Biochemistry Involving Carbon-Fluorine Bonds

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Downloaded by GEORGETOWN UNIV on August 25, 2015 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0028.ch004

Thymidylate Synthetase: Interaction with 5-Fluoro and 5-Trifluoromethyl-2'-Deoxyuridylic A c i d DANIEL V. SANTI, ALFONSO L. POGOLOTTI, THOMAS L. JAMES, YUSUKE WATAYA, KATHRYN M. IVANETICH, and STELLA S. M. LAM Department of Biochemistry and Biophysics, and Department of Pharmaceutical Chemistry, University of California, San Francisco, Calif. 94143

Thymidylate synthetase catalyzes the reductive methylation of 2'-deoxyuridylate (dUMP) to thymidylate (TMP) with the concomitant conversion of 5, 10-methylene tetrahydrofolate (CH FAH ) to 7, 8-dihydrofolate (FAH ) as depicted in F i gure 1 (for a recent review, see reference 1). In this process the hydrogen at C-6 of FAH is directly transferred to the methyl group of TMP (2). One of our objectives over the past few years has been to establish the mechanism of catalysis of thymidylate synthetase. Extensive investigations of chemical counterparts (3-6) have in dicated that the reaction is initiated by attack of a nucleophile at the 6-position of dUMP and that many, if not all, reactions along the pathway are facilitated by analogous nucleophilic catalysis. The proposed mechanism of this enzyme, as derived from investigations of chemical models is illustrated in Figure 2. It is proposed that the reaction is initiated by attack of a nucleophilic group of the enzyme to the 6-position of dUMP. In this manner, the 5-position of dUMP could be made sufficient ly nucleophilic (viz I, Figure 2) to react with CH FAH or an equivalent reactive species of formaldehyde. Thus, the initial condensation product between dUMP and CH F A H is now generally accepted (1, 7) to be one which is covalently bound to the enzyme and saturated across the 5, 6-double bond of dUMP (II). Proton abstraction from II would give the intermediate 2

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enolate III. As with the chemical models, III should readily undergo a β-elimination to produce the highly reactive exocyclic methylene intermediate IV and FAH , bound to the enzyme in close proximity. Intermolecular hydride transfer from FAH to IV would yield dTMP, FAH , and the native enzyme. It should be emphasized that all of the aforementioned reactions and inter mediates have direct chemical counterparts, and are in complete accord with all available biochemical data. With the availability of a stable enzyme from an amethop terin resistant strain of Lactobacillus casei (8, 9) and facile methods for its purification (9-11), we undertook studies which 4

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57 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Figure 2. Suggested sequence for the thymidylate synthetase reaction. All pyrimidine structures have a l-(5-phospho-2'-deoxyribosyl) substituent and R = CH NHC H COGlu. t

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might p r o v i d e d i r e c t s u p p o r t f o r p r o p o s a l s w h i c h w e r e b a s e d on the a f o r e m e n t i o n e d n o n e n z y m i c m o d e l s . A l t h o u g h a n u m b e r of d i r e c t i o n s h a v e b e e n p u r s u e d t o w a r d s t h i s o b j e c t i v e , the f o l l o w i n g s u m m a r i z e s o u r i n v e s t i g a t i o n s of the i n t e r a c t i o n of t h y m i d y l a t e synthetase w i t h 5 - f l u o r o - 2 ' - d e o x y u r i d y l a t e ( F d U M P ) and 5 - t r i f l u o r o m e t h y l - 2 - d e o x y u r i d y l a t e ( C R d U M P ) . Studies of these two f l u o r i n a t e d n u c l e o t i d e s have p r o v i d e d c o n v i n c i n g e v i d e n c e for the m e c h a n i s m of this e n z y m e ; i n a d d i t i o n , they h a v e p r o v i d e d i n s i g h t into how these d r u g s act, and how the r e a c t i v i t y of f l u o r i n a t e d m o l e c u l e s m i g h t be u t i l i z e d i n the d e s i g n of e n z y m e i n h i b i t o r s . We e m p h a s i z e that a n u m b e r of other l a b o r a t o r i e s have been engaged i n s i m i l a r i n v e s t i g a t i o n s , and r e g r e t that s p a c e does not p e r m i t c o m p l e t e c i t a t i o n of a l l the e x c e l l e n t s t u d i e s p e r f o r m e d i n this a r e a .


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5 - F l u o r o - 2 ' - d e o x y u r i d y l a t e ( F d U M P ) . It h a s been known for s o m e t i m e ( 12, 13) that F d U M P is an e x t r e m e l y potent i n h i b i t o r of t h y m i d y l a t e s y n t h e t a s e , but the n a t u r e of i n h i b i t i o n has b e e n the topic of c o n s i d e r a b l e c o n t r o v e r s y (_1_4). S i n c e the 6 - p o s i t i o n of 1 - s u b s t i t u t e d 5 - f l u o r o u r a c i l s i s quite s u s c e p t i b l e t o w a r d n u c l e o p h i l i c attack ( 15- 17), we s u s p e c t e d that F d U M P m i g h t e x e r t its i n h i b i t o r y effect by r e a c t i o n w i t h the p r o p o s e d n u c l e o p h i l i c c a t a l y s t of t h y m i d y l a t e s y n t h e t a s e . Studies f r o m this (18, 19) and o t h e r (20, 21 ) l a b o r a t o r i e s h a v e s i n c e d e m o n s t r a t e d this to be the c a s e . A s i m p l i f i e d d e p i c t i o n of the i n t e r a c t i o n s of F d U M P and C H ^ F A H ^ is g i v e n below i n F i g u r e 3.

