Multifunctional Photoaffinity Labeling Molecular Probes:Design,Synthesis And The Application In Pesticide Development

Target identification of bioactive compounds is the key step in biomedical research and drug discovery.An ideal strategy in probe design is to take use of the photoaffinity labeling technology and bioorthogonal reactions.The strategy can be used to investigate the protein-small molecule interactions in situ(e.g.,in living cells)and at the same time identify unknown targets of small molecules.Ideal multifunctional probes should contain three functional moieties,namely a photoaffinity group,an active constituent and a reporter,and have the ability to covalently bind with target proteins in high efficiency,specificity,and sensitivity.On the basis of the relevant reports,we design and synthesize a series of respective functional modules,and assemble them into multifunctional probes via the formation of amide linkages.The molecular structures of these probes were confirmed by 1H NMR,13C NMR,HRMS,and IR.We also investigated the performance relative properties of these probes such as photolysis reaction and bioorthogonal reaction as well as the preliminary application in live plants.The main works are listed as follows:1.The first chapter outlines the target identification technologies,the theory of photoaffinity labeling,and the applications in target localization,imaging,isolation and identification.On the basis of this outline,the research topic of this thesis is put forward,that is the design and synthesis multifunctional photoaffinity labeling molecular probes.The outcomes of this work are expected to provide some foundation for the research of herbicide design and target protein identification.2.According to the relevant literatures and the availability of starting materials,two aliphatic diazirine compounds(2-2a and 2-2b)were synthesized,and their molecular structures confirmed.Taking use of UV-vis spectroscopy,the photolysis processes were monitored,and the photophysical parameters such as their maximum absorption wavelength λmax,molar absorption coefficient ε,and photolysis kinetics constant k determined.3.Respective functional modules were assembled into multifunctional probes by take the advantage of amide linkage,orthogonal reaction,and click chemistry strategy.The probes and their respective reference compounds were confirmed structurally by 1H NMR,13C NMR,HRMS,and IR.The probe molecular scalffold can be used as a universal platform for the rapid and convenient construction of various probes by changing the bioactive component.4.Several fluorophore tags were screened and some fluorescent compounds with turn on-off effect synthesized.It turned out that BODIPYs as the fluorescent tags have the remarkable turn on-off effects.Click reaction kinetics of the functional cyclopropylene and tetrazine compounds was also studied using UV-vis spectroscopy,giving a secondary kinetic constant k = 2.73 ± 0.2 M-· s-1.5.Auxin antagonist α-tert-butoxycarbonylaminohexyl-indole-3-acetic was choosed as the bioactive component and introduced into the probe molecule for potoaffinity labeling.Molecular docking analysis indicateed that the binding pocket is large enough to accommodate this photoaffinity tag appended to the bioactive molecular scaffold.By using the method of indoor petri dish test,the herbicidal activity of the probes was assayed.The results demonstrated that all probes showed similar inhibitory activity compared to the original auxin antagonist and do not affect the biological activity of small molecule.One of the probes was applied to image the possible binding sites in Arabidopsis,and thus the distribution of TIR1 protein in arabidopsis root was preliminary identified.