Synthesis and Evaluation of Novel Nucleoside Analogues: Development of Fluorescent, Bioorthogonal, and Antiviral Tool Compounds

Abstract

The advent of modern biology was preceded by a century’s long search for the chemical basis of how genetic information is made, copied, and translated into life. These discoveries started with the identification of “nuclein,” and the smallest units that make up our genetic material, i.e. nucleotides, and their chemical composition and structure. While a seemingly small undertaking by modern standards, these basic discoveries were the beginning of a whole series of investigations into the chemical principles that now define nucleotide chemistry and biology. The studies on the chemistry of nucleotides led to the discovery of not only the construction of DNA, but also its tertiary structure – the now famous “double helix” – setting off another century’s worth of research. As one of biology’s fundamental metabolites, nucleotides and nucleotide-like small molecules play a role not only as components of DNA and RNA, but also as enzyme cofactors and substrates. For this reason, tools to study nucleoside, nucleotide, and oligonucleotide-related chemistry and biology are valuable. Therefore, scientists have taken advantage of molecules that are structurally similar to the nucleotides found in our genetic code – adenosine (A), cytidine (C), guanosine (G), thymidine (T), and uridine (U). Whether these nucleotide analogues are natural products or synthetic variants invented by chemists, they can mimic or replace endogenous nucleotides in order to study or interfere with natural processes. This thesis will focus on the development of nucleoside analogue tools and bioactive molecules. Chapter 2 will discuss the development of novel nucleoside fluorophores. In order to limit the risk of perturbing the natural state of DNA, the development of a small and isomorphic, i.e. a chemical structure resembling a purine or pyrimidine skeleton, nucleoside fluorophore was determined to be advantageous. Therefore, 4-cyanoindole (4CI), originally reported as a component of a fluorescent tryptophan amino acid mimic, was adapted to make a fluorescent nucleoside. The synthesis, incorporation into DNA, and evaluation of the fluorescent properties of the fluorescent nucleoside are described. Chapter 3 discusses the expansion of the novel indole isomorphic nucleoside family and the investigations of enzymatic incorporation of an indole fluorophore into DNA. Chapter 4 introduces a novel indole-based bioorthogonal nucleoside probe. Using an isonitrile, the novel 4-isocyanoindole (4ICI) serves to expand beyond common azide/alkyne-modified nucleotides to bioorthogonally label DNAs. Chapter 5 describes efforts to modify the naturally occurring antiviral small molecule 3ʹ-deoxy-3ʹ,4ʹ-didehydro-cytidine triphosphate (ddhCTP) in order to improve its therapeutic efficacy. This was done by developing a prodrug form of ddhCTP in order to improve upon ddhCTP’s poor pharmacokinetic properties. Biological evaluation provides evidence of this novel molecule’s potent antiviral activity and mechanism of action.