Free Radicals In Cells To Detect The Design Synthesis And Application Of New Fluorescent Probes

During the metabolic processes of living organisms, a variety of active substances,such as reactive oxygen species, reactive nitrogen and reactive chlorine molecules,and a variety of small molecule metabolites, etc. are involved in energy transfer andsignal transduction. They play important roles in physiological and pathologicalprocesses of cells. Organisms maintain their stable redox state by regulating thecontent of thiols and reactive oxygen species, which can avoid oxidative damage,oxidative stress and reductive stress. Therefore, study on redox cycles mediated by theintracellular oxidizing substances （such as reactive oxygen species, etc.） has importantbiological significance.Hypochlorous acid （HOCl） is an important active molecular in vivo. It is generallyproduced by reaction of H2O2and Cl-under the catalysis of myeloperoxidase （MPO）.HOCl, which has natural anti-bacterial ability and is considered to be a pro-nuclearnon-free radical oxidant, exerts important influence on the physiological function ofcells. Recently, fluorescent probe based fluorescence analysis has made some progressin intracellular HOCl detection, but the development of a reversible fluorescent probefor HOCl in living cells is still limited, restricting the in-depth study on the cellualrfunction of HOCl involved in the intracellular redox cycling.Peroxynitrite （ONOO^-） is an extremely important reactive oxygen radical inbiological organisms. Intracellular ONOO^-is generated by reaction of O2- and NO.. Itis a double-edged sword for cells, and has both beneficial and adverse influence. Theuse of fluorescent probes to detect ONOO^-has made great progress. However, amongthe reported fluorescent probes, there is still limited probes can be used to reversiblydetect ONOO^-in living cells. Therefore, the design of organic fluorescent probes withreversible response to ONOO^-is of great significance.Based on the design, synthesis and cell imaging of reversible organic fluorescentprobes, this thesis carried out the following two aspects of research work:First, this paper presents the design, synthesis and cell imaging analysis of afluorescent probe for HOCl. Boron dipyrromethene （BODIPY） is a kind of common fluorescent dye, which is generally used as a basis for probe fabrication. We foundthat the C=N double bond in BODIPY can be reduced by Na₂S2O₄, generatinghydrogenated BODIPY with C-N single bond, accompanied by fluorescence decrease.After adding HOCl, the hydrogenated product can be selectively oxidized toreproduce BODIPY, accompanied by fluorescence recovery. This reversible processcan be repeated at least four cycles. Under the simulated physiological conditions,using Br-BODIPY as a fluorescence probe, the redox cycle by Na₂S2O₄/HOCl isaccomplished. The selectivity, stability, and repeatability is studied. Using thisinteresting and solid reacton, the redox cycle relating HOCl in murine macrophages（Raw 264.7） and HL-60 cells are fluorescently monitored and the fluorescent imagesare obtained.Second, this paper presents the design, synthesis and cell imaging of a newfluorescent probe for ONOO^-. Based on the antioxidant activity of sulfur （S）, which isin the same family with selenium （Se）, and a near-infrared fluorescent dye Cy.7.Cl, afluorescent probe is developed for reversible detection of ONOO^-in living cells. Theprobe shows good selectivity, reversibility, and stability toward ONOO^-/GSH redoxcycles. Based on this, the redox cycle relating ONOO^-in murine macrophages （Raw264.7） is fluorescently monitored and the fluorescent images are obtained.