Probing Bioactive Mechanisms - American Chemical Society

Chapter 10 Critical Differences in the Binding of Aryl Phosphate and Carbamate Inhibitors of Acetylcholinesterases Philip S. Magee BIOSAR Research Project, Vallejo, CA 94591 and School of Medicine, Downloaded by EAST CAROLINA UNIV on March 7, 2017 | http://pubs.acs.org Publication Date: November 14, 1989 | doi: 10.1021/bk-1989-0413.ch010

University of California, San Francisco, CA 94143 A study of bulk tolerance in the ring substituents of commercial aryl carbamate and phosphate acetylcholin esterase inhibitors strongly suggests that active site binding must be different for these related classes. This is confirmed by transition state modelling of the serine hydroxyl ion raction with the N-methylcarbamoyl and dimethyl phosphoryl derivatives of 3,4dimethyl-phenol. Distance measurements from the esteratic site (serine oxygen) to the meta- and para-methyl groups show that binding must be different in both spacing and direction. Meta-alkyl groups of aryl carbamates bind in the lipophilic region adjacent to the anionic site. The compounds are efficiently held for reaction with the serine hydroxyl ion. To react with similar efficiency, the aryl ring of a phosphate must bind about 1.0 Å further from the esteratic site, placing the meta position beyond the lipophilic site used by the aryl carbamates. Many differences between aryl carbamate and phosphate inhibitors are clarified by this new binding model. I n h i b i t i o n o f a c e t y l c h o l i n e s t e r a s e s by organophosphate (OP) and organocarbamate (OC) i n h i b i t o r s p r o c e e d s by r e v e r s i b l e b i n d i n g f o l l o w e d by s u b s t a n t i a l l y i r r e v e r s i b l e b l o c k i n g o f t h e a c t i v e s i t e serine hydroxyl (1,2). T h i s i s i r r e f u t a b l e , and whether i n h i b i t e d f o r hours by c a r b a m o y l a t i o n o r days by p h o s p h o r y l a t i o n , t h e n e u r a l r e s p o n s e mechanism depending on m i c r o s e c o n d c l e a r a n c e o f a c e t y l choline i s e f f e c t i v e l y blocked. I n t h e case o f a r y l phosphates and carbamates, t h e r e i s a l s o no q u e s t i o n t h a t the p h e n o l a t e a n i o n i s an e l e c t r o n e g a t i v e l e a v i n g group ( 3 , 4 ) . Cross-resistance of 0C s to phosphate r e s i s t a n t house f l i e s p r o v i d e s a d d i t i o n a l m e c h a n i s t i c o v e r l a p (5). The g e n e r a l p r i n c i p l e s i n v o l v e d have l e d t o d e s i g n o f many commercial O C s and many more O P s f o r crop p r o t e c t i o n , a n i m a l h e a l t h and human d i s e a s e v e c t o r c o n t r o l (6,7). W i t h so many o b v i o u s s i m i l a r i t i e s i n mechanism and g e n e r a l s t r u c t u r a l f e a t u r e s , one would f

1

1

0O97-6156/89/0413-O147$06.O0/0 c 1989 American Chemical Society

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

148

PROBING BIOACTIVE MECHANISMS

expect many more p a r a l l e l s when examined i n d e t a i l . T h i s i s not the case f o r e i t h e r symptomology or l o c a l m o l e c u l a r s t r u c t u r e . S t u d i e s by M i l l e r and co-workers show c l e a r d i f f e r e n c e s i n the t e m p o r a l b e h a v i o r of f l i e s p o i s o n e d by O C s and OP's ( 8 , 9 ) . This o c c u r s d e s p i t e s i m i l a r r a t e s o f e n t r y and appears to be a fundamen t a l d i f f e r e n c e i n mechanism "not w h o l l y e x p l a i n a b l e by c h o l i n e s t e r a s e i n h i b i t i o n " (authors quote)(9). I t might be e x p l a i n a b l e , however, by c h o l i n e s t e r a s e s e l e c t i v i t y . The h o u s e f l y i s known to c o n t a i n a t l e a s t n i n e AChE isozymes t h a t respond q u i t e d i f f e r e n t l y to i n h i b i t i o n by s t a n d a r d O P s ( 1 0 ) . I f these isozymes are a s s i g n e d s p e c i f i c a l l y (one on one) to d i f f e r e n t n e u r a l f u n c t i o n s , r a t h e r than random l y d i s t r i b u t e d , then d i f f e r e n t i a l shutdown by OP's and 0 C s i s under standable. A l l t h a t would be r e q u i r e d f o r a d i f f e r e n t symptomology would be a d i f f e r e n t sequence o f i n h i b i t i o n of the AChE i s o z y m e s . T h i s i s c l e a r l y a c h i e v a b l e by the s e l e c t i v i t y b u i l t i n t o two p a r a l l e l , but not i d e n t i c a l , i n h i b i t i o n mechanisms. f

