Synthesis And Properties Of Small Molecule Biothiols Fluorescent Probe Based On Coumarin Structure

Small molecule biothiols,such as cysteine(Cys),homocysteine(Hcy)and glutathione(GSH),are important biomarkers and play vital roles in many physiological processes.Currently,the main methods for the detection of biothiols have been reported,such as high performance liquid chromatography(HPLC),capillary electrophoresis,mass spectrometry(MS),electrochemical analysis,gas chromatography-mass spectrometry series and fluorescent probe.Among the various detection methods,fluorescent probes have been widely studied and applied due to its advantages of high sensitivity,strong selectivity,short response time and simple equipment.In recent years,biothiols fluorescent probes based on various fluorophiles have been reported.These fluorescent probes are mainly designed and synthesized by utilizing the strong nucleophilicity of the sulfhydryl group.The main reaction mechanisms include Michael addition,cyclization reaction with aldehydes,nucleophilic substitution and cleavage reactions.Coumarin has some excellent biological and physical properties,such as large Stokes' shift,strong light stability,strong oxidation resistance,good water solubility,high fluorescence quantum yields and fluorescence intensity.In this study,three small molecule biothiols fluorescent probe based on coumarin were synthesized,and their properties were studied.The main contents of this thesis are as follows:In the first chapter,the importance of biothiols,the recognition mechanism of fluorescent probe,the research progress of biothiols fluorescent probe and the structure and application of coumarin are briefly introduced.In the second chapter,probe 1 was synthesized with morpholine substituted coumarin as the fluorescent group and NBD as the recognition group.Results showed that the probe 1 could selectively identify Cys/Hcy and GSH from other amino acids by naked eyes with color changes.When the excitation wavelength is 400 nm,in the presence of Cys/Hcy and GSH,significant fluorescence emission appeared at 490 nm,and the fluorescent color of the solution changed from dark to blue.When the excitation wavelength is 470 nm,in the presence of Cys/Hcy,significant fluorescenceemission appeared at 580 nm,and the fluorescent color of the solution changed from dark to green,These results demonstrated that the probe 1 can simultaneously detect and distinguish Cys/Hcy and GSH by dual emission.Moreover,imaging experiments indicated that the probe 1 could be used for simultaneous detection and discrimination of Cys and GSH in living cells,and colocalization experiments demonstrated that probe 1 has the property of targeting lysosome.In the third chapter,the probe 2 was synthesized with morpholine substituted coumarin as the fluorescent group and acrylate as the recognition group,and its properties were studied.When the excitation wavelength was 390 nm,in the presence of Cys,there was an obvious fluorescence emission peak at 490 nm,and the fluorescent color of the solution changed from dark to blue.The results showed that the probe 2 could selectively identify Cys from other analytes(including Hcy and GSH)with high selectivity and sensitivity,and the detection limit was as low as 6.8n M.In the fourth chapter,the probe 3 was synthesized with triphenylphosphine substituted coumarin as fluorophasebased and acrylate as recognition group.When the excitation wavelength was 400 nm,in the presence of Cys,there was an obvious fluorescence emission peak at 475 nm,and the fluorescent color of the solution changed from dark to blue.The results showed that the probe 3 could selectively identify Cys from other analytes(including Hcy and GSH)with high selectivity and sensitivity.