手性农药氯氟醚菌唑是广谱高效的新型三唑类杀菌剂，具有广阔的应用前景，但目前就其对映体水平上的研究甚少，缺乏系统和全面的研究，限制了其高效低毒的光学纯农药的开发与应用。本论文以氯氟醚菌唑对映选择性生物效应及降解行为研究为切入点，建立了氯氟醚菌唑手性对映体的分离检测方法并明确其绝对构型，进而研究了其靶标生物活性、非靶标生物生态毒性对映选择性及其在田间蔬菜和天然水体中的对映选择性降解行为；此外，以大鼠肝微粒体和斑马鱼为模型从药物代谢水平阐释了氯氟醚菌唑在生物体中的对映选择性生物富集及代谢行为。研究结果可为氯氟醚菌唑对映体水平的风险评估提供数据支撑，对氯氟醚菌唑高效、低风险、低残留异构体的开发及其科学、合理使用提供科学指导作用。主要研究结果如下：

1、利用超高效液相色谱串联质谱（UPLC-MS/MS）技术建立了在Chiralpak IG-3手性柱上氯氟醚菌唑对映体分离的方法：当流动相为0.1%甲酸水溶液/乙腈=10/90（v/v）、流速0.5 mL/min、柱温35 ℃时，氯氟醚菌唑对映体可在10 min内实现基线分离，分离度为1.82。通过旋光检测器和电子圆二色光谱仪确定了对映体的旋光性和绝对构型，明确了氯氟醚菌唑对映体在Chiralpak IG-3手性柱上的洗脱顺序为R-(-)-氯氟醚菌唑和S-(+)-氯氟醚菌唑。

2、利用菌丝生长速率抑制法研究了氯氟醚菌唑对映体对6种靶标真菌的生物活性。结果表明，R-氯氟醚菌唑为高效体，其抑菌活性为S-氯氟醚菌唑的5.3~473.1倍，为外消旋体的1.5~2.1倍。与S-氯氟醚菌唑相比，R-氯氟醚菌唑可以更加有效地抑制番茄灰霉病菌麦角甾醇的生物合成。通过同源建模技术结合分子对接技术研究了氯氟醚菌唑对映体与番茄灰霉病菌靶标蛋白（CYP51B）之间的结合亲和力差异，结果表明R-氯氟醚菌唑能与CYP51B蛋白形成更稳定的结合构象，结合能更低，对靶标酶的抑制作用更强，可能是导致氯氟醚菌唑产生对映选择性生物活性的主要因素。

3、以斑马鱼、蛋白核小球藻和紫背浮萍为供试非靶标生物，研究了氯氟醚菌唑对映体对水生生物的急性毒性差异。结果表明，氯氟醚菌唑对不同发育时期的斑马鱼的毒性顺序均表现为S-氯氟醚菌唑>Rac-氯氟醚菌唑>R-氯氟醚菌唑，且S-氯氟醚菌唑对斑马鱼胚胎、仔鱼和成鱼的毒性分别是R-氯氟醚菌唑的2.5、2.4和2.0倍。氯氟醚菌唑对蛋白核小球藻和紫背浮萍的生长抑制限度试验结果表明，其对水生植物蛋白核小球藻和紫背浮萍不存在明显的毒副作用。

4、通过田间试验研究了氯氟醚菌唑在黄瓜、辣椒和土壤中的对映选择性降解行为。结果表明，氯氟醚菌唑对映体在植物中的降解遵循一级动力学方程，在黄瓜样品中，S-氯氟醚菌唑优先发生降解，其对映体分数（EF）值由初始的0.50显著增加到0.53，而在辣椒样品中，氯氟醚菌唑两对映体之间不存在选择性降解。田间土壤中的氯氟醚菌唑在施药后35 d内未发生显著性降解行为。此外，开展了长江、黄河、巢湖和黄海水域水样在好氧和灭菌条件下的室内孵育实验。结果表明，灭菌条件下，氯氟醚菌唑在四种水体中均未发生显著性降解行为；而在好氧条件下，氯氟醚菌唑在4种水体中的降解遵循一级动力学方程，降解速率大小为黄海水>巢湖水>长江水>黄河水，且对映体之间不存在对映选择性降解，表明微生物可能是其发生降解的重要因素。

