WO2025154779A1 - Method for producing peptide, compound, and peptide synthesis reagent - Google Patents

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

Method for producing peptides, compounds, and peptide synthesis reagents

The present disclosure relates to a method for producing peptides, compounds, and peptide synthesis reagents.

Methods for producing peptides include the solid-phase method, the liquid-phase method, etc. The liquid-phase method has good reactivity and allows purification of intermediate peptides by extraction, washing, isolation, etc. after the condensation reaction.
In the production of peptides, 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 a peptide containing aspartic acid/glutamic acid, the presence of peptides in which these residues have been dehydrated as impurities can complicate purification or make it impossible to obtain the desired peptide. For example, Non-Patent Document 1 reports that the formation of aspartimide in the synthesis of a peptide containing aspartic acid reduces the yield of the desired peptide.

Patent Document 1 discloses that aspartic acid/glutamic acid derivatives having linear side chain protecting groups can suppress the formation of aspartimide/glutarimide.

RSC Chem. Biol. , 2023, 4, 292-299

European Patent Publication No. EP2886531

It has been found that the method described in Patent Document 1 is insufficient in 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 suppress aspartimide/glutarimide formation.
Another problem to be solved by another embodiment of the present disclosure is to provide a novel compound that exhibits a high inhibitory effect on the formation of aspartimide/glutarimide.
A 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.

As a result of extensive research to solve the above problems, the inventors discovered that a high effect of inhibiting the formation of aspartimide/glutarimide can be achieved by introducing a branched structure into the protecting group of the aspartic acid side chain, and thus completed the present invention. The present disclosure provides the following aspects.

<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 an amino acid or an amino terminal of a peptide:
In formula (1) and formula (2), n is 1 or 2; R ₁ , R ₂ and R ₃ each independently represent 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 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 above formula (1) or 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 formula (1) or (2) with a C-terminal protected amino acid or a C-terminal protected peptide; and
a precipitation step of precipitating the N-terminal protected and C-terminal protected peptide obtained in the peptide chain elongation step;
The method for producing the peptide according to <1> further comprises:
<3> After the above precipitation step,
deprotecting the N-terminus of the obtained N-terminus-protected C-terminus-protected peptide;
a step of condensing an N-terminal protected amino acid or an N-terminal protected peptide to the N-terminus of the obtained C-terminal protected peptide; and
The method for producing a peptide according to <2>, further comprising a step of precipitating the obtained N-terminally protected and C-terminally protected peptide in this order at least once.
<4> The method for producing the peptide according to <2>, further comprising a C-terminal deprotection step of deprotecting a C-terminal protecting group, the deprotection being carried out using a trifluoroacetic acid solution of 10% by volume or less.
<5> The method for producing a peptide according to any one of <2> to <4>, wherein the C-terminal protected amino acid or the C-terminal protected peptide has a C-terminal protecting group which has an aliphatic hydrocarbon group having 12 or more carbon atoms.
<6> The 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> The 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> The method for producing the peptide according to any one of <1> to <7>, wherein the substituent not containing an aromatic ring is cyclohexyl, tetrahydrapyranyl, or alkoxy having 1 to 6 carbon atoms.
<9> The method for producing the peptide according to any one of <1> to <8>, wherein R ₄ is 9-fluorenylmethyloxycarbonyl or benzyloxycarbonyl.
<10> The method for producing a peptide according to any one of <1> to <9>, wherein at least one of R ₁ , R ₂ and R ₃ is a butyl group having a branched structure which may have a substituent not containing an aromatic ring.
<11> The 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 the following formula (2):
In formula (1) and formula (2), n is 1 or 2; R ₁ , R ₂ and R ₃ each independently represent 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 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 <12>, wherein the aliphatic hydrocarbon group which may have a substituent and does not contain 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, tetrahydrapyranyl, or alkoxy having 1 to 6 carbon atoms.
<16> The compound according to any one of <12> to <15>, wherein R ₄ is 9-fluorenylmethyloxycarbonyl or benzyloxycarbonyl.
<17> The compound according to any one of <12> to <16>, wherein at least one of R ₁ , R ₂ and R ₃ is a butyl group having a branched structure which may have a substituent not containing an aromatic ring.
<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>.

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 the formation of aspartimide/glutarimide can be provided.
Furthermore, 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.

