Stapled peptides provide an opportunity to target protein-protein interactions, that are otherwise considered ‘undruggable’, by rigidifying flexible peptides to improve binding specificity, in vivo half-life, and cell permeability. However, after optimising a constrained peptide, further modification is required to enable tracking within a biological environment. Here a new peptide modification is presented that defines helical secondary structure and is fluorescent, allowing it to be directly monitored by fluorescence microscopy.1 Mono- or dibromobimane is reacted with suitably positioned thiols in peptides to define secondary structure as a macrocycle; or in a sequence dependent manner as a side-chain decoration.2 We introduce the bimane moiety into a diverse range of peptides, by on-resin alkylation; or to an unprotected peptide in buffer. CD and NMR revealed 310-helical structure in an i-i+4 bimane constrained sequence known to bind Estrogen Receptor alpha (ERα) as an α-helix.2 In silico docking indicated the peptide adopts an α-helix on interaction with the ERα surface, suggesting bimane constraint still allows the peptide backbone flexibility. Additionally, 310-helical structure is stabilised in linear peptides, in a sequence dependent manner, when a bimane appended to a cysteine is six residues from a tryptophan.
We then compared the bimane constraint to four other common bis-alkylation linkers to stabilise a 310-helical turn in a p21-derived sequence that targets PCNA.3 The bimane-constrained p21 macrocycle was the most potent PCNA binder (KD=570 nM).4 X-ray crystallography and computational modelling studies revealed the bimane-constrained peptide was the only macrocycle to adopt the canonical 310-helical binding structure upon interaction with PCNA. Confocal microscopy of cells treated with this inherently fluorescent macrocycle revealed it was cell permeable, in contrast to a linear analogue. These studies demonstrate the fluorescent bimane modification as a powerful tool to influence peptide structure and enable concomitant imaging, thereby allowing therapeutically interesting molecules to be directly interrogated.