Construction And Application Of Fluorescent Probes For Detecting The Up-regulated Peroxynitrite In Inflammation And Drug-induced Liver Injury

As a short-lived and highly reactive nitrogen specie,peroxynitrite（ONOO^-）plays essential roles on various physiological and pathological processes of living systems.It is generated through the diffusion control chemical transformation reaction of nitric oxide（NO）and superoxide(O₂^·-)in mitochondria,that’s the reason why ONOO^?have properties both of ROS and RNS.As an important signaling molecule,ONOO^?plays an important role in regulating cellular immune signal response.However,large numbers of biological species including nucleic acids,lipids and proteins could react with peroxynitrite when it it is overexpressed,and eventually result in apoptosis,which could induce oxidative/nitrative stress,eventually damage the body.Until now,mounting evidences have been improved that excessive ONOO^-could cause many diseases,such as inflammatory,drug induced liver injury,neurodegenerative,Alzheimer’s disease,Parkinson’s disease,cardiovascular,ischemic reperfusion injury and cancer.Therefore,it is of great importance to construct effective tools for exploring the role of ONOO^-in related pathophysiological processes,as well monitoring the fluctuation of ONOO^-to diagnose related diseases.To date,fluorescent analytical method is one of the most useful tools for widely applying in chemical analysis,bioanalysis,and medicine study owing to its outstanding advantages of rapid response,real-time visualization,excellent sensitivity and noninvasive nature.Although numerous fluorescent probes for tracing ONOO^-in living system have been reported,there still exist some limitations:several probes showe poor stability because of the high oxidative capacity of ONOO^-,which may not suitable for long-time tracting ONOO^?under physiological conditions;probes have absorption/emission in the short-wavelength will inevitably subject to biological interference from autofluorescence,which has limited the application in bioimaging.Therefore,the construction of long-wavelength fluorescent probes with high stability for ONOO^-is still urgently needed.According to the previous literature and the work of our research group,two excellent dyes resorufin and BODIPY are chose as the fluorophore by installing with different recognition groups to construct a series long-wavelength fluorescent probe.It is expected that the probes can realize the specific detection of ONOO^?and diagnosis of Inflammation/Drug-Induced Liver Injury by detecting the fluctuation of ONOO^-in vivo.In the second Chapter,two fluorescent probes RFP and RFAc were successfully constructed with diphenylphosphinate andα-ketoamide as recognition targeting groups based on the resorufin dyes.After successful obtained two detectors,the preliminary solution test showed that both probes could selective response and withstand the strong oxidizing properties of high concentrations of ONOO^?.Under simulated physiological conditions,probes RFP and RFAc can fast detect ONOO^?（within 20 and 30 min respectively）;in the concentration titration experiments,the probes RFP and RFAc showed good linearity in the concentration range of 0-35μM and 0-30μM,respectively.The detection limit of probes RFP and RFAc were 238 n M and 220 n M respectively,which confirmed that they can sensitively detect ONOO^?.Besides,the probes RFP and RFAc showed highly selectivity,good anti-interference and p H stability when detecting ONOO^–.In addition,the detection mechanism of the probes RFP and RFAc for ONOO^?was verified by ESI-MS;density functional theory（DFT）calculations indicated that the fluorescence quenching of two probes was due to the ICT process.Cell experiments also proved that the probes RFP and RFAc have low toxicity to biological samples and can be applied to detect the fluctuation of intracellular and extracellular ONOO^?.More importantly,with the fluorescence increase induced by the expression of ONOO^?,the probes realized detecting the inflammation in mouse modle,In the third chapter,a fluorescent probe BDPP was constructed for detecting ONOO^?which based on the diphenylphosphinate group as the specific trigger,BODIPY dyes was selected as the fluorophore in order to prolong the emission wavelength of the probe.According the performances of BDPP reponsed to ONOO^?in simulated physiological conditions,we found that it has better solubility and longer emission wavelength（613 nm?590 nm）,faster response speed（10 min?20 min）,better anti-interference and other properties compared with RFP and RFAc.These more excellent properties enable the probe BDPP to detect endogenous/exogenous peroxynitrite rapidly and specifically in cells and zebrafish with effectively shield the interference of other reactive oxygen species.More importantly,the probe BDPP realized the specific detection of APAP-induced hepatotoxicity and evaluate the remediation of NAC in Hep G2 and QSG-7701 cell.