Method for producing amino acids or peptides, reagents for forming protecting groups, and compounds

Abstract

The present invention addresses the problem of providing: a highly efficient peptide production method by which a protecting group of a side chain can be de-protected via a weak acid; a reagent for forming a protecting group, the reagent having an excellent de-protection property; and a compound. According to the present invention, a method for producing a peptide is provided, the method comprising: a peptide chain elongation step in which a first amino acid or peptide having a side chain protected by a first protecting group represented by formula (1) and a second amino acid or peptide are reacted to obtain a third amino acid or peptide; and a first de-protection step in which the first protecting group of the third amino acid or peptide is de-protected, wherein during the first de-protection step, de-protection is implemented using a de-protection solution with a trifluoroacetic acid content of 10 mass% or less. In the formula, R11 is an aryl group having a substituent, R21 and R22 are each independently a hydrogen atom or a substituent, and n is an integer of 1-6.

Description

[Technical Field]

[0001] This disclosure relates to methods for producing amino acids or peptides, reagents for forming protecting groups, and compounds. [Background technology]

[0002] Methods for producing peptides include solid-phase methods and liquid-phase methods. The liquid-phase method offers good reactivity, allowing for the purification of intermediate peptides after condensation reactions through extraction, washing, and isolation. However, depending on the peptide being synthesized, existing side-chain protecting groups may be insufficient. Conventional reagents for forming protecting groups include the compounds described in Patent Documents 1 and 2. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2-36197 [Patent Document 2] Japanese Patent Application Publication No. 4-360899 [Overview of the project] [Problems that the invention aims to solve]

[0004] However, the protecting groups disclosed in Patent Documents 1 and 2 were insufficient in their performance. One embodiment of this disclosure aims to solve the problem of providing a highly efficient method for producing amino acids or peptides in which the protecting group of the side chain can be deprotected with a weak acid. Another embodiment of this disclosure aims to solve the problem of providing a protecting group forming reagent with excellent deprotection properties and a compound protected therewith. [Means for solving the problem]

[0005] The means for solving the above problems include the following embodiments. <1> A peptide chain elongation step in which a first amino acid or peptide whose side chain is protected by a first protecting group represented by the following formula (1) is reacted with a second amino acid or peptide to obtain a third amino acid or peptide. The process includes a first deprotection step of deprotecting the primary protecting group of a tertiary amino acid or peptide, The first deprotection step involves deprotection using a deprotection solution containing 10% by mass or less of trifluoroacetic acid. A method for producing peptides. [ka] In the formula, R 11 R is an aryl group having a substituent, 21 and R 22 Each of these is independently a hydrogen atom or substituent, n is an integer from 1 to 6, and the asterisk indicates a linkage. <2> The deprotection solution contains 2% by mass or less of trifluoroacetic acid. <1> A method for producing the peptide described above. <3> A C-terminal protection step in which a C-terminal protective agent is used to protect the carboxyl group or amide group of an N-terminal protected amino acid or peptide. An N-terminal deprotection step is performed to deprotect the N-terminal protecting group of the N-terminal C-terminal protected amino acid or peptide obtained in the above C-terminal protection step. A peptide chain extension step is performed in which an N-terminally protected amino acid or peptide is condensed to the N-terminus of the C-terminally protected amino acid or peptide obtained in the above N-terminal deprotection step, and The method further includes a C-terminal deprotection step of deprotecting the C-terminal protecting group, Either the C-terminally protected amino acid or peptide, or the N-terminally protected amino acid or peptide, is a primary amino acid or peptide protected by the primary protecting group described above. <1> or <2> A method for producing the peptide described above. <4> R 11 The substituents on the aryl group are electron-donating groups, and if there are multiple substituents, they may be the same or different. <1> ~ <3> A method for producing the peptide described in any one of the following. <5> The electron-donating group is an amino group, an alkoxyalkyl group, an alkoxy group, or an alkyl group, and these may further have substituents. The method for producing a peptide according to any one of <1> to <4>. <6> The electron-donating group is an alkoxy group having 1 to 10 carbon atoms. The method for producing a peptide according to any one of <1> to <4>. <7> The aryl group is a phenyl group or a naphthyl group. The method for producing a peptide according to any one of <1> to <6>. <8> The first protecting group is a protecting group for the guanidino group of arginine. The method for producing a peptide according to any one of <1> to <7>. <9> A protecting step of reacting a first amino acid or peptide with a protecting group-forming reagent represented by the following formula (2) to obtain a first protected amino acid or peptide in which the side chain is protected by a first protecting group. A method for producing an amino acid or peptide comprising

Chemical formula

Chemical formula

[0006] According to one embodiment of the present invention, a highly efficient method for producing peptides can be provided in which the protecting group of the side chain can be deprotected with a weak acid. Furthermore, according to another embodiment of the present invention, a protecting group forming reagent with excellent deprotection properties and a compound protected therewith can be provided. [Modes for carrying out the invention]

[0007] The contents of this disclosure are described in detail below. The descriptions of the constituent elements described below may be based on representative embodiments of this disclosure, but this disclosure is not limited to such embodiments. In this specification, unless otherwise specified, the following terms have the meanings set forth below. A numerical range represented using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively. In numerical ranges described stepwise within this specification, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described stepwise. Furthermore, in numerical ranges described within this specification, the upper or lower limit of that range may be replaced with the values ​​shown in the examples. The term "process" includes not only independent processes, but also any process that cannot be clearly distinguished from other processes, as long as its intended purpose is achieved. In the notation of a group (atomic group), when substitution and unsubstituted are not indicated, the notation includes both those with and without substituents. For example, "alkyl group" includes not only alkyl groups without substituents (unsubstituted alkyl groups) but also alkyl groups with substituents (substituted alkyl groups). Chemical structural formulas are sometimes written using simplified structural formulas that omit hydrogen atoms. "Mass%" and "Weight%" are synonymous, and "Parts of Mass" and "Parts of Weight" are synonymous. A combination of two or more preferred embodiments is a more preferred embodiment.

