2,2-联吡啶链接的小檗碱二聚体的合成及其对金属离子和DNA的选择性荧光检测

发布时间：2018-03-30 10:34

本文选题：荧光探针　切入点：小檗碱二聚体　出处：《南方医科大学》2015年硕士论文

【摘要】：在过去的十年中,研究开发用于生物大分子检测的荧光探针受到了越来越广泛的关注,主要是因为此类生物荧光探针具有灵敏度和选择性高、成本低、操作简便等优点,此外,其在临床疾病诊断与治疗方面也有一定的应用前景。目前,一些已报道的荧光探针,例如：香豆素、罗丹明等在有机溶剂中对一些阳离子、阴离子以及小分子化合物具有良好的检测功能。但是,由于这些荧光探针分子缺乏一定的水溶性,限制了它们在生物大分子检测方面的应用。因此,设计并合成具有一定水溶性的新型荧光分子探针,成为了一个热门的研究领域。本论文基于具有一定水溶性的天然产物小檗碱,设计合成了具有较好水溶性的2,2'-联吡啶链接的小檗碱二聚体1,并系统研究了化合物1对生物体内重要的金属离子、生物大分子G-四链体和HIV病毒核酸分子DNA的荧光探针功能。主要研究结果如下：1.化合物1的合成、表征与性质设计合成了由2,2’-联吡啶链接的小檗碱二聚体1,通过1H NMR、13C NMR、 ESI-MS和HR-MS进行了结构表征。化合物1具有较好水溶性,能溶于含1‰DMSO的水溶液：化合物1具有聚集诱导发光性能,在DMSO/H2O溶液中,随着水的含量升高,荧光逐渐增强。化合物1的荧光强度受pH值影响,当pH值从2.0升高到7.0时,荧光逐渐增强,而pH从7.0升高到10.5时,荧光则逐渐减弱,并且化合物的荧光变化具有pH值变化可逆性,说明在不同pH环境中,骨架结构并未发生变化,可能是由于其质子化程度不同导致荧光强度发生变化。2.化合物1识别金属离子的探针功能研究紫外吸收和荧光光谱实验证明化合物1与Cu2+的结合比为2：1,因此,化合物1能够在pH 7.0的5 mM Tris-HCl缓冲溶液中选择性识别Cu2+,且不受其他金属离子干扰,检测极限为1.08μM。化合物1对金属离子Cu2+, Ni2+和Co2+表现出了不同的时间响应,荧光完全淬灭响应时间分别为瞬时、2h和10 h。化合物1与Co2+和Ni2+的的反应速率常数k分别为9.83×10-3min-1和9.60×10-2min-1,Ni2+与化合物1的反应速率常数为Co2+的10倍。3.化合物1识别G-四链体的探针功能研究荧光和紫外滴定结果表明化合物1与G-四链体(G4s)、单链DNA和双链DNA均有较强的结合能力,结合常数为105～106 M-1,但是G-四链体对化合物1的紫外和荧光呈现增强的趋势,而单、双链DNA则呈现减弱趋势,荧光变化可以通过肉眼识别。化合物1对G4平行结构的结合能力高于G4反平行结构。此外,化合物1对不含有金属离子的人体端粒无序序列22AG具有较强的结合能力并能诱导其形成反平行结构。CD melting实验结果表明化合物1对平行c-kit2、反平行22AG (Na+)和无序22AG三种G4s DNA作用的(△Tm)分别为24℃、1.5℃和14℃,进一步证明了化合物1对平行G4s的稳定性能。此外,化合物1对G4/hemin配合物过氧化酶的抑制活性实验证明,化合物1与平行G-四链体主要是通过π-π堆积的方式结合。化合物1还可以对Hela细胞内的核酸进行荧光成像。4.化合物1对靶向HIV DNA的探针功能研究化合物1的结构中含有芳香环和季铵盐正电荷中心,这些官能团可能与DNA产生π-π、静电、氢键等作用。为了验证我们的想法,我们研究了化合物1检测HIV DNA的探针功能。研究结果表明,化合物1能够通过π-π、静电、或氢键作用将FAM荧光标记的单链DNA荧光淬灭,淬灭率高达92%,有趣的是,加入与单链DNA互补的靶向HIV双螺旋DNA后,荧光基本恢复,复苏率RE为7.4,化合物1能够特异性的识别HIV DNA,检测限为0.11nM。
[Abstract]:In the past ten years, the research and development of fluorescent probe for Biomacromolecule detection has attracted more and more attention, mainly because of such biological fluorescent probe with high sensitivity and selectivity, low cost, simple operation and other advantages, in addition, it also has a certain application prospect in clinical diagnosis and treatment of diseases at present. Some have been reported, such as fluorescent probe, coumarin, Luo Danming in organic solvent for cations, anions and small molecule compounds have good detection function. However, due to the lack of a fluorescent probe into water soluble, limit their application in the detection of biological molecules. Therefore, a novel fluorescence molecular probes were designed and synthesized with certain water soluble, has become a hot research field. This paper has some water soluble natural product of berberine based on Design Has a good water solubility of berberine 2,2'- bipyridine links two dimer 1, and studied 1 compounds of metal ions in vivo important, fluorescence probe function of macromolecular chain of G- four and HIV virus nucleic acid molecules of DNA. The main results are as follows: 1. the synthesis of 1 compounds by berberine 2,2 '- bipyridine linked two dimer 1 synthesis and characterization of design, through the 1H NMR, 13C NMR, ESI-MS and HR-MS were used to characterize the structures. Compound 1 has good solubility in water, soluble in aqueous solution containing 1 DMSO%: compound 1 exhibited aggregation induced luminescence properties in DMSO/H2O solution with the increase in water