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WO2023127331A1 - Procédé de production de peptide, procédé d'élimination de groupe protecteur, agent d'élimination et composé benzylique - Google Patents

Procédé de production de peptide, procédé d'élimination de groupe protecteur, agent d'élimination et composé benzylique Download PDF

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Publication number
WO2023127331A1
WO2023127331A1 PCT/JP2022/042265 JP2022042265W WO2023127331A1 WO 2023127331 A1 WO2023127331 A1 WO 2023127331A1 JP 2022042265 W JP2022042265 W JP 2022042265W WO 2023127331 A1 WO2023127331 A1 WO 2023127331A1
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group
compound
represented
peptide
solvent
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Japanese (ja)
Inventor
宗江 葛西
康嗣 下田
誠 佐藤
杏梨 上原
圭介 松浦
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Tokuyama Corp
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Tokuyama Corp
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Priority to CN202280074943.1A priority Critical patent/CN118234736A/zh
Priority to JP2022578817A priority patent/JP7260725B1/ja
Publication of WO2023127331A1 publication Critical patent/WO2023127331A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/23—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • One aspect of the present invention relates to a novel benzyl compound, particularly to a novel benzyl compound used in a peptide synthesis method using a tag synthesis method, and a peptide synthesis method using the benzyl compound.
  • Another aspect of the present invention is to efficiently capture a by-product having a fulvene skeleton generated during the deprotection reaction of a protective group having a fluorene skeleton in liquid-phase peptide synthesis, and to efficiently remove the by-product by washing. It also relates to methods, methods of producing peptides using the methods, and the like.
  • solid phase peptide synthesis method SPPS method
  • liquid phase peptide synthesis method LPPS method
  • the solid-phase peptide synthesis method purifies the peptide by filtering out unnecessary substances in the condensation reaction of amino acids, so that the peptide can be synthesized relatively easily, and therefore, it is suitable for the synthesis of long-chain peptides.
  • the solid-phase peptide synthesis method uses an excessive amount of amino acids and washing solvents, resulting in a high synthesis cost and is considered unsuitable for large-scale peptide synthesis.
  • liquid-phase peptide synthesis method is used for large-scale peptide synthesis.
  • the elongation of the peptide chain tends to become difficult when the peptide chain becomes long, and there is a problem in long-chain peptide synthesis.
  • the synthesis method is a method of synthesizing a peptide using a protective group soluble in a solution (a C-terminal protective group of an amino acid, hereinafter also referred to as a "tag").
  • a peptide is synthesized by binding amino acids to the tag possessed and sequentially repeating the elongation reaction.
  • this synthetic method by solidifying (for example, crystallizing) only the peptide bound to the tag at each elongation step, the solidified substance can be easily separated and purified.
  • a benzyl alcohol tag having linear alkyl groups each having 18 carbon atoms at the 3, 4, and 5 positions disclosed in Patent Document 4 and a tag at the 3 and 5 positions disclosed in Patent Document 2
  • the benzyl alcohol tags each having a linear alkyl chain of 22 carbon atoms have low solubility in organic solvents that satisfy the above conditions, and thus only precipitate from the reaction system during peptide synthesis. Moreover, it was often difficult to separate and purify the compound after the reaction.
  • Patent Document 3 describes an organic group having at least one aliphatic hydrocarbon group having one or more branched chains, a total number of branched chains of 3 or more, and a total carbon number of 14 or more and 300 or less. Branched chain-containing aromatic compounds having substituents are disclosed.
  • Patent Document 6 discloses a benzyl compound having a group containing a -O-Si- structure at its terminal.
  • the benzyl compound described in Patent Document 6 is highly soluble in organic solvents and can be effectively used for liquid phase synthesis.
  • the technique of using the Fmoc group as an amino group-protecting group of amino acids is often used.
  • a by-product derived from the Fmoc group dibenzofulvene (DBF)
  • DBF dibenzofulvene
  • a technique is known in which a reagent for removing DBF is used, and an adduct of DBF and a removing reagent (hereinafter sometimes abbreviated as DBF-capture) is removed by liquid separation washing.
  • Patent Document 1 describes a method in which a thiol-containing carboxylic acid or a thiol-containing sulfonic acid is added during deprotection of an Fmoc group to generate a DBF-trapper, which is then removed by washing with an alkaline solution.
  • the thiol-containing carboxylic acid added as a scavenger reacts with the amino acid to produce a thioester. Since this thioester is an active ester, it may react with the amino group generated after removing Fmoc to form a double hit product.
  • the capture of DBF with a thiol compound reacts with the thiol structure at the side chain site of the cysteine residue contained in the peptide sequence, producing other byproducts. there is a risk of
  • Patent Document 2 DBF generated in the deprotection of the Fmoc group is reacted with a divalent or higher water-soluble amine (for example, N-methylpiperazine) to obtain a DBF-captured product, which is then removed by acidic liquid separation washing. method is described.
  • a divalent or higher water-soluble amine for example, N-methylpiperazine
  • Patent Document 9 and Non-Patent Document 4 disclose that the N-terminal Fmoc group is deprotected under basic conditions in the presence of a reagent such as diethylamine.
  • the branched chain-containing aromatic compound described in Patent Document 3 requires an expensive noble metal reduction catalyst such as platinum-carbon to produce the branched chain, so when applied to mass production, the cost increases. Therefore, there is room for improvement.
  • the benzyl compound described in Patent Document 6 has a possibility that the O—Si bond is cut under conditions such as acidic liquid separation, resulting in considerable decomposition, and there is room for improvement (see also Non-Patent Document 5). see).
  • a divalent or higher water-soluble amine is used, and there are operational problems such as the need to use a large amount of acid for neutralization.
  • water-soluble divalent amines represented by N-methylpiperazine and diethylamine used in Patent Document 10 and Non-Patent Document 4 have sufficient basicity. Therefore, when a water-soluble divalent amine is added for the purpose of inactivating amino acid active esters generated from surplus amino acids and condensing agents during the condensation reaction, an unintended deprotection reaction of the Fmoc group may proceed. There is As a result, other side reactions such as the formation of diketopiperazine may proceed.
  • one of the objects of the present invention is to solve the above problems and improve the solubility of the tag in organic solvents so that it can be precipitated or insolubilized during the peptide synthesis or during liquid separation after the reaction.
  • the object of the present invention is to provide a tag that does not contain a soluble tag, a method for producing the same, and a method for synthesizing a peptide using the tag.
  • one of the objects of the present invention is to suppress the formation of double-hit compounds and diketopiperazines, and to facilitate production of a compound having a fulvene skeleton, which is a by-product that can be generated after deprotection of a protective group having a fluorene skeleton. It is an object of the present invention to provide a method for removing a protective group, which enables easy separation of the trapped substance from the by-product, a method for producing a peptide comprising the step of removing the protective group, and an agent for removing the protective group.
  • the present inventors have made intensive studies to solve the above problems, and found that an aromatic ring compound having a substituent, or an alkyl group and an aralkyl group are introduced into a benzene ring having a benzyl alcohol via an oxyarene group.
  • the present inventors have found that a benzyl compound that is highly soluble in an organic solvent and highly hydrophobic can be provided, and completed one embodiment of the present invention.
  • benzyl compound according to one aspect of the present invention has the following formula (X1):
  • m Q 1 and Q 2 are each an oxygen atom
  • m R 1 are each independently an alkylene group
  • 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
  • m is an integer of 2 or 3
  • k represents an integer from 0 to (5-m).
  • X1 is a benzyl compound (X1) represented by
  • the present inventors found that by using a highly hydrophobic tag in addition to being highly soluble in an organic solvent, peptides can be synthesized more uniformly in an organic solvent that is immiscible with the aqueous layer. Therefore, when unnecessary substances are removed by liquid separation through the aqueous layer after the reaction, the peptide bound to the tag does not precipitate or become insoluble, and the loss of the peptide bound to the tag to the aqueous layer is suppressed. Based on the idea that it might lead to an improvement in yield, the inventors conducted intensive studies.
  • benzyl compound according to one aspect of the present invention has the following formula (Y1):
  • m Q's each represent an oxygen atom
  • m R 1 are each independently represented by the following formula (YA):
  • R 1a , R 1b , R 1c , R 1d and R 1e each independently represent a hydrogen atom or an alkyl group
  • n 1 represents an integer of 0 to 6
  • n 2 represents an integer of 0 or more and 6 or less
  • R 1a , R 1b , R 1c and R 1d are hydrogen atoms.
  • 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
  • X represents a hydroxyl group
  • m represents an integer of 2 or 3
  • k represents an integer of 0 or more (5-m) or less
  • At least one of m [QR 1 ] is substituted at the meta position with respect to the substituent containing X.
  • ] is a benzyl compound (Y1) represented by
  • the present inventors have made intensive studies and found that, among cyclic amines, a specific cyclic amine containing only one nitrogen atom in particular is used as a scavenger to obtain a fluorene skeleton such as an Fmoc group. Capturing a by-product having a fulvene skeleton such as DBF generated in the deprotection reaction of a protecting group having and separating the trapped body with the by-product from the reaction system can easily remove the by-product and completed one aspect of the present invention.
  • the method for producing a peptide according to one aspect of the present invention is In an organic solvent, an amino group-containing compound protected with a protective group having a fluorene skeleton at the N-terminus is brought into contact with a scavenger represented by the following formula (Z1) to obtain a compound having a fulvene skeleton derived from the protective group. obtaining a capturing body in which the by-product and the capturing agent are bound; a step of separating the captured body obtained from the organic solvent; including, It is a peptide manufacturing method.
  • N is a nitrogen atom
  • H is a hydrogen atom
  • X is a divalent group represented by —CH 2 —, —O—, —S—, or —(SO 2 )—
  • n 1 R 1a , n 1 R 1b , n 2 R 2a , n 2 R 2b , n 3 R 3a , and n 3 R 3b are each independently H , —OH, —OR (R is an alkyl group), —SH, —SR (R is as defined for —OR above.), —(SO 2 )H, or —(SO 2 )
  • R is the same as that of -OR above
  • R 2a or R 2b and R 3a or R 3b may be bonded to each other to form a ring together with the carbon atoms to which they are bonded
  • n 1 , n 2 and n 3 are each independently 1 or 2
  • m is an integer of 0 or
  • a method for removing a protecting group comprises, in an organic solvent, an amino group-containing compound whose N-terminus is protected with a protecting group having a fluorene skeleton, and a scavenger represented by the following formula (Z1): and a step of contacting with to obtain a capturing body in which a by-product having a fulvene skeleton derived from the protecting group and the capturing agent are bound; a step of separating the captured body obtained from the organic solvent; including, A method for removing protecting groups.
  • N is a nitrogen atom
  • H is a hydrogen atom
  • X is a divalent group represented by —CH 2 —, —O—, —S—, or —(SO 2 )—
  • n 1 R 1a , n 1 R 1b , n 2 R 2a , n 2 R 2b , n 3 R 3a , and n 3 R 3b are each independently H , —OH, —OR (R is an alkyl group), —SH, —SR (R is as defined for —OR above.), —(SO 2 )H, or —(SO 2 )
  • R is the same as that of -OR above
  • R 2a or R 2b and R 3a or R 3b may be bonded to each other to form a ring together with the carbon atoms to which they are bonded
  • n 1 , n 2 and n 3 are each independently 1 or 2
  • m is an integer of 0 or
  • the remover according to one aspect of the present invention is an agent for removing a protecting group having a fluorene skeleton, containing a scavenger represented by the following formula (Z1) and a basic deprotecting agent.
  • N is a nitrogen atom
  • H is a hydrogen atom
  • X is a divalent group represented by —CH 2 —, —O—, —S—, or —(SO 2 )—
  • n 1 R 1a , n 1 R 1b , n 2 R 2a , n 2 R 2b , n 3 R 3a , and n 3 R 3b are each independently H , —OH, —OR (R is an alkyl group), —SH, —SR (R is as defined for —OR above.), —(SO 2 )H, or —(SO 2 )
  • R is the same as that of -OR above
  • R 2a or R 2b and R 3a or R 3b may be bonded to each other to form a ring together with the carbon atoms to which they are bonded
  • n 1 , n 2 and n 3 are each independently 1 or 2
  • m is an integer of 0 or
  • a benzyl compound that has improved solubility in an organic solvent and that can be easily separated and purified after a peptide condensation reaction by liquid-liquid layer separation. can be done.
  • DBF can be easily captured and the DBF-captured body can be easily removed.
  • the cyclic amine used in the present invention has low basicity, unintended deprotection of the Fmoc group can be suppressed, and the progress of side reactions can be suppressed.
  • m represents the number of substituents (-[Q 1 -R 1 -Q 2 -R 2 ]).
  • m is an integer of 2 or 3;
  • the substituents (-[Q 1 -R 1 -Q 2 -R 2 ]) are preferably present at the 3- and 5-positions or at the 2- and 4-positions.
  • the two substituents (-[Q 1 -R 1 -Q 2 -R 2 ]) are more preferably present at the 2- and 4-positions.
  • m is 3 they are preferably present at adjacent positions.
  • the three substituents (-[Q 1 -R 1 -Q 2 -R 2 ]) 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 or more and (5-m) or less. Specifically, when m is 2, k is an integer of 0 or more and 3 or less, and when m is 3, k is an integer of 0 or more and 2 or less.
  • n Q 1 and Q 2 each represent an oxygen atom.
  • X represents a hydroxyl group.
  • R 1 is independently an alkylene group.
  • R 1 is a linear or branched alkylene group having 2 to 16 carbon atoms.
  • the number of carbon atoms in the alkylene group is preferably 2 or more, more preferably 6 or more, and even more preferably 8 or more, from the viewpoint of improving the solubility of the peptide bound to the benzyl compound (X1) of the present invention in an organic solvent. Also, it is preferably 16 or less, more preferably 14 or less, and even more preferably 12 or less.
  • alkylene group examples include ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nanomethylene, decamethylene, undecamethylene, dodecamethylene, tetra decamethylene group, tridecamethylene group, tetradecamethylene group, pentadecamethylene group, hexadecamethylene group and the like.
  • Each of m R 2 is independently an optionally substituted alkyl group, an optionally substituted aralkyl group, or an optionally substituted aryl group.
  • 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 and having a substituent.
  • the number of carbon atoms in the alkyl group is preferably 5 or more, more preferably 6 or more, and 7 or more from the viewpoint of improving the solubility of the peptide bound to the benzyl compound (X1) according to the present embodiment in an organic solvent. It is more preferably 28 or less, more preferably 24 or less, and even more preferably 22 or less.
  • alkyl group is a linear alkyl group (that is, an alkyl group having no branched chain) or an alkyl group having a total of 1 or 2 branched chains, and specifically, the following formula (XA ):
  • n1 is an integer of 0 or more and 16 or less
  • n2 is an integer of 0 or more and 16 or less.
  • n1 is an integer of 0 or more and 6 or less
  • n2 is an integer of 0 or more and 13 or less.
  • R 2a , R 2b , R 2c , R 2d and R 2e are each independently a hydrogen atom or an alkyl group.
  • the alkyl group may have a substituent, and the substituent is, for example, a halogen atom such as fluorine, chlorine, bromine, or iodine.
  • At least two or more of R 2a , R 2b , R 2c and R 2d are hydrogen atoms.
  • groups represented by the formula (XA) include pentyl group, octyl group, isooctyl group, nonyl group, decyl group, undecyl group, dodecyl group, 1-methyl-1-dodecyl group, 1-methyl-1 -hexadecyl group, 1-ethyl-1-heptadecyl group, 1-propyl-1-decyl group, 1-butyl-1-decyl group, 2-methyl-1-dodecyl group, 2-methyl-1-hexadecyl group, 2 -butyl-1-octyl group, 2-butyl-1-dodecyl group, 2-butyl-1-octadecyl group, 2-hexyl-1-decyl group, 2-hexyl-1-dodecyl group, 2-heptyl-1- dodecyl group, 2-octyl-1-dodecyl group,
  • the alkyl group includes pentyl group, octyl group, isooctyl group, nonyl group, decyl group, undecyl group, dodecyl group, 2-butyl-1-octyl group, 2-hexyl-1-dodecyl group, 2-octyl- Preferred examples include 1-dodecyl group, 2-decyl-1-tetradecyl group, 2-dodecyl-1-hexadecyl group and the like.
