WO2020189621A1 - ペプチド化合物の製造方法 - Google Patents
ペプチド化合物の製造方法 Download PDFInfo
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- WO2020189621A1 WO2020189621A1 PCT/JP2020/011420 JP2020011420W WO2020189621A1 WO 2020189621 A1 WO2020189621 A1 WO 2020189621A1 JP 2020011420 W JP2020011420 W JP 2020011420W WO 2020189621 A1 WO2020189621 A1 WO 2020189621A1
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- 0 CC(C)C1*CCC1 Chemical compound CC(C)C1*CCC1 0.000 description 13
- BEWBGJSWIQNHKP-ZDUSSCGKSA-N CC(C)(C)OC(N(C)[C@@H](Cc1ccccc1)C(OC)=O)=O Chemical compound CC(C)(C)OC(N(C)[C@@H](Cc1ccccc1)C(OC)=O)=O BEWBGJSWIQNHKP-ZDUSSCGKSA-N 0.000 description 1
- MCNUMEAACFTUFP-HOTGVXAUSA-N CC(C)(C)OC(N[C@@H](CSCc1ccccc1)C(N(CCC1)[C@@H]1C(O)=O)=O)=O Chemical compound CC(C)(C)OC(N[C@@H](CSCc1ccccc1)C(N(CCC1)[C@@H]1C(O)=O)=O)=O MCNUMEAACFTUFP-HOTGVXAUSA-N 0.000 description 1
- IFVORPLRHYROAA-LBPRGKRZSA-N CC(C)(C)OC(N[C@@H](CSCc1ccccc1)C(O)=O)=O Chemical compound CC(C)(C)OC(N[C@@H](CSCc1ccccc1)C(O)=O)=O IFVORPLRHYROAA-LBPRGKRZSA-N 0.000 description 1
- SDBKSBUGAVTHJY-UHFFFAOYSA-N CCC(CC)C(C(C)(C)C)OC(Cl)=O Chemical compound CCC(CC)C(C(C)(C)C)OC(Cl)=O SDBKSBUGAVTHJY-UHFFFAOYSA-N 0.000 description 1
- ZZQHYOTYLZRWOJ-VUZXFBFESA-N C[C@@H](C(N[C@@H](CC1C=CC(O[Si](C)(C)C)=CC1C)C(O[Si](C)(C)C)=O)=O)N(C)C([C@H](Cc1ccccc1)N(C)C(OC(C)(C)C)=O)=O Chemical compound C[C@@H](C(N[C@@H](CC1C=CC(O[Si](C)(C)C)=CC1C)C(O[Si](C)(C)C)=O)=O)N(C)C([C@H](Cc1ccccc1)N(C)C(OC(C)(C)C)=O)=O ZZQHYOTYLZRWOJ-VUZXFBFESA-N 0.000 description 1
- OBHWWKQRVSHXTC-TZYHBYERSA-N C[C@@H](C(N[C@@H](Cc(cc1)ccc1O)C(O)=O)=O)N(C)C([C@H](Cc1ccccc1)N(C)C(OC(C)(C)C)=O)=O Chemical compound C[C@@H](C(N[C@@H](Cc(cc1)ccc1O)C(O)=O)=O)N(C)C([C@H](Cc1ccccc1)N(C)C(OC(C)(C)C)=O)=O OBHWWKQRVSHXTC-TZYHBYERSA-N 0.000 description 1
- SZSQMENJUOSUKV-NEWSRXKRSA-N C[C@H](C1=CC=CC1)SC[C@@H](C(N(CCC1)[C@@H]1C(O[Si](C)(C)C)=O)=O)NC(OC(C)(C)C)=O Chemical compound C[C@H](C1=CC=CC1)SC[C@@H](C(N(CCC1)[C@@H]1C(O[Si](C)(C)C)=O)=O)NC(OC(C)(C)C)=O SZSQMENJUOSUKV-NEWSRXKRSA-N 0.000 description 1
- VWGXGSJXTCSLLI-INIZCTEOSA-N OC([C@H](Cc1ccccc1)NC(CNCc1ccccc1)=O)=O Chemical compound OC([C@H](Cc1ccccc1)NC(CNCc1ccccc1)=O)=O VWGXGSJXTCSLLI-INIZCTEOSA-N 0.000 description 1
Classifications
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- 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
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- 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
- C07K1/08—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 using activating agents
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- C—CHEMISTRY; METALLURGY
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/96—Esters of carbonic or haloformic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
- C07K5/06052—Val-amino acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
- C07K5/06147—Dipeptides with the first amino acid being heterocyclic and His-amino acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0815—Tripeptides with the first amino acid being basic
- C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1021—Tetrapeptides with the first amino acid being acidic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- 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 invention relates to a method for producing a peptide having an N-alkyl amino acid.
- a method for producing a peptide containing an N-alkyl amino acid for example, the following method is known.
- Non-Patent Document 2 -Activates the C-terminus of the N-terminal protective peptide with 1- [bis (dimethylamino) methylene] -1H-1,2,3, -triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU) A method of reacting with N-methylalanine (see, for example, Patent Document 2).
- Patent Document 2 uses HATU containing an explosive triazole structure as a condensing agent, and is not always applicable in the production of industrial peptides.
- Patent Document 1 and Non-Patent Document 3 the C-terminal of the peptide to be produced is protected, and a deprotection step is further required to obtain a peptide having an unprotected C-terminal. Therefore, since a deprotection step always occurs in addition to the condensation step, it has not been applicable as an efficient method for producing a peptide.
- the present invention provides a method for producing a peptide containing an N-alkyl amino acid and having an unprotected C-terminal.
- the present invention also provides a method for producing a peptide using an amino acid or peptide containing an N-alkyl group and whose N-terminal and C-terminal are not protected and an N-terminal protected amino acid or peptide as materials.
- the present inventors mixed an unprotected amino acid or peptide containing an N-alkyl group and whose N-terminal and C-terminal were not protected with a silylating agent, and further specified an N-terminal protected amino acid or peptide.
- a carboxylic acid activator having a structure, and have completed the present invention. That is, the present invention is characterized by the following.
- PA 1- OH in the formula, P is an N-terminal protective group, A 1 is an amino acid-derived group, NC 1-6 alkyl amino acid-derived group (C 1-6 alkyl is).
- An N-terminal protected amino acid or N-terminal protected peptide represented by (which may have a substituent) or represents a peptide-derived group) is represented by the formula (II).
- X represents a halogen atom
- R 1 represents a secondary or tertiary aliphatic hydrocarbon group having 5 or more carbon atoms and may have a substituent, or has a carbon number of carbons.
- a primary aliphatic hydrocarbon group having 4 or more and having a substituent (here, the substituent of the primary aliphatic hydrocarbon group exists on a carbon atom bonded to carbonyl carbon).
- X represents a halogen atom
- R 2 represents a secondary aliphatic hydrocarbon group having 5 or more carbon atoms and may have a substituent
- an alkyl halide represented by an alkyl halide represented by an alkyl halide.
- a 2 is a group derived from an NC 1-6 alkyl amino acid (C 1-6 alkyl may have a substituent), or 4- A group derived from a 6-membered cyclic secondary amino acid (the 4-6-membered ring is fused with a cyclic compound selected from the group consisting of a C 6-14 aryl ring, a C 6-14 haloaryl ring and a C 3-8 cycloalkyl ring.
- an N-C 1-6 alkyl amino acid (C 1-6 alkyl may have a substituent) or a 4-6-membered cyclic secondary amino acid (which may have a substituent).
- the 4-6-membered ring may be fused to a cyclic compound selected from the group consisting of C 6-14 aryl ring, C 6-14 haloaryl ring and C 3-8 cycloalkyl ring)).
- a step of mixing an amino acid or peptide represented by) with a silylating agent (3) The step of mixing the product obtained in the step (1) and the product obtained in the step (2), A method for producing a peptide containing.
- a primary aliphatic hydrocarbon group having 4 or more and having a substituent (here, the substituent of the primary aliphatic hydrocarbon group exists on a carbon atom bonded to carbonyl carbon).
- a carboxylate halide represented by formula (III) (In the formula, X represents a halogen atom, R 2 represents a secondary aliphatic hydrocarbon group having 5 or more carbon atoms and may have a substituent), and an alkyl halide represented by an alkyl halide.
- a 2 has a group derived from N-methylamino acid, a group derived from NC 1-6 alkylglycine (C 1-6 alkyl has a substituent).
- a group derived from a 4-6-membered cyclic secondary amino acid, or an N-terminal residue is an N-methyl amino acid, NC 1-6 alkylglycine (C 1-6 alkyl has a substituent).
- PA 1- OH or formula (V) PA 3- OH (in the formula, P is an N-terminal protective group, and A 1 and A 3 are peptide-derived groups, respectively.
- the amino acid located at the C-terminal in the N-terminal protected peptide represented by) is an NC 1-6 alkyl amino acid (C 1-6 alkyl may have a substituent) or 4-6 members.
- the 4-6-membered ring may be fused with a cyclic compound selected from the group consisting of a C 6-14 aryl ring, a C 6-14 haloaryl ring and a C 3-8 cycloalkyl ring.
- the amino acid located at the C-terminal in the N-terminal protected amino acid represented by the formula (I) or the N-terminal protected peptide represented by the formula (I) is an ⁇ -amino acid, ⁇ -amino acid or ⁇ -amino acid.
- the amino acid located at the C-terminal in the N-terminal protected amino acid represented by the formula (I) or the N-terminal protected peptide represented by the formula (I) is an ⁇ -amino acid, according to the above [8]. Method for producing peptide of.
- the amino acid represented by the formula (IV) or the amino acid located at the N-terminal in the peptide represented by the formula (IV) is NC 1-6 alkyl- ⁇ -amino acid (C 1-6 alkyl).
- the amino acid represented by the formula (IV) or the amino acid located at the N-terminal in the peptide represented by the formula (IV) is an N-methyl- ⁇ -amino acid or an N-ethyl- ⁇ -amino acid (N-).
- the activator is a carboxylic acid halide represented by the formula (II), R 1 has 5 to 20 carbon atoms, and X is a chlorine atom.
- the method for producing a peptide according to any one.
- the silylating agent is N, O-bis (trimethylsilyl) acetamide, N, N'-bis (trimethylsilyl) urea or N, O-bis (trimethylsilyl) trifluoroacetamide, as described above [1] to [19]. ], The method for producing a peptide according to any one of.
- a novel method for producing a peptide containing an N-alkyl group whose N-terminal and C-terminal are not protected and an N-terminal protected amino acid or peptide as materials and whose C-terminal is unprotected can be obtained. I was able to provide it. According to the production method of the present invention, regardless of the type of N-alkyl amino acid to be introduced and the N-terminal residue of the peptide to be introduced, the purpose is The peptide to be obtained can be obtained in a satisfactory yield.
- n- is normal, “s-” is secondary, “t-” and “tert-” are tertiary, “Me” is methyl, “Et” is ethyl, “Pr” is propyl, and “"Bu” is butyl, “Ph” is phenyl, “Bn” is benzyl, “Boc” is t-butoxycarbonyl, “Cbz” is benzyloxycarbonyl, “Fmoc” is 9-fluorenylmethoxycarbonyl, “Trt” is Trityl, “TMS” means trimethylsilyl, “TFA” means trifluoroacetic acid.
- Halogen atom means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
- alkyl group means a linear or branched chain monovalent group of saturated aliphatic hydrocarbons.
- C 1-6 alkyl group means a linear or branched alkyl group having 1 to 6 carbon atoms, and specific examples thereof include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group.
- n-butyl group isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 3-pentyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1, Examples thereof include 2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, n-hexyl group and 3,3-dimethylbutane-2-yl group.
- the "secondary or tertiary C 5-40 alkyl group” is a saturated aliphatic hydrocarbon having 5 to 40 carbon atoms and containing at least one secondary or tertiary carbon atom. It means a monovalent group from which hydrogen on a tertiary or tertiary carbon atom has been removed, and specific examples thereof include 2-methylbutane-2-yl group, 3-methylbutane-2-yl group, and 3,3-dimethyl.
- Butane-2-yl group, 3-pentyl group, 2,2,4-trimethylpentane-3-yl group, 2,4-dimethylpentane-3-yl group, 4-ethyl-2,2-dimethylhexane-3 -Il group, 3-heptyl group, 2,2,4,8,10,10-hexamethylundecane-5-yl group and the like can be mentioned.
- the "secondary or tertiary C 5-20 alkyl group” means a secondary or tertiary alkyl group having 5 to 20 carbon atoms.
- the "primary C 4-40 alkyl group” is a linear or branched saturated aliphatic hydrocarbon having 4 to 40 carbon atoms from which hydrogen on the primary carbon atom has been removed. Means a valent group, n-butyl group, isobutyl group, n-pentyl group, 2-methylbutyl group or 3-methylbutyl group, or n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group.
- the "primary C 4-20 alkyl group” means a primary alkyl group having 4 to 20 carbon atoms.
- alkenyl group means a monovalent group of unsaturated aliphatic hydrocarbons containing at least one carbon-carbon double bond in a straight chain or branched chain.
- a “secondary or tertiary alkenyl group” is a secondary from an unsaturated aliphatic hydrocarbon containing at least one secondary or tertiary carbon atom and at least one carbon-carbon double bond.
- it means a monovalent group from which hydrogen on a tertiary carbon atom has been removed, and specific examples thereof include an isopropenyl group and a 1-methyl-1-propenyl group.
- the "secondary or tertiary C 5-40 alkenyl group” has 5 to 40 carbon atoms, and the “secondary or tertiary C 5-20 alkenyl group” has 5 to 40 carbon atoms. It means 20 secondary or tertiary alkenyl groups.
- a “primary C 4-40 alkenyl group” is an unsaturated aliphatic hydrocarbon containing at least one carbon-carbon double bond in the form of a linear or branched chain having 4 to 40 carbon atoms. It means a monovalent group from which hydrogen on the primary carbon atom has been removed, and examples thereof include a 2-butenyl group, a 3-butenyl group, and a 2-pentenyl group.
- the "primary C 4-20 alkenyl group” means a primary alkenyl group having 4 to 20 carbon atoms.
- the "C 6-14 aryl group” means an aromatic hydrocarbon group having 6 to 14 carbon atoms, and specific examples thereof include a phenyl group, a 1-naphthyl group, a 2-naphthyl group, and a 1-. Examples thereof include an anthryl group, a 2-anthryl group, a 9-antryl group and a biphenyl group. Further, the "C 6-14 aryl ring” means an aromatic hydrocarbon ring having 6 to 14 carbon atoms.
- the "C 6-14 haloaryl group” means an aromatic hydrocarbon group having 6 to 14 carbon atoms substituted with one or more halogen atoms, and specific examples thereof include a 4-chlorophenyl group. 2,4-Dichlorophenyl group, 5-fluoro-1-naphthyl group, 6-bromo-2-naphthyl group, 6,7-diiodo-1-anthryl group, 10-bromo-9-anthryl group, 4'-chloro- Examples thereof include (1,1'-biphenyl) -2-yl group.
- the "C 6-14 haloaryl ring” means an aromatic hydrocarbon ring having 6 to 14 carbon atoms substituted with one or more halogen atoms.
- the "C 6-14 aryloxy group” means an aryloxy group having 6 to 14 carbon atoms, and specific examples thereof include a phenoxy group, a 1-naphthyloxy group, a 2-naphthyloxy group, and a 1-. Examples thereof include an anthryloxy group, a 2-anthrioxy group, a 9-anthrioxy group, and a biphenyloxy group.
- the "5-10-membered heterocyclic group” has 5 to 10 atoms constituting the ring and is independent of the group consisting of nitrogen atom, oxygen atom and sulfur atom in the atom constituting the ring. It means a heterocyclic group of a monocyclic system or a fused ring system containing 1 to 4 heteroatoms selected from the above.
- the heterocyclic group may be saturated, partially unsaturated or unsaturated, and specific examples thereof include a pyrrolidinyl group, a tetrahydrofuryl group, a tetrahydrothienyl group, a piperidyl group, a tetrahydropyranyl group and a tetrahydrothiopyranyl group.
- C 1-6 alkoxy group means a linear or branched alkoxy group having 1 to 6 carbon atoms, and specific examples thereof include a methoxy group, an ethoxy group, an n-propoxy group, and an iso. Examples thereof include a propoxy group, an n-butoxy group, an isobutoxy group, a t-butoxy group, an n-pentyloxy group and an n-hexyloxy group.
- C 3-6 cycloalkyl group means a monovalent group of a cyclic saturated aliphatic hydrocarbon having 3 to 6 carbon atoms, and specific examples thereof include a cyclopropyl group, a cyclobutyl group, and a cyclopentyl group. Groups, cyclohexyl groups and the like can be mentioned.
- the "C 3-8 cycloalkyl group” means a cycloalkyl group having 3 to 8 carbon atoms, and specific examples thereof include cycloheptyl in addition to the above-mentioned "C 3-6 cycloalkyl group”. Examples include a group and a cyclooctyl group.
- the "C 5-8 cycloalkyl group” is a cycloalkyl group having 5 to 8 carbon atoms
- the "C 5-6 cycloalkyl group” is a cycloalkyl group having 5 to 6 carbon atoms.
- the "C 3-8 cycloalkyl ring” means a cycloalkyl ring having 3 to 8 carbon atoms.
- C 3-6 cycloalkoxy group means a cycloalkyloxy group having 3 to 6 carbon atoms, and specific examples thereof include a cyclopropoxy group, a cyclobutoxy group, a cyclopentyloxy group, and a cyclohexyloxy group. Can be mentioned.
- the "mono-C 1-6 alkylamino group” means a group in which one "C 1-6 alkyl group” is bonded to an amino group, and specific examples thereof include a monomethylamino group and a monoethylamino group.
- Mono-n-propylamino group, monoisopropylamino group, mono-n-butylamino group, monoisobutylamino group, mono-t-butylamino group, mono-n-pentylamino group, mono-n-hexylamino group, etc. Can be mentioned.
- di C 1-6 alkylamino group means a group in which two identical or different "C 1-6 alkyl groups” are bonded to an amino group, and specific examples thereof include a dimethylamino group and a diethylamino group.
- Di-n-propylamino group diisopropylamino group, di-n-butylamino group, diisobutylamino group, di-t-butylamino group, di-n-pentylamino group, di-n-hexylamino group, N -Ethyl-N-methylamino group, N-methyl-Nn-propylamino group, N-isopropyl-N-methylamino group, Nn-butyl-N-methylamino group, N-isobutyl-N-methyl Amino group, Nt-butyl-N-methylamino group, N-methyl-Nn-pentylamino group, Nn-hexyl-N-methylamino group, N-ethyl-Nn-propylamino group , N-ethyl-N-isopropylamino group, Nn-butyl-N-ethylamino group,
- the “C 1-6 alkoxycarbonyl group” means a linear or branched alkoxycarbonyl group having 1 to 6 carbon atoms, and specific examples thereof include a methoxycarbonyl group, an ethoxycarbonyl group, and n ⁇ . Examples thereof include a propoxycarbonyl group, an isopropoxycarbonyl group, an n-butoxycarbonyl group, an isobutoxycarbonyl group, a t-butoxycarbonyl group, an n-pentyloxycarbonyl group, and an n-hexyloxycarbonyl group.
- tri-C 1-6 alkylsilyl group means a group in which the same or different three "C 1-6 alkyl groups" are bonded to a silyl group, and specific examples thereof include a trimethylsilyl (TMS) group. Examples thereof include a triethylsilyl group, a triisopropylsilyl group, a t-butyldimethylsilyl group and a di-t-butylisobutylsilyl group.
- tri-C 1-6 alkylsilyloxy group means a group in which the same or different three "C 1-6 alkyl groups" are bonded to a silyloxy group, and specific examples thereof include a trimethylsilyloxy group and a triethyl. Examples thereof include a silyloxy group, a triisopropylsilyloxy group, a t-butyldimethylsilyloxy group, and a di-t-butylisobutylsilyloxy group.
- the "bicycloalkyl group” means a monovalent group of a saturated aliphatic hydrocarbon containing two bridgehead carbons and having two rings, and as a specific example, an octahydroinden-3-yl group. , Octahydronaphthalene-4-yl group, bicyclo [2.2.1] heptane-1-yl group or bicyclo [2.2.1] heptane-2-yl group and the like.
- the "C 5-10 bicycloalkyl group” means a bicycloalkyl group having 5 to 10 carbon atoms
- the "C 7-10 bicycloalkyl group” means a bicycloalkyl group having 7 to 10 carbon atoms. To do.
- tricycloalkyl group means a monovalent group of a saturated aliphatic hydrocarbon containing at least 3 bridge carbons and having 3 rings, and a specific example thereof is tricyclo [3.3. 1.1 3,7 ] Decane-1-yl (adamantan-1-yl) group or tricyclo [3.3.1.1 3,7 ] decane-2-yl (adamantan-2-yl) group, etc. Be done.
- the "C 5-15 tricycloalkyl group” has 5 to 15 carbon atoms, and the "C 7-15 tricycloalkyl group” has 7 to 15 carbon atoms. Means a group.
- a "secondary or tertiary aliphatic hydrocarbon group” is a branched or cyclic, saturated or unsaturated aliphatic hydrocarbon containing at least one secondary or tertiary carbon atom in a hydrocarbon chain.
- Examples thereof include an alkenyl group, and specific examples thereof include a secondary or tertiary alkyl group having 5 or more carbon atoms, a bicycloalkyl group, a tricycloalkyl group, a secondary or tertiary alkenyl group, and the like, which is preferable.
- Examples include a secondary or tertiary C 5-40 alkyl group, a C 5-10 bicycloalkyl group, a C 5-15 tricycloalkyl group, a secondary or tertiary C 5-40 alkenyl group, and the like, more preferably.
- Examples include a secondary or tertiary C 5-20 alkyl group, a C 7-10 bicycloalkyl group, a C 7-15 tricycloalkyl group, a secondary or tertiary C 5-20 alkenyl group, and the like.
- a "secondary aliphatic hydrocarbon group” is a branched or cyclic, saturated or unsaturated aliphatic hydrocarbon containing at least one secondary carbon atom in a hydrocarbon chain. It is a monovalent group from which hydrogen on the carbon atom of the carbon atom has been removed, and examples thereof include a secondary alkyl group, a cycloalkyl group, and a secondary alkenyl group. Specific examples thereof are secondary groups having 5 or more carbon atoms. Alkyl group, cycloalkyl group, secondary alkenyl group and the like, preferably secondary C 5-40 alkyl group, C 3-8 cycloalkyl group, secondary C 5-40 alkenyl group and the like. More preferably, a secondary C 5-20 alkyl group, a C 3-6 cycloalkyl group, a secondary C 5-20 alkenyl group and the like can be mentioned.
- a "primary aliphatic hydrocarbon group” is a monovalent group in which hydrogen on a primary carbon atom is removed from a linear or branched, saturated or unsaturated aliphatic hydrocarbon.
- Examples thereof include a primary alkyl group and a primary alkenyl group. Specific examples thereof include a primary alkyl group having 4 or more carbon atoms and a primary alkenyl group, and a primary alkenyl group is preferable.
- Examples thereof include a C 4-40 alkyl group, a primary C 4-40 alkenyl group, and more preferably a primary C 4-20 alkyl group and a primary C 4-20 alkenyl group.
- arbitrary substituent is not particularly limited as long as it is a substituent that does not adversely affect the reaction targeted by the present invention.
- the C 1-6 alkyl group may have a substituent
- substituents examples include a C 6-14 aryl group, a C 6-14 haloaryl group, and a C 6-14 aryloxy group.
- the "substituent" in the "secondary or tertiary aliphatic hydrocarbon group which may have a substituent” or the "primary aliphatic hydrocarbon group having a substituent” for example, C 6-14 aryl group, C 6-14 haloaryl group, C 6-14 aryloxy group, 5-10-membered heterocyclic group, hydroxy group, C 1-6 alkoxy group, C 3-6 cycloalkoxy group, acetoxy group , Benzoyloxy group, mono C 1-6 alkylamino group, N-acetylamino group, diC 1-6 alkylamino group, halogen atom, C 1-6 alkoxycarbonyl group, phenoxycarbonyl group, N-methylcarbamoyl group, Examples thereof include N-phenylcarbamoyl group, tri C 1-6 alkylsilyl group, tri C 1-6 alkylsilyloxy group, C 3-8 cycloalkyl group, cyano
- N-terminal protected amino acid and N-terminal protected peptide mean amino acids and peptides in which the N-terminal amino group is protected and the C-terminal carboxy group is not protected, respectively.
- C-terminal protected amino acid and C-terminal protected peptide mean amino acids and peptides in which the C-terminal carboxy group is protected and the N-terminal amino group is not protected, respectively.
- amino acids with unprotected N-terminal and C-terminal and peptides with unprotected N-terminal and C-terminal are amino acids with unprotected N-terminal amino group and C-terminal carboxy group, respectively. Means a peptide.
- the functional group may or may not be protected. .. It is preferably a side chain protected amino acid.
