Session: Nucleic Acids, Carbohydrates and Peptides:

Fluorescent nucleoside analogues are important in advancing knowledge of the genetic code, especially in DNA replication, expression, and maintenance. Tricyclic cytidine analogs, such as 8-DEA-tC (characterized by a diethyl amino group atop a tricyclic cytidine scaffold), exhibit a range of fluorescent properties and are utilized in a multitude of assays including DNA/RNA polymerase studies, measuring DNA–protein interactions, and FRET experiments. However, the differences in the fluorescent properties of these nucleoside analogues should be further examined to aid the development of predictable fluorogenic probes. As a free nucleoside, 8-DEA-tC is nearly non-fluorescent (Φ_em = 0.006) and increases quantum yield of emission up to Φ_em = 0.12 when based-paired with guanosine in double-stranded DNA. We are synthesizing a related compound, the oxygen-containing analog 8-DEA-tC^Oto further examine the mechanism for the fluorescence turn-on. The six-step synthesis of 8-DEA-tC^O includes the challenging step of adding a protected N⁴-Boc-O¹-TBS-aminophenol (made in three steps) to a protected 5-bromouridine, with activation by POCl₃ and 1H-triazole. Once 8-DEA-tC^O is synthesized, a phosphoramidite derivative of the nucleoside will be made in two steps to insert intooligonucleotides for further testing. Finally, 8-DEA-tC^O and 8-DEA-tC will be compared to better examine the photophysical properties as a free nucleoside in single-stranded and double-stranded DNA. These studies provide a greater understanding of the relationship between structural and photophysical properties of analogues and allow for better design of highly predictable fluorescent probes for studies on nucleic acid structure and dynamics.

Byron Purse

Associate Professor, San Diego State University

Appearances