A fluorescence polarization assay is described that measures the binding of fluorescently labeled erythromycin to 70S ribosomes from Escherichia coli and the displacement of the erythromycin from these ribosomes. The assay has been validated with several macrolide derivatives and other known antibiotics. We demonstrate that this assay is suitable for determining the dissociation constants of novel compounds that have binding sites overlapping those of macrolides. This homogeneous binding assay provides a valuable tool for defining structure-activity relationships among compounds during the discovery and development of new ribosometargeting drugs.Macrolide antibiotics comprise a large group of clinically useful compounds, characterized by having a 14-, 15-, or 16-membered lactone ring with two or more sugar groups attached. Of particular importance are the 14-membered macrolide erythromycin and its newer-generation derivatives clarithromycin, roxithromycin, and azithromycin (a 15-membered macrolide), which are valuable therapeutic agents for the treatment of community-acquired respiratory tract infections. These antibiotics selectively inhibit bacterial protein synthesis by binding reversibly to the ribosome. They have been shown to interact with domain V of 23S rRNA near the peptidyl transferase center (8). Recent structural work (12, 21) has confirmed that these antibiotics exert their inhibitory effect by blocking the entrance to the polypeptide exit tunnel on the 50S ribosomal subunit and thus preventing the extrusion of nascent polypeptides. Sixteen-membered macrolides, such as tylosin, spiramycin, and carbomycin, also bind so as to block the peptide exit tunnel, but in addition these antibiotics have a mycaminose-mycarose disaccharide side chain that protrudes towards the peptidyl transferase center and more directly inhibit the peptidyl transferase reaction (12).Since the introduction of erythromycin nearly 50 years ago, antibacterial resistance to macrolides has been an increasingly persistent threat to public health. One major resistance mechanism is a form of target modification, wherein the dimethylation of A2058 of the 23S rRNA by erm gene-encoded ribosomal methylases results in cross-resistance to macrolides, to the structurally related lincosamides, and to group B streptogramins (commonly referred to as the MLS B phenotype). The emergence of these resistant bacterial pathogens has been of particular concern and has fueled the search for newer and more potent antibiotics with activity against these organisms.For years the interaction of erythromycin and other macrolides with the bacterial ribosome has been the focus of intense investigation, and a number of experimental approaches have been developed to characterize the equilibria of binding and the kinetics of these drugs with their ribosomal target. In many of these studies, the binding of macrolides to ribosomes was investigated using a radiolabeled antibiotic as a ligand (6,7,10,11,19,22). Since binding was measured by filtration, RNA footprintin...

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Bei der Incubierung von Porphobilinogen (I) mit einem Enzym‐System aus Vogel‐Erythrocyten wird Phyriaporphyrin‐III′ gebildet, für das die Struktur (IIb) ermittelt wird: Von den 4 möglichen Strukturen (IIa)‐(IIIb) für dieses Porphyrin können 2 [(IIIa) und (IIIb)] durch Versuche mit "C‐dotiertem (I) und 13C‐NMR‐ Analyse des erhaltenen Produkts ausgeschlossen werden.

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PorPhYrinogen (5) to (6)* phPin-IX ( ' 1 produced enzpically four bridges gave four separate 13C n.m.r. signals. labelled PBG also holds true for uroporphyrinogen-I11 Earlier work had shown that the dimethyl ester of , .