The Construction And Biosensing Study Of Organic Fluorescent Probes For Peroxynitrite

Peroxynitrite (ONOO-), a powerful oxidizing and nitrating agent in living systems, is formed by reaction between nitric oxide (NO) and superoxide (O2·-) under diffusion control without the need of enzymatic catalysis. Due to its strong chemical reactivity, peroxynitrite and its derivatives (·OH、CO3·- and ·NO2) can react with a wide array of biomolecules, such as proteins, lipids and nucleic acids, resulting in cell death. As a result, ONOO- has been implicated in a variety of disease states. However, recent research shows that peroxynitrite play neglected functions in the regulation of redox potential. Meanwhile, evidences also revealed that ONOO- plays a positive role in either signal transduction via nitrating tyrosine residues or immunogenic response against invading pathogens. Therefore, methods for detection of cellular ONOO- levels are of considerable importance for both disease diagnosis and exploration of its various pathophysiology.Fluorescent probes have been considered as the efficient molecular tools which can monitor and visualize trace amounts of samples in live cells and tissues.In particular, the exploitation of the reaction-based fluorescent probes have been attracted increasing attention in recent years due to the higher selectivity and large spectroscopic response. To date, a number of small-molecule fluorescent probes with the improved performance were exploited based on the ONOO--triggered specific reactions, most of which still have little selectivity among various ROS. In addition, although mitochondrial has been recognized to be the major site for formation and reaction of intracellular ONOO-, as far as we know, only one mitochondria-targetable fluorescent probe for ONOO- was reported to date. Hence, it is still a challenge to develop ONOO- fluorescent probe with high selectivity and sensitivity, but also the mitochondria-targetable characteristic. The above concerns encouraged us to develop following research.(1) We presented a novel fluorescence probe 2-1 by the direct conjugation of a pyronin dye with a ONOO- sensitive methyl(4-hydroxyphenyl)amino reaction group. The probe was proved to be able to selectively and rapidly sense ONOO- over other ROS by ONOO- triggered oxidative N-dearylation reaction to release the fluorescent aminopyronin. Assisted by laser scanning confocal microscope, we demonstrated that the probe is mitochondria-targetable, and could detect the exogenous and endogenous ONOO- in RAW264.7 cells.(2) We constructed a novel fluorescent Probe 3-1 by directly connecting a pyronin fluorophore and a 2,4-dimethylpyrrole moiety acting as a novel recognition group. We examined the selectivity of fluorescent probe 3-1 among various kinds of ROS/RNS and the results show that only ONOO- has caused fluorescence enhancement. Assisted by laser scanning confocal microscope, we demonstrated that the probe have a good penetration in cells, and was successfully applied in detecting endogenous ONOO- in RAW264.7 cells...