Design, Synthesis And Imaging Application Research Of Ratiometric Fluorescent Probe For Peroxynitrite Anion

Drug-induced liver injury（DILI）refers to liver damage caused by the drug itself or its metabolites.It has become one of the main reasons for drug withdrawal and drug development failure.DILI is a complex process,in which process,the content of various related biological enzymes and active species（active oxygen,active nitrogen,active sulfur,etc.）changes.Among them,peroxynitrosyl anion（ONOO^-）increased significantly in the early stage of DILI.Therefore,the accurate detection of ONOO^-concentration changes in cells and in vivo is of great significance for the research and prevention of DILI mechanism.However,ONOO^-has the characteristics of high activity and short life span,and conventional methods are difficult to achieve non-destructive monitoring of ONOO^-in cells and in vivo.The small molecule fluorescent probe has the advantages of high sensitivity,non-invasiveness and real-time monitoring,and has become a reliable detection method for the detection of biologically active molecules in cells and in vivo.Based on this,through a combination of screening and design strategies,we developed probes that can be used for intracellular ONOO^-ratio fluorescence imaging and DILI research.The specific work is as follows:（1）Conventional ONOO^-probes are susceptible to interference from other reactive oxygen species,such as H₂O₂,leading to problems such as limited selectivity.In response to this problem,we combined screening and design strategies to develop ONOO^-fluorescent probes with high selectivity,and studied its"structure-activity"relationship in detail.We first synthesized four types of pyran salt-based fluorescent probes by introducing groups with different"donating-withdrawing"electron abilities into the phenyl 4’-position of 2-phenylpyran salt.The results of the spectroscopic experiment showed that the substituents significantly affect the selectivity and sensitivity of the probe to ONOO^-.As the electron donating ability of the substituents on the probe decreases,the selectivity of the probe gradually improves,while the sensitivity decreases.When the substituent is an electron-donating amino group,probe 2-2 can avoid the interference of H₂O₂,and specifically respond to ONOO^-,with good selectivity.（2）Since the fluorescence of the probes screened out in the previous work is reduced after responding to ONOO^-,bioimaging applications are not used.Therefore,we introduced the benzothiazole group with ESIPT effect into the probe structure,and synthesized the probe AHC and AHMC with ratio response to ONOO^-.After the probe AHC responds to ONOO^-,its maximum emission wavelength is blue-shifted from 626 nm to 462 nm.It has a good ratio response（detection limit as low as 1.8n M）and is not interfered by active species such as H₂O₂.Cell experiment results show that the probe AHMC can be used for ratio imaging of endogenous and exogenous ONOO-in cells.We further used the probe AHMC for DILI cell imaging research.The results show that as the concentration of drugs（such as INH）increases,the intrahepatic fluorescence ratio(I₄₆₂/I₆₂₆)also shows an increasing trend,indicating that the probe can be used for drug-induced liver injury imaging research.（3）In the previous work,due to the introduction of hydroxyl groups in the probe AHC,the probe has a strong solvation effect,which is not conducive to accurate imaging applications.For this reason,we used coumarin to replace the phenyl group in the 2-phenylpyran salt,and at the same time introduced a julonidine group on the pyran salt,and synthesized the ratio-type two-photon fluorescent probe RTFP.The results of in vitro experiments show that the probe RTFP has the advantages of high sensitivity（detection limit as low as 4.1 n M）and good selectivity.We used this probe to detect ONOO^-in the process of free fatty acid（FFA）-induced non-alcoholic fatty liver（NAFLD）,APAP-induced liver injury and mixed liver injury,and found that cytochrome P450 2E1（CYP2E1）is in NAFLD,Both DILI and mixed liver injury were significantly up-regulated.