Method for producing peptide, compound, and peptide synthesis reagent

Abstract

The present invention addresses the problem of providing: a method for producing a peptide, in which aspartimide / glutarimide formation can be sufficiently suppressed; a novel compound exhibiting a high aspartimide / glutarimide formation suppressing effect; and a peptide synthesis reagent capable of sufficiently suppressing aspartimide / glutarimide formation. The present invention provides a method for producing a peptide, the method comprising a step for reacting a compound represented by formula (1) or formula (2) with an amino terminal of an amino acid or a peptide. In formula (1) and formula (2), n is 1 or 2; R1, R2, and R3 each independently represent an aliphatic hydrocarbon group which may have a substituent containing no aromatic ring, wherein at least one of R1, R2, and R3 has a branched structure, and each of two groups selected from among R1, R2, and R3 may form a ring; and R4 represents a protecting group for an amino group.

Description

Peptide production method, compound, and peptide synthesis reagent

[0001] The present disclosure relates to methods for producing peptides, compounds, and peptide synthesis reagents.

[0002] Methods for producing peptides include solid-phase and liquid-phase methods. Liquid-phase methods have good reactivity and allow for purification of intermediate peptides by extraction, washing, isolation, etc. after the condensation reaction. In peptide production, it is known that combinations of aspartic acid or glutamic acid with other amino acids (Asp / Glu-X) form aspartimide / glutarimide through intramolecular dehydration. It has been reported that when synthesizing peptides containing aspartic acid / glutamic acid, the presence of peptides in which these residues have been dehydrated as impurities can complicate purification or prevent the desired product from being obtained. For example, Non-Patent Document 1 reports that the formation of aspartimide in the synthesis of peptides containing aspartic acid reduces the yield of the desired peptide.

[0003] Patent Document 1 discloses that aspartic acid / glutamic acid derivatives having chain-like side chain protecting groups can suppress the formation of aspartimide / glutarimide.

[0004] RSC Chem. Biol. , 2023, 4, 292-299

[0005] European Patent No. EP2886531

[0006] It has been found that the method described in Patent Document 1 is insufficient in its effect of inhibiting the formation of aspartimide / glutarimide. An object of one embodiment of the present disclosure is to provide a method for producing a peptide that can sufficiently inhibit the formation of aspartimide / glutarimide. Another object of another embodiment of the present disclosure is to provide a novel compound that exhibits a high effect of inhibiting the formation of aspartimide / glutarimide. Another object of yet another embodiment of the present disclosure is to provide a peptide synthesis reagent that can sufficiently inhibit the formation of aspartimide / glutarimide.

[0007] As a result of extensive research aimed at solving the above problems, the present inventors discovered that a high inhibitory effect on aspartimide / glutarimide formation can be achieved by introducing a branched structure into the protecting group of the aspartic acid side chain, and thus completed the present invention.