[0008] The alkyl group may be linear or branched, and may be substituted with halogen atoms or the like. The alkyl group is preferably an alkyl group having 1 to 10 carbon atoms (also called the number of carbon atoms). The number of carbon atoms of the alkyl group is more preferably 1 to 6, more preferably 1 to 4, and 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. A specific example is 1-propenyl. The "aryl group" is preferably an aryl group having 6 to 14 carbon atoms, and examples include phenyl group, 1-naphthyl group, 2-naphthyl group, biphenylyl group, and 2-anthuryl group. Among these, an aryl group having 6 to 10 carbon atoms is more preferred, and the phenyl group is particularly preferred. Examples of "silyl groups" include trimethylsilyl, triethylsilyl, dimethylphenylsilyl, tert-butyldimethylsilyl, and tert-butyldiethylsilyl. Examples of "halogen atoms" include fluorine, chlorine, bromine, and iodine atoms. As the "alkoxy group," an alkoxy group having 1 to 10 carbon atoms is preferred. The number of carbon atoms in the alkyl group is more preferably 1 to 6, more preferably 1 to 4, and more preferably 1 or 2. Specific examples include methoxy, ethoxy, and propoxy. As for the "arylalkyl group," arylalkyl groups having 7 to 20 carbon atoms are preferred. The number of carbon atoms is preferably 7 to 12. A specific example is benzyl. As for the "heteroarylalkyl group," a heteroarylalkyl group having 6 to 20 carbon atoms is preferred. The number of carbon atoms is preferably 6 to 12. As the "acyl group," an acyl group having 1 to 6 carbon atoms is preferred. Specific examples include acetyl and propionyl. The "arylalkylcarbonyl group" is preferably an arylalkylcarbonyl group having 7 to 10 carbon atoms. A specific example is benzylcarbonyl. As for the "alkoxycarbonyl group," an alkoxycarbonyl group having 1 to 6 carbon atoms is preferred. Specific examples include methoxycarbonyl, ethoxycarbonyl, and Boc groups. The Boc group refers to the tert-butoxycarbonyl group. As the "arylalkyloxycarbonyl group," an arylalkyloxycarbonyl group having 7 to 20 carbon atoms is preferred. The number of carbon atoms is preferably 8 to 20, and more preferably 8 to 14. Specific examples include benzyloxycarbonyl (hereinafter also referred to as the Cbz group or Z group) and the Fmoc group. The Fmoc group refers to the 9-fluorenylmethoxycarbonyl group.

[0009] If the amino acids and peptides relating to this disclosure have a hydroxyl group, an amino group (-NH2 or -NHR, where R represents an alkyl or aryl group), a carboxyl group, a carbonyl group, an amide group, a guanidyl group, a mercapto group, etc., these groups may be protected, and the target compound can be obtained by removing the protecting group as needed after the reaction. Examples of protecting groups for hydroxyl groups include alkyl groups, aryl groups, trityl groups, arylalkyl groups having 7 to 10 carbon atoms, formyl groups, acyl groups having 1 to 6 carbon atoms, benzoyl groups, arylalkylcarbonyl groups having 7 to 10 carbon atoms, 2-tetrahydropyranyl groups, 2-tetrahydrofuranyl groups, silyl groups, and alkenyl groups having 2 to 6 carbon atoms. These groups may be substituted with one to three 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.

[0010] Examples of protecting groups for amino groups include formyl, acyl groups having 1 to 6 carbon atoms, alkoxycarbonyl groups having 1 to 6 carbon atoms, benzoyl, arylalkylcarbonyl groups having 7 to 10 carbon atoms, arylalkyloxycarbonyl groups having 7 to 14 carbon atoms, trityl, monomethoxytrityl, 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl, phthaloyl, N,N-dimethylaminomethylene, silyl, and alkenyl groups having 2 to 6 carbon atoms. These groups may be substituted with 1 to 3 substituents selected from the group consisting of halogen atoms, alkoxy groups having 1 to 6 carbon atoms, and nitro groups. Examples of protecting groups for carboxyl groups include the hydroxyl group protecting group and the trityl group. Examples of protecting groups for amide groups include the trityl group. Examples of protecting groups for carbonyl groups include cyclic acetals (e.g., 1,3-dioxane) and acyclic acetals (e.g., di(1-6 C1 alkyl) acetals). Examples of protecting groups for the guanidino group include the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group, the 2,3,4,5,6-pentamethylbenzenesulfonyl group, the tosyl group, and the nitro group. Examples of protecting groups for mercapto groups (thiol groups) include trityl group, 4-methylbenzyl group, acetylaminomethyl group, t-butyl group, and t-butylthio group.

[0011] The above-mentioned protecting group can be removed by known methods, such as those described in Protective Groups in Organic Synthesis, published by John Wiley and Sons (1980). Methods using acids, bases, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylsilyl halide, or reduction methods are used.

[0012] "Amino acids" refer to α, β, or γ amino acids, and are not limited to naturally occurring amino acids; they may also be unnatural amino acids such as N-methyl amino acids. They may also be amino acid analogs such as hydroxycarboxylic acids.

[0013] (Reagents for forming protecting groups, compounds containing arginine or arginine residues) The protective group-forming reagent relating to this disclosure is represented by the following formula (2), and is preferably represented by the following formula (2a). Such a compound is used in the method for producing amino acids or peptides of the present invention. [ka]

[0014] In equations (2) and (2a), R 11 R is an aryl group which may have substituents, 21 and R 22 Each of these is independently a hydrogen atom or a substituent, and n is an integer from 1 to 6. R 11The substituents on the aryl group are preferably electron-donating groups, such as amino groups, alkoxyalkyl groups, alkoxy groups, or alkyl groups. These substituents may have substituents, and if there are multiple substituents, they may be the same or different. The electron-donating group is preferably an alkoxy group, more preferably an alkoxy group having 1 to 10 carbon atoms, and even more preferably an alkoxy group having 1 to 6 carbon atoms. The electron-donating group is preferably one that does not have substituents. The aryl group is preferably a phenyl group or a naphthyl group. The substituents protected by the protecting group-forming reagent are preferably amino groups, imino groups, or guanidino groups (amino groups and imino groups), with the guanidino group of arginine being more preferred.

