Isomalathion and related compounds as active site probes of acetylcholinesterase

To examine the interactions of asymmetric organophosphorus compounds within the steric confines of the acetylcholinesterase (AChE) active site, the synthesis of the stereoisomers of isomalathion [S-(1,2-dicarboethoxyethyl) O,S-dimethyl phosphorodithiolate] was conducted. The methodology used was also adapted for the first synthesis of the stereoisomers of the novel isoparathion thiosuccinate phosphorothiolate (ITP) compound [ S-(1,2-dicarboethoxyethyl) O-methyl O-(4-nitro-phenyl) phosphorothiolate]. The kinetic parameters k_i, K_D, and k_p for the inhibition of electric eel AChE (EEAChE) and soluble recombinant mouse AChE (rMAChE) by the isomalathion and ITP stereoisomers were determined. EEAChE demonstrated a 4-fold difference in anti-AChE potency between the strongest, S _PR_C, and weakest, S_PS_C, isomalathion isomers and an 8-fold difference in anti-AChE potency between the strongest ITP stereoisomer, R_PR_C, and the weakest, S_PS_C. rMAChE demonstrated a 40-fold difference in anti-AChE potency between the weakest isomalathion isomer, S_PS_C, and the most potent, R_PR_C, and a 24-fold difference in anti-AChE potency between the strongest ITP stereoisomer, R_PR_C, and the weakest, S_PS_C, thus demonstrating both stereochemical and species differences in the inhibitory profiles of the two compounds. The rate constants for reactivation, k₃, both spontaneous and in the presence of the oximes 2-PAM and TMB-4, were determined for both enzyme sources inhibited by the stereoisomers of both compounds. Both sources of AChE inhibited by isomalathion stereoisomers that were R at phosphorus demonstrated both spontaneous and oxime mediated reactivation while inhibition by the isomers that were S at phosphorus resulted in phosphorylated adducts resistant to reactivation even in the presence of an oxime suggesting distinct postinhibitory pathways dependent upon the stereochemical orientation at phosphorus. This is in contrast with the results obtained following inhibition with the ITP stereoisomers where the postinhibitory mechanisms appeared to be dependent upon the stereochemical orientation of the asymmetric carbon present in the diethylmalonate portion of the molecule. These observations suggest that despite the similarities of the two compounds their steric and electrostatic differences cause them to interact with the AChE active center by distinct mechanisms.