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

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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.