Unprotected Peptide Macrocyclization and Stapling via A Fluorine-Thiol Displacement Reaction

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 linker promoting alpha helicity of a variety of peptide substrates, as corroborated by molecular dynamics simulations. 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 Axin analogues demonstrated better inhibition of cancer cell growth than the RCM-stapled analogues. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/290379v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@103a978org.highwire.dtl.DTLVardef@16b1323org.highwire.dtl.DTLVardef@b565b6org.highwire.dtl.DTLVardef@128c989_HPS_FORMAT_FIGEXP M_FIG C_FIG