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Diazirines have been traditionally associated with chemical biology and materials chemistry, but their utility has been limited in organic chemistry. Recently in the Journal of the American Chemical Society, Arnold and co-workers employed diazirines as precursors to hemoprotein-derived metal carbenoid species.

The asymmetric reduction of ketones to chiral hydroxyl compounds by alcohol dehydrogenases (ADHs) is an established strategy for the provision of valuable precursors for fine chemicals and pharmaceutics. However, most ADHs favor linear aliphatic and aromatic carbonyl compounds, and suitable biocatalysts with preference for cyclic ketones and diketones are still scarce. Among the few candidates, the alcohol dehydrogenase from Thauera aromatica (ThaADH) stands out with a high activity for the reduction of the cyclic α-diketone 1,2-cyclohexanedione to the corresponding α-hydroxy ketone. This study elucidates catalytic and structural features of the enzyme. ThaADH showed a remarkable thermal and pH stability as well as stability in the presence of polar solvents. A thorough description of the substrate scope combined with the resolution and description of the crystal structure, demonstrated a strong preference of ThaADH for cyclic α-substituted cyclohexanones, and indicated structural determinants responsible for the unique substrate acceptance.

The synthesis of secondary and tertiary amines through the reductive amination of carbonyl compounds is one of the most significant reactions in synthetic chemistry. Asymmetric reductive amination for the formation of chiral amines, which are required for the synthesis of pharmaceuticals and other bioactive molecules, is often achieved through transition metal catalysis, but biocatalytic methods of chiral amine production have also been a focus of interest owing to their selectivity and sustainability. The discovery of asymmetric reductive amination by imine reductase (IRED) and reductive aminase (RedAm) enzymes has served as the starting point for a new industrial approach to the production of chiral amines, leading from laboratory-scale milligram transformations to ton-scale reactions that are now described in the public domain. In this perspective we trace the development of the IRED-catalyzed reductive amination reaction from its discovery to its industrial application on kg-ton scale. In addition to surveying examples of the synthetic chemistry that has been achieved with the enzymes, the contribution of structure and protein engineering to the understanding of IRED-catalyzed reductive amination is described, and the consequent benefits for activity, selectivity and stability in the design of process suitable catalysts.

Native Amine Dehydrogenases (nat-AmDHs) have recently emerged as a potentially valuable new reservoir of enzymes for the sustainable and selective synthesis of chiral amines, catalyzing the NAD(P)H-dependent ammoniation of carbonyl compounds with high activity and selectivity. MATOUAmDH2, recently identified from the Marine Atlas of Tara Oceans Unigenes (MATOUv1) database of eukaryotic genes, displays exceptional catalytic performance against its best identified substrate, isobutyraldehyde, as well as broader substrate scope than other nat-AmDHs. In the interests of providing a platform for the rational engineering of this and other nat-AmDHs, we have determined the structure of MATOUAmDH2 in complex with NADP+ and also with the cofactor and cyclohexylamine. Monomers within the structure are representative of more open and closed conformations of the enzyme and illustrate the profound changes undergone by nat-AmDHs during the catalytic cycle. An alanine screen of active site residues revealed that M215A and L180A are more active than the wild-type enzyme for the amination of cyclohexanone with ammonia and methylamine respectively, the latter suggesting that AmDHs have the potential to be engineered for the improved production of secondary amines.

Small optically active molecules, and more particularly short-chain chiral amines, are key 20 compounds in the chemical industry and precursors of various pharmaceuticals. Their chemo-21 biocatalytic production on a commercial scale is already established, mainly through lipase-22 catalyzed resolutions leading to ChiProsTM products among others. Nevertheless, their 23 biocatalytic synthesis still remains challenging for very short-chain C4 to C5 amines due to low 24 enantiomeric excess. To complement the possibilities recently offered by transaminases, this 25 work describes alternative biocatalytic access using amine dehydrogenases (AmDHs). Without 26 any protein engineering, some of the already described wild-type AmDHs (CfusAmDH, 27 MsmeAmDH, MicroAmDH and MATOUAmDH2) were shown to be efficient for the synthesis 28 of hydroxylated or unfunctionalized small 2-aminoalkanes. Conversions up to 97.1% were 29 reached at 50 mM, and moderate to high enantioselectivities were obtained, especially for (S)-30 1-methoxypropan-2-amine (98.1%), (S)-3-aminobutan-1-ol (99.5%), (3S)-3-aminobutan-2-ol 31 (99.4%) and the small (S)-butan-2-amine (93.6%) with MsmeAmDH. Semi-preparative scale 32 up experiments were successfully performed at 150 mM substrate concentrations for the 33 synthesis of (S)-butan-2-amine and (S)-1-methoxypropan-2-amine, the latter known as “(S)-34 MOIPA”. Modelling studies provided some preliminary results explaining the basis for the 35 challenging discrimination between similarly sized substituents in the active sites of these 36 enzymes.

Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (R)-selective IRED from Myxococcus stipitatus (NADH-IRED-Ms) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (S)-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of NADH-IRED-Ms. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2- methylpyrroline compared to the wild-type and afforded the (S)- amine product with >99% conversion and 91% enantiomeric excess. We also report the crystal-structures of the NADPH-dependent (R)- IRED-Ms wild-type enzyme and the NADH-dependent NADH-IREDMs variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.