Hexafluorobenzene undergoes 1,4‐selective thiol‐fluoride disubstitution and is an attractive disulfide crosslinking reagent for peptide cyclisation and stapling. Little attention has been directed toward understanding the scope of this reaction. Traditional reaction optimisation relies on a one‐variable‐at‐a‐time approach, which can exclude important combined effects of reaction variables. This study initially explored base and solvent effects to inform a subsequent two‐level factorial design approach to understand how to control the reactivity and product selectivity in a model reaction of hexafluorobenzene. We describe new conditions that selectively afford higher order substitution products for example, 1,2,4,5‐tetrasubstitution, making hexafluorobenzene a possible suitable scaffold for future branched or multicyclic peptide systems. Moreover, our new conditions provide an improved rapid (<1 minute) and selective peptide disulfide stapling and cyclisation approach under peptide‐compatible conditions.

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Polyfluorinated aromatic reagents readily react with thiolates via nucleophilic aromatic substitution (SNAr) and provide excellent scaffolds for peptide cyclisation. Here we report a robust and versatile platform for peptide stapling and multicyclisation templated by 5,10,15,20‐tetrakis(pentafluorophenyl)porphyrin, opening the door to the next generation of functional scaffolds for 3D peptide architectures. We demonstrate that stapling and multicyclisation occurs with a range of non‐protected peptides under peptide‐compatible conditions, exhibiting chemoselectivity and wide‐applicability. Peptides containing two cysteine residues are readily stapled, and the remaining perfluoroaryl groups permit the introduction of a second peptide in a modular fashion to access bicyclic peptides. Similarly, peptides with more than two cysteine residues can afford multicyclic products containing up to three peptide ‘loops’. Finally, we demonstrate that a porphyrin‐templated stapled peptide containing the Skin Penetrating and Cell Entering (SPACE) peptide affords a skin cell penetrating conjugate with intrinsic fluorescence.