Design,Synthesis And Application Of Three Two-Photon

In recent years,with the development of imaging technology,fluorescence imaging technology based on fluorescence probes has become an ideal tool for visual monitoring of objects,which has the advantages of simple operation,high sensitivity and high temporal and spatial resolution,so it has been widely concerned and applied.Two-photon fluorescence imaging based on two-photon fluorescence probes uses femtosecond（fs）pulsed laser to generate two photons simultaneously in the near-infrared region（700-900 nm）to excite the fluorophore.Compared with one-photon fluorescence imaging,this technology can improve the visualization depth of target samples,and reduce spontaneous fluorescence and light scattering.Therefore,two-photon fluorescence imaging has attracted much attention in the field of chemical biology.In this thesis,three two-photon fluorescence probes were designed and synthesized,and their fluorescence response and bio-imaging were studied,with two-photon fluorescence probes as the main research direction and bioactive species as the research object.（1）Employing rhodol-HBT coupling dye as the fluorophore and o-phenylene-diamine as the recognition group,a ratiometric two-photon fluorescence probe TP-NO-P1 for nitrous oxide based on ESIPT effect and rhodamine open-closed loop mechanism was designed and synthesized.The probe can specifically respond to NO with low detection limit（51.3 n M）,low cytotoxicity and good photostability.The results of confocal imaging show that the probe can be applied to detect exogenous and endogenous NO in cells,and can also be used to monitor the change of NO content in wound inflammation model tissues,indicating that the probe can be used as a candidate material for detecting the change of NO in vivo.（2）Using coumarin as the two-photon fluorophore,quinolinium ion as the mitochondrial targeting group and diphenylphosphinyl group as the O₂^·-recognition group,a ratiometric two-photon fluorescence probe HMDP for superoxide anion free radical(O₂^·-)with mitochondria-targeting function was designed and synthesized.The probe has a rapid response to O₂^·-,low detection limit（37.2 n M）and good selectivity.The probe HMDP can be used to detect the change of O₂^·-content in OGD/R cell model,and successfully applied to the bio-imaging in mouse stroke model,showing that the probe can be used to monitor the change of O₂^·-in vivo.（3）With GCTPOC as the two-photon fluorophore and1-methylindoline-2,3-dione as the recognition group of ONOO^-,a two-photon fluorescence probe GCT-ONOO for nitrite peroxide was designed and synthesized.The probe has a rapid response to ONOO^-,low detection limit（13.5 n M）and high selectivity.The GCT-ONOO probe was successfully applied to detect the changes of endogenous and exogenous ONOO^-content in cells.