Stereoselectivity in glycosylation by means of 2-azido ... - Springer Link

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Use of chloride (IiI) obtained immediately prior to glycosylation from bromide (V) and ..... fled mixture obtained as a result of the addition of chlorine azide to 3,4 ...
STEHEOSELECTIVITY OF

IN

2-A ZIDO-2-DE

DERIVATIVES

SOX Y - D -

AND

DETERMINATIVE GROUP

A,

TYPE

GLYCOSYLATION

THE

BY M E A N S

GALA C TOPYRANOSE

SYNTHESIS

OLIGOSACCHARIDE

OF

THE

OF

BLOOD

1 UDC 542.91 : 547.458

N. V. Bovin, S. 1~. Z u r a b y a n , and A. Ya. Khorlin

O l i g o s a c c h a r i d e s with 2 - d e s o x y - a - D - g l y c o s y l u n i t s a r e v e r y c o m m o n in n a t u r e and, in p a r t i c u l a r , the d e t e r m i n a t i v e o l i g o s a c c h a r i d e of blood g r o u p A, type 1 (I) is 2 - a c e t a m i d e - 2 - d e s o x y - a - D - g a l a e t o s i d e D-Gal p NAc-cr (i ~ 3) \ / L-Fuc p-co(t -~ 2)

D-Gal p-~ (t ~ 3)-D-GlcNAc (I)

F o r m a t i o n of the a - h e x o s a m i n i d e bond r e m a i n s a d i f f i c u l t p r o b l e m in the s y n t h e s i s of a - g l y c o s i d e s . The m o s t p r o m i s i n g c u r r e n t m e t h o d for the s y n t h e s i s of a - h e x o s a m i n i d e s is the " a z i d e m e t h o d " [1], which has b e e n u s e d to p r e p a r e a s e r i e s of h e t e r o o l i g o s a c c h a r i d e s with 2 - a e e t a m i d e - 2 - d e s o x y - a - D - g l y e o s y l u n i t s . The m e t h ods f o r p r e p a r a t i o n of the s t a r t i n g g l y c o s y l a t i n g d e r i v a t i v e s of 2 - a z i d o - 2 - d e s o x y h e x o s e s have r e c e n t l y b e e n s i g n i f i c a n t l y s i m p l i f i e d [2-5]. Two m e t h o d s have b e e n d e v e l o p e d in the p a s t few y e a r s for the s y n t h e s i s of a - g l y c o s a m i n i d e s f r o m 2a z i d o - 2 - d e s o x y s u g a r d e r i v a t i v e s . The f i r s t m e t h o d l i e s in g l y c o s y l a t i o n by m e a n s of 2 - a z i d o - 2 - d e s o x y - ~ D - g l y e o s y l c h l o r i d e s such a s (iII) and (IV) [6] in the p r e s e n c e of a m i x t u r e of Ag2CO 3 and AgC104. The s t e r i c s e l e c t i v i t y of t h i s r e a c t i o n is 8 5 - 9 0 ~ [1, 7-11]. The second m e t h o d e n t a i l s u s e of 2 - a z i d o - 2 - d e s o x y - . c ~ - D g l y c o s y l b r o m i d e s such a s ( V ) a n d (Vi) [6] u n d e r H e l f f e r i e h g l y c o s y l a t i o n e o n d i t i o n s [12-14]. H o w e v e r , c r i t e r i a for s e l e c t i n g the m o r e s u i t a b l e m e t h o d w e r e n o t g i v e n . M o d e l e x p e r i m e n t s [2] have shown the l a c k of s t e r i c s e l e c t i v i t y in the g l y c o s y l a t i o n of b e n z y l a l c o h o l a n d t e r t - b u t y l a l c o h o l by c h l o r i d e (IH) and b r o m i d e (V). F i n a l l y , the r e c e n t l y p r o p o s e d " i m i d a t e m e t h o d " for the s y n t h e s i s of 1 , 2 - c i s - g l y c o s i d e s in the c a s e of a z i d e (VII) d o e s n o t give s a t i s f a c t o r y r e s u l t s in the g l y e o s y l a t i o n of s u g a r s a t s e c o n d a r y h y d r o x y l g r o u p s [15]

,oo

N3

Na

(VII)

R = R" = Ac, X = 13-C1 (III); R = Bzl, R' = Ac, X = }-C1 {IV); R = R' = Ac, (V); lq = Bzl, R" = Ac, X = cr (VI); R = B' = Ac, X = l~-Br (VIII); B = R' = Bzl, X = a-Br (XI); R = R' = Bzl, X = ~-C1 (XI1); R = R' = Bzl, 9 X = OAc (XIII)

X = a-Br

D e s p i t e s o m e d e f i n i t e s u c c e s s in the s y n t h e s i s of h e t e r o o l i g o s a c c h a r i d e s with 2 - a e e t a m i d o - 2 - d e s o x y a - D - g l y c o s y l r e s i d u e s , s y s t e m a t i c s t u d i e s have n o t b e e n c a r r i e d o u t on p r e p a r a t i o n of the ( ~ - h e x o s a m i n i d e bond r e l a t i v e to the s t r u c t u r e of the g l y c o s y l a t i n g a g e n t and the r e a c t i o n c o n d i t i o n s . * In the p r e s e n t w o r k on * A f t e r this w o r k was a c c e p t e d for p u b l i c a t i o n , the w o r k of P a u l s e n a n d Kol~r [16] a p p e a r e d on the effect of the c a t a l y s t on the r a t e of g l y c o s i d e s y n t h e s i s , but no data w e r e g i v e n on the s t e r e o c h e m i s t r y of the g l y c o s y l a t i o n . M. l~I. S h e m y a k i n i n s t i t u t e of B i o o r g a n i c C h e m i s t r y , A c a d e m y of S c i e n c e s of the USSH, Moscow. T r a n s l a t e d f r o m [ z v e s t i y a A k a d e m i i Nauk SSSR, S e r i y a K h i m i c h e s k a y a , No. 5, pp. 1 1 4 8 - 1 1 5 6 , May, 1982. O r i g i n a l a r t i c l e s u b m i t t e d J u l y 16, 1981.

