Processing

Please wait...

Settings

Settings

Goto Application

1. WO1996041166 - DETECTION OF TRANSMEMBRANE POTENTIALS BY OPTICAL METHODS

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

WHAT IS CLAIMED IS:

1. A method of determining the electrical
potential across a membrane comprising:
(a) introducing a first reagent comprising a hydrophobic fluorescent ion capable of redistributing from a first face of the membrane to a second face of the membrane in response to changes in the membrane potential;
(b) introducing a second reagent which labels the first face or the second face of the membrane, which second reagent compriseε a chromophore capable of undergoing energy transfer by either (i) donating excited state energy to the fluorescent ion, or (ii) accepting excited state energy from the fluorescent ion;
(c) exposing the membrane to excitation light;
(d) measuring energy transfer between the
fluoresent ion and the second reagent; and
(e) relating the energy transfer to the membrane potential.

2. The method of Claim 1, wherein energy transfer between the fluorescent ion and the second reagent is by fluorescent resonance energy transfer (FRET) .

3. The method of Claim 1, wherein the membrane is a plasma membrane of a biological cell.

4. The method of Claim 3, wherein the cell is a mammalian cell.

5. The method of Claim 4, wherein the membrane is in an intracellular organelle.

6. The method of Claim 4, wherein the cell is selected from the group consisting of L-M (TK~) cells, neuroblastoma cells, astrocytoma cells and neonatal cardiac myocytes.

7. The method of Claim 1, wherein the membrane comprises a phospholipid bilayer.

8. The method of Claim 1, wherein the ion is an anion.

9. The method of Claim 8 , wherein the anion bears a single charge.

10. The method of Claim 8, wherein the anion is selected from the group consiεting of polymethine oxonolε, tetraaryl borates and complexes of transition metals.

11. The method of Claim 10, wherein the anion is an oxonol of the formula


wherein:
R is independently selected from the group
consisting of H, hydrocarbyl and heteroalkyl;
X is oxygen or sulfur; and
n is an integer from 1 to 3 ;
and salts thereof.

12. The method of Claim 11, wherein X is εulfur.

13. The method of Claim 11, wherein:
each R is identical and is a hydrocarbyl group selected from C^^ alkyl groups; and
n = 2.

14. The method of Claim 10, wherein the anion is a tetraaryl borate of the formula
[ (Ar1)3B-Ar2-Y-FLU]~
wherein:
Ar1 is an aryl group;
Ar2 is an arylene group;
B is boron;
Y iε oxygen or sulfur; and
FLU is a neutral fluorophore;
and derivatives thereof.

15. The method of Claim 14, wherein:
Ar1 is trifluoromethylphenyl;
Ar2 is tetrafluorophenyl; and
Y is oxygen.

16. The method of Claim 14, wherein the neutral fluorophore is selected from the group consiεting of bimanes, difluoroboradiazaindacenes and cou arinε.

17. The method of Claim 16, wherein the neutral fluorophore is a bimane of the formula


wherein:
each R5, which may be the same or different, is independently H, lower alkyl or an alkylene attachment point.

18. The method of Claim 16, wherein the neutral fluorophore is a difluoroboradiazaindacene of the formula


wherein:
each R1, which may be the same or different, is independently selected from the group consisting of H, lower alkyl, aryl, heteroaro atic, aralkenyl and an alkylene attachment point;
each R2, which may be the same or different, is independently selected from the group consisting of H, lower alkyl, phenyl and an alkylene attachment point.

19. The method of Claim 16, wherein the neutral fluorophore is a coumarin of the formulas


wherein:
each R3, which may be the same or different, is independently selected from the group consiεting of H, halogen, lower alkyl, CN, CF3 and OR5;
each R4, which may be the same or different, iε selected from the group consisting of H, OR5 and an alkylene attachment point; and
Z is 0, S or NR"; and
R5 is selected from the group consisting of H, lower alkyl and an alkylene attachment point.

20. The method of Claim 16, wherein the neutral fluorophore is a complex of a transition metal of the formula


wherein:
Ln = Tb, Eu, or Sm;
R is independently H, C1-C8 alkyl, C1-C8 cycloalkyl or C1-C4 perfluoroalkyl;
X and Y are independently H, F, Cl, Br, I, N02, CF3, lower (C1-C4) alkyl, CN, Ph, 0-(lower alkyl), or OPh; or X and Y together are -CH=CH-; and
Z = alkylenediyl, heteroalkylenediyl or
heterocyclodiyl .

