选择性识别儿茶酚胺的硼酸类荧光探针的合成及初步活性研究
发布时间：2018-12-07 09:04
【摘要】：儿茶酚胺类物质主要包括去甲肾上腺素、多巴胺、肾上腺素、异丙肾上腺素、5-羟色胺、丁羟色胺及其衍生物。儿茶酚胺类物质调节人体基本的生理功能,是生理过程中重要的信号介质,在病理过程中也随之发生相应的含量变化。在医学研究领域可用于辅助诊断甲亢、高血压、神经母细胞瘤和嗜铬细胞瘤等内分泌类相关疾病。儿茶酚物质广泛存在体内组织和器官,特别是儿茶酚胺类物质(如多巴胺,左旋多巴,肾上腺素等)与中枢神经系统类疾病和体内心血管类疾病有重多关联。因此开发一种高选择性和灵敏度的儿茶酚胺类物质的检测方法具有重要医学研究意义和实用价值。荧光探针技术具有高灵敏度、操作简便、快速、检出限低和高选择性等特点,在生物化学传感、光学新材料研究以及生物分子检测和识别等领域获得了广泛的应用。在水溶液中,苯硼酸及其衍生物可与二羟基化合物可逆性的结合形成五元或六元环状酯,基于硼酸的这种亲和性,从而实现了对儿茶酚胺类、糖类、糖蛋白、细菌和细胞等的分析检测。2-(4-二羟基硼烷)苯基喹啉-4-羧酸(PBAQA)是一种新型水溶性硼酸荧光探针。结构中的喹啉结构通过一个苯环与硼酸基团相连,结构中存在游离的羧基,可作为修饰位点引入其它官能团或结构。PBAQA结构中硼酸可与儿茶酚胺结构中的邻苯二酚结构通过共价键结合,通过PET机理实现荧光淬灭,从而实现对儿茶酚胺类结构的选择性识别。但PBAQA化合物对多巴胺和左旋多巴等结构类似的儿茶酚胺类物质选择性不佳。本文通过在PBAQA结构的基础上引入另一个硼酸基团构建了一种双硼酸识别位点的荧光探针,并进行了初步的活性测试,考察了两识别位点的空间距离,不同取代基电子效应以及位置对荧光探针活性的影响。先导化合物PBAQA的合成方法未见文献公开报道,一般通过购买并进行衍生化。但由于价格昂贵,不利于系统的衍生化。我们利用课题组前期工作中的硼酸化合物合成方法,在此基础上,对合成工艺进行优化。即以对溴苯乙酮、靛红为起始原料,通过Pfitzinger Reaction、羧基酯化、钯催化、水解等步骤合成PBAQA,考察了溶剂选择、反应温度和反应时间对PBAQA收率的影响。然后通过酰胺缩合的方法将另一硼酸位点通过Linker结合到PBAQA母核上,设计了一条简单高效的双硼酸位点荧光探针的合成路线。首先利用二碳酸二叔丁酯单保护二胺化合物,然后与使用SOCl2活化的羧基酰胺缩合,通过盐酸法脱去BOC基团,形成盐酸盐沉淀析出,纯度可达98%。第二步酰胺缩合反应使用碳二亚胺类缩合剂法,将羧基苯硼酸化合物溶于DMF中,利用DCC和HOBT活化羧基,然后与上一步的PBAQA衍生物酰胺缩合。相比酰氯法,该步避免了SOCl2的使用,减少刺鼻气体和废液的产生,绿色环保而且操作简便高效。采用控制变量的方法研究了溶剂选择、反应温度等对二硼酸目标化合物收率的影响。本文合成了新化合物39个,其中二硼酸目标化合物35个、中间体4个。39个新化合物经TLC硼酸显色、荧光实验验证并通过核磁共振氢谱、核磁共振碳谱或高分辨质谱的确证。活性测试结果表明,化合物12h与盐酸多巴胺的结合常数是邻苯二酚和左旋多巴结合常数的两倍。化合物11g与盐酸多巴胺的结合常数相比PBAQA升高约6倍;化合物11g与邻苯二酚的结合常数相比PBAQA升高约4倍;化合物11g与左旋多巴的结合常数相比PBAQA升高约5倍,说明化合物11g的空间结构和氯原子电子效应有利于提高对儿茶酚胺类物质的亲和力。同时初步测试了目标化合物与部分糖类物质的荧光光谱活性。初步筛选出具有一定选择性的荧光探针。
[Abstract]:Catecholamines include norepinephrine, dopamine, epinephrine, isoproterenol, 5-hydroxytryptamine, hydroxytryptamine and its derivatives. Catecholamines regulate the basic physiological function of the human body, which is an important signal medium in the physiological process, and the corresponding content changes in the pathological process. It can be used in the field of medical research to assist in the diagnosis of endocrine-related diseases such as hyperthyroidism, hypertension, neuroblastoma and pheochromocytoma. Catechol substances are widely present in in-vivo tissues and organs, in particular catecholamines (such as dopamine, levodopa, epinephrine, etc.), which are associated with central nervous system diseases and in-vivo cardiovascular diseases. Therefore, it is of great significance and practical value to develop a method for the detection of catecholamines with high selectivity and sensitivity. the fluorescence probe technology has the characteristics of high sensitivity, simple and convenient operation, high speed, low detection limit and high selectivity, and has wide application in the fields of biochemical sensing, optical new material research and biological molecule detection and identification. In the aqueous solution, the phenylboronic acid and its derivatives can be combined with the reversible combination of the dihydroxy compound to form a five-or six-membered cyclic ester, based on the affinity of the boric acid, The analysis and detection of bacteria and cells and the like. 2-(4-dihydroxyboron) phenyl-1-4-coumaric acid (PBAQA) is a new kind of water-soluble fluorescent probe. The dendritic structure in the structure is connected with a boric acid group through a benzene ring, and free radicals exist in the structure, and other functional groups or structures can be introduced as a modification site. The boracic acid in the PBAQA structure can be combined with the catechol structure in the catecholamine structure through a covalent bond, and the fluorescence quenching can be realized by the PET mechanism, so that the selective identification of the catecholamines structure can be realized. however, that PBAQA compound is not selective for catecholamine such as dopamine and levodopa. In this paper, a double-boronic acid recognition site-based fluorescent probe was constructed by introducing another boronic acid group on the basis of the PBAQA structure, and a preliminary active test was carried out. The spatial distance of the two recognition sites, the electron effect of different substituents and the effect of the position on the activity of the fluorescent probe were investigated. The synthetic method