Bruno Combourieu

Biodegradation pathway of mesotrione: Complementarities of NMR, LC–NMR and LC–MS for qualitative and quantitative metabolic profiling

Enhanced knowledge of pesticide transformation products formed in the environment could lead to both accurate estimates of the overall effects of these compounds on environmental ecosystems and human health and improved removal processes. These compounds can present chemical and environmental behaviours completely different from the starting active ingredient. The difficulty lies on their identification or/and their quantification due to the lack of analytical reference standards. In this context, ex situ Nuclear Magnetic Resonance (NMR) and Liquid Chromatography-NMR (LC-NMR) were used as complementary tools to LC-Mass Spectrometry (MS) to define the metabolic pathway of mesotrione, an emergent herbicide, by the bacterial strain Bacillus sp. 3B6. The complementarities of ex situ and LC-NMR allowed us to unambiguously identify six metabolites whereas the structures of only four metabolites were suggested by LC-MS. The presence of a new metabolic pathway was evidenced by NMR. These results demonstrate that NMR and LC-NMR spectroscopy provided unambiguous structural information for xenobiotic metabolic profiling, even at moderate magnetic field and allowed direct absolute quantification despite the lack of commercial or synthetic standards, required for LC-MS techniques.

First isolation and characterization of a bacterial strain that biotransforms the herbicide mesotrione.

Aim:u2002 The aim of this study was to find and characterize a fungal or bacterial strain capable of metabolizing mesotrione, a new selective herbicide for control of broad‐leaved weeds in maize.

Degradation of morpholine and thiomorpholine by an environmental Mycobacterium involves a cytochrome P450. Direct evidence of intermediates by in situ 1H NMR

Abstract A strain of Mycobacterium sp. RP1 was isolated from a contaminated activated sludge. It was capable of utilizing morpholine, a waste of chemical industry, as sole source of carbon, nitrogen and energy. The kinetic of biodegradation of morpholine was followed directly on the incubation medium using in situ 1 H NMR. This technic allowed to identify two intermediates of the degradative pathway: glycolate and 2-(2-aminoethoxy)acetate. The inhibitory effects of metyrapone on the degradative abilities of the strain RP1 indicated the involvement of a cytochrome P 450. This observation was confirmed by spectrophotometric analysis and 1 H NMR. Metyrapol, a known metabolite of metyrapone, was also found to be an inhibitor. The same study of degradation of thiomorpholine showed the formation of sulfoxide, which confirmed the presence of a cytochrome P 450.

Thiomorpholine and morpholine oxidation by a cytochrome P450 in Mycobacterium aurum MO1. Evidence of the intermediates by in situ 1H NMR.

Spectrophotometric assays of Mycobacterium aurum MO1 cells extracts gave evidence of a soluble cytochrome P450, involved in the degradative pathway of morpholine, a waste product from the chemical industry. In order to get further information, the kinetics of the biodegradation of the sulfur analogue thiomorpholine was monitored by using in situ nuclear magnetic resonance (NMR). This technique allowed the identification of two intermediates: the sulfoxide of thiomorpholine resulting from S-oxidation and thiodiglycolic acid owing to ring cleavage. The S-oxidation (S → SO) represents one of the well-known reactions catalyzed by cytochromes P450. The inhibitory effect of metyrapone, a cytochrome P450 inhibitor, on the thiomorpholine and morpholine degradative abilities of M. aurum MO1 confirmed the involvement of a cytochrome P450. These results and the decrease of the rate of formation of the first intermediate during the morpholine degradation, 2-(2-aminoethoxy) acetate, proved the key role of the cytochrome P450 in the early events of the biodegradation, i.e, in the C–-N bond cleavage.

Differentiation of mobile and immobile pesticides on anionic clays by 1H HR MAS NMR spectroscopy

Adsorbed vs. intercalated MCPA (4-chloro-2-methylphenoxyacetic acid) in highly hydrated clays taken as a soil model were clearly distinguished by 1H HR MAS NMR; adsorbed herbicide gave sharp signals indicating high mobility while intercalated herbicide gave very wide unresolved spectra due to its strong interaction with the solid matrix.

1H NMR: a tool to study the fate of pollutants in the environment

Abstract In situ 1 H NMR, directly performed on biological fluids is a very powerful tool to study the fate of pollutants in the environment. The biodegradation of 2-aminobenzothiazole by Rhodococcus rhodochrous was monitored by reverse phase HPLC and by in situ 1 H NMR, methods performed directly on culture media without purification. The xenobiotic was biotransformed into a hydroxylated derivative. The chemical structure of this metabolite was determined by a long-range 1 H– 15 N heteronuclear shift correlation without any previous 15 N enrichment of the compound. This approach allowed the assignment of the metabolite structure to 2-amino-6-hydroxybenzothiazole.