a v e d  produce the s t r a i g h t chain The  by m i l d  polysaccharide  disadvantage of p a r t i a l  yields  of o l i g o s a c c h a r i d e s  better  y i e l d s a modified  acidic  "backbone".  hydrolysis i s that  are r e a l i z e d .  apparatus s i m i l a r  G a l a n o s e t a l " ^ was e m p l o y e d h e r e .  c o n d i t i o n s to  very  In an a t t e m p t  poor  to produc  t o t h a t u s e d by  The p r i n c i p l e  of continuous  removal  of  hydrolysis coal  to  column  table  subsequent  the  monosaccharides  is  gel was  amount  of  In partial  by  ion  was  from by  form. to  column the  paper  II.6  (45  a number The  one  of  be  (90  In  mg)  gram  size  by  P-2.  sieve  were gel  Two  were,  through  K17,  this twice  generated  as  polysaccharide. from  chromatographic from and in  the  neutral the  milliliter  techni-  hydrolysis portions  chloride  individually  filtration  after  of  sui-  when  obtained  1-X2  then  felt  fact,  was  of  acidic  Dowex  char-  accomplished  produced  into  total The  i t is  hydrolysis  standard  material  separated  Bio-Gel  theory.  since  oligosaccharides  molecular  or  separated  chromatography  fractions collected  1yophilization,  monitored  term  Oxidation  "classical  oxidation  investigations."^ (generally system This  as  in  the  sodium)  shown  are  method" area  Aqueous  reaction  which  1,2-diol  from  prevent  chromatography.  periodate  drates  mg)  portions  molecular  The  diol  can  partial  These  of  apparatus  chromatography,  Periodate  salts  the  purify  first  basic  tetrasaccharide  employed.  according a  to  exchange  formate  on  acid  the  to  chromatography.  to  acidic  hydrolysis  apparatus  our  filtration  order  were  in  is  s e pa r a t i o n ;  applied  aldobiouronic  ques  removed  monosaccharide  apparatus  - 29 from s o l u t i o n  oligosaccharides  on  will page  i s well  would  well  be  used  polysaccharide  solutions cleave  of  periodic  the  bond  to  describ  structural acid  between  and a  its 1,2-  30. suited  polyhydroxylic  structures  of  could  then  by be  to  the  nature oxidized  water-soluble and to  whose a  carbohy  cyclic  straight  chain  -  30 -  +  CHOH  +  HC = 0  CHOH R  a,  oj-d i a l d e h y de .  drate polyol  polymer  uses  analytical periodate on can  of periodate  sodium  sugar  from  the carbohy-  borohydride  using  oxidation small  solutions to maintain of periodate  be d e t e r m i n e d  linkages,  with  product  to yield the  of handling.  technique  t h e number  zable  The p o l y a l d e h y d e  i s reduced  f o r ease The  R  unit,  sugar  a n d 1-6 1 i n k e d , i i n - ch a i n  atory  data  on l i n k a g e  sites  to that  Secondly  a n d more  synthetic  technique  isolating  o l i g o s a c c h a r i d e fragments  done  sugars  with  in a  i s consumed f o rthose  This  found  important  and d i l u t e Information  polysaccharide per o x i d i with  of a non-reducing  sugars)  analysis.  oxidation.  except  i n t h e case  One i s an  of m a t e r i a l  selective  of periodate  i n the repeating  a n d two m o l e s  amounts  sensitive  (one mole  are twofold.  yields  1-6  terminal confirm-  i n methylation  i s a preparative or  l a r g e r q u a n t i t i e s with composed  t h e aim o f  of periodate  52 insensitive  sugar  to  oligomeric units.  the  liberate  more a c i d  dized  sugar  components.  labile  residues  Smith This  acetal linkages compared  degradation method  takes  resulting  i s employed advantage of  from  t o t h e more s t a b l e  the oxi-  intact  -  31 -  — G l c ~ ~ G lc —^ Glc A — ? Rha —? R h a — 1  CH OH  1  1  1  CH OH  2  2  HO  ~  "polysaccharide"  * = site of periodate attack CH OH  CH OH  2  COOH  2  OH  II  ||  0  OH  II  0  0  II  II --J*  V  0  ^ W  O0H  "polyaldehyde"  0  BH; CH OH  CH OH  COOH  0.  h~0.  2  ?  HO / ~ 0  HO/dT^s CH.  ^CH V-O 3  HO'  I OH  H~C C H I I HO OH 2  HJC d I2| I " HO OH  0  2  OH  2  HO  'polyalcohol'  Smith degradation  m ^ T ^ o — r t -  6H'OH ^CH  HO  OH  2 x glycolaldehyde 1.2-propanediol Figure II.8  Periodate-Smith polysaccharide.  OH  "oligomer'  CH 0H 2  glyceraldehyde D-erythronic acid  degradation  of K l e b s i e l l a K44  glycosidic  linkages  demonstrates periodate  how  achieved  in  to  et  this  comparing  date  oxidation  rate  constant  that  of  technique  approach  is  the  indicated  attack  ones.  was u s e d  periodate  application  of  sugar  residues  are  others.  This  second-order  can  more be  rate  methyl  a-L-rhamnopyranoside  3-D-glucopyranoside.  repulsion  inhibitive  between  the  effect  uronate  shown  For  Also  quantitafor  seven  fact  because  anion  and  from  perio-  example,  is  the  Smith  susceptible  constants  for  an  selective  communication  glycopy ranosides.  show  the  oxidation  a recent  methyl  polysaccharides  II.8  in  in  than the  to  of  methyl  Figure  K44.  a l " ^ certain  by  static  of  -  nonoxidized  synthetic  study  As  periodate  tively  the  interesting  degradation. Painter  this  oxidation  Another  of  32  times  that  of  the  the  acidic  electro-  attacking  54 periodate  ion  K17j s o d i u m prevent (see  was u s e d  under-oxidation  in  the  side-chain  by  for  in  K17  II.9,  the  fact  in  the page  K17  experiment  of  Figure  aided  versatility  perchldrate  experimental  added  (See  adds  to  the  33).  that  the  target  chain"  residue.  on  the  where  branching several  sugar  attempts  the  of  for  success the  However, oxidize  other  the  salt  the  the  was  units.  attack  also  was a m o r e an  "in-  Smith  linkage  methylation had  was  than  selective  side-chain  hydrolysis  polymer  experiment  rather  and  rhamnose  oxidizable  sugar of  of  no  periodate  For  repulsion  oxidation  subsequent  partial  oxidations.  this  particular  terminal  yielded  after at  6-8' ) .  This  accessible side-chain  conclusively  suppress  "total"  presence  easily  degradation  to  periodate  selectively  page  However,  to  site  analysis,  failed.  -^Glc —^Rha -r^GlcA -^RhJ 1  1  1  CX  CK  Rha CH 0H 2  ~0 *  most vulnerable site for periodate attack  1. 0.027 M 10". 2 h 2. BH7  H O * OH  H t 2  CH  3. 0.5 M trifluoroacetic acid, 2  •17 h, R.T.  HO OH  OH U. methylation 5. carboxyl reduction 6. hydrolysis 2,3,5-tri -O-methylglucose 3,4-di-O-methylrhamnose  gu r e I I . 9  Selective K17  2.3- di-O-methylglucose 2.4- di-O-methylrhamnose  p e r i o d a t e -Smith  polysaccharide.  degradation  of  - 34 11.7  Anomeric Linkage  Determination  The a s s i g n m e n t o f a n o m e r i c c o n f i g u r a t i o n t o s p e c i f i c glycosidic linkages in a polysaccharide the  simpler nuclear  i s accomplished  magnetic resonance s p e c t r a of t h e i r o l i 1  gomeric s u b u n i t s . discussed this  The t e c h n i q u e  (Section lated  This  in Section  o l i g o s a c c h a r i d e s from  long  information  will  give  regard  the wealth  b u t no d e t a i l  of C o n f i g u r a t i o n  (partially  (alditol  methylated  anomeric  would  cannot  magnetic  the  compete  resonance  I I . 1 , page 3 5 ) .  and r o u t i n e l y by c i r c u l a r  studies  chemists.  concerning  t h i s method  in a nuclear  Use i s made o f d e r i v a t i v e s a l r e a d y tation  II.4).  r o t a t i o n measurement  o f Component  The D and L c o n f i g u r a t i o n s o f component mined s i m p l y  (Section  A s e r i e s of oligosaccharides  of i n f o r m a t i o n  Determination  f o r methy-  of carbohydrate  but u n d e r s t a n d a b l y  (See T a b l e  were  i n d i c a t i o n s of the o v e r a l l  of a polysaccharide  In  acid hydrolysis  degradation  of o p t i c a l  the f r i e n d  sugar r e s i d u e s .  in this  11.8  specific  technique  been c o n s i d e r e d  experiment.  acetates)  alditol  d e r i v a t i v e s are conveniently and  II.2.2.  I I . 5 ) and "*"H n.m.r. s p e c t r a were o b t a i n e d  individual  with  C n.m.r. h a s been  f o r o l i g o s a c c h a r i d e s from p a r t i a l  configuration  aid  H and  i n terms o f p o l y s a c c h a r i d e s  The c l a s s i c a l has  of  13  i n v e s t i g a t i o n "*"H n.m.r. and "^C n.m.r. s p e c t r a  obtained  through  a v a i l a b l e from or methylation  measurements. ^ sugar  quanti-  studies  These v o l a t i l e g . l . c .  c o l l e c t e d as t h e y  e l u t e d from a s u i t a b l e c o l u m n .  sugars are deter-  dichroism  acetates).  Sugars  (Sections  are separated II.3.2  and I I . 2 . 1 ) ;  TABLE  I I . 1 C a l c u l a t e d and M e a s u r e d V a l u e s f o r S p e c i f i c R o t a t i o n s and K44 P o l y - and O l i g o s a c c h a r i d e s  Compound  of K l e b s i e l l a  K17  Optical Rotation at S p e c i f i c Cone W a v e l e n g t h (nm) " Na589 FTTgTTS FTg54~S PTgTTJ^ FT7gT63 j j c ^ m e a s .  „ A [a] calc? A  3  K  D  K17 capsular  polysaccharide  polysaccharide  P I  ( N a ) 0.57  +0.17  +0.18  +0.20  +0.33  +0.48  + 30  + 15  +15  1.81  +0.80  +0.83  +0.94  +1.55  +2.27  +444  + 34  +10  1 . 38  +0 . 52  +0.55  +0 . 62  1.12  +1.24  +1.30  +1.46  0.82  +0.03  +0.03  +0 . 0 4 3  +0 . 0 48  +0. 0 58 +0 . 080  1 .23  +0 . 10  +0. 11  +0 . 12  +0. 18  1 .95  + 0 .48  +0 . 50  + 0 .56  1 . 53  +0 . 62  + 0 .65  1 . 44  -0 .27  0 . 46 0 .87  +  6  tetrasaccharide aldobiouronic  acid  disaccharide  capsular  polysaccharide  capsular  polysaccharide  pentasaccharide f tetrasaccharide trisaccharide^ aldobiouronic  acid  disaccharide^  ( N a ) 1 .08 +  +1.02 +2.43  +1.54 +3.69  page  9;8. ^ F r o m (  periodate  oxidation;  +24  + 77  +34  +3.7  -1Z5  +16  + 4.0  -  8  + 12  +0 . 21  +8 . 1  -  8  + 16  + 0 .90  + 1. 22  + 25  +  7  + 18  +0 . 73  + 1. 19  + 1. 74  + 41  +  24  + 17  - 0 . 28  -0 .31  -0 . 53  - 0 . 77  -19  - 31  + 12  -0 .032  -0 .032  -0 .0 3 5  -0 .0 62 - 0 . 11  -7.0  -  + 7  -0 .38  -0 .40  - 0 . 74  -44  - 48  i n g/100 ml w a t e r . 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STRUCTURAL  INVESTIGATION  SEROTYPE K17 CAPSULAR  OF  Klebsiella  POLYSACCHARIDE  - 41 III.1  Abstract Klebsiella  gated  -  by  K17  capsular  the t e c h n i q u e s  periodate  polysaccharide  of m e t h y l a t i o n ,  o x i d a t i o n , and  uronic acid  In a d d i t i o n ,  oxidation-Smith  degradation  was  e m p l o y e d t o remove a rhamnose s i d e c h a i n .  was  f o u n d t o c o n s i s t of t h e  polysaccharide  derived  through  pentasaccharide  A n o m e r i c c o n f i g u r a t i o n s of t h e "'"H and  and  degradative  ^C  X  investi-  degradation.  c o n c e p t of s e l e c t i v e  were d e t e r m i n e d by  been  partial hydrolysis,  the  shown b e l o w .  periodate  has  The  repeating unit sugar  n.m.r. s p e c t r o s c o p y  p o l y - and  structure  residues on  the  oligosaccharides  obtained  procedures.  _ B - D - G l c p - ( l ~ - 2 ) - - L - R h a p - ( 1*4) - O-D-G a  t  3  1 a-L-Rhap  1 cAp-( 1*3) - 6-L-R hap-(  1*  - 42 111.2  -  In t r o d u c t i o n Of  the  approximately  80  serologically different 1 2  strains  of K l e b s i e l l a  according  t o the  polysaccharides, the  b a c t e r i a that  q u a l i t a t i v e composition  of t h e i r  capsular  a s m a l l g r o u p of t h e s e s e r o t y p e s  consists  sugars D-glucuronic  firstthought  3  capsular  a c i d , D - g l u c o s e , and  to i n c l u d e o n l y  shown t h a t K23  have been g r o u p e d '  K17  and  K44,  L-rhamnose.  recent  The  the  and  K45  polysaccharide  s t r u c t u r e of t h e  p r e d i c t i o n , ^ ' ^ from  r e a c t i o n s of K l e b s i e l l a and  f i n d i n g s have  are has  part  of t h i s  group a l s o .  anti-Klebsiella  capsular  We  polysaccharide  immunochemical  t h a t K17  this  study.  now  capsular  report  of t y p e  r e l a t i o n s h i p s by  in antipneumococcal,  sera,  Each  a u n i q u e s t r u c t u r e , however,  K17. cross-  anti-Salmonella, polysaccharide  would have a n o n r e d u c i n g L - r h a m n o s e e n d - g r o u p has by  At  4  which determines i t s s e r o l o g i c a l s p e c i f i c i t y . on  of  been  confirmed  - 43 111 . 3  Results  Composition  and D i s c u s s i o n  and n.m.r.  Klebsiella sular  K17 was grown on an a g a r medium; t h e cap-  polysaccharide  purified  spectra  was i s o l a t e d  by C e t a v l o n  precipitation.  moved as one band d u r i n g The  molecular  gel  chromatography An  charide  The p u r i f i e d  was d e t e r m i n e d by  t o be 9.4 x 10"'.  acid hydrolyzate  of the carboxyl-reduced^  rhamnose and g l u c o s e  3:2, a n a l y z e d  chromatography  material  e l e c t r o p h o r e s i s and had C°G Q +30° .  weight of the p o l y s a c c h a r i d e  contained  portions  by u 1 t r a c e n t r i f u g a t i o n and  as t h e i r  (g.l.c).  i n the r e l a t i v e  alditol  acetates  Rhamnose was c o n f i r m e d  L c o n f i g u r a t i o n and g l u c o s e  polysacpro-  by g a s - l i q u i d t o be o f t h e  o f t h e D c o n f i g u r a t i o n by  circular  8 dichroism  ( c d . ) measurement  The has  five  on t h e i r  "^C n.m.r. s p e c t r u m  anomeric carbon  attributable not  indicates that  sugar r e s i d u e s  C-6 s i g n a l .  The r e l a t i v e l y  one  downfield  shift  since of the  l a r g e s i g n a l a t 17.4 p.p.m. can  t o 0-6 o f rhamnose (CH^) i n d i c a t i n g t h a t more than  rhamnose r e s i d u e  Finally,  103.2,  the glucose i s  at the 6 p o s i t i o n  result in a significant  assigned  the p o l y s a c c h a r i  s i g n a l s a t 105.0,  l i n k a g e would 9  be  acetates.  The s i g n a l a t 61.5 p.p.m.,  t o C-6 o f g l u c o s e ,  l i n k e d to other  i n d i c a t e s that  atoms w i t h  101.3, 100.8 and 96.4 p.p.m.  alditol  i s present  i n the repeating  u n i t o f K17.  t h e s i g n a l a t 175 p.p.m. was due t o t h e c a r b o x y l  of g l u c u r o n i c  acid.  group  A s s i g n m e n t o f t h e a n o m e r i c s i g n a l s was  made a f t e r e x a m i n a t i o n  o f t h e ^C  p o l y - and o l i g o s a c c h a r i d e s  X  n.m.r. s p e c t r a  (see Table  of derived  III.lpage44).  TABLE I I I . l  N.m.r. d a t a and  of K l e b s i e l l a  K17 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-  oligosaccharides  C ompoun d  .  1  _C 6  G lc^-Rha-OH  (J-, ) , i n t e g r a l , 1>^  5. 36 4. 84 4. 63 1. 28  p  GlcA^-^Rha-OH a  9  assignment  (1), 0.5, H - l a-Rha-OH <T<1), 0.5, H - l B-Rha-OH (7), 1, H - l 3 - G l c ( J , , 6 ) , 3, H-6 Rha  5. 