Synthesis of Oligosaccharides Containing 3-Deoxy-D-manno-2

6 Synthesis of Oligosaccharides Containing 3-Deoxy-D-manno-2-octulopyranosylono (KDO) Residues P. W A L D S T Ä T T E N , R. C H R I S T I A N , G . S C H U L Z , and F. M. U N G E R — Sandoz-Forschungsinstitut Vienna, A-1235 Vienna 23, Austria P. KOSMA—Institut für Chemie der Universität für Bodenkultur Vienna, A-1180 Vienna 18, Austria C. KRATKY—Institut für Physikalische Chemie der Universität Graz, A-8010 Graz, Austria

Downloaded by FUDAN UNIV on February 6, 2017 | http://pubs.acs.org Publication Date: September 29, 1983 | doi: 10.1021/bk-1983-0231.ch006

H . PAULSEN—Institut für Organische Chemie und Biochemie der Universität Hamburg, D-2000 Hamburg, Federal Republic of Germany

3-Deoxy-D-manno-2-octulosonic acid (KDO) is a characteristic, ketosidically-linked constituent of the lipopolysaccharide (LPS) in practically all the strains of Gram-negative bacteria examined so far (1, 2). Additionally, in an increasing number of cas es, KDO has been found as a constituent of capsular polysaccharides from Εscherichia coli (3) or Neisser ia strains (4). The significance of these bacterial, cell-surface macromolecules in the pathophysiology and diagnosis of Gram-negative bacterial infections has stimulated investigations into the biosynthesis and immunochemistry of structures containing KDO. In this context, model oligosaccharides involving KDO residues and representing partial structures of LPS or capsular polysaccharides have become of interest as artificial haptens, model substances for spectroscopy, or biochemical probes. In the present chapter, the state of efforts to synthesize KDO-containing oligo saccharides is briefly reviewed. KDO-containing of the 'inner

oligosaccharides core' of LPS.

as partial

structures

Following the identification of KDO as a constit uent of LPS, studies by Osborn and her group (5) have revealed that, in LPS, KDO (or 'the KDO region') is located at the reducing end of the polysaccharide chain, linking the core segment to lipid A. Later, the application of differential color reactions based on thiobarbituric acid (TBA tests) (6, 7) has led to the view that KDO is present, in LPS from Salmonella or E. coli, in the form of a branched trisaccharide (Fig. 1). In this model, a branchpoint KDO residue (KDO I), ketosidically-linked to the second glucosaminyl residue of lipid A, is substituted in position 4 or 5 by a 0097-6156/83/0231-0121$06.25/0 © 1983 American Chemical Society

Anderson and Unger; Bacterial Lipopolysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

