Exploration of the Substrate Diversity of the Amine Transaminase 3FCR‐4M and its Engineered Variants

Chemoenzymatic Synthesis of Chiral Piperidines Using the Immobilized Transaminase ATA‐117 in Continuous Flow Reactors

Nowadays, there is a growing need for streamlined and sustainable strategies to access chiral nitrogen heterocycles, whose stereodefined frameworks underpin their broad functional and pharmaceutical relevance. Herein, we report a robust continuous flow enzymatic protocol for the stereoselective synthesis of the natural alkaloid (–)‐pinidinone and demonstrate its applicability to the preparation of a small family of structurally diverse chiral 2,6‐disubstituted piperidines. The strategy relies on the enantioselective transamination of tailor‐made α , β ‐unsaturated ketones catalyzed by an immobilized form of ATA‐117, which triggers a spontaneous intramolecular aza‐Michael reaction, enabling rapid access to chiral cis ‐(2 R ,6 R )‐piperidine scaffolds under mild conditions. The enzyme, successfully immobilized on Eupergit C, showed good operational and storage stability while a dual‐flow feed configuration minimized the formation of imine and Michael‐type by‐products observed when a single stock solution of α , β ‐unsaturated ketone acceptor and isopropylamine (IPA) amino donor was employed. The optimized conditions (20 mM substrate, 200 mM IPA, 60 min residence time, 30 °C) allowed for complete conversion and high stereoselectivity, while an integrated in‐line work‐up improved automation and reduced manual handling. This study establishes a robust biocatalytic flow platform for the asymmetric synthesis of valuable nitrogen‐containing heterocycles.

Enantiodivergent C3-Selective Reduction of Arylvinyl-Substituted 3,5-Dicarbonyl Esters Enabled by Engineered Ketoreductases

Chiral arylvinyl-substituted 3-hydroxy-5-oxo esters are valuable motifs in pharmaceuticals, yet their enantiocontrolled synthesis remains challenging. Herein, we report a biocatalytic system using ketoreductases (KREDs) for the highly C3-selective reduction of arylvinyl-substituted 3,5-dicarbonyl esters. Through semirational protein engineering, RasADH-E189D afforded the representative (S)-products in 89–99% conversion and 93–99% ee. Complementary (R)-products were obtained using KmCR2 and several other variants with up to 97% conversion and >99% ee, including a key intermediate for the synthesis of pitavastatin. Preparative-scale reactions (greater than 100 mg) further demonstrated the practicality of this platform. Protein–ligand docking among the substrate, cofactor, and KREDs provided mechanistic insights for great stereoselectivity.

A biocatalytic platform for the stereoselective synthesis of diverse atropisomeric compounds using engineered metalloenzymes

Atropisomeric compounds are widely distributed in natural products and pharmaceuticals and serve as key scaffolds in asymmetric catalysis. Despite their remarkable structural diversity, current synthetic efforts have primarily focused on C–C biaryl atropisomers, while the synthesis of other classes of atropisomeric compounds has been much less explored. Biocatalysis offers precise control over the stereoselectivity of reactions, however, its application in the synthesis of atropisomeric compounds remains at an early stage. In this work, we report a versatile biocatalytic platform that integrates metalloenzyme-catalyzed reduction reactions to access a broad array of structurally diverse atropisomeric compounds, including C–N atropisomers, diaryl ethers, and styrene derivatives, with high yields and excellent enantioselectivities. Molecular dynamics simulations provided mechanistic insights into the origin of the high stereoselectivity, and this study expands the repertoire of metalloenzyme-catalyzed, new-to-nature transformations for the synthesis of valuable molecules.

Biocatalytic Dynamic Kinetic Resolutions Enable Enantiodivergent Access to Axially Chiral Biaryl Dimethanols via Transient Seven-Membered Lactols

Axially chiral biaryl dimethanols are ubiquitous and multifunctional intermediates for the synthesis of valuable atropisomeric molecules in advanced materials, drugs, natural products, and organocatalysts. Despite their broad synthetic utility, direct enantiodivergent catalytic access to this specific class of compounds remains largely unexplored, particularly in biocatalysis. Herein, we report a biocatalytic platform for enantiodivergent and atroposelective dynamic kinetic carbonyl reduction via transient seven-membered cyclic lactol intermediates. These engineered alcohol dehydrogenase (ADH)-driven transformations deliver up to 99% yield and enantioselectivity (>99:1 e.r. and < 1:99 e.r). This strategy demonstrates broad substrate compatibility, extending even to complementary “flipped” substrate series. Furthermore, the protocol is readily scalable to gram quantities and accommodates diverse downstream derivatizations. Mechanistic experiments and theoretical calculations delineate the pathway and illuminate the origin of enzymatic stereocontrol.

Transaminases engineering and their cascade systems for natural product synthesis.

Reprogramming ω -Transaminase for Enantioselective Assembly of Axially Chiral Spiro[3.3]heptane Building Blocks

Axially chiral amines embedded in rigid spirocyclic frameworks are valuable motifs in medicinal chemistry, yet their synthesis by direct asymmetric amination of sterically congested ketones remains highly challenging. Here, we report an engineered ω-transaminase variant for the direct asymmetric amination of 2,6-disubstituted spiro[3.3]heptanones, providing access to enantioenriched axially chiral spiro amines under mild aqueous conditions. Starting from a weakly active wild-type ω-transaminase from Arthrobacter sp. KNK168 (ArTA), semi-rational engineering identified a double variant, ArTA-M2 (G136M/S223P), that substantially improved both catalytic activity and stereocontrol. This variant enables the asymmetric amination of a broad range of 2,6-disubstituted spiro[3.3]heptanone ketones with high conversions (up to 91%) and enantioselectivities (up to 99% ee). The preparative utility of ArTA-M2 was further demonstrated through the synthesis of pharmaceutically relevant building blocks and gram-scale whole-cell biotransformations. Molecular docking and molecular dynamics simulations suggest that the beneficial mutations reshape the binding pocket to promote a productive and stereodiscriminating binding mode. This work establishes a streamlined biocatalytic pathway to a challenging class of axially chiral building blocks.