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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
  • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/30—Extraction; Separation; Purification by precipitation
  • C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
  • C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
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  • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C227/38—Separation; Purification; Stabilisation; Use of additives
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  • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
  • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
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  • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
  • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
  • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
  • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
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  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C269/06—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups by reactions not involving the formation of carbamate groups
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  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C269/08—Separation; Purification; Stabilisation; Use of additives
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  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C271/06—Esters of carbamic acids
  • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
  • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
  • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/30—Extraction; Separation; Purification by precipitation
  • C07K1/303—Extraction; Separation; Purification by precipitation by salting out
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
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  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/10—Nitrogen as only ring hetero atom
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• the present invention relates to a method for producing an amino acid salt, a peptide compound salt, or a solvate thereof, which includes a step of precipitating a lithium salt.
• Amino acid salts, peptide compound salts, and solvates thereof are important compounds that can serve as drug candidate molecules or synthetic intermediates thereof.
• Known methods for purifying amino acid salts, peptide compound salts, or solvates thereof include protecting or modifying their terminals and then purifying them.
• Patent Document 1 describes optically active aziridine-2-carboxylic acid in which an amino group is protected with a benzenesulfonyl group substituted with a nitro group at the 2- and/or 4-position using an optically active 3-chloroalanine derivative. It is disclosed that a derivative or an optically active 3-haloalanine derivative in which an amino group is protected with a benzenesulfonyl group substituted with a nitro group at the 2- and/or 4-positions is used.
• Patent Document 2 discloses that ⁇ -hydroxy- ⁇ -aminocarboxylic acid (however, the basicity of the amino group at the ⁇ -position is not blocked by the presence of a substituent on the amino group) or a salt with that acid is A method for producing ⁇ -halogeno- ⁇ -aminocarboxylic acid or a salt thereof is disclosed, which is characterized by halogenating a hydroxyl group by treatment with a halogenating agent.
• Non-Patent Document 1 states that when a peptide compound is protected with the Fmoc group, Fmoc- ⁇ - It has been shown that alanine is produced and that Fmoc- ⁇ -alanine is derived from Fmoc-OSu (Fmoc N-hydroxysuccinimide ester). Fmoc-OSu is one of the raw materials commonly used when protecting amino acids or peptide compounds with an Fmoc group.
• an object of the present invention is to provide a method for obtaining highly pure amino acid salts, peptide compound salts, or solvates thereof.
• a method for producing an amino acid salt, a peptide compound salt, or a solvate thereof including the following steps (A) and (B): Step (A); a step of bringing a lithium-containing substance into contact with the object to be purified, which is a mixture of the object (i) below, which is the object to be purified, and the object (ii) below, which is an impurity; (i) the above-mentioned amino acid having a protecting group at the N-terminus or the above-mentioned peptide compound having a protecting group at the N-terminus; (ii) a compound other than the above-mentioned purification target; Step (B): A step of precipitating the lithium salt of the object to be purified.
• a method for removing impurities from a substance to be purified including the following steps (A) and (B): Step (A); a step of bringing a lithium-containing substance into contact with the object to be purified, which is a mixture of the object (i) below, which is the object to be purified, and the object (ii) below, which is an impurity; (i) the above-mentioned amino acid having a protecting group at the N-terminus or the above-mentioned peptide compound having a protecting group at the N-terminus; (ii) a compound other than the above-mentioned purification target; Step (B): A step of precipitating the lithium salt of the object to be purified.
• the above impurities contain a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group as a protecting group.
• the method according to any one of [1] to [9], wherein the purification target product and the impurity have the same protecting group.
• the first organic solvent comprises nitriles, ethers, ketones, amides, ureas, sulfoxides, sulfones, alkanes, aromatic compounds, esters, and alkyl halides.
• the first organic solvent is a nitrile or an ether.
• the first organic solvent is acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, tetrahydrofuran, 1,4- Dioxane, 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, 1,2-dimethoxyethane, acetone, methyl ethyl ketone, methyl isobutyl ketone, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, 1,3 -dimethyl-2-imidazolidinone, dimethyl sulfoxide, sulfolane, heptane, methylcyclohexane, hexane, cyclohexane, pentane, benzene, toluene, 1,2-dimethyl
• the second organic solvent may include nitriles, alcohols, ethers, ketones, amides, ureas, sulfoxides, sulfones, alkanes, aromatic compounds, esters, and alkyl halides.
• the method according to [27] or [28], wherein the method is at least one selected from the group consisting of: [30] The method according to any one of [27] to [29], wherein the second organic solvent is a nitrile or an ether.
• the second organic solvent is acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, tetrahydrofuran, 1,4- Dioxane, 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, 1,2-dimethoxyethane, acetone, methyl ethyl ketone, methyl isobutyl ketone, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, 1,3 -dimethyl-2-imidazolidinone, dimethyl sulfoxide, sulfolane, heptane, methylcyclohexane, hexane, cyclohexane, pentane, benzene, toluene, 1,2-dimethyl
• the above steps (A) and (B) are performed in the presence of a first organic solvent,
• the volume of the first organic solvent per 1 g of the object to be purified is 1 ml or more and 40 ml or less throughout the step (A) and the step (B).
• Method. [42]
• the volume of the first organic solvent per 1 g of the object to be purified at the end of the step (A) is 1 ml or more when the proportion of the second organic solvent is increased in the step (B).
• the volume of the first organic solvent per 1 g of the object to be purified at the end of the step (A) is 1 ml or more if the proportion of the second organic solvent is not increased in the step (B).
• the above steps (A) and (B) are performed in the presence of a second organic solvent, and the volume of the second organic solvent per 1 g of the object to be purified is the same as that of the above step (A) and the above step (B).
• the amount of the lithium-containing substance in the total liquid component at the end of the step (A) is 0.5 equivalent or more and 2.0 equivalent or less, based on the amount of the purification target. , the method according to any one of [1] to [50].
• the lithium-containing substance includes lithium hydroxide, lithium carbonate, lithium hydride, trilithium phosphate, lithium methoxide, lithium ethoxide, lithium isopropoxide, lithium tert-butoxide methyllithium, n-butyllithium, At least one member selected from the group consisting of sec-butyllithium, tert-butyllithium, lithium amide, lithium diisopropylamide, lithium hexamethyldisilazide, and lithium tetramethylpiperidide, [1] to [51] The method described in any of the above.
• n represents a number from 1 to 3
• R 1 and R 2 are a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, a heteroaryl group, or a substituent in which two or more of these substituents are bonded.
• R 1 and R 2 may combine with N and A to form a ring, in which case R 1 and R 2 have one hydrogen atom subtracted from the structure of the substituent when not involved in ring formation.
• the N-terminal amino acid constituting the above amino acid or the above peptide compound is (S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoic acid, 3-(tert-butoxycarbonylamino)propanoic acid, 3 -(benzyloxycarbonylamino)propanoic acid, (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid, (S)-2-(benzyloxycarbonyl(methyl)amino)-3- Phenylpropanoic acid, 3-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid, (R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenyl-propanoic
• the polypeptide has 90% or more sequence identity in either or both of the N-terminus and C-terminus with a sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified.
• the above polypeptide contains one or more selected from the group consisting of a streptavidin-binding peptide tag sequence and a His tag sequence at either or both of the N-terminus and C-terminus, [58] to [62] The method described in any of the above. [64] The method according to any one of [58] to [63], wherein the polypeptide has 300 or more and 400 or less amino acid residues. [65] The method according to any one of [1] to [64], wherein the purity of the precipitate obtained in the step (B) is 95 mol% or more. [66] The method according to any one of [1] to [65], wherein the precipitate obtained in the step (B) has an apparent purity of 95% or more.
• [74] A method for producing a peptide compound, a salt thereof, or a solvate thereof, comprising the step according to any one of [1] to [73].
• [75] A cyclic peptide compound comprising the method according to any one of [1] to [74], and comprising a step of binding the amino acid or the peptide compound, or a solvate thereof, to the amino acid or the peptide compound. manufacturing method.
• the method according to any one of [73] to [75] comprising the step of cyclizing the peptide compound or the peptide compound obtained by bonding.
