Fluorinated Carbohydrates - American Chemical Society

Chapter 3

Chemoenzymatic

Synthesis

of Fluorosugars

C.-H. Wong, D. G. Dreckhammer, and H. M. Sweers

Downloaded by IOWA STATE UNIV on February 21, 2017 | http://pubs.acs.org Publication Date: August 2, 1988 | doi: 10.1021/bk-1988-0374.ch003

Departments of Chemistry and of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843 S e v e r a l p r a c t i c a l enzymatic procedures have been developed f o r the s y n t h e s i s of fluorocarbohydrates. The enzymes f r u c t o s e - 1 , 6 diphosphate a l d o l a s e and glucose isomerase are used i n the s y n t h e s i s of 6-deoxy-6fluorohexoses. Hexokinase i s used f o r the p r e p a r a t i o n of a number of fluorohexose-6phosphates, two of them are f u r t h e r converted t o 2-deoxy-2-fluoropentose-5 phosphates. When ATP is r e q u i r e d , it is regenerated from ADP c a t a l y z e d by pyruvate kinase i n the presence of phosphoenol pyruvate. Lipases are used as c a t a l y s t s i n s e l e c t i v e d e p r o t e c t i o n of p e r a c y l a t e d sugars, the products of which are converted to fluorosugars by r e a c t i o n with diethylaminosulfur triflouride. Incorporation of F i n t o o l i g o s a c c h a r i d e is illustrated by a gram-scale s y n t h e s i s of 6 ' - d e o x y - 6 ' f l u o r o s u c r o s e c a t a l y z e d by sucrose synthetase coupled with enzymatic regenerations of UDP, UTP and UDP-glucose. F l u o r i n a t e d Sugars i n which one of the hydroxyl groups i s r e p l a c e d with the f l u o r i n e group can be strong i n h i b i t o r s of the n o n f l u o r i n a t e d species i n biochemical processes and are of i n t e r e s t as p o t e n t i a l pharmaceuticals or pharmacological probes (1-4). The i n h i b i t i o n s are mainly due to the d i f f e r e n c e of C-F and C-OH i n r e a c t i v i t y and the s i m i l a r i t y of both groups i n p o l a r i t y and bond l e n g t h . Examples have been shown i n which f l u o r i n a t e d monosaccharides i n h i b i t the g l y c o s y l a t i o n of glycoproteins. 6 - F l u o r o - L - f u c o s e (5), 6-deoxy-6-fluorog a l a c t o s e (5), 4-deoxy-4-fluoromannose (6), and 2-deoxy2-fluoromannnose have been shown to i n h i b i t the

0097-6156/88/0374-0029$06.00/0 © 1988 American Chemical Society

TAYLOR; Fluorinated Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1988.


30

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

i n c o r p o r a t i o n of the corresponding n o n f l u o r i n a t e d sugars i n t o g l y c o p r o t e i n s . M e c h a n i s t i c s t u d i e s on t h e g l y c o s y l a t i o n o f some v i r a l g l y c o p r o t e i n s h a v e i n d i c a t e d that the fluorosugars are converted i n t o the corresponding n u c l e o s i d e diphosphates which i n h i b i t the assembly of the l i p i d - l i n k e d o l i g o s a c c h a r i d e s c a t a l y z e d by t h e a p p r o p r i a t e g l y c o s y l t r a n s f e r a s e s ( 5 - 8 ) . The s y n t h e s i s o f f l u o r o c a r b o h y d r a t e s c u r r e n t l y d e p e n d s on c h e m i c a l p r o c e d u r e s (9-14) w h i c h v e r y o f t e n r e q u i r e m u l t i p l e p r o t e c t i o n and d e p r o t e c t i o n s t e p s t o o v e r c o m e the problems o f s t e r e o s e l e c t i v i t y . Recent r e s e a r c h has b e e n f o c u s e d on t h e d e v e l o p m e n t o f m i l d e r and more s e l e c t i v e methods f o r t h e i n c o r p o r a t i o n o f f l u o r i n e i n t o carbohydrates. T h i s r e v i e w i s o n l y f o c u s e d on t h e r e c e n t developments i n the enzymatic approach t o the s y n t h e s i s of f l u o r i n a t e d carbohydrates. The a p p r o a c h h a s t h e a d v a n t a g e s t h a t t h e r e a c t i o n s a r e c a t a l y t i c , s t e r e o s e l e c t i v e and t h a t t h e y a r e c a r r i e d o u t u n d e r m i l d c o n d i t i o n s i n aqueous s o l u t i o n , w i t h minimum p r o t e c t i o n of the f u u n c i o n a l groups. Aldolase/Isomerase-Catalyzed