17 ( b ) ,  1.6,  H - l a - G l c A and H - l a-Rha-OH 4. 87 ( b ) , 0.4, H - l B-Rha-OH 1. 33 ( J .6), 3, H-6 Rha c  5, 6  Gl c^-^R h a ^ — - G l cA-^—R h a-OH D  Oo  5. 17 ( b ) ,  LX  5. 11 4. 87 4. 57 1. 31  H - l a-GlcA and H - l a-Rha-OH (2), 1, H - l a-Rha (5), 0.5, H - l B-Rha-OH (7-8), 1, H - l B - G l c ( J . , 6 ) , 6, H-6 Rha 5, 6  1.5,  p.p.m.  105. 1 93. 7 61. 4 17. 6  assignment  C-1 C-1 C-6 C-6  B-Glc aY6-RhaGlc Rha  96. 7 96. 3 94. 4 17 .7  C-1 a-Rha-OH C-1 B-Rha-OH C-1 - G l c A C-6 Rha  105. 0 100. 5 96. 6 96. 2 94. 4 61. 4 17. 3  C-1 B - G l c C-1 - R h a C-1 a-Rha-OH C-1 B-Rha-OH C-1 a-Gl cA C-6 G l c C-6 Rha  a  a  TABLE  III.l  (continued)  Compound  1^ 6 (J^  integral,  a  4  12 1C  HA  14 V ° 1  13 C A  HA  1  5.17 5.11 4.87 4. 56 1.34 1.27  V  Pla  13^, assignment  ( 3 ) , 1, H - l a-GlcA ( s ) , 1, H - l a-Rha ( s ) , 1, H - l B-Rha ( 7 . 5 ) , 1, H--1 B - G l c ( 6 ) , 3, H-6 Rha ( 6 ) , . 3 , H-6 'Rha  p.p.m.  assignment  6  C-1 C-1 C-1 C-1 C-6 C-6  104. 8 101. 3 100 .3 96. 1 61. 4 17. 5  B-Gl c B-Rha a-Rha a-GlcA of G l c of Rha Un  4 12 14 1 3 1 -^G1 c^r^R h a^-^G 1 c A ^ ^ R h a ^ — • a a  B  a  B  1  Rha capsular polysaccharide  a  Chemical  shift  relative  silapentane-1-su1fon ate ^Values  i n Hz; b, b r o a d ;  Chemical ^A p a i r  shift  relative  of unresolved  A triplet  5.15 5.04 4.85 4.60 4.51 1.30  to internal  ( 3 - 4 ) , 1, H - l a-GlcA ( ) , 2, H - l a-Rha,a-Rha ( s ) , 1, H - l B-Rha ( 1 0 ) , 1, H - l B - G l c and ( 7 ) , 1, H-5 a-GlcA ( 6 ) , 9, H-6 Rha d  e  105.0 103.2 101. 3 100.8 96.4 61.5 17.4 sodium  C-1 B - G l c C-1 a - R h a ( t e r m i n a l ) C-1 B-Rha "C-1 a-Rha C-1 a-GdcA-. C-6 G l c C-6 Rha  acetone;  52.23 d o w n f i e l d from  4, 4-dim et h y l - 4-  acetone;  31.07 p.p.m. d o w n f i e l d from D.S.S.  (D.S.S.) s, s i n g l e t . to internal  singlets.  with coupling constant  of unknown  origin.  -  Initially to  interpret.  -  46  t h e "'"H n.m.r. s p e c t r u m  The d i f f i c u l t y  arose  discerned  i n the anomeric region  spectrum  from a 2 50 MHz i n s t r u m e n t  Appendix,  s p e c t r u m no. 1 ) .  was a c h i e v e d  by e x a m i n a t i o n  oligosaccharides derived during  the s t r u c t u r a l  Studies  done i n t h i s  possible  and e s p e c i a l l y  field  approaching  present  glucuronic acid.  (64.5-5.5). shows t h i s  from t h e c a p s u l a r  polysaccharide  l a b o r a t o r y " ' ' ^ have p o i n t e d  of a - D - g l u c u r o n i c  H-l  o f a 8 - D - g l u c o s e component.  of g l u c u r o n i c  a c i d , t o a p p e a r down( 64.5).  a t 64.51 has been a s s i g n e d  Therefore,  at  t o H-5 o f B-D-  also that the s i g n a l  constant  44)  out that i t i s  o f t h e H-5 p r o t o n  a rather large coupling  a t 64.60  (10 Hz) f o r a s s i g n m e n t t o  The i n t e g r a l  t o rhamnose m e t h y l p r o t o n s  presence of three  c l e a r l y (see  i n v e s t i g a t i o n ( s e e T a b l e 111.1, page  I t i s notable  anomeric protons  The "'"H n.m.r.  of "*"H n.m.r. s p e c t r a o f p o l y - and  has  The  when s i x s i g n a l s c o u l d be  the anomeric region  the s i g n a l  difficult  Assignment of the anomeric s i g n a l s  f o r t h e n.m.r. s i g n a l  acid,  o f K17 was  ratio  o f 5:9 f o r  confirms the  rhamnose r e s i d u e s p-e r -r e p e a L i n g .urnfitt.  absence of pyruvate  and a c e t a t e  i s d e m o n s t r a t e d by t h e "*"H n.m.r. These t h r e e  s e t s of data  be  composed o f t h r e e  one  of D-glucuronic  residues  i n K17 c a p s u l a r  polysacchari  spectrum. show K17 p o l y s a c c h a r i d e t o  o f L-rhamnose, one o f D - g l u c o s e ,  a c i d , and t o have t h r e e  a- and two  B-glycosidic linkages. Methylation  analysis 11  Methylation quent r e d u c t i o n  with  12 '  of the a c i d i c  polysaccharide,  sodium b o r o h y d r i d e ,  subse-  h y d r o l y s i s , and  - 47  derivatization composed  as a l d i t o l  residue  repeating  t o a branch point  to the terminal  sugar  methylg1ucose i s the only from t h e D - g l u c u r o n i c  u n i t (see Table  methylation  results.  acetates  in this  and t h a t  III.2,  o f a t r i m e t h y l rhamnose  of a s i d e  chain.  d e r i v a t i v e which  a c i d component.  of a l l t h e c o n s t i t u e n t  readily  i n d i c a t e d t h a t K17 i s  The p r e s e n c e of a m o n o m e t h y l rhamnose r e s i d u e i s  attributable  and,  acetates  of a p e n t a s a c c h a r i d e  page 4 8 ) .  alditol  -  The 2,3-di^O-  could  have a r i s e n  The p y r a n o s i d i c  rhamnose s u g a r s i s d e m o n s t r a t e d by t h e P o s i t i o n s of m e t h y l a t i o n  i n the derived  were d e t e r m i n e d by mass s p e c t r o m e t r y  investigation, individual  identified  nature  by t h e i r  stereoisomers  characteristic  (m.s.)^  3  were  r e t e n t i o n t i m e s on  g.l.c. The p r e s e n c e of 3, 4 - d i - O - m e t h y l r h a m n o s e i n t h e methylation hydrolysis acidic  a n a l y s i s can be e x p l a i n e d (~25?o) t a k i n g p l a c e upon  ion-exchange r e s i n  by a s m a l l  prolonged  when c o n v e r t i n g  initial  amount o f  exposure t o the  the i s o l a t e d  saccharide  t o the a c i d form.  side  w o u l d t h u s be removed and s u b s e q u e n t m e t h y l a t i o n  the  chain  branch point  (position the  branch  oxidation  3).  rhamnose w o u l d o c c u r  The s i t e o f s i d e c h a i n  point  rhamnose i s s u p p o r t e d  analysis.  (see Table  labile  at t h e l i n k a g e  by l a t e r  of  site  periodate  a n a l y s i s showed  when t h e t i m e of c o n t a c t III.3,  rhamnose  l i n k a g e to p o s i t i o n 3 of  A second m e t h y l a t i o n  no h y d r o l y s i s o c c u r r e d was r e d u c e d ,  The r e l a t i v e l y  poly-  column I, page  with  50).  that  the r e s i n  TABLE  III.2  Methylation  analysis  of o r i g i n a l  Methylated sugars (as a l d i t o l acetates)  K17  capsular  polysaccharide  Mole  T' Column B (OV-17)  Column C (OS-138)  d  e  Column D Column D (ECNSS-M) (ECNSS-M)  Column  19. 3  f  9  B  %  Column  D  f  Column  20 .9  13 .9  3.7  4.4  2,3,4-Rha  0. 54  0 . 53  0.46  0. 51  3,4-Rha  0.89  0.86  0.91  0.91  5.1  2,4-Rha  1.00  1.00  1.00  1.00  22 . 5  2 5.6  23.8  4-Rha  1.41  1.40  1.71  1. 51  15. 8  17 .8  15.5  2, 3, 6 - G l c  1.77  2.02  2.48  1.94  20 . 3  20 . 4  19 . 7  2, 3 - G l c  2 . 42  3.58  5. 40  3.14  17. 1  11.6  17. 6  a  2,3,4-Rha  =  b  Retention  time  c  Values 29 et-al  are corrected  1 , 3 , 5 - t r i - O - a c e t y l - 2 - 4 - d i - 0 - m e t h y 1 - L - r h a mn i t o 1  to  using  e f f e c t i ve  the  carbon  ot .  Peak  areas  f o r column  for 8  min  d  Program;  175°  e  Program;  190°C  isothermal,  f  Program: 170 C  isothermal,  g  Program;  180°and  h  Probably  due  U  then  h  - 2 , 3 , 4 - t r i -0-methy1-L-rhamnitol, e t c .  1,5-di-0-acetyl relative  h  and  C vi/ere n  then  2°/min  to h y d r o l y s i s of  to  response  factors givaiby  Albershiem  available,  to  210°C.  2°/min  220 C. U  rhamnose  side  chain  by  IR-120  resin  (see t e x t  for detail)  D  9  - 49 Partial  hydrolysis Partial,  was by  -  followed  by  acidic  h y d r o l y s i s of  separation  of t h e  ion-exchange chromatography.  separation  by  saccharides,  gel  mainly  oligomers  (2_ and  of the  5,6).  (see  a reducing  King  Methylation, acetates  acidic  Table I I I . l ,  3-anomeric  by  neutral fractions  neutral fraction,  fraction  signal  (_1) .  and  and  two  Bishop  to  "'"H and  contain  (see Appendix,  s t u d i e s ' on ( ^ H ) , ^ and  this  (see  Table  Colson  g.1.c.-m.s.  III.3,  13  column V,  1 i s thus e s t a b l i s h e d  one  Compound 2_, [[of] ^ +111°, was  due  reducing  (see T a b l e to the  III.l,  attribuno.'s  of  and the  King  ( C) . 3  alditol  3,4-di-0-  page 5 0 ) .  The  as  Q)  shown by  page 44)  13 H and  C n.m.r,  t o g i v e one  anomeric  combined s i g n a l s of a n o n - r e d u c i n g  a - g l y c o s i d i c l i n k a g e and  non-  spectra  1  signal  acidic  compound have been  3-D-*Glcp-(l*>2)-L-Rhap  spectroscopy  Chroma-  ^Cn.m.r.  anomeric s i g n a l s  gave 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y 1 g l u c o s e and  s t r u c t u r e of  mono-  y i e l d e d two  shown by  page 44)  6-deoxyhexose  h y d r o l y s i s , and  methylrhamnose  after  contained  a disaccharide  +3.7°, was  n  N.m.r. s p e c t r o s c o p i c  reported  and  polysaccharide  _3) .  Compound ^1 , [of]  t a b l e to  The  rhamnose, and  separation  reducing,  acidic  f i l t r a t i o n chromatography,  tographic  spectroscopy  the n a t i v e  another anomeric  and  signal  a  TABLE I I I . 3  Methylation and  analyses  o f K17 c a p s u l a r p o l y s a c c h a r i d e  T  b T  I  Mole %  .  II  2,3,4-Rha  0 . 48  3,4-Rha  0 . 79  2,4-Rha  0.89  2,3,4, 6 - G l c  1.00  4-Rha  1.25  21.8(15.8)  2, 3, 6 - G l c  1 . 57  20.2(20.3)  2, 3, 4 - G l c  1.61  2, 3 - G l c  2.15  2,3,4-Rha =  an OV-17  T  I,  (5.1) 21.1(22.5)  7.2  f  23.9  26. 3  27.9  27.9  49 .9 48.2 50 .1  30.6 24.2 51.8 15.4(17.1)  column prog ramm ed a t 17 5 by use  0  f o r 8 min  of e f f e c t i v e  a c i- dA i- c c a p s u l a r p o l y s a c c h a r i d e ,  16.8  15.2  c o m p a r i s o n , numbers i n b r a c k e t s  and  then 2° /min  to 210 C. U  carbon response f a c t o r s g i v e n  second m e t h y l a t i o n  t e t r a s a c c h a r i d e ;3| IV, a l d o b i o u r o n i c a c i d For  e  IV  r e l a t i v e t o 1, 5- d i - 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0- m e t h y l - D - g l u c i t o l  °Values a r e c o r r e c t e d d  21.6(19.3)  III  1 , 5 - d i - 0 - a c e t y l - 2 , 3 , 4 - t r i - O - m e t h y l - L-rhamn i t o l , e t c .  ^R e t e n t i o n t i m e on  poly-  oligosaccharides  a Methylated sugar (as a l d i t o l a c e t a t e )  a  and d e r i v e d  ana l y s i s ;  by  I I , PI;  2; and V, n e u t r a l d i s a c c h a r i d e  r e f e r t o the f i r s t  methylation  Albersheim I I I a, c i d i c 1.  r e s u l t s , Table  III.2  page 4 8 ( S e e t e x t f o r d e t a i l s ) . A s m a l l amount o f 2 - 0 - a c e t y 1 - 1 , 3 , 4 - t r i - 0 - m e t h y 1 e r y t h r i t o l  a l s o appears i n t h e chromatogram  - 51 attributable  to a r e d u c i n g  s p e c t r a no.'s with as  lithium  alditol  7,8).  acetates  0 - m e t h y l g l u c o s e and X  2_, t h e  3-6-deoxyhexose  Methylation  aluminum  g.1.c.-m.s. ^ ( s e e  -  hydride,  of  2_  and  (see  subsequent  h y d r o l y s i s , and  gave p r o d u c t s  Appendix,  derivatization  corresponding  to 2 , 3 , 4 - t r i -  2 , 4 - d i - 0 - m e t h y 1 - r h a m n o s e , as Table I I I . 3 ,  column  IV,  reduction  page  analyzed 50).  by  Compound  a l d o b i o u ron i c a c i d , i s t h u s e s t a b l i s h e d as h a v i n g  the  s t r u ctu re  a-D-GlcAp-(1>3)-L-Rhap  This  i s the  first  occurrence,  acid  i n a Kleb s i el1 a b a c t e r i a l Compound 3^, [of] ^  spectroscopy in  the  cosyl  (see  and  and  shown by  page  44)  characteristic  glucose 3-D  unit  signal  s p e c t r a no.'s compound 2)  one  9,10.  "''H and  aldobiouronic  ''""'n.m.r.  to produce four  (unresolved), to a  i n the  the  large coupling  "'"H n.m.r. s p e c t r u m " ^ ) .  Methylation  a n a l y s i s (as  See  2,3-di-0-methylglucose,  nose  III.3,  Table  column  I I I , page  and 50) •  non-  reducing non-  constant Appendix,  described  y i e l d e d 2 , 3, 4, 6 - t e t r a - 0 - m e t h y l g l u c o s e ,  a-gly-  one  B - g l y c o s y l bond a t t r i b u t a b l e t o a ( i m p l i e d by  signals  of a n o n - r e d u c i n g  g - g l y c o s y l bond due  methylrhamnose, (see  above  polysaccharide.  +38°, was  a - g l y c o s y l bond, a  6 - deo xy h exo s e ,  of the  of the  a reducing a - g l y c o s y l linkage  reducing  reducing  to date,  Table I I I . l ,  anomeric region  (2)  for  3,4-di-0-  2,4-di-0-methylrhamHaving  established  - 52 the  s t r u c t u r e s o f _1 and  ture of 3  B-D^  1_ i t i s p o s s i b l e t o a s s i g n  the s t r u c -  as  lcp-(l*2)-a-L-Rhap-(l*-4)- -D-GlcAp-(l*-3)-L-Rhap  (3)  a  Periodate  oxidation  A complete p e r i o d a t e was  -  achieved  after  two  o x i d a t i o n o f K17  treatments  with  polysaccharide  sodium periodate"*"^  and  18 sodium p e r c h l o r a t e the  .  Carboxyl  sodium b o r o h y d r i d e - r e d u c e d ,  reduction  and  h y d r o l y s i s of  periodate-oxidized  gave e q u i m o l a r amounts o f  erythritol  and  as t h e i r  by  These r e s u l t s  alditol  complementary  acetates  data  g.l.c.  to t h e m e t h y l a t i o n  to e s t a b l i s h the l e n g t h  the  p o s i t i o n on  that  analyzed provide  a n a l y s i s of the  saccharide. In o r d e r r e s i d u e , and  rhamnose  polymer  of the  poly-  side chain  r e s i d u e , to which t h e  and  terminal  19 rhamnose i s a t t a c h e d , formed.  Knowing t h e  possibilities  f o r the  selective  oxidation  s t r u c t u r e O f compound _3, two s t r u c t u r e of the  — G l c^-^R h a^-^-Gl cA^-^R h a ^ a 3 1 Rha  periodate  or  was  per-  distinct  r e p e a t i n g u n i t o f K17  -R h a^-^G 1 c A^-^R a a 6  GI ? • i  1 Rha  h a—  exi  - 53 The  periodate  i.e.  sensitive  components o f t h e p o l y s a c c h a r i d e ,  1 , 4 - l i n k e d 3-D-glucopy ranose,  terminal  rhamnose, p r o v i d e  oxidation  to occur.  g l u c u r o n i c a c i d , and  excellent opportunity  The l o w r e a c t i v i t i e s  for partial  of 1,4-linked 19  3-D-glu co py ran o se , due t o s t e r i c  hindrance  acid,  due t o e l e c t r o s t a t i c  anion  and t h e a t t a c k i n g p e r i o d a t e  high  reactivitiy  r e p u l s i o n between  of a terminal  A selective,  partial  the uronate  i o n } ^ a r e opposed by t h e  a - L - rh amn opy ran ose .  periodate  o x i d a t i o n of t h e i n t a c t  p o l y s a c c h a r i d e u s i n g 0.05 M_ s o d i u m p e r i o d a t e with  2.6 m o l e s o f p e r i o d a t e b e i n g  unit  after  tion).  , and g l u c u r o n i c  100 min ( t h e o r e t i c a l  proceeded  rapidly  consumed p e r mole o f r e p e a t i n g  = 4 moles, f o r complete  oxida-  An a n a l y s i s o f t h e s u g a r components o f t h e sodium  borohydride-reduced, the p e r a c e t a t e s  p e r i o d a t e - o x i d i z e d product  of e r y t h r i t o l ,  rhamnitol  and g l u c i t o l  These r e s u l t s  that  were  present  i n the r a t i o  partial  o x i d a t i o n p r o c e e d e d beyond t h a t d e s i r e d , i . e . e r y t h -  ritol  1:3.2:1.9.  