Design And Application Of Fixable Fluorescence Probes Targeting Subcellular Organelles

With the rapid development of optical imaging technology,fluorescent probes have been widely used in biomedicine.However,most of the fluorescent probes do not stably localize in subcellular organelles,and there are short absorption or emission wavelengths of fluorescent dyes.In view of these deficiencies,this paper designs the organic molecules to target the Snap-tag fusion proteins,near infrared fluorescent dyes to label the cells,and the probes to fix on the proteins in subcellular organelles via covalent bonds.1.The general intermediates,named PYBG and ADBG,are synthesized.And the fluorescent dyes or probes,named PYBG-TMR,PYBG-BODIPY,ADBG-Np-N,ADBG-Np-N+,ADBG-Np-OH,PYBG-SF,PYBG-NO,PYBG-CIO,and PYBG-Vis,are obtained via'Click' and 'Sonogashira Coupling' reaction between traditional dyes and the general intermediates.PYBG-TMR,PYBG-BODIPY and ADBG-Np-OH have been used to specifically label Snap-tag proteins in the subcellular organelles.PYBG-NO is successful to detect NO in the living cells.And PYBG-CIO also successfully detects hypochlorite in living cells.PYBG-Vis detects the difference of the viscosity around Snap-tag proteins in different subcellular organelles via fluorescence lifetime imaging.2.A series probes for cellular microenvironment,named N-Vis,Cl-N-Vis,M-Vis-A,M-Vis-B,CM-Vis-A,M-O-A and M-O-B,are synthesized.There are good linear relationships between the fluorescence lifetimes of the probes(N-Vis,Cl-N-Vis,M-Vis-A,M-Vis-B,and CM-Vis-A)and the viscosity.N-Vis cand Cl-N-Vis localize in the lipid droplets of MCF-7 cells and HL7702 cells.And N-Vis monitors the changes of viscosity in the lipid droplets of HL7702 cells which are cultured in alcohol and palmitic acid via fluorescence lifetime imaging.M-Vis-A and M-Vis-B accurately target the mitochondria.And the probe,M-Vis-A,contains a reactive aldehyde group,which can react with the amino group to generate stable covalent bond to localize stably in the mitochondria.The fluorescent probe,M-Vis-A,monitors the increased viscosity after the cells are stimulated by rotenone or CCCP.The probes,M-O-A and M-O-B,are sensitive to oxygen and accurately target the mitochondria.The probes,M-O-A,contain benzyl chloride which can react with the proteins to generate stable covalent bonds.And M-O-A successfully monitors the changes of oxygen concentration via ratio fluorescence imaging in living cells.3.The near infrared fluorescent dyes,named N-NIR,S-NIR and K-NIR,are synthesized by designing the structures of indole,quinoline and thiazole.The fluorescent dyes,N-NIR and S-NIR,have high molar extinction coefficients and high fluorescence quantum yields;the maximum absorption wavelength of fluorescent dye,K-NIR,is close to 800 nm.And the platform dye which contains a bromo,named Br-N-NIR,is also obtained.The near infrared Snap-tag substrate,named PYBG-NIR,is also obtained from the reaction between Br-N-NIR and PYBG.PYBG-NIR can specifically label Snap-tag proteins in living cells.The near infrared fluorescent probes,named pH-A-NIR and pH-B-NIR for pH are successfully synthesized.And they are successfully applied to monitor the change of pH in the subcellular organelles.