Pseudo-native chemical ligation

Abstract

The present invention concerns methods and compositions for extending the technique of native chemical ligation to permit the ligation of a wider range of peptides, polypeptides, other polymers and other molecules via an amide bond. The invention further provides methods and uses for such proteins and derivatized proteins. The invention is particularly suitable for use in the synthesis of optionally polymer-modified, synthetic bioactive proteins, and of pharmaceutical compositions that contain such proteins.

1 . A synthetic protein containing a pseudo-amino acid residue whose side chain has the formula: —S—R aa , where R aa  is an optionally substituted terminal portion of a ribosomally-specified amino acid side chain, or an analog of said terminal portion of a ribosomally-specified amino acid side chain.  
     
     
         2 . The synthetic protein of claim l, wherein said side chain —S—R aa  has the same chain length as the side chain of said ribosomally-specified amino acid.  
     
     
         3 . The synthetic protein of  claim 1 , wherein said side chain —S—R aa  has a greater chain length than the side chain of said ribosomally-specified amino acid.  
     
     
         4 . The synthetic protein of  claim 1 , wherein said protein has a biological activity possessed by a ribosomally-specified bioactive protein.  
     
     
         5 . The synthetic protein of  claim 4 , wherein said side chain —S—R aa  of said pseudo amino acid residue mimics the structure or function of a ribosomally-specified amino acid at a corresponding position in said bioactive protein.  
     
     
         6 . The synthetic protein of  claim 4 , wherein said side chain —S—R aa  of said pseudo amino acid residue alters the structure or function of a ribosomally-specified amino acid at a corresponding position in said bioactive protein.  
     
     
         7 . The synthetic protein of  claim 4 , wherein said synthetic protein has a monomer molecular weight greater than about 25 kDa.  
     
     
         8 . The synthetic protein of  claim 4 , wherein said ribosomally produced bioactive protein contains a cysteine residue.  
     
     
         9 . The synthetic protein of  claim 8 , wherein said pseudo amino acid residue is at a position other than one corresponding to the position of said cysteine residue in said ribosomally produced bioactive protein.  
     
     
         10 . The synthetic protein of  claim 1 , wherein a plurality of amino acid residues of said protein are bound to adjacent amino acid residues by an amide bond.  
     
     
         11 . The synthetic protein of  claim 10 , wherein each amino acid residue is bonded to its adjacent amino acid residues by amide bonds.  
     
     
         12 . The synthetic protein of  claim 1 , wherein said protein is composed of a plurality of amino acid residues wherein at least one of such amino acid residues is bonded to an adjacent amino acid residue by a non-amide bond.  
     
     
         13 . The synthetic protein of  claim 12 , wherein said non-amide bond is selected from the group consisting of a thioester bond, a thioether bond, and an oxime bond.  
     
     
         14 . The synthetic protein of  claim 1 , wherein said pseudo-amino acid residue is a D-configuration amino acid residue.  
     
     
         15 . The synthetic protein of  claim 1 , wherein said pseudo-amino acid residue is an L-configuration amino acid residue.  
     
     
         16 . The synthetic protein of  claim 1 , wherein said pseudo-amino acid residue is selected from the group consisting of a pseudo-arginine; a pseudo-asparagine; a pseudo-aspartate; a pseudo-dopamine; a pseudo-glutamate; a pseudo-glutamine; a pseudo-histidine; a pseudo-isoleucine; a pseudo-leucine; a pseudo-lysine; a pseudo-methionine; a pseudo-phenyalanine; a pseudo-serine; a pseudo-threonine; a pseudo-tryptophan; a pseudo-tyrosine; and a pseudo-valine.  
     
     
         17 . The synthetic protein of  claim 16  wherein said pseudo-amino acid residue is a pseudo-glutamate.  
     
     
         18 . The synthetic protein of  claim 4 , wherein said ribosomally produced bioactive protein is a mammalian protein.  
     
     
         19 . The synthetic protein of  claim 18 , wherein said mammalian protein is selected from the group consisting of a human, simian, bovine, murine, porcine, ovine, and equine protein.  
     
     
         20 . The synthetic protein of  claim 18 , wherein said mammalian protein is a human protein.  
     
     
         21 . The synthetic protein of any of claims  19 - 20 , wherein said protein has a bioactivity selected of a protein receptor or fragment thereof, of a protein receptor ligand or fragment thereof, or of a cytokine.  
     
     
         22 . The synthetic protein of  claim 21 , wherein said synthetic bioactive protein has a bioactivity of a cytokine.  
     