[0015] R 12 These may be an aryl group having substituents, a heteroaliphatic ring group having substituents, or a heteroaryl group having substituents, and they may be linked via an oxygen atom (O) or a sulfur atom (S). The aryl group is preferably a phenyl group or a naphthyl group. As the heteroaliphatic ring group, a nitrogen-containing heteroaliphatic ring group is preferred, and a succinimide group is more preferred. As the heteroaryl group, a monocyclic nitrogen-containing heteroaryl group is preferred, and imidazolyl, pyridyl, pyrazolyl, and triazolyl are more preferred. These may be linked by nitrogen atoms. 12 The substituents of the aryl group, heteroaliphatic ring group, or heteroaryl group are preferably electron-withdrawing groups, and if there are multiple substituents, they may be the same or different. The electron-withdrawing groups are preferably carbonyl groups, nitro groups, or halogen atoms, and more preferably nitro groups or fluoro groups.

[0016] R 21 and R 22 Examples of substituents include halogen atoms and alkyl groups. Specific examples of protecting group-forming reagents are described below. In the formulas, R represents an electron-donating group, and m represents an integer from 0 to 5. [ka]

[0017] The compound protected by the above-mentioned protecting group forming reagent is preferably a compound containing arginine or an arginine residue (preferably a peptide). Specifically, the protected compound is arginine or a compound containing an arginine residue having a protecting group represented by the following formula (1), and may further have an Fmoc protecting group (9-fluorenylmethoxycarbonyl group). The protecting group represented by the following formula (1) is preferably the protecting group represented by the following formula (1a). [ka] In the above formula, R 11 R is an aryl group which may have substituents, 21 and R 22 Each of the following is independently a hydrogen atom or a substituent, n is an integer from 1 to 6, and the asterisk in the formula indicates a linkage. In the above formula, R 11 , R 21 and R 22 The preferred values ​​are the same as those in formula (2), and the same applies to the following descriptions.

[0018] (Method for producing peptides) The method for producing the peptide according to this disclosure uses a protecting group forming reagent represented by formula (2). The step of using the protecting group forming reagent represented by formula (2) is preferably a side chain protection step that protects the side chain of the amino acid or peptide. Arginine is preferred as the amino acid, and a peptide containing an arginine residue is preferred as the peptide. The protecting group-forming reagent represented by formula (2) can be used as a protecting group for amino acids, and is more preferably used as a protecting group for the guanidino group, amino group, or imino group that are the side chains of amino acids, and is even more preferably used as a protecting group for the guanidino group. From the viewpoint of ease of peptide synthesis and yield, the method for producing peptides according to this disclosure more preferably further includes, in addition to the side chain protection step, an N-terminal deprotection step for deprotecting the N-terminus of the N-terminal protected C-terminal amino acid or N-terminal protected C-terminal peptide obtained through the side chain protection step, and a peptide chain elongation step for condensing the N-terminal protected amino acid or 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. It is more preferable to further include a precipitation step in which the N-terminal and C-terminal protected peptides obtained in the peptide chain elongation step are precipitated. It is more preferable to further include, one or more steps in this order after the precipitation step, a step of deprotecting the N-terminus of the obtained N-terminal and C-terminal 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 a step of precipitating the obtained N-terminal and C-terminal protected peptide.

[0019] In a preferred embodiment, the method for producing the peptide according to the Disclosure is: A C-terminal protection step in which a C-terminal protective agent is used to protect the carboxyl group or amide group of an N-terminal protected amino acid or peptide. An N-terminal deprotection step is performed to deprotect the N-terminal protecting group of the N-terminal C-terminal protected amino acid or peptide obtained in the above C-terminal protection step. A peptide chain extension step is performed in which an N-terminally protected amino acid or peptide is condensed to the N-terminus of the C-terminally protected amino acid or peptide obtained in the above N-terminal deprotection step, and The method further includes a C-terminal deprotection step of deprotecting the C-terminal protecting group, Either the C-terminally protected amino acid or peptide, or the N-terminally protected amino acid or peptide, is a primary amino acid or peptide protected by the primary protecting group described above.

[0020] Furthermore, an N-terminally protected amino acid or N-terminally protected peptide is an amino acid or peptide in which only the N-terminus is protected, while an N-terminal or C-terminally protected amino acid or peptide is an amino acid or peptide in which both the N-terminus and C-terminus are protected. The peptide bond formation reaction (condensation reaction) between the amino group and the carboxyl group in each step, and the deprotection of the protecting group, can be carried out by known methods. For example, International Publication No. 2020 / 175473 is referenced and incorporated herein by reference. The following provides a detailed explanation of each of the processes mentioned above.

[0021] <Side chain protection process> The present disclosure preferably includes a side-chain protection step in which the amino group of the side chain of the amino acid or peptide is protected with a protecting group forming reagent represented by formula (2) above. That is, the present invention provides a method for producing an amino acid or peptide, which includes a protection step in which a primary amino acid or peptide is reacted with a protecting group forming reagent represented by formula (2) above to obtain a primary protected amino acid or peptide in which the side chain is protected with a primary protecting group. The amino acid to be protected is preferably arginine, but the side chain, amino group, or carboxyl group of other amino acids may also be protected. Furthermore, if the side chain of the peptide is protected, it is preferable that the side chain of the portion derived from arginine is protected. In the side-chain protection step, a primary amino acid or peptide is obtained in which the side chain is protected with a primary protecting group represented by the following formula (1). The present invention relates to a method for producing an amino acid or peptide, which includes such a protection step. [ka] The side-chain protection step is preferably carried out in a solvent that does not affect the reaction and under basic conditions. As solvents, general organic solvents can be used in the reaction. Specifically, examples include halogenated hydrocarbons such as chloroform and dichloromethane; and nonpolar organic solvents such as 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether. Two or more of these solvents may be used in mixtures. In addition, 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 (NMP); and sulfoxides such as dimethyl sulfoxide may be used in mixtures with the above halogenated hydrocarbons or nonpolar organic solvents. NMP is particularly preferred.

