New “clickable” reagents for bioconjugate chemistry
thesisposted on 03.03.2017 by O'Malley, William
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
Bioorthogonal “click” chemistries represent a small but growing class of reliable “spring-loaded” reactions that occur exclusively between a pair of specific reaction partners, without interference from any of the native functional groups found in biological systems. These chemistries can be used to efficiently introduce a whole host of entities into biomolecules, ranging from targeting groups through to detectable molecules and functionalities that alter biodistribution characteristics. This thesis describes the synthesis, utilisation and assessment of five new “clickable” imaging probes, spanning three different designs, with each bearing an alkyne functionality to facilitate highly-site specific tagging of azide-bearing (bio)molecules via the Cu(I)-catalysed click reaction. The first set of tags are luminescent terbium(III) complexes incorporating cyclen-based chelators. Their utility for luminescent labelling was demonstrated though successful click conjugation to a small model azide compound as well as E.coli aspartate/glutamate-binding protein incorporating a genetically encoded p-azido-L-phenylalanine or p-(azidomethyl)-L-phenylalanine residue. Upon conjugation, one of the complexes displayed a significant luminescent enhancement (“light-up” effect), providing a simple indicator of successful ligation. The tags should prove useful for time-gated assay luminescence applications. A second set of tags, also cyclen-based, were designed for potential bimodal imaging applications. These feature a fluorescent alkynyl-napthalimide group for optical detection, and a chelated Gd(III) ion to provide image contrast in MRI. As proof-of-concept, one of the complexes was clicked to a lipidated cell-penetrating peptide and uptake into tongue squamous carcinoma cells successfully visualised by fluorescence microscopy. The naphthalimide group was also found to impart photo-cytotoxic activity to the tagged peptide, suggesting that the tag may be a useful prototype building block for the production of theranostic agents. The final class of tags, based upon the TACN macrocycle, were designed for chelation of the radionuclide 64Cu and thereby the production of radiolabelled conjugates for imaging of tumours via positron emission tomography. Model 64Cu chelates proved to be highly stable under physiological conditions, including in the presence of serum. By clicking one of the tags to a fluorescently-labelled bombesin peptide, a radiotracer designed for targeted imaging of prostate cancer was produced and evaluated in a small animal model. Although the peptide conjugate ultimately proved to have poor biodistribution characteristics, the “clickable” tags should prove useful for the development of better performing PET tracers in the future.