The 6-methoxymethyl derivative of pyrrolo-dC for selective fluorometric detection of guanosine-containing sequences.

The beta-cyanoethyl phosphosphoramidite derivatives of 6-methyl- and 6-methoxymethyl-3-(2-deoxy-beta-D-ribofuranosyl)-3H-pyrrolo[2,3-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 (Delta Tm = +1.3 degrees C) whereas the known 6-methylpyrrolocytidine is destabilizing (Delta Tm = -4.7 degrees 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.