Reactions

We h a v e d e v e l o p e d p r e p a r a t i v e e n z y m a t i c s y n t h e s e s o f s e v e r a l unusual hexoketoses u s i n g f r u c t o s e - 1 , 6 d i p h o s p h a t e a l d o l a s e ( F D P - a l d o l a s e , E.C.4.1.2.13) a s c a t a l y s t and 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) and an a l d e h y d e a s s u b s t r a t e s (15) . The enzyme a p p e a r s t o be v e r y s p e c i f i c f o r DHAP b u t w i l l a c c e p t a v a r i e t y o f a l d e h y d e s as a c c e p t o r s . The k e t o s e - 1 - p h o s p h a t e s prepared are converted t o the phosphate f r e e ketoses a f t e r removal o f t h e p h o s p h a t e g r o u p 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 hydrolysis. The k e t o s e s c a n be i s o m e r i z e d s t e r e o s p e c i f i c a l l y t o a l d o s e s c a t a l y z e d by g l u c o s e i s o m e r a s e (E.C.5.3.1.5.) f r o m F l a v o b a c t e r i u u m arborescens. The e q u i l i b r i u m m i x t u r e s o f a l d o s e s and k e t o s e s a r e t h e n 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 Dowex 50 ( B a ) o r Dowex 1 ( H S 0 ~ ) . Figure 1 i l l u s t r a t e s the preparation of a mixture of 6-deoxy-6-fluoro-D-fructose (5) and 6 - d e o x y - 6 - f l u o r o - L - s o r b o s e (6), the former i s s e l e c t i v e l y isomerized t o 6-deoxy-6-fluoro-D-glucose (7) i n the presence of glucose isomerase. The e q u i l i b r i u m m i x t u r e o f (5) and (7) c o n t a i n s 80% o f ( 7 ) . D e t a i l e d p r o c e d u r e s f o r t h e s e p r e p a r a t i o n s were r e p o r t e d ( 1 5 ) . The s u b s t r a t e 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) c a n be p r e p a r e d e n z y m a t i c a l l y (15) o r c h e m i c a l l y ( 1 5 , 1 6 ) . A l t e r n a t i v e l y , i t c a n be r e p l a c e d w i t h 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 arsenate. The p r o p o s e d mechanism f o r t h i s i n t e r e s t i n g p r o c e s s i s shown i n F i g u r e 2. In s o l u t i o n , d i h y d r o x y a c e t o n e r e v e r s i b l y r e a c t s w i t h a r s e n a t e t o form d i h y d r o x y a c e t o n e a r s e n a t e w h i c h i s a n a l o g o u s t o DHAP and t h u s a c c e p t e d by t h e enzyme a s a s u b s t r a t e . The a r s e n a t e moiety of the a l d o l product i s then d i s s o c i a t e d + +

3



WONG ET AL.

