Method for making chromogenic and/or fluorogenic substrates for use in monitoring catalytic or enzymatic activity

US4529796A · kind A · utility

Assignee

• Baker Instruments Corporation · US

Inventors

• Jemo Kang · Princeton, US
• Glen L. Tolman · Chelmsford, US

Key dates

• Filing date: Feb 22, 1982
• Grant date: Jul 16, 1985
• Expiry date: Feb 22, 2002

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C07H19/04
• WIPO field: Organic fine chemistry
• WIPO sector: Chemistry

Abstract

A method for preparing a substrate capable of undergoing catalytic-induced hydrolysis of the phosphate ester at the 3' position, to yield a species capable of being monitored spectrophotometrically or fluorometrically, comprises PA0 (a) forming a mononucleotide 20', 3' cyclic phosphate of the formula: ##STR1## wherein B is a nucleotide base, and wherein the CH.sub.2 OH group at the 4'-position is either cis or trans to the cyclic phosphate; PA0 (b) blocking said mononucleotide 2', 3'-cyclic phosphate at the 5'-hydroxyl with a suitable 5'-blocking member to form a mononucleotide 5'-O-blocked-2', 3'-cyclic phosphate; PA0 (c) opening said cyclic phosphate so that essentially only a mononucleotide 5'-O-blocked-2'-hydroxyl-3'-phosphate is formed under conditions leaving the 5'-position essentially blocked; PA0 (d) blocking said mononucleotide 5'-O-blocked 3'-phosphate at the 2'-hydroxyl with a suitable 2'-O-blocking member to form a mononucleotide 2'-blocked-5'-blocked-3'-phosphate; and PA0 (e) forming a 2'-O-blocked-5'-O-blocked phosphodiester by bonding said mononucleotide 2'-O-blocked-5'-O-blocked-3'-phosphate with a moiety selected from the group consisting of a chromophore or fluor…