Methods for Producing Polypeptide Compounds

Abstract

This method comprises: (i) a step for forming an amide bond between a Ta-substituted carboxyl group located on the right side in formula (R1) in an N-terminal-protected amino acid or peptide ester represented by formula (R1) and an amino group amino group located on the left side in formula (R2) in an amino acid or peptide represented by formula (R2) or an ester compound thereof to produce an N-terminal-protected peptide represented by formula (S1); (ii) a step for deprotecting the N-terminal of the compound represented by formula (S1) produced in step (i) to produce a peptide compound represented by formula (P1); and (iii) a step for repeatedly performing the steps (i) and (ii) using the compound represented by formula (P1) produced in step (ii) as the compound represented by formula (R2) in step (i) to extend a peptide chain by amidation. In formulae (R1), (R2), (S1) and (P1), each symbol is as defined in the claims.

Description

[Technical field]

[0001] The present invention relates to a novel method for producing a polypeptide compound. [Background technology]

[0002] Traditionally, amide compounds, such as peptides, have been used in a wide range of fields, including pharmaceuticals, cosmetics, and functional foods, and the development of methods for synthesizing them has been vigorously pursued as an important research topic in synthetic chemistry (Non-Patent Documents 1-3). However, for amidation, which is the most important step in peptide synthesis, there are almost no truly effective catalysts or reactants other than carboxylic acid activators. Therefore, a reaction mode that produces a large amount of by-products must be used, and peptide synthesis, which involves repeated multi-step reactions, is extremely inefficient from the standpoint of atom economy (atomic yield), and the by-products are produced in huge quantities, and there are few effective purification methods. As a result, the cost of disposing of and purifying the by-products accounts for most of the necessary expenses in peptide synthesis, which is one of the biggest obstacles to the development of this field.

[0003] In peptide synthesis using amino acids or their derivatives as raw materials, highly stereoselective amidation is required. An example of highly stereoselective amidation is an enzyme reaction in vivo. For example, peptides are synthesized in vivo with extremely high stereoselectivity by skillfully utilizing enzymes and hydrogen bonds. However, enzyme reactions are not suitable for mass production, and applying them to synthetic chemistry requires huge financial and time costs.

[0004] In synthetic chemistry, amidation using a catalyst has also been investigated. However, in conventional methods, amide bonds are mainly formed by activating carboxylic acids, which leads to rapid racemization, making it difficult to synthesize amide compounds efficiently and with high stereoselectivity.

[0005] Furthermore, in conventional methods, it is extremely difficult to chemically ligate a peptide formed by linking a plurality of amino acids or derivatives thereof with an amide bond, or to ligate two or more peptides with an amide bond. As amidation methods for ligating such peptides, there are known a method in which an amino acid having a sulfur atom is used to perform ligation utilizing the high reactivity of the sulfur atom (Non-Patent Document 4), and a method in which a hydroxylamine of an amino acid is synthesized and ligation is performed utilizing the high reactivity of the hydroxylamine (Non-Patent Document 5). However, the former method requires the difficulty of synthesizing an amino acid having a sulfur atom, and the latter method requires the separate synthesis of hydroxylamine over several steps, so that both methods are time-consuming, costly, and inefficient.

[0006] The present inventors have developed technologies for synthesizing amide compounds with high chemoselectivity, such as a method of amidating a carboxylic acid / ester compound having a hydroxy group at the β-position in the presence of a specific metal catalyst (Patent Document 1), a method of using a hydroxyamino / imino compound as an amino acid precursor, amidating this in the presence of a specific metal catalyst, and then reducing it in the presence of a specific metal catalyst (Patent Document 2), and a method of amidating a carboxylic acid / ester compound in the presence of a specific metal catalyst (Patent Document 3). Furthermore, they have also developed a technique for synthesizing peptides consisting of various amino acid residues with high efficiency and high selectivity by amidating the carboxyl group of an N-terminal protected amino acid / peptide with the amino group of a C-terminal protected amino acid / peptide in the presence of a specific silylating agent (and a Lewis acid catalyst, which is optionally used in combination), followed by deprotection (Patent Document 4); a technique for synthesizing peptides consisting of various amino acid residues with high efficiency and high selectivity by amidating the carboxyl group of an N-terminal protected or unprotected amino acid / peptide with the amino group of a C-terminal protected or unprotected amino acid / peptide in the presence of a specific silylating agent, followed by deprotection (Patent Documents 5 and 6); a technique for performing an amidation reaction using a Brønsted acid as a catalyst (Patent Document 7); a novel silane-containing fused ring dipeptide compound and a novel method for synthesizing peptides using the compound (Patent Document 8); and a novel method for synthesizing peptides by site-selective CN bond cleavage of lactam (Non-Patent Document 6).

[0007] Recently, there is an increasing demand for a method for synthesizing peptides inexpensively, efficiently, and quickly. In particular, if such peptide synthesis can be performed continuously by a flow reaction, the steps of treating the solvent during the reaction and purifying the compounds can be omitted, and the reaction intermediates can be monitored as necessary, and further, all operations can be performed automatically.

[0008] However, the peptide bond formation reaction is less reactive than a normal amidation reaction, and it is difficult to complete the reaction in the flow channel. In addition, the use of a condensing agent, which was essential for conventional peptide synthesis reactions, can cause clogging in the flow path. Furthermore, undesired side reactions may occur at the same time, such as unreacted amino acids mixed in at the previous stage reacting unintentionally with amino acids to be the target of the amidation reaction at the subsequent stage. For this reason, it has been extremely difficult to continuously carry out peptide synthesis reactions by flow reactions in the past. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2017 / 204144 [Patent Document 2] International Publication No. 2018 / 199146 [Patent Document 3] International Publication No. 2018 / 199147 [Patent Document 4] International Publication No. 2019 / 208731 [Patent Document 5] International Publication No. 2021 / 085635 [Patent Document 6] International Publication No. 2021 / 085636 [Patent Document 7] International Publication No. 2021 / 149814 [Patent Document 8] International Publication No. 2022 / 190486 [Non-patent literature]

[0010] [Non-Patent Document 1] Chem. Rev., 2011, Vol.111, p.6557-6602 [Non-Patent Document 2] Org. Process Res. Dev., 2016, Vol.20, No.2, p.140-177 [Non-Patent Document 3] Chem. Rev., 2016, Vol.116, p.12029-12122 [Non-Patent Document 4] Science, 1992, Vol.256, p.221-225 [Non-Patent Document 5] Angew. Chem. Int. Ed., 2006, Vol.45, p.1248-1252 [Non-Patent Document 6] Chem. Sci., (2022), Vol.13, pp.6309-6315 [Non-Patent Document 7] Chem. Eur. J., 2019, Vol.25, p.15759-15764 [Non-Patent Document 8] J. Med. Chem., 2001, Vol.44, p.3896-3903 [Non-Patent Document 9] Org. Biomol. Chem., 2003, Vol.1, p.965-972 [Non-Patent Document 10] J. Org. Chem., 1995, 60, 6, 1733-1740 Summary of the Invention [Problem to be solved by the invention]

[0011] In view of the above, there has been a demand for a method for efficiently synthesizing a desired peptide chain by continuously carrying out peptide chain elongation through a peptide bonding reaction. [Means for solving the problem]

[0012] As a result of extensive investigations, the present inventors have found that a monovalent aromatic hydrocarbon group or heterocyclic group T aThe inventors have found that by using an N-terminal protected amino acid or peptide ester (R1) whose C-terminus has been esterified by the above formula as an electrophilic compound, mixing this with a nucleophilic compound, an amino acid or peptide or its ester (R2), to carry out a peptide bond reaction, deprotecting the N-terminus of the N-terminal protected amino acid or peptide (S1) obtained by the reaction, and subsequently subjecting the deprotected amino acid or peptide (P1) as the nucleophilic compound (R2) again to a peptide bond reaction with the electrophilic compound (R1), it is possible to continuously carry out elongation of a peptide chain by a peptide bond reaction, thereby enabling efficient synthesis of a desired peptide chain, and have arrived at the present invention.

[0013] That is, the gist of the present invention is as follows. [Item 1] A method for producing a polypeptide compound, comprising: (i) forming an amide bond between a substituted carboxyl group on the right side of an N-terminal protected amino acid ester or peptide ester compound represented by the following formula (R1) and an amino group on the left side of an amino acid or peptide, or an amino acid ester or peptide ester compound represented by the following formula (R2), to obtain an N-terminal protected peptide compound represented by the following formula (S1); (ii) deprotecting the N-terminus of the compound of formula (S1) obtained in step (i) to obtain a peptide compound represented by the following formula (P1): (iii) The compound of formula (P1) obtained in step (ii) is used as the compound of formula (R2) in step (i) to repeatedly carry out steps (i) and (ii) to elongate the peptide chain by amidation. A manufacturing method comprising: [ka] In formula (R1), T a represents a monovalent aromatic hydrocarbon group or heterocyclic group having one or more electron-withdrawing substituents, PG a represents a monovalent protecting group, R 11 and R12 each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, or a thiol group, or an amino group, a monovalent aliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, or a monovalent heterocyclic group, each of which may have one or more substituents; R 13 represents a hydrogen atom, a carboxyl group, a hydroxyl group, or a monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group which may have one or more substituents, in which case the monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group may be bonded to a nitrogen atom via a linking group, Or, R 11 and R 13 and are bonded to each other to form R 11 The carbon atom to which R is bonded 13 may form a heterocycle together with the nitrogen atom to which it is bonded which may have one or more substituents, A 11 and A 12 each independently represents a divalent aliphatic hydrocarbon group having 1 to 3 carbon atoms which may have one or more substituents, p11 and p12 each independently represent 0 or 1; n 1 is an integer of 1 or more and represents the number of structural units represented by the structure in [ ]. 1 When is 2 or more, the multiple constitutional units represented by the structure in [ ] may be the same or different. [ka] In formula (R2), T b represents a hydrogen atom or a monovalent substituent, R 21 and R 22 each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, or a thiol group, or an amino group, a monovalent aliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, or a monovalent heterocyclic group, each of which may have one or more substituents; R23 represents a hydrogen atom, a carboxyl group, a hydroxyl group, or a monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group which may have one or more substituents, in which case the monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group may be bonded to a nitrogen atom via a linking group, Or, R 21 and R 23 and are bonded to each other to form R 21 The carbon atom to which R is bonded 23 may form a heterocycle together with the nitrogen atom to which it is bonded which may have one or more substituents, A 21 and A 22 each independently represents a divalent aliphatic hydrocarbon group having 1 to 3 carbon atoms which may have one or more substituents, p21 and p22 each independently represent 0 or 1; n 2 is an integer of 1 or more and represents the number of structural units represented by the structure in [ ]. 2 When is 2 or more, the multiple constitutional units represented by the structure in [ ] may be the same or different. [ka] In formula (S1), PG a , R 11 , R 12 , R 13 , A 11 , A 12 , p11, p12, and n 1 represents the same group as defined in the above formula (R1), R 21 , R 22 , R 23 , A 21 , A 22 , p21, p22, n 2 , and T b represents the same group as defined in the above formula (R2). [ka] In formula (P1), R 11 , R 12 , R 13 , A 11 , A 12 , p11, p12, and n 1 represents the same group as defined in the above formula (R1), R 21 , R 22 , R 23 , A 21 , A 22 , p21, p22, n 2 , and T b represents the same group as defined in the above formula (R2). [Item 2] The method according to item 1, wherein steps (i) and (ii) are carried out continuously as a flow reaction. [Item 3] The method according to item 1 or 2, wherein in the reaction of step (i), the compound of formula (R1) and the compound of formula (R2) are used in an approximately equimolar ratio. [Item 4] Group T of formula (R1) a is a group selected from aromatic hydrocarbon groups having one or more substituents selected from a halogen atom, a halogen-substituted alkyl group, a halogen-substituted alkoxy group, a nitro group, an acetyl group, an ester group, a sulfonate ester group, and an amide group. [Item 5] Protecting group PG of formula (R1) a is a group selected from a monovalent hydrocarbon group, an acyl group, a hydrocarbonoxycarbonyl group, a hydrocarbonsulfonyl group, and an amide group, each of which may have one or more substituents. [Item 6] The method according to any one of Items 1 to 5, wherein the deprotection of the N-terminal protecting group of formula (S1) in step (ii) is carried out by passing the compound of formula (S1) through a column packed with a basic ion exchange resin. [Item 7] The method according to Item 6, wherein the basic ion exchange resin is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) resins, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) resins, 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) resins, piperazine resins, dimethylaminopyridine resins, and ammonium resins. Effect of the Invention

