Quinone-masked probes as labeling reagents for cell uptake measurements
View Patent ↗Provided are labeling reagents and methods of using the reagents for cell uptake measurements. The labeling reagents can be quinone-masked probes including fluorophores and/or luminophores.
1. A compound of formula (I), or a salt thereof,
wherein
A is a reporter moiety;
R 14 is H, alkyl, hydroxyalkyl, alkoxy, carboxyalkyl, or amidoalkyl;
R 9 and R 10 are independently selected from alkyl;
R 11 , R 12 and R 13 are independently selected from alkyl;
X is O;
L is —(CH 2 ) m C(R 17 ) 2 (CH 2 ) n —Y—C(O)—;
R 17 is independently H, 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 ;
R 5 is -alkyl-amide, wherein the -alkyl-amide is -alkyl-CON(R 32 )(R 33 ) or -alkyl-(CO)—NR 34 —(CR a R b ) p —NR 35 (CO)-T, wherein
R 32 and R 33 are each independently selected from hydrogen, alkyl, carboxy, aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heterocycle, alkene, polyol, alkenylpolylol, a peptide, a drug, a derivative of a drug, a biologically active moiety, and a dye;
R 34 and R 35 are each independently selected from hydrogen, and alkyl;
R a and R b are each independently selected from hydrogen, alkyl, and carboxy;
p is 0 to 6; and
T is selected from aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hydroxyalkyl, protein, polypeptide, polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof;
or
R 15 is polyalkoxyalkyl, wherein the polyalkoxyalkyl is —(C 2 -C 6 -alkoxy) x -alkyl-CON(R 32 )(R 33 ) or —(C 2 -C 6 -alkoxy) x -alkyl-(CO)—NR 34 -(CR a R b ) p —NR 35 (CO)-T;
x is an integer selected from 1 to 20;
R 32 and R 33 are each independently selected from hydrogen, alkyl, carboxy, aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hydroxyalkyl, protein, polypeptide, polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof;
R 34 and R 35 are each independently selected from hydrogen, and alkyl;
R a and R b are each independently selected from hydrogen, alkyl, and carboxy;
p is 0 to 6; and
T is selected from aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hydroxyalkyl, protein, polypeptide, polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof;
or
R 15 is
wherein
R 40 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, and cycloalkyl, wherein said aryl, heteroaryl, heterocyclyl, and cycloalkyl are unsubstituted or substituted with one or more suitable substituents; and
x is an integer selected from 1 to 20.
2. The compound of claim 1 , wherein R 15 is polyalkoxyalkyl, wherein the polyalkoxyalkyl is —(C 2 -C 6 -alkoxy) x -alkyl-CON(R 32 )(R 33 ) or —(C 2 -C 6 -alkoxy) x -alkyl-(CO)—NR 34 —(CR a R b ) p —NR 35 (CO)-T;
x is an integer selected from 1 to 20;
R 32 and R 33 are each independently selected from hydrogen, alkyl, carboxy, aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hydroxyalkyl, protein, polypeptide, polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof;
R 34 and R 35 are each independently selected from hydrogen, and alkyl;
R a and R b are each independently selected from hydrogen, alkyl, and carboxy;
p is 0 to 6; and
T is selected from aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hydroxyalkyl, protein, polypeptide, polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof.
3. The compound of claim 1 , wherein R 15 is
wherein
T is selected from aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hydroxyalkyl, protein, polypeptide, polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof; and
x is an integer selected from 1 to 20.
4. The compound of claim 1 , wherein R 15 is -alkyl-amide, wherein the -alkyl-amide is -alkyl-CON(R 32 )(R 33 ) or -alkyl-(CO)—NR 34 —(CR a R b ) p —NR 35 (CO)-T, wherein
R 32 and R 33 are each independently selected from hydrogen, alkyl, carboxy, aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heterocycle, alkene, polyol, alkenylpolylol, a peptide, a drug, a derivative of a drug, a biologically active moiety, and a dye;
R 34 and R 35 are each independently selected from hydrogen, and alkyl;
R a and R b are each independently selected from hydrogen, alkyl, and carboxy;
p is 0 to 6; and
T is selected from aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hydroxyalkyl, protein, polypeptide, polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof.