21. The method of Claim 20, wherein
Z = 1,2-ethanediyl, 1, 3-propanediyl, 2, 3-butanediyl, 1,2-cyclohexanediyl, 1, 2-cyclopentanediyl, 1,2- cycloheptanediyl, 1,2-phenylenediyl, 3-oxa-l, 5-pentanediyl, 3- aza-3-(lower alkyl) -1, 5-pentanediyl, pyridine-2, 6-bis (methylene) or tetrahydrofuran-2,5-bis (methylene) .

22. The method of Claim 16, wherein the neutral fluorophore is a complex of a transition metal of the formula


wherein:
Ln = Tb, Eu, or Sm;
R is independently H, C1-C8 alkyl, C1-C8 cycloalkyl or C1-C4 perfluoroalkyl;
X' and Y' are independently H, F, Cl, Br, I, N02 , CF3, lower (C1-C4) alkyl, CN, Ph, 0-(lower alkyl), or OPh; or X' and Y' together are -CH=CH-; and
Z' is independently a valence bond, CR2, pyridine-2-6-diyl or tetrahydofuran-2 , 5-diyl.

23. The method of Claim 1, wherein the second
reagent is a fluorophore.

24. The method of Claim 23, wherein the second reagent is selected from the group consisting of lectins, lipids, carbohydrates, cytochromes and antibodies, each being labelled with a fluorophore.

25. The method of Claim 24, wherein the fluorophore is selected from the group consisting of xanthenes, cyanines and coumarins.

26. The method of Claim 24, wherein the second reagent is a lipid which is a phospholipid.

27. The method of Claim 1, wherein the first
reagent and the second reagent are covalently joined by a linker.

28. The method of Claim 27, wherein the linker is a compound of the formula:
X-(CH2)m-Zq-(CH2)m.-Z'q.-(CH2)m.,-Z"g..-Y
wherein:
X is a hydrophobic fluorescent anion selected from the group consisting of polymethine oxonols and fluorescent tetraaryl borates;
Y is a fluorescent second reagent selected from the group consisting of lectins and phospholipidε;
Z, Z', Z" are independently 0, S, SS, CO, COO;
m, m' and m" are integerε from 0 to about 32;
q, q', and q" are independently 0 or 1; and
m + q + m' + q' + m" + q" is from about 20 to 40.

29. The method of Claim 28, wherein the linker is a thioether.

30. A kit comprising:
(a) a first reagent comprising a hydrophobic fluorescent ion capable of redistributing from a first face of a membrane to a second face of the membrane in response to changes in the membrane potential; and
(b) a second reagent which labels the first face or the second face of the membrane, which second reagent
comprises a chromophore capable of undergoing energy transfer by either (i) donating excited state energy to the fluorescent ion, or (ii) accepting excited state energy from the
fluorescent ion.

31. The kit of Claim 30, wherein:
the first reagent is selected from the group consisting of polymethine oxonols, tetraaryl borates and complexes of transition metals; and
the second reagent is εelected from the group conεiεting of lectinε, lipidε, carbohydrateε , cytochromeε and antibodies, each being labelled with a fluorophore.

32. The kit of Claim 30, further comprising a solubilizing agent.

33. A compound of the formula A-L-B wherein:
A is independently a polymethine oxonol or a tetraaryl borate linked to a fluorophore;
L is a linker; and
B is a membrane-impermeant fluorophore or a
membrane-impermeant conjugate of a fluorophore.

34. The compound of Claim 33 wherein A is a polymethine oxonol of the formula:


wherein:
R is independently selected from the group consisting of H, hydrocarbyl and heteroalkyl;
X is oxygen or sulfur; and
n is an integer from 1 to 3 ;
provided that at least one R is an alkylene group.

35. A compound of a formula Cou-PE wherein:
Cou is a coumarin of formula:


wherein:
each R3, which may be the same or different, is independently selected from the group consisting of H, halogen, lower alkyl, CN, CF3, COOR5, CON(R5)2, OR5, and an attachment point;
R4 is selected from the group consisting of OR5 and N(R5)2;
Z is 0, S or NR5; and
each R5, which may be the same or different, is independently selected from the group consisting of H, lower alkyl and an alkylene attachment point; and
PE is an N-linked phosphatidylethanolamine.