[0022] Examples of bases include tertiary amines such as triethylamine and diisopropylethylamine, 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). The amount of base used is preferably more than 1 molar equivalent and 20 molar equivalents or less, and more preferably 2 to 10 molar equivalents, relative to 1 molar equivalent of the protecting group forming reagent shown in formula (2) above. Furthermore, when protecting the guanidino group of arginine, it is preferable to protect the amino and carboxyl groups of arginine with Alloc (allyloxycarbonyl group) and Allyl (allyl group), etc.

[0023] <Deprotection process of side chains> The present invention provides a peptide production method comprising a peptide chain elongation step in which a first amino acid or peptide protected by a first protecting group is reacted with a second amino acid or peptide to obtain a third amino acid or peptide. The process includes a first deprotection step of deprotecting the primary protecting group of a tertiary amino acid or peptide, The first deprotection step involves deprotection using a deprotection solution containing 10% by mass or less of trifluoroacetic acid (TFA). The TFA content of the deprotection solution is preferably 7% by mass or less, preferably 5% by mass or less, preferably 2% by mass or less, preferably 1% by mass or less, preferably 0.5% by mass or less, preferably 0.1% by mass or less. The TFA content of the deprotection solution is preferably 0.01% by mass or more, preferably 0.1% by mass or more. If the TFA content is high, the resulting peptide will decompose, by-products will be generated, and the production efficiency will decrease. If it is low, the deprotection will take time and the production efficiency will decrease. In the present invention, it is possible to achieve both deprotection with a weak acid and blocking of the side chain (especially the guanidino group).

[0024] <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 peptide (either the above-mentioned first amino acid or peptide or the second amino acid or peptide) with a C-terminal protecting group. As the C-terminal protecting group, a compound having an aliphatic hydrocarbon group with 12 or more carbon atoms is 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 their carbon atoms is preferably 30 to 80, and more preferably 36 to 80. The C-terminal protecting group preferably has a ring structure, preferably 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 International Publication No. 2020 / 175473 is preferable. 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 formula (1) of International Publication No. 2020 / 175472 is also preferable. Such a compound is referred to International Publication No. 2020 / 175472 and incorporated herein by reference. As the C-terminal protecting group, a compound represented by formula (1) of International Publication No. 2020 / 262259 may also be used. International Publication No. 2020 / 262259 is referred to and incorporated herein by reference. There are no particular restrictions on the amino acids or peptides used in the above C-terminal protection step, but N-terminal protected amino acids or N-terminal protected peptides are preferred, and Fmoc protected amino acids or Fmoc protected peptides are more preferred. Furthermore, it is preferable that hydroxyl groups, amino groups, carbonyl groups, carboxyl groups, amide groups, imidazole groups, indole groups, guanidyl groups, mercapto groups, etc., other than the C-terminal portion of the amino acid or peptide used in the above C-terminal protection step are protected by protecting groups.

[0025] When the binding site of the C-terminal protecting group is -OH or -SH, it is preferable to add the condensing agent in a solvent that does not affect the reaction, in the presence of a condensation additive (condensation activator), or to carry out the reaction in an acid catalyst.

[0026] The binding site for the C-terminal protecting group is -NHR 18 (R 18 When (a) is a hydrogen atom, alkyl group, arylalkyl group, or heteroarylalkyl group, it is preferable to add the condensing agent in the presence of a condensation additive (condensation activator) or to react the condensing agent with a base. For solvents, condensation additives (condensation activators), condensing agents, acid catalysts, and bases, International Publication Nos. 2020 / 175472, 2020 / 175473, and 2020 / 262259 are referenced and incorporated herein by reference.

[0027] As the solvent, a nonpolar organic solvent is preferred, and THF is more preferred. Alternatively, solvents described in Organic Process Research & Development, 2017, 21, 3, 365-369 may be used. As a coupling agent, coupling agents commonly used in peptide synthesis can be used without limitation in this disclosure, for example, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorphonium chloride (DMT-MM), O-(benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU(6-Cl)), O-(benzotriazole-1-yl)- Examples include 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-(6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), its hydrochloride salt (EDC·HCl), and hexafluorophosphate (benzotriazole-1-yloxy)tripyrrolidinophosphonium (PyBop). Among these, DIC, EDC, EDC·HCl, DMT-MM, HBTU, HATU, or COMU are preferred. The amount of condensing agent used is preferably 1 to 10 molar equivalents, and more preferably 1 to 5 molar equivalents, per 1 molar equivalent of the substrate.

[0028] As the acid catalyst used in the condensation reaction, any acid catalyst commonly used in peptide synthesis can be used without restriction. Examples include methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, and acetic acid. Among these, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, and acetic acid are preferred. The amount of acid catalyst used is preferably 1 to 10 molar equivalents, and more preferably 0.01 to 5 molar equivalents, per 1 molar equivalent of substrate.

[0029] In the C-terminus protection step described above, it is preferable to add a condensation activator to promote the reaction and suppress side reactions such as racemization. A condensation activator is a reagent that, when used in conjunction with a condensation agent, facilitates the formation of peptide bonds (amide bonds) by guiding amino acids to their corresponding active esters, symmetric acid anhydrides, etc. As condensation activators, any activators commonly used in peptide synthesis can be used without limitation. Examples 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-benzotriazodine-4(3H)-one (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), pentafluorophenol, and ethyl (hydroxyimino)cyanoacetate (Oxyma). Among these, 4-dimethylaminopyridine, HOBt, HOCt, HOAt, HOOBT, HOSu, HONb, or Oxyma are preferred. The amount of condensation activator used is preferably greater than 0 molar equivalents and 4.0 molar equivalents, and more preferably between 0.1 and 1.5 molar equivalents, relative to 1 molar equivalent of the substrate.