The contents of the present disclosure will be described in detail below. The following description of the components may be based on a representative embodiment of the present disclosure, but the present disclosure is not limited to such an embodiment.
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 lower and upper limits.
In the present specification, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range. In addition, in the present specification, the upper or lower limit of the numerical range may be replaced with a value shown in the examples.
The term "process" includes not only an independent process, but also a process that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved.
In the notation of a group (atomic group), if there is no indication of substituted or unsubstituted, it includes both unsubstituted and substituted groups. For example, an "alkyl group" includes not only an alkyl group that does not have a substituent (an unsubstituted alkyl group) but also an alkyl group that has a substituent (a substituted alkyl group).
Chemical structures may be written as simplified structures in which hydrogen atoms are omitted.
Combinations of two or more preferred aspects are more preferred aspects.

When the amino acids and peptides disclosed herein 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.

Hydroxy-protecting groups include, for example, 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.

Examples of the protecting group for the amino 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, an 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 carboxy-protecting group include the above-mentioned hydroxy-protecting groups, trityl group, and the like.
Examples of the carbonyl-protecting group include cyclic acetals (eg, 1,3-dioxane), non-cyclic acetals (eg, di(alkyl having 1 to 6 carbon atoms)acetals), and the like.
An example of the protecting group for the amide group is a trityl group.
Examples of the protecting group for the guanidyl group include a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group, a 2,3,4,5,6-pentamethylbenzenesulfonyl group, a tosyl group, and a nitro group.
Examples of the protecting group for a mercapto group (thiol group) include a trityl group, a 4-methylbenzyl group, an acetylaminomethyl group, a t-butyl group, and a t-butylthio group.

The above-mentioned protecting groups can be removed in accordance with known methods, such as those described in Protective Groups in Organic Synthesis, John Wiley and Sons (1980). Methods using acids, bases, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylsilyl halides, and reduction methods can be used.

"Amino acid" refers to α, β, or γ amino acids, and is not limited to naturally occurring amino acids, but may also be non-naturally occurring amino acids. It may also be an amino acid analogue such as a hydroxycarboxylic acid.

<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; R ₁ , R ₂ and R ₃ each independently represent 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 R ₁ , R ₂ and R ₃ may form a ring; and R ₄ represents a protecting group for an amino group.

The aliphatic hydrocarbon group may be saturated or unsaturated and may be linear, branched, or cyclic, provided that at least one of R ₁ , R ₂ , and R ₃ has a branched structure.
Preferably, at least one of R ₁ , R ₂ and R ₃ is a butyl group having a branched structure which may have a substituent not containing an aromatic ring.

The aliphatic hydrocarbon group is preferably an alkyl group or an alkenyl group, and more preferably an alkyl group.
The alkyl group is preferably an alkyl group having a carbon number (also referred to as the "number of carbon atoms") of 1 to 10. The number of carbon atoms in the alkyl group is more preferably 1 to 6, more preferably 1 to 4, and still more preferably 1 or 2. 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 being 1-propenyl.

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 not containing an aromatic ring is not particularly limited, but may be a cyclic aliphatic hydrocarbon group, a cyclic aliphatic 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 in the alkoxy group is more preferably 1 to 6, more preferably 1 to 4, and still more preferably 1 or 2. Specific examples include methoxy, ethoxy, and propoxy.

Specific examples of particularly preferred substituents that do not contain an aromatic ring include cyclohexyl, tetrahydropyranyl, or alkoxy having 1 to 6 carbon atoms.

Examples of the amino-protecting group represented by R ₄ 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 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 amino-protecting group represented by R ₄ include 9-fluorenylmethyloxycarbonyl or benzyloxycarbonyl.

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

Specific examples of compounds represented by formula (1) or the following formula (2) are shown below.

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

<Method for producing the compound represented by formula (1) or formula (2)>
The method for producing the 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 the compounds represented by formula (1) or formula (2) may be commercially available compounds or may be produced according to known methods or methods equivalent thereto.
If necessary, the produced compound represented by formula (1) or formula (2) may be purified by a known purification method, such as a method of isolating and purifying by recrystallization, column chromatography, or the like, or a method of purifying by reprecipitation by changing the solution temperature or solution composition, or the like.

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

wherein R ₁ -R ₄ and n are as defined herein, DCC denotes N,N-dicyclohexylcarbodiimide, DMAP denotes 4-dimethylaminopyridine, and rt denotes room temperature.

<Method of 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 terminus of an amino acid or peptide. The amino acid or peptide may be bound to a solid support such as a resin.

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,
a peptide chain elongation step of condensing the compound represented by formula (1) or (2) with a C-terminal protected amino acid or a C-terminal protected peptide; and
a precipitation step of precipitating the N-terminal protected and C-terminal protected peptide obtained in the peptide chain elongation step;
It is preferred that the compound further comprises:

In the present invention, after the above precipitation step,
deprotecting the N-terminus of the obtained N-terminus-protected C-terminus-protected peptide;
a step of condensing an N-terminal protected amino acid or an N-terminal protected peptide to the N-terminus of the obtained C-terminal protected peptide; and
It is preferred that the method further comprises the step of precipitating the obtained N-terminally protected C-terminally protected peptide at least once in this order.

