Stereoselective bioactivity of the chiral triazole fungicide prothioconazole and its metabolite.

Chiral triazole fungicides have played a significant role in plant pathogen control. Although their enantiomers often exhibit different bioactivity, the mechanism of the stereoselectivity has not been well studied. The stereoselective bioactivity and mechanisms of prothioconazole and its chiral metabolite against plant pathogenic fungi were investigated. The results indicated that the metabolite exerted more fungicidal activities than the activities of the parent compound. R-Prothioconazole and R-prothioconazole-desthio were 6-262 and 19-954 times more potent against pathogenic fungi than the S-enantiomers, respectively. The R-enantiomers were more effective than in inhibiting the biosynthesis of ergosterol and deoxynivalenol the S-enantiomer. Homology modeling and molecular docking suggested that the R-enantiomers of prothioconazole and prothioconazole-desthio possessed better binding modes than S-enantiomers to CYP51B. Moreover, exposure to prothioconazole and its metabolite enantiomers significantly changed the transcription levels of the CYP51 (CYP 51A, CYP51B, CYP 51C) and Tri (Tri5, Tri6, Tri12) genes. The results showed that application of the R-prothioconazole could require a smaller application amount to eliminate the carcinogenic mycotoxins and any environmental risks.

Biocatalytic Asymmetric Phosphorylation Catalyzed by Recombinant Glycerate-2-Kinase.

The efficient synthesis of pure d-glycerate-2-phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one-step biocatalytic phosphorylation of glyceric acid. Glycerate-2-kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate-2-kinase-catalyzed phosphorylation was followed by 31 P NMR and showed excellent enantioselectivity towards phosphorylation of the d-enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d-glycerate 2-phosphate. This phosphorylation reaction, using recombinant glycerate-2-kinase, yielded d-glycerate 2-phosphate in fewer reaction steps and with higher purity than chemical routes.

Metabolism of esfenvalerate in tomato plants (Solanum lycopersicum).

The metabolic fate of esfenvalerate (1), 14C-labeled at the chlorophenyl or phenoxyphenyl ring, in tomato plants was investigated by spraying it three times at 15 g/ha. The overall metabolic trend of 1 was similar in foliage and fruit. The applied 1 gradually penetrated into the foliage/fruit, and approximately 30% of the total radioactive residue (TRR) distributed within the plant. The applied radioactivity remained mostly intact on the plant surface, while its degradation proceeded via ester cleavage to produce two corresponding acids derived from the chlorophenyl and phenoxyphenyl moieties, followed by saccharide conjugation at the inner tissues (each <5%TRR). While 1 retained its optical configuration (2S,αS) on the plant surface and in the fruit, a very slight isomerization at the α-cyanobenzyl carbon occurred to form a (2S,αR) isomer in the foliage (≤1%TRR). The isomerization at another asymmetric carbon C2 in the isovaleric acid moiety did not proceed on/in the plant for 1 or its metabolite.

Stereoselective endocrine-disrupting effects of the chiral triazole fungicide prothioconazole and its chiral metabolite.

The wide use of chiral fungicides has generated interest in the stereoselectivity of their ecotoxicological effects. However, there are few studies about the potential endocrine-disrupting effects (EDEs) of chiral fungicides. This study evaluated the hormone receptor activities of the chiral triazole fungicide prothioconazole and its metabolite using reporter gene assays. The results indicated that prothioconazole and its metabolite possessed EDEs, and the metabolite exerted more activities than the activities of the parent compound, suggesting that the metabolic process is toxification. Stereoselective EDEs were observed, and the S-enantiomers possessed greater hormonal effects than those possessed by the R-enantiomers; the REC20 values ranged from 7.9 × 10-10 to 6.4 × 10-7 M for the thyroid hormone effects and from 3.2 × 10-9 to 7.8 × 10-8 M for the estrogenic effects. The molecular docking results revealed that the stereoselective EDEs of prothioconazole and its metabolite were partially attributed to enantiospecific receptor binding affinities. Overall, our results reveal that prothioconazole and its metabolite might disrupt the balance of the endocrine system by affecting the function of multiple nuclear hormone receptors and that they have the potential to affect the developmental and reproductive systems in humans.

Enantioselective degradation and transformation of the chiral fungicide prothioconazole and its chiral metabolite in soils.

