【作者】 周珊珊；

【导师】 刘维屏；

【作者基本信息】 浙江大学 ， 环境科学， 2009， 博士

【摘要】 我国对5种高毒有机磷农药(organophosphorus pesticides，OPs)的全面禁用将导致国内在农药使用上的巨大空缺，因此急需开发和生产既经济有效，又环境友好的可替代品。OPs在结构上有两个特点：第一，容易因亲核试剂或其他因素而发生变化，保存难度高；第二，有40％都是手性的，而手性OPs的不同旋光异构体的药效、毒性、神经系统干扰和环境归趋等均有可能不同。因此，在新OPs的开发和生态风险评价中，稳定性和手征性问题引起了广泛的关注，是农药化学和环境科学研究的共同热点，已成为一个世界性的研究课题。本文以两种我国目前推荐使用的高毒农药的替代品——氯胺磷(CP)和蔬果磷为研究对象做了以下几个方面的研究：(1)对CP唯一的商品化制剂——30％氯胺磷乳油(CP EC)的常温贮存过程进行监控，发现其不稳定性并解释相关机制。结果表明，氯胺磷乳油的不稳定性与其助溶剂甲醇有关，引发两个后果：一方面，大部分CP与甲醇通过亲核加成生成一个新的有机磷化合物——甲基氯胺磷(MCP)，此化合物后被证实稳定且高效；另一方面，CP分解生成甲胺磷致使制剂的毒性增加。由此，30％氯胺磷乳油的应用受到很大的限制，我们提出两条解决思路：第一是从外因出发，开发氯胺磷新剂型，使其避免过于极性的环境；第二是从内因出发，以甲基氯胺磷替代氯胺磷。(2)鉴于环糊精在改善农药稳定性中的应用，本人制备、表征了CP/β-环糊精的包合物，并分别从环糊精对CP的稳定性和毒性效应的影响来论证此包合物作为CP新剂型活性成分的开发前景。结果表明，简单的固相研磨法可制得环糊精与CP的包合物，当CP包结于β-CD后，其热稳定性提高3．6倍，同时环糊精的包合对CP的活性和毒性均未产生明显影响。由此我们认为，与环糊精包合可能是开发氯胺磷新剂型的一条较好的途径，具有一定的应用前景。(3)旋光纯异构体的分离和制备是研究手性农药立体特异性构效关系和环境行为的首要前提和基本保障。本文借助多糖类商品化手性柱在HPLC上拆分并制备CP、MCP和蔬果磷的旋光异构体标样。结果表明，CP和MCP的四个立体异构体在Chiralpak AD柱上均得到基线分离，结合前人的研究我们进一步发现Chiralpak AD柱对含碳、磷两个不对称中心的OPs的手性拆分具有普遍的适用性。多糖类手性固定相对蔬果磷的手性识别能力很强，Chiralpak AD、Chiralcel OD和Chiralcel OJ三根手性柱均能有效分离蔬果磷的两个对映体，其中Chiralpak AD的分离效果最好，分离度为4．14。由上，本文采用Chiralpak AD柱制备以上三种有机磷化合物的单一旋光异构体，得到的旋光异构体标样的纯度均＞98％，可用于生物学实验。(4)在得到旋光纯异构体标样的基础上，我们进一步研究了以上三种有机磷化合物在不同毒性效应上的立体选择性。①实验首先测定了CP外消旋体，各立体异构体(峰1～峰4)及等摩尔对映体混合物(pair 1和pair 2)的体外乙酰胆碱酯酶(AChE)抑制活性及体内大型蚤毒性，均发现了明显的立体选择性。其中，异构间对AChE的抑制活性大小为峰4＞峰3＞峰2＞峰1，差异在1．1～18．1倍之间，对大型蚤的毒性大小为峰4＞峰3＞峰2＞峰1，差异在1．1～21．4之间。进一步的酶抑制动力学研究表明不同异构体对AChE的空间取向和磷酰化性能均不相同。②对MCP毒性的立体特异性研究开始关注农药对人体健康的影响，通过在体外模式下对人成神经细胞瘤SH-SY5Y的AChE和轴突生长的抑制的测定，分别评价了MCP对人体的急性和迟发性神经毒性的立体选择性。研究进一步结合MCP在药效上的立体选择性探讨外消旋MCP及其手性富集产品作为农药的可能性和价值。结果表明，外消旋MCP稳定、高效、急性毒性低，但有可能引发迟发性的神经中毒，因此作为农药使用有一定的限制。MCP各旋光异构体的急性毒性都很低，无明显差异，但在杀虫活性和迟发性神经毒性上则具有很明显的立体选择性。峰1、峰2、峰3、峰4的杀虫活性分别为其外消旋体的3．1、0．6、0．5和1．0倍，相应的迟发性神经毒性分别为其外消旋的＜0．7、0．7、42．9和9．5倍。由此我们得出，在MCP的四个立体异构体中，峰1是其外消旋体的最佳替代品。若以峰1替代外消旋MCP，农药用量节省2/3，同时人体遭受迟发性神经中毒的风险将下降。此外，峰1和峰3的等摩尔混合物pair 1的杀虫活性、急性毒性以及迟发性神经毒性均与峰1相当，从经济和技术上考虑，pair 1的开发价值大，应用前景佳，是CP的优良替代品。③蔬果磷及其制剂的稳定性已有不少研究，因此我们仅关注其生物行为对映体选择性。本文评价了蔬果磷在体外AChE、丁酰胆碱酯酶(BChE)抑制，体内大型蚤毒性及细胞模式下对人体急、慢性神经毒性的对映体选择性。结果表明，蔬果磷的体外AChE抑制活性和迟发性神经毒性很低，但对体外BChE、细胞AChE的抑制活性以及对大型蚤的毒性则较高，且具有明显的对映体选择性。但总体来讲，蔬果磷在多种毒性作用上的对映体选择性均不强烈，最大差异也小于5倍，目前无手性转化的必要。另外，蔬果磷的两个对映体在多种毒性作用上都表现出明显的拮抗效应，因此由外消旋体得到的毒性数据可能会造成对其生态风险的低估，而且，随着蔬果磷在环境中的对映体选择性降解，其对环境的毒性将会增加。综合以上，本论文有两个方面的主要成果：一方面，我们对氯胺磷制剂的贮存不稳定性给出了一个可靠而合理的解释，提出解决问题的思路并从内外因上分别给予具体尝试，为氯胺磷今后的使用提供了有效参考。另一方面，通过对氯胺磷、甲基氯胺磷和蔬果磷三个有机磷化合物的手性层面的研究，并结合其它研究者的工作，我们得到对于手性有机磷化合物的生物效应来说，立体选择性可能是个普遍现象。因此，有关手性农药的研究，只有深入到手性层面，才能反映其真实的环境行为学状况，制定出准确的环境政策、法规和标准。此外，化合物靶标药效和非靶标毒性的立体选择性的比较研究对其手性转换价值和可能性的评价也非常有效。更多还原

【Abstract】 Since the five highly toxic organophosphorus pesticides (OPs) have been banned,the demand for pesticides in China will exceed supply in the following several years.Therefore, it is greatly expected to exploit some economically viable andenvironment-friendly alternatives. OPs have two properties in their configurations:firstly, they are susceptible to react with nucleophilic reagents or others, and thus easyto change during storage; Secondly, 40% of OPs are chiral and the biological actionssuch as activity, toxicity, neurological disruption and fate in the environment of chiralOPs are stereoselective. As a result, both the stability and chirality of the new createdOPs attract worldwide attention.Our studies focused on chloramines phosphorus (CP) and salithion, two OPscurrently recommended to instead the highly toxic ones, and the detailed researchcontents and results are as follows:(1) Changes in composition and toxicity of the exclusively commercialformulation of CP, i.e., 30% emulsifiable concentrates (EC) during its storage