【作者】 周大刚；

【导师】 唐明生； 朱艳艳；

【作者基本信息】 郑州大学 ， 工业催化， 2013， 硕士

【摘要】 由于金属核酸酶小分子对DNA具有较好的切割性,越来越多的人们开始关注和设计此类核酸酶小分子。人们逐渐的发现虽然其对DNA有着很高的切割能力,但是它们的选择特异性很差,在切割病态DNA的同时对正常的DNA也有很大的威胁。为此人们迫切需要一种选择特异性好的核酸酶,此时锌指蛋白的出现为这个问题的解决带来了曙光。锌指蛋白不仅能够识别不同的DNA碱基序列,而且能够稳定的结合在DNA的大沟中。在锌指蛋白的帮助下,金属核酸酶小分子不仅通过对附近糖环中氢的抽取实现了对DNA的特异性切割,而且还具有识别特定DNA碱基序列的功能。这引起了广大研究者的兴趣。在本论文中,主要做了以下三部分工作：一.采用分子对接方法研究了锌指蛋白识别结合DNA的主要影响因素。通过改变DNA序列来考察锌指蛋白对DNA结合强弱的影响。通过对两者结合能的监测,发现当第一个锌指结合位点的碱基序列由GCCC改变为第二个锌指所识别的碱基序列CCGC时导致其结合能的降低,而当改变为第三个锌指所识别的CCCC碱基序列时,其结合能也随之降低。但是对比两个突变的碱基序列CCGC和CCCC时,发现后面三个碱基序列是紧相连着的C碱基时,其结合能要高于含G序列的。因此得出第一个锌指在识别的过程中侧重于识别其天然存在的GCCC序列,而后再识别CCGC和CCCC这两个序列。二.锌指核酸酶是通过连接域把锌指蛋白和人工设计的金属核酸酶小分子连接起来,锌指核酸酶不仅能够识别特定的DNA,还能促进DNA的解离。然而,由于目前在实验上很多锌指核酸酶的晶体结构很难得到,这样就限制了锌指核酸酶和DNA相互作用的理论研究。在本文中,通过HADDOCK软件来预测锌指蛋白核酸酶Sp1-Cu(BPA)和DNA的结合模式；然后进行了一个25ns的分子动力学模拟。模拟的结果表明Spl的氨基酸残基和DNA碱基通过氢键等作用力稳定的作用在DNA大沟中,Cu(BPA)在模拟的过程中能稳定的作用于DNA小沟内,其方位对抽取邻近糖环上的氢是有利的,使得金属核酸酶小分子Cu(BPA)能够在特定区域促进DNA分子的断裂。这些计算结果说明,DNA识别剂的锌指蛋白的引入,很好地提升含铜核酸酶的DNA切割特异性。三.本部分主要研究了两个有机反应机理。第一个反应是芳基叠氮化合物和二丁基三氟甲磺酸酯反应生成N-烷基化苯胺的反应。实验上合成N-烷基化苯胺有很多方法,但是这些方法需要的实验条件都是比较苛刻的,不是在高温下就是使用有毒的或者腐蚀性的烷基化试剂。本文对Nagula教授提出的反应机理用密度泛函的理论进行计算,来验证其的正确性。对所有的反应物,过渡态,中间体和产物结构都在DFT理论中B3LYP/6-31+G(d,p)的水平下进行优化以确保所的结果的正确性,并对所有过渡态进行了IRC的计算,以确保过渡态是连接着预期的反应物和产物。计算结果表明,叠氮化物在二烷基三氟甲磺酸酯的作用下,不仅能够比较好的得到预期产物,而且反应条件温和,在室温下反应能发生。这些计算结果对设计新的反应提供了理论上的支持和帮助。第二个反应是多组分合成多取代的中氮茚。由于中氮茚衍生物在具有生物活性的天然产物中关键的亚基结构,有机荧光材料和药物物质等方面的广泛应用而引起了人们对其的极大重视。多组分反应在有机化学,合成化学和药物化学领域是一种强有力的和高效率的成键工具,三组分反应已经被证明是一种来合成中氮茚衍生物的比较直接的方法。Zhenjun Mao研究组采用吡啶、苯甲酰甲基溴和乙基乙醛酸为反应物,以乙腈为溶剂研究通过简洁和高效的四组分串联方法来合成多取代中氮茚衍生物,反应涉及到吡啶盐的叶立德形成,a,β-不饱和酮以及后面的1,3-偶极环加成反应和芳构化反应。所有的反应物,过渡态,中间体和产物的结构都在M05/6-31+G(d,p)的水平下进行优化。通过对反应的研究不仅可以揭示反应的机理,还可以进一步为新反应的设计提供理论的指导。更多还原