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U s i n g the i s o t o p e t r a p p i n g m e t h o d (2_2), we have found that the two l i g a n d s , F d U M P and C H ^ F A H ^ , i n t e r a c t w i t h the e n z y m e i n a r a n d o m f a s h i o n , a n d that f o r m a t i o n of the i n i t i a l t e r n a r y c o m p l e x ( E ^ F d U M P ^ C H ^ F A H ^ ) i s at l e a s t p a r t i a l l y r a t e d e t e r mining. F r o m e q u i l i b r i u m b i n d i n g techniques we h a v e a s c e r tained that the d i s s o c i a t i o n constantes of both b i n a r y c o m p l e x e s (K^ a n d K ^ ) a r e a p p r o x i m a t e l y 10" M . T h e f i r s t t e r n a r y c o m plex which is f o r m e d with F d U M P , C H ^ F A H . and enzyme is d e p i c t e d as E - F d U M P ' C H ^ F A H ^ a n d does not i n v o l v e c o v a l e n t bonds. Interestingly, analogous r e v e r s i b l e t e r n a r y complexes m a y be f o r m e d u s i n g a n a l o g s of the c o f a c t o r . T h r o u g h s t u d i e s of the i n t e r a c t i o n of F d U M P and a n a l o g s of C H ^ F A H ^ we h a v e a s c e r t a i n e d that t h e r e i s a s t r i k i n g s y n e r g i s m i n b i n d i n g of l i g a n d s to this p r o t e i n . T h a t i s , the a f f i n i t y of e i t h e r l i g a n d f o r the cognate b i n a r y c o m p l e x i s c a . two o r d e r s of m a g n i t u d e g r e a t e r than the a f f i n i t y f o r the f r e e e n z y m e . W i t h m a n y a n a l o g s of C H ^ F A H ^ , this t e r n a r y c o m p l e x i s s u f f i c i e n t l y stable that i t m a y be p h y s i c a l l y s e p a r a t e d f r o m o t h e r s p e c i e s (J_9). T h e s e t e r n a r y c o m p l e x e s show i n t e r e s t i n g u l t r a v i o l e t d i f f e r e n c e s p e c t r a w h i c h m a y be u s e d f o r t h e i r q u a n t i t a t i o n a n d characterization. Shown i n F i g u r e 4 a r e d i f f e r e n c e s p e c t r a o b tained w i t h C H 2 F A H ^ a n d 5, 8 - d e a z a f o l i c a c i d ; s i m i l a r d i f f e r e n c e s p e c t r a h a v e a l s o b e e n o b t a i n e d w i t h a n u m b e r of other a n a l o g s of f o l i c a c i d . C h a r a c t e r i s t i c of these d i f f e r e n c e s p e c t r a i s a peak at c a . 330 n m a n d , u s u a l l y , a t r o u g h at c a . 290 n m . A l t h o u g h the e x a c t r e a s o n f o r the s p e c t r a l changes w h i c h o c c u r upon f o r m a t i o n of the r e v e r s i b l e t e r n a r y c o m p l e x e s i s y e t u n k n o w n , we s u g g e s t that they r e s u l t f r o m p e r t u r b a t i o n s of the p a m i n o b e n z o y l g l u t a m a t e m o i e t y of the c o f a c t o r a n a l o g s w h i c h r e sult f r o m t h e i r e n v i r o n m e n t w i t h i n the t e r n a r y c o m p l e x . T h i s p e r t u r b a t i o n i s b e l i e v e d to be a m a n i f e s t a t i o n of a c o n f o r m a t i o n a l change w h i c h i s r e l a t e d to the a f o r e m e n t i o n e d s y n e r g i s m i n b i n d i n g of the two l i g a n d s . T h e d i f f e r e n c e s p e c t r u m of the E » F d U M P C H F A H c o m p l e x ( F i g u r e 4) i s s i m i l a r to those o b s e r v e d f o r the c o f a c t o r a n a l o g s , s u g g e s t i n g that s i m i l a r c h a n ges i n e n v i r o n m e n t o c c u r w i t h the n a t u r a l c o f a c t o r , C H ^ F A H . . T h e r e i s one s t r i k i n g d i f f e r e n c e i n that t h e r e i s a l o s s of d i f f e r e n t i a l a b s o r b a n c e at 269 n m i n the c o m p l e x f o r m e d w i t h C H ^ F A H ^ (18, 1 9 ) w h i c h w e h a v e not o b s e r v e d i n c o m p l e x e s f o r m e d w i t h c o f a c t o r a n a l o g s ; the r e a s o n f o r this w i l l b e c o m e a p p a r e n t i n the e n s u i n g d i s c u s s i o n . e

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The complex f o r m e d with thymidylate synthetase, F d U M P , and the n a t u r a l c o f a c t o r C H J A H . , h a s b e e n e x t e n s i v e l y i n v e s t i g a t e d . T h i s c o m p l e x i s e x t r e m e l y tight a n d m a y r e a d i l y be i s o l a t e d b y a v a r i e t y of t e c h n i q u e s (18, 1 9 , 21, 23, 24). Using r a d i o a c t i v e F d U M P and C H , F A H ^ , the e n z y m e h a s b e e n t i t r a t e d a n d shown to p o s s e s s two F d U M P a n d two c o f a c t o r b i n d i n g s i t e s p e r m o l e ( 1 9 ) . T h e s t o i c h i o m e t r y of b i n d i n g h a s b e e n v e r i f i e d i n a n u m b e r of l a b o r a t o r i e s b y a v a r i e t y of m e thods ( 11, 25, 26). T h i s i s i n a c c o r d w i t h the e a r l i e r f i n d i n g


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that t h y m i d y l a t e s y n t h e t a s e f r o m L . c a s e i has two a p p a r e n t l y i d e n t i c a l subunits of M W 35, 000 e a c h (9, 27). Studies of the r a t e of a s s o c i a t i o n of F d U M P w i t h the E - C H - , F A H . c o m p l e x (k ) and its d i s s o c i a t i o n (k ) have 2 4 ^ on off ' v

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off a l l o w e d us t o ^ c ^ l c u l a t e the d i s s o c i a t i o n c o n s t a n t of the c o m p l e x to be c a . 10 M . T h i s p r o v i d e s a n e x p l a n a t i o n f o r the d i s c r e p a n c i e s i n R e v a l u e s r e p o r t e d f o r F d U M P in the l i t e r a t u r e ; n a m e l y , p r e v i o u s e x p e r i m e n t s w e r e u s i n g c o n c e n t r a t i o n s of e n z y m e h i g h e r than the and w e r e i n c o n c e n t r a t i o n r a n g e s w h e r e F d U M P was b e h a v i n g as a s t o i c h i o m e t r i c i n h i b i t o r . The kinetically determined i s a p p r o x i m a t e l y 10 - f o l d l o w e r than that f o r the b i n a r y c o m p l e x ; i n e f f e c t , the p r e s e n c e of the c o f a c t o r i n c r e a s e s the a f f i n i t y of the e n z y m e f o r F d U M P b y o v e r 10 k c a l / m o l i n b i n d i n g e n e r g y . C l e a r l y , a m o s t pertinent fea t u r e of the i n t e r a c t i o n of F d U M P and t h y m i d y l a t e s y n t h e t a s e i n v o l v e s changes w h i c h o c c u r w i t h i n the bound t e r n a r y c o m p l e x . S e v e r a l l i n e s of e v i d e n c e d e m o n s t r a t e r a t h e r c o n c l u s i v e l y that a c o v a l e n t bond i s f o r m e d b e t w e e n F d U M P and t h y m i d y l a t e s y n t h e t a s e w i t h i n the c o m p l e x , (a) T h e E - ^ l F d U M P ^ C r L F A H ^ c o m p l e x m a y be t r e a t e d w i t h a n u m b e r of p r o t e i n d é n a t u r a n t s ( u r e a , g u a n i d i n e h y d r o c h l o r i d e , e t c . ) without a p p a r e n t l o s s of p r o t e i n - b o u n d r a d i o a c t i v i t y . W i t h few e x c e p t i o n s , s u c h t r e a t m e n t i s s u f f i c i e n t to d i s r u p t n o n c o v a l e n t i n t e r a c tions b e t w e e n low m o l e c u l a r w e i g h t l i g a n d s and t h e i r p r o t e i n receptors, (b) U p o n f o r m a t i o n of the c o m p l e x , t h e r e i s a d e c r e a s e of a b s o r b a n c e at 269 n m w h i c h c o r r e s p o n d s to s t o i c h i o m e t r i c l o s s of the p y r i m i d i n e c h r o m o p h o r e of F d U M P . This res u l t s t r o n g l y s u g g e s t s that the 5, 6 - d o u b l e bond of the p y r i m i dine is s a t u r a t e d i n the bound c o m p l e x , (c) T h e r a t e of d i s s o c i a t i o n of [6- H ] F d U M P f r o m the c o m p l e x shows a s e c o n d a r y t r i t i u m i s o t o p e effect ( k ^ / k ^ ) of 1.23. T h i s would c o r r e s p o n d to k £ j / k j 3 1. 15 and c l e a r l y α errions traite s that the 6 - c a r b o n of the h e t e r o c y c l e u n d e r g o e s sp to sp r e h y b r i d i z a t i o n d u r i n g the p r o c e s s as r e q u i r e d i f the 5, 6 - d o u b l e bond of F d U M P i s s a t u r a t e d i n the c o m p l e x , (d) P r o t e o l y t i c d i g e s t i o n of the c o m p l e x y i e l d s a p e p t i d e w h i c h i s c o v a l e n t l y bound to both F d U M P and C H ^ F A H ^ . T h e u l t r a v i o l e t and f l u o r e s c e n c e s p e c t r a of this p e p t i d e a r e c h a r a c t e r i s t i c of 5 - a l k y l t e t r a h y d r o f o l a t e s a n d , as w i t h the n a t i v e c o m p l e x , t h e r e i s no e v i d e n c e of u l t r a v i o l e t a b s o r p t i o n of the F d U M P c h r o m o p h o r e .