[0014] According to the method of the present invention, it is possible to continuously carry out peptide chain elongation through a peptide bonding reaction, thereby efficiently synthesizing a desired peptide chain. [Brief description of the drawings]

[0015] [Figure 1] 1 is a schematic diagram showing the procedure for synthesizing a polypeptide consisting of the amino acid sequence H2N-AAn-AAn-1-(···)-AA2-AA1-O-tBu (wherein AA1, AA2, ···AAn-1, and AAn each represent an amino acid residue) using a preferred embodiment of the production method of the present invention by flow reaction. Note that a pentafluorophenyl (Pfp) group is used as the Ta group in formula (R1), an Fmoc group is used as the PGa group, and a t-butyl (tBu) group is used as the Tb group in formula (R2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments, and can be embodied in any form without departing from the spirit of the present invention.

[0017] All patent publications, published patent applications, and non-patent publications cited in this disclosure are hereby incorporated by reference in their entirety into this disclosure for all purposes.

[0018] [I. Definitions of Terms] In the present disclosure, "amino acid" refers to a compound having a carboxyl group and an amino group. Unless otherwise specified, the type of amino acid is not particularly limited. For example, from the viewpoint of optical isomerism, it may be D-form, L-form, or racemic. In addition, from the viewpoint of the relative position of the carboxyl group and the amino group, it may be any of α-amino acid, β-amino acid, γ-amino acid, δ-amino acid, ω-amino acid, etc. Examples of amino acids include, but are not limited to, natural amino acids that constitute proteins, and specific examples include valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, serine, etc.

[0019] In the present disclosure, "peptide" refers to a compound in which multiple amino acids are linked via peptide bonds. Unless otherwise specified, the multiple amino acid units constituting a peptide may be the same type of amino acid unit, or may be two or more different types of amino acid units. The number of amino acids constituting a peptide is not particularly limited as long as it is two or more. Examples include 2 (also called "dipeptide"), 3 (also called "tripeptide"), 4 (also called "tetrapeptide"), 5 (also called "pentapeptide"), 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or more. In addition, the term "polypeptide" may refer to a peptide that is tripeptide or more.

[0020] In the present disclosure, the term "amino group" refers to a functional group represented by the formula -NH2, -NRH, or -NRR' (wherein R and R' each represent a substituent) obtained by removing hydrogen from ammonia, a primary amine, or a secondary amine, respectively.

[0021] In the present disclosure, unless otherwise specified, the hydrocarbon group may be aliphatic or aromatic. The aliphatic hydrocarbon group may be linear or cyclic. The linear hydrocarbon group may be linear or branched. The cyclic hydrocarbon group may be monocyclic, bridged, or spirocyclic. The hydrocarbon group may be saturated or unsaturated, in other words, may contain one or more carbon-carbon double bonds and / or triple bonds. That is, the hydrocarbon group is a concept including alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups, aryl groups, arylalkyl groups, alkylaryl groups, and the like. In addition, unless otherwise specified, one or more hydrogen atoms of the hydrocarbon group may be substituted with any substituent, and one or more carbon atoms of the hydrocarbon group may be substituted with any heteroatom depending on the valence.

[0022] In the present disclosure, the term "hydrocarbonoxy group" refers to a group in which a hydrocarbon group as defined above is linked to one bond of an oxy group (-O-). In other words, the term "hydrocarbonoxy group" encompasses alkyloxy groups, alkenyloxy groups, alkynyloxy groups, cycloalkyloxy groups, cycloalkenyloxy groups, cycloalkynyloxy groups, aryloxy groups, and the like.

[0023] In the present disclosure, the term "hydrocarbon carbonyl group" refers to a group in which a hydrocarbon group as defined above is linked to one bond of a carbonyl group (-C(=O)-). In other words, the term "hydrocarbon carbonyl group" encompasses alkylcarbonyl groups, alkenylcarbonyl groups, alkynylcarbonyl groups, cycloalkylcarbonyl groups, cycloalkenylcarbonyl groups, cycloalkynylcarbonyl groups, arylcarbonyl groups, and the like.

[0024] In the present disclosure, the term "hydrocarbon sulfonyl group" refers to a group in which a hydrocarbon group as defined above is linked to one bond of a sulfonyl group (-S(=O)-). In other words, the term "hydrocarbon sulfonyl group" encompasses alkylsulfonyl groups, alkenylsulfonyl groups, alkynylsulfonyl groups, cycloalkylsulfonyl groups, cycloalkenylsulfonyl groups, cycloalkynylsulfonyl groups, arylsulfonyl groups, and the like.

[0025] In the present disclosure, a "heterocyclic group" may be saturated or unsaturated, in other words, it may contain one or more carbon-carbon double bonds and / or triple bonds. In addition, a heterocyclic group may be a monocyclic, bridged, or spirocyclic group. In addition, the heteroatoms contained in the heterocyclic ring of a heterocyclic group are not limited, and examples thereof include nitrogen, oxygen, sulfur, phosphorus, silicon, etc.

[0026] In the present disclosure, the term "heterocyclic oxy group" refers to a group in which a heterocyclic group as defined above is linked to one bond of an oxy group (-O-).

[0027] In the present disclosure, the term "heterocyclic carbonyl group" refers to a group in which a heterocyclic group as defined above is linked to one bond of a carbonyl group (-C(=O)-).

[0028] In the present disclosure, the term "heterocyclic sulfonyl group" refers to a group in which a heterocyclic group as defined above is linked to one bond of a sulfonyl group (-S(=O)2-).

[0029] In the present disclosure, a "metaloxy group" (which may have one or more substituents) is a group represented by the formula (R n In the formula, M represents an arbitrary metal element, R represents an arbitrary substituent, and n represents an integer of 0 to 8 that can be taken depending on the coordination number of the metal element M.

[0030] In the present disclosure, the term "substituent" refers to any substituent that is not particularly limited as long as the amidation step in the production method of the present invention proceeds, unless otherwise specified. Examples of the substituent include, but are not limited to, a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, a thiol group, a sulfonic acid group, an amino group, an amido group, an imino group, an imido group, a hydrocarbon group, a heterocyclic group, a hydrocarbonoxy group, a hydrocarboncarbonyl group (acyl group), a hydrocarbonoxycarbonyl group, a hydrocarboncarbonyloxy group, a hydrocarbon-substituted amino group, a hydrocarbon-substituted aminocarbonyl group, a hydrocarboncarbonyl-substituted amino group, a hydrocarbon-substituted thiol group, a hydrocarbonsulfonyl group, a hydrocarbonoxysulfonyl group, a hydrocarbonsulfonyloxy group, a heterocyclicoxy group, a heterocycliccarbonyl group, a heterocyclicoxycarbonyl group, a heterocycliccarbonyloxy group, a heterocyclicamino group, a heterocyclicaminocarbonyl group, a heterocycliccarbonyl-substituted amino group, a heterocyclic-substituted thiol group, a heterocyclicsulfonyl group, a heterocyclicoxysulfonyl group, a heterocyclicsulfonyloxy group, and the like. In addition, as long as the valence and physicochemical properties of these functional groups allow, functional groups further substituted with these functional groups are also included in the "substituent" in the present disclosure. When a functional group has a substituent, the number of the substituents is not particularly limited as long as the valence and physicochemical properties allow. In addition, when multiple substituents are present, these substituents may be the same or different from each other.

[0031] The main abbreviations used in this disclosure are shown in Tables 1-1 and 1-2 below. [Table 1-1] [Table 1-2]

[0032] In this disclosure, amino acids and their residues may be represented by three-letter abbreviations well known to those skilled in the art. The three-letter abbreviations of the main amino acids used in this disclosure are shown in the following table. [Table 2]

[0033] In this disclosure, β-homo amino acids and residues thereof may be referred to by adding "Ho" before the three letter abbreviation of the corresponding α-amino acid.

[0034] [II. Method for producing polypeptide compounds] ·overview One aspect of the present invention relates to a method for producing a polypeptide compound, the method comprising at least the following steps (i) to (iii) (hereinafter appropriately referred to as the "method for producing the peptide compound of the present invention" or simply the "method for producing the peptide compound of the present invention"). (i) The right side of the N-terminal protected amino acid or peptide compound represented by the following formula (R1) a An amide bond is formed between the substituted carbonyl group and the amino group on the left side of an amino acid or peptide, or an amino acid ester or peptide ester compound represented by the following formula (R2), to obtain an N-terminal protected peptide compound represented by the following formula (S1) (amide bond forming reaction, condensation reaction). (ii) The N-terminus of the compound of formula (S1) obtained in step (i) is deprotected to obtain a peptide compound represented by the following formula (P1) (deprotection reaction). (iii) The compound of formula (P1) obtained in step (ii) is used as the compound of formula (R2) in step (i), and steps (i) and (ii) are repeatedly carried out to elongate the peptide chain by amidation.

[0035] [ka] [ka] [ka] [ka]

[0036] That is, in the production method of the present invention, an aromatic hydrocarbon group or a heterocyclic group T having an electron-withdrawing substituent is a The N-terminal protected amino acid or peptide ester (R1) whose C-terminus has been esterified by the above method is used as an electrophilic compound, which is mixed with a nucleophilic compound, an amino acid or peptide or its ester (R2), to form a peptide bond (condensation reaction) (step (i)), the N-terminus of the N-terminal protected amino acid or peptide (S1) obtained by the reaction is deprotected (step (ii)), and the deprotected amino acid or peptide (P1) is then used as the nucleophilic compound (R2) again in a peptide bond reaction with the electrophilic compound (R1) (step (iii)). By repeating the above steps, the peptide chain can be continuously elongated by the peptide bond reaction, making it possible to efficiently synthesize the desired peptide chain.

