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WO2025229966A1 - Procédé d'élimination d'un groupe fmoc, procédé de production de peptide et procédé de production d'un composé - Google Patents

Procédé d'élimination d'un groupe fmoc, procédé de production de peptide et procédé de production d'un composé

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Publication number
WO2025229966A1
WO2025229966A1 PCT/JP2025/016301 JP2025016301W WO2025229966A1 WO 2025229966 A1 WO2025229966 A1 WO 2025229966A1 JP 2025016301 W JP2025016301 W JP 2025016301W WO 2025229966 A1 WO2025229966 A1 WO 2025229966A1
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WIPO (PCT)
Prior art keywords
group
formula
peptide
fmoc
groups
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English (en)
Japanese (ja)
Inventor
研史 白兼
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Fujifilm Corp
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Fujifilm Corp
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Publication of WO2025229966A1 publication Critical patent/WO2025229966A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B51/00—Introduction of protecting groups or activating groups, not provided for in the preceding groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
    • C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
    • C07C227/20—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters by hydrolysis of N-acylated amino-acids or derivatives thereof, e.g. hydrolysis of carbamates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/38—Separation; Purification; Stabilisation; Use of additives
    • C07C227/44—Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/56—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • 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

  • the present disclosure relates to a method for removing an Fmoc group, a method for producing a peptide, and a method for producing a compound represented by formula (2) defined herein.
  • the 9-fluorenylmethyloxycarbonyl group (also known as the Fmoc group) is an important protecting group for amino acid and peptide amino groups in peptide synthesis. Removal of the Fmoc group produces dibenzofulvene (DBF) or a DBF derivative as a by-product. Furthermore, removal of the Fmoc group is generally performed using a base. For example, when an amine is used to remove the Fmoc group, an adduct of DBF and the amine (also known as a DBF-amine adduct) is produced as a DBF derivative by-product. Continuing peptide synthesis with DBF or a DBF derivative remaining can result in side reactions, so it is desirable to efficiently remove DBF or a DBF derivative.
  • Patent Document 1 describes a method for removing DBF or a DBF derivative, which includes the steps of mixing a compound represented by HS-L-COOH (wherein L represents a C1-8 alkylene group optionally having a substituent), an amino-group-containing compound protected with an Fmoc group, and a base to obtain a reaction mixture containing a compound represented by Fm-S-L-COOH (wherein Fm represents a 9-fluorenylmethyl group and L is as defined above) and the amino-group-containing compound, and washing the resulting reaction mixture with a basic aqueous solution to remove the compound represented by Fm-S-L-COOH.
  • HS-L-COOH wherein L represents a C1-8 alkylene group optionally having a substituent
  • Fm-S-L-COOH wherein Fm represents a 9-fluorenylmethyl group and L is as defined above
  • Patent Document 2 also describes the use of a trapping agent for DBF (dibenzofulvene) and an adduct of DBF with an amine (DBF-amine adduct) produced by the Fmoc removal reaction, and describes a mercapto compound such as 3-mercaptopropionic acid as the trapping agent.
  • Patent Document 3 describes that if DBF, which is produced during Fmoc removal in the peptide elongation step, is captured with HS-L-COOH (where L is a C1-8 alkylene group), DBF can be removed by separation using a basic aqueous solution.
  • Patent No. 6136934 International Publication No. WO2023/033017 Patent No. 7063408
  • L 1 includes one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 1 , —O—, and —S—, and the above groups may have a substituent, and includes at least one of >NR 1 , —O—, and —S— in the main chain;
  • R 1 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR 2 ;
  • R2 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkyny
  • Formula (2) Fm-S-L 1 -COOH (In formula (2), Fm represents a 9-fluorenylmethyl group, and L1 has the same definition as in formula (1)). and a step D of separating the amino group-containing compound from which the Fmoc group has been removed from the compound represented by formula (2), Methods for removing the Fmoc group.
  • Methods for removing the Fmoc group ⁇ 2> The method for removing an Fmoc group according to ⁇ 1>, wherein L 1 includes at least one of >NR 1 or —O—.
  • ⁇ 3> The method for removing an Fmoc group according to ⁇ 1> or ⁇ 2>, wherein the step B and the step C are carried out in one pot.
  • ⁇ 4> The method for removing an Fmoc group according to any one of ⁇ 1> to ⁇ 3>, wherein in the step D, the amino group-containing compound from which the Fmoc group has been removed is separated from the compound represented by formula (2) by crystallization or liquid separation.
  • ⁇ 5> The method for removing an Fmoc group according to any one of ⁇ 1> to ⁇ 4>, wherein in the step D, the amino group-containing compound from which the Fmoc group has been removed is separated from the compound represented by formula (2) by liquid separation using a basic aqueous solution.
  • Step A condensing an amino acid or peptide whose N-terminus is protected with an Fmoc group with an amino acid or peptide whose C-terminus is protected;
  • Step B deprotecting the peptide obtained in Step A to produce a peptide from which the Fmoc group has been removed; Dibenzofulvene produced in step B above, Formula (1): HS-L 1 -COOH (In formula (1), L 1 includes one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 1 , —O—, and —S—, and the above groups may have a substituent, and includes at least one of >NR 1 , —O—, and —S— in the main chain; R 1 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group,
  • Formula (2) Fm-S-L 1 -COOH (In formula (2), Fm represents a 9-fluorenylmethyl group, and L1 has the same definition as in formula (1)). and a step D of separating the peptide from which the Fmoc group has been removed from the compound represented by formula (2).
  • a method for producing a peptide comprising: ⁇ 7> The method for producing a peptide according to ⁇ 6>, wherein the protecting group in the C-terminal protected amino acid or peptide is linked to the carbonyl group of the amino acid or peptide via an ester or amide bond.
  • Y represents -OR C , -NR C R, or -SR C ;
  • R represents a hydrogen atom, an alkyl group, an arylalkyl group, a heteroarylalkyl group, or a 9-fluorenylmethyloxycarbonyl group;
  • m represents 1 or 2; and
  • n represents an integer of 1 to 5.
  • R and B each independently represent an aliphatic hydrocarbon group; RC represents the binding site to the C-terminal amino acid or peptide; Each R is independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group, at least one of the aliphatic hydrocarbon groups having 12 or more carbon atoms, and the benzene ring in formula (3) may further have a substituent in addition to R.
