Construction And Biological Application Of Subcellular Organelles Fluorescent Probe For Sulfur Dioxide

Endogenous sulfur dioxide(SO₂)could enhance the antioxidant effect of cells and exert the signal transduction role.High concentrations of SO₂in organisms could cause major diseases such as lung cancer.An important biosynthetic pathway is that cysteine(Cys)is metabolized into endogenous SO₂by the catalysis of enzymes,which directly affects its concentration in the body.Therefore,the real-time monitoring of the physiological concentration of SO₂and the visualization of its biosynthetic pathway have important biological and medical significance.Organic small molecule fluorescent probes have become an essential research method in the fields of biological small molecule detection due to these unique features including of small size,easy modification of structure,and easy regulation of luminescence performance.Herein,with benzopyrylium-coumarin organic small molecule fluorescent probes in hand,we have successfully achieved ultra-fast detection of cell-level SO₂,and the visualization of Cys metabolizing into SO₂in subcellular organelles.The details are as follows:1.We have rationally developed a near-infrared(NIR)fluorescent probe2-1 through the organic combination of benzopyrylium-coumarin system to lengthen the conjugation structure.The probe 2-1 processed an ultra-fast response time(10 s)for detecting SO₂and overcome physiological interferences from long-term exposure of bio-thiols,which could satisfy the real-time detection requirement of SO₂as a transient metabolite in living cells.When the?-conjugated system of the benzopyronium unit in probe 2-1 was broken due to the unsaturated C=C double bond attacked by SO₂,the fluorescence emission blue shifted to 480 nm.Moreover,under the confocal fluorescence biological imaging,we have successfully applied probe 2-1 to detect SO₂in mitochondria of SW480 cells with a ratiometric fluorescence.2.On the basis of the above research,we have constructed a novel bi-reversible reaction sites fluorescent probe 3-1 to simultaneously distinguish and detect Cys and SO₂,and realized the visual fluorescence trace of Cys metabolizing SO₂in cells.The?,?-unsaturated C=C double bond in coumarin moiety was selected as the reversible recognition site toward Cys and the addition compound as the energy donor of FRET mechanism.Meanwhile,benzopyrylium unit could not only served as energy receptor,but also showed the reversible recognition for SO₂.Based on the FRET sensing mechanism,probe 3-1 showed multi-fluorescence signal patterns toward different targeting analytes(Cys?SO₂?Cys-SO₂),and successfully achieved the real-time visualization of the biological pathway of Cys metabolizing into SO₂in subcellular organelles and living mice tumor biological models.