Synthesis And Bioimaging Application Of Peroxynitrite Fluorescent Probe

Peroxynitrite（ONOO^-）,as a highly reactive oxygen and nitrogen species（RONS）in living systems,has the extremely high oxidization and nitrification abilities.ONOO^-is easy to react with biomacromolecules,such as proteins,lipids and DNA,resulting in protein oxidation,lipid oxidation and DNA mutation,which can lead to some diseases including cancer,inflammation,cardiovascular diseases as well as neurodegenerative diseases.Fluorescent probes combined with fluorescence imaging technology have become powerful tools for detecting and imaging ONOO^-in living bio-system,due to its excellent sensitivity,simple operation,high spatiotemporal resolution and in situ real-time detection.This paper aims to construct a series of ONOO^-fluorescent probes with high sensitivity and good selectivity through rational design,and further apply them for ferroptosis imaging and visual diagnosis of tumors.The main research contents are as follows:1.A fluorescent probe BTMO-PN using benzothiazolyl derivatives as the fluorophore and phenylboronic acid ester as the ONOO^-recognition site was designed and synthesized for visual imaging ferroptosis and tumor tissue.BTMO-PN exhibited a rapid and significant fluorescence enhancement signal（～560-fold）toward ONOO^-at 477 nm,with the detection limit of 37.8 n M,and good selectivity.BTMO-PN was capable of sensitive imaging endogenous/exogenous ONOO^-changes in live cells,and the up-generation of ONOO^-levels in cancer cells during ferroptosis was observed.In addition,BTMO-PN has been successfully applied for distinguishing tumor tissue from normal tissues.2.Based on twisted intramolecular charge transfer（TICT）and excited-state intramolecular charge transfer（ESICT）sensing mechanisms,a multifunctional fluorescent probe MQA-P using diphenyl phosphinate as the ONOO^-recognition site,was designed and synthesized for simultaneous detecting ONOO^-,viscosity and polarity.MQA-P was highly sensitive to viscosity/polarity changes in NIR channel withλ_em>704 nm,and could quickly and sensitively detect ONOO^-at 645 nm in the green channel with approximately233-fold fluorescence enhancement,and the detection limit of 23.0 n M.Due to the presence of quinolone cations,MQA-P possessed mitochondria-targeting ability in He La cells.MQA-P was applied to image microenvironments and endogenous/exogenous ONOO^-changes at the cellular level.Most importantly,using MQA-P the simultaneous visualization of ONOO^-,viscosity/polarity in cancer cells during ferroptosis was first observed through dual-channel images,revealing the overexpressed of ONOO^-,increase of viscosity（or decrease of polarity）during ferroptosis.In addition,the multifunctional fluorescent probe has also been used to observe the abnormal changes of ONOO^-,viscosity/polarity in cancer cells/tumor tissue/tumor mice model via dual-channel images,realizing tumor diagnosis in vitro and in vivo.3.Using phenylboronic acid ester and C=C double bond as the two specific ONOO^-recognition sites,a ratiometric fluorescent probe DHX-BE based on oxanthene backbone,was designed and synthesized for detecting ONOO^-.DHX-BE itself displayed a red fluorescence emission at 658 nm.Upon addition of ONOO^-,the ONOO^--triggered the oxidative hydrolysis of phenylboronic acid ester and the cleavage of C=C double bond lead to the quenching of red fluorescence at 658 nm,whereas a new strong green fluorescence emission appears at 554 nm,with the emission peak shift up to 104 nm.DHX-BE showed a significant ratiometric characteristic toward ONOO^-,with an enhanced fluorescence intensity ratio I_{554 nm}/I_{658 nm}more than 727 times,and a detection limit of 20.4 n M.Moreover,DHX-BE also exhibited a fast response and high selectivity to ONOO^-,which can achieve rapid,efficient and specific detection of ONOO^-.