Asymmetric Reactions and Processes in Chemistry - American

15 Asymmetric Synthesis Using Cofactor-Requiring Enzymes

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 5, 2018 | https://pubs.acs.org Publication Date: April 28, 1982 | doi: 10.1021/bk-1982-0185.ch015

GEORGE M. WHITESIDES, CHI-HUEY WONG, and A L F R E D POLLAK Massachusetts Institute of Technology, Department of Chemistry, Cambridge, MA 02139

The use of cofactor-requiring enzymes as catalysts for large-scale requires efficient and economical procedures for in situ regeneration of these co factors. This manuscript summarizes the procedures which are now available for cofactor preparation and regeneration. ATP can be effectively regenerated from ADP (and AMP) using acetyl phosphate and acetate kinase (and adenylate kinase), and it can be prepared inexpen sively from RNA. Use of ATP-requiring enzymes is now routine (at least as far as the ATP regeneration is concerned). The use of the nicotinamide cofactors is more difficult, because these materials decompose in solution. The best procedure for regenerating NAD(P)H from NAD(P) are those based on formate/formate dehydro genase, glucose 6-phosphate/glucose-6-phosphate dehydro genase, and ethanol/alcohol dehydrogenase/aldehyde de hydrogenase. The best procedures for regenerating NAD(P) from NAD(P)H use dioxygen/methyl viologen or ketoglutarate/glutamic dehydrogenase. +

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Although enzymes can be e f f e c t i v e c a t a l y s t s f o r enantios e l e c t i v e r e a c t i o n s , they have been r e l a t i v e l y l i t t l e used f o r t h i s purpose i n p r a c t i c a l organic s y n t h e s i s . The r e l a t i v e i n d i f f e r e n c e of s y n t h e t i c chemists t o the p o t e n t i a l of t h i s group o f c a t a l y s t s i s a consequence of a number o f circumstances. F i r s t , enzymes are u n f a m i l i a r : they r e q u i r e aqueous environments; they are prepared, c h a r a c t e r i z e d , and manipulated using s p e c i a l i z e d techniques having l i t t l e i n common with techniques used i n other areas o f s y n t h e t i c organic chemistry; and they appear to be unstable. Second, c e r t a i n g e n e r a l l y i n t e r e s t i n g c l a s s e s of enzymatic r e a c t i o n s ( i n c l u d i n g many r e a c t i o n s which form bonds between organic molecules and most r e a c t i o n s which i n v o l v e o x i d a t i o n or reduction) i n v o l v e c o f a c t o r s ; these r e a c t i o n s a r e expensive. T h i r d , the s u b s t r a t e s e l e c t i v i t y

0097-6156/82/0185-0205$05.00/0 © 1982 American Chemical Society

Eliel and Otsuka; Asymmetric Reactions and Processes in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


