Solid-state NMR experiments with stable isotope-labeled Staphylococcus aureus have provided insight into the structure of the peptidoglycan binding site of a potent fluorobiphenyl derivative of chloroeremomycin (Eli Lilly LY329332). Rotational-echo double resonance (REDOR) NMR provided internuclear distances from the 19F of this glycopeptide antibiotic to natural-abundance 31P and to specific 13C and 15N labels biosynthetically incorporated into the bacteria from labeled alanine, glycine, or lysine in the growth medium. Results from experiments with intact late log phase bacteria and cell walls indicated homogeneous drug-peptidoglycan binding. Drug dimers were not detected in situ, and the hydrophobic fluorobiphenyl group of LY329332 did not insert into the bilayer membrane. A model of the binding site consistent with the REDOR results positions the vancomycin cleft around an un-cross-linked D-Ala-D-Ala peptide stem with the fluorobiphenyl moiety of the antibiotic near the base of a second, proximate stem in a locally ordered peptidoglycan matrix.

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Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the D-Ala-D-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan D-Ala-D-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.

Cross-polarization magic-angle spinning and rotational-echo double resonance 13C and 15N NMR experiments have been performed on intact cells of Staphylococcus aureus labeled with D-[1-13C]alanine and [15N]glycine or with [1-13C]glycine and L-[epsilon-15N]lysine. The cells were harvested during stationary or exponential growth conditions, the latter in media with and without the addition of vancomycin. The results of these experiments allowed the in situ determination of the relative concentrations of peptidoglycan cross-links (the number of peptide-stem D-alanines covalently linked to a pentaglycyl bridge) and bridge-links (the number of peptide-stem lysines covalently linked to a pentaglycyl bridge). The concentration of cross-links remained constant in the presence of vancomycin, whereas the number of bridge-links decreased. These changes suggest that vancomycin (at therapeutic levels) interrupts peptidoglycan synthesis in S. aureus by interference with transglycosylation.

Solid-state NMR has been used to examine isolated cell walls and intact whole cells of Staphylococcus aureus complexed to 5 different vancomycin, eremomycin, and chloroeremomycin derivatives. The cell walls and whole cells were specifically labeled with D-[1-13 C]alanine, or a combination of [1-13 C]glycine and [ε-15 N]lysine. Each of the bound glycopeptides had a 19 F-labeled substituent at either its C-terminus or disaccharide position. The 13 C{ 19 F} rotational-echo doubleresonance (REDOR) dephasing for the cell-wall 13 C-labeled bridging pentaglycyl segment connecting a glycopeptide-complexed peptidoglycan stem with its neighboring stem indicates that the fluorine labels for all bound glycopeptides are positioned at one end or the other of the bridge. An exception is N'-(p-trifluoromethoxybenzyl)chloroeremomycin whose hydrophobic substituent differs in length by one phenyl group compared to that of oritavancin, N'-4- [(4-chlorophenyl) benzyl)]chloroeremomycin. For this drug the fluorine label is near the middle of the pentaglycyl segment. The 15 N{ 19 F} REDOR dephasing shows proximity of the fluorine to the bridge-link site of the pentaglycyl bridge for C-terminus-substituted moieties, and to the cross-link site for disaccharide-substituted moieties. Full-echo REDOR spectra of cell-wall complexes from cells labeled by D-[1-13 C]alanine (in the presence of an alanine racemase inhibitor) reveal three different carbonyl-carbon chemical-shift environments, arising from the D-Ala-D-Ala binding site and the DAla-Gly-1 cross-link site. The REDOR results indicate a single fluorine dephasing center in each peptidoglycan complex. Molecular models of the mature cell-wall complexes that are consistent with internuclear distances obtained from 13 C{ 19 F} and 15 N{ 19 F} REDOR dephasing allow a correlation of structure and antimicrobial activity of the glycopeptides. KeywordsDipolar coupling; glycopeptide antibiotic; magic-angle spinning; peptidoglycan; solid-state NMR; transglycosylase Vancomycin is a potent antibiotic that is effective against multi-drug-resistant Gram-positive bacteria including methicillin-resistant S. aureus. As many as 60% of clinically isolated strains of S. aureus are methicillin resistant (1), which means that vancomycin is one of the most important antibiotics in use today. Vancomycin inhibits the transglycosylation step in the † This paper is based on work supported by the National Institutes of Health under grant number EB02058. * Jacob Schaefer, NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 August 9. The emergence of vancomycin-resistant enterococci (VRE) has limited vancomycin usage against methicillin-resistant S. aureus. In 2002, vancomycin-resistant S. aureus (VRSA) with a minimum inhibitory concentration (MIC) of greater than 128 μg/mL was recovered from a patient in Michigan who was being treated with multiple antibiotics (5). E. faecalis, a vancomycin-resistant enterococcus, was also recovered from the patient. The VRSA isolat...