Benzoxazole-based fluorescent metal ion indicators

1. A compound of 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.

2. A compound of 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

wherein n is 1, 2, or 3;

R 6 is —CH 2 COOR 6 , —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.

3. The compound of claim 1 or 2 wherein R 4 is substituted with an alkyl having 1-6 carbons, an alkoxy having 1-6 carbons, —OH, —COOH, COO − , oxygen, halogen, —SO 2 NH 2 , or —N—R 10 R 11 , wherein R 10 and R 11 are independently H or an alkyl group having 1-6 carbon atoms.

4. The compound of claim 1 or 2 wherein R 10 and R 11 are independently methyl or ethyl.

5. The compound of claim 1 or 2 wherein R 4 comprises a heteroatom selected from O, N, and S.

6. The compound of claim 1 or 2 wherein R 4 is phenyl or a substituted phenyl moiety.

7. The compound of claim 1 or 2 wherein R 4 is selected from p-H 2 NSO 2 —C 6 H 4 —, p-KOOC—C 6 H 4 —, p-CH 3 O—C 6 H 4 —, p-(CH 3 ) 2 N—C 6 H 4 —,

wherein K is H or a counterion.

8. The compound of claim 1 or 2 wherein R 4 is selected from the group consisting of

9. The compound of claim 1 or 2 wherein R 4 is

10. The compound of claim 1 or 2 wherein R 4 is selected from Ph-CH═CH—, p-CH 3 O—C 6 H 4 —CH═CH—,

11. The compound of claim 1 or 2 wherein R 4 is selected from Ph-CH═CH—, p-CH 3 O—C 6 H 4 —CH═CH—, p-(CH 3 ) 2 N—C 6 H 4 —CH═CH—, and

12. The compound of claim 1 or 2 wherein R 4 is

13. The compound of claim 1 or 2 wherein M is —N(CH 2 COOH) 2 or —N(CH 2 OCOCH 3 ) 2 .

14. The compound of claim 2 having the formula

15. A compound of the formula

or a salt thereof,

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

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 compound of claim 1 or 2 , further comprising a counterion selected from Na + , Li + , K + , Tl + , trialkylammonium and tetraalkylammonium.

17. A complex comprising a compound of claim 1 or 2 and a polycationic metal ion.

18. The complex of claim 17 wherein the polycationic metal ion is a divalent metal ion.

19. The complex of claim 17 wherein the polycationic metal ion is Ca 2+ , Zn 2+ , Mg 2+ , Pb 2+ , Hg 2+ , or Pb 2+ .

20. A method of measuring the concentration of a polycationic metal ion in a sample, comprising:

a) combining a compound of any one of claims 1 - 16 with a sample 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; and

c) observing the absorbance or emission response.

21. The method of claim 20 wherein a fluorometer, fluorescence microscope, laser scanner, flow cytometer is used to observe the absorbance or emission response.

22. The method of claim 20 wherein the polycationic metal ion is Ca 2+ .

23. The method of claim 20 wherein the sample comprises living cells or biological fluids.

24. The method of claim 20 , further comprising quantifying the fluorescence excitation or emission response.

25. The method of claim 20 wherein the compound exhibits a change in emission wavelength maximum upon binding to the polycationic metal ion.

26. The method of claim 20 wherein the compound exhibits a change in excitation wavelength maximum upon binding to the polycationic metal ion.

27. A kit for measuring the concentration of a polycationic metal ion in a sample comprising a compound of any one of claims 1 - 16 .

28. The kit of claim 27 , further comprising a component selected from a salt solution having biological osmolarity, a buffer, and a metal ion solution.