US 12,351,601 B2Granted

Method for producing peptide continuously

AssigneeAjinomoto Co., Inc.

InventorsDaisuke Takahashi, Metten Bert, Kimio Kawajiri, Ryotaro Nakaya

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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.

Claims15

Dependent claims

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

8 more elements

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 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-unprotected C-protected peptide by partitioning in a continuous flow by an oil-water separation and separating an organic layer comprising the N-unprotected C-protected peptide under the concentration condition being 2- to 100-fold dilution with respect to the N-unprotected C-protected peptide as a substrate;
wherein said method prior to the washing step in A or B, comprises producing a reaction mix comprising the peptide protected by a pseudo-solid-phase group by flow synthesis in a flow reactor;
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.

28 more elements

Dependencies are inferred from the claim text.

Facts

Application: 17/506,934Filed 21 Oct 2021 · continuation
Priority: JP 2019-08465225 Apr 2019
Published: US 2022/0041648 A110 Feb 2022
Granted: 8 Jul 2025
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

C07K 1/062
Using protecting groups: for alpha- or omega-carboxy functions
Y02P 20/55
Improvements relating to the production of bulk chemicals: design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
C07K
Peptides
Y02P
Low-carbon manufacturing