Engineering Protease-Resistant Peptides via Non-Canonical Amino Acids: Design Strategies and Biosynthetic Advances

Peptide therapeutics offer high target selectivity and low toxicity, but their clinical utility remains constrained by rapid proteolysis in vivo and negligible oral bioavailability. Incorporating non-canonical amino acids (ncAAs) provides a robust molecular engineering framework to overcome these pharmacokinetic bottlenecks. This review analyzes the structural and biophysical design rules of ncAA-mediated peptide stabilization, categorizing them into side-chain steric shielding, backbone conformational constraint, and stereochemical evasion of L-specific proteases. We systematically evaluate the biosynthetic milestones enabling this field, focusing on engineered orthogonal translation systems (tRNA/synthetase pairs, orthogonal ribosomes, quadruplet codons) and metabolic engineering strategies that supply fluorinated and other ncAA precursors de novo. Furthermore, we examine the translation of these technologies into clinical candidates (e.g., modified antimicrobial peptides, antibody–drug conjugates, and PROTACs) and identify scaling, immunogenicity, and computational modeling as key bottlenecks. This review serves as a technical reference for designing next-generation, hyper-stable peptide therapeutics.