Benzylamine compounds, methods for producing peptides, and C-terminal protected peptides

Abstract

This invention provides a benzylamine compound that can be used as a C-terminal tag with excellent acid resistance. [Solution] It is expressed by the following formula (1), where R 1 R is an organic group having at least one selected from the group consisting of aromatic hydrocarbon groups, heteroaromatic groups, and aliphatic hydrocarbon groups, 2 and R 3 Each is independently selected from the group consisting of alkyl groups which may have substituents and heteroalkyl groups which may have substituents, Y 2 and Y 3 A benzylamine compound characterized in that each of the elements is independently selected from the group consisting of -O-, -S-, -CONH-, -NHCO-, and -CH2-. [Formula 1] TIFF2026166126000054.tif37142

Description

[Technical Field]

[0001] This disclosure relates to benzylamine compounds, methods for producing peptides, and C-terminally protected peptides. [Background technology]

[0002] Conventionally, a known method for synthesizing peptides involves introducing a solvent-soluble protecting group (hereinafter also called a "tag") to the C-terminus of an amino acid, and then extending the N-terminus of that amino acid. For example, a tag having a long alkyl group is attached to an amino acid, and peptides are synthesized by repeatedly performing the extension reaction. With this synthesis method, at each extension stage, only the peptide bound to the tag is solidified (e.g., crystallized), making it easy to separate and purify the solidified substance.

[0003] As a tag to protect the C-terminus of a peptide, Non-Patent Document 1 discloses a bisbenzylamine compound in which the 2nd and 4th positions of the benzyl group are substituted with oxyalkyl groups. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kitada, S.; Fujita, S.; Okada, Y.; Kim, S.; Chiba, K. Total synthesis of α-conotoxin MII using a soluble-tag-assisted method. Tetrahedron 2013, 69, 2555-2559. [Overview of the project] [Problems that the invention aims to solve]

[0005] In the tag described in Non-Patent Document 1, in which oxyalkyl groups are bonded to the 2nd and 4th positions of the benzyl group, the methylene group contained in the benzyl group has weak acid resistance. Therefore, in peptide synthesis using this tag, the tag may be cleaved at an unintended time, resulting in deprotection of the peptide's C-terminus. Benzylamine-type tags, such as those described in Non-Patent Document 1, are considered useful in the synthesis of C-terminal amide peptides having an amide group at the C-terminus, but as mentioned above, there is room for improvement in their acid resistance.

[0006] One aspect of this disclosure aims to provide a benzylamine compound that can be used as a C-terminal tag having excellent acid resistance. [Means for solving the problem]

[0007] To solve the above problems, a benzylamine compound according to one aspect of this disclosure is represented by the following formula (1), where R 1 R is an organic group having at least one selected from the group consisting of aromatic hydrocarbon groups, heteroaromatic groups, and aliphatic hydrocarbon groups, 2 and R 3 Each is independently selected from the group consisting of alkyl groups which may have substituents and heteroalkyl groups which may have substituents, Y 2 and Y 3 Each of these is independently selected from the group consisting of -O-, -S-, -CONH-, -NHCO-, and -CH2-.

[0008] [ka] [Effects of the Invention]

[0009] According to one aspect of this disclosure, a benzylamine compound is provided that can be used as a C-terminal tag having excellent acid resistance. [Modes for carrying out the invention]

[0010] <Benzylamine Compound> One aspect of the present disclosure relates to a benzylamine compound. The benzylamine compound according to one aspect of the present disclosure is represented by the following formula (1), wherein R 1 is an organic group having at least one selected from the group consisting of an aromatic hydrocarbon group, a heteroaromatic group, and an aliphatic hydrocarbon group, and R 2 and R 3 are each independently selected from the group consisting of an optionally substituted alkyl group and an optionally substituted heteroalkyl group, and Y 2 and Y 3 are each independently selected from the group consisting of -O-, -S-, -CONH-, -NHCO-, and -CH2-.

[0011] [Chemical Formula]

[0012] The benzylamine compound according to one aspect of the present disclosure can be used as a C-terminal tag. Hereinafter, the benzylamine compound according to one aspect of the present disclosure may be simply referred to as "the present benzylamine".

[0013] Due to having substituents -Y 2 R 2 and -Y 3 R 3 at the 3-position and 4-position of the benzyl group, the bond between the N atom shown in formula (1) and the benzyl moiety (-CH2-C6H6(Y 2 R 2 )(Y 3 R 3 )) is less likely to be cleaved under acidic conditions. Therefore, the present benzylamine has excellent acid resistance.

[0014] In addition, when the C-terminus of a peptide is protected using the present benzylamine, -CO-N(-R 1 )-CH2-C6H6(Y 2 R 2 )(Y 3 R 3A C-terminally protected peptide is obtained, which has the following properties: ) After extending this peptide toward the N-terminus, the C-terminus is deprotected, and the bond between the N atom and the methylene group in the benzyl group shown in formula (1) is cleaved. After cleavage, the peptide that was bound to the N atom can be recovered as a C-terminal amide peptide.

[0015] Note that R shown in equation (1) 1 R 1 Depending on the design, the bond to the N atom continues even after C-terminal deprotection, and therefore the C-terminus of the resulting peptide has a secondary amide (-CONHR 1 ) may be provided. Alternatively, R shown in formula (1) 1 R 1 Depending on the design, the C-terminal deprotection may cleave the N atom, thus providing a primary amide (-CONH2) at the C-terminus of the resulting peptide.

[0016] (R 2 , R 3 ) In formula (1), R 2 and R 3 Each is independently selected from the group consisting of alkyl groups which may have substituents and heteroalkyl groups which may have substituents. 2 and R 3 These may be the same or different. From the viewpoint of easily producing this benzylamine, R 2 and R 3 They are preferably the same.

[0017] R 2 and R 3 The alkyl groups in each are independently linear alkyl groups, branched alkyl groups, or cyclic alkyl groups, preferably linear alkyl groups or branched alkyl groups, and more preferably linear alkyl groups. The number of branched chains in a branched alkyl group is 1 or more, preferably 1 to 30. The number of ring-forming carbons in a cyclic alkyl group is 3 or more, preferably 3 to 10.

[0018] R 2and R 3 The number of carbon atoms in the alkyl group can be selected as appropriate. From the viewpoint of improving solubility in organic solvents, R 2 and R 3 The number of carbon atoms in each alkyl group is preferably 1 to 30, more preferably 1 to 24, even more preferably 1 to 22, and even more preferably 1 to 18.

[0019] R 2 and R 3 The heteroalkyl groups in each are independently linear heteroalkyl groups, branched heteroalkyl groups, or cyclic heteroalkyl groups, preferably linear heteroalkyl groups or branched heteroalkyl groups, and more preferably linear heteroalkyl groups. The number of branched chains in a branched heteroalkyl group is 1 or more, preferably 1 to 10. The number of ring-forming carbons in a cyclic heteroalkyl group is 2 or more, preferably 2 to 10.

[0020] R 2 and R 3 The number of carbon atoms in the heteroalkyl group can be appropriately selected. From the viewpoint of improving solubility in organic solvents, the number of carbon atoms in each heteroalkyl group is preferably 1 to 30, more preferably 1 to 26, even more preferably 1 to 24, and even more preferably 1 to 22.

[0021] R 2 and R 3 The heteroatom of the heteroalkyl group in is at least one selected from the group consisting of O, S, and N, preferably at least one selected from the group consisting of O and S.

[0022] R 2 and R 3 The substituents that the alkyl group and heteroalkyl group in the above are each independently halogens selected from the group consisting of F, Cl, Br, and I.

[0023] R 2 and R 3 The total number of carbon atoms is preferably 2 to 60, more preferably 2 to 48, even more preferably 2 to 44, and even more preferably 2 to 36.

[0024] Among them, R 2 and R 3 Each of these is independently: preferably selected from the group consisting of linear alkyl groups having 1 to 30 carbon atoms, branched alkyl groups having 3 to 30 carbon atoms, linear heteroalkyl groups having 1 to 30 carbon atoms, and branched heteroalkyl groups having 3 to 30 carbon atoms; more preferably selected from the group consisting of linear alkyl groups having 1 to 24 carbon atoms and branched alkyl groups having 3 to 24 carbon atoms; even more preferably selected from the group consisting of linear alkyl groups having 1 to 22 carbon atoms; even more preferably selected from the group consisting of linear alkyl groups having 1 to 20 carbon atoms; and particularly preferably selected from the group consisting of linear alkyl groups having 1 to 18 carbon atoms.

[0025] R 2 and R 3Examples include: methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, henicosyl group, docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, no Linear alkyl groups such as nacosyl group and triacontyl group; branched alkyl groups formed by attaching one or more of the aforementioned linear alkyl groups as side chains to a main chain consisting of any of the aforementioned linear alkyl groups, for example isooctyl group, 1-methyl-1-dodecyl group, 1-methyl-1-hexadecyl group, 1-ethyl-1-heptadecyl group, 1-propyl-1-decyl group, 1-butyl-1-decyl group, 2-methyl-1-dodecyl group, 2-methyl-1-hexa Decyl group, 2-butyl-1-octyl group, 2-butyl-1-dodecyl group, 2-butyl-1-octadecyl group, 2-hexyl-1-decyl group, 2-hexyl-1-dodecyl group, 2-heptyl-1-dodecyl group, 2-octyl-1-dodecyl group, 2-octyl-1-tetradecyl group, 2-decyl-1-tetradecyl group, 2-dodecyl-1-tetradecyl group, 2-dodecyl-1-hexadecyl group, 3-methyl-1-tetradecyl group, 4-ethyl- Examples include 1-octyl group, 6-methyl-1-heptyl group, 9-methyl-1-dodecyl group, 12-methyl-1-tridecyl group, 15-methyl-1-hexadecyl group, 2-tetradecyl-1-octadecyl group; linear heteroalkyl groups obtained by substituting one or more carbon atoms constituting the aforementioned linear alkyl groups with heteroatoms; and branched heteroalkyl groups obtained by substituting one or more carbon atoms constituting the aforementioned branched alkyl groups with heteroatoms.

[0026] (Y 2 , Y 3 ) In formula (1), Y 2 and Y 3 Each of these is independently selected from the group consisting of -O-, -S-, -CONH-, -NHCO-, and -CH2-. From the viewpoint of facilitating the synthesis of this benzylamine, Y 2and Y 3 Each of these is independently selected, preferably from the group consisting of -O-, -S-, -CONH-, and -NHCO-, more preferably from the group consisting of -O- and -S-, and even more preferably -O-.

