Peptide Conjugates and Fluorescence Detection Methods for Intracellular Caspase Assay

摘要

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.

主权项

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 R13, R14, R15, and R16 are selected from hydrogen, C1-C12 alkyl, C1-C12 alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, a water-solubilizing group and a linkage; R13 when taken together with R14 is C2-C8 alkyldiyl; R15 when taken together with R16 is C2-C8 alkyldiyl; R13 when taken together with R17 is C2-C8 alkyldiyl; R15 when taken together with R22 is C2-C8 alkyldiyl; R17, R18, R19, R20, R21, R22, R23, R24, R25, and R26 are selected from hydrogen, fluorine, chlorine, bromine, C1-C8 alkyl, carboxylate, sulfate, sulfonate, alkylsulfonate, aminomethyl (—CH2NH2), aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—CH2OH), methoxy (—OCH3), hydroxyalkyl (—ROH), thiomethyl (—CH2SH), thioalkyl (—RSH), alkylsulfone (—SO2R), arylthio (—SAr), arylsulfone (—SO2Ar), sulfonamide (—SO2NR2), alkylsulfoxide (—SOR), arylsulfoxide (—SOAr), primary amino (—NH2), ammonium (—NH3+), secondary amino (—NHR), tertiary amino (—NR2), quaternary amine (—NR3+), amido (—CONR2), nitrile (—CN), nitro (—NO2), C1-C8 alkoxy (—OR), phenoxy, phenolic, tolyl, phenyl, C5-C20 aryl, benzyl, heterocycle, phosphonate, phosphate, sulfate, polyethyleneoxy, a water-solubilizing group, and a linkage, wherein R is selected from C1-C6 alkyl and C1-C6 alkyldiyl, and Ar is C5-C14 aryl; R19 and R20, when taken together, are C5-C14 aryleno; R24 and R25, when taken together, are C5-C14 aryleno; with the proviso that at least one of R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, and R26 is the first linkage or the second linkage; and the formula: wherein R1, R2, R3, and R4 are selected from hydrogen, C1-C12 alkyl, C1-C12 alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, a water-solubilizing group, and a linkage; R1 when taken together with R2 is C2-C8 alkyldiyl; R3 when taken together with R4 is C2-C8 alkyldiyl; R5, R6, R7, R8, R9, R10, R11, and R12 are selected from hydrogen, fluorine, chlorine, bromine, C1-C8 alkyl, carboxylate, sulfate, sulfonate, alkylsulfonate, aminomethyl (—CH2NH2), aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—CH2OH), methoxy (—OCH3), hydroxyalkyl (—ROH), thiomethyl (—CH2SH), thioalkyl (—RSH), alkylsulfone (—SO2R), arylthio (—SAr), arylsulfone (—SO2Ar), sulfonamide (—SO2NR2), alkylsulfoxide (—SOR), arylsulfoxide (—SOAr), primary amino (—NH2), ammonium (—NH3+), secondary amino (—NHR), tertiary amino (—NR2), quaternary amine (—NR3+), amido (—CONR2), nitrile (—CN), nitro (—NO2), C1-C8 alkoxy (—OR), phenoxy, phenolic, tolyl, phenyl, C5-C20 aryl, benzyl, heterocycle, phosphonate, phosphate, sulfate, polyethyleneoxy, a water-solubilizing group, and a linkage, R is selected from C1-C6 alkyl and C1-C6 alkyldiyl, and Ar is C5-C14 aryl; R6 and R7, when taken together, is C5-C14 aryleno; R10 and R11, when taken together, is C5-C14 aryleno; with the proviso that at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, and R12 is the second linkage.