Chemoenzymatic Synthesisof Fluorosugars

7 F i g u r e 1. P r e p a r a t i o n o f f l u o r o s u g a r s v i a e n z y m a t i c a l d o l r e a c t i o n s , a) C £ H C N / H 0 - b) 1, K H F H . c) 1, F D P - a l d o l a s e ; 2, H . d) g l u c o s e i s o m e r a s e . 5

2

2

2 /

F i g u r e 2. U s e o f d i h y d r o x y a c e t o n e a n d a r s e n a t e a s s u b s t r a t e i nFDP-aldolase r e a c t i o n s . The r a t e c o n s t a n t w a s d e t e r m i n e d i n F i g u r e 3.



32


s p o n t a n e o u s l y a n d u s e d r e p e a t e d l y . The e v i d e n c e w h i c h s u p p o r t s t h i s mechanism i s t h e f o l l o w i n g . Both i n o r g a n i c a r s e n a t e a n d d i h y d r o x y a c e t o n e do n o t i n h i b i t t h e r e a c t i o n o f DHAP i n t h e a l d o l a s e - c a t a l y z e d c o n d e n s a t i o n s , i n d i c a t i n g a preformation o f dihydroxyacetone arsenate e s t e r which i s then e n t e r i n g t h e a c t i v e s i t e . The r a t e c o n s t a n t f o r t h e spontaneous f o r m a t i o n 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 was d e t e r m i n e d b y t h e glycerophosphate dehydrogenase-catalyzed reduction o f dihydroxyacetone i n the presence o f inorganic arsenate. A l i n e a r plot of the reciprocal of the rate versus the r e c i p r o c a l o f t h e enzyme c o n c e n t r a t i o n shows a n o n - z e r o intercept. T h i s intercept corresponds t o t h e rate a t i n f i n i t e enzyme ( F i g u r e 3) c o n c e n t r a t i o n w h i c h i s e q u a l to the rate of formation o f the dihydroxyacetone ester (2.4 X 1 0 ~ M s " ) . A p r e v i o u s s t u d y on g l u c o s e - 6 p h o s p h a t e d e h y d r o g e n a s e showed t h a t g l u c o s e - 6 - a r s e n a t e was f o r m e d a n d t h e r a t e c o n s t a n t was 6.3 X 1 0 ~ M ^ s " (17). I n o u r s t u d y on t h e s u b s t r a t e s p e c i f i c i t y o f FDP a l d o l a s e , t h e compound 3-deoxy-3-fluorohydroxyacetone-1p h o s p h a t e was f o u n d n o t a s u b s t r a t e f o r t h e enzyme; i t was a n i n h i b i t o r f o r t h e e n z y m a t i c c l e a v a g e o f f r u c t o s e 1,6-diphosphate. T h e k^ was d e t e r m i n e d t o be 3 mM ( F i g u r e 4 ) . When s o d i u m b o r o h y d r i d e was a d d e d t o t h e m i x t u r e o f a l d o l a s e and 3 - d e o x y - 3 - f l u o r o h y d r o x y a c e t o n e - 1 p h o s p h a t e , no i n a c t i v a t i o n o f a l d o l a s e was o b s e r v e d , i n d i c a t i n g t h a t a S c h i f f base i s n o t formed. The S c h i f f b a s e i n t e r m e d i a t e i n t h e m i x t u r e o f DHAP a n d a l d o l a s e h a s p r e v i o u s l y been t r a p p e d by r e d u c t i o n w i t h sodium b o r o h y d r i d e t o i n a c t i v a t e t h e enzyme ( 8 ) . T h e i n h i b i t o r may b e u s e f u l f o r s t u d y o f t h e a c t i v e s i t e o f t h e enzyme. T h e r e h a v e b e e n more t h a n 10 d i f f e r e n t a l d o l a s e s i s o l a t e d ; they c a t a l y z e a l d o l condensations u s i n g d i f f e r e n t s u b s t r a t e s t o g i v e sugar d e r i v a t i v e s (19). T h e s e enzymes w o u l d be u s e f u l f o r t h e s y n t h e s i s o f f l u o r i n a t e d s u g a r s i f t h e y would a c c e p t f l u o r i n a t e d m o l e c u l e s a s s u b s t r a t e s . We a r e i n t h e p r o c e s s o f e x p l o r i n g other types o f a l d o l a s e s as s y n t h e t i c catalysts. 3