werediscovered and the corresponding mechanism was carefully investigated. The resultsshowed that the storage instability of CP EC was probably attributable to its cosolventmethanol: On the one hand, majority of the active ingredient CP was reacted withmethanol by nucleophilic addition, forming a stable and highly active neworganophosphorus compound——O,S-dimethyl-[2,2,2-trichloro-1-methoxyethyl]phosphoramidothioate (MCP); On theother hand, some CP decomposed to methamidophos, resulting in toxic potentiationof CP EC during storage. These results give us two information: first, the use of 30%CP EC is advised to be limited: second, either reformulation of CP in a lesshydrophilic environment or using MCP instead of CP may be available for resolvingthe problem.(2) In view of cyclodextrins ability to improve the stability of pesticides,CP-β-cyclodextrin (CP-β-CD) complex was prepared and identified, and the effects ofthe encapsulated process ofβ-CD to the thermal stability, activity and toxicity of CPwere also evaluated. The results showed that the degradation rate of CP in 14-daysincubation at 54±1℃was slowed by a factor of 3.6 when it was inclusion complexed withβ-CD. Meanwhile, no significant adverse effects on the bio-efficacyof CP were caused by the encapsulation process. All these results indicate thatCP-β-CD is promising to be produced as the active ingredients of various formulationadditives of CP for its continuous application.(3) Preparation of optical pure isomers is the prerequisite for studying thestereoselectivities of chiral pesticides in their structure-activity relationships andenvironmental behaviours. In this thesis, milligram scale amount of individual opticalisomers of CP, MCP and salithion were prepared on several polysaccharide modelchiral stationary phases (CSPs). Separation of all the four stereoisomers of CP andMCP were coincidentally accomplished on the Chiralpak AD column, with theresolutions (R_s) between either two adjacent peaks＞1.5 which suggests a baselineresolution. Addition to the previous studies, we further found that Chiralpak ADcolumn seems commonly available to separate stereoisomers of chiralorganophosphorus compounds with two chiral centres on the phosphorus and carbonatoms. The enantiomers of salithion were then successfully isolated on the ChiralpakAD, Chiralcel OD and Chiralcel OJ columns, among which the Chiralpak AD columnalso offered the highest R_s. Therefore, in our study we chose the Chiralpak ADcolumn to prepare the individual optical isomers of the above three organophosphoruscompounds. The purity was then calculated to be more than 98% for all isomersprepared for toxicity assays.(4) Based on the successful preparation of the individual optical isomers, thestereoselectivities in various biological activities of CP, MCP. and salithion werestudied.①The inhibition on acetylcholinesterases (AChE, in vitro) and the acuteaquatic toxicity to Daphnia magna (in vivo) tested with optically pure isomers of CPshowed its stereoselectivity. The inhibitory potentials toward AChE decreased in theorder of pk 4＞pk 3＞pk 2＞pk 1. In comparison, the acute toxicity to D. magna wasin the order of pk 3＞pk 2＞pk 1＞pk 4. The stereoselectivity was found to beisomer-dependent, with 1.1-18.1-fold differences (in vitro) and 1.1-21.4-fold differences (in vivo) among the stereoisomers. The enzyme inhibition dynamic assaysfurther indicated that both the spatial orientations and phosphorylation properties ofdifferent stereoisomers towards AChE were different.