Non-ribosomal peptide cyclase-directed chemoenzymatic synthesis of lariat lipopeptides

Lariat-shaped lipopeptides are important antimicrobial agents; however, their complex structures pose synthetic challenges that hamper efficient structural diversification. Here we report a new chemoenzymatic approach that facilitates access to lariat-shaped macrocycles. Unprotected, branched peptides bearing multiple nucleophiles, including a native amino terminus and a pseudo-amino terminus, were site-selectively cyclized using versatile non-ribosomal peptide cyclases, generating an array of lariat peptides with diverse sequences and ring sizes. The generality of this strategy was demonstrated using two penicillin-binding protein-type thioesterases, SurE and WolJ, as well as one type-I thioesterase, TycC thioesterase. Furthermore, the remaining nucleophile, which was not involved in the cyclization process, was exploited as a reactive handle for subsequent diversification via a site-selective acylation reaction (that is, Ser/Thr ligation). The tandem cyclization–acylation strategy enabled the one-pot, modular synthesis of lariat-shaped lipopeptides equipped with various acyl groups. Biological screening revealed that the site-selective acylation endowed the macrocyclic scaffolds with antimycobacterial activity and led to the identification of lipopeptides that inhibit 50% of growth at concentrations of 8–16 µg ml−1. Non-ribosomal peptide cyclases that catalyse head-to-tail macrocyclization are repurposed here to enable regioselective macrocyclization of branched peptides with multiple nucleophiles, affording lariat-shaped peptides with various sequences and ring sizes. Coupled with subsequent site-selective acylation chemistry, this chemoenzymatic approach facilitates modular access to structurally diverse lariat lipopeptides.