Design,Synthesis And Application Of The Novel Fluorescent Ligands Acting At Binding Site Of Insecticide Chlorantraniliprole

Novel insecticides acting on ryanodine receptor features with unique chemical structure,high efficiency against pest insects and good environment safety is the hot focus of pesticide discovery at present.However,ryanodine receptor is a macrobiomolecule across membrane,and it is very difficult to obtain its 3-D configurations.Thus,it is limited to deeply study the relationship between chemical structure of active molecule and its insecticidal activity.In this dissatation,propargyl substituted intermediates were obtained through replacing methyl on the N-methyl amide of chlorantraniliprole and cyantraniliprole by propargly group,and then reacted with an azide group-containing hydroxycoumarin to get fluorescent labeled compounds of fluorescent ligands.The ryanodine receptor in leg of roach was extracted,and Western Blot was employed to confirm the existence of ryanodine receptor in extract.Insecticidal activity of fluorescent ligands were tested,and affinity binding to ryanodine receptor in housefly and roack were studied by[3H]Chlo assay and fluorescence polarization,respectively.An assay based on fluorescence polarization was established to study the affinity binding of active compounds to ryanodine receptor,the specific innovation results were obtained as follows:1?Upon the skeleton structure of Chloranitraniliprole and Cyantraniliprole,two derivatives containing terminal alkynyl were designed and synthesized,meanwhile 3-hydroxycoumarin molecule containing azide group was also designed and synthesized,and then two fluorencent ligands containing skeleton structure of chloranitraniliprole and cyantraniliporle were prepared by Click reaction(Figure 1),four new compounds Prop-Cl,Prop-CN,Fluor-Cl and Fluor-CN were confirmed by NMR and HRMS.Figure 1 Fluorencent ligands containing skeleton structure of antranilic amide insecticides2?Bioactivities of four synthesized compunds against Plutella xylostella were tested.Bioassay results showed that all these compounds had good insecticidal activities with IC50's for Prop-C1 and Prop-CN of 0.08 mg/L and 0.34 mg/L,respectively.After incorporating the fluorescent hydroxycoumarin fluorophore,the ligands were 22-32 fold less potent than the propargyl compounds,with IC50's of 2.6-7.6 mg/L.The new compounds retain good insecticidal activity despite the propargyl and large polar fluorescent substituents were introduced.Most importantly,the fluorescent probes are very potent in vitro in binding assays,although less so in vivo with penetration barriers.3?Synthesized anthranilic diamide analogs and fluorescent labeled compounds can effectively suppressaffinity binding of isotope labeled probe[3H]Chlo to housefly ryanodine receptor.Prop-Cl and Prop-CN inhibited[3H]Chlo binding with IC50's 16-40 nM,Fluor-Cl and Fluor-CN inhibited[3H]Chlo binding with IC50's of 34-39 nM,compared to 6-14 nM for Chlo and Cyan,which indicated that they all with chlorantraniliprole work at the same binding site on Musca RyRs.In addition,Fluor-Cl and Fluor-CN stimulated[3H]Ry binding with EC50's of 5-7 nM,which is consistent with the reported value of 3-15 nM for Chlo and Cyan.4?This study extracted the membranescontaining ryanodine receptor in leg of roach,and Western Blot was employed to confirm the existence of ryanodine receptor in extract.5?Through studies on various parameters,one assay based on fluorescence polarization was developed to study the affinity binding of various active compounds to ryanodine receptor.With developed method,it was verified again that chloranitraniliprole and its analogue binds the same site on ryanodine receptor.However,flubendiamide and ryanodine could not reduce the binding of fluorescent ligands on ryanodine receptor.This elucidated clearly that two classes of diamide insecticides act on different binding sites on ryanodine receptor.Through the test of new compounds,some compounds can act at binding site of insecticide chlorantraniliprole on ryanodine receptor,they also displayed good insecticidal activity against Plutella xylostella,and the results from developed assay is close related with those from biological assay against Plutella xylostella.The developed assay in this study could replace radio-labeled assay and monitoring calcium ion flux method at certain extent to study the affinity binding of insecticidal active compounds on ryanodine receptor,and could promote deep study of relevant researches.Meanwhile,it is very significant to promofe new molecules discovery acting on ryanodine receptorconveniently,safely and quickly.