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of enzyme-catalyzed r e a c t i o n s o f t e n l i m i t s the g e n e r a l i t y of t h e i r a p p l i c a t i o n . Nonetheless, i n these r e a c t i o n s i n which they are a p p l i c a b l e , they can be very e f f i c i e n t c a t a l y s t s , and t h e i r a b i l i t y to c a t a l y z e r e a c t i o n s of n a t u r a l l y - o c c u r r i n g substances (which are, of course, products of and reactants i n the r e a c t i o n s which take place i n l i f e ) makes them of p a r t i c u l a r i n t e r e s t i n pharmaceutical, food, and a g r i c u l t u r a l chemistry. The research summarized i n t h i s manuscript was d i r e c t e d toward one p a r t i c u l a r problem i n enzymology: that i s , the development of techniques which would make p o s s i b l e the use of c o f a c t o r - r e q u i r i n g enzymes i n organic s y n t h e s i s . The c e n t r a l problem i n t h i s area has been one of expense. ATP costs approximately $800/mole when pur chased i n mole q u a n t i t i e s ; the costs of the nicotinamide c o f a c t o r s range from $1500/mole ( f o r NAD *) to $250,000/mole ( f o r NADPH). There are few organic r e a c t i o n s which can t o l e r a t e costs of t h i s magnitude f o r s t o i c h i o m e t r i c reagents. The s o l u t i o n to t h i s prob lem of cost i s , i n p r i n c i p l e , s t r a i g h t f o r w a r d , and has been the subject of extensive previous work. The most e f f i c i e n t way of lowering the e f f e c t i v e cost of the c o f a c t o r s i s to develop proce dures which make p o s s i b l e t h e i r regeneration from inexpensive reagents in situ (Figure 1) Among the c o n s i d e r a t i o n s which determine the usefulness of a s y n t h e t i c sequence which i n v o l v e s a c o f a c t o r - r e q u i r i n g enzymatic step a r e : 1) The character of the r e a c t i o n used f o r regeneration of the c o f a c t o r . The reagent A should be r e a d i l y a v a i l a b l e , inexpensive, and s t a b l e ; the product Β should not complicate workup; the e q u i l i b r i u m constant f o r the r e a c t i o n A + X Β + Y should l i e f a r to the r i g h t ; the enzymes used ( i f any) should have low c o s t , high s t a b i l i t y , and high s p e c i f i c a c t i v i t y . 2) The i n t r i n s i c s t a b i l i t i e s of the c o f a c t o r s X and Y under the c o n d i t i o n s of the r e a c t i o n . 3) The o r i g i n a l cost of the c o f a c t o r . 4) The o p e r a t i o n a l s i m p l i c i t y of the regeneration scheme. Here we d i v i d e the d i s c u s s i o n of approaches to c o f a c t o r r e generation i n t o three s e c t i o n s : one each f o r ATP, o x i d i z e d n i c o tinamide c o f a c t o r s (NAD " and NADP ), and reduced nicotinamide cof a c t o r s (NADH and NADPH). Most of the other c o f a c t o r s which appear i n biochemistry are e i t h e r e a s i l y regenerated or of l i t t l e importance, and we s h a l l not discuss t h e i r regeneration here. Although e i t h e r p u r e l y chemical or enzymatic procedures might be used to e f f e c t the regeneration r e a c t i o n s , i n general enzymatic procedures are s u p e r i o r . To be able to r e c y c l e the c o f a c t o r s a l a r g e number of times i t i s necessary to have high y i e l d s f o r the r e a c t i o n s which regenerate them. Thus, to have 50% of the cof a c t o r remaining a f t e r 100 c y c l e s of r e a c t i o n and regeneration, the y i e l d f o r each c y c l e must be 99.3% (100 l o g 0.993 = l o g 0.50), and f o r 1000 c y c l e s , the corresponding y i e l d must be 99.9%. This type of s e l e c t i v i t y i s most e a s i l y obtained by enzymatic c a t a l y s i s , and we have t h e r e f o r e used only enzymatic methods i n our work. 4

4


WHITESIDES E T A L .

Cofactor-Requiring

Enzymes


15.

Figure 1. General scheme for cofactor regeneration (top) and structures of adenosine and nicotinamide cofactors (bottom). Key: X, Y, cofactors; A, regenerating agent; and B, product from this reagent.


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ATP Regeneration. Most b i o c h e m i c a l r e a c t i o n s which i n v o l v e ATP as a c o f a c t o r convert i t t o ADP o r AMP; adenosine i t s e l f i s important only as a product of the s m a l l group o f r e a c t i o n s which proceed through S-adenosyl methionine. Thus, i t i s necessary to have regeneration procedures which w i l l convert both AMP and ADP t o ATP. Chemical methods f o r these phosphorylation r e a c t i o n s can be r e j e c t e d out of hand: they a r e incompatible with the enzymes which would be present i n the system as c a t a l y s t s f o r r e a c t i o n s which use the ATP, and l a c k the s p e c i f i c i t y r e q u i r e d to give high y i e l d s and h i g h t o t a l turnover numbers (TTN) f o r the ATP (TTN = moles o f product produced i n the r e a c t i o n per mole of c o f a c t o r or enzyme p r e s e n t ) . The s t a b i l i t y o f ATP i s good: the h y d r o l y s i s o f ATP at pH 6-8 i s slow compared with any s y n t h e t i c r e a c t i o n o f practical interest. The choice of phosphorylating agents which might, i n p r i n c i p l e , be used t o convert AMP or ADP t o ATP i s l i m i t e d . Table I summarizes values of AG°'for the r e a c t i o n XP + ADP Y + ATP f o r those compounds XP which are ( r e l a t i v e l y ) r e a d i l y a v a i l a b l e and exergonic with respect t o phosphorylation of ADP. Of these, PEP Table I .

Free energy o f phosphorylation o f ADP t o ATP e

XP

AG ' (kcal/mole)

Phosphoenolpyruvate

(PEP)

-7.5

Carbamyl phosphate

-5.0

A c e t y l phosphate (AcP)

-3.0

Pyrophosphate (PP.)

