Pseudo native chemical ligation
View Patent ↗The present invention concerns methods and compositions for extending the technique of native chemical ligation of a wider range of peptides, polypeptides, other polymers and other molecules via an amide bond (see FIG. 1 ). 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 method for synthesizing a desired polypeptide of formula:
aa NH 2 -Q-aa x -aa y -W-aa COOH
wherein 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 wherein aa y is a non-ribosomally synthesized amino acid residue 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, 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 comprises 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.
2. The method of claim 1 , wherein X is a halogen.
3. The method of claim 2 , wherein said halogen is F, I or Br.
4. The method of claim 1 , 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.
5. The method of claim 1 , wherein R is an aryl, benzyl, or alkyl group.