Fluorescent substrate for detection of enzymatic activity of nitrile-related enzyme
View Patent ↗The object of the present invention is to provide a fluorescent substrate for detecting the enzymatic activity of a nitrile-related enzyme. The present invention provides a compound represented by formula (I) and a fluorescent substrate for detecting the enzymatic activity of a nitrile-related enzyme, which comprises the compound.
1. A compound consisting of formula (I), a salt thereof, or a hydrate thereof:
wherein R 1 is —CN, —CONH 2 , —CH═CH—CN: or —CH═CH—CONH 2 ,
R 2 is a C 1-4 alkyl group,
R 3 , and R 4 are each independently a hydrogen atom, a halogen atom or a C 1-4 alkyl group, and
R 5 is a hydrogen atom, a C 1-4 alkylcarbonyl group, or a C 1-4 alkylcarbonyloxymethyl group.
2. A fluorescent substrate, according to claim 1 , wherein the substrate is suitable for detecting an enzymatic activity of a nitrile-related enzyme wherein the nitrile-related enzyme is selected from the group consisting of nitrilase, nitrile hydratase, and amidase.
3. The substrate of claim 2 , wherein R 1 in formula (I) is —CN or —CH═CH—CN, and the nitrile-related enzyme is nitrilase or nitrile hydratase.
4. The substrate of claim 2 , wherein R 1 in formula (I) is —CONH 2 or —CH═CH—CONH 2 , and the nitrile-related enzyme is amidase.
5. A kit comprising the compound of claim 1 .
6. The kit of claim 5 , further comprising a cell lysis solution, a buffer and/or instructions for use.
7. A method for detecting enzymatic activity of a nitrile-related enzyme, comprising:
(a) contacting the nitrile-related enzyme with the compound of claim 1 , wherein the nitrile-related enzyme is selected from the group consisting of nitrilase, nitrile hydratase, and amidase and
(b) measuring the optical intensity of an emitted wavelength generated as a result of step (a), wherein an increase in the intensity of the emitted wavelength positively correlates to nitrile-related enzymatic activity.
8. The method of claim 7 , wherein R 1 in formula (I) is —CN or —CH═CH—CN, and the nitrile-related enzyme is nitrilase or nitrile hydratase.
9. The method of claim 7 , wherein R 1 in formula (I) is —CONH 2 or —CH═CH—CONH 2 , and the nitrile-related enzyme is amidase.
10. The method of claim 7 , wherein measuring step (b) comprises flow cytometry.