BACTERIAL LIPOPOLYSACCHARIDES

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k e t o s i d i c a l l y - l i n k e d KDO r e s i d u e (KDO II) which f r e q u e n t l y c a r r i e s a 7-0-phosphorylethanolamine group; a t h i r d KDO r e s i d u e (KDO I I I ) , b e a r i n g on i t s 0-5 the remainder of the ' c o r e oligosaccharide together with the O - a n t i g e n i c c h a i n , w o u l d t h e n be k e t o s i d i c a l l y l i n k e d t o 0-7 o r 0-8 o f KDO I ( F i g . 1). Due t o t h e l i m i t a t i o n s o f t h e TBA a s s a y s ( 8 ) , i t has n o t b e e n p o s s i b l e , on t h i s b a s i s , t o d e f i n e more c l e a r l y t h e c o n s t i t u t i o n o f t h e KDO t r i s a c c h a r i d e . On the o t h e r hand, d e t a i l e d s p e c t r o s c o p i c a n a l y s e s of the KDO r e g i o n o f LPS h a v e n o t b e e n r e p o r t e d . I n f a c t , the a n o m e r i c c o n f i g u r a t i o n s o f t h e KDO r e s i d u e s i n LPS are s t i l l unknown, w h e r e a s s u c h c o n f i g u r a t i o n s h a v e b e e n a s s i g n e d t o t h e KDO r e s i d u e s p r e s e n t i n a number o f capsular polysaccharides (9^, J 3 , 30 ( c f . the chapter by J a n n i n t h i s v o l u m e ) . O n l y r e c e n t l y , p r o g r e s s has b e e n made t o w a r d t h e e l u c i d a t i o n o f s t r u c t u r e o f t h e KDO r e g i o n o f LPS by p h y s i c a l m e t h o d s . T h u s , n.m.r. m e a s u r e m e n t s seem t o i n d i c a t e t h a t i n LPS f r o m s e v e r a l s t r a i n s , two rather t h a n t h r e e KDO r e s i d u e s are present (JJ)). Brade, et a l . (11) h a v e d e t e c t e d what i s p r e s u m a b l y a d i s a c c h a r i d e K D 0 p 2 — » 4 o r 5 KDO, f o l l o w i n g h y d r o l y s i s (acetate buf fer, pH 4.5) o f LPS f r o m s e v e r a l s t r a i n s o f Salmonella. The f i n d i n g t h a t t h e p o l y s a c c h a r i d e c h a i n i s n o t r e l e a s e d f r o m l i p i d A u n d e r t h e s e c o n d i t i o n s has l e d B r a d e , e t a l . t o s p e c u l a t e t h a t o n l y one KDO residue i s l o c a t e d w i t h i n the main p o l y s a c c h a r i d e c h a i n , and t h a t t h e r e m a i n i n g two a r e p a r t o f a d i s a c c h a r i d e , side branch ( F i g . 2a). According to t h i s i n t e r p r e t a t ion, the K D O - t r i s a c c h a r i d e o f t h e h e p t o s e - l e s s , R 595 m u t a n t o f S. m i n n e s o t a w o u l d be a l i n e a r , r a t h e r t h a n a b r a n c h e d , t r i s a c c h a r i d e . Both the o r i g i n a l l y - p r o p o s e d , b r a n c h e d t r i s a c c h a r i d e ( F i g s . 1 and 2 b ) , and the l i n e a r s t r u c t u r e s u g g e s t e d r e c e n t l y , would have i n common a d i s a c c h a r i d e o f t h e c o n s t i t u t i o n K D 0 p 2 — » - 4 K D 0 , as f o u n d by P r e h m , e t a l . f o r t h e LPS o f t h e ' d e e p r o u g h ' , E. c o l i m u t a n t BB 12 (7.). The KD0j>2—*4KD0 l i n k a g e has a l s o b e e n p r o p o s e d ( J 2 ) f o r a product con t a i n i n g two KDO r e s i d u e s , i s o l a t e d f o l l o w i n g the enz yme-catalyzed t r a n s f e r iri v i t r o of r a d i o a c t i v e KDO f r o m CMP-KDO o n t o a l i p i d - A p r e c u r s o r m o l e c u l e . R e c e n t work i n t h e a u t h o r s ' l a b o r a t o r y has b e e n d i r e c t e d at the d e f i n i t i v e e l u c i d a t i o n of s t r u c t u r e of the K D O - t r i s a c c h a r i d e by n . m . r . - s p e c t r o s c o p i c m e t h o d s . F o r t h i s p u r p o s e , t h e LPS f r o m t h e h e p t o s e - l e s s , S». m i n n e s o t a R 595 m u t a n t i s b e i n g s t u d i e d , w h i c h has b e e n r e p o r t e d ( 6 J t o c o n t a i n , a s i d e f r o m l i p i d A, o n l y three KDO r e s i d u e s . The p r e p a r a t i o n o f a h o m o g e n e o u s p s e u d o t e t r a s a c c h a r i d e , c o n t a i n i n g b e s i d e s t h e t h r e e KDO re-


1



6. WALDSTATTEN ET AL.

Oligosaccharides Containing KDO Residues 123

Figure 1. Linkages of the KDO region of LPS from Salmonella strains as proposed in Refs. 5 and 6.

1 5 Hepcc

2 KDO 4 |

2> KDO Aor5 l

3 -GlcN

. 1 5 Hepa - — ^ KDO

2 7or8 — - ~ K DO 4or5

2 3 — GlcN

2 KDO

2 I KDO

Figure 2. Hypothetical linkages of KDO in Salmonella LPS: (left) according to Brade et al. (11); (right) according to Refs. 5 and 6; cf. Figure 1.