0 5 6 8 - 5 2 3 0 / 8 2 / 3 1 0 5 - 1023507.50 9 1982 P l e n u m P u b l i s h i n g C o r p o r a t i o n

1023

TA BLE 1. The G l y c o s y l a t i o n o f C y e l o h e x a n o l by A c y l h a l o g e n o s e s (III), (V), a n d (VIII) IAcylExpt. haloNo. genose

6 7 8 9

(v) (v) (v) (v) (v) HI)~ III)~ [II) ~ (v)

t0

(v)

tl t2

(v) (v)

13

(v)

14

(v)

Catalyst and/or ace epto r Hg (CN)~+HgBr2 Hg (CN)2+HgBr2 Ag2COa AgCIO~a BuCNBr+i-Pr2NEt Ag2COs+AgC1Q Ag~C0a+AgC10~ Ag2C0a+AgC10r AgO.SO~CFa+eollinine AgOSO,o,EF3+ collidinet AgOTs+collidine Dowex 50W-X4. 9(Ag+)+ collidine Ag0SO~CFa+-collidine AgOS2CHa-~cotlidine dine

Molar ratio of halogenose to cataIy, t and/o aeeeptor 1:2:0,5 1:2:0,5 t:2 t:2 t:3:2 t:2:0,2 t:2:0,2 t:2:0,2 t:2:2 t:2:2 t:2:2 t:t0:2

[

= 1.$ ~

Solvent

~ .~ 24 24 24 1

72 4 4 4 4 4 72 120

Ratio of c~:g glycosides

26H6+MeN0a (1:t) 9:9t CHIC12 1t :89 CH2C12 43 : 57 C6H6 62 : 38, 36H6+.DMFc (t : t) 77 : 23 t~ CH2Cl2 73 : 27 CH2C12 77 : 23 8t : 19 CH2Clz 36:64 CH2C12 CH~CI~