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F r o m t h e s e l i n e s of e v i d e n c e , together w i t h i n f o r m a t i o n g a t h e r e d f r o m m o d e l c h e m i c a l c o u n t e r p a r t s , the s t r u c t u r e of the e n z y m e ' F d U M P ' C H ^ F A H ^ c o m p l e x i s c u r r e n t l y b e l i e v e d to be as d e p i c t e d i n F i g u r e 5. H e r e , a n u c l e o p h i l e of the e n z y m e has a d d e d to the 6 - p o s i t i o n of F d U M P , and the 5 - p o s i t i o n of the p y r i m i d i n e i s c o u p l e d to the 5 - p o s i t i o n of F A H . v i a the m e t h y l e n e


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WAVELENGTH (nm) Figure 4. Dashed line: Ultraviolet difference spectra of FdUMP, CH FAH and thymidylate synthetase vs. CHgFAHi and thymidylate synthetase. Solid line: FdUMP, 5,8-deazofohte and thymidylate synthetase vs. enzyme and 5,8-deazafolate. t

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Structure of the FdUMP · CH · F A t f j thymidylate synthetase ternary complex where X represents a nucleophile of one of the enzyme amino acids t


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g r o u p of the c o f a c t o r . A s i m i l a r structure was proposed f r o m e v i d e n c e o b t a i n e d i n d e p e n d e n t l y i n another l a b o r a t o r y (20). R e f e r r i n g to F i g u r e 5, it i s noted that the a s s i g n e d s t r u c ture f o r the E F d U M P * C H > F A r J c o m p l e x i s a n a l o g o u s to one of the p r o p o s e d s t e a d y state i n t e r m e d i a t e s of the n o r m a l e n z y m i c r e a c t i o n ( v i z II, F i g u r e 2). T h e y d i f f e r i n that II p o s s e s s e s a p r o t o n at the 5 - p o s i t i o n of the n u c l e o t i d e w h i c h i s a b s t r a c ted i n a s u b s e q u e n t s t e p , w h e r e a s the E ' F d U M P - C H ^ F A H ^ c o m p l e x ( F i g u r e 5) p o s s e s s e s a s t a b l e f l u o r i n e at the c o r r e s p o n d i n g position. T h u s , i t a p p e a r s that F d U M P behaves as a ' q u a s i substrate f o r this r e a c t i o n . T h a t i s , it e n t e r s into the c a t a l y t i c r e a c t i o n as d e p i c t e d f o r the s u b s t r a t e d U M P i n F i g u r e 2 up to the p o i n t w h e r e a n i n t e r m e d i a t e i s f o r m e d w h i c h c a n p r o c e e d no f u r t h e r ; i n effect, a c o m p l e x i s t r a p p e d w h i c h r e s e m b l e s a steady state i n t e r m e d i a t e ( v i z II, F i g u r e 2) of the n o r m a l catalytic reaction. e

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W e h a v e r e c e n t l y o b t a i n e d the f l u o r i n e - 1 9 n m r s p e c t r u m of a n FdUMP* C r L F A H . * peptide o b t a i n e d u p o n p r o t e o l y t i c d i g e s t i o n of the F O U M P M C H ^ A H ^ • t h y m i c j v l a t e s y n t h e t a s e c o m p l e x . A s shown i n F i g u r e 6, the 94 M H z F s p e c t r u m c o n s i s t s o f a doublet of t r i p l e t s l o c a t e d 87. 2 p p m u p f i e l d of the e x t e r n a l r e f e r e n c e / t r i f l u o r o a c e t i c a c i d . O u r c u r r e n t i n t e r p r e t a t i o n of this s p e c t r u m i s as f o l l o w s : T h e d o u b l e t i s c a u s e d b y s p l i t t i n g of the F r e s o n a n c e b y the p r o t o n at the 6 - p o s i t i o n of the u r a c i l r i n g (H . ) w i t h a c o u p l i n g constant ^ of 32. 5 H z . E a c h c o m p o n e n t of m e d o u b l e t i s s p l i t f u r t h e r into a t r i p l e t ( i n t e n s i t y r a t i o (1:2:1) c a u s e d b y c o u p l i n g of the f l u o r i n e w i t h the a d j a c e n t m e t h y l e n e p r o t o n s ( H ^ ) of the c o f a c t o r w i t h the m a g n i t u d e of the c o u p l i n g constant b e i n g Λ 9 . 2 H z . T h e i n n e r m o s t l i n e s of the t r i p l e t s o v e r l a p s o the F r e s o n a n c e a p p e a r s to be a quintet w i t h i n t e n s i t y r a t i o 1:2:2:2:1. It h a s b e e n w e l l e s t a b l i s h e d that the t r i g o n a l g e o m e t r y of the c a r b o n y l a t o m s i n u r a c i l d e r i v a t i v e s s a t u r a t e d a c r o s s the 5, 6 - d o u b l e bond r e s u l t s i n a h a l f - c h a i r c o n f o r m a t i o n w i t h s u b stituents o n c a r b o n a t o m s 5 a n d 6 s t a g g e r e d (28-30) as c o m m o n l y fowjid i n c y c l o h e x a n e . The F spectrum, in conjunction with p r e v i o u s l y reported u l t r a v i o l e t and f l u o r e s c e n c e s p e c t r a l data (3J.), of the F d U M P » C H ^ F A H . * peptide y i e l d s d e f i n i t i v e e v i d e n c e f o r i t s s t r u c t u r e . T h e d o u b l e t of t r i p l e t s i m p l i e s that the f l u o r i n e - b o n d e d c a r b o n i s f l a n k e d b y C H a n d C H g r o u p s ( i . e. C H C F C P ^ ) . T h e C H , of c o u r s e , o c c u r s at the 6 - p o s i t i o n of the n u c l e o t i d e w h i c h i s a t t a c h e d to the n u c l e o p h i l e of the e n z y m e . T h e m o s t l o g i c a l a s s i g n m e n t f o r the C H - , i s the b r i d g i n g g r o u p between the n u c l e o t i d e and c o f a c t o r a s d e p i c t e d f o r the n a t i v e c o m p l e x i n F i g u r e 5. B a s e d o n the s t a b i l i t y of the F d U M P ' C r ^ F A H . » peptide i n the a b s e n c e o f a n t i o x i d a n t s , we h a v e s u r m i s e d that the C F ^ g r o u p b r i d g e s the n u c l e o t i d e to the 5 - a n d n o t to the 1 0 - n i t r o g e n o f the cofactor. It i s p o s s i b l e to a s s i g n the s t e r e o c h e m i s t r y of the 2