124


sidues only 2,5-anhydromannito1, i s c u r r e n t l y being p e r f o r m e d as f o l l o w s . E x h a u s t i v e h y d r a z i n o l y s i s o f t h e m u t a n t LPS ( p r e v i o u s l y p u r i f i e d by c o n v e n t i o n a l met h o d s ) (6>) w o u l d f r e e t h e c a r b o h y d r a t e ' b a c k b o n e ' o f JD-f a t t y - a c y l , JJj-f a t t y - a c y 1, a n d O^-phosphono g r o u p s (cf. t h e c h a p t e r by R i e t s c h e l i n t h i s v o l u m e ) . T r e a t ment w i t h n i t r o u s a c i d o f t h e r e s u l t i n g , d i a m i n o - d i d e o x y - p e n t a s a c c h a r i d e would then a f f o r d the d e s i r e d pseudotetrasaccharide, suitable f o r spectroscopic s t u d i e s . A s i m i l a r d e g r a d a t i o n has p r e v i o u s l y been p e r f o r m e d by M i r e i l l e R i e t s c h e l a t t h e M a x - P l a n c k Institut i n Freiburg (unpublished r e s u l t s ) . Prelimin ary r e s Y l t s from the a u t h o r s ' l a b o r a t o r y (observation of t h e H-n.m.r.-signals a t t r i b u t a b l e to the deoxyp r o t o n s o f KDO r e s i d u e s , & 1.80 - 2.40 p.p.m., a n d TBA a s s a y s ) i n d i c a t e t h a t , a s e x p e c t e d , t h e KDO p o r t i o n o f t h e LPS f r o m S. m i n n e s o t a R 595 i s s t a b l e t o t h e c o n d i t i o n s o f h y d r a z i n o l y s i s and nitrous-acid-deamination. I t i s h o p e d t h a t n . m . r . - s p e c t r a l a n a l y s i s o f t h e samp les t h u s o b t a i n e d , i n c o m p a r i s o n t o t h e m o d e l com p o u n d s whose s y n t h e s e s a r e d e s c r i b e d b e l o w , w i l l lead t o a d e f i n i t i v e e l u c i d a t i o n o f s t r u c t u r e o f t h e 'KDO r e g i o n ' i n the near f u t u r e . S e l e c t i o n of t a r g e t s t r u c t u r e s f o r s y n t h e s e s of model oligosaccharides c o r r e s p o n d i n g t o t h e KDO r e g i o n o f L P S . As a f i r s t a p p r o a c h , t h e s y n t h e s i s o f a 2—*-4l i n k e d , d i s a c c h a r i d e o f KDO r e s i d u e s was a t t e m p t e d . I n the absence of c o n c l u s i v e d a t a e s t a b l i s h i n g the anom e r i c c o n f i g u r a t i o n ( s ) o f t h e KDO r e s i d u e s i n L P S , t h e disaccharide h a v i n g t h e KDO r e s i d u e s i n t h e fi-anom e r i c c o n f i g u r a t i o n , was c h o s e n c o n s i d e r i n g t h e e s t a b l i s h e d (9^, J 3 ) B - a n o m e r i c c o n f i g u r a t i o n o f t h e KDO r e sidues i n the capsular p o l y s a c c h a r i d e s f r o m N. m e n i n g i t i d i s 29e (JJ3) and E . c o l i 06 K13 HI (9) ( c f . the c h a p t e r by J a n n i n t h i s v o l u m e ) . An a g l y c o n i c d e r i v a t i v e o f KDO, h a v i n g 0-7 a n d 08 temporarily b l o c k e d , was r e q u i r e d f o r t h e s y n t h e s i s of t h e model d i s a c c h a r i d e K o e n i g s - K n o r r r e a c t i o n of 3 w i t h a s u i t a b l e h a l i d e d e r i v a t i v e o f KDO w o u l d be e x p e c t e d t o o c c u r e x c l u s i v e l y a t t h e ( e q u a t o r i a l ) 0-4 w h i l e 0H-5, b e i n g a x i a l l y - d i s p o s e d and experiencing a d d i t i o n a l s t e r i c s h i e l d i n g by H-7, w o u l d be p r a c t i c a l l y u n r e a c t i v e . X-ray c r y s t a l s t r u c t u r e a n a l y s i s of KDO d e r i v a t i v e s ( J 4 , 2J>) h a s shown t h a t H-7 i s o r i e n t ed p a r a l l e l t o t h e a x i a l s u b s t i t u e n t s o f t h e r i n g c a r bon atoms. Synthesis of the d i s a c c h a r i d e The r e a c t i o n o f t h e known ( 1 3 ) m e t h y l k e t o s i d e - m e t h y l e s t e r d e r i v a t i v e 1^


6. WALDSTATTEN ET AL.

125

Oligosaccharides Containing KDO Residues H -C

HO*

>

HO HO


7—0 H HO- \~-CH20H Ho\ C

, H , O-S—CH

I

/ H

°'C-CH

2

oA

HO\

OMe

2

C0 Me

C0 Me

C0 Me

2

2

2

'C-CH2OAC AcO' 0^ \ AcO\

AcO^S^^^M. 5.