revealed  came from t h e u n w a n t e d p e r i o d a t e  glucuronic  show t h a t t h e  o x i d a t i o n of glucose or  acid. 20  Smith degradation  of the reduced, o x i d i z e d  u s i n g 0.5 M_ t r i f l u o r o a ce t i c a c i d yielded  a polysaccharide  o f P_l_ i n d i c a t e d o n l y 100.0  designated  Pl_.  temperature,  The "^C n.m.r. s p e c t r u m  f o u r a n o m e r i c s i g n a l s a t 104.8,  101.2,  and 96.0 p.p.m. as w e l l as two s i g n a l s a t 17.6 and 17.4  p.p.m. a t t r i b u t a b l e ever,  f o r 17 h a t room  product  t o t h e C-6 o f two rhamnose u n i t s .  s i g n a l s a t 63.4, 61.5, and 61.2 p.p.m. were a l s o  confirming  the presence of e r y t h r i t o l  Howobserved  i n the polymer ( s e e  A p p e n d i x , s p e c t r u m no. 4 ) . M e t h y l a t i o n  a n a l y s i s o f P_l y i e l d e d  - 54 t h e s u g a r d e r i v a t i v e s shown i n T a b l e In p a r t i c u l a r , instead  and  that the t e r m i n a l  branch p o i n t  o f K17 p o l y s a c c h a r i d e  rhamnose.  polysaccharide  (See F i g u r e  (0.014 M_) .  120 m i n . S m i t h d e g r a d a t i o n  saccharide  (Pla) having  sugar r e p e a t i n g u n i t . "'"H and  revealed  o x i d i z e d product  that of the o r i g i n a l  as j u d g e d  III.l,  page 4 8 ) .  polysaccharide,  corresponding  to the anomeric  Thus, t h e t e r m i n a l  a t 0-1 t o t h e g l u c u r o n i c a c i d  is  B-linked.  The i n c r e a s e  the  original  polysaccharide  in optical  residue  r o t a t i o n from +30° f o r  t o +44° f o r P l a i s i n a g r e e m e n t  assignments.  The  establish  four-  i s a - l i n k e d and t h e r e f o r e , by e l i m i n a t i o n ,  rhamnose a t t a c h e d  hydrolysis  occurred  (see Table  o f an a - l i n k e d 6 - d e o x y h e x o s e .  these  chain,  ( s e e A p p e n d i x , s p e c t r u m n o . 3) o f P1 a,  the absence of a s i g n a l  rhamnose r e s i d u e  yielded a poly-  an u n b r a n c h e d , s t r a i g h t  n.m.r. s p e c t r o s c o p y  when compared w i t h  p e r mole o f r e p e a t i n g  (same as f o r P_l) o f t h e  No o v e r - o x i d a t i o n  "*"H n.m.r. s p e c t r u m  with  a t h r e e - f o l d excess of  The r e a c t i o n p r o c e e d e d more  sodium b o r o h y d r i d e - r e d u c e d ,  the  o x i d a t i o n of the i n t a c t  c o n s u m i n g 1.1 m o l e s o f p e r i o d a t e  after  proton  only  t o 0-3 o f t h e  I I . 9 , page 3 3 ) .  periodate  was done u s i n g  sodium p e r i o d a t e  The  and  c o n s i s t s o f one u n i t  rhamnose i s a t t a c h e d  Another s e l e c t i v e  by  I I , page 50  3 , 4 - d i - 0 - m e t h y 1rhamnose a p p e a r e d , e s t a b l i s h i n g t h a t  side chain  unit  column  no 4-0-methy1rhamnose was g e n e r a t e d  the  slowly  III.3,  results  obtained  and p e r i o d a t e  from m e t h y l a t i o n  analysis, partial  o x i d a t i o n s t u d i e s are s u f f i c i e n t to  t h e s t r u c t u r e o f K17 p o l y s a c c h a r i d e .  However,  uronic  - 55 acid  degradation  s t u d i e s are  supply  confirmation  of the  Uronic  acid  degradation  The  permethylated  -  presented  as w e l l b e c a u s e  they  proposed s t r u c t u r e .  polysaccharide  was  subjected  to a  21 base-catalyzed anion. and  uronic acid degradation  After neutralization,  then  re-alkylated using  H y d r o l y s i s o f the of  the p r o d u c t s  the  results  the  by  g.1.c.-m.s.^^ of  shown i n T a b l e I I I . 4 ,  polysaccharide  the  under the with  r e a c t i o n mixture 22  degraded m a t e r i a l  .  column  The  is  p r e s e n c e of  t h a t i t was  Since not  (2).  e t h y l a t e d , i t may  formed d u r i n g suggesting appropriate involves  Columns I and  I I , page  56..  p e r f o r m e d on  the  prior  isolation  was  essentially  amount of t h e  permethy-  before directly  of  the  the  same  ethylated (see  Table  compound III.4,  3-0-ethyl-2,4-di-O-methylrhamnose  the  residue the  a l l the be  attached  t o 0-1  s t r u c t u r e of t h e  2 , 4-d  case.  a c i d treatment  and  o se  component  ethers  cleaved,  would have been  L o s s of g l u c u r o n i c a c i d  of t h e main c h a i n  the  aldobiouronic  enol  e l i m i n a t i o n r e a c t i o n are not 21  in t h i s  of  i-0-rn e t hy 1 rh amn  assumed t h a t a l l the  t h a t some m i l d  cleavage  the  56).  glucuronic acid, confirming acid  gave  a r e a c t i o n c l o s e r to completion  I I I page  indicates  acetates  same c o n d i t i o n s as  result  oxide.  alditol  was  without  The  the  isolated  analysis  ethyl iodide occurred  except f o r a l a r g e r r e l a t i v e indicating  silver  e t h y l a t e d , d e g r a d e d m a t e r i a l and  except that a l k y l a t i o n in  dime t h y 1 s u f i n y 1  d e g r a d e d m a t e r i a l was  e t h y l i o d i d e and  Another u r o n i c a c i d degradation lated  using  was  residue  a c c o m p a n i e d by  some  TABLE  III.4  G.l.c. analysis  Methylated sugars (as a l d i t o l a c e t a t e s )  o f K17 p o l y s a c c h a r i d e a f t e r U r o n i c A c i d  T  Degradation  Mole ? o  b  Column B (OU-17)  C o l u mn E (HIEFF - I B )  I  0. 54  0. 42  0. 57  2,4-Rha  6  C  II  III  16. 6  19. 3  17 . 6  0. 37  19. 9  19.0  22 . 5  1.00  1.00  17. 6  16.6  11. 7  4-Rha  1. 41  1.55  14. 1  11.9  16.8  2, 3, 6 - G l c  1.78  1.94  31. 8  33.2  31. 4  2 , 3 , 4-R h a 3-Et-2,4-Rha  a  9  2,3,4-Rha = 1 , 5 - d i - 0 - a c e t y 1 - 2 , 3 , 4 - 1 r i - 0 - m e t h y 1 - L - r h a m n i t o l ,  ^Retention c  time  relative  t o 200°C. then  175° f o r 8 min and t h e n I, u r o n i c acid  ethylation,  column  saccharide with d i r e c t g  etc.  to 1,3,5-1ri-0-acetyl-2,4-di-0-methy1-L-rhamnitol .  V a l u e s a r e c o r r e c t e d u s i n g e f f e c t i v e carbon  ^Program:  f  2°/min  degradation  response  t o 210 °'C.  e  factor  g i v e n by A l b e r s h e i m  P r o g r a m : 1 6 0 f o r 8 min t h e n u  of p o l y s a c c h a r i d e , i s o l a t i o n  B; I I , same, column E; I I I , u r o n i c a c i d ethylation,  column B ( s e e t e x t  r  o f p r o d u c t s , and page72 ) .  rhamn i t o l .  2  2°/min  degradation of poly-  for details,  3 - E - t - 2 , 4-Rha = 1, 5 - d i - 0 - a c e t y 1 - 3 - 0 - e t h y l - 2 , 4 - d i - 0 - m e t h y l - L  et a l  - 57 further lower  degradation  relative  o f e x p o s e d r e d u c i n g - g r o u p s as seen by  amounts o f 4 - 0 - m e t h y l r h a m n o s e and 2 , 3 , 4 - t r i -  O-methylrhamnose i n T a b l e rhamnose r e s i d u e  III.4.  i s more l i k e l y  of t h e g l u c u r o n i c a c i d ( t h r o u g h titative  g.l.c.  Therefore, attached 0-4)  the  branching  to the other  judging  side  by t h e quan-  data.  The r e s u l t s  reported  capsular polysaccharide  here demonstrate that the  from K l e b s i e l l a  composed o f p e n t a s a c c h a r i d e  serotype  K17 i s  repeating u n i t s having  the  following structure.  • 4 ) - B-D-Glcp-( 1+-2) - a- L-R h ap-( 1*4) - a-D - G l cAp-( 1 * 3 ) - 6-L-R h ap-( 1*. t  3  1 a-L -R h ap  Of t h e K l e b s i e l l a  capsular  polysaccharide  reported  to  23 date,  t h e s t r u c t u r e o f K62  unit with  , having  a s i n g l e u n i t s i d e chain  a five-sugar  repeating  and g l u c u r o n i c a c i d i n  the main c h a i n , i s t h e c l o s e s t a n a l o g u e t o K17. The p r e s e n c e o f a g - l i n k e d L-rhamnose r e s i d u e has been reported  i n only  one o t h e r  Klebsiella  capsular  polysaccharide,  24 that of serotype  K32.  However, i n t h e p r e s e n t i n v e s t i g a t i o n ,  t h e a b s e n c e o f an i s o l a t e d linkage to  intact  and t h e u n u s u a l l y  the c o n c l u s i o n  tible  oligosaccharide with high  yield  the  g-L-rhamnose  o f compound  _3 l e a d s  t h a t t h e g-L-rhamnose l i n k a g e i s more s u s c e p -  t o a c i d h y d r o l y s i s t h a n t h e c o m p a r a b l e main  chain  -  58  -  ex-L-rhamn ose l i n k a g e . The "*"H n.m.r. s p e c t r u m six  o f K17 p o l y s a c c h a r i d e  s i g n a l s i n the anomeric region  repeating  unit, illustrating  when i n t e r p r e t i n g t h i s s c o p y , as w e l l ,  that  from a caution  type of data.  pentasaccharide must be e x e r c i s e d  In "^C n.m.r.  a clear dividing line  includes  spectro-  for distinguishing  a n o m e r i c c a r b o n atoms f r o m a- and g-L-rhamnose r e s i d u e s not  appear to e x i s t .  Assuming t h a t  a change i n s u b s t i t u e n t s  does n o t make a l a r g e d i f f e r e n c e i n c h e m i c a l signal  does  a t 103.2 p.p.m. has been a s s i g n e d  s h i f t s , the  t o C-1 o f an a-L-  rhamnose component, s u b s t a n t i a l l y d o w n f i e l d maximum o f 101 p.p.m. f o r a - l i n k e d h e x o s e s .  from a n o m i n a l The  g-L-rhamnose  a n o m e r i c s i g n a l a t 101.3 p.p.m. a p p e a r s between t h o s e o f t h e two a-L-rhamnose s i g n a l s . distinguishes  Of c o u r s e ,  "''H n.m.r.  t h e two anomers o f L-rhamnose  spectroscopy  easily.  - 59 III.4  E xperiroen t a l  General  methods Descending paper  Whatman No.  1 p a p e r and  (A) e t h y l a c e t a t e (18:3:1:4);  chromatography  - a c e t i c acid - formic  (150 X 3 cm o r 100 columns  were i r r i g a t e d  (1000:10:4)  at a flow  were c o l l e c t e d , weighed  in tared  Analytical  dual  r a t e of -10  t u b e s and a  a c i d - water  was  c o n d u c t e d on  P-2  (100-200  ml/h.  the r e s u l t s  5710A gas c h r o m a t o g r a p h detectors.  were u s e d w i t h  fitted  Stainless steel  a carrier  gas f l o w  (100-130  mesh);  (B) 3% o f OV-17  on Gas  (C) 10% o f OS-138 on t h e same s u p p o r t ;  (F)  Chrom Q (D)  g . l . c . was  (1.8 m X 6.3  dual mm)  performed using  thermal  mesh), a n a l a g o u s t o column  A,  with  columns r a t e o f 20 Supelcoport  (100-120);  (80-100  was  10C  on Gas  used  the  mesh).  an F and M model 7 20  conductivity detectors.  5% o f S i l a r  a  5% o f ECNSS-M on  (E) 3% o f HIEFF-1B on Chrom W(HP)  with  ml)  graph  were p e r f o r m e d u s i n g  Columns u s e d were (A) 3?o o f SP-2340 on  instrument  The  on p a p e r or  were p l o t t e d on  ml/min.  Preparative  mesh).  F r a c t i o n s (2.0-2.5  chromatographed  g.l.c. separations  flame i o n i z a t i o n  same s u p p o r t ;  columns  chromatogram.  P a c k a r d model  (1.8 m X 3 mm)  (v/v):  water - p y r i d i n e - a c e t i c a c i d  f r e e z e - d r i e d , and  paper to produce  Hewlett  of B i o - G e l  with  systems  using  (8:2:1). ?5 s i l v e r n i t r a t e . "~  chromatography  X 3 cm)  out  - p y r i d i n e - water  C h r o m a t o g r a m s were d e v e l o p e d u s i n g filtration  carried  the f o l l o w i n g s o l v e n t  (B) e t h y l a c e t a t e  Gel  was  Chrom Q  Column (100-120  for preparative  separations.  An I n f o t r o n i c s CRS-100 e l e c t r o n i c i n t e g r a t o r was u s e d t o m e a s u r e peak  areas.  -60G.l.c.-m.s. was p e r f orm ed ,.u s i n g a M i c r o m a s s 12 i n s t - i : rument f i t t e d recorded and  with  a Watson-Biemann  a t 70 and 20 eV w i t h  an i o n s o u r c e  separator.  an i o n i z a t i o n  dried  or four times  measured a g a i n s t sulfonate  ^C  as  99.1% D^0  internal  99.1% D^O. A c e t o n e  90°C.  using  standard.  Spectra  and were o b t a i n e d  at ambient  as s o l v e n t and a c e t o n e  ''"H n.m.r. s p e c t r a  on a V a r i a n temperature  of t h e p o l y s a c c h a r i d e  220 MHz NMR  Center,  courtesy  o f D r . A.  Department of M e d i c a l  University  "*"H and ^C  n.m.r. s p e c t r a o f t h e p o l y s a c c h a r i d e  o f T o r o n t o on a V a r i a n  HR-220  instrument.  were  obtained  o f D r . M. V i g n o n , CERMAV/CNRS, G r e n o b l e , F r a n c e on  a Cameca 2 50 MHz Circular  cell  at ambient  (31.07 p.p.m. from DSS)  Genetics,  Jasco  methylated  standard.  A. G r e y , C a n a d i a n  courtesy  of  Spectra  C D C l ^ as s o l v e n t w i t h TMS as  o l i g o s a c c h a r i d e s 2_ and 3. were o b t a i n e d  X  ( §2.23,  on t h e U.B.C. 270 MHz i n s t r u m e n t  In a d d i t i o n , and  using  n.m.r. s p e c t r a were r e c o r d e d  X  CFT-20 i n s t r u m e n t using  i n D2O were e x c h a n g e d and f r e e z  at approximately  standard  temperature.  XL-100  aqueous sodium 2 , 2 - d im e t h y 1 - 2-s i l a pen t an e-5-  d e r i v a t i v e s were o b t a i n e d internal  on a V a r i a n  ( D S S ) ) was u s e d as t h e i n t e r n a l  were r e c o r d e d  o f 100 uA  U  Samples d i s s o l v e d  three  current  were  temperature of 200 C.  "*"H n.m.r. s p e c t r a were r e c o r d e d instrument.  Spectra  instrument. dichroism  J-20 a u t o m a t i c of path  m e a s u r e m e n t s were made u s i n g a  recording  l e n g t h 0.01 cm.  a t 23 - 2 C on a P e r k i n - E l m e r U  spectropolarimeter Optical  with  a quart  r o t a t i o n s were measured  model 241 MC p o l a r i m e t e r  using  - 61 a 10 cm c e l l .  -  I n f r a r e d s p e c t r a were r e c o r d e d  E l m e r model 457  temperatures not exceeding  Preparation  under reduced  K17  K17 c a p s u l a r  ( 2 0 0 5 / 4 9 ) was  from D r . I . JZlrskov, Copenhagen, and was grown e x t r a c t - a g a r medium composed o f N a C l  MgS0 .7 H 0 4  yeast  e x t r a c t (5 g ) , and a g a r  cells  were h a r v e s t e d  containing  l?o phenol,  after  obtained  (5 g ) , K^PO^  -  (2.5 g ) ,  (75 g ) , Bacto  ( 3 7 . 5 g) i n w a t e r  3 days, d i l u t e d  polysaccharide  on a 3% s u c r o s e  (0.62 g ) , CaSO^ ( 1 . 2 5 g ) , s u c r o s e  2  pressure  40°C.  and p r o p e r t i e s o f K l e b s i e l l a  A c u l t u r e of K l e b s i e l l a  yeast  a Perkin-  spectrophotometer.  S o l u t i o n s were c o n c e n t r a t e d at bath  using  ( 2 . 5 1 ) . The  to 2 1 with  water  and c e n t r i f u g e d i n b a t c h e s f o r 3 h a t  34,000 r.p.m. i n a Beckman model L3-50 u 1 t r a c e n t r i f u g e w i t h rotor  type  combined solvent  The c l e a r ,  supernatant  (^1300 m l ) , and p r e c i p i t a t e d  by d e c a n t a t i o n  (800 ml) and then  solution.  precipitated  i n 4 H NaCl  was  decanted,  into  10 1 o f  was d i s s o l v e d i n  w i t h a 10%  isolated  Cetavlon  by c e n t r i f u g a t i o n ,  (800 m l ) , and r e p r e c i p i t a t e d by  into Solvent  IK ( 8 1 ) .  