<i>In Vitro</i> Double Oxidation of <i>n</i>‐Heptane with Direct Cofactor Regeneration

Abstract

Abstract A novel concept for the direct oxidation of cycloalkanes to the corresponding cyclic ketones in a one‐pot synthesis in water with molecular oxygen as sole oxidizing agent was reported recently. Based on this concept we have developed a new strategy for the double oxidation of n ‐heptane to enable a biocatalytic resolution for the direct synthesis of heptanone and ( R )‐heptanols in a one‐pot reaction. The bicatalytic cascade employs an NADH driven P450 BM3 monooxygenase variant (WT NADH , 19A12 NADH or CM1 NADH ) and an ( S )‐enantioselective alcohol dehydrogenase (RE‐ADH). In the initial step n ‐heptane is hydroxylated under consumption of NADH to produce ( R / S )‐heptanol. In the second oxidation step the ( S )‐heptanol enantiomers are transformed to the corresponding ketones, reducing and thereby regenerating the cofactor. Characterization of initial hydroxylation step revealed high turnover frequencies (TOF) of up to 600 min −1 , as well as high coupling efficiencies using NADH as cofactor (up to 44%). In the cascade reaction a nearly 2‐fold improved product formation was achieved, compared to the single hydroxylation reaction. The total product concentration reached 1.1 mM, corresponding to a total turnover number (TTN) of 2500. Implementation of an additional cofactor regeneration system ( D ‐glucose/glucose dehydrogenase) enabled a further enhancement in product formation with a total product concentration of 1.8 mM and a TTN of 3500.