  • the number of carbon atoms in the aralkyl group which may have a substituent is preferably 5 or more, and preferably 6 or more, from the viewpoint of improving the solubility of the peptide bound to the benzyl compound (X1) according to the present embodiment in an organic solvent. is more preferable, 7 or more is more preferable, 28 or less is preferable, 24 or less is more preferable, and 22 or less is even more preferable.
  • the aryl substituent is preferably a substituent containing a halogen atom.
  • alkyl group examples include 6-phenyl-1-hexyl group, 8-phenyl-1-octyl group, 10-phenyl-1-decyl group, 12-phenyl-1-dodecyl group and the like.
  • the number of carbon atoms in the aryl group which may have a substituent is preferably 6 or more, and preferably 7 or more, from the viewpoint of improving the solubility of the peptide bound to the benzyl compound (X1) according to the present embodiment in an organic solvent. is more preferable, 8 or more is more preferable, 16 or less is preferable, 14 or less is more preferable, and 12 or less is even more preferable.
  • R 2 is preferably aryl having a substituent containing a halogen atom.
  • Halogen atoms include fluorine, chlorine and bromine, with fluorine being particularly preferred.
  • aryl group examples include a 3-trifluoromethylphenyl group, a 3,5-bistrifluoromethylphenyl group, a 4-fluoro-3-trifluoromethylphenyl group, a 4-chloro-2-fluorophenyl group, a 2- isopropylphenyl group, 2,6-isopropylphenyl group, 2-sec-butylphenyl group, 5-isopropyl-2-methylphenyl group and the like.
  • Each of the k R3 's is independently a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom.
  • 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 group, ethyl group, n-propyl group, isopropyl group, n-butyl group, tert-butyl group and the like, of which methyl group is particularly preferred.
  • alkoxy group examples include methoxy group, ethoxy group, n-propyloxy group, isopropyloxy group, n-butyloxy group, tert-butyloxy group and the like, among which methoxy group is particularly preferred.
  • the halogen atom includes a fluorine atom, a chlorine atom and a bromine atom, of which the fluorine atom is particularly preferred.
  • benzyl compounds As the benzyl compound (X1) represented by the above formula (X1), compounds represented by the following formulas (X1A) to (X1D) can be mentioned as preferable ones in view of their usefulness.
  • the benzyl compound represented by the formula (X1A) is a benzyl compound represented by the formula (X1) in which three substituents represented by the above formula (XB1) are substituted at positions 3, 4 and 5. (3,4,5-tris(11-(3,5-bis(trifluoromethyl)phenoxy)undecyl)oxy)phenyl)methanol).
  • the benzyl compound represented by the formula (X1B) is a benzyl compound represented by the formula (X1) in which two substituents represented by the above formula (XB2) are substituted at positions 2 and 4. (2,4-bis((12-((2-octyldodecyl)oxy)dodecyl)oxy)phenyl)methanol).
  • the benzyl compound represented by the formula (X1C) is a benzyl compound represented by the formula (X1) in which three substituents represented by the above formula (XB2) are substituted at positions 3, 4 and 5.
  • the resulting compound (3,4,5-tris((12-((2-octyldodecyl)oxy)phenyl)methanol)).
  • the benzyl compound represented by the formula (X1D) is a benzyl compound represented by the formula (X1) in which two substituents represented by the above formula (XB3) are substituted at positions 2 and 4. (2,4-bis((12-((2-decyltetradecyl)oxy)dodecyl)oxy)phenyl)methanol).
  • Method for producing benzyl compound (X1) in which R 2 is a substituent other than an aryl group is not particularly limited. An example is described below. For example, a dialkyl bromide and an alkyl alcohol are dissolved in a suitable solvent and heated in the presence of a base to obtain an alkyl-etherified monobromide (hereinafter also referred to as "Step Xa1").
  • Step Xa2 A compound or a benzester compound is obtained (hereinafter also referred to as “Step Xa2”).
  • the alkyl-etherified benzaldehyde compound or benzester compound is dissolved in an appropriate solvent, and the formyl group or ester group is reduced using a reducing agent such as a metal hydride (hereinafter also referred to as “step Xa3”). ), and a method of obtaining as a benzyl alcohol compound.
  • the base used for the reaction between the dialkyl bromide and the alkyl alcohol includes an organic base such as lithium diisopropylamide (LDA), lithium hexamethyldisilazide (LHMDS), sodium bis(trimethylsilyl)amide (NaHMDS). , sodium hydride (NaH), and lithium hydride (LiH).
  • LDA lithium diisopropylamide
  • LHMDS lithium hexamethyldisilazide
  • NaHMDS sodium bis(trimethylsilyl)amide
  • NaH sodium hydride
  • LiH lithium hydride
  • the amount of the base used is not particularly limited, but it is preferable to use 1.0 mol or more and 10 mol or less, more preferably 1.0 mol or more and 5 mol or less, relative to 1 mol of the alkyl alcohol. preferable.
  • Solvents include hydrocarbons such as hexane and heptane, diisopropyl ether, tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), 4-methyltetrahydropyran (MTHP), ethers such as dioxane, dimethylformamide (DMF), dimethyl
  • hydrocarbons such as hexane and heptane
  • diisopropyl ether such as tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), 4-methyltetrahydropyran (MTHP), ethers such as dioxane, dimethylformamide (DMF), dimethyl
  • amides such as acetamide, sulfoxides such as dimethylsulfoxide (DMSO), lactams such as N-methylpyrrolidone, aromatic hydrocarbons such as toluene and xylene, and mixed solvents thereof.
  • toluene
  • the amount of solvent used is not particularly limited, but it is preferable to use 5 mL or more and 100 mL or less, more preferably 10 mL or more and 50 mL or less, per 1 g of alkyl alcohol. Moreover, when a mixed solvent is used, the total amount of the mixed solvent should satisfy the above range. Hereinafter, similar description may be omitted.
  • reaction temperature is not particularly limited, it may be carried out in the range of 70°C to 150°C, for example.
  • reaction time is not particularly limited, but may be, for example, 1 hour to 24 hours.
  • Step Xa2 the base used for the reaction between the alkyl-etherified monobromide and the benzaldehyde compound or benzester compound includes triethylamine (TEA), diisopropylethylamine (DIPEA), 1,8-diazabicyclo [5.4.
  • DBU 1,5-diazabicyclo[4.3.0]non-5-ene
  • DBN 1,4-diazabicyclo[2.2.2]octane
  • pyridine imidazole, 4-(dimethylamino)pyridine (DMAP), LDA, sodium acetate (NaOAc), sodium methoxide (MeONa), potassium methoxide (MeOK), lithium hexamethyldisilazide (LHMDS), sodium bis(trimethylsilyl ) organic bases such as amides (NaHMDS), sodium carbonate ( Na2CO3 ), sodium hydrogen carbonate (NaHCO3) , potassium carbonate ( K2CO3 ), cesium carbonate ( Cs2CO3 ), sodium hydride (NaH) and inorganic bases such as Among these, K 2 CO 3 is preferably used as the base in terms of allowing the reaction to proceed smoothly.
  • the amount of the base used is not particularly limited, but it is preferable to use 1 mol or more and 10 mol or less, more preferably 2 mol or more and 8 mol or less, per 1 mol of the benzaldehyde compound or benzester compound. preferable.
  • the solvent described in step Xa1 may be used.
  • DMF or a mixed solvent of DMF and CPME or MTHP is preferably used, and DMF is more preferably used, in order to allow the reaction to proceed smoothly.
  • the amount of the solvent used is not particularly limited, but it is preferably 30 mL or more and 200 mL or less, more preferably 50 mL or more and 180 mL or less, per 1 g of the benzaldehyde compound or benzester compound.
  • reaction temperature is not particularly limited, it may be carried out in the range of 50°C to 150°C, for example.
  • reaction time is not particularly limited, but may be, for example, 1 hour to 24 hours.
  • the reducing agent used for reducing the formyl group of the alkyl-etherified benzaldehyde compound or the ester group of the benzester compound to obtain the benzyl alcohol compound includes sodium borohydride, lithium borohydride, lithium triethylborohydride, lithium aluminum hydride, 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 benzester compound includes sodium borohydride, lithium borohydride, Lithium triethylborohydride, lithium aluminum hydride, aluminum hydride, sodium bis(2-methoxyethoxy)aluminum hydride, diisobutylaluminum hydride are preferred.
  • sodium borohydride is used as a reducing agent, for example, iodine, sulfuric acid, borane trifluoroetherate (BF 3 Et 2 O), etc. may be allowed to coexist in order to increase the reducing power of the reducing agent. is preferred.
  • Solvents include hydrocarbons such as hexane and heptane, alcohols such as methanol and ethanol, ethers such as diethyl ether, isopropyl ether, THF, CPME, MTHP and dioxane, aromatic hydrocarbons such as toluene and xylene, Alternatively, a mixed solvent thereof may be used.
  • a mixed solvent of alcohols and ethers is preferably used from the viewpoint of allowing the reaction to proceed smoothly.
  • the amount of the solvent used is not particularly limited, but it is preferably 1 mL or more and 100 mL or less, more preferably 5 mL or more and 50 mL or less, per 1 g of the benzaldehyde compound. Moreover, when using the mixed solvent of alcohols and ethers, it is preferable to use 1 mL or more and 10 mL or less of ethers with respect to 1 mL of alcohols.
  • Solvents used for reduction of ester groups include ethers such as diethyl ether, isopropyl ether, THF, CPME, MTHP and dioxane, aromatic hydrocarbons such as toluene and xylene, and mixed solvents thereof.
  • THF, CPME, and MTHP are preferably used as the solvent for reducing the methyl ester group from the viewpoint of smooth progress of the reaction.
  • the amount of the solvent used is not particularly limited, but it is preferably 1 mL or more and 100 mL or less, more preferably 5 mL or more and 50 mL or less, per 1 g of the ester compound.
  • reaction temperature is not particularly limited, it may be carried out in the range of -10°C to 90°C, for example.
  • reaction time is not particularly limited, but may be, for example, 1 hour to 24 hours.
  • Step Xb1 Method for producing a benzyl compound (X1) in which R 2 in formula (X1) is an aryl group
  • the method for producing a benzyl compound in which R 2 in formula (X1) has an aryl group is particularly limited. isn't it. An example is described below. For example, a bromoalkyl alcohol and a 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 Xb1”).
  • step Xb2 a bromoalkylaryl ether compound in which the hydroxyl group is substituted with a bromine atom is obtained.
  • Step Xb3 The obtained bromoalkylaryl ether compound and the hydroxybenzaldehyde compound or hydroxybenzester compound are heated in the presence of a base such as potassium carbonate to obtain an alkyl-etherified benzaldehyde compound or benzester compound (hereinafter referred to as “Step Xb3 ”).
  • a base such as potassium carbonate
  • Step Xb4 the alkyl-etherified benzaldehyde compound or benzester compound
  • the compound is dissolved in an appropriate solvent, and the formyl group or ester group is reduced using a reducing agent (hereinafter also referred to as “step Xb4”. ) and benzyl alcohol.
  • the base used for the reaction between the bromoalkyl alcohol and the substituted phenol compound includes TEA, DIPEA, DBU, DBN, DABCO, pyridine, imidazole, DMAP, LDA, NaOAc, MeONa, MeOK, and LHMDS. , NaHMDS, and inorganic bases such as Na 2 CO 3 , NaHCO 3 , K 2 CO 3 , Cs 2 CO 3 , and NaH.
  • K 2 CO 3 is preferably used as the base in terms of allowing the reaction to proceed smoothly.
  • the amount of the base used is not particularly limited, but it is preferable to use 1 mol or more and 10 mol or less, more preferably 1 mol or more and 5 mol or less, per 1 mol of the phenol compound having a substituent. preferable.
  • the solvent described in step Xa1 may be used.
  • DMF is preferably used as the solvent in terms of allowing the reaction to proceed smoothly.
  • the amount of the solvent used is not particularly limited, but it is preferably 2 mL or more and 100 mL or less, more preferably 4 mL or more and 50 mL or less, per 1 g of the phenol compound having a substituent.
  • reaction temperature is not particularly limited, it may be carried out in the range of 40°C to 150°C, for example.
  • reaction time is not particularly limited, but may be, for example, 1 hour to 24 hours.
  • the reagent used in the reaction for converting the hydroxyl group of the aryl-etherified bromoalkyl alcohol into a bromine atom includes a reagent containing triphenylphosphine and carbon tetrabromide, a reagent containing hydrobromic acid, and the like. is mentioned.
  • the amount of the reagent used is not particularly limited, but it is preferably 0.5 mol or more and 10 mol or less, and 1 mol or more and 5 mol or less, per 1 mol of the aryl-etherified bromoalkyl alcohol. It is more preferable to use
  • solvents include the solvents described in step Xa1, halogenated hydrocarbons such as dichloromethane and chloroform, and mixed solvents thereof.
  • Halogenated hydrocarbons are preferably used as the solvent in terms of allowing the reaction to proceed smoothly.
  • the amount of the solvent used is not particularly limited, but it is preferably 0.5 mL or more and 100 mL or less, more preferably 1 mL or more and 50 mL or less, per 1 g of the aryl-etherified bromoalkyl alcohol. .
  • reaction temperature is not particularly limited, it may be carried out in the range of 20°C to 150°C, for example.
  • reaction time is not particularly limited, but may be, for example, 1 hour to 24 hours.
  • Step Xb3 As the base used for the reaction between the bromoalkylaryl ether and the benzaldehyde compound or benzester compound, the base explained in step Xb1 can be used. In step X3b, the same base as that used in step X1b may be used, or a different base may be used. However, it is preferable to use the same base as the base used in step X1b in terms of production efficiency and cost reduction.
  • the amount of the base used is not particularly limited, but it is preferable to use 1 mol or more and 50 mol or less, more preferably 3 mol or more and 30 mol or less, per 1 mol of the benzaldehyde compound or benzester compound. preferable.
  • the solvent described in step Xa1 may be used.
  • DMF is preferably used as the solvent in terms of allowing the reaction to proceed smoothly.
  • the amount of the solvent used is not particularly limited, but it is preferably 30 mL or more and 200 mL or less, more preferably 50 mL or more and 180 mL or less, per 1 g of the benzester compound.
  • reaction temperature is not particularly limited, it may be carried out in the range of 40°C to 150°C, for example.
  • reaction time is not particularly limited, but may be, for example, 1 hour to 24 hours.
  • Step Xb4 As the reducing agent used to obtain the benzyl alcohol compound by reducing the formyl group of the alkyl-etherified benzaldehyde compound or the ester group of the benzester compound, the reducing agent used in the above step Xa3 can be used.
  • Solvents include hydrocarbons such as hexane and heptane, alcohols such as methanol and ethanol, ethers such as diethyl ether, isopropyl ether, THF, CPME, MTHP and dioxane, aromatic hydrocarbons such as toluene and xylene, Alternatively, a mixed solvent thereof may be used.
  • hydrocarbons such as hexane and heptane
  • alcohols such as methanol and ethanol
  • ethers such as diethyl ether, isopropyl ether, THF, CPME, MTHP and dioxane
  • aromatic hydrocarbons such as toluene and xylene
  • a mixed solvent thereof may be used.
  • reaction temperature is not particularly limited, it may be carried out in the range of -10°C to 90°C, for example.
  • reaction time is not particularly limited, but may be, for example, 1 hour to 24 hours.
  • a peptide synthesis method using the benzyl compound (X1) of the present invention as a protective group at the C-terminal of an amino acid is, for example, a production method comprising the following steps (X1) to (X5).
  • the C-terminal protected peptide obtained in each condensation step can be separated by liquid-liquid separation, which facilitates the purification step.
  • Step (X1) a step of dissolving the benzyl compound (X1) of the present invention in a soluble solvent (dissolving 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 and a reaction substrate (condensation reaction step);
  • Step (X3) A base is added to the reaction solvent containing the condensate obtained above to scavenge the amino acid active ester, which is an unreacted substance, and deprotect the peptide N-terminal protecting group, and the protecting group a step of scavenging the derived by-products with a base (deprotection and scavenging reaction step);
  • Step (X4) An acidic aqueous solution is added to the reaction solution containing the condensate and the scavenged product obtained in the above step for washing, and the layers are separated to remove the scavenged product and unreacted substances (condensing agent, activ
  • Step (X5) A step of removing into layers (layer splitting step), and Step (X5) A step of removing the benzyl compound (X1) according to the present embodiment and the protective group of the peptide side chain from the C-terminal of the peptide and performing purification to obtain the target peptide (deprotection and purification step).
