Method for producing peptide continuously

Patent metadata

Field	Value
Publication number	US-12351601-B2
Application number	US-202117506934-A
Country	US
Kind code	B2
Filing date	Oct 21, 2021
Priority date	Apr 25, 2019
Publication date	Jul 8, 2025
Grant date	Jul 8, 2025

Abstract

Peptides may be continuously produced by a method including the following steps (1), (A), (2), and (B):
(1) performing a condensation reaction in a flow reactor to obtain an N-protected C-protected peptide in which an N-terminal amino group and C-terminal are protected by protecting groups, a side chain functional group is optionally further protected by a protecting group, and at least one of the C-terminal or the side chain functional group is protected by a pseudo-solid-phase protecting group (N-protected C-protected peptide), (A) washing a reaction mixture containing an N-protected C-protected peptide in a flow reactor and separating oil and water to separate an organic layer containing the N-protected C-protected peptide, (2) subjecting an organic layer containing an N-protected C-protected peptide to a reaction to remove a protecting group of an N-terminal amino group in a flow reactor to obtain a C-protected peptide in which an N-terminal amino group is not protected, C-terminal is protected by a protecting group, a side chain functional group is optionally further protected by a protecting group, and at least one of the C-terminal or the side chain functional group is protected by a pseudo-solid-phase protecting group (N-unprotected C-protected peptide), (B) subjecting a reaction mixture containing an N-unprotected C-protected peptide to washing and oil-water separation in a flow reactor to separate an organic layer containing the N-unprotected C-protected peptide.

First claim

The invention claimed is:

1. A method for producing a peptide by flow synthesis in a flow reactor wherein the peptide is protected by a pseudo-solid phase protecting group to maintain solubility of the peptide as it elongates, comprising the following (A) or (B), or a combination thereof:
(A) producing a reaction mix comprising the peptide protected by a pseudo-solid-phase group in a flow reactor; wherein the peptide comprises an N-protected C-protected peptide having an amino acid residue number of from 5 to 100, in which an N-terminal amino group and C-terminal are protected by protecting groups;
washing the reaction mix that contains the peptide with water and/or a hydrophilic organic solvent
and then
separating the peptide into an organic layer via an oil and water type phase separation comprising water or the hydrophilic organic solvent and a solvent immiscible with water or the hydrophilic organic solvent, thereby recovering the protected peptide,
wherein protected peptide solution is diluted 5- to 100-fold compared to the initial concentration of the substrate peptide in the immiscible solvent
(B) producing a reaction mix comprising the peptide protected by a pseudo-solid-phase group in a flow reactor; wherein the peptide comprises an N-terminal amino group that is not protected, a C-terminal that is protected by a protecting group having an amino acid residue number of from 5 to 100, in which an N-terminal amino group is not protected and C-terminal are protected by protecting groups:
washing the reaction mix that contains the peptide with water and/or a hydrophilic organic solvent; separating the peptide into an organic layer via an oil and water type phase separation comprising water or the hydrophilic organic solvent and a second solvent immiscible with water or immiscible with the hydrophilic organic solvent, thereby recovering the protected peptide,
wherein protected peptide solution is diluted 2- to 100-fold compared to the initial concentration of the substrate peptide in the immiscible solvent

2. The production method according to claim 1 , wherein the oil-water separation is conducted by a continuous layer separation using a filter, or by a Gravity continuous layer separation.

3. The production method according to claim 1 , wherein the protecting group of the amino group is a 9-fluorenylmethyloxycarbonyl group, a tert-butoxycarbonyl group, or a benzyloxycarbonyl group.

4. The production method according to claim 3 , wherein the protecting group of the amino group is a 9-fluorenylmethyloxycarbonyl group.

5. The production method according to claim 1 , further comprising obtaining the organic layer comprising the N-protected C-protected peptide obtained in the (A), or the N-unprotected C-protected peptide obtained in the (B), and then removing all protecting groups.

6. The production method according to claim 1 , wherein the pseudo-solid-phase protecting group is selected from the group consisting of:
(4′,4′-bis (2,3-dihydrophytyloxy) phenyl) methylamine);
3,4,5-tri (2′,3′-dihydrophytyloxy) benzyl alcohol;
2-[3,4,5-tri (2′,3′-dihydrophytyloxy) benzyloxy]-4-methoxybenzylalcohol; and
3,4,5-tri (octadecyloxy) cyclohexanemethanol.

