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The theory and practice of proton microspeciation based on NMR-pH titrations are surveyed. Principles of bi-, tri-, tetra-, and n-protic microequilibrium systems are discussed. Evaluation methods are exemplified by case studies on bi- and tetraprotic biomolecules. Selection criteria and properties of 'reporter' NMR nuclei are described. Literature data on complete microspeciations of small ligands and site-specific basicity characterizations of peptides and proteins are critically reviewed.

The site-specific basicities of imatinib (Gleevec, a new signal transduction inhibitor drug of chronic myeloid leukemia) and two of its fragment compounds were quantitated in terms of protonation macroconstants, microconstants, and group constants by NMR-pH and pH-potentiometric titrations. Sequential protonation of imatinib follows the N(34), N(11), N(31), N(13) order, in which N(11) and N(31) show commensurable basicity, but negligible intramolecular interaction. Fragment compounds include two "halves" of imatinib, and their moiety-specific basicities confirm the NMR-based protonation sequence of the parent compound. NMR-pH profiles, macro- and/or microscopic protonation schemes, and species-specific distribution diagrams are presented. On the basis of these data, imatinib is shown to be predominantly neutral, monocationic, and tricationic at intestinal, blood, and gastric pH, respectively. The molecular hypotheses on imatinib binding to the Bcr-Abl oncogene fusion protein are interpreted at the site-specific level in view of the moiety basicities of imatinib.

The first complete microequilibrium analysis of a tetrabasic acid is presented. A total of 18 protonation microconstants of glutathione disulfide (GSSG) were determined, 16 of them quantitate the overlapping equilibria in acidic medium, where 4 carboxylate sites protonate. The related, pH-dependent concentrations of 16 coexisting microspecies were also obtained, including the 10 different ones and their 6 identical twins of symmetry origin. The GSSG acid-base chemistry in basic medium is also characterized. It is shown for tetra-and further multibasic acids that the number of traditional microconstants mandatorily exceeds that of the pieces of independent information available by any state-of-the-art methodology, making the complete microequilibrium resolution not only highly complex but theoretically impossible on the basis of hitherto reported principles. The GSSG microspeciation was made feasible here by introducing cumulative microconstants, a new equilibrium parameter, and the invariance of the interactivity coefficient, a plausible simplifying principle, on the experimental basis of 1 H NMR-pH titrations in H 2 O/D 2 O 9/1 media. The methods and results were verified by corresponding data of GSSG-(glycyl)-diethyl-ester, a model compound of reduced microequilibrium complexity. The obtained log k U ) 2.44 and log k Y ) 3.59 "core" microconstants of the respective glutamyl and glycyl carboxylates of GSSG are close to those of the sulfhydryl (GSH) form of glutathione. The GSSG intermoiety interactions were found to be weak, apparently of Coulombic nature. The pH-dependent distribution of the 18 microspecies is depicted.

Pressure-assisted CE (PACE) was applied to determine the previously inaccessible complete set of pK values for folic acid and eight related multiprotic compounds. PACE allowed the determination of all acidity macroconstants at low (

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