Enzyme-Catalyzed Synthesis of Carbohydrates - ACS Symposium

Chapter 18

Enzyme-Catalyzed Synthesis of Carbohydrates

Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: December 30, 1989 | doi: 10.1021/bk-1989-0386.ch018

C.-H. Wong, D. G. Drueckhammer, J . R. Durrwachter, B. Lacher, C. J . Chauvet, Y.-F. Wang, H. M . Sweers, G. L. Smith, L. J.-S. Yang, and W. J . Hennen Department of Chemistry, Texas A&M University, College Station, TX 77843 Several enzymatic procedures have been developed f o r the s y n t h e s i s o f carbohydrates from a c y c l i c precursors. A l d o l a s e s appear t o be u s e f u l c a t a l y s t s f o r the c o n s t r u c t i o n o f sugars through asymmeteric C-C bond formation. 2-deoxy-KDO, 2-deoxy-2-fluoro-KDO, 9-0-acetyl sialic a c i d and s e v e r a l unusual sugars were prepared by a combined chemical and enzymatic approach. A l c o h o l dehydrogenases and l i p a s e s have been used i n the p r e p a r a t i o n o f chiral furans, hydroxyaldehydes, and g l y c e r o l acetonide which are u s e f u l as b u i l d i n g blocks i n carbohydrate s y n t h e s i s . Recent developments i n t h e enzymatic s y n t h e s i s o f c a r b o h y d r a t e s c a n b e c l a s s i f i e d i n t o f o u r a p p r o a c h e s : 1) a s y m m e t r i c C-C bond f o r m a t i o n c a t a l y z e d b y a l d o l a s e s ( 1 10) ; 2) e n z y m a t i c s y n t h e s i s o f c a r b o h y d r a t e s y n t h o n s ( 1 0 11) ; 3) a s y m m e t r i c g l y c o s i d i c f o r m a t i o n c a t a l y z e d b y g l y c o s i d a s e s (12-17) a n d g l y c o s y l t r a n s f e r a s e s ( 1 8 - 2 3 ) ; and 4) r e g i o s e l e c t i v e t r a n s f o r m a t i o n s o f s u g a r s a n d d e r i v a t i v e s (24-25). These enzymatic t r a n s f o r m a t i o n s a r e s t e r e o s e l e c t i v e and c a r r i e d out under m i l d c o n d i t i o n s w i t h minimum p r o t e c t i o n o f f u n c t i o n a l g r o u p s . They h o l d promise i n preparative carbohydrate s y n t h e s i s . I n c o n n e c t i o n w i t h t h i s book, we f o c u s o n t h e f i r s t two approaches. Aldol

Condensations

FDP A l d o l a s e . T h e r e h a v e b e e n a p p r o x i m a t e l y 15 a l d o l a s e s i s o l a t e d , each o f which c a t a l y z e s a d i s t i n c t a l d o l r e a c t i o n (25-26). The a l d o l a s e s which have been s t u d i e d t h e most a s s y n t h e t i c c a t a l y s t s a r e f r u c t o s e - 1 , 6 -

c

0097-6156/89/0386-0317$06.00/0 1989 American Chemical Society

In Trends in Synthetic Carbohydrate Chemistry; Horton, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


