Site-, Stereo-, and Chemoselective Enzymatic Halogenation of Terpenoids by a Substrate Masquerade

Enzymatic halogenation of C–H bonds is a promising approach to synthesize chlorine-containing compounds. However, few halogenases chlorinate C(sp³)–H bonds of molecules lacking a carrier protein, and only a small subset readily accommodate non-native substrates. Competitive oxygenation of non-native substrates makes their halogenation a challenge to achieve. Herein, we report a strategy for the halogenation of unnatural substrates by which an anchoring group leads them to masquerade as the native substrate. By this approach, a series of terpenoids connected to an indole moiety, undergo enzymatic halogenation catalyzed by WelO5*, a non-heme, α-ketoglutarate-dependent halogenase. By in silico substrate evaluation and mutagenesis, we generated WelO5* variants that catalyze the chlorination of C(sp³)–H bonds in a series of non-native substrates with high selectivity for chlorination over oxygenation and with excellent stereoselectivity, as well as variants that catalyze bromination and azidation. Studies that vary the anchoring group showed that a series of heteroaromatic and aromatic groups can enhance reactivity and can influence the degree of chlorination of the anchored menthol substrate. Cleavage of the ester tethering the indole anchoring group to the terpenoid gives the free halogenated compound.