  • ⁇ 9> The method for producing the peptide according to ⁇ 8>, wherein the total number of carbon atoms in all aliphatic hydrocarbon groups possessed by all R 1 A is 36 to 80.
  • ⁇ 10> The method for producing the peptide according to any one of ⁇ 6> to ⁇ 9>, wherein L 1 includes at least one of >NR 1 or —O—.
  • ⁇ 11> The method for producing the peptide according to any one of ⁇ 6> to ⁇ 10>, wherein the step B and the step C are carried out in one pot.
  • ⁇ 12> The method for producing the peptide according to any one of ⁇ 6> to ⁇ 11>, wherein in the step D, the amino group-containing compound from which the Fmoc group has been removed is separated from the compound represented by formula (2) by crystallization or liquid separation.
  • ⁇ 13> The method for producing the peptide according to any one of ⁇ 6> to ⁇ 12>, wherein in the step D, the amino group-containing compound from which the Fmoc group has been removed is separated from the compound represented by formula (2) by liquid separation using a basic aqueous solution.
  • step D the amino group-containing compound from which the Fmoc group has been removed is separated from the compound represented by formula (2) by liquid separation using a basic aqueous solution.
  • ⁇ 14> The method for producing the peptide according to any one of ⁇ 6> to ⁇ 13>, comprising, between the step A and the step B, a step of trapping the amino acid activated ester produced in the step A.
  • ⁇ 15> The method for producing a peptide according to ⁇ 14>, wherein the step of trapping the amino acid activated ester produced in the step A is a step of mixing the amino acid activated ester produced in the step A with an amine represented by formula (4) to trap the amino acid activated ester.
  • L 1 contains one or more groups selected from a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 3 , ⁇ N—, —CO—NR a —, —CS—NR a —, —O—, and —S—, and these groups may have a substituent;
  • R 3 represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group;
  • R a represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR 4 ;
  • R1 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR4 , and R4 represents a hydrogen
  • L 1 includes one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 1 , —O—, and —S—, and the above groups may have a substituent, and includes at least one of >NR 1 , —O—, and —S— in the main chain;
  • R 1 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR 2 ;
  • R2 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group.
  • Formula (2) Fm-S-L 1 -COOH (In formula (2), Fm represents a 9-fluorenylmethyl group, and L1 has the same definition as in formula (1)).
  • a method for producing a compound represented by formula (2) comprising the step of producing a compound represented by formula (2).
  • L 1 includes at least one of >NR 1 or —O—.
  • a method for removing an Fmoc group can be provided that can highly efficiently remove dibenzofulvene generated during peptide synthesis.
  • a numerical range expressed using "to” means a range that includes the numerical values before and after "to" as the lower and upper limits.
  • the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range.
  • the upper or lower limit of a numerical range may be replaced with a value shown in the examples.
  • the term "process” includes not only an independent process but also a process that cannot be clearly distinguished from other processes as long as the intended purpose of the process is achieved.
  • an "alkyl group” encompasses not only alkyl groups without a substituent (unsubstituted alkyl groups) but also alkyl groups with a substituent (substituted alkyl groups).
  • Chemical structural formulas may be written as simplified structural formulas in which hydrogen atoms are omitted. Combinations of two or more preferred aspects are more preferred aspects.
  • amino acids and peptides disclosed herein contain hydroxyl groups, amino groups, carboxyl groups, carbonyl groups, amide groups, guanidyl groups, mercapto groups, etc., these groups may be protected, and the target compounds can be obtained by removing the protecting groups as necessary after the reaction.
  • hydroxy-protecting groups include alkyl groups, aryl groups, trityl groups, arylalkyl groups having 7 to 10 carbon atoms, formyl groups, acyl groups having 1 to 6 carbon atoms, benzoyl groups, arylalkylcarbonyl groups having 7 to 10 carbon atoms, 2-tetrahydropyranyl groups, 2-tetrahydrofuranyl groups, silyl groups, and alkenyl groups having 2 to 6 carbon atoms. These groups may be substituted with one to three substituents selected from the group consisting of halogen atoms, alkyl groups having 1 to 6 carbon atoms, alkoxy groups having 1 to 6 carbon atoms, and nitro groups.
  • protecting groups for amino groups include formyl, acyl groups having 1 to 6 carbon atoms, alkoxycarbonyl groups having 1 to 6 carbon atoms, benzoyl groups, arylalkylcarbonyl groups having 7 to 10 carbon atoms, arylalkyloxycarbonyl groups having 7 to 14 carbon atoms, trityl groups, monomethoxytrityl groups, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl groups, phthaloyl groups, N,N-dimethylaminomethylene groups, silyl groups, alkenyl groups having 2 to 6 carbon atoms, etc.
  • These groups may be substituted with 1 to 3 substituents selected from the group consisting of halogen atoms, alkoxy groups having 1 to 6 carbon atoms, and nitro groups.
  • the protecting group for the carboxy group include the above-mentioned protecting groups for the hydroxy group, a trityl group, and the like.
  • the carbonyl-protecting group include cyclic acetals (eg, 1,3-dioxane), acyclic acetals (eg, di(alkyl having 1 to 6 carbon atoms)acetals), and the like.
  • Examples of the protecting group for the amide group include a trityl group.
  • Examples of the protecting group for the guanidyl group include a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group, a 2,3,4,5,6-pentamethylbenzenesulfonyl group, a tosyl group, and a nitro group.
  • Examples of the protective group for a mercapto group (thiol group) include a trityl group, a 4-methylbenzyl group, an acetylaminomethyl group, a t-butyl group, and a t-butylthio group.
  • protecting groups can be removed by known methods, such as those described in Protective Groups in Organic Synthesis, John Wiley and Sons (1980). Methods that use acids, bases, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylsilyl halides, and reduction methods can be used.
  • amino acid refers to ⁇ , ⁇ , or ⁇ amino acids, and is not limited to naturally occurring amino acids; it may also be a non-naturally occurring amino acid. It may also be an amino acid analogue such as a hydroxycarboxylic acid.
  • the alkyl group may be linear or branched and may be substituted.
  • substituents include a halogen atom, a C1-6 alkyl group, a C3-8 cycloalkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C1-6 acyl group, a C1-6 alkoxy group, a C6-20 aryl group, a C5-20 heteroaryl group, and a heterocyclic group.