BIOCHEMISTRY

64


19

Figure 6.

F

INVOLVING

CARBON-FLUORINE

BONDS

NMR

94 MHz

Fluorine-19 nmr spectrum

of the

FdUMP

· CH FAH t

k

· peptide

substituents w h i c h have a d d e d a c r o s s the 5, 6 - d o u b l e bond of F d U M P to g i v e the t e r n a r y c o m p l e x by c o m p a r i n g the o b s e r v e d c o u p l i n g constants w i t h those f r o m e x t e n s i v e l y s t u d i e d m o d e l s . A s shown i n F i g u r e 7, the 5 - f l u o r o and 6 - h y d r o g e n of the F d U M P * C H ^ F A H ^ * peptide a r e p r o p o s e d to be i n a t r a n s p s e u d o axial conformation. T h e e n z y m e n u c l e o p h i l e and c o f a c t o r a r e t h e r e f o r e trans_ p s e u d ο e q u a t o r i a l . T h e a d d i t i o n of a n u c l e o p h i l e of t h y m i d y l a t e s y n t h e t a s e to the 6 - p o s i t i o n of d U M P is a p r i m a r y event i n the e n z y m e - c a t a l y z e d r e a c t i o n ( F i g u r e 8). T h e r e s u l t a n t c a r b a n i o n (1) r e a c t s w i t h C H , F A H ^ to p r o d u c e a n i n t e r m e d i a t e w i t h a s t r u c t u r e analogous to tnat of the t e r n a r y c o m p l e x f o r m e d w i t h F d U M P and the c o f a c t o r (2, 3). A b s t r a c t i o n of the 5 - h y d r o g e n f o l l o w e d by a s e r i e s of steps i n v o l v i n g r e d u c t i o n of the one c a r b o n u n i t a n d e l i m i n a t i o n of the n u c l e o p h i l e r e s u l t s i n the o b s e r v e d p r o ducts of the r e a c t i o n . T h e s e steps h a v e b e e n p r e v i o u s l y d e p i c ted i n d e t a i l i n F i g u r e 2. W i t h the l o g i c a l a s s u m p t i o n that the n o r m a l e n z y m e - c a t a l y z e d r e a c t i o n o c c u r s i n a m a n n e r s i m i l a r to the f o r m a t i o n of the t h y m i d y l a t e s y n t h e t a s e « F d U M P · C r | F A H ^ c o m p l e x , s e v e r a l d e t a i l s c o n c e r n i n g the m e c h a n i s m of the n o r m a l e n z y m i c r e a c t i o n m a y be i n f e r r e d . F i r s t , the o v e r a l l s t e r e o c h e m i c a l pathway of the e n z y m e c a t a l y z e d r e a c t i o n m a y be d e d u c e d . T h e a d d i t i o n of the n u c l e o p h i l e and c o f a c t o r a c r o s s the 5, 6 - d o u b l e bond m u s t o c c u r i n a t r a n s f a s h i o n . C o n s e q u e n t l y , the s u b s e q u e n t e l i m i n a t i o n of the 5 - h y d r o g e n and the e n z y m i c n u c l e o p h i l e m u s t o c c u r as a c i s elimination .


SANTi E T A L .

Thymidyhte

Synthetase

R

Figure 7. Stereochemical projection of the FdUMP · CH FAH · peptide as determined hy its F nmr spectrum; R = 5-phospho-2'deoxyribosyl


t

h

19

Figure 8



66

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

S e c o n d , the s t e r e o c h e m i s t r y c o n c e r n i n g the t r a n s i e n t i n t e r m e d i a t e s shown i n F i g u r e 8 m a y be i n f e r r e d . T h e m e c h a n i s tic d e t a i l s d i s c u s s e d below f o l l o w f r o m the p r i n c i p l e that a g r o u p r e a c t i n g w i t h the π - s y s t e m of the u r a c i l h e t e r o c y c l e ap p r o a c h e s a p p r o x i m a t e l y p e r p e n d i c u l a r to the plane of the r i n g ; by m i c r o s c o p i c r e v e r s i b i l i t y , a s i m i l a r o r i e n t a t i o n i s r e q u i r e d w h e n a g r o u p d e p a r t s to r e f o r m the π - s y s t e m . T h u s , the i n i t i a l attack of the n u c l e o p h i l e of the e n z y m e at the e l e c t r o p h i l i c 6 - c a r b o n of d U M P s h o u l d be p e r p e n d i c u l a r to the plane of the heterocycle. T h e r e s u l t a n t c a r b a n i o n (1) w i l l be d e l ^ c a l i z e d throughout the c a r b o n y l g r o u p s and w i l l be h i g h i n sp c h a r a c ter. T h e a p p r o a c h of C H ^ F A H ^ to the 5 - p o s i t i o n w o u l d ^ p e r p e n d i c u l a r to the plane of the r i n g a n d , b a s e d on the F data p r e s e n t e d a b o v e , t r a n s to the n u c l e o p h i l e attached to the 6 - p o s i t i o n . A s a r e s u l t , as shown i n s t r u c t u r e 2, the c o f a c t o r w o u l d e x i s t i n a p s e u d o a x i a l p o s i t i o n , and the 5 - h y d r o g e n w o u l d be p s e u d o e q u a t o r i a l . F o r the s u b s e q u e n t e l i m i n a t i o n r e a c t i o n , the p r o t o n f r o m the 5 - p o s i t i o n m u s t be i n the p s e u d o a x i a l p o s i t i o n (3) p r i o r to its a b s t r a c t i o n to f o r m the c a r b a n i o n (4). This mechanistic interpretation necessitates a previously u n r e c o g n i z e d c o n f o r m a t i o n a l c h a n g e , w h i c h o c c u r s a f t e r a d d i t i o n of the c o f a c t o r but b e f o r e a b s t r a c t i o n of the 5 - p r o t o n ; and r e s u l t s i n r i n g i n v e r s i o n about the 5 - a n d 6 - p o s i t i o n s of the n u c l e o t i d e 19

