基于半花菁与黄酮醇荧光团的比率荧光探针的设计、合成与应用

发布时间：2018-04-03 18:43

本文选题：比率荧光探针　切入点：FRET　出处：《山东大学》2017年博士论文

【摘要】：在生物体内,二氧化硫(SO_2)可产生自含硫氨基酸的分解、硫化氢(H_2S)的氧化等酶促过程。研究发现低浓度的SO_2在血压调节、炎症反应等生理与病理过程中发挥重要的作用。与一氧化氮(NO)分子类似,SO_2也被认为是一种生物气体信号分子。由于SO_2易溶于水形成亚硫酸盐与亚硫酸氢盐,因此其生理作用是通过这类SO_2衍生物实现的。另一方面,SO_2衍生物广泛应用于食品抗氧化剂、抗菌剂,例如SO_2作为葡萄酒的抗菌剂使用等。但是由于现有检测方法的限制,SO_2及其衍生物具体的生理作用及转化过程尚不十分明确。因此,亟待开发高灵敏的、高分辨率的、实时原位的检测方法。荧光分析法具有良好的生物兼容性,在生物及医学领域得到了广泛关注。有机小分子荧光探针具有开发周期短,光稳定性好,细胞毒性低、操作简单及信号直观等优点,近年来得以飞速发展,包括许多基于有机荧光小分子的SO_2衍生物探针也有报道。目前报道的SO_2衍生物荧光探针多是基于单波长荧光强度的变化(增强或减弱)实现检测的。单一信号容易受到测试体系、探针浓度、仪器参数等外界因素的干扰而影响分析结果的准确度。比率荧光探针具有两个荧光发射峰,在SO_2衍生物的作用下,至少一个荧光峰的强度发生变化。通过探针分子内部的相互参照,能有效地消除来自外界因素的干扰,从而提高分析结果的准确度。比率荧光探针一般含有一个荧光基团或者两个不同的荧光基团。单荧光基团的比率荧光探针的信号调控机理主要有分子内电荷转移(ICT)和激发态分子内质子转移(ESIPT)等,双荧光团的比率荧光探针信号的主要调控机理有荧光共振能量转移(FRET)和跨键能量转移(TBET)等。黄酮醇结构是ESIPT型荧光染料的典型代表。黄酮醇由于具有大的Stokes位移,好的生物膜透性和低的细胞毒性而受到广泛关注。基于黄酮醇及其衍生物的荧光探针大多是通过控制其ESIPT过程的"开-关"实现的。FRET过程的实现必须要有两个荧光基团的共同参与,其中一个作为能量供体,另一个作为能量受体。半花菁荧光团具有摩尔消光系数大、荧光量子产率高、可见光有效激发、长波光区域发射、结构容易修饰等特点,有作为FRET体系中能量受体使用的潜质。本论文中,我们致力于研究比率荧光探针的设计、合成与应用。以FRET和ESIPT为主要比率信号变化机理,以半花菁和黄酮醇结构为主要的荧光报告基团,设计合成了四个SO_2衍生物探针以及一个H_2S探针,并研究了它们的荧光性质和在生物检测方面的应用。第一章,简要介绍了荧光探针的基本概念,荧光信号的变化类型及其调控原理;总结了 SO_2衍生物荧光探针的最新进展;综述了基于半花菁和黄酮醇结构的荧光探针的最新进展。第二章,设计并合成了以半花菁为"能量-检测对象"双受体的FRET新模板并应用与SO_2衍生物的比率检测。该FRET体系具有高的能量传递效率与显著的比率特性。同时探针HCy-D可靶向细胞内线粒体,并通过对该细胞内生亚硫酸氢盐的检测,成功区分正常肝细胞(L-02)与癌变的肝细胞(HepG2)。第三章,合成了一例基于FRET新模板的比率荧光及比色探针HCy-NBD,以检测SO_2衍生物。该探针以半花菁基团为受体,以NBD基团为供体,可实现灵敏,快速的检测及裸眼识别。第四章,以FRET模板为基础,引入具有ESIPT过程的黄酮醇基团作为能量供体,从而将ESIPT过程与FRET过程联系起来。由于ESIPT效应,供体黄酮醇分子的Stokes位移高达185 nm;而由于ESIPT与FRET效应的共同作用,受体会产生很大的伪Stokes位移值,故基于该模板的探针在与检测对象作用前后都将体现大的(伪)Stokes位移。探针L-HF1选取对SO_2衍生物有高度反应专一性的半花菁作为能量受体,具有好的选择性与灵敏度。将黄酮醇结构中的羟基转化为酯基以抑制ESIPT过程,所得化合物L-HF2分子中没有检测到FRET过程,这进一步印证了 EISPT对FRET过程的重要辅助作用。同样,该探针可应用于细胞内亚硫酸氢盐的成像,体现了良好的比率特性与低的细胞毒性。第五章,依托ESIPT辅助的FRET模板,为有效减少供体发射峰与受体发射峰的重叠,增加两个荧光发射峰的分辨率,设计合成了探针L-HF3。由于受体荧光团TCF的长波或近红外发射特性,该探针具有更低的荧光检测背景与更小的光谱重叠程度,因此有更大程度的比率荧光变化及更可靠的检测结果。此外,探针L-HF3可有效地检测SO_2有机供体原位生成的SO_2及其衍生物。第六章,基于对ESIPT过程"开-关"状态的调控,以黄酮醇为荧光团,以二硫键与酯基为反应活性位点,设计合成了一例H_2S荧光探针HF-PBA。该探针本身由于ESIPT过程被阻断,只在400 nm波长处有微弱的荧光。H_2S进攻二硫键引发的二步连串反应将具有ESIPT效应的黄酮醇分子游离出来,从而产生酮式(强)与醇式(弱)两个结构的荧光发射峰。该探针具有高的反应活性、高的灵敏度与低的细胞毒性,可应用于细胞内H_2S的成像研究。本论文主要创新点:1.以半花菁、黄酮醇等结构为基础,以FRET、ESIPT与ICT等原理为信号调控方式,以化学反应为识别方式,设计合成了一系列在生理条件下检测SO_2衍生物比率荧光探针及一个H_2S比率荧光探针。并成功地将这些探针应用于细胞内成像研究。2.首次将半花菁荧光基团引入FRET体系中,半花菁作为供体能量及检测对象的受体,构建出新的FRET模板。该FRET模板是通过对受体结构内ICT过程的阻断来实现比率调控的,具有高的能量传递效率以及较大的Stokes位移。通过对细胞内源亚硫酸氢盐的检测,首次揭示了肝癌细胞与正常肝细胞的内源亚硫酸氢盐浓度具有较大差异,为在细胞水平上诊断肝癌提供了新的思路。3.首次将ESIPT过程与FRET过程联系起来,构建了 ESIPT辅助的FRET模板,具有很大的(伪)Stokes位移值与高的能量转移效率,并且丰富了 FRET型比率探针供体的结构多样性。
[Abstract]:In vivo, sulfur dioxide (SO_2) can be produced from decomposition of sulfur-containing amino acids, hydrogen sulfide (H_2S) oxidation enzymatic process. The study found that low concentration of SO_2 in blood pressure regulation, play an important role in inflammation and other physiological and pathological processes. And nitric oxide (NO) molecules are similar to SO_2, is also considered a biological gas signal molecule. Because the SO_2 soluble form of sulfite and bisulfite in water, so its physiological function is realized by this kind of SO_2 derivatives. On the other hand, SO_2 derivatives are widely used in food antioxidant, antibacterial agents, such as SO_2 Wine as antibacterial agent and so on. But because of the existing detection methods limit, physiological function and transformation process of SO_2 and its derivatives concrete is not very clear. Therefore, the development needs of high sensitivity, high resolution, real time detection method for in situ analysis has good fluorescent light. Good biological compatibility, has received wide attention in the field of biology and