Design, synthesis, and evaluation of nucleoside fluorophores for nucleic acid base replacement
Dunlap, Vincent Kelly
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With the development of the field of genomics has come an increased need to develop analytical tools for the large variety of studies that genomics has introduced. The field of fluorescence spectroscopy has provided convenient tools for assistance in the analysis of a wide array of these biological studies including those of nucleic acid structure due to its high sensitivity and good reproducibility. The standard method for using fluorescent probe molecules with nucleic acids is to append the molecule through a linker chain to a nucleobase. This method can be improved by instead modifying the nucleobase so as to render it fluorescent. Such changes can result in more sensitive nucleic acid probes of the nucleic acid structure as the fluorophore is now located in the intimate environment of the nucleic acid structure. A series of three 3 H -isoindolo-[2,1-α]purin-2-one-based fluorescent ribo-and 2'-deoxyribonucleosides have been developed and synthesized as 5-aminocytosine derivatives in order to achieve this goal. The synthetic strategy utilized involves condensation with o -phthalaldehyde, an agent previously utilized for the derivativization of highly fluorogenic detection tools of amino acids. A series of NMR studies (DEPT, 2-D ROESY) were conducted to aid in determination of the regioselectivity of the fluorophore condensation and the tautomeric form of the heterocyclic product. These compounds were then synthesized as monomers for nucleic acid chemical synthesis and were incorporated into DNA for thermodynamic and fluorescence studies. In addition, a series of three purine and pteridine-based nucleosides also containing a cytosine H-bonding face and originally synthesized as antiviral and anticancer agents, were also incorporated into DNA owing to their fluorescent properties. These compounds were also analyzed for their thermodynamic and fluorescent properties which were compared with those of the other series. Enzyme inhibition and cytotoxicity analysis of these compounds proved negative. The fluorophore-incorporated oligodeoxynucleotides (ODN's) were analyzed by duplex thermal denaturation, or melting, to determine duplex stability and base pairing specificity of the fluorophores. Fluorescence intensity comparisons between hybridized and unhybridized ODN's were conducted in order to determine the fluorescent signaling specificity upon duplex formation in order to evaluate the fluorophores' applicability as a detector of single base mismatching. Furthermore, analysis of the solvaltochromicity of the fluorophore nucleosides was conducted in a small range of polar solvents along with the analysis of the pH-dependence of fluorescence. The results of the pH analysis coupled with thermal denaturation and ODN fluorescence studies indicate that some of the 3 H -isoindolo-[2,1-α]purin-2-one derivatives undergo tautomeric shift in basic pH. With the exception of ODN's containing Compound 55 , the fluorescence was largely quenched upon incorporation into the ODN and further quenched upon hybridization thus making hybridization-dependent applications undesirable. The compounds were then evaluated for their potential utility in a variety of fluorescence-related applications as well as other potential applications outside of fluorescence such as medicinal chemistry.