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 are incorporated herein by reference.
Each of the above steps will now be described in detail.

<C-terminal protection process>
The method for producing a peptide according to the present disclosure preferably includes a C-terminal protection step of protecting a carboxy group or an amide group of an amino acid or peptide 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. When the C-terminal protecting group has a plurality of aliphatic hydrocarbon groups, the total number of carbon atoms therein is preferably 30 to 80, and more preferably 36 to 80. The C-terminal protecting group preferably has a ring structure, and more 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 formula (1) of WO 2020/175473 is preferred. As such a compound, WO 2020/175473 is referred to and incorporated herein by reference.
As the C-terminal protecting group, a condensed polycyclic aromatic hydrocarbon compound represented by formula (1) in WO 2020/175472 is also preferred. As such a compound, WO 2020/175472 is referred to and 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), and International Publication No. 2020/262259 (Japanese Patent Application No. 2019-122492 and a patent application based thereon) is 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 an N-terminal protected peptide is preferred, and an Fmoc-protected amino acid or an Fmoc-protected peptide is more preferred.
In addition, in the amino acid or peptide used in the above-mentioned C-terminal protection step, hydroxyl groups, amino groups, carbonyl groups, carboxyl groups, amide groups, imidazole groups, indole groups, guanidyl groups, mercapto groups and the like other than the C-terminal portion are preferably protected with protecting groups.

If the binding 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 affect the reaction, in the presence of a condensation additive (condensation activator), by adding a condensation agent, or in the presence of an acid catalyst.

When the binding 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 or react the condensing agent with a base in the presence of a condensation additive. As for the condensation activator, condensing agent, and acid catalyst, WO 2020/175473 and WO 2020/175473 are incorporated by reference and are incorporated herein by reference.

Condensing agents that are generally used in peptide synthesis can be used without limitation in the present disclosure. Examples of condensing agents 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-(benzotriazol-1 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), its hydrochloride (EDC.HCl), and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop).
Among these, DIC, EDC, EDC.HCl, DMT-MM, HBTU, HATU, or COMU is preferred.
The amount of the condensing agent used is preferably 1 to 10 molar equivalents, and more preferably 1 to 5 molar equivalents, relative to 1 molar equivalent of the substrate.

As the acid catalyst used in the condensation reaction, any acid catalyst generally used in peptide synthesis can be used without limitation, and examples thereof include methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, and the like.
Of these, methanesulfonic acid and p-toluenesulfonic acid are preferable.
The amount of the acid catalyst used is preferably 1 to 10 molar equivalents, and more preferably 1 to 5 molar equivalents, per 1 molar equivalent of the substrate.

In the above C-terminal protection step, it is preferable to add a condensation activator in order to promote the reaction and suppress side reactions such as racemization.
A condensation activator is a reagent that, in the presence of a condensation agent, converts an amino acid into a corresponding active ester, acid anhydride, or the like, thereby facilitating the formation of a peptide bond (amide bond).
As the condensation activating agent, any activating agent generally used in peptide synthesis can be used 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), and the like. Of these, 4-dimethylaminopyridine, HOBt, HOCt, HOAt, HOOBt, HOSu, HONb, or Oxyma is preferred.
The amount of the activator used is preferably more than 0 and 4.0 molar equivalents, and more preferably 0.1 to 1.5 molar equivalents, per 1 molar equivalent of the substrate.

As the solvent, a general organic solvent can be used in the reaction. Specific examples include halogenated hydrocarbons such as chloroform and dichloromethane; and non-polar organic solvents such as 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether. Two or more of these solvents may be mixed for use. In addition, the above-mentioned halogenated carbons and non-polar 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; and 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.

The N-terminally protected amino acid or N-terminally protected C-terminally protected peptide obtained in the above C-terminal protection step may be purified.
For example, the obtained N-terminally and C-terminally protected amino acid compound or N-terminally and C-terminally protected peptide is dissolved in a solvent (a reaction solvent, for example, THF), and the desired organic synthesis reaction is carried out, followed by isolating the obtained product. Then, the solvent in which the N-terminally and C-terminally protected amino acid compound or the N-terminally and C-terminally protected peptide is dissolved is changed (for example, the solvent composition is changed, or the type of the solvent is changed), and the product is reprecipitated.
Specifically, the reaction is carried out under conditions in which the N-terminal-protected, C-terminal-protected amino acid or the N-terminal-protected, C-terminal-protected peptide is dissolved. After the reaction, the solvent is removed by distillation and then replaced with a new solvent, or a polar solvent is added to the reaction system without removing the solvent, thereby precipitating the aggregates and removing the impurities.
As the replacement solvent or polar solvent, a polar organic solvent such as methanol, acetonitrile, water, etc. is used alone or in combination. That is, the reaction is carried out under conditions in which the N-terminal-protected, C-terminal-protected amino acid or the N-terminal-protected, C-terminal-protected peptide is dissolved, and after the reaction, for example, a halogenated solvent, THF, etc. is used for the solvent replacement for dissolution, and a polar organic solvent such as methanol, acetonitrile, water, etc. is used for precipitation.

