Stability And Stereoselective Biological Activities Of Chiral Organophosphorus Pesticides

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.