Design And Synthesis Of Benzoheterocyclic Fluorescent Probes Based On Structural And Conformational Changes And Their Application In Biochemical Analysis

Fluorescent probe is a kind of substance with special optical properties.It can respond with the detection substance specifically to cause the change of molecular structure and conformation.When excited by light,it can emit a specific response signal with high sensitivity and adjustable wavelength.It can emit a specific response signal with high sensitivity and adjustable wavelength after being stimulated by light.Therefore,fluorescent probes have become a kind of important means in modern biochemical analysis and detection,and have been widely used in analytical chemistry,life science and medical diagnosis and many other fields.In the process of life activities,fluorescent probes can realize the detection of biomarkers in abnormal environments（such as hypoxia,oxidative damage,etc.）.For example,abnormal increase of nitro reductase in tumor cells under hypoxia;hypochlorous acid with abnormal changes in content during oxidative stress;DNA and mt DNA damage sites caused by oxidative damage-AP sites,etc.However,the reported fluorescent probes still have some problems in the detection of these markers:（1）Nitroreductase probes targeting specific organelles are still lacking;（2）Specific selectivity and real-time monitoring of hypochlorous acid in organisms by fluorescent probe;（3）Fluorescent probes used to detect specific domains（AP sites）in nucleic acids cannot be accurately monitored in situ and respond to the problem of single signal;（4）Fluorescent probes used for biochemical analysis cannot achieve specific and ultra-sensitive monitoring of analytes at the single-cell level.In this paper,the molecular structure change,conformational change and synergistic change of the fluorescent probe are specifically triggered by the target recognition,such as the activity and domain of the measured substance to change the energy release pathway of the excited state,regulate the energy between the ground state and the excited state.Finally,the improvement and enhancement of the response signal performance of the fluorescent probe is realized,and the function of the fluorescent probe is enhanced.Realize its specific biochemical analysis and bioimaging applications such as:specific targeting,precise detection and analysis,response signal difference,real-time,sensitivity,etc.The first work aimed at the lack of specific detection of nitroreductase（NTR）probes in the mitochondria of subcellular organelles.Benzimidazole was used as the fluorophore,and nitroreductase catalyzed the reaction activity-nitro as the recognition group.Designed and synthesized a fluorescent probe MBI-NO₂ for detecting nitroreductase based on changes in molecular structure.In the presence of the electron donor NADH,fluorescent probes can be reduced by NTR to generate amino groups.Spectroscopy experiments show that there is a good linear relationship between MBI-NO₂ and NTR concentration,and the detection limit is as low as 145μg/L.In addition,the probe has good water solubility,photostability,physiological p H stability and low cytotoxicity.The probe has been successfully applied to the fluorescence imaging analysis of hypoxic tumor cells and solid tumors in vivo.The second work addressed the specific selection of hypochlorous acid for abnormal changes in levels during oxidative stress.Based on the difference of redox reactivity among-NOH,-CHO and-COOH,a fluorescent hypochlorite probe MBI-NOH was designed and synthesized by introducing hypochlorite acid specific reactive group-aldoxime into the probe to trigger electron transfer by structural change.Based on the specific reaction promoted by hypochlorous acid,the specific detection ability of the probe for hypochlorous acid was achieved,and the detection limit was as low as 5.9 nmol/L.Fluorescent probe induced oxidative bond breaking through intracellular environment-ultra-trace hypochlorous acid,and gradually reduced step by step to generate aldehyde and carboxyl groups.The progressive fluorescence response signal between three wavelengths is realized to monitor and image the exogenous and endogenous hypochlorous acid levels in cells.The third work mainly studied the identification of DNA oxidative damage site–AP site at the single cell level.In this paper,a novel probe design strategy based on rate-limiting hydrolysis reaction of benzylacrylonitrile induced synergistic changes in molecular structure and conformation is reported.Fluorescent probes that can be used to identify the defect space of biological macromolecules were designed,and more than 30 cases of 2-（4-vinylbenzylidene）malononitrile derivatives（BMN-Fluors）were synthesized.BMN-Fluors can activate its specific recognition reaction in the AP site cavity in a short time-the hydrolysis reaction of benzylidene malononitrile.Each of these molecules can be fixed in the cavity and can be self-contained in turn adjust to form different stable conformations according to the size of the cavity,complete the synergistic effect of structural changes to define conformational changes,and finally form hydrolysates of different stable conformations to emit off-on,ratio and multicolor fluorescent signals.Efficient recognition of AP sites at the single cell level was achieved according to different fluorescence signals.The fourth work is based on the above research,focusing on how to accurately detect AP sites in single-cell mt DNA（2-10 groups）with high selectivity and sensitivity.Using the strategy of conformational change limited by the molecular structure changes in the above work,a proportional fluorescence signal was achieved by adjusting the degree of torsion.A super-sensitive（0.14/1×10~5 bp）fluorescent probe（BTBM-CN₂）targeting mt DNA for AP sites was designed and synthesized using benzothiazole as the mother.The fluorescent probe enhanced its function of site-specific recognition of mt DNA damage sites by targeting mitochondrial organelles.BTBM-CN₂ can have recognition reaction and rate-limiting reaction successively with AP sites in mt DNA in a short time to realize proportional fluorescence signal monitoring.According to the proportional fluorescence signal,the probe was used to rapidly and accurately screen and classify mt DNA damaged cells by flow cytometry and to evaluate the extent of mt DNA damage in real time by comet assay.