②Studies of stereoselective toxicities of MCP became focus on human health.The acute cholinergic crisis and organophosphate-induced delayed neuropathy(OPIDN) of racemic MCP and its four stereoisomers to humans were evaluated by theinhibitory potentials to acetylcholinesterase (AChE) and axon-like outgrowth inSH-SY5Y human neuroblastoma cells, respectively. Their insecticidal activities wereadditionally measured in order to evaluate the possibility and worth of racemic MCPor its single- or enriched-enantiomer products to be a new pesticide. The resultsshowed that racemic MCP was stable, high active against insects and low acute toxictowards humans. However, the high threat to OPIDN makes its pesticidal use doubtful.The acute toxicities of the four stereoisomers to humans were all low and unselective.However, their insecticidal activities and delayed neurotoxicities were extremelydifferent. Hereinto, the relative insecticidal activities between the racemate and thestereoisomers were 3.1-, 0.6-, 0.5-, and 1.0-fold for pk1, pk2, pk 3, and pk 4,respectively. While the corresponding ratios in the delayed neurotoxicities were＜0.7,0.7, 42.9, and 9.5-times, respectively. In view of its highest activity and lowesttoxicity, pk 1 is the optimal substitute for the racemate among the four stereoisomers.It can be determined that if racemic MCP is replaced by pk 1, two-thirds of the usualpesticide amount can be saved in addition to a better prevention for humans from thedelayed neurotoxic risks. Furthermore, the insecticidal activity and the potencies ofcholinergic toxicity and neuropathic hazard of pair 1. i.e., an equimolar mixture of pk1 and pk 3, were all comparable with those of pk 1. Considering the absence of theeconomically feasible manufacturing of plants of single stereoisomers, pair 1 of MCPshows considerable worth for future applications and may be proper to instead of CP.③Enantioselective toxicities of salithion were also evaluated. In this study, wechose five biological endpoints, including the toxicities towards AChE (in vitro),butyrylcholinesterase (BChE, in vitro) and D. Magna (in vivo), and the inhibitorypotentials against AChE and axon growth in SH-SY5Y. The restults showed that both the in vitro anti-AChE potentials and delayed neurotoxicities of the racemic salithionand its enantiomers were low, while their toxicities towards BChE, cell AChE, and D.Magna were high and selective. More concretely, the enantioselectivities of salithionin various biological behaviours were all less than a factor of five, suggesting that thechiral switch of salithion may be not necessary. Moreover, an antagonistic interactionis likely to be exist between the enantiomers of salithion during many toxic actions,which may result in an underestimation of ecological risks when using the dataderived from the racemate. It can also be predicted that the toxicity of salithion to theenvironment will increase during its enantioselective degradation.To sum up, our work gained two achievements: First, we offered a credible andrational explanation for the instability of CP EC during storage, and this is useful for acontinuous application of CP; Second, data of the stereoselective bioassays of CP,MCP and salithion implied that stereoselectivity might be ubiquitous for thebiological actions of chiral OPs. Therefore, we suggest that only the studies whichtake the stereoselective environmental behaviors into account can offer acomprehensive assessment of the ecotoxicology risks of chiral pesticides. Moreover,comparative studies of the stereospecific toxicities against the target and non-targetorganisms are also useful to assess the worth and possibility of chiral switch ofpesticides.更多还原