Method for producing peptide, compound, and peptide synthesis reagent

Abstract

To provide 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.SOLUTION: A method for producing a peptide, the method comprising a step of reacting a compound represented by the following formula (1) or the following formula (2) with an amino terminal of an amino acid or a peptide. 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, at least one of R1, R2, and R3 has a branched structure, two groups selected from among R1, R2, and R3 may form a ring, and R4 represents a protecting group for an amino group.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a method for producing a peptide, a compound, and a peptide synthesis reagent.

Background Art

[0002] As methods for producing peptides, there are solid-phase methods, liquid-phase methods, and the like. The liquid-phase method has good reactivity, and after the condensation reaction, the intermediate peptide can be purified by extraction, washing, isolation, etc. In the production of peptides, combinations of aspartic acid or glutamic acid with other amino acids (Asp / Glu-X) are known to form aspartimide / glutarimide by an intramolecular dehydration reaction. When synthesizing a peptide containing aspartic acid / glutamic acid, it has been reported that the presence of dehydrated peptides of these residues as impurities complicates purification or makes it impossible to obtain the target product. For example, Non-Patent Document 1 reports that the formation of aspartimide in the synthesis of a peptide containing aspartic acid results in a decrease in the yield of the desired peptide.

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

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] It has been found that the method described in Patent Document 1 has an insufficient effect of suppressing the formation of aspartimide / glutarimide. The problem to be solved by one embodiment of the present disclosure is to provide a method for producing a peptide capable of sufficiently suppressing the formation of aspartimide / glutarimide. Another problem to be solved by one embodiment of the present disclosure is to provide a novel compound exhibiting a high aspartimide / glutarimide formation suppressing effect. Another problem to be solved by yet another embodiment of the present disclosure is to provide a peptide synthesis reagent capable of sufficiently suppressing the formation of aspartimide / glutarimide.

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that by introducing a branched structure into the protecting group of the aspartic acid side chain, a high aspartimide / glutarimide formation suppressing effect can be obtained, and thus the present invention has been completed. According to the present disclosure, the following aspects are provided.

[0008] <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.

Chemical Formula

Chemical formula

Advantages of the Invention

[0009] According to one embodiment of the present invention, a method for producing a peptide capable of sufficiently suppressing aspartimid / glutarimide formation can be provided. Also, according to another embodiment of the present invention, it is possible to provide a novel compound showing a high aspartimide / glutarimide formation inhibitory effect. Also, according to still another embodiment of the present invention, it is possible to provide a peptide synthesis reagent capable of sufficiently suppressing the formation of aspartimide / glutarimide.

Mode for Carrying Out the Invention

[0010] Hereinafter, the content of the present disclosure will be described in detail. The description of the constituent elements described below may be based on typical 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 represented using "~" means a range including the numerical values described before and after "~" as the lower limit value and the upper limit value. In the numerical ranges described stepwise in this specification, the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of the numerical ranges described in other stepwise descriptions. Also, in the numerical ranges described in this specification, the upper limit value or the lower limit value of the numerical range may be replaced with the value shown in the examples. The term "step" includes not only an independent step but also cases where it cannot be clearly distinguished from other steps, as long as the intended purpose of the step is achieved. In the notation of a group (atomic group), a notation without indicating substitution or non-substitution includes both those having no substituent and those having a substituent. For example, the term "alkyl group" includes not only an alkyl group having no substituent (unsubstituted alkyl group) but also an alkyl group having a substituent (substituted alkyl group). The chemical structural formula may sometimes be described as a simplified structural formula omitting hydrogen atoms. 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, amino group, carboxy group, carbonyl group, amide group, guanidyl group, mercapto group, etc., 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 the protecting group for the hydroxy group include an alkyl group, aryl group, trityl group, arylalkyl group having 7 to 10 carbon atoms, formyl group, acyl group having 1 to 6 carbon atoms, benzoyl group, arylalkylcarbonyl group having 7 to 10 carbon atoms, 2-tetrahydropyranyl group, 2-tetrahydrofuranyl group, silyl group, alkenyl group having 2 to 6 carbon atoms, etc. These groups may be substituted with 1 to 3 substituents selected from the group consisting of a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, and a nitro group.

