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

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 NH₂ 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.