Zinc sensors for cellular imaging
View Patent ↗The present invention provides compounds for use as zinc ligand sensors in cellular imaging. These compounds are sensitive imaging agents that can be used with techniques such a laser scanning microscopy in intact cells in culture or in tissue preparations.
1. A method of detecting zinc ion (Zn 2+ ) release by a cell comprising:
(a) contacting a cell with a compound having the formula:
wherein
R 1 and R 4 are independently H, or —CH 2 (CH 2 ) a COOH, CH 2 (CH 2 ) b SO 3 H, wherein
a = is 0, 1 or 2 and b, 1, 2 or 3;
R 2 and R 3 are independently H, or linear or branched C 1-20 alkyl;
R 5 and R 6 are independently H, F, Cl, Br;
R7 is H, or linear or branched C 1-4 alkyl or acetate;
R8 and R9 are independently H, or linear or branched C 1-4 alkyl or acetate; and
m is 1, 2 or 3;
n is 1, 2, 3 or 4; and
o is 1,2, 3 or 4;
and pharmaceutically acceptable salts thereof, and
(b) detecting fluorescence of said compound following zinc release by said cell and binding of zinc by said compound.
2. The method of claim 1 , wherein said cell is pancreatic beta cell, a submandibular salivary gland cell, a prostate epithelial cell, a paneth cell, a mast cell, a granulocyte, a pituitary cell, a or a CNS neuron.
3. The method of claim 1 , wherein detecting comprises confocal laser scanning microscopy, two photon laser scanning microscopy, or total internal reflection fluorescence microscopy.
4. The method of claim 1 , wherein said cell is an isolated cell.
5. The method of claim 1 , wherein said cell is in an intact tissue.
6. The method of claim 5 , wherein said tissue is an isolated pancreatic islet.
7. The method of claim 1 , further comprising detecting fluorescence at multiple time points.
8. The method of claim 1 , further comprising stimulating said cell with a zinc ion release agent.
9. The method of claim. 1 , wherein the concentration of said compound is between 1 nM and 100 μM.
10. The method of claim 1 , wherein the concentration is between 5 nM and 1 μM.
11. The method of claim 1 , wherein R 5 , R 6 , R 7 , R 8 and R 9 are H, in and n are 1 and o is 2.
12. The method of claim 11 wherein R 1 and R 4 are the same.
13. The method of claim 11 , wherein R 2 and R 3 are the same.
14. The method of claim 12 , wherein R 2 and R 3 are the same.
15. The method of claim 14 , wherein R 1 and R 4 are —CH 2 COOH, and R 2 and R 3 are —CH 2 CH 3 or —(CH 2 ) 11 CH 3 .
16. A compound having the formula:
wherein
R 1 and R 4 are independently H, or —CH 2 (CH 2 ) a COOH, CH 2 (CH 2 ) b SO 3 H, wherein a is 0, 1 or 2 and b 1, 2 or 3;
R 2 and R 3 are independently H, or linear or branched C 1-20 alkyl;
R 5 and R 6 are independently H, F, Cl, Br;
R7 is H, or linear or branched C 1-4 alkyl or acetate;
R8 and R9 are independently H, or linear or branched C 1-4 alkyl or acetate; and
m is 1, 2 or 3;
n is 1, 2, 3 or 4; and
o is 1 2, 3 or 4;
and pharmaceutically acceptable salts thereof.
17. The compound of claim 16 , wherein R 5 , R 6 , R 7 , R 8 and R 9 are H, m and n are 1 and o is 2.
18. The compound of claim 17 , wherein R 1 and R 4 are the same.
19. The compound of claim 17 , wherein R 2 and R 3 are the same.
20. The compound of claim 18 , wherein R 2 and R 3 are the same
21. The compound of claim 20 , wherein R 1 and R 4 are —CH 2 COOH and R 2 and R 3 are —CH 2 CH 3 or —(CH 2 ) 11 CH 3 .
22. A ligand metal complex having the formula:
wherein
R 1 and R 4 are independently H, or —CH 2 (CH 2 ) a COOH, CH 2 (CH 2 ) b SO 3 H, wherein a is 0, 1 or 2 and b 1, 2 or 3;
R 2 and R 3 are independently H, or linear or branched C 1-20 alkyl;
R 5 and R 6 are independently H, F, Cl, Br;
R7 is H, or linear or branched C 1-4 alkyl or acetate;
R8 and R9 are independently H, or linear or branched C 1-4 alkyl or acetate; and
m is 1, 2 or 3;
n is 1, 2, 3 or 4; and
o is 1, 2, 3 or 4;
and pharmaceutically acceptable salts thereof.
23. The ligand metal complex of claim 22 , wherein R 5 , R 6 , R 7 , R 8 and R 9 are H, m and n are 1 and o is 2.
24. The ligand metal complex of claim 23 , wherein R 1 and R 4 are the same.
25. The ligand metal complex of claim 23 , wherein R 2 and R 3 are the same.
26. The ligand metal complex of claim 24 . wherein R 2 and R 3 are the same
27. The ligand metal complex of claim 26 , wherein R 1 and R 4 are —CH 2 COOH and R 2 and R 3 are —CH 2 CH 3 or —(CH 2 ) 11 CH 3 .