1

f

Downloaded by EAST CAROLINA UNIV on March 7, 2017 | http://pubs.acs.org Publication Date: November 14, 1989 | doi: 10.1021/bk-1989-0413.ch010

f

In terms o f m o l e c u l a r s t r u c t u r e , some d i f f e r e n c e s between OC's and OP's a r e v e r y c l e a r . The e a r l i e s t s t r u c t u r e - a c t i v i t y s t u d i e s o f d i e t h y l a r y l phosphates u n e q u i v o c a l l y d e f i n e P-0 bond b r e a k i n g w i t h a p h e n o l a t e l e a v i n g group as the p h o s p h o r y l a t i o n s t e p ( 3 ) . This f o l l o w s from the dominant dependence o f pI50 (HF head AChE) on Hammett s sigma w i t h p o s i t i v e rho ( 3 ) . L a t e r s t u d i e s have m o d i f i e d sigma to sigma minus and r e v e a l e d the p r e s e n c e o f a s t e r i c e f f e c t w i t h o u t a l t e r i n g the b a s i c c o n c e p t ( 1 1 ) . The m e c h a n i s t i c simplicity of OP i n h i b i t i o n ( 1 2 ) , was r e f l e c t e d i n e a r l y s t r u c t u r e a c t i v i t y s t u d i e s on OC i n h i b i t i o n (13, 14, 15). E l e c t r o n i c e f f e c t s i n OC i n h i b i t i o n were much weaker and o p p o s i t e l y d i r e c t e d ( n e g a t i v e r h o ) . Ortho s u b s t i t u t e d carbamates, however, d i s p l a y e d a s t r o n g p o s i t i v e rho s i m i l a r to the phosphates ( 1 5 ) . L a t e r QSAR s t u d i e s r e v e a l e d e x c e p t i o n a l l y complex r e l a t i o n s f o r b o t h i n s e c t s (16) and i s o l a t e d i n s e c t AChE (16, 17). In b o t h s t u d i e s , e l e c t r o n e u t r a l r a t h e r than e l e c t r o n e g a t i v e s u b s t i t u e n t s a r e f a v o r e d f o r maximum a c t i v i t y . This i s due p a r t l y to the s e n s i t i v i t y of a r o m a t i c OC's to d e g r a d a t i o n by s i m p l e h y d r o l y s i s , a f a c t o r l e s s i m p o r t a n t i n r e l a t e d OP's and f a r l e s s i m p o r t a n t f o r the p r o - i n s e c t i c i d a l t h i o n o p h o s p h a t e s . Despite the l a c k of a c l e a r - c u t e l e c t r o n i c e f f e c t to s u p p o r t the mechanism, t h e r e i s no q u e s t i o n t h a t c a r b a m o y l a t i o n o c c u r s w i t h a p h e n o l a t e l e a v i n g group. The two i n h i b i t i o n mechanisms a r e i d e n t i c a l i n t h i s respect. 1

