METHOD FOR MAKING CHROMOGENIC AND/OR FLUOROGENIC SUBSTRATES FOR USE IN MONITORING CATALYTIC OR ENZYMATIC ACTIVITY

ABSTRACT A method for preparing a substrate capable of under-going catalytic-induced hydrolysis of the phosphate ester at the 3'-position to yield a species capable of being monitored spectrophotometrically or fluorometrically and thus useful in carrying out various non-isotopic immunoassays, comprises (a) blocking a mononucleosids of the formula: wherein B is a nucleotide base, and wherein the CH2OH group at the 4'-position is either cis or trans to said base, with a silyl blocking member at both the 2'- and 5'-hydroxyls of said mononucleoside to form a 2'-, 5'-diblocked mononucleoside; and (b) forming a 2'-, 5'-diblocked mononucleotide 3'-phosphodiester by bonding said 2'-, 5'-diblocked mononucleoside with a moiety selected from the group consisting of a chromophore or fluorophore; said silyl blocking member at the 2'-hydroxyl being capable of at least essentially blocking medium-induced hydro-lysis of the phosphodiester at the 3'-position, and said silyl blocking member at least at the 2'-hydroxyl being capable of being removed to provide a substrate characterized by the abil-ity to undergo catalytic-induced hydrolysis of the phosphate ester at the 3'-position to yield a species capable of being monitored spectrophotometrically or fluorometrically. The silyl blocking member at least at the 2'-hydroxyl is removed so as to provide a substrate characterized by the ability to undergo cat-alytic induced hydrolysis of said phosphodiester to yield a species capable of being monitored spectrophotometrically or fluorometrically.