Studies On Stereoselectivity Of Chiral Insecticide Flufiprole Enantiomers

The enantiomers of chiral pesticides have similar physical and chemical properties in achiral environments,however,the stereoisomers always differ significantly with respect to their biological activity,toxicity and environmental behavior.Investigation based on the only racemic form of chiral pesticide would result in inaccurate environment risk assessment.In recent years,stereoselectivity of chiral pesticides have attracted increasing attention.Flufiprole,a significant substitute for fipronil,is a widely used chiral phenylpyrazole pesticide,and there is still no report on the stereoselectivity of flufiprole enantiomers.The research purpose of this paper is to fill the relevant knowledge gap through systematic assessments of different features of flufiprole enantiomers in chiral environment.The main results were listed as followed:1.The separation of flufiprole enantiomers was performed using reversed-phase HPLC on a Lux Cellulose-2 column.The influences of different mobile phase composition on separation were discussed.Satisfactory separation of which the resolution factor reached 6.28 was obtained using a mobile phase of acetonitrile/water(55:45,v/v)at ultraviolet(UV)detection 230 nm,the flow rate was 0.7 mL/min,and the column temperature was set as 30 °C.The absolute configuration of flufiprole enantiomers was determined through the comparison of experimental and predicted ECD spectra,the specific optical rotation was measured to confirm the levo and dextro isomers.The first enantiomer eluted from Lux Cellulose-2 column was confirmed to be(S)-(-)-flufiprole and the second was(R)-(+)-flufiprole.2.A simple and reliable method for the simultaneous determination of flufiprole enantiomers in fruits,vegetables,and soil samples was established.An Alumina-N solid phase extraction(SPE)column was used in the cleanup of the samples.The method was evaluated by the specificity,matrix effect,linearity,precision and accuracy.The matrix effect could be negligible and good linearity(R2>0.998)was obtained for all matrix calibration curves within the range of 0.2-20 mg/L.The limit of detections(LODs)for two enantiomers in the six matrices were 0.007-0.008 mg/kg,whereas the limit of quantifications(LOQs)were 0.021-0.025 mg/kg.The mean recoveries of two enantiomers ranged from 86.8-98.9%,with 1.1-6.4%intra-day relative standard deviation(RSD)and 1.2-5.8%inter-day RSD.The accuracy,precision and sensitivity of the analysis meet the requirement for simultaneous determination of flufiprole enantiomers in food and environmental samples.3.The stereoselective bioactivity and acute toxicity of flufiprole enantiomers were first studied.Four types of representative insects(Plutella xylostella,Nilaparvata lugens,Mythiwmna separata and Acyrthosiphon pisum)were used to investigate enantioselective insecticidal activity.Acute toxicities of flufiprole enantiomers toward two non-target organisms(Scenedesrmus obliquus and Trichogramma japonicum Ashmead)were also evaluated.The bioactivity of(R)-flufiprole was 1.9-5.1 times higher than that of(S)-fluflprole and(R)-isomer also showed 3.7-5.7 times higher acute toxicity to non-target organisms than(S)-isomer.4.Stereoselective degradation in soil and four vegetables(pak choi,spinach,cucumber and tomato)under field conditions was studied to achieve a comprehensive assessment for the environmental behavior of flufiprole enantiomers.The dissipation of flufiprole enantiomers in soil accorded with first order kinetics(R2>0.97)and their half-lives were the same(9.76 d).No significant stereoselectivity was observed.However,the degradation of two stereoisomers in four vegetables complied with first order kinetics(R2=0.9164-0.9638)as well,and significant differences in the half-lives of stereoisomers were observed.A reversed enantioselectivity degradation tendency for flufiprole enantiomers occurred in four vegetables.The half-lives of(S)-flufiprole in pak choi,spinach,cucumber and tomato were 4.07,3.76,4.33 and 7.79 d,respectively,while the half-lives of(R)-flufiprole were 5.19,6.15,3.46 and 6.73 d,respectively.The EF values increase from 0.50 to 0.70 in pak choi and 0.81 in spinach finally,(S)-flufiprole was preferentially degraded;by contrast,the EF values decreased gradually from 0.50 to 0.37 in cucumber and 0.42 in tomato,respectively,the degradation(R)-flufiprole was faster than its antipode.5.Stereoselective kinetic dissipation and enzyme kinetics of flufiprole enantiomers in rat liver microsomes were investigated.The metabolism of flufiprole enantiomers also aligned with the first-order kinetics equation(R2=0.9533-0.,582).More rapid metabolism of(S)-isomer was catalyzed in the incubation system.The in vitro elimination half-lives(t1/2)of(S)flufiprole and(R)-flufiprole were 14.1 min and 19.3 min,respectively,and the EF values reached 0.69 finally.The Michaelis-Menten model was applied to evaluate metabolic rate constants,and the Vmax and Km of(S)-flufiprole was approximately 1.7 and 1.6 times of(R)-flufiprole,respectively.In addition,CLint of(S)-isomer was also higher than its antipode.The results suggested that the rat liver microsomes have a stronger metabolic ability to(S)-flufiprole,and there was no chiral conversion between the two enantiomers in the reaction process.6.The mechanism of stereoselective bioactivity of flufiprole enantiomers was illustrated using molecular simulations.A homopentamer model for fruit fly RDL was generated by homology modeling,and molecular docking was applied to investigate interactions between the receptor and flufiprole enantiomers.The docking result showed that hydrophobic interaction between(R)-flufiprole and the amino acid residues was significantly stronger than that of(S)-flufiprole,which will lead to a better Glide Score(-5.82 kcal/mol)for(R)-isomer than the value(-5.11 kcal/mol)for(S)-isomer.Because of a large difference in the docking modes of receptor protein for each enantiomer,(R)-flufiprole was better able to block the passage of chloride ions in GABAR.The results are consistent with these of the bioactivity tests.