Synthesis And Properties Of NIR Fluorescent Probes For Detection Of Intracellular Microenvironment And Small Molecules

The microenvironment in the cell,including parameters such as temperature,viscosity,and pH,is an important factor affecting the physical and chemical behavior of molecules in the cell,and plays a vital role in controlling molecular diffusion,transport,and interaction.It have shown that abnormal microenvironmental parameters in living organisms can be used as a signal for the judgment of disease occurrence.Therefore,reasonable design are required to achieve efficient,convenient,and accurate fluorescence sensors for the detection of the microenvironment,and the exploration of the physiological and pathological changes associated with the microenvironment and disease has important research significance.The research of fluorescent probes for hypoxia recognition was first carried out.Based on the trimethyl lock quinone recognition group,four different fluorophore-linked near-infrared fluorescent probes Dia1,Dia2,Dia3,and Dia4 were used to monitor hNQO1.Among them,Dia1 showed higher selectivity for luteinase.After the addition with luteinase,the fluorescence intensity of Dia1 increased by 3.9 fold,with the advantages of high sensitivity and strong selectivity,fast response,and long wavelength emission.In the in vitro spectrum tests and in vivo bioimaging experiments,Dia1 can track the endogenous luteinase produced under hypoxic conditions in the cell,and indirectly monitor the degree of hypoxia in the cell by the spectrum changes,Dia1is a good near-infrared fluorescent probe for hypoxia recognition.With azo bond as a recognition group,the anticancer drug nitrogen mustard was introduced to make a fluorescent probe（Azo-M）targeting azoreductase was synthesized.The peodrug w released in situ to achieve a therapeutic effect on the tumor.In the in vitro spectroscopic experiments,Sn²⁺was used to break the azo bond and release the fluorophore,and the fluorescence intensity of the solution increased by 46times.Azo-M can perform fluorescence imaging of azoreductase endogenously produced by cells and nematodes under hypoxic conditions,effective fluorescence imaging of three azoreductase-rich bacteria（E.coli,S.aureus,and P.aeruginosa）were carried out.After the treatment of Azo-M to tumor-bearing mice,tumors in the mice were significantly inhibited after three weeks to achieve a therapeutic effect.Experiments have shown that Azo-M can specifically recognize azoreductase,indirectly measure the degree of hypoxia in cells and tumors through fluorescence changes,Azo-M is a good azoreductase fluorescent probre,which can achieve drug release under hypoxic condition and treatment of tumors in situ.Based on the near-infrared fluorophores of rhodamine,xanthracene and cyanine,three different near-infrared fluorescent probes RhopH1,RhopH2 and RhopH3 were synthesized for pH detection.Among them,RhopH1 and RhopH3 showed high sensitivity and selectivity to pH changes.Due to the acidity,the structures of the fluorescent probes changed from a spiral ring to an open ring,which led to the fluorescence intensity increase of 32 times and 30 times for RhopH1 and RhopH3,respectively.RhopH1 has a pH detection range of 1.7-7.4 and RhopH3 has a pH detection range of 2.2-7.4,both probes can achieve fluorescence imaging of pH changes in Hela cells.The experimental results showed that RhopH1 and RhopH3 are near-infrared fluorescent probes that can specifically identify the pH change in cells.Based on the fluorophores of rhodamine and hemicyanine derivative,three near-infrared fluorescent probes HemGSH1,HemGSH2 and HemGSH3 for the detection of GSH were synthesized.The HemGSH3 probe uses 2,4-dinitrobenzenesulfonyl as a thiol recognition group and was connected to the hydroxyl group of the hemicyanine derivative.In the presence of GSH,the SNAr reaction cleaveed 2,4-dinitrobenzenesulfonyl to generated the free fluorophore with a turn on fluorescence.HemGSH3 has many characteristics,such as strong specificity,high sensitivity,and low detection limit（0.6?M）,for GSH among many small thiol molecules.HemGSH3is a high-sensitivity near-infrared fluorescent probe that can be successfully used for GSH imaging in Hela cells.In summary,we synthesized a series of near-infrared fluorescent probes for detecton of the in vivo levels of hypoxia,pH environment and GSH in living organisms,providing an effective method for sense of related signals,and exploring the integration of hypoxia tracer,drug release and in situ treatment Integrated research of diagnosis and treatment,achieved good results.