Nerve Agent Analogues That Produce Authentic Soman, Sarin, Tabun, and Cyclohexyl Methylphosphonate-Modified Human Butyrylcholinesterase

Abstract

The goal was to test 14 nerve agent model compounds of soman, sarin, tabun, and cyclohexyl methylphosphonofluoridate (GF) for their suitability as substitutes for true nerve agents. We wanted to know whether the model compounds would form the identical covalent adduct with human butyrylcholinesterase that is produced by reaction with true nerve agents. Nerve agent model compounds containing thiocholine or thiomethyl in place of fluorine or cyanide were synthesized as Sp and Rp stereoisomers. Purified human butyrylcholinesterase was treated with a 45-fold molar excess of nerve agent analog at pH 7.4 for 17 h at 21°C. The protein was denatured by boiling and digested with trypsin. Aged and non-aged active site peptide adducts were quantified by MALDI-TOF mass spectrometry of the tryptic digest mixture. The active site peptides were isolated by HPLC and analyzed by MALDI-TOF-TOF mass spectrometry. Serine 198 of butyrylcholinesterase was covalently modified by all 14 compounds. Thiocholine was the leaving group in all compounds that had thiocholine in place of fluorine or cyanide. Thiomethyl was the leaving group in the GF thiomethyl compounds. However, sarin thiomethyl compounds released either thiomethyl or isopropyl, while soman thiomethyl compounds released either thiomethyl or pinacolyl. Thiocholine compounds reacted more rapidly with butyrylcholinesterase than thiomethyl compounds. Labeling with the model compounds resulted in aged adducts that had lost the O-alkyl group (O-ethyl for tabun, O-cyclohexyl for GF, isopropyl for sarin, and pinacolyl for soman) in addition to the thiocholine or thiomethyl group. The nerve agent model compounds containing thiocholine, and the GF thiomethyl analog were found to be suitable substitutes for true soman, sarin, tabun, and GF in terms of the adduct they produced with human butyrylcholinesterase. However, the soman and sarin thiomethyl compounds yielded two types of adducts, one of which was thiomethyl phosphonate, a modification not found after treatment with authentic soman and sarin.

Human Carboxylesterase 1 Stereoselectively Binds the Nerve Agent Cyclosarin and Spontaneously Hydrolyzes the Nerve Agent Sarin

“…For complexes of P R organophosphates and hCE1, we propose that they remain at the acyl-enzyme intermediate stage of the enzyme's reaction mechanism. This state has been observed in the hCE1-P R soman and hCE1-P R cyclosarin crystal structures and via matrix-assisted laser desorption ionization/time of flight analysis of BuChE inhibited by P R analogs of sarin, soman, and cyclosarin (Gilley et al, 2009). For the 40% of P S -sarin analog complexes with hCE1 that do not undergo reactivation, we propose that an alternate Ser221 state is created.…”

“…5). These analogs have been shown by matrix-assisted laser desorption ionization/time of flight mass spectrometry to yield, among other products, acyl-enzyme adducts identical with those from authentic agents when used to inhibit butyrylcholinesterase (Gilley et al, 2009). The thiomethyl-leaving group that replaces the fluoride atom found in nerve agents decreases the toxicity of the analogs by slowing the rate of phosphonylation (k 2 ) compared with real agents; however, the dissociation constants (K d ) remain similar (Forsberg and Puu, 1984).…”

“…For the 40% of P S -sarin analog complexes with hCE1 that do not undergo reactivation, we propose that an alternate Ser221 state is created. In addition to forming adducts identical with authentic agents, the thiomethyl-nerve agent analogs used in this study have been shown to undergo a unique reaction, in which the O-alkoxy rather than the thiomethyl group is displaced upon collapse of the original pentahedral intermediate (Gilley et al, 2009). Therefore, dehydroalanine formation at the catalytic serine might also occur with these nerve agent analogs.…”

A structural examination of agrochemical processing by human carboxylesterase 1

“…These analogs have been previously shown to form adducts identical to authentic agents. 13,14) This second-order inhibition constant (k i ϭk 2 /K m ) can be broken down into individual components. The rates of phosphonylation (k 2 ) between P R and P S isomers were similar, while the binding constants (K m ) differed by at least three-orders of magnitude (Fig.…”

Identification of Human Butyrylcholinesterase Organophosphate-Resistant Variants through a Novel Mammalian Enzyme Functional Screen

“…In this article, we describe the development and validation of a novel screening method that identified hBChE variants with a decreased rate of inhibition by OPs. Because the use of chemical warfare agents is strictly regulated, we used nerve-agent model compounds that have been shown to have similar functional properties to authentic OP compounds (Barakat et al, 2009;Gilley et al, 2009). OP model compounds not only served as a useful means for screening hBChE variants in molecular evolution screening, but also were used in kinetic studies of variants identified (Ralph et al, 2011).…”