CN118234736A - Method for producing peptide, method for removing protecting group, remover, and benzyl compound - Google Patents

Method for producing peptide, method for removing protecting group, remover, and benzyl compound Download PDF

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CN118234736A
CN118234736A CN202280074943.1A CN202280074943A CN118234736A CN 118234736 A CN118234736 A CN 118234736A CN 202280074943 A CN202280074943 A CN 202280074943A CN 118234736 A CN118234736 A CN 118234736A
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group
compound
formula
peptide
represented
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葛西宗江
下田康嗣
佐藤诚
本山杏梨
松浦圭介
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Tokuyama Corp
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Tokuyama Corp
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Priority claimed from PCT/JP2022/042265 external-priority patent/WO2023127331A1/en
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Abstract

One of the problems to be solved by the present invention is to provide a method for removing a protecting group, which suppresses the formation of a double-attacker or diketopiperazine, is easy to trap a byproduct that may be formed after deprotection of a protecting group having a fluorene skeleton, i.e., a compound having a fulvene skeleton, and is capable of easily separating a trap formed with the byproduct, a method for producing a peptide including a protecting group removing step, and a protecting group remover. The method for producing a peptide according to an aspect of the present invention comprises: a step of bringing an amino group-containing compound having an N-terminal protected with a protecting group having a fluorene skeleton into contact with a capturing agent represented by the following formula (Z1) in an organic solvent, wherein a capturing agent is obtained by bonding a byproduct having an fulvene skeleton derived from the protecting group to the capturing agent; and a step of separating the capture body obtained from the organic solvent;

Description

Method for producing peptide, method for removing protecting group, remover, and benzyl compound
Technical Field
One aspect of the present invention relates to a novel benzyl compound, and in particular, to a novel benzyl compound used in a method for synthesizing a peptide using a tag synthesis method, and a peptide synthesis method using the benzyl compound. Further, an aspect of the present invention relates to a method of efficiently capturing a byproduct having a fulvene skeleton generated in a deprotection reaction of a protecting group having a fluorene skeleton in liquid phase peptide synthesis and efficiently removing the byproduct by washing, a peptide manufacturing method using the same, and the like.
Background
Conventionally, a solid-phase peptide synthesis method (SPPS method) and a liquid-phase peptide synthesis method (LPPS method) are well-known peptide synthesis methods. Solid-phase peptide synthesis is suitable for long-chain peptide synthesis because in the condensation reaction of amino acids, peptides can be synthesized relatively easily by purifying the peptides by filtering unwanted substances. However, the solid-phase peptide synthesis method is considered unsuitable for large-scale peptide synthesis because of high synthesis cost due to the use of an excessive amount of amino acids and a washing solvent.
In contrast, liquid phase peptide synthesis can be used for large amounts of peptide synthesis. However, the liquid phase peptide synthesis method has a problem in synthesizing a long chain peptide because extension of a peptide chain becomes difficult easily after the peptide chain becomes long.
Therefore, a synthesis method combining the advantages of the above solid-phase peptide synthesis method and liquid-phase peptide synthesis method has been studied (for example, refer to patent document 4). This synthesis method is a method of synthesizing a peptide using a soluble protecting group (protecting group at the C-terminal of an amino acid, hereinafter also referred to as "tag") in a solution, for example, by bonding an amino acid to a tag having a long chain alkyl group and repeating continuous extension reaction. According to this synthesis method, only the peptide bonded to the tag is solidified (e.g., crystallized) in each extension stage, so that separation and purification of the solidified substance can be easily performed.
In addition, there is known a method of selectively dissolving only a peptide component bonded to a dissolution tag in a specific phase along with phase separation of a liquid, thereby separating from other unwanted components (hereinafter also referred to as "liquid phase tag method". For example, refer to patent documents 1 to 9 and non-patent documents 1 to 3). According to this method, unnecessary components can be separated without solidifying, and thus the reaction process can be facilitated to be speeded up and simplified.
However, since the liquid phase labeling method is a one-pot method continuously carried out in an organic solvent, it is preferable to use an organic solvent having a specific gravity of 1 or less and not mixed with the aqueous layer. In addition, from the viewpoint of environmental protection, an organic solvent other than halogen is preferably used.
However, the benzyl alcohol type tags having straight-chain alkyl groups each formed of 18 carbon atoms at the 3,4, and 5 positions disclosed in patent document 4 and the benzyl alcohol type tags having straight-chain alkyl chains each formed of 22 carbon atoms at the 3, 5 positions disclosed in patent document 2 have low solubility in an organic solvent satisfying the above conditions, and therefore, not only are precipitated from the reaction system during peptide synthesis, but also separation and purification of the compounds after the reaction tend to become difficult.
In view of this, a tag having high solubility and hydrophobicity in an organic solvent has been developed as a tag used in a liquid-phase tag method (for example, refer to patent documents 3 and 6). For example, patent document 3 discloses a branched aromatic compound having at least one aliphatic hydrocarbon group having one or more branches, having three or more total branches, and having an organic group having 14 to 300 total carbon atoms as a substituent in the branches.
Patent document 6 discloses a benzyl compound having a group having an-O-Si-structure at the terminal. The benzyl compound described in patent document 6 has high solubility in an organic solvent, and is useful for a liquid phase synthesis method.
In the liquid phase peptide synthesis methods such as the liquid phase labeling method, a method using an Fmoc group as an amino protecting group of an amino acid is widely used. Deprotection of the Fmoc group with base yields the byproduct Dibenzofulvene (DBF) from the Fmoc group. In order to remove such by-products, a method is known in which a DBF removing reagent is used and an adduct of DBF and the removing reagent (hereinafter, sometimes simply referred to as DBF-trap) is washed by liquid separation.
For example, patent document 1 describes a method in which thiol-containing carboxylic acid or thiol-containing sulfonic acid is added to produce a DBF-trap during deprotection of an Fmoc group, and the DBF-trap is removed by alkaline liquid separation washing.
However, in this method, the thioester is formed by reacting the thiol-containing carboxylic acid added as a capturing agent with an amino acid. Since this thioester is an active ester, it may react with an amino group formed after Fmoc removal to form a double-attacker. In addition, if a cysteine residue is particularly contained in the peptide sequence, capture of DBF with a thiol compound may react with the thiol structure of the side chain site of the cysteine residue contained in the peptide sequence, thereby generating other by-products.
In this regard, a method of inhibiting the formation of double-attacked species by reacting thioesters with amino groups of peptides whose Fmoc groups have been deprotected has been proposed (for example, refer to patent document 2). Patent document 2 describes a method in which DBF generated during deprotection of an Fmoc group is reacted with a water-soluble amine having a valence of two or more (for example, N-methylpiperazine) to obtain a DBF-trap, and then the DBF-trap is removed by acidic liquid separation washing.
Patent document 9 and non-patent document 4 disclose that the basic condition for deprotection of the Fmoc group as the N-terminal is in the presence of a reagent such as diethylamine.
Prior art literature
Patent literature
Patent document 1: japanese patent No. 6136934
Patent document 2: japanese patent No. 6703668
Patent document 3: japanese patent No. 5929756
Patent document 4: japanese patent laid-open No. 2000-44493
Patent document 5: japanese patent No. 7063408
Patent document 6: japanese patent No. 6116782
Patent document 7: japanese patent No. 6703669
Patent document 8: japanese patent No. 5768712
Patent document 9: japanese patent No. 5201764
Patent document 10: international publication No. 2019/069978
Non-patent literature
Non-patent document 1: livingston et al, angew.chem.int.ed.60 (2021) 7786-7795.
Non-patent document 2: qin et al, org. Lett.22 (2020) 3323-3328.
Non-patent document 3: Y.Okada, K.Chiba et al, org.process.Res.Dev.23 (2019) 2576-2581.
Non-patent document 4: n. Nishizawa et al, org. Process. Res. Dev.25 (2021) 2029-2038.
Non-patent document 5: J.chem.Soc.Perkin Trans.1,3043,1992.
Disclosure of Invention
Problems to be solved by the invention
However, the branched aromatic compound described in patent document 3 requires the use of an expensive noble metal reduction catalyst (platinum carbon or the like) to produce the branched chain, and thus there is room for improvement because the cost increases when it is used in mass production. In addition, the benzyl compound described in patent document 6 has room for improvement because the o—si bond may be cleaved under acidic conditions such as liquid separation and the like, and there is a possibility of slight decomposition (refer to non-patent document 5).
In view of the above, it is desired to develop a tag having a structure other than a branched structure or a structure containing an o—si bond, which can be used in a liquid phase tag method.
Further, according to the method described in patent document 2, there is a problem in that an operation of neutralization or the like using a water-soluble amine having a valence of two or more is required by using a large amount of acid. In addition, water-soluble divalent amines typified by N-methylpiperazine, and diethylamine used in patent document 10 and non-patent document 4 have sufficient basicity. Thus, in the condensation reaction, when a water-soluble divalent amine is added in order to inactivate the amino acid active ester formed by the excess amino acid and the condensing agent, an unexpected deprotection reaction of the Fmoc group may occur. This may lead to other side reactions that produce diketopiperazines and the like.
Accordingly, an object of the present invention is to solve the above-mentioned problems and to improve the solubility of a tag in an organic solvent, thereby providing a tag which does not precipitate or dissolve during the liquid separation after peptide synthesis and reaction, a method for producing the same, and a method for synthesizing a peptide using the tag.
Accordingly, an object of the present invention is to provide a method for removing a protecting group, which suppresses the formation of a double-attacker or diketopiperazine, facilitates capturing, and can easily separate a capturing body formed with the by-product, a method for producing a peptide including a protecting group removing step, and a protecting group remover.
Technical scheme for solving problems
In order to solve the above problems, the inventors have conducted intensive studies and found that a benzyl compound having excellent solubility in an organic solvent and high hydrophobicity can be provided by introducing an aromatic ring compound having a substituent, or an alkyl group and an aralkyl group, into a benzene ring having benzyl alcohol via an oxyarene group, thereby completing one aspect of the present invention.
That is, the benzyl compound according to one aspect of the present invention is a benzyl compound (X1) represented by the following formula (X1):
In the formula (X1), the amino acid sequence of the formula (X),
M Q 1 and Q 2 are each an oxygen atom,
M R 1 are each independently alkylene,
M R 2 are each independently an optionally substituted alkyl group, an optionally substituted aralkyl group or an optionally substituted aryl group,
K R 3 are each independently a hydrogen atom, an alkyl group, an alkoxy group or a halogen atom,
X is a hydroxyl group, and is a hydroxyl group,
M is an integer of 2 or 3,
K represents an integer of 0 to (5-m).
Furthermore, the inventors have conducted intensive studies based on the following ideas: in addition to the fact that a peptide can be more uniformly synthesized in an organic solvent which is not mixed with an aqueous layer using a tag having high solubility in an organic solvent and also having high hydrophobicity, when the reaction is followed by liquid separation through the aqueous layer to remove unwanted substances, the peptide bonded to the tag is not precipitated or insoluble, and the loss of the peptide bonded to the tag in the aqueous layer is suppressed, thereby improving the yield.
However, the present invention has been completed based on the finding that, as a result, it was unexpectedly found that a tag having a higher hydrophobicity is not necessarily more suitable for the separation of long-chain peptides, i.e., a tag having a hydrophobicity below a certain level is suitable for the separation and purification of long-chain peptides by a liquid separation method.
That is, the benzyl compound according to one aspect of the present invention is a benzyl compound (Y1) represented by the following formula (Y1):
in the formula (Y1), the amino acid sequence of the formula (I),
M Q's each represent an oxygen atom,
M R 1 are each independently a group represented by the following formula (YA):
in the formula (YA),
* The bonding position is indicated by the number of the bonding sites,
R 1a、R1b、R1c、R1d and R 1e each independently represent a hydrogen atom or an alkyl group,
N 1 represents an integer of 0 to 6, and when n 1 is 1 or more, the repeating unit represented in parentheses with n 1 is an alkylene group,
N 2 represents an integer of 0 to 6, and when n 2 is 1 or more, the repeating unit represented in parentheses with n 2 is an alkylene group,
At least two or more of R 1a、R1b、R1c and R 1d are hydrogen atoms;
In the formula (Y1), k R 2 each independently represents a hydrogen atom, an alkyl group, a cycloalkyl group, an alkoxy group, an aryl group, an aralkyl group or a halogen atom,
In the formula (Y1), X represents a hydroxyl group,
In the formula (Y1), m represents an integer of 2 or 3,
In the formula (Y1), k represents an integer of 0 to (5-m) inclusive,
In the formula (Y1), at least one of m [ Q-R 1 ] is substituted in the meta position with respect to the substituent containing the X.
Further, as a result of intensive studies in view of the above-mentioned problems, the inventors have found that by using a specific cyclic amine having only one nitrogen atom in the cyclic amine as a capturing agent, a by-product having a fulvene skeleton such as DBF generated in a deprotection reaction of a protecting group having a fluorene skeleton such as Fmoc group can be captured, and the by-product can be easily removed by separating a capturing body formed by the by-product from the reaction system, thereby completing an aspect of the present invention.
That is, a method for producing a peptide according to an aspect of the present invention comprises:
a step of bringing an amino group-containing compound having an N-terminal protected with a protecting group having a fluorene skeleton into contact with a capturing agent represented by the following formula (Z1) in an organic solvent, wherein a capturing agent is obtained by bonding a byproduct having an fulvene skeleton derived from the protecting group to the capturing agent; and
And separating the obtained capturing body from the organic solvent.
In the above-mentioned formula (Z1),
N is a nitrogen atom and is preferably a nitrogen atom,
H is a hydrogen atom and is preferably a hydrogen atom,
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented by,
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -H- (SO 2) OR- (SO 2) R (R is synonymous with-OR),
R 2a or R 2b are bonded to each other with R 3a or R 3b, or may form a ring together with the carbon atoms to which they are bonded,
N 1、n2 and n 3 are each independently 1 or 2,
M is an integer of 0 or 1.
In addition, a method for removing a protecting group according to an aspect of the present invention includes: a step of bringing an amino group-containing compound having an N-terminal protected with a protecting group having a fluorene skeleton into contact with a capturing agent represented by the following formula (Z1) in an organic solvent, wherein a capturing agent is obtained by bonding a byproduct having an fulvene skeleton derived from the protecting group to the capturing agent; and
And separating the obtained capturing body from the organic solvent.
In the above-mentioned formula (Z1),
N is a nitrogen atom and is preferably a nitrogen atom,
H is a hydrogen atom and is preferably a hydrogen atom,
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented by,
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -H- (SO 2) OR- (SO 2) R (R is synonymous with-OR),
R 2a or R 2b are bonded to each other with R 3a or R 3b, or may form a ring together with the carbon atoms to which they are bonded,
N 1、n2 and n 3 are each independently 1 or 2,
M is an integer of 0 or 1.
Further, the remover according to one aspect of the present invention is a remover having a protecting group of fluorene skeleton, which includes a capturing agent represented by the following formula (Z1) and an alkaline deprotecting agent.
In the above-mentioned formula (Z1),
N is a nitrogen atom and is preferably a nitrogen atom,
H is a hydrogen atom and is preferably a hydrogen atom,
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented by,
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -H- (SO 2) OR- (SO 2) R (R is synonymous with-OR),
R 2a or R 2b are bonded to each other with R 3a or R 3b, or may form a ring together with the carbon atoms to which they are bonded,
N 1、n2 and n 3 are each independently 1 or 2,
M is an integer of 0 or 1.
Effects of the invention
According to an aspect of the present invention, there can be provided a benzyl compound which is easily separated and purified by a liquid-liquid layer separation operation after a peptide condensation reaction, in addition to an improvement in solubility in an organic solvent.
Furthermore, according to an aspect of the present invention, DBF can be easily captured and DBF-trap can be easily removed. Furthermore, the cyclic amine used in the present invention has low basicity, and thus can suppress unexpected deprotection of Fmoc group and suppress progress of side reaction.
Detailed Description
[ Embodiment 1]
[ Benzyl Compound ]
The benzyl compound according to embodiment 1 of the present invention is a benzyl compound (X1) represented by the following formula (X1):
m represents the number of substituents (- [ Q 1-R1-Q2-R2 ]). m is an integer of 2 or 3. When m is 2, the substituent (- [ Q 1-R1-Q2-R2 ]) is preferably present at the 3-and 5-positions or at the 2-and 4-positions. In this case, the two substituents (- [ Q 1-R1-Q2-R2 ]) are more preferably located at the 2-and 4-positions. When m is 3, it is preferably present in an adjacent position. In this case, three substituents (- [ Q 1-R1-Q2-R2 ]) are more preferably present at the 3-, 4-and 5-positions.
K represents the number of substituents (-R 3). k is an integer of 0 to (5-m). Specifically, when m is 2, k is an integer of 0 to 3, and when m is 3, k is an integer of 0 to 2.
M numbers of Q 1 and Q 2 each represent an oxygen atom.
X represents a hydroxyl group.
M R 1 are each independently alkylene. For example, R 1 is a straight-chain or branched alkylene group having 2 to 16 carbon atoms. The number of carbon atoms of the alkylene group is preferably 2 or more, more preferably 6 or more, further preferably 8 or more, further preferably 16 or less, more preferably 14 or less, further preferably 12 or less, from the viewpoint of improving the solubility of the peptide bonded to the benzyl compound (X1) of the present invention in an organic solvent. Specific examples of the alkylene group include ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, decamethylene, undecamethylene, dodecamethylene, tetradecamethylene, tridecamethylene, tetradecamethylene, pentadecamethylene, hexadecamethylene and the like.
M R 2 are each independently an alkyl group which may have a substituent, an aralkyl group which may have a substituent or an aryl group which may have a substituent. For example, R 2 is an alkyl group having 5 to 28 carbon atoms, an aralkyl group having 5 to 28 carbon atoms which may have a substituent, or an aryl group having 7 to 12 carbon atoms which may have a substituent.
In view of improving the solubility of the peptide bonded to the benzyl compound (X1) in the organic solvent in this embodiment, the number of carbon atoms of the alkyl group is preferably 5 or more, more preferably 6 or more, further preferably 7 or more, further preferably 28 or less, more preferably 24 or less, further preferably 22 or less.
Further, as the alkyl group, it is a linear alkyl group (i.e., an alkyl group having no branching), or an alkyl group having a total of 1 or 2 branching, specifically, a group represented by the following formula (XA) is preferable:
In the formula (XA) of the present invention,
* Indicating the bonding position to the adjacent Q 2. Hereinafter, the same description may be omitted. n 1 is an integer of 0 to 16, and n 2 is an integer of 0 to 16. n 1 is preferably an integer of 0 to 6, and n 2 is preferably an integer of 0 to 13.
R 2a、R2b、R2c、R2d and R 2e are each independently a hydrogen atom or an alkyl group. The alkyl group may have a substituent such as a halogen atom of fluorine, chlorine, bromine, iodine or the like. At least two or more of R 2a、R2b、R2c and R 2d are hydrogen atoms.
Specific examples of the group represented by the above formula (XA) include pentyl, octyl, isooctyl, nonyl, decyl, undecyl, dodecyl, 1-methyl-1-hexadecyl, 1-ethyl-1-heptadecyl, 1-propyl-1-decyl, 1-butyl-1-decyl, 2-methyl-1-dodecyl, 2-methyl-1-hexadecyl, 2-butyl-1-octyl, 2-butyl-1-dodecyl, 2-butyl-1-octadecyl, 2-hexyl-1-decyl, 2-hexyl-1-dodecyl, 2-heptyl-1-dodecyl, 2-octyl-1-tetradecyl, 2-decyl-1-tetradecyl, 2-dodecyl-1-hexadecyl, 3-methyl-1-tetradecyl, 4-ethyl-1-octyl, 6-methyl-1-heptyl, 9-methyl-1-dodecyl, 12-hexyl-1-dodecyl, 2-heptyl-1-tetradecyl and 2-dodecyl.
Preferred examples of the alkyl group include pentyl, octyl, isooctyl, nonyl, decyl, undecyl, dodecyl, 2-butyl-1-octyl, 2-hexyl-1-dodecyl, 2-octyl-1-dodecyl, 2-decyl-1-tetradecyl, and 2-dodecyl-1-hexadecyl.
The number of carbon atoms of the aralkyl group which may have a substituent is preferably 5 or more, more preferably 6 or more, further preferably 7 or more, and further preferably 28 or less, more preferably 24 or less, further preferably 22 or less, from the viewpoint of improving the solubility of the peptide bonded to the benzyl compound (X1) according to the present embodiment in an organic solvent. Further, as the aryl substituent, a substituent containing a halogen atom is preferable. Specific examples of the alkyl group include 6-phenyl-1-hexyl, 8-phenyl-1-octyl, 10-phenyl-1-decyl, and 12-phenyl-1-dodecyl.
The number of carbon atoms of the aryl group which may have a substituent is preferably 6 or more, more preferably 7 or more, further preferably 8 or more, and further preferably 16 or less, more preferably 14 or less, further preferably 12 or less, from the viewpoint of improving the solubility of the peptide bonded to the benzyl compound (X1) according to the present embodiment in an organic solvent. The substituent of the aryl group is preferably an alkyl group which may have a substituent, and more preferably an alkyl group containing a halogen atom. In other words, R 2 is preferably an aryl group having a substituent containing a halogen atom. Examples of the halogen atom include fluorine, chlorine and bromine, and fluorine is particularly preferable.
Specific examples of the aryl group include 3-trifluoromethylphenyl, 3, 5-bistrifluoromethylphenyl, 4-fluoro-3-trifluoromethylphenyl, 4-chloro-2-fluorophenyl, 2-isopropylphenyl, 2, 6-isopropylphenyl, 2-sec-butylphenyl, 5-isopropyl-2-methylphenyl and the like.
K R 3 are each independently a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. For example, R 3 is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, or a halogen atom. Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, and t-butyl, and among them, methyl is particularly preferred. Specific examples of the alkoxy group include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and tert-butoxy, and methoxy is particularly preferable. The halogen atom may be a fluorine atom, a chlorine atom or a bromine atom, and among these, a fluorine atom is particularly preferable.
Specific examples of benzyl Compounds
(Appropriate substituent (- [ Q 1-R1-Q2-R2 ]))
As the above substituent (- [ Q 1-R1-Q2-R2 ]), substituents represented by the following formulas (XB 1) to (XB 3) can be listed as preferable substituents in view of their usefulness. In the formulae (XB 1) to (XB 3), "" indicates a position bonded to a carbon atom constituting the benzene ring in the formula (X1).
