Site-selective functionalization of proteins using traceless affinity labels

Abstract

The present disclosure relates to site-selective labeling compounds, and methods of using such compounds.

1 . A compound comprising:
 i. a peptide (W);   ii. a first orthogonally-reactive moiety (R); and   iii. a covalent attachment between W and R, comprising an electrophilic moiety;   wherein the electrophilic moiety is capable of reacting with an amino group of a protein, thereby breaking the covalent attachment between W and R.   
     
     
         2 . The compound of  claim 1 , wherein W is a peptide of 1-25, 4-15, 6-10 or 4-8 amino acid residues. 
     
     
         3 . e compound of  claim 1 , wherein the covalent attachment between W and R comprises an electrophilic moiety selected from —C(O)O—, —C(O)S—, —C(O)Se—, —SO 2 O—, —SO 2 S- and —SO 2 Se—. 
     
     
         4 . The compound of  claim 2 , wherein no more than four positions of the peptide are fixed with W, F, Y, I, or L between the 3- and C-terminal position. 
     
     
         5 . The compound of  claim 1 , wherein R comprises a moiety selected from the group consisting of an alkyne, cycloalkyne, azide, 1,3-diene, nitrile oxide, nitrone, tetrazine, trans-cyclooctene, and carbonyl (e.g., aldehyde). 
     
     
         6 . The compound of  claim 1 , having the structure of formula (II): 
       
         
           
           
               
               
           
         
         wherein 
         E is a moiety selected from the group consisting of halogen, —CN, —NO 2 , —SO 2 , —SO 2 NHW 4 , —S(O)C 1 -C 6  alkyl, —S(O)aryl, and —C 1 -C 6  alkyl; 
         X is O, S or Se; 
         Y is a linker selected from the group consisting of alkyl, polyalkylenoxide, and combinations thereof; 
         R is selected from the group consisting of an alkyne, cycloalkyne, azide, 1,3-diene, nitrile oxide, nitrone, tetrazine, trans-cyclooctene, and carbonyl; and 
         R′ is —C(O)W 1 , —SO 2 W 1 , —CH 2 W 2 , —C(O)W 3 , —SO 2 W 3 , —C(O)W 4  or —SO 2 W 4 ; wherein 
         W 1  comprises a linear peptide of 1-25 amino acid residues, attached to C(O) or SO 2  at the N-terminus; 
         W 2  comprises a peptide of 1-25 amino acid residues, attached to CH 2  at an O-tyrosine or S-thiotyrosine residue; 
         W 3  comprises a peptide of 1-25 amino acid residues, attached to C(O) or SO 2  at the P-amino group of a 2,P-diamino-n-alkanoic acid residue, wherein P is 3, 4, 5, 6, 7 or 8; 
         W 4  comprises a peptide of up to 25 residues, linked to C(O) or SO 2  at the N-terminus; and 
         q is 0, 1, 2 3 or 4. 
       
     
     
         7 . The compound of  claim 6 , wherein Y has the formula:
   —(CH 2 CH 2 O) m (CH 2 ) n —
   wherein m is 0-30 and n is 1-20.   
     
     
         8 . The compound of  claim 6 , wherein W 1 , W 2 , W 3  and W 4  independently comprise peptides of 4-8 or 6-10 amino acid residues. 
     
     
         9 . The compound of  claim 1 , having the structure of formula (III): 
       
         
           
           
               
               
           
         
         wherein 
         E is a moiety selected from the group consisting of halogen, —CN, —NO 2 , —SO 2 , —SO 2 NHW 4 , —S(O)C 1 -C 6  alkyl, —S(O)aryl, and —C 1 -C 6  alkyl; 
         X is O, S or Se; 
         Y is a linker selected from the group consisting of alkyl, polyalkylenoxide, and combinations thereof; 
         R is selected from the group consisting of an alkyne, cycloalkyne, azide, 1,3-diene, nitrile oxide, nitrone, tetrazine, trans-cyclooctene, and carbonyl; and 
       
       R′ is —C(O)W 1 , —SO 2 W 1 , —CH 2 W 2 , —C(O)W 3 , —SO 2 W 3 , —C(O)W 4  or —SO 2 W 4 ; wherein
 W 1  comprises a linear peptide of 1-25 amino acid residues, attached to C(O) or SO 2  at the N-terminus; 
 W 2  comprises a peptide of 1-25 amino acid residues, attached to CH 2  at an O-tyrosine or S-thiotyrosine residue; 
 W 3  comprises a peptide of 1-25 amino acid residues, attached to C(O) or SO 2  at the P-amino group of a 2,P-diamino-n-alkanoic acid residue, wherein P is 3, 4, 5, 6, 7 or 8; 
 W 4  comprises a peptide of up to 25 residues, linked to C(O) or SO 2  at the N-terminus; and 
 q is 0, 1, 2 3 or 4. 
 
