Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor

Dr. Leonard G. Luyt

Abstract

This dissertation is in an integrated format discussing three major projects centered around probe development. In the first project, novel metal-chelated and fluorinated GLP-1 derivatives were prepared all containing D-Ala-8, a modification known to improve resistance towards degradation by dipeptidyl-peptidase IV. The effect of increased distance between DOTA and the peptide chain was investigated using a spacer, in order to reduce steric effects imposed by DOTA. Placement of linker and DOTA moieties were varied within the GLP-1 sequence to test for optimal metal-complex location. Binding affinity of peptide derivatives was determined in vitro with CHO/GLP-1R cell line and shown to be in the nM range. In vivo imaging was carried out using C57BL/6 mice. Modifications made to the peptide backbone, based on charge distribution, were shown to improve pharmacokinetics. Our results suggest developed GLP-1 tracers to be potential candidates for the non-invasive imaging of pancreatic islets in vivo.

In the second project, Gallium-chelated protoporphyrin IX (PPIX) derivatives containing the tripeptide RGD αvβ3-targeting moiety were prepared and evaluated. Reaction conditions for both naturally occurring 69/71Ga-labeling as well as 68Ga-radiolabeling were optimized using a microwave reactor. Optical properties were evaluated in order to study the effects of ligand conjugation and gallium chelation on absorption/emission properties of PPIX. Quantum yields pre- and post gallium chelation were comparable, indicating that the presence of gallium does not quench the inherent fluorescence of PPIX. The targeted compound was preferentially taken up by αvβ3 integrin-expressing cancer cells versus the non-targeted form, as assessed by fluorescence microscopy. Our results suggest that gallium-PPIX conjugates could be used as dual modality positron emission tomography/fluorescence microscopy probes and can assist in bridging imaging agent discovery from in vitro microscopy through to in vivo imaging.

In the third and final project, a novel high-throughput cell-based technique was developed for screening one-bead-one-compound (OBOC) libraries. In order to validate this technique, a small library consisting of heptameric peptides, and the αvβ3 integrins targeting RGD sequence, was synthesized on Tentagel beads via a photo-labile linker. Compounds were screened against the αvβ3-expressing MD 435 cell line. After fluorescent-based sorting, peptides were photolytically cleaved off resin and analyzed using MALDI-TOF/TOF. Deconvolution of all peptide sequences was carried out successfully indicating this screening process to be a facile and efficient technique for discovery of novel targeting entities.


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