Method for producing peptide

Abstract

The present invention addresses the problem of providing a method for producing a peptide, which enables the improvement of amino acid removal efficiency and therefore enables the production of a peptide having a high purity. According to the present invention, a method for producing a peptide is provided, the method comprising a step for mixing an amino acid active ester with an amine represented by formula (1) so as to trap the amino acid active ester, wherein the amino acid active ester is produced during the condensation of an N-terminal-protected amino acid or peptide with a C-terminal-protected amino acid or peptide. In formula (1), each symbol is as defined in the description.

Description

Method for producing peptides

[0001] The present disclosure relates to methods for producing peptides.

[0002] Methods for producing peptides include solid-phase methods, liquid-phase methods, etc. The liquid-phase method has good reactivity and allows the intermediate peptide to be purified by extraction, washing, isolation, etc. after the condensation reaction.

[0003] Patent Document 1 describes a method of condensing an amino acid, peptide, or amino acid amide bound to a support for liquid-phase peptide synthesis with an amino acid or peptide whose amino group is protected by an amino-protecting group in a solvent containing an organic solvent, and then removing the amino acid activated ester by using a compound selected from aminosulfonic acids, aminosulfuric acids, aminophosphonic acids, aminophosphoric acids, and aminoalcohols as a scavenger for the amino acid activated ester remaining without condensation.

[0004] Patent Document 2 describes a method for synthesizing a peptide, which includes the steps of condensing, in the presence of a condensing agent, an N-Fmoc-protected amino acid with a peptide whose C-terminus is protected with a carrier that crystallizes as the composition of the solvent in which the amino acid is dissolved to obtain an N-Fmoc-C-carrier-protected peptide; adding an alkylamine or hydroxylamine having 1 to 14 carbon atoms to the reaction system; deprotecting the N-terminus; and changing the composition of the solvent in which the C-carrier-protected peptide is dissolved to crystallize and separate the peptide.

[0005] Patent Document 3 describes that a specific scavenger is used to inactivate an amino acid active ester generated during a condensation reaction, thereby preventing double hits of amino acids during Fmoc removal.

[0006] Japanese Patent No. 7063408 International Publication No. WO2016 / 140232 Japanese Patent No. 6703668

[0007] The problem to be solved by the present disclosure is to provide a method for producing peptides that improves the efficiency of amino acid removal and enables the production of highly pure peptides.

[0008] As a result of extensive research aimed at solving the above problems, the present inventors have discovered that quenching an amino acid activated ester produced during peptide synthesis with a specific amine compound improves the efficiency of amino acid removal, enabling the synthesis of highly pure peptides, and have thus completed the present disclosure. The present disclosure provides the following aspects.

[0009] <1> A method for producing a peptide, comprising a step of mixing an amino acid activated ester produced upon condensation of an N-terminally protected amino acid or peptide with a C-terminally protected amino acid or peptide, and trapping the amino acid activated ester with an amine represented by formula (1). In formula (1), L １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C=O, >C=S, >NR ３ ,=N-,-CO-NR ａ -, -CS-NR ａ -, -O-, and -S-, each of which may have a substituent; R ３ represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group; R ａ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR ４ indicates, R １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR ４ indicates R ４ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group; R ２ represents a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, or an ammonium group; R ２ If there are multiple R ２ may be the same or different, L １ When the main chain of L contains a heteroatom-containing aliphatic cyclic structure or heteroaryl, l and m each represent an integer of 0 or more (with the proviso that l+m≧1), １When R does not contain a heteroatom-containing aliphatic cyclic structure or heteroaryl in the main chain, l represents an integer of 4 or more. １ <3> The method for producing a peptide according to <1>, wherein in the formula (1), L is a hydrogen atom. １ represents a hydrogen atom, an alkyl group, an alkenyl group, a heteroaryl group, ═N—, —CO—NR ａ - and -O-, and the groups may have a substituent; L １ When the main chain contains a heteroatom-containing aliphatic cyclic structure, l and m each represent an integer of 0 or more (with the proviso that l+m≧1), １ <4> The method for producing a peptide according to any one of <1> to <3>, wherein, when the main chain does not contain a heteroatom-containing aliphatic cyclic structure, 1 is an integer of 4 or more. <5> The method for producing a peptide according to any one of <1> to <3>, wherein the amine represented by formula (1) is a compound represented by formula (2). In formula (2), R ５ ~R １２ each independently represents an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C=O, >C=S, or >NR １３ , -O-, -S-, -OH, -OR １４ , one or more groups selected from a phosphate group, a sulfonic acid group, a carboxylic acid group, and an ammonium group, and one —NH ２ and R １３ represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group; R ５ ~R １２ In the formula, at least one of —OH, —OR １４ , phosphate group, sulfonic acid group, carboxylic acid group, and ammonium group are present, and R １４ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group; X represents —O—, —S—, or >NR １３ indicates R １３represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group, and m represents 1 or 2. <5> The method for producing a peptide according to any one of <1> to <4>, comprising condensing an N-terminally protected amino acid or peptide with a C-terminally protected amino acid or peptide before the trapping step. <6> The method for producing a peptide according to <5>, in which the protecting group of the N-terminally protected amino acid or peptide is a 9-fluorenylmethyloxycarbonyl group, a benzyloxycarbonyl group, a tert-butoxycarbonyl group, or an acetyl group. <7> The method for producing a peptide according to <5> or <6>, in which the protecting group of the C-terminally protected amino acid or peptide is linked to the carbonyl group of the amino acid or peptide via an ester or amide bond. <8> The method for producing a peptide according to any one of <5> to <7>, in which the protecting group of the C-terminally protected amino acid or peptide is represented by formula (3). In formula (3), Y is -OR Ｃ , -NR Ｃ R, or -SR Ｃ R represents a hydrogen atom, an alkyl group, an arylalkyl group, a heteroarylalkyl group, or a 9-fluorenylmethyloxycarbonyl group, m represents 1 or 2, and n represents an integer of 1 to 5; Ｂ each independently represents an aliphatic hydrocarbon group; Ｃ represents the binding site to the C-terminal amino acid or peptide, R Ａ are each independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group, at least one of the aliphatic hydrocarbon groups having 12 or more carbon atoms, and the benzene ring in formula (2) is R Ａ <9> All of R Ａ<8> The method for producing the peptide according to <8>, wherein the total number of carbon atoms in all of the aliphatic hydrocarbon groups is 36 to 80. <10> The method for producing the peptide according to any one of <1> to <9>, further comprising, after the trapping step, a step of separating the peptide obtained by the condensation. <11> The method for producing the peptide according to <10>, wherein the peptide obtained by the condensation is separated by crystallization or liquid separation.

[0010] According to the present disclosure, it is possible to provide a method for producing a peptide, which improves the efficiency of removing amino acids and enables the synthesis of a highly pure peptide.

[0011] The contents of the present disclosure are described in detail below. The following description of the constituent elements may be based on representative embodiments of the present disclosure, but the present disclosure is not limited to such embodiments. In this specification, unless otherwise specified, each term has the following meaning. A numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the upper and lower limits. In numerical ranges described in stages in this specification, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in numerical ranges described in this specification, the upper or lower limit of that numerical range may be replaced with a value shown in the examples. The term "process" includes not only independent processes, but also processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. When describing a group (atomic group), a notation that does not specify whether it is substituted or unsubstituted encompasses both unsubstituted and substituted groups. For example, the term "alkyl group" encompasses not only alkyl groups without substituents (unsubstituted alkyl groups) but also alkyl groups with substituents (substituted alkyl groups). The chemical structural formula may be expressed as a simplified structural formula in which hydrogen atoms are omitted. A combination of two or more preferred embodiments is a more preferred embodiment.

