Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor

Dr. Hudson

Abstract

This thesis focused on nucleobase modified peptide nucleic acid (PNA) and DNA to expand the repertoire of available nucleobase analogs used in the study of nucleic acids. More specifically, we are interested in modification of the nucleobases such that they retain their ability to undergo Watson-Crick base pairing. Moreover, our goal is to modify the nucleobases in order to engender them with useful photophysical properties such that they may be used as biomolecular tools to investigate nucleic acid structure and dynamics.

To that end, a derivative of 5-aminouracil (5-AU) labelled with the amine-reactive chromophore 9-chloroacridine was prepared for the purpose of investigating its potential as a base-discriminating fluorophore. This modified nucleobase was incorporated into PNA by fluorenylmethyloxycarbonyl (Fmoc)-based oligomerization chemistry. During study of its hybridization to complementary DNA (cDNA), the 5-substitution was found to be thermally labile and hydrolyzed to a small degree in neutral aqueous solution, thus liberating the highly fluorescent acridone moiety.

Moreover, three novel fluorescent 7-deaza-2´-deoxyadenosine analogs were successfully synthesized via the Sonogashira cross-coupling reaction of 7-iodo-7-deaza-2´-deoxyadenosine with 1-ethynylpyrene, 2-ethynyl-6-methoxynaphthalene, and 9-ethynylphenanthrene. These analogs were photophysically characterized in dioxane, EtOH, and H2O to evaluate their potential for use as environmentally sensitive fluorescent probes. All three analogs displayed high solvatofluorochromicity in H2O, relative to their emission wavelengths in dioxane and EtOH. Moreover, all three analogs exhibited moderate to high fluorescence quantum yields in dioxane and EtOH, and significantly lower fluorescence quantum yields in H2O, indicating that these analogs display microenvironment sensitivity.

Furthermore, the synthesis towards a novel tricyclic adenine analog was attempted by extending the substrate scope of the Sonogashira cross-coupling/heteroannulation chemistry developed in the Hudson groupfor cytidine analogs to adenine analogs. However, unlike the cytidine scaffold, the 7-deazaadenine substrate did not cyclize under the Sonogashira cross-coupling/heteroannulation reaction conditions to give the tricyclic adenine and the straight cross-coupled product was obtained in moderate yield.

Lastly, an intrinsic nucleobase quencher was synthesized via the azo coupling reaction between 5-diazouracil and N,N-dimethylaniline. Fluorescence quenching experiments were performed between the nucleobase quencher and the fluorophores phenylpyrrolocytidine (pC), and pyrene to determine viable FRET pairs for use in molecular beacon constructs.

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