The reductive aminase from Aspergillus oryzae (AspRedAm) was combined with a single alcohol dehydrogenase (either metagenomic ADH-150, an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-neutral cascade for the biocatalytic alkylation of amines using primary and secondary alcohols. Aliphatic and aromatic secondary amines were obtained in up to 99 % conversion, as well as chiral amines directly from the racemic alcohol precursors in up to >97 % ee, releasing water as the only byproduct.

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.

Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthesis of various cyclic and acyclic amines. Herein a number of guidelines and considerations toward the development and scale-up of IRED catalyzed reactions have been determined based on the reductive amination of cyclohexanone (1) with cyclopropylamine (2). A Design of Experiments (DoE) strategy has been followed to study the different reaction parameters, facilitating resourceefficient and informative screening. Enzyme stability was identified to be the limiting factor. By moving from batch to fed-batch, it was possible to double the concentration of the substrate and turnover number (TON). Kinetic studies revealed that IRED-33 was the best enzyme for the reaction with respect to both activity and stability. Under the optimal reaction conditions, it was possible to react 1 and 2 at 750 mM concentration and reach 100% conversion to the desired amine (>90% isolated yield) in the space of 8 h. Hence, excellent volumetric productivity of 12.9 g L −1 h −1 and TON above 48 000 were achieved.

Reaction of the C2-symmetric "Trost modular ligand" with cationic Pd(II) allyl fragments allows isolation of air- and bench-stable pro-catalysts for the asymmetric allylic alkylation of racemic cycloalkenyl esters. In solution, three distinct complexation modes are observed. When mixed in a ligand/Pd ratio of 1/2, a binuclear bis-P,O-chelate complex is generated. This species does not induce enantioselectivity in the reaction. In contrast, with a ligand/Pd ratio of 1/1, a highly enantioselective, P,P-coordinated pro-catalyst system is generated in which there are two basic coordination modes: monomeric and oligomeric. The monomeric form is mononuclear and exists as two 13-membered chelates, isomeric through loss of C2-symmetry in the ligand. The oligomeric form is polynuclear and forms chains and rings of alternating ligand and cationic Pd(allyl) units, one of which was identified by single-crystal X-ray diffraction. In solution, the monomeric and oligomeric species are in dynamic equilibrium with populations and interconversion rates controlled by concentration, temperature, and counterion. Isotopic desymmetrization analysis suggests that the monomer-oligomer equilibrium plays a crucial role in both the selectivity and efficiency of the asymmetric allylic alkylation reaction.

The identification and 3D structural characterization of a homolog of the (R)-selective transaminase (RTA) from Aspergillus terreus (AtRTA), from the thermotolerant fungus Thermomyces stellatus (TsRTA) is here reported. The thermostability of TsRTA (40% retained activity after 7 days at 40 • C) was initially attributed to its tetrameric form in solution, however subsequent studies of AtRTA revealed it also exists predominantly as a tetramer yet, at 40 • C, it is inactivated within 48 h. The engineering of a cysteine residue to promote disulfide bond formation across the dimer-dimer interface stabilized both enzymes, with TsRTA_G205C retaining almost full activity after incubation at 50 • C for 7 days. Thus, the role of this mutation was elucidated and the importance of stabilizing the tetramer for overall stability of RTAs is highlighted. TsRTA accepts the common amine donors (R)-methylbenzylamine, isopropylamine, and D-alanine as well as aromatic and aliphatic ketones and aldehydes.