Peptide Conjugates and Fluorescence Detection Methods for Intarcellular 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(—NH3 + ), 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, 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 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 first linkage or the second linkage.
2 - 54 . (canceled)
55 . An assay method comprising:
a) delivering the peptide conjugate of claim 1 inside a plurality of eukaryotic cells;
b) illuminating the cells; and
c) detecting fluorescence.
56 - 76 . (canceled)
77 . A method of detecting caspase activity comprising:
a) suspending the peptide conjugate on a solid support of claim 49 in an aqueous solution containing a caspase in a vessel;
b) illuminating the vessel; and
c) detecting fluorescence in the vessel;
wherein the peptide conjugate has a caspase cleavage site.
78 - 85 . (canceled)
86 . A method of synthesizing a peptide conjugate comprising:
a) synthesizing a polypeptide on a solid support;
b) reacting the polypeptide on a solid support with a first dye labelling reagent;
c) cleaving the polypeptide from the solid support; and
d) reacting the cleaved polypeptide with a second dye labelling reagent;
wherein the first dye labelling reagent and the second dye labelling reagent are 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 linking moiety;
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 9 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 linking moiety, 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 linking moiety; 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 linking moiety;
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 linking moiety, 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 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 a linking moiety;
whereby a peptide conjugate is formed.
87 - 88 . (canceled)
89 . A kit comprising the peptide conjugate of claim 1 and an apoptosis inducer.
90 . A kit comprising the peptide conjugate of claim 1 and a caspase inhibitor.
91 . A kit comprising the peptide conjugate of claim 1 and a plurality of eukaryotic cells.
92 . A kit comprising a polypeptide and a first dye labelling reagent and/or a second dye labelling reagent according to claim 86.