Studies of Reversible Interactions in Biomaterials and Biomacromolecules

Abstract

This thesis focusses on the studies of biomaterials and biomacromolecules looking at the reversible interactions between molecules that control the overall structure, either to create pHresponsive materials or to investigate an enzyme that is particularly sensitive to surroundings conditions. The first part of studies of biomaterials involved demonstration of boronic ester chemistry, using a range of flexible building blocks (boronic acids and diols) to create boronic esters that are pH-responsive and can make nanostructures. The boronic ester chemistry was demonstrated with phenylboronic acid and catechol as a model system using 1H NMR spectroscopy, and the extend of ester formation as a function of concentration of the diol and boronic acid and pH was optimised. We found that boronic ester was formed as phenyl boronic acid interacted with high concentration of catechol at pH 7.40. It was also revealed that at relatively low concentration of catechol, the proportion of boronic ester formation has increased at high pH and it was shown to be reversible as the pH was changed. Then, more substituted boronic acids were investigated, that proved that with 1, 4-phenyl di-boronic acid and 1, 3, 5-phenyl triboronic acid and catechol reversible boronic esters can be formed with multiple branching points from the boronic acid, allowing the development of higher-dimensional structures. The results not only showed that these building blocks as flexible in forming mixture of esters but at the same time the possibility of forming higher dimensional structures that can be reversible. The model systems were then expanded by investigating the use of more complex diol molecules to link between the boronic acids – using polyalanine peptides, functionalised with or without positive charge and with a conjugated diol built in to the molecule, and 1, 4-phenyl di-boronic acid and 1, 3, 5-phenyl tri-boronic acid as building blocks. Atomic force microscopy was used to demonstrate nanostructure formation in the optimised conditions and the formed nanostructures were found to be two dimensional provided the peptide linkers were positively charged. The nanostructures formed using Polyalanine peptide and 1, 3, 5-phenyl tri-boronic acid were nano-networks, whereas nano-rings were formed with 1, 4-phenyl di-boronic acid. The structures were found to be pH responsive, forming in an optimised pH window dictated by the pKAs of the boronic acid and the peptide. These flexible and reversible two dimensional nanostructures could potentially be used for drug delivery or as a molecular template. The second part of studies of biomacromolecules reviews the use of NMR spectroscopy as a tool to study a superfamily of enzymes called the non-haem Fe (II) and 2-oxoglutarate (2OG)- dependent oxygenases. This included enzyme kinetics, ligand binding and use of protein NMR spectroscopy as a complementary technique to X-ray crystallography in studying the structure, dynamics and conformational changes of 2-oxoglutarate oxygenases in solution. Then the small-angle X-ray scattering was used as a solution-based method to determine the lowresolution solution structure of PHD2, an examples of this class, and the conformational change this protein undergoes upon substrate binding of the hypoxia-inducible factor prolyl hydroxylase 2 which is theorised to be relevant to the protein’s function.