Compounds to co-localize luminophores with luminescent proteins
View Patent ↗A method of measuring the enzymatic activity of a luciferase includes contacting a luminogenic protein, such as a luciferase, with a protected luminophore to form a composition; and detecting light produced from the composition. The protected luminophore provides increased stability and improved signal-to-background ratios relative to the corresponding unmodified coelenterazine.
1. A compound of formula (XII)
wherein
R 7 is H, alkyl, heteroalkyl, aryl, or —CH 2 —C 6 H 4 OR 14 ;
R 8 is H, alkyl, heteroalkyl, or aryl;
R 9 is H, alkyl, heteroalkyl, aryl, or —C 6 H 4 OR 15 ;
R 10 is —H, —CH 3 , or —CH(CH 3 ) 2 ;
R 14 and R 15 are each independently a protecting group; and
R 11 together with the oxygen atom to which it is attached is an ester or an acyloxymethyl ether;
with the proviso that R 11 , R 14 , and R 15 are not all acetyl groups.
2. The compound of claim 1 , wherein
R 7 is —CH 2 —C 6 H 5 , naphthyl, —CH 2 —C 6 H 4 OH, —CH 2 —C 6 H 4 F, or —CH 2 —C 6 H 4 OR 14 ;
R 8 is —CH 2 C 6 H 5 , —CH 2 C 6 H 11 , —CH 2 C 5 H 9 , or —(CH 2 ) 3 NHC(═NH)NH 2 ; and
R 9 is phenyl, indolyl, —C 6 H 4 OH, —C 6 H 4 NH 2 , —C 6 H 4 F, or —C 6 H 4 OR 15 .
3. The compound of claim 1 , wherein —OR 11 , —OR 14 , and —OR 15 are each independently esters.
4. The compound of claim 1 , wherein
R 11 is acetyl; and
R 14 and R 15 are independently butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, or pivaloyloxymethyl.
5. The compound of claim 1 , wherein
R 11 is butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, or pivaloyloxymethyl; and
R 14 and R 15 are independently acetyl, butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, or pivaloyloxymethyl.
6. The compound of claim 5 , wherein
R 7 is —CH 2 —C 6 H 5 , naphthyl, —CH 2 —C 6 H 4 OH, —CH 2 —C 6 H 4 F, or —CH 2 —C 6 H 4 OR 14 ; and
R 8 is —CH 2 C 6 H 5 , —CH 2 C 6 H 11 , —CH 2 C 5 H 9 , or —(CH 2 ) 3 NHC(═NH)NH 2 ; and R 9 is —C 6 H 4 OR 15 .
7. The compound of claim 5 of the formula:
8. A composition, comprising:
the compound of claim 1 in solution.
9. The composition of claim 8 , wherein the solution is an aqueous solution.
10. The composition of claim 8 , wherein the solution comprises DMSO or alcohol.
11. The compound of claim 1 , wherein
R 14 and R 15 are each independently selected from the group consisting of an alkyl group containing from 1-20 carbon atoms and a heteroalkyl group containing from 1-20 carbon atoms.
12. The compound of claim 1 , wherein
R 14 and R 15 are independently selected from the group consisting of an alkyl group containing from 1-15 carbon atoms and a heteroalkyl group containing from 1-15 carbon atoms.
13. The compound of claim 1 , wherein
R 14 and R 15 are independently a heteroalkyl group containing from 1-20 carbon atoms, and wherein —OR 14 and —OR 15 are each independently an ester group or an ether group.
14. The compound of claim 1 , wherein the protecting group is selected from the group consisting of ester, ether, phosphoryl and glucosyl.
15. The compound of claim 1 of the formula:
16. The compound of claim 1 wherein R 11 , R 14 , and R 15 are each independently selected from the group consisting of acetyl, butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, pivaloyloxymethyl and t-butyryl.
17. The compound of claim 16 , wherein
R 7 is —CH 2 —C 6 H 5 , naphthyl, —CH 2 —C 6 H 4 OH, —CH 2 —C 6 H 4 F, or —CH 2 —C 6 H 4 OR 14 ;
R 8 is —CH 2 C 6 H 5 , —CH 2 C 6 H 11 , —CH 2 C 5 H 9 , or —(CH 2 ) 3 NHC(═NH)NH 2 ; and
R 9 is phenyl, indolyl, —C 6 H 4 OH, —C 6 H 4 NH 2 , —C 6 H 4 F, or —C 6 H 4 OR 15 .
18. A compound of formula (XII)
wherein R 7 is H, alkyl, heteroalkyl, aryl, or —CH 2 —C 6 H 4 OR 14 ;
R 8 is H, alkyl, heteroalkyl, or aryl;
R 9 is H, alkyl, heteroalkyl, aryl, or —C 6 H 4 OR 15 ;
R 10 is —H, —CH 3 , or —CH(CH 3 ) 2 ; and
R 14 and R 15 are each independently a protecting group
R 11 together with the oxygen atom to which it is attached is an ester or an acyloxymethyl ether; and
wherein the concentration of the compound in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. is reduced by less than 50% after 45 minutes.
19. The compound of claim 18 , wherein
R 7 is —CH 2 —C 6 H 5 , naphthyl, —CH 2 —C 6 H 4 OH, —CH 2 —C 6 H 4 F, or —CH 2 —C 6 H 4 OR 14 ;
R 8 is —CH 2 C 6 H 5 , —CH 2 C 6 H 11 , —CH 2 C 5 H 9 , or —(CH 2 ) 3 NHC(═NH)NH 2 ; and
R 9 is phenyl, indolyl, —C 6 H 4 OH, —C 6 H 4 NH 2 , —C 6 H 4 F, or —C 6 H 4 OR 15 .