• a lithium salt of an amino acid, a lithium salt of a peptide compound, or a solvate thereof contains a lithium salt of an amino acid represented by the above general formula (1) with a purity of 99 mol% or more or an apparent purity of 99% or more
• the above lithium salt of a peptide compound is a lithium salt of an amino acid or a peptide containing a lithium salt of a peptide compound having a residue of an amino acid represented by the above general formula (1) with a purity of 99 mol% or more or an apparent purity of 99%.
• Lithium salts of compounds or solvates thereof [78] The lithium salt of an amino acid or the lithium salt of a peptide compound or a solvate thereof according to [77], which is a crystal.
• one or more means a number of one or more.
• substituents of a group the term means one up to the maximum number of substituents allowed by the group.
• one or more includes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and/or a larger number.
• ⁇ indicating a range includes the values at both ends thereof; for example, “A to B” means a range that is greater than or equal to A and less than or equal to B.
• the term "about" when used in conjunction with a numerical value means a value range of +10% and 10% of that numerical value.
• the meaning of the term “and/or” includes any combination of “and” and “or” as appropriate.
• “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.
• One embodiment of the present invention is a method for producing an amino acid salt, a peptide compound salt, or a solvate thereof, which includes the following steps (A) and (B).
• Step (A) a step of bringing a lithium-containing substance into contact with the object to be purified, which is a mixture of the object (i) below, which is the object to be purified, and the object (ii) below, which is an impurity;
• Step (B) Step of precipitating the lithium salt of the purification target product
• Another embodiment of the present invention is a method for removing impurities from a substance to be purified, which includes the above steps (A) and (B).
• Yet another embodiment of the present invention is a lithium salt of an amino acid, a lithium salt of a peptide compound, or a solvate thereof, wherein the lithium salt of an amino acid is a lithium salt of an amino acid represented by the general formula (1).
• the lithium salt of the peptide compound contains a salt with a purity of 99 mol% or more or an apparent purity of 99% or more, and the lithium salt of the peptide compound having a residue of an amino acid represented by the general formula (1) is 99 mol % or more in purity. % or more or with an apparent purity of 99%, a lithium salt of an amino acid, a lithium salt of a peptide compound, or a solvate thereof.
• the object of purification is used as a raw material for peptide compound, protein synthesis, and the like.
• Obtaining highly pure peptide compounds and proteins is important in order to suppress side effects caused by impurities when they are used as pharmaceuticals. That is, it is important to refine the raw materials constituting them to a high degree of purity.
• the present inventors surprisingly discovered that it is possible to purify the target product to a high degree of purity by converting it into a lithium salt. This improves the usefulness of peptide compounds and protein synthesis.
• the salt of an amino acid or the salt of a peptidic compound or a solvate thereof is the object of purification. It is obtained by treating the following steps (A) and (B).
• the object to be purified in the present invention is a mixture of the object to be purified and the impurity.
• the purification target refers to the amino acid having a protecting group at the N-terminus or the peptide compound having a protecting group at the N-terminus.
• An example of the object of purification is the amino acid represented by the above general formula (1).
• impurity refers to a compound other than the object to be purified, which is contained in the object to be purified.
• the impurities include quasi-specific impurities (amino acids having a protecting group at the N-terminus or peptide compounds having a protecting group at the N-terminus, other than the object of purification). More specifically, ⁇ -alanine or a derivative thereof is exemplified.
• a derivative is a compound that has been modified, such as introduction of a functional group, oxidation, reduction, or substitution of atoms, to an extent that does not significantly change the structure or properties of the base, when considering an organic compound as a base.
• amino acid as used herein includes natural amino acids and unnatural amino acids (sometimes referred to as amino acid derivatives).
• amino acid residue includes natural amino acid residues and unnatural amino acid (amino acid derivative) residues.
• Natural amino acids include glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), Tryptophan (Trp), histidine (His), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), cysteine (Cys), methionine (Met), lysine (Lys), arginine (Arg) , and proline (Pro).
• Unnatural amino acids are not particularly limited, but include ⁇ -amino acids, D-type amino acids, N-substituted amino acids (excluding Pro), ⁇ , ⁇ -disubstituted amino acids, amino acids whose side chains differ from natural amino acids, and hydroxycarbons. Examples include acids.
• non-natural N-substituted amino acids refer to N-substituted amino acids other than Pro.
• any steric configuration is acceptable for the amino acids herein.
• side chains of amino acids include alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, heteroaralkyl groups, cycloalkyl groups, and spiro bonds.
• cycloalkyl groups Each may be provided with a substituent, and these substituents are not limited, for example, any substituent containing a halogen atom, an O atom, an S atom, a N atom, a B atom, a Si atom, or a P atom.
• One or more may be independently selected from the following.
• examples thereof include optionally substituted alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, and oxo, aminocarbonyl, and halogen atoms.
• the amino acid according to one embodiment may be a compound having a carboxy group and an amino group in the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in the amino acid).
• Step (A) is a step of bringing a lithium-containing substance into contact with the object to be purified. Further, in step (A), a lithium salt of the purification target may be formed, and a lithium salt of the impurity may be formed. Step (B) is a step of precipitating the lithium salt of the purification target. Further, a part of step (A) and a part of step (B) may be performed temporally overlappingly. Note that, before step (A), the object to be purified may be purified by a method other than the present invention. For example, purification can also be achieved by precipitating the purification target in a free form without forming a lithium salt of the purification target. At least one of step (A) and step (B) can be performed in the presence of a solvent, and in this specification, the formation of a solid from the solvent is referred to as "precipitation.”
• the protecting group for the purification target may be a carbamate group.
• the carbamate group refers to a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group; It may be a nylmethoxycarbonyl group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group, it may be a fluorenylmethoxycarbonyl group or a benzyloxycarbonyl group, it may be a fluorenylmethoxycarbonyl group, a benzyloxycarbonyl group It may be a base.
• the protecting group for impurities may be a carbamate group.
• the carbamate group refers to a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group; It may be a nylmethoxycarbonyl group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group, it may be a fluorenylmethoxycarbonyl group or a benzyloxycarbonyl group, it may be a fluorenylmethoxycarbonyl group, a benzyloxycarbonyl group It may be a base.
• These protecting groups may be those that protect the N-terminus of impurities.
• the protecting group for the purification target and the protecting group for the impurity may be different or the same. It is preferable that the protecting group of the purification target product and the protecting group of the impurity are the same.
• Step (A) may be performed in the presence of the first organic solvent.
• the first organic solvent is a solvent that can dissolve the purification target. It is preferable that the solubility of the purification target substance in the first organic solvent is 20 g/L or more. It is preferable that the solubility of the lithium salt of the purification target in the first organic solvent is 20 g/L or less. It is preferable that the solubility of the impurity in the first organic solvent is 20 g/L or more.
• the first organic solvent examples include nitriles such as acetonitrile, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, and tetrahydrofuran.
• nitriles such as acetonitrile
• alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, and tetrahydrofuran.
• 1,4-dioxane 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, ethers such as 1,2-dimethoxyethane, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, dimethyl formamide, dimethyl Acetamide, amides such as N-methyl-2-pyrrolidone, ureas such as 1,3-dimethyl-2-imidazolidinone, sulfoxides such as dimethyl sulfoxide, sulfones such as sulfolane, heptane, methylcyclohexane, hexane, Alkanes such as cyclohexane and pentane, aromatic compounds such as benzene, toluene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, and 1,4-dimethylbenzane
• Step (A) may be performed in the presence of water.
• Step (B) may be performed in the presence of the second organic solvent.
• the second organic solvent is a solvent in which the lithium salt of the purification target has a low solubility, that is, a solvent that functions as a poor solvent for the lithium salt of the purification target.
• the solubility of the lithium salt of the object to be purified in the second organic solvent is preferably 10 g/L or less.
• Examples of the second organic solvent include nitriles such as acetonitrile, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, and tetrahydrofuran.
• nitriles such as acetonitrile
• alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, and tetrahydrofuran.
