Method for quantifying unprotected amino acid in amino acid raw material

Abstract

The present invention relates to a method for quantifying an unprotected amino acid in a material containing an amino acid protected by a protective group, the method including a step for obtaining a quantification value of the unprotected amino acid in the material using a charged aerosol detector. The quantification value of the unprotected amino acid is obtained on the basis of a corrected response value that is calculated by applying a sensitivity correction coefficient to a response value of the unprotected amino acid in the material which is obtained by the charged aerosol detector. The sensitivity correction coefficient is a value that is calculated on the basis of a response value of a reference standard of the unprotected amino acid and a response value of a reference standard for quantification both of which are obtained using the charged aerosol detector.

Description

Method for quantifying unprotected amino acids in amino acid raw materials

[0001] The present invention relates to a method for quantifying unprotected amino acids in amino acid raw materials, and a method for producing peptide compounds using the quantification method.

[0002] In recent years, drug discovery technologies that enable the discovery of drugs targeting tough targets, such as protein-protein interaction inhibitors, agonists, and molecular chaperones, using medium-molecular-weight peptide compounds (molecular weight 500-2000), have attracted considerable attention. These compounds are generally synthesized by extending amino acids, whose N-terminus and C-terminus are protected by various protecting groups such as Fmoc, Boc, and Cbz groups, as raw materials. In pharmaceutical manufacturing, evaluating impurities in amino acid raw materials is extremely important from a quality control perspective, and ICH Q11 requires the establishment of appropriate quality control items, especially for raw materials designated as GMP starting materials. When using amino acids containing protecting groups as raw materials (including starting materials), it is necessary to evaluate the content of amino acids without protecting groups ("unprotected amino acids") as one of the related substances that are impurities. Unprotected amino acids can become raw materials for unwanted side reactions in the synthesis process and may remain in the pharmaceutical active pharmaceutical ingredient as related substances such as over-extended products. Since it is not always possible to remove the impurities in subsequent processes, a high level of quality control is required at the raw material stage.

[0003] Generally, in the evaluation of related substances, a liquid chromatography apparatus equipped with a UV / VIS detector is often used. If the protecting group contains structures with UV absorption, such as aromatic rings, it can be evaluated with a UV / VIS detector. However, for unprotected amino acids, if their side chains do not contain structures with UV absorption, the detection sensitivity is extremely low and evaluation is not possible with a UV detector. When evaluation is not possible with a UV / VIS detector, mass spectrometer (MS), evaporative light scattering detector (ELSD), charged aerosol detector (CAD) can be considered. However, considering detection sensitivity, measurable concentration range, standardization of test conditions, and versatility, using CAD is considered one of the simplest methods that best achieves quality control objectives. When quantifying related substances that are impurities more accurately using CAD, it is necessary to use the related substance itself, whose content is known, as a standard. Therefore, when there are multiple compounds to be quantified, it is necessary to prepare a standard sample for each compound to be quantified, but this preparation has the disadvantage of requiring a great deal of effort. When there are multiple related substances and it is difficult to obtain a standard sample for a specific related substance, it is known that the main component compound can be used as a substitute for the standard sample to quantify related substances by calculating a sensitivity coefficient that shows the difference in CAD responsiveness between the main component compound and its related substances, and correcting the peak area with the sensitivity coefficient (Non-Patent Literature 1).

[0004] Huang et al., “Development of HPLC-CAD method for simultaneous quantification of nine related substances in ursodeoxycholic acid and identification of two unknown impurities by HPLC-Q-TOF-MS” journal of pharmaceutical and biomedical analysis 229, 2023, 115357

[0005] Generally, the synthesis of peptide compounds often involves using several to more than ten types of amino acids, which can result in a large number of unprotected amino acids being produced as impurities. Preparing and maintaining samples for all of these impurities has been a major challenge in development. Furthermore, the method described in Non-Patent Document 1 still requires the cumbersome preparation of the main component compound as a sample each time. Therefore, there was a need for a simpler and more versatile evaluation test system.

[0006] The object of the present invention is to provide a novel method for quantifying unprotected amino acids in a material containing amino acids protected by a protecting group.

[0007] In one aspect of the present invention, the following inventions are provided: [1] A method for quantifying unprotected amino acids in a material containing amino acids protected by a protecting group, comprising the step of obtaining a quantitative value of the unprotected amino acids in the material using a charged particle detector, wherein the quantitative value of the unprotected amino acids is obtained based on a corrected response value calculated by applying a sensitivity correction coefficient to the response value of the unprotected amino acids in the material by the charged particle detector, and the sensitivity correction coefficient is a value calculated based on the response values ​​of the unprotected amino acid standard and the quantitative standard, respectively, by the charged particle detector. [2] The method according to [1], wherein the quantitative standard is a natural amino acid. [3] The method according to [1] or [2], wherein the quantitative standard is either isoleucine (Ile) or proline (Pro). [4] The method according to [3], wherein the quantitative standard is isoleucine (Ile). [5] The method according to [3], wherein the quantitative standard is proline (Pro). [6] The method according to any one of [1] to [5], wherein the unprotected amino acid is a non-natural amino acid. [7] The method according to any one of [1] to [6], wherein the unprotected amino acid is an unprotected amino acid with low UV detection sensitivity. [8] The method according to any one of [1] to [7], wherein the protecting group is at least one of Fmoc, Cbz, or t-Bu. [9] The method according to any one of [1] to [8], wherein the sensitivity correction coefficient is set based on the response ratio of the unprotected amino acid standard and the quantitative standard.

[10] The method according to [9], wherein the sensitivity correction coefficient is set as the average of the response ratios at different concentrations.

[11] The method according to

[10] , wherein the sensitivity correction coefficient is set as the average of the response ratios at different concentrations from 5 μg / mL to 50 μg / mL.

[12] The method according to any one of [1] to

[11] , wherein the material comprises two or more types of unprotected amino acids, and the sensitivity correction coefficient is set for each combination of the quantitative standard and the unprotected amino acid standard.

[13] The method according to any one of [1] to

[12] , wherein the difference between the quantitative value and the theoretical value of the unprotected amino acid is within ±20%.

[14] The method according to any one of [1] to

[13] , wherein the material is used for the synthesis of a cyclic peptide compound.

[15] A method for quantifying unprotected amino acids in a material containing protecting amino acids, comprising the following steps: (1) measuring the response value of a standard of the unprotected amino acid and the response value of a quantification standard using a charged particle detector; (2) calculating the response ratio between the standard of the unprotected amino acid and the quantification standard; (3) setting a sensitivity correction coefficient based on the response ratio; and (4) obtaining a quantitative value of the unprotected amino acid in the material based on a corrected response value calculated by applying the sensitivity correction coefficient to the response value of the unprotected amino acid in the material and the response value of the quantification standard.

[16] The method according to

[15] , wherein the quantification standard is a natural amino acid.

[17] The method according to

[15] or

[16] , wherein the quantification standard is either isoleucine (Ile) or proline (Pro).

[18] The method according to

[17] , wherein the quantitative standard is isoleucine (Ile).

[19] The method according to

[17] , wherein the quantitative standard is proline (Pro).

[20] The method according to any one of

[15] to

[19] , wherein the unprotected amino acid is a non-natural amino acid.

[21] The method according to any one of

[15] to

[20] , wherein the unprotected amino acid is an unprotected amino acid with low UV detection sensitivity.

[22] The method according to any one of

[15] to

[21] , wherein the protecting group is at least one of Fmoc, Cbz, or t-Bu.

