Method for producing amino acid n-carboxyanhydride (NCA)

Abstract

It is possible to provide a method for producing an amino acid N-carboxyanhydride (NCA) in a short time without using water as a solvent by including a step A in which a first amino acid in which an amino group is protected by a carbamate protecting group and a first halogenating agent that is thionyl halide or oxalyl chloride are reacted in the presence of a first tertiary amine.

Description

Method for producing amino acid N-carboxyanhydride (NCA)

[0001] The present invention relates to a method for producing amino acid N-carboxyanhydrides (NCAs).

[0002] Amino acid N-carboxyanhydrides (hereinafter sometimes referred to as "NCAs") are frequently used as raw material monomers for polypeptides, which are important as pharmaceuticals and pharmaceutical carriers.

[0003] Although several methods for synthesizing NCAs are known, the Leuchs method, in which an amino acid whose N-terminus is carbamate-protected is cyclized by the action of an electrophile such as thionyl chloride, is one of the most widely used NCA synthesis methods (e.g., Non-Patent Document 1). Since the Leuchs method was first reported by Leuchs et al. in 1906, many improved methods have been proposed. For example, in 1992, a method was reported in which NCAs were obtained at room temperature in a few hours using a combination of triphosgene and triethylamine (Non-Patent Document 2). However, from the perspective of realizing flow synthesis, a reaction time of several hours is too long, and further reduction of the reaction time is desired.

[0004] The present inventors have reported a method for synthesizing NCAs by the Fuchs method using unprotected amino acids, which allows NCAs to be obtained on a time scale of seconds (Non-Patent Document 3). However, the method of Non-Patent Document 3 requires water as a solvent, and therefore has the problem that, for example, if the subsequent organic synthesis reaction is averse to water, the prepared NCA cannot be used directly in the subsequent reaction. Since many organic synthesis reactions are averse to water, the method of Non-Patent Document 3 is disadvantageous for large-scale synthesis.

[0005] For the above reasons, there is a need to develop a novel method for synthesizing NCA that is suitable for flow synthesis.

[0006] Chem. Ges. 1906, 39, 857-861. J. Org. Chem. 1992, 57, 2755-2756. Angew. Chem. Int. Ed. 2018, 57, 11389-11393.

[0007] The present invention has been made in view of the current state of the prior art as described above, and a main object of the present invention is to provide a method for producing amino acid N-carboxyanhydride (NCA) in a short period of time without requiring water as a solvent.

[0008] The present inventors have conducted extensive research to achieve the above-mentioned object. As a result, they have found that an amino acid N-carboxyanhydride (NCA) can be obtained in a short time without the need for water as a solvent by reacting an amino acid whose amino group is protected with a carbamate-based protecting group with a specific halogenating agent in the presence of a tertiary amine. Based on this finding, the present inventors have conducted further research and completed the present invention. That is, the present invention encompasses the following features.

[0009] Item 1. A method for producing an amino acid N-carboxyanhydride (NCA), comprising: Step A: reacting a first amino acid, the amino group of which is protected with a carbamate-protecting group, with a first halogenating agent in the presence of a first tertiary amine; and the first halogenating agent contains a thionyl halide and / or oxalyl chloride.

[0010] Item 2. The amino acid N-carboxyanhydride (NCA) is represented by the general formula (1):

[0011] [In the formula, R １ indicates the side chain of an amino acid residue. １ may form a ring together with the adjacent nitrogen atom via the carbon atom to which it is bonded. ２ represents a hydrogen atom or a monovalent organic group. n represents 1 or 2, and when n is 2, two R １ and two R ２ may be the same or different.]

[0012] Item 3. The first amino acid is represented by general formula (2):

[0013] [In the formula, R １ indicates the side chain of an amino acid residue. １may form a ring together with the adjacent nitrogen atom via the carbon atom to which it is bonded. ２ represents a hydrogen atom or a monovalent organic group. n represents 1 or 2, and when n is 2, two R １ and two R ２ may be the same or different. ３ represents a carbamate protecting group.] The method for producing an amino acid according to Item 1 or 2.

[0014] Item 4. The first tertiary amine is represented by general formula (3):

[0015] [In the formula, R ４ , R ５ and R ６ are the same or different and represent an alkyl group, an aryl group, or a heteroaryl group. ４ , R ５ and R ６ wherein two or more of the groups may be linked to have one or more heteroatoms or substituents, or may form a ring.

[0016] Item 5. The production method according to any one of Items 1 to 4, wherein the first halogenating agent is thionyl chloride.

[0017] Item 6. The n is 1, and the R ２ Item 4. The method according to Item 2 or 3, wherein R is a hydrogen atom.

[0018] Item 7. The production method according to any one of Items 1 to 6, wherein the amount of the first tertiary amine used is 0.5 moles to 3 moles per mole of the first amino acid.

[0019] Item 8. The method according to any one of Items 1 to 7, wherein step A is carried out by a flow method.

[0020] Item 9. A method for producing an acylated amino acid N-carboxyanhydride (NCA), comprising a step B of reacting the amino acid N-carboxyanhydride (NCA) obtained by the production method according to any one of Items 1 to 8 with a halide of a second amino acid or peptide.

[0021] Item 10. A method for producing a two-residue-extended peptide, comprising a step C of reacting the acylated NCA obtained by the production method according to Item 9 with a third amino acid or peptide.

[0022] According to the method of the present invention, amino acid N-carboxyanhydride (NCA) can be obtained in a short time without requiring water as a solvent.

[0023] FIG. 1 is a schematic diagram showing a general configuration of a microflow reactor.

[0024] In this specification, the term "containing" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of."

[0025] In this specification, the expression "A to B" indicating a range of values ​​means "greater than or equal to A and less than or equal to B."

[0026] 1. Method for Producing NCA Amino acid N-carboxyanhydride (NCA), the target of the present invention, can be produced by a production method including Step A, in which a first amino acid having an amino group protected with a carbamate protecting group is reacted with a first halogenating agent in the presence of a first tertiary amine, wherein the first halogenating agent contains a thionyl halide or oxalyl chloride. This method for synthesizing NCA using a combination of a tertiary amine and the halogenating agent is a novel synthesis method that has not been reported previously.

[0027] According to the method of the present invention, epimerization of amino acids is unlikely to occur even when amino acids that are prone to epimerization are included. Furthermore, according to the method of the present invention, it is possible to safely and easily provide NCA on a large scale using inexpensive commercially available raw materials. In particular, when the microflow method is used in the method of the present invention, it is possible to obtain high-purity NCA in high yield by appropriately selecting a tertiary amine.

[0028] 1-1. NCA The NCA is not particularly limited, and any of known NCAs and NCAs derived from known amino acids can be widely used. Among them, NCAs represented by the general formula (1):

[0029] [In the formula, R １ indicates the side chain of an amino acid residue. １ may form a ring together with the adjacent nitrogen atom via the carbon atom to which it is bonded. ２ represents a hydrogen atom or a monovalent organic group. n represents 1 or 2, and when n is 2, two R １ and two R ２ may be the same or different.] is preferred.

[0030] In general formula (1), R １ is a side chain of an amino acid residue. In this specification, the side chain of an amino acid residue is defined as a group consisting of an amino acid having an amino group and a carboxy group in one molecule.

[0031] It means a monovalent group excluding a group represented by the following formula:

[0032] The side chain of the amino acid residue may be either a natural amino acid residue or a synthetic amino acid residue, and examples thereof include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (ILE), phenylalanine (Phe), serine (Ser), threonine (Thr), lysine (Lys), 5-hydroxylysine (Hyl), arginine (Arg), aspartic acid (Asp), asparagine (Asn), glutamine, and the like. Examples of the side chains include those of natural amino acid residues such as hydroxyl group (Glu), glutamine (Gln), cysteine ​​(CySH), cystine (Cyss), cysteic acid (Cya), methionine (Met), tyrosine (Tyr), thyroxine (Thy), proline (Pro), hydroxyproline (Hyp), tryptophan (Trp), histidine (His), β-alanine, N-methyl-β-alanine, sarcosine, γ-aminobutyric acid, kainic acid, and derivatives thereof. As will be apparent to those skilled in the art, for example, when the side chain of the amino acid residue is the side chain of glycine, R １ is a hydrogen atom, and when it is the side chain of alanine, R １ is a methyl group.