(Suitable benzyl Compounds)
As the benzyl compound (X1) represented by the above formula (X1), compounds represented by the following formulas (X1A) to (X1D) can be listed as preferable compounds in view of their usefulness.
The benzyl compound represented by the formula (X1A) is a compound (3, 4, 5-tris (11- (3, 5-bis (trifluoromethyl) phenoxy) undecyloxy) phenyl) methanol) in which, in the benzyl compound represented by the formula (X1), three substituents represented by the above formula (XB 1) are substituted at the positions of the 3-position, 4-position and 5-position.
The benzyl compound represented by the formula (X1B) is a compound (2, 4-bis ((12- ((2-octyldodecyl) oxy) dodecyl) oxy) phenyl) methanol in which two substituents represented by the above formula (XB 2) are substituted at the positions of the 2-and 4-positions in the benzyl compound represented by the formula (X1).
The benzyl compound represented by the formula (X1C) is a compound (3, 4, 5-tris ((12- ((2-octyldodecyl) oxy) phenyl) methanol) in which three substituents represented by the above formula (XB 2) are substituted at the positions of the 3-position, 4-position and 5-position in the benzyl compound represented by the formula (X1).
The benzyl compound represented by the formula (X1D) is a compound (2, 4-bis ((12- ((2-decyltetradecyl) oxy) dodecyl) oxy) phenyl) methanol in which two substituents represented by the above formula (XB 3) are substituted at the positions of the 2-and 4-positions in the benzyl compound represented by the formula (X1).
[ Method for producing benzyl Compound ]
Next, a method for producing the benzyl compound (X1) will be described in detail. In the following description, a method for producing a benzyl compound (X1) in which R 2 in the formula (X1) is a substituent other than an aryl group, and a method for producing a benzyl compound (X1) in which R 2 in the formula (X1) is an aryl group will be described.
[1] Process for producing benzyl compound (X1) wherein R 2 is a substituent other than aryl
The method for producing the benzyl compound (X1) according to the present embodiment is not particularly limited. An example is described below. For example, the following methods can be cited: dialkyl bromide and alkyl alcohol are dissolved in a suitable solvent and heated in the presence of a base to obtain alkyl etherified monobromide (hereinafter also referred to as "step Xa 1"). The monobromide and the hydroxybenzoyl compound (including the hydroxybenzaldehyde compound and the hydroxybenzene compound) obtained are dissolved in a suitable solvent, and heated in the presence of a base such as potassium carbonate to obtain an alkyl etherified benzaldehyde compound or a phenyl ester compound (hereinafter also referred to as "step Xa 2"). Then, the alkyl etherified benzaldehyde compound or the phenyl ester compound is dissolved in an appropriate solvent, and the formyl group or the ester group is reduced with a reducing agent such as a metal hydride (hereinafter also referred to as "step Xa 3") to obtain a benzyl alcohol compound.
(Process Xa 1)
In the step Xa1, examples of the base used for the reaction between the dialkyl bromide and the alkyl alcohol include organic bases such as Lithium Diisopropylamide (LDA), lithium Hexamethyldisilazide (LHMDS), and sodium bis (trimethylsilyl) amide (NaHMDS), and inorganic bases such as sodium hydride (NaH) and lithium hydride (LiH). The amount of the base to be used is not particularly limited, but is preferably 1.0 mol or more and 10 mol or less, more preferably 1.0 mol or more and 5 mol or less per mol of the alkyl alcohol.
Examples of the solvent include hydrocarbons such as N-hexane and heptane, esters such as diisopropyl ether, tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), 4-Methyltetrahydrofuran (MTHP) and dioxane, amides such as Dimethylformamide (DMF) and dimethylacetamide, sulfones such as Dimethylsulfoxide (DMSO), lactams such as N-methylpyrrolidone, aromatic hydrocarbons such as toluene and xylene, and mixed solvents thereof. Among them, toluene is preferably used from the viewpoint of smooth progress of the reaction.
The amount of the solvent to be used is not particularly limited, but is preferably 5mL or more and 100mL or less, more preferably 10mL or more and 50mL or less, relative to 1g of the alkyl alcohol. When a mixed solvent is used, the total amount of the mixed solvent may be within the above range. Hereinafter, the same description may be omitted.
The reaction temperature is not particularly limited, and may be, for example, in the range of 70℃to 150 ℃. The reaction time is not particularly limited, and may be, for example, 1 to 24 hours.
(Process Xa 2)
In the step Xa2, as a base used when the alkyl etherified monobromide is reacted with the benzaldehyde compound or the phenyl ester compound, there may be mentioned organic bases such as Triethylamine (TEA), diisopropylethylamine (DIPEA), 1, 8-diazabicyclo [5.4.0] undec-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), bis (trimethylsilyl) sodium amide (NaHMDS), inorganic bases such as sodium carbonate (Na 2CO3), sodium bicarbonate (NaHCO 3), potassium carbonate (K 2CO3), cesium carbonate (Cs 2CO3), sodium hydride (NaH), and the like. Among them, K 2CO3 is preferably used from the viewpoint of smooth progress of the reaction. The amount of the base to be used is not particularly limited, but is preferably 1 to 10 moles, more preferably 2 to 8 moles, per mole of the benzaldehyde compound or phenyl ester compound.
As the solvent, the solvents described in the procedure Xa1 can be used. As the solvent, DMF is preferably used, or a mixed solvent of DMF and CPME or MTHP is more preferably used, from the viewpoint of smooth progress of the reaction. The amount of the solvent to be used is not particularly limited, but is preferably 30mL or more and 200mL or less, more preferably 50mL or more and 180mL or less, based on 1g of the benzaldehyde compound or phenyl ester compound.
The reaction temperature is not particularly limited, and may be, for example, 50℃to 150 ℃. The reaction time is not particularly limited, and may be, for example, 1 to 24 hours.
(Process Xa 3)
In the above step Xa3, examples of the reducing agent used in the reduction of the formyl group of the alkyl etherified benzaldehyde compound or the ester group of the phenyl ester compound to obtain the benzyl alcohol compound include sodium borohydride, lithium triethylborohydride, lithium aluminum hydride, sodium bis (2-methoxyethoxy) aluminum hydride, and diisobutylaluminum hydride. The reducing agent used for reducing the formyl group of the benzaldehyde compound is preferably sodium borohydride, and the reducing agent used for reducing the ester group of the phenyl ester compound is preferably sodium borohydride, lithium triethylborohydride, lithium aluminum hydride, sodium bis (2-methoxyethoxy) aluminum hydride, or diisobutylaluminum hydride. In addition, when sodium borohydride is used as the reducing agent, for example, in order to increase the reducing power of the reducing agent, it is preferable to react with iodine, sulfuric acid, boron trifluoroacetate (BF 3·Et2 O), or the like in a coexisting manner.
Examples of the solvent include hydrocarbons such as n-hexane and heptane, alcohols such as methanol and ethanol, esters such as diethyl ether, isopropyl ether, THF, CPME, MTHP and dioxane, aromatic hydrocarbons such as toluene and xylene, and mixed solvents thereof. As the solvent used in the reduction of the formyl group, a mixed solvent of an alcohol and an ester is preferably used in view of the smooth progress of the reaction. The amount of the solvent to be used is not particularly limited, but is preferably 1mL or more and 100mL or less, more preferably 5mL or more and 50mL or less, based on 1g of the benzaldehyde compound. When a mixed solvent of an alcohol and an ester is used, it is preferable to use 1mL or more and 10mL or less of the ester with respect to 1mL of the alcohol.
Examples of the solvent used for reducing the ester group include esters such as diethyl ether, isopropyl ether, THF, CPME, MTHP, and dioxane, aromatic hydrocarbons such as toluene and xylene, and mixed solvents thereof. As the solvent used in the reduction of the methyl ester group, THF, CPME, MTHP is preferably used in view of smooth progress of the reaction. The amount of the solvent to be used is not particularly limited, but is preferably 1mL or more and 100mL or less, more preferably 5mL or more and 50mL or less, based on 1g of the ester compound.
The reaction temperature is not particularly limited, and may be carried out, for example, in the range of-10℃to 90 ℃. The reaction time is not particularly limited, and may be, for example, 1 to 24 hours.
[2] Process for producing benzyl compound (X1) wherein R 2 in formula (X1) is aryl
The method for producing the benzyl compound having an aryl group in R 2 of the formula (X1) is not particularly limited. An example is described below. For example, the following methods can be cited: the bromoalkyl alcohol and the phenol compound having a substituent are heated in the presence of a base to obtain an aryl etherified bromoalkyl alcohol compound (hereinafter also referred to as "step Xb 1"). After the aryl etherified bromoalkyl alcohol is obtained, the hydroxyl group is substituted with a bromine atom to obtain a bromoalkylaryl ether compound (hereinafter also referred to as "step Xb 2").
The obtained bromoalkylaryl ether compound and hydroxybenzaldehyde compound or hydroxyphenylester compound are heated in the presence of a base such as potassium carbonate to obtain an alkyl etherified benzaldehyde compound or phenylester compound (hereinafter also referred to as "step Xb 3"). After obtaining an alkyl etherified benzaldehyde compound or a phenyl ester compound, the compound is dissolved in an appropriate solvent, and a formyl group or an ester group is reduced with a reducing agent (hereinafter also referred to as "step Xb 4") to obtain benzyl alcohol.
(Process Xb 1)
In the step Xb1, examples of the base used in the reaction of the bromoalkyl alcohol and the phenol compound having a substituent include organic bases such as TEA, DIPEA, DBU, DBN, DABCO, pyridine, imidazole and DMAP, LDA, naOAc, meONa, meOK, LHMDS, naHMDS, and inorganic bases such as Na 2CO3、NaHCO3,K2CO3、Cs2CO3 and NaH. Among them, K 2CO3 is preferably used as a base from the viewpoint of smooth progress of the reaction. The amount of the base to be used is not particularly limited, but is preferably 1 to 10 moles, more preferably 1 to 5 moles, based on 1 mole of the phenol compound having a substituent.
As the solvent, the solvents described in the procedure Xa1 can be used. DMF is preferably used as the solvent from the viewpoint of smooth progress of the reaction. The amount of the solvent to be used is not particularly limited, but is preferably 2mL or more and 100mL or less, more preferably 4mL or more and 50mL or less, based on 1g of the phenol compound having a substituent.
The reaction temperature is not particularly limited, and may be, for example, 40℃to 150 ℃. The reaction time is not particularly limited, and may be, for example, 1 to 24 hours.
(Process Xb 2)
In the step Xb2, examples of the reagent used in the reaction for converting the hydroxyl group of the aryl etherified bromoalkyl alcohol into a bromine atom include a reagent containing triphenylphosphine and carbon tetrabromide, a reagent containing hydrobromic acid, and the like. The amount of the reagent to be used is not particularly limited, but is preferably from 0.5 to 10 mol, more preferably from 1 to 5mol, based on 1mol of the aryl etherified bromoalkyl alcohol.
The solvent may be any of the solvents described in step Xa1, halogenated hydrocarbons such as methylene chloride and chloroform, and a mixed solvent thereof. As the solvent, halogenated hydrocarbons are preferably used from the viewpoint of smooth progress of the reaction. The amount of the solvent to be used is not particularly limited, but is preferably 0.5mL or more and 100mL or less, more preferably 1mL or more and 50mL or less, based on 1g of the aryl etherified bromoalkyl alcohol.
The reaction temperature is not particularly limited, and may be, for example, 20℃to 150 ℃. The reaction time is not particularly limited, and may be, for example, 1 to 24 hours.
(Process Xb 3)
In the above step Xb3, as the base used in the reaction of the bromoalkylaryl ether and the benzaldehyde compound or the phenyl ester compound, the base described in the step Xb1 can be used. In step X3b, the same base as in step X1b may be used, or a different base may be used. However, from the viewpoint of improving the production efficiency and reducing the cost, the same base as in step X1b is preferably used. The amount of the base to be used is not particularly limited, but is preferably 1 to 50 mol, more preferably 3 to 30 mol, based on 1 mol of the benzaldehyde compound or phenyl ester compound.
As the solvent, the solvents described in the procedure Xa1 can be used. DMF is preferably used as the solvent from the viewpoint of smooth progress of the reaction. The amount of the solvent to be used is not particularly limited, but is preferably 30mL or more and 200mL or less, more preferably 50mL or more and 180mL or less, based on 1g of the phenyl ester compound.
The reaction temperature is not particularly limited, and may be, for example, 40℃to 150 ℃. The reaction time is not particularly limited, and may be, for example, 1 to 24 hours.
(Process Xb 4)
As the reducing agent used in the reduction of the formyl group of the alkyl etherified benzaldehyde compound or the ester group of the phenyl ester compound to obtain the benzyl alcohol compound, the reducing agent used in the above-mentioned step Xa3 can be used.
Examples of the solvent include hydrocarbons such as n-hexane and heptane, alcohols such as methanol and ethanol, esters such as diethyl ether, isopropyl ether, THF, CPME, MTHP and dioxane, aromatic hydrocarbons such as toluene and xylene, and mixed solvents thereof.
The reaction temperature is not particularly limited, and may be carried out, for example, in the range of-10℃to 90 ℃. The reaction time is not particularly limited, and may be, for example, 1 to 24 hours.
[ Peptide Synthesis ]
The method for synthesizing a peptide using the benzyl compound (X1) of the present invention as a protecting group for the C-terminal end of an amino acid is, for example, a method comprising the following steps (X1) to (X5). The peptide synthesis method makes the purification process easier because the C-terminal protected peptide obtained in each condensation process can be isolated by liquid-liquid separation.
Step (X1) a step of dissolving the benzyl compound (X1) of the present invention in a soluble solvent (dissolution step),
Step (X2) a step of condensing the benzyl compound (X1) according to the present embodiment dissolved in the solvent obtained in the above step with a reaction substrate (condensation reaction step),
Step (X3) a step of adding a base to the reaction solvent containing the condensate obtained as described above to capture (remove) the amino acid active ester which is an unnecessary substance for the reaction and deprotect the N-terminal protecting group of the peptide, and capturing (removing) by-products derived from the protecting group with a base (deprotection and removal reaction step),
Step (X4) of adding an acidic aqueous solution to the reaction solution containing the condensate and the scavenged product obtained in the above step, washing and layering, removing the scavenged product and the substances (condensing agent, activator, alkali) unnecessary for the reaction to the aqueous layer (layering step), and
Step (X5) a step of removing the benzyl compound (X1) and the protecting group of the side chain of the peptide according to the present embodiment from the C-terminal end of the peptide and purifying the resulting product to obtain the target peptide (deprotection and purification step).
Hereinafter, each step will be described separately. In the following description, introduction of N (N represents an amino group at the α -position of an amino acid) -9-fluorenylmethoxycarbonyl (N-Fmoc) protected amino acid into a benzyl compound (X1) (hereinafter also referred to as "tag X") according to the present embodiment, and condensation reaction of a tag-protected peptide with the N-Fmoc protected amino acid are described as examples. The N-Fmoc protected amino acid used may also have protecting groups on the side chains. The Fmoc group is exemplified as the protecting group at the N-terminal end of the amino acid, but the protecting group of the amino acid is not limited thereto. Examples thereof include benzyloxycarbonyl (Cbz group), t-butyloxycarbonyl (Boc group), and allyloxycarbonyl (Alloc group).
[ Process (X1) (dissolution Process) ]
This step is a step of dissolving the tag X in a soluble solvent.
As the soluble solvent, an organic solvent commonly used in peptide synthesis can be used for the reaction. Examples thereof include diethyl ether, THF, 2-methyltetrahydrofuran, 1, 4-dioxane, methyl tert-butyl ether, CPME, MTHP and other esters, ethyl acetate, isopropyl acetate and other acetates, chloroform, methylene chloride and other halogenated hydrocarbons, toluene, xylene and other aromatic hydrocarbons, n-hexane, heptane, cyclohexane and other hydrocarbons. From the viewpoint of good reactivity and liquid separation and suitability for industrial use, methyl tert-butyl ether, CPME, MTHP, isopropyl acetate, chloroform, toluene are preferable, CPME, MTHP, isopropyl acetate, toluene are more preferable, and CPME and MTHP are particularly preferable.
In order to improve the solubility of the substrate during the reaction, to improve the solubility of the unreacted materials and by-products in the aqueous layer during the extraction, or to improve the liquid separation, it is preferable to use a polar solvent such as DMF, dimethylacetamide, DMSO, sulfolane, N-methylpyrrolidone, N' -Dimethylpropylurea (DMPU), acetonitrile or the like in a suitable ratio in the above soluble solvent.
[ Step (X2) (condensation reaction step) ]
The step is a step of introducing an N-Fmoc-protected amino acid into the tag X dissolved in the soluble solvent obtained in the step (X1) to perform an esterification reaction, and introducing an N-Fmoc-protected amino acid into the tag X-protected peptide to perform an amidation reaction.
The amount of the N-Fmoc-protected amino acid to be used is 1 to 4 moles, preferably 1 to 2 moles, particularly preferably 1.05 to 1.3 moles, based on 1 mole of the tag X.
In the reaction of introducing an N-Fmoc protected amino acid into the tag X, a condensing agent is added under the catalysis of Dimethylaminopyridine (DMAP) in a solvent which does not affect the reaction to form an ester bond.
In addition, in the condensation reaction of the tag X-protected peptide and the N-Fmoc protected amino acid, a condensing agent and an activating agent are added to a solvent which does not affect the reaction, thereby forming an amide bond.
The condensing agent is not limited as long as the reaction proceeds, and condensing agents commonly used in peptide synthesis can be used.
For example, the number of the cells to be processed, 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine ammonium chloride (DMT-MM), O- (benzotriazol-1-yl) -1, 3-tetramethyluronium Hexafluorophosphate (HBTU), O- (7-azabenzotriazol-1-yl) -1, 3-tetramethyluronium Hexafluorophosphate (HATU), O- (6-benzotriazol-1-yl) -1, 3-tetramethyluronium hexafluorophosphate (HBTU (6-Cl)), O- (benzotriazol-1-yl) -1, 3-tetramethyluronium tetrafluoroborate (TBTU) O- (6-Chloropropanacil-1-yl) -1, 3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholino-carbonium hexafluorophosphate (COMU), diisopropylcarbodiimide (DIPCI), dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI. HCl), preferably DMT-MM, HBTU, HATU, COMU, EDCI or EDCI HCl condensing agent, the protecting peptide is usually used in an amount of 1 to 4 equivalents, preferably 1 to 2 equivalents, more preferably 1.05 to 1.5 equivalents, and even more preferably 1.05 to 1.3 equivalents, relative to the tag X or the tag X.
In the condensation reaction step, an activator is preferably added to promote the reaction and suppress side reactions such as racemization. The activator is a reagent which, when it is present with a condensing agent, leads an amino acid to a corresponding active ester, symmetrical acid anhydride or the like, and facilitates the formation of a peptide bond (amide bond). As the activator, an activator commonly used in peptide synthesis can be used, and the present invention is not particularly limited, and examples thereof include 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), ethyl 1-hydroxy-1H-1, 2, 3-triazole-4-carboxylate (HOCt), 3-hydroxy-1, 2, 3-benzotriazin-4 (3H) -one (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornene-2, 3-dicarboximide (HONb), pentafluorophenol, cyano (hydroxyimino) ethyl acetate (Oxyma) and the like, and HOBt, HOAt, HOCt, HOOBt, HONb, HOSu, oxyma are preferable. The amount of the activator to be used is usually 0.1 to 2 equivalents, preferably 0.2 to 1.5 equivalents, more preferably 0.3 to 1.0 equivalents, based on the tag X protecting peptide.
The solvent used in the condensation reaction step may be any solvent commonly used in peptide synthesis, and examples thereof include, but are not limited to, the aforementioned soluble solvents or mixed solvents of soluble solvents and polar solvents.
The amount of the solvent to be used is not particularly limited as long as the reaction proceeds, and the concentration of the dissolved tag X protecting peptide or the like is usually 0.1 mM-1M, preferably 1 mM-0.5M.
As the reaction temperature, the present invention can use a temperature usually used in peptide synthesis, for example, usually in the range of-20 to 40℃and preferably in the range of 0 to 30 ℃. The reaction time is usually 0.5 to 30 hours (condensation time of one residue).
[ Procedure (X3) (deprotection and removal reaction procedure) ]
This step is to add a first base to the reaction solvent after the condensation reaction step of the amino acid, thereby capturing (scavenging) the unreacted amino acid active ester to form a capturing body and inactivating it. Further by adding the first base and the second base, fmoc group removal reaction of the N-Fmoc protected peptide is advanced, and a trap is formed and deactivated from the first base for the by-product dibenzofulvene from Fmoc group.
The amount of the first base used for removing the unreacted amino acid active ester is not particularly limited, but is usually 1 to 5 equivalents, preferably 1 to 3 equivalents, relative to the remaining amino acid equivalents.
The amount of the second base used for Fmoc group deprotection of the N-Fmoc protected peptide is preferably 1to 12 equivalents, more preferably 2to 10 equivalents, particularly preferably 3 to 8 equivalents, relative to the Fmoc groups present in the reaction system.
The amount of the first base used for removing dibenzofulvene from the Fmoc group is preferably 5to 50 equivalents, more preferably 8 to 40 equivalents, and particularly preferably 10 to 35 equivalents relative to the Fmoc group present in the reaction system.
[ Procedure (X4) (layering procedure) ]
The step is a step of adding an acidic aqueous solution to the solution of the step (X3) to neutralize the solution, and further adding an acidic solution to remove the first base trap and the substances (condensing agent, activator, base) unnecessary for the reaction to the aqueous layer. The amino acid active ester and dibenzofulvene scavenged by the first base can be readily removed to the aqueous layer by acid washing.
The acid used in the neutralization is not particularly limited as long as it can neutralize the alkali in the reaction solution, and examples thereof include aqueous solutions of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and the like. For example, when hydrochloric acid is used, 1M to 12M, preferably 3M to 12M, and more preferably 5M to 12M hydrochloric acid is used. The neutralization means that the pH of the reaction solution becomes neutral, or the pH may be 7.0 or less.
Further adding an acidic aqueous solution to the acid-neutralized reaction solution, washing, separating the solution, removing the aqueous layer, and recovering the organic layer.
The acidic aqueous solution to be used is not particularly limited, and examples thereof include aqueous hydrochloric acid, aqueous dilute sulfuric acid, aqueous phosphoric acid, and aqueous acetic acid, and aqueous hydrochloric acid is preferable. The pH of the acidic aqueous solution is 1 to 5, preferably 1 to 4, more preferably 1 to 3.
