Enzymatic bromination of native peptides for late-stage structural diversification via Suzuki-Miyaura coupling

Flavin-dependent halogenases provide a biocatalytic approach for site-selective halogenation of aromatic compounds, but their use in late-stage functionalization of peptides has remained limited. Here, we show that the tryptophan (Trp) 7-halogenase RebH and an engineered variant (4V) originally optimized for larger small-molecule scaffolds can brominate peptidyl-Trp residues across a broad range of sequence and positional contexts. Through extensive analysis of diverse substrate sequences, we define features that enable RebH activity and reveal 4V’s expanded sequence tolerance. We applied 4V for enzymatic bromination of diverse bioactive peptide scaffolds, including an antimicrobial peptide, a cell-penetrating peptide, and a G protein-coupled receptor agonist, without the need for sequence modification. These brominated peptides served as substrates for Suzuki-Miyaura coupling, enabling installation of functional groups that conferred new functional properties or tuned the biological activity of these peptides. Our results expand the substrate landscape of FDHs and establish bromination-enabled cross-coupling as a general approach for late-stage diversification of bioactive peptides.