兰州大学机构库 >化学化工学院
选择性检测生物活性分子的荧光探针的构建 及其应用研究
Alternative TitleConstruction and Application Research of Fluorescent Probes for Selective Detecting Bioactive Molecules
张芳
Subtype博士
Thesis Advisor房建国
2023-08-30
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学博士
Degree Discipline化学
Keyword荧光探针 Fluorescent probes 谷胱甘肽 glutathione 碳酸酐酶 carbonic anhydrases Baylis-Hillman衍生物 Baylis-Hillman derivative 基于亲和力标记蛋白 affinity-based protein labeling 脂质过氧化 lipid peroxidation 共轭二烯烃 conjugated diene
Abstract

生命体中的小分子物质以及包括蛋白质、核酸等在内的生物大分子在整个细胞周期中维持生命体系的正常运行发挥着必不可少的作用,他们的异常表达或者活性缺失都会引起相关的病理性疾病。基于分子识别这一概念,荧光探针在与大分子受体特异性结合以及对小分子底物选择性识别方面取得了明显的进展。荧光探针凭借操作简便,选择性好,灵敏度高及生物兼容性好等优点,已经成为生物传感和生物成像技术中必不可少的利器。与此同时,在化学、医学、生物学、材料学等领域,它也成为当前研究的热门焦点之一。结合待测底物的性质及荧光探针的结构,本论文设计合成了一系列小分子荧光探针,能够在复杂的生物系统内选择性地检测和可视化生物活性分子,同时反映了生物活性分子在不同病理模型中的表达,这不仅阐明了探针设计与生物应用之间的相互关系,还初步揭示了生物活性分子与相关病理过程之间的作用机制,为之后的生物研究提供了简便快捷的工具。本论文的具体内容如下:
第一章 绪论部分,本章内容简要介绍了荧光探针的基本概念和多种类型的发光机理。之后汇总了探针在谷胱甘肽(Glutathione, GSH)检测,碳酸酐酶(Carbonic anhydrases, CAs)检测,基于亲和力蛋白标记以及脂质过氧化(Lipidperoxidation, LPO)检测方面的研究进展。
第二章 设计合成了基于萘酰亚胺骨架的GSH 荧光探针R13,用于简便准确地量化GSH。探针结构中以乙酸甲酯磺酰基作为识别位点,相比于Cys 和Hcy,探针对GSH 显示出了较好的选择性。该探针被成功应用于细胞和组织中GSH 的准确定量,揭示了x 射线辐照导致小鼠肝脏中氧化型GSH(GSSG)的增加和还原型GSH 的消耗以及帕金森小鼠模型脑中GSH 的减少。使用探针定量生物样品中GSH 的便捷性有助于我们进一步了解相关疾病中GSH/GSSG 比值的波动。
第三章 采用环境敏感型荧光团和CAs 抑制剂相结合的策略,成功开发了选择性检测CAs 的红色荧光探针FMRs-CA。当存在CAs 时,探针FMRs-CA 的芳磺酰胺部位将探针分子带入CAs 的疏水口袋,限制了荧光团上单键的自由旋转进而释放出荧光信号。探针FMRs-CA 对CAs 展现出了较好的选择性,不仅可以可视化成像细胞异常状态下CAs 的变化,还可以应用于小鼠体内成像,并首次研究了CAs 在正常小鼠各器官中的分布,揭示了探针FMRs-CA 主要在肝脏内富集,并逐渐被肝脏代谢。借助探针FMRs-CA,我们首次从荧光成像角度观察到癫痫小鼠大脑中CAs 的异常表达,将CAs 水平的紊乱与癫痫联系起来,证实了癫痫可使大脑中CAs 水平升高。
第四章 采用氨基香豆素做荧光信号基团,Baylis-Hillman(BH)衍生物的单巯基加合物做识别位点,利用BH 衍生物和硫醇之间双亲核加成的反应性设计合成了一系列基于亲和力的蛋白标记探针。探针Cou-S10 在多种细胞裂解液中可清晰地标记独立的蛋白条带,该条带中共有四种疾病相关的邻二巯基蛋白(共济失调蛋白-10 (Ataxin-10 ), α- 烯醇化酶( -enolase ), 磷酸甘油酸激酶1
(Phosphoglycerate kinase 1),真核起始因子4A-1(Eukaryoticinitiation factor 4A-1))被探针Cou-S10 以共价结合的方式标记到。在这里,我们提供了一种结构多样化的基于亲和力的蛋白质组学探针库的构建策略,可产生多种靶向蛋白的功能蛋白质组学探针,从而丰富可用于识别人类疾病的新诊断标志物和治疗靶点的化学分子库。
第五章 通过共轭二烯链结构将荧光团和N,N-二甲基苯环相连,增大共轭体系,释放出红色荧光。由于共轭二烯烃结构只对氢过氧化物形成的自由基比较敏感,因此容易受脂质过氧化影响,导致共轭结构消失,荧光发射峰消失。我们基于BODIPY 以外的荧光团成功构建出脂质过氧化探针Cou-LPO 和Qui-LPO,并且使用低成本试剂直接合成投入使用。实验初步证明该探针同样可以特异性检测脂质过氧化,并生成相应的氧化产物。选择BODIPY 以外的荧光团作为脂质过氧化探针的信号单元,可以很好的避免BODIPY 类荧光染料存在的一些弊端。在此基础上可以更方便地对探针进行进一步修饰,以开发各种功能性的脂质过氧化探针。
第六章 对全文研究工作进行总结,并且提出探针分子在其他活性物质检测方面的展望。