[0037] In particular, in the production method of the present invention, an aromatic hydrocarbon group or a heterocyclic group T having an electron-withdrawing substituent is a By using an N-terminal protected amino acid or peptide ester (R1) whose C-terminus has been esterified by the above formula as an electrophilic species, the reactivity of the electrophilic species amino acid can be dramatically improved, and the reaction can be quantitatively progressed even when the molar ratio of electrophilic species amino acid to nucleophilic species amino acid used is close to 1:1. This makes it possible to prevent the generation of excess amino acid that is not involved in the reaction, and to avoid side reactions due to unreacted substances.

[0038] According to a preferred embodiment of the production method of the present invention, the N-terminal protecting group PG of the electrophilic amino acid or peptide ester (R1) is aAs a protective group, a specific protective group that can be deprotected by passing through a basic ion exchange resin can be used. This makes it possible to continuously carry out a peptide bond reaction (step (i)) by mixing an electrophilic N-terminal amino acid or peptide ester (R1) with a nucleophilic amino acid or peptide (R2) and deprotecting the N-terminal protected amino acid or peptide (S1) obtained by the reaction with a basic ion exchange resin (step (ii)) as a series of reactions. Furthermore, the deprotected amino acid or peptide (P1) is again subjected to a peptide bond reaction with the electrophilic compound (R1) as a nucleophilic compound (R2) (step (iii)). By repeating the above, the peptide chain can be continuously extended by a flow reaction through a peptide bond reaction, and the desired peptide chain can be efficiently synthesized.

[0039] In addition, conventionally, the amino acid T a As a peptide bond formation reaction using a compound corresponding to an ester as an electrophilic species, there are reports of the use of a pentafluorophenyl ester of an amino acid (hereinafter referred to as H-AA-O-Pfp) (Non-Patent Document 7: J. Med. Chem., 2001, 44, 3896-3903; Non-Patent Document 8: Chem. Eur. J., 2019, 25, 15759-15764; Non-Patent Document 9: Org. Biomol. Chem., 2003, 1, 965-972; Non-Patent Document 10: J. Org. Chem., 1995, 60, 6, 1733-1740), which is known as a useful method. However, there have been no cases in which such electrophilic amino acids and nucleophilic amino acids have been used in a molar ratio of 1:1, and there have been no cases of application to flow reactions.

[0040] ·Substrate compound In the above general formulas (R1) and (R2), the definitions of each symbol are as follows.

[0041] R 11 , R 12 , R 21 , and R 22Each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, or a thiol group, or a monovalent hydrocarbon group or heterocyclic group which may have one or more substituents. When these groups have a substituent, the type of the substituent is as described above. Specific examples of the number of substituents are, for example, 5, 4, 3, 2, 1, or 0.

[0042] Also, R 11 , R 12 , R 21 , and / or R 22 is a monovalent hydrocarbon group or heterocyclic group which may have one or more substituents, a linking group may be present between the hydrocarbon group or heterocyclic group and the carbon atom to which it is bonded. Such linking groups are not limited, but may be each independently selected from the structures shown below (note that in the chemical formulas below, each A independently represents a monovalent hydrocarbon group or heterocyclic group which may have one or more substituents. When there are two A's in the same group, they may be the same or different).

[0043] [ka]

[0044] The number of carbon atoms in the hydrocarbon group (including the substituent if any) is not particularly limited, but the upper limit is, for example, 20 or less, 15 or less, 10 or less, 8 or less, or 6 or less. The lower limit varies depending on the type of hydrocarbon group, but is 1 or more for an alkyl group, 2 or more for an alkenyl group or alkynyl group, and 3 or more for a cycloalkyl group, for example, 4 or more or 5 or more. Specific examples of the number of atoms are, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0045] The total number of carbon atoms and heteroatoms (including substituents, if any) in the heterocyclic group is not particularly limited, but the upper limit is, for example, 20 or less, 15 or less, 10 or less, 8 or less, or 6 or less. The lower limit varies depending on the type of heterocyclic structure, but is usually 3 or more, for example, 4 or more, or 5 or more. Specific examples of the number of atoms are, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0046] Among them, R 11 , R 12 , R 21 , and R 22 are preferably each independently a hydrogen atom, a hydroxyl group, a thiol group, a carboxyl group, a nitro group, a cyano group, or a halogen atom, or an amino group, an alkyl group, an alkenyl group, a cycloalkyl group, an alkoxy group, an aryl group, an aryloxy group, an acyl group, a heterocyclic group, or a heterocyclic oxy group, which may have one or more substituents.

[0047] R 11 , R 12 , R 21 , and R 22 Specific examples include, but are not limited to, the following:

[0048] Hydrogen atoms, hydroxyl groups, thiol groups, carboxyl groups, nitro groups, cyano groups; Halogen atoms such as fluorine, chlorine, bromine, and iodine atoms; Alkyl groups such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, decyl, and nonyl; Alkenyl groups such as ethenyl, propenyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, and octenyl; Alkynyl groups, such as propargyl groups; Cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclooctyl, and spirooctyl groups; Alkoxy groups such as methoxy, ethoxy, propoxy, butoxy, sec-butoxy, and tert-butoxy; Aryl groups such as phenyl, benzyl, tolyl, naphthyl, and anthracenyl; Aryloxy groups such as phenyloxy, benzyloxy, and naphthyloxy; Acyl groups such as acetyl, propionyl, benzoyl, p-methoxybenzoyl, and cinnamoyl; Unsubstituted amino groups and substituted amino groups such as dimethylamino groups, benzylamino groups, and triphenylmethylamino groups; Furanyl group, thiophenyl group, pyranyl group, pyrrolinyl group, pyrrolyl group, 2,3-dihydro-1H-pyrrolyl group, piperidinyl group, piperazinyl group, homopiperazinyl group, morpholino group, thiomorpholino group, 1,2,4,6-tetrahydropyridyl group, hexahydropyrimidyl group, hexahydropyridazyl group, 1,2,4,6-tetrahydropyridyl group, 1,2,4,6-tetrahydropyridazyl group, 3,4-dihydropyridyl group, imidazolyl group, 4,5-dihydro-1H-imidazolyl group heterocyclic groups such as an alkyl group, a 2,3-dihydro-1H-imidazolyl group, a pyrazolyl group, a 4,5-dihydro-1H-pyrazolyl group, a 2,3-dihydro-1H-pyrazolyl group, an oxazolyl group, a 4,5-dihydro-1,3-oxazolyl group, a 2,3-dihydro-1,3-oxazolyl group, a 2,5-dihydro-1,3-oxazolyl group, a thiazolyl group, a 4,5-dihydro-1,3-thiazolyl group, a 2,3-dihydro-1,3-thiazolyl group, a 2,5-dihydro-1,3-thiazolyl group, and a carbazolyl group; Heterocyclic oxy groups such as a furanyloxy group, a pyrrolyloxy group, an indolyloxy group, a quinolyloxy group, etc.

[0049] Among the above groups, the group having a carboxyl group may or may not have a protecting group. Although it depends on the reactivity between the compound (R1) and the compound (R2) used in the reaction, when the group having a carboxyl group among the above groups has a protecting group, the reaction selectivity with the carboxylate group on the right side in the formula of the compound (R2) is usually improved compared to the reaction selectivity with the carboxyl groups present in other substituents.

[0050] R 13 and R 23 Each independently represents a hydrogen atom, a carboxyl group, or a hydroxyl group, or a monovalent hydrocarbon group or heterocyclic group which may have one or more substituents. When the group has a substituent, the type of the substituent is as described above. Specific examples of the number of the substituents are, for example, 5, 4, 3, 2, 1, or 0.

[0051] Also, R 13 and / or R 23 is a monovalent hydrocarbon group or heterocyclic group which may have one or more substituents, a linking group may be present between the hydrocarbon group or heterocyclic group and the nitrogen atom to which it is bonded. Such linking groups are not limited, but may be each independently selected from the structures shown below (note that in the chemical formulas below, each A independently represents a monovalent hydrocarbon group or heterocyclic group which may have one or more substituents. When there are two A's in the same group, they may be the same or different).

[0052] [ka]

[0053] The upper limit of the number of carbon atoms in the hydrocarbon group (including the substituent if any) is, for example, 20 or less, 15 or less, 10 or less, 8 or less, or 6 or less. The lower limit varies depending on the type of hydrocarbon group, but is 1 or more for an alkyl group, 2 or more for an alkenyl group or alkynyl group, and 3 or more for a cycloalkyl group, for example, 4 or more or 5 or more. Specific examples of the number of atoms are, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0054] The upper limit of the total number of carbon atoms and heteroatoms (including substituents, if any) in the heterocyclic group is, for example, 20 or less, 15 or less, 10 or less, 8 or less, or 6 or less. The lower limit varies depending on the type of heterocyclic structure, but is usually 3 or more, for example, 4 or more, or 5 or more. Specific examples of the number of atoms are, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0055] Among them, R 13 and R 23 are preferably each independently a hydrogen atom, a hydroxyl group, or a carboxyl group, or an alkyl group, an alkenyl group, a cycloalkyl group, an alkoxy group, an aryl group, an aryloxy group, an acyl group, a heterocyclic group, or a heterocyclic oxy group, each of which may have one or more substituents.

[0056] R 13 and R 23 Specific examples include, but are not limited to, the following:

[0057] Hydrogen atoms, hydroxyl groups, carboxyl groups; Alkyl groups such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, decyl, and nonyl; Alkenyl groups such as ethenyl, propenyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, and octenyl; Alkynyl groups, such as propargyl groups; Cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclooctyl, and spirooctyl groups; Aryl groups such as phenyl, benzyl, tolyl, naphthyl, and anthracenyl; Furanyl, thiophenyl, pyranyl, pyrrolinyl, pyrrolyl, 2,3-dihydro-1H-pyrrolyl, piperidinyl, piperazinyl, homopiperazinyl, morpholino, thiomorpholino, 1,2,4,6-tetrahydropyridyl, hexahydropyrimidyl, hexahydropyridazyl, 1,2,4,6-tetrahydropyridyl, 1,2,4,6-tetrahydropyridazyl, 3,4-dihydropyridyl, imidazolyl, 4,5-dihydro-1H-imidazolyl heterocyclic groups such as a 2,3-dihydro-1H-imidazolyl group, a pyrazolyl group, a 4,5-dihydro-1H-pyrazolyl group, a 2,3-dihydro-1H-pyrazolyl group, an oxazolyl group, a 4,5-dihydro-1,3-oxazolyl group, a 2,3-dihydro-1,3-oxazolyl group, a 2,5-dihydro-1,3-oxazolyl group, a thiazolyl group, a 4,5-dihydro-1,3-thiazolyl group, a 2,3-dihydro-1,3-thiazolyl group, a 2,5-dihydro-1,3-thiazolyl group, a carbazolyl group, and the like;

[0058] In addition, R 11 and R 13 and are bonded to each other to form R 11 The carbon atom to which R is bonded 13 may form a heterocycle optionally having one or more substituents together with the nitrogen atom to which R is bonded, 21 and R 23 and are bonded to each other to form R 21 The carbon atom to which R is bonded 23may form a heterocycle which may have one or more substituents together with the nitrogen atom to which it is bonded. When it has a substituent, the type of the substituent is as described above. Specific examples of the number of the substituents are, for example, 5, 4, 3, 2, 1, or 0.