t6 : 84 6i : 39 g

79 : 2i

t:2:2

24

CH2C]2 CH~CI~ or dioxane MeOCII2CH2OMe

t:2:2

24

MeOCH2CH2OMe

73 : 27

a) G l y c o s y l a t i o n by the d i p h e n y l c y e l o p r o p e n y l m e t h o d [17]. b) The y i e l d o f g l y c o s i d e s w a s > 2 0 g ; the g l y e o s i d e y i e l d in the r e m a i n i n g e a s e s ( e x c e p t f o r e x p e r i m e n t No. 12) w a s > 90%. e ) O b t a i n e d f r o m (V) by the a c t i o n of Bu4NC1. d)Crystalline compound. e ) o b t a i n e d f r o m (V) by the a c t i o n of AgOSO2CF3, then Bu4NBr. f)At - 3 0 ~ the r e m a i n i n g c a s e s w e r e a t 20~ g)The reaction does not proceed. the s y n t h e s i s of d e t e r m i n a t i o n o l i g o s a e c h a r i d e o f blood g r o u p A t y p e 1 ([) and i t s f r a g m e n t , D - G a l p N A c - c~(1 3 ) - D - G a l p - f i ( l ~ 3 ) - D - G l e N A c (II), we a l s o s t u d i e d the s t e r e o c h e m i c a l d i r e c t i o n of the g l y e o s y l a t i o n of a m o d e l a l c o h o l by 2 - a z i d o - 2 - d e s o x y g l y c o s y l h a l i d e s . C a r e should c e r t a i n l y be t a k e n in a p p l y i n g the r e s u l t s o b t a i n e d f o r the g l y c o s y l a t i o n o f m o d e l a l c o h o l f o r the s y n t h e s i s o f o l i g o s a c c h a r i d e s s i n c e the s t e r i c s e l e c t i v i t y of the r e a c t i o n m a y be a l t e r e d . N e v e r t h e l e s s , the s t u d y of c o r r e c t l y c h o s e n m o d e l r e a c t i o n s m a y i n d i c a t e the n a t u r e of the r e a c t i o n s t e r e o c h e m i s t r y r e l a rive to the n a t u r e o f the g l y c o s y l a t i n g a g e n t s and the s y n t h e s i s c o n d i t i o n s , in o u r w o r k , we s t u d i e d the g l y c o s y l a t i o n o f c y c l o h e x a n o l . The h y d r o x y l g r o u p o f t h i s c o m p o u n d is v e r y s i m i l a r in r e a c t i v i t y to t y p i c a l c a r b o h y d r a t e s . C y c l o h e x a n o l a l s o p e r m i t s q u a n t i t a t i v e a n a l y s i s o f m i x t u r e s of a n o m e r i e g l y c o s i d e s by g a s liquid c h r o m a t o g r a p h y (GLC). T a b l e 1 g i v e s the r e s u l t s f o r the g l y e o s y l a t i o n o f e y c l o h e x a n o l by a c y l h a l o g e n o s e s (III), (V), and (VIII). The s t e r e o c h e m i s t r y of the r e a c t i o n v a r i e s w i d e l y f r o m 91% f i - g l y c o s i d e to 81g c ~ - g l y c o s i d e . M a i n l y , p - g l y co s i d e s a r e o b t a i n e d f r o m c ~ - b r o m i d e {V) in the H e l f f e r i c h r e a c t i o n [with Hg(CN)2 in a b e n z e n e - n i t r o m e t h a n e m i x t u r e in CHIC12, e x p e r i m e n t s N o s . 1 a n d 2], a l t h o u g h p r e v i o u s s t u d y of s i m i l a r h a l o g e n o s e s showed high c ~ - s t e r e o s e l e c t i v i t y [12-14] o r its a b s e n c e [2]. A s l i g h t p r e d o m i n a n c e o f the a - a n o m e r w a s found u s i n g the d i p h e n y l e y e l o p r o p e n y l m e t h o d [17] ( e x p e r i m e n t No. 4). In the h a l i d e - c a t a l y z e d L e m i e u x r e a c t i o n [18], the c~ : p - g l y c o s i d e r a t i o is r a t h e r high ( e x p e r i m e n t No. 5), a l t h o u g h the r a t e o f g l y c o s i d e f o r m a t i o n is low. The u s e o f f l - g l y c o s y l h a l i d e s u n d e r c o n d i t i o n s of the K o e n i g s - K n o r r r e a c t i o n l e a d s to a n i n e r e a s e in the a m o u n t of ~ - g l y c o s i d e . Use o f c h l o r i d e (IiI) o b t a i n e d i m m e d i a t e l y p r i o r to g l y c o s y l a t i o n f r o m b r o m i d e (V) a n d c h l o r i d e ion [6], the c~ : p - g l y c o s i d e r a t i o is 73 : 27 ( e x p e r i m e n t No. 6). A s i m i l a r r e s u l t is found f o r u s e o f the c r y s t a l l i n e c h l o r i d e (liD. S i m i l a r to the a b o v e , b r o m i d e {VIII) w a s o b t a i n e d by the a c t i o n o f s i l v e r t r i f l a t e ( t r i f l u o r o m e t h a n e s u l f o n a t e ) a t - 7 8 ~ on b r o m i d e (V) w h i c h i s c o n v e r t e d to the c~ - g l y c o s y l t r i f l a t e (see the w o r k o f L e r o u x a n d P e r l i n [19]). The a c t i o n o f b r o m i d e ion on t h i s p r o d u c t r e a d i l y g i v e s b r o m i d e (VIII) w i t h i n v e r s i o n o f c o n f i g u r a t i o n w h i c h c o n t a i n s m o r e t h a n 9 0 g / 3 - a n o m e r (5 4.7 and 6.0 p p m , J1,2 = 9.0 and 4.0 Hz f o r /3- a n d c ~ - a n o m e r s , r e s p e c t i v e l y , in the P M R s p e c t r u m ) . The u s e o f (VIII) in the g l y c o s y l a t i o n r e a c t i o n a l s o l e a d s to the p r e d o m i n a n c e of the c ~ - a n o m e r ( e x p e r i m e n t No. 8).

1024

Glyeosylation by glyeosyl sulfonates was studied in detail for neutral s u g a r s [20] and the ratio of the glycosides formed was found to depend on the nature of the protective groups, nature of the tearing suifonyl group, r e a c t i v i t y of the alcohol, the alcohol : sulfonate ratio, and solvent. The high c~-stereoselectivity is facilitated by the acyl substituents at O-6 and O-4 of the glycosylating agent and the use of solvents with high donor capacity and low d i e l e c t r i c constant such as ethers. In addition, the reaction r e s u l t s lead to lower s t e r i c selectivity. This behavior is also found for 2 - a z i d o - 2 - d e s o x y derivatives of D - g a l a c t o s e , as seen in Table 1. The use of glyeosyl triflate in CH2C12 at 20~ leads to the predominance of the ~-glycoside (experiment No. 9), the f i - s t e r e o s e l e c t i v i t y i n c r e a s e s with d e c r e a s i n g t e m p e r a t u r e (experiment No. 10). The f r a c tion of the ~ - a n o m e r i n c r e a s e s sharply in going to the less reactive gtycosyl tosylate (experiment No. 11), though the r e a c t i o n rate in this case is an o r d e r of magnitude lower than in the other cases studied. R e p l a c e ment of silver tosylate with Dowex 50W-X4 (Ag +) p o l y m e r a c c e p t e r obtained by analogy to the procedure of E s e h e n f e l e r et al. [21] leads to the complete absence of glycosides in the reaction products. Finally, the fraction of ~ - a n o m e r i n c r e a s e s sharply by replacing CH2C12 with 1,2-dimethoxyethane which has good donor p r o p e r t i e s (experiments Nos. 13 and 14). Thus, the model glycosylation indicates two s e r i e s of more successful experiments based on the use of fl-halides (experiments Nos. 6-8) and glyeosyl sulfonates in 1,2-dimethoxyethane (experiments Nos. 13 and 14). These conditions were used for the glyeoslyation of 1,2, 95, G - d i - O - i s o p r o p y l i d e n e - a - D - g a l a e t o f u r a n o s e (IX). N this c a s e , glyeosyl sulfonates in 1,2-dimethoxyethane do not give glycosides at all, while the ~-halides give high yields of the aglycone acetate (X) instead of the expected glycosides, Analogous r e s u l t s were obtained in the glyeosylation of m o r e complex compounds (see below). Thus, it is better to avoid use of acetylated glycosyl