[0033] Furthermore, as the side chain of the amino acid residue, a side chain of an amino acid residue having a protecting group can be adopted depending on the type of functional group to be protected. When the amino acid residue has an amino group or a carboxy group in the side chain, the side chain of the amino acid residue having a protecting group is preferred from the viewpoint of avoiding side reactions. On the other hand, when the amino acid residue consists only of amino acid residues having functional groups other than an amino group or a carboxy group in the side chain, the side chain of the amino acid residue not having a protecting group is preferred from the viewpoint of eliminating the deprotection step, improving atom economy, and reducing waste. According to the method of the present invention, when step A is performed by a flow method (preferably a microflow method), side reactions derived from the side chain are unlikely to occur even if the side chain of the amino acid residue does not have a protecting group.

[0034] The protecting group carried by the side chain of an amino acid residue may be any protecting group known to be carried by the side chain of an amino acid residue, and examples thereof include alkyl protecting groups such as a tert-butyl (t-Bu) group, a benzyl group, an allyl group, a methyl group, and a triphenylmethyl group (Trt); silyl protecting groups such as a tert-butyldimethylsilyl group (TBS); aryl protecting groups such as a p-methoxybenzyl group (PMB) and a p-methoxyphenyl group (PMP); amide protecting groups such as a formyl group and an acetyl group (Ac); phthalimide protecting groups such as a phthaloyl group (Phth); Examples of protecting groups include carbamate-type protecting groups such as aryloxycarbonyl group (Cbz), tert-amyloxycarbonyl group (Aoc), 9-fluorenylmethoxycarbonyl group (Fmoc), tert-butyloxycarbonyl group (Boc), allyloxycarbonyl group (Alloc), and 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as 3-nitro-2-pyridinesulfenyl group (Npys), 2-nitrobenzenesulfonyl group (Ns), and (2-trimethylsilyl)-ethanesulfonyl group (SES).

[0035] In general formula (1), R ２is a hydrogen atom or an organic group. The organic group is not particularly limited, and examples thereof include hydrocarbon groups such as alkyl groups, aryl groups, and heteroaryl groups.

[0036] The alkyl group is not particularly limited, and examples thereof include chain alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as methyl, ethyl, n-propyl, n-butyl, and n-pentyl groups; branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as isopropyl, isobutyl, sec-butyl, and tert-butyl groups; and cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8), such as cyclopentyl and cyclohexyl groups.

[0037] The aryl group is not particularly limited, and any of a monocyclic aryl group, a fused ring aryl group, and a polycyclic aryl group can be used. Examples thereof include aryl groups having 6 to 18 carbon atoms (particularly 6 to 14 carbon atoms), such as a phenyl group, a hydroxyphenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, a biphenyl group, a terphenyl group, a fluorenyl group, an indolyl group, an imidazolyl group, a pyrenyl group, and a triphenylenyl group.

[0038] The heteroaryl group is not particularly limited, and both a monocyclic heteroaryl group and a polycyclic heteroaryl group can be employed. Examples thereof include a pyrrolidyl group, a pyrrolyl group, a tetrahydrothienyl group, a thienyl group, an oxolanyl group, a furanyl group, an imidazolyl group, an N-methylimidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, an N,N-dimethyl-4-aminopyridyl group, a pyrazyl group, an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, and a quinoxalyl group.

[0039] The organic group (a hydrocarbon group such as an alkyl group, an aryl group, or a heteroaryl group) may have a substituent. Examples of the substituent include, but are not particularly limited to, a hydroxyl group, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), the alkyl group, the aryl group, an alkoxy group (a methoxy group, an ethoxy group, a propoxy group, etc.), a carboxy group, and an amino group. When the organic group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

[0040] The substituent of the hydrocarbon group may have a protecting group. As the protecting group, any protecting group known as a protecting group for the above-mentioned substituent can be used, and examples thereof include alkyl-type protecting groups such as tert-butyl (t-Bu) group, benzyl group, allyl group, methyl group, and triphenylmethyl group (Trt); silyl-type protecting groups such as tert-butyldimethylsilyl group (TBS); aryl-type protecting groups such as p-methoxybenzyl group (PMB) and p-methoxyphenyl group (PMP); amide-type protecting groups such as formyl group and acetyl group (Ac); phthalimide-type protecting groups such as phthaloyl group (Phth); benzyl Examples of protecting groups include carbamate-type protecting groups such as an oxycarbonyl group (Cbz), a tert-amyloxycarbonyl group (Aoc), a 9-fluorenylmethoxycarbonyl group (Fmoc), a tert-butyloxycarbonyl group (Boc), an allyloxycarbonyl group (Alloc), and a 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns), and a (2-trimethylsilyl)-ethanesulfonyl group (SES). When protecting groups are present, the number of protecting groups is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

[0041] In general formula (1), R ２ From the viewpoint of improving the yield and suppressing side reactions, the group represented by the formula (I) is preferably a hydrogen atom or a methyl group, and more preferably a hydrogen atom.

[0042] In the general formula (1), n ​​is 1 or 2. From the viewpoint of improving the yield and suppressing side reactions, n is preferably 1. When n is 2, two R １and two R ２ may be the same or different. As will be apparent to those skilled in the art, when n is 1, the NCA obtained by the method of the present invention is an α-amino acid N-carboxyanhydride (α-NCA), and when n is 2, it is a β-NCA.

[0043] The amino acid residues constituting the NCA may be either the L- or D-optical isomer. The production method of the present invention can suppress epimerization.

[0044] From the above, specific examples of NCA include, for example:

[0045] etc.

[0046] The NCA can be obtained singly or in combination of two or more kinds.

[0047] The NCA, the object of the present invention, is useful as a raw material for peptide and polypeptide synthesis, and for the synthesis of amino acid derivatives and peptide analogs. It is common knowledge that NCA is generally difficult to store. However, there are limited methods for producing large amounts of NCA in a short period of time. Therefore, the method of the present invention is useful in opening the way to on-site production of NCA. Furthermore, the method of the present invention is applicable to the continuous multi-step synthesis of useful molecules using NCA.

[0048] 1-2. Step A In step A, a first amino acid whose amino group is protected with a carbamate protecting group is reacted with a first halogenating agent containing thionyl halide and / or oxalyl chloride in the presence of a first tertiary amine. In step A, if the first tertiary amine is not present, an NCA cannot be obtained.

[0049] Step A can be carried out by either a batch method or a flow method. From the viewpoints of high safety and ease of scale-up, the flow method is more preferred, and from the viewpoints of the ability to precisely control the reaction temperature and reaction time, the microflow method is even more preferred.

[0050] In this specification, the term "batch method" refers to a batch operation, specifically a series of operations in which the substances to be reacted are charged into a reaction vessel or reactor at once, reacted, and then removed after equilibrium or a certain reaction rate is reached.

[0051] In this specification, the term "flow method" refers to a continuous operation, specifically, an operation in which raw materials and substances required for their processing are continuously fed into and discharged from an apparatus at a constant rate (flow rate). In this specification, the term "microflow method" refers to a flow method (continuous operation) using a microflow reactor.

[0052] In particular, the reaction step of the present invention can be carried out using a microflow reactor, which includes, for example, a flow path for transporting a fluid containing a raw material or an intermediate to be used in the reaction, a pump for feeding the fluid into the flow path, and a mixer for mixing the fluid.

[0053] The configuration of the microflow reactor and the method for producing NCA of the present invention using the same will be described below with reference to Fig. 1. However, it goes without saying that the production method of the present invention is not limited to the embodiment shown in Fig. 1.

[0054] 1 is a schematic diagram showing a general configuration of a microflow reactor 1. In one embodiment, the microflow reactor 1 can include a syringe pump 11 that contains a first liquid, a syringe pump 12 that contains a second liquid, a syringe pump 13 that contains a third liquid, a syringe pump 14 that contains a fourth liquid, a syringe pump 15 that contains a fifth liquid, and a syringe pump 16 that contains a sixth liquid.

[0055] In one embodiment, the microflow reactor 1 can include flow paths F1 to F11 for transporting fluids. The inner diameter of the flow paths may be, for example, 0.1 mm to 50 mm, or 0.3 mm to 1 mm. In one embodiment, the microflow reactor 1 can include mixers M1 to M5 for mixing fluids. The mixers are not particularly limited, but for example, a V-shaped mixer, a T-shaped mixer, or the like, in which fluids are mixed by being introduced into the mixer. In such mixers, the interfacial area between the two liquids is increased by the entrainment flow generated within the flow paths, making it easy to mix two or more reaction liquids within several milliseconds. The inner diameter of the flow paths within the mixers can be, for example, 0.1 mm to 50 mm, or 0.2 mm to 1 mm.