  • the alkyl group includes an alkyl group having 1 to 30 carbon atoms (also referred to as "number of carbon atoms"), and may be an alkyl group having 1 to 20 carbon atoms, an alkyl group having 1 to 10 carbon atoms, or an alkyl group having 1 to 6 carbon atoms.
  • Examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, and tert-butyl.
  • the cycloalkyl group may be substituted with a halogen atom, an alkoxy group, etc.
  • the alkyl group may be a cycloalkyl group having 3 to 10 carbon atoms or an alkyl group having 3 to 6 carbon atoms.
  • the arylalkyl group may be an arylalkyl group having 7 to 30 carbon atoms, or may be an arylalkyl group having 7 to 20 carbon atoms, an arylalkyl group having 7 to 16 carbon atoms (for example, an aryl group having 6 to 10 carbon atoms bonded to an alkylene group having 1 to 6 carbon atoms), or an arylalkyl group having 7 to 10 carbon atoms.
  • Specific examples include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, and 1-naphthylpropyl groups, with benzyl being preferred.
  • the alkenyl group preferably has 2 to 30 carbon atoms, more preferably 2 to 20, even more preferably 2 to 10, and even more preferably 2 to 6.
  • alkenyl groups include pentenyl, hexenyl, and oleyl groups.
  • the alkynyl group preferably has 2 to 30 carbon atoms, more preferably 2 to 20, even more preferably 2 to 10, and even more preferably 2 to 6.
  • alkynyl groups include 4-pentynyl and 5-hexenyl groups.
  • Aliphatic hydrocarbon groups include alkyl groups, cycloalkyl groups, alkenyl groups, and alkynyl groups. Aliphatic hydrocarbon groups may be linear or branched, and may be substituted. Substituents are the same as those for alkyl groups.
  • the aliphatic hydrocarbon group preferably has 1 to 30 carbon atoms, more preferably 1 to 20, even more preferably 1 to 10, and even more preferably 1 to 6.
  • the aryl group preferably has 6 to 30 carbon atoms, more preferably 6 to 20, even more preferably 6 to 14, and even more preferably 6 to 10.
  • Specific examples include phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, and 2-anthryl groups. Of these, phenyl is preferred.
  • the heteroaryl group preferably has 5 to 30 constituent atoms, more preferably 5 to 20, even more preferably 5 to 14, and even more preferably 5 to 10.
  • the heteroaryl group is preferably a monocyclic or bicyclic heteroaryl group.
  • Examples of the monocyclic heteroaryl group include a monocyclic nitrogen-containing heteroaryl group, a monocyclic oxygen-containing heteroaryl group, a monocyclic sulfur-containing heteroaryl group, a monocyclic nitrogen- and oxygen-containing heteroaryl group, and a monocyclic nitrogen- and sulfur-containing heteroaryl group.
  • Examples of the bicyclic heteroaryl group include a bicyclic nitrogen-containing heteroaryl group, a bicyclic oxygen-containing heteroaryl group, a bicyclic sulfur-containing heteroaryl group, a bicyclic nitrogen- and oxygen-containing heteroaryl group, and a bicyclic nitrogen- and sulfur-containing heteroaryl group.
  • heteroaryl groups include an indolyl group, a benzofuranyl group, a thiophenyl group, a benzothiophenyl group, a furfuryl group, a benzofuranylmethyl group, a thiophenylmethyl group, and a benzothiophenylmethyl group.
  • Heteroarylalkyl groups include the above-mentioned alkyl groups substituted with the above-mentioned heteroaryl groups.
  • halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • Alkoxy groups include methoxy, ethoxy, propoxy, and the like.
  • examples of the acyl group include acetyl and propionyl.
  • Arylalkylcarbonyl groups include benzylcarbonyl.
  • Examples of the alkoxycarbonyl group include methoxycarbonyl, ethoxycarbonyl, and Boc groups, where Boc group means a tert-butoxycarbonyl group.
  • Step B deprotecting the amino group-containing compound having an Fmoc group to produce an amino group-containing compound from which the Fmoc group has been removed; Dibenzofulvene produced in step B above, Formula (1): HS-L 1 -COOH (In formula (1), L 1 includes one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 1 , —O—, and —S—, and the above groups may have a substituent, and includes at least one of >NR 1 , —O—, and —S— in the main chain; R 1 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or
  • Formula (2) Fm-S-L 1 -COOH (In formula (2), Fm represents a 9-fluorenylmethyl group, and L1 has the same definition as in formula (1)). and a step D of separating the amino group-containing compound from which the Fmoc group has been removed from the compound represented by formula (2), This relates to a method for removing the Fmoc group.
  • L 1 includes one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 1 , —O—, and —S—, and the above groups may have a substituent, and includes at least one of >NR 1 , —O—, and —S— in the main chain;
  • R 1 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR 2 ;
  • R2 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group.
  • Formula (2) Fm-S-L 1 -COOH (In formula (2), Fm represents a 9-fluorenylmethyl group, and L1 has the same definition as in formula (1)).
  • the present invention relates to a method for producing a compound represented by formula (2), comprising the step of producing a compound represented by formula (2).
  • step B an amino group-containing compound having an Fmoc group is deprotected to produce an amino group-containing compound from which the Fmoc group has been removed.
  • the amino group-containing compound having an Fmoc group can be deprotected by treating it with a base.
  • the deprotection reaction is carried out in a solvent that does not affect the reaction.
  • Bases include secondary amines such as dimethylamine and diethylamine, and non-nucleophilic organic bases such as 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 1,5-diazabicyclo[4.3.0]-5-nonene (DBN).
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • DABCO 1,4-diazabicyclo[2.2.2]octane
  • DBN 1,5-diazabicyclo[4.3.0]-5-nonene
  • General organic solvents can be used as solvents for the reaction.
  • Specific examples include halogenated hydrocarbons such as chloroform and dichloromethane; and nonpolar organic solvents such as 4-methyltetrahydropyran, 2-methyltetrahydrofuran, 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether. Two or more of these solvents may be mixed together.
  • halogenated hydrocarbons and nonpolar organic solvents may be mixed with aromatic hydrocarbons such as benzene, toluene, and xylene; nitriles such as acetonitrile and propionitrile; ketones such as acetone and 2-butanone; amides such as N,N-dimethylformamide and N-methylpyrrolidone; and sulfoxides such as dimethyl sulfoxide.