R = H (IX); R = Ac (X). halides (tII) and (V) and go to the completely benzylated analogs (Xt) and (XH). The synthesis of (X][) and (XID was c a r r i e d out starting with 1 - O - a c e t a t e (NIH) obtained by the haloazide method [5]. For this purpose, an a n o m e r i e mixture of (X[[I) was hydrolyzed with 0.8 M HC1 in dioxane to the c o r r e s p o n d i n g l - h y d r o x i d e which was converted to the l~O-(p-nitrobenzoate) by the action of p-nitrobenzoyi chloride in pyridine. This product is converted to c~-bromide (XI) by t r e a t m e n t with H e r . Both c r y s t a l l i n e (XI) and the fl-chloride (XII) generated immediately p r i o r to use (by the reaction with t r i e t h y l b e n z y t a m m o n i um chloride in acetonitriIe using the minimum optical rotation) were used in the glyeosylation reaction_. D i - a n d t r i s a e e h a r i d e derivatives into which we must introduce a 2 - a e e t a m i d o - 2 - d e s o x y - c ~ - D - g a l a e t o pyranose unit in the final step of the synthesis of blood group A o l i g o s a c e h a r i d e s were synthesized starting from d i s a c c h a r i d e (k:IV) [11]. P a r t i a l ehloroaeetylation of diol (X[V) was used to obtain chloroacetate (XV). The conditions for the preparation of (XV) and its physical indices somewhat differ from those given by P a u l sen and t(oI~ir [11]. Chloroacetate (gV) was subjected to acetylatien and d e - O - c h l o r o a e e t y l a t i o n by the action of thiourea [22, 23] to yield a d i s a c c h a r i d e with a free hydroxyl group at C-3 (XVI). The s t r u c t u r e of (X[) and, thereby, of ehloroaeetate ~ V ) was proven by convergent synthesis from the methylorthoaeetate of D - g a l a c tose (XVII) [24] Ph-"x--"~CH2

Ph /OCH 2

\oR,/

Ph--v-~CH2

\k?

OR

+ NI-IAc

/-0.CH2

-+

ONBz

(XIV)-(XVI)

Ph

(XIX)