[0030] As the solvent, the solvent described above can be suitably used in the dissolution step described above. 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.

[0031] Further, the N-terminal C-terminal protected amino acid or N-terminal C-terminal protected peptide obtained by the above C-terminal protection step may be purified. For example, the obtained N-terminal C-terminal protected amino acid compound or N-terminal C-terminal protected peptide is dissolved in a solvent (reaction solvent, such as THF), and the product obtained after performing a desired organic synthesis reaction is isolated. Then, the solvent in which the N-terminal C-terminal protected amino acid compound or N-terminal C-terminal protected peptide is dissolved is changed (e.g., change in solvent composition, change in solvent type), and re-precipitation is performed. Specifically, the reaction is carried out under conditions such that the N-terminal C-terminal protected amino acid or N-terminal C-terminal protected peptide dissolves. After the reaction, the solvent is distilled off, and then the solvent is replaced, or a polar solvent is added to the reaction system without distilling off the solvent, thereby precipitating the aggregate and eliminating impurities. As the substitution solvent or polar solvent, polar organic solvents such as methanol, acetonitrile, and water are used alone or in combination. 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.

[0032] <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 amino groups 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.

[0033] 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.

[0034] Examples of bases 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 solvent described above can be suitably used in the dissolution step described above.

[0035] <Peptide chain elongation process> The method for producing peptides according to this disclosure preferably includes a peptide chain elongation step in which an N-terminally protected amino acid or peptide is condensed onto the N-terminus of the C-terminally protected amino acid or peptide obtained in the above N-terminal deprotection step. Either the C-terminally protected amino acid or peptide, or the N-terminally protected amino acid or peptide, has its side chain protected by the above first protecting group. The first protecting group is then deprotected at an appropriate time. The peptide chain elongation step described above is suitably carried out under peptide synthesis conditions commonly used in the field of peptide chemistry, using the aforementioned condensing agents, condensation additives, etc. There are no particular restrictions on the N-terminally protected amino acid or N-terminally protected peptide, but Fmoc-protected amino acids or Fmoc-protected peptides are preferred.

[0036] <Precipitation process> The method for producing the peptide according to this disclosure preferably further includes a precipitation step of precipitating the N-terminally protected C-terminally protected peptide obtained in the peptide chain elongation step. The precipitation step can be carried out in the same manner as the purification (reprecipitation) step of the C-terminus protection step described above. Specifically, without distilling off the reaction solvent after the reaction in the previous stage, 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 the case of this usage 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.

[0037] <Chain elongation> The method for producing a peptide according to the present disclosure preferably further includes, after the precipitation step described above, a step of deprotecting the N-terminal of the obtained N-terminal protected C-terminal protected peptide, a step of condensing an N-terminal protected amino acid or an N-terminal protected peptide to the N-terminal of the obtained C-terminal protected peptide, and a step of precipitating the obtained N-terminal protected C-terminal protected peptide in this order one or more times. By repeating the above three steps, the chain elongation of the obtained peptide can be easily performed. Each step in the above three steps can be performed in the same manner as the corresponding steps described above.

[0038] <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, the peptide as the final target product can be obtained by removing the C-terminal protecting group in the C-terminal protected peptide having the desired number of amino acid residues. As a method for removing the C-terminal protecting group, a deprotection method using an acidic compound is preferably mentioned. For example, methods include adding an acid catalyst or hydrogenation using a metal catalyst. Examples of acid catalysts include trifluoroacetic acid (TFA), hydrochloric acid, trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and acetic acid. For peptides that do not decompose with strong acids, TFA is preferred, while for peptides that do decompose with strong acids, TFE, HFIP, or acetic acid is preferred. The acid concentration can be appropriately selected depending on the side chain protecting group of the elongated amino acid and the deprotection conditions. For example, the concentration of TFA is preferably 50% by volume or less, more preferably 30% by volume or less, more preferably 10% by volume or less, more preferably 5% by volume or less, and particularly preferably 1% by volume or less, relative to the total volume of the solvent used. The lower limit is preferably 0.01% by volume, more preferably 0.1% by volume, and more preferably 0.5% by volume. The deprotection time is preferably 5 hours or less, more preferably 3 hours or less, and even more preferably 1 hour or less.

[0039] In this disclosure, deprotection of the C-terminal protecting group and side-chain protecting group is possible even under weak acid conditions, and side reactions of the resulting peptide can be suppressed. Peptides suitable for deprotection of the C-terminal protecting group under weakly acidic conditions (i.e., acid-sensitive peptides) include, for example, peptides having an N-alkylamide structure. From the viewpoint of suppressing side reactions of the resulting peptide and ensuring long-term stability, the peptide production method according to this disclosure is preferably applied to a method for producing acid-sensitive peptides, and more preferably to a method for producing peptides having an N-alkylamide structure.

[0040] <Peptide> The final target peptide obtained by the peptide production method according to this disclosure can be isolated and purified according to methods commonly used in peptide chemistry. For example, the final target peptide can be isolated and purified from the reaction mixture by extraction washing, crystallization, chromatography, etc. The type of peptide produced by the peptide production method according to this disclosure is not particularly limited, but it is preferable that the number of amino acid residues of the peptide is, for example, several tens or less (specifically, 50 or less, 40 or less, 30 or less, or 20 or less). The peptide obtained by the peptide production method according to this disclosure can be used in various fields, such as pharmaceuticals, food, cosmetics, electronic materials, and biosensors, as well as existing or unknown synthetic peptides and natural peptides. The peptide production method according to this disclosure may also be used to appropriately omit the precipitation step, provided that it does not affect the reaction in the next step. [Examples]

[0041] The embodiments of the present invention will be described in more detail below with reference to examples. The materials, amounts used, proportions, processing content, 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, "parts" and "%" are based on mass. Room temperature means 25°C. In the chemical formula, Me represents a methyl group.

