Used For Detection Of Intracellular Reactive Oxygen Free Radicals Can Be Seen Far And Near-infrared Fluorescent Probes Were Designed And Synthesized And Applied Research

The levels of various reactive oxygen species (ROS) in organism have been associated with biologic physiology and pathology. Recognition and quantifying of free radicals are a premise to study ROS biological functions. In this dissertation, we designed and synthesized a series of fluorescein- and naphthofluorescein-fluorescent probes for detecting and imaging superoxide anion radical (O2-·) and peroxide hydrogen (H2O2) in living cells or aqueous solutions, based on a nonredox mechanism; and performed the simultaneous optical measurement of superoxide anion radical and peroxide hydrogen within living cells from Wistar rats of bleomycin-induced lung fibrosis.The main results of this dissertation were shown as follows:1. Based on the nucleophilicity of O2-·, a novel green fluorescent probe, bis(diphenylphosphinacyl) fluorescein (PF-1), was synthesized and characterized with elemental analysis, IR, 1H NMR, 31P NMR, and chemical ionization mass spectrometry (CIMS). The synthesis method is simple and original. Upon treatment with O2-?, PF-1, a closed, colorless, and non-fluorescent lactone, was transformed into an open, colored, and fluorescent product (λex/em = 490/530 nm). The fluorimetric experiments of the probe showed that the sensitive fluorogenic reagent features a high selectivity for O2-? over other intracellular ROS and biological compounds, wide response ranges (1×10-10 - 1×10-8 mol l-1 or 1×10-6 - 8×10-6 mol l-1) and low detection limit (4.6 pM) owing to its nucleophilic mechanism. Using fluorescence microscope, cell-derived O2-? were located in live cells. The specific response of PF-1 to O2-? was confirmed by adding a nonenzymatic superoxide scavenger. These experimental results show that PF-1 is an excellent fluorescent probe, which possesses good selectivity, high sensitivity, good water solubility, and prompt reactivity.2. A near-IR fluorescent probe, bis(diphenylphosphinacyl) naphthofluorescein (PNF-1), was synthesized and characterized with elemental analysis, IR, 1H NMR, 31P NMR, and chemical ionization mass spectrometry (CIMS). The synthesis method is simple and original. The design strategy for the probe is based on the nucleophilic mechanism of O2-·to mediate deprotection of the probe to naphthofluorescein (λex/em = 602/662 nm), while the emission spectrum of naphthofluorescein is just in a spectral region of low background fluorescence interference in biological systems. Upon reaction with O2-·, the probe exhibits a strong fluorescence response and high selectivity for O2-·only, rather than the other reactive oxygen species and some biological compounds. PNF-1 linear calibration curve was obtained and the detection limit was 0.1 nM. The phosphinate-based probe, a new type of fluorescent probe, is cell permeable and can detect nanomolar changes in O2-·concentrations in living cells, using confocal microscopy.3. Bis(diphenylphosphinacyl)-5(6)-carboxylnaphthofluorescein was synthesized and characterized with elemental analysis, IR and 1H NMR. The design strategy for the probe is based on the nucleophilic mechanism of O2-·to mediate deprotection of the probe to naphthofluorescein (λex/em = 588/667nm). Upon reaction with O2-·, the probe, a closed, colorless, and non-fluorescent lactone, was transformed into an open, colored, and fluorescent product. The fluorimetric experiments of the probe showed that the sensitive fluorogenic reagent features a strong fluorescence response and high selectivity for O2-·in a aqueous solution.4. In this chapter, we presented the synthesis, fluorescence properties, and biological applications of naphthofluorescein disulfonates (NFDS-1 and NFDS-2), as near-IR fluorescent imaging probes to detect intracellular H2O2. The probe NFDS-1 features high selectivity for H2O2 over competing with other intracellular ROS and some biological compounds, a wide dynamic response range (6.0×10-9-4.0×10-6 mol l-1) and low detection limit (81.5 pM) owing to its nonredox mechanism, and far-visible excitation and near-IR fluorescence emission profiles to minimize cell and tissue damage while avoiding native fluorescence from native cellular species. Furthermore, we have demonstrated the value of this probe by measuring living cell-derived H2O2 and the nanomolar concentration of exogenous H2O2 within living macrophages. We found that NFDS-1 is an excellent fluorescence reagent in detecting intracellular H2O2 and can respond to nanomolar change in H2O2 concentrations within living cells. 5. We designed and synthesized fluorescein disulfonates (FS-1 and FS-2) as fluorescence imaging probes for intracellular H2O2, which were characterized with elemental analysis, IR, 1HNMR. FS-1 and FS-2 are the closed, colorless, and non-fluorescent lactones. Upon treatment with H2O2, hydrolytic deprotection of the probes generated subsequently open, colored, and fluorescent products. The fluorescein-based reagents feature excellent selectivity toward H2O2 over competing with cellular ROS (O2.-, .OH, NO and ONOO-) and some biological compounds (HQ, GSH, Vc and esterase), large dynamic response ranges owing to their dual colorimetric/fluorometric detection mechanism, and long-wavelength visible excitation and emission profiles to minimize cell and tissue damage. The sulfonate-based probes are cell-permeable and can detect mircomolar changes in O2-·concentrations in living cells, using confocal microscopy.6. The concentrations of H2O2 and O2-·in the clear solutions, the macrophages from the pleural lavage fluid and the lung cells of Wistar rats of bleomycin-induced lung fibrosis were simultaneously detected by applying the two probes, naphthofluorescein disulfonates (NFDS-1) and bis(diphenylphosphinacyl) fluorescein (PF-1). Compared with the clear solutions from normal lung and fibrosis lung, the qualitative relation between the grades of lung fibrosis and the levels of H2O2 and O2-·was expounded.