     
     
         10 . The compound of  claim 9 , wherein Y has the formula:
   —(CH 2 CH 2 O) m (CH 2 ) n —
   wherein m is 0-30 and n is 1-20.   
     
     
         11 . The compound of  claim 9 , wherein W 1 , W 2 , W 3  and W 4  independently comprise peptides of 4-8 or 6-10 amino acid residues. 
     
     
         12 . The compound of  claim 1  having the structure of formula (IV): 
       
         
           
           
               
               
           
         
         wherein 
         E is selected from the group of: 
       
       
         
           
           
               
               
           
         
         R 1 , R 2 , R 3 , R 4 , and R 5  independently, are selected from H, F, Cl, NO 2 , CN and L, provided that only one of R 1 , R 2 , R 3 , R 4 , and R 5  is L; 
         L is R—(CH 2 CH 2 O) q (CH 2 ) r —C(O)—X— or R—(CH 2 CH 2 O) q (CH 2 ) r —X—SO 2 —; 
         R is selected from the group consisting of an alkyne, cycloalkyne, azide, 1,3-diene, nitrile oxide, nitrone, tetrazine, trans-cyclooctene, and carbonyl (e.g., aldehyde); 
         X is O or S; 
         X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7  and X 8  independently are selected from residues of natural amino acids and 2,3-diaminopropionic acid, thiotyrosine, 4-benzoylphenylalanine, 2-thiazole-alanine, norvaline, 1-naphthylalanine, 2-naphthylalanine, 3-naphthylalanine, N N-□□carbamyl-lysine, 2-thienylalanine, 3-aminopyrrolidine-4-carboxylic acid, 2′,4′-phenylalanine, 2′,5′-phenylalanine, 2′,6′-phenylalanine, 3′,4′-phenylalanine, —OH, —O—(C 1 -C 6 )alkyl, —O-aryl, amino, —(C 1 -C 6 )alkylamino, di-(C 1 -C 6 )alkylamino, or are absent, provided that at least one of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7  and X 8  are present; 
         Q is an electron-withdrawing substituent (e.g., haloalkyl, —F, —NO 2  or —CN); 
         g is 0-8; 
         m is 0 or 1; 
         n is 0-8; 
         p is 1-8; 
         r is 1-20; and 
         q is 0-100. 
       
     
     
         13 . The compound of  claim 12 , wherein X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7  and X 8  independently are amino acid residues selected from the group of: glutamic acid, glutamine, aspartic acid, asparagine, arginine, methionine, serine, tyrosine, leucine, isoleucine, alanine, glycine, threonine, valine, proline, phenylalanine, tryptophan, 2,3-diaminopropionic acid, thiotyrosine, 4-benzoylphenylalanine, 2-thiazole-alanine, norvaline, 1-naphthylalanine, 2-naphthylalanine, 3-naphthylalanine, N-□□carbamyl-lysine, 2-thienylalanine, 3-aminopyrrolidine-4-carboxylic acid, 2′,4′-phenylalanine, 2′,5′-phenylalanine, 2′,6′-phenylalanine, 3′,4′-phenylalanine, —OH, —O—(C 1 -C 6 )alkyl, —O-aryl, amino, —(C 1 -C 6 )alkylamino, di-(C 1 -C 6 )alkylamino, or are absent, provided that at least one of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7  and X 8  are present. 
     
     
         14 . The compound of  claim 12 , wherein at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or 8 of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7  and X 8  are present. 
     
     
         15 . The compound of  claim 12 , wherein at least four of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7  and X 8  independently are an aromatic ring-containing amino acid, leucine, valine or isoleucine. 
     
     
         16 . A method for site-selectively functionalizing an amino group of a protein, comprising:
 contacting the protein with a compound according to  claim 1 ;   forming a covalent attachment between the amino group of the protein and the first orthogonally-reactive moiety of the compound; and   breaking the covalent attachment between the first orthogonally-reactive moiety and the peptide;   to obtain a site-selectively functionalized protein.   
     
     
         17 . The method of  claim 15 , wherein the first orthogonally-reactive moiety comprises an azide. 
     
     
         18 . The method of  claim 15 , wherein the protein is an antibody and the amino group is located in the Fab region of the antibody. 
     
     
         19 . The method of  claim 18  wherein the antibody is a therapeutic antibody useful for the treatment of cancer or autoimmune diseases. 
     
     
         20 . The method of  claim 15 , wherein the compound has an affinity for a conserved region of the protein. 
     
     
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