多肽荧光化学传感器的合成及生物应用研究

发布时间：2018-04-16 04:19

本文选题：多肽 + 荧光化学传感器　；参考：《兰州大学》2017年博士论文

【摘要】：在细胞中,蛋白质通过氨基酸侧链与特定的金属离子结合形成特有的结构域(Motif),这种结构域在维持细胞正常的生物学功能中起着至关重要的作用。比如锌指蛋白(Zinc finger)是蛋白中的组氨酸和半胱氨酸结合锌离子形成的一个手指形折叠蛋白质,它与基因的转录和调控密切相关;EF手形(EF-hand),一个螺旋-环-螺旋的结构基序,由天冬氨酸和谷氨酸提供和钙配位的螯合位点,它参与了细胞增殖、分化、凋亡等重要的生物学过程;金属蛋白酶(Metalloproteins)的活性中心含有金属离子,失去这些金属离子的酶将失去催化活性;铁离子和铜离子是光合作用和氧化磷酸化电子传递链的重要组成部分。这些重要的生物大分子结构特征给我们提供了大量的化学信息,通过模仿天然蛋白与金属离子结合位点进行多肽荧光化学传感器的设计,是研究细胞中金属离子重要作用的有效手段之一。近几年,多肽荧光化学传感器的设计与应用得到了研究者的广泛关注,国内外多个课题组相继报道了多种类型的多肽荧光化学传感器。相比于其他荧光传感器,多肽荧光化学传感器具有自身的优势:(1)固相多肽合成与侧链修饰技术比较成熟,多肽荧光化学传感器的灵敏度和选择性可以通过改变肽骨架来优化;(2)多肽荧光化学传感器的水溶性、生物相容性和低毒性都优于有机小分子传感器,可以实现纯水检测和生物应用;(3)多肽荧光化学传感器的识别模式很容易通过不同的识别机理来实现,如光诱导电子转移(PET)、荧光共振能量转移(FRET)和螯合增强荧光(CHEF)等。基于这些优势,我们设计合成了一系列新颖的多肽荧光化学传感器,并研究了其对生物小分子(Zn~(2+)、Cd~(2+)、Cu~(2+)、S2-、H2S)的识别选择性及其在细胞成像和斑马鱼成像中的应用。本文共分为七个部分。第一章:简述了荧光化学传感器的设计原理和研究背景,并着重总结了多肽荧光化学传感器的研究进展。第二章:基于Fmoc-Lys(Fmoc)-OH的骨架机构,我们设计合成出一个新颖的多肽荧光化学传感器L1,实现了纯水体系中Zn~(2+)、Cu~(2+)和S2-的荧光响应,并成功在细胞内实现应用。第三章:在L1的基础上,通过改变氨基酸,基于光诱导电子转移机理,我们合成了在纯水体系中识别Cd~(2+)的多肽荧光化学传感器L2,L2具有良好的细胞渗透性和低毒性,实现了HeLa细胞中Cd~(2+)的检测。第四章:基于光诱导电子转移机理,我们报道了一个四肽荧光化学传感器L3,该传感器在纯水体系中实现了“Turn-On”型Zn~(2+)检测,进一步,我们利用螯合剂EDTA置换出了L3-Zn配合物体系中的Zn~(2+),实现了EDTA的检测。L3能够穿透HeLa细胞,从而实现了HeLa活细胞内的Zn~(2+)和EDTA的成像检测。第五章:我们设计并合成了一个多肽荧光化学传感器L4,L4在纯水体系和活细胞中可以利用不同激发实现Zn~(2+)的检测。在290 nm激发下,基于荧光共振能量转移效应(FRET),荧光呈比率型增强模式;在330 nm激发下,基于荧光螯合增强效应(CHEF),荧光呈“Turn-On”型增强模式。第六章:我们成功地开发出一种新颖的多功能四肽荧光传感器L5,在纯水体系中,随着Cu~(2+)和S2-的添加,L5呈现出“On-Off-On”型响应模式。L5具有良好的细胞渗透性和低毒性,可在HeLa活细胞内实现“On-Off-On”型连续性检测。第七章:在L5的基础上,通过荧光团的改变,我们报道了一个新颖的多肽荧光化学传感器L6,基于Cu~(2+)的淬灭传感机制,L6提供和Cu~(2+)配位的螯合位点而形成L6-Cu体系,从而实现荧光快速淬灭;当向L6-Cu体系加入H2S形成CuS沉淀之后,L6的荧光实现恢复。因此,L6-Cu体系既可以在纯水溶液中检测H2S,也能在活细胞和斑马鱼幼崽模型中实现生物成像。
[Abstract]:In the cell, protein domain specific amino acid side chain through binding with specific metal ion (Motif), plays a vital role in maintaining the biological function of this domain in normal cells. Such as zinc finger protein (Zinc finger) is a group of cysteine finger ammonia and acid protein in combination with zinc ion the formation of the folded protein, gene transcription and regulation and it is closely related to the hand shape; EF (EF-hand), a helix loop - helix structure based sequence, composed of aspartic acid and glutamic acid and calcium ligand chelating sites, it is involved in cell proliferation, differentiation, apoptosis and other important biological processes; metalloproteinase (Metalloproteins) activity center containing metal ions, the loss of these metal ions will lose enzyme catalytic activity; iron ion and copper ion is an important part of photosynthesis and oxidative phosphorylation electron transfer chain These important points. The biological characteristics of molecular structure provides a large amount of chemical information to us, through the design of imitation natural protein and metal ion binding sites of polypeptide fluorescent chemical sensor, is one of the effective means of the important role of metal ions in the cell. In recent years, the design and application of polypeptide fluorescent chemical sensor has been widely the attention of researchers, a number of domestic and foreign research group reported a polypeptide fluorescent chemical sensor types. Compared with other fluorescent sensors, polypeptide fluorescent chemical sensor has its own advantages: (1) synthesis and solid phase peptide side chain modification technology is relatively mature, the polypeptide fluorescent chemical sensor