基于非对称咪唑衍生物双核金属配合物的RNA人工酯酶研究

Investigations on the phosphate diester cleavage of RNA structure is of great significance for the understanding of the physiological activity of RNA, and the simulation of RNA nucleases is an vital route for exploring this mechanism. Based on our previous study concerning the transesterication of HPNP as classic RNA model mediated by two water-soluble mononuclear zinc(II) and cobalt(II) complexes with tridentate imidazole derivative (biap), the present project mainly focuses on the design of novel symmetric or unsymmetric hexadentate imidazole derivatives as potential ligands for the preparation of the relevant homodinuclear metal complexes. Furthermore, the reactivity of the as-prepared complexes will be evaluated towards the catalytic cleavage of varied RNA model substrates including HPNP as a classic model substrate..One highly important aspect is that investigating the distinguishable reactivity of metal complexes with unsymmetric ligands for comparing to their analogues with symmetric ones. Moreover, it will also be one of important surveys that studying the subject-object matching relationship between substrate and the designed complexes as well as the bimetallic synergetic effect in the catalytic pathway. In this case, this project also especially carries out the expand designs of RNA model substrates besides HPNP via the introducing of varied groups with different geometry, which is beneficial to the understanding of the cleaved mechanism of the phosphate ester bond in synthesized RNA models by analyzing the products and intermediates. Especially, effects of the introducing of unsymmetric gemini surfactants will be explored on the cleavage of RNA models. Effects of spacer length, chain-length ratio and rigid-flexible property of desired unsymmetric gemini surfactants will be evaluated on the micelle-catalytic cleavage of RNA models, which probably contribute the empirical evidence which is related to the introducing of micelles into the ester bond cleavage of RNA analogues.

对RNA磷酸酯键剪切的研究对认识RNA参与生理活动机制具有重大意义，RNA核酸酶的人工模拟是了解该机制的一种重要途径。基于水溶性三齿咪唑衍生物单核锌（II）、钴（II）配合物催化模型物HPNP酯转移研究，本课题拟设计新型含三齿咪唑衍生物单元的对称/非对称六齿配体及其双核金属配合物，考察其在非对称Gemini表面活性剂胶束体系中催化包含HPNP在内的RNA模型物酯键断裂的反应性。探索非对称与对称配体的双核金属配合物催化RNA模型物酯键剪切的差异性及结构所决定的反应性变化规律，阐述配合物与RNA模型物间主客体匹配关系以及双金属协同效应。经调变底物分子结构中2’-OH邻位基团物理体积大小实现RNA模型物的扩展设计，通过产物及中间体分析揭示不同RNA模型物酯键剪切机理。探索非对称Gemini表面活性剂链长比、联接基长短、刚柔性等对反应的影响趋势，丰富胶束引入RNA类似物磷酯键催化断裂反应的实证。

本课题利用便利的“一锅法”策略和分步法成功合成了不同联结基的对称和非对称六齿咪唑衍生物，经单晶解析了部分配体及双核金属配合物结构。对称咪唑衍生物双核金属配合物酯水解活性研究发现，因较大金属间距而未呈现协同效应。双核配合物活性仅为单核配合物活性的1.2-1.9倍，也证实了双金属协同的缺失。但加入H2O2，对称咪唑衍生物双核Cu(II)配合物催化HPNP剪切速率显著增加。柔性联结的双核铜(II)配合物表现出比刚性对苯二亚甲基联结的双核铜(II) 配合物更好的活性。非对称配体的配合物活性相比对称类似物并无明显活性提升。胶束影响发现非对称氧桥双核铜(II)配合物在CTAB胶束中表现出更好的酯转移活性，且其异双核CuFeL和同双核La2L、Fe2L配合物能较好地催化HPNP酯转移。.合成了带不同疏水链的三齿咪唑衍生物，发现其原位形成的单核镧(III)配合物能将HPNP酯转移速率提升3个数量级，活性比曾报道的亲水三齿单核Zn(II)配合物高出22至56倍。其单核铜(II)配合物亦有2个数量级的加速，β-环糊精的加入通过主客体作用加速了HPNP酯转移反应。竞争吸附实验显示，加入竞争试剂可一定程度上降低CTAB胶束中HPNP酯转移反应速度（降幅10~14%左右），其可能与竞争试剂在CTAB胶束界面区域排挤出原分布于此的HPNP分子而降低了HPNP局部浓度有关，以上结果为“三明治吸附模式”提供了证据。.合成了系列氨基酸（苏氨酸、丝氨酸等）还原Schiff 碱铜(II)配合物，发现它们可加速PNPP和PNPA水解分别达3个或2个数量级。相对非胶束体系，其在16-6-16及CTAB胶束体系中分别呈现约2-3倍和2-4倍的速率增加。在LSS和Brij35胶束中反应受到一定程度抑制。另外，发现配体中5-位取代基对配合物活性影响并不显著。还原Schiff碱铜(II)配合物比未还原类似物活性提升近10倍。

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