Design And Synthesis Of Peroxynitrite Fluorescent Probes And Their Imaging Studies In Physiological And Pathological Models

Peroxynitrite(ONOO^-),as a potent biological oxidant,is formed by the reaction of oxide radical(·NO)and superoxide(O₂^·-).ONOO^-oxidizes and destroys the structure of components such as lipids,proteins and DNA in cells,leading to cell dysfunction.Previous studies have shown that abnormal levels of ONOO^-can lead to cardiovascular disease,ischemic stroke,nervous system disease,cancer,rheumatoid arthritis,inflammation and autoimmune diseases and many other diseases.In the process of primary immune response,immune-activated phagocytes can produce large amounts of reactive oxygen species(ROS)through respiratory burst to kill the invading organisms and early cancerous cells.As one kind of representative ROS,ONOO^-plays an important role in regulating key signaling pathways,so studying the ONOO^-related physiological and pathological process plays a great significance in the diagnosis and treatment of related diseases.Due to the unique biological roles of ONOO^-in the physiological and pathological environment,the efficient detection of ONOO^-still remains a certain challenge.Fluorescence imaging,by virture of its low tissue damage,high detection sensitivity,visualization and non-invasion,has been designed by more and more researchers to conduct detecting and imaging for the detection of targets of interest.Researchers have synthesized a large number of specific ONOO^-fluorescent probes based on fluorescence methods,and these probes well illustrated the roles of ONOO^-in specific biological processes.However,the biological imaging of peroxynitrite is limited due to the lack of excellent photochemical properties in some probes.It is concluded that the chemical properties of fine probes usually have a longer emission wavelength,large stokes shift,low biological light damage,strong penetrating ability and low background,so compared with the visible light emission fluorescent probes,the near infrared emission fluorescent probes exhibits promising imaging capacity.Fluorescent probes with single emission peak can be easily interfered by instrument performance,uneven load or changes of microenvironment in the cells around the probe.However,fluorescence probes with ratio emission can avoid the interference of the above unrelated factors,thus improving the measurement accuracy.In order to improve the photostability,selectivity and response time of the probe and thus improve the determination accuracy,four novel fluorescent molecules were designed and synthesized based on modified rhodamine and cyanine fluorophore.The chemical structures of the fluorescent molecules were characterized by HR-MS and NMR.The response capacity and mechanism of fluorescent molecules toward peroxynitrite was studied by UV-Vis spectroscopy,steady-transient state fluorescence spectroscopy,liquid chromatography,dynamic light scattering,transmission electron microscopy and theoretical calculation.The imaging effects were studied by cell imaging,tissue and in vivo imaging.The main innovative results are as follows:(1)Two ratiometric fluorescent molecules Ratio-A and Ratio-B were synthesized by modified rhodamine fluorophore and cinnamaldehyde derivatives.The spectrum and LC-MS experiments showed that the ratio fluorescence detection of ONOO^-could be performed by both probes,and the detection mechanism was confirmed as oxidation of diene bonds.This work studied the response ability of probes to intracellular ROS,cell experiments showed that Ratio-A could be used to detect ONOO^-in cells with specific fluorescence ratio mode.With the aid of Ratio-A,this work realized ONOO^-imaging in acute inflammation mice model,and also succeeded in evaluation of ONOO^-mediated rheumatoid arthritis diagnosis and MTX treatment effect.Therefore,the probe has potential clinical application value in the study of diagnosis and treatment evaluation of rheumatoid arthritis.(2)Two cases of cyanine-based fluorescent molecules Cy717 and Cy683 were designed and synthesized.Fluorescence titration experiments show that,due to the different installing groups,there are significant fluorescence responses toward ONOO^-:Cy717 shows ratio fluorescence responses toward ONOO^-,and Cy683shows intensity based fluorescence responses toward ONOO^-.LC-MS and TEM results confirmed that the reaction mechanisms toward ONOO^-was nonoxidative nucleophilic substitution.With the assistance of our developed probes,this study realized the detection of ONOO^-levels in immune stimulated phagocytes,as well as the evaluation of ONOO^-imaging capacity in acute peritonitis model,and the visualization of ONOO^-variations in the process of tumorigenesis.The established nonoxidative strategy in this study provides a promising molecular tool for early tumor diagnosis and evaluation of tumor inhibition during immune response without interfering with ROS signal transduction function.