was i s o l a t e d  by c e n t r i f u g a t i o n ,  pouring  The C e t a v l o n - p u r i f i e d p o l y s a c c h a r i d e  2 days a g a i n s t running  solution  were  by p o u r i n g  and c e n t r i f u g a t i o n ,  The p r e c i p i t a t e  redissolved  yzed  liquors  IK ( e t h a n o l - m e t h a n o l , 95:5) C r u d e p o l y s a c c h a r i d e ,  isolated water  35.  d i s s o l v e d i n w a t e r , and  tap-water.  Freeze-drying  y i e l d e d 7.4 g o f t h e sodium s a l t  dial-  of t h i s  of the p o l y s a c c h a r i d e  (approximately  2.5 g p e r 2.5 1 o f medium), [a] ^ + 3 0 ( £ 0.57,  water).  of the p o l y s a c c h a r i d e  Purity  phoreis using  u  a l?o  solution  was  on c e l l u l o s e  c h e c k e d by acetate  electro-  strips  - 62 (Sepraphore  I I I ; 15 x 2.5 cm) i n v e r o n a l b u f f e r  ( L K B - P r o d u k t e r AB, S t o c k h o l m and  then  ethanol also  12, Sweden) a t 300 V f o r 90 min  development i n e i t h e r  alcian  (pH4) o r p e r i o d a t e - S c h i f f  cular  weight The  blue i n c i t r a t e buffered  reagent.  c o n f i r m e d by g e l c h r o m a t o g r a p h y  Churms, U n i v e r s i t y  Homogeneity  Africa,  o f K17 p o l y s a c c h a r i d e d e t e r m i n e d  by p a s s i n g a s o l u t i o n  form  and t h e m o l e t o be 9.4 x 10^.  o f t h e p o l y s a c c h a r i d e was o b t a i n e d  o f t h e sodium  A m b e r l i t e 1R-120 (H ) r e s i n , Spectroscopic  was  c o u r t e s y o f D r . S. C.  o f Capetown, S o u t h  free-acid  pH8.6  salt  through  a column o f  f o l l o w e d by f r e e z e - d r y i n g .  a n a l y s e s were p e r f o r m e d  on K17 p o l y -  s a c c h a r i d e and t h e d e r i v e d p o l y s a c c h a r i d e t h a t had been partially  depolymerized  by m i l d h y d r o l y s i s  e v a p o r a t i o n under reduced  several  The polysaccharide  T h i s was a c h i e v e d  n.m.r. s p e c t r u m  p r e s s u r e and  of p a r t i a l l y  x  2  region,  observed  relating  10 Hz), 4.51 (1H, c o u p l i n g c o n s t a n t 7 H z ) . lved  d o u b l e t s due t o t h e CH^ o f rhamnose a p p e a r e d  (J,  6 Hz) and i n t e g r a t e d  z>, 6  page 44, and A p p e n d i x , The polysaccharide  n.m.r.  to nine protons.  spectrum spectrum  2^~^  4.60 (1H,  Three  K17  tos i x  a t 65.15 (1H,  b r o a d ) , 4.85 (1H, s i n g l e t ) ,  washing  acid.  depolymerized  i n D^0 a t 90°C showed s i g n a l s  5.04 (2H, J  f o r 15 min  times to e l i m i n a t e t r i f l u o r o a e e t i c  protons i n the anomeric Hz),  viscosity.  i n 0.4 M_ t r i f 1 u or oa ce t i c a c i d  a t 95°C, and then w i t h water  to reduce  2  sets of unresoaround  (See T a b l e  61.30 III.l  no. 1.) of p a r t i a l l y  confirmed the presence  depolymerized  of more than  K17  one rham-  - 63 nose r e s i d u e signal  (large signal  f o r C-6 a t 17.4 p.p.m.).  One  a t t r i b u t a b l e t o C-6 o f a h e x o s e was o b s e r v e d  a t 61.5  p.p.m.  In t h e a n o m e r i c r e g i o n , f i v e  guished  a t 105.0, 103.2, 101.3, 100.8, and 96.4 p.p.m. ( s e e  Table I I I . l , Analysis  page  44, and A p p e n d i x , s p e c t r u m n o .  of c o n s t i t u e n t Methanolysis  capsular and  subsequent treatment  with  (1:1)  pentaacetate  with  3?o m e t h a n o l i c  with  hydrochloric acid  sodium b o r o h y d r i d e  i n anhydrous  i n the polysaccharide.7  2 M_ t r i f l u o r o a c e t i c a c i d o v e r n i g h t a t  of the l i b e r a t e d  sodium b o r o h y d r i d e ,  pyridine  sugars  the uronic acid residues  Total h y d r o l y s i s with 95°C, r e d u c t i o n  2).  o f a s a m p l e (30 mg) o f K l e b s i e l l a K17  polysaccharide  methanol reduced  s i g n a l s c o u l d be d i s t i n -  monosaccharides to a l d i t o l s  and a c e t y l a t i o n w i t h  a t room t e m p e r a t u r e  a c e t i c anhydride  overnight  and D - g l u c i t o l h e x a a c e t a t e  yielded  i n the r a t i o  ( c o l u m n A; programmed a t 1 9 5 f o r 4 min and then a t u  260°C).  Circular  MiCN showed Ae j ^ - l . l  a n  2  after  dichroism  collection  of the r h a m n i t o l  ^ *-he g l u c i t o l  of the a l d i t o l  hexaacetate acetates  K17  a n a l y s i s of n a t i v e polysaccharide  o f 1.6:1.0 2 °/min t o  MeCN  +0»32  by p r e p a r a t i v e g . l . c .  (column F; programmed a t 210°and then a t 4 /'min Methylation  L-rhamnitol  pentaacetate As  t o 250°C).  polysaccharide  ( 2 8 5 mg) i n t h e a c i d form was  11 12 methylated  '  i n dimethyl  s u l f o x i d e (20 m l ) by t r e a t m e n t  dim e t h y l s u 1 f i n y l  anion  (4 m l ) f o r 1 h.  A f t e r removal of excess reagents  the methylated  (10 m l ) f o r 4 h, and t h e n m e t h y l  polysaccharide  was i s o l a t e d  -  with  iodide  by d i a l y s i s ,  by f r e e z e - d r y i n g  - 64 26 (yield oxide ride  283 mg).  t h a t showed no h y d r o x y l  material (100  with  (yield  297 mg).  was r e d u c e d  overnight  with  sodium  H y d r o l y s i s of the methylated,  polysacchai n the i n f r a -  reduced  with  a c e t y l a t i o n of the a l d i t o l s .  borohydride  ( 1 : 1 ) a t room  overnight  reduction of the h y d r o l y z a t e  isothermal:  silver  A s a m p l e ( 3 5 mg) o f t h i s  2 lj_ t r i f l u o r o a c e t i c a c i d  then  with  group a b s o r p t i o n  mg) i n t e t r a h y d r o f u r a n - e t h a n o l  ture.  tempera-  polysaccharide  a t 95°C was f o l l o w e d  sodium b o r o h y d r i d e  G.l.c.  and  ( c o l u m n C; 190°C  column D; 1 7 0 C i s o t h e r m a l : column D; programmed u  a t 180 °C and then  2°/min t o 220 G) and g . l . c . - m . s . a n a l y s i s U  ( c o l u m n B) o f m e t h y l a t e d the assignments given  alditol  i n Table  A second m e t h y l a t i o n on  treatment  and m e t h y l i o d i d e y i e l d e d a p e r m e t h y l a t e d  red spectrum  by  Subsequent P u r d i e  acetates  III.2,  produced  allowed  page 48.  o f K17 p o l y s a c c h a r i d e  was done  a s a m p l e (200 mg) w h i c h had been d e i o n i z e d by s t i r r i n g i n  solution  with  IR-120(Ht) r e s i n  and  freeze-dried (yield  and  one P u r d i e  hydroxyl  methylated tive  2 days, f i l t e r e d ,  A f t e r one H a k o m o r i  the methylated  product  i n the i n f r a r e d  dialyzed, treatment  showed no  spectrum.  Reduction,  r e d u c t i o n , and a c e t y l a t i o n y i e l d e d a m i x t u r e o f acetates  and g . l . c . - m . s .  t h a t were i d e n t i f i e d (see Table  III.3,  by compara-  column  I , page 5 0 ) .  a c i d i c 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 Acidic  lyzed  treatment  alditol  g.l.c.  Partial,  163 mg).  group a b s o r p t i o n  hydrolysis,  over  polysaccharide  i n 0.1 II t r i f l u o r o a c e t i c  (942 mg) was p a r t i a l l y acid  hydro-  a t 95 °C f o r 3 d a y s u s i n g  - 65 an  apparatus s i m i l a r to that  described  by G a l a n o s and c o -  27 workers.  A f t e r each 24 h p e r i o d  changed and t h e d i a l y z e d m a t e r i a l of  t h e w a t e r by e v a p o r a t i o n  freeze-drying  obtained dual  a large  concentration  1 0 0 % a f t e r 48 h. sac.  oligosaccharides,  o f rhamnose.  The t o t a l  eluted  yielding  with  water  As  8 0 % was No  resi-  Paper material  g l u c o s e and  amount o f d i a l y -  was n e u t r a l i z e d ( 0 . 1 M_ NaOH) and a p p l i e d  oligosaccharides,  to a  The column  ( 1 1 ) , y i e l d i n g n e u t r a l mono- and  and then w i t h  10% f o r m i c  the a c i d i c oligosaccharides  acid  (100 m l ) ,  (shown t o be f r e e o f  components by p a p e r c h r o m a t o g r a p h y , s o l v e n t B ) . Gel  neutral  flask.  (30 X 1.5 cm) o f B i o - R a d A G l - X 2 ( C l " ) r e s i n .  was f i r s t  neutral  and then  ( s o l v e n t A) o f t h e d i a l y z e d , h y d r o l y z e d  material  column  o f most  recovered,  remained i n t h e d i a l y s i s  showed t h e p r e s e n c e o f s e v e r a l  zable  solution i n a tared  a f t e r 24 h and v i r t u a l l y  chromatography  pressure  of d i a l y z a b l e m a t e r i a l  polysaccharide  s o l u t i o n was  i s o l a t e d by r e m o v a l  under reduced  the concentrated  j u d g e d by q u a n t i t y  the d i a l y s i s  chromatography  (Bio-Gel  components p r o v i d e d  a neutral  1_ had [ajp +3.7 ( c 0.82, w a t e r ) . u  90 °C) showed a d o u b l e t  P-2; 100 X 3 cm) o f t h e disaccharide  1_ (9 mg).Compound  The H n.m.r. s p e c t r u m ( 0 ^ 0 , 1  ( J , ,6 Hz) a t 61.28 c h a r a c t e r i s t i c o f  j  6  CH-j o f rhamnose, and a n o m e r i c s i g n a l s a t §4.63 ( I H , J ^ ^1 H z ) , 4.84(0.5H, J <1 H z ) , and 5.36 (0.5H, J 1 Hz). See l, z l, z 1 4  Table I I I . l , 1 3  C  of  page 44 and A p p e n d i x , s p e c t r u m no. 5.  n.m.r. s p e c t r u m  showed s i g n a l s a t 17.6 p.p.m. from C r 6  rhamnose, 61.4 p.p.m. from C-6 o f g l u c o s e ,  anomeric r e g i o n ,  The  93.7 and 105.1 p.p.m.  and i n t h e  In a d d i t i o n  a signal  - 66 a t 81.7 p.p.m. i s a t t r i b u t a b l e t o t h e l i n k a g e Table  III.1,  page  44 and A p p e n d i x ,  Hakomori m e t h y l a t i o n dimethyl  iodide  to a separatory  neutralized  with  chloroform back  dim e t h y l s u 1 f i n y l  funnel  10% a c e t i c a c i d  (3 X 10 m l ) .  extracted  with  s p e c t r u m n o . 6.  (1 m l , 1 h ) .  transferred  The m i x t u r e with  (2 m l ) ,  The c o m b i n e d  H^O  water  was t h e n  freeze-drying  (15 ml) s e v e r a l  disaccharide  with  with  e x t r a c t s were  ( 4 X 10 m l ) and e v a p o r a t e d  water  (2 m l ,  (15 ml),  chloroform  Any r e s i d u a l s o l v e n t  the m e t h y l a t e d  anion  and e x t r a c t e d  under reduced p r e s s u r e . with  See  o f 1_ (6 mg) was p e r f o r m e d i n  s u l f o x i d e (4 ml) u s i n g  4 h) and m e t h y l  carbon.^  to dryness  was removed by  times.  H y d r o l y s i s of  t r i f l u o ro a ce t i c a c i d (2 M_,  16  h, 95°C), r e d u c t i o n  of  3 , 4 - d i - 0 - m e t h y 1 r h a m n o s e and 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e  in  the r a t i o  the  and a c e t y l a t i o n y i e l d e d a l d i t o l  1:1 ( g . l . c :  column B ) . G . l . c . - m . s .  a s s i g n m e n t s o f t h e above components ( s e e  column V , page50 f o r r e l a t i v e  confirmed  Table  amounts and column  acetates  III.3,  details).  Compound _1 i s t h e r e f o r e 2 - 0 - 3 - D - g l u copy ran o sy 1 - L - rhamn op y r a n o s e Gel  chromatography  components p r o v i d e d acidic  [a]  D  an a l d o b i o u r o n i c  tetrasaccharide The  ( B i o - G e l ; 150 X 3 cm) o f t h e a c i d i c a c i d 2 (47 mg) and an  3_ (92 mg) .  aldobiouronic  a c i d 2 (R , 0.87, s o l v e n t — glc '  +111° ( c 1 . 1 2 , w a t e r ) .  The H n.m.r. s p e c t r u m 1  showed a n o m e r i c s i g n a l s a t 65.17 (1.6H, (0.4H, J , 61.33  abroad),  1 5 ^  (D 0, 90 C) U  2  b r o a d ) and 4.87  a s w e l l a s , two u n r e s o l v e d  ( 3 H , J r , 6 Hz) r e p r e s e n t i n g j, 6  2  A) had  doublets  around  t h e CH^ o f rhamnose ( s e e J  -  Table I I I . 1 , methylation acid  which  refluxing  -  67  page 44 and A p p e n d i x , o f 2 ( 2 4 mg) y i e l d e d  was then  reduced  spectrum  no. 8 ) .  Hakomori  permethylated a l d o b i o u r o n i c  with l i t h i u m  aluminum h y d r i d e i n  t e t r a h y d r o f u r a n . The n e u t r a l , m e t h y l a t e d  was h y d r o l y z e d , r e d u c e d ,  and a c e t y l a t e d  yielding  disaccharid  equimolar  amounts o f 2 , 4 - d i - 0 - m e t h y 1 - r h a m n o s e and 2 , 3 , 4 - t r i - O - m e t h y l g l u c o s e as t h e i r page  50).  alditol  Identification  g.l.c.-m.s.  analysis  g l c  0.48,  column I V ,  g . l . c . and  B). s o l v e n t A) h a d [ a ]  The "''H n.m.r. s p e c t r u m  the anomeric  III.3,  «/as made by c o m p a r a t i v e  (column  Compound J5 ( R water).  a c e t a t e s (see Table  D  +38° (c  1.38,  (D2O, 90°C) showed s i g n a l s i n  r e g i o n a t 65.17 ( 1 . 5 H , b r o a d ) , 5.11 ( 1 H , J  „ 2 Hz 1 > ^  4.87 (0.5H, s i n g l e t ) , a group  of d o u b l e t s around  rhamnose ( s e e T a b l e 9).  and 4.57 (1H, J  • 1 > ^  61.31 (6H, 3^ ^ 6 Hz) from  I I I . 1, page 44, and A p p e n d i x ,  The "^C n.m.r. s p e c t r u m  carbon  7-8 H z ) , as w e l l a s , CH^ o f  spectrum no.  showed s i g n a l s due t o  anomeric  atoms a t 105.0, 100.5, 9 6 . 6 , 9 6 . 2 , and 94.4 p.p.m., due  t o C-6 o f g l u c o s e a t 61.4 p.p.m., and due t o C-6 rhamnose (two 44  c o m p o n e n t s ) a t 17.7 and 17.3 p.p.m.(see T a b l e and A p p e n d i x ,  spectrum  no. 1 0 ) .  2_ ( 3 5 mg) gave t h e p e r m e t h y l a t e d to a l d i t o l nents. being  the a l d i t o l  derivative  (column  a c e t a t e s of  which  small  (see Table  III.3,  was c o n v e r t e d f o u r compor- : . :  B) i d e n t i f i e d  t h e s e as  3,4-di-0-methylrhamnose,2,4-di-  0-methylrhamnose, 2 , 3 , 4 , 6 - t e t r a - O - m e t h y l g l u c o s e , methy1-glucose  page  Hakomori m e t h y l a t i o n o f  a c e t a t e s ( a s f o r compound 2) y i e l d i n g  G . l . c . and g . l . c . - m . s .  III.l,  column  and 2 , 3 - d i - 0 -  I I I , page 5 0 ) .  amount o f 2 , 3-d i-0-m e t h y 1 g l u c o s e i s most l i k e l y  The due t o  - 68 an un d e r - r e d u ced o f 2-  p r o d u c t of the methyl  ester after methylation  Compound 3 i s , t h e r e f o r e , 3-D-Gl c p - ( 1 - 2 )-ct-L-Rhap-(1-4)  - a-D-Gl cA p - ( 1 - 3 )-L-Rh ap , k n o w i n g what t h e pounds 1_ and Periodate  s t r u c t u r e s of  com-  i n the  salt  2_ a r e .  oxidation Capsular  of  polysaccharide ^ 1  polysaccharide  (529  d i s s o l v e d i n water  (100  solution  (100  0.1  M_ of sodium p e r i o d a t e  The  s o l u t i o n was  w h i c h was  and  sodium  form was  ml)  ml)  mg)  t o t h i s was  added a and  0.4  18  H of sodium p e r c h l o r a t e a t 4°C. the  A f t e r 120  s o l u t i o n was  Following addition and  h,  dialysis,  of p o l y m e r i c To  the  dialysis  sodium b o r o h y d r i d e  o x i d a t i o n was  sodium p e r c h l o r a t e d a r k a t 4°C  was  added  dark and  f o r 3 days. (1 g)  f o r 24  excess hydride  h,  (pH  6),  f r e e z e - d r i e d y i e l d i n g 3 50  and  (0.4  M_) was h.  freeze-drying  treatment with  d i s s o l v e d in water  added.  