The Peroxynitrite Fluorescent Probe Was Constructed Based On The Recognition Groups Of Formamide And Lactam

Peroxynitrite（ONOO^-）is an important reactive oxygen species,which is endogenously generated by reaction of nitric oxide（NO）and superoxide anion(O₂^·-).Intracellular ONOO^-can assist organisms to kill invading microbes and play an important role in host immune response.Moreover,ONOO^-is also a signal molecule involved in the intracellular signal transduction.Nevertheless,owing to its extremely oxidizing and nitrating capabilities,excess ONOO^-can react with a wide variety of biomacromolecules of organism,including protein,lipids and nucleic acid,etc.,leading to their structural change and dysfunction,which eventually results in cell apoptosis or death.Increasing researches fully proves that ONOO^-has been associated with the occurrence and development of many diseases,including neurodegenerative disease,chronic inflammation,ischemia reperfusion injury,antoimmune disease and cancer,etc..Therefore,it is great significance for elucidating its physiological and pathological mechanisms,as well as for the diagnosis,treatment and pathogenesis of related diseases to study the production,distribution and concentration changes of ONOO^-in cells and organism.The generated ONOO^-was in situ visualized in cultured cells and vivo by using probe-based fluorescence imaging technology,which is an effective tool for studying ONOO^--related chemical biology and pathology.Although researchers have developed a variety of ONOO^-fluorescent probes,selective recognition and bioimaging of ONOO^-are still challenging due to（1）the lack of highly specific recognition groups,（2）the tedious and low-yielding synthesis of current sophisticated probes,and（3）the unavailability of versatile chemical strategy.Two series of novel molecular fluorescent probes（FA and LT series）based on two novel recognition groups（formamide and lactam）of ONOO^-were constructed for in situ fluorescent imaging of ONOO^-in cells and vivo,and a real-time fluorescent detection method for concentration fluctuation of ONOO^-in pathological process was established.The specific research work is as follows:（1）Formamide was used as a new recognition group of ONOO^-.Four fluorescent probes containing formamide groups（FA-Blue,FA-Green,FA-Yellow and FA-Red）were prepared by one-step formylation of four common amino-containing fluorophores（coumarin,aminonaphthalene,naphthalimide and benzophenoxazine）.It was found that the four probes could react with ONOO^-in our research,which results in the breaking of amide bonds and release of corresponding amino-containing fluorophores,and emits blue,green,yellow and red fluorescence respectively.The four probes react with ONOO^-rapidly,with good selectivity and high sensitivity,and can also perform in situ fluorescence imaging of ONOO^-in living cells.In addition,using FA-Yellow,we found that the levels of ONOO^-in paraquat-treated cells and major organs of mouse（heart,liver,lung and kidney）were significantly increased by two-photon fluorescence imaging and flow cytometry analysis.Therefore,it is speculated that ONOO^-may be an important factor in organ damage induced by paraquat.These results indicated that ONOO^-induced formamide deformamide is a facile,versatile,and highly efficient strategy for ONOO^-recognition and bioimaging,which will promote the research progress of ONOO^-related pathophysiology.（2）Lactam was used as a new recognition group of ONOO^-.LT-Green and LT-Yellow were prepared by substitution-cyclization reaction of two amino-containing fluorophores（aminophenol and naphthalimide）with diethyl keto-malonate,respectively.Studies have indicated that both probes with rapid response and high sensitivity can specifically recognize ONOO^-and lead to fluorescence of probes enhanced.In addition,combined witn two-photo techniques,both probes can perform imaging of ONOO^-in living cells.The reaction mechanism of between the probes and ONOO^-and the biological application of the probes are being further study.In summary,two kinds of molecular fluorescent probes have been developed by using formamide and lactam as ONOO^-recognition groups respectively in this paper.Both types of probes can rapidly respond to ONOO^-with high sensitivity and selectivity,and were successfully applied in the detection of ONOO^-in vitro.Furthermore,the production,distribution and concentration changes of ONOO^-in cell and animal models were monitored successfully by using these probes,which provides an efficient in situ fluorescence imaging tool for the study of pathophysiology of ONOO^-and the pathogenesis of ONOO^-related diseases.