Helicogenic Amino Acid Increases Passive Permeability and Oral Bioavailability of Macrocycles by Preorganizing γ-Turn

Macrocyclic peptides (MCPs) are promising drug candidates but often exhibit poor passive membrane permeability due to their polar backbone. This study demonstrates that incorporation of α-aminoisobutyric acid (Aib), a helicogenic residue, can enhance MCP permeability in a scaffold-dependent manner by enforcing γ-turns and promoting saddle-shaped conformations that stabilize intramolecular hydrogen bonds (IMHBs). Systematic Pro → Aib substitutions across several representative 6-8-residue scaffolds improved lipophilicity, reduced polar surface area, and minimized conformational rearrangements between aqueous and membrane environments, as confirmed by NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulations. However, Aib incorporation did not increase permeability in all scaffolds, particularly those that were already highly preorganized. Permeability improvements were most pronounced in flexible scaffolds, with up to 8-fold gains and increased oral bioavailability in mice, primarily through enhanced absorption. Mechanistically, Aib and aromatic residues contribute additively to hydrophobic patch formation, promoting membrane affinity. These findings establish Aib as a valuable design element for optimizing passive permeability and oral exposure of macrocyclic peptides.