Pseudo-native chemical ligation
AssigneeAmylin Pharmaceuticals, Inc.
InventorsChristie Hunter, Paolo Botti, James Bradburne, Shiah-yun Chen, Sonya Cressman, Stephen Kent, Gerd Kochendoerfer, Donald Low
Life of this patent
Estimating the term…
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.
Claims62
A synthetic protein containing a pseudo-amino acid residue whose side chain has the formula:
- —S—Raa, where Raa 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.
The synthetic protein of claim l, wherein said side chain —S—Raa has the same chain length as the side chain of said ribosomally-specified amino acid.
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—Raa, where Raa is selected from the group consisting of an optionally substituted terminal portion of a ribosomally-specified amino acid side chain, or an analog thereof.
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—Raa where Raa 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.
A method for synthesizing a desired polypeptide of formula:
- aaNH2-Q-aax-aay-W-aaCOOH
- where Q and W each denote the optional presence of one or more additional amino acid residues, aaNH2 denotes the N-terminal amino acid residue of the polypeptide;
- aax and aay denote internal adjacent amino acid residues, having side chains x and y, respectively, and aaCOOH denotes the C-terminal amino acid residue of the polypeptide;
- said method comprising:
- (A) ligating a first peptide having the formula:
- aaNH2-Q-aax-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-aaCOOH to thereby form the polypeptide:
- aaNH2-Q-aax-Cys-W-aaCOOH;
- and
- (B) incubating said polypeptide in the presence of a reagent Raa—X, where Raa is a group whose structure mimics the terminal portion of the side chain of amino acid residue aay;
- and X is a good leaving group;
- said incubation being under conditions sufficient to form said desired polypeptide.
A polypeptide of formula:
- aaNH2-Q-aax-aay-W-aaCOOH
- where Q and W each denote the optional presence of one or more additional amino acid residues, aaNH2 denotes the N-terminal amino acid residue of the polypeptide;
- aax and aay denote internal adjacent amino acid residues, having side chains x and y, respectively, and aaCOOH 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:
- aaNH2-Q-aax-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-aaCOOH to thereby form the polypeptide:
- aaNH2-Q-aax-Cys-W-aaCOOH;
- and
- (B) incubating said polypeptide in the presence of a reagent Raa—X, where Raa is a group whose structure mimics the terminal portion of the side chain of amino acid residue aay;
- and X is a good leaving group;
- said incubation being under conditions sufficient to form said desired polypeptide.
Dependencies are inferred from the claim text.