Methods and compositions for site-specific labeling of peptides and proteins

Abstract

Methods and compositions are provided for covalently linking a chemical species to a recombinant or synthetic polypeptide. The methods involve the reaction of a thioester-comprising polypeptide with a reagent comprising a reactive amino-thiol group connected to the chemical species which is to be covalently linked to the polypeptide, via a linker. Such chemical species can be a functional group, a label or tag molecule, a biological molecule, a ligand, or a solid support. Efficient and catalyst-free methods for C-terminal protein labeling are also provided. The methods expand current capabilities in the area of protein functionalization, providing useful and complementary tools for the isolation, detection, characterization, and analysis of proteins in a variety of in vitro and in vivo applications.

what is claimed is: 
     
         1 . A method for forming a covalent linkage between a polypeptide and a chemical species, the method comprising the steps of:
 a) providing a polypeptide, wherein the polypeptide comprises a thioester group and/or wherein the polypeptide is C-terminally fused to an intein;   b) providing a chemical reagent of formula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII):   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           or a salt of the chemical reagent, wherein:
 i) R is a chemical species to be covalently linked to the polypeptide, 
 ii) R 1  is hydrogen, a substituted or non-substituted aliphatic group, or a substituted or non-substituted aryl group, 
 iii) X, Y, W, and Z are hydrogen and/or non-hydrogen substituents selected from the group consisting of alkyl, heteroatom-comprising alkyl, alkenyl, heteroatom-comprising alkenyl, alkynyl, heteroatom-comprising alkynyl, aryl, heteroatom-comprising aryl, alkoxy, heteroatom-comprising alkoxy, aryloxy, heteroatom-comprising aryloxy, halo, —OH, —OR′, —SR′, —COOH, —COOR′, —CONR′ 2 , —NR′ 2 , —NO 2 , —SO 3 R′, —SO 2 NR 2 ′, —C≡N, —O—C≡N, —P(O) k R′ where k is 2 or 3, and —S—C≡N, wherein each R′ is independently H, alkyl, or substituted alkyl, 
 iv) n is 2 or 3; and 
 v) L is a linker or a linker group selected from the group consisting of a single bond, C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-comprising alkyl, C 1 -C 24  substituted heteroatom-comprising alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-comprising alkenyl, C 2 -C 24  substituted heteroatom-comprising alkenyl, C 2 -C 24  alkynyl, C 2 -C 24  substituted alkynyl, C 2 -C 24  substituted heteroatom-comprising alkynyl, C 2 -C 24  substituted heteroatom-comprising alkynyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-comprising aryl, C 5 -C 24  substituted heteroatom-comprising aryl, C 1 -C 24  alkoxy, C 5 -C 24  aryloxy, —O—, —S—, —NR′—, —C(O)—, —C(S)—, —C(O)NR′—, —C(S)NR′—, —N(R′)C(O)—, —S(O) k — where k is 1, 2, or 3, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—, wherein each R′ is independently an H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; and 
 
         
         c) allowing the polypeptide to react with the chemical reagent so that a covalent linkage between the reagent and the polypeptide is formed. 
       
     
     
         2 . The method of  claim 1 , wherein R is a functional group, a label molecule, a tag molecule, an affinity label molecule, a photoaffinity label, a dye, a chromophore, a fluorescent molecule, a phosphorescent molecule, a chemiluminescent molecule, an energy transfer agent, a photocrosslinker molecule, a redox-active molecule, an isotopic label molecule, a spin label molecule, a metal chelator, a metal-comprising moiety, a heavy atom-comprising-moiety, a radioactive moiety, a contrast agent molecule, a MRI contrast agent, an isotopically labeled molecule, a PET agent, a photocaged moiety, a photoisomerizable moiety, a chemically cleavable group, a photocleavable group, an electron dense group, a magnetic group, an amino acid, a polypeptide, an antibody or antibody fragment, a carbohydrate, a monosaccharide, a polysaccharide, a nucleotide, a nucleoside, a DNA, a RNA, a siRNA, a polynucleotide, an antisense polynucleotide, a peptide nucleic acid (PNA), a fatty acid, a lipid, a cofactor, biotin, a biotin analogue, a biomaterial, a polymer, a water-soluble polymer, a polyethylene glycol derivative, a water-soluble dendrimer, a cyclodextrin, a small molecule, a protein-, nucleic acid-, or receptor-binding molecule, a biologically active molecule, a drug or drug candidate, a cytotoxic molecule, a solid support, a surface, a resin, a nanoparticle, or a quantum dot, or any combination thereof. 
     