1

6

Hexokinase-Catalyzed

1

Reactions

To i n t r o d u c e t h e p h o s p h a t e m o i e t y t o f l u o r i n a t e d s u g a r s , we h a v e d e v e l o p e d p r a c t i c a l e n z y m a t i c p r o c e d u r e s b a s e d on ATP-requiring hexokinase-catalyzed reactions coupled with an ATP r e g e n e r a t i o n s y s t e m c a t a l y z e d b y p y r u v a t e k i n a s e ( F i g u r e 5) (20). The p h o s p h o r y l a t i n g a g e n t p h o s p h o e n o l p y r u v a t e u s e d i n t h e r e g e n e r a t i o n o f ATP c a n be e a s i l y p r e p a r e d b a s e d on t h e i m p r o v e d p r o c e d u r e i n w h i c h t h e m o n o p o t a s s i u m s a l t o f p h o s p h o e n o l p y r u v a t e was o b t a i n e d a s c r y s t a l s (2JL) . T h e r e l a t i v e a c t i v i t i e s o f h e x o k i n a s e on f l u o r i n a t e d h e x o s e s a r e shown i n F i g u r e 5. Both y e a s t hexokinase and p y r u v a t e k i n a s e a r e q u i t e s t a b l e i n t h e i m m o b i l i z e d f o r m s (20) . Phosphoenol



3.

WONGETAL.

ChemoenzymaticSynthesisofFluorosugars

0.0

1

1

100

1

1

200

l/[Enzyme] x 1 0

300

33

r

400

2

F i g u r e 3. R e c i p r o c a l o f t h e r a t e (M/min) o f glycerophosphate dehydrogenase-catalyzed reduction of d i h y d r o x y a c e t o n e i n t h e p r e s e n c e o f 5 mM a r s e n a t e v s t h e r e c i p r o c a l o f enzyme c o n c e n t r a t i o n (mg/mL). R e a c t i o n s were c a r r i e d o u t i n T r i s b u f f e r (20 mM, pH 8.0) c o n t a i n i n g NADH (0.1 mM), d i h y d r o x y a c e t o n e (50 roM)/ g l y c e r o p h o s p h a t e d e h y d r o g e n a s e a n d s o d i u m a r s e n a t e (5 mM). T h e d e c r e a s e i n a b s o r b a n c e a t 340 nm was m o n i t o r e d v e r s u s t i m e .

p y r u v a t e i s a s t r o n g p h o s p h o r y l a t i n g agent and i s q u i t e s t a b l e i n s o l u t i o n (A G ° f o r h y d r o l y s i s = 16 k c a l / m o l , t l / 2 a t PH 7 = 30 d a y s ) ( 1 9 ) . T h i s a l l o w s u s t o s y n t h e s i z e f l u o r i n a t e d h e x o s e - 6 - p h o s p h a t e s on gram s c a l e s w i t h o u t d i f f i c u l t y a l t h o u g h some f l u o r o s u g a r s a r e weak substrates f o r hexokinase. Of t h e f l u o r o h e x o s e p h o s p h a t e s p r e p a r e d , two o f them h a v e b e e n c o n v e r t e d t o 2-deoxy-2-fluoropentose-5-phosphates. The d e t a i l e d r e a c t i o n s a r e shown i n F i g u r e 6. The p h o s p h a t e m o i e t y not o n l y s e r v e s as a p r o t e c t i n g group i n t h e t r a n s f o r m a t i o n o f hexose phosphate t o pentose phosphate, i t a l s o l o c k s t h e p e n t o s e phosphates i n t h e f u r a n o s e form which i s d e s i r e d i n t h e s y n t h e s i s o f n u c l e o s i d e monophosphates. Lipase-Catalyzed

