Compounds and methods for assaying redox state of metabolically active cells and methods for measuring NAD(P)/NAD(P)H

Patent metadata

Field	Value
Publication number	US-9951372-B2
Application number	US-201314032420-A
Country	US
Kind code	B2
Filing date	Sep 20, 2013
Priority date	Sep 2, 2011
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

The present invention provides compounds and methods for assaying redox state of metabolically active cells and methods for assaying enzyme activity and/or metabolite level by coupling to redox defining co-factor NAD(P)/NAD(P)H measurement.

First claim

The invention claimed is:

1. A method for detecting cellular metabolites in a sample comprising,
a. contacting the sample with a compound of formula (II), (III), or (IV), a dehydrogenase amplification enzyme system, NAD or NADP, a diaphorase that utilizes NADH or NADPH as a co-factor and the compound of formula (II), (III), or (IV) as a diaphorase substrate, and a luciferase reaction mixture; and
b. detecting bioluminescence;
wherein the compound of formula (II) is

wherein R 1 is H, C 1-4 alkyl, C 1-4 hydroxylalkyl, C 3-7 cyclic ring, aryl, benzyl or substituted benzyl ring, heterocycle, heteroaryl or —(CH 2 ) n′ —P(Ph) 3 ; R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl; and n′ is an integer from 2-7;
the compound of formula (III) is

wherein R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl;
the compound of formula (IV) is

wherein R 2 is —CH 2 -aryl or —CH 2 -heteroaryl; R 5 is —CH 2 -aryl or —CH 2 -heteroaryl; and R 7 is aryl or heteroaryl;
wherein R 14 is H, C 1-4 alkyl, C 1-4 alkoxyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide;
R 9 and R 10 are independently selected from C 1-4 alkyl;
R 11 , R 12 and R 13 are independently selected from H, C 1-4 alkyl, C 1-4 alkoxyl, bromo, chloro or amino, or R 11 and R 12 can form a fused phenyl ring;
X is O, NH or a direct bond;
L is a direct bond or —C 6 (R 16 ) 4 CH 2 — or —(CH 2 ) m C(R 17 ) 2 (CH 2 ) n —Y—C(O)—;
wherein at least one of X or L is not a direct bond when the compound has formula (II);
R 16 is independently H, halogen, CH 3 , OCH 3 , or NO 2 ;
R 17 is independently H, C 1-4 alkyl or both R 17 together can form an alkyl ring having from 3-7 carbons;
m is an integer from 0-2;
n is an integer from 0-2;
Y is O or NR 15 ; and
R 15 is H, C 1-4 alkyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide.

2. The method of claim 1 , wherein the compound, dehydrogenase amplification enzyme, diaphorase, NAD or NADP and luciferase reaction mixture are in a single composition.

3. A method of detecting NADH or NADPH in a sample comprising:
a. contacting the sample with a compound of formula (II), (III), or (IV), a diaphorase that utilizes NADH or NADPH as a co-factor and the compound of formula (II), (III), or (IV) as a diaphorase substrate and a luciferase reaction mixture; and
b. detecting bioluminescence;
wherein the compound of formula (II) is

wherein R 1 is H, C 1-4 alkyl, C 1-4 hydroxylalkyl, C 3-7 cyclic ring, aryl, benzyl or substituted benzyl ring, heterocycle, heteroaryl or —(CH 2 ) n′ —P(Ph) 3 ; R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl; and n′ is an integer from 2-7;
the compound of formula (III) is

wherein R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl;
the compound of formula (IV) is

wherein R 2 is —CH 2 -aryl or —CH 2 -heteroaryl; R 5 is —CH 2 -aryl or —CH 2 -heteroaryl; and R 7 is aryl or heteroaryl;
wherein R 14 is H, C 1-4 alkyl, C 1-4 alkoxyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide;
R 9 and R 10 are independently selected from C 1-4 alkyl;
R 11 , R 12 and R 13 are independently selected from H, C 1-4 alkyl, C 1-4 alkoxyl, bromo, chloro or amino, or R 11 and R 12 can form a fused phenyl ring;
X is O, NH or a direct bond;
L is a direct bond or —C 6 (R 16 ) 4 CH 2 — or —(CH 2 ) m C(R 17 ) 2 (CH 2 ) n —Y—C(O)—;
wherein at least one of X or L is not a direct bond when the compound has formula (II);
R 16 is independently H, halogen, CH 3 , OCH 3 , or NO 2 ;
R 17 is independently H, C 1-4 alkyl or both R 17 together can form an alkyl ring having from 3-7 carbons;
m is an integer from 0-2;
n is an integer from 0-2;
Y is O or NR 15 ; and
R 15 is H, C 1-4 alkyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide.

4. The method of claim 3 , wherein the sample is further contacted with a diaphorase inhibitor.

5. The method of claim 3 , wherein the compound, diaphorase and luciferase reaction mixture are in a single composition.

6. A method of determining total NAD(P)/NAD(P)H in a sample comprising:
a. contacting the sample with a dehydrogenase amplification enzyme system, a compound of formula (II), (III) or (IV), a diaphorase that utilizes NADH or NADPH as a co-factor and the compound of formula (II), (III), or (IV) as a diaphorase substrate, and a luciferase reaction mixture; and
b. detecting bioluminescence;
wherein the compound of formula (II) is

wherein R 1 is H, C 1-4 alkyl, C 1-4 hydroxylalkyl, C 3-7 cyclic ring, aryl, benzyl or substituted benzyl ring, heterocycle, heteroaryl or —(CH 2 ) n′ —P(Ph) 3 ; R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl; and n′ is an integer from 2-7;
the compound of formula (III) is

wherein R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl;
the compound of formula (IV) is

wherein R 2 is —CH 2 -aryl or —CH 2 -heteroaryl; R 5 is —CH 2 -aryl or —CH 2 -heteroaryl; and R 7 is aryl or heteroaryl;
wherein R 14 is H, C 1-4 alkyl, C 1-4 alkoxyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide;
R 9 and R 10 are independently selected from C 1-4 alkyl;
R 11 , R 12 and R 13 are independently selected from H, C 1-4 alkyl, C 1-4 alkoxyl, bromo, chloro or amino, or R 11 and R 12 can form a fused phenyl ring;
X is O, NH or a direct bond;
L is a direct bond or —C 6 (R 16 ) 4 CH 2 — or —(CH 2 ) m C(R 17 ) 2 (CH 2 ) n —Y—C(O)—;
wherein at least one of X or L is not a direct bond when the compound has formula (II);
R 16 is independently H, halogen, CH 3 , OCH 3 , or NO 2 ;
R 17 is independently H, C 1-4 alkyl or both R 17 together can form an alkyl ring having from 3-7 carbons;
m is an integer from 0-2;
n is an integer from 0-2;
Y is O or NR 15 ; and
R 15 is H, C 1-4 alkyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide.

7. The method of claim 6 , wh

Assignees

• Promega Corp

Inventors

• Zhou Wenhui
• Vidugiriene Jolanta
• Benink Helene A
• Cali James J
• Duellman Sarah
• Klaubert Dieter
• Leippe Donna
• O'Brien Martha
• Shultz John
• Sobol Mary

Classifications

• G01N33/582

  with fluorescent label · CPC title

• C12Q1/32 Primary

  involving dehydrogenase · CPC title

• C07D417/04

  directly linked by a ring-member-to-ring-member bond · CPC title

• C07D487/04 Primary

  Ortho-condensed systems · CPC title

• G01N33/5735

  co-enzymes or co-factors, e.g. NAD or ATP · CPC title