US2008274907A1PendingUtilityA1
Fluorogenic ph sensitive dyes and their method of use
Est. expiryOct 27, 2026(~0.3 yrs left)· nominal 20-yr term from priority
G01N 33/84G01N 33/582C07D 209/14G01N 33/5005C09B 11/12G01N 31/22G01N 33/5058C09B 69/00C09K 11/06C07D 311/82G01N 2333/245C07D 311/90C07K 1/13C09B 57/00C09B 11/28C07D 405/12C09B 7/00Y10T436/143333C09B 11/24C07D 491/22G01N 33/5091C07F 5/02C07D 413/04G01N 33/80
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Claims
Abstract
A new class of pH sensitive fluorescent dyes and assays relating thereto are described. The dyes and assays are particularly suited for biological applications including phagocytosis and monitoring intracellular processes. The pH sensitive fluorescent dyes of the present invention include compounds of Formula I: wherein the variables are described throughout the application.
Claims
exact text as granted — not AI-modified1 - 87 . (canceled)
88 . A compound of Formula I:
wherein,
R 1 , R 2 , R 3 and R 6 are each independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
X is a fluorophore;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof;
with the proviso that at least one of Y or Z is present.
89 . The compound of claim 88 , wherein X is selected from the group consisting of a xanthene, an indole and a borapolyazaindacine.
90 . The compound of claim 88 , wherein X is:
wherein,
R 7 , R 8 , R 9 and R 10 are each independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and
R 10 and R 11 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or
R 11 and R 14 are taken together with R 7 and R 8 to form a fused ring; and R 12 and R 13 are taken together with R 9 and R 10 to form a fused ring.
91 . The compound of claim 90 , wherein R 7 , R 8 , R 9 and R 10 are alkyl.
92 . The compound of claim 91 , wherein R 7 , R 8 , R 9 and R 10 are methyl.
93 . The compound of claim 90 , wherein R 11 , R 12 , R 13 and R 14 are H.
94 . The compound of claim 90 , wherein R 11 and R 14 are taken together with R 7 and R 8 to form a fused ring; and R 12 and R 13 are taken together with R 9 and R 10 to form a fused ring; wherein the fused ring has the following structure:
wherein,
R 27 and R 23 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO 3 —, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
95 . The compound of claim 88 , wherein X is:
R 15 , R 16 , R 17 , R 18 , R 19 and R 20 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
96 . The compound of claim 95 , wherein R 16 and R 19 are H.
97 . The compound of claim 95 , wherein R 15 , R 17 , R 13 and R 20 are methyl.
98 . The compound of claim 88 , wherein X is:
R 21 is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and
R 22 , R 23 , R 24 , R 25 and R 26 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
99 . The compound of claim 98 , wherein R 21 , R 22 , R 24 , R 25 and R 26 are H.
100 . The compound of claim 98 , wherein R 23 is a carboxyl ester.
101 . The compound of claim 98 , wherein R 23 is —CO 2 CH 3 .
102 . The compound of claim 88 , wherein R 1 is —OCH 3 .
103 . The compound of claim 102 , wherein R 2 and R 6 are H.
104 . The compound of claim 88 , wherein the EDG is selected from the group consisting of alkoxy, substituted alkoxy, amino, substituted amino, thiol, alkylthio, hydroxy, acylamino, and (carboxyl ester)oxy.
105 . The compound of claim 88 , wherein R 1 and R 3 are —OCH 3 or —N(CH 3 ) 2 .
106 . The compound of claim 88 , wherein the EDG is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted alkyl, heterocyclyl, substituted heterocyclyl, heteroaryl, and substituted heteroaryl.
107 . The compound of claim 88 , wherein R 4 and R 5 are alkyl or substituted alkyl.
108 . The compound of claim 107 , wherein R 5 is —(CH 2 ) n —CO 2 (CH 2 ) m H, wherein n is 0, 1, 2, 3, 4 or 5; and m is 0, 1, 2 or 3.
109 . The compound of claim 108 , wherein R 5 is ═CH(substituted amino).
110 . The compound of claim 109 , wherein R 4 is ethyl.
111 . The compound of claim 88 , wherein the pKa of the compound is about 5 to about 8.
112 . A compound of Formula II:
wherein,
R 1 , R 2 , R 3 and R 6 are independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 7 , R 8 , R 9 and R 10 are each independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and
R 11 , R 12 , R 13 and R 14 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or
R 11 and R 14 are taken together with R 7 and R 8 to form a fused ring; and R 12 and R 13 are taken together with R 9 and R 10 to form a fused ring;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof;
with the proviso that at least one of Y or Z is present.
