In-Tab化合物的设计合成及其作为生物荧光探针的研究
发布时间：2018-08-15 11:58
【摘要】：4-(三甲基铵基)苯硫酚(TabHPF6, Tab= 4-(trimethyammonio)benzenethiolate),是一种季铵盐型两性硫酚配体,它能够与金属反应,得到结构新颖的配位金属化合物。近年来,我们课题组一直致力于这类化合物的设计合成和反应性研究工作,并尝试将其应用到先进材料、生物功能模型、有机催化等领域,逐步挖掘它们的实际应用价值。另一方面,目前已经有越来越多的报道介绍了无机材料和有机小分子化合物在DNA生物荧光探针方面的应用,这些材料为核酸序列的检测工作提供了非常重要的理论和现实依据。由于TabHPFe配体本身的结构特点,所构筑的金属-Tab化合物往往具有良好的水溶性、稳定性和可调控的结构骨架,使其有望被设计成为新型DNA生物荧光探针,为进一步研究简单金属配合物作为病毒核酸分子检测剂提供可能性。本文中,我们选取金属铟,开展了In(Ⅲ)-Tab-辅助配体化合物的构筑和应用探索工作。分离得到五个In-Tab-Phen/Dpphen化合物：[In(Tab)4](OAc)3 (1), [In(Tab)2(Phen)2](PF6)3 (2), [In(Tab)2(Phen)2-In(Tab)4(Phen)](OAc)6 (3) [In(Tab)4(Phen)](OAc)3 (4)和[In(Tab)2(Dpphen)2](PF6)3 (5),详细地探讨了它们在液相和固相条件下的反应性,并研究了化合物2,4和5在识别检测DNA序列以及稳定和诱导G-四链体DNA结构方面的性能。主要研究工作包括以下几个部分：1)通过常规液相反应和固相转化反应,设计合成了五个In-Tab化合物,并对它们进行了X-射线单晶衍射、红外、紫外、元素分析、电喷雾质谱和核磁测试,细致地探究了In(OAc)3、TabHPF6以及Phen/Dpphen的反应性。研究发现,在固相反应的条件下向In-Tab体系引入Phen,能够分离得到三个不同产物2,3和4,且投入的Phen越多,产物中参与配位的Phen个数反而越少。而液相反应中,调节Phen的投入量,只能够得到唯一产物4。这一发现充分地体现了固相反应对于液相反应来说不可替代的优势。在使用配体Dpphen替代Phen进一步探究反应性时,由于Dpphen占据了较大的横向空间位阻,固相反应只能够得到唯一产物5。这一结果从实例上证明了固相反应在化合物构建上的独特之处。2)将化合物2,4和5应用于艾滋病病毒(HIV)和苏丹埃博拉病毒(Sudan Ebolavirus)核酸序列的生物探针研究。化合物通过π-π堆积,静电和氢键作用,与荧光标记的探针DNA(P-DNA)结合,形成稳定的化合物@P-DNA并使P-DNA的荧光淬灭。加入P-DNA的互补目标DNA链,P-DNA与互补目标DNA链形成双链或三链,从化合物中游离出来并伴随P-DNA的荧光恢复。以荧光淬灭和复苏情况为指标,实现了对HIV-1 ds-DNA和Sudan Ebolavirus RNA两种病毒核酸序列的检测,其检测限分别达到14 nM(2).1.7 nM(4).1.2 nM(5)以及11 nM(2).0.37 nM (4).0.81 nM(5)。此外,通过化合物@P-DNA与非互补碱基序列结合作用的竞争实验发现,化合物对于目标DNA具有良好的选择特异性和识别性。3)将化合物2,4和5应用于诱导和稳定G-四链体结构的生物活性研究。筛选出7种不同的DNA序列,通过紫外吸收滴定实验和荧光滴定实验,考察化合物与DNA之间的结合能力。其中,化合物5与G-四链体之间的结合常数可以达到107M-1。CD光谱实验表明,化合物能够诱导无序状态的22AG和G2T1形成反平行结构,且能够与22AG(K+).22AG(Na+).c-kit2(K+)以及G2T1(Na+)几种G-四链体形成复合物,进一步稳定G-四链体的结构。
[Abstract]:4 - (trimethylammonium) thiophenol (TabHPF6, Tab = 4 - (trimethyammonio) benzenethiolate) is a quaternary ammonium salt amphoteric thiophenol ligand. It can react with metals to obtain novel coordination metal compounds. In recent years, our research group has been devoted to the design, synthesis and reactivity of these compounds, and try to make it. On the other hand, there have been more and more reports on the application of inorganic materials and organic small molecule compounds in DNA biofluorescent probes. These materials provide very important information for the detection of nucleic acid sequences. Because of the structural characteristics of TabHPFe ligands, the metal-Tab compounds often have good water-solubility, stability and adjustable structural skeleton, so they are expected to be designed as novel DNA biofluorescent probes for further study of simple metal complexes as viral nucleic acid molecular detectors. In this paper, five in-Tab-Phen/Dpphen compounds were separated and obtained: in (Tab) 4 (OAc) 3 (1), in (Tab) 2 (Phen) 2] (PF6) 3 (2), in (Tab) 2 (Phen) 2-in (Tab) 4 (Phen) 4 (OAc) and in (Tab) 6 (c). 