[0059] The upper limit of the total number of carbon atoms and heteroatoms (including substituents, if any) in the heterocyclic group is, for example, 20 or less, 15 or less, 10 or less, 8 or less, or 6 or less. The lower limit varies depending on the type of heterocyclic structure, but is usually 3 or more, for example, 4 or more, or 5 or more. Specific examples of the number of atoms are, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0060] Specific examples of such heterocycles include, but are not limited to, a pyrrolinyl group, a pyrrolyl group, a 2,3-dihydro-1H-pyrrolyl group, a piperidinyl group, a piperazinyl group, a homopiperazinyl group, a morpholino group, a thiomorpholino group, a 1,2,4,6-tetrahydropyridyl group, a hexahydropyrimidyl group, a hexahydropyridazyl group, a 1,2,4,6-tetrahydropyridyl group, a 1,2,4,6-tetrahydropyridazyl group, a 3,4-dihydropyridyl group, an imidazolyl group, a 4,5-dihydropyridyl group, a 5,6-dihydropyridyl group, a 6,7-dihydropyridyl group, a 7,8-dihydropyridyl group, a 8,9-dihydropyridyl group, a 9,10-dihydropyridyl group, a 11,12-dihydropyridyl group, a 12,13-dihydropyridyl group, a 13,14-dihydropyridyl group, a 14,15-dihydropyridyl group, a 15,16-dihydropyridyl group, a 17,18-dihydropyridyl group, a 19,19-dihydropyridyl group, a 20,11-dihydropyridyl group, a 21,12-dihydropyridyl group, a 22,13-dihydropyridyl group, a 23,14-dihydropyridyl group, a 24,15-dihydropyridyl group, a 25,16-dihydropyridyl group, a 26,17-dihydropyridyl group, a 27,18-dihydropyridyl group, a 28,19-dihydropyridyl group, a 29,11-dihydropy -1H-imidazolyl group, 2,3-dihydro-1H-imidazolyl group, pyrazolyl group, 4,5-dihydro-1H-pyrazolyl group, 2,3-dihydro-1H-pyrazolyl group, oxazolyl group, 4,5-dihydro-1,3-oxazolyl group, 2,3-dihydro-1,3-oxazolyl group, 2,5-dihydro-1,3-oxazolyl group, thiazolyl group, 4,5-dihydro-1,3-thiazolyl group, 2,3-dihydro-1,3-thiazolyl group, and 2,5-dihydro-1,3-thiazolyl group.

[0061] A 11 , A 12 , A 21 , and A 22each independently represents a divalent aliphatic hydrocarbon group having 1 to 3 carbon atoms which may have one or more substituents. Specific examples include, but are not limited to, a methylene group, an ethylene group, a propylene group, an isopropylene group, and the like, as well as groups in which these groups are substituted with one or more of the above-mentioned substituents. Specific examples of the number of substituents are, for example, 3, 2, 1, or 0.

[0062] p11, p12, p21, and p22 each independently represent 0 or 1.

[0063] n 1 is an integer of 1 or more, which represents the number of amino acid units in the brackets [ ] of general formula (R1). 1 When n is 1, compound (R1) is an amino acid, and 1 When n is 2 or more, the compound (R1) is a peptide. 1 The upper limit of n is not particularly limited as long as the amination step proceeds, but is, for example, 100 or less, 80 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 12 or less, or 10 or less. 1 Specific examples are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, etc.

[0064] n 2 is an integer of 1 or more, which represents the number of amino acid units in the brackets [ ] of general formula (R2). 2 When n is 1, compound (R2) is an amino acid, and 2 When n is 2 or more, the compound (R2) is a peptide. 2 The upper limit of is not particularly limited as long as the amination step proceeds, and examples thereof include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the like.

[0065] Needless to say, n 1 If is 2 or more, R that defines the structure in [ ] 11 , R 12 , R 13, A 11 , A 12 , p11, and p12 may be the same or different among multiple amino acid units. 2 If is 2 or more, R that defines the structure in [ ] 21 , R 22 , R 23 , A 21 , A 22 , p21, and p22 may be the same or different among multiple amino acid units. That is, when compound (R1) and / or compound (R2) are peptides, multiple amino acid units constituting the peptide may be the same or different.

[0066] In compound (R1), T a represents a monovalent aromatic hydrocarbon group or heterocyclic group having one or more electron-withdrawing substituents. a Details will be provided later.

[0067] In compound (R2), T b represents a hydrogen atom or a monovalent substituent. In the case of a monovalent substituent, the type thereof is not particularly limited, but R 13 and R 23 In addition to the above-mentioned groups, a protecting group for a carboxyl group (hereinafter referred to as PG b The protecting group for the carboxyl group, PG b There are no particular limitations on the protecting group PG as long as it can protect the carboxyl group from reaction in the amidation reaction and can be converted to a carboxyl group by deprotection after the reaction. b Details will be provided later.

[0068] In compound (R2), the amino group on the left side of the formula may form a salt with another acid. In this case, the other acid may include, but is not limited to, aliphatic carboxylic acids having 1 to 5 carbon atoms such as acetic acid and propionic acid; trifluoroacetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, sulfonic acid, etc.

[0069] The above-mentioned substrate compounds (R1) and (R2) may each be used as a single compound, or as a mixture of two or more compounds in any combination and ratio.

[0070] In addition, a part or all of the above-mentioned substrate compound (R1) or (R2) may be linked and fixed to a support such as a substrate or resin at any of the substituents. In this case, the type of the support such as the substrate or resin is not limited. Any conventionally known support such as a substrate or resin can be used without substantially inhibiting the amide bond reaction in the production method of the present invention and within the scope of the present invention. The mode of linking and fixing the substrate compound to a support such as a substrate or resin is not limited in any way, but it is preferable to form a covalent bond between any of the substituents of the substrate compound and the substituents present on the support such as the substrate or resin. The type of each substituent and the method of forming the covalent bond are also not limited in any way. Any conventionally known type of substituent and method of forming the covalent bond can be used without substantially inhibiting the amide bond reaction in the production method of the present invention and within the scope of the present invention. The substrate compound may be linked and fixed to a support such as a substrate or resin by a covalent bond using a carboxyl group or amino group (other than the carboxylate group or amino group that is the target of the amide bond reaction) of the substrate compound. This embodiment can be considered similar to an embodiment in which a carboxyl group or an amino group (other than a carboxylate group or an amino group to be the target of amide bond formation) of a substrate compound is protected by introducing a protecting group.

[0071] ·C-terminal substituent T of electrophilic substrate compound (R1) a : In compound (R1), T a represents a monovalent aromatic hydrocarbon group or heterocyclic group having one or more electron-withdrawing substituents.

[0072] Examples of the monovalent aromatic hydrocarbon group include, but are not limited to, a phenyl group, a benzyl group, a tolyl group, a naphthyl group, an anthracenyl group, etc. The number of carbon atoms is not limited, but is usually 6 or more, and usually 14 or less, or 10 or less.

[0073] Examples of the monovalent heterocyclic group include, but are not limited to, a furanyl group, a thiophenyl group, a pyranyl group, a pyrrolinyl group, a pyrrolyl group, a 2,3-dihydro-1H-pyrrolyl group, a piperidinyl group, a piperazinyl group, a homopiperazinyl group, a morpholino group, a thiomorpholino group, a 1,2,4,6-tetrahydropyridyl group, a hexahydropyrimidyl group, a hexahydropyridazyl group, a 1,2,4,6-tetrahydropyridyl group, a 1,2,4,6-tetrahydropyridazyl group, a 3,4-dihydropyridyl group, an imidazolyl group, a 4,5 -dihydro-1H-imidazolyl group, 2,3-dihydro-1H-imidazolyl group, pyrazolyl group, 4,5-dihydro-1H-pyrazolyl group, 2,3-dihydro-1H-pyrazolyl group, oxazolyl group, 4,5-dihydro-1,3-oxazolyl group, 2,3-dihydro-1,3-oxazolyl group, 2,5-dihydro-1,3-oxazolyl group, thiazolyl group, 4,5-dihydro-1,3-thiazolyl group, 2,3-dihydro-1,3-thiazolyl group, 2,5-dihydro-1,3-thiazolyl group, carbazolyl group, etc. The number of ring-constituting elements is not limited, but is usually 5 or more, and usually 14 or less, or 10 or less.

[0074] Examples of the electron-withdrawing substituent that the monovalent aromatic hydrocarbon group or heterocyclic group has include, but are not limited to, the following: halogen atoms (e.g. fluorine, chlorine, bromine, iodine, etc., among which fluorine or chlorine, etc. are preferred); Halogen-substituted alkyl groups (the above-mentioned alkyl groups substituted with one or more of the above-mentioned halogen atoms); Halogen-substituted alkoxy groups (groups in which any of the above halogen-substituted alkyl groups are linked to an oxy group (-O-)); Nitro group; · Acetyl group; · Carboxylic groups (-C(=O)-OH); Carboxy ester group (a group in which the above-mentioned monovalent aliphatic or aromatic hydrocarbon group (R), which may have one or more substituents, is linked to a carboxy group (-C(=O)-OH) (-C(=O)-OR)); ·Sulfoxy group (-S(=O)2-OH); sulfoxy ester group (a group (-S(=O)2-OR) in which the above-mentioned monovalent aliphatic or aromatic hydrocarbon group (R), which may have one or more substituents, is linked to a sulfoxy group (-S(=O)2-OH); · Amide group (-C(=O)-NH2); Substituted amide groups (groups in which one or two monovalent aliphatic or aromatic hydrocarbon groups (R), which may have one or more of the above-mentioned substituents, are linked to an amide group (-C(=O)-NH2) (-C(=O)-NRH or -C(=O)-NR2), etc.

[0075] T a The number of electron-withdrawing substituents in the aromatic hydrocarbon group or heterocyclic group is 1 or more. There is no particular upper limit, and the number may be equal to or less than the upper limit determined depending on the type of the monovalent aromatic hydrocarbon group or heterocyclic group, and may be, for example, 5 or less.

[0076] Amino protecting groups: Amino protecting group PG aA wide variety of known linking groups are known. Examples include monovalent hydrocarbon groups which may have one or more substituents, and monovalent heterocyclic groups which may have one or more substituents. Among them, monovalent hydrocarbon groups which may have one or more substituents are preferred. However, a linking group may be present between such a hydrocarbon group or heterocyclic group and the nitrogen atom of the amino group it protects. Such linking groups are not limited, but are each independently selected from the linking groups shown below (note that in the following chemical formula, A each independently represents a monovalent hydrocarbon group or heterocyclic group which may have one or more substituents. When there are two A's in the same group, they may be the same or different).

[0077] [ka]

[0078] The protecting group usually has 1 or more, or 3 or more, and usually has 20 or less, or 15 or less.

[0079] Among these, the protecting group for the amino group is preferably one or more groups selected from the group consisting of monovalent hydrocarbon groups, acyl groups, hydrocarbonoxycarbonyl groups, hydrocarbonsulfonyl groups, and amide groups, each of which may have one or more substituents.

[0080] Specific examples of the protecting group for the amino group are listed below. Note that the name of the protecting group for the amino group includes not only the name of the functional group bonded to the nitrogen atom of the amino group, but also the name including the nitrogen atom, and both are included in the following names.