[23] The method according to any one of

[15] to

[22] , wherein the sensitivity correction coefficient is set based on the response ratio of the unprotected amino acid standard and the quantitative standard.

[24] The method according to

[23] , wherein the sensitivity correction coefficient is set as the average of the response ratios at different concentrations.

[25] The method according to

[24] , wherein the sensitivity correction coefficient is set as the average of the response ratios at different concentrations from 5 μg / mL to 50 μg / mL.

[26] The method according to any one of

[15] to

[25] , wherein the material comprises two or more types of unprotected amino acids, and the sensitivity correction coefficient is set for each combination of the quantitative standard and the unprotected amino acid standard.

[27] The method according to any one of

[15] to

[26] , wherein the difference between the quantitative value and the theoretical value of the unprotected amino acid is within ±20%.

[28] The method according to any one of

[15] to

[27] , wherein the material is used for the synthesis of a cyclic peptide compound.

[29] A method for producing a peptide compound comprising the following steps (1) to (3): (1) a step of quantifying the unprotected amino acid in a material containing an amino acid protected by a protecting group by the method according to any one of [1] to

[28] ; (2) a step of selecting a material in which the content of the unprotected amino acid is less than or equal to a predetermined value; and (3) a step of linking the amino acid protected by a protecting group in the selected material with another amino acid or peptide compound.

[0008] According to the present invention, a simple and versatile method can be provided for quantifying unprotected amino acids in a material containing amino acids protected by a protecting group.

[0009] This graph shows the calibration curves for each quantitative standard in the examples.

[0010] The present invention will be described in detail below, including definitions of symbols and terms used herein, and embodiments of the present invention.

[0011] In this specification, the "~" indicating a numerical range includes the values ​​at both ends of the range; for example, "A~B" means a numerical range where A is greater than or equal to B and B is less than or equal to B.

[0012] In this specification, the meaning of the term "and / or" includes any combination of "and" and "or" as appropriate. Specifically, for example, "A, B, and / or C" includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, (vii) A, B, and C.

[0013] As used herein, "one or more" means one or two or more. When "one or more" is used for the number of substituents of a certain group, it means a number from one to the maximum number of substituents that the group can tolerate. Specifically, examples of "one or more" include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or a number greater than that.

[0014] As used herein, "alkyl" refers to a linear or branched monovalent saturated hydrocarbon group derived by removing one arbitrary hydrogen atom from an aliphatic saturated hydrocarbon, which does not contain a heteroatom (an atom other than a carbon atom and a hydrogen atom) and an unsaturated carbon-carbon bond in the skeleton, and is a hydrocarbyl containing a hydrogen atom and a carbon atom, or a group having a subset of the hydrocarbon group structure. Examples of alkyl include, for example, alkyl having 1 to 20 carbon atoms (C １ ~C ２０ , hereinafter in the specification, "C ｐ ~C ｑ " means having p to q carbon atoms), and C １ ~C １５ alkyl is preferred, C １ ~C １０ alkyl is more preferred, and C １ ~C ６ alkyl is most preferred. Specific examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, and the like.

[0015] As used herein, "alkenyl" refers to a linear or branched monovalent unsaturated hydrocarbon group having one or more carbon-carbon double bonds (a bond formed by two adjacent sp ２ carbon atoms). sp ２Depending on the arrangement of the atoms or groups of atoms bonded to the carbon atom, the geometric form of the double bond can be entgegen (E) or tuzamen (Z), cis or trans configuration. Examples of alkenyls include C ２ ~C １０ It is an alkenyl, C ２ ~C ８ Alkenyl is preferred, C ２ ~C ７ Alkenyl is more preferred, C ２ ~C ６ Alkenyls are most preferred. Specific examples of alkenyls include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, 1-butenyl, 2-butenyl (including cis and trans forms), 3-butenyl, pentenyl, hexenyl, and the like.

[0016] In this specification, "alkynyl" refers to a linear or branched monovalent unsaturated hydrocarbon group having one or more carbon-carbon triple bonds (bonds formed by two adjacent sp carbon atoms). Examples of alkynyls include C ２ ~C １０ It is alkinyl, C ２ ~C ８ Alkinyl is preferred, C ２ ~C ７ Alkinyl is more preferred, C ２ ~C ６ Alkynnyl is most preferred. Specific examples of alkynyl include ethynyl, 1-propynyl, propargyl(2-propynyl), 1-butynyl, 2-butynyl, 3-butynyl, pentynyl, hexynyl, and the like.

[0017] In this specification, "aryl" refers to a monovalent aromatic hydrocarbon ring group consisting of a monocyclic or fused ring exhibiting monovalent aromaticity. In this specification, aryls consisting of a monocyclic ring are referred to as monocyclic aryls, and aryls consisting of a fused ring are referred to as fused-ring aryls. Examples of aryls include C ６ ~C １４ It is aryl, C ６ Ariel, C １０ Aryl and C １４ Aryl is preferred, C ６Aryl and C １０ Aryl is more preferred, C ６ Aryls are most preferred. Specific examples of aryls include phenyl, 1-naphthyl, and 2-naphthyl. These aryls may also have substituents, such as tolyl, xylyl, and 2,4-dinitrophenyl.

[0018] In this specification, "heteroaryl" refers to a monovalent aromatic heterocyclic group comprising a monocyclic or fused ring that contains at least one heteroatom in addition to a carbon atom and exhibits aromaticity. In this specification, a heteroaryl composed of a monocyclic ring is referred to as a monocyclic heteroaryl, and a heteroaryl composed of a fused ring is referred to as a fused-ring heteroaryl. The number of atoms constituting the ring of the heteroaryl is, for example, 5 to 14 (5 to 14-membered heteroaryl), preferably 5 to 13 (5 to 13-membered heteroaryl), more preferably 5 to 10 (5 to 10-membered heteroaryl), and most preferably 5 to 7 (5 to 7-membered heteroaryl). Examples of heteroaryl compounds include five-membered heteroaryls such as furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, triazolyl, and tetrazolyl; six-membered heteroaryls such as pyridyl, pyrimidyl, pyridadinyl, pyrazinyl, and triazinyl; nine-membered heteroaryls such as benzofuranil, benzothienyl, benzothiazolyl, benzimidazolyl, benzotriazolyl, indolyl, indazolyl, and pyrazolopyridyl; and ten-membered heteroaryls such as quinolyl, isoquinolyl, sinnolinyl, quinazolinyl, and quinoxalinyl.

