Target Oriented Design,Synthesis And Bioactivity Study Of New EBF Analogues

As the important pests in agriculture,aphids are endowed with many characteristics,such as great variety of species,breed rapidly,extensive host plants and so on.Aphids are the primary insect vectors of plant viruses.The honeydew,secreted by aphid,can induce plant mycosis and has an effect on plant photosynthesis.Therefore,it is necessary to control and prevent effectively aphids.However,the over application of aphicides in farm resulted in serious problems on the security of food and the ecological environment.Due to the problems caused by traditional aphicides such as resistance and toxicity to non-target organisms,especially honeybee,the development of new strategy for aphid control is of great significance.Aphid alarm pheromone,with main component of(E)-?-farnesene(abbrev as EBF),can let the same kind of other individuals to leave the host plants and stop feeding.In addition to alarm activity,EBF also has toxicity to aphids at high dosage,and synergistic effect on aphid when mixed with insecticides.EBF has aroused people' s attention thanks to its advantages such as strong selectivity,low dosage and good safety to non-target organism.However,its application in field is hindered by its shortcomings of easy oxidation and instability due to the conjugated double bonds.Therefore,it is necessary to develop novel EBF analogues with good activity and stability.In recent years,as the constant development on molecular mechanism of insect olfactory system,people realized that insects can percept and recognize odor molecules in the environment and produce the corresponding behavioral responses,which maybe play an important role in the regulation of insect behavior and pest control.Studies have shown that aphid odorant binding proteins(OBPs)and olfactory receptors(ORs),which might be the target of EBF,are closely related to the aphids' behavior.Especially,the crystal structures of two aphid OBPs(MvicOBP3 and NribOBP3)have been identified in recent years.This is a good basis for the rational design of the new EBF analogues and the study of new green aphids control agent.In the early research period in our group,some EBF analogues with high activity have been found following by the structure modification and optimization in various aspects.Based on the previous result,in this paper,oriented by the key target OBPs and lead EBF,several new types of EBF analogues were rationally designed and synthesized for the purpose of discovering new green aphid control agent with excellent activity.The biological activities of all EBF analogues were carried out and their mechanism was preliminarily studied through the protein binding experiment and molecular docking technology.In the meantime,the activity of the high active EBF analogues was deeply studied both in greenhouse and field.The main innovative results of this thesis are presented as follows:(1)Based on the latest research results of OBPs and the early work in our lab,four types of target molecules were rationally designed and synthesized using the principles of connecting sub-structure and bioisosterism.?To further study the role of heterocycles on activity,different types of heterocyclic rings such as oxadiazine,imidazole,furan,thiophene,pyrrole,pyridine and 1,3,5-hexahydrotriazine were introduced to replace the conjugated double bonds in EBF.33 EBF analogues ? containing heterocycles were designed and synthesized;?The prophase results of molecular docking indicate that hydrophobic effect is the main interaction between small molecule and protein OBP3,and hydrophobic ester is favorable to binding activity.At the same time,the literature reported that methyl salicylate had an apparent repellent effect on aphids.Therefore,29 EBF analogues ? containing salicylic acid and ester group were designed and synthesized;?According to the principle of bioisosterism,19 EBF analogues ? containing salicylic acid and amide group were designed and synthesized by replacing-O-in target II structure with bioisosteric group-NH-;? In the view of the excellent insecticidal activity and antimicrobial activity of the isonicotinic acid(INA),INA was introduced to replace the unstable conjugated double bonds in EBF in terms of the principles of connecting sub-structure.And 31 EBF analogues ? containing INA were designed and synthesized,in which the CH2 connected with conjugated double bonds in the EBF was replaced with the bioisosteric group of O,NH or N-R.(2)The repellent activity of EBF analogues was studied.Meanwhile,to avoid leaving screening out,the insecticidal activity,anti-TMV activity in vivo and fungicidal activity of target compounds were measured.The bioactive test results showed that:?all four series compounds had repellent activity against Myzus persicae.In particular,introduced salicylic acid and ester,series ? compounds are prominent in repellent activity.For example,the compounds,?