Other Abstract

Small molecules in organism and biomacromolecules including protein and nucleic acids play an essential role in maintaining the normal operation of living system in the whole cell cycle, their abnormal expression or lack of activity will cause related
pathological diseases. Based on the concept of molecular recognition, fluorescent probes have made remarkable progress in specific binding with macromolecular receptors and selective recognition of small molecular substrates. With the advantages of simple operation, high sensitivity, excellent selectivity and desirable biocompatibility, fluorescent probes have become indispensable tools in biosensing and biological
imaging technology, and have become one of the hot points in chemistry, medicine, biology, materials science and other fields. Combined with the properties of the substrate to be tested and the structure of fluorescent probes, this dissertation designed and synthesized a series of small molecular fluorescent probes. These probes enable selective detection and visualization of bioactive molecules within complex biological systems, simultaneously reflecting the expression of bioactive molecules in different pathological models. This not only elucidated the interrelation between probe design and biological applications, but also preliminarily revealed the mechanism of interactions between bioactive molecules and relevant pathological processes, providing a simple and quick tool for subsequent biological research. The specific contents of this dissertation are as follows:
Chapter 1. Introduction. This chapter briefly introduced basic concepts of fluorescent probes and multiple types of fluorescent mechanism. Then advancements of glutathione (GSH) detection, carbonic anhydrases (CAs) detection, affinity-based protein labeling and lipid peroxidation (LPO) detection were summarized.
Chapter 2. A GSH fluorescent probe based on the naphthalimide skeleton was designed and synthesized to simply and accurately quantify GSH. The recognition site of the probe is methyl acetate sulfonyl group, which has higher selectivity preference for GSH compared with Cys and Hcy. It was successfully applied in quantifying GSH in cells and tissues, revealing that radiation-induced oxidative stress led to an increase in oxidized GSH (GSSG) and GSH consumption in the liver of mice, and a decrease in GSH content in the brains of Parkinson's mouse was detected. The convenience of the
probe in quantifying GSH in biological samples facilitates further understanding of the fluctuation of the GSH/GSSG ratio in diseases.
Chapter 3. Fluorescent probe used to selectively detect CAs was successfully constructed by adopting the strategy of combing environment-sensitive fluorophore with inhibitor of CAs. When CAs are present, the aromatic sulfonamide moiety of the probe FMRs-CA bringing the probe close to the hydrophobic pocket of the protein, constraining the free rotation of the single bonds on the probe and further releasing
fluorescence. The probe FMRs-CA shows excellent selectivity to CAs, which not only could visualize the changes of CAs in cells under abnormal conditions, but also be used for in vivo imaging in mice. The distribution of CAs in various organs of normal mice was studied for the first time, revealing that probe FMRs-CA was mainly enriched in the liver and gradually metabolized by the liver. With the help of probe FMRs-CA, we first observed the abnormal expression of CAs in the brains of epilepsy mouse and connected the disorder of CAs level with epilepsy, confirming that epilepsy can lead to the increase of CAs in brain.
Chapter 4. A series of affinity-based protein labeling probes were designed and synthesized by using coumarin as the fluorescence signal group and the monothiol adduct of the Baylis-Hillman (BH) derivative as the recognition site. The probe Cou-S10 could clearly mark independent protein bands in various cell lysates, in which a total of four disease-related vicinal dithiol-containing proteins were covalently labeled
by the probe. These proteins were ataxin-10, a-enolase, phosphoglycerate kinase 1, and eukaryotic initiation factor 4A-1. Here, we provided a strategy for the construction of a
structurally diverse affinity-based proteomics probe library that can yield a variety of functional proteomic probes targeting proteins, thereby enriching the chemical molecular library that can be used to identify new diagnostic markers and therapeutic targets for human diseases.
Chapter 5. The fluorophore was linked with N, N- dimethylbenzene by conjugated diene chain structure, and the conjugated system was expanded to release red fluorescence. As conjugated diene structure is only sensitive to free radicals formed by hydroperoxide, it is easily influenced by lipid peroxidation, resulting in the disappearance of conjugated structure and fluorescence emission. We successfully
constructed lipid peroxidation probes Cou-LPO and Qui-LPO based on fluorophores other than BODIPY, and were directly synthesized with low-cost reagents and then put into use. Preliminary experiments showed that these probes could detect lipid peroxidation specifically and generate corresponding oxidation products. Some disadvantages of BODIPY dyes can be avoided by using non-BODIPY fluorophores as the signal units of LPO probes. On this basis, the probes can be further modified to develop a variety of functional LPO probes.
Chapter 6. The works of this dissertation were summarized and the prospects of probe in detection of other active substances were put forward.

Subject Area化学生物学
MOST Discipline Catalogue理学 - 化学 - 有机化学
URL查看原文
Language中文
Other Code262010_120190903790
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/538056
Collection化学化工学院
Affiliation
兰州大学化学化工学院
Recommended Citation
GB/T 7714
张芳. 选择性检测生物活性分子的荧光探针的构建 及其应用研究[D]. 兰州. 兰州大学,2023.
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