[0056] The inner diameter of the flow path may be the diameter of the inner portion of the flow path (the portion through which the fluid passes) in the cross section of the flow path in a direction perpendicular to the longitudinal direction of the flow path. If the shape of the inner portion of the flow path is not a perfect circle, the inner diameter of the flow path may be the diameter of the shape of the inner portion of the flow path converted into a perfect circle on an area basis.

[0057] The syringe pumps 11 to 16 and the flow paths F1 to F11 may be made of, for example, a resin such as plastic or elastomer, glass, metal, or ceramic.

[0058] In one embodiment, the NCA production method of the present invention can use the syringe pumps 13 and 14, flow paths F3, F4, and F8, and mixer M2 shown in FIG. 1 . The third liquid can also contain a first amino acid and a first tertiary amine, and the fourth liquid can contain a first halogenating agent. In this embodiment, the third liquid can also contain the first amino acid, and the fourth liquid can contain the first tertiary amine and a first halogenating agent, but the present invention is not limited to either embodiment. In the NCA production method of the present invention, the former case, i.e., the third liquid contains the first amino acid and the first tertiary amine, and the fourth liquid contains the first halogenating agent, is preferred.

[0059] By operating the syringe pump 13, the third liquid can move through flow path F3 and flow into mixer M2. By operating the syringe pump 14, the fourth liquid can move through flow path F4 and flow into mixer M2. The third liquid and the fourth liquid are then mixed by mixer M2 to form a second mixed liquid, which can be sent to flow path F8. The second mixed liquid containing NCA moves through flow path F8 and flows into mixer M3, where it can be used in a subsequent reaction, stored in any test tube, or subjected to isolation, purification processes, and in-line analysis.

[0060] The microflow reactor 1 described above makes it easy to increase the area for heat exchange per volume of reaction solution. In addition, it is easy to control the reaction time by adjusting the flow rate and the length of the flow path. This makes it easy to strictly control the reaction solution, which in turn makes it easy to minimize the progress of undesired side reactions and improve the yield of the target product.

[0061] After the reaction using a microflow reactor is completed, the target compound can be obtained in high purity and high yield through normal isolation and purification steps as needed. Furthermore, when the target compound is obtained in high yield through the reaction using a microflow reactor, the isolation and purification steps can be omitted or simplified, and the target compound can be directly subjected to crystallization or analysis.

[0062] The method for producing an NCA of the present invention is not limited to one performed using a microflow reactor, and may also use, for example, a batch vessel having a small volume and capable of achieving a high stirring speed. The volume of the mixing section of the batch vessel may be 1 mL to 100 mL, or may be 5 mL to 50 mL.

[0063] As described above, the method for producing NCA of the present invention can be carried out by a liquid phase method, and therefore can be easily scaled up.

[0064] The reaction temperature in step A is preferably −80° C. to 100° C., more preferably 0° C. to 80° C., and even more preferably 0° C. to 60° C., from the viewpoints of suppressing epimerization and shortening the reaction time.

[0065] The reaction time in step A can be the time required for the NCA synthesis reaction to proceed, and from the viewpoint of suppressing epimerization, etc., it is preferably 0.01 seconds to 12 hours, more preferably 1 second to 6 hours, and even more preferably 10 seconds to 1 hour. When the reaction step of the present invention is carried out by a microflow method, the reaction time can be preferably less than 10 minutes, more preferably less than 5 minutes, and even more preferably less than 1 minute.

[0066] The solvent used in step A is not particularly limited, and for example, any organic solvent in which the substrate is soluble can be used. From the viewpoint of the solubility of the compound, however, it is preferable that the solvent contains an aprotic polar solvent.

[0067] The aprotic polar solvent is not particularly limited, but examples thereof include halogenated solvents such as dichloromethane and chloroform; ketone solvents such as acetone and methyl ethyl ketone; ether solvents such as 1,4-dioxane, tetrahydrofuran, and diethyl ether; amide solvents such as N,N-dimethylformamide and N,N-diethylformamide; sulfoxide solvents such as dimethyl sulfoxide; nitrile solvents such as acetonitrile; and mixtures thereof. Of these, from the viewpoints of substrate solubility, low reactivity, ease of removal, etc., halogenated solvents, nitrile solvents, etc. are preferred, and dichloromethane, acetonitrile, etc. are more preferred. These solvents can be used alone or in combination of two or more.

[0068] The solvent used in step A may contain components other than the organic solvent, such as water, as long as the effects of the present invention are not significantly impaired. However, if the obtained NCA is used directly in a subsequent reaction and the subsequent reaction is averse to water, it is preferable that the solvent used in step A be substantially free of water. As used herein, the phrase "substantially free of water" means that the water content in the solvent is preferably less than 5% by volume, more preferably less than 1% by volume, even more preferably less than 0.1% by volume, and particularly preferably 0% by volume, relative to the total volume of the solvent. In this case, the NCA obtained by the production method of the present invention can be used in the subsequent reaction without isolation and / or purification, which is advantageous for large-scale synthesis. The production method of the present invention enables NCA to be produced in a short period of time without the need for water as a solvent.

[0069] In the method of the present invention, in addition to the above components, known additives used in NCA synthesis can be used as appropriate within the range that does not impair the effects of the present invention.

[0070] 1-2-1. First Amino Acid The first amino acid whose amino group is protected with a carbamate protecting group is not particularly limited, and any known amino acid corresponding to the above NCA whose amino group is protected with a carbamate protecting group can be widely used. Among them, the first amino acid whose amino group is protected with a carbamate protecting group is represented by the general formula (2):

[0071] [In the formula, R ３ represents a carbamate protecting group. The other symbols are the same as above.] is preferred.

[0072] In general formula (1), R ３is a carbamate protecting group. In this specification, a carbamate protecting group refers to a protecting group that forms a carbamate (urethane) together with the amino group to which it is bonded. Examples of carbamate protecting groups include a tert-butyloxycarbonyl group (Boc), an allyloxycarbonyl group (Alloc), a benzyloxycarbonyl group (Cbz), a 2,2,2-triethoxycarbonyl group (Troc), a 9-fluorenylmethoxycarbonyl group (Fmoc), and a 2-(trimethylsilyl)ethoxycarbonyl group (Teoc). That is, specific examples of carbamate protecting groups include those represented by the general formula (4):

[0073] [In the formula, R ３ａ represents a tert-butyl group, an allyl group, a benzyl group, a 2,2,2-trichloroethyl group, a 9-fluorenylmethyl group, or a 2-(trimethylsilyl)ethyl group. Of these, preferred carbamate protecting groups are a tert-butyloxycarbonyl group (Boc) and an allyloxycarbonyl group (Alloc), with a tert-butyloxycarbonyl group (Boc) being more preferred.

[0074] From the above, specific examples of the first amino acid include, for example,

[0075] etc.

[0076] The first amino acid may be used alone or in combination of two or more kinds.

[0077] The first amino acid is not particularly limited, but from the viewpoints of yield improvement, economy, etc., it is preferably used as a 0.001 M to 10 M solution, more preferably a 0.01 M to 5 M solution, and even more preferably a 0.1 M to 1 M solution.

[0078] 1-2-2. First tertiary amine The first tertiary amine is not particularly limited, and any known tertiary amine can be widely used. Among them, the first tertiary amine is preferably a tertiary amine represented by the general formula (3):

[0079] [In the formula, R４ , R ５ and R ６ are the same or different and represent an alkyl group, an aryl group, or a heteroaryl group. ４ , R ５ and R ６ and two or more of these may be linked to have one or more hetero atoms or substituents, or may form a ring.

[0080] As the alkyl group, aryl group and heteroaryl group, the alkyl group, aryl group and heteroaryl group defined in 1-1 above can be used, respectively.

[0081] The alkyl group, aryl group, and heteroaryl group may each have a substituent, and as the substituent, the substituents defined in 1-1 above can be used.

[0082] As the first tertiary amine, a bulky tertiary amine is preferred from the viewpoint of efficiently proceeding with the desired reaction while suppressing self-polymerization of the obtained NCA. Specifically, the first tertiary amine is preferably a tertiary amine having a structure represented by the formula: ４ , R ５ and R ６ Preferably, the first tertiary amine is a tertiary amine having at least one carbon atom of R ４ , R ５ and R ６ The sum of the carbon atoms of R is preferably 6 or more. ４ , R ５ and R ６ More preferred are tertiary amines in which the sum of the carbon numbers of the above is 6 to 10. When such tertiary amines are used, it is easy to suppress self-polymerization of the obtained NCA and it is easy to allow the desired reaction to proceed efficiently.

