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Reaction monitoring with 1H-31P HSQC NMR spectroscopy

1D 1H-31P HSQC spectra monitoring GF hydrolysis in solution1D 1H-31P HSQC spectra monitoring GF hydrolysis in solution

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.

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