  • aromatic hydrocarbons such as benzene, toluene, and xylene
  • nitriles such as acetonitrile and propionitrile
  • ketones such as acetone and 2-butanone
  • amides such as N,N-dimethylformamide and N-methylpyrrolidone
  • sulfoxides such as dimethyl sulfoxide.
  • reaction temperature is preferably between -10°C and 80°C, more preferably between 0°C and 60°C, and even more preferably between 0°C and 40°C.
  • reaction time is preferably between 10 minutes and 30 hours.
  • L 1 includes one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 1 , —O—, and —S—, and the above groups may have a substituent, and includes at least one of >NR 1 , —O—, and —S— in the main chain;
  • R 1 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR 2 ;
  • R2 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group.
  • Formula (2) Fm-S-L 1 -COOH (In formula (2), Fm represents a 9-fluorenylmethyl group, and L1 has the same definition as in formula (1)).
  • Steps B and C may be carried out in one pot. That is, step C may be carried out as a one-pot reaction using the reaction solution obtained in step B without isolation or purification.
  • the alkyl group, alkenyl group, alkynyl group, aryl group or heteroaryl group in L1 may have a substituent, and examples of the substituent include an alkyl group, an aryl group or a heteroaryl group, and a combination of one or more groups selected from -CO-, -NH-, -O-, -COO-, -OCO-, -SO 2 -, -COOH, -NO 2 , ⁇ NH, ⁇ O, -NH 2 , -N(CH 3 ) 2 and -N + (CH 3 ) 3 , and the alkyl group may be substituted with -COOH or -NHCOCH 3 .
  • L 1 preferably contains at least one of >NR 1 or —O—.
  • R 1 preferably represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, or —OR 2 , and more preferably represents a hydrogen atom.
  • R2 preferably represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group, and more preferably represents a hydrogen atom.
  • L1 is more preferably composed of a combination of two alkyl groups and -CONH-.
  • L 1 is preferably composed of a combination of an alkyl group, an aryl group, a heteroaryl group, and >C ⁇ O.
  • the base used in step B may be used as is.
  • the base include secondary amines such as dimethylamine and diethylamine, and non-nucleophilic organic bases such as 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 1,5-diazabicyclo[4.3.0]-5-nonene (DBN).
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • DABCO 1,4-diazabicyclo[2.2.2]octane
  • DBN 1,5-diazabicyclo[4.3.0]-5-nonene
  • any common organic solvent can be used as the solvent for the reaction, and the solvent used in Step B may be used as is.
  • Specific examples include halogenated hydrocarbons such as chloroform and dichloromethane; and nonpolar organic solvents such as 4-methyltetrahydropyran, 2-methyltetrahydrofuran, 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether. Two or more of these solvents may be mixed together.
  • halogenated hydrocarbons or nonpolar organic solvents may be mixed with aromatic hydrocarbons such as benzene, toluene, and xylene; nitriles such as acetonitrile and propionitrile; ketones such as acetone and 2-butanone; amides such as N,N-dimethylformamide and N-methylpyrrolidone; or sulfoxides such as dimethyl sulfoxide.
  • aromatic hydrocarbons such as benzene, toluene, and xylene
  • nitriles such as acetonitrile and propionitrile
  • ketones such as acetone and 2-butanone
  • amides such as N,N-dimethylformamide and N-methylpyrrolidone
  • sulfoxides such as dimethyl sulfoxide.
  • reaction temperature is preferably between -10°C and 80°C, more preferably between 0°C and 60°C, and even more preferably between 0°C and 40°C.
  • reaction time is preferably between 10 minutes and 30 hours.
  • step D the amino group-containing compound from which the Fmoc group has been removed is separated from the compound represented by formula (2).
  • the amino group-containing compound from which the Fmoc group has been removed can be separated from the compound represented by formula (2) by crystallization or liquid separation.
  • the amino group-containing compound from which the Fmoc group has been removed can be separated from the compound represented by formula (2) by liquid separation using a basic aqueous solution.
  • the basic aqueous solution may be, for example, a mixture of an aqueous Na2CO3 solution and an aqueous NaCl solution.
  • the mixture of an aqueous Na2CO3 solution and an aqueous NaCl solution may be added to the reaction solution, and the organic layer may be recovered, thereby separating the amino group-containing compound from which the Fmoc group has been removed from the compound represented by formula (2).
  • the separation may be performed once, or may be performed two or more times.
  • Step A condensing an amino acid or peptide whose N-terminus is protected with an Fmoc group with an amino acid or peptide whose C-terminus is protected;
  • Step B deprotecting the peptide obtained in Step A to produce a peptide from which the Fmoc group has been removed; Dibenzofulvene produced in step B above, Formula (1): HS-L 1 -COOH (In formula (1), L 1 includes one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 1 , —O—, and —S—, and the above groups may have a substituent, and includes at least one of >NR 1 , —O—, and —S— in the main chain; R 1 represents a hydrogen atom,
  • Formula (2) Fm-S-L 1 -COOH (In formula (2), Fm represents a 9-fluorenylmethyl group, and L1 has the same definition as in formula (1)). and a step D of separating the peptide from which the Fmoc group has been removed from the compound represented by formula (2).
  • the present invention relates to a method for producing a peptide, comprising:
  • step A of the peptide production method an amino acid or peptide whose N-terminus is protected with an Fmoc group is condensed with an amino acid or peptide whose C-terminus is protected.
  • the protecting group in the C-terminally protected amino acid or peptide is linked to the carbonyl group of the amino acid or peptide via an ester or amide bond.
  • the C-terminal protecting group in an amino acid or peptide with a protected C-terminus preferably has an aliphatic hydrocarbon group with 12 or more carbon atoms, preferably 15 or more carbon atoms, and more preferably 20 to 30 carbon atoms.
  • the total number of carbon atoms is preferably 30 to 80, and more preferably 36 to 80.
  • the C-terminal protecting group preferably has a ring structure, and preferably has a fused polycyclic ring, aromatic heterocyclic ring, or naphthalene ring.
  • the C-terminal protecting group is preferably an aromatic heterocyclic compound represented by formula (1) in WO 2020/175473. WO 2020/175473 is incorporated herein by reference.
