Peptide Conjugates and Fluorescence Detection Methods for Intarcellular Caspase Assay

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.

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.