• 1,4-dioxane 2-methyltetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, diethyl ether, ethers such as 1,2-dimethoxyethane, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, dimethyl formamide, dimethyl Acetamide, amides such as N-methyl-2-pyrrolidone, ureas such as 1,3-dimethyl-2-imidazolidinone, sulfoxides such as dimethyl sulfoxide, sulfones such as sulfolane, heptane, methylcyclohexane, hexane, Alkanes such as cyclohexane and pentane, aromatic compounds such as benzene, toluene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, and 1,4-dimethylbenzane
• Step (B) may be carried out in the presence of water, and is preferably carried out in the presence of water from the viewpoint of dissolving the impurity lithium salt.
• the solubility of the impurity lithium salt in water is preferably 10 g/L or more.
• the second organic solvent may be used together with the first organic solvent in step (A).
• step (B) the lithium salt of the purification target is precipitated.
• the second organic solvent may be added to the first organic solvent used in step (A).
• the ratio of the first organic solvent or the ratio of the second organic solvent is the volume calculated by dividing the volume of the first organic solvent or the second organic solvent by the mass of the purification target. / mass ratio (ml/g, hereinafter also referred to as v/w).
• the first organic solvent and the second organic solvent may be the same solvent. Also good. In this case, there is no need to add the second organic solvent in step (B). In this case, when the lithium-containing substance is brought into contact with the object to be purified in step (A), step (B) is immediately started, and the lithium salt of the object to be purified is precipitated.
• the lithium salt to be purified is preferably precipitated as a solid.
• the solid here includes crystal or amorphous. Moreover, it is more preferable to precipitate (crystallize) as crystals.
• step (B) can also be called a step of crystallizing the lithium salt of the purification target.
• the volume of the first organic solvent per 1 g of the object to be purified may be 1 ml or more, 2 ml or more, or 3 ml or more, and 100 ml or less, 80 ml or less, 60 ml or more throughout step (A) and step (B). It may be 40 ml or less.
• the capacity of the first organic solvent per 1 g of the object to be purified is 1 ml or more and 100 ml or less, 1 ml or more and 80 ml or less, 2 ml or more and 60 ml or less, or 3 ml or more and 40 ml or less throughout step (A) and step (B). It's fine.
• the capacity of the first organic solvent (initial concentration of the first organic solvent) with respect to 1 g of the object to be purified at the end of step (A) increases the proportion of the second organic solvent in step (B).
• the amount may be 1 ml or more, 2 ml or more, or 3 ml or more, and may be 50 ml or less, 40 ml or less, 30 ml or less, or 20 ml or less.
• the initial concentration of the first organic solvent may be from 1 ml to 50 ml, from 1 ml to 40 ml, from 2 ml to 30 ml, or from 3 ml to 20 ml.
• the capacity of the first organic solvent (initial concentration of the first organic solvent) with respect to 1 g of the object to be purified at the end of step (A) does not increase the proportion of the second organic solvent in step (B).
• the volume may be 1 ml or more, 10 ml or more, 20 ml or more, 30 ml or more, or 35 ml or more, and may be 100 ml or less, 75 ml or less, 50 ml or less, or 37 ml or less.
• the initial concentration of the first organic solvent may be from 1 ml to 100 ml, from 10 ml to 75 ml, from 20 ml to 50 ml, from 30 ml to 37 ml, or from 35 ml to 37 ml.
• the volume of the second organic solvent per 1 g of the object to be purified may be 0 ml or more, 40 ml or less, 60 ml or less, 80 ml or less, or 100 ml or less throughout steps (A) and (B).
• the volume of the second organic solvent per 1 g of the object to be purified may be 0 ml or more and 100 ml or less, 0 ml or more and 80 ml or less, 1 ml or more and 60 ml or less, or 1 ml or more and 40 ml or less throughout steps (A) and (B).
• the volume of the second organic solvent per 1 g of the object to be purified at the end of step (B) may be 0 ml or more, 40 ml or less, 60 ml or less, 80 ml or less, or 100 ml or less.
• the volume of the second organic solvent per 1 g of the object to be purified at the end of step (B) may be 0 ml or more and 100 ml or less, 0 ml or more and 80 ml or less, 0 ml or more and 60 ml or less, or 0 ml or more and 40 ml or less.
• the volume of the water per 1 g of the purification target may be 0.1 ml or more, 0.3 ml or more, or 0.5 ml or more, and 50 ml or less, 30 ml or less, or 10 ml throughout steps (A) and (B). It may be less than or equal to 5 ml.
• the volume of water in the solvent is 0.1 ml to 50 ml, 0.1 ml to 30 ml, 0.3 ml to 10 ml, or 0.5 ml to 5 ml throughout steps (A) and (B). good.
• the volume of the water (initial concentration of water) per 1 g of the object to be purified may be 0.1 ml or more, 0.3 ml or more, or 0.5 ml or more, and 50 ml or less, 30 ml. Below, it may be 10 ml or less or 4 ml or less.
• the initial concentration of water may be 0.1 ml or more and 50 ml or less, 0.1 ml or more and 30 ml or less, 0.3 ml or more and 10 ml or less, or 0.5 ml or more and 4 ml or less.
• the volume of the water (initial concentration of water) per 1 g of the object to be purified may be 0.1 ml or more, 0.3 ml or more, or 0.5 ml or more, and 50 ml or less, 30 ml. Below, it may be 10 ml or less or 4 ml or less.
• the initial concentration of water may be 0.1 ml or more and 50 ml or less, 0.1 ml or more and 30 ml or less, 0.3 ml or more and 10 ml or less, or 0.5 ml or more and 4 ml or less.
• Step (A) and step (B) may be performed in the presence of a solvent other than the first organic solvent and the second organic solvent.
• solvents include phosphate buffer, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), Tris (trishydroxymethylaminomethane), bicine (N,N-di(2-hydroxyethyl)glycine), etc.
• An example is a buffer solution of
• the temperature at which step (A) is performed may be -20°C or higher, 0°C or higher, 10°C or higher, or 20°C or higher.
• the temperature at which step (A) is carried out may be 100°C or lower, 80°C or lower, 60°C or lower, 40°C or lower, or 30°C or lower.
• the temperature at which step (A) is carried out may be -20 to 100°C, may be 0 to 80°C, may be 10 to 60°C, may be 10 to 40°C, may be 20 to 30°C. It may be °C.
• the temperature at which step (B) is carried out may be -20°C or higher, 0°C or higher, 10°C or higher, 20°C or higher, 30°C or higher. The temperature may be 35°C or higher.
• the temperature at which step (A) is carried out may be 100°C or lower, 80°C or lower, 60°C or lower, 40°C or lower, or 30°C or lower. .
• the temperature at which step (A) is carried out may be -20 to 100°C, may be 0 to 80°C, may be 10 to 60°C, may be 10 to 40°C, may be 20 to 30°C. It may be °C.
• Conditions such as temperature and concentration of substances contained in various solutions in step (A) and step (B) may be the same or different. If these conditions are different between the two, conditions selected from the various conditions of step (A) described above may be combined. Further, the amount of the lithium-containing substance at the end of the step (A) is 0.5 equivalent or more, 0.8 equivalent or more, or 1.0 equivalent or more based on the amount of the substance to be purified. Often, it may be 2.0 equivalents or less, 1.5 equivalents or less, or 1.1 equivalents or less. The content of the lithium-containing substance based on the mass of the reactant is 0.5 equivalent or more and 2.0 equivalent or less, 0.8 equivalent or more and 1.5 equivalent or less, or 1.0 equivalent or more and 1.1 equivalent or less. It's good.
• the lithium-containing substance in this specification refers to a lithium-containing substance to be brought into contact with the purification target in step (A).
• the lithium-containing substance used in step (A) include lithium hydroxide, lithium tert-butoxide, lithium hydroxide, lithium carbonate, lithium hydride, trilithium phosphate, lithium methoxide, lithium ethoxide, and lithium isopropoxy.
• lithium tert-butoxide methyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium, lithium amide, lithium diisopropylamide, lithium hexamethyldisilazide, and lithium tetramethylpiperidide. At least one type is mentioned.
• the object to be purified and the lithium-containing substance can be brought into contact in the presence of the first organic solvent.
• the object to be purified, the lithium-containing substance, and the first organic solvent may be mixed in the same container from the beginning, or the object to be purified and the first organic solvent are mixed, and the resulting mixture is mixed with lithium.
• the lithium-containing substance may be added, or the lithium-containing substance and the first organic solvent may be mixed, and the object to be purified may be added to the resulting mixed liquid.
