Secondary structure engineering of an amphipathic arginine-rich CPP using non-proteinogenic amino acids for enhanced...

Cell-penetrating peptides (CPPs) represent a powerful class of drug delivery systems for transporting impermeable cargo into the intracellular space. While the B-peptide (Sequence: RXRRBRRXRRBRXB; B = β-alanine, X = 1-aminohexanoic acid) has shown significant potential for nucleic acid delivery, its inherent conformational flexibility-stemming from the long alkyl chains of Ahx (X)-often results in an unstable secondary structure that limits delivery efficiency. We hypothesized that rigidifying the B-peptide scaffold while maintaining its hydrophobic-cationic balance would yield superior delivery vehicles. In this study, we rationally designed and synthesized a series of B-peptide derivatives incorporating an α,α-disubstituted amino acid (Ac₅c) and a cyclic β-amino acid (ACPC) to induce helical stabilization. Circular dichroism (CD) analysis and computational modeling confirmed that the synergistic incorporation of Ac₅c and ACPC successfully transitioned the disordered B-peptide into a stable α/β-hybrid helical fold. The optimized derivative, FAM-8, demonstrated an approximately 7-fold enhancement in cell-membrane permeability compared to the original lead peptide. Furthermore, FAM-8 exhibited significantly improved proteolytic stability, retaining 40% integrity after 16 h of trypsin exposure, whereas the lead peptide was completely degraded. Fluorescence microscopy confirmed that these structural modifications maintained the endocytic internalization pathway while vastly improving uptake efficiency. Our findings provide a robust design principle for engineering structurally stabilized and metabolically resilient B-peptides, establishing a promising platform for the delivery of diverse therapeutic modalities.