Novel Fluorescent Probes For Bioimaging Of SO₂ Derivatives And Methionine Sulfoxide Reductase

Sulfur dioxide?SO₂?is a major air pollutant,which can be converted into derivative sulfite(SO₃^2-)and bisulfite?HSO₃^-?in water.Epidemiological studies have shown that exposure to high doses of SO₂ or its derivatives may lead to serious health problems in the respiratory,cardiovascular and nervous systems.On the other hand,endogenous SO₂ is produced by enzymatic oxidation of hydrogen sulfide or sulfur-containing amino acids in living cells and participates in a variety of normal physiological processes.Abnormal levels of SO₂ in the body are closely related to varius diseases in the respiratory system,cardiovascular system and nervous system.Methionine sulfoxide reductase is a protein-repairing and antioxidant enzyme in a diversity of organisms,which plays an important role in biological redox system.It can specifically turn the free or protein-bound methionine sulfoxide into methionine,regulating the protein functions.Therefore,the research and development of sensitive,specific,rapid fluorescence SO₂ probes and methionine sulfoxide reductase probes is of great significance for tracking changes their levels in living cells or tissues and further understanding their physiological and pathological functions.The main work carried out in this thesis is as follows:A mitochondria-targeted near-infrared fluorescent probe?NDMBT?for ratiometric detection of SO₂ was designed based on Michael addition and propared from 2-methylbenzothiazole,N,N-dimethylformamide,6-bromo-2-naphthol,methyl iodide and dimethylamine.The structures of intermediates and the target product were characterized by ¹H NMR,¹³C NMR and MS.The probe displays a strong absorption peak at 488 nm,which disappears rapidly upon addition of HSO₃^-/SO₃^2-.Under excitation at 488 nm,the probe exhibits a strong fluorescence peak emission at 690 nm.Upon addition of HSO₃^-/SO₃^2-,when excited at 405 nm,a strong peak fluorescence emission at 430 nm appears.The probe shows good selectivity,fast response?less than10 s?and high sensitivity with a linear response range of 0-90?mol/L and a detection limit of 43 nmol/L.The laser-scanning confocal experiment shows that the probe can target mitochondria and visualize both exogenously and endogenously produced SO₂derivatives in living cells.Three ICT-based fluorescent probes?Msr-M,Msr-P and Msr-B?for methionine sulfoxide reductase were designed and synthesized from 2'-hydroxyacetophenone,1,1,2-trimethyl-1H-benzo[e]indole,3-Chloroperoxybenzoic acid,ethyl acetate,methyl iodide,malononitrile,4-?methylthio?benzaldehyde and 4-methylpyridine.The structures of the intermediates and target products were characterized by ¹H NMR,¹³C NMR and MS.According to the response of probe towards methionine sulfoxide reductase,Msr-P was identified as the best fluorescent probe.When excited with light of 405 nm,the probe was almost non-fluorescent.However,when the probe was incubated with methionine sulfoxide reductase at 37?for 2 h,and irradiated with the same excitation light,the solution dispay a clear green fluorescence signal with a maximum emission wavelength of 505 nm.The fluorescence intensity in 505 nm was linearly correlated with the concentration of methionine sulfoxide reductase with a linear response range of 0.1-1.5?mol/L and a detection limit of 0.26?mol/L.The probe showed low toxicity,good selectivity and sensitivity and high stability in the range of pH 7.0-8.0.Laser confocal imaging experiments show that the probe can be used for the detection of methionine sulfoxide reductase activity in living cells.