Crosslinking reagents, methods, and compositions for studying protein-protein interactions

Abstract

The invention provides reagents, methods, and compositions for studying protein-protein interactions. The inventive system and methods allow the analysis of protein-protein interactions in vivo and in vitro. Advantages offered by various embodiments of the inventive system and methods compared to existing photocrosslinking approaches include, for example, (i) novel reversible crosslinking reagents that allow easy isolation, purification, and enrichment of the crosslinked products; (ii) trifluoromethyl phenyldiazirine- or perfluorinated phenylazide-based photocrosslinking reagents that provide high specific labeling, no side product, and higher photocrosslinking efficiency; (iii) versatile spacer groups that allow systematic contact site mapping; (iv) novel methods for isolating, purifying, and detecting crosslinked products based on the reversible-link chemistry; and (v) the ability to study the interaction sites in vitro, in situ, or in vivo.

1 . A crosslinking reagent having the Formula (I)
   FG2-SP1-W  (I)
   wherein   FG2 is aminooxy;   SP1 is a spacer group selected from the group consisting of a bond, a substituted or unsubstituted (C 1 -C 24 ) alkyl group, a substituted or unsubstituted (C 1 -C 24 ) heteroalkyl group, a polyethyleneglycol group, a polyalcohol group, a polyamine group, a polyester group, a polyphosphodiester group, a peptide, and a peptidomimetics group; and   W is a chemical- or photocrosslinking group selected from the group consisting of an aryl ketone group, an azide group, a diazo group, a diazirene group, a ketene group, an olefin group, a dicarbonyl group, an epoxide group, an organosilane group, an isothiocyanate group, an isocyanate group, an acyl azide group, an active ester group, a sulfonyl chloride group, a carbonate group, an imidoester group, an anhydride group, a haloacetyl group, an alkyl halide group, a maleimide group, a vinylsulfone group, a thioester group, a disulfide group, and a sulfhydryl group,   
     
     
         2 . The crosslinking reagent of  claim 1 , wherein W is 3-aryl-3-(trifluoromethyl)diazirine group. 
     
     
         3 . The crosslinking reagent of  claim 1 , wherein W is a perfluorinated phenyl azide group. 
     
     
         4 . The crosslinking reagent of  claim 1 , wherein W is a haloacetyl group, an alkyl halide group, a maleimide group, or an N-hydroxylsuccinimide ester group. 
     
     
         5 . The crosslinking reagent of  claim 1 , having the structural Formula (A): 
       
         
           
           
               
               
           
         
         wherein R 6  is selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive; R 8  is H, or an alkyl group. 
       
     
     
         6 . The crosslinking reagent of  claim 1 , having the structural Formula (B): 
       
         
           
           
               
               
           
         
         wherein each of R 5  and R 6  are independently selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive; R 8  is H, or an alkyl group. 
       
     
     
         7 . The crosslinking reagent of  claim 1 , having the structural Formula (C): 
       
         
           
           
               
               
           
         
         wherein each of R 5  and R 6  are independently selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —, wherein each n is an integer independently selected from between 1 and 16 inclusive. 
       
     
     
         8 . The crosslinking reagent of  claim 1 , having the structural Formula (D): 
       
         
           
           
               
               
           
         
         wherein R 5  is selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive; R 5  is H, or an alkyl group. 
       
     
     
         9 . The crosslinking reagent of  claim 1 , having the structural Formula (E): 
       
         
           
           
               
               
           
         
         wherein each R is independently an amino acid side group. 
       
     
     
         10 . A crosslinking reagent having the Formula (II) 
       
         
           
           
               
               
           
         
       
       wherein
 R 1  and R 2  are independently a H, an alkyl group, or an aryl group; 
 SP1 is a spacer group selected from the group consisting of a bond, a substituted or unsubstituted (C 1 -C 24 ) alkyl group, a substituted or unsubstituted (C 1 -C 24 ) heteroalkyl group, a polyethyleneglycol group, a polyalcohol group, a polyamine group, a polyester group, a polyphosphodiester group, a peptide, and a peptidomimetic group; and 
 W is a chemical or photo crosslinking group selected from the group consisting of an aryl ketone group, an azide group, a diazo group, a diazirene group, a ketene group, an olefin group, a dicarbonyl group, an epoxide group, an organosilane group, an isothiocyanate group, an isocyanate group, an acyl azide group, an N-hydroxylsuccinimide ester group, a fluorophenyl ester group, a sulfonyl chloride group, a carbonate group, an imidoester group, an anhydride group, a haloacetyl group, an alkyl halide group, a maleimide group, a vinylsulfone group, a thioester group, a disulfide group, and a sulfhydryl group. 
 
     
     
         11 . The crosslinking reagent of  claim 10 , wherein W is 3-aryl-3-(trifluoromethyl)diazirine group. 
     
     
         12 . The crosslinking reagent of  claim 10 , wherein W is a perfluorinated phenyl azide group. 
     
     
         13 . The crosslinking reagent of  claim 10 , wherein W is a haloacetyl group, an alkyl halide group, a maleimide group, or an N-hydroxylsuccinimide ester group. 
     
