The application of Dendrimers for catalysis and purification and analysis for protein binding

Abstract

The aim of this thesis was two-fold. Firstly, to demonstrate how dendrimers, and internally functionalized dendrimers could be used to speed up the rate of a reaction. Secondly, to develop a new non-covalent methodology towards selective or specific protein binding. The results for each are summarised below: For the catalysis systems we synthesised a series of neutral water soluble dendrimers as model enzyme systems. These PAMAM dendrimers significantly increased the rate of the ester cleavage reaction (trans-esterification) in water, using the hydrophobic PNPA substrate. These rate accelerations did not require the use of acid or base, and could take place at neutral pH. In this respect, they resemble the action and properties of an enzyme. Dendrimers with 4, 8 and 16 end groups were studied (G 0.5, 1.5 and 2.5 respectively), and it was clear that the larger dendrimers were able to speed up the reaction to a greater extent. This was due to the fact the larger dendrimers had a more hydrophobic interior that was more ordered and could bind the PNPA substrate more tightly, and in closer proximity to the larger number of internal and external functionality (required for reaction and stabilisation), The catalytic process was also studied at pH 7.5, 8.0 and 8.5. In all cases, the rates were significantly faster, with the fastest occurring at pH 8.5. Over this pH range, we also observed that the change in relative rate was significantly lower for the dendrimers when compared to the simple control reaction (without dendrimers). In an attempt to develop a synthetic metallo-enzyme, copper was inserted into each of the dendrimers by reacting them with excess copper sulphate. The copper stoichiometry per dendrimer was determined by Job plots, which identified a stoichiometry of 1, 2 and 4 coppers for the G0.5, G1.5 and G 2.5 dendrimers respectively. These copper dendrimers were subjected to the same rate studies carried out for the non-functionalised dendrimers, and similar results and trends were obtained across all pHs studied (with the copper dendrimers being slightly faster). This included the ability to significantly increase rates at neutral pH, and the very small increase in relative rate with respect to pH (compared to the control). Overall, we concluded that the dendrimers were able to speed up PNPA cleavage through an initial hydrophobic binding, which placed the substrate in close proximity to the dendrimer’s reactive and stabilising groups. The pH rate experiments confirmed that the dendrimer reactions proceeded by a completely different mechanism, and gave a different product, to those occurring in the simple hydrolysis reaction (the control). The second area of study was aimed at generating new macromolecules as inhibitors to protein-protein binding and for use in protein purification. To achieve this, neutral OH ended PAMAM dendrimers were used as host molecules to support a number of different functional groups on its surface. The functional groups would be attached to linear chains, which would bind via a series of non-covalent interactions (hydrophobic and H-bonding) to the interior of the dendrimer. The dynamic nature of these interactions would allow the functionalised chains to move and maximise their interactions with a protein surface. In effect, the protein would template its optimum dendrimer based ligand, with the relative position of the linear chains and their functional groups controlled by the protein surface. 3

A tyrosine and valine linear chain was synthesis, as these groups are known to respectively strengthen and weaken binding to a number of target proteins. The chains were synthesised using a standard Boc peptide methodology to generate beta-alanine based chains with three hydrogen bonding amides in the backbone (designed to interact with the internal amides of the PAMAM dendrimers). The initial stages of the synthesis progressed well, but due to solubility problems in the later stages, and despite many purification attempts, we were not able to obtain the desired chains in good purity. Nevertheless, we used the crude material to generate dendrimer-linear chain complexes (with the tyrosine and valine chains). Inhibition experiments were used to assess binding to chymotrypsin, with strong inhibition be directly related to strong binding. In our experiments we observed that the tyrosine based complex had a Ki value of 1.23 M, which was 25% higher than the valine derived complex. Control studies confirmed that the dendrimer and the linear chains were required for binding and that no inhibition/binding occurred when either were used on their own.