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Elements of the ribosome exit tunnel can interact with the nascent peptide chain, resulting (in some cases) in the arrest of translation. One approach to study the principles of peptide chain recognition by the ribosome exit tunnel, as well as related mechanisms of translation regulation – is development and use of the molecular probes that are based on ribosome-targeting antibiotics, whose structure includes amino acid and peptide residues mimicking the nascent polypeptide chain [1,2]. Such compounds bound to the peptidyl transferase center or ribosome exit tunnel allow to identify contacts between amino acid residues of the peptide part of the molecule and walls of the ribosome exit tunnel by using a variety of biochemical methods. For example, if dichloroacetic moiety of chloramphenicol molecule is replaced with amino acid residue or a short peptide, it should be oriented towards the ribosome exit tunnel (similarly to the growing polypeptide chain), while the unchanged amphenicol moiety will anchor the entire molecule in the A site of the peptidyl transferase center [2,3]. Furthermore, such compounds could be of general pharmaceutical interest as potential antibacterials. In this work, several novel amino acid analogues of chloramphenicol were synthesized. The amino acid moieties of these compounds are represented by L-, D- and some unnatural residues. The ability of these compounds to bind to bacterial ribosomes was studied by displacement of the fluorescent analogue of erythromycin from its complex with E. coli ribosomes. Compounds containing lysyl and histidyl residues showed the highest affinities to the ribosome that are comparable or even exceeding the affinity of chloramphenicol. Also, we have determined the X-ray crystal structures of the Thermus thermophilus 70S ribosome with A-, P-, and E-site tRNAs bound and in complex with chloramphenicol analogues. These structures revealed specific contacts between the amino acid moieties of the chloramphenicol derivatives and the elements of the ribosome exit tunnel. Finally, our chloramphenicol analogues were tested for their ability to inhibit translation both in vitro and in vivo. This study was supported by the Russian Foundation for Basic Research (Grant 16- 04-00709) and the Russian Science Foundation (Grant 14-24-00061)