Characterization of imine reductases in reductive amination for the exploration of structure-activity relationships

Abstract

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.

Enzymatic strategies for asymmetric synthesis

“…125 Overall, sequence-activity relationships with imine reductases are highly substrate dependent and no general rule could be delineated to anticipate the outcome of a particular reaction or from specific pairs of substrate/enzyme. 126 IREDs can be further creatively combined with chemical steps, as shown by combining enzymatic imine reduction with a base-mediated rearrangement to generate chiral 2,2-disubstituted azepanes and benzazepines. 127 An example of application of IREDs in industry was reported by GSK for the synthesis of a molecule currently in clinical trials for use in the treatment of leukemia.…”

“…Overall, sequence-activity relationships with imine reductases are highly substrate-dependent and no general rule could be delineated to anticipate the outcome of a particular reaction or from specific pairs of substrate/enzyme. 126 …”

Production of High Value Amine Intermediates via Biocatalytic Cascades in Continuous Flow

“…To further improve the productivity of this reaction a more suitable RedAm was selected from a metagenomics panel. 37 For the generation of N-butylcylohexlyamine 10, IR-79 was selected and immobilized on the EziG amber support (100 mg, 10 wt%), with 85% conversion to the secondary amine 10 observed and maintained for three hours at steady state (Table 2). This represented a total system residence time of 21 mins (STY: 1.87 g L -1 h -1 ), and only required two equivalents of butylamine 7 (20 mM butylamine and 10 mM ketone in RedAm module).…”

NAD(P)H‐Dependent Enzymes for Reductive Amination: Active Site Description and Carbonyl‐Containing Compound Spectrum

“…Further work from researchers at Johnson-Matthey reported a study of 95 IRED sequences (corresponding enzymes named here JM_IRXX) including homologs from bacteria, fungi and plants. [56] The study unearthed further enzymes, such as JM_IR23, with improved activity for the reductive amination of cyclohexanone 3 with only two equivalents of aniline e. Homology modelling led to the identification of residues potentially interacting with substrate/product. However, mutation work or in silico analysis (residues 170, 176, 180, 210 and 244, AspRedAm numbering) did not reveal critical determinants for activity toward specific substrates, except for a possible role of residue 244 in catalysis and selectivity following the slight increased conversion obtained with aniline e with the mutant F246L in JM_IR48.…”

“…Significant differences in other active‐site residues (177, 179, 180, 210, 240) prompted the hypothesis that some of these IREDs such as Pfizer_IR91 may catalyze the reductive amination reaction in a different way. Further work from researchers at Johnson‐Matthey reported a study of 95 IRED sequences (corresponding enzymes named here JM_IRXX) including homologs from bacteria, fungi and plants [56] . The study unearthed further enzymes, such as JM_IR23, with improved activity for the reductive amination of cyclohexanone 3 with only two equivalents of aniline e .…”