Construction Of Fluorescent Probes For Active Nitrides Of Nitric Oxide And Peroxynitrite And Biosensing

Nitric oxide?NO?is a single electron radical which can freely diffuse through the biofilm systems.The metabolites of NO in organisms include N₂O₃,HNO,NO₂^-and NO₃^-which can also react with O₂·^-to produce peroxynitrite?ONOO^-?.NO regulates normal physiological functions of cells by reacting with metal proteins,free radicals,and protein thiols in cells.ONOO^-regulates normal physiological functions of cells by oxidizing and nitrifying amino acids.Therefore,NO and ONOO^-are important signal molecules in the body.Fluorescent probes have become important detection tools for NO and ONOO in recent years.Although many types of NO and ONOO^-fluorescent probes have been reported,there are also deficiencies.In this dissertation,starting from changing the reaction mechanism,a series of new reaction types of NO,ONOO^-fluorescence probes were designed and synthesized.The following work was done:?1?We have created a new strategy utilizing the reaction of nitrosation of aromatic secondary amines with NO.By applying the above stategy a new NO fluorescent probe 2.1 with BODIPY as a fluorophore and N-benzyl-4-hydroxyaniline as a recognition group was developed.Based on the structure of 2.1,by introducing the triphenylphosphorane salt?TPP?,a new Mitochondrial NO fluorescence probe 2.2 was developed.The simple one-step reaction of probe 2.1 with NO and the electron donating ability of the hydroxy substituents allowed the reaction to complete in a few seconds.Moreover in the presence of a large amount of GSH,Hcy,Cys,there was no interference with the fluorescent signal after the reaction between 2.1 and NO.The special stratey solved the problem of probe response speed.The UV absorption spectra and fluorescence emission spectra demonstrated that the probe 2.1 did not react with other ROS?ClO^-,H₂O₂,O2·^-,¹O₂,·OH?.Further we studied the reaction between the probe 2.1 and the highly active ClO^-by LC-MS analysis and the result indicated clearly that there was surely no reaction between the two compounds.Due to the special structure of 2.1,DHA/AA/MGO interference was avoided.Therefore,we not only solved the problem of the selectivity of the probes for imaging NO,but also solved the problem of potential consumption of probes in biological systems.By titrating NO with probe 2.1,we calculated that the detection limit of 2.1 was as low as 4.8 nM.The probe 2.2 was co-localized with commercial mitochondrial probes with a Pearson coefficient as high as 0.92.At last Probes 2.1 and 2.2 respectively imaged exogenous and endogenous NO in HeLa cells and RAW 264.7 cells.?2?We have created a new strategy utilizing the reaction between aromatic tertiary amines and ONOO^-producing corresponding N-nitroso,nitrogen-oxygen compounds.Applying the above strategy a new ONOO^-fluorescent probe 3.1 with BODIPY as a fluorophore and N,N-dibenzyl-4-methoxyaniline as a recognition group was developed.By introducing the TPP on the basis of the structure of 3.1,we developed the ONOO^-fluorescent probe Mito-3.1 capable of targeting mitochondria,and by introducing morpholine ONOO^-fluorescence probe Lyso-3.1 capable of targeting lysosome was developed for the first time.The specific reaction of probe 3.1 with ONOO^-and the electron donating ability of the methoxy group enabled the probe to complete the reaction in seconds and there was no interference with the fluorescence signal in the presence of 1 mM carbon dioxide and 3.1.This stratege solved the problem of probe response speed.Through ultraviolet absorption spectrum and fluorescence emission spectrum,it was proved that 3.1 did not react with other ROS(ClO^-,H₂O₂,O2·^-,^?16?O2,·OH).In the presence of ROS and 3.1,there was no interference with the fluorescence signal after the probe reacted with ONOO^-.The above stratege solved the problem of the probes recognizing the selectivity of ONOO^-and the potential consumption of probes in biological systems.Through the 3.1titration of ONOO^-,the detection limit of 3.1 was calculated to be as low as0.15 nM.Mito-3.1 and Lyso-3.1 were co-localized with commercial mitochondrial and lysosomal probes with the Pearson coefficient 0.92 and0.84,respectively.Probe 3.1,Mito-3.1,and Lyso-3.1 imaged HeLa cells,exogenous and endogenous ONOO^-in RAW264.7 cells.Further 3.1 was first used to detect ONOO^-in a diabetic mouse model?kidney section,Liver section?.?3?Using the established mechanism of the specific reaction of aromatic tertiary amines with ONOO^-and the mechanism of PET fluorescence switch,we developed a new ONOO^-near-infrared fluorescent probe 4.1.The silicon rhodamine with good water solubility and strong photostability was used as the fluorophore and N,N-dibenzyl-4-methoxyaniline as the recognition group.4.1 inherited the excellent properties of 3.1.It could react with ONOO^-in seconds and was not affected by other ROS(ClO^-,H₂O₂,O2·^-,^?16?O2,·OH).In the presence of carbon dioxide,there is no interference with the fluorescence signal after reaction of 4.1 with ONOO^-.The limit of detection is as low as 3 nM.4.1 irradiated by laser in HeLa cells for 60minutes did not induce any fluorescence intensity,indicating that 4.1 is resistant to photooxidation.The product of the reaction between 4.1 and ONOO^-was also irradiated by laser for 60 min in the cells and the fluorescence intensity was almost not attenuated,indicating that the product has strong anti-photobleaching ability.Probe 4.1 visualized exogenous and endogenous ONOO^-in HeLa cells and RAW 264.7 cells,respectively.And4.1 also imaged STZ-stimulated ONOO^-in islet?-cell and diabetic mouse models?renal sections,liver sections?.At the same time,the therapeutic effect of phenol antioxidants on ischemia-reperfusion models of EA.hy926cells was evaluated by using 4.1.