Method of synthesizing peptides, proteins and bioconjugates

Patent metadata

Field	Value
Publication number	US-9453044-B2
Application number	US-201314376675-A
Country	US
Kind code	B2
Filing date	Feb 6, 2013
Priority date	Feb 7, 2012
Publication date	Sep 27, 2016
Grant date	Sep 27, 2016

Abstract

The invention relates to the synthesis of peptides, proteins and related bioconjugates, and in particular, to such synthesis using a peptide ligation method whereby a C-terminal salicylaldehyde ester peptide is reacted with an aminoacyl-N-hydroxl peptide. The invention also relates to the synthesis of cyclic peptides, including serinyl- or threonyl-containing cyclic peptides. The invention further relates to a solid phase synthesis of C-terminal salicylaldehyde ester peptides.

First claim

The invention claimed is:

1. A method of preparing a polypeptide or protein of formula (3)

comprising:
reacting a C-terminal salicylaldehyde ester peptide of formula (1)

with an aminoacyl-N-hydroxyl peptide of formula (2)

wherein the reaction is carried out in a pyridine/organic acid mixture to form an intermediate compound having a 1,2,5-oxadiazinane ring structure; and
adding a reducing agent to cleave the 1,2,5-oxadiazinane ring structure of the intermediate compound to form the polypeptide or protein of formula (3), wherein each of R 1 , R 2 and R 3 is independently selected from the group consisting of H, a substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 2 -C 15 alkenyl, substituted or unsubstituted C 2 -C 15 alkynyl, substituted or unsubstituted C 3 -C 15 cycloalkyl, substituted or unsubstituted C 3 -C 15 cycloalkenyl, substituted or unsubstituted C 3 -C 15 heterocycloalkyl, substituted or unsubstituted C 3 -C 15 heterocycloalkenyl, substituted or unsubstituted C 6 -C 15 aryl, or substituted or unsubstituted C 6 -C 15 heteroaryl, and a side chain of an amino acid; or
R 1 is C 1 -C 10 alkylyl that forms together with the nitrogen of the adjacent backbone imino group, a heteroalicyclic ring.

2. The method of claim 1 , wherein at least one of R 1 and R 3 is independently selected from the group consisting of H, CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 , (CH 2 ) 2 —S—CH 3 , CH 2 —SH, CH 2 —OH, CHOH—CH 3 , CH 2 -(3-indolyl), CH 2 -phenyl, (CH 2 ) 4 —NH 2 , (CH 2 ) 3 —NH—C(—NH)NH 2 , CH 2 -1H-imidazol-4-yl, CH 2 -(p-hydroxy-phenyl), CH 2 —C(O)NH 2 , (CH 2 ) 2 —C(O)NH 2 , CH 2 —COOH, and (CH 2 ) 2 —COOH, or R 1 is (CH 2 ) 3 that forms together with the nitrogen of the adjacent backbone imino group a 5-membered heteroalicyclic ring.

3. The method of claim 1 , wherein R 2 is selected from the group consisting of H, CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 , (CH 2 ) 2 —S—CH 3 , CH 2 —SH, CH 2 —OH, CHOH—CH 3 , CH 2 -(3-indolyl), CH 2 -phenyl, (CH 2 ) 4 —NH 2 , (CH 2 ) 3 —NH—C(═NH)NH 2 , CH 2 -1H-imidazol-4-yl, CH 2 -(p-hydroxy-phenyl), CH 2 —C(O)NH 2 , (CH 2 ) 2 —C(O)NH 2 , CH 2 —COOH, and (CH 2 ) 2 —COOH.

4. The method of claim 1 , wherein the reducing agent is samarium (II) iodide (SmI 2 ) solution.

5. The method of claim 4 , wherein the reducing agent is SmI 2 solution in tetrahydrofuran (THF) or SmI 2 solution in trifluoroethanol (TFE).

6. The method of claim 1 , wherein the organic acid is acetic acid, propionic acid, isobutyric acid or butyric acid.

7. The method of claim 1 , wherein the intermediate compound has the following formula (4):

8. The method of claim 2 , wherein at least one of R 1 , R 2 and R 3 is independently selected from the group consisting of CH 3 , CH 2 -phenyl, CH 2 CH(CH 3 ) 2 , CH(CH 3 ) 2 and H.

