Synthesis And Application Of Cellular Fluorescent Probes

As an important technical mean, fluorescent probes have been widely used in molecular biology, cell biology, biochemistry, chemical engineering and medicine. At present, Fluorescent probes used to be marked have fluorescein derivatives, rhodamine derivatives, coumarin derivatives, o-phthalaldehyde derivatives and so on. Fluorescein and its derivatives have been a hot spot of research in the field of chemical and biological analysis. Fluorescein derivatives entered into the cell were enzymolysised to fluorescien, which was used to research structural features of protein and cell detections, due to its high extinction coeffieient, high fluorescence quantum yield in aqueous solution, non-toxic, low cost.Cells are the basic units of life activity and morphology of organisms. Studying the properties of specific cells through fluorescent probes has been an advancing topic in the field. Probes used for labeling living cells must have the following conditions:(1) Probes can combine with specific intracellular substrate;(2) Signals of the product labeled by probes can be enough strong to distinguish unlabeled cells;(3) Probes can’t injure the target cells;(4)There is a little interference of fluorescent background, that’s to say, the fluorescent products can’t leak from the cells. Fluorescein diacetate, aminofluorescein and isothiocyanate fluorescein were used to label living cells, but the leakage of their fluorescent products has not been satisfactorily resolved.In this thesis, a series of fluorescein derivatives were synthesized based on fluorescein, in order to modificate their lipophilicity and binding specificit and reduce the reverse osmosis of fluorescein, which were applied to the detection of cellular activity. The studies in this thesis were summarized as follows:1.Progress of fluorescent probes and application in cytology were reviewed.2.Fluorescein and fluorescin were synthesized and esterified, then synthesis of fluorescin was optimized. Stability of the fluorescein diacetate and fluorescin diacetate was studied to explore optimal conditions by single factor experiments, which were pH6-7,the temperature of0℃,the solvent of acetone and the concentration of5x10-4g/L3.Nitrofluorescein, aminofluorescein, bromomethyl acetate, and aminofluorescein derivatives of which the lipotropy was modified were synthesized and identified their structure. Aminofluorescein derivatives are as follows:5-acetamidofluorescein diacetate(AF-DA),5-succinamidofluorescein (SAF),5-succinimidylfluorescein(SF),5-succinamidofluorescein diacetate (SAF-DA),5-succinamidofluorescein diacetate (SAF-DA),5-succinamidofluorescein acetoxymethyl ester (SAF-AM).4.New fluorescein derivatives were applied in dyeing HepG2cell and U266cell, and the optimal conditions of fluorescein derivatives on labeling cells were studied, which were the concentration of1mg/L and the marking time of20min. AF-DA, SAF-DA, SAF-AM were the optimal fluorescein derivatives on labeling cells, compared with FDA.