Much g r e a t e r d i f f e r e n c e s are o b s e r v e d when b u l k t o l e r a n c e s a r e c o n s i d e r e d i n the b i n d i n g s t e p p r i o r to i r r e v e r s i b l e i n h i b i t i o n . Though not the s u b j e c t o f t h i s paper, d i f f e r e n c e s i n b u l k t o l e r a n c e at the e s t e r a t i c s i t e between OC's and OP's are s i m p l y immense i n m o l e c u l a r terms. The carbamate N - a l k y l group i s l i m i t e d i n s i z e to m e t h y l f o r commercial a c t i v i t y w h i l e p h o s p h a t e s , p h o s p h o r a m i d a t e s , and phosphonates t y p i c a l l y accommodate i s o p r o p y l and p h e n y l g r o u p s . The v a r i a t i o n i s e x t e n s i v e , however, w i t h some e s t e r a t i c s i t e s (OP r e s i s t a n t m i t e s ) ( 1 8 ) , u n a b l e to a c c e p t an 0 , 0 - d i m e t h y l p h o s p h o r y l group w h i l e o t h e r s ( e l e c t r i c e e l AChE) a r e a b l e to b i n d a d i p h e n y l p h o s p h i n y l group ( 1 9 ) . B u l k t o l e r a n c e of the r i n g s u b s t i t u e n t s i s a d i r e c t c o n c e r n o f t h i s study. T a b l e s 1 and 2 l i s t the a r o m a t i c s u b s t i t u e n t s on commer c i a l OP's and OC's h a v i n g p h e n o l l e a v i n g g r o u p s ( 6 ) . The l a r g e s t groups accommodated i n the v a r i o u s p o s i t i o n s are summarized a t the


10. MAGEE

Inhibitors ofAcetylcholinesterases


Table

1.

149

A r o m a t i c S u b s t i t u e n t s on Commercial Organophosphate I n s e c t i c i d e s

X = 0, S Rp

R

2

« OR, SR, E t , Ph

R - C, - C

C

3

Alkyl

A

B

bromophos

CI

H

Br

CI

chlorthiophos

CI

H

SCH

CI

cyanofenphos

H

H

CN

H

CN

H

Common Name

cyanophos

H

H

dicapthon

CI

H

dichlofenthion

CI

H

EPN

H

H

fenitrothion

H

CH

fensulfothion

H

H

fenthion

H

CH

iodofenphos

CI

H

N0

D

3

H

2

CI

3

N0

2

N0

2

H H

H

S(0)CH 3

SCH

3

3

H H CI

I

CI

leptophos

CI

H

parathion

H

H

Br

profenofos

CI

H

Br

H

prothiofos

CI

H

CI

H

ronnel

CI

H

CI

CI

sulprofos

H

H

SCH

trichloronate

CI

H

CI

CI

L a r g e s t Group

CI

CHn

S(0)CHo

CI

N0

H

2

3

H


150



T a b l e 2.

A r o m a t i c S u b s t i t u e n t s on Commercial Organocarbamate I n s e c t i c i d e s

Common Name

A

aminocarb

H

bendiocarb

-0-C(CH ) - 0 -

BMPC

C H

3

s-C H 4

N(CH )

3

3

2

H

9

bufencarb

H

s-C H

butacarb

H

t-C H

5

4

u

9

H

H

H

H

H

H

t-C H H H

carbaryl

-CH-CH-CH =CH-

carbofuran

-0-C(CH ) 2CH2—

H

3

CI

fenethacarb

H

methiocarb

H

promecarb propoxur

C

H

H

H

G

2 5 CH

i-C H

H

3

-0-i-C H 3

matacil

H

CH

zectran

H

CH

Largest

Group

s-C^Hg

?

H

7

s-

C

H

3

3

5 11

4

3

H

3

H

H 3

H

2

N(CH CH*CH ) 2

2

CH

2

N(CH CH=CH ) 2

H

2 5

CH

3

N(CH ) 2

9

H C

CH

SCH

3

H

2

H

H

CPMC

D

C

B

3

t-C H 2


4

9

10. MAGEE


151

1


end o f each t a b l e . F o r O P s w i t h h e t e r o c y c l i c l e a v i n g groups, sub s t i t u e n t s c a n be l a r g e r than those l i s t e d i n T a b l e 1. A good example i s p y r i d a p h e n t h i o n (6) which has a p h e n y l group i n t h e m e t a - e q u i v a l e n t p o s i t i o n . Ring n i t r o g e n s , however, i n c r e a s e t h e l i k e l i h o o d o f s p e c i f i c b i n d i n g t h a t d i f f e r s from t h e simple a r o m a t i c s and h e t e r o c y c l i c examples a r e n o t c o n s i d e r e d i n t h i s s t u d y . P h y s i c a l Nature o f t h e AChE B i n d i n g S i t e . The o f t e n quoted d i s t a n c e from t h e e s t e r a t i c s i t e ( s e r i n e h y d r o x y l ) t o the a n i o n i c s i t e ( c a r boxy l a t e group) i s r e l e v a n t o n l y f o r a c e t y l c h o l i n e (ACh) and mimics with charged amino-residues. None o f these a r e important commercial i n h i b i t o r s as c a t i o n i c s t r u c t u r e s do n o t t r a n s p o r t w e l l t h r o u g h p h o s p h o l i p i d membranes i n l i v i n g t a r g e t s . The n a t u r a l p r o c e s s w i t h ACh i s a d i f f u s i o n c o n t r o l l e d i o n - p a i r i n g r e a c t i o n o f v e r y h i g h v e l o c i t y , a n e c e s s a r y r e q u i r e m e n t f o r a c y c l i c a l m i c r o s e c o n d response (1). The f a c t t h a t a l d i c a r b (Temik) (6) and ACh have n e a r l y i d e n t i c a l c a r b o n y l t o t e r t i a r y c e n t e r d i s t a n c e s (extended) i s i r r e l e v a n t as a l d i b a r b cannot b i n d t o a c a r b o x y l a t e s i t e . Nevertheless, the d i s t a n c e a n a l o g y has been v a l u a b l e i n b o t h OP and OC d e s i g n .