The amount of the acidic aqueous solution to be used for the washing is not particularly limited as long as it has a washing effect, and is preferably 0.1 to 4 times, more preferably 0.3 to 3 times, and still more preferably 0.5 to 2 times, the amount of the reaction solution.
The number of times of washing, separating and discarding the aqueous layer is not particularly limited, and may be one time or a plurality of times. The number of times may be appropriately selected depending on the kind of the compound in the reaction system, the amount of the unnecessary substance, and the like.
The temperature at which the washing is carried out is not particularly limited, but is usually 10 to 50 ℃, preferably 15 to 45 ℃, more preferably 20 to 40 ℃.
This step basically removes the first alkali trap and the unnecessary substances by the acidic aqueous solution, but other washing steps may be added in addition to the washing with the acidic aqueous solution. For example, a weak alkaline washing or a brine washing may be cited.
Examples of the weakly alkaline aqueous solution include an aqueous sodium bicarbonate solution, an aqueous sodium carbonate solution, and an aqueous potassium carbonate solution having a pH of 8 to 12.
As the brine, 5wt% to saturated brine can be mentioned.
[ Procedure (X5) (deprotection and purification procedure) ]
This step is a step of removing the protecting group of the tag X and the side chain of the peptide from the C-terminal of the peptide to obtain the target peptide.
The method of removing the protecting group of the tag X and the side chain of the peptide from the C-terminal of the peptide is not particularly limited, and any known deprotection method may be used, and an acid treatment method is preferably used. Deprotection may be performed, for example, using trifluoroacetic acid (TFA).
Depending on the amino acid sequence, TFA may also be combined with water, thioanisole, 1, 2-ethanedithiol, phenol, triisopropylsilane, and the like to form a suitable composition.
The deprotected peptide may be isolated and purified according to purification methods commonly used in peptide synthesis. For example, the target peptide may be isolated and purified by extractive washing, crystallization, chromatography.
[ Embodiment 2]
[ Benzyl Compound ]
The benzyl compound according to embodiment 2 of the present invention is a benzyl compound (Y1) represented by the following formula (Y1):
in the formula (Y1), the amino acid sequence of the formula (I),
M Q's each represent an oxygen atom.
The total number of carbon atoms of the benzyl compound represented by the formula (Y1) is preferably 30 to 80, more preferably 40 to 60.
(R1)
When synthesizing a peptide by a liquid phase labeling method using a benzyl compound represented by the above formula (Y1), the inventors found that, when peptide synthesis is performed using a benzyl compound having particularly high hydrophobicity, liquid separation defects (long time is required to re-separate an organic layer and an aqueous layer after stirring the organic layer and the aqueous layer) occur in a liquid separation operation involving a separation and purification step after completion of the reaction (refer to the "liquid separation step" described below) as the length of the peptide chain increases (refer to comparative examples Y3 and table Y4 described below).
It is assumed that the reason for this poor separation is that an emulsion (micelle structure) is formed when the organic layer and the aqueous layer containing the component of the highly hydrophobic benzyl compound bonded to the peptide are separated. Therefore, the hydrophobicity of the benzyl compound is preferably not too high from the viewpoint of performing the liquid separation operation satisfactorily. The hydrophobicity of the benzyl compound can be adjusted, for example, by the number of carbon atoms in R 1 and R 2.
In this embodiment, for example, the number of carbon atoms of m R 1 in the above formula (Y1) (hereinafter also referred to as "side chain carbon atoms") is preferably in the range of 24 to 84, more preferably 30 to 72, and particularly preferably 36 to 48. The total number of branches of each of the m R 1 is 1 or 2. In addition, the m R 1 are each alkyl.
That is, R 1 is an alkyl group having one or two total branches, preferably an alkyl group having one or two total branches and having 24 to 84 total carbon atoms, more preferably an alkyl group having one or two total branches and having 30 to 72 carbon atoms in a side chain, and particularly preferably an alkyl group having 36 to 48 carbon atoms in a side chain.
Among the above, R 1 is particularly preferably an alkyl group having one total number of branches, and the position of the branch is preferably 1 to 7 positions relative to Q, more preferably 1 to 4 positions relative to Q, and still more preferably 2 positions relative to Q. In addition, when R 1 has two branches, these two branches are present at a distance of 0 to 6 carbon atoms, preferably 0 to 3 carbon atoms.
The branched chain may be an alkyl group which may have a substituent, an aralkyl group which may have a substituent, or an aryl group which may have a substituent. Among them, the branched chain is preferably an alkyl group having 2 to 12 carbon atoms which may have a substituent, more preferably an alkyl group having 4 to 10 carbon atoms which may have a substituent. Examples of such substituents include halogen atoms such as fluorine and chlorine.
In view of the above, specifically, it is preferable that m R 1 are each independently a group represented by the following formula (YA):
In the formula (YA),
* Indicating the bonding location. Hereinafter, the same description may be omitted.
N 1 is an integer of 0 to 6, and n 2 is an integer of 0 to 6. Preferably, n 1 represents an integer of 0 to 3, and n 2 is an integer of 0 to 3. When n 1 is 1 or more, the repeating unit shown in parentheses with n 1 is an alkylene group, and when n 2 is 1 or more, the repeating unit shown in parentheses with n 2 is an alkylene group.
R 1a、R1b、R1c、R1d and R 1e are each independently a hydrogen atom or an alkyl group. The alkyl group may have a substituent such as a halogen atom such as fluorine or chlorine. At least two or more of R 1a、R1b、R1c and R 1d are hydrogen atoms. Here, R 1a、R1b、R1c and R 1d may both be hydrogen atoms, but preferably R 1a、R1b、R1c and R 1d are not both hydrogen atoms. Specific examples of the group represented by the above formula (YA) are the same as those of the group represented by the above formula (XA), and therefore, description thereof is omitted.
In particular, R 1 is preferably an organic radical having a branching which is present in the 2-position with respect to Q. That is, preferably, m R 1 are each independently a group represented by the following formula (YA'):
Here, the difference between the number of carbon atoms of R 1f and the number of carbon atoms of R 1g is preferably 2. Further, R 1f is preferably a linear alkyl group having 4 to 12 carbon atoms which may have a substituent, more preferably a linear alkyl group having 4 to 10 carbon atoms which may have a substituent, and most preferably a linear alkyl group having 6 carbon atoms which may have a substituent. Examples of such substituents include halogen atoms such as fluorine and chlorine.
R 1g is preferably a linear alkyl group having 6 to 14 carbon atoms which may have a substituent, more preferably a linear alkyl group having 6 to 12 carbon atoms which may have a substituent, and most preferably a linear alkyl group having 8 carbon atoms which may have a substituent. Examples of such substituents include halogen atoms such as fluorine, chlorine, bromine and iodine.
Specifically, preferable examples of the group represented by the above (YA') include 2-n-butyl-1-octyl, 2-hexyl-1-decyl, 2-octyl-1-dodecyl, 2-decyl-1-tetradecyl, and 2-dodecyl-1-hexadecyl.
(R2)
K R 2 are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an alkoxy group, an aryl group, an aralkyl group, or a halogen atom. Specific examples of k R 2 are the same as those described for k R 3 in embodiment mode 1. More preferably, k R 2 are hydrogen atoms.
X represents a hydroxyl group.
M represents an integer of 2 or 3.
K represents an integer of 0 to (5-m) inclusive,
At least one of m [ Q-R 1 ] is preferably substituted in the meta-position with respect to the substituent containing said X.
The benzyl compound represented by formula (Y1) is particularly preferably used as a tag in long-chain peptide synthesis. For example, the benzyl compound represented by the formula (Y1) is preferably used for synthesis of a peptide having 5 or more residues, more preferably for synthesis of a peptide having 7 or more residues, and even more preferably for synthesis of a peptide having 10 or more residues.
Specific examples of benzyl Compounds
(Appropriate substituent (- [ Q-R 1 ])
As the above substituent (- [ Q-R 1 ]), substituents represented by the following formulas (YB 1) to (YB 3) can be listed as preferred compounds in view of their usefulness. In the formulae (YB 1) to (YB 3), the "×" indicates the carbon atom bonding position constituting the benzene ring in the formula (Y1).
(Suitable benzyl Compounds)
As the benzyl compound (Y1) represented by the above formula (Y1), compounds represented by the following formulas (Y1A) to (Y1D) are listed as preferable compounds in view of their usefulness.
The benzyl compound represented by the formula (Y1A) is a compound (3, 4, 5-tris ((2-butyloctyl) oxy) phenyl) methanol in which three substituents represented by the above formula (YB 1) are substituted at the 3-position, 4-position and 5-position with respect to a hydroxyl group (-OH) in the benzyl compound represented by the formula (Y1).
The benzyl compound represented by the formula (Y1B) is a compound (3, 4, 5-tris ((2-hexyldecyl) oxy) phenyl) methanol in which three substituents represented by the above formula (YB 2) are substituted at the 3-position, 4-position and 5-position with respect to a hydroxyl group (-OH) in the benzyl compound represented by the formula (Y1).
The benzyl compound represented by the formula (Y1C) is a compound (3, 4, 5-tris ((2-decyltetradecyl) oxy) phenyl) methanol in which three substituents represented by the above formula (YB 3) are substituted at the 3-position, 4-position and 5-position with respect to a hydroxyl group (-OH) in the benzyl compound represented by the formula (Y1).
The benzyl compound represented by the formula (Y1D) is a compound (3, 5-bis ((2-decyltetradecyl) oxy) phenyl) methanol in which three substituents represented by the above formula (YB 3) are substituted at the 3-position and the 5-position with respect to the hydroxyl group (-OH) in the benzyl compound represented by the formula (Y1).
[ Method for producing benzyl Compound ]
Next, a method for producing the benzyl compound (Y1) will be described in detail.
The method for producing the benzyl compound (Y1) according to the present embodiment is not particularly limited. An example is described below. For example, the following methods can be cited: the alkyl halide and the hydroxyphenylester compound are dissolved in a suitable solvent, and heated in the presence of a base such as potassium carbonate to obtain an alkyl etherified phenylester compound (hereinafter also referred to as "step Ya 1"). The alkyl halide is a compound in which an alkyl terminal is bonded to a halogen atom. Examples of such halogen atoms include chlorine, bromine, and iodine, and bromine and iodine are preferable in view of reactivity, and bromine is more preferable in view of cost. That is, the alkyl halide is preferably alkyl bromide or alkyl iodide, and more preferably alkyl bromide. The alkyl halide may be a commercially available product, or may be obtained by halogenating a hydroxyl group corresponding to the alkyl halide starting material by a known method.
In this embodiment, an alkyl bromide and a hydroxyphenylester compound are dissolved in a suitable solvent, and heated in the presence of a base such as potassium carbonate to obtain an alkyl etherified phenylester compound. As the alkyl bromide, a commercially available product may be used, or a product obtained by halogenating a hydroxyl group corresponding to the alkyl bromide starting material by a known method may be used. Then, the alkyl etherified phenyl ester compound is dissolved in an appropriate solvent, and the ester group is reduced with a reducing agent such as a metal hydride (hereinafter also referred to as "step Ya 2") to obtain the benzyl alcohol compound.
(Process Ya 1)
Examples of the base used in the reaction of the alkyl bromide and the hydroxyphenylester compound in the step Ya1 include organic bases such as Triethylamine (TEA), diisopropylethylamine (DIPEA), 1, 8-diazabicyclo [5.4.0] undec-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), lithium Diisopropylamide (LDA), sodium acetate (NaOAc), sodium methoxide (MeONa), potassium methoxide (MeOK), lithium Hexamethyldisilazide (LHMDS), sodium bis (trimethylsilyl) amide (NaHMDS), and inorganic bases such as sodium carbonate (Na 2CO3), sodium bicarbonate (NaHCO 3), potassium carbonate (K 2CO3), cesium carbonate (Cs 2CO3) and sodium hydride (NaH). Among them, K 2CO3 is preferably used as a base from the viewpoint of smooth progress of the reaction. The amount of the base to be used is not particularly limited, but is preferably 1 to 10 moles, more preferably 2 to 8 moles, based on 1 mole of the hydroxyphenylester compound.
Examples of the solvent include hydrocarbons such as N-hexane and heptane, esters such as diisopropyl ether, tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), 4-Methyltetrahydrofuran (MTHP) and dioxane, amides such as Dimethylformamide (DMF) and dimethylacetamide, sulfones such as Dimethylsulfoxide (DMSO), lactams such as N-methylpyrrolidone, aromatic hydrocarbons such as toluene and xylene, and mixed solvents thereof. Among them, DMF or a mixed solvent of DMF and CPME is preferably used from the viewpoint of smooth progress of the reaction. The amount of the solvent to be used is not particularly limited, but is preferably 10mL or more and 200mL or less, more preferably 15mL or more and 150mL or less, relative to 1g of the hydroxyphenyl ester compound.
The reaction temperature is not particularly limited, and may be, for example, 50℃to 150 ℃. The reaction time is not particularly limited, and may be, for example, 1 to 24 hours.
(Process Ya 2)
In the step Ya2, as the reducing agent used in the reduction of the ester group of the alkyl etherified phenyl ester compound to obtain the benzyl alcohol compound, for example, a metal hydrogen compound may be used. The metal hydrogen compound is preferably a group 13 alkali metal hydride. Specifically, examples of the alkali metal borohydride compound include sodium borohydride, lithium borohydride, and lithium triethylborohydride, and aluminum hydride compounds include lithium aluminum hydride, sodium bis (2-methoxyethoxy) aluminum hydride, and diisobutylaluminum hydride. The amount of the reducing agent to be used is not particularly limited, but is preferably 1 to 10 moles, more preferably 2 to 5 moles, based on 1 mole of the alkyl etherified phenyl ester compound.
In addition, when sodium borohydride is used as the reducing agent, for example, in order to increase the reducing power of the reducing agent, it is preferable to react with iodine, sulfuric acid, boron trifluoroacetate (BF 3·Et2 O), or the like in a coexisting manner.
Examples of the solvent used for reducing the ester group of the alkyl etherified phenyl ester compound include esters such as diethyl ether, diisopropyl ether, THF, CPME, MTHP and dioxane, aromatic hydrocarbons such as toluene and xylene, and mixed solvents thereof. Among them, THF, CPME, MTHP is particularly preferable as a solvent used in reducing the methyl ester group from the viewpoint of smooth progress of the reaction. The amount of the solvent to be used is not particularly limited, but is preferably 1mL or more and 100mL or less, more preferably 5mL or more and 50mL or less, based on 1g of the ester compound.
The reaction temperature is not particularly limited, and may be carried out, for example, in the range of-10℃to 90 ℃. The reaction time is not particularly limited, and may be, for example, 1 to 24 hours.
[ Peptide Synthesis ]
The peptide synthesis method using the benzyl compound (Y1) of the present invention as a protecting group (i.e., tag) at the C-terminal end of an amino acid includes, for example, the following steps (Y1) to (Y6) as a method for producing the same. This peptide synthesis method facilitates the purification step, because the peptide with the protected C-terminal end of the amino acid (hereinafter also referred to as "C-terminal protected peptide") obtained in the condensation reaction step and the peptide extension step can be separated by liquid-liquid separation.
Step (Y1) a step of dissolving the benzyl compound (Y1) of the present invention in a soluble solvent (dissolution step),
A step (Y2) of adding a condensing agent and an activating agent to the soluble solvent obtained in the above step, condensing the benzyl compound (Y1) dissolved in the soluble solvent and a first amino acid whose N-terminal is protected by an N-terminal protecting group (hereinafter also referred to as "N-terminal protecting amino acid") to form a first condensate (condensation reaction step),
A step (Y3) of adding a first base to a soluble solvent (reaction solvent) containing the first condensate (the substance obtained by condensing the N-terminal protected amino acid and the benzyl compound (Y1) after removing the-OH group) obtained in the above step (Y2), removing (trapping) the remaining amino acid active ester (the substance obtained by reacting the carboxylic acid and the condensing agent at the C-terminal of the remaining amino acid in the step (Y2) and then reacting with the activator), further adding a first base and a second base to the soluble solvent (reaction solvent), deprotecting the N-terminal protecting group of the N-terminal protected amino acid constituting the first condensate, and removing the by-product (dibenzofulvene) from the N-terminal protecting group by the first base,
A step (Y4) of adding an acidic aqueous solution to a reaction solution (soluble solvent) containing the second condensate (condensate after removing the N-terminal protecting group from the first condensate), the capturing body (a substance bonded by a first base and an amino-active ester, and a substance bonded by a first base and a dibenzofuran) and a substance unnecessary for the reaction (by-product from the condensing agent, an activator, a base, and a water-soluble organic solvent) obtained in the above step (Y3), washing the mixture, separating the mixture into an aqueous layer and an organic layer, removing the capturing body and the substance unnecessary for the reaction to the aqueous layer, then deprotecting the N-terminal protecting group from the first condensate in the above step (Y3) in the organic layer separated from the aqueous layer (reaction solvent) to obtain a second condensate (i.e., a C-terminal protecting peptide),
The step (Y5) of adding the second amino acid having the protected N-terminal to the reaction solvent containing the second condensate obtained in the step (Y4), continuing the condensation reaction in the same manner as in the step (Y2) to condense the second condensate with the second amino acid having the protected N-terminal to obtain a third condensate, and performing the deprotection of the N-terminal protecting group from the third condensate in the same manner as in the steps (Y3) and (Y4) to obtain a fourth condensate (peptide extension step).
The step (Y5) includes the following sub-steps (Y5-1) to (Y5-3) repeatedly performed:
In the step (Y5-1), in the 2N-th condensate containing an N-terminal unprotected amino acid and a C-terminal protected amino acid of the benzyl compound (Y1), a total number (also referred to as "number of residues") of N (N is a natural number of 2 or more, preferably a natural number of 5 or more), the N-terminal protected N-th amino acid is condensed to produce a (2n+1) -th condensate containing the number of residues (n+1) -th amino acid,
Step (Y5-2) of removing the remaining amino acid active ester, followed by deprotection of the N-terminal protecting group from the (2n+1) th condensate, step of removing by-products from the N-terminal protecting group, and
Step (Y5-3) of adding an acidic aqueous solution to the soluble solvent, washing and separating the mixture, removing the capturing substance and the substance not required for the reaction to an aqueous layer, and obtaining the (2n+2) th condensate (deprotected condensate of the (2n+1) th condensate from which the N-terminal protecting group was removed) obtained in the above step (Y5-2) in an organic layer (reaction solvent), and
Step (Y6) a step of removing the benzyl compound (Y1) and the protecting group of the side chain of the peptide from the C-terminal of the peptide obtained in step (Y5) and purifying the resulting product to obtain the target peptide (deprotection and purification step).
The step (Y5) may be performed by repeating the steps (Y2) to (Y4). In other words, the process (Y2) may include the sub-process (Y5-1), the process (Y3) may include the sub-process (Y5-2), the process (Y4) may include the sub-process (Y5-3), and the processes (Y5) including the sub-processes (Y5-1) to (Y5-3) may be performed by repeating the processes (Y2) to (Y4).
Hereinafter, each step will be described. In the following description, a case where the benzyl compound (Y1) (hereinafter also referred to as "tag Y") is used instead of the benzyl compound (X1) according to embodiment 1 is described as an example.
[ Process (Y1) (dissolution Process) ]
This step is a step of dissolving the benzyl compound (Y1) in a soluble solvent. The step (Y1) can be performed in the same manner as the step (X1), and thus a detailed description thereof will be omitted.
[ Step (Y2) (condensation reaction step) ]
The step (B) is a step of introducing an N-Fmoc-protected amino acid into the tag Y dissolved in the soluble solvent obtained in the step (Y1), performing an esterification reaction, and introducing an N-Fmoc-protected amino acid into the tag Y-protected peptide to perform an amidation reaction. The step (Y2) can be performed in the same manner as the step (X2), and thus a detailed description thereof will be omitted.
[ Step (Y3) (deprotection and removal reaction step) ]
This step is to add a first base to the reaction solvent after the condensation reaction step of the amino acid, thereby capturing (scavenging) the unreacted amino acid active ester to form a capturing body and inactivating it. Further by adding the first base and the second base, fmoc-group removal of the N-Fmoc protected amino acid is advanced, and the capture body is also formed and deactivated from the first base for the by-product dibenzofulvene from Fmoc group. The step (Y3) can be performed in the same manner as the step (X3), and thus a detailed description thereof will be omitted.
[ Process (Y4) (liquid separation Process) ]
The step is a step of adding an acidic aqueous solution to the solution of the step (Y3) to neutralize the solution, and removing the first alkali capturing substance and the substances (condensing agent, activator, alkali, water-soluble organic solvent) unnecessary for the reaction from the solution to an aqueous layer by separation. The amino acid active ester and dibenzofulvene scavenged by the first base can be removed to the aqueous layer by acid washing.
The acid used in the neutralization is not particularly limited as long as it can neutralize the alkali in the reaction solution, and examples thereof include aqueous solutions of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and the like. For example, when hydrochloric acid is used, hydrochloric acid of 0.5M to 12M, preferably 1M to 12M, more preferably 1M to 6M is used. The neutralization means that the reaction solution is neutral in pH, and may be 7.0 or less in pH. In order to optimize the liquid separation, a ketone liquid separation promoting solvent such as acetone or methyl ethyl ketone may be further added. As a cause of low liquid separation, the inventors speculate that the tag Y-peptide molecules combine with each other to form a micelle structure due to hydrophobic interaction and hydrogen bonding, which makes liquid separation low. It is presumed that the addition of the above-mentioned liquid separation promoting solvent weakens hydrophobic interactions between side chains of the tag Y or hydrogen bonds between peptide molecules, suppresses formation of micelle structures, and improves liquid separation. In addition, the step of heating the solution of step (Y3) instead of or simultaneously with the step of adding the liquid separation promoting solvent is also effective for improving the liquid separation.
An acidic aqueous solution was further added to the reaction solution neutralized with the above acid and washed, followed by separation, removal of the aqueous layer, and recovery of the organic layer. The washing with the acidic aqueous solution can be performed in the same manner as in the above-described step (X4), and therefore, a detailed description thereof will be omitted.
In this step (Y4), the first alkali captured body and the unnecessary substance are substantially removed by the acidic aqueous solution, but if the unnecessary substance which is difficult to be removed by the acidic aqueous solution is present, another washing step may be added before and after the acidic aqueous solution washing. For example, alkaline aqueous solution washing or brine washing may be cited.
