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Chloramphenicol (CHL) is a well-known broad-spectrum antibiotic which inhibits translation in a wide range of Gram-positive and Gram-negative bacteria. Its inhibitory effect on bacterial protein synthesis is due to competition with the amino acid side chains of incoming aminoacyl-tRNAs (aa-tRNAs) for binding to the ribosome A-site crevice, the wedge-shaped gap formed by the bases of nucleotides U2504, A2451, and C2452 of the 23S rRNA. It binds to the A-site crevice of the 50S subunit, and its nitrobenzyl ring forms a π-stacking interaction with the base of C2452 of the 23S rRNA. The rapid spread of antibiotic resistance among pathogenic microorganisms greatly limits the medical utility of the existing arsenal of antibiotics and poses a serious healthcare threat, warranting the need for the search and development of new compounds and improvement of the existing drugs. Chloramphenicol analogues with replacement of the dichloroacetyl moiety with amino acid and peptide residues and other groups capable of interacting with elements of the ribosomal nascent peptide exit tunnel (NPET) are known [1,2,3]. Such compounds bind to ribosome like chloramphenicol and are therefore of interest as molecular probes to study the interactions of ligands (peptides and antibiotics) with elements of NPET, as well as potential antibacterial drugs. Here, we report chemical synthesis and characterization of three triphenylphosphonium (TPP) analogues of CLM. Chemical synthesis of CLM-analogues is based on acylation of the amino group of chloramphenicol amine (CAM), an inactive CLM-derivative, with alkyl(triphenyl)phosphonium acids: (4-carboxybutyl)(triphenyl)phosphonium, (CAM-C4-TPP); (10-carboxydecyl)(triphenyl)phosphonium, (CAM-C10-TPP); and 4-{[10-(triphenylphosphanyl)decanoyl]amino}butanoic acid, (CAM-C14-TPP). It was supposed that the triphenylphosphonium group will provide the transport of the compound inside the cell and, possibly, interactions with phosphate groups of nucleotide residues in the NPET. On the other hand, it is known that triphenylphosphonium derivatives exhibit an antibacterial effect due to the decrease of the potential on the bacterial membrane [4]. At the same time, triphenylphosphonium derivatives can be pumped out of bacterial cells by the efflux systems causing antimicrobial multidrug resistance (MDR) [5]. Using competition binding with BODIPY-erythromycin analogue we investigated the affinity of CAM-Cn-TPP for the bacterial 70S ribosome E. coli. CAM-C4-TPP and CAM-C10-TPP were found to bind to the ribosome 40-70 times better than CLM. Molecular docking of CAM-Cn-TPP into E. coli ribosome performed by means of the AutoDock Vina software revealed that the nitrophenyl group of all compounds was located in the chloramphenicol binding site, while alkyl-chains were oriented not towards the exit of NPET, but in that way that triphenylphosphonium moiety was located in a side “cave” of NPET in the region of the nucleotide C2610. We used a double reporter system with pDualrep2 to assess the abilities of CLM-analogues to inhibit protein synthesis. This reporter system allows to test compounds for antimicrobial activity with simultaneous classification by the mechanisms of their action: the red fluorescent protein RFP is induced in the presence of DNA-damaging antibiotics, while the far-red fluorescent protein Katushka2S is induced by translation stalling agents. It was shown that the action of triphenylphosphonium analogues was similar to chloramphenicol and was associated with the translation stalling. Experiments with various bacteria demonstrated that triphenylphosphonium analogues of chloramphenicol similarly to alkyl-TPP inhibited growth of the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and Mycobacterium sp., and exhibited less antibiotic activity towards Escherichia coli due to the presence of the highly effective multidrug resistance pump AcrAB-TolC. E. coli mutants lacking AcrB-TolC showed similar CAM-Cn-TPP sensitivity, as Gram-positive bacteria. We also examined the effect of triphenylphosphonium analogues of CLM on B. subtilis membrane potential. It turned out that CAM-Cn-TPP reduce the membrane potential like alkyl(triphenyl)phosphonium compounds. Collapsing membrane potential by triphenylphosphonium analogues of chloramphenicol, as well as stalling of the bacterial protein translation might be involved in the mechanism of their bactericidal action. This work was supported by the grant RFBR (16-04-00709).