     
         3 . The method of  claim 1 , wherein:
 R is a bioorthogonal functional group selected from the group consisting of —NR′NR′ 2 , —C(O)NR′NR′ 2 , —ONH 2 , —N 3 , —C≡CR′, —CR′═CR′ 2 , —PR′ 2 , 2-cyanobenzothiazole, tetrazole, tetrazine, aziridine, dihydroazirine, and norbornadiene groups, and   each R′ is independently an H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group.   
     
     
         4 . The method of  claim 1 , wherein R is a fluorescent molecule selected from the group consisting of a coumarin derivative, a naphthalene derivative, a pyrene derivative, a fluorescein derivative, a rhodamine derivative, a naphthoxanthene derivative, a phenanthridine derivative, a boron difluoride dipyrromethene (BODIPY) derivative, a cyanine derivatives, a phthalocyanine derivative, and an oxazine derivative. 
     
     
         5 . The method of  claim 1 , wherein R is biotin, a biotin analogue, a poly(ethyleneglycol) molecule, or a perfluorinated alkyl chain CF 3 —(CF 2 ) m — where m=3-15. 
     
     
         6 . The method of  claim 1 , wherein R is a resin, a nanoparticle, a functionalized surface, or a microarray. 
     
     
         7 . The method of  claim 1 , wherein the intein is a naturally occurring intein, an engineered variant of a naturally occurring intein, a fusion of the N-terminal and C-terminal fragments of a naturally occurring split intein, or a fusion of the N-terminal and C-terminal fragments of an artificial split intein. 
     
     
         8 . The method of  claim 1 , wherein the intein is a polypeptide of SEQ ID NO:1-76, or an engineered variant thereof. 
     
     
         9 . The method of  claim 8 , wherein:
 the C-terminal terminal asparagine, aspartic acid, or glutamine residue in the intein is mutated to an amino acid other than asparagine, aspartic acid, or glutamine, or   the N-terminal serine is mutated to a cysteine residue and the C-terminal asparagine, aspartic acid, or glutamine residue in the intein is mutated to an amino acid other than asparagine, aspartic acid, or glutamine.   
     
     
         10 . The method of  claim 9 , wherein the intein is C-terminally fused to a polypeptide affinity tag selected from the group consisting of polyhistidine tag, Avi-Tag, FLAG tag, Strep-tag II, c-myc tag, S-Tag, calmodulin-binding peptide, streptavidin-binding peptide, chitin-binding domain, glutathione S-transferase, and maltose-binding protein. 
     
     
         11 . The method of  claim 1 , wherein the polypeptide C-terminally fused to the intein comprises one or a plurality of the features selected from the group consisting of: the residue at position 1 prior to the intein (hereinafter “intein-1” or “I-1”) being F, Y, A, T, W, N, R or Q; the residue at position 2 prior to the intein (hereinafter “intein-2” or “I-2”) being G, P, or S; and the residue at position 3 prior to the intein (hereinafter “intein-3” or “I-3”) being G or S. 
     
     
         12 . The method of  claim 1 , wherein the intein-fused polypeptide is inside a cell or associated with the exterior surface of a cell membrane. 
     
     
         13 . The method of  claim 12 , wherein the cell is a prokaryotic or eukaryotic cell. 
     
     
         14 . The method of  claim 1 , wherein:
 R 1 , X, Y, and Z are hydrogen atoms,   L is selected from the group consisting of —C(O)NR′—, —C(O)NR′CH 2 C(O)—, —C(O)NR′(CH 2 )n-, and —C(O)NR′(CH 2 —CH 2 —O)n-,   R′ is a hydrogen, alkyl or aryl group, and   n is an integer number from 1 to 15.   
     
     
         15 . The method of  claim 14 , wherein R is selected from the group consisting of biotin, a biotin analogue, and a coumarin derivative. 
     