     
         23 . The synthetic protein of  claim 22 , wherein said cytokine is selected from the group consisting of an interleukin, a lymphokine, a RANTES protein, an erythropoiesis stimulating protein, tumor necrosis factor (TNF), an interferon, a growth factors and a single peptide hormone.  
     
     
         24 . The synthetic protein of  claim 23 , wherein said cytokine is an erythropoiesis stimulating protein.  
     
     
         25 . The synthetic protein of  claim 24 , wherein said erythropoiesis stimulating protein is erythropoietin.  
     
     
         26 . The synthetic protein of  claim 25 , wherein said synthetic bioactive protein is selected from the group consisting of SEP-0, SEP-1, and SEP-3.  
     
     
         27 . The synthetic protein of  claim 22 , wherein said cytokine is a RANTES protein.  
     
     
         28 . The synthetic protein of  claim 22 , wherein said cytokine is a growth factor.  
     
     
         29 . The synthetic protein of  claim 28 , wherein said growth factor is G-CSF.  
     
     
         30 . The synthetic protein of  claim 1 , wherein said synthetic protein comprises one or more amino acid residues that are modified by one or more polymer adducts.  
     
     
         31 . The synthetic protein of  claim 4 , wherein said ribosomally produced bioactive protein is glycosylated at one or more glycosylation sites.  
     
     
         32 . The synthetic protein of  claim 31 , wherein said synthetic protein comprises one or more amino acid residues that are modified by one or more polymer adducts at amino acid residue(s) that correspond to at least one of said glycosylation sites of said ribosomally produced bioactive protein.  
     
     
         33 . A molecularly homogeneous pharmaceutical composition comprising a synthetic protein, wherein said protein possesses a biological activity that mimics a biological activity associated with a ribosomally-specified bioactive mammalian protein, and has a monomer molecular weight greater than about 25 kDa, and contains a pseudo-amino acid residue whose side chain has the formula: —S—R aa , where R aa  is selected from the group consisting of an optionally substituted terminal portion of a ribosomally-specified amino acid side chain, or an analog thereof.  
     
     
         34 . The pharmaceutical composition of  claim 33 , wherein said composition comprises a mixture of at least two of said molecularly homogeneous pharmaceutical compositions.  
     
     
         35 . The pharmaceutical composition of  claim 33 , wherein said ribosomally-specified bioactive mammalian protein is selected from the group consisting of a human, simian, bovine, murine, porcine, ovine, and equine protein.  
     
     
         36 . The pharmaceutical composition of  claim 33 , wherein said ribosomally-specified bioactive mammalian protein is a human protein.  
     
     
         37 . The pharmaceutical composition of  claim 36 , wherein said synthetic protein has a biological activity of a protein receptor or fragment thereof, of a protein receptor ligand or fragment thereof, or of a cytokine.  
     
     
         38 . The pharmaceutical composition of  claim 37 , wherein said synthetic protein has a biological activity of a cytokine.  
     
     
         39 . The pharmaceutical composition of  claim 38 , wherein said cytokine is selected from the group consisting of an interleukin, a RANTES protein, a lymphokine, an erythropoiesis stimulating protein, tumor necrosis factor (TNF), an interferon, a growth factor and a single peptide hormone.  
     
     
         40 . The pharmaceutical composition of  claim 39 , wherein said cytokine is an erythropoiesis stimulating protein.  
     
     
         41 . The pharmaceutical composition of  claim 40 , wherein said erythropoiesis stimulating protein is erythropoietin.  
     
     
         42 . The pharmaceutical composition of  claim 41 , wherein said synthetic protein is selected from the group consisting of SEP-0, SEP-1, and SEP-3.  
     
     
         43 . The pharmaceutical composition of  claim 38 , wherein said cytokine is a RANTES protein.  
     
     
         44 . The pharmaceutical composition of  claim 39 , wherein said cytokine is a growth factor.  
     
     
         45 . The pharmaceutical composition of  claim 44 , wherein said growth factor is G-CSF.  
     
     
         46 . A method of treating a human disease or condition that comprises administering to an individual in need of such treatment an effective amount of a pharmaceutical composition comprising one or more molecularly homogeneous pharmaceutical compositions each comprising a synthetic protein, wherein said synthetic protein has a monomer molecular weight greater than about 25 kDa, and contains a pseudo-amino acid residue whose side chain has the formula: —S—R aa  where R aa  is selected from the group consisting of an optionally substituted terminal portion of a ribosomally-specified amino acid side chain, or an analog thereof; said synthetic protein possessing a biological activity that mimics a biological activity of a ribosomally-specified bioactive human protein receptor or fragment thereof, protein receptor ligand or fragment thereof, or a cytokine.  
     
     
         47 . The method of  claim 46 , wherein said synthetic protein has a biological activity of a cytokine.  
     