[0027] Y 2 and Y 3 These may be the same or different. From the viewpoint of easily producing this benzylamine, Y 2 and Y 3 They are preferably the same. Also, -Y 2 R 2 and -Y 3 R 3 These may be the same or different, and from the viewpoint of easily producing this benzylamine, -Y 2 R 2 and -Y 3 R 3 They are preferably the same.

[0028] (R 1 ) In formula (1), R 1 This is an organic group having at least one selected from the group consisting of aromatic hydrocarbon groups, heteroaromatic groups, and aliphatic hydrocarbon groups.

[0029] In this disclosure, "organic group" refers to a functional group in which carbon C, oxygen O, and nitrogen N make up more than half of the atoms constituting the functional group, excluding H. "Organic group having X group" (where X is any functional group) refers to an organic group that contains X as part of the structure of the functional group, or an organic group consisting of X.

[0030] R 1 The aliphatic hydrocarbon group in this compound may be linear, branched, or alicyclic. Furthermore, the aliphatic hydrocarbon group may be saturated or unsaturated.

[0031] R 1Examples of aliphatic hydrocarbon groups in this context include: methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group, octyl group, decyl group, hexadecyl group, octadecyl group, eicosyl group, tetracosyl group, lauryl group, tridecyl group, myristyl group, isostearyl group, cyclopentyl group, cyclohexyl group, isobornyl group, tricyclodecanyl Examples include alkyl groups such as the group; alkylene groups obtained by removing one hydrogen atom from those alkyl groups; alkenyl groups obtained by substituting one of the carbon-carbon single bonds of those alkyl groups with a double bond, such as vinyl group, 1-propenyl group, allyl group, isopropenyl group, butenyl group, isobutenyl group, etc.; alkynyl groups obtained by substituting one of the carbon-carbon single bonds of those alkyl groups with a triple bond, such as 4-pentinyl group, 5-hexynyl group, etc.

[0032] The alicyclic aliphatic hydrocarbon group may be condensed, linked, or crosslinked with at least one of the aromatic hydrocarbon group and the heteroaromatic group.

[0033] R 1 The aromatic hydrocarbon group and the heteroaromatic group in each may independently be either a monocyclic or polycyclic group. The polycyclic group may be either a fused ring or a linking ring. 1 In this, the aromatic hydrocarbon group and the heteroaromatic group may form a polycyclic group by condensation or linkage. The heteroatom of the heteroaromatic group is preferably at least one selected from the group consisting of O, S, and N.

[0034] R 1 Examples of aromatic hydrocarbon groups in R include benzene rings, naphthalene rings, anthracene rings, fluorene rings, phenanthrene rings, tetracene rings, triphenylene rings, pyrene rings, chrysene rings, etc. 1Examples of the heteroaromatic group in the above include a pyridine ring, quinoline ring, isoquinoline ring, benzothiophene ring, furan ring, benzofuran ring, xanthene ring, pyrrole ring, indole ring, carbazole ring, pyrazole ring, indazole ring, thiophene ring, and the like.

[0035] R 1 The organic group represented by preferably has at least one selected from the group consisting of an aromatic hydrocarbon group and a heteroaromatic group, and more preferably has an aromatic hydrocarbon group. Further, R 1 in the above, it is preferable that the aromatic hydrocarbon group or the heteroaromatic group is bonded to the N atom represented by formula (1) via an alkylene group. The number of carbon atoms of the alkylene group is preferably 1 to 8, more preferably 1 to 3, and still more preferably 1.

[0036] R 1 may further have a moiety other than the aromatic hydrocarbon group, the heteroaromatic group, and the aliphatic hydrocarbon group. Examples of the moiety other than the aromatic hydrocarbon group, the heteroaromatic group, and the aliphatic hydrocarbon group include -O-, -S-, -CONH-, -OCONH-, -COO-, and the like.

[0037] R 1 The total number of carbon atoms in the above is preferably 9 to 97, more preferably 9 to 85, still more preferably 9 to 79, even more preferably 9 to 73, and particularly preferably 9 to 61. R 1 The total number of heteroatoms in the above is preferably 1 or more, more preferably 2 or more. R 1 The total number of heteroatoms in is preferably 2 to 12, more preferably 2 to 8, and still more preferably 2 to 6.

[0038] In formula (1), R 1 is preferably represented by the following formula (2). R represented by formula (2) 1 may be one in which the bond to the N atom is cleaved by C-terminal deprotection, thus providing a primary amide (-CONH2) at the C-terminus of the resulting peptide.

[0039] [ka]

[0040] In equation (2), * represents the bond position with N as shown in equation (1).

[0041] (Ring A) In formula (2), ring A is an aryl group which may have substituents. The number of carbon atoms in the aryl group is preferably 6 to 14, more preferably 6 to 12, and even more preferably 6 to 10. Examples of aryl groups include phenyl, naphthyl, fluorenyl, and anthracenyl groups.

[0042] The number of substituents that ring A may have is preferably 0 (i.e., ring A is unsubstituted), or 1 to 8, more preferably 2 to 6, and even more preferably 2 to 4. When the number of substituents is 2 or more, the 2 or more substituents may be the same or different, and from the viewpoint of facilitating the production of the benzylamine, the 2 or more substituents are preferably the same.

[0043] The substituents that ring A may have are selected from the group consisting of halogens, organic groups having aliphatic hydrocarbon groups, and organic groups having halogenated hydrocarbon groups. The aliphatic hydrocarbon groups in the substituents may independently be linear, branched, or alicyclic. The aliphatic hydrocarbon groups in the substituents may independently be saturated or unsaturated, and are preferably saturated aliphatic hydrocarbon groups.

[0044] The number of carbon atoms in a substituent that ring A may have is 0 if the substituent is a halogen. The number of carbon atoms in a substituent is preferably 1 to 30, more preferably 1 to 26, and even more preferably 1 to 24, when the substituent is an organic group having an aliphatic hydrocarbon group.

[0045] The substituents that ring A may have may further have portions other than aliphatic hydrocarbon groups. Examples of portions other than aliphatic hydrocarbon groups include -O-, -S-, -CONH-, -OCONH-, -COO-, aromatic hydrocarbon groups, heteroaromatic groups, etc. In the substituent, it is preferable that the portion other than the aliphatic hydrocarbon group connects ring A to the aliphatic hydrocarbon group of the substituent, and it is more preferable that -O-, -S-, -CONH- or -NHCO- connect ring A to the aliphatic hydrocarbon group of the substituent.

[0046] Examples of substituents that ring A may have include alkyl groups, heteroalkyl groups, arylalkyl groups, and heteroarylalkyl groups. Examples of alkyl groups include linear alkyl groups, branched alkyl groups, and alicyclic alkyl groups. Examples of heteroalkyl groups include alkoxy groups and poly(oxyalkylene)-alkyl groups. Examples of arylalkyl groups include alkoxyaryl groups. Examples of heteroarylalkyl groups include alkoxyheteroaryl groups.

[0047] When ring A is a phenyl ring, it is preferable that substituents are attached to the ring-forming carbons at positions 3 and 4 of the benzyl group formed by ring A and the methylene group shown in formula (2).

[0048] In formula (2), the number of carbon atoms in ring A is 8 to 78, preferably 8 to 72, more preferably 8 to 66, and even more preferably 8 to 60. The number of carbon atoms in ring A here refers to the sum of the number of carbon atoms in the aryl group and the number of carbon atoms in the substituent.

[0049] In formula (1), R 1 R is more preferably represented by the following formula (3). 1 This may involve deprotection of the C-terminus, which cleaves the bond with the N atom, thus providing a primary amide (-CONH2) at the C-terminus of the resulting peptide.

[0050] [ka]

[0051] In formula (3), * represents the bonding position to N shown in formula (1).

[0052] In formula (3), m is an integer of 1 to 3, preferably an integer of 1 to 2, and more preferably 2.

[0053] (R A ) In formula (3), the m R A groups are each independently selected from the group consisting of an optionally substituted alkyl group and an optionally substituted heteroalkyl group. The m R A groups may be the same or different. From the viewpoint of easily producing the present benzylamine, the m R A groups are preferably the same.

[0054] The alkyl group in the m R A groups is each independently a linear alkyl group, a branched alkyl group or a cyclic alkyl group, preferably a linear alkyl group or a branched alkyl group, and more preferably a linear alkyl group. The number of branched chains that the branched alkyl group has is 1 or more, preferably 1 to 30. The number of ring-forming carbon atoms in the cyclic alkyl group is 3 or more, preferably 3 to 10.

[0055] The number of carbon atoms of the alkyl group in the m R A groups can be appropriately selected. From the viewpoint of improving the solubility in an organic solvent, the number of carbon atoms of the alkyl group in each R A group is each independently preferably 1 to 30, more preferably 1 to 24, still more preferably 1 to 22, and even more preferably 1 to 18.

[0056] The m R AThe heteroalkyl groups in each are independently linear heteroalkyl groups, branched heteroalkyl groups, or cyclic heteroalkyl groups, preferably linear heteroalkyl groups or branched heteroalkyl groups, and more preferably linear heteroalkyl groups. The number of branched chains in a branched heteroalkyl group is 1 or more, preferably 1 to 10. The number of ring-forming carbons in a cyclic heteroalkyl group is 2 or more, preferably 2 to 10.

[0057] R A The number of carbon atoms in the heteroalkyl group can be appropriately selected. From the viewpoint of improving solubility in organic solvents, the number of carbon atoms in each heteroalkyl group is preferably 1 to 30, more preferably 1 to 26, even more preferably 1 to 24, and even more preferably 1 to 22.

[0058] m R A Each heteroatom in the heteroalkyl group is independently selected from the group consisting of O, S, and N, and preferably at least one selected from the group consisting of O and S.

[0059] m R A The substituents that the alkyl group and heteroalkyl group in the above are each independently halogens selected from the group consisting of F, Cl, Br, and I.

[0060] m R A The total number of carbon atoms is preferably 2 to 60, more preferably 2 to 48, even more preferably 2 to 44, and even more preferably 2 to 36.

[0061] Among them, R AEach of these is independently: preferably selected from the group consisting of linear alkyl groups having 1 to 30 carbon atoms, branched alkyl groups having 3 to 30 carbon atoms, linear heteroalkyl groups having 1 to 30 carbon atoms, and branched heteroalkyl groups having 3 to 30 carbon atoms; more preferably selected from the group consisting of linear alkyl groups having 1 to 24 carbon atoms and branched alkyl groups having 3 to 24 carbon atoms; even more preferably selected from the group consisting of linear alkyl groups having 1 to 22 carbon atoms; even more preferably selected from the group consisting of linear alkyl groups having 1 to 20 carbon atoms; and particularly preferably selected from the group consisting of linear alkyl groups having 1 to 18 carbon atoms.