CH.

0

0

CH COCH CH N-CH3 3

2

3

to N

+

- 5.05

3

C H

3

C-0

CHo

CH NHCON=CH-C-SCH

2

I

3

C - 0 t o t-C - 5.04

1

T a b l e 3 g i v e s some examples t h a t we have m o d e l l e d . B i n d i n g o f these t e r t i a r y c e n t e r s i s c r i t i c a l f o r OC a c t i v i t y and must o c c u r i n a l i p o p h i l i c region adjacent to the a n i o n i c s i t e . Mapping o f t h e l i p o p h i l i c r e g i o n s near t h e a n i o n i c s i t e has been c a r r i e d o u t by s e v e r a l i n v e s t i g a t o r s . The e a r l i e s t work by Kabachnik e t a l . i s the most e x t e n s i v e and c l e a r l y i n d i c a t e s two b i n d i n g r e g i o n s , one s u r r o u n d i n g t h e a n i o n i c s i t e and one beyond i t t h a t c a n accommodate an 8-carbon c h a i n ( 2 0 ) . T h e i r work was done e n t i r e l y w i t h a l k y l - s u b s t i t u t e d phosphates and phosphonates h a v i n g t o t a l molecular f l e x i b i l i t y . Thus, t h e p o s i t i o n o f t h e r e g i o n "beyond" the a n i o n i c s i t e i s n o t d e f i n e d . Moreover, t h e i r work w i t h bovine e r y t h r o c y t e AChE may n o t t r a n s l a t e i n d e t a i l t o i n s e c t AChE s. L a t e r , S t e i n b e r g and co-workers used r i g i d , r e v e r s i b l e i n h i b i t o r s t o probe an a r e a a d j a c e n t t o t h e a n i o n i c s i t e d e s c r i b e d as "a conformat i o n a l l y f l e x i b l e , h y d r o p h o b i c ( l i p o p h i l i c ) a r e a which tends r e a d i l y to assume a near p l a n a r form" ( 2 1 ) . This i s c l e a r l y a region that c o u l d accommodate an OC o r OP a r y l o x y - g r o u p . Studies using s p i n l a b e l l e d ACh a n a l o g s l e d Abou-Donia and co-workers t o d e s c r i b e a p l a n a r , l i p o p h i l i c b i n d i n g s i t e o f l a r g e r a d i u s o f c u r v a t u r e (>10 A) i n g e n e r a l agreement w i t h S t e i n b e r g ( 2 2 ) . 1

0

Thus, i t i s c l e a r t h a t r e g i o n s s u i t a b l e f o r t h e b i n d i n g o f OC and OP a r y l o x y - g r o u p s e x i s t near t h e a n i o n i c s i t e . The purpose o f t h i s study i s t o d e c i d e i f t h i s a r e a i s used i d e n t i c a l l y by b o t h classes of i n h i b i t o r s .


