摘要
荧光探针在分子生物学、微生物学、生物化学、分析化学、化工和医药工业等领域有着广泛地应用。同时它的发展与生命科学、化学、生物技术、诊断医学等学科紧密相关。随着相关技术的发展与普及,荧光探针的应用越来越广泛,与此同时对荧光探针染料性能的要求也越来越高。荧光探针染料的合成与应用研究已发展成为生命科学和化学交叉的前沿研究领域。
荧光素具有高的消光系数,激发和发射波长都在可见光区,在水中具有较高的荧光量子产率,且无毒等优点。另外还可以通过对母体的修饰满足不同使用条件(例如:最大吸收波长,最大发射波长,高的荧光量子产率等)的需要。所以该类荧光探针染料已广泛的应用于生物活体内DNA的检测和蛋白质标记复杂生物体系的研究等领域。
本论文围绕新型荧光素基单克隆抗体荧光探针染料的设计、合成及免疫荧光组织化学应用展开,主要内容有以下几个方面。
1.对荧光素前体2,5-二氯对二甲苯的合成条件进行了优化。提出一种未见文献报道制备2,5-二氯对二甲苯的新方法--微波促进合成法,并对其合成条件进行了研究。对荧光素中间体3,6-二氯-1,2,4-苯三酸酐合成进行了研究,并对文献报道方法进行了优化改进。
2.设计并合成了2种未见文献报道的荧光素类化合物4,7,4',5'-四氯-5-羧基荧光素和4,7,4',5'-四氯-6-羧基荧光素。对5(6)-羧基氯代荧光素的合成条件进行了优化,并提出了一种有效的异构体混合物的分离方法,混和物经酯化、成盐、重结晶,成功地进行了分离。对所有异构体结构都进行了表征。
3.合成了4,7,2',7'-四氯-5(6)-羧基荧光素并对5(6)-羧基异构体成功地进行了分离,首次报道了的4,7,2',7'-四氯-5-羧基荧光素新戊酸酯的晶体结构。
4.合成了4种具有空间连接链的荧光素基单克隆抗体DNA荧光探针染料,其中2种未见文献报道,并对其结构进行了表征。
5.合成了4种未见文献报道的具有空间连接链的新型荧光素基单克隆抗体蛋白荧光标记探针染料,并对其结构进行了表征。
6.分别用新型单克隆抗体荧光探针染料TET2477 acid及TET4457 acid对抗生蛋白链菌素单克隆抗体(免疫球蛋白G,IgG)进行标记,制备了单克隆抗体荧光探针。经过测定两种荧光探针染料对抗体的取代度均在2～4之间,标记有效,取代度适中。用该单克隆荧光探针进行免疫反应,利用共聚焦荧光显微成像技术得到了牛肺部动脉内皮细胞α-微管蛋白及牛肺部内皮细胞肌动蛋白标记细胞图像。对U2OS(人体骨肉瘤上皮细胞),Hela(人宫颈癌上皮细胞)用直接法进行标记,得到了标记细胞图像。免疫荧光组织化学应用表明新型单克隆抗体荧光探针染料具有良好的荧光性能和生物特异性,是一种性能良好的单克隆抗体荧光染料。
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.
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