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α-Chiral amines are of great relevance in pharmaceutical manufacturing as they display a variety of biological activities. The enzymatic reductive amination of carbonyl-containing compounds using amine dehydrogenases (AmDHs) provides a sustainable synthetic alternative compared with the traditional synthetic chemistry procedures (e.g., elevated atom economy, mild reaction conditions). The diversity of substrate scope and reactivity among the known engineered AmDHs is, unfortunately, poor. In this work, via rational enzyme engineering, we created new AmDHs for the synthesis of (R)-configured amines from ketones; these enzymes offer several advantages compare with the previously generated AmDHs (i.e., complementary substrate scope, increased enantioselectivity and thermostability, reduced product inhibition). The applicability of these novel enzymes was further demonstrated in the kinetic resolution of racemic mixtures of amines by using a tandem AmDH NOx system, which yields pharmaceutically relevant (S)-configured amines. Furthermore, the scarce availability of (S) selective AmDHs motivated us to develop a high-throughput screening methodology that enables to detect the formation of (S) configured amines in aqueous solution. Notably, we have also shown that AmDHs are promiscuous enzymes that can enable the synthesis of secondary and tertiary amines in enantioenriched form using small aliphatic or cyclic aliphatic amines as amino donors. In contrast, AmDHs were previously supposed to accept only ammonia as amino donor. Through the combination of practical lab experiments and computational simulations, we gained new insights into the catalytic mechanism of these AmDHs. Finally, by controlling the conditions of the AmDH-catalyzed enzymatic transformation, we could obtain AmDHs capable of producing exclusively primary alcohols in high yields.

Asymmetric catalytic cascade processes offer direct access to complex chiral molecules from simple substrates and in a single step. In biocatalysis, cascades are generally designed by combining multiple enzymes, each catalyzing individual steps of a sequence. Herein, we report a different strategy for biocascades based on a single multifunctional enzyme that can promote multiple stereoselective steps of a domino process by mastering distinct catalytic mechanisms of substrate activation in a sequential way. Specifically, we have used an engineered 4-oxalocrotonate tautomerase (4-OT) enzyme with the ability to form both enamines and iminium ions and combine their mechanisms of catalysis in a complex sequence. This approach allowed us to activate aldehydes and enals toward the synthesis of enantiopure cyclohexene carbaldehydes. The multifunctional 4-OT enzymes could promote both a two-component reaction and a triple cascade characterized by different mechanisms and activation sequences.