Compositions and methods for stabilizing luminogenic substrates
View Patent ↗Disclosed herein are compositions and methods for stabilizing a luminogenic substrate such as coelenterazine or a functional analog thereof. The functional analog may be furimazine. The composition may include the luminogenic substrate, a thionucleoside, and an organic solvent, in which the thionucleoside is present in an amount effective to stabilize the luminogenic composition against decomposition. The method provided herein stabilizes the luminogenic substrate against decomposition by contacting the luminogenic substrate with an effective amount of the thionucleoside in the presence of the organic solvent. Also provided herein is a kit containing the composition.
1. A composition comprising
(a) a luminogenic substrate;
(b) an effective amount of a compound of formula (I) or a tautomer thereof,
wherein
R 1 is hydrogen, alkyl, substituted alkyl, alkyl-aryl, alkyl-heteroaryl, cycloalkyl, aryl, heteroaryl, carboxylic acid, ester, NR a R b , imine, hydroxyl, or oxo;
R 2 is hydrogen, NR a R b , imine, alkyl, or aryl;
R a and R b are each independently hydrogen, alkyl, or aryl; and
(c) an organic solvent;
wherein the luminogenic substrate is coelenterazine or a functional analog thereof.
2. The composition of claim 1 , wherein the composition does not contain a luminogenic enzyme.
3. The composition of claim 1 , wherein the luminogenic substrate is stabilized against decomposition in the presence of light, in the absence of light, or at temperatures from −80° C. to 60° C.
4. The composition of claim 1 , wherein the functional analog of coelenterazine is furimazine.
5. The composition of claim 1 , wherein the effective amount of the compound of formula (I) is greater than 0.1 mM.
6. The composition of claim 1 , wherein the compound of formula (I) is selected from the group consisting of: ATT, ATCA, 3-(4-Amino-5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoic acid, tetrahydro-2-methyl-3-thioxo-1,2,4-triazine-5,6-dione, 4-((2-furylmethylene)amino)-3-mercapto-6-methyl-1,2,4-triazin-5(41-1)-one, 6-benzyl-3-sulfanyl-1,2,4-triazin-5-ol, 4-amino-3-mercapto-6-methyl-1,2,4-triazin-5(4H)-one, 3-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoic acid, (E)-6-methyl-4-((thiophen-2-ylmethylene)amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one, (E)-6-methyl-4-((3-nitrobenzylidene)amino)-3-thioxo -3,4-dihydro-1,2,4-triazin-5(2H)-one, (E)-4-((4-(diethylamino)benzylidene)amino)-6-methyl-3-thioxo -3,4-dihydro-1,2,4-triazin-5(2H)-one, ATCA ethyl ester, TAK-0014, TAK-0002, TAK-0021, TAK -0020, TAK-0018, TAK-0009, TAK-0007, TAK-0008, TAK-0003, TAK-0004, 3-thioxo-6-(trifluoromethyl)-3,4-dihydro-1,2,4-triazin-5(2H)-one, 6-cyclopropyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one, and 6-(hydroxymethyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H) -one.
7. The composition of claim 6 , wherein the compound of formula (I) is ATT and wherein the effective amount of ATT is greater than 32 mM.
8. The composition of claim 1 , wherein the organic solvent is selected from the group consisting of alcohol, propylene glycol, dimethyl sulfoxide (DMSO), acetonitrile, glycerol, and any combination thereof.
9. The composition of claim 8 , wherein the organic solvent is a combination of ethanol and propylene glycol or a combination of ethanol and glycerol.
10. A method for stabilizing a luminogenic substrate, the method comprising contacting the luminogenic substrate with an effective amount of a compound of formula (I) or a tautomer thereof in the presence of an organic solvent, whereby the luminogenic substrate is stabilized against decomposition,
wherein the luminogenic substrate is coelenterazine or a functional analog thereof;
wherein the compound of formula (I) is
wherein
R 1 is hydrogen, alkyl, substituted alkyl, alkyl-aryl, alkyl-heteroaryl, cycloalkyl, aryl, heteroaryl, carboxylic acid, ester, NR a R b , imine, hydroxyl, or oxo;
R 2 is hydrogen, NR a R b , imine, alkyl, or aryl; and
R a and R b are each independently hydrogen, alkyl, or aryl.
11. The method of claim 10 , wherein the effective amount of the compound of formula (I) is greater than 0.1 mM.
12. The method of claim 10 , wherein the compound of formula (I) is selected from the group consisting of: ATT, ATCA, 3-(4-Amino-5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoic acid, tetrahydro-2-methyl-3-thioxo-1,2,4-triazine-5,6-dione, 4-((2-furylmethylene)amino)-3-mercapto-6-methyl-1,2,4-triazin-5(4H)-one, 6-benzyl-3-sulfanyl-1,2,4-triazin-5-ol, 4-amino-3-mercapto-6-methyl-1,2,4-triazin-5(4H)-one, 3-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoic acid, (E)-6-methyl-4-((thiophen-2-ylmethylene)amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one, (E)-6-methyl-4-((3-nitrobenzylidene)amino)-3-thioxo -3,4-dihydro-1,2,4-triazin-5(2H)-one, (E)-4-((4-(diethylamino)benzylidene)amino)-6-methyl-3-thioxo -3,4-dihydro-1,2,4-triazin-5(2H)-one, ATCA ethyl ester, TAK-0014, TAK-0002, TAK-0021, TAK -0020, TAK-0018, TAK-0009, TAK-0007, TAK-0008, TAK-0003, TAK-0004, 3-thioxo-6-(trifluoromethyl)-3,4-dihydro-1,2,4-triazin-5(2H) -one, 6-cyclopropyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H) -one, and 6-(hydroxymethyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H) -one.
13. The method of claim 12 , wherein the compound of formula (I) is ATT and wherein the effective amount of the ATT is greater than 32 mM.
14. The method of claim 10 , wherein luminogenic substrate is stabilized against decomposition in the presence of light, in the absence of light, or at temperatures from −80° C. to 60° C.
15. The method of claim 10 , wherein the functional analog of coelenterazine is furimazine.
16. The method of claim 10 , wherein the organic solvent is selected from the group consisting of alcohol, propylene glycol, dimethyl sulfoxide (DMSO), acetonitrile, glycerol, and any combination thereof.
17. A kit comprising the composition of claim 1 in a single container, wherein the compound of formula (I) stabilizes the luminogenic substrate.