113 . A compound of Formula III:
wherein,
R 3 is an electron donating group (EDG); and
R 4 and R 5 are each independently selected from the group consisting of H, alkyl, substituted alkyl, acyl, ═CH(amino), ═CH(substituted amino), ═CH(alkyl), and ═CH(substituted alkyl).
114 . A compound of Formula IV:
wherein,
R 1 , R 2 , R 3 and R 6 are independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 15 , R 16 , R 17 , R 18 , R 19 and R 20 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof.
115 . A compound of Formula V:
wherein,
R 1 , R 2 , R 3 and R 6 are independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 21 is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;
R 22 , R 23 , R 24 , R 25 and R 26 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof.
116 . A method for determining the pH of a sample, the method comprising:
(a) contacting the sample with a compound of Formula I, to form a contacted sample, wherein the Formula I is
wherein,
R 1 , R 2 , R 3 and R 6 are each independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
X is a fluorophore;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof;
with the proviso that at least one of Y or Z is present;
(b) incubating the contacted sample for an appropriate amount of time to form an incubated sample;
(c) illuminating the incubated sample with an appropriate wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated sample;
wherein the fluorescent emissions are used to determine the pH of the sample.
117 . The method of claim 116 , wherein the sample comprises cells.
118 . The method of claim 117 , wherein the contacted sample is incubated for a sufficient amount of time for the dye or compound to enter the cell.
119 . The method of claim 116 , wherein the sample comprises live cells, intracellular fluids, extracellular fluids, sera, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions biological fluids or chemical reactors, blood cells, immune cells, cultured cells, muscle tissue, neurons, extracellular vesicles; vascular tissue, blood fluids, saliva, urine, water, soil, waste water, sea water; pharmaceuticals, foodstuffs or beverages.
120 . The method of claim 116 , wherein the compound becomes fluorescent when the pH of the sample drops below the pKa of the compound.
121 . The method of claim 116 , wherein the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, and on a microfluidic chip.
122 . The method of claim 116 , wherein the contacting step further comprises contacting the sample with a second dye.
123 . The method of claim 122 , wherein the second dye is a pH sensitive dye having a different fluorescent emission spectra from the dye or compound.
124 . The method of claim 123 , wherein the second dye has a different pKa from the pKa of the compound.
125 . A method for monitoring the pH inside a live cell, the method comprising:
(a) contacting the cell with a compound of Formula I to form a contacted cell, wherein the Formula I is
wherein,
R 1 , R 2 , R 3 and R 6 are each independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
X is a fluorophore;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof;
with the proviso that at least one of Y or Z is present;
(b) incubating the contacted cell for a sufficient amount of time for the compound to enter the cell to form a labeled cell;
(c) illuminating the labeled cell with an appropriate wavelength whereby fluorescence is measured, thereby monitoring the pH inside the cell.
126 . The method of claim 125 , wherein a change in the pH inside the cell corresponds to a cellular process.
127 . The method of claim 125 , wherein the compound is conjugated to a protein, nucleic acid or lipid.
128 . A method of synthesizing a compound of Formula II:
wherein,
R 1 , R 2 , R 3 and R 6 are independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 7 , R 8 , R 9 and R 10 are each independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and
R 11 , R 12 , R 13 and R 14 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or
R 11 and R 14 are taken together with R 7 and R 8 to form a fused ring; and
R 12 and R 13 are taken together with R 9 and R 10 to form a fused ring;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
with the proviso that at least one of Y or Z is present;
the method comprising:
(a) contacting a compound of Formula IIA:
with a compound of Formula IIC:
to form a compound of Formula II.
129 . The method of claim 128 , further comprising synthesizing the compound of Formula IIA, the method comprising:
(b) contacting a compound of Formula IIB
with a chlorinating agent, to form a compound of Formula IIA.
130 . The method of claim 129 , wherein the chlorinating agent is oxalyl chloride.
131 . A method of synthesizing a compound of Formula IV:
wherein,
R 1 , R 2 , R 3 and R 6 are independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 15 , R 16 , R 17 , R 18 , R 19 and R 20 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
the method comprising:
(a) contacting a compound of Formula IVA:
with a compound of Formula IVB and/or IVC:
and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione (cloranil);
and BF 3 Et 2 O;
to form a compound of Formula IV.
