Design,Synthesis And Application Of Fluorescent Probe For The Detection Of Peroxynitrite

Peroxynitrite is one of the most important reactive oxygen species(ROS)in living systems generated by reaction of nitric oxide(NO)and superoxide radical anion(O₂^-?).It features a relative higher reactivity in physiological and pathological processes.Excessive peroxynitrite will cause critical damage to cellular biomolecules,such as tyrosine residues or thiols containing proteins,DNA,and unsaturated fatty-acid-containing lipids by oxidation or nitrification,leading to a series of diseases.Recent research has revealed that peroxynitrite is also involved in cellular signal transduction as well as cell apoptosis.Thus,the monitoring of cellular peroxynitrite has captured lots of attention in order to elucidate its cell physiological function and pathological mechanism that lead to peroxynitrite-related disease diagnosis.Among the methods available for peroxynitrite detection,small molecule fluorescent probes have attracted much attention due to their manipulation simplicity,high selectivity,noninvasive nature and their widespread application in realtime fluorescence imaging.A large number of fluorescent probes for detecting peroxynitrite are reported,most of which are based on sensing mechanisms such as photo-induced electron transfer(PET),intramolecular charge transfer(ICT)and fluorescence resonance energy transfer(FRET).Excited state intramolecular proton transfer(ESIPT),another photophysical process has been attracting considerable interest since the past few years in the field of chemsensors because of their desirable unique photophysical properties.In generally,when ESIPT takes place,it implies low quantum yield(low background),which is exploited by some fluorescent probes.However,ESIPT-based peroxynitrite fluorescence probes are rare.Therefore,we designed and synthesized a naphthalimide fluorescent probe N-CBT based on the ESIPT mechanism to selectively detect the exogenous and endogenous peroxynitrite in living cells.The probe molecule uses 1,8-naphthalimide as a fluorophore and the incorporation of2-benzothiazoleacetonitrile offers N-CBT a rare pre-existing eight-membered ring hydrogen bonding configuration,which is able to generate two types of emission of naphthalimide.Confirmed by calculation results,fast proton transfer from hydroxyl group to carbonyl group occurs along with the excited-state energy transfer via the intramolecular H-bond,leading to a tautomeric transformation from the excited enol form to the excited keto form.In aqueous solution,the formation of intermolecular hydrogen bonding with water perturbs ESIPT and destroys the stable planar construction.By breaking the cyano carbon-carbon double bond in the presence of peroxynitrite,green fluorescence can be regenerated efficiently.As results,34-fold fluorescence enhancement at 518 nm was observed in response,and it showed a good linear relationship in the range of 1 to 14?M with a detection limit of 37 n M.Subsquently,N-CBT was applied in visualizing cellular peroxynitrite,and it demonstrated a great potential in selectively visualizing endo/exogenous peroxynitrite(ONOO^-)in living cells.