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Stable adducts of nerve agents with buffer compounds like TRIS and HEPES

Reaction mechanism of adduct formation with nerve agents

Mechanism of adduct formation

Buffer compounds are used to maintain a certain pH in solution. Besides buffer capacity and pH stability over prolonged times the non-reactivity with other components of the solution is an important factor for buffer selection. In a new publication in the Journal of Chrmatography B we report the formation of stable adducts of nerve agents like Sarin, Soman or Cyclosain with common and widely employed buffer compounds like TRIS, TES or HEPES. The reaction proceeds in competition with spontaneous hydrolysis in aqueous solution and yields can reach up to 40% based on the organophosphate/phosphonate present. Highest yields are achieved with high buffer concentrations and high pH.

Using a recently presented NMR method to monitor the degradation of nerve agents by enzymes in buffered solution we were able to observe the formation of new, phosphorus containing and stable compounds. Using LC-ESI-MS/MS we were able to show that the compounds are adducts of the buffer and the nerve agent. Buffer compounds like TES or TRIS can act both as nitrogen nucleophiles (via the amino group) and as oxygen nucleophiles (via the oxygen atoms of the hydroxyl groups). The identification of the adducts as phosphodiesters (“O-adducts”) was finally achieved by NMR spectroscopy.

As a potential reaction mechanism we propose that the amino group of the buffer acts as an intramolecular proton acceptor, which can accept a proton from a hydroxyl group of the buffer. This increases the nucleophilicity of the hydroxyl oxygen atom attacking the phosphorus atom of the warfare agent leading to the formation of a phosphodiester (with organophosphonates like Sarin, Soman and Cyclosarin). As alternative buffer compounds for work with nerve agents we propose MOPS (pK = 7.2), CHES (pK = 9.3) und MES (pK = 6.15). These compounds do not contain a combination of hydroxyl and amino groups and do not show any adducts formation in solution.

Stable adducts of nerve agents sarin, soman and cyclosarin with TRIS, TES and related buffer compounds–Characterization by LC-ESI-MS/MS and NMR and implications for analytical chemistry.
Gäb J, John H, Melzer M, Blum MM.
J. Chromatogr. B 2010; 878(17-18):1382-1390.

Buffering compounds like TRIS are frequently used in chemical, biochemical and biomedical applications to control pH in solution. One of the prerequisites of a buffer compound, in addition to sufficient buffering capacity and pH stability over time, is its non-reactivity with other constituents of the solution. This is especially important in the field of analytical chemistry where analytes are to be determined quantitatively. Investigating the enzymatic hydrolysis of G-type nerve agents sarin, soman and cyclosarin in buffered solution we have identified stable buffer adducts of TRIS, TES and other buffer compounds with the nerve agents. We identified the molecular structure of these adducts as phosphonic diesters using 1D 1H-31P HSQC NMR and LC-ESI-MS/MS techniques. Reaction rates with TRIS and TES are fast enough to compete with spontaneous hydrolysis in aqueous solution and to yield substantial amounts (up to 20-40%) of buffer adduct over the course of several hours. A reaction mechanism is proposed in which the amino function of the buffer serves as an intramolecular proton acceptor rendering the buffer hydroxyl groups nucleophilic enough for attack on the phosphorus atom of the agents. Results show that similar buffer adducts are formed with a range of hydroxyl and amino function containing buffers including TES, BES, TRIS, BIS-TRIS, BIS-TRIS propane, Tricine, Bicine, HEPES and triethanol amine. It is recommended to use alternative buffers like MOPS, MES and CHES when working with G-type nerve agents especially at higher concentrations and over prolonged times.


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