- the amino acid used in the present invention is an organic compound having both functional groups of an amino group and a carboxy group, and means natural and non-natural amino acids. It is preferably an ⁇ -, ⁇ - or ⁇ -amino acid, or a homoamino acid, and more preferably an ⁇ -amino acid. If these amino acids have two or more amino groups (eg, arginine, lysine, 2,3-diaminopropionic acid (Dap), etc.) and two or more carboxy groups (eg, glutamic acid, aspartic acid).
- amino groups eg, arginine, lysine, 2,3-diaminopropionic acid (Dap), etc.
- carboxy groups eg, glutamic acid, aspartic acid
- the amino acids used in the present invention are amino, carboxy and non-peptide formation.
- reactive functional groups eg, cysteine, serine, tyrosine, glutamine, histidine, tryptophan, etc.
- the amino acids used in the present invention are amino, carboxy and non-peptide formation.
- reactive functional groups also include protected and / or modified amino acids.
- the "4-6-membered cyclic secondary amino acid" used in the present invention is an amino acid in which a nitrogen atom of an amino group and two alkyl groups bonded to the amino group are combined to form a 4-6-membered ring.
- proline can be mentioned.
- the 4-6-membered ring is fused to a cyclic compound selected from the group consisting of C 6-14 aryl rings, C 6-14 haloaryl rings and C 3-8 cycloalkyl rings, the preferred cyclic compound is C 6-. It is a 14 aryl ring, more preferably benzene. Therefore, specific examples of the case where a 4-6-membered cyclic secondary amino acid is condensed with a cyclic compound include 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic).
- the NC 1-6 alkyl amino acid used in the present invention is an amino acid in which the amino group of the amino acid is substituted with a C 1-6 alkyl group which may have a substituent, and is preferably the amino of the amino acid.
- the groups are C 6-14 aryl group, C 6-14 haloaryl group, C 1-6 alkoxy group, di C 1-6 alkylamino group, tri C 1-6 alkylsilyl group, tri C 1-6 alkylsilyloxy.
- the amino group of the amino acid is an amino acid substituted with a methyl group, an ethyl group, a propyl group, a butyl group, a benzyl group or a cyclohexylmethyl group, and even more preferably, the amino group of the amino acid is a methyl group or an ethyl group. It is a substituted amino acid, and particularly preferably, the amino group of the amino acid is an amino acid substituted with a methyl group.
- amino acid derived group used in the present invention, "group derived from N-C 1-6 alkylamino acid", N-C 1-6 alkylamino amino or N-C 1-6 alkyl amino acids of an amino acid It means a divalent group in which a hydrogen atom is removed from the nitrogen atom of the group and a hydroxy group is removed from the carboxy group.
- group derived from N-methylamino acid is a divalent group in which the hydrogen atom is removed from the nitrogen atom of the N-methylamino group of the N-methylamino acid and the hydroxy group is removed from the carboxy group.
- the "group derived from NC 1-6 alkyl glycine” includes a hydrogen atom removed from the nitrogen atom of the NC 1-6 alkyl amino group of NC 1-6 alkyl glycine, and a hydroxy group from the carboxy group.
- the divalent group from which is removed has a hydrogen atom removed from the nitrogen atom of the secondary amino group of the cyclic secondary amino acid, and a hydroxy group removed from the carboxy group.
- the obtained divalent group, "group derived from a 4-6-membered cyclic secondary amino acid” has a hydrogen atom removed from the nitrogen atom of the secondary amino group of the 4-6-membered cyclic secondary amino acid. Moreover, it means a divalent group in which a hydroxy group is removed from a carboxy group.
- amino acids constituting the peptide used in the present invention are the above-mentioned amino acids.
- the "peptide-derived group" used in the present invention has a hydrogen atom removed from the primary or secondary amino group of various amino acids constituting the N-terminal, and a hydroxy group from the carboxy group of the amino acid constituting the C-terminal. Means the excluded divalent group.
- the three-dimensional structure of the amino acid is not particularly limited, but it is preferably L-form.
- N-terminal protecting group represented by P in the formulas (I) and (V) is a protecting group on the N-terminal side when carrying out a peptide extension reaction (amidation reaction).
- known protecting groups can be used.
- carbamate protecting groups (9-fluorenylmethoxycarbonyl group, t-butoxycarbonyl group, benzyloxycarbonyl group, allyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, 2-( p-biphenyl) isopropyloxycarbonyl group, etc.
- amide protecting group acetyl group, trifluoroacetyl group, etc.
- imide protecting group phthaloyl group, etc.
- sulfonamide protecting group p-toluenesulfonyl group, 2- Nitrobenzenesulfonyl group and the like
- benzyl group and the like preferably 9-fluorenylmethoxycarbonyl group, t-butoxycarbonyl group, benzyloxycarbonyl group and the like.
- each step (1) to (5) of the method for producing a peptide of the present invention will be described below.
- the production of the peptide of the present invention comprises the respective unit steps described as the following steps (1) to (5).
- the peptide of the present invention can be produced by all or a combination of the unit steps described as the following steps (1) to (5) as appropriate. This specific description will be described based on the following.
- R 1 and R 2 in the description of steps (1) to (5) have the same meanings as described above.
- the specific conditions of the reaction are not particularly limited as long as the production of the peptide of the present invention is achieved. Preferred conditions for each reaction will be described in detail as appropriate.
- the solvent described in each reaction may be used alone or in combination of two or more.
- This step is a step of mixing an N-terminal protected amino acid or an N-terminal protected peptide with a carboxylic acid halide or an alkyl halide formic acid.
- This step is a step of activating the C-terminal of an N-terminal protected amino acid or N-terminal protected peptide with a carboxylic acid halide or an alkyl halide formic acid.
- formula (I) PA 1- OH in the formula, P is an N-terminal protecting group, A 1 is an amino acid-derived group, NC 1-6 alkyl amino acid-derived group.
- N-terminal protected amino acid or N-terminal protected peptide is represented by a carboxylic acid halide or an alkyl halide. It is a process of mixing with.
- 3 -OH Formula (V) P-A (wherein, P is N-terminal protecting group, A 3 represents.
- a group of derived peptides) N represented by This is a step of mixing the terminal protecting peptide with a carboxylic acid halide.
- the N-terminal protected amino acid or N-terminal protected peptide is the above-mentioned amino acid or peptide that is N-terminal protected, and specifically, an N-terminal protected amino acid or an N-C 1-6 alkyl amino acid (N-terminal protected NC 1-6 alkyl amino acid).
- C 1-6 alkyl is a peptide which is also be) or N-terminal protected have a substituent.
- the C-terminal amino acid is an NC 1-6 alkyl amino acid (C 1-6 alkyl may have a substituent) or 4-6 members.
- the 4-6-membered ring may be fused with a cyclic compound selected from the group consisting of a C 6-14 aryl ring, a C 6-14 haloaryl ring and a C 3-8 cycloalkyl ring. ), which is an N-terminal protective peptide.
- the carboxylic acid halide is represented by the following formula (II).
- R 1 represents a secondary or tertiary aliphatic hydrocarbon group having 5 or more carbon atoms and may have a substituent, or has a carbon number of carbons.
- a primary aliphatic hydrocarbon group having 4 or more and having a substituent here, the substituent of the primary aliphatic hydrocarbon group exists on a carbon atom bonded to carbonyl carbon). Represent.
- the carboxylic acid halide represented by the formula (II) preferably has a secondary or tertiary C 5-40 alkyl group in which R 1 may have a substituent, even if it has a substituent.
- the substituent of the C 4-40 alkenyl group is a carboxylic acid halide which is (existing on the carbon atom bonded to the carbonyl carbon), more preferably a secondary in which R 1 may have a substituent. Alternatively , it has a tertiary C 5-20 alkyl group, a C 7-10 bicycloalkyl group which may have a substituent, a C 7-15 tricycloalkyl group which may have a substituent, and a substituent.
- R 1 may have a secondary or tertiary C 5-20 alkyl group which may have a substituent, a C 7-10 bicycloalkyl group which may have a substituent, and a substituent.
- a carboxylic group that is a C 7-15 tricycloalkyl group that may have a substituent, a secondary or tertiary C 5-20 alkenyl group that may have a substituent, or an isobutyl group that has a substituent. It is an acid chloride, and more preferably selected from the following compound group.
- the alkyl halide formic acid is represented by the following formula (III).
- X represents a halogen atom
- R 2 represents a secondary aliphatic hydrocarbon group having 5 or more carbon atoms and may have a substituent.
- the alkyl halide represented by the formula (III) is preferably a secondary C 5-40 alkyl group in which R 2 may have a substituent, and C 5 in which R 2 may have a substituent.
- R 2 has a secondary C 5-20 alkyl group which may have a substituent, a C 5-6 cycloalkyl group which may have a substituent, and a substituent. It is an alkyl chloronate which is a secondary C 5-20 alkenyl group which may be used, and is particularly preferably selected from the following compound group.
- the number of carbon atoms in R 1 or R 2 is the sum of the number of carbon atoms having R 1 or R 2, respectively, when the R 1 or R 2 has a substituent, the number of carbon atoms in the substituent Is also included.
- the base used in this step is not particularly limited, and examples thereof include aliphatic amines (eg, triethylamine, N, N-diisopropylethylamine, N-methylmorpholin), aromatic amines (eg, pyridine, imidazole, etc.). N, N-dimethyl-4-aminopyridine), amidine (eg, diazabicycloundecene), alkali metal salts (eg, sodium hydrogencarbonate, potassium carbonate) and the like. It is preferably an aliphatic amine, more preferably N, N-diisopropylethylamine, triethylamine or N-methylmorpholine.
- the amount of the carboxylate halide or alkyl halide used in this step is preferably 0.2 to 50 equivalents, more preferably 0.5 equivalents or more, relative to the N-terminal protected amino acid or N-terminal protected peptide. It is 20 equivalents, more preferably 0.8 equivalents to 5 equivalents.
- the amount of the base used in this step is preferably 0.2 to 50 equivalents, more preferably 0.5 to 20 equivalents, and even more preferably 0. Equivalent to the carboxylic acid halide or alkyl halide formic acid. It is 8 equivalents to 5 equivalents.
- the solvent used in this step is not particularly limited as long as it does not interfere with the activation reaction, and examples thereof include halogen-containing hydrocarbon solvents (for example, dichloromethane and chloroform) and aromatic hydrocarbon solvents (for example, toluene and xylene).
- Ether solvent eg, tetrahydrofuran, 1,4-dioxane, cyclopentylmethyl ether, methyl-t-butyl ether
- amide solvent eg, N, N-dimethylformamide, N, N-dimethylacetamide
- nitrile solvent eg, eg.
- Acetonitrile ketone solvents (eg acetone, methyl ethyl ketone), aliphatic hydrocarbon solvents (eg hexane, heptane, cyclohexane), ester solvents (eg ethyl acetate) and the like. It is preferably a nitrile solvent, an amide solvent, or an ether solvent, and more preferably acetonitrile, tetrahydrofuran, or N, N-dimethylacetamide.
- ketone solvents eg acetone, methyl ethyl ketone
- aliphatic hydrocarbon solvents eg hexane, heptane, cyclohexane
- ester solvents eg ethyl acetate
- It is preferably a nitrile solvent, an amide solvent, or an ether solvent, and more preferably acetonitrile, tetrahydrofuran, or N
- the amount of the solvent used in this step is preferably 100 times by mass or less, more preferably 1 to 50 times by mass, still more preferably 3 times by mass or less with respect to the carboxylic acid halide or alkyl halide formic acid. It is a mass times to 20 mass times.
- the N-terminal protected amino acid or N-terminal protected peptide is mixed with the carboxylic acid halide or alkyl halide formic acid, if necessary, in the presence of a solvent and / or base.
- the temperature of the obtained mixture is controlled by using an oil bath or a cooling bath, if necessary.
- the temperature of the mixture is not particularly limited, but is preferably from ⁇ 40 ° C. to the reflux temperature of the mixture, more preferably ⁇ 20 ° C. to 50 ° C., still more preferably ⁇ 10 ° C. to 30 ° C.
- This step forms an N-terminal protected amino acid or N-terminal protected peptide with the C-terminal activated.
- the product obtained by this step means an N-terminal protected amino acid or N-terminal protected peptide with C-terminal activation, or a mixture containing either of them.
- the C-terminally activated N-terminal protected amino acid or N-terminal protected peptide thus obtained is isolated as a reaction solution or as a (crude) purified product without undergoing a purification step, followed by a subsequent step. It may be used for (3).
- N-alkyl amino acid means an N-alkyl amino acid in which the N-terminal amino group and the C-terminal carboxy group are not protected, and "a peptide having an N-alkyl amino acid at the N-terminal”. Means a peptide having an N-alkyl amino acid at the N-terminus and unprotected N-terminal amino group and C-terminal carboxy group.
- the N-alkyl amino acid or a peptide having an N-alkyl amino acid at the N-terminal is reacted with a silylating agent to cause the C-terminal, N-terminal and / or hydroxy (if any) of the amino acid or peptide.
- a silylating agent to cause the C-terminal, N-terminal and / or hydroxy (if any) of the amino acid or peptide.
- a peptide in which at least a part of a functional group such as a group has a trialkylsilylated N-alkyl amino acid or an N-alkyl amino acid at the N-terminal (hereinafter, also referred to as "trialkylsilylated amino acid or peptide"). This is the process of obtaining.
- a 2 is a group derived from an NC 1-6 alkyl amino acid (C 1-6 alkyl has a substituent). (May be), or a group derived from a 4-6-membered cyclic secondary amino acid (the 4-6-membered ring consists of a C 6-14 aryl ring, a C 6-14 haloaryl ring and a C 3-8 cycloalkyl ring). (May be condensed with a cyclic compound selected from), or the N-terminal residue is an NC 1-6 alkyl amino acid (C 1-6 alkyl may have a substituent) or 4-6 members.
- the 4-6-membered ring may be fused with a cyclic compound selected from the group consisting of a C 6-14 aryl ring, a C 6-14 haloaryl ring and a C 3-8 cycloalkyl ring.
- a 2 ' is, N- methyl amino acids derived from groups, N-C 1-6 alkyl glycine group derived from (C 1-6 alkyl may have a substituent) or represents a group derived from a 4-6-membered cyclic secondary amino acid] N-terminal amino group and C-terminal carboxy group Is a step of mixing an unprotected amino acid with a silylating agent.
- the N-alkyl amino acid in this step or the N-terminal amino acid in the peptide having an N-alkyl amino acid at the N-terminal is preferably an NC 1-6 alkyl amino acid (C 1-6 alkyl is substituted with cyclohexyl or phenyl). It may be), more preferably N-methyl amino acid, N-ethyl amino acid, N-propyl amino acid, N-butyl amino acid, N-pentyl amino acid, N-cyclohexylmethyl amino acid or N-benzyl amino acid, and further. It is preferably an N-methyl amino acid or an N-ethyl amino acid, and particularly preferably an N-methyl amino acid.
- the silylating agent in this step is not particularly limited, and examples thereof include trimethylsilyl chloride, trimethylsilyl cyanide, 1,1,1,3,3,3-hexamethyldisilazane, N-trimethylsilylacetamide, and N. , N'-bis (trimethylsilyl) urea, N-methyl-N-trimethylsilyltrifluoroacetamide, N, O-bis (trimethylsilyl) acetamido, N, O-bis (trimethylsilyl) trifluoroacetamide and other trimethylsilylating agents, N- (Tert-Butyldimethylsilyl) -N-methyltrifluoroacetamide and the like can be mentioned.
- it is trimethylsilyl chloride, N, O-bis (trimethylsilyl) acetamide, N, N'-bis (trimethylsilyl) urea or N, O-bis (trimethylsilyl) trifluoroacetamide, and more preferably N, O-bis. (Trimethylsilyl) acetamide.
- the amount of the silylating agent used is preferably 0.01 to 50 equivalents, more preferably 0.1 to 20 equivalents, relative to the N-alkyl amino acid or the peptide having the N-alkyl amino acid at the N-terminus. More preferably, it is 0.2 to 5 equivalents.
- silylation reaction can be carried out in the presence of a base and / or a solvent, if necessary.
- the base used is not particularly limited, and examples thereof include aliphatic amines (eg, dicyclohexylamine, piperidine, triethylamine, N, N-diisopropylethylamine, N-methylmorpholin), aromatic amines (eg, pyridine, etc.). Imidazole, N, N-dimethyl-4-aminopyridine), alkali metal salts (for example, sodium hydrogen carbonate, potassium carbonate) and the like can be mentioned. It is preferably an aliphatic amine, and more preferably triethylamine or N, N-diisopropylethylamine.
- aliphatic amines eg, dicyclohexylamine, piperidine, triethylamine, N, N-diisopropylethylamine, N-methylmorpholin
- aromatic amines eg, pyridine, etc.
- the amount of the base used is preferably 0.01 to 50 equivalents, more preferably 0.1 to 20 equivalents, still more preferably 0.1 equivalents, relative to the N-alkyl amino acid or the peptide having the N-alkyl amino acid at the N-terminal. Is 0.2 equivalent to 5 equivalent.
- the solvent used in this step is not particularly limited as long as it does not interfere with the silylation reaction, and examples thereof include halogen-containing hydrocarbon solvents (for example, dichloromethane and chloroform) and aromatic hydrocarbon solvents (for example, toluene and xylene).
- Ether solvent eg, tetrahydrofuran, 1,4-dioxane, cyclopentylmethyl ether, methyl-t-butyl ether
- amide solvent eg, N, N-dimethylformamide
- nitrile solvent eg, acetonitrile
- It is preferably a nitrile solvent, an amide solvent, or an ether solvent, and more preferably acetonitrile, tetrahydrofuran, or N, N-dimethylacetamide.
- the amount of the solvent used in this step is preferably 100 times by mass or less, more preferably 1 to 50 times by mass, that of the N-alkyl amino acid or the peptide having an N-alkyl amino acid at the N-terminal. Yes, more preferably 3 to 20 times by mass.
- a peptide having an N-alkyl amino acid or an N-alkyl amino acid at the N-terminal is mixed with a silylating agent in the presence of a solvent and / or a base.
- the temperature of the obtained mixture is controlled by using an oil bath or a cooling bath, if necessary.
- the temperature of the mixture is not particularly limited, but is preferably from 0 ° C. to the reflux temperature of the mixture, more preferably 10 ° C. to 100 ° C., still more preferably 20 ° C. to 80 ° C.
- the mixture may also be exposed to microwave irradiation.
- a trialkylsilylated N-alkyl amino acid or a peptide having an N-alkyl amino acid at the N-terminal is formed.
- the product obtained by this step means a mixture containing a trialkylsilylated N-alkyl amino acid or a peptide having an N-alkyl amino acid at the N-terminus.
- the trialkylsilylated amino acid or peptide thus obtained is isolated as a reaction solution or as a (crude) purified product without undergoing a purification step, and is used in the subsequent step (3). May be good.
- the trialkylsilylated N-alkyl amino acid or the peptide having an N-alkyl amino acid at the N-terminal can be analyzed by an analytical instrument such as NMR.
- Process (3) This step is a step of mixing the product obtained in the step (1) and the product obtained in the step (2).
- the C-terminally activated N-terminal protected amino acid or N-terminal protected peptide obtained in step (1) is reacted with the trialkylsilylated amino acid or peptide obtained in step (2).
- This is a peptide extension step, and is preferably carried out by mixing and stirring the reaction solution obtained in the step (1) and the reaction solution obtained in the step (2).
- peptide extension by reacting the C-terminally activated N-terminal protected amino acid obtained in step (1) with the trialkylsilylated amino acid or peptide obtained in step (2). It is a process.
- peptide extension by reacting the C-terminally activated N-terminal protected peptide obtained in step (1) with the trialkylsilylated amino acid obtained in step (2). It is a process.
- the temperature of the obtained mixture is controlled by using an oil bath or a cooling bath as needed.
- the temperature of the mixture is not particularly limited, but is preferably from ⁇ 40 ° C. to the reflux temperature of the reaction mixture, more preferably ⁇ 20 ° C. to 50 ° C., still more preferably ⁇ 10 ° C. to 30 ° C.
- the peptide chain can be further extended by repeating the following steps (4) to (5) a desired number of times with respect to the peptide obtained in the step (3). .. (4) A step of removing the N-terminal protecting group of the peptide obtained in step (3) or (5). (5) A step of reacting the N-terminal of the peptide obtained in step (4) with an N-terminal protected amino acid or an N-terminal protected peptide. Step (5) can be carried out by the same operation as in steps (1), (2) and (3) above, or by a general peptide synthesis reaction.
- the purification steps of steps (1) to (5) can be appropriately omitted as long as the reaction of the next step is not affected.
- Step (4) N-terminal deprotection step
- the N-terminal protecting group is removed from the peptide obtained in the above step (3) or (5), and the N-terminal and C-terminal are unprotected peptides. Is the process of obtaining.
- the deprotection reagent used in this step is appropriately selected according to the protecting group used.
- examples include acids (eg, trifluoroacetic acid, hydrochloric acid, Lewis acid), secondary or tertiary amines (eg, pyrrolidine, piperidine, morpholine, triethylamine), hydrogenation (eg, palladium catalyst / hydrogenation) and the like.
- acids eg, trifluoroacetic acid, hydrochloric acid, Lewis acid
- secondary or tertiary amines eg, pyrrolidine, piperidine, morpholine, triethylamine
- hydrogenation eg, palladium catalyst / hydrogenation
- the deprotection conditions used in this step are appropriately selected depending on the type of N-terminal protecting group.
- a 9-fluorenylmethoxycarbonyl group it is carried out by treating with a base, and t-butoxycarbonyl.
- a group it is carried out by treatment with an acid, and in the case of a benzyloxycarbonyl group or an allyloxycarbonyl group, it is neutral, for example, by hydrogenation in the presence of a metal catalyst.
- reaction substrate has a hydroxy group, a mercapto group, an amino group, a carboxy group or a carbonyl group (particularly when the side chain of the amino acid or peptide has a functional group), these groups are generally used in peptide chemistry and the like.
- a protective group such as that used in the above may be introduced, and the target compound can be obtained by removing the protective group if necessary after the reaction.
- Protection and deprotection use commonly known protecting groups to protect and deprotect reactions (eg, Protective Group in Organic Synthesis, Fourth edition). , TWGreene, John Wiley & Sons Inc. (2006), etc.).
- amino acid A The N-terminal amino acid in the N-terminal protected amino acid or N-terminal protected peptide used in step (1)
- amino acid B The N-terminal amino acid in the amino acid or peptide used in step (2)
- amino acid A and B are ⁇ -, ⁇ - or ⁇ -amino acids, respectively, and more preferably either amino acid A or amino acid B is an ⁇ -amino acid.
- amino acid A is ⁇ -amino acid and amino acid B is ⁇ -amino acid, ⁇ -amino acid or ⁇ -amino acid, or amino acid A is ⁇ -amino acid, ⁇ -amino acid or ⁇ -amino acid.
- Amino acid B is ⁇ -amino acid, particularly preferably amino acid A is ⁇ -amino acid, and amino acid B is ⁇ -amino acid, ⁇ -amino acid or ⁇ -amino acid.
- the N-terminal amino acid in the peptide used in step (2) is preferably ⁇ -amino acid.
- Initiator + manufactured by Biotage was used as the microwave reactor.
- the proton nuclear magnetic resonance ( 1 H-NMR) of the synthesis example is JNM-ECP300 manufactured by JEOL, or JNM-ECX300 manufactured by JEOL, or Bruker. Measured in deuterated chloroform or deuterated dimethylsulfoxide solvent using Ascend TM 500 manufactured by the company, the chemical shift was shown by the ⁇ value (ppm) when tetramethylsilane was used as the internal standard (0.0 ppm).
- high performance liquid chromatography / mass spectrometry is either ACQUITY UPLC H-Class / QDa manufactured by Waters, ACQUITY UPLC H-Class / SQD2 manufactured by Waters, or LC-20AD / Triple Tof5600 manufactured by Shimadzu. Was measured using.
- ESI + means a positive mode of electrospray ionization
- M + H means a proton adduct
- M + Na means a sodium adduct.
- ESI- is a negative mode of electrospray ionization
- MH means a proton deficient.
- silica gel column chromatography used either a Hi-Flash column manufactured by Yamazen, SNAP Ultra Silka Cartridge manufactured by Biotage, silica gel 60 manufactured by Merck, or PSQ60B manufactured by Fuji Silysia Chemical Ltd.
- the yield or quantitative yield may exceed 100%. All of these exceed 100% due to measurement error, the influence of the purity of the raw material or product, or other factors based on common general knowledge. In the following examples, the causes when the yield exceeds 100% are not individually mentioned, but those skilled in the art can fully understand the scientific validity of these examples.
- 2,2,4-trimethylpentane-3-ol (5.25 g, 40.3 mmol) and pyridine (3.67 g, 46.3 mmol) were mixed with carbon tetrachloride (40 mL) and cooled to 0 ° C.
