| About 40% of current-use pesticides are chiral, and this ratio is yet increasing as compounds with more complex structures are introduced. Due to their stereoselective interactions in biological systems, enantiomers often behave drastically differently in their toxicity and environmental fate. Therefore, in order to reduce pesticide use and protect the environment from unintended effects, it is greatly advantageous to separate and semi-prepare the enantiomers of chiral pesticides. It is the aim of this study. In this study, we choose two type pesticides as the study target. The first one is organophosphorus pesticides (OPs), which represented by five synthesized 1-(substituted phenoxyacetoxy) alkylphosphonates herbicides and one commercial insecticides trichlorfon. The other one is organochlorine pesticides (OCPs), represented by acetofenate (AF). AF is the only one OCP which was commercial in China. In this article, enantiomeric separation and preparation of chiral pesticides by high performance liquid chromatography (HPLC) were investigated. After preparation of pure isomers, we also investigated the enantioselective toxicity of these two type pesticides in vitro and in vivo model. The detail steps were listed as follows.With four commercial polysaccharide-based chiral stationary phases (CSPs: Chiralpak AD, Chiralpak AS, Chiralcel OD and Chiralcel OJ), we investigated the enantiomeric separation of five alkylphosphonates by HPLC. All the analytes investigated obtained baseline resolution (Rs > 1.5) on Chiralpak AD column, which showed best chiral separation capacity. Further investigation was carried out on Chiralpak AD to evaluate the effects of the mobile phase composition and column temperature. The results showed that compounds which have different structure could have different chiral discrimination. And the decrease of organic additive content showed a dramatic increase in retention, but a better resolution factor. The thermodynamic parameters also provide an understanding of the thermosynamic driving forces for enantiomeric separation. The chromatographic condition that isopropanol as the organic modifier at lower temperatures was found to be the preferred set of conditions for separating the enantiomers of chiral OPs. The resolved enantiomers were distinguished by their signs of circular dichroism.We also prepared the pure optical enantiomers of five alkylphosphonates under the best chromatographic condition which have been shown in the former part. Then 48h acute aquatic toxicity of enantiomers and racemate to Daphnia magna (D. magna) were assessed. It was found that these five OPs have the similar structure, but toxicities to D. magna differ largely. Especially for compound 3, it was about 2~148.5 times more toxic than the other four analogues to D. magna. Furthermore, in the research of the inhibition of target enzyme acetylcholinesterase (AChE, in vitro), it is interesting to find that these five OPs showed lower inhibition to AChE. And compound 3 which show the highest toxic to D. magna has little inhibition to AChE. But compound 5 which show the lower toxic to D. magna has highest inhibition to AChE.We also prepared the pure optical enantiomers of trichlorfon on Chiralcel OJ column by HPLC. And target enzyme AChE was used as the in vitro model to investigated inhibition potential of trichlorfon and its enantiomer. Stereoselectivity was found to be on the inhibition of AChE by trichlorfon, and the toxic order was (–)–trichlorfon﹥(+)–trichlorfon﹥racemate. Furthermore, we calculated the dissociation constant of the intermediate complex (Kd), phosphorylation (kp) and bimolecular inhibition (ki), and the constants indicated that both the spatial orientations and phosphorylation properties of different stereoisomers towards AChE were different.At last part of the study, we investigated the possibility of enantioselective oxidative stress and cytotoxicity induction by AF on the in vitro model PC12 cells. After exposure of highest AF for 24h, the results showed that S-(+)-AF, R-(–)-AF and (±)-AF led to decrease in cell viability by 50.7%, 27.5% and 38.2%, respectively. And AF also showed enantioselective oxidative stress to PC12. Especially for the highest concentration, ROS induced by S-(+)-AF was 1.17 times higher than R-(–)-AF; the SOD activities of PC12 cells decreased by 32.9%, 16.8% and 30.2% when exposed to 10-5 mol/L of S-(+)-AF, R-(–)-AF and rac-AF; the activity of CAT in cells treated with S-(+)-AF was significantly lower than in cells with R-(–)-AF and rac-AF; S-(+)-AF, R-(–)-AF and rac-AF increased the MDA levels in cells by 1.46-, 1.17- and 1.23-fold, respectively.Therefore, when we develop new chiral pesticides or use commercialized chiral pesticides, it is important to investigate the enatioselectivity in their fate and effects in the environment in order to obtain higher activity but lower toxicity enantiomers. It not only decreases the environmental contaminant but also economize on energy use. It meets the requirements of sustainable development and cleaner production.
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