• the content of the purification target in the purification target which is the standard for determining the amounts of the first organic solvent, the second organic solvent, and the lithium-containing substance, may be an actual value, an estimated value, or a calculated value. It may be an actual value or a calculated value, and is most preferably an actual value.
• the amino acids having a protecting group at the N-terminus or the amino acids constituting the peptide compound having a protecting group at the N-terminus that are contained in the purification target include the amino acids represented by the above general formula (1) (also referred to as amino acids (1)). ), hydrophobic amino acids, aliphatic amino acids, aromatic amino acids, etc.
• n represents a number of 1 or more and 3 or less, and may be an integer of 1 or more and 3 or less. n may be 1, 2 or 3.
• n 2 or 3
• R 2 and the two R 1s is not a hydrogen atom, and even if R 2 and two or more of the two R 1s are not hydrogen atoms. Often, R 2 and all three of the two R 1s may not be hydrogen atoms.
• R 1 and R 2 are a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heterocyclyl group, a heteroaryl group, or a substituent in which two or more of these substituents are bonded. (for example, a benzyl group), and these groups may have a substituent, and these groups may be saturated or unsaturated.
• A represents a carbon atom
• B represents a hydrogen atom or a bonding point with an amino acid or a peptide compound.
• Z represents a fluorenylmethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or a 2-(trimethylsilyl)ethoxycarbonyl group.
• R 1 and R 2 may be combined with N and A to form a ring.
• R 1 and R 2 are substituents having a structure in which one hydrogen atom has been extracted from the structure of the substituent when it is not involved in ring formation.
• alkyl is a monovalent group derived from an aliphatic hydrocarbon by removing one arbitrary hydrogen atom, and has a heteroatom (atom other than carbon and hydrogen atoms) in the skeleton. ) or have a subset of hydrocarbyl or hydrocarbon group structures that do not contain unsaturated carbon-carbon bonds and contain hydrogen and carbon atoms.
• Alkyl includes not only linear ones but also branched ones. Specifically, the alkyl is an alkyl having 1 to 20 carbon atoms (C 1 to C 20 , hereinafter "C p to C q " means the number of carbon atoms is p to q), Preferably C 1 -C 10 alkyl, more preferably C 1 -C 6 alkyl.
• alkyl examples 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, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl , 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,
• alkylene refers to a divalent group derived from the above “alkyl” by further removing one hydrogen atom, and C 4 -C 8 alkylene is preferred.
• alkylene includes -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -CH(CH 3 )CH 2 -, -C(CH 3 ) 2 -, -(CH 2 ) 4 -, -CH(CH 3 )CH 2 CH 2 -, -C(CH 3 ) 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 C(CH 3 ) 2 - , -CH 2 CH 2 CH (CH 3 )-, -(CH 2 ) 5 -, -(CH 2 ) 6 -, -(CH 2 ) 7 -, -(CH 2 ) 8 -, and the like.
• alkenyl is a monovalent group having at least one double bond (two adjacent SP 2 carbon atoms). Depending on the configuration of the double bond and substituents (if present), the geometry of the double bond can be Entadel (E) or Caribbean (Z), cis or trans configuration.
• Alkenyl includes not only straight-chained alkenyl but also branched alkenyl.
• the alkenyl includes C 2 -C 10 alkenyl, more preferably C 2 -C 6 alkenyl, and specifically, for example, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl. (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, and the like.
• alkynyl is a monovalent group having at least one triple bond (two adjacent SP carbon atoms).
• Alkynyl includes not only straight chain but also branched chain.
• Alkynyl preferably includes C 2 -C 10 alkynyl, more preferably C 2 -C 6 alkynyl, and specifically includes, for example, ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl.
• cycloalkyl means a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, and includes a monocyclic ring, a bicyclo ring, and a spiro ring.
• cycloalkyl include C 3 -C 8 cycloalkyl, specifically, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[ 3.3] heptyl and the like.
• aryl means a monovalent aromatic hydrocarbon ring, and preferably includes C 6 to C 10 aryl.
• aryl include phenyl, naphthyl (eg, 1-naphthyl, 2-naphthyl), and the like.
• heterocyclyl means a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. Heterocyclyl may have double and/or triple bonds in the ring, carbon atoms in the ring may be oxidized to form carbonyl, and may be a single ring or a fused ring.
• the number of atoms constituting the ring is preferably 4 to 10 (4 to 10 membered heterocyclyl), more preferably 4 to 7 (4 to 7 membered heterocyclyl).
• heterocyclyl examples include azetidinyl, oxiranyl, oxetanyl, azetidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, Pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,2-thiazinane, thiadiazolidinyl, azetidinyl, oxazolidone, benzodioxanyl, benzoxazolyl, dioxolanyl, dioxanyl, Tetrahydro
• heteroaryl means an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms.
• the ring may be a monocyclic ring, a fused ring with another ring, or a partially saturated ring.
• the number of atoms constituting the ring may be 5 to 12 (5- to 12-membered heteroaryl), 6 to 10 (5- to 12-membered heteroaryl), and 6 to 7 (6- to 7-membered heteroaryl). ).
• heteroaryl examples include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl.
• benzothiadiazolyl benzothiazolyl
• benzoxazolyl benzoxadiazolyl
• benzimidazolyl indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, imidazopyridyl, and the like.
• “may be substituted” means “may have a substituent", and means that a certain group may be substituted with any substituent. do.
• the substituent includes an alkyl group, an alkoxy group, a fluoroalkyl group, a fluoroalkoxy group, an oxo group, an aminocarbonyl group, an alkylsulfonyl group, an alkylsulfonylamino group, a cycloalkyl group, an aryl group, and a heteroaryl group.
• Examples include a group, a heterocyclyl group, an arylalkyl group, a heteroarylalkyl group, a halogen group, a nitro group, an amino group, a monoalkylamino group, a dialkylamino group, a cyano group, a carboxyl group, an alkoxycarbonyl group, and a formyl group.
• the N-terminal amino acid constituting the amino acid or peptide compound can be obtained by a reductive amination reaction catalyzed by a polypeptide containing an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1. .
• Identity with the amino acid sequence represented by SEQ ID NO: 1 is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more , 99% or more, or 100%.
• BLAST Basic Local Alignment Search Tool
• NCBI National Center for Biotechnology Information
• the polypeptide according to this embodiment may be a polypeptide in which one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 have been modified, such as 1 to 20, 1 to 15, or 1 to 10. 1 to 7 or 1 to 5 amino acid residues may be modified.
• the number of amino acid residues that have been modified may be up to 3, up to 2, or up to 1.
• the modification may be one or more selected from the group consisting of substitution, deletion, and insertion, and may be a substitution. Modifications may be conservative modifications.
• a conservative modification means a modification of an amino acid residue that does not reduce the desired catalytic activity compared to the polypeptide before modification.
• the modification may be a substitution with a natural amino acid that is different from the amino acid residue before modification.
• the natural amino acids used for the substitution include glycine, alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, asparagine, glutamic acid, aspartic acid, cysteine, methionine, lysine, arginine, and It may be one or more selected from the group consisting of proline.
• the polypeptide according to this embodiment includes a histidine residue at position 44, a phenylalanine residue at position 117, a methionine residue at position 141, a threonine residue at position 156, and a threonine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1.
• Amino acid residues located at positions corresponding to one or more amino acid residues selected from the group consisting of histidine residue, glutamine residue at position 186, tryptophan residue at position 253, and lysine residue at position 260 are modified.
• the polypeptide may include a sequence in which an amino acid residue located at a site corresponding to one or more amino acid residues selected from the group consisting of lysine residue at position 260 has been modified, and further, a methionine residue at position 141.
• a polypeptide containing a sequence in which an amino acid residue located at a position corresponding to one or more amino acid residues selected from the group consisting of a tryptophan residue at position 253, and a lysine residue at position 260 is modified. It's fine.
• the polypeptide according to this embodiment has a sequence in which the amino acid residue located at the position corresponding to the histidine residue at position 44 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than histidine residue. It may be a polypeptide containing.
• the polypeptide may be a polypeptide containing the amino acid sequence represented by SEQ ID NO:2.
