Protein melt analysis using dipyrrometheneboron difluoride compounds

Abstract

According to the present teachings, systems, compositions, kits and methods for protein melt analysis are provided that utilizing a dye that is a dipyrrometheneboron difluoride compound. In some embodiments, a method comprises preparing a sample by mixing at least one protein with a dye, and applying a controlled heating, while recording the fluorescence emission of the sample.

What is claimed is: 
     
         1 . A method comprising:
 (a) forming a sample solution mixture comprising at least one protein and a dye of formula (I):   
       
         
           
           
               
               
           
         
         wherein:
 R 1  to R 6  is independently selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination, 
 wherein o, m and p are independently selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination, 
 wherein an alkenyl substituent is substituted or unsubstituted, 
 wherein the alkenyl group is ethenyl, dienyl, or trienyl, 
 wherein the one or more substituents for an alkenyl group is selected from the group consisting of: hydrogen, halogen, alkyl (1-5 carbon atoms), cyano, carboxylate ester, carboxamide, aryl, heteroaryl, and any combination thereof, 
 wherein an aryl is selected from phenyl, 1-naphthyl, 2-naphthyl, 1-pyrenyl, 9-anthryl, pyridyl, quinolyl, and alkoxy derivatives thereof, 
 wherein an aryl or heteroaryl group is substituted or unsubstituted, 
 wherein the one or more substituents for an aryl or heteroaryl group is selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6) alkyl (1-5 carbons), alkoxy(wherein the alkyl portion is 1-4 carbon atoms), and any combination thereof, 
 wherein any alkyl or arylalkyl group is substituted or unsubstituted, 
 wherein the one or more substituents for an alkyl or arylalkyl group is selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination, 
 wherein the alkyl group in any substituent of an aryl group may be further substituted by an ester or amide substituent, and 
 wherein L x  is a linker, where x=0 to 6, and wherein the linker may be selected from alkyl (1-6 carbons), and heteroalkyl, (1-6 atoms); and 
 
         (b) applying a controlled heating to the mixture; and 
         (c) measuring fluorescence emitted over a temperature range. 
       
     
     
         2 . The method of  claim 1 , wherein the dye in the protein-dye mixture is 4,4-difluoro-1,3,5,7-tetramethyl-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene. 
     
     
         3 . The method of  claim 1 , wherein the dye in the protein-dye mixture is 4,4-difluoro-3,5-bis(4-methoxyphenyl)-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene. 
     
     
         4 . The method of  claim 1 , wherein the controlled heating is a thermal ramp. 
     
     
         5 . The method of  claim 4 , wherein the thermal ramp is between about 20° C. to about 95° C. 
     
     
         6 . The method of  claim 1 , wherein the controlled heating is isothermal heating. 
     
     
         7 . The method of  claim 1 , wherein the mixture further comprises a buffer. 
     
     
         8 . The method of  claim 1 , wherein the mixture further comprises a surfactant. 
     
     
         9 . The method of  claim 8 , wherein the surfactant is at or above the critical micelle concentration. 
     
     
         10 . The method of  claim 1 , wherein the mixture further comprises a polyol. 
     
     
         11 . The method of  claim 10 , wherein the polyol is glycerol. 
     
     
         12 . The method of  claim 10 , wherein the polyol is a polysaccharide. 
     
     
         13 . A composition comprising:
 (a) at least one protein and a dye of formula (I):   
       
         
           
           
               
               
           
         
         wherein:
 R 1  to R 6  is independently selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination, 
 wherein o, m and p are independently selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination, 
 wherein an alkenyl substituent is substituted or unsubstituted, 
 wherein the alkenyl group is ethenyl, dienyl, or trienyl, 
 wherein the one or more substituents for an alkenyl group is selected from the group consisting of: hydrogen, halogen, alkyl (1-5 carbon atoms), cyano, carboxylate ester, carboxamide, aryl, heteroaryl, and any combination thereof, 
 wherein an aryl is selected from phenyl, 1-naphthyl, 2-naphthyl, 1-pyrenyl, 9-anthryl, pyridyl, quinolyl, and alkoxy derivatives thereof, 
 wherein an aryl or heteroaryl group is substituted or unsubstituted, 
 wherein the one or more substituents for an aryl or heteroaryl group is selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6) alkyl (1-5 carbons), alkoxy(wherein the alkyl portion is 1-4 carbon atoms), and any combination thereof, 
 wherein any alkyl or arylalkyl group is substituted or unsubstituted, 
 wherein the one or more substituents for an alkyl or arylalkyl group is selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination, 
 wherein the alkyl group in any substituent of an aryl group may be further substituted by an ester or amide substituent, and 
 wherein L x  is a linker, where x=0 to 6, and wherein the linker may be selected from alkyl (1-6 carbons), and heteroalkyl, (1-6 atoms). 
 
