Synthesis,performance And Biological Applications Research Of Novel PH Fluorescent Probes

As one of the most important evaluation indicators in enzyme,cell and tissue activities,pH played a key role in maintaining normal physiological processes such as cell growth,proliferation,apoptosis and signal transduction.In addition,micro-changes of pH could also affect a variety of nervous system activities such as synaptic signaling,excitation of nerve cells,and multicellular coupling due to inter-cell signaling pathways and gap junctions.The optimal pH of the human body was 7.35~7.45,however,when pH fluctuated up and down,it might cause cell function disorder,which might cause nerves,heart and lungs and other related diseases,even could seriously threaten life safety.Therefore,monitoring the dynamic changes of pH in cells was important for analyzing the mechanism of action of understanding the physiological functions of cells.Fluorescent probes had become an widely used method for monitoring intracellular pH due to their high sensitivity,good selectivity,fast response and real-time monitoring of intracellular pH changes.In this paper,based on the research status of pH fluorescent probes,the pH probes with O-(2-benzothiazolyl)phenol(HBT),naphthalimide and 7-hydroxycoumarin as fluorescence platforms were successfully designed and synthesized.The needles were used for in situ pH detection of cells and endoplasmic reticulum acidification under ER stress conditions,and their spectral properties and biological applications were studied in depth.Firstly,the ratiometric fluorescent probe BtyC-1 was designed using O-(2-benzothiazolyl)phenol as a fluorescent platform,and BtyC-1 regulated the fluorescence response by proton transfer between the ortho-hydroxyl group of the benzothiazole and the nitrogen atom based on excited state intramolecular proton transfer(ESIPT)process.The probe exhibited green fluorescence at 546 nm under acidic conditions due to the inability of the hydroxyl hydrogen to ionize;and under alkaline conditions,intramolecular proton transfer occurs rapidly between the hydroxyl hydrogen and the nitrogen atom to exhibit strong blue fluorescence at 473 nm and weak green fluorescence at 546 nm.Besides,biological experiments demonstrated that the probe BtyC-1 could be successfully applied for the ratiometric imaging of cellular pH,the NH4Cl-induced pH changes in living cells and tissue imaging.Secondly,a new ER-targeting enhanced pH fluorescent probe Na-pH was designed and synthesized by naphthalimide as the fluorescent platform.And p-methylbenzenesulfonamide was used for the endoplasmic reticulum localization,and the piperazine group was designed as the pH response site.Protonation of the probe's nitrogen atom on piperazine with decrease in pH leaded to inhibition of the PET process,thereby exhibiting a sensitive fluorescence enhancement,and further indicated that the probe was sensitive to acid,and could be expected to be used for cell imaging of ER acidification.Besides,Na-pH was successfully used to track ER acidification during ER-stress in biological experiments.In addition,Na-p H was also used for two-photon imaging of the pH of living tissue and zebrafish.Thirdly,based on the ICT-PET-FRET mechanism for two-site detecting the pH in the ER ratio type fluorescent probe CNER-pH was designed and synthesized through naphthalimide and 7-hydroxycoumarin as the fluorescent signal platform.Piperazine was used as the acidic environment response site,hydroxy group was used as the alkaline response site,and p-methylbenzenesulfonamide was used as ER-targeting group.Under acidic conditions,the piperazine of naphthalimide moiety protonized to inhibit the PET process,emitting green fluorescence;under alkaline conditions,hydroxyl hydrogen of coumarin occurred ionization to form strong push-pull electronic system leading to the blue fluorescence enhanced,and at this time,the PET progress of naphthalimide moiety is recovered due to the deprotonation process and the fluorescence quenched.The probe showed highly sensitive response to pH in the range 5.0~7.2,and could be applied for the quantitative measurement of the pH values in ER during ER stress and dexamethanose-induced stimulation by cell imaging and flow cytometry.