Methods of measuring luminogenic activity with a protected coelenterazine

1. A method of measuring luminogenic activity of a luminogenic protein comprising:

contacting a luminogenic protein, a deprotecting enzyme, and a protected luminophore in solution to form a composition; and

detecting light produced from the composition;

wherein the protected luminophore includes an imidazolone oxygen protected with a protecting group, and the protecting group together with the imidazolone oxygen to which it is attached is an ester, an acetyl, a butyryl, an acetoxymethyl, a propanoyloxymethyl, a butyryloxymethyl, a pivaloyloxymethyl group or —C(═O)—C(CH 3 ) 3 ;

wherein the deprotecting enzyme is capable of removing the protecting group from the protected luminophore.

2. The method of claim 1 , wherein the luminogenic protein is Renilla luciferase.

3. The method of claim 1 , wherein the protected luminophore is 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, an acetyl, a butyryl, an acetoxymethyl, a propanoyloxymethyl, a butyryloxymethyl, a pivaloyloxymethyl group or —C(═O)—C(CH 3 ) 3 .

4. The method of claim 3 , 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 .

5. The method of claim 3 , wherein —OR 11 , —OR 14 , and —OR 15 are each independently esters.

6. The method of claim 3 , wherein R 11 , R 14 , and R 15 are independently acetyl, butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, or pivaloyloxymethyl.

7. The method of claim 6 wherein the protected luminophore is a compound of formula:

8. The method of claim 3 , wherein the protected luminophore is a compound of formula:

9. The method of claim 1 , wherein the protected luminophore is 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 each independently a protecting group; and

R 11 together with the oxygen atom to which it is attached is an ester, an acetyl, a butyryl, an acetoxymethyl, a propanoyloxymethyl, a butyryloxymethyl, a pivaloyloxymethyl group or —C(═O)—C(CH 3 ) 3 .

10. The method of claim 9 , 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 .

11. The method of claim 9 , wherein —OR 11 , —OR 14 , and —OR 16 are each independently esters.

12. The method of claim 9 , wherein R 11 , R 14 , and R 16 are independently acetyl, butyryl, acetoxymethyl, propanoyloxymethyl, butyryloxymethyl, or pivaloyloxymethyl.

13. The method of claim 9 , wherein n is 1.

14. The method of claim 1 , wherein the composition comprises a cell.

15. The method of claim 1 , wherein the composition comprises a cell which contains the deprotecting enzyme.

16. The method of claim 15 , wherein detecting light produced from the composition indicates the location of the deprotecting enzyme in a cell.

17. The method of claim 1 , wherein the composition comprises a cell lysate.

18. The method of claim 1 , wherein the deprotecting enzyme is an esterase.

19. The method of claim 1 , wherein the solution is an aqueous solution.

20. The method of claim 1 , wherein the solution comprises DMSO.

21. The method of claim 1 , wherein the protected luminophore is a modified coelenterazine;

wherein the enol group has been converted to an ester or an ether comprising an enzyme-removable group.

22. A method of generating luminescence in a living cell comprising a luciferase, the method comprising:

contacting the cell in solution with a protected luminophore;

wherein the protected luminophore includes an imidazolone oxygen protected with a protecting group, and the protecting group together with the imidazolone oxygen to which it is attached is an ester, an acetyl, a butyryl, an acetoxymethyl, a propanoyloxymethyl, a butyryloxymethyl, a pivaloyloxymethyl group or —C(═O)—C(CH 3 ) 3 .

23. The method of claim 22 , wherein the protected luminophore is a modified coelenterazine;

wherein the enol group has been converted to an ester or an ether comprising an enzyme-removable group.

24. The method of claim 22 , wherein the protected luminophore is 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; and

R 11 together with the oxygen atom to which it is attached is an ester, an acetyl, a butyryl, an acetoxymethyl, a propanoyloxymethyl, a butyryloxymethyl, a pivaloyloxymethyl group or —C(═O)—C(CH 3 ) 3 .

25. The method of claim 22 , wherein the protected luminophore is 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 each independently —H, —OH, alkyl, heteroalkyl, aryl, or —OR 16 ;

n is 0, 1, or 2;

R 14 and R 16 are each independently a protecting group; and

R 11 together with the oxygen atom to which it is attached is an ester, an acetyl, a butyryl, an acetoxymethyl, a propanoyloxymethyl, a butyryloxymethyl, a pivaloyloxymethyl group or —C(═O)—C(CH 3 ) 3 .