s The Site-specific Protonation Constants of Spectinomycin, Characterized by ¹H and ¹⁵N NMR Methods

Spectinomycin, a tricyclic aminoglycoside antibiotic with peculiar chemical structure and pharmacological profile was characterized in terms of microscopic protonation constants. ¹H-¹⁵N HMBC-pH titrations were carried out to allocate the order of basicities of the two similar methylamino functions of spectinomycin, and ¹H NMR-pH titrations were performed on spectinomycin and actinamine, its symmetrical model compound to determine the basicity of the amino sites. It was found that the methylamino moiety in position 3 is of some 60% higher basicity than its counterpart in position 1, and protonation at one site decreases the basicity of the other site by 1.17 logk units. Both secondary amino sites as such are of relatively low basicity, due to the adjacent, electron-withdrawing 7 oxygens. At the pH of blood nearly equal amounts of di- and monocationic species coexist, while less than 1% of neutral spectinomycin occurs at this pH. The pHdependent distribution of the microspecies is depicted.