Daptomycin, an FDA-approved gram-positive antibiotic, is a 13-membered lipodepsipeptide macrocyclised through a depsipeptide bond between the Thr4 hydroxyl and alpha-carboxylic acid of Kyn13. Several syntheses of daptomycin have been reported, including our Boc solid-phase peptide synthesis (SPPS) that included on-resin reductive ozonolysis converting Trp13 to the requisite Kyn13.1 Here, we present a step efficient Fmoc SPPS adaptation, featuring a selective on-resin ozonolysis step. The Asp9 side-chain, anchored to high-acid labile resin, is elongated to the N-terminal decanoyl-capping before formation of the key depsipeptide bond between Thr4 and Fmoc-L-Trp-OH, further elongation, and macrolactamisation between Gly8 and Asp9. Finally, selective ozonolysis of Trp13 to Kyn13, in the presence of Trp1, and cleavage from the resin yields daptomycin. This approach reduces the number of synthetic steps and orthogonal protecting groups required.
Depsipeptide bond formation was optimised in a simplified system, and indicated reaction solvent is a key determinant of success. The location of the reactive hydroxyl within the peptide sequence, and its position relative to the resin, were the next most important considerations. Amino-acid stereochemistry and choice of side-chain protecting groups also impacted the success of depsipeptide formation.
The second key step, selective ozonolysis of Trp13, was first optimised in a model peptide. A reaction time of less than two minutes, including in situ reduction by pyridine,2 was needed for complete conversion to Kyn. A peptide containing the tertiary Boc-protected indole, Trp(Boc), exhibited lower conversion to Kyn after two minutes than the secondary indole of unprotected Trp. This observation was harnessed to achieve selectivity for reaction of Trp over Trp(Boc). The conversion was slowed to maximise selectivity by adding sacrificial reagent Fmoc-Trp(Boc)-OH to the reaction mixture, where 10 equiv proved optimal. This enabled selective ozonolysis of unprotected Trp13 to Kyn13, in the presence of a Boc-protected Trp at position 1, to access daptomycin.