Chemical Synthesis of Two Series of Nerve Agent Model Compounds and Their Stereoselective Interaction with Human Acetylcholinesterase and Human Butyrylcholinesterase

Abstract

Both G-and V-type nerve agents possess a center of chirality about phosphorus. The S p -enantiomers are generally more potent inhibitors than their R p -counterparts toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). To develop model compounds with defined centers of chirality that mimic the target nerve agent structures, we synthesized both the S p and R p stereoisomers of two series of G-type nerve agent model compounds in enantiomerically enriched form. The two series of model compounds contained identical substituents on the phosphorus as the G-type agents, except that thiomethyl (CH 3 -S-) and thiocholine ((CH 3 ) 3 NCH 2 CH 2 -S-) groups were used to replace the traditional nerve agent leaving groups (i.e., fluoro for GB, GF, and GD; and cyano for GA). Inhibition kinetic studies of the thiomethyl-and thiocholine-substituted series of nerve agent model compounds revealed that the S p enantiomers of both series of compounds showed greater inhibition potency toward AChE and BChE. The level of stereoselectivity, as indicated by the ratio of the bimolecular inhibition rate constants between S p and R p enantiomers, was greatest for the GF model compounds in both series. The thiocholine analogs were much more potent than the corresponding thiomethyl analogs. With the exception of the GA model compounds, both series showed greater potency against AChE than BChE. The stereoselectivity (i.e., S p > R p ), enzyme selectivity, and dynamic range of inhibition potency contributed from these two series of compounds suggest that the combined application of these model compounds will provide useful research tools for understanding interactions of nerve agents with cholinesterase and other enzymes involved in nerve agent and organophosphate pharmacology. The potential of and limitations for using these model compounds in the development of biological therapeutics against nerve agent toxicity are also discussed.

Immunodetection of Serum Albumin Adducts as Biomarkers for Organophosphorus Exposure

“…13. Time course for inhibition of rat plasma cholinesterase functional activity by nerve agent model compound Sp-GD tertiary amine (Barakat et al, 2009) or GD after administration to rats. (A) Comparison of inhibition of rat plasma cholinesterase by nerve agent model compound Sp-GD (u) (250 mg/kg, i.p.)…”

“…(B) Comparison of time course (0-10 days) for inhibition of rat plasma cholinesterase activity by nerve agent model compound Sp-GD tertiary amine (u) (250 mg/kg, i.p.) (Barakat et al, 2009) and GD (◯) (25.5 mg/kg, i.p.) (modified from Jovic, 1974).…”

“…Buffers, reagents, and solvents were purchased from VWR Scientific, Inc. (San Diego, CA) in the highest purity commercially available. The nerve agent model compounds and their corresponding Sp and Rp isomers were synthesized as previously described (Berman and Leonard, 1989;Barakat et al, 2009). The nerve agent model compounds are toxic and should be handled with extreme care.…”

“…One of the acute toxic effects of nerve agents is the cholinesterase inhibition in the blood. We therefore investigated the time course of cholinesterase inhibition with a GD model compound Rp-GD tertiary amine (Barakat et al, 2009) and compared it to one reported for GD . As shown in 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).…”