Site- and stereoselective enzymatic halogenation of terpenoids by substrate anchoring and protein engineering

Enzymatic halogenation of C–H bonds is a promising approach to synthesize chlorine-containing compounds because it enables the precise modification of complex molecules without extensive synthetic steps. However, few halogenases are known to chlorinate C(sp3)–H bonds of free-standing, complex molecules and a smaller subset have been shown to readily accommodate non-native substrates. Moreover, the competitive formation of oxygenated products makes the evolution of these halogenases for selective chlorination of non-native substrates a challenge. Herein, we report an anchoring-group strategy for the halogenation of unnatural substrates. Specifically, we show that various terpenoids can be enzymatically halogenated when connected to a removable indole moiety, which is present on the native substrates of WelO5*, a non-heme, iron-containing, alpha-ketoglutarate-dependent halogenase. By in-silico substrate evaluation, rational mutagenesis, and site-saturation mutagenesis, we have created WelO5* variants that catalyze the chlorination of C(sp3)–H bonds in a series of these non-native substrates with high selectivity for chlorination over oxygenation and high selectivity for chlorination at a single site with excellent stereoselectivity. Cleavage of the ester tethering the indole anchoring group to the terpenoid gives the free halogenated compound.