Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

First Advisor

Robert Paul Hammer


Non-hydrogen bonding nucleosides with iodo, nitro, propynyl and thiazolyl substituted at the 4-position of pyrazole were prepared. These nucleosides were converted to their corresponding nucleoside phosphoramidites and incorporated into a series of complementary oligonucleotides in order to determine the effect that varying size, charge distribution and polarizability has on duplex stability and structure. The self-complementary Dickerson dodecamer sequence 5'-CGCXAATTYGCG-3', as well as the non-self complementary sequence 5'-CAAAATGGTGGCCAAGT-3 ' previously investigated by Brown, were used to determine the duplex stabilization and thermodynamic consequences of placing 5-nitroindole and 4-substituted pyrazole nucleosides across from each natural base. In the pyrazole series, the largest and smallest duplex destabilization in all cases studied was found with cytosine and adenosine, respectively, while the 4-thiazolyl substitution was determined to form the most stable duplex regardless of the complementary base. We investigated the directing ability of the pyrazole analogs for incorporation of dNTP's. The DNA polymerases chosen were Taq and Pfu exo-, which lack 3' --5' proofreading exonuclease activity, and Vent, Deep Vent, Pfu which contain exonuclease activity. The enzymes were able to incorporate natural nucleotides across from our modification as detected using the polymerase chain reaction. The Sanger method of dideoxysequencing was used to determine the natural base incorporated across from the modification. The non-proofreading enzymes mainly incorporated deoxyadenosine across from our modification where as the proofreading enzymes removed our modification but not with total efficiency. Automated DNA synthesis procedures might modifying the 5-nitroindole base via electrophilic aromatic substitution by replacing the nitro group or addition of iodine to the ring system. An alternative oxidation reagent, CSO, was used to prepare a series of sequences for comparison with sequences prepared using standard oxidation conditions. Results from MALDI-MS did not provide conclusive evidence for either substitution or iodination of the bicyclic ring system. Examination of the thermodynamic results led to the preparation of thiazole-C-nucleoside, which historically is more difficult to synthesize. The thiazole ring offers a site for the formation of an N-oxide. It has been suggested this group is responsible for enzyme recognition. We prepared the thiazole and thiazole-N-oxide nucleosides for incorporation into oligonucleotides for thermodynamic and enzymic investigations.