Water-soluble, fluorescent compounds for detection of potassium ions
View Patent ↗The invention provides chromoionophore compounds comprising a triazacryptand (TAC) K + ionophore conjugated to at least a first chromophoric moiety (e.g., xanthylium dyes and derivatives thereof). In related embodiments, the chromoionophore compounds further comprise a second chromophoric moiety which is insensitive to potassium binding by the TAC ionophore, thus providing for dual wavelength detection and absolute determination of K + concentration. The invention further provides methods and kits for the determination of K + concentrations in biological systems, either in vitro or in vivo, using embodiments of inventive chromoionophores.
1. A potassium-sensitive chromoionophore comprising the formula:
wherein
F 1 and F 2 each represent a chromophoric moiety, where F 2 may be present or absent;
R′ 1 and R′ 2 are lower alkyls and R′ 3 and R′ 4 independently selected from H or a lower alkyl; or, when F 2 is absent, R′ 1 and R′ 2 are independently selected from a lower alkyl or F 3 , and R′ 3 and R′ 4 are independently H or lower alkyls; or when F 2 is absent, R′ 1 and R′ 2 are lower alkyls, and R′ 3 and R′ 4 are independently selected from a lower alkyl or F 3 , wherein F 3 is of the formula
X′—P H —F′ 3
where
X′ is a reactive group, selected from an amine, amide, succinimidyl ester, or aldehyde,
P H is selected from a hydrophilic, water-soluble polymer; and
F′ 3 is a chromophoric moiety insensitive to pH and to potassium binding by the chromoionophore, where F′ 3 provides a detectable signal that is different from a detectable signal of F 1 ;
R′ 5 is a substituted or unsubstituted alkyl, alkoxy, alkoxyalkoxy, alkoxyaryl, t-alkyl ester of carboxyalkoxy, t-alkyl ester of carboxyalkoxyalkoxy, succinimidylester of carboxyalkoxy, succinimidylester of carboxyalkoxyalkoxy, aminoalkoxy, aminoalkoxyalkoxy, mercaptoalkoxy, or mercaptoalkoxyalkoxy;
L is a linker selected from a substituted or unsubstituted lower alkyl of the formula —(CH 2 ) v — or —(CH 2 ) w —NH—, where v is 0, 1, or 2 and w is 1 or 2; a substituted phenyl group; or a bifunctional group; and
F 1 is a chromophoric moiety which provides a detectable fluorescent signal upon excitation when potassium is bound to the ionophore,
wherein when F 2 and F 3 are absent, F 1 comprises the formula:
where
L x indicates binding to the linker L;
R 1 and R 14 are each independently selected from H or a lower alkyl;
R 2 and R 13 are each independently selected from H or a lower alkyl unless:
R 2 and R 13 are joined to form substituted or unsubstituted quinolizine ring systems that include R 3 , R 6 , and R 7 , and R 8 , R 9 , and R 10 , respectively, to provide one or two quinolizine ring systems in the compound; or
R 2 and R 13 are joined to form in the compound pyridine or thieno-pyridine ring systems that include R 3 and R 10 , respectively, to provide one or two pyridine ring systems or one or two thieno-pyridine ring systems in the compound, and wherein the thieno-pyridine ring systems are thieno-quinoline ring systems;
wherein when R 2 and R 13 are present in ring structures, at least one of the ring structures contains a positively charged amine, and when R 2 and R 13 are joined to form one or two pyridine ring systems that include R 3 and R 10 , respectively, the pyridine ring systems are substituted by a methylene or polymethylene group that is substituted by an anionic moiety;
R 4 , R 5 , R 11 , and R 12 are independently selected from H or a lower alkyl, and are present when R 2 and R 13 are joined in a ring structure with R 3 and R 10 , respectively, otherwise R 4 , R 5 , R 11 , and R 12 are absent;
R 3 , R 6 , R 9 , and R 10 are independently selected from a lower alkyl if “N” is not part of a ring structure; and
R 7 and R 8 are independently selected from H or a lower alkyl if R 7 and R 8 are not part of a ring structure with R 6 and R 9 , respectively; and
Z is of the formula:
wherein Y are each independently halides, and L x indicates binding to the linker L, and Z is present when R 2 and R 13 are joined to form pyridine ring systems that include R 3 and R 10 , respectively; and
F 2 , when present, is of the formula X—P H —F 2 ′, where
X is an alkyl benzamide, succinimidyl ester, or aldehyde;
P H is a hydrophilic, water-soluble polymer, and
F 2 is a chromophoric moiety that provides a stable detectable signal insensitive to potassium binding and pH, where F′ 2 provides a detectable signal distinguishable from the detectable signal of F′ 1 ;
and pharmaceutically acceptable salts thereof.