[0081] Specific examples of unsubstituted or substituted hydrocarbon groups include alkyl groups such as methyl, ethyl, and propyl groups; alkenyl groups such as ethenyl, propenyl, and allyl groups; alkynyl groups such as propargyl groups; cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups; aryl groups such as phenyl, benzyl, paramethoxybenzyl, tolyl, and triphenylmethyl (toroc) groups; and substituted hydrocarbon groups such as cyanomethyl groups. The number of carbon atoms is usually 1 or more, or 3 or more, and usually 20 or less, or 15 or less.

[0082] Specific examples of the unsubstituted or substituted acyl group include a benzoyl group (Bz), an ortho-methoxybenzoyl group, a 2,6-dimethoxybenzoyl group, a para-methoxybenzoyl group (PMPCO), a cinnamoyl group, and a phthaloyl group (Phth).

[0083] Specific examples of unsubstituted or substituted hydrocarbonoxycarbonyl groups include a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Cbz or Z), a methoxycarbonyl group, an ethoxycarbonyl group, a 2-trimethylsilylethoxycarbonyl group, a 2-phenylethoxycarbonyl group, a 1-(1-adamantyl)-1-methylethoxycarbonyl group, a 1-(3,5-di-t-butylphenyl)-1-methylethoxycarbonyl group, a vinyloxycarbonyl group, an allyloxycarbonyl group, Examples of such groups include an allyl group (Alloc), an N-hydroxypiperidinyloxycarbonyl group, a p-methoxybenzyloxycarbonyl group, a p-nitrobenzyloxycarbonyl group, a 2-(1,3-dithianyl)methoxycarbonyl group, a m-nitrophenoxycarbonyl group, a 3,5-dimethoxybenzyloxycarbonyl group, an o-nitrobenzyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group (Troc), and a 9-fluorenylmethyloxycarbonyl group (Fmoc).

[0084] Specific examples of unsubstituted or substituted hydrocarbon sulfonyl groups include a methanesulfonyl group (Ms), a toluenesulfonyl group (Ts), and a 2- or 4-nitrobenzenesulfonyl group (Ns).

[0085] Specific examples of unsubstituted or substituted amide groups include acetamide, o-(benzoyloxymethyl)benzamide, 2-[(t-butyldiphenylsiloxy)methyl]benzamide, 2-toluenesulfonamide, 4-toluenesulfonamide, 2-nitrobenzenesulfonamide, 4-nitrobenzenesulfonamide, tert-butylsulfinylamide, 4-toluenesulfonamide, 2-(trimethylsilyl)ethanesulfonamide, benzylsulfonamide, and the like.

[0086] From the viewpoint of the deprotection technique, examples of the protecting group for the amino group include a protecting group that can be deprotected by at least one technique selected from the group consisting of deprotection by hydrogenation, deprotection by a weak acid, deprotection by a fluoride ion, deprotection by a one-electron oxidizing agent, deprotection by hydrazine, and deprotection by oxygen. In particular, in the production method of the present invention, the protecting group PG of the terminal amino group of the electrophilic amino acid or peptide ester (R1) is a It is preferable to use a protecting group that can be deprotected by passing through a basic ion exchange resin.

[0087] Preferred specific examples of the protecting group for the amino group include a mesyl group (Ms), a tert-butoxycarbonyl group (Boc), a benzyl group (Bn or Bzl), a benzyloxycarbonyl group (Cbz), a benzoyl group (Bz), a paramethoxybenzyl group (PMB), a 2,2,2-trichloroethoxycarbonyl group (Troc), an allyloxycarbonyl group (Alloc), a 2,4-dinitrophenyl group (2,4-DNP), a phthaloyl group (Phth), a paramethoxybenzoyl group (PMPCO), a cinnamoyl group, a toluenesulfonyl group (Ts), a 2- or 4-nitrobenzenesulfonyl group (Ns), a cyanomethyl group, a 9-fluorenylmethyloxycarbonyl group (Fmoc), etc. This is because these protecting groups can easily protect an amino group and can be removed under relatively mild conditions, as described above.

[0088] More preferred specific examples of the amino-protecting group include a mesyl group (Ms), a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Cbz), a benzyl group (Bn), a paramethoxybenzyl group (PMB), a 2,2,2-trichloroethoxycarbonyl group (Troc), an allyloxycarbonyl group (Alloc), a paramethoxybenzoyl group (PMPCO), a benzoyl group (Bz), a cyanomethyl group, a cinnamoyl group, a 2- or 4-nitrobenzenesulfonyl group (Ns), a toluenesulfonyl group (Ts), a phthaloyl group (Phth), a 2,4-dinitrophenyl group (2,4-DNP), a 9-fluorenylmethyloxycarbonyl group (Fmoc), and the like.

[0089] In particular, the protecting group PG of the terminal amino group of the electrophilic amino acid or peptide ester (R1) a As described above, the protecting group is preferably a protecting group that can be removed by passing through a basic ion exchange resin. Specific examples thereof include a 9-fluorenylmethyloxycarbonyl group (Fmoc group), a benzyloxycarbonyl group (Cbz group), a tert-butoxycarbonyl group (Boc group), an allyloxycarbonyl group (Alloc group), a p-methoxybenzyl group (PMB group), and the like. Among these, the Fmoc group and the like are particularly preferred.

[0090] Carboxyl protecting groups: Carboxyl protecting group PG b Various types are known as the substituent. Examples include monovalent hydrocarbon groups or heterocyclic groups which may have one or more substituents. In addition, when the substituent is present, the type is as described above. Specific examples of the number of the substituents are, for example, 5, 4, 3, 2, 1, or 0.

[0091] The upper limit of the number of carbon atoms in the hydrocarbon group (including the substituent if any) is, for example, 20 or less, 15 or less, 10 or less, 8 or less, or 6 or less. The lower limit varies depending on the type of hydrocarbon group, but is 1 or more for an alkyl group, 2 or more for an alkenyl group or alkynyl group, and 3 or more for a cycloalkyl group, for example, 4 or more or 5 or more. Specific examples of the number of atoms are, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0092] The upper limit of the total number of carbon atoms and heteroatoms (including substituents, if any) in the heterocyclic group is, for example, 20 or less, 15 or less, 10 or less, 8 or less, or 6 or less. The lower limit varies depending on the type of heterocyclic structure, but is usually 3 or more, for example, 4 or more, or 5 or more. Specific examples of the number of atoms are, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0093] Specific examples of the protecting group for the carboxyl group include, but are not limited to, the following:

[0094] Alkyl groups such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, decyl, and nonyl; Alkenyl groups such as ethenyl, propenyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, and octenyl; Alkynyl groups, such as propargyl groups; Cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclooctyl, and spirooctyl groups; Aryl groups such as phenyl, benzyl, tolyl, naphthyl, and anthracenyl; Furanyl group, thiophenyl group, pyranyl group, pyrrolinyl group, pyrrolyl group, 2,3-dihydro-1H-pyrrolyl group, piperidinyl group, piperazinyl group, homopiperazinyl group, morpholino group, thiomorpholino group, 1,2,4,6-tetrahydropyridyl group, hexahydropyrimidyl group, hexahydropyridazyl group, 1,2,4,6-tetrahydropyridyl group, 1,2,4,6-tetrahydropyridazyl group, 3,4-dihydropyridyl group, imidazolyl group, 4,5-dihydro-1H-imidazolyl group heterocyclic groups such as an alkyl group, a 2,3-dihydro-1H-imidazolyl group, a pyrazolyl group, a 4,5-dihydro-1H-pyrazolyl group, a 2,3-dihydro-1H-pyrazolyl group, an oxazolyl group, a 4,5-dihydro-1,3-oxazolyl group, a 2,3-dihydro-1,3-oxazolyl group, a 2,5-dihydro-1,3-oxazolyl group, a thiazolyl group, a 4,5-dihydro-1,3-thiazolyl group, a 2,3-dihydro-1,3-thiazolyl group, a 2,5-dihydro-1,3-thiazolyl group, and a carbazolyl group; Silicon-based protecting groups such as a trimethylsilyl (TMS) group, a triethylsilyl (TES) group, a triisopropylsilyl (TIPS) group, a tritert-butylsilyl (TBS) group, a tert-butyldiphenylsilyl (TBDPS) group, a tris(trialkylsilyl)silyl group, and the like.

[0095] Silane compounds: In the production method of the present invention, a silane compound may be present in the reaction system. By carrying out the reaction in the presence of a silane compound in the reaction system, various advantages such as improved reaction yield and improved stereoselectivity may be obtained.

[0096] Examples of the silane compound include various tris{halo (preferably fluorine)-substituted alkyl}silanes such as HSi(OCH(CF3)2)3, HSi(OCH2CF3)3, HSi(OCH2CF2CF2H)3, and HSi(OCH2CF2CF2CF2CF2H)3, as well as trimethylsilyl trifluoromethanesulfonate (TMS-OTf), 1-(trimethylsilyl)imidazole (TMSIM), dimethylethylsilylimidazole (DMESI), dimethylisopropylsilylimidazole (DMIPSI), 1-(tert-butyldimethylsilyl)imidazole (TBSIM), 1-(trimethylsilyl)triazole, 1-(ter t-butyldimethylsilyl)triazole, dimethylsilylimidazole, dimethylsilyl(2-methyl)imidazole, trimethylbromosilane (TMBS), trimethylchlorosilane (TMCS), N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), N,O-bis(trimethylsilyl)acetamide (BSA), N-(trimethylsilyl)dimethylamine (TMSDMA), N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA), hexamethyldisilazane (HMDS), etc. These may be used alone or in any combination and ratio of two or more.

[0097] However, when the production method of the present invention is carried out by a flow reaction, it is preferable not to use such a silane compound from the viewpoint of omitting a step of removing unnecessary components after the reaction and improving efficiency.

[0098] Lewis acid catalyst: In the production method of the present invention, a Lewis acid catalyst may be present in the reaction system. By carrying out the reaction in the presence of a Lewis acid catalyst in the reaction system, various advantages such as improved reaction yield and improved stereoselectivity may be obtained. However, when a Lewis acid catalyst is used, it may be necessary to separate and remove the Lewis acid catalyst from the reaction product. Therefore, it is preferable to appropriately determine whether or not to use a Lewis acid catalyst, taking into consideration the purpose of using the production method of the present invention.

[0099] When using a Lewis acid catalyst in the production method of the present invention, the type is not limited, but it is preferable that the catalyst is a metal compound that functions as a Lewis acid. Metal elements constituting the metal compound include various metals belonging to Groups 2 to 15 of the Periodic Table of Elements. Specific examples of metal elements include boron, magnesium, gallium, indium, silicon, calcium, lead, bismuth, mercury, transition metals, lanthanoid elements, etc. Specific examples of transition metals include scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, tin, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, thallium, etc. Specific examples of lanthanoid elements include lanthanum, cerium, neodymium, samarium, europium, gadolinium, holmium, erbium, thulium, ytterbium, etc. Among these, from the viewpoint of exerting an excellent reaction promotion effect and producing an amide compound with high stereoselectivity, one or more selected from titanium, zirconium, hafnium, tantalum, niobium, boron, vanadium, tungsten, neodymium, iron, lead, cobalt, copper, silver, palladium, tin, thallium, etc. are preferred, and one or more selected from titanium, zirconium, hafnium, tantalum, niobium, etc. are preferred. The metal compound may contain one or more metal elements. When the metal compound contains two or more metal elements, these may be the same type of element, or two or more different metal elements.