-02,?-05,?-10,?-17and ?-25,exhibited repellent activity of over 70%against Myzus persicae.Although they still showed lower activity than the lead EBF,they have higher stability for further research;Under the same substituent,the repellent activity of salicylic acid amide EBF analogues ? were lower than that of salicylic acid ester EBF analogue ?,which proved that the ester truly plays an important role in repellent activity;?All the EBF analogues showed some insecticidal activity against aphids.Some of these compounds showed obvious aphicidal activity.For instance,the compound I-A07 displayed 100%aphicidal activity,which was higher than that of control pymetrozine(70.9%)at the concentration of 150?g/mL;?The anti-TMV activity in vivo(protective effect,curative effect and inactivation effect)results indicated that most of the target compounds showed anti-TMV activity under the three modes,in which protective effect is better one.The compounds,?-05,?-17 and ?-25 displayed anti-TMV activity of 55%,higher than that of ribavirin 48%;?The series of target compounds ? showed obvious fungicidal activity against Fusarium graminearum and Botrytis cinerea.(3)The interaction characteristics between EBF analogues and OBPs were studied.?9 aphid odorant binding proteins with his-tag(ApisOBP1,OBP3,OBP4,OBP5,OBP6,OBP7,OBP8,OBP9 and OBP10)were obtained through molecular biology technology.Two of them,ApisOBP5 and ApisOBP9,were firstly obtained;?The binding activity of series I-B EBF analogues with eight OBPs have been studied preliminarily by the fluorescence competition method.The results showed that these compounds had good binding activity with three proteins(ApisOBP3,ApisOBP7 and ApisOBP9);?In order to further clarify the binding specificity of EBF analogues with the above three kinds of OBPs,their pure proteins without his-tag were obtained using molecular biology techniques via designing different primers and choosing different carrier.The binding activity between all series compounds and the three proteins was systematically studied.The binding results exhibited that EBF and some EBF analogues truly had binding affinity with ApisOBP3,ApisOBP7 and ApisOBP9.The EBF analogue,which showed good repellent activity against aphids,can be identified and combined by these OBPs.This implied that these three proteins are the potential target for EBF analogues.As the first expressed and purified odorant binding proteins from aphid,ApisOBP9 is able to recognize EBF and its analogues.Therefore,ApisOBP9 presumably work as a new potential target;?Further molecular docking studies manifested that the interaction between EBF analogues and amino acid residues of MvicOBP3 are mainly hydrophobic effect,hydrogen bonding and ?-? stacking effect.Especially,the hydrophobic effect is the main one.This result provides a reasonable explanation for the reason why the EBF analogues containing ester group had higher repellent activity.(4)The activity of the representative EBF analogue ?-05 and/or ?-02 was deeply studied.The control effect experiments were carried out in the greenhouse aphids,wheat aphids,Chinese rose aphids and the plant virus carried by aphids,respectively.The results showed that:?the compounds ?-02 and ?-05 exhibited good effect against wheat aphids,Chinese rose aphids under the low concentration in the field;?After the concentration of the two EBF analogues was reduced,the aphid control effect was not decreased.This was obviously different from the traditional aphid control agents;?The effect was not obvious after applying after 1 day.However,the control effect appeared gradually clear after 3 days.The control effect of these EBF analogues is similar to imidacloprid at low concentrations,and showed potential value for further application;?After treated with compound ?-05,the soybean leaves showed the decreased disease index of the infected soybean Mosaic virus(SMV),which indicated that the compound ?-05 had certain anti-SMV activity.In conclusion,four series of total 112 EBF analogues(105 was not reported)were designed and synthesized.All the structures of target compounds were identified through1H NMR or 13C NMR,IR and HRMS.Some target compounds showed favorable bioactivities,especially,two representative compounds ?-02 and ?-05 were found to be candidate with higher activity.Through expressing and purifying aphid odorant binding proteins,the new potential target ApisOBP9 was discovered by the fluorescence competition binding experiment.The structure-activity relationship was systematically explored by molecular docking,which could be further instructive for the creation of new aphid control agents.