Backbone Steric Constraints Underlie High Passive Membrane Permeability of <i>N</i> ‐Alkyl Peptides

Abstract

ABSTRACT 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 C α‐substituents, we isolated the backbone steric effects independently of amide hydrogen removal. N ‐Alkyl peptides bearing an N / C α‐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.