[0019] In this specification, "aralkyl (arylalkyl)" refers to a group in which one or more hydrogen atoms of an "alkyl" group are substituted with "aryl" atoms. Examples of aralkyls include C ７ ~C ２０ It is Aralkir, C ７ ~C １８ Aralkyl is preferred, C ７ ~C １６ Aralkir is more preferred, C ７ ~C １４ Aralquil is the most preferable. C ７ ~C２０ As the aralkyl, for example, C ６ - C １０ aryl C １ - C １０ alkyl, and C ６ - C １０ aryl C １ - C ８ alkyl is preferred, and C ６ aryl C １ - C ８ alkyl or C １０ aryl C １ - C ８ alkyl is more preferred, and C ６ aryl C １ - C ８ alkyl is most preferred. C ７ - C １８ As the aralkyl, for example, C ６ - C １０ aryl C １ - C ８ alkyl, and C ６ - C １０ aryl C １ - C ６ alkyl is preferred, and C ６ aryl C １ - C ６ alkyl or C １０ aryl C １ - C ６ alkyl is more preferred, and C ６ aryl C １ - C ６ alkyl is most preferred. C ７ - C １６ As the aralkyl, for example, C ６ - C １０ aryl C １ - C ６ alkyl, and C ６ - C １０ aryl C １ - C ４ alkyl is preferred, and C ６ aryl C １ - C ４ alkyl or C １０ aryl C １ - C ４ alkyl is more preferred, and C ６Aryl C １ ~C ４ Alkyl is most preferred. ７ ~C １４ For example, C ６ ~C １０ Aryl C １ ~C ４ It is alkyl, C ６ ~C １０ Aryl C １ ~C ３ Alkyl is preferred, C ６ Aryl C １ ~C ３ Alkyl or C １０ Aryl C １ ~C ３ Alkyl is more preferred, C ６ Aryl C １ ~C ３ Alkyl is most preferred. Specific examples of aralkyls include benzyl, phenethyl, and 3-phenylpropyl.

[0020] In this specification, "heteroarylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" group are substituted with a "heteroaryl" group. A preferred heteroarylalkyl group is, for example, a group in which one hydrogen atom of an alkyl group is substituted with a heteroaryl group. An example of a heteroarylalkyl group is a 5-10 membered heteroaryl C group. １ ~C ６ Alkyl, 5-10 membered heteroaryl C １ ~C ４ Alkyl is preferred, and 5-10 membered heteroaryl C １ ~C ３ Alkyl is more preferred, and 5-10 membered heteroaryl C １ ~C ２Alkyl is most preferred. Examples of heteroarylalkyls include 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-furanylmethyl, 2-thienylmethyl, 3-thienylmethyl, 4-thiazolylmethyl, 2-(2-pyridyl)ethyl, 2-(3-pyridyl)ethyl, 2-(4-pyridyl)ethyl, 2-(6-quinolyl)ethyl, 2-(7-quinolyl)ethyl, 2-(6-indolyl)ethyl, 2-(5-indolyl)ethyl, and 2-(5-benzofuranyl)ethyl.

[0021] In this specification, "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent non-aromatic hydrocarbon ring group (alicyclic ring group). The carbon atoms constituting the ring may be oxidized to include carbonyl atoms. Cycloalkyls may be selected from the group consisting of monocycles, fused rings, and spirocycles. In this specification, cycloalkyls containing a monocycle are referred to as monocyclic cycloalkyls, i.e., monocyclic alicyclic ring groups. Cycloalkyls containing a fused ring are referred to as fused cycloalkyls, i.e., fused alicyclic ring groups. Cycloalkyls containing a spirocycle are referred to as spirocyclic cycloalkyls, i.e., spirocyclic alicyclic ring groups. Cycloalkyls may form fused rings with saturated alicyclic rings such as cyclopentane rings or cyclohexane rings, unsaturated alicyclic rings such as cyclopentene rings or cyclohexene rings, or aromatic hydrocarbon rings such as benzene rings or naphthalene rings. Cycloalkyls may form spiro rings with saturated alicyclic rings such as cyclopropane rings, cyclobutane rings, cyclopentane rings, and cyclohexane rings. Examples of cycloalkyls include C ３ ~C １０ It is a cycloalkyl, C ３ ~C ８ Cycloalkyl is preferred, C ３ ~C ７ Cycloalkyl is more preferred, C ３ ~C ６ Cycloalkyls are most preferred. Specific examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, and cyclohexenyl.

[0022] In this specification, "amino acid" includes natural amino acids and non-natural amino acids. In this specification, "amino acid" may mean an amino acid residue. In this specification, "natural amino acids" are Gly (glycine), as well as L-Ala (alanine), L-Ser (serine), L-Thr (threonine), L-Val (valine), L-Leu (leucine), L-Ile (isoleucine), L-Phe (phenylalanine), L-Tyr (tyrosine), L-Trp (tryptophan), L-His (histidine), L-Glu (glutamic acid), L-Asp (aspartic acid), L-Gln (glutamine), L-Asn (asparagine), L-Cys (cysteine), L-Met (methionine), L-Lys (lysine), L-Arg (arginine), and L-Pro (proline). In this specification, L-type amino acids included in natural amino acids may be written without the "L-". In other words, the natural amino acid L-Ala is sometimes written as Ala. In this specification, "non-natural amino acid" refers to an amino acid other than a natural amino acid. Examples of non-natural amino acids include β-amino acids, γ-amino acids, D-type amino acids, N-substituted amino acids other than Pro, α,α-disubstituted amino acids, and amino acids whose side chains differ from those of natural amino acids. Amino acids in this specification may have any stereochemistry. There are no particular restrictions on the side chains of amino acids, but each can be freely selected from groups such as alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, cycloalkyl, spiro-bonded cycloalkyl, and / or atoms such as hydrogen atoms. Each group may be further substituted. In one non-limiting embodiment, an amino acid in this specification may be a compound having both a carboxyl group and an amino group in the same molecule. Even in this case, proline, hydroxyproline, azetidine-2-carboxylic acid, etc., in which the nitrogen atom of the amino group and any atom of the amino acid's side chain form a ring together, are also included as amino acids.

[0023] In some embodiments, the “amino acids” as used herein are isotope-labeled by substituting one or more atoms within them with atoms having the same atomic number (number of protons) but different mass numbers (sum of the number of protons and neutrons) in a ratio different from their natural abundance. Such isotope-labeled (e.g., radiolabeled) amino acids are included in the “amino acids” as used herein. Examples of isotopes included in the “amino acids” as used herein include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine atoms, for example. ２ H, ３ H, １３ C, １４ C, １５ N, １７ O, １８ O, ３２ P, ３５ S, １８ F, ３６ It contains chlorine, etc.

[0024] In this specification, "protected amino acid" refers to a compound in which a functional group contained in an "amino acid" is protected by any protecting group. The functional groups that can be protected are selected from one to three functional groups from the group consisting of the amino group of the amino acid main chain, the carboxyl group of the amino acid main chain, and the side chain of the amino acid. Specifically, examples include amino acids having protecting groups on the amino group of the amino acid main chain, the carboxyl group of the amino acid main chain, and the side chain of the amino acid, or amino acids having protecting groups on the amino group of the amino acid main chain and the side chain of the amino acid, or amino acids having protecting groups on the amino group of the amino acid main chain and the carboxyl group of the amino acid main chain, or amino acids having protecting groups on the carboxyl group of the amino acid main chain and the side chain of the amino acid, or amino acids having a protecting group on the amino group of the amino acid main chain, or amino acids having a protecting group on the carboxyl group of the amino acid main chain, or amino acids having a protecting group on the side chain of the amino acid. Such methods for selecting and desorbing protecting groups can be found, for example, in "Greene's, 'Protective Groups in Organic Synthesis' (5th edition, John Wiley & Sons 2014)," and these can be used as appropriate depending on the reaction conditions. Furthermore, the order of reaction steps, such as the introduction of substituents, can be changed as needed.

[0025] Examples of functional groups to which protecting groups can be introduced include carboxyl, amino, hydroxyl, and heterocyclyl groups, which are referred to as protected carboxyl, protected amino, protected hydroxyl, and protected heterocyclyl groups, respectively. Examples of "carboxyl protecting groups" include alkyl ester protecting groups, benzyl ester protecting groups, and substituted alkyl ester protecting groups. Examples of "amino protecting groups" include carbamate protecting groups, amide protecting groups, arylsulfonamide protecting groups, alkylamine protecting groups, and imide protecting groups. Examples of "hydroxyl protecting groups" include alkyl ether protecting groups, aralkyl ether protecting groups, silyl ether protecting groups, and carbonate ester protecting groups. Examples of "heterocyclyl protecting groups" include carbamate protecting groups, amide protecting groups, and arylsulfonamide protecting groups.