Examples of the alkaline aqueous solution include an aqueous sodium bicarbonate solution, an aqueous sodium carbonate solution, and an aqueous potassium carbonate solution having a pH of 8 to 13.
As the brine, 5wt% to saturated brine can be mentioned. After being washed by acidic aqueous solution, the solution is washed by alkaline aqueous solution, so that the pH value of the solution is neutral to weak alkaline. The aqueous alkaline solution may be the aqueous solution.
[ Process (Y5) (peptide extension Process) ]
The step is a step of adding an N-terminal protected amino acid to the reaction solvent containing the tag Y-protected peptide obtained in the above step, and repeating the above steps (Y5-1) to (Y5-3) to extend the peptide. However, in the condensation reaction in this step, DMAP used in the step (Y2) is not used, but an activator defined below is used.
The amount of the N-Fmoc-protected amino acid to be used is 1 to 4 moles, preferably 1 to 2 moles, particularly preferably 1.05 to 1.5 moles, based on 1 mole of the benzyl compound (Y1).
The condensing agent used in the peptide extension step may be the same as that described in step (Y2).
In order to promote the peptide condensation reaction and suppress side reactions such as racemization, an activator is preferably added. The activator herein refers to a reagent which, when it is present with a condensing agent, leads an amino acid to a corresponding active ester, symmetrical acid anhydride or the like, and facilitates the formation of a peptide bond (amide bond). As the activator, an activator commonly used in peptide synthesis can be used, and the present invention is not particularly limited, and examples thereof include 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), ethyl 1-hydroxy-1H-1, 2, 3-triazole-4-carboxylate (HOCt), 3-hydroxy-1, 2, 3-benzotriazin-4 (3H) -one (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornene-2, 3-dicarboximide (HONb), pentafluorophenol, cyano (hydroxyimino) ethyl acetate (Oxyma) and the like, and HOBt, HOAt, HOCt, HOOBt, HONb, HOSu, oxyma are preferable. The amount of the activator to be used is usually 0.1 to 2 equivalents, preferably 0.2 to 1.5 equivalents, more preferably 0.3 to 1.0 equivalents, based on the tag Y-protecting peptide.
The solvent used in the peptide extension step may be any solvent commonly used in peptide synthesis, and examples thereof include, but are not limited to, the soluble solvents described in step (Y1) and the mixed solvents of the soluble solvents and the polar solvents.
The amount of the solvent to be used is not particularly limited as long as the reaction proceeds, and the concentration of the dissolved tag Y protecting peptide or the like is usually 0.1 mM-1M, preferably 1 mM-0.5M.
The reaction temperature may be a temperature usually used for peptide synthesis in the present invention, and is, for example, usually in the range of-20 to 40℃and preferably in the range of 0 to 30 ℃. The reaction time is usually 0.5 to 30 hours (condensation time of one residue).
[ Step (Y6) (deprotection and purification step) ]
This step is a step of removing the benzyl compound (Y1) and the protecting group of the peptide side chain from the C-terminal end of the peptide to obtain the target peptide. The step (Y6) can be performed in the same manner as the step (X5), and thus a detailed description thereof will be omitted.
[ Embodiment 3]
Embodiment 3 according to the present invention will be described below. The following embodiments are examples of suitable embodiments for carrying out the present invention, and although some portions describe examples of technical preferred technical matters, the technical scope of the present invention is not limited to these specific aspects.
For convenience of explanation, the following will be described in detail with respect to the order of < 1 > a method for removing a protecting group from an amino group-containing compound having an N-terminal protected with a protecting group having a fluorene skeleton, < 2 > a method for producing a peptide including the protecting group removing step, and < 3 > a removing agent for the protecting group.
Method for removing protecting group with the number of < 1>
The method for removing a protecting group according to the present embodiment includes: a step of bringing an amino group-containing compound having an N-terminal protected with a protecting group having a fluorene skeleton into contact with a capturing agent in an organic solvent, a step of bonding a capturing agent to a byproduct having a fulvene skeleton derived from the protecting group, and a step of separating the capturing agent obtained from the organic solvent.
(Amino group-containing Compound)
Amino group-containing compounds refer to compounds having a primary or secondary amino group. Amino group-containing compounds include, for example, single amino acids, peptides formed by peptide bonding of multiple amino acids, and the like.
(Protecting group having fluorene skeleton)
The protecting group having a fluorene skeleton is a group that is bonded to a nitrogen atom in an amino group at the N-terminus of an amino group-containing compound to protect the N-terminus of the amino group-containing compound. The protecting group is a monovalent protecting group having a fluorene structure represented by the following formula (Z2).
In the formula (Z2), R 4 is an alkoxycarbonyl group having 1 to 6 carbon atoms which may have a substituent. R 5a～R5d and R 6a～R6d are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may have a substituent, an alkoxy group having 1 to 6 carbon atoms which may have a substituent, a sulfone group which may have a substituent, or a sulfo group which may have a substituent. Examples of the substituent include a halogen atom and an alkyl group having 1 to 3 carbon atoms. "x" means the position of bonding to the amino group at the N-terminus of an amino-containing compound.
R 4 is preferably an alkoxycarbonyl group having 1 to 3 carbon atoms, and more preferably a methoxycarbonyl group. R 5a～R5d and R 6a～R6d are each preferably a hydrogen atom.
That is, as the protecting group having a fluorene skeleton represented by the above formula (Z2), 9-fluorenylmethoxycarbonyl (Fmoc group) may be cited as a suitable example in view of its usefulness.
An amino group-containing compound whose N-terminal is protected by a protecting group having a fluorene skeleton means a compound in which at least one of a primary amino group or a secondary amino group that the amino group-containing compound has is protected by the protecting group having a fluorene skeleton.
(Capture agent)
The capturing agent is a reactant that is bonded to a protecting group that has been deprotected from the amino group-containing compound to capture the protecting group, thereby producing a capturing body (reference formula (Z3)). The capturing agent has a function of capturing a deprotected protecting group and also has a function of deprotecting the protecting group from an amino group-containing compound protected by the protecting group. The capturing agent is a compound represented by the following formula (Z1). That is, the capturing agent is at least one compound selected from the group consisting of a cyclic amine containing one nitrogen atom (reference formula (Z1A)), and a hydrochloride containing the cyclic amine (reference formula (Z1B)).
In the formula (Z1), N is a nitrogen atom. H is a hydrogen atom.
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented. X is preferably-O-or- (SO 2) -. By including an oxygen atom in X, the basicity is reduced, and thus side reactions can be suppressed. X is most preferably-O-.
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently a monovalent group represented by H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -H, - (SO 2) OR- (SO 2) R (R is synonymous with-OR). In addition, R 2a or R 2b are bonded to each other with R 3a or R 3b, and may form a ring together with the carbon atoms to which they are bonded.
N 1 R 1a、n1 R 1b、n2 R 2a and n 2 R 2b, the preferred bond to the nitrogen atom and the adjacent carbon atom is a hydrogen atom. this is because the stability of the capture agent is improved. In addition, in the case of the optical fiber, n 1 pairs of R 1a and R 1b、n2 pairs of R 2a and R 2b and n 3 pairs of R 3a and R 3b, Preferably, one pair is a hydrogen atom. In the best case of the circumstances, n 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each a hydrogen atom.
N 1、n2 and n 3 are each independently 1 or 2. The sum of n 1、n2 and n 3 is preferably 3 or 4. That is, as the capturing agent represented by the formula (Z1), it is preferable to be represented by the following formula (Z1 a) or formula (Z1 b). The capturing agent represented by the formula (Z1 a) is a compound in which the sum of n 1、n2 and n 3 in the formula (Z1) is 3. The capturing agent represented by the formula (Z1 b) is a compound in which the sum of n 1、n2 and n 3 in the formula (Z1) is 4. m is an integer of 0 or 1.
,N、H、X、R1a、R1b、R2a、R2b、R3a、R3b、n1、n2、n3 And m in the above formulas (Z1 a) and (Z1 b) are synonymous with those in formula (Z1).
When m is 0, the capturing agent is a cyclic amine containing one nitrogen atom represented by the following formula (Z1A). When m is 1, the capturing agent is a hydrochloride represented by the following formula (Z1B). In addition, preferably, m is 0.
In the above formulas (Z1A) and (Z1B), N, H, X, R 1a、R1b、R2a、R2b、R3a、R3b、n1、n2, and n 3 are synonymous with those in formula (Z1).
That is, the capturing agent is a cyclic amine containing at least one element selected from the group consisting of an oxygen element and a sulfur element. The amine is preferably water soluble. In addition, the capture agent is preferably a primary or secondary amine.
(Suitable Capture agent)
The capturing agent represented by the above formula (Z1) is preferably a cyclic amine having one amino group. The capturing agent is, for example, at least one selected from the group consisting of morpholine, piperidine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide, preferably at least one selected from the group consisting of morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide, more preferably at least one selected from the group consisting of morpholine, 3-hydroxypiperidine and 4-hydroxypiperidine, most preferably morpholine.
The amount of the capturing agent to be added is 5 to 100 equivalents, preferably 5 to 50 equivalents, more preferably 10 to 30 equivalents, relative to the amount of the protecting group present in the reaction system. If the addition amount of the capturing agent is less than this range, capturing of a by-product having a fulvene skeleton generated by deprotection reaction of a protecting group having a fluorene skeleton becomes insufficient, and impurities become difficult to remove by acidic liquid separation washing. If the addition amount of the trapping agent is larger than this range, the trapping agent remains in the organic layer during the acidic liquid separation washing, and there is a risk of side reactions.
(Organic solvent)
The contacting of the amino group-containing compound and the capturing agent represented by the above formula (Z1) is performed in an organic solvent. The organic solvent is preferably the same as the reaction solvent used in the peptide extension (synthesis) reaction in the liquid phase labeling method described below. This is to ensure that the removal of the protecting group and the peptide extension do not adversely affect each other when the peptide extension reaction is repeated in sequence, and also to facilitate handling. As the organic solvent, the same solvent as the soluble solvent described above in the step (X1) can be used, and thus a detailed description thereof will be omitted.
(Contact method)
The method of contacting the components is not particularly limited. For example, the components may be mixed in a reaction vessel equipped with a stirring mechanism. By mixing the components, the amino group-containing compound and the capturing agent can be brought into contact. The order in which the components are mixed is not particularly limited. For example, after synthesizing an amino group-containing compound in an organic solvent, a capturing agent may be mixed in the organic solvent (hereinafter also referred to as "reaction solution") containing the amino group-containing compound.
[ Deprotection agent ]
In order to promote the deprotection reaction of the protecting group, a deprotection agent may be further added. The amino group-containing compound and the deprotecting agent may be contacted by mixing the deprotecting agent. The deprotecting agent includes at least one base selected from the group consisting of organic bases such as1, 8-diazabicyclo [5.4.0] -7-undecene (DBU), 1, 5-diazabicyclo [4.3.0] -5-nonene (DBN), 1, 4-diazabicyclo [2.2.2] octane (DABCO) triethylamine and tributylamine, and inorganic bases such as potassium tert-butoxide and sodium tert-butoxide, more preferably 1, 8-diazabicyclo [5.4.0] -7-undecene (DBU), 1, 5-diazabicyclo [4.3.0] -5-nonene (DBN), and 1, 4-diazabicyclo [2.2.2] octane (DABCO), and still more preferably 1, 8-diazabicyclo [5.4.0] -7-undecene (DBU).
The amount of the deprotection agent to be added in this step is preferably 1 to 12 equivalents, more preferably 2 to 10 equivalents, and particularly preferably 3 to 8 equivalents, based on the protecting group present in the reaction system.
The order of adding the deprotection agent is not particularly limited. For example, the capturing agent may be mixed into the reaction solution, or the deprotecting agent may be mixed into the reaction solution before the capturing agent is mixed into the reaction solution.
(Capture body)
The capture body represented by the following formula (Z3) is obtained by contacting an amino group-containing compound with a capture agent represented by the above formula (Z1). The capturing body is formed by bonding a byproduct having a fulvene skeleton represented by the following formula (Z2') and a capturing agent represented by the above formula (Z1).
The by-product represented by the following formula (Z2') is produced by deprotection of a protecting group represented by the formula (Z2).
,N、H、X、R1a、R1b、R2a、R2b、R3a、R3b、R5a、R5b、R5c、R5d、R6a、R6b、R6c、R6d、n1、n2 And n 3 in the above formulae (Z2') and (Z3) are synonymous with those in the formulae (Z1) and (Z2).
(Capture separation step)
Next, a step of separating the obtained capture bodies, that is, a step of taking out the capture bodies from the reaction solution will be described. The method shown below is one example of a method for separating a capturing body, and is not limited to the following method.
For example, an acidic aqueous solution is added to the reaction solution to neutralize, and the capturing body is induced to an aqueous layer by separation. The capture bodies may be induced to the aqueous layer by acid washing, and the capture bodies separated from the reaction solution.
The acid used in the neutralization is not particularly limited as long as it can neutralize the alkali in the reaction solution, and examples thereof include aqueous solutions of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and the like. For example, when hydrochloric acid is used, 0.5M ("M" means mol/L, and the same explanation is omitted below) to 12M, preferably 1M to 12M, and more preferably 1M to 6M hydrochloric acid is used. The neutralization means that the reaction solution is neutral in pH, and may be 7.0 or less in pH. In order to improve the liquid separation, a ketone liquid separation promoting solvent such as acetone or methyl ethyl ketone may be further added. As a cause of low liquid separation, the inventors speculate that the tag-peptide molecules are bound to each other to form a micelle structure due to hydrophobic interactions and hydrogen bonds. It is presumed that the addition of the above-mentioned liquid separation promoting solvent weakens hydrophobic interactions between side chains of the tag or hydrogen bonds between peptide molecules, suppresses formation of micelle structures, and improves liquid separation. In addition, the step of heating the reaction solution is also effective for improving the liquid separation, instead of or simultaneously with the step of adding the liquid separation promoting solvent.
An acidic aqueous solution is further added to the acid-neutralized reaction solution and washed, followed by separation of the aqueous layer and recovery of the organic layer.
The acidic aqueous solution to be used is not particularly limited, and examples thereof include aqueous hydrochloric acid, aqueous dilute sulfuric acid, aqueous phosphoric acid, and aqueous acetic acid, and aqueous hydrochloric acid is preferable. The pH of the acidic aqueous solution is 1 to 5, preferably 1 to 4, more preferably 1 to 3.
The amount of the acidic aqueous solution to be used for the washing is not particularly limited as long as the washing effect is exhibited, and the amount is 0.1 to 4 times, preferably 0.3 to 3 times, more preferably 0.5 to 2 times, the amount of the reaction solution.
The number of times of washing, separating and discarding the aqueous layer is not particularly limited, and may be one time or a plurality of times. The number of times may be appropriately selected depending on the kind of the compound in the reaction system, the amount of the capturing body, and the like.
The temperature at which the washing is carried out is not particularly limited, and may be 10℃to 50℃and preferably 15℃to 45℃and more preferably 20℃to 40 ℃.
In this step, the capturing body is basically removed by the acidic aqueous solution, but if the capturing body which is difficult to be removed by the acidic aqueous solution is present, another washing step may be added before and after the washing with the acidic aqueous solution. For example, alkaline aqueous solution washing or brine washing may be cited.
Examples of the alkaline aqueous solution include an aqueous sodium bicarbonate solution, an aqueous sodium carbonate solution, and an aqueous potassium carbonate solution having a pH of 8 to 13.
As the brine, 5wt% to saturated brine can be mentioned.
After being washed by acidic aqueous solution, the solution is washed by alkaline aqueous solution, so that the pH value of the solution is neutral to weak alkaline. The aqueous alkaline solution may be the aqueous solution.
[ Fmoc group removal method ]
Next, as a suitable method for removing the protecting group, a case where the protecting group is an Fmoc group will be described in detail. The Fmoc group removal method according to the present embodiment is characterized by comprising: a step of obtaining a capturing body (hereinafter also referred to as "DBF-capturing body") by bonding Dibenzofulvene (DBF) produced by deprotection of Fmoc group and a cyclic amine by using the cyclic amine as a capturing agent and the Fmoc group-protected amino group-containing compound as a protecting group and mixing with an optional deprotecting agent, and obtaining the Fmoc group-deprotected amino group-containing compound; and a step of washing the obtained reaction mixture with an acidic aqueous solution to remove the DBF-trap, thereby obtaining an amino-containing compound. DBF-capture bodies are one example of "capture bodies" of the present invention.
Fmoc group-protected amino group-containing compound means a compound having at least one of a primary amino group or a secondary amino group which the amino group-containing compound has been protected by Fmoc group.
The cyclic amine in the present specification means a cyclic amine having one amino group, and examples thereof include morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide, preferably morpholine, 3-hydroxypiperidine and 4-hydroxypiperidine, more preferably morpholine, as described above.
The amount of cyclic amine added in the Fmoc group removal step is 5 to 100 equivalents, preferably 5 to 50 equivalents, more preferably 10 to 30 equivalents, based on the amount of Fmoc group present in the reaction system. If the added amount of cyclic amine is less than this range, the capturing of DBF produced by Fmoc group deprotection reaction is insufficient, and impurities are difficult to remove by acidic liquid separation washing.
The deprotecting agent is a reagent for removing Fmoc group from Fmoc group-protected amino group-containing compound. As the deprotecting agent in the Fmoc group removing step, as described above, for example, 1, 8-diazabicyclo [5.4.0] -7-undecene (DBU), 1, 5-diazabicyclo [4.3.0] -5-nonene (DBN), organic bases such as1, 4-diazabicyclo [2.2.2] octane (DABCO), triethylamine and tributylamine, inorganic bases such as potassium tert-butoxide and sodium tert-butoxide are mentioned, more preferably 1, 8-diazabicyclo [5.4.0] -7-undecene (DBU), 1, 5-diazabicyclo [4.3.0] -5-nonene (DBN), 1, 4-diazabicyclo [2.2.2] octane (DABCO), and still more preferably 1, 8-diazabicyclo [5.4.0] -7-undecene (DBU).
The amount of the deprotecting agent used for deprotection of the Fmoc group is preferably 1 to 12 equivalents, more preferably 2 to 10 equivalents, particularly preferably 3 to 8 equivalents, relative to the Fmoc group present in the reaction system. The deprotection agent is not necessarily a reactant, and may not be added. However, the use of the capturing agent together with the deprotecting agent is preferable because the use of the deprotecting agent accelerates the reaction rate of the deprotection reaction of Fmoc group.
The acidic aqueous solution to be used is not particularly limited, and examples thereof include aqueous hydrochloric acid, aqueous dilute sulfuric acid, aqueous phosphoric acid, and aqueous acetic acid, and aqueous hydrochloric acid is preferable. The pH of the acidic aqueous solution is 1 to 5, preferably 1 to 4, more preferably 1 to 3.
By concentrating the solution obtained by the Fmoc group deprotection reaction of the present invention, fmoc group deprotected amino group-containing compound can be isolated. Further, the resulting amino group-containing compound solution can be directly used as a raw material for the following method for producing a peptide by a liquid phase synthesis method.
< 2 > Process for producing peptide comprising protecting group removal step
[ Method for producing peptide by liquid phase Synthesis method ]
When the amino group-containing compound protected by the protecting group is a C-protected peptide or the like having a C-terminal end bonded to a soluble carrier, the method for removing the protecting group of the present invention can be suitably used in a method for producing a peptide by liquid phase synthesis.
The peptide synthesis method including the method for removing the protecting group will be described below.
The peptide synthesis method comprises the following steps: a step (step Z1) of condensing an amino group-containing compound (hereinafter, also referred to as "C-terminal carrier protecting peptide") having a C-terminal end protected with a carrier for specific liquid-phase peptide synthesis, a peptide, an amino acid amide or the like with an amino group-containing compound (hereinafter, also referred to as "N-terminal protecting peptide") having an N-terminal end protected with the protecting group (see formula (Z2)); a step (step Z2) of quenching the active ester remaining after the condensation reaction; a deprotection step (step Z3) of removing a protecting group from the condensed peptide (hereinafter also referred to as "N-terminal protected-C-terminal carrier protecting peptide"); a step of washing the reaction solution with an acidic aqueous solution (step Z4); and a step (step Z5) of deprotecting the C-terminal carrier and the side chain protecting group. The above peptide synthesis method is characterized in that it comprises capturing a byproduct having a fulvene skeleton, which is produced by deprotecting an N-terminal protecting group, with a cyclic amine.
Hereinafter, for convenience of explanation, a liquid phase synthesis method of a peptide including a method of removing Fmoc group will be described in detail by taking Fmoc as an example of a protecting group. The protecting group is not limited to Fmoc group, and the method shown below can be applied to an amino group-containing compound protected by a protecting group represented by the above formula (Z2).
(Process Z1: condensation Process)
The C-terminal carrier-protected peptide protected by the carrier for liquid phase peptide synthesis and the amino acid or peptide having the N-terminal protected by the Fmoc group (hereinafter also referred to as "N-Fmoc-protected amino acid or peptide") are condensed in an organic solvent in the presence of a condensing agent to obtain a peptide having an extended amino acid residue (hereinafter also referred to as "N-Fmoc-protected-C-terminal carrier-protected peptide").
[ Carrier for liquid phase peptide Synthesis ]
Examples of the carrier for liquid phase peptide synthesis used in step Z1 of "1" include the following compounds.
That is, an example of the carrier for liquid phase peptide synthesis is the benzyl compound (X1) represented by the above formula (X1). The configuration of the benzyl compound (X1) is the same as that of embodiment 1, and thus a detailed description thereof will be omitted.
As the benzyl compound (X1) represented by the above formula (X1), compounds represented by the following formulas (X1A) to (X1D) may be listed as preferable compounds in view of their usefulness.
Another example of a carrier for liquid phase peptide synthesis of the formula (Y1) below is a benzyl compound (Y1). The configuration of the benzyl compound (Y1) is the same as that of embodiment 2, and thus a detailed description thereof will be omitted.
As the benzyl compound (Y1) represented by the above formula (Y1), compounds represented by the following formulas (Y1A) to (Y1D) can be listed as preferable compounds in view of their usefulness.
The carrier for liquid phase peptide synthesis used in step Z1 is not limited to the benzyl compound (X1) represented by formula (X1) and the benzyl compound (Y1) represented by formula (Y1), and may be a compound other than these benzyl compounds.
As other examples of the carrier for liquid phase peptide synthesis, the compounds represented by the following formula (Z6) can be listed as preferred compounds.
As other examples of the carrier for liquid phase peptide synthesis of 4, there may be mentioned a compound represented by the following formula (Z7).