sensitivity and selectivity can be optimized by changing the the peptide backbone; (2) peptide fluorescence chemical sensor water solubility, biocompatibility and low toxicity are better than organic small molecular sensor, can The water detection and biological applications; (3) peptide fluorescence chemical sensor identification model is easier to achieve recognition through different mechanisms, such as photoinduced electron transfer (PET), fluorescence resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF). Based on these advantages, we designed and synthesized a series of novel the polypeptide fluorescent chemical sensor, and Study on its biological small molecules (Zn~ (2+), Cd~ (2+), Cu~ (2+), S2-, H2S) selective recognition and its application in cell imaging and imaging in zebrafish. This paper is divided into seven parts. The first chapter summarizes the design principle and the research background of fluorescence chemical sensor, and emphatically summarized the research progress of peptide fluorescence chemical sensor. The second chapter: Based on the Fmoc-Lys (Fmoc) framework of -OH, we designed and synthesized a novel polypeptide fluorescent chemical sensor L1, has realized the water system in Zn~ (2+), Cu~ ( 2+) fluorescence response and S2-, and successfully applied in the cells. The third chapter: on the basis of L1, by changing the amino acid, the photoinduced electron transfer mechanism based on Cd~, we synthesized recognition in pure water system (2+) of the peptide fluorescence chemical sensor L2, L2 has low toxicity and good cell the HeLa cell infiltration, Cd~ (2+) detection. The fourth chapter: Based on the mechanism of photoinduced electron transfer, we reported a four peptide fluorescence chemical sensor L3, the sensor to achieve the "Turn-On" type Zn~ in pure water system (2+) detection, further, we use EDTA replacement chelating agent the L3-Zn complexes in Zn~ (2+), the detection of.L3 EDTA can penetrate HeLa cells, so as to realize the HeLa in the living cell Zn~ (2+) imaging and EDTA. The fifth chapter: we design a fluorescent chemical sensor and polypeptide synthesis of L4, L4 in pure water Department of living cells and can be used in different excitation Zn~ (2+) detection. Under 290 nm excitation, fluorescence resonance energy transfer effect based on (FRET), ratio of fluorescence was enhanced; under 330 nm excitation, the fluorescence enhancement effect of chelate (CHEF), based on the fluorescence enhancement mode is "Turn-On". The sixth chapter: we successfully developed a multifunctional peptide fluorescence sensor L5 four novel, in the pure water system, with Cu~ (2+) and the addition of S2-, L5 showed "On-Off-On" type response mode.L5 has low toxicity and good cell infiltration, continuous detection of HeLa in living cells the realization of "On-Off-On". The seventh chapter: on the basis of L5, the fluorescence change, we report a novel polypeptide fluorescent chemical sensor L6, based on Cu~ (2+) quenching sensing mechanism, L6 and Cu~ (2+) ligand chelate sites and the formation of L6-Cu system, and real Fluorescence is rapidly quenched. When L6-Cu is added to H2S to form CuS precipitates, the fluorescence of L6 is restored. Therefore, L6-Cu system can detect H2S in pure aqueous solution, and can also achieve biological imaging in living cells and zebrafish pups.

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

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