[0012] When the amino acids and peptides according to the present disclosure have a hydroxy group, an amino group, a carboxy group, a carbonyl group, an amide group, a guanidyl group, a mercapto group, or the like, these groups may be protected, and the target compound can be obtained by removing the protecting group as necessary after the reaction.

[0013] Examples of protective groups for hydroxy groups include alkyl groups, aryl groups, trityl groups, arylalkyl groups having 7 to 10 carbon atoms, formyl groups, acyl groups having 1 to 6 carbon atoms, benzoyl groups, arylalkylcarbonyl groups having 7 to 10 carbon atoms, 2-tetrahydropyranyl groups, 2-tetrahydrofuranyl groups, silyl groups, alkenyl groups having 2 to 6 carbon atoms, etc. These groups may be substituted with 1 to 3 substituents selected from the group consisting of halogen atoms, alkyl groups having 1 to 6 carbon atoms, alkoxy groups having 1 to 6 carbon atoms, and nitro groups.

[0014] Examples of the amino-protecting group include a formyl group, an acyl group having 1 to 6 carbon atoms, an alkoxycarbonyl group having 1 to 6 carbon atoms, a benzoyl group, an arylalkylcarbonyl group having 7 to 10 carbon atoms, an arylalkyloxycarbonyl group having 7 to 14 carbon atoms, a trityl group, a monomethoxytrityl group, a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl group, a phthaloyl group, an N,N-dimethylaminomethylene group, a silyl group, and an alkenyl group having 2 to 6 carbon atoms. These groups may be substituted with one to three substituents selected from the group consisting of a halogen atom, an alkoxy group having 1 to 6 carbon atoms, and a nitro group. Examples of the carboxy-protecting group include the above-mentioned hydroxy-protecting group and a trityl group. Examples of protecting groups for carbonyl groups include cyclic acetals (e.g., 1,3-dioxane) and acyclic acetals (e.g., di(alkyl having 1 to 6 carbon atoms)acetals). Examples of protecting groups for amide groups include a trityl group. Examples of protecting groups for guanidyl groups include a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group, a 2,3,4,5,6-pentamethylbenzenesulfonyl group, a tosyl group, and a nitro group. Examples of protecting groups for mercapto groups (thiol groups) include a trityl group, a 4-methylbenzyl group, an acetylaminomethyl group, a t-butyl group, and a t-butylthio group.

[0015] The protecting group can be removed by a known method, for example, a method similar to that described in Protective Groups in Organic Synthesis, John Wiley and Sons (1980), etc. Methods that use an acid, a base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, or a trialkylsilyl halide, or a reduction method can be used.

[0016] The term "amino acid" refers to α, β, or γ amino acids, and is not limited to naturally occurring amino acids, but may also include non-naturally occurring amino acids, or amino acid analogs such as hydroxycarboxylic acids.

[0017] The alkyl group may be linear or branched, and may be substituted. Examples of the substituent include a halogen atom, a C １－６ Alkyl group, C ３－８ Cycloalkyl group, C ２－６ Alkenyl group, C ２－６ Alkynyl group, C １－６ Acyl group, C １－６ Alkoxy group, C ６－２０ Aryl group, C ５－２０ Examples of the alkyl group include a heteroaryl group and a heterocyclic group. Unless otherwise specified, the alkyl group may be an alkyl group having 1 to 30 carbon atoms (also referred to as "number of carbon atoms"), and may be an alkyl group having 1 to 20 carbon atoms, an alkyl group having 1 to 10 carbon atoms, or an alkyl group having 1 to 6 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, and tert-butyl. The cycloalkyl group may be substituted with a halogen atom, an alkoxy group, or the like. The alkyl group may be a cycloalkyl group having 3 to 10 carbon atoms, or an alkyl group having 3 to 6 carbon atoms.

[0018] Examples of the arylalkyl group include an arylalkyl group having 7 to 30 carbon atoms, and may be an arylalkyl group having 7 to 20 carbon atoms, an arylalkyl group having 7 to 16 carbon atoms (for example, a group in which an alkylene group having 1 to 6 carbon atoms is bonded to an aryl group having 6 to 10 carbon atoms), or an arylalkyl group having 7 to 10 carbon atoms. Specific examples include a benzyl group, a 1-phenylethyl group, a 2-phenylethyl group, a 1-phenylpropyl group, a naphthylmethyl group, a 1-naphthylethyl group, and a 1-naphthylpropyl group, with a benzyl group being preferred.

[0019] The alkenyl group preferably has 2 to 30 carbon atoms, more preferably 2 to 20, even more preferably 2 to 10, and still more preferably 2 to 6. Examples of the alkenyl group include a pentenyl group, a hexenyl group, and an oleyl group.

[0020] The alkynyl group preferably has 2 to 30 carbon atoms, more preferably 2 to 20, even more preferably 2 to 10, and still more preferably 2 to 6. Examples of the alkynyl group include a 4-pentynyl group and a 5-hexenyl group.

[0021] Examples of the aliphatic hydrocarbon group include an alkyl group, a cycloalkyl group, an alkenyl group, and an alkynyl group. The aliphatic hydrocarbon group may be linear or branched, and may be substituted. The substituents are the same as those of the alkyl group. The aliphatic hydrocarbon group preferably has 1 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, even more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms.

[0022] The aryl group preferably has 6 to 30 carbon atoms, more preferably 6 to 20, even more preferably 6 to 14, and still more preferably 6 to 10. Specific examples include a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a biphenylyl group, and a 2-anthryl group. Of these, a phenyl group is preferred.

[0023] The heteroaryl group preferably has 5 to 30 constituent atoms, more preferably 5 to 20, even more preferably 5 to 14, and still more preferably 5 to 10. The heteroaryl group is preferably a monocyclic or bicyclic heteroaryl group.

[0024] Examples of monocyclic heteroaryl groups include monocyclic nitrogen-containing heteroaryl groups, monocyclic oxygen-containing heteroaryl groups, monocyclic sulfur-containing heteroaryl groups, monocyclic nitrogen- and oxygen-containing heteroaryl groups, and monocyclic nitrogen- and sulfur-containing heteroaryl groups. Examples of bicyclic heteroaryl groups include bicyclic nitrogen-containing heteroaryl groups, bicyclic oxygen-containing heteroaryl groups, bicyclic sulfur-containing heteroaryl groups, bicyclic nitrogen- and oxygen-containing heteroaryl groups, and bicyclic nitrogen- and sulfur-containing heteroaryl groups. Preferred examples include an indolyl group, a benzofuranyl group, a thiophenyl group, a benzothiophenyl group, a furfuryl group, a benzofuranylmethyl group, a thiophenylmethyl group, and a benzothiophenylmethyl group.

[0025] Examples of the heteroarylalkyl group include the above-mentioned alkyl groups substituted with the above-mentioned heteroaryl groups.

[0026] Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms. Examples of alkoxy groups include methoxy, ethoxy, and propoxy. Examples of acyl groups include acetyl and propionyl. Examples of arylalkylcarbonyl groups include benzylcarbonyl. Examples of alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, and Boc groups. The Boc group refers to a tert-butoxycarbonyl group.