318

TRENDS IN SYNTHETIC CARBOHYDRATE CHEMISTRY

d i p h o s p h a t e a l d o l a s e (FDP a l d o l a s e , EC 4.1.1.13) f r o m r a b b i t m u s c l e (1-4) a n d N - a c e t y l n e u r a m i n i c a c i d a l d o l a s e (NANA a l d o l a s e , EC 4.1.3.3.) f r o m C l o s t r i d i a ( 5 ) . From t h e r e s u l t s we a n d o t h e r g r o u p s h a v e o b t a i n e d s o f a r , i t a p p e a r s t h a t t h e enzyme FDP a l d o l a s e i s q u i t e s p e c i f i c f o r t h e a l d o l d o n o r d i h y d r o x y a c e t o n e p h o s p h a t e (DHAP), but w i l l accept a v a r i e t y o f aldehydes as t h e acceptor (1-4). T h e s t e r e o c h e m i s t r y o f C-C bond f o r m a t i o n i s n o t a f f e c t e d by t h e s u b s t i t u e n t s a t t h e carbon c e n t e r next t o the aldehyde group. The α - s u b s t i t u e n t s , however, do a f f e c t t h e r a t e o f C-C bond f o r m a t i o n . Using the s t r u c t u r a l r e p r e s e n t a t i o n o f t h e a l d e h y d e a c c e p t o r shown i n F i g u r e 1, a n a l d e h y d e w i t h R b e i n g t h e l a r g e s t a n d R' t h e second l a r g e s t group a t t h e c h i r a l c e n t e r would a l l o w t h e r e a c t i o n t o t a k e p l a c e much f a s t e r i n r e l a t i o n t o i t s e n a n t i o m e r . However, u n d e r t o t a l l y r e v e r s i b l e , c a t a l y t i c c o n d i t i o n s , t h e product d i s t r i b u t i o n i s thermodynamically controlled. T y p i c a l a l d o l r e a c t i o n s c a n b e c a r r i e d o u t on a 10100 mmol s c a l e w i t h f r e e o r i m m o b i l i z e d enzyme. After removal o f t h e phosphate group from t h e a l d o l p r o d u c t by acido r phosphatase-catalyzed h y d r o l y s i s , a free ketose can be p r e p a r e d . Several unusual ketoses with d i f f e r e n t s u b s t i t u e n t s a t R and R' h a v e b e e n p r e p a r e d . These i n c l u d e d e o x y s u g a r s , f l u o r o s u g a r s , O - a l k y l s u g a r s (1-3) and o t h e r h i g h e r m o n o s a c c h a r i d e s ( 4 ) . T h e s e k e t o s e s , 3, may be f u r t h e r t r a n s f e r r e d t o t h e c o r r e s p o n d i n g a l d o s e s 4 upon t r e a t m e n t w i t h t h e enzyme g l u c o s e i s o m e r a s e (EC 5.3.1.5) (3.), a n i n d u s t r i a l enzyme u s e d i n t h e manufacture o f high f r u c t o s e corn syrup. T h i s combined a l d o l a s e / i s o m e r a s e c a t a l y s i s p r o v i d e s a new r o u t e t o a v a r i e t y o f s u g a r s and d e r i v a t i v e s which a r e u s e f u l f o r o t h e r a p p l i c a t i o n s (2-3.) . The s u b s t r a t e DHAP r e q u i r e d i n t h e s e e n z y m a t i c s y n t h e s e s c a n be p r e p a r e d c h e m i c a l l y (27.) o r e n z y m a t i c a l l y (1-4). I t c a n a l s o be g e n e r a t e d i n s i t u f r o m FDP ( 1 - 4 ) . P o t e n t i a l p r o b l e m s w i t h t h e u s e o f DHAP i n t h e s e p r o c e d u r e s i n c l u d e t h e s t a b i l i t y o f DHAP i n s o l u t i o n ( t i / o ~ 15 h a t pH 7.5) a n d t h e t h e n o n - t r i v i a l p r e p a r a t i o n o f DHAP. We h a v e f o u n d t h a t a m i x t u r e o f d i h y d r o x y a c e t o n e and a s m a l l amount o f i n o r g a n i c a r s e n a t e c a n be u s e d t o r e p l a c e DHAP i n t h e a l d o l r e a c t i o n s i n w h i c h t h e i n o r g a n i c a r s e n a t e i s r e c y c l e d a b o u t 50 t i m e s d u r i n g t h e r e a c t i o n s ( 3 ) . T h i s e l i m i n a t e s t h e need f o r t h e p r e p a r a t i o n o f DHAP. From t h e r e s u l t s o f k i n e t i c a n d c o m p e t i t i o n s t u d i e s , we c o n c l u d e t h a t t h e r e a c t i o n s proceed through dihydroxyacetone a r s e n a t e monoester which i s a n a l o g o u s t o DHAP a n d i s a c c e p t e d b y t h e enzyme a s a substrate (Figure 2). T h e C-C bond f o r m a t i o n i s t h u s c o m p l e t e l y d e t e r m i n e d b y t h e enzyme w h i c h o n l y a c c e p t s the a r s e n a t e monoester as a s u b s t r a t e , a l t h o u g h t h e r e v e r s i b l e f o r m a t i o n o f a r s e n a t e e s t e r s i n aqueous s o l u t i o n i s n o n s e l e c t i v e and s e v e r a l e n z y m a t i c a l l y i n a c t i v e a r s e n a t e s p e c i e s may e x i s t i n s o l u t i o n . Under


