Peptide Conjugates and Fluorescence Detection Methods for Intracellular Caspase Assay
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.
1 . 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 and the acceptor 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 8 alkyldiyl;
R 15 when taken together with R 16 is C 2 -C 8 alkyldiyl;
R 13 when taken together with R 17 is C 2 -C 8 alkyldiyl;
R 15 when taken together with R 22 is C 2 -C 8 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, wherein R is selected from C 1 -C 6 alkyl and C 1 -C 6 alkyldiyl, and Ar is C 5 -C 14 aryl;
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 or the second linkage; and 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 is selected from C 1 -C 6 alkyl and C 1 -C 6 alkyldiyl, and Ar is C 5 -C 14 aryl;
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, or is a homolog of the amino acid sequence: Asp-Glu-Val-Asp-Gly.
4 . (canceled)
5 . The peptide conjugate of claim 1 wherein the polypeptide comprises the amino acid sequence: Gly-Asp-Glu-Val-Asp-Gly-Iso-Lys, or a homolog of the amino acid sequence: Gly-Asp-Glu-Val-Asp-Gly-Iso-Lys.
6 . (canceled)
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, a protected aspartic acid side-chain, a protected lysine side-chain, or a protected cysteine 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 - 11 . (canceled)
12 . The peptide conjugate of claim 1 wherein the first linkage is attached to the polypeptide at the side-chain of an amino acid, the amino terminus, or the carboxy terminus.
13 . The peptide conjugate of claim 12 wherein the amino acid is lysine, or cysteine.
14 - 16 . (canceled)
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, an amide bond, or benzyliyl.
19 - 20 . (canceled)
21 . The peptide conjugate of claim 1 wherein the second linkage is attached to the polypeptide at the side-chain of an amino acid, the amino terminus, or the carboxy terminus.
22 . The peptide conjugate of claim 21 wherein the amino acid is lysine.
23 - 24 . (canceled)
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, an amide bond, or benzyldiyl.
27 - 28 . (canceled)
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 - 47 . (canceled)
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, a polyethyleneoxy/polystyrene graft copolymer, or a bead.
52 - 53 . (canceled)
54 . The peptide conjugate of claim 49 wherein the solid support is a substantially planar surface.
55 - 92 . (canceled)