Complete resolution of the microscopic protonation equilibria of N-methyl-d-aspartic acid and related compounds

Protonation equilibria of N-methyl-d-aspartate (NMDA, a specific glutamate receptor agonist) and its derivatives are characterized at the macroscopic and microscopic levels. 1H NMR-pH and pH-potentiometric titrations were carried out to determine the macroconstants. Microconstants were obtained by appropriate combination of acidity and NMR parameters of the parent compound and its three synthetic derivatives. These derivatives were close models of the NMDA minor microspecies, allowing the calculation of all the 12 microconstants, the 8 microspecies concentrations and 3 site interactivity parameters. Reliability of the microconstants was assessed by three independent test methods. It was found that protonation of the secondary amino site decreases the β- and α-carboxylate basicities almost exactly by one and two orders of magnitude, respectively, whereas protonation of one of the carboxylates lessens the basicity of the other one by a factor of 3. NMR-pH profiles, macro- and microscopic protonation schemes and species-specific distribution diagrams are presented.