As other examples of the carrier for liquid phase peptide synthesis of the formula (Z8-1), (Z8-2) and (Z8-3) below are mentioned.
As other examples of the carrier for liquid phase peptide synthesis of 6, there can be mentioned a compound represented by the following formula (Z9).
Further examples of the carrier for liquid phase peptide synthesis of 7 include compounds represented by the following formula (Z10).
[ Amino acids ]
N-Fmoc-protected C-terminal carrier protecting peptide and the like and C-terminal carrier protecting peptide, and the amino acid constituting the basic structure of the N-Fmoc-protected amino acid may be a natural amino acid or a non-natural amino acid. Furthermore, the amino acid may be L-or D-form. As natural amino acids, arg, lys, asp, asn, glu, gln, his, pro, tyr, trp, ser, thr, gly, ala, met, cys, phe, leu, val, ile, beta-Ala and the like can be mentioned. Examples of the unnatural amino acid include Tle (tertiary leucine).
Amino acids may also have side chain functionality. The amino group of the side chain is preferably protected by a protecting group other than Fmoc group (e.g., boc group, cbz group, alloc group, ac group, etc.).
Examples of the protecting group for the side chain carboxyl group include alkyl groups such as methyl, ethyl and t-butyl, and benzyl substituents such as benzyl and p-methoxybenzyl. Examples of the protecting group for an amide group include a trityl (Tr) group and the like. Examples of the protecting group for the side chain hydroxyl group include benzyl and tert-butyl. Examples of the protecting group for the side chain imidazolyl group include a Boc group, a Trt group, a Bom (benzyloxymethyl) group, and the like. Examples of the protecting group for the side chain guanidine group include a nitro group and a Pbf group. Examples of the protecting group for thiol group include Trt group, acm group, dpm group, ddm group, t-butyl group, S-t-butyl group, mmt group, and Npys group.
The amount of the N-Fmoc-protected amino acid to be used is 1 to 4 moles, preferably 1 to 2 moles, particularly preferably 1.05 to 1.3 moles, based on 1 mole of the carrier for liquid phase peptide synthesis.
[ Condensing agent ]
The condensing agent is not limited as long as the reaction proceeds, and condensing agents commonly used in peptide synthesis can be used. The condensing agent described in the step (X2) (condensation reaction step) can be used, and thus a detailed description thereof will be omitted.
In order to promote the peptide condensation reaction and suppress side reactions such as racemization, an activator is preferably added. The activator described in the step (X2) (condensation reaction step) can be used as the activator, and thus a detailed description thereof will be omitted.
[ Reaction solvent ]
The reaction solvent (hereinafter also simply referred to as "solvent") used in the condensation reaction step may be any solvent commonly used in peptide synthesis, and is not limited thereto, and examples thereof include a soluble solvent or a mixed solvent of a soluble solvent and a polar solvent. The solvent described in the step (X1) (dissolution step) can be used as the soluble solvent, and thus a detailed description thereof will be omitted.
In order to improve the solubility of the substrate during the reaction, to improve the solubility of the unreacted materials and by-products in the aqueous layer during the extraction, or to improve the liquid separation, it is preferable to mix a polar solvent such as DMF, dimethylacetamide, DMSO, sulfolane, N-methylpyrrolidone, N' -Dimethylpropourea (DMPU), acetonitrile or the like in a proper ratio into the above soluble solvent. The mixing ratio is not particularly limited as long as the reaction proceeds, and the ratio of the soluble solvent to the polar solvent is (50:50) to (95:5), preferably (70:30) to (90:10).
The amount of the solvent to be used is not particularly limited as long as the reaction proceeds, and the concentration after dissolution of the tag is usually 0.1 mM-1M, preferably 1 mM-0.5M.
The reaction temperature of the present invention is a temperature usually used in peptide synthesis, for example, usually in the range of-20 to 40℃and preferably in the range of 0 to 30 ℃. The reaction time is usually 0.5 to 30 hours (condensation time of one residue).
The reaction solvent is an example of the "organic solvent" of the present invention, and can be used in the following steps Z3 and Z4.
(Process Z2: active ester quenching Process)
An amine is added to the reaction solvent containing the N-Fmoc-protected C-terminal carrier protecting peptide obtained in the step Z1 to capture (remove) the remaining amino acid active ester (a substance in which the carboxylic acid at the C-terminal end of the remaining amino acid in the step Z1 reacts with a condensing agent and then with an activating agent). The amine used in this step is sometimes referred to as a first scavenger.
The amine used as the first scavenger in the step Z2 is preferably a water-soluble primary or secondary amine, and examples thereof include morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine dioxide, 1-methylpiperazine, 4-aminopiperidine, N-dimethylethylenediamine and ethylenediamine, preferably morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide, more preferably morpholine, 3-hydroxypiperidine and 4-hydroxypiperidine, and still more preferably morpholine.
The amount of amine to be added as a scavenger in the step Z2 is not particularly limited, but is usually 1 to 5 equivalents, preferably 1 to 3 equivalents, relative to the amino acid equivalent which remains theoretically.
(Process Z3: fmoc group deprotection and Capture Process)
Deprotection of the N-terminal Fmoc group is performed on the N-Fmoc-C terminal carrier protecting peptide by adding a deprotecting agent to the reaction solution obtained in the above step Z2. Further, the present process includes a process of capturing a by-product (DBF) from the Fmoc group by a second scavenger. In addition, the reaction solution is an example of the "organic solvent" of the present invention. In addition, a second scavenger is one example of a "capture agent" of the present invention. The process of capturing DBF by the second scavenger is one example of the "process of obtaining a capturing body" of the present invention.
The amount of the deprotection agent added in this step is preferably 1 to 12 equivalents, more preferably 2 to 10 equivalents, and particularly preferably 3 to 8 equivalents, relative to Fmoc groups present in the reaction system.
The deprotecting agent is not particularly limited, and examples thereof include 1, 8-diazabicyclo [5.4.0] -7-undecene (DBU), 1, 5-diazabicyclo [4.3.0] -5-nonene, 1, 4-diazabicyclo [2.2.2] octane, potassium tert-butoxide, sodium tert-butoxide, triethylamine and tributylamine, and DBU is preferable.
The amount of the second scavenger used for capturing DBF from Fmoc group removal is preferably 5 to 50 equivalents, more preferably 8 to 40 equivalents, particularly preferably 10 to 35 equivalents, relative to Fmoc group present in the reaction system.
The second scavenger that can be used in procedure Z3 is an amine that acts as a DBF trap. As described above, the amine is cyclic and contains at least one element selected from the group consisting of an oxygen element and a sulfur element, preferably a water-soluble primary amine or secondary amine, and for example, morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide, preferably morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, and more preferably morpholine, can be cited.
In addition, the second scavenger in this step may be the same as or different from the first scavenger added in step Z2 (active ester quenching step). However, in order to simplify the operation and reduce the use of the scavenger, the second scavenger used in the present process Z3 is preferably the same as the first scavenger added in the process Z2. In this case, in the step Z2, the amine is preferably a cyclic amine such as morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine dioxide or the like.
(Process Z4: cleaning Process with an acidic aqueous solution)
The step Z4 is an example of the "separation step" of the present invention. Specifically, this step is a step of adding an acidic aqueous solution to the reaction solution of step Z3 to neutralize the reaction solution, and separating the reaction solution to remove the first scavenger and the substance (hereinafter, also referred to as "amino acid active ester-capturing substance") that is not necessary for the reaction (here, the substance that is not necessary for the reaction means a condensing agent, an activating agent, a deprotecting agent, a polar solvent among the above-mentioned reaction solvents, and the like) into an aqueous layer. The amino acid active ester (i.e., amino acid active ester-capturing body) removed by the first scavenger and the DBF (i.e., DBF-capturing body) removed by the second scavenger are guided to the aqueous layer by acid washing, and these substances are separated from the reaction solution.
The acid used in the neutralization may be the acid described in the step (Y4) (the liquid separation step), and therefore, a detailed description thereof will be omitted.
Further adding an acidic aqueous solution to the acid-neutralized reaction solution, washing, separating the solution, removing the aqueous layer, and recovering the organic layer.
The acidic aqueous solution to be used is not particularly limited, and examples thereof include aqueous hydrochloric acid, aqueous dilute sulfuric acid, aqueous phosphoric acid, and aqueous acetic acid, and aqueous hydrochloric acid is preferable. The pH of the acidic aqueous solution is 1 to 5, preferably 1 to 4, more preferably 1 to 3.
The amount of the acidic aqueous solution to be used for the washing is not particularly limited as long as the washing effect is exhibited, and the amount is 0.1 to 4 times, preferably 0.3 to 3 times, more preferably 0.5 to 2 times, the amount of the reaction solution.
The number of times of washing, separating and discarding the aqueous layer is not particularly limited, and may be one time or a plurality of times. The number of times may be appropriately selected depending on the kind of the compound in the reaction system, the amount of the substance unnecessary for the reaction, and the like.
The cleaning temperature is not particularly limited, and may be 10℃to 50℃and preferably 15℃to 45℃and more preferably 20℃to 40 ℃.
This step basically removes the amino acid active ester-capturing body, the DBF-capturing body and the substances unnecessary for the reaction by the acidic aqueous solution, but if the substances unnecessary for the reaction which are difficult to be removed by the acidic aqueous solution are present, another washing step may be added before and after the washing with the acidic aqueous solution. For example, alkaline aqueous solution washing or brine washing can be cited.
Examples of the alkaline aqueous solution include an aqueous sodium bicarbonate solution, an aqueous sodium carbonate solution, and an aqueous potassium carbonate solution having a pH of 8 to 13.
As the brine, 5wt% to saturated brine can be mentioned. After the acidic aqueous solution is washed, the solution is washed by an alkaline aqueous solution, so that the pH value of the solution is neutral to weak alkaline. The aqueous alkaline solution may be the aqueous solution.
In the peptide synthesis using the method of the present invention, the C-terminal carrier-protected peptide may be solidified (crystallized) at a stage after deprotection of the Fmoc group, and the C-terminal carrier-protected peptide may be recovered by a solid-liquid separation operation. The solidification may be carried out by changing the composition of the solvent in which the carrier-protecting peptide is dissolved, and the specific method may be referred to as known methods as appropriate, and for example, a hydrocarbon solvent such as methanol, acetonitrile, n-hexane may be added to the solution in which the carrier-protecting peptide is dissolved, and the composition of the solution may be changed, and the solution may be a stock solution or a concentrated solution.
By repeating the above steps Z1 to Z4, a C-terminal carrier-protecting peptide having a desired amino acid residue can be obtained. Finally, the final target peptide is obtained by a C-terminal carrier and side chain protecting group removal step [ step Z5 ].
(Process Z5: deprotection and purification Process)
This step is a step of removing the protecting groups of the carrier and the peptide side chains from the C-terminal end of the peptide to obtain the target peptide.
The method for removing the protecting group of the carrier and the side chain of the peptide from the C-terminal of the peptide is not particularly limited, and a known deprotection method can be used, but is preferably carried out by acid treatment. Deprotection methods using trifluoroacetic acid (TFA), for example, may be used.
Depending on the amino acid sequence, TFA may be combined with molecules such as water, thioanisole, 1, 2-ethanedithiol, phenol, triisopropylsilane, and the like to form suitable compositions.
The carrier and the peptide whose protecting group of the side chain of the peptide is deprotected may be isolated and purified according to a purification method commonly used in peptide synthesis. For example, the target peptide may be isolated and purified by extraction washing, crystallization, chromatography.
If the Fmoc group removal method is used for synthesizing the peptide, the intermediate peptide obtained after Fmoc group deprotection can be used for the next condensation process without separation. Thus, the one-pot synthesis of the peptide can be realized, and the method is particularly suitable for industrial production.
< 3 > Removing agent for protecting group
The removing agent for the protecting group represented by the above formula (Z2) is a composition comprising a capturing agent represented by the above formula (Z1) and an alkaline deprotecting agent. The capturing agent and the deprotecting agent are as described above, and detailed description thereof is omitted here.
[ Summary ]
As can be appreciated from the above, the peptide production method according to the first aspect of the present invention comprises: a step of bringing an amino group-containing compound having an N-terminal protected with a protecting group having a fluorene skeleton into contact with a capturing agent represented by the following formula (Z1) in an organic solvent, wherein a capturing agent is obtained by bonding a byproduct having an fulvene skeleton derived from the protecting group to the capturing agent; and
A step of separating the capture body obtained from the organic solvent;
in the above-mentioned formula (Z1),
N is a nitrogen atom and is preferably a nitrogen atom,
H is a hydrogen atom and is preferably a hydrogen atom,
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented by,
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -SO 2 H, OR- (SO 2) R (R is synonymous with-OR),
R 2a or R 2b and R 3a or R 3b may be bonded to each other or may form a ring together with the carbon atom to which they are bonded,
N 1、n2 and n 3 are each independently 1 or 2,
M is an integer of 0 or 1.
The peptide production method according to the second aspect of the present invention further comprises a step of contacting the amino group-containing compound having the N-terminal protected with the protecting group with a deprotecting agent, in addition to the configuration of the peptide production method according to the first aspect.
In the peptide production method according to the third aspect of the present invention, in addition to the constitution of the peptide production method according to the second aspect, the deprotecting agent is at least one base selected from the group consisting of 1, 8-diazabicyclo [5.4.0] -7-undecene (DBU), 1, 5-diazabicyclo [4.3.0] -5-nonene (DBN), 1, 4-diazabicyclo [2.2.2] octane (DABCO), potassium t-butoxide, sodium t-butoxide, triethylamine and tributylamine.
In the peptide production method according to a fourth aspect of the present invention, in addition to the configuration of the peptide production method according to any one of the first to third aspects, the trap separation step includes adding an acidic aqueous solution to the organic solvent, washing the organic solvent, separating the organic solvent into an aqueous layer and an organic layer, and separating the separated aqueous layer.
In the peptide production method according to the fifth aspect of the present invention, in addition to the configuration of the peptide production method according to any one of the first to fourth aspects, the capturing agent represented by the formula (Z1) is at least one selected from the group consisting of morpholine, piperidine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide.
In the peptide production method according to a sixth aspect of the present invention, in addition to the configuration of the peptide production method according to any one of the first to fifth aspects, the capturing agent represented by the formula (Z1) is at least one selected from the group consisting of morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide.
The method for removing a protecting group according to the seventh aspect of the present invention comprises: a step of bringing an amino group-containing compound having an N-terminal protected with a protecting group having a fluorene skeleton into contact with a capturing agent represented by the following formula (Z1) in an organic solvent, wherein a capturing agent is obtained by bonding a byproduct having an fulvene skeleton derived from the protecting group to the capturing agent; and
A step of separating the capture body obtained from the organic solvent;
in the above-mentioned formula (Z1),
N is a nitrogen atom and is preferably a nitrogen atom,
H is a hydrogen atom and is preferably a hydrogen atom,
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented by,
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -SO 2 H, OR- (SO 2) R (R is synonymous with-OR),
R 2a or R 2b and R 3a or R 3b may be bonded to each other or may form a ring together with the carbon atom to which they are bonded,
N 1、n2 and n 3 are each independently 1 or 2,
M is an integer of 0 or 1.
The remover according to the eighth aspect of the present invention is a remover comprising a capturing agent represented by the following formula (Z1) and an alkaline deprotecting agent, the remover having a protecting group of fluorene skeleton:
in the above-mentioned formula (Z1),
N is a nitrogen atom and is preferably a nitrogen atom,
H is a hydrogen atom and is preferably a hydrogen atom,
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented by,
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -SO 2 H, OR- (SO 2) R (R is synonymous with-OR),
R 2a or R 2b are bonded to each other with R 3a or R 3b, or may form a ring together with the carbon atoms to which they are bonded,
N 1、n2 and n 3 are each independently 1 or 2,
M is an integer of 0 or 1.
In the peptide production method according to the ninth aspect of the present invention, in addition to the configuration of the remover according to the eighth aspect, the capturing agent is at least one selected from the group consisting of morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide.
In the method for producing a peptide according to a tenth aspect of the present invention, in addition to the composition of the removing agent according to the eighth or ninth aspect, the removing agent is at least one base selected from the group consisting of 1, 8-diazabicyclo [5.4.0] -7-undecene, 1, 5-diazabicyclo [4.3.0] -5-nonene, 1, 4-diazabicyclo [2.2.2] octane, potassium t-butoxide, sodium t-butoxide, triethylamine and tributylamine.
The benzyl compound (Y1) according to the eleventh aspect of the present invention is represented by the following formula (Y1):
in the formula (Y1), the amino acid sequence of the formula (I),
M Q's each represent an oxygen atom,
M R 1 are each independently a group represented by the following formula (YA):
in the formula (YA),
* The bonding position is indicated by the number of the bonding sites,
R 1a、R1b、R1c、R1d and R 1e each independently represent a hydrogen atom or an alkyl group,
N 1 represents an integer of 0 to 6, and when n 1 is 1 or more, the repeating unit represented in parentheses with n 1 is an alkylene group,
N 2 represents an integer of 0 to 6, and when n 2 is 1 or more, the repeating unit represented in parentheses with n 2 is an alkylene group,
But at least two or more of R 1a、R1b、R1c and R 1d are hydrogen atoms;
In the formula (Y1), k R 2 each independently represents a hydrogen atom, an alkyl group, a cycloalkyl group, an alkoxy group, an aryl group, an aralkyl group or a halogen atom,
In the formula (Y1), X represents a hydroxyl group,
In the formula (Y1), m represents an integer of 2 or 3,
In the formula (Y1), k represents an integer of 0 to (5-m) inclusive,
In the formula (Y1), at least one of m [ Q-R 1 ] is substituted in the meta position with respect to the substituent containing the X.
In the benzyl compound (Y1) according to the twelfth aspect of the present invention, the total number of carbon atoms is 40 to 60 in addition to the configuration of the benzyl compound (Y1) according to the eleventh aspect.
In the benzyl compound (Y1) according to the thirteenth aspect of the present invention, in addition to the constitution of the benzyl compound (Y1) according to the eleventh or twelfth aspect, each of the m R 1 is independently an organic group having one side chain,
Which is a group represented by the following formula (YA'):
In the formula (YA '), the catalyst is represented by the formula (YA'),
* The bonding position is indicated by the number of the bonding sites,
R 1f is a linear alkyl group having 4 or more and 12 or less carbon atoms,
R 1g is a linear alkyl group having 6 or more and 14 or less carbon atoms.
In the benzyl compound (Y1) according to the fourteenth aspect of the present invention, in addition to the composition of the benzyl compound (Y1) according to the thirteenth aspect, R 1f is a linear alkyl group having 4 to 10 carbon atoms,
The R 1g is a linear alkyl group having 6 or more and 12 or less carbon atoms.
The method for producing a peptide according to the fifteenth aspect of the present invention comprises: a dissolving step of dissolving the benzyl compound according to any one of the eleventh to fourteenth aspects in a soluble solvent;
A condensation reaction step of condensing the dissolved benzyl compound with an amino acid having an N-terminal protected by an N-terminal protecting group to form a first condensate;
A scavenging step of adding a first base to the soluble solvent containing the first condensate to scavenge an amino acid active ester, further adding the first base and a second base to the soluble solvent, deprotecting the N-terminal protecting group of the first condensate, and scavenging by-products derived from the N-terminal protecting group by the first base; and
And a liquid separation step of adding an acidic aqueous solution to the soluble solvent containing the capturing substance captured in the removal step, and washing the mixture, wherein the liquid is separated into an aqueous layer and an organic layer, the capturing substance and unnecessary substances are removed to the aqueous layer, and the first condensate is deprotected at the N-terminal protecting group in the organic layer to obtain a second condensate.
In the method for producing a peptide according to a sixteenth aspect of the present invention, in addition to the configuration of the method for producing a peptide according to the fifteenth aspect, the condensation reaction step includes: condensing the N-terminal-protected nth amino acid into a 2n-th condensate to form a (2n+1) -th condensate, wherein the 2n-th condensate contains an N-terminal-unprotected amino acid and a C-terminal-protected amino acid with the benzyl compound and contains N-number of amino acids,
The scavenging step includes a step of deprotecting the N-terminal protecting group of the (2n+1) -th condensate,
The liquid separation step includes a step of deprotecting the (2n+1) th condensate in the organic layer to obtain a (2n+2) th condensate,
And n is a natural number of 2 or more.
In the method for producing a peptide according to the seventeenth aspect of the present invention, in addition to the configuration of the method for producing a peptide according to the sixteenth aspect, n is 5 or more.
In the method for producing a peptide according to an eighteenth aspect of the present invention, in addition to the method for producing a peptide according to any one of the fifteenth to seventeenth aspects, the step of separating the liquid further includes a step of adding a ketone-based liquid separation promoting solvent to the soluble solvent.
The benzyl compound (X1) according to the nineteenth aspect of the present invention is represented by the following formula (X1):
In the formula (X1), the amino acid sequence of the formula (X),
M Q 1 and Q 2 are each an oxygen atom,
M R 1 are each independently alkylene,
M R 2 are each independently an optionally substituted alkyl group, an optionally substituted aralkyl group or an optionally substituted aryl group,
K R 3 are each independently a hydrogen atom, an alkyl group, an alkoxy group or a halogen atom,
X is a hydroxyl group, and is a hydroxyl group,
M is an integer of 2 or 3,
K represents an integer of 0 to (5-m).
In the benzyl compound (X1) according to the twentieth aspect of the present invention, in addition to the configuration of the benzyl compound (X1) according to the nineteenth aspect, the m R 1 are alkylene groups having 2 to 16 carbon atoms.
In the benzyl compound (X1) according to the twentieth aspect of the present invention, in addition to the configuration of the benzyl compound (X1) according to the nineteenth or twentieth aspect, the m R 2 are aryl groups having a substituent containing a halogen atom.
In the benzyl compound (X1) according to the twenty-second aspect of the present invention, in addition to the configuration of the benzyl compound (X1) according to the nineteenth or twentieth aspect, the m R 2 are alkyl groups having 5to 28 carbon atoms.
In the benzyl compound (X1) according to the twenty-third aspect of the present invention, in addition to the constitution of the benzyl compound (X1) according to the twenty-second aspect described above, the m R 2 are linear alkyl groups or alkyl groups having a total number of branches of 1 or 2, and are groups represented by the following formula (XA):
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In the formula (XA),
* The bonding position is indicated by the number of the bonding sites,
R 2a、R2b、R2c、R2d and R 2e each independently represent a hydrogen atom or an alkyl group,
N 1 represents an integer of 0 to 16,
N 2 represents an integer of 0 to 16 inclusive.