[0008] <1> A method for producing a peptide, comprising a step of reacting a compound represented by the following formula (1) or (2) with an amino acid or the amino terminal of a peptide: In formula (1) and formula (2), n is 1 or 2, and R １ , R ２ and R ３ are each independently an aliphatic hydrocarbon group that does not contain an aromatic ring and may have a substituent, and R １ , R ２ and R ３ At least one of R has a branched structure, １ , R ２ and R ３ Two groups may form a ring, and R ４represents an amino-protecting group. <2> The method for producing the peptide according to <1>, wherein the compound represented by formula (1) or (2) is an N-terminally protected amino acid or an N-terminally protected peptide, and further comprising: a peptide chain elongation step of condensing the compound represented by formula (1) or (2) with a C-terminally protected amino acid or a C-terminally protected peptide, and a precipitation step of precipitating the N-terminally protected and C-terminally protected peptide obtained in the peptide chain elongation step. <3> The method for producing the peptide according to <2>, further comprising, after the precipitation step, one or more of the following steps in this order: deprotecting the N-terminus of the obtained N-terminally protected and C-terminally protected peptide, condensing the N-terminus of the obtained C-terminally protected peptide with an N-terminally protected amino acid or an N-terminally protected peptide, and precipitating the obtained N-terminally protected and C-terminally protected peptide. <4> The method for producing the peptide according to <2>, further comprising a C-terminal deprotection step of deprotecting the C-terminal protecting group, wherein the deprotection is performed using a trifluoroacetic acid solution of 10% by volume or less. <5> A method for producing the peptide according to any one of <2> to <4>, wherein the C-terminal protecting group of the C-terminal protected amino acid or C-terminal protected peptide has an aliphatic hydrocarbon group having 12 or more carbon atoms. <6> A method for producing the peptide according to any one of <1> to <5>, wherein the aliphatic hydrocarbon group which may have a substituent not containing an aromatic ring is an aliphatic hydrocarbon group which may contain -O-. <7> A method for producing the peptide according to any one of <1> to <6>, wherein the substituent not containing an aromatic ring is a cyclic aliphatic hydrocarbon group, a cyclic aliphatic hydrocarbon group containing -O- in the ring, or an alkoxy group. <8> A method for producing the peptide according to any one of <1> to <7>, wherein the substituent not containing an aromatic ring is cyclohexyl, tetrahydropyranyl, or alkoxy having 1 to 6 carbon atoms. <9> R ４ <10> The method for producing the peptide according to any one of <1> to <8>, wherein R is 9-fluorenylmethyloxycarbonyl or benzyloxycarbonyl. １ , R ２ and R ３<11> A method for producing the peptide according to any one of <1> to <10>, wherein n is 1. <12> A compound represented by the following formula (1) or (2): In formula (1) and formula (2), n is 1 or 2, and R １ , R ２ and R ３ are each independently an aliphatic hydrocarbon group that does not contain an aromatic ring and may have a substituent, and R １ , R ２ and R ３ At least one of R has a branched structure, １ , R ２ and R ３ Two groups may form a ring, and R ４ represents a protecting group for an amino group. <13> The compound according to <12>, wherein the aliphatic hydrocarbon group which may have a substituent not containing an aromatic ring is an aliphatic hydrocarbon group which may contain -O-. <14> The compound according to <12> or <13>, wherein the substituent not containing an aromatic ring is a cyclic aliphatic hydrocarbon group, a cyclic aliphatic hydrocarbon group containing -O- in the ring, or an alkoxy group. <15> The compound according to any one of <12> to <14>, wherein the substituent not containing an aromatic ring is cyclohexyl, tetrahydropyranyl, or alkoxy having 1 to 6 carbon atoms. <16> R ４ <17> The compound according to any one of <12> to <15>, wherein R is 9-fluorenylmethyloxycarbonyl or benzyloxycarbonyl. １ , R ２ and R ３ <16> The compound according to any one of <12> to <16>, wherein at least one of n is a butyl group having a branched structure, which may have a substituent not containing an aromatic ring. <17> The compound according to any one of <12> to <16>, wherein n is 1. <18> The compound according to any one of <12> to <17>, wherein n is 1. <19> A peptide synthesis reagent comprising the compound according to any one of <12> to <18>.

[0009] According to one embodiment of the present invention, a method for producing a peptide capable of sufficiently suppressing aspartimide / glutarimide formation can be provided. According to another embodiment of the present invention, a novel compound exhibiting a high inhibitory effect on aspartimide / glutarimide formation can be provided. According to yet another embodiment of the present invention, a peptide synthesis reagent capable of sufficiently suppressing aspartimide / glutarimide formation can be provided.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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 trityl groups. Examples of protecting groups for guanidyl groups include 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl groups, 2,3,4,5,6-pentamethylbenzenesulfonyl groups, tosyl groups, and nitro groups. Examples of protecting groups for mercapto groups (thiol groups) include trityl groups, 4-methylbenzyl groups, acetylaminomethyl groups, t-butyl groups, and t-butylthio groups.

[0014] 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.

[0015] 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.

[0016] <Compound Represented by Formula (1) or the Following Formula (2)> In the method for producing a peptide of the present invention, a compound represented by the following formula (1) or (2) is used. In formula (1) and formula (2), n is 1 or 2, and R １ , R ２ and R ３ are each independently an aliphatic hydrocarbon group that does not contain an aromatic ring and may have a substituent, and R １ , R ２ and R ３ At least one of R has a branched structure, １ , R ２ and R ３ Two groups may form a ring, and R ４ indicates a protecting group for an amino group.

