Abstract:
The behaviour of 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP, H4L) was studied in the aqueous medium and at the hydroxyapatite interface as a model of bone. In solution, the pH-dependency of the various protonated forms of HEDP was studied using nuclear magnetic resonance (NMR) spectroscopy of various nuclei and from this comparable pKa values could be obtained from the 31P chemical shift curve. The Raman spectra of the aqueous samples were measured and each protonated form was identified by unique vibrational bands. Multivariate curve resolution analysis was used to redetermine the species distribution diagram, as well as pure component spectra of each protonated form. Molecular modelling was employed to determine the most probable conformer present in solution and also to calculate the theoretical vibrational spectrum of each conformer. Comparison of the theoretical and experimental data allowed the assignment of the different Raman bands observed. The species present at low pH were the most problematic to analyse due to the strong inter- and intramolecular hydrogen bonding indirectly observed in the data. The interaction of HEDP at low and high concentrations with hydroxyapatite, bovine bone and CaHPO4 was investigated in situ by means of Raman spectroscopy and it was found that two Ca-HEDP complexes are sequentially formed at both concentrations, and that the order of formation of these two complexes can be explained from the species distribution diagrams of Ca-HEDP complexes. One complex, CaHEDP•2H2O, was successfully isolated and characterised by means of single-crystal X-ray Diffraction (XRD) methods and Raman spectroscopy. Theoretically generated Raman spectra were used to assist in the assignment of the solid-state Raman spectrum of CaHEDP•2H2O. It is postulated that the unknown complex is the monoprotonated Ca-HEDP complex. Using the Raman spectra of the complexes and HEDP as references, it was determined that HEDP(aq) interacts similarly with hydroxyapatite, bovine bone and CaHPO4 and thus hydroxyapatite can be substituted for bone in the Raman spectroscopic study of HEDP with bone. HEDP interaction was also studied at pH values of 5.0 and 7.4 to understand the nature of the interaction at the pH values at which the diprotonated (H2L2-) form is predominantly present, as well as at the pH of human blood plasma, which is slightly basic. HEDP exists as a monohydrate at room temperature and the single-crystal structure was redetermined, during which the hydrogen positions were experimentally obtained for the first time by means of X-ray diffraction methods. The anhydrous form of HEDP exists above 70°C and Rietveld refinement of the powder X-ray pattern of anhydrous HEDP was used to solve its crystal structure. The complexity of contributory factors allowed only for the non-hydrogen atom positions to be determined. Fourier-transform infrared (FTIR) and Raman spectroscopy were performed on both phases and there is evidence in the Raman spectrum that hydrogen bonding still plays a predominant role in the anhydrous solid state. All these studies led to a better understanding of the nature of bisphosphonate interaction with bone and the results can therefore be applied in future medical studies for drug screening regarding bone cancer research.