• the amino acid residues represented by X in SEQ ID NO: 2 are alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, isoleucine residue, lysine residue, leucine residue, and methionine residue.
• an asparagine residue a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue, or a tyrosine residue.
• the amino acid residue located at the position corresponding to the histidine residue at position 44 may include a sequence substituted with a methionine residue. That is, the polypeptide according to this embodiment has an amino acid sequence (H44M) in which the amino acid residue located at the site corresponding to the histidine residue at position 44 in the amino acid sequence represented by SEQ ID NO: 1 is replaced with a methionine residue. It may be included.
• the polypeptide according to this embodiment has a sequence in which the amino acid residue located at the site corresponding to the phenylalanine residue at position 117 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than the phenylalanine residue. It may be a polypeptide containing.
• the polypeptide may be a polypeptide containing the amino acid sequence represented by SEQ ID NO:3.
• the amino acid residues represented by X in SEQ ID NO: 3 are alanine residue, aspartic acid residue, glutamic acid residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, and methionine residue.
• an asparagine residue a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue, or a tyrosine residue.
• the amino acid residue located at the site corresponding to the phenylalanine residue at position 117 may be substituted with a leucine residue. That is, the polypeptide according to this embodiment has an amino acid sequence (F117L) in which the amino acid residue located at the site corresponding to the phenylalanine residue at position 117 in the amino acid sequence represented by SEQ ID NO: 1 is replaced with a leucine residue. It may be included.
• the polypeptide according to this embodiment has a sequence in which the amino acid residue located at the site corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than the methionine residue. It may be a polypeptide containing.
• the polypeptide may be a polypeptide containing the amino acid sequence represented by SEQ ID NO:4.
• the amino acid residues represented by X in SEQ ID NO: 4 are alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, and leucine residue.
• an asparagine residue a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue, or a tyrosine residue.
• the amino acid residues located at the site corresponding to the methionine residue at position 141 are tyrosine residues, tryptophan residues, valine residues, threonine residues, A sequence substituted with one or more amino acid residues selected from the group consisting of serine residue, arginine residue, leucine residue, lysine residue, isoleucine residue, histidine residue, phenylalanine residue, and alanine residue. It may be included.
• the amino acid residue located at the site corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is a tyrosine residue, a tryptophan residue, a valine residue, Amino acid sequences substituted with threonine, serine, arginine, leucine, lysine, isoleucine, histidine, phenylalanine, or alanine residues (M141Y, M141W, M141V, M141T, M141S, M141R, M141L, M141K, M141I, M141H, M141F or M141A).
• amino acid residue located at the site corresponding to the methionine residue at position 141 is a group consisting of tyrosine residue, tryptophan residue, valine residue, lysine residue, isoleucine residue, phenylalanine residue, and alanine residue.
• the sequence may include a sequence substituted with one or more amino acid residues selected from the following.
• the amino acid residue located at the site corresponding to the methionine residue at position 141 in the amino acid sequence represented by SEQ ID NO: 1 is a tyrosine residue, a tryptophan residue, a valine residue, It may contain an amino acid sequence (M141Y, M141W, M141V, M141K, M141I, M141H, M141F, or M141A) substituted with a lysine residue, isoleucine residue, histidine residue, phenylalanine residue, or alanine residue.
• the polypeptide according to this embodiment has a sequence in which the amino acid residue located at the site corresponding to the threonine residue at position 156 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than the threonine residue. It may be a polypeptide containing.
• the polypeptide may be a polypeptide containing the amino acid sequence represented by SEQ ID NO:5.
• the amino acid residues represented by X in SEQ ID NO: 5 are alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, and leucine residue.
• an asparagine residue a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue, or a tyrosine residue.
• the amino acid residue located at the site corresponding to the threonine residue at position 156 may include a sequence substituted with a serine residue. That is, the polypeptide according to this embodiment has an amino acid sequence (T156S) in which the amino acid residue located at the site corresponding to the threonine residue at position 156 in the amino acid sequence represented by SEQ ID NO: 1 is replaced with a serine residue. It may be included.
• the polypeptide according to this embodiment has a sequence in which the amino acid residue located at the position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than histidine residue. It may be a polypeptide containing.
• the polypeptide may be a polypeptide containing the amino acid sequence represented by SEQ ID NO: 6.
• the amino acid residues represented by X in SEQ ID NO: 6 are alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, isoleucine residue, lysine residue, leucine residue, and methionine residue.
• an asparagine residue a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue, or a tyrosine residue.
• the amino acid residue located at the position corresponding to the histidine residue at position 182 is a tyrosine residue, a glutamine residue, a methionine residue, a leucine residue, It may include a sequence substituted with one or more amino acid residues selected from the group consisting of glycine residues, phenylalanine residues, and alanine residues. That is, in the polypeptide according to the present embodiment, the amino acid residue located at the site corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is a tyrosine residue, a glutamine residue, or a methionine residue.
• a leucine residue, a glycine residue, a phenylalanine residue, or an alanine residue H182Y, H182Q, H182M, H182L, H182G, H182F, or H182A.
• a sequence in which the amino acid residue located at the position corresponding to the histidine residue at position 182 is replaced with one or more amino acid residues selected from the group consisting of methionine residue, leucine residue, and phenylalanine residue. may contain.
• the amino acid residue located at the position corresponding to the histidine residue at position 182 in the amino acid sequence represented by SEQ ID NO: 1 is a methionine residue, a leucine residue, or a phenylalanine residue.
• the amino acid sequence may include a substituted amino acid sequence (H182M, H182L, or H182F).
• the polypeptide according to this embodiment has a sequence in which the amino acid residue located at the site corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than the glutamine residue. It may be a polypeptide containing.
• the polypeptide may be a polypeptide containing the amino acid sequence represented by SEQ ID NO:7.
• the amino acid residues represented by X in SEQ ID NO: 7 are alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, and leucine residue. , methionine residue, asparagine residue, proline residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue, or tyrosine residue.
• the amino acid residue located at the position corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is combined with a methionine residue and a glutamic acid residue.
• the amino acid residue may contain a sequence substituted with one or more amino acid residues selected from the group consisting of: That is, the polypeptide according to this embodiment is an amino acid in which the amino acid residue located at the site corresponding to the glutamine residue at position 186 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a methionine residue or a glutamic acid residue. (Q186M or Q186E).
• the polypeptide according to this embodiment has a sequence in which the amino acid residue located at the site corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than the tryptophan residue. It may be a polypeptide containing.
• the polypeptide may be a polypeptide containing the amino acid sequence represented by SEQ ID NO:8.
• the amino acid residues represented by X in SEQ ID NO: 8 are alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, and leucine residue. , methionine residue, asparagine residue, proline residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, or tyrosine residue.
• the amino acid residue located at the site corresponding to the tryptophan residue at position 253 is a tyrosine residue, a valine residue, a threonine residue, a serine residue,
• the amino acid residue located at the site corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is a tyrosine residue, a valine residue, or a threonine residue.
• amino acid residue located at the site corresponding to the tryptophan residue at position 253 is substituted with one or more amino acid residues selected from the group consisting of leucine residue, isoleucine residue, and histidine residue. It may be included. That is, in the polypeptide according to the present embodiment, the amino acid residue located at the site corresponding to the tryptophan residue at position 253 in the amino acid sequence represented by SEQ ID NO: 1 is a leucine residue, an isoleucine residue, or a histidine residue. may contain an amino acid sequence substituted with (W253L, W253I, or W253H).
• the polypeptide according to this embodiment has a sequence in which the amino acid residue located at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than the lysine residue. It may be a polypeptide containing.
• the polypeptide may be a polypeptide containing the amino acid sequence represented by SEQ ID NO:9.
• the amino acid residues represented by X in SEQ ID NO: 9 are alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, leucine residue, and methionine residue.
• an asparagine residue a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue, or a tyrosine residue.
• the amino acid residue located at the site corresponding to the lysine residue at position 260 is a tyrosine residue, a tryptophan residue, a threonine residue, a serine residue, and an arginine residue.
• the amino acid residue located at the site corresponding to the lysine residue at position 260 in the amino acid sequence represented by SEQ ID NO: 1 is a tyrosine residue, a tryptophan residue, or a threonine residue.
