基于二硫键的荧光探针的设计合成及应用

发布时间：2018-06-05 07:28

本文选题：二硫键 + 前药　；参考：《山东师范大学》2016年硕士论文

【摘要】：二硫键作为我们所熟知的官能团,存在于众多的化学和生物试剂中,在化学和生物学中发挥着重要作用。比如,在氧化型谷胱甘肽(GSSG)和一些蛋白(如血小板反应蛋白TSP-1、蛋白质二硫键异构酶PDI),甚至一些天然的药物(如mitomycin disulfides、leinamycin)中均含有二硫键。生物体中的二硫键一般由两个半胱氨酸残基共价交联而成,氧化型谷胱甘肽(GSSG)与还原型谷胱甘肽(GSH)维持着细胞内的氧化还原平衡,而蛋白中的二硫键能够维持蛋白质构象的的稳定、保证相应蛋白的生物活性。二硫键在硫醇存在下可生物降解,且对不同浓度的GSH刺激有较强的响应性。在人体中,细胞内含有大量游离的硫醇。谷胱甘肽(GSH)作为生物体中含量最多的非蛋白巯基源,细胞内浓度可达到毫摩尔级别(1-10 mM),但在一些细胞外的液体(如血浆以及其他体液)中为微摩尔级别(20 40μM)。更有趣的是,癌细胞中GSH的浓度远远高于与之对应的正常细胞,这一特点在抗癌药物输送系统的发展中发挥重要作用。本论文中构建了一种新型的药物输送系统,通过荧光共聚焦成像对药物的释放进行实时示踪。当今社会的不断进步伴随着环境的污染,导致癌症的发病率越来越高,亚硒酸钠(Na2SeO3)能够有效杀死肿瘤细胞且对正常细胞损害较小,但其抗癌机制却还不清楚。有文献报道,Na2SeO3诱导肿瘤细胞凋亡过程中会代谢产生H2Se,为了观察Na2SeO3代谢过程中H2Se的累积,我们利用含二硫键的六元环做为官能团,设计了一种检测硒化氢的荧光探针,这将为进一步研究硒酸钠抗癌机制提供必要的前提。总之,本论文主要开展了以下两方面的工作:(一)我们采用较新颖的方法合成了近红外的阳离子型氨基部花菁,并与临床化疗药物喜树碱经二硫化物相连,组成了一种新型的线粒体定位的近红外前药。模拟生理条件下,该前药能特异性地被硫醇打断,在GSH 0 40 eq.范围内呈良好的线性(线性方程F=5021.5+516.39[GSH])。由于癌细胞中GSH的浓度远远高于与之对应的正常细胞,前药被癌细胞中谷胱甘肽裂解并重排,释放出活性抗癌药物喜树碱(CPT)的同时在702nm处发出强荧光,实现了药物释放剂量的实时示踪。由于其激发发射均在近红外区,对细胞以及活体伤害较小且生物背景干扰较少。(二)在该部分,我们用较易合成的氨基半花菁为荧光母体,以含二硫键的六元环(氧化型DTT)为识别基团,合成了一种新型的特异性检测硒化氢的荧光探针。在模拟生理条件下,该探针对H2Se具有较高的选择性,且在H2Se浓度0-100μM范围内呈良好的线性关系(线性方程F=79.3733+3.4596[H2Se])。在常氧(20%pO2)和乏氧(1%pO2)情况下,该探针被成功用于亚硒酸钠诱导肝癌细胞凋亡过程中H2Se的成像分析,为进一步探究Na2SeO3诱导肿瘤细胞凋亡机理提供了必要的前提。
[Abstract]:As a well known functional group, disulfide bonds exist in many chemical and biological reagents and play an important role in chemistry and biology. For example, disulfide bonds are found in oxidized glutathione (GSSG) and some proteins (such as platelet reactive protein TSP-1, protein disulfide isomerase PDIX, and even some natural drugs, such as mitomycin disulfideslein amycin). Disulfide bonds in organisms are generally covalently crosslinked by two cysteine residues. Oxidized glutathione (GSSGG) and reduced glutathione (GSH) maintain redox equilibrium in cells. The disulfide bond in the protein can maintain the conformation stability of the protein and guarantee the biological activity of the corresponding protein. The disulfide bond can be biodegradable in the presence of mercaptan and has a strong response to different concentrations of GSH stimulation. In the human body, the cells contain a large amount of free mercaptan. Glutathione (GSH) is the most abundant nonprotein sulfhydryl source in organisms. The intracellular concentration of GSH can reach the millimole level of 1-10 mM-1, but in some extracellular liquids (such as plasma and other body fluids), it is a micro-molar level of 20 ~ 40 渭 M-1. More interestingly, the concentration of GSH in cancer cells is much higher than that in normal cells, a feature that plays an important role in the development of anticancer drug delivery systems. In this paper, a novel drug delivery system was constructed to trace drug release in real time by fluorescence confocal imaging. With the environment pollution, the incidence of cancer is higher and higher. Sodium selenite Na _ 2SeO _ 3) can kill tumor cells effectively and damage to normal cells less, but its anti-cancer mechanism is not clear. In order to observe the accumulation of H2Se in the process of Na2SeO3 metabolism, we designed a fluorescence probe for the detection of hydrogen selenide by using a six-member ring containing disulfide bonds as a functional group in the process of apoptosis of tumor cells induced by Na _ 2SeO _ 3.In order to observe the accumulation of H2Se in the process of Na2SeO3 metabolism, we designed a fluorescence probe for the detection of hydrogen selenide. This will provide a necessary prerequisite for further study of sodium selenate anti-cancer mechanism. In conclusion, in this paper, we have mainly carried out the following two aspects of work: (1) We have synthesized the cationic anthocyanine by a novel method, and connected with the clinical chemotherapeutic drug camptothecin through disulfide. A new Mitochondrial localization near infrared prodrug was formed. Under simulated physiological conditions, the prodrug was specifically interrupted by mercaptan at GSH 0 ~ 40 eq. The linear equation FV 5021.5 516.39 [GSH] is linear in the range. Because the concentration of GSH in cancer cells is much higher than that in the corresponding normal cells, prodrug is cleavage and redischarged by glutathione in cancer cells, releasing active anticancer drug camptothecin (CPT) and emitting strong fluorescence at 702nm. Real-time tracer of drug release dose was realized. Because its excitation emission is in the near infrared region, it has less damage to the cells and living body and less interference with the biological background. (2) in this part, we have synthesized a novel fluorescent probe for the detection of hydrogen selenide by using an easily synthesized amino-hemicyanine as the fluorescence matrix and a six-member ring (oxidized DTT) containing disulfide bond as the recognition group. Under simulated physiological conditions, the probe was highly selective to H2Se and showed a good linear relationship (linear equation F 79.3733 3.4596 [H2Se]) in the range of 0-100 渭 M of H2Se concentration. Under the condition of normoxic and hypoxic oxygen, the probe was successfully used in the imaging analysis of H2Se in the process of sodium selenite induced apoptosis of hepatoma cells, which provided a necessary prerequisite for further exploring the mechanism of apoptosis induced by Na2SeO3.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3

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