Synthesis And Application Of Novel Fluorescent Probes Targeting Biothiols In Mitochondria

Fluorescent dyes have the advantages of strong penetration,low background interference,and good biocompatibility,they have gradually replaced the radioisotope method as the main tool of bioanalysis.At present,fluorescent probes are widely used in the fields of biomolecular detection,early cancer diagnosis and fluorescent in vivo imaging,which can realize visualization and low-damage detection in the process of biological analysis.As an important fluorescent dye,cyanine dye not only has a variety of structures,but also exhibits good selectivity,wide absorption region,small background interference and superior sensitivity.Currently,many near-infrared fluorescent probes that use cyano dyes as fluorophores have been developed and widely used in environmental analysis,intracellular small molecule labeling,in vivo imaging and clinical diagnostics.Biothiols not only are closely related to protein synthesis,cell metabolism and other life activities,but also play an important role in maintaining the normal physiological function balance and antioxidant system in cells.Cysteine(Cys),homocysteine(Hcy)and glutathione(GSH)are the three main biothiols in cells.Although their levels and specific functions in cells are different,they are extremely important for maintaining the balance of normal physiological functions in cells.As we all know,the life activities of cells rely on mitochondria to provide energy.Mitochondria are essential for cellular ATP synthesis,cell death and other activities.Its abnormality usually leads to an imbalance of redox in cells,leading to cell apoptosis and dead.Besides,biothiols of mitochondria also have an important impact on maintaining normal physiological functionsin cells.Cys and GSH in mitochondria are both vital reducing agents in mitochondria.One of their main functions is to scavenge active free radicals produced by mitochondria and protect cells from free radical oxidation.The Hcy in mitochondria is essential for maintaining the normal function of mitochondria.Abnormal mitochondrial content can cause liver damage and other diseases.At present,the reaction sites used to detect cysteine,homocysteine and glutathione are mainly sulfonyl groups,halogenated acetyl groups,acrylates and aldehyde groups,with few reaction types and high tissue permeability,stable near-infraredfluorescent probes are still scarce.In view of the important role of small thiol molecules in maintaining the normal physiological activities of cells,under physiological conditions,new reaction sites and fluorescent probes with high selectivity and specificity for biothiols,especially in mitochondria have been developed.Acupuncture has important research significance for detecting abnormal levels of biothiols in cells and understanding its role in the occurrence and development of diseases.The main tasks as follows:1.In the chapter,a new type of organic small molecule fluorescent probe Cy-FN was synthesized using 5-nitro-2-furanate as a new reaction site for targeting biothiols,the probe uses hemicyanine with lipophilic indole cation as the fluorophore.In the process of probe synthesis,the research describe the characteristics of the final probe and intermediate products in detail,and the possible reaction mechanism was verified by mass detection method.The probe has not only good selectivity for Cys/Hcy and GSH,but also a low detection limit and p H changes in the physiological range.Other amino acids will not interfere with the detection.In addition,ultraviolet absorption spectrum and fluorescence spectrum are employed to test the probe and biothiols reaction system,and optimize the reaction conditions.Finally,fluorescence imaging and co-localization experiments of biothiols in Hep G2 cell confirmed that the probe can be successfully used for fluorescence imaging to detect biothiols in cell mitochondria.2.In this chapter,the chloroacetyl group is used as the sulfhydryl reaction site,and the cyanidone is used as the fluorophore to synthesize a new type of fluorescent probe Cy-CC,which can visually detect biothiols.In the process of probe synthesis,the probes and intermediate products have been characterized in detail by mass spectrometry,and through chemical system tests,it is found that Cy-CC probes can selectively recognize biothiols.The upper sulfhydryl group will specifically attack the intramolecular ester bond of the fluorescent probe,resulting in the cleavage of the intramolecular ester bond and the conversion of the fluorophore from the enol type to the ketone structure with strong fluorescence.In order to explore whether the probe can be used to detect biothiols in cells,this chapter conducted acytotoxicity analysis,the experimental results show that the Cy-CC probe has good biocompatibility with cells.