In-Tab化合物的設(shè)計合成及其作為生物熒光探針的研究
發(fā)布時間：2018-08-15 11:58
【摘要】：4-(三甲基銨基)苯硫酚(TabHPF6, Tab= 4-(trimethyammonio)benzenethiolate),是一種季銨鹽型兩性硫酚配體,它能夠與金屬反應(yīng),得到結(jié)構(gòu)新穎的配位金屬化合物。近年來,我們課題組一直致力于這類化合物的設(shè)計合成和反應(yīng)性研究工作,并嘗試將其應(yīng)用到先進材料、生物功能模型、有機催化等領(lǐng)域,逐步挖掘它們的實際應(yīng)用價值。另一方面,目前已經(jīng)有越來越多的報道介紹了無機材料和有機小分子化合物在DNA生物熒光探針方面的應(yīng)用,這些材料為核酸序列的檢測工作提供了非常重要的理論和現(xiàn)實依據(jù)。由于TabHPFe配體本身的結(jié)構(gòu)特點,所構(gòu)筑的金屬-Tab化合物往往具有良好的水溶性、穩(wěn)定性和可調(diào)控的結(jié)構(gòu)骨架,使其有望被設(shè)計成為新型DNA生物熒光探針,為進一步研究簡單金屬配合物作為病毒核酸分子檢測劑提供可能性。本文中,我們選取金屬銦,開展了In(Ⅲ)-Tab-輔助配體化合物的構(gòu)筑和應(yīng)用探索工作。分離得到五個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),詳細地探討了它們在液相和固相條件下的反應(yīng)性,并研究了化合物2,4和5在識別檢測DNA序列以及穩(wěn)定和誘導(dǎo)G-四鏈體DNA結(jié)構(gòu)方面的性能。主要研究工作包括以下幾個部分：1)通過常規(guī)液相反應(yīng)和固相轉(zhuǎn)化反應(yīng),設(shè)計合成了五個In-Tab化合物,并對它們進行了X-射線單晶衍射、紅外、紫外、元素分析、電噴霧質(zhì)譜和核磁測試,細致地探究了In(OAc)3、TabHPF6以及Phen/Dpphen的反應(yīng)性。研究發(fā)現(xiàn),在固相反應(yīng)的條件下向In-Tab體系引入Phen,能夠分離得到三個不同產(chǎn)物2,3和4,且投入的Phen越多,產(chǎn)物中參與配位的Phen個數(shù)反而越少。而液相反應(yīng)中,調(diào)節(jié)Phen的投入量,只能夠得到唯一產(chǎn)物4。這一發(fā)現(xiàn)充分地體現(xiàn)了固相反應(yīng)對于液相反應(yīng)來說不可替代的優(yōu)勢。在使用配體Dpphen替代Phen進一步探究反應(yīng)性時,由于Dpphen占據(jù)了較大的橫向空間位阻,固相反應(yīng)只能夠得到唯一產(chǎn)物5。這一結(jié)果從實例上證明了固相反應(yīng)在化合物構(gòu)建上的獨特之處。2)將化合物2,4和5應(yīng)用于艾滋病病毒(HIV)和蘇丹埃博拉病毒(Sudan Ebolavirus)核酸序列的生物探針研究�；衔锿ㄟ^π-π堆積,靜電和氫鍵作用,與熒光標(biāo)記的探針DNA(P-DNA)結(jié)合,形成穩(wěn)定的化合物@P-DNA并使P-DNA的熒光淬滅。加入P-DNA的互補目標(biāo)DNA鏈,P-DNA與互補目標(biāo)DNA鏈形成雙鏈或三鏈,從化合物中游離出來并伴隨P-DNA的熒光恢復(fù)。以熒光淬滅和復(fù)蘇情況為指標(biāo),實現(xiàn)了對HIV-1 ds-DNA和Sudan Ebolavirus RNA兩種病毒核酸序列的檢測,其檢測限分別達到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,通過化合物@P-DNA與非互補堿基序列結(jié)合作用的競爭實驗發(fā)現(xiàn),化合物對于目標(biāo)DNA具有良好的選擇特異性和識別性。3)將化合物2,4和5應(yīng)用于誘導(dǎo)和穩(wěn)定G-四鏈體結(jié)構(gòu)的生物活性研究。篩選出7種不同的DNA序列,通過紫外吸收滴定實驗和熒光滴定實驗,考察化合物與DNA之間的結(jié)合能力。其中,化合物5與G-四鏈體之間的結(jié)合常數(shù)可以達到107M-1。CD光譜實驗表明,化合物能夠誘導(dǎo)無序狀態(tài)的22AG和G2T1形成反平行結(jié)構(gòu),且能夠與22AG(K+).22AG(Na+).c-kit2(K+)以及G2T1(Na+)幾種G-四鏈體形成復(fù)合物,進一步穩(wěn)定G-四鏈體的結(jié)構(gòu)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：O657.3
本文編號：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：O657.3
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1 厲芙伶;In-Tab化合物的設(shè)計合成及其作為生物熒光探針的研究[D];蘇州大學(xué);2016年
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