Stereochemistry of One-Carbon Transfer Reactions - ACS Publications

17 Stereochemistry of One-Carbon Transfer Reactions

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 26, 2016 | http://pubs.acs.org Publication Date: April 28, 1982 | doi: 10.1021/bk-1982-0185.ch017

HEINZ G. FLOSS Purdue University, Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, West Lafayette, IN 47907

The steric course of a number of biological one-carbon transfer reactions has been studied by means of stereo specifically isotope-labeled substrates. These reactions in clude the transfer of the methylene group of serine to tetra hydrofolate catalyzed by serine transhydroxymethylase, the fur ther utilization of the methylene group of methylene-tetrahydro folate for the generation of the methyl group of thymidylic acid catalyzed by thymidylate synthetase, the transfer of the S-meth yl group of S-adenosylmethionine to various acceptors catalyzed by a number of different methyl transferases, and the transfer of a methyl group from dimethylnitrosamine to DNA or model nucleo philes, a process thought to initiate carcinogenic cell trans formation. As part of a broader interest in stereochemical aspects of biological processes, our laboratory has recently carried out a variety of studies on the stereochemistry of biological one -carbon transfer reactions. Since biologically important single carbon units, like methyl groups, are not per se chiral, this work has required the use of one-carbon centers made chiral by virtue of isotopic substitution; for example, methyl groups which are chiral by virtue of the presence of normal hydrogen, deuterium and tritium. The synthesis of such species is not particularly difficult; it can be accomplished essentially by an extension of methods used widely to generate stereospecifically labeled prochiral centers. However, the configurational analy sis, i.e., the determination whether an unknown sample represents a methyl group of R- or S- configuration presented a conceptually new problem. This was solved by the pioneering work carried out in the laboratories of Cornforth (1) and Arigoni (2). These au thors developed a method which involves conversion of the methyl group in the form of acetic acid into acetyl-CoA followed by con densation with glyoxylate, catalyzed by malate synthase, to give malate, and equilibration with fumarase. Based on an isotope ef fect in the malate synthase reaction, the percentage tritium re tention in the fumarase reaction, called the F value, indicates 0097-6156/82/0185-0229$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.


