The macrolide antibiotic erythromycin interacts with bacterial 23S ribosomal RNA (rRNA) making contacts that are limited to hairpin 35 in domain II of the rRNA and to the peptidyl transferase loop in domain V. These two regions are probably folded close together in the 23S rRNA tertiary structure and form a binding pocket for macrolides and other drug types. Erythromycin has been derivatized by replacing the L‐cladinose moiety at position 3 by a keto group (forming the ketolide antibiotics) and by an alkyl‐aryl extension at positions 11/12 of the lactone ring. All the drugs footprint identically within the peptidyl transferase loop, giving protection against chemical modification at A2058, A2059 and G2505, and enhancing the accessibility of A2062. However, the ketolide derivatives bind to ribosomes with widely varying affinities compared with erythromycin. This variation correlates with differences in the hairpin 35 footprints. Erythromycin enhances the modification at position A752. Removal of cladinose lowers drug binding 70‐fold, with concomitant loss of the A752 footprint. However, the 11/12 extension strengthens binding 10‐fold, and position A752 becomes protected. These findings indicate how drug derivatization can improve the inhibition of bacteria that have macrolide resistance conferred by changes in the peptidyl transferase loop.

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The macrolide antibiotic erythromycin and its 6‐O‐methyl derivative (clarithromycin) bind to bacterial ribosomes primarily through interactions with nucleotides in domains II and V of 23S rRNA. The domain II interaction occurs between nucleotide A752 and the macrolide 3‐cladinose moiety. Removal of the cladinose, and substitution of a 3‐keto group (forming the ketolide RU 56006), results in loss of the A752 interaction and an ≈ 100‐fold drop in drug binding affinity. Within domain V, the key determinant of drug binding is nucleotide A2058 and substitution of G at this position is the major cause of drug resistance in some clinical pathogens. The 2058G mutation disrupts the drug‐domain V contact and leads to a further > 25 000‐fold decrease in the binding of RU 56006. Drug binding to resistant ribosomes can be improved over 3000‐fold by forming an alternative and more effective contact to A752 via alkyl–aryl groups linked to a carbamate at the drug 11/12 position (in the ketolide antibiotics HMR 3647 and HMR 3004). The data indicate that simultaneous drug interactions with domains II and V strengthen binding and that the domain II contact is of particular importance to achieve binding to the ribosomes of resistant pathogens in which the domain V interaction is perturbed.