N-Alkyl amino acids are invaluable building blocks for ribosomally synthesized peptides because they can increase proteolytic stability and cell-permeability of peptides. However, previous studies showed that N-alkyl amino acids bearing an electrically charged side-chain cannot be directly incorporated into ribosomally synthesized peptides. Here we report a simple and robust method for ribosomal synthesis of peptides containing charged N-alkyl amino acids. In this method, an N-alkyl amino acid precursor, in which the charge of the translation-incompatible charged N-alkyl amino acid is masked, is ribosomally incorporated into a peptide. Subsequently, the precursor is post-translationally converted into a charged N-alkyl amino acid by bio-orthogonal chemical or enzymatic reaction. Using this method, we demonstrate the efficient incorporation of amine- or carboxylate-containing N-alkyl amino acids into ribosomally synthesized peptides and its compatibility with our recently developed in vitro transcription–translation coupled with association of puromycin-linker (TRAP) display. This study represents an important step toward in vitro display selection from structurally diverse N-alkyl-peptide libraries that will facilitate the discovery of proteolytically stable and cell-permeable peptidomimetics from libraries with high diversity.

Cyclic structures can increase the proteolytic stability and conformational rigidity of peptides, and N-alkylation of the peptide backbone can make peptides more cell-permeable and resistant to proteolysis. Therefore, cyclic N-alkyl amino acids are expected to be useful building blocks to increase simultaneously these pharmacological properties of peptides. In this study, we screened various cyclic N-alkyl amino acids for their ribosomal incorporation into peptides and identified cyclic N-alkyl amino acids that can be efficiently and successively incorporated. We also demonstrated genetic code reprogramming for reassigning 16 NNU codons to 16 different cyclic N-alkyl amino acids with high fidelity to synthesize highly N-alkylated polycyclic peptidomimetics and an mRNA-displayed library of completely N-alkylated polycyclic peptidomimetics by using our recently developed TRAP (transcription/translation coupled with association of puromycin linker) display. In vitro selection from a highly diverse library of such completely N-alkylated polycyclic peptidomimetics could become a powerful means to discover small-molecule ligands such as drug candidates that can be targeted to biomolecules inside living cells.

N-Alkyl amino acids are invaluable building blocks for ribosomally synthesized peptides because they can increase proteolytic stability and cell-permeability of peptides. However, previous studies showed that N-alkyl amino acids bearing an electrically charged side-chain cannot be directly incorporated into ribosomally synthesized peptides. Here we report a simple and robust method for ribosomal synthesis of peptides containing charged N-alkyl amino acids. In this method, an N-alkyl amino acid precursor, in which the charge of the translation-incompatible charged N-alkyl amino acid is masked, is ribosomally incorporated into a peptide. Subsequently, the precursor is post-translationally converted into a charged N-alkyl amino acid by bio-orthogonal chemical or enzymatic reaction. Using this method, we demonstrate the efficient incorporation of amine- or carboxylate-containing N-alkyl amino acids into ribosomally synthesized peptides and its compatibility with our recently developed in vitro transcription–translation coupled with association of puromycin-linker (TRAP) display. This study represents an important step toward in vitro display selection from structurally diverse N-alkyl-peptide libraries that will facilitate the discovery of proteolytically stable and cell-permeable peptidomimetics from libraries with high diversity.