- the obtained reaction solution was washed twice with water (50 mL) and sequentially with saturated aqueous sodium chloride solution (50 mL).
- the obtained organic layer was concentrated to obtain 2,2,4-trimethylpentane-3-ylcarbonochloride (6.06 g, 78% yield) as a colorless transparent liquid. This compound was used in the next step without further purification.
- Synthesis Example 7 Synthesis of 3-methylbutano-2-ylcarbonochloridate 3-Methylbutano-2-ol (3.97 g, 45.0 mmol) and pyridine (4.09 g, 51.8 mmol) were mixed with carbon tetrachloride (40 mL) and cooled to 0 ° C. A solution in which triphosgene (5.47 g, 18.5 mmol) and carbon tetrachloride (20 mL) were separately mixed was added to this solution, and the mixture was further heated to 60 ° C. and stirred for 8 hours. The obtained reaction solution was washed twice with water (50 mL) and sequentially with saturated aqueous sodium chloride solution (50 mL). The obtained organic layer was concentrated to obtain 3-methylbutano-2-ylcarbonochloridate (5.10 g, yield 95%) as a colorless transparent liquid. This compound was used in the next step without further purification.
- Boc-Phe-OH (0.066 g, 0.25 mmol) and triethylamine (0.033 g, 0.32 mmol) were mixed with tetrahydrofuran (5 mL) and 2,2-dimethylbutanoyl chloride (0.040 g) at 0 ° C. , 0.30 mmol) and stirred for 1 hour.
- H-MePhe-OH (0.067 g, 0.38 mmol), N, O-bis (trimethylsilyl) acetamide (0.161 g, 0.750 mmol) and acetonitrile (4 mL) are separately mixed with this solution and irradiated with microwaves. The solution prepared by stirring at 75 ° C.
- Fmoc-Phe-OH (0.097 g, 0.25 mmol) and N-methylmorpholine (0.033 g, 0.33 mmol) were mixed with tetrahydrofuran (5.0 mL) and 4-ethyl-2,2 at 0 ° C. -Dimethylhexane-3-ylcarbonochloride (0.066 g, 0.30 mmol) was added, and the mixture was stirred for 1 hour.
- Fmoc-Phe-OH (0.097 g, 0.25 mmol) and N-methylmorpholine (0.033 g, 0.33 mmol) were mixed with N, N-dimethylacetamide (5.0 mL) and 4- at 0 ° C.
- Ethyl-2,2-dimethylhexane-3-ylcarbonochloride (0.066 g, 0.30 mmol) was added, and the mixture was stirred for 1 hour.
- reaction solution was diluted with ethyl acetate (30 mL) and washed successively with 10 mass% citric acid aqueous solution (50 mL), 10 mass% sodium chloride aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL).
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Fmoc-Phe-MePhe-OH (0.137 g, yield 100%) as a white solid.
- Fmoc-Phe-OH (0.194 g, 0.500 mmol) and triethylamine (0.0607 g, 0.60 mmol) were mixed with tetrahydrofuran (10 mL) and 2,2-dimethylbutanoyl chloride (0.074 g) at 0 ° C. , 0.550 mmol) and stirred for 1 hour.
- H-MePhe-OH (0.108 g, 0.600 mmol
- N, N'-bis (trimethylsilyl) urea (0.250 g, 1.20 mmol)
- acetonitrile (4.0 mL) were separately mixed with this solution.
- Fmoc-Phe-OH (0.097 g, 0.250 mmol) and triethylamine (0.0304 g, 0.300 mmol) were mixed with tetrahydrofuran (5 mL) and at 0 ° C. 2,2-dimethylbutanoyl chloride (0.0371 g). , 0.275 mmol) and stirred for 1 hour.
- H-MePhe-OH (0.0538 g, 0.300 mmol), N, O-bis (trimethylsilyl) trifluoroacetamide (0.155 g, 0.601 mmol) and acetonitrile (4.0 mL) are separately mixed with this solution. , The solution prepared by stirring at 75 ° C.
- Boc-MePhe-OH (0.070 g, 0.250 mmol) and triethylamine (0.033 g, 0.32 mmol) were mixed with tetrahydrofuran (5 mL) and 2,2-dimethylbutanoyl chloride (0.040 g) at 0 ° C. , 0.30 mmol) and stirred for 1 hour.
- H-MePhe-OH (0.067 g, 0.38 mmol), N, O-bis (trimethylsilyl) acetamide (0.161 g, 0.774 mmol) and acetonitrile (4.0 mL) were separately mixed with this solution, and 75 The prepared solution was added by stirring at ° C.
- reaction solution was concentrated, diluted with ethyl acetate (20 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL).
- the obtained organic layer was washed successively with a 10 mass% citric acid aqueous solution (20 mL) and a saturated sodium chloride aqueous solution (20 mL).
- Boc-MePhe-OH (1.40 g, 5.00 mmol) and N-methylmorpholine (0.556 g, 5.50 mmol) are mixed with tetrahydrofuran (50 mL) and isopropyl chloroformate (0.643 g, 5) at 0 ° C. .25 mmol) was added and the mixture was stirred for 15 minutes.
- H-Phe-OH (0.991 g, 6.00 mmol), N, O-bis (trimethylsilyl) acetamide (2.57 g, 12.4 mmol) and acetonitrile (15 mL) were separately added to this solution under microwave irradiation 75. Stir at ° C.
- reaction solution was diluted with ethyl acetate (200 mL) and washed successively with 10 mass% citric acid aqueous solution (75 mL), 5 mass% sodium chloride aqueous solution (75 mL) and saturated sodium chloride aqueous solution (75 mL).
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Boc-MePhe-Phe-OH (2.20 g, yield 103%) as a white solid.
- Fmoc-MePhe-OH (2.00 g, 5.00 mmol) and N-methylmorpholine (0.556 g, 5.50 mmol) are mixed with tetrahydrofuran (30 mL) and isopropyl chloroformate (0.663 g, 5) at 0 ° C. .25 mmol) was added and the mixture was stirred for 30 minutes.
- H-Phe-OH (0.991 g, 6.00 mmol), N, O-bis (trimethylsilyl) acetamide (2.68 g, 12.9 mmol) and acetonitrile (15 mL) were separately mixed with this solution and heated to 75 ° C.
- the obtained reaction solution was diluted with ethyl acetate (75 mL) and washed successively with 10 mass% citric acid aqueous solution (50 mL), 5 mass% sodium chloride aqueous solution (50 mL) and saturated sodium chloride aqueous solution (50 mL).
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Fmoc-MePhe-Phe-OH (2.85 g, yield 98%) as a white solid.
- Boc-MePhe-Phe-OH (0.213 g, 0.500 mmol) was mixed with 4M-HCl / ethyl acetate (10 mL) and stirred at 25 ° C. for 1 hour.
- the obtained reaction solution was concentrated, diisopropyl ether was added and suspended, and the produced solid was collected by filtration through a Kiriyama funnel, dried, and H-MePhe-Phe-OH ⁇ HCl (0.164 g, yield 91%) was added. Obtained as a white solid.
- the obtained reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution.
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Boc-Phe-MePhe-MePhe-MePhe-OH (0.532 g, 90% yield) as a white solid.
- Acetonitrile (10 mL) and N, O-bis (trimethylsilyl) acetamide (0.430 g, 2.07 mmol) were mixed with the obtained residue, and the mixture was stirred at 25 ° C. for 20 minutes to obtain a colorless and transparent solution.
- the obtained reaction solution was diluted with ethyl acetate (20 mL) and washed successively with 10 mass% citric acid aqueous solution (20 mL), 10 mass% sodium chloride aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL).
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Boc-Phe-MePhe-MePhe-Phe-OH (0.453 g, yield 92%) as a white solid.
- Acetonitrile (10 mL) and N, O-bis (trimethylsilyl) acetamide (0.621 g, 2.99 mmol) were mixed with the obtained residue, and the mixture was stirred at 25 ° C. for 20 minutes to obtain a colorless and transparent solution.
- the obtained reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution.
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Boc-MePhe-MePhe-Phe-OH (0.701 g, yield 103%) as a brown solid.
- Boc-MePhe-OH (1.40 g, 5.00 mmol) and N-methylmorpholine (0.556 g, 5.50 mmol) were mixed with tetrahydrofuran (30 mL) and 3,3-dimethylbutane-2- at 0 ° C.
- Ilcarbonochloridate (0.864 g, 5.25 mmol) was added, and the mixture was stirred for 1 hour.
- H-Pro-OH (0.691 g, 6.00 mmol), N, O-bis (trimethylsilyl) acetamide (3.85 g, 18.6 mmol) and acetonitrile (12 mL) were separately mixed with this solution and heated to 70 ° C.
- the obtained reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution.
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Boc-MePhe-MePhe-Pro-OH (1.71 g, 90% yield) as a white solid.
- Boc-MePhe-MePhe-Pro-OH (1.61 g, 3.00 mmol) was mixed with 4M-HCl / ethyl acetate (15 mL) and stirred at 25 ° C. for 90 minutes.
- the obtained reaction solution was concentrated, ethyl acetate (5 mL) and diisopropyl ether (20 mL) were added and suspended, and the produced solid was collected by filtration through a Kiriyama funnel. Washed with diisopropyl ether (10 mL) and dried to give H-MePhe-MePhe-Pro-OH ⁇ HCl (1.30 g, 91% yield) as a white solid. The obtained solid was used in the next step.
- Condensation step Solution B was mixed while the solution A was cooled to 0 ° C, and the mixture was further stirred at 25 ° C for 1 hour.
- the obtained reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution.
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Boc-Tyr-MePhe-MePhe-Pro-OH (0.698 g, yield 99%) as a white solid.
- the obtained reaction solution was concentrated, diluted with ethyl acetate (30 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL).
- the obtained organic layer was washed successively with 10% by mass aqueous citric acid solution (20 mL) and saturated aqueous sodium chloride solution.
- Boc-MePhe-MeAla-Tyr-OH (0.474 g, 0.898 mmol) was mixed with 4M-HCl / ethyl acetate (5 mL) and stirred at 25 ° C. for 60 minutes.
- the obtained reaction solution was concentrated, acetonitrile (5 mL) and N, N-diisopropylethylamine (1.74 g, 13.5 mmol) were added and suspended, and the produced solid was collected by filtration through a Kiriyama funnel.
- Boc-Phe-OH (0.125 g, 0.470 mmol) and triethylamine (0.057 g, 0.564 mmol) were mixed with tetrahydrofuran (5.0 mL) and 2,2-dimethylbutanoyl chloride (0) at 0 ° C. .070 g, 0.52 mmol) was added, and the mixture was stirred for 45 minutes.
- H-MePhe-MeAla-Tyr-OH (0.221 g, 0.517 mmol), N, O-bis (trimethylsilyl) acetamide (0.332 g, 1.60 mmol) and acetonitrile (5.0 mL) were separately added to this solution.
- Boc-Phe-MePhe-MeAla-Tyr-OH (0.317 g, 0.470 mmol) was mixed with trifluoroacetic acid (1.45 mL) and stirred at 25 ° C. for 15 minutes.
- the obtained reaction solution was concentrated, acetonitrile (7 mL) and triethylamine (0.476 g, 4.70 mmol) were added and suspended, and the produced solid was collected by filtration through a Kiriyama funnel.
- Boc-MePhe-OH (0.092 g, 0.33 mmol) and triethylamine (0.040 g, 0.396 mmol) were mixed with tetrahydrofuran (5.0 mL) and 2,2-dimethylbutanoyl chloride (0) at 0 ° C. .0489 g, 0.363 mmol) was added, and the mixture was stirred for 45 minutes.
- the obtained reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with 10 mass% citric acid aqueous solution and saturated sodium chloride aqueous solution.
- Boc-MePhe-OH (0.559 g, 2.00 mmol) and triethylamine (0.243 g, 2.40 mmol) were mixed with tetrahydrofuran (30 mL) and 2,2-dimethylbutanoyl chloride (0.296 g) at 0 ° C. 2.20 mmol) was added and the mixture was stirred for 45 minutes.
- H-MeAla-Phe-OH (0.601 g, 2.40 mmol), N, O-bis (trimethylsilyl) acetoamide (1.03 g, 4.95 mmol) and acetonitrile (20 mL) were separately mixed with this solution, and 25 The prepared solution was added by stirring at ° C.
- Boc-MePhe-MeAla-Phe-OH (1.06 g, 2.08 mmol) was mixed with 4M-HCl / ethyl acetate (20 mL) and stirred at 25 ° C. for 60 minutes. The obtained reaction solution was concentrated, diisopropyl ether (20 mL) was added and suspended, and the produced solid was collected by filtration with a Kiriyama funnel. Washing and drying with diisopropyl ether (10 mL) gave H-MePhe-MeAla-Phe-OH ⁇ HCl (0.886 g, 95% yield) as a white solid.
- the obtained reaction solution was concentrated, diluted with ethyl acetate (20 mL), and washed twice with saturated aqueous sodium hydrogen carbonate solution (30 mL), water (30 mL), and saturated aqueous sodium chloride solution (12 mL).
- the obtained organic layer was washed successively with a 10 mass% citric acid aqueous solution (20 mL) and a saturated sodium chloride aqueous solution (20 mL).
- the resulting organic layer was concentrated and the residue was dissolved in ethyl acetate (5.0 mL) and poured into hexane (95 mL).
- Solution B Mix H-MePhe-MeAla-Tyr-OH (0.171 g, 0.400 mmol), N, N-diisopropylethylamine (0.431 g, 3.33 mmol) and acetonitrile (20 mL) and concentrate the solution to prepare the solvent. Removed. Acetonitrile (10 mL) and N, O-bis (trimethylsilyl) acetamide (0.343 g, 1.65 mmol) were mixed with the obtained residue, and the mixture was stirred at 25 ° C. for 40 minutes to obtain a colorless and transparent solution.
- the obtained reaction solution was concentrated, diluted with ethyl acetate (20 mL), and washed twice with saturated aqueous sodium hydrogen carbonate solution (15 mL), water (15 mL), and saturated aqueous sodium chloride solution (7.5 mL).
- the obtained organic layer was washed successively with a 10 mass% citric acid aqueous solution (20 mL) and a saturated sodium chloride aqueous solution (20 mL).
- Fmoc-BnGly-OH (0.387 g, 1.00 mmol) and N-methylmorpholine (0.111 g, 1.10 mmol) were mixed with tetrahydrofuran (20 mL) and isopropyl chloroformate (0.129 g, 1) at 0 ° C. 0.05 mmol) was added and the mixture was stirred for 5 minutes.
- This solution is separately mixed with H-Phe-OH (0.198 g, 1.20 mmol), N, O-bis (trimethylsilyl) acetamide (0.519 g, 2.48 mmol), and acetonitrile (5 mL) and irradiated with microwaves.
- Boc-Phe-OH (0.066 g, 0.25 mmol) and triethylamine (0.030 g, 0.30 mmol) were mixed with tetrahydrofuran (5 mL) and 2,2-dimethylbutanoyl chloride (0.037 g) at 0 ° C. , 0.28 mmol) and stirred for 45 minutes.
- H-BnGly-Phe-OH (0.093 g, 0.30 mmol), N, O-bis (trimethylsilyl) acetamide (0.129 g, 0.616 mmol) and acetonitrile (4 mL) were separately mixed with this solution, and 25 The prepared solution was added by stirring at ° C.
- reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The obtained organic layer was washed successively with a 10 mass% citric acid aqueous solution and a saturated sodium chloride aqueous solution (20 mL).
- reaction solution was concentrated, diluted with ethyl acetate (20 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The obtained organic layer was washed twice with a 10 mass% citric acid aqueous solution (20 mL) and a saturated sodium chloride aqueous solution (20 mL).
- Boc-Phe-OH (0.066 g, 0.25 mmol) and triethylamine (0.030 g, 0.300 mmol) were mixed with tetrahydrofuran (5.0 mL) and 2,2-dimethylbutanoyl chloride (0) at 0 ° C. .037 g, 0.27 mmol) was added, and the mixture was stirred for 45 minutes.
- Hn-PrGly-Phe-OH (0.066 g, 0.25 mmol), N, O-bis (trimethylsilyl) acetamide (0.118 g, 0.564 mmol), and acetonitrile (4.0 mL) were separately added to this solution. The solution was mixed, stirred at 25 ° C.
- reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The obtained organic layer was washed successively with a 10 mass% citric acid aqueous solution and a saturated sodium chloride aqueous solution.
- Fmoc-MePhe-OH (0.080 g, 0.20 mmol) and triethylamine (0.024 g, 0.24 mmol) were mixed with tetrahydrofuran (5 mL) and 2,2-dimethylbutanoyl chloride (0.030 g) at 0 ° C. , 0.22 mmol) and stirred for 45 minutes.
- Hn-PrGly-Phe-OH (0.063 g, 0.24 mmol), N, O-bis (trimethylsilyl) acetamide (0.104 g, 0.50 mmol) and acetonitrile (2 mL) are separately mixed with this solution.
- Boc-MePhe-OH (0.084 g, 0.30 mmol) and triethylamine (0.036 g, 0.36 mmol) were mixed with tetrahydrofuran (6.0 mL) and 2,2-dimethylbutanoyl chloride (0) at 0 ° C. .044 g, 0.331 mmol) was added and the mixture was stirred for 1 hour.
- reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL).
- the obtained organic layer was washed successively with a 10 mass% citric acid aqueous solution and a saturated sodium chloride aqueous solution.
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Boc-MePhe-MePhe-MeAla-Tyr-OH (0.177 g, yield 85%) as a white solid.
- Acetonitrile (5.0 mL) and N, O-bis (trimethylsilyl) acetamide (0.167 g, 0.80 mmol) were mixed with the obtained residue, and the mixture was stirred at 25 ° C. for 5 minutes to obtain a colorless and transparent solution.
- the obtained reaction solution was concentrated, diluted with ethyl acetate (30 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL).
- the obtained organic layer was concentrated to obtain Fmoc-Glu (tBu) -MePhe-MePhe-MeAla-Tyr-OH (0.192 g, 90% yield) as a white solid.
- Fmoc-Cys (Trt) -OH (0.147 g, 0.251 mmol) and triethylamine (0.031 g, 0.30 mmol) were mixed with tetrahydrofuran (5.0 mL) and 2,2-dimethylbutanoyl at 0 ° C.
- Chloride (0.037 g, 0.28 mmol) was added and the mixture was stirred for 1 hour.
- H-MePhe-OH (0.054 g, 0.30 mmol), N, O-bis (trimethylsilyl) acetamide (0.130 g, 0.62 mmol) and acetonitrile (4.0 mL) were separately mixed with this solution, and 75 The prepared solution was added by stirring at ° C.
- reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with 10 mass% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate solution, water and saturated aqueous sodium chloride solution.
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Fmoc-Cys (Trt) -MePhe-OH (0.187 g, 100% yield) as a white solid.
- Boc-Arg (Cbz) 2- OH (0.136 g, 0.250 mmol) and triethylamine (0.030 g, 0.30 mmol) were mixed with tetrahydrofuran (5.0 mL) and 2,2-dimethylbuta at 0 ° C.
- Noyl chloride (0.037 g, 0.28 mmol) was added and the mixture was stirred for 45 minutes.
- H-MePhe-OH 0.054 g, 0.30 mmol
- N, O-bis (trimethylsilyl) acetamide (0.128 g, 0.62 mmol) and acetonitrile (4.0 mL) were separately mixed with this solution, and 75 The prepared solution was added by stirring at ° C.
- reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The obtained organic layer was washed successively with a 10 mass% citric acid aqueous solution and a saturated sodium chloride aqueous solution.
- Boc-Arg (Cbz) 2- OH (0.136 g, 0.251 mmol) and triethylamine (0.030 g, 0.30 mmol) were mixed with tetrahydrofuran (5.0 mL) and 2,2-dimethylbuta at 0 ° C.
- Noyl chloride (0.037 g, 0.28 mmol) was added, and the mixture was stirred for 1 hour.
- H-MeAla-Phe-OH (0.075 g, 0.30 mmol), N, O-bis (trimethylsilyl) acetoamide (0.130 g, 0.621 mmol) and acetonitrile (4.0 mL) are separately mixed with this solution.
- reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The obtained organic layer was washed successively with a 10 mass% citric acid aqueous solution and a saturated sodium chloride aqueous solution.
- Fmoc-His (Boc) -OH (0.120 g, 0.251 mmol) and N-methylmorpholine (0.033 g, 0.33 mmol) were mixed with tetrahydrofuran (5.0 mL) and 4-ethyl- at 0 ° C. 2,2-Dimethylhexane-3-ylcarbonochloride (0.067 g, 0.30 mmol) was added, and the mixture was stirred for 2 hours.
- the obtained reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with 10 mass% citric acid aqueous solution and saturated sodium chloride aqueous solution.
- the obtained organic layer was concentrated and then purified by silica gel column chromatography to obtain Fmoc-MeHis (Trt) -Leu-OH (1.37 g, yield 92%) as a pale yellow solid.
- Fmoc-Phe-OH (0.078 g, 0.20 mmol) and triethylamine (0.024 g, 0.24 mmol) were mixed with tetrahydrofuran (5.0 mL) and 2,2-dimethylbutanoyl chloride (0) at 0 ° C. .030 g, 0.22 mmol) was added and the mixture was stirred for 1 hour.
- reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with 10 mass% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate solution, water and saturated aqueous sodium chloride solution.
- the obtained organic layer was concentrated to obtain Fmoc-Phe-MeHis (Trt) -Leu-OH (0.154 g, yield 86%) as a white solid.
- Fmoc-Val-OH (0.101 g, 0.298 mmol) and triethylamine (0.053 mL, 0.383 mmol) were mixed with tetrahydrofuran (1.0 g) and pivaloyl chloride (0.043 mL, 0) at 0 ° C. .354 mmol) was added and the mixture was stirred for 1 hour.
- H-MePhe-OH (0.080 g, 0.446 mmol), trimethylsilyl chloride (0.070 mL, 0.554 mmol), triethylamine (0.082 mL, 0.592 mmol), acetonitrile (0.80 g) were separately added to this solution.
- the obtained reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with a 10 mass% citric acid aqueous solution (3.0 g) and a saturated salt aqueous solution (2.0 g).
- the quantitative yield was 11%.
- the ratio of the raw material Fmoc-Val-OH to the product Fmoc-Val-MePhe-OH was calculated by analysis ⁇ analysis condition 4> using high performance liquid chromatography.
- Synthesis Example 53 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100 g, 0.295 mmol) and triethylamine (0.053 mL, 0.38 mmol) were mixed with tetrahydrofuran (1.0 g) and 0. Pivaloyl chloride (0.043 mL, 0.35 mmol) was added at ° C., and the mixture was stirred for 1 hour.
- Synthesis Example 54 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100 g, 0.295 mmol) and N-methylmorpholine (0.042 mL, 0.383 mmol) are mixed with tetrahydrofuran (1.0 g). Then, isobutylcarbonochloridate (0.046 mL, 0.35 mmol) was added at 0 ° C., and the mixture was stirred for 5 minutes.
- Synthesis Example 55 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100 g, 0.295 mmol) and triethylamine (0.053 mL, 0.38 mmol) were mixed with tetrahydrofuran (1.0 g) and 0. 2-Ethylbutanoyl chloride (0.048 mL, 0.35 mmol) was added at ° C., and the mixture was stirred for 1 hour.
- Synthesis Example 56 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100 g, 0.295 mmol) and triethylamine (0.053 mL, 0.38 mmol) were mixed with tetrahydrofuran (1.0 g) and 0. 2,2-Dimethylbutanoyl chloride (0.049 mL, 0.35 mmol) was added at ° C., and the mixture was stirred for 1 hour.
- Synthesis Example 57 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100 g, 0.295 mmol) and N, N-diisopropylethylamine (0.058 mL, 0.32 mmol) were added to acetonitrile (1.0 g). At 0 ° C., a 50 mass% toluene solution of 2- (4,4-dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride (0.107 g, 0.354 mmol) was added. Stirred for 2 hours.
- reaction solution was quenched with methanol (0.5 mL) and N, N-diisopropylethylamine (0.05 mL), diluted with ethyl acetate (5.0 g), and diluted with 10 mass% aqueous citric acid solution (2.0 g). And washed twice with saturated aqueous saline solution (1.0 g). The quantitative yield of Fmoc-Val-MePhe-OH in the collected organic layer was 96%.
- Synthesis Example 58 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100 g, 0.295 mmol) and N-methylmorpholine (0.071 mL, 0.648 mmol) were added to N, N-dimethylacetamide (5). .9 mL) was mixed, 2,4-dimethylpentane-3-yl carbonochloridet (0.105 g, 0.589 mmol) was added at 0 ° C., and the mixture was stirred for 2 hours.
- reaction solution was quenched with methanol (5.0 mL) and N, N-diisopropylethylamine (0.50 mL), diluted with ethyl acetate (30.0 g), and diluted with 10 mass% aqueous citric acid solution (18.0 g). And washed twice with saturated aqueous saline solution (6.0 g). The quantitative yield of Fmoc-Val-MePhe-OH in the collected organic layer was 85%.
