Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/101469
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Type: Journal article
Title: Improving the monooxygenase activity and the regio- and stereoselectivity of terpenoid hydroxylation using ester directing groups
Author: Hall, E.
Sarkar, M.
Lee, J.
Munday, S.
Bell, S.
Citation: ACS Catalysis, 2016; 6(9):6306-6317
Publisher: ACS Publications
Issue Date: 2016
ISSN: 2155-5435
2155-5435
Statement of
Responsibility: 
Emma A. Hall, Raihan Sarkar, Joel H. Z. Lee, Samuel D. Munday, and Stephen G. Bell
Abstract: 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.
Keywords: Biocatalysis; norisoprenoids; terpenoids; esters; substrate engineering; directing groups; C−H bond oxidation
Rights: © 2016 American Chemical Society
DOI: 10.1021/acscatal.6b01882
Grant ID: http://purl.org/au-research/grants/arc/FT140100355
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