Two-Step Biocatalytic Synthesis of (1S)-Nor(pseudo)ephedrine Catalyzed by Immobilized Enzymes

Nor(pseudo)ephedrines [N(P)Es] are naturally occurring compounds showing sympathomimetic activity that have many applications especially in the chemical industry and in the pharmaceutical field. Recently a two-step biocatalytic cascade for the preparation of (1S)-N(P)E was designed, consisting of a benzoin-type condensation catalyzed by the (S)-selective acetoin:dichlorophenolindophenol oxidoreductase (Ao : DCPIP OR) followed by a transamination mediated by either a (S)- or (R)-selective amine transaminase (ATA). In this study, we successfully immobilized both Ao : DCPIP OR and two ATAs of opposite enantioselectivity and used them in the biosynthetic cascade to N(P)Es. Immobilization yield, activity recovery, and stability of the immobilized enzymes, both after use (enzyme recycling) and storage (shelf-life), were assessed. Ao : DCPIP OR immobilized on glyoxyl-agarose exhibited remarkable stability under storage conditions throughout the 30-days monitoring period. Moreover, it was successfully reused in the synthesis of (S)-phenylacetylcarbinol [(S)-PAC] yielding high conversion (>94 %) and enantiomeric excess (>99 %). The (R)-selective At-ATA from Aspergillus terreus, immobilized on Eupergit (R) C, demonstrated a slightly higher storage stability when compared to the (S)-selective Sbv333-ATA from Streptomyces sp. Bv333 immobilized on the same carrier. Remarkably, both enzymes were effectively reused for ten reaction cycles in the synthesis of N(P)Es, starting from the enzymatically synthesized (S)-PAC, achieving complete conversions and excellent diastereoselectivity (>99 %).