Design,Synthesis And Optical Control Of Biological Activities Against Phytopathogen Of 1,3,4-oxadiazole-containing Azobenzene Derivatives

Agriculture accounts for a large proportion in national economy of china,and pesticides are an important tool to guarantee agricultural production.However,the increasingly common problem of resistance has restricted modern crop production.The persistent usage of a high dose of active microbicides causes their continuous accumulation in the environment,inevitably causing problems such as resistance and environmental toxicity.Therefore,how to skillfully design activity-controllable molecules and accurately control the activity of these molecules,so that they do not affect other environments after exerting their effects,is of great significance for safety application of microbicides and mitigation of resistance.The development of photocontrollable microbicides may provide a new insight to reduce the problems of side effects and resistance.The use of"photoswitches"in the field of medicine to control the biological activity of molecules can reduce the environmental toxicity of drugs and the resistance of bacteria to achieve unprecedented precision treatment.To this end,the design of photocontrollable microbicidal molecules is bound to promote the development of environment-friendly microbicides,effectively solving the existing defects of microbicidal residues and resistance.In this study,an antimicrobial active fragment 1,3,4-oxadiazole was combined with a photoswitch of azobenzene to design a series of novel 1,3,4-oxadiazole-containing azobenzene derivatives.The photoisomerization of the target compound and the reversibility of the process were studied.On this basis,the in vitro photocontrolled antimicrobial activities of the compounds against several phytopathogens were tested,and the compounds with obviously photocontrolled effect were selected to optically control the crop diseases in vivo.The photocontrolled antifungal mechanism of 1,3,4-oxadiazole-containing azobenzene derivatives was further studied by cell membrane permeability measurement and mycelial morphology observation,and by computational simulation.The detailed research contents and experimental results are as follows:?1?The raw material of 4-nitrobenzoyl hydrazide was reacted with 4-fluorobenzaldehyde and 3,4,5-trimethoxybenzaldehyde to form acylhydrazone.Followed by the reaction of oxidative ring closure,reduction,diazotization,coupling,and methylation,eleven novel 1,3,4-oxadiazole-containing azobenzene derivatives were synthesized.The structures of all compounds were confirmed by ¹H NMR,¹³C NMR,and HRMS.?2?The photoisomerization properties of the compounds were qualitatively and quantitatively studied using an ultraviolet-visible spectrophotometer and a nuclear magnetic resonance spectrometer.By comparing the changes of UV-Vis spectra before and after illumination,it was found that the intensity of?-?^*transition absorption peaks of all compounds decreased after illumination,indicating that the compounds had been photoisomerized from trans to cis forms,and the spectrum changes were influenced by the substituents of the ortho position of the bond of azo.By comparing the changes of the ¹H NMR spectrum before and after irradiation,it was found that the compounds generated new signal peaks.The proportion of cis isomers obtained by the illumination of the 3,4,5-trimethoxy series of compounds were less than those of the 4-fluoro series compounds,indicating the effect of steric hindrance on the cis-trans isomerization.By analyzing the changes of the UV-Vis spectrum over time after reaching the cis photo stationary state?PSS?,it was found that the process of cis-trans isomerization of compounds was reversible.The half-life of isomerization from cis to trans form related to whether the ortho position of azo were replaced with-OCH₃and the number of-OCH₃ on other substitution sites.?3?The photocontrolled antimicrobial activities of the target compounds against Xac and B.cinerea in vitro were determined by turbidity method and mycelial growth inhibition method,respectively.The auto-inactivation effects were also investigated.The results indicated that compound 4 showed relatively significant photoactivation against Xac,and its activity of cis isomer was increased by 17.2%relative to the trans isomer at 100?g/mL.In addition,the molecule was spontaneously inactivated over time after being activated.Compound 2,4,5,10,11 showed significant photoactivation against B.cinerea.At a concentration of 200?g/mL,the inhibition rates of cis-PSS of compounds 4 and 5 against B.cinerea were 4 times higher than those of the trans PSS.Moreover,the compounds can be automatically inactivated over time after activation by photoactivation,which can reduce the accumulation of active fungicides in the environment.On this basis,compound 4 was selected as an activity-controllable antifungal agent to investigate its optical control of gray mold in vivo on tomato and cucumber.The results showed that compound 4 had photocontrollable properties against gray mold on tomato and cucumber,and the protection efficacy of the irradiated group was significantly better than that of the unirradiated group.?5?The damage to the cell membrane of B.cinerea after treated with different isomers of the compounds 4 was investigated by the determination of the conductivity of the mycelium solution and the observation of morphology of the mycelium.The results showed that the cis-PSS of compound 4 leads more damage to the membrane of B.cinerea compared with trans-PSS.Meanwhile,the observation of hyphal morphology also showed that the cis-PSS could cause more obvious morphological changes on the cell surface.The molecular surface property changes and the difference in binding to the receptor BcCYP51 accompanying the isomerization of compound 4 were further studied by computational simulation.The results showed that compound 4 caused changes in the physical and chemical properties such as molecular surface configuration,the interatomic distance,charge distribution,and lipophilicity after photoisomerization.The docking results indicated that the docking scores of the compounds with BcCYP51 were basically consistent with the results of the activity test.Compared with the trans-PSS,the cis-PSS of compound 4 displayed more strongly interaction with the target enzyme,which forms hydrogen bonds and?-?stacking interactions with residues around the active pocket.