     
         16 . The method of  claim 1 , wherein the reagent is:
 a) a compound of formula (I), wherein:   R 1 , X, Y, and Z are hydrogen atoms,   R is —ONH 2  or —N 3 , and   L is a single bond;   b) a compound of formula (I), wherein:   R 1 , X, Y, and Z are hydrogen atoms,   R is —ONH 2 , and   L is a linker or linker group of formula   
       
         
           
           
               
               
           
         
         c) a compound of formula (I), wherein: 
         R 1 , X, Y, and Z are hydrogen atoms, 
         R is 7-amino-4-(trifluoromethyl)-2H-chromen-2-one, and 
         L is —C(O)NHCH 2 C(O)—; or 
         d) a compound of formula (I), wherein: 
         R 1 , X, Y, and Z are hydrogen atoms, 
         R is biotin, and 
         L is —C(O)NH(CH 2 ) 3 NH—. 
       
     
     
         17 . A kit for forming a covalent linkage between a polypeptide and a chemical species, the kit comprising:
 a) at least one chemical reagent of formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a salt of the reagent; and   b) one or a plurality of containers, wherein at least one container comprises a pre-selected or desired amount of at least one of the chemical reagents of formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a salt of the reagent, wherein:
 i) R is the chemical species which is to be covalently linked to the polypeptide, 
 ii) R 1  is hydrogen, a substituted or non-substituted aliphatic group, or a substituted or non-substituted aryl group, 
 iii) X, Y, W, and Z are hydrogen and/or non-hydrogen substituents selected from the group consisting of alkyl, heteroatom-comprising alkyl, alkenyl, heteroatom-comprising alkenyl, alkynyl, heteroatom-comprising alkynyl, aryl, heteroatom-comprising aryl, alkoxy, heteroatom-comprising alkoxy, aryloxy, heteroatom-comprising aryloxy, halo, —OH, —OR′, —SR′, —COOH, —COOR′, —CONR′ 2 , —NR′ 2 , —NO 2 , —SO 3 R′, —SO 2 NR 2 ′, —C≡N, —O—C≡N, —P(O) k R′ where k is 2 or 3, and —S—C≡N, wherein each R′ is independently an H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group, 
 iv) n is 2 or 3, and 
 v) L is a linker or a linker group selected from the group consisting of a single bond, C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-comprising alkyl, C 1 -C 24  substituted heteroatom-comprising alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-comprising alkenyl, C 2 -C 24  substituted heteroatom-comprising alkenyl, C 2 -C 24  alkynyl, C 2 -C 24  substituted alkynyl, C 2 -C 24  substituted heteroatom-comprising alkynyl, C 2 -C 24  substituted heteroatom-comprising alkynyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-comprising aryl, C 5 -C 24  substituted heteroatom-comprising aryl, C 1 -C 24  alkoxy, C 5 -C 24  aryloxy, —O—, —S—, —NR′—, —C(O)—, —C(S)—, —C(O)NR′—, —C(S)NR′—, —N(R′)C(O)—, —S(O) k — where k is 1, 2, or 3, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—, wherein each R′ is independently an H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group. 
   
     
     
         18 . The kit of  claim 17 , wherein R is a functional group, a label molecule, a tag molecule, an affinity label molecule, a photoaffinity label, a dye, a chromophore, a fluorescent molecule, a phosphorescent molecule, a chemiluminescent molecule, an energy transfer agent, a photocrosslinker molecule, a redox-active molecule, an isotopic label molecule, a spin label molecule, a metal chelator, a metal-comprising moiety, a heavy atom-comprising-moiety, a radioactive moiety, a contrast agent molecule, a MRI contrast agent, an isotopically labeled molecule, a PET agent, a photocaged moiety, a photoisomerizable moiety, a chemically cleavable group, a photocleavable group, an electron dense group, a magnetic group, an amino acid, a polypeptide, an antibody or antibody fragment, a carbohydrate, a monosaccharide, a polysaccharide, a nucleotide, a nucleoside, a DNA, a RNA, a siRNA, a polynucleotide, an antisense polynucleotide, a peptide nucleic acid (PNA), a fatty acid, a lipid, a cofactor, biotin, a biotin analogue, a biomaterial, a polymer, a water-soluble polymer, a polyethylene glycol derivative, a water-soluble dendrimer, a cyclodextrin, a small molecule, a protein-, nucleic acid-, or receptor-binding molecule, a biologically active molecule, a drug or drug candidate, a cytotoxic molecule, a solid support, a surface, a resin, a nanoparticle, or a quantum dot, or any combination thereof. 
     
