Dyes for analysis of protein aggregation

Abstract

Provided are dyes and compositions which are useful in a number of applications, such as the detection and monitoring protein aggregation, kinetic studies of protein aggregation, neurofibrillary plaques analysis, evaluation of protein formulation stability, and analysis of molecular chaperone activity.

What is claimed is: 
     
         1 . A compound comprising the structure 
       
         
           
           
               
               
           
         
         wherein m and n are independently 1, 2 or 3; 
       
       wherein L is a linker arm comprising carbon, sulfur, oxygen, nitrogen, or any combination thereof;
 wherein R 1 , R 2 , R 3 , R 4 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 19 , R 20 , R 21  and R 22  are independently hydrogen, halogen, amino, ammonium, nitro, sulfo, sulfonamide, carboxy, ester, cyano, phenyl, benzyl, an alkyl group wherein the alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein the alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken in combination R 1  and R 2 , or R 3  and R 4 , or R 9  and R 10 , or R 11  and R 12 , or R 13  and R 14 , or R 15  and R 16 , or R 19  and R 20 , or R 21  and R 22  form a five or six membered ring wherein the ring is saturated or unsaturated, substituted or unsubstituted, and wherein R 9  and R 10 , or R 11  and R 12 , or R 13  and R 14 , or R 15  and R 16  can comprise alkyl chains that are joined together, wherein a quinoline moiety can be formed; 
 
       wherein R 7 , R 8 , R 17  and R 18  are independently hydrogen, Z, an alkyl group wherein the alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein the alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together, R 7  and R 8  and R 17  and R 18 , may form a 5 or 6 membered ring wherein the ring is saturated or unsaturated, substituted or unsubstituted;
 wherein Z comprises a carboxyl group (CO 2   − ), a carbonate ester (COER 25 ), a sulfonate (SO 3   − ), a sulfonate ester (SO 2 ER 25 ), a sulfoxide (SOR 25 ), a sulfone (SO 2 CR 25 R 26 R 27 ), a sulfonamide (SO2NR 25 R 26 ), a phosphate (PO 4   = ), a phosphate monoester (PO 3   − ER 25 ), a phosphate diester (PO 2 ER 25 ER 26 ), a phosphonate (PO 3   = ) a phosphonate monoester (PO 2   − ER 25 ) a phosphonate diester (POER 25 ER 26 ), a thiophosphate (PSO 3   = ), a thiophosphate monoester (PSO 2   − ER 25 ) a thiophosphate diester (PSOER 25 ER 26 ), a thiophosphonate (PSO 2   = ), a thiophosphonate monoester (PSO − ER 25 ) a thiophosphonate diester (PSER 25 ER 26 ), a phosphonamide (PONR 25 R 26 NR 28 R 29 ), its thioanalogue (PSNR 25 R 26 NR 28 R 29 ), a phosphoramide (PONR 25 R 26 NR 27 NR 28 R 29 ), its thioanalogue (PSNR 25 R 26 NR 27 NR 28 R 29 ), a phosphoramidite (PO 2 R 25 NR 28 R 29 ) or its thioanalogue (POSR 25 NR 28 R 29 ) wherein E is independently O or S;
 wherein R 25 , R 26 , R 27 , R 28 , and R 29  are independently a hydrogen, an unsubstituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group, a substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2  groups are substituted with an O atom, N atom, S atom, or NH group, or an unsubstituted or substituted aromatic group; 
 wherein Z is attached directly, or indirectly through a second linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein the second linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted or any combinations thereof; 
 
 wherein R 5 , R 6 , R 23  and R 24  can independently be hydrogen or an alkyl group wherein the alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, or when taken in combination R 5  and R 6  or R 2  and R 5  or R 3  and R 6  or R 23  and R 24  or R 22  and R 23  or R 20  and R 24  form a five or six membered ring wherein the ring is saturated or unsaturated, substituted or unsubstituted; and 
 wherein said compound is modified to comprise a reactive group which is an isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal or aldehyde group. 
 
     
     
         2 . The compound of  claim 1 , wherein the compound exhibits increased fluorescence in the presence of an aggregated form of a protein when compared to the fluorescence exhibited when the compound is in the presence of the unaggregated form of the protein. 
     
     
         3 . The compound of  claim 1 , comprising the structure 
       
         
           
           
               
               
           
         
         modified to comprise the reactive group, or 
       
       
         
           
           
               
               
           
         
         modified to comprise the reactive group. 
       
     
     
         4 . The compound of  claim 4 , wherein each of R 5 , R 6 , R 23  and R 24  are a methyl or an ethyl moiety. 
     
     
         5 . The compound of  claim 1 , wherein the compound is S25, S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135, YAT2324 or YAT2150, modified to comprise the reactive group.