[0026] In this specification, "protected amino" means an amino group protected with any protecting group. Specific examples of protected aminos include Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethyloxycarbonyl), Cbz (benzyloxycarbonyl), Troc (2,2,2-trichloroethoxycarbonyl), Alloc (allyloxycarbonyl), Teoc (2-(trimethylsilyl)ethoxycarbonyl), or aminos protected with a protecting group such as trifluoroacetyl.

[0027] In this specification, "protected hydroxyl" means a hydroxyl group protected by any protecting group. Examples of hydroxyl group protecting groups include methoxymethyl (MOM), tetrahydropyranyl (THP), t-butyl (t-Bu), benzyl (Bn), trityl (Tr), cumyl, methoxytrityl, trimethylsilyl (TMS), and t-butyldimethylsilyl.

[0028] In this specification, "protected heterocyclyl" refers to a heterocyclyl in which one or more functional groups, such as an amino group, are protected by an arbitrary protecting group. Examples of protected heterocyclyls include protected 3- to 14-membered heterocyclyls, which may be protected 3- to 12-membered heterocyclyls, protected 3- to 10-membered heterocyclyls, or protected 4- to 7-membered heterocyclyls. Specific examples of protecting groups include Boc, Fmoc, Cbz, Troc, Alloc, etc.

[0029] In this specification, "amino acid backbone" means the chain portion consisting of an amino group, an α-carbon, and a carboxyl group in the case of α-amino acids, the chain portion consisting of an amino group, a β-carbon, an α-carbon, and a carboxyl group in the case of β-amino acids, and the chain portion consisting of an amino group, a γ-carbon, a β-carbon, an α-carbon, and a carboxyl group in the case of γ-amino acids. In this specification, "α-amino acid skeleton" means the chain portion consisting of an amino group, an α-carbon, and a carboxyl group, "β-amino acid skeleton" means the chain portion consisting of an amino group, a β-carbon, an α-carbon, and a carboxyl group, and "γ-amino acid skeleton" means the chain portion consisting of an amino group, a γ-carbon, a β-carbon, an α-carbon, and a carboxyl group. In this specification, an amino acid having a "β-amino acid skeleton" as a whole or as a partial structure may be referred to as an "amino acid having a β-amino acid skeleton". For example, L-aspartic acid has a chain-like portion (β-amino acid skeleton) composed of an amino group, a β-carbon, an α-carbon, and a carboxyl group, and therefore falls under the category of "amino acids having a β-amino acid skeleton."

[0030] In this specification, "amino acid side chain" refers to, in the case of α-amino acids, a group and / or atom bonded to the carbon atom to which the amino group and carboxyl group are bonded (α-carbon). For example, the methyl group of Ala is an amino acid side chain. In the case of β-amino acids, a group and / or atom bonded to the α-carbon and / or β-carbon becomes the amino acid side chain, and in the case of γ-amino acids, a group and / or atom bonded to the α-carbon, β-carbon and / or γ-carbon may become the amino acid side chain.

[0031] In this specification, "amino group of the amino acid backbone" refers to an amino group bonded to a carbon atom in the backbone of an amino acid. The amino group of the amino acid backbone is unsubstituted (NH ２The amino group in the main chain of an amino acid may be substituted, or it may be a substituted group. If the amino group is substituted, it can be represented as a -NHR group, where R may be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group, and one or two non-adjacent methylene groups in these groups may be an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO-). ２ It may be substituted with -). R may form a 4- to 10-membered saturated heterocycle including the nitrogen atom to which R is bonded and the carbon atom of the main chain of the amino acid to which the nitrogen atom is bonded, and the 4- to 10-membered saturated heterocycle may have substituents. The substituents that are permissible for R are selected in the same way as substituents in the amino acid side chain described above. Amino acids in which such a main chain amino group is substituted are referred to as "N-substituted amino acids" in this specification. Examples of "N-substituted amino acids" in this specification are N-alkyl amino acids and N-C １ ~C ２０ Alkyl amino acids (main chain amino group is C) １ ~C ２０ (Alkyl-substituted amino acids) are preferred, N-C １ ~C ６ Alkyl amino acids are more preferred, N-C １ ~C ４ Alkyl amino acids are most preferred. Examples of N-substituted amino acids include, but are not limited to, N-methyl amino acids, N-ethyl amino acids, and proline.

[0032] In this specification, "peptide compound" refers to a compound in which two or more amino acids are linked by an amide bond. Peptide compounds may also contain bonds other than amide bonds. For example, peptides containing bonds selected from the group consisting of COC bonds, C(O)-O bonds, C(S)-O bonds utilizing oxygen atoms, C(O)-S bonds, C(S)-S bonds, CSSC bonds, CSC bonds, CS(O)-C bonds, CS(O)2-C bonds utilizing sulfur atoms, and CNC bonds, C=NC bonds, NC(O)-N bonds, NC(S)N bonds, C(S)-N bonds, and CC bonds utilizing nitrogen atoms are also included in the definition of "peptide compound" in this specification. The number of amino acid residues in a peptide compound is, for example, 5 to 30 residues, preferably 5 to 25 residues, more preferably 7 to 20 residues, and most preferably 8 to 14 residues. The number of N-substituted amino acid residues in a peptide compound is, for example, at least 3 residues, preferably at least 4 residues, more preferably at least 5 residues, and most preferably at least 6 residues. These N-substituted amino acids may be present continuously or discontinuously in the peptide compound. The number of amide bonds in the peptide compound is at least five, preferably at least seven, more preferably at least nine, and most preferably at least eleven. The peptide compound in this disclosure may be linear, branched, or cyclic, with cyclic peptide compounds being preferred.

[0033] In this specification, "cyclic peptide compound" means a peptide compound having a cyclic structure composed of four or more amino acid residues. The cyclic structure of a cyclic peptide compound may include bonds other than amide bonds, for example, bonds selected from the group consisting of COC bonds, C(O)-O bonds, C(S)-O bonds utilizing oxygen atoms, C(O)-S bonds, C(S)-S bonds, CSSC bonds, CSC bonds, CS(O)-C bonds, CS(O2)-C bonds utilizing sulfur atoms, and CNC bonds, C=NC bonds, NC(O)-N bonds, NC(S)N bonds, C(S)-N bonds, and CC bonds utilizing nitrogen atoms. In addition to the cyclic structure, a cyclic peptide compound may have amino acids or chain-like peptide structures that are not included in the cyclic structure. Furthermore, it may have structures other than amino acids or chain-like peptide structures. The cyclization of the cyclic peptide compound can take any form, such as cyclization via a carbon-nitrogen bond like an amide bond, cyclization via a carbon-oxygen bond like an ester bond or an ether bond, cyclization via a carbon-sulfur bond like a thioether bond, cyclization via a carbon-carbon bond, or cyclization by heterocycle construction. Of these, cyclization via covalent bonds such as an amide bond, carbon-sulfur bond, or carbon-carbon bond is preferred. Cyclization via an amide bond is particularly preferred, and the position of the carboxyl group or amino group used in the cyclization may be on the main chain or on the side chain. More preferably, cyclization is via an amide bond between a carboxyl group on the side chain and an amino group on the N-terminal main chain. The number of amino acid residues contained in the cyclic peptide compound is, for example, 5 to 30 residues, preferably 5 to 25 residues, more preferably 7 to 20 residues, and most preferably 8 to 14 residues. The number of amino acid residues contained in the cyclic portion of the cyclic peptide compound is, for example, 4 to 25 residues, preferably 4 to 20 residues, more preferably 5 to 15 residues, and most preferably 6 to 14 residues. The number of N-substituted amino acid residues in the cyclic peptide compound is, for example, at least three residues, preferably at least four, more preferably at least five, and most preferably at least six. These N-substituted amino acids may be present consecutively or discontinuously in the cyclic peptide compound.The number of amide bonds in the cyclic peptide compound is at least 5, preferably at least 7, more preferably at least 9, and most preferably at least 11.

