Design,Synthesis And Properties Investigation Of Reactive Fluorescent Probe Molecules Based On Flavonols

Objective: There are huge numbers of small molecules in organisms that are very essential for biological system.These small molecules include amino acids,glucose,H2 S and a variety of metal ions.Levels of these small molecules can reflect the physiological state of the cell or organism.In order to detect these small molecules,fluorescent probes are becoming important means for biological research.It can be used for quantitative detection by analyzing the fluorescence intensity,and visualize the analyte directly in vivo.This work aimed to design and synthesize three fluorescent probes based on the fluorescent core of 2-（4-（diethylamino）phenyl）-3-hydroxy-4H-chromen-4-one.These three fluorescent probes were used to detect Cys,H2 S and Cu²⁺,respectively.And visualize these analytes in living cells.Methods:（1）Synthesis fluorescent probes.4-Diethylaminobenzaldehyde,2-Hydroxy acetophenone,Acrylylchloride,3,3-Dithiodipropionic acid,2-Picolinic acid as raw material,and 2-（4-（diethylamino）phenyl）-3-hydroxy-4H-chromen-4-one was used as a fluorescent core.Three fluorescent probes were designed and synthesized for the detection of Cys,H2 S,Cu²⁺.The purities of three fluorescent probes were determined by UPLC and structures were identified by 1H-NMR,13C-NMR and UPLC-QTOF-MS.（2）Fluorescence probe spectroscopy.The stability of the probes were investigated.UV-vis and fluorescence spectra of probes were determinated with and without targeted small molecules.Furthermore,relationship between fluorescence intensity and concentration of analytes were investigated.The minimum detection limits were calculated by the formula MDI=3S/K.The fluorescence intensity of probe in the presence of interfering ions or amino acids were also measured.（3）Fluorescent probe imaging in live cells.Cys,H2 S,Cu²⁺ were added to A549 human lung adenocarcinoma cells respectively,and probes were applied afterward.Fluorescence in living cells were then observed and captured.Results:（1）In current word,we designed and synthesized three types of fluorescent probes with 2-（4-（diethylamino）phenyl）-3-hydroxy-4H-chromen-4-one as the fluorescent emission group.Structures were identified by LC-MS 1H NMR and 13 C NMR.Their purities were determined to be 94.74%、 97.5%、 93.31% by UPLC.（2）When the detection molecules were added,the color of the probe solution changed remarkably,the fluorescence intensity was enhanced significantly and the maximum emission wavelength was shifted to red.There was a good linear relationship between the molecular concentration and fluorescence intensity.Furthermore,the probes still work well when the interference ions and amino acids were present.The detection limits of Probe A1,A2,A3 were 0.047μM,1.54μM,9.37μM,respectively.（3）Obvious fluorescence was observed in the cells when the probes were added,bright fluorescence could be observed under the fluorescence microscope.Conclusions:In current work,three kinds of fluorescent probes were successfully designed and synthesized for the detection of Cys,H2 S,Cu²⁺.These three fluorescent probes have the advantages of fast reaction,high sensitivity and high selectivity.The results of intracellular fluorescence imaging showed that the probe had good cell membrane permeability,which could be applied to living cells successfully.It indicates that the probe has great potential for biological applications.To the best of our knowledge,the three new structures fluorescent probes have not been reported in the literature,we believe that these new probe will be of great benefit for many researchers engaging in the study of the effects of Cys、H₂S、Cu²⁺ on biological systems.