US2010273943A1PendingUtilityA1
Peptide conjugates and fluorescence detection methods for intracellular caspase assay
Est. expiryMay 21, 2021(expired)· nominal 20-yr term from priority
G01N 33/542C12Q 1/37G01N 2510/00C07K 5/1021C07K 5/101C07K 7/06
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Claims
Abstract
Polypeptides labelled with a donor and acceptor pair of dyes selected from a dibenzorhodamine dye and a diamino-benzophenoxazine dye are peptide conjugates which are useful for intracellular and bead-based assays with fluorescence detection. Peptide conjugates with a caspase-recognition site undergo cleavage into peptide fragments which may be detected, located, and quantitated by the changes in fluorescence. Intracellular cleavage of peptide conjugates is correlated with apoptosis.
Claims
exact text as granted — not AI-modified1 . A peptide conjugate comprised of:
a polypeptide covalently attached to a donor dye through a first linkage and to an acceptor dye through a second linkage, wherein the donor dye is selected from the formula:
wherein R 13 , R 14 , R 15 , and R 16 are selected from hydrogen, C 1 -C 12 alkyl, C 1 -C 12 alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, a water-solubilizing group and a linkage;
R 13 when taken together with R 14 is C 2 -C 9 alkyldiyl;
R 15 when taken together with R 16 is C 2 -C 9 alkyldiyl;
R 13 when taken together with R 17 is C 2 -C 9 alkyldiyl;
R 15 when taken together with R 22 is C 2 -C 9 alkyldiyl;
R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , and R 26 are selected from hydrogen, fluorine, chlorine, bromine, C 1 -C 8 alkyl, carboxylate, sulfate, sulfonate, alkylsulfonate, aminomethyl (—CH 2 NH 2 ), aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—CH 2 OH), methoxy (—OCH 3 ), hydroxyalkyl (—ROH), thiomethyl (—CH 2 SH), thioalkyl (—RSH), alkylsulfone (−SO 2 R), arylthio (—SAr), arylsulfone (—SO 2 Ar), sulfonamide (−SO 2 NR 2 ), alkylsulfoxide (−SOR), arylsulfoxide (—SOAr), primary amino (—NH 2 ), ammonium (—NH 3 + ), secondary amino (—NHR), tertiary amino (—NR 2 ), quaternary amine (—NR 3 + ), amido (—CONR 2 ), nitrile (—CN), nitro (—NO 2 ), C 1 -C 8 alkoxy (—OR), phenoxy, phenolic, tolyl, phenyl, C 5 -C 20 aryl, benzyl, heterocycle, phosphonate, phosphate, sulfate, polyethyleneoxy, a water-solubilizing group, and a linkage;
R 19 and R 20 , when taken together, are C 5 -C 14 aryleno;
R 24 and R 25 , when taken together, are C 5 -C 14 aryleno;
with the proviso that at least one of R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , and R 26 is the first linkage; and wherein the acceptor dye is selected from the formula:
wherein R 1 , R 2 , R 3 , and R 4 are selected from hydrogen, C 1 -C 12 alkyl, C 1 -C 12 alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, a water-solubilizing group, and a linkage;
R 1 when taken together with R 2 is C 2 -C 8 alkyldiyl;
R 3 when taken together with R 4 is C 2 -C 8 alkyldiyl;
R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are selected from hydrogen, fluorine, chlorine, bromine, C 1 -C 8 alkyl, carboxylate, sulfate, sulfonate, alkylsulfonate, aminomethyl (—CH 2 NH 2 ), aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—CH 2 OH), methoxy (—OCH 3 ), hydroxyalkyl (—ROH), thiomethyl (—CH 2 SH), thioalkyl (—RSH), alkylsulfone (−SO 2 R), arylthio (—SAr), arylsulfone (—SO 2 Ar), sulfonamide (−SO 2 NR 2 ), alkylsulfoxide (−SOR), arylsulfoxide (—SOAr), primary amino (—NH 2 ), ammonium (—NH 3 + ), secondary amino (—NHR), tertiary amino (—NR 2 ), quaternary amine (—NR 3 + ), amido (—CONR 2 ), nitrile (—CN), nitro (—NO 2 ), C 1 -C 8 alkoxy (—OR), phenoxy, phenolic, tolyl, phenyl, C 5 -C 20 aryl, benzyl, heterocycle, phosphonate, phosphate, sulfate, polyethyleneoxy, a water-solubilizing group, and a linkage;
R 6 and R 7 , when taken together, is C 5 -C 14 aryleno;
R 10 and R 11 , when taken together, is C 5 -C 14 aryleno;
with the proviso that at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is the second linkage.