[0027] <Trapping of amino acid activated ester> The present disclosure relates to a method for producing a peptide, which includes a step of mixing an amino acid activated ester produced upon condensation of an N-terminally protected amino acid or peptide with a C-terminally protected amino acid or peptide, with an amine represented by formula (1), to trap the amino acid activated ester. In formula (1), L １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C=O, >C=S, >NR ３ ,=N-,-CO-NR ａ -, -CS-NR ａ -, -O-, and -S-, each of which may have a substituent; R ３ represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group; R ａ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR ４ indicates, R １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR ４ indicates R ４ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group; R ２ represents a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, or an ammonium group; R ２If there are multiple R ２ may be the same or different, L １ When the main chain of L contains a heteroatom-containing aliphatic cyclic structure or heteroaryl, l and m each represent an integer of 0 or more (with the proviso that l+m≧1), １ When the main chain does not contain a heteroatom-containing aliphatic cyclic structure or heteroaryl, 1 is an integer of 4 or more. The amine represented by formula (1) may form a salt.

[0028] L １ The alkyl group, alkenyl group or heteroaryl group represented by may have a substituent, and examples of the substituent include -NH ２ , a halogen atom, —O—(CH ２ ) ２ -O-CH ３ , —NHCO—CH ３ , -OCH ３ Examples include:

[0029] Preferably, L １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, >C=O, >C=S, >NR ３ ,=N-,-CO-NR ａ -, -CS-NR ａ -, -O-, and -S-, each of which may have a substituent; R ３ represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group, and preferably represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group. ａ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, or —OR ４ Shows.

[0030] Preferably, R １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, or —OR ４ indicates R ４ represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group. ２ represents a phosphate group, a sulfonate group, or an ammonium group.

[0031] Preferably, R １ is a hydrogen atom. Preferably, in formula (1), L１ represents a hydrogen atom, an alkyl group, an alkenyl group, a heteroaryl group, ═N—, —CO—NR ａ - and -O-, and the groups may have a substituent; L １ When the main chain contains a heteroatom-containing aliphatic cyclic structure, l and m each represent an integer of 0 or more (with the proviso that l+m≧1), １ When the main chain does not contain a heteroatom-containing aliphatic cyclic structure, l is an integer of 4 or more.

[0032] In one embodiment, the amine of formula (1) is a compound of formula (2). In formula (2), R ５ ~R １２ each independently represents an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C=O, >C=S, or >NR １３ , -O-, -S-, -OH, -OR １４ , one or more groups selected from a phosphate group, a sulfonic acid group, a carboxylic acid group, and an ammonium group, and one —NH ２ and R １３ represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group; R ５ ~R １２ In the formula, at least one of —OH, —OR １４ , phosphate group, sulfonic acid group, carboxylic acid group, and ammonium group are present, and R １４ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group; X represents —O—, —S—, or >NR １３ indicates R １３ represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group; and m represents 1 or 2.

[0033] Specific examples of the compound represented by formula (1) are shown below. The compounds shown below may form salts such as hydrochlorides.

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] <Condensation of an N-terminally protected amino acid or peptide with a C-terminally protected amino acid or peptide> The method for producing a peptide according to the present disclosure may include condensing an N-terminally protected amino acid or peptide with a C-terminally protected amino acid or peptide prior to the trapping step.

[0043] The protecting group for the N-terminally protected amino acid or peptide is preferably a 9-fluorenylmethyloxycarbonyl group, a benzyloxycarbonyl group, a tert-butoxycarbonyl group, or an acetyl group, and particularly preferably a 9-fluorenylmethyloxycarbonyl group.

[0044] Preferably, the protecting group in the C-terminally protected amino acid or peptide is linked to the carbonyl group of the amino acid or peptide by an ester or amide bond.

[0045] The C-terminal protecting group in the C-terminal protected amino acid or peptide preferably has an aliphatic hydrocarbon group having 12 or more carbon atoms, preferably 15 or more carbon atoms, and more preferably 20 to 30 carbon atoms. When the C-terminal protecting group has multiple aliphatic hydrocarbon groups, the total number of carbon atoms therein is preferably 30 to 80, and more preferably 36 to 80. The C-terminal protecting group preferably has a ring structure, and preferably has a fused polycyclic ring, an aromatic heterocyclic ring, or a naphthalene ring.

[0046] Preferred C-terminal protecting groups are aromatic heterocyclic compounds represented by formula (1) in WO 2020 / 175473. Reference is made to WO 2020 / 175473 for such compounds, which are incorporated herein by reference. Preferred C-terminal protecting groups are also fused polycyclic aromatic hydrocarbon compounds represented by formula (1) in WO 2020 / 175472. Reference is made to WO 2020 / 175472 for such compounds, which are incorporated herein by reference. The C-terminal protecting group may also be a compound disclosed in WO 2020 / 262259 (Japanese Patent Application No. 2019-122492 and a patent application based thereon), which are incorporated herein by reference.

[0047] Preferably, the protecting group of the C-terminally protected amino acid or peptide is represented by formula (3). In formula (3), Y is -OR Ｃ , -NR Ｃ R, or -SR Ｃ R represents a hydrogen atom, an alkyl group, an arylalkyl group, a heteroarylalkyl group, or a 9-fluorenylmethyloxycarbonyl group, m represents 1 or 2, and n represents an integer of 1 to 5; Ｂ each independently represents an aliphatic hydrocarbon group; Ｃ represents the binding site to the C-terminal amino acid or peptide, R Ａ are each independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group, at least one of the aliphatic hydrocarbon groups having 12 or more carbon atoms, and the benzene ring in formula (2) is R Ａ In addition, the group may further have a substituent.

[0048] The substituted benzyl compound represented by formula (3) is R Ｂ are each independently an aromatic group (excluding a phenyl group). Ｂ The aromatic group R may have a substituent. Ｂis preferably an aryl group or a heteroaryl group, more preferably an aryl group having 10 or more carbon atoms, and particularly preferably a naphthalene group. The heteroaryl group is preferably a monocyclic or bicyclic ring. More preferably, it is a heteroaryl group of a pyridine ring, a pyrazine ring, a triazine ring, a benzothiophene ring, a furan ring, a benzofuran ring, a pyrrole ring, an indole ring, a carbazole ring, a pyrazole ring, an indazole ring, or a thiophene ring.

[0049] R is preferably a hydrogen atom, an amino protecting group, an alkyl group having 1 to 6 carbon atoms, or an arylalkyl group having 7 to 16 carbon atoms, more preferably a hydrogen atom, a methyl group, an ethyl group, or a benzyl group, and even more preferably a hydrogen atom.

[0050] R in formula (3) Ｂ The number m of substitutions in R is 1 or 2, and more preferably 1, from the viewpoints of deprotection rate, solvent solubility, and yield. Ｂ is preferably an alkyl group having 1 to 6 carbon atoms or an arylalkyl group having 7 to 16 carbon atoms, more preferably a methyl group, an ethyl group or a benzyl group, still more preferably a methyl group or an ethyl group, and particularly preferably a methyl group.

[0051] R on the benzene ring in formula (3) Ａ The number of substitutions, n, is an integer of 1 to 5, more preferably an integer of 1 to 4, further preferably 2 or 3, and particularly preferably 3, from the viewpoints of deprotection rate, solvent solubility, and yield.

[0052] R Ａ are each independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group. Ａ The "organic group having an aliphatic hydrocarbon group" in the above means a monovalent organic group (having one bond to the benzene ring) that has an aliphatic hydrocarbon group in its molecular structure.

