A simple approach to sense codon-templated synthesis of natural/unnatural hybrid peptides.

In the presence of Phe-SA, the stable sulfamoyl analogue of phenylalanyl adenylate, the codon (UUU/UUC) for phenylalanine (Phe) can be reassigned to naphthylalanine (Nap) bound to tRNA(Phe). The efficiency and selectivity of this Phe-to-Nap reassignment induced by the "orthogonal reacylation stalling" method was demonstrated at the single-codon level in the translation of mRNAs of dihydrofolate reductase (DHFR) and a 24-mer oligopeptide. In the prokaryotic translation system with essential preincubation, the endogenous precharged phenylalanyl-tRNA(Phe) undergoes deacylation and reacylation of the resulting tRNA(Phe) is inhibited by the action of Phe-SA to kill the phenylalanyl-tRNA synthetase activity. The significance of the present small-molecule-based approach to sense-codon templated natural-unnatural peptides is discussed.