- Synthesis Example 59 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100 g, 0.295 mmol) and N-methylmorpholine (0.071 mL, 0.648 mmol) were added to N, N-dimethylacetamide (5). .9 mL) was mixed, 2,2,4-tritylpentane-3-yl carbonochloridet (0.114 g, 0.589 mmol) was added at 0 ° C., and the mixture was stirred for 2 hours.
- reaction solution was quenched with methanol (5.0 mL) and N, N-diisopropylethylamine (0.50 mL), diluted with ethyl acetate (30.0 g), and diluted with 10 mass% aqueous citric acid solution (18.0 g). And washed twice with saturated aqueous saline solution (6.0 g). The quantitative yield of Fmoc-Val-MePhe-OH in the collected organic layer was 88%.
- Synthesis Example 60 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100 g, 0.295 mmol) and N, N-diisopropylethylamine (0.065 mL, 0.38 mmol) were added in tetrahydrofuran (1.0 g). 1-adamantane carbonyl chloride (0.070 g, 0.35 mmol) was added at 0 ° C., and the mixture was stirred for 1 hour.
- Synthesis Example 61 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.10 g, 0.30 mmol) and N-methylmorpholine (0.071 mL, 0.65 mmol) were added to N, N-dimethylacetamide (5). .9 mL) was mixed, 4-ethyl-2,2-dimethylhexane-3-ylcarbonochloride (0.13 g, 0.59 mmol) was added at 0 ° C., and the mixture was stirred for 2 hours.
- reaction solution was quenched with methanol (5.0 mL) and N, N-diisopropylethylamine (0.50 mL), diluted with ethyl acetate (30.0 g), and diluted with 10 mass% aqueous citric acid solution (18.0 g). , Washed twice with saturated aqueous salt solution (10.0 g) and saturated aqueous salt solution (6.0 g). The quantitative yield of Fmoc-Val-MePhe-OH in the collected organic layer was 83%.
- Synthesis Example 62 Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100 g, 0.295 mmol) and triethylamine (0.053 mL, 0.383 mmol) were mixed with tetrahydrofuran (1.0 g) and 0. 2-Ethylhexanoyl chloride (0.061 mL, 0.354 mmol) was added at ° C., and the mixture was stirred for 1 hour.
- the ratio of the raw material Cbz-Phe-OH to the product Cbz-Phe-MePhe-Phe-OH was calculated by analysis using high performance liquid chromatography ⁇ Analysis Condition 5>.
- the reaction mixture is concentrated under reduced pressure, diluted with methanol (300 g), filtered, and the obtained filtrate is diluted again with methanol (300 g), and the operation of filtering is repeated three times, and all the obtained filtrates are collected.
- the mixture was concentrated, diluted with acetonitrile (100 g) and concentrated again to obtain H-MeOH-Phe-OH (1.35 g, yield 65%) as a white solid.
- Synthesis Example 67 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g) and N-methylmorpholine (20 mg, 0.20 mmol) are mixed and mixed. Isobutyl carbonate chloride (27 mg, 0.20 mmol) was added at 0 ° C., and the mixture was stirred for 3 hours. H-MePhe-Phe-OH (82 mg, 0.25 mmol), N, O-bis (trimethylsilyl) acetoamide (97 mg, 0.48 mmol) and acetonitrile (0.82 g) were separately mixed with this solution and heated to 50 ° C.
- the obtained reaction solution was diluted with ethyl acetate (2.5 g), separated with a 10 mass% citric acid aqueous solution (1.0 g), and washed twice with a 10 mass% sodium chloride aqueous solution (1.0 g).
- the quantitative yield of Cbz-Phe-MePhe-Phe-OH of the obtained organic layer was 43%.
- Synthesis Example 68 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g) and triethylamine (20 mg, 0.20 mmol) are mixed and brought to 0 ° C. 2,2-Dimethylbutanoyl chloride (27 mg, 0.20 mmol) was added, and the mixture was stirred for 2 hours. H-MePhe-Phe-OH (82 mg, 0.25 mmol), N, O-bis (trimethylsilyl) acetoamide (97 mg, 0.48 mmol) and acetonitrile (0.82 g) were separately mixed with this solution and heated to 50 ° C.
- the obtained reaction solution was diluted with ethyl acetate (2.5 g), separated with a 10 mass% citric acid aqueous solution (1.0 g), and washed twice with a 10 mass% sodium chloride aqueous solution (1.0 g).
- the quantitative yield of Cbz-Phe-MePhe-Phe-OH of the obtained organic layer was 95%.
- Synthesis Example 69 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g) and triethylamine (20 mg, 0.20 mmol) are mixed and brought to 0 ° C. 2-Ethylbutanoyl chloride (27 mg, 0.20 mmol) was added, and the mixture was stirred for 2 hours. H-MePhe-Phe-OH (82 mg, 0.25 mmol), N, O-bis (trimethylsilyl) acetoamide (97 mg, 0.48 mmol) and acetonitrile (0.82 g) were separately mixed with this solution and heated to 50 ° C.
- the obtained reaction solution was diluted with ethyl acetate (2.5 g), separated with a 10 mass% citric acid aqueous solution (1.0 g), and washed twice with a 10 mass% sodium chloride aqueous solution (1.0 g).
- the quantitative yield of Cbz-Phe-MePhe-Phe-OH of the obtained organic layer was 102%.
- Synthesis Example 70 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g) and triethylamine (20 mg, 0.20 mmol) are mixed and heated to 25 ° C. 2- (4,4-Dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride in a 50 mass% toluene solution (124 mg, 0.20 mmol) was added, and the mixture was stirred for 9 hours.
- Synthesis Example 71 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (99 mg, 0.33 mmol), acetonitrile (1.0 g), N, N-diisopropylethylamine (52 mg, 0.40 mmol) are mixed. Then, at room temperature, a 50 mass% toluene solution (250 mg, 0.41 mmol) of 2- (4,4-dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride was added, and the mixture was stirred for 2 hours.
- Synthesis Example 72 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), N, N-dimethylacetamide (0.5 g), N-methylmorpholine (22 mg, 0) .22 mmol) was mixed, 2,2,4-trimethylpentan-3-yl carbonochloridet (39 mg, 0.20 mmol) was added at 0 ° C., and the mixture was stirred for 2 hours.
- Synthesis Example 73 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), N, N-dimethylacetamide (0.5 g), N-methylmorpholine (22 mg, 0.22 mmol) ) was mixed, 2,4-dimethylpentane-3-ylcarbonochloride (39 mg, 0.20 mmol) was added at 0 ° C., and the mixture was stirred for 1.5 hours.
- Synthesis Example 74 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), N, N-diisopropylethylamine (30 mg, 0.23 mmol) are mixed. Then, 1-adamantane carbonyl chloride (51 mg, 0.26 mmol) was added at 0 ° C., and the mixture was stirred for 1 hour.
- H-MePhe-Phe-OH 110 mg, 1.0 mmol
- N, O-bis (trimethylsilyl) acetoamide 129 mg, 0.63 mmol
- acetonitrile 1.1 g
- the obtained reaction solution was diluted with ethyl acetate (3.0 g), separated with a 10 mass% citric acid aqueous solution (3.0 g), and washed twice with a saturated aqueous sodium chloride solution (1.0 g).
- the obtained organic layer was quantified to obtain Cbz-Phe-MePhe-Phe-OH with a quantified yield of 98%.
- Synthesis Example 75 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (0.050 g, 0.17 mmol), N, N-dimethylacetamide (3.0 mL), N-methylmorpholine (0.040 mL) , 0.37 mmol) was mixed, 4-ethyl-2,2-dimethylhexane-3-ylcarbonochloride (0.074 g, 0.33 mmol) was added at 0 ° C., and the mixture was stirred for 1 and a half hours.
- Synthesis Example 76 Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), N, N-dimethylacetamide (0.5 g), N-methylmorpholine (22 mg, 0.22 mmol) ) was mixed, 3,3-dimethylbutane-2-ylcarbonochloride (39 mg, 0.20 mmol) was added at 0 ° C., and the mixture was stirred for 1.5 hours.
- H-MePhe-Phe-OH (82 mg, 0.25 mmol), N, O-bis (trimethylsilyl) acetoamide (97 mg, 0.48 mmol) and acetonitrile (0.82 g) were separately mixed with this solution and heated to 50 ° C.
- Boc-Val-OH (128 mg, 0.59 mmol), tetrahydrofuran (1.3 g), N-methylmorpholine (60 mg, 0.59 mmol) were mixed and isobutyl carbonochloridet (72 mg, 0.59 mol) at 0 ° C. ) was added and the mixture was stirred for 2.5 hours.
- MASS (ESI +) m / z; (M + H) +315.20
- the ratio of the raw material Boc-Val-OH to the product Boc-Val-Pro-OH was calculated by analysis ⁇ analytical condition 5> using high performance liquid chromatography.
- Synthesis Example 78 Synthesis of Boc-Val-Pro-OH Boc-Val-OH (128 mg, 0.59 mmol), acetonitrile (1.3 g), N, N-diisopropylethylamine (91 mg, 0.71 mmol) were mixed and 2- (4,4-dimethylpentane) was mixed at 25 ° C. A 50 mass% toluene solution (0.43 g, 0.71 mol) of ⁇ 2-yl) -5,7,7-trimethyloctanoyl chloride was added, and the mixture was stirred for 4 hours.
- Synthesis Example 79 Synthesis of Boc-Val-Pro-OH Boc-Val-OH (50 mg, 0.23 mmol), N, N-dimethylacetamide (1.0 g), N-methylmorpholine (30 mg, 0.30 mmol) The mixture was mixed, 2,2,4-trimethylpentan-3-yl carbonochloridet (53 mg, 0.28 mmol) was added at 0 ° C., and the mixture was stirred for 2.5 hours. H-Pro-OH (34 mg, 0.77 mmol), N, O-bis (trimethylsilyl) acetoamide (0.80 g, 1.0 mmol) and acetonitrile (0.50 g) were separately mixed with this solution and heated to 50 ° C.
- the obtained reaction solution was diluted with ethyl acetate (5 mL), separated with a 10 mass% citric acid aqueous solution (2 mL), and washed twice with a 10 mass% sodium chloride aqueous solution (1.0 g).
- the quantitative yield of Boc-Val-Pro-OH in the obtained organic layer and aqueous layer was 90%.
- Synthesis Example 80 Synthesis of Boc-Val-Pro-OH Boc-Val-OH (100 mg, 0.46 mmol), tetrahydrofuran (1.0 g), N, N-diisopropylethylamine (77 mg, 0.60 mmol) are mixed and mixed. 2,2-Dimethylbutanoyl chloride (74 mg, 0.55 mol) was added at 0 ° C., and the mixture was stirred for 2 hours. H-Pro-OH (64 mg, 0.55 mmol), N, O-bis (trimethylsilyl) acetoamide (0.18 g, 0.87 mmol) and acetonitrile (1.0 g) were separately mixed with this solution and heated to 50 ° C.
- the obtained reaction solution was diluted with ethyl acetate (10 mL), separated with water (2 mL) and 10 mass% citric acid aqueous solution (2 mL), and washed twice with 10 mass% sodium chloride aqueous solution (2 mL).
- the quantitative yield of Boc-Val-Pro-OH in the obtained organic layer was 99%.
- Synthesis Example 81 Synthesis of Boc-Val-Pro-OH Boc-Val-OH (100 mg, 0.46 mmol), tetrahydrofuran (1.0 g), N, N-diisopropylethylamine (77 mg, 0.60 mmol) are mixed and mixed. 2-Ethylbutanoyl chloride (74 mg, 0.55 mol) was added at 0 ° C., and the mixture was stirred for 2 hours. H-Pro-OH (64 mg, 0.55 mmol), N, O-bis (trimethylsilyl) acetoamide (0.18 g, 0.87 mmol) and acetonitrile (1.0 g) were separately mixed with this solution and heated to 50 ° C.
- the obtained reaction solution was diluted with ethyl acetate (10 mL), separated with water (2 mL) and 10 mass% citric acid aqueous solution (2 mL), and washed twice with 10 mass% sodium chloride aqueous solution (2 mL).
- the quantitative yield of Boc-Val-Pro-OH in the obtained organic layer was 99%.
- Synthesis Example 82 Synthesis of Boc-Val-Pro-OH Boc-Val-OH (100 mg, 0.46 mmol), N, N-dimethylacetamide (1.0 g), N-methylmorpholine (61 mg, 0.60 mmol) The mixture was mixed, 2,4-dimethylpentane-3-ylcarbonochloride (99 mg, 0.55 mol) was added at 0 ° C., and the mixture was stirred for 2.5 hours. H-Pro-OH (64 mg, 0.55 mmol), N, O-bis (trimethylsilyl) acetoamide (0.18 g, 0.88 mmol) and acetonitrile (1.0 g) were separately mixed with this solution and heated to 50 ° C.
- the obtained reaction solution was diluted with ethyl acetate (5 mL), separated with a 10 mass% citric acid aqueous solution (2 mL), and washed twice with a 10 mass% sodium chloride aqueous solution (1.0 g).
- the quantitative yield of Boc-Val-Pro-OH in the obtained organic layer and aqueous layer was 89%.
- the ratio of the raw material Fmoc-Val-OH to the product Fmoc-Val-MeGly-OH was calculated by analysis ⁇ analysis condition 5> using high performance liquid chromatography.
- Synthesis Example 84 Synthesis of Fmoc-Val-MeGly-OH Fmoc-Val-OH (200 mg, 0.59 mmol), acetonitrile (2.0 g), N, N-diisopropylethylamine (91 mg, 0.71 mmol) are mixed and mixed. Add a 50 mass% toluene solution (0.43 g, 0.71 mol) of 2- (4,5,4-dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride at 25 ° C. and stir for 4 hours. did.
- Synthesis Example 85 Synthesis of Fmoc-Val-MeGly-OH Fmoc-Val-OH (200 mg, 0.59 mmol), N, N-dimethylacetamide (2.0 g), N-methylmorpholine (78 mg, 0.77 mmol) The mixture was mixed, 2,2,4-trimethylpentane-3-yl carbonochloridet (0.14 g, 0.71 mol) was added at 0 ° C., and the mixture was stirred for 2.5 hours.
- Synthesis Example 86 Synthesis of Fmoc-Val-MeGly-OH Fmoc-Val-OH (100 mg, 0.29 mmol), tetrahydrofuran (1.0 g), N, N-diisopropylethylamine (50 mg, 0.38 mmol) are mixed and mixed. 2,2-Dimethylbutanoyl chloride (48 mg, 0.35 mol) was added at 0 ° C., and the mixture was stirred for 2 hours.
- Synthesis Example 87 Synthesis of Fmoc-Val-MeGly-OH Fmoc-Val-OH (100 mg, 0.29 mmol), tetrahydrofuran (1.0 g), N, N-diisopropylethylamine (50 mg, 0.38 mmol) were mixed and mixed. 2-Ethylbutanoyl chloride (48 mg, 0.35 mol) was added at 0 ° C., and the mixture was stirred for 2 hours.
- Synthesis Example 88 Synthesis of Fmoc-Val-MeGly-OH Fmoc-Val-OH (100 mg, 0.29 mmol), N, N-dimethylacetamide (1.0 g), N-methylmorpholine (39 mg, 0.38 mmol) The mixture was mixed, 2,4-dimethylpentane-3-ylcarbonochloride (63 mg, 0.35 mmol) was added at 0 ° C., and the mixture was stirred for 2 hours.
- Boc-Cys (Bn) -OH 200 mg, 0.64 mmol
- chloroform 1.3 mL
- triethylamine 67 mg, 0.66 mmol
- pivaloyl chloride 81 mg, 0.67 mol was mixed at ⁇ 20 ° C.
- H-Pro-OH 96 mg, 0.84 mmol
- triethylamine 0.16 g, 1.6 mmol
- trimethylsilyl chloride (0.11 g, 1.0 mmol
- dichloromethane 1.6 mL
- N Mix N-dimethylformamide (0.32 mL), stir at 40 ° C.
- the ratio of the raw material Boc-Cys (Bn) -OH to the product Boc-Cys (Bn) -Pro-OH was calculated by analysis using high performance liquid chromatography ⁇ Analysis Condition 6>. ..
- Synthesis Example 90 Synthesis of Boc-Cys (Bn) -Pro-OH Boc-Cys (Bn) -OH (100 mg, 0.32 mmol), acetonitrile (1.0 g), N, N-diisopropylethylamine (50 mg, 0. 39 mmol) was mixed, and a 50 mass% toluene solution (231 mg, 0.39 mol) of 2- (4,4-dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride was added at 25 ° C. Stirred for 2 minutes.
- Synthesis Example 91 Synthesis of Boc-Cys (Bn) -Pro-OH Boc-Cys (Bn) -OH (100 mg, 0.32 mmol), N, N-dimethylacetamide (1.0 g), N-methylmorpholine (42 mg) , 0.42 mmol) was mixed, 2,2,4-trimethylpentane-3-yl carbonochloridete (74 mg, 0.39 mol) was added at 0 ° C., and the mixture was stirred for 2 hours.
- Synthesis Example 92 Synthesis of Boc-Cys (Bn) -Pro-OH Boc-Cys (Bn) -OH (100 mg, 0.32 mmol), acetonitrile (1.0 g), N, N-diisopropylethylamine (54 mg, 0. 41 mmol) was mixed, 2,2-dimethylbutanoyl chloride (52 mg, 0.38 mol) was added at 0 ° C., and the mixture was stirred for 2 minutes.
- Synthesis Example 93 Synthesis of Boc-Cys (Bn) -Pro-OH Boc-Cys (Bn) -OH (100 mg, 0.32 mmol), acetonitrile (1.0 g), N, N-diisopropylethylamine (54 mg, 0. 41 mmol) was mixed, 2-ethylbutanoyl chloride (52 mg, 0.38 mol) was added at 0 ° C., and the mixture was stirred for 2 minutes.
- Synthesis Example 94 Synthesis of Boc-Cys (Bn) -Pro-OH Boc-Cys (Bn) -OH (100 mg, 0.32 mmol), N, N-dimethylacetamide (1.0 g), N-methylmorpholine (42 mg) , 0.42 mmol) was mixed, 2,4-dimethylpentane-3-ylcarbonochloride (69 mg, 0.38 mol) was added at 0 ° C., and the mixture was stirred for 2 hours.
- the ratio of the raw material Cbz-Phe-Phe-OH to the product Cbz-Phe-Phe-MePhe-OH was calculated by analysis ⁇ analytical condition 7> using high performance liquid chromatography.
- Synthesis Example 96 Synthesis of Cbz-Phe-Phe-MePhe-OH 2-chlorotrityl chloride resin (200-400 mesh) (0.10 g, 0.13 mmol), dichloromethane (1.0 mL), Fmoc-MePhe-OH ( 0.062 g (0.15 mmol) and N, N-diisopropylethylamine (0.11 mL, 0.61 mmol) were added, and the mixture was stirred overnight. The following solutions were sequentially added to the obtained suspension and filtered each time.
- the reaction solution was filtered and the resin was washed 10 times with N-methylpyrrolidone. Then, the above condensation / washing operation was carried out again, and after adding 20% piperidine / N-methylpyrrolidone, stirring for 20 minutes and washing with N-methylpyrrolidone were performed 10 times.
- synthesis examples 1 to 7, 66 and 96 are reference examples relating to the synthesis of the raw materials used in the examples, while synthesis examples 1 and 2 and synthesis examples 3 to 4 relate to the compound of the present application. It is also an embodiment of the invention.
- Synthesis Examples 8 to 13, 18 to 28, 32 to 35, 38, 41 to 48, 51, 55 to 63, 68 to 76, 78 to 82, 84 to 88, 90 to 95 are methods for producing the peptides of the present application.
- Examples of the invention according to the present invention, and Synthesis Examples 14 to 17, 29 to 31, 36 to 37, 39 to 40, 49 to 50, 52 to 54, 64 to 65, 67, 77, 83, 89 and 96 are the examples thereof. This is a comparative example.
- Fmoc-Val-OH (0.102 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), triethylamine (0.050 mL, 0.36 mmol) and pivaloyl chloride (0.041 mL, 0) at 0 ° C. .33 mmol) was added, and the mixture was stirred at 0 ° C. for 2 hours.
- H-MeTyr (tBu) -OH (0.090 g, 0.36 mmol), N, O-bis (trimethylsilyl) acetamide (0.19 mL, 0.72 mmol) and acetonitrile (1.5 mL) are separately mixed with this solution.
- reaction solution was diluted with ethyl acetate (20 mL) and washed successively with saturated aqueous hydrogen carbonate solution (20 mL), water (5 mL) and saturated aqueous sodium chloride solution (5 mL). After concentrating the obtained organic layer, Fmoc-Val-MeTyr (tBu) -OH (0.18 g, yield 106%) was obtained as a pale yellow solid.
- H-MeTyr (tBu) -OH 0.090 g, 0.36 mmol
- N, O-bis (trimethylsilyl) acetamide (0.185 mL, 0.72 mmol) and acetonitrile (1.5 mL) are separately mixed with this solution.
- reaction solution was diluted with ethyl acetate (5 mL), saturated aqueous sodium hydrogen carbonate solution (5 mL) was added, the mixture was stirred for 1 hour, and then washed successively with water (5 mL) and saturated aqueous sodium chloride solution (5 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val-MeTyr (tBu) -OH (0.170 g, yield 99%) as a white solid.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.050 mL, 0.36 mmol), pivaloyl chloride (0.041 mL, 0) at 0 ° C. .33 mmol) was added and the mixture was stirred for 2 hours.
- H-MeVal-OH (0.047 g, 0.36 mmol), N, O-bis (trimethylsilyl) acetamide (0.19 mL, 0.72 mmol) and acetonitrile (1.5 mL) were separately mixed with this solution, and 50 The mixture was stirred at ° C.
- reaction solution was diluted with ethyl acetate (5 mL) and washed successively with saturated aqueous hydrogen carbonate solution (5 mL), 10 mass% citric acid aqueous solution (5 mL), water (5 mL) and saturated aqueous sodium chloride solution (5 mL). After concentrating the obtained organic layer, Fmoc-Val-MeVal-OH (0.16 g, yield 116%) was obtained as a pale yellow solid.
- Synthesis Example 100 Synthesis of Fmoc-Val-MeVal-OH Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL) and triethylamine (0.033 mL, 0. 24 mmol), 2- (4,4-dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) was added, and the mixture was stirred for 2 hours.
- reaction solution was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate solution (5 mL) was added, the mixture was stirred for 1 hour, and then washed successively with water (5 mL) and saturated aqueous sodium chloride solution (5 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val-MeVal-OH (0.092 g, yield 92%) as a white solid.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.050 mL, 0.36 mmol), pivaloyl chloride (0.041 mL, 0) at 0 ° C. .33 mmol) was added and the mixture was stirred for 1 hour.
- H-MeSer (tBu) -OH (0.063 g, 0.36 mmol), N, O-bis (trimethylsilyl) acetamide (0.19 mL, 0.72 mmol) and acetonitrile (1.5 mL) are separately mixed with this solution. The mixture was stirred at 50 ° C.
- Synthesis Example 102 Synthesis of Fmoc-Val-MeSer (tBu) -OH Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.033 mL) was mixed at 0 ° C. , 0.24 mmol), 2- (4,4-dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) was added and stirred for 2 hours.
- H-MeSer (tBu) -OH 0.045 g, 0.26 mmol
- N, O-bis (trimethylsilyl) acetoamide (0.13 mL, 0.52 mmol) and acetonitrile (1.0 mL) are separately mixed with this solution.
- reaction solution was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate solution (5 mL) was added, the mixture was stirred for 1 hour, and then washed successively with water (5 mL) and saturated aqueous sodium chloride solution (5 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val-MeSer (tBu) -OH (0.095 g, yield 96%) as a white solid.
- Fmoc-Val-OH (0.034 g, 0.1 mmol) was mixed with tetrahydrofuran (1.0 mL) and triethylamine (0.017 mL, 0.12 mmol), pivaloyl chloride (0.014 mL, 0) at 0 ° C. .11 mmol) was added and the mixture was stirred for 1 hour.
- N ⁇ -Boc-N ⁇ -methyl-2,3-diaminopropionic acid H-MeDap (Boc) -OH
- N, O-bis (trimethylsilyl) acetonitrile 0.62 mL, 0.24 mmol
- acetonitrile 1.0 mL
- Synthesis Example 104 Synthesis of Fmoc-Val-MeDap (Boc) -OH Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.033 mL) was mixed at 0 ° C. , 0.24 mmol), 2- (4,4-dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) was added and stirred for 2 hours.
- H-MeDap (Boc) -OH 0.052 g, 0.24 mmol
- acetonitrile 1.0 mL
- reaction solution was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate solution (5 mL) was added, the mixture was stirred for 1 hour, and then washed successively with water (5 mL) and saturated aqueous sodium chloride solution (5 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val-MeDap (Boc) -OH (0.107 g, yield 99%) as a white solid.
