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, cellular uptake, and inhibition of cancer cells. This approach enabled selective modification of the orthogonal fluoroacetamide side chains in unprotected peptides in the presence of intrinsic cysteines. The identified benzenedimethanethiol linker greatly promoted the alpha helicity of a variety of peptide substrates, as corroborated by molecular dynamics simulations. The cellular uptake of benzenedimethanethiol stapled peptides appeared to be 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 in a distinct pattern in comparison to peptides stapled by RCM. Consistent with the improved cell permeability, the FTDR-stapled lead Axin and p53 peptide analogues demonstrated enhanced inhibition of cancer cells over the RCM-stapled analogues and the unstapled peptides.

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Global detection and identification of protein post-translational modification (PTM) is a major bottleneck due to its dynamic property and rather low abundance. Tremendous efforts have been since made to develop antibody-based immunoaffinity enrichment or bioorthogonal chemistry-based chemical reporter approach but both suffer from inherent limitations. Following our previously reported steric-free tagging strategy, we hereby report the invention of selenol as a new generation of fluorine-displacement probe. The fluorine-selenol based displacement reaction enabled us to efficiently label and image acetylation and glycosylation at cellular level. We further pursued FSeDR in tandem with SILAC based quantitative proteomics to globally profile acetylation substrate proteins in a representative prostate cancer cell line PC3. Our results unraveled the fluorine-based toolbox for powerful chemical biology probing and allow for the future study of PTMs in a systemic manner.