0

0

M~---e~ t\ B~19 / R6 Xr____J/

NHAe 2)-D-Galp-[~ (~ --->3)-D-GlcNAc

(xxvi) Thus, the use of a new g l y c o s y l a t i n g agent, chloride ~ I I ) , prevents the formation of side products r e lated to i n t e r m o l e e u l a r t r a n s a c e t y l a t i o n and p e r m i t s the preparation of complex h e t e r o o l i g o s a c c h a r i d e s with a 2 - a c e t a m i d o - 2 - d e s o x y - a - D - g a l a c t o syl unit. EXPERIMENTAL The melting points were d e t e r m i n e d using a Boetius instrument. The t h i n - l a y e r c h r o m a t o g r a p h y was c a r r i e d out on M e r c k 60 F - 2 5 4 silica gel plates. The column c h r o m a t o g r a p h y was c a r r i e d out on 40-100 ~m silica gel L supplied by the Czechoslovakian f i r m , Chemapol. The PMR spectra were taken on a Varian X L 100 s p e c t r o m e t e r at 100 MHz with TMS as the internal standard. The optical rotation was m e a s u r e d on a P e r k i n - E l m e r 141-M p o l a r i m e t e r . Gas--liquid c h r o m a t o g r a p h y (GLC) was c a r r i e d out on a H e w l e t t - P a c k a r d 5710A c h r o m a t o g r a p h with a flame ionization detector using a 50 m • 0.24 mm capillary column packed with SE-30 at 140-200~ (2 deg/min) and helium gas c a r r i e r at 1.2 atm. Analytical ion-exchange c h r o m a t o g r a p h y was c a r r i e d out on a Biotronic a n a l y z e r . The solvents were evaporated in vacuum at 30-40~ 2 - A z i d o - 3 , 4 , 6 - t r i - O - A c e t y l - 2 - d e s o x y - f i - D - g a l a c t o p y r a n o s y l Chloride (IIl). A sample of 2.78 g ~ 0 mmoles) t e t r a b u t y l a m m 0 n i u m chloride was added to a solution of 1.97 g (5 mmoles) 2 - a z i d o - 3 , 4 , 6 - t r i - O a c e t y l - 2 - d e s o x y - a - D - g a l a c t o p y r a n o s y l bromide (V) [6] in 40 ml CH3CN. A f t e r 30 min, the solution was diluted with 200 ml benzene, t h r i c e washed with water, dried o v e r MgSO~, and evaporated. Crystallization of the residue from ether yielded 1.26 g (72~) (HI) with mp 98-99~ and [~]t~ 0 - 1 6 ~ (C 1, CHC13) [2]. 2 - A z i d o - 3 , 4 , 6 - t r i - O - b e n z y l - 2 - d e s o x y - a - D - g a l a c t o p y r a n o s y l Bromide (XI). A sample of 10 g of an a n o m e r i c mixture of a c e t a t e s 0fII[) obtained as described in our previous work [5] was heated at reflux for 4 h in a mixture of 400 ml dioxane and 50 ml 0.5 M HC1 and then evaporated and subjected to c h r o m a t o g r a p h y using 9 : 1 t o l u e n e - a c e t o n e eluent. In another variant, an analogous hydrolysis w a s c a r r i e d outon an unpurifled mixture obtained as a r e s u l t of the addition of chlorine azide to 3 , 4 , 6 - t r i - O - b e n z y l - D - g a l a c t a l and s u b s e qu.ent t r e a t m e n t with Hg(OAc)2 [5]. The mixture of 1-hydroxides formed was dissolved in 150 ml CH2CI 2 and 20 ml pyridine. Then, a sample of 6 g p - n i t r o p b e n z o y l chloride was added batchwise at 20~ A f t e r 15 h, the mixture was washed with w a t e r , 500 ml 0.5 M HCI, aq. NaHCO3, and water, dried over CaCI2, and evaporated. The 1 - O - ( p - n i t r o b e n z o a t e ) f o r m e d was maintained for 5 h without purification at 0~ in 100 ml of a solution formed by saturation of HBr in CH2CI 2 at 0-5~ This solution was evaporated at 20~ and the residue, benzene was added to the r e s i d u e and then distilled off. Another sample of benzene was added to the resultant residue and the precipitate was r e m o v e d by filtration. The filtrate was evaporated and the residue was d i s s o l v e d i n l 5 ml CH2CI 2. Heptane was added until the solution became turbid. After 10 min, 2 g silica gel was added and the mixture was v i g o r o u s l y shaken and rapidly filtered. The c o l o r l e s s solution gave 6.24 g (XI) in c r y s t a l l i n e f o r m (60~ yield relative to the acetate) with mp 92-93~ laiD2~ + 180 ~ (C 1, CH3CN), + 145 ~ (C 1, CHC13). PMR s p e c t r u m (6, ppm), CC14): 6.35 d (1 H, J1.2 = 2.7 Hz, H - l ) , 7.35 m (]5 H, 3 Ph). Found: C 60.30; H 5.19; Br 14.72; N 7.80~. C27H28BrN304: C 60.22; H 5.25; Br 14.84; N 7,81%. Glycosylation of Cyclohexanol. In all c a s e s , we used 0.1 mmole acylhalogenose and 0.2 mmole c y c l o hexanol (or its 2 , 3 - d l p h e n y l - 2 - e y z l o p r o p e n - l - y l ether) in 1-2 ml solvent. The amount of catalyst, reaction t e m p e r a t u r e , and time for the glycosylation a r e given in Table 1. At the end of the reaction, the mb;ture was diluted with c h l o r o f o r m , filtered, and the filtrate was washed with 0.5 M HC1, aq. NaHCO3, dried, evaporated, and deacetylated a c c o r d i n g to Semplen. The a n o m e r ratio was found by t h i n - l a y e r c h r o m a t o g r a p h y a f t e r the standard silylation p r o c e d u r e (see Table 1). The pure cyclohexylglycoside a n o m e r s which served as GLC standards were s e p a r a t e d by column c h r o m a t o g r a p h y of the reaction mixture in 7 : 3 e t h e r - h e x a n e .