5、利用体外代谢模型大鼠肝微粒体探究了氯氟醚菌唑对映体在动物中的代谢行为差异。结果表明，氯氟醚菌唑对映体在大鼠肝微粒体中的代谢遵循一级动力学方程，S-氯氟醚菌唑具有较短的半衰期而优先被代谢。其酶反应动力学实验结果进一步表明S-氯氟醚菌唑与底物酶具有更好的结合而促进其代谢的发生。此外，以斑马鱼为模式动物，研究了氯氟醚菌唑在斑马鱼体内的对映选择性富集及代谢行为。结果表明，在富集阶段，高浓度下R-氯氟醚菌唑和S-氯氟醚菌唑富集系数分别为8.38和3.41，低浓度下分别为1.02和0.62，均表现为R-氯氟醚菌唑优先在斑马鱼体内被富集；在清除阶段，低浓度处理组中R-氯氟醚菌唑具有较快的代谢速率和较短的半衰期而优先被代谢排出体外，而高浓度条件下则未表现出对映选择性代谢行为。同时，氯氟醚菌唑两对映体均会在一定程度上引起斑马鱼体内CAT酶活性显著下调和MDA含量显著上调，从而诱导其氧化应激反应。进一步通过高分辨质谱技术鉴定了氯氟醚菌唑在动物体内和体外代谢相关的9种代谢产物，推测氯氟醚菌唑在斑马鱼体内主要代谢途径是通过羟基化反应生成Ⅰ相代谢物，进而在磺基转移酶的作用下与硫酸基团结合生成硫酸酯化合物，此外也能与葡萄糖醛基结合生成葡萄糖醛基络合物；但是在大鼠肝微粒体的体外代谢过程中，未发现Ⅱ相代谢产物。

The chiral pesticide mefentrifluconazole is a new type of triazole fungicide with broad spectrum and high efficiency, which has been used widely over the world. At present, there were few studies at the enantiomer level and lacked of systematic and comprehensive researches, which hindered the development and application of optically pure pesticides with high efficiency and low toxicity. In this papper, starting from the enantioselective biological effects and degradation behavior of mefentrifluconazole, we established a method for the separation and clarify the absolute configuration of mefentrifluconazole enantiomers. On this basis, two enantiomers of mefentrifluconazole were prepared for the study on enantioselective bioactivity, ecotoxicity and degradation in vegetables and water. In addition, rat liver microsomes and zebrafish were used to study enantioselective bioaccumulation and metabolism of mefentrifluconazole in organisms based on pharmacokinetics. Results in this study not only provide reference datas for the risk assessment of mefentrifluconazole at the enantiomer level, but also provide scientific guidance for the development of new pesticides products with high efficiency, low risk and low residual as well as its scientific and rational application. The main findings of this study are as follows:

1. A method for the separation of mefentrifluconazole enantiomers on Chiralpak IG-3 chiral column was established by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). When the composition of mobile phasethe was 0.1% formic acid aqueous solution/acetonitrile=10/90 (v/v), the flow rate was 0.5 mL/min, and the column temperature was 35 ℃, enantiomers of mefentrifluconazole could achieve the baseline separated completely within 10 minutes, and resolution was 1.82. The optical rotation and absolute configuration of the enantiomers were determined by optical rotation detector and electron circular dichroism spectrometer. Finally, it was clarified that the elution order of the enantiomers of mefentrifluconazole on the Chiralpak IG-3 chiral column was R- (-) -mefentrifluconazole and S- (+) -mefentrifluconazole.

2. The bioactivities of mefentrifluconazole enantiomers against six target fungi were determined by the growth rate method. The results showed that R-mefentrifluconazole had more effectively antibacterial activity, which was 5.3~473.1 times than that of S-mefentrifluconazole and 1.5~2.1 times than that of Rac-mefentrifluconazole. Compared with S-mefentrifluconazole, R-mefentrifluconazole can more effectively inhibit the biosynthesis of ergosterol in Botrytis cinerea. The binding affinity between the enantiomers of mefentrifluconazole and the target protein (CYP51B) of Botrytis cinerea were studied by homology modeling technology combined with molecular docking technology. The results showed that R-mefentrifluconazole could form a more stable binding conformation, lower binding energy and stronger inhibition with CYP51B, which may be the main factor leading to the enantioselective bioactivity of mefentrifluconazole enantiomers.

