Helix stabilization by i,i + 7 amine-containing hydrocarbon Staples: Effects of length, stereochemistry, and orientation

We previously introduced the i,i + 4 amine-containing hydrocarbon (ACH) stapling system as a helix-stabilizing alternative to conventional all-hydrocarbon (AHC) staples, offering improved aqueous compatibility and chemical tunability. Here, we extend this approach to the i,i + 7 topology, which spans two helical turns and enables long-range conformational control. Systematic variation of cross-link length, stereochemistry, and orientation identified a 13-atom butylaminoalkenyl tether with SS configuration as the most effective helix-stabilizing i,i + 7 ACH staple. Notably, orientation reversal substantially enhanced helicity relative to the canonical arrangement, and this effect proved transferable across helical registers. Furthermore, the orientation-optimized staple not only reinforced α-helical conformation but also conferred significant proteolytic resistance, thereby revealing a direct link between structural preorganization and biochemical resilience. Taken together with earlier i,i + 4 variants, i,i + 7 ACH staples constitute a coherent design platform that expands the chemical and functional space of peptide stapling, with broad implications for structurally reinforced, therapeutically relevant α-helical peptides.