230

ASYMMETRIC

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PROCESSES IN

CHEMISTRY

t h e c o n f i g u r a t i o n a n d d e g r e e o f p u r i t y o f t h e a c e t a t e methyl g r o u p . An F v a l u e o f 79 c o r r e s p o n d s t o an o p t i c a l l y pure R_methy l g r o u p , a n F v a l u e o f 21 i s shown b y a c h i r a l l y pure methyl g r o u p ( 3 ) . T h i s a n a l y t i c a l m e t h o d o l o g y was e m p l o y e d i n most o f the s t u d i e s t o be r e p o r t e d here. Our f i r s t s t u d y o f a n e n z y m a t i c o n e - c a r b o n t r a n s f e r r e a c t i o n a c t u a l l y i n v o l v e d n o t t h e t r a n s f e r o f a methyl g r o u p b u t r a t h e r o f a m e t h y l e n e g r o u p a n d was c a r r i e d o u t i n c o l l a b o r a t i o n w i t h t h e l a b o r a t o r y o f B e n k o v i c ( 4 ) . T h e p y r i d o x a l p h o s p h a t e enzyme serine transhydroxymethylase catalyzes the conversion o f serine and t e t r a h y d r o f o l a t e i n t o g l y c i n e and m e t h y l e n e - t e t r a h y d r o f o l a t e as shown i n Scheme I . M e c h a n i s t i c c o n s i d e r a t i o n s s u g g e s t e d t h a t f r e e o r enzyme-bound f o r m a l d e h y d e must b e a r e a c t i o n i n t e r m e d i a t e . T o p r o b e t h i s q u e s t i o n , we c a r r i e d o u t t h e r e a c t i o n w i t h s e r i n e s t e r e o s p e c i f i c a l l y t r i t i a t e d i n t h e 3 p o s i t i o n and t r a p p e d t h e m e t h y l e n e - t e t r a h y d r o f o l a t e g e n e r a t e d i m m e d i a t e l y by f u r t h e r dehydrogenation t o m e t h e n y l - t e t r a h y d r o f o l a t e c a t a l y z e d by methylene t e t r a h y d r o f o l a t e dehydrogenase. The s t e r e o s p e c i f i c removal o f one h y d r o g e n f r o m t h e m e t h y l e n e g r o u p b y t h i s enzyme simultaneously served t o determine the t r i t i u m d i s t r i b u t i o n between t h e two m e t h y l e n e h y d r o g e n s o f m e t h y l e n e - t e t r a h y d r o f o l a t e . S t a r t i n g f r o m s e r i n e c a r r y i n g 100% o f i t s t r i t i u m i n o n e d i a s t e r e o t o p i c h y d r o g e n we o b t a i n e d , u n d e r s i n g l e t u r n o v e r c o n d i t i o n s , m e t h y l e n e - t e t r a h y d r o f o l a t e c o n t a i n i n g 76% o f i t s t r i t i u m i n one m e t h y l e n e h y d r o g e n a n d 2 4 % i n t h e o t h e r . I f t h e l a b e l i n s e r i n e was i n t h e o t h e r d i a s t e r e o t o p i c h y d r o g e n , t h e m i r r o r image t r i t i u m d i s t r i b u t i o n i n m e t h y l e n e - t e t r a h y d r o f o l a t e was g e n e r a t e d . I f t h e r e a c t i o n was a l l o w e d t o go b a c k and f o r t h s e v e r a l t i m e s , t h e m e t h y l e n e g r o u p was r a n d o m l y l a b e l e d . T h i s c h a r a c t e r i s t i c r e a c t i o n - d e p e n d e n t s c r a m b l i n g c a n be e x p l a i n e d i n e i t h e r o f two ways. F o r m a l d e h y d e may be a r e a c t i o n i n t e r m e d i a t e w h i c h r e m a i n s enzyme bound d u r i n g i t s t r a n s i e n t e x i s t e n c e e x c e p t f o r a few m o l e c u l e s w h i c h d i s s o c i a t e f r o m t h e enzyme a n d r e b i n d b e f o r e r e a c t i n g w i t h t e t r a h y d r o f o l a t e . A l t e r n a t i v e l y , t h e enzyme may b i n d s e r i n e i n two c o n f o r m a t i o n s a r o u n d t h e a,3 bond w i t h each c o n f o r m a t i o n r e a c t i n g s t e r e o s p e c i f i c a l l y a s i l l u s t r a t e d i n Scheme I I . I t i s n o t p o s s i b l e a t t h e moment t o d i s t i n g u i s h between t h e s e two a l t e r n a t i v e s , a l t h o u g h c i r c u m s t a n c i a l e v i d e n c e f a v o r s the second p o s s i b i l i t y . The a b s o l u t e s t e r i c course o f the r e a c t i o n was n o t a p p a r e n t a t t h e t i m e b u t c a n now b e w r i t t e n a s shown i n Scheme I I I b a s e d on t h e r e c e n t d e t e r m i n a t i o n o f t h e a b s o l u t e c o n f i g u r a t i o n o f t e t r a h y d r o f o l a t e and a d d i t i o n a l s t u d i e s in the l a b o r a t o r y o f Benkovic. Scheme I I I shows t h e e x p e r i m e n t a l a r r a n g e m e n t t o s t u d y t h e s e c o n d o n e - c a r b o n t r a n s f e r r e a c t i o n we i n v e s t i g a t e d , t h e f o r m a t i o n o f t h y m i d y l i c a c i d from u r i d y l i c a c i d c a t a l y z e d by thymidyl a t e s y n t h e t a s e . I n t h i s r e a c t i o n , t h e methyl g r o u p o f t h y m i d y l a t e i s d e r i v e d f r o m t h e c a r b o n and t h e two h y d r o g e n s o f t h e m e t h y l e n e b r i d g e p l u s H-6 o f m e t h y l e n e - t e t r a h y d r o f o l a t e . T o s t u d y t h e s t e r e o c h e m i s t r y o f t h i s r e a c t i o n , we ( 5 ) s y n t h e s i z e d s e r i n e s t e r e o s p e c i f i c a l l y l a b e l e d w i t h t r i t i u m and d e u t e r i u m a t



17.