But at least two or more of R 2a、R2b、R2c and R 2d are hydrogen atoms.
Examples
The present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
First, the example of embodiment 1 of the present invention described above is exemplified.
< Manufacturing example >)
Example X1
Synthesis of Compound (X1-4)
Examples (X1-a)
25G (100 mmol) of 3, 5-bistrifluoromethylphenol were dissolved in 125mL of DMF, 27.6g (120 mmol) of 11-bromoundecanol and 27.6g (200 mmol) of potassium carbonate were added and stirred at 60℃for 4 hours. The reaction solution was cooled to room temperature, and solids were removed by filtration. To the filtrate, 150mL of toluene and 75mL of 1M hydrochloric acid were added, and the mixture was washed with a portion of 1M hydrochloric acid (75 mL) and 100mL of saturated brine, and the organic layer was further washed. 50g of sodium sulfate was added to dry the organic layer, and then the solvent was removed under reduced pressure, whereby 37.2g (yield 93%) of compound (X1-1) was obtained.
1H-NMR(400MHz,CDCl3)δ1.10-1.60(m,16H),1.81-1.90(m,2H),3.63(t,2H,J＝2.0Hz),4.04(t,1H,J＝6.8Hz)、7.28(s,2H),7.43(s,1H)
Examples (X1-b)
24G (60 mmol) of the compound (X1-1) was dissolved in 240mL of methylene chloride, and 20.5g (78 mmol) of triphenylphosphine and 26.0g (78 mmol) of carbon tetrabromide were added thereto, followed by stirring at room temperature for 2 hours. 36g of silica gel was added to the reaction solution to remove the solvent, and the residue was adsorbed on the silica gel. The silica gel was placed on a tung mountain funnel lined with filter paper, and the target product was eluted by washing the silica gel with 480mL of an organic solvent (n-hexane: ethyl acetate=90:10). The solvent was removed under reduced pressure to give 27.0g (yield 97%) of compound (X1-2).
1H-NMR(400MHz,CDCl3)δ1.10-1.60(m,12H),1.81-1.90(m,4H),3.41(t,2H,J＝6.8Hz),4.02(t,1H,J＝6.4Hz)、7.29(s,2H),7.44(s,1H)
Examples (X1-c)
1.16G (6.33 mmol) of methyl gallate was dissolved in 150mL of DMF, 14.1g (30.4 mmol) of compound (X1-2) and 16.6g (120 mmol) of potassium carbonate were added thereto, and the mixture was stirred at 60℃for 18 hours. After potassium carbonate was removed by filtration, 100mL of 1M hydrochloric acid and 100mL of n-hexane were added to the reaction solution, and the solution was washed with separated liquid, and the organic layer was further washed with 100mL of 5% sodium bicarbonate and 20% brine. The organic layer was dried over sodium sulfate, and then the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane: ethyl acetate=98:2 to 85:15) to give 6.7g of compound (X1-3) (yield 79.6%).
1H-NMR(400MHz,CDCl3)δ1.20-1.60(m,48H),1.81-1.90(m,6H),3.87(s,3H),3.95-4.05(m、12H)、7.21(s,2H)、7.28(s,6H),7.42(s,3H)
Examples (X1-d)
6.2G (4.66 mmol) of compound (X1-3) was dissolved in 150mL of THF, and 20.96mL (1M solution in THF, 20.96 mmol) of lithium triethylborohydride was added under ice-cooling and stirred at room temperature for 2 hours. To the reaction solution, water (50 mL) and 1M hydrochloric acid (20 mL) were added and the reaction was stopped, and then 200mL of ethyl acetate was added to conduct liquid separation washing, and the organic layer was further washed 2 times with 100mL of water. After drying the organic layer over sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane: ethyl acetate=90:10 to 60:40) to give 3.8g of compound (X1-4) (yield 61.2%).
1H-NMR(400MHz,CDCl3)δ1.20-1.60(m,48H),1.81-1.90(m,6H),3.90-4.05(m、12H)、4.59(d、2H)、6.54(s、2H)、7.28(s,6H),7.42(s,3H)
ESI-MS：1303.77〔M+〕
Example X2
Synthesis of Compound (X2-3)
Examples (X2-a)
8G (26.79 mmol) of 2-n-octyl-1-dodecanol was dissolved in 210mL of toluene (anhydrous), 17.6g (53.59 mmol) of dibromododecane and 2.14g (53.59 mmol) of NaH were added thereto, and the mixture was stirred at 105℃overnight. The reaction solution was cooled to room temperature, and 10mL of 1M hydrochloric acid was added thereto and stirred for 10 minutes. To the reaction solution, 90mL of 1M hydrochloric acid was added and the mixture was washed with a solution of saturated brine (100 mL), and the organic layer was washed 3 times with saturated brine and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane to n-hexane: ethyl acetate=90:10) to give 8.8g (yield 60.1%) of compound (X2-1).
1H-NMR(400MHz,CDCl3)δ0.88(t,6H,J＝7.6Hz),1.20-1.59(m,51H),1.81-1.89(m,2H),3.25(d,2H,J＝6.0Hz),3.35-3.43(m,4H)
Examples (X2-b)
0.77G (5.61 mmol) of 2, 4-dihydroxybenzaldehyde was dissolved in DMF: to 116mL of a mixed solvent of cyclopentylmethyl ether (1:1), 7.6g (14.03 mmol) of the compound (X2-1) and 3.9g (28.06 mmol) of potassium carbonate were added, and the mixture was stirred at 90℃for 3 hours. After potassium carbonate was removed by filtration, 100mL of 1M hydrochloric acid and 100mL of n-hexane were added to the reaction solution, and the solution was washed with separated liquid, and the organic layer was further washed with 100mL of 5% sodium bicarbonate and 20% brine. After drying the organic layer over sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane to n-hexane: ethyl acetate=90:10) to give 4.4g of compound (X2-2) (yield 73.9%).
1H-NMR(400MHz,CDCl3)δ0.88(t,12H,J＝6.6Hz),1.20-1.59(m,102H),1.74-1.88(m,4H),3.25(d,4H,J＝6.6Hz),3.36(t,4H,J＝6.6Hz),3.98-4.05(m,4H),6.41(d,1H,J＝2.4Hz),6.51(dd,1H,J＝2.4Hz,8.6Hz),7.79(d,1H,J＝8.6Hz),10.33(s,1H)
Examples (X2-c)
4.4G (4.08 mmol) of compound (X2-2) was dissolved in THF (anhydrous): to 65mL of a mixed solvent of methanol (10:3), 0.31g (8.17 mmol) of sodium borohydride was added under ice-cooling conditions, and the mixture was stirred for 10 minutes, and the mixture was taken out of the ice bath and stirred at room temperature for 1 hour. The reaction was stopped by adding 5mL of acetone to the reaction mixture, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane: ethyl acetate=96:4 to n-hexane: ethyl acetate=90:10) to give 3.9g of compound (X2-3) (yield 89.9%).
1H-NMR(400MHz,CDCl3)δ0.88(t,12H,J＝6.5Hz),1.20-1.59(m,102H),1.72-1.85(m,4H),2.24(t,1H),3.25(d,4H,J＝6.5Hz),3.36(t,4H,J＝6.5Hz),3.90-4.01(m,4H),4.61(d,2H,J＝6.5Hz),6.41(dd,1H,J＝2.7Hz,8.4Hz),6.45(d,1H,8.4Hz),7.13(d,1H)
ESI-MS：1069.13〔M+〕
Example X3
Compound (Synthesis of X3-2)
Examples (X3-a)
0.79G (4.29 mmol) of methyl gallate is dissolved in DMF: MTHP (1:1) 132mL of the mixed solvent, 8.8g (16.12 mmol) of the compound (X2-1) produced in example (X2-a) and 2.65g (19.17 mmol) of potassium carbonate were added, and the mixture was stirred at 90℃overnight. After potassium carbonate was removed by filtration, 100mL of 1M hydrochloric acid and 100mL of n-hexane were added to the reaction solution, and the solution was washed with separated liquid, and the organic layer was further washed with 100mL of 5% sodium bicarbonate and 20% brine. After drying the organic layer over sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane to n-hexane: ethyl acetate=90:10) to give 4.1g (yield 61.1%) of compound (X3-1).
1H-NMR(400MHz,CDCl3)δ0.88(t,18H,J＝6.3Hz),1.20-1.86(m,147H),3.24(d,6H,J＝6.3Hz),3.36(t,6H,J＝6.3Hz),3.88(s,3H),3.98-4.40(m,6H),7.24(s,2H)
Examples (X3-b)
3.2G (2.04 mmol) of the compound (X3-1) was dissolved in 30mL of THF (anhydrous), and 4.1mL (1.5M toluene solution, 6.16 mmol) of diisobutylaluminum hydride was added thereto under ice-cooling, and stirred for 1 hour, and further stirred at room temperature for 2 hours. To the reaction solution, 5mL of acetone was added and the reaction was stopped, then 30g of silica gel was added, and the mixture was stirred at room temperature for 15 minutes, the reaction solution was filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane: ethyl acetate=96:4 to n-hexane: ethyl acetate=85:15) to give 2.3g of compound (X3-2) (yield 72.5%).
1H-NMR(400MHz,CDCl3)δ0.88(t,18H,J＝6.6Hz),1.18-1.84(m,147H),3.25(d,6H,J＝6.6Hz),3.36(t,6H,J＝6.6Hz),3.60-3.66(m,1H),3.90-4.00(m,6H),4.59(d,2H,J＝6.6Hz),6.55(s,2H)
ESI-MS：1549.41〔M+〕
Example X4
Synthesis of Compound (X4-3)
Example (X4-a)
1G (2.81 mmol) of 2-decyl-1-tetradecanol was dissolved in 20mL of toluene (anhydrous), and 1.85g (5.63 mmol) of dibromododecane and 0.226g (5.63 mmol) of NaH were added thereto and stirred at 95℃overnight. The reaction solution was cooled to room temperature, and 10mL of 1M hydrochloric acid was added thereto and stirred for 10 minutes. N-hexane (20 mL) was added thereto, and the mixture was washed with a solution of saturated brine (40 mL) and the organic layer was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane to n-hexane: ethyl acetate=90:10) to give 1.18g of compound (X4-1) (yield 70.0%).
1H-NMR(400MHz,CDCl3)δ0.88(t,6H,J＝6.8Hz),1.15-1.60(m,55H),1.81-1.89(m,2H),3.25(d,2H,J＝6.3Hz),3.35-3.42(m,4H)
Examples (X4-b)
0.106G (0.77 mmol) of 2, 4-dihydroxybenzaldehyde was dissolved in DMF: to 17.4mL of a mixed solvent of cyclopentylmethyl ether (1:1), 1.16g (1.92 mmol) of the compound (X4-1) and 0.532g (3.85 mmol) of potassium carbonate were added, and the mixture was stirred at 90℃for 4 hours. After potassium carbonate was removed by filtration, the reaction mixture was washed with 20mL of n-hexane and 36mL of 1M hydrochloric acid by liquid separation, and the organic layer was further washed with 18mL of 5% sodium hydrogencarbonate and 18mL of 20% brine for 2 times. After the organic layer was dried over a proper amount of sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane-n-hexane: ethyl acetate=90:10) to give 0.33g of compound (X4-2) (yield 72.6%).
1H-NMR(400MHz,CDCl3)δ0.88(t,12H,J＝7.5Hz),1.15-1.60(m,118H),1.74-1.88(m,4H),3.25(d,4H,J＝6.2Hz),3.36(t,4H,J＝6.2Hz),3.98-4.05(m,4H),6.41(d,1H,J＝2.5Hz),6.51(dd,1H,J＝3.1Hz,9.2Hz),7.79(d,1H,J＝8.9Hz),10.33(s,1H)
Examples (X4-c)
1.0G (0.837 mmol) of compound (X4-2) was dissolved in THF (anhydrous): to 19.5mL of a mixed solvent of methanol (10:3), 0.070g (1.850 mmol) of sodium borohydride was added under ice-cooling conditions, and the mixture was stirred for 10 minutes, taken out of the ice bath, and stirred at room temperature for 1 hour. The reaction was stopped by adding 4mL of acetone to the reaction mixture, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane: ethyl acetate=90:10) to give 0.55g (yield 55.6%) of compound (X4-3).
1H-NMR(400MHz,CDCl3)δ0.87(t,12H,J＝6.5Hz),1.16-1.58(m,118H),1.71-1.83(m,4H),2.24(t,1H,J＝6.5Hz),3.24(d,4H,J＝6.2Hz),3.35(t,4H,J＝6.2Hz),3.88-4.00(m,4H),4.59(d,2H,J＝6.5Hz),6.40(dd,1H,J＝2.2Hz,8.2Hz),6.44(d,1H,J＝2.2Hz),7.11(d,1H,J＝8.2Hz)
ESI-MS：1181.07〔M+〕
Example X5
< Confirmation of solubility of tag in organic solvent >
The solubility (25 ℃) of the compound (X1-4) produced in example X1, the compound (X2-3) produced in example X2, the compound (X3-2) produced in example X3, and the compound (X4-3) produced in example X4 in various solvents was measured.
[ Experimental methods ]
The compounds of examples X1 to X4 and the linear chain-containing compounds as comparative examples (comparative examples X1 and X2 of reference Table X2) were dissolved in each solvent at 25℃in a saturated manner, and the solubilities (unit: wt%) thereof were measured (tables X1 and X2). The solvent used was CPME, MTHP, toluene and chloroform.
The linear C 18H37 compound shown in comparative example X1 was synthesized by the method described in examples of Japanese patent application laid-open No. 2000-44494, and the linear C 22H45 compound shown in comparative example X2 was synthesized by the method described in Bioorganic & MEDICINAL CHEMISTRY LETTERS,21, (2011), 4476-4479.
Table X1 shows the results of the solubility of example X1 (compound (X1-4)), example X2 (compound (X2-3)), example X3 (compound (X3-2)), and example X4 (compound (X4-3)). Table X2 shows the results of the solubilities of comparative examples X1 and X2. The values in parentheses in table X2 indicate how many times the solubilities of the compounds of examples X1 to X4 (i.e., 50 divided by the solubility) are equivalent when the solubilities of the compounds of examples X1 to X4 are each 50 (wt%).
Table X1
Table X2
[ Experimental results ]
The solubility of the compounds of examples X1 to X4 is a value of more than 50% by weight. In contrast, the compounds of comparative examples X1 and X2 were each less than 50 (mass%) and the maximum was only 20.0 (mass%) (comparative example X2, chloroform). As shown in table X2, the solubility of the compounds of examples X1 to X4 was at least 2.5 times greater than the solubility of the compounds of comparative examples X1 and X2 (refer to comparative example X2-chloroform), and more than 17.2 times greater than the solubility of the compounds of comparative examples X1 and X2 (refer to comparative example X1-toluene).
As described above, the compounds of examples X1 to X4 were confirmed to exhibit a high solubility of more than 2.5 to 17 times in each solvent, compared with the compounds of comparative examples X1 and X2, in which the corresponding side chains were linear. From these results, it was found that the above-mentioned compounds can function as excellent tags in peptide synthesis in the peptide production method.
Example X6-1
< Hydrophobicity of confirmation tag >
The hydrophobicity of the compounds of examples X1 to X4 of the present invention was evaluated using liquid chromatography (HPLC).
[ Experimental methods ]
The labels according to one aspect of the present invention (the compounds of examples X1 to X4), the linear C 18H37 compound of comparative example X1 and the linear C 22H45 compound of comparative example X2 were prepared in 10mg/10mL (THF) solutions, respectively, and the retention times (elution times) of liquid chromatography (HPLC) under the following conditions were compared (Table X3).
Table X3
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Liquid Chromatography (HPLC) conditions:
chromatographic column: inert Sustand C18 (3 μm, 4.6X1125 mm)
Mobile phase B: THF, mobile phase a:0.1% aqueous trifluoroacetic acid solution
Eluent: mobile phase a/mobile phase b=25/75 (isocratic)
Flow rate: 1.0mL/min
Column temperature: 40 DEG C
A detector: ultraviolet visible spectrum detector (lambda=220 nm)
[ Experimental results ]
As shown in Table X3, the holding times of the compounds of comparative examples X1 and X2 were 13.6 minutes and 12.1 minutes, respectively, whereas examples X1 to X4 were 8.4 minutes, 21.8 minutes, 60.2 minutes and 33.2 minutes, respectively. The holding times of examples X2 to X4 were all much longer than those of comparative examples X1 and X2. The value of example X1 is smaller than the holding time of comparative examples X1 and X2, but is generally kept at the same level.
As described above, the compounds of examples X1 to X4 each exhibited an equivalent or higher holding time as compared with the compounds of comparative examples X1 and X2 in which the corresponding side chains were linear, and it was found that: in the method for producing a peptide, the above-mentioned compound functions as an excellent tag in peptide synthesis.
Example X6-2
< Acid resistance >
The side chain moiety of the compound of the present invention was evaluated for stability to acid using the compound (X1-4) produced in example X1 and the compound (X3-2) produced in example X3.
[ Experimental methods ]
50Mg of each of the compound (X1-4) and the compound (X3-2) of the present invention was measured in a closed sample tube container, 0.5mL of 1wt% HCl/CPME was added thereto, and the change in the purity (%) of each compound solution with time was confirmed while stirring at room temperature, and the stability was evaluated. In addition, a solution of the same concentration as described above, in which only CPME was contained and hydrogen chloride-containing gas was not contained, was prepared, and the purity (%) of the solution was taken as an initial value. Table X4 shows the initial values and the HPLC purity% of the compounds (X1-4) and (X3-2) of the present invention after stirring for 3 hours.
Table X4
Liquid Chromatography (HPLC) conditions:
chromatographic column: inert Sustand C18 (3 μm, 4.6X1125 mm)
Mobile phase B: THF, mobile phase a:0.1% aqueous trifluoroacetic acid solution
Eluent: mobile phase a/mobile phase B: the measurement was performed under gradient conditions shown in table X5.
Table X5
Flow rate: 1.0mL/min
Column temperature: 40 DEG C
A detector: ultraviolet visible spectrum detector (lambda=220 nm)
[ Experimental results ]
As shown in table X4, the compounds of example X1 and example X3 were both acid stable over 3 hours. Non-patent document 5 discloses that the half-life of triisopropylsilyl groups in an acidic solvent (1 wt% hydrochloric acid/methanol) is 55 minutes. This is considered as follows: the compounds of examples X1 and X3 are more stable to acids than compounds having at least O-Si bonds.
As described above, when the peak changes with time of the compounds of examples X1 and X3 were confirmed by liquid chromatography, no decomposition was observed, indicating that the above compounds can function as excellent labels stable to acids.
< Peptide Synthesis example >)
Peptide synthesis was carried out using the compound (X1-4) and the compound (X2-3) of the present invention. Hereinafter, the compound (X1-4) in the formula is also referred to as HO-Tag (X1-4), and the compound (X2-3) is also referred to as HO-Tag (X2-3). Specifically, tag (X1-4) represents a moiety after removing-OH from the compound (X1-4), and Tag (X2-3) represents a moiety after removing-OH from the compound (X2-3).
Example X7: synthesis of H-Tyr (OtBu) -Ile-Leu-OTag (X1-4)
Example X7-1: synthesis of HO-Leu-OTag (X1-4)
1.3G (1.0 mmol) of the compound (X1-4) was dissolved in 20mL of a mixture of MTHP/DMF (8/2), and 0.46g (1.3 mmol) of Fmoc-Leu-OH, 0.25g (1.3 mmol) of EDCI. HCl and 0.012g (0.1 mmol) of DMAP were added and stirred at room temperature for 4 hours. Morpholine 39 μl (0.4 mmol) was added and stirred at room temperature for 30 minutes. Morpholine 1.74mL (20.0 mmol) and DBU 1.04mL (7.0 mmol) were added and stirred at room temperature for 1 hour. Under ice-cooling conditions, 8.3mL of 6M hydrochloric acid was added, and 23.4mL of 0.1M hydrochloric acid was further added to separate the solution. The organic layer was washed with 10mL of 2M hydrochloric acid and 23.4mL of a 0.5M aqueous sodium hydrogencarbonate solution, separated, dried over a proper amount of sodium sulfate, and filtered to give an amino acid condensate (HO-Leu-OTag (X1-4)) solution.
Example X7-2: synthesis of HO-Ile-Leu-OTag (X1-4)
To the solution of HO-Leu-OTag (X1-4) obtained above were added DMF 4mL, fmoc-Ile-OH 0.46g (1.3 mmol), EDCI. HCl 0.25g (1.3 mmol) and Oxyma 0.046g (0.3 mmol) and stirred at room temperature for 1 hour. Morpholine 39 μl (0.4 mmol) was added and stirred at room temperature for 30 min. Morpholine 1.74mL (20.0 mmol) and DBU 1.04mL (7.0 mmol) were added and stirred at room temperature for 1 hour. Under ice-cooling conditions, 8.3mL of 6M hydrochloric acid was added, and 23.4mL of 0.1M hydrochloric acid was further added to separate the solution. The organic layer was washed with 10mL of 2M hydrochloric acid and 23.4mL of a 0.5M aqueous sodium hydrogencarbonate solution, separated, dried over a proper amount of sodium sulfate, and filtered to give an amino acid condensate (HO-Ile-Leu-OTag (X1-4)) solution.
Example X7-3: synthesis of H-Tyr (OtBu) -Ile-Leu-OTag (X1-4)
The same operations as in example X7-2 were conducted except that Fmoc-Tyr (OtBu) -OH was used in the condensed amino acid, to obtain a solution of H-Tyr (OtBu) -Ile-Leu-OTag (X1-4). The resulting organic layer was removed with solvent under reduced pressure to give 1.58g (yield 90.3%) of H-Tyr (OtBu) -Ile-Leu-OTag (X1-4).
ESI-MS：1749.13〔M+H〕+
It was confirmed that H-Tyr (OtBu) -Ile-Leu-OTag (X1-4) was obtained in high yield, and by-products could be easily removed by using a specific cyclic amine containing only one nitrogen atom such as morpholine as a capturing agent.
Using a small amount of this compound in trifluoroacetic acid (TFA): water = 9.5: when the label was subjected to deprotection analysis in 0.5 mixed solution, ESI-MS of H-Tyr-Ile-Leu-OH was confirmed: 408.24 [ M+H ] +.
