By using infrared and 19F nuclear magnetic resonance spectroscopies, we localized the binding site and measured the affinity of magnesium for six fluoroquinolones. It was proven that magnesium is situated between the ketone and the carboxylate groups. We determined the binding constants for the 1:1 Mg2e-drug complex in solution. Sparfloxacin and pefloxacin, with affinity constants (K.) of (10.1 + 0.6) x 102 M1 and (21 1) x 102 M-', respectively, were the least and the most bound, respectively. The trend of the affinities of the assayed fluoroquinolones for magnesium was correlated with their antimicrobial activities against four bacteria and with their accumulation by these bacteria. The reference strain, Escherichia coli KL16, and two resistant mutants, NalA (gyrase mutation) and NalB (uptake defect), plus Staphylococcus aureus 209P were used. It appeared that, in every case, an impairment of accumulation is responsible for the increase in the MICs observed upon the addition of magnesium.The advantages of fluoroquinolone antibiotics include their excellent activity against various bacteria, a low frequency of adverse effects, and good absorption on oral administration. However, different studies (5,11,14,22) have shown that some cations, such as magnesium and aluminum, cause malabsorption of most fluoroquinolones, which may result in therapeutic failure. Moreover, the MIC of an antibacterial agent is the result of two steps: the entry of the molecule into the bacterial cell and its interaction with its target within the cell. The rate of penetration of fluoroquinolones across the bacterial cell envelope depends on various physical properties of the drug (6,12,18,20), with its hydrophobicity (3) and the presence of magnesium (7, 13) being major factors. While conflicting reports have been accumulating in the literature on the way in which quinolone antibacterial agents inhibit DNA gyrasecatalyzed supercoiling (17,24,25,32,34), magnesium is also implicated in the binding of the drug to DNA (17,21,33). To better understand the role of magnesium in these various features of quinolone activity, we aimed to determine the site of the binding and to measure the affinity of this ion for some fluoroquinolones. Our approach includes infrared (IR) and '9F nuclear magnetic resonance (NMR) spectroscopies, which have never been used for this purpose, and a comparative study of the effect of magnesium concentration on the in vitro activities of the assayed fluoroquinolones and on their uptake by four bacterial strains.( IR spectra. The solutions of pefloxacin (1 mM) and sparfloxacin (5 mM) were prepared from 10 mM stock solutions either in 100% D20 or in buffered D20 propanesulfonic acid (MOPS), 20 mM KCl) at pD 7.4. Because the spectra were differential spectra (the buffer was subtracted), their qualities may vary with the compound, and in order to ensure better-quality spectra, the concentration of sparfloxacin had to be increased to 5 mM. The pH meter reading was adjusted by DCl or KOD at 0.4 unit less than the required val...

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Substituent effects of methyl and amino groups on the chemical shifts of pyrimidine have been investigated by'H and "C n.m.r. and compared with similar data obtained for benzene and pyridine. Takbg into account pairwise interactions, the chemical shifts calculated by using an additivity relationship are in very good agreement with the experimental results, except for some hindered Pyrimidines. This study enabled us to assign the "C nmr. spectra of some trisubstituted pyrimidines.Les effets de substituant des groupes mCthyle et amino sur les denlacements chimiques de la pyrimidine ont CtC CtudiCs par RMN du proton et du carbone-13 et cornparts h ceux observes pour le benzkne et la pyridine. Compte-tenu des interactions mutuelles des substituants, les dkplacements chimiques calculCs B l'aide d'un schCma d'additivitb sont en trbs bon accord avec les dkplacements chimiques observCs, sauf dans quelques cas concernant des pyrimidines encombrbes. Cette Ctude des effets de substituant a permis l'attribution sans equivoque des spectres du carbone-13 de pyrimidines trisubstitukes.

Nuclear spin relaxation rates associated with cross-correlated, dipole-dipole interactions are enlisted to help characterize the solution state dynamics of a small heptapeptide, deltorphin-I. A simple two-site jump model can be used to interpret the data obtained on two specific (13)C labeled residues, D-alanine and glycine. The influence of temperature and solvent upon the observed dynamics is investigated. Similarly, relaxation rates associated with dipole-shielding anisotropy interferences are used to examine the magnitude and orientation of various chemical shielding tensors within the D-alanine and glycine residues.