Addition  The  gave 324  mg  sodium b o r o h y d r i d e  y i e l d e d 303  mg  from  (50  mg  of  (0.1  and  FO  an  and  kept i n  g l y c o l (5  ml),  polyaldehyde.  (0.2  g),  dialysis  o f p o l y o l w h i c h was  considered  oxidation.  d e r i v a t i v e s were p r e p a r e d w i t h  p o l y o l by m e t h a n o l y s i s w i t h  the  ml)  m i x t u r e was  of e t h y l e n e  t o have u n d e r g o n e c o m p l e t e p e r i o d a t e Usual  product  s o l u t i o n of sodium p e r i o d a t e  f o r 94  freeze-drying  of t h e  ml)  ensure complete o x i d a t i o n , the  amount o f t h e  and  with  kept i n the  material.  equal  Further  1 h before  s o l u t i o n was  periodate  dialysis,  g l y c o l (10  a c e t i c a c i d to destroy  first  the  ethylene  stirred  reduction of 10%  .  a s a m p l e (15  3% m e t h a n o l i c  mg)  hydrochloric  - 69 acid,  reduction with  hydrolysis, by  sodium b o r o h y d r i d e  r e d u c t i o n and  comparative  g.l.c.  i n anhydrous  acetylation.  ( c o l u m n A;  and  then  two  compounds were p r e s e n t ,  acetate  -  The  product  methanol, was  analyzed  programmed a t 150°for 4  a t 4 °/min to 260°C) w i t h a u t h e n t i c s t a n d a r d s .  and  identified  rhamnitol pentaccetate  as e r y t h r i t o l  i n the  ratio  min Only  tetra-  1:1. 19  Selective  periodate  o x i d a t i o n of  A s a m p l e (525 salt  form was  a solution was  mg)  polysaccharide  of p o l y s a c c h a r i d e  d i s s o l v e d i n water  (100  ml)  and  i n the  sodium  t o i t was  added  o f 0.1  allowed  consumption  M_ s o d i u m p e r i o d a t e (100 m l ) . The r e a c t i o n o t o p r o c e e d a t 4 C i n t h e d a r k and t h e p e r i o d a t e  was  monitored  by  r e m o v i n g 1 ml  a l i q u o t s which  were  28 analyzed reached K17  by  the F l e u r y - L a n g e  a level  after  ethylene  only glycol  of 2.6 1.7 (10  ml).  isolated  and  The  volatile  acetates  periodate  was  reduction mg  with  with of  ration  data  rhamnitol,  (column  A)  and  1:3.2:1.9 r e s p e c t i v e l y .  glycol  pressure  f o r sugar  reduction,  G.l.c.  of e r y t h r i t o l ,  of e t h y l e n e  analyzed  methanolysis,  acetylation.  i n the  were l o s t u n d e r d i m i n i s h e d The  terminated  f r e e z e - d r y i n g , 396  m a t e r i a l (17 mg)  r e d u c t i o n , and  were p r e s e n t  r e a c t i o n was  Klebsiella  isolated.  showed t h a t the p e r a c e t a t e s glucitol  consumption  r e p e a t i n g u n i t of  the  components i n t h e u s u a l manner: hydrolysis,  Periodate  Following d i a l y s i s ,  dialysis,  m a t e r i a l was The  m o l e s per  h,whereupon  sodium b o r o h y d r i d e , polymeric  method.  and  during  1,2-propanediol  work-up.  o x i d i z e d m a t e r i a l (120  mg)  was  subjected  20 to Smith degradation  u s i n g 0.5  M_ t r i f l u o r o a c e t i c  acid  a t room  t e m p e r a t u r e f o r 17 h.  1 3  C  in  Following  59 mg), t h e r e s u l t a n t  (yield  7o:  dialysis  signals  i n the  n.m.r. s p e c t r u m a t 104.8, 101.2, 100.0, and 96.0  p.p.m.  the anomeric  61.5,  region.  polymer  group  P_l gave  S i g n a l s were a l s o o b s e r v e d a t 6 3 . 4 ,  and 61.2 p.p.m. a t t r i b u t a b l e  alcohol  to carbons of a primary  from C-6 o f a h e x o s e  and C - l and C-4 o f e r y t h r i t o l .  two s i g n a l s a t 17.6 and 17.4 p.p.m. were a s s i g n e d t o  Finally,  CH-j o f two 6-deoxy s u g a r s ( S e e A p p e n d i x , The  oxidized  m e t h y l a t e d under treatment  lithium lzed,  and d e g r a d e d  Hakomori  absorption  that  the a l d i t o l  2,4-di-0-methylrhamnose, 0-methy1glucose  with  hydro-  and g . l . c . - m . s .  3,4-di-0-methy1rhamnose,  2 , 3 , 6 - t r i - 0 - m e t h y 1 g l u c o s e , and 2 , 3 - d i -  (See T a b l e I I I . 3 ,  as w e l l  a s , minor peaks  column  corres-  and 2 , 3 , 4 - 1 r i - 0 - m e t h y 1 -  I I , page 50 f o r r e l a t i v e  details.)  A subsequent by d i s s o l v i n g  in the i n f r a r e d  A n a l y s i s by g . l . c .  acetates of  were o b t a i n e d ,  amounts and column  by a P u r d i e  tetrahydrofuran,  ponding to 1 , 3 , 4 - 1 r i - 0 - m e t h y 1 e r y t h r i t o l rhamnose.  Pl_ was t h e n  was r e d u c e d o v e r n i g h t  hydride in refluxing  r e d u c e d , and a c e t y l a t e d .  revealed  polysaccharide  This methylated material  aluminum  s p e c t r u m no. 4 ) .  conditions followed  (absence of h y d r o x y l  spectrum).  (50  and f r e e z e - d r y i n g  selective  periodate  K17 p o l y s a c c h a r i d e  m l ) , and a d d i n g a t h r e e - f o l d  (289  oxidation  mg, s a l t  was p e r f o r m e d  form)  e x c e s s o f sodium  i n water  periodate  ( 0 . 0 2 8 M, 50 m l ) . R e a c t i o n c o n d i t i o n s and a n a l y s i s o f p e r i o d a t e consumption reaction  were t h e same as d e s c r i b e d  above.  was s t o p p e d by a d d i n g e t h y l e n e g l y c o l ,  consumption  had r e a c h e d 1.1  moles  After  2 h the  periodate  per r e p e a t i n g u n i t .  Isolation  - 71 of t h e p o l y o l a s d e s c r i b e d The  p o l y o l (90 mg) was t h e n  above p r o d u c e d 238 mg o f m a t e r i a l . subjected  to Smith  degradation  using  0. 5 M_ t r i f 1 u o r o a c e t i c a c i d a t room t e m p e r a t u r e f o r 17 h.  After  dialysis  considered  and f r e e z e - d r y i n g , t h e p o l y s a c c h a r i d e  t o be n e a r l y q u a n t i t a t i v e l y f r e e o f t e r m i n a l  nose branch u n i t s , This to  i . e . a s t r a i g h t chain  polysaccharide  be i n a p u r e r  periodate  form t h a n  P^L from t h e i n i t i a l  (D2O, 9 0 C ) o f p a r t i a l l y U  taken  no o v e r - o x i d a t i o n The "*"H n.m.r. P l a (0.4 t r i -  two o v e r l a p p i n g  t h e CH^ o f two rhamnose c o m p o n e n t s , and a n o m e r i c  singlet),  7.5 H z ) ,  4.87 (1H, s i n g l e t ) ,  and 5.17 (1H, J , „ 3 H z ) .  page 44, and A p p e n d i x , s p e c t r u m n o . 3. of P l a c o n t a i n e d  a large signal  C-6 o f more than  from C-6 o f g l u c o s e , at 96.1,  76 mg).  selective  depolymerized  a c i d , 20 m i n , 95°C) c o n t a i n e d  s i g n a l s a t 64.56 (1H, J  the  (yield  a t 61-27 (3H, J , , 6 Hz) and 1.34 (3H, J , , 6 Hz)  representing  (1H,  rham-  i n t h e above manner was  as j u d g e d by t h e n.m.r. s p e c t r a .  fluoroacetic doublets  PI a o b t a i n e d  polymer  o x i d a t i o n experiment, i . e . v i r t u a l l y  took p l a c e , spectrum  PI a was  The  13  one rhamnose, a s i g n a l  III.l,  C n.m.r.  a t 17.5 p.p.m.  depolymerized  spectrum  representing  a t 61.4 p.p.m.  and f o u r s i g n a l s i n t h e a n o m e r i c  100.3, 101.3, and 104.8 p.p.m.  page 4 4 ) . P a r t i a l l y  See T a b l e  5.11  (see Table  region  III.l,  P l a had £ofj ^ + 44° • (_c 1.81,  water) . Base-catalyzed acid  d e q r a d a t ion  degradation;  of methylated  (Uronic  '  A sample of c a r e f u l l y (99  polysaccharide  mg) and t o l u e n e - p - s u 1 f o n i c  d r i e d , methylated  polysaccharide  a c i d ( t r a c e ) were s e a l e d  ina  - 72 flask  with  sulfoxide The  a r u b b e r cap. (19  ml)  m i x t u r e was  and  stirred  the  stirred  lowing  the  the  t o 6,  pH  extracted  (10  ml)  with  the  to  silver and  oxide.  a n a l y s i s by  adjust  d i l u t e d with  water  (50  ml)  and  with  The  water  combined  (4 X 25 ml) and  of a p o r t i o n  g . l . c . and  and  alditol  c o l u m n s I and  I I , page 56 f  Another  e t h y l a t i o n of t h e  analysis of t h e  as a l d i t o l  with  ethylated  acetates  A second u r o n i c methylated the  K17  relative  r  components m e n t i o n e d  that  and  change t h e  gave  3-0-ethyl4-0-methyl-  amounts and and  material  acetates  Table  material  d i d not  III.4, column d e t a i l s )  subsequent relative  amounts  above.  acid degradation  polysaccharide  exception  (see  Half  ethylated  2,4-di-0-methylrhamnose, 2,4-di-0-methy1rhamnose, 2,3,6-tri-0-methylglucose  evapor-  ethyl iodide  peaks c o r r e s p o n d i n g to 2,3,4-tri-0-methy1rhamnose,  rhamnose, and  chloroform  freeze-dried.  of t h e  g . l . c . - m . s . as  o  Fol-  to  ethylated using  Hydrolysis  and  ml)  (3 :x 25 m l ) .  was  syringe  room t e m p e r a t u r e .  dryness under reduced, pressure  degraded m a t e r i a l  added by  hours.  (~10  s o l u t i o n was  chlorofdrm  were added.  for several  then  h at  (1 ml)  dimethyl-  50% a c e t i c a c i d  e x t r a c t s were back e x t r a c t e d ated  was  f o r 18  s l o w a d d i t i o n of the  a i d of a s y r i n g e ,  under n i t r o g e n  anion  was  With the  2,2-dimethoxypropane  Dimethylsu1finy1 reaction  -  (36  mg)  was  using  e t h y l a t i o n was  performed  the  above  done d i r e c t l y  on  procedure without  22 isolation with  of the  degraded m a t e r i a l .  d i m e t h y l s u l f i n y l anion  added d r o p w i s e d i r e c t l y and  external  added s l o w l y  f o r 18  i n t o the  cooling.  After  to a d j u s t  the  pH  1 h, to  That i s , a f t e r h,  ethyl  basic 50?o  6.  iodide  (3 ml)  solution using acetic acid  reaction  (10  a ml)  was syringe was  E x t r a c t i o n , back e x t r a c t i o n ,  - 73 and f r e e z e - d r y i n g Hydrolysis,  yielded  reduction,  the ethylated,  acetylation,  a n a l y s e s showed t h e same components uronic  acid degradation.  2,4-di-0-methylrhamnose  degraded  and g . l . c .  (See T a b l e  amounts and column  as i n t h e  to 2,4-di-0-methylrhamnose  I I I . 4 , column details.)  g.l.c.-m.s.  r a t i o of  even t h o u g h n o t a l l t h e 2 , 4 - d i - 0 - m e t h y 1 r h a m n o s e lated.  and  were p r e s e n t  However, a h i g h e r  material.  first  3-0-ethyl-  i s indicated  had been  I I I , page56 f o r r e l a t i v e  ethy-  - 74 111. 5  Bibliography  f o r Sect-i ori'- J I I  1.  W. N i m m i c h , A c t a B i o l .  2.  W. Nimmich, Z. M i c r o b i o l .  3.  G.G.S. D u t t o n , K. L. 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S h a p i r o , and P. A l b e r s h e i m , R e s . , 40, 217 ( 1 9 7 5 ) .  Chem., 1_, 435( 1 9 6 2 ) . Carbohyd.  STRUCTURAL  INVESTIGATION  SEROTYPE K44 CAPSULAR  OF  Klebsiella  POLYSACCHARIDE  - 77 IV . 1  -  Abstract The  techniques  degradation,  of m e t h y l a t i o n  periodate  o x i d a t i o n , and  used to i n v e s t i g a t e the capsular scopy was  polysaccharide.  Nuclear  g r o u p was  approximate r a t i o structure  was  repeating  unit.  e s t a b l i s h e d , but per  anomeric  fragments i s o l a t e d  procedures used.  o f one  h y d r o l y s i s were  magnetic resonance  used e x t e n s i v e l y to determine the  various degradative  +. 3) - 8-D-G  partial  acid  s t r u c t u r e o f K l eb s i e l l a s e r o t y p e  of g l y c o s i d e l i n k a g e s i n t h e  0-acetyl  analysis, uronic  The  existence  present  fifteen  shown t o c o n s i s t of t h e  from  only  in  sugar r e s i d u e s . following  K44  spectronature the of  an  the The  pentasaccharide  l c p - ( 1*4) - o u D - G l c p - ( 1*4) - B.-D-GlcAp-( 1*2) -o-L-Rhap-  (l*3)-a-L-Rhap-(l-  - 78 IV . 2  In t ro du c t i o n In a q u a l i t a t i v e  a n a l y s i s of the c a p s u l a r  polysaccharide  1 2 from K l e b s i e l 1 a K44, Nimmich glucuronic acid, glucose, Nimmich  3  residues  reported  and rhamnose.  p r e d i c t e d t h a t two o r t h r e e  the presence of Heidelberger  consecutive  w o u l d be f o u n d i n K44, a c c o r d i n g  r e l a t i o n s h i p s between bacteria.  '  specificity  we now  determination  of serotype  K44 c a p s u l a r  I I I , page  Klebsiella  structure with  r e p o r t on t h e s t r u c t u r a l polysaccharide.  40) and 2 3 , whose  have t h e same q u a l i t a t i v e  as K44, have been  to immunochemical  to c o r r e l a t e chemical  immunological  polysaccharides  rhamnose  t h e f a m i l i e s o f p n e u m o c o c c i and  In an e f f o r t  t y p e s 17 ( see S e c t i o n  and  investigated previously.  4  sugar  K-  capsular  composition  - 79 IV . 3  R e s u l t s and D i s c u s s i o n  Composition  and n.m.r.  Isolation previously  spectra  and p u r i f i c a t i o n  described  (Section  of t h e p o l y s a c c h a r i d e ,  III.4),  provided  p o l y m e r as i n d i c a t e d by e l e c t r o p h o r e s i s . C j" Q a  +  spectrum"''^' i n d i c a t e d a s h a r p s i n g l e t  The "''H n.m.r.  a t 62.17,  o f 0 - a c e t y l , and t h e a b s e n c e o f p y r u v a t e . the  acetyl:sugar  ratio  signal.  signal  In t h e a n o m e r i c r e g i o n  s i g n a l s were o b s e r v e d , due t o t h e m e t h y l apparent 20,21).  C  ment w i t h  Determination  ( 64.5-5.5),  g r o u p s o f two 6-deoxy  of the  five  sugars,  group  proton  a t 61.28, was a l s o  80 and a p p e n d i x s p e c t r a n o . ' s  n.m.r. s p e c t r o s c o p i c  t h e "'"H n.m.r. d a t a ,  of  and t h e rhamnose m e t h y l  and a s i x - p r o t o n d o u b l e t  (See T a b l e I V . 1 , page 1 3  characteristic  (1:1.5) was made by c o m p a r i s o n  i n t e g r a l s f o r the acetate  had  w e i g h t o f 2.6 x  U  lO"' a s d e t e r m i n e d by g e l c h r o m a t o g r a p h y .  a homogeneous  The p r o d u c t  4.0 (£ 1.08, w a t e r ) and a m o l e c u l a r  as  i n f o r m a t i o n ^ was i n a g r e e -  and i n a d d i t i o n , showed t h e  p r e s e n c e o f two h e x o s e s (two s i g n a l s were o b s e r v e d a t 61.6 and  60.2 p.p.m., i n d i c a t i v e o f t h e s i g n a l s from C-6 o f two  h e x o s e s ; see T a b l e I V . 1 and A p p e n d i x s p e c t r u m no. 2 2 ) . n.m.r. and  1 3  C  n.m.r. s p e c t r a i n d i c a t e t h e p r e s e n c e o f two  rhamnose and two h e x o s e r e s i d u e s . composition  o f K44 c a p s u l a r  t o deduce t h a t t h e r e m a i n i n g unit  From t h e q u a l i t a t i v e  polysaccharide,  i t i s possible  sugar r e s i d u e  i n the repeating  i s that of g l u c u r o n i c a c i d . Methanolysis  of the product  with  o f K l eb s i e l 1 a K44 p o l y s a c c h a r i d e , sodium b o r o h y d r i d e  reduction  i n d r y m e t h a n o l , and  TABLE I V . 1  N.M.R. d a t a  of K l e b s i e l l a  K44 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  oligosaccharides 1  Compound  13  H  L  6 ( c o u p l i n g con s t a n t ) ^, i h t e g r a l , a s s i g n m e n t (Hz) a  Rha  13 12 G1 c—r—e a 8  rythritol 7  GlcA^-Rha-OH  G 1 c A^Q-^-R h a - i - ^ R h a - 0 H  p 3a  a  E  5. 