[0017] The aliphatic hydrocarbon group may be saturated or unsaturated, and may be linear, branched, or cyclic. １ , R ２ and R ３ At least one of R has a branched structure. １ , R ２ and R ３ At least one of the groups is a butyl group having a branched structure which may have a substituent not containing an aromatic ring.

[0018] The aliphatic hydrocarbon group is preferably an alkyl group or an alkenyl group, more preferably an alkyl group. The alkyl group is preferably an alkyl group having 1 to 10 carbon atoms (also referred to as "number of carbon atoms"). The alkyl group preferably has 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, and even more preferably 1 or 2 carbon atoms. Specific examples include methyl, ethyl, propyl, isopropyl, butyl, and tert-butyl. The alkenyl group is preferably an alkenyl group having 2 to 6 carbon atoms. A specific example is 1-propenyl.

[0019] The aliphatic hydrocarbon group may have a substituent that does not contain an aromatic ring. The aliphatic hydrocarbon group that may have a substituent that does not contain an aromatic ring may be an aliphatic hydrocarbon group that may contain -O-. The substituent that does not contain an aromatic ring is not particularly limited, but may be a cyclic aliphatic hydrocarbon group, a cyclic aliphatic hydrocarbon group that contains -O- in the ring, or an alkoxy group. The alkoxy group is preferably an alkoxy group having 1 to 10 carbon atoms. The number of carbon atoms in the alkoxy group is more preferably 1 to 6, more preferably 1 to 4, and even more preferably 1 or 2. Specific examples include methoxy, ethoxy, and propoxy.

[0020] Particularly preferred examples of the substituent not containing an aromatic ring include cyclohexyl, tetrahydropyranyl, and alkoxy having 1 to 6 carbon atoms.

[0021] R ４ Examples of the amino-protecting group represented by R 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, an alkenyl group having 2 to 6 carbon atoms, and the like. These groups may be substituted with 1 to 3 substituents selected from the group consisting of a halogen atom, an alkoxy group having 1 to 6 carbon atoms, and a nitro group. ４Particularly preferred examples of the amino-protecting group represented by the formula (I) include 9-fluorenylmethyloxycarbonyl and benzyloxycarbonyl.

[0022] In formula (1) and formula (2), n is 1 or 2, and preferably n is 1.

[0023] Specific examples of the compound represented by formula (1) or the following formula (2) are shown below.

[0024]

[0025]

[0026]

[0027] The compound represented by formula (1) or (2) can be used as a reagent for peptide synthesis. Peptide synthesis will be described later.

[0028] <Method for Producing a Compound Represented by Formula (1) or Formula (2)> The method for producing a compound represented by formula (1) or formula (2) is not particularly limited, and the compound can be produced by referring to known methods. Unless otherwise specified, the starting compounds used in the production of a compound represented by formula (1) or formula (2) may be commercially available compounds, or may be produced by known methods or methods equivalent thereto. Furthermore, if necessary, the produced compound represented by formula (1) or formula (2) may be purified by known purification methods. For example, methods of isolation and purification by recrystallization, column chromatography, etc., and methods of purification by reprecipitation by changing the solution temperature or solution composition, etc., can be used.

[0029] The method for synthesizing the compound represented by formula (1) or formula (2) is not particularly limited, but for example, the compound can be synthesized according to the following scheme.

[0030]

[0031] In the formula, R １ ~R ４and n are as defined herein, DCC refers to N,N-dicyclohexylcarbodiimide, DMAP refers to 4-dimethylaminopyridine, and rt refers to room temperature.

[0032] <Method for Producing Peptide> The method for producing a peptide according to the present disclosure includes a step of reacting a compound represented by formula (1) or formula (2) with the amino terminal of an amino acid or peptide. The amino acid or peptide may be bound to a solid support such as a resin.

[0033] In the method for producing a peptide according to the present disclosure, preferably, the compound represented by formula (1) or formula (2) is an N-terminal protected amino acid or an N-terminal protected peptide, and the method preferably further comprises a peptide chain elongation step of condensing the compound represented by formula (1) or formula (2) with a C-terminal protected amino acid or a C-terminal protected peptide, and a precipitation step of precipitating the N-terminal protected C-terminal protected peptide obtained in the peptide chain elongation step.