• an amino acid sequence substituted with a serine residue, arginine residue, glutamine residue, asparagine residue, methionine residue, leucine residue, histidine residue, glycine residue, phenylalanine residue, glutamic acid residue or alanine residue K260Y, K260W, K260T, K260S, K260R, K260Q, K260N, K260M, K260L, K260H, K260G, K260F, K260E, or K260A).
• the amino acid residue located at the site corresponding to the lysine residue at position 260 is substituted with one or more amino acid residues selected from the group consisting of glutamine residue, methionine residue, glutamic acid residue, and asparagine residue.
• the polypeptide according to this embodiment has an amino acid sequence a1 (mutation: M141V), which is represented by SEQ ID NO: 4, and the amino acid residue represented by X is a valine residue; Amino acid sequence a2 (mutation: M141Y) in which the amino acid residue represented by Amino acid sequence a4 (mutation: W253H) represented by number 8 and the amino acid residue represented by X is a histidine residue, or represented by SEQ ID NO: 9 and the amino acid residue represented by X is a glutamic acid residue It can contain a certain amino acid sequence a5 (mutation: K260E).
• the polypeptide according to this embodiment can include a sequence having 90% or more sequence identity with the amino acid sequence a1, a2, a3, a4, or a5.
• the sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and 100% or more. It's good.
• the polypeptide according to this embodiment may be a polypeptide comprising a sequence in which one or more amino acid residues in the amino acid sequence a1, a2, a3, a4, or a5 are modified, and 1 to 20, 1 to 15 1-10, 1-7 or 1-5 amino acid residues may be modified.
• the number of amino acid residues that have been modified may be up to 3, up to 2, or up to 1.
• the polypeptide according to this embodiment may be fused with other polypeptides or proteins. That is, the polypeptide according to this embodiment has a sequence in which one amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has been modified at either or both of the N-terminus and C-terminus, and 90% or more of the sequence. It may have an amino acid sequence (another amino acid sequence) other than the sequence having identity.
• the other amino acid sequence may be, for example, a tag sequence.
• polypeptides or proteins having other amino acid sequences include His tags (6XHis, 10XHis, etc.), which are tags consisting of several (for example, 6, 10, etc.) His (histidine) residues, streptavidin, etc.
• Streptavidin-binding peptide tag SBP tag containing an amino acid sequence capable of binding to the biotin-binding site of MBP (maltose binding protein), FLAG (Hopp, T.P.
• influenza agglutinin HA
• human c-myc fragment VSV-GP fragment
• examples include p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, Ick tag, ⁇ -tubulin fragment, B-tag, Protein C fragment, Stag, StrepTag, HaloTag, and the like.
• the His tag may be, for example, the amino acid sequence represented by SEQ ID NO: 11.
• the SBP tag may be, for example, the amino acid sequence represented by SEQ ID NO: 12.
• the polypeptide according to this embodiment may include one or more selected from the group consisting of a streptavidin-binding peptide tag sequence and a His tag sequence at either or both of the N-terminus and C-terminus, and the C-terminus It may include a streptavidin-binding peptide tag sequence (SBP tag) and a His tag sequence.
• SBP tag streptavidin-binding peptide tag sequence
• the polypeptide according to this embodiment has an amino acid sequence a1 (mutation: M141V), which is represented by SEQ ID NO: 4, and the amino acid residue represented by X is a valine residue; Amino acid sequence a2 (mutation: M141Y) in which the amino acid residue represented by Amino acid sequence a4 (mutation: W253H) represented by number 8 and the amino acid residue represented by X is a histidine residue, or represented by SEQ ID NO: 9 and the amino acid residue represented by X is a glutamic acid residue It can have a certain amino acid sequence a5 (mutation: K260E) and a tag sequence.
• the polypeptide according to this embodiment connects the amino acid sequence a1, a2, a3, a4, or a5 and the tag sequence in addition to the amino acid sequence a1, a2, a3, a4, or a5 and the tag sequence. It may have a linker sequence.
• the linker sequence may have, for example, an amino acid sequence represented by GGSS or GGS.
• the polypeptide has an amino acid sequence a1, a2, a3, a4, or a5, and a linker sequence that is bonded to the C-terminus of the amino acid sequence a1, a2, a3, a4, or a5, and is represented by the amino acid sequence: GGSS. and a His tag bound to the linker sequence and represented by SEQ ID NO: 11.
• One or more amino acid residues in polypeptide X1 may be modified, with 1-20, 1-15, 1-10, 1-7 or 1-5 amino acid residues modified. It's fine. In polypeptide X1, the number of modified amino acid residues may be up to 3, up to 2, or up to 1.
• the polypeptide according to one embodiment has an amino acid sequence a1, a2, a3, a4 or a5, and an SBP tag represented by SEQ ID NO: 12 bonded to the C-terminus of the amino acid sequence a1, a2, a3, a4 or a5,
• the polypeptide X2 may be composed of a His tag represented by SEQ ID NO: 11 and a linker sequence connecting the His tag and SBP tag and represented by the amino acid sequence GGS.
• One or more amino acid residues in polypeptide It's fine.
• the number of modified amino acid residues may be up to 3, up to 2, or up to 1.
• polypeptide according to this embodiment may be used as a mixture with other polypeptides, or may be used in an isolated/produced state.
• the desired amino acid can be obtained through a step of isolating and producing the desired product.
• the number of amino acid residues may be 300 or more, 310 or more, 320 or more, or 325 or more, and may be 330. Moreover, it may be 400 or less, 390 or less, 380 or less, or 375 or less. The number of amino acid residues in the polypeptide may be 300 or more and 400 or less.
• the number of amino acid residues of the polypeptide according to this embodiment is 300 or more, 310 or more, 320 or more, or 325 or more. It may be 330. Moreover, it may be 360 or less, 350 or less, 340 or less, or 335 or less.
• the number of amino acid residues of the polypeptide according to this embodiment is 340 or more, 350 or more, 360 or more, or It may be 370 or more, and may be 374. Moreover, it may be 400 or less, 390 or less, 380 or less, or 375 or less.
• polypeptide according to this embodiment may be used as a monomer, or may be used in a form in which two or more monomers are associated with each other. Furthermore, the polypeptide according to this embodiment may be a homodimer.
• the number of amino acid residues is twice that when it exists as a monomer.
• Peptide compounds having a protecting group at the N-terminus are 30 residues or less, 25 residues or less, 20 residues or less, 15 residues or less, 14 residues or less, 13 residues or less, 12 residues or less, 11 residues or less , up to 10 residues, up to 9 residues, up to 8 residues, up to 7 residues, up to 6 residues, up to 5 residues, up to 4 residues, up to 3 residues, or up to 2 residues, and up to 2 residues Groups or more, 3 or more residues, 4 or more residues, 5 or more residues, 6 or more residues, 7 or more residues, 8 or more residues, 9 or more residues, 10 or more residues, 11 or more residues, 12 residues It may be a peptide compound consisting of at least 13 amino acid residues, 13 or more residues, 14 or more residues, or 15 or more amino acid residues.
• the peptide compound having a protecting group at the N-terminus may be a peptide compound consisting of 2 to 10 amino acid residues, 2 to 8 residues, 2 to 6 residues, or 2 to 4 amino acid residues.
• peptide compounds having a protecting group at the N-terminus can be made from amino acids of 5 to 30 residues, 7 to 25 residues, 8 to 15 residues, 9 to 14 residues, 10 to 13 residues, or 11 residues. It may be a peptide compound, etc. Also included are peptide compounds containing the residue of amino acid (1) at the N-terminus.
• the purified product may be precipitated such that the content of quasi-specific impurities contained in the precipitate (refined product) obtained in step (B) is reduced.
• the purified product may be precipitated such that the purity of the purified product is 95 mol% or more.
• the purity is more preferably 96 mol% or more, even more preferably 97 mol% or more, even more preferably 98 mol% or more, particularly preferably 99 mol% or more, and 100 mol% or more. Most preferably it is mole %.
• purity means, in a broad sense, the proportion of the target substance contained in the substance whose purity is to be measured, and in a narrow sense, it means the proportion of the target substance contained in the purified product. It means the ratio of the amount of lithium salt in the purification target based on the total amount of lithium salt and lithium salt as a specific impurity.
