Preparation,Anion Recognition And Lipid Droplet Imaging Of Coumarin/Aurone Derivatives

In this thesis,small molecular fluorescent probes based on coumarin/aurone skeleton were investigated.Coumarin-based fluorescent probes have been widely paid attention because of their good luminescence properties and excellent biocompatibility.Many coumarin-derived fluorescent probes have been developed to monitor analytes.Even aurone derivatives also have good fluorescence properties and excellent biocompatibility,there are few aurone probes reported for ion detection,which need be further developed.Lipid droplet organelles play a key role in regulating the storage and metabolism of neutral fat,protein degradation,membrane construction and maintenance,and signal transduction.Lipid droplets are related to cancer,obesity,diabetes and inflammation.Therefore,confocal imaging of lipid droplets in vivo is of great significance.Based on the important role of bisulfite and hypochlorite in the organism,strong fluorescence and excellent biocompatibility of coumarin and citrone derivatives,four kinds of small molecule fluorescent probes based on coumarin/aurone derivatives were designed and synthesized.Their photophysical properties and ion recognition properties were discussed in detail,and their recognition mechanism and intracellular imaging were studied.Their application in confocal imaging of lipid droplets was discussed.（1）Coumarin cyanopyridine derivatives 1 and 2（2）Tetrastyrene borate ester derivatives 3-5（3）Aurone derivatives 6-8（4）Coumarin aurone derivative 9In the system of coumarin cyanopyridine derivatives,compounds 1 and 2 were confirmed by ¹H NMR,¹³C NMR and high resolution mass spectrometry.Two kinds of crystal 1a（blue block,triclinic system,P-1 space group）and 1b（red plate,monoclinic system,P21/n space group）were obtained from acetonitrile（1a）and dichloromethane（1b）by solvent evaporation method,respectively.Compound 1 has a high fluorescence quantum yield（0.22）in n-hexane.The fluorescence peaks in different solvents are linearly related to solvent polarity,and the linear correlation coefficient is high,which can track the environmental polarity.Compound 1 can identify bisulfite（HSO₃^-）in HEPES buffer solution.In situ ¹H NMR and mass spectrometry analysis show that when HSO₃^-is added to the C=C bond,the charge transfer from diethylamine coumarin to pyridine group is interrupted,resulting in strong blue fluorescence of coumarin.Compared with compound 1,compound 2 has weak fluorescence emission and low quantum yield,as well as fluorescence quenching response to HSO₃^-,which limits its application.Compound 3-5,three fluorescent probes with tetrastyrene structure,were designed and synthesized.Due to the presence of tetrastyrene structure,all three compounds show aggregation-induced emission（AIE）characteristics,with the highest fluorescence intensity and red shift of fluorescence peak in water.Compounds 3-5 all emit green fluorescence,and the absolute fluorescence quantum yields are 30.89%,63.02%and 22.46%,respectively.The green fluorescence emission of compounds 3-5 in the methanol-water mixture increases with the increase of the proportion of poor solvent water.The green fluorescence emission of compounds 3-5 in the methanol-glycerol mixed system gradually increases with the increase of glycerol proportion,indicating that the environmental viscosity restricting the intramolecular motion and leading to enhanced fluorescence.Further selectivity and titration experiments showed that all the three compounds could recognize ClO^-in PBS buffer solution,and compounds 3-5 were found to be fluorescence quenching probes by titration test.All the three compounds have good selectivity and sensitivity,among which,compound 5 has a low fluorescence detection limit and achieves stability in the shortest time.Therefore,compound 5has a better recognition property for ClO^-.The recognition mechanism is that ClO^-promotes the oxidation and hydrolysis of borate ester,then the group of borate ester is removed and the hydroxyl group is exposed,which leads to the increased water solubility of the molecule,the disintegrated aggregation state of the molecule,and then quenched fluorescence.Three two-photon fluorescence compounds 6-8 were designed and synthesized as efficient lipid droplets（LDs）imaging probes in the system of aurone derivatives.Compounds6-8 exhibit distinct solvation discoloration effect from blue to orange emission（about 140 nm red-shift）in various solvents.The compounds have low toxicity to living cells,high photobleaching resistance and excellent photostability,and can target LDs with high specificity.In addition,compound 6 was used to monitor the morphological changes of LDs and the interaction between LDs and mitochondria in real time under specific physiological conditions（e.g.,starvation）.We have for the first time observed the dynamic changes between mitochondria and LDs when excessive H₂O₂causes mitochondrial damage in a short period of time.A compound 9 was designed and synthesized from the coumarin aurone derivatives system.In compound 9,aminoaurone is used as the energy donor,diethylaminocoumarin as the energy acceptor and C=N bond as conjugated bridge.The fluorescence peak of compound9 is vibrated under short wavelength excitation by changing different excitation wavelength.Compound 9 can recognize ClO^-in the mixed system of methanol/water（9/1,v/v）.After ClO^-is added to the probe,the solution color changes significantly.Under natural light,the color of the solution changes from yellow to colorless,and under 365 nm ultraviolet lamp,it changes from yellow to blue.When ClO^-interacts with compound 9,C=N bond breaks,carboxyl group and amino group are exposed,so the fluorescence peak is blue-shifted and the blue fluorescence is enhanced.