Synthesis and structural studies of nucleic acids

Abstract

The first project focused on the synthesis of 4-15N-2′-deoxycytidine as a tool to study the B- to Z-DNA transition using 15N NMR. This study investigated alternative methods for synthesizing 15N enriched 2′-deoxycytidine at the 4-NH2 position. As a model study, ammonium chloride was used as the 15N source to synthesize 5-methyl-2′-deoxycytidine from thymidine. This chemistry was successfully applied to the synthesis 4-15N-2′-deoxycytidine from 2′-deoxyuridine. In addition, the 15N NMR detection limit was also established to determine the feasibility of studying DNA structures using 15N 1D NMR. The second project involved finding ‘difficult sequences’ in solid-phase oligonucleotide synthesis through phosphoramidite chemistry. Using 18-mers as models, all mathematically possible repeating trinucleotides, dinucleotide, and mononucleotide sequences were synthesized, and the overall yields were analyzed by anion exchange high performance liquid chromatography. It was concluded that depurination was a major contributor to decreased yields in purine-rich sequences. In addition, certain dinucleotide-repeat sequences, especially d(CG) and d(GC), were associated with low overall yields for reasons unknown at this time. The last project involved the identification of alternative detritylation composition for industrial solid-phase oligonucleotide synthesis. These compositions include acids, solvents, and carbocation scavengers. The acid currently used in industrial oligonucleotide solid-phase synthesis is dichloroacetic acid, which has a short shelf life and contains impurities that react with the desired reaction product. For solvents used in the detritylation reaction, toluene and dichloromethane are currently used. While dichloromethane is not preferred in industry, toluene has been known to swell the polystyrene solid support creating an issue during detritylation. Furthermore, the use of carbocation scavengers helps to remove the cleaved DMTr+, preventing it from re-protecting 5′-OH. However, some scavengers have been reported as problematic for this purpose. The results of this project provided a potential alternative acid, namely difluoroacetic acid, investigated and explained the reason for the current solvent choice, and studied the feasibility of scavengers used in the literature.