  • the benzyl compound (X1) (hereinafter also referred to as “tag X”) according to the present embodiment has N (N represents an amino group at the ⁇ -position of an amino acid)-9-fluorenylmethyloxycarbonyl
  • N represents an amino group at the ⁇ -position of an amino acid
  • the introduction of (N-Fmoc) protected amino acids and the condensation reaction of N-Fmoc protected amino acids to tag-protected peptides are described as examples.
  • the N-Fmoc protected amino acids used may have side chain protecting groups.
  • the Fmoc group is used as an example of the amino acid N-terminal protective group, the amino acid protective group is not limited to this. Examples include benzyloxycarbonyl group (Cbz group), tert-butoxycarbonyl group (Boc group), allyloxycarbonyl group (Alloc group) and the like.
  • Step (X1) (dissolving step)
  • This step is a step of dissolving tag X in a soluble solvent.
  • a soluble solvent a general organic solvent used for peptide synthesis can be used for the reaction.
  • ethers such as diethyl ether, THF, 2-methyltetrahydrofuran, 1,4-dioxane, methyl-t-butyl ether, CPME and MTHP
  • acetic esters such as ethyl acetate and isopropyl acetate
  • halogenation such as chloroform and dichloromethane
  • Examples include hydrocarbons, aromatic hydrocarbons such as toluene and xylene, and hydrocarbons such as hexane, heptane and cyclohexane.
  • methyl-t-butyl ether, CPME, MTHP, isopropyl acetate, chloroform and toluene are preferred, and CPME, MTHP, isopropyl acetate and toluene are more preferred. CPME and MTHP are particularly preferred.
  • the soluble solvent is used to improve the solubility of the substrate in the reaction, to improve the solubility of unreacted substances and by-products in the aqueous layer during extraction, or to improve the liquid separation.
  • DMF dimethylacetamide, DMSO, sulfolane, N-methylpyrrolidone, N,N'-dimethylpropylene urea (DMPU), acetonitrile, etc., in a suitable ratio.
  • Step (X2) (condensation reaction step)
  • an N-Fmoc protected amino acid is introduced into the tag X dissolved in the soluble solvent obtained in the above step (X1), an esterification reaction and an N-Fmoc protected amino acid are introduced into the tag X-protected peptide, This is the step of carrying out the amidation reaction.
  • the amount of the N-Fmoc-protected amino acid used is 1 to 4 mol, preferably 1 to 2 mol, particularly preferably 1.05 to 1.3 mol, relative to 1 mol of tag X.
  • an ester bond is formed by adding a condensing agent under the presence of dimethylaminopyridine (DMAP) catalyst in a solvent that does not affect the reaction.
  • DMAP dimethylaminopyridine
  • an amide bond is formed by adding a condensing agent and an activating agent in a solvent that does not affect the reaction.
  • the condensing agent is not particularly limited as long as the reaction proceeds, and condensing agents commonly used in peptide synthesis can be used.
  • DMT-MM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorphonium chloride
  • HBTU O-(benzotriazol-1-yl)-1, 1,3,3-tetramethyluronium hexafluorophosphate
  • HATU O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
  • HBTU(6-Cl) O-(benzotriazol-1-yl)-1 , 1,3,3-tetramethyluronium hexafluorophosphate
  • TBTU 1,3,3-tetramethyluronium tetrafluoroborate
  • TCTU O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
  • the amount of the condensing agent used is usually 1 to 4 equivalents, preferably 1 to 2 equivalents, more preferably 1.05 to 1.5 equivalents, still more preferably 1.05 to 1.05 equivalents, relative to the tag X or the tag X-protected peptide. 1.3 equivalents.
  • an activator is preferably added in order to promote the reaction and suppress side reactions such as racemization.
  • the activating agent is a reagent that facilitates the formation of a peptide bond (amide bond) by leading an amino acid to a corresponding active ester, symmetrical acid anhydride, or the like in coexistence with a condensing agent.
  • activators commonly used in peptide synthesis can also be used in the present invention without limitation.
  • HOAt ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate
  • HOBt 3-hydroxy-1,2,3-benzotriazin-4(3H)-one
  • HOOBt N -hydroxysuccinimide
  • HPht N-hydroxyphthalimide
  • HONb N-hydroxy-5-norbornene-2,3-dicarboximide
  • pentafluorophenol ethyl cyano(hydroxyimino)acetate
  • Oxyma ethyl cyano(hydroxyimino)acetate
  • the amount of the activating agent to be used is generally 0.1-2 equivalents, preferably 0.2-1.5 equivalents, more preferably 0.3-1.0 equivalents, relative to the tag X-protected peptide.
  • Solvents commonly used in peptide synthesis can be used without limitation for the solvent used in the condensation reaction step, and examples thereof include, but are not limited to, the above-described soluble solvents or mixed solvents of soluble solvents and polar solvents. be done.
  • the amount of the solvent to be used is not particularly limited as long as the reaction proceeds, but it is an amount such that the concentration of the dissolved tag X-protected peptide or the like is usually 0.1 mM to 1 M, preferably 1 mM to 0.5 M. is the amount to be
  • reaction temperature the temperature generally used in peptide synthesis is also used in the present invention.
  • the reaction time is usually 0.5 to 30 hours (condensation time for one residue).
  • Step (X3) (deprotection and scavenging reaction step)
  • the first base is added to the reaction solvent to capture (scavenge) unreacted amino acid active esters to form captured bodies and inactivate them.
  • the removal of the Fmoc group from the N-Fmoc-protected peptide proceeds, and the first base also acts as a scavenger for dibenzofulvene, which is a by-product derived from the Fmoc group. form and inactivate.
  • the amount of the first base used to scavenge unreacted amino acid active esters is not particularly limited, but is usually 1 to 5 equivalents, preferably 1 to 3 equivalents, relative to the theoretically remaining amino acid equivalents. .
  • the amount of the second base necessary for deprotecting the Fmoc group of the N-Fmoc protected peptide is preferably 1 to 12 equivalents, preferably 2 to 10 equivalents, relative to the Fmoc group present in the reaction system. More preferably, 3 equivalents to 8 equivalents are particularly preferred.
  • the amount of the first base used to scavenge the dibenzofulvene derived from the de-Fmoc group is preferably 5 equivalents to 50 equivalents, more preferably 8 equivalents to 40 equivalents, relative to the Fmoc groups present in the reaction system. 10 equivalents to 35 equivalents are particularly preferred.
  • Step (X4) (layer splitting step)
  • an acidic aqueous solution is added to the solution of the above step (X3) to neutralize it, and an acidic solution is added to remove the scavenger of the first base and the unreacted substances (condensing agent, activating agent, base).
  • This is the step of removing to the water layer.
  • the amino acid active ester and dibenzofulvene scavenged by the first base can be easily removed to the aqueous layer by acid washing.
  • the acid used for neutralization is not limited as long as it can neutralize the base in the reaction solution, but examples include aqueous solutions of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and the like.
  • hydrochloric acid 1M to 12M, preferably 3M to 12M, more preferably 5M to 12M hydrochloric acid is used.
  • Neutralization here means that the reaction solution should have a neutral pH, and the pH may be 7.0 or less.
  • An acidic aqueous solution is further added to the reaction solution neutralized with the acid for washing, followed by liquid separation, removing the aqueous layer, and recovering the organic layer.
  • the acidic aqueous solution to be used is not particularly limited, but examples thereof include hydrochloric acid aqueous solution, dilute sulfuric acid aqueous solution, phosphoric acid aqueous solution, and acetic acid aqueous solution, preferably hydrochloric acid aqueous solution.
  • the pH of the acidic aqueous solution is 1-5, preferably 1-4, more preferably 1-3.
  • the amount of the acidic aqueous solution used for washing is not particularly limited as long as it exhibits a washing effect. ⁇ 2 times the amount.
  • the number of washings, liquid separations, and discarding of the aqueous layer there are no particular restrictions on the number of washings, liquid separations, and discarding of the aqueous layer, and it may be performed once or multiple times. The number of times is appropriately selected depending on the type of compound in the reaction system, the amount of unnecessary substances, and the like.
  • the temperature for washing is not particularly limited, but is 10°C to 50°C, preferably 15°C to 45°C, more preferably 20°C to 40°C.
  • this step basically, the capturing body of the first base and unnecessary substances are removed with an acidic aqueous solution, but other cleaning steps may be added in addition to the cleaning with the acidic aqueous solution. For example, washing with a weak base and washing with a saline solution can be mentioned.
  • Examples of the weakly basic aqueous solution include an aqueous sodium hydrogen carbonate solution, an aqueous sodium carbonate solution, an aqueous potassium carbonate solution, etc., having a pH of 8 to 12.
  • salt solution 5 wt% to saturated salt solution can be mentioned.
  • Step (X5) (deprotection, purification step)] This step is a step of removing the tag X and the protective group of the peptide side chain from the C-terminal of the peptide to obtain the target peptide.
  • the method for removing the tag X and the protective group of the peptide side chain from the peptide C-terminus is not particularly limited, and a known deprotection method may be used, preferably by acid treatment.
  • a known deprotection method may be used, preferably by acid treatment.
  • deprotection methods using trifluoroacetic acid (TFA) can be used.
  • TFA may be used in combination with molecules such as water, thioanisole, 1,2-ethanedithiol, phenol, and triisopropylsilane in an appropriate composition.
  • the deprotected peptide can be isolated and purified according to purification methods commonly used in peptide synthesis.
  • the target peptide can be isolated and purified by extraction washing, crystallization, and chromatography.
  • benzyl compound (Y1) represented by.
  • m Q's each represent an oxygen atom.
  • the total carbon number of the benzyl compound represented by formula (Y1) is preferably 30 or more and 80 or less, more preferably 40 or more and 60 or less.
  • this poor liquid separation is due to the formation of an emulsion (micelle structure) at the time of liquid separation between the organic layer and the aqueous layer containing a component in which a peptide is bound to a highly hydrophobic benzyl compound. Therefore, it is preferable that the hydrophobicity of the benzyl compound is not too high from the viewpoint of performing the liquid separation operation well.
  • the hydrophobicity of benzylic compounds can be adjusted, for example, by the number of carbons contained in R 1 and R 2 .
  • the number of carbon atoms of m R 1 in the above formula (Y1) (hereinafter also referred to as “side chain carbon number”) is preferably in the range of 24 to 84, and the side chain More preferably, the number of carbon atoms in the chain is 30-72, and it is particularly preferable that the number of carbon atoms in the side chain is 36-48.
  • each of the m R 1 's has a total of 1 or 2 branched chains.
  • each of the m R 1 is an alkyl group.
  • R 1 is an alkyl group having a total of 1 or 2 branched chains, preferably an alkyl group having a total of 1 or 2 branched chains and a total of 24 to 84 carbon atoms, It is more preferably an alkyl group having a total of 1 or 2 branched chains and a side chain carbon number of 30 to 72, having a total of 1 or 2 branched chains and a side chain carbon number of 36. ⁇ 48 alkyl groups are particularly preferred.
  • R 1 is particularly preferably an alkyl group having one branched chain in total, and the position of the branched chain is 1 to 7 with respect to Q. is preferred, it is more preferably present at positions 1 to 4 with respect to Q, and more preferably at position 2 with respect to Q. Also, when R 1 has two branches, the two branches are separated from each other by 0-6 carbons, preferably 0-3 carbons.
  • the above branched chain is an optionally substituted alkyl group, an optionally substituted aralkyl group, or an optionally substituted aryl group.
  • the branched chain is preferably an optionally substituted alkyl group having 2 to 12 carbon atoms, more preferably an optionally substituted alkyl group having 4 to 10 carbon atoms.
  • This substituent is, for example, a halogen atom such as fluorine or chlorine.
  • m R 1 are specifically each independently represented by the following formula (YA):
  • n1 is an integer of 0 or more and 6 or less
  • n2 is an integer of 0 or more and 6 or less
  • n1 represents an integer of 0 or more and 3 or less
  • n2 is an integer of 0 or more and 3 or less.
  • R 1a , R 1b , R 1c , R 1d and R 1e are each independently a hydrogen atom or an alkyl group.
  • the above alkyl group may have a substituent, and the substituent is, for example, a halogen atom such as fluorine or chlorine.
  • at least two or more of R 1a , R 1b , R 1c and R 1d are hydrogen atoms.
  • R 1a , R 1b , R 1c and R 1d may all be hydrogen atoms, but preferably all of R 1a , R 1b , R 1c and R 1d are hydrogen atoms. except.
  • Specific examples of the group represented by the above formula (YA) have the same meanings as specific examples of the group represented by the above formula (XA), and thus description thereof is omitted.
  • R 1 is preferably an organic group having one branched chain, and the branched chain is present at the 2-position relative to Q. That is, m R 1 are each independently represented by the following formula (YA'):
  • R 1f is preferably a linear alkyl group having 4 to 12 carbon atoms which may have a substituent; It is more preferably a straight-chain alkyl group, and most preferably a straight-chain alkyl group having 6 carbon atoms, which may have a substituent.
  • This substituent is, for example, a halogen atom such as fluorine or chlorine.
  • R 1g is preferably a linear alkyl group having 6 to 14 carbon atoms, which may have a substituent, and a linear alkyl group having 6 to 12 carbon atoms, which may have a substituent It is more preferably a linear alkyl group, and most preferably a linear alkyl group having 8 carbon atoms, which may have a substituent. Examples of this substituent include halogen atoms such as fluorine, chlorine, bromine and iodine.
  • the group represented by (YA′) above includes a 2-n-butyl-1-octyl group, a 2-hexyl-1-decyl group, a 2-octyl-1-dodecyl group, a 2-decyl-1- Preferred examples include a tetradecyl group and a 2-dodecyl-1-hexadecyl group.
  • Each of the k R 2 is 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 the k R 2 are the same as the specific examples of the k R 3 in the first embodiment.
  • the k R 2 are more preferably hydrogen atoms.
  • X represents a hydroxyl group.
  • m represents an integer of 2 or 3;
  • k represents an integer of 0 or more (5-m) or less, At least one of m [QR 1 ] is preferably meta-substituted with respect to the substituent containing X.
  • the benzyl compound represented by formula (Y1) is particularly preferably used as a tag in long-chain peptide synthesis.
  • the benzyl compound represented by formula (Y1) is preferably used for synthesizing a peptide having 5 or more residues, and preferably used for synthesizing a peptide having 7 or more residues. More preferably, it is used for synthesizing ten or more peptides.
  • benzyl compounds As the benzyl compound (Y1) represented by the above formula (Y1), compounds represented by the following formulas (Y1A) to (Y1D) can be mentioned as preferable ones in view of their usefulness.
  • the benzyl compound represented by the formula (Y1A) is a benzyl compound represented by the formula (Y1) in which three substituents represented by the above formula (YB1) are located at the 3-position relative to the hydroxyl group (—OH). , (3,4,5-tris((2-butyloctyl)oxy)phenyl)methanol substituted at the 4- and 5-positions, respectively.
  • the benzyl compound represented by the formula (Y1B) is a benzyl compound represented by the formula (Y1) in which the three substituents represented by the above formula (YB2) are at the 3-position with respect to the hydroxyl group (—OH). , (3,4,5-tris((2-hexyldecyl)oxy)phenyl)methanol substituted at the 4- and 5-positions, respectively.
  • the benzyl compound represented by the formula (Y1C) is a benzyl compound represented by the formula (Y1) in which three substituents represented by the above formula (YB3) are located at the 3-position relative to the hydroxyl group (—OH). , (3,4,5-tris((2-decyltetradecyl)oxy)phenyl)methanol substituted at the 4- and 5-positions, respectively.
  • the benzyl compound represented by the formula (Y1D) is a benzyl compound represented by the formula (Y1) in which the three substituents represented by the above formula (YB3) are at the 3-position with respect to the hydroxyl group (--OH). and a compound (3,5-bis((2-decyltetradecyl)oxy)phenyl)methanol substituted at the 5-position, respectively.
  • an alkyl halide and a hydroxybenzester compound are dissolved in an appropriate solvent and heated in the presence of a base such as potassium carbonate to obtain an alkyl-etherified benzester compound (hereinafter also referred to as "step Ya1").
