On the Transport of Tripeptide Antibiotics in Bacteria

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Abstract
The 2 tripeptide antibiotics L-2-amino-4-methylphosphinobutyryl-alanyl-alanine (L-phosphinothricyl-alanyl-alanine) and L-(N5-phosphono)methionine-S-sulfoximinyl-alanyl-alanine, both inhibitors of the glutamine synthetase,are transported in the cell of Escherichia coli K 12 via the oligopeptide transport system. The uptake by this sytem is proved first of all by cross-resistance with tri-L-ornithine using oligopeptide-transport-deficient mutants, and secondly by antagonism tests demonstrating competitive reversal of the action of the antibiotic by several peptides which are transported via the oligopeptide transport system, e.g., tri-L-alanine, tetra-L-alanine, tri-L-lysine, tri-L-serine, tri-glycine, glycyl-glycyl-L-alanine and the synthetic tripeptide L-azaadenylaminohexanoyl-alanyl-alanine. There is no effect on the action of the antibiotic in antagonism tests with compounds which use different transport systems, such as L-alanyl-alanine, L-lysyl-lysine, glutathione and the synthetic amino acid azaadenylaminohexanoic acid, i.e., 2-amino-6-(7-amino-3H-v-triazolo-[4,5-d]-pyrimidin-3-yl)hexanoic acid. Another inhibitor of the glutamine synthetase, L-methionine-S-dioxide (methioninesulfone) could be converted into a tripeptide form by linkage to L-alanyl-alanine analogously to the tripeptide antibiotics described above. Whereas the free L-methionine-S-dioxide seems to be transported via the methionine transport system, the tripeptide form is transported via the oligopeptide transport system. This glutamine synthetase inhibitor can be taken up by the cell via 2 different transport mechanisms; this could provide a synergistic effect. The syntheses of the new tripeptides L-azaadenylaminohexanoyl-alanyl-alanine and L-methionine-S-dioxidyl-alanyl-alanine were performed by dicyclohexylcarbodiimide couplings of the unusual N-protected L-.alpha.-amino acids azaadenylaminohexanoic acid and L-methionine-S-dioxide to L-alanylalanine-tert-butyl ester followed by common deprotection steps. Tri L-ornithine was synthesized without carboxyl protection via 2 successive couplings of hdroxybenzotriazol esters of N.alpha.-butoxycarbonyl-N.delta.-benzyloxycarbonyl-L-ornithine to N.delta.-benzyloxycarbonyl-L-ornithine.