Biocatalytic Cascade for Nor(pseudo)ephedrine Synthesis: Enzyme Immobilization for Enhanced Efficiency and Scalability

Nor(pseudo)ephedrines (N(P)Es) are vicinal amino alcohols with sympathomimetic activity, widely utilized in pharmaceuticals. However, large-scale isolation of N(P)Es from natural sources is unfeasible, and existing chemical asymmetric syntheses are complex, requiring multi-step procedures that affect both yield and optical purity [1] . A two-step biocatalytic cascade for the synthesis of 1(S)-N(P)Es (Figure 1) and their analogues has recently been developed by some of the Authors. This approach combines an acyloin condensation catalyzed by the (S)-selective acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) from Bacillus licheniformis with a subsequent transamination mediated by an amine transaminase ((S)- or (R)-ATA)[2]. Nevertheless the use of free enzymes presents limitations in terms of recyclability and downstream processing, especially for large-scale applications. In this study, we successfully immobilized Ao:DCPIP OR and ATAs and integrated them into the biosynthetic cascade for N(P)E production. Immobilization studies were performed to investigate the immobilization efficiency, the activity recovery and the enzyme stability under operational and storage conditions. Finally, the two-step biotransformation was implemented on a semi-preparative scale, achieving moderate conversions and high optical purity for both (1S,2S)-NPE and (1S,2R)-NE[3] . [1] T. Sehl; Z. Maugeri; D. Rother, Journal of Molecular Catalysis B: Enzymatic 2015, 65-71. [2] N. Fracchiolla; S. Patti; F. Sangalli; D. Monti; F. Presini; P.P. Giovannini; F. Parmeggiani; E. Brenna; D. Tessaro; E. E. Ferrandi, ChemCatChem 2024, 16, e202301199. [3] S. Patti; I. Magrini Alunno; R. Semproli; D. Tessaro; D. Monti; S. Riva; D. Ubiali; E. E. Ferrandi, ChemCatChem 2024, e202400861.