Convergent synthesis of proteins by kinetically controlled ligation

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.

2. The method of claim 1 wherein one or more of the first C-activated peptide, the first N-deprotected peptide, the second C-activated peptide, the second N-deprotected peptide, the first ligation product, the second ligation product, the N-deprotected ligation product, the C-activated ligation product, the third ligation product, or the polypeptide each independently contain one or more internal residues with protected side chains.

3. The method of claim 2 , wherein the internal residues with protected side chains are protected cysteinyl residues.

4. The method of claim 3 , wherein the protected cysteinyl residues are protected with a protection group selected from the group consisting of acetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), and phenylacetamidomethyl (Phacm).

5. The method of claim 4 , wherein the protection group is Acm.

6. The method of claim 2 , wherein one or more of the first C-activated peptide, the first N-deprotected peptide, the second C-activated peptide, the second N-deprotected peptide, the first ligation product, the second ligation product, the N-deprotected ligation product, the C-activated ligation product, the third ligation product, or the polypeptide is selectively desulfurized to convert cysteinyl residues into alanyl residues, whereby neither the internal residues with protected side chains nor the thiol-based leaving groups are desulfurized.

7. The method of claim 2 , further comprising deprotecting the protected side chains from one or more one or more of the first C-activated peptide, the first N-deprotected peptide, the second C-activated peptide, the second N-deprotected peptide, the first ligation product, the second ligation product, the N-deprotected ligation product, the C-activated ligation product, the third ligation product, or the polypeptide.

8. The method of claim 1 , wherein any of the good C-terminal thiol-based leaving groups is —SR 1 , wherein R 1 is a substituted or unsubstituted C 6 -C 15 -aryl.

9. The method of claim 8 , wherein R 1 is chosen from the group consisting of —C 6 H 4 COOH, —C 6 H 4 COO − , —C 6 H 4 Cl, —C 6 H 4 NO 2 , —C 6 H 4 CH 3 , —C 6 H 4 OH, —C 6 H 4 OCOCH 3 , —C 6 H 4 COOCH 3 , —C 6 H 4 CH 2 COOH, —C 6 H 4 CH 2 COO − , —C 6 H 4 CONH 2 , and —C 6 H 4 CH 2 CONH 2 .

10. The method of claim 9 , wherein at least one of the good C-terminal thiol-based leaving groups is C 6 H 4 CH 2 COOH or —C 6 H 4 CH 2 COO.

11. The method of claim 1 , wherein the weak C-terminal thiol-based leaving group is —SH, —S − or —SR 2 , wherein R 2 represents a substituted or unsubstituted version of C 1 -C 15 -alkyl or C 1 -C 15 -aralkyl.

12. The method of claim 11 , wherein R 2 is —CH 2 CH 2 SO 3 Na, —CH 2 SO 3 Na, —CH 2 CH 2 CONHCH(R 3 )COOH, —CH 2 CONHCH(R 3 )COOH, —CH 2 CH 2 CONHCH(R 3 )COO − , —CH 2 CONHCH(R 3 )COO − , —CH 2 CH 2 CONHCH(R 3 )CONH 2 , —CH 2 CONHCH(R 3 )CONH 2 , —CH 2 CH 2 CO(Arg) n OH, or —CH 2 CH 2 CO(Arg) n NH 2 ; wherein R 3 is H, amino, hydroxyl, halo, —NHR 4 , —NR 4 R 5 , —OR 4 , or a substituted or unsubstituted version of C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl, C 2 -C 10 -alkynyl, C 6 -C 10 -aryl, C 7 -C 10 -aralkyl, C 10 -heteroaryl, C 2 -C 10 -heteroaralkyl, or C 1 -C 10 -acyl; wherein R 4 and R 5 are each independently H or a substituted or unsubstituted version of C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl, C 2 -C 10 -alkynyl, C 6 -C 10 -aryl, C 7 -C 10 -aralkyl, C 1 -C 10 -heteroaryl, C 2 -C 10 -heteroaralkyl, or C 1 -C 10 -acyl; further wherein Arg is an arginyl residue, n is an integer from 1 to 6 and (Arg) 4-6 is SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36, respectively.

13. The method of claim 1 , wherein the N-terminal protected cysteinyl residue has a linking group connecting its thiol group to its amino group.