5. The compound of claim 1 , wherein R 5 is
wherein
T is selected from aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heterocycle, hydroxyalkyl, protein, polypeptide, polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof; and
x is 0 to 20.
6. The compound of claim 1 , wherein R 15 is
wherein
R 40 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, and cycloalkyl, wherein said aryl, heteroaryl, heterocyclyl, and cycloalkyl are unsubstituted or substituted with one or more suitable substituents; and
x is an integer selected from 1 to 20.
7. The compound of claim 6 , wherein R 40 is a 5- or 6-membered heterocyclyl, having 1, 2, or 3 heteroatoms independently selected from the group consisting of N, O, and S.
8. The compound of claim 6 , having formula (I-vi), or a salt thereof,
9. The compound of claim 8 , wherein
R 9 , R 10 , R 11 , R 12 , R 13 , and R 14 are methyl;
x is 3;
R 40 is hydrogen; and
A is
10. The compound of claim 1 , selected from the group consisting of:
or a salt thereof.
11. The compound of claim 1 , wherein the reporter moiety comprises a bioluminescent reporter moiety or fluorescent reporter moiety.
12. The compound of claim 11 , wherein the bioluminescent reporter moiety comprises a substrate for a luciferase.
13. The compound of claim 11 , wherein the fluorescent reporter moiety comprises a fluorophore.
14. A method for evaluating cellular uptake of an agent, the method comprising:
a) contacting a sample with a labeled agent, wherein the labeled agent comprises a compound of claim 1 , wherein the sample comprises a cell; and
b) detecting light emission,
whereby the detection of light emission indicates cellular uptake of the agent.
15. The method of claim 14 , wherein the cellular uptake of the agent results in the reduction of the compound and the generation of a released reporter moiety.
16. The method of claim 15 , wherein the reporter moiety comprises a fluorescent reporter moiety.
17. The method of claim 16 , wherein detecting light emission comprises exposing the sample to a wavelength of light and detecting fluorescence level in the sample, wherein an increase in fluorescence or a change in fluorescence wavelength as compared to the fluorescence or fluorescence wavelength of a control sample indicates cellular uptake of the agent.
18. The method of claim 15 , wherein the reporter moiety comprises a bioluminescent reporter moiety.
19. The method of claim 18 , wherein the bioluminescent reporter moiety comprises a substrate for a luciferase.
20. The method of claim 19 , further comprising contacting the sample with a luciferase and wherein detecting light emission comprises detecting the luminescence produced by the luciferase utilizing the released reporter moiety.
21. The method of claim 14 , wherein the cell is a eukaryotic cell or a prokaryotic cell.
22. A labeled agent comprising a compound according to claim 1 conjugated to an agent.
23. The labeled agent of claim 22 , wherein the agent is selected from the group consisting of a nucleoside, a nucleotide, a polynucleotide, a polypeptide, a polypeptide-based toxin, an amino acid, a lipid, a carbohydrate, an enzyme substrate, and combinations thereof.
24. The labeled agent of claim 22 , wherein the agent is selected from the group consisting of a peptide, an antibody, a lipoprotein, a sugar, a fatty acid, and a detergent.
25. The labeled agent of claim 22 , wherein the agent is selected from the group consisting of a therapeutic drug, a small molecule, and a nanoparticle, and combinations thereof.
26. A kit comprising the compound of claim 1 , an agent, or any combination thereof.
27. The kit of claim 26 , further comprising a detection reagent.
28. The kit of claim 27 , wherein the detection reagent comprises a luciferase enzyme.
29. A kit comprising a labeled agent according to claim 22 and at least one additional component.
30. The kit of claim 29 , wherein the at least one additional component comprises a detection reagent.
31. The kit of claim 30 , wherein the detection reagent comprises a luciferase enzyme.