[0062] R AExamples include: methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, henicosyl group, docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, no Linear alkyl groups such as nacosyl group and triacontyl group; branched alkyl groups formed by attaching one or more of the aforementioned linear alkyl groups as side chains to a main chain consisting of any of the aforementioned linear alkyl groups, for example isooctyl group, 1-methyl-1-dodecyl group, 1-methyl-1-hexadecyl group, 1-ethyl-1-heptadecyl group, 1-propyl-1-decyl group, 1-butyl-1-decyl group, 2-methyl-1-dodecyl group, 2-methyl-1-hexa Decyl group, 2-butyl-1-octyl group, 2-butyl-1-dodecyl group, 2-butyl-1-octadecyl group, 2-hexyl-1-decyl group, 2-hexyl-1-dodecyl group, 2-heptyl-1-dodecyl group, 2-octyl-1-dodecyl group, 2-octyl-1-tetradecyl group, 2-decyl-1-tetradecyl group, 2-dodecyl-1-tetradecyl group, 2-dodecyl-1-hexadecyl group, 3-methyl-1-tetradecyl group, 4-ethyl- Examples include 1-octyl group, 6-methyl-1-heptyl group, 9-methyl-1-dodecyl group, 12-methyl-1-tridecyl group, 15-methyl-1-hexadecyl group, 2-tetradecyl-1-octadecyl group; linear heteroalkyl groups formed by substituting one or more carbon atoms constituting the aforementioned linear alkyl groups with heteroatoms; and branched heteroalkyl groups formed by substituting one or more carbon atoms constituting the aforementioned branched alkyl groups with heteroatoms.

[0063] In equation (3), m R A It is preferable that the compound is bonded to at least one selected from the group consisting of the 3rd, 4th, and 5th positions of the benzyl group, more preferably to at least one of the 3rd and 4th positions of the benzyl group, and even more preferably to both the 3rd and 4th positions of the benzyl group.

[0064] (Y A ) In equation (1), m Y A Each of these is independently selected from the group consisting of -O-, -S-, -CONH-, and -NHCO-. From the viewpoint of facilitating the synthesis of this benzylamine, m Y A Each of these is independently selected from the group consisting of -O- and -S-, and more preferably -O-.

[0065] m Y A These may be the same or different. From the viewpoint of easily producing this benzylamine, m units of Y A They are preferably identical. Also, m -Y A R A These may be the same or different, and from the viewpoint of easily producing this benzylamine, m units of -Y A R A They are preferably the same.

[0066] (Preferred examples of benzylamine compounds) The benzylamine is preferably represented by the following formula (4).

[0067] [ka]

[0068] (R 2 , R 3 , R 4 , R 5 ) In formula (4), R 2 , R 3 , R 4 and R 5 Each of these is independently selected from the group consisting of alkyl groups having 1 to 24 carbon atoms, which may have substituents, and is preferably selected from the group consisting of linear alkyl groups having 1 to 24 carbon atoms and branched alkyl groups having 3 to 24 carbon atoms.

[0069] R 2 , R3 , R 4 and R 5 These may be the same or different. From the viewpoint of easily producing this benzylamine, R 2 and R 3 They are identical, R 4 and R 5 It is preferable that they are the same, R 2 , R 3 , R 4 and R 5 It is more preferable that they are identical.

[0070] R shown in equation (4) 2 and R 3 The explanation for this is given by R shown in equation (1). 2 and R 3 Since the explanation is the same as that given, we will not repeat it. Also, R shown in equation (4) 4 For the purpose of explaining this, R shown in equation (1) 3 The explanation regarding this will be used, but will not be repeated. R shown in equation (4) 5 For the purpose of explaining this, R shown in equation (1) 2 Referencing the explanation regarding that point, and not repeating that explanation.

[0071] R shown in equation (4) 2 , R 3 , R 4 and R 5 The explanation of substituents that may have substituents is given by R shown in formula (1). 2 and R 3 Since this is the same as the explanation for substituents that may have substituents, that explanation will not be repeated.

[0072] R 2 , R 3 , R 4 and R 5 The total number of carbon atoms is preferably 4 to 120, more preferably 4 to 96, more preferably 4 to 88, and even more preferably 4 to 72.

[0073] (Y 2 , Y 3, Y 4 , Y 5 ) In formula (4), Y 2 , Y 3 , Y 4 and Y 5 Each of these is independently selected from the group consisting of -O-, -S-, -CONH-, and -NHCO-, and preferably selected from the group consisting of -O- and -S-.

[0074] Y 2 , Y 3 , Y 4 and Y 5 These may be the same or different. From the viewpoint of easily producing this benzylamine, Y 2 and Y 3 They are identical, Y 4 and Y 5 It is preferable that they are the same, Y 2 , Y 3 , Y 4 and Y 5 It is more preferable that they are identical.

[0075] Also, -Y 2 R 2 ,-Y 3 R 3 ,-Y 4 R 4 and -Y 5 R 5 These may be the same or different. From the viewpoint of easily producing this benzylamine, -Y 2 R 2 and -Y 3 R 3 They are identical, -Y 4 R 4 and -Y 5 R 5 It is preferable that they are the same, -Y 2 R 2 ,-Y 3 R 3 ,-Y 4 R 4 and -Y 5 R 5 It is more preferable that they are identical.

[0076] Y shown in equation (4) 2 and Y 3 The explanation for this is given by Y shown in equation (1). 2 and Y 3 Since the explanation is the same as that given, we will not repeat it. Also, Y shown in equation (4) 4 For the purpose of explaining this, the Y shown in equation (1) 3 The explanation regarding this will be used, but will not be repeated. Y shown in equation (4) 5 For the purpose of explaining this, the Y shown in equation (1) 2 Referencing the explanation regarding that point, and not repeating that explanation.

[0077] A preferred specific example of this benzylamine is represented by the following formula, but this disclosure is not limited to the following formula.

[0078] [ka]

[0079] <Method for producing benzylamine compounds> As an example, this benzylamine is produced by using 3,4-dihydroxybenzaldehyde as a starting material, alkylating it with an alkyl bromide, aminating the resulting dialkoxybenzaldehyde, and then converting the resulting dialkoxybenzylamine into aldehyde R. 1 It can be produced by condensing with -CHO and reducing the resulting dialkoxybenzylimine. Each reaction can be carried out using known reagents and solvents.

[0080] [ka]

[0081] In formula (1), Y 2 and Y 3when is -S-, -CONH-, -NHCO- or -CH2-, the starting material may be appropriately modified. Further, when producing the present benzylamine represented by formula (4), the above dialkoxybenzaldehyde and dialkoxybenzylamine may be condensed and refluxed.

[0082] Further, if necessary, the present benzylamine may be purified by a known purification method. As the purification method, for example, a method of isolation and purification by recrystallization, column chromatography or the like, or a method of purification by reprecipitation by a technique of changing the solution temperature, a means of changing the solution composition, or the like may be adopted.

[0083] <C-terminally protected peptide> One aspect of the present disclosure relates to a C-terminally protected peptide. In the C-terminally protected peptide according to one aspect of the present disclosure, the above-described present benzylamine is introduced as a protecting group at the C-terminus.

[0084] The C-terminally protected peptide according to one aspect of the present disclosure is represented by the following formula (101).

[0085]

Chemical Formula

[0086] In formula (101), R 1 , R 2 , R 3 , Y 2 and Y 3 have the same definitions as those described above for the present benzylamine, and AA represents a peptide. In formula (101), AA and N are bonded via the C-terminal carbonyl group of the peptide AA.

[0087] In formula (101), the peptide represented by AA may be an amino acid, i.e., a one-residue peptide, or a peptide having multiple residues, for example, a 2, 3, 4, 5, 6, 7, 8, 9, or 10-residue peptide. In other words, the C-terminally protected peptide may be a C-terminally protected amino acid having one residue. For the sake of brevity, in this disclosure, a C-terminally protected amino acid having one residue and a C-terminally protected peptide having two or more residues are collectively referred to as a "C-terminally protected peptide."

[0088] The number of amino acid residues constituting the C-terminal protective peptide is preferably 1 to 50, more preferably 1 to 30, and even more preferably 1 to 15. The number of amino acid residues may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0089] There are no particular restrictions on the types of amino acid residues that constitute a C-terminally protected peptide. From the viewpoint of ease of synthesis, a C-terminally protected peptide preferably has at least one amino acid residue selected from the group consisting of amino acids having a protecting group; more preferably has at least one amino acid residue selected from the group consisting of amino acids having a protecting group at the N-terminus. Each of the amino acid residues may be a D-isomer or an L-isomer.

[0090] C-terminally protected peptides may have an N-terminal protecting group at their N-terminus. The N-terminal protecting group may be one known in the art. Examples of N-terminal protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc) group, tert-butoxycarbonyl (Boc) group, allyloxycarbonyl (Alloc) group, benzyloxycarbonyl (Cbz) group, 2,2,2-trichloroethoxycarbonyl (Troc) group, 2-(trimethylsilyl)ethoxycarbonyl (Teoc) group, acetyl (Ac) group, benzoyl (Bz) group, and the like.

[0091] C-terminally protected peptides may have a side-chain protecting group on the side chain of an amino acid residue. The side-chain protecting group may be one known in the art and can be appropriately selected according to the type of side chain. Examples of side-chain protecting groups include the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) group, trityl group, tert-butyl group, Boc group, and benzyl group.

[0092] The C-terminally protected peptide may have substituents on the side chain of the amino acid residue. There are no particular restrictions on the type of substituent, and any known substituent may be used. Examples of substituents include: an amino acid residue that binds to the side chain, for example, cysteine ​​that binds to the side chain of a cysteine ​​residue via a disulfide bond; and so on.

[0093] A C-terminally protected peptide according to one aspect of this disclosure is preferably represented by the following formula (102).

[0094] [ka]

[0095] In formula (102), ring A has the same definition as described above for this benzylamine.

[0096] A C-terminally protected peptide according to one aspect of this disclosure is preferably represented by the following formula (103).

[0097] [ka]

[0098] In formula (103), R A , Y A And m have the same definitions as described above for this benzylamine.

[0099] A C-terminally protected peptide according to one aspect of this disclosure is preferably represented by the following formula (104).

[0100]

Chem.

[0101] In formula (104), R 4 , R 5 , Y 4 and Y 5 have the same definitions as those described above for the present benzylamine.

[0102] The C-terminus protected peptide according to one aspect of the present disclosure is preferably represented by the following formula.

[0103]

Chem.

[0104] <Method for producing C-terminus protected peptide> The above-described C-terminus protected peptide can be produced as an intermediate when carrying out the peptide production method described later. As an example, in the peptide production method described later, by carrying out each step that at least includes the condensation reaction step (step B2) using the present benzylamine compound without carrying out the C-terminus deprotection step (A1), the C-terminus protected peptide can be produced.

