Finally, to explore internal peptide 'stapling' using this strategy, we selected a region of a BH3 protein -BID80-102, that binds MCL-1 to regulate apoptosis. This PPI is known to play a significant role in cancer development and progression. Three sequences (55-57) were synthesised with alGly and Cys positioned to afford either an internal i,i+4 staple (58, 60; SI) or i,i+7 staple (59; SI), fixing the length of either one or two full helical turns of the peptide, respectively (Figure ). In addition, peptide macrocycle 26 is a head-to-tail macrocycle representing the i,i+4 binding region of these longer sequences. The staple for all three peptides was successfully formed under the standard conditions, albeit in lower yield than the N-to Cterminal cyclisations previously explored (58-60). The alpha-helical structure of peptide 60 was assessed via CD spectroscopy. While macrocyclisation did increase the helicity by a few percent compared to the native BH3 sequence (22% compared to 19%), the starting peptide carrying the allyl glycine residue had a relatively high helical content (38%, Figure ). This can be rationalised by considering strain on the macrocycle imposed by the linker, and the fact that the native residues glutamine (Gln) and Ser were switched for alGly and ORCID: Content not peer-reviewed by ChemRxiv. License: BY 4.0 Cys, respectively. These original residues have higher helical propensities relative to their replacements. Competitive inhibition studies (measured via fluorescence anisotropy) using MCL-1 and the fluorophore-labelled WT BH3 sequence demonstrated slightly lower inhibitory potency (26±4 mM) for 60 compared to BID-wt (7.4±0.9 mM) (Figure ).