[0034] In the present invention, after the above-mentioned precipitation step, it is preferable to further include the following steps, in this order, once or more times: a step of deprotecting the N-terminus of the obtained N-terminus-protected C-terminus-protected peptide; a step of condensing an N-terminus-protected amino acid or an N-terminus-protected peptide to the N-terminus of the obtained C-terminus-protected peptide; and a step of precipitating the obtained N-terminus-protected C-terminus-protected peptide.

[0035] Note that an N-terminally protected amino acid or an N-terminally protected peptide is an amino acid or peptide in which only the N-terminus is protected, and an N-terminally protected C-terminally protected amino acid or peptide is an amino acid or peptide in which both the N-terminus and the C-terminus are protected. The peptide bond formation reaction (condensation reaction) between the amino group and the carboxy group and the deprotection of the protecting group in each step can be carried out by known methods. For example, WO 2020 / 175473 and WO 2020 / 175473 are incorporated by reference and incorporated herein by reference. Each of the above-mentioned steps will be described in detail below.

[0036] <C-Terminal Protection Step> The method for producing a peptide according to the present disclosure preferably includes a C-terminal protection step in which the carboxyl group or amide group of an amino acid or peptide is protected with a C-terminal protecting group. The C-terminal protecting group 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 contains multiple aliphatic hydrocarbon groups, the total carbon number of these groups is preferably 30 to 80, 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. A preferred C-terminal protecting group is an aromatic heterocyclic compound represented by formula (1) in WO 2020 / 175473. WO 2020 / 175473 is incorporated herein by reference for such compounds. A preferred C-terminal protecting group is also a fused polycyclic aromatic hydrocarbon compound represented by formula (1) in WO 2020 / 175472. For such compounds, see International Publication No. 2020 / 175472, which is incorporated herein by reference. The C-terminal protecting group may be a compound disclosed in International Publication No. 2020 / 262259 (Japanese Patent Application No. 2019-122492 and a patent application based thereon), which is incorporated herein by reference. The amino acid or peptide used in the C-terminal protection step is not particularly limited, but an N-terminal protected amino acid or an N-terminal protected peptide is preferred, and an Fmoc-protected amino acid or an Fmoc-protected peptide is more preferred. Furthermore, it is preferable that the hydroxy group, amino group, carbonyl group, carboxyl group, amide group, imidazole group, indole group, guanidyl group, mercapto group, etc. other than the C-terminal portion of the amino acid or peptide used in the C-terminal protection step are protected by a protecting group.

[0037] When the bonding site of the C-terminal protecting group is —OH or —SH, it is preferable to carry out the reaction in a solvent that does not influence the reaction, in the presence of a condensation additive (condensation activator), by adding a condensation agent, or in the presence of an acid catalyst.

[0038] The C-terminal protecting group is attached at -NHR１８ (R １８ is a hydrogen atom, an alkyl group, an arylalkyl group, or a heteroarylalkyl group), it is preferable to add a condensing agent in the presence of a condensation additive, or to react the condensing agent with a base. WO 2020 / 175473 and WO 2020 / 175473 are incorporated by reference as examples of condensation activators, condensing agents, and acid catalysts.

[0039] 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.

[0040] The acid catalyst used in the condensation reaction can be any acid catalyst commonly used in peptide synthesis, without limitation, and examples thereof include methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, etc. Of these, methanesulfonic acid and p-toluenesulfonic acid are preferred. The amount of the acid catalyst used is preferably 1 to 10 molar equivalents, and more preferably 1 to 5 molar equivalents, relative to 1 molar equivalent of the substrate.

[0041] In the above C-terminal protection step, 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, in the presence of a condensation agent, converts an amino acid into a corresponding activated ester, acid anhydride, or the like, thereby facilitating the formation of a peptide bond (amide bond). 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 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.

[0042] 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 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether. Two or more of these solvents may be mixed together. Furthermore, the above-mentioned 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. The reaction temperature is not particularly limited, but is preferably −10°C to 80°C, and more preferably 0°C to 40°C. The reaction time is not particularly limited, but is preferably 1 hour to 30 hours.