[0105] <Method for producing peptide> One aspect of the present disclosure relates to a method for producing a peptide. The method for producing a peptide according to one aspect of the present disclosure uses a C-terminus protected peptide in which the benzylamine compound according to one aspect of the present disclosure is introduced to the C-terminus as a protecting group. Hereinafter, the method for producing a peptide according to one aspect of the present disclosure may be simply referred to as "the present production method".

[0106] According to the present production method, a peptide is produced using the present benzylamine as a C-terminus tag. The present production method can be particularly useful for producing a peptide in the form of a C-terminal amide compound.

[0107] This manufacturing method uses a C-terminally protected peptide in which the benzylamine compound is introduced as a protecting group at the C-terminus. The C-terminally protected peptide used may be, for example, the C-terminally protected peptide described above, but is not limited thereto. In this manufacturing method, the C-terminally protected peptide may be used as an intermediate produced in the manufacturing process, or as a starting material.

[0108] This manufacturing method typically includes a C-terminal deprotection step (step A1). Furthermore, this manufacturing method may further include a dissolution step (step B1), a condensation reaction step (step B2), an N-terminal deprotection step (step B3), a liquid-liquid separatory step (step B4), and a peptide extension step (step B5) prior to step A1. Steps B1 to B5 are steps for producing the C-terminally protected peptide to be used in step A1.

[0109] [C-terminal deprotection step (Step A1)] Step A1 is a step in which a C-terminally protected peptide is brought into contact with a deprotection solution containing a deprotection component to obtain a peptide with a deprotected C-terminus.

[0110] Step A1 is carried out by contacting the C-terminally protecting peptide with a deprotection solution. Contact may be carried out by mixing the solution containing the C-terminally protecting peptide with a pre-prepared deprotection solution, or by adding each component of the deprotection solution described later sequentially or simultaneously to the solution containing the C-terminally protecting peptide. When each component is added sequentially or simultaneously to the solution containing the C-terminally protecting peptide, the total amount of added components is considered to be the deprotection solution, and the amount of each component contained in the considered deprotection solution is considered to be the amount of each component used.

[0111] The deprotective components in the deprotection solution only need to act on the C-terminus to deprotect it. Examples of deprotective components include: acids such as methanesulfonic acid (MSA), trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid, p-toluenesulfonic acid, and trifluoromethanesulfonic acid; and bases such as sodium hydroxide, potassium hydroxide, barium hydroxide, lithium hydroxide, and sodium methoxide. The deprotective components may be a single type or a combination of multiple types.

[0112] In this manufacturing method, the protecting group introduced at the C-terminus exhibits excellent acid resistance. Therefore, when using an acid as the deprotection component and carrying out step A1 by acid treatment, it is preferable to use a strong acid. Examples of strong acids include MSA, TFA, p-toluenesulfonic acid, and trifluoromethanesulfonic acid.

[0113] From the viewpoint of obtaining a C-terminal amide peptide having -CONH2 at the C-terminus in step A1, the deprotection solution preferably contains a strong acid with a pKa of -2.0 or less. In this disclosure, the pKa value of the acid refers to the value measured in water at 25°C.

[0114] If the C-terminal carboxylic acid form is produced even though the peptide to be manufactured is a C-terminal amide form, several additional steps are required to convert it to the C-terminal amide form, thus increasing manufacturing costs. Obtaining the C-terminal amide form directly through step A1 is advantageous in terms of manufacturing costs.

[0115] Examples of acids with a pKa of 2.0 or less include MSA, p-toluenesulfonic acid, and trifluoromethanesulfonic acid, with preferred examples being MSA and p-toluenesulfonic acid. The acid with a pKa of 2.0 or less may be a single type or a combination of multiple types. The deprotection solution may contain a combination of an acid with a pKa of 2.0 or less and an acid with a pKa greater than 2.0.

[0116] From the viewpoint of obtaining a C-terminal amide peptide having -CONH2 at the C-terminus, the content of acid with a pKa of 2.0 or less in the deprotection solution is preferably 50 v / v% or more, more preferably 60 v / v% or more, and even more preferably 80 v / v% or more. There is no particular upper limit to the content of acid with a pKa of 2.0 or less, and it is 100 v / v% or less.

[0117] From the viewpoint of obtaining a C-terminal amide peptide having -CONH2 at the C-terminus in step A1 while suppressing side reactions by radical cations, it is preferable that the deprotection solution further contains a radical cation scavenging component. Examples of radical cation scavenging components include alkylbenzenes and heteroalkylbenzenes.

[0118] The deprotection solution preferably contains a compound represented by the following formula (5) as a radical cation scavenging component.

[0119] [ka]

[0120] In equation (5), k is an integer between 1 and 5, preferably between 2 and 4, and more preferably 3. When k is 3, 3 R B Preferably, each of these is bonded to the 1st, 3rd, and 5th positions of the benzene ring.

[0121] Equation (5), k R B Each of these is independently selected from the group consisting of optionally substituted alkyl groups and optionally substituted heteroalkyl groups. k R B They may be the same or they may be different.

[0122] R B Examples of alkyl and heteroalkyl groups in this context include methyl, ethyl, and methoxy groups.

[0123] R BThe substituents that the alkyl group and heteroalkyl group in the above are each independently halogens selected from the group consisting of F, Cl, Br, and I.

[0124] Preferred examples of compounds represented by formula (5) include mesitylene and tetramethylbenzene, with mesitylene being a more preferred example.

[0125] The content of the compound represented by formula (5) in the deprotection solution is preferably 2 moles to 100 moles, more preferably 2 moles to 50 moles, and even more preferably 2 moles to 10 moles, per mole of the C-terminally protected peptide.

[0126] The deprotection solution may further contain a solvent. The solvent may be a compound known in the art for use in deprotecting the C-terminus of a peptide. Examples of solvents include: water; ethers such as diethyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, 1,4-dioxane, methyl-t-butyl ether, cyclopentyl methyl ether (CPME), and 4-methyltetrahydropyran (MTHP); acetic acid esters such as ethyl acetate and isopropyl acetate; halogenated hydrocarbons such as chloroform and dichloromethane (DCM); aromatic hydrocarbons such as toluene and xylene; and hydrocarbons such as hexane, heptane, and cyclohexane. The amount of solvent used is preferably 1,000 mL to 10,000 mL, more preferably 1,000 mL to 5,000 mL, and even more preferably 1,000 mL to 2,500 mL per mole of the C-terminus-protected peptide.

[0127] The temperature of step A1 can be adjusted as appropriate, and for example, it is between 0°C and 40°C. From the viewpoint of proceeding with C-terminal deprotection at a faster reaction rate, the temperature of step A1 is preferably 10°C or higher, and more preferably 20°C or higher. Furthermore, from the viewpoint of reducing the hydrolysis of the amide group of the C-terminal amide peptide produced by deprotection, which converts the peptide to a carboxylic acid form, the temperature of step A1 is preferably 40°C or lower, and more preferably 30°C or lower.

[0128] The duration of step A1 can be adjusted as appropriate, and as an example, it is between 1 hour and 48 hours. From the viewpoint of sufficiently advancing the deprotection of the C-terminus, the duration of step A1 is preferably 3 hours or more, and more preferably 6 hours or more. Furthermore, from the viewpoint of reducing the hydrolysis of the amide group of the C-terminal amide peptide produced by deprotection, which converts the peptide to a carboxylic acid, the duration of step A1 is preferably 36 hours or less, and more preferably 24 hours or less.

[0129] The atmosphere in process A1 may be an inert atmosphere such as argon or nitrogen.

[0130] The peptide whose C-terminus has been deprotected in step A1 can be isolated or purified according to the purification methods commonly used in peptide synthesis. Examples of such purification methods include extraction washing, crystallization, and chromatography.

[0131] [Dissolution process (process B1)] Step B1 is a step of dissolving the benzylamine as a C-terminal tag, or a C-terminally protected peptide in which the benzylamine is introduced at the C-terminus, in a solvent.

[0132] As for the solvent, the same solvents described above can be used as the solvent for the deprotection solution in the deprotection step (step A1). From the viewpoint of good reactivity, liquid-liquid properties, and industrial applicability, the solvent is preferably at least one selected from the group consisting of methyl-t-butyl ether, CPME, MTHP, isopropyl acetate, chloroform, and toluene; more preferably at least one selected from the group consisting of CPME, MTHP, isopropyl acetate, and toluene; and even more preferably at least one selected from the group consisting of CPME and MTHP.

[0133] Furthermore, in order to improve the solubility of the reaction substrate, to improve the solubility of unreacted substances and by-products in the aqueous layer during extraction, or to improve liquid-liquid properties, the above solvent may be mixed with polar solvents such as dimethylformamide (DMF), dimethylacetamide, dimethyl sulfoxide (DMSO), sulfolane, N-methylpyrrolidone, N,N'-dimethylpropylene urea (DMPU), and acetonitrile in appropriate proportions.

[0134] [Condensation reaction step (step B2)] Step B2 is a step in which the C-terminal tag or C-terminally protected peptide dissolved in Step B1 is condensed with the N-terminally protected peptide, which is the reaction substrate, to obtain the C-terminal and N-terminally protected peptides.

[0135] The number of amino acid residues constituting an N-terminally protected peptide may be one or two or more. In other words, an N-terminally protected peptide may be an N-terminally protected amino acid having one residue. For the sake of brevity, in this disclosure, an N-terminally protected amino acid having one residue and an N-terminally protected peptide having two or more residues are collectively referred to as an "N-terminally protected peptide."

[0136] The amount of N-terminally protected peptide used is, for example, 1.0 mole to 3.0 moles, preferably 1.2 moles to 2.0 moles, per mole of the C-terminal tag or C-terminally protected peptide.

[0137] In step B2, a condensing agent, catalyst, or activator may be added to the reaction solution, provided that it does not adversely affect the condensation reaction.

[0138] As for the coupling agent, there are no particular restrictions as long as the reaction proceeds, and coupling agents commonly used in peptide synthesis can be used. Examples of coupling agents include 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorphonium chloride (DMT-MM), O-(benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU(6-Cl)), and O-(benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU(6-Cl)). Examples include methyluronium tetrafluoroborate (TBTU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl). The coupling agent is preferably DIC, DMT-MM, HBTU, HATU, COMU, EDCI, or EDCI·HCl. The amount of condensing agent used is, for example, 1.0 mole to 3.0 moles, preferably 1.2 moles to 2.0 moles, per mole of the C-terminal tag or C-terminal protective peptide.