- Fmoc-Val-OH (0.33 g, 0.10 mmol) was mixed with tetrahydrofuran (0.5 mL) and at 0 ° C. triethylamine (0.017 mL, 0.12 mmol), pivaloyl chloride (0.014 mL, 0). .11 mmol) was added and the mixture was stirred for 1 hour.
- H-MeGln (Trt) -OH (0.048 g, 0.12 mmol), N, O-bis (trimethylsilyl) acetoamide (0.062 mL, 0.24 mmol) and acetonitrile (1.5 mL) are separately mixed with this solution. The mixture was stirred at 50 ° C.
- Synthesis Example 106 Synthesis of Fmoc-Val-MeGln (Trt) -OH Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.033 mL) was mixed at 0 ° C. , 0.24 mmol), 2- (4,4-dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) was added and stirred for 2 hours.
- reaction solution was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate solution (5 mL) was added, the mixture was stirred for 1 hour, and then washed successively with water (5 mL) and saturated aqueous sodium chloride solution (5 mL).
- the obtained organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGln (Trt) -OH (0.151 g, yield 104%) as a white solid.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.050 mL, 0.36 mmol), pivaloyl chloride (0.041 mL, 0) at 0 ° C. .33 mmol) was added and the mixture was stirred for 1 hour.
- H-MeGlu (OtBu) -OH (0.098 g, 0.45 mmol), N, O-bis (trimethylsilyl) acetamide (0.23 mL, 0.90 mmol) and acetonitrile (1.5 mL) are separately mixed with this solution. The mixture was stirred at 50 ° C.
- reaction solution was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate solution (5 mL) was added, the mixture was stirred for 1 hour, and then washed successively with water (5 mL) and saturated aqueous sodium chloride solution (5 mL). After concentrating the obtained organic layer, Fmoc-Val-MeGlu (OtBu) -OH (0.16 g, yield 100%) was obtained as a pale yellow solid.
- H-MeGlu (OtBu) -OH 0.078 g, 0.36 mmol
- N, O-bis (trimethylsilyl) acetamide (0.185 mL, 0.72 mmol) and acetonitrile (1.5 mL) are separately mixed with this solution.
- reaction solution was diluted with ethyl acetate (5 mL), saturated aqueous sodium hydrogen carbonate solution (5 mL) was added, the mixture was stirred for 1 hour, and then washed successively with water (5 mL) and saturated aqueous sodium chloride solution (5 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val-MeGlu (OtBu) -OH (0.176 g, yield 109%) as a white solid.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.049 mL, 0.35 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.11 g, 0.32 mmol) was added, and the mixture was stirred for 1 hour.
- the acetonitrile solution was diluted with ethyl acetate (6.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (5.0 mL). After concentrating the obtained organic layer, Fmoc-Val-EtAla-OH (0.13 g, yield 104%) was obtained as a pale yellow solid.
- Fmoc-Ala-OH (0.10 g, 0.32 mmol) was mixed with tetrahydrofuran (1.6 mL) and triethylamine (0.054 mL, 0.39 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.13 g, 0.35 mmol) was added, and the mixture was stirred for 1 hour.
- Fmoc-Ala-OH (0.10 g, 0.32 mmol) was mixed with tetrahydrofuran (1.6 mL) and triethylamine (0.054 mL, 0.39 mmol), pivaloyl chloride (0.44 mL, 0) at 0 ° C. .35 mmol) was added and the mixture was stirred for 1 hour.
- H-BnAla-OH (0.069 g, 0.39 mmol), N, O-bis (trimethylsilyl) acetamide (0.19 mL, 0.77 mmol) and acetonitrile (1.6 mL) were separately mixed with this solution, and 25 The prepared solution was added by stirring at ° C.
- reaction solution is concentrated, diluted with ethyl acetate (8.0 mL), and washed successively with saturated aqueous sodium hydrogen carbonate solution (6.0 mL), water (6.0 mL), and saturated aqueous sodium chloride solution (6.0 mL). did.
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Ala-BnAla-OH (0.12 g, 80%) as a white solid.
- Fmoc-Gly-OH (0.020 g, 0.067 mmol) was mixed with tetrahydrofuran (0.42 mL) and triethylamine (0.011 mL, 0.080 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.026 g, 0.074 mmol) was added, and the mixture was stirred for 1 hour.
- reaction solution was diluted with ethyl acetate (5.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (5.0 mL), water (5.0 mL) and saturated aqueous sodium chloride solution (5.0 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Gly-EtAla-Phe-OH (0.040 g, yield 109%) as a white solid.
- Fmoc-Gly-OH (0.025 g, 0.084 mmol) was mixed with tetrahydrofuran (0.42 mL), triethylamine (0.014 mL, 0.10 mmol) and pivaloyl chloride (0.011 mL, 0) at 0 ° C. .092 mmol) was added and the mixture was stirred for 1 hour.
- H-EtAla-Phe-OH (0.027 g, 0.10 mmol), N, O-bis (trimethylsilyl) acetoamide (0.049 mL, 0.20 mmol) and acetonitrile (0.42 mL) are separately mixed with this solution. , Stirred at 25 ° C.
- reaction solution was diluted with ethyl acetate (5.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (5.0 mL), water (5.0 mL) and saturated aqueous sodium chloride solution (5.0 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Gly-EtAla-Phe-OH (0.050 g, yield 109%) as a white solid.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.049 mL, 0.35 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.098 g, 0.32 mmol) was added, and the mixture was stirred for 2 hours.
- the acetonitrile solution was diluted with ethyl acetate (10 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (10 mL), 1 M aqueous hydrochloric acid solution (10 mL), water (10 mL) and saturated aqueous sodium chloride solution (12 mL). After concentrating the obtained organic layer, Fmoc-Val-cHexmGly-OH (0.15 g, yield 104%) was obtained as a white solid.
- Fmoc-Val-OH (0.030 g, 0.088 mmol) was mixed with tetrahydrofuran (0.44 mL), triethylamine (0.015 mL, 0.11 mmol) and pivaloyl chloride (0.012 mL, 0) at 0 ° C. .097 mmol) was added and the mixture was stirred for 1 hour.
- H-cHexmGly-OH (0.018 g, 0.11 mmol), N, O-bis (trimethylsilyl) acetoamide (0.052 mL, 0.21 mmol) and acetonitrile (0.44 mL) were separately mixed with this solution, and 50 The mixture was stirred at ° C.
- reaction solution was concentrated, diluted with ethyl acetate (5.0 mL), saturated aqueous sodium hydrogen carbonate solution (5.0 mL), 1 M hydrochloric acid (5.0 mL), water (6.0 mL), saturated aqueous sodium chloride solution. It was washed sequentially with (6.0 mL). After concentrating the obtained organic layer, Fmoc-Val-cHexmGly-OH (0.048 g, yield 109%) was obtained as a white solid.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol), 2,2-dimethylbutanoyl chloride (0.049 mL, 0.35 mmol) at 0 ° C. 0.045 mL (0.32 mmol) was added, and the mixture was stirred for 1 hour.
- reaction solution was diluted with ethyl acetate (10 mL), saturated aqueous sodium hydrogen carbonate solution (8.0 mL), 10 mass% citric acid aqueous solution (8.0 mL), water (10 mL), saturated aqueous sodium chloride solution (10 mL).
- ethyl acetate 10 mL
- saturated aqueous sodium hydrogen carbonate solution 8.0 mL
- 10 mass% citric acid aqueous solution 8.0 mL
- water 10 mL
- saturated aqueous sodium chloride solution 10 mL
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and at 0 ° C., triethylamine (0.049 mL, 0.35 mmol), pivaloyl chloride (0.040 mL, 0.32 mmol) was added, and the mixture was stirred for 1 hour.
- H-Tic-OH (0.063 g, 0.35 mmol), N, O-bis (trimethylsilyl) acetoamide (0.17 mL, 0.71 mmol) and acetonitrile (1.5 mL) were separately mixed with this solution, and 25 The prepared solution was added by stirring at ° C.
- reaction solution was diluted with ethyl acetate (10 mL), saturated aqueous sodium hydrogen carbonate solution (8.0 mL), 10 mass% citric acid aqueous solution (8.0 mL), water (10 mL), saturated aqueous sodium chloride solution (10 mL). Was washed sequentially with. After concentrating the collected organic layer, Fmoc-Val-Tic-OH (0.14 g, yield 97%) was obtained as a white solid.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.049 mL, 0.35 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl bromide (0.13 g, 0.32 mmol) was added, and the mixture was stirred for 1 hour.
- H-MeGlu (OtBu) -OH 0.077 g, 0.35 mmol
- N, O-bis (trimethylsilyl) acetamide (0.17 mL, 0.71 mmol) and acetonitrile (1.5 mL) are separately mixed with this solution.
- the obtained reaction solution was concentrated, diluted with acetonitrile (8.0 mL), hexane (8.0 mL) was added, and the mixture was washed twice.
- the acetonitrile solution was diluted with ethyl acetate (8.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (7.0 mL), 1 M aqueous hydrochloric acid (7.0 mL), water (10 mL) and saturated aqueous sodium chloride solution (10 mL). After concentrating the obtained organic layer, Fmoc-Val-MeGlu (OtBu) -OH (0.16 g, yield 98%) was obtained as a white solid.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) is mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.058 mL, 0.41 mmol), 2- (4,4-dimethylpentane) at 0 ° C. ⁇ 2-Il) -5,7,7-trimethyloctanoyl chloride (0.12 g, 0.38 mmol) was added, and the mixture was stirred for 2 hours.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.058 mL, 0.41 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.12 mL, 0.38 mmol) was added, and the mixture was stirred for 2 hours.
- H-MeGABA-OH hydrochloride (0.063 g, 0.41 mmol), N, O-bis (trimethylsilyl) acetoamide (0.38 mL, 1.5 mmol) and acetonitrile (1.5 mL) are separately mixed with this solution.
- the obtained reaction solution was diluted with ethyl acetate (4.0 mL), saturated aqueous sodium hydrogen carbonate solution (4.0 mL) was added, and the mixture was stirred at 25 ° C.
- Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), triethylamine (0.049 mL, 0.35 mmol) and pivaloyl chloride (0.040 mL, 0) at 0 ° C. .32 mmol) was added and the mixture was stirred for 1 hour.
- H-MeGABA-OH hydrochloride 0.054 g, 0.35 mmol
- N, O-bis (trimethylsilyl) acetoamide (0.35 mL, 1.4 mmol
- acetonitrile 1.5 mL
- Fmoc-Val-OH (0.100 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.049 mL, 0.35 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.12 g, 0.32 mmol) was added, and the mixture was stirred for 1 hour.
- reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (3.0 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val- (Me) ⁇ Ala-MePhe-OH (0.18 g, yield 107%) as a white solid.
- reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (3.0 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val- (Me) ⁇ Ala-MePhe-OH (0.19 g, yield 109%) as a white solid.
- Fmoc-Val-OH (0.100 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.049 mL, 0.35 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.12 g, 0.32 mmol) was added, and the mixture was stirred for 1 hour.
- H-MeGABA-MePhe-OH 0.098 g, 0.35 mmol
- N, O-bis (trimethylsilyl) acetamide (0.17 mL, 0.71 mmol)
- acetonitrile 1.5 mL
- reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (3.0 mL).
- the obtained organic layer was concentrated and then washed with hexane to give Fmoc-Val-MeGABA-MePhe-OH (0.18 g, yield 107%) as a white solid.
- Fmoc-Val-OH (0.100 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.049 mL, 0.35 mmol), pivaloyl chloride (0.040 mL, 0) at 0 ° C. .32 mmol) was added and the mixture was stirred for 1 hour.
- H-MeGABA-MePhe-OH (0.098 g, 0.35 mmol), N, O-bis (trimethylsilyl) acetamide (0.17 mL, 0.71 mmol) and acetonitrile (1.5 mL) are separately mixed with this solution. , The solution prepared by stirring at 25 ° C.
- reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (3.0 mL).
- the obtained organic layer was concentrated and then washed with hexane to give Fmoc-Val-MeGABA-MePhe-OH (0.19 g, yield 108%) as a white solid.
- Fmoc-Val-OH (0.030 g, 0.11 mmol) was mixed with tetrahydrofuran (0.44 mL) and triethylamine (0.015 mL, 0.11 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.035 g, 0.097 mmol) was added, and the mixture was stirred for 2 hours.
- reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (3.0 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val- (Me) ⁇ homomoTrp (1-Me) -OH (0.056 g, yield 111%) as a white solid.
- Fmoc-Val-OH (0.070 g, 0.21 mmol) was mixed with tetrahydrofuran (1.0 mL), triethylamine (0.035 mL, 0.11 mmol) and pivaloyl chloride (0.028 mL, 0) at 0 ° C. .23 mmol) was added and the mixture was stirred for 1 hour.
- reaction solution was diluted with ethyl acetate (4.0 mL), saturated aqueous sodium hydrogen carbonate solution (4.0 mL) was added, and the mixture was stirred at 25 ° C. for 1 hour.
- the obtained organic layer was washed successively with 10% by mass aqueous citric acid solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (3.0 mL).
- the collected organic layer was concentrated and then washed with hexane to obtain Fmoc-Val- (Me) ⁇ homomoTrp (1-Me) -OH (0.090 g, 77% yield) as a white solid.
- Fmoc-Val-OH (0.050 g, 0.15 mmol) was mixed with tetrahydrofuran (0.74 mL) and triethylamine (0.025 mL, 0.18 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.049 g, 0.16 mmol) was added, and the mixture was stirred for 1 hour.
- H- ⁇ homotropic (1-Me) -OH hydrochloride 0.048 g, 0.18 mmol
- N, O-bis (trimethylsilyl) acetamide (0.17 mL, 0.71 mmol)
- acetonitrile (0. 74 mL)
- reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (3.0 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val- ⁇ homoTrp (1-Me) -OH (0.11 g, yield 130%) as a white solid.
- Fmoc-Val-OH (0.050 g, 0.15 mmol) was mixed with tetrahydrofuran (0.74 mL), triethylamine (0.025 mL, 0.18 mmol), pivaloyl chloride (0.020 g, 0) at 0 ° C. .16 mmol) was added and the mixture was stirred for 1 hour.
- H- ⁇ homotropic (1-Me) -OH hydrochloride (0.048 g, 0.18 mmol), N, O-bis (trimethylsilyl) acetamide (0.17 mL, 0.71 mmol), acetonitrile (0. 74 mL) was mixed, stirred at 50 ° C.
- reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (3.0 mL).
- the obtained organic layer was concentrated and then washed with hexane to obtain Fmoc-Val- ⁇ homoTrp (1-Me) -OH (0.079 g, yield 97%) as a white solid.
- Fmoc-Val-OH (0.030 g, 0.088 mmol) was mixed with tetrahydrofuran (0.44 mL) and triethylamine (0.015 mL, 0.11 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.035 g, 0.097 mmol) was added, and the mixture was stirred for 2 hours.
- reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (4.0 mL), water (3.0 mL) and saturated aqueous sodium chloride solution (3.0 mL).
- the obtained organic layer was concentrated and then washed with a mixed solution of hexane and 10% isopropyl ether / hexane to obtain Fmoc-Val- (Me) ⁇ homoLeu-OH (0.045 g, yield 106%) as a white solid. ..
- Fmoc-Val-OH (0.070 g, 0.21 mmol) was mixed with tetrahydrofuran (1.0 mL), triethylamine (0.035 mL, 0.25 mmol) and pivaloyl chloride (0.028 mL, 0) at 0 ° C. .23 mmol) was added and the mixture was stirred for 1 hour.
- H- (Me) ⁇ homoleu-OH hydrochloride (0.048 g, 0.25 mmol), N, O-bis (trimethylsilyl) acetamide (0.24 mL, 0.99 mmol), acetonitrile (1.0 mL) was mixed, stirred at 25 ° C.
- Fmoc- (Me) ⁇ Ala-OH (1.0 g, 3.1 mmol) is mixed with tetrahydrofuran (15 mL) and triethylamine (0.51 mL, 3.7 mmol), 2- (4,4-dimethylpentane) at 0 ° C. -2-yl) -5,7,7-trimethyloctanoyl chloride (1.2 g, 3.4 mmol) was added, and the mixture was stirred for 1 hour.
- Fmoc- (Me) ⁇ Ala-OH (0.10 g, 0.31 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.051 mL, 0.37 mmol), pivaloyl chloride (0. 041 mL, 0.34 mmol) was added, and the mixture was stirred for 1 hour.
- H-MePhe-OH (0.066 g, 0.37 mmol), N, O-bis (trimethylsilyl) acetamide (0.18 mL, 0.74 mmol) and acetonitrile (1.5 mL) were separately mixed with this solution, and 25 The prepared solution was added by stirring at ° C.
- reaction solution was diluted with ethyl acetate (10 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (8.0 mL), 1 M hydrochloric acid (8.0 mL), water (10 mL) and saturated aqueous sodium chloride solution (10 mL). .. After concentrating the collected organic layer, Fmoc- (Me) ⁇ Ala-MePhe-OH (0.16 g, yield 107%) was obtained as a white solid.
- Fmoc-MeGABA-OH (1.0 g, 3.0 mmol) was mixed with tetrahydrofuran (15 mL) and triethylamine (0.50 mL, 3.5 mmol), 2- (4,4-dimethylpentane-2-) at 0 ° C. (Il) -5,7,7-trimethyloctanoyl chloride (1.2 g, 3.2 mmol) was added, and the mixture was stirred for 1 hour.
- Fmoc-MeGABA-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.049 mL, 0.35 mmol), pivaloyl chloride (0.040 mL, 0) at 0 ° C. .32 mmol) was added and the mixture was stirred for 1 hour.
- H-MePhe-OH (0.063 g, 0.35 mmol), N, O-bis (trimethylsilyl) acetamide (0.17 mL, 0.71 mmol) and acetonitrile (1.5 mL) were separately mixed with this solution, and 25 The prepared solution was added by stirring at ° C.
- reaction solution was diluted with ethyl acetate (10 mL) and washed successively with saturated aqueous sodium hydrogen carbonate solution (8.0 mL), 1 M hydrochloric acid (8.0 mL), water (10 mL) and saturated aqueous sodium chloride solution (10 mL). .. After concentrating the collected organic layer, Fmoc-MeGABA-MePhe-OH (0.15 g, yield 105%) was obtained as a white solid.
- Fmoc- (2SMe) ⁇ Ala-OH (0.050 g, 0.15 mmol) was mixed with tetrahydrofuran (0.42 mL) and triethylamine (0.026 mL, 0.18 mmol), 2- (4,4-) at 0 ° C.
- Dimethylpentane-2-yl) -5,7,7-trimethyloctanoyl chloride (0.051 g, 0.17 mmol) was added, and the mixture was stirred for 1 hour.
- Fmoc-GABA-OH (0.10 g, 0.31 mmol) was mixed with tetrahydrofuran (1.5 mL) and triethylamine (0.051 mL, 0.37 mmol), 2- (4,4-dimethylpentane-) at 0 ° C. 2-Il) -5,7,7-trimethyloctanoyl chloride (0.12 g, 0.34 mmol) was added, and the mixture was stirred for 1 hour.
- Fmoc-GABA-OH (0.10 g, 0.31 mmol) was mixed with tetrahydrofuran (1.5 mL), triethylamine (0.051 mL, 0.37 mmol) and pivaloyl chloride (0.042 mL, 0) at 0 ° C. .34 mmol) was added and the mixture was stirred for 1 hour.
- H- (Me) ⁇ Ala-OH hydrochloride 0.052 g, 0.37 mmol
- N, O-bis (trimethylsilyl) acetamide (0.36 mL, 1.5 mmol
- acetonitrile 1.5 mL
- synthesis example 118 is a reference example relating to the synthesis of the raw materials used in the examples.
- Synthesis Examples 98, 100, 102, 104, 106, 108 to 110, 112, 114, 116, 119, 120, 122, 124, 126, 128, 132, 134, 136, 138 and 140 are the peptides of the present application.
- Examples of the invention relating to the production method Synthesis Examples 97, 99, 101, 103, 105, 107, 111, 113, 115, 117, 121, 123, 125, 127, 129, 133, 135, 137, 139.
- And 141 are comparative examples thereof, and synthetic examples 130 and 131 are reference examples.
