Structural and Biochemical Insights into the Inhibition of Human Acetylcholinesterase by G-Series Nerve Agents and Subsequent Reactivation by HI-6.

The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and soman-inhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.

Structural Insights of Stereospecific Inhibition of Human Acetylcholinesterase by VX and Subsequent Reactivation by HI-6.

Over 50 years ago, the toxicity of irreversible organophosphate inhibitors targeting human acetylcholinesterase (hAChE) was observed to be stereospecific. The therapeutic reversal of hAChE inhibition by reactivators has also been shown to depend on the stereochemistry of the inhibitor. To gain clarity on the mechanism of stereospecific inhibition, the X-ray crystallographic structures of hAChE inhibited by a racemic mixture of VX (P R/S) and its enantiomers were obtained. Beyond identifying hAChE structural features that lend themselves to stereospecific inhibition, structures of the reactivator HI-6 bound to hAChE inhibited by VX enantiomers of varying toxicity, or in its uninhibited state, were obtained. Comparison of hAChE in these pre-reactivation and post-reactivation states along with enzymatic data reveals the potential influence of unproductive reactivator poses on the efficacy of these types of therapeutics. The recognition of structural features related to hAChE's stereospecificity toward VX shed light on the molecular influences of toxicity and their effect on reactivators. In addition to providing a better understanding of the innate issues with current reactivators, an avenue for improvement of reactivators is envisioned.

Mechanistic insights into the hydrolysis of 2-chloroethyl ethyl sulfide: the expanded roles of sulfonium salts.

The hydrolysis of 2-chloroethyl ethyl sulfide has been examined in an effort to better understand its mechanism under more concentrated conditions. Two salts formed during hydrolysis were synthesized, and an emphasis was placed on determining their effect on the reaction as it proceeded. Unexpected changes in mechanism were seen when excess chloride was added to the reaction. By measuring rates and product distributions as the products were added back into the hydrolysis, a mechanism was developed. The formation of these sulfonium salts represents additional products in the disappearance of 2-chloroethyl ethyl sulfide with k3 in particular causing a deviation away from expected first-order behavior. Sulfonium salts 3 and 4 do not appear to interconvert, and the system as a whole had fewer pathways available than previously proposed. Initial conditions for studying the hydrolysis were very important and could lead to different conclusions depending on the conditions used. This work will aid in better understanding the hydrolysis of the very toxic chemical warfare agent mustard (bis(2-chloroethyl)sulfide) in the environment and during its decontamination.

Liquid chromatography-time-of-flight mass spectrometry analysis of 1-(2-chloroethoxy)-2-[(2-chloroethyl)thio] ethane and related compounds: separation of an eleven component mixture.

A method of separation for an eleven component mixture comprised of 1-(2-chloroethoxy)-2-[(2-chloroethyl)thio] ethane (4) and its derivatives has been developed using LC-time-of-flight-MS. All analytical figures of merit for compounds 1-11 have been determined. Compound 4 was examined in a substrate extraction study consisting of different sand and soil matrices, and a hydrolysis study of 4 on sand revealed an extremely complex degradation pathway which appeared to be concentration dependent. Substrate extraction and hydrolysis results where compared with sulfur mustard (HD).

Trace Level Analysis of Sarin and VX in Food Using Normal Phase Silica Gel and Ultra-Performance Liquid Chromatography-Time-of-Flight Mass Spectrometry (UPLC-TOF-MS).

Ultra-Performance Liquid Chromatography/electrospray ionization mass spectrometry was used for the trace level determination of isopropyl methylphosphonofluoridate (Sarin, GB) and ( O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) after extraction from various foods. A method utilizing normal phase silica gel was developed for the sample preparation and extraction of VX and GB from food. The extraction efficiencies of the normal phase silica gel method for VX was compared to those of other commercial solid phase extraction media and was found to be comparable. Sarin was found to be incompatible with both the mixed mode cation exchange (MCX) sorbents and QuEChERS methods that are commercially available but was successful with the normal phase silica gel method. The linear range of quantitation for VX was 0.1-330 ng/mL and for GB was 20-1200 ng/mL. The average recoveries of VX and GB from the various food matrices along with the corresponding relative standard deviations (RSDs) are reported.

Structural and rate studies of the 1,2-additions of lithium phenylacetylide to lithiated quinazolinones: influence of mixed aggregates on the reaction mechanism.

The 1,2-addition of lithium phenylacetylide (PhCCLi) to quinazolinones was investigated using a combination of structural and rate studies. (6)Li, (13)C, and (19)F NMR spectroscopies show that deprotonation of quinazolinones and phenylacetylene in THF/pentane solutions with lithium hexamethyldisilazide affords a mixture of lithium quinazolinide/PhCCLi mixed dimer and mixed tetramer along with PhCCLi dimer. Although the mixed tetramer dominates at high mixed aggregate concentrations and low temperatures used for the structural studies, the mixed dimer is the dominant form at the low total mixed aggregate concentrations, high THF concentrations, and ambient temperatures used to investigate the 1,2-addition. Monitoring the reaction rates using (19)F NMR spectroscopy revealed a first-order dependence on mixed dimer, a zeroth-order dependence on THF, and a half-order dependence on the PhCCLi concentration. The rate law is consistent with the addition of a disolvated PhCCLi monomer to the mixed dimer. Investigation of the 1,2-addition of PhCCLi to an O-protected quinazolinone implicates reaction via trisolvated PhCCLi monomers.