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Benzy~2~acetamid~4,6~Benzy~iden~3~(4,6~benzy~iden~3~h~r~a~ety~D~ga~a~topyran~ syl)2 - d e s o x y - ~ - D - g l u c o p y r a n o s i d e (XV). A sample of 8.0 ml (60 mmoles) sym-collidine was added to a solution of 19.5 g (30 mmoles) d i s a c c h a r i d e (XIV) [11] in 1.5 liter abs. dioxane and then, a f t e r 24 h at 20~ ~ 4.5 ml (~ 60 mmoles) c h l o r o a c e t y l chloride was added and the reaction was monitored by t h i n - l a y e r c h r o m a t o g r a p h y . At the end of the reaction, the m o i s t u r e was evaporated and the residue was dissolved in CHC13, washed with w a t e r , 0.5 Iv[ HC1, aq. NaHCO3, and w a t e r , dried and evaporated. Crystallization of the residue from n i t r o methane yielded 13.1 g (60o/~) (XV) with mp 297~ [C~]D2~+115 ~ (C 0.5, CHCl3). Paulsen [11] r e p o r t e d mp 271~ and [~]D22 +103 ~ (C 0.5, CHC13); the PMR spectrum c o r r e s p o n d s to that r e p o r t e d by Paulsen [11]. Benzyl-2-a•etamid•-3-•-(2-O-acety•-4,6-•-benzy•iden-•-D-ga•actopyran•sy•)-4,6-•-benzy•idene-2d e s o x y - a - D - g l u c o p y r a n o s i d e (XVI). Method A. A sample of 1.45 g (2.0 mmoles) c h l o r o a c e t a t e (XV) was t r e a t e d with a mixture of 10 ml acetic anhydride and 10 ml pyridine at 20~ After 2 h, the mixture was diluted with c h l o r o f o r m , washed with water, 0.5 M HC1, aq. NaHCO3, and dried over CaC12. After evaporation, the residue was dissolved in 40 ml 3 : 1 p y r i d i n e - m e t h a n o l a n d heated at reflux for 30 rain with 0.18 g (2.4 mmoles) thiourea. The mixture was evaporated and the residue was subjected to c h r o m a t o g r a p h y with 3 9 1 c h l o r o f o r m - a c e t o n e eluent to yield 1.11 g (80~) (XV[) with mp 258~ (from n i t r o m e t h a n e - e t h e r ) , [~]D 2~ +56 ~ (C 0.5, CHC13). Found: C 64.29; H 6.02; N 1.96G. Calculated for C37H4~NO12: C 64.23; H 5.99; H 2.03~. Method ]3. A sample of 10.9 g (30 mmoles) 3 , 4 , 6 - t r i - O - a e e t y l - l , 2 - m e t h y l o r t h o a c e t y l - a - D - g a l a c t o pyranose (XVH) obtained a c c o r d i n g to Lemieux [24] was deaeetylated a e e o r d i n g t o Semplen. The solution was evaporated to d r y n e s s and the residue was dissolved in a mixture of 50 ml pyridine and 100 mt CH2C12. A sample of 17.9 g (105 mmoles) (C1CH2CO)20 was added to this solution at 0~ A f t e r 2 h, the mixture was diluted with 500 ml c h l o r o f o r m , washed with water and 0.5 M HCI, and evaporated. The residue was dissolved in 100 ml 90G acetic acid and, a f t e r 10 rain, evaporated. The residue was acetylated with 100 ml acetic a n h y dride in 100 ml pyridine and the mixture was diluted with CHCI 3 and washed by the usual procedure. Column c h r o m a t o g r a p h y with 9 : 1 t o l u e n e - a c e t o n e eluent gave 4.74 g 1 , 2 - d i - O - a c e t y l - 3 , 4 , 6 - t r i - O - e h l o r o a c e t y l - ~ - D g a l a c t o p y r a n o s e (XVIK) (32G yield) as a syrup. PMR s p e c t r u m (6, ppm, CDCI~): 2.02 s (3 H, Ae), 2.17 s (3 H, Ac), 4.01 s (2 H, ClCH2) , 4.06 s (2 H, CICH2), 4.18 s (2 H, CICH2) , 6.43 d (1 H, J1,2 = 2.9 Hz, H-l). The sample of (XVHI) obtained was dissolved in 100 ml 30G HBr in acetic acid and maintained for 15 h at 0~ The solution was dissolved in cold CHC13 and washed with cold water, aq. NaHCO3, and water and dried o v e r CaC12. The halogenose obtained after evaporation was r e a c t e d with b e n z y l - 2 - a c e t y l a m i d o - 4 , 6 - O - b e n z y l i d e n e - 2 d e s o x y - a - D - g l u c o p y r a n o s i d e [25] as d e s c r i b e d by Paulsen [9] for 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l - a - D - g a l a c t o p y r a n o s y l bromide. The protected d i s a e c h a r i d e was separated by c h r o m a t o g r a p h y with 55 : 9 : 1 C H C 1 3 - e t h e r methanol eluent and treated with 1.4 g thiourea as described in method A. A f t e r evaporation of the r e a c t i o n mixture without purification, the residue was t r e a t e d with 15 ml benzaldehyde in the p r e s e n c e of 1 g ZnCI 2 for 15 h. C h r o m a t o g r a p h y using 3 : 1 CHC13-acetone eluent yielded 1.87 g (XVI) identical to that obtained by method A. The yield of (XVI) was 99 relative to o r t h o e s t e r (KVK). B e n z y l - 2 - a c e t a m i d o - 3 - O - ( 2 - O - [ 2 - O - b e n z y l - 3,4,di-O-(p-nitro benzene) - a - L - f u c o p y r a n o s y l ] - 4 , 6 - 0 b e n z y l i d e n e - ~ - g a l a c t o p y r a n o s y l ] - 4 , 6 - O - b e n z y l i d e n - 2 - d e s o x y - a - D - g l u c o p y r a n o s i d e (XX). A solution of 0.73 g (1.0 mmole) (XV), 0.51 g(2.0 m m o l e s ) Hg(CH)2 , and 0.11 g (0.3 mmoles) HgBr 2 in a mixture of 35 ml n i t r o methane and 35 ml benzene was e v a p o r a t e d to o n e - q u a r t e r the original volume and after 1 h, 0.37 g (0.6 mmole) 2 - O - b e n z y l - 3 , 4 - d i - O - ( p - n i t r o b e n z e n e ) - a - L - f u c o p y r a n o s y l bromide (XIX) [23, 26] was added at 50~ and then after 1 h, another 0.37 g (0.6 mmole) (XIX) was added without heating. The mixture was maintained for 24 h at 20~ and then diluted with c h l o r o f o r m , washed with water and aq. NaHCO3, and dried o v e r CaCl 2. A f t e r evaporation, the residue was heated at reflux with 0.09 g (1.2 mmole) thiourea in a mixture of 45 ml pyridine and 15 ml ethanol for 1 h. Evaporation and c h r o m a t o g r a p h y with 17 : 3 t o l u e n e - a c e t o n e eluent yielded 0.77 g (65G) t r i s a e c h a r i d e (XX) with mp 166-169~ [a]D2~ - 4 3 ~ (C 1, CHC13). PMR s p e c t r u m (5,ppm, CDCl3): 1.14 d (3 H J~",C' ----6.6 Hz, focuse CH3) , 1.95 s (3 H, Ac), 2.94 s (1 H, OH), 6.40 m (20 H, 4Ph), 6.907.50 2 A A ' B B ' ( 8 H , 2C6H4). Found: C 62.83; H 5.02; N 3.42G. Calculated for CG2H61N3021: C 62.88; H 5.20; N 3.55~. B e n z y l - 2- a c e t a m i d o - 3 - O - [2-O- ( 2 , 3 , 4 - t r i - O - b e n z y l - a - L - f u c o p y r a n o s y l ) - 4 , 6 - O - b e n z y l i d i n e - / 3 - D - g a l a c t o p y r a n o s y l ] - 4 , 6 - O - b e n z y l i d e n e - 2 - d e s o x y - c ~ - D - g l u c o p y r a n o s i d e (XXI). A sample of 2.66 ml (20 mmoles) s y m - c o l l i d i n e was added to a solution of 2.37 g (2 mmoles) (XX) in 20 ml THF. The r e a g e n t obtained f r o m 20 ml THF, 1.62 ml (20 mmoles) SO2C12 and 2.79 ml (20 mmoles) triethylamine [27] was added batchwise at 0~ o v e r 1 h. A f t e r 2 h, the residue was filtered off, the filtrate was evaporated, and the residue was d e a c y l ated a c c o r d i n g to Semplen (a g r e a t e r than usual amount of CH3ONa was required since some amount of the hydrochloride salt of the amine r e m a i n s in the mixture). After evaporation, the residue was dissolved in

