Syntheses And Characterization Of Fluorescein Derivatives

The synthesis of fluorescein was first reported by Bayer in 1871. Fluorescein was widely used in the biological analysis, due to its high extinction coefficient, high fluorescence quantum yield in aqueous solution, its excitation wavelength and emission wavelength in the visible range and innoxiousness. However, it's some intrinsic properties also limit its further applications in many fields. To improve fluorescein's performance, a wide variety of fluorescein derivatives have been prepared by structural modification and used as fluorescent reagents favorable for detecting the organism.In this thesis, a series of fluorescein derivatives such as fluorescein esters of straight chain carboxylic acid, fluorescein derivatives with reactive group, chlorofluorescein derivatives, dinaphthafluorescein derivatives and unsymmetrical fluorescein derivatives, were designed and synthesized based on fluorescein, in order to modificate their lipophilicity, binding specificity, pH-sensitive range, longer-wavelengths and the large stokes shift. Then their fluorescence properities were investigated by UV-Vis spectra and fluorescence spectra and some relationship between their structures and photophysical properties were summerized.Firstly, a series of fluorescein esters with 2~18 carbon chain were prepared. The general method for synthesis of fluorescein esters with long carbon chain was established. In addition, the hydrolytic properties of flourescein esters including the rate of spontaneous hydrolysis and the rate of enzymatic hydrolysis were investigated. And the complicated relationship between the chain length of fluorescein esters and their hydrolytic properties had been found. The excellent fluorescent probe for the assay of lipase was filtrated among the fluorescein esters.Secondly, the synthetic methods of fluorescein derivatives with reactive group were studies and improved, and these regioisomers with reactive group at 5 and 6 position were successfully separated by chromatography with the mixture of ammonia and alcohol. Then, the fluorescence properties were investigated by comparison with each other.Thirdly, the five chlorofluorescein derivatives were designed and obtained by the traditional synthetic method of fluorescein. Their fluorescence properties were examined and found their absorption and emission maxima at longer wavelength, highfluorescence quantum yield. Furthermore, the relationships between their fluorescence properties and pH value were examined. The results shew chlorofluorescein derivatives were strongly pH dependent and the pH-sensitive ranges of them shifted to the acid condition with the increase of number of chlorine. Therefore, chlorofluorescein derivatives could be used as pH-sensitive fluorescent probe to measure intracellular pH and could detect the sample with the maximal fluorescence intensity at the physiological pH.Fourthly, dinaphthafluorescein derivatives with a big conjugated structure were prepared and examined by UV-Vis spectra and fluorescence spectra. These derivatives were found to shift absorption spectra and emission spectra to longer-wavelengths than fluorescein. These substrates could be used as near-infrared fluorescent probe because their emission wavelengths were over 600nm.Finally, a series of unsymmetrical fluorescein derivatives were designed and prepared by two-step synthetic route, and their photophysical properties were investigated both in acid and base condition. The results shew the unsymmetrical fluorescein derivatives had some excellent properties, including a big stokes shift, two absorption and emission wavelengths and pH-dependence. Due to the above performance, unsymmetrical fluorescein derivatives could be used to measure pH quantitatively and could be applied in other biological analysis fields.According to the above preparation and detection, the better fluorescent probes were selected, such as fluorescent probe for the assay of lipase---fluorescein dilaurate, pH fluorescent probe with high fluorescence quantum yield---4(7)-chlorofluorescein, pH fluorescent probe to examine the acid condition---2',7'-dichloro-4,5,6,7-tetrachloro -fluorescein, near-infrared fluorescent probe---dinaphthafluorescein and near-infrared fluorescent probe with reactive group---5(6)-carboxy-dinaphthafluorescein, dual emission pH probes--- unsymmetrical fluorescein 7.1, 7.4, 7.6, and so on.