3-Furylalanine (Fua) is a reactive amino acid that occurs naturally in a limited number of cyclopeptides. For example, hepatotoxic rhizonins are produced by bacterial endosymbionts of Rhizopus microsporus,^[1] and their toxicity critically depends on the Fua residues.^[2] Fua moieties are also found in endolides, where the presence of Fua moieties confers receptor binding specificity.^[3] Beyond, furyl-substituted peptides are amenable to bioorthogonal Diels-Alder-type derivatization.

Despite its relevance, Fua biosynthesis and incorporation by nonribosomal peptide (NRP) synthetases has remained elusive. By genome sequencing and gene inactivation, we discovered the gene cluster responsible for rhizonin biosynthesis. Mutational analysis and heterologous reconstitution identified the dioxygenase RhzB as a Fua synthase, and isotope labeling pinpointed tyrosine and L-DOPA as Fua precursors.^[4] Interestingly, RhzB-like Fua synthases form a novel clade of heme-dependent aromatic oxygenases and are encoded in the genomes of phylogenetically distant bacterial phyla.

The prevalence of cryptic Fua-containing NRPs motivated us to devise a strategy to rapidly access novel clickable Fua-harboring bioactives. First, we screened various RhzB orthologs for Fua formation in a recombinant strain. After treating the native hosts" culture extracts with a dienophilic maleimide, LC-MS/MS analysis identified Diels-Alder-derivatized Fua-carrying compounds. This way, we discovered two groups of strongly antiproliferative Fua-containing cyclopeptides from Actinobacteria. We translated this approach to a tailor-made maleimide resin that allows the solid-support-based purification of Fua-harboring peptides.

The presented work provides insight into the biosynthesis and incorporation of the Fua pharmacophore, which permits bioorthogonal labelling, and shows the power of combining genome mining with reactivity-based screening to discover novel antiproliferative cyclopeptides.

[1] L. P. Partida-Martinez, C. F. D. Looß, K. Ishida, M. Ishida, M. Roth, K. Buder, C. Hertweck, Appl. Environ. Microbiol. 2007, 73, 793–797.

[2] K. Shimokawa, K. Yamada, D. Uemura, Bioorg. Med. Chem. Lett. 2009, 19, 867–869.

[3] C. Almeida, F. E. Maddah, S. Kehraus, G. Schnakenburg, G. M. König, Org. Lett. 2016, 18, 528–531.

[4] F. J. Ehinger, S. P. Niehs, B. Dose, M. Dell, J. Krabbe, S. J. Pidot, T. P. Stinear, K. Scherlach, C. Ross, G. Lackner, C. Hertweck, Angew. Chem. Int. Ed. 2023, e202308540.