  • a base such as potassium carbonate
  • an alkyl halide is a compound in which a halogen atom is bonded to the tip of an alkyl group. Examples of the halogen atom include chlorine, bromine, and iodine.
  • the alkyl halide is preferably alkyl bromide or alkyl iodine, more preferably alkyl bromide.
  • the alkyl halide a commercially available product may be used, or a hydroxyl compound corresponding to the starting material of the alkyl halide may be halogenated by a known method.
  • an alkyl bromide and a hydroxybenzester compound are dissolved in an appropriate solvent and heated in the presence of a base such as potassium carbonate to obtain an alkyl-etherified benzester compound.
  • a base such as potassium carbonate
  • the alkyl bromide a commercially available product may be used, or a hydroxyl derivative corresponding to the raw material of the alkyl bromide may be brominated by a known method.
  • the alkyl-etherified benzester compound is dissolved in an appropriate solvent, and the ester group is reduced using a reducing agent such as a metal hydride (hereinafter also referred to as “step Ya2”) to obtain a benzyl alcohol compound.
  • a reducing agent such as a metal hydride
  • the base used for the reaction between the alkyl bromide and the hydroxybenzester compound includes triethylamine (TEA), diisopropylethylamine (DIPEA), 1,8-diazabicyclo[5.4.0]undec-7-ene.
  • TAA triethylamine
  • DIPEA diisopropylethylamine
  • 1,8-diazabicyclo[5.4.0]undec-7-ene 1,8-diazabicyclo[5.4.0]undec-7-ene.
  • DBU 1,5-diazabicyclo[4.3.0]non-5-ene
  • DABCO 1,4-diazabicyclo[2.2.2]octane
  • pyridine imidazole, 4-(dimethyl amino)pyridine (DMAP), lithium diisopropylamide (LDA), sodium acetate (NaOAc), sodium methoxide (MeONa), potassium methoxide (MeOK), lithium hexamethyldisilazide (LHMDS), sodium bis(trimethylsilyl)amide organic bases such as (NaHMDS), sodium carbonate ( Na2CO3 ), sodium hydrogencarbonate (NaHCO3), potassium carbonate (K2CO3), cesium carbonate (Cs2CO3 ) , sodium hydride ( NaH), etc.
  • Inorganic bases can be mentioned.
  • K 2 CO 3 is preferably used as the base in terms of allowing the reaction to proceed smoothly.
  • the amount of the base used is not particularly limited, but it is preferably used in an amount of 1 to 10 mol, more preferably 2 to 8 mol, per 1 mol of the hydroxybenzester compound.
  • Solvents include hydrocarbons such as hexane and heptane, diisopropyl ether, tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), 4-methyltetrahydropyran (MTHP), ethers such as dioxane, dimethylformamide (DMF), dimethyl
  • Examples include amides such as acetamide, sulfoxides such as dimethylsulfoxide (DMSO), lactams such as N-methylpyrrolidone, aromatic hydrocarbons such as toluene and xylene, and mixed solvents thereof.
  • DMF or a mixed solvent of DMF and CPME in terms of allowing the reaction to proceed smoothly.
  • the amount of the solvent used is not particularly limited, but it is preferably 10 mL or more and 200 mL or less, more preferably 15 mL or more and 150 mL or less, per 1 g of the hydroxybenzester compound.
  • reaction temperature is not particularly limited, it may be carried out in the range of 50°C to 150°C, for example.
  • reaction time is not particularly limited, but may be, for example, 1 hour to 24 hours.
  • a metal hydrogen compound may be used as the reducing agent used to reduce the ester group of the alkyl-etherified benzester compound to obtain the benzyl alcohol compound.
  • a metal hydride a hydrogenated Group 13 alkali metal compound is preferred.
  • alkali metal borohydride compounds such as sodium borohydride, lithium borohydride, and lithium triethylborohydride, lithium aluminum hydride, aluminum hydride, and bis(2-methoxyethoxy)aluminum hydride
  • aluminum hydride compounds such as sodium and diisobutylaluminum hydride.
  • the amount of the reducing agent used is not particularly limited, but it is preferably used in an amount of 1 mol or more and 10 mol or less, preferably 2 mol or more and 5 mol or less, relative to 1 mol of the alkyl-etherified benzester compound. is more preferable.
  • sodium borohydride for example, iodine, sulfuric acid, borane trifluoroetherate (BF 3 Et 2 O), etc. may be allowed to coexist in order to increase the reducing power of the reducing agent. is preferred.
  • Solvents used for reducing the ester group of the alkyl-etherified benzester compound include ethers such as diethyl ether, diisopropyl ether, THF, CPME, MTHP and dioxane, aromatic hydrocarbons such as toluene and xylene, or Mixed solvents of these are included.
  • THF, CPME, and MTHP are particularly preferable as the solvent used for reducing the methyl ester group from the viewpoint of smooth progress of the reaction.
  • the amount of the solvent used is not particularly limited, but it is preferably 1 mL or more and 100 mL or less, more preferably 5 mL or more and 50 mL or less, per 1 g of the ester compound.
  • reaction temperature is not particularly limited, it may be carried out in the range of -10°C to 90°C, for example.
  • reaction time is not particularly limited, but may be, for example, 1 hour to 24 hours.
  • a method for synthesizing a peptide using the benzyl compound (Y1) of the present invention as a protective group (that is, tag) at the C-terminal of an amino acid is, for example, a production method including the following steps (Y1) to (Y6).
  • This peptide synthesis method is capable of liquid-liquid separation of peptides with C-terminal protected amino acids (hereinafter also referred to as "C-terminal protected peptides”) obtained in the condensation reaction step and the peptide elongation step. Therefore, the purification process is facilitated.
  • Step (Y1) a step of dissolving the benzyl compound (Y1) of the present invention in a soluble solvent (dissolving step),
  • Step (Y2) A condensing agent and an activating agent are added to the soluble solvent obtained in the above step, and the benzyl compound (Y1) dissolved in the soluble solvent and the second 1 amino acid (hereinafter also referred to as "N-terminal protected amino acid") to produce a first condensate (condensation reaction step);
  • Step (Y3) Soluble containing the first condensate obtained in the step (Y2) (condensation of the N-terminal protected amino acid and the benzyl compound (Y1) from which the —OH group has been removed)
  • a first base is added to a solvent (reaction solvent) to scavenge the remaining amino acid active ester (resulting from the reaction of the C-terminal carboxylic acid of the surplus amino acid in step (Y2) with a condensing agent and then with an activating agent).
  • scavenging the by-product (dibenzofulvene) from the N-terminal protecting group with a first base deprotection and scavenging reaction step);
  • Step (Y4) The second condensate obtained in the above step (Y3) (meaning a condensate in which the N-terminal protective group is removed from the first condensate), a scavenger (first base and amino active ester and is bound, and the first base and dibenzolfulvene are bound) and unreacted substances (by-products derived from condensing agents, activators, bases, water-soluble organic solvents)
  • An acidic aqueous solution is added to the reaction solution (soluble solvent) to wash the solution, and the aqueous layer and the organic layer are separated to remove the capturing bodies and unreacted substances into the aqueous layer, and the organic layer separated from the aqueous layer.
  • a step of obtaining a second condensate i.e., C-terminal protected peptide obtained by deprotecting the N-terminal protecting group from the first condensate in step (Y3) above (reaction solvent) (liquid separation step);
  • Step (Y5) A second amino acid having a protected N-terminus is added to the reaction solvent containing the second condensate obtained in the above step (Y4), and a second amino acid is produced in the same manner as in step (Y2).
  • Step (Y5) includes repeating the following sub-steps (Y5-1) to (Y5-3):
  • Step (Y5-2) A step of scavenging the remaining amino acid active ester and then deprotecting the N-terminal protecting group from the (2n+1) condensate to scavenge the by-product derived from the N-terminal protecting group. ,as well as,
  • Step (Y5-3) Wash by adding an acidic aqueous solution to the soluble solvent, separate the phases, remove the captured body and unreacted substances into the aqueous layer, and transfer to the organic layer (reaction solvent) in the above step (Y5-2).
  • a step of obtaining the obtained (2n+2)th condensate (referring to a deprotected condensate obtained by removing the N-terminal protecting group from the (2n+1)th condensate), and
  • Step (Y6) A step of removing the benzyl compound (Y1) and the protective group of the peptide side chain from the C-terminal of the peptide obtained in step (Y5) and purifying to obtain the target peptide (deprotection and purification step) .
  • step (Y5) may be performed by repeating steps (Y2) to (Y4).
  • step (Y2) includes sub-step (Y5-1)
  • step (Y3) includes sub-step (Y5-2)
  • step (Y4) includes sub-step (Y5-3).
  • step (Y5) including substeps (Y5-1) to (Y5-3) may be performed by repeating such steps (Y2) to (Y4).
  • Step (Y1) (dissolving step)] This step is a step of dissolving the benzyl compound (Y1) in a soluble solvent. Since step (Y1) can be performed in the same manner as step (X1) described above, detailed description thereof will be omitted.
  • Step (Y2) (condensation reaction step)
  • the tag Y and the N-Fmoc protected amino acid dissolved in the soluble solvent obtained in the above step (Y1) are introduced, the esterification reaction is performed, and the N-Fmoc protected amino acid is introduced into the tag Y-protected peptide to form an amide.
  • step (Y2) can be performed in the same manner as step (X2) described above, detailed description thereof will be omitted.
  • Step (Y3) (deprotection and scavenging reaction step)]
  • the first base is added to the reaction solvent to capture (scavenge) unreacted amino acid active esters to form captured bodies and inactivate them.
  • the first base also acts as a scavenger for dibenzofulvene, which is a by-product derived from the Fmoc group. form and inactivate. Since step (Y3) can be performed in the same manner as step (X3) described above, detailed description thereof will be omitted.
  • Step (Y4) liquid separation step
  • an acidic aqueous solution is added to the solution of the above step (Y3) for neutralization, and the scavenger of the first base and unreacted substances (condensing agent, activating agent, base, water-soluble organic solvent) are separated. It is a step of removing to the water layer by The amino acid active ester and dibenzofulvene scavenged by the first base can be removed to the aqueous layer by an acid wash.
  • the acid used for neutralization is not limited as long as it can neutralize the base in the reaction solution, but examples include aqueous solutions of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and the like.
  • hydrochloric acid 0.5M to 12M, preferably 1M to 12M, more preferably 1M to 6M hydrochloric acid is used.
  • Neutralization here means that the reaction solution should have a neutral pH, and the pH may be 7.0 or less.
  • a ketone-based liquid separation promoting solvent such as acetone or methyl ethyl ketone may be further added.
  • the inventors speculate that the reason for the decrease in liquid separation is that tag Y-peptide molecules associate with each other through hydrophobic interaction and hydrogen bonding to form a micelle structure, thereby reducing liquid separation. .
  • the hydrophobic interaction between the side chains of the tag Y or the hydrogen bond between the peptide molecules is weakened, and the formation of the micelle structure is suppressed, thereby improving the liquid separation. It is estimated to be. It is also effective in improving the liquid separation property to perform the step of heating the solution in the step (Y3) instead of or together with the step of adding the liquid separation promoting solvent.
  • An acidic aqueous solution is further added to the reaction solution neutralized with the acid for washing, followed by liquid separation, removing the aqueous layer, and recovering the organic layer. Since the cleaning using the acidic aqueous solution can be performed in the same manner as the cleaning in step (X4) described above, detailed description thereof will be omitted.
  • the step (Y4) basically removes the capturing body of the first base and unnecessary substances with an acidic aqueous solution. may be added before or after washing with Examples include washing with a basic aqueous solution and washing with a saline solution.
  • a sodium bicarbonate aqueous solution for example, a sodium bicarbonate aqueous solution, a sodium carbonate aqueous solution, or a potassium carbonate aqueous solution having a pH of 8 to 13 can be mentioned.
  • salt solution 5 wt% to saturated salt solution can be mentioned.
  • washing with a basic aqueous solution is performed to make the pH of the solution neutral to weakly basic.
  • the basic aqueous solution include the aqueous solutions described above.
  • Step (Y5) (peptide elongation step)
  • an N-terminally protected amino acid is added to the reaction solvent containing the tagged Y-protected peptide obtained in the above step, and the above steps (Y5-1) to (Y5-3) are repeated to obtain the peptide.
  • This is the process of elongation.
  • the DMAP used in the step (Y2) is not used, but the activating agent specified below is used.
  • the amount of the N-Fmoc-protected amino acid to be used is 1-4 mol, preferably 1-2 mol, particularly preferably 1.05-1.5 mol, per 1 mol of the benzyl compound (Y1).
  • the condensing agent used can be the same as the condensing agent described in step (Y2).
  • an activator is preferably added to promote the peptide condensation reaction and suppress side reactions such as racemization.
  • the activating agent is a reagent that facilitates the formation of a peptide bond (amide bond) by leading an amino acid to a corresponding active ester, symmetrical acid anhydride, or the like in coexistence with a condensing agent.
  • activators commonly used in peptide synthesis can also be used in the present invention without limitation.
  • HOAt ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate
  • HOBt 3-hydroxy-1,2,3-benzotriazin-4(3H)-one
  • HOOBt N -hydroxysuccinimide
  • HPht N-hydroxyphthalimide
  • HONb N-hydroxy-5-norbornene-2,3-dicarboximide
  • pentafluorophenol ethyl cyano(hydroxyimino)acetate
  • Oxyma ethyl cyano(hydroxyimino)acetate
  • the amount of the activating agent used is generally 0.1-2 equivalents, preferably 0.2-1.5 equivalents, more preferably 0.3-1.0 equivalents, relative to the tag Y-protected peptide.
  • the solvent used in the peptide elongation step can be any solvent commonly used in peptide synthesis without limitation, and is not limited thereto. and a mixed solvent of
  • the amount of the solvent to be used is not particularly limited as long as the reaction proceeds, but it is an amount such that the concentration of the dissolved tag Y-protected peptide or the like is usually 0.1 mM to 1 M, preferably 1 mM to 0.5 M. is the amount to be
  • reaction temperature the temperature generally used in peptide synthesis is also used in the present invention.
  • the reaction time is usually 0.5 to 30 hours (condensation time for one residue).
  • Step (Y6) (deprotection, purification step)] This step is a step of removing the benzyl compound (Y1) and protecting groups of peptide side chains from the C-terminus of the peptide to obtain the desired peptide. Since step (Y6) can be performed in the same manner as step (X5) described above, detailed description thereof will be omitted.
  • ⁇ 1> a removal method of removing the protecting group from an amino group-containing compound protected with a protecting group having a fluorene skeleton at the N-terminus
  • ⁇ 2> a step of removing the protecting group. Details will be described in the order of ⁇ 3> the removing agent for the protecting group.
  • an amino group-containing compound whose N-terminus is protected by a protective group having a fluorene skeleton is brought into contact with a scavenger in an organic solvent. obtaining a scavenger in which a by-product having a fulvene skeleton derived from the protecting group and the scavenger are bound; and separating the obtained scavenger from the organic solvent.
  • amino group-containing compound means a compound having a primary amino group or a secondary amino group.
  • Amino group-containing compounds include, for example, single amino acids, peptides formed by peptide bonds of a plurality of amino acids, amino acids, and the like.
  • a protective group having a fluorene skeleton is a group that binds to the nitrogen atom in the N-terminal amino group of an amino group-containing compound to protect the N-terminal of the amino group-containing compound.
  • This protecting group is a monovalent protecting group containing a fluorene structure represented by the following formula (Z2).
  • R 4 is an optionally substituted alkyloxycarbonyl group having 1 to 6 carbon atoms.
  • R 5a to R 5d and R 6a to R 6d are each independently a hydrogen atom, an optionally substituted C 1-6 alkyl group, an optionally substituted C 1 It is an alkoxy group of up to 6, a sulfone group which may have a substituent, or a sulfonyl group which may have a substituent.
  • substituents include halogen atoms and alkyl groups having 1 to 3 carbon atoms.
  • "*" represents the bonding position with the N-terminal amino group of the amino group-containing compound.
  • R 4 is preferably an alkyloxycarbonyl group having 1 to 3 carbon atoms, and more preferably a methyloxycarbonyl group.
  • R 5a to R 5d and R 6a to R 6d are each preferably hydrogen atoms.