152



Experimental Section Modelling. Two-dimensional models o f a c e t y l c h o l i n e , a l d i c a r b (Temik), b u f e n c a r b (Bux) and f e n i t r o t h i o n (Sumithion) were c r e a t e d i n t h e draw mode o f a MACCS database and t r a n s f e r r e d t o t h e PRXBLD m o d e l l i n g program f o r approximate energy m i n i m i z a t i o n . Identical o p e r a t i o n s were c a r r i e d o u t on t h e N-methylcarbamoyl and d i m e t h y l phosphoryl d e r i v a t i v e s of 3,4-dimethylphenol. A l l s o f t w a r e programs were a c c e s s e d on a Prime 9950 r e s i d i n g a t M o l e c u l a r D e s i g n L t d . (MDL) i n San Leandro, C a l i f o r n i a , t h r o u g h an E n v i s i o n 230 g r a p h i c s t e r m i nal. While t h e PRXBLD program i s much l e s s p r e c i s e than MM2, i t has the advantage o f h a n d l i n g u n u s u a l groups such as p h o s p h o r y l . More over, a l l o f the s t r u c t u r e s modelled a r e o f s u f f i c i e n t s i m p l i c i t y t h a t f u r t h e r r e f i n e m e n t i s u n l i k e l y t o y i e l d new i n f o r m a t i o n . F o r p h y s i c a l comparison, a l l s t r u c t u r e s were m o d e l l e d be s e e d i n g PRXBLD i n t h e c o n f o r m a t i o n s i n d i c a t e d i n T a b l e 3. T h i s i s extended f o r t h e a l i p h a t i c s and s y n - p l a n a r f o r t h e a r o m a t i c s . A f t e r t h e m o d e l l i n g p r o c e s s , minor adjustments were made by s i m p l e bond r o t a t i o n s i n t h e MDL DISP program. While t h e s e may n o t be t h e p r e c i s e c o n f o r m a t i o n s during b i o a c t i v i t y , i t provides standard conformations f o r c r i t i c a l d i s t a n c e comparisons. Distances to the t e r t i a r y center or alphac a r b o n c e n t e r from t h e c a r b o n y l o r p h o s p h o r y l group were measured by the LOOK program i n DISP. The m o d e l l i n g p r o c e s s i s s u b j e c t t o s m a l l p o s i t i o n a l e r r o r s , b u t t h e d i s t a n c e measurements a r e p r e c i s e . R e s u l t s a r e shown i n T a b l e 3.

T a b l e 3.

D i s t a n c e from C a r b o n y l o r P h o s p h o r y l to P o s s i b l e B i n d i n g C e n t e r

Compound acetylcholine^ aldicarb

N(CH ) 3

b

bufencarb

3

3,4-dimethylphenyl 0

5.41

m-CH

3

5.24

m-CH

3

5.50

3

6.89

p-CH

3,4-dimethylphenyl phosphate

p-CH

3

7

3

5.48

3

6.88

m-CH 0

5.04

3

m-CH(CH )(C H ) 3

0

N-methyl c a r b a m a t e

dimethyl

5.05

3

C(CH )2SCH c

fenitrothion

°a Distance, A

Center

Measured from t h e c a r b o n y l C o r p h o s p h o r y l P atoms. ^Extended

conformation.

°Syn c o n f o r m a t i o n .

Carbonyl or phosphoryl planar with


ring.

10. MAGEE

153


T r a n s i t i o n s t a t e models o f s e r i n e h y d r o x y l a n i o n r e a c t i n g w i t h the 3»4-dimethylphenyl carbamate and phosphate were c r e a t e d from t h e 3-D s t r u c t u r e s w i t h t h e f o l l o w i n g a s s u m p t i o n s . The carbamate i n t e r mediate i s assumed t o be t e t r a h e d r a l w i t h normal C-0 bond l e n g t h s o

(1.43 A ) . The p h o s p h o r y l i n t e r m e d i a t e i s assumed t o be b i p y r a m i d a l ( l i n e a r d i s p l a c e m e n t ) w i t h normal P-0 bonds (1.57 A) ( 2 3 ) . Figure 1 shows the c o n s t r u c t i o n o f these models. The t e t r a h e d r a l carbamate s t r u c t u r e was m o d e l l e d d i r e c t l y by PRXBLD, then r o t a t e d i n DISP t o b r i n g t h e C-0 bond c o p l a n a r w i t h the r i n g . The b i p y r a m i d a l phos phate i n t e r m e d i a t e r e q u i r e d mapping on graph paper as t h e p e n t a c o v a l e n t P atom was n o t a c c e p t a b l e i n t h e m o d e l l i n g program. Using the s e r i n e oxygen atom as a f i x e d s i t e , d i s t a n c e s were measured t o the meta and p a r a - m e t h y l groups as shown i n F i g u r e 1. A g r a p h i c a l s o l u t i o n by t r i a n g u l a t i o n was used t o measure t h e phosphate i n t e r m e diate distances. These v a l u e s have a somewhat l a r g e r e r r o r than t h e OC measurements.