2 . The peptide conjugate of claim 1 wherein the polypeptide comprises a caspase cleavage site.
3 . The peptide conjugate of claim 1 wherein the polypeptide comprises the amino acid sequence: Asp-Glu-Val-Asp-Gly.
4 . The peptide conjugate of claim 3 wherein the polypeptide is a homolog of the amino acid sequence: Asp-Glu-Val-Asp-Gly.
5 . The peptide conjugate of claim 1 wherein the polypeptide comprises the amino acid sequence: Gly-Asp-Glu-Val-Asp-Gly-Iso-Lys.
6 . The peptide conjugate of claim 5 wherein the polypeptide is a homolog of the amino acid sequence: Gly-Asp-Glu-Val-Asp-Gly-Iso-Lys.
7 . The peptide conjugate of claim 1 wherein the polypeptide comprises a protected amino acid side-chain.
8 . The peptide conjugate of claim 7 wherein the protected amino acid side-chain is a protected glutamic acid side-chain or a protected aspartic acid side-chain.
9 . The peptide conjugate of claim 8 wherein the glutamic acid side-chain or aspartic acid side-chain is protected as a tert-butyl ester.
10 . The peptide conjugate of claim 7 wherein the protected amino acid side-chain is a protected lysine side-chain.
11 . The peptide conjugate of claim 7 wherein the protected amino acid side-chain is a protected cysteine side-chain.
12 . The peptide conjugate of claim 1 wherein the first linkage is attached to the polypeptide at the side-chain of an amino acid.
13 . The peptide conjugate of claim 12 wherein the amino acid is lysine.
14 . The peptide conjugate of claim 12 wherein the amino acid is cysteine.
15 . The peptide conjugate of claim 1 wherein the first linkage is attached to the polypeptide at the amino terminus.
16 . The peptide conjugate of claim 1 wherein the first linkage is attached to the polypeptide at the carboxyl terminus.
17 . The peptide conjugate of claim 1 wherein the first linkage is attached to the donor dye at R 13 , R 14 , R 15 or R 16 .
18 . The peptide conjugate of claim 1 wherein the first linkage comprises C 1 -C 12 alkyldiyl.
19 . The peptide conjugate of claim 1 wherein the first linkage comprises an amide bond.
20 . The peptide conjugate of claim 1 wherein the first linkage comprises benzyldiyl.
21 . The peptide conjugate of claim 1 wherein the second linkage is attached to the polypeptide at the side-chain of an amino acid.
22 . The peptide conjugate of claim 21 wherein the amino acid is lysine.
23 . The peptide conjugate of claim 1 wherein the second linkage is attached to the polypeptide at the amino terminus.
24 . The peptide conjugate of claim 1 wherein the second linkage is attached to the polypeptide at the carboxyl terminus.
25 . The peptide conjugate of claim 1 wherein the second linkage is attached to the acceptor dye at R 1 , R 2 , R 3 or R 4 .
26 . The peptide conjugate of claim 1 wherein the second linkage comprises C 1 -C 12 alkyldiyl.
27 . The peptide conjugate of claim 1 wherein the second linkage comprises an amide bond.
28 . The peptide conjugate of claim 1 wherein the second linkage comprises benzyldiyl.
29 . The peptide conjugate of claim 1 wherein R 1 and R 2 are each C 1 -C 6 alkyl; R 3 is hydrogen and R 4 is a linkage.
30 . The peptide conjugate of claim 1 wherein R 13 and R 14 are each C 1 -C 6 alkyl; R 15 is hydrogen and R 16 is a linkage.
31 . The peptide conjugate of claim 1 wherein the donor dye is selected from the formula:
32 . The peptide conjugate of claim 31 wherein R 18 , R 19 , R 20 , R 21 , R 23 , R 24 , R 25 , and R 26 are hydrogen.