1 8 ( s ) , 1H, 4. 60(8 ), 1H, 1. 2 8 ( 6 ) , 3H,  H - l a-Rha H-l B-Glc H-6 Rha  p.p.m.°  assignment  103. 3 C-1 B - G l c 101. 9 C-1 a-Rha 63. ? C-6 G l c 62 . 0 C-1 e r y t h r i t o l 61. 6J [C-4 e r y t h r i t o l 17. 3 C-6 Rha  5. 3 4 ( b ) , 4. 8 5 ( s ) , 4. 7 0 ( 6 ) , 1. 2 8 ( 6 ) ,  0.6H, H - l a-Rha-OH 0.4H, H - l B-Rha-OH IH,-, H - l 3 _ G l c A 3H, H-6 Rha  105. 0 93. 8 17. 6  C-1 B-G I c A C-1 a , B-Rha C-6 Rha  5. 3 2 ( s ) , 5. 1 K b ) , 4. 8 8 ( s ) , 4. 6 9 ( 7 ) , 1. 2 9 ( 6 ) ,  IH, H- 1 a-Rha 0 . 4H, H -1 a-Rha-OH 0.6H, H -1 B-Rha-0H IH, H- 1 B -G 1 cA 6H, H- 6 Rha  105. 1 101. 6 94. 8 94. 2 17 . 5  C- 1 C- 1 C- 1 C- 1 C- 6  B - G l cA a-Rha a-Rha-OH B-Rha-OH Rha  TABLE  Gl  IV . 1 ( c o n t i n u e d )  \l  cH  c A — -^Rha-0H  e  4a  Glc  1 4, G l c- — G l c A — •^R h a — ^flha-OH a  IH, H- 1 a-G l c 0.6H, H -1 a-Rha- OH 0 . 4H , H -1 6-Rha- OH IH, H- 1 B - G l c A 1H, H- 1 3 - G l c 3H, H- 6 Rha  105. 1 10 3. 3 99 .3 93. 8 61. 4 60 .2J 17 .6  5. 4 1 ( 3 ) , 5. 2 9 ( b ) , 5. 0 9 ( b ) , 4. 8 6 ( s ) , 4. 6 9 ( 8 ) , 4. 5 3 ( 7 ) , 1. 2 7 ( 6 ) ,  IH, H -1 a - G l c IH, H -1 - R h a 0 . 5H, H- 1 a-Rha -OH 0 . 5H, H- 1 3-Rha -OH IH, H -1 8 - G l c A IH, H -1 8 - G l c 6H, H -6 Rha  10 5. 2 C-1 3 - G l c A 103. 2 C-1 3 - G l c C-1 a-Rha 101. 7 C-1 - G } c 99 .4 C-1 - R h a - 0 H 94. 8 94. 2 C-1 3-Rha-OH 61. 5) fC-6 a - G l c 60 .2J [C-6 3-G l c 17 .4 C-6 Rha  a  1  C- 1 C- 1 C- 1 C- 1 6 6 Lcc- 6  3-GIcA 3-Glc a-Glc a, 3-Rha a-Glc '3-Glc Rha  5. 4 2 ( 3 ) , 5. 3 0 ( b ) , 4. Q 3 ( s ) , 4. 6 9 ( 8 ) , 4. 5 3 ( 7 ) , 1. 2 8 ( 6 ) ,  a  a  TABLE I V . 1 ( c o n t i n u e d )  3 1 4 1 . 4 , ,1 2 ,. 1 3 . l —G1 c G1 c G1 c A =R h a Rh a P 1  r i  p  r  a  n  D  B  a  a  K44 p o l y s a c c h a r i d e  5.41(4.5), IH, H - l - G l c 5 . 2 8 ( ~ 1 ) , I H , H - l a,Rha 5 . 1 5 ( 1 . 5 ) , I H , H - l a-Rha 4.71(8), IH, H - l B-GlcA 4.54(8), IH, H - l B - G l c a  'l.28(6.5),  6H, H-6 Rha  105. 2 103. 2 101. 8 99 . 8 61. 6 60 .2 17. 4  C -1 C -1 c -1 c -1 -6 -6 c -6  rip  B-GIcA B-Glc a-Rha(2) a-Glc B-Glc a-Glc Rha  CO  ho I  Footnotes: g  Chemical  shift relative  to internal  a c e t o n e ; 6 2.23 d o w n f i e l d from  D.S.S.  ^ s , s i n g l e t ; b, b r o a d , u n a b l e t o a s s i g n a c c u r a t e c o u p l i n g c o n s t a n t . F o r H-6 of Rha coupling constant refers- t o J5,6. C h e m i c a l s h i f t r e l a t i v e t o i n t e r n a l a c e t o n e ; 31.07 p.p.m. dovi/nfield from D.S.S. ^Two u n r e s o l v e d d o u b l e t s ; s e e A p p e n d i x ,  s p e c t r a no. 20,21.  e O l i g o s a c c h a r i d e s 3 and 4 were n o t c o m p l e t e l y p u r e ; m i n o r n.m.r. s i g n a l s a r e omitted f o r c l a r i t y (see text f o r d e t a i l s ) .  83  -  then  h y d r o l y s i s of the carboxyl-reduced  a mixture  o f rhamnose  Identification acetates. and  -  and g l u c o s e  in the proportion  45:55.  was made by g . l . c .  a n a l y s i s of t h e i r  alditol  Rhamnose and g l u c o s e  were shown  t o be of t h e L  D c o n f i g u r a t i o n r e s p e c t i v e l y by t h e c i r c u l a r  curves  of the c o r r e s p o n d i n g These r e s u l t s  polysaccharide  alditol  show t h a t  i s composed  the r e p e a t i n g  a n o m e r i c l i n k a g e s were d e t e r m i n e d  Methylation  derived  u n i t o f K44  o f two r e s i d u e s o f L-rhamnose,  and two 3 - g l y c o s i d i c l i n k a g e s .  oligosaccharides  dichroism^  acetates.  two o f D - g l u c o s e , one o f D - g l u c u r o n i c a-  material, yielded  from  a c i d , and has  Final after  three  assignments of the examination  the o r i g i n a l  of  polysaccharide.  analysis  9 Methylation confirmed  analysis  t h a t K44  of the a c i d i c  i s composed  of a pentasaccharide  u n i t and i n d i c a t e d t h a t i t i s a l i n e a r T a b l e I V . 2 , c o l u m n s 1 and I I , page o f a l l p e a k s from partially  methylated  columns o f OV-17 glucose  the g . l . c .  residue  alditol  and OV-225. than  expected  because of the incomplete ronosyl methyl Uronic  acid  polysaccharide repeating  polysaccharide  8 4).  Complete r e s o l u t i o n  a n a l y s i s of the mixture acetates  (see  was  of  accomplished  on  Lower amounts  of t h e  dimethyl  were o b t a i n e d  most',  likely  carboxyl  reduction  of t h e g l u c u -  ester.  degradation  A sample o f m e t h y l a t e d  K44  capsular  polysaccharide  was  TABLE IV.2  Methylation analysis  of o r i g i n a l  Methylated sugars (as a l d i t o l a c e t a t e s )  b  3  2-E t - 3 , 4 - R h a  c M  Column B  2,3,4-Rha  and d e g r a d e d K44 c a p s u l a r p o l y s a c c h a r i d e  d  Column C  e  I  f  o  l  e  II  °° III  0 . 48  49 .8 0. 50  g  IV  26:4  3, 4.-Rha  0 . 77  0.89  19 . 9  2,4-Rha  0 .90  0. 95  22 . 4  31.9  2 , 4, 6 - G l c  1.56  1. 51  22.0  34.0  2,3,6-Glc  1. 56  1.72  21.8  7.7  2,3-Glc  2.14  2.41  13.9  33. 5  3  2,3,4-Rha = 1, 5 - d i - 0 - a c e t y 1 - 2 , 3 , 4 - 1 r i - 0 - m e t h y 1 - L - r h a m n i t o l , e t c . Retention time r e l a t i v e to 1,5-di-0-acety1-2,3,4,6-tetra-0-methy1-D-glucitol. c  V a l u e s . c o r r e c t e d by u s e o f e f f e c t i v e OV-17  carbon  r e s p o n s e f a c t o r s g i v e n by A l b e r s h e i m  c o l u m n , programmed a t 175° f o r 8 min and t h e n  et a l  24  2 / m i n t o 210°C. u  0 V - 2 2 5 c o l u m n , programmed a t 180°for 4 min t h e n a t 2 / m i n t o 230°C. f I , o r i g i n a l p o l y s a c c h a r i d e , column B; I I , o r i g i n a l p o l y s a c c h a r i d e , column C; I I I , p o l y s a c c h a r i d e a f t e r u r o n i c a c i d d e g r a d a t i o n , column C; IV, p o l y s a c c h a r i d e a f t e r p e r i o d a t e o x i d a t i o n - S m i t h d e g r a d a t i o n , compound jL,. column B. l,5-di-0-acetyl-2-0-ethyl-3,4-di-O-methyl-L-rhamn i t o l .  e  u  9  ^Remainder of t h e t o t a l  i s made up o f 2 - 0 - a c e t y 1 - 1 , 3 , 4 - t r i - 0 - m e t h y l - D - e r y t h r i t o l , 16.7%.  - 85 subjected  to a uronic acid degradation ,  and t h e p r o d u c t  1 0  was d i r e c t l y  a l k y l a t e d with  ethyl i o d i d e  1 1  .  H y d r o l y s i s of 12  the  d e g r a d e d , e t h y l a t e d m a t e r i a l and g.1.c.-m,s. . a n a l y s i s  of t h e a l d i t o l  acetates  T a b l e I V . 2 , column residues  y i e l d e d t h e compounds l i s t e d i n  I I I , page 84 .  i n v o l v e s cleavage  of the l i n e a r  p a n i e d , by f u r t h e r d e g r a d a t i o n witnessed glucose halted  by a l m o s t  residue.  Loss of g l u c u r o n i c a c i d chain  o f e x p o s e d r e d u c i n g - g r o u p s as  c o m p l e t e l o s s o f t h e 2 , 3, 6 - 1 r i - 0 - m e t h y 1 -  Still  f u r t h e r degradation  at t h i s - p o i n t because of f o r m a t i o n  o r " p e e l i n g " was of the base-stable,  3-deoxy-hex-2-enopyranose u n i t (see S e c t i o n and  Figure  I I . 7,  and was accom-  page  27).  I I . 4 , page 24  The a p p e a r a n c e o f 2 - 0 - e t h y l - 3 , 4-  d i - O - m e t h y l r h a m n o s e , a c c o m p a n i e d by t h e d i s a p p e a r a n c e o f t h e 3,4-di-0-methy1rhamnose r e s i d u e , d e t e r m i n e s t h e sugar to  0-1 o f g l u c u r o n i c a c i d .  Therefore,  sugars of the pentasaccharide saccharide Periodate  t h e sequence of t h r e e  r e p e a t i n g u n i t o f K44 p o l y -  i s *4)-D-Glcp-(l-»-4)-D-GlcAp-(l*2)-L-Rhap-(l-»-. oxidation  Periodate polysaccharide  oxidation " 1  (see Figure  with  3.6 m o l e s o f p e r i o d a t e  unit  after  5  of p a r t i a l l y  I I . 8 , page  24 h ( t h e o r e t i c a l  being  depolymerized  31) p r o c e e d e d  Smith  1 3  indicated  C  n.m.r. s p e c t r o s c o p y  rapidly  degradation ^ 1  periodate-oxidized  f o l l o w e d by g e l c h r o m a t o g r a p h y y i e l d e d o l i g o m e r and  K44  consumed p e r mole o f r e p e a t i n g  = 3 moles).  of t h e sodium b o r o h y d r i d e - r e d u c e d ,  *H  linked  of 1 (see Table  t h e p r e s e n c e o f two n o n - r e d u c i n g  product  1_, Cofjp -44°. IV.1,  anomeric  page 80) signals;  - 86 one 3 - h e x o s e s i g n a l attributable no.'s  one  8  Hz) and a n o t h e r  t o an ot-L-rhamn ose  11,12).  carbon  (J^ ^  signals  In a d d i t i o n ,  large signal  accounted  n.m.r. s p e c t r u m  1 3  16 s i g n a l s  f o r two carbon  t h r e e s i g n a l s a t 61-63 p.p.m. ( i n d i c a t i v e signifying''.that,  besides  (singlet)  r e s i d u e (see Appendix,  the C  (theoretically  signal  showed 15  should appear but atoms),  including  o f -CH^OH  t h e two h e x o s e s u g a r s ,  f r a gm en t (e r y.th r i t o l ) was p r e s e n t  spectra  groups),  a four  on t h e r e d u c i n g  carbon  terminus.  12 Methylation  o f j^,  hydrolysis,  and g . l . c . - m . s .  a c e t a t e s gave 2 , 3 , 4 - t r i - 0 - m e t h y 1 r h a m n o s e ,  of the a l d i t o l  2,4,6-tri-O-methyl-  g l u c o s e , and 1 , 3 , 4 - t r i - 0 - m e t h y 1 e r y t h r i t o l ( s e e T a b l e I V . 2 , column  IV, page  8 4 ) . The s t r u c t u r e  o f compound 1 i s t h u s  established as: a-L-Rhap-(l->-3) - 3-D-G l c p - ( 1*2) -D-ery t h r i t o l  The  results  obtained  from  uronic acid  periodate oxidation are s u f f i c i e n t of t h e r e p e a t i n g u n i t  (j.)  d e g r a d a t i o n and  to e s t a b l i s h  t h e sequence  o f K44 p o l y s a c c h a r i d e as b e i n g  *3)-L-  Rhap-(1*3)-D-G 1 c p - ( 1 * 4 ) - D - G 1 c p - ( 1 * 4 ) - D - G 1 c A p - ( 1 * 2 ) -L-R hap-( 1-^. In o r d e r t o a s s i g n t h e r e m a i n i n g  anomeric c o n f i g u r a t i o n s to  g l y c o s i d i c l i n k a g e s , examination  of the products  h y d r o l y s i s was Partial  from  partial  undertaken.  hydrolysis Partial,  acidic hydrolysis  o f K44 p o l y s a c c h a r i d e .and  - 87 separation  of a c i d i c  and n e u t r a l  components by i o n e x c h a n g e  chromatography  yielded a mixture  of a c i d i c  were s e p a r a t e d  by g e l f i l t r a t i o n  chromatography.  oligomer  (_5)  and u n r e s o l v e d  Compound _5, C 3 Q spectroscopy  (see Table  components (>)  25°,  +  a  oligomers  IV.1,  that  A pure  were  was shown by "'"H and  obtained. 1 3  C  n.m.r.  page 80 and A p p e n d i x ,  spectra  no. 's 18,19) t o g e n e r a t e two n on - redu c i n g , ^ - a n o m e r i c s i g n a l s , two  n on - redu c i n g, a - a n o m e r i c s i g n a l s , and two s i g n a l s  butable  to a reducing  6-deoxyhexose.  Methylation,  attri-  reduction 12  with l i t h i u m  aluminum h y d r i d e ,  a n a l y s i s of the a l d i t o l in  Table  I V . 3 , column  F r a c t i o n _3>  acetates  H p  "» i n i t i a l l y  t r i m e r as w e l l .  identical  of t h e m i x t u r e one  reducing  rhamnose u n i t  also observed and  indicated that  (see Table  relative  the major  IV.1).  C-6 a t 60.9 p.p.m.) n.m.r. s p e c t r a  (as described  column  of 3 a r e d i s c u s s e d  1 3  C  n.m.r.  component ( 3 a )  rhamnose, and a  M i n o r s i g n a l s were 1 3  C  ( C - 1 a t 99.6  indicative  s p e c t r a no.'s 14.15).  o f an  Methylation  f o r compound _5) showed t h e r e s i d u e s  proportions  Table IV.3,  (~20?o) o f a n o t h e r  The "'"H and  ( 6 5 . 4 3 , 0.2 H) and  1  analysis  (see  by t h i s method.  i n the H  (see Appendix,  t o be a p u r e  c h r o m a t o g r a p h y and,  8 - l i n k e d h e x o s e , one a - l i n k e d  a-hexose  and  thought  a c e r t a i n amount  properties in gel f i l t r a t i o n  contains  listed  These two compounds had e s s e n t i a l l y  h e n c e , were i n s e p a r a b l e spectra  p r o d u c e d t h e compounds  I , page 8 8 .  a l do t r i o u r on i c a c i d , c o n t a i n e d acidic  h y d r o l y s i s , and g . l . c . - m . s .  o f t h e two t r i s a c c h a r i d e s i n v o l v e d III).  later.  The s t r u c t u r e s o f t h e components  TABLE IV. 3  Methylation hydrolysis  Methylated sugar (as a l d i t o l a c e t a t e ) 3  analysis  of a c i d i c o l i g o s a c c h a r i d e s i s o l a t e d  partial,  acid  o f K44 c a p s u l a r p o l y s a c c h a r i d e  T  b  ^d  ^ j  Mole %°  3, 4-Rha  0 .77  24.8  27 . 5  2,4-Rha  0.90  19 .9  7. 5  2,3,4, 6 - G l c  1.00  22 . 6  28 .0  2,3,6-Glc  1 . 56  20 . 4  18 . 1  2, 3, 4 - G l c  1.62  2, 3 - G l c  2 . 14  g  after  36.2  61.3  22.9 6.7 27.0  12 .3  18 .9  38 .7  7.2  3,4-Rha = 1 , 2 , 5 - t r i - 0 - a c e t y 1 - 3 , 4 - d i - 0 - m e t h y 1 - L - r h a m n i t o l , e t c . Retention time r e l a t i v e t o 1,5-di-0-acety1-2,3,4,6-tetra-0-methy1-D-glucitol c o l u m n , programmed a t 175° f o r 8 min and t h e n 2 / m i n t o 210°C.  on an OV-17  u  c  Values  c o r r e c t e d by u s e o f e f f e c t i v e  I , p e n t a s a c c h a r i d e , compound _5> U > IV, a l d o b i o u r o n i c a c i d ,  component2;  carbon  response  tetrasaccharide, compound 2_.  factors  g i v e n by A l b e r s h e i m  et a l  component 4-; I I I , t r i s a cch a r i d e,  24  - 89 Another p a r t i a l performed  in order  separation  h y d r o l y s i s of t h e  to i s o l a t e  of a c i d i c  and  p o n e n t s were s e p a r a t e d chromatogram is  produced  shown i n F i g u r e  and  _5,  -  the  n e u t r a l components, the  by  gel f i l t r a t i o n  page 80.  Compound 2,  [>]  spectroscopy  (see Table  attributable  t o one  - 7.0°, IV.1)  ( s e e A p p e n d i x s p e c t r u m no. reduction  with l i t h i u m  derivatization  was  13).  one  acetates  column  Thus, the  collected  another  "*"H and  reducing  1 3  C  n.m.r.  rhamnose u n i t o f 2,  subsequent  h y d r o l y s i s , and 4-di-0-methyl-  (see  Table  IV.3,  s t r u c t u r e of compound 2_ i s shown to  !-D-GlcAp-(l+-2)-L-Rhap  s a c c h a r i d e s has  The  anomeric s i g n a l s  gave 3,  2,3,4-tri-O-methylglucose  occurrence  2 and  Methylation  rhamnose and IV).  fractions  shown by  aluminum h y d r i d e ,  as a l d i t o l  com-  obtained.  to generate  8 - n e x o s e and  acidic  In a d d i t i o n t o components _3  s m a l l amount of a l d o b i o u r o n i c a c i d  a  After  chromatography.  from t h e w e i g h t o f t h e  IV.1,  was  aldobiouronic acid.  m i x t u r e , _4, o f t e t r a s a c c h a r i d e s were  The  polysaccharide  (2)  of t h i s a l d o b i o u r o n i c a c i d been r e p o r t e d  be:  in b a c t e r i a l  poly-  f o r K l eb s i e l l a K - t y p e s 36 "* 1  and  18 . 