-0.7

i s r e l a t i v e l y expensive (although r e g e n e r a t i o n systems based on PEP have many advantages i n s i m p l i c i t y ) , carbamyl phosphate has very poor s t a b i l i t y i n s o l u t i o n , and pyrophosphate i s only a weak phosphorylating agent and r e q u i r e s enzymes which are a v a i l a b l e only w i t h d i f f i c u l t y . A c e t y l phosphate (AcP) i s a reagent which o f f e r s a p r a c t i c a l combination i n i t s c h a r a c t e r i s t i c s : i t i s prepared e a s i l y from inexpensive reagents; the enzymes i t r e q u i r e s f o r use i n ATP regeneration are commercially a v a i l a b l e and acceptably s t a b l e ; i t i s a good phosphorylating agent. In t h i s manus c r i p t we focus a t t e n t i o n on procedures f o r ATP regeneration based on AcP. The only other procedure which seems u s e f u l f o r l a b o r a t o r y - s c a l e i s that based on PEP; d e t a i l s of t h i s procedure w i l l be p u b l i s h e d elsewhere. AcP can be prepared e a s i l y by a c y l a t i o n o f phosphoric a c i d with a c e t i c anhydride or w i t h ketene, and i s o l a t e d as a f a i r l y



15.

WHITESIDES

E T AL.

Cojactor-Requiring

209

Enzymes

s t a b l e ammonium s a i t (1,2). Acetate kinase (the enzyme which c a t a l y z e s the r e a c t i o n of a c e t y l phosphate with ADP) and adenylate kinase (the enzyme which c a t a l y z e s phosphate t r a n s f e r between ATP and AMP) a r e r e a d i l y a v a i l a b l e and inexpensive. The ATP regener a t i o n schemes based on these enzymes a r e shown i n Figure 2 (3,4,5). These schemes have now been used t o prepare organic m a t e r i a l s on s c a l e s of s e v e r a l moles. An example r e l e v a n t t o asymmetric synthesis i s the g l y c e r o l k i n a s e - c a t a l y z e d phosphorylation o f g l y c e r o l (equation i ) ( 6 ) .

Η χ ^ Ο Η

H.^0H n yvn

(i) ADP

AcP

This r e a c t i o n y i e l d s e n a n t i o m e r i c a l l y pure s n - g l y c e r o l - 3 phosphate ( ( R ) - g l y c e r o l - l - p h o s p h a t e , a compound having the c o r r e c t c o n f i g u r a t i o n to serve as the b a s i s f o r the synthesis of phospho l i p i d s ) . The turnover numbers (TTN = moles product per mole c o f a c t o r ) achieved i n these syntheses (TTN - 100) have been l i m i t e d p r i m a r i l y by convenience: we normally use a r e l a t i v e l y l a r g e quantity of ATP, t o keep r e a c t i o n r a t e s h i g h . The ATP i s , how ever, e s s e n t i a l l y a l l s t i l l present at the c o n c l u s i o n of the r e a c t i o n . For l a b o r a t o r y - s c a l e s y n t h e s i s of f i n e chemicals, the methods shown i n Figure 2 represent an e f f e c t i v e s o l u t i o n t o the problem o f ATP regeneration. Synthesis. A f i n a l problem r e l a t e d t o ATP u t i l i z a t i o n i s that of o b t a i n i n g the i n i t i a l q u a n t i t y o f ATP t o be used i n the r e a c t i o n . ATP as a pure biochemical i s expensive. A m a t e r i a l s u i t a b l e f o r use i n r e c y c l i n g can be obtained from RNA (approxi mately $80/kg) by the process o u t l i n e d i n equation i i ( 7 ) .


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210


15.

WHiTESiDES E T A L .

Cojactor-Requiring

Enzymes

211

RNA


l

Nuclease Ρ,

AMP + U M P + G M P + C M P AcP

N

Acetate

kinase

Α ,CN

2

•OiO

ATP

+

CONH

HO

Ac

OH

N A D Figure 3. Combined chemical and enzymatic synthesis of NAD* (27). Key: AcK, acetate kinase; AdK, adenylate kinase; NAD-PP, NAD pyrophosphorylase; PPase, pyrophosphorylase: NADK, NAD kinase; r-5-Ρ, ribose-5-phosphate; rA-5-P, rihosylamine-5-phosphate; NMN, nicotinamide mononucleotide; AcP, acetyl phosphate; PP%, pyrophosphate; and NDC, N J2,4-dinitrophenyl)-3-carbamoylpyridinium chloride. t


2

15.

WHiTESiDES

E T AL.

