Design,Synthesis And Application Of Fluorescent Probes For Hemicyanine Esterase

Esterase participates in many physiological processes and metabolism in human life activities,and also palys important roles in the activation and detoxification of many drugs.The abnormal level/activity of esterases in human body will lead to diseases.Traditional detection methods for enzymes including high performance liquid chromatography（HPLC）,UV-vis spectrophotometry,GC-MS,ELISA,chemiluminescence method etc.,usually suffer from time-consuming,low limit of detection,complex sample pretreatment,lack of reproducibility and expensive equipments.In contrast,fluorescent probes are the most promising techniques for researchers to study the physiological functions and distributions of enzymes due to its simple and common equipment requirements,simple operation process,quick analysis ability and high sensitivity.Considering the high significance of enzymes and the unique advantage of fluorescent probe,here is the dissertation,two fluorescent probe based on the hemicyanine derivatives were designed and synthesiszed for the detection of carboxylesterases（CaE）and penicillin G amylase（PGA）,respectively.The main results are as follows:1.A fluorescent probe（HCyNAc）with donor-π-acceptor（D-π-A）structure was synthesized,based on naphthalene and hemicyanine derivatives for the two-photon imaging of CaE in living cells.The protection of the hydroxyl group of HCyN by reaction with acetyl chloride yielded HCyNAc,which also silenced the fluorescence.However,after hydrolysis by CaE,HCyNAc was converted to HCyN,and a substantial fluorescence turn-on response was observed.HCyNAcexhibited a fast reponse（7 min）toward CaE.Moreover,a good linearity between the fluorescence intensity and the concentration of CaE in the range of 0.02-0.07 U mL^-1 was obtained,resulting in a detection limit of 1.8 U L^-1.Compared to other reported fluorescent probes for CaE,the probe HCyNAc displayed unique advantages of high selectivity toward CaE,insensitivity to pH in the physiologival range,high stability and low cytotoxicity.The successful application of HCyNAc for the two photon-excited imaging of CaE in living cells was also realized.2.A light-up and red emissive fluorescent probe HCyNB for PGA detection and imaging in penicillinase-producing Bacillus cereus was also fabricated in a simple two-step processbased on the hemicyanine and naphthaline derivatives.The probe HCyNB,with a phenylacetyl group possesses weak emission in aqueous solution due to the weak intramolecular charge transfer（ICT）effect.However,after the enzymatic reaction,HCyN was generated with a strong ICT effect,thus boosting strong red emission.HCyNB exhibited a rapid response（10 min）,good stability,pH independence,high sensitivity(detection limit of 0.067 U L^-1)and satisfactory selectivity toward PGA over other biologically-relevant analytes.Application of HCyNB in bioimaging of endogenous PGA was confirmed in PGA-producing Bacillus cereus.