Fluorescent substrate for detection of enzymatic activity of nitrile-related enzyme
View Patent ↗The object of the invention is to provide a fluorescent substrate for detecting the enzymatic activity of a nitrile-related enzyme and compound represented by formula (I) and a fluorescent substrate for detecting the enzymatic activity of a nitrile-related enzyme, which includes 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 halobenzyl 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 compound consisting of formula (II), a salt thereof, or a hydrate thereof:
3. A method for detecting nitrile-related enzymatic activity of a test substance, the method comprising:
(a) reacting the test substance with the compound of claim 1 , a salt thereof or a hydrate thereof; and
(b) measuring optical intensity of an emitted wavelength generated-as a result of the reacting,
wherein an increase in the measured optical intensity compared to the compound without reacting to the test substance is indicative of nitrile-related enzymatic activity.
4. The method of claim 3 , wherein the nitrile-related enzymatic activity is an activity of one enzyme selected from the group consisting of nitrilase, nitrile hydratase, and amidase.
5. The method of claim 3 , wherein:
R 1 is —CN or —CH═CH—CN; and
the enzymatic activity is the activity of nitrilase or nitrile hydratase.
6. The method of claim 3 , wherein:
R 1 is —CONH 2 or —CH═CH—CONH 2 ; and
the enzymatic activity is the activity of amidase.
7. The method of claim 3 , wherein the measuring comprises flow cytometry.
8. A fluorescent substrate comprising the compound of claim 1 , a salt thereof, or a hydrate thereof, wherein the substrate is suitable for detecting an enzymatic activity of a nitrile-related enzyme.
9. The substrate of claim 8 , wherein the nitrile-related enzyme is a member selected from the group consisting of nitrilase, nitrile hydratase, and amidase.
10. The substrate of claim 8 , wherein:
R 1 is —CN or —CH═CH—CN; and
the nitrile-related enzyme is nitrilase or nitrile hydratase.
11. The substrate of claim 8 , wherein:
R 1 is —CONH 2 or —CH═CH—CONH 2 ; and
the nitrile-related enzyme is amidase.
12. A kit, comprising the compound of claim 1 .
13. The kit of claim 12 , further comprising a cell lysis solution, a buffer and instructions.
14. A method for detecting nitrile-related enzymatic activity of a test substance, the method comprising:
(a) reacting the test substance with the compound of claim 2 , a salt thereof or a hydrate thereof; and
(b) measuring optical intensity of an emitted wavelength generated-as a result of the reacting,
wherein an increase in the measured optical intensity compared to the compound without reacting to the test substance is indicative of nitrile-related enzymatic activity.
15. A fluorescent substrate comprising the compound of claim 2 , a salt thereof, or a hydrate thereof, wherein the substrate is suitable for detecting an enzymatic activity of a nitrile-related enzyme.
16. A kit, comprising the compound of claim 2 .
17. The kit of claim 16 , further comprising a cell lysis solution, a buffer, or a combination thereof and instructions.