FLOSS

Scheme I.

One-Carbon

Serine

Transfer

231

Reactions

transhydroxymethylase mechanism stereochemical analysis.

and experimental

setup


for


232

A S Y M M E T R I C REACTIONS A N D PROCESSES IN CHEMISTRY

S

\ / E

=

N

\

/ -

c

(?)

\

±

L-serine

/ E = N

\

^ S

H

L-serine

F ^ J ^ F ^ Ï fllllllllllllllllllllir

ΤΤΤΤΤΤΤΤΤΤΤΠΤΤΤΤΤΤΤΤΓ

H - folate

^

4

Η S

^ C - H - folate 4

Η R

^C-H -folate 4

5,10-methylenetetra- 5,10-methylenetetrahydrofolate hydrofolate Scheme II.

Stereochemical

mechanism of serine

transhydroxymethylase.



FLOSS

Scheme

One-Carbon

111. Stereochemistry

Transfer

of

Reactions

serine transhydroxymethylase synthetase.

233

and

thymidylate



234

ASYMMETRIC

REACTIONS

A N D PROCESSES IN

CHEMISTRY

C-3 a n d c o n v e r t e d i t i n a c o u p l e d r e a c t i o n s e q u e n c e i n t o t h y m i d y l i c a c i d w h i c h was t h e n d e g r a d e d t o r e c o v e r t h e m e t h y l g r o u p a s a c e t i c a c i d . C h i r a l i t y a n a l y s i s o f t h e a c e t i c a c i d showed t h a t i t was i n d e e d c h i r a l a n d had t h e c o n f i g u r a t i o n shown i n Scheme I I I . G e n e r a t i o n o f t h e m e t h y l g r o u p o f t h y m i d y l a t e f r o m methyl e n e - t e t r a h y d r o f o l a t e i n v o l v e s f o u r s e q u e n t i a l bond b r e a k i n g and forming steps a t the s t e r e o s p e c i f i c a l l y l a b e l e d one-carbon u n i t ; t h e r e s u l t s show t h a t e a c h o f t h e s e s t e p s o c c u r s i n a h i g h l y s t e r e o s p e c i f i c manner. However, b e c a u s e o f t h e m u l t i t u d e o f s t e p s i n v o l v e d , t h e r e s u l t does n o t y e t a l l o w us t o d e s c r i b e t h e s t e r i c course o f each i n d i v i d u a l s t e p . The mode o f f o r m a t i o n o f a m e t h y l g r o u p s e e n i n t h y m i d y l a t e i s e x c e p t i o n a l ; most m e t h y l g r o u p s i n b i o l o g i c a l m o l e c u l e s a r i s e f r o m t h e S-methyl g r o u p o f m e t h i o n i n e . O u r n e x t g o a l was t o d e termine the s t e r i c c o u r s e o f the t r a n s f e r o f a methyl group from m e t h i o n i n e o r S - a d e n o s y l m e t h i o n i n e (AdoMet) t o v a r i o u s C-, N-, o r 0-atoms i n b i o l o g i c a l m o l e c u l e s c a t a l y z e d by m e t h y l t r a n s f e r a s e enzymes. P u r s u i t o f t h i s g o a l i n v o l v e d t h e f o l l o w i n g t a s k s : 1) S y n t h e s i s o f m e t h i o n i n e and AdoMet c a r r y i n g a c h i r a l m e t h y l g r o u p o f known c o n f i g u r a t i o n . 2) E n z y m a t i c t r a n s f e r o f t h e m e t h y l g r o u p t o t h e s u b s t r a t e . 