2. The chromoionophore of claim 1 , wherein R′ 5 is of the formula:
—[OCH 2 CH 2 ] n OCH 3 ;
—[OCH 2 CH 2 ] n O—(CH 2 ) m —COO- t -butyl;
—[OCH 2 CH 2 ] n O—(CH 2 ) m —NH 2 ; or
—[OCH 2 CH 2 ] n O—(CH 2 ) m —SH
n is 0 or 1, and m is an integer from 1 to 6.
3. The chromoionophore of claim 1 , wherein P H of X′—P H —F′ 3 or X—P H —F′ 2 is dextran, polyethylene oxide, polyethyleneimine (PEI), polylactide, polyglycolide, or polylactic polylactide glycolide acid (PLGA).
4. The chromoionophore of claim 1 , wherein an X′ of the formula X′—P H —F′ 3 or X or the formula X—P H —F′ 2 is
[CH 2 ] q O—(CH 2 ) s —CO—NH— or —O—[CH 2 ]s -CO—NH—
wherein q is an integer from 1 to 2; and s is an integer from 2 to 6.
5. The chromoionophore of claim 1 , wherein F 2 is absent, R′ 1 and R′ 2 are lower alkyls, R′ 3 and R′ 4 are H or lower alkyls, and F 1 comprises the formula:
where:
R 1 and R 14 are each independently selected from H or a lower alkyl;
R 2 and R 13 are each independently selected from H or a lower alkyl unless:
R 2 and R 13 are joined to form substituted or unsubstituted quinolizine ring systems that include R 3 , R 6 , and R 7 , and R 8 , R 9 , and R 10 , respectively, to provide one or two quinolizine ring systems in the compound; or
R 2 and R 13 are joined to form in the compound pyridine or thieno-pyridine ring systems that include R 3 and R 10 , respectively, to provide one or two pyridine ring systems or one or two thieno-pyridine ring systems in the compound, and wherein the thieno-pyridine ring systems are thieno-quinoline ring systems;
wherein when R 2 and R 13 are present in ring structure, at least one of the ring structures contains a positively charged amine;
R 4 , R 5 , R 11 , and R 12 are independently selected from H or a lower alkyl, and are present when R 2 and R 13 are joined in a ring structure with R 3 and R 10 , respectively, otherwise R 4 , R 5 , R 11 , and R 12 are absent;
R 3 , R 6 , R 9 , and R 10 are independently selected from a lower alkyl if “N” is not part of a ring structure;
R 7 and R 8 are independently selected from H or a lower alkyl if R 7 and R 8 are not part of a ring structure with R 6 and R 9 , respectively; and
I K+ represents the position of binding to the ionophore through L.
6. The chromoionophore of claim 5 , wherein
R 1 , R 2 , R 7 , R 8 , R 13 , and R 14 are each independently selected from H or a lower alkyl;
R 3 , R 6 , R 9 , and R 10 are independently selected from a lower alkyl; and
R 4 , R 5 , R 11 , and R 12 are absent.
7. The chromoionophore of claim 5 , wherein
R 1 and R 14 are each independently selected from H or a lower alkyl;
R 2 and R 13 are joined to form quinolizine ring systems that include R 3 , R 6 , and R 7 , and R 8 , R 9 , and R 10 , respectively, to provide one or two quinolizine ring systems in the compound; and R 4 , R 5 , R 11 , and R 12 are independently selected from H or a lower alkyl.