X = CI

6.

X = Br 0

AcO- r AcO C

C H 2

°

H0\

A C

OMe

AcO C0 Me

C0 Me 2

2

AcOCH

1 H 2 C R0-Q-CH20R R 0 - - \ CH2OR A v ^ R0\ R0>

2

\ OAc

OAc

R

C0 Me

OMe

OMe

C0 Me 2

2

9 , R = Ac Anderson and Unger; Bacterial Lipopolysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1983.



126

w i t h 4 - n i t r o p h e n y 1 c h l o r o f ormate (J6) i n p y r i d i n e at low t e m p e r a t u r e a f f o r d e d p r e p o n d e r a n t l y t h e crystal l i n e b i s - c a r b o n y l d e r i v a t i v e 4^, b u t a l s o some o f the s y r u p y 7 , 8 - 0 - m o n o c a r b o n y 1 d e r i v a t i v e ^3. Compounds 3 and 4, w e r e e a s i l y s e p a r a t e d by c h r o m a t o g r a p h y on sili c a g e l . The u n w a n t e d compound 4, may be r e - c y c l e d i n t o t h e s t a r t i n g m a t e r i a l 2 by Z e m p l e n s a p o n i f i c a t i o n . The modified Koenigs-Knorr r e a c t i o n (J7, J8) (silver car b o n a t e , D r i e r i t e , d i c h l o r o m e t h a n e , room t e m p e r a t u r e , o v e r n i g h t ) of 3 w i t h m e t h y l (3-deoxy-4,5,7,8-tetra0-acetyl--D-manno-2-octulopyranosy1)onate chloride 5, ( J 3 ) a f f o r d e d a v e r y low y i e l d o f s e v e r a l materials w h i c h were too l i t t l e f o r f u r t h e r p r o c e s s i n g and char a c t e r i z a t i o n . When, i n t h e K o e n i g s - K n o r r g l y c o s i d a t i o n o f ^3, f o l l o w i n g a s u g g e s t i o n by P a u l s e n ( J 9 ) , t h e k e t o p y r a n o s y 1 c h l o r i d e ^5 was r e p l a c e d by t h e b r o m i d e j6 ( a v a i l a b l e i n e x c e l l e n t y i e l d f r o m t h e r e a c t i o n o f methyl 3-deoxy-2,4,5,7,8-penta-0-acety1-oc-D-manno-2o c t u l o p y r a n o s o n a t e (19-21) with t i t a n i u m tetrabromide) a f a i r y i e l d of a ca. 10:1 m i x t u r e o f t h e B and « - a n o m e r s £ and 8, o f m e t h y l [ m e t h y l 7 , 8 - 0 - c a r b o n y 1 - 3 - d e o x y 4-0-methy1 ( 3 - d e o x y - 4 , 5 , 7 , 8 - t e t r a - Q - a c e t y l - D - m a n n o - 2 octulopyranosyl)ono-fi-D-manno-2-octulopyranosidj onate was obtained. Separation o f t h e a n o m e r s by h.p.l.c. (22) f o l l o w e d by Z e m p l e n s a p o n i f i c a t i o n o f t h e B,Bd e r i v a t i v e jP, a f f o r d e d a m e t h y l d i c a r b o x y l a t e disac c h a r i d e w h i c h was per-0-acetylated (acetic anhydridep y r i d i n e - 4 - d i m e t h y l a m i n o p y r i d i n e ) to g i v e the h e p t a -0-acetyl derivative, C o n v e r s i o n o f 9, i n t o t h e t a r g e t compound \^ was p e r f o r m e d by s e q u e n t i a l Zemplen s a p o n i f i c a t i o n and t r e a t m e n t w i t h O.2 N a q u e o u s s o d i u m h y d r o x i d e , as p r e v i o u s l y d e s c r i b e d f o r t h e c o r r e s p o n d ing monosaccharide d e r i v a t i v e s (J3, 2 1). C o n s t i t u t i o n a l and c o n f i g u r a t i o n a l c h a r a c t e r i z a t i o n by H - n . m . r . ^ s p e c t r o s c o p y of the d i s a c c h a r i d e derivat ive The H-n.m. r . - s p e c t r u m o f 9, (250 MHz, CDCK) c a n be i n t e r p r e t e d t o i n d i c a t e b o t h t h e c o n s t i t u t i o n o f t h e d i s a c c h a r i d e and t h e a n o m e r i c c o n f i g u r a t i o n o f t h e n e w l y - i n t r o d u c e d KDO residue (KDO II, 2—^4-linka g e ) , as f o l l o w s . R e g a r d i n g c o n s t i t u t i o n , t h e p r e s e n c e of seven l i n e s a t t r i b u t a b l e to Q - a c e t v l groups, of one s i g n a l a t t r i b u t a b l e to a k e t o s i d i c methoxy group ( s , 3 H, h 3.37 p.p.m.), and o f two s i g n a l s ( 2 s , 6 H, 3.80 and 3.86 p.p.m.) a t t r i b u t a b l e t o e s t e r m e t h o x y g r o u p s , i s i n k e e p i n g w i t h t h e o v e r - a l l s t r u c t u r e o f ^9. T h e r e a r e n i n e s i g n a l s due t o h y d r o g e n atoms a d j a c e n t t o a c e t o x y g r o u p s , o f w h i c h , by f i r s t - o r d e r a n a l y s i s , two e a c h a r e a s s i g n e d t o t h e g e m i n a l h y d r o g e n atoms o f C-8 and C-8 (4 H ) , t o H-7 and H-7 (2 H ) , and t o H-5 and f