2 (Dpphen) 2] (PF6) 3 (5) and their reactivity in liquid and solid phases were investigated in detail. The properties of compounds 2, 4 and 5 in identifying and detecting DNA sequences and stabilizing and inducing the structure of G-quadruplex DNA were also studied. The main research work includes the following parts: 1) Designing and synthesizing compounds by conventional liquid phase reaction and solid phase transformation reaction. Five in-Tab compounds were synthesized and characterized by X-ray single crystal diffraction, infrared spectroscopy, ultraviolet spectroscopy, elemental analysis, electrospray ionization mass spectrometry and nuclear magnetic resonance. The reactivity of in(OAc)3, TabHPF6 and Phen/Dpphen was investigated in detail. It was found that three different products 2,3 could be separated by introducing Phen into in-Tab system under the condition of solid state reaction. And 4, and the more he put in, the less he participated in the coordination of the products. In the liquid phase reaction, only the only product 4 can be obtained by adjusting the amount of Phen input. This discovery fully reflects the irreplaceable advantages of the solid phase reaction for the liquid phase reaction. Dpphen occupies a large transverse steric hindrance and only one product can be obtained by solid-state reaction. This result demonstrates the uniqueness of solid-state reaction in the construction of compounds. 2. Compounds 2, 4 and 5 were used in the study of biological probes for nucleic acid sequences of HIV and Sudan Ebolavirus. By pi-pi stacking, electrostatic and hydrogen bonding, the compounds bind to fluorescent probe DNA (P-DNA) to form a stable compound @P-DNA and quench the fluorescence of P-DNA. By adding complementary target DNA strands of P-DNA, P-DNA and complementary target DNA strands form double or triple strands, which are separated from the compounds and recovered with the fluorescence of P-DNA. Recovery was used as an indicator to detect the nucleic acid sequences of HIV-1 ds-DNA and Suan Ebolavirus RNA. The detection limits were 14 nM(2). 1.7 nM(4). 1.2 nM(5) and 11 nM(2). 0.37 nM(4). 0.81 nM(5). In addition, the competitive experiments of the binding of compound @P-DNA and non-complementary base sequences showed that the compound was suitable for the target. 3) Compounds 2, 4 and 5 were used to induce and stabilize the G-quadruplex structure. Seven different DNA sequences were screened and their binding ability to DNA was investigated by UV absorption titration and fluorescence titration. The binding constants can reach 107M-1. CD spectra show that the compounds can induce disordered 22AG and G2T1 to form antiparallel structures, and can form complexes with 22AG (K +).22AG (Na +).C-kit2 (K +) and G2T1 (Na +) G-tetrads to further stabilize the structure of G-tetrads.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3
本文编号：2184130
[Abstract]:4 - (trimethylammonium) thiophenol (TabHPF6, Tab = 4 - (trimethyammonio) benzenethiolate) is a quaternary ammonium salt amphoteric thiophenol ligand. It can react with metals to obtain novel coordination metal compounds. In recent years, our research group has been devoted to the design, synthesis and reactivity of these compounds, and try to make it. On the other hand, there have been more and more reports on the application of inorganic materials and organic small molecule compounds in DNA biofluorescent probes. These materials provide very important information for the detection of nucleic acid sequences. Because of the structural characteristics of TabHPFe ligands, the metal-Tab compounds often have good water-solubility, stability and adjustable structural skeleton, so they are expected to be designed as novel DNA biofluorescent probes for further study of simple metal complexes as viral nucleic acid molecular detectors. In this paper, five in-Tab-Phen/Dpphen compounds were separated and obtained: in (Tab) 4 (OAc) 3 (1), in (Tab) 2 (Phen) 2] (PF6) 3 (2), in (Tab) 2 (Phen) 2-in (Tab) 4 (Phen) 4 (OAc) and in (Tab) 6 (c). 