Functionalised Dendrimers and Graphene Oxide for Application as Macromolecular Inhibitors of Protein–Protein Binding

This research focused on the development of dendrimers and graphene oxide (GO) as protein-binding macromolecular systems. Both materials were functionalised with amino acids to enhance their binding potential and specificity. While the dendrimer studies were primarily exploratory and focused on system design and functionalisation, experimental evaluation of protein binding was carried out using GO.
Initially, the study focused on developing improved hydrophobic linear chains (HLCs) with fewer hydrogen bonding groups to enhance synthetic accessibility, followed by their non-covalent encapsulation within PAMAM–OH dendrimers. Tyrosine- and valine-terminated HLCs were efficiently encapsulated in G2.5 and G3.5 dendrimers, with average loadings of five and seven chains, respectively. Only the tyrosine chain showed measurable retention in G1.5, likely due to stabilising interactions from its phenolic group. Building on this, structurally similar HLCs with terminal acidic groups were synthesised using microwave-assisted Fmoc-based solid-phase peptide synthesis (SPPS) to enable a more controlled and scalable route for chain assembly. Although synthesis was successful, the overall yield was low (~20%) due to structural constraints and challenges in the cleavage and purification steps.
The second part of the study focused on developing functionalised GO to explore protein binding interactions with α-chymotrypsin, where the degree of inhibition was used as an indicator of GO–protein binding affinity. GO was functionalised with anthracene–amino acid ligands through non-covalent π–π interactions and compared with covalently modified analogues synthesised via Diels–Alder cycloaddition. In the non-covalent system, enzyme inhibition was influenced by amino acid side chains; tyrosine and glutamic acid showed the lowest residual enzymatic activities (36% and 31%, respectively). Covalent GO systems demonstrated more effective inhibition and higher protein binding affinity. For example, phenyl aniline-functionalised GO achieved 29% residual activity in the covalent form, compared to 52% in the non-covalent form. This limitation is likely due to steric hindrance limiting ligand mobility in the dynamic non-covalent system, whereas improved inhibition in the covalent system is attributed to higher surface density. Although increased adsorption was observed, hetero-functionalised GO showed lower inhibition than homo-functionalised GO, potentially due to intramolecular competition and steric effects.