[0139] The catalyst can be one that is commonly used in peptide synthesis as a catalyst for condensation reactions. Examples of catalysts include dimethylaminopyridine (DMAP) and pyridine. The amount of catalyst used is, for example, 0.01 moles to 0.5 moles, preferably 0.05 moles to 0.1 moles, per mole of C-terminal tag or C-terminal protected peptide.

[0140] Activators are added to promote condensation reactions and suppress side reactions such as racemization. Here, an activator is a reagent that, in the presence of a condensing agent, induces amino acids or peptides into corresponding active esters, symmetric acid anhydrides, etc., thereby promoting the formation of amide bonds. Any activator commonly used in peptide synthesis can be used without restriction. Examples of activators include 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxy-1H-1,2,3-triazole-4-carboxylate ethyl (HOCt)3-hydroxy-1,2,3-benzotriazine-4(3H)-one (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), pentafluorophenol, and ethyl cyano(hydroxyimino)ethyl acetate (Oxyma). The activator is preferably HOBt, HOAt, HOCt, HOOBt, HONb, HOSu, or Oxyma. The amount of activator used is, for example, 0.1 moles to 2.0 moles, preferably 0.3 moles to 1.0 mole, per mole of the C-terminal tag or C-terminal protected peptide.

[0141] The solvent used in step B2 can be any solvent commonly used in peptide synthesis, without restriction. For example, the solvent described above for step B1, or a mixed solvent of a solvent and a polar solvent, may be used. The amount of solvent used is not particularly limited as long as the reaction proceeds, but it may be adjusted so that the concentration of the C-terminal tag or C-terminally protected peptide is, for example, between 40 mM and 200 mM.

[0142] The reaction temperature in step B2 may be any temperature commonly used in peptide synthesis, for example, between -20°C and 40°C, and preferably between 0°C and 30°C. The reaction time for one step B2 is, for example, between 0.5 hours and 30 hours.

[0143] [N-terminal deprotection step (Step B3)] Step B3 is a step that follows step B2, in which a base is added to the reaction solution containing the C-terminus and N-terminus protected peptides to deprotect the N-terminus and obtain the C-terminus protected peptide.

[0144] The base is selected appropriately depending on the type of N-terminal protecting group. Examples of bases include: organic bases such as triethylamine (TEA), diisopropylethylamine (DIPEA), 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,4-diazabicyclo[2.2.2]octane (DABCO), pyridine, imidazole, 4-(dimethylamino)pyridine (DMAP), LDA, sodium acetate (NaOAc), sodium methoxide (MeONa), potassium methoxide (MeOK), lithium hexamethyldisilazide (LHMDS), and sodium bis(trimethylsilyl)amide (NaHMDS); and inorganic bases such as sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), potassium carbonate (K2CO3), cesium carbonate (Cs2CO3), and sodium hydride (NaH). The amount of base used is, for example, 1 mole to 20 moles, preferably 3 moles to 10 moles, per mole of N-terminal protecting group present in the reaction solution.

[0145] In step B3, a base may be added not only to deprotect the N-terminal protecting group, but also to capture unreacted active ester and by-products generated by deprotection.

[0146] [Liquid separation process (process B4)] Step B4 involves neutralizing the reaction solution with an acidic aqueous solution, washing, and separating the solution to remove by-products and unwanted reaction materials into the aqueous layer.

[0147] The acidic aqueous solution used for neutralization is not limited as long as it can neutralize the base in the reaction solution. Neutralization here means that the reaction solution has a neutral pH, and the pH may be 7.0 or lower. Examples of acidic aqueous solutions include aqueous solutions of hydrochloric acid, sulfuric acid, phosphoric acid, and acetic acid. For example, when using hydrochloric acid, it is preferable to use hydrochloric acid with a concentration of 0.5 M to 12 M, preferably 1 M to 12 M, and more preferably 1 M to 6 M. In addition, to improve liquid-liquid separation, a ketone-based liquid-liquid separation promoting solvent such as acetone or methyl ethyl ketone may be added.

[0148] The reaction solution neutralized with the above acid is further washed with an acidic aqueous solution, then separated, the aqueous layer is removed, and the organic layer is recovered. The acidic aqueous solution used for washing is not particularly limited, but examples include hydrochloric acid aqueous solution, dilute sulfuric acid aqueous solution, phosphoric acid aqueous solution, and acetic acid aqueous solution, with hydrochloric acid aqueous solution being preferred. The pH of the acidic aqueous solution is, for example, 1 to 5, preferably 1 to 4, and more preferably 1 to 3. The amount of acidic aqueous solution used for washing is not particularly limited as long as it shows a washing effect, but for example, it is 0.1 to 4 times the amount of the reaction solution, preferably 0.3 to 3 times, and more preferably 0.5 to 2 times.

[0149] There are no particular restrictions on the number of washes, separatory steps, and aqueous layer discards; they can be done once or multiple times. The number of steps should be appropriately selected depending on the type of compound in the reaction system, the amount of waste material, etc.

[0150] There are no particular restrictions on the washing temperature, but as an example, it is 10°C to 50°C, preferably 15°C to 45°C, and more preferably 20°C to 40°C.

[0151] Step B4 basically uses an acidic aqueous solution to remove by-products and unwanted reaction materials. However, the disclosure is not limited to this configuration, and if the reaction solution contains components that are difficult to remove by washing with an acidic aqueous solution, other washing steps may be added before or after washing with an acidic aqueous solution. For example, washing with a basic aqueous solution or saline solution may be performed.

[0152] [Peptide elongation process (process B5)] Step B5 is a process in which the C-terminally protected peptide obtained in step B4 is repeatedly condensed with the N-terminally protected peptide, which is the reaction substrate, to extend the C-terminally protected peptide toward the N-terminus. Step B5 may also be carried out by repeating steps B2, B3, and B4 described above.

[0153] After obtaining a C-terminally protected peptide having the desired amino acid residues by step B5, the C-terminal amide peptide is produced by performing step A1 described above.

[0154] <Summary> As can be understood from the above description, this disclosure encompasses the following aspects:

[0155] Embodiment 1: Represented by the following formula (1), [ka] In the formula, R 1 R is an organic group having at least one selected from the group consisting of aromatic hydrocarbon groups, heteroaromatic groups, and aliphatic hydrocarbon groups, 2 and R 3 Each is independently selected from the group consisting of alkyl groups which may have substituents and heteroalkyl groups which may have substituents, Y 2 and Y 3 A benzylamine compound characterized in that each of the elements is independently selected from the group consisting of -O-, -S-, -CONH-, -NHCO-, and -CH2-. According to Embodiment 1, a benzylamine compound is provided that can be used as a C-terminal tag having excellent acid resistance.

[0156] Appearance 2: R 1 This is expressed by the following equation (2): [ka] In the formula, A is an aryl group which may have substituents, the number of carbon atoms of A is 8 to 78, and * represents the bond position with N shown in formula (1), wherein this is the benzylamine compound according to Embodiment 1. According to Embodiment 2, a benzylamine compound that is more preferably usable as a C-terminal tag having excellent acid resistance is provided.

[0157] Appearance 3: R 1 This is expressed by the following equation (3): [ka] In the formula, m R A Each is independently selected from the group consisting of alkyl groups which may have substituents and heteroalkyl groups which may have substituents, and m Y A Each of the following is independently selected from the group consisting of -O-, -S-, -CONH-, and -NHCO-, m is an integer between 1 and 3, and * represents the bond position with N shown in formula (1), wherein the benzylamine compound is according to embodiment 1 or 2. According to embodiment 3, a benzylamine compound is provided that is more preferably usable as a C-terminal tag having excellent acid resistance.

[0158] Embodiment 4: The benzylamine compound is represented by the following formula (4), [ka] In the formula, R 2 , R 3 , R 4 and R 5 Each is independently selected from the group consisting of alkyl groups having 1 to 24 carbon atoms, which may have substituents, Y 2 , Y 3 , Y 4 and Y 5Each is independently selected from the group consisting of -O-, -S-, -CONH-, and -NHCO-, and is a benzylamine compound according to any one of embodiments 1 to 3. According to embodiment 4, a benzylamine compound is provided that is more preferably usable as a C-terminal tag having excellent acid resistance.

[0159] Aspect 5: In the formula, R 2 , R 3 , R 4 and R 5 Each is independently selected from the group consisting of a linear alkyl group having 1 to 24 carbon atoms and a branched alkyl group having 3 to 24 carbon atoms, Y 2 , Y 3 , Y 4 and Y 5 This is a benzylamine compound according to Embodiment 4, selected from the group consisting of -O- and -S-. According to Embodiment 5, a benzylamine compound that is more preferably usable as a C-terminal tag having excellent acid resistance is provided.

[0160] Embodiment 6: The benzylamine compound is selected from the group consisting of the following: [ka] A benzylamine compound according to any one of embodiments 1 to 5. According to embodiment 6, a benzylamine compound is provided that is more preferably usable as a C-terminal tag having excellent acid resistance.

[0161] Embodiment 7: A method for producing a peptide using a C-terminally protected peptide in which a benzylamine compound according to any one of Embodiments 1 to 6 is introduced as a protecting group at the C-terminus. According to Embodiment 7, a peptide is produced using a benzylamine compound according to one embodiment of the present disclosure as a C-terminal tag.

[0162] Embodiment 8: A method for producing a peptide according to Embodiment 7, comprising a C-terminal deprotection step of contacting the C-terminally protected peptide with a deprotection solution containing 50 v / v% or more of an acid with a pKa of -2.0 or less to obtain a C-terminally amide peptide having -CONH2 at the C-terminus. According to Embodiment 8, a C-terminally amide peptide is produced using a C-terminal tag with excellent acid resistance.

[0163] Embodiment 9: The deprotection solution further comprises a compound represented by the following formula (5), [ka] In the formula, k R B A method for producing a peptide according to embodiment 7 or 8, wherein each is independently selected from the group consisting of optionally substituted alkyl groups and optionally substituted heteroalkyl groups, and k is an integer between 1 and 5. According to embodiment 9, C-terminal deprotection can be carried out at a faster reaction rate.

[0164] Embodiment 10: A C-terminally protected peptide in which a benzylamine compound according to any one of Embodiments 1 to 6 is introduced at the C-terminus as a protecting group. Embodiment 10 provides a C-terminally protected peptide that can be used as a precursor for a C-terminally amide peptide.

[0165] <Additional Notes> This disclosure is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of this disclosure. [Examples]

[0166] One embodiment of this disclosure is described below.