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30 ml DMF and treated with 0.18 g (6 mmoles) 80g Nail and 0.69 ml (6 mmoles) CGHsCH2C1. After !5 rain, the m i x t u r e was diluted with c h l o r o f o r m , washed with water, and evaporated. The residue was maintained for 15 h at 20~ in 50 ml 3 : I :1 A c O H - T H F - w a t e r and then evaporated. C h r o m a t o g r a p h y with 5 : 2 h e x a n e acetone eluent yielded 1.79 g (80g) a m o r p h o u s t r i s a e c h a r i d e (XXI) with [a]D 2~ +20 ~ (C 1, CHC13) [11]. B e n z y I - 2 - aeetaraido-4,6- O - b e n z y l i d e n e - 3 - O - { 3 - O - ( 2 - a z i d o - 3 , 4 , 6 - t r i - O - b e n z y l - 2 - d e soxy-c~-D- gal ac~op y r a n o s y l ) - 4 , 6 - O - b e n z y l i d e n e - 2 - O - [ 1 - O - b e n z y l - 3 , 4 - d i - O ( p - n i t r o b e n z e n e )-c~- L - f u c o p y r a n o s y l ] - f i - D - g a l a c t o pyranosyl}-2-desoxy-o~-D-glueopyranoside (XXIII). A solution of 0.46 g (2.0 m m o l e s ) t r i e t h y l b e n z y l a m m o n i u m chloride in 4 ml CH3CN was added to a solution of 0.54 g (1.0 mmole) bromide (XI) in 45 ml CH~CN. After 9 min when the minimum [c~]D2~ +35 ~ was attained, the mixture was diluted with 100 ml CH2C12, t h r i c e washed with cold water, dried o v e r MgSO~, and evaporated. The chloride (XII) obtained is a syrup eontaining a small impurity of the starting bromide as show~ by t h i n - l a y e r c h r o m a t o g r a p h y with h e x a n e - a c e t o n e eluent. This product was added with s t i r r i n g over 30 min to a mixture of 0.59 g (0.5 mmole) t r i s a c e h a r i d e (XX). 2.5 g Ag2CO a, 70 mg AgC104, and 5 g roasted 4-~ m o l e c u l a r sieves in 40 ml CH2CI2. A f t e r 30 mtn, the mixture was filtered, washed with water and aq. NaHCO3, dried o v e r CaCI 2 and evaporated. Chromatography of the residue with 17 : 3 b e n z e n e - e t h y l acetate eluent yielded 0.67 g (82g) t e t r a s a c c h a r i d e (XXIII) with mp 151-.153~ (from methanol), [a]D2~ +9 ~ (C t , CHCla), PMR s p e c t r u m (6, ppm, CDClq): 0.66 d (3 H, J~",6" = 6.0 Hz, focuse CHa) , 1.78 s (3 H, Ac), 5.~2 s (1 H, PhCH__), 5.56 s (1 H, PhCH_), 7.6-8.4 2 A A ' B B ' (8 H, 2CGH4). Found: C 65.00; H 5.52; N 5.09g. Calculated for Cs.~H88N602[: C 65.10; H 5.41; N 5.12g. Benzyl- 2 - a c e t a m i d o - 4 , 6 - O - b e n z y l i d e n e - 3-O- [ 3 - O - ( 2 - a z i d o - 3 , 4 , 6 - t r i - O - b e n z y l - 2 - d e s o x y - c ~ -D-galacto pyrano syl)-4,6-O-benzylidene-2-O-(2,3,4-tri-O-benzyl-~L-fucopyrano syl)-fi-D-galacto pyrano sy I]- 2-desoxv_7 a - D - g l u c o p y r a n o s i d e (XXIV). This t e t r a s a c c h a r i d e was obtained by analogy with the above p r o c e d u r e from t r i s a c c h a r i d e (XXD and chloride (X][I) as a syrup in 60g yield with [~]D 2~ +26 ~ (C 1, CHCla). PMR s p e c t r u m (5, ppm, CDC13): 1.04 d (3 H, J5",6" = 6.0 Hz, fucose CHa), 1.46 s (3 H, Ae), 5.39 s (1 H, PhCH), 5.5 s (1 H, PhCH), 7.3 m (4~ H, 9Ph). Found: C 70.09; H 6.23; N 3.60g. Ca!oulated for Cs.qHq4N4Ol.q: C 70.14; H 6.23; N 3.68g.

