本文選題：熒光探針 + 半菁染料��； 參考：《延邊大學(xué)》2017年碩士論文

【摘要】：生物硫醇在人體的生理進(jìn)程中,扮演者重要的角色。一旦其濃度出現(xiàn)異常,就會(huì)給人們帶來(lái)一些疾病。所以定性、定量的去檢測(cè)生物硫醇(包括:Cys、Hcy、GSH),是非常迫切需要解決的問(wèn)題。而在眾多的檢測(cè)方法中,由于熒光探針?lè)ú僮骱?jiǎn)單、高選擇性、高靈敏度、實(shí)時(shí)監(jiān)測(cè)、不容易損害細(xì)胞等優(yōu)點(diǎn),在檢測(cè)生物硫醇方面?zhèn)涫苎芯空咔嗖A。此外,我們通過(guò)對(duì)前人工作的總結(jié)研究發(fā)現(xiàn),之前所報(bào)道的關(guān)于檢測(cè)生物硫醇的文章,大多數(shù)都是單一的檢測(cè)一種生物硫醇。因此發(fā)展一種小分子熒光探針,實(shí)現(xiàn)從不同的發(fā)射波長(zhǎng)來(lái)檢測(cè)兩種或三種生物硫醇是具有很高價(jià)值和挑戰(zhàn)性的�？紤]以上問(wèn)題,我們?cè)O(shè)計(jì)并合成了一種新型的熒光探針1,并對(duì)其結(jié)構(gòu)進(jìn)行了相關(guān)結(jié)構(gòu)表征(核磁、碳譜、以及質(zhì)譜),最終確定化合物結(jié)構(gòu)。在探針1的結(jié)構(gòu)設(shè)計(jì)上,我們以含有香豆素-吲哚結(jié)構(gòu)單元的半菁作為熒光發(fā)光基團(tuán),以3,4-二甲氧基-苯硫酚作為一種通過(guò)光誘導(dǎo)電子轉(zhuǎn)移(PET)確保一個(gè)低背景熒光的熒光猝滅劑和離去基團(tuán)。探針1可以同時(shí)在不同發(fā)射波長(zhǎng)下來(lái)選擇性的檢測(cè)GSH和Cys,并且可以將其與其它氨基酸和必須陽(yáng)離子區(qū)分開(kāi)來(lái)。在長(zhǎng)波長(zhǎng)(651 nm)、純水體系下,探針1與GSH反應(yīng),只發(fā)生一步芳香取代反應(yīng),生成硫代香豆素半菁染料(1-GSH),表現(xiàn)為熒光增強(qiáng)。探針1與GSH反應(yīng)非�？焖�,熒光響應(yīng)時(shí)間為10s。探針1對(duì)GSH濃度的變化而導(dǎo)致的熒光強(qiáng)度的改變極為靈敏,最低檢測(cè)極限濃度達(dá)到了 8.8 nM。探針1與Cys反應(yīng),第一步發(fā)生芳香取代反應(yīng),生成巰基香豆素半菁染料(2a),隨后發(fā)生分子內(nèi)重排反應(yīng),生成氨基香豆素半菁染料(3a),之后又發(fā)生分子內(nèi)的環(huán)化反應(yīng),生成化合物4a,從而使其在457 nm處熒光明顯增強(qiáng)。相較于Cys,探針1與Hcy反應(yīng)最終發(fā)生環(huán)化反應(yīng)生成八元環(huán),這在動(dòng)力學(xué)上是不利的,以及吲哚鹽氮連接的兩個(gè)甲基的空間位阻作用。最終導(dǎo)致探針1與Hcy反應(yīng)所生成的大部分化合物停留在分子內(nèi)重排反應(yīng)后的產(chǎn)物3b。所以探針1可以用來(lái)選擇性檢測(cè)Cys區(qū)別于Hcy/GSH以及其他分析物。尤其是探針1對(duì)Cys的競(jìng)爭(zhēng)實(shí)驗(yàn),充分體現(xiàn)了探針1對(duì)于Cys的檢測(cè)具有很強(qiáng)的抗干擾能力。最后,進(jìn)行了探針1對(duì)于識(shí)別細(xì)胞中生物硫醇的細(xì)胞成像實(shí)驗(yàn)。表明了探針1可以在不同的有機(jī)溶劑濃度和發(fā)射通道下分別對(duì)活體細(xì)胞中的GSH和Cys選擇性識(shí)別,且具有一定的潛在生物應(yīng)用價(jià)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.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O657.3

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本文編號(hào)：2025300