18. WONG ET A L



H R

f-

r-r-..

Lys-E

Η

HO ^L^OP N

3 D Η 2) Glucose Isomerase

FDP Aldolase

Hs Ο Η

θ

λ

^ Ο Η Ο

OH

0

H,0

0

FDP Aldolase 2) Η

μ ρ / HO

Γ

^ ^

0

Η

F i g u r e 1. S y n t h e s i s o f u s u a l a n d u n u s u a l s u g a r s u s i n g FDP a l d o l a s e a n d g l u c o s e i s o m e r a s e a s catalysts.


319

320


H O v J l .OH RCHO


Aldolase

°v^k^

0 H

- ΗΟ^^Λ^ΟΑβοΓ

• HOAsO,"

• Η,Ο

NAOH GlyctroP DH

ΜΟ^Λ^ΟΑιΟ,

2

4 [Enzyme] χ 10

6 2

F i g u r e 2. Mechanism o f d i h y d r o x y a c e t o n e / a r s e n a t e r e a c t i o n w i t h FDP a l d o l a s e . Both dihydroxyacetone and i n o r g a n i c a r s e n a t e a r e n o t t h e i n h i b i t o r o f t h e aldolase reactions. The r a t e c o n s t a n t f o r t h e arsenate e s t e r formation i s determined enzymatically (a p l o t o f 1/v v s 1/E g i v e s a n o n - z e r o i n t e r c e p t w h i c h i s a t t r i b u t e d t o t h e r a t e a t i n f i n i t e enzyme c o n c e n t r a t i o n and t h a t r a t e c o r r e s p o n d s t o t h e r a t e of nonenzymatic formation o f t h e a r s e n a t e e s t e r ) .


