Synthesis Of Novel Fluorescent Probe Dyes For Monoclonal Antibody And Its Application In Immunofluorescene Histochemistry

Fluorescent probes have been widely used in molecular biology, microbiology, biochemistry, analytical chemistry, chemical engineering and medicine engineering. At the same time, the request for the properties of fluorescent probes is becoming more and more rigid. Therefore, the synthesis and application research of fluorescent probe dyes become a hot point in interdisciplinary of life science and chemistry.Fluorescien derivatives are prized for their long excitation wavelength, high extinction coefficient, high fluorescence quantum yield in aqueous solution and the ability of their fluorescence characteristics to be tailored for a particular application. As a result, they are used as fluorescent probe dyes widely.In this thesis, a series of novel monoclonal antibody fluorescent probe dyes based on fluorescien were designed and synthesized. Tow of the dyes were investigated for their application in immunofluorescence histochemistry.(1) The synthesis conditions of 2,5-dichloro-p-xylene were studied and optimized. A new method of synthesis of 2,5-dichloro-p-xylene under microwave was studied. The synthesis conditions of 3,6-dichloro-trimellitic anhydride were studied and optimized.(2) Two novel compounds (4,7,4',5'-tetrachloro-6-carboxyfluorescein and 4,7, 4',5'-tetrachloro-5-carboxyfluorescein) were designed and synthesized. The structures of the isomers were identified by IR and MS. The synthesis conditions of 5(6)-carboxyl chlorofluorescein derivatives were studied and optimized. 5(and 6)-carboxyl chlorofluorescein isomers were separated successfully. All of the isomers were determined by ~1HNMR, IR, elemental analysis and MS.(3) 4,7,2',7'-tetrachloro-5(and6)-carboxylfluorescein isomers were synthesized and separated successfully. The crystal structure of 4,7,2',7'-tetrachloro-5-carboxy fluorescein NHS ester was determined by X-ray diffraction firstly.(4) Four chlorofluorescein-based monoclonal antibody fluorescent probe dyes (two of them were novel) with 6-amiohexanol linker were synthesized as dye-labeled primers in DNA sequence analysis. All of the dyes isomers were determined by ~1HNMR, IR, elemental analysis and MS.(5) The synthesis of four novel chlorofluorescein-based monoclonal antibody fluorescent probe dyes with 6-amiohexal acid linker were researched. All of the dyes isomers were determined by ~1HNMR, IR, elemental analysis and MS.(6) Two of the novel dyes (TET2477 acid and TET4457acid) were investigated for application in immunofluorescence histochemistry. It was found that the novel dyes (TET2477 acid and TET4457 acid) are excellent fluorescent probe dyes that can be used to label a variety of monoclonal antibody and other biopolymers.