     
         19 . The kit of  claim 17 , wherein R is a bioorthogonal functional group selected from the group consisting of —NR′NR′ 2 , —C(O)NR′NR′ 2 , —ONH 2 , —N 3 , —C≡CR′, —CR′═CR′ 2 , —PR′ 2 , 2-cyanobenzothiazole, tetrazole, tetrazine, aziridine, dihydroazirine, norbornadiene groups, wherein each R is independently H, aliphatic, substituted aliphatic, aryl, or substituted aryl group. 
     
     
         20 . The kit of  claims 17 , wherein R is a fluorescent molecule selected from the group consisting of a coumarin derivative, a naphthalene derivative, a pyrene derivative, a fluorescein derivative, a rhodamine derivative, a naphthoxanthene derivative, a phenanthridine derivative, a boron difluoride dipyrromethene (BODIPY) derivative, a cyanine derivatives, a phthalocyanine derivative, and a oxazine derivative. 
     
     
         21 . The kit of  claim 17 , wherein R is biotin, a biotin analogue, or a perfluorinated alkyl chain CF 3 —(CF 2 ) m — where m=3-15. 
     
     
         22 . The kit of  claim 17 , wherein the at least one reagent comprises at least one compound selected from the group consisting of:
 a) a compound of formula (I), wherein:   R 1 , X, Y, and Z are hydrogen atoms,   R is —ONH 2  or —N 3 , and   L is a single bond:   b) a compound of formula (I), wherein:   R 1 , X, Y, and Z are hydrogen atoms,   R is —ONH 2 , and   L is a linker or linker group of formula   
       
         
           
           
               
               
           
         
         c) a compound of formula (I), wherein: 
         R 1 , X, Y, and Z are hydrogen atoms, 
         R is 7-amino-4-(trifluoromethyl)-2H-chromen-2-one, and 
         L is —C(O)NHCH 2 C(O)—; or 
         d) a compound of formula (I), wherein: 
         R 1 , X, Y, and Z are hydrogen atoms, 
         R is biotin, and 
         L is —C(O)NH(CH 2 ) 3 NH—. 
       
     
     
         23 . The kit of  claim 17  further comprising a functionalized solid support with which the functional group R reacts. 
     
     
         24 . The kit of  claim 18 , wherein the solid support is a resin, a nanoparticle, a surface, or a microarray. 
     
     
         25 . A compound having the formula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
         i) R is a bioorthogonal functional group, a label molecule, a tag molecule, an affinity label molecule, a photoaffinity label, a dye, a chromophore, a fluorescent molecule, a phosphorescent molecule, a chemiluminescent molecule, an energy transfer agent, a photocrosslinker molecule, a redox-active molecule, a spin label molecule, a metal chelator, a metal-comprising moiety, a heavy atom-comprising-moiety, a radioactive moiety, a contrast agent molecule, a MRI contrast agent, an isotopically labeled molecule, a PET agent, a photocaged moiety, a photoisomerizable moiety, a chemically cleavable group, a photocleavable group, an electron dense group, a magnetic group, an amino acid, a polypeptide, an antibody or antibody fragment, a carbohydrate, a monosaccharide, a polysaccharide, a nucleotide, a nucleoside, a DNA, a RNA, a siRNA, a polynucleotide, an antisense polynucleotide, a peptide nucleic acid (PNA), a fatty acid, a lipid, a cofactor, biotin, a biotin analogue, a biomaterial, a polymer, a water-soluble polymer, a polyethylene glycol derivative, a water-soluble dendrimer, a cyclodextrin, a small molecule, a protein-, nucleic acid-, or receptor-binding molecule, a biologically active molecule, a drug or drug candidate, a cytotoxic molecule, a solid support, a surface, a resin, a nanoparticle, a quantum dot, or any combination thereof, 
         ii) R 1  is hydrogen, a substituted or non-substituted aliphatic group, or a substituted or non-substituted aryl group, 
         iii) X, Y, W, and Z are hydrogen and/or non-hydrogen substituents selected from the group consisting of alkyl, heteroatom-comprising alkyl, alkenyl, heteroatom-comprising alkenyl, alkynyl, heteroatom-comprising alkynyl, aryl, heteroatom-comprising aryl, alkoxy, heteroatom-comprising alkoxy, aryloxy, heteroatom-comprising aryloxy, halo, —OH, —OR′, —SR′, —COOH, —COOR′, —CONR′ 2 , —NR′ 2 , —NO 2 , —SO 3 R′, —SO 2 NR 2 ′, —C≡N, —O—C≡N, —P(O) k R′ where k is 2 or 3, and —S—C≡N, wherein each R′ is independently H, alkyl, or substituted alkyl, 
         iv) n is 2 or 3; and 
         v) L is a linker or a linker group selected from the group consisting of a single bond, C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-comprising alkyl, C 1 -C 24  substituted heteroatom-comprising alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-comprising alkenyl, C 2 -C 24  substituted heteroatom-comprising alkenyl, C 2 -C 24  alkynyl, C 2 -C 24  substituted alkynyl, C 2 -C 24  substituted heteroatom-comprising alkynyl, C 2 -C 24  substituted heteroatom-comprising alkynyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-comprising aryl, C 5 -C 24  substituted heteroatom-comprising aryl, C 1 -C 24  alkoxy, C 5 -C 24  aryloxy, —O—, —S—, —NR′—, —C(O)—, —C(S)—, —C(O)NR′—, —C(S)NR′—, —N(R′)C(O)—, —S(O) k — where k is 1, 2, or 3, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—, where each R′ is independently an H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group, 
         the compound being reactive with a polypeptide,
 wherein the polypeptide comprises a thioester group and/or wherein the polypeptide is C-terminally fused to an intein, and 
 wherein reaction of the compound with the polypeptide forms a covalent linkage between the compound and the polypeptide. 
 