Example X8: synthesis of H-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (OtBu) -Ile-Leu-OTag (X2-3)
The same operations as in examples X7-1 to X7-3 were carried out using 0.5g (0.46 mmol) of the compound (X2-3) and the amino acids at residues 1 to 6 shown below as tags,
A solution of H-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (OtBu) -Ile-Leu-OTag (X2-3) was obtained.
Table X6
Amino acids used in condensation
Residue 1 Fmoc-Leu-OH
Residue 2 Fmoc-Ile-OH
Residue 3 Fmoc-Tyr(OtBu)-OH
Residue 4 Fmoc-Pro-OH
Residue 5 Fmoc-Arg(Pbf)-OH
Residue 6 Fmoc-Arg(Pbf)-OH
The solvent was removed from the obtained organic layer under reduced pressure to obtain 0.80g (yield: 70.6%) of H-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (OtBu) -Ile-Leu-OTag (X2-3).
ESI-MS：2430.23〔M+H〕+
Using a small amount of this compound, in trifluoroacetic acid (TFA): water: thioanisole: 1, 2-ethylene glycol: phenol=10: 0.5:0.5:0.25: when the label was subjected to deprotection analysis in 0.75 mixed solution, ESI-MS of H-Arg-Arg-Pro-Tyr-Ile-Leu-OH was confirmed: 817.50 [ M+H ] +.
[ Production of tag ]
Next, an example of embodiment 2 of the present invention described above is exemplified.
Example Y1 >
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Examples (Y1-a)
1.20G (6.516 mmol) of methyl gallate was dissolved in 12mL of DMF and 12mL of cyclopentylmethyl ether (CPME), 7.15g (29.322 mmol) of 5-bromomethylundecane and 4.50g (32.580 mmol) of potassium carbonate were added and stirred at 110℃for 10 hours. The reaction solution was cooled to room temperature, and solids were removed by filtration. To the filtrate, CPME 20mL and 1M hydrochloric acid 20mL were added for washing by liquid separation, and further 5% sodium hydrogencarbonate 20mL and 20% brine 20mL were added for washing. After the organic layer was dried over sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=97:3 to 90:10) to give 3.8g of compound (Y1-1) (yield 84.6%).
1H-NMR(400MHz,CDCl3)δ0.84-0.96(m,18H),1.20-1.60(m,66H),1.71-1.85(m,3H),3.86-3.92(m,9H),7.24(s,2H)
Examples (Y1-b)
2.42G (3.511 mmol) of compound (Y1-1) was dissolved in 48mL of THF, 7.0mL (1.5M toluene solution, 10.533 mmol) of diisobutylaluminum hydride was added dropwise thereto under ice-cooling, and the mixture was stirred under ice-cooling for 2 hours. To the reaction mixture was added 10mL of 0.2M hydrochloric acid to stop the reaction, and the solvent was removed under reduced pressure. After 100mL of ethyl acetate was added to the residue, the mixture was washed 2 times with 75mL of 1M hydrochloric acid, and then 50mL of 5% sodium hydrogencarbonate and 50mL of 20% brine were used for washing. After drying the organic layer with an appropriate amount of sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=95:5 to 90:10) to give 1.86g of compound (Y1-2) (yield 80.2%).
1H-NMR(400MHz,CDCl3)δ0.84-0.94(m,18H),1.22-1.64(m,66H),1.72-1.84(m,3H),3.75-3.88(dd,J＝7.2Hz,14.4Hz,6H)、4.60(d、J＝7.2Hz,2H)、6.54(s,2H)
ESI-MS：683.54[M+Na]+
Example Y2 >, an
Example (Y2-a)
Methyl gallate 3.01g (16.357 mmol) was dissolved in DMF 30mL and CPME 30mL, 7-bromomethylpentadecane 19.98g (65.428 mmol) and potassium carbonate 11.30g (81.785 mmol) were added, and the mixture was stirred at 110℃for 12 hours. The reaction solution was cooled to room temperature, and solids were removed by filtration. To the filtrate, 30mL of CPME and 60mL of 1M hydrochloric acid were added and the mixture was washed by liquid separation, followed by washing with 60mL of 5% sodium bicarbonate and 60mL of 20% brine. After the organic layer was dried over sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=96:4) to give 10.09g of compound (Y2-1) (yield 71.9%).
1H-NMR(400MHz,CDCl3)δ0.83-0.94(m,18H),1.20-1.58(m,72H),1.70-1.85(m,3H),3.85-3.92(m,9H),7.24(s,2H)
Example (Y2-b)
10.08G (11.755 mmol) of compound (Y2-1) was dissolved in 120mL of anhydrous THF, 23.5mL (1.5M toluene solution, 35.267 mmol) of diisobutylaluminum hydride was added dropwise thereto under ice-cooling, and the mixture was stirred under ice-cooling for 2 hours. To the reaction mixture was added 10mL of 0.2M hydrochloric acid to stop the reaction, and the solvent was removed under reduced pressure. After 150mL of ethyl acetate was added to the residue, the mixture was washed 2 times with 75mL of 1M hydrochloric acid, and then washed with 75mL of 5% sodium hydrogencarbonate and 75mL of 20% brine. After the organic layer was dried over a suitable amount of sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=95:5 to 85:15) to give 7.43g of compound (Y2-2) (yield 76.4%).
1H-NMR(400MHz,CDCl3)δ0.88(t,J＝6.0Hz,18H),1.20-1.60(m,72H),1.72-1.84(m,3H),3.75-3.88(dd,J＝6.0Hz,12.0Hz,6H)、4.60(d、J＝6.0Hz,2H)、6.54(s,2H)
ESI-MS：829.74[M+H]+
Example Y3 >
Example (Y3-a)
Methyl gallate 0.50g (2.715 mmol) was dissolved in DMF 5mL and CPME 5mL, and 4.62g (11.064 mmol) of 11-bromomethyl trichloroethane and 1.88g (13.602 mmol) of potassium carbonate were added thereto and stirred at 110℃for 10 hours. The reaction solution was cooled to room temperature, and solids were removed by filtration. To the filtrate, 20mL of n-hexane and 20mL of 1M hydrochloric acid were added, followed by washing with a solution of 5% sodium hydrogencarbonate (20 mL) and 20mL of 20% brine. After the organic layer was dried over sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=98:2 to 95:5) to give 2.64g (yield 81.5%) of compound (Y3-1).
1H-NMR(400MHz,CDCl3)δ0.84-0.91(m,18H),1.20-1.56(m,120H),1.70-1.84(m,3H),3.85-3.92(m,9H),7.24(s,2H)
Example (Y3-b)
2.60G (2.177 mmol) of compound (Y3-1) was dissolved in 40mL of THF, and 4.4mL (1.5M toluene solution, 6.531 mmol) of diisobutylaluminum hydride was added dropwise thereto under ice-cooling, and stirred under ice-cooling for 2 hours. To the reaction mixture was added 4mL of 0.2M hydrochloric acid to stop the reaction, and the solvent was removed under reduced pressure. After 40mL of ethyl acetate was added to the residue, the mixture was washed 2 times with 20mL of 1M hydrochloric acid, and then washed with 20mL of 5% sodium hydrogencarbonate and 20mL of 20% brine. After the organic layer was dried over a proper amount of sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=95:5 to 90:10) to give 2.08g of compound (Y3-2) (yield 82.3%).
1H-NMR(400MHz,CDCl3)δ0.84-0.92(m,18H),1.20-1.60(m,72H),1.72-1.84(m,3H),3.80(dd,J＝7.0Hz,14.0Hz,6H)、4.60(d、J＝7.0Hz,2H)、6.54(s,2H)
ESI-MS：1166.10[M++H]
Example Y4 >, an
Example (Y4-a)
1.35G (8.0 mmol) of methyl 3, 5-dihydroxybenzoate was dissolved in 70mL of DMF, 8.0g (19.2 mmol) of 11-bromomethyl trichloroethane and 3.32g (24.0 mmol) of potassium carbonate were added thereto, and the mixture was stirred at 90℃for 7 hours. After potassium carbonate was removed by filtration, 100mL of water and 100mL of ethyl acetate were added to the filtrate, and the mixture was washed with water (100 mL) and 20% brine (100 mL) in this order. After drying the organic layer over sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane: ethyl acetate=98:2 to 80:20) to give 4.8g (yield 71.0%) of compound (Y4-1).
1H-NMR(400MHz,CDCl3)δ0.83-1.00(m,12H),1.32-1.61(m,80H),1.81-1.90(m,2H),3.75-3.85(m、4H)、3.90(s,3H),6.34(s,1H),7.15(s,2H)
Example (Y4-b)
4.8G (5.68 mmol) of compound (Y4-1) was dissolved in 80mL of THF, 11.4mL (1.5M toluene solution, 17.0 mmol) of diisopropylaluminum hydride was added under ice-cooling, and the mixture was stirred at room temperature for 3 hours. To the reaction mixture was added 10% rochelle salt solution (100 mL) to stop the reaction, and then 200mL of ethyl acetate was added to wash the mixture in a liquid-separated manner, and the organic layer was further washed 2 times with 100mL of water. After drying the organic layer over sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane: ethyl acetate=95:5 to 80:20) to give 3.5g (yield 75.7%) of compound (Y4-2).
1H-NMR(400MHz,CDCl3)δ0.83-1.00(m,12H),1.32-1.61(m,80H),1.81-1.90(m,2H),3.80(d、4H、J＝5.2Hz)、4.62(s,2H),6.38(s,1H),6.50(s,2H)
ESI-MS：813.74[M+H]+
[ Confirmation of solubility of tag in organic solvent ]
Example Y5 >
The solubility (25 ℃) of the compound (Y1-2) produced in example Y1, the compound (Y2-2) produced in example Y2, the compound (Y3-2) produced in example Y3 and the compound (Y4-2) produced in example Y4 in various solvents was measured.
[ Experimental methods ]
The compounds (Y1-2), (Y2-2), (Y3-2) and (Y4-2) of examples Y1 to Y4 and the linear chain-containing compound (comparative examples Y1 and Y2 of reference Table Y2) as comparative examples were dissolved in each solvent at 25℃in a saturated manner, and the solubility (unit: weight%) was measured (tables Y1, Y2). The solvent used was CPME, MTHP, toluene and chloroform.
The linear C 18H37 compound shown in comparative example Y1 was synthesized by the method described in examples of Japanese patent application laid-open No. 2000-44493, and the linear C 22H45 compound shown in comparative example Y2 was synthesized by the method described in Bio organic & MEDICINAL CHEMISTRY LETTERS,21, (2011), 4476-4479.
Table Y1 shows the results of the solubility of example Y1 (compound (Y1-2)), example Y2 (compound (Y2-2)), example Y3 (compound (Y3-2)) and example Y4 (compound (Y4-2)). Table Y2 shows the results of the solubilities of comparative examples Y1 and Y2. The values in parentheses in table Y2 indicate how many times the solubilities of the compounds of examples Y1 to Y4 (i.e., 50 divided by the solubility) are equivalent, assuming that the solubilities of the compounds of examples Y1 to Y4 are 50 (wt%).
Table Y1
Table Y2
[ Experimental results ]
The solubility of the compounds of examples Y1 to Y4 is a value of more than 50% by weight. In contrast, the compounds of comparative examples Y1 and Y2 were each less than 50 (mass%) and the maximum was only 12.4 (mass%) (comparative example Y1, chloroform). As shown in table Y2, the solubility of the compounds of examples Y1 to Y4 was at least 4.0 times greater than the solubility of the compounds of comparative examples Y1 and Y2 (refer to comparative example Y1-chloroform), and greater than 38.5 times greater than the solubility of the compounds of comparative example Y2-toluene, even if the difference was small.
As described above, the compounds of examples Y1 to Y4 were confirmed to exhibit high solubility of more than 4.0 to 38.5 times in all solvents, compared with the compounds described in comparative examples Y1 and Y2, in which the corresponding side chains were linear. From these results, it was found that the above-mentioned compounds can function as excellent tags in peptide synthesis in the peptide production method.
[ Confirmation of hydrophobicity of tag ]
Example Y6 >
The hydrophobicity of the compounds (Y1-2), (Y2-2), (Y3-2) and (Y4-2) of examples Y1 to Y4 was evaluated using liquid chromatography (HPLC).
[ Experimental methods ]
10Mg/10mL (THF) solutions of each of the compounds (Y1-2), (Y2-2), (Y3-2) and (Y4-2) of examples Y1 to Y4, the linear C 18H37 compound of comparative example Y1 and the linear C 22H45 compound of comparative example Y2 were prepared, and the retention times (elution times) of liquid chromatography (HPLC) under the following conditions were compared (Table Y3).
Table Y3
Liquid Chromatography (HPLC) conditions:
chromatographic column: inert Sustand C18 (3 μm, 4.6X1125 mm)
Mobile phase B: THF, mobile phase a:0.1% aqueous trifluoroacetic acid solution
Eluent: mobile phase a/mobile phase b=25/75 (isocratic)
Flow rate: 1.0mL/min
Column temperature: 40 DEG C
A detector: ultraviolet visible spectrum detector (lambda=220 nm)
[ Experimental results ]
As shown in Table Y3, the compounds (Y3-2) and (Y4-2) of examples Y3 and Y4 were higher in hydrophobicity than the compounds of comparative examples Y1 and Y2. The hydrophobicity of the compound (Y2-2) according to example Y2 was substantially equivalent to that of comparative examples Y1 and Y2. In contrast, the hydrophobicity of the compound (Y1-2) according to example Y1 was not higher than that of the compounds according to comparative examples Y1 and Y2.
Peptide Synthesis example
Peptide synthesis was performed using the compound (Y2-2) and the compound (Y3-2). In the following formula, the compound (Y2-2) is also referred to as HO-Tag (Y2-2), and the compound (Y3-2) is also referred to as HO-Tag (Y3-2). Specifically, tag (Y2-2) represents a moiety after removing-OH from the compound (Y2-2), and Tag (Y3-2) represents a moiety after removing-OH from the compound (Y3-2).
Example Y7：PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Y2-2) Synthesis
Example Y7-1: synthesis of HO-Leu-OTag (Y1-4)
1.0G (1.21 mmol) of compound (Y2-2) was dissolved in 25mL of a mixture of MTHP/acetonitrile (8/2), and 0.554g (1.57 mmol) of Fmoc-Leu-OH, 0.30g (1.57 mmol) of EDCI. HCl and 0.0147g (0.121 mmol) of DMAP were added and stirred at room temperature for 2 hours. Morpholine 42.2 μl (0.482 mmol) was added and stirred at room temperature for 30 minutes. Morpholine 2.11mL (24.1 mmol) and DBU 1.26mL (8.44 mmol) were added and stirred at room temperature for 1 hour. The reaction solution was transferred to a separatory funnel, washed with 2 times 18mL of 20% brine, and separated. The organic layer was washed with a further 3 times of 18mL of 2M hydrochloric acid and separated, and further washed with 18mL of 0.5M aqueous sodium bicarbonate and separated. After drying the organic layer with an appropriate amount of sodium sulfate, the solution of the amino acid condensate (HO-Leu-OTag (Y2-2)) was obtained by washing with an appropriate amount of MTHP and filtering.
Example Y7-2: synthesis of HO-Ile-Leu-OTag (Y2-2)
To the solution of HO-Leu-OTag (Y2-2) obtained above, 5.0mL of acetonitrile, 0.511g (1.45 mmol) of Fmoc-Ile-OH, 0.277g (1.45 mmol) of EDCI. HCl and 0.0514g (0.362 mmol) of Oxyma were added and stirred at room temperature for 1 hour. Morpholine 42.2 μl (0.482 mmol) was added and stirred at room temperature for 30 minutes. Morpholine 2.11mL (24.1 mmol) and DBU 1.26mL (8.44 mmol) were added and stirred at room temperature for 1 hour. The reaction solution was transferred to a separatory funnel, washed with 2 times 18mL of 20% brine, and separated. The organic layer was further washed with 18mL of 2M hydrochloric acid 3 times and separated, and further washed with 18mL of 0.5M aqueous sodium hydrogencarbonate solution and separated, and the organic layer was dried over an appropriate amount of sodium sulfate, and then filtered while washing with an appropriate amount of MTHP to obtain an amino acid condensate (HO-Ile-Leu-OTag (Y2-2)) solution.
Example Y7-3: synthesis of H-Tyr (tBu) -Ile-Leu-OTag (Y2-2)
The same operations as in example Y7-2 were conducted except that Fmoc-Tyr (tBu) -OH was used in the condensed amino acid, to obtain a solution of H-Tyr (tBu) -Ile-Leu-OTag (Y2-2).
Examples Y7-4: synthesis of H-Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2)
The same operation as in example Y7-2 was performed except that Fmoc-Pro-OH was used in the condensed amino acid, to obtain a solution of H-Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2).
Examples Y7-5: synthesis of H-Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2)
The same operations as in example Y7-2 were conducted except that Fmoc-Arg (Pbf) -OH and MTHP/DMF (8/2) were used as a reaction solvent in the condensed amino acid, to give a H-Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2) solution.
Examples Y7-6: synthesis of H-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2)
The same operations as in example Y7-2 were conducted except that Fmoc-Arg (Pbf) -OH and MTHP/DMF (8/2) were used as a reaction solvent in the condensed amino acid, to give a H-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2) solution.
Examples Y7-7: synthesis of H-Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2)
The same operations as in example Y7-2 were conducted except that Fmoc-Pro-OH and 2mL of acetone was added to the condensed amino acid for pipetting, and the pipetting was conducted, whereby a H-Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2) solution was obtained.
Examples Y7-8: synthesis of H-Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2)
The same operations as in example Y7-2 were performed except that Fmoc-Lys (Boc) -OH and 2mL of acetone were added to the condensed amino acid to separate the amino acid, thereby obtaining a solution of H-Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2).
Examples Y7-9: synthesis of H-Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2)
The same operation as in example Y7-2 was performed except that Fmoc-Asn (Trt) -OH was used in the condensed amino acid, to give a solution of H-Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2).
Examples Y7-10: synthesis of H-Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2)
The same operations as in example Y7-2 were performed, except that Fmoc-Glu (OtBu) -OH was used in the condensed amino acid, to give a solution of H-Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2).
Examples Y7-11: synthesis of H-Tyr (tBu) -Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2)
The same operation as in example Y7-2 was performed except that Fmoc-Tyr (tBu) -OH was used in the condensed amino acid, to give a solution of H-Tyr (tBu) -Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2).
Example Y7-12：H-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Y2-2) Synthesis
The same operation as in example Y7-2 was performed except that Fmoc-Leu-OH was used in the condensed amino acid, to give a solution of H-Leu-Tyr (tBu) -Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y2-2).
Example Y7-13：H-PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Y2-2) Synthesis
The same operations as in example Y7-2 were conducted except that Fmoc-pyroGlu-OH was used in the condensed amino acid, to obtain H-PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Y2-2) solution.
The solvent was removed from the obtained organic layer under reduced pressure, 120mL of an aqueous 80% acetonitrile solution was added to the residue under ice-cooling, and the obtained precipitate was filtered and dried under reduced pressure to obtain H-PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Y2-2)(1.401g、0.40mmol、 yield 33.1%. (yield was calculated from starting compound (Y2-2) (1.21 mmol)
ESI-MS：3497.94〔M〕+
Even though the 13-peptide extension reaction ,H-PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Y2-2) composed of examples Y7-1 to Y7-13 was carried out, the yield was high, and therefore it was confirmed that by-products could be easily removed when a specific cyclic amine containing only one nitrogen atom such as morpholine was used as a capturing agent.
Using a small amount of this compound, trifluoroacetic acid (TFA) was added: water: triisopropylsilane (TIS) =9.5: 2.5:2.5 for 3 hours at room temperature, and deprotecting the protecting groups of the tag and amino acid side chains to confirm ESI-MS of H-pyroGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH: 1672.77 [ M+H ] +.
Example Y8: synthesis of H-Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y3-2)
Peptide synthesis was carried out in the same manner as in examples Y7-1 to Y7-6 using 0.5g (0.428 mmol) of the compound (Y3-2) as a tag. In the fraction obtained by condensing Fmoc-Lys (Boc) -OH at residue 8, it was confirmed that it took a long time to separate the organic layer from the aqueous layer when the fraction was taken with 2M hydrochloric acid. The synthesized solution was allowed to stand for one day and night, and the mass spectrometry was performed on the organic layer separated after the standing, to confirm the mass number of the target H-Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Y3-2).
ESI-MS：2849.49〔M〕+
Comparative example Y3
Synthesis of H-Leu-OTag (comparative example Y1)
The synthesis was carried out in the same manner as in example Y7-1 using 1.0g (1.09 mmol) of the compound shown in comparative example Y1 as a tag. After Fmoc-Leu-OH was condensed, a large amount of solid was precipitated in the solution, which resulted in difficulty in the solution separation, and thus it was confirmed that the peptide was difficult to synthesize.
[ Concerning peptide Synthesis results ]
Table Y4 summarizes the peptide synthesis results (number of residues) of example Y7, example Y8 and comparative example Y3, and the solubility and hydrophobicity of the benzyl compounds used. When comparing the compound (Y2-2) and the compound (Y3-2) which are examples of the benzyl compound of the present invention with the linear C 18H37 compound of comparative example Y1, it was confirmed that the higher the solubility of the benzyl compound in an organic solvent, the more the number of peptide synthesis residues. In addition, when the compound (Y2-2) and the compound (Y3-2) were compared, it was confirmed that the number of peptide synthesis residues in the compound (Y2-2) having moderate hydrophobicity was most increased.
From these results, it is clear that a tag having high solubility in an organic solvent, low hydrophobicity and moderate hydrophobicity is suitable as a tag for producing a long-chain peptide. As such tags, the benzyl compounds (Y1-2), (Y2-2), (Y3-2) and (Y4-2)) of examples Y1 to Y4 are useful in the synthesis of long chain peptides by the liquid phase tag method, among which particularly useful are compounds (Y2-2) and (Y3-2) of examples Y2 and Y3, and more useful is compound (Y2-2) of example Y2.
Table Y4
Next, an example of embodiment 3 of the present invention described above is given. The following shows a method for synthesizing a peptide having the sequence shown below as an example, but the present invention is not limited thereto.
[ Deprotection of Fmoc group ]
The Fmoc removal protocol used in this example is shown below. The use of the above-mentioned Y1B (also referred to as carrier (ZA)) as a carrier compound is exemplified, but the carrier compound usable in the method of the present invention is not limited to Y1B. The addition amount of each reagent is also merely an example, and is not limited thereto. In addition, the carrier compound Y1B is the same as the compound Y2-2.
