FtmOx1-catalyzed diverse C(sp3)-H oxyfunctionalization enables versatile late-stage diversification of phenethylamines and tetrahydroisoquinolines

The enzymatic late-stage functionalization of C(sp3)-H bonds has emerged as a transformative strategy in medicinal chemistry. Verruculogen synthase (FtmOx1) is known to catalyze the endoperoxidation of its native substrate, fumitremorgin B. Here, we demonstrate the catalytic versatility of FtmOx1 by showing its ability to mediate diverse C(sp3)-H transformations on non-native substrates—phenethylamine and tetrahydroisoquinoline derivatives (1) —including β-hydroxylation, β-ketone formation, dealkylation, and α,β-desaturation. A total of 48 structurally diverse substrates prove suitable for this method, substantially expanding the biocatalytic repertoire of FtmOx1. Mechanistic studies, combined with crystallographic and mutagenesis analyses, reveal the distinct molecular basis for this diverse C(sp3)-H oxyfunctionalization compared to native pathway. Notably, the relative flexibility of the phenethyl and benzyl chains of 1, assisted by specific substitutions that allow the two chains to swing, enables high β-selectivity for hydroxylation and prevents secondary oxidation. This C(sp3)-H activation platform efficiently generates structurally diverse oxyfunctionalized derivatives, including 1,2-amino alcohols (2), 1,2-amino ketones (3), and enamines (4), accelerating lead discovery for modulators of the human relaxin family peptide receptor 4 (RXFP4). Collectively, our work establishes FtmOx1 as a powerful biocatalytic tool, expanding the frontiers of C–H functionalization in drug discovery. Enzymatic late-stage functionalization of C(sp3)-H bonds is an important strategy in medicinal chemistry, and verruculogen synthase (FtmOx1) is an enzyme that catalyses endoperoxidation of its native substrate, fumitremorgin B. Here, the authors report the catalytic versatility of FtmOx1 in mediating diverse C(sp3)-H transformations of non-native substrates, including phenethylamines and tetrahydroisoquinolines, through β-hydroxylation, β-ketone formation, dealkylation, and α, β-desaturation.