  • the C-terminal protecting group is also preferably a fused polycyclic aromatic hydrocarbon compound represented by formula (1) in WO 2020/175472. WO 2020/175472 is incorporated herein by reference.
  • the C-terminal protecting group may be a compound disclosed in WO 2020/262259 (Japanese Patent Application No. 2019-122492 and a patent application based thereon), and WO 2020/262259 (Japanese Patent Application No. 2019-122492 and a patent application based thereon) is incorporated herein by reference.
  • the protecting group of the C-terminally protected amino acid or peptide is a group represented by formula (3).
  • Y represents -OR C , -NR C R, or -SR C ;
  • R represents a hydrogen atom, an alkyl group, an arylalkyl group, a heteroarylalkyl group, or a 9-fluorenylmethyloxycarbonyl group;
  • m represents 1 or 2; and
  • n represents an integer of 1 to 5.
  • R and B each independently represent an aliphatic hydrocarbon group; RC represents the binding site to the C-terminal amino acid or peptide; Each R is independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group, at least one of the aliphatic hydrocarbon groups having 12 or more carbon atoms, and the benzene ring in formula (3) may further have a substituent in addition to R.
  • the substituted benzyl compound represented by formula (3) may be a compound in which R and B are each independently an aromatic group (excluding a phenyl group). Everything except R and B is the same as above, and the aromatic group may have a substituent.
  • the aromatic group R B is preferably an aryl group or a heteroaryl group, more preferably an aryl group having 10 or more carbon atoms, and particularly preferably a naphthalene group.
  • the heteroaryl group is preferably a monocyclic or bicyclic ring. More preferably, it is a heteroaryl group of a pyridine ring, a pyrazine ring, a triazine ring, a benzothiophene ring, a furan ring, a benzofuran ring, a pyrrole ring, an indole ring, a carbazole ring, a pyrazole ring, an indazole ring, or a thiophene ring.
  • R is preferably a hydrogen atom, an amino protecting group, an alkyl group having 1 to 6 carbon atoms, or an arylalkyl group having 7 to 16 carbon atoms, more preferably a hydrogen atom, a methyl group, an ethyl group, or a benzyl group, and even more preferably a hydrogen atom.
  • m which is the number of substitutions of R 1 B , is 1 or 2, and is more preferably 1, from the viewpoints of deprotection rate, solvent solubility, and yield.
  • R 1 B is preferably an alkyl group having 1 to 6 carbon atoms or an arylalkyl group having 7 to 16 carbon atoms, more preferably a methyl group, an ethyl group or a benzyl group, still more preferably a methyl group or an ethyl group, and particularly preferably a methyl group.
  • n which is the number of substitutions of R A on the benzene ring, is an integer of 1 to 5, more preferably an integer of 1 to 4, further preferably 2 or 3, and particularly preferably 3, from the viewpoints of deprotection rate, solvent solubility, and yield.
  • Each R is independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group.
  • the "organic group having an aliphatic hydrocarbon group" in R refers to a monovalent organic group (having one bond to a benzene ring) having an aliphatic hydrocarbon group in its molecular structure.
  • the "aliphatic hydrocarbon group” and the “aliphatic hydrocarbon group” in the "organic group having an aliphatic hydrocarbon group” refer to a linear, branched, or cyclic saturated or unsaturated aliphatic hydrocarbon group, preferably an aliphatic hydrocarbon group having 5 or more carbon atoms, more preferably an aliphatic hydrocarbon group having 5 to 60 carbon atoms, even more preferably an aliphatic hydrocarbon group having 5 to 30 carbon atoms, and particularly preferably an aliphatic hydrocarbon group having 10 to 30 carbon atoms.
  • the lower limit for the number of carbon atoms in the aliphatic hydrocarbon group is preferably 12, more preferably 15, and even more preferably 18.
  • the upper limit is preferably 30, more preferably 26, and even more preferably 24.
  • the portion of the "aliphatic hydrocarbon group" in the "organic group having an aliphatic hydrocarbon group” is not particularly limited, and may be present at the terminal (monovalent group) or at another portion (for example, a divalent group).
  • the "aliphatic hydrocarbon group” includes an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, and the like.
  • alkyl group preferably has 5 to 30 carbon atoms, more preferably 12 to 30, even more preferably 16 to 26, and still more preferably 18 to 24.
  • Specific examples include pentyl, hexyl, octyl, 2-ethylhexyl, decyl, hexadecyl, octadecyl, icosyl, docosyl, tetracosyl, lauryl, tridecyl, myristyl, and isostearyl groups, with octadecyl, icosyl, docosyl, and tetracosyl groups being preferred, and icosyl, docosyl, and tetracosyl groups being more preferred.
  • cycloalkyl group is preferably, for example, a cycloalkyl group having 5 to 30 carbon atoms, such as a cyclopentyl group, a cyclohexyl group, an isobornyl group, a tricyclodecanyl group, etc. These may be repeatedly linked together, or may have a condensed ring structure of two or more rings.
  • the "alkenyl group” preferably has 5 to 30 carbon atoms, more preferably 12 to 30, even more preferably 16 to 26, and still more preferably 18 to 24. Specific examples include a pentenyl group, a hexenyl group, and an oleyl group.
  • the "alkynyl group” preferably has 5 to 30 carbon atoms, more preferably 12 to 30, even more preferably 16 to 26, and still more preferably 18 to 24. Specific examples include a 4-pentynyl group and a 5-hexenyl group.
  • steroid group for example, a group having a cholesterol structure or a group having an estradiol structure is preferred.
  • the above organic group may be further substituted with one or more substituents selected from a silyl group, a hydrocarbon group having a silyloxy structure, and an organic group having a perfluoroalkyl structure.
  • the silyl group is preferably a trialkylsilyl group, and more preferably a silyl group having three alkyl groups each having 1 to 3 carbon atoms.
  • the silyloxy structure in the hydrocarbon group having a silyloxy structure is preferably a trialkylsilyloxy structure, and more preferably a silyloxy structure having three alkyl groups having 1 to 3 carbon atoms.
  • the hydrocarbon group having a silyloxy structure preferably has 1 to 3 silyloxy structures.
  • the hydrocarbon group having the silyloxy structure preferably has 10 or more carbon atoms, more preferably 10 to 100 carbon atoms, and particularly preferably 16 to 50 carbon atoms.