3) D e g r a d a t i o n o f t h e p r o d u c t t o c a r v e o u t t h e c h i r a l methy l g r o u p and c o n v e r t i t i n t o a compound s u i t a b l e f o r c o n f i g u r a t i o n a l a n a l y s i s , u s i n g o n l y s t e r e o s p e c i f i c r e a c t i o n s o f known steric course. 4) C o n f i g u r a t i o n a l a n a l y s i s o f the methyl group. The s y n t h e s i s o f m e t h i o n i n e and AdoMet c a r r y i n g a c h i r a l m e t h y l g r o u p s t a r t e d f r o m c h i r a l a c e t a t e , w h i c h had been p r e p a r e d as shown i n Scheme IV ( 6 ) . T h e c o n v e r s i o n i n t o m e t h i o n i n e (Scheme V) i n v o l v e d a S c h m i d t r e a c t i o n , known t o p r o c e e d w i t h r e t e n t i o n o f c o n f i g u r a t i o n , t o g i v e methylamine, which, i n the f o r m o f i t s d i t o s y l i m i d e , was t h e n u s e d t o a l k y l a t e t h e S - a n i o n o f h o m o c y s t e i n e ( 6 ) . T h e l a t t e r r e a c t i o n was e x p e c t e d t o p r o c e e d w i t h i n v e r s i o n o f c o n f i g u r a t i o n o f the methyl group; the o n l y p l a u s i b l e a l t e r n a t i v e , r a c e m i z a t i o n due t o a n S*.l mechanism, i s r u l e d o u t b y t h e s u b s e q u e n t f i n d i n g t h a t t h e m e t h y l g r o u p was i n deed s t i l l c h i r a l . E n z y m a t i c a c t i v a t i o n o f t h e two samples o f m e t h i o n i n e ( 7 ) t h e n gave AdoMet. The f i r s t t r a n s m e t h y l a t i o n s t u d i e d was t h a t c a t a l y z e d b y c a t e c h o l - 0 - m e t h y l t r a n s f e r a s e (COMT) u s i n g e i t h e r e p i n e p h r i n e ( l a ) or 3,4-dihydroxybenzoic a c i d ( l b ) as s u b s t r a t e . The products,~~ m e t a n e p h r i n e ( 2 a ) a n d 4 - h y d r o x y - 3 - m e t h o x y b e n z o i c a c i d (2fe)> were d e g r a d e d b y t h e ~ s t e r e o s p e c i f i c r e a c t i o n s e q u e n c e shown i n Scheme VI t o g i v e a c e t i c a c i d c a r r y i n g t h e c h i r a l m e t h y l g r o u p . I t w i l l be n o t e d t h a t t h e d e g r a d a t i o n s e q u e n c e i n v o l v e s o n e i n v e r s i o n o f the c o n f i g u r a t i o n o f the methyl group i n the c y a n i d e d i s p l a c e m e n t step ( 8 ) . C o n f i g u r a t i o n a l a n a l y s i s o f t h e v a r i o u s a c e t i c a c i d samples showed t h a t AdoMet s y n t h e s i z e d f r o m a c e t a t e o f F=28 gave 2a and 2b w h i c h , upon d e g r a d a t i o n , p r o d u c e d a c e t a t e o f F=68 and 5 7 , r e s p e c t i v e l y . I n t h e o t h e r e n a n t i o m e r i c s e r i e s , t h e v a l u e s were



•

Λ

xX

Scheme IV.

1

M

E

R

E

2

2

© h

COOH

acetate.