1 6  The in  components 3. were i d e n t i c a l  the p r e v i o u s and  13  C  partial  to t h o s e  h y d r o l y s i s experiment,  n.m.r. s p e c t r o s c o p y .  The  obtained as  judged  by  f o l l o w i n g s t r u c t u r e s of  12n  pentaColumn: Bio-Gel P-2 (100 x 3 cm). Flow rate: 10 m l / h .  10H  Collection: 4 f r a c t i o n s / h after void volume of 150 ml. 8WT. (mg) 6j M3  O  4H  40  20  '  7o  FRACTION NO. F i q u re I V . 1  Gelfiltration from p a r t i a l acid,  chromatography  hydrolysis  l h , 95 C). U  separation  of K l e b s i e l l a  of a mixture  ilo  loo"  80  of saccharides  K 44 p o l y s a c c h a r i d e  (0.5  obtained  M trifluoroaceti c  - 91 t h e m a j o r component _3_a and m i n o r component _3b a r e c o n s i s t e n t with  the methylation  and w i t h  and n.m.r. s p e c t r o s c o p i c  data  obtained,  2.  t h e known s t r u c t u r e o f compound  3-D-GlcAp-(l*2)-a-L-Rhap-(l*3)-L-Rhap  (3a)  and a - D - G l c p - ( l * 4 ) - 3-D-GlcAp-( 1*2)-L-Rhap  The a l d o t r i o u ron i c a c i d 3a.  n  the  structural  a  been i s o l a t e d  s  i n v e s t i g a t i o n s of K l e b s i e l l a  Fraction  4-, [oQ  +41 °, was a g a i n  n  by t h e g e l f i l t r a t i o n  technique.  component 4-a i s d i s c e r n a b l e . (see  two 3 - l i n k e d  component c o n t a i n s h e x o s e per r e d u c i n g were o b s e r v e d i n  1  spectra indicative  x  1  inseparable  1 3  C  3_, a m a j o r  n.m.r.  spectra  i n d i c a t e d that the major h e x o s e s and one  a-linked  S i g n a l s of a minor  H) and  Methylation  y i e l d e d t h e r e s u l t s shown data  1  nature  13 C (101.7 p.p.m.) n.m.r.  o f an a - l i n k e d rhamnose ( s e e A p p e n d i x ,  s p e c t r a no.'s 16,17).  From t h e s e  K36 '' and K S l ^ .  As f o r f r a c t i o n  rhamnose u n i t .  H (°5.10, 0.2  previously in  a mixture  The "'"H and  Table IV.1) o f t h e m i x t u r e  before  (3b)  a n a l y s i s as  described  i n T a b l e I V . 3 , column I I .  i t i s p o s s i b l e t o propose s t r u c t u r e s f o r the  m a j o r component _4a and t h e m i n o r component j4b_, knowing t h e s t r u c t u r e s o f compounds  2_, _3a, and _3b_, as f o l l o w s :  3 -D-G l c p - ( l * 4 ) - a - D - G l c p - ( 1*4) - 3 - D - G l c A p - ( 1*2 )-L-Rhap and  (_4a)  - 92 o - D - G l c p - ( l * 4 ) - 3 - D - G l c p - ( 1*2)- a - L - R h a p - ( 1 * 3 )-L-Rhap  Component _5 was i d e n t i c a l previous  partial  spectroscopy. s c o p i c data discussed  to that  obtained  i n the  h y d r o l y s i s e x p e r i m e n t , as j u d g e d by "'"H n.m.r.  Based on t h e m e t h y l a t i o n  and n.m.r.  and k n o w l e d g e of o l i g o s a c c h a r i d e  above,  (4b)  spectro-  structures  t h e s t r u c t u r e of compound _5 i s e s t a b l i s h e d a s :  3 - D - G l c p - ( l * 4 ) - a - D - G l c p - ( 1 * 4 ) - 3-D-G I c A p - ( 1 * 2 ) - a-L-R hap(_5) (1*3)-L-Rhap  The is  evidence presented  consistent with  for Klebsiella  K44  polysaccharide  the f o l l o w i n g s t r u c t u r e .  *3)-3-D-Glcp-(l*4)-a-D-Glcp-(1*4)-  3-D-GlcAp-(1*2)-a-L-  Rhap-(l*3)-°<-L-Rhap-(l*  The repeating previous  existence  of an 0 - a c e t y l  u n i t i s n o t uncommon structural  polysaccharides,  g r o u p on o t h e r  of K20  18  every  and has been d e m o n s t r a t e d i n  i n v e s t i g a t i o n s of K l e b s i e l l a  those  than  and K59  19  capsular  - 93 IV . 4  Experimental  General  methods Instrumentation  (see S e c t i o n tography, ethyl  III.4,  the  u s e d has  page 5 9 ) .  ethyl  g.l.c.  For  descending  - formic acid  a c e t a t e - p y r i d i n e - water  m X 3 mm)  - water  mesh);  (B)  3%  (C)  3% of OV-225 on  3%  of 0V-17 SPT  on Gas  (18:3.1:4);  Analytical  Preparation  and  10C  on Gas  for K l e b s i e l l a  Chrom  K17  was  grown and  (see  Section  isolated III.4,  i n the s o d i u m s a l t  water).  of the p o l y s a c c h a r i d e  p h o r e s i s u s i n g a l?o s o l u t i o n ( S e p r a p h o r e I I I ; 15 X 2.5 Produkter  AB,  on  cm)  S t o c k h o l m 12,  (pH4)  a l s o confirmed  m X 6.3  obtained  f o r m , had was  Sweden) a t 300  g e l chromatography  polysaccharide  by  courtesy previously  The  isolated  [of] ^ +4.0° (£  c h e c k e d by acetate  electro-  8.6  V f o r 90  in c i t r a t e  min  (LKBand  buffered  Homogeneity  courtesy  1.08,  strips  i n v e r o n a l b u f f e r pH  blue  mm)  capsular  page 59).  or p e r i o d a t e - S c h i f f r e a g e n t . by  (100-  g.l.c.  as d e s c r i b e d  cellulose  development i n e i t h e r a l c i a n  ethanol  K44  (7730),  polysaccharide, Purity  ml/  Q (100-120 mesh).  p r o p e r t i e s of K l e b s i e l l a  I. 0rskov,  r a t e of 20  Preparative  a l a r g e r (1.8  A c u l t u r e o f K l e b s i e l l a K44 of Dr.  steel  Chrom Q (100-120 mesh);  (100-120 mesh).  5% of S i l a r  (D)  flow  (A)  of SP-2340 on S u p e l c o p o r t  s e p a r a t i o n s were p e r f o r m e d u s i n g column  gas  chroma-  were u s e d :  (8:2:1).  with a c a r r i e r  Columns u s e d were (A)  130  then  paper  s e p a r a t i o n s were p e r f o r m e d u s i n g s t a i n l e s s  c o l u m n s (1.8 min.  been d e s c r i b e d p r e v i o u s l y  f o l l o w i n g s o l v e n t systems (v/v)  acetate - a c e t i c acid  (B)  -  o f Dr.  S.  was C.  Churms,  - 94 University weight  o f CapeTown, S o u t h  Africa,  and t h e m o l e c u l a r  o f K44 p o l y s a c c h a r i d e d e t e r m i n e d The  ''"H n.m.r. s p e c t r u m  polysaccharide i n D^O  (0.4 M t r i f l u o r o a c e t i c  (IH, J  doublets  K44  15 m i n , 95°C),  to five  anomeric  ~ 1 H z ) , 5.15  8 H z ) , and 4.54 ( I H , J 1,  , Z  Two u n r e s o l v e d  acetic acid,  4.5 H z ) , 5.28 ( 1 H , J  1.5 H z ) , 4.71 ( I H , J 1  depolymerized  corresponding  u  a t 65.41 ( I H , J  5  of p a r t i a l l y  a t 9 0 C, r e v e a l e d s i g n a l s  protons  t o be 2.6 X 1 0 .  Z  centered  a t £1.28 (6H, J ,  8 Hz). 1,  Z  , 6.5 Hz)  5» &  were s i g n a l s a t t r i b u t a b l e hexose r e s i d u e s . 20,21). one  A sharp  proton  Migration  to the methyl  (see Table singlet  I V . 1 and A p p e n d i x , s p e c t r a n o . ' s  a t <$2.17 i n t e g r a t e d t o a p p r o x i m a t e l y  and was a s s i g n e d  to the methyl  of the a c e t a t e ' s s i g n a l  sodium h y d r o x i d e The  1 3  C  polysaccharide  g r o u p s o f two 6-deoxy-  confirmed  of p a r t i a l l y  showed f o u r s i g n a l s  of an a c e t a t e .  t o 61-91 upon a d d i t i o n o f  t o t h e sample t u b e  n.m.r. s p e c t r u m  group  this  depolymerized  signals.  K44  i n the anomeric region at  105.2, 103.2, 101.8, and 99.8 p.p.m. w i t h t h e s i g n a l p.p.m. b e i n g a p p r o x i m a t e l y  assignment.  a t 101.8  t w i c e the h e i g h t of the other  three  S i g n a l s a t 61.6 and 60.2 p.p.m. due t o C-6 o f two  h e x o s e u n i t s and a t 17.4 p.p.m. due t o C-6 o f a 6 - d e o x y h e x o s e unit  a l s o appeared.  distinguishable.  No s i g n a l  See T a b l e  from  I V . 1 , page  spectrum  n o . 22.  Analysis  of sugar c o n s t i t u e n t s  S u g a r a n a l y s i s was p e r f o r m e d ( s e e S e c t i o n I I I . 4, page  t h e CH-j o f a c e t a t e was 80 and A p p e n d i x ,  as p r e v i o u s l y d e s c r i b e d  59). The a l d i t o l  a c e t a t e s o f rhamnose and  - 95 glucose  were i d e n t i f i e d  by g . l . c . (column A; programmed a t  195° f o r 4 min and then present  i n the r a t i o  a t 2 °/min t o 260 °C) and f o u n d t o be  o f 45:55 r e s p e c t i v e l y .  (column D; programmed  a t 210° and then  to i s o l a t e  t h e d e r i v a t i v e s and t h e n c . d .  MeCN ^ 215  ^  e  the  glucitol  Methylation  ^he r h a m n i t o l  o r  Preparative g.l.c,  at 4 /min  t o 250 °C)  u  measurement showed MeCN andAe^^^ +0.30 f o r  pentaacetate  hexaacetate. analysis of native  A sample o f i s o l a t e d  polysaccharide  K44 p o l y s a c c h a r i d e  t h r o u g h a column o f A m b e r l i t e  IR-120(H ) resin +  was p a s s e d and t h e n  methy-  20 lated 59).  by t h e llak om o r i Methylation  procedure  was i n c o m p l e t e  (see Section  and t r e a t m e n t  III.4, with  page  Purdie's  21 reagents  was r e q u i r e d  gible absorption of t h e i n f r a r e d  in the hydroxyl spectrum.  lated  polysaccharide  furan  - ethanol  acid,  and r e d u c t i o n  acetylation  to obtain  with  a product  group r e g i o n  Carboxyl  sodium b o r o h y d r i d e  methylated  sodium b o r o h y d r i d e  alditol  -  o f t h e methyin tetrahydro-  2 M. t r i f l u o r o a c e t i c  i n p y r i d i n e - a c e t i c anyhdride  of p a r t i a l l y  negli-  (~3500 cm"'")  reduction  (1:1), h y d r o l y s i s with with  t h a t showed  acetates  was f o l l o w e d by (1:1).  The m i x t u r e  was a n a l y z e d  by g . l . c .  12 m.s.  .  The a l d i t o l  acetates  o f 3 , 4 - d i - 0 - m e t h y l r h a m n o s e , 2,4-  di-0-methylrhamnose, 2,4,6-tri-0-methylglucose, methylglucose,  and 2 , 3 - d i - 0 - m e t h y l g l u c o s e were i d e n t i f i e d ( s e e  T a b l e I V . 2 , c o l u m n s I and I I , page 8 4 f o r column suu s e d ) .  2,3,6-tri-0-  exact  r a t i o s and  - 96 Uronic acid  degradation  Methylated fully  and  acid,  dissolved  and  10 , 11  p o l y s a c c h a r i d e (37 mg)  The  in a mixture  base,  (20 ml)  alkylated  Following neutralization  w i t h 5Q%  the e t h y l a t e d , degraded  partition  between  chloroform  isolated  and  product  g.l.c.-m.s. a n a l y s i s  yielded  anion  (10 m l ) , was  a m i n o r peak due  IV. 2,  18 h,  product  isolated  was  addition by  t h e aqueous s o l u t i o n .  Hydro-  u s i n g 2 M. t r i f l u o r a ce t i c a c i d  to  acetate  derivatives  2-0-ethy1-3,4-di-0-methy12,3,6-tri-0-methylglucose,  to 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e .  column I I I , page :8'4  the iodide.  and  of t h e a l d i t o l  peaks c o r r e s p o n d i n g  After  added  acetic acid  rhamnose, 2 , A - d i - 0 - m e t h y 1rhamnose, and  care-  dimethylsu1foxide  directly"'""'" w i t h e t h y l  of w a t e r ,  of the  dried  (19:1) under n i t r o g e n i n a s e a l e d  dimethy1su1finy1  d e g r a d e d m a t e r i a l was  and  of  a l l o w e d t o r e a c t a t room t e m p e r a t u r e .  lysis  was  a l o n g w i t h a t r a c e of t o l u en e-p-su 1 f on i c  2,2-dimethoxy-propane  flask. and  then,  K44  -  f o r exact  r a t i o s and  (See  column  Table  used.)  P e r i o d a t e o x i d a t i o n of K44 p o l y s a c c h a r i d e ' " K44 (50 ml) (50 m l ) .  polysaccharide  and  the s o l u t i o n  The  reaction  dark  and  1 ml  a l i q u o t s which  (300 was  was  mg)  was  dissolved  m i x e d w i t h 0.1  M. sodium  allowed to proceed  the p e r i o d a t e consumption  was  in  at 4 C  f o l l o w e d by  water periodate i n the  U  removing 22  were a n a l y z e d  by  the Fleury-Lange  method,  *In order to conserve m a t e r i a l , p a r t i a l l y d e p o l y m e r i z e d K44 p o l y s a c c h a r i d e , r e c o v e r e d from an a t t e m p t e d p a r t i a l h y d r o l y s i s e x p e r i m e n t , was u s e d i n t h e p e r i o d a t e s t u d y . The s a m p l e o f p o l y s a c c h a r i d e had u n d e r g o n e t r e a t m e n t w i t h 0.01 M_ t r i f l u o r o a c e t i c a c i d , f o r 70 h a t 95°C and then f o r t h e r e m a i n d e r o f s i x days at room t e m p e r a t u r e , i n t h e G a l a n o s appa r a t u s 42 3J) N e g l i g i b l e amounts of o l i g o s a c c h a r i d e s were o b t a i n e d and t h e p o l y s a c c h a r i d e r e c o v e r e d from t h e d i a l y s i s was c o n s i d e r e d tco l-.b:e e s s e n t i a l l y unchanged.  - 97 Periodate  consumption  was r a p i d  moles per r e p e a t i n g u n i t moles) a f t e r dialysis,  24 h.  freeze-drying,  of K l e b s i e l l a  Following  reduction  and r e a c h e d a l e v e l  with  K44 ( t h e o r t i c a l  the addition  sodium  the polyalcohol  o f 3.6 = 3  of ethylene g l y c o l ,  b o r o h y d r i d e , r e - d i a l y s i s , and ( p o l y o l ) was o b t a i n e d  (yield  192 mg). 14 Smith d e g r a d a t i o n  ( 0 . 5 IA_ t r i f l u o r o a c e t i c  room t e m p e r a t u r e ) o f t h e p o l y o l  ( 1 0 5 mg) y i e l d e d  p r o d u c t s w h i c h were s e p a r a t e d by g e l f i l t r a t i o n ( B i o - G e l P-2; 100 x 3 cm c o l u m n ) . w e i g h t 82 mg) were o b t a i n e d , p u r e compound,  collected  contained  fractions,  a mixture of chromatography  fractions  (total  one was f o u n d t o be a  from t h e w e i g h t o f t h e f r e e z e p u r e o l i g o s a c c h a r i d e 1_ (6 mg) was  i n t u b e numbers 72-76  Compound J. (R water).  but only  17 h,  as j u d g e d by "''H n.m.r. s p e c t r o s c o p y . A c c o r d i n g  t o t h e chromatogram produced dried,  Several  acid,  l  inclusive.  0.44, s o l v e n t B) had [of]  c  D  -44° ( £ 0 . 8 7 ,  In t h e "''H n.m.r. s p e c t r u m , s i g n a l s a t t r i b u t a b l e t o  a n o m e r i c p r o t o n s were o b s e r v e d a t 65.18 ( I H , s i n g l e t ) and 4.60 (IH,  j \ „ 8 H z ) , as w e l l a s , 1.28 ( 3 H , J . , 6 Hz) due t o t h e 1  5,6  , Z  m e t h y l g r o u p o f rhamnose ( s e e T a b l e I V . 1 , page 80 and A p p e n d i x , s p e c t r u m no. 1 1 ) . The  1 3  C  n.m.r. s p e c t r u m  103.3 and 101.9 p.p.m. due t o a n o m e r i c 62.0,  showed s i g n a l s a t  c a r b o n atoms, a t 6 3 . 2 ,  and 61.6 p.p.m. due t o C-6 o f g l u c o s e and C-1 and C-4  of e r y t h r i t o l ,  and a t 17.3 p.p.m. from C-6 o f rhamnose ( s e e  T a b l e I V . 1 and A p p e n d i x , (see S e c t i o n reduction  III.4,  s p e c t r u m no. 1 2 ) . H a k o m o r i m e t h y l a t i o n  page 5 9 ) o f 1 f o l l o w e d  and a c e t y l a t i o n  yielded  by h y d r o l y s i s ,  the a l d i t o l  acetates  2,3,4-  - 98  -  t r i - ) - m e t h y l r h a m n o s e and  2,4,6-tri-O-methylglucose,  analysed  Only a m i n o r amount of  by  g.l.c.-m.s.  1,3,4-tri-O-methylerythritol tility  under reduced  column  IV,  Partial,  page 84  pressure  present  during  for relative  polysaccharide  (666  2-0-acetyl-  b e c a u s e of  work-up.  amounts and  a c i d i c h y d r o l y s i s of K44  K44 0.1  was  as  See  i t s volaIV. 