• the purified product may be precipitated such that the impurity content in the purified product is 5 mol% or less.
• the impurity content is preferably 4 mol% or less, more preferably 3% mol or less, even more preferably 2 mol% or less, particularly preferably 1 mol% or less, Most preferably, it is 0 mol%.
• impurity content refers to the amount of the lithium salt as a specific impurity, based on the total amount of the lithium salt as the purification target and the lithium salt as the specific impurity, contained in the purified product. means.
• Purity or impurity content can be measured, for example, by nuclear magnetic resonance, gas chromatography, or high performance liquid chromatography. When using high performance liquid chromatography, it can be calculated from the area ratio of UV absorption peaks. Equation 1a below shows a formula for determining purity, and Equation 2a below shows a formula for determining impurity content.
• the amount of substance per unit UV absorption peak area is the value obtained by dividing the UV absorption peak area of the object of purification or specific impurity by the amount of substance.
• the purified product may be precipitated such that the apparent purity of the purified product is 95% or more.
• the apparent purity is more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, particularly preferably 99% or more, and 100%. is most preferred.
• "apparent purity” refers to purification based on the sum of the UV absorption peak areas at specific wavelengths of the purified product and specific impurities, which are measured after separating the purified product by high-performance liquid chromatography. It means the ratio of the UV absorption peak area of the target object at the specific wavelength.
• the purified product may be precipitated such that the apparent impurity content of the purified product is 5% or less.
• the apparent impurity content is preferably 4% or less, more preferably 3% or less, even more preferably 2% or less, particularly preferably 1% or less, and 0%. is most preferable.
• "apparent impurity content” is based on the sum of the UV absorption peak areas at specific wavelengths of the purified product and specific impurities, which are measured after separating the purified product into the purified target product and specific impurities using high performance liquid chromatography. , means the ratio of the UV absorption peak area of a specific impurity at a specific wavelength.
• the following formula 1b shows a formula for determining the apparent purity
• the following formula 2b shows a formula for determining the apparent impurity content.
• the specific wavelength is a wavelength within 10% of the maximum absorption wavelength exhibited by the protecting group of the object of purification.
• any maximum absorption wavelength may be selected as the maximum absorption wavelength as long as it exhibits an absorption intensity of 30% or more based on the absorption intensity at the maximum absorption wavelength among them. .
• the influence of noise can be reduced by selecting the long wavelength side among the plurality of maximum absorption wavelengths.
• the sample concentration can be adjusted depending on the absorption intensity of the selected maximum absorption wavelength. At the time of measurement, the maximum absorption wavelength that is considered to be the most preferable from the viewpoint of ease of adjusting sample concentration and noise reduction can be selected.
• UV light of a specific wavelength can be 254 nm for Fmoc group, 197 nm for Boc group, and 210 nm for Cbz group.
• the content of ⁇ -alanine or its derivatives in the object to be purified can be reduced by obtaining the lithium salt.
• the production method according to the present embodiment may include a step of reducing the content of ⁇ -alanine or its derivative.
• the manufacturing method according to the present embodiment may further include a step of forming a salt other than a lithium salt.
• a free amino acid or a free peptide compound can be obtained by desalting the lithium salt obtained by the production method according to one embodiment. Desalting can be performed by known methods. Furthermore, salts other than lithium salts can be produced from the obtained free amino acid or peptide compound.
• Salts other than lithium salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, Salts with organic acids such as benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid; salts with alkali metals (sodium or potassium); salts with alkaline earth metals such as calcium and magnesium; ammonium salts etc. can be exemplified.
• the obtained free amino acid or peptide compound is linked with an amino acid or peptide compound by a conventional method to produce a peptide compound having the desired number of amino acid residues, for example, a cyclic portion.
• Peptide compounds cyclic peptide compounds
• the number of amino acid residues of the cyclic peptide compound according to the present embodiment is 30 or less, 25 or less, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 Below, it may be 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
• the cyclic peptide compound according to the present embodiment includes 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more , or a peptide compound consisting of 15 or more amino acids.
• the number of amino acid residues of the cyclic peptide compound according to the present embodiment may be 2 or more and 10 or less, 2 or more and 8 or less, 2 or more and 6 or less, or 2 or more and 4 or less. Further, the number of amino acid residues of the cyclic peptide compound according to the present embodiment may be 5 or more and 30 or less, 7 or more and 25 or less, 8 or more and 15 or less, 9 or more and 14 or less, 10 or more and 13 or less, or 11.
• the "cyclic part" of a cyclic peptide compound means a cyclic part formed by connecting two or more amino acid residues.
• the number of amino acid residues constituting the cyclic part of the cyclic peptide compound is 5 or more and 14 or less, 6 or more and 14 or less, 7 or more and 14 or less, 8 or more and 14 or less, 9 or more and 14 or less, 10 or more and 14 or less, 5 or more and 13 or less, 6 to 13, 7 to 13, 8 to 13, 9 to 13, 10 to 13, 5 to 12, 6 to 12, 7 to 12, 8 to 12, 9 to 12, It may be 10 or more and 12 or less, 5 or more and 11 or less, 6 or more and 11 or less, 7 or more and 11 or less, 8 or more and 11 or less, 9 or more and 11 or less, 10 or more and 11 or less, or 11.
• the cyclic portion is preferably formed through a covalent bond such as an amide bond, a carbon-carbon bond forming reaction, an SS bond, a thioether bond, or a triazole bond, and is a linear peptide compound in which amino acid residues are linked. It can be formed by bonding a group on the N-terminal side and a group on the C-terminal side. Specifically, for example, it can be formed by bonding the amino group at the N-terminus and the carboxyl group at the C-terminus of a linear peptide compound in which amino acid residues are linked.
• Cyclization includes cyclization by carbon-nitrogen bonds such as amide bonds, cyclization by carbon-oxygen bonds such as ester bonds and ether bonds, cyclization by carbon-sulfur bonds such as thioether bonds, and carbon-carbon bonds. Any form may be used, such as cyclization by , cyclization by a sulfur-sulfur bond, or cyclization by constructing a heterocyclic ring. Among these, cyclization via a covalent bond such as an amide bond or a carbon-carbon bond is preferred, and cyclization via an amide bond between a carboxy group in a side chain and an amino group in the main chain is more preferred.
• the positions of the carboxy group, amino group, etc. used for cyclization may be on the main chain or on the side chain, and are not particularly limited as long as they are at positions that allow cyclization.
• the cyclic peptide compound according to this embodiment may have a ring atom number of 15 or more and 46 or less.
• number of ring atoms means the number of atoms (ring atoms) of a cyclic compound including the innermost part of the ring, and if the compound has multiple rings, the number of atoms is the largest. Defined as the number of atoms in the ring. In addition, when two rings share some atoms, the number of ring atoms of each ring is calculated using the one with the smaller number of atoms in common.
• the number of ring atoms of tetrahydrofuran (THF) is 5
• the number of ring atoms of tacrolimus (FK506) is 21.
• the number of ring atoms of the cyclic peptide compound according to the present embodiment may be, for example, 34 or more and 46 or less, 34 or more and 43 or less, 34 or more and 40 or less, 34 or more and 37 or less, 34 or more and 36 or less, or 34.
• the ring atoms used to calculate the number of ring atoms may be selected from the group consisting of carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms, sulfur atoms, phosphorus atoms, and silicon atoms; It may be selected from the group consisting of nitrogen atoms and oxygen atoms.
• the cyclic peptide compound according to this embodiment may have a linear part in addition to the cyclic part.
• the specific embodiment of the number of amino acid residues of the cyclic peptide compound is the same as the specific embodiment of the number of amino acid residues of the peptide compound described above.
• the cyclic peptide compound has a linear portion, it is preferable that the total number of amino acid residues in the cyclic portion and the linear portion fall within the same range.
• the number of amino acid residues constituting the cyclic part is 5 to 15, 6 to 15, 6 to 14, 7 to 14, 8 to 14, or
• the number of amino acid residues constituting the linear part is preferably 7 or more and 13 or less, more preferably 7 or more and 12 or less, or 8 or more and 11 or less, even more preferably 9 or more and 11 or less, particularly preferably 10 or 11, and the number of amino acid residues forming the linear part is 1 or more. It is preferably 8 or less, 1 or more and 7 or less, 1 or more and 6 or less, 1 or more and 5 or less, or 1 or more and 4 or less, and more preferably 1 or more and 3 or less.