  • hydrocarbon group having a silyloxy structure examples include groups represented by the following formula (Si):
  • R si1 represents a single bond or an alkylene group having 1 to 3 carbon atoms
  • R si2 represents an alkylene group having 1 to 3 carbon atoms
  • R si3 and R si4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or —OSiR si5 R si6 R si7
  • R si5 to R si7 each independently represent an alkyl group having 1 to 6 carbon atoms or an aryl group.
  • R si5 to R si7 are each independently preferably an alkyl group having 1 to 6 carbon atoms or a phenyl group, more preferably an alkyl group having 1 to 6 carbon atoms, and particularly preferably a linear or branched alkyl group having 1 to 4 carbon atoms.
  • the perfluoroalkyl structure in the organic group having a perfluoroalkyl structure is preferably a perfluoroalkyl structure having 1 to 20 carbon atoms, more preferably a perfluoroalkyl structure having 5 to 20 carbon atoms, and particularly preferably a perfluoroalkyl structure having 7 to 16 carbon atoms.
  • the perfluoroalkyl structure may be linear, branched, or have a cyclic structure.
  • the organic group having a perfluoroalkyl structure is preferably a perfluoroalkyl group, an alkyl group having a perfluoroalkyl structure, or an alkyl group having a perfluoroalkyl structure and an amide bond in the alkyl chain.
  • the organic group having a perfluoroalkyl structure preferably has 5 or more carbon atoms, more preferably 10 or more carbon atoms, further preferably 10 to 100 carbon atoms, and particularly preferably 16 to 50 carbon atoms.
  • Preferred examples of the organic group having a perfluoroalkyl structure include the groups shown below.
  • the moiety other than the "aliphatic hydrocarbon group" in the "organic group having an aliphatic hydrocarbon group” can be set arbitrarily. For example, it may have a moiety such as -O-, -S-, -COO-, -OCONH-, -CONH-, or a hydrocarbon group (monovalent group or divalent group) other than the "aliphatic hydrocarbon group.”
  • hydrocarbon groups other than “aliphatic hydrocarbon groups” include aromatic hydrocarbon groups, and specifically, for example, monovalent groups such as aryl groups and divalent groups derived therefrom are used.
  • the aliphatic hydrocarbon groups and hydrocarbon groups other than the aliphatic hydrocarbon groups may be substituted with a substituent selected from a halogen atom, an oxo group, and the like.
  • the bond (substitution) of the "organic group having an aliphatic hydrocarbon group" to the benzene ring may be via the "aliphatic hydrocarbon group” or the "hydrocarbon group” present in the above R A , i.e., may be via a direct carbon-carbon bond, or may be via a moiety such as -O-, -S-, -COO-, -OCONH-, -CONH-, etc. present in the above R A. From the viewpoint of ease of synthesis of the compound, it is preferable that it is via -O-, -S-, -COO- or -CONH-, and particularly preferable that it is via -O-.
  • the total number of carbon atoms in all aliphatic hydrocarbon groups possessed by all R A is preferably 24 or more, more preferably 24 to 200, even more preferably 32 to 100, particularly preferably 34 to 80, and most preferably 36 to 80, from the viewpoints of solvent solubility, crystallization property, and yield.
  • the protecting group represented by formula (3) has at least one aliphatic hydrocarbon group having 12 or more carbon atoms in at least one R A, and from the viewpoints of solvent solubility, crystallization property, and yield, it preferably has at least one aliphatic hydrocarbon group having 12 to 100 carbon atoms in at least one R A , more preferably has at least one aliphatic hydrocarbon group having 18 to 40 carbon atoms, and even more preferably has at least one aliphatic hydrocarbon group having 20 to 36 carbon atoms.
  • the aliphatic hydrocarbon group is preferably an alkyl group, and more preferably a linear alkyl group. Furthermore, the number of carbon atoms in each R A is preferably 12 to 200, more preferably 18 to 150, even more preferably 18 to 100, and particularly preferably 20 to 80, from the viewpoints of solvent solubility, crystallization property, and yield.
  • the substituent that the protecting group represented by formula (3) may have on the benzene ring is not particularly limited, and examples thereof include an alkoxy group, an aryloxy group, a halogen atom, an alkyl group, a halogenated alkyl group, an aryl group, an acyl group, an acyloxy group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylthio group, an arylthio group, R st -CO-NR st -, -CON(R st ) 2 , a dialkylamino group, an alkylarylamino group, a diarylamino group, and a group formed by combining two or more of these, where R st represents a hydrogen atom, an alkyl group, or an aryl group.
  • the molecular weight of the protecting group represented by formula (3) is preferably 340 to 3,000, more preferably 400 to 2,000, even more preferably 500 to 1,500, and particularly preferably 800 to 1,300. Furthermore, if the molecular weight is 3,000 or less, the proportion of formula (1) in the target product is appropriate, and the proportion of the compound obtained by deprotecting formula (1) is not reduced, resulting in excellent productivity.
  • Condensation of an amino acid or peptide whose N-terminus is protected with an Fmoc group with an amino acid or peptide whose C-terminus is protected can be carried out using a condensing agent.
  • Condensing agents that are commonly used in peptide synthesis can be used without limitation in the present disclosure, and include, but are not limited to, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorphonium chloride (DMT-MM), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU(6-Cl)), O-(benzotriazole ...
  • DMT-MM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorphonium chloride
  • HBTU O-
  • O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), its hydrochloride salt (EDC.HCl), and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop).
  • DCC dicyclohe
  • the amount of the condensing agent used is preferably 1 to 10 molar equivalents, more preferably 1 to 5 molar equivalents, relative to 1 molar equivalent of the substrate.
  • a condensation activator is a reagent that, in the presence of a condensation agent, converts an amino acid into the corresponding activated ester, acid anhydride, or the like, thereby facilitating the formation of a peptide bond (amide bond).
  • the condensation activating agent can be any activating agent commonly used in peptide synthesis, without limitation.
  • Examples include 4-dimethylaminopyridine, N-methylimidazole, boronic acid derivatives, 1-hydroxybenzotriazole (HOBt), ethyl 1-hydroxytriazole-4-carboxylate (HOCt), 1-hydroxy-7-azabenzotriazole (HOAt), 3-hydroxy-1,2,3-benzotriazodin-4(3H)-one (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), pentafluorophenol, and ethyl(hydroxyimino)cyanoacetate (Oxyma).
