选择性检测细胞内还原性物质荧光探针的合成及其生物应用

发布时间：2018-03-09 17:19

本文选题：荧光探针　切入点：吖啶　出处：《山东师范大学》2010年硕士论文　论文类型：学位论文

【摘要】： 硫醇是生物体中许多蛋白质和小分子的重要组成部分,在细胞的抗氧化系统中具有重要的作用。细胞内硫醇的水平与白细胞缺失、牛皮癣、肝损伤、癌症以及艾滋病等许多疾病有着直接的联系。对细胞内硫醇类物质的检测有助于阐明还原体系在整个细胞氧化还原网络的重要调控作用。近年来,检测细胞内硫醇类物质的研究越来越多,其中荧光法简单易行,便于操作,具有高灵敏度、高选择性和广泛可用的荧光染料等特点。更加吸引人的是探针分子可以在不破坏生物活性的情况下进入细胞,与活细胞内的硫醇结合生成强荧光物质,并借助于激光共聚焦成像技术,实现细胞内硫醇的“可视化”,从而对它们在生命体内进行“实时在线”的检测。目前,用于检测生物体内硫醇的荧光探针发展迅速,然而能够用于生理条件下实时快速检测硫醇的荧光探针少有报道,因此我们的主要目的在于设计高选择性、高灵敏度的,能够快速检测细胞内硫醇的荧光探针。本论文基于荧光探针与硫醇类物质特异性作用后荧光光谱性质的改变从而实现对其的检测。本文主要开展了以下两方面的研究工作: (一)基于马来酰亚胺基团对巯基的特异性响应,以吖啶衍生物为母体设计合成了一种新的含有两个马来酰亚胺基团、可以快速检测非蛋白硫醇的荧光探针。探针本身荧光较弱,与非蛋白硫醇以1:2的比例反应后荧光增强明显,激发和发射波长分别位于380 nm和440 nm。由于探针分子中两个马来酰亚胺基团是邻位取代的,空间距离很近,当探针与蛋白硫醇以1:1的比例反应之后,受体积较大的蛋白硫醇影响,未反应的马来酰亚胺基团空间位阻变大,很难继续与其他蛋白硫醇反应,从而继续猝灭母体荧光团的荧光,导致探针荧光无法恢复,因此探针对蛋白硫醇没有响应;非蛋白硫醇由于体积较小,可以与探针分子以2:1的比例反应而使探针荧光明显增强。在室温、生理pH值条件下探针能够快速检测非蛋白硫醇,1.5μM探针与1.0μM谷胱甘肽在小于1 min内即反应结束,蛋白硫醇、生物体内的活性氧、金属离子及其他抗氧化剂均对测定没有干扰。在1.0×10~(-8)-1.5×10~(-6)M范围内谷胱甘肽的浓度与荧光强度呈良好的线性关系,方法的检测限为7.7 nM。为了比较两个马来酰亚胺基团之间的空间距离对反应速率的影响,本文同时合成了间二马来酰亚胺取代的化合物作为对比,结果发现两个巯基响应基团处于间位时与非蛋白硫醇反应速率比邻位时速率慢很多,推测是由于邻位取代时两个取代基团空间距离太近而存在空间位阻效应(空间位阻效应可以使反应速率加快),使得探针本身能量较高而不如间位取代的化合物稳定,从而更易与谷胱甘肽反应。实验过程中发现探针溶液在室温下放置三周后,溶液颜色由浅黄色变为黄绿色(探针在冰箱中放置数月稳定),而间位取代的化合物却没有任何变化,证明探针不如间位取代的化合物稳定。 (二)以罗丹明110为母体设计合成一种含有两个马来酰胺基团的化合物预期能够快速检测特异性小肽。
[Abstract]:Mercaptan is an important component of many proteins and small molecules in organisms, plays an important role in the antioxidant system in cells. Intracellular thiol levels and leukocyte deficiency, psoriasis, liver damage, cancer and AIDS many diseases is a direct link. The detection of intracellular thiols may help to elucidate restore the system restore an important role in the regulation network in the whole cell oxidation. In recent years, more and more research on detection of intracellular thiols, the fluorescence method is simple, easy to operate, with high sensitivity, high selectivity and wide availability of fluorescent dyes and other characteristics. More attractive is the probe molecules can enter the cell in the do not destroy the biological activity under the condition of thiols and in living cells are combined to generate a strong fluorescent material, and with the help of confocal laser imaging technology, intracellular thiols visibility And then they do "real-time online" detection in their lives.
At present, for the detection of biological thiols fluorescent probe developed rapidly, however, can be used under physiological conditions of fluorescent probe detection of thiols is rarely reported, so our main purpose is to design a high selectivity, high sensitivity, can rapid detection of intracellular thiol fluorescent probe. The fluorescence probe with thiols specificity after the change of fluorescence spectra to detect the based on. In this paper, the main work includes the following two aspects:
(a) maleimide group specificity of thiol responses based on a novel containing two maleimide groups were synthesized by acridine derivatives as matrix design, can rapid detection of non protein thiol fluorescent probe. The probe itself weak fluorescence, and non protein thiol to 1: ratio of 2 after the reaction of fluorescence enhanced obviously, excitation and emission wavelengths were 380 nm and 440 nm. for two maleimide groups in the ortho substituted probe molecule is, the space distance is very near, when the probe and protein thiol to the ratio of 1:1 reaction by larger protein thiols, unreacted maleimide groups steric change large, it is difficult to continue to react with other protein thiols, fluorescence quenching of fluorophores to continue maternal, leading to fluorescence cannot be recovered, so the probe did not respond to protein thiols; non protein thiol because of small volume, Can response to the ratio of 2:1 and the fluorescent probe molecules significantly enhanced. At room temperature, physiological pH conditions can quickly detect non protein thiol probe, 1.5 M and 1 M probe glutathione in less than 1 min in the end of the reaction, protein thiol, active oxygen in vivo, metal ions and other antioxidants no interference on the determination. In 1 * 10~ (-8) -1.5 * 10~ (-6) M in the range of glutathione concentration showed a good linear relationship with the fluorescence intensity, the detection limit was 7.7 nM. for comparison between the two maleimide group space distance effect on the reaction rate, at the same time, the synthesis of compounds the bismaleimide substituted for comparison, found that two sulfhydryl groups response reaction and non protein thiol in a rate much slower rate than ortho, presumably due to the two generation of the ortho substituted group The space is too close and steric effect (steric effect can accelerate the reaction rate), makes the probe itself with high energy and stable compounds substituted a better, thus more likely to react with glutathione. The experiment is allowed to stand for three weeks in the probe solution at room temperature, the solution color changed from pale yellow to yellow green (probe placed in the refrigerator for a few months, and stable) m-substituted compounds without any change, as a probe that stable compounds substituted.
(two) a compound containing two maleamide groups was designed and synthesized by Luo Danming 110 as the mother. It is expected to be able to quickly detect the specific peptide.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341;O657.3

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