[0013] Examples of the protecting group for the amino group include a formyl group, acyl group having 1 to 6 carbon atoms, alkoxycarbonyl group having 1 to 6 carbon atoms, benzoyl group, arylalkylcarbonyl group having 7 to 10 carbon atoms, arylalkyloxycarbonyl group having 7 to 14 carbon atoms, trityl group, monomethoxytrityl group, 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl group, phthaloyl group, N,N-dimethylaminomethylene group, silyl group, alkenyl group having 2 to 6 carbon atoms, etc. 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. Examples of the protecting group for the carboxy group include the protecting group for the above hydroxy group, trityl group, etc. Examples of the protecting group for the carbonyl group include cyclic acetals (e.g., 1,3-dioxane), acyclic acetals (e.g., di(alkyl having 1 to 6 carbon atoms) acetal), etc. Examples of the protecting group for the amide group include trityl group, etc. Examples of the protecting group for the guanidyl group include 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group, 2,3,4,5,6-pentamethylbenzenesulfonyl group, tosyl group, nitro group and the like. Examples of the protecting group for the mercapto group (thiol group) include trityl group, 4-methylbenzyl group, acetylaminomethyl group, t-butyl group, t-butylthio group and the like.

[0014] The method for removing the above protecting group may be carried out according to a known method, for example, the method described in Protective Groups in Organic Synthesis, published by John Wiley and Sons (1980). Methods using an acid, a base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylsilyl halide, and a reduction method are used.

[0015] The "amino acid" is an α, β or γ amino acid, and is not limited to natural amino acids, and may be an unnatural amino acid. It may also be an amino acid analog such as hydroxycarboxylic acid.

[0016] <Compound represented by formula (1) or the following formula (2)> In the method for producing the peptide of the present invention, a compound represented by the following formula (1) or the following formula (2) is used. [Chemical formula] In formula (1) and formula (2), n is 1 or 2, R1, R2 and R3 are each independently an aliphatic hydrocarbon group which may have a substituent not containing an aromatic ring, at least one of R1, R2 and R3 has a branched structure, two of the groups among R1, R2 and R3 may form a ring, and R4 represents a protecting group for an amino group.

[0017] The aliphatic hydrocarbon group may be saturated or unsaturated, and may be linear, branched or cyclic. However, at least one of R1, R2 and R3 has a branched structure. Preferably, at least one of R1, R2, and R3 is a butyl group having a branched structure, which may have a substituent that does not contain 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 "the number of carbon atoms"). The number of carbon atoms of the alkyl group is more preferably 1 to 6, still more preferably 1 to 4, and even more preferably 1 or 2. Specific examples include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, etc. The alkenyl group is preferably an alkenyl group having 2 to 6 carbon atoms. Specific examples include 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 is a cycloaliphatic hydrocarbon group, a cycloaliphatic hydrocarbon group containing -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 of the alkoxy group is more preferably 1 to 6, still more preferably 1 to 4, and even more preferably 1 or 2. Specific examples include methoxy, ethoxy, and propoxy.

[0020] Particularly preferred specific examples of the substituent that does not contain an aromatic ring are cyclohexyl, tetrahydropyranyl, or an alkoxy having 1 to 6 carbon atoms.

[0021] Examples of the protecting group for the amino group represented by R4 include, for example, 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. Particularly preferred specific examples of the protecting group for the amino group represented by R4 include 9-fluorenylmethyloxycarbonyl or 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]

Chemical formula

[0025]

Chemical formula

[0026]

Chemical formula

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

[0028] <Method for producing the compound represented by formula (1) or formula (2)> The method for producing the compound represented by the formula (1) or the formula (2) is not particularly limited, but it can be produced with reference to known methods. Unless otherwise specified, the starting compounds used for the production of the compound represented by the formula (1) or the formula (2) may be commercially available ones, or may be produced according to known methods or methods analogous thereto. Furthermore, if necessary, the produced compound represented by the formula (1) or the 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 means of changing the solution temperature, changing the solution composition, etc. can be carried out.

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

[0030]

Chemical formula

[0031] In the formula, R1 to R4 and n are as defined in this specification. DCC represents N,N-dicyclohexylcarbodiimide, DMAP represents 4-dimethylaminopyridine, and r.t. represents 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 the formula (1) or the formula (2) with the amino terminus of an amino acid or a peptide. The amino acid or peptide may be bound to a solid-phase carrier such as a resin.

[0033] In the method for producing a peptide according to the present disclosure, preferably, the compound represented by the formula (1) or the formula (2) is an N-terminal protected amino acid or an N-terminal protected peptide, a peptide chain elongation step of condensing the compound represented by the above formula (1) or the 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 above peptide chain extension step, is preferably further included.

