Amines constitute the major targets for the production of a plethora of chemical compounds that have applications in the pharmaceutical, agrochemical and bulk chemical industries. However, the asymmetric synthesis of α-chiral amines with elevated catalytic efficiency and atom economy is still a very challenging synthetic problem. Here, we investigated the biocatalytic reductive amination of carbonyl compounds employing a rising class of enzymes for amine synthesis: amine dehydrogenases (AmDHs). The three AmDHs from this study – operating in tandem with a formate dehydrogenase from Candida boidinii (Cb-FDH) for the recycling of the nicotinamide coenzyme – performed the efficient amination of a range of diverse aromatic and aliphatic ketones and aldehydes with up to quantitative conversion and elevated turnover numbers (TONs). Moreover, the reductive amination of prochiral ketones proceeded with perfect stereoselectivity, always affording the (R)-configured amines with more than 99% enantiomeric excess. The most suitable amine dehydrogenase, the optimised catalyst loading and the required reaction time were determined for each substrate. The biocatalytic reductive amination with this dual-enzyme system (AmDH–Cb-FDH) possesses elevated atom efficiency as it utilizes the ammonium formate buffer as the source of both nitrogen and reducing equivalents. Inorganic carbonate is the sole by-product.

In this study, two stereocomplementary ω-transaminases from Arthrobacter sp. (AsR-ωTA) and Chromobacterium violaceum (Cv-ωTA) were immobilized via iron cation affinity binding onto polymer-coated controlled porosity glass beads (EziG™). The immobilization procedure was studied with different types of carrier materials and immobilization buffers of varying compositions, concentrations, pHs and cofactor (PLP) concentrations. Notably, concentrations of PLP above 0.1 mM were correlated with a dramatic decrease of the immobilization yield. The highest catalytic activity, along with quantitative immobilization, was obtained in MOPS buffer (100mM, pH 8.0, PLP 0.1 mM, incubation time 2 h). Leaching of the immobilized enzyme was not observed within 3 days of incubation. EziG-immobilized AsR-ωTA and Cv-ωTA retained elevated activity when tested for the kinetic resolution of rac-α-methylbenzylamine (rac-α-MBA) in single batch experiments. Recycling studies demonstrated that immobilized EziG 3 -AsR-ωTA could be recycled for at least 16 consecutive cycles (15min per cycle) and always affording quantitative conversion (TON ca. 14,400). Finally, the kinetic resolution of rac-α-MBA with EziG 3 -AsR-ωTA was tested in a continuous flow packed-bed reactor (157 µL reactor volume), which produced more than 5 g of ( S )-α-MBA (> 49% conversion, > 99% ee) in 96 h with no detectable loss of catalytic activity. The calculated TON was more than 110,000 along with a space-time yield of 335 g L −1 h −1 .

The amination of alcohols is an important transformation in chemistry. The redox-neutral (i.e., hydrogen-borrowing) asymmetric amination of alcohols is enabled by the combination of an alcohol dehydrogenase (ADH) with an amine dehydrogenase (AmDH). In this work, we enhanced the efficiency of hydrogen-borrowing biocatalytic amination by co-immobilizing both dehydrogenases on controlled porosity glass FeIII ion-affinity beads. The recyclability of the dual-enzyme system was demonstrated (5 cycles) with total turnover numbers of >4000 and >1000 for ADH and AmDH, respectively. A set of (S)-configured alcohol substrates was aminated with up to 95% conversion and >99%ee (R). Preparative-scale amination of (S)-phenylpropan-2-ol resulted in 90% conversion and 80% yield of the product in 24 h.

Compared with biocatalysis in aqueous media, the use of enzymes in neat organic solvents enables increased solubility of hydrophobic substrates and can lead to more favorable thermodynamic equilibria, avoidance of possible hydrolytic side reactions and easier product recovery. ω-Transaminases from Arthrobacter sp. (AsRÀ ωTA) and Chromobacterium violaceum (CvÀ ωTA) were immobilized on controlled porosity glass metal-ion affinity beads (EziG) and applied in neat organic solvents for the amination of 1phenoxypropan-2-one with 2-propylamine. The reaction system was investigated in terms of type of carrier material, organic solvents and reaction temperature. Optimal conditions were found with more hydrophobic carrier materials and toluene as reaction solvent. The system's water activity (a w ) was controlled via salt hydrate pairs during both the biocatalyst immobilization step and the progress of the reaction in different nonpolar solvents. Notably, the two immobilized ωTAs displayed different optimal values of a w , namely 0.7 for EziG 3 À AsRÀ ωTA and 0.2 for EziG 3 À CvÀ ωTA. In general, high catalytic activity was observed in various organic solvents even when a high substrate concentration (450-550 mM) and only one equivalent of 2propylamine were applied. Under batch conditions, a chemical turnover (TTN) above 13000 was obtained over four subsequent reaction cycles with the same batch of EziG-immobilized ωTA. Finally, the applicability of the immobilized biocatalyst in neat organic solvents was further demonstrated in a continuous flow packedbed reactor. The flow reactor showed excellent performance without observable loss of enzymatic catalytic activity over several days of operation. In general, ca. 70% conversion was obtained in 72 hours using a 1.82 mL flow reactor and toluene as flow solvent, thus affording a space-time yield of 1.99 g L À 1 h À 1 . Conversion reached above 90% when the reaction was run up to 120 hours.