i n t e r m e d i a t e s {i.e. 2-*\3). T h e r e s u l t s of F n m r studies d e s c r i b e d h e r e a r e p r e l i m i n a r y ; a d e t a i l e d n m r study of the i n t e r a c t i o n of F d U M P w i t h t h y m i d y l a t e synthetase w i l l be p u b lished elsewhere. 5 - T r i f l u o r o m e t h y l - 2 ' - d e o x y u r i d y l a t e (C ξ d U M P ) . CEdUMP i s a potent i n h i b i t o r of t h y m i d y l a t e s y n t h e t a s e . R e y e s aria H e i d e l b e r g e r have r e p o r t e d that u p o n p r e i n c u b a t i o n C E ^ d U M P c a u s e s i r r e v e r s i b l e i n h i b i t i o n of t h y m i d y l a t e synthetase f r o m E h r l i c h A c s i t e s c e l l s (32). B a s e d on the o b s e r v a t i o n that t r i f l u o r o m e t h y l u r a c i l ( C F ~ U ) a c y l a t e s a m i n e s i n aqueous media to give u r a c i l - 5 - c a r b o x a m i d e s (33), it was s u g g e s t e d that the i r r e v e r s i b l e i n a c t i v a t i o n of t h y m i d y l a t e synthetase m i g h t r e s u l t f r o m a s i m i l a r a c y l a t i o n of a n a m i n o g r o u p at o r n e a r the a c t i v e site of the e n z y m e as shown i n F i g u r e 9 (32). A q u e s t i o n that a r o s e i s why the tr i f l u o r o m e t h y l g r o u p at the 5 - p o s i t i o n of u r a c i l d e r i v a t i v e s s h o u l d be at a l l s u s c e p t i b l e to these r e a c t i o n s . T h e c a r b o n - f l u o r i n e bond i s quite s t r o n g (34) and a n outstanding c h a r a c t e r i s t i c of m o s t t r i f l u o r o m e t h y l groups is their unusual r e s i s t a n c e toward c h e m i c a l d e g r a d a t i o n . A s r e l e v a n t e x a m p l e s , it is noted that b e n z o t r i f l u o r i d e s , d e r i v a t i v e s of 6 - t r i f l u o r o m e t h y l u r a c i l s (35), d e r i v a t i v e s of 2t r i f l u o r o m e t h y l - 4 - o x o p y r i m i d i n e s (36) and 5 - t r i f l u o r o m e thy Ι ό - a z a u r a c i l (37) a r e quite stable t o w a r d h y d r o l y t i c r e a c t i o n s . In c o n t r a s t , U is r a p i d l y c o n v e r t e d into 5 - c a r b o x y u r a c i l ( C U ) (33 ) i n b a s i c m e d i a a n d , although s o m e w h a t s l o w e r , n u c l e o s i d e s of C F ^ U a r e c o n v e r t e d into the c o r r e s p o n d i n g


4.

SANTi E T A L .

Thymidylate

Synthetase


Figure 9

n u c l e o s i d e s of C U (38-40). In v i v o , the m e t a b o l i s m of C F ^ U and C F g d U R p r o v i d e s C U and not the n o r m a l p r o d u c t s expected f r o m p y r i m i d i n e s (41). A n u m b e r of other c o m p o u n d s have been r e p o r t e d to h a v e r e a c t i v e C - F g r o u p s (see r e f e r e n c e 42). T h e r e a c t i v i t y of C - F bonds i n m o s t c a s e s has b e e n a t t r i b u t e d to h y p e r c o n j u g a t i v e e f f e c t s (43, 44), h y d r o g e n bonding effects (44, 45), and d i r e c t d i s p l a c e m e n t (S.^2) r e a c t i o n s (46). M o d e l r e a c t i o n s i n v o l v i n g the h y d r o l y s i s o f c o m p o u n d s p o s s e s s i n g C - F bonds w e r e e x a m i n e d in this l a b o t a t o r y i n an a t t e m p t to u n d e r s t a n d the u n d e r l y i n g f e a t a r e s w h i c h r e s u l t e d i n the r e a c t i v i t y of s o m e of these m o l e c u l e s (42). F r o m s u c h s t u d i e s we p r o p o s e d that C - F bond l a b i l i z a t i o n u s u a l l y i n v o l v e s one of s e v e r a l g e n e r a l m e c h a n i s m s , (a) A s d e p i c t e d i n F i g u r e 10a, p r o t o n r e m o v a l i s at an a t o m a to the c a r b o n b e a r i n g the f l u o r i n e a t o m w i t h the r e s u l t a n t negative c h a r g e , e i t h e r i n a s t e p w i s e o r c o n c e r t e d m a n n e r , a i d i n g i n the f o r m a t i o n of an i n t e r m e d i a t e (fluoro) a l k e n e . D e p e n d i n g on the s t a b i l i t y of the a l k e n e , it m a y o r m a y not react with solvent, (b) T h e p r o t o n m a y be s i t u a t e d on a n a t o m s u c h that the n e g a t i v e c h a r g e r e s u l t i n g f r o m the i o n i z a t i o n of the p r o t o n can e x e r t its i n f l u e n c e t h r o u g h an extended π - s y s t e m ( F i g u r e 10b). (c) W h e n the c o m p o u n d i s an a l l y l i c f l u o r i d e i n c a pable of u n d e r g o i n g either of the m e c h a n i s m s d e s c r i b e d a b o v e , it m a y u n d e r g o n u c l e o p h i l i c ( M i c h a e l - t y p e ) attack at the β - c a r bon w i t h a s s i s t a n c e by the d e v e l o p i n g c a r b a n i o n to give an i n t e r m e d i a t e s i m i l a r to those p r e v i o u s l y d e s c r i b e d ( F i g u r e 10c). In any of the a b o v e , t r i f l u o r o m e t h y l g r o u p s give c a r b o x y l i c a c i d s o r d e r i v a t i v e s , d i f l u o r o m e t h y l g r o u p s give a l d e h y d e s or k e t o n e s , and f l u o r o m e t h y l g r o u p s give a l c o h o l s or alkenes. M o s t of the C - F bond c l e a v a g e s thus f a r r e p o r t e d c a n be e x p l a i n e d then i n t e r m s of the a f o r e m e n t i o n e d m e c h a n i s m s ; the a b i l i t y of the c o m p o u n d s to f o r m o l e f i n i c i n t e r m e d i a t e s of the type d e s c r i b e d a p p e a r s n e c e s s a r y f o r s u c h r e a c t i o n s to o c c u r . T h e m e c h a n i s m ( s ) b y w h i c h the o l e f i n i c i n t e r m e d i a t e s a r e t r a n s f o r m e d to p r o d u c t s i s b e l i e v e d to i n v o l v e a l t e r n a t e a d d i t i o n of


68

BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS

H

I I — X-C—F

5=t -X^-C-^F —X=C

—product

(a)

