Nature has evolved halogenase enzymes to regioselectively halogenate a diverse range of biosynthetic precursors, with the halogens introduced often having a profound effect on the biological activity of the resulting natural products. Synthetic endeavours to create non-natural bioactive small molecules for pharmaceutical and agrochemical applications have also arrived at a similar conclusion -halogens can dramatically improve the properties of organic molecules for selective modulation of biological targets in vivo. Consequently a high proportion of pharmaceuticals and agrochemicals on the market today possess halogens. Halogenated organic compounds are also common intermediates in synthesis and are particularly valuable in metal catalyzed cross-coupling reactions.Despite the potential utility of organohalogens, traditional non-enzymatic halogenation chemistry utilizes deleterious reagents and often lacks regiocontrol. Reliable, facile and cleaner methods for the regioselective halogenation of organic compounds are therefore essential in the development of economical and environmentally-friendly industrial processes. A potential avenue towards such methods is the use of halogenase enzymes, responsible for the biosynthesis of halogenated natural products, as biocatalysts. This review will discuss the advances towards this goal thus far, in addition to untapped potential sources of such biocatalysts and how further development of the enzymes may be focused in order to achieve the goal of industrial scale biohalogenation.

Targeted mutagenesis increases the activity and alters the regioselectivity of flavin-dependent halogenases.

Despite major recent advances in C–H activation, discrimination between two similar, unactivated C–H positions is beyond the scope of current chemocatalytic methods. Here we demonstrate that integration of regioselective halogenase enzymes with Pd-catalysed cross-coupling chemistry, in one-pot reactions, successfully addresses this problem for the indole heterocycle. The resultant ‘chemobio-transformation' delivers a range of functionally diverse arylated products that are impossible to access using separate enzymatic or chemocatalytic C–H activation, under mild, aqueous conditions. This use of different biocatalysts to select different C–H positions contrasts with the prevailing substrate-control approach to the area, and presents opportunities for new pathways in C–H activation chemistry. The issues of enzyme and transition metal compatibility are overcome through membrane compartmentalization, with the optimized process requiring no intermediate work-up or purification steps.

Flavin‐dependent halogenases are potentially useful biocatalysts for the regioselective halogenation of aromatic compounds. Haloaromatic compounds can be utilised in the synthesis and biosynthesis of pharmaceuticals and other valuable products. Here we report the first X‐ray crystal structure of a tryptophan 6‐halogenase (SttH), which enabled key residues that contribute to the regioselectivity in tryptophan halogenases to be identified. Structure‐guided mutagenesis resulted in a triple mutant (L460F/P461E/P462T) that exhibited a complete switch in regioselectivity; with the substrate 3‐indolepropionate 75 % 5‐chlorination was observed with the mutant in comparison to 90 % 6‐chlorination for the wild‐type SttH. This is the first clear example of how regiocomplementary halogenases can be created from a single parent enzyme. The biocatalytic repertoire of SttH was also expanded to include a range of indolic and non‐indolic substrates.