Ribosome-mediated incorporation of hydrazinophenylalanine into modified peptide and protein analogues

(S)-α-Hydrazinophenylalanyl-tRNAPhe, an aminoacyl-tRNA derivative containing the unnatural amino acid (S)-α-hydrazinophenylalanine, was prepared in an effort to examine the stereochemical requirements of the A-site of the ribosome during in vitro protein synthesis. The (S)-α-hydrazinophenylalanine moiety was of interest because it contains two nucleophilic centers, the secondary nitrogen attached to Cα, which is normally acylated during the course of peptide bond formation, and the sterically less hindered primary nitrogen. To determine the position of acylation, (S)-α-hydrazinophenylalanyl-tRNAPhe was tested in an Escherichia coli in vitro protein biosynthesizing system lacking elongation factor G, such that only dipeptide products were formed. The dipeptide product mixture was analyzed by HPLC in direct comparison with authentic synthetic standards. The dipeptide assay utilizing (S)-α-hydrazinophenylalanyl-tRNAPhe as the A-site tRNA established that the analogue functioned well as an acceptor tRNA; HPLC...