Site-specific protonation microequilibria of penicillin and cephalosporin beta-lactam core molecules

Acid-base chemistry of 6-aminopenicillanic acid (6-APA) and 7-aminocephalosporanic acid (7-ACA), the fundamental units of the two classical antibiotic families is characterised at the macroscopic and microscopic levels. 1H NMR-pH and pH-potentiometric titrations were combined to monitor the previously unreported extent of the site-specific protonation of 6-APA and 7-ACA. Microscopic protonation constants were derived either from direct multiple fittings of NMR-pH titration curves of adjacent carbon-bound 1H nuclei, using in situ pH-indicator molecules, or from newly synthesised methyl ester derivatives. The results indicate that even the amino protonation of both structures occurs well below pH 5, the major protonation route includes the zwitterionic forms, and protonation at the carboxylate site brings about a three-fold diminish in the amino basicity and vice versa.