2,  Table  column  details.  polysaccharide  mg)  was  partially  M_ t r i f l u o r a c e t i c a c i d a t 9 5° C u s i n g  an  hydrolyzed  in  apparatus s i m i l a r 23  to that  described  most of t h e (40  mg)  of t h e 1.5  of  G a l a n o s and  m a t e r i a l had  coworkers  been d i a l y z e d ,  polysaccharide  dialyzed material  cm)  remained was  ml)  (5-10 and  were e l u t e d  ml).  eliminate  freeze-dried  with  formic  The j u d g e d by  in the  10%  formic  acid  pressure  a c i d , and  340 (200  then  dialysis on  void  volume (160  freeze-drying .  (Bio-Gel  Every t h i r d  as  All  (30  P-2;  No  with  t u b e was  as  water  A c i d i c compounds to  water  (yield  to  220  mg) . as  was  separated  100  X 3 cm)  by  using  (1000:10:4) f o r material  dextran) a f t e r  the  X  volume  evaporated  B),  ( 2 - 2 . 5 ml)  t u b e s , f r e e z e - d r i e d , w e i g h e d , and a chromatogram.  ml),  acid  ml/h.  blue  Fractions  mg) .  (solvent  of 10  ml;  sac.  from n e u t r a l compounds  a s o l u t i o n of w a t e r - p y r i d i n e - a c e t i c rate  small  h,  amount  a column  freeze-dried  free  chromatography  at a f l o w  a small  s e v e r a l times with  paper c h r o m a t o g r a p h y  irrigation  A f t e r 38  compounds were e l u t e d  (Yield  acidic fraction,  gel f i l t r a t i o n  and  only  then a p p l i e d  Neutral  d r y n e s s under reduced  in the  .  of B i o - R a d AG1-X2(C1 ) r e s i n i n as  possible (1600  by  was  present  collection  were c o l l e c t e d i n  weights p l o t t e d to analyzed  by  paper  tared  produce chroma-  - 99 tography  (solvent A).  A major  component (j?, 32 mg) w i t h R g ^  A) and C°Q n +25° spectrum at  (c_ 1.95, w a t e r )  was o b t a i n e d .  3 H z ) , 5.29 ( I H , b r o a d ) ,  4.86 (0.5H, s i n g l e t ) , A doublet  4.69 ( I H , J  1 3  C  n.m.r. s p e c t r u m ,  a t 105. 2, 103. 2, anomeric  protons  5.09 ( 0 . 5H, b r o a d ) ,  8 H z ) , and 4.53 ( I H ,  ( J ^ ^ 6 Hz) a t $1.27 (6H) was a l s o  ( s e e T a b l e I V . 1 , page 80 and A p p e n d i x , the  "'"H n.m.r.  o f _5 showed s i g n a l s a t t r i b u t a b l e t o a n o m e r i c  65.41 ( I H , J  7 Hz).  0.14 ( s o l v e n t  c  spectrum  four non-reducing  J  apparent  n o . 1 8 ) . In  anomeric  signals  101 .7, and 99.4 p.p.m. and two r e d u c i n g  s i g n a l s a t 94.8 and 94.2 p.p.m. were o b s e r v e d .  Two  s i g n a l s a t 61.5 and 60.2 p.p.m. due t o C-6 o f two g l u c o s e  units  and  units  a l a r g e one a t 17.4 p.p.m. due t o C-6 o f two rhamnose  were a l s o 19).  evident (see Table  A portion  spectrum no.  (16 mg) o f compound jj was m e t h y l a t e d  Hakomori c o n d i t i o n s . reduced  IV . 1 and A p p e n d i x ,  with l i t h i u m  The perm e t h y l a t ed p r o d u c t was then aluminum  hydride i n reluxing tetrahydro-  fu ran o v e r n i g h t , h y d r o l y z e d w i t h 2 M. t r i f l u o r a c e t i c reduced acetic  w i t h sodium anhydride  presence  b o r o h y d r i d e and a c e t y l a t e d  (1:1).  G.l.c.-m.s. a n a l y s i s  of 3,4-di-0-methylrhamnose,  2,3,4,6-tetra-O-methylglucose,  See. T a b l e  I , page 88 f o r r e l a t i v e  column  indicated the  alditol  acetate  derivatives.  amounts and  used. A minor  this  in pyridine -  2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e , and  as t h e i r  column  acid,  2,4-di-0-methylrhamnose,  2,3-di-0-methylglucose IV. 3,  under  fraction  technique, with R  (.3; 15 mg), a m i x t u r e i n s e p a r a b l e by q  1  c  0.77 ( s o l v e n t A) and [of] ^ -19°  - 100 (c  1.44, w a t e r ) was a l s o  •*"H n.m.r. s p e c t r u m in t h e anomeric (see  The m a j o r  A small  signal  (see Appendix,  n.m.r. s p e c t r u m , and  a t 65.43 w h i c h  and 4.69  101.6 p.p.m. and two r e d u c i n g a n o m e r i c  and 94.2 p.p.m., as w e l l  integrates  a-D-glucose  s p e c t r u m n o . 1 4 ) . In t h e  two n o n - r e d u c i n g a n o m e r i c  rhamnose m e t h y l g r o u p  i n the  and a t 6i.29 due t o CH^ o f rhamnose  t o <~0.2 p r o t o n i n d i c a t e s t h e p r e s e n c e o f an component a l s o  signals  o c c u r r e d a t 65 . 32 , 5. 11, 4 . 88,  region  Table IV.1).  obtained.  as, a s i g n a l  were o b s e r v e d  1 3  C  s i g n a l s a t 105.1 s i g n a l s a t 94.8  a t 17.5 p.p.m. from a  (see Table IV.1).  Other,  l e s s i n t e n s e s i g n a l s were p r e s e n t a t 99.6 (n o n - r e d u c i n g a-Dglucose),  93.7 ( r e d u c i n g  glucose).  See A p p e n d i x ,  of  rhamnose),  and 60.9 p.p.m. ( C - 6 o f  s p e c t r u m no. 15;  Hakomori m e t h y l a t i o n  3.> f o l l o w e d by ca rbo x y l - redu ct i o n , h y d r o l y s i s and d e r i v a -  tization pound _5. acetates  were c a r r i e d  out under  G.l.c.-m.s. a n a l y s i s  t h e same c o n d i t i o n s as comidentified  a mixture of a l d i t o l  c o r r e s p o n d i n g t o 3,4-di-0-methylrhamnose,  methylrhamnose,  2,4-di-0-  2,3,4,6-tetra-0-methylglucose, 2,3,4-tri-0-  m e t h y l g l u c o s e , and 2 , 3 - d i - 0 - m e t h y l g l u c o s e i n t h e a p p r o x i m a t e ration for  1.25:1:0.25:1:0.25 ( s e e T a b l e I V . 3 , column  e x a c t r a t i o s and column Another  partial  on K44 p o l y s a c c h a r i d e  details).  hydrolysis  experiment  (240 mg) u n d e r  a c i d a t 9 5°C f o r 1 h.  acid  successive evaporations with  lization  w i t h 0.1 \A sodium  was s e p a r a t e d i n t o n e u t r a l  was p e r f o r m e d  t h e c o n d i t i o n s o f 0.5 M.  trifluoroacetic by s e v e r a l  I I I , page 8 8 ,  After  removal  of the  w a t e r and n e u t r a -  hydroxide, the hydrolyzed material and a c i d i c  f r a c t i o n s u s i n g AG1-X2(C1 )  -101resin. gel  The a c i d i c  components  chromatography  provided charide  (same  procedure  an a l d o b i o u r o n i c a c i d mixture  separated  (2_; 7 m g ) , an a c i d i c  pentamer  by  as d e s c r i b e d p r e v i o u s l y ) ,  (3_; 17 m g ) , an a c i d i c  (4-; 17 mg) , a n d an a c i d i c IV.1,  ( 2 0 0 m g ) , when  trisac-  tetrasaccharide  (_5; 39 m g ) .  mixture  See F i g u r e  page 9 0 . The  water).  aldobiouronic acid  T h e "*"H n . m . r .  (0.6H, b r o a d ) ,  spectrum  4.85 ( 0 . 4 H ,  J-^-J _7.o  2^^,16 contained  singlet),  (g_ 0 . 4 6 ,  u  s i g n a l s a t 65.34  a n d 4.70 ( I H J  6 Hz) 1? ^  in  the anomeric  due  to the methyl  spectrum in  region,  (reducing) IV.1). tion,  group  no. 1 3 ) .  the anomeric  as w e l l  o f rhamnose  In t h e  region  1 3  C  n.m.r.  (see Table spectrum,  a t 1 0 5 . 0 (n on - r e d u c i n g)  o f Z, f o l l o w e d  h y d r o l y s i s and d e r i v a t i z a t i o n the a l d i t o l  acetates  2,3,4-1ri-0-methy 1glucose  signals  occurred  a n d 9 3 . 8 p.p.m.  by  (see Table  carboxy1-reduc-  in the usual  manner  o f 3 , 4 - d i - O - m e t h y l r h a m n o s e and  (g.l.c.-m.s.;  Table  I V . 3 , column I V ,  88) . F r a c t i o n ~b_ was t h e same m i x t u r e  the  Hz)  I V . 1 and A p p e n d i x ,  a n d a t 1 7 . 6 p.p.m. f o r C-6 o f r h a m n o s e  Hakomori m e t h y l a t i o n  produced  page  a s , a t 6 1.28 ( 3 H , 3^.6  first  obtained  partial from  "''H a n d  Fraction completely  n.m.r.  from spectra  spectroscopy.  by g e l c h r o m a t o g r a p h y ,  T h e "'"H n . m . r .  a t 65.42,  unresolved  C  1 3  obtained  by t h e i d e n t i c a l  4-, a t e t r a s a c c h a r i d e m i x t u r e  separated  1. 5 3 , w a t e r ) . signals  h y d r o l y s i s , as j u d g e d  as t h a t  spectrum  which  was n o t  h a d []oTJ Q +41° ( c  contained  major  anomeric  5 . 3 0 , 4 . 8 3 , 4 . 6 9 , a n d 4.53 a n d t w o s e t s o f  doublets  (J  R  c  6 Hz) c e n t e r e d  a t 61.28 ( s e e T a b l e  - 102 IV.1).  An a d d i t i o n a l  signal  (~0.2H) i s a p p a r e n t a t 65.10  s u g g e s t i n g a m i n o r a - L - r h a m n o s e component as an i m p u r i t y ( s e e Appendix,  s p e c t r u m no. 1 6 ) . F o u r m a j o r  present i n the C 1 3  93.8 and  p.p.m., a l o n g w i t h  some f o r m , as an i m p u r i t y of 4- i n t o  nose,  103.3, 9 9 . 3 , and 60.2 (C-6 g l u c o s e )  i n T a b l e I V . 1 . The  (see Appendix,  spectrum no. 1 7 ) .  p a r t i a l l y methylated a l d i t o l acetates  t h e u s u a l manner p r o d u c e d  corresponding  and  61.4 (C-6 g l u c o s e ) ,  signals are  a t 101.7 p.p.m. c o n f i r m s t h e p r e s e n c e o f an a - L - r h a m n o s e ,  Derivatization in  a t 105.1,  17.6 p.p.m. (C-6 rhamnose) as l i s t e d  signal in  n.m.r. s p e c t r u m  anomeric  a m i x t u r e , i d e n t i f i e d by  to 3,4-di-0-methy1rhamnose,  g.l.c.-m.s.,  2,4-di-0-methylrham-  2 , 3 , 4 , 6-1 e t ra-0-m e t hy 1 g l u c os e , 2 , 3 , 6-1 r i - 0 - m e t hy 1 g l u co se ,  2 , 3 - d i - 0 - m e t h y l g l u c o s e ( s e e T a b l e I V . 3 , column I I ) . Compound _5> t h e a c i d i c  spectrum first  p e n t a s a c c h a r i d e , gave a "''H n.m.r.  i d e n t i c a l t o t h a t from t h e pentamer i s o l a t e d  partial hydrolysis  experiment.  a f t e r the  - 103 IV . 5  Bibliography  f o r S e c t i o n IV  1.  W. Nimmich, Z. M i c r o b i o l .  Immunol., 154, 1 1 7 ( 1 9 6 8 ) .  2.  W. Nimmich, A c t a B i o l .  3.  M. H e i d e l b e r g e r and W. N i m m i c h , Immun o ch emi s t r y , (1976) .  4.  G.G.S. D u t t o n , K. L. M a c k i e , A. V. S a v a g e , and M. D. S t e p h e n s o n , C a r b o h y d . R e s . , 6_6, 125( 1978 ).  5.  Y. M. Choy, G.G.S. D u t t o n , A. M. S t e p h e n , A n a l . L e t t . , 5, 6 7 5 ( 1 9 7 2 ) .  6.  G. M. B e b a u l t , Y. M. Choy, G.G.S. D u t t o n , N. F u n n e l l , A. M. S t e p h e n , and M. T. Yang, J . B a c t e r i o l . , 1_13, 1345( 1 9 7 3 ) .  7.  J . M. B e r r y , G.G.S. D u t t o n , L. D. H a l l , C a r b o h y d . R e s . , b±, C 8 ( 1 9 7 7 ) .  8.  G. M. B e b a u l t , J . M. B e r r y , Y. M. Choy, G.G.S. D u t t o n , N. F u n n e l l , L. D. Hayward, and A. M. S t e p h e n , Can J . Chem., 51, 3 2 4 ( 1 9 7 3 ) .  9.  H. B j o r n d a l , C.G. H e l l e r q v i s t , B. L i n d b e r g , and S. S v e n s s o n , Angew. Chem. I n t . E d . E n g l . , 9, 6 1 0 ( 1 9 7 0 ) .  Med. G e r . ,  2_6, 397( 1 9 7 1 ) . 1_3, 67  and M. T. Yang,  and K. L. M a c k i e ,  10.  B. L i n d b e r g , J . L o n n g r e n , Res, 2_8, 351( 1 9 7 3 ) .  and J . L. Thompson,  11.  G. 0. A s p i n a l l (1977) .  12.  J . L o n n g r e n and S. S v e n s s o n , 29_, 4 1 ( 1 9 7 4 ) .  13.  G. W. Hay, B. A. L e w i s , and F. S m i t h , Methods C a r b o h y d . 5, 357( 1 9 6 5 ) .  14.  I . J . G o l d s t e i n , G. W. Hay, B. A. L e w i s , and F. S m i t h , Methods C a r b o h y d . Chem., 5, 3 6 1 ( 1 9 6 5 ) .  15.  G.G.S. D u t t o n  16.  G.G.S. D u t t o n , K. L. M a c k i e , and M. T. Yang, C a r b o h y d . R e s . , 251(1978).  17.  M. C u r v a l l , B. L i n d b e r g , J . L o n n g r e n , C a r b o h y d Res., 42, 7 3 ( 1 9 7 5 ) .  and K. -G . R o s e l l ,  Carbohyd.  Carbohyd.  R e s . , _57,  Adv. Carbohyd.Chem.  and K. L. M a c k i e , C a r b o h y d .  c23  Biochem., Chem.,  R e s . , 5J5, 49 ( 1977) .  and W.  Nimmich,  - 104 18.  Y. M. Choy and G.G.S. D u t t o n , Can. J . Chem., _51, 3 0 1 5 ( 1 9 7 3 ) .  19.  B. L i n d b e r g , J . L o n n g r e n , U. Ruden, and W. Nimmich, R e s . , 42, 8 3 ( 1 9 7 5 ) .  20.  S. H a k o m o r i ,  21.  E. L. H i r s t 287(1965).  22.  R. D. G u t h r i e ,  23.  C. G a l a n o s , 0. L i / d e r i t z , B i o c h e m . , 8, 3 3 2 ( 1 9 6 9 ) .  24.  D. P. Sweet, R. H. S h a p i r o , R e s . , 40, 2 1 7 ( 1 9 7 5 ) .  J . Biochem.  Carbohyd  ( T o k y o ) , 55, 2 0 5 ( 1 9 6 4 ) .  and E. P e r c i v a l ,  Methods C a r b o h y d . Chem., _5,  Methods C a r b o h y d . Chem., _1, 435( 1 9 6 2 ) . and K. H i m m e l s p a c h ,  Eur. J .  and P. A l b e r s h e i m , C a r b o h y d .  APPENDIX:  N.M.R. SPECTRA  K17  polysaccharide  Solvent  D^O  Tern p •  8 0 °C  S.W.  3000  3  -, 0  4  65. 15  5.0 Mo.  4.35 4. 60  6.0 Spectrum  1 . 30  Hz  1  4  K17  polysaccharide  S.W.  8000  Hz  A.T.  0.5  P.W.  18  P.D.  0  N .T .  357, 900  sec ysec sec  K17  polysaccharide  Solvent  D^O  Temp.  9 0 °C  S.W.  1000  Hz  Pla  K17 n e u t r a l  disaccharide . 28  Glc-^-Rha-^OH S o l v e n t D ^0 Temp.  95°C  S.W.  1000 Hz  ••6 5.  S p e c t r u m No. 5  17  Spectrum  No. 6  . 6  K17  aldobiouronic  GlcA—-Rha~0H a Solvent  D^O  Temp.  85 C  S.W.  1000  U  Hz 1.33  6 5. 17  S p e c t r u m No. 7  K17  aldobiouronic  GlcA-i-^Rha-OH et',  S.W.  8 000  Spectrum  Hz  No. 8  acid  (47 mg/ml)  K17  acidic  tetrasaccharide  G1 c-i-^-R h a-^-^G 1 cA^-^R h a~0 H 8 a a Solvent  D^O  Temp.  35°C  S.W.  1000 Hz  4. 52 4. 57 65.17? !  5.11 4.87  S p e c t r u m - No. 9  K17 a c i d i c  tetrasaccharide  (9 2 mg/ml)  G 1 c-i-^R h a - — G l cA^-^R h a~OH B a a S.W.  8000 Hz  A.T.  0.507 s e c  P.W.  7 Usee  P.D.  0 sec  N.T.  160,300  a c e t,o n e 105  17 .  61. 4  Spectrum  No.  10  i  K44 Rha  Smith 13  G1 c  a  degradation 12  product  e rythritol  3  Solvent  D^O  Temp.  95°C  S.W.  1000 Hz 1. 28  S p e c t r u m No. 11  K44 S m i t h d e g r a d a t i o n p r o d u c t 13 12 R h a — — G 1 c—|—e r y t h r i t o l s. A . T.  8000 Hz 0.5 s e c  P.W.  15 p s e c  P.D.  0 sec  N.T.  90,709  - 118 -  i  i  K44  trisaccharide  Solvent  D0 2  Temp.  9 5 °C  S.W.  1000  Hz  j  Spectrum  (impure)  No.  14  .j  i  !  K44 a l d o t r i o u r o n i c G l cA-^r^-R h a-—^-R h a~0H  S.W.  8000Hz  A.T.  0.5 s e c  P.W.  18 u s e e  P.D.  0 sec  N.T.  3 43,622  Spectrum  No.  15  acid  (impure)  KA4  tetrasaccharide  Solvent  D^0  Temp.  9 5° C  S.W.  1000  Spectrum  No.  Hz  16  (impure)  K44  tetrasaccharide  (impure)  1 CA^T^-R h  G 1 c^-A  1 c^-^G  s . W.  8000  Hz  A . T.  0.50 7  sec  P .W.  20  P .D .  0  N.f 7  142,000  a~0H  ysec  sec  i  !  ;101.  Spectrum N o . 1 7  7  K44  pentasaccharide  Solvent  D^O  Temp  95 °C  S.W.  1000  S p e c t r u m No.  Hz  18  K44  pentasaccharide  Glc—^ G l c ^ c i c A S , W . 8000 A  . T .  0.5  P  .W .  18  Hz sec  ysec  P.D.  0  N.T.  438 , 90 0..  sec  S p e c t r u m No.  19  1  K44  polysaccharide  Solvent  D0  Temp  90 C  ~  S.W.  2 500  Hz  2  u  S p e c t r u m No.  20  K44  polysaccharide  Solvent D Temp.  90°C  S.W.  500 Hz  Offset  720 Hz  S p e c t r u m No. 21  K4A  polysaccharide  S.W.  8000  A.T.  0.5  P.W.  18  P.D.  0  Hz sec  ysec sec  ISt.T . 220 , 300 :  S p e c t r u m No.  22  

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