2

Τ

3 |

COOH H(D)

.„ (H)D3

D(H)

\ HQI γ_|ΟΗ OH

N

C*

sN

Cr0 pyruvate (H)D kinose,excess 'OH H ® dehydro- H(D) Ilactate I U \ # I U I C uciiyuiw-

Q

,

Jgenose,H 0(D20)

Γ\/Ι_Ι\ D(H,

O

U

; —• phosphofructokinase, D 0(H 0)

S

U

Synthesis of chiral

X

COOH

(OH V T O H HO\_/ (DH

2

CH 0 ®

COOH phosphogly- ® 0 aldolase, \P*^ cerate mutaseJ triose phosphate (H)D enolose isomerase, glyceraldehyde Η 3- phosphate 0® dehydrogenase, orsenate

( O H VT.OH ΗΟγ_γ OH

UA

2

CH 0H

®0




236

D

H,J

*"C

N0N3

R

COONo

H S0 2

Hj •

4

d)-S-CH CH CHC00H, I NH 2

\

NaOH

NH,

y \

NH-Ts

2

H

2

N

2+ ATP, Mg;

Νο,ΗΜΡΑ INVERSION

H N. 9

H Scheme

V.

COOH

Synthesis of chiral

H

(S)-adenosylmethionine

COOH from

chiral

acetate.


Ts


17.

FLOSS

One-Carbon

Transfer

237

Reactions

°«Js "Γ

OH

S-Ad

OH

OH

COMT

H N2

H

COOH

I ^a:X=

-C0 H 2

Ce(NH ) (N0 )

= - CH O H - C H 2 N H C H 3

NaH /

(J

4

a

3

6

S-CI > 5 . OH

KCN/HMPA

1. HoOo/OH^ CN

Scheme VI.

2

H2O

— • 2. NaN0fc/H S0 2

4

Degradation of the products from the COMT chiral methyl group as acetic acid.

D///JS /^C0 H ?

