Imine reductases (IREDs) have emerged as a valuable new set of biocatalysts for the asymmetric synthesis of optically active amines. The development of bioinformatics tools and searchable databases has led to the identification of a diverse range of new IRED biocatalysts that have been characterised and employed in different synthetic processes. This review describes the latest developments in the structural and mechanistic aspects of IREDs, together with synthetic applications of these enzymes, and identifies ongoing and future challenges in the field.

Reductive aminases (RedAms) catalyze the asymmetric reductive amination of ketones with primary amines to give secondary amine products. RedAms have great potential for the synthesis of bioactive chiral amines; however, insights into their mechanism are currently limited. Comparative studies on reductive amination of cyclohexanone with allylamine in the presence of RedAms, imine reductases (IREDs), or NaBH3CN support the distinctive activity of RedAms in catalyzing both imine formation and reduction in the reaction. Structures of AtRedAm from Aspergillus terreus, in complex with NADPH and ketone and amine substrates, along with kinetic analysis of active-site mutants, reveal modes of substrate binding, the basis for the specificity of RedAms for reduction of imines over ketones, and the importance of domain flexibility in bringing the reactive participants together for the reaction. This information is used to propose a mechanism for their action and also to expand the substrate specificity of RedAms using protein engineering.

The alkylation of amines with either alcohols or carboxylic acids represents a mild and safe alternative to the use of genotoxic alkyl halides and sulfonate esters. Here we report two complementary one-pot systems in which the reductive aminase (RedAm) from Aspergillus oryzae is combined with either (i) a 1° alcohol/alcohol oxidase (AO) or (ii) carboxylic acid/carboxylic acid reductase (CAR) to affect N-alkylation reactions. The application of both approaches has been exemplified with respect to substrate scope and also preparative scale synthesis. These new biocatalytic methods address issues facing alternative traditional synthetic protocols such as harsh conditions, overalkylation and complicated workup procedures.

Imine reductases (IREDs) have shown great potential as catalysts for the asymmetric synthesis of industrially relevant chiral amines, but a limited understanding of sequence activity relationships makes rational engineering challenging. Here, we describe the characterization of 80 putative and 15 previously described IREDs across 10 different transformations and confirm that reductive amination catalysis is not limited to any particular subgroup or sequence motif. Furthermore, we have identified another dehydrogenase subgroup with chemoselectivity for imine reduction. Enantioselectivities were determined for the reduction of the model substrate 2-phenylpiperideine, and the effect of changing the reaction conditions was also studied for the reductive aminations of 1-indanone, acetophenone, and 4-methoxyphenylacetone. We have performed sequence-structure analysis to help explain clusters in activity across a phylogenetic tree and to inform rational engineering, which, in one case, has conferred a change in chemoselectivity that had not been previously observed.

Fungal reductive aminases as effective biocatalysts for the preparation of chiral primary amines.