[0083] R ４ , R ５ and R ６two or more of these may be linked to form a ring which may have one or more heteroatoms or substituents. The ring is not particularly limited, and examples thereof include an aziridine ring, an azirine ring, a diaziridine ring, a diazirine ring, an azetidine ring, an azeto ring, a diazeto ring, a pyrrolidine ring, a pyrrole ring, an imidazolidine ring, an imidazole ring, a pyridine ring, a piperidine ring, a piperazine ring, and a morpholine ring.

[0084] The first tertiary amine is, for example, triethylamine (Et ３ N), diisopropylethylamine (i-Pr ２ NEt), dimethylbenzylamine (Me ２ Examples of suitable amines include aliphatic amines such as N-methylmorpholine (NBn), N-methylmorpholine (NMM), and / or heterocyclic aromatic amines such as pyridine, 4-dimethylaminopyridine (DMAP), and N-methylimidazole (NMI).

[0085] As the first tertiary amine, from the viewpoint of efficiently proceeding with the desired reaction while suppressing self-polymerization of the obtained NCA, a tertiary amine having a pKaH of 8 to 14 is preferred, a tertiary amine having a pKaH of 8.5 to 13 is more preferred, and a tertiary amine having a pKaH of 8.9 to 12 is even more preferred. Here, "pKaH" refers to the pKa of the conjugate acid of the tertiary amine in water. Note that when the pKaH of the first tertiary amine is 10 or more and the obtained NCA is used as is as a nucleophile in the subsequent reaction, the reaction time in step A is preferably 10 seconds or more, more preferably 30 seconds or more, and even more preferably 50 seconds or more.

[0086] From the above, specific examples of the first tertiary amine include, for example,

[0087] etc.

[0088] The first tertiary amine may be used alone or in combination of two or more kinds.

[0089] The amount of the first tertiary amine used in step A is not particularly limited, but from the viewpoints of improving yield, suppressing epimerization, economic efficiency, etc., it is usually preferably about 0.1 mole to about 10 moles, more preferably about 1 mole to about 2 moles, and even more preferably about 1 mole to about 1.2 moles, relative to 1 mole of the first amino acid. When two or more first tertiary amines are used in combination, it is preferable to adjust the total amount thereof to be within the above range.

[0090] 1-2-3. First Halogenating Agent The production method of the present invention uses a first halogenating agent containing thionyl halide and / or oxalyl chloride (sometimes simply referred to as "first halogenating agent" in this specification). According to the method of the present invention, the use of a strong halogenating agent makes it possible to obtain NCA in a short period of time.

[0091] Here, the thionyl halide is a compound represented by the general formula (5):

[0092] [wherein X represents a halogen atom].

[0093] In general formula (5), X is a halogen atom. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. From the viewpoints of yield improvement, economy, safety, and the like, a chlorine atom or a bromine atom is preferred, and a chlorine atom is more preferred.

[0094] From the above, specific examples of thionyl halide include, for example,

[0095] Among them, the thionyl halide is preferably thionyl chloride or thionyl bromide, and more preferably thionyl chloride.

[0096] The thionyl halides may be used alone or in combination of two or more.

[0097] As a reminder, oxalyl chloride has the formula: or a first halogenating agent that is oxalyl chloride, which has the formula: (COCl) ２or formula: Cl(CO) ２ It can also be expressed as Cl.

[0098] Among these, thionyl halide is preferred as the first halogenating agent from the viewpoints of high reactivity, cost, etc.

[0099] In Step A, the first halogenating agent does not contain triphosgene. From a safety perspective, Step A is preferably performed in the absence of triphosgene. Despite its high toxicity, triphosgene has traditionally been considered safe to handle as long as its properties and reactivity are understood. However, in Org. Process Res. Dev. 2017, 21, 1439-1446, Livius et al. suggested the dangers of industrial use of triphosgene. Specifically, Livius et al. reported that because triphosgene readily sublimes at room temperature, triphosgene at levels exceeding toxic levels can fill the environment without users realizing it. The production method of the present invention enables safe production of NCA without using reagents that are difficult to handle.

[0100] The amount of the first halogenating agent used in step A is not particularly limited. From the viewpoint of suppressing the formation of contaminants and improving the yield, the amount is usually preferably about 0.1 mol to about 10 mol, more preferably about 0.5 mol to about 5 mol, and even more preferably about 1 mol to about 3 mol, relative to 1 mol of the first amino acid.

[0101] 2. Method for Producing Acylated NCA Acylated amino acid N-carboxyanhydride (acylated NCA), another object of the present invention, can be produced by reacting the above-mentioned NCA with a halide of a second amino acid or peptide, step B. That is, the acylated NCA can be produced by sequentially carrying out steps A and B of the present invention.

[0102] Conventionally, acylated NCAs derived from normal amino acid N-carboxyanhydrides (NCAs), rather than dehydroamino acid N-carboxyanhydrides (dehydroNCAs), have been thought to be unstable and therefore impossible to synthesize ( Organic Synthetic Chemistry, 1989, Vol. 47, No. 9, pp. 782-794). In contrast, the production method of the present invention allows for the synthesis of acylated NCAs from the above-mentioned NCAs, which can then be used in peptide synthesis. Furthermore, the production method of the present invention also allows for the isolation of the acylated NCAs that may be produced as intermediates. Thus, the production method of the present invention allows for the sequential production of highly reactive reaction intermediates in a short period of time, making it easy to suppress epimerization of each amino acid residue that constitutes the acylated NCA.

[0103] According to the method of the present invention, epimerization of amino acid residues is unlikely to occur even when an amino acid that is prone to epimerization is included. Furthermore, according to the method of the present invention, it is possible to provide acylated NCA safely and easily on a large scale using inexpensive commercially available raw materials. In particular, when the microflow method is used in the method of the present invention, it is possible to obtain high-purity acylated NCA in high yield by appropriately selecting a tertiary amine.

[0104] Alternatively, a halide of the second amino acid or peptide can be produced by reacting the second amino acid or peptide with a second halogenating agent in step B', which is preferably carried out in the presence of a second tertiary amine.

[0105] The acylated NCA is not particularly limited, and any of known NCAs and known acylated NCAs derived from amino acids or peptides can be widely used. Among them, the acylated NCA is preferably an NCA represented by the general formula (6):

[0106] [In the formula, R １ａ indicates the side chain of an amino acid residue. １ａ may form a ring together with the adjacent nitrogen atom via the carbon atom to which it is bonded. ２ａ represents a hydrogen atom or a monovalent organic group. ７ａ represents a hydrogen atom or an alkyl group. １represents a hydrogen atom, a protecting group for an amino group, a tag, or a solid phase support. n1 represents an integer of 1 or more, and when n1 is an integer of 2 or more, n1 R １ａ , R ２ａ and R ７ａ may be the same or different, and other symbols are the same as above.] is preferred.

[0107] The side chain and organic group of the amino acid residue may be the same as those defined in 1-1 above.

[0108] In general formula (6), R ７ａ is a hydrogen atom or an alkyl group. Here, the alkyl group may be an alkyl group defined in 1-1 above. ７ａ From the viewpoint of improving yield, the group represented by the formula: is preferably a hydrogen atom or a methyl group, and more preferably a hydrogen atom.

[0109] In addition, in the general formula (6), P １ is a protecting group for a hydrogen atom or an amino group, a tag, or a solid phase support. From the viewpoint of avoiding side reactions, it is preferably a protecting group for an amino group. On the other hand, from the viewpoint of reducing the deprotection step, improving atom efficiency, and reducing waste, P １ On the other hand, when n1 is large in general formula (6), that is, when the second amino acid or peptide is a long-chain peptide, from the viewpoint of enabling the application of the production method of the present invention, the group represented by P １ Preferably, the group represented by is a tag or solid support.

[0110] As the protecting group for the amino group, any protecting group known as a protecting group for an amino group can be used, and examples thereof include alkyl-type protecting groups such as tert-butyl (t-Bu) group, benzyl group, allyl group, methyl group, and triphenylmethyl group (Trt); silyl-type protecting groups such as tert-butyldimethylsilyl group (TBS); aryl-type protecting groups such as p-methoxybenzyl group (PMB) and p-methoxyphenyl group (PMP); amide-type protecting groups such as formyl group and acetyl group (Ac); phthalimide-type protecting groups such as phthaloyl group (Phth); benzyl Examples of protecting groups include carbamate-type protecting groups such as an oxycarbonyl group (Cbz), a tert-amyloxycarbonyl group (Aoc), a 9-fluorenylmethoxycarbonyl group (Fmoc), a tert-butyloxycarbonyl group (Boc), an allyloxycarbonyl group (Alloc), and a 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns), and a (2-trimethylsilyl)-ethanesulfonyl group (SES).