3. The acute toxicity difference of mefentrifluconazole to aquatic organisms was studied towards zebrafish, Chlorella pyrenoides and Spirodela polyrrhiza. The results showed that the order of the toxicity of mefentrifluconazole to zebrafish at different growth stages was as follows: S-mefentrifluconazole > Rac-mefentrifluconazole > R-mefentrifluconazole. The toxicity of S-mefentrifluconazole to zebrafish embryos, larve and adults is 2.5, 2.4 and 2.0 times higher than that of R-mefentrifluconazole, respectively. However, the results of limiting test on Chlorella pyrenoides and Spirodela polyrrhiza  exposure to mefentrifluconazole showed that there was no toxicity on the two species of aquatic plants .

4. The enantioselective degradation behavior of mefentrifluconazole in cucumber, cowpea, pepper and soil were studied with field trials. The results showed that the degradation of the enantiomers of mefentrifluconazole in plants followed a first-order kinetic equation. In cucumber samples, S-mefentrifluconazole showed the behavior of priority degradation, and the EF value increased significantly from the initial 0.50 to 0.53. While in cowpea and pepper samples, there was no significant difference in selective degradation between the two enantiomers. There was no significant degradation of mefentrifluconazole in soil within 35 days. In addition, the incubation experiments of water samples from the Yangtze River, Yellow River, Chaohu Lake and Yellow Sea under aerobic and sterilizing conditions were carried out indoor. The results showed that there was no significant degradation behavior of mefentrifluconazole occurred in any of the four water samples under sterilization conditions, while the degradation followed the first-order kinetic equation under aerobic conditions. The datas of half-life showed that the degradation rates were in the order of Yellow Sea > Chaohu Lake > Yangtze River > Yellow River, and there was no enantioselective degradation between the two enantiomers in these four types of water. Which may indicate that microbial activity is an important factor in promoting the degradation of pesticide compounds.

5. The enantioselective metabolic behavior of mefentrifluconazole enantiomers in animals was investigated with the in vitro metabolic model—rat liver microsomes. The results showed that the metabolism of mefentrifluconazole enantiomers followed a first-order kinetic equation in rat liver microsomes, S-mefentrifluconazole had a shorter half-life and was preferentially metabolized. The experimental results of the enzyme reaction kinetics further showed that S-mefentrifluconazole had better binding with the substrate enzyme to promote its metabolism. In addition, the enantioselective absorption and metabolism of mefentrifluconazole in zebrafish were also studied. The BCFs of R-mefentrifluconazole and S-mefentrifluconazole were 8.38 and 3.41 while 1.02 and 0.62 exposed to high and low concentration of mefentrifluconazole, respectively. And the results showed that there was a significant enantioselective absorption behavior in zebrafish, indicated that R- mefentrifluconazole was preferentially enriched in zebrafish during the exposure stage. Only in the low-concentration treatment group that R-mefentrifluconazole can discharge from the body preferentially with a faster metabolic rate and a shorter half-life during the clean-up stage. While in the high-concentration condition, there was no enantioselective metabolic behavior. At the same time, both enantiomers of mefentrifluconazole can significantly down-regulate the activities of CAT enzymes and up-regulate the content of MDA in zebrafish to some extent, thereby induced oxidative stress reaction. Further, nine metabolites related to the metabolism of mefentrifluconazole in vivo and in vitro were identified by LC-Q-TOF-MS/MS. It was speculated that the main metabolic pathways of mefentrifluconazole were through hydroxylation reaction for phase Ⅰ, and then combined with sulfate groups to generate sulfate compounds under the action of sulfotransferase. In addition, it can also combine with glucuronic group to form glucuronic complex. However, no metabolites for phase Ⅱ were found in rat liver microsomes.

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