Peptide Conjugates and Fluorescence Detection Methods for Intracellular 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 (—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)