[0053] The "aliphatic hydrocarbon group" and the "aliphatic hydrocarbon group" in the "organic group having an aliphatic hydrocarbon group" refer to a linear, branched, or cyclic saturated or unsaturated aliphatic hydrocarbon group, preferably an aliphatic hydrocarbon group having 5 or more carbon atoms, more preferably an aliphatic hydrocarbon group having 5 to 60 carbon atoms, even more preferably an aliphatic hydrocarbon group having 5 to 30 carbon atoms, and particularly preferably an aliphatic hydrocarbon group having 10 to 30 carbon atoms. The lower limit of the number of carbon atoms in the aliphatic hydrocarbon group is preferably 12, more preferably 15, and even more preferably 18. The upper limit is preferably 30, more preferably 26, and even more preferably 24.

[0054] The "aliphatic hydrocarbon group" in the "organic group having an aliphatic hydrocarbon group" is not particularly limited, and may be present at a terminal (monovalent group) or at another position (for example, a divalent group). Examples of the "aliphatic hydrocarbon group" include an alkyl group, a cycloalkyl group, an alkenyl group, and an alkynyl group.

[0055] The "alkyl group" preferably has 5 to 30 carbon atoms, more preferably 12 to 30, even more preferably 16 to 26, and even more preferably 18 to 24. Specific examples include pentyl, hexyl, octyl, 2-ethylhexyl, decyl, hexadecyl, octadecyl, icosyl, docosyl, tetracosyl, lauryl, tridecyl, myristyl, and isostearyl groups. Octadecyl, icosyl, docosyl, and tetracosyl groups are preferred, and icosyl, docosyl, and tetracosyl groups are more preferred. The "cycloalkyl group" is preferably, for example, a cycloalkyl group having 5 to 30 carbon atoms, such as a cyclopentyl group, a cyclohexyl group, an isobornyl group, and a tricyclodecanyl group. These groups may be repeatedly linked together, or may form a fused ring structure of two or more rings.

[0056] The "alkenyl group" preferably has 5 to 30 carbon atoms, more preferably 12 to 30 carbon atoms, even more preferably 16 to 26 carbon atoms, and even more preferably 18 to 24 carbon atoms. Specific examples include a pentenyl group, a hexenyl group, and an oleyl group. The "alkynyl group" preferably has 5 to 30 carbon atoms, more preferably 12 to 30 carbon atoms, even more preferably 16 to 26 carbon atoms, and even more preferably 18 to 24 carbon atoms. Specific examples include a 4-pentynyl group and a 5-hexenyl group. The "steroid group" preferably has, for example, a group having a cholesterol structure or an estradiol structure.

[0057] The organic group may be further substituted with one or more substituents selected from a silyl group, a hydrocarbon group having a silyloxy structure, and an organic group having a perfluoroalkyl structure.

[0058] The silyl group is preferably a trialkylsilyl group, and more preferably a silyl group having three alkyl groups of 1 to 3 carbon atoms. The silyloxy structure in the hydrocarbon group having a silyloxy structure is preferably a trialkylsilyloxy structure, and more preferably a silyloxy structure having three alkyl groups of 1 to 3 carbon atoms. The hydrocarbon group having a silyloxy structure preferably has 1 to 3 silyloxy structures. The number of carbon atoms in the hydrocarbon group having a silyloxy structure is preferably 10 or more, more preferably 10 to 100, and particularly preferably 16 to 50.

[0059] Preferred examples of the hydrocarbon group having a silyloxy structure include groups represented by the following formula (Si):

[0060]

[0061] In formula (Si), R ｓｉ１ represents a single bond or an alkylene group having 1 to 3 carbon atoms, R ｓｉ２ represents an alkylene group having 1 to 3 carbon atoms, and R ｓｉ３ and R ｓｉ４ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or —OSiRｓｉ５ R ｓｉ６ R ｓｉ７ represents R ｓｉ５ ~R ｓｉ７ each independently represents an alkyl group having 1 to 6 carbon atoms or an aryl group.

[0062] R in formula (Si) ｓｉ５ ~R ｓｉ７ are each independently preferably an alkyl group having 1 to 6 carbon atoms or a phenyl group, more preferably an alkyl group having 1 to 6 carbon atoms, and particularly preferably a linear or branched alkyl group having 1 to 4 carbon atoms.

[0063] The perfluoroalkyl structure in the organic group having a perfluoroalkyl structure is preferably a perfluoroalkyl structure having 1 to 20 carbon atoms, more preferably a perfluoroalkyl structure having 5 to 20 carbon atoms, and particularly preferably a perfluoroalkyl structure having 7 to 16 carbon atoms. The perfluoroalkyl structure may be linear, branched, or have a cyclic structure. The organic group having a perfluoroalkyl structure is preferably a perfluoroalkyl group, an alkyl group having a perfluoroalkyl structure, or an alkyl group having a perfluoroalkyl structure and an amide bond in the alkyl chain.

[0064] The number of carbon atoms in the organic group having a perfluoroalkyl structure is preferably 5 or more, more preferably 10 or more, even more preferably 10 to 100, and particularly preferably 16 to 50. Preferred examples of the organic group having a perfluoroalkyl structure include the groups shown below.

[0065]

[0066] The moiety other than the "aliphatic hydrocarbon group" in the "organic group having an aliphatic hydrocarbon group" can be set arbitrarily. For example, it may have a moiety such as -O-, -S-, -COO-, -OCONH-, -CONH-, or a hydrocarbon group (monovalent group or divalent group) other than the "aliphatic hydrocarbon group". Examples of the "hydrocarbon group" other than the "aliphatic hydrocarbon group" include aromatic hydrocarbon groups, and specific examples include monovalent groups such as aryl groups, and divalent groups derived therefrom. Furthermore, the aliphatic hydrocarbon group and the hydrocarbon group other than the above aliphatic hydrocarbon group may be substituted with a substituent selected from a halogen atom, an oxo group, and the like.

[0067] The bond (substitution) of the "organic group having an aliphatic hydrocarbon group" to the benzene ring is as defined above in R Ａ Even if the "aliphatic hydrocarbon group" present in the R Ａ In view of ease of synthesis of the compound, it is preferable that the bond is formed via —O—, —S—, —COO—, —OCONH—, —CONH—, or the like moiety present in the alkyl group. Preferably, the bond is formed via —O—, —S—, —COO—, or —CONH—, and it is particularly preferable that the bond is formed via —O—.

[0068] In the protecting group represented by formula (3), all R Ａ From the viewpoints of solvent solubility, crystallization property, and yield, the total number of carbon atoms in all of the aliphatic hydrocarbon groups is preferably 24 or more, more preferably 24 to 200, even more preferably 32 to 100, particularly preferably 34 to 80, and most preferably 36 to 80.

[0069] The protecting group represented by formula (3) is a group having at least one R Ａ and from the viewpoints of solvent solubility, crystallization property, and yield, at least one R ＡIn the formula (I), it is preferable that the compound (I) has at least one aliphatic hydrocarbon group having 12 to 100 carbon atoms, it is more preferable that the compound (I) has at least one aliphatic hydrocarbon group having 18 to 40 carbon atoms, and it is even more preferable that the compound (I) has at least one aliphatic hydrocarbon group having 20 to 36 carbon atoms.

[0070] From the viewpoint of crystallization property and yield, the aliphatic hydrocarbon group is preferably an alkyl group, more preferably a linear alkyl group. Ａ are each independently, from the viewpoints of solvent solubility, crystallization property, and yield, preferably have 12 to 200 carbon atoms, more preferably have 18 to 150 carbon atoms, further preferably have 18 to 100 carbon atoms, and particularly preferably have 20 to 80 carbon atoms.