[0034] In this specification, "cyclization of a peptide compound" means forming a cyclic structure of a cyclic portion containing four or more amino acid residues. The number of amino acids contained in the cyclic portion of the cyclic peptide compound in this specification is, for example, 4 to 25 residues, preferably 4 to 20 residues, more preferably 5 to 15 residues, and most preferably 6 to 14 residues. A method for converting a linear peptide compound into a cyclic peptide compound can be carried out by performing an intramolecular bond formation reaction as described in Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition (by RC Larock), or March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th Edition (by MB Smith, J. March), etc. A functional group transformation reaction can also be performed after the bond formation reaction. Examples of bond-forming reactions include C(O)-N bonds formed from carboxylic acids and amines, COC bonds, C(O)-O bonds, and C(S)-O bonds utilizing oxygen atoms, C(O)-S bonds, C(S)-S bonds, CSSC bonds, CSC bonds, CS(O)-C bonds, and CS(O2)-C bonds utilizing sulfur atoms, and CNC bonds, C=NC bonds, NC(O)-N bonds, NC(S)N bonds, and C(S)-N bonds utilizing nitrogen atoms. Furthermore, transition metal-catalyzed CC bond formation reactions such as the Suzuki reaction, Heck reaction, and Sonogashira reaction can be cited. For peptide cyclization methods, refer to WO2013100132, WO2017181061, J. Chem. Soc., Perkin Trans. 1, 2001, 471-484, etc. Examples of functional group transformation reactions performed after bond-forming reactions include oxidation reactions and reduction reactions. Specifically, examples include reactions that oxidize sulfur atoms to convert them into sulfoxide or sulfone groups. Also, examples include reduction reactions that reduce triple or double carbon-carbon bonds to convert them back into double or single bonds.When two amino acids are joined in the main chain of an amino acid, a ring-closed structure is formed by a peptide bond. However, a covalent bond may also be formed between the two amino acids through bonding of their side chains, or between the side chains and the main chain.

[0035] The compounds described herein, their salts, or their solvates include all of their isomers (e.g., stereoisomers exemplified by enantiomers and diastereomers, as well as cis-trans isomers, atrop isomers, tautomers, etc.). This also includes racemates of the optical isomers and mixtures of other isomers.

[0036] The compounds described herein may, in certain embodiments, be salts thereof or solvates thereof, preferably pharmaceutically acceptable salts thereof or pharmaceutically acceptable solvates thereof. A pharmaceutically acceptable salt, solvate, or solvate of a salt may be any salt, any solvate, or any solvate of any salt, provided that it retains the desired biological activity of the compound and does not have excessive undesirable toxicity, for example, those described in J. Pharmaceutical Sciences, 1977, 66(1), 1-19, or Drug Discov. Today, 2021, 26(2), 384-398. In this specification, “compound, or salt thereof, or solvate thereof” includes the compound, the salt of the compound, the solvate of the compound, and the solvate of the salt of the compound. Examples of compound salts include hydrochloride salts; hydrobromide salts; hydroiodide salts; phosphate salts; phosphonate salts; sulfate salts; sulfonates such as methanesulfonate and p-toluenesulfonate; carboxylate salts such as acetate, citrate, malate, tartrate, succinate, salicylate, and adipic acid; alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as magnesium salts and calcium salts; and ammonium salts such as ammonium salts, alkylammonium salts, dialkylammonium salts, trialkylammonium salts, and tetraalkylammonium salts. These salts can be produced, for example, by contacting the compound with an acid or a base. In this specification, a solvate means a compound that forms a molecular group with a solvent, and is not limited to solvates with one type of solvent, but also includes solvates formed with multiple solvents for one compound molecule, or solvates formed with multiple types of solvents for one compound molecule, and is not particularly limited as long as it is a solvate formed with a solvent that is permissible to ingest in conjunction with the administration of a drug. Specific examples of solvates include solvates with water, alcohols (ethanol, methanol, 1-propanol, 2-propanol, etc.), and dimethyl sulfoxide, and in particular, compounds that form a solvate with water are called hydrates.

[0037] [Method for Quantifying Unprotected Amino Acids] The method according to this embodiment is a method for quantifying unprotected amino acids in a material containing amino acids protected by a protecting group. The method includes a step of obtaining a quantitative value of unprotected amino acids in the material using a charged particle detector. The quantitative value of unprotected amino acids is obtained based on a corrected response value calculated by applying a sensitivity correction coefficient to the response value of the unprotected amino acids in the material measured by the charged particle detector. The sensitivity correction coefficient is a value calculated based on the response values ​​of the unprotected amino acid standard and the quantitative standard measured by the charged particle detector, respectively.

[0038] The method according to this embodiment can be carried out by a method that includes the following steps (1) to (4) in this order: (1) measuring the response value of an unprotected amino acid standard and the response value of a quantitative standard using a charged particle detector; (2) calculating the response ratio between the unprotected amino acid standard and the quantitative standard; (3) setting a sensitivity correction coefficient based on the response ratio; and (4) obtaining a quantitative value of unprotected amino acids in the material based on a corrected response value calculated by applying the sensitivity correction coefficient to the response value of the unprotected amino acids in the material and the response value of the quantitative standard.

[0039] In this specification, "material containing protecting group-protected amino acids" refers to a material whose main component is a protecting group-protected amino acid, and which may contain unprotected amino acids in a quantity below a predetermined value. Here, "main component" means that the content of protecting group-protected amino acids relative to the total mass of the material is 95% by mass or more. The content of protecting group-protected amino acids in a material containing protecting group-protected amino acids is 95% by mass or more, preferably 97% by mass or more, more preferably 99% by mass or more, even more preferably 99.8% by mass or more, and most preferably 99.9% by mass or more, based on the total mass of the material.

[0040] Materials containing amino acids protected by protecting groups may be used as raw materials for the synthesis of various compounds. For example, such materials may be used in the synthesis of peptide compounds, or in the synthesis of cyclic peptide compounds.

[0041] Protected amino acids include compounds in which the same or different amino acids are linked by amide bonds (peptide compounds) and then protected by a protecting group. The number of amino acid residues in a protected amino acid may be 1 to 3 or 1 to 2. Protected amino acids may be protected natural amino acids or protected non-natural amino acids.

[0042] In this specification, "unprotected amino acids in the material" refers to compounds present as impurities in the material, which are amino acids in which at least one protecting group of a protecting amino acid has been deprotected. Unprotected amino acids include not only amino acids in which all protecting groups of a protecting amino acid have been deprotected, but also amino acids in which only some of the protecting groups have been deprotected. That is, if the functional groups of a protecting amino acid in the material are protected by two or more protecting groups, the material may contain two or more types of unprotected amino acids.