[0111] Tags that can be used to improve peptide solubility include a wide range of tags, such as hydrophobic tags consisting of an aromatic ring substituted with multiple long-chain alkoxy groups. This allows the use of reactions established in solid-phase peptide synthesis. Here, "solubility" refers to the ease with which a molecule dissolves in a solvent.

[0112] As the solid phase support, a solid phase support used in a conventional solid phase peptide synthesis method can be used.

[0113] In general formula (6), n1 is an integer of 1 or more, and is usually an integer of 1 to 100. From the viewpoint of improving yield and suppressing side reactions, an integer of 1 to 50 is preferred, an integer of 1 to 10 is more preferred, and an integer of 1 to 5 is even more preferred. When n1 is an integer of 2 or more, n1 R １ａ , R ２ａ and R ７ａ may be the same or different.

[0114] The amino acid residues constituting the acylated NCA may be either L- or D-optical isomers. The production method of the present invention can suppress epimerization of the amino acid residues.

[0115] From the above, specific examples of acylated NCA include, for example,

[0116] etc.

[0117] The acylated NCA can be obtained singly or in combination of two or more kinds.

[0118] Acylated NCAs, another object of the present invention, are useful as starting materials for the synthesis of peptides and peptide analogs.

[0119] 2-2. Steps B and B' In step B', a second amino acid or peptide is reacted with a second halogenating agent. Step B' is preferably carried out in the presence of a second tertiary amine.

[0120] In step B, the NCA obtained in step A is reacted with a halide of a second amino acid or peptide. Step B is preferably carried out in the presence of a second tertiary amine. The halide of the second amino acid or peptide used in step B is preferably the one obtained in step B'.

[0121] Step B and step B′ can be carried out by either a batch method or a flow method, as in step A. From the viewpoints of high safety and ease of scale-up, the flow method is more preferable, and from the viewpoints of enabling precise control of the reaction temperature and reaction time, the microflow method is even more preferable.

[0122] Hereinafter, the method for producing an acylated NCA and a halide of a second amino acid or peptide of the present invention using a microflow reactor will be described with reference to Fig. 1. However, it goes without saying that the production method of the present invention is not limited to the embodiment shown in Fig. 1.

[0123] The syringe pump, flow path, and mixer in Fig. 1 may be those defined in 1-2 above, but are not limited thereto.

[0124] In one embodiment, the method for producing an acylated NCA and a halide of a second amino acid or peptide of the present invention can use syringe pumps 11-15, flow paths F1-F5 and F7-F9, and mixers M1-M4 shown in FIG. 1 . The first liquid can contain a second amino acid or peptide and a second tertiary amine, the second liquid can contain a second halogenating agent, the third liquid can contain a first amino acid and a first tertiary amine, the fourth liquid can contain a first halogenating agent, and the fifth liquid can contain a second tertiary amine. As another example, in the above example, the first liquid can contain a second amino acid or peptide, and the second liquid can contain a second halogenating agent and a second tertiary amine, but the present invention is not limited to any of these embodiments. In the method for producing a halide of an amino acid or peptide of the present invention, the former case, i.e., the first liquid can contain a second amino acid or peptide and a second tertiary amine, and the second liquid can contain a second halogenating agent, is preferred. Furthermore, it is preferable that the second tertiary amine contained in the fifth liquid is different from the second tertiary amine contained in the first liquid or the second liquid, or that the fifth liquid contains two or more types of tertiary amines.

[0125] By operating syringe pump 11, the first liquid can move through flow path F1 and flow into mixer M1. By operating syringe pump 12, the second liquid can move through flow path F2 and flow into mixer M1. The first and second liquids are then mixed by mixer M1 to form a first mixed liquid, which can be sent to flow path F7. The first mixed liquid containing the second amino acid or peptide halide moves through flow path F7 and flows into mixer M3, where it can be used in a subsequent reaction, stored in any test tube, or subjected to isolation, purification, and in-line analysis.

[0126] Meanwhile, as described in 1-2 above, the second mixed solution can be sent to flow path F8 by operating syringe pumps 13 and 14. The second mixed solution containing NCA moves through flow path F8 and flows into mixer M3, where it is mixed with the first mixed solution to form a third mixed solution, which can be sent to flow path F9. The fifth solution moves through flow path F5 and flows into mixer M4, where it is mixed with the third mixed solution to form a fourth mixed solution, which can be sent to flow path F10. The fourth mixed solution containing the acylated NCA moves through flow path F10 and flows into mixer M5, where it can be used in a subsequent reaction, stored in any test tube, or subjected to isolation, purification, and in-line analysis.

[0127] Here, a halide of the second amino acid or peptide may be prepared in situ using the first and second liquids by a microflow method and reacted with the NCA, or a halide synthesized by a method other than the microflow method may be reacted with the NCA. From the viewpoints of improving reaction efficiency and suppressing side reactions, the halide of the second amino acid or peptide is preferably synthesized by a microflow method. That is, in step B, it is preferable to use the halide of the second amino acid or peptide obtained in step B'.

[0128] The method for producing an acylated NCA and a halide of a second amino acid or peptide of the present invention is not limited to being performed using a microflow reactor, and may be performed using, for example, a batch vessel having a small volume and capable of achieving a high stirring speed. The volume of the mixing section of the batch vessel may be 1 mL to 100 mL, or may be 5 mL to 50 mL.

[0129] As described above, the method of the present invention for producing an acylated NCA and a halide of a second amino acid or peptide can be carried out by a liquid phase method, and therefore can be easily scaled up.

[0130] In Step B and Step B', the reaction temperature, reaction time, solvent and additives in Step A can be applied.

[0131] 2-2-1. Halide of Second Amino Acid or Peptide The halide of the second amino acid or peptide is not particularly limited, and any halide corresponding to a known amino acid or peptide can be widely used. Among them, the halide of the second amino acid or peptide is preferably a halide of the second amino acid or peptide represented by the general formula (7):

[0132] [wherein each symbol is the same as defined above] is preferred.

[0133] The second amino acid or peptide halide may be used alone or in combination of two or more.

[0134] The amount of the halide of the second amino acid or peptide used in step B is not particularly limited. From the viewpoint of suppressing the formation of contaminants and improving the yield, the amount is usually preferably about 0.1 mol to about 10 mol, more preferably about 0.2 mol to about 2 mol, and even more preferably about 0.5 mol to about 1 mol, relative to 1 mol of the NCA.

[0135] 2-2-2. Second Amino Acid or Peptide The second amino acid or peptide is not particularly limited, and any known amino acid or peptide can be widely used. Among them, the second amino acid or peptide is preferably a compound represented by the general formula (8):

[0136] [wherein each symbol is the same as defined above] is preferred.

[0137] From the above, specific examples of the second amino acid or peptide include, for example,

[0138] etc.

[0139] 2-2-3. Second tertiary amine The second tertiary amine is not particularly limited, and any known tertiary amine can be widely used. In particular, the first tertiary amine defined in 1-2-2 above can be used as the second tertiary amine.

[0140] Here, the second tertiary amine used in step B may be the first tertiary amine used in step A or the second tertiary amine used in step B' that has been recovered and reused, or a tertiary amine different from the first tertiary amine used in step A and the second tertiary amine used in step B' may be newly added.

[0141] Among these, the second tertiary amine preferably includes an aliphatic amine and a heterocyclic aromatic amine from the viewpoint of improving yield, etc., and more preferably includes triethylamine (Et ３ N), diisopropylethylamine (i-Pr ２ NEt) and dimethylbenzylamine (Me ２ More preferably, the solvent contains at least one aliphatic amine selected from the group consisting of pyridine, 4-dimethylaminopyridine (DMAP), and N-methylimidazole (NMI), and at least one heterocyclic aromatic amine selected from the group consisting of triethylamine (Et ３ N), diisopropylethylamine (i-Pr ２ NEt) and dimethylbenzylamine (Me ２ It is particularly preferred that the heterocyclic aromatic amine contains at least one aliphatic amine selected from the group consisting of N-methylimidazole (NMI), N-methylimidazole (NBn), and N-methylimidazole (NMI).