[0071] The substituent that the protecting group represented by formula (3) may have on the benzene ring is not particularly limited, and examples thereof include an alkoxy group, an aryloxy group, a halogen atom, an alkyl group, a halogenated alkyl group, an aryl group, an acyl group, an acyloxy group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylthio group, an arylthio group, R ｓｔ -CO-NR ｓｔ -, -CON(R ｓｔ ) ２ , dialkylamino groups, alkylarylamino groups, diarylamino groups, and groups in which two or more of these groups are combined. ｓｔ represents a hydrogen atom, an alkyl group, or an aryl group.

[0072] The molecular weight of the protecting group represented by formula (3) is not particularly limited, but from the viewpoints of deprotection rate, crystallization property, solvent solubility, and yield, it is preferably 340 to 3,000, more preferably 400 to 2,000, even more preferably 500 to 1,500, and particularly preferably 800 to 1,300.

[0073] Details of the protecting group represented by formula (3), preferred examples, and a production method thereof are described in WO2023 / 106356, and the contents of the present specification relating to the protecting group represented by formula (3) described in WO2023 / 106356 are incorporated herein by reference.

[0074] The condensation of an N-terminally protected amino acid or peptide with a C-terminally protected amino acid or peptide can be carried out using a condensing agent.

[0075] Condensing agents that are commonly used in peptide synthesis can be used without limitation in the present disclosure, and include, but are not limited to, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorphonium chloride (DMT-MM), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU(6-Cl)), O-(benzotriazole ... Examples of suitable benzotriazol-1-yloxytrimethyluronium tetrafluoroborate include (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), its hydrochloride salt (EDC·HCl), and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop). Among these, DIC, EDC, EDC·HCl, DMT-MM, HBTU, HATU, and COMU are preferred. The amount of the condensing agent used is preferably 1 to 10 molar equivalents, more preferably 1 to 5 molar equivalents, relative to 1 molar equivalent of the substrate.

[0076] It is preferable to add a condensation activator to promote the reaction and suppress side reactions such as racemization. The condensation activator is a reagent that converts amino acids into corresponding activated esters, acid anhydrides, etc., in the presence of a condensation agent, thereby facilitating the formation of peptide bonds (amide bonds). The condensation activating agent can be any activating agent commonly used in peptide synthesis, without limitation, and examples thereof include 4-dimethylaminopyridine, N-methylimidazole, boronic acid derivatives, 1-hydroxybenzotriazole (HOBt), ethyl 1-hydroxytriazole-4-carboxylate (HOCt), 1-hydroxy-7-azabenzotriazole (HOAt), 3-hydroxy-1,2,3-benzotriazodin-4(3H)-one (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), pentafluorophenol, ethyl (hydroxyimino)cyanoacetate (Oxyma), etc. Among these, 4-dimethylaminopyridine, HOBt, HOCt, HOAt, HOOBt, HOSu, HONb, or Oxyma is preferred. The amount of the condensation activator used is preferably more than 0 molar equivalents and 4.0 molar equivalents, and more preferably 0.1 to 1.5 molar equivalents, relative to 1 molar equivalent of the substrate.

[0077] As the solvent, a common organic solvent can be used in the reaction. Specific examples include halogenated hydrocarbons such as chloroform and dichloromethane; and nonpolar organic solvents such as 4-methyltetrahydropyran, 2-methyltetrahydrofuran, 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether. Two or more of these solvents may be mixed for use. Furthermore, the halogenated hydrocarbons or nonpolar organic solvents may be mixed with aromatic hydrocarbons such as benzene, toluene, and xylene; nitriles such as acetonitrile and propionitrile; ketones such as acetone and 2-butanone; amides such as N,N-dimethylformamide and N-methylpyrrolidone; or sulfoxides such as dimethyl sulfoxide.

[0078] The reaction temperature is not particularly limited, but is preferably −10° C. to 80° C., more preferably 0° C. to 60° C., and more preferably 0° C. to 40° C. The reaction time is not particularly limited, but is preferably 10 minutes to 30 hours.

[0079] <Trapping of amino acid activated ester> In one aspect of the method for producing a peptide according to the present disclosure, after performing step A in which an amino acid or peptide having a protected N-terminus is condensed with an amino acid or peptide having a protected C-terminus, the amino acid activated ester produced during the condensation can be mixed with an amine represented by formula (11) to trap the amino acid activated ester.

[0080] One aspect of the method for producing a peptide of the present disclosure includes a step B of deprotecting the peptide protected with a 9-fluorenylmethyloxycarbonyl group (also referred to as an Fmoc group) obtained in the above step A to produce an amino group-containing compound from which the Fmoc group as a protecting group has been removed; １ -COOH (in formula (11), L １ represents an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C=O, >C=S, >NR １ , —O—, and —S—, and the above groups may have a substituent, and at least >NR １ , —O—, —S—, R １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR ２ indicates, R ２ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group), and a base, １ —COOH (in formula (12), Fm represents a 9-fluorenylmethyl group, L １ is defined as in formula (11)), and a step D of separating the amino group-containing compound from which the Fmoc group has been removed, from the compound represented by formula (12).

[0081] In step B, the amino group-containing compound having an Fmoc group is deprotected to produce an amino group-containing compound from which the Fmoc group has been removed. The amino group-containing compound having an Fmoc group can be deprotected by treating it with a base. The deprotection reaction is carried out in a solvent that does not affect the reaction.

[0082] Examples of the base include secondary amines such as dimethylamine and diethylamine, and non-nucleophilic organic bases such as 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 1,5-diazabicyclo[4.3.0]-5-nonene (DBN).

[0083] As the solvent, a common organic solvent can be used in the reaction. Specific examples include halogenated hydrocarbons such as chloroform and dichloromethane; and nonpolar organic solvents such as 4-methyltetrahydropyran, 2-methyltetrahydropyran, 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether. Two or more of these solvents may be mixed for use. Furthermore, the halogenated hydrocarbons or nonpolar organic solvents may be mixed with aromatic hydrocarbons such as benzene, toluene, and xylene; nitriles such as acetonitrile and propionitrile; ketones such as acetone and 2-butanone; amides such as N,N-dimethylformamide and N-methylpyrrolidone; or sulfoxides such as dimethyl sulfoxide.

[0084] The reaction temperature is not particularly limited, but is preferably −10° C. to 80° C., more preferably 0° C. to 60° C., and more preferably 0° C. to 40° C. The reaction time is not particularly limited, but is preferably 10 minutes to 30 hours.

[0085] In step C, dibenzofulvene and a compound represented by formula (11): HS-L １ -COOH (in formula (11), L １ represents an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C=O, >C=S, >NR １, —O—, and —S—, and the above groups may have a substituent, and at least >NR １ , —O—, —S—, R １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR ２ indicates, R ２ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group), and a base, １ —COOH (in formula (12), Fm represents a 9-fluorenylmethyl group, L １ is defined as in formula (11)) to produce a compound represented by the formula:

[0086] The main chain refers to the group connecting the HS group and the COOH group. When two or more COOH groups are present, the longer chain is the main chain. Steps B and C may be carried out in one pot. That is, step C may be carried out as a one-pot reaction using the reaction solution obtained in step B without isolation or purification.

[0087] L １ The alkyl group, alkenyl group, alkynyl group, aryl group or heteroaryl group in the formula (I) may have a substituent, and examples of the substituent include an alkyl group, an aryl group or a heteroaryl group, —CO—, —NH—, —O—, —COO—, —OCO—, —SO ２ -, -COOH, -NO ２ , =NH, =O, -NH ２ , -N(CH ３ ) ２ , -N ＋ (CH ３ ) ３ The alkyl group may be a combination of one or more groups selected from the group consisting of —COOH and —NHCOCH ３ may be substituted with.