[0043] The unprotected amino acids in the material may be natural amino acids (unprotected natural amino acids) or unnatural amino acids (unprotected unnatural amino acids).

[0044] The unprotected amino acids in the material may be unprotected amino acids with low UV detection sensitivity. An example of an unprotected amino acid with low UV detection sensitivity is an unprotected amino acid that does not contain a substructure with absorption in the UV region (e.g., a benzene ring) within the molecule. Because the method according to this embodiment uses a charged particle detector, even unprotected amino acids with low UV detection sensitivity can be quantified. The unprotected amino acids in the material may be natural amino acids with low UV detection sensitivity, or unnatural amino acids with low UV detection sensitivity.

[0045] The content of unprotected amino acids in the material may be 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0.9% by mass or less, 0.8% by mass or less, 0.7% by mass or less, 0.6% by mass or less, 0.5% by mass or less, 0.4% by mass or less, 0.3% by mass or less, 0.2% by mass or less, or 0.1% by mass or less, based on the total mass of the material.

[0046] Specific examples of combinations of amino acids protected by protecting groups in the material and unprotected amino acids (amino acids whose protecting groups have been removed) include, for example, the combination of Cbz-MeNva-OH and H-MeNva-OH, the combination of Cbz-Ile-OH・DCHA (dicyclohexylamine) and H-Ile-OH, the combination of H-Pro-OtBu・HCl and H-Pro-OH, and Fmoc-MeAlGly-OH. , combination with H-MeAlGly-OH, combination with H-MeGly-OtBu·HCl and H-MeGly-OH, combination with Cbz-Hyp(Et)-OH·DCHA and H-Hyp(Et)-OH, combination with Cbz-MecVal-OH and H-MecVal-OH, combination with Cbz-MeGly(cPent)-OH and H-MeGly(cPent)-OH, and Cbz-MeAsp ２ -OtBu-OH・DCHA and H-MeAsp ２ One example is the combination with -OtBu-OH.

[0047] <Step (1)> In Step (1), the response value of the unprotected amino acid standard and the response value of the quantitative standard are measured using a charged particle detector.

[0048] A charged aerosol detector (CAD) is a type of detector used in liquid chromatography. It works by atomizing and drying compounds separated by liquid chromatography to create microparticles, which are then charged for detection.

[0049] The response value can be measured using CAD by liquid chromatography with CAD as the detector. Examples of liquid chromatography methods include high-performance liquid chromatography and ultrafast separation liquid chromatography. The various conditions of the liquid chromatography method, such as the type of apparatus, column, and mobile phase, the elution method, and the mobile phase flow rate, are not particularly limited and can be appropriately set from known conditions, for example.

[0050] The unprotected amino acid standard is a material whose main component is an unprotected amino acid. The purity of the unprotected amino acid standard is, for example, 95% or higher, preferably 97% or higher, more preferably 99% or higher, even more preferably 99.8% or higher, and most preferably 99.9% or higher.

[0051] A quantitative standard is a standard used for the quantitative determination of unprotected amino acids in a material. The quantitative standard is selected based on factors such as the type of unprotected amino acid to be quantified, availability, and measurement sensitivity. The quantitative standard can be the same type or a different type of amino acid as the unprotected amino acid being quantified.

[0052] The purity of the quantitative standard is, for example, 95% or higher, preferably 97% or higher, more preferably 99% or higher, even more preferably 99.8% or higher, and most preferably 99.9% or higher.

[0053] The quantitative analysis standard is preferably a natural amino acid because it is easier to prepare in large quantities and is more readily available. The quantitative analysis standard is preferably one selected from the group consisting of Trp, Ile, Thr, Leu, Ser, Gly, and Pro, and more preferably either Ile or Pro, from the viewpoint of availability and other factors.

[0054] Step (1) can be carried out by a method that includes preparing a standard for quantification and a standard of unprotected amino acids, preparing a standard solution containing each of the standard for quantification and the standard of unprotected amino acids, and measuring the peak area of ​​the standard solution as the response value of the standard solution by CAD.

[0055] When measuring response values, multiple standard solutions with different concentrations are used. The standard concentration in the standard solution used for response value measurement can be set in the range of 5 to 50 μg / mL, for example, with a lower limit of 5 μg / mL, which corresponds to 0.05% of unprotected amino acids (impurities) relative to amino acids protected by protecting groups, and an upper limit of 50 μg / mL, which corresponds to 0.5%. The standard concentration in the standard solution may be 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, or 50 μg / mL based on the total volume of the standard solution.

[0056] <Step (2)> In step (2), the response ratio between the unprotected amino acid standard and the quantitative standard is calculated. The response ratio is the ratio of the peak area of ​​the quantitative standard to the peak area of ​​the unprotected amino acid standard (quantitative standard / unprotected amino acid).

[0057] In step (2), the response ratio is calculated from the peak area ratio between a standard solution containing an unprotected amino acid standard and a standard solution containing a quantitative standard prepared to the same concentration (μg / mL) as the standard solution. The response ratio is calculated for each standard concentration in the standard solution.

[0058] <Step (3)> In step (3), a sensitivity correction coefficient is set based on the response ratio. Preferably, the sensitivity correction coefficient is set as the average of the above response ratios at different concentrations.

[0059] Step (3) can be carried out, for example, by the following method. First, the average value of the response ratio calculated for each standard concentration in the standard solution is obtained. This is taken as the sensitivity correction coefficient for day 1. The above operation is repeated to obtain the sensitivity correction coefficient for day 2 and the sensitivity correction coefficient for day 3. The measurement is performed for three consecutive days. The average value of the sensitivity correction coefficients for days 1 to 3 is taken as the sensitivity correction coefficient (final sensitivity correction coefficient) for obtaining the quantitative value of unprotected amino acids. The measurement period for the sensitivity correction coefficient is preferably carried out for three consecutive days, but is not limited to this.

[0060] Sensitivity correction factors may be set for each combination of quantitative standard and unprotected amino acid. The combination of quantitative standard and unprotected amino acid is one quantitative standard selected from the group consisting of tryptophan (Trp), isoleucine (Ile), threonine (Thr), leucine (Leu), serine (Ser), glycine (Gly), and proline (Pro), and H-MeNva-OH, H-Ile-OH, H-Pro-OH, H-MeAlGly-OH, H-MeGly-OH, H-Hyp(Et)-OH, H-MecVal-OH, H-MeGly(cPent)-OH, and H-MeAsp ２The combination may be one unprotected amino acid selected from the group consisting of -OtBu-OH, for example, Ile (quantitative standard) and one unprotected amino acid selected from the group consisting of H-MeNva-OH, H-Ile-OH, H-MeAlGly-OH, H-Hyp(Et)-OH, and H-MeCpg-OH, or Pro (quantitative standard) and H-Pro-OH, H-Sar-OH, H-MecVal-OH, and H-MeAsp ２ The combination may be with one unprotected amino acid selected from the group consisting of -OtBu-OH. When the combination of the quantitative standard and the unprotected amino acid is set using the sensitivity correction coefficient described above, a more accurate quantitative value of the unprotected amino acid can be obtained.

[0061] <Step (4)> In step (4), the quantitative value of unprotected amino acids in the material is obtained based on the corrected response value calculated by applying a sensitivity correction coefficient to the response value of unprotected amino acids in the material, and the response value of the quantitative standard.