reaction

to recover the


ASYMMETRIC


238

REACTIONS

AND

PROCESSES IN

CHEMISTRY

F=68 f o r t h e s t a r t i n g a c e t a t e , a n d F=39 and 44 f o r t h e a c e t a t e s a m p l e s f r o m t h e d e g r a d a t i o n o f £ a and £ b , r e s p e c t i v e l y . T h u s , t h e r e i s a n odd number o f i n v e r s i o n s i n g o i n g f r o m t h e s t a r t i n g a c e t a t e (Scheme V , u p p e r l e f t ) t o t h e f i n a l p r o d u c t (Scheme V , l o w e r r i g h t ) . S i n c e b o t h t h e s y n t h e s i s and t h e d e g r a d a t i o n e a c h i n v o l v e one i n v e r s i o n , i t f o l l o w s t h a t the enzymatic t r a n s f e r o f t h e m e t h y l g r o u p c a t a l y z e d b y COMT must have o c c u r r e d w i t h i n v e r sion o f configuration (8). The same s t e r e o c h e m i c a l c o u r s e was a l s o o b s e r v e d f o r a n o t h e r methyl t r a n s f e r t o oxygen, the m e t h y l a t i o n o f the p o l y g a l a c t u r o n i c a c i d c a r b o x y l g r o u p s o f p e c t i n c a t a l y z e d b y a n enzyme p r e p a r a t i o n f r o m mung bean s h o o t s . The m e t h y l g r o u p i n t h i s c a s e was r e c o v e r e d by d i r e c t c y a n o l y s i s o f t h e p e c t i n t o g i v e a c e t o n i t r i l e ( w i t h i n v e r s i o n ) , w h i c h was t h e n c o n v e r t e d t o a c e t a t e f o r a n a l y s i s . A g a i n , t h e s t a r t i n g and t h e f i n a l a c e t a t e s a m p l e s had o p p o s i t e c o n f i g u r a t i o n s (F=28 + F=62; F=68 -> F=32) ( 9 ) . In a m i c r o b i a l s y s t e m , S t r e p t o m y c e s g r i s e u s , we s t u d i e d s i m u l t a n e o u s l y two m e t h y l t r a n s f e r s , o n e t o c a r b o n and one t o n i t r o g e n , which are i n v o l v e d i n the b i o s y n t h e s i s o f the a n t i b i o t i c i n d o l m y c i n ( 1 0 ) . I n t h i s c a s e , c h i r a l m e t h i o n i n e was added d i r e c t l y t o t h e c u l t u r e s and t h e r e s u l t i n g i n d o l m y c i n and i n d o l m y c e n i c a c i d were d e g r a d e d a s shown i n Scheme V I I . T h e r e s u l t s again i n d i c a t e d enzymatic t r a n s f e r o f the methyl group, both t o c a r b o n and t o n i t r o g e n , w i t h i n v e r s i o n o f c o n f i g u r a t i o n ( 6 ) . E a r l i e r work f r o m o u r l a b o r a t o r y had shown t h a t , i n t h e C-methyl a t i o n r e a c t i o n l e a d i n g t o i n d o l m y c i n , a h y d r o g e n a t C-3 o f i n d o l e - p v r u v a t e i s r e p l a c e d by the methyl group i n a r e t e n t i o n mode ( 1 1 ) . Thus t h e s t e r e o c h e m i s t r y o f i n d o l m y c i n b i o s y n t h e s i s i n S t r e p t o m y c e s g r i s e u s can be summarized a s shown i n Scheme V I I I . In c o n c l u s i o n , a l l e n z y m a t i c m e t h y l t r a n s f e r r e a c t i o n s s t u d i e d s o f a r proceed with net i n v e r s i o n o f c o n f i g u r a t i o n o f the m e t h y l g r o u p . A l l t h e s e m e t h y l t r a n s f e r a s e s t h e r e f o r e i n v o l v e an uneven number o f t r a n s f e r s o f t h e m e t h y l g r o u p , most l i k e l y a s i n g l e , d i r e c t t r a n s f e r f r o m t h e s u l f u r o f AdoMet t o t h e a c c e p t o r atom i n a n S 2 - t y p e r e a c t i o n . P i n g - p o n g mechanisms i n w h i c h a g r o u p i n t h e enzyme a c t i v e s i t e i s t r a n s i e n t l y m e t h y l a t e d can be e x c l u d e d . T h e two s u b s t r a t e s must be o r i e n t e d i n t h e enzyme a c t i v e s i t e such t h a t i n the t r a n s i t i o n s t a t e the s u l f u r , the m e t h y l c a r b o n and t h e a c c e p t o r atom f o r m a l i n e a r a r r a y . Methyl t r a n s f e r a s e s are not o n l y important i n v a r i o u s metab o l i c p r o c e s s e s , b u t a l s o i n t h e p r o c e s s i n g o f i n f o r m a t i o n a l macr o m o l e c u l e s ; f o r e x a m p l e , DNA b y r e s t r i c t i o n m e t h y l a s e s . Aberrat i o n s i n t h i s p r o c e s s i n g , a s o c c u r i n t h e m e t h y l a t i o n by c a r c i n o gens l i k e d i m e t h y l n i t r o s a m i n e , a r e p r o b a b l y i n v o l v e d i n t h e t r a n s f o r m a t i o n o f t h e c e l l i n t o a tumor c e l l . T h i s p r o c e s s i n v o l v e s m e t a b o l i c a c t i v a t i o n o f d i m e t h y l n i t r o s a m i n e , i n t h e manner shown i n Scheme IX, t o g e n e r a t e t h e u l t i m a t e c a r c i n o g e n , a methyldiazoniurn i o n which then t r a n s f e r s i t s methyl group t o v a r i o u s n u c l e o p h i l i c s i t e s on DNA. T h i s l a t t e r p r o c e s s i s p r e sumably n o t e n z y m e - c o n t r o l l e d and s h o u l d t h e r e f o r e f o l l o w t h e same r u l e s a s t h e same p r o c e s s i n a n a b i o l o g i c a l s y s t e m . K e e p i n g N



17.