content, fluorescence gradually. The fluorescence intensity of compound 1 is affected by the pH value, when the pH value increased from 2 to 7, the fluorescence gradually increased, while pH increased from 7 to 10.5, the fluorescence decreases, and the fluorescence change of compounds with pH Changes in the value of the reversibility, explains the different pH environment, the skeleton structure has not changed, may be due to the combination of different degree of protonation leads to probe function of purple fluorescence intensity changes of.2. compound 1 recognition of metal ions absorption and fluorescence spectra experiments show that compound 1 and Cu2+ ratio is 2:1, therefore, compound 1 can be selective identification of Cu2+ in pH 7 mM 5 Tris-HCl buffer solution, and no other metal ion interference, the detection limit is 1.08 M. 1 compounds for metal ions Cu2+, Ni2+ and Co2+ showed different fluorescence quenching response time, complete response time were instantaneous, 2h and 10 h. compounds with Co2+ and 1 the Ni2+ of the reaction rate constant K was 9.83 * 10-3min-1 and 9.60 * 10-2min-1, the reaction rate constants of Ni2+ and compound 1 as a probe function 10 times.3. Co2+ 1 G- four compound identification chain research fluorescence And UV titration results showed that compounds 1 and four G- chain (G4s), single stranded DNA and double stranded DNA have strong binding ability, the binding constant was 105~106 M-1, but the G- four chain body shows the trend of compound 1 UV and fluorescence of single, double stranded DNA showed a weakening trend fluorescence, changes can be identified by the naked eye. The binding ability of compound 1 on G4 parallel structure is higher than that of the G4 anti parallel structure. In addition, 1 compounds of metal ion containing human telomeric sequence of random 22AG has stronger binding ability and can induce the formation of anti parallel structure of.CD melting showed that 1 compounds of c-kit2 parallel, anti parallel 22AG (Na+) three G4s and disordered 22AG the role of DNA (delta Tm) respectively for 24 degrees, 1.5 degrees and 14 degrees, further proved that the compound 1 pairs of parallel G4s stable performance. In addition, compound 1 complexes with G4/hemin oxidase inhibitory activity Experiments show that compounds 1 and four parallel G- chain is mainly through the combination of stacking way. Compound 1 can also nucleic acid on Hela cells for fluorescence imaging of.4. compound structure of the compound function of 1 probe targeting HIV DNA 1 containing aromatic ring and quaternary ammonium positive charge center, these functional groups may produce Pi Pi, and DNA electrostatic, hydrogen bond effects. In order to verify our idea, we studied 1 probe compounds for detection of HIV DNA. The results show that the compound 1 can by tt-tt, electrostatic, hydrogen bonding or single stranded DNA fluorescence quenching of FAM fluorescence labeled inactivated, quenching rate 92%, interestingly, adding complementary and single stranded DNA targeting HIV double helix DNA, fluorescence recovered, the recovery rate of RE was 7.4, compound 1 is able to identify HIV specific DNA, the detection limit is 0.11nM.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R914

【共引文献】