18. WONG ET AL·


321


the r e a c t i o n conditions, the arsenate formation i s t h e r a t e d e t e r m i n i n g s t e p and t h e a l d o l r e a c t i o n s t e p i s v i r t u a l l y i r r e v e r s i b l e . T h i s i s c o n t r a r y t o t h e normal r e v e r s i b l e a l d o l r e a c t i o n s w i t h DHAP where t h e C-C bond formation i s rate determining. T h e s e r e s u l t s were s u p p o r t e d by t h e f a c t t h a t a h i g h d i a s t e r e o s e l e c t i v i t y was o b s e r v e d i n t h e r e a c t i o n o f DHAP w i t h a n e x c e s s o f a r a c e m i c a l d e h y d e , whereas i n t h e r e a c t i o n w i t h d i h y d r o x y a c e t o n e / a r s e n a t e (DHA/As) no s i g n i f i c a n t s e l e c t i v i t y was o b s e r v e d ( F i g u r e 3 ) . I n a n a t t e m p t t o use i n o r g a n i c vanadate i n s t e a d o f a r s e n a t e i n t h e a l d o l a s e r e a c t i o n s no a p p r e c i a b l e a l d o l c o n d e n s a t i o n was o b s e r v e d , p r e s u m a b l y due t o t h e u n d e s i r e d r e d o x r e a c t i o n between DHA a n d i n o r g a n i c v a n a d a t e . KDO-8 P h o s p h a t e S y n t h e t a s e . KDO (3-deoxy-a-D-manno-2o c t u l o p y r a n o s o n i c a c i d ) i s a component o f c a p s u l a r p o l y s a c c h a r i d e s ( k - a n t i g e n s ) and l i p o p o l y s a c c h a r i d e s (LPS, a l s o known a s e n d o t o x i n s ) f o u n d i n G r a m - n e g a t i v e b a c t e r i a (28)· T h e s e K D O - c o n t a i n i n g membrane components a r e u n i q u e t o g r a m - n e g a t i v e b a c t e r i a and a r e c r u c i a l t o the s u r v i v a l o f t h e organism. S i n c e KDO i s n o t f o u n d i n mammalian t i s s u e s , i t h a s become a p r i m a r y t a r g e t f o r i n v e s t i g a t i o n b y m e d i c i n a l a n d s y n t h e t i c c h e m i s t s (28-31) f o r t h e development o f a n t i b a c t e r i a l agents e f f e c t i v e a g a i n s t G r a m - n e g a t i v e b a c t e r i a . 0-2-Deoxy KDO (32) a n d t h e 8 - d i p e p t i d y l d e r i v a t i v e o f 8-amino-2,8-dideoxy-0-KDO (33) h a v e b e e n shown t o be p o t e n t i n h i b i t o r s o f t h e enzyme CMP-KDO s y n t h e t a s e w h i c h u t i l i z e s t h e 0 - p y r a n o s e f o r m o f KDO a s s u b s t r a t e (34)· U n l i k e 2-deoxy-^-KDO which cannot p e n e t r a t e i n t a c t b a c t e r i a , t h e g l y c o p e p t i d e a n a l o g c a n be t r a n s p o r t e d t h r o u g h t h e c e l l membrane a n d h y d r o l y z e d by a p e p t i d a s e i n s i d e t h e c e l l t o r e l e a s e t h e i n h i b i t o r ( 3 1 ) . S e v e r a l C - g l y c o s i d i c d e r i v a t i v e s o f KDO h a v e r e c e n t l y b e e n p r e p a r e d from p r o t e c t e d KDO ( 3 5 ) . We h a v e b e e n s u c c e s s f u l l y i n i m m o b i l i z i n g t h e enzyme KD0-8-phosphate s y n t h e t a s e w h i c h was i s o l a t e d f r o m E . c o l i Β c e l l s (ATCC 11303) (36)· A b o u t 400 U o f t h e enzyme c a n be p r o d u c e d from 500 g o f wet c e l l s . The u s e o f t h i s enzyme p r e p a r a t i o n i s i l l u s t r a t e d i n F i g u r e 4. W i t h t h e u s e o f a s y s t e m o f c o - i m m o b i l i z e d enzymes on polyacrylamide g e l s prepared according t o p r e v i o u s l y d e s c r i b e d p r o c e d u r e s ( 3 7 ) , KDO-8-phosphate was p r e p a r e d on a 10-50 g s c a l e s t a r t i n g w i t h D - a r a b i n o s e a n d p h o s p h o e n o l p y r u v a t e (38)· A f t e r r e m o v a l o f t h e phosphate moiety by a c i d - o r p h o s p h a t a s e - c a t a l y z e d h y d r o l y s i s , t h e p r o d u c t was c o n v e r t e d c h e m i c a l l y t o 2deoxy-0-KDO a n d 2 - d e o x y - 2 - f l u o r o KDO ( s e e F i g u r e 5 ) . The p r o c e d u r e f o r t h e i n t r o d u c t i o n o f t h e F g r o u p was s i m i l a r to t h a t r e p o r t e d p r e v i o u s l y (39). A f t e r completing t h e work, we n o t i c e d t h a t KDO-8-phosphate was a l s o p r e p a r e d by W h i t e s i d e s ' g r o u p i n a s i m i l a r way w i t h t h r e e enzymes e n c l o s e d i n a d i a l y s i s membrane ( 4 0 ) .



