The 6-Methoxymethyl Derivative Of Pyrrolo-Dc For Selective Fluorometric Detection Of Guanosine-Containing Sequences

The β-cyanoethyl phosphosphoramidite derivatives of 6-methyl- and 6-methoxymethyl-3-(2-deoxy-β-D-ribofuranosyl)-3H-pyrrolo2,3-d]pyrimidin-2-one have been synthesized. These monomers have been employed for oligodeoxynucleotide synthesis to evaluate their effect on duplex stability and ability to fluorometrically report on hybridization. The structurally conservative 6-methoxymethyl-substitution results in a pyrrolocytidine that is stabilizing toward hybrid formation (Δ Tm = +1.3 °C) whereas the known 6-methylpyrrolocytidine is destabilizing (Δ Tm = ?4.7 °C), in the sequence examined. The 6-methoxymethylpyrrolocytidine retains excellent mismatch discrimination and its fluorescence is selectively quenched when hybridized to a match oligodeoxynucleotide sequence. The quenching of fluorescence for an internal position is approximately three-fold, whereas a terminal position (5′-end or 3′-end) experienced approximately two-fold decrease in the fluorescence intensity.