• the molecular weight of the cyclic peptide compound according to this embodiment is not particularly limited, and includes, for example, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, or 950 or more, preferably 1000 or more, 1100 or more, 1200 or more, 1300 or more, or 1400 or more.
• the upper limit of the molecular weight of the peptide compound according to this embodiment is not particularly limited, but preferably 5000 or less, 4000 or less, 3000 or less, 2500 or less, or 2000 or less.
• Molecular weight in this specification means the sum of the atomic weights of atoms constituting a compound molecule (unit: "g/mol"), and is obtained by calculating the sum of the atomic weights of atoms included in the molecular structural formula (unit: g/mol). "g/mol”). In this specification, the unit of molecular weight may be omitted. Note that the molecular weight of the peptide compound can be measured by liquid chromatography mass spectrometry (LC/MS) as described in Examples.
• LC/MS liquid chromatography mass spectrometry
• the peptide compound containing one or more N-substituted amino acid residues is a peptide compound containing one or more N-substituted amino acid residues, as long as it is a peptide compound containing one or more N-substituted amino acid residues.
• Specific embodiments can be applied without restriction.
• the cyclic peptide compound according to this embodiment may contain one or more N-substituted amino acid residues, preferably contains at least three N-substituted amino acid residues, and preferably contains at least four N-substituted amino acid residues. More preferably, it contains at least 5 N-substituted amino acid residues.
• the N-substituted amino acid residues may be present continuously or discontinuously in the N-substituted cyclic peptide compound.
• Raw materials were obtained from commercial suppliers and used without purification unless otherwise noted.
• the abbreviations listed in Tables 1 and 2 were used in the examples.
• Preparation Example 1 W253H -SBP -HIS preparation Semorable Semorable, represented by amino acid sequence, which X is HIS, and at the C -termin, STREPTTAVIDIN -BINDINDING PEPTIDE tag array Q), linker sequence (GGS) and HIS tag sequences ( HHHHHH) was synthesized and cloned into an E. coli expression vector.
• This expression vector was introduced into BL21 (DE3) E. coli strain (Novagen) and cultured at 18°C for 2 days using Overnight Express Instant TB Medium (Novagen) to express the final preparation of the target protein (sequence No. 10, W253H).
• 2-cyclopentyl-2-oxo-acetic acid sodium salt (3.9 g, 24 mmol), methylamine hydrochloride (8.1 g, 120 mmol), D-glucose (8.7 g, 48 mmol) and N,N-di(2- Hydroxyethyl)glycine (7.8 g, 48 mmol) was dissolved in distilled water (50 mL), and the external temperature of the reaction vessel was set at 25°C. A 5M aqueous sodium hydroxide solution (6.0 mL) was added to adjust the pH to 8.8.
• a 50 w/v% aqueous sodium hydroxide solution (1.5 mL) was added and stirred for 3 hours.
• N-carbobenzoxyoxysuccinimide (1.2 g, 4.8 mmol) was added and stirred for 90 minutes.
• a 50 w/v% aqueous sodium hydroxide solution (0.5 mL) was added and stirred for 18 hours.
• Concentrated hydrochloric acid (8.0 mL) was added, the target product was extracted into the organic layer, and the obtained organic layer was concentrated to dryness under reduced pressure at an external temperature of the reaction vessel of 40°C.
• Methyl tert-butyl ether (15 mL) was added, and the organic layer was washed twice with a 5% aqueous disodium hydrogen phosphate solution (10 mL x 2) and once with a 2M aqueous sodium hydroxide solution (10 mL), and the target product was added to the aqueous layer. Extracted. The resulting aqueous layers were combined and methyl tert-butyl ether (20 mL) and concentrated hydrochloric acid (1.5 mL) were added to re-extract the target product into the organic layer. The obtained organic layer was concentrated to dryness under reduced pressure at an external temperature of the reaction vessel of 40°C.
• 2-cyclopentyl-2-oxo-acetic acid sodium salt (5.3 g, 33 mmol), methylamine hydrochloride (11 g, 160 mmol), D-glucose (12 g, 65 mmol), N,N-di(2-hydroxyethyl)glycine (11 g, 65 mmol) was dissolved in distilled water (69 mL), and the external temperature of the reaction vessel was set at 25°C. A 50 w/v% aqueous sodium hydroxide solution (4.8 mL) was added to adjust the pH to 9.2.
• NADP+ (0.25g, 0.33mmol
• D-glucose dehydrogenase 26mg, 1300U, 50U/mg
• NMAADH modified enzyme solution final preparation, 2.0mL, equivalent to 53mg as enzyme, 1.0% by mass
• Concentrated hydrochloric acid (7.5 mL) was added to adjust the pH to 1.9, and the mixture was stirred for 1 hour.
• Insoluble matter was filtered through a Celite pad, and the Celite pad was washed with distilled water (11 mL) to obtain a (S)-2-cyclopentyl-2-(methylamino)acetic acid solution (130 g).
• Methyl tert-butyl ether (5.3 mL), acetonitrile (2.7 mL), and N-carbobenzoxyoxysuccinimide (1.8 g, 7.2 mmol) were added and stirred for 105 minutes.
• Acetonitrile (1.6 mL) was added and the mixture was further stirred for 30 minutes.
• a 50 w/v% aqueous sodium hydroxide solution (0.8 mL) was added to adjust the pH to 9.3, and the mixture was stirred for 105 minutes.
• N-carbobenzoxyoxysuccinimide (1.0 g, 3.9 mmol) was added and stirred for 105 minutes.
• Synthesis Example 3 Synthesis method of (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-lithium acetate salt seed crystals (S)-2-(benzyloxycarbonyl(methyl)amino)-2- Cyclopentyl-acetic acid (1.0 g, 3.4 mmol, commercially available) was dissolved in tetrahydrofuran (5.0 ml, 5.0 v/w) and stirred at an external temperature of the reaction vessel of 25°C. Lithium tert-butoxide (0.30 g, 3.8 mmol) was added, and precipitation of crystals was confirmed.
• Comparative Example 1 and Example 1 Purification of amino acid derivative in free form and purification with lithium salt Purification of amino acid derivative in free form (carboxylic acid) and purification with lithium salt were compared. Amino acid derivatives containing impurities were isolated as free forms or lithium salts, and their apparent purity was compared. The apparent purity or apparent impurity content was calculated from the UV absorption peak area ratio of high performance liquid chromatography. The apparent purity was calculated using Formula 1b, and the apparent impurity content was calculated using Formula 2b.
• Heptane (1.0 mL, 0.6 v/w), seed crystals ((S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid (free form) crystals, commercially available product, 5.0 mg ) was added and stirred for 15 minutes, and precipitation of crystals was confirmed.
• Example 1-1 Purification of (S)-2-(benzyloxycarbonyl(methyl)amino)-2-cyclopentyl-acetic acid with lithium salt
• Acetonitrile (5.0 ml, 10 v/w) was added over 10 minutes, seed crystals (synthesized according to Synthesis Example 3, 5.0 mg) were added and stirred for 40 minutes, and precipitation of crystals was confirmed.
• the external temperature of the reaction vessel was set at 25°C, and the mixture was stirred for 3 hours.
• the external temperature of the reaction vessel was set to 0° C., and the mixture was stirred for 30 minutes.
• Acetonitrile (2.5 ml, 5.0 v/w) was added over 5 minutes, and the mixture was further stirred for 30 minutes.
• Acetonitrile (5.0 ml, 10 v/w) was added over 5 minutes and stirred for 30 minutes.
• Example 2 Purification of amino acid derivatives by lithium chloride The effect of purifying amino acid derivatives by lithium chloride was evaluated. 3-aminopropanoic acid ( ⁇ -alanine) having the same protecting group was added as an impurity to an amino acid derivative having a specific protecting group to form a mixture. The mixture was isolated and purified by lithium chloride, and the purification effect was verified by comparing the apparent purity of the mixture and the isolated compound. The apparent impurity content was calculated using the same method as described in Example 1.

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• 2023
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