[0034] In the present invention, after the above precipitation step, a step of deprotecting the N-terminus 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 with the N-terminus of the obtained C-terminal protected peptide, and a step of precipitating the obtained N-terminal protected C-terminal protected peptide are preferably further included one or more times in this order.

[0035] Note that the N-terminal protected amino acid or the N-terminal protected peptide is an amino acid or a peptide in which only the N-terminus is protected, and the N-terminal protected C-terminal protected amino acid or peptide is an amino acid or a peptide in which both the N-terminus and the C-terminus are protected. The formation reaction (condensation reaction) of the peptide bond 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, International Publication No. 2020 / 175473 and International Publication No. 2020 / 175473 are referred to and incorporated herein by reference. Hereinafter, each of the above-described steps will be described in detail.

[0036] <C-Terminal Protection Step> The method for producing a peptide according to the present disclosure preferably includes a C-terminal protection step of protecting the carboxy group or amide group of an amino acid or a peptide with a C-terminal protecting group. As the C-terminal protecting group, those having an aliphatic hydrocarbon group having 12 or more carbon atoms are preferable, the preferable number of carbon atoms is 15 or more, and more preferably 20 to 30. When there are a plurality of aliphatic hydrocarbon groups in the C-terminal protecting group, the total number of carbon atoms thereof 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 condensed polycyclic ring, an aromatic heterocyclic ring or a naphthalene ring. As the C-terminal protecting group, an aromatic heterocyclic compound represented by the formula (1) in International Publication No. 2020 / 175473 is preferred. Such a compound is referred to International Publication No. 2020 / 175473 and incorporated herein by reference. As the C-terminal protecting group, a condensed polycyclic aromatic hydrocarbon compound represented by the formula (1) in International Publication No. 2020 / 175472 is also preferred. Such a compound is referred to International Publication No. 2020 / 175472 and incorporated herein by reference. As the C-terminal protecting group, a compound disclosed in International Publication No. 2020 / 262259 (Japanese Patent Application No. 2019-122492 and patent applications based thereon) may also be used. International Publication No. 2020 / 262259 (Japanese Patent Application No. 2019-122492 and patent applications based thereon) is referred to and incorporated herein by reference. The amino acid or peptide used in the above C-terminal protection step is not particularly limited, but an N-terminal protected amino acid or N-terminal protected peptide is preferred, and an Fmoc protected amino acid or Fmoc protected peptide is more preferred. In addition, hydroxy groups, amino groups, carbonyl groups, carboxyl groups, amide groups, imidazole groups, indole groups, guanidyl groups, mercapto groups, etc. other than the C-terminal portion in the amino acid or peptide used in the above C-terminal protection step are preferably protected by protecting groups.

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

[0038] When the bonding site of the C-terminal protecting group is -NHR 18 (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 condensing additive, or to react a condensing agent with a base. The condensing activator, condensing agent, and acid catalyst are referred to and incorporated herein by reference to International Publication No. 2020 / 175473 and International Publication No. 2020 / 175473.

[0039] As the condensing agent, condensing agents generally used in peptide synthesis can be used without limitation in the present disclosure. Examples include, but are not limited to, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium 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-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbene hexafluorophosphate (COMU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), its hydrochloride (EDC·HCl), and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop), etc. Among them, DIC, EDC, EDC·HCl, DMT-MM, HBTU, HATU, or COMU is preferred. The usage amount of the condensing agent is preferably 1 to 10 molar equivalents, more preferably 1 to 5 molar equivalents, relative to 1 molar equivalent of the substrate.

[0040] As the acid catalyst used in the condensation reaction, acid catalysts generally used in peptide synthesis can be used without limitation. Examples include methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, etc. Among them, methanesulfonic acid and p-toluenesulfonic acid are preferred. The amount of the acid catalyst used is preferably 1 to 10 molar equivalents, more preferably 1 to 5 molar equivalents, relative to 1 molar equivalent of the substrate.