f


WALDSTATTEN ET AL.

6.

Oligosaccharides Containing KDO Residues 127


1

1

H-5 (2 H ) . The r e m a i n i n g s i g n a l i s a t t r i b u t e d to H-4 (1 H ) . T h e r e f o r e , o f t h e c a r b o h y d r a t e hydroxyl funct i o n s , o n l y 0-4 i s n o t p a r t o f an e s t e r g r o u p i n g and w o u l d be e n g a g e d i n t h e i n t e r g l y c o s i d i c l i n k a g e , as c o n f i r m e d by t h e c h e m i c a l s h i f t o f H-4 (3.95 p.p.m.). The a s s i g n m e n t o f t h i s s i g n a l t o H-4 has b e e n v e r i f i e d by INDOR w i t h H-5 (22 ) . The H-n . m. r . - s p e c t r u m o f i n a d d i t i o n , s u p p l i e s evidence f o r the B-anomeric con f i g u r a t i o n o f KDO I I . E m p i r i c a l c o r r e l a t i o n s among t h e s h i f t v a l u e s o f H-4 i n the n . m . r . - s p e c t r a of k e t o s i d i c , 0 - a c e t y l a t e d KDO d e r i v a t i v e s show t h a t , f o r t h e 0$-ketosides, t h e s h i f t v a l u e s (8) of the H-4-signals a r e s i g n i f i c a n t l y l a r g e r (by c a . O.4 p.p.m.) t h a n f o r the B - k e t o s i d e s . T h i s d i f f e y e n c e i s c l e a r l y apparent from the comparison of the H-n.m.r.-spectra of the two a n o m e r i c , O - a c e t y l a t e d k e t o s i d e e s t e r s and H. ( 1 3 , 21) ( S H-4 i n t h e o c - k e t o s i d e 10: 5.36, in th~Bk e t o s i d e 1J,: 4.91 p . p . m . ) . The s h i f t d i f f e r e n c e c a n be e x p l a i n e d by d e - s h i e l d i n g o f t h e H-4, in o6-ketosidic d e r i v a t i v e s , t h r o u g h 1 , 3 - s y n - i n t e r a c t i o n w i t h the ano m e r i c o x y g e n atoms. In the s p e c t r u m o f d i s a c c h a r i d e the s i g n a l c o r r e s p o n d i n g t o H-4 o c c u r s a t

Synthesis of Oligosaccharides Containing 3-Deoxy-D-manno-2

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