Backbone Steric Constraints Underlie High Passive Membrane Permeability of N-Alkyl Peptides.

N-Alkyl peptides have emerged as promising drug modalities, yet the structural determinants governing passive membrane permeability beyond amide hydrogen removal remain poorly understood. Here, we show that sterically constrained N-alkyl peptide backbones, generated by dual substitution at the amide nitrogen and the α-carbon, play a critical role in promoting passive membrane permeability. By directly comparing N-alkyl peptides with oligo(N-alkyl glycines) lacking Cα-substituents, we isolated the backbone steric effects independently of amide hydrogen removal. N-Alkyl peptides bearing an N/Cα-dually substituted backbone architecture consistently exhibited enhanced permeability across a broad range of lipophilicity and diverse sequences. Molecular dynamics simulations revealed two cooperative mechanisms: conformational restriction that favors less hydrated states and steric limitation of backbone hydration by β-carbons during membrane permeation. Together, these findings uncover a previously unrecognized structural basis for the high passive membrane permeability of N-alkyl peptides.