Continuous Flow Bioamination of Ketones in Organic Solvents at Controlled Water Activity using Immobilized ω‐Transaminases

Abstract

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.

Is it time for biocatalysis in fragment-based drug discovery?

“…66,67 The use of ow chemistry for biocatalysis continues to offer solutions to problems that have proven long-standing and hard to overcome, such as enzyme stability with immobilization, 68 improving the kinetics of an enzymatic reaction by novel reactor designs, 69 and the easier integration of organic solvents. 70 Other technological solutions, including the use of non-aqueous medias such as deep eutectic solvents (DESs), 71 or membrane separation for reaction compartmentalization, 72 provide a glimpse as to some of the potential ways biocatalysts could be applied to circumvent issues with their application.…”

Parallel Interconnected Kinetic Asymmetric Transformation (PIKAT) with an Immobilized ω-Transaminase in Neat Organic Solvent

“…Toluene was used as the reaction solvent, and the water activity (a w ) was controlled using a hydrate salt pair (i.e., Na 2 HPO 3 •5H 2 O/Na 2 HPO 4 •7H 2 O, 1:1 w w −1 ), as reported in our previous study [35]. AsR-ωTA-a recombinant (R)-selective enzyme originated from Arthrobacter sp.-was immobilized on EziG 3 (Fe Amber) controlled porosity glass beads and prepared for reactions in organic solvent, as previously reported [35,51]. The reactions were performed in dark glass vials (2 mL) with screw caps and shaken in a thermomixer at 25 • C.…”

“…As mentioned above, recurrent challenges in ωTA-catalyzed reactions using the "classical" alanine (or another α-amino acid) as the sacrificial amine donor in an aqueous environment are unfavorable reaction equilibrium and frequent enzyme inhibition by the amine product and pyruvate co-product [11,[25][26][27][28][29][30][31][32]. However, our group and others have demonstrated that these limitations can be addressed (or at least mitigated) by performing the transamination reaction in a neat organic solvent at controlled water activity (a w ) and using inexpensive 2-propylamine as the amine donor [33][34][35]. Biocatalyst recyclability and long-time stability under process conditions are also critical factors for the successful implementation of enzymes such as ω-TAs in industry, particularly in continuous flow processes [36].…”

“…Our group recently reported a highly efficient kinetic resolution of a racemic α-chiral amine in both batch and continuous flow using ω-TAs immobilized on polymer-coated controlled porosity glass beads (EziG) [51]. Notably, the same immobilized EziG 3 -ωTA biocatalyst also led to excellent activities for the asymmetric synthesis of α-chiral amines from prochiral ketones in neat organic solvents when the system's water activity (a w ) was controlled with hydrate salt pairs [35]. In particular, we obtained conversions of 80% or up to 96% for the asymmetric synthesis of (R)-phenoxy-2-propylamine from phenoxyacetone by using only one or two equivalents of 2-propylamine as the amine donor, respectively.…”

“…(EziG 3 -AsR-ωTA) in a neat organic solvent. The immobilization of AsR-ωTA on EziG controlled porosity glass beads was previously investigated and exhibited high retention of enzyme activity and excellent stability in both aqueous and organic media [35,51]. Herein, the same biocatalyst preparation was employed in the organic solvent for the one-pot asymmetric formal reductive amination of a prochiral ketone combined with an anti-parallel kinetic resolution of a racemic α-chiral primary amine (Scheme 1).…”