SYNTHESIS AND P-31 CHEMICAL-SHIFT IDENTIFICATION OF TRIPEPTIDE ACTIVE-SITE MODELS THAT REPRESENT HUMAN SERUM ACETYLCHOLINESTERASE COVALENTLY MODIFIED AT SERINE BY CERTAIN ORGANOPHOSPHATES

Most organophosphorus (OF) insecticides impart their toxic action via inhibition of cholinesterases by reacting at an essential serine hydro xyl group. The inhibition process is dependent upon the reactivity, st ereochemistry, leaving group, and the mechanism of phosphorylation and /or reactivation (or aging) inherent to the OP compound under consider ation. Because a wide array of phosphorylated structures are possible following inhibition by an OF, a simple model system was sought to inv estigate the mechanistic details of these and related reactions. In th e present study, the tripeptide N-CBZ-Glu-Ser(OH)-Ala-OEt (chosen as a truncated form of human serum cholinesterase) was chemically modified at the serine hydroxyl group by various O-methyl phosphate groups and the P-31 NMR chemical shift recorded. Six tripeptides, representing ( a) phosphorylation by dimethyl phosphorothionates (N-CBZ-Glu-Ser[O-P(S )(OMe)(2)]Ala-OEt; 5), (b) phosphorylation by dimethyl phosphates (N-C BZ-Glu-Ser[O-P(O)(OMe)(2)]Ala-OEt; 6), (c) phosphorylation by O,S-dime thyl phosphorothiolates (N-CBZ-Glu-Ser[O-P(O)(OMe)(SMe)]Ala-OEt; 7), ( d) aging following inhibition by dimethyl phosphorothionates (N-CBZ-Gl u-Ser[O-P(O)(OMe)(S-)]Ala-OEt 8), (e) aging following inhibition by di methyl phosphates (N-CBZ-Glu-Ser[O-P(O)(OMe)(O-)]Ala-OEt; 9), and (f) phosphorylation by (R/S)(P)S-c-isomalathion stereoisomers -P(O)(OMe)(S CH(CO(2)CO(2)Et)CH(2)CO(2)ET)]Ala-OEt; 10) have been synthesized. Trip eptides 5 and 6 were prepared via preliminary formation of an intermed iate tripeptide phosphite followed by direct conversion to 5 using Ss or to 6 with m-CPBA, respectively. Tripeptides 8 and 9 were prepared b y dealkylation of 5 and 6, respectively. Tripeptides 7 and 10 were pre pared by reaction of 8 with dimethyl sulfate and (R)- or (S)-diethyl ( trifluoromethanesulfonyl)malate, respectively.