The late-stage oxidative functionalisation of C-H bonds can be used to circumvent synthetic route re-design and to generate molecules not easily accessible via conventional methods. Such reactions can be problematic due to a lack of site-selectivity and the poor intrinsic reactivity of the C-H bond. Enzymes may provide a solution to both these issues, and enhancements in activity, selectivity, temperature stability and organic solvent tolerance over the wild-type enzyme are achievable by protein engineering. The stereoselective chlorination of unactivated hydrocarbons is challenging from a synthetic perspective, yet this type of reaction is performed in nature by enzymes of the iron and 2-oxoglutarate (2OG)-dependent halogenase family. By utilising a radical mechanism, these enzymes remove the requirement of existing chlorination biocatalysts for inherent substrate activation (in the form of aromatic or alkene functionalities), and demonstrate the potential for the evolution of new and interesting C-H functionalisation capabilities. Historically, enzymes from the iron and 2OG- dependent halogenase family were unsuitable for biocatalysis due to the necessity of a substrate-bound carrier protein for activity. Identified in 2014, WelO5 was the first enzyme from this family shown to be capable of carrier protein-independent chlorination. This thesis has evaluated the tractability of developing synthetically useful biocatalysts based on the 2OG-dependent halogenase WelO5, with the aim of adding new reaction capabilities to the synthetic toolbox of the future. To achieve this, WelO5 was produced in high yields and purity for characterisation and crystallisation trials. Three novel WelO5 crystal structures were determined, which aided the rational selection of active site residues for replacement. The natural substrate for WelO5, (+)-12-epi-fischerindole U isonitrile, was synthetically produced and used for the development of a screening assay for assessing WelO5 variant activity against substrate-like analogues. A total of 40 WelO5 variants were produced and tested for activity towards the natural substrate and structurally similar analogues. Variant I161A was found to introduce a new hydroxylation activity to the enzyme. Activity was also seen for WelO5 and variants against two structurally unrelated compounds. Overall, the generated variants of WelO5 have shown promise as biocatalysts, with the main limitation to further progress being the throughput of the available screening methods. Novel activities have been discovered which merit further investigation into small molecule 2OG-halogenases as biocatalysts and this thesis provides the tools with which to do so.