Construction Of Cysteine And Sulfur Dioxide Fluorescent Probes And Their Biological Application

Active sulfur species play a vital role in the antioxidant system of the organism.Cysteine（Cys）and sulfur dioxide（SO₂）two small molecules participate in many important physiological processes.SO₂ is a common air pollutant.Excessive intake will not only damage the human eyeballs and upper respiratory tract mucosa,but also cause cardiovascular diseases,neurological diseases,cancer and other diseases.However,SO₂,as the fourth gas signal molecule,can maintain the redox homeostasis in the body,prevent and reduce tissue damage,and regulate vasodilation in the body.In cells,SO₂ is mainly produced by the metabolism of sulfur-containing amino acids.Cys is the only amino acid in the human body that contains a reducing sulfhydryl group.It is an important reducing substance.It can regulate cell functions in the organism,maintain redox homeostasis,and participate in metabolism.Under aerobic conditions,Cys in the organism will metabolize to produce SO₂.Abnormal Cys levels can cause developmental delay,brain damage,edema and other diseases.Therefore,the development of fluorescent probes that specifically detect SO₂ and Cys is of far-reaching significance for better research on the mechanism of action of SO₂ and Cys in physiology and pathology.In the first part of the thesis,based on the reversibility of the Michael addition reaction and the two conditions under which SO₂ can react with FA,a fluorescent probe for the reversible detection of SO₂/FA based on the benzocoumarin-benzopyran structure was designed（DC-BP）.The benzopyrylium cation moiety can locate both mitochondria and the reaction site of SO₂.When SO₂ attacksα,β-unsaturated double bonds,it reacts with the probe,and the probe appears a new emission peak at 574 nm.And FA can capture the free HSO₃^-in the solution,promote the dissociation of the DC-BP-SO₃H adduct,the recovery of the probe,and the disappearance of fluorescence.In the Et OH/HEPES buffer solution（5%Et OH,HEPES 10 m M,p H 7.4）,the probe can complete the response within 50 s with a detection limit of 0.051μM,which has the advantages of high sensitivity and fast response.The probe has also been successfully applied to detect the real-time reversible dynamic changes of SO₂/FA in cells.In the second part of the paper,a dual-excitation and dual-emission ratio fluorescent probe（BCT）based on the structure of a coumarin derivative for detecting SO₂ was developed.Due to the rigid benzene ring,the probe molecule itself has a largeπ-conjugated system with an emission wavelength of 640 nm;when SO₂ attacks the pyrylium ring,the probe’s conjugation is interrupted and the emission wavelength blue shifts to 428 nm.The probe has a large emission displacement（greater than 200 nm）,which avoids the measurement error caused by the two emission peaks being too close or overlapping between the two peaks.In DMSO aqueous solution,the probe can quickly respond to SO₂（within 2 min）,and the detection limit is 0.072μM.After testing,the probe molecule has excellent selectivity and has good luminescence characteristics under biologically compatible p H conditions.Therefore,the probe has also been successfully applied to the imaging of SO₂ at different concentrations in cells.In the third part of the paper,a fluorescent probe（DAnt-Cys）for specifically detecting Cys based on the mitochondrial localization of the anthocyanin derivative structure was constructed.The probe uses acrylate as the response site of Cys.When Cys is added,Cys will undergo a Michael addition reaction with the acrylate part,the cyclization will be removed,and the probe molecule will release strong fluorescence after the ester bond is broken.In DMF aqueous solution,after the probe responds,a new emission peak appears at 620 nm,producing red fluorescence.The probe can not only be used to monitor endogenous and exogenous Cys in living cells,but also can realize the visualization of oxidative stress response of mitochondria in cells.