Nerve agents are highly toxic organophosphorus compounds (OPs) that are used as chemical warfare agents. Developing a catalytic bioscavenger to efficiently detoxify nerve agents in the bloodstream of affected individuals has been recognized as an attractive approach to prevent nerve agent toxicity. However, the search for nerve agent catalysts has been hindered by the lack of efficient direct assays for nerve agent hydrolysis. In addition, authentic nerve agents are restricted and access to use for experiments by the general research community is prohibited. Herein we report development of a method that combines use of novel nerve agent model compounds possessing a thiocholine leaving group that reacts with the fluorescent thio-detection probe, BES-Thio, to afford detection of sub-micromolar amounts of nerve agent model compounds hydrolysis products. The detection sensitivity of BES-Thio assay was approximately 10 times better than the Ellman assay. This developed method is useful as a direct, sensitive screening method for evaluating OP hydrolysis efficiency from catalytic cholinesterases. When the assay was assembled in the presence of oxime, OP-inhibited cholinesterases that were able to be reactivated by specific oxime showed oxime-assisted enzyme-mediated OP hydrolysis. Therefore, this method is also useful to screen oxime analogs to identify novel agents that can reactivate OP-inhibited cholinesterases or to screen various enzymes to identify pseudo-catalytic bioscavengers that can be readily reactivated by clinically approved oximes.
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