14. The method of claim 13 , wherein the linking group is —CH 2 —, —CH(R 6 )—, or —C(R 6 )(R 7 )—, wherein R 6 and R 7 are each independently, amino, hydroxyl, halo, —NHR 8 , —NR 8 R 9 , —OR 8 , or a substituted or unsubstituted version of C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl, C 2 -C 10 -alkynyl, C 6 -C 10 -aryl, C 7 -C 10 -aralkyl, C 1 -C 10 -heteroaryl, C 2 -C 10 -heteroaralkyl, or C 1 -C 10 -acyl; wherein R 8 and R 9 are each independently H or a substituted or unsubstituted version of C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl, C 2 -C 10 -alkynyl, C 6 -C 10 -aryl, C 7 -C 10 -aralkyl, C 1 -C 10 -heteroaryl, C 2 -C 10 -heteroaralkyl, or C 1 -C 10 -acyl.

15. The method of claim 1 , further comprising

obtaining a third C-activated peptide having an N-terminal-protected cysteinyl residue and a good C-terminal thiol-based leaving group;

deprotecting the N-terminal-protected cysteinyl residue of the third ligation product to form a further N-deprotected ligation product, and

combining the third C-activated peptide with the further N-deprotected ligation product to form a fourth ligation product having an amide bond between the α-carbon of the C-terminal residue of the third C-activated peptide and the α-carbon of the N-terminal of the further N-deprotected ligation product;

whereby the fourth ligation product is a polypeptide.

16. The method of claim 1 , further comprising

obtaining a third N-deprotected peptide having an N-terminal unprotected cysteinyl residue and one of the weak C-terminal thiol-based leaving groups;

replacing the weak C-terminal thiol-based leaving group of the third ligation product with a good C-terminal thiol-based leaving group to form a further C-activated ligation product, and

combining the further C-activated ligation product with the third N-deprotected peptide to form a fifth ligation product having an amide bond between the α-carbon of the C-terminal residue of the further C-activated ligation product and the α-carbon of the N-terminal of the third N-deprotected peptide;

whereby the fifth ligation product is a polypeptide.

17. The method of claim 1 , wherein one or both of the first C-activated peptide and the second C-activated peptide is a C-activated ligation product, formed prior to its use in the method of claim 1 .

18. The method of claim 1 , wherein one or both of the first N-deprotected peptide and the second N-deprotected peptide is a N-deprotected ligation product, formed prior to its use in the method of claim 1 .

19. The method of claim 1 , wherein one or more of

the combining of the first C-activated peptide with the first N-deprotected peptide to form a first ligation product,

the combining of the second C-activated peptide with the second N-deprotected peptide to form a second ligation product, or

the combining of the C-activated ligation product with the first N-deprotected peptide to form a third ligation product,

occurs in an aqueous buffer having a pH between 6 and 8.

20. The method of claim 1 , further comprising deprotecting one or more of the N-terminal protected cysteinyl residue of the first ligation product, the second ligation product, or the third ligation product with methoxyamine-hydrochloride, carboxymethoxylamine-hemihydrochloride, o-phenylhydroxylamine-hydrochloride, o-benzylhydroxylamine-hydrochloride, o-4-nitrobenzylhydroxylamine-hydrochloride, and hydroxylamine-hydrochloride.

21. The method of claim 20 , wherein deprotecting with methoxyamine-hydrochloride occurs in aqueous buffer at a pH from 3 to 5.

22. A method for synthesizing a polypeptide comprising:

a) reacting a first N—C a peptide with a first N a —C peptide to form a first N—C ligation product;

b) reacting a second N—C a peptide with a second N a —C peptide to form a second N—C ligation product;

c) converting the first N—C ligation product, into a N—C a ligation product;

d) converting the second N—C ligation product, into a N a —C ligation product, and

e) reacting the N—C a ligation product with the N a —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—C a peptide, first N a —C peptide, second N—C a peptide, nor the

second N a —C peptide contain any internal residues with protected side chains;

N and N a represent the N-terminal residue;

C and C a represent the C-terminal residue;

C a is more reactive than C, and

N a is more reactive than N,

further wherein one or more of steps a, b and e occurs in the presence of 4-mercaptophenyl acetic acid.

23. A method of synthesizing a polypeptide comprising:

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.

24. 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 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.

25. 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.