Deprotection of Acylated

Sugars

S e l e c t i v e deprotection o f peracylated sugars i s o f i n t e r e s t i n carbohydrate chemistry. T h e f r e e OH g r o u p o f t h e d e p r o t e c t e d s u g a r c a n be c o n v e r t e d t o t h e F group by



34

FLUORINATED CARBOHYDRATES: C H E M I C A L AND BIOCHEMICAL ASPECTS

0

2

4

6

8

10

l / [ F r u c t o s e - l , 6 - d l p h o s p h a t e ] , mM

F i g u r e 4. Reciprocal p l o t s of aldolase a c t i v i t y at d i f f e r e n t c o n c e n t r a t i o n s o f 3-deoxy-3fluorohydroxyacetone-l-phosphate. The r e a c t i o n was coupled with glycerophosphate dehydrogenase i n the p r e s e n c e o f NADH t o d e t e r m i n e t h e enzyme a c t i v i t y , (see r e f . 15). Inserted, slope of the r e c i p r o c a l p l o t s of a l d o l a s e a c t i v i t y vs 3-deoxy-3-fluorohydroxyacetone-l-phosphate.

r e a c t i o n w i t h d i e t h y l a m i n o s u l f u r t r i f l u o r i d e (DAST) (22.) . Of t h e d i f f e r e n t a c y l a t e d s u g a r s t e s t e d , an a c y l g r o u p with a longer chain i s a better substrate than that with s h o r t e r c h a i n and t h e p r i m a r y p o s i t i o n i s s e l e c t i v e l y h y d r o l y z e d (22). I n a t y p i c a l 100-mmol s c a l e r e a c t i o n , the tetrapentanoyl d e r i v a t i v e o f a - m e t h y l Dg l u c o p y r a n o s i d e was s e l e c t i v e l y h y d r o l y z e d by t h e l i p a s e f r o m C a n d i d a c v l i n d r a c e a e t o t h e 6-OH derivative in >90% yield. S i m i l a r 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 h y d r o l y s i s o f t h e o t h e r a c y l d e r i v a t i v e s o f a - and pm e t h y l D - h e x o p y r a n o s i d e s i n c l u d i n g g a l a c t o s e , mannose, N a c e t y l g l u c o s a m i n e and N - a c e t y l m a n n o s a m i n e . (22) T h e s e 6OH s u g a r d e r i v a t i v e s c a n be u s e d f o r s y n t h e s i s o f 6-F h e x o s e s by r e a c t i o n w i t h DAST. Table I indicates r e l a t i v e and s p e c i f i c a c t i v i t i e s o f C a n d i d a l i p a s e c a t a l y z e d h y d r o l y s i s of d i f f e r e n t sugar e s t e r s at C-6 position. A l l t h e p r o d u c t s were i s o l a t e d by s i l i c a g e l


3.

WONGETAL.


Cmpd

ChemoenzymaticSynthesisof Fluorosugars

M

N

a

OH

b

F

c

H

F

d

F

F

e f

H

O

P

H

Q

OH

R

OH

X

H

K

m

0

•

F

H F

9

F

h

H

(mM)

Vmax (Umg" )

0.17

300

1

0.19

15 0

0.41

260

0.13

1 60

82

30

70

30

84

30

-

3

F

35

F i g u r e 5. H e x o k i n a s e - c a t a l y z e d p h o s p h o r y l a t i o n o f f l u o r i n a t e d h e x o s e s c o u p l e d w i t h a n ATP r e g e n e r a t i o n system u s i n g phosphoenol pyruvate as p h o s p h o r y l a t i n g agent and pyruvate k i n a s e as c a t a l y s t .