[0043] The N-terminally protected C-terminally protected amino acid or N-terminally protected C-terminally protected peptide obtained by the above-mentioned C-terminal protection step may be purified. For example, the obtained N-terminally protected C-terminally protected amino acid compound or N-terminally protected C-terminally protected peptide is dissolved in a solvent (reaction solvent, e.g., THF), and the desired organic synthesis reaction is carried out, followed by isolation of the resulting product. The solvent in which the N-terminally protected C-terminally protected amino acid compound or N-terminally protected C-terminally protected peptide is then changed (e.g., by changing the solvent composition or type of solvent), and reprecipitation is carried out. Specifically, the reaction is carried out under conditions in which the N-terminally protected C-terminally protected amino acid or N-terminally protected C-terminally protected peptide dissolves. After the reaction, the solvent is distilled off and then solvent replacement is carried out, or a polar solvent is added to the reaction system without distilling off the solvent, thereby precipitating the aggregates and removing impurities. Polar organic solvents such as methanol, acetonitrile, and water are used alone or in combination as the replacement solvent or polar solvent. That is, the reaction is carried out under conditions in which the N-terminal-protected and C-terminal-protected amino acid or the N-terminal-protected and C-terminal-protected peptide is dissolved, and after the reaction, solvent substitution is carried out using, for example, a halogenated solvent, THF, or the like for dissolution, and a polar organic solvent such as methanol, acetonitrile, or water for precipitation.

[0044] <N-Terminal Deprotection Step> The method for producing a peptide according to the present disclosure preferably includes an N-terminal deprotection step of deprotecting the N-terminal protecting group of the N-terminally protected amino acid or N-terminally protected peptide obtained in the above-mentioned C-terminal protection step. As the N-terminal protecting group, an amino group protecting group described below that is commonly used in technical fields such as peptide chemistry can be used. In the present disclosure, a Boc group, a Cbz group, or an Fmoc group is preferred.

[0045] The deprotection conditions are appropriately selected depending on the type of the temporary protecting group. For example, in the case of an Fmoc group, deprotection is carried out by treatment with a base, and in the case of a Boc group, deprotection is carried out by treatment with an acid. The reaction is carried out in a solvent that does not affect the reaction.

[0046] 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). As the solvent, the solvents described above in the <C-terminal protection step> can be suitably used.

[0047] <Peptide Chain Elongation Step> The method for producing a peptide according to the present disclosure preferably includes a peptide chain elongation step of condensing an N-terminal protected amino acid or an N-terminal protected peptide to the N-terminus of the C-terminal protected amino acid or C-terminal protected peptide obtained in the N-terminal deprotection step. The peptide chain elongation step is preferably carried out using the above-mentioned condensing agent, condensation additive, etc., under peptide synthesis conditions generally used in the field of peptide chemistry. The N-terminal protected amino acid or N-terminal protected peptide is not particularly limited, and an Fmoc-protected amino acid or an Fmoc-protected peptide is preferred.

[0048] <Precipitation Step> The method for producing a peptide according to the present disclosure preferably further includes a precipitation step of precipitating the N-terminal-protected, C-terminal-protected peptide obtained in the peptide chain elongation step. The precipitation step can be carried out in the same manner as the purification (reprecipitation) in the C-terminal protection step. Specifically, after the reaction in the previous step, a polar solvent is added to the reaction system without distilling off the reaction solvent. In this case, THF is used as the nonpolar organic solvent and acetonitrile is used as the polar solvent for the reaction solvent. The ratio (by volume) of the nonpolar organic solvent to the polar solvent used is preferably 1:1 to 1:100, more preferably 1:3 to 1:50, and even more preferably 1:5 to 1:20. With this ratio, the N-terminal-protected, C-terminal-protected amino acid compound or the N-terminal-protected, C-terminal-protected peptide can be efficiently precipitated, allowing the target product to be efficiently purified.

[0049] <Chain elongation> The method for producing a peptide according to the present disclosure preferably further comprises, after the precipitation step, one or more of the following steps, in this order: deprotecting the N-terminus of the obtained N-terminally protected and C-terminally protected peptide; condensing an N-terminally protected amino acid or an N-terminally protected peptide to the N-terminus of the obtained C-terminally protected peptide; and precipitating the obtained N-terminally protected and C-terminally protected peptide. By repeating the above three steps, chain elongation of the obtained peptide can be easily carried out. Each step in the above three steps can be carried out in the same manner as the corresponding step described above.

[0050] <C-Terminal Deprotection Step> The method for producing a peptide according to the present disclosure preferably further comprises 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 of adding an acid catalyst and 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 appropriately selected depending on the side chain protecting group of the elongating amino acid 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.

