Convergent synthesis of proteins by kinetically controlled ligation
First Claim
1. A method of synthesizing a polypeptide comprising:
- obtaining a first C-activated peptide, having (1) an N-terminal-protected cysteinyl residue or an N-terminal non-cysteinyl residue and (2) a good C-terminal thiol-based leaving group;
a first N-deprotected peptide, having an N-terminal unprotected cysteinyl residue and a weak C-terminal thiol-based leaving group;
a second C-activated peptide, having an N-terminal-protected cysteinyl residue and a good C-terminal thiol-based leaving group, wherein the good C-terminal thiol-based leaving groups are the same or different;
a second N-deprotected peptide, having (1) an N-terminal unprotected cysteinyl residue and (2) a C-terminal α
-carboxylate, α
-carboxamide, or a weak C-terminal thiol-based leaving group, wherein the weak C-terminal thiol-based leaving groups are the same or different;
combining the first C-activated peptide with the first N-deprotected peptide to form a first ligation product having an amide bond between the α
-carbon of the C-terminal residue of the first C-activated peptide and the α
-carbon of the N-terminal of the first N-deprotected peptide;
combining the second C-activated peptide with the second N-deprotected peptide to form a second ligation product having an amide bond between the α
-carbon of the C-terminal residue of the second C-activated peptide and the α
-carbon of the N-terminal of the second N-deprotected peptide;
replacing the weak C-terminal thiol-based leaving group of the first ligation product or the second ligation product with a good C-terminal thiol-based leaving group to form a C-activated ligation product, and deprotecting the N-terminal-protected cysteinyl residue of the first ligation product or the second ligation product to form a N-deprotected ligation product;
combining the C-activated ligation product with the N-deprotected ligation product to form a third ligation product having an amide bond between the α
-carbon of the C-terminal residue of the C-activated ligation product and the α
-carbon of the N-terminal of the N-deprotected ligation product;
whereby the third ligation product is a polypeptide.
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Accused Products
Abstract
The present invention concerns methods and compositions for synthesizing a polypeptide using kinetically controlled reactions involving fragments of the polypeptide for a fully convergent process. In more specific embodiments, a ligation involves reacting a first peptide having a protected cysteyl group at its N-terminal and a phenylthioester at its C-terminal with a second peptide having a cysteine residue at its N-termini and a thioester at its C-termini to form a ligation product. Subsequent reactions may involve deprotecting the cysteyl group of the resulting ligation product and/or converting the thioester into a thiophenylester.
24 Citations
28 Claims
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1. A method of synthesizing a polypeptide comprising:
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obtaining a first C-activated peptide, having (1) an N-terminal-protected cysteinyl residue or an N-terminal non-cysteinyl residue and (2) a good C-terminal thiol-based leaving group;
a first N-deprotected peptide, having an N-terminal unprotected cysteinyl residue and a weak C-terminal thiol-based leaving group;
a second C-activated peptide, having an N-terminal-protected cysteinyl residue and a good C-terminal thiol-based leaving group, wherein the good C-terminal thiol-based leaving groups are the same or different;
a second N-deprotected peptide, having (1) an N-terminal unprotected cysteinyl residue and (2) a C-terminal α
-carboxylate, α
-carboxamide, or a weak C-terminal thiol-based leaving group, wherein the weak C-terminal thiol-based leaving groups are the same or different;
combining the first C-activated peptide with the first N-deprotected peptide to form a first ligation product having an amide bond between the α
-carbon of the C-terminal residue of the first C-activated peptide and the α
-carbon of the N-terminal of the first N-deprotected peptide;
combining the second C-activated peptide with the second N-deprotected peptide to form a second ligation product having an amide bond between the α
-carbon of the C-terminal residue of the second C-activated peptide and the α
-carbon of the N-terminal of the second N-deprotected peptide;
replacing the weak C-terminal thiol-based leaving group of the first ligation product or the second ligation product with a good C-terminal thiol-based leaving group to form a C-activated ligation product, and deprotecting the N-terminal-protected cysteinyl residue of the first ligation product or the second ligation product to form a N-deprotected ligation product;
combining the C-activated ligation product with the N-deprotected ligation product to form a third ligation product having an amide bond between the α
-carbon of the C-terminal residue of the C-activated ligation product and the α
