Development of cobalamin surrogates as probes and carriers through synthetic and chemical biology approaches

合成生物学具有通过代谢酶的组合重组来扩大天然产物化学库的潜力。虽然预计这种方法将使模块化构建的抗生素（如聚酮类）库升级，但也应考虑采用类似的方法修饰辅酶和辅因子。这种方法可以产生一种化合物，它可以作为强大的抑制剂，干扰特定的细胞过程。为此，我们概述了一种方法的修改钴胺素，维生素B12，这将导致合成一些类似物。这些类似物将通过重新设计钴胺素生物合成途径来构建，从而分离稳定的中间体。然后，这些将受到化学生物学方法的影响，其中具有扩展活性的辅因子将允许将新的官能团引入科林斯大环。最初，该项目的第一阶段将是制造钴胺素的金属同分异构体，其中中心的钴离子将被铑或铱取代。这将通过使用大肠杆菌的基因工程菌株来制造氢化硼酸a，c-二胺来实现。然后，替代金属将被非酶性地螯合成这种化合物。电化学方法将允许金属的腺苷化产生腺苷酸等价物。酶酰胺化将允许转化为腺苷酸对应物，它可以通过化学反应或通过使用合适的基因工程菌株的生物转化转化为最终的辅酶形式。为了补充这项工作，我们还将构建一些钴胺素变体，这些变体将通过酶组合方法或采用含有扩展官能团的s -腺苷甲硫氨酸SAM类似物产生。例如，通过解剖和重组酶，有可能使氢酸失去C5甲基或在C8位置上含有内酯。通过使用含有烯基、炔基和酮基取代基的SAM类似物，可以激活corrin大环的C5和C15位置。corrin分子的这种修饰将允许一系列化学品/药物的附着。所有的钴胺素替代物将被研究它们与钴胺素结合蛋白相互作用的能力以及被一系列细胞吸收的能力。它们对钴胺依赖酶的影响也将被研究，它们与钴胺素核开关相互作用的能力也将被研究。这样一项全面的研究将使我们能够确定这些分子作为抗菌药物和生物治疗药物的潜力。

Synthetic biology holds the potential to expand the chemical repertoire of natural products through a combinatorial reshuffling of metabolic enzymes. Whilst such an approach has been predicted to escalate the library of modular-constructed antibiotics, such as polyketides, a similar approach for the modification of coenzymes and cofactors should also be considered. This approach could produce compounds that act as powerful inhibitors that interfere with specific cellular processes. To this end we have outlined a method for the modification of cobalamin, vitamin B12, which will lead to the synthesis of a number of analogues. These analogues will be constructed through re-engineering the cobalamin biosynthetic pathway allowing the isolation of stabilised intermediates. These will then be subject to chemical biology approaches whereby cofactors with expanded activity will allow the introduction of new functional groups into the corrin macrocycle. Initially, the first phase of the project will be to make metal isosteres of cobalamin, whereby the central cobtalt ion will be replaced by either rhodium or iridium. This will be achieved by using genetically engineered strains of E. coli to make hydrogenobyrinic acid a,c-diamide. The alternative metal will then be chelated into this compound non-enzymatically. An electrochemical approach will allow the adenosylation of the metal to generate the adenosylcobyrinic acid equivalent. Enzymatic amidation will allow the transformation into the adenosylcobyric acid counterpart, which can be converted into the final coenzyme form by either a chemical reaction or through biotransformation using a suitable genetically engineered strain.To complement this work we will also construct a number of cobalamin variants that will be generated either through an enzymatic combinatorial approach or by employing S-adenosylmethionine SAM analogues containing extended functional groups. For instance, by dissecting and shuffling enzymes it is possible to make hydrogenobyrinic acid missing the C5 methyl group or containing a lactone on the C8 position. By using SAM analogues containing alkenyl, alkynyl and keto substituents it will be possible to activate the C5 and C15 positions of the corrin macrocycle. Such modification of the corrin molecule will allow for attachment of a range of chemicals/drugs.All the cobalamin surrogates will be investigated for their ability to interact with cobalamin binding proteins and to be taken up by a range of cells. Their effect on cobalamin-dependent enzymes will also be examined as will their ability to interact with cobalamin riboswitches. Such a comprehensive study will allow us to determine the potential of these molecules as anti-bacterial agents and biotherapeutics.