【作者】 罗来斌；

【导师】 陈慧兰； 唐雯霞；

【作者基本信息】 南京大学 ， 无机化学， 1996， 博士

【摘要】 辅酶B₁₂作为辅因子参加生物体内多种酶反应，这些反应的共同特点是底物氢原子和邻近碳原子上的基团发生1，2位分子内交换。上述反应最关键的步骤之一是在酶诱导下咕啉环构型改变，从而发生的钴-碳键断裂产生脱氧腺苷自由基。研究表明当辅酶B₁₂和酶键合后钴-碳键断裂速度增加约10¹⁰倍。蛋白如何加速钴碳-键断裂?以及辅酶B₁₂参与的这种反应机理仍然不清楚。因此围绕辅酶B₁₂结构与功能的关系，大量的模型化合物被合成和研究。最近，研究人工辅酶B₁₂／酶模型成为关注的热点。本文开展了以下几个方面的工作： （1）作为辅酶B₁₂／酶模型的α环糊精／烷基钴肟超分子化合物的研究 环糊精是由n个D葡萄糖以α（1，4）糖苷键结合而形成的一类环状低聚糖（n=6，α，n=7，β，n=8，γ），具有疏水内腔，可包结各种底物，已用于人工酶模型的研究。我们合成了一系列新型α环糊精／烷基钴肟超分子化合物(α-CD／H₂OCo（DH）₂R，R=n-C₃H₇，i-C₄H₉，n-C₄H₉，n-C₅H₁₁)，其中环糊精作为主体，客体烷基钴肟是研究最深入的一类辅酶B₁₂模型。元素分析确定其组成为1∶1。一维核磁共振研究表明形成超分子后，α环糊精腔内的3，5位质子化学位移移向了高场，烷基钴肟上质子化学位移大部分移向了低场。在二维NOE核磁谱中，观察到烷基和α环糊精H5之间的联结信号，说明烷基插入了α-CD腔内，钴肟平面靠近α-CD大口端，二者形成了1∶1包结化合物。随包结物浓度的变化，化学位移的变化也发生了改变。据此我们测定了包结物形成常数。发现烷基碳链越长，插入腔内越深。因此，主客体分子之间的疏水和范德华力相互作用越大。我们测定了客体化合物H₂OCo（DH）₂n-C₃H₇和它的包结物的晶体结构。发现氢键也是形成稳定包结物的重要因素之一。比较这两个化合物的结构数据，我们可以看到形成包结物后，H₂OCo（DH）₂n-C₃H₇发生了一些结构和构象变化，即：（1）平面配体折叠角从1°增大为10°。（2）轴向Co-O（H₂O）键长增加。（3）Co周围的键角增大（水平）或减少（轴向）等。这些变化可能是由于正丙基和α环糊精空腔之间的疏水作用，以及α-CD和烷基钴肟平面配体的位阻相互作用引起的。该变化也十分类似于酶诱导的咕啉环平面的折叠以及咕啉环与5’-脱氧腺苷基的位阻相互作用。因而，此类超分子可能是一类新的辅酶B₁₂全酶更多还原

【Abstract】 Coenzyme B₁₂（5’-deoxyadenosylcobalamin, AdoCbl） serves as a cofactor for various enzymatic reactions. A common feature of these reactions is the 1,2-interchange of a hydrogen atom and another substituent on adjacent carbon atoms of the substrate. One of the key steps in those reactions is that the distortion of corrin ring of the cofactor is induced by enzymes, and then the cobalt-carbon bond is broken to produce a 5’-deoxyadenosyl radical. The rate of homolytic cleavage of the cobalt-carbon bond in the enzyme-bound cofactor is about 10¹⁰ times larger than in free cofactor. However, how the protein accelerates the Co-C bond cleavage and the mechanisms for the coenzyme B₁₂-dependent enzymatic reactions are still unclear now. Therefore, efforts have been made for investigation on a large number of coenzyme B₁₂ models encompassing the structure and function relationship for the coenzyme B₁₂. Recently, the special interests are paid for the artificial coenzyme B₁₂/enzyme models. In this thesis, the following themes that relate to the mechanism for coenzyme B₁₂-dependent rearrangements are discussed:（1）.Investigation on a-cyclodextrin/alkyl（aqua） cobaloxime inclusion complexes as a new kind of coenzyme B₁₂/enzyme modelsCyclodextrins（CDs）, a series of oligosaccharides consisting of six or more D-glucopyranose residues α -1,4-linked in a cyclic array, are one of the most fascinating naturally occurring molecular receptors. They have been taken as artificial enzyme models. In this work, a series of novel 1:1 inclusion complexes α-cyclodextrin/ alkyl（aqua）cobaloxime (α-CD/H₂OCo（DH）₂R, R = n-C₃H₇, i-C₄H₉, n-C₄H₉, n-C₅H₁₁), in which alkylcobaloxime is a kind of widely studied coenzyme B₁₂ modes, has been synthesized and characterized by elemental analysis. One-dimensional NMR investigation revealed that upfield shifts are induced for H-3, H-5 at α-CD and downfield shifts are induced for most hydrogens at alkylcobaloximes. Connectivity signal in two-dimensional NOE spectroscopy between alkyls and H-5 of α-CD have been observed. It suggested that alkyl groups were included into α-CD. According to that the chemical shifts for the inclusion complexes vary on changing the concentration of the 1:1 adduct, we have determined the formation constant of inclusion complexes. It was revealed that the longer the alkyl chain is, the deeper inclusion of the R group更多还原