BiocatalyticReduction of Heterocyclic Imines in ContinuousFlow with Immobilized Enzymes

Heterocyclic amines are a key structural motif for the synthesis of pharmaceuticals (e.g., antibiotics) as well as pesticides and flavors. In this regard, imine reductases (IREDs) have recently emerged as a highly selective and sustainable alternative for asymmetric reductive amination reactions. Herein, we have applied six IREDs, two of which were newly identified, in the reduction of heterocyclic imines with either a <i>N</i>, <i>S</i>, or <i>O</i> substitution at C-4. Since IREDs are NADPH-dependent enzymes, a commercially available, supported glucose dehydrogenase was added as a cofactor-regenerating system. IREDs were then immobilized on porous microparticles to further improve the efficiency and sustainability of the system. The strategic combination of bioinformatic analysis and immobilization screening resulted in immobilized biocatalysts with 95% retained activity. This enabled the integration of the bienzymatic system into a continuous-flow reactor leading to >90% conversion of 50 mM of the <i>S</i>-heterocyclic amine, 5-methyl-3,6-dihydro-2<i>H</i>-1,4-thiazine, with a residence time of 30 min, and reaching space-time yields up to 14.3 g L^–1 h^–1. In addition, (<i>S</i>)- or (<i>R</i>)-stereoselectivity of the biocatalytic reduction of the 1,4-disubstituted heterocyclic imines was achieved by using the newly identified IREDs fromGoodfellowiella coeruleoviolaceaandLabilithrix luteola, respectively.