The starting material was dissolved to 18v/w in a mixture of MTHP/DMF (8/2), morpholine (20.0 eq) and DBU (7.0 eq) were added and stirred at room temperature for 1 hour. The reaction solution was transferred to a separating funnel, and 2N hydrochloric acid was added for washing and separation to obtain a solution for removing Fmoc.
[ Peptide Synthesis method Using Cyclic amine Capture agent ]
Fmoc-AA-OH as starting material was dissolved to 18v/w in MTHP/DMF (8/2) mixture, fmoc amino acid (1.3 eq), EDCI. HCl (1.3 eq) and Oxyma 0.0514g (0.1 eq) were added and stirred at room temperature for 1 hour. Morpholine (0.4 eq) was added and stirred at room temperature for 30 minutes. Morpholine (20.0 eq) and DBU (7.0 eq) were added and stirred at room temperature for 1 hour. The reaction solution was transferred to a separatory funnel, washed with 2 times 20% brine (18 v/w) and separated. The organic layer was further washed with 3 times 2M hydrochloric acid (18 v/w) and separated, and further washed with 0.5M aqueous sodium hydrogencarbonate (18 v/w) and separated, and the organic layer was dried over an appropriate amount of sodium sulfate, and then filtered while washing with an appropriate amount of MTHP to obtain an amino acid condensate solution.
Example Z1：PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z1-13) Synthesis
Example Z1-1: synthesis of HO-Leu-OTag (Z1-1)
An amino acid condensate (HO-Leu-OTag (Z1-1)) solution was obtained in the same manner as in example Y7-1. The amino acid condensate (HO-Leu-OTag (Z1-1)) was identical to the amino acid condensate (HO-Leu-OTag (Y2-2)) synthesized in example Y7-1.
Examples Z1-2: synthesis of HO-Ile-Leu-OTag (Z1-2)
An amino acid condensate (HO-Ile-Leu-OTag (Z1-2)) solution was obtained in the same manner as in example Y7-2. The amino acid condensate (HO-Ile-Leu-OTag (Z1-2)) was identical to the amino acid condensate (HO-Leu-OTag (Y2-2)) synthesized in example Y7-2.
Examples Z1-3: synthesis of H-Tyr (tBu) -Ile-Leu-OTag (Z1-3)
The same operations as in example Z1-2 were performed, except that Fmoc-Tyr (tBu) -OH was used in the condensed amino acid, to give a solution of H-Tyr (tBu) -Ile-Leu-OTag (Z1-3).
Examples Z1 to 4: synthesis of H-Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-4)
The same operation as in example Z1-2 was performed except that Fmoc-Pro-OH was used in the condensed amino acid, to give a solution of H-Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-4).
Examples Z1 to 5: synthesis of H-Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-5)
The same operation as in example Z1-2 was performed except that Fmoc-Arg (Pbf) -OH and MTHP/DMF (8/2) was used as a reaction solvent in the condensed amino acid, to obtain a H-Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-5) solution.
Examples Z1 to 6: synthesis of H-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-6)
The same operation as in example Z1-2 was performed except that Fmoc-Arg (Pbf) -OH and MTHP/DMF (8/2) was used as a reaction solvent in the condensed amino acid, to give a H-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-6) solution.
Examples Z1 to 7: synthesis of H-Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-7)
The same operations as in example Z1-2 were performed, except that Fmoc-Pro-OH was used in the condensed amino acid and 2mL of acetone was added to carry out the pipetting in 3 rd 2M hydrochloric acid, to thereby obtain a H-Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-7) solution.
Examples Z1 to 8: synthesis of H-Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-8)
The same operations as in examples Z1-8 were performed, except that Fmoc-Lys (Boc) -OH and 2mL of acetone were added to the condensed amino acid to separate the amino acid, thereby obtaining a solution of H-Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-8).
Examples Z1 to 9: synthesis of H-Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-9)
The same operations as in examples Z1-2 were performed, except that Fmoc-Asn (Trt) -OH was used in the condensed amino acid, to give a solution of H-Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-9).
Examples Z1 to 10: synthesis of H-Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-10)
The same operations as in examples Z1-2 were performed, except that Fmoc-Glu (OtBu) -OH was used in the condensed amino acid, to give a solution of H-Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-10).
Examples Z1 to 11: synthesis of H-Tyr (tBu) -Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-11)
The same operations as in examples Z1-2 were performed, except that Fmoc-Tyr (tBu) -OH was used in the condensed amino acid, to give a solution of H-Tyr (tBu) -Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-11).
Example Z1-12：H-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z1-12) Synthesis
The same operation as in example Z1-2 was performed except that Fmoc-Leu-OH was used in the condensed amino acid, to give a solution of H-Leu-Tyr (tBu) -Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z1-12).
Example Z1-13：PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z1-13) Synthesis
The same operations as in example Z1-2 were performed, except that Fmoc-pyroGlu-OH was used in the condensed amino acid, to obtain H-PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z1-13) solution.
Examples Z1 to 14: deprotection of C-terminal support and side chain functionalities
The solvent was removed from the obtained organic layer under reduced pressure, 120mL of an 80% acetonitrile aqueous solution was added to the residue under ice-cooling, and the obtained precipitate was filtered and dried under reduced pressure to obtain PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z1-13)(1.401g、0.40mmol、 yield 33.1%).
ESI-MS：3497.94〔M〕+
Using a small amount of this compound, trifluoroacetic acid (TFA) was added: water: triisopropylsilane (TIS) =95: 2.5:2.5, and after deprotecting the protecting groups of the C-terminal carrier and the amino acid side chain, confirming the ESI-MS of PyroGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH: 1672.77 [ M+H ] +.
Example Z2: synthesis Using C-terminal vector B PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z2-13)
The C-terminal carrier B used in this example Z2 was the following compound (Z7).
Example Z2-1: synthesis of H-Tyr (tBu) -Ile-Leu-OTag (Z2-2)
To a solution of H-Ile-Leu-OTag (Z2-1) (1.32 mmol) in MTHP.4 mL were added DMF 3.6mL, fmoc-Tyr (tBu) -OH 0.789g (1.72 mmol), EDCI. HCl 0.329g (1.72 mmol) and Oxyma 0.0563g (0.396 mmol) and stirred at room temperature for 1 hour. Morpholine 45.7 μl (0.528 mmol) was added and stirred at room temperature for 30 min. Morpholine 2.28mL (26.4 mmol) and dbu1.38mL (9.24 mmol) were added and stirred at room temperature for 1 hour. Under ice-cooling, the reaction solution after adding 10.89mL of 6M hydrochloric acid was transferred to a separating funnel, and 18mL of 0.1M hydrochloric acid was added for washing and liquid separation. To the organic layer was further added 18mL of 2M hydrochloric acid and washed and separated, and further washed with 18mL of 0.5M aqueous sodium hydrogencarbonate solution and separated. After drying the organic layer with an appropriate amount of sodium sulfate, the solution of the amino acid condensate (H-Tyr (tBu) -Ile-Leu-OTag (Z2-2)) was obtained by washing with an appropriate amount of MTHP and filtering.
Example Z2-2: synthesis of H-Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-3)
The same operation as in example Z2-1 was performed except that Fmoc-Pro-OH was used in the condensed amino acid, to give a solution of H-Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-3).
Examples Z2-3: synthesis of H-Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-4)
The same operation as in example Z2-1 was performed except that Fmoc-Arg (Pbf) -OH was used in the condensed amino acid, to give a solution of H-Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-4).
Examples Z2-4: synthesis of H-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-5)
The same operation as in example Z2-1 was performed except that Fmoc-Arg (Pbf) -OH was used in the condensed amino acid, to give a H-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-5) solution.
Examples Z2-5: synthesis of H-Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-6)
The same operation as in example Z2-1 was performed except that Fmoc-Pro-OH was used in the condensed amino acid, to give a H-Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-6) solution.
Examples Z2-6: synthesis of H-Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-7)
The same operation as in example Z2-1 was performed except that Fmoc-Lys (Boc) -OH was used in the condensed amino acid and the liquid was separated as follows. The reaction solution was transferred to a separatory funnel, washed with 2 times 18mL of 20% brine and separated. The organic layer was further washed with 18mL of 2M hydrochloric acid 3 times and separated, and further washed with 18mL of 0.5M aqueous sodium hydrogencarbonate solution and separated, and after drying the organic layer with an appropriate amount of sodium sulfate, the organic layer was filtered while washing with an appropriate amount of MTHP to obtain an amino acid condensate (H-Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-7)) solution.
Examples Z2-7: synthesis of H-Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-8)
The same operation as in example Z2-1 was performed except that Fmoc-Asn (Trt) -OH was used in the condensed amino acid, to give a solution of H-Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-8).
Examples Z2-8: synthesis of H-Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-9)
The same operation as in example Z2-1 was performed except for using Fmoc-Glu (OtBu) -OH in the condensed amino acid, to give a solution of H-Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-9).
Examples Z2-9: synthesis of H-Tyr (tBu) -Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-10)
The same operations as in examples Z2-6 were performed, except that Fmoc-Tyr (tBu) -OH was used in the condensed amino acid, to give a solution of H-Tyr (tBu) -Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-10).
Example Z2-10：H-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z2-11) Synthesis
The same operation as in example Z2-1 was performed except that Fmoc-Leu-OH was used in the condensed amino acid, to give a solution of H-Leu-Tyr (tBu) -Glu (OtBu) -Asn (Trt) -Lys (Boc) -Pro-Arg (Pbf) -Arg (Pbf) -Pro-Tyr (tBu) -Ile-Leu-OTag (Z2-11).
Example Z2-11：PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z2-12) Synthesis
The same operations as in example Z2-1 were performed, except that Fmoc-pyroGlu-OH was used in the condensed amino acid, to obtain PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z2-12) solution.
The solvent was removed from the obtained organic layer under reduced pressure, 45mL of an aqueous 80% acetonitrile solution was added to the residue under ice-cooling, and the obtained precipitate was filtered and dried under reduced pressure to obtain PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z2-12)(1.4g、0.86mmol、 yield 54%). (yield was calculated from starting compound (Z2-1) (1.32 mmol)
ESI-MS：3497.94〔M〕+
Using a small amount of this compound, trifluoroacetic acid (TFA) was added: water: triisopropylsilane (TIS) =9.5: 2.5:2.5, and stirring the mixture at room temperature for 3 hours, and subjecting the tag and the protecting group of the amino acid side chain to deprotection analysis, and confirming the formation of PyroGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Pro-Tyr-Ile-Leu-OH based on ESI-MS. ESI-MS:1672.77 [ M+H ] +
Example Z3: comparison of the amount of diketopiperazine produced by various bases
To a MTHP/DMF solution (8:2) of Fmoc-Tyr (tBu) -Leu-OTag (Z3-1) was added morpholine (Z3-2) in 5 equivalents and after stirring at room temperature for 2 hours, quantitative analysis was performed by HPLC. The amount of diketopiperazine produced was equal to the amount of Tag-OH (Z3-8) produced at the same time as the diketopiperazine was produced. Therefore, the production rate of each compound was calculated by calculating the area ratio of each compound in the total area of Fmoc-Tyr (tBu) -Leu-OTag (Z3-1), H-Tyr (tBu) -Leu-OTag (Fmoc-deprotected, Z3-7) and carrier compound (Z3-8) produced when diketopiperazine was produced.
Liquid Chromatography (HPLC) conditions:
chromatographic column: inert Sustand C18 (3 μm, 4.6X1125 mm)
Mobile phase B: THF, mobile phase a:0.1% aqueous trifluoroacetic acid solution
Eluent: mobile phase a/mobile phase B: the measurement was performed under gradient conditions shown in table Z5.
Table Z1
Flow rate: 1.0mL/min
Column temperature: 40 DEG C
A detector: ultraviolet visible spectrum detector (lambda=220 nm)
Comparative example Z1
The same operation as in example Z3 was performed and the same quantitative analysis was performed, except that 5 equivalents of diethylamine (Z3-4, described in patent document 9 and non-patent document 4) was added instead of 5 equivalents of morpholine.
Comparative example Z2
The same operation as in example Z3 was performed and the same quantitative analysis was performed, except that 5 equivalents of N-methylpiperazine (Z3-5, described in patent document 2) was added instead of 5 equivalents of morpholine.
[ Experimental results ]
As shown in table Z2, the Fmoc group deprotection reaction of morpholine proceeds more slowly than other compounds such as the compound described in patent document 2 (N-methylpiperazine, Z3-5), resulting in a small amount of diketopiperazine. It was thus found that the cyclic amine of the present invention can function as an excellent capture molecule in peptide synthesis.
Table Z2
As shown in Table Z2, morpholine was able to suppress the rate of diketopiperazine formation compared to diethylamine and N-methylpiperazine. Specifically, when morpholine is used, the ratio of diketopiperazine to various compounds produced is suppressed to about 6 times that of diethylamine and about half that of N-methylpiperazine. Therefore, it was confirmed that progress of the side reaction was suppressed. This is considered to be because the cyclic amine used in the present invention is less basic, and thus can suppress unexpected deprotection of Fmoc group.

Claims (23)

1. A method of producing a peptide, comprising: a step of bringing an amino group-containing compound having an N-terminal protected with a protecting group having a fluorene skeleton into contact with a capturing agent represented by the following formula (Z1) in an organic solvent, wherein a capturing agent is obtained by bonding a byproduct having an fulvene skeleton derived from the protecting group to the capturing agent; and
A step of separating the capture body obtained from the organic solvent;
in the above-mentioned formula (Z1),
N is a nitrogen atom and is preferably a nitrogen atom,
H is a hydrogen atom and is preferably a hydrogen atom,
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented by,
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -H- (SO 2) OR- (SO 2) R (R is synonymous with-OR),
R 2a or R 2b and R 3a or R 3b may be bonded to each other or may form a ring together with the carbon atom to which they are bonded,
N 1、n2 and n 3 are each independently 1 or 2,
M is an integer of 0 or 1.
2. The method for producing a peptide according to claim 1, further comprising: and contacting the amino group-containing compound having the N-terminal protected with the protecting group with a deprotecting agent.
3. The method for producing a peptide according to claim 2, wherein the deprotecting agent is at least one base selected from the group consisting of 1, 8-diazabicyclo [5.4.0] -7-undecene (DBU), 1, 5-diazabicyclo [4.3.0] -5-nonene (DBN), 1, 4-diazabicyclo [2.2.2] octane (DABCO), potassium t-butoxide, sodium t-butoxide, triethylamine and tributylamine.
4. The method for producing a peptide according to claim 1 or 2, wherein the step of separating the capturing body comprises: adding an acidic aqueous solution into the organic solvent, washing, separating the organic solvent into an aqueous layer and an organic layer, and separating the separated aqueous layer.
5. The method for producing a peptide according to claim 4, wherein the capturing agent represented by the formula (Z1) is at least one selected from the group consisting of morpholine, piperidine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide.
6. The method for producing a peptide according to claim 1 or 2, wherein the capturing agent represented by the formula (Z1) is at least one selected from the group consisting of morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide.
7. A method of removing a protecting group comprising: a step of bringing an amino group-containing compound having an N-terminal protected with a protecting group having a fluorene skeleton into contact with a capturing agent represented by the following formula (Z1) in an organic solvent, wherein a capturing agent is obtained by bonding a byproduct having an fulvene skeleton derived from the protecting group to the capturing agent; and
A step of separating the capture body obtained from the organic solvent;
in the above-mentioned formula (Z1),
N is a nitrogen atom and is preferably a nitrogen atom,
H is a hydrogen atom and is preferably a hydrogen atom,
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented by,
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -H- (SO 2) OR- (SO 2) R (R is synonymous with-OR),
R 2a or R 2b and R 3a or R 3b may be bonded to each other or may form a ring together with the carbon atom to which they are bonded,
N 1、n2 and n 3 are each independently 1 or 2,
M is an integer of 0 or 1.
8. A remover for a protecting group having a fluorene skeleton, comprising a capturing agent represented by the following formula (Z1) and an alkaline deprotecting agent,
In the above-mentioned formula (Z1),
N is a nitrogen atom and is preferably a nitrogen atom,
H is a hydrogen atom and is preferably a hydrogen atom,
X is-CH 2 -, -O-, -S-or- (SO 2) -a divalent group represented by,
N 1 R 1a、n1 R 1b、n2 R 2a、n2 R 2b、n3 R 3a and n 3 R 3b are each independently H, -OH, -OR (R is alkyl), -SH, -SR (R is synonymous with-OR), -H- (SO 2) OR- (SO 2) R (R is synonymous with-OR),
R 2a or R 2b and R 3a or R 3b may be bonded to each other or may form a ring together with the carbon atom to which they are bonded,
N 1、n2 and n 3 are each independently 1 or 2,
M is an integer of 0 or 1.
9. The remover of claim 8, wherein the capture agent is at least one selected from the group consisting of morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine, and thiomorpholine dioxide.
10. The remover according to claim 8 or 9, wherein the deprotecting agent is at least one base selected from the group consisting of 1, 8-diazabicyclo [5.4.0] -7-undecene, 1, 5-diazabicyclo [4.3.0] -5-nonene, 1, 4-diazabicyclo [2.2.2] octane, potassium t-butoxide, sodium t-butoxide, triethylamine and tributylamine.
11. A benzyl compound (Y1) represented by the following formula (Y1),
In the formula (Y1), the amino acid sequence of the formula (I),
M Q's each represent an oxygen atom,
M R 1 are each independently a group represented by the following formula (YA):
in the formula (YA),
* The bonding position is indicated by the number of the bonding sites,
R 1a、R1b、R1c、R1d and R 1e each independently represent a hydrogen atom or an alkyl group,
N 1 represents an integer of 0 to 6, and when n 1 is 1 or more, the repeating unit represented in parentheses with n 1 is an alkylene group,
N 2 represents an integer of 0 to 6, and when n 2 is 1 or more, the repeating unit represented in parentheses with n 2 is an alkylene group,
At least two or more of R 1a、R1b、R1c and R 1d are hydrogen atoms;
In the formula (Y1), k R 2 each independently represents a hydrogen atom, an alkyl group, a cycloalkyl group, an alkoxy group, an aryl group, an aralkyl group or a halogen atom,
In the formula (Y1), X represents a hydroxyl group,
In the formula (Y1), m represents an integer of 2 or 3,
In the formula (Y1), k represents an integer of 0 to (5-m) inclusive,
In the formula (Y1), at least one of m [ Q-R 1 ] is substituted in the meta position with respect to the substituent containing the X.
12. The benzyl compound (Y1) according to claim 11, wherein the total number of carbon atoms is 40 or more and 60 or less.
13. The benzyl compound (Y1) according to claim 11, wherein each of the m R 1 is independently an organic group having one side chain, which is a group represented by the following formula (YA'),
In the formula (YA '), the catalyst is represented by the formula (YA'),
* The bonding position is indicated by the number of the bonding sites,
R 1f is a linear alkyl group having 4 or more and 12 or less carbon atoms,
R 1g is a linear alkyl group having 6 or more and 14 or less carbon atoms.
14. The benzyl compound (Y1) according to claim 13, wherein R 1f is a linear alkyl group having 4 or more and 10 or less carbon atoms,
The R 1g is a linear alkyl group having 6 or more and 12 or less carbon atoms.
15. A method of producing a peptide, comprising: a dissolving step of dissolving the benzyl compound according to any one of claims 11 to 14 in a soluble solvent;
A condensation reaction step of condensing the dissolved benzyl compound with an amino acid having an N-terminal protected by an N-terminal protecting group to form a first condensate;
A scavenging step of adding a first base to the soluble solvent containing the first condensate to scavenge an amino acid active ester, further adding the first base and a second base to the soluble solvent, deprotecting the N-terminal protecting group of the first condensate, and scavenging by-products derived from the N-terminal protecting group by the first base; and
And a liquid separation step of adding an acidic aqueous solution to the soluble solvent containing the capturing substance captured in the removal step, and washing the mixture, wherein the liquid is separated into an aqueous layer and an organic layer, the capturing substance and unnecessary substances are removed to the aqueous layer, and the first condensate is deprotected at the N-terminal protecting group in the organic layer to obtain a second condensate.
16. The method for producing a peptide according to claim 15, wherein the condensation reaction step comprises: condensing the N-terminal-protected nth amino acid into a 2n-th condensate to form a (2n+1) -th condensate, wherein the 2n-th condensate contains an N-terminal-unprotected amino acid and a C-terminal-protected amino acid with the benzyl compound and contains N-number of amino acids,
The scavenging step includes a step of deprotecting the N-terminal protecting group of the (2n+1) -th condensate,
The liquid separation step includes a step of deprotecting the (2n+1) th condensate in the organic layer to obtain a (2n+2) th condensate,
And n is a natural number of 2 or more.
17. The method for producing a peptide according to claim 16, wherein n is 5 or more.
18. The method for producing a peptide according to claim 15, wherein the step of separating the liquid further comprises a step of adding a ketone liquid separation promoting solvent to the soluble solvent.
19. A benzyl compound (X1) represented by the following formula (X1):
In the formula (X1), the amino acid sequence of the formula (X),
M Q 1 and Q 2 are each an oxygen atom,
M R 1 are each independently alkylene,
M R 2 are each independently an optionally substituted alkyl group, an optionally substituted aralkyl group or an optionally substituted aryl group,
K R 3 are each independently a hydrogen atom, an alkyl group, an alkoxy group or a halogen atom,
X is a hydroxyl group, and is a hydroxyl group,
M is an integer of 2 or 3,
K represents an integer of 0 to (5-m).
20. The benzyl compound (X1) according to claim 19, wherein the m R 1 are alkylene groups having 2 or more and 16 or less carbon atoms.
21. The benzyl compound (X1) of claim 19 or 20, wherein the m R 2 are aryl groups having substituents containing halogen atoms.
22. The benzyl compound (X1) according to claim 19 or 20, wherein the m R 2 are alkyl groups having 5 or more and 28 or less carbon atoms.
23. The benzyl compound (X1) of claim 22, wherein the m R 2 are linear alkyl groups or alkyl groups having a total number of branches of 1 or 2, and are groups represented by the following formula (XA):
In the formula (XA),
* The bonding position is indicated by the number of the bonding sites,
R 2a、R2b、R2c、R2d and R 2e each independently represent a hydrogen atom or an alkyl group,
N 1 represents an integer of 0 to 16,
N 2 represents an integer of 0 to 16,
At least two or more of R 2a、R2b、R2c and R 2d are hydrogen atoms.
CN202280074943.1A 2021-12-27 2022-11-14 Method for producing peptide, method for removing protecting group, remover, and benzyl compound Pending CN118234736A (en)

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