Peptide stapling reactions represent powerful methods for structuring native α-helices to improve their bioactivity in targeting protein-protein inetractions (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 Fluorescent Isoindole Crosslink (FlICk). This methodology offers positional selectivity favouring 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 in vitro 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 with appreciable quantum yields that enable direct use for cell imaging in the assessment of cell permeability, thus bridging therapeutic potential with cytological probe development.

中文翻译：

---

未受保护的肽的化学选择性、区域选择性和位置选择性荧光吻合，用于细胞摄取和直接细胞成像


肽装订反应是构建天然 α-螺旋以提高其靶向蛋白质-蛋白质内酯 （PPI） 的生物活性的强大方法。鉴于对涉及未保护状态下天然氨基酸残基的区域和位置选择性吻合方法的需求不断增长，我们报道了一种快速、温和且高度化学选择性的三组分吻合反应，使用一类基于 2-芳基酮苯甲醛 （ArKBCHO） 的分子关键，产生荧光短纤维，以下简称荧光异吲哚交联 （FlICk）。该方法提供了位置选择性，有利于由赖氨酸和半胱氨酸组成的 i，i+4 螺旋主食合物，在未受保护的肽上存在竞争性亲核试剂。在我们进一步验证这种化学反应的努力中，我们已经成功地证明了 FlICk 肽 （IC50 = 5.10±1.27 μM） 的体外细胞毒性，与烯烃接合物同系物等效。在利用巯基-异吲哚的先天荧光时，我们报道了具有可观量子产率的新型蓝绿色荧光团，可以直接用于细胞成像以评估细胞通透性，从而将治疗潜力与细胞学探针开发联系起来。