20. The compound of claim 18 , wherein —OR 11 , —OR 14 , and —OR 15 are independently esters.
21. The compound of claim 18 , wherein R 11 , R 14 , and R 15 are independently acetyl, butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, or pivaloyloxymethyl.
22. A composition, comprising:
the compound of claim 18 , in solution.
23. The composition of claim 22 , wherein the solution is an aqueous solution.
24. The composition of claim 22 , wherein the solution comprises DMSO or alcohol.
25. A compound of formula (XII)
wherein R 7 is H, alkyl, heteroalkyl, aryl, or —CH 2 —C 6 H 4 OR 14 ;
R 8 is H, alkyl, heteroalkyl, or aryl;
R 9 is H, alkyl, heteroalkyl, aryl, or —C 6 H 4 OR 15 ;
R 10 is —H, —CH 3 , or —CH(CH 3 ) 2 ; and
R 11 , R 14 , and R 15 are each independently a protecting group;
R 11 together with the oxygen atom to which it is attached is an ester or an acyloxymethyl ether; and
wherein the removal of at least one protecting group provides a parent compound; and
wherein the time necessary for the concentration of the compound in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. to be reduced by 50% is greater than the time necessary for the concentration of the parent compound in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. to be reduced by 50%.
26. The compound of claim 25 , wherein the removal of at least two protecting groups provides the parent compound.
27. The compound of claim 25 , wherein the removal of all protecting groups provides the parent compound.
28. The compound of claim 25 , wherein
R 7 is —CH 2 —C 6 H 5 , naphthyl, —CH 2 —C 6 H 4 OH, —CH 2 —C 6 H 4 F, or —CH 2 —C 6 H 4 OR 14 ;
R 8 is —CH 2 C 6 H 5 , —CH 2 C 6 H 11 , —CH 2 C 5 H 9 , or —(CH 2 ) 3 NHC(═NH)NH 2 ; and
R 9 is phenyl, indolyl, —C 6 H 4 OH, —C 6 H 4 NH 2 , —C 6 H 4 F, or —C 6 H 4 OR 15 .
29. The compound of claim 25 , wherein —OR 11 , —OR 14 , and —OR 15 are each independently esters.
30. The compound of claim 25 , wherein R 11 , R 14 , and R 15 are each independently acetyl, butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, or pivaloyloxymethyl.
31. A composition, comprising:
the compound of claim 25 , in solution.
32. The composition of claim 31 , wherein the solution is an aqueous solution.
33. The composition of claim 31 , wherein the solution comprises DMSO or alcohol.
34. The compound of claim 25 , wherein
R 14 and R 15 are independently selected from the group consisting of an alkyl group containing from 1-20 carbon atoms and a heteroalkyl group containing from 1-20 carbon atoms.
35. The compound of claim 25 , wherein
R 14 and R 15 are independently a heteroalkyl group containing from 1-20 carbon atoms, and wherein —OR 14 and —OR 15 are each independently an ester group or an ether group.
36. A compound of formula (XIII) or (XIV)
wherein R 7 is H, alkyl, heteroalkyl, aryl, or —CH 2 —C 6 H 4 OR 14 ;
R 8 is H, alkyl, heteroalkyl, or aryl;
R 12 and R 13 are independently —H, —OH, alkyl, heteroalkyl, aryl, or —OR 16 ;
n is 0, 1, or 2;
R 14 and R 16 are independently a protecting group; and
R 11 together with the oxygen atom to which it is attached is an ester or an acyloxymethyl ether.
37. The compound of claim 36 , wherein
R 7 is —CH 2 —C 6 H 5 , naphthyl, —CH 2 —C 6 H 4 OH, —CH 2 —C 6 H 4 F, or —CH 2 —C 6 H 4 OR 14 ; and
R 8 is —CH 2 C 6 H 5 , —CH 2 C 6 H 11 , —CH 2 C 5 H 9 , or —(CH 2 ) 3 NHC(═NH)NH 2 .
38. The compound of claim 36 , wherein —OR 11 , —OR 14 , and —OR 15 are each independently esters.
39. The compound of claim 36 , wherein R 11 , R 14 , and R 16 are each independently acetyl, butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, or pivaloyloxymethyl.
40. The compound of claim 36 , wherein n is 1.
41. A composition, comprising:
the compound of claim 36 , in solution.
42. The composition of claim 41 , wherein the solution is an aqueous solution.
43. The composition of claim 41 , wherein the solution comprises DMSO or alcohol.
44. The compound of claim 36 , wherein
R 14 and R 16 are independently selected from the group consisting of an alkyl group containing from 1-20 carbon atoms and a heteroalkyl group containing from 1-20 carbon atoms.
45. The compound of claim 36 , wherein
R 14 and R 16 are independently a heteroalkyl group containing from 1-20 carbon atoms, and wherein —OR 14 and —OR 15 are each independently an ester group or an ether group.
46. A protected luminophore, which is
a protected coelenterazine that includes an imidazolo oxygen; protected with a protecting group;
wherein the protecting group together with the imidazole oxygen to which it is attached, form an ester or an acyloxymethyl ether;
wherein subsequent removal of said protecting group provides a parent coelenterazine; and
wherein the time necessary for the concentration of the protected coelenterazine in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. to be reduced by 50% is greater than the time necessary for the concentration of the parent coelenterazine in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. to be reduced by 50%.
47. A compound of the formula:
where R 11 together with the oxygen atom to which it is attached is an ester or an acyloxymethyl ether;
R 15 is a heteroalkyl group containing from 1-20 carbon atoms, and wherein —OR 15 is an ester group or an ether group.
48. The compound of claim 47 , wherein R 11 and R 15 are each independently selected from the group consisting of acetyl, butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, pivaloyloxymethyl and t-butyryl.