Design,Synthesis And Application Of Several Fluorescent Probes For Small Molecules And Bioenzymes

Small-molecule fluorescent probes show great potential in environmental analysis,biomarkers,cell and tissue imaging and clinical diagnosis,due to their advantages of controllable structure,sensitive response,high selectivity and visual monitoring.The application of small-molecule fluorescent probes in response to chemical/biological molecules is of great significance for environmental quality monitoring and the relevant physiological and pathological processes studying.Nevertheless,due to the complexity and instability of environmental samples and biological systems,the design and synthesis of small molecule fluorescent probes with better selectivity,higher sensitivity and faster response is still an important and challenging task.Ratiometric fluorescent probes can effectively eliminate the interference of environmental factors through the ratio of two emissions at different wavelengths,thus improving the detection accuracy and sensitivity.Meanwhile,most of ratiometric fluorescence probes have visualization capability by displaying continuous color change.Therefore,ratiometric fluorescent probes have a good application prospect.In addition,near-infrared fluorescent probes can effectively avoid the organism's self-absorption and spontaneous fluorescence,reduce the damage to living tissues and enhance the penetrating ability,which has great advantages and application prospects in bioimaging.In this thesis,the human life and health closely related small molecules and enzymes were taken as analytes.In order to improve the detection efficiency in environmental and biological samples,several ratiometric and near-infrared fluorescence probes were developed based on the excited state intramolecular proton transfer?ESIPT?and intramolecular charge transfer?ICT?mechanism.The probes were used to detect phosgene,hydrazine?N₂H₄?,alkaline phosphatase?ALP?and?-galactosidase??-gal?,respectively.Moreover,they were successfully applied to environmental sample detection and biological imaging analysis.The main contents and results are as follows:?1?A fluorescent probe 2-?1H-phenanthro[9,10-d]imidazol-2-yl?phenol?Pi?was designed and synthesized,bearing hydroxyl and imidazole moieties as recognition sites,and employ it for the excited-state intramolecular proton transfer based?ESIPT-based?detection of phosgene.In the presence of phosgene,Pi undergoes sequential nucleophilic substitution and cyclization reactions that facilitate rapid response?30 s?,high selectivity,and excellent sensitivity,the linear range was 0-4?M,and the detection limit was 0.13?M.Furthermore,a fluorescent test strip?FTS-Pi?was fabricated for the detection of phosgene in the gas phase that undergoes a fluorescence color change,from green to blue,under365-nm UV light in the presence of phosgene,which is easily observed with the naked eye.It can provide a method for spot and rapid detection of phosgene.?2?A colorimetric and ratiometric fluorescent probe?E?-3-?4-?dimethylamino?phenyl?-1-?2-hydroxyphenyl?prop-2-en-1-one?DH?was designed and synthesized for the detection of N₂H₄ based on the mechanism of ESIPT.The?,?-unsaturation carbonyl group in DH was used as a recognition group for N₂H₄.The probe can detect hydrazine by colorimetric and ratiometric response with high selectivity and sensitivity,the linear range was 8-100?M,and the detection limit of N₂H₄ was 0.06?M.Furthermore,DH was applied to detect N₂H₄ in real water samples.?3?A biocompatible N₂H₄ near-infrared fluorescent probe2-??6-hydroxy-2,3-dihydro-1H-xanthen-4-yl?methylene?malononitrile?HM?was developed for the detection and imaging of N₂H₄.Near-infrared fluorescent dye was selected as the fluorophore,and methylene malononitrile was taken as the recognition group of N₂H₄.Methylene malononitrile with strong electron withdrawing ability can react with N₂H₄ to form hydrazone,which significantly weakens the electron withdrawing ability,changes the original push-pull electronic structure of HM,and blocks the intramolecular charge transfer?ICT?process.As a result,the ultraviolet absorption spectrum and fluorescence emission spectrum of the probe are blue shifted,and the color of the solution gradually fades from dark blue to light yellow,thus realizing colorimetric and ratio fluorescence dual-signal detection of N₂H₄.The probe has high selectivity,and good sensitivity to detect hydrazine,the linear range was 1-900?M,and the minimum detection limit was 0.09?M.Furthermore,due to the advantage of low background and good biocompatibility in cell imaging,the study of N₂H₄ imaging in human normal liver cells?LO2?has been successfully realized.?4?A ratiometric fluorescent probe 2-?benzo[d]thiazol-2-yl?phenyl dihydrogen phosphate?PHT?was designed and synthesized to monitor endogenous alkaline phosphatase?ALP?activity in living cells.PHT was designed and synthesized by using 2-?2'-hydroxyphenyl?benzothiazole as the fluorophore,and based on the specific enzymatic hydrolysis reaction of ALP,phosphate group was used as the identification site of ALP.In the presence of ALP,PBT generates HBT by enzymatic hydrolysis,and the fluorescence is on due to the typical ESIPT characteristics of HBT,so as to realize the detection of ALP based on ESIPT mechanism.The probe has a high selectivity and sensitivity for ALP detection,with a linear range of 1-80 U/L and a detection lower limit of 0.8 U/L.Furthermore,it has been successfully applied to fluorescence imaging of ALP in LO2 and human hepatocellular carcinoma?HepG2?.?5?A near-infrared fluorescent probe 2-?5,5-dimethyl-3-??E?-4-???2R,3S,4R,5S,6S?-3,4,5-trihydroxy-6-?hydroxymethyl?tetrahydro-2H-py ran-2-yl?oxy?styryl?cyclohex-2-en-1-ylidene?malononitrile?DCM-?-gal?was designed and synthesized to monitor the endogenous activity of?-galactosidase??-gal?in living cells.DCM-?-gal was designed and synthesized by using near-infrared fluorescent dye?E?-2-?3-?4-hydroxystyryl?-5,5-dimethylcyclohex-2-en-1-ylidene?malononitrile?DCM-OH?as the fluorophore,and based on the specific enzymatic hydrolysis reaction of?-gal,galactosyl used as the identification site of?-gal.The probe has the advantages of good selectivity and high sensitivity for the detection of?-gal,with the linear ranges of 0.08-1 and1-4 U/mL,and the detection lower limit of 1.6×10^-3 U/mL.Furthermore,it has been successfully applied to the fluorescence imaging of ovarian cancer cells?SKOV-3?.