H

ι

I

Α Γ \ Ι Α - X - * ( C = C ) i-C-L-F — •

— X-(C=C) - C - F

— X=C—(C=C) —C=C

> - » product


n l

ι

(b)

—c=Q-c—F -*• —c—cic-ί —»N u :

>

ι

ι

/

—C—C=C —»—^-product

I

(c)

\

Nu

Figure 10

—C—CF. I J

ι

— C = C

/ \

H

II

F

HO

I II

—C—COH -*• — C — C — F

II _ ! =

yl /

\ c

O

H

,

v

H

\

I

^ - U o

Figure 11

n u c l e o p h i l e (or solvent) to the i n t e r m e d i a t e , and e l i m i n a t i o n of f l u o r i d e i o n . A p o s s i b l e m e c h a n i s m f o r h y d r o l y s i s of the C F ^ g r o u p is d e p i c t e d i n F i g u r e 11 a n d , as shown, m a y i n v o l v e the i n t e r m e d i a c y of a c y l f l u o r i d e s and k e t e n e s i n the t r a n s f o r m a tion of a t r i f l u o r o m e t h y l g r o u p to a c a r b o x y l a t e f u n c t i o n , a l though these i n t e r m e d i a t e s h a v e , as yet, not been d e t e c t e d .



4.

SANTi E T A L .

Thymidylate

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T h e above p r o v i d e d i n s i g h t into the p o s s i b l e m e c h a n i s m s by w h i c h C F ^ d U M P m i g h t a c t as an a c y l a t i n g agent. T o o b tain d i r e c t s u p p o r t i n g e v i d e n c e , the m e c h a n i s m s of h y d r o l y t i c r e a c t i o n s of 5 - t r i f l u o r o m e t h y l u r a c i l a n d its N - a l k y l a t e d d e r i v a t i v e s w e r e e x a m i n e d i n d e t a i l (47). T h e r e s u l t s of these studies a r e s u m m a r i z e d b e l o w , and d e p i c t e d i n F i g u r e s 12-14. A l l r e a c t i o n s a p p e a r to p r o c e e d by f o r m a t i o n of a h i g h l y reactive intermediate having an exocyclic difluoromethylene g r o u p at the 5 - p o s i t i o n w h i c h s u b s e q u e n t l y r e a c t s w i t h w a t e r o r h y d r o x i d e i o n i n a s e r i e s of r a p i d steps to give c o r r e s p o n ding 5 - c a r b o x y u r a c i l s . F o r those d e r i v a t i v e s w h i c h p o s s e s s an i o n i z a b l e p r o t o n at the 1 - p o s i t i o n , the p r e d o m i n a n t m e c h a n i s m i n v o l v e s i o n i z a t i o n to the conjugate b a s e and a s s i s t a n c e by the 1 - a n i o n i n the e x p u l s i o n of f l u o r i d e i o n ( F i g u r e 12). W h e n i o n i z a t i o n at the 1 - p o s i t i o n i s p r e c l u d e d by the p r e s e n c e of a n a l k y l substituent ( F i g u r e 13), a c y l a t i o n r e a c t i o n s p r o c e e d by r a t e d e t e r m i n i n g attack of h y d r o x i d e i o n at the 6 - p o s i t i o n of the n e u t r a l o r n e g a t i v e l y c h a r g e d ( 3 - a n i o n ) h e t e r o c y c l e to p r o v i d e the r e a c t i v e i n t e r m e d i a t e . In o r d e r to o b t a i n s u i t a b l e i n t r a m o l e c u l a r m o d e l s , and to v e r i f y the p r i m a r y s i t e of r e a c t i o n of 1 - s u b s t i t u t e d d e r i v a t i v e s , a s e r i e s of 1-(a>-aminoalkyl)trif l u o r o m e t h y l u r a c i l s w e r e p r e p a r e d and t h e i r h y d r o l y s e s e x a m i n e d ( F i g u r e 14). N e i g h b o r i n g g r o u p p a r t i c i p a t i o n was a p p a r e n t w h e r e attack of the a m i n o g r o u p on the 6 - p o s i t i o n of the h e t e r o c y c l e r e s u l t s i n the f o r m a t i o n of f i v e - , s i x - , and s e v e n - m e m b e r e d r i n g s ; i n the c a s e of 1 - ( 3 - a m i n o p r o p y l ) - 5 - t r i f l u o r o m e t h y l u r a c i l , apparent f i r s t - o r d e r constants w e r e m o r e than 10 t i m e s g r e a t e r than s i m p l e 1 - a l k y l d e r i v a t i v e s not possessing a neighboring nucleophile. W i t h r e g a r d to the e n z y m i c r e a c t i o n , the s a l i e n t f e a t u r e of these studies i s that the t r i f l u o r o m e t h y l g r o u p of C F ^ d U M P d e r i v a t i v e s w o u l d o n l y b e h a v e as an a c y l a t i n g agent when a s e c o n d a r y d r i v i n g f o r c e i s f u r n i s h e d by r e a c t i o n s w h i c h o c c u r at other p a r t s of the h e t e r o c y c l e . T h a t i s , it i s n e c e s s a r y that a n u c l e o p h i l e i s a d d e d to the 6 - p o s i t i o n of the h e t e r o c y c l e ; i n this m a n n e r , the n o r m a l l y i n e r t t r i f l u o r o m e t h y l g r o u p w o u l d be c o n v e r t e d into a h i g h l y r e a c t i v e e x o c y c l i c d i f l u o r o m e t h y l e n e i n t e r m e d i a t e w h i c h m i g h t then a c y l a t e a n u c l e o p h i l i c g r o u p of the e n z y m e . In a c c o r d w i t h p r o p o s a l s f o r the i n v o l v e m e n t of n u c l e o p h i l i c c a t a l y s i s i n the e n z y m i c r e a c t i o n (viz I, F i g u r e 2), these studies l e d us to p r o p o s e a r e l a t e d m i n i m a l m e c h a n i s m f o r the r e p o r t e d i r r e v e r s i b l e i n a c t i v a t i o n of t h y m i d y l a t e synthetase b y CFjdUMP. In the pathway d e p i c t e d i n F i g u r e 15, it was s u g g e s t e d that j u x t a p o s e d w i t h i n the a c t i v e s i t e , a n u c l e o p h i l i c g r o u p of the e n z y m e (:Z) adds to the 6 - p o s i t i o n of C F ~ d U M P , p r o m o t i n g the e x p u l s i o n of f l u o r i d e i o n and the f o r m a t i o n of a r e a c t i v e e x o c y c l i c d i f l u o r o m e t h y l e n e i n t e r m e d i a t e s i m i l a r to those e n c o u n t e r e d i n o u r m o d e l s t u d i e s . The reactive interm e d i a t e w o u l d then be t r a p p e d by a n u c l e o p h i l i c g r o u p of