  • the amount of the condensation activator used is preferably more than 0 molar equivalents and 4.0 molar equivalents, and more preferably 0.1 to 1.5 molar equivalents, relative to 1 molar equivalent of the substrate.
  • General organic solvents can be used as solvents for the reaction.
  • Specific examples include halogenated hydrocarbons such as chloroform and dichloromethane; and nonpolar organic solvents such as 4-methyltetrahydropyran, 2-methyltetrahydrofuran, 1,4-dioxane, tetrahydrofuran (THF), and cyclopentyl methyl ether. Two or more of these solvents may be mixed together.
  • halogenated hydrocarbons and nonpolar organic solvents may be mixed with aromatic hydrocarbons such as benzene, toluene, and xylene; nitriles such as acetonitrile and propionitrile; ketones such as acetone and 2-butanone; amides such as N,N-dimethylformamide and N-methylpyrrolidone; and sulfoxides such as dimethyl sulfoxide.
  • aromatic hydrocarbons such as benzene, toluene, and xylene
  • nitriles such as acetonitrile and propionitrile
  • ketones such as acetone and 2-butanone
  • amides such as N,N-dimethylformamide and N-methylpyrrolidone
  • sulfoxides such as dimethyl sulfoxide.
  • reaction temperature is preferably between -10°C and 80°C, more preferably between 0°C and 60°C, and even more preferably between 0°C and 40°C.
  • reaction time is preferably between 10 minutes and 30 hours.
  • Steps B, C, and D in the peptide production method can be carried out in the same manner as steps B, C, and D in the Fmoc group removal method.
  • the method for producing a peptide according to the present disclosure may include, between step A and step B, a step of trapping the amino acid activated ester produced in step A.
  • An example of the step of trapping the amino acid activated ester produced in step A is a step of mixing the amino acid activated ester produced in step A with an amine represented by formula (4) to trap the amino acid activated ester.
  • L 1 contains one or more groups selected from a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 3 , ⁇ N—, —CO—NR a —, —CS—NR a —, —O—, and —S—, and these groups may have a substituent;
  • R 3 represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group;
  • R a represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR 4 ;
  • R1 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or —OR4 , and R4 represents a hydrogen
  • the alkyl group, alkenyl group or heteroaryl group represented by L 1 may have a substituent, and examples of the substituent include -NH 2 , a halogen atom, -O-(CH 2 ) 2 -O-CH 3 , -NHCO-CH 3 , and -OCH 3 .
  • L 1 contains one or more groups selected from a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, >C ⁇ O, >C ⁇ S, >NR 3 , ⁇ N—, —CO—NR a —, —CS—NR a —, —O—, and —S—, and these groups may have a substituent;
  • R 3 represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group; and preferably, R a represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, or —OR 4 .
  • R 1 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, or —OR 4
  • R 4 represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group.
  • R2 represents a phosphate group, a sulfonate group, or an ammonium group.
  • R 1 is a hydrogen atom.
  • L 1 contains one or more groups selected from a hydrogen atom, an alkyl group, an alkenyl group, a heteroaryl group, ⁇ N—, —CO—NR a —, and —O—, and the above groups may have a substituent;
  • L1 contains a heteroatom-containing aliphatic cyclic structure in the main chain
  • l and m each represent an integer of 0 or more (with the proviso that l+m ⁇ 1)
  • L1 does not contain a heteroatom-containing aliphatic cyclic structure in the main chain
  • l represents an integer of 4 or more.
  • the amine represented by formula (4) is a compound represented by the following formula (2):
  • R 5 to R 12 each independently represent one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, >C ⁇ O, >C ⁇ S, >NR 13 , —O—, —S—, —OH, —OR 14 , a phosphate group, a sulfonate group, a carboxylate group, and an ammonium group, and one —NH 2 group;
  • R 13 represents an amide group, a sulfonamide group, a phosphoamide group, or an oxide group;
  • R 5 to R 12 each contain at least one of —OH, —OR 14 , a phosphate group, a sulfonic acid group, a carboxylate group, or an ammonium group, and
  • R 14 represents a hydrogen atom, an alkyl group, an alkenyl group,
  • Amino group-containing compounds other than the amine represented by formula (4) above can also be used as compounds for quenching amino acid activated esters (also known as amino acid activated ester scavengers).
  • amino acid activated ester scavengers particularly preferred are amino group-containing compounds selected from divalent or higher water-soluble amines, alkylamines, aromatic amines, hydroxylamines, aminosulfonic acids, aminosulfuric acids, aminophosphonic acids, aminophosphoric acids, and aminoalcohols.
  • Examples of divalent or higher water-soluble amines include 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N,N-dimethylethylenediamine, ethylenediamine, and piperazine.
  • Preferred are 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, N,N-dimethylethylenediamine, and ethylenediamine. More preferred are 1-methylpiperazine, 4-aminopiperidine, N,N-dimethylethylenediamine, and diethylenetriamine, and even more preferred is 1-methylpiperazine.
  • alkylamines examples include alkylamines having 1 to 14 carbon atoms, preferably alkylamines having 2 to 10 carbon atoms, more preferably alkylamines having 2 to 8 carbon atoms, and even more preferably alkylamines having 3 to 4 carbon atoms.
  • aromatic amines examples include aromatic amines having 1 to 14 carbon atoms, preferably aromatic amines having 6 to 10 carbon atoms.
  • alkylamines, aromatic amines, and hydroxylamines include, but are not limited to, propylamine, butylamine, hexylamine, aniline, toluidine, 2,4,6-trimethylaniline, anisidine, phenetidine, and hydroxylamine, with propylamine being particularly preferred.
  • Aminosulfonic acids, aminosulfuric acids, aminophosphonic acids, aminophosphoric acids, and aminoalcohols are preferably those represented by the following general formula:
  • Aminosulfonic acids and aminosulfuric acids represented by formula (11); H 2 NR 1 -X 1 -SO 3 H (11)
  • R 1 represents a divalent organic group having 1 to 10 carbon atoms
  • X 1 represents a single bond or an oxygen atom.
  • R 2 represents a divalent organic group having 1 to 10 carbon atoms
  • X 2 represents a single bond or an oxygen atom.
  • n represents an integer of 0 to 20
  • R 3 and R 4 each independently represent a hydrogen atom, a methyl group, an ethyl group, or a hydroxymethyl group.