[0042] Unless otherwise specified, purification by column chromatography was performed using the ISOLERA automated purification system (Biotage) or the YFLC-Wprep2XY.N medium-pressure liquid chromatograph (Yamazen Corporation). Unless otherwise specified, the silica gel column chromatography used was SNAPKP-Sil Cartridge (Biotage), with high-flash columns W001, W002, W003, W004, or W005 (Yamazen Corporation). The mixing ratio of the eluent used in column chromatography is expressed as a volume ratio. For example, "Hexane:ethyl acetate gradient elution = 50:50 to 0:100" means that the eluent is ultimately changed from 50% hexane / 50% ethyl acetate to 0% hexane / 100% ethyl acetate. Furthermore, 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 was changed from 50% hexane / 50% ethyl acetate to 0% hexane / 100% ethyl acetate, and finally changed to 20% methanol / 80% ethyl acetate.

[0043] MS spectra were measured using the ACQUITY SQD LC / MS System (Waters, ionization method: ESI (ElectroSpray Ionization)).

[0044] NMR spectra were measured using a Bruker AV300 (Bruker, 300 MHz) or Bruker AV400 (Bruker, 400 MHz) with tetramethylsilane as the internal reference, and the total δ values ​​were expressed in ppm. HPLC purity was measured using ACQUITY UPLC (Waters, column: CSH C18 1.7μm).

[0045] <Examples> <Synthesis of Protecting Group Forming Reagent (1)> [ka]

[0046] <Synthesis of intermediate (1-1)> To a solution of methyl 4-methoxy-2-methylbenzoate (5.00 g, 27.2 mmol) in tetrahydrofuran (25 ml), a solution of 70% bis(2-methoxyethoxy)aluminum sodium hydride (20.2 g, 122.5 mmol) in toluene was added dropwise under an ice bath, and the mixture was stirred at room temperature for 30 minutes. After adding acetone (3 ml) to the reaction mixture, 20% aqueous potassium sodium tartrate (50 mL) and ethyl acetate (50 mL) were added, and the mixture was stirred for 10 minutes. After separating the organic layer, the mixture was concentrated under reduced pressure to obtain 4.81 g of a colorless oily intermediate (1-1).

[0047] <Synthesis of Protecting Group Forming Reagent (1)> To a solution of intermediate (1-1) (4.20 g, 27.6 mmol) in tetrahydrofuran (42 ml), 1,1'-carbonyldiimidazole (4.70 g, 29.0 mmol) was added under an ice bath and stirred at room temperature for 30 minutes. After concentrating the reaction mixture, a hexane / ethyl acetate mixture (1 / 1) (42 mL) and water (42 mL) were added and stirred for 5 minutes. The organic layer was sequentially washed with water (42 mL) and saturated saline (10 mL), then dried over sodium sulfate and filtered. By concentrating the filtrate under reduced pressure, 6.50 g of the protecting group forming reagent (1-1) was obtained (2 steps, 86%). 1 H-NMR (DMSOd 6 ,400MHz)δ=8.13-8.10(1H,m),7.40(1H,t,J=1.5Hz)7.33(1H,d,J=8.4Hz),7.05(1H,dd,1.5,0 .9Hz),6.79(1H,d,J=2.5Hz),6.75(1H,dd,8.4,2.8Hz),5.40(2H,s),3.81(3H,s),2.41(3H,s)

[0048] <Synthesis of protecting group forming reagents (2), (3), and (4)> [ka]

[0049] <Reagent for protecting group formation (2)> The reagent was synthesized in the same manner as the protecting group forming reagent (1), except that intermediate (1-1) was replaced with 4-methoxybenzyl alcohol.

[0050] <Reagent for forming protecting groups (3)> The reagent was synthesized in the same manner as the protecting group forming reagent (1), except that intermediate (1-1) was replaced with 2-methoxybenzyl alcohol.

[0051] <Reagent for protecting group formation (4)> The reagent was synthesized in the same manner as the protecting group forming reagent (1), except that intermediate (1-1) was replaced with 2,4-dimethoxybenzyl alcohol.

[0052] <Synthesis of intermediates (2-3)> [ka]

[0053] <Synthesis of intermediate (2-1)> To a solution of Fmoc-Arg(Pbf)-OH (30.0 g, 46.2 mmol) in N-methylpyrrolidone (150 ml), allyl bromide (5.99 mL, 69.4 mmol) and potassium carbonate (19.2 g, 138 mmol) were added at room temperature, and the mixture was stirred for 5 hours. Water (150 mL) and ethyl acetate (300 mL) were added to the reaction mixture to separate the organic layer. The organic layer was then washed twice with water (100 mL) and saturated brine (50 mL). After drying with sodium sulfate and filtering, the filtrate was concentrated under reduced pressure to obtain 31.2 g of a pale yellow solid intermediate (2-1). ESI-MS(+)=689.4

[0054] <Synthesis of intermediate (2-2)> To a solution of intermediate (2-1) (31.2 g, 45.3 mmol) in tetrahydrofuran (150 ml), diazabicycloundecene (6.77 g, 45.3 mmol) was added at room temperature and the mixture was stirred for 30 minutes at room temperature. N-(allyloxycarbonyloxy)succinimide (9.0 mL, 45.3 mmol) was added to the reaction mixture and stirred for 1.5 hours, after which 1 mol / L hydrochloric acid (100 mL) and ethyl acetate (300 mL) were added. The organic layer was sequentially washed with water (100 mL), saturated sodium bicarbonate solution (100 mL), and saturated saline solution (10 mL), then dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain 18.3 g of intermediate (2-2) (73% in 2 steps). ESI-MS(+)=551.3

[0055] <Synthesis of intermediates (2-3)> To a solution of intermediate (2-2) (12.5 g, 22.7 mmol) in methylene chloride (57 ml), triisopropylsilane (9.3 mL, 45.4 mmol) and trifluoroacetic acid (57 mL) were added at room temperature, and the mixture was stirred for 1 hour. After concentrating the reaction mixture, methyl tert-butyl ether (100 mL) was added, and the mixture was suspended and stirred for 30 minutes. The solid was filtered off, and 8.6 g of the pale yellow intermediate (2-3) was obtained as trifluoroacetate. ESI-MS(+)=298.8