<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 amino acid or peptide obtained in the above C-terminal protection step.
As the N-terminal protecting group, an amino protecting group generally used in technical fields such as peptide chemistry, which will be described later, can be used. In the present disclosure, a Boc group, a Cbz group, or an Fmoc group is preferred.

The deprotection conditions are appropriately selected depending on the type of 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.

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 mentioned above in the <C-terminal protection step> can be suitably used.

<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 suitably carried out using the above-mentioned condensation 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.

<Precipitation step>
The method for producing a peptide according to the present disclosure preferably further comprises 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 above-mentioned C-terminal protection step.
Specifically, after the previous reaction, the reaction solvent is not distilled off, and a polar solvent is added to the reaction system. In this case, THF is used as the nonpolar organic solvent, and acetonitrile is used as the polar 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. In the case of 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, and the target product can be efficiently purified.

<Chain extension>
It is preferable that the method for producing a peptide according to the present disclosure further comprises, after the above-mentioned precipitation step, one or more steps of deprotecting the N-terminus of the obtained N-terminus-protected C-terminus-protected peptide, 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 precipitating the obtained N-terminus-protected C-terminus-protected peptide, in this order.
By repeating the above three steps, the chain of the resulting peptide can be easily extended.
Each step in the above three steps can be carried out in the same manner as the corresponding step described above.

<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 the C-terminal protecting group.
In the C-terminal deprotection step, the C-terminal protecting group in the C-terminal protected peptide having the desired number of amino acid residues is removed to obtain the final target peptide.
A preferred method for removing the C-terminal protecting group is a deprotection method using an acidic compound.
For example, a method of adding an acid catalyst or a method of hydrogenation using a metal catalyst can be mentioned. Examples of the acid catalyst include trifluoroacetic acid (TFA), hydrochloric acid, trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), acetic acid, etc. 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 depending on the side chain protecting group of the amino acid to be elongated and the deprotection conditions.
For example, the concentration of TFA is preferably 50% by volume or less, more preferably 30% by volume or less, more preferably 10% by volume or less, more preferably 5% by volume or less, and particularly preferably 1% by volume or less, 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 more preferably 0.5% by volume.
The deprotection time is preferably 5 hours or less, more preferably 3 hours or less, and even more preferably 1 hour or less.

The final target peptide obtained by the method for producing a peptide according to the present disclosure can be isolated and purified according to a method commonly used in peptide chemistry. For example, the final target peptide can be isolated and purified by subjecting the reaction mixture to extraction and washing, crystallization, chromatography, etc.
The type of peptide produced by the method for producing a peptide according to the present disclosure is not particularly limited, but the number of amino acid residues of the peptide is preferably, for example, about several tens or less. 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, medicine, food, cosmetics, electronic materials, biosensors, and the like, like existing or unknown synthetic peptides and natural peptides.
In the method for producing a peptide according to the present disclosure, the above precipitation step can be omitted as appropriate to the extent that it does not affect the reaction in the subsequent step.

The following examples further illustrate the embodiments of the present invention. The materials, amounts used, ratios, processing details, and processing procedures shown in the following examples can be modified 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.

Unless otherwise specified, purification by column chromatography was performed using an automatic purification apparatus ISOLERA (Biotage) or a medium pressure liquid chromatograph YFLC-Wprep2XY.N (Yamazen Co., Ltd.).
Unless otherwise specified, the carrier used in silica gel column chromatography was SNAPPKP-Sil Cartridge (manufactured by Biotage) or Hi-Flash Column W001, W002, W003, W004 or W005 (manufactured by Yamazen Corporation).
The mixing 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.

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 ACQUITY UPLC (Waters, column: CSH C18 1.7 μm).

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

Synthesis of Compound of Example 1

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 eggplant flask, and a n-butyllithium hexane solution (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 an extraction operation was performed with ethyl acetate. The resulting organic layer was washed with saturated saline, dried using sodium sulfate, and 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.

