生物标识荧光染料及游离基氯化反应的研究

【作者】 彭正红；

【导师】 彭孝军；

【作者基本信息】 大连理工大学 ， 应用化学， 2004， 硕士

【摘要】 本论文的主要工作是合成了四个用于生物标识的荧光染料，以及对游离基氯取代苯环上硝基和游离基氯取代苯环上甲基氢的的相对反应速度进行了研究。 第一：采用了一种实用的路线合成提纯6-羧基荧光素化合物。本文采用间苯二酚或4-氯间苯二酚和偏苯三甲酸酐缩合得到5-位和6-位羧基荧光素异构体。荧光素类衍生物由于在普通溶剂中的溶解性很差，并且存在内酯环和游离羧基两种状态，增加了分离提纯的难度。上面介绍的两个缺点能够通过在吡啶溶液中和特戊酰氯反应得到特戊酰氯的羧基荧光素而解决，特戊酰氯的羧基荧光素重新溶解在乙醇中，加入二异丙胺，特戊酰基的6-羧基荧光素二异丙胺盐(特戊酰基的2′，7′-6-羧基荧光素二异丙胺盐)在乙醇中析出来。在稀盐酸中水解，和N，N′-二琥珀酰亚胺碳酸酯反应得到相应的NHS酯，能够直接用于蛋白质标记。 第二：合成了两个新的BODIPY染料。对羧基苯甲醛或对羟基苯甲醛和2，4-二甲基吡咯缩合得到二吡咯的甲基化合物，在四氯苯醌的条件下脱氢得到二吡咯的亚甲基化合物。再与三氟化硼和三乙胺反应后得到总收率为9.6％羧基BODIPY染料和总收率为35.7％的羟基BODIPY染料。羧基BODIPY能够直接用来标识蛋白而羟基BODIPY能够用于标识核酸。 第三：对两类游离基氯化反应的相对反应速度进行了研究。实验结果表明游离基氯取代苯环上硝基的速度比游离基氯单取代苯环上甲基氢的速度慢，但是比游离基氯二取代苯环上甲基氢的速度快。更多还原

【Abstract】 In this thesis, four different fluorescent dyes for labeling biological molecules have been synthesized and the mechanism of free radical chlorination of nitrotoluenes have been investigated.Firstly: A practical and reliable method for the production of multigram quantities of carboxy fluorescein is described. The poor solubility of fluorescein derivatives and their existence as lactone or free acid tautomers complicate 5- and 6-isomeric separation.Both of the problems are circumvented by the reaction of pivaloyl chloride and carboxyfluorescein in pyridine to give a crude mixture of dipivalates. After resolved in ethanol and treated with diisopropylamine, pure 6-carboxy fluorescein Dipivalates diisopropylamine salt is obtained. After treated with dilute hydrochloric acid and N,N-disuccinimidyl carbonate, the corresponding 6-carboxyfluorescein dipivalate succinimidyl ester which is suitable for labeling protein is synthesized.Secondly:Two novel BODIPY dyes are designed and synthesized. The p-substituted benzaldehyde condense with two equivalent 2, 4~dimethylpyrrole give dipyrromethane. Subsequent treatment of dipyrromethane with p-chloranil affords a black reaction mixture. Treatment of the black mixture with triethylamine and BF.,-etherate affords the carboxy BODIPY dyes and the hydroxy BODIPY dye in 9.6% and 35.7% yield respectively. The NHS ester of the former can be used for labeling protein, and the latter is suitable for labeling oligonucletides.Thirdly: The relative reactivities of two kinds of radical chlorination have been investigated. The result shows that the reactivity of denitrochlorination is much less than the first a -chlorination of methyl group but more than the second a-chlorination.更多还原