[0056] <Synthesis method of compound 1 in example> [ka]

[0057] <Synthesis of intermediates (2-4)> To a solution of intermediate (2-3) (7.80 g, 19.7 mmol) in N-methylpyrrolidone (39 ml), diazabicycloundecene (14.7 mL, 98.4 mmol) and protecting group forming reagent (1) (29.1 g, 118 mmol) were added at room temperature, and the mixture was stirred at room temperature for 4 hours. Saturated ammonium chloride aqueous solution (40 mL) and ethyl acetate (80 mL) were added to the reaction mixture, and the organic layer was washed twice with water (40 mL) and sequentially with saturated saline solution (10 mL). The mixture was dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain 5.03 g of intermediate (2-4).

[0058] <Synthesis of intermediates (2-5)> To a solution of intermediate (2-4) (3.00 g, 4.58 mmol) in methylene chloride (30 ml), triphenylsilane (1.1 mL, 9.16 mmol) and tetrakistriphenylphosphine palladium (0.265 g, 5 mol%) were added at room temperature and the mixture was stirred for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain 2.00 g of intermediate (2-5). ESI-MS(+)=531.1

[0059] <Synthesis of Example Compound (1)> To a mixed solution of intermediate (2-5) (2.00 g, 3.77 mmol) in tetrahydrofuran (19 ml) / water (19 mL), sodium carbonate (1.32 g, 12.4 mmol) and N-(9-fluorenylmethoxycarbonyloxy)succinimide (1.40 g, 4.15 mmol) were added at room temperature and the mixture was stirred for 1 hour. Saturated ammonium chloride aqueous solution (19 mL) and ethyl acetate (19 mL) were added to the reaction mixture, and the organic layer was washed with saturated brine (5 mL), dried over sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain 1.73 g of example compound (1). 1 H-NMR (DMSOd 6 ,400MHz)δ=11.58(1H,s),8.46-8.35(1H,m),7.88(2H,d,J=7.5Hz),7.70(2H,d,7.5Hz),7.44- 7.36(2H,m),7.35-7.25(3H,m),7.19(1H,d,J=8.3Hz),6.85-6.67(4H,m),5.15(2H,s),4.92(2H ,s),4.31-4.17(3H,m),3.90-3.81(1H,m),3.74(3H,s),3.71(3H,s),3.53-3.45(1H,m),3.45- 3.38(1H,m),2.30(3H,s),2.24(3H,s),1.77-1.42(4H,m),1.30-1.21(1H,m),0.90-0.79(1H,m) ESI-MS(+)=753.0,(-)751.1

[0060] <Comparative Compounds 1 and 2> [ka] Comparative compound 1 and comparative compound 2 were commercially available products manufactured by Watanabe Chemical Industry Co., Ltd.

[0061] (Rating 1) <Deprotection rate> For the protected amino acids synthesized above, the deprotection rate of the protected side chain was determined as follows. 50 mg of a side-chain protected amino acid using Example Compound 1 (reagent for protecting groups) or a side-chain protected amino acid using Comparative Compounds 1 and 2 was mixed with an equimolar amount of Fmoc-Gly-OH (internal standard) to which the side-chain protected amino acid belonged. Dichloromethane / trifluoroethanol / trifluoroacetic acid (100 / 10 / 1:vol ratio) was then added to the mixture so that the substrate concentration based on the side-chain protected amino acid was 0.025 M, and the mixture was stirred at 30°C for 60 minutes. 5 μL of the reaction solution was dissolved in 400 μL of MeOH (methanol), and the deprotection rate (%) was determined by quantifying the ratio of Fmoc-amino acids produced by deprotecting the side-chain protected amino acids to Fmoc-Gly-OH using Ultra Performance LC (Waters, model number: ACQUITY), and evaluated based on the following criteria. The column and measurement conditions used for ultra-performance liquid chromatography are described below. Column: Waters, Model: BEH C18, 1.7μm, 2.1mm x 30mm Flow rate: 0.5mL / min Solvents: Solution A: 0.1% formic acid-water, Solution B: 0.1% formic acid-acetonitrile Gradient cycle: 0.00 min (Solution A / Solution B = 95 / 5), 2.00 min (Solution A / Solution B = 5 / 95), 3.00 min (Solution A / Solution B = 95 / 5) Detection wavelength: 254nm

[0062] For the evaluation of the deprotection rate, a rating of "B" or higher was considered acceptable. The results are shown in Table 1. Furthermore, a higher deprotection rate indicates a faster deprotection speed, which is preferable. -Evaluation Criteria- "A": The deprotection rate is 90% or higher. "B": The deprotection rate is between 50% and 90%. "C": The deprotection rate is between 10% and 50%. "D": The deprotection rate is less than 10%.

[0063] [Table 1]

[0064] Table 1 shows that Example Compound 1 (protecting group forming reagent) used in Example 1 exhibits superior deprotection rate compared to the comparative compounds in Comparative Examples 1 and 2. Therefore, it can be said to be suitable for the synthesis of acid-unstable peptides. Example Compound 1 also exhibited excellent sealing properties.

