選擇性識別兒茶酚胺的硼酸類熒光探針的合成及初步活性研究
發(fā)布時間：2018-12-07 09:04
【摘要】：兒茶酚胺類物質(zhì)主要包括去甲腎上腺素、多巴胺、腎上腺素、異丙腎上腺素、5-羥色胺、丁羥色胺及其衍生物。兒茶酚胺類物質(zhì)調(diào)節(jié)人體基本的生理功能,是生理過程中重要的信號介質(zhì),在病理過程中也隨之發(fā)生相應(yīng)的含量變化。在醫(yī)學(xué)研究領(lǐng)域可用于輔助診斷甲亢、高血壓、神經(jīng)母細(xì)胞瘤和嗜鉻細(xì)胞瘤等內(nèi)分泌類相關(guān)疾病。兒茶酚物質(zhì)廣泛存在體內(nèi)組織和器官,特別是兒茶酚胺類物質(zhì)(如多巴胺,左旋多巴,腎上腺素等)與中樞神經(jīng)系統(tǒng)類疾病和體內(nèi)心血管類疾病有重多關(guān)聯(lián)。因此開發(fā)一種高選擇性和靈敏度的兒茶酚胺類物質(zhì)的檢測方法具有重要醫(yī)學(xué)研究意義和實用價值。熒光探針技術(shù)具有高靈敏度、操作簡便、快速、檢出限低和高選擇性等特點,在生物化學(xué)傳感、光學(xué)新材料研究以及生物分子檢測和識別等領(lǐng)域獲得了廣泛的應(yīng)用。在水溶液中,苯硼酸及其衍生物可與二羥基化合物可逆性的結(jié)合形成五元或六元環(huán)狀酯,基于硼酸的這種親和性,從而實現(xiàn)了對兒茶酚胺類、糖類、糖蛋白、細(xì)菌和細(xì)胞等的分析檢測。2-(4-二羥基硼烷)苯基喹啉-4-羧酸(PBAQA)是一種新型水溶性硼酸熒光探針。結(jié)構(gòu)中的喹啉結(jié)構(gòu)通過一個苯環(huán)與硼酸基團(tuán)相連,結(jié)構(gòu)中存在游離的羧基,可作為修飾位點引入其它官能團(tuán)或結(jié)構(gòu)。PBAQA結(jié)構(gòu)中硼酸可與兒茶酚胺結(jié)構(gòu)中的鄰苯二酚結(jié)構(gòu)通過共價鍵結(jié)合,通過PET機理實現(xiàn)熒光淬滅,從而實現(xiàn)對兒茶酚胺類結(jié)構(gòu)的選擇性識別。但PBAQA化合物對多巴胺和左旋多巴等結(jié)構(gòu)類似的兒茶酚胺類物質(zhì)選擇性不佳。本文通過在PBAQA結(jié)構(gòu)的基礎(chǔ)上引入另一個硼酸基團(tuán)構(gòu)建了一種雙硼酸識別位點的熒光探針,并進(jìn)行了初步的活性測試,考察了兩識別位點的空間距離,不同取代基電子效應(yīng)以及位置對熒光探針活性的影響。先導(dǎo)化合物PBAQA的合成方法未見文獻(xiàn)公開報道,一般通過購買并進(jìn)行衍生化。但由于價格昂貴,不利于系統(tǒng)的衍生化。我們利用課題組前期工作中的硼酸化合物合成方法,在此基礎(chǔ)上,對合成工藝進(jìn)行優(yōu)化。即以對溴苯乙酮、靛紅為起始原料,通過Pfitzinger Reaction、羧基酯化、鈀催化、水解等步驟合成PBAQA,考察了溶劑選擇、反應(yīng)溫度和反應(yīng)時間對PBAQA收率的影響。然后通過酰胺縮合的方法將另一硼酸位點通過Linker結(jié)合到PBAQA母核上,設(shè)計了一條簡單高效的雙硼酸位點熒光探針的合成路線。首先利用二碳酸二叔丁酯單保護(hù)二胺化合物,然后與使用SOCl2活化的羧基酰胺縮合,通過鹽酸法脫去BOC基團(tuán),形成鹽酸鹽沉淀析出,純度可達(dá)98%。第二步酰胺縮合反應(yīng)使用碳二亞胺類縮合劑法,將羧基苯硼酸化合物溶于DMF中,利用DCC和HOBT活化羧基,然后與上一步的PBAQA衍生物酰胺縮合。相比酰氯法,該步避免了SOCl2的使用,減少刺鼻氣體和廢液的產(chǎn)生,綠色環(huán)保而且操作簡便高效。采用控制變量的方法研究了溶劑選擇、反應(yīng)溫度等對二硼酸目標(biāo)化合物收率的影響。本文合成了新化合物39個,其中二硼酸目標(biāo)化合物35個、中間體4個。39個新化合物經(jīng)TLC硼酸顯色、熒光實驗驗證并通過核磁共振氫譜、核磁共振碳譜或高分辨質(zhì)譜的確證�；钚詼y試結(jié)果表明,化合物12h與鹽酸多巴胺的結(jié)合常數(shù)是鄰苯二酚和左旋多巴結(jié)合常數(shù)的兩倍�；衔�11g與鹽酸多巴胺的結(jié)合常數(shù)相比PBAQA升高約6倍;化合物11g與鄰苯二酚的結(jié)合常數(shù)相比PBAQA升高約4倍;化合物11g與左旋多巴的結(jié)合常數(shù)相比PBAQA升高約5倍,說明化合物11g的空間結(jié)構(gòu)和氯原子電子效應(yīng)有利于提高對兒茶酚胺類物質(zhì)的親和力。同時初步測試了目標(biāo)化合物與部分糖類物質(zhì)的熒光光譜活性。初步篩選出具有一定選擇性的熒光探針。
[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.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446;O657.3
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