[0051] The final 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 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, food, cosmetics, electronic materials, biosensors, etc. In the peptide production method according to the present disclosure, the above-mentioned precipitation step can be omitted as appropriate to the extent that it does not affect the reaction in the subsequent step.

[0052] The following examples further illustrate the embodiments of the present invention. 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 invention. Therefore, the scope of the embodiments of the present invention is not limited to the specific examples shown below. Unless otherwise specified, "%" is based on mass. Furthermore, room temperature means 25°C.

[0053] Unless otherwise specified, purification by column chromatography was performed using an automatic purification system ISOLERA (Biotage) or a medium-pressure liquid chromatograph YFLC-Wprep2XY.N (Yamazen Corporation). Unless otherwise specified, the carrier used in silica gel column chromatography was SNAPKP-Sil Cartridge (Biotage) or Hi-Flash Column W001, W002, W003, W004, or W005 (Yamazen Corporation). The mixture ratio of the eluent used in column chromatography is a volume ratio. For example, "gradient elution of hexane:ethyl acetate = 50:50 to 0:100" means that the eluent of 50% hexane / 50% ethyl acetate was finally changed to an eluent of 0% hexane / 100% ethyl acetate. For example, "gradient elution of hexane:ethyl acetate = 50:50 to 0:100, gradient elution of methanol:ethyl acetate = 0:100 to 20:80" means that the eluent of 50% hexane / 50% ethyl acetate was changed to an eluent of 0% hexane / 100% ethyl acetate, and then finally changed to an eluent of 20% methanol / 80% ethyl acetate.

[0054] MS spectra were measured using an ACQUITY SQD LC / MS System (manufactured by Waters, ionization method: ESI (ElectroSpray Ionization)). HPLC purity was measured using an ACQUITY UPLC (manufactured by Waters, column: CSH C18 1.7 μm).

[0055] The following compounds were used: The compound of Comparative Example 1 was purchased from Tokyo Chemical Industry Co., Ltd. (catalog number: B3150), and the compound of Comparative Example 2 was purchased from Sigma-Aldrich Co., Ltd. (catalog number: 8.52418).

[0056]

[0057] <Synthesis of Compound of Example 1>

[0058] Synthesis of Compound (1-2) 2.03 g of isovaleryl chloride (Compound 1-1, manufactured by Tokyo Chemical Industry Co., Ltd.) and 40 mL of tetrahydrofuran (ultra-dehydrated grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were placed in a 200 mL three-necked recovery flask, and a hexane solution of n-butyllithium (1.6 mol / L, manufactured by Kanto Chemical Co., Ltd.) was added dropwise at −78°C, followed by stirring at room temperature for 1 hour. After completion of the reaction, a saturated aqueous solution of ammonium chloride was added, and extraction with ethyl acetate was performed. The resulting organic layer was washed with saturated saline and dried over sodium sulfate, and then insoluble matter was filtered off. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 10 / 1) and dried to obtain 2.78 g of compound (1-2) as a colorless, transparent syrup.

[0059] Synthesis of Compound (1-3) 800 mg of compound (1-2), 500 mg of N-benzyloxycarbonyl-L-aspartate 4-benzyl (Tokyo Chemical Industry Co., Ltd.), 34 mg of 4-dimethylaminopyridine (Tokyo Chemical Industry Co., Ltd.), and 10 mL of dichloromethane (Fujifilm Wako Pure Chemical Industries, Ltd.) were placed in a 50 mL recovery flask, and 1.17 g of N,N-dicyclohexylcarbodiimide (Fujifilm Wako Pure Chemical Industries, Ltd.) was added in four portions at hourly intervals at room temperature. After stirring for an additional hour at room temperature, 247 μL of acetic acid was added. After filtering off insoluble matter, the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 10 / 1) and dried to obtain 111 mg of compound (1-3) as a colorless, transparent syrup. Mass of the resulting compound (1-3): [M+Na] 563