Q

Serine Oxyl to Methyl D i s t a n c e , Angstroms

Figure

1.

Meta

5.48

Para

7.03

Meta

6.20

Para

8.10

T r a n s i t i o n S t a t e Models o f S e r i n e H y d r o x y l D i s p l a c e m e n t o f 3 , 4 - D i m e t h y l p h e n o l a t e I o n from the N-Methylcarbamate and D i m e t h y l p h o s p h a t e

Discussion I f b i n d i n g were t h e o n l y i s s u e , t h e r e s u l t s o f T a b l e 3 would s u p p o r t s i m i l a r b i n d i n g f o r a r y l carbamates and p h o s p h a t e s . Models o f t h e analogs a r e superimposible. A l d i c a r b i s a n e a r l y p e r f e c t model f o r a c e t y l c h o l i n e i n t h e extended form, and i t i s easy t o v i s u a l i z e a l i p o p h i l i c r e g i o n adjacent to the a n i o n i c s i t e with s t r u c t u r e s favorable for binding t e r t i a r y centers. Moreover, t h e a r y l c a r b a mates i n t h e s y n - p l a n a r c o n f i g u r a t i o n a r e c l o s e enough i n c a r b o n y l meta-ct-carbon d i s t a n c e t o r e a s o n a b l y b i n d t o the same s i t e . I n s u p p o r t o f t h i s h y p o t h e s i s , t h e r e i s no s i g n i f i c a n t movement r e q u i r e d of t h e bound carbamate d u r i n g t h e t e t r a h e d r a l a d d i t i o n o f the s e r i n e



154


h y d r o x y l i o n ( F i g u r e 1). The meta-a-carbon remains w i t h i n 5.5 A o f the e s t e r a t i c s i t e . Hence, the g e n e r a l l o r e o f f a v o r a b l e s e c o n d a r y and t e r t i a r y - a l k y I s i n the m e t a - p o s i t i o n o f a r y l carbamates i s s u p p o r t e d by a f a v o r a b l e sequence o f b i n d i n g and r e a c t i v i t y . Table 2 shows the h i g h f r e q u e n c y o f m e t a - a l k y l groups i n commercial a r y l OC's. Good i n h i b i t o r s w i t h meta groups as l a r g e as h e x y l and h e p t y l a r e known ( 2 4 ) . The s i t u a t i o n w i t h a r y l OP's i s q u i t e d i f f e r e n t . Table 1 r e v e a l s t h a t meta-groups o f any type a r e uncommon and tend to m o d i f y r a t h e r than promote b i o a c t i v i t y . B u f e n c a r b (Bux) has no a c t i v i t y w i t h o u t the m e t a - a l k y l group w h i l e f e n i t r o t h i o n ( S u m i t h i o n ) s i m p l y r e v e r t s to m e t h y l p a r a t h i o n , a n o t h e r commercial OP. As the f u n c t i o n o f the m e t a - a l k y l group i s c l e a r l y d i f f e r e n t f o r OP's and OC's, t h e r e i s no n e c e s s a r y c o n d i t i o n f o r i d e n t i c a l b i n d i n g . Another major f a c t o r i s b u l k t o l e r a n c e a t the m e t a - p o s t i o n i n a r y l OC's and OP's. A study o f T a b l e s 1 and 2 r e v e a l s some s t a r t l i n g d i f f e r e n c e s i n the l e a v i n g groups t h a t have l i t t l e to do w i t h e l e c t r o n i c e f f e c t s . Meta-groups i n commercial a r y l OC's a r e b o t h common and l a r g e , a consequence o f f a v o r a b l e b i n d i n g . Meta-groups i n commercial a r y l OP's a r e b o t h r a r e and s m a l l , h a v i n g l i t t l e to do w i t h enhancement of a c t i v i t y . The g e n e r a l absence o f l a r g e r meta-groups from the a r y l OP l i t e r a t u r e f o r the l a s t f o r t y y e a r s i s a c l e a r message t h a t these groups do not enhance b i n d i n g and may, i n f a c t , be non-binding. T h i s c o n c l u s i o n i s c o m p l e t e l y u n r e a s o n a b l e i f the OC's and OP's occupy e x a c t l y the same b i n d i n g s i t e . A f i n a l argument f o r d i f f e r e n t a r y l b i n d i n g s i t e s depends on the r e a c t i o n t r a n s i t i o n s t a t e . The v e r s a t i l i t y o f phosphorus do r b i t a l s a l l o w s the p o t e n t i a l o f a n o n - l i n e a r d i s p l a c e m e n t by s e r i n e hydroxyl anion. However, the normal model of d i s p l a c e m e n t i s by i n v e r s i o n , presumably t h r o u g h an i n t e r m e d i a t e o f the P C l ^ s t r u c t u r e (25-28). I f we assume a r y l OP to b i n d a t the same l o c a t i o n as a r e l a t e d OC, then one o f two t h i n g s must o c c u r d u r i n g r e a c t i o n . o