S w X

OH

3

HAs0 4

=

Aldolase

+

Η

OH

60%

97%

3 ° ^ ^ ο OH

OH

3

3%

40%

OH

CH

F i g u r e 3. S e l e c t i v i t y o f t h e F D P - a l d o l a s e r e a c t i o n s u s i n g DHAP v s . d i h y d r o x y a c e t o n e / a r s e n a t e a s a substrate. I n t h e f o r m e r c a s e , t h e more s t a b l e sugar i s o b t a i n e d due t o t h e r e v e r s i b l e n a t u r e o f the r e a c t i o n . I n t h e l a t e r c a s e , b o t h s u g a r s were o b t a i n e d i n n e a r l y e q u a l amounts, b e c a u s e t h e r e a c t i o n was f o u n d t o b e v i r t u a l l y i r r e v e r s i b l e a n d t h e f o r m a t i o n o f t h e a r s e n a t e e s t e r was r a t e limiting.

HO

+ HO

= 1)Aldolase Jl^OP0 2)H

HO


OH

HO OH

ο

η η

Η

H



18. WONG ET AL.

F i g u r e 4. S y n t h e s i s of KDO-8-phosphate. h e x o k i n a s e ; PK, p y r u v a t e k i n a s e .

HK,


323



324

2-Deoxy-2-Fluoro-KDO Methyl Ester F i g u r e 5. Chemical conversion KDO and 2 - d e o x y - 2 - f l u o r o - K D O

2-Deoxy-KDO

o f KDO t o 2-deoxy-0



18. WONG ET AL.


325

N - A c e t y l n e u r a m i n i c A c i d A l d o l a s e . A new p r o c e d u r e h a s a l s o been d e v e l o p e d f o r t h e s y n t h e s i s o f 9 - 0 - a c e t y l - N acetylneuraminic acid using the aldolase catalyzed r e a c t i o n m e t h o d o l o g y . T h i s compound i s a n u n u s u a l s i a l i c a c i d f o u n d i n a number o f tumor c e l l s a n d i n f l u e n z a v i r u s C g l y c o p r o t e i n s ( 4 1 ) . The a l d o l a c c e p t o r , 6 - 0 - a c e t y l - D mannosamine was p r e p a r e d i n 70% i s o l a t e d y i e l d f r o m i s o p r o p e n y l a c e t a t e and N-acetyl-D-mannosamine c a t a l y z e d b y p r o t e a s e Ν f r o m B a c i l l u s s u b t i l i s ( f r o m Amano). T h e 60 - a c e t y l h e x o s e was p r e v i o u s l y p r e p a r e d b y a c o m p l i c a t e d c h e m i c a l p r o c e d u r e (42.) . T h e t a r g e t m o l e c u l e was o b t a i n e d i n 90% y i e l d v i a t h e c o n d e n s a t i o n o f t h e 6-0a c e t y l s u g a r a n d p y r u v a t e c a t a l y z e d b y NANA a l d o l a s e ( F i g u r e 6 ) . W i t h s i m i l a r p r o c e d u r e s a p p l i e d t o KDO, 2deoxy-NANA a n d 2 - d e o x y - 2 - f l u o r o - N A N A were p r e p a r e d f r o m NANA. Carbohydrate