medicine. Organic small molecule fluorescent probe has a short development cycle, good light stability, low toxicity, simple operation and visual signal and other advantages, in recent years the rapid development of SO_2, including many organic small molecule fluorescent probe derivatives based on SO_2 have also been reported. The reported derivatives fluorescent probe is based on single wavelength fluorescence intensity change (increase or decrease) detection. A single signal is susceptible to test system, probe concentration, instrument parameters of external factors such as interference and affect the accuracy of the analysis results. The ratio of fluorescence probe with two fluorescence emission peaks, in SO_2 derivatives under the effect of changes in at least one fluorescence peak intensity. By cross referencing the probe molecule inside, can effectively eliminate the interference from external factors, so as to improve the analysis The accuracy ratio fluorescence probe contains a fluorophore or two different fluorophores. The signal regulation mechanism of ratiometric fluorescent probes of single fluorophores of intramolecular charge transfer (ICT) and excited state intramolecular proton transfer (ESIPT), the main control signal ratio fluorescent probe double fluorescence mechanism the group has the fluorescence resonance energy transfer (FRET) and cross bond energy transfer (TBET). Flaonols structure is a typical ESIPT type fluorescent dyes. Because of its large displacement Stokes flavonols, biofilm good permeability and low toxicity and attracted widespread attention. Based on the fluorescence probe of flavonols and its derivatives the most common is controlled by the ESIPT involved in the process of "on-off" implementation of the.FRET process must have two fluorescent groups, one as the energy donor and another half flowers as energy acceptor. Cyanine fluorophore with high extinction coefficient, high fluorescence quantum yield, visible light excitation and emission characteristics of long wavelength region, easy structure modification, as the use of energy in the system of FRET receptor potential. In this thesis, we are committed to the study of ratio fluorescent probe design, synthesis and application. The main mechanism of signal ratio changes in FRET and ESIPT, with hemicyanine and flavonols as the main structure of the fluorescent probe, four SO_2 derivatives and a H_2S probe was designed and synthesized, and studied their fluorescence properties and the application in biological detection. The first chapter briefly introduces the basic concept of the fluorescence probe changes the type and regulation principle of fluorescence signal; summarizes the latest progress of SO_2 derivatives as fluorescent probe; reviewed the latest progress of fluorescent probe hemicyanine and flavonols based on the structure. In the second chapter, design and synthesis The ratio of detection into a hemicyanine as "energy detection object" double receptor FRET new templates and application and SO_2 derivatives. The FRET system has the characteristics of high efficiency ratio and significant energy transfer. At the same time probe HCy-D targets mitochondria, and through the detection of the cell endogenous hydrogen sub salt sulfuric acid, distinguish between normal liver cells (L-02) and cancerous liver cells (HepG2). In the third chapter, the synthesis of a new template FRET cases ratiometric fluorescent and colorimetric probe based on HCy-NBD to detect SO_2 derivatives. The probe with hemicyanine groups as receptors, with NBD group as the donor, can be realized sensitive, rapid detection and identification of naked eye. In the fourth chapter, the