Synthesis and 31P chemical shift identification of tripeptide active site models that represent human serum acetylcholinesterase covalently modified at serine by certain organophosphates.

Most organophosphorus (OP) insecticides impart their toxic action via inhibition of cholinesterases by reacting at an essential serine hydroxyl group. The inhibition process is dependent upon the reactivity, stereochemistry, leaving group, and the mechanism of phosphorylation and/or reactivation (or aging) inherent to the OP compound under consideration. Because a wide array of phosphorylated structures are possible following inhibition by an OP, a simple model system was sought to investigate the mechanistic details of these and related reactions. In the 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 31P 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-CBZ-Glu-Ser[O-P(O)(OMe)2] Ala-Oet; 6), (c) phosphorylation by O,S-dimethyl phosphorothiolates (N-CBZ-Glu-Ser[O-P(O)(OMe)(SMe)]Ala-OEt; 7), (d) aging following inhibition by dimethyl phosphorothionates (N-CBZ-Glu-Ser[O-P(O)(OMe)(S-)]Ala-OEt; 8), (e) aging following inhibition by dimethyl phosphates (N-CBZ-Glu-Ser[O-P(O)(OMe)(O-)]Ala-OEt; 9), and (f) phosphorylation by R/S)PSc-isomalathion stereoisomers (N-CBZ-Glu-Ser[O-P(O)(OMe)(SCH(CO2CO2Et)CH2-CO2Et)]Ala-OEt; 10) have been synthesized. Tripeptides 5 and 6 were prepared via preliminary formation of an intermediate tripeptide phosphite followed by direct conversion to 5 using S8 or to 6 with m-CPBA, respectively. Tripeptides 8 and 9 were prepared by dealkylation of 5 and 6, respectively. Tripeptides 7 and 10 were prepared by reaction of 8 with dimethyl sulfate and (R)- or (S)-diethyl (trifluoromethanesulfonyl)malate, respectively.