The transfer of genetic information from nucleotide sequences to proteins is the central defining feature of life on Earth. With the vast majority of the human genome composed of non-coding sequences, DNA has been regarded by most as a uniform double helix and a passive library of genetic information. However, DNA is a highly dynamic molecule and non-duplex DNA structures play important roles in the regulation of gene expression and chromosome stability. Cytosine-rich sequences can self-assemble into four-stranded structures called i-motifs that contain hemi-protonated C-C+ base pairs. The presence of this unique architecture in vivo is still a source of debate, since i-motif folding requires slightly acidic conditions (pH ≤ 6) to promote protonation of the cytosine residues. To detect i-motif structures in vivo, we envisioned the utilization of a minimally disruptive fluorescent nucleoside analog. For this purpose, we designed and evaluated a new family of fluorescent cytosine (1) and thymidine (2) nucleosides containing a quinazoline fluorophore. To create “push-pull" fluorophores, electron-donating groups were added to position C6 of quinazoline, which was selected for its negligible impact on the physicochemical properties of N3 due to the lack of conjugation. The targeted compounds were prepared using Pd-catalyzed Suzuki-Miyaura or Buchwald-Hartwig cross-couplings reactions, and stereoselective N-glycosylation for the assembly of the nucleobase (5) and the furanose ring (4). Chapter 8: Summary 185 To facilitate synthesis of the target compounds, an efficient method for the N-2-deoxyribosylation of modified nucleobases by thioriboside donors was developed. In this approach, thioethers are selectively activated by iodination to produce five-membered ring oxocarbenium ions. The conformation preferences of these intermediates were tuned using various functional groups on the furanose ring to induce stereochemistry at C1 (α/β = 1.0:4.0 to 4.5:1.0) upon condensation with an activated nucleobase. This strategy enabled the stereo- and regioselective synthesis of various pyrimidine nucleosides, including ring-expanded pyrimidine derivatives containing sulfur and bromine atoms.