Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates

This paper reviews previously published data and presents new results to address the hypothesis that fluorinated aminophosphonates (FAPs), (RO)2P(O)C(CF3)2NHS(O)2C6H5, R = alkyl, inhibit serine esterases by scission of the P–C bond. Kinetics studies demonstrated that FAPs are progressive irreversible inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7.), butyrylcholinesterase (BChE, EC 3.1.1.8.), carboxylesterase (CaE, EC 3.1.1.1.), and neuropathy target esterase (NTE, EC 3.1.1.5.), consistent with P–C bond breakage. Chemical reactivity experiments showed that diMe-FAP and diEt-FAP react with water to yield the corresponding dialkylphosphates and (CF3)2CHNHS(O)2C6H5, indicating lability of the P–C bond. X-ray crystallography of diEt-FAP revealed an elongated (and therefore weaker) P–C bond (1.8797 (13) Å) compared to P–C bonds in dialkylphosphonates lacking α-CF3 groups (1.805–1.822 Å). Semi-empirical and non-empirical molecular modeling of diEt-FAP and (EtO)2P(O)C(CH3)2NHS(O)2C6H5 (diEt-AP), which lacks CF3 groups, indicated lengthening and destabilization of the P–C bond in diEt-FAP compared to diEt-AP. Active site peptide adducts formed by reacting diEt-FAP with BChE and diBu-FAP with NTE catalytic domain (NEST) were identified using peptide mass mapping with mass spectrometry (MS). Mass shifts (mean ± SE, average mass) for peaks corresponding to active site peptides with diethylphosphoryl and monoethylphosphoryl adducts on BChE were 136.1 ± 0.1 and 108.0 ± 0.1 Da, respectively. Corresponding mass shifts for dibutylphosphoryl and monobutylphosphoryl adducts on NEST were 191.8 ± 0.2 and 135.5 ± 0.1 Da, respectively. Each of these values was statistically identical to the theoretical mass shift for each dialkylphosphoryl and monoalkylphosphoryl species. The MS results demonstrate that inhibition of BChE and NEST by FAPs yields dialkylphosphoryl and monoalkylphosphoryl adducts, consistent with phosphorylation via P–C bond cleavage and aging by net dealkylation. Taken together, predictions from enzyme kinetics, chemical reactivity, X-ray crystallography, and molecular modeling were confirmed by MS and support the hypothesis that FAPs inhibit serine esterases via scission of the P–C bond.