Isothermal Titration Calorimetric Studies of Complexation Reactions

The objective of this work has been to study the binding of metal ions to complex ligands expressing two or more metal binding sites, in terms of the thermodynamics of the binding events, and to use this information to contribute to the understanding of both the metal binding processes and the properties of the metal/ligand complexes. Two ligands have formed the basis of the study, one a naturally occurring enzyme and the other a newly synthesised ligand with two distinct binding sites (to form supramolecular complexes). The principle technique used to determine thermodynamic parameters has been isothermal titration calorimetry (ITC).

The first system studied involved the enzyme β-lactamase II from Bacillus cereus 569/H/9 (BcII). The binding of zinc (II), cobalt (II) and cadmium (II) was investigated in aqueous solution at pH’s from 5.2 to 7.2. By using three or more buffers at each pH it has been possible to determine not only the binding constants of the metal ions at these pH’s but also the molar enthalpies of binding (and hence molar entropies). In addition, the dissociation of hydrogen ions from the enzyme on metal binding was also observed. In the case of all three metal ions, one metal ion per enzyme appears to bind at low pH and two metal ions at high pH (within the pH 5.20-7.20 range). In the case of zinc and cobalt ions two metal ions seem to bind per mole of enzyme in a cooperative mechanism. In contrast two cadmium ions appear to bind to the active sites of the enzyme non-cooperatively. For all three metal ions, models have been developed to describe the two binding sites and the sources of H+ ions that dissociate on metal ion binding.

The second system studied involved the supramolecular synthesised ligand bipy-aza-crown-4. This ligand has two metal binding sites, one at the 2,2-bipyridine and the other at the aza-crown-4. The objective was to compare the way copper (II) binds in acetonitrile solution with the way in which the copper (II) complex with this ligand is crystallised from methanol solution with progressively increased copper (II) concentration, as revealed by single crystal x-ray diffraction.

Smaller ligands that represent the two individual complexation regions of the synthesised ligand were also used for comparison. Both ITC and solution spectroscopic measurements were used to compare the binding model that was proposed in methanol solution. It was demonstrated in acetonitrile solution that copper (II) binds to the 2,2-bipyridine site first in a 1:2 (Cu2+:L) ratio, then binds to the aza-crown site in a 1:1 (Cu2+:L) ratio . So it was demonstrated that the overall stoichiometric ratio of copper ion to bipy-aza-crown-4 in the fully formed complex is 3:2 (Cu2+: L). Possibly the main conclusion drawn is that the order in which the binding sites are occupied in acetonitrile solution is different to that in which they are occupied in methanol.