[0088] In formula (11), L １ At least >NR １ It is preferable that R contains either —O— or —O—.１ is preferably a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, or —OR ２ R represents, more preferably, a hydrogen atom. ２ preferably represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group, and more preferably represents a hydrogen atom.

[0089] In one aspect, L １ is preferably composed of a combination of one or two alkyl groups, >C=O, and >NH. １ More preferably, L is composed of a combination of two alkyl groups and -CONH-. １ is also preferably composed of a combination of an alkyl group, a heteroaryl group, and >C═O.

[0090] Specific examples of the compound represented by formula (11) are shown below.

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] Among the above, the more preferred compounds are as follows:

[0099] Among the above, the following compounds are more preferred.

[0100] Mercapto compounds other than the compounds described above can also be used as the compound for trapping the dibenzofulvene produced in step B. The mercapto compound that can be used is not particularly limited as long as it has a mercapto group and the compound that reacts with dibenzofulvene is water-soluble, but examples include mercapto compounds having an alkyl group having 1 to 10 carbon atoms and having one or more substituents selected from sulfonic acid, alkali metal salts of sulfonic acid, phosphonic acid, alkali metal salts of phosphonic acid, carboxylic acid, and alkali metal salts of carboxylic acid, and examples thereof include compounds represented by the following formula (21), (22), or (23):

[0101] HS-L １ -SO ３ M Formula (21) HS-L ２ -PO ３ M ２ Formula (22) HS-L ２ -CO ２ M Formula (23) (wherein, L １ and L ２ each represents a divalent organic group, and M represents a hydrogen atom or an alkali metal.

[0102] In formula (21), (22) or (23), L １ and L ２ Each of the groups represents a divalent organic group. The divalent organic group is preferably a divalent organic group having 1 to 10 carbon atoms, and more preferably a linear or branched alkylene group having 1 to 10 carbon atoms which may have a mercapto group, an arylene group having 6 to 10 carbon atoms which may have a mercapto group, or a heteroarylene group having 4 to 9 carbon atoms which may have a mercapto group. Even more preferably, in formula (21) or (22), L １ and L ２is a linear or branched alkylene group having 1 to 6 carbon atoms, more preferably a linear alkylene group having 1, 2 or 4 carbon atoms, and even more preferably an alkylene group having 1 or 2 carbon atoms. Specific examples include a methylene group, an ethylene group, a trimethylene group, a propylene group, a mercaptotrimethylene group, a mercaptopropylene group, a tetramethylene group, a butylene group, a pentamethylene group, a phenylene group, a naphthylene group, an indole group, a benzimidazole group, a quinolyl group, and an isoquinoline group.

[0103] M represents a hydrogen atom or an alkali metal, specifically, a hydrogen atom, sodium, or potassium.

[0104] Specific examples of the compound represented by formula (21), (22), or (23) include sodium mercaptomethanesulfonate, sodium 2-mercaptoethanesulfonate, 2-mercaptoethanesulfonic acid, 3-mercaptopropanesulfonic acid, sodium 3-mercaptopropanesulfonate, 1,3-dimercaptopropanesulfonic acid, sodium 2-mercaptobenzimidazole-5-sulfonate, sodium mercaptomethanephosphonate, mercaptoethanephosphonic acid, sodium 3-mercaptopropanephosphonate, sodium 1,3-dimercaptopropanephosphonate, 3-mercaptopropionic acid, thiomalic acid, and cysteine.

[0105] As the base, the base used in Step B may be used as it is, and examples thereof include secondary amines such as dimethylamine and diethylamine, and non-nucleophilic organic bases such as 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 1,5-diazabicyclo[4.3.0]-5-nonene (DBN).

[0106] As the solvent, a common organic solvent can be used in the reaction, and the solvent used in step B may be used as is. Specific examples include halogenated hydrocarbons such as chloroform and dichloromethane; and nonpolar organic solvents such as 4-methyltetrahydropyran, 2-methyltetrahydrofuran, 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether. Two or more of these solvents may be mixed for use. Furthermore, the halogenated hydrocarbons or nonpolar organic solvents may be mixed with aromatic hydrocarbons such as benzene, toluene, and xylene; nitriles such as acetonitrile and propionitrile; ketones such as acetone and 2-butanone; amides such as N,N-dimethylformamide and N-methylpyrrolidone; or sulfoxides such as dimethyl sulfoxide.

[0107] The reaction temperature is not particularly limited, but is preferably −10° C. to 80° C., more preferably 0° C. to 60° C., and more preferably 0° C. to 40° C. The reaction time is not particularly limited, but is preferably 10 minutes to 30 hours.

[0108] In step D, the peptide obtained by condensation is separated. That is, the method for producing a peptide of the present disclosure may further include a step of separating the peptide obtained by condensation after the trapping step. Preferably, the peptide obtained by condensation can be separated by crystallization or liquid separation.

[0109] More preferably, the peptide obtained by condensation can be separated by separation using a basic aqueous solution. ２ CO ３ A mixture of an aqueous solution of NaCl and an aqueous solution of NaCl can be used. ２ CO ３ The peptide obtained by condensation can be separated by adding a mixture of aqueous solution and aqueous NaCl solution and recovering the organic layer. The separation may be carried out once or twice or more times.

[0110] <Peptide Chain Extension> In the method for producing a peptide according to the present disclosure, it is preferable to perform the step of deprotecting the N-terminus of an N-terminally protected, C-terminally protected peptide and the step of condensing an N-terminally protected amino acid or an N-terminally protected peptide to the N-terminus of the obtained C-terminally protected peptide, in this order, twice or more times. That is, the peptide from which the Fmoc group separated in step D has been removed can be used as the peptide whose C-terminus has been protected in step A. As described above, by repeatedly performing steps A to D, the chain of the obtained peptide can be extended.

[0111] <C-Terminal Deprotection Step> The method for producing a peptide according to the present disclosure may further include a C-terminal deprotection step of deprotecting a C-terminal protecting group. In the C-terminal deprotection step, the C-terminal protecting group in a C-terminal protected peptide having a desired number of amino acid residues is removed to obtain the final target peptide. Preferred methods for removing the C-terminal protecting group include deprotection methods using an acidic compound. Examples include a method using an acid catalyst or a method of hydrogenation using a metal catalyst. Examples of acid catalysts include trifluoroacetic acid (TFA), hydrochloric acid, trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and acetic acid. TFA is preferred for peptides that do not decompose in strong acids, and TFE, HFIP, or acetic acid is preferred for peptides that decompose in strong acids. The acid concentration can be selected appropriately depending on the side chain protecting group of the amino acid to be elongated and the deprotection conditions. For example, the concentration of TFA is preferably 50% by volume or less, more preferably 30% by volume or less, more preferably 10% by volume or less, more preferably 5% by volume or less, and particularly preferably 1% by volume or less, relative to the total volume of the solvent used. The lower limit is preferably 0.01% by volume, more preferably 0.1% by volume, and more preferably 0.5% by volume. The deprotection time is preferably 5 hours or less, more preferably 3 hours or less, and more preferably 1 hour or less.

[0112] The final target peptide obtained by the peptide production method according to the present disclosure can be isolated and purified according to methods commonly used in peptide chemistry. For example, the final target peptide can be isolated and purified by subjecting the reaction mixture to extraction and washing, crystallization, chromatography, etc. The type of peptide produced by the peptide production method according to the present disclosure is not particularly limited, but it is preferable that the number of amino acid residues of the peptide is, for example, several tens or less. The peptides obtained by the peptide production method according to the present disclosure, like existing or unknown synthetic peptides and natural peptides, can be used in various fields, including, but not limited to, pharmaceuticals, foods, cosmetics, electronic materials, biosensors, etc.

