一种同时检测GSH和Cys的小分子荧光探针的设计、合成及其性质研究

发布时间：2018-06-16 04:25

本文选题：荧光探针 + 半菁染料　；参考：《延边大学》2017年硕士论文

【摘要】：生物硫醇在人体的生理进程中,扮演者重要的角色。一旦其浓度出现异常,就会给人们带来一些疾病。所以定性、定量的去检测生物硫醇(包括:Cys、Hcy、GSH),是非常迫切需要解决的问题。而在众多的检测方法中,由于荧光探针法操作简单、高选择性、高灵敏度、实时监测、不容易损害细胞等优点,在检测生物硫醇方面备受研究者青睐。此外,我们通过对前人工作的总结研究发现,之前所报道的关于检测生物硫醇的文章,大多数都是单一的检测一种生物硫醇。因此发展一种小分子荧光探针,实现从不同的发射波长来检测两种或三种生物硫醇是具有很高价值和挑战性的。考虑以上问题,我们设计并合成了一种新型的荧光探针1,并对其结构进行了相关结构表征(核磁、碳谱、以及质谱),最终确定化合物结构。在探针1的结构设计上,我们以含有香豆素-吲哚结构单元的半菁作为荧光发光基团,以3,4-二甲氧基-苯硫酚作为一种通过光诱导电子转移(PET)确保一个低背景荧光的荧光猝灭剂和离去基团。探针1可以同时在不同发射波长下来选择性的检测GSH和Cys,并且可以将其与其它氨基酸和必须阳离子区分开来。在长波长(651 nm)、纯水体系下,探针1与GSH反应,只发生一步芳香取代反应,生成硫代香豆素半菁染料(1-GSH),表现为荧光增强。探针1与GSH反应非常快速,荧光响应时间为10s。探针1对GSH浓度的变化而导致的荧光强度的改变极为灵敏,最低检测极限浓度达到了 8.8 nM。探针1与Cys反应,第一步发生芳香取代反应,生成巯基香豆素半菁染料(2a),随后发生分子内重排反应,生成氨基香豆素半菁染料(3a),之后又发生分子内的环化反应,生成化合物4a,从而使其在457 nm处荧光明显增强。相较于Cys,探针1与Hcy反应最终发生环化反应生成八元环,这在动力学上是不利的,以及吲哚盐氮连接的两个甲基的空间位阻作用。最终导致探针1与Hcy反应所生成的大部分化合物停留在分子内重排反应后的产物3b。所以探针1可以用来选择性检测Cys区别于Hcy/GSH以及其他分析物。尤其是探针1对Cys的竞争实验,充分体现了探针1对于Cys的检测具有很强的抗干扰能力。最后,进行了探针1对于识别细胞中生物硫醇的细胞成像实验。表明了探针1可以在不同的有机溶剂浓度和发射通道下分别对活体细胞中的GSH和Cys选择性识别,且具有一定的潜在生物应用价值。
[Abstract]:Biological mercaptan plays an important role in the physiological process of human body. Once its concentration is abnormal, it will bring some diseases to people. So qualitative and quantitative detection of biological mercaptan (including: cystol GSH) is a very urgent problem to be solved. Among the many detection methods, the fluorescence probe method has many advantages, such as simple operation, high selectivity, high sensitivity, real-time monitoring and not easy to damage the cells, so it is very popular in the detection of biological mercaptan. In addition, we have found that most of the previous papers on the detection of biological mercaptan are single detection of a biological mercaptan. Therefore, the development of a small molecular fluorescence probe to detect two or three biological mercaptans from different emission wavelengths is of great value and challenge. Considering the above problems we have designed and synthesized a novel fluorescent probe 1 and characterized its structure (NMR carbon spectrum and mass spectrometry) to determine the structure of the compound. In the structural design of probe 1, we use the semicyanine containing coumarin-indole structure unit as the fluorescence luminescent group. A fluorescence quenching agent and a departure group with low background fluorescence were obtained by using 3O4-dimethoxy-phenylthiophenol as a fluorescence quenching agent via photoinduced electron transfer (PET). Probe 1 can selectively detect GSH and Cys1 at different emission wavelengths and distinguish them from other amino acids and essential cations. In the system of long wavelength of 651 nm ~ (-1) and pure water, only one step of aromatic substitution reaction occurred in the reaction of probe 1 with GSH, resulting in the formation of thiocoumarin hemicyanine dye (1-GSH), which showed enhanced fluorescence. The fluorescence response time of probe 1 was 10 s. Probe 1 is very sensitive to the change of fluorescence intensity caused by the change of GSH concentration, and the lowest detection limit concentration is 8.8 nm. When probe 1 reacted with Cys, aromatic substitution reaction occurred in the first step to produce thiogoumarin hemicyanine dye 2 a, followed by intramolecular rearrangement to form amino coumarin hemicyanine dye 3 a, followed by intramolecular cyclization. A compound of 4a was formed, which enhanced the fluorescence at 457 nm. Compared with Cys1, the cyclization reaction between probe 1 and Hcy resulted in the formation of octa-ring, which was not beneficial to the kinetics and the steric resistance of two methyl groups connected by indole nitrogen. In the end, most of the compounds produced by the reaction of probe 1 with Hcy stay in the product of 3b. after the intramolecular rearrangement reaction. So probe 1 can be used to selectively detect Cys as distinct from Hcy / GSH and other analytes. Especially, the competitive experiment of probe 1 on Cys fully shows that probe 1 has strong anti-interference ability for Cys detection. Finally, a cell imaging experiment was carried out to identify biological mercaptan in cells with probe 1. The results showed that probe 1 could selectively recognize GSH and Cys in living cells under different organic solvent concentrations and emission channels, and had potential biological application value.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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