[0142] The amount of the second tertiary amine used in step B' is not particularly limited, but from the viewpoints of improving yield, suppressing epimerization, economic efficiency, etc., it is usually preferably about 0.1 mol to about 10 mol, more preferably about 0.2 mol to about 5 mol, and even more preferably about 0.5 mol to about 3 mol, per mol of the second amino acid or peptide. When two or more second tertiary amines are used in combination, it is preferable to adjust the total amount to be within the above range.

[0143] 2-2-4. Second Halogenating Agent The second halogenating agent used in step B' is an electrophilic halogenating agent.

[0144] Examples of electrophilic halogenating agents include PX ３ [wherein X is the same as above. The same applies hereinafter in this specification.] or PX５ Phosphorus halides represented by the formula: SOX ２ Thionyl halides represented by the formula: SO ２ X ２ sulfuryl halide represented by (COX) ２ Examples thereof include oxalyl halides represented by the following formula:

[0145] Among them, thionyl halide is preferred as the second halogenating agent from the viewpoints of high reactivity, cost, etc. As the thionyl halide, the thionyl halide defined in 1-2-3 above can be used.

[0146] The second halogenating agent may be used alone or in combination of two or more kinds.

[0147] The amount of the second halogenating agent used in step B' is not particularly limited, but from the viewpoint of suppressing the formation of contaminants and improving the yield, it is usually preferably about 0.1 mol to about 10 mol, more preferably about 0.2 mol to about 5 mol, and even more preferably about 0.5 mol to about 3 mol, per mol of the second amino acid or peptide. When two or more second halogenating agents that can be used in the present invention are used in combination, it is preferable to adjust the total amount to be within the above range.

[0148] 3. Method for Producing Two-Residue-Extended Peptides Another object of the present invention, a two-residue-extended peptide, can be produced by reacting the acylated NCA with a third amino acid or peptide in step C. That is, a two-residue-extended peptide can be produced by sequentially carrying out steps A, B, and C of the present invention.

[0149] Conventionally, acylated NCAs derived from normal amino acid N-carboxyanhydrides (NCAs), rather than dehydroamino acid N-carboxyanhydrides (dehydroNCAs), have been thought to be unstable and therefore impossible to synthesize ( Organic Synthetic Chemistry, 1989, Vol. 47, No. 9, pp. 782-794). In contrast, the production method of the present invention allows for the synthesis of acylated NCAs from the above-mentioned NCAs, which can then be used in peptide synthesis. Furthermore, the production method of the present invention also allows for the isolation of the acylated NCAs that may be produced as intermediates. Thus, the production method of the present invention allows for the sequential production of highly reactive reaction intermediates in a short period of time, making it easy to suppress epimerization of each amino acid residue constituting a two-residue-elongated peptide.

[0150] According to the method of the present invention, epimerization of amino acid residues is unlikely to occur even when the peptide contains an amino acid that is prone to epimerization. Furthermore, according to the method of the present invention, it is possible to provide two-residue-elongated peptides safely and simply on a large scale using inexpensive commercially available raw materials. In particular, when the microflow method is used in the method of the present invention, it is possible to obtain highly pure two-residue-elongated peptides in high yield by appropriately selecting a tertiary amine.

[0151] 3-1. Two-residue-extended peptide The two-residue-extended peptide is not particularly limited, and any known peptide can be widely used. Among them, the two-residue-extended peptide is preferably a peptide represented by the general formula (9):

[0152] [In the formula, R １ｂ indicates the side chain of an amino acid residue. １ｂ may form a ring together with the adjacent nitrogen atom via the carbon atom to which it is bonded. ２ｂ represents a hydrogen atom or a monovalent organic group. ７ｂ represents a hydrogen atom or an alkyl group. ２ represents a hydrogen atom, a protecting group for a carboxy group, a tag, or a solid phase support. n2 represents an integer of 1 or more, and when n2 is an integer of 2 or more, n2 R １ｂ , R ２ｂ and R ７ｂmay be the same or different, and other symbols are the same as above.] is preferred.

[0153] The side chain and organic group of the amino acid residue may be the same as those defined in 1-1 above.

[0154] In general formula (9), R ７ｂ is a hydrogen atom or an alkyl group. Here, the alkyl group may be an alkyl group defined in 1-1 above. ７ｂ From the viewpoint of improving yield, the group represented by the formula: is preferably a hydrogen atom or a methyl group, and more preferably a hydrogen atom.

[0155] In addition, in the general formula (9), P ２ is a protecting group for a hydrogen atom or a carboxy group, a tag, or a solid phase support. From the viewpoint of avoiding side reactions, it is preferably a protecting group for a carboxy group. On the other hand, from the viewpoint of reducing the deprotection step, improving atom efficiency, and reducing waste, P ２ On the other hand, when n2 is large in general formula (9), that is, when the peptide extended by two residues is a long-chain peptide, from the viewpoint of enabling the application of the production method of the present invention, it is preferable that P ２ Preferably, the group represented by is a tag or solid support.

[0156] The protecting group for the carboxy group can be any protecting group known as a protecting group for a carboxy group, and examples thereof include alkyl protecting groups such as a tert-butyl (t-Bu) group, a benzyl group, an allyl group, a methyl group, and a triphenylmethyl group (Trt); silyl protecting groups such as a tert-butyldimethylsilyl group (TBS); aryl protecting groups such as a p-methoxybenzyl group (PMB) and a p-methoxyphenyl group (PMP); amide protecting groups such as a formyl group and an acetyl group (Ac); phthalimide protecting groups such as a phthaloyl group (Phth); Examples of protecting groups include carbamate-type protecting groups such as a tert-amyloxycarbonyl group (Cbz), a tert-amyloxycarbonyl group (Aoc), a 9-fluorenylmethoxycarbonyl group (Fmoc), a tert-butyloxycarbonyl group (Boc), an allyloxycarbonyl group (Alloc), and a 2,2,2-triethoxycarbonyl group (Troc); and sulfonamide-type protecting groups such as a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns), and a (2-trimethylsilyl)-ethanesulfonyl group (SES).

[0157] As the tag and solid phase carrier, the tag and solid phase carrier defined in 2-1 above can be used, respectively.

[0158] In general formula (9), n2 is an integer of 1 or more, and is usually an integer of 1 to 100. From the viewpoint of improving yield and suppressing side reactions, an integer of 1 to 50 is preferred, an integer of 1 to 10 is more preferred, and an integer of 1 to 5 is even more preferred. When n2 is an integer of 2 or more, n2 R １ｂ , R ２ｂ and R ７ｂ may be the same or different.

[0159] The amino acid residues constituting the two-residue-elongated peptide may each be either the L- or D-optical isomer. The production method of the present invention can suppress epimerization of the amino acid residues.

[0160] The two-residue-extended peptide, which is another object of the present invention, is useful as a raw material or intermediate for peptide synthesis, a pharmaceutical, a pharmaceutical carrier, or a functional material.

[0161] 3-2. Step C In step C, the acylated NCA obtained in step B is reacted with a third amino acid or peptide. Step C is preferably carried out in the presence of a third tertiary amine.

[0162] Step C can be carried out by either a batch method or a flow method. From the viewpoints of high safety and ease of scale-up, the flow method is more preferred, and from the viewpoints of precise control of the reaction temperature and reaction time, the microflow method is even more preferred.

[0163] Hereinafter, a method for producing a two-residue-elongated peptide of the present invention using a microflow reactor will be described with reference to Fig. 1. However, it goes without saying that the production method of the present invention is not limited to the embodiment shown in Fig. 1.

[0164] The syringe pump, flow path, and mixer in Fig. 1 may be those defined in 1-2 above, but are not limited thereto.

[0165] In one embodiment, the method for producing a two-residue-extended peptide of the present invention can use syringe pumps 11-16, flow paths F1-F11, and mixers M1-M5 shown in FIG. 1 . The first liquid can contain a second amino acid or peptide and a second tertiary amine, the second liquid can contain a second halogenating agent, the third liquid can contain a first amino acid and a first tertiary amine, the fourth liquid can contain a first halogenating agent, the fifth liquid can contain a second tertiary amine, and the sixth liquid can contain a third amino acid or peptide. As another example, in the above example, the first liquid can contain a second amino acid or peptide, and the second liquid can contain a second halogenating agent and a second tertiary amine, but the present invention is not limited to any of these embodiments. In the method for producing a halide of an amino acid or peptide of the present invention, the former case, i.e., the first liquid can contain a second amino acid or peptide and a second tertiary amine, and the second liquid can contain a second halogenating agent, is preferred. Furthermore, it is preferable that the second tertiary amine contained in the fifth liquid is different from the second tertiary amine contained in the first liquid or the second liquid, or that the fifth liquid contains two or more types of tertiary amines.

