Fluorescent probes for monitoring voltage by photo-induced electron transfer

Patent metadata

Field	Value
Publication number	US-9651494-B2
Application number	US-201214118836-A
Country	US
Kind code	B2
Filing date	May 21, 2012
Priority date	May 19, 2011
Publication date	May 16, 2017
Grant date	May 16, 2017

Abstract

Compounds and methods for determining transmembrane potential, monitoring changes in transmembrane potential, and/or drug screening are provided. In one aspect, compounds of the invention have a structure according to the formula: E-M-A, wherein A is a fluorophore, selected from xanthenes, coumarins, cyanines, bimanes, and difluoroboradizaindacenes, charged at physiological pH; M is a molecular wire; and E is a hydrophobic moiety, wherein A and E are capable of being involved in a photo-induced, intramolecular electron transfer that quenches the fluorescence of A in response to a voltage condition. When in use, compounds of the invention are membrane-impermeant and oriented within the cell membrane such that the charged moiety localizes at the outer leaflet of the lipid bilayer and the hydrophobic moiety and molecular wire associate with the hydrophobic portion of the lipid bilayer. The rate of electron transfer, fluorescence intensity, and quenching are altered in response to changes in transmembrane potential.

First claim

What is claimed is:

1. A compound having a structure according to the formula:
E-M-A
wherein
A is a fluorophore selected from the group consisting of xanthenes, coumarins, cyanines, and bimanes, wherein A is charged at physiological pH;
M is a molecular wire comprising a carbon containing ring and/or chain that permits a flow of electrons from one end to the other end of the wire; and
E is a hydrophobic moiety, wherein A and E are involved in a photo-induced electron transfer (PeT) through M that quenches the fluorescence of A via PeT in response to a voltage condition of a cellular membrane.

2. The compound of claim 1 , wherein A is an electron acceptor and E is an electron donor in said photo-induced electron transfer.

3. The compound of claim 1 , wherein A is an electron donor and E is an electron acceptor in said photo-induced electron transfer.

4. The compound of claim 1 , wherein A is negatively charged at physiological pH.

5. The compound of claim 1 , wherein A is a xanthene.

6. The compound of claim 1 , wherein A has a structure according to the formula:

wherein R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, H, NO 2 , CN, SO 3 H, NR 11 R 12 , C(Z 1 )R 13 and Z 2 R 12 ;
X 1 is selected from Z 2 R 12 and NR 11 R 12 ;
X 2 is selected from NR 13 R 14 and O;
R 12 is selected from H, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, and C(Z 3 )R 15 ;
R 13 and R 15 are independently selected from alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, OR 16 and NR 17 R 18 ;
R 16 is selected from H, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl and C(O)R 19 ;
R 19 is selected from alkyl, substituted alkyl, heteroalkyl and substituted heteroalkyl;
R 11 , R 13 , R 14 , R 17 and R 18 are independently selected from H, alkyl, substituted alkyl, heteroalkyl and substituted heteroalkyl;
Z 1 and Z 3 are independently selected from O, S and NH; and
Z 2 is selected from O and S.

7. The compound of claim 6 , wherein A has a structure according to the formula:

wherein R 7 , R 8 , R 9 , and R 10 are independently selected from alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, H, NO 2 , CN, SO 3 H, NR 11 R 12 , C(Z 1 )R 13 and Z 2 R 12 .

8. The compound of claim 6 , wherein X 1 is OH, X 2 is O, R 2 is Cl, R 5 is Cl and R 10 is SO 3 H.

9. The compound of claim 6 , wherein R 1 , R 3 , R 4 , and R 6 are H.

10. The compound of claim 6 , wherein R 7 , R 8 and R 9 are H.

11. The compound of claim 1 , wherein E is selected from a substituted aryl and substituted heteroaryl.

12. The compound of claim 1 , wherein E is phenyl substituted with an amine.

13. The compound of claim 1 , wherein E has the structure according to the formula:

wherein E 1 and E 2 are independently alkyl.

14. The compound of claim 13 , wherein E 1 and E 2 are independently selected from unsubstituted C 1 -C 6 alkyl.

15. The compound of claim 14 , wherein E 1 and E 2 are independently selected from methyl or butyl.

16. The compound of claim 15 , wherein E 1 and E 2 are butyl.

17. The compound of claim 16 , wherein said butyl is n-butyl.

18. The compound of claim 1 , wherein M is selected from alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarylene and substituted heteroarylene.

19. The compound of claim 1 , wherein M has a structure according to the formula:
(-M 1 -M 2 -) m -(M 1 ) n
wherein M 1 is alkylene,
M 2 is arylene,
m is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20,
n is an integer selected from 0 and 1, and
at least one of m and n is greater than 0.

20. The compound of claim 1 , wherein M has a structure according to the formula:

wherein m is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

21. The compound of claim 1 , wherein M is a conjugated system.

22. The compound of claim 1 , wherein M is hydrophobic.

23. The compound of claim 1 , having a structure selected from:

24. The compound of claim 1 , having a structure selected from:

25. The compound of claim 1 , substituted with a targeting moiety.

26. The compound of claim 25 , wherein the targeting moiety is selected from a nucleic acid, a peptide, a saccharide, a lipid and a combination thereof.

27. The compound of claim 25 , wherein the targeting moiety is specific for an excitable cell type.

28. The compound of claim 27 , wherein said excitable cell type is selected from a neuron, a nerve cell, an endocrine secretory cell, a neuroendocrine secretory cell, a cardiac muscle cell, a smooth muscle cell, and a skeletal muscle cell.

29. An amphipathic compound having the structure:
E-M-A
wherein
A is a charged fluorophore selected from the group consisting of xanthenes, coumarins, cyanines, and bimanes;
M is a molecular wire comprising a carbon containing ring and/or chain that permits a flow of electrons from one end to the other end of the wire; and
E is an electron-rich, hydrophobic moiety involved in a photo-induced electron transfer to A through the molecular wire, which quenches the fluorescence of A in response to a voltage condition of a cellular membrane.

30. The compound of claim 29 , wherein A is a positively charged fluorophore.

31. The compound of claim 29 , wherein A is a negatively charged fluorophore.

32. The compound of claim 29 , wherein A is a xanthene.

33. The compound of claim 29 , wherein A has a structure according to the formula:

wherein R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, H, NO 2 , CN, SO 3 H, NR 11 R 12 , C(Z 1 )R 13

Assignees

• Miller Evan Walker
• Tsien Roger Y
• Univ California

Inventors

• Miller Evan Walker
• Tsien Roger Y

Classifications

• C09K11/06

  containing organic luminescent materials · CPC title

• G01N33/5008 Primary

  for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics · CPC title

• C09K2211/1011

  Condensed systems · CPC title

• C09K2211/1007

  Non-condensed systems · CPC title

• G01N21/6486 Primary

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title