[0041] In the above C-terminal protection step, in order to promote the reaction and suppress side reactions such as racemization, it is preferable to add a condensation activator. A condensation activator is a reagent that, in coexistence with a condensing agent, guides an amino acid to a corresponding active ester, acid anhydride, etc., and facilitates the formation of a peptide bond (amide bond). As the condensation activator, activators generally used in peptide synthesis can be used without limitation. For example, 4-dimethylaminopyridine, N-methylimidazole, boronic acid derivatives, 1-hydroxybenzotriazole (HOBt), ethyl 1-hydroxy-1,2,3-triazole-4-carboxylate (HOCt), 1-hydroxy-7-azabenzotriazole (HOAt), 3-hydroxy-1,2,3-benzotriazinedione-4(3H)-one (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), pentafluorophenol, ethyl (hydroxyimino)cyanoacetate (Oxyma), etc. can be mentioned. Among them, 4-dimethylaminopyridine, HOBt, HOCt, HOAt, HOOBt, HOSu, HONb, or Oxyma is preferable. The amount of the activator used is preferably more than 0 molar equivalent and 4.0 molar equivalents or less, more preferably 0.1 to 1.5 molar equivalents, relative to 1 molar equivalent of the substrate.

[0042] As the solvent, common organic solvents can be used in the reaction. Specifically, halogenated hydrocarbons such as chloroform and dichloromethane; non-polar organic solvents such as 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether, etc. can be mentioned. These solvents may be used as a mixture of two or more. Also, 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; sulfoxides such as dimethyl sulfoxide can be mixed with the above-mentioned halogenated hydrocarbons and non-polar organic solvents and used. The reaction temperature is not particularly limited, but it is preferably -10°C to 80°C, and more preferably 0°C to 40°C. The reaction time is not particularly limited, but it is preferably 1 hour to 30 hours.

[0043] Also, the N-terminal protected C-terminal protected amino acid or N-terminal protected C-terminal protected peptide obtained by the above C-terminal protection step may be purified. For example, the obtained N-terminal protected C-terminal protected amino acid compound or N-terminal protected C-terminal protected peptide is dissolved in a solvent (reaction solvent, such as THF), and after performing a desired organic synthesis reaction, the resulting product is isolated. Then, the solvent in which the N-terminal protected C-terminal protected amino acid compound or N-terminal protected C-terminal protected peptide is dissolved is changed (e.g., change in solvent composition, change in solvent type), and reprecipitation is carried out. Specifically, the reaction is carried out under conditions such that the N-terminal protected C-terminal protected amino acid or N-terminal protected C-terminal protected peptide dissolves. After the reaction, the solvent is distilled off, and then the solvent is replaced, or without distilling off the solvent, a polar solvent is added to the reaction system to precipitate aggregates and eliminate impurities. As the substitution solvent or polar solvent, polar organic solvents such as methanol, acetonitrile, and water are used alone or as a mixture. That is, the reaction is carried out under conditions such that the N-terminal protected C-terminal protected amino acid or N-terminal protected C-terminal protected peptide dissolves. After the reaction, for solvent replacement, for example, halogenated solvents, THF, etc. are used for dissolution, and polar organic solvents such as methanol, acetonitrile, and water are used 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 - terminal protected C - terminal protected amino acid or N - terminal protected C - terminal protected peptide obtained in the above C - terminal protection step. As the protecting group for the N - terminal, the protecting groups for the amino group described below, which are generally 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 preferable.

[0045] The deprotection conditions are appropriately selected according to the type of the temporary protecting group. For example, in the case of an Fmoc group, it is carried out by treatment with a base, and in the case of a Boc group, it 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 preferably used.

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

[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 extension step. The precipitation step can be carried out in the same manner as the purification (re-precipitation) in the C-terminal protection step. Specifically, without distilling off the reaction solvent after the previous reaction, a polar solvent is added to the reaction system. In this case, THF is used as the non-polar organic solvent for the reaction solvent, and acetonitrile is used as the polar solvent. The usage ratio (volume basis) of the non-polar organic solvent to the polar solvent is preferably 1:1 to 1:100, more preferably 1:3 to 1:50, and even more preferably 1:5 to 1:20. In this case of the usage ratio, the N-terminal protected C-terminal protected amino acid compound or N-terminal protected C-terminal protected peptide can be efficiently precipitated, and the target product can be efficiently purified.

[0049] <Chain extension> The method for producing a peptide according to the present disclosure preferably further includes, 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 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 in this order one or more times. By repeating the above three steps, the chain extension 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 steps described above.