9. The method of claim 2 , wherein:
R 1 is CH 3 and R 2 is CH 3 ;
R 1 is CH 3 and R 2 is H;
R 1 is CH 3 and R 2 is CH 2 -phenyl;
R 1 is CH 3 and R 2 is CH 2 CH(CH 3 ) 2 ;
R 1 is CH 2 -phenyl and R 2 is CH 3 ;
R 1 is CH 2 CH(CH 3 ) 2 and R 2 is CH 3 ;
R 1 is CH(CH 3 ) 2 and R 2 is CH 3 ;
R 1 is CH(CH 3 ) 2 and R 2 is H;
R 1 is CH 2 CH(CH 3 ) 2 and R 2 is CH 2 CH(CH 3 ) 2 ; or
R 1 is CH 2 -phenyl and R 2 is CH 2 CH(CH 3 ) 2 .

10. A method of preparing a cyclic peptide of formula (5)

where AA n represents an amino acid at a n th position of the peptide, comprising:
reacting the C-terminal salicylaldehyde ester peptide of formula (1A)

in a pyridine/organic acid mixture, where “ - - - ” represents additional amino acids in the chain of the peptide; and
adding a reducing agent to form the cyclic peptide of formula (5).

11. The method of claim 10 , wherein the reducing agent is SmI 2 solution.

12. The method of claim 10 , wherein the reducing agent is SmI 2 solution in THF or SmI 2 solution in TFE.

13. The method of claim 10 , wherein the organic acid is acetic acid, propionic acid, isobutyric acid or butyric acid.

14. A method of preparing a serinyl- or threonyl-containing cyclic peptide of formula (6)

where the peptide contains serine (Ser) or threonine (Thr), comprising:
reacting the C-terminal Ser- or Thr-containing salicylaldehyde ester peptide of formula (1B)

in a pyridine/organic acid mixture; and
adding an acidic deprotecting agent to form the serinyl- or threonyl-containing cyclic peptide of formula (6),
wherein R is a substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 2 -C 15 alkenyl, substituted or unsubstituted C 2 -C 15 alkynyl, substituted or unsubstituted C 3 -C 15 cycloalkyl, substituted or unsubstituted C 3 -C 15 cycloalkenyl, substituted or unsubstituted C 3 -C 15 heterocycloalkyl, substituted or unsubstituted C 3 -C 15 heterocycloalkenyl, substituted or unsubstituted C 6 -C 15 aryl, or substituted or unsubstituted C 6 -C 15 heteroaryl, or a side chain of an amino acid.

15. The method of claim 14 , wherein the acidic deprotecting agent to deprotect the oxazolidine ring is trifluoroacetic acid (TFA).

16. The method of claim 14 , wherein the organic acid is acetic acid, propionic acid, isobutyric acid, or butyric acid.

17. A method of preparing a C-terminal salicylaldehyde ester peptide in solid phase, comprising:
(i) mixing 2-hydroxycinnamic acid and 4-methylbenzhydrylamine hydrochloride salt (MBHA) resin in the presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and a tertiary amine to form a 2-hydroxycinnamamide linker on the resin;
(ii) coupling a Boc-amino acid to the 2-hydroxycinnamamide linker-resin, followed by standard Boc solid phase peptide synthesis to form a peptidyl 2-hydroxycinnamamide phenolic ester on the resin, where Boc represents tert-butoxycarbonyl protecting group;
(iii) adding a cleavage agent to release the peptidyl 2-hydroxycinnamamide phenolic ester from the resin; and
(iv) ozonizing the C═C bond of the peptidyl 2-hydroxycinnamamide phenolic ester to obtain the C-terminal salicylaldehyde ester peptide.

18. The method of claim 17 , wherein the tertiary amine is diisopropylethylamine (DIEA).

19. The method of claim 17 , wherein the cleavage agent is hydrofluoric acid or trifluoromethanesulfonic acid-trifluoroacetic acid mixture.

20. A method of preparing a polypeptide or protein of formula (7)

comprising reacting a compound of formula (4)

Assignees

• Univ Nanyang Tech

Inventors

• Liu Chuan Fa
• Zhao Junfeng

Classifications

• C07K1/10 Primary

  using coupling agents {(C07K1/006 takes precedence)} · CPC title

• C07K7/64

  Cyclic peptides containing only normal peptide links · CPC title

• C07K1/026

  by fragment condensation in solution · CPC title

• C07K1/04

  on carriers {(C07K1/003, C07K1/006 take precedence)} · CPC title

• C07K1/02 Primary

  in solution {(C07K1/003, C07K1/006 take precedence)} · CPC title