Effect of Conformation on the Rate of Deamidation of Vancomycin in Aqueous Solutions

The instability of vancomycin, a glycopeptide antibiotic, limits its shelf‐life because the deamidation of its asparagine residue results in the formation of a zwitterion with limited aqueous solubility. Analysis of the pH–rate profile for vancomycin indicates that the deamidation reaction is notably sensitive to the ionic state of the molecule. This observation results in a hypothesis in which the ionic state of vancomycin may influence the conformation of the molecule and therefore affect its reactivity. Two‐dimensional nuclear magnetic resonance (NMR), homonuclear Hartmann–Hahn (HOHAHA) and rotating frame Overhauser enhancement spectroscopy (ROESY) information combined with molecular dynamic simulations were used to estimate the apparent conformation of vancomycin in aqueous solution at pH 4 and pH 9 where the molecule exists primarily as a monocation and monoanion, respectively. The apparent conformation for vancomycin at pH 4 is compact, and the proximity of the backbone amide nitrogen to the side chain carbonyl carbon of asparagine is favorable for the rapid formation of the cyclic imide intermediate, thus increasing its reactivity. The apparent conformation for vancomycin at pH 9, however, is expanded in comparison with the conformation at pH 4, and the increase in distance between the reacting atoms leads to slower cyclic imide formation and thus decreased intrinsic reactivity. That cyclic imide formation was rate limiting at both pH values was confirmed by cyclic imide isolation and stability estimation. It becomes apparent from the analysis of the pH–rate and conformational profiles of vancomycin that the deamidation rate of vancomycin is largely influenced by the ionization state of the N‐methyl leucine nitrogen. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 742–750, 2000