FLOSS

One-Carbon

Transfer

D NaOH

239

Reactions

D l)NoOH/TsCl%

H

""'c

w

*

\ 1

2)NaH/TsCI V MU.

c

\

I

/

ν

d

T

s

N

Ts

KCN/HMPA

Η

"r

\OOH

D

% D Scheme

VIL

ι

C=N

>

NaN0 ,

/NaOH

2

COOH

Z

*

Degradation of indolmycin and indolmycenic chiral methyl groups as acetic acid.

D

CONH

5

acid to recover


the

ASYMMETRIC

REACTIONS

AND

PROCESSES IN

CHEMISTRY


240



17.

FLOSS

One-Carbon

Transfer

241

Reactions

a l l o t h e r f a c t o r s c o n s t a n t , t h e s t e r e o c h e m i s t r y o f t h i s methyl t r a n s f e r should be s e n s i t i v e t o the p o l a r i t y o f the r e a c t i o n environment. T h e r e f o r e , a c o m p a r i s o n o f t h e i n v i t r o and t h e i n v i v o process s h o u l d enable us t o probe whether the r e a c t i o n i n the i n t a c t c e l l takes p l a c e i n a hydrophobic environment, l i k e t h e n u c l e a r membrane, o r i n an aqueous s u r r o u n d i n g . T o l a y t h e groundwork f o r e x p e r i m e n t s p r o b i n g t h i s q u e s t i o n , we s y n t h e s i z e d d i m e t h y l n i t r o s a m i n e c a r r y i n g a c h i r a l methyl group by the r e a c t i o n sequence shown i n Scheme X. Methods f o r t h e a l k y l a t i o n o f DNA and f o r t h e r e c o v e r y o f t h e methyl g r o u p f r o m t h e most p r o m i n e n t m o d i f i e d b a s e , 7 - m e t h y l g u a n i n e , have been worked o u t , b u t r e s u l t s f r o m t h e s t e r e o c h e m i c a l a n a l y s i s o f t h e s e samples a r e n o t y e t a v a i l a b l e . We h a v e , however, c o m p l e t e d t h e s t e r e o c h e m i c a l a n a l y s i s o f t h e a l k y l a t i o n o f a model n u c l e o p h i l e , 3,4d i c h l o r o t h i o p h e n o l , by d i m e t h y l n i t r o s a m i n e a c t i v a t e d w i t h r a t l i v e r m i c r o s o m e s ( 1 2 ) . The r e a c t i o n sequence i s shown i n Scheme X I . Based on t h e s t r u c t u r e o f t h e a l k y l group i n t h i s r e a c t i o n and t h e n a t u r e o f t h e n u c l e o p h i l e , we e x p e c t e d t o s s e t r a n s f e r o f t h e methyl g r o u p w i t h a h i g h d e g r e e o f i n v e r s i o n o f c o n f i g u r a t i o n . However, t h e f i r s t s e t o f a n a l y s e s o f t h e a c e t a t e samples f r o m t h e d e g r a d a t i o n o f t h e a l k y l a t e d m a t e r i a l shown i n T a b l e I s u g g e s t s t r a n s f e r o f t h e methyl g r o u p w i t h c o m p l e t e r e t e n t i o n o f c o n f i g u r a t i o n . T h i s s u r p r i s i n g r e s u l t may i n d i c a t e t h a t even i n t h i s i n v i t r o s y s t e m , t h e r e a c t i o n t a k e s p l a c e e n t i r e l y /in t h e l i p o p h i l i c m i c r o s o m e s and t h e r e f o r e p r o c e e d s e x c l u s i v e l y b y an i o n p a i r mechanism w i t h i n t e r n a l r e t u r n . A l t e r n a t i v e l y , t h e mechanism o f d i m e t h y l n i t r o s a m i n e a c t i v a t i o n and a l k y l t r a n s f e r may be more complex than i s c u r r e n t l y e n v i s i o n e d and may, f o r example, i n v o l v e a double displacement p r o c e s s . F u r t h e r e x p e r i ments a r e under way t o v e r i f y t h e i n i t i a l r e s u l t and t o s t u d y t h i s problem f u r t h e r . Table I.