       
     
     
         14 . The composition of  claim 13 , wherein the dye in the protein-dye mixture is 4,4-difluoro-1,3,5,7-tetramethyl-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene. 
     
     
         15 . The composition of  claim 13 , wherein the dye in the protein-dye mixture is 4,4-difluoro-3,5-bis(4-methoxyphenyl)-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene. 
     
     
         16 . The composition of  claim 13 , wherein the composition further comprises a buffer. 
     
     
         17 . The composition of  claim 13 , wherein the composition further comprises a surfactant. 
     
     
         18 . The composition of  claim 17 , wherein the surfactant is at or above the critical micelle concentration. 
     
     
         19 . The composition of  claim 13 , wherein the composition further comprises a polyol. 
     
     
         20 . The composition of  claim 19 , wherein the polyol is glycerol. 
     
     
         21 . The composition of  claim 19 , wherein the polyol is a polysaccharide. 
     
     
         22 . A kit comprising:
 (a) a dye of formula (I):   
       
         
           
           
               
               
           
         
         wherein:
 R 1  to R 6  is independently selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination, 
 wherein o, m and p are independently selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination, 
 wherein an alkenyl substituent is substituted or unsubstituted, 
 wherein the alkenyl group is ethenyl, dienyl, or trienyl, 
 wherein the one or more substituents for an alkenyl group is selected from the group consisting of: hydrogen, halogen, alkyl (1-5 carbon atoms), cyano, carboxylate ester, carboxamide, aryl, heteroaryl, and any combination thereof, 
 wherein an aryl is selected from phenyl, 1-naphthyl, 2-naphthyl, 1-pyrenyl, 9-anthryl, pyridyl, quinolyl, and alkoxy derivatives thereof, 
 wherein an aryl or heteroaryl group is substituted or unsubstituted, 
 wherein the one or more substituents for an aryl or heteroaryl group is selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6) alkyl (1-5 carbons), alkoxy(wherein the alkyl portion is 1-4 carbon atoms), and any combination thereof, 
 wherein any alkyl or arylalkyl group is substituted or unsubstituted, 
 wherein the one or more substituents for an alkyl or arylalkyl group is selected from the group consisting of: hydrogen, halogen, —(CH 2 ) n CO 2 H (wherein n=0 to 6), —(CH 2 ) n CO 2 R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination, 
 wherein the alkyl group in any substituent of an aryl group may be further substituted by an ester or amide substituent, and 
 wherein L x  is a linker, where x=0 to 6, and wherein the linker may be selected from alkyl (1-6 carbons), and heteroalkyl, (1-6 atoms). 
 
       
     
     
         23 . The kit of  claim 22 , wherein the dye in the protein-dye mixture is 4,4-difluoro-1,3,5,7-tetramethyl-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene. 
     
     
         24 . The kit of  claim 22 , wherein the dye in the protein-dye mixture is 4,4-difluoro-3,5-bis(4-methoxyphenyl)-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene. 
     
     
         25 . The kit of  claim 22 , wherein the kit further comprises a buffer. 
     
     
         26 . The kit of  claim 22 , wherein the kit further comprises a surfactant. 
     
     
         27 . The kit of  claim 26 , wherein the surfactant is at or above the critical micelle concentration. 
     
     
         28 . The kit of  claim 22 , wherein the kit further comprises a polyol. 
     
     
         29 . The kit of  claim 28 , wherein the polyol is glycerol. 
     
     
         30 . The kit of  claim 28 , wherein the polyol is a polysaccharide.