33 . The peptide conjugate of claim 1 wherein the donor dye is selected from the formula:
wherein R 15 is a linkage.
34 . The peptide conjugate of claim 1 wherein the acceptor dye is selected from the formula:
wherein R 4 is a linkage.
35 . The peptide conjugate of claim 1 wherein at least one of R 1 , R 2 , R 3 and R 4 is selected from the structures:
—(CH 2 ) n —NR 2 —(CH 2 ) n — + NR 3 —(CH 2 ) n — + NR 2 —(CH 2 ) n —NR 2 —(CH 2 ) n — + NR 2 —(CH 2 ) n — + NR 3 wherein each n is independently 2 or 3, and each occurrence of R is independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkyldiyl, and a linkage.
36 . The peptide conjugate of claim 1 wherein at least one of R 1 , R 2 , R 3 , R 4 , R 13 , R 14 , R 15 and R 16 is C 1 -C 6 alkylsulfonate or C 4 -C 10 arylsulfonate.
37 . The peptide conjugate of claim 36 wherein the C 4 -C 10 arylsulfonate is:
wherein n is 0 or 1.
38 . The peptide conjugate of claim 1 wherein at least one of R 1 , R 2 , R 3 , R 4 , R 13 , R 14 , R 15 and R 16 is C 1 -C 6 alkylcarboxylate or C 4 -C 10 arylcarboxylate.
39 . The peptide conjugate of claim 38 wherein the C 4 -C 10 arylcarboxylate is:
wherein n is 0 or 1.
40 . The peptide conjugate of claim 1 wherein the alkyldiyl, substituted phenyl, substituted benzyl, substituted biphenyl, substituted heterocycle and substituted naphthyl is substituted with sulfonate.
41 . The peptide conjugate of claim 1 wherein the alkyldiyl, substituted phenyl, substituted benzyl, substituted biphenyl, substituted heterocycle and substituted naphthyl is substituted with carboxyl.
42 . The peptide conjugate of claim 1 wherein the water-solubilizing group is carboxylate, sulfonate, phosphonate, phosphate, quaternary amine, sulfate, polyhydroxyl, or water-soluble polymer.
43 . The peptide conjugate compound of claim 1 wherein R 6 and R 7 , when taken together, are [1,2]benzeno, [1,2]naphthaleno or [2,3]naphthaleno.
44 . The peptide conjugate compound of claim 1 wherein R 10 and R 11 , when taken together, are [1,2]benzeno, [1,2]naphthaleno or [2,3]naphthaleno.
45 . The peptide conjugate compound of claim 1 wherein R 19 and R 20 , when taken together, are [1,2]benzeno, [1,2]naphthaleno or [2,3]naphthaleno.
46 . The peptide conjugate compound of claim 1 wherein R 24 and R 25 , when taken together, are [1,2]benzeno, [1,2]naphthaleno or [2,3]naphthaleno.
47 . The peptide conjugate of claim 1 wherein the heterocycle is pyrrole, indole, furan, benzofuran, thiophene, benzothiophene, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 2-imidazole, 4-imidazole, 3-pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3-(1,2,4-N)-triazolyl, 5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O,3-N)-oxazole, 5-(1-O,3-N)-oxazole, 4-(1-S,3-N)-thiazole, 5-(1-S,3-N)-thiazole, 2-benzoxazole, 2-benzothiazole, 4-(1,2,3-N)-benzotriazole, or benzimidazole.
48 . The peptide conjugate of claim 1 wherein the donor dye is capable of absorbing light at a first wavelength and emitting excitation energy in response thereto; and the acceptor dye is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.
49 . The peptide conjugate of claim 1 wherein the peptide is covalently attached by a linkage to a solid support.
50 . The peptide conjugate of claim 49 wherein the peptide is covalently attached at the carboxyl terminus to the solid support.
51 . The peptide conjugate of claim 49 wherein the solid support is polystyrene.
52 . The peptide conjugate of claim 49 wherein the solid support is a polyethyleneoxy/polystyrene graft copolymer.
53 . The peptide conjugate of claim 49 wherein the solid support is a bead.
54 . The peptide conjugate of claim 49 wherein the solid support is a substantially planar surface.
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