[0060] Synthesis of Compound (1-4) 111 mg of compound (1-3), 28 mg of 10% palladium-activated carbon (approximately 55% water-wet product, Fujifilm Wako Pure Chemical Industries, Ltd.), 1.0 mL of tetrahydrofuran (Fujifilm Wako Pure Chemical Industries, Ltd.), and 1.2 mL of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) were placed in a 50 mL pressure-resistant reaction vessel and stirred under a hydrogen atmosphere at room temperature for 10 hours. After completion of the reaction, insoluble matter was removed by filtration using Celite. The obtained filtrate was concentrated under reduced pressure and dried to obtain 65 mg of compound (1-4) as a white solid. Mass of the obtained compound (1-4): [M-H] 314

[0061] Synthesis of Example (1) 65 mg of compound (1-4), 90 mg of sodium carbonate (Fujifilm Wako Pure Chemical Industries, Ltd.), 1.3 mL of tetrahydrofuran (Fujifilm Wako Pure Chemical Industries, Ltd.), and 1.3 mL of distilled water were placed in a 50 mL recovery flask, and 105 mg of N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide (Fujifilm Wako Pure Chemical Industries, Ltd.) was added at room temperature and stirred for 1 hour. After completion of the reaction, a saturated aqueous solution of ammonium chloride and chloroform were added, and the organic layer was separated and then dried over sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 10 / 1) and dried to obtain 59 mg of the compound of Example 1 as a white amorphous substance. Mass of the obtained compound of Example 1: [M+Na] 561

[0062] <Comparative Data> The compounds of Examples, Comparative Examples 1 and 2 were used as compounds, and H ２ The yield of aspartimide (Asi) and purity of the target product during the synthesis of N-Asp-Gly-Gly-Phe-OH were determined by calculating the area value of ultra-performance liquid chromatography (UPLC). The model sequence can be synthesized using the method of introducing a substituted benzyl compound to the C-terminus, as described in International Publication WO 2023 / 106356.

[0063] Aspartimide (Asi) production rate Rating A: Asi production rate is less than 1% Rating B: Asi production rate is 1% or more and less than 3% Rating C: Asi production rate is 3% or more and less than 5% Rating D: Asi production rate is 5% or more

[0064] Purity of target substance Rating A: Purity of target substance is 95% or more Rating B: Purity of target substance is 93% or more and less than 95% Rating C: Purity of target substance is 90% or more and less than 93% Rating D: Purity of target substance is less than 90%

[0065] The results of evaluating the aspartimide (Asi) production rate and purity of the target product are shown in the table below.

[0066]

[0067] Protected peptide (four-residue peptide: H ２ Synthesis of N-Asp(X)-Gly-Gly-Phe-OH> Details of each abbreviation other than those mentioned above are as follows: Phe: phenylalanine residue Gly: glycine residue Asp(X): asparagine residue having a protecting group of the present invention

[0068] (Synthesis of Fmoc-Phe-O-TAG)

[0069] Starting material (1) (10.0 g, 10.78 mmol) synthesized according to the example in International Publication WO 2023 / 106356 was dissolved in dichloromethane (110 mL), and Fmoc-Phe-OH (1.5 molar equivalents), 4-dimethylaminopyridine (0.2 molar equivalents), and diisopropylcarbodiimide (1.5 molar equivalents) were added and stirred. After completion of the condensation reaction, an 80% aqueous methanol solution (550 mL) was added and stirred. The precipitate was collected by filtration and dried under reduced pressure to obtain Fmoc-Phe-O-TAG (14.0 g, 100% yield).

[0070] (H ２ Synthesis of N-Asp(X)-Gly-Gly-Phe-O-TAG) Using Fmoc-Phe-O-TAG, the peptide sequence was elongated by repeatedly removing the Fmoc group and condensing the protected amino acids shown in Table 2 below.

[0071]

[0072]

[0073] (Deprotection of peptide) H ２ Trifluoroacetic acid / 3,6-dioxa-1,8-octanedithiol / triisopropylsilane / water (92.5 / 2.5 / 2.5 / 2.5:vol%, 10 mL) was added to N-Asp(X)-Gly-Gly-Phe-O-TAG (384 mg) at room temperature, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was added dropwise to ice-cooled cyclopropyl methyl ether (50 mL) to precipitate the product. The supernatant was removed and the product was washed with cyclopropyl methyl ether repeatedly, and the precipitate was collected by H ２ N-Asp(X)-Gly-Gly-Phe-OH (102 mg) was obtained. HPLC purity (220 nm): 95% MS (ESI, m / Z): 395 (M+H)

[0074]

Claims

1. A method for producing a peptide, comprising a step of reacting a compound represented by the following formula (1) or the following formula (2) with the amino terminus of an amino acid or a peptide. In formula (1) and formula (2), n is 1 or 2, and R １ , R ２ and R ３ are each independently an aliphatic hydrocarbon group which may have a substituent not containing an aromatic ring, and at least one of R １ , R ２ and R ３ has a branched structure, and two of the groups of R １ , R ２ and R ３ may form a ring, and R ４ represents a protecting group for an amino group.

