BioChem - DOS

In Vitro Double Oxidation of n-Heptane with Direct Cofactor Regeneration

Advanced synthesis & catalysis

Mü ller, Christina A.; Akkapurathu, Beneeta; Winkler, Till; Staudt, Svenja; Hummel, Werner; Grö ger, Harald; Schwaneberg, Ulrich

<P><B>Abstract</B></P><P>A novel concept for the direct oxidation of cycloalkanes to the corresponding cyclic ketones in a one&#8208;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 <I>n</I>&#8208;heptane to enable a biocatalytic resolution for the direct synthesis of heptanone and (<I>R</I>)&#8208;heptanols in a one&#8208;pot reaction. The bicatalytic cascade employs an NADH driven P450 BM3 monooxygenase variant (WT<SUP>NADH</SUP>, 19A12<SUP>NADH</SUP> or CM1<SUP>NADH</SUP>) and an (<I>S</I>)&#8208;enantioselective alcohol dehydrogenase (RE&#8208;ADH). In the initial step <I>n</I>&#8208;heptane is hydroxylated under consumption of NADH to produce (<I>R</I>/<I>S</I>)&#8208;heptanol. In the second oxidation step the (<I>S</I>)&#8208;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&#8197;min<SUP>&minus;1</SUP>, as well as high coupling efficiencies using NADH as cofactor (up to 44%). In the cascade reaction a nearly 2&#8208;fold improved product formation was achieved, compared to the single hydroxylation reaction. The total product concentration reached 1.1&#8197;mM, corresponding to a total turnover number (TTN) of 2500. Implementation of an additional cofactor regeneration system (<SMALL>D</SMALL>&#8208;glucose/glucose dehydrogenase) enabled a further enhancement in product formation with a total product concentration of 1.8&#8197;mM and a TTN of 3500.</P>

2013

1615-4150

1615-4169

355

9

pp.1787-1798

10.1002/adsc.201300143

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART66410498

alkanes; cascade reactions; C&#63743; H activation; directed evolution; enzymatic resolution; oxidation; P450 BM3;