[0166] As described above in 1-1 and 1-2, the third mixture containing an amino acid or peptide halide can be sent to flow path F10 by operating syringe pumps 11 to 15. Meanwhile, the sixth liquid moves through flow path F6 and flows into mixer M5, where it is mixed with the fourth mixture to form a fifth mixture, which can then be sent to flow path F11. The fifth mixture containing the two-residue-elongated peptide can be stored in any test tube, or can be subjected to isolation and purification steps and in-line analysis.

[0167] The method for producing a two-residue-elongated peptide of the present invention is not limited to those carried out using a microflow reactor, and may also be carried out using, for example, a batch vessel having a small volume and capable of achieving a high stirring speed. The volume of the mixing section of the batch vessel may be 1 mL to 100 mL, or may be 5 mL to 50 mL.

[0168] As described above, the method for producing a two-residue-extended peptide of the present invention can be carried out by a liquid phase method, and therefore can be easily scaled up.

[0169] In step C, the reaction temperature, reaction time, solvent and additives in step A can be applied.

[0170] 3-2-1. Third Amino Acid or Peptide The third amino acid or peptide is not particularly limited, and any known amino acid or peptide can be widely used. Among them, the third amino acid or peptide is preferably a compound represented by the general formula (10):

[0171] [wherein each symbol is the same as defined above] is preferred.

[0172] From the above, specific examples of the third amino acid or peptide include, for example,

[0173] etc.

[0174] 3-2-2. Third tertiary amine The third tertiary amine is not particularly limited, and any known tertiary amine can be widely used. In particular, the first tertiary amine defined in 1-2-2 above can be used as the third tertiary amine.

[0175] The amount of the third tertiary amine used in step C is not particularly limited, but from the viewpoints of improving yield, suppressing epimerization, economic efficiency, etc., it is usually preferably about 0.1 mol to about 10 mol, more preferably about 0.2 mol to about 5 mol, and even more preferably about 0.5 mol to about 3 mol, relative to 1 mol of the acylated NCA. When two or more third tertiary amines are used in combination, it is preferable to adjust the total amount so that it is within the above range.

[0176] EXAMPLES Hereinafter, examples and comparative examples will be shown to further clarify the features of the present invention, but the present invention is not limited to the following examples.

[0177] The symbols and abbreviations used in the examples have the following meanings: Boc: benzyloxycarbonyl group Fmoc: 9-fluorenylmethoxycarbonyl group Phe: phenylalanine NMM: N-methylmorpholine Me ２ NBn: N,N-dimethylbenzylamine i-Pr ２ NEt: N,N-diisopropylethylamine NMI: N-methylimidazole THF: tetrahydrofuran

[0178] [Example 1] Investigation of the range of halogenating agents and tertiary amines As shown in Table 1, thionyl chloride or oxalyl chloride was used as the halogenating agent, and NMM, Me, ２ NBn or i-Pr ２ NCA was synthesized using NEt. Specifically, NCA was synthesized by the following method.

[0179]

[0180] A dichloromethane solution (CH ) of a protected amino acid represented by Boc-Phe-OH (0.180 M, 1.00 molar equivalent) and a tertiary amine (X × 0.180 M, X molar equivalent) was prepared. ２Cl ２ A solution of thionyl chloride (0.216 M, 1.2 molar equivalents) or oxalyl chloride (0.216 M, 1.2 molar equivalents) in dichloromethane (flow rate: 2.00 mL / min) was introduced into a T-shaped mixer at 20°C using a syringe pump. The resulting mixture was introduced into reaction tube 1 (inner diameter: 0.800 mm, reaction time: T seconds) at the same temperature. After reaching a steady state over 60 seconds, the reaction mixture was poured into 1 M hydrochloric acid (1.5 mL) and dichloromethane (6 mL) at room temperature for 25 seconds. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and concentrated under vacuum at room temperature. The yield was determined by NMR. The results are shown in Table 1.

[0181] NMM, Me ２ NBn and i-Pr ２ When any of the tertiary amines of NEt was used, the desired Phe-NCA was obtained (Examples 1-1 to 1-11). ２ From a comparison with the Examples using NEt, it can be seen that the yield improves when the amount of tertiary amine used is 2 moles or less per mole of amino acid (Examples 1-6 to 1-8).

[0182] Furthermore, the desired Phe-NCA was obtained when either thionyl chloride or oxalyl chloride was used as the halogenating agent (Examples 1-1 to 1-11). This indicates that thionyl chloride and oxalyl chloride have equivalent effects in the method of the present invention.

[0183] On the other hand, when a tertiary amine was not used, an NCA was not obtained (Comparative Example 1-1). Non-Patent Document 1 reports that an NCA can be obtained by reacting a carbazole-protected amino acid with thionyl chloride under acidic conditions at high temperature for a long period of time, but no NCA synthesis method has been reported to date that uses thionyl chloride and a tertiary amine in combination. According to the method of the present invention, by using thionyl chloride and a tertiary amine in combination, an amino acid N-carboxyanhydride (NCA) can be produced in a short time without the need for water as a solvent.

[0184] L-Phe-NCA(Angew. Chem. Int. Ed., 2018, 57, 11389-11393.): 1 H NMR (400 MHz, CDCl3): δ7.37-7.28 (m, 3H), 7.19-7.17 (m, 2H), 6.37 (brs, 1H), 4.53 (dd, J = 4.4, 7.6 Hz, 1H), 3.26 (dd, J = 4.4, 14.2 Hz, 1H), 3.01 (dd, J = 7.6, 14.2, 1H) ppm.

[0185] [Example 2] Application to peptide synthesis We investigated whether the NCA obtained by the method of the present invention can be used in subsequent reactions. Specifically, NCA was synthesized by the following method and applied to peptide synthesis.

[0186]

[0187] A protected amino acid represented by Boc-Phe-OH (0.180 M, 1.00 molar equivalent) and a tertiary amine (X １ x 0.180M,X １ molar equivalent) in dichloromethane (CH ２ Cl ２ A solution of thionyl chloride (0.216 M, 1.2 molar equivalents) in dichloromethane (flow rate: 2.00 mL / min) was introduced into a T-shaped mixer at 20°C using a syringe pump. The resulting mixture 1 was added to reaction tube 1 (inner diameter: 0.800 mm, reaction time: T １ Next, an N-terminal protected amino acid represented by Fmoc-L-Phe-OH (0.300 M, 1.00 molar equivalent) and i-Pr ２A dichloromethane solution of NEt (0.360 M, 1.2 molar equivalents) (flow rate: 1.20 mL / min) and a dichloromethane solution of thionyl chloride (0.216 M, 1.20 molar equivalents) (flow rate: 2.00 mL / min) were introduced into a T-shaped mixer at 20°C using a syringe pump. The resulting mixture 2 was introduced into reaction tube 2 (inner diameter: 0.800 mm, length: 1061 mm, volume: 533 μL, reaction time: 10 seconds) at the same temperature. Mixture 1 in reaction tube 1 and mixture 2 in reaction tube 2 were introduced into a T-shaped mixer at 20°C using a syringe pump. The resulting mixture 3 was introduced into reaction tube 3 (inner diameter: 0.800 mm, length: 72.9 mm, volume: 37 μL, reaction time: 0.50 seconds) at the same temperature. The resulting mixture 3 and i-Pr ２ NET (Y １ ×0.216M,Y １ A dichloromethane solution (flow rate: 2.00 mL / min) of NMI (0.180 M, 1.00 molar equivalents) and NMI (0.180 M, 1.00 molar equivalents) was injected into a T-type mixer at 20°C using a syringe pump. The resulting mixture 4 was added to a reaction tube 4 (inner diameter: 0.800 mm, reaction time: T ２ The resulting mixture 4 was mixed with a C-terminal protected amino acid represented by HL-Ala-Ot-Bu (1.2 molar equivalents) and i-Pr ２ A dichloromethane solution of NEt (1.2 molar equivalents) (flow rate: 1.20 mL / min) was injected into a T-shaped mixer at 20°C using a syringe pump. The resulting mixture 5 was introduced into a reaction tube 5 (inner diameter: 0.800 mm, reaction time: 20 seconds) at the same temperature. After reaching a steady state over approximately 120-190 seconds, the reaction mixture was poured into 1 M hydrochloric acid (1.5 mL) and dichloromethane (8 mL) at room temperature for 25 seconds. The aqueous layer was extracted with dichloromethane, and the organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and concentrated under vacuum at room temperature. The yield of the resulting tripeptide represented by Fmoc-L-Phe-L-Phe-L-Ala-Ot-Bu was determined by HPLC-UV analysis using a calibration curve (column: COSMOSIL 5C). １８ -AR-II 4.6 mm ID x 150 mm, solvent: acetonitrile + 0.1% formic acid / H ２O+0.1% acid (0-20 points: 30~100%, 20-25 points: 100%, 25-27 points: 30%, 27-30 points: 30%), flow rate: 1.0mL / min, wavelength: 254nm, temperature: 40°C, holding time: 17.3 minutes) It is decided by によって. The results are shown in Table 2.