     
         14 . The crosslinking reagent of  claim 10 , having the structural Formula (F) 
       
         
           
           
               
               
           
         
         wherein R 6  is selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive; R 8  is H, or an alkyl group. 
       
     
     
         15 . The crosslinking reagent of  claim 10 , having the structural Formula (G): 
       
         
           
           
               
               
           
         
         wherein each of R 5  and R 6  are independently selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —, wherein each n is an integer independently selected from between 1 and 16 inclusive. 
       
     
     
         16 . The crosslinking reagent of  claim 10 , having the structural Formula (H): 
       
         
           
           
               
               
           
         
         wherein R 5  is selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive; R 8  is H, or an alkyl group. 
       
     
     
         17 . The crosslinking reagent of  claim 10 , having the structural Formula (I′): 
       
         
           
           
               
               
           
         
         wherein each R is independently selected from an amino acid side group. 
       
     
     
         18 . A crosslinking reagent having the formula (III)
   V—SP2-L-SP1-W  (III)
   wherein   V is a chemical crosslinking group selected from the group consisting of an isothiocyanate group, an isocyanate group, an acyl azide group, an ester group, a sulfonyl chloride group, a carbonate group, an imidoester group, an anhydride group, a haloacetyl group, an alkyl halide group, a maleimide group, a vinylsulfone group, and a thioester group;   W is a chemical- or photo-crosslinking group selected from the group consisting of an aryl ketone group, an azide group, a diazo group, a diazirene group, a ketene group, an olefin group, a dicarbonyl group, an epoxide group, an organosilane group, an isothiocyanate group, an isocyanate group, an acyl azide group, an ester group, a sulfonyl chloride group, a carbonate group, an imidoester group, an anhydride group, a haloacetyl group, an alkyl halide group, a maleimide group, a vinylsulfone group, a thioester group, a disulfide group, and a sulfhydryl group;   each of SP1 and SP2 is independently a spacer group selected from the group consisting of a bond, a substituted or unsubstituted (C 1 -C 24 ) alkyl group, a substituted or unsubstituted (C 1 -C 24 ) heteroalkyl group, a polyethyleneglycol group, a polyalcohol group, a polyamine group, a polyester group, a polyphosphodiester group, a peptide, and a peptidomimetics group; and   L is an oxime bond.   
     
     
         19 . The crosslinking reagent of  claim 18 , wherein W is 3-aryl-3-(trifluoromethyl)diazirine group. 
     
     
         20 . The crosslinking reagent of  claim 18 , wherein W is a perfluorinated phenyl azide group. 
     
     
         21 . The crosslinking reagent of  claim 18 , wherein V is a haloacetyl group, an alkyl halide group, a maleimide group, or an N-hydroxylsuccinimide ester group. 
     
     
         22 . The crosslinking reagent of  claim 18 , having the structural Formula (J) 
       
         
           
           
               
               
           
         
       
       wherein V is a haloacetyl group, an alkyl halide group, a maleimide group, or an N-hydroxylsuccinimide ester group; each of R 6  and R 7  is independently selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive; R 8  is H, or an alkyl group. 
     
     
         23 . The crosslinking reagent of  claim 22 , having the structural Formula (J′) 
       
         
           
           
               
               
           
         
         wherein each of R 6  and R 7  is independently selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive; R 8  is H, or an alkyl group. 
       
     
     
         24 . The crosslinking reagent of  claim 18 , having the structural Formula (K): 
       
         
           
           
               
               
           
         
         wherein V is a haloacetyl group, an alkyl halide group, a maleimide group, or an N-hydroxylsuccinimide ester group; each of R 5 , R 6  and R 7  is independently selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive. 
       
     
     
         25 . The crosslinking reagent of  claim 24 , having the structural Formula (K′): 
       
         
           
           
               
               
           
         
         wherein each of R 5 , R 6 , and R 7  are independently selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —, wherein each n is an integer independently selected from between 1 and 16 inclusive. 
       
     
     
         26 . The crosslinking reagent of  claim 18 , having the structural Formula (L): 
       
         
           
           
               
               
           
         
         wherein V is a haloacetyl group, an alkyl halide group, a maleimide group, or an N-hydroxylsuccinimide ester group; each of R 5  and R 7  is independently selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive; R 8  is H, or an alkyl group. 
       
     
     
         27 . The crosslinking reagent of  claim 26 , having the structural Formula (L′): 
       
         
           
           
               
               
           
         
         wherein each of R 5  and R 7  is independently selected from —(CH 2 ) n — and —O(CH 2 CH 2 ) n —; and wherein each n is an integer independently selected from between 1 and 16 inclusive; R 8  is H, or an alkyl group. 
       
     
     
         28 . The crosslinking reagent of  claim 18 , having the structural Formula (M): 
       
         
           
           
               
               
           
         
         wherein V is a haloacetyl group, an alkyl halide group, a maleimide group, or an N-hydroxylsuccinimide ester group; each R is independently an amino acid side group. 
       
     
     
         29 . The crosslinking reagent of  claim 28 , having the structural Formula (M′): 
       
         
           
           
               
               
           
         
         wherein each R is independently selected from an amino acid side group. 
       
     
     
         30 - 34 . (canceled)