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.

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.
http://dx.doi.org/10.1016/j.jchromb.2010.01.043

Abstract:
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.

A new publication in Analytical and Bioanalytical Chemistry describes the use of 1H-31P HSQC NMR spectroscopy to monitor of the degradation of highly toxic organophosphorus compounds by the enzyme DFPase. The method can be used for methylphosphonates, a group of compounds including nerve agents sarin (GB), soman (GD), cyclosarin (GF) and also VX. The limit of quantitation (LOQ) of the method is around 100 μM when using a 400 MHz NMR spectrometer. The work is founded on previous results from Koskela et al. (Koskela et al., Anal. Chem. 2007; 79:9098-9106) who were able to use the method to detect agents and hydrolysis products in comlex decontamination fluids. We were now able to show that the procedure works not only in the static case but also for dynamic reaction monitoring.

The method is of special relevance for reaction monitoring in complex media. We were able to show that monitoring is also possible in multi-phase systems by using a biodiesel based bicontinuous microemulsion as a model system. Other methods like pH-stat titration or the use of fluoride sensitive electrodes regularly fail in these complex fluids.

Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of disopropyl fluorophosphatase (DFPase) with 1H-31P HSQC NMR spectroscopy.
Gäb J, Melzer M, Kehe K, Wellert S, Hellweg T, Blum MM.
Anal. Bioanal. Chem. 2009;396(3):1213-1221.
http://dx.doi.org/10.1007/s00216-009-3299-2

Abstract:
The enzyme diisopropyl fluorophosphatase (DFPase, EC 3.1.8.2) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Until now, determination of kinetic data has been achieved by use of techniques such as pH-stat titration, ion-selective electrodes, and a recently introduced method based on in situ Fourier-transform infrared (FTIR) spectroscopy. We report the use of 1D 1H-31P HSQC NMR spectroscopy as a new method for real-time quantification of the hydrolysis of toxic organophosphonates by DFPase. The method is demonstrated for the agents sarin (GB), soman (GD), and cyclosarin (GD) but can also be used for V-type nerve agents, for example VX. Besides buffered aqueous solutions the method was used to determine enzymatic activities in a biodiesel-based bicontinuous microemulsion that serves as an example of complex decontamination media, for which other established techniques often fail. The method is non-invasive and requires only limited manual handling of small volumes of liquid (700 μl), which adds to work safety when handling highly toxic organophosphorus compounds. Limits of detection are slightly below 100 μM on a 400 MHz spectrometer with 16 FIDs added for a single time frame. The method is not restricted to DFPase but can be used with other phosphotriesterases, for example paraxonase (PON), and even reactive chemicals, for example oximes and other nucleophiles, as long as the reaction components are compatible with the NMR experiment.

Experimental setup with ReactIR 4000

A new publication in the journal Analytical Biochemistry describes the use of in-situ FTIR spectroscopy to monitor the degradation of toxic organophosphorus componds by the enzyme DFPase. The use of Attenuated Total Reflexion (ATR) allows direct meassurements in the reaction vessel without the need for cuvettes. In comparison to established methods the total reaction volume can be significantly reduced, which also leads to a substantial reduction in the required ammount of toxic substrate and therefore to an increase in work safety. Detection is based on the different IR absorption bands of the organophosphorus compound and its hydrolysis product. We were able to show that changes in the IR bands are directly proportional to changes of substrate and product concentrations in solution. The Lambert-Beer law therefore holds. The limit of quantitation (LOQ) of the methods is around 1 mM. This is a significant progress compared to older work in the field of in-situ FTIR spectroscopy. Dadd and co-workers used FTIR spectroscopy to monitor the reaction of nitriles (Dadd et al., J. Microbiol. Methods 2000; 41:69-75), but the required substrate concentration were in the range of 250 mM. For the experiments we used a ReactIR 4000 made by Mettler-Toledo.

Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy.
Gäb J, Melzer M, Kehe K, Richardt A, Blum MM.
Anal Biochem. 2009; 385(2):187-193.
http://dx.doi.org/10.1016/j.ab.2008.11.012

Abstract:
The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Up to now, the determination of kinetic data has been achieved by techniques such as pH-stat titration, ion-selective electrodes, and fluorogenic substrate analogs. We report a new assaying method using in situ Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR) for the real-time determination of reaction rates. The method employs changes in the P–O–R stretching vibration of DFP and nerve agent substrates when hydrolyzed to their corresponding phosphoric and phosphonic acids. It is shown that the Lambert–Beer law holds and that changes in absorbance can be directly related to changes in concentration. Compared with other methods, the use of in situ FTIR spectroscopy results in a substantially reduced reaction volume that adds extra work safety when handling highly toxic substrates. In addition, the new method allows the noninvasive measurement of buffered solutions with varying ionic strengths complementing existing methods. Because the assay is independent of the used enzyme, it should also be applicable to other phosphotriesterase enzymes such as organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), and paraoxonase (PON).