Practical Synthesis of N-Formylmethionylated Peptidyl-tRNA Mimics

[Image: see text] Hydrolysis-resistant RNA-peptide conjugates that mimic peptidyl-tRNAs are frequently needed for structural and functional studies of protein synthesis in the ribosome. Such conjugates are accessible by chemical solid-phase synthesis, allowing for the utmost flexibility of both the peptide and the RNA sequence. Commonly used protection group strategies, however, have severe limitations with respect to generating the characteristic N(α)-formylmethionyl terminus because the formyl group of the conjugate synthesized at the solid support is easily cleaved during the final basic deprotection/release step. In this study, we demonstrate a simple solution to the problem by coupling appropriately activated N(α)-formyl methionine to the fully deprotected conjugate. The structural integrity of the obtained N(α)-formylmethionyl conjugate—and hence the chemoselectivity of the reaction—were verified by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry sequence analysis. Additionally, we confirmed the applicability of our procedure for structural studies by obtaining two structures of the ribosome in complex with either fMAI-nh-ACCA or fMFI-nh-ACCA in the P site and ACC-PMN in the A site of the bacterial ribosome at 2.65 and 2.60 Å resolution, respectively. In summary, our approach for hydrolysis-resistant N(α)-formylated RNA-peptide conjugates is synthetically straightforward and opens up new avenues to explore ribosomal translation with high-precision substrate mimics.