NMR analysis and site-specific protonation constants of streptomycin

Streptomycin, the classical aminoglycoside antibiotic, generally considered the most basic drug compound was characterized in terms of protonation macro- and microconstants.1H NMR–pH and 1H–13C HSQC–pH titrations were carried out on streptomycin and streptidine, a symmetrical constituent compound of reduced complexity to monitor the proton-binding processes of the basic sites. Accurate, undistorted, electrodeless pH measurement was ensured by a new set of in tube indicators.The microscopic protonation constants of the two guanidino groups of streptomycin were calculated by evaluating the various NMR–pH data and transferring the pair-interactivity parameter from streptidine to streptomycin. Inherent guanidino basicities fall in the range of 13.03–13.39 log k units, which drop to 12.48–12.85 upon protonation of the other site. pH-dependent distribution of the major microspecies and charge-related biological consequences are provided.

► Accurate, unbiased protonation constants of streptomycin and streptidine. ► Pair interactivity parameter between two guanidino groups. ► Microconstants of the guanidino groups in streptomycin. ► Error analysis of the microconstants.