Approaches to Asymmetric Oxidations of Nonpolar Reactants Catalyzed by Cyt P450 3A4 Baculosome Films in a CTAB Microemulsion

Cytochrome (cyt) P450 3A4 catalyzes regioselective- and enantioselective oxidations of diverse organic reactants, but its synthetic use is limited by the cost of enzyme and NADPH activation. Here, we compare alternative strategies for biocatalytic oxidations of water-insoluble reactants using cyt P450 3A4 baculosome film in CTAB microemulsions: (1) direct electrochemical activation of the LbL enzyme films on pyrolytic graphite electrodes and (2) H2O2-mediated activation of the LbL enzyme films on magnetic beads, with H2O2 supplied by O2 reduction using an electrocatalyst. Cyt P450 3A4 baculosome films were stable in the microemulsion, enabling oxidations by solubilizing nonpolar reactants insoluble in water. Both approaches drove asymmetric oxidations of reactants, including methyl phenylacetate, ethylbenzene, thiochroman-4-one, styrene, propionic acid, and 2-naphthol. Both strategies gave good stereoselectivity, but bead-based activation by electrocatalytically produced H2O2 provided slightly higher conversions and percentage of enantiomeric excess (% ee). Biocatalytic efficiency was consistently much better at 37 °C than at 25 °C, giving higher yields and % ee values. Results establish the first direct benchmark of electrode-driven vs peroxide-driven cyt P450 3A4 asymmetric catalysis by baculosome films in a microemulsion under identical conditions, revealing practical advantages for scalable asymmetric syntheses of nonpolar reactants.