E i t h e r the s e r i n e h y d r o x y l i o n must undergo n e a r l y 1 A o f d i s t o r t i o n or the a r y l r i n g must d e s o r b and move a comparable d i s t a n c e away from the i n i t i a l b i n d i n g s i t e ( F i g u r e 1 ) . P o s i t i o n i n g i s a l s o important as the models show a d i f f e r e n c e between the m e t a - p o s i t i o n (OP - OC * o

o

0.72 A) and the p a r a - p o s i t i o n (OP - OC « 1.07 A ) . This indicates the most f a v o r a b l e b i n d i n g l o c a t i o n f o r the OP would be b o t h f u r t h e r from the e s t e r a t i c s i t e and i n a s h a r p l y d i f f e r e n t d i r e c t i o n , i . e . , o

into a t o t a l l y different e s t e r a t i c s i t e , i t i s no

region. At a d i s t a n c e o f 6.20 longer s u r p r i s i n g that m-alkyl

A from groups

the fail

o

to s u p p o r t b i n d i n g i n a r e g i o n 5.0-5.5 A away. I t i s a l s o c l e a r that a b i n d i n g p o s i t i o n f u r t h e r away from the e s t e r a t i c s i t e i s c o n s i s t e n t w i t h the g r e a t e r b u l k t o l e r a n c e f o r s u b s t i t u e n t s on phosphorus, a previously unexplainable f a c t . F u r t h e r d i s c u s s i o n i s s p e c u l a t i v e and u n s u p p o r t e d by e v i d e n c e . However, we can ask what f a c t o r s might cause the a r y l r i n g o f an OP to s e l e c t a b i n d i n g r e g i o n t h a t d i f f e r s i n d i s t a n c e and d i r e c t i o n from t h a t s e l e c t e d by an a r y l OC. As mapped by the s t u d i e s o f K a b a c h n i k (20) and o t h e r s (21,22) the l i p o p h i l i c r e g i o n s n e a r the a n i o n i c s i t e can accommodate an 8-carbon c h a i n . Abou-Donia and c o o

workers s u g g e s t a l a r g e r a d i u s o f c u r v a t u r e

(>10

A)

for

this


10. MAGEE


155


r e g i o n (22). These a r e a s were mapped by f l e x i b l e a l i p h a t i c c h a i n s and c o u l d d e s c r i b e e l l i p t i c a l r e g i o n s w i t h d i s s i m i l a r a x e s . As seen by a s t u d y o f T a b l e s 1 and 2, t h e d i f f e r e n t l e a v i n g group r e q u i r e ments s e p a r a t e the OC and OP a r y l s i n t o two d i s t i n c t c l a s s e s . Car bamate a r y l s a r e l i p o p h i l i c and o f low d i p o l a r i t y , i d e a l f o r b i n d i n g i n the d e s c r i b e d r e g i o n . By c o n t r a s t , the phosphate a r y l s a r e much l e s s l i p o p h i l i c and h i g h l y d i p o l a r . We c a n s p e c u l a t e then t h a t t h e e n e r g e t i c s o f a r y l phosphate b i n d i n g a r e enhanced when the d i p o l a r r i n g s t r e t c h e s a c r o s s a l o n g b u t narrow l i p o p h i l i c r e g i o n toward a more c o m p a t i b l e d i p o l a r a r e a . Firm evidence f o r t h i s s p e c u l a t i o n w i l l r e q u i r e more e x t e n s i v e mapping s t u d i e s o r b e t t e r , s e q u e n c i n g and m o d e l l i n g o f a p u r i f i e d AChE. Acknowledgment The a u t h o r g r a t e f u l l y thanks M o l e c u l a r D e s i g n L t d . (San L e a n d r o , C a l i f o r n i a ) f o r a c c e s s t o t h e i r m o d e l l i n g programs and f o r a generous g r a n t - i n - a i d to support t h i s study.