[0113] The following examples will further illustrate the embodiments of the present disclosure. The materials, amounts used, ratios, processing details, processing procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the embodiments of the present disclosure. Therefore, the scope of the embodiments of the present disclosure is not limited to the specific examples shown below. Unless otherwise specified, "%" is based on mass. Furthermore, room temperature means 25°C.

[0114] Example 1 According to the following scheme, Fmoc-Cys(Trt)-OH (N-[(9H-fluoren-9-ylmethoxy)carbonyl]-S-(triphenylmethyl)-D-cysteine) was condensed with a polyhydric alcohol, and the liquid separation and removability of the amino acid derivative after removal of the Fmoc group was evaluated.

[0115]

[0116] Synthesis of Amino Acid Derivatives Fmoc-Cys(Trt)-OH (N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-cysteine ​​5-tert-butyl ester) (100 mg, 0.17 mmol), 4-MTHP (4-methyltetrahydropyran) (6.8 mL), and DMF (N,N-dimethylformamide) (0.68 mL) were mixed at room temperature, and then added with DIPEA (N,N-diisopropylethylamine) (119 μL, 0.68 mmol), DMS Compound (1-1) (46.4 mg, 0.26 mmol) dissolved in 0 (dimethyl sulfoxide) (0.68 mL), oxyma (ethyl cyano(hydroxyimino)acetate) (36.4 mg, 0.26 mmol), and COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate) (110 mg, 0.26 mmol) were added and stirred at 30°C for 30 minutes. DBU (diazabicycloundecene) (191 μL, 1.28 mmol) was added and stirred at 30°C for 30 minutes. The resulting reaction solution was diluted with 10% Na ２ CO ３ Aqueous solution / 20% NaCl aqueous solution (0.68 mL / 6.12 mL) was added and the layers were separated.

[0117] Compounds (1-2) to (1-7) were synthesized in the same manner except that compounds (1-2) to (1-7) were used instead of compound (1-1), and the organic layer after separation was measured by HPLC.

[0118] HPLC analysis conditions Column: ACQUITY UPLC CSH C18 2.1 x 100 mm Column (Waters) Mobile phase A: Aqueous solution containing 0.1% TFA Mobile phase B: 80% THF 20% acetonitrile solution containing 0.1% TFA Flow rate: 0.3 mL / min Column temperature: 40°C Detection wavelength: 220 nm Gradient conditions: 25% B (0 min) → 25% B (0.5 min) → 100% B (7.5 min) → 100% B (9.5 min) → 25% B (9.6 min) → 25% B (15 min)

[0119] Since the added reagent and the amine adduct are removed in the aqueous layer, it is preferable that only DBF produced by removal of the Fmoc group remains in the organic layer after separation.

[0120] Evaluation criteria A: 95 area% or more B: 80 area% or more but less than 95 area% C: Less than 80 area%

[0121]

[0122] In Comparative Examples 1-1 and 1-2, although the compounds were water-soluble, their hydrophilicity was insufficient, resulting in a low removal rate of amine adducts. As in Examples 1-1 and 1-2, tetrahydric or higher alcohols are capable of removing amino acid adducts with high efficiency. Furthermore, when a cyclic ether structure is contained, as in Example 1-5, even a divalent hydroxyl group can be used to remove amino acid adducts with high efficiency.

[0123] Example 2 Compound (3-5) H-Asn-Cys-Glu-OH was synthesized using compound (2-1) described in WO 2023 / 106356 according to the following scheme.

[0124]

[0125] Synthesis of Compound (3-1) Compound (2-1) (5.0 g, 5.39 mmol), Fmoc-Glu(Trt)-OH (N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-glutamic acid 5-tert-butyl ester) (3.59 g, 8.10 mmol), and THF (tetrahydrofuran) (53.9 mL) were mixed at room temperature, and DMAP (4-dimethylaminopyridine) (132 mg, 1.08 mmol) and DIC (diisopropylcarbodiimide) (2.90 mL, 18.9 mmol) were added. The reaction solution was stirred at 30°C for 1 hour, and the insoluble matter was filtered off. The resulting reaction solution was then added to methanol (260 mL). The precipitated solid was collected by filtration and dried under reduced pressure to obtain compound (3-1) (7.00 g).

[0126] Synthesis of Compound (3-2) Compound (3-1) (4.32 g, 3.23 mmol) and THF (tetrahydrofuran) (32.3 mL) were mixed at room temperature, DBU (diazabicycloundecene) (580 μL, 3.88 mmol) was added, and the mixture was stirred at 30°C for 1 hour. NMM (N-methylmorpholine) (711 μL, 6.47 mmol) and MsOH (methanesulfonic acid) (273 μL, 4.20 mmol) were added in that order, and the reaction solution was added to methanol (162 mL). The precipitated solid was collected by filtration and dried under reduced pressure to obtain compound (3-2) (3.32 g).

[0127] Synthesis of Compound (3-3) Compound (3-2) (1.0 g, 0.90 mmol), 4-MTHP (4-methyltetrahydropyran) (36 mL), and DMF (N,N-dimethylformamide) (3.6 mL) were mixed at room temperature, and the resulting mixture was mixed with DIPEA (N,N-diisopropylethylamine) (626 μL, 3.59 mmol), Fmoc-Cys(Trt)-OH(N-[(9H-fluoren-9-ylmethoxy)carbonyl]- S-trityl-L-cysteine) (632 mg, 1.08 mmol), oxyma (ethyl cyano(hydroxyimino)acetate) (153 mg, 1.08 mmol), and COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate) (462 mg, 1.08 mmol) were added and stirred at 30°C for 30 minutes. D-(+)-glucosamine hydrochloride (58.1 mg, 0.27 mmol) dissolved in DMSO (dimethyl sulfoxide) (2.7 mL) and DBU (diazabicycloundecene) (44.3 μL, 0.30 mmol) was added and stirred at 30°C for 15 minutes, after which DBU (diazabicycloundecene) (872 μL, 5.84 mmol) was added and stirred at 30°C for 30 minutes. Subsequently, tiopronin (880 mg, 5.39 mmol) was added and stirred at 30° C. for 30 minutes. The resulting reaction solution was diluted with 10% Na ２ CO ３ The separation by adding aqueous solution / 20% NaCl aqueous solution (3.6 mL / 32.4 mL) was repeated twice, and the organic layer was used in the next step.

[0128] Synthesis of Compound (3-4) The organic layer obtained when compound (3-3) was synthesized was mixed with DMF (N,N-dimethylformamide) (3.6 mL) at room temperature, and the resulting mixture was mixed with DIPEA (N,N-diisopropylethylamine) (614 μL, 3.52 mmol), Fmoc-Asn(Trt)-OH(N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N γ -trityl-L-asparagine) (631 mg, 1.06 mmol), oxyma (ethyl cyano(hydroxyimino)acetate) (150 mg, 1.06 mmol), and COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate) (453 mg, 1.06 mmol) were added and stirred at 30°C for 30 minutes. D-(+)-glucosamine hydrochloride (57.0 mg, 0.26 mmol) dissolved in DMSO (dimethyl sulfoxide) (2.7 mL) and DBU (diazabicycloundecene) (43.4 μL, 0.29 mmol) was added and stirred at 30°C for 15 minutes, after which DBU (diazabicycloundecene) (854 μL, 5.72 mmol) was added and stirred at 30°C for 30 minutes. Subsequently, tiopronin (862 mg, 5.28 mmol) was added and stirred at 30° C. for 30 minutes. The resulting reaction solution was diluted with 10% Na ２ CO ３ The separation by adding aqueous solution / 20% NaCl aqueous solution (3.6 mL / 32.4 mL) was repeated twice, and the organic layer was used in the next step.

