CYP101B1 from Novosphingobium aromaticivorans oxidises ionone derivatives and phenylcyclohexane with high activity and regioselectivity.

The monooxygenase enzyme CYP101B1, from Novosphingobium aromaticivorans DSM12444, binds norisoprenoids more tightly than monoterpenoids and oxidized these substrates with high regioselectivity. Ionols bound less tightly to CYP101B1 than ionones, but the levels of product formation remained high and the selectivity of oxidation was similar to that observed for the parent norisoprenoid. The structurally related sesquiterpene lactone (+)-sclareolide (9) was stereoselectively hydroxylated by CYP101B1 to (S)-(+)-3-hydroxysclareolide (9a). The turnover of monoterpenoid derivatives showed low levels of product formation and selectivity despite promising binding data. CYP101B1 catalyzed the selective oxidation of (1R)-(−)-nopol (14) and cis-jasmone (15), generating >90% (1R)-(−)-5-hydroxynopol (14a) and 4-hydroxy-cis-jasmone (15a), respectively. To develop strategies for the efficient and selective oxidation of monoterpenoid-based substrates using CYP101B1, we investigated the binding and catalytic properties of terpenoid acetates. The ester functional group of these substrates mimicked the carbonyl moiety of norisoprenoids and anchored the monoterpenoid acetates in the active site of CYP101B1 with high affinity for the monoterpenoid acetates. The oxidation of these substrates by CYP101B1 occurred with product formation rates in excess of 1000 min–1 and total turnover numbers of greater than 5000 being observed in all but one instance. Critically, the oxidations were regioselective, with several being stereoselective. (−)-Myrtenyl acetate (20) was oxidized regioselectively (>95%) to yield cis-4-hydroxy-myrtenyl acetate (20a), which was further oxidized to 4-oxomyrtenyl acetate (20b) using a whole-cell system, providing a biocatalytic route to generate intermediates used in the production of cannabinoid derivatives. The ester carbonyl moiety could also be used as a directing group also to enhance the activity and control the selectivity of P450-catalyzed reactions; for example, the turnover of l-(−)-bornyl acetate (18) and isobornyl acetate (19) by CYP101B1 generated 9-hydroxybornyl acetate (18a) and 5-exo-hydroxyisobornyl acetate (19a), respectively, as the sole products.

In vivo and In vitro hydroxylation of Cineole and Camphor by Cytochromes P450CYP101A1, CYP101B1 and N242A CYP176A1

Adamantane, 1‐ and 2‐adamantanol and 2‐adamantanone, were poor substrates for the cytochrome P450 enzyme CYP101B1. The CYP101B1 catalysed oxidation of 1‐adamantyl methyl ketone, and methyl 2‐(1‐adamantyl acetate), were more active generating a majority of the 4‐hydroxy metabolite. Substrate engineering using acetate and isobutyrate ester directing groups significantly increased the affinity, activity and coupling efficiency of CYP101B1 for the esters compared to the parent adamantanols, resulting in enhanced product formation rates (720 to 1350 nmol.(nmol‐P450)−1.min−1). The majority of the turnovers were selective for C−H bond hydroxylation with 4‐hydroxy‐1‐adamantyl isobutyrate and the 5‐hydroxy‐2‐adamantyl esters being generated as the sole majority product, 97 %, with high total turnover numbers, ranging from 4130 to 16500. In addition N‐(1‐adamantyl)acetamide, was oxidised by CYP101B1 whereas 1‐adamantylamine, was not. Whole‐cell biocatalytic reactions were used to generate the products in good yield. Overall the use of ester protecting groups and the modification of the amine to an amide enabled the more efficient and selective biocatalytic oxidation of adamantane frameworks.

Oxidation of polyaromatic hydrocarbons by P450s can be lowered by redox cycling but CYP101B1 regioselectively hydroxylated substituted naphthalenes and biphenyls.

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