Ribosomal Elongation of Trans-4-Aminocrotonic Acids into Nascent Peptide Chain

Expanding the structural diversity of ribosomally synthesized peptides is critical for the development of novel peptide therapeutics. Trans-4-aminocrotonic acid derivatives (Xcro), α,β-unsaturated nonproteinogenic γ-amino acids observed in marine natural products, impart peptides with enhanced lipophilicity, conformational rigidity, and protease resistance. Thus, these features would grant better pharmacological profiles to such peptides. However, the ribosomal incorporation of Xcro into nascent peptide chains has been considered challenging, based on the poor ribosomal compatibility of γ-aminoacyl-tRNA to undergo self-hydrolysis. Here, we report successful incorporation of Xcro using Xcro-tRNA^Pro1E2 prepared by flexizymes in the custom-made flexible <i>in vitro</i> translation (FIT) system. Our findings establish Xcro is a privileged γ-amino acid scaffold for expanding the chemical space of ribosomally encoded peptides, paving the way for the de novo discovery of peptide ligands with a unique structural backbone and post-translational modification functionality.