       
     
     
         26 . The compound of  claim 25 , wherein:
 R is a bioorthogonal functional group selected from the group consisting of —NR′NR′ 2 , —C(O)NR′NR′ 2 , —ONH 2 , —N 3 , —C≡CR′, —CR′═CR′ 2 , —PR′ 2 , 2-cyanobenzothiazole, tetrazole, tetrazine, aziridine, dihydroazirine, and norbornadiene groups, and   each R′ is independently an H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group.   
     
     
         27 . The compound of  claim 25 , wherein R is a fluorescent molecule selected from the group consisting of a coumarin derivative, a naphthalene derivative, a pyrene derivative, a fluorescein derivative, a rhodamine derivative, a naphthoxanthene derivative, a phenanthridine derivative, a boron difluoride dipyrromethene (BODIPY) derivative, a cyanine derivatives, a phthalocyanine derivative, and an oxazine derivative. 
     
     
         28 . The compound of  claim 25 , wherein R is biotin, a biotin analogue, a poly(ethyleneglycol) molecule, or a perfluorinated alkyl chain CF 3 —(CF 2 ) m — where m=3-15. 
     
     
         29 . The compound of  claim 25 , wherein R is a resin, a nanoparticle, a functionalized surface, or a microarray. 
     
     
         30 . The compound of  claim 25 , wherein:
 R 1 , X, Y, and Z are hydrogen atoms,   L is selected from the group consisting of —C(O)NR′—, —C(O)NR′CH 2 C(O)—, —C(O)NR′(CH 2 )n-, and —C(O)NR′(CH 2 —CH 2 —O)n-,   R′ is a hydrogen, alkyl or aryl group, and   n is an integer number from 1 to 15.   
     
     
         31 . The compound of  claim 25 , wherein R is selected from the group consisting of biotin, a biotin analogue, and a coumarin derivative. 
     
     
         32 . The compound of  claim 25  having formula (I), wherein:
 a) R 1 , X, Y, and Z are hydrogen atoms, 
 R is —ONH 2  or —N 3 , and 
 L is a single bond; 
 b) R 1 , X, Y, and Z are hydrogen atoms, 
 R is —ONH 2 , and 
 L is a linker or linker group of formula 
 
       
         
           
           
               
               
           
         
         c) R 1 , X, Y, and Z are hydrogen atoms, 
         R is 7-amino-4-(trifluoromethyl)-2H-chromen-2-one, and 
         L is —C(O)NHCH 2 C(O)—; or 
         d) R 1 , X, Y, and Z are hydrogen atoms, 
         R is biotin, and 
         L is —C(O)NH(CH 2 ) 3 NH—.