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Abstract
Description
(1)生成物のC末端が保護されているペプチド
-N末端保護アミノ酸のC末端を塩化ピバロイルで活性化し、N-メチルアミノ酸のベンジルエステルを反応させる方法(例えば、特許文献1参照)。
(2)生成物のC末端が無保護のペプチド
-N末端保護アミノ酸のC末端をイソブチルクロロホルメートで活性化し、シリル化されたN-メチルグリシン(サルコシン)やプロリンを反応させる方法(例えば、非特許文献2参照)。
-N末端保護ペプチドのC末端を1-[ビス(ジメチルアミノ)メチレン]-1H-1,2,3,-トリアゾロ[4,5-b]ピリジニウム 3-オキシドヘキサフルオロホスファート(HATU)で活性化し、N-メチルアラニンを反応させる方法(例えば、特許文献2参照)。
一方、特許文献1、非特許文献3に記載の方法では、生成するペプチドのC末端が保護されており、C末端が無保護のペプチドを得るには、さらに脱保護工程が必要となる。従って、縮合工程に加えて必ず脱保護工程が発生するため、ペプチドの効率的な製造法として適用できるものではなかった。
(1)
式(I)P-A1-OH(式中、PはN末端保護基であり、A1は、アミノ酸由来の基、N-C1-6アルキルアミノ酸由来の基(C1-6アルキルは置換基を有していてもよい)またはペプチド由来の基を表す)で表されるN末端保護アミノ酸またはN末端保護ペプチドを
式(II)
(式中、Xはハロゲン原子を表し、R1は炭素数が5以上であり、置換基を有していてもよい2級または3級の脂肪族炭化水素基を表すか、あるいは炭素数が4以上であり、置換基を有している1級の脂肪族炭化水素基(ここで、1級の脂肪族炭化水素基の置換基は、カルボニル炭素に結合する炭素原子上に存在する)を表す)で表されるカルボン酸ハロゲン化物、および式(III)
(式中、Xはハロゲン原子を表し、R2は炭素数が5以上であり、置換基を有していてもよい2級の脂肪族炭化水素基を表す)で表されるハロゲン化ギ酸アルキルよりなる群から選択される活性化剤と混合する工程;
(2)
式(IV)H-A2-OH(式中、A2は、N-C1-6アルキルアミノ酸由来の基(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸由来の基(4-6員環は、C6-14アリール環、C6-14ハロアリール環およびC3-8シクロアルキル環からなる群から選ばれる環状化合物と縮合していてもよい)、あるいはN末端残基がN-C1-6アルキルアミノ酸(C1-6アルキルは置換基を有していてもよい)または4-6員の環状の2級アミノ酸(4-6員環は、C6-14アリール環、C6-14ハロアリール環およびC3-8シクロアルキル環からなる群から選ばれる環状化合物と縮合していてもよい)であるペプチド由来の基を表す)で表されるアミノ酸またはペプチドをシリル化剤と混合する工程;
(3)
工程(1)で得られた生成物と工程(2)で得られた生成物とを混合する工程、
を含むペプチドの製造方法。
(1)
式(I)P-A1-OH(式中、PはN末端保護基であり、A1は、アミノ酸由来の基またはN-C1-6アルキルアミノ酸由来の基(C1-6アルキルは置換基を有していてもよい)を表す)で表されるN末端保護アミノ酸を
式(II)
(式中、Xはハロゲン原子を表し、R1は炭素数が5以上であり、置換基を有していてもよい2級または3級の脂肪族炭化水素基を表すか、あるいは炭素数が4以上であり、置換基を有している1級の脂肪族炭化水素基(ここで、1級の脂肪族炭化水素基の置換基は、カルボニル炭素に結合する炭素原子上に存在する)を表す)で表されるカルボン酸ハロゲン化物および
式(III)
(式中、Xはハロゲン原子を表し、R2は炭素数が5以上であり、置換基を有していてもよい2級の脂肪族炭化水素基を表す)で表されるハロゲン化ギ酸アルキルよりなる群から選択される活性化剤と混合する工程;
(2)
式(IV)H-A2-OH(式中、A2は、N-メチルアミノ酸由来の基、N-C1-6アルキルグリシン由来の基(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸由来の基、あるいはN末端残基がN-メチルアミノ酸、N-C1-6アルキルグリシン(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸であるペプチド由来の基を表す)で表されるアミノ酸またはペプチドをシリル化剤と混合する工程;
(3)
工程(1)で得られた生成物と工程(2)で得られた生成物とを混合する工程、
を含むペプチドの製造方法。
(1)
式(V)P-A3-OH(式中、PはN末端保護基であり、A3は、ペプチド由来の基を表す)で表されるN末端保護ペプチドを
式(II)
(式中、Xはハロゲン原子を表し、R1は炭素数が5以上であり、置換基を有していてもよい2級または3級の脂肪族炭化水素基を表すか、あるいは炭素数が4以上であり、置換基を有している1級の脂肪族炭化水素基(ここで、1級の脂肪族炭化水素基の置換基は、カルボニル炭素に結合する炭素原子上に存在する)を表す)で表されるカルボン酸ハロゲン化物と混合する工程;
(2)
式(IV')H-A2’-OH(式中、A2’は、N-メチルアミノ酸由来の基、N-C1-6アルキルグリシン由来の基(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸由来の基を表す)で表されるアミノ酸をシリル化剤と混合する工程;
(3)
工程(1)で得られた生成物と工程(2)で得られた生成物とを混合する工程、
を含むペプチドの製造方法。
(4)
工程(3)または(5)で得られたペプチドのN末端の保護基を除去する工程;
(5)
工程(4)で得られたペプチドのN末端に、N末端保護アミノ酸またはN末端保護ペプチドを反応させる工程、
の繰り返しを1以上含む、上記[1]乃至[3]のいずれかに記載のペプチドの製造方法。
本明細書における「n-」はノルマル、「s-」はセカンダリー、「t-」および「tert-」はターシャリー、「Me」はメチル、「Et」はエチル、「Pr」はプロピル、「Bu」はブチル、「Ph」はフェニル、「Bn」はベンジル、「Boc」はt-ブトキシカルボニル、「Cbz」はベンジルオキシカルボニル、「Fmoc」は9-フルオレニルメトキシカルボニル、「Trt」はトリチル、「TMS」はトリメチルシリル、「TFA」はトリフルオロ酢酸を意味する。
以下に本発明のペプチドの製造法の各工程(1)乃至(5)について説明する。
一つの態様として、本発明のペプチドの製造は、以下の工程(1)乃至(5)として記載されるそれぞれの単位工程により構成される。
一つの態様として、本発明のペプチドの製造は、以下の工程(1)乃至(5)として記載される単位工程を、すべてまたは適宜組み合わせることで行うことができる。
なお、本具体的な説明は以下に基づき説明される。
(a)工程(1)乃至(5)の記載におけるR1およびR2は、上記と同義である。
(b)反応の具体的な条件は、本発明のペプチドの製造が達成される限りにおいて特に制限されない。各反応における好ましい条件は適宜詳述される。
(c)各反応で記載される溶媒は、単独で用いても、2種類以上を混合して用いても良い。
本工程は、N末端保護アミノ酸またはN末端保護ペプチドをカルボン酸ハロゲン化物またはハロゲン化ギ酸アルキルと混合する工程である。本工程は、N末端保護アミノ酸またはN末端保護ペプチドのC末端をカルボン酸ハロゲン化物またはハロゲン化ギ酸アルキルで活性化する工程である。本発明の一態様では、式(I)P-A1-OH(式中、PはN末端保護基であり、A1は、アミノ酸由来の基、N-C1-6アルキルアミノ酸由来の基(C1-6アルキルは置換基を有していてもよい)またはペプチド由来の基を表す。)で表されるN末端保護アミノ酸またはN末端保護ペプチドを、カルボン酸ハロゲン化物またはハロゲン化ギ酸アルキルと混合する工程である。また本発明の別の一態様では、式(V)P-A3-OH(式中、PはN末端保護基であり、A3は、ペプチド由来の基を表す。)で表されるN末端保護ペプチドを、カルボン酸ハロゲン化物と混合する工程である。
(式中、Xはハロゲン原子を表し、R1は炭素数が5以上であり、置換基を有していてもよい2級または3級の脂肪族炭化水素基を表すか、または炭素数が4以上であり、置換基を有している1級の脂肪族炭化水素基(ここで、1級の脂肪族炭化水素基の置換基は、カルボニル炭素に結合する炭素原子上に存在する)を表す。)
本工程は、N-アルキルアミノ酸またはN末端にN-アルキルアミノ酸を有するペプチドをシリル化剤と混合する工程である。なお、本工程において、「N-アルキルアミノ酸」は、N末端のアミノ基およびC末端のカルボキシ基が保護されていないN-アルキルアミノ酸を意味し、「N末端にN-アルキルアミノ酸を有するペプチド」は、N末端にN-アルキルアミノ酸を有しN末端のアミノ基およびC末端のカルボキシ基が保護されていないペプチドを意味する。本工程は、N-アルキルアミノ酸またはN末端にN-アルキルアミノ酸を有するペプチドをシリル化剤と反応させることにより、当該アミノ酸またはペプチドのC末端、N末端および/または(存在する場合には)ヒドロキシ基等の官能基の少なくとも一部が、トリアルキルシリル化されたN-アルキルアミノ酸またはN末端にN-アルキルアミノ酸を有するペプチド(以下、「トリアルキルシリル化されたアミノ酸またはペプチド」ともいう)を得る工程である。本発明の一態様では、式(IV)H-A2-OH[式中、A2は、N-C1-6アルキルアミノ酸由来の基(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸由来の基(4-6員環は、C6-14アリール環、C6-14ハロアリール環およびC3-8シクロアルキル環からなる群から選ばれる環状化合物と縮合していてもよい)、あるいはN末端残基がN-C1-6アルキルアミノ酸(C1-6アルキルは置換基を有していてもよい)または4-6員の環状の2級アミノ酸(4-6員環は、C6-14アリール環、C6-14ハロアリール環およびC3-8シクロアルキル環からなる群から選ばれる環状化合物と縮合していてもよい)であるペプチド由来の基を表す]で表されるN末端のアミド基およびC末端のカルボキシ基が保護されていないアミノ酸またはN末端のアミド基およびC末端のカルボキシ基が保護されていないペプチドをシリル化剤と混合する工程である。また本発明の別の一態様では、式(IV')H-A2’-OH[式中、A2’は、N-メチルアミノ酸由来の基、N-C1-6アルキルグリシン由来の基(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸由来の基を表す]で表されるN末端のアミノ基およびC末端のカルボキシ基が保護されていないアミノ酸をシリル化剤と混合する工程である。
本工程は、工程(1)で得られた生成物と工程(2)で得られた生成物とを混合する工程である。本工程は、工程(1)で得られたC末端が活性化されたN末端保護アミノ酸またはN末端保護ペプチドと、工程(2)で得られたトリアルキルシリル化されたアミノ酸またはペプチドを反応させるペプチド伸長工程であり、好ましくは、工程(1)で得られた反応液と、工程(2)で得られた反応液とを混合、撹拌することにより実施することができる。本発明の一態様では、工程(1)で得られたC末端が活性化されたN末端保護アミノ酸と、工程(2)で得られたトリアルキルシリル化されたアミノ酸またはペプチドを反応させるペプチド伸長工程である。本発明の別の一態様では、工程(1)で得られたC末端が活性化されたN末端保護ペプチドと、工程(2)で得られたトリアルキルシリル化されたアミノ酸を反応させるペプチド伸長工程である。
(4)工程(3)または(5)で得られたペプチドのN末端の保護基を除去する工程。
(5)工程(4)で得られたペプチドのN末端に、N末端保護アミノ酸またはN末端保護ペプチドを反応させる工程。
工程(5)は、上記工程(1)、(2)および(3)と同様の操作、または一般的なペプチド合成反応により実施することができる。
本工程は、上記工程(3)または(5)で得られたペプチドから、N末端の保護基を除去し、N末端及びC末端が無保護のペプチドを得る工程である。
また、工程(2)で使用されるペプチドにおけるN末端アミノ酸は、α-アミノ酸が好ましい。
<分析条件1>
高速液体クロマトグラフィー:Waters製 ACQUITY UPLD H-Class/SQD2
カラム:Phenomenex 社製 Kinetex EVO C18 (1.7μm, 2.1 x 50 mm)
カラムオーブン温度:60℃
溶離液:アセトニトリル:0.025 vol%トリフルオロ酢酸水溶液
5:95(0-2.1分)、95:5(2.1-2.84分)(v/v)
溶離液速度:0.6 mL/分
検出波長:220 nm
<分析条件2>
高速液体クロマトグラフィー:Waters製 ACQUITY UPLD H-Class/SQD2
カラム:Waters社製 ACQUITY BEH C18 (1.7μm, 2.1 x 100 mm)
カラムオーブン温度:60℃
溶離液:アセトニトリル:0.025 vol% トリフルオロ酢酸水溶液
5:95(0-3.7分)、95:5(3.7-4.81分)(v/v)
溶離液速度:0.6 mL/分
検出波長:220 nm
<分析条件3>
高速液体クロマトグラフィー:Waters製 ACQUITY UPLD H-Class/SQD2
カラム:Waters社製 ACQUITY BEH C18 (1.7μm, 2.1 x 100 mm)
カラムオーブン温度:40℃
溶離液:アセトニトリル:0.025 vol% トリフルオロ酢酸水溶液
5:95(0-5.56分)、95:5(5.56-7.22分)(v/v)
溶離液速度:0.4 mL/分
検出波長:220 nm
δppm:0.88-0.94(9H+4H, m), 1.06-1.16(1H, m),1.27(1H,d,J=6.0Hz),1.29-1.38(2H,m),1.40-1.48(1H,m),1.52-1.58(1H,m),3.24(1H,d,J=6.0Hz).
δppm:0.90-0.94(6H,m),0.97(9H,s),1.10-1.20(1H,m),1.25-1.35(1H,m),1.45-1.53(2H,m),1.59-1.67(1H,m),4.72(1H,d,J=2.0Hz).
δppm:0.91(3H,d,J=7.0Hz),0.94(9H,s),1.01 (3H,d,J=7.0Hz),1.32(1H,d,J=6.5Hz),1.91-1.99(1H,m),3.11(1H,dd,J=6.5Hz,2.0Hz).
δppm:0.97(3H,d,J=7.0Hz),0.99(9H,s),1.02 (3H,d,J=7.0Hz),2.04-2.13(1H,m),4.58(1H,d,J=3.0Hz).
δppm:0.96(9H,s),1.29(3H,d,J=6.5Hz),4.74 (1H,q,J=6.5Hz).
δppm:0.94(6H,d,J=2.5Hz),0.95(6H,d,J=2.5Hz),1.94-2.04(2H,m)4.60(1H,t,J=6.0Hz).
3-メチルブタン-2-オール(3.97g、45.0mmol)、ピリジン(4.09 g、51.8mmol)を四塩化炭素(40mL)と混合させ、0℃に冷却した。この溶液に、別途トリホスゲン(5.47g、18.5mmol)、四塩化炭素(20mL)を混合した溶液を加え、さらに60℃に加熱して8時間撹拌した。得られた反応液を水(50mL)で二回、飽和塩化ナトリウム水溶液(50mL)で順次洗浄した。得られた有機層を濃縮し、3-メチルブタン-2-イルカルボノクロリデート(5.10g、収率95%)を無色透明液体として得た。本化合物はこれ以上精製せずに次工程で用いた。
δppm:0.95(3H,d,J=4.0Hz),0.97(3H,d,J=4.0Hz),1.31(3H,d,J=6.5Hz),1.87-1.94(1H,m),4.80(1H,sept,J=6.5Hz).
MASS(ESI+)m/z;(M+H)+427.3
MASS(ESI+)m/z;(M+H)+549.3
MASS(ESI+)m/z;(M+H)+549.3
MASS(ESI+)m/z;(M+H)+549.4
MASS(ESI+)m/z;(M+H)+549.4
MASS(ESI+)m/z;(M+H)+441.4
MASS(ESI+)m/z;(M+H)+427.3
MASS(ESI+)m/z;(M+H)+549.4
MASS(ESI+)m/z;(M+H)+327.3
MASS(ESI+)m/z;(M+H)+327.3
Boc-MePhe-OH(0.280g、1.00mmol)、N-メチルモルホリン(0.112g、1.10mmol)をテトラヒドロフラン(10mL)と混合させ、0℃にて2,2,4-トリメチルペンタン-3-イルカルボノクロリデート(0.203g、1.05mmol)を加え1時間撹拌した。
Boc-MePhe-Phe-OH(0.449g、1.05mmol)を4M-HCl/シクロペンチルメチルエーテル(5mL)と混合させ、25℃にて30分撹拌した。得られた反応液を濃縮し、酢酸エチル(20mL)を加えて濃縮し、N,N-ジイソプロピルエチルアミン(1.30g、10.0mmol)とアセトニトリル(8mL)を加えて濃縮した。得られた残渣にアセトニトリル(10mL)、N,O-ビス(トリメチルシリル)アセトアミド(0.644g、3.11mmol)を混合させ、25℃にて20分間撹拌して無色透明溶液を得た。
溶液Aを0℃に冷却したまま溶液Bを混合し、さらに25℃のまま18時間撹拌した(原料:目的物=1:21(分析条件3))。得られた反応液を酢酸エチル(40mL)で希釈し、10質量%クエン酸水溶液、飽和塩化ナトリウム水溶液で順次洗浄した。得られた有機層を濃縮後、シリカゲルカラムクロマトグラフィーで精製し、Boc-Phe-MePhe-MePhe-MePhe-OH(0.532g、収率90%)を白色固体として得た。MASS(ESI+)m/z;(M+H)+588.4
Boc-Phe-OH(0.177g、0.669mmol)、N-メチルモルホリン(0.744g、0.736mmol)をテトラヒドロフラン(10mL)と混合させ、0℃にて2,2,4-トリメチルペンタン-3-イルカルボノクロリデート(0.135g、0.702mmol)を加え1時間撹拌した。
Boc-MePhe-MePhe-Phe-OH(0.413g、0.702mmol)を4M-HCl/シクロペンチルメチルエーテル(10mL)と混合させ、25℃にて1時間撹拌した。得られた反応液を濃縮し、酢酸エチル(20mL)を加えて濃縮し、N,N-ジイソプロピルエチルアミン(0.865g、6.69mmol)とアセトニトリル(8mL)を加えて濃縮した。得られた残渣にアセトニトリル(10mL)、N,O-ビス(トリメチルシリル)アセトアミド(0.430g、2.07mmol)を混合させ、25℃にて20分間撹拌して無色透明溶液を得た。
溶液Aを0℃に冷却したまま溶液Bを混合し、さらに25℃のまま18時間撹拌した(原料:目的物=0:100(分析条件3))。得られた反応液を酢酸エチル(20mL)で希釈し、10質量%クエン酸水溶液(20mL)、10質量%塩化ナトリウム水溶液(20mL)、飽和塩化ナトリウム水溶液(20mL)で順次洗浄した。得られた有機層を濃縮後、シリカゲルカラムクロマトグラフィーで精製し、Boc-Phe-MePhe-MePhe-Phe-OH(0.453g、収率92%)を白色固体として得た。
MASS(ESI+)m/z;(M+H)+735.5
Boc-MePhe-OH(0.324g、1.16mmol)、トリエチルアミン(0.141g、1.39mmol)をテトラヒドロフラン(10mL)と混合させ、0℃にて2,2-ジメチルブタノイルクロリド(0.172g、1.28mmol)を加え1時間撹拌した。
Boc-MePhe-Phe-OH(0.544g、1.28mmol)を4M-HCl/シクロペンチルメチルエーテル(20mL)と混合させ、25℃にて1時間撹拌した。得られた反応液を濃縮し、シクロペンチルメチルエーテル(20mL)を加えて濃縮し、N,N-ジイソプロピルエチルアミン(1.50g、11.6mmol)とアセトニトリル(8mL)を加えて濃縮した。得られた残渣にアセトニトリル(10mL)、N,O-ビス(トリメチルシリル)アセトアミド(0.621g、2.99mmol)を混合させ、25℃にて20分間撹拌して無色透明溶液を得た。
溶液Aを0℃に冷却したまま溶液Bを混合し、さらに25℃のまま48時間撹拌した(原料:目的物=1:37(分析条件3))。得られた反応液を酢酸エチル(40mL)で希釈し、10質量%クエン酸水溶液、飽和塩化ナトリウム水溶液で順次洗浄した。得られた有機層を濃縮後、シリカゲルカラムクロマトグラフィーで精製し、Boc-MePhe-MePhe-Phe-OH(0.701g、収率103%)を褐色固体として得た。
MASS(ESI+)m/z;(M+H)+588.4
MASS(ESI+)m/z;(M+H)+377.3
Boc-MePhe-OH(0.838g、3.00mmol)、トリエチルアミン(0.364g、3.60mmol)をテトラヒドロフラン(60mL)と混合させ、0℃にて2,2-ジメチルブタノイルクロリド(0.444g、3.30mmol)を加え30分撹拌した。
Boc-MePhe-Pro-OH(1.24g、3.30mmol)を4M-HCl/シクロペンチルメチルエーテル(20mL)と混合させ、25℃にて1時間撹拌した。得られた反応液を濃縮し、アセトニトリル(20mL)を加えて濃縮し、N,N-ジイソプロピルエチルアミン(0.865g、6.69mmol)とアセトニトリル(20mL)を加えて濃縮した。得られた残渣にアセトニトリル(20mL)、N,O-ビス(トリメチルシリル)アセトアミド(2.31g、11.1mmol)を混合させ、25℃にて20分間撹拌して無色透明溶液を得た。
溶液Aを0℃に冷却したまま溶液Bを混合し、さらに25℃のまま48時間撹拌した(原料:目的物=1:24(分析条件3))。得られた反応液を酢酸エチル(40mL)で希釈し、10質量%クエン酸水溶液、飽和塩化ナトリウム水溶液で順次洗浄した。得られた有機層を濃縮後、シリカゲルカラムクロマトグラフィーで精製し、Boc-MePhe-MePhe-Pro-OH(1.71g、収率90%)を白色固体として得た。
MASS(ESI+)m/z;(M+H)+538.5
Boc-Tyr-OH(0.282g、1.00mmol)、トリエチルアミン(0.122g、1.20mmol)をテトラヒドロフラン(10mL)と混合させ、0℃にて2,2-ジメチルブタノイルクロリド(0.148g、1.10mmol)を加え1時間撹拌した。
H-MePhe-MePhe-Pro-OH・HCl(0.482g、1.02mmol)をN,N-ジイソプロピルエチルアミン(2mL)と混合させ、25℃にて2分撹拌した。得られた反応液を濃縮した。得られた残渣にアセトニトリル(6mL)、N,O-ビス(トリメチルシリル)アセトアミド(0.773g、3.74mmol)を混合させ、25℃にて20分間撹拌して無色透明溶液を得た。
溶液Aを0℃に冷却したまま溶液Bを混合し、さらに25℃のまま1時間撹拌した。得られた反応液を酢酸エチル(40mL)で希釈し、10質量%クエン酸水溶液、飽和塩化ナトリウム水溶液で順次洗浄した。得られた有機層を濃縮後、シリカゲルカラムクロマトグラフィーで精製し、Boc-Tyr-MePhe-MePhe-Pro-OH(0.698g、収率99%)を白色固体として得た。
MASS(ESI+)m/z;(M+H)+701.5
Boc-MePhe-OH(0.894g、3.20mmol)、トリエチルアミン(0.389g、3.84mmol)をテトラヒドロフラン(25mL)と混合させ、0℃にて2,2-ジメチルブタノイルクロリド(0.474g、3.52mmol)を加え45分撹拌した。
H-MeAla-Tyr-OH(0.937g、3.52mmol)にアセトニトリル(10mL)、N,O-ビス(トリメチルシリル)アセトアミド(2.28g、10.9mmol)を混合させ、25℃にて15分間撹拌して無色透明溶液を得た。
溶液Aを0℃に冷却したまま溶液Bを混合し、さらに25℃のまま2時間撹拌した(原料:目的物=0:100(分析条件1))。得られた反応液を濃縮後、酢酸エチル(30mL)で希釈し、飽和炭酸水素ナトリウム水溶液(20mL)、水(20mL)、飽和塩化ナトリウム水溶液(10mL)で順次洗浄した。得られた有機層を10質量%クエン酸水溶液(20mL)、飽和塩化ナトリウム水溶液で順次洗浄した。得られた有機層を濃縮後、シリカゲルカラムクロマトグラフィーで精製し、Boc-MePhe-MeAla-Tyr-OH(1.67g、収率94%)を白色固体として得た。
MASS(ESI+)m/z;(M+H)+528.3
MASS(ESI+)m/z;(M+H)+428.8
MASS(ESI+)m/z;(M+H)+675.5
MASS(ESI+)m/z;(M+H)+575.4
MASS(ESI+)m/z;(M+H)+836.5
MASS(ESI+)m/z;(M+H)+385.3
MASS(ESI+)m/z;(M+H)+251.2
MASS(ESI+)m/z;(M+H)+512.3
MASS(ESI+)m/z;(M+H)+411.5
MASS(ESI+)m/z;(M+H)+920.5
Fmoc-Gln(Trt)-OH(0.204g、0.333mmol)、トリエチルアミン(0.040g、0.40mmol)をテトラヒドロフラン(20mL)と混合させ、0℃にて2,2-ジメチルブタノイルクロリド(0.049g、0.37 mmol)を加え45分間撹拌した。
H-MePhe-MeAla-Tyr-OH(0.171g、0.400mmol)、N,N-ジイソプロピルエチルアミン(0.431g、3.33mmol)、アセトニトリル(20mL)を混合させ、溶液を濃縮して溶媒を除去した。得られた残渣にアセトニトリル(10mL)、N,O-ビス(トリメチルシリル)アセトアミド(0.343g、1.65mmol)を混合させ、25℃にて40分間撹拌して無色透明溶液を得た。
溶液Aを0℃に冷却したまま溶液Bを混合し、さらに25℃のまま23時間撹拌した(原料:目的物=1:5(分析条件1))。得られた反応液を濃縮後、酢酸エチル(20mL)で希釈し、飽和炭酸水素ナトリウム水溶液(15mL)、水(15mL)、飽和塩化ナトリウム水溶液(7.5mL)で2回ずつ順次洗浄した。得られた有機層を10質量%クエン酸水溶液(20mL)、飽和塩化ナトリウム水溶液(20mL)で順次洗浄した。得られた有機層を濃縮し、Fmoc-Gln(Trt)-MePhe-MeAla-Tyr-OH(0.209g、収率62%)を白色固体として得た。
MASS(ESI+)m/z;(M+H)+1020.6
MASS(ESI+)m/z;(M+H)+535.3
MASS(ESI+)m/z;(M+H)+313.2
MASS(ESI+)m/z;(M+H)+560.4
MASS(ESI+)m/z;(M+H)+487.3
MASS(ESI+)m/z;(M+H)+265.2
MASS(ESI+)m/z;(M+H)+512.4
MASS(ESI+)m/z;(M+H)+648.4
MASS(ESI+)m/z;(M+H)+689.5
Fmoc-Glu(tBu)-OH(0.091g、0.21mmol)、トリエチルアミン(0.026g、0.257mmol)をテトラヒドロフラン(5.0mL)と混合させ、0℃にて2,2-ジメチルブタノイルクロリド(0.032g、0.24mmol)を加え1時間撹拌した。
Boc-MePhe-MePhe-MeAla-Tyr-OH(0.177g、0.257mmol)、トリフルオロ酢酸(3.0mL)を混合させ、さらに25℃のまま30分間撹拌した。溶液を濃縮してトリフルオロ酢酸を除去し、アセトニトリル(20mL)を混合させ濃縮した。得られた残渣にアセトニトリル(8.0mL)、トリエチルアミン(0.026g、0.257mmol)を加え、溶液を濃縮して溶媒とトリエチルアミンを除去した。得られた残渣にアセトニトリル(5.0mL)、N,O-ビス(トリメチルシリル)アセトアミド(0.167g、0.80mmol)を混合させ、25℃にて5分間撹拌して無色透明溶液を得た。
溶液Aを0℃に冷却したまま溶液Bを混合し、さらに25℃のまま17時間撹拌した(原料:目的物=1:5(分析条件3))。得られた反応液を濃縮後、酢酸エチル(30mL)で希釈し、飽和炭酸水素ナトリウム水溶液(20mL)、水(20mL)、飽和塩化ナトリウム水溶液(10mL)で順次洗浄した。得られた有機層を濃縮し、Fmoc-Glu(tBu)-MePhe-MePhe-MeAla-Tyr-OH(0.192g、収率90%)を白色固体として得た。
MASS(ESI+)m/z;(M+H)+730.4,+996.5
MASS(ESI+)m/z;(M+H)+243.2(トリチルカチオン)、+747.2
MASS(ESI+)m/z;(M+H)+704.5
MASS(ESI+)m/z;(M+H)+775.5
MASS(ESI+)m/z;(M+H)+539.6,+639.4
Fmoc-MeHis(Trt)-OH(1.27g、2.00mmol)、N,N-ジメチルホルムアミド(0.015g、0.20mmol)をテトラヒドロフラン(30mL)と混合させ、0℃にて塩化チオニル(1.19g、10.0mmol)を加え25℃で1時間撹拌した。溶液を濃縮して塩化チオニルと溶媒を除去し、テトラヒドロフラン(10mL)を混合させ濃縮した。得られた残渣にテトラヒドロフラン(10mL)を加え、淡黄色透明の酸塩化物溶液を得た。
