Stapled peptides serve as a powerful tool for probing protein-protein interactions, but its application has been largely impeded by the limited cellular uptake. Here we report the discovery of a facile peptide macrocyclization and stapling strategy based on a fluorine thiol displacement reaction (FTDR), which renders a class of peptide analogues with enhanced stability, affinity, and cell permeability. This new approach enabled selective modification of the orthogonal fluoroacetamide side chains in unprotected peptides, with the identified 1,3-benzenedimethanethiol and 1,4-benzenedimethanethiol linkers promoting the alpha helicity of i, i+4 and i, i+7 stapled peptides respectively. The cellular uptake of these stapled peptides was universally enhanced compared to the classic ring-closing metathesis (RCM) stapled peptides. Pilot mechanism studies suggested that the uptake of FTDR-stapled peptides may involve multiple endocytosis pathways. Consistent with the improved cell permeability, the FTDR-stapled lead analogues demonstrated better inhibition of cancer cell growth than the RCM-stapled analogues. In addition, the gene reporter assays demonstrated FTDR analogues can inhibit Axin-betacatenine and p53-MDM2 interactions. In summary, our FTDR stapling approach may provide a novel class of cell permeable peptides to probe intracellular protein-protein interactions.

Alpha fluoroacetamide-based novel peptide stapling to probe intra-cellular protein-protein interactions

ACS Spring 2022

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