The synthesis of potential drug candidates based on the PEE-G dendrimer scaffold

Polyethoxyethyl glycinamide (PEE-G) dendrimer scaffolds were developed by Rendle et al. in consideration of the regulatory requirements for pharmaceutical applications. The aim of this PhD project was to demonstrate the potential of these scaffolds for use in three different applications.

First, the PEE-G scaffolds were utilised for conjugation of multiple peptide units. These multivalent structures can provide multiple interactions at a target site and improve the binding affinity of the peptide. The present study used a heptapeptide SNTSESF which was previously studied for its anti-tumour activity by blockade of the PD-1 receptor mediated signalling pathway. The PEE-G scaffolds with two, four, and eight peptide units were synthesised in more than 95% purity. The peptide SNTSESF and the PEE-G dendritic conjugates with two and four peptide units were examined by an in vivo assay that used a C57BL/6 mouse model implanted with GL261 tumours. This study suggested that having more than two copies of peptide SNTSESF on the dendritic scaffold does not increase the anti-tumour activity of the peptide.

Next, the PEE-G scaffolds were appended with a fluorescent tag for their potential application in bioimaging. A cyclooctyne-functionalised BODIPY fluorophore was used to label an N-acetylated PEE-G scaffold and a peptide SNTSESF-capped G1 PEE-G conjugate that contained an azide functionality. The fluorescence quantum yield of the labelled products was measured. Additionally, this approach also demonstrated that the PEE-G scaffolds could be designed to be multifunctional.

Lastly, the PEE-G scaffolds were functionalised with Gd(III) complexes. Gd(III)-based contrast agents are used to enhance the proton relaxation rate and improve sensitivity in magnetic resonance imaging. To further improve efficiency and to minimize Gd(III)-associated toxicity, the lower molecular weight contrast agents have been attached to scaffolds such as dendrimers. A preliminary screening process found a macrocyclic ligand, DOTAGA, as the optimal Gd(III) chelating agent. Multiple copies of the ligand were conjugated to the PEE-G polyamine surface and subjected to Gd(III) complexation. An optimized synthetic method used a PEE-G scaffold with an azide functionality and multiple Gd(III) complexes. This dendritic conjugate was attached to a bis cyclooctyne-functionalised PEE-G scaffold by strain-promoted azide-alkyne cycloaddition reaction. Furthermore, it was investigated whether the PEE-G scaffolds could be designed to accommodate both Gd(III) complexes and targeting moieties for site-specific imaging. A Janus dendrimer that could present Gd(III) complexes on one side and targeting moieties on the other side, and a mono Gd(III) complex conjugated PEE-G scaffold that could present targeting moieties on the multivalent surface were synthesized. The resulting dendritic conjugates were characterised by liquid chromatography, mass spectrometry, combustion analysis, and inductively coupled plasma mass spectrometry. Performance of those conjugates were measured in terms of relaxivity at two different magnetic fields. In comparison to representative commercially available contrast agents, the PEE-G dendritic conjugates demonstrated two to three times higher relaxivity. The solutions of these compounds were further screened by an NMR microimaging procedure.

Therefore, this PhD study successfully demonstrated three applications of the PEE-G dendrimer scaffolds for use as potential pharmaceuticals.