Side‐Chain Macrocyclization in Ahp‐Bicyclodepsipeptides Biosynthesis Involves Cytochrome P450‐Catalyzed Sequential Aromatic Hydroxylation and C─N Coupling

Abstract We report the biosynthesis of FR901277 ( 1 ) and delmomycin A2 ( 2 ), two 3‐amino‐6‐hydroxypiperidone (Ahp)‐containing bicyclodepsipeptides featuring an N‐C bridge linking the citrulline and tyrosine residues. This intriguing side‐chain macrocyclization is catalyzed by Dlm16, a cytochrome P450 monooxygenase (CYP450), through a sequential process initiated by ortho‐hydroxylation of the tyrosine ring, followed by intramolecular C─N coupling between the resulting catechol moiety and the terminal NH 2 of the ureido group. Structure‐function analyses and site‐directed mutagenesis confirmed the catalytic importance of identified key residues, enabling the proposal of plausible macrocyclization mechanisms. Functional characterization of eight additional Dlm16 homologs further revealed a CYP450 subfamily capable of catalyzing C─N bond formation, underscoring the prevalence of this unusual macrocyclization in cyclodepsipeptide biosynthesis. Our work highlights nature's strategies for macrocycle construction and provides another example of CYP450‐catalyzed C─N coupling via direct C─H functionalization.