-carbon of the N-terminal of the N-deprotected ligation product;
whereby the third ligation product is a polypeptide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for synthesizing a polypeptide comprising:
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a) reacting a first N—
Ca peptide with a first Na—
C peptide to form a first N—
C ligation product;
b) reacting a second N—
Ca peptide with a second Na—
C peptide to form a second N—
C ligation product;
c) converting the first N—
C ligation product, into a N—
Ca ligation product;
d) converting the second N—
C ligation product, into a Na—
C ligation product, ande) reacting the N—
Ca ligation product with the Na—
C ligation product to form a third N—
C ligation product, whereby the third N—
C ligation product is a polypeptide;
wherein neither the first N—
Ca peptide, first Na—
C peptide, second N—
Ca peptide, nor the second Na—
C peptide contain any internal residues with protected side chains;
N and Na represent the N-terminal residue;
C and Ca represent the C-terminal residue;
Ca is more reactive than C, and Na is more reactive than N. - View Dependent Claims (23, 24, 25)
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26. A method of synthesizing a polypeptide comprising:
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obtaining a first C-activated peptide, having an N-terminal-protected cysteinyl residue and a good C-terminal thioester-based leaving group;
a first N-deprotected peptide, having an N-terminal unprotected cysteinyl residue and a weak C-terminal thioester-based leaving group;
a second C-activated peptide, having (1) an N-terminal-protected cysteinyl residue or an N-terminal non-cysteinyl residue and (2) a good C-terminal thioester-based leaving group, wherein the good C-terminal thioester-based leaving groups are the same or different;
combining the first C-activated peptide with the first N-deprotected peptide to form a first ligation product having an amide bond between the α
-carbon of the C-terminal residue of the first C-activated peptide and the α
-carbon of the N-terminal of the first N-deprotected peptide;
deprotecting the N-terminal-protected cysteinyl residue of the first ligation product to form an N-deprotected ligation product, and combining the second C-activated peptide with the N-deprotected ligation product to form a second ligation product having an amide bond between the α
-carbon of the C-terminal residue of the second C-activated peptide and the α
-carbon of the N-terminal of the N-deprotected ligation product;
wherein the side chains of the internal residues of the first C-activated peptide, the first N-deprotected peptide, and the second C-activated peptide are unprotected, and whereby the second ligation product is a polypeptide.
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27. A method of synthesizing a polypeptide comprising:
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obtaining a first C-activated peptide, having (1) an N-terminal-protected cysteinyl residue or an N-terminal non-cysteinyl residue and (2) a good C-terminal thioester-based leaving group;
a first N-deprotected peptide, having an N-terminal unprotected cysteinyl residue and a weak C-terminal thioester-based leaving group;
a second N-deprotected peptide, having an N-terminal unprotected cysteinyl residue and a weak C-terminal thioester-based leaving group, wherein the weak C-terminal thioester-based leaving groups are the same or different;
combining the first C-activated peptide with the first N-deprotected peptide to form a first ligation product having an amide bond between the α
-carbon of the C-terminal residue of the first C-activated peptide and the α
-carbon of the N-terminal of the first N-deprotected peptide;
replacing the weak C-terminal thioester-based leaving group of the first ligation product with a good C-terminal thioester-based leaving group to form a C-activated ligation product, and combining the second N-deprotected peptide with the C-activated ligation product to form a second ligation product having an amide bond between the α
-carbon of the C-terminal residue of the C-activated ligation product and the α
-carbon of the N-terminal of the second N-deprotected peptide,wherein the side chains of the internal residues of the first C-activated peptide, the first N-deprotected peptide, and the second N-deprotected peptide are unprotected, and whereby the second ligation product is a polypeptide.
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28. A method of selectively desulfurizing a polypeptide, comprising:
- obtaining a polypeptide containing both cysteinyl residues with protected side chains and cysteinyl residues with unprotected side chains, and reacting said polypeptide with a desulfurizing agent to convert the cysteinyl residues with unprotected side chains into alanyl residues, whereby the cysteinyl residues with protected side chains are not desulfurized, and wherein the protected side chains are protected with a protection group selected from the group consisting of Acm, Tacm, and Phacm.
Specification