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Figure 13


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Figure 15


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the e n z y m e to g i v e , a f t e r a n u m b e r of s t e p s , the a c y l a t e d enzyme. Subsequent to c o m p l e t i o n of these m o d e l s t u d i e s , the i n t e r a c t i o n of C F ^ d U M P w i t h t h y m i d y l a t e synthetase was r e i n v e s tigated i n another l a b o r a t o r y u s i n g the e n z y m e f r o m L . c a s e i (21). T h e s e w o r k e r s o b s e r v e d that C F ^ U M P , C H . F A F L t h y m i d y l a t e synthetase f o r m e d a tight t e r n a r y c o m p l e x w n i c h was i s o l a t a b l e by d i s c g e l e l e c t r o p h o r e s i s u n d e r n o n - d e n a t u r i n g c o n d i t i o n s . H o w e v e r , u n l i k e the F d U M P * C H ^ F A H ^ e n z y m e c o m p l e x , no change i n the d i f f e r e n c e s p e c t r a w a s o b s e r v e d w h e n C F g d U M P was u s e d . F u r t h e r m o r e , g e l e l e c t r o p h o r e s i s i n the p r e s e n c e of a p r o t e i n d é n a t u r a n t r e s u l t e d i n a p p a r e n t d e s t r u c t i o n of the c o m p l e x . A f t e r d e n a t u r a t i o n of the c o m p l e x , the n u c l e o t i d e p r o d u c t was o b s e r v e d to m i g r a t e i d e n t i c a l l y w i t h authentic C F ^ d U M P on D E A E - c e l l u l o s e p a p e r . F r o m these r e s u l t s , i t was c o n c l u d e d that C - F bonds of the n u c l e o t i d e w e r e not c l e a v e d by the e n z y m e and a n u c l e o p h i l e of the e n z y m e d i d not a d d to the 6 - p o s i t i o n of C F dUMP. R e c e n t r e s u l t s o b t a i n e d i n this l a b o r a t o r y a r e not i n a c c o r d w i t h t h e s e f i n d i n g s . A l t h o u g h the m e c h a n i s m of r e a c t i o n of C F ^ d U M P w i t h t h y m i d y l a t e synthetase has not b e e n a s c e r t a i n e d at this t i m e , i t a p p e a r s c e r t a i n that C - F bond c l e a v a g e i s c a t a l y z e d by the e n z y m e , p r o b a b l y v i a n u c l e o p h i l i c c a t a l y s i s . O u r p r e l i m i n a r y r e s u l t s w h i c h l e a d to this c o n c l u s i o n are s u m m a r i z e d below. C o n t r a r y to the p r e v i o u s r e p o r t (2 1), we o b s e r v e that s u b t r a c t i o n of the u l t r a v i o l e t s p e c t r u m of e n z y m e a n d C F L F A F L f r o m that of the e n z y m e , C H ^ F A H ^ a n d C F ^ d U M P p r o d u c e s a d i f f e r e n c e s p e c t r a ( F i g u r e 16a) w h i c h i s v e r y s i m i l a r to that o b s e r v e d w i t h the F d U M Ρ · C H ^ F A H ^ · e n z y m e t e r n a r y c o m p l e x ( F i g u r e 4). A s w i t h the t h y m i a y l a t e s y n t h e t a s e ^ F d U M P C H ^ F A H ^ c o m p l e x , t h e r e i s the c h a r a c t e r i s t i c i n c r e a s e of a b s o r b a n c e at 330 n m and a d e c r e a s e at 261 n m ; the l a t t e r is i n a c c o r d w i t h s a t u r a t i o n of the 5, 6 - d o u b l e bond of the n u c l e o t i d e . U p o n a d d i t i o n of s o d i u m d o d e c y l s u l f a t e , the o n l y d i f f e r e n t i a l a b s o r b a n c e i s that c h a r a c t e r i s t i c of a n u c l e o t i d e w i t h λ » 267 n m ; although this i s u p f i e l d f r o m the m a x i m u m of C F ^ d U M P , we have not y e t a s c e r t a i n e d w h e t h e r a n a l t e r a t i o n of s t r u c t u r e i s i n v o l v e d o r whether the s h i f t i s a r t i f a c t u a l . In the a b s e n c e of C H ^ F A H the i n c u b a t i o n of t h y m i d y l a t e s y n t h e t a s e and C F ^ d U M F ^ ( r a t i o 1:6) f o r 20 m i n u t e s at 2 2 ° r e s u l t s i n 89% i n a c t i v a t i o n of the e n z y m e . T h i s i s i n a c c o r d w i t h p r e v i o u s r e p o r t s on the effect of this n u c l e o t i d e on the e n z y m e f r o m E h r l i c h a s c i t e s c e l l s (32). The difference spec t r a of C F g d U M P and e n z y m e vs e n z y m e i s shown i n F i g u r e 16b. T h e m a x i m u m of C F ^ d U M P at 261 n m d e c r e a s e s , and a t r a n sient b r o a d peak a p p e a r s w h i c h has a b s o r b t i o n u p to c a . 340 n m . A f t e r 1 h o u r , the f i n a l s p e c t r u m e x h i b i t s a m a x i m a at 276 n m , r e s e m b l i n g 5 - a c y l d e r i v a t i v e s of d U M P . Paper


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ι 260

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W A V E L E N G T H Figure 16. Ultraviolet difference spectra, (a) Dashed line: CF dUM? CH FAH and thymidylate synthetase vs. CH FAH and thymidylate synthetase. Solid line: after treatment with sodium dodecyl sulfate, (b) Dashed line: CF dUMP and thymidylate syn thetase vs. thymidylate synthetase after 20 seconds. Solid line: after 1 hr. Broken line: ultraviolet spectrum of CF dUMP. s

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s


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c h r o m a t o g r a p h y of this r e a c t i o n m i x t u r e shows a s i n g l e spot w h i c h m o v e s s l i g h t l y s l o w e r than the s t a r t i n g m a t e r i a l (C^dUMP). A l t h o u g h this p r o d u c t h a s not yet b e e n i d e n t i f i e d , i t i s not 5 - c a r b o x y - d U M P . W h e n C R d U M P was t r e a t e d w i t h a l i m i t i n g a m o u n t of t h y m i d y l a t e synthetase (50:1) f o r 23 h o u r s at 2 2 ° , we w e r e a b l e to d e t e c t that at l e a s t 0.3 e q u i v a l e n t s of F ~ w e r e r e l e a s e d , d e m o n s t r a t i n g that C - F bonds of the n u c l e o tide w e r e i n d e e d l a b i l i z e d . A l t h o u g h these r e s u l t s a r e too p r e l i m i n a r y to p e r m i t d e f i n i tive i n t e r p r e t a t i o n , c e r t a i n c o n c l u s i o n s m a y be r e a c h e d a n d s p e c u l a t i o n s m a y be f o r w a r d e d . It i s c l e a r that the i n t e r a c t i o n of C I ^ d U M P and t h y m i d y l a t e synthetase i n the a b s e n c e of c o f a c tor m a y r e s u l t i n c l e a v a g e of C - F bonds of the n u c l e o t i d e as w e l l as i n a c t i v a t i o n of the e n z y m e . F r o m the a f o r e m e n t i o n e d m o d e l s t u d i e s , it i s m o s t r e a s o n a b l e to p r o p o s e that a c t i v a t i o n of the C - F bond r e q u i r e s a d d i t i o n of a n u c l e o p h i l e of the e n z y m e tu the 6 - p o s i t i o n of the n u c l e o t i d e . T h e m e c h a n i s m of e n z y m e i n a c t i v a t i o n i s not k n o w n , but it a p p e a r s to be s l o w e r ( 10%) than the r e a c t i o n l e a d i n g to C - F bond c l e a v a g e . It i s a l s o a p p a r e n t that the p r e s e n c e of C H ^ F A H ^ m o d u l a t e s this r e a c t i o n i n s o m e y e t unknown m a n n e r ; tne e n z y m e * C F ^ d U M P C H 2 F A H ^ c o m p l e x h a s u l t r a v i o l e t s p e c t r a l q u a l i t i e s quite s i m i l a r to those of the c o m p l e x f o r m e d w i t h F d U M P , i n d i c a t i n g that the 5, 6 - d o u b l e bond of d U M P i s s a t u r a t e d i n the ternary complex. Further experiments are i n progress which a i m to e l u c i d a t e the m e c h a n i s m of i n t e r a c t i o n of d U M P with thymidylate synthetase. #