[0065] <Synthesis of protective peptide (5-residue peptide: Fmoc-MeNle-MeNle-Arg(X)-Cys(Mmt)-Gly-NH2)> Details of the abbreviations other than those mentioned above are shown below. MeNle: N-methylnorleucine residue Arg(X): Arginine residue having the protecting group of the present invention Cys(Mmt): Mmt-protected cysteine ​​residues Mmt: Methoxytrityl group

[0066] <Example 2> (Synthesis of Fmoc-Gly-NH-IndoTAG) [ka] Starting material (1) (5.00 g, 4.02 mmol), synthesized according to the example in International Publication No. 2020 / 262259, was dissolved in tetrahydrofuran (40 mL), and diazabicycloundecene (DBU) (1.20 mL, 8.04 mmol) was added and the mixture was stirred. After the deprotection reaction was complete, N-methylmorpholine (0.906 mL, 8.24 mmol) and methanesulfonic acid (0.522 mL, 8.04 mmol) were added, followed by the addition of Fmoc-Gly-OH (1.43 g, 4.82 mmol) and (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU) (2.07 g, 4.82 mmol), and the mixture was stirred. After the condensation reaction was complete, acetonitrile (400 mL) was added and stirred. The precipitate was filtered, washed with acetonitrile, and dried under reduced pressure to obtain Fmoc-Gly-NH-IndoTAG (5.13 g, yield 98.1%).

[0067] (Synthesis of Fmoc-MeNle-MeNle-Arg(X)-Cys(Mmt)-Gly-NH-IndoTAG) The peptide sequence was extended by repeatedly removing the Fmoc group and condensing it with the protective amino acids shown in Table 2 using Fmoc-Gly-NH-IndoTAG. X above is Example Compound 1.

[0068] [Table 2]

[0069] (Peptide deprotection) To Fmoc-MeNle-MeNle-Arg(X)-Cys(Mmt)-Gly-NH-IndoTag (50.0 mg), trifluoroacetic acid / hexafluoroisopropanol / dichloromethane (1 / 10 / 100:vol%, 2.0 mL), triisopropylsilane (10.0 molar equivalent), and 3,6-dioxa-1,8-octanedithiol (10.0 molar equivalent) were added at room temperature and the mixture was stirred for 1 hour at room temperature. The reaction mixture was then added dropwise to Tert-butyl methyl ether (20 mL) under ice cooling to precipitate, and the supernatant was removed and the mixture was washed with Tert-butyl methyl ether repeatedly to obtain Fmoc-MeNle-Arg(X)-Cys(Mmt)-Gly-NH-IndoTag (13.8 mg). HPLC purity (220nm): 84% MS(ESI,m / Z):810.4(M+H)

[0070] The peptide production method described herein allows for the synthesis of peptides in high yield. Furthermore, since the protecting group of the side chain can be deprotected even under weak acid conditions, even peptides that are particularly unstable to acid can be synthesized with high purity.

Claims

1. A peptide chain elongation step in which a first amino acid or peptide, whose side-chain amino group is protected by a first protecting group represented by the following formula (1), is reacted with a second amino acid or peptide to obtain a third amino acid or peptide. The process includes a first deprotection step of deprotecting the primary protecting group of a tertiary amino acid or peptide, The first deprotection step involves deprotection using a deprotection solution containing 10% by mass or less of trifluoroacetic acid. A method for producing a peptide, wherein the first amino acid or peptide contains arginine, and two of the nitrogen atoms of the guanidino group of the side chain of the arginine are protected with a protecting group represented by formula (1). 【Chemistry 1】 In the formula, R １１ R is a phenyl group having one or more substituents selected from alkoxy groups or alkyl groups, ２１ and R ２２ is a hydrogen atom, n is 1, and the asterisk indicates a linkage.

2. A method for producing a peptide according to claim 1, wherein the trifluoroacetic acid content of the deprotection solution is 2% by mass or less.

3. A C-terminal protection step in which the carboxyl group or amide group of an N-terminal protected amino acid or peptide is protected by a C-terminal protective agent. An N-terminal deprotection step is performed to deprotect the N-terminal protecting group of the N-terminal C-terminal protected amino acid or peptide obtained in the C-terminal protection step. A peptide chain extension step is performed in which an N-terminally protected amino acid or peptide is condensed to the N-terminus of the C-terminally protected amino acid or peptide obtained in the N-terminal deprotection step, and The method further includes a C-terminal deprotection step of deprotecting the C-terminal protecting group, Either the C-terminally protected amino acid or peptide, or the N-terminally protected amino acid or peptide, is a primary amino acid or peptide protected by the primary protecting group. A method for producing a peptide according to claim 1 or 2.

4. R １１ A method for producing a peptide according to any one of claims 1 to 3, wherein the substituent of the phenyl group is an alkoxy group having 1 to 10 carbon atoms.

5. A protection step is to react a primary amino acid or peptide with a protecting group forming reagent shown in formula (2) below to obtain a primary protected amino acid or peptide in which the side chain amino group is protected by a primary protecting group. A method for producing an amino acid or peptide comprising the above, wherein the first amino acid or peptide comprises arginine, and two of the nitrogen atoms of the guanidino group of the side chain of the arginine are protected with a protecting group forming reagent represented by formula (2). 【Chemistry 2】 In the formula, R １１ R is a phenyl group having one or more substituents selected from alkoxy groups or alkyl groups, １２ This is an aryl group which may have one or more substituents selected from a carbonyl group, a nitro group or a halogen atom, a heteroaliphatic ring group which may have one or more substituents selected from a carbonyl group, a nitro group or a halogen atom, or a heteroaryl group which may have one or more substituents selected from a carbonyl group, a nitro group or a halogen atom, and these may be linked via an oxygen atom or a sulfur atom, R ２１ and R ２２ is a hydrogen atom, and n is 1.

6. A compound containing arginine or an arginine residue having a protecting group represented by the following formula (1), wherein two of the nitrogen atoms of the guanidino group in the side chain of the arginine are protected by the protecting group represented by formula (1). 【Transformation 3】 In the formula, R １１ is a phenyl group having one or more substituents selected from an alkoxy group or an alkyl group. When there are a plurality of electron-donating groups, they may be the same or different. R ２１ and R ２２ are hydrogen atoms, n is 1, and the asterisk indicates the connecting portion.

7. R １１ A compound containing arginine or an arginine residue according to claim 6, wherein the substituent of the phenyl group is an alkoxy group having 1 to 10 carbon atoms.

8. A compound further comprising arginine or an arginine residue according to claim 6 or 7, having a 9-fluorenylmethoxycarbonyl group.

Images