  • a protective group having a fluorene skeleton represented by the above formula (Z2) a 9-fluorenylmethyloxycarbonyl group (Fmoc group) can be mentioned as a suitable example in consideration of its usefulness. .
  • An amino group-containing compound whose N-terminus is protected by a protecting group having a fluorene skeleton is a protecting group in which at least one of the primary amino group or secondary amino group possessed by the amino group-containing compound is the above-described protecting group having a fluorene skeleton. means a compound protected by
  • the scavenging agent is a reactive agent that binds to the protective group deprotected from the amino group-containing compound to capture the protective group to form a scavenging compound (see formula (Z3)).
  • the scavenger also has the function of deprotecting the protecting group from the amino group-containing compound protected with the protecting group.
  • the scavenger is a compound represented by the following formula (Z1). That is, the scavenger is at least one compound selected from the group consisting of a cyclic amine containing one nitrogen atom (see formula (Z1A)) and a hydrochloride containing the cyclic amine (see formula (Z1B)). .
  • N is a nitrogen atom.
  • H is a hydrogen atom.
  • X is a divalent group represented by -CH 2 -, -O-, -S- or -(SO 2 )-.
  • X is preferably -O- or -(SO 2 )-.
  • Including an oxygen atom in X lowers the basicity, thereby suppressing side reactions.
  • X is optimally -O-.
  • n 1 R 1a , n 1 R 1b , n 2 R 2a , n 2 R 2b , n 3 R 3a , and n 3 R 3b are each independently H , —OH, —OR (R is an alkyl group), —SH, —SR (R is as defined for —OR above.), —(SO 2 )H, or —(SO 2 ) It is a monovalent group represented by R (R is the same as defined above for —OR). Also, R 2a or R 2b and R 3a or R 3b may be bonded to each other to form a ring together with the carbon atoms to which they are bonded.
  • n 1 R 1a , n 1 R 1b , n 2 R 2a and n 2 R 2b the one bonded to the carbon atom adjacent to the nitrogen atom is preferably a hydrogen atom. . This is because the stability of the scavenger increases.
  • one of n 1 pair of R 1a and R 1b , n 2 pair of R 2a and R 2b and n 3 pair of R 3a and R 3b is preferably a hydrogen atom.
  • n 1 R 1a , n 1 R 1b , n 2 R 2a , n 2 R 2b , n 3 R 3a , and n 3 R 3b are each hydrogen is an atom.
  • n 1 , n 2 and n 3 are each independently 1 or 2;
  • the sum of n 1 , n 2 and n 3 is preferably 3 or 4. That is, the scavenger represented by formula (Z1) is preferably represented by formula (Z1a) or formula (Z1b) below.
  • the trapping agent represented by formula (Z1a) is a compound in which the sum of n 1 , n 2 and n 3 is 3 in formula (Z1).
  • the trapping agent represented by formula (Z1b) is a compound in which the sum of n 1 , n 2 and n 3 is 4 in formula (Z1).
  • m is an integer of 0 or 1;
  • N, H, X, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , n 1 , n 2 , n 3 and m are represented by formula (Z1 ) is synonymous with
  • the scavenger is a cyclic amine containing one nitrogen atom represented by the following formula (Z1A).
  • the scavenger is hydrochloride represented by the following formula (Z1B). Note that m is preferably 0.
  • N, H, X, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , n 1 , n 2 , and n 3 are represented by formula (Z1) is synonymous with
  • the scavenger is an amine that is cyclic and contains at least one element selected from the group consisting of oxygen element and sulfur element.
  • the amine is water soluble.
  • the scavenger is preferably a primary or secondary amine.
  • the scavenger represented by formula (Z1) above is preferably a cyclic amine having one amino group.
  • the scavenger is, for example, at least one selected from the group consisting of morpholine, piperidine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine, and thiomorpholine dioxide, preferably morpholine, 3-hydroxypiperidine , 4-hydroxypiperidine, thiomorpholine, and at least one selected from the group consisting of thiomorpholine dioxide, more preferably selected from the group consisting of morpholine, 3-hydroxypiperidine, and 4-hydroxypiperidine At least one, more preferably morpholine.
  • the amount of the scavenger to be added is 5 to 100 equivalents, preferably 5 to 50 equivalents, more preferably 10 to 30 equivalents, relative to the amount of protective groups present in the reaction system. If the amount of the scavenger to be added is less than this range, the by-product having a fulvene skeleton generated by the deprotection reaction of the protective group having a fluorene skeleton will be insufficiently captured, making it difficult to remove impurities by acidic liquid separation washing. If the amount of the scavenger added is more than this range, the scavenger may remain in the organic layer during acidic liquid-separating washing, which may cause side reactions.
  • organic solvent The contact between the amino group-containing compound and the scavenger represented by formula (Z1) is carried out in an organic solvent.
  • this organic solvent it is preferable to use the same solvent as the reaction solvent used for the elongation (synthesis) reaction of the peptide in the liquid phase tagging method described below. This is because when the peptide elongation reaction is repeated sequentially, the removal of the protective group and the elongation of the peptide do not adversely affect each other, and the operation is facilitated.
  • the organic solvent the same soluble solvent as described above for step (X1) can be used, and therefore detailed description thereof will be omitted.
  • the method of contacting each component is not particularly limited.
  • the ingredients may be mixed in a reaction vessel equipped with a stirring mechanism.
  • the amino group-containing compound and the scavenger can be brought into contact with each other.
  • the procedure for mixing each component is not particularly limited.
  • a scavenger may be mixed with the organic solvent containing the amino group-containing compound (hereinafter also referred to as "reaction solution").
  • a deprotecting agent may be further mixed in order to accelerate the deprotection reaction of the protecting group.
  • Deprotecting agents include 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1.5-diazabicyclo[4.3.0]-5-nonene (DBN), 1,4- At least one base selected from the group consisting of organic bases such as 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), 1,4-diazabicyclo[2.2.2]octane (DABCO),
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • DBN
  • the amount of the deprotecting agent added in this step is preferably 1 to 12 equivalents, more preferably 2 to 10 equivalents, particularly preferably 3 to 8 equivalents, relative to the protective groups present in the reaction system.
  • the procedure for adding the deprotecting agent is not particularly limited.
  • the scavenger may be mixed with the reaction solution, or the deprotecting agent may be mixed with the reaction solution before the scavenger is mixed with the reaction solution.
  • a scavenger represented by the following formula (Z3) is obtained.
  • This scavenger is a combination of a by-product having a fulvene skeleton represented by the following formula (Z2') and a scavenger represented by the above formula (Z1).
  • a by-product represented by the following formula (Z2') is produced by deprotection of the protective group represented by the formula (Z2).
  • N, H, X, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 5a , R 5b , R 5c , R 5d , R 6a , R 6b , R 6c , R 6d , n 1 , n 2 , and n 3 have the same meanings as in Formula (Z1) and Formula (Z2).
  • Step of separating capture bodies Next, the step of separating the captured bodies obtained, that is, the step of removing them from the reaction solution will be described.
  • the method shown below is an example of a method for separating captured bodies, and is not limited to the method shown below.
  • an acidic aqueous solution is added to the above reaction solution to neutralize it, and the capturing body is guided to the aqueous layer by liquid separation.
  • the scavengers can be guided into the aqueous layer and separated from the reaction solution by acid washing.
  • the acid used for neutralization is not limited as long as it can neutralize the base in the reaction solution, but examples include aqueous solutions of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and the like.
  • hydrochloric acid 0.5 M (“M” indicates mol/L.
  • M indicates mol/L.
  • hydrochloric acid 0.5 M (“M” indicates mol/L.
  • M indicates mol/L.
  • M indicates mol/L.
  • 12 M preferably 1 M to 12 M, more preferably 1 M to 6 M hydrochloric acid
  • Use Neutralization here means that the reaction solution should have a neutral pH, and the pH may be 7.0 or less.
  • a ketone-based liquid separation promoting solvent such as acetone or methyl ethyl ketone may be further added.
  • tag-peptide molecules associate with each other through hydrophobic interaction and hydrogen bonding to form a micelle structure, which causes the liquid separation to decrease.
  • the hydrophobic interaction between the side chains of the tag or the hydrogen bond between the peptide molecules is weakened, and the formation of the micelle structure is suppressed, so that the liquid separation is improved. Presumed. It is also effective in improving the liquid separation property to perform a step of heating the reaction solution instead of or together with the step of adding the liquid separation promoting solvent.
  • an acidic aqueous solution is added to the reaction solution neutralized with the above acid for washing, then the solution is separated, the aqueous layer is separated, and the organic layer is recovered.
  • the acidic aqueous solution to be used is not particularly limited, but examples thereof include hydrochloric acid aqueous solution, dilute sulfuric acid aqueous solution, phosphoric acid aqueous solution, and acetic acid aqueous solution, preferably hydrochloric acid aqueous solution.
  • the pH of the acidic aqueous solution is 1-5, preferably 1-4, more preferably 1-3.
  • the amount of the acidic aqueous solution used for washing is not particularly limited as long as it exhibits a washing effect. ⁇ 2 times the amount.
  • the number of washings, liquid separations, and discarding of the aqueous layer there are no particular restrictions on the number of washings, liquid separations, and discarding of the aqueous layer, and it may be performed once or multiple times. The number of times is appropriately selected depending on the type of compound in the reaction system, the amount of trapping material, and the like.
  • the temperature for washing is not particularly limited, but is 10°C to 50°C, preferably 15°C to 45°C, more preferably 20°C to 40°C.
  • the captured material is basically removed with an acidic aqueous solution.
  • other washing processes may be added before and after washing with the acidic aqueous solution. Examples include washing with a basic aqueous solution and washing with a saline solution.
  • a sodium bicarbonate aqueous solution for example, a sodium bicarbonate aqueous solution, a sodium carbonate aqueous solution, or a potassium carbonate aqueous solution having a pH of 8 to 13 can be mentioned.
  • salt water examples include 5 wt % to saturated salt water.
  • washing with a basic aqueous solution is performed to make the pH of the solution neutral to weakly basic.
  • the basic aqueous solution examples include the aqueous solutions described above.
  • the method for removing the Fmoc group involves mixing a cyclic amine as a scavenger, an amino group-containing compound protected with an Fmoc group as a protecting group, and an optional deprotecting agent to deprotect the Fmoc group.
  • DBF-trapper is an example of a "trapper" of the present invention.
  • An amino group-containing compound protected by an Fmoc group means a compound in which at least one of the primary amino group or secondary amino group of the amino group-containing compound is protected by an Fmoc group.
  • Cyclic amines herein are cyclic amines having one amino group, such as morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine, and thiomorpholine dioxide, as described above.
  • morpholine, 3-hydroxypiperidine and 4-hydroxypiperidine are preferred, and morpholine is more preferred.
  • the amount of the 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, relative to the amount of Fmoc groups present in the reaction system. be. If the amount of the cyclic amine added is less than this range, the capture of DBF produced by the Fmoc group deprotection reaction becomes insufficient, making it difficult to remove impurities by acidic liquid separation washing.
  • a deprotecting agent is a reactive agent that removes the Fmoc group from an amino group-containing compound protected with an Fmoc group.
  • Deprotecting agents in the Fmoc group removal step include, as described above, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1.5-diazabicyclo[4.3.0] organic bases such as -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.
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • DBN 1,4-diazabicyclo[2.2.2]octane
  • triethylamine and tributylamine
  • inorganic bases such as potassium tert-butoxide and sodium tert-
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • DBN 1.5-diazabicyclo[4.3.0]-5-nonene
  • DABCO 4-diazabicyclo[2.2.2]octane
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • the amount of the deprotecting agent necessary for deprotecting the Fmoc group is preferably 1 to 12 equivalents, more preferably 2 to 10 equivalents, 3 to 8 equivalents, relative to the Fmoc group present in the reaction system. Equivalents are particularly preferred.
  • the deprotecting agent is not an essential reactant and does not necessarily have to be added. However, it is preferable to use a deprotecting agent because the reaction rate of the deprotection reaction of the Fmoc group can be increased by using the deprotecting agent together with the scavenger.
  • the acidic aqueous solution to be used is not particularly limited, but examples thereof include hydrochloric acid aqueous solution, dilute sulfuric acid aqueous solution, phosphoric acid aqueous solution, and acetic acid aqueous solution, preferably hydrochloric acid aqueous solution.
  • the pH of the acidic aqueous solution is 1-5, preferably 1-4, more preferably 1-3.
  • an amino group-containing compound in which the Fmoc group has been deprotected can be isolated. Furthermore, the obtained solution of the amino group-containing compound can be used as it is as a raw material for the peptide production method by the liquid phase synthesis method described below.
  • This peptide synthesis method comprises an amino group-containing compound such as an amino acid, peptide, or amino acid amide whose C-terminal is protected with a specific carrier for liquid-phase peptide synthesis (hereinafter also referred to as "C-terminal carrier-protected peptide"), A step of condensing an amino group-containing compound (hereinafter also referred to as an “N-terminal protected peptide”) whose N-terminus is protected with the above-described protecting group (see formula (Z2)) (step Z1), and remaining after the condensation reaction A step of quenching the active ester (step Z2), a step of deprotecting the protective group from the condensed peptide (hereinafter also referred to as “N-terminal protected-C-terminal carrier-protected peptide”) (step Z3), and adding an acidic aqueous solution to the reaction solution.
  • C-terminal carrier-protected peptide a specific carrier for liquid-phase peptide synthesis
  • Step Z4 deprotecting the C-terminal carrier and side chain protecting groups
  • Step Z5 deprotecting the C-terminal carrier and side chain protecting groups
  • liquid-phase peptide synthesis method including the method for removing the Fmoc group will be described in detail below, taking Fmoc as an example of the protective group.
  • the protective group is not limited to the Fmoc group, and the method shown below can also be applied to an amino group-containing compound protected with a protective group represented by formula (Z2) above.
  • Step Z1 condensation step
  • a condensing agent a C-terminal carrier-protected peptide protected with a carrier for liquid-phase peptide synthesis
  • an amino acid or peptide whose N-terminus is protected with an Fmoc group hereinafter referred to as "N-Fmoc-protected amino acid or Also referred to as "peptide”
  • N-Fmoc-protected amino acid or Also referred to as "peptide” is condensed to obtain a peptide with extended amino acid residues (hereinafter also referred to as "N-Fmoc-protected-C-terminal carrier-protected peptide").
  • Carriers for liquid-phase peptide synthesis used in step Z1 include, for example, the following compounds.
  • the carrier for liquid-phase peptide synthesis is the benzyl compound (X1) represented by the above formula (X1). Since the structure of the benzyl compound (X1) is the same as that of Embodiment 1 described above, detailed description thereof will be omitted.
  • a benzyl compound (Y1) represented by the following formula (Y1). Since the structure of the benzyl compound (Y1) is the same as that of Embodiment 2 described above, detailed description thereof will be omitted.
  • benzyl compound (Y1) represented by the above formula (Y1) compounds represented by the following formulas (Y1A) to (Y1D) can be mentioned as preferable ones 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). It may be a compound.
  • N-Fmoc-protected C-terminal carrier-protected peptides, etc., C-terminal carrier-protected peptides, and amino acids forming the basic structure of N-Fmoc-protected amino acids may be either natural amino acids or non-natural amino acids. Also, this amino acid may be either L- or D-form.
  • Natural amino acids include Arg, Lys, Asp, Asn, Glu, Gln, His, Pro, Tyr, Trp, Ser, Thr, Gly, Ala, Met, Cys, Phe, Leu, Val, Ile, ⁇ -Ala, and the like. mentioned.
  • Unnatural amino acids include Tle (tert-leucine) and the like.
  • Amino acids may have side chain functional groups.
  • the side chain amino group is desirably protected by a protective group other than the Fmoc group, such as a Boc group, a Cbz group, an Alloc group, an Ac group, or the like.
  • Examples of protective groups for side chain carboxy groups include alkyl groups such as methyl groups, ethyl groups and tBu groups, and benzylic substituents such as benzyl groups and p-methoxybenzyl groups.
  • a trityl (Tr) group etc. are mentioned as a protection group of an amide group.
  • a benzyl group, a tBu group, etc. are mentioned as a side-chain hydroxy-protecting group.
  • Examples of side-chain imidazole-protecting groups include Boc, Trt, and Bom (benzyloxymethyl) groups.
