Terpenes are a large and diverse class of plant metabolites which possess a variety of different industrial applications, such as antibacterial soaps and cosmetics, precursors of aroma and flavor chemicals, among others. Due to their structural diversity, their functionalization leads to a multitude of products that can be turned into building blocks for fine and bulk chemicals. In order to achieve highly desirable enantio- and regioselective products and circumvent the use of multiple synthetic procedures and harsh oxidative conditions, biological systems have proved to be versatile and mild catalysts in the chemoselective functionalization of a variety of small molecules, in addition to being flexible engineering platforms through site-directed mutagenesis. One such system is the hemoglobin from the marine worm Amphitrite ornata, named dehaloperoxidase (DHP), which acts as a catalytic globin in the oxidation of toxic metabolites found in A. ornata’s environment. DHP possesses a wide substrate scope, which includes native and non-native metabolites, such as halophenols, haloindoles, pyrroles, nitrophenols, guaiacols and cresols. These substrates are oxidized by DHP via one or more of its four known oxidative functions: peroxidase, peroxygenase, oxygenase and oxidase. Here, we examined seven monoterpenes as new possible substrates for DHP: R-(+)- and S-(-)-limonene, α- and γ-terpinene, terpinolene, ortho- and isothymol. Reactivity studies were performed using chromatographic and spectroscopic methods (GC-MS, LC-MS, HPLC, UV-Vis and stopped-flow UV-Vis), which together showed that DHP was able to oxidize the aforementioned substrates. Results show that α-terpinene is the most reactive substrate (93% conversion) and the limonene isomers the least reactive (~15% conversion). The oxidation of the limonene enantiomers yielded the cis- and trans-limonene oxide as the only products with different diastereomic ratios (cis:trans), (R-(+), 60:40) and (S-(-), 80:20). In addition, preliminary results using designed DHP mutants, e.g. Y38F and F21W, showed different effects on reactivity and enantioselectivity. Taken together, these results demonstrate that DHP can activate sp2-hybridized C-H bonds in the enantio- and regioselective oxidation of terpenes, thus making DHP a unique biocatalyst and protein engineering platform for the chemoenzymatic synthesis of relevant building blocks.

The multifunctional catalytic globin dehaloperoxidase as an unspecific peroxygenase in the oxidation of monoterpenes

ACS Spring Meeting 2021

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