Method for producing peptide

Patent metadata

Field	Value
Publication number	US-9353147-B2
Application number	US-201314089853-A
Country	US
Kind code	B2
Filing date	Nov 26, 2013
Priority date	May 31, 2011
Publication date	May 31, 2016
Grant date	May 31, 2016

Abstract

The present invention provides a production method of peptide, which includes the following step (1).
(1) removing N-terminal Fmoc group of N-Fmoc C-protected amino acid or N-Fmoc C-protected peptide wherein a C-terminal carboxy group is protected by an anchor group derived from an anchor soluble in halogenated solvents or ether solvents, insoluble in polar solvents and having a molecular weight of not less than 300, with a non-nucleophilic organic base in a halogenated solvent or ether solvent to give a C-protected amino acid or C-protected peptide, neutralizing with an acid, adding N-Fmoc amino acid or N-Fmoc peptide, a condensing agent and a condensation accelerator to the reaction solution after neutralization, and condensing the N-terminal of the C-protected amino acid or C-protected peptide with N-Fmoc amino acid or N-Fmoc peptide to give an N-Fmoc C-protected peptide.

First claim

The invention claimed is:

1. A method of producing a peptide, comprising:
(1) removing an N-terminal Fmoc group from (a) N-Fmoc C-protected amino acid or (b) an N-Fmoc C-protected peptide, with a non-nucleophilic organic base in a halogenated solvent or ether solvent to obtain a mixture comprising (a′) a C-protected amino acid or (b′) a C-protected peptide;
(2) neutralizing said mixture with an acid, to obtain a neutralized mixture;
(3) adding (c) an N-Fmoc amino acid or (d) an N-Fmoc peptide, a condensing agent and a condensation accelerator to said neutralized mixture; and
(4) condensing the N-terminal of either of said (a′) C-protected amino acid or said (b′) C-protected peptide with either of said (c) N-Fmoc amino acid or said (d) N-Fmoc peptide to obtain (e) an N-Fmoc C-protected peptide,
wherein, when said (1) removing an N-terminal Fmoc group comprises removing an N-terminal Fmoc group from (b) an N-Fmoc C-protected peptide, said (b) N-Fmoc C-protected peptide is different from said (e) N-Fmoc C-protected peptide,
wherein a C-terminal carboxy group either of said (a) N-Fmoc C-protected amino acid or said (b) N-Fmoc C-protected peptide is protected by an anchor group, and
wherein said (1) removing, said (2) neutralizing, said (3) adding, and said (4) condensing are performed without any intervening isolation of said (a′) C-protected amino acid or said (b′) C-protected peptide, and
wherein said anchor group is a group represented by formula (II):

wherein:
R 1 is a hydrogen atom or, when R b is a group represented by the following formula (a), optionally forms a single bond together with R 3 to form a fluorene ring together with ring A and ring B;
each R 2 is independently an organic group having an aliphatic hydrocarbon group;
p is an integer of 1 to 4;
ring A optionally further has, in addition to OR 2 , a substituent selected from the group consisting of a halogen atom, a C 1-6 alkyl group optionally substituted by a halogen atom, and a C 1-6 alkoxy group optionally substituted by a halogen atom;
R a is a hydrogen atom, or a phenyl group optionally substituted by a halogen atom; and
R b is a hydrogen atom, or a group represented by formula (a):

wherein:
* is the point of binding to the remainder of the molecule;
r is an integer of 0 to 4;
each R 4 is independently an organic group having an aliphatic hydrocarbon group;
R 3 is a hydrogen atom, or optionally forms a single bond together with R 1 to form a fluorene ring together with ring A and ring B; and
ring B optionally further has, in addition to OR 4 , a substituent selected from the group consisting of a halogen atom, a C 1-6 alkyl group optionally substituted by a halogen atom, and a C 1-6 alkoxy group optionally substituted by a halogen atom; and
L represents a connection to the carbonyl group of said C terminus and is —O— or —N(R)—, wherein R is a hydrogen atom, an alkyl group, or an aralkyl group.

2. The method according to claim 1 , further comprising:
(5) precipitating said (e) N-Fmoc C-protected peptide with a polar solvent; and
(6) obtaining said (e) N-Fmoc C-protected peptide by solid-liquid separation.

3. The method according to claim 2 , further comprising:
(7) removing N-terminal Fmoc group and/or C-terminal anchor group of said (e) N-Fmoc C-protected peptide.

4. The method according to claim 1 , wherein said anchor group is derived from an anchor compound selected from the group consisting of:
3,4,5-tri(octadecyloxy)benzyl alcohol,
2,4-di(docosyloxy)benzyl alcohol,
4-methoxy-2-[3′,4′,5′-tri(octadecyloxy)benzyloxy]benzyl alcohol,
4-methoxy-2-[3′,4′,5′-tri(octadecyloxy)-cyclohexylmethyloxy]benzyl alcohol,
2-methoxy-4-[3′,4′,5′-tri(octadecyloxy)-cyclohexylmethyloxy]benzyl alcohol,
4-[3′,4′,5′-tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
3,5-dimethoxy-4-[3′,4′,5′-tri(octadecyloxy)-cyclohexylmethyloxy]benzyl alcohol,
2,4-di(dodecyloxy)benzyl alcohol,
3,4,5-tri(octadecyloxy)benzylamine,
bis(4-docosyloxyphenyl)methanol,
bis(4-docosyloxyphenyl)methylamine, and
2-(12-docosyloxy-dodecyloxy)-9-(3-fluorophenyl)-9-bromofluorene.

5. The method according to claim 1 , wherein said non-nucleophilic organic base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]-7-undecene, 1,4-diazabicyclo[2.2.2]octane, and 1,5-diazabicyclo[4.3.0]-5-nonene.

6. The method according to claim 1 , wherein said non-nucleophilic organic base is 1,8-diazabicyclo[5.4.0]-7-undecene.

7. The method according to claim 1 , wherein said acid is selected from the group consisting of methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid anhydride, sulfuric acid, and a hydrogen chloride/ether solution.

8. The method according to claim 1 , wherein said acid is selected from the group consisting of methanesulfonic acid, trifluoromethanesulfonic acid, hydrogen chloride/diethyl ether, and hydrogen chloride/cyclopentyl methyl ether.

9. The method according to claim 1 , wherein said condensation accelerator is 1-hydroxybenzotriazole, ethyl 1-hydroxy-1H-1,2,3-triazole-5-carboxylate, 1-hydroxy-7-azabenzotriazole, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, or O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate.

10. The method according to claim 1 , wherein said N-Fmoc peptide has a length of not more than 100 amino acid residues.

11. The method according to claim 1 , wherein said N-Fmoc peptide has a length of not more than 50 amino acid residues.

12. The method according to claim 1 , wherein said N-Fmoc peptide has a length of not more than 30 amino acid residues.

13. The method according to claim 1 , which comprises removing an N-terminal Fmoc group from (b) an N-Fmoc C-protected dipeptide, with a base which consists of a non-nucleophilic base.

Assignees

• Ajinomoto Kk

Inventors

• Takahashi Daisuke

Classifications

• C07K1/062 Primary

  for alpha- or omega-carboxy functions · CPC title

• C07K1/02

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

• C07K1/061 Primary

  using protecting groups · CPC title

• C07K5/0806

  the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala · CPC title