Construction Of Red/Near-Infrared Fluorescent Probes For Detection Of Peroxynitrte And Its Imaging Application In Inflammation,Fatty Liver And Diabetes

Peroxynitrite（ONOO^-）is a typical endogenous reactive oxygen species（ROS）and reactive nitrogen species（RNS）,usually produced by the reaction of highly active nitric oxide（NO）and superoxide anion(O₂^·-).Compared with other active substances,ONOO^-has strong oxidizing and nucleophilic properties,making it participate in various biochemical reactions in living organisms.It has been reported that ONOO^-can affect cellular oxidative stress,inflammation and immune response,and trigger cell death through various signaling pathways.Therefore,abnormal concentrations of ONOO^-are involved in many diseases and pathological conditions,and are considered to be biomarkers of inflammation,fatty liver,diabetes and other diseases.Exploring the concentration of ONOO^-in disease-related processes can help diagnose and evaluate various diseases.In recent years,fluorescence imaging technology has been widely used in the fields of biology,physiology and environmental science because of its advantages of non-invasive,in situ and real-time imaging of biological samples,high sensitivity,simple operation and good selectivity.So far,although many fluorescent probes for the detection of ONOO^-have been reported,they still have some limitations:The solubility/water solubility of some probes is poor,which limits the probes in biological applications;Some probes have relatively short emission wavelengths,which is not conducive to in situ imaging in vivo;Some probes are not stable enough to be suitable for long-term fluorescence imaging in organisms.Therefore,the development of fluorescent probes with good water solubility,long emission wavelength,high stability and high specificity for the detection of ONOO^-will help improve the diagnostic efficiency of diseases and provide more powerful help for the prevention and in-depth understanding of the pathogenesis of various diseases.In this paper,based on the literature research and the preliminary work of our group in fluorescent probes,we first selected resorufin as the fluorophore to improve solubility by introducing alkyl chains and diethylene glycol monomethyl ether chains in the identification group indole-2,3-dione derivatives,and constructing red fluorescent probes with good water solubility.Then,we used near-infrared BODIPY dye（BDP-NIR）and the classical ONOO^-identification group phenylboronic acid pinacol ester to construct a near-infrared fluorescent probe,hoping that the probe could achieve early diagnosis of inflammation,fatty liver and diabetes by detecting the change of ONOO^-concentration in vivo.In the second chapter,we used indole-2,3-dione derivatives as the identification group,introduced long alkyl chains and diethylene glycol monomethyl ether chains on the N atom of the identification group to improve solubility,and successfully constructed two fluorescent probes RF-IT-OC and RF-IT-EG that specifically detect ONOO^-.The response of both probes to ONOO^-in vitro solution was systematically tested,and the results showed that both probes could achieve selective response to ONOO^-.However,we found that the probe RF-IT-EG had better water solubility（3.2 mg/L）,faster response（2 min vs 5 min）,higher signal-to-noise ratio（103-fold vs 91-fold）,and lower detection limits（87 n M vs 142 n M）.In addition,the detection mechanism of RF-IT-EG for ONOO^-was verified by HRMS.At the same time,density functional theory（DFT）also verified that the fluorescence quenching of the probe RF-IT-EG is caused by blocking the ICT process.Moreover,the probe RF-IT-EG had low cytotoxicity and could be used to detect the production of endogenous ONOO^-in cells.More importantly,the probe RF-IT-EG could quickly monitor the production of ONOO^-in leg inflammation of LPS-stimulated mice,indicating that the probe could achieve early diagnosis of inflammation.In the third chapter,in order to further extend the emission wavelength of the probe,we selected BODIPY dye with easy structure modification as the fluorophore,introduced a parallel ring structure on the BODIPY parent nucleus,and constructed a fluorescent probe BDP-NIR-Py⁺with near-infrared emission wavelength by using the classical ONOO^-identification group phenylboronic acid pinacol ester,and systematically tested its performance in the detection of ONOO^-in vitro solution.Compared with the fluorescent probes RF-IT-OC and RF-IT-EG constructed based on halorin dyes in the second chapter,the probe BDP-NIR-Py⁺had a longer emission wavelength（661 nm vs 590 nm）and faster response（60 s vs120 s）.In addition,the probe BDP-NIR-Py⁺could rapidly and specifically detected changes in endogenous ONOO^-levels in cells and zebrafish.More importantly,we found that the dye BDP-NIR could target LDs after the probe reacts with ONOO^-,and could accurately track the dynamic changes of LDs as well as 3D imaging,and visualized the changes in ONOO^-levels in different stages of NAFLD.Compared with serum marker detection and tissue section staining detection,the probe BDP-NIR-Py⁺two-parameter analysis method had obvious advantages in early NAFLD diagnosis.In addition,the probe BDP-NIR-Py⁺utilized the increased fluorescence intensity caused by elevated ONOO^-levels to diagnosed and evaluated type Ⅰ and type Ⅱ diabetes.