8. The chromoionophore of claim 5 , wherein:
R 1 and R 14 are each independently selected from H or a lower alkyl;
R 2 and R 13 are'joined to form in the compound pyridine or thieno-pyridine ring systems that include R 3 and R 10 , respectively, to provide one or two pyridine ring systems or one or two thieno-pyridine ring systems in the compound, and wherein the thieno-pyridine ring systems are thieno-quinoline ring systems; and
R 4 , R 5 , R 11 , and R 12 are independently selected from H or a lower alkyl.
9. The chromoionophore of claim 1 , wherein R′ 1 and R′ 2 are each methyl, and R′ 5 is —O(CH 2 ) 2 O—CH 3 .
10. The chromoionophore of claim 8 , wherein F 2 is absent.
11. The chromoionophore of claim 10 , wherein F 1 is in the para position.
12. The chromoionophore of claim 1 , wherein the chromoionophore comprises the formula:
13. A potassium-sensitive chromoionophore comprising the formula:
wherein
F 1 and F 2 each represent a chromophoric moiety, where F 2 may be present or absent;
R′ 1 and R′ 2 are lower alkyls and R′ 3 and R′ 4 independently selected from H or a lower alkyl; or, when F 2 is absent, R′ 1 and R′ 2 are independently selected from a lower alkyl or F 3 , and R′ 3 and R′ 4 are independently H or lower alkyls; or when F 2 is absent, R′ 1 and R′ 2 are lower alkyls, and R′ 3 and R′ 4 are independently selected from a lower alkyl or F 3 , wherein F 3 is of the formula
X′—P H —F′ 3
where X′ is a reactive group, selected from an amine, amide, succinimidyl ester, or aldehyde; P H is selected from a hydrophilic, water-soluble polymer; and F′ 3 is a chromophoric moiety insensitive to pH and to potassium binding by the chromoionophore, where F′ 3 provides a detectable signal that is different from a detectable signal of F 1 ;
R′ 5 is a substituted or unsubstituted alkyl, alkoxy, alkoxyalkoxy, alkoxyaryl, t-alkyl ester of carboxyalkoxy, t-alkyl ester of carboxyalkoxyalkoxy, succinimidylester of carboxyalkoxy, succinimidylester of carboxyalkoxyalkoxy, aminoalkoxy, aminoalkoxyalkoxy, mercaptoalkoxy, or mercaptoalkoxyalkoxy;
L is a linker selected from a substituted or unsubstituted lower alkyl of the formula —(CH 2 ) v — or —(CH 2 ) w —NH—, where v is 0, 1, or 2 and w is 1 or 2; and
F 1 is a chromophoric moiety which provides a detectable fluorescent signal upon excitation when potassium is bound to the ionophore, wherein when F 2 and F 3 are absent, F 1 is selected from a moiety of the formula:
where:
each of R 15 , R 16 , R 17 , R 18 , R 19 and R 20 is independently selected from H or lower alkyl, and wherein R 15 and R 20 are further independently selected from an alkali metal cation,
n is an integer from 0 to 6;
where:
each of R 15 , R 16 , R 17 , R 18 , and R 19 is independently selected from H or lower alkyl, and wherein R 15 is further independently selected from an alkali metal cation,
n is an integer from 0 to 6;
I K+ represents the position of binding to the ionophore through L; and
X′—P H —F′ 3
is defined above; and
where:
each of R 21 , R 22 , R 23 , R 24 , and R 25 is independently selected from H or lower alkyl, and R 25 is further independently selected from an alkali metal cation,
n is an integer from 0 to 6; and
I K+ represents the position of binding to the chromoionophore through L; and
F 2 , when present, is of the formula X—P H —F 2 ′, where
X is an alkyl benzamide, succinimidyl ester, or aldehyde;
P H is a hydrophilic, water-soluble polymer, and
F′ 2 is a chromophoric moiety that provides a stable detectable signal insensitive to potassium binding and pH, where F′ 2 provides a detectable signal distinguishable from the detectable signal of F′ 1 ;
and pharmaceutically acceptable salts thereof.