Prothioconazole is a widely used chiral triazole fungicide. In this work, the enantioselective degradation and transformation of prothioconazole and its chiral metabolite prothioconazole-desthio in five kinds of soils were investigated under native and sterile conditions using reversed phase liquid chromatography tandem mass spectrometry with a Lux-cellulose-1 column. The results showed that an enantioselective degradation was observed with R-prothioconazole preferentially degraded in the five soils and enantiomeric fraction values that ranged from 0.32 to 0.41 under native conditions. Furthermore, the major metabolite prothioconazole-desthio was formed rapidly during prothioconazole dissipation. The prothioconazole-desthio enantiomers were degraded slowly, and there was a slight enantioselectivity with enantiomeric fraction values that ranged from 0.45 to 0.51 in the Nanjing and Jilin soils. Under sterile conditions, prothioconazole and its metabolite enantiomers were more slowly degraded with no enantioselectivity. The result of the incubation experiment with single enantiomers verified that R- and S-prothioconazole were transformed to R- and S-prothioconazole-desthio, respectively. No enantiomerization for prothioconazole and its chiral metabolite was observed. In addition, the excellent correlation between organic matter content and degradation rate indicated that organic matter could promote the degradation of prothioconazole and its metabolite enantiomers. The data in this study provide the experimental evidence of the stereoselective degradation and metabolism of both prothioconazole and its chiral metabolite in the environment.

Simultaneous Enantioselective Determination of the Chiral Fungicide Prothioconazole and Its Major Chiral Metabolite Prothioconazole-Desthio in Food and Environmental Samples by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

An efficient and sensitive chiral analytical method was established for the determination of the chiral fungicide prothioconazole and its major chiral metabolite prothioconazole-desthio in agricultural and environmental samples using ultraperformance liquid chromatography-tandem mass spectrometry. The optical rotation and absolute configuration of enantiomers were identified by optical rotation detector and electronic circular dichroism spectra. The elution order of prothioconazole and its chiral metabolite enantiomers was R-(+)-prothioconazole-desthio, S-(-)-prothioconazole-desthio, R-(-)-prothioconazole, and S-(+)-prothioconazole. The mean recoveries from the samples was 71.8-102.0% with intraday relative standard deviations (RSDs) of 0.3-11.9% and interday RSDs of 0.9-10.6%. The formation of prothioconazole-desthio was studied in soil under field conditions and enantioselective degradation was observed for chiral prothioconazole. Remarkable enantioselective degradation was observed: R-prothioconazole degraded preferentially with EF values from 0.48 to 0.37. Although prothioconazole-desthio is the most remarkably bioactive metabolite, no obvious enantioselective behavior was observed in soil. These results may help to systematically evaluate prothioconazole and its metabolites in food and environmental safety.

Simultaneous enantioselective determination of phenylpyrazole insecticide flufiprole and its chiral metabolite in paddy field ecosystem by ultra-high performance liquid chromatography/tandem mass spectrometry.

A novel and sensitive ultra-high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous enantioselective determination of flufiprole and its hydrolysis metabolite in paddy field ecosystem. The separation and determination were performed using reversed-phase chromatography on a novel cellulose chiral stationary phase, a Lux Cellulose-4 (150 mm × 2.0 mm) column, under isocratic conditions at 0.25 mL/min flow rate. The effects of other four different polysaccharide-based chiral stationary phases (CSPs) on the separation and simultaneous enantioseparation of the two target compounds were also evaluated. The elution orders of the eluting enantiomers were identified by an optical rotation detector. Modified QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) method and solid-phase extraction (SPE) were used for the enrichment and cleanup of paddy water, rice straw, brown rice and paddy soil samples, respectively. Parameters including the matrix effect, linearity, precision, accuracy and stability were evaluated. Under the optimal conditions, the mean recoveries for all enantiomers from the above four sample matrix were ranged from 83.6% to 107%, with relative standard deviations (RSD) in the range of 1.0-5.8%. Coefficients of determination R(2)≥0.998 were achieved for each enantiomer in paddy water, rice straw, brown rice and paddy soil matrix calibration curves within the range of 5-500 µg/kg. The limits of quantification (LOQ) for all stereoisomers in the above four matrices were all below 2.0 µg/kg. The methodology was successfully applied for simultaneously enantioselective analysis of flufiprole enantiomers and their chiral metabolite in the real samples, indicating its efficacy in investigating the environmental stereochemistry of flufiprole in paddy field ecosystem.