【Abstract】 Because of the highly cutting of metal nuclease to DNA, more and more people begin to pay attention and design these nuclease moleculars. Although with a very high cutting capacity, it always has a great threat to normal DNA while cutting morbid DNA according to its poor specificity of their choice. To end this, there is an urgent need for a nuclease with good select specificity. Zinc finger proteins can solve this problem and bring the dawn, which not only can be able identify the type of the DNA nucleotide sequence,but also incorporate in the DNA major groove stably. With the help of zinc finger protein, the metal nuclease not only achieves a specificity of DNA cutted by the extractionof hydrogen in the vicinity of sugar ring, but also has the function to identify a specific DNA nucleotide sequence, which aroused the interest of the major researchers.In this paper, we mainly do three parts work:Firstly, we take the method of molecular docking to study the main factors in the identification and binding of zinc finger to DNA and examine the zinc finger protein DNA binding strength through changing the DNA sequence. By monitoring the binding energy, we found that the decrease in the binding energy when the first zinc finger DNA binding sites from sequences GUA、CYT、CYT and CYT change into the DNA sequence of second zinc finger identifying CYT、CYT、GUA and CYT or another sequence of third zinc finger recognizing CYT、CYT、CYT and CYT. But contrasting the two mutations in the nucleotide sequence of CCGC and CCCC, the former has a lower binding energy than the latter. So we can conclude that the first zinc finger in the process of identifying focuses on the identification of their naturally occurring sequence of GCCC, and then recognizing others, such as CCGC and CCCC.Secondly, the zinc finger nuclease is the combination of zinc finger protein and the artificial metallonuclease through a linked field, which can recognize special DNA sequences and result in DNA scission. However, since crystal structure of the zinc finger nuclease has not been obtained in experiments, theoretical studies on interactions of zinc finger nuclease with DNA remain very limited. In this work, firstly, the binding modes of the zinc finger nuclease Spl-Cu(BPA) with B-DNA were predicted using the HadDock program, and then,25ns molecular dynamics simulations were performed. The simulated results reveal that the amino acid residues of Spl interact with the major groove of the DNA nucleotide through a hydrogen bond force, and Cu(BPA) can stay in the minor groove steadily, whose orientation is advantageous for extracting hydrogen from adjacent sugar ring, in order to make the nuclease Cu(BPA) deducing DNA strand scission in some certain regions. The results of these calculations illustrate the introduction of zinc finger protein of DNA detection agent to enhance the copper nuclease DNA cleavage specificity.Thirdly, this section consists of two organic reaction mechanism. The first reaction is that the Aryl azide interact with the Bu2BOTf to generate the N-alkylated aniline. There are many methods to synthesis N-alkylated aniline in experiment. But these methods require that the experimental conditions are more stringent,either at higher temperature or using toxic or corrosive alkylating reagent. In this paper, we use the density functional theory to calculate the reaction mechanism, professor Nagula Shankaraiah proposed, to verify its correctness. All structures of the reactants, transition states, intermediates and products were fully optimized at the B3LYP/6-31+G(d,p) level of theory to ensure we have the corrected results. By the calculating the IRC, we can assure that the transition state is the bridge connecting reactant and expected product. The calculating results show that the azide under the effect of dialkyl triflate, not only be able to better obtain the desired product and the restrictions on the reaction conditions are more flexible, such as reacting at room temperature. The calculated results show that it can provide theoretical support and help on the design of a new reaction. The second reaction is a multi-component synthesis of poly-substituted indolizine. The pyrindine derivative have received great attention as they widely occur as key structural subunits of manybioactive natural products, organic fluorescent materials, and pharmaceutical substances. Multi-component reactions (MCRs) have emerged as powerful and bond-forming efficient tools in organic, combinatorial, and medicinal chemistry. Actually, three-component reactions have been demonstrated as a straightforward approach to the synthesis of indolizines. Mao’s seminar began their investigations by treating pyridine and phenacyl bromide with ethyl glyoxalate through concise and efficient four-component tandem approaches to polysubstituted indolizine derivatives involving the formation of pyridinium ylides and α, β-unsaturated ketones with subsequent1,3-dipolar cycloaddition and aromatization reaction. All structures of reactants, transition states, intermediates and products are optimized in the M05/6-31+G (d, p) level. The investigation not only can reveal the reaction mechanism, but also can provide theoretical significance for the design of subsequent reactions.更多还原