[0167] <Synthesis Example 1-1: Synthesis of bis(3,4-didodecoxybenzyl)amine (benzylamine 1)>

[0168] [ka]

[0169] Step 1: 3,4-Dihydroxybenzaldehyde (10.0 g, 72.4 mmol), dodecyl bromide (45.1 g, 181 mmol), and potassium carbonate (50.0 g, 362 mmol) were added to N,N'-dimethylacetamide (100 mL), and the mixture was heated to 110°C and stirred for 4 hours. 150 mL of ethyl acetate was added to the reaction solution, and the mixture was filtered while hot. 100 mL of water was added to the filtrate, and liquid separation washing at 60°C was performed twice. The reaction solution was concentrated under reduced pressure and recrystallized from 300 mL of hexane to obtain 3,4-didodecoxybenzaldehyde with a yield of 93.5% (32.1 g, 67.7 mmol).

[0170]

Chemical Formula

[0171] Step 2: 3,4-Didodecoxybenzaldehyde (4.54 g, 9.56 mmol), hydroxylamine hydrochloride (5.98 g, 86.1 mmol), and triethylamine (9.68 g, 95.6 mmol) were added to 50 mL of tetrahydrofuran, and the mixture was stirred at room temperature for 3 days. The reaction solution was diluted with 50 mL of toluene, washed by liquid separation with 50 mL of water, and the organic layer was concentrated under reduced pressure to obtain 3,4-didodecoxybenzaldoxime with a yield of 98.6% (4.62 g, 9.43 mmol).

[0172]

Chemical Formula

[0173] Step 3: Under a nitrogen atmosphere, 80 mL of tetrahydrofuran was cooled in an ice bath, lithium aluminum hydride (1.25 g, 33.0 mmol) was added and suspended, and a solution of 3,4-didodecoxybenzaldoxime (4.62 g, 9.43 mmol) dissolved in 20 mL of tetrahydrofuran was added dropwise. The reaction mixture was stirred at room temperature for 24 hours. Sodium sulfate decahydrate (4.25 g, 13.2 mmol) was added to the reaction mixture to stop the reaction, filtered through Celite, and concentrated under reduced pressure to obtain 3,4-didodecoxybenzylamine in 93.8% yield (4.21 g, 8.85 mmol).

[0174] [ka]

[0175] Step 4: 3,4-didodecoxybenzylamine (4.21 g, 8.85 mmol) and 3,4-didodecoxybenzaldehyde (4.20 g, 8.85 mmol) were dissolved in 80 mL of toluene and heated under reflux for 2.5 hours in a reaction vessel equipped with a Dean-Stark apparatus. The reaction mixture was concentrated under reduced pressure to obtain bis(3,4-didodecoxybenzyl)imine.

[0176] [ka]

[0177] Step 5: Add 115 mL of tetrahydrofuran / ethanol (75:40) to bis(3,4-didodecoxybenzyl)imine, heat to dissolve the imine, add sodium borohydride (1.00 g, 26.5 mmol), and stir for 16 hours. Dilute the reaction mixture with 100 mL of toluene and perform two separatory washes with 50 mL of water. The organic layer was concentrated under reduced pressure, and the resulting solid was recrystallized over hexane and then over ethyl acetate to obtain bis(3,4-didodecoxybenzyl)amine in a two-step (Steps 4 and 5) yield of 93.8% (6.54 g, 6.89 mmol). 1H-NMR (600 MHz, CDCl3) δ 0.85 - 0.88 (t, J = 7.2 Hz, 12H), 1.24 - 1.34 (m, 64H), 1.41 - 1.47 (m, 8H), 1.76 - 1.84 (m, 8H), 3.67 (s, 4H), 3.96 - 3.99 (dd, J=7.2 Hz, 14.4 Hz, 8H), 6.78 - 6.82 (m, 4H), 6.88 (s, 2H).

[0178] <Synthesis Example 1-2: Synthesis of N-3,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylamine (benzylamine 2)>

[0179] [ka]

[0180] Step 1: 3,4-dimethoxybenzylamine (2.11 g, 12.6 mmol) and 3,4-didodecoxybenzaldehyde (5.00 g, 10.5 mmol) were dissolved in 50 mL of toluene and heated under reflux for 2.5 hours in a reaction vessel equipped with a Dean-Stark apparatus. The reaction mixture was concentrated under reduced pressure to obtain N-3,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylimine.

[0181] [ka]

[0182] Step 2: 60 mL of tetrahydrofuran / ethanol (40:20) was added to N-3,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylimine, and the mixture was heated to dissolve the imine. Sodium borohydride (1.20 g, 31.6 mmol) was added, and the mixture was stirred for 12 hours. The reaction solution was diluted with 100 mL of toluene, and liquid separation washing with 50 mL of water was performed twice. The organic layer was concentrated under reduced pressure, and the obtained solid was recrystallized from hexane to obtain N-3,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylamine with a yield of 72.0% (4.87 g, 7.59 mmol). 1 H-NMR (600 MHz, CDCl3) δ 0.85 - 0.88 (t, J = 7.2 Hz, 6H), 1.24 - 1.34 (m, 32H), 1.38 - 1.45 (m, 4H), 1.72 - 1.77 (m, 4H), 3.67 (s, 2H), 3.72 (s, 2H), 3.85 (s, 3H), 3.87 (s, 3H), 6.38 - 6.40 (dd, J = 7.8 Hz, 2.4 Hz, 1H), 6.43 (d, J = 2.4Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H), 6.82 - 6.83 (dd, J = 7.8 Hz, 2.4 Hz, 1H), 6.89 (d, J = 1.8 Hz, 1H).

[0183] <Synthesis Example 1-3: Synthesis of bis[3,4-bis(2-hexyl)decoxybenzyl]amine (benzylamine 3)>

[0184] [Chemical Formula]

[0185] Step 1: 3,4-Dihydroxybenzaldehyde (10.0 g, 72.4 mmol), 7-Bromohemethylpentadecane (77.4 g, 253 mmol), and potassium carbonate (50.0 g, 362 mmol) were added to 100 mL of N,N'-dimethylformamide. The mixture was heated to 80°C and stirred for 11 hours. 50 mL of water was added to the reaction mixture, and two liquid-liquid washes were performed. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (hexane / ethyl acetate = 100:0 to 70:30) to obtain 3,4-bis(2-hexyl)decoxybenzaldehyde in 84.7% yield (36.0 g, 61.3 mmol).

[0186] [ka]

[0187] Step 2: 3,4-Bis(2-hexyl)decoxybenzaldehyde (5.00 g, 8.52 mmol), hydroxylamine hydrochloride (5.34 g, 76.8 mmol), and triethylamine (10.4 g, 102 mmol) were added to 50 mL of tetrahydrofuran and stirred at room temperature for 3 days. The reaction mixture was filtered, diluted with 100 mL of hexane, and washed twice with 30 mL of water. The organic layer was concentrated under reduced pressure to obtain 3,4-bis(2-hexyl)decoxybenzaldehyde.

[0188] [ka]

[0189] Step 3: Under a nitrogen atmosphere, 100 mL of tetrahydrofuran was cooled in an ice bath, lithium aluminum hydride (0.647 g, 17.0 mmol) was added and suspended, and a solution of 3,4-bis(2-hexyl)decoxybenzaldoxime dissolved in 20 mL of tetrahydrofuran was added dropwise. The reaction mixture was stirred at room temperature for 22 hours. Sodium sulfate decahydrate (2.74 g, 8.52 mmol) was added to the reaction mixture to stop the reaction, filtered through Celite, and concentrated under reduced pressure to obtain 3,4-bis(2-hexyl)decoxybenzylamine.

[0190] [ka]

[0191] Step 4: 3,4-bis(2-hexyl)decoxybenzylamine and 3,4-bis(2-hexyl)decoxybenzaldehyde (3.39 g, 5.78 mmol) were dissolved in 80 mL of toluene and heated under reflux for 5 hours in a reaction vessel equipped with a Dean-Stark apparatus. The reaction mixture was concentrated under reduced pressure to obtain bis[3,4-bis(2-hexyl)decoxybenzyl]imine.

[0192] [ka]

[0193] Step 5: Add 80 mL of ethanol to bis[3,4-bis(2-hexyl)decoxybenzyl]imine, heat to dissolve the imine, add sodium borohydride (0.656 g, 17.3 mmol), and stir for 18 hours. Dilute the reaction mixture with 100 mL of hexane and perform two separate washes with 50 mL of water. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 90:10~0:100) to obtain bis[3,4-bis(2-hexyl)decoxybenzyl]amine in 4 steps (Steps 2-5) with a yield of 69.8% (4.68 g, 4.04 mmol). 1 H-NMR (600 MHz, CDCl3) δ 0.85 - 0.89 (t, J = 7.2 Hz, 24H), 1.25 - 1.48 (m, 104H), 1.75 - 1.80 (m, 4H), 3.69 (s, 4H), 6.77 - 6.80 (m, 4H), 6.85 (d, J = 1.8 Hz, 2H).

[0194] <Synthesis Example 1-4: Synthesis of N-3,4-bis(2-hexyl)decoxybenzyl-N'-3,4-dimethoxybenzylamine (benzylamine 4)> [ka]

[0195] Step 1: 3,4-dimethoxybenzylamine (1.71 g, 10.2 mmol) and 3,4-bis(2-hexyl)decoxybenzylbenzaldehyde (5.00 g, 8.52 mmol) were dissolved in 50 mL of toluene and heated under reflux for 2.5 hours in a reaction vessel equipped with a Dean-Stark apparatus. The reaction mixture was concentrated under reduced pressure to obtain N-3,4-bis(2-hexyl)decoxybenzyl-N'-3,4-dimethoxybenzylimine.

[0196] [ka]

[0197] Step 2: Add 50 mL of ethanol to N-3,4-bis(2-hexyl)decoxybenzyl-N'-3,4-dimethoxybenzylimine and heat to dissolve the imine. Add sodium borohydride (0.967 g, 25.6 mmol) and stir for 15 hours. Dilute the reaction mixture with 100 mL of hexane and perform two separate washes with 50 mL of water. The organic layer was concentrated under reduced pressure and purified by silica gel column chromatography (hexane / acetone = 95:5) to obtain N-3,4-bis(2-hexyl)decoxybenzyl-N'-3,4-dimethoxybenzylamine in a yield of 89.0% (5.72 g, 7.58 mmol) from the two steps (Steps 1 and 2). 1 H-NMR (600 MHz, CDCl3) δ 0.85 - 0.87 (t, J = 7.2 Hz, 12H), 1.26 - 1.50 (m, 52H), 1.75 - 1.80 (m, 2H), 3.70 (s, 2H), 3.72 (s, 2H), 3.81 - 3.84 (dd, J = 7.2 Hz, 5.4 Hz, 4H) 3.86 (s,3H), 3.87 (s, 3H), 6.78 - 6.88 (m, 6H).

