Construction Of ONOO~- Long Wavelength Fluorescent Probe Based On BODIPY Dye And Its Application In Imaging Inflammation And Liver Injury

Peroxynitrite（ONOO^-）is a typical endogenous reactive oxygen species（ROS）and active nitrogen（RNS）,which is generated by nitric oxide（NO）and superoxide anion(O₂^·-)through free radical coupling reaction.ONOO^-has high oxidation and nitrification effects,under normal physiological conditions,ONOO^-as a signaling molecule can regulate a series of physiological homeostasis and help to resist the invasion of pathogens;when the level of ONOO^-exceeds the reasonable value,it will enhance the oxidation modification of internal lipids,proteins and nucleic acids,but also can irreversibly damage proteins and nucleic acids,and accelerate protein hydrolysis.Ultimately,the buildup of oxidized proteins can lead to serious inflammation and diseases such as cardiovascular disease,neurodegenerative diseases,Alzheimer’s disease,liver damage,and cancer.Therefore,the development of a direct,rapid,sensitive and selective method for the detection of ONOO^- can help us better study the dynamic role of ONOO^-in biochemistry and achieve early diagnosis and treatment of diseases,which is of profound significance.Fluorescence imaging technology is widely used because of its advantages of high sensitivity,good selectivity,fast response time and simple operation.Although the reported fluorescence probes for the detection of ONOO^-had good performance,there are still some limitations:the selectivity of the probes were poor,and they would be affected by other analytes,so they could not perform good biological imaging;most probes were applied to only one or two cell lines and did not detect ONOO^-in multicell lines.Therefore,the construction of a highly specific fluorescent probe for the detection of ONOO^-is of great significance for the detection of its related diseases.Based on previous literature research and previous work of our research group,we first selected DM-BDP,a BODIPY dye with excellent performance as a fluorophore,and constructed a long-wavelength fluorescence probe BDP-PN withα-ketoamide as the recognition group.The probe showed excellent performance of fast response（10 min）,good specificity and high sensitivity（LOD=220 n M）to ONOO^-in vitro solution.The PET mechanism and detection mechanism of the probe were verified by Density Functional Theory calculation（DFT）and High-Resolution Mass spectrometry.The probe had low cytotoxicity and good biocompatibility and could detect endogenous and exogenous ONOO^-in different cell lines（Hep G2,He La,RAW 264.7,QSG-7701）.And the probe was able to differentiate between normal cells and cancer cells based on different levels of ONOO^-expression.More importantly,in APAP-induced cellular and mouse models of drug-induced liver injury,the probe BDP-PN could detect up-regulated ONOO^-and evaluated the repair effect of the hepatoprotective drug N-acetylcysteine（NAC）.In Chapter 3,we constructed two near-infrared fluorescent dyes BDP-ENE-S-Me/BDP-ENE-O-Me by regulating the structure of BODIPY dyes.Two near-infrared fluorescent probes BDP-ENE-S-Py⁺/BDP-ENE-O-Py⁺were constructed by using the most commonly used phenyl borate group as the identification group of ONOO^-and the dyes were connected in the form of pyridine quaternary ammonium salt.The results of in vitro solution test showed that the two probes could rapidly respond to ONOO^-（BDP-ENE-S-Py⁺:30 s;BDP-ENE-O-Py⁺:50 s）,and had good selectivity,anti-interference and p H stability.In addition,the results of cell experiments showed that both probes could detect ONOO^-in cells without interference from other reactive oxygen species,and the fluorescence intensity gradually increased with the increase of the concentration of exogenous releasers.More importantly,the probe was successfully used to detect ONOO^-in a mouse model of peritonitis induced by LPS.