Design Of Novel Fluorescent Probes Based On Peroxynitrite Detection And Biological Imaging Research

Peroxynitrite(ONOO^-)is a powerful biological oxidant involved in many physiological and pathological processes,and a key pathological intermediate for various diseases.The development of ONOO^-probes with excellent performance is extremely important for understanding the relevant physiological and pathological processes.Although many ONOO^-probes have been reported,problems such as poor selectivity,low sensitivity,long reaction time,high fluorescence background,and high cytotoxicity still exist.On the other hand,most probes are based on single emission mode,which is not conducive to accurate quantitative analysis.Based on this,this paper through the rational modification and design of fluorescent dyes,three novel organic small-molecule fluorescent probes were constructed,and they were applied to the detection of ONOO^-in living cells and tissues perfectly.1.Using rhodamine as the fluorophore,a high signal-to-noise ratio fluorescent probe for detecting ONOO^-was constructed.The sensing site of ONOO^---boronate is introduced at position 3 of rhodamine to achieve the effect of quenching the fluorescence.The addition of ONOO^-leads to a significant change in the fluorescence intensity.At the same time,different substituents(-CHO,-H,-Cl,-CN,-Et)were introduced at 2 position to adjustment the photophysical properties of the probe.After performance screening,The probe Rhod-CN-B with the-CN substituent shows excellent sensing performance for ONOO^-:high sensitivity(LOD=34 n M),high signal-to-noise ratio(S/N=130),fast response(within 10 s).The probe Rhod-CN-B was successfully used for imaging studies of live cells and mouse liver models of ischemia-reperfusion.2.The study found that the covalent linkage with 2-hydro-pyran-2-imine on rhodamine has specific recognition ability for ONOO^-,developed a new recognition mechanism with different substituents(-CN,-SN,-CONH₂,-COOH)ratiometric fluorescent probes for specific detection of ONOO^-.Due to the covalent cross-linking of 2-hydro-pyran-2-imine,the conjugation of rhodamine is extended,and the probe exhibits an emission at 650 nm.When the probe responds to ONOO^-,2-hydro-pyran-2-imine is susceptible to the nucleophilic attack of ONOO^-and oxidatively cleaved.The conjugation structure is destroyed,and the spectrum blue shifts to emit rhodamine fluorescence at 580 nm,and then realize the ratiometric detection of ONOO^-.Through the sensing performance,it is found that the probe Rhod-Pr-CN has better recognition ability for ONOO^-,the linear response range is0-20?M,and the detection limit is 54 n M.Rhod-Pr-CN was successfully applied to the imaging study of exogenous and endogenous ONOO^-in A549 cells and the imaging study of ONOO^-in xenograft 4T1 tumor mice.3.In order to explore the universality of the recognition unit2-hydro-pyran-2-imine,synthesized a ratiometric fluorescent probe(DCN-CN)with dicyanoisophorone derivatives as fluorophore.The fluorescence emission of the probe itself is at 565 nm.after ONOO^-is added,2-hydro-pyran-2-imine is oxidized and cleaved,and the conjugation structure is destroyed.The fluorescence emission peak at565 nm is continuously weakened,and the spectrum is red-shifted to the near-infrared emission at 690 nm,achieving the purpose of ratiometric detection.The probe has a large emission shift(125 nm)and the response range of ONOO^-is 0-20?M,and the detection limit is 78 n M.The research shows 2-hydropyran-2-imine provides a new sensing strategy for the development of new ONOO^-probes.