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.

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One of the primary goals of protein design is to engineer proteins with improved stability. Protein stability is a key issue for chemical, biotechnology and pharmaceutical industries. The development of robust proteins/enzymes with the ability to withstand the potentially harsh conditions of industrial operations is of high importance. A number of strategies are currently being employed to achieve this goal. Two particular approaches, (i) directed evolution and (ii) computational protein design, are quite powerful yet have only recently been combined or applied and analyzed in parallel. In directed evolution, libraries of variants are searched experimentally for clones possessing the desired properties. With computational methods, protein design algorithms are utilized to perform in silico screening for stable protein sequences. Here, we used gene libraries of an unstable variant of streptococcal protein G (Gbeta1) and an in vivo screening method to identify stabilized variants. Many variants with notably increased thermal stabilities were isolated and characterized. Concomitantly, computational techniques and protein design algorithms were used to perform in silico screening of the same destabilized variant of Gbeta1. The combined use, and critical analysis, of these methods promises to advance the field of protein design.