S t e r e o c h e m i c a l a n a l y s i s o f t h e a l k y l a t i o n o f 3,4dichlorothiophenol by m e t a b o l i c a l l y a c t i v a t e d dimethylnitrosamine. F-VALUE

STARTING ACETATE

28

CONFIGURATION S

F-VALUE 68

CONFIGURATION R

DIMETHYLNITROSAMINE

S

R

3,4-DICHLOROTHIOPHENOL METHYL ETHER

S

R

ACETATE FROM DEGRADATION

R

33




242

NaN /H S0 3

CHDT-COONa

2

4

• CHDT-NH

TsCI ^ CH I ^ » CHDT-NH-Ts 2.5 Ν Ν 2.5NNaOH Steam 3

2

g

ά

CHDT \ . N

T

s

CHDT V

3 I % H Ç ^

/

CHDT \

/

CH3

HOAc

CH3

Scheme X.

Synthesis

CN® HMPA Q Scheme XL

D

»

H

Alkylation

of dimethylnitrosamine

\

7

Ι)Η 0 /0Η 2

C

-

C

N

carrying

Θ

2

of 3 4-dichlorothiophenol f

D

2

2)HN0

CH3

»

H

a chiral

M

.

M

-

0

/

methyl

group.

\

7 /

C

"

C

by chiral

0

0

H

dimethylnitrosamine.


17.

FLOSS

One-Carbon

Transfer

Reactions

243

Acknowledgements T h i s work was s u p p o r t e d b y t h e 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 I wish t o acknowledge w i t h g r a t i t u d e the e n t h u s i a s t i c c o n t r i b u t i o n s o f numerous c o w o r k e r s and c o l l a b o r a t o r s whose names a p p e a r on t h e p u b l i c a t i o n s l i s t e d .


Literature Cited 1 Cornforth, J.W.; Redmond, J.W.; Eggerer, H.; Buckel, W.; Gutschow, C. Eur. J. Biochem., 1970, 14, 1. 2 Lüthy, J.; Rétey, J.; Arigoni, D. Nature (Lond.), 1969, 221, 1213. 3 For a review see: Floss, H.G.,; Tsai, M.D. Adv. Enzymol., 1979, 50, 243. 4 Tatum, C.M.; Benkovic, P.Α.; Benkovic, S.J.; Potts, R.; Schleicher, E.; Floss, H.G. Biochemistry, 1977, 16, 1093. 5 Tatum, C.; Vederas, J.; Schleicher, E.; Benkovic, S.J.; Floss, H.G. J. Chem. Soc. Chem. Commun., 1977, 218. 6 Woodard, R.W.; Mascaro, L . ; Hörhammer, R.; Ei,senstein, S.; Floss, H.G. J. Amer. Chem. Soc., 1980, 102, 6314. 7 Cantoni, G.L. Biochem. Prep., 1957, 5, 58. 8 Woodard, R.W.; Tsai, M.D.; Floss, H.G.; Crooks, P.Α.; Coward, J.K. J. Biol. Chem., 1980, 255, 9124. 9 Woodard, R.W.; Weaver, J.; Floss, H.G. Arch. Biochem. Biophys., 1981, 207, 51. 10 Hornemann, U.; Hurley, L.H.; Speedie, M.K.; Floss, H.G. J. Amer. Chem. Soc., 1971, 93, 3029. 11 Zee, L . ; Hornemann, U.; Floss, H.G. Biochem. Physiol. Pflanzen (Jena), 1975, 168, 19. 12 Shen, S.J.; Tsai, M.D.; Floss, H.G., unpublished results. RECEIVEDDecember 14, 1981.


Stereochemistry of One-Carbon Transfer Reactions - ACS Publications

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