132 . A method of synthesizing a compound of Formula V:
wherein,
R 1 , R 2 , R 3 and R 6 are independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 21 is H; and
R 22 , R 23 , R 24 , R 25 and R 26 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
the method comprising:
(a) contacting a compound of Formula VA:
with P(OEt) 3 , to form the compound of Formula V.
133 . The method of claim 132 , further comprising synthesizing the compound of Formula VA, the method comprising:
(a) contacting a compound of Formula IVA:
with a compound of Formula VB:
to form the compound of Formula VA.
134 . The method claim 133 further comprising a purifying step.
135 . The method of claim 134 , wherein said purifying step comprises at least one of:
column chromatography, trituration, recrystallization, filtration, or aqueous separation.
136 . A composition comprising:
(a) a compound of Formula I, wherein the Formula I is
wherein,
R 1 , R 2 , R 3 and R 6 are each independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
X is a fluorophore;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof;
with the proviso that at least one of Y or Z is present; and
(b) an analyte.
137 . The composition of claim 136 , wherein the analyte is a cell and the compound is located inside the cell.
138 . The composition of claim 136 , wherein the analyte is a protein, lipid or nucleic acid.
139 . The composition of claim 138 , wherein the compound is conjugated to a carrier molecule.
140 . A kit for determining the pH of a sample comprising:
(a) a compound of Formula I, wherein the Formula I is
wherein,
R 1 , R 2 , R 3 and R 6 are each independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
X is a fluorophore;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof;
with the proviso that at least one of Y or Z is present; and
(b) instructions for determining the pH of the sample.
141 . The kit according to claim 140 , further comprising one or more of the following: a buffering agent, a purification medium, a vial comprising the sample, or an organic solvent.
142 . A fluorescent pH sensitive dye comprising the compound of Formula I wherein the Formula I is
wherein,
R 1 , R 2 , R 3 and R 6 are each independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
X is a fluorophore;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof;
with the proviso that at least one of Y or Z is present.
143 . The fluorescent pH sensitive dye of claim 142 , wherein the pKa of the compound is about 5 to about 8.
144 . The fluorescent pH sensitive dye of claim 142 , wherein the pKa of the compound is about 6 to about 7.
145 . A method for detecting phagocytosis of a carrier molecule in solution, the method comprising:
(a) conjugating a carrier molecule to a fluorescent pH sensitive dye to form a carrier conjugate; (b) contacting the carrier conjugate with a cell to form a contacted cell; (c) incubating the contacted cell to form an incubated solution; (d) illuminating the incubated solution to form an illuminated solution; and (e) detecting fluorescent emissions from the illuminated solution; wherein fluorescent emissions indicate phagocytosis of the carrier molecule.
146 . The method of claim 145 , wherein the carrier molecule is an E. coli bioparticle.
147 . A method for detecting a pH related intracellular process, the method comprising:
(a) contacting a compound of Formula I with a cell to form a contacted cell, wherein the Formula I is
wherein,
R 1 , R 2 , R 3 and R 6 are each independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
X is a fluorophore;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof;
with the proviso that at least one of Y or Z is present;
(b) incubating the contacted cell to form an incubated solution;
(c) illuminating the incubated solution to form an illuminated solution; and
(d) detecting fluorescent emissions from the illuminated solution;
wherein increased fluorescent emissions indicates activation of the intracellular process.
148 . The method of claim 147 , wherein the intracellular process is opening of an ion channel.
149 . The method of claim 148 , wherein the ion channel is calcium.
150 . The method of claim 147 , wherein the compound is internalized after being incubated to the cytosol of the cell.
151 . The method of claim 147 , wherein the cell is a neuron.
152 . A method for identifying a target cell within a population of cells whereby the target cell is differentially labeled relative to neighboring cells within the population, the method comprising:
a) contacting a compound of Formula I with the population of cells to form a contacted cell population, wherein the Formula I is
wherein,
R 1 , R 2 , R 3 and R 6 are each independently H, Z, or an electron donating group (EDG);
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
X is a fluorophore;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino;
or a stereoisomer, tautomer, or salt thereof;
with the proviso that at least one of Y or Z is present;
b) incubating the contacted cell population for a period of time sufficient for the compound to enter the target cell, thereby forming an incubated cell population; and
c) illuminating the incubated cell population, wherein the target cell is identified by a differential label relative to neighboring cells within the population.
153 . The method of claim 152 , wherein the target cell is a neuronal cell.
154 . The method of claim 153 , wherein the differential label comprises increased fluorescence.Join the waitlist — get patent alerts
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