Leu-OH(0.315g、2.40mmol)、N,O-ビス(トリメチルシリル)アセトアミド(1.30g、6.26mmol)、アセトニトリル(5mL)を混合させ、80℃にて1時間撹拌して無色透明溶液を得た。
溶液Aを0℃に冷却したまま溶液Bを混合し、さらに0℃のまま1時間撹拌した(原料:目的物=1:25(分析条件3))。得られた反応液を濃縮後、酢酸エチル(40mL)で希釈し、10質量%クエン酸水溶液、飽和塩化ナトリウム水溶液で順次洗浄した。得られた有機層を濃縮後、シリカゲルカラムクロマトグラフィーで精製し、Fmoc-MeHis(Trt)-Leu-OH(1.37g、収率92%)を淡黄色固体として得た。
MASS(ESI+)m/z;(M+H)+747.4
MASS(ESI+)m/z;(M+H)+525.8
MASS(ESI+)m/z;(M+H)+894.5
高速液体クロマトグラフィー:SHIMADZU製 HPLC LC-20A
カラム:Agilent製Poroshell 120EC-C18(2.7μm、3.0×100 mm)
カラムオーブン温度:40℃
溶離液:アセトニトリル:0.05 vol% リン酸水溶液
50:50(0-15分)、50:50-95:5(15-18分)、95:5(18-22分)(v/v)
溶離液速度:0.7 mL/分
検出波長:210 nm
標準物質:合成例57に記載の方法で得られたFmoc-Val-MePhe-OHをシリカゲルクロマトグラフィーにて精製し標準物質とした。
MASS(ESI+)m/z;(M+H)+501.22
定量方法:絶対検量法
Fmoc-Val-OH(0.100g、0.295mmol)、トリエチルアミン(0.053mL、0.38mmol)をテトラヒドロフラン(1.0g)と混合させ、0℃にてピバロイルクロリド(0.043mL、0.35mmol)を加え1時間撹拌した。この溶液に、別途H-MePhe-OH(0.079g、0.44mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.135mL、0.552mmol)、アセトニトリル(0.81g)を混合させ、50℃にて1.5時間撹拌して調製した溶液を加え、さらに0℃のまま70時間撹拌した(原料:目的物=1:7)。得られた反応液を酢酸エチル(5.0g)で希釈し、10質量%クエン酸水溶液(2.0g)および飽和食塩水溶液(1.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は79%であった。
Fmoc-Val-OH(0.100g、0.295mmol)、N-メチルモルホリン(0.042mL、0.383mmol)をテトラヒドロフラン(1.0g)と混合させ、0℃にてイソブチルカルボノクロリデート(0.046mL、0.35mmol)を加え5分間撹拌した。この溶液に、別途H-MePhe-OH(0.079g、0.44mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.135mL、0.552mmol)、アセトニトリル(0.79g)を混合させ、50℃にて1時間撹拌して調製した溶液を加え、さらに0℃のまま21時間撹拌した(原料:目的物=3.5:1)。得られた反応液を酢酸エチル(5.0g)で希釈し、10質量%クエン酸水溶液(2.0g)および飽和食塩水溶液(1.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は18%であった。
Fmoc-Val-OH(0.100g、0.295mmol)、トリエチルアミン(0.053mL、0.38mmol)をテトラヒドロフラン(1.0g)と混合させ、0℃にて2-エチルブタノイルクロリド(0.048mL、0.35mmol)を加え1時間撹拌した。この溶液に、別途H-MePhe-OH(0.079g、0.44mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.135mL、0.552mmol)、アセトニトリル(0.79g)を混合させ、50℃にて1時間撹拌して調製した溶液を加え、さらに0℃のまま110時間撹拌した(原料:目的物=1:13)。得られた反応液を酢酸エチル(5.0g)で希釈し、10質量%クエン酸水溶液(2.0g)および飽和食塩水溶液(1.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は87%であった。
Fmoc-Val-OH(0.100g、0.295mmol)、トリエチルアミン(0.053mL、0.38mmol)をテトラヒドロフラン(1.0g)と混合させ、0℃にて2,2-ジメチルブタノイルクロリド(0.049mL、0.35mmol)を加え1時間撹拌した。この溶液に、別途H-MePhe-OH(0.079g、0.44mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.135mL、0.552mmol)、アセトニトリル(0.79g)を混合させ、50℃にて1時間撹拌して調製した溶液を加え、さらに0℃のまま95時間撹拌した(原料:目的物=1:18)。得られた反応液を酢酸エチル(5.0g)で希釈し、10質量%クエン酸水溶液(2.0g)および飽和食塩水溶液(1.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は89%であった。
Fmoc-Val-OH(0.100g、0.295mmol)、N,N-ジイソプロピルエチルアミン(0.058mL、0.32mmol)をアセトニトリル(1.0g)と混合させ、0℃にて2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリドの50質量%トルエン溶液(0.107g、0.354mmol)を加え2時間撹拌した。この溶液に、別途H-MePhe-OH(0.079g、0.442mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.135mL、0.552mmol)、アセトニトリル(0.79g)を混合させ、50℃にて1時間撹拌して調製した溶液を加え、さらに0℃のまま67時間、20℃で7時間撹拌した(原料:目的物=1:35)。得られた反応液をメタノール(0.5mL)、N,N-ジイソプロピルエチルアミン(0.05mL)でクエンチ後、酢酸エチル(5.0g)で希釈し、10質量%クエン酸水溶液(2.0g)および飽和食塩水溶液(1.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は96%であった。
Fmoc-Val-OH(0.100g、0.295mmol)、N-メチルモルホリン(0.071mL、0.648mmol)をN,N-ジメチルアセトアミド(5.9mL)と混合させ、0℃にて2,4-ジメチルペンタン-3-イルカルボノクロリデート(0.105g、0.589mmol)を加え2時間撹拌した。この溶液に、別途H-MePhe-OH(0.106g、0.589mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.288mL、1.179mmol)、アセトニトリル(7.9mL)を混合させ、75℃にて15分間撹拌して調製した溶液を加え、さらに0℃のまま114時間、20℃で4時間撹拌した(原料:目的物=1:11)。得られた反応液をメタノール(5.0mL)、N,N-ジイソプロピルエチルアミン(0.50mL)でクエンチ後、酢酸エチル(30.0g)で希釈し、10質量%クエン酸水溶液(18.0g)および飽和食塩水溶液(6.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は85%であった。
Fmoc-Val-OH(0.100g、0.295mmol)、N-メチルモルホリン(0.071mL、0.648mmol)をN,N-ジメチルアセトアミド(5.9mL)と混合させ、0℃にて2,2,4-トリチルペンタン-3-イルカルボノクロリデート(0.114g、0.589mmol)を加え2時間撹拌した。この溶液に、別途H-MePhe-OH(0.106g、0.589mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.288mL、1.179mmol)、アセトニトリル(7.9mL)を混合させ、75℃にて15分間撹拌して調製した溶液を加え、さらに0℃のまま91時間、20℃で7時間撹拌した(原料:目的物=1:10)。得られた反応液をメタノール(5.0mL)、N,N-ジイソプロピルエチルアミン(0.50mL)でクエンチ後、酢酸エチル(30.0g)で希釈し、10質量%クエン酸水溶液(18.0g)および飽和食塩水溶液(6.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は88%であった。
Fmoc-Val-OH(0.100g、0.295mmol)、N,N-ジイソプロピルエチルアミン(0.065mL、0.38mmol)をテトラヒドロフラン(1.0g)と混合させ、0℃にて1-アダマンタンカルボニルクロリド(0.070g、0.35mmol)を加え1時間撹拌した。この溶液に、別途H-MePhe-OH(0.079g、0.44mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.135mL、0.552mmol)、アセトニトリル(0.79g)を混合させ、50℃にて1時間撹拌して調製した溶液を加え、さらに0℃のまま96時間撹拌した(原料:目的物=1:10)。得られた反応液を酢酸エチル(5.0g)で希釈し、10質量%クエン酸水溶液(2.0g)および飽和食塩水溶液(1.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は90%であった。
Fmoc-Val-OH(0.10g、0.30mmol)、N-メチルモルホリン(0.071mL、0.65mmol)をN,N-ジメチルアセトアミド(5.9mL)と混合させ、0℃にて4-エチル-2,2-ジメチルヘキサン-3-イルカルボノクロリデート(0.13g、0.59mmol)を加え2時間撹拌した。この溶液に、別途H-MePhe-OH(0.106g、0.59mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.29mL、1.18mmol)、アセトニトリル(7.9mL)を混合させ、75℃にて15分間撹拌して調製した溶液を加え、さらに0℃のまま164時間撹拌した(原料:目的物=1:9)。得られた反応液をメタノール(5.0mL)、N,N-ジイソプロピルエチルアミン(0.50mL)でクエンチ後、酢酸エチル(30.0g)で希釈し、10質量%クエン酸水溶液(18.0g)、飽和食塩水溶液(10.0g)および飽和食塩水溶液(6.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は83%であった。
Fmoc-Val-OH(0.100g、0.295mmol)、トリエチルアミン(0.053mL、0.383mmol)をテトラヒドロフラン(1.0g)と混合させ、0℃にて2-エチルヘキサノイルクロリド(0.061mL、0.354mmol)を加え1時間撹拌した。この溶液に、別途H-MePhe-OH(0.079g、0.442mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.135mL、0.552mmol)、アセトニトリル(0.79g)を混合させ、50℃にて1時間撹拌して調製した溶液を加え、さらに0℃のまま111時間撹拌した(原料:目的物=1:11)。得られた反応液を酢酸エチル(5.0g)で希釈し、10質量%クエン酸水溶液(2.0g)および飽和食塩水溶液(1.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は90%であった。
Fmoc-Val-OH(0.100g、0.295mmol)、トリエチルアミン(0.053mL、0.383mmol)をテトラヒドロフラン(1.0g)と混合させ、0℃にて2-(4-クロロフェニル)-3-メチルブタノイルクロリド(0.070mL、0.354mmol)を加え1時間撹拌した。この溶液に、別途H-MePhe-OH(0.079g、0.442mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.135mL、0.552mmol)、アセトニトリル(0.79g)を混合させ、50℃にて1時間撹拌して調製した溶液を加え、さらに0℃のまま66時間撹拌した(原料:目的物=1:10)。得られた反応液を酢酸エチル(5.0g)で希釈し、10質量%クエン酸水溶液(2.0g)および飽和食塩水溶液(1.0g)で2回洗浄した。集めた有機層のFmoc-Val-MePhe-OHの定量収率は84%であった。
MASS(ESI+)m/z;(M+H)+608.44
高速液体クロマトグラフィー:SHIMADZU製 HPLC -20A
カラム:Agilent製Poroshell 120EC-C18(2.7 μm、3.0×100 mm)
カラムオ-ブン温度:50℃
溶離液:0.2vоl%リン酸 アセトニトリル溶液:0.2vоl% リン酸水溶液
12:88-95:5(0-15分)、95:5(15-19分)、(v/v)
溶離液速度:0.7 mL/分
検出波長:214 nm
標準物質:合成例71に記載の方法にて合成したCbz-Phe-MePhe-Phe-OHをシリカゲルクロマトグラフィーにて精製し、標準物質とした。
標準物質のNMRおよびMASSを示す。
1H NMR(300MHz,DMSO-d6):
δppm:2.64(3H,s),2.67-3.33(6H,m),4.27-4.45(3H,m),4.89(2H,s),5.21(1H,m),7.04-7.33(20H,m)
MASS(ESI+)m/z;(M+H)+608.44
定量方法:絶対検量法
MASS(ESI+)m/z;(M+H)+327.2
Cbz-Phe-OH(50mg、0.17mmol)、テトラヒドロフラン(0.5g)、N-メチルモルホリン(20mg、0.20mmol)を混合させ、0℃にてイソブチルカルボノクロリデート(27mg、0.20mmol)を加え3時間攪拌した。この溶液に、別途H-MePhe-Phe-OH(82mg、0.25mmol)、N,O-ビス(トリメチルシリル)アセトアミド(97mg、0.48mmol)、アセトニトリル(0.82g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃に昇温し3時間攪拌した(原料:目的物=1:1.6)。得られた反応液を酢酸エチル(2.5g)で希釈し、10質量%クエン酸水溶液(1.0g)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層のCbz-Phe-MePhe-Phe-OHの定量収率は43%であった。
Cbz-Phe-OH(50mg、0.17mmol)、テトラヒドロフラン(0.5g)、トリエチルアミン(20mg、0.20mmol)を混合させ、0℃にて2,2-ジメチルブタノイルクロリド(27mg、0.20mmol)を加え2時間攪拌した。この溶液に、別途H-MePhe-Phe-OH(82mg、0.25mmol)、N,O-ビス(トリメチルシリル)アセトアミド(97mg、0.48mmol)、アセトニトリル(0.82g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃に昇温し21時間攪拌した(原料:目的物=1:24)。得られた反応液を酢酸エチル(2.5g)で希釈し、10質量%クエン酸水溶液(1.0g)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層のCbz-Phe-MePhe-Phe-OHの定量収率は95%であった。
Cbz-Phe-OH(50mg、0.17mmol)、テトラヒドロフラン(0.5g)、トリエチルアミン(20mg、0.20mmol)を混合させ、0℃にて2-エチルブタノイルクロリド(27mg、0.20mmol)を加え2時間攪拌した。この溶液に、別途H-MePhe-Phe-OH(82mg、0.25mmol)、N,O-ビス(トリメチルシリル)アセトアミド(97mg、0.48mmol)、アセトニトリル(0.82g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃に昇温し27時間攪拌した(原料:目的物=1:30)。得られた反応液を酢酸エチル(2.5g)で希釈し、10質量%クエン酸水溶液(1.0g)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層のCbz-Phe-MePhe-Phe-OHの定量収率は102%であった。
Cbz-Phe-OH(50mg、0.17mmol)、テトラヒドロフラン(0.5g)、トリエチルアミン(20mg、0.20mmol)を混合させ、25℃にて2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリドの50質量%トルエン溶液(124mg、0.20mmol)を加え9時間攪拌した。この溶液に、別途H-MePhe-Phe-OH(82mg、0.25mmol)、N,O-ビス(トリメチルシリル)アセトアミド(97mg、0.48mmol)、アセトニトリル(0.82g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃に昇温し14時間攪拌した(原料:目的物=1:80)。得られた反応液を酢酸エチル(2.5g)で希釈し、10質量%クエン酸水溶液(1.0g)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層のCbz-Phe-MePhe-Phe-OHの定量収率は100%であった。
Cbz-Phe-OH(99mg、0.33mmol)、アセトニトリル(1.0g)、N,N-ジイソプロピルエチルアミン(52mg、0.40mmol)を混合させ、室温にて2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリドの50質量%トルエン溶液(250mg、0.41mmol)を加え2時間攪拌した。この溶液に、別途H-MePhe-Phe-OH(164mg、0.50mmol)、N,O-ビス(トリメチルシリル)アセトアミド(195mg、0.96mmol)、アセトニトリル(1.6g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃で21時間攪拌した(原料:目的物=1:335)。得られた反応液を酢酸エチル(5.0g)で希釈し、10質量%クエン酸水溶液(3.0g)で分液後、飽和塩化ナトリウム水溶液(2.0g)で2回洗浄した。得られた有機層のCbz-Phe-MePhe-Phe-OHの定量収率は100%であった。
Cbz-Phe-OH(50mg、0.17mmol)、N,N-ジメチルアセトアミド(0.5 g)、N-メチルモルホリン(22 mg、0.22 mmol)を混合させ、0℃にて2,2,4-トリメチルペンタン-3-イルカルボノクロリデート(39mg、0.20mmol)を加え2時間攪拌した。この溶液に、別途H-MePhe-Phe-OH(82mg、0.25mmol)、N,O-ビス(トリメチルシリル)アセトアミド(97mg、0.48mmol)、アセトニトリル(0.82g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃に昇温し3時間攪拌した(原料:目的物=1:32)。得られた反応液を酢酸エチル(2.5g)で希釈し、10質量%クエン酸水溶液(1.0g)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層のCbz-Phe-MePhe-Phe-OHの定量収率は99%であった。
Cbz-Phe-OH(50mg、0.17mmol)、N,N-ジメチルアセトアミド(0.5g)、N-メチルモルホリン(22mg、0.22mmol)を混合させ、0℃にて2,4-ジメチルペンタン-3-イルカルボノクロリデート(39mg、0.20mmol)を加え1.5時間攪拌した。この溶液に、別途H-MePhe-Phe-OH(82mg、0.25mmol)、N,O-ビス(トリメチルシリル)アセトアミド(97mg、0.48mmol)、アセトニトリル(0.82g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃に昇温し29時間攪拌した(原料:目的物=1:15)。得られた反応液を酢酸エチル(2.5g)で希釈し、10質量%クエン酸水溶液(1.0g)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層のCbz-Phe-MePhe-Phe-OHの定量収率は83%であった。
Cbz-Phe-OH(50mg、0.17mmol)、テトラヒドロフラン(0.5g)、N,N-ジイソプロピルエチルアミン(30mg、0.23mmol)を混合させ、0℃にて1-アダマンタンカルボニルクロリド(51mg、0.26mmol)を加え1時間攪拌した。この溶液に、別途H-MePhe-Phe-OH(110mg、1.0mmol)、N,O-ビス(トリメチルシリル)アセトアミド(129mg、0.63mmol)、アセトニトリル(1.1g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、0℃で28時間攪拌した(原料:目的物=1:58)。得られた反応液を酢酸エチル(3.0g)で希釈し、10質量%クエン酸水溶液(3.0g)で分液後、飽和塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層を定量し、定量収率98%でCbz-Phe-MePhe-Phe-OHを得た。
Cbz-Phe-OH(0.050g、0.17mmol)、N,N-ジメチルアセトアミド(3.0mL)、N-メチルモルホリン(0.040mL、0.37mmol)を混合させ、0℃にて4-エチル-2,2-ジメチルヘキサン-3-イルカルボノクロリデート(0.074g、0.33mmol)を加え1時間半攪拌した。この溶液に、別途H-MePhe-Phe-OH(0.109g、0.334mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.16mL、0.67mmol)、アセトニトリル(4.0mL)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、室温に昇温し21時間攪拌した(原料:目的物=1:43)。得られた反応液を酢酸エチル(15g)で希釈し、10質量%クエン酸水溶液(9.0g)および飽和食塩水溶液(5.0g)で分液後、飽和食塩水溶液(3.0g)で2回洗浄した。得られた有機層のCbz-Phe-MePhe-Phe-OHの定量収率は92%であった。
Cbz-Phe-OH(50mg、0.17mmol)、N,N-ジメチルアセトアミド(0.5g)、N-メチルモルホリン(22mg、0.22mmol)を混合させ、0℃にて3,3-ジメチルブタン-2-イルカルボノクロリデート(39mg、0.20mmol)を加え1.5時間攪拌した。この溶液に、別途H-MePhe-Phe-OH(82mg、0.25mmol)、N,O-ビス(トリメチルシリル)アセトアミド(97mg、0.48mmol)、アセトニトリル(0.82g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃に昇温し16時間攪拌した(原料:目的物=1:1.2)。
MASS(ESI+)m/z;(M+H)+315.20
Boc-Val-OH(128mg、0.59mmol)、アセトニトリル(1.3g)、N,N-ジイソプロピルエチルアミン(91mg、0.71mmol)を混合させ、25℃にて2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリドの50質量%トルエン溶液(0.43g、0.71mol)を加え、4時間攪拌した。この溶液に、別途H-Pro-OH(71mg、0.62mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.22g、1.1mmol)、アセトニトリル(1.3g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃のまま15時間攪拌した(原料:目的物=1:731)。得られた反応液を酢酸エチル(10mL)で希釈し、水(2mL)、10質量%クエン酸水溶液(2mL)で分液後、5質量%塩化ナトリウム水溶液(5mL)、水(5mL)で順次洗浄した。得られた有機層のBoc-Val-Pro-OHの定量収率は94%であった。
標準物質:インディアン ジャーナル オブ ケミストリー 2004年、43B巻、1282頁を参考に別途単離精製したBoc-Val-Pro-OHを標準物質とした。
標準物質のMASSを示す。
MASS(ESI+)m/z;(M+H)+315.20
定量方法:絶対検量法
Boc-Val-OH(50mg、0.23mmol)、N,N-ジメチルアセトアミド(1.0g)、N-メチルモルホリン(30mg、0.30mmol)を混合させ、0℃にて2,2,4-トリメチルペンタン-3-イルカルボノクロリデート(53mg、0.28mmol)を加え2.5時間攪拌した。この溶液に、別途H-Pro-OH(34mg、0.77mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.80g、1.0mmol)、アセトニトリル(0.50g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、0℃のまま15時間攪拌した(原料:目的物=1:99)。得られた反応液を酢酸エチル(5mL)で希釈し、10質量%クエン酸水溶液(2mL)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層および水層のBoc-Val-Pro-OHの定量収率は90%であった。
Boc-Val-OH(100mg、0.46mmol)、テトラヒドロフラン(1.0g)、N,N-ジイソプロピルエチルアミン(77mg、0.60mmol)を混合させ、0℃にて2,2-ジメチルブタノイルクロリド(74mg、0.55mol)を加え、2時間攪拌した。この溶液に、別途H-Pro-OH(64mg、0.55mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.18g、0.87mmol)、アセトニトリル(1.0g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、0℃のまま21時間攪拌した(原料:目的物=1:520)。得られた反応液を酢酸エチル(10mL)で希釈し、水(2mL)、10質量%クエン酸水溶液(2mL)で分液後、10質量%塩化ナトリウム水溶液(2mL)にて2回洗浄した。得られた有機層のBoc-Val-Pro-OHの定量収率は99%であった。
Boc-Val-OH(100mg、0.46mmol)、テトラヒドロフラン(1.0g)、N,N-ジイソプロピルエチルアミン(77mg、0.60mmol)を混合させ、0℃にて2-エチルブタノイルクロリド(74mg、0.55mol)を加え、2時間攪拌した。この溶液に、別途H-Pro-OH(64mg、0.55mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.18g、0.87mmol)、アセトニトリル(1.0g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、0℃のまま21時間攪拌した(原料:目的物=1:248)。得られた反応液を酢酸エチル(10mL)で希釈し、水(2mL)、10質量%クエン酸水溶液(2mL)で分液後、10質量%塩化ナトリウム水溶液(2mL)にて2回洗浄した。得られた有機層のBoc-Val-Pro-OHの定量収率は99%であった。
Boc-Val-OH(100mg、0.46mmol)、N,N-ジメチルアセトアミド(1.0g)、N-メチルモルホリン(61mg、0.60mmol)を混合させ、0℃にて2,4-ジメチルペンタン-3-イルカルボノクロリデート(99mg、0.55mol)を加え2.5時間攪拌した。この溶液に、別途H-Pro-OH(64mg、0.55mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.18g、0.88mmol)、アセトニトリル(1.0g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、0℃のまま21時間攪拌した(原料:目的物=1:179)。得られた反応液を酢酸エチル(5mL)で希釈し、10質量%クエン酸水溶液(2mL)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層および水層のBoc-Val-Pro-OHの定量収率は89%であった。
MASS(ESI+)m/z;(M+H)+411.28
