Novel Fluorescent Probes For Bioimaging Of Intracellular PH And Histone Deacetylase

pH homeostasis is essential for human body to carry out a series of physiological and pathological processes.Disruption of pH homeostasis are closely associated with various health problems.Internal pH values ranging from alkaline to highly acidic levels can be observed for various cells,subcellular organelles and intercellular matrices as well as body fluids.Precise measurement of pH values in specific tissues,cells and organelles are of great use for further study on relevant pathophysiological processes.Histone deacetylases?HDACs?are a series of important enzymes in living mammalian cells that regulate gene expression,cell cycle and tumorigenesis.HDACs ha ve been reported to be overexpressed in tumor cells,which results in hypoacetylation of histone and inhibition of genes expression involved in cell apoptosis and differentiation,and eventually leads to cell hyperproliferation,making them important targets for cancer therapy.Fluorescent pH and HDACs probes with high selectivity,fast response,and good biocompatibility are powerful tools for bioimaging of pH and HDACs in living cells,and can provide scientific evidence for the diagnosis and treatment of related diseases.The main work carried out in this thesis is as follows:Fluorescent pH probe DDXC was designed and synthesized with phosphorous tribromide,Cs₂CO₃,4-?diethylamino?-salicylaldehyde,N,N-dimethylformamide,and cyclohexanone as the main raw materials.The synthesized intermediates and the target product were characterized by ¹H NMR,¹³C NMR,and HRMS.The carbonyl group in DDXC can be effectively protonated in an extremely acidic environment owing to the stabilizing effect of the following keto-enol tautomerization,and switch the fluorescence from orange to green,enabling the probe to ratiometrically measure the pH values of aqueous solutions and visualize the intracellular pH levels.In the UV-vis absorption titration,when pH value decreased from 6.8 to 1.2,the absorbance of the probe at 490 nm decreased significantly,and a new peak appeared at around 400 nm.Simultaneously,the fluorescence intensity of the fluorescence spectra at 580 nm decreased,while the fluorescence intensity at 512 nm increased gradually,with pK_a value being 3.11.The blue shift of the absorption and fluorescence emission triggered by the pH change indicates that probe DDXC can effectively respond to pH changes.The probe exhibits high sensitivity,excellent sele ctivity and good reversibility in extremely acidic environment,making it suitable for in vitro pH detection and live-cell pH imaging.A two-photon fluorescent probe?TP-H?for HDACs was designed and prepared from N,N-dimethylformamide,6-acetamidocaproic acid,trimethylsilyl cyanide,6-bromo-2-naphthol and dimethylamine.The synthesized intermediates and the target product were characterized by ¹H NMR,¹³C NMR,HRMS.Under the excitation of 380 nm,the probe has a very weak fluorescence.After incubating the probe with histone deacetylase for 2 h at 37?,the probe is excited by the same excitation light and a strong fluorescence emission peak appeared at 525 nm.The fluorescence intensity was linearly correlated with the concentration of histone deacetylase.The linear response range was 0.1-9.0 ug/mL and the detection limit was 0.3?g/mL.In the stability test the probe exhibited stable optical properties in the pH range from 6.8 to 8.5.In addition,probe TP-H was successfully applied to bioimaging of HDACs in living cells and tissues.