  • Examples of side-chain guanidyl-protecting groups include a nitro group and a Pbf group.
  • Examples of thiol-protecting groups include Trt group, Acm group, Dpm group, Ddm group, tBu group, S-tBu group, Mmt group and Npys group.
  • the amount of the N-Fmoc-protected amino acid used is 1 to 4 mol, preferably 1 to 2 mol, particularly preferably 1.05 to 1.3 mol, per 1 mol of the carrier for liquid phase peptide synthesis. .
  • the condensing agent is not particularly limited as long as the reaction proceeds, and condensing agents commonly used in peptide synthesis can be used.
  • the condensing agent the one described in the context of step (X2) (condensation reaction step) can be used, and therefore detailed description thereof is omitted.
  • An activator is preferably added to promote the peptide condensation reaction and suppress side reactions such as racemization.
  • the activating agent the one described in the context of step (X2) (condensation reaction step) can be used, and therefore detailed description thereof is omitted.
  • reaction solvent As the reaction solvent used in the condensation reaction step (hereinafter also referred to simply as "solvent"), any solvent commonly used in peptide synthesis can be used without limitation. A mixed solvent of a solvent and a polar solvent can be mentioned. As the soluble solvent, the one described in the context of step (X1) (dissolving step) can be used, and therefore detailed description thereof will be omitted.
  • the soluble solvent is used to improve the solubility of the substrate in the reaction, to improve the solubility of unreacted substances and by-products in the aqueous layer during extraction, or to improve the liquid separation.
  • DMF dimethylacetamide, DMSO, sulfolane, N-methylpyrrolidone, N,N'-dimethylpropylene urea (DMPU), acetonitrile, etc.
  • the mixing ratio is not particularly limited as long as the reaction proceeds, but the amount is such that the ratio of the soluble solvent and the polar solvent is 50:50 to 95:5, preferably 70:30 to 90:10. quantity.
  • the amount of the solvent to be used is not particularly limited as long as the reaction proceeds, but it is an amount such that the concentration of the dissolved tag is usually 0.1 mM to 1 M, preferably 1 mM to 0.5 M. be.
  • reaction temperature the temperature generally used in peptide synthesis is also used in the present invention.
  • the reaction time is usually 0.5 to 30 hours (condensation time for one residue).
  • This reaction solvent is an example of the "organic solvent" in the present invention, and can be used in steps Z3 and Z4 described below.
  • Step Z2 Active ester quenching step
  • An amine is added to the reaction solvent containing the N-Fmoc-protected C-terminal carrier-protected peptide obtained in step Z1 above, and the remaining amino acid active ester (condensing agent, Then, scavenging is performed.
  • the amine used in this step may be referred to as the first scavenger.
  • Amines as first scavengers that can be used in step Z2 are preferably primary or secondary water-soluble amines, for example morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine, thiomorpholine di oxide, 1-methylpiperazine, 4-aminopiperidine, N,N-dimethylethylenediamine, ethylenediamine, preferably morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine, thiomorpholine dioxide. , more preferably morpholine, 3-hydroxypiperidine and 4-hydroxypiperidine, and still more preferably morpholine.
  • the amount of amine added as a scavenger in step Z2 is not particularly limited, but is usually 1 to 5 equivalents, preferably 1 to 3 equivalents, relative to the theoretically remaining amino acid equivalents.
  • Step Z3 Fmoc group deprotection and capture step
  • a deprotecting agent is added to the reaction solution obtained in step Z2 to deprotect the N-terminal Fmoc group from the N-Fmoc-protected-C-terminal carrier-protected peptide.
  • this step includes a step of catching Fmoc group-derived by-products (DBF) with a second scavenger.
  • the reaction solution is an example of the "organic solvent” of the present invention.
  • the second scavenger is an example of the "trapping agent" of the present invention.
  • the step of scavenging DBF with a second scavenger is an example of the "step of obtaining a scavenger" of the present invention.
  • the amount of the deprotecting agent added in this step is preferably 1 to 12 equivalents, more preferably 2 to 10 equivalents, particularly preferably 3 to 8 equivalents, relative to the Fmoc groups present in the reaction system.
  • the deprotecting agent is not particularly limited, but 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, preferably DBU.
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • 1.5-diazabicyclo[4.3.0]-5-nonene 1,4 -diazabicyclo[2.2.2]octane
  • potassium tert-butoxide sodium tert-butoxide
  • triethylamine and tributylamine preferably DBU.
  • the amount of the second scavenger used to scavenge DBF derived from the de-Fmoc group is preferably 5 equivalents to 50 equivalents, more preferably 8 equivalents to 40 equivalents, more preferably 10 equivalents, relative to the Fmoc groups present in the reaction system. Equivalents to 35 equivalents are particularly preferred.
  • a second scavenger that can be used in step Z3 is an amine as a DBF scavenger.
  • the amine as described above, is cyclic and contains at least one element selected from the group consisting of an oxygen element or a sulfur element, and is preferably a primary or secondary water-soluble amine, Examples include morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide, preferably morpholine, 3-hydroxypiperidine and 4-hydroxypiperidine, more preferably morpholine is.
  • 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).
  • the second scavenger used in step Z3 is preferably the same as the first scavenger added in step Z2.
  • cyclic amines such as morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine and thiomorpholine dioxide are preferably used.
  • Step Z4 is an example of the "separating step" of the present invention. That is, in this step, an acidic aqueous solution is added to the reaction solution of the above step Z3 to neutralize it, and the first scavenger and amino acid active ester are combined (hereinafter also referred to as "amino acid active ester-capturing body"). , and unreacted substances (here, the unreacted substances are condensing agents, activating agents, deprotecting agents, polar solvents among the reaction solvents described above, etc.) are removed into the aqueous layer by liquid separation.
  • the amino acid active ester scavenged by the first scavenger i.e. amino acid active ester-capture
  • the DBF scavenged by the second scavenger i.e. DBF-capture
  • the acid used for neutralization can be the one described in the context of step (Y4) (liquid separation step), so a detailed description thereof will be omitted.
  • an acidic aqueous solution is added to the reaction solution neutralized with the above acid for washing, followed by liquid separation, removing the aqueous layer, and recovering the organic layer.
  • the acidic aqueous solution to be used is not particularly limited, but examples thereof include hydrochloric acid aqueous solution, dilute sulfuric acid aqueous solution, phosphoric acid aqueous solution, and acetic acid aqueous solution, preferably hydrochloric acid aqueous solution.
  • the pH of the acidic aqueous solution is 1-5, preferably 1-4, more preferably 1-3.
  • the amount of the acidic aqueous solution used for washing is not particularly limited as long as it exhibits a washing effect. ⁇ 2 times the amount.
  • washings There are no particular restrictions on the number of washings, liquid separations, and discarding of the aqueous layer, and it may be performed once or multiple times. The number of times is appropriately selected depending on the type of compound in the reaction system, the amount of unreacted substances, and the like.
  • the temperature for washing is not particularly limited, but is 10°C to 50°C, preferably 15°C to 45°C, more preferably 20°C to 40°C.
  • the amino acid active ester-captured body, the DBF-captured body, and unreacted substances are basically removed with an acidic aqueous solution.
  • an acidic aqueous solution may be added before or after washing with the acidic aqueous solution. Examples include washing with a basic aqueous solution and washing with a saline solution.
  • a sodium bicarbonate aqueous solution for example, a sodium bicarbonate aqueous solution, a sodium carbonate aqueous solution, or a potassium carbonate aqueous solution having a pH of 8 to 13 can be mentioned.
  • salt solution 5 wt% to saturated salt solution can be mentioned.
  • washing with a basic aqueous solution is performed to make the pH of the solution neutral to weakly basic.
  • the basic aqueous solution include the aqueous solutions described above.
  • the C-terminal carrier-protected peptide is solidified (crystallized) in the stage after deprotection of the Fmoc group, and the C-terminal carrier-protected peptide is solid-liquid separated. may be recovered.
  • Solidification can be carried out by appropriately referring to known methods by changing the composition of the solvent in which the carrier-protected peptide is dissolved. After that, it can be carried out by adding a hydrocarbon solvent such as methanol, acetonitrile, or hexane to change the composition of the solution.
  • Step Z5 deprotection, purification step
  • This step is a step of removing the carrier and protecting group of the side chain of the peptide from the C-terminus of the peptide to obtain the desired peptide.
  • the method for removing the carrier and peptide side chain protecting groups from the peptide C-terminus is not particularly limited, and a known deprotection method may be used, preferably by acid treatment.
  • a known deprotection method may be used, preferably by acid treatment.
  • deprotection methods using trifluoroacetic acid (TFA) can be used.
  • TFA may be used in combination with molecules such as water, thioanisole, 1,2-ethanedithiol, phenol, and triisopropylsilane in an appropriate composition.
  • the peptide from which the carrier and the protecting group of the peptide side chain have been deprotected can be isolated and purified according to a purification method commonly used in peptide synthesis.
  • the target peptide can be isolated and purified by extraction washing, crystallization, and chromatography.
  • the intermediate peptide obtained after deprotection of the Fmoc group can be used in the next condensation step without isolation. This enables one-pot synthesis of peptides and is particularly suitable for industrial production.
  • the protecting group remover represented by the above formula (Z2) is a composition containing the scavenger represented by the above formula (Z1) and a basic deprotecting agent. .
  • the scavenger and deprotection agents are described above and will not be described in detail here.
  • the method for producing a peptide according to the first aspect of the present invention comprises, in an organic solvent, an amino group-containing compound whose N-terminus is protected with a protecting group having a fluorene skeleton, and the following formula (Z1): a step of contacting with a capturing agent represented by to obtain a capturing body in which a by-product having a fulvene skeleton derived from the protecting group and the capturing agent are bound; a step of separating the captured body obtained from the organic solvent; including, Peptide production method:
  • N is a nitrogen atom
  • H is a hydrogen atom
  • X is a divalent group represented by —CH 2 —, —O—, —S—, or —(SO 2 )—; n 1 R 1a , n 1 R 1b , n 2 R 2a , n 2 R 2b , n 3 R 3a , and n 3 R
  • the method for producing a peptide according to the second aspect of the present invention comprises, in addition to the configuration of the method for producing a peptide according to the first aspect, an amino group-containing compound having the N-terminus protected with the protecting group and a deprotecting agent. and the step of contacting with.
  • the deprotecting agent is 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 tert-butoxide, At least one base selected from the group consisting of sodium tert-butoxide, triethylamine, and tributylamine.
  • DBU 1,8-diazabicyclo[5.4.0] -7-undecene
  • DBN 1,5-diazabicyclo[4.3.0]-5-nonene
  • DABCO 1,4-diazabicyclo[2.2.2]octane
  • potassium tert-butoxide At least one base selected from the group consisting of sodium tert-butoxide, triethylamine, and tributylamine.
  • the step of separating the captured body comprises: After washing by adding an acidic aqueous solution to the organic solvent, the organic solvent is separated into an aqueous layer and an organic layer, and then the separated aqueous layer is separated.
  • the scavenger is at least one selected from the group consisting of morpholine, piperidine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine, and thiomorpholine dioxide.
  • the scavenger 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, in an organic solvent, an amino group-containing compound whose N-terminus is protected by a protecting group having a fluorene skeleton, and a scavenger represented by the following formula (Z1): and a step of contacting with to obtain a capturing body in which a by-product having a fulvene skeleton derived from the protecting group and the capturing agent are bound; a step of separating the captured body obtained from the organic solvent; including, Methods for removing protecting groups:
  • N is a nitrogen atom
  • H is a hydrogen atom
  • X is a divalent group represented by —CH 2 —, —O—, —S—, or —(SO 2 )—
  • n 1 R 1a , n 1 R 1b , n 2 R 2a , n 2 R 2b , n 3 R 3a , and n 3 R 3b are each independently H
  • the remover according to the eighth aspect of the present invention is an agent for removing a protecting group having a fluorene skeleton, containing a scavenger represented by the following formula (Z1) and a basic deprotecting agent:
  • N is a nitrogen atom
  • H is a hydrogen atom
  • X is a divalent group represented by —CH 2 —, —O—, —S—, or —(SO 2 )—
  • n 1 R 1a , n 1 R 1b , n 2 R 2a , n 2 R 2b , n 3 R 3a , and n 3 R 3b are each independently H , —OH, —OR (R is an alkyl group), —SH, —SR (R is as defined for —OR above.), —(SO 2 )H, or —(SO 2 )
  • R is the same as that of -OR above
  • the scavenger is morpholine, 3-hydroxypiperidine, 4-hydroxypiperidine, thiomorpholine , and thiomorpholine dioxide.
  • the deprotecting agent comprises 1,8-diazabicyclo[5. 4.0]-7-undecene, 1,5-diazabicyclo[4.3.0]-5-nonene, 1,4-diazabicyclo[2.2.2]octane, potassium tert-butoxide, sodium tert-butoxide, At least one base selected from the group consisting of triethylamine and tributylamine.
  • the benzyl compound (Y1) has the following formula (Y1): [In the formula, m Q's each represent an oxygen atom, m R 1 are each independently represented by the following formula (YA): (In the formula, * indicates the binding position, R 1a , R 1b , R 1c , R 1d 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 shown in parentheses to which n 1 is attached is an alkylene group, n 2 represents an integer of 0 or more and 6 or less, and when n 2 is 1 or more, the repeating unit shown in parentheses to which n 2 is attached is an alkylene group, However, at least two or more of R 1a , R 1b , R 1c and R 1d are hydrogen atoms.
  • 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
  • X represents a hydroxyl group
  • m represents an integer of 2 or 3
  • k represents an integer of 0 or more (5-m) or less
  • At least one of m [QR 1 ] is substituted at the meta position with respect to the substituent containing X. ] is represented by
  • the benzyl compound (Y1) according to the twelfth aspect of the present invention has a total carbon number of 40 or more and 60 or less in addition to the structure of the benzyl compound (Y1) according to the eleventh aspect described above.
  • each of the m R 1 independently one branched organic group,
  • R 1f is a linear chain having 4 to 10 carbon atoms is an alkyl group in the form of R 1g is a linear alkyl group having 6 or more and 12 or less carbon atoms.
  • a 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 above-described eleventh to fourteenth aspects in a soluble solvent, Next, a condensation reaction step of condensing the dissolved benzyl compound and an amino acid whose N-terminus is protected by an N-terminal protecting group to produce a first condensate; Then, a first base is added to the soluble solvent containing the first condensate to scavenge the amino acid active ester, the first base and the second base are added to the soluble solvent, and the second base is added to the soluble solvent.
  • a liquid separation step of obtaining a second condensate in which the N-terminal protecting group is deprotected from the first condensate in the organic layer including.
  • the N-terminally unprotected amino acid and The 2n-th condensate comprising an amino acid whose C-terminus is protected by the benzyl compound and the number of residues is n, is condensed with the n-th amino acid whose N-terminus is protected to the ( 2n+1) forming a condensate of the step of scavenging comprises performing deprotection of the N-terminal protecting group from the (2n+1)th condensate;
  • the liquid separation step includes a step of obtaining a (2n+2)th condensate in which the N-terminal protecting group is deprotected from the (2n+1)th condensate in the organic layer, Said n is a natural number of 2 or more.
  • n is 5 or more in addition to the configuration of the method for producing a peptide according to the 16th aspect described above.
  • the separation step includes: The step of adding a ketone-based liquid separation promoting solvent to the soluble solvent is further included.
  • the benzyl compound (X1) has the following formula (X1): [In the formula, m Q 1 and Q 2 are each an oxygen atom, m R 1 are each independently an alkylene group, 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, m is an integer of 2 or 3, k represents an integer from 0 to (5-m). ] is represented by
  • the m R 1s have 2 to 16 carbon atoms. is an alkylene group of
  • the m R 2 are halogen An aryl group having substituents containing atoms.
  • the m R 2 are carbon It is an alkyl group having a number of 5 or more and 28 or less.
  • the m R 2 are linear alkyl groups , or an alkyl group having a total of 1 or 2 branched chains, represented by the following formula (XA):
  • R 2a , R 2b , R 2c , R 2d and R 2e each independently represent a hydrogen atom or an alkyl group
  • n 1 represents an integer of 0 or more and 16 or less
  • n2 represents an integer of 0 or more and 16 or less.