column chromatography e l u t e d w i t h CHC1 : MeOH (10:1) a n d t h e s t r u c t u r e s p r o v e n b y m i c r o a n a l y s i s a n d C-NMR a n a l y s i s u s i n g t h e d e u t e r i u m i n d u c e d s h i f t s (DIS) t e c h n i q u e (24.) • T h e a d d i t i o n o f d i m e t h y l f o r m a m i d e (10%) t o t h e Candida l i p a s e - c a t a l y z e d h y d r o l y s i s o f g l u c o s e p e n t a a c e t a t e changes t h e r e g i o s e l e c t i v i t y . In reaction with glucose pentaacetate, 1,2,3-tri-O-acetyl-a-D-glucose and 1 , 2 , 3 , 4 - t e t r a - O - a c e t y l - ^ - D - g l u c o s e were i s o l a t e d i n 73% a n d 27% y i e l d r e s p e c t i v e l y . T h e s t r u c t u r e s o f t h e compounds a r e shown i n T a b l e I I I . A l l s h i f t s a r e i n ppm f r o m TMS. T h e DIS numbers a r e i n p a r e n t h e s e s . Negative values indicate upfield s h i f t s . B o t h compounds w e r e i d e n t i f i e d b y u s i n g t h e DIS t e c h n i q u e s ( T a b l e I I ) . T h e y show e x p e c t e d u p f i e l d s h i f t o f t h e d e a c y l a t e d c a r b o n . When t h e p e r a c e t y l a t e d s u g a r s were h y d r o l y z e d b y p o r c i n e p a n c r e a s l i p a s e i n t h e p r e s e n c e o r a b s e n c e o f 10% DMF, t h e same r e g i o s e l e c t i v i t y was o b s e r v e d , a l l b e i n g hydrolyzed a t C - l p o s i t i o n p r e f e r e n t i a l l y (Table I I I ) . The p r o d u c t s were i s o l a t e d b y e x t r a c t i o n w i t h e t h y l 3

13



36


F i g u r e 6. Preparation of 2-deoxy-2-fluoro-arabinose5 - p h o s p h a t e ( u p p e r scheme) and 2 - d e o x y - 2 - D - r i b o s e - 5 p h o s p h a t e ( l o w e r scheme). U p p e r scheme: a) 1, p y r i d i u m d i c h r o m a t e . 2, NaBH /70% a q . e t h a n o l , 92% yield, b) D A S T / C H C l / p y r i d i n e , 88 % y i e l d . c) Dowex 50 ( H + ) / H 0 92% Y i e l d . d) ATP/hexokinase/phosphoenol pyruvate/pyruvate kinase, 90% y i e l d , e) P b ( 0 A c ) / H , 64% y i e l d . Lower scheme: a) P h C C l / p y r i d i n e 70 % y i e l d , b) D A S T / C H C 1 , 50% yield. c) 1.80% AcOH/Dowex 50 ( H ) ; 2. A T P / h e x o k i n a s e s y s t e m a s above, 90% y i e l d , d) P b ( O A c ) / H , 60 % y i e l d . 4

2

2

2

/

+

4

3

/

2

2

+

+

4


3.

37

ChemoenzymaticSynthesisofFluorosugars

WONGETAL.

Table I. Lipase-Catalyzed Selective Hydrolysis of D i f f e r e n t S u g a r E s t e r s a t C-6 p o s i t i o n Specific Activity

Substrate


Me a - D - g l u c o p y r a n o s i d e , t e t r a p e n t a n o a t e Me a - D - g l u c o p y r a n o s i d e , t e t r a o c t a n o a t e Me B - D - g l u c o p y r a n o s i d e , t e t r a o c t a n o a t e Me a - D - g a l a c t o p y r a n o s i d e , t e t r a p e n t a n o a t e Me a-D-mannopyranoside, t e t r a p e n t a n o a t e aD-glucose, pentaacetate B-D-glucose, p e n t a a c e t a t e Me N - A c e t y l g l u c o s a m i n e , t r i p e n t a n o a t e Me N-acetylmanosamine, t r i p e n t a n o a t e

0.,020 0,.153 0..044 0..017 0..015 0..00 0.,04b

Fluorinated Carbohydrates - American Chemical Society

Recommend Documents