[0100] The ligand constituting the metal compound is appropriately selected depending on the type of metal. Specific examples of the ligand include substituted or unsubstituted linear or branched alkoxy groups having 1 to 10 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a trifluoroethoxy group, or a trichloroethoxy group; halogen atoms, such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom; an aryloxy group having 1 to 10 carbon atoms; an acetylacetonate group (acac), an acetoxy group (AcO), or a trifluoromethanesulfonate group (TfO); a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms; a phenyl group, an oxygen atom, a sulfur atom, a -SR group (wherein R is a substituent, and examples of the substituent include a substituted or unsubstituted hydrocarbon group having about 1 to 20 carbon atoms), a -NRR' group (wherein R and R' are each independently a hydrogen atom or a substituent, and examples of the substituent include a substituted or unsubstituted hydrocarbon group having about 1 to 20 carbon atoms), and a cyclopentadienyl (Cp) group.

[0101] Among them, the metal compound is preferably a titanium compound, a zirconium compound, a hafnium compound, a tantalum compound, or a niobium compound. Specific examples of each are given below. These may be used alone or in any combination and ratio of two or more.

[0102] A specific example of a titanium compound is TiX. 1 4 (but 4 X 1 Each of X is independently a ligand exemplified above. 1 X may be the same ligand or may be different from each other. 1 When X is an alkoxy group, it is preferably a straight or branched alkoxy group having 1 to 10 carbon atoms, more preferably a straight or branched alkoxy group having 1 to 5 carbon atoms, and even more preferably a straight or branched alkoxy group having 1 to 4 carbon atoms. 1When X is an aryloxy group, it is preferably an aryloxy group having 1 to 20 carbon atoms, more preferably an aryloxy group having 1 to 15 carbon atoms, and even more preferably an aryloxy group having 1 to 10 carbon atoms. These ligands may further have a substituent. 1 When is a halogen atom, preferred examples include a chlorine atom, a bromine atom, etc. Among these, for example, Ti(OMe)4, Ti(OEt)4, Ti(OPr)4, Ti(Oi-Pr)4, Ti(OBu)4, Ti(Ot-Bu)4, Ti(OCH2CH(Et)Bu)4, CpTiCl3, Cp2TiCl2, Cp2Ti(OTf)2, (i-PrO)2TiCl2, (i-PrO)3TiCl, etc. are preferred.

[0103] Specific examples of zirconium compounds include ZrX 2 4 (but 4 X 2 Each of X is independently a ligand exemplified above. 2 X may be the same ligand or may be different from each other. 2 When X is an alkoxy group, it is preferably a straight or branched alkoxy group having 1 to 10 carbon atoms, more preferably a straight or branched alkoxy group having 1 to 5 carbon atoms, and even more preferably a straight or branched alkoxy group having 1 to 4 carbon atoms. 2 When X is an aryloxy group, it is preferably an aryloxy group having 1 to 20 carbon atoms, more preferably an aryloxy group having 1 to 15 carbon atoms, and even more preferably an aryloxy group having 1 to 10 carbon atoms. These ligands may further have a substituent. 2 When is a halogen atom, preferred examples include a chlorine atom, a bromine atom, etc. Among these, for example, Zr(OMe)4, Zr(OEt)4, Zr(OPr)4, Zr(Oi-Pr)4, Zr(OBu)4, Zr(Ot-Bu)4, Zr(OCH2CH(Et)Bu)4, CpZrCl3, Cp2ZrCl2, Cp2Zr(OTf)2, (i-PrO)2ZrCl2, (i-PrO)3ZrCl, etc. are preferred.

[0104] Specific examples of hafnium compounds include HfX 3 4 (but 4 X 3Each of X is independently a ligand exemplified above. 3 may be the same ligand or may be different from each other. 3 When X is an alkoxy group, it is preferably a straight or branched alkoxy group having 1 to 10 carbon atoms, more preferably a straight or branched alkoxy group having 1 to 5 carbon atoms, and even more preferably a straight or branched alkoxy group having 1 to 4 carbon atoms. 3 When X is an aryloxy group, it is preferably an aryloxy group having 1 to 20 carbon atoms, more preferably an aryloxy group having 1 to 15 carbon atoms, and even more preferably an aryloxy group having 1 to 10 carbon atoms. These ligands may further have a substituent. 3 When is a halogen atom, preferred examples include a chlorine atom, a bromine atom, etc. Among these, for example, HfCp2Cl2, HfCpCl3, HfCl4, etc. are preferred.

[0105] Specific examples of tantalum compounds include TaX 4 5 (However, 5 X 4 Each of X is independently a ligand exemplified above. 4 X may be the same ligand or may be different from each other. 4 When X is an alkoxy group, it is preferably a straight or branched alkoxy group having 1 to 10 carbon atoms, more preferably a straight or branched alkoxy group having 1 to 5 carbon atoms, and even more preferably a straight or branched alkoxy group having 1 to 3 carbon atoms. 4 When X is an aryloxy group, it is preferably an aryloxy group having 1 to 20 carbon atoms, more preferably an aryloxy group having 1 to 15 carbon atoms, and even more preferably an aryloxy group having 1 to 10 carbon atoms. These ligands may further have a substituent. 4 When X is a halogen atom, preferred examples include a chlorine atom and a bromine atom. Among these, tantalum alkoxide compounds (e.g., X 4 is an alkoxy group), and the like are preferable, for example, Ta(OMe)5, Ta(OEt)5, Ta(OBu)5, Ta(NMe2)5, Ta(acac)(OEt)4, TaCl5, TaCl4(THF), TaBr5, and the like are preferable.4 Compounds in which is oxygen, i.e., Ta2O5, can also be used.

[0106] Specific examples of niobium compounds include NbX 5 5 (However, 5 X 5 Each of X is independently a ligand exemplified above. 5 X may be the same ligand or may be different from each other. 5 When X is an alkoxy group, it is preferably a straight or branched alkoxy group having 1 to 10 carbon atoms, more preferably a straight or branched alkoxy group having 1 to 5 carbon atoms, and even more preferably a straight or branched alkoxy group having 1 to 3 carbon atoms. 5 When X is an aryloxy group, it is preferably an aryloxy group having 1 to 20 carbon atoms, more preferably an aryloxy group having 1 to 15 carbon atoms, and even more preferably an aryloxy group having 1 to 10 carbon atoms. These ligands may further have a substituent. 5 When X is a halogen atom, preferred examples include a chlorine atom and a bromine atom. Among these, niobium alkoxide compounds (e.g., X 5 is an alkoxy group), for example, NbCl4(THF), NbCl5, Nb(OMe)5, Nb(OEt)5, etc. are preferred. 5 Compounds in which Nb2O5 is oxygen can also be used.

[0107] The Lewis acid catalyst may be supported on a carrier. The carrier for supporting the Lewis acid catalyst is not particularly limited, and any known carrier can be used. Also, any known method can be used as a method for supporting the Lewis acid catalyst on a carrier.

[0108] However, when the production method of the present invention is carried out by a flow reaction, it is preferable not to use such a Lewis acid catalyst from the viewpoint of omitting a step of removing unnecessary components after the reaction and improving efficiency.

[0109] Other Ingredients: In the production method of the present invention, other components may be present in the reaction system. Examples of such other components include, but are not limited to, conventional catalysts (other than Lewis acid catalysts) that can be used in amidation reactions, bases, phosphorus compounds, solvents, etc. Any one of these may be used alone, or two or more of them may be used in any combination and ratio.

[0110] Examples of the catalyst (other than the Lewis acid catalyst) include methylaluminum bis(4-bromo-2,6-di-tert-butylphenoxide) (MABR), trimethylsilyl trifluoromethanesulfonate (TMS-OTf), methylaluminum bis(2,6-di-tert-butylphenoxide) (MAD), etc. Any one of these may be used alone, or two or more of them may be used in any combination and ratio.

[0111] The type of base is not limited, and a known base that is known to improve the reaction efficiency can be used. Examples of such bases include amines having 1 to 4 linear or branched alkyl groups having 1 to 10 carbon atoms, such as tetrabutylammonium fluoride (TBAF), triethylamine (Et3N), diisopropylamine (i-Pr2NH), and diisopropylethylamine (i-Pr2EtN), and inorganic bases such as cesium fluoride. These may be used alone or in any combination and ratio of two or more.

[0112] Examples of phosphorus compounds include phosphine compounds (e.g., trimethylphosphine, triethylphosphine, tripropylphosphine, trimethyloxyphosphine, triethyloxyphosphine, tripropyloxyphosphine, triphenylphosphine, trinaphthylphosphine, triphenyloxyphosphine, tris(4-methylphenyl)phosphine, tris(4-methoxyphenyl)phosphine, tris(4-fluorophenyl)phosphine, tris(4-methylphenyloxy)phosphine, tris(4-methoxyphenyloxy)phosphine, tris(4-fluorophenyloxy)phosphine, etc.), phosphate compounds (e.g., trimethyl phosphate, triethyl phosphate, tripropyl phosphate, trimethyloxy phosphate, , triethyloxy phosphate, tripropyloxy phosphate, triphenyl phosphate, trinaphthyl phosphate, triphenyloxy phosphate, tris(4-methylphenyl)phosphate, tris(4-methoxyphenyl)phosphate, tris(4-fluorophenyl)phosphate, tris(4-methylphenyloxy)phosphate, tris(4-methoxyphenyloxy)phosphate, tris(4-fluorophenyloxy)phosphate, etc.), polyvalent phosphine compounds or polyvalent phosphate compounds (e.g., 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP), 5,5'-bis(diphenylphosphino)-4,4'-bi-1,3-benzodioxole (SEGPHOS), etc.). Any one of these may be used alone, or two or more may be used in any combination and ratio.

[0113] However, when the production method of the present invention is carried out by a flow reaction, it is preferable not to use such other components in order to omit the step of removing unnecessary components after the reaction and to improve efficiency.

[0114] Reaction solvent: In order to increase the reaction efficiency, a solvent may be used during the reaction, particularly when the production method of the present invention is carried out by a flow reaction.

[0115] Examples of the solvent include, but are not limited to, aqueous solvents and organic solvents. Examples of the organic solvent include, but are not limited to, aromatic hydrocarbons such as toluene and xylene, ethers such as pentane, petroleum ether, tetrahydrofuran (THF), 1-methyltetrahydrofuran (1-MeTHF), diisopropyl ether (i-Pr2O), diethyl ether (Et2O), and cyclopentyl methyl ether (CPME), nitrogen-based organic solvents such as acetonitrile (MeCN), chlorine-based organic solvents such as dichloromethane (DCM), esters such as ethyl acetate (AcOEt), and organic acids such as acetic acid. These solvents may be used alone or in combination of two or more.

[0116] The solvent used in the amide bond forming reaction (step (i)) and the solvent used in the deprotection (step (ii)) may be the same or different. When different solvents are used, it is necessary to exchange the solvent of the product after the amide bond forming reaction (step (i)) and then subject it to the deprotection (step (ii). Therefore, when the production method of the present invention is carried out by a flow reaction, it is preferable from the viewpoint of efficiency that the solvent used in the amide bond forming reaction (step (i)) and the solvent used in the deprotection (step (ii)) are the same solvent. Examples of solvents that can be suitably used in both the amide bond forming reaction (step (i)) and the deprotection (step (ii)) include THF, dichloromethane, chloroform, acetonitrile, CPME, etc. Among them, THF is preferable.

