Aminoacyl-and peptidyl-tRNA are specific biomolecules involved in many biological processes, from ribosomal protein synthesis to the synthesis of peptidoglycan precursors. Here, we report a post-synthetic approach based on traceless Staudinger ligation for the synthesis of a stable amide bond to access aminoacyl-or peptidyl-di-nucleotide. A series of amino-acid and peptide ester phenyl phosphines were synthetized, and their reactivity was studied on a 2′-N 3 di-nucleotide. The corresponding 2′-amide di-nucleotides were obtained and characterized by LC-HRMS, and mechanistic interpretations of the influence of the amino acid phenyl ester phosphine were proposed. We also demonstrated that enzymatic 5′-OH phosphorylation is compatible with the acylated di-nucleotide, allowing the possibility to access stable aminoacylated-tRNA.