Fluorescein lactone ion indicators and their applications

1. A method of detecting intracellular calcium, the method comprising:

contacting a sample comprising a cell with a compound;

incubating the sample for a time sufficient for the compound to be loaded into the cell and an indicator compound to be generated intracellularly;

illuminating the sample at a wavelength that generates a fluorescence response from the indicator compound;

detecting a fluorescence response from the indicator compound;

wherein the compound is described by of one of the following formulas 1, 2, 3 or 4:

wherein R 1 -R 12 are independently H, halogen, carboxy, alkoxy, arlyoxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionally substituted one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; A is an alkyl having 1-10 carbons; and Z is an acyloxymethyl having 1-10 carbons.

2. The method of claim 1 , wherein A is methyl and Z is acetoxymethyl.

3. The method of claim 2 , wherein R 1 and R 3 , R 4 and R 6 -R 8 are each hydrogen; R 9 -R 12 are independently hydrogen, halogen, nitro or alkyl; and R 2 and R 5 are independently hydrogen, fluoro or chloro.

4. A method of monitoring intracellular calcium comprising:

a) contacting a sample containing a cell with a compound;

b) incubating the sample for a time sufficient for the compound to be loaded into the cell and an indicator compound to be generated intracellularly;

c) illuminating the sample at a wavelength that generates a fluorescence response from the indicator compound; and

d) detecting the fluorescence response from the indicator compound;

e) monitoring changes in the intensity of the fluorescence response from the indicator compound; and

f) correlating the changes in fluorescence intensity with changes in intracellular calcium levels;

wherein the compound is of one of the following formulas 1, 2, 3 or 4:

wherein R 1 -R 12 are independently H, halogen, carboxy, alkoxy, arlyoxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionally substituted one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; A is an alkyl having 1-10 carbons; and Z is an acyloxymethyl having 1-10 carbons.

5. The method of claim 4 , further comprising stimulating the cell.

6. The method of claim 5 , further comprising adding a cell-impermeant and a non-fluorescent dye to the sample.

7. The method of claim 2 , wherein R 1 and R 3 , R 4 and R 6 -R 12 are each hydrogen; and R 2 and R 5 are independently hydrogen, fluoro or chloro.

8. The method of claim 7 , wherein R 2 and R 5 are each fluoro.

9. The method of claim 7 , wherein the compound is of the formula:

10. The method of claim 4 , wherein A is methyl and Z is acetoxymethyl.

11. The method of claim 10 , wherein R 1 and R 3 , R 4 and R 6 -R 8 are each hydrogen; R 9 -R 12 are independently hydrogen halogen, nitro or alkyl; and R 2 and R 5 are independently hydrogen, fluoro or chloro.

12. The method of claim 11 , wherein R 1 and R 3 , R 4 and R 6 -R 12 are each hydrogen; and R 2 and R 5 are independently hydrogen, fluoro or chloro.

13. The method of claim 12 , wherein R 2 and R 5 are each fluoro.

14. The method of claim 12 , wherein the compound is of the formula:

15. The method of claim 7 , wherein R 2 and R 5 are each hydrogen.

16. The method of claim 3 , wherein R 10 is methyl and R 9 , R 11 and R 12 are each hydrogen.

17. The method of claim 12 , wherein R 2 and R 5 are each hydrogen.

18. The method of claim 11 , wherein R 10 is methyl and R 9 , R 11 and R 12 are each hydrogen.