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US 6,207,365 B1Granted
Fluorescent enzyme substrates and method for determining enzyme activities
AssigneeLaboratory of Molecular Biophotonics
InventorsHirofumi Shiono, Hitoshi Nohta, Chika Utsuyama
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Abstract
The enzyme substrate according to this invention has within its molecule both a group to be cleaved by an enzyme reaction and a group that forms a strongly fluorescent coumarin derivative through intramolecular lactonization when cleaved by the enzyme reaction. Furthermore, the method for determining an enzyme activity according to this invention comprises conducting the enzyme reaction by the use of the enzyme substrate of the invention and determining the enzyme activity by means of the measurement of fluorescence of the coumarin derivative formed.
Claims21
An enzyme substrate represented by the following formula:
- (BLOCK-O)-Xcu
- wherein BLOCK is a blocking group selected from the group consisting of:
- a monovalent blocking group derivable by removal of one hydroxyl from a phosphoric acid group, a sulfuric acid group, or a salt thereof;
- and a monovalent blocking group derivable by removal of a hydroxyl from an aliphatic carboxyl, an aromatic carboxyl, an amino acid carboxyl, or a peptide carboxyl, wherein said BLOCK is cleaved from said substrate by the action of an enzyme to yield a HO—Xcu product, and further, said HO—Xcu product intramolecularly forms a lactone ring to provide a coumarin derivative;
- wherein Xcu has a structure represented by the following formula and is covalently bound to oxygen O at C1 carbon,
- wherein L represents H, NH, alkyl having 1 to 4 carbons, tetralkylammonium having 1 to 4 carbons, or an alkaline metal or an alkaline earth metal;
- and further,
- wherein said coumarin derivative has a structure represented by the following formula:
5 more elements
A method for detecting an enzyme activity, said method comprising the steps of:
- (A) contacting a sample containing an enzyme having an activity to be detected, with an enzyme substrate represented by the following formula:
- (BLOCK-O)-Xcu
- wherein BLOCK is a blocking group selected from the group consisting of:
- a monovalent blocking group derivable by removal of one hydroxyl from a phosphoric acid group, a sulfuric acid group, or a salt thereof;
- and a monovalent blocking group derivable by removal of a hydroxyl from an aliphatic carboxyl, an aromatic carboxyl, an amino acid carboxyl, or a peptide carboxyl, wherein said BLOCK is cleaved from said substrate by the action of an enzyme to yield a HO—Xcu product, and further, said HO—Xcu product intramolecularly forms a lactone ring to provide a coumarin derivative;
- wherein Xcu has a structure represented by the following formula, the C7-C2 bond and the C8-C9 bond have cis-configuration with respect to the C7═C8 double bond, and the Xcu is covalently bound to oxygen O at the C1 carbon,
- wherein L represents H, NH, alkyl having 1 to 4 carbons, tetralkylammonium having 1 to 4 carbons, or an alkaline metal or an alkaline earth metal;
- and further,
- wherein said coumarin derivative has a structure represented by the following formula:
- (B) detecting said coumarin derivative, and correlating the detected coumarin derivative to detect the enzyme activity.
7 more elements
A method for detecting an enzyme activity, said method comprising the steps of:
- (A) contacting a sample containing an enzyme having an activity to be detected, under irradiation conditions, with an enzyme substrate represented by the following formula:
- (BLOCK-O)-Xcu
- wherein BLOCK is a blocking group selected from the group consisting of:
- a monovalent blocking group derivable by removal of one hydroxyl from a phosphoric acid group, a sulfuric acid group, or a salt thereof;
- and a monovalent blocking group derivable by removal of a hydroxyl from an aliphatic carboxyl, an aromatic carboxyl, an amino acid carboxyl, or a peptide carboxyl, wherein said BLOCK is cleaved from said substrate by the action of an enzyme to yield a HO—Xcu product, and further, said HO—Xcu product intramolecularly forms a lactone ring to provide a coumarin derivative;
- wherein Xcu has a structure represented by the following formula, the C7-C2 bond and the C8-C9 bond have trans-configuration with respect to the C7═C8 double bond, and the Xcu is covalently bound to oxygen O at the C1 carbon,
- wherein L represents H, NH, alkyl having 1 to 4 carbons, tetralkylammonium having 1 to 4 carbons, or an alkaline metal or an alkaline earth metal;
- and further,
- wherein said coumarin derivative has a structure represented by the following formula:
- (B) detecting said coumarin derivative, and correlating the detected coumarin derivative to detect the enzyme activity.
7 more elements
Dependencies are inferred from the claim text.