Literature Cited 1. Friedkin, Μ., (1973), Advan. Enzymol. 38, 235. 2. Pastore, E. J., and Friedkin, M. (1962), J. Biol. Chem. 237, 3802. 3. Santi, D. V., and Brewer, C. F. (1973), Biochemistry 12, 2416. 4. Pogolotti, A. L., and Santi, D. V. (1974), Biochemistry 13, 456. 5. Santi, D. V., and Brewer, C. F. (1968), J. Amer. Chem. Soc. 90, 6236. 6. Kalman, T.I. (1971), Biochemistry 10, 2567. 7. Benkovic, S. J., and Bullard, W. P. (1973), Prog. Bioorg. Chem. 2, 134. 8. Crusberg, T.C., Leary, R., and Kisliuk, R. L. (1970), J. Biol. Chem. 245, 5292. 9. Dunlap, R. Β., Harding, N. G. L., and Huennekens, F. M. (1971), Biochemistry 10, 88. 10. Leary, R. P., and Kisliuk, R. L. (1971), Prep. Biochem. 1, 47. 11. Galivan, J. H., Maley, G. F., and Maley, F. (1975), Bio chemistry 14, 3338.



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SANTI E T A L .

Thymidylate

Synthetase

75

12. Cohen, S. S., Flaks, J. G., Barner, H. D., Loeb, M. R., and Lichenstein, J. (1958), Proc. Nat. Acad. Sci. U. S. 44, 1004. 13. Heidelberger, C., Kaldor, G., Mukherjee, K. L., and Danenberg, P. B. (1960), Cancer Res. 20, 903. 14. Blakley, R. L. (1969). The Biochemistry of Folic Acid and Related Pteridines, New York, Ν. Υ., American Elsevier. 15. Fox, J. J., Miller, N.C., and Cushley, R.J. (1966), Tetrahedron Lett., 4927 16. Otter, B.A., Falco, Ε. A., and Fox, J.J. (1969), J. Org. Chem. 34, 1390. 17. Reist, E. J., Benitez, Α., and Goodman, L. (1964), J. Org. Chem. 29, 554. 18. Santi, D. V., and McHenry, C.S. (1972), Proc. Nat. Acad. Sci. U.S. 69, 1855. 19. Santi, D. V., McHenry, C.S., and Sommer, H. (1974), Biochemistry 13, 471. 20. Langenbach, R. J., Danenberg, P. V., and Heidelberger, C. (1972), Biochem. Biophys. Res. Commun. 48, 1565. 21. Danenberg, P. V., Langenbach, R. J., and Heidelberger, C. (1974), Biochemistry 13, 926. 22. Rose, I. Α., O'Connell, E. L., Litwin, S., and Bar Tana, J. (1974), J. Biol. Chem. 249, 5163. 23. Santi, D. V., McHenry, C.S., and Perriard, E.R. (1974), Biochemistry 13, 467. 24. Aull, J. L., Lyon, J. A., and Dunlap, R. B. (1973), Bio chem. Jour. 19, 210. 25. Sharma, R. Κ., and Kisliuk, R. L. (1973), Fed. Proc., Fed. Amer. Soc. Exp. Biol. 31, 591. 26. Aull, J. L., Lyon, J.A., and Dunlap, R. B. (1974), Arch. Biochem. Biophys. 165, 805. 27. Loeble, R.B., and Dunlap, R. B. (1972), Biochem. Bio phys. Res. Commun. 49, 1671. 28. Rouillier, P., Delmau, J., and Nofre, C. (1966), Bull. Soc. Chim. (France), 3515. 29. Furburg, S., and Janson, L. H. (1968), J. Amer. Chem. Soc. 90, 470. 30. Katritzky, A. R., Nesbit, M.R., Kurtev, B. J., Lyapova, Μ., and Pojarlieff, I. G. (1969), Tetrahedron 25, 3807. 31. Wahba, A. J., and Friedkin, M. (1961), J. Biol. Chem. 236, PC 11. 32. Reyes, P., and Heidelberger, C. (1965), Mol. Pharma col. 1, 14. 33. Heidelberger, C., Parsons, D. G., and Remy, D. C. (1964), J. Med. Chem. 7, 1. 34. Pauling, L. (1960), The Nature of the Chemical Bond. Ithaca, Ν. Υ., Cornell University Press, 85. 35. Giner-Sorolla, Α., and Bendich, A. (1958), J. Amer. Chem. Soc. 80, 5744. 36. Barone, J.A. ( 1963), J. Med. Chem. 6, 39.



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BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS

37. Dipple, Α., and Heidelberger, C. (1966), J. Med. Chem. 9, 715. 38. Shen, T. Y., Ruyle, W. V., and Lewis, Η. M. (1965), J. Org. Chem. 30, 835. 39. Khwaja, Τ. A., and Heidelberger, C. (1969), J. Med. Chem. 12, 543. 40. Ryan, K. J., Acton, Ε. Μ., and Goodman, L. (1966), J. Org. Chem. 31, 1181. 41. Heidelberger, C., Boohar, J., and Kampschroer, D. (1965), Cancer Res. 25, 377. 42. Sakai, Τ. Τ., and Santi, D. V. (1973), J. Med, Chem. 1079. 43. Roberts, J. D., Webb, R. L., and McElhill, E. A. (1950), J. Amer. Chem. Soc. 72, 408. 44. Filler, R., and Novar, H. (1960), Chem. Ind. (London), 1273. 45. Jones, R. (1947), J. Amer. Chem. Soc. 69, 2347. 46. Nestler, H. J., and Garrett, E.R. (1968), J. Pharm. Sci. 57, 1117. 47. Santi, D. V., and Sakai, T. T. (1971), Biochemistry 10, 3598.


Biochemistry Involving Carbon-Fluorine Bonds

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