Synthesis of Fluorescence Turn-On DNA Hybridization Probe Using the DEA tC 2'-Deoxycytidine Analog.

Abstract

^DEA tC is a tricyclic 2'-deoxycytidine analog that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with a guanine base in a DNA-DNA duplex. The synthesis of ^DEA tC begins with 5-amino-2-methylbenzothiazole and provides the ^DEA tC nucleobase analog over five synthetic steps. This nucleobase analog is then silylated using N,O-bis(trimethylsilyl)acetamide and conjugated to Hoffer's chlorosugar to provide the protected ^DEA tC nucleoside in good yield. Following protective-group removal and chromatographic isolation of the β-anomer, dimethoxytritylation and phosphoramidite synthesis offer the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the ^DEA tC amidite and a short deprotection time are employed to maximize efficiency. By following the protocols described in this unit, the ^DEA tC fluorescent probe can be synthesized and can be incorporated into any desired synthetic DNA oligonucleotide. © 2018 by John Wiley & Sons, Inc.