[0188] Fmoc-L-Phe-L-Phe-L-Ala-Ot-Bu: Purification method: silica gel column chromatography (CH2Cl 2 : CH3OH = 98 : 2 to 87 : 13) and recrystallization from CH2Cl2 / hexane White solid, mp 162-164 ℃, IR (neat): 3286, 1732, 1704, 1644, 1539, 1451, 1254, 1146, 741 cm -1 ; [α] 25 D = -21.7 (c 0.88, CHCl3); 1 H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 7.6 Hz, 2H), 7.50 (dd, J = 7.6, 7.6 Hz, 2H), 7.39 (dd, J = 7.6, 7.6 Hz, 2H), 7.31-7.07 (m, 12H), 6.67 (brs, 1H), 6.54 (brs, 1H), 5.44 (d, J = 6.0 Hz, 1H), 4.68 (d, J = 6.4 Hz, 1H), 4.46 (brs, 1H), 4.42-4.38 (m, 1H), 4.34-4.31 (m, 1H), 4.24 (brs, 1H), 4.14 (t, J = 6.8 Hz, 1H), 3.01-2.99 (m, 4H), 1.43 (s, 9H), 1.28 (d, J= 6.0 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3): δ 171.6, 170.8, 169.8, 156.1, 143.8, 141.4, 136.3, 129.4, 128.8, 128.6, 127.9, 127.2, 127.1, 125.2, HRMS (ESI): calcd for [C 40 H 43 N3O6+Na] + 684.3044, found 684.3041.

[0189] As shown in Table 2, when NMM was used as the tertiary amine for NCA synthesis, the yield of tripeptide decreased (Examples 2-1 and 2-2). ２ When NEt was used, the yield of the tripeptide was improved (Examples 2-3 and 2-4). Considering the results of Example 1, it is possible that the Phe-NCA obtained using NMM has low reactivity with the chloride of Fmoc-L-Phe-OH contained in the above-mentioned mixture 2. Therefore, it can be understood that in the method of the present invention, it is preferable to use a highly basic tertiary amine in order to obtain an NCA that can be used as a nucleophile in the subsequent reaction.

[0190] However, even in Examples 2-4, the tripeptide yield was only 43%. To further improve the tripeptide yield, it is thought that it would be necessary to supply a larger amount of NCA that can be used as a nucleophile by the method of the present invention. Therefore, in the next example, the amount of NCA required to sufficiently proceed with the subsequent reaction was investigated.

[0191] Example 3: Investigation of NCA supply amount In order to improve the yield of tripeptide, the amounts of the compounds used in the method of the present invention, and the amounts of the third amino acid and third tertiary amine used were investigated.

[0192] Specifically, the amount of the protected amino acid represented by Boc-Phe-OH used in NCA synthesis is X２ The amount of tertiary amine used is X molar equivalents. ２ × 1.2 molar equivalents, and the amount of thionyl chloride used is X ２ × 1.2 molar equivalents of i-Pr mixed with Mixture 3 ２ The amount of NET used is X ２ × 2.4 molar equivalents, and a C-terminal protected amino acid represented by HL-Ala-Ot-Bu and i-Pr ２ The amount of NEt used is Y ２ NCA synthesis and tripeptide synthesis were carried out in the same manner as in Example 2, except that the molar equivalents were changed.

[0193]

[0194] As shown in Table 3, a certain improvement in the yield of the tripeptide was confirmed by increasing the amount of compounds used in NCA synthesis (Examples 3-1 to 3-3). On the other hand, increasing the amount of C-terminal protected amino acid used did not increase the yield of the tripeptide (Examples 3-4 and 3-5). Therefore, it is possible that the reaction between Phe-NCA obtained by the method of the present invention and the chloride of Fmoc-L-Phe-OH did not proceed sufficiently. For this reason, in the next example, the reaction time required for sufficient progression of the subsequent reaction was investigated.

[0195] Example 4: Investigation of reaction time of NCA synthesis The influence of the reaction time of the NCA synthesis reaction on the subsequent reaction was investigated.

[0196] Specifically, the amount of the protected amino acid represented by Boc-Phe-OH used is X ２ and 1.5 molar equivalents of the C-terminal protected amino acid represented by HL-Ala-Ot-Bu. ２ is set to 1.2 molar equivalents, and the reaction time of the NCA synthesis reaction T １ NCA synthesis and tripeptide synthesis were carried out in the same manner as in Example 3, except that the time was changed to 10, 20, 50 or 90 seconds.

[0197]

[0198] As shown in Table 4, the reaction time T １Increasing the time to 50 seconds or more improved the yield of the tripeptide (Examples 4-1 to 4-4). Furthermore, by applying column purification and recrystallization, the tripeptide could be isolated in high yield (Examples 4-3 and 4-4). Furthermore, the tripeptide obtained was free of racemization (Example 4-3).

[0199] Example 5: Investigation of solvents for NCA synthesis The influence of the solvents used in the present invention on the reaction of the present invention and subsequent reactions was investigated.

[0200] Specifically, the reaction time T １ The synthesis of NCA and the synthesis of tripeptide were carried out in the same manner as in Example 4, except that the amount of HCl was 50 and the solvent used in the NCA synthesis reaction was changed from dichloromethane to acetonitrile or THF.

[0201]

[0202] As shown in Table 5, in the method for producing NCA of the present invention, the yield was not significantly affected by the type of solvent (Examples 5-1 to 5-3).

Claims

1. A method for producing an amino acid N-carboxyanhydride (NCA), comprising the steps of: a first amino acid whose amino group is protected with a carbamate protecting group; a first halogenating agent; In the presence of a first tertiary amine, Step A: obtaining the amino acid N-carboxyanhydride (NCA) by reacting the the first halogenating agent comprises thionyl halide and / or oxalyl chloride; The amino acid N-carboxyanhydride (NCA) has the general formula (1): 【Chemistry 1】 [In the formula, R １ indicates the side chain of an amino acid residue. ２ represents a hydrogen atom or a monovalent organic group. n represents 1 or 2, and when n is 2, two R １ and two R ２ may be the same or different.] is an amino acid N-carboxyanhydride (NCA) represented by Manufacturing method.

2. (delete)

3. The first amino acid has the general formula (2): 【Chemistry 2】 [In the formula, R １ indicates the side chain of an amino acid residue. ２ represents a hydrogen atom or a monovalent organic group. n represents 1 or 2, and when n is 2, two R １ and two R ２ may be the same or different. ３ represents a carbamate protecting group. The method according to claim 1, wherein the amino acid is an amino acid represented by the formula:

4. The first tertiary amine is represented by the general formula (3): 【Transformation 3】 [In the formula, R ４ , R ５ and R ６ are the same or different and represent an alkyl group, an aryl group, or a heteroaryl group. ４ , R ５ and R ６ Two or more of these may be linked to have one or more heteroatoms or substituents, or may form a ring. The method according to claim 1, wherein the tertiary amine is represented by the formula:

5. The method of claim 1 , wherein the first halogenating agent is thionyl chloride.

6. The n is 1, and the R ２ The method according to claim 1 or 3, wherein is a hydrogen atom.

7. The method according to any one of claims 1 and 3 to 5, wherein the amount of the first tertiary amine used is 0.5 moles to 3 moles per mole of the first amino acid.

8. The method according to any one of claims 1 and 3 to 5, wherein step A is carried out by a flow method in the absence of triphosgene.

9. 1. A method for producing an acylated amino acid N-carboxyanhydride (NCA), comprising: The amino acid N-carboxyanhydride (NCA) obtained by the production method according to any one of claims 1 and 3 to 5 is a step B of reacting with a halide of a second amino acid or peptide.

10. A method for producing a two-residue extended peptide, comprising the steps of: The acylated NCA obtained by the production method according to claim 9 is and step C. reacting with a third amino acid or peptide.

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