[0198] <Synthesis Example 1-5: Synthesis of N-2,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylamine (benzylamine 5)>

[0199] [ka]

[0200] Step 1: 2,4-dihydroxybenzaldehyde (10.0 g, 72.4 mmol), dodecyl bromide (45.1 g, 181 mmol), and potassium carbonate (50.0 g, 362 mmol) were added to 100 mL of N,N'-dimethylacetamide. The mixture was heated to 110°C and stirred for 1 hour. 100 mL of toluene and 200 mL of water were added to the reaction mixture, and a liquid-liquid wash was performed at 60°C. The organic layer was further washed twice with 100 mL of water at 60°C. The organic layer was concentrated under reduced pressure and recrystallized with 300 mL of hexane to obtain 2,4-didodecoxybenzaldehyde in 84.3% yield (29.0 g, 61.0 mmol).

[0201] [ka]

[0202] Step 2: 3,4-dimethoxybenzylamine (2.14 g, 12.8 mmol) and 2,4-didodecoxybenzaldehyde (5.07 g, 10.7 mmol) were dissolved in 50 mL of toluene and heated under reflux for 2.5 hours in a reaction vessel equipped with a Dean-Stark apparatus. The reaction mixture was concentrated under reduced pressure to obtain N-2,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylimine.

[0203] [ka]

[0204] Step 3: Add 70 mL of tetrahydrofuran / ethanol (5:2) to N-3,4-didodecoxybenzyl-N'-2,4-dimethoxybenzylimine and heat to dissolve the imine. Add sodium borohydride (1.21 g, 32.0 mmol) and stir for 2 hours. Dilute the reaction mixture with 100 mL of toluene and perform two separate washes with 50 mL of water. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / acetone = 95:5) to obtain N-2,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylamine in a yield of 86.5% (5.78 g, 9.23 mmol) from two steps (Steps 2 and 3). 1 H-NMR (600 MHz, CDCl3) δ 0.85 - 0.87 (t, J = 7.2 Hz, 6H), 1.17 - 1.43 (m, 36H), 1.73 - 1.75 (m, 4H), 3.60 - 3.93 (m, 14H), 6.38 - 6.46 (m, 2H), 6.56 - 6.88 (m, 3H), 7.06 - 7.09 (m, 1H).

[0205] <Synthesis Example 1-6: Synthesis of N-3,4-diocadecoxybenzyl-N'-2,4-dimethoxybenzylamine (benzylamine 6)>

[0206] [ka]

[0207] Step 1: 3,4-Dihydroxybenzaldehyde (5.00 g, 36.2 mmol), 1-Bromooctadecane (30.2 g, 90.5 mmol), and potassium carbonate (25.0 g, 181 mmol) were added to 100 mL of N,N'-dimethylacetamide. The mixture was heated to 110°C and stirred for 3 hours. 100 mL of water was added to the reaction mixture, and the solid was filtered off. 200 mL of tetrahydrofuran was added to the obtained solid, heated to dissolve, and 200 mL of methanol was added dropwise. The precipitated solid was filtered off, and two liquid-liquid washes were performed at 60°C. The reaction mixture was concentrated under reduced pressure and recrystallized with 300 mL of hexane to obtain 3,4-Dioctadecoxybenzaldehyde in 100% yield (23.3 g, 36.2 mmol).

[0208] [ka]

[0209] Step 2: 2,4-Dimethoxybenzylamine (2.22 g, 13.3 mmol) and 3,4-Diocadecoxybenzaldehyde (7.00 g, 10.9 mmol) were dissolved in 100 mL of toluene and heated under reflux for 17 hours in a reaction vessel equipped with a Dean-Stark apparatus. The reaction mixture was concentrated under reduced pressure to obtain N-3,4-Diocadecoxybenzyl-N'-2,4-Dimethoxybenzylimine.

[0210] [ka]

[0211] Step 3: Add 120 mL of tetrahydrofuran / ethanol (70:50) to N-3,4-dioctadecoxybenzyl-N'-2,4-dimethoxybenzylimine and heat to dissolve the imine. Add sodium borohydride (1.24 g, 32.7 mmol) and stir for 17 hours. Dilute the reaction mixture with 50 mL of toluene and perform two separate washes with 30 mL of water. The organic layer was concentrated under reduced pressure, and the resulting solid was recrystallized with ethyl acetate to obtain N-3,4-didodecoxybenzyl-N'-2,4-dimethoxybenzylamine in a two-step (Steps 2 and 3) yield of 93.9% (8.10 g, 10.2 mmol). 1 H-NMR (600MHz, CDCl3) 0.85 - 0.88 (t, J = 7.2 Hz, 6H), 1.24 - 1.46 (m, 64H), 1.75 - 1.81 (m, 4H), 3.66 (s, 2H), 3.72 (s, 2H), 3.78 (s, 3H), 3.79 (s, 3H), 3.94 - 3.98 (dt, J = 10.2 Hz, 6.6 Hz, 4H), 6.41 - 6.43 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 6.80 (s, 1H), 6.89 (2, 1H), 7.10 - 7.11 (d, J = 1.8 Hz, 1H).

[0212] <Synthesis Example 2-1: Synthesis of N-benzoylphenylalanine-bis(3,4-didodecoxybenzyl)amide (C-terminally protected peptide 1)>

[0213] [ka]

[0214] Bis(3,4-didodecoxybenzyl)amine (benzylamine 1, 2.06 g, 2.20 mmol), N-benzoylphenylalanine (1.37 g, 5.07 mmol), EDCI·HCl (1.06 g, 5.51 mmol), and oxyma (0.031 g, 0.22 mmol) were dissolved in MTHP / MeCN (4:1, 20 mL) and stirred at room temperature for 3 days. The reaction mixture was diluted with 30 mL of toluene, separated by liquid-liquid washing with 30 mL of 1 M hydrochloric acid, and the organic layer was further separated by liquid-liquid washing with 30 mL of 0.5 M aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. Methanol was added to the resulting residue, and the mixture was suspended, stirred, and a sample was taken. The resulting solid was dissolved in toluene, the solvent was removed by distillation under reduced pressure, methanol was added, and the mixture was suspended, stirred, and a sample was taken. The obtained solid was recrystallized with hexane to obtain N-benzoylphenylalanine-bis(3,4-didodecoxybenzyl)amide in 88.5% yield (2.34 g, 1.95 mmol).

[0215] <Synthesis Example 2-2: Synthesis of N-benzoylphenylalanine-N-3,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylamide (C-terminally protected peptide 2)>

[0216] [ka]

[0217] Step 1: Dissolve N-3,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylamine (benzylamine 2, 1.03 g, 1.60 mmol), N-fluorenylmethyloxycarbonylphenylalanine (0.75 g, 1.92 mmol), EDCI·HCl (0.368 g, 1.92 mmol), and oxyma (0.068 g, 0.48 mmol) in MTHP / MeCN (4:1, 20 mL) and stir at room temperature for 3 hours. Add morpholine (2.79 g, 32.0 mmol) and DBU (1.71 g, 11.2 mmol) to the reaction mixture and stir for 1 hour. Add 26 mL of 2 M hydrochloric acid to the reaction mixture and separate the solutions. The organic layer was then separated and washed with 26 mL of 0.5 M sodium bicarbonate aqueous solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain phenylalanine-N-3,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylamide. This was used in the next reaction without purification.

[0218] [ka]

[0219] Step 2: Phenylalanine-N-3,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylamide was dissolved in 20 mL of MTHP, and benzoyl chloride (0.27 g, 1.92 mmol) and triethylamine (0.194 g, 1.92 mmol) were added. The mixture was stirred at room temperature for 18 hours. 20 mL of saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and the mixture was separated and washed. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain N-benzoylphenylalanine-N-3,4-didodecoxybenzyl-N'-3,4-dimethoxybenzylamide in a yield of 95.4% (1.34 g, 1.53 mmol) from two steps (Steps 1 and 2).

[0220] <Synthesis Example 2-3: Synthesis of N-benzoylphenylalanine-N-3,4-diocadecoxybenzyl-N'-2,4-dimethoxybenzylamide (C-terminally protected peptide 3)>

[0221] [ka]

[0222] Step 1: Dissolve N-3,4-diocadecoxybenzyl-N'-2,4-dimethoxybenzylamine (benzylamine 6, 0.72 g, 0.89 mmol), N-fluorenylmethyloxycarbonylphenylalanine (0.41 g, 1.07 mmol), EDCI·HCl (0.20 g, 1.07 mmol), and oxyma (0.12 g, 0.89 mmol) in MTHP / MeCN (4:1, 20 mL) and stir at room temperature for 1 day. Add morpholine (1.55 g, 17.8 mmol) and DBU (0.95 g, 6.22 mmol) to the reaction mixture and stir for 1 hour. Add 17 mL of 2 M hydrochloric acid to the reaction mixture and separate the washes, and then separate the organic layer with 20 mL of 0.5 M sodium bicarbonate aqueous solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain phenylalanine-N-3,4-dioctadecoxybenzyl-N'-2,4-dimethoxybenzylamide. This was used in the next reaction without purification.

[0223] [ka]

[0224] Step 2: Phenylalanine-N-3,4-diocadecoxbenzyl-N'-2,4-dimethoxybenzylamide was dissolved in 20 mL of MTHP, and benzoyl chloride (0.15 g, 1.07 mmol) and triethylamine (0.11 g, 1.07 mmol) were added. The mixture was stirred at room temperature for 18 hours. 20 mL of saturated sodium bicarbonate aqueous solution was added to the reaction mixture and the mixture was separated and washed. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain N-benzoylphenylalanine-N-3,4-diocadecoxbenzyl-N'-2,4-dimethoxybenzylamide in 100% yield (0.93 g, 0.89 mmol).

[0225] <Synthesis Example 2-C1: Synthesis of N-benzoylphenylalanine-N-2,4-didocosaoxybenzyl-N'-2,4-dimethoxybenzylamide (C-terminally protected peptide C1)>

[0226] [ka]

[0227] Step 1: Dissolve N-2,4-didocosaoxybenzyl-N'-2,4-dimethoxybenzylamine (1.00 g, 1.08 mmol), N-fluorenylmethyloxycarbonylphenylalanine (0.50 g, 1.30 mmol), EDCI·HCl (0.25 g, 1.30 mmol), and oxyma (0.046 g, 0.33 mmol) in MTHP / MeCN (4:1, 20 mL) and stir at room temperature for 18 hours. Add morpholine (1.89 g, 21.7 mmol) and DBU (1.14 g, 1.08 mmol) to the reaction mixture and stir for 1 hour. Add 17 mL of 2 M hydrochloric acid to the reaction mixture and separate the washes, then separate the organic layer with 20 mL of 0.5 M sodium bicarbonate aqueous solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain phenylalanine-N-2,4-didocosaoxybenzyl-N'-2,4-dimethoxybenzylamide. This was used in the next reaction without purification.