14. The chromoionophore of claim 13 , wherein X′ of the formula X′—P H —F′ 3 or X or the formula X—P H —F′ 2 is —[CH 2 ] q O—(CH 2 ) s —CO—NH—,
—O—[CH 2 ]s -CO—NH—, or —[CH 2 ]s -CO—NH—, where q is an integer from 1 to 2 and s is an integer from 2 to 6.
15. The chromoionophore of claim 13 , wherein the chromoionophore comprises the formula:
16. The chromoionophore of claim 13 , wherein the chromoionophore comprises the formula:
17. A potassium-sensitive chromoionophore comprising a conjugate of an ionophoric moiety, a first chromophoric moiety and a second chromophoric moiety, the chromoionophore comprising the formula:
where
I K+ is a triazacryptand (TAC) ionophoric moiety;
p is 0, 1 or 2;
L 1 is a first linker selected from a substituted aryl group, an unsubstituted aryl group or —CO—(CH2)-COO-t-butyl;
L 2 is a second linker selected from a substituted or unsubstituted aryl group;
P H is a hydrophilic, water soluble polymer; and
D 1 and D 2 are different chromophoric moieties having detectably distinct emission wavelengths, where D 1 is potassium sensitive chromophoric moiety and D 2 is a potassium insensitive chromophoric moiety.
18. The chromoionophore of claim 17 , wherein:
p is 1;
L 1 and L 2 are independently a substituted or unsubstituted phenyl group; and
P H is bound to L 2 through a nitrogen of an amide group.
19. The chromoionophore of claim 18 , wherein L 1 and L 2 are unsubstituted phenyl groups.
20. The chromoionophore of claim 17 , wherein the compound is of the formula:
where
I K+ is a triazacryptand (TAC) ionophoric moiety;
n and p are independently selected from 0, at least 1, or at least 2;
P H is a water soluble polymer, wherein L 2 and P H are covalently bound through a secondary amine; and
D 1 and D 2 are different chromophoric moieties having detectably distinct emission wavelengths, where D 1 is potassium sensitive chromophoric moiety and D 2 is a potassium insensitive chromophoric moiety.
21. The chromoionophore of claim 17 , wherein D 1 comprises the formula:
where
I K+ represents a covalent linkage to the ionophore;
R 1 and R 14 are each independently selected from H or a lower alkyl;
R 2 and R 13 are each independently selected from H or a lower alkyl unless:
R 2 and R 13 are joined to form substituted or unsubstituted quinolizine ring systems that include R 3 , R 6 , and R 7 , and R 8 , R 9 , and R 10 , respectively, to provide one or two quinolizine ring systems in the compound; or
R 2 and R 13 are joined to form in the compound pyridine or thieno-pyridine ring systems that include R 3 and R 10 , respectively, to provide one or two pyridine ring systems or one or two thieno-pyridine ring systems in the compound, and wherein the thieno-pyridine ring systems are thieno-quinoline ring systems;
wherein when R 2 and R 13 are present in ring structures, at least one of the ring structures contains a positively charged amine, and when R 2 and R 13 are joined to form pyridine ring systems that include R 3 and R 10 , respectively, the pyridine ring systems are substituted by a methylene or polymethylene group that is substituted by an anionic moiety;
R 4 , R 5 , R 11 , and R 12 are independently selected from H or a lower alkyl, and are present when R 2 and R 13 are joined in a ring structure with R 3 and R 10 , respectively, otherwise R 4 , R 5 , R 11 , and R 12 are absent;
R 3 , R 6 , R 9 , and R 10 are independently selected from a lower alkyl if “N” is not part of a ring structure; and
R 7 and R 8 are independently selected from H or a lower alkyl if R 7 and R 8 are not part of a ring structure with R 6 and R 9 , respectively.
22. The chromoionophore of claim 21 , wherein
R 1 , R 2 , R 7 , R 8 , R 13 , and R 14 are each independently selected from H or a lower alkyl;
R 3 , R 6 , R 9 , and R 10 are independently selected from a lower alkyl; and
R 4 , R 5 , R 11 , and R 12 are absent.