[0117] Amide bond forming reactions: In the production method of the present invention, an amide bond is formed between the electrophilic substrate, an N-terminal protected amino acid or peptide ester (R1), and a nucleophilic substrate, an amino acid or peptide or its ester (R2), to obtain an N-terminal protected peptide (S1) (step (i)). The procedure and conditions for such a reaction are not limited, but are preferably as follows.

[0118] The ratio of the amount of the electrophilic substrate compound (R1) to the amount of the nucleophilic substrate compound (R2) is not particularly limited, but the amount of the nucleophilic substrate compound (R2) can be, for example, 0.1 moles or more, or 0.2 moles or more, or 0.3 moles or more, or 0.4 moles or more, or 0.5 moles or more, and for example, 20 moles or less, or 10 moles or less, or 5 moles or less, or 4 moles or less, or 3 moles or less, relative to 1 mole of the electrophilic substrate compound (R1). Of course, it is necessary to use 1 mole or more of each of the substrate compounds (R1) and (R2) relative to the target production amount of the compound to be produced (S1).

[0119] In particular, when the production method of the present invention is carried out by a flow reaction, it is preferable to use the substrate compounds (R1) and (R2) in a ratio as close to an equivalent ratio as possible. Specifically, in the stage of synthesizing a dipeptide using both amino acids as the substrate compounds (R1) and (R2), it is preferable to use the other substrate compound in a range of, for example, 1.5 moles or less, 1.4 moles or less, 1.3 moles or less, 1.2 moles or less, 1.1 moles or less, or even 1.05 moles or less per mole of one substrate compound. On the other hand, in the stage of synthesizing a tripeptide or more peptide using an amino acid as the substrate compound (R1) and a dipeptide or more peptide as the substrate compound (R2), all the substrate amino acids (R1) become electrophilic species amino acids, and it is considered that the unreacted electrophilic species amino acids are immediately converted into inactive diketopiperazines by self-condensation even if they are deprotected, so that even if a slightly excessive amount is added, there is little adverse effect on the future reaction. In this case, therefore, the amount of the other substrate compound used per 1 mole of either substrate compound can be within the range of, for example, 3 moles or less, 2.5 moles or less, 2.0 moles or less, or 1.5 moles or less.

[0120] When a silane compound is used, the amount used is not particularly limited, but when the amount of the compound of formula (R1) used is taken as 100 mol%, for example, 0.1 mol% or more, or 0.2 mol% or more, or 0.3 mol% or more, and for example, 50 mol% or less, or 30 mol% or less, or 20 mol% or less, or 15 mol% or less of the silane compound can be used.

[0121] When a Lewis acid catalyst is used, the amount used is not particularly limited, but when the amount of the compound of formula (R1) used is taken as 100 mol%, for example, 0.1 mol% or more, or 0.2 mol% or more, or 0.3 mol% or more, and for example, 50 mol% or less, or 30 mol% or less, or 20 mol% or less, or 15 mol% or less of the Lewis acid catalyst can be used.

[0122] When other optional components are used, the amounts used may be appropriately adjusted with reference to conventional knowledge and the like, for example, in the past patent documents (Patent Documents 1 to 8) of the present inventors and others.

[0123] In addition, for any of the above components, the total amount may be added to the system at once, or may be added to the system in several batches, or may be added to the system continuously in small amounts. In particular, in the case of a flow reaction, solutions containing the substrate compounds (R1) and (R2) in a reaction solvent may be continuously supplied by a liquid delivery pump or the like, and contacted and reacted while flowing through the reaction path.

[0124] The reaction temperature is not limited as long as the reaction proceeds, but can be, for example, 0°C or higher, or 10°C or higher, or 20°C or higher, and can be, for example, 100°C or lower, or 80°C or lower, or 60°C or lower.

[0125] The reaction pressure is not limited as long as the reaction proceeds, and the reaction may be carried out under reduced pressure, normal pressure, or increased pressure, but is usually carried out under normal pressure.

[0126] The reaction atmosphere is not limited as long as the reaction proceeds, but the reaction is usually carried out in an atmosphere of an inert gas such as argon or nitrogen.

[0127] In the case of a flow reaction, the flow rate of each solution of the substrate compounds (R1) and (R2) is not limited as long as the reaction proceeds. From the viewpoint of allowing the reaction to proceed sufficiently and efficiently, the flow rate can be, for example, 0.01 mL / min or more, or 0.05 mL / min or more, or 0.1 mL / min or more, and can be, for example, 100 mL / min or less, or 50 mL / min or less, or 20 mL / min or less.

[0128] The reaction time is not limited as long as the reaction proceeds, but from the viewpoint of allowing the reaction to proceed sufficiently, it can be, for example, 5 minutes or more, 10 minutes or more, 20 minutes or more, or 30 minutes or more. The upper limit is not particularly limited, but from the viewpoint of efficiency, it can be, for example, 50 hours or less, 20 hours or less, 5 hours or less, or 1 hour or less. In the case of a flow reaction, the time for contacting the solutions of the substrate compounds (R1) and (R2) in the reaction path may be adjusted within the above range so that the reaction proceeds sufficiently and efficiently.

[0129] Deprotection of N-terminal protected peptides: In the production method of the present invention, the N-terminus of the N-terminal protected peptide (S1) obtained by the above reaction is deprotected to obtain a peptide compound represented by the following formula (P1) (step (ii)). The deprotection method is not particularly limited, and the protecting group PG a An appropriate method may be selected depending on the type of the compound. The deprotection method is as described above.

[0130] In particular, in the production method of the present invention, it is preferable to carry out the deprotection by passing the compound of formula (S1) through a column packed with a basic ion exchange resin. By carrying out the deprotection by this method, the deprotection (step (ii)) following the amide bond formation reaction (step (i)) can also be carried out by a flow reaction, and thus the entire production method of the present invention can be carried out by a flow reaction.

[0131] When the N-terminal deprotection of the compound of formula (S1) is carried out using a column packed with a basic ion exchange resin, the type of the basic ion exchange resin is not particularly limited, and the protecting group PG a The resin may be appropriately selected depending on the type of resin and the manufacturing conditions, etc. Examples include 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-based resins, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN)-based resins, 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-based resins, piperazine-based resins, dimethylaminopyridine-based resins, and ammonium-based resins.

[0132] The procedure for deprotection is not particularly limited, and the compound of formula (S1) obtained in the above-mentioned amide bond formation reaction (step (i)) may be passed through a column packed with a basic ion exchange resin. In particular, in the case of a flow reaction, the reaction solution containing the compound of formula (S1) obtained in the above-mentioned amide bond formation reaction (step (i)) may be continuously sent as it is by a liquid sending pump or the like to pass through a column packed with a basic ion exchange resin.

[0133] The temperature of the column during deprotection is not limited as long as the deprotection proceeds, but can be, for example, 0°C or higher, or 10°C or higher, or 20°C or higher, and can be, for example, 100°C or lower, or 80°C or lower, or 60°C or lower.

[0134] The pressure during deprotection is not limited as long as the deprotection proceeds, and the deprotection may be carried out under reduced pressure, normal pressure, or increased pressure, but is usually carried out under normal pressure.

[0135] In the case of a flow reaction, the flow rate of the reaction solution containing the compound of formula (S1) is not limited as long as the deprotection proceeds; however, from the viewpoint of allowing the deprotection to proceed sufficiently and efficiently, the flow rate can be, for example, 0.01 mL / min or more, or 0.05 mL / min or more, or 0.1 mL / min or more, and can be, for example, 100 mL / min or less, or 50 mL / min or less, or 20 mL / min or less.

[0136] The deprotection time is not limited as long as the deprotection proceeds, but from the viewpoint of allowing the deprotection to proceed sufficiently and efficiently, it can be, for example, 10 minutes or more, 20 minutes or more, or 30 minutes or more, and for example, 80 hours or less, 60 hours or less, or 50 hours or less. In the case of a flow reaction, the deprotection time can be adjusted to the above range by adjusting the flow rate of the reaction solution containing the compound of formula (S1) and the length of the column packed with the basic ion exchange resin.

[0137] - Peptide chain elongation by repeated reaction and deprotection In the production method of the present invention, the compound of formula (P1) obtained in the deprotection (step (ii)) is used as the nucleophilic substrate compound of formula (R2) and the amide bond formation reaction (step (i)) and deprotection (step (ii)) are repeatedly carried out (step (iii)). This allows the desired amino acids to be linked sequentially through amide bonds to elongate the peptide chain and produce the desired peptide compound (P1). In particular, by sequentially repeating this procedure while selecting appropriate compounds as the substrate compounds (R1) and (R2), it is theoretically possible to synthesize a polypeptide having any number of amino acid residues and any amino acid sequence.

[0138] For example, using a preferred embodiment of the production method of the present invention by flow reaction, H2N-AA n -AA n-1 -(···)-AA 2 -AA 1 A polypeptide with n amino acid residues consisting of the sequence AA 1 , A.A. 2 , AA n-1 , A.A. n In the case of synthesizing an electrophilic substrate compound (R1), AA 2 The N-terminal protected amino acid ester of formula (R1) (e.g., Fmoc-AA 2 -Ot-OPfp) was used as the nucleophilic substrate compound (R2) 1 The corresponding amino acid ester (H2N-AA 1The amide bond formation reaction of step (i) is carried out using Fmoc-AA 2 -AA 1 Then, the compound of formula (S1) is passed through an ion exchange resin, and the N-terminus is deprotected in step (ii) to produce a dipeptide of formula (P1) with an unsubstituted N-terminus (HN-AA 2 -AA 1 Then, the dipeptide of formula (P1) is used as a new nucleophilic substrate compound (R2) to obtain the next amino acid residue AA 3 The amide bond formation reaction in step (i) and the deprotection reaction in step (ii) are carried out using an N-terminal protected amino acid ester of formula (R1) corresponding to the following formula: n -AA n-1 -(···)-AA 2 -AA 1 -O-tBu can be synthesized.

[0139] Post-processing (refining, recovery, etc.): The peptide compound (P1) obtained by the production method of the present invention may be subjected to various post-treatments. For example, the produced peptide compound (P1) can be isolated and purified by a standard method such as column chromatography or recrystallization. In addition, when the produced peptide compound (P1) has an amino group and / or a carboxyl group protected by a protecting group or the like, the compound can be deprotected by the method described below.

[0140] [III. Other] The production method of the present invention may be carried out as a sequential method (batch method) or a continuous method (flow method). The specific details of the procedure for carrying out the sequential method (batch method) and the continuous method (flow method) are known in the art. However, as described above in detail, the production method of the present invention can be carried out as a continuous method (flow method), which is preferable because it provides various advantages.

[0141] The peptide compound (P1) obtained by the production method of the present invention may be further subjected to various post-treatments. For example, the peptide compound (P1) obtained by the above-mentioned production method can be isolated and purified by standard methods such as column chromatography and recrystallization.

