【作者】 周春琼；

【导师】 杨频；

【作者基本信息】 山西大学 ， 无机化学， 2003， 硕士

【摘要】 近年来，多核金属配合物应用作核酸切割剂的研究在切割反应的条件、时间、效率、特异性等方面的优越性取得了引人注目的进展，其中重要的推动原因是人们从结构上逐步认识到与核酸有关的多种天然酶的活性部位含有两个或三个协同作用的金属离子，如Zn（Ⅱ）、Mg（Ⅱ）、Ni（Ⅱ）、Mn（Ⅱ）、Fe（Ⅲ）等。与此同时，有关稀土及其配合物作为人工酶的研究越来越受到人们的重视，因为它们能以水解方式断裂核酸中的磷酸二酯键，并且断裂效率较高。 基于以上两点，我们模拟天然金属蛋白中的多核活性中心结构，合成了能为两个金属离子提供相同配位环境的配体Hbbimp，并合成了一系列的双核金属配合物（L表示配体）：Mg₂L、Mn₂L、Cd₂L、Ni₂L、Zn₂L、Nd₂L、Eu₂L。用¹HNMR谱、IR光谱、UV光谱、摩尔电导、EDTA滴定法测金属离子含量、元素分析，基本确定了所合成的七种双核金属配合物的结构。更可喜的是：得到了双核镍金属配合物的单晶，这对于确定该类配合物体系的主体结构提供了可靠的依据。 通过电泳筛选实验，在37℃，pH 7.23时，两种稀土（铕和钕）配合物切割质粒DNA效果最好，同时对其最佳反应条件进行了摸索，其最佳反应条件为：50℃，pH 8.0，可将大部分CCC带转化为OC带，无线性产物出现。当[配合物]：[双氧水]=1:20，[双氧水]＜1mmol／L时，在配合物浓度较小时，即可将CCC带全部转化为OC带，无线性产物出现，其切割效果明显强于单独的稀土配合物切割DNA。用UV光谱法、DNA热变性实验、荧光光谱法研究了两种稀士配合物与CT DNA的作用。随着浓度的增加，CT DNA在最大吸收峰处的吸光度值逐渐减少；DNA热变性实验中，变性过程与降解过程共存；配合物的加入使EB-DNA体系的荧光强度减弱，这些实验结果表明：配合物与DNA螺旋进入了相对强的外部结合状态，配合物阳离子有效地替代了DNA外层阳离子，进而引起DNA构象的变化。配合物与活化的磷酸二酯键模型物BDNPP，在恰当的浓度，不同的温度，不同的pH值条件下的动力学实验表明，两种稀土配合物是断裂BDNPP中P-O键的很好的试剂。更多还原

【Abstract】 Recently, research on using several nucleus metal ion complexes as nucleic acid - cleavage reagents has gotten rapid development on condition, time, efficiency, sequence specificity during the cleavage reaction, whose important reason is that people slowly realize that in according to the structures that the active position of a number of natural enzymes in relation to nucleic acid has two or three cooperative metal ions ,for example Zn(II),Mg(II),Mn(II),Ni(II), Fe(III) etc. Meanwhile, research about using rare-earth ions and their complexes as artificial restriction enzymes has gotten more and more recognition, as they can hydrolytically cleave the phosphate diester bond of nucleic acid, and their, cleavage efficiencies are higher.On the base of the two above points, we simulate several nucleus active center structure of natural metal albumen and synthesize the ligand Hbbimp (where Hbbimp is 2,6-bis[bis(2-benzimidazolylmethyl)aminomethyl]-4-methylphenol). The preparation of dinuclear metal complexes of the ligand with Mg(II), Mn(II), Cd(II), Ni(II) Zn(II), Nd(III), Eu(III) are described. We have ascertained their rough structures with infrared spectra, nuclear magnetic resonance spectra, UV-Vis spectra, molar electrolytic conductivity and elemental analysis. All of the happiest is that we get the crystal structure of binuclear nickel (II) complex [Ni2(bbimp)(CH3CH2OH)2Cl2]Cl . 4H2O ( bbimp = 2,6-bis [bis ( 2-benzimidazolylmethyl ) ] aminomethyl -4- methylphenoxo ) , which gives a credible evidence to the main body structure of this kind of the complexes.In according of the experiment of the electrophoresis filtration, we find that two rare-earth(Eu(III) and Nd(III)) complexes has better efficiencies when cleaving DNA at 37℃, pH 7.23. Then we research the best conditions of their reactions, and they are at 50’C, pH 8.0, when a majority of CCC are chaged into OC outcome ,and Linear outcome don’t appear. At [complexes]: [H2O2]=l:20, [H2O2]< 1mM, CCC in the pBR322DNA are chaged into OC and Linear outcome don’t also appear when the concentrations of the complexes are small, whose cleaving-DNA efficiencies apparently are better than the solely complexes cleaving DNA. We also research the action of two rare-earth ion complexes and CT-DNA with UV-Vis spectra, DNA heat-denaturalization experiment, fluorescene spectra. When the concentrations of the complexes are increased, A260 are gradually reduced; in the DNA heat-denaturalization experiment, the denaturalization and decompose processes coexist; When the complexes are placed into EB-DNA, its intension of fluorescene spectra is gradually weaken. At the appropriate concentrations, the different temperature and pH, we research the action of the complexes and an Activated Phosphate Diester BDNPP. The experiment indicates that the complexes are the good reagents cleaving an Activated Phosphate Diester BDNPP.更多还原