[0129] Synthesis of Compound (3-5) The organic layer obtained during the synthesis of compound (3-4) was mixed with DMF (N,N-dimethylformamide) (3.6 mL) at room temperature, and DIPEA (N,N-diisopropylethylamine) (601 μL, 3.45 mmol), Fmoc-Ala-OH (N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine) (322 mg, 1.04 mmol), oxyma (ethyl cyano(hydroxyimino)acetate) (147 mg, 1.04 mmol), and COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate) (444 mg, 1.04 mmol) were added, followed by stirring at 30° C. for 30 minutes. D-(+)-glucosamine hydrochloride (55.8 mg, 0.26 mmol) dissolved in DMSO (dimethyl sulfoxide) (2.7 mL) and DBU (diazabicycloundecene) (42.5 μL, 0.28 mmol) was added and stirred at 30°C for 15 minutes, after which DBU (diazabicycloundecene) (837 μL, 5.61 mmol) was added and stirred at 30°C for 30 minutes. Subsequently, tiopronin (845 mg, 5.18 mmol) was added and stirred at 30°C for 30 minutes. The resulting reaction solution was diluted with 10% Na ２ CO ３ The separation by adding aqueous solution / 20% NaCl aqueous solution (3.6 mL / 32.4 mL) was repeated twice, and the organic layer was used in the next step.

[0130] Synthesis of Compound (3-6) The organic layer obtained during the synthesis of compound (3-5) was mixed with DMF (N,N-dimethylformamide) (3.6 mL) at room temperature, and DIPEA (N,N-diisopropylethylamine) (601 μL, 3.45 mmol), Fmoc-Asp(OtBu)-OH (N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-4-tert-butyl aspartate) (426 mg, 1.04 mmol), oxyma (ethyl cyano(hydroxyimino)acetate) (147 mg, 1.04 mmol), and COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate) (444 mg, 1.04 mmol) were added, followed by stirring at 30° C. for 30 minutes. D-(+)-glucosamine hydrochloride (55.8 mg, 0.26 mmol) dissolved in DMSO (dimethyl sulfoxide) (2.7 mL) and DBU (diazabicycloundecene) (42.5 μL, 0.28 mmol) was added and stirred at 30°C for 15 minutes, after which DBU (diazabicycloundecene) (837 μL, 5.61 mmol) was added and stirred at 30°C for 30 minutes. Subsequently, tiopronin (845 mg, 5.18 mmol) was added and stirred at 30°C for 30 minutes. The resulting reaction solution was diluted with 10% Na ２ CO ３ The organic layer was separated twice by adding a mixture of 20% NaCl aqueous solution (3.6 mL / 32.4 mL) and methanol (144 mL) to the organic layer, and the precipitated solid was collected by filtration and dried under reduced pressure to obtain compound (3-6) (1.5 g).

[0131] Synthesis of compound (3-7) TFA / TIPS / H ２ Compound (1-6) (450 mg, 0.22 mmol) was added to 0 / DODT (7.4 mL / 220 μL / 220 μL / 880 μL) under ice-cooling, and the mixture was stirred at 30° C. for 1.5 hours. MTBE (tert-butyl methyl ether) (44 mL) was added to the resulting reaction solution under ice-cooling, and the precipitated solid was collected by filtration and dried under reduced pressure to obtain compound (1-7) (138 mg). The purity of the resulting compound (3-7) was 90.4%, and the results of MS measurement were as follows. MS (ESI m / z): (M+H+)+ = 551, (M−H+)− = 549

Claims

1. A method for producing a peptide, comprising the step of mixing an amino acid activated ester produced upon condensation of an N-terminally protected amino acid or peptide with a C-terminally protected amino acid or peptide, with an amine represented by formula (1) to trap the amino acid activated ester. In formula (1), L １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C=O, >C=S, >NR ３ ,=N-,-CO-NR ａ -, -CS-NR ａ -, -O-, and -S-, each of which may have a substituent; R ３ represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group; R ａ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR ４ indicates, R １ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR ４ indicates R ４ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group; R ２ represents a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, or an ammonium group; R ２ If there are multiple R ２ may be the same or different, L １ When the main chain of L contains a heteroatom-containing aliphatic cyclic structure or heteroaryl, l and m each represent an integer of 0 or more (with the proviso that l+m≧1), １ When the main chain does not contain a heteroatom-containing aliphatic cyclic structure or heteroaryl, 1 represents an integer of 4 or more.

2. R １ The method for producing a peptide according to claim 1, wherein is a hydrogen atom.

3. In the formula (1), L １ represents a hydrogen atom, an alkyl group, an alkenyl group, a heteroaryl group, ═N—, —CO—NR ａ - and -O-, and the groups may have a substituent; L １ When the main chain contains a heteroatom-containing aliphatic cyclic structure, l and m each represent an integer of 0 or more (with the proviso that l+m≧1), １ 2. The method for producing a peptide according to claim 1, wherein when does not contain a heteroatom-containing aliphatic cyclic structure in the main chain, 1 represents an integer of 4 or more.

4. The method for producing a peptide according to claim 1, wherein the amine represented by formula (1) is a compound represented by formula (2). In formula (2), R ５ ~R １２ each independently represents an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C=O, >C=S, or >NR １３ , -O-, -S-, -OH, -OR １４ , one or more groups selected from a phosphate group, a sulfonic acid group, a carboxylic acid group, and an ammonium group, and one —NH ２ and R １３ represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group; R ５ ~R １２ In the formula, at least one of —OH, —OR １４ , phosphate group, sulfonic acid group, carboxylic acid group, and ammonium group are present, and R １４ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group; X represents —O—, —S—, or >NR １３ indicates R １３ represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group; and m represents 1 or 2.

5. The method for producing a peptide according to claim 1, which comprises condensing an N-terminally protected amino acid or peptide with a C-terminally protected amino acid or peptide before the trapping step.

6. The method for producing a peptide according to claim 5, wherein the protecting group of the N-terminally protected amino acid or peptide is a 9-fluorenylmethyloxycarbonyl group, a benzyloxycarbonyl group, a tert-butoxycarbonyl group, or an acetyl group.

7. The method for producing a peptide according to claim 5, wherein the protecting group of the C-terminally protected amino acid or peptide is linked to the carbonyl group of the amino acid or peptide via an ester or amide bond.

8. The method for producing a peptide according to claim 5, wherein the protecting group of the C-terminal protected amino acid or peptide is represented by formula (3). In formula (3), Y is -OR Ｃ , -NR Ｃ R, or -SR Ｃ R represents a hydrogen atom, an alkyl group, an arylalkyl group, a heteroarylalkyl group, or a 9-fluorenylmethyloxycarbonyl group, m represents 1 or 2, and n represents an integer of 1 to 5; Ｂ each independently represents an aliphatic hydrocarbon group; Ｃ represents the binding site to the C-terminal amino acid or peptide, R Ａ are each independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group, at least one of the aliphatic hydrocarbon groups having 12 or more carbon atoms, and the benzene ring in formula (2) is R Ａ In addition, the group may further have a substituent.

9. All R Ａ The method for producing a peptide according to claim 8, wherein the total number of carbon atoms in all aliphatic hydrocarbon groups contained in 10. The method for producing the peptide according to claim 1, further comprising the step of separating the peptide obtained by the condensation after the trapping step.

11. The method for producing the peptide according to claim 10, wherein the peptide obtained by the condensation is separated by crystallization or liquid separation.

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