Study On The Enantioselectivity Of Chiral Fungicide Prothioconazole And Its Metabolite

Chiral pesticide enantiomers have the same physicochemical properties,but the enantiomers often show different biological activities,ecotoxicity and environmental behavior in the chiral environment.Therefore,chiral pesticides enantiomers were evaluated as the same compound,and the results are inaccurate and unscientific.Prothioconazole is a broad-spectrum chiral triazole fungicide that was officially registered for use in China at the end of 2018.Prothioconazole is rapidly degraded in soil,plants,and animals and formed to its metabolite prothioconazole-desthio.Prothioconazole and metabolites have one chiral center,respectively,consisted with a pair of enantiomers.In this work,a chiral analysis method for simultaneous separation and detection of enantiomers of prothioconazole and its metabolites was established,and the stereoselective activity,toxicity and environmental behavior of prothioconazole and its metabolites were invetigated.On the theoretical level,the molecular mechanism of biological effects of prothioconazole enantiomers was revealed.On the application level,based on the research on the biological effect differences of enantiomers,the efficient and safe enantiomer of prothioconazole was screened to minimize and control the damage of pesticide residues to the ecosystem,as well as to provide scientific guidance for the creation of environmentally friendly pesticides.1.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 ultra-performance liquid chromatography tandem mass spectrometry.Baseline separation of prothioconazole and its metabolite enantiomers was achieved with acetonitrile and water(40:60,v/v)as mobile phase at the flow rate of 0.8 mL min-1 and cliromatographic column temperature of 25℃ within 20 min.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 matrix-matched calibration curves from 5 to 500 μg kg-1 showed excellent linearity of all enantiomers with R2>0.9902.The LODs were estimated at 0.0025-0.0087 μg kg-1,and the LOQs were 0.0083-0.0290 μg kg-1.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%.2.The stereoselective bioactivity and mechanisms of prothioconazole and its chiral metabolites 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.3.The hormone receptor activities of the chiral triazole fungicide prothioconazole and its metabolite was investigated 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 and the S-enantiomer performed better binding with receptor.4.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.The results showed that an enantioselective degradation was observed with R-prothioconazole preferentially degraded in all 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 slowlv 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 major 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.5.Enantioselective metabolism of prothioconazole and prothioconazole in rat liver microsomes was investigated using LC-MS/MS.The metabolism of the prothioconazole and its metabolite enantiomers was well described by first-order kinetics with a R2 ranged from 0.9366-0.9233.There was no stereoselectivity for prothioconazole.However,remarkable stereoselective metabolism was observed for the metabolite of prothioconazole with the EF value ranged from 0.50-0.38.The results of enzyme kinetic revealed different affinity between the enantiomers and metabolic enzymes.In addition,homologous modeling and molecular docking res ults indicated that enantioselectivity of prothioconazole-desthio were partially to enantiospecific binding affinities with CYP2D2.6.Stereoselective uptake and metabolism of PTA and its metabolite in zebrafish was investigated using LC-MS/MS.The acute toxicity of the major metabolite prothioconazole-desthio,with an LC50 of 1.31 mg L-1,was 3.5-fold more toxic than the precursor compound,indicating that the metabolism of PTA in zebrafish was toxic.Remarkable enantioselectivity was observed:S-PTA and S-PTA-desthio exhibited uptake rate constants of 8.22 and 8.15 d-1,respectively,at 0.5 mg L-1 exposure and were preferentially taken up,and the R-enantiomers were preferentially metabolized.PTA-desthio was rapidly formed during the uptake processes of PTA.The antioxidant enzyme activities in the zebrafish changed significantly,and these effects were reversible.A metabolic pathway including 13 phase I metabolites and 2 phase II metabolites(glucuronic acid and sulfate conjugate)was identified in zebrafish.