Fmoc-Val-OH(200mg、0.59mmol)、アセトニトリル(2.0g)、N,N-ジイソプロピルエチルアミン(91mg、0.71mmol)を混合させ、25℃にて2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリドの50質量%トルエン溶液(0.43g、0.71mol)を加え、4時間攪拌した。この溶液に、別途H-MeGly-OH(79mg、0.88mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.22g、1.1mmol)、アセトニトリル(2.0g)を混合させ、75℃にて1時間攪拌して調製した溶液を加え、25℃のまま15時間攪拌した(原料:目的物=1:61)。得られた反応液を酢酸エチル(3.0g)で希釈し、10質量%クエン酸水溶液(2.0g)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層のFmoc-Val-MeGly-OHの定量収率は97%であった。
標準物質:インディアン ジャーナル オブ ケミストリー 2004年、43B巻、1282頁を参考に別途単離精製したFmoc-Val-MeGly-OHを標準物質とした。
標準物質のMASSを示す。
MASS(ESI+)m/z;(M+H)+411.28
定量方法:絶対検量法
Fmoc-Val-OH(200mg、0.59mmol)、N,N-ジメチルアセトアミド(2.0g)、N-メチルモルホリン(78mg、0.77mmol)を混合させ、0℃にて2,2,4-トリメチルペンタン-3-イルカルボノクロリデート(0.14g、0.71mol)を加え2.5時間攪拌した。この溶液に、別途H-MeGly-OH(79mg、0.88mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.22g、1.1mmol)、アセトニトリル(2.0g)を混合させ、75℃にて1時間攪拌して調製した溶液を加え、0℃のまま15時間攪拌した(原料:目的物=1:64)。得られた反応液を酢酸エチル(10mL)で希釈し、水(5mL)、10質量%クエン酸水溶液(5mL)で分液後、水層を酢酸エチル(10mL)で抽出した。有機層を混合し、10質量%塩化ナトリウム水溶液で洗浄した。得られた有機層のFmoc-Val-MeGly-OHの定量収率は97%であった。
Fmoc-Val-OH(100mg、0.29mmol)、テトラヒドロフラン(1.0g)、N,N-ジイソプロピルエチルアミン(50mg、0.38mmol)を混合させ、0℃にて2,2-ジメチルブタノイルクロリド(48mg、0.35mol)を加え、2時間攪拌した。この溶液に、別途H-MeGly-OH(32mg、0.35mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.11g、0.55mmol)、アセトニトリル(1.0g)を混合させ、75℃にて1時間攪拌して調製した溶液を加え、0℃のまま20時間攪拌した(原料:目的物=1:48)。得られた反応液を酢酸エチル(5.0mL)で希釈し、10質量%クエン酸水溶液(2.0mL)で分液後、10質量%塩化ナトリウム水溶液(2.0mL)で2回洗浄した。得られた有機層のFmoc-Val-MeGly-OHの定量収率は95%であった。
Fmoc-Val-OH(100mg、0.29mmol)、テトラヒドロフラン(1.0g)、N,N-ジイソプロピルエチルアミン(50mg、0.38mmol)を混合させ、0℃にて2-エチルブタノイルクロリド(48mg、0.35mol)を加え、2時間攪拌した。この溶液に、別途H-MeGly-OH(32mg、0.35mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.11g、0.55mmol)、アセトニトリル(1.0g)を混合させ、75℃にて1時間攪拌して調製した溶液を加え、0℃のまま20時間攪拌した(原料:目的物=1:20)。得られた反応液を酢酸エチル(5.0mL)で希釈し、10質量%クエン酸水溶液(2.0mL)で分液後、10質量%塩化ナトリウム水溶液(2.0mL)で2回洗浄した。得られた有機層のFmoc-Val-MeGly-OHの定量収率は92%であった。
Fmoc-Val-OH(100mg、0.29mmol)、N,N-ジメチルアセトアミド(1.0g)、N-メチルモルホリン(39mg、0.38mmol)を混合させ、0℃にて2,4-ジメチルペンタン-3-イルカルボノクロリデート(63mg、0.35mmol)を加え2時間攪拌した。この溶液に、別途H-MeGly-OH(32mg、0.35mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.11g、0.55mmol)、アセトニトリル(1.0g)を混合させ、75℃にて1時間攪拌して調製した溶液を加え、0℃のまま20時間攪拌した(原料:目的物=1:33)。得られた反応液を酢酸エチル(5.0mL)で希釈し、10質量%クエン酸水溶液(2.0mL)で分液後、10質量%塩化ナトリウム水溶液(2.0mL)で2回洗浄した。得られた有機層および水層のFmoc-Val-MeGly-OHの定量収率は88%であった。
MASS(ESI+)m/z;(M+H)+409.26
高速液体クロマトグラフィー:SHIMADZU製 HPLC -20A
カラム:Agilent製Poroshell 120EC-C18(2.7μm、3.0×100mm)
カラムオ-ブン温度:50℃
溶離液:0.2vоl%リン酸 アセトニトリル溶液:0.2vоl% リン酸水溶液
35:65(7分)、35:65-95:5(7-14分)、95:5(14-18分)、(v/v)
溶離液速度:0.7mL/分
検出波長:214nm
Boc-Cys(Bn)-OH(100mg、0.32mmol)、アセトニトリル(1.0g)、N,N-ジイソプロピルエチルアミン(50mg、0.39mmol)を混合させ、25℃にて2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリドの50質量%トルエン溶液(231mg、0.39mol)を加え2間攪拌した。この溶液に、別途H-Pro-OH(48mg、0.42mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.12g、0.59mmol)、アセトニトリル(1.0g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、25℃のまま23時間攪拌した(原料:目的物=1:265)。得られた反応液を酢酸エチル(3.0g)で希釈し、10質量%クエン酸水溶液(2.0g)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層および水層を定量し、定量収率97%でBoc-Cys(Bn)-Pro-OHを得た。
標準物質:ケミストリー オブ ネイチャーコンパウンズ 1992年、28巻、344頁を参考に別途単離精製したBoc-Cys(Bn)-Pro-OHを標準物質とした。
標準物質のMASSを示す。
MASS(ESI+)m/z;(M+H)+409.26
定量方法:絶対検量法
Boc-Cys(Bn)-OH(100mg、0.32mmol)、N,N-ジメチルアセトアミド(1.0g)、N-メチルモルホリン(42mg、0.42mmol)を混合させ、0℃にて2、2、4-トリメチルペンタン-3-イルカルボノクロリデート(74mg、0.39mol)を加え2時間攪拌した。この溶液に、別途H-Pro-OH(48mg、0.42mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.12g、0.59mmol)、アセトニトリル(1.0g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、0℃のまま15時間攪拌した(原料:目的物=1:461)。得られた反応液を酢酸エチル(3.0g)で希釈し、10質量%クエン酸水溶液(2.0g)で分液後、10質量%塩化ナトリウム水溶液(1.0g)で2回洗浄した。得られた有機層および水層を定量し、定量収率96%でBoc-Cys(Bn)-Pro-OHを得た。
Boc-Cys(Bn)-OH(100mg、0.32mmol)、アセトニトリル(1.0g)、N,N-ジイソプロピルエチルアミン(54mg、0.41mmol)を混合させ、0℃にて2,2-ジメチルブタノイルクロリド(52mg、0.38mol)を加え2間攪拌した。この溶液に、別途H-Pro-OH(44mg、0.38mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.12g、0.61mmol)、アセトニトリル(1.0g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、0℃のまま19時間攪拌した(原料:目的物=1:31)。得られた反応液を酢酸エチル(5.0mL)で希釈し、10質量%クエン酸水溶液(2.0mL)で分液後、10質量%塩化ナトリウム水溶液(2.0mL)で2回洗浄した。得られた有機層を定量し、定量収率97%でBoc-Cys(Bn)-Pro-OHを得た。
Boc-Cys(Bn)-OH(100mg、0.32mmol)、アセトニトリル(1.0g)、N,N-ジイソプロピルエチルアミン(54mg、0.41mmol)を混合させ、0℃にて2-エチルブタノイルクロリド(52mg、0.38mol)を加え2間攪拌した。この溶液に、別途H-Pro-OH(44mg、0.38mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.12g、0.61mmol)、アセトニトリル(1.0g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、0℃のまま19時間攪拌した(原料:目的物=1:30)。得られた反応液を酢酸エチル(5.0mL)で希釈し、10質量%クエン酸水溶液(2.0mL)で分液後、10質量%塩化ナトリウム水溶液(2.0mL)で2回洗浄した。得られた有機層を定量し、定量収率97%でBoc-Cys(Bn)-Pro-OHを得た。
Boc-Cys(Bn)-OH(100mg、0.32mmol)、N、N-ジメチルアセトアミド(1.0g)、N-メチルモルホリン(42mg、0.42mmol)を混合させ、0℃にて2,4-ジメチルペンタン-3-イルカルボノクロリデート(69mg、0.38mol)を加え2時間攪拌した。この溶液に、別途H-Pro-OH(48mg、0.42mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.12g、0.59mmol)、アセトニトリル(1.0g)を混合させ、50℃にて1時間攪拌して調製した溶液を加え、0℃のまま19時間攪拌した(原料:目的物=1:60)。得られた反応液を酢酸エチル(5.0mL)で希釈し、10質量%クエン酸水溶液(2.0mL)で分液後、10質量%塩化ナトリウム水溶液(2.0g)で2回洗浄した。得られた有機層を定量し、定量収率98%でBoc-Cys(Bn)-Pro-OHを得た。
高速液体クロマトグラフィー:SHIMADZU製 HPLC LC-20A
カラム:Agilent製Poroshell120EC-C18(2.7μm、3.0×100mm)
カラムオーブン温度:50℃
溶離液:0.2vоl%TFA/アセトニトリル:メタノール=1:1
0.2vоl%TFA水溶液
55:45(0-20分)、55:45-95:5(20-20.1分)、95:5(20.1-24分)Post time5分(v/v)
溶離液速度:0.7mL/分
検出波長:214nm
標準物質:合成例96にて合成したCbz-Phe-Phe-MePhe-OHを標準物質とした。
標準物質のMASSを示す。
MASS(ESI+)m/z;(M+H)+608.4
定量方法:絶対検量法
2-クロロトリチルクロリドレジン(200-400メッシュ)(0.10g、0.13mmol)にジクロロメタン(1.0mL)、Fmoc-MePhe-OH(0.062g、0.15mmol)、N,N-ジイソプロピルエチルアミン(0.11mL、0.61mmol)を加えて終夜撹拌した。得られた懸濁液に下記溶液を順次加え、都度ろ過した。(1)(ジクロロメタン/メタノール/N,N-ジイソプロピルエチルアミン=17/2/1)の混合溶液を3回、(2)ジクロロメタンを3回、(3)N-メチルピロリドンを2回、(4)ジクロロメタンを2回、(5)メタノールを5回。ろ過後のレジンが十分浸かるようにN-メチルピロリドンを加え、15分撹拌した。その後ろ過を行い、レジンが十分浸かるように20%ピペリジン/N-メチルピロリドン溶液を加え20分撹拌した。続いてろ過を実施し、レジンをN-メチルピロリドンで10回洗浄した。得られたレジン(0.10g、0.050mmol)に0.5M エチルシアノ(ヒドロキシイミノ)アセテート/N-メチルピロリドン溶液を0.80mL、Fmoc-Phe-OH(0.078mg、0.20mmol)、N-[1-(シアノ-2-エトキシ-2-オキソエチリデンアミノオキシ)ジメチルアミノ(モルホリノ)]ウロニウム ヘキサフルオロホスフェート(0.086g、0.20mmol)、N,N-ジイソプロピルエチルアミン(0.085mL、0.50mmol)を加えて2時間撹拌した。反応溶液をろ過し、レジンをN-メチルピロリドンで10回洗浄した。その後、上記縮合・洗浄操作を再度実施し、20%ピペリジン/N-メチルピロリドンを添加後20分の撹拌とN-メチルピロリドンでの洗浄を10回行った。得られたレジンに0.5M エチルシアノ(ヒドロキシイミノ)アセテート/N-メチルピロリドン溶液を0.400mL、Cbz-Phe-OH(0.060mg、0.20mmol)、N,N-ジイソプロピルカルボジイミド(0.032mL、0.20mmol)を加えて終夜撹拌した。反応液をろ過後、N-メチルピロリドンで10回、メタノールで10回洗浄し、30%ヘキサフルオロイソプロパノールをレジンが十分浸るように加え、10分撹拌後ろ過の操作を5回実施した。集めた溶液を減圧濃縮した結果、Cbz-Phe-Phe-MePhe-OH(0.031g、100%)を得た。
MASS(ESI+)m/z;(M+H)+608.4
Fmoc-Val-OH(0.068g、0.20mmol)をテトラヒドロフラン(1.0mL)と混合させ、0℃にてトリエチルアミン(0.033mL、0.24mmol)、2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリド(0.66g、0.22mmol)を加え2時間撹拌した。この溶液に、別途H-MeTyr(tBu)-OH(0.090g、0.36mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.185mL、0.72mmol)、アセトニトリル(1.5mL)を混合させ、50℃にて1時間撹拌して、調製した溶液を加え、さらに0℃にて1時間攪拌したのちに、25℃にて24時間撹拌した(原料:目的物=1:20(分析条件3))。得られた反応液を酢酸エチル(5mL)で希釈し、飽和炭酸水素ナトリウム水溶液(5mL)を加え、1時間攪拌したのちに、水(5mL)、飽和塩化ナトリウム水溶液(5mL)で順次洗浄した。得られた有機層を濃縮後、ヘキサンで洗浄し、Fmoc-Val-MeTyr(tBu)-OH(0.170g、収率99%)を白色固体として得た。
Fmoc-Val-OH(0.068g、0.20mmol)をテトラヒドロフラン(1.0mL)と混合させ、0℃にてトリエチルアミン(0.033mL、0.24mmol)、2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリド(0.66g、0.22mmol)を加え2時間撹拌した。この溶液に、別途H-MeVal-OH(0.032g、0.24mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.12mL、0.48mmol)、アセトニトリル(1.0mL)を混合させ、50℃にて1時間撹拌して、調製した溶液を加え、さらに0℃にて2時間攪拌したのちに、25℃にて36時間撹拌した(原料:目的物=1:16(分析条件3))。得られた反応液を酢酸エチル(5mL)で希釈し、飽和炭酸水素水溶液(5mL)を加え、1時間攪拌したのちに、水(5mL)、飽和塩化ナトリウム水溶液(5mL)で順次洗浄した。得られた有機層を濃縮後、ヘキサンで洗浄し、Fmoc-Val-MeVal-OH(0.092g、収率92%)を白色固体として得た。
Fmoc-Val-OH(0.068g、0.20mmol)をテトラヒドロフラン(1.0mL)と混合させ、0℃にてトリエチルアミン(0.033mL、0.24mmol)、2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリド(0.66g、0.22mmol)を加え2時間撹拌した。この溶液に、別途H-MeSer(tBu)-OH(0.045g、0.26mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.13mL、0.52mmol)、アセトニトリル(1.0mL)を混合させ、50℃にて1時間撹拌して、調製した溶液を加え、さらに0℃にて1時間攪拌したのちに、25℃にて15時間撹拌した(原料:目的物=1:25(分析条件3))。得られた反応液を酢酸エチル(5mL)で希釈し、飽和炭酸水素水溶液(5mL)を加え、1時間攪拌したのちに、水(5mL)、飽和塩化ナトリウム水溶液(5mL)で順次洗浄した。得られた有機層を濃縮後、ヘキサンで洗浄し、Fmoc-Val-MeSer(tBu)-OH(0.095g、収率96%)を白色固体として得た。
Fmoc-Val-OH(0.068g、0.20mmol)をテトラヒドロフラン(1.0mL)と混合させ、0℃にてトリエチルアミン(0.033mL、0.24mmol)、2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリド(0.66g、0.22mmol)を加え2時間撹拌した。この溶液に、別途H-MeDap(Boc)-OH(0.052g、0.24mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.12mL、0.48mmol)、アセトニトリル(1.0mL)を混合させ、50℃にて1時間撹拌して、調製した溶液を加え、さらに0℃にて1時間攪拌したのちに、25℃にて36時間撹拌した(原料:目的物=1:20(分析条件3))。得られた反応液を酢酸エチル(5mL)で希釈し、飽和炭酸水素水溶液(5mL)を加え、1時間攪拌したのちに、水(5mL)、飽和塩化ナトリウム水溶液(5mL)で順次洗浄した。得られた有機層を濃縮後、ヘキサンで洗浄し、Fmoc-Val-MeDap(Boc)-OH(0.107g、収率99%)を白色固体として得た。
Fmoc-Val-OH(0.068g、0.20mmol)をテトラヒドロフラン(1.0mL)と混合させ、0℃にてトリエチルアミン(0.033mL、0.24mmol)、2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリド(0.66g、0.22mmol)を加え2時間撹拌した。この溶液に、別途H-MeGln(Trt)-OH(0.097g、0.24mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.12mL、0.48mmol)、アセトニトリル(1.0mL)を混合させ、50℃にて1時間撹拌して、調製した溶液を加え、さらに0℃にて1時間攪拌したのちに、25℃にて44時間撹拌した(原料:目的物=1:21(分析条件3))。得られた反応液を酢酸エチル(5mL)で希釈し、飽和炭酸水素水溶液(5mL)を加え、1時間攪拌したのちに、水(5mL)、飽和塩化ナトリウム水溶液(5mL)で順次洗浄した。得られた有機層を濃縮後、ヘキサンで洗浄し、Fmoc-Val-MeGln(Trt)-OH(0.151g、収率104%)を白色固体として得た。
Fmoc-Val-OH(0.068g、0.20mmol)をテトラヒドロフラン(1.0mL)と混合させ、0℃にてトリエチルアミン(0.033mL、0.24mmol)、2-(4,4-ジメチルペンタン-2-イル)-5,7,7-トリメチルオクタノイルクロリド(0.66g、0.22mmol)を加え2時間撹拌した。この溶液に、別途H-MeGlu(OtBu)-OH(0.078g、0.36mmol)、N,O-ビス(トリメチルシリル)アセトアミド(0.185mL、0.72mmol)、アセトニトリル(1.5mL)を混合させ、50℃にて1時間撹拌して、調製した溶液を加え、さらに0℃にて1時間攪拌したのちに、25℃にて24時間撹拌した(原料:目的物=1:25(分析条件3))。得られた反応液を酢酸エチル(5mL)で希釈し、飽和炭酸水素ナトリウム水溶液(5mL)を加え、1時間攪拌したのちに、水(5mL)、飽和塩化ナトリウム水溶液(5mL)で順次洗浄した。得られた有機層を濃縮後、ヘキサンで洗浄し、Fmoc-Val-MeGlu(OtBu)-OH(0.176g、収率109%)を白色固体として得た。
Claims (24)
- 下記工程(1)乃至(3):
(1)
式(I)P-A1-OH(式中、PはN末端保護基であり、A1は、アミノ酸由来の基、N-C1-6アルキルアミノ酸由来の基(C1-6アルキルは置換基を有していてもよい)またはペプチド由来の基を表す)で表されるN末端保護アミノ酸またはN末端保護ペプチドを
式(II)
(式中、Xはハロゲン原子を表し、R1は炭素数が5以上であり、置換基を有していてもよい2級または3級の脂肪族炭化水素基を表すか、あるいは炭素数が4以上であり、置換基を有している1級の脂肪族炭化水素基(ここで、1級の脂肪族炭化水素基の置換基は、カルボニル炭素に結合する炭素原子上に存在する)を表す)で表されるカルボン酸ハロゲン化物、および式(III)
(式中、Xはハロゲン原子を表し、R2は炭素数が5以上であり、置換基を有していてもよい2級の脂肪族炭化水素基を表す)で表されるハロゲン化ギ酸アルキルよりなる群から選択される活性化剤と混合する工程;
(2)
式(IV)H-A2-OH(式中、A2は、N-C1-6アルキルアミノ酸由来の基(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸由来の基(4-6員環は、C6-14アリール環、C6-14ハロアリール環およびC3-8シクロアルキル環からなる群から選ばれる環状化合物と縮合していてもよい)、あるいはN末端残基がN-C1-6アルキルアミノ酸(C1-6アルキルは置換基を有していてもよい)または4-6員の環状の2級アミノ酸(4-6員環は、C6-14アリール環、C6-14ハロアリール環およびC3-8シクロアルキル環からなる群から選ばれる環状化合物と縮合していてもよい)であるペプチド由来の基を表す)で表されるアミノ酸またはペプチドをシリル化剤と混合する工程;
(3)
工程(1)で得られた生成物と工程(2)で得られた生成物とを混合する工程、
を含むペプチドの製造方法。 - 下記工程(1)乃至(3):
(1)
式(I)P-A1-OH(式中、PはN末端保護基であり、A1は、アミノ酸由来の基またはN-C1-6アルキルアミノ酸由来の基(C1-6アルキルは置換基を有していてもよい)を表す)で表されるN末端保護アミノ酸を
式(II)
(式中、Xはハロゲン原子を表し、R1は炭素数が5以上であり、置換基を有していてもよい2級または3級の脂肪族炭化水素基を表すか、あるいは炭素数が4以上であり、置換基を有している1級の脂肪族炭化水素基(ここで、1級の脂肪族炭化水素基の置換基は、カルボニル炭素に結合する炭素原子上に存在する)を表す)で表されるカルボン酸ハロゲン化物および
式(III)
(式中、Xはハロゲン原子を表し、R2は炭素数が5以上であり、置換基を有していてもよい2級の脂肪族炭化水素基を表す)で表されるハロゲン化ギ酸アルキルよりなる群から選択される活性化剤と混合する工程;
(2)
式(IV)H-A2-OH(式中、A2は、N-メチルアミノ酸由来の基、N-C1-6アルキルグリシン由来の基(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸由来の基、あるいはN末端残基がN-メチルアミノ酸、N-C1-6アルキルグリシン(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸であるペプチド由来の基を表す)で表されるアミノ酸またはペプチドをシリル化剤と混合する工程;
(3)
工程(1)で得られた生成物と工程(2)で得られた生成物とを混合する工程、
を含むペプチドの製造方法。 - 下記工程(1)乃至(3):
(1)
式(V)P-A3-OH(式中、PはN末端保護基であり、A3は、ペプチド由来の基を表す)で表されるN末端保護ペプチドを
式(II)
(式中、Xはハロゲン原子を表し、R1は炭素数が5以上であり、置換基を有していてもよい2級または3級の脂肪族炭化水素基を表すか、あるいは炭素数が4以上であり、置換基を有している1級の脂肪族炭化水素基(ここで、1級の脂肪族炭化水素基の置換基は、カルボニル炭素に結合する炭素原子上に存在する)を表す)で表されるカルボン酸ハロゲン化物と混合する工程;
(2)
式(IV')H-A2’-OH(式中、A2’は、N-メチルアミノ酸由来の基、N-C1-6アルキルグリシン由来の基(C1-6アルキルは置換基を有していてもよい)、または4-6員の環状の2級アミノ酸由来の基を表す)で表されるアミノ酸をシリル化剤と混合する工程;
(3)
工程(1)で得られた生成物と工程(2)で得られた生成物とを混合する工程、
を含むペプチドの製造方法。 - 工程(3)で得られたペプチドのN末端の保護基を除去する工程を含む、請求項1乃至3のいずれか一項に記載のペプチドの製造方法。
- さらに下記工程(4)および(5):。
(4)
工程(3)または(5)で得られたペプチドのN末端の保護基を除去する工程;
(5)
工程(4)で得られたペプチドのN末端に、N末端保護アミノ酸またはN末端保護ペプチドを反応させる工程、
の繰り返しを1以上含む、請求項1乃至3のいずれか一項に記載のペプチドの製造方法。 - 式(I)P-A1-OHまたは式(V)P-A3-OH(式中、PはN末端保護基であり、A1およびA3は、それぞれペプチド由来の基を表す)で表されるN末端保護ペプチド中のC末端に位置するアミノ酸が、N-C1-6アルキルアミノ酸(C1-6アルキルは置換基を有していてもよい)または4-6員の環状の2級アミノ酸(4-6員環は、C6-14アリール環、C6-14ハロアリール環およびC3-8シクロアルキル環からなる群から選ばれる環状化合物と縮合していてもよい)以外のアミノ酸である、請求項1または3に記載のペプチドの製造方法。
- A1が、アミノ酸由来の基である、請求項1または2に記載のペプチドの製造方法。
- 式(I)で表されるN末端保護アミノ酸または式(I)で表されるN末端保護ペプチド中のC末端に位置するアミノ酸が、α-アミノ酸、β-アミノ酸またはγ-アミノ酸である、請求項1または2に記載のペプチドの製造方法。
- 式(I)で表されるN末端保護アミノ酸又は式(I)で表されるN末端保護ペプチド中のC末端に位置するアミノ酸が、α-アミノ酸である、請求項8に記載のペプチドの製造方法。
- 式(IV)で表されるアミノ酸または式(IV)で表されるペプチド中のN末端に位置するアミノ酸が、N-C1-6アルキル-α-アミノ酸(C1-6アルキルは置換基を有していてもよい)または4-6員の環状の2級-α-アミノ酸である、請求項1に記載のペプチドの製造方法。
- 式(IV)で表されるアミノ酸または式(IV)で表されるペプチド中のN末端に位置するアミノ酸が、N-メチル-α-アミノ酸もしくはN-エチル-α-アミノ酸(N-メチル、N-エチルはそれぞれ置換基を有していてもよい)または4-6員の環状の2級-α-アミノ酸である、請求項1に記載のペプチドの製造方法。
- 活性化剤が式(II)で表されるカルボン酸ハロゲン化物であり、R1の炭素数が5乃至20であり、Xが塩素原子である、請求項1乃至11のいずれか一項に記載のペプチドの製造方法。
- 活性化剤が式(III)で表されるハロゲン化ギ酸アルキルであり、Xが塩素原子である、請求項1または2に記載のペプチドの製造方法。
- シリル化剤がトリメチルシリル化剤である請求項1乃至18のいずれか1項に記載のペプチドの製造方法。
- シリル化剤が、N,O-ビス(トリメチルシリル)アセトアミド、N,N′-ビス(トリメチルシリル)尿素またはN,O-ビス(トリメチルシリル)トリフルオロアセトアミドである、請求項1乃至19のいずれか1項に記載の、ペプチドの製造方法。
- シリル化剤が、N,O-ビス(トリメチルシリル)アセトアミドである、請求項1乃至20のいずれか1項に記載の、ペプチドの製造方法。
- 式(IV)で表されるアミノ酸またはペプチドが、それぞれ、プロリン以外のアミノ酸またはN末端残基がプロリン以外のアミノ酸残基であるペプチドである、請求項1に記載のペプチドの製造方法。
- 式(IV’)で表されるアミノ酸が、プロリン以外のアミノ酸である請求項3に記載のペプチドの製造方法。
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