Modularization of Immobilized Multienzyme Cascades for Continuous‐Flow Enantioselective C−H Amination

Abstract Multienzyme cascades (MECs) have gained much attention in synthetic chemistry but remain far from being a reliable synthetic tool. Here we report a four‐enzyme cascade comprising a cofactor‐independent and a cofactor self‐sustaining bienzymatic modules for the enantioselective benzylic C−H amination of arylalkanes, a challenging transformation from bulk chemicals to high value‐added chiral amines. The two modules were subsequently optimized by enzyme co‐immobilization with microenvironmental tuning, and finally integrated in a gas‐liquid segmented flow system, resulting in simultaneous improvements in enzyme performance, mass transfer, system compatibility, and productivity. The flow system enabled continuous C−H amination of arylalkanes (up to 100 mM) utilizing the sole cofactor NADH (0.5 mM) in >90 % conversion, achieving a high space‐time yield (STY) of 3.6 g ⋅ L −1 ⋅ h −1 , which is a 90‐fold increase over the highest value previously reported.