Stapled peptides have received increasing attention as potential therapeutic agents. Stapling can improve essential properties like selectivity, binding affinity, and bioavailability. The current work involves the stapling of peptides through a novel two-component peptide stapling strategy, capitalising on a staple that contains two α-bromoketone moieties and peptides with two 1,2-aminothiol functional groups.
Previous work in the Nitsche group has shown that 1,2-aminothiols can be selectively introduced into peptides for biocompatible peptide stapling using 2,6-dicyanopyridine.1 The current work expands this principle towards a new range of staples that selectively form six-membered dihydrothiazine rings. The reaction combines an S-alkylation and imine formation to form the dihydrothiazine ring at each end of the staple, stapling the peptide. Stapling occurs in aqueous solution at pH 7.5 and typically completes in less than five minutes. We have demonstrated this stapling approach using a variety of staples and peptide substrates, providing a versatile toolbox for constraining biologically active peptides.
Two different strategies for the introduction of the 1,2-aminothiol motif into peptides have been investigated. The motif can either be resembled by canonical N-terminal cysteine in the peptide main chain or introduced in the side chain as a “pseudo-cysteine” residue with varying linker length.1 The latter can be incorporated either as fully protected amino-acid building block or directly assembled on the solid support during standard Fmoc solid-phase peptide synthesis.
This approach is fully compatible with standard Fmoc solid-phase peptide synthesis and the stapled peptide can be isolated by HPLC purification. The new stapling approach described in this study has proven to be a remarkably simple and effective tool for rapid peptide stapling with obvious future applications in peptide-based drug design.