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 at room temperature in four portions at one-hour intervals. After stirring at room temperature for one hour, 247 μL of acetic acid was added. After filtering out insoluble matter, 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 111 mg of compound (1-3) as a colorless transparent syrup.
Mass of the obtained compound (1-3): [M+Na] 563

Synthesis of Compound (1-4) In a 50 mL pressure-resistant reaction vessel, 111 mg of compound (1-3), 28 mg of 10% palladium-activated carbon (about 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 and stirred for 10 hours at room temperature under a hydrogen atmosphere. 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

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 using sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure to obtain a residue, which 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 compound of Example 1 obtained: [M+Na] 561

<Comparative data>
The compound of Example, Comparative Example 1, or Comparative Example 2 was used as the compound, and the aspartimide (Asi ₎ production rate and the purity of the target product were obtained by calculating the area value of ultra-performance liquid chromatography (UPLC) when H 2 N-Asp-Gly-Gly-Phe-OH was synthesized as the model sequence. The model sequence can be synthesized by the method of introducing a substituted benzyl compound to the C-terminus described in International Publication WO2023/106356.

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

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

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

<Synthesis of Protected Peptide (Four-Residue Peptide: H ₂ N-Asp(X)-Gly-Gly-Phe-OH>
Details of each abbreviation other than those mentioned above are given below.
Phe: phenylalanine residue Gly: glycine residue Asp(X): asparagine residue having a protecting group of the present invention

(Synthesis of Fmoc-Phe-O-TAG)

The raw material (1) (10.0 g, 10.78 mmol) synthesized according to the example in International Publication 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 the condensation reaction was completed, 80% aqueous methanol solution (550 mL) was added and stirred, and the precipitate was filtered and dried under reduced pressure to obtain Fmoc-Phe-O-TAG (14.0 g, 100% yield).

(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 elongate the peptide sequence.

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

Claims (19)

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 an amino terminal of a peptide:
In formula (1) and formula (2), n is 1 or 2; R ₁ , R ₂ and R ₃ each independently represent 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 R ₁ , R ₂ and R ₃ may form a ring; and R ₄ represents a protecting group for an amino group. the compound represented by formula (1) or (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 formula (1) or (2) with a C-terminal protected amino acid or a C-terminal protected peptide; and
a precipitation step of precipitating the N-terminal protected and C-terminal protected peptide obtained in the peptide chain elongation step;
The method for producing the peptide according to claim 1, further comprising: After the precipitation step,
deprotecting the N-terminus of the obtained N-terminus-protected C-terminus-protected peptide;
a step of condensing an N-terminal protected amino acid or an N-terminal protected peptide to the N-terminus of the obtained C-terminal protected peptide; and
The method for producing the peptide according to claim 2, further comprising the step of precipitating the obtained N-terminally protected and C-terminally protected peptide, in this order, one or more times. The method for producing the peptide according to claim 2 further comprises a C-terminal deprotection step of deprotecting the C-terminal protecting group, and the deprotection is carried out using a trifluoroacetic acid solution of 10% by volume or less. The method for producing a peptide according to claim 2, wherein the C-terminal protected amino acid or the C-terminal protected peptide has a C-terminal protecting group that has an aliphatic hydrocarbon group having 12 or more carbon atoms. 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-. 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 cyclic aliphatic hydrocarbon group, a cyclic aliphatic hydrocarbon group containing -O- in the ring, or an alkoxy group. 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 alkoxy having 1 to 6 carbon atoms. The method for producing the peptide according to any one of claims 1 to 5, wherein R ₄ is 9-fluorenylmethyloxycarbonyl or benzyloxycarbonyl. 6. The method for producing a peptide according to claim 1, wherein at least one of R ₁ , R ₂ and R ₃ is a butyl group having a branched structure which may have a substituent not containing an aromatic ring. The method for producing the peptide according to any one of claims 1 to 5, wherein n is 1. A compound represented by the following formula (1) or the following formula (2):
In formula (1) and formula (2), n is 1 or 2; R ₁ , R ₂ and R ₃ each independently represent 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 R ₁ , R ₂ and R ₃ may form a ring; and R ₄ represents a protecting group for an amino group. 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-. 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. 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. The compound according to any one of claims 12 to 15, wherein _R4 is 9-fluorenylmethyloxycarbonyl or benzyloxycarbonyl. The compound according to any one of claims 12 to 15, wherein at least one of R ₁ , R ₂ and R ₃ is a butyl group having a branched structure which may have a substituent not containing an aromatic ring. The compound according to any one of claims 12 to 15, wherein n is 1. A peptide synthesis reagent comprising a compound according to any one of claims 12 to 15.