of the lead compound PBAQA is not reported in the literature, and is generally obtained by purchasing and derivatization. However, because of the high price, it is not conducive to the derivatization of the system. We make use of the synthesis of boric acid compounds in the preliminary work of the research group, and on this basis, the synthesis process is optimized. The effects of solvent selection, reaction temperature and reaction time on the yield of PBAQA were investigated. and then the other boric acid site is bound to the PBAQA mother nucleus by means of a method for condensing the amine, and a simple and high-efficiency synthetic route of the double-boric acid site fluorescent probe is designed. First, the didi-tert-butyl dicarbonate is used to protect the diamine compound, and then the diamine compound activated with the SOCl2 is condensed, the BOC group is removed by the hydrochloric acid method, the precipitate of the hydrochloride is formed, and the purity can be up to 98 percent. In the second step, the melamine condensation reaction is carried out by using a carbodiimide type condensing agent method, and the phenyl-based phenylboronic acid compound is dissolved in DMF and activated by DCC and HOBT, and then is condensed with the last step of the PBAQA derivative. Compared with the chlorine method, the step avoids the use of the SOCl2, reduces the generation of the pungent and nasal gas and the waste liquid, is green and environment-friendly, and is simple and efficient to operate. The effect of solvent selection and reaction temperature on the yield of diboronic acid target compound was studied by using control variable method. In this paper, 39 new compounds were synthesized, including 35 target compounds and 4 intermediates. 39 new compounds were developed by TLC-boric acid, and the fluorescence experiments were verified and confirmed by nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum or high-resolution mass spectrometry. The activity test results show that the binding constant of the compound 12h and the dopamine in the hydrochloric acid is twice the binding constant of the catechol and the levodopa. The binding constant of compound 11g to dopamine in hydrochloric acid is about 6 times higher than that of PBAQA; the binding constant of compound 11g to catechol is about 4 times higher than that of PBAQA; the binding constant of compound 11g to levodopa is about 5 times higher than that of PBAQA, The spatial structure of the compound 11g and the electron effect of the chlorine atom can be used to improve the affinity of the catecholamines. The fluorescence spectrum of the target compound and some of the saccharide was also tested. a fluorescence probe with a certain selectivity was initially screened.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446;O657.3
[Abstract]:Catecholamines include norepinephrine, dopamine, epinephrine, isoproterenol, 5-hydroxytryptamine, hydroxytryptamine and its derivatives. Catecholamines regulate the basic physiological function of the human body, which is an important signal medium in the physiological process, and the corresponding content changes in the pathological process. It can be used in the field of medical research to assist in the diagnosis of endocrine-related diseases such as hyperthyroidism, hypertension, neuroblastoma and pheochromocytoma. Catechol substances are widely present in in-vivo tissues and organs, in particular catecholamines (such as dopamine, levodopa, epinephrine, etc.), which are associated with central nervous system diseases and in-vivo cardiovascular diseases. Therefore, it is of great significance and practical value to develop a method for the detection of catecholamines with high selectivity and sensitivity. the fluorescence probe technology has the characteristics of high sensitivity, simple and convenient operation, high speed, low detection limit and high selectivity, and has wide application in the fields of biochemical sensing, optical new material research and biological molecule detection and identification. In the aqueous solution, the phenylboronic acid and its derivatives can be combined with the reversible combination of the