23. The chromoionophore of claim 22 , wherein
R 1 and R 14 are each independently selected from H or a lower alkyl;
R 2 and R 13 are joined to form quinolizine ring systems that include R 3 , R 6 , and R 7 , and R 8 , R 9 , and R 10 , respectively, to provide one or two quinolizine ring systems in the compound; and
R 4 , R 5 , R 11 , and R 12 are independently selected from H or a lower alkyl.
24. The chromoionophore of claim 22 , wherein:
R 1 and R 14 are each independently selected from H or a lower alkyl;
R 2 and R 13 are joined to form in the compound pyridine or thieno-pyridine ring systems that include R 3 and R 10 , respectively, to provide one or two pyridine ring systems or one or two thieno-pyridine ring systems in the compound, and wherein the thieno-pyridine ring systems are thieno-quinoline ring systems; and
R 4 , R 5 , R 11 , and R 12 are independently selected from H or a lower alkyl.
25. The chromoionophore of claim 17 , wherein D 1 comprises the formula:
where Z 1 and Z 2 are O or S, and I K+ represents the position of covalent attachment to L 1 .
26. The chromoionophore of claim 17 , where the TAC ionophoric moiety comprises the formula:
where: R′ 1 , R′ 2 are independently H or a lower alkyl;
R′ 5 is —[OCH 2 CH 2 ] n OCH 3 , where n=1, and
X represents the position at which the TAC ionophoric moiety is bound in the chromoionophore.
27. The chromoionophore of claim 26 , wherein the TAC ionophoric moiety comprises the formula:
where X represents the position at which the TAC ionophoric moiety is bound in the chromoionophore.
28. The compound of claim 21 , wherein the TAC ionophoric moiety is bound in the chromoionophore in the para position relative to the amine group.
29. The chromoionophore of claim 17 , wherein the chromoionophore comprises the formula:
where
n is 0 or at least 1;
P H is a water soluble polymer; and
D 2 is a chromophoric moiety having a emission wavelength different from the chromophoric moiety at position D 1 .
30. A potassium-sensitive chromoionophore comprising a conjugate of an ionophoric moiety, a first chromophoric moiety and a second chromophoric moiety, the chromoionophore comprising the formula:
I K+ —(CH 2 ) p -D 1 -(CH 2 ) p —NH—P H -D 2
where
I K+ is a triazacryptand (TAC) ionophoric moiety;
p is 0, 1 or 2;
P H is a hydrophilic, water soluble polymer; and
D 1 and D 2 are different chromophoric moieties having detectably distinct emission wavelengths, where D 1 is potassium sensitive chromophoric moiety and D 2 is a potassium insensitive chromophoric moiety.
31. The chromoionophore of claim 30 , wherein the TAC ionophoric moiety comprises the formula:
where X represents the position at which the TAC ionophoric moiety is bound in the chromoionophore.
32. The chromoionophore of claim 31 , wherein the TAC ionophoric moiety is bound in the chromoionophore in the para position relative to the amine group.
33. The chromoionophore of claim 30 , wherein the chromoionophore comprises the formula:
where
P H is a water soluble polymer; and
D 2 is a chromophoric moiety having a emission wavelength different from the chromophoric moiety at position D 1 .
34. A composition for assessing extracellular potassium ion concentrations, the composition comprising a physiologically compatible solution and a chromoionophore of claim 1 .
35. A method for assessing extracellular potassium ion concentrations in vivo, the method comprising:
delivering a chromoionophore according to claim 1 to an extracellular fluid compartment of a subject; and
detecting the presence of absence of a potassium-sensitive detectable signal emitted from the chromoionophore,
wherein intensity of the detectable signal is indicative of the concentration of potassium ions in the compartment.
36. The method of claim 35 , wherein said detecting is by imaging through one or more tissues of the subject.
37. The method of claim 35 , wherein the method further comprises administering a candidate agent to the subject to assess the effect of the candidate agent upon extracellular potassium concentrations in the extracellular fluid compartment.
38. The method of claim 35 , wherein the chromoionophore is contained in a physiologically acceptable solution comprising one or more components of a biological sample of the subject.
