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Chloramphenicol is a widely used antibiotic that inhibits protein synthesis by binding to bacterial ribosomes in the A-site of the peptidyl transferase center. X-ray data of the ribosome – chloramphenicol complexes showed that dichloroacetic moiety of the antibiotic is directed to the ribosome tunnel [1], and, consequently, if being replaced with amino acid or peptide residue could mimic nascent peptide chain. In continuation of our previous studies in which we have designed and synthesized a number of peptide derivatives of macrolides where the peptide part modeled the growing chain, while the antibiotic served as an “anchor” for positioning the peptide at the specific site of ribosomal tunnel [2, 3], now we report chloramphenicol peptide derivatives as analogues of the peptidyl-tRNA. These derivatives are of interest both as antibacterial agents and as tools for investigation of the interactions of nascent peptide chain with the specific sites of the ribosomal tunnel and their influence on the translation. Previously [4] some amino acid and peptide derivatives of chloramphenicol were described. Virtual screening for binding of all amino acid, di- and tripeptide chloramphenicol derivatives to the E. coli ribosome was performed. These derivatives were constructed by removing of dichloroacetic moiety from chloramphenicol structure and attaching of amino acid or peptide by its carboxyl group to vacant amino function of chloramphenicol residue. Optimized 3D structures of the chloramphenicol peptide derivatives were generated from SMILES strings using the OpenBabel program. Docking-based virtual screening was carried out by means of the AutoDock Vina software [5], using the rigid structure of the ribosome [6] and flexible structures of ligands. Molecule conformations obtained by means of the molecular docking were ranged both by their binding energies, and by the RMSD of chloramphenicol moiety from corresponding X-ray data. The most interesting compounds including chloramphenicol derivatives modified with short “stop-peptides” (MRL, IRA and TRP) were synthesized. The new chloramphenicol derivatives were examined for their binding ability to bacterial ribosomes by displacement of fluorescently labeled erythromycin from their complexes with E. coli ribosome and for their power to inhibit translation in vitro and in vivo.