A method for preparing a mononucleotide-3'-phosphodiester-based substrate

A method for preparing a substrate capable of undergoing catalytic-induced hydrolysis of the phosphate ester at the 3 min -position to yield a species capable of being monitored spectrophotometrically or fluorometrically, comprises (a) blocking a mononucleoside of the formula: wherein B is a nucleotide base, and wherein the CH2OH group at the 4 min -position is either cis or trans to said base, with a silyl blocking member at both the 2 min -and 5 min -hydroxyls of said mononucleoside to form a 2 min -, 5 min -diblocked mononucleoside; and (b) forming a 2 min -, 5 min -diblocked mononucleotide 3 min -phosphodiester by bonding said 2 min -, 5 min -diblocked mononucleoside with a moiety selected from the group consisting of a chromophore or fluorophore; said silyl blocking member at the 2 min -hydroxyl being capable of at least essentially blocking medium-induced hydrolysis of the phosphodiester at the 3 min -position, and said silyl blocking member at least at the 2 min -hydroxyl being capable of being removed to provide a substrate characterized by the ability to undergo catalytic-induced hydrolysis of the phosphate ester at the 3 min -position to yield a species capable of being monitored spectrophotometrically or fluorometrically. The silyl blocking member at least at the 2 min -hydroxyl is removed so as to provide a substrate characterized by the ability to undergo catalytic induced hydrolysis of said phosphodiester to yield a species capable of being monitored spectrophotometrically or fluorometrically.