2 (Dpphen) 2] (PF6) 3 (5) and their reactivity in liquid and solid phases were investigated in detail. The properties of compounds 2, 4 and 5 in identifying and detecting DNA sequences and stabilizing and inducing the structure of G-quadruplex DNA were also studied. The main research work includes the following parts: 1) Designing and synthesizing compounds by conventional liquid phase reaction and solid phase transformation reaction. Five in-Tab compounds were synthesized and characterized by X-ray single crystal diffraction, infrared spectroscopy, ultraviolet spectroscopy, elemental analysis, electrospray ionization mass spectrometry and nuclear magnetic resonance. The reactivity of in(OAc)3, TabHPF6 and Phen/Dpphen was investigated in detail. It was found that three different products 2,3 could be separated by introducing Phen into in-Tab system under the condition of solid state reaction. And 4, and the more he put in, the less he participated in the coordination of the products. In the liquid phase reaction, only the only product 4 can be obtained by adjusting the amount of Phen input. This discovery fully reflects the irreplaceable advantages of the solid phase reaction for the liquid phase reaction. Dpphen occupies a large transverse steric hindrance and only one product can be obtained by solid-state reaction. This result demonstrates the uniqueness of solid-state reaction in the construction of compounds. 2. Compounds 2, 4 and 5 were used in the study of biological probes for nucleic acid sequences of HIV and Sudan Ebolavirus. By pi-pi stacking, electrostatic and hydrogen bonding, the compounds bind to fluorescent probe DNA (P-DNA) to form a stable compound @P-DNA and quench the fluorescence of P-DNA. By adding complementary target DNA strands of P-DNA, P-DNA and complementary target DNA strands form double or triple strands, which are separated from the compounds and recovered with the fluorescence of P-DNA. Recovery was used as an indicator to detect the nucleic acid sequences of HIV-1 ds-DNA and Suan Ebolavirus RNA. The detection limits were 14 nM(2). 1.7 nM(4). 1.2 nM(5) and 11 nM(2). 0.37 nM(4). 0.81 nM(5). In addition, the competitive experiments of the binding of compound @P-DNA and non-complementary base sequences showed that the compound was suitable for the target. 3) Compounds 2, 4 and 5 were used to induce and stabilize the G-quadruplex structure. Seven different DNA sequences were screened and their binding ability to DNA was investigated by UV absorption titration and fluorescence titration. The binding constants can reach 107M-1. CD spectra show that the compounds can induce disordered 22AG and G2T1 to form antiparallel structures, and can form complexes with 22AG (K +).22AG (Na +).C-kit2 (K +) and G2T1 (Na +) G-tetrads to further stabilize the structure of G-tetrads.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3
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1 厉芙伶;In-Tab化合物的设计合成及其作为生物荧光探针的研究[D];苏州大学;2016年
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