Cofactor-Requiring

217

Enzymes

ribose-5-phosphate t o NAD"* i n t h i s procedure i s approximately 60% on s m a l l s c a l e ; that from AMP t o NAIf* v i a ATP i s e s s e n t i a l l y q u a n t i t a t i v e . The procedure i n v o l v e s only one i s o l a t i o n (that of ribose-5-phosphate). The s o l u t i o n c o n t a i n i n g the NAD can be used d i r e c t l y f o r c o f a c t o r r e c y c l i n g : whatever components a r e present as i m p u r i t i e s i n t h i s s o l u t i o n apparently do not i n a c t i v a t e o r i n h i b i t enzymes. T h i s procedure ( a f t e r development) or some r e l a t e d procedure may provide the best hope f o r reducing the cost of the nicotinamide c o f a c t o r s .


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Acknowlegement s Names of many of our coworkers who c o n t r i b u t e d t o t h i s work are l i s t e d i n the r e f e r e n c e s . The research was supported by the N a t i o n a l I n s t i t u t e s o f H e a l t h , Grant GM 26543, and by the Monsanto Corporation.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Whitesides, G. M.; Siegal, M.; Garrett, P. J. Org. Chem. 1975, 40, 2516-9. Lewis, J. M.; Haynie, S. L . ; Whitesides, G. M. J. Org. Chem. 1979, 44, 864-5. Pollak, Α.; Baughn, R. L . ; Whitesides, G. M. J. Am. Chem. Soc. 1977, 99, 2366-7. Shih, Y. S.; Whitesides, G. M. J. Org. Chem. 1977, 42, 4165-6. Baughn, R. L . ; Adalsteinsson, O; Whitesides, G. M. J. Am. Chem. Soc. 1978, 100, 304-6. Rios-Mercadillo, V. M.; Whitesides, G. M. J . Am. Chem. Soc. 1979, 101, 5828-9. Leuchs, H. J.; Lewis, J. M.; Rios-Mercadillo, V. M.; Whitesides, G. M. J. Am. Chem. Soc. 1979, 101, 5829-30. Johnson, S. L . ; Morrison, D. L. Biochemistry 1970, 9, 1460-70. Johnson, S. L.; Tuazon, P. T. Biochemistry 1977, 16, 1175-83. Wong, C.-H.; Whitesides, G. M. J. Am. Chem. Soc. 1981, 103, 4890-99. Wong, C.-H.; McCurry, S. D.; Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 7938-9. Shaked, Z.; Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 7104. Schutte, H.; Flossdorf, J . ; Sahm, H.; Kula, M. R. Eur. J. Biochem. 1976, 62, 151-60. Levy, H. R.; Adv. Enzymol. 1979, 48, 141-3. Jones, J. B.; Beck, J. F. "Application of Biochemical Systems in Organic Chemistry" Jones, J. B.; Perlman, D.; Sih, C. J. Ed.; Wiley-Interscience, New York, 1976, p 107-401. Wang, S. S.; King, C. K. Adv. Biochem. Eng. 1979, 12, 119-46.


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17. 18. 19. 20.


21. 22. 23. 24. 25. 26. 27.

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Chemical and Engineering News, Feb. 25, 1974, p 19. Wong, C. H.; Pollak, Α.; Whitesides, G. M. J. Am. Chem. Soc. submitted. Wratten, C. C.; Cleland, W. W. Biochemistry 1963, 2, 935-41. Lamed, R. J.; Keinan, E . ; Zeikus, J. G. Enzyme. Microb. Technol. 1981, 3, 144-8. Wong, C. H.; Lacy, D.; Orme-Johnson, W. H.; Whitesides, G. M. J. Am. Chem. Soc. in press. DiCosimo, R.; Wong, C. H.; Lacy, D.; Whitesides, G. M. J. Org. Chem. in press. Shaked, Z.; Barber, J. J.; Whitesides, G. M. J. Org. Chem. in press. Jones, J. B. "Enzymic and Non-enzymic Catalysis", Dunill, P.; Wiseman, Α.; Blakebrough, N. Ed.,; Ellis Horwood, Ltd.; Chichester, England, 1980, p 54-81. Nakayama, K.; Sato, Z.; Tanaka, H. Kinoshita, S. Agr. Biol. Chem. 1968, 32, 1331-6. Sakai, T.; Uchida, T.; Chibata, I. Agr. Biol. Chem. 1973, 37, 1041-8. Walt, D. R.; Rios-Mercadillo, V. M.; Auge, J.; Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 7805-6.

RECEIVED December 14, 1981.


Asymmetric Reactions and Processes in Chemistry - American

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