Synthesis Of Novel Fluorescent Probes For Monoclonal Antibody And PH Probes And Application In Immunofluorescene Histochemistry

Fluorescent probe is an important technical mean, which is used to monitor biomolecule real-time by a fluorescence microscope. Fluorescent probes are widely used because of high sensitivity, strong visibility, no damage and little interference on the target cells or biological macromolecules. Fluorescent probes have been widely used in molecular biology, microbiology, biochemistry, analytical chemistry, chemical engineering and medicine engineering. Chemists and biologists continue to design and synthesis of new types of fluorescent probe dye to meet different application requirements and higher fluorescence performance requirements. The synthesis and application research of fluorescent probe dyes become a hot point in interdisciplinary of life science and chemistry.In a variety of fluorescent probe dye, fluorescein dyes are most widely used. Because of easy synthesis, easy isolation and purification, with high fluorescence quantum yield and non-toxic, fluorescein has become an important probe. Fluorescein derivatives are easily obtained by modifying fluorescein in order to meet different needs. Fluorescein dyes are widely used in DNA sequencing, antibody protein staining and other biological research.Design, synthesis and application in immunofluorescence of monoclonal antibody fluorescent dye were discussed in this thesis. Design, synthesis and spectral properties of new pH probes were also discussed in this thesis. Summarized as follows:1. Compound 2,4-dichloro-resorcinol which was an important material for synthesis of fluorescein was study in detail. A simple synthetic method of 2,4-dichloro-resorcinol was reported and reaction conditions was optimized. Compared with the traditional synthesis methods, the new synthesis method has many advantages. The synthesis of N-hydroxysuccinimidyl trifluoroacacetate (NHS-TFA) was reported in this thesis.2. Two novel chlorinated fluoresceins 4,7-dichlorofluorescein and 2',4',5',7'-Tetrachloro-6-(5-carboxyl)-4,7-dichlorofluorescein were synthesized. Structures of target compounds and intermediates were determined via IR, MS,1H NMR and element analysis. The effects of reaction conditions was discussed and reaction conditions was optimized. The relationship between fluorescence properties and the number of chlorine atoms was studied in detail.6-carboxyl-4,7-dichlorofluorescein and 2',4',5',7'-Tetrachloro-6-carboxyl-4, 7-dichlorofluorescein were isolated as their diisopropylamine salt, which can be converted to the free acid. Structures of target compounds and intermediates were determined via IR, MS,1H NMR and element analysis.3. One chlorofluorescein-based monoclonal antibody fluorescent probe dyeswith 6-amiohexanol linker was synthesized as dye-labeled primers in DNA sequence analysis. All of the dyes isomers were determined by 1HNMR, IR, elemental analysis and MS.Ten chlorofluorescein-based monoclonal antibody fluorescent probe dyes with amino acid linker were synthesized as fluorescent probes for labeling proteins. Structures of target compounds and intermediates were determined via IR, MS,1H NMR and element analysis. Spectral properties were studied in detail. All the probes for labeling proteins have not been reported in references.4. One of the novel dyes (8dh) were investigated for application in immunofluorescence histochemistry. The compound 8dh was used to label fixed U2OS cell. Fluorescence images of U2OS cells in 96-well costar black plate were taken by fluorescence confocal microscopy. It was found that the novel dye (8dh) is excellent green fluorescent probe dyes that can be used to label a variety of monoclonal antibody and other biopolymers.5. The research progress of pH value fluorescent probes was overviewed in this thesis. Eight chlorinated fluoresceins have been synthesized by the reaction of chlorinated resorcinols with 4,5,6,7-tetrachlorophthalic anhydride or 3,6-dichloro-4-carboxyphthalic anhydride in the presence of methanesulfonic acid. The spectral properties of the chlorinated fluoresceins were studied. It was found that they have absorption and emission maxima at long wavelengths and high fluorescence quantum yields. Emission spectra of chlorinated fluoresceins shifted towards long wavelength with increase of chlorine. pH-Dependent properties of chlorinated fluoresceins were studied in detail. These compounds showed strongly pH-sensitive range of 3.0～7.0. These chlorinated fluoresceins will be used as pH probes for pH measurement of the cell because of the high quantum yield and the strong pH-sensitivity.