Chemoselective, regioselective, and positionally selective fluorogenic stapling of unprotected peptides for cellular uptake and direct cell imaging

Peptide stapling reactions represent powerful methods for structuring native α-helices to improve their bioactivity in targeting protein-protein interactions (PPIs). In light of a growing need for regio- and positionally selective stapling methods involving natural amino acid residues in their unprotected states, we report a rapid, mild, and highly chemoselective three-component stapling reation using a class of molecular linchpins based on 2-arylketobenzaldehydes (ArKBCHOs) that create a fluorescent staple, hereafter referred to as a Fluorescent Isoindole Crosslink (FlICk). This methodology offers positional selectivity favouring <i>i</i>, <i>i</i> + 4 helical staples comprising a lysine and cysteine, in the presence of competing nucleophiles on unprotected peptides. In our efforts to further validate this chemistry, we have successfully shown <i>in vitro</i> cytotoxicity of a FlICk-ed peptide (IC₅₀ = 5.10 ± 1.27 μM), equipotent to an olefin-stapled congener. In harnessing the innate fluorescence of the thiol-isoindole, we report new blue-green fluorophores, which arise as a consequence of stapling, with appreciable quantum yields that enable direct cellular imaging in the assessment of cell permeability, thus bridging therapeutic potential with cytological probe development.