  • R 1 in formula (11) and R 2 in formula (12) are independently a divalent organic group having 1 to 10 carbon atoms, preferably a linear or branched alkylene group having 1 to 10 carbon atoms, or an arylene group having 6 to 10 carbon atoms. Specific examples include a methylene group, ethylene group, trimethylene group, propylene group, tetramethylene group, butylene group, pentamethylene group, phenylene group, and naphthylene group.
  • a linear or branched alkylene group having 1 to 6 carbon atoms and an arylene group having 6 to 8 carbon atoms are more preferred, a linear or branched alkylene group having 1 to 6 carbon atoms is even more preferred, a linear or branched alkylene group having 1 to 5 carbon atoms is even more preferred, a linear alkylene group having 1 to 3 carbon atoms is even more preferred, and a linear alkylene group having 1 or 2 carbon atoms is most preferred.
  • n represents an integer of 0 to 20. Among these, n is preferably 0 or an integer of 2 to 20, more preferably 0 or an integer of 2 to 6, and even more preferably 0 or an integer of 2 to 4.
  • R 3 and R 4 are preferably a hydrogen atom or a hydroxylmethyl group.
  • peptide chain extension> In the method for producing a peptide according to the present disclosure, it is preferable to perform the step of deprotecting the N-terminus of an N-terminally protected, C-terminally protected peptide and the step of condensing an N-terminally protected amino acid or an N-terminally protected peptide to the N-terminus of the obtained C-terminally protected peptide, in this order, twice or more times. That is, the peptide from which the Fmoc group has been separated in step D can be used as the peptide whose C-terminus has been protected in step A. As described above, by repeatedly performing steps A to D, the obtained peptide can be chain-elongated.
  • the method for producing a peptide according to the present disclosure may further include a C-terminal deprotection step of deprotecting the C-terminal protecting group.
  • the C-terminal deprotection step the C-terminal protecting group of the C-terminal protected peptide having the desired number of amino acid residues is removed to obtain the final target peptide.
  • a preferred method for removing the C-terminal protecting group is a deprotection method using an acidic compound. Examples include a method of adding an acid catalyst and a method of hydrogenating using a metal catalyst.
  • acid catalysts examples include trifluoroacetic acid (TFA), hydrochloric acid, trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and acetic acid.
  • TFA trifluoroacetic acid
  • TFE trifluoroethanol
  • HFIP hexafluoroisopropanol
  • acetic acid examples include trifluoroacetic acid (TFA), hydrochloric acid, trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and acetic acid.
  • TFA trifluoroacetic acid
  • TFE trifluoroethanol
  • HFIP hexafluoroisopropanol
  • acetic acid acetic acid.
  • concentration of the acid can be appropriately selected depending on the side chain protecting group of the elongating amino acid and the deprotection conditions.
  • the concentration of TFA is preferably 50% by volume or less, more preferably 30% by volume or less, more preferably 10% by volume or less, more preferably 5% by volume or less, and particularly preferably 1% by volume or less, based on the total volume of the solvent used.
  • the lower limit is preferably 0.01% by volume, more preferably 0.1% by volume, and more preferably 0.5% by volume.
  • the deprotection time is preferably 5 hours or less, more preferably 3 hours or less, and even more preferably 1 hour or less.
  • the final target peptide obtained by the peptide production method according to the present disclosure can be isolated and purified according to methods commonly used in peptide chemistry, for example, by subjecting the reaction mixture to extraction and washing, crystallization, chromatography, etc.
  • the type of peptide produced by the method for producing a peptide according to the present disclosure is not particularly limited, it is preferable that the number of amino acid residues of the peptide is, for example, not more than several tens.
  • the peptides obtained by the method for producing a peptide according to the present disclosure can be used in various fields, including, but not limited to, pharmaceuticals, foods, cosmetics, electronic materials, biosensors, and the like.
  • Example 1 Compound (1-7) H-Asp-Ala-Asn-Cys-Glu-OH was synthesized using compound (2-1) described in WO 2023/106356 according to the following scheme.
  • reaction solution was stirred at 30°C for 1 hour, and the insoluble matter was filtered off.
  • the resulting reaction solution was then added to methanol (260 mL).
  • the precipitated solid was collected by filtration and dried under reduced pressure to obtain compound (1-1) (7.00 g).
  • Tiopronin has an amide bond in its main chain and is a more hydrophilic compound than 3-mercaptopropionic acid.
  • the tiopronin adduct of DBF is also thought to be hydrophilic and more likely to migrate into the aqueous phase.

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  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
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  • Biochemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

La présente invention aborde le problème consistant à fournir : un procédé d'élimination d'un groupe Fmoc, au moyen duquel il devient possible d'éliminer de manière hautement efficace le dibenzofulvène généré pendant la synthèse d'un peptide ; un procédé de production d'un peptide ; et un procédé de production d'un composé thiol qui est protégé par un groupe 9-fluorénylméthyle. La présente invention concerne un procédé d'élimination d'un groupe Fmoc, le procédé comprenant : une étape B dans laquelle un composé contenant un groupe amino ayant un groupe Fmoc est déprotégé pour produire le composé contenant un groupe amino à partir duquel le groupe Fmoc a été éliminé ; une étape C dans laquelle du dibenzofulvène généré à l'étape B, un composé représenté par la formule HS-L1-COOH (L1 étant tel que défini dans la description), et une base sont mélanges ensemble pour produire un composé représenté par la formule Fm-S-L1-COOH (Fm représentant un groupe 9-fluorénylméthyle) ; et une étape D dans laquelle le composé contenant un groupe amino à partir duquel le groupe Fmoc a été éliminé est séparé.
PCT/JP2025/016301 2024-04-30 2025-04-30 Procédé d'élimination d'un groupe fmoc, procédé de production de peptide et procédé de production d'un composé Pending WO2025229966A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6136934B2 (ja) * 2011-12-15 2017-05-31 味の素株式会社 Ｆｍｏｃ基の除去方法
JP7063408B1 (ja) * 2021-07-02 2022-05-09 ペプチスター株式会社 液相ペプチド製造方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6136934B2 (ja) * 2011-12-15 2017-05-31 味の素株式会社 Ｆｍｏｃ基の除去方法
JP7063408B1 (ja) * 2021-07-02 2022-05-09 ペプチスター株式会社 液相ペプチド製造方法

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