dihydroxy compound to form a five-or six-membered cyclic ester, based on the affinity of the boric acid, The analysis and detection of bacteria and cells and the like. 2-(4-dihydroxyboron) phenyl-1-4-coumaric acid (PBAQA) is a new kind of water-soluble fluorescent probe. The dendritic structure in the structure is connected with a boric acid group through a benzene ring, and free radicals exist in the structure, and other functional groups or structures can be introduced as a modification site. The boracic acid in the PBAQA structure can be combined with the catechol structure in the catecholamine structure through a covalent bond, and the fluorescence quenching can be realized by the PET mechanism, so that the selective identification of the catecholamines structure can be realized. however, that PBAQA compound is not selective for catecholamine such as dopamine and levodopa. In this paper, a double-boronic acid recognition site-based fluorescent probe was constructed by introducing another boronic acid group on the basis of the PBAQA structure, and a preliminary active test was carried out. The spatial distance of the two recognition sites, the electron effect of different substituents and the effect of the position on the activity of the fluorescent probe were investigated. The synthetic method of the lead compound PBAQA is not reported in the literature, and is generally obtained by purchasing and derivatization. However, because of the high price, it is not conducive to the derivatization of the system. We make use of the synthesis of boric acid compounds in the preliminary work of the research group, and on this basis, the synthesis process is optimized. The effects of solvent selection, reaction temperature and reaction time on the yield of PBAQA were investigated. and then the other boric acid site is bound to the PBAQA mother nucleus by means of a method for condensing the amine, and a simple and high-efficiency synthetic route of the double-boric acid site fluorescent probe is designed. First, the didi-tert-butyl dicarbonate is used to protect the diamine compound, and then the diamine compound activated with the SOCl2 is condensed, the BOC group is removed by the hydrochloric acid method, the precipitate of the hydrochloride is formed, and the purity can be up to 98 percent. In the second step, the melamine condensation reaction is carried out by using a carbodiimide type condensing agent method, and the phenyl-based phenylboronic acid compound is dissolved in DMF and activated by DCC and HOBT, and then is condensed with the last step of the PBAQA derivative. Compared with the chlorine method, the step avoids the use of the SOCl2, reduces the generation of the pungent and nasal gas and the waste liquid, is green and environment-friendly, and is simple and efficient to operate. The effect of solvent selection and reaction temperature on the yield of diboronic acid target compound was studied by using control variable method. In this paper, 39 new compounds were synthesized, including 35 target compounds and 4 intermediates. 39 new compounds were developed by TLC-boric acid, and the fluorescence experiments were verified and confirmed by nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum or high-resolution mass spectrometry. The activity test results show that the binding constant of the compound 12h and the dopamine in the hydrochloric acid is twice the binding constant of the catechol and the levodopa. The binding constant of compound 11g to dopamine in hydrochloric acid is about 6 times higher than that of PBAQA; the binding constant of compound 11g to catechol is about 4 times higher than that of PBAQA; the binding constant of compound 11g to levodopa is about 5 times higher than that of PBAQA, The spatial structure of the compound 11g and the electron effect of the chlorine atom can be used to improve the affinity of the catecholamines. The fluorescence spectrum of the target compound and some of the saccharide was also tested. a fluorescence probe with a certain selectivity was initially screened.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446;O657.3
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