[0062] In the method according to this embodiment, since correction is performed by applying a sensitivity correction coefficient, it is not necessarily required to select a quantitative standard for each material being measured. Furthermore, once the sensitivity correction coefficient has been determined, even when performing impurity analysis in multiple types of materials (materials with different types of unprotected amino acids that may be contained as impurities), it is possible to more accurately measure unprotected amino acids contained in trace amounts as impurities using a limited number of quantitative standards (for example, one or two types). The method according to this embodiment has the advantages of minimizing the number of quantitative standards to be managed, significantly reducing the time required for sample preparation during quality evaluation, and not necessarily requiring separation studies of compounds that can be used as quantitative standards for each material being measured.

[0063] In peptide compounds containing multiple types of non-natural amino acids as amino acid residues, there is no need to select a quantitative standard with similar sensitivity for each non-natural amino acid to be measured, nor is there a need to prepare a large quantity of non-natural amino acids for the quantitative standard. Therefore, the method according to this embodiment can be particularly suitably used in situations where quality control of large quantities of amino acids, including non-natural amino acids, is required.

[0064] The response values ​​of unprotected amino acids in the material can be measured using the same method as the liquid chromatography method using CAD described above.

[0065] The corrected response value can be calculated by multiplying the peak area, which is the response value of unprotected amino acids in the material measured by CAD, by a sensitivity correction coefficient.

[0066] The quantitative value of unprotected amino acids is calculated based on the corrected response value and the response value of the quantitative standard. Specifically, the quantitative value of unprotected amino acids is calculated by applying the corrected response value to a calibration curve that shows the relationship between the response value of the quantitative standard and concentration, which has been prepared in advance. The calibration curve is a quadratic curve (y = ax) with the vertical axis (y axis) representing the response value and the horizontal axis (x axis) representing the concentration (unit: μg / mL). ２ The formula is (a + bx + c, where a, b, and c are coefficients). A calibration curve can be prepared using a standard solution containing the quantitative standard described above. The quantitative value of unprotected amino acids can be obtained by substituting the corrected response value for y in the calibration curve.

[0067] The measurement of the response value of unprotected amino acids in the material to obtain a quantitative value of unprotected amino acids may be carried out over several days (for example, three consecutive days).

[0068] The difference between the quantitative value and the theoretical value of unprotected amino acids may be within ±20%, more preferably within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, or ±3%. The difference between the quantitative value and the theoretical value of unprotected amino acids is calculated using the following formula: Formula: (Quantitative value of unprotected amino acids (μg / mL) - Theoretical value of unprotected amino acids (μg / mL)) / Quantitative value of unprotected amino acids (μg / mL) × 100

[0069] The theoretical value of unprotected amino acids is the actual concentration of unprotected amino acids calculated from the weighed value and purity of the unprotected amino acid standard.

[0070] [Method for producing peptide compounds] The method for producing peptide compounds according to this embodiment includes the following steps (1) to (3): (1) a step of quantifying the amount of unprotected amino acids in the materials used to produce the peptide compound by the method for quantifying unprotected amino acids described above; (2) a step of selecting materials in which the amount of unprotected amino acids is less than or equal to a predetermined value; and (3) a step of linking the amino acids protected by protecting groups in the selected materials with another amino acid or peptide.

[0071] Other amino acids and peptides in step (3) may each be protected with a protecting group. The other amino acids and peptides protected with a protecting group may be amino acids or peptides selected by carrying out steps (1) and (2) in the same manner as the amino acids protected with a protecting group in the selected material.

[0072] In materials where the content of unprotected amino acids is below a predetermined value, the content of unprotected amino acids may be 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0.9% by mass or less, 0.8% by mass or less, 0.7% by mass or less, 0.6% by mass or less, 0.5% by mass or less, 0.4% by mass or less, 0.3% by mass or less, 0.2% by mass or less, or 0.1% by mass or less, based on the total mass of the material.

[0073] The linking process may be carried out by a solid-phase synthesis method or a liquid-phase synthesis method.

[0074] In this specification, "solid-phase synthesis" refers to a method of synthesizing a target compound by binding the compound to a "solid support" and chemically reacting the compound with a reagent on the solid support. Solid-phase synthesis of peptides is a method of synthesizing a peptide by binding a desired amino acid or peptide to a solid support, and then sequentially linking further desired amino acids or peptides to the amino acid or peptide bound to the solid support to extend the peptide chain. The target peptide can be obtained by detaching the peptide bound to the solid support from the solid support. Peptide arrays used in the production of peptide libraries are also a form of solid-phase synthesis.

[0075] In this specification, the term "solid support" is not particularly limited as long as it can be used for the synthesis of peptide compounds by solid-phase synthesis. Specific examples of such solid supports include those that can be removed under acidic conditions, such as CTC resin, NovaSyn TGT resin (TGT resin), Wang resin, SASRIN resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin), and 4-methoxytrityl chloride resin (Mmt resin). The solid support can be appropriately selected according to the functional group of the amino acid used. For example, when using a carboxyl group (main chain carboxyl group, or side chain carboxyl group represented by Asp or Glu) or a hydroxyl group on an aromatic ring (phenol group represented by Tyr) as the functional group of the amino acid, it is preferable to use trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (CTC resin) as the solid support. When using an aliphatic hydroxyl group (such as Ser or Thr, which are aliphatic alcohol groups) as the functional group of an amino acid, it is preferable to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (CTC resin), or 4-methyltrityl chloride resin (Mtt resin) as the solid support. In this specification, the solid support may also be referred to as resin. The type of polymer constituting the solid support is not particularly limited. In the case of a resin composed of polystyrene, either 100-200 mesh or 200-400 mesh may be used. The crosslinking ratio is also not particularly limited, but 1% DVB (divinylbenzene) crosslinking is preferred. Examples of polymers constituting the solid support include TentaGel® or ChemMatrix®.

[0076] In this specification, "liquid-phase synthesis" refers to a method of synthesizing a target compound by chemically reacting compounds in a liquid phase (solution) without using a solid support. In the liquid-phase synthesis of peptides, amino acids and peptides that are not bound to a solid support can be used. Such liquid-phase synthesis of peptides also includes a method in which a functional group (tag) that solubilizes the peptide in the solvent is attached to the target peptide chain during the peptide synthesis reaction, and then a poor solvent is added during the isolation of the target peptide to isolate the target peptide chain as a solid with the tag attached (sometimes called the liquid-phase tag method or tag method). Examples of liquid-phase tagging methods include the following methods using hydrophobic tags (see: Y. Okada, H. Suzuki, T. Nakae, S. Fujita, H. Abe, K. Nagano, T. Yamada, N. Ebata, S. Kim and K. Chiba, Tag-Assisted Liquid-Phase Peptide Synthesis Using Hydrophobic Benzyl Alcohols as Supports, J. Org. Chem., 2013, 78, 320-327, or S. Yano et al., Molecules 2021, 26(12), 3497).

[0077] The present invention will be further illustrated by the following examples, but the present invention is not limited thereto. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods.

[0078] High-performance liquid chromatography (HPLC) analysis using a charged particle detector (CAD) was performed under the analytical conditions described below.

[0079] For quantitative analysis, we used readily available natural amino acids such as tryptophan (Trp), isoleucine (Ile), threonine (Thr), leucine (Leu), serine (Ser), glycine (Gly), or proline (Pro) as quantitative analysis standards.