[0228] [ka]

[0229] Step 2: Phenylalanine-N-3,4-didocosaoxybenzyl-N'-3,4-dimethoxybenzylamide was dissolved in 20 mL of MTHP, and benzoyl chloride (0.17 g, 1.19 mmol) and triethylamine (0.12 g, 1.19 mmol) were added. The mixture was stirred at room temperature for 18 hours. 20 mL of saturated sodium bicarbonate aqueous solution was added to the reaction mixture and the mixture was separated and washed. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain N-benzoylphenylalanine-N-2,4-didocosaoxybenzyl-N'-2,4-dimethoxybenzylamide in a two-step (Steps 1 and 2) yield of 90.0% (1.13 g, 0.98 mmol).

[0230] <Example 1: C-terminal deprotection of C-terminal protected peptide 1 (No. 1)> C-terminally protected peptide 1 (201 mg, 1.00 equiv.) was packed into a test tube, and trifluoroacetic acid (TFA, 3.8 mL, 95 v / v%), triisopropylsilane (TIPS, 0.1 mL, 2.5 v / v%), and water (H2O, 0.1 mL, 2.5 v / v%) were added. The mixture was stirred at room temperature (25°C) for 7 hours. A portion of the reaction mixture was withdrawn, diluted, and analyzed by assay by HPLC. The yield of N-benzoylphenylalaninamide (Bz-Phe-NH2) was 0.0%, and the yield of N-benzoylphenylalanine (Bz-Phe-OH) was 8.4%.

[0231] The yield of each product was calculated using calibration curves prepared from standard solutions of Bz-Phe-NH2 and Bz-Phe-OH, respectively, using HPLC.

[0232] The procedure was performed under various conditions, with the deprotection conditions modified as shown in Table 1. <Example 2: C-terminal deprotection of C-terminal protected peptide 2 (No. 18)> C-terminally protected peptide 2 (51.9 mg, 1.00 equiv.) was packed into a test tube, and trifluoroacetic acid (TFA, 0.5 mL, 50 v / v%) and methanesulfonic acid (MSA, 0.5 mL, 50 v / v%) were added. The mixture was stirred at room temperature (25°C) for 2 hours. A portion of the reaction mixture was withdrawn, diluted, and analyzed by assay by HPLC. The yield of Bz-Phe-NH2 was 8.1%, and the yield of Bz-Phe-OH was 2.8%.

[0233] The procedure was performed under various conditions, with the deprotection conditions modified as shown in Table 1.

[0234] <Example 3: C-terminal deprotection of C-terminal protected peptide 3 (No. 24)> C-terminally protected peptide 3 (103 mg, 1.00 equiv.) was packed into a test tube, and trifluoroacetic acid (TFA, 3.8 mL, 95 v / v%), methanesulfonic acid (MSA, 0.1 mL, 2.5 v / v%), and triisopropylsilane (TIPS, 0.1 mL, 2.5 v / v%) were added. The mixture was stirred at room temperature (25°C) for 2 hours. A portion of the reaction mixture was withdrawn, diluted, and analyzed by assay by HPLC. The yield of Bz-Phe-NH2 was 0%, and the yield of Bz-Phe-OH was 0%.

[0235] The procedure was performed under various conditions, with the deprotection conditions modified as shown in Table 1.

[0236] <Comparative Example 1: C-terminal deprotection of C-terminal protected peptide C1 (No. 27)> A test tube was filled with C-terminally protected peptide C1 (212 mg, 1.00 equiv.) as shown in the formula below, and trifluoroacetic acid (TFA, 3.8 mL, 95 v / v%), triisopropylsilane (TIPS, 0.1 mL, 2.5 v / v%), and water (H2O, 0.1 mL, 2.5 v / v%) were added. The mixture was stirred at room temperature (25°C) for 18 hours. After the reaction was complete, the filtrate was collected by suction filtration. This filtrate was subjected to assay analysis. The yield of Bz-Phe-NH2 was 37.6%, and the yield of Bz-Phe-OH was 26.5%.

[0237] The procedure was performed under various conditions, with the deprotection conditions modified as shown in Table 1.

[0238] The results for Examples 1 and 2 and Comparative Example 1 are shown in Table 1. In Table 1, * indicates the equivalent amount per mole of C-terminally protected peptide.

[0239] [Table 1]

[0240] A comparison of numbers 1 and 27 shows that the C-terminal tag according to one embodiment of this disclosure is less prone to deprotection under acidic conditions and therefore exhibits excellent acid resistance. Furthermore, the results from numbers 24 to 26 show that the degree of deprotection can be controlled by appropriately adjusting the components of the deprotection solution.

[0241] <Synthesis Example 2-1: Synthesis of Ala-Arg-benzylamine 1 (C-terminally protected peptide 4)> Step 1: Bis(3,4-didodecoxybenzyl)amine (3.65 g, 3.91 mmol), Fmoc-Arg(Pbf)-OH (5.32 g, 8.20 mmol), EDCI·HCl (1.57 g, 8.20 mmol), and oxyma (0.17 g, 1.17 mmol) were dissolved in MTHP / MeCN (4:1, 73 mL) and stirred at room temperature for 1 day. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 66:34~50:50~0:100). The purified product was dissolved in MTHP / MeCN (4:1, 73 mL), morpholine (6.81 g, 78.1 mmol), and DBU (4.16 g, 27.3 mmol) were added, and the mixture was stirred for 1 hour. 63 mL of 2 M hydrochloric acid was added to the reaction mixture and the mixture was separated and washed. The organic layer was then separated and washed again with 63 mL of 0.5 M aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain Arg(Pbf)-benzylamine 1 in 53.4% ​​yield (2.80 g, 2.08 mmol).

[0242] Step 2: Arg(Pbf)-benzylamine 1 (2.80 g, 2.08 mmol), Fmoc-Ala-OH (1.04 g, 3.34 mmol), EDCI·HCl (0.64 g, 3.34 mmol), and oxyma (0.09 g, 0.625 mmol) were dissolved in MTHP / MeCN (4:1, 56 mL) and stirred at room temperature for 4 hours. Morpholine (3.63 g, 41.7 mmol) and DBU (2.22 g, 14.6 mmol) were added to the reaction mixture and stirred for 1 hour. 33 mL of 2 M hydrochloric acid was added to the reaction mixture and the mixture was separated and washed. The organic layer was then separated and washed again with 33 mL of 0.5 M aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and methanol was added to the resulting residue to solidify it and obtain Ala-Arg(Pbf)-benzylamine 1. This was used in the next reaction without purification.

[0243] <Synthesis Example 2-2: Synthesis of Ala-Arg-NH2> Ala-Arg(Pbf)-benzylamine 1 (C-terminally protected peptide 4, 51.9 mg, 1.00 equiv.) was packed into a test tube, and methanesulfonic acid (MSA, 0.5 mL, 100 v / v%) and mesitylene (51.9 mg, 10 equiv.) were added. The mixture was stirred at room temperature (25°C) for 24 hours. A portion of the reaction solution was withdrawn, diluted, and analyzed by LC / MS to confirm the formation of Ala-Arg-NH2. ESI-MS: 245.16 [M+H] + [Industrial applicability]

[0244] This disclosure is usable as a C-terminal tag in peptide synthesis.

Claims

1. It is expressed by the following formula (1), 【Chemistry 1】 During the ceremony, R １ This is an organic group having at least one selected from the group consisting of aromatic hydrocarbon groups, heteroaromatic groups, and aliphatic hydrocarbon groups. R ２ and R ３ Each is independently selected from the group consisting of alkyl groups which may have substituents and heteroalkyl groups which may have substituents. Y ２ and Y ３ These are independently -O-, -S-, -CONH-, -NHCO-, and -CH ２ - Selected from the group consisting of, A benzylamine compound characterized by the following features.

2. R １ This is expressed by the following equation (2): 【Chemistry 2】 During the ceremony, A is an aryl group which may have substituents, The number of carbon atoms in A is between 8 and 78. * represents the bonding position with N as shown in formula (1) above. The benzylamine compound according to claim 1.

3. R １ This is expressed by the following equation (3): 【Transformation 3】 During the ceremony, m R groups Ａ are each independently selected from the group consisting of an optionally substituted alkyl group and an optionally substituted heteroalkyl group, m pieces of Y Ａ Each of these is independently selected from the group consisting of -O-, -S-, -CONH-, and -NHCO-, m is an integer between 1 and 3, * represents the bonding position with N as shown in formula (1) above. The benzylamine compound according to claim 1 or 2.

4. The benzylamine compound is represented by the following formula (4): 【Chemistry 4】 During the ceremony, R ２ , R ３ , R ４ and R ５ Each of these is independently selected from the group consisting of alkyl groups having 1 to 24 carbon atoms, which may have substituents. Y ２ , Y ３ , Y ４ and Y ５ Each of these is independently selected from the group consisting of -O-, -S-, -CONH-, and -NHCO-. The benzylamine compound according to any one of claims 1 to 3.

5. During the ceremony, R ２ , R ３ , R ４ and R ５ Each of these is independently selected from the group consisting of linear alkyl groups having 1 to 24 carbon atoms and branched alkyl groups having 3 to 24 carbon atoms. Y ２ , Y ３ , Y ４ and Y ５ This is selected from the group consisting of -O- and -S-. The benzylamine compound according to claim 4.

6. The benzylamine compound is selected from the group consisting of the following: 【Transformation 5】 The benzylamine compound according to claim 1.

7. A method for producing a peptide, using a C-terminally protected peptide in which a benzylamine compound according to any one of claims 1 to 6 is introduced at the C-terminus as a protecting group.

8. The C-terminally protected peptide is brought into contact with a deprotection solution containing 50 v / v% or more of an acid with a pKa of -2.0 or less, and -CONH is added to the C-terminus. ２ A C-terminal deprotection step is included to obtain a C-terminal amide peptide having the following characteristics: A method for producing the peptide according to claim 7.

9. The deprotection solution further comprises a compound represented by the following formula (5): 【Transformation 6】 During the ceremony, k R Ｂ Each is independently selected from the group consisting of alkyl groups which may have substituents and heteroalkyl groups which may have substituents. k is an integer between 1 and 5 (inclusive). A method for producing the peptide according to claim 8.

10. A C-terminally protected peptide having a benzylamine compound according to any one of claims 1 to 6 introduced as a protecting group at its C-terminus.

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