Determination of the pKa value of the hydroxyl group in the alpha-hydroxycarboxylates citrate, malate and lactate by 13C NMR: implications for metal coordination in biological systems.

Citric acid is an important metal chelator of biological relevance. Citric acid helps solubilizing metals, increasing their bioavailability for plants and microbes and it is also thought to be a constituent of both the extracellular and cytoplasmic low molecular iron pools occurring in plants and vertebrates. Metal coordination by citric acid involves coordination both by the carboxylate and hydroxyl groups, of particular interest is its alpha-hydroxycarboxylate function. This structural feature is highly conserved in siderophores produced by evolutionarily distant species and seems to confer specificity toward Fe(III) binding. In order to understand the mechanism of metal coordination by alpha-hydroxycarboxylates and correctly evaluate the respective complex stability constants, it is essential to improve the knowledge about the ionisation of the alcohol group in these compounds. We have evaluated the hydroxyl pKa value of citric, malic and lactic acids with the objective of understanding the influence of alpha-carbon substitution. Studies at high pH values, utilizing (13)C NMR, permitted estimation of the pKa values for the three acids. The pKa (alcohol) values (14.4 for citric acid, 14.5 for malic acid, and 15.1 for lactic acid) are considerably higher than the previously reported value for citric acid (11.6) but still lower than the value of 15.5 for methanol. A comparative analysis of the three compounds indicates that different substitutions on the alpha-carbon introduce changes to the inductive effect experienced by the hydroxyl group thereby modulating its ionisation behaviour. Comparison with the siderophore rhizoferrin, which pKa (alcohol) values were confirmed to be 10 and 11.3, suggests that intra-molecular hydrogen bonding may also aid in the hydroxyl ionisation by stabilizing the resulting anion. Studies of metal coordination by alpha-hydroxycarboxylates should take these factors into account.