     
         48 . The method of  claim 47 , wherein said cytokine is selected from the group consisting of an interleukin, a lymphokine, a RANTES protein, an erythropoiesis stimulating protein, tumor necrosis factor (TNF), an interferon, a growth factor and a single peptide hormone.  
     
     
         49 . The method of  claim 48 , wherein said cytokine is an erythropoiesis stimulating protein.  
     
     
         50 . The method of  claim 49 , wherein said erythropoiesis stimulating protein is erythropoietin.  
     
     
         51 . The method of  claim 50 , wherein said synthetic bioactive protein is selected from the group consisting of SEP-0, SEP-1, and SEP-3.  
     
     
         52 . The method of  claim 48 , wherein said cytokine is a RANTES protein.  
     
     
         53 . The method of  claim 48 , wherein said cytokine is a growth factor.  
     
     
         54 . The method of  claim 53 , wherein said growth factor is G-CSF.  
     
     
         55 . A method for synthesizing a desired polypeptide of formula:  
         aa NH     2   -Q-aa x -aa y -W-aa COOH    
       where Q and W each denote the optional presence of one or more additional amino acid residues, aa NH     2    denotes the N-terminal amino acid residue of the polypeptide; aa x  and aa y  denote internal adjacent amino acid residues, having side chains x and y, respectively, and aa COOH  denotes the C-terminal amino acid residue of the polypeptide; 
 said method comprising:  
 (A) ligating a first peptide having the formula: aa NH     2   -Q-aa x -COSR, wherein R is any group compatible with the thioester group, including, but not limited to, aryl, benzyl, and alkyl groups, to a second peptide having the formula: Cys-W-aa COOH  to thereby form the polypeptide: aa NH     2   -Q-aa x -Cys-W-aa COOH ; and  
 (B) incubating said polypeptide in the presence of a reagent R aa —X, where R aa  is a group whose structure mimics the terminal portion of the side chain of amino acid residue aa y ; and X is a good leaving group; said incubation being under conditions sufficient to form said desired polypeptide.  
 
     
     
         56 . The method of  claim 55 , wherein X is a halogen.  
     
     
         57 . The method of  claim 56 , wherein said halogen is F, I or Br.  
     
     
         58 . The method of  claim 55 , wherein said amino acid residue aa y  is selected from the group consisting of a pseudo-arginine; a pseudo-asparagine; a pseudo-aspartate; a pseudo-dopamine; a pseudo-glutamate; a pseudo-glutamine; a pseudo-histidine; a pseudo-isoleucine; a pseudo-leucine; a pseudo-lysine; a pseudo-methionine; a pseudo-phenyalanine; a pseudo-serine; a pseudo-threonine; a pseudo-tryptophan; a pseudo-tyrosine; and a pseudo-valine.  
     
     
         59 . A polypeptide of formula:  
         aa NH     2   -Q-aa x -aa y -W-aa COOH    
       where Q and W each denote the optional presence of one or more additional amino acid residues, aa NH     2    denotes the N-terminal amino acid residue of the polypeptide; aa x  and aa y  denote internal adjacent amino acid residues, having side chains x and y, respectively, and aa COOH  denotes the C-terminal amino acid residue of the polypeptide; 
 wherein said polypeptide is produced by a method comprising:  
 (A) ligating a first peptide having the formula: aa NH     2   -Q-aa x -COSR, wherein R is any group compatible with the thioester group, including, but not limited to, aryl, benzyl, and alkyl groups, to a second peptide having the formula: Cys-W-aa COOH  to thereby form the polypeptide: aa NH     2   -Q-aa x -Cys-W-aa COOH ; and  
 (B) incubating said polypeptide in the presence of a reagent R aa —X, where R aa  is a group whose structure mimics the terminal portion of the side chain of amino acid residue aa y ; and X is a good leaving group; said incubation being under conditions sufficient to form said desired polypeptide.  
 
     
     
         60 . The polypeptide of  claim 59 , wherein X is a halogen.  
     
     
         61 . The polypeptide of  claim 60 , wherein said halogen is F, I or Br.  
     
     
         62 . The polypeptide of  claim 59 , wherein said amino acid residue aa y  is selected from the group consisting of a pseudo-arginine; a pseudo-asparagine; a pseudo-aspartate; a pseudo-dopamine; a pseudo-glutamate; a pseudo-glutamine; a pseudo-histidine; a pseudo-isoleucine; a pseudo-leucine; a pseudo-lysine; a pseudo-methionine; a pseudo-phenyalanine; a pseudo-serine; a pseudo-threonine; a pseudo-tryptophan; a pseudo-tyrosine; and a pseudo-valine.