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Engelhard, N.; Prchal, K.; Nenner, M. Angew. Chem. Internat. Edit.; 1967, 6, 615. O'Brien, R. D. In Insecticide Biochemistry and Physiology; Wilkinson, C. F . , Ed.; Plenum Press, New York and London, 1976, Chapter 7. Fukuto, T. R.; Metcalf, R. L. J . Agr. Food Chem., 1956, 4, 930. Hastings, F. L.; Main, A. R.; Iverson, F. J . Agr. Food Chem., 1970, 18, 497, and references cited therein. Tripathi, R. K. Pest. Biochem. Physiol., 1976, 6, 30. The Agrochemicals Handbook, Royal Society of Chemistry, Unwin Brothers Ltd.; Hartley, D.; Kidd, H. Eds.; Old Woking, Surrey, England, 1983. The Merck Index; Windholz, M.; Budavari, S.; Blumetti, R. F . ; Otterbein, E. S., Eds.; Merck & Co., Inc., Rahway, New Jersey, Tenth Edition, 1983. Miller, T.; Kennedy, J . M.; Collins, C . ; Fukuto, T. R. Pest. Biochem. Physiol., 1973, 3, 447. Miller, T. Pest. Biochem. Physiol., 1976, 6, 307. Tripathi, R. K.; O'Brien, R. D. Pest. Biochem. Physiol., 1973, 2, 418. Hansch, C. J . Org. Chem., 1970, 35, 620. Fukuto, T. R. Bull. Wld. Hlth. Org., 1971, 44, 31. Metcalf, R. L.; Fukuto, T. R.; Frederickson, M. J. Agr. Food Chem., 1964, 12, 231. Metcalf, R. L.; Fukuto, T. R. J . Agr. Food Chem., 1965, 13, 220. Hansch, C.; Deutsch, E. W. Biochem. Biophys. Acta, 1966, 126, 117. Kamoshita, K.; Ohno, I.; Kasamatsu, K.; Fujita, T.; Nakajima, M. Pest. Biochem. Physiol., 1979, 11, 104. Goldblum, A.; Yoshimoto, M.; Hansch, C. J . Agr. Food Chem., 1981, 29, 277. Zahavi, N.; Tahori, A. S.; Klimer, F. Mol. Pharmacol., 1971, 7, 611.


156 19. 20. 21. 22. 23.


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Lieske, C. N.; Clark, J . H . ; Meyer, N. G.; Lowe, J . R. Pest. Biochem. Physiol., 1980, 13, 205. Kabachnik, M. I.; Brestkin, A. P.; Godovikov, N. N.; Michelson, M. J.; Rozengart, E. V . , Rozengart, V. I. Pharmacol. Rev., 1970, 22, 355. Steinberg, G. M.; Mednick, M. L.; Maddox, J.; Rice, R. J . Med. Chem., 1975, 18, 1056. Abou-Donia, M. B.; Rosen, G. M.; Paxton, J . Int. J . Biochem., 1976, 7, 371. Baughman, R. G.; Jacobsen, R. A. J . Ag. Food Chem., 1976, 24, 1036. Average P-O distance in bromophos = 1.57 A. Kohn, G. K.; Ospenson, J . N.; Moore, J . E. J. Ag. Food Chem., 1965, 13, 232. Aaron, H. S.; Uyeda, R. I.; Frack, H. F . ; Miller, J . I. J . Am. Chem Soc., 1962, 84, 617. Green, M.; Hudson, R. F. J . Chem. Soc., 1963, 3883. Michalski, J.; Mikolajczyk, M.; Mlotkowska, B.; Omelanczuk, J . Tetrahedron, 1969, 25, 1743. Wadsworth, W. S. J r . ; Tsay, Y.-G. J . Org. Chem., 1974, 39, 984.

RECEIVED June 14, 1989


Probing Bioactive Mechanisms - American Chemical Society

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