Benzoxazole-based fluorescent metal ion indicators

1. A method of measuring the concentration of metal ions in a sample, comprising:

a) combining a fluorescent compound comprising a 2-substituted benzoxazole group attached to a metal ion binding group with a sample known or suspected to contain a metal ion in an amount sufficient to generate a detectable fluorescent response to the metal ion;

b) illuminating the sample to generate a fluorescence excitation or emission response;

c) observing the absorbance or emission response; and

d) quantifying the fluorescence excitation or emission response of the compound to determine the concentration of metal ions in the sample.

2. The method of claim 1 , wherein the compound generates a fluorescence excitation or emission response in the visible region of the electromagnetic spectrum.

3. The method of claim 1 , wherein the compound exhibits a change in emission wavelength maximum upon binding to the metal ion.

4. The method of claim 1 , wherein the compound exhibits a change in excitation wavelength maximum upon binding to the metal ion.

5. The method of claim 3 or 4 , wherein the excitation or emission wavelength maximum shifts about 20 nm or greater upon metal ion binding.

6. The method of claim 1 , wherein the compound comprises a BAPTA crown ether or cryptand group.

7. The method of claim 1 , wherein the metal ion is monovalent.

8. The method of claim 7 , wherein the monovalent metal ion is selected from the group consisting of Na + , Li + , K + , Tl + , trialkylammonium and tetraalkylammonium.

9. The method of claim 1 , wherein the metal ion is divalent.

10. The method of claim 9 , wherein the divalent metal ion is selected from the group consisting of Ca 2+ , Zn 2+ , Mg 2+, Pb 2+ , Hg 2+ and Pb 2+ .

11. The method of clam 1 , wherein the fluorescent compound is water-soluble.

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

13. The method of claim 1 , wherein the sample comprises living cells or biological fluids.

14. The method of claim 1 , wherein the fluorescent compound is permeant to membranes of biological cells.

15. The method of claim 1 , wherein the compound has the formula:

or a salt thereof,

wherein M is —NCH 2 -Py or —N(CH 2 COOR 5 ) 2 , wherein R 5 is H, an alkyl having 1-6 carbons, —CH 2 OCOCH 3 , or a counterion;

Y is —CH 3 , —CH 2 COOH, —CH 2 COOPy, or

X is —N(CH 2 COOR 5 ) 2 , —NCH 2 Py, —OCH 2 Py, or —OCH 2 COOR 5 ;

R 7 , R 8 , R 9 , and R 10 are independently selected from the group consisting of an alkyl having 1-6 carbons, halogen, amino, nitro, cyano, trifluoromethyl, sulfo, and sulfonamide;

R 1 and R 2 are independently H, an alkyl having 1-6 carbons, halogen, or sulfo;

R 4 is a 5 or 6-membered aromatic ring or a fused ring system comprising at least one 6-membered aromatic ring;

L is an alkylene having 2-6 carbons; and

p is 0 or 1.

16. The method of claim 1 , wherein the compound has the formula:

or a salt thereof,

wherein M is —NR 3 —, wherein R 3 is —CH 2 -Py, —CH 2 Py, —CH 2 CH 2 OR 5 , or —CH 2 COOR 5 , wherein R 5 is H, an alkyl having 1-6 carbons, —CH 2 OCOCH 3 , or a counterion;

Z is

 or

 wherein n is 1, 2, or 3;

R 6 is —CH 2 COOR 5 , —CH 2 CH 2 OR 5 , —CH 2 Py, or, when taken in combination with M, forms a structure

 wherein m is 1, 2, or 3;

R 7 , R 8 , R 9 , and R 10 are independently selected from the group consisting of an alkyl having 1-6 carbons, halogen, amino, nitro, cyano, trifluoromethyl, sulfo, and sulfonamide;

R 1 and R 2 are independently H, an alkyl having 1-6 carbons, halogen, or sulfo;

R 4 is a 5 or 6-membered aromatic ring or a fused ring system comprising at least one 6-membered aromatic ring;

L is an alkylene having 2-6 carbons; and

p is 0 or 1.

17. A method of measuring the concentration of intracellular metal ions in a sample, comprising:

a) combining a fluorescent compound comprising a 2-substituted benzoxazole group attached to a metal ion binding group with a sample containing biological cells, wherein the cells are known or suspected to contain metal ions, in an amount sufficient to generate a detectable fluorescent response to a metal ion;

b) illuminating the sample to generate a fluorescence excitation or emission response;

c) observing the absorbance or emission response; and

d) quantifying the fluorescence excitation or emission response of the compound to determine the concentration of the metal ions within the cells.

18. The method of claim 17 , wherein the biological cells are living cells.

19. The method of claim 17 , wherein the fluorescent compound is permeant to membranes of the biological cells.