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Adducts of nerve agents in phosphate buffer and their hydrolysis

Reaction pathway of the formation of buffer adducts with nerve agents

Adduct formation in phosphate buffer (shown here with GF)

We have recently reported about the formation of stable adducts of G-type nerve agents like Sarin, Soman and Cyclosarin with buffering compounds that are aminoalcohols like TRIS, TES or HEPES. The formation of the phosphorus diester adduts that are stable to hydrolysis is dependent on the concentrations of both agent and buffer compound. We have recommended to avoid these buffer compounds for analytical work and resort to buffer compounds like MES, MOPS and CHES that do not form adducts.

An other buffer compund that is widely employed in biological, biochemical and medical research, especially when pH is to be controlled at the physiological pH of 7.4, is inorganic phosphate. We were interested if the phosphate species present at this pH can react as nucleophiles with G-type nerve agents an form adducts as well. This is indeed the case. We were able to show that agents hydrolyse much more quickly in phosphate buffer than in MOPS buffer at the same pH. We were also able to to detect the formation of significant amounts of pyro-phosphate like adducts (phosphorylated methylphosphonates). These hydrolyzed slowly with a kinetic following pseudo-0th order. This resulted in a complex mixture of phosphorus containing species with changing concentrations over time. The molecular structures of these adducts were determined by 1D 1H–31P HSQC NMR and LC–ESI-MS/MS techniques. The rates of formation of the adducts is similar to the the rate of hydrolysis of the agents (leading to the primary hydrolysis products) and leads to the accumulation of significant amounts of the adducts within just a few minutes.

We propose the hydrogen phosphate ion to be the active nucleophile. The other dominating species at pH 7.4 is dihyrogen phosphate and is less nucleophilic. The observed pseudo-0th order hydrolysis kinetic of the adducts can be explained by the fact that only a very small amount is present as a neutral species at pH 7.4 , while the anionic species is protected from hydrolysis (like for example phosphorus diesters) and is present in large excess. This leads to a constant concentration of the neutral species for a long time and therefore results in a pseudo-0th order kinetic. For the competing hydrolysis reaction leading to the primary hydrolysis products we assume that the hydrogenphosphate dianion functions as a base and the reaction is base catalyzed, leading to rate increase compared to the rate determined in MOPS buffer at the same pH.

Formation of pyrophosphate-like adducts from nerve agents sarin, soman and cyclosarin in phosphate buffer: Implications for analytical and toxicological investigations.
Gäb J, John H, Blum MM.
Toxicol. Lett. 2011; 200(1):34-40.
http://dx.doi.org/10.1016/j.toxlet.2010.10.011

Abstract:
Phosphate buffer is frequently used in biological, biochemical and biomedical applications especially when pH is to be controlled around the physiological value of 7.4. One of the prerequisites of a buffer compound among good buffering capacity and pH stability over time is its non-reactivity with other con- stituents of the solution. This is especially important for quantitative analytical or toxicological assays. Previous work has identified a number of amino alcohol buffers like TRIS to react with G-type nerve agents sarin, soman and cyclosarin to form stable phosphonic diesters. In case of phosphate buffer we were able to confirm not only the rapid hydrolysis of these agents to the respective alkyl methylphosphonates but also the formation of substantial amounts of pyrophosphate-like adducts (phosphorylated methylphos- phonates), which very slowly hydrolyzed following zero-order kinetics. This led to a complex mixture of phosphorus containing species with changing concentrations over time. We identified the molecular structure of these buffer adducts using 1D 1H–31P HSQC NMR and LC–ESI-MS/MS techniques. Reaction rates of adduct formation are fast enough to compete with hydrolysis in aqueous solution and to yield substantial amounts of buffer adduct over the course of just a couple of minutes. Possible reaction mechanisms are discussed with respect to the formation and subsequent hydrolysis of the pyrophosphate-like compounds as well as the increased rate of hydrolysis of the nerve agent to the corresponding alkyl methylphosphonates. In summary, the use of phosphate buffer for the development of new assays with sarin, soman and cyclosarin is discouraged. Already existing protocols should be carefully reexamined on an individual basis.

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