[0142] In addition, in the peptide compound (P1) obtained by the above-mentioned production method, the amino group protected by a protecting group can also be deprotected. The method for deprotecting the protected amino group is not particularly limited, and various methods can be used depending on the type of the protecting group. Examples include deprotection by hydrogenation, deprotection by weak acid, deprotection by fluoride ion, deprotection by one-electron oxidizing agent, deprotection by hydrazine, and deprotection by oxygen. In the case of deprotection by hydrogenation, examples include (a) a method of deprotection by reduction using a metal catalyst such as palladium, palladium-carbon, palladium hydroxide, palladium hydroxide-carbon, etc. as a reduction catalyst in the presence of hydrogen gas, and (b) a method of deprotection by reduction using a hydrogenation reducing agent such as sodium borohydride, lithium aluminum hydride, lithium borohydride, and diborane in the presence of a metal catalyst such as palladium, palladium-carbon, palladium hydroxide, palladium hydroxide-carbon, etc.

[0143] In addition, in the peptide compound (P1) obtained by the above-mentioned production method, the carboxyl group protected by a protecting group can also be deprotected. The method for deprotecting the protected carboxyl group is not particularly limited, and various methods can be used depending on the type of the protecting group. Examples include deprotection by hydrogenation, deprotection by a base, and deprotection by a weak acid. In the case of deprotection by a base, examples include a method of deprotection using a strong base such as lithium hydroxide, sodium hydroxide, or potassium hydroxide as the base.

[0144] The present inventors have filed the following prior patent applications relating to amidation reactions for linking amino acids or peptides and methods for producing polypeptides using the same, and the methods for producing various polypeptides of the present invention can be carried out in appropriate combination with the amidation reactions and methods for producing polypeptides described in these prior patent applications, and / or can be modified as appropriate in consideration of the conditions for the amidation reactions and methods for producing polypeptides described in these prior patent applications. The descriptions of these prior patent applications are incorporated herein in their entirety by reference. (1) International Publication No. 2017 / 204144 (Patent Document 1) (2) International Publication No. 2018 / 199146 (Patent Document 2) (3) International Publication No. 2018 / 199147 (Patent Document 3) (4) International Publication No. 2019 / 208731 (Patent Document 4 mentioned above) (5) International Publication No. 2021 / 085635 (Patent Document 5) (6) International Publication No. 2021 / 085636 (Patent Document 6) (7) International Publication No. 2021 / 149814 (Patent Document 7) (8) International Publication No. 2022 / 190486 (Patent Document 8) (9) Chem. Sci., (2022), Vol.13, pp.6309-6315 (Non-Patent Document 6) EXAMPLES

[0145] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative and are not intended to limit the present invention in any way.

[0146] The reaction system shown in Figure 1 was constructed, and the tripeptide H2N-AA was synthesized according to the above synthesis procedure. 3 -AA 2 -AA 1 -OtBu was synthesized (AA 1 ,AA 2 ,AA 3Each represents an amino acid residue.) As shown in FIG. 1, in this embodiment, T a The pentafluorophenyl (Pfp) group is used as the group, and the PG a Fmoc group as a group, T b Although a t-butyl (tBu) group is used as the group, the present invention is not limited to the embodiments shown in the examples and figures.

[0147] The following parts and devices were used to construct the reaction system. Path: PTFE tube, 0.1cm diameter DBU polymer: Sigma-Aldrich 595128 Cartridge: Tokyo Rikakikai 278800, diameter 1.02 cm, length 10 cm or 30 cm Pump: Tokyo Rikakikai TR-278550 Temperature control device: Tokyo Rikakikai MCR-1000, product code 267950

[0148] In the following description, "reaction time" refers to the time from when the reaction mixture enters the flow machine until just before it enters the deprotection cartridge, in the case of the amide bond formation reaction in step (i), and refers to the time from when the reaction mixture enters the deprotection cartridge until it flows out of the deprotection cartridge, in the case of the deprotection reaction in step (ii).

[0149] Pentafluorophenyl esters of amino acids with N-terminus protected by Fmoc (Fmoc-AA 2 A 1.25 mL solution of 0.1 M of H-OPfp (0.125 mmol) in tetrahydrofuran (THF) was prepared using tert-butyl amino acid esters (H-AA 1 A THF solution (concentration: 0.1 M) of Fmoc-AA (-OtBu) was pumped to each of the two solutions (flow rate: 1.0 mL / min) and merged inside the pathway, and the amide bond formation reaction of step (i) was carried out (reaction temperature: room temperature, reaction time: <5 min). 2 -AA 1 -OtBu) was obtained. The resulting N-terminal protected dipeptide ester (Fmoc-AA 2 -AA1 The deprotection reaction of step (ii) was carried out by pumping (flow rate 2.0 mL / min) through a cartridge (length 10 cm) filled with DBU polymer (reaction temperature 50° C., reaction time <5 min) to obtain a dipeptide ester (HN-AA 2 -AA 1 -OtBu) was obtained.

[0150] The resulting dipeptide ester (H2N-AA 2 -AA 1 -OtBu) in THF (0.05 M) and another pentafluorophenyl ester of an amino acid N-terminally protected with Fmoc (Fmoc-AA 3 A THF solution (concentration: 0.05 M) of Fmoc-OPfp was pumped to each of the channels (flow rate: 1.0 mL / min) to join the channels, and the amide bond formation reaction of step (i) was carried out. 3 -AA 2 -AA 1 -OtBu) was obtained.

[0151] Amino Acid AA 1 ,AA 2 ,AA 3 The N-terminal protected tripeptide Fmoc-H2N-AA was synthesized according to the above synthesis procedure, with various types of 3 -AA 2 -AA 1 The results are shown in the table below. In the table, "quant." means actual equivalent (approximately 100%).

[0152] [Table 3]

[0153] [ka]

[0154] These N-terminal protected tripeptides Fmoc-AA 3 -AA2 -AA 1 The deprotection reaction of step (ii) was carried out by passing -OtBu through the cartridge filled with DBU polymer by the same procedure as above (flow rate 1.0 mL / min) and passing it through the cartridge (length 30 cm) filled with DBU polymer. This resulted in the corresponding tripeptide ester (HN-AA 3 -AA 2 -AA 1 -OtBu) can be obtained.

[0155] In addition, the tripeptide ester H2N-AA synthesized by the above procedure 3 -AA 2 -AA 1 -OtBu plus one more amino acid residue, AA 4 The tripeptide ester HN-AA was synthesized as follows: 3 -AA 2 -AA 1 A THF solution of -OtBu (concentration 0.05 M) and a pentafluorophenyl ester of another amino acid N-terminally protected with Fmoc (Fmoc-AA 4 A THF solution (concentration 0.05 M) of Fmoc-L-Ala-L-Ala-L-Ala-L-Ala-OtBu (i.e., amino acid residues AA-OPfp) is pumped (flow rate 1.0 mL / min) to join the inside of the pathway, and the amide bond formation reaction of step (i) is carried out. 1 , A.A. 2 , A.A. 3 , and A.A. 4 We also succeeded in synthesizing L-alanine (both in 92% yield).

Claims

1. 1. A method for producing a polypeptide compound, comprising: (i) forming an amide bond between a substituted carboxyl group on the right side of an N-terminal protected amino acid ester or peptide ester compound represented by the following formula (R1) and an amino group on the left side of an amino acid or peptide, or an amino acid ester or peptide ester compound represented by the following formula (R2), thereby obtaining an N-terminal protected peptide compound represented by the following formula (S1); (ii) deprotecting the N-terminus of the compound of formula (S1) obtained in step (i) to obtain a peptide compound represented by the following formula (P1): (iii) The compound of formula (P1) obtained in step (ii) is used as the compound of formula (R2) in step (i), and steps (i) and (ii) are repeatedly carried out to elongate the peptide chain by amidation. This includes: The process, wherein steps (i) and (ii) are carried out continuously as a flow reaction. 【Chemistry 1】 In formula (R1), T a represents a pentafluorophenyl group, P.G. a represents a monovalent protecting group, R 11 and R 12 each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, or a thiol group, or an amino group, a monovalent aliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, or a monovalent heterocyclic group, each of which may have one or more substituents; R 13 represents a hydrogen atom, a carboxyl group, a hydroxyl group, or a monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group which may have one or more substituents, in which case the monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group may be bonded to a nitrogen atom via a linking group, Or, R 11 and R 13 and are bonded to each other to form R 11 and the carbon atom to which R 13 may form, together with the nitrogen atom to which it is bonded, a heterocycle which may have one or more substituents, A 11 and A 12 each independently represents a divalent aliphatic hydrocarbon group having 1 to 3 carbon atoms which may have one or more substituents, p11 and p12 each independently represent 0 or 1; n 1 is an integer of 1 or more and represents the number of constitutional units represented by the structure in [ ]. 1 When the number is 2 or more, the multiple constitutional units represented by the structure in [ ] may be the same or different. 【Chemistry 2】 In formula (R2), T b represents a hydrogen atom or a monovalent substituent, R 21 and R 22 each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, or a thiol group, or an amino group, a monovalent aliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, or a monovalent heterocyclic group, each of which may have one or more substituents; R 23 represents a hydrogen atom, a carboxyl group, a hydroxyl group, or a monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group which may have one or more substituents, in which case the monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group may be bonded to a nitrogen atom via a linking group, Or, R 21 and R 23 and are bonded to each other to form R 21 and the carbon atom to which R 23 may form, together with the nitrogen atom to which it is bonded, a heterocycle which may have one or more substituents, A 21 and A 22 each independently represents a divalent aliphatic hydrocarbon group having 1 to 3 carbon atoms which may have one or more substituents, p21 and p22 each independently represent 0 or 1; n 2 is an integer of 1 or more and represents the number of constitutional units represented by the structure in [ ]. 2 When the number is 2 or more, the multiple constitutional units represented by the structure in [ ] may be the same or different. 【Chemistry 3】 In formula (S1), P.G. a , R 11 , R 12 , R 13 , A 11 , A 12 , p11, p12, and n 1 represents the same group as defined in formula (R1), R 21 , R 22 , R 23 , A 21 , A 22 , p21, p22, n 2 , and T b represents the same group as defined in formula (R2). 【Chemistry 4】 In formula (P1), R 11 , R 12 , R 13 , A 11 , A 12 , p11, p12, and n 1 represents the same group as defined in formula (R1), R 21 , R 22 , R 23 , A 21 , A 22 , p21, p22, n 2 , and T b represents the same group as defined in formula (R2).

2. The method according to claim 1, wherein in the reaction of step (i), the compound of formula (R1) and the compound of formula (R2) are used in an approximately equimolar ratio.

3. The protecting group PG of formula (R1) ａ is a group selected from a monovalent hydrocarbon group, an acyl group, a hydrocarbonoxycarbonyl group, a hydrocarbonsulfonyl group, and an amide group, each of which may have one or more substituents.

4. The method according to any one of claims 1 to 3, wherein the deprotection of the N-terminal protecting group of formula (S1) in step (ii) is carried out by passing the compound of formula (S1) through a column packed with a basic ion exchange resin.

5. The method according to claim 4, wherein the basic ion exchange resin is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-based resins, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN)-based resins, 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-based resins, piperazine-based resins, dimethylaminopyridine-based resins, and ammonium-based resins.

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