36. A compound of a formula


wherein:
Ln = Tb, Eu, or Sm;
R is independently H, C1-C8 alkyl, C1-C8 cycloalkyl or C1-C4 perfluoroalkyl;
X and Y are independently H, F, Cl, Br, I, N02, CF3, lower (C1-C4) alkyl, CN, Ph, 0-(lower alkyl), or OPh; or X and Y together are -CH=CH-; and
Z = alkylenediyl, heteroalkylenediyl or
heterocyclodiy1.

37. The compound of Claim 36, wherein:
Z = 1,2-ethanediyl, 1, 3-propanediyl, 2 , 3-butanediyl, 1,2-cyclohexanediyl, 1, 2-cyclopentanediyl, 1,2-cycloheptanediyl , 1,2-phenylenediyl, 3-oxa-l, 5-pentanediyl, 3-aza-3-(lower alkyl) -1, 5-pentanediyl, pyridine-2 , 6-bis (methylene) or tetrahydrofuran-2 , 5-bis (methylene) .

38. A compound of a formula


wherein:
Ln = Tb, Eu, or Sm;
R is independently H, C1-C8 alkyl, C1-C8 cycloalkyl or C1-C4 perfluoroalkyl;

X' and Y' are independently H, F, Cl, Br, I, N02 , CF3, lower (C1-C4) alkyl, CN, Ph, 0-(lower alkyl), or OPh; or X' and Y' together are -CH=CH-; and
Z' is independently a valence bond, CR2, pyridine-2-6-diyl or tetrahydofuran-2,5-diyl.

39. A polymethine oxonol of the formula


wherein:
X is oxygen or sulfur;
R is a C_12 hydrocarbyl;
n is an integer from 1 to 3 ;
provided that when n is 1, R is a C7_12 hydrocarbyl;
and salts thereof.

40. A compound of the formula:


wherein PE represents an N-linked phosphatidylethanolamine.

41. A method of identifying a teεt εample which affects membrane potential in a cell, comprising:
(a) loading the cells with a first and second reagents, which together determine the membrane potential by the method of Claim 1;
(b) exposing the membrane to the test sample;
(c) determining the potential of the membrane; and

(d) comparing the potential in (c) to the potential in the absence of the test sample, thereby determining the effect of the test sample on the membrane potential.

42. The method of Claim 41, further comprising:
(e) exposing the membrane to a stimuluε which modulateε the activity of an ion channel, pump or exchanger;
(f) determining the membrane potential;
(g) redetermining the membrane potential in the presence of the test sample; and
(h) comparing the membrane potentials in (f) and (g) to determine the effect of the test sample on the
stimulus.

43. The methods of Claimε 41 or 42, wherein the cell iε a mammalian cell.

44. A method of εcreening test samples to identify a compound which modulates the activity of an ion channel, pump or exchanger in a membrane, comprising:
(a) loading a first set and a second set of cells with first and second reagents which together measure membrane potential by the method of Claim 1;
(b) optionally, exposing both the first and second set of cells to a stimuluε which modulates an ion channel, pump or exchanger;
(c) exposing the first set of cells to a test sample;
(d) measuring the membrane potential of the first and second sets of cells; and (e) relating the difference in membrane potentials between the first and second sets of cells to the ability of a compound in the test sample to modulate the activity of an ion channel, pump or exchanger in the membrane.

45. A method of synthesizing a fluorescent tetraryl borate of formula
[ (Ar1)3B-Ar2-Y-FLU]"
wherein:
Ar1 is an aryl group;
Ar2 is an arylene group;
B is boron;
Y is oxygen or sulfur; and
FLU iε a neutral fluorophore;
said method comprising:
(a) treating a triaryl borane (Ar1)3B with an organometallic reagent of formula (P-Y-Ar2)-M, wherein P is a protecting group and M is a metal to form a protected
tetraaryl borate (Ar1) 3B-Ar2-Y-P;
(b) removing the protecting group from the
protected tetraaryl borate to form (Ar1) 3B-Ar2-YH; and
(c) treating (Ar1) 3B-Ar2-YH with a fluorophore FLU bearing a leaving group to form a fluorescent tetraryl borate.

46. A compound of the formula

wherein:
each R1 iε independently H, hydrocarbyl, halogen, CF3 or a linker group;
n is an integer from 0 to 5;
each X is independently H, halogen or CF3;
m is an integer from 0 to 4 ;
Y is oxygen or sulfur; and
FLU is a neutral fluorophore selected from the group consisting of xanthenes, coumarins, cyanines, bimanes and difluoroboradiazaindacenes .