Benzyl-2-aeetamido- 4,6-O-benzylidene- 3-0- [3-O-(2-azido- 3,4,6-tri-O-benzyl-2-desoxy-~-D-ga !acto__% pyran~syl)-4~6- 9 (XXV). Method A. The glycosylation of disaccharide (XVI) using chloride (XH) was carried out as in the case of trisaccharide (XX). The reaction mixture was deacylated according to Semplen and subjected to chromatography with 3 : 2 CHCI 3ethyl acetate eluent to yield 74~ (XXV) with mp 265~ (dec., from methanol-nitromethane), [(~ ]D 2~ + 91 ~ (C I, CHCI3). PMR spectrum (6, ppm, CD2C12): 1.90 s (3 HAc), 5.59 s (2 H, 2PhCH), 6.44d (I H, JNH,2 = 8.5 Hz, NH), 7.35 m (30 H, 6Ph). Found: C 67.31; H 6.11; N 4.98~. Calculated for CGzH~6N4015: C 67.25; H 6.02; N 5.06~. Method B. A solution of chloride (XH) obtained from 1.0 mmole bromide (XD was added o v e r 3 h to a mixture of 0.65 g (1.0 mmole) diol (XIV), 2.5 g Ag2CO2, 70 mg AgCIO4, and 5 g 4-A m o l e c u l a r sieves in 200 m[ CHzC12. A f t e r 30 rain, the mixture was filtered and the filtrate was washed with water and aq. NaHCO3, dried and subjected to c h r o m a t o g r a p h y as in method A. The yield of (XXV) was 0.36 g (33~). O - 2 - A c e t a m i d o - 2 - d e s o x y - ~ - D - g a l a e t o p y r a n o s y l - ( 1 - 3)-[O-c~-L-fucopyranosyl-(1 ~ 2)]-O-fi-D-ga!actop y r a n o s y l - ( 1 - - 3 ) - 2 - a c e t a m i d o - 2 - d e s o x y - D - g l u c o s e (D- Method A. A sample of 0.82 t e t r a s a e c h a r i d e (XT~[II) was deaeylated a c c o r d i n g to Semplen in 3 : 1 m e t h a n o l - b e n z e n e . The solution was neutralized with acetic acid and evaporated. The product was hydrogenated for 2 days in methanol at 50~ on 10g P d / C . The mixture was cooled, filtered, and 10 eq. acetic anhydride was added to the filtrate. A f t e r 24 h, the mixture was evaporated at 20~ and the residue was subjected to c h r o m a t o g r a p h y on a column packed with P - 2 biogel with water eluent to yield 0.33 g (90~) amorphous t e t r a s a c c h a r i d e ([) with [CqD2~ * 49 ~ (C 1, methanol) [11]. The PMR s p e c t r a l data were identical to those of Paulsen [11]. Method B. By analogy with the above p r o c e d u r e , but excluding the deacyIation step, t e t r a s a c o h a r i d e (I) was obtained from the protected derivative (XXIV). O-2-Acetamido-2-desoxy-c~-D-galactopyranosyl-(1 -- 3)-O-fi-D-galactopyranosyl-(1 ~ 3)-2-acetamido2-desoxy-D-glucose (If). By analogy with the above procedure (method B), amorphous trisaccharide (If) with [~]D 2~ +130 ~ (C 1, water) [9] was obtained from trisaccharide (X~V). PMR spectrum (300 MHz, 6, ppm, CD3OD): 2.00 s (6 H, 2Ac), 5.08 d (1 H, J1,,, 2" = 3.2 Hz, H = 1"), 5.18 d (1 H, JI,2 = 3.4 Hz, H-I).

O-2-L-Fucopyranosyl-(l ~ 2)-O-fi-D-galactopyranosyl-(1 ~ 3)-2-acetamido-2-desoxy-D-glucose (XXV[). A sample of 0.59 g t r i s a c c h a r i d e (XX) was deacylated and hydrogenated as described for the preparation of ([) by method A. The product was dissolved in water, washed with benzene, the aqueous solution was treated with

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[R-120 (H +) a m b e r l i t e , and evaporated to dryness to yield 0.26 g ( ~ 100~ amorphous t r i s a c c h a r i d e (XXVI) with [a]D2~ - 1 8 ~ (C 1, methanol) [11]. The PMR s p e c t r a l data a r e identical to those obtained by Paulsen [11]. CONCLUSIONS 1. The s t e r e o s e l e c t i v i t y of the glyeosylation of 2 - a z i d o - 2 - d e s o x y - D - g a l a e t o p y r a n o s y l halides was studied. 2. 2 - A z i d o - 3 , 4 , 6 - t r i - O - b e n z y l - 2 - d e s o x y - f i - D - g a l a e t o p y r a n o s y l chloride was proposed as a new glycosylating agent for the synthesis of a - g a l a c t o s a m i n i d e s . The fol!~)wing determinative group specific o l i g o s a c c h a r i d e s were synthesized: D-GalpNAc-~(I - - 3 ) [ L - F u c p - a (1 -- 2)]-D-Galp-fi (1 - - 3)-D-GlcNAc and L - Y u c p - a (1 - - 2)-D-Galp-fi (1 - - 3)-D-GleNAc. LITERATURE 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

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CITED

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