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In Vitro Double Oxidation of n ‐Heptane with Direct Cofactor Regeneration
Author(s) -
Müller Christina A.,
Akkapurathu Beneeta,
Winkler Till,
Staudt Svenja,
Hummel Werner,
Gröger Harald,
Schwaneberg Ulrich
Publication year - 2013
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201300143
Subject(s) - chemistry , cofactor , hydroxylation , alcohol dehydrogenase , cascade reaction , turnover number , monooxygenase , stereochemistry , enantiomeric excess , heptane , oxidizing agent , nad+ kinase , combinatorial chemistry , organic chemistry , enantioselective synthesis , alcohol , catalysis , enzyme , cytochrome p450
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.