[0050] <C-terminal deprotection step> The method for producing a peptide according to the present disclosure preferably further includes a C-terminal deprotection step of deprotecting the C-terminal protecting group. In the above C-terminal deprotection step, by removing the C-terminal protecting group in the C-terminal protected peptide having the desired number of amino acid residues, the peptide as the final target product can be obtained. As a method for removing the C-terminal protecting group, a deprotection method using an acidic compound is preferably mentioned. For example, methods such as adding an acid catalyst and hydrogenating using a metal catalyst can be mentioned. Examples of the acid catalyst include trifluoroacetic acid (TFA), hydrochloric acid, trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), acetic acid, and the like. For peptides that are not decomposed by strong acids, TFA is preferred, and for peptides that are decomposed by strong acids, TFE, HFIP, or acetic acid is preferred. The concentration of the acid can be appropriately selected according to 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, still more preferably 10% by volume or less, yet more preferably 5% by volume or less, and particularly preferably 1% by volume or less, based on the total volume of the solvent used. The lower limit is preferably 0.01% by volume, more preferably 0.1% by volume, and still more preferably 0.5% by volume. The deprotection time is preferably 5 hours or less, more preferably 3 hours or less, and still more preferably 1 hour or less.

[0051] The peptide, which is the final target obtained by the method for producing a peptide according to the present disclosure, can be isolated and purified according to methods commonly used in peptide chemistry. For example, the peptide that is the final target can be isolated and purified from the reaction mixture by extraction washing, crystallization, chromatography, and the like. The type of peptide produced by the method for producing a peptide according to the present disclosure is not particularly limited, but it is preferably, for example, about several tens or less in terms of the number of amino acid residues of the peptide. The peptide obtained by the method for producing a peptide according to the present disclosure can be used in various fields, such as, but not limited to, the fields of medicine, food, cosmetics, electronic materials, biosensors, etc., in the same manner as existing or unknown synthetic peptides and natural peptides. The method for producing a peptide according to the present disclosure can also appropriately omit the above precipitation step as long as it does not affect the reaction in the next step.

Examples

[0052] Examples are given below to explain the embodiments of the present invention in more detail. The materials, amounts used, ratios, treatment details, treatment procedures, etc. shown in the following examples can be appropriately changed as long as they do not depart 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. Also, room temperature means 25°C.

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

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

[0055] The following compounds were used. The compound of Comparative Example 1 was purchased from Tokyo Chemical Industry Co., Ltd. (Catalog No.: B3150), and the compound of Comparative Example 2 was purchased from Sigma-Aldrich Co., LLC (Catalog No.: 8.52418).

[0056]

Chemical formula

[0057] <Synthesis of the compound of Example 1>

Chemical formula

[0058] Synthesis of compound (1-2) Into a 200 mL three-necked eggplant flask, 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 Corporation) were added. After dropping an n-butyllithium hexane solution (1.6 mol / L, manufactured by Kanto Chemical Co., Inc.) at -78°C, the mixture was stirred at room temperature for 1 hour. After completion of the reaction, a saturated aqueous ammonium chloride solution was added, and extraction was performed with ethyl acetate. The obtained organic layer was washed with saturated brine, dried using sodium sulfate, and then the insoluble matter was filtered off. The residue obtained by concentrating the filtrate under reduced pressure 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) Into a 50 mL eggplant-shaped flask, 800 mg of compound (1-2), 500 mg of N-benzyloxycarbonyl-L-aspartic acid 4-benzyl (manufactured by Tokyo Chemical Industry Co., Ltd.), 34 mg of 4-dimethylaminopyridine (manufactured by Tokyo Chemical Industry Co., Ltd.), and 10 mL of dichloromethane (manufactured by Fujifilm Wako Pure Chemical Corporation) were added. At room temperature, 1.17 g of N,N-dicyclohexylcarbodiimide (manufactured by Fujifilm Wako Pure Chemical Corporation) was added in 4 portions at one-hour intervals. After further stirring at room temperature for 1 hour, 247 μL of acetic acid was added. After filtering off the insoluble matter, the residue obtained by concentrating the filtrate under reduced pressure 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 obtained compound (1-3): [M+Na] 563