7. The method of claim 1 that comprises (A).

8. The method of claim 1 , that comprises (B).

9. The method of claim 1 , wherein (A) or (B) a side chain functional group is further protected by a protecting group, and at least one of the C-terminal or the side chain functional group is protected by a pseudo-solid-phase protecting group.

10. The method of claim 1 , wherein the water or hydrophilic organic solvent comprises a nitril, a ketone, an amide, or a sulfoxide.

11. The method of claim 1 , wherein the water or hydrophilic organic solvent comprises acetonitrile, N,N-dimethylformamide (DMF), or N-methylpyrrolidone (NMP).

12. The method of claim 1 , wherein the organic solvent immiscible with water comprises a hydrocarbon, an aromatic hydrocarbon or a halogenated hydrocarbon.

13. The method of claim 1 , wherein the second organic solvent immiscible with water comprises chloroform, dichloromethane, tetrahydrofuran (THF) or cyclopentylmethylether (CPME).

14. The method of claim 1 , wherein the pseudo-solid phase protecting group is at least one selected from the group consisting of:
(4′,4′-bis (2,3-dihydrophytyloxy) phenyl) methylamine);
3,4,5-tri (2′,3′-dihydrophytyloxy) benzyl alcohol;
2-[3,4,5-tri (2′,3′-dihydrophytyloxy) benzyloxy]-4-methoxybenzyl alcohol;
3,4,5-tri (octadecyloxy) cyclohexanemethanol;
[bis-(4-docosoxy-phenyl)-methyl]-amine;
3,4,5-tri (octadecyloxy) benzyl alcohol;
4-methoxy-2-[3′,4′,5′-tris (octadecyloxy) benzyloxy) benzyl alcohol;
4-methoxy-2-[3′,4′,5′-tris (octadecyloxy) cyclohexylmethyloxy] benzyl alcohol;
2-docosyloxy-9-(4-chlorophenyl)-9-fluorenol;
2-docosyloxy-9-(4-chlorophenyl)-9-bromofluorene;
2,7-didocosyloxy-9-(4-chlorophenyl)-9-bromofluorene;
2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluorene;
1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-fluorenol)-dodecyloxy]-dodecane;
1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-bromofluorene)-dodecyloxy]-dodecane;
2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol;
2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bromofluorene;
9-(4-chlorophenyl)-2-(3,4,5-tris (octadecyloxy)-cyclohexylmethoxy)-9-fluorenol;
9-(4-chlorophenyl)-2-(3,4,5-tris (octadecyloxy)-cyclohexylmethoxy)-9-bromofluorene;
3,5-didocosyloxybenzyl alcohol;
2,4-didocosyloxybenzyl alcohol;
2,4-bis octadecyloxybenzyl alcohol;
3-didocosylaminobenzyl alcohol;
3-diphytylaminobenzyl alcohol;
N-(2′,3′-dihydrophytyl)-N-(3-hydroxymethylphenyl) acetamide;
N-triacontyl-N-(3-hydroxymethylphenyl) acetamide;
3-(aminomethyl)-N,N-didocosylaniline;

wherein TIPS is a triisopropylsilyl group, and TBDPS is a tert-butyldiphenylsilyl group.

15. A method for producing a peptide, comprising the following (A) or (B), or a combination thereof:
(A) washing a reaction mixture comprising an N-protected C-protected peptide having an amino acid residue number of from 5 to 100, in which an N-terminal amino group and C-terminal are protected by protecting groups, a side chain functional group is optionally further protected by a protecting group, and at least one of the C-terminal or the side chain functional group is protected by a pseudo-solid-phase protecting group in a flow reactor with water and/or a hydrophilic organic solvent in a continuous flow, and then purifying the N-protected C-protected peptide by partitioning in a continuous flow by an oil-water separation and separating an organic layer comprising the N-protected C-protected peptide, under a concentration condition being 5- to 100-fold dilution with respect to the N-protected C-protected peptide as a substrate;
(B) washing a reaction mixture comprising an N-unprotected C-protected peptide having an amino acid residue number of from 5 to 100, in which an N-terminal amino group is not protected, C-terminal is protected by a protecting group, a side chain functional group is optionally further protected by a protecting group, and at le

Assignees

• Ajinomoto Kk

Inventors

• Takahashi Daisuke
• Bert Metten
• Kawajiri Kimio
• Nakaya Ryotaro

Classifications

• Y02P20/55

  Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups · CPC title

• C07K1/062 Primary

  for alpha- or omega-carboxy functions · CPC title