SYNTHESIS AND PROPERTIES OF ORGANOCOBALT COMPLEXES

关于B12依赖者的重要未决问题 酶的过程是：什么是构象变化 其促进Co-C键的均裂断裂速率 辅酶B12的10-10倍，有利于均裂 比B12全酶的异裂作用更强 为什么是Co- N（DMBz）键长，DMBz在其中的作用是什么？ 过程？ 如果酶对重排的贡献 工艺（例如甲基丙二酰辅酶A到琥珀酰辅酶A）受到限制 为了确保长寿命底物自由基的存在，如何 这些酶阻止了B12的自由基结合？ 这些和 由于若干原因，其他问题没有得到充分回答。 B12依赖酶很大，使X射线和一些 光谱学研究困难。 辅因子通常紧密地位于 结合在探针无法接近的受保护酶口袋中 试剂 只有四种烷基钴卟啉的X射线结构， 已经报道了辅酶B12和甲基B12。 的分子 是复杂和独特的，并且光谱方法受到以下因素的限制： 这种复杂性和缺乏背景数据。 因此 研究充分的辅酶类似物的构象是未知的。 这种有限信息的一个后果是， 结构和光谱之间的清晰关系。 而且 仍然是一个不充分的背景，以评估立体的 导致Co-C键断裂的因素。 继续处理 这些问题，我们提出了一个三管齐下的办法。 详细 （i）烷基钴可啉，（ii）合成有机钴 物种，和（iii）核糖核苷酸还原酶（RR）。 烷基钴的可能构象范围为： 通过合成修饰的或空间应变的 烷基钴卟啉（钴卟啉、钴酰胺和其它侧链- 改性衍生物）和另外的有机钴化合物， 空间或电子改性的赤道配体， Cosaloph和Costa系统。 结构的影响 将通过X射线评估这些系统中引起的变化 晶体学，现代光谱方法（CD，2D和 HMQC NMR，拉曼）和通过反应速率和机理 研究（DMBz解离，配体交换）。 的应用 这些方法中的一些已经产生了丰富的信息， 在这段时间里提供了新的见解。 这些方法将 可以扩展到RR的研究。 RR相对较小， 松散结合可接近，B12位点和作用机制 与铁酶有关。 因此，RR具有特殊的优势， 意义 Co和Co之间的烷基键中的空间应变 辅酶类似物中的腺嘌呤也将变化， 类似物和辅酶B12与RR的相互作用将是 研究了 对于NMR研究，我们将使用钴酸盐 含有同位素富集位点。 光谱（CD，维斯， NMR（2D，HMQC）），待开发的背景通常将是 可用于其他B12全酶和B12转运的研究 系统.

Important unanswered questions concerning B12-dependent enzymic processes are: What are the conformational changes which promote the rate of homolytic cleavage of the Co-C bond of coenzyme B12 by a factor of 10-10 and which favor homolytic over heterolytic cleavage in B12 holoenzymes? Why is the Co- N(DMBz) bond so long and what is the role of DMBz in these processes? If the contribution of the enzyme to rearrangement processes (e.g. methylmalonyl-CoA to succinylCoA) is restricted to insuring the existence of long-lived substrate radicals, how do the enzymes prevent radical combination with B12? These and other questions have not been fully answered for several reasons. The B12-dependent enzymes are large, making X-ray and some spectroscopic studies difficult. The cofactor generally lies tightly bound in a protected enzyme pocket inaccessible to probe reagents. Only four alkylcobaltcorrin X-ray structures including coenzyme B12 and methyl B12 have been reported. The molecules are complex and unique and spectroscopic methods are limited by this complexity and by a lack of background data. Therefore, the conformations of well studied coenzyme analogues are unknown. One consequence of this limited information is that there are few clear relationships between structure and spectra. Also, there remains an inadequate background with which to assess the steric factors leading to Co-C bond cleavage. To continue to address these questions, we propose a three-pronged approach. Detailed studies of (i) alkylcobaltcorrins, (ii) synthetic organocobalt species, and (iii) ribonucleotide reductase (RR) are contemplated. The range of conformations possible for alkylcobalamins will be defined via the synthesis of modified or sterically strained alkylcobaltcorrins (cobalamins, cobinamides and other side chain- modified derivatives) and additional organocobalt compounds with sterically or electronically modified equatorial ligands based on the Cosaloph and "Costa" systems. The effect of structural changes induced in these systems will be assessed by X-ray crystallography, modern spectroscopic methods (CD, 2D and HMQC NMR, Raman) and by reaction rates and mechanistic studies (DMBz dissociation, ligand exchange). Application of some of these methods has produced a wealth of information and provided new insight during this grant period. These methods will be extended to the study of RR. RR is relatively small, with a loosely bound accessible, B12 site and a mechanism of action related to the Fe enzymes. Thus, RR has special advantages and significance. Steric strain in the alkyl linkage between Co and adenine in coenzyme analogues will also be varied and the interaction of the analogues and coenzyme B12 with RR will be investigated. For NMR studies, we will employ cobalamins containing isotopically enriched sites. The spectroscopic (CD, vis, NMR (2D, HMQC)), background to be developed will be generally useful in studies on other B12 holoenzymes and on B12 transport systems.