Synthesis and characterisation of zincobalamin the zinc analogue of vitamin B12

Cobalamin or vitamin B12 is one of the most complex small molecules produced by prokaryotic organisms. B12 is a member of the metabolically diverse group of compounds called tetrapyrroles, whose members include haem, chlorophyll, sirohaem and co-enzyme F430. The metabolic activity of B12 is dependent upon the cobalt ion located at the centre of a contracted corrin macrocycle. Replacement of this central cobalt ion with an alternative metal ion results in the formation of a compound that structurally resembles B12 but shares none of its vitamin function. Zincobalamin (Zbl), the zinc analogue of B12, was synthesised from an intermediate of B12 biosynthesis, hydrogenobyrinic acid a, c diamide (HBAd). This intermediate is the last metal-free intermediate of B12 biosynthesis. The synthesis of Zbl from HBAd involves a combination of chemical synthesis and biochemical modification of the starting material. Regiospecific amidation of 4 out the 5 carboxylic acid sidechains decorating the macrocycle of HBAd is required in order to produce Zbl. During B12 biosynthesis these amidations are catalysed by a single enzyme CobQ. Previously characterised CobQs are highly specific for a cobalt containing intermediate of B12 biosynthesis. A CobQ capable of acting "out of turn" with respect to the normal order of B12 biosynthesis was identified within the genome of Allochromatium vinosum. This enzyme was used to good effect in the conversion of HBAd into hexa-amidated compound called hydrogenobyric acid (Hby), the metal free analogue of a late intermediate of B12 biosynthesis cobyric acid. A structural reason as to why A. vinousm CobQ is able to recognise HBAd as a substrate was investigated through sequence alignment and structural prediction. The remaining steps towards Zbl synthesis were completed chemically through the abiotic insertion of zinc into the macrocycle of Hby and the attachment of the pre-fabricated lower nucleotide loop. As both Hby and its zinc containing counterpart have not been previously described in the literature all three compounds including Zbl were subject to biochemical characterisation by UV-visible, NMR and mass spectroscopy. The influence of these analogues on the growth of a strain of Salmonella enterica dependent on exogenous B12 for growth, was investigated.