Synthons

E n z y m a t i c r e d u c t i o n o f c a r b o n y l compounds a n d e n z y m a t i c e n a n t i o s e l e c t i v e t r a n s f o r m a t i o n o f r a c e m i c o r meso a l c o h o l s (25,42) a r e two m e t h o d o l o g i e s t h a t h a v e p r o v e n t o be b e n e f i c i a l i n t h e p r e p a r a t i o n o f o p t i c a l l y a c t i v e h y d r o x y l compounds, k e y c h i r a l b u i l d i n g b l o c k s u s e d i n c a r b o h y d r a t e a n d n a t u r a l p r o d u c t s y n t h e s e s ( 4 4 - 4 5 ) . Our i n t e r e s t i n t h i s area i s t o develop enzymatic routes t o o p t i c a l l y a c t i v e g l y c e r o l and f u r a n d e r i v a t i v e s , and hydroxyaldehydes. C h i r a l F u r a n s . The a l k y l f u r y l c a r b i n o l 5 i s a u s e f u l b u i l d i n g b l o c k f o r t h e s y n t h e s i s o f d e o x y - L - s u g a r s (46) ( F i g u r e 7 ) . T h i s synthon c a n be p r e p a r e d from a c y l furans v i a enzymatic r e d u c t i o n s c a t a l y z e d by t h e a l c o h o l dehydrogenase from Thermoanaerobium b r o c k i i . o r from an esterase-catalyzed k i n e t i c r e s o l u t i o n o f t h e racemic a l c o h o l (11). Both approaches p r o v i d e t h e c h i r a l b u i l d i n g b l o c k i n > 90% e e u s i n g q u i t e s t r a i g h t f o r w a r d p r o c e d u r e s on g s c a l e s (Figure 8). C h i r a l Hvdroxvaldehvdes. S i m i l a r l y , o p t i c a l l y a c t i v e l a c t a l d e h y d e and α-hydroxybutyraldehyde, b o t h u s e f u l a l d o l a c c e p t o r s , c a n b e p r e p a r e d e n z y m a t i c a l l y (10) a c c o r d i n g t o t h e scheme shown i n F i g u r e 9. Chiral Glycerol Derivatives. I r r e v e r s i b l e T r a n s e s t e r i f i c a t i o n . A new p r e p a r a t i o n o f c h i r a l glycerol acetonide (2,2-dimethyl-l,3-dioxolane-4m e t h a n o l ) i n v o l v i n g a n e n a n t i o s e l e c t i v e h y d r o l y s i s o f 20 - b e n z y l y c e r o l d i a c e t a t e t o che (R)-monoacetate c a t a l y z e d by a l i p o p r o t e i n l i p a s e (4_7) h a s r e c e n t l y b e e n d e v e l o p e d . I n a n e f f o r t t o p r e p a r e t h e ( S ) - e n a n t i o m e r , we h a v e u s e d the aforementioned i r r e v e r s i b l e t r a n s e s t e r i f i c a t i o n r e a c t i o n u s i n g i s o p r o p e n y l a c e t a t e as an a c y l a t i n g r e a g e n t , w h i c h upon r e a c t i o n g i v e s a c e t o n e a s a




β ο ^

-μ

rH ι >ι >ι C Ό Λ Η 0) 0) ίΟ 0 · Η ^ (0 M (d Ο U

u ο Ο Λ Ό W - Ρ ω ο α) Cu•Η · Η

ω 55 υ υ C Φ to (0 W (0 Η >irH (1) Ο) -μ Ο c - μ υ Η •Η Ο

ι ι

si

υ < 10 3 Φ Ζ

^ α) (0 H) OH ο (Α Ό «J Η £ t0

(0 «3 ω ο ·Η ι Ο Ι 0) ο > Ι · Η υ -μ «3 I £ c 10 σ» Ε-» c (0Ό W ϋ

Λ

ο ω •Η m ω Λ .μ c

· Ό ·Η υ to

Η Ν fl) C >ι > ι Ό · Η •Ρ Η Π β

ο) id ο id ϋ 4J ϋ U

ί0 υ I · Η 21 c β >ι·Η Ο Μ (0 «-ι

, -μ Ο ϋ 10 I 551

to to ο ο* α-μ c CJ> α)

Enzyme-Catalyzed Synthesis of Carbohydrates - ACS Symposium

Recommend Documents