2. The compound represented by the formula (1) or formula (2) is an N-terminal protected amino acid or an N-terminal protected peptide, a peptide chain extension step of condensing the compound represented by the formula (1) or formula (2) with a C-terminal protected amino acid or a C-terminal protected peptide, and a precipitation step of precipitating the N-terminal protected C-terminal protected peptide obtained in the peptide chain extension step. The method for producing a peptide according to claim 1, further comprising:

3. After the precipitation step, a step of deprotecting the N-terminal of the obtained N-terminal protected C-terminal protected peptide, a step of condensing an N-terminal protected amino acid or an N-terminal protected peptide to the N-terminal of the obtained C-terminal protected peptide, and a step of precipitating the obtained N-terminal protected C-terminal protected peptide. The method for producing a peptide according to claim 2, further comprising the above steps in this order one or more times.

4. Further comprising a C-terminal deprotection step of deprotecting the C-terminal protecting group, and the deprotection is performed using a trifluoroacetic acid solution of 10% by volume or less. The method for producing a peptide according to claim 2.

5. The C-terminal protecting group of the C-terminal protected amino acid or C-terminal protected peptide has an aliphatic hydrocarbon group having 12 or more carbon atoms. The method for producing a peptide according to claim 2.

6. The method for producing a peptide according to any one of claims 1 to 5, wherein the aliphatic hydrocarbon group which may have a substituent not containing an aromatic ring is an aliphatic hydrocarbon group which may contain -O-.

7. The method for producing a peptide according to any one of claims 1 to 5, wherein the substituent not containing an aromatic ring is a cycloaliphatic hydrocarbon group, a cycloaliphatic hydrocarbon group containing -O- in the ring, or an alkoxy group.

8. The method for producing a peptide according to any one of claims 1 to 5, wherein the substituent not containing an aromatic ring is cyclohexyl, tetrahydropyranyl, or an alkoxy having 1 to 6 carbon atoms.

9. R ４ The method for producing a peptide according to any one of claims 1 to 5, wherein R is 9-fluorenylmethyloxycarbonyl or benzyloxycarbonyl.

10. R １ , R ２ and R ３ At least one of which may have a substituent not containing an aromatic ring and is a branched butyl group, the method for producing a peptide according to any one of claims 1 to 5.

11. n is 1. The method for producing a peptide according to any one of claims 1 to 5.

12. A compound represented by the following formula (1) or the following formula (2): In formula (1) and formula (2), n is 1 or 2, and R １ , R ２ and R ３ are each independently an aliphatic hydrocarbon group which may have a substituent not containing an aromatic ring, and at least one of R １ , R ２ and R ３ has a branched structure, and two of the groups of R １ , R ２ and R ３ may form a ring, and R ４ represents a protecting group for an amino group.

13. The compound according to claim 12, wherein the aliphatic hydrocarbon group which may have a substituent not containing an aromatic ring is an aliphatic hydrocarbon group which may contain -O-.

14. The compound according to claim 12, wherein the substituent not containing an aromatic ring is a cycloaliphatic hydrocarbon group, a cycloaliphatic hydrocarbon group containing -O- in the ring, or an alkoxy group.

15. The compound according to claim 12, wherein the substituent not containing an aromatic ring is cyclohexyl, tetrahydropyranyl, or an alkoxy having 1 to 6 carbon atoms.

16. R ４ The compound according to any one of claims 12 to 15, wherein R is 9-fluorenylmethyloxycarbonyl or benzyloxycarbonyl.

17. R １ , R ２ and R ３ and at least one of R ３ is a branched butyl group which may have a substituent not containing an aromatic ring, the compound according to any one of claims 12 to 15.

18. The compound according to any one of claims 12 to 15, wherein n is 1.

19. A peptide synthesis reagent comprising the compound according to any one of claims 12 to 15.

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