  • at least two or more of R 2a , R 2b , R 2c and R 2d are hydrogen atoms.
  • Embodiment 1 of the present invention First, an example of Embodiment 1 of the present invention described above will be given.
  • Example (X1-a) 25 g (100 mmol) of 3,5-bistrifluoromethylphenol was dissolved in 125 mL of DMF, 27.6 g (120 mmol) of 11-bromoundecanol and 27.6 g (200 mmol) of potassium carbonate were added, and the mixture was stirred at 60° C. for 4 hours. The reaction solution was returned to room temperature, and solid content was removed by filtration. 150 mL of toluene and 75 mL of 1M hydrochloric acid were added to the filtrate for liquid separation and washing, and the organic layer was further washed with 75 mL of 1M hydrochloric acid and 100 mL of saturated brine.
  • Example (X1-c) 1.16 g (6.33 mmol) of methyl gallate was dissolved in 150 mL of DMF, 14.1 g (30.4 mmol) of compound (X1-2) and 16.6 g (120 mmol) of potassium carbonate were added, and the mixture was stirred at 60° C. for 18 hours. . After the potassium carbonate was removed by filtration, 100 mL of 1M hydrochloric acid and 100 mL of hexane were added to the reaction solution for liquid separation and washing, and the organic layer was further washed with 100 mL of 5% sodium hydrogen carbonate and 20% brine. After drying the organic layer with sodium sulfate, the solvent was distilled off under reduced pressure.
  • Example (X1-d) 6.2 g (4.66 mmol) of compound (X1-3) was dissolved in 150 mL of THF, 20.96 mL of lithium triethylborohydride (1 MTHF solution, 20.96 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 2 hours. Water (50 mL) and 20 mL of 1 M hydrochloric acid were added to the reaction solution to stop the reaction, and 200 mL of ethyl acetate was added to perform liquid separation and washing, and the organic layer was washed twice with 100 mL of water. After drying the organic layer with sodium sulfate, the solvent was distilled off under reduced pressure.
  • Example (X2-a) Dissolve 8 g (26.79 mmol) of 2-n-octyl-1-dodecanol in 210 mL of toluene (anhydrous), add 17.6 g (53.59 mmol) of dibromododecane and 2.14 g (53.59 mmol) of NaH, and stir at 105°C. Stirred overnight. The reaction solution was returned to room temperature, 10 mL of 1M hydrochloric acid was added, and the mixture was stirred for 10 minutes.
  • Example (X2-b) 0.77 g (5.61 mmol) of 2,4-dihydroxybenzaldehyde was dissolved in 116 mL of a mixed solvent of DMF:cyclopentyl methyl ether (1:1), and 7.6 g (14.03 mmol) of compound (X2-1) and potassium carbonate were dissolved. 3.9 g (28.06 mmol) was added and stirred at 90° C. for 3 hours. After the potassium carbonate was removed by filtration, 100 mL of 1M hydrochloric acid and 100 mL of hexane were added to the reaction solution for liquid separation and washing, and the organic layer was further washed with 100 mL of 5% sodium hydrogen carbonate and 20% brine.
  • Example (X2-c) 4.4 g (4.08 mmol) of compound (X2-2) was dissolved in 65 mL of a mixed solvent of THF (anhydrous):methanol (10:3), and 0.31 g (8.17 mmol) of sodium borohydride was dissolved under ice-cooling. was added and stirred for 10 minutes, the ice bath was removed, and the mixture was stirred at room temperature for 1 hour. 5 mL of acetone was added to the reaction solution to stop the reaction, and the solvent was distilled off under reduced pressure.
  • Example (X3-a) 0.79 g (4.29 mmol) of methyl gallate was dissolved in 132 mL of a mixed solvent of DMF:MTHP (1:1), and 8.8 g of compound (X2-1) prepared in Example (X2-a) (16. 12 mmol) and 2.65 g (19.17 mmol) of potassium carbonate were added, and the mixture was stirred at 90°C overnight. After the potassium carbonate was removed by filtration, 100 mL of 1M hydrochloric acid and 100 mL of hexane were added to the reaction solution for liquid separation and washing, and the organic layer was further washed with 100 mL of 5% sodium hydrogen carbonate and 20% brine.
  • Example (X3-b) 3.2 g (2.04 mmol) of compound (X3-1) was dissolved in 30 mL of THF (anhydrous), and 4.1 mL of diisobutylaluminum hydride (1.5 M toluene solution, 6.16 mmol) was added under ice-cooling. The mixture was stirred for 1 hour and stirred at room temperature for an additional 2 hours. After 5 mL of acetone was added to the reaction solution to stop the reaction, 30 g 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 distilled off under reduced pressure.
  • Example (X4-a) Dissolve 1 g (2.81 mmol) of 2-decyl-1-tetradecanol in 20 mL of toluene (anhydrous), add 1.85 g (5.63 mmol) of dibromododecane and 0.226 g (5.63 mmol) of NaH, and stir at 95°C. Stirred overnight. The reaction solution was returned to room temperature, 10 mL of 1M hydrochloric acid was added, and the mixture was stirred for 10 minutes.
  • Example (X4-b) 0.106 g (0.77 mmol) of 2,4-dihydroxybenzaldehyde was dissolved in 17.4 mL of a mixed solvent of DMF: cyclopentyl methyl ether (1:1) to obtain 1.16 g (1.92 mmol) of compound (X4-1), 0.532 g (3.85 mmol) of potassium carbonate was added and stirred at 90° C. for 4 hours. After potassium carbonate was removed by filtration, 20 mL of hexane and 36 mL of 1M hydrochloric acid were added to the reaction solution for liquid separation and washing, and the organic layer was washed twice with 18 mL of 5% sodium hydrogen carbonate and 18 mL of 20% brine.
  • Example (X4-c) 1.0 g (0.837 mmol) of compound (X4-2) was dissolved in 19.5 mL of a mixed solvent of THF (anhydrous):methanol (10:3), and 0.070 g of sodium borohydride (1.0 g of sodium borohydride) was dissolved under ice-cooling. 850 mmol) was added and stirred for 10 minutes, the ice bath was removed, and the mixture was stirred at room temperature for 1 hour. 4 mL of acetone was added to the reaction solution to terminate the reaction, and the solvent was distilled off under reduced pressure.
  • Example X5 ⁇ Confirmation of solubility of tag in organic solvent> Compound (X1-4) produced in Example X1, compound (X2-3) produced in Example X2, compound (X3-2) produced in Example X3, and compound (X4- The solubility (25°C) in various solvents of 3) was measured.
  • the linear C 18 H 37 compound shown in Comparative Example X1 was prepared by the method described in Examples of JP - A-2000-44494. , (2011), 4476-4479 was synthesized with reference to the method described in 4476-4479 was used.
  • Table X1 shows Example X1 (Compound (X1-4)), Example X2 (Compound (X2-3)), Example X3 (Compound (X3-2)), and Example X4 (Compound (X4-3 )) solubility results.
  • Table X2 shows the solubility results of Comparative Example X1 and Comparative Example X2. The value in parentheses in Table X2 indicates how many times the solubility of Examples X1 to X4 corresponds to the solubility of the compounds of Examples X1 to X4 when it is assumed that the solubility of the compounds of Examples X1 to X4 is uniformly 50 (% by weight) (i.e., 50 divided by the solubility).
  • Example X6-1 Confirmation of hydrophobicity of tag> Liquid chromatography (HPLC) was used to evaluate the hydrophobicity of the compounds of Examples X1 to X4 of the present invention.
  • Example X3 As shown in Table X3, the compounds of Comparative Examples X1 and X2 had retention times of 13.6 minutes and 12.1 minutes, respectively, while Examples X1-X4 had retention times of 8.4 minutes, respectively. minutes, 21.8 minutes, 60.2 minutes, and 33.2 minutes. All of Examples X2 to X4 greatly exceeded the retention times of Comparative Examples X1 and X2. In Example X1, although the retention times were smaller than those of Comparative Examples X1 and X2, they were kept at approximately the same level.
  • the compounds of Examples X1 to X4 all showed retention times equal to or longer than those of the compounds of Comparative Examples X1 and X2, in which the corresponding side chains were linear chains.
  • the compounds described above can serve as excellent tags in peptide synthesis.
  • Example X6-2 ⁇ Acid resistance> The stability of the side chain portion of the compound of the present invention against acid was evaluated using the compound (X1-4) produced in Example X1 and the compound (X3-2) produced in Example X3.
  • Non-Patent Document 5 discloses that the half-life of a triisopropylsilyl group to an acidic solvent (1 wt % hydrochloric acid/methanol) is 55 minutes. It was suggested that the compounds of Examples X1 and X3 are more stable to acid than compounds having at least an O--Si bond.
  • Example X7 Synthesis of H-Tyr(OtBu)-Ile-Leu-OTag (X1-4)
  • Example X7-1 Synthesis of HO-Leu-OTag (X1-4)
  • Compound (X1-4) 1.3 g (1.0 mmol) was dissolved in 20 mL of a mixed solution of MTHP/DMF (8/2), Fmoc-Leu-OH 0.46 g (1.3 mmol), EDCI.HCl 0.25 g (1.3 mmol) and DMAP 0.012 g ( 0.1 mmol) was added and stirred at room temperature for 4 hours. 39 ⁇ L (0.4 mmol) of morpholine was added and stirred at room temperature for 30 minutes.
  • Example X7-2 Synthesis of HO-Ile-Leu-OTag (X1-4) To the solution of HO-Leu-OTag (X1-4) obtained above, 4 mL of DMF, 0.46 g of Fmoc-Ile-OH (1. 3 mmol), 0.25 g (1.3 mmol) of EDCI.HCl and 0.046 g (0.3 mmol) of Oxyma were added and stirred at room temperature for 1 hour. 39 ⁇ L (0.4 mmol) of morpholine was added and stirred at room temperature for 30 minutes. 1.74 mL (20.0 mmol) of morpholine and 1.04 mL (7.0 mmol) of DBU were added and stirred at room temperature for 1 hour.
  • Example X7-3 Synthesis of H-Tyr(OtBu)-Ile-Leu-OTag (X1-4) Same as Example X7-2 except that Fmoc-Tyr(OtBu)-OH was used as the amino acid to be condensed. Working up, H-Tyr(OtBu)-Ile-Leu-OTag (X1-4) was obtained as a solution. The solvent was distilled off from the resulting organic layer under reduced pressure to obtain 1.58 g of H-Tyr(OtBu)-Ile-Leu-OTag (X1-4) (yield 90.3%). ESI-MS: 1749.13 [M+H] +
  • H-Tyr(OtBu)-Ile-Leu-OTag (X1-4) was obtained in high yield, by using a specific cyclic amine containing only one nitrogen atom such as morpholine as a scavenger, It was confirmed that the by-product could be easily removed.
  • Example X8 Synthesis of H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(OtBu)-Ile-Leu-OTag(X2-3) 0.5 g (0.46 mmol) of compound (X2-3) as a tag ) and the amino acids shown below from the 1st residue to the 6th residue, performing the same operations as in Examples X7-1 to Example X7-3, H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(OtBu)-Ile-Leu-OTag (X2-3) was obtained as a solution.
  • Example (Y1-a) 1.20 g (6.516 mmol) of methyl gallate was dissolved in 12 mL of DMF and 12 mL of cyclopentyl methyl ether (CPME), and 7.15 g (29.322 mmol) of 5-bromomethylundecane and 4.50 g (32.580 mmol) of potassium carbonate were added. The mixture was added and stirred at 110° C. for 10 hours. The reaction liquid was returned to room temperature, and the solid content was removed by filtration.
  • CPME cyclopentyl methyl ether
  • Example (Y2-a) 3.01 g (16.357 mmol) of methyl gallate was dissolved in 30 mL of DMF and 30 mL of CPME, 19.98 g (65.428 mmol) of 7-bromomethylpentadecane and 11.30 g (81.785 mmol) of potassium carbonate were added, and the mixture was stirred at 110°C for 12 hours. Stirred for hours. The reaction liquid was returned to room temperature, and the solid content was removed by filtration. 30 mL of CPME and 60 mL of 1M hydrochloric acid were added to the filtrate to perform liquid separation washing, and further, 60 mL of 5% sodium hydrogen carbonate and 60 mL of 20% brine were washed.
  • Example (Y2-b) 10.08 g (11.755 mmol) of compound (Y2-1) was dissolved in 120 mL of anhydrous THF, and 23.5 mL of diisobutylaluminum hydride (1.5 M toluene solution, 35.267 mmol) was added dropwise under ice cooling. , and stirred for 2 hours. 10 mL of 0.2 M hydrochloric acid was added to the reaction solution to terminate the reaction, and the solvent was distilled off under reduced pressure.
  • Example (Y3-a) 0.50 g (2.715 mmol) of methyl gallate was dissolved in 5 mL of DMF and 5 mL of CPME, 4.62 g (11.064 mmol) of 11-bromomethyltricosane and 1.88 g (13.602 mmol) of potassium carbonate were added, and the mixture was heated at 110°C. Stirred for 10 hours. The reaction liquid was returned to room temperature, and the solid content was removed by filtration. 20 mL of hexane and 20 mL of 1M hydrochloric acid were added to the filtrate to perform liquid separation washing, and the filtrate was further washed with 20 mL of 5% sodium hydrogen carbonate and 20 mL of 20% brine.
  • Example (Y4-a) 1.35 g (8.0 mmol) of methyl 3,5-dihydroxybenzoate was dissolved in 70 mL of DMF, 8.0 g (19.2 mmol) of 11-bromomethyltricosane and 3.32 g (24.0 mmol) of potassium carbonate were added, Stirred at 90° C. for 7 hours. After potassium carbonate was removed by filtration, 100 mL of water and 100 mL of ethyl acetate were added to the filtrate to separate and wash, and the organic layer was successively washed with 100 mL of water and 100 mL of 20% brine. After drying the organic layer with sodium sulfate, the solvent was distilled off under reduced pressure.
  • Example (Y4-b) 4.8 g (5.68 mmol) of compound (Y4-1) was dissolved in 80 mL of THF, 11.4 mL of diisopropylaluminum hydride (1.5 M toluene solution, 17.0 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 3 hours. . A 10% Rochelle salt aqueous solution (100 mL) was added to the reaction solution to stop the reaction, and then 200 mL of ethyl acetate was added to perform liquid separation and washing, and the organic layer was washed twice with 100 mL of water. After drying the organic layer with sodium sulfate, the solvent was distilled off under reduced pressure.
  • Example Y5 [Confirmation of solubility of tag in organic solvent] ⁇ Example Y5> 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- The solubility (25°C) in various solvents of 2) was measured.
  • the linear C 18 H 37 compound shown in Comparative Example Y1 was produced by the method described in Examples of JP-A-2000-44493, and the linear C 22 H 45 compound shown in Comparative Example Y2 was produced by the method described in Bioorganic & Medicinal chemistry letters, 21. , (2011), 4476-4479 was synthesized with reference to the method described in 4476-4479 was used.
  • Table Y1 shows Example Y1 (Compound (Y1-2)), Example Y2 (Compound (Y2-2)), Example Y3 (Compound (Y3-2)), and Example Y4 (Compound (Y4-2 )) solubility results.
  • Table Y2 shows the solubility results of Comparative Examples Y1 and Y2. The values in parentheses in Table Y2 indicate how many times the solubility of Examples Y1 to Y4 corresponds to the solubility of the compounds of Examples Y1 to Y4 when it is assumed that the solubility of the compounds of Examples Y1 to Y4 is uniformly 50 (% by weight) (that is, 50 divided by the solubility).
  • Example Y7-1 Synthesis of HO-Leu-OTag (Y1-4) 1.0 g (1.21 mmol) of compound (Y2-2) was dissolved in 25 mL of a mixture of MTHP/acetonitrile (8/2), and Fmoc -Leu-OH 0.554 g (1.57 mmol), EDCI.HCl 0.30 g (1.57 mmol) and DMAP 0.0147 g (0.121 mmol) were added and stirred at room temperature for 2 hours. 42.2 ⁇ L (0.482 mmol) of morpholine was added and stirred at room temperature for 30 minutes.