[0060] Synthesis of compound (1-4) Into a 50 mL pressure-resistant reaction vessel, 111 mg of compound (1-3), 28 mg of 10% palladium-activated carbon (about 55% water-wetted product, manufactured by Fujifilm Wako Pure Chemical Corporation), 1.0 mL of tetrahydrofuran (manufactured by Fujifilm Wako Pure Chemical Corporation), and 1.2 mL of ethanol (manufactured by Fujifilm Wako Pure Chemical Corporation) were added. Under a hydrogen atmosphere, the mixture was stirred at room temperature for 10 hours. After completion of the reaction, the insoluble matter was filtered off by a filtration operation 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) To a 50 mL eggplant flask, add 65 mg of compound (1-4), 90 mg of sodium carbonate (manufactured by Fujifilm Wako Pure Chemical Corporation), 1.3 mL of tetrahydrofuran (manufactured by Fujifilm Wako Pure Chemical Corporation), and 1.3 mL of distilled water. At room temperature, add 105 mg of N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide (manufactured by Fujifilm Wako Pure Chemical Corporation) and stir for 1 hour. After completion of the reaction, add saturated aqueous ammonium chloride solution and chloroform, separate the organic layer, and dry it over sodium sulfate. Filter off the insoluble matter, concentrate the filtrate under reduced pressure, and purify the resulting residue by silica gel column chromatography (chloroform / methanol = 10 / 1), and dry to obtain 59 mg of the compound of Example 1 as a white amorphous solid. Mass of the obtained compound of Example 1: [M+Na] 561

[0062] <Comparative data> When using the compound of Example, Comparative Example 1 or Comparative Example 2 as the compound and synthesizing H2N-Asp-Gly-Gly-Phe-OH as the model sequence, the aspartimide (Asi) formation rate and the purity of the target product were obtained by calculating the area values of ultra performance liquid chromatography (UPLC). For the synthesis of the model sequence, the method of introducing a substituted benzyl compound described in International Publication WO2023 / 106356 can be used at the C-terminus.

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

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

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

[0066]

Table 1

[0067] <Synthesis of protected peptide (4-residue peptide: H2N-Asp(X)-Gly-Gly-Phe-OH)> In addition, the details of each abbreviation other than those described above are shown below. Phe: Phenylalanine residue Gly: Glycine residue Asp(X): Asparagine residue having the protecting group of the present invention

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

Chemical formula

[0069] The raw material (1) (10.0 g, 10.78 mmol) synthesized according to the example of International Publication No. WO2023 / 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, 80% aqueous methanol solution (550 mL) was added and stirred, and the precipitate was collected by filtration and dried under reduced pressure to obtain Fmoc-Phe-O-TAG (14.0 g, yield 100%).

[0070] (Synthesis of H2N-Asp(X)-Gly-Gly-Phe-O-TAG) Using Fmoc-Phe-O-TAG, the removal of the Fmoc group and the condensation reaction of the protected amino acids shown in Table 2 below were repeated to extend the peptide sequence.

[0071]

Chemical formula

[0072]

Table 2

[0073] (Deprotection of Peptide) To H2N-Asp(X)-Gly-Gly-Phe-O-TAG (384 mg), trifluoroacetic acid / 3,6-dioxo-1,8-octanedithiol / triisopropylsilane / water (92.5 / 2.5 / 2.5 / 2.5: vol%, 10 mL) was added at room temperature, and the mixture was stirred at room temperature for 1 hour. The reaction solution was dropped into ice-cooled cyclopropylmethyl ether (50 mL) to precipitate, and the removal of the supernatant and washing with cyclopropylmethyl ether were repeated to obtain H2N-Asp(X)-Gly-Gly-Phe-OH (102 mg). HPLC purity (220 nm): 95% MS (ESI, m / Z): 395 (M + H)

[0074]

Chemical Structure

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. 【Chemical 1】 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 the above steps.

3. After the precipitation step, a step of deprotecting the N-terminus 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 with the N-terminus 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 one or more times in this order.

4. The method for producing a peptide according to claim 2, 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.

5. The method for producing a peptide according to claim 2, 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. 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 containing no 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 containing no 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 containing no 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 that does not contain an aromatic ring and is a butyl group having a branched structure, the method for producing a peptide according to any one of claims 1 to 5.

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

12. A compound represented by the following formula (1) or the following formula (2): 【Chemical 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. At least one of R １ , R ２ and R ３ has a branched structure. Two of the groups of R １ , R ２ and R ３ may form a ring. 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 containing no 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 cyclic aliphatic hydrocarbon group, a cyclic aliphatic 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 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 At least one of which may have a substituent that does not contain an aromatic ring and is a branched butyl group, the compound according to any one of claims 12 to 15.

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

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

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