Synthesis of Fluorescence Turn‐On DNA Hybridization Probe Using the DEAtC 2′‐Deoxycytidine Analog

DEAtC is a tricyclic 2’-deoxycytidine analogue that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with guanine base in a DNA–DNA duplex. The synthesis of DEAtC begins with 5-amino-2-methylbenzothiazole and provides the DEAtC nucleobase analogue over four synthetic steps. This nucleobase analogue is then silylated using BSA and conjugated to Hoffer’s chlorosugar to provide the protected DEAtC nucleoside in good yield. Following protective group removal and chromatographic isolation of the β-anomer, dimethoxytritylation and phosphoramidite synthesis offered the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the DEAtC amidite and a short deprotection time are used to maximize efficiency. By following the protocol described in this unit, the DEAtC fluorescent probe can be synthesized and incorporated into any desired synthetic DNA oligonucleotide.