[0080] Standard unprotected amino acids include H-MeNva-OH, H-Ile-OH, H-Pro-OH, H-MeAlGly-OH, H-MeGly-OH, H-Hyp(Et)-OH, H-MecVal-OH, H-MeGly(cPent)-OH, and H-MeAsp shown in Table 1. ２ -OtBu-OH was used.

[0081] HPLC-CAD Analysis Conditions (Analysis Conditions-1) Instrument: Thermo Fisher Scientific Vanquish UHPLC Detector: Charged Aerosol Detector Column: Intrada Amino Acid (Imtakt), 3.0 mm ID × 100 mm, 3 μm Mobile Phase: 0.1% Formic Acid (FA) / Acetonitrile (MeCN) (A), 10 mM NH4COOH aq. (B) Elution Method: Pump right B) 14% (3 min) → 100% (10 min) → 14% (10.1 min) → 14% (14 min) Pump left B) 14% (3.445 min) → 100% (10.445 min) → 14% (10.545 min) → 14% (14.445 min) Flow Rate: 1.0 mL / min Column Temperature: 35℃ Evaporator Temperature: 35℃ Filter constant: 5 s

[0082] [Example 1] Preparation of Calibration Curves for Quantitative Standards For each quantitative standard, a calibration curve was prepared from the peak area of ​​the chromatogram at concentrations of 5, 10, 20, 30, 40, and 50 μg / mL, and a quadratic regression equation was calculated. As shown in Figure 1, the calibration curves for each quantitative standard showed a high correlation with the quadratic curve calibration curve in the concentration range of 5 to 50 μg / mL. Furthermore, it was confirmed that there are differences in sensitivity characteristics among the various amino acids, with Pro showing high sensitivity and Ile showing low sensitivity.

[0083] Calculation of Sensitivity Correction Factors for Unprotected Amino Acid Standards Compared to Quantitative Standards The peak areas of chromatograms of unprotected amino acids at concentrations of 5, 10, 20, 30, 40, and 50 μg / mL were calculated, and the response ratio at each concentration was calculated by dividing these peak areas by the peak areas of the chromatograms of the quantitative amino acid standard at the same concentrations (response ratio at each concentration = peak area of ​​quantitative standard ÷ peak area of ​​unprotected amino acid). The average response ratio was obtained by averaging the response ratios at each concentration. This measurement was performed for three consecutive days, and the average response ratio obtained for each day was further averaged to obtain the sensitivity correction factor. The results are shown in Table 2.

[0084]

[0085] Determination of Unprotected Amino Acids Using Sensitivity Correction Factors The peak areas of chromatograms were calculated for unprotected amino acids at concentrations of 5, 10, 20, 30, 40, and 50 μg / mL, and the peak areas were corrected by multiplying them by a sensitivity correction factor. The concentration of unprotected amino acids was quantified by substituting the corrected peak area values ​​into the quadratic regression equation of the quantification standard. This quantification was performed for three consecutive days. Compared to quantification without using a sensitivity correction factor, the difference between the theoretical value and the quantitative value was significantly reduced in this example using a sensitivity correction factor. The results are shown in Tables 3-1 to 3-4 and 4-1 to 4-4. In these tables, results where the difference between the theoretical value and the quantitative value is ±10% or less are shown without coloring, results where the difference is greater than ±10% but within ±20% are shown in light gray, and results where the difference is greater than ±20% are shown in dark gray.

[0086]

[0087] When a sensitivity correction factor was used, the difference between the theoretical value and the quantitative value was approximately within ±20%, and in particular, when either Ile or Pro was used as the quantitative standard, it was confirmed that the difference between the theoretical value and the quantitative value was within ±10% for each concentration of all unprotected amino acids (bold boxes in Tables 3-1 to 3-4).

[0088] [Example 2] Further quantification was performed on two unnatural, unprotected amino acids, MecVal and MeCpg, using two natural amino acids, Ile and Pro, as quantitative standards. The method for calculating the sensitivity correction factor and the method for quantifying the concentration of the unprotected amino acids were the same as in Example 1. The results are shown in Tables 5 to 7. In Tables 6 to 7, results where the difference between the theoretical value and the quantitative value is ±10% or less are shown without coloring, results where the difference is greater than ±10% but within ±20% are shown in light gray, and results where the difference is greater than ±20% are shown in dark gray. As shown in Tables 6 to 7, it was confirmed that when either Ile or Pro is used as a quantitative standard, the difference between the theoretical value and the quantitative value can be suppressed at each concentration of the unprotected amino acid (bold frame in Table 6).

Claims

1. A method for quantifying unprotected amino acids in a material containing amino acids protected by a protecting group, comprising the step of obtaining a quantitative value of the unprotected amino acids in the material using a charged particle detector, wherein the quantitative value of the unprotected amino acids is obtained based on a corrected response value calculated by applying a sensitivity correction coefficient to the response value of the unprotected amino acids in the material measured by the charged particle detector, and the sensitivity correction coefficient is a value calculated based on the response values ​​of the unprotected amino acid standard and the quantitative standard measured by the charged particle detector, respectively.

2. The method according to claim 1, wherein the quantitative analysis standard is a natural amino acid.

3. The method according to claim 1 or 2, wherein the quantitative analysis standard is either isoleucine (Ile) or proline (Pro).

4. The method according to claim 3, wherein the quantitative analysis standard is isoleucine (Ile).

5. The method according to claim 3, wherein the quantitative analysis sample is proline (Pro).

6. The method according to any one of claims 1 to 5, wherein the unprotected amino acid is a non-natural amino acid.

7. The method according to any one of claims 1 to 6, wherein the unprotected amino acid is a non-natural amino acid with low UV detection sensitivity.

8. The method according to any one of claims 1 to 7, wherein the protecting group is at least one of Fmoc, Cbz, or t-Bu.

9. The method according to any one of claims 1 to 8, wherein the sensitivity correction coefficient is set based on the response ratio of the unprotected amino acid standard and the quantitative standard.

10. The method according to claim 9, wherein the sensitivity correction coefficient is set as the average of the response ratios at different concentrations.

11. The method according to any one of claims 1 to 10, wherein the material comprises two or more types of unprotected amino acids, and the sensitivity correction coefficient is set for each combination of the quantitative standard and the unprotected amino acid standard.

12. The method according to any one of claims 1 to 11, wherein the difference between the quantitative value and the theoretical value of the unprotected amino acid is within ±20%.

13. The method according to any one of claims 1 to 12, wherein the material is used for the synthesis of a cyclic peptide compound.

14. A method for quantifying unprotected amino acids in a material containing amino acids protected by a protecting group, comprising the following steps: (1) measuring the response values ​​of a standard of the unprotected amino acid and a standard for quantification using a charged particle detector; (2) calculating the response ratio between the standard of the unprotected amino acid and the standard for quantification; (3) setting a sensitivity correction coefficient based on the response ratio; and (4) obtaining a quantitative value of the unprotected amino acid in the material based on a corrected response value calculated by applying the sensitivity correction coefficient to the response value of the unprotected amino acid in the material and the response value of the standard for quantification.

15. A method for producing a peptide compound, comprising the following steps (1) to (3): (1) a step of quantifying the amount of unprotected amino acids in a material containing amino acids protected by a protecting group by the method of any one of claims 1 to 14; (2) a step of selecting a material in which the content of the unprotected amino acids is less than or equal to a predetermined value; and (3) a step of linking the amino acids protected by a protecting group in the selected material with another amino acid or peptide compound.

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