FRET template based introduction of flavonol group has ESIPT as the energy donor, and thus linked to ESIPT process and FRET process. Due to the ESIPT effect, the displacement of Stokes donor flavonol molecules up to 185 Nm; and due to the combined effect of ESIPT and FRET effect, by the experience of pseudo Stokes displacement of large value, it is based on the template effect before and after the probe and detection of objects will be reflected in (pseudo) Stokes L-HF1 probe displacement. The selection of SO_2 derivatives with high selectivity of the reaction of hemicyanine as energy acceptor. Has good selectivity and sensitivity. The structure of the flavonols into hydroxyl ester to inhibit ESIPT process, not the compound L-HF2 molecule was detected in the FRET process, which further confirms the important role of EISPT assisted FRET process. Also, the imaging probe can be applied to the intracellular hydrogen sulfate, reflects the the ratio of good characteristics and low toxicity. In the fifth chapter, based on the ESIPT auxiliary FRET template, in order to effectively reduce the donor emission peaks overlap with the receptor of emission peaks, two increase in fluorescence emission peak resolution, design A probe of the L-HF3. due to the long wave acceptor fluorophore TCF or near infrared emission characteristics, spectral overlap and background fluorescence detection of the probe with smaller lower, so the ratio of fluorescence changes more and more reliable detection results. In addition, the probe L-HF3 can effectively detect SO_2 SO_2 in situ organic donor and its derivatives the sixth chapter, based on the regulation of ESIPT during the "on-off" state, with flavonols as fluorophore, with two disulfide bond and ester reactive sites, designed and synthesized a H_2S fluorescent probe HF-PBA. was blocked by the probe itself in the ESIPT process, the flavonol molecule two step consecutive reaction only at 400 nm wavelength with fluorescent.H_2S weak offensive of the two disulfide bonds with ESIPT effect free, resulting in the keto alcohol (strong) and type two (weak) structure of the emission peak. The probe with high response Activity, high sensitivity and low cytotoxicity, imaging studies can be applied to the H_2S cells. The main innovations of this dissertation: 1. hemicyanine, flavonol structure based on FRET, ESIPT and ICT principle of signal regulation, the chemical reaction for the identification, design and synthesis. In a series of physiological conditions to detect SO_2 derivative ratio fluorescence probe and a H_2S ratio of fluorescence probe. And successfully applied to these probes to study the intracellular.2. imaging for the first time hemicyanine fluorophore introduced in FRET system, hemicyanine as donor and energy detection object receptor, construct new FRET templates the FRET template by blocking the receptor structure of ICT in the process to achieve the ratio of regulation, has the advantages of high energy transfer efficiency and large displacement of Stokes. Through the detection of endogenous bisulfite, revealed for the first time in hepatocellular carcinoma cells With large differences in endogenous bisulfite concentration and normal liver cells, for the diagnosis of liver cancer at the cellular level provides a new idea of.3. for the first time will be linked to the ESIPT process and FRET process, construct the ESIPT assisted FRET template has great (pseudo) Stokes displacement efficiency and high energy transfer, and the rich the FRET type of structural diversity. The ratio of donor probe

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3

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