  • Example Y7-2 Synthesis of HO-Ile-Leu-OTag (Y2-2) To the solution of HO-Leu-OTag (Y2-2) obtained above was added 5.0 mL of acetonitrile and 0.511 g of Fmoc-Ile-OH. (1.45 mmol), 0.277 g (1.45 mmol) of EDCI.HCl and 0.0514 g (0.362 mmol) of Oxyma were added and stirred at room temperature for 1 hour. 42.2 ⁇ L (0.482 mmol) of morpholine was added and stirred at room temperature for 30 minutes.
  • Example Y7-3 Synthesis of H-Tyr(tBu)-Ile-Leu-OTag (Y2-2) Same as Example Y7-2 except that Fmoc-Tyr(tBu)-OH was used as the amino acid to be condensed. After working up, H-Tyr(tBu)-Ile-Leu-OTag (Y2-2) was obtained as a solution.
  • Example Y7-4 Synthesis of H-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) The same procedure as in Example Y7-2 except that Fmoc-Pro-OH was used as the amino acid to be condensed. was carried out to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) as a solution.
  • Example Y7-5 Synthesis of H-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) The same operation as in Example Y7-2 was performed except that MTHP/DMF (8/2) was used as the solvent, and H-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2 ) was obtained as a solution.
  • Example Y7-6 Synthesis of H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2)
  • MTHP/DMF (8/2) was used as the reaction solvent
  • H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu) -Ile-Leu-OTag (Y2-2) was obtained as a solution.
  • Example Y7-7 Synthesis of H-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) Using Fmoc-Pro-OH as the amino acid to be condensed , In addition, the same operation as in Example Y7-2 was performed except that 2 mL of acetone was added during the third 2M hydrochloric acid separation to separate the liquids, and H-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr (tBu)-Ile-Leu-OTag (Y2-2) was obtained as a solution.
  • Example Y7-8 Synthesis of H-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Y2-2)
  • Lys(Boc)-OH was used, and 2 mL of acetone was added during the third 2M hydrochloric acid separation to separate the liquids
  • H-Lys(Boc)-Pro- Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) was obtained as a solution.
  • Example Y7-9 Synthesis of H-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) Condensation
  • Fmoc-Asn(Trt)-OH was used as the amino acid to be used
  • H-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf )-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) was obtained as a solution.
  • Example Y7-10 H-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Y2- 2) Synthesis H-Glu(OtBu)-Asn(Trt)-Lys(Boc)- Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) was obtained as a solution.
  • Example Y7-11 H-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu - Synthesis of OTag (Y2-2)
  • Fmoc-Tyr(tBu)-OH was used as the amino acid to be condensed
  • H-Tyr(tBu)-Glu(OtBu)- Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) was obtained as a solution.
  • Example Y7-12 H-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile -Synthesis of Leu-OTag (Y2-2) H-Leu-Tyr(tBu)-Glu(OtBu) was prepared in the same manner as in Example Y7-2, except that Fmoc-Leu-OH was used as the amino acid to be condensed.
  • Example Y7-13 H-PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu) -Synthesis of Ile-Leu-OTag (Y2-2)
  • Fmoc-PyroGlu-OH was used as the amino acid to be condensed
  • H-PyroGlu-Leu-Tyr(tBu)- Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y2-2) was obtained as a solution.
  • Example Y8 Synthesis of H-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Y3-2) Compound (Y3-2) as tag Using 0.5 g (0.428 mmol), peptide synthesis was carried out in the same manner as in Examples Y7-1 to Y7-6. It was confirmed that in liquid separation after condensation of Fmoc-Lys(Boc)-OH at the 8th residue, it took a long time to separate the organic layer and the aqueous layer during liquid separation with 2M hydrochloric acid.
  • Comparative example Y3 Synthesis of H-Leu-OTag (Comparative Example Y1) Using 1.0 g (1.09 mmol) of the compound shown in Comparative Example Y1 as a tag, synthesis was carried out in the same manner as in Example Y7-1. It was confirmed that a large amount of solid precipitated in liquid separation after condensation of Fmoc-Leu-OH, making liquid separation difficult and further peptide synthesis difficult.
  • 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.
  • the solubility of the benzyl compound in an organic solvent was It was confirmed that the number of peptide synthesis residues increases as the temperature increases.
  • compound (Y2-2) and compound (Y3-2) it was confirmed that compound (Y2-2), which has moderate hydrophobicity, increased the number of peptide synthesis residues the most. bottom.
  • tags with high solubility in organic solvents and moderate hydrophobicity, not too high are suitable as tags for producing long-chain peptides.
  • the benzyl compounds (compounds (Y1-2), (Y2-2), (Y3-2) and (Y4-2)) according to Examples Y1 to Y4 above are used as long-chain tags in the liquid phase tagging method.
  • Compounds (Y2-2) and (Y3-2) according to Examples Y2 and Y3 are particularly useful for peptide synthesis, and compound (Y2-2) according to Example Y2 is more useful. Confirmed that there is.
  • Embodiment 3 of the present invention described above will be given.
  • the synthesis method will be described below using peptides having the sequences shown below as examples, but the present invention is not limited to these.
  • the starting material was dissolved in a mixed solution of MTHP/DMF (8/2) so as to be 18 v/w, morpholine (20.0 equiv) and DBU (7.0 equiv) were added, and the mixture was stirred at room temperature for 1 hour.
  • the reaction solution was transferred to a separating funnel, and 2N hydrochloric acid was added to wash and separate the liquids to obtain the de-Fmoc compound as a solution.
  • the starting material, Fmoc-AA-OH was dissolved in a mixture of MTHP/DMF (8/2) to a concentration of 18 v/w, and Fmoc amino acid (1.3 equiv), EDCI.HCl (1.3 equiv) and Oxyma0. 0514 g (0.1 equiv) was added and stirred at room temperature for 1 hour. Morpholine (0.4 equiv) was added and stirred at room temperature for 30 minutes. Morpholine (20.0 equiv) and DBU (7.0 equiv) were added and stirred at room temperature for 1 hour. The reaction solution was transferred to a separating funnel, and 20% saline (18 v/w) was added twice to wash and separate the solution.
  • the organic layer was washed with 2M hydrochloric acid (18v/w) ⁇ 3 times, and separated, and further washed with 0.5M sodium hydrogen carbonate aqueous solution (18v/w), separated, and the organic layer was diluted with an appropriate amount of sulfuric acid. After drying with sodium, it was filtered while washing with an appropriate amount of MTHP to obtain an amino acid condensate as a solution.
  • Example Z1 PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu - Synthesis of OTag (Z1-13)
  • Example Z1-1 Synthesis of HO-Leu-OTag (Z1-1)
  • the amino acid condensate (HO-Leu-OTag(Z1-1)) is the same as the amino acid condensate (HO-Leu-OTag(Y2-2)) synthesized in Example Y7-1.
  • Example Z1-2 Synthesis of HO-Ile-Leu-OTag (Z1-2)
  • an amino acid condensate (HO-Ile-Leu-OTag (Z1-2)) was prepared in solution. obtained as The amino acid condensate (HO-Ile-Leu-OTag (Z1-2)) is the same as the amino acid condensate (HO-Leu-OTag (Y2-2)) synthesized in Example Y7-2.
  • Example Z1-3 Synthesis of H-Tyr(tBu)-Ile-Leu-OTag (Z1-3) The same procedure as in Example Z1-2 except that Fmoc-Tyr(tBu)-OH was used as the amino acid to be condensed. After working up, H-Tyr(tBu)-Ile-Leu-OTag (Z1-3) was obtained as a solution.
  • Example Z1-4 Synthesis of H-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-4) The same procedure as in Example Z1-2 except that Fmoc-Pro-OH was used as the amino acid to be condensed. was carried out to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-4) as a solution.
  • Example Z1-5 Synthesis of H-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-5) H-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-5 ) was obtained as a solution.
  • Example Z1-6 Synthesis of H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-6)
  • MTHP/DMF (8/2) was used as the reaction solvent
  • H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu) -Ile-Leu-OTag (Z1-6) was obtained as a solution.
  • Example Z1-7 Synthesis of H-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-7) , In addition, the same operation as in Example Z1-2 was performed except that 2 mL of acetone was added during the third 2M hydrochloric acid separation to separate the liquids, and H-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr (tBu)-Ile-Leu-OTag (Z1-7) was obtained as a solution.
  • Example Z1-8 Synthesis of H-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z1-8) The same procedure as in Example Z1-8 was carried out except that Lys(Boc)-OH was used, and 2 mL of acetone was added during the third 2M hydrochloric acid separation to separate the liquids, and H-Lys(Boc)-Pro- Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-8) was obtained as a solution.
  • Example Z1-9 Synthesis of H-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-9) Condensation The same operation as in Example Z1-2 was performed except that Fmoc-Asn(Trt)-OH was used as the amino acid to be used, and H-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf )-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-9) was obtained as a solution.
  • Examples Z1-10 H-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z1- 10) Synthesis H-Glu(OtBu)-Asn(Trt)-Lys(Boc)- Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-10) was obtained as a solution.
  • Example Z1-11 H-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu Synthesis of -OTag (Z1-11)
  • Fmoc-Tyr(tBu)-OH was used as the amino acid to be condensed
  • H-Tyr(tBu)-Glu(OtBu)- Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-11) was obtained as a solution.
  • Examples Z1-12 H-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile - Synthesis of Leu-OTag (Z1-12)
  • the same operation as in Example Z1-2 was performed except that Fmoc-Leu-OH was used as the amino acid to be condensed, and H-Leu-Tyr(tBu)-Glu(OtBu) -Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-12) was obtained as a solution.
  • Examples Z1-13 PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile - Synthesis of Leu-OTag (Z1-13)
  • the same operation as in Example Z1-2 was performed except that Fmoc-PyroGlu-OH was used as the amino acid to be condensed, and H-PyroGlu-Leu-Tyr(tBu)-Glu( OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z1-13) was obtained as a solution.
  • Example Z1-14 Deprotection of C-Terminal Carrier and Side Chain Functional Group
  • the solvent was distilled off from the resulting organic layer under reduced pressure, and 120 mL of an 80% aqueous acetonitrile solution was added to the residue under ice-cooling to obtain The precipitate is filtered, dried under reduced pressure, and PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu )-Ile-Leu-OTag (Z1-13) (1.401 g, 0.40 mmol, 33.1% yield).
  • Example Z2 PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr with C-Terminal Support B Synthesis of (tBu)-Ile-Leu-OTag (Z2-13)
  • the C-terminal carrier B used in Example Z2 is the following compound (Z7).
  • Example Z2-1 Synthesis of H-Tyr(tBu)-Ile-Leu-OTag (Z2-2) 6 mL, Fmoc-Tyr(tBu)-OH 0.789 g (1.72 mmol), EDCI.HCl 0.329 g (1.72 mmol) and Oxyma 0.0563 g (0.396 mmol) were added and stirred at room temperature for 1 hour. 45.7 ⁇ L (0.528 mmol) of morpholine was added and stirred at room temperature for 30 minutes. 2.28 mL (26.4 mmol) of morpholine and 1.38 mL (9.24 mmol) of DBU were added and stirred at room temperature for 1 hour.
  • Example Z2-2 Synthesis of H-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-3) The same procedure as in Example Z2-1 except that Fmoc-Pro-OH was used as the amino acid to be condensed. was carried out to obtain H-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-3) as a solution.
  • Example Z2-3 Synthesis of H-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-4) The procedure was carried out except that Fmoc-Arg(Pbf)-OH was used as the amino acid to be condensed. By performing the same operation as in Example Z2-1, H-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-4) was obtained as a solution.
  • Example Z2-4 Synthesis of H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-5) H-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-5) was obtained as a solution by performing the same operation as in Example Z2-1 except that .
  • Example 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 H-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-6) was obtained as a solution. rice field.
  • Example 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 Lys(Boc)-OH was used and liquid separation was performed by the following method. The reaction solution was transferred to a separating funnel, and 18 mL of 20% saline solution was added twice to wash and separate the solution.
  • the organic layer is washed with 18 mL of 2M hydrochloric acid 3 times and separated, further washed with 18 mL of 0.5M sodium hydrogen carbonate aqueous solution, separated, and the organic layer is dried with an appropriate amount of sodium sulfate, and then added with an appropriate amount of MTHP. While washing with, filter, amino acid condensate (H-Lys (Boc)-Pro-Arg (Pbf)-Arg (Pbf)-Pro-Tyr (tBu)-Ile-Leu-OTag (Z2-7) ) was obtained as a solution.
  • amino acid condensate H-Lys (Boc)-Pro-Arg (Pbf)-Arg (Pbf)-Pro-Tyr (tBu)-Ile-Leu-OTag (Z2-7)
  • Example Z2-7 Synthesis of H-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-8) Condensation
  • Fmoc-Asn(Trt)-OH was used as the amino acid to be used
  • H-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf )-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-8) was obtained as a solution.
  • Example Z2-8 H-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag(Z2- 9) Synthesis H-Glu(OtBu)-Asn(Trt)-Lys(Boc)- Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-9) was obtained as a solution.
  • Example Z2-9 H-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu - Synthesis of OTag (Z2-10)
  • Fmoc-Tyr(tBu)-OH was used as the amino acid to be condensed
  • H-Tyr(tBu)-Glu(OtBu)- Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-10) was obtained as a solution.
  • Example Z2-10 H-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile -Synthesis of Leu-OTag (Z2-11)
  • Fmoc-Leu-OH was used as the amino acid to be condensed
  • H-Leu-Tyr(tBu)-Glu(OtBu) -Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile-Leu-OTag (Z2-11) was obtained as a solution.
  • Example Z2-11 PyroGlu-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Lys(Boc)-Pro-Arg(Pbf)-Arg(Pbf)-Pro-Tyr(tBu)-Ile -Synthesis of Leu-OTag (Z2-12) PyroGlu-Leu-Tyr(tBu)-Glu(OtBu) was prepared in the same manner as in Example Z2-1 except that Fmoc-PyroGlu-OH was used as the amino acid to be condensed.
  • Example Z3 Comparison of amounts of diketopiperazine produced by various bases To an MTHP/DMF solution (8:2) of Fmoc-Tyr(tBu)-Leu-OTag (Z3-1), 5 equiv of morpholine (Z3-2) was added. After stirring for 2 hours at room temperature, quantitative analysis by HPLC was performed. The amount of diketopiperazine produced is equal to the amount of Tag-OH (Z3-8) produced simultaneously with the production of diketopiperazine.
  • Comparative example Z2 Instead of 5 equiv of morpholine, 5 equiv of N-methylpiperazine (Z3-5, described in Patent Document 2)] was added, but the same operation as in Example Z3 was performed and the same quantitative analysis was performed.
  • morpholine was able to suppress the production rate of diketopiperazine compared to diethylamine and N-methylpiperazine. Specifically, when morpholine is used, the ratio of diketopiperazine formation to the various compounds produced is suppressed to about 60% when diethylamine is used, and about half when N-methylpiperazine is used. could be suppressed. Thus, it was confirmed that the progress of side reactions was suppressed. This is probably because unintended deprotection of the Fmoc group could be suppressed because the cyclic amine used in the present invention has low basicity.

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Abstract

Un problème à résoudre par la présente invention est de fournir : un procédé d'élimination de groupe protecteur capable de supprimer la génération de corps à double impact et de la dicétopipérazine, de piéger facilement un composé comprenant un squelette fulvène qui est un sous-produit potentiellement généré après déprotection d'un groupe protecteur ayant un squelette fluoré, et d'isoler facilement un corps piégé du sous-produit ; un procédé de production de peptide comprenant une étape d'élimination d'un groupe protecteur ; et un agent d'élimination de groupe protecteur. Un procédé de production de peptide selon un aspect de la présente invention comprend : une étape qui consiste à mettre en contact, dans un solvant organique, un agent de piégeage représenté par la formule (Z1) avec un composé contenant un groupe amino, l'extrémité N-terminale étant protégée par un groupe protecteur ayant un squelette fluoré, pour obtenir un corps piégé, l'agent de piégeage étant lié à un sous-produit ayant un squelette fulvène dérivé du groupe protecteur ; et une étape qui consiste à séparer le corps piégé obtenu du solvant organique.
PCT/JP2022/042265 2021-12-27 2022-11-14 Procédé de production de peptide, procédé d'élimination de groupe protecteur, agent d'élimination et composé benzylique Ceased WO2023127331A1 (fr)

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WO2025229967A1 (fr) * 2024-04-30 2025-11-06 富士フイルム株式会社 Procédé de production de peptide

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