Design And Synthesis Of NIR Xanthene Dyes And Application In Ratiometric Bioimaging

Fluorescence analysis is widely well received by biochemists due to its simple operation,high sensitivity,and real-time detection.However,most fluorescent probes excite and emit in the visible region,which has some problems such as large interference of biological background,deep tissue penetration,minimum photo-damage,etc.Because of these advantages,the development of near infrared fluorescent dyes with high fluorescence quantum yield,high stability and low toxicity has attracted much attention.Moreover,most probes response to an analyte of interest with optical signal changes only in fluorescent intensity,which is easily interfered by the concentration of the probe itself and other factors such as the external environment,so it would reduce the accuracy of the measurement.The ratio fluorescence probe can eliminate the errors caused by the interference of the probe’s own concentration,the efficiency of the instrument and the difference of environmental factors through the ratio detection of two channels,which has a broad prospect of application in biological imaging.In this thesis,we have designed and synthesized a series of novel fluorescent dyes with near infrared emission wavelength,and studied their optical properties in detail.Finally,based on these dyes,two novel ratio fluorescent probes were developed for the detection of nitroreductase and L-cysteine,the main works are as follows:(1)Because of the problems of poor stability and low fluorescence quantum yield of 2,4,6-triphenylpyrylium salt(TPP),a series of triphenylpyran salt fluorescent dyes with good stability were designed and synthesized.And the optical properties are studied in detail.These dyes have optimized stability,quantum yield and stokes shift compared to TPP dyes.The fluorescence emission wavelength of dyes can be regulated by modifying their amino groups.We further developed a ratiometric fluorescent probe for detection of nitroreductase(NTR)using TPPF7,and the probe can be used for the detection of NTR in cells under different hypoxia conditions.(2)We have developed a class of novel smart near infrared fluorescent dyes(HDFL)by hybridization of the fluorescein and HD NIR dye.And these dyes have high chemical stability,photostability and near infrared emission.Moreover,by tuning the hydroxyl groups of the dyes,the emission wavelength of dyes shifted significantly and the product had positive charge with target site.We further developed a novel near-infrared ratiometric probe HDFL-Cys,the probe not only can detect L-cysteine selectivityly and sensitively,but also bioimaging studies showed that HDFL-Cys can first accumulate in mitochondria and then react with the analyte Cys.Furthermore,HDFL-Cys can also be used to distinguish normal and cancer cells by monitoring the cysteine levels.