Work

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

Year: 2026

Type: article

Abstract: The enzymatic late-stage functionalization of C(sp 3 )-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 remarkable catalytic versatility of FtmOx1 in mediating diverse C(sp 3 )-H transformations of non-native substrates, including phenethylamines and tetrahydroisoquinolines, through β-hydroxylation, β-carbonylation, dehydrogenation, and α,β-desaturation—with β-C(sp 3 )-H oxyfunctionalization being the predominant pathway. Mechanistic studies, combined with crystallographic and mutagenesis analyses, reveal that non-native substrates adopt a distinct C(sp 3 )-H activation mode compared to the native pathway. Specifically, substrates 1t and 1w trigger selective β-C(sp 3 )-H activation by binding to three hydrophobic pockets, one of which (P3) is not utilized in the native catalytic cycle. The hydroxylated product state, FtmOx1•succinate• 2t , shows that the β-C-OH group moves away from the iron center after the first hydroxylation, preventing further oxidation. Notably, non-native substrates induce two conformations of residue Y224—flipping toward or away from the iron center—while mutagenesis studies indicate that Y224’s flexibility facilitates substrate binding but does not participate in hydrogen atom transfer (HAT). Additionally, residues L115 and F222, positioned near the β-C(sp 3 )-H, modulate the first hydroxylation and subsequent oxidation steps. This C(sp 3 )-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 RXFP4 modulators. Our work establishes FtmOx1 as a powerful biocatalytic tool, expanding the frontiers of C–H functionalization in drug discovery.

Source: Nature Communications

DOI: 10.1038/s41467-026-78405-z