39. A method for assessing extracellular potassium ion concentrations in a cell or tissue culture, the method comprising:
contacting a cell or tissue culture with a chromoionophore of claim 1 ; and
detecting the presence or absence of a potassium-sensitive detectable signal emitted from the chromoionophore;
wherein intensity of the detectable signal is indicative of the extracellular concentration of potassium ions in the culture.
40. The method of claim 39 , wherein the method further comprises contacting the cell or tissue culture with a candidate agent to assess the effect of the candidate agent upon extracellular potassium concentrations.
41. A kit for the assessing extracellular potassium ion concentrations, the kit comprising a chromoionophore of claim 1 in a container.
42. A composition for assessing extracellular potassium ion concentrations, the composition comprising a physiologically compatible solution and a chromoionophore of claim 13 .
43. A method for assessing extracellular potassium ion concentrations in vivo, the method comprising:
delivering a chromoionophore according to claim 13 to an extracellular fluid compartment of a subject; and
detecting the presence or absence of a potassium-sensitive detectable signal emitted from the chromoionophore,
wherein intensity of the detectable signal is indicative of the concentration of potassium ions in the compartment.
44. The method of claim 43 , wherein said detecting is by imaging through one or more tissues of the subject.
45. The method of claim 43 , wherein the method further comprises administering a candidate agent to the subject to assess the effect of the candidate agent upon extracellular potassium concentrations in the extracellular fluid compartment.
46. The method of claim 43 , wherein the chromoionophore is contained in a physiologically acceptable solution comprising one or more components of a biological sample of the subject.
47. A method for assessing extracellular potassium ion concentrations in a cell or tissue culture, the method comprising:
contacting a cell or tissue culture with a chromoionophore of claim 13 ; and
detecting the presence of absence of a potassium-sensitive detectable signal emitted from the chromoionophore;
wherein intensity of the detectable signal is indicative of the extracellular concentration of potassium ions in the culture.
48. The method of claim 47 , wherein the method further comprises contacting the cell or tissue culture with a candidate agent to assess the effect of the candidate agent upon extracellular potassium concentrations.
49. A kit for the assessing extracellular potassium ion concentrations, the kit comprising a chromoionophore of claim 13 in a container.
50. A hydrophilic potassium-sensitive chromoionophore, wherein the chromoionophore comprises the formula:
where:
R′ 1 and R′ 2 are independently selected from a lower alkyl;
L is a linker selected from a substituted or unsubstituted lower alkyl of the formula —(CH 2 ) v — or —(CH 2 ) w —NH—, where v is 0, 1, or 2 and w is 1 or 2;
R′ 5 is a substituted or unsubstituted alkyl, alkoxy, alkoxyalkoxy, alkoxyaryl, t-alkyl ester of carboxyalkoxy, t-alkyl ester of carboxyalkoxyalkoxy, succinimidylester of carboxyalkoxy, succinimidylester of carboxyalkoxyalkoxy, aminoalkoxy, aminoalkoxyalkoxy, mercaptoalkoxy, or mercaptoalkoxyalkoxy; and
D is a chromophore moiety, and
wherein a water-soluble polymer is covalently bound to D, and wherein the water-soluble polymer is dextran.
51. The chromoionophore of claim 50 , wherein R′ 5 is of the formula:
—[OCH 2 CH 2 ] n OCH 3 ;
—[OCH 2 CH 2 ] n O—(CH 2 ) m —COO- t -butyl;
—[OCH 2 CH 2 ] n O—(CH 2 ) m —NH 2 ; or
—[OCH 2 CH 2 ] n O—(CH 2 ) m —SH;
n is 0 or 1; and m is an integer from 1 to 6.
52. The chromoionophore of claim 50 , wherein:
R′ 1 and R′ 2 are each methyl;
R′ 5 is